1 Abstract 2 Abstract 3 Abstract 4

Bangladesh Society of Microbiologists (BSM) 1st International Conference (28th AGM), 2015

Abstract 2 Microbial quality analysis of irradiated and frozen non-spotted indo-pacific king mackerel fish during preservation Najmun Naher Orin*, Badrut Tamam Ahmed Department of Microbiology, Stamford University Bangladesh *Corresponding author. E-mail: [email protected], [email protected]

Aquaculture products can harbor pathogenic bacteria which are part of the natural micro-flora of the environment. Radiation has been used as a fungicidal and bactericidal treatment in the seafood. An investigation was undertaken for the isolation and identification of fish-borne microorganisms from non-spotted Indo-Pacific King Mackerel (Scomberomorus guttatus) collected from Tongi Bazar at Gazipur of Dhaka, Bangladesh. Fish samples were irradiated with 3 kGy of gamma radiation and kept at -20°C.Three types of samples were taken for the analysis of bacteria and fungi, these are packet washed water, fished washed water and fish blend. The microbial load was higher in non-irradiated fish samples than irradiated fish samples.In terms of non-irradiated fish samples Vibrio cholerae count(4.5×106) and listeria (4.4×105) were highest in fish blend samples where as salmonella (3.7×106) and fungal count(3.6×106) were highest in packet washed water samples. The similar result pattern were observed in irradiated fish samples.The highest listeria count(3.0×105) was observed in fish blend samples and salmonella count(1.0×106) and fungal count (1.1×106) were highest in packet washed water samples.But there was one exception in terms of Vibrio cholerae count which was highest in irradiated packet washed water (1.4×105) than fish blend (2.1×104).The results indicate that the load of pathogenic organisms will decrease if more dose applied in the sample.

Abstract 3 Molecular characterization of hepatitis B virus and mutation analysis Modhusudon Shaha*, Sheikh Ariful Hoque, Sabita Rezwana Rahman National Institute of Biotechnology (NIB), Savar, Dhaka-1349, Bangladesh. *Corresponding author. E-mail: [email protected]

Hepatitis B virus (HBV) is highly contagious and causes liver diseases. Globally more than 350 million people are chronically infected and among them above 80% are from developing countries like Bangladesh. Resistance to existing drugs and vaccines are common phenomenon due to mutations in ‘a’ determinant. Due to lack of data about mutations and subtypes of HBV genome in Bangladesh this study demands to be documented. The aim of this study is to determine the genotypes and subtypes of HBV prevalent in Bangladesh, and their significant mutations associated with vaccine and drug resistance. Blood samples were collected from 385 patients with jaundice like illness during July to December 2013 from different hospitals. HBV-DNA was extracted (by Stratec molecular kit, Berlin, Germany) and detected by Polymerase chain reaction (PCR). Partial S gene was sequenced using ABI PRISM® 3500xL Genetic Analyzer. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5.0. A total of 54 samples (14%) were found HBV positive and predominant genotype was D (73.7%), followed by genotype A (15.8%), and genotype C (10.5%). Here, most prevalent subtype observed was ayw3 (47.4%), followed by ayw2 (26.3%), adw2 (15.8%) and adr (10.5%). A significant number of mutations (T118V, T125M, T126I, P127T, A128V, T/S143L/M) were observed in ‘a’ determinant region which probably have impact on the structure and function of that gene. Phylogenetic analysis showed that these viruses are from various origin. This is the first comprehensive study from Bangladesh reporting mutations and subtypes which has clinical importance like disease diagnosis and treatment.

Abstract 4 1


Investigation of beta lactamase activity and determination of macrolide antibiotic resistance mediators in clinical Escherichia coli Mayen Uddin and Sunjukta Ahsan* Department of Microbiology, University of Dhaka, Bangladesh *Corresponding author. Email: [email protected]

This study investigated the susceptibility of clinical E. coli to selected antibiotics including a Carbapenem Imipenem, β lactams – Ceftriaxone and Ceftazidime and a macrolide – Azithromycin. The study also analyzed mediators of resistance that encompassed enzyme production, efflux pump activity and presence of mobile genetic elements. Test isolates were variably resistant to Ceftazidime and Azithromycin. All of them were resistant to Ceftriaxone. In contrast, most of the isolates were sensitive to Imipenem. The MICs of Ceftazidime and Azithromycin were found to be between 128 µg/ml to 256 µg/ml with some showing MICs ranging between 1024 µg/ml and 2048 µg/ml. The MIC for Imipenem was 0.50 µg/ml and 2 µg/ml for intermediately resistant isolates. The prevalence of β lactamase producers was 57.89%. ESBL activity was exhibited by 36.84% of the isolates. Some exhibited activity against Ceftazidime and Ceftriaxone (21.05%, n=19), while others were active against either Ceftriaxone (10.52%, n=19) or Ceftazidime (5.26%, n=19). Except two clinical isolates all test isolates harbored plasmids of various sizes in different numbers. No specific correlation could be found between plasmid sizes and antibiotic resistance patterns. Efflux pump was found to be involved in Azithromycin resistance in 63.15% of the tested isolates. Of these, 9 (75% of efflux pump mediators) also contained mph(A) (phosphotransferase) and 1 (8.33%) contained erm(A) (esterase); however, none of them contained the gene for erm(C).In the present study, the erm(A) and erm(C) genes were detected in 10.53% of the test isolates each. The isolates that contained erm(A) gene were different from those that contained erm(C) gene. The gene for phosphotransferase, mph(A) was found to be the most common among the macrolide modifying genes. It was detected in 73.68% (14 out of 19) of the isolates. Both the isolates that contained erm(C) also contained mph(A), whereas only one isolate (C6) contained both erm(A) and mph(A).This study concluded that clinical E. coli can be resistant to multiple classes of antibiotics and use various mechanisms of resistance to combat them.

Abstract 05 Determination of antibiotic susceptibility profile and pathotypes of Escherichia coli of environmental and clinical origin isolated from Dhaka, Bangladesh Asmaul Husna2, Sunjukta Ahsan1,*, Marufa Zerin Akhter1, Md. Shahidul Kabir3, Shamima Begum2, Sheikh Shahidul Islam4 and Arif Ahmed Khan4 1

Department of MIcrobiology, University of Dhaka, Bangladesh, 2Department of Microbiology, Jagannath University, Dhaka, Bangladesh, 3Department of Micrbiology, Stamford University, Dhaka, Bangladesh, 4 Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Dhaka Cantonment *Corresponding author. E-mail: [email protected]

Pathogenic forms of Escherichia coli are commonly known to cause a variety of diarrheal diseases in hosts. This study was carried out to investigate the distribution of pathogenic Escherichia coli isolates from environmental samples such as water, food and clinical samples such as stool, pus, tracheal swab etc. by multiplex PCR and observe their antibiotic resistance patterns to eighteen commercial antibiotics. For this study, a total of 48, environmental (n= 28) and clinical (n = 20) E. coli were used. All of the E. coli isolates revealed the same morphological, cultural and biochemical characteristics. All isolates of E. coli showed metallic sheen on EMB agar plate. After initial biochemical characterization the prevalence of E. coli pathotypes (ETEC, EPEC, EIEC, and EAEC) was determined by multiplex PCR. Out of forty eight reconfirmed E. coli it was observed that the prevalence of ETEC was 22.92 % (11/48), EAEC was 4.17% (2/48), and EPEC was 4.17 % (2/48). When calculated individually, clinical isolates exhibited 35 % (7/20) ETEC and 5% (1/20) EAEC. As for environmental samples the prevalence of ETEC was 14.28 % (4/28), EAEC was 3.57% (1/28) and EPEC was

2


7.14% (2/28). Eighteen commercial antibiotics were used to study the antibiogramof the isolated E. coli. In this study, clinical E. coli were70-100% resistant to most of the antibiotics tested except Chloramphenicol, Meropenem, Nitrofuratoin, Piperacillin/Tazobactam and Amoxycillin. Greatest resistance (100%) was observed against Ampicillin, Metronidazole and Clindamycin. On the other hand, environmental E. coli showed greatest resistance to Metronidazole and Clindamycin (100% for each) and 10-35% resistance to most of the antibiotics except Imipenem, Azithromycin and Ampicillin. All except three environmental isolates contained the genetic element characteristic of Class 1 integron, a common carrier of antibiotic resistance genes the Asian subcontinent. There was no correlation between the number of plasmid and antibiotic resistance pattern. This was reflected by the finding that bacteria showing resistance to more than one antibiotic carried only one plasmid and isolates carrying multiple plasmids were not as drug resistant. The present study suggests that drug resistant E. coli are prevalent in the environment and in clinical samples isolated from Dhaka city. Environmental E. coli may play a role in antibiotic resistance gene transfer and causing diseases in human, a matter of great public health significance.

Abstract 6 Source tracking of E. coli of clinical and environmental origin isolated from Dhaka, Bangladesh Reshma Sultana and Sunjukta Ahsan* Department of Microbiology, University of Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

The present study investigated the phylogenetic groups and molecular types of clinical and environmental E. coli. The detection and abundance of Escherichia coli in the environment is used to monitor and mandate the quality of drinking and recreational water. Distinguishing commensal waterborne E. coli isolates from those that cause diarrhea or extra-intestinal disease in humans is important for assessing human health risks. Phylogenetic analysis of E. coli forms part of microbial source tracking by which the source of the contamination can be identified. PCR analysis of three marker genes chuA, yjaA and the DNA fragment TspE4.C2 in 41 clinical and environmentalE. coli was used for phylotyping in the present study. Of the 41 test bacteria, 36.58% contained chuA, 46.34% carried yjaA and 56.09% amplified the TspE4.C2 fragment. These strains were assigned to four main phylogenetic groups, A, B1, B2 and D, which can be divided into eight subgroups (A0, A1, B11, B12, B22, B23, D1 and D2), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Most isolates belonged to the phylogenetic group B1 (41.5%) followed by group A (22%), then group D (19.5%) and finally group B2 (17%). The source of clinical isolates was tracked to commensal groups A1 and B1 (63.12%), while the rest belonged to extra-intestinal pathogens B2 and D comprising 26.32% and 10.53%, respectively. On a similar note, environmental isolates were tracked back to 60% commensal and 40% extra-intestinal pathogens. New phylogenetic subgroups (B11 and B12) were defined in the present study for group B1 to improvise for yjaA+ and yjaA- markers. The genetic diversity among the different phylogenetic groups was investigated by ERIC-PCR with subsequent dendogram construction. Genetic diversity among the isolates was found to be less heterogeneous. With some exceptions, environmental isolates shared the same cluster with each other as did clinical isolates. The present study determined the source of E. coli to be commensal or extra-intestinal (pathogens) irrespective of the nature of the sample (clinical or environmental). The presence of both commensal and pathogenic E. coli in clinical and environmental samples may be explained by extensive gene transfer among the members of this species.

Abstract 7 Azithromycin resistance and mediators in clinical isolates of Salmonella enterica serovars Typhi and Paratyphi Sahida Rahman1, Sunjukta Ahsan1,* and M Shahidul Kabir2

3

Bangladesh Society of Microbiologists (BSM) 1st International Conference (28th AGM), 2015 1

Department of MIcrobiology, University of Dhaka, Bangladesh 2Department of MIcrobiology, Stamford University, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

Salmonellaenterica is an important cause of morbidity and mortality worldwide. Salmonellaenterica serovar Typhi and Paratyphi are the etiologic agents of typhoid and paratyphoid fever while non-typhoid Salmonella spp. are associated with gastroenteritis and invasive infections in children, the elderly and immune compromised. This study was carried out to investigate macrolide resistance and identify mediators of resistance in clinical isolates of Salmonellaenterica serovar Typhi and Paratyphi. Genetic diversity among the isolates was investigated by ERIC and RAPD PCR assays. By antibiotic susceptibility testing it was observed that 95% of the isolates were resistant to Azithromycin and 100% were resistant to Clindamycin and Metronidazole. MIC values of azithromycin ranged between 32 µg/ml and 128 µg/ml. Small and mega plasmids were not found in any isolates but class1 integrons were present. Efflux pumps were present in 36 isolates (96%) out of 40 samples (including Salmonellaenterica serovar Typhi and Paratyphi). The gene erm(B) for methylase involved in macrolide resistance was found in 25 isolates (62.5%). Other resistance genes viz. mph(A)and mph(B) for phosphotransferase, ere(A), ere(B) and erm(C) for esteraseswerenotfoundbymultiplexPCR. The efflux pump gene mef(A) was not found in the test isolates. The Minimum Regrowth Concentration (MRC) of Azithromycin active against Salmonella enterica serovars Typhi and Paratyphi was found to be 5 to 10 times greater than the corresponding MIC values found for the test isolates. Molecular characterization suggested that ERIC-PCR was unable to bring out a serovar specific differentiation among the isolates. In contrast, RAPD was better in differentiation of the test isolates.

Abstract 8 Biodecolorization of a textile reactive dye by fungal isolate Aspergillus fumigatus EF-1 M Ekramul Karim*, Kartik Dhar and M Towhid Hossain Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh. *Corresponding author. E-mail:[email protected]

In view of compliance with environmental legislation and sustainable environmental development, it is necessary to consider viable remediation methods of industrial effluent than the conventional treatment methods. Hence, the present study deals with screening and characterization of dye decolorizing fungi isolated from dying effluents and optimization of different cultural conditions to maximize bioremediation of a commercially available reactive dye. Screening of dye decolorization was performed in Czapek Dox Agar (CDA) medium amended with the test dye C.I. Reactive Blue 268 (0.1 g.L-1) based on clear zone formation. Decolorization was assayed using spectrophotometric method. A number of different cultural conditions such as inoculums age, temperature, pH, different carbon and nitrogen sources were optimized. Among the fungal isolates, Aspergillus fumigatus showed potential activity in dye decolorization which was finally selected for detail study in Czapek Dox broth (CDB) medium. Complete decolorization of the test dye was recorded within 6 days of static incubation at 27°C. However, the isolate was unable to utilize the dye as a sole source of energy in CDA and CDB in absence of sucrose and obligate requirement of a labile carbon source, i.e., sucrose needed for induction of decolorization. Biosorption seems to play the privotal role in decolorization as evident by coloring of the fungal biomass as that of dye color. The optimal conditions for the highest decolorization of the test dye were found at 30°C and pH 6.0 with 6 days old inoculums supplemented with sucrose (10 g.L-1) and ammonium chloride (2 g.L-1) as a carbon and nitrogen source, respectively. Surprisingly, about 65% of dye decolorization was recorded with heat inactivated biomass powder within 6 days of static incubation. Results of this study have established the candidature of the isolate for biotechnological removal of dyes from disreputable dying effluents. Also, dead fungal biomass seems to be a better alternative to treat dying effluent than commercial physical adsorbent like activated carbon as soon as it is proved that the decolorization level is at least as good as with activated carbon.

4


Abstract 9 Inhibition of biofilm forming multi-drug resistant Pseudomonas aeruginosa from wound infection by Lactobacillus sp. Sohana Al Sanjee*, Mohammod Mahmudul Hasan and M Abul Manchur Prime Asia University, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

Bacterial wound infections are a global problem among the hospital acquired infections (HAI) associated with morbidity and mortality. The primary aim of this study was to evaluate the antibiogram profile of multidrug resistant (MDR) Pseudomonas aeruginosa from wound infection and the antagonistic effect of Lactobacillus against it. Firstly, a total of 15 isolates were selected as the representative pathogens of the wound infection (Surgical Site and burn) using Cetrimide Agar. Later, three isolates were finally selected on the basis of their multiple antibiotic resistances (MAR index) after observing antibiotic susceptibility pattern by Kirby Bauer Disc Method (Bauer et al., 1966) and identified as P. aeruginosa according to ‘‘Bergey’s Manual of Determinative Bacteriology’’. 17 antibiotics from β lactam, aminoglycosides, quinolones, cephalosporins, macrolide, carbapenem, sulfonamides, tetracycline and others were used. One isolate (P3) was found resistant to all the antibiotics. The Meropenem, Imipenem and Ceftriaxone were found effective against the two other isolates (P1 and P2). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Meropenem and Ceftriaxone has been determined by “Microdilution” method for the three isolates. Though one of the isolates (P3) was resistant to all of the antibiotics according to the “CLSI Standards for Antimicrobial Susceptibility Testing”, its MIC and MBC has also been done because of its zone of inhibition (ZoI) against those antibiotics. All the selected isolates were biofilm former in microtiter plate assay and test tube assay but in CRA method, only one isolate (P2) showed positive result. Anti-pseudomonal lactic acid bacteria (LAB) was screened by agar cup method with the isolates L. coryniformis sub sp.coryniformis, L. plantarum, L. homohiochii, L. delbruckii and L. xylosus. Only L. coryniformis sub sp. coryniformisshowed significant results towards the planktonic as well as biofilm of the isolates. The potent LAB was bacteriocin like substance producer and H2O2 and organic acids production increased with the incubation period. The remarkable inhibition was caused by organic acids and also by bacteriocin. H2O2 did not cause any appreciable inhibitory activity. The inhibitory activity was lost after treatment with papain indicating bacteriocin like substance as protein and the antimicrobial activity was checked after chloroform extraction and ammonium sulfate precipitation method. Though Meropenem showed the best result, LAB showed more significant result i.e. zone of inhibition than Ceftriaxone which can show us a new path for the treatment of wound infection associated with the MDR microorganisms.

Abstract 10 Multi-drug resistant bacteria in hospital wastewater in Bangladesh Jahidul Alam*, Syeda Tasneem Towhid, Nazratan Naeem and Suvamoy Datta Department of Microbiology, Primeasia University, Dhaka1213, Bangladesh. *Corresponding author. E-mail: [email protected]

Use of antibiotics is indispensable in modern medical service raising the risk of antibiotic-resistance in hospital environment and hospital wastes. This research work aims to report the distribution of bacteria in hospital effluents, occurrence of antibiotic-resistant bacteria in it and the risk it poses to the population exposed to the effluent. Hospital effluent was collected from three state-run general hospitals and the bacterial species present there were identified by cultural and biochemical methods. E. coli, Proteus vulgaris, Salmonella sp., Pseudomonas sp., Klebsiella sp., Vibrio sp. and Enterobactor sp. were found to be predominant in the effluent. At least 75%, 20% and 60% of E coli isolates were resistant against sulfomethoxazole, ampicillin and erythromycin respectively; 84% and 56% of Klebsiella isolates were resistant against ampicillin and erythromycin respectively; 60% and 40% of Vibrio isolates were resistant against ampicillin and erythromycin

5


respectively and 66% of Salmonella isolates were resistant against ampicillin. In conclusion, release of effluent with multi-drug resistant bacteria can cause hard-to-treat infections. Therefore better waste treatment and surveillance is needed for the sake of public health.

Abstract 11 Bacterial skin and soft tissue infection in Dhaka, Bangladesh Tahmina Aktar, Syeda Tasneem Towhid, K M Shahidul Islam, Kaniz Fatema, Parimal Majumder, Suvamoy Datta* Department of Microbiology, Primeasia University, Dhaka-1213, Bangladesh. *Corresponding author. E-mail: [email protected]

Specimen from 300 patients with different kinds of skin and soft tissue infections were collected and cultured on blood agar, MacConkey agar and chocolate agar. Colonies cultured in these media were identified by biochemical tests and then checked for antibiotic susceptibility. These data taken together with the patient history provided the prevalence of skin and soft tissue infections in Dhaka, Bangladesh. 32.42% of the skin and soft tissue infections were due to bacterial agents, with Escherichia coli (52%), Staphylococcus aureus (27%), Pseudomonas (18%), Acinetobacter (3%), Candida (1%), Serratia (1%) and Proteus (2%) being the major pathogens. Men within the age group of 40-60 years had the highest number (41.89%) of infections, with E coli (21.62%) being the most common pathogen. Women within the reproductive age (18-45 years) were infected by E coli, S aureus and Pseudomonas (16.67%) while E coli (23.33%) was the predominant cause of infections in post-menopausal women. 12.24% males and 28.57% female patients were diabetic. The rate of post-surgical nosocomial infection was 6.86% while 9.8% contracted nosocomial infections from non-surgical sources. The infections were recurrent in 25.49% cases. A wide-spread resistance against amoxicillins and β-lactams, azithromycin and second generation cephalosporins was found.

Abstract 12 Detection of Extra Pulmonary Tuberculosis (EPTB) by GeneXpert MTB/RIF Paul Daru1*, Mostofa Kamal2, Suvamoy Datta1 1

Department of Microbiology, Primeasia University, Dhaka-1213, Bangladesh. 2 National Tuberculosis Reference Laboratory (NTRL), Mohakhali, Dhaka, Bangladesh. *Corresponding author. E-mail:[email protected]

Tuberculosis (commonly shortened to TB) is a chronic bacterial infection, caused by the bacterium Mycobacterium tuberculosis, which most commonly affects the lungs (pulmonary TB) but can also affect the extra-pulmonary system. Extra-pulmonary tuberculosis (EPTB) refers to disease outside the lungs like central nervous system (meningitis), lymphatic system, circulatory system (miliary TB), genitourinary system, bones and joints. Extra pulmonary tuberculosis is not uncommon. As the lesion is in the pocy-bacillary, microscopy was not sufficient for bacteriological evidence. Xpert MTB/RIF and liquid culture were found to be effective for early bacteriological diagnosis of EPTB. During the period of August 2014 to October 2014 a total of 152 patients with different extra-pulmonary lesion attended in the NTRL. Of these 70(46%) were male and 82(54%) were female. Young and adult age group attended more than the other. Majority specimen (Pus and lymph node) were from lymph node. Among 152 patient 46(30.26%) were positive by GeneXpert and culture positive cases were positive in 40(26%) cases. Compare to culture, the sensitivity, specificity and positive and negative predictive value were 95%, 92%, 82% and 98% respectively. Therefore GeneXpert appears as an accurate tool for the diagnosis of Extra pulmonary tuberculosis.

Abstract 13 6


Comparative analysis of antibiotic sensitivity pattern in Streptococcusmutans isolated from dental carries of different age & sex groups in Dhaka city, Bangladesh 1

Tasnim Sharmin1, Priyanka Dutta2, Suvamoy Datta1, * Department of Microbiology, Primeasia University, 9 Banani, Dhaka 1213. 2 Department of Public Health, American Intrnational University of Bangladesh (AIUB), Kemal Attaturk Avenue, Banani, Dhaka 1213. *Corresponding author. E-mail: [email protected]

Streptococcus mutans plays a significant role in dental caries and control of its activities can promote prevention of dental caries. Dental diseases are recognized as a major public health problem throughout the world. Teeth and their supporting structure the gum (gingival) are subjected to infection by cariogenic bacteria that causes cavity and pyorrhea, which if left untreated can eventually lead to gingivitis. It was seen that 0% (Amikacin, Meropenem) to 96.52% (Penicillin) of the S. mutans strains are resistant to the antibiotics used against them. Amikacin & Meropenem (100 % sensitive) are the best drugs for the treatment of S. mutans infection followed by Imipenem (97.5% sensitive). Elder male and female samples exhibited a higher resistance rate than isolates from young male and female. Two of the samples (5%) were resistant to 8 antibiotics, Eight of the samples (20%) were resistant to 7 antibiotics, Eleven of the samples (27.5%) were resistant to 6 antibiotics, and Eight of the samples (20%) were resistant to at least 8 antibiotics. Over 72.5% of the strains are Multidrug resistant (resistant to 5 or more of the antibiotics tested). From this study, Amikacin & Meropenem (100 % sensitive) are the best drugs for the treatment of S. mutans infection followed by Imipenem (97.5% sensitive). A more conservative approach to using antibiotics. Maintaining or upgrading oral hygiene practices, and good infection prevention and control measures such as hand washing /teeth brushing. Developing new lines of antibiotics that are effective against S. mutans.

Abstract 14 Isolation, identification and antibiotic sensitivity pattern of Salmonella typhi and Salmonella paratyphi A isolated from blood samples of patients in Dhaka city Bangladesh M Saiful Islam, Smritimoy Datta, Suvamoy Datta* Department of Microbiology, Primeasia University, Dhaka 1213, Bangladesh. *Corresponding author. E-mail: [email protected]

In recent years, there has been a significant rise in the prevalence of multidrug resistance Salmonella typhi and salmonella paratyphi A in Dhaka city. It is therefore a subject of interest to observe the number of incidence and antimicrobial resistant pattern of typhoidal Salmonella in patients. To study the prevalence rate and identification of typhoidal Salmonella typhi & S. paratyphi A among the patients attending the at a hospital by conventional and serologic test. To determine the antimicrobial sensitivity pattern of clinically isolated S. typhi & S. paratyphi A by antibiogram. Ampicillin; Azithromycin; Cefixime; Ceftriaxone; Chloramphenicol; Gentamycin; Nalidixic acid; Cotrimoxazole and Ciprofloaxin were used in this study.During August 01 to October 31, 2014; a total of 4106 blood samples of typhoid suspected patients were tested at Ibne Sina Hospital, Dhaka. Amongst these 395 samples came out positive which is 9.62% of the total samples studied. Amongst those 395 samples 313 isolates were found to be Salmonella typhi (79.24%) and 82 isolates as Salmonella paratyphi A (20.76%). Both the organisms were found to be totally susceptible to Gentamycin and Ceftriaxone but were found totally resistant to Nalidixic acid. Ampicillin (26.84%), Cotrimoxazole (13.10%), Chloramphenicol (12.46%), Azithromycin (10.12%) and Ciprofloxacin (1.28%) are resistance to Salmonella typhi. Salmonella paratyphi A are resistance to Ampicillin (69.51%), Cotrimoxazole (1.22%), Chloramphenicol (1.22%), Azithromycin (26.83%) and Ciprofloxacin (1.22%). Typhoid fever occurs infrequently in developed counties but massively in developing countries. Traditionally the drugs of choice were chloramphenicol, ampicillin, and cotrimoxazole but unfortunately the emergence of multidrug resistant strains of S. typhi &S. paratyphi A introduced the use of ciprofloxacin among the patients. However, now a day’s ciprofloxacin used

7


frequently as a drug of choice but this study, isolated S. typhi & S. paratyphi A are sensitive to ciprofloxacin (92.01 % and 68.29% respectively), that twisted the circumstances into different directions.

Abstract 15 Extraction, purification and characterization of pyocyanin from Pseudomonas aeruginosaand study of its antimicrobial activity against pathogenic microbes Popy Devnath, Md Towhid Hossain* and M A Manchur Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh *Corresponding author. E-mail: [email protected]

Pyocyanin is a secondary metabolite, which has a broad range of antimicrobial activity, produced by Pseudomonas aeruginosa. In the present study, primarily 16 isolates of P. aeruginosa were isolated from various clinical samples (skin swab, urine and pus) using cetrimide agar as a selective medium and observed for pyocyanin production. Among them, 5 isolates were finally selected as potential pyocyanin producer. The isolates were provisionally identified on the basis of their microscopic features, physiological and biochemical characteristics. The isolates were cultured on pseudomonas broth for the production of pyocyanin. Then the productwas extracted using chloroform and the presence of pyocyanin was further confirmed by the addition of 0.1N HCl. The amount of pyocyanin pigment produced in per ml of culture broth was quantified spectrophotometrically. The culture conditions were optimized for maximum yield of pyocyanin during their growth in broth. The optimum incubation period was 72 hours at 37oC. Pseudomonas broth medium was modified with asparagine or alanine and better yield was found in alanine rich medium. Partial purification of the pigment was done by column chromatography. After column chromatography,Thin layer chromatography (TLC) was done to check its purity. The Rf value was found around 0.81 for all the isolates. To characterize the sample as pyocyanin, it was further subjected to FT-IR and UV-visible spectrophotometric analysis. FT-IR analysis assigned different functional groups (-OH, -C=N, -CH3 etc.) which belongs to the aromatic structure of pyocyanin. In UV-Vis spectrophotometric analysis, a maximum absorption was observed at 270-271 nm. The partially purified pyocyanin pigment was subjected to antibacterial activity against human pathogens such as Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Salmonella enteric (NCTC 6017), Bacillus cereus and Klebsiella pneumoniae by using cross streak, and agar cup method. All the test pathogens showed sensitivity to pyocyanin pigment in cross streak method. Both crude extract and partially purified pyocyanin were used in agar cup method. These studies revealed that pyocyanin from various isolates of P. aeruginosa showed significant antibacterial activity against S. aureus (ATCC 6538), E. coli (ATCC 8739), and S. enterica (NCTC 6017) and B. cereus. Among the test pathogens, S. aureus was highly sensitive against pyocyanin of all isolates. Antifungal activity of pyocyanin also determined against ochratoxin producer Aspergillus ochraceus as test fungi, where the crude extract of P. aeruginosa (PS1) showed maximum inhibition (67%).

Abstract 16 Evaluation of Gene X-pert by conventional method (Lowenstein-Jensen LJ-DST) for early detection of MTB/RIF resistant tuberculosis M Anwar Hossain1*, Armand Van Deun2, Suvamoy Datta1 1

Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213. 2 Mycobacteriology Unit, Institute of Tropical Medicine, B 2000 Antwerpen, Belgium. *Corresponding author. E-mail: [email protected]

Anti-tuberculosis (TB) drug resistance is more emergences and spread of MDR-TB/ XDR-TB is threatening to the global health in TB care and control worldwide. Rapid detection of MDR TB and proper treatment initiation of all patients for resource limited countries like Bangladesh need to search for methods to detect DR TB that

8


are affordable, faster. From the evaluation of DF- Bangladesh strains of 2013 have been compared for R resistant strains to see accuracy of the modern technique X-pert. Gene X-pert DST with LJ –DST has been analyzed. Total 324 sputa were detected as MTB by Gene X-sputa were cultured in DF-Netrakona Ref. Lab/ SRL Antwerp for setting in DST on LJ which is known as gold standard. Analytical result:- LJ-DST detected 358 strains as MTB where as Gene X-pert detected among 152 true resistant by LJ DST confirmed by Xpert DST. Total true susceptible 189, false resistant 12, false susceptible 05 Accuracy stands to 95.3% by X-pert compare to LJ-DST. True Susceptable-189 & False Susceptible -05 by X-pert. Sensitivity of the X-pert DST 96.8% Specificity of of X-pert 94.0% & Predictive value resistant 92.7%, Predictive value susceptible is 97.4% So it is highly efficient for Bangladesh NTP program for early detection of MDR-TB. It shows that this test is highly appreciated because of its accuracy compare to gold standard LJ-DST

Abstract 17 Antibiotic sensitivity pattern of Escherichia coli isolated from the UTI patients of different age & sex groups in Chittagong area, Bangladesh M Maksudur Rahman, Smritimoy Datta, Suvamoy Datta* Department of Microbiology, Primeasia University, Dhaka 1213, Bangladesh. *Corresponding author. E-mail: [email protected]

Urinary tract infection (UTI) is a serious health problem and it has been estimated that about six million patients visit outpatient departments and about 300,000 are treated in the wards every year for UTI worldwide. With the constantly shifting trends in drug resistance, antibiotic options, and multiplying microorganisms, UTI implies both microbial colonization of the urine and invasion of the lower or upper urinary tract by microorganisms. For this study consecutive urine samples of 120 of person of both sexes and various age groups were taken in a hospital setting of both out patients and inpatients. There was marked gender variations is all age groups, which comprised 0-90 years of age of the member of urine sample requested for examination only 33.33% of specimens yielded E. coli positive culture. It has been reported earlier that E.coli is the most important cause of UTI. This is also corroborated in this study. But the sensitivity pattern of the organisms has shown much change from the earlier studies. In this study the organisms are resistant to common urinary antibiotics which were used before through oral route. In this study incidence of infection by E. coli was much higher in the patient between 21-40 years of age as compared to other age groups and the incidence of disease is higher in female (70%) than male (30%). More than 90% sensitivity found for Imipenem, Amikacin & Nitrofurantoin against E. coli. Now most of these antibiotics can not be used as the organisms have developed resistance to these antibiotics due to indiscriminate use. Now it is imperative that indiscriminate use of antibiotics on the clinical diagnosis of UTI, should be taken with caution and full course of treatment should be given after full culture and sensitivity test only.

Abstract 18 Multidrug resistance pattern of Staphylococcus aureus isolated from pus samples of different age and sex groups in Dhaka city, Bangladesh M Rubel Sikder, Suvamoy Datta* Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213 *Corresponding author. E-mail: [email protected]

Staphylococcus aureus is an important human pathogen that causes skin and soft tissue abscesses. Abscess formation is not unique to staphylococcal infection and purulent discharge has been widely considered a physiological feature of healing and tissue repair. S. aureus is also an important pathogen due to a combination of toxin-mediated virulence, invasiveness and antibiotic resistance. This study aims to find out multidrug resistance pattern of isolated Staphylococcus aureus from pus samples. It was seen that 5% (Amikacin &

9


Gentamycin) to 100% (Penicillin & amoxycillin) of the S. aureus strains are resistant to the antibiotics used against them. Amikacin & Gentamycin (100% sensitive) are the best drugs for the treatment of S. aureus infection followed by Ciprofloxacin (77.5% sensitive) and Tetracycline (60 % sensitive). Cloxacillin is partially sensitive to both elder male and female samples but totally resistance to both young male and female samples. Six of the samples (15%) were resistant to 9 antibiotics, Three of the samples (7.5%) were resistant to 8 antibiotics, Three of the samples (7.5%) were resistant to 7 antibiotics, Seven of the samples (14.5%) were resistant to 6 antibiotics and Seven of the samples (17.5%) were resistant to at least 5 antibiotics. Overall 65% of the strains are Multidrug resistant (resistant to 5 or more of the antibiotics tested). Pus infection has been a major concern among health care practitioners not only in terms of increased trauma to the patient but also in view of its burden on financial resources and the increasing requirement for cost-effective management within the health care system.

Abstract 19 Multidrug resistance pattern of Beta-Haemolytic Streptococci isolated from throat swab of different age and sex groups M Anwar Hossain, Kaniz Fatema, Bushra Zannat, Suvamoy Datta* Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213 *Corresponding author. E-mail: [email protected]

Beta haemolytic Streptococci is an important human pathogen responsible for a myriad of infections such as pharyngo-tonsillitis, pyoderma, scarlet fever, necrotizing fasciitis, toxic shock syndrome and septicemia. Molecular mimicry between streptococcal and host antigens results in generation of cross-reactive antibodies which target host tissues leading to non-suppurative sequelae such as rheumatic fever (RF), rheumatic heart disease (RHD), post streptococcal glomerulonephritis (PSGN), reactive arthritis and other brain disorders. It was seen that 27.5% (Doxycycline) to 91.25% (Bacitracin) of the Beta-haemolytic Streptococci (SBH) strains are resistant to the antibiotics used against them.Doxycycline (72.5 % sensitive) are the best drugs for the treatment of Beta-haemolytic Streptococci (SBH) infection followed by Amoxycillin (50. 7% sensitive) and Gentamycin(50 % sensitive). Bacitracin is partially sensitive to both young male and female samples but totally resistance to both elder male and female samples. The rising resistance rates of SHB, especially to macrolides, tetracycline and quinolones are probable due to the extensive use of these antibiotics. Confirmation of the presence of S. pyogenes at the site of infection by bacteriological cultures, serological grouping and susceptibility testing are recommended to avoid inappropriate antibiotic treatment and increased drug resistance. There is also a definite need to control over- the-counter drug-abuse. Self-administration of antibiotics in dosages sufficient to subside symptoms may not eliminate the pathogen completely, resulting in chronic carriage or recurrent infections.

Abstract 20 Effect of chitosan on physical status of mammals and in vivo antimicrobial effect on pathogenic bacteria Sazin Islam, Smritimoy Datta, Maruf Abony, Suvamoy Datta* Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213 *Corresponding author. E-mail:[email protected]

The shells as raw materials was extracted from shrimp (Metapenous Monoceros) to extract chitosan in this experiment. Demineralizations process was carrieded out by 4% HCl at room temperature in the ratio of 1:14(w/v).. The deproteinization process was initiated by 5% NaOH at 90°C for 24 hours with a solvent to solid ratio of 12:1(v/w). Removal of acetyl groups from the chitin was achieved by using 70% NaOH solution with a solid to solvent ratio of 1:14 (w/v) at room temperature for 72 hours. Extracted chitosan was soluble in

10


1% acetic acid. Rabbit were treated with extracted chitosan to determine in vivoeffects. 2.74g of chitosan was up taken by each rabbits per day. 80gm of standard food was given to each rabbit during experiments. Physical status was observed and stool samples were collected to observe antimicrobial before chitosan treatment and after chitosan treatment. Presence of pathogenic bacteria was detected by streaking loop-full sample on three specific media. Coliform detection was initiated by MacConkey Agar. Salmonella-Shigella detection was initiated by S-S Agar. Pseudomonas detection was initiated by Cetrimide Agar. Before chitosan treatment 2300g body weight was found, stool status was rigid and physical movement was normal. After chitosan treatment a continuous bacterial growth in the 7th, 10th, 15th day but none of Coliform and SalmonellaShigella growth occurred that indicates a ratio of growth of normal microbial flora. The physical status of the rabbit was change during chitosan treatment. Continuous reduction in weight and looseness of stool was observed, but physical movement was normal.

Abstract 21 Microbiological qualities of some foods sold in the street and in the mid-level and high-level restaurants Asma Binte Afzal1, M. Mahboob Hossain2,*, Naiyyum Choudhury1 1

Biotechnology Programme, MNS Department, BRAC University, Mohakhali, Dhaka-1212, Bangladesh. 2Microbiology Programme, MNS Department, BRAC University, Mohakhali, Dhaka-1212, Bangladesh *Corresponding author. E-mail: [email protected], [email protected]

Street foods and restaurant foods play an important role in people’s daily food options as well as their regular nutritional requirements. Over the years, many food-borne diseases have been reported due to contaminated non-homemade food consumption. The present study was conducted to analyze the microbiological quality of foods which are sold by the street side vendors, mid-level restaurants and high-level restaurants and analyzed the microbiological quality of seven most commonly consumed food items i.e rice, dal, ruti, cake, biscuit, sugarcane juice, and laddu, of street side carts, mid-level restaurants and high-level restaurants of Paribagh and Kawran Bazaar areas of Dhaka city, Bangladesh. Total viable count (TVC), Coliform and Enteric pathogen count (CEC), Staphylococci count (SC), Fungal count (FC) and also the presence of urinary tract infection causing pathogens, Salmonella spp., Shigella spp. were observed in this study. Microbiological quality of cooked food items was better than raw food items. Mid-level restaurant foods showed less microbial load than street side cart and high-level restaurant foods. According to the biochemical test results 27% invasive pathogens, 61% opportunistic pathogens and 12% rare pathogens were found in food samples depending on the source. Invasive food pathogens were Staphylococcus aureus (54.54%), Bacillus cereus(27.27%), Shigella dysentriae (9.09%), Proteus mirabilis (9.09%) and also positively identified some opportunistic and rare pathogens from obtained food samples. This study specifically highlighted the microbiological quality of nonhomemade food items found in three different categories vending places. Finally, this study recommends some preventive measures which the government and food-maker together should follow, and also maintain the good hygienic practice to prepare, cook and handle foods. Implementation of such measures particularly on street food vendors and also on restaurant foods are highly crucial to maintain the hygienic condition as well as to avoid spreading of harmful organisms through consumption of contaminated foods.

Abstract 22 Isolation of yeasts from natural sources for bioethanol production from vegetable peels and the role of cellulose degrading bacteria (Bacillus subtilis) on ethanol production Salman Khan Promon1, M. Mahboob Hossain2,*, Naiyyum Choudhury1 1

Biotechnology Programme, MNS Department, BRAC University, Mohakhali, Dhaka-1212, Bangladesh. 2Microbiology Programme, MNS Department, BRAC University, Mohakhali, Dhaka-1212, Bangladesh *Corresponding author. E-mail:[email protected], [email protected]

11


Microbial production of bioethanol can replace the conventional fossil fuel with green energy. In this study, local yeast isolates were used for the production of bioethanol using cellulosic vegetable wastes as substrate. Wild-type yeast isolated form sugarcane juice (SC1) and date juice (DJ1) were used as ethanol producing organisms. After proper isolation, identification and characterization of stress tolerance (thermo-, ethanol-, pH-, osmo- & sugar tolerance), detailed characterization and optimization of physiochemical parameters for ethanol production of the strain was done. Very inexpensive and easily available raw materials (vegetable peel kitchen wastes) were then used as fermentation media. The overall objective of this study was to meet the demand for an inexpensive and highly efficient integrated anaerobic Saccharomyces spp. Fermentation process to produce ethanol as an energy source directly from insoluble lignocellulosic substrate (kitchen-waste). Fermentation was optimized with respect to temperature, reducing sugar concentration and pH. Analysis of fermentation characteristics under different substrate and environmental conditions, it was observed that temperature of 30°C and pH 6.0 were optimum for fermentation with maximum yield of ethanol. The maximum percentage of ethanol produced by yeast using kitchen waste as substrate was 5.34%. However, when the kitchen waste was inoculated with yeast isolates after being incubated with Bacillus subtilis for 24 hours, the ethanol production rate went to 17.39% after 48 hours incubation at 30°C.

Abstract 23 Isolation of diesel and kerosene degrading bacteria from soil samples and determination of optimum growth conditions Mashiat Nawar Chowdhury1, Tasmin Naila1, M. Mahboob Hossain2*, Naiyyum Choudhury1 1

Biotechnology Programme, MNS Department, BRAC University, Dhaka, Bangladesh, 2Microbiology Programme, MNS Department, BRAC University, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected], [email protected]

Oil spills are global catastrophes transpiring annually from minute to substantial amounts, threatening the lives of plants, animals and humans. Current physical and chemical management systems are very costly and less efficient. Bioremediation through utilization of oil-degrading bacteria proves to be an auspicious hope for the future. Diesel and kerosene are petroleum products consisting of high percentage of aromatic compounds and the BTEX conglomerate. Hydrocarbons in these oils have been linked to detrimental effects on human health and contribute to extremely noxious pollutions during spills. In this study, several bacterial isolates from four soil samples were assessed for their ability to degrade kerosene and diesel. These isolates were incubated on mineral salts broth for 7 days at 35°C in the absence of any carbon source apart from kerosene or diesel. Growths of some organisms were observed by visible turbidity and these were enumerated by CFU/mL on mineral salts agar. The genera of eight isolates were identified as Nocardia, Corynebacterium, Bacillus, Pseudomonas and Arthrobacter by morphological characterization and biochemical test results compared to standard references. Pseudomonas sp. exhibited to be the most promising amongst the isolated microbes at utilizing the oils, followed by Bacillus sp., both of which were isolated from soils that were previously contaminated by oil. Isolates such as Nocardia and Corynebacterium taken from soil samples dearth of prior exposure to oil contamination were unable to grow in the presence of kerosene or diesel. Pseudomonas sp. tolerated kerosene as high as 6%, and displayed optimum growth at 3% kerosene (v/v). This isolate also showed optimal growth at 3% (v/v) diesel concentration, especially with agitation at 120 rpm. Ammonium sulfate of 1 g/L was observed to be optimum nitrogen source concentration for growth in media with kerosene as sole carbon source. Near neutral pH proved to be conducive for the growth of the isolate in both diesel and kerosene.

Abstract 24 Genomic diversity analyses of indigenous Bacillus thuringiensis of Bangladesh by PCR-RAPD method

12


Safirun Pervin1*, Asaduzzaman Shishir1, Shakila N. Khan2 and Md. Mozammel Hoq2 1

Department of Microbiology, Primeasia University, Dhaka, Bangladesh. 2Department of Microbiology, University of Dhaka, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

The study aims to determine the genomic diversity of Bacillus thuringiensis strains by PCR-RAPD (Randomly Amplified Polymorphic DNA) method. Bacillus thuringiensis is a ubiquitous, Gram-positive and spore-forming bacterium, recognized as an eco-friendly pest control agent and known to have diversity among them within and between the environments. About 212 isolates from different locations of Bangladesh were collected and confirmed by starch hydrolysis test and categorized based on their biochemical properties as biotypes. PCRRAPD analysis performed with 212 Bt strains and amplicons were analyzed by agarose gel (1.5%) electrophoresis. For diversity analysis genotyping was done based on the various sizes of RAPD products. There were 23 genotype where genotype II was found to be the most prevalent (n=60) followed by genotype V (n=27), genotype I (n=24), Genotype XX (n=10) and so on. Diversity was also analyzed based on biotypes and locations. From the phylogenetic trees, it was observed that, diversity was present not only between the isolates from different biotypes and locations, but also within the isolates of same biotype and location. Maximum diversity was observed among isolates from biotype indiana (0.869) followed by kurstaki (0.826) and thuringiensis (0.565). Again isolates from Dhaka (0.826) followed by Jamalpur (0.652 and Natore (0.434) have maximum diversity and isolates of Cox’s Bazar and Chapainawabgonj have little diversity. This study will facilitate the research on Bacillus thuringiensis in Bangladesh by providing valuable information to find out diverse Bt strains those which can be used against resistant pest and for biotechnological purpose.

Abstract 25 Seroprevalence and detection of avian influenza type A in ducks at Nikli and Bajitpur upazila of Bangladesh M. Z. Hassan1*, B. C. Das2, M. S. Mahmud1, M. A. Amin2, M. A. Yousuf1, M. Jaber2, S. M. S. H. Belal2, M. A. Hasan1, A. Hossen1, M. R. Karim1, M. S. Rahman3 and M. F. Hoque3 Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh. 2Department of Livestock Services, Dhaka, Bangladesh. 3Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh. *Corresponding author. E-mail: [email protected] 1

Waterfowl are the natural reservoir of avian influenza viruses and ducks may play a role in the maintenance of avian influenza type A. The aim of the present study was to investigate the seroprevalence and detection of avian influenza virus (AIV) type A in duck. This study was carried out during July 2013 to December 2013 on AIV type A from semi-scavenging farm at Nikli and Bajitpur upazila of Kishoregonj district in Bangladesh. A total of 368 blood samples were collected from duck and tested by indirect ELISA for seroprevalence. For detection of AIV type A, The cloacal swabs were collected from 75 duck and subjected to RNA extraction and real time RT-PCR (rRT-PCR) with specific primer and probe for detection of matrix (M) gene. The average seroprevalance of AIV type A in seven different age groups was found to be 90.21%. The highest (25.81 %) seroprevalence was found in 5 months age of birds and the lowest (2.44 %) was found in 12 months age of birds. As regard to area distribution, the average degree of seroprevalence was 93.51% from Nikli had the highest order than Bajitpur (86.88%) upazila of Bangladesh. In case of cloacal sample by using rRT–PCR, out of 15 pooling cloacal samples, two pooling samples (13.33%) that contain 10 samples were positive and 13 pooling samples showed negative (86.67%) for AIV type A in duck. It can be concluded that the long distance movement of duck flocks, may influence outbreak of avian influenza virus (AIV) type A among different poultry species in Bangladesh. Therefore, it needs to develop control strategy for future dissemination of AIV in duck population.

Abstract 26 13


Abstinence from injecting drugs is associated with decline in inflammatory makers but not with monocyte activation markers Salequl Islam1,2*, Jacquie Astemborski2, Huifen Li3, Sean X. Leng3, Damani Piggott2, Shruti H. Mehta2, and Gregory D. Kirk2 1

Department of Microbiology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh. 2 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD. 3Division of Geriatric Medicine and Gerontology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD *Corresponding author. E-mail: [email protected]; [email protected]

Chronic inflammation has been associated with HIV/AIDS disease progression and non-AIDS comorbidities among persons who inject drugs (PWID). Yet, sparse data exist on the relationship of longitudinal inflammation trajectories to injection use. We examined the effects of changes in injecting behavior on changes in interleukin6 (IL-6), soluble tumor necrosis factor receptor-2 (sTNFR-2), soluble CD14 (sCD14) and soluble CD163 (sCD163). Study participants were HIV-infected and uninfected persons in the AIDS Linked to the Intravenous Experience cohort with high frequency injection drug use (>daily) at baseline, and injecting drug cessation at subsequent 6 and 12 month visits. Serum IL-6, sCD14, sCD163 and sTNFR-2 levels were measured by ELISA. Paired t-test was used to analyze biomarker level differences at 6 and 12 month visits compared to baseline. The Wilcoxon rank-sum test was employed to examine HIV effects. Among 100 participants (50 HIV-positive and 50 HIV-negative), IL-6 and TNFR-2 levels decreased significantly after 12-months of cessation (p=0.004 and 0.037). No reduction in sCD14 or sCD163 was observed. sTNFR-2, sCD14 and sCD163 levels were higher among HIV-positive individuals (p90%) with other previously published isolates. Comparison of all these study sequences of VP1 genes from the Asia 1 and serotype O isolates revealed no amino acid substitution. However, including the outcome of the present study, further studies will be helpful for the development of vaccine.

Abstract 30 Association of Shigella dysenteriae with Anabaena variabilis inlaboratory microcosms Mehedee Hasan1 and Md. Abdul Karim1*, Zahid Hayat Mahmud2 and Md. Sirajul Islam2 1

Laboratory of Microbiology, Department of Botany, University of Dhaka, Dhaka 1000. 2Environmental Microbiology Laboratory, Centre for Food and Waterborne Diseases, ICDDR’B, Dhaka. *Corresponding author. E-mail: [email protected]

Shigella spp. are the causative agent of shigellosis with Shigella dysenteriae being one of the most prevalent in many developing countries. It is the principal cause of endemic diarrheas and is generally associated with a severe, longer duration of diarrhea with blood in the stools. Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act as foci of infection. The present investigation was aimed to determine the association of S. dysenteriae with cyanobacteriain laboratory microcosms. A reference strain of S. dysenteriaeand the cyanobacteria Anabaena variabilis were used in this study. Survival of culturable S. dysenteriae inmicrocosms was monitored using simple drop plate method on MacConkey and Salmonella Shigella (SS) agar medium. S. dysenteriae fromthe reference strain were detected using Polymerase Chain Reaction (PCR). S. dysenteriae could be isolated for up to 10 days in aculturable form in association with cyanobacteria, up to 8 days in water with algae and 5 days in water without algae. The non-culturable S.dysenteriae was also detected in the microcosms through PCR technique which showed that S.dysenteriae survived better in non-culturable state with A. variabilis than the control and algal water. These results, therefore, suggest that A. variabilis can act as a long-term reservoir of S.dysenteriae in an aquatic environment. Moreover, the association of S.dysenteriae with A. variabilis demonstrated in this study may be important for understanding the epidemiology of shigellosis.

Abstract 31 Isolation and molecular identification of Aeromonas hydrophila from diseased shing (Heteropneustes fossilis) with antibiogram study Tanvir Ahammed, M Alimul Islam and SM Lutful Kabir* Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh *Corresponding author. E-mail: [email protected]

Fish play a crucial role in the Bangladeshi diet, providing more than 60% of animal source food, representing a crucial source of micro-nutrients, and possessing an extremely strong cultural attachment. In this study we isolated and initially identified Aeromionas hydrophila was done by studying cultural properties, gram staining and biochemical properties of isolates of Shing fish (Heteropneustes fossilis) of different upazillas of Mymensingh district. Antibiogram profile of the isolated bacteria was studied by using wide range of

16


commercially available antibiotics.Quantitative study of bacteria isolated from diseased Shing fish showed variation of number in different organ. Total bacterial load was found to be 1.90 × 105, 1.19 × 105, 3.21 × 105, 2.18 × 106 and 3.14 × 105 cfu/g in lesions; 2.52 × 107, 2.34 × 108, 5.41 × 108, 2.54 × 109 and 5.21 × 109 cfu/g in liver; 2.54 × 108, 2.41 × 108, 1.90 × 107, 3.65 × 107 and 3.45 × 108 cfu/g in spleen; 3.51 × 107, 5.28 × 107, 3.14 × 106, 1.85 × 107 and 4.52× 107 cfu/g in kidney in diseased Shing of Mymensingh sadar, Muktagacha, Tarakanda, Gouripur and Fulpur upazillas, respectively under Mymensingh districts. Aeromonas hydrophila was initially identified by their specific morphological, physiological and biochemical characteristics. Then molecular detection of A. hydrophila was done by PCR. PCR products of desired 760 bp were obtained for A. hydrophila. The results of the antibiotic sensitivity test is exhibited that most of the bacterial samples were sensitive against ciprofloxacin (92%) and lvofloxacin (84%), intermediate sensitive against gentamycin (40%) and resistance against novobiocine (84%), ampicillin (100%) and penicillin (92%).

Abstract 32 Isolation, molecular identification and antibiogram profiles of Campylobacter species, Salmonella species and Escherichia coli from broiler meat sold in different upazila markets of Mymensingh, Sherpur and Gazipur districts of Bangladesh SM Lutful Kabir*, Abu Saim Al-Salauddin and Sukumar Saha Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh *Corresponding author. E-mail:[email protected]

The study was conducted to investigate the presence of Campylobacter species. Salmonella species, Esherichia coli from broiler meat collected from different upazila markets of Mymensingh, Gazipur and Sherpur districts and to evaluate their antimicrobial profiles. A total of 60 samples were collected from 20 upazila live bird markets for 6 months study period. It was found that among 60 samples, most of them were contaminated with Campylobacter species (86%).and 32% samples were contaminated with Salmonella and all samples were contaminated with E. coli. Total 52 isolates of Campylobacter spp. 19 Salmonella and 60 isolates of E coli were confirmed from broiler meat by conventional cultural techniques, biochemical test followed by PCR (Polymerase chain reaction). The Campylobacter spp.were isolated and identified by culturing on blood base agar with 5% sheep blood where Campylobacter spp.produced gray color spreading colonies and positive to catalase, oxidase and hippurate hydrolysis test. Campylobacter specific 16S rRNA gene, were amplified from all isolates. The Salmonella spp.were identified by observing pink colonies with black centre on XLD agar, positive to MR test and negative to Indole and VP test as well as amplification of histidine transport operon gene were positive in PCR.All the isolates of Escherichia coli showed metallic sheen on EMB agar and produced rose pink colonies on MacConkey agar, positive to Indole and MR, but negative to VP test and fermented all five basic sugars and produced both acid and gas. In PCR all the isolates of E coli were positive to the amplification of 16S rRNA gene. Antimicrobial susceptibility test was performed to know the susceptibility and resistance pattern of the isolates to different antimicrobial agents. Almost all isolates of Campylobacter species, Salmonella species and E. coli showed their highest susceptibility to gentamicin, ciprofloxacin and norfloxacin whereas most isolates were resistant to amoxicillin and erythromycin. The findings of the study revealed the presence of multidrug resistant isolates of Campylobacter, Salmonella and Escherichia coli in broiler meat. Results of this study demonstrated the high levels of microbial contamination and occurrence of pathogenic bacteria that reflect the poor hygienic quality of dressed broiler meat sold in different upazila markets of Bangladesh.

Abstract 33 Occurrence of Salmonella and Vibrio species in fresh fishes collected from different markets of Mymensingh, Gazipur and Sherpur districts and their characterization Amit Dutta, S. M. Lutful Kabir* and Muhammad Tofazzal Hossain

17


Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh *Corresponding author. E-mail: [email protected]

Fish is known to harbour bacteria of public health significance. Aquatic environments are known to influence the bacterial loads in the harvested fish. Present work was undertaken to determine total viable count (TVC), total salmonella count (TSC) and total vibrio count (TVibC) in fresh fishes collected from markets of Mymensingh, Gazipur and Sherpur districts. Isolation and identification of Salmonella spp. and Vibrio spp. was also done from pangas (n=20), tilapia (n=20) and koi (n=20) collected from different markets of Mymensingh, Gazipur and Sherpur districts. Samples were cultured on plate count agar to determine TVC, xylose-lysine deoxycholate agar (XLD) to determine TSC and thiosulfate citrate bile salt sucrose agar (TCBS) to determine TVibC. The mean values of TVC, TSC and TVibC in pangas were log 9.09±0.616, log 5.32±0.391 & log 3.14±0.557 CFU/g; log 8.46±0.441, log 5.26±0.589 & log 3.59±0.823 CFU/g and log 7.58±0.466, log 3.28±0.493 & log 2.88±0.386 CFU/g of Mymensingh, Gazipur and Sherpur respectively. Similarly, the mean values of TVC, TSC and TVibC in tilapia were log 6.60±0.790, log 3.59±0.388 & log 3.75±0.176 CFU/g; log 6.55±0.553, log 3.26±0.502 & log 3.67±0.021 CFU/g and log 6.74±0.372, log 3.44±0.411 & log 3.05±0.609 CFU/g of Mymensingh, Gazipur and Sherpur respectively and in koi were log 7.51±0.537, log 3.49±0.459 & log 3.35±0.390 CFU/g; log 7.66±0.752, log 3.25±0.465 & log 3.59±0.581 CFU/g and log 7.13±0.393, log 3.27±0.384 & log 3.43±0.297 CFU/g of Mymensingh, Gazipur and Sherpur respectively. The targeted Vibrio spp. and Salmonella spp. were isolated and identified from collected fishes. All the isolates of Salmonella were confirmed by targeting genus specific gene histidine transport operon gene. Antimicrobial sensitivity test was done for all the isolates of Salmonella and Vibrio species by disc diffusion method. Out of forty five isolates of Salmonella, seven were found multi drug resistant. The bacteria isolated from fish were of public health significance as well as responsible for spoilage of fish. Proper hygienic measures should be taken during harvesting, selling and processing of fish to safeguard public health.

Abstract 34 Bacterial assessment of milk collected from different markets of Mymensingh, Gazipur and Sherpur districts with particular emphasis on the molecular detection and antimicrobial resistance of the isolated bacteria Mohammad Farhad Hossain, S. M. Lutful Kabir* and Md. Tanvir Rahman Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh *Corresponding author. E-mail: [email protected]

The study was conducted to determine the total viable count (TVC) and total coliform count (TCC) of unpasteurized, pasteurized and UHT milk samples. A total of 60 samples were collected from different markets of Mymensingh, Gazipur and Sherpur districts. The TVC of milk was performed to know the bacterial load in supplied milk samples and TCC of milk was performed to know the coliform bacterial load in supplied milk samples. Milk samples were cultured onto various selective media for the isolation of bacteria. The isolated bacteria were identified by cultural properties on different selective media, biochemical tests and finally by PCR. Out of 60 samples 20 unpasteurized milk samples from different markets were found positive for Staphylococcus aureus and 19 unpasteurized milk samples were found positive for Escherichia coli.S. aureus specific 16S rRNA gene was amplified from all isolates. Out of 19 isolates of E. coli, 15 isolates were amplified by 16S rRNA gene based PCR. Results of antimicrobial susceptibility test showed that most of the isolates of S. aureus and E .coli were susceptible to azithromycin, streptomycin, gentamicin, norfloxacin, tetracycline and ciprofloxacin but resistant to amoxicillin and erythromycin. The results of this study indicate that pasteurized and UHT milk is safe for human consumption but the unpasteurized milk from markets without any treatment has the public health importance as well as zoonotic importance.

Abstract 35 18


Bacteriological assessment of tap water collected from different markets of Mymensingh, Gazipur and Sherpur districts with special focus on the molecular detection and antimicrobial resistance of the isolated E. coli M Shihab Hassan, SM Lutful Kabir* and M Tanvir Rahman Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh *Corresponding author. E-mail:[email protected]

The objectives of this study were to assess the bacteriological quality of tap water samples obtained from different markets of different upazilas of Mymensingh, Sherpur & Gazipur district. For achieving the above mentioned objectives, methods of heterotrophic plate count (HPC) and total coliform count (TCC) were applied. Moreover, isolated E. coli from tap water samples were characterized by using biochemical test, molecular method and antimicrobial susceptibility tests. HPC was highest in market tap water collected from Kaligonj and TCC was highest in market tap water of collected from Mymensingh sadar. The geometric mean of HPC of Mymensingh, Gazipur and Sherpur districts water was 8.4x105, 2.5 x106 and 6.8 x105 C.F.U/100 ml. Out of 20 isolates of E. coli, 20 isolates were amplified by using 16S rRNA gene based PCR. In respect to antimicrobial susceptibility testing, most of the E. coli isolates were susceptible to, norfloxacin, ampicilin, tetracycline, streptomycin and ciprofloxacin. Furthermore, a few E. coli isolates were intermediate resistant to gentamycin and ciprofloxacin. However, a few of the E. coli isolates were resistant to erythromycin and amoxycilin. Moreover, out of 20 E. coli isolates 3 (15%) isolates were detected as multidrug resistant. This study indicated the presence of multidrug resistant E. coli isolates in tap water in Mymensingh, Sherpur and Gazipur districts that warrants particular attention.

Abstract 36 Characterization of wild, mutant and recombinant keratinase of Bacillus licheniformis Tabassum Tasnim Auroni, Mukitu Nahar, Md. Asaduzzaman Shishir, Shakila Nargis Khan and Md. Mozammel Hoq* Department of Microbiology, University of Dhaka, Dhaka, Bangladesh. *Corresponding author. E-mail: [email protected]

Bacterial keratinase has large scale usage worldwide for several biotechnological applications. In this study, the keratinolytic activity of naturally occurring keratinase producing strain Bacillus licheniformis MZK05 was compared to previously developed mutant strain BlM9 and a recombinant strain E. coli BL21 for identifying a much broader activity for industrial application. BlM9 differed from MZK05 in colony morphology and some biochemical tests. Mutant showed higher rate of casein hydrolysis on Skim Milk Agar medium than wild. Enzymes, obtained from culture supernatant, were assayed by using azocasein as substrate and the keratinolytic activity of BlM9 (167.6 U/ml) was found 2.3 times higher than MZK05 (73.8 U/ml). To confirm the keratinolytic activity of BlM9; it was important to detect kerA gene in the organism. PCR was carried out with two sets of newly designed primers selected from conserved sequences of kerA for the detection. After purifying the PCR products for both organisms and sequencing; it was found that at position 91, MZK05 had Glutamic acid (E) but BlM9 had Aspartic acid (D). Determining the optimum pH and temperature of keratinase activity, it was deduced that wild and mutant strains showed their highest keratinase activity at pH 8.0 and at 40°C in feather meal broth, while recombinant BL21 showed their highest activity at pH 8.0 and at 50°C in LB broth. After determining soluble protein concentration, mutant showed highest specific activity. In presence of Ca2+, Mg2+ and Mn2+ the keratinolytic activity resulted in an increase, while in presence of Co2+, Zn2+ and Cu2+ it decreased. Observation for 7 days revealed that enzyme preparation from MZK05 dissolved 57.67% of native poultry feather; whereas mutant dissolved 72% and recombinant dissolved 64.77%. Further study would require determining all other possible locations of mutations in kerA gene of BlM9.

19


Abstract 37 Epidemiology of Hepatitis B virus and syphilis of immigrant workers of Bangladesh Sattya Narayan Talukdar1,2*, Sudip Paul2, Md. Bokhtiar Rahman1 1

Dept.of Biochemistry, School of Science, Primeasia University, Dhaka, Bangladesh. Dept.of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka, Bangladesh. *Corresponding author. E-mail: [email protected]

2

Hepatitis B and Syphilis caused by Hepatitis B virus (HBV) and Treponema pallidum, respectively, are globally considered as alarming sexually transmitted disease. General health status of Bangladeshi workers is under enormous risk in home and abroad, specifically, because of increasing predominance of HBV in Bangladesh. The study was designed to evaluate the prevalence of syphilis and AIDS by collecting blood samples from Bangladeshi migrant workers of Bangladesh. A total of 215 Bangladeshi workers including 177 males and 38 females from 21 to 45 were taken and categorized into three age groups for this study. The study was carried out in Al-Riyadh Medical Checkup, Banani, Dhaka, Bangladesh from August, 2015 to October, 2015. A questionnaire was filled either by the donor or one of the authors of this studyEvery worker was screened for HbsAg to detect Hepatitis B by Enzyme linked immunosorbent assay (ELISA). Two tests including venereal disease research laboratory test (VDRL) and Treponema pallidum hemagglutination assay (TPHA) were performed for the diagnosis of syphilis. No workers showed positivity in VDRL and TPHA indicating 0% prevalence of syphilis.The rate of recurrence of hepatitis B was found to be 12 (5.58%) and frequency was higher in female (7.89%) than male (5.08%). Age group 21-30 in both sex showed higher prevalence of HBV than remaining two groups. Although Syphilis prevalence is satisfactory but HBV prevalence of these workers in both sexes is higher than current prevalence over Bangladesh. This alarming predominance of HBV refers strict selection procedure and increasing public awareness along with public and private initiatives against these diseases.

Abstract 38 Cytotoxic activity induced by ctx gene negative Vibrio fluvialis organisms isolated from environmental sources Anica Tasnim Protity, Tahsina Jainab, Somen Kumar Mistri,Mahmuda Yasmin, Jamalunnesa, Chowdhury Rafiqul Ahsan* Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh *Corresponding author. E-mail: [email protected]

Vibrio fluvialis is a halophilic Vibrio species commonly found in river and estuarine water. Considering recent increase in numbers of diarrheal outbreaks, V. fluvialis has been considered as an emerging pathogen. The clinical features of the disease closely resembled cholera, but it does not carry cholera toxin (ctx) gene. This study aims at sample collection, isolation and biochemical identification of V. fluvialis from environmental samples, precipitation of bacterial crude protein product by ammonium sulfate precipitation method, and cytotoxicity assay of the crude protein using HeLa and MDCK cell lines. V.fluvialis was collected from different environmental sources such as water and hyacinth root sample of Buriganga and Turag River, shrimp field of Satkhira and pond of Khulna. Samples were tested for the isolation and identification by biochemical characterization, and were later re-confirmed by API 20E kit. A chemically defined medium was prepared for the growth of V. fluvialis. The crude protein was collected by ammonium sulfate precipitation method. A striking findingwith the crude protein product of V. fluvialis was the capability of the bacterium to evoke cytotoxic and vacuolation effects on HeLa and MDCK cells. Ammonium sulfate precipitated crude protein preparation ofV. fluvialis containing 240 ng protein per well gave a complete destruction of monolayer on both MDCK and HeLa cells after 18 hours of incubation. End point titers showing cytotoxic effects on 10% HeLa and MDCK cells were ranged from neat, 2, 4, 8 to 64 dilution of this invasive cytotoxin. Crude protein product of V. fluvialis confered 95% cytotoxicity whereas culture filtrate of Escherichia coli 0517:H7 conferredupto

20


60% cytotoxicity. Although the bacterium is ctx gene negative, its cytotoxin can destroy HeLa and MDCK cell monoloayer. Therefore, there is a risk of developing a severe invasive cytotoxic disease upon infection with V. fluvialis from environmental sources.

Abstract 39 Jeopardy of Hepatitis B Surface Antigenemia in Bangladesh M Abdul Halim1, Md. Rubel Mia1, M Moyen Uddin PK1*and Year Kabir2 1

Department of Biochemistry, Primeasia University, Bangladesh. 2Department of Biochemistry & Molecular Biology, University of Dhaka, Bangladesh *Corresponding Author. E-mail: [email protected]

Hepatitis B virus (HBV) infection is one of the most mutual public health difficulties worldwide. The transmission of the hepatitis B virus (HBV) is parenteral, sexual and perinatal. In this study, jeopardy of hepatitis B surface antigenemia has been piloted among adult Bangladeshi hepatitis B patients. This research work was carried out in collaboration between Primeasia University and Dhaka Hospital Ltd., Bangladesh from January to June, 2015. In this study, 171 adult patients aged of 20-65y were conscripted. HBV surface antigenemia was reconfirmed by using Enzyme Linked Immunosorbent Assay confirmatory (Bio-X-Act of Finland, HBsAg ELISA Kit/TMB). The mean age of HBV patients was 33.25±11.25y and the mean of hepatitis B surface antigenemia found 5753.53±3407.07 IU/L. As ascribed, 29.2% female accounted for this study. The highest prevalence of hepatitis B surface antigenemia was in ≥40y (25.1%) followed by 24%, 23.4%, 15.8% and 11.7% in 20-24y, 25-29y, 30-34y and 35-39y age groups respectively. About 45% patients were job holder tailed by students (21.6%), housewife (19.9%) and business (13.5%) respectively. In blood groups stratifications, 42.1 % patients raised in O+ve while 22.2% patients were in B+ve. The rest of 21.1 % and 14.6 % found for A+ve and AB+ve blood type patients respectively. In respect to blood transfusion history, 5.3 % patient experienced to previous blood transfusion. The mean difference of HBsAg titer distributions in blood groups and age groups found statistically significant (P B. animalis VKL. Probiotic strain survival in the macrophages depended on the bacterial cell wall elasticity and on the time of their joint cultivation. LAB and bifidobacteria strains stimulate immunemodulatory cytokines and active oxygen and nitrogen oxide compound production in macrophages. Strains with a more elastic cell wall according to AFM data demonstrated higher resistance to intracellular digestion in macrophages and higher level of their activation. AFM might be considered as a fast and accurate method to assess parameters of probiotic strain cell wall to predict their immune-modulatory properties.

Abstract 73 Distribution of mecA genes from vancomycin-resistant Staphylococcus aureus worldwide and natural products that can bind to Van A from resistant Staphylococci Syeda Tasneem Towhid Department of Microbiology, University of Dhaka *Corresponding E-mail: [email protected]

Vancomycin is a glycosylated heptapeptide naturally produced by bacilli and actinomycetes, which was introduced in 1954 in clinical use to tackle methicillin-resistant Staphylococcus aureus. Despite its numerous side-effects, vancomycin remained the last resort for treating multi-drug resistant Gram positive bacteria. Unfortunately, staphylococci acquired mec gene family, which encoded a number of proteins that allow the staphylococci to render vancomycin ineffective by target modification and target removal. VanA protein is encoded by mecA gene, which, together with VanH protein, confers resistance to vancomycin. All full-length VanA protein sequences retrieved from the protein database of NCBI (34 sequences till November 15, 2015) show sufficient similarity among isolates from North America, Europe, Asia and Australia collected between 2010 to 2015 to cluster them in a single group, indicating that resistant mecA is not mutating fast. This could bring hope in development of new anti-microbial agents because one active VanA blocker in combination with vancomycin could control vancomycin resistant Staphylococcusaureus. Construction of 3D structure of one representative VanA (EIK20085) followed by docking of natural products from Pubchem show that quinazolinone , Ceftaroline and Legume isolectin I can bind to VanH. The alpha and beta chains of legume isolectin I bind to VanA from MRSA in presence of high concentrations of Ca and Mn. Chemical modification of isolectin I targeting stable binding with VanA at relevant serum concentrations of Ca and Mn could pave way for new antibiotic against vancomycin-resistant Staphylococci.

Abstract 74 Anti quorum sensing and biofilm inhibitor screening by using star anise (Illicium verum Hook. f.) as a novel platform : Vivo efficacy in food matrix Md Ramim Tanver Rahman 1,2, Zaixiang Lou *1,2,3, Jun Zhang1,2, Md. Saifullah4, Md Furkanur Rahaman Mizan5, Md. Salahuddin 6 1

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R.China, 2 National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, P.R. China, 3Department of Food Science and Technology, University of California, Davis, USA, 4Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, Selangor, 43400, Malaysia, 5School of

37


Food Science and Technology, Chung-Ang University, 72-1 Nae-Ri, Daedeok-Myun, Anseong-Si, Gyunggido 456-756, South Korea, 6Faculty of Medicine, University of Hongkong, Pokfulam Road, Hongkong * Corresponding author. E-mail: [email protected]

Quorum sensing (QS) regulated bacterial biofilm formation can cause serious problems in clinical, industrial, food settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. We evaluated the potentiality of a green product, a popular spice named star anise (SA) - as anti-QS and anti-biofilm agent and foundpossible application in milk safety. Biosensor strain Choromobacterium violaceum ATCC 12472 was used as anti QS assay and cristal violet assay was used in biofilm determination.The yield of SA extract was 25.90±0.2% (w/w).SA extract inhibited the production of violacein up to 45%, 52%, 65% and 89% when the dose of SA extract were 0.5 , 1, 2 and 4 mg/mL respectively. MIC value for Staphylococcus aureus, Salmonella Typhimurium and Pseudomonas aeruginosa was 2- 4, ≤0.5 and 1-2 mg/mL respectively. The extract also inhibited the formation of biofilm on dose dependent manner up to 87%, 60% , 48 %, respectively. Exopolysaccharide (EPS) production had an inhibition of70.45% , 42.82%and 35.66%at concentration 4 mg/mL. About 95.9% swarming motility of S. aureus was reduced by 4 mg/mL extract concentration.Confocal laser scanning microscopy analysis confirmed the hampered biofilm architecture. This Study found, SA extract can delay the spoilage of milk.Taken together, SA extract can be potential as natural/green food preservative by disrupting the QS circuit.

Abstract 75 Molecular identification and characterization of Probiotic Properties of Lactic Acid Bacteria (LAB) from Yogurt and Goat Milk Iqbal Hossain*, Santonu Kumar Sanyal and Iqbal Kabir Jahid* Department of Microbiology, Jessore University of Science and Technology, Jessore, Bangladesh *Corresponding Author. E-mail: [email protected]

Lactic acid bacteria (LAB) have been used in food production for centuries without posing any health risks, they are designated as generally regarded as safe (GRAS) microorganisms. Probiotic strains can be isolated from various animal sources which milk and milk productswhich have ancient history of nutritive and antimicrobial properties. This study was designed to identifyy and characterize potential LAB strains that produce broad spectrum bacteriocin like substances for possible applications in the food industry.To reveal this, arrays of microbiological, biochemical and molecular biological approaches were performed using standard procedures. A total of 40 LAB strains were isolated from five yogurt and five goat milk samples on the basis of their cultural characterics. Nine LAB isolates that showed antagonistic properties were identified as Pediococcus acidilactici (five) and . Enterococcus faecium (four) strains by biochemical methods and 16S rDNA gene sequencing. These isolates showed tolerance against 0.3% bile concentration,6% NaCl concentration and 0.4% phenol concentration. The strains were non-heamolytic. These nine isolates were all non hemolytic and showed coagulase positive properties. Enterococcus faecium strain 12/1showed antimicrobial activity against pathogenic Vibrio cholerae and Salmonellatyphi respectively. Another isolates E. faecium strain 14/1 showed antimicrobial activity against Salmonellatyphi.Pediococcus acidilactici strain B1 showed antimicrobial activity against pathogenic Salmonellatyphi, Stapylococcus aureus,and Shigella sp.This study concludedthat goat milk traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. All nine isolates would be as potential probiotic strains by further field analysis.

Abstract 76 Incidence of Vibrio cholerae in Hilsha (Tenualosa ilisha) of Bangladesh Zenat Zebin Hossain1,2, Israt Farhana1*, Suhella MohanTulsiani2,3, Peter Kjær Mackie Jensen2,3, Anowara Begum1,*

38

Bangladesh Society of Microbiologists (BSM) 1st International Conference (28th AGM), 2015 1

Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Institute of Public Health, University of Copenhagen, Copenhagen 1014, Denmark 3 Copenhagen Centre for Disaster Research, University of Copenhagen, Copenhagen 1014, Denmark *Corresponding author. E-mail: [email protected] 2

In this study, the incidence of Vibrio cholerae was investigated in the commonly fished and consumed, Hilsha fish (Tenualosa ilisha) which exhibit a life cycle in both, fresh and marine environments of Bangladesh. In the period between October, 2014 and October 2015, gills, rectum, intestine and outer scale surface of 48 fish were analyzed for each month, collected at both, local market of capital Dhaka and from the river, Padma. Storage ice was also investigated for the local market fish. V. cholerae species-specific PCR targeting ompW gene on total DNA template of the samples revealed 34 of 48 fish (70.8%) were positive in outer swab samples and respectively 33 (68.75%), 29 (60.41%) and 12 fish(25%) were positive for gills, rectum and intestine. The storage ice samples of 70.8% (17 of 24) local fish were also found positive in PCR. Prevalence of V. cholerae showed distinctive dual seasonal peak (March-May and August-October) pattern in the 12 months of sampling. Virulence gene profile analysis of the ompW PCR positive total DNA exhibited 11.57% and 4.17% of samples were positive for rfbO1 and rfbO139 gene of V. cholerae. The major cholera toxin genes like tcp, cep and ace were present whilst ctxB, zot were not found in any sample. Real time PCR analysis demonstrated 3.7% of the sample contains cholera causing ctxA gene. In conclusion, Hilsha fish may act as a source of contamination of coastal bacteria V. cholerae from the Bay of Bengal via river system to the households of major cities like Dhaka. Presence of virulence genes in fishes evokes potential public health risk and demands precautions during processing and cooking. Further investigation into transmission mechanisms from Hilsha to the household, need to be elucidated.

Abstract 77 Prevalence of Pathotypes of Escherichia coli in Drinking Water of Dhaka, Bangladesh Ridwan Bin Rashid1*, Jannatul Ferdous1, 2, Suhella Tulsiani2,3, Sabera Saima1,Peter Mackie Kjaer Jensen2,3, Anowara Begum1,* 1

Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Section for Global Health, Institute of Public Health, University of Copenhagen, Denmark 1014 3 Copenhagen Centre for Disaster Research, University of Copenhagen, Denmark 1014 *Corresponding author. E-mail: [email protected]

2

Escherichia coli is ubiquitous in nature and originates from the colon of warm blooded animals. Although most are harmless, some strains might cause significant morbidity and can cause epidemics. One hundred twenty isolates isolated from drinking water samples (household and main source) were analyzed for their virulence properties by Polymerase Chain Reaction. Of these, 20 isolates (16.67%) indicated presence of eltB gene and 49 isolates (40.83%) had the estAgene, both of which are representative of ETEC. The genes for verotoxin 1 and 2 were each present in one isolate only. The intimin gene eaeA, which is present in EHEC and EPEC, was detected in 24 isolates (20%). None of the isolates had the bfpA and ial genes. The pCVD region and ipaH was detected in 1 and 2 isolates, respectively. Out of the 54 household isolates, 38 were commensals. The most prevalent virotype was ETEC (10 isolates). Two of each EHEC and EIEC was detected and one EAEC was isolated. Of the 66 isolates from the main source water, 21 were commensals. The number of EPEC and ETEC was 22 and 23, respectively. No EIEC, EHEC and EAEC was found. Hence, in terms of the spatial distribution, 68.1% of the isolates from the main source was pathogenic compared to 29.6% of isolates from household water. The χ² value was 17.696 and P value was 2.6 E-5 thus proving to be statistically significant at 0.1% level. Contamination of main source with clinical waste and rapid death rate of pathotypes might be accountable for this difference. Enterotoxigenic Escherichia coli was the most prevalent virulotype and is most likely to be the etiological agent for diarrhea. The presence of commensals indicates fecal contamination and might be due to poor hygiene practices.

Abstract 78 39


Evaluation of molecular typing methods- ERIC PCR and REP PCR to reveal genetic heterogeneity of Vibrio cholerae in water from river Turag Jannatul Ferdous1, 2, Mohammed Tasminuzzaman1*, Suhella Tulsiani2,3, Sumaiya Zaman1, Humaira Akhter1, Peter Mackie Kjaer Jensen2,3, Anowara Begum1,* 1

Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Section for Global Health, Institute of Public Health, University of Copenhagen, Denmark 1014 3 Copenhagen Centre for Disaster Research, University of Copenhagen, Denmark 1014 *Corresponding author. E-mail: [email protected]

2

Vibrio cholerae, the causative agent of cholera remains a major public health concern in developing countries like Bangladesh due to its potential to lead epidemics and pandemics. For epidemiological investigation, current molecular subtyping methods such as pulsedfield gel electrophoresis (PFGE), ribotyping, Multilocus Sequence typing (MLST) are traditionally available to measure the genetic diversity and to trace the global spread of clones. However, these methods are labor-intensive, expensive and time consuming compared with other simpler, non-sequencing PCR-based DNA fingerprinting approaches. In this study, 38 isolates of Vibrio cholerae recovered from two different locations of river Turag in Dhaka were examined to investigate the genetic diversity and relatedness using two PCR-based fingerprinting methods: Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR, and Repetitive Extragenic Palindromic (REP) PCR. A total of 22 and 16 fingerprint patterns were detected in ERIC and REP PCR respectively. Three fragment patterns of ERIC- 1kb, 0.6kb, 0.48kb and four fragment patterns of REP 1.4 kb, 0.8 kb, 0.75 kb, 0.5 kb were found to be most common among the isolates. It was possible to detect unique bands which ranged from 0.1kb to 2.8kb in ERIC PCR and from 0.1kb to 2.3 kb in REP PCR. Further, cluster analysis and fingerprint patterns revealed that ERIC PCR is more effective than REP PCR due to reproducibility, typeability and discriminatory attributes that facilitate rapid typing of Vibrio cholerae subtypes in environmental samples to study the transmission dynamics of outbreaks.

Abstract 79 Virulence of uropathogenic Escherichia coli isolates and their potential to be diarrheagenic Marium Khaleque1*, Selina Akter2, Humaira Akhter1, Sirajul Islam Khan1 and Anowara Begum1 1

Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Department of Microbiology, Jessore Science and technology University, Jessore, Bangladesh *Corresponding author. E-mail: [email protected]

2

Whether the Escherichia coli strains responsible for urinary tract infection (UPEC) carry virulence properties of diarrheagenic E. coli (DEC), 56 UPEC strains, collected from urine sample of urinary tract infection patients, were examined for the presence of DEC and UPEC characteristics (e.g. biofilm formation, hemolysis activity, virulence genes). Among 56 UPEC strains, 21 showed capable of biofilm formation and only 5 showed hemolysis activity on sheep blood agar. On assessment of virulence genes related to uropathogenesis; majority of these strains (42%) was found positive for papC gene, 27% was fim1 positive, 11% was afa positive and none was found positive for sfa. Most of the isolates found carrying none of eight diarrhea- associated (e.g. estA, eltB, vt1,vt2, eaeA, ea, ial and bfpA) as expected. Interestingly seven isolates found to harbor these genes: five genes i.e., vt2, ial, eltB, bfpA and ea were found in five different isolates and two isolates were positive for estA, among these two, one was found positive for fim1, papCalong with estA, a UPEC strain containing characteristic of ETEC strain. One isolate was found carryingfim1and vt2 showing the property of EHEC and another isolate was found positive for fim1 and ial, the characteristic of EIEC. One isolate harboring bfpA gene characterized as EPEC and the other onefound to harbor eagene, characterized as EAEC. This study observed that most UPEC strains are unique to uropathogenesis, still some may carry the diarrheagenic property or alternatively some diarrheagenic E. coli may evolve UPEC virulence property.

40


Abstract 80

Incidence of proximatelatrine enhance the possibility ofmicrobialcontamination of tubewell water Md. Mominur Rahman, Santonu Kumar Sanyal and Iqbal Kabir Jahid* Department of Microbiology, Jessore University of Science and technology, Jessore-7408 *Corresponding author. E-mail: [email protected]

Contamination of shallow tubewell might be contaminated by pit latrine in Bangladesh. The present study was conducted to measure enteric bacterial contamination of tubewell water and the relationship with distance of pit latrine. 60 tube wells were randomly selected from Jessore district and single water samples from each tube well were collected by maintaining standard aseptic techniques. Tubewell water samples were assessed for total aerobic bacterial count, faecal coliform and facecal Streptococci. Escherichiacoli were isolated from faecal coliform positive plates. The total aerobic bacterial count was done using the direct plate count methods while feacal coliform and faecal Streptococci were done by standard membrane filtration techniques.A questionnaire was made for the sampling survey and at that time distance of the tube well from the nearest latrine pit, condition of latrine; occurrence of diseases and other information were collected from the house holder of the tube well. There was significant relation with the distance and coliform counts (P< 0.0001, R2=0.4538) and faecal streptococci(P< 0.0001, R2=0.2429) while non-significant relationship existed for total bacterial counts (P= 0.0799, R2=0.0519) In this study, among 60 samples 13 samples (21.67%) were E. coli positive and 33 samples (55%) were faecal Streptococci positive. Among the 20 isolates, 13 isolates (65%) were biofilm producer and 7 isolates (35%) were biofilm negative. The results indicated that most of the pathogenic strains of microorganism including pathogenic E. coli produce biofilm and is characterized by increased tolerance to stress, biocides (including antibiotics). So the contaminated water could be a large route for diarrheal disease transmission. Hence, unhygienic practices must be stopped to prevent spread of faecal-oral diseases among human beings due to contaminated water. Abstract 81

Influence of plant growth promoting bacteria on growth and yield of rice M.S. Islam, A.R.M. Solaiman* and M.S. Alam Department of Soil Science , Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh *Corresponding author. E-mail:[email protected]

A laboratory experiment was conducted in the Department of Soil Science of the Bangabandhu Sheikh MujiburRahman Agricultural University, Gazipur, Bangladesh to characterize twenty RhizobiumstrainsisolatedfromlentilBU Le 11 , BU Le 12, BU Le 13, BU Le 14 and BU Le 15 ; grasspea BU Ls 19, BU Ls 20, BU Ls 21, BU Ls 22, BU Ls 23, BU Ls 24 and BU Ls 25 ; chickpea BU Ca 9, BU Ca 10 ,BU Ca 11, BU Ca 12 and BU Ca 13 ;peaBU Ps 3 , BU Ps 4 and BU Ps 5. The strains showed standard pattern of reactions in respect of growth on Yeast Extract Mannitol (YEM) agar incorporated with congo red, peptone glucose agar, colony characteristics on YEM agar, YEM agar containing bromothymol blue (BTB) indicator, growth in YEM broth, cellulose degradation, KOH test, IAA production, P solubilizing activity and Gram staining. Following the laboratory experiment a pot experiment was conducted during boro rice growing season of 2014 at the Bangabandhu Sheikh MujiburRahman Agricultural University Farm, Gazipur to assess the effect of previously studied three Rhizobium strains viz. BU Ls 2, BU Ls 6 and BU Le 6 in combination with different doses of urea on growth and yield of rice. Broth culture of Rhizobium was inoculated as root soaking and also applied in root zone after transplantation of rice up to tillering stage @ 1 ml plant-1 at one month interval. Rhizobium strains were inoculated in combination with 50% and 75% recommended dose of nitrogen as urea. At vegetative stage all the growth parameters increased significantly over control. The highest leaf greenness (SPAD value), plant height, number of tiller, root length, root volume, biomass yield, nutrient content and

41


uptake were obtained from the treatment receiving 75% N from urea + Rhizobium (BU Ls 6). Treatments receiving 50% N from urea + Rhizobium (BU Ls 6 ) also performed similarly. The effect of treatment receiving 100% nitrogen was also more or less similar as the treatments mentioned above. At harvesting stage all the growth parameters, yield and yield contributing characters were also increased significantly over control.

Abstract 82 Variability of biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and distribution of genes involved in biofilm formation Md Furkanur Rahaman Mizan*, Iqbal Kabir Jahid, Hyeon-Jo Bang, Mohammad Sadekuzzaman, Sang-Do Ha School of Food Science and Technology, Chung-Ang University, 72-1 Naeri, Ansung, Gyunggi-do 456-756, South Korea *Corresponding author. E-mail: [email protected]

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, twenty-twoV. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with motility, cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field Emission Scanning Electron Microscopy (FESEM) showed that strong-biofilm-forming strains established thick three-dimensional structures, whereas weak-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and type I pilus in the V. parahaemolyticus seafood isolates been examined. Biofilm-associated genes were present in almost all of the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and ensuing diseases.

Abstract 83 Detection and characterization of genes for anticancer protein, Parasporin, from indigenous Bacillus thuringiensis Nuzhat Jerin, Naurin Rahman, Md. Asaduzzaman Shishir, Shakila Nargis Khan and Md Mozammel Hoq* Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh. *Corresponding author. E-mail: [email protected]

In addition to insecticidal Cry proteins, Bacillus thuringiensis is also known to have non-insecticidal Cry proteins, parasporin, which have cytocidal activity exclusively on human cancer cells. In the present study, twenty nine indigenous non hemolyticBt isolates were selected from previously isolated stock for the detection of parasporin genes ps1, ps2, ps3 and ps4. Of 29 isolates tested, 10 isolates were found to be positive in PCRbased screening using parasporin specific primers (ps1, ps2, ps3, ps4). PCR amplified presumptive ps amplicons were sequenced and the homology search of the sequences with the database revealed that the isolates: DSf2 and DSa1; RhSc3 and DSd2; DSf2, DSf3, FhSa1 and FhSb1; and SgSp2 and DSa1 belong tops1, ps2, ps3 and ps4 respectively. Phylogenetic analyses revealed that the amplicon sequences of the isolates have homology with more than one ps genes. Following partial purification exclusively for parasporal crystal protein and treatment with alkaline solution, the solubilized crystal protein of all 10 isolates were subjected to denaturing gel electrophoresis with a view to identifying Cry proteins of desired sizes: 81, 37, 88 and 31-34 kDa for ps1, ps2, ps3 and ps4 gene products respectively.Upon proteinase K treatment, the putative proteins from the positive Bt isolates harbouring ps genes exhibited differences in molecular size suggesting as active fractions of cytocidal parasporin. These results thus will be an useful basis for developing therapeutic agent in the field of cancer therapy.

42


Abstract 84 Microencapsulated viable probiotic bacteria preserved orange juice Md. Shahid Hossain, Md. Abdul Alim Al-Bari*, Mir Imam Ibne Wahed Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh. *Correspondig author. E-mail: [email protected] Biopreservation refers to the use of a natural microflora and/or its antimicrobial metabolites to extend the shelf life and improve the safety of food. In recent years, microencapsulation of food processing is an innocuous and ecological approach for food preservation. Microencapsulation process ensures the stability of probiotics in foods remains cutting edge technology, which leads to the development of functional food with diverse environmental stress factors. For viable cfu count pour plate method is followed with serial dilution procedure using MRS agar media. Average count of three plates is calculated for viable cfu of each commercial probiotic product and compared that is claimed by manufacturer. Morphological, physiological and biochemical tests are performed for identification and isolation of LAB including microscopic visual colony, gram staining, milk coagulation, catalase, effect of temperature for growth, salt and pH tolerance, and carbohydrate utilization tests. For antimicrobial activity, cell free supernatant (CFS) is used from cultured LAB in MRS broth against food spoilage pathogenic microbes and finding out the activity of bacteriocin like inhibitory substance (BLIS) from CFS against pathogenic microbes. Finally isolated LAB cells are used for immobilization prepared by extrusion method to form calcium-alginate beads applied in orange juices (OJ) and compared to free LAB cells used in OJ for counting viability of LAB cells after periodically five weeks. From the experimental data, it is shown promisingly that (i) the viability of cfu count from commercial probiotic products are less than their original counts that are recommended by manufacturers. (ii) the isolated LAB are Lactobacillus acidophyllus, Lactobacillus bulgaricus, Lactococcus lactis and Biffidobacterium biffidum after comparing their morphological, physiological and biochemical tests. (iii) all of the isolated LAB showed their antimicrobial activities against common food spoilage pathogenic microbes.(iv) viablity count of LAB from microencapsulated is found in higher numbers compared to free cells in OJ indicating the capability of immobilized cells to protect the OJ for long time than free cells from spoilage owing to restrict the growth of pathogenic microorganisms in the OJ. In the near future, the microencapsulated probiotics will be used in preservation of food and beverage industry in place of chemical preservatives in the food processing and final products. Hopefully, biopreserved OJ will ensure beneficial and protective role in human health as a functional food.

Abstract 85 Cloning of truncated cry1Aa-type gene of Bacillus thuringiensis strain JSc1 for activated endotoxin delivery Umme Salma Sumi, Md. Asaduzzaman Shishir, Shakila N. Khan and Md. Mozammel Hoq* Department of Microbiology, University of Dhaka, Dhaka – 1000, Bangladesh *Corresponding author. E-mail: [email protected]

As an attractive alternative to the application of chemical pesticides, it has been demonstrated from both in lab and field research that the indigenous Bacillus thuringiensis JSc1 (Bt JSc1) strain harboring cry1Aa-typegene encoding 133 kDa delta-endotoxin was effective in controlling lepidopteran pests affecting cauliflower, cabbage and tea plants. With a view to circumvent toxin activation step and to bypass the pest resistance mechanism (viz. reduction in crystal solubilization and alteration of protoxin proteolytic cleavage in the larvae gut) prevalent against others, the protease resistant core toxin consisting of residues 29 to 618 from the protoxin sequence of the Bt JSc1 was selected for cloning and heterologous expression in this study. Sequence for activated protein was obtained by 3D structure analysis of 3-domain matured Cry1Aa-type protein by Swiss-modelling. Primers were designed based on the sequence and desired amplicon of 1792 bp was obtained. Following double

43


digestion with BamHI and XhoI, the vector pGEX-6P-2 and the amplicon were ligated and transformed into Escherichia coli DH5α for gene cloning. Transformants obtained both at 1:3 and 1:1 vector-insert ratio indicates the successful cloning of the truncated gene which should further be screened by PCR and enzymatic digestion.

Abstract 86 Diversity of plasmid profile in multidrug resistant non-E.coli normal flora lacking correlation with resistance phenomenon Nazneen Jahan1,3,Jamil Mahmud1,2, Fatema Akter1, Salma Akter1 and M. Hasibur Rahman1* 1

Department of Microbiology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh Present address: Department of Microbiology, Jagannath University, Dhaka 1100, Bangladesh. 3 Present address: Department of Mathematics and Natural Science, BRAC University, Mohakhali, Dhaka 1212, Bangladesh. *Corresponding author. E-mail: [email protected] 2

Fecal specimens were collected from healthy adults of student dormitories and antimicrobial resistance pattern of 90 non- E. coli isolates were determined. Majority of the isolated non- E. coli (81%; 73 of 90) showed resistance to one or more antibiotic and 13.33% (12 of 90) showed multi-drug resistance phenotype. Highest percentage of resistance phenotype was observed against amoxicillin (66.6%) followed by cefixime (27.5%), gentamicin (24.4%), sulfamethoxazole-trimethoprim (15.5%) and tetracycline (14.4%), while ciprofloxacin (7.7%) resistance was found to be least frequent. Plasmid extraction revealed that 62.22% (56 of 90) were found to contain plasmid with 34 different profiles. The number of plasmids varied from 1 to 6 with the molecular size ranges from ~1.4 to 140 Mdal.High molecular weight plasmids (>20 Mdal) were observed in 36% (32 of 90) of the isolates and 51% contained (46 of 90) more than one plasmid. Four isolates (of 90; 4.4 %) carried a large plasmid of approximately 140 Mdal size indicating possible presence of large virulence plasmid. The widespread occurrence and diversity of plasmid profile indicates that intestinal non-E.coli community isolates may serves as a reservoir of heterogeneous plasmid population. Nine plasmidless isolates were found to be resistant to several antibiotics, such as amoxicillin, cefixime, tetracycline, ciprofloxacin and gentamicin. On the other hand 9 Isolates carried 1 to 3 Plasmid were sensitive to all the antibiotics under investigation. Occurrence of plasmids in sensitive isolates and multidrug resistance property exhibited by plasmidless strains showed lack of correlation between presence of plasmid and antibiotic resistance.

Abstract 87 Occurrence of heterogeneous plasmid population in multidrug resistant environmental E. coli lacking correlation with resistance phenomenon Fatema Akter1,Nazneen Jahan1,2 , Salma Akter1, and M. Hasibur Rahman1* 1

Department of Microbiology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh Present address: Department of Mathematics and Natural Science, BRAC University, Mohakhali, Dhaka 1212, Bangladesh. *Corresponding author E-mail: [email protected]

2

A total of 50 surface water samples were collected from 20 different ponds and lakes in Savar, Dhaka and cultured onto MacConkey agar plates for selective isolation of Gram negative bacteria. A total of 110 (of 190) isolates were confirmed as E. coli. Majority of the E. coli isolates (84 of 110; 76.3%) showed resistance to one or more antibiotic in disc diffusion and MIC assay. A notable portion of the isolates (41.8%; 46 of 110) showed multi-drug resistance (resistance to three or more antibiotic) phenotype. Higher degree of resistance phenotype was observed against amoxicillin (55.4%), followed by cefixime (42.7%), sulfamethoxazole-trimethoprim (42.7%), ciprofloxacin (33.6%), tetracycline (28.18%) and gentamicin (26.3%). Plasmid extraction revealed that 77.27% (85 of 110) were found to contain plasmids with 48 different profiles. The number of plasmids varied

44


from 1 to 7 with the molecular size ranges from ~1.2 to 140 Mdal.High molecular weight plasmids (>20 Mdal) were observed in 56.3% (62 of 110) of the isolates and 47.2% (52 of 110) contained more than one plasmid. Nineteen isolates (of 110; 17.27%) carried a large plasmid of approximately 140 Mdal size indicating possible presence of invasive plasmid. For the diverse plasmid as observed in this study indicate that environmental E.coli is a heterogenous population and may serves as a reservoir for plasmids of diverse origin. Fifteen isolates that were sensitive to all the 6 antibiotics tested, carried 1 to 6 plasmids. Conversely, 8 multi drug resistant isolates carried no plasmid. Lack of correlation between presence of plasmids and antibiotic resistance would imply a mechanism of resistance based on chromosomally located genes.

Abstract 88 Characterization of Pseudomonas sp. that is able to degrade recalcitrant compounds of different concentrations to resolve environmental pollution Shamsun Nahar1 *, Dr.Kamruzzaman Pramanik2, Tahmin Afroze3 1

Environmental Biotechnology Division, National Institute of Biotechnology, Savar, Dhaka, Bangladesh, 2Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Savar, Dhaka, Bangladesh, 3Tahmin Afroze, Apex Biotechnology Lab, Apex Holdings Limited, Bangladesh *Corresponding author. E-mail: [email protected]

A bacterium Pseudomonas pseudomellei was investigated for its ability to grow and degrade different concentrations of phenol as a sole carbon source. After identification of this strain, assessment of phenol degradation was observed. The parameters which affected the phenol degradation by adaptation of those bacteria to phenol and density of cell mass at particular temperature were investigated. The degradation effect was observed for 600 ppm, 800 ppm, 1000 ppm and 1200 ppm at 37°C up to 144 hours at 6 and 18 hours interval. Best degradation effect was found for 600 ppm and 800 ppm concentration comparing to 1000 ppm and 1200 ppm at that same parameters. Using the substrate degradation model, inoculum size was also increased. This work focuses on our growing understanding of biological agent to detoxify the hydrocarbon containing polluted water under aerobic condition by bioremediation process being a great concern in the field of biotechnology.

Abstract 89 Comparative analysis of phytochemical screening and antibacterial assay of ethanolic, methanolic and aqueous extracts of Camellia sinensis Ilma Rahman, Jebunnesa Chowdhury Department of Mathematics and Natural Sciences, BRAC University, Dhaka. *Corresponding author. E-mail: [email protected]

Camellia sinensis, the tea plant belongs to the Theaceae botanical family. Tea industry is the second highest foreign exchange earner agro-based, labor-intensive, export oriented industry in Bangladesh. Increased consumption of green tea is due to its medicinal properties including antimicrobial, antifungal, antioxidant and anticarcinogenic activities. Hence, this study was carried out using the first and only internationally certified organic green tea produced in Bangladesh. Ethanolic, methanolic and aqueous extracts of green tea (Camellia sinensis) were collected. Preliminary screening was conducted to identify the presence of phytoconstituents: alkaloids, flavonoids, steroids, saponins, tannins, phenols, cardiac glycosides and gums. Comparative analysis of the antimicrobial activity of the extracts was investigated against four bacterial strains Escherichia coli,Enterotoxigenic Escherichia coli (ETEC), Shigella flexneri, and Staphylococcus aureus. This was done using agar-gel diffusion method and ampicillin, ciprofloxacin, nitrofurantoin/cefoxitin discs were used as positive control and to further examine the synergistic activity of tea and antibiotics. The crude extracts exhibiting the most antimicrobial activity was further tested for synergistic activity with the same antibiotics. Oxacillin and tetracyclin disks were used to show the clinical isolate of S.aureus was methicillin-resistant

45


S.aureus (MRSA) due to its characteristic multi-drug resistance pattern. It was observed that repeated subculturing had changed the virulence pattern of S.aureus from the clinical isolate. The sensitive S.aureus alone exhibited sensitivity to all tested concentrations of each extract. It can be concluded from the results that methanolic and ethanolic extracts of Camellia sinensis are effective antibacterial agents against S.flexneri, ETEC, E.coli and S.aureus and may even inhibit MRSA. Aqueous extract had inhibitory effect on S.flexneri and S.aureus. Extracts did not demonstrate synergisitic activity with any of the drugs. Zone of inhibition was measured in millimeters to check sensitivity of the extracts as well as the antibiotic disks.

Abstract 90 Antibacterial Activity of Silver Nanoparticles Synthesis by Vitis vinifera Extract A.K.M. Asaduzzaman1, A S M Tanbirul Haque2, Byung-Soo Chun2 and Syed Rashel Kabir1,* 1

Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh, 2Department of Food Science and Technology, Pukyong National University , 45 Yongso-ro, Nam-Gu, Busan 608-737, Republic of Korea *Corresponding author. E-mail: [email protected]; [email protected]

Nano-biotechnology is an emerging field of biomedical and pharmaceutical areas due to the boost properties of materials in the form of nano-sized particles. There are many noble metals found naturally which are palladium, silver, platinum and gold etc. Among those noble metals, silver has widely application in jewellery, dental alloy and health additive in traditional Bangladeshi, Chinese and Indian Ayurvedic medicine.Recently, green synthesis of nanoparticles has proven to be better methods due to slower kinetics. The present study was to synthesize silver nanoparticles (AgNPs) by fruits extract of green colour Vitis vinifera and to investigate its antibacterial properties. Synthesized AgNPs was analyzed by UV-visible spectra at 250-700 nm and found a sharp peak at 449 nm. Atomic force microscopy (AFM) images were obtained to analyze the surface morphology of the synthesized AgNPs. AgNPs shape was spherical and nano size particle was measured by transmission electron microscopy (TEM). The presence of silver in synthesized AgNPs was confirmed by energy dispersive X-ray (EDX) spectrophotometer. Functional groups of AgNPs were identified by fourier transform infrared (FTIR) spectroscopy. Five pathogenic bacteria were used for antibacterial activity of AgNPs. Among them streptococcus aureus was the most sensitive bacteria towards assisted Vitis vinifera AgNPs. That was also confirmed by the zone of inhibition study. AgNPs were tested against Pseudomonas aeuroginosa and Klebsiella pneumonia for antibiofilm activity. No biofilm was observed for K. pneumonia and P. aeuroginosa at synthesized AgNPsup to 96 and 120 h, respectively.

Abstract 91 Exploring textile dye from microorganisms, an eco-friendly alternative Shovon Lal Sarkar*, Prianka Saha, Nigarin Sultana, Selina Akter Department of Microbiology, Jessore University of Science and Technology *Corresponding Author. E-mail: [email protected]

Dyeing is an ancient art and the use of natural resource as a dyeing material is neither a noble issue. To develop a green and sustainable world and to reduce the world pollution level the natural resources are now common sought. In microbiological perspective microorganisms can be used as a proliferative resource of textile dye as some of them have pigment producing capability. In this study screening of pigment producing microorganisms and extracting of their pigments were the prime concern. After screening of three environmental sources for one year a few pigment producing bacteria and several pigmented fungus were isolated. After solvent extraction, only two colors were extracted having greater solubility and dyeing capability. By microscopic observation, the isolates were preliminarily identified as Aspergillus sp., the green color producing fungus and were and Penicillum sp., the red color producing fungus respectively. Two types of fabric, cotton and silk, were dyed with the extracted color. The dying was satisfactory in respect of wash fastness. The extracted colors also

46


showed antimicrobial activity against several pathogenic microorganisms, i.e., E. coli O157:H7, Shigella sonnie andKlebsiella pneumonia. Dyes were also subjected to patch test and found to be non allergic to human volunteers. However, these dyes have potential to be used in sophisticated garment dyeing for hyper allergic patients and infants. As the biomass yielding capability of isolated fungus was very low in the designed media. Further modification in growth media and environmental conditions are to be optimized to maximize the biomass production, hence large-scale color production.

Abstract 92 Isolation and identification of meropenem resistance bacteria from Bangladeshi currency Prianka Saha*, Shovon Lal Sarkar, Selina Akter* Department of Microbiology, Jessore University of Science and Technology. E-mail: [email protected] *Corresponding Author. E-mail: [email protected]

Paper currency is enormously used and transferred for having goods and services in countries worldwide and it was first developed in China. An individual living unhygienic conditions or having unhygienic habits will contaminate the notes with and will act as a vehicle of delivering bacteria to contaminate the hands of the next user. Improper hand washing after using the toilet, counting paper notes using saliva, droplet from coughing and sneezing are eventually getting access to money, and keeping or storage of paper notes on shabby surfaces leads to the contamination. Paper notes of currency, handled by an uncountable number of people, under a range of personal and environmental conditions thus increase the likelihood of acting environmental vehicle for the transmission of potential pathogenic microorganisms. As the pathogens responsible for infections are frequently resistant to most currently available antimicrobials, they are extremely difficult to treat. The study was undertaken to screen the meropenem resistance bacteria from Bangladeshi taka (paper currency and coin) sample by using standard procedure. Various types of organisms were found in this study and Minimal Inhibitory Concentration (MIC) of meropenem resistant isolates was measured by broth microdillution method. Among these 55% were coagulase negativeStaphylococcus (CNS) 33.33% were Bacillus anthrasis, Bacillus subtilis 14.814% and Bacillus cereus were 3.70%.According to the antimicrobial susceptibility results of this study it was found that more than 67.7% of the isolates were highly resistant. Highest frequency of resistance showed in two taka note and 100 taka note also showed harborage of significant resistant bacteria. Biofilm formation of isolates measured by the quantitative biofilm assay, there was observed strong relation between biofilm formation and drug resistance. PCR were performed to identify carbapenemase genes (blaKPC-1, blaKPC2, blaVIM, blaNDM) but band seems to be absent. A number of non specific bands were observed. The findings revealed that meropenem resistant organisms are common in taka sample. Further monitoring and effective measures are required to track the gene responsible for the resistance in order to, prevent contamination and transmission of resistant bacteria for improper handling.

Abstract 93 Immuno Modulatory Effects of Zearalenone as Food Borne Contaminant for Safety Evaluation Mohammad Rafiqul Islam1,2*, Yoon-Seok Roh2, Ara Cho2, Chae-Woong Lim2,Bumseok Kim2 1

Bangladesh Livestock Research Institute, Savar, Dhaka, 2Laboratory of Pathology, College of Veterinary Medicine and Biosafety Research Institute, Chonbuk National University, Jeonju, South Korea. *Corresponding author. E-mail: [email protected]

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by various Fusarium species. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. However, the immunomodulatory effects of ZEN in mice have not been yet elucidated. In the present study, we have investigated the the

47


immunomodulatory effects of ZEN as food borne contaminat in female BALB/c mice. ZEN (5 or 20 mg Kg-1 b.w.) was administered orally to seven weeks old female BALB/c mice for 14 days and various immunotoxicity tests with standard protocols were performed. The population of CD4+, CD8+ and CD11c+ cells in the spleen; and CD4+, CD8+ and F4/80+ cells in the MLN were significantly decreased. However, CD19+ and CD11c+ cells were significantly increased in the MLN. Both in the spleen and MLN, CD4+CD25+Foxp3+ cells were significantly deceased. There were differential changes on immune cells in the small intestine. In a cell proliferation assay, the increased proliferative capacities of ConA-induced splenocytes; LPS-induced splenocytes and MLN cells were noticeable. Along with the functional activity of T and B cells, IFN-γ production was significantly increased in the cell supernatant of ConA-induced splenocytes; LPS-induced splenocytes and MLN cells. The level of serum IgM was reduced but serum IgE was increased in ZEN-treated mice. Mucosal IgA concentration was significantly decreased in the duodenum and vagina. TNF-α level was significantly decreased, whereas, IL-6 level was significantly inceased in serum of ZEN-treated mice. Furthermore, ZEN administration induced apoptosis in the spleen and Peyer’s patches of mice, promoted by the change in the ratio of Bax/Bcl-2 activities. After priming of RAW 264.7 macrophage cell line by different TLR ligands, it was observed that ZEN differentially modulated TLR signaling by increased or decreased production og IL-1β, IL-10 and TNF-α. This is the first report of ZEN that causes different immune modulatory effects in the lymphoid organs and blood of female BALB/c mice and alteration of TLR signaling by cytokines production in RAW 264.7 macrophage cell lines that altered the normal functions of the immune system, might offer other pathogens to infect.

Abstract 94 Gut bacterial community and their effect on the longevity of melon Fly, Bactrocera cucurbitae (Coq.) Md. Kamruzzaman Pramanik1, Abdullah-Al-Mahin1, Mahfuza Khan2*, Kajla Seheli2 and Shakil A. Khan2 1

Microbiology and Industrial Irradiation Division Insect Biotechnology Division, Institute of Food and Radiation Biology (IFRB) Atomic Energy Research Establishment (AERE), Ganak bari-1349, Savar, Dhaka-1000, Bangladesh. *Corresponding author. E-mail: [email protected] 2

Tephritid fruit flies usually harbour different bacterial symbionts in their digestive system. The identification of these symbiotic bacteria is of utmost importance in understanding their true role and utility in different fitness parameters of flies. In the present study the mid-gut bacterial community of the adult Melon fly, Bactrocera cucurbitae (Coq.) (Diptera:Tephritidae) were isolated and identified using conventional techniques. Morphologically different isolates were analyzed for Gram reaction, motility, and different biochemical tests. On the basis of those tested results isolated gut-bacteria were primarily identified as Hafnia alvei, Escherichia coli, Salmonella pullorum, Citrobacter freundii, Erwinia herbicola, Proteus rettgeri, Klebsiella oxytoca and Levinea spp. which largely belong to Enterobacteriaceae family. Total viable counts were revealed as 6.7 x 105 (cfu/sample) in the mid gut of B. cucurbitae. To study the effect of bacteria supplemented diets on the longevity of B. cucurbitae gut-bacteria spp. P. rettgeri and K. oxytoca were incorporated with protein (casein:yeast extract:sugar, 1:1:2) and sugar diets. For each gut bacteria species 2 ml active bacterial isolates (7.6 x108 cfu/ml, in 0.8% saline water) were added to 40 gm protein and 40 ml sugar diets. The percentage mortality was recorded as 25.2±2.5, 21.7±2.0, 53.5±3.7, 72.0±2.5, 29.5±1.5, and 52.7±2.0 over 20 days for B. cucurbitae fed on P. rettgeri +protein, K. oxytoca + protein, P. rettgeri +sugar, K. oxytoca +sugar, only protein, and only sugar diets, respectively. Incorporation of gut bacteria P. rettgeri and K. oxytoca in protein diet observed to enhance the longevity of B. cucurbitae under controlled laboratory condition.

Abstract 95 Microbiological Analyses of Water Quality in a Northeast Texas Creek

48


Nurul Alam1,*, Amanda Thompson2, Daniel Simoneau2, Mary Hearron3,and David Allard1 1

Department of Biology; College of Science, Technology, Engineering, and Mathematics (STEM); Texas A&M University-Texarkana, 7101 University Avenue, Texarkana, Texas, TX 75701, USA, 2Former Undergraduate Students; Texas A&M University, Texas, TX 75701, USA 3 Department of Biology,Northeast Texas Community College (NTCC), 2866 FM 1735, Mt. Pleasant, TX 75455, Texas, USA *corresponding author. E-mail: [email protected]

Water is essential for life. Human beings cannot live for more than a few days without water. Safe and readily available water is important for public health, whether it is used for drinking, domestic use, food production or recreational purposes. Water of high quality does not come easily, and must look for microbes as part of the problems. Amongst the various strategies for managing water resources, continuous water monitoring is very important for compliance with the government standard and in the interest of the public. The main objective of this study was to analyze the microbiological quality of two selected sites on Tankersley Creek near Mount Pleasant, Texas, and to determine the antibiotic resistance profiles of the selected isolates. Water samples were collected weekly during the recreational season of June and July and analyzed for fecal coliform and antibiotic resistance against five commonly used antibiotics. Our data showed that all 16 samples were positive for fecal coliform and exceeded the US-EPA and Texas surface water quality standards. Fecal coliform count ranged from 0.438 to 1.2X103 CFU/100 ml, which is 2.19 to 60 times higher than the Texas surface water quality standards. None of the isolates from site-1 showed resistance against the antibiotics tested whereas site-2 isolates showed resistance to all but one antibiotics. When compared with available data, our results indicated that the bacteriological quality of water in these water bodies has deteriorated drastically. The results of this study will incorporate new information on microbiological conditions into the existing database for reassessment of water quality in Tankersley Creek.

Abstract 96 Optimization of Real Time PCR for detection of, and virulence gene contents in naturally occurring Vibrio cholerae in Bangladesh Md. Deen Islam1*, Md. Tarequl Islam2, Dr. Majibur Rahman1, Dr. Munirul Alam2 1

Department of Microbiology, University of Dhaka, Dhaka 1000, 2International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR,B), Mohakhali 1212 *Corresponding author. E-mail: [email protected]

Vibrio cholerae serogroups O1 and O139 have been responsible for cholera; a deadly diarrheal disease capable of killing many people in developing countries where safe drinking water is scarce and people uses surface water for drinking. V. cholerae has been a resident flora of the natural aquatic environment, and rapid detection of this bacterium from surface water is crucial for prediction of upcoming cholera to prevent people from ominous infection. In the present study, a duplex real-time (RT)-PCR has been optimized with the aim to detect V. cholerae O1 and non-O1/O139, and distinguish them from each other. The method was validated by testing a series of V. cholerae O1 and non-O1 from old collection, including control V. cholerae O1 El Tor and classical biotype strains, N16961 and 0395, respectively. With the validated RT-PCR, V. cholerae was not detected directly from environmental water samples that were not enriched at least for 3 hours at 37°C. The sensitivity of the RT-PCR in detecting V. cholerae was determined to be 115 CFU, while the minimum number for detecting V. cholerae by conventional PCR is 4800 CFU. A total of 23 V. cholerae O1 strains isolated from enriched surface water samples were studied to determine their biotype, presence of different virulence markers, antibiotic resistance, and presence of genetic elements and their possible links to antibiotic resistance of the bacterium. PCR with gene specific primers confirmed all of the 23 V. cholerae O1 strains to be altered El Tor carrying ctxBCL and El Tor type rstR under ET biotype background. Ca. 97% of V. cholerae O1 strains carried the CTXΦ in chromosome-I, confirming their ET biotype traits. Of 16 V. cholerae O1 tested, 56% of the strains were carrying intact VSP-I, while 50% were carrying intact VSP-II. Of 16V. cholerae O1 tested, 56% of the strains were found carrying both of the intact VSP-I and VSP-II; two of the isolates had maximum deletion of

49


open reading frames (ORFs). All 16 strains were determined to be VSP-II variants, and were confirmed to be CIRS101 type. Antibiotic sensitivity assay with 22 strains revealed all to be resistant to Sulfamethoxazole/Ttrimethoprim (SXT), 22.73% to Ampicillin (AMP), and 72.73% to Tetracycline (TE).Ca. 90.91% and 100% were sensitive to Ciprofloxacin (CIP) and Gentamycin (CN), respectively; and 27.27% of V. cholerae O1 strains were multi-drug resistant (MDR) in the present study. Rapid detection method of toxigenic V. cholerae from the environment and data from molecular analyses of the isolates presented in this study might help in effective monitoring and understanding the ecology, epidemiology and evolution of the deadly pathogen.

Abstract 97 Trend in antimicrobial susceptibility among gram negative clinical isolates from Bangladeshi children during 2004 to 2012 Md. Akram Hossain1*, Maksuda Islam2, Samir Kumar Saha2, Donald James Gomes1 1

Department of Microbiology, University of Dhaka, Dhaka, Bangladesh, 2Dhaka Shishu Hospital, Sher-E-Bangla Nogor, Dhaka , Bangladesh *Corresponding author. E-mail: [email protected]

An increase in blood stream infections in children caused by Gram negative organisms has been reported in the recent years in Bangladesh. Gram-negative bacteria are micro organisms that possess an “external cell envelope” or outer membrane (OM), which differs markedly from other bacterial strains in accordance with their structure and function. Severe infections including meningitis, pneumonia, wound or surgical site infections, and bloodstream infections are caused by Gram negative bacteria in children. These bacteria are multiple drug resistant and are increasingly acquiring resistance to most of the available antibiotics. Just as drug resistance is obviously an acquired property; it can be lost in due course of time also. That’s why the resistance pattern of some drugs towards a unique pathogen shows downfalls and rises with course of time. Here, in this study, an effective gold standard method known as Broth Microdilution method has been performed to determine interpretive MIC breakpoint values for 8 different antibiotics to determine the trend of antimicrobial susceptibility and resistance pattern of those gram negative clinical isolates collected from the blood samples of children from seven different places of Bangladesh. 70% of the total collected samples were from Neonates aged from 0-30 days which denotes thatNeonates are predominantly infected by gram negative bacteria.262 samples are of Klebsiella pneumoniae among 374 total samples educe the fact that Klebsiella pneumoniae is predominant in neonatal sepsis infections. 100% samples were sensitive to Meropenem and Trimethoprim. 100% and almost 96% samples were resistant against Amoxicillin and Ceftriaxone respectively which concludes a remark of not using these antibiotics in the treatment for E. coli and K. pneumoniae infections in children.A gradual increasing trend in antimicrobial resistance against Ciprofloxacin in progression of years from 2004 to 2012 was observed. 80% of the samples from KWMCH were resistant against Ciprofloxacin whereas only 52.69% and 66.67% of the samples from CMOSH and DSH respectively were resistant against Ciprofloxacin. So, it is contended that the rural children population were harboring more Ciprofloxacin resistant Gram-negative organisms than the urban children population. 15% of the samples from DMC were sensitive to Ciprofloxacin whereas almost 30% of the samples from CMCH were sensitive to Ciprofloxacin. So, it is contended that Dhaka city children population were harboring less Ciprofloxacin sensitive Gram-negative organisms than the Chittagong city children population.

Abstract 98 Optimization of amylase fermentation by Bacillus licheniformis MZK05M9 based on Response Surface Methodology Md. Mahmuduzzaman Mian1*, Muhammad Al Mamun1, Arafat Al Mamun2, Mohammad Saifuddin3, ShakilaNargis Khan1and Md. Mozammel Hoq1 1

Department of Microbiology, University of Dhaka, Dhaka-1000, 2Centre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka-1000, 3Bangladesh University, Mohammadpur, Dhaka-1207

50


E-mail: [email protected]

Microbial amylase is one of the most widely used industrial enzymes in various sectors such as pharmaceuticals, food, textile and detergents, etc. FourBacillus strains (Bacillus licheniformis MZK05, Bacillus licheniformis 250-0-1, Bacillus licheniformis MZK05M9, and Bacillus amyloliquefaciens) were tested for their amylase activity through hydrolysis of starch on starch agar medium followed by the enzyme production capability in shake flask cultures. With the best producer, B. licheniformis MZK05M9, optimization of fermentation conditions of amylase production was carried out using Response Surface Methodology (RSM) based on Central Composite Design (CCD) model. The soluble starch as carbon source, yeast extract as nitrogen source, salt MgSO4 and K2HPO4 were selected to design an effective medium for production of amylase by the strain at pH 7.5, agitation 150 rpm and temperature 35°C. The optimum values for the tested variables for the maximum amylase production was found as starch (1.13%, w/v), yeast extract (0.606%, w/v), MgSO4 (1.0%, w/v), and K2HPO4 (0.445%, w/v) predicted by statistical software and the amylase activity obtained was 34.5 U/ml, 50.61 U/ml and 55 U/ml in the initial medium, predicted value of the software (Minitab Version 17) and in the optimized medium respectively. Thus the statistical optimization by RSM resulted in 1.59 fold increase in the enzyme production by the B.licheniformis MZK05M9 mutant. The productivity in shake culture was 1145 U/L/hr while 1650 U/L/hr was in bioreactor cultivation using optimized medium. Ammonium sulphate fractionation and ultrafiltration resulted the enzyme purified to 9.07 fold with specific activity of 3219.6 U/mg of protein. The molecular mass of the enzyme was about 46 kDa as judged by SDS-PAGE.

Abstract 99 Isolation and Characterization of Nitrogen-Fixing Bacteria from Sugarcane Rhizosphere K. M. Alam1,*, T. Zhang2, Y. Yan2, W. Zhang2, M. Lin2 and W. Lu2 1

Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China *Corresponding author. E-mail: [email protected]

2

The seven nitrogen fixing bacterial strains were isolated from rhizosphere soil and surface sterilized roots of sugarcane. The aimed of this study were to isolate nitrogen-fixing bacteria, determine the nitrogen-fixing ability and test the indole acetic acid production. It was found that the five isolated species were distributed in the two genera as Klebsiella and Burkholderia based on 16S rRNA sequence. The PCR amplification of nifH gene showed that seven produced the expected 360-bp amplification product. The molecular identification results from 16S rRNA analyses of these bacteria were also corroborated with the morphological and biochemical data. The nifH gene sequences from the strains belonging to Klebsiella showed that the strains clustered with Klebsiella sp but the nifH gene for Burkholderia was not detected. The ability to fix nitrogen was verified by the acetylene reduction assay and the variation of nitrogenase activities were 1.29+0.4 to 29.63+0.3 nmol C2H4/h/mg protein. The highest nitrogenase activity found in Gp47, the type strain Klebsiella pneumoniae strain sctcc295; was 29.63+0.3 nmolC2H4/h/mg protein. Diazotrophic strains were assessed for plant-growthpromoting trait such as indole acetic acid production. The highest indole acetic acid production was found in Gp10, the type strain Klebsiella sp. strain zmmo which was 99.0+7 µg/ml.

Abstract 100 Bioelectricity Generation through decoulrization of textile dye by chromium resistant bacteria in a mediator less Microbial fuel cell (MFC) Reaz Mohammad Mazumdar*, Mala Khan, Md. Moniruzzaman, Mamudul Hasan Razu, Rana Karmakar Designated Reference Institute for Chemical Measurements, BCSIR, Dhaka-1205, Bangladesh. *Corresponding author. E-mail: [email protected]

51


Target of Microbial Fuel Cell (MFC) technology is to generate electricity from industrial effluent and waste water. Dye in textile effluent is a major concern for the environment. Degradation of the dyes in effluent MFC can generate electricity as well as improve the scenario. A two chambered Microbial fuel Cell (MFC), 200ml each, was constructed to produce electricity by degrading textile dye using a chromium resistant bacterium. Chromium (VI) resistant (1000 ppm) Isolates from tannery was studied for dye decoulorization activity. Among them a Pseudomonas spp. denoted as CrSp-11 was selected for the purpose. Fucozol Red UCX (Reactive Red 195) dye (100 ppm) containing basal medium was used as anolyte and phosphate buffer (PH-7) was as catholyte. Carbon plate was used as electrode. 3% KCL agar salt bridge was used to connect the chambers. 24 h enriched inoculum as 1ml/100ml added in the cathode. Performance of MFC was studied by determining the voltage. 170mV of electricity generated continuously for 120hr with 100% decolorization of the dye. Result showed that chromium resistant Pseudomonas spp. can be used to generate electricity along with degradation of textile dye.

Abstract 101 A Comprehensive Study of FMDV Genotypes Using FMDV Genotyping Tool SM Sabbir Alam 1, Arafat Rahman1†, Sadikur Rahman1, Saddam Hossain2, Munawar Sultana1 and M Anwar Hossain1,* 1

Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh, 2 Bio-Bio-1 Bioinformatics R&D Foundation, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

FMD virus causes Foot and Mouth disease in bovids, ovids, suids and other cloven hoofed animals. It is one of the most devastating diseases of cattle’s and causes great economic losses every year. FMDV is a positive-sense RNA virus and has seven immunologically distinct serotype: O, A, C, Asia-1, SAT-1, SAT-2, SAT-3. Conventionally, FMDV genotyping was performed by phylogenetic analysis of VP1 sequences. This is a time consuming, error prone and non-universal process. With a view to solve this problem, FMDV genotyping tool, a viro-informatics program is being developed. After cluster exploration in each serotype based on sequence variability and mutational pattern analysis, different genotypes have been ascribed that can be used as a basis of common genotyping framework. Phylogenetic analysis has been carried out to confirm these genotype-specific grouping. This framework has been employed in the development of Python based FMDV genotyping tool which uses a sliding window method and blast2 program to find similarity with reference sequences of different serotype. Also a pattern matching algorithm has been employed to predict query sequence’s genotype based on the framework. This viro-informatics program enables computational detection of FMDV serotype, recombination analysis as well as genotype prediction. This study provides a comprehensive description of FMDV genotypes along with a GUI-based cross-platform program to do rapid genotype analysis, which may have implications in FMDV surveillance of detection of emerging new genotypes and switching of occurring genotypes.

Abstract 102 Efficacy of Sanitizing Agents on Pseudomonas fluorescenceMigula 1895 Biofilm on Food Processing Surfaces Ovinu Kibria Islam1,2, Dirk Bockmuehl2, Munawar Sultana1, M Anwar Hossain1,* 1

Department of Microbiology, University of Dhaka, Dhaka, Bangladesh, 2 Department of Hygiene and Microbiology, Rhine Waal University of Applied Science, Kleve, Germany * Corresponding author. E-mail: [email protected]

52


Bacteria can produce persistent biofilms on wide range of surfaces that can be a source of contamination and food spoilage. Pseudomonas fluorescence Migula 1895 was investigated for its biofilm forming ability on food processing surfaces like plastic, glass and polypropylene. Overnight culture of P. fluorescence was applied on each surface and was washed with sterile water thrice to remove the loosely attached cells. Cells were stained with live and dead stain to observe biofilm concentration and viability under epifluorescent microscope. Diluted tryptic soya broth (TSB)was used for the growth of the organism. The surface chips containing biofilms were observed under epifluorescent microscope at variable time intervals. Experiments showed that lower (5%) TSB concentration facilitated well attachment of cells and biofilm formation. Initial cell concentration of ca. 106 cfu/ml showed better biofilm formation ability. Robust biofilms were observed on plastic surface after 24 hours of incubation, but after longer incubation, glass surface seemed to have more attached cells. Different sanitizing agents such as 70% ethanol, 0.1% SDS (sodium dodecyl sulfate) and household detergents were applied to the biofilms on chips of different surfaces. It was observed that 70% ethanol had more lethal effect on the cells than 0.1% SDS. Ethanol killed the cells in the upper layer of the biofilm, but viable cells were detected beneath the upper layer. Both 0.1% SDS and household detergents could remove loosely attached bacteria after treatment on plastic and glass surfaces, but the surface texture of polypropylene chopping board seemed to contain more bacteria. Household detergents did not show any lethal effect on biofilm, while nonviable cells were increased when treated with 0.1% SDS. Food processing surfaces can be modified or engineered as well as effective sanitizing agents should be used to prevent bacterial biofilm formation to reduce source of contamination of food.

Abstract 103 Contribution of efflux pumps, qnrS and DNA gyrase alteration in ciprofloxacin resistance in Escherichia spp. Ram Prosad Chakrabarty, Munawar Sultana, Saadlee Shehreen+, Selina Akter++, and M. Anwar Hossain* Department of Microbiology, University of Dhaka, + Present address: Department of Genetic Engineering and Biotechnology, University of Dhaka, Bangladesh ++ Present address: Department of Microbiology, Jessore University of Science and Technology, Jessore, Bangladesh *Corresponding author. E-mail: [email protected]

Ciprofloxacin (CIP) is a second generation fluoroquinolone and widely used in the treatment of a wide range of infections caused by Enterobacteriaceae, and Pseudomonas aeruginosa. However, frequent reports about the association of the emergence of the resistance with previous exposure have created a conundrum regarding its use. The present study investigated ciprofloxacin (CIP) resistance mechanisms in 26 selected multidrug resistant (MDR) Escherichia spp. isolated from clinical wastewater (CWW) (12 isolates) and urine samples of patients with urinary tract infection (UTI) (14 isolates). MICCIP for these isolates were in the range of 4-512 µg/mL and 69.23% Escherichia spp. has the range of MICCIP: 128-512 µg/mL. A widely occurring variant of quinolone resistance gene, qnrS was present in 73.08% (19/26) isolates; and among them, 10 highly resistant isolates (MICCIP: 256-512 µg/mL) were selected for locating qnrS gene as well as for detecting and analyzing the contribution of efflux pump and mutation in QRDR region of gyrA to the emergence of CIP resistant phenotypes. Southern blot hybridization using qnrS gene probe confirmed that 4 out of 10 Escherichia spp. harbored qnrS gene in plasmid. The acrA, acrB and tolC genes encoding AcrAB-TolC efflux pump were present in all 10 Escherichia isolates and the efflux pump inhibition experiment using RND family efflux pump inhibitor, 1-(1-Naphthylmethyl)-piperazine (NMP) showed an additive (7-isolates) or synergistic (3-isolates) effect on CIP action. In addition, 8 Escherichia spp. contained double amino acid substitutions (S83L and D87N) in QRDR of GyrA altering the conformation of putative CIP binding pocket of GyrA to induce resistance. Escherichia sp. CR1 contained single amino acid substitution (S83L) and Escherichia sp. NCX9 contained no amino acid substitution but showed MICCIP 256 µg/mL. In contrast to earlier report(s), these results inferred that efflux pump played the major role in Enterobacteriaceae for acquiring high CIP resistant phenotypes.

53


Abstract 104 Screening and Molecular Characterization of Multidrug Resistant Salmonella spp. from Poultry Samples Khandokar Fahmida Sultana, Mohammad Anwar Siddique, Munawar Sultana and M. Anwar Hossain* Department of Microbiology, University of Dhaka, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

Salmonella spp. are the most frequently reported cause of food-borne illnesses worldwide that are closely associated with the consumption of contaminated poultry and egg products. The present study was designed to isolate and identify salmonella and evaluate their prevalence in poultry samples. A total of 23 poultry samples were collected from three farm houses at Narayanganj, Bangladesh on October 2015. The poultry samples included cloacal swabs, droppings, handler swabs, feed samples, swabsfrom ovarian follicle of dead and diseased chicken.The samples were enriched in Rappaport vassiliadis Broth and Tetrathionate Broth media and was subsequently spread onto the Nutrient Agar, MacConkey Agar and selective Xylose lysine deoxycholate agar (X.L.D) plates and also on Lysine Iron Agar and Triple Sugar Iron Agar slants. A total of 18 samples out of 23 (78%) were culture positive for Salmonella spp. from which 60 presumptive Salmonella isolates were selected from Xylose lysine deoxycholate agar (XLD) plates. Cultured Salmonella spp. were retrieved prevalently from dropping samples (7 out of 9; 78%). Antibiogram of the isolates were examined by following CLSI methods against Ceftiofur, Ceftriaxone, Oxacillin, Ciprofloxacin, Ampicillin and Tetracycline antibiotics. Isolates were resistant to three or more antimicrobials so considered as multidrug resistant (MDR).Genomic DNA of each isolates was subjected to Salmonella-specific gene (invA) PCR and was confirmed as Salmonellapositive by the predicted amplicon of 284-bp .Molecular profiling of the isolates were determined by amplified ribosomal DNA restriction analysis (ARDRA) of 16S-23S rRNA internal spacer region (ISR) through restriction cleavage with Alu I and Hha I followed by sequencing of each ARDRA types. Furthermore, multilocus sequence typing (MLST) of the Salmonella spp. were done based on the sequencing of seven housekeeping genes (thrA, purE , sucA , hisD , aroC , hemD and dnaN ) to determine the circulatory sequence types (STs) of Salmonella spp. that are transmissible within the poultry farms. The data of this study showed a higher prevalence of Salmonella in broilers with interfarm transmission of common Salmonella STs and underscored the need for detail epidemiological investigation as well as strict hygienic practices in the poultry farms all over Bangladesh.

Abstract 105 Mechanism of Carbapenem Resistance in clinical Pseudomonas spp. in Bangladesh Sabrin Bashar1*, Santonu Kumar Sanyal2+, Sumaiya Sharmin1, M. Anwar Hossain1, Munawar Sultana1,* 1

Department of Microbiology, University of Dhaka, Dhaka, Bangladesh, 2Department of Microbiology, Jessore University of Science and Technology, Jessore, Bangladesh *Corresponding author. E-mail: [email protected]

The aim of the present investigation was to explore Carbapenem resistance mechanism within Pseudomonas isolates from clinical origin. A total of 18 presumptive clinical Pseudomonas spp. were selected retrieved from diabetic, non-diabetic as well as UTI patients. Approximately seven clinical (39%) Pseudomonas spp. showed 100 % resistant to Imipenem, Meropenem and Doripenem. All of these Carbapenem resistant isolates showed resistance to 8 or more groups of antibiotics with 100% resistance to Ampicillin, Oxacillin, Nitrofurantoin, Vancomycin and Cephalosporin 1st to 4th generation antibiotics. The isolates showed significant resistance to other antibiotics such as 42.86% for Gentamycin, Amikacin, 57% for Tetracycline, 85.7% for Azithromycin, Doxycycline, Aztreonam and 85.7% for Floruroquinolone 1st to 3rd generation antibiotics. All Carbapenem resistant isolates were sensitive to Colistin. Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence identified the Pseudomonas isolates within 3-genotypes such as P. stutzeri (ID:

54


40/D/Mac1, 40/D/Mac2, 40/D/Swab1, 40/D/CIP+Cefo1), P. aeruginosa (ID: 54/D/Mac3, 3C CIP+Cefo2) and P. hibiscicola (ID: 50/D/Swab1). Production of carbapenamase within the resistant isolates were confirmed by both blue carba test and combined disc assay and gene specific PCR of metallo-β-lactamase (MBL) (blaVIM-2, blaNDM-1and blaIMP-3 and blaIMP-4). The finds confirmed 4 clinical P. stutzeri isolates (ID: 40/D/Mac1, 40/D/Mac2, 40/D/Swab1, 40/D/CIP+Cefo1) as MBL producers and possessing blaVIM-2 gene encoded within an integron gene cassette Int1. All of the isolate contains Resistance-Nodulation-Division (RND)efflux pump mexE gene, but inhibition experiment using checkerboard method with specific RND efflux pump inhibitor, 1(1-Naphthylmethyl)-piperazine had no significant effect on MICIMP(FIC index=1.0). This investigation concludes the emergence of Int1 gene cassette mediated blaVIM-2Carbapenemresistant determinant in clinical Pseudomonas spp.

Abstract 106 Isolation and Molecular Profiling of Arsenotrophic Bacteria from Groundwater and Soils of Aresnic Prone Areas in Bangladesh Farzana diba1,2,Santonu Kumar Sanyal1,+, Sadikur Rahman1, Mala Khan2, M. Anwar Hossain1, Munawar Sultana1,* 1

Department of Microbiology, University of Dhaka, Dhaka, Bangladesh Department of Microbiology, Jessore University of Science and Technology, Jessore-7408, Bangladesh 2 Designated Reference Institute of Chemical Measurement, Bangladesh Council of Scientific and Industrial Research (BCSIR) *Corresponding author. E-mail: [email protected] +

Arsenic (As) pollution in ground water and soil exerted one of the largest mass poisoning in the history of Bangladesh. About 35 to 77 million people incredibly exposed to high concentration of As (> 50 ppb) through drinking water and irrigation soil. Arsenotrophic bacteria and their functional genes contribute largely to the fate of arsenic compounds in the polluted environment. The present study aims to isolate arsenite metabolizing bacteria and to investigate their arsenotrophic genes (aio, ars, arr) from As contaminated ground water and soil. Nine ground water and seven soil samples were collected from arsenic prone area of Munshiganj and Bogura in Bangladesh. Total As contents was detected using Flame Atomic Absorption Spectrometry (FAAS). As contents of all groundwater samples were detected to be >50 ppb and those of soil samples was above 20ppm Exceeding the World health organization (WHO) standard for Bangladesh. A total of 80 heterotrophic and 62 autotrophic arsenite resistant bacteria were screened on respective minimal salt media (MSM) supplemented with 2 mM sodium arsenite. Phenotypic detection of arsenite oxidation was observed using KMnO4 and AgNO3 assays. Representative isolates were genotypically categorized by 16S rRNA PCR and amplified ribosomal DNA restriction analysis (ARDRA) followed by sequencing of representative genotypes. Presumed gene fragments for arsenite efflux pumps (arsB, acr3P), arsenite oxidase (aio) and arsenite respiratory reductase (arr) were amplified from each genotypes of the isolates. Five groundwater isolates belonging to Burkholderia spp. showed arsenite oxidation capacity and was positive for the presence of aio gene. Arsenotrophic bacteria found in this study could play a potential role in biogeocycling of aresnic and aerobic detoxification of highly toxic arsenite to less toxic arsenate in arsenic prone area.

Abstract 108 Validation of a real time PCR assay for detection and quantification of Salmonella spp. Juthika Mandal*, Mohammad Anwar Siddique, Munawar Sultana and M. Anwar Hossain Department of Microbiology, University of Dhaka, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

Salmonella spp. is a notorious pathogen responsible for typhoid, paratyphoid fever, and food-borne gastroenteritis in humans. Conventional detection of Salmonella is time consuming and labor intensive. The

55


present study aims to develop a rapid and reproducible method for detecting Salmonellaspp. by quantitative realtime PCR (qPCR) assay. To develop a SYBR green real time PCR, invA gene was amplified producing 284 bp amplicon and its specificity and reproducibility was confirmed by using template DNA from SalmonellaEnteritidis S9 and other related Enterobacteriacae. invA gene is present as a single copy number in Salmonella and quantification of Salmonella spp. is possible through quantification of copy number of invA. Three types of template standards — purified 284 amplicon products, cloned invA amplicon (284 bp) in the TOPO TA vector and gDNA were used for the development of standard template (Salmonella Enteritidis IFO 3313). The standard curve was constructed using the mean threshold cycle and various concentrations of recombinant TOPO TA-invA amplicon (ranging from 10 to 107 copies per reaction) showed good linearity (R2 _ 0.97) and efficiency of the assay was found to be 99%. The specificity of the reaction was confirmed by the Tm, which was consistently specific for the amplicon obtained; the mean peak Tmobtained with curves specific for Salmonella was 83.5°C. After laboratory validation, the method was further validated by using egg samples collected from Dhaka city and deliberate spiking of known concentration of Salmonella Enteritidis S9. The results of this study demonstrate that the SYBR Green real-time PCR constitutes an effective and easy-toperform method for detecting Salmonella spp. from a large volume of samples.

Abstract 109 Prediction of Peptide Vaccine Epitopes for Foot-and-Mouth-Disease Virus Prevalent in Bangladesh Samina Momtaz, Arafat Rahman, Munawar Sultana and M Anwar Hossain* †

Department of Microbiology, University of Dhaka, Dhaka Present address: Department of Microbiology, Noakhali Science and Technology University, Bangladesh. *Corresponding author. E-mail: [email protected]

Foot-and-mouth disease (FMD) is endemic in Bangladesh and is predominantly due to FMDV serotype O. There is no cross-protection among FMDV serotypes and vaccination escape mutation may happen. Peptide vaccines containing discrete selected epitopes could be an alternative that avoid the manipulation of the infectious virus. The aim of this study is to predict and map the B and T cell epitopes of VP1 proteins of FMDV serotypes O and A that were circulating in Bangladesh from 2012 to 2013. Epidemiological analysis of circulatory FMDV in 2012-13 revealed the confirmed emergence of two distinct serotypes, A (in Chittagong and Gazipur districts) and O (Pabna, Faridpur and Natore) in Bangladesh. Evidence of positive selection was identified in both serotype O and A sequences on different codon positions suggesting that arising antigenic variants benefit from such selective advantage in their interaction with the immune system. Using different bioinformatics tools (BCPred, BepiPred, DiscoTope, ElliPro, and ProPred-I, IEDB analysis for MHC-I prediction), total 11 B and T cell epitopes were predicted. Instead, homology modeling of VP1 protein (SWISS3D MODEL, PyMol and YASARAforce field minimization server) showed that five epitopes located on N- and C-termini can be considered as good vaccine candidates whereas epitopes on the G–H loop can be served as receptor recognition sites for peptide vaccine design. These predicted epitopes could be considered as candidates for designing novel peptide vaccines, which could provide specific protection against FMDV serotypes O and A found in Bangladesh.

Abstract 110 Characterization of starch and xylose fermenting microbes from various natural sources of Bangladesh for bioethanol production Roni Miah1, Ayesha Siddiqa1, Md. Masud Rana1, Nadim Sharif1, Jamsheda Ferdouse Tuli1, Muntarin Jannat Oishee1, Mamoru Yamada2 and Ali Azam Talukder1* 1

Department of Microbiology, Jahangirnagar University, Dhaka-1342, Bangladesh, 2Department of Biological Chemistry, Yamaguchi University, Yamaguchi-755, Japan

56

Bangladesh Society of Microbiologists (BSM) 1st International Conference (28th AGM), 2015 *

Corresponding Author. E-mail: [email protected], [email protected]

Ethanol fermentation process has a long history in the production of alcoholic drinks and medicine. However, at present greater scale production of ethanol is now required to enable its use as a substituent of gasoline fuel called biofuel. Because, worldwide depletion of fossil fuels, rising fuel prices, environmental pollutions and pressures for fossil fuel dependence are creating a strong demand for biofuels. Approximately eleven microorganisms were isolated from the natural fermented sources of Bangladesh [rumen part of cows gut (Y), termites gut (TM) decomposing woods (XM), papaya trees (PM), and grasses (GM)]. Our main focus was to identify some microbes which are capable to ferment starch and xylose. In aspects of Bangladesh, potato may be used as a raw material for bioethanol production. Because, potatoes are frequent availability and low cost price. Moreover, starch is naturally the second most abundant carbohydrate next to cellulose. Starch is bio-renewable, bio-degradable, environmentally friendly and easy to process. Cultural, morphological, physiological and biochemical analysis were carried out under various physiological conditions. Our results conclude that some potential thermotolerant strains could ferment starch and xylose under high temperature conditions.

Abstract 111 Optimization of Acetic acid production rate from newly isolated Acetobacter spp from decomposed fruit materials Roni Miah*1, Ayesha Siddiqa1, Aukhil Uddin, Md. Masud Rana1, Mamoru Yamada2 and Ali Azam Talukder1 1

Department of Microbiology, Jahangirnagar University, Dhaka-1342, Bangladesh, 2Department of Biological Chemistry, Yamaguchi University, Yamaguchi-755, Japan * Corresponding Author. E-mail: [email protected], [email protected]

Acetic acid is an organic compound with the chemical formula CH3COOH is an important industrial chemical with approximately millions of tons/year being produced and consumed worldwide. The biological route accounts for only about 10% of world production of acetic acid, but it remains important for the production of vinegar, as many food purity laws stipulate that vinegar used in foods must be of biological origin. In our previous study, we have identified several acetic acid producing microorganisms from decomposed fruit materials for vinegar production (1). In this study, we would like to discuss our recent results based on optimum physiological conditions for acetic acid production from newly isolated Acetobacter strain (F-10). Abstract 112

Optimization of biofuel producing yeasts from date palm juice Ayesha Siddiqa*1, Roni Miah1, Aukhil Uddin, Md. Masud Rana1, Mamoru Yamada2 and Ali Azam Talukder1 1

Department of Microbiology, Jahangirnagar University, Dhaka-1342, Bangladesh, 2Department of Biological Chemistry, Yamaguchi University, Yamaguchi-755, Japan * Corresponding Author. E-mail: [email protected], [email protected]

Bioethanol or biofuel as an alternative to fossil fuels has been expanded in the last few decades in the world. Use of bioethanol as a renewable vehicles fuel will minimize the amounts of fossil-derived carbon dioxide (CO2) to the Earth’s atmosphere. The main objective of this research work was to optimize thermo-sensitive five carbon degrading, high potential ethanol producing yeast strains isolated from date palm juice (Phoenix dectylofera). Date palm juice is a natural fermented product, which have various amounts of carbohydrate, lipids, sugars, vitamins and other resources including alcoholic beverages were reported. Based on our previous and recent results under various physiological conditions, here we concluded that the strain 5a encoded Debaryomyces nepalensiswould be potential candidate for optimization of bioethanol production.

Abstract 113 57


Molecular characterization of rhizobial isolates from Sesbania bispinosa Mir Salma Akter, *Najmunnahar, Anowara Begum, Humaira Akhter Department of Microbiology, University of Dhaka, Dhaka-1000 *Corresponding author. E-mail: [email protected]

Amongst the soil bacteria one unique group of bacteria, the Rhizobia has a beneficial effect on the growth of plants. It can live either in the soil or within the root nodules of host legumes. The bacteria within root nodules converts atmospheric nitrogen to ammonia and provides organic nitrogenous compounds to the plants. The symbiotic association between rhizobia and leguminous plants is one of the major contributors to the total biological nitrogen fixation, which is an alternative to the use of nitrogen fertilizers that lead to unacceptable pollution levels. Of the different legumes, one well-known legume of Bangladesh, Dhaincha (Sesbania bispinosa) have generally been considered as an important green manure among the farmers. Thus it was important to study the rhizobial isolate of this less studied legume in order to learn its association to increase soil fertility. The test isolates were previously isolated and characterized and identified through biochemical tests. They gave the correct size amplification product for the nifH gene and nodC gene, which indicated their possession of nodulation and nitrogen fixation ability. This was further confirmed when the same isolates was found to produce nodules in plant infection test in the laboratory. Furthermore, the amplification band of nifH gene was observed only in plasmid, further proving that the symbiotic genes might be plasmid borne. Lipopolysaccharide profiling showed variation in some bands indicating certain diversity prevails among the rhizobial isolates with respect to LPS expression. They were observed further and found to be capable of phosphate solubilization. These characteristics made these rhizobia a suitable choice for use as in symbiotic association with Sesbania bispinosa to work as biofertilizer. Biological Nitrogen fixation (BNF) technology can play an important role in substituting for commercially available Nitrogen fertilizer. The test isolates from Sesbania bispinosa can be an environmental friendly solution in the face of N-fertilizer pollution. Thus it’s important to further characterize these isolates in the molecular level to choose the ideal strain of rhizobia for Sesbania bispinosa.

Abstract 114 Analysis of protective immune responses of E. coli O157:H7 Hly E toxin in mice model against four different enteropathogenic bacteria Nasif Sayeed, Saeed Salehin Akhand, Mahmuda Yasmin, Jamalun Nessa and Chowdhury Rafiqul Ahsan* Department of Microbiology, University of Dhaka, Dhaka-1000 *Corresponding author. E-mail: [email protected]

Hemolysin E (hlyE) has been identified as a major virulence factor among different enteropathogenic bacteria and causes hemolysis of mammalian blood cells. Presence of highly conserved region, detected by multiple sequence alignment, among the HlyE protein sequence of E.coli O157:H7, Salmonella typhi, Salmonella paratyphi A and Shigella flexneri inspired us to analyze the protective immune response of the HlyE in mice model against these four HlyE producing enteropathogens. E.coli O157:H7 was grown in Brain Heart Infusion (BHI) medium and the culture supernatant was concentrated to 50 times using Amicon concentrator. Presence of the HlyE in the concentrated supernatant was confirmed by tube hemolysis method and SDS-PAGE. Toxic activity of the HlyE in the concentrated supernatant was inactivated by formalin treatment.Mice were immunized, three times at two week intervals, with the inactivated HlyE and challenged with live E.coli O157:H7, Salmonella typhi, Salmonella paratyphi A or Shigella flexneri seven days after the last immunization. Control mice were immunized with the BHI only and later challenged with the live organisms. All mice were observed for 30 days and any physiological change was recorded. Mice which were immunized with the inactivated HlyE and later challenged with E.coli O157:H7 or S.flexneri, showed 33% and 34% more protective activity, respectively, when compared with the control groups. However, mice challenged with the live S. typhi or S.paratyphi A did not show any protective activity.

58


Abstract 115 Charaterization of lactobacilli isolated from broiler chicken gastrointestinal tract and used as probiotic poultry feed Md Mizanur Rahaman1, Nantu Chandra Das 2, Md Nur Hossain1*, Monzur Morshed Ahmed1 1

Industrial Microbiology Laboratory, Institute of Food Science and Technology (IFST), Bangladesh Council of Scientificand Industrial Research (BCSIR), Dhaka, Bangladesh. 2 Department of Microbiology & Biotechnology, Jagannath University, Dhaka, Bangladesh. *Corresponding Author. E-mail: [email protected]

This research was conducted in order to evaluate probiotic properties of lactobacilli isolated from broiler chicken gastrointestinal tract from Dhaka, Bangladesh. Fourteen Lactobacillus strains were isolated. Probiotics are the health promoting viable microorganisms that exhibit a beneficial effect on the health of poultry by improving the intestinal microbial flora. On the basis of low PH (2.5), 2% bile salts, 2% NaCl & gastric juice (0.5% Bile Salt, 0.2% NaCl, 0.32% pepsin) tolerance test; four isolates have been selected for antimicrobial activity, antibiotic susceptibility, protease activity & shelf life study on freeze dried condition. The results showed these four isolates were highly responded to some probiotic criteria and were identified as Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus fermentum according to Bergey's Manual of Systematic Bacteriology. All the strains were showed protease activity on skim milk agar. These strains were resistance to commercial antibiotic vancomycin (30µg), ciprofloxacin (5µg), ampicillin (10µg), chloramphenicol (30µg), penicillin G units (10µg), neomycin (10µg) nalidicxic acid (10µg) but sensitive to nitrofurantoin (300µg) tetracycline (30µg); also have antimicrobial activity against common pathogen such as ATCC of Listeria monocytogenes, Staphylococcus aureus, Shigella flexneri, Klebsiella pneumonia, Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Aspergillus flavus and Candida albicans. Four isolated Lactobacilli were able to maintain their probiotic properties in freeze dried condition over 120 day’s storage. This study concludes that these isolates may be used as potential candidate as probiotic poultry feed.

Abstract 116 Insight in bacterial arsenic cycling in contaminated groundwaters in Bangladesh Zahid Hassan1, 2*, Munawar Sultana2, Rob J. M. van Spanning1, Sirajul I. Khan2, Martin Braster1, Wilfred F.M. Röling1†, Hans V. Westerhoff1, 3, 4 1

Department of Molecular Cell Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, the Netherlands, 2Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh, 3Manchester Centre for Integrative Systems Biology (MCISB), Manchester Institute of Biotechnology, School of Chemical Engineering and Analytical Sciences (CEAS), the University of Manchester, Manchester, United Kingdom, 4Synthetic Systems Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands, †deceased 25 September 2015 * Corresponding author. E-mail: [email protected]

The activities of arsenite-oxidizing and arsenate-reducing microorganisms impact the fate of arsenic in groundwater. Phylogenetic properties provide insufficient information for inferring the potential of the microbial population of aquifers for arsenic oxidation or reduction. Therefore, we complemented a previous cultivationindependent microbial community survey covering 22 arsenic-contaminated drinking water wells in Bangladesh with new information relating more directly to activity. This involved the characterization of enrichments of aerobic and denitrifying chemolithoautotrophic arsenite oxidizers, and included aerobic and anaerobic heterotrophic arsenite oxidizers and anaerobic arsenate reducers. Nearly all investigated enrichments revealed a potential for microbial arsenite oxidation and arsenate reduction. Enrichments were further analyzed by 16S rRNA and functional gene amplification. The microbial communities in the aquifers were phylogenetically diverse within and between enrichments. Enrichments revealed a larger diversity of arsenic-cycling

59


microorganisms compared to their original water samples. The arsenite-oxidizing enrichments were dominated by several 16S rRNA gene sequences most closely related to Hydrogenophaga,Acinetobacter, Comamonas, Rhizobium/Agrobacterium and Dechloromonas. This was in addition to the various arsenite oxidase (AioA)containing well-known chemolithoautotrophic (Paracoccus sp. SY) and heterotrophic arsenite-oxidizing strains that we found evidence for. The enrichments also recovered other and more AioA phylotypes (Bosea sp. WAO, Hydrogenophaga sp. Cl3/Thiobacillus sp. S1/Ancylobacter sp. OL1 and Achromobacter sp NT-10/Alcaligenes sp.S46). These had not been detected in our previous molecular survey of groundwater samples. Anaerobic enrichments disclosed a beta diversity of arsenite oxidizing AioA phylotypes between the different types of enrichments. Anaerobic-arsenate reducing enrichment revealed a limited 16S rRNA and arrA gene diversity, most closely relating to known arsenate-reducing Sulfurospirillum spp. The chemolithoautotrophic and heterotrophic arsenite oxidizers we identified should be of interest fortheemerging in situ or ex situ arsenic bioremediation technologyfor the cleansing of drinking water in Bangladesh.

Abstract 117 Prevalence of Metallo-β-lactamase producing nonfermentative Pseudomonas species from clinical isolates in Dhaka, Bangladesh Nazrul Islam1, 2* Dilruba Ahmed1, M Anowar Hossain1, Chowdhury R Ahsan 2 and Mahmuda Yasmin2 1

Clinical Microbiology Laboratory, Clinical Laboratory Services, International Centre for Diarrhoea Disease Research, Bangladesh; GPO Box 128, Dhaka 1000, Bangladesh 2 Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh * Corresponding author. E-mail: [email protected]

Antimicrobial drug resistance, a global concern, has been increasingunpredictably in microorganism causing human infections specially among Gram negative non-fermenting Pseudomonas spp. Carbapenems antibiotics are the most potent and effective drug and usually kept reserved for treating the multi-drug resistant Psedomonas spp. It is also used in treating infections caused by organisms producing Extended Spectrum Beta Lactamase (ESBL) and AmpC. Clinical utility of carbapenem will reduce when resistant bacteria evolve due to production of carbapenem hydrolyzing metallo-beta-lactamase (MBL) which confers high-level resistance to all beta-lactam antibiotics except aztreonam. The various reports on the prevalence of MBLs are available from many countries but few from Bangladesh. We investigated the prevalence of MBL production in these nonfermentative Gram negative bacteria obtained from clinical sources in an uraban setting in Dhaka, Bangladesh. Culture results of 29,136 specimens were reviewed for presence of Psedomonas spp from January 2011 and December 2012. A total of 2,340 (12%) were isolated and identified asPseudomonas spp. Of the identified Pseudomonas spp, 238 (57.6%) were from tracheal aspirate, 216 (40.4%) from sputum, 902 (36.7%)from other body fluids, 463 (4.1%)from blood, and 521 (3.6%) from urine samples. From 2,340 Pseudomonas species, by selective sampling, imipenem-meropenem resistant and intermediate susceptible 100 strains were tested for MBL production and 92 were found positive. Tracheal aspirate showed 38%, other body fluids 30%, Urine 17%, sputum 4% and blood 3% MBL production respectively. Irrespective of the sources of isolates/specimens, Pseudomonas spp showed 71% resistance to cefixime, 70% to ceftriaxone, 64% to gentamicin, 56% to piperecillin+tazobactam, 50% to ciprofloxacin, 49% to amikacin, 46% to netilmicin, 45% to ceftazidime, 30% to meropenem, 26% to imipenem and 19% to polymyxin B. The high drug resistance pattern is alarming and needs further evaluaion to identify new antibiotics that could be recognized as the appropriate drug of choice for the therapy for patients infected with Pseudomonas spp. As multi-drug resistant Pseudomonas showed high level of (92%) MBL production, so MBL detection testing facility may be a useful battery to determine MDR Pseudomonas species from clinical isolates.

Abstract 118 Monitoring of Environmental and Personal Hygiene in Production plants of Akij Food and Beverage Limited, Bangladesh

60


Sm Sajidur Rahman, Selina Akter Jessore University of Science and Technology (JUST) *Corresponding author. E-mail: [email protected] The environmental monitoring is a key process to get an overview of good manufacturing and operation practices. In this study, microbiological quality of industrial environment of ‘Akij Food and Beverage Limited’ was assesses. National and international guidelines were followed to select the methodologies and compared with the national standard. Environmental monitoring was not found well practiced. The air samples of most of the production plants were not up to the mark of international standard as assessed by settle plate method. The plant for production of dry food items chips was found to be the most contaminated area. Machine surfaces, doors of the plats close to the production belt and operators working with finished products were also assessed. Effectiveness of hand washing facilities was evaluated and found effective but operators should have more careful for personal hygiene practices. In Bangladesh, a standard for environmental monitoring of food and beverage industries is insufficient. Industries usually set their own standard according to the international guideline and BSTI regulation. A unified protocol and standard should be issued and proper monitoring should be practiced to ensure safe food chain.

Abstract 119 Antigenic profile of Burkholderia pseudomallei whole cell extract against patient sera Jannat Jahan Jamini*, Shariful Alam Jilani, Shashwata Biswas, Mahmuda Yasmin, Jamalun Nessa and Chowdhury Rafiqul Ahsan Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh *Corresponding author. E-mail: [email protected]

Burkholderia pseudomallei is a saprophytic soil bacterium and potential bio threat agent causing the infectious disease melioidosis (fatal bacterial pneumonia and sepsis), which is naturally acquired through environmental contact with the bacterium. For last couple of years, although rare, however, a number of cases of melioidosis have been detected in patients coming to the hospitals with other ailments in Bangladesh. In most of the cases, the detection methods were based on colony morphology, biochemical and ELISA techniques. In this study, we used immunoblot analysis to understand the antigenic profile of the B. pseudomallei whole cell extract against patient sera for more specific detection of the patients infected with the organism. A reference clinical strain of B. pseudomallei were grown in Ashdown medium and sonicated whole cell extract of the organisms was run on SDS-PAGE. Protein bands in the gel were transferred to a nitrocellulose membrane and run against sera of blood collected from confirmed melioidosis patients in the immunoblot analysis. A number of antigenic bands, including two prominent bands of 83 and 45 kDa molecular weight, were detected on the membrane. However, these two prominent antigenic bands along with other bands were not found in control healthy human sera. Therefore, these two bands of 83 and 45 kDa molecular weight of the B. pseudomallei, may be used as diagnostic markers for melioidosis.

Abstract 120 Antibacterial and bacterial agglutination activities of Snake guard seeds lectin Md. Rezaul Karim, Syed Rashel Kabir* Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh. *Corresponding author. E-mail: [email protected]; [email protected]

A novel lectin was purified from the Snake guard seeds with the molecular weight of 56±2 kD (Kabir et al, 2012). The lectin showed potent antitumor properties against Ehrlich ascites carcinoma cells. In the present study bacterial agglutination activity against Listeria monocytogenes, Salmonella enteritidis, Shigella flexneri,

61


Staphylococcus aureus, Shigella boydii, Pseudomonas aeruginosa were checked. T first the bacteria were grown at 37oC for 18 h in liquid nutrition medium and centrifuged at 1,027 g for 5 min and washed with 10 mM TrisHCl buffer saline, pH 7.8. The bacteria were re-suspended in the same buffer with a turbidity of 2.3 at A630. Then the lectin was serially diluted with hemagglutination beffer and 50 µl of each bacterial suspension was mixed to a final volume of 100 µl in 96-well microtiter plates. The plate was agitated for 2 min and kept at room temperature for 60 min. Finally, light microscope was used to monitor the bacterial agglutinating activity. The lectin agglutinated Salmonella enteritidis, Shigella flexneri,Staphylococcus aureus among the six. Bacterial growth inhibition was studied against Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus. The bacteria were growing overnight in nutrient broths at 37ºC and the absorbance was adjusted to 0.180.2 at A630 with liquid nutrient medium. Then the lectin was serially diluted in nutrient broth in 96-well microtiter plates and 50 µl of each bacterial suspension was mixed to a final volume of 100 µl. Four wells without lectin for each bacterium were used as control. The reading was taken at A630 after the plates were agitated for 8 h at 32oC by using temperature controlled titer plate shaker. According to the following formula percent of bacterial growth inhibition was determined. Inhibition (%) = {(Absorbance of control - Absorbance of test) /Absorbance of control} × 100. The lectin inhibited the growth of Salmonella enteritidis and Staphylococcus aureus partially.

Abstract 121 Strawberry mediated silver nanoparticles are potent antibacterialagents Md. Musfikur Rahman, A.K.M. Asaduzzaman, Syed Rashel Kabir* Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh *Corresponding author. E-mail: [email protected]; [email protected]

Nanotechnology is an emerging field for generating new applications in biotechnology and nanomedicine. Silver nanoparticles (AgNPs) are interesting candidate in nanotechnology due to their strong effectiveness as alternative to antibiotics against resistance bacteria. In the present study strawberry fruit juice extract mediated AgNPs were synthesized by mixing AgNO3 solution to the strawberry fruit juice that was primarily confirmed by colour changed and UV-visible spectrum. Shape was confirmed by Transmission electron microscopic (TEM) image and the sized was measured from the image by using ‘ImageJ’ software. The presence of silver in the AgNPs was confirmed by Energy dispersive X-ray (EDX) spectrophotometer and the functional groups were identified by Fourier-transform infrared spectroscopy (FTIR). Surface was further characterized by atomic force microscope (AFM). Biofilm formation by multidrug resistance bacteria P. aeuroginosa and K. pneumonia werestrongly inhibited by AgNPs at 37 and 20 µg/ml up to 108 h and 72 h respectively. Antibacterial activities against three pathogenic bacteria (S. enterica, E. coli and S. typhi)were checked by turbidity methods and it was found that the AgNPs inhibited the bacterial growth partially. That was also confirmed by disc diffusion method. From the above observation we can conclude that the strawberry juice extract mediated AgNPs have the high potentiality as an alternative antibacterial agents.

Abstract 122 Application of microorganisms enriched organic fertilizersto improve the soil fertility and crops yield Sunzid Ahmed1*, Maisha Mosharrat Chowdhury2, M Nazim Uddin3, M. Shabuddin3, and Md Latiful Bari1 1

Center for Advanced Research in Sciences, University of Dhaka Dhaka-1000, Bangladesh, 2Department of Mathematics & Natural Sciences, BRAC University, Mohakhali, Dhaka, Bangladesh, 3Horticulture Research Center, Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh

The experiment was carried out to evaluate the improvement of soil fertility and crop yield by using microorganism rich organic fertilizers in a Randomized Complete Block Design (RCBD)from October2008toApril2015and the test crop was eggplant and tomato. Five plots were prepared, three of which

62


received organic fertilizers (microorganisms enriched organic fertilizers named as EMOFER) at a rate of 15 t/ha each year, one plot used conventional fertilizers and the other plot served as control (Natural vegetation as control). The physicochemical properties, behaviour and persistence of plant beneficial microorganisms including nitrogen fixing bacterium, (e.g.Rhizobium sp., Azotobacter sp.), phosphate solubilising bacteria e.g. (Pseudomonas sp., Phosphobacteria) and Trichoderma sp., in the treated/non treated plot were evaluated each year. Results revealed that conventional plots had consistently higher Nitrogen (N), Potassium (K), Phosphorus (P) contents than the organic fertilizer used plots. However, a gradual increase of N, P, K contents was observed every year in organic fertilizer used plots.On the other hand organic plots had consistently higher microbial population and organic matter carbon (C)than conventional and control plots. Efficient N-fixation and P solubilisation rate in vitro using isolated microorganisms from organic plots showed bio-availability. Conventional fertilizers used plot resulted in consistently higher crops yield throughout the 7 years of study periods. On the other hand, gradual increase of crops yield was observed and the yield differences are minimum at the last year of this study. Thus, higher crops yield compare to conventional practices could be possible if organic practices are continued with essential microorganisms enriched organic fertilizers. Microbial processes are important for the management of farming system, improvement of soil quality and environmental protection.

Abstract 123 Screening of arsenite tolerant and arsenite oxidizing bacteria from arsenic contaminated soil and water from Dohar, Dhaka Fahmida Sultana, Molla Rabiul Islam, Ishteak Ahmed, Syeda Moriam Liza and Taslin Jahan Mou* Department of Microbiology, Jahangirnagar University, Dhaka-1342, Banagladesh. *Corresponding author. E-mail: [email protected]

Arsenic contamination has been emerged as a major public health concern throughout the world including Bangladesh grappled with the largest mass poisoning of a population in history. The present investigation focused on the presence of arsenic tolerant bacteria in soil and water along with screening of arsenite oxidizing bacteria for transformation of more toxic arsenite (III) to less toxic arsenate (V). For this purpose three water samples and three soil samples were collected from previously reported arsenic contaminated area of Dohar, Dhaka. A total of 50 arsenite tolerant bacteria were preliminary isolated in minimal salt media supplemented with 2mM sodium arsenite. Among them 25 isolates were from soil samples and 25 were from water samples. Gram staining revealed the prevalence of gram positive bacteria (92%) in the soil and water samples. Arsenite tolerance assay determined the level of arsenite tolerance of the isolates with the highest level of 10mM in 72% isolates. Biochemical tests were performed for presumptive identification of isolates. According to the result E. coli, Pseudomonas, Klebsiella and Bacillus were assumed to be prevalent in the arsenic contaminated soil and water. Antimicrobial susceptibility pattern of the isolates was examined to determine the correlation between arsenite and antibiotic resistance. Among the arsenite tolerant bacteria 100% organisms were susceptible to Gentamycin and Nalidixic Acid, 14% were resistant to tetracycline and 98% were susceptible to Chloramphenicol. Phenotypic screening of the arsenite oxidizing bacteria was done by AgNO3 and KMnO4 test which showed arsenite oxidation potential in four isolates biochemically identified as Pseudomonas. The arsenite oxidation potential of the isolates might be explored for arsenic bioremediation by further molecular analysis.

Abstract 124 Growth in biofilm mode leads to enhanced potential to form new biofilm in clinical isolates of Klebsiella pneumonia Abdul Razak Al-Harbi, Abdullah Alatig, Abdul Rahman Al-Ateeq Muhammad Manjurul Karim and Ashfaque Hossain*

63


College of Medicine, University of Hail, Hail, Saudi Arabia *Corresponding author. E-mail: [email protected]

Biofilm is a bacterial community embedded in a matrix of exopolysaccharides and other macromolecules. Biofilm growth is considered as a survival mode of lifestyle of bacteria and is produced by bacteria in response to physical and chemical stress such as nutrient limitation and exposure to antibiotics. Bacteria in biofilm is reported to be up to 1000 times more resistant to antibiotics and other disinfectants in comparison to their planktonic (free floating) counterparts. Many bacteria produce biofilm and production of biofilm is reported to be associated with more than 60 % of all infections reported, according to an estimate of National Institute of Health (NIH), USA. The influence of growth in biofilm in formation of new biofilm by clinical isolates of Klebsiellapenumoniae was investigated in this study. The K. pneumoniae strains used in this study were obtained from the King Khaled Hospital, Hail. Saudi Arabia. Trypticase soy broth (TSB) and trypticase soy agar plates were used for growth of the bacterial strains as required. Bacterial sample taken from biofilm or planktonic culture were used to initiate the formation of new biofilm for comparison. Crystal violet dye binding spectrophotometric assay was used for quantitation of biofilm. Sequential passage of K. pneumoniae isolates in biofilm culture in trypticase soy broth (TSB) resulted in gradually increased amount of biofilm production. On the other hand, passage of the same isolates in planktonic culture did not result in enhanced biofilm production. Passage induced enhanced biofilm production reached maximum level at passage 3 in 4 out 5 K. pneumoniae strains tested; while biofilm production by the other strain was maximal at passage 4. The increase in biofilm ranged from 22.3 % to 57.9 % for the strain tested in comparison to their planktonic counterparts. Further passage did not result in any further increase in biofilm production. The effect of Normal human serum (NHS) on passage induced biofilm formation was investigated by growing and passaging the bacterial strains in TSB containing 20 % NHS. NHS was found to further increase biofilm production by K. pneumoniae strains grown in biofilm mode, in comparison to their counterparts grown in planktonic state. The results of this study indicate that growth of K. pneumoniae in biofilm enhances its potential to form new biofilm and biofilm formation is further increased by the presence of NHS in the growth medium.

Abstract 125 Characterization of salt-tolerant Azotobacter spp for its potential application as biofertilizer in coastal zone of Bangladesh under changing climate conditions Samia Parveen1, Sumonta C Paul1, Sirajul Hoq2 and Muhammad Manjurul Karim1,* 1

Department of Microbiology, 2Department of Soil, Water and Environment, University of Dhaka, Dhaka 1000 *Corresponding author. E-mail: [email protected]

Salinity intrusion as a result of sea level rise in the mainland of the coastal zone is thought to have one of the many effects of climate change inflicting Bangladesh in its broader coast line. Over 30% of the net cultivable areas of Bangladesh, belonging to the coastal zone are prone to be affected with varying degrees of salinity with the consequent reduction of normal crop production. In order to find out an eco-friendly remedy to bring these vulnerable areas into agriculture under changing climatic condition, this study attempts to isolate and identify salt tolerant, agriculturally-important bacteria, Azotobacter spp. Soil samples from rice fields were collected both from salinity-prone and non-saline areas of Bangladesh from which Azotobacter spp. were isolated based on microscopic and cultural properties, and their tolerance to salt stress ranging from 0.63% to 15% NaCl, supplemented in a medium, Azotobacter broth that selectively support growth of nitrogen-fixing bacteria. Amplified ribosomal DNA restriction analyses (ARDRA) followed by gene sequencing of respective ribosomal RNAs revealed that the isolates belonged to six different clusters each indicating separate Azotobacter species, viz. A. vinelandii, A. chroococcum, A. salinestris, A. beijernickii, A. armenicus and A. nigricans. In order to characterize their ability to remain proactive under conditions of salinity stress, representative isolates from each ARDRA group were subjected to test their plant growth-promoting ability. The Azotobacter spp isolated from saline zones exhibited remarkable efficiency in fixing atmospheric nitrogen (0.015% under 15% salinity); producing Indole-3-acetic acid (IAA) (up to 46 µg/ml at 5 to 7.5% salt), and releasing soluble phosphorus from

64


inorganic matter (up to 155.55 µg/ml even at 15% salt condition); the abilities which are way too high from their non-saline counterparts. Further, while non-saline Azotobacter isolates exhibited super-sensitivity to drugs belonging to 15 different groups, their saline counterparts showed shear resistance to most of the drugs (estimated 84%), a property which was not attributed by plasmids. The presence of thick exopolysaccharide, found only in saline isolates as revealed by scanning electron microscopy could advocate these tolerance properties. Overall, Azotobacter spp. are indeed agriculturally-important bacteria which are capable to promote plant growth even under salinity stress, a trait that can be well exploited in combating the adverse effects of salinity intrusion for sustainable agriculture under changing climate conditions.

Abstract 126 Characterization of serologically cross-reactive environmental Shigella-like bacteria Rabeya Bilkis1,2*, Nafisa Azmuda1,2, Nils-Kåre Birkeland3, S.I. Khan1 1

Department of Microbiology, University of Dhaka, Dhaka-1000,Bangladesh, 2Department of Microbiology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh, 3Department of Biology, University of Bergen, N-5020,Bergen, Norway *Corresponding author. E-mail: [email protected]

An integrated approach comprising cultural and immunological techniques for isolation of environmental Shigella spp. was employed and this resulted in twenty-two Shigella-like isolates. In the present study, these isolates were phenotypically characterised using conventional microscopy, cultural and biochemical techniques and grouped into 13 biotypes. The isolates were confirmed by API20E and API20NE testsand those did not show a close similarity to any bacteria (99%) with the sequence of S. sclerotiorum. Different management options including eco-friendly cultural means were practiced to control the disease. A Trichoderma-based biofungicide, and Bacillus-based formulation were found effective against the disease in in-vitro and pot house assay. Intercropping with short duration crops was found to be best in field condition.

Abstract 161 Effect of Bradyrhizobium inoculation and chemical fertilizers on mungbean at southern region of Bangladesh M.A.H. Bhuiyan1*, M.B. Banu2 and M.R. Islam3 1

2

Principal Scientific Officer and Scientific Officer, Soil Science Division, BARI, Gazipur-1701, Bangladesh and 3Senior Scientific Officer, Soil Science Division, RARS, BARI, Rahmatpur, Barisal, Bangladesh *Corresponding author. E-mail: [email protected]

A field experiments was conducted at the Regional Agricultural Research Station (RARS) during late rabi season of 2011 and 2012 with the objectives to find out the efficiency of bacterial inoculum and chemical fertilizers on mungbean. The experiment was designed in RCBD having four replications in each treatment. The variety BARI Mung-6 and peat based bradyrhizobial inoculum (BARI RVr-402) was used for the experiment. There were six treatments (T1: NPKSZn, T2: PKSZn + Inoculum, T3: PKSZn, T4: N, T5: Inoculum, T6: Control). Rhizobial inoculum was used @ I.5 kg ha-1. Chemical fertilizers @ 50 kg N ha-1 from urea, 22 kg P ha-1 from TSP, 42 kg K ha-1 from MoP, 20 kg S ha-1 from gypsum and 5 kg Zn ha-1 from ZnSO4 were used where necessary. It was observed that plants receiving all chemical fertilizers along with inoculum except nitrogen gave significantly higher number of nodules (23.33 plant-1 in 2011and 23.45 plant- 1 in 2012) and nodule weight (30.00 mg plant-1 in 2011 and 27.50 mg plant-1 in 2012). The highest seed yield (1.53 t ha-1, 35.4% higher over control in 2011 and 1.19 t ha-1, 48.8% higher over control in 2012) was obtained with the same treatment which was identical with full doses of chemical fertilizers. The inoculum was more profitable than other chemical fertilizers based on benefit cost ratio.

81


Abstract 162 Performance of Bradyrhizobium inoculation on four varieties of soybean at northern parts of Bangladesh M.A.H. Bhuiyan1, F. Alam2, M.W. Rahman3 and M.B. Banu2 1*

Principal Scientific Officer, 2Scientific Officer, Soil Microbiology Laboratory, BARI, Gazipur-1701, Bangladesh; and 3 Scientific Officer, BSPC, BARI, Debigonj, Panchagarh, Bangladesh *Corresponding author. E-mail: [email protected]

A field experiment with bradyrhizobial inoculum on different soybean varieties was conducted at Breeder Seed Production Centre (BSPC), BARI, Debigonj, Panchagarh during 2011-2012 with the objectives to find out the response of Bradyrhizobium inoculation with different plant genotypes of soybean. Two soybean varieties viz. Shohag and BARI Soybean-6, and two advance lines MTD-10 and BGM-02026 were studied with and without bradyrhizobial inoculum in Factorial Randomized Complete Block Design (RCBD) with four replications. Peat based bradyrhizobial inoculum (strain BARI RGm-901) was used at the rate of 1.5 kg ha-1 as seed inoculant. Bradyrhizobium inoculation significantly increased nodulation, dry matter production, seed and stover yields of soybean. Nodulation was higher in BARI Soybean-6 and this variety also performed better in respect of yield. Among the varieties and advance lines, the highest seed yield of 1.44 t ha-1 was noted in BARI Soybean-6. Interaction effects revealed that inoculated BGM-02026 gave the highest seed yield.

Abstract 163 Response of Rhizobium strains on chickpea M.A.H. Bhuiyan1*, M.E. Ali2, F. Alam3 and M.B. Banu3 Principal Scientific Officer, 2Senior Scientific Officer and 3Scientific Officer, Soil Microbiology Laboratory, BARI, Gazipur-1701, Bangladesh *Corresponding author. E-mail: [email protected]

1

A field experiment was carried out at BARI Central Farm, Gazipur during rabi season of 2011-2012 to find out the efficiency of Rhizobium strains on chickpea. BARI Chola-5 and Rhizobium inoculum (newly isolated strains BARI RCa-220, BARI RCa-259, BARI RCa-204 and BARI RCa-205) and a mixed culture using these above four strains were used. A basal dose of P, K, S, Zn @ 22, 42, 20, 5 kg ha-1, respectively for all treatments were used in the experiment. Plants receiving mixed culture produced significantly higher nodule number and nodule weight but strain BARI RCa-205 produced the highest seed yield (1.34 t ha-1 which was 50.6% higher over noninoculated control).

Abstract 164 Effect of biofertilizer, vermicompost and chemical fertilizers on bushbean M.A.H. Bhuiyan1*, M.B. Banu2 and F. Alam3 1*

2

Principal Scientific Officer, Scientific Officer and 3Senior Scientific Officer, Soil Microbiology Laboratory, BARI, Gazipur-1701, Bangladesh *Corresponding author. E-mail: [email protected]

A field experiment was conducted at BARI Central Farm, Joydebpur, Gazipur to evaluate the effect of Rhizobium biofertilizer, vermicompost and chemical fertilizers on bushbean during the rabi season of 20112012. The crop variety was BARI Jharseem-1 and Rhizobium strain was BARI RPv-701. There were nine

82


treatments viz. T1: Control, T2: Vermicompost (VC) @ 2.5 t ha-1, T3: VC @ 5 t ha-1, T4: VC @ 2.5 t ha-1 + Integrated Plant Nutrient System (IPNS) based NPKSZnB, T5: VC @ 5 t ha-1 + IPNS based NPKSZnB, T6: VC @ 2.5 t ha-1+ Rhizobium+ IPNS based PKSZnB, T7: VC @ 5 t ha-1 + Rhizobium+ IPNS based PKSZnB, T8: 100%NPKSZnB, T9: Rhizobium+ 100%PKSZnB which were replicated four times. Peat based rhizobial inoculum was used at the rate of 1.5 kg ha-1 as seed inoculant. The Rhizobium inoculated jharseem with VC 5 t ha-1 and IPNS based PKSZnB increased nodule number (12.91 plant-1), nodule weight (19.30 mg plant-1) and 1000-seed weight. It was observed that full doses of NPKSZnB fertilizers produced the highest green pod yield (11.24 t ha-1, 72.9% higher over control) and seed yield (1.37 t ha-1, 93.0% higher over control) of jharseem. This green pod yield was statistically identical with all other treatments except control treatment, VC @ 2.5 t ha1 and VC @ 5.0 t ha-1, and seed yield was identical with VC @ 2.50 t ha-1 + IPNS based NPKSZnB, VC @ 2.5 t ha-1+ Rhizobium+ IPNS based PKSZnB, VC @ 5.0 t ha-1 + Rhizobium+ IPNS based PKSZnB and Rhizobium + 100%PKSZnB fertilizers but differed from other treatments. This indicates that application of vermicopost @ 5 t ha-1 plus Rhizobium can reduce a considerable amount of chemical fertlilizers. Vermicompost exhibited better performance in jharseem. To improve soil health and to maintain it as well as to improve crop production, we have to reduce chemical fertilizer. Benefit cost ratio (BCR) revealed that the highest BCR (15.19) was found in T9 treatment followed by T8, T6 and T4 treatment. As the yield was higher in T8 and T9 treatment, poor farmer can adopt these treatments. From one year trial, it can be concluded that full doses of chemical fertilizers and Rhizobium along with IPNS based chemical fertilizers except N may be recommended for jharseem cultivation in Grey Terrace Soil of Joydebpur (AEZ-28). From economic point of view, full doses of chemical fertilizers along with rhizobial inoculum are the best treatment. Further trials at Joydebpur and different AEZs, and economic analysis are required for final recommendation.

Abstract 165 Isolation of possible bioactive secondary metabolites from the endophytic fungi of Sonneratia apetala and Heritiera fomes from the Sundarbans Tauhidur Rahman Nurunnabi1*, Ashraful Alam2, Mahmuda Akter1, Md. Hossain Sohrab2, Md. Morsaline Billah1, S M Mahbubur Rahman1 1

Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh, 2Department of Plant Pathology, Patuakhali Science and Technology University, Dumki, Patuakhali, Bangladesh, 3Pharmaceutical Science Research Division, BCSIR Laboratories, Dhaka-1205, Bangladesh * Corresponding Author, E-mail: [email protected]

Mangrove plants have adapted to a unique but harsh habitat with muddy saline waters, anaerobic soil, brackish tidal activities and high microbial and faunal competition. Endophytic fungal association with mangrove plants confers protection from adverse environmental conditions and allows them to successfully compete with saprobic fungi decomposing senescent parts. They are known to be unique in nature for producing interesting bioactive secondary metabolites in plants. The plant Sonneratia apetala is locally known as Keora and Heritiera fomes as Sundari. They have not been reported so far for the presence of active endophytic fungi, respectively and constitute an interesting avenue of investigation. A total of 210 samples comprising healthy and mature plant parts such as leaves, barks and roots (showing no visual disease symptom) of these plantsfrom the three different zones of the Sundarbans based on salinity (Less, Moderate and Strong saline zone) was selected for investigation. From these samples, 17 Pestalotia, 23 Colletotrichum and 16 Aspergillus genus were presumptively identified based on microscopic observation. Further studies were directed employing the coculture properties (2 Colletotrichum, 1 Pestalotia and 1 Aspergillus) fungi were selected for secondary metabolite isolation purposes in potato dextrose broth (PDB). For every 1L of PDB culture, secondary metabolites was estimated average 0.46g of Colletotrichum, 0.38g of Pestalotia and 0.52g of Aspergillus to be present. Metabolites need to further test for different types of bioactivities towards the isolation of active principles and subsequent structural elucidation.

Abstract 166 83


Disinfection of archieved materials and cultural heritage artefacts by radiation processing technologies Md Kamruzzaman Pramanik, Md Firoz Mortuza, Abdul Bathen Miah and Md Khorshed Alam* Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Bangladesh Atomic Energy Commission, Dhaka, Bangladesh *Corresponding author. E-mail: [email protected]

The culture of Bangladesh is a composite of various music, drama, art, folklore languages and literature, philosophy and religion, festivals as well as in a distinct cuisine and culinary tradition. The aim of the study is to preserve paper-based archived material for a long period of time using ionizing radiation/nuclear technique. To conduct this research, note-pad samples were selected as tentative archived material. Four sets of samples were prepared and irradiated at a series of radiation doses e.g. 0, 2.0, 4.0, 6.0, 8.0, 10.0 and 14.0 kGy at a dose rate of 12.8 kGy/h from panaromic Batch type 80 kCi 60Co source. After irradiation different quality parameters such as microbiological (Total Viable Bacterial Count, Total Fungal Count), mechanical (Tensile Strength, Percent of Elongation at Break and Elastic Modulus) and color properties (L-value, a-value and b-value) of one set of samples were assessed to observe the immediate effect of ionizing radiation on this material and rest of the sample sets were stored for further analysis at a long interval. Results showed that the total bacterial count of unirradiated (control) paper were 4.0X102 cfu/g and radiattion dose of 2.0 kGy was enough to eliminate the microbial load completely. Among mechanical properties, TS of unirradiated sample was 16.23 MPa and it was gradually increased as the dose increased and finally reached 18.99 MPa at a dose of 14 kGy causing the TSchange above significant level (p < 0.05). Though changes of EB due to irradiation was insignificant, EM increased as the radiation dose increased gradually. EM of non irradiated sample was 381.85N/m2 and it started changing significantly from 6.0 kGy and finally reaches upto 477.03 N/m2 at 14.0 kGy. Results showed that Lvalue of colour parameter changed very slightly though a and b-value changed significantly from 6.0 kGy. From these findings it can be inferred that this nuclear technology might be used to conserve the cultural heritage including valuable paper-based archived materials.

Abstract 167 Antibiofilm and antimicrobial activity of some medicinal plants against multidrug resistance Pseudomonas isolates Muslima Jahan* and M. Minnatul Karim Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh *Corresponding author. E-mail: [email protected]

Pseudomonas aeruginosa is a human opportunistic pathogen and it has been emerging as a primary source of nosocomial infections including infections of artificial implants, contact lenses, and urinary catheter tubes. Pseudomonas aeruginosa isable to form biofilms on different biotic and abiotic surfaces. More than 60% of hospital-acquired infections are biofilm-related. Quorum sensing, the process of bacterial cell to cell communication; play an important role in biofilm formation. Bacteria associated with biofilms are much more difficult to kill with antibiotics and remove from surfaces than planktonic organisms. The objective of this study was to evaluate antiquorum sensing and antimicrobial activity of six medicinal plants (Centella asiatica, Sygizium aromaticum, Cinnamomum zeylanicum,Mentha spicata, Azadirachta indica, Psidium guajava) against multidrug resistance Pseudomonasaeruginosa. Pseudomonas aeruginosa was isolated form hospital waste of Islamic University, Kushtia. Identification was carried through microscopy and biochemical tests. Microscopy and biochemical tests results suggested that it was Pseudomonasaeruginosa. Disk diffusion test was carried out with commercially available antibiotic discs to know the antibiotic resistance pattern of isolated Pseudomonas aeruginosa. It was found that 10 strains were multidrug resistant among the isolated 18 strains. Biofilm assay was performed through tube assay method and it was observed that all of the Pseudomonas strains can form biofilm on the surface of test tube, though biofilm formation was varied from strain to strain. Collection of plant

84


extracts was performed with different solvents, methanol, ethyl acetate and chloroform. The extracts were subjected to test antibiofilm and antimicrobial activity against isolated high biofilm forming multidrug resistance Pseudomonas strains using the well diffusion and test tube method respectively. Ethyl acetate extracts showed higher antimicrobial activity than methanolic extracts whereas, there was no considerable antimicrobial activity of chloroform extracts. Anti biofilm assay result suggested that Cinnamon, Mentha and Cloves have high antibiofilm activity. Bioactive compounds from these medicinal plants could be the target to develop natural antibacterial and antibiofilm agents to fight against many infectious diseases.

Abstract 169 Identification of Stress Proteins in Toxic and Non-toxic strains of Vibrio parahaemolyticus under Variable Environmental Conditions Farhin Rahman*1, Nayyium Choudhury1, Chowdhury Rafiqul Ahsan2 1

Department of Mathematics and Natural Sciences, BRAC University, Mohakhali, Dhaka-1213, Bangladesh, 2Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh *Corresponding author. E-mail: [email protected]

Vibrio parahaemolyticus has been recognized as the leading cause of food-poisoning associated with seafood in Asia and the United States. The Gram negative halophilic organism is predominantly found in brackish, marine and estuarine environment, although studies have reported its isolation from freshwater as well. The changes in the habitats and different seasons, subject the organism to fluctuations in salinity and temperature respectively; these become a constant challenge to its adaptive capacities which are vital for its survival under such stress conditions. This study was designed to identify stress proteins of V.parahaemolyticus expressed under various salinity and temperatures. A trh+ve and a trh-ve strain of V.parahaemolyticus was grown under 1%, 3%, 7%, 1-7% and 7-1% salinity and 30°C, 35°C , 40°C, 30-40°C and 40-30°C temperatures. Their respective surface proteins were obtained by water-based extraction of proteins and SDS-PAGE was performed. Overall, surface protein profile for trh+ve strain was very different from that of trh-ve strain. However, distinctively, trh+ve strain showed bands for two stress proteins, a 40 KDa protein at 1% and a 20 KDa protein at 7% salinity, while no such observation was made for trh-ve strain.

Abstract 171 The microbial and fungal counts of dried and semi-dried foods collected from Dhaka, Bangladesh and methods of decreasing its load Farahnaaz Feroz1,2, Miho Mori2, and Yoshikazu Sakagami2* 1

Department of Microbiology, Stamford University Bangladesh, 2Department of Agriculture , Kindai University, Nara, Japan * Corresponding Author. E-mail: [email protected]

Food is important for our daily life, but it serves as a major reservoir of infectious pathogens. It is responsible for various food-borne illnesses as a result of bacterial and fungal contamination, posing a serious threat to public health and safety. The need for developing safe, healthy and cost effective mechanisms of decontamination prior to consumption continues to be important. Dried foods, such as spices, herbs, nuts, whole foods and semi-dried foods in Bangladesh have long been known to pose high bacterial and fungal numbers. Forty six samples of dried and semi-dried foods were tested with microbial and fungal loads of 2.5 to 3.0 (log values). Current study examined the effects of heat and low-pressure plasma treatment on the microbial and fungal growths in the foods. Log reductions after heat treatment (30, 60 and 120 minutes) were between 0.08 to 4.50. Microbial reductions were higher than that of fungal reductions. Dried spices and herbs expressed the lowest reduction rates, whereas whole foods expressed higher reduction rates. Plasma treatments (5, 10, 20, 40

85


minutes) had significantly higher log reduction rates, raging from 2.1 to 5.9. Establishing a method of decontamination, which is cost effective and poses no harm to human health, will positively impact food safety. Both of heat and plasma treatments, especially plasma treatment, will be considered as effective decontamination methods.

Abstract 172 Comparison in Bio-Degradation State of Crude Oil with Bacterial Index Balasundaram M1*, Gomathi C2, Shanmuga Priya T3 1

Faculty of Medicine, AIMST University, Semeling, Kedah, Malaysia, 2Dept. of Microbiology, JJ College of Arts & Science, Pudukkottai, India, 3Dept. of Botany, Meenakshi College, Madurai, India *Corresponding author. E-mail: [email protected]

Biodegradation rates of crude oil obtained from Nagapattinam shores, India were studied and evaluated. Hydrocarbon (HC)-degradation was compared among 3-methods of electrochemical, biological & integrated (bio-electro-chemical) procedures. Common bacterial species were isolated from enrichment oil-sludge soil, namely, bacillus, micrococcus and pseudomonas. Mineral salt medium supplemented with crude oil was used for biodegradation experiments where the effect of pH, nutrients & oxygen (O2) content on HC-degradation were assessed. A microbial count of 2.5 to 5.6 x105 cfu/g were used as the biodegradation index for pseudomonas and micrococcus spp. Moreover, pseudomonas spp. were more capable to degrade crude oil substantially in due course of time. HC-degradation rate was initially seen at low velocity but increased after 3hrs using electrochemical method. Findings of electrochemical and biological methods though differed apparently, it didn’t reveal statistically significance (p4096 µg/ml (2/20 isolates). In contrast, 16 isolates had an MIC of Imipenem at 1 µg/ml with 1 isolate having MIC of 8 µg/ml and 3 isolates with MIC of 2048 µg/ml. Eight out of 20 (40%) isolates were ESBL producers, 14 (70%) were β lactamase producers.Two isolates exhibited ESBL activity against Cefotaxime and Ceftriaxone, 3 against only Cefotaxime and 3 against only Ceftriaxone. Stability assays to investigate the stability of Ceftriaxone resistance found 10 randomly picked isolates to stably retain resistance for 21days. The present study reflected on the prevalence of β lactam antibiotics in clinical samples. Considering the findings of the present study, it appears that Imipenem still remains the drug of choice for E. coli infections; alternatively, combination therapy may need to be prescribed. The long stability of Ceftriaxone resistance in vitro raises concern from a public health point of view as persistence of the isolate in the environment may aid antibiotic resistance transfer to other bacteria.

Abstract 183 Spreads microbiology in association with product matrix, structure and chemistry Khan, I.C.* and Dodd, C.E.D University of Nottingham, UK *Corresponding author. E-mail: [email protected] The key hurdle factor in dairy spreads to prevent microbial growth is water droplet size. The four key microbes resulting spoilage usually are Bacillus spp, Staphylococcus spp, yeasts and moulds. The major sources of these bacteria are usually the ingredients as buttermilk, skimmed milk and environment as water or aerosols. The presence of these organisms resulted in an off flavour by feeding on the oil element of the recipe, containing high level C12, thus generating methyl ketones. Spreads showing off flavour had a droplet size distribution of 95%