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Contribution of the MexX-MexY-OprM Efflux System to. Intrinsic Resistance in Pseudomonas aeruginosa. Antimicrob. Agents. Chemother. 44(9): 2242–2246.
Republic of Iraq Ministry of Higher Education and Scientific Research University of Baghdad College of Science

Role of oprD Gene in Biofilm Formation and Imipenem Resistance in Pseudomoanas aeruginosa

A Thesis Submitted to the College of Science/University of Baghdad in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology/Microbiology

By Hadeel Kareem Musafer B.Sc in Microbiology/ Al-Mustansiriya University, College of Science 2005 MSc. in Microbiology/ Al-Mustansiriya University, College of Science 2007

Supervised by Dr. Harith J. Fahad Al-Mathkhury Assistant professor

November 2013

Muharrem 1434

Committee Certification We certify that we have read this thesis entitled “Role of oprD Gene in Biofilm Formation and Imipenem Resistance in Pseudomoanas aeruginosa” and as the examination committee examined the student in its content and in our opinion it adequate for award of the doctorate of philosophy in Biology/Microbiology. Signature Signature Name: Dr. Abdel Kareem Al- Qazaz Name: Dr. Mouruj Abd Alsatar Scientific Degree: Assistant professor Scientific Degree: Assistant professor Member Member Date: / / 2013 Date: / / 2013

Signature Name: Dr. Haifa Hadi Hassani Scientific Degree: Professor Member Date: / / 2013

Signature Name: Dr. Hanaa Saleem Yossef Scientific Degree: Assistant professor Member Date: / / 2013

Signature Name: Dr. Harith J. F. Al-Mathkhury Scientific Degree: Professor Member/advisor Date: / / 2013

Signature Name: Dr. Rajwa Hasan Essa Scientific Degree: Professor Chairman Date: / / 2013

Approved by the deanery of the college of science: Signature: Name: Dr. Saleh Mahdi Ali Scientific Degree: Professor Address: Dean of the College of Science Date: / /2013

Linguistic Certification

I certify that this thesis entitled “Biofilm Formation by Psudomonas aeruginosa in Respect of Imipenem Resistance” was prepared by Hadeel Kareem Musafer, under my linguistic supervision. It was amended to meet the style of the English Language.

Dr. Ayaid Khadem Zgair

Biology Department/ College of Science/ University of Baghdad / / 2013

In view of the available recommendations, I forward this thesis for Debate by the examination committee

‫�ﺴﻢ ﷲ �اﻟﺮ ْ َﲪ ِﻦ �اﻟﺮ ِﺣ ِﲓ‬ ‫))‬ ‫ا� � ِا� َ�ﻦ آ ٓ َﻣنُﻮا ِﻣنْ ُ ْﲂ َو � ِا� َ�ﻦ �آوﺗُﻮا اﻟْ ِﻌ ْ َﲅ د ََر َ�ﺎتٍ‬ ‫َ‬ ‫�َ ْﺮﻓﻊ ِ � ُ‬ ‫((‬ ‫ﻮن َﺧبِﲑٌ‬ ‫َو � ُ‬ ‫ا� ِﺑ َﻤﺎ ﺗَ ْﻌ َﻤﻠُ َ‬

‫ﺻﺪق ﷲ اﻟﻌﻈﲓ‬ ‫آٓﻳﺔ )‪ (١١‬ﻣﻦ ﺳﻮرة ا�ﺎد�‬

Acknowledgment I praise and thank Allah for clarifying my way so I could have the aptitude to accomplish this modest effort. I gratefully acknowledge dean of college of science and head of department of Biology/University of Baghdad, to permit me to complete my Ph. D. study. I would like to express appreciation and deepest thanks to the Iraqi Ministry of Higher education and scientific research for the financial support to complete my project. I am grateful to my supervisor Dr. Harith Jabar Fahad Al-Mathkhury for his guidance, patience and scientific directions. My deep appreciation to Dr. George A. O’tool, Geisel School of Medicine, Dartmouth College / USA, for his patience, scientific directions and partially funding my thesis. I would like to express my special grateful thanks to Dr. Sherry L. Kuchma, microbiology and immunology department, Geisel School of Medicine, Dartmouth College / USA for her kind and scientific assistance during six months. Special thanks for all members of Microbiology and Immunology department\ Geisel School of Medicine, Dartmouth College/ USA for their assistant. Also I would like to thank my colleagues in University of Baghdad and AlMustansyria University for their kind help.

Summary Fifty eight Pseudomonas aeruginosa strains evaluated with E test; forty seven (81.03%), two (3.4٥%), and nine (15.5۲%) strains were susceptible, intermediate, and resistant to imipenem, respectively. All the resistant and intermediate susceptible strains, previously isolated from patients with cystic fibrosis, showed no carbapenemase activity as confirmed by the Hodge test. The relationship between biofilm formation, imipenem resistance, and oprD expression were assessed in some imipenem resistant clinical P. aeruginosa

strains

(SMC631C,

SMC631F,

SMC631J,

SMC631H,

SMC631K and SMC 4974). All resistant strains showed low oprD expression and low biofilm values in comparison to the sensitive ones. The results in this study was revealed that oprD::isphoA/hah resistant strain developed significantly lower biofilm formation capacity than PAO1 wild type. Moreover, this mutation triggered Imipenem resistance (P < 0.001 by comparison to PAO1 strain). During this work, an strain with point mutation (SMC631F-ImR) in oprD gene was obtained. This mutant was resistant to imipenem (MIC> 32 mg/l) and weak biofilm former (OD550= 0.06) compared with the parent SMC631 biofilm (OD550=0.19). Expression of poprD::isphoA/hah plasmid in the oprD mutant fully restores the biofilm defect observed for the oprD mutant. There were no significant differences (P>0.05) between PAO1 and complemented strain; nevertheless, significant differences (P0.05)

between them. However, significant

differences were found between the complemented strain and the mutant oprD::isphoA/hah with empty vector. Similar results were obtained with the clinical strain SMC631. An SMC631F-ImR/poprD plasmid introduced into the oprD mutant. Again, expression of SMC631F-ImR / poprD+ in the oprD mutant fully restores the biofilm defect observed for the oprD mutant relative to the empty vector as control. Taking together, these results confirm that the inactivation of oprD is responsible for the observed defect of biofilm phenotype. mariner transposon mutagenesis was performed in two clinical strains comprised the highest biofilm former. Additionally, biofilm deficient strains were screened by microtiter plate assay, in the mutant derivatives (originated from SMC576 and SMC214). We mapped four different genes, pilY1, pilW, pslI and algC. The results revealed that pilY::Mar mutants and pilW::Mar19 mutant showed significantly deficient biofilm formation in comparison to the parent SMC576, pilY1::Mar represented five independent mutant strains (five insertion sites in pilY1 gene) and one strain with insertion in pilW gene. Moreover, mutation of pilY1 and pilW in the SMC576 background lead to loss of twitching motility. The swarm phenotypes of SMC576 mutants were shown. The pilY1::Mar mutants and pilW::Mar19 mutant exhibit a pattern of swarming motility in comparison with SMC576 (weak swarm, shorter and fewer tendrils). The second highest biofilm is formed by SMC214; were mapped two different genes pilW, pilX I that responsible for biofilm deficient. Those

biofilms formed by the pilX::Mar and pilW::Mar mutants were significantly lower (P < 0.001) than the biofilm of the SMC214 parent strain. The pilW::Mar and pilX::Mar mutants exhibit swarming phenotypes that closely resembled the SMC214 parent strain; while the pilW::Mar and pilX::Mar showed strong suppressor of twitching motility. We introduced constructed plasmid, +ppilY1 plasmid into the pilY1::Mir8 mutant, pilY1:Mir8 expression restored the higher biofilm formation to the levels comparable to the SMC576. Furthermore, the expression of +ppilY1 in the pilY1::Mir8 mutant fully restored the swarming defect observed for the pilY1::Mir8 mutant relative to the vector control. Regarding twitching motility, the expression of PilY1 was observed to complement the twitching defect of the pilY1::Mir8 mutant. Collectively, these results confirmed the inactivation of pilY1 alone which responsible for the observed suppression of SMC576 mutant phenotypes. The map of mutants pslI::Mar and algC::Mar showed the differences in Psl that produced by pslI::Mar and algC::Mar mutants which were highly deficient biofilm.

List of contents

Subject Summary List of Contents 1. Introduction and literature review 1.1. Introduction 1.2. Literature review 1.2.1. Pseudomonas aeruginosa: general characteristics 1.2.2. Pseudomonas aeruginosa and cystic fibrosis 1.2.3 Antibiotic resistance in P. aeruginosa 1.2.4 Carbapenem resistance in P. aeruginosa 1.2.4.1 Carbapenems 1.2.4.2 Carbapenemase 1.2.4.3 Porin oprD 1.2.4.4 Molecular Mechanisms of OprD-Mediated Resistance 1.2.5. Biofilm 1.2.5.1. Stages of biofilm development 1.2.5.2. Roles of extracellular polymeric substances in P. aeruginosa biofilms 1.2.5.3. Extracellular DNA 1.2.6. Pseudomonas aeruginosa motility on surface 1.2.6.1 Swarming motility 1.2.6.2 Twitching motility 2. Materials and Methods 2.1. Materials 2.1.1. Apparatuses and Equipment 2.1.2. Chemicals and biological materials 2.1.3. Culture media 2.1.4. Kits

Page I V 1 1 3 3 4 5 7 7 8 10 10 11 13 15 19 20 20 22 28 28 28 29 31 31

2.1.5. Standard strains quality control bacteria 2.1.6. Primers 2.1.6.1. Primers used in oprD genetic complementation and sequencing 2.1.6.2. Quantification real time PCR primers 2.1.6.3. Primer used in Arbitrary PCR 2.1.6.4. Primers used in pilY1 genetic complementation and sequencing 2.1.7. Plasmids and vectors used in this study 2.1.8. Buffers and Solutions 2.1.8.1. Magnesium sulfate MgSO4 1 M 2.1.8.2. Glucose20% 2.1.8.3. Lazy Bones Solution 2.1.8.4. Sodium Borate (SB) buffer 20X 2.1. 8.5. Ethylendiaminetetraacetic Acid (EDTA), 0.5 and 0.05 M 2.1. 8.6. Tris buffer (1M) pH 8 2.1. 8.7. EDTA (TE) buffer 2.1. 8.8. loadeing buffer 6X dye 2.1. 8.9. Ethanol (70%) 2.1. 8.10. Glycial acetic acid 30% 2.1.9.11. Casamino acids 20% (CAA) 2.2. Methods 2.2.1. Sterilization 2.2.2. Laboratory prepared culture media 2.2.2.1. Yeast peptone dextrose (YPD) broth media 2.2.2.2. Yeast peptone dextrose (YPD) agar plate 2.2.2.3. Lysogeny broth (LB) broth 2.2.2.4. Lysogeny broth (LB) agar plates 2.2.2.5. Minimal salts medium 5X M63 2.2.2.6. Minimal salts medium 5X M8 2.2.2.7. Minus uracil medium 2.2.2.8. Glycerol for -80ºC 2.2.2.9. Stabs 2.2.3. McFarland Standard (no. 0.5) Preparation

32 33 33 33 34 34 34 35 35 35 35 35 35 35 35 36 36 36 36 36 36 36 37 37 37 37 37 37 38 38 38

2.2.4. Single Stranded Carrier DNA 2.2.5. Preservation of bacterial strains 2.2.6. DNA agarose gel electrophoresis 2.2.7. Imipenem stock preparation 2.2.8. Antibiotic stocks used in this study 2.2.9. Biofilm Media 2.2.10. Swarming motility media 2.2.11. Twitching motility media 2.2.12. Standard and clinical strains culture 2.2.13. Determination of MIC of Imipenem for strains 2.2.13. 1. The E-test method 2.2.13. 2. Microdilution method 2.2.14. Modified Hodge Test (MHT) 2.2.15. Biofilm formation assay 2.2.16. Extraction of Genomic DNA 2.2.17. Amplification of oprD gene by polymerase chain reaction 2.2.18. Sequencing 2.2.19. Quantitative reverse transcription-PCR (qRT-PCR) (Kuchma 2.2.19.1. Bacterial Harvest 2.2.19.2. RNA extraction and cDNA 2.2.20. Genetic complementation steps of oprD mutant strain 2.2.20.1. Digestion of PMQ72 2.2.20. 2. Yeast transformation 2.2.20. 3. Electroporation 2.2.21. mariner transposon mutagenesis of the Highest biofilm producers; 576 and 214 2.2.21.1. Conjugation and selection for mutants 2.2.21.2. Storing/screening the library 2.2.21.3. Screening for Biofilm deficient 2.2.21.4. To store the library 2.2.21.5. Mapping Mariner transposons by Arbitrary PCR (ARB PCR) 2.2.22. Construction of mutant strains and plasmids 2.2.23. Twitching assays 2.2.24. Swarming motility

39 39 39 40 41 41 41 41 41 42 42 42 43 44 45 45 46 47 47 47 48 48 49 50 50 51

52 55 55 56

2.2.25. Estimation of polysaccharide extracts ۲.3. Statistical analysis 3. Results and Discussion 3.1. Imipenem susceptibility and carbapenemase detection 3.2. Assessing biofilm formation and imipenem resistance in clinical strains 3.3. Analysis of Sequencing of oprD gene 3.4. Analysis of oprD expression 3.5. OprD participates in biofilm formation. 3.6. Genes required for biofilm formation in clinical strains are conserved 3.7. Biofilm defective mutants of clinical strains SMC576 and SMC214 are sensitive to imipenem. Conclusion Recommendations REFERENCES

56 56 58 58 59 61 65 66 70 78 81 82 83

chapter one iNTROducTiON and literature review

2. Introduction and literature review 2.1. Introduction Pseudomonas aeruginosa is an important opportunistic human pathogen that can cause life-threatening infections, especially in patients with cystic fibrosis (CF) and individuals with a compromised immune system. This environmental bacterium is able to survive both in free-swimming planktonic form and in surface-associated communities known as biofilms.

P.

aeruginosa biofilms can be formed on both biotic and abiotic surfaces, thus likely contributing to this microbe’s ability to cause disease in clinical settings (Davies et al, 1998; O’Toole et al., 2000). Although there are several antimicrobial agents that continue to be effective against P. aeruginosa (i.e., carbapenem, cefepime, ceftazidime, tobramycin and amikacin), in the last few years this bacterium’s increasing resistance to antibiotics has been reported (Sanchez-Romero et al, 2007; Ruiz-Martinez et al, 2011). The emergence and spread of acquired carbapenem resistance in this species have challenged the success of therapeutic and control efforts. Therefore, investigation of the molecular mechanisms leading to resistance is crucial (Riera et al, 2011). The OprD porin of P. aeruginosa facilitates the uptake across the outer membrane of basic amino acids, small peptides that contain these amino acids, and their structural analogue, the antibiotic imipenem. Indeed, prolonged treatment of patients with P. aeruginosa infections with this antibiotic leads to imipenem resistant mutants that either lack OprD or Metallo-β-lactamase (Wolter et al., 2009).

A biofilm is a structured consortium of bacteria embedded in a selfproduced polymer matrix. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics (Høiby et al., 2010). The goal of the present study was to investigating the link between imipenem resistance, due to oprD dysfunction, and biofilm formation in laboratory and clinical isolates of P. aeruginosa, the steps of the study are listed below:

1. Detecting the Imipenem resistance isolates in P. aeruginosa associated with cystic fibrosis by E test strip. 2. Investigating the mechanism of imipenem resistance whether it is related to carbapenemase by Hodge test, or to mutation in oprD gene, by sequence analysis of oprD gene. 3. Quanatification of Biofilm formation in wild type PAO1 and clinical isolates. 4. Assessing the expression of oprD gene by quantification reverse transcription time PCR (qRT-PCR) and compared the expression with the biofilm values. 5. Genetic complementation of oprD into imipenem resistance isolate restores imipenem sensitivity and biofilm formation. 6. Explaining why the clinical isolate forms high biofilm by using transposon mutagenesis with mariner transpose and genetic screening for the genes responsible for high biofilm formation in clinical isolates by Arbitrary PCR and sequence analysis for these genes. 7. Perceiving the relationship between biofilm and Twitching test, polysaccharide synthesis, and swarming motility.

8. Genetic complementation of mutant gene pilY1responsible for high biofilm. 9. Estimation of polysaccharide production by ELISA test.

1.2 Literature review 1.2.1 Pseudomonas aeruginosa: general characteristics P. aeruginosa is a gram negative, uniformly stained, straight or slightly curved rods, measuring 0.5 to 1.0 μm by 1.5 to 5.0 μm in length. They are aerobic, non-spore forming, motile by one or more polar flagella. They are either incapable of utilizing carbohydrates as source of energy or degrade them “oxidatively” rather than fermentative pathway (de Freitas and Luis Barth, 2002). P. aeruginosa is a member of the Gamma Proteobacteria class of bacteria, belonging to the bacterial family Pseudomonadaceae. Since the revision taxonomy based on conserved macromolecules (e.g. 16S ribosomal RNA) the family includes only members of the genus Pseudomonas which are cleaved into eight groups. P. aeruginosa is the type species of its group (Hall et al., 2004). Biochemically, they are oxidase and catalase positive, motile, grows well at 42˚C, P. aeruginosa, utilizes glucose oxidatively, reduces nitrate to nitrite, Methyl red/Voges Proskauer test is negative, do not decarboxylate lysine and ornithine, but dihydrolyze agrinine, mannitol not fermented, produce alkaline slant/alkaline butt with no gas and no H2S in TSI, indole negative, utilize citrate, do not hydrolyze urea, do not produce phenyl pyruvic acid, liquefies gelatin, do not hydrolyze aesculin and utilize acetamide (Trautmann et al., 2008). The typical Pseudomonas bacterium in nature might be found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a

unicellular

organism,

actively

swimming

by

means

of

its

flagellum. Pseudomonas is one of the most vigorous, fast-swimming bacteria seen in hay infusions and pond water samples. In its natural habitat P. aeruginosa is not particularly distinctive as a pseudomonad, but it does have a combination of physiological traits that are noteworthy and may relate to its pathogenesis (Bodey et al., 1983). On

nutrient

agar,

colonies

are

large

and

pigmented

pyocyanin/fluorescence and on 5% sheep blood agar, they produce βhemolytic, large flat spreading, mucoid, rough, pigmented colonies with characteristic metallic sheen (high carbon, low nitrogen content). Many strains may produce a fruity, sweety, musty or grape like odor due to the presence of 2-aminoacetophenone. On MacConkey agar, they produce non lactose fermenting colonies with green pigmentation and metallic sheen. Colonies from respiratory tract infection samples produce large amounts of alginate, an exopolysaccharide consisting of mannuronic and guluronic acids which aids in forming mucoid colonies. Cetrimide agar is a selective and differential medium for the identification of P. aeruginosa in which Cetrimide acts as detergent which inhibits most bacteria and enhances the production of two pigments pyocyanin, pyoverdine. However, about 4% of clinical strains of P. aeruginosa do not produce pyocyanin (Mackie and MacCartney. 1996). 1.2.2 P. aeruginosa and cystic fibrosis Cystic fibrosis (CF) is the most common inherited lethal genetic disorder, Individuals suffering from CF harbor mutations in a gene on the long arm of chromosome 7 (Riordan et al., 1989). The gene product is the cystic fibrosis transmembrane conductance regulator (CFTR) which

regulates and facilitates transport of electrolytes across epithelial cell and other membranes. The mucus in the CF airways is highly viscid, sulphated and readily forms aggregates (Chace et al., 1985; Welsh and Smith, 1993). In the CF lung, the viscid mucous cannot be propelled so easily and the escalator fails, leading to an accumulation of mucus and trapped bacteria (Bals et al., 1999). P. aeruginosa is a major pathogen in the CF lung. Chronic colonization with P. aeruginosa is associated with a more rapid decline in lung function, especially if the isolate becomes mucoid (Emerson et al., 2002). Although most patients are initially infected with nonmucoid P. aeruginosa, it later transitions to a mucoid state (Li et al., 2004). Mucoidy results from an overproduction of alginate which is thought to play a protective role in the relatively harsh environment of the CF lung, perhaps by enhancing the formation of biofilms, The link between CF-derived mucoid P. aeruginosa isolates and their biofilm lifestyle, has led to the assumption that alginate is the key secreted polysaccharide in biofilms of both mucoid and nonmucoid strains (Hentzer et al., 2001). The studies examining the role of alginate in the initiation or maturation of biofilms often involved the comparison of mucoid strains isolated from the CF lung with nonisogenic, nonmucoid strains; these studies generally compared the ability of these strains to form biofilms and the antibiotic resistance of established biofilms (Mai et al., 1993). 1.2.3 Antibiotic resistance in P. aeruginosa P. aeruginosa has become an important and frequent opportunistic nosocomial pathogen. This organism is characterized by an innate resistance to multiple classes of antimicrobials, causing difficult-to-treat infections,

which are therefore associated with significant morbidity and mortality (Obritsch et al., 2004). Infections by P. aeruginosa are a serious clinical problem, particularly in immune compromised hosts in hospital settings (Fujitani et al, 2011). Moreover, the treatment of these infections is often difficult because of the limited number of effective antimicrobial agents, due to the intrinsic resistance of P. aeruginosa strains and their different modes of growth (Khan et al, 2010). This resistance reflects the synergy between the bacterium’s low outer-membrane permeability, its chromosomally encoded AmpC β-lactamase, and its broadly specific drug efflux pump (Masuda et al, 2000). Furthermore, P. aeruginosa readily acquires resistance to most antimicrobials through mutations in its chromosomal genes and through extrachromosomal elements carrying resistance determinants (Qiu et al, 2009; Livermore and Yang, 1987). The broad-spectrum resistance of P. aeruginosa is mainly due to a combination of different factors: (i) low outer membrane permeability (Nikaido , 1994), (ii) Presence of the inducible AmpC chromosomal ßlactamase (Lister et al., 2009), (iii) synergistic action of several multidrug efflux systems (Poole, 2004), and (iv) prevalence of transferable resistance determinants, in particular, carbapenemhydrolyzing enzymes (mainly metallo-ß-lactamases (MBL) (Gutiérrez et al., 2007). Although there are several antimicrobials (carbapenems, cefepime, ceftazidime, tobramycin and amikacin) that continue to be effective against P. aeruginosa, in the last few years the bacterium’s increasing resistance to many others has been reported (Sanchez-Romero et al, 2007; Ruiz-Martinez et al, 2011).

Carbapenems are good antimicrobial activity against P. aeruginosa but the emergence and spread of acquired carbapenem resistance in this species have challenged the success of therapeutic and control efforts. Since carbapenems, especially imipenen, are widely used in the clinical setting (Riera et al, 2011), investigation of the molecular mechanisms leading to resistance is crucial. 1.2.4 Carbapenem resistance in P. aeruginosa 1.2.4.1 Carbapenems Carbapenems are frequently used to treat P. aeruginosa; however, resistance to the carbapenems is emerging rapidly (Zavascki et al., 2005). The introduction of carbapenem into clinical practice represented a great advancement for the treatment of β-lactam resistant bacteria. Due to their broad spectrum of activity and stability to hydrolysis by most β-lactamase, the carbapenems have been the drugs of choice for treatment of infections caused by penicillin or cephalosporin resistant gram negative bacilli (Jesudason et al., 2005). Carbapenems

exert

peptidoglycan-assembling

their

action

transpeptidases

primarily

by

inhibiting

(penicillin-binding

the

proteins

[PBP]) located on the outer face of the cytoplasmic membrane. In general, carbapenems can efficiently cross the outer membrane of the bacterium, as they are small hydrophilic antibiotics. They enter the cell by passing through the aqueous channels provided by porin proteins (Huang et al., 1995). Among the several mutation-mediated resistance mechanisms existing in P. aeruginosa are those conferring decreased susceptibility or resistance to carbapenems. These antimicrobial agents are commonly used to treat infections produced by multiresistant strains of P. aeruginosa, as they are

stable against most clinically relevant ß-lactamases (including broadspectrum,

extendedspectrum,

and

AmpC-type

enzymes).

Although

carbapenems remain effective antibiotics for therapy of infections caused by multidrug resistance (MDR) P. aeruginosa isolates, development of high carbapenem resistance rates in P. aeruginosa isolates has been reported worldwide (Davies et al., 2007). 1.2.4.2 Carbapenemase Metallo-β-lactamase (MBL) was first detected in 1960, in Bacillus cereus which was chromosomal in location. Then, first plasmid mediated MBL isolates was found in P. aeruginosa in 1991 in Japan. Since early 1990s, MBL encoding genes have been reported all over the world in clinically important pathogens, such as Pseudomonas spp., Acinetobacter spp., and members of the Enterobacteriaceae family (Picao et al., 2008). MBL in gram negative bacilli is becoming a therapeutic challenge, as these enzymes usually possess a broad hydrolysis profile that includes all β-lactam antibiotics including carbapenems (Galani et al., 2008). The carbapenemases MBLs are the most feared because of their ability to hydrolyze virtually all drugs in that class, including the carbapenems (Walsh et al., 2002). In addition to their resistance to all β-lactams, the MBL producing strains are frequently resistant to aminoglycosides and fluoroquinolones, Unlike carbapenem resistance due to several other mechanisms, the resistance due to MBL and other carbapenemase production has a potential for rapid dissemination, as it is often plasmid mediated (Walsh et al., 2005). Consequently, the rapid detection of carbapenemase production is necessary to initiate effective infection control measures to prevent their dissemination. Various methods like Modified

Hodge test, EDTA disk synergy (EDS) test (Lee et al., 2001), MBL E-test, EDTA-based microbiological assay are used for the detection of MBLs, Nevertheless, detection of genes coding for carbapenem resistance by PCR, usually give reliable and satisfactory results, but this method is of limited practical use for daily application in clinical laboratories because of the cost (Marchiaro et al., 2005). MBLs spread easily on plasmids and cause nosocomial infections and outbreaks. Such infections mainly concern patients admitted to Intensive Care Units with several co-morbidities and a history of prolonged administration of antibiotics (Maltezou, 2009). Moreover, MBL producing isolates are also associated with higher morbidity and mortality (Walsh et al., 2005). Early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their interhospital and intrahospital dissemination (Picao et al., 2008). Class 1 integron-containing P. aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands (Martinez et al., 2012). The transmissible MBLs confer high-level resistance to all carbapenems and are found worldwide (Walsh et al., 2005). AmpC, the chromosomal β-lactamase, has been found to have very little activity against carbapenems but can work in synergy with other resistance mechanisms (Quale et al., 2006). In the absence of carbapenem-hydrolyzing enzymes, the mechanism leading to carbapenem resistance is mostly mediated by OprD loss, which primarily confers resistance to imipenem but also confers low grade resistance to meropenem (Köhler et al., 1999; Livermore, 1992).

1.2.4.3 Porin oprD The main porin for uptake of carbapenems in P. aeruginosa is the outer membrane protein (OMP) OprD, a specialized porin which has a specific role in the uptake of positively charged amino acids such as lysine and glutamate (Huang et al., 1995). Other routes for carbapenem uptake have been proposed (Pérez et al., 1996), but their actual relevance has not been consistently proved. Inactivating mutations in OprD have been documented to confer resistance to imipenem and to a lesser extent to meropenem and doripenem (Sanbongi et al., 2009). It is also remarkable that mutations leading to the upregulation of the MexAB-OprM active efflux system may increase the resistance to meropenem and doripenem but with no effect on the susceptibility of P. aeruginosa to imipenem, which is not a substrate for this system (Köhler et al., 1999). Porin OprD of P. aeruginosa facilitates the uptake across the outer membrane of basic amino acids, small peptides that contain these amino acids, and their structural analogue imipenem. Indeed, prolonged imipenem treatment of patients with P. aeruginosa infections leads to imipenem resistant mutants that either lack OprD due to an oprD gene mutation (Lynch et al., 1987) or have strongly reduced OprD levels due to an nfxC-type mutation (mexT) which suppresses oprD expression at the same time as upregulation of the mexEF-oprN multidrug efflux operon (Fukuda et al., 1995; Kohler et al., 1997).

1.2.4.4 Molecular Mechanisms of OprD-Mediated Resistance The pathway to OprD-mediated resistance can involve mechanisms that decrease the transcriptional expression of oprD and/or mutations that disrupt the translational production of a functional porin for the outer membrane. At the level of oprD transcription, characterized mechanisms include (i) disruptions of the oprD promoter, (ii) premature termination of oprD transcription, (iii) coregulation with mechanisms of trace metal resistance, (iv) salicylate-mediated reduction, and (v) decreased transcriptional expression through mechanisms of coregulation with the multidrug efflux pump encoded by mexEF-oprN. oprD promoter disruptions have occurred as a result of deletions or insertions within the upstream region of oprD. Yoneyama and Nakae (1993) reported the association of a large deletion encompassing the putative promoter and initiation codon that prevented transcription of oprD. IS1394 and an ISPa16-like insertion (IS) element have been described upstream of the oprD coding region for imipenem-resistant isolates of P. aeruginosa exhibiting decreased oprD expression (Wolter et al., 2008; Wolter et al., 2009). El Amin et al. (2005) observed that premature termination of transcription was occurring in clinical isolate, potentially due to mutations within the structural gene sequence. The most complex mechanisms are the transcription of oprD that are linked to the regulation of expression of the mexEF-oprN efflux pump (Ochs et al., 1999). These mechanisms of coregulation are, showed the complexity by which P. aeruginosa is able to regulate expression of resistance mechanisms and why it is sometimes so difficult to definitively link

phenotypes to changes in one specific mechanism (Wolter et al., 2004; Evans and Segal, 2007). 1.2.5 Biofilm Biofilms are surface-attached communities of bacteria embedded in an extracellular matrix of biopolymeric substances and are involved in many types of chronic infections (Costerton et al., 1995). Biofilm bacteria are physiologically distinct from free-swimming bacteria of the same species. Wild-type, nonmucoid P. aeruginosa biofilm formation proceeds through distinct developmental steps. After initial attachment of single cells to a surface, the bacteria move on the surface by twitching motility to form clumps of cells or microcolonies (O’Toole and Kolter, 1998). Figure 1-1 explains the stages of biofilm formation.

Figure 1-1: Essential steps of bacterial biofilm formation inspect with swarming motility (http://www.pasteur.fr/recherche/RAR/RAR2006/Ggb-en.html).

Common examples of biofilms include dental plaque, endocarditis, and slime on river stones. Biofilms are increasingly recognized as contributing to disease pathogenesis in cystic fibrosis and in other bacterial diseases (Parsek et al., 2003). Bacteria in a biofilm state exhibit increased resistance to antibiotics (Prince et al., 2002) and host defense factors (Jesaitis et al., 2003). Communal bacteria in a biofilm can survive antibiotic concentrations as much as 1000-fold higher than the same bacteria in an individual, freeliving, planktonic state (Høiby, 2001). Therefore, clinically attainable antibiotic concentrations may not adequately clear biofilm infections, allowing the bacterial population to recover, persist, and spread (Singh et al., 2000). P. aeruginosa environmental bacterium is capable of living planktonically or in surface-associated communities known as biofilms. P. aeruginosa biofilms can form on a variety of surfaces, including in mucus plugs of the CF lung and abiotic surfaces, such as contact lenses and catheters (Davies et al., 1998; O’Toole et al., 2000). 1.2.5.1. Stages of biofilm development ·

Attachment Motile (planktonic) bacteria transform to the sessile form prior to

biofilm formation as they adhere to a favourable surface; such as a medical device or the host tissue. In some cases initial adhesion of biofilm forming microorganisms is achieved by means of adhesins located on specialised organelles such as fimbriae (pili) (Sauer et al, 2002; Lasaro et al, 2009).

· Formation of microcolonies The cells aggregate as they divide on adhesion to a surface but the daughter cells multiply outward and upward from the point of attachment to form cell clusters. The dividing cells produce quorum sensing molecules and extracellular polymeric substances, or polymer matrix. The matrix houses the aggregating cells in microcolonies and also attaches the biofilm to the surface on which it is forme, Microcolonies become larger as the number of organisms increase and the quantity of polymer matrix produced also increases (Watnick and Kolter, 1999). More signalling molecules and polymer matrix are produced by the organisms within the microcolonies at this stage (Tolker - Neilsen et al, 2000; Malic et al, 2009). The fully mature biofilm structure comprises of bacterial cells, the polymer matrix, and interstitial water channels that facilitate the exchange of nutrients and wastes in and out of the biofilm into the surrounding environment, P. aeruginosa displayed multiple phenotypes during biofilm development and biofilm cells at dispersion were found to be similar to the planktonic cells in phenotypic expression (Sauer et al, 2007). · Detachment and dispersal of biofilm organisms The biofilm environment is innately regulated and studies have shown that high population density within a mature biofilm induced programmed detachment of bacteria from biofilm through the secretion of chemical substances by the organisms (O'Toole et al, 2000). Studies have shown that detachment occurs when the organisms respond to chemical substances secreted by them such as signalling molecules (Stoodley et al, 2005), proteins and degradative enzymes (Barraud et al, 2006) and oxidative or nitrosative stress-inducing molecules such as nitric oxide (NO) produced as

a result of metabolic processes within a biofilm (Schlag et al, 2007). It has also been shown that alginate lyase; a degradative enzyme produced by biofilm organisms cleaves the polymer matrix into short oligosaccharides. The cleavage antagonises the attachment characteristics of alginate leading to increased detachment of biofilm organisms (Barraud et al, 2006). 1.2.5.2 Roles of extracellular polymeric substances in P. aeruginosa biofilms Exopolysaccharides are an important component of the microbial biofilm extracellular matrix, since they contribute to overall biofilm architecture and to the resistance phenotype of bacteria in biofilms. Several species

have

been

shown

to

produce

a

matrix

consisting

of

exopolysaccharides, proteins and nucleic acids (Pamp et al., 2007). The major functions ascribed to the matrix are its role as a structural scaffolding for biofilm cells and as a protective barrier against some antimicrobials. Matrix production can dictate pattern formation in biofilms in a number of ways. In some cases where biofilm populations consist of motile and nonmotile subpopulations, matrix production can facilitate the transition from surface motility to sessility (Merritt et al., 2007; Kuchma et al., 2007). The common theme in several species, where motility and matrix production are inversely regulated by intracellular levels of the signalling molecule cyclic dimeric guanosine monophosphate (c-di-GMP), Although the matrix in general is considered to spatially fix the cells in a biofilm, evidence has been presented that matrix components in some cases can guide migration of the cells (Barken et al., 2008). Matrix production can also influence average cell-to-cell distances between members of a biofilm community. The formation of large, tightly packed aggregates is a feature

sometimes observed in a biofilm population overproducing secreted components of the biofilm matrix (Stapper et al., 2004). In P. aeruginosa, three major secreted polysaccharides have been implicated in pattern formation in biofilms; alginate, Psl, and Pel (Colvin et al., 2012). · Alginate AlgC appears to be crucial for general exopolysaccharide biosynthesis, not just alginate, as it is also required for precursor synthesis of Psl, as well as LPS and rhamnolipids (Goldberg et al., 1993; Olvera et al., 1999). Alginate is composed of the uronic acids, mannuronic acid, and its epimer, guluronic acid (Govan and, Deretic, 1996). In non-mucoid strains, alginate does not appear to be an important component of pattern formation/community structure (Wozniak et al., 2003). In clinical biofilms, it appears to be produced, where its expression is induced under conditions of low oxygen tension (Worlitzsch et al., 2002). In the airways of people suffering from Cystic Fibrosis, P. aeruginosa is seen to undergo a transition to a mucoid phenotype (Govan and, Deretic, 1996). Mucoidy is characterized by alginate overproduction and its impact on pattern formation in biofilm communities is great (Stapper et al., 2004). The chemical environment is key in this regard. Extracellular calcium acts as a cation bridge between the negatively charged alginate polymers (Sarkisova et al., 2005). In the absence of calcium, alginate overproduction results in the production of aggregates of tightly packed bacterial cells in the biofilm. However, in the presence of low calcium (µM–mM), the secreted alginate forms a gel. This results in individual cells being suspended within the gel, increasing the average intercellular distance (Hentzer et al., 2001; Sarkisova

et al., 2005). A functional consequence of alginate overproduction in a biofilm is increased tolerance to antibiotics such as tobramycin (Hentzer et al., 2001).

· Psl The psl gene cluster contains 15 cotranscribed genes (pslA to pslO) encoding proteins predicted to synthesize the Psl EPS, which is important to initiate and maintain biofilm structure by providing cell-cell and cell-surface interactions The pslH- and pslI-encoded proteins exhibit homology to galactosyltransferases and mannosyltransferases, respectively. That means the Psl EPS is composed mainly of mannose and galactose and that Psl is indeed a matrix component of the biofilm (Ma et al., 2007), Overhage and colleagues (2005) mapped the psl operon promoter 41 bp upstream of the pslA start codon. Ma et al. (2007) observed the frame deletions of pslH (strain WFPA818) and pslI (strain WFPA819). The biofilm formation capacities of these strains were compared with those of wild-type and psl-deficient strains in a rapid attachment assay. Loss of either PslH or PslI function results in a profound attachment defect, similar to that observed with the psl null strain WFPA800. The attachment defect of WFPA818 and WFPA819 was restored when a plasmid expressing either pslH (pMA10) or pslI (pMA11) was introduced into the respective strain; these data indicate that PslH and PslI are key proteins for Psl EPS synthesis. In a prior transposon mutagenesis screen, pslH and pslI mutants also exhibited reduced biofilm formation (Friedman, and Kolter, 2004).

Overproduction of the Psl polysaccharide led to enhanced cell-surface and intercellular adhesion of P. aeruginosa. This translated into significant changes in the architecture of the biofilm. Consequently, it was proposed that Psl has an important role in P. aeruginosa adhesion, which is critical for initiation and maintenance of the biofilm structure. The ability to form biofilms in the airways of people suffering from cystic fibrosis is a critical element of P. aeruginosa pathogenesis. The 15-gene psl operon encodes a putative polysaccharide that plays an important role in biofilm initiation in nonmucoid P. aeruginosa strains. Biofilm initiation by a P. aeruginosa PAO1 strain with disruption of pslA and pslB (ΔpslAB) was severely compromised, indicating that psl has a role in cell-surface interactions. In previous study showed the adherence properties of this ΔpslAB mutant using biotic surfaces (epithelial cells and mucin-coated surfaces) and abiotic surfaces. Accordingly, psl is required for attachment to a variety of surfaces, independent of the carbon source (Ma et al., 2006). The actual structure of Psl is not known, although it is rich in rhamnose, mannose, and glucose monomers (Ma et al., 2007). The primary function for Psl in non-mucoid strains is attachment; Strains defective for Psl production are defective in surface attachment on many different surface types. However, Psl contributes to maintaining biofilm structure at later stages in development (Matsukawa and Greenberg, 2004). Ma et al. (2006) demonstrated that a strain that conditionally produces Psl formed mature biofilms that eroded away once Psl expression was disrupted.

Psl was seen to preferentially localize to the exterior of

aggregates or microcolonies, forming a shell (Wozniak et al., 2003). The interesting Psl staining pattern suggests that the polysaccharide plays a key structural role encasing and ultimately holding together cells in

an aggregate. Like alginate/mucoidy, genetic variants that overproduce Psl have been observed in CF sputum samples (Haussler et al., 2003). Psl producing strains produce distinctive colony morphology on solid medium, called rough, small colony variants (RSCVs). Like mucoidy, the CF airways select for this phenotype (Smith et al., 2006). RSCVs are characterized by autoaggregation in liquid culture and hyper-attachment to surfaces (Kirisits et al., 2005). Overhage et al. (2005) stated that psl expression was localized to the centers of microcolonies within biofilms. In addition, Kirisits et al. (2005) showed that the expression of psl and pel was elevated in variants isolated from aging P. aeruginosa PAO1 biofilms. It has been suggested that the mechanistic basis for psl and pel overproduction in these variants, as well as other autoaggregative variants, involves elevated levels of the c-diGMP. P. aeruginosa has several loci capable of modulating the c-diGMP level, including the wsp, LadS, and retS signal transduction systems (Kuchma et al., 2010). · Pel The last major exopolysaccharide produced by P. aeruginosa is Pel. The pel gene cluster consists of seven genes, which encode the enzymatic activities required for synthesis of the hydrophobic glucose rich Pel exopolysaccharide (Friedman and Kolter, 2004). Unlike Psl, Pel does not appear to play a role in attachment, although it is important in maintaining mature biofilm structures (Vasseur et al., 2005). Its contribution to the cellular distribution patterns found in biofilms, and where it is produced in a biofilm is unclear. However, like Psl, Pel is found to be overproduced in many RSCVs (Kirisits et al., 2005).

1.2.5.3 Extracellular DNA Extracellular DNA was shown to be present in high concentrations in the outer part of the microcolonies in young P. aeruginosa biofilms and between the stalk-forming and cap-forming subpopulations in mature glucose grown biofilms (Allesen-Holm et al., 2006). Type IV pili bind with high affinity to DNA (Aas et al., 2002; van Schaik et al., 2005), and evidence has been presented that the high concentration of extracellular DNA on the microcolonies in developing P. aeruginosa biofilms may cause accumulation of the migrating piliated cells and thereby facilitate formation of the mushroom caps (Barken et al., 2008). Production of extracellular DNA during P. aeruginosa biofilm development has been shown to be dependent on the quorum-sensing system (Allesen-Holm et al., 2006). Expression of the pqs-genes in developing P. aeruginosa biofilms was shown to occur specifically in the outer layer of the stalk forming microcolonies, correlating with the location of the extracellular DNA (Yang et al., 2007). 1.2.6 P. aeruginosa motility on surface 1.2.6.1 Swarming motility Swarming motility is operationally defined as a rapid multicellular bacterial surface movement powered by rotating flagella. Although simple, accurate, and mechanistically meaningful, the definition does not do justice to the wide array of phenotypes associated with swarming motility, nor does it emphasize all that remains unknown about this behavior (Copeland et al., 2010). A. Factores important for swarming motility · Flagella

During swarming, P. aeruginosa retains its polar flagella but synthesizes an alternative motor specifically required to propel movement on surfaces and through viscous environments, Thus the expression of alternative motors is at least one other way to facilitate swarming motility besides use of peritrichous flagella (Toutain et al., 2005). · Rafting Whereas bacteria swim as individuals, swarming bacteria move in sideby-side cell groups called rafts (Copeland et al., 2010). Raft formation is dynamic: cells recruited a raft move with the group whereas cells lost from a raft quickly become non-motile. The dynamism in cell recruitment and loss suggests that no substance or matrix maintains raft stability save perhaps the flagella themselves. Indeed, scanning electron microscopy of a swarm of Proteus mirabilis revealed extensive rafting and perhaps intercellular bundling of flagella (Jones et al., 2004). As with hyperflagellation, the reason that swarming motility requires raft formation is at present unclear. · Surfactant synthesis Initial characterization of P. aeruginosa implicated rhamnolipids as the swarming surfactant (Köhler et al., 2000). Di-rhamnolipid is composed of two rhamnose sugars attached to the complex fatty acid β-hydroxydecanoylβ-hydroxydecanoate (HAA) (Caiazza et al., 2005). Subsequent investigation has shown that the di-rhamnolipid precursors HAA and mono-rhamnolipid also act as surfactants to promote swarm expansion (Tremblay et al., 2007). Surfactant production is commonly regulated by quorum sensing (Ochsner and Reiser, 1995). Surfactants are shared secreted resources and are only effective at high concentration. Therefore, quorum sensing may have evolved to regulate surfactant production to ensure that the surfactants

are only made when there are sufficient bacteria present to make surfactants beneficial (Kato et al., 1999). The

flagellum

and

the

chemotaxis

system,

consisting

of

chemoreceptors and a signal relay system similar to that of E. coli, allow the bacterium to respond to attractants and repellents (Kato et al., 1999). Swarmer cells, which are usually elongated and hyperflagellated, differentiate from vegetative cells probably by sensing the viscosity of the surface or in response to nutritional signals (Harshey, 1994). The swarming of the normally polar, monotrichously flagellated bacterium P. aeruginosa is induced on 0.5 to 0.7% agar when certain amino acids are provided as the sole source of nitrogen. The swarmer cells of P. aeruginosa are elongated and can possess two polar flagella. Unlike all other swarming bacteria, P. aeruginosa also requires type IV pili for this type of motility. B. Biofilm and swarming motility inversely regulate by c-diGMP Cyclic-di-GMP is a ubiquitous second messenger in bacteria, the c-diGMP antagonistically controls motility and virulence of single, planktonic cells on one hand and cell adhesion and persistence of multicellular communities on the other has spurred interest in this regulatory compound (Aldridge et al., 2003). C-di-GMP controls cellular processes associated with the sessile-motile transition in eubacteria, including exopolysaccharide (EPS) production, attachment, and motility. Low concentrations of c-di-GMP are associated with cells that move by virtue of flagellar motors or retracting pili. In contrast, increasing concentration of c-di-GMP promote the expression of adhesive matrix components and results in multicellular behavior and

biofilm formation that mean coordinate regulation of these two surface behaviors depends upon common downstream effectors, such as regulation of flagellar function and production of the Pel-derived EPS (Lim et al., 2006). Although Events during early biofilm formation by P. aeruginosa PA14 require proper control of flagellar function for the reversible to irreversible attachment phase, as well as robust production of the Pel EPS (Caiazza et al., 2007; Merritt et al., 2007). C-di-GMP-mediated regulation can occur through a variety of mechanisms, including stimulation of exopolysaccharide (EPS) production, cell surface adhesin expression/ localization, and/or repression of various forms of motility (Newell et al., 2009). Conversely, reduced levels of c-di-GMP generally lead to stimulate of motile behaviors concomitant with reduced biofilm formation and hence promote the switch to a motile lifestyle (Hengge, 2009). Swarming motility is a flagellum-driven process and therefore the sensitive to changes in flagellar function (Caiazza et al., 2007; Merritt et al., 2007). Moreover, strains defective for production of the Pel EPS show enhanced swarming relative to the wild type (WT), indicating that production of the polysaccharide negatively impacts on swarming motility in P. aeruginosa PA14 (Caiazza et al., 2007). Strains with mutations in the bifA gene in PA14 elevate the levels of cdi-GMP, and this accumulation is largely dependent upon the cyclase activities of both SadC and RoeA, the resulting excess c-di-GMP produced in the bifA mutant leads to hyper-biofilm formation and repression of swarming motility (Kuchma et al., 2007, Merritt et al., 2010). Moreover, it was shown that enhanced production of the Pel EPS is required for the hyper-biofilm-forming phenotype but contributes only

marginally to the swarming defect of the bifA mutant (Kuchma et al., 2007). Always the question was witch factors are required for repression of swarming motility when c-di-GMP levels are elevated, (Kuchma et al., 2010) performed a genetic screen to identify suppressors in the bifA background that restored the ability to swarm. They identified a role for the pilY1 gene in c-di-GMP-mediated repression of swarming motility. Strains with mutations in the pilY1 gene show robust suppression of both the swarming deficiency as well as the hyper-biofilm-forming phenotype exhibited by the bifA mutant. 1.2.6.2 Twitching motility Twitching motility is believed to result from the extension and retraction of the pilus filament, which propels the cells across a surface. Pilus synthesis and assembly require at least 40 genes which are located in several unlinked regions on the chromosome (Hobbs et al., 1993). It is clear that active extension and retraction of type IV pili is involved in twitching motility (Skerker and Berg, 2001). These pili are about 6 nm in diameter and up to 4 µm in length; they are typically found at one or both cell poles (Bradley, 1972). Radial expansion rates of colonies via twitching motility can approach 0.3 µm/s (Mattick, 2002), involved in biofilm formation (O’Toole and Kolter, 1998). Twiching motility like swarmer cells, rafts or spearhead-like clusters of aggregated cells in P. aeruginosa have been observed during twitching motility. Within the rafts, cells are highly aligned in close cell-cell contact. The rafts move radially outward, following the long axis of the cells. Cells from one group join into another and form a lattice, many like swarming bacteria. Such cells at first move end forward toward the other cells until

they touch with their poles and then rapidly snap into an aligned position, which accounts for the characteristic jerky twitching motion observed with this form of motility (Mattick, 2002). Although twitching motility is primarily a social activity, individual cells can show limited movement when in contact with inert substrates or on agar at low concentrations (Mattick, 2002). In vitro and in vivo studies show that mutants lacking functional type IV pili have a significant reduction in colonization, biofilm formation, and ability to spread (O’Toole and Kolter, 1998; Wozniak and Keyser, 2004). Control of type IV pili expression and twiching motility is complex. One system that controls twiching motility is a sensor kinase and a response regulator pair referred to as FimS/AlgR (Whitchurch et al., 1996). However, type IV pili are polar organelles that are composed of a single protein subunit, PilA. PilA is exported out of the cell and polymerized to form the surface fimbrial strand. Prepilin genes are located in one of the many v gene clusters and appear to play a role in type IV pili assembly, export, localization, and maturation and the general efficiency of the type IV pili machinery (Mattick, 2002). A microarray study revealed that the fimUpilVWXY1Y2E prepilin cluster is under the control of algR (Lizewski et al., 2004). The pilY1 gene from P. aeruginosa was discovered by Richard Alm and colleagues at the University of Queensland in 1996, it was identified at the same time as three other previously uncharacterized genes, pilW, pilX, and pilY2, all of which were initially implicated in pilus biogenesis. PilY1 is a large (127 kDa) protein with ~1163 amino acids (value varies between different strains).

However, mutation of the pilA gene, encoding the major subunit of the type IV pilus, showed only weak suppression of the bifA swarming and biofilm defects, indicating that it is loss of PilY1 specifically, and not loss of pili, that fully suppresses the bifA mutant defects (Kuchma et al. 2010). PilY1 protein plays two distinct cellular roles: one role promoting pilus assembly and a second role repressing swarming motility. Also the minor pilins, PilW and PilX, impact both pilus assembly and swarming motility, and this subset of pilus assembly proteins functions to allow P. aeruginosa to coordinately regulate two distinct motility behaviors, swarming and twitching, when associated with a surface (Kuchma et al., 2012). Finally Surface induction of PilY1 not only facilitates pilus biogenesis but also stimulates synthesis of the intracellular signaling molecule, c-diGMP, via an interaction with the SadC diguanylate cyclase (figure 1-3). These data are consistent with the minor pilins, PilX and PilW, also participating in the modulation of c-di-GMP levels, either in conjunction with PilY1 or possibly via a separate pathway. Increased synthesis of c-diGMP then leads to repression of swarming motility, most likely by influencing flagellar function as previously proposed (Caiazza et al., 2007). Elevated c-di-GMP levels also stimulate biofilm formation (Caiazza et al., 2007; Kuchma et al., 2007; Merritt et al., 2007), allowing P. aeruginosa cells to coordinately regulate all three of these distinct surface behaviors (Kuchma et al., 2012).

Figure 1-3: Model for coordinate regulation of surface-based swarming and twitching motility, surface growth induces expression of pilus assembly proteins, including PilY1, PilX, and PilA, likely to prepare cells for twitching motility (Kuchma et al., 2012).

chapter Two mATERiALs ANd mETHOds

2. Materials and Methods 2.1. Materials 2.1.1. Equipment Apparatuses and equipment used in this study were listed in table 2-1. Table 2-1: Apparatuses and equipment used in this study Id 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Equipment 96 well flat bottom plate (poly styrene) 96 well U bottom plate (Vinyl) ABI 7500 Fast System Autoclave Centrifuge Deep-freezer Digital camera Electrical balance Electroporation unit Eppendorf tubes Gel electrophoresis unit Hot plate with magnetic stirrer Incubator Laminar air flow hood Membrane filters (0.22µm) Microcentrifuge Micropipette Microwave Microwave oven Nanodrop Oven PCR Unit pH meter Power supply Refrigerator Screw capped test tubes

Manufacturing Company/ Origin Coastar/ USA Coastar/ USA Fischer / USA Hirayama /Japan Hettich /Germany Sanyo/Japan Canon /Japan Fischer /USA Fischer /USA Fischer / USA Fischer / USA IKA /Germany Memmert /Germany Fischer / USA Schleicher and Schuel / USA Hettich /Germany Brand /Germany Fischer / USA Siga / USA Fischer / USA SherWood /USA IKA /Germany Fischer / USA Fischer / USA Fischer / USA BBL /USA

27 28

Spectrophotometer Stericup filter unit

Shimadzu /Japan Millipore/ USA

Table 2-1 continued

Id

Equipment

29

Ultraviolet light

30 31 32

Vortex Water bath Water distillatory

Manufacturing Company/ Origin Ultraviolet products institute /USA Labcoo /Germany Memmert /Germany Fischer / USA

2.1.2. Chemicals and biological materials Chemicals and biological material used in this study are listed in table 2.2. Table 2-2: Chemical and biological materials Id 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Chemical and biological materials 1 KB plus DNA Ladder 5X HF Buffer Agarose gel Ammonium sulfate (NH4)2SO4 Ampicillin (Ap) Arabinose Barium chloride (BaCl2. 2H2O ) Boric acid Bromothymol blue dye Carbincillin (CA) Cassamino acid Crystal violet Dipotassium phosphate (K2HPO4) Disodium phosphate (Na2HPO4.7H2O) DMSO EDTA Ethanol Absolute

Company (origin) Invitrogen/USA Ivitrogene / USA Promega /USA Difco /USA Siga/ Aldrich /USA Himedia /India Difco/USA Difco /USA BDH/UK Siga-Aldrich /USA Difco /USA Merck /Germany Siga- Aldrich /USA Difco /USA Difco /USA Difco /USA Merck /Germany

18 19 20 21

Gentamicin (Gm) Glacial acetic acid Glucose Glycerol

Siga- Aldrich /USA Difco / USA Difco/ USA Siga- Aldrich /USA

Table 2-2 continued

Id 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Chemical and biological materials Imipenem powder 25mg Ladder 1 Kb Lithium acetate Magnesium sulfat (MgSO4) Monopotassium phosphate (KH2PO4) Nalidixic acid (NA) Nucleotide mix dNTP Pepton Phusion enzyme Polyethylene glycol Sodium chloride (NaCl) Sodium hydroxide (NaOH ) Sodium phosphate (NaHPO4) Sucrose Sulphuric acid (H2SO4) Syper safe stain Taq polymerase

Company (origin) Siga-Aldrich / USA Promega / USA

39

Taq polymerase 5U/µl Tri-HCl Tris base Tryptone Yeast extract Yeast Nitrogen Base

Promega / USA

40 41 42 43 44

Difco / USA Merck / Germany Difco / USA Siga-Aldrich /USA Roch-Germany /USA Difco / USA Invitrogene /USA Siga /USA Difco / USA Difco /USA Difco / USA Fluka-Swizerland/ Germany Difco /USA Invetrogen / USA BioLabs / USA Difco / USA Difco / USA Difco / USA Biomerieux /France Difco / USA

2.1.3 Culture media All culture media used for the isolation and identification of bacteria through this study are listed in table 2- 3. Table 2-3: Ready to use culture media

Id 1 2 3 4

Medium Cetrimide Agar Base MacConkey Agar Muller Hinton Agar Muller Hinton Broth

Company(Origion) Difco (USA)

2.1.4. Kits All kits used in this study are listed in table 2-4. Table 2-4: Kits used in the present work ID

The kit E-test strip kit of Imipenem (from 0.002 to 32 μg/ml) Gentra Puregen Yeast/Bactacteria Kit

Company (Origin) BioMérieux (France)

Invitrogen (USA)

4

Phusion enzyme kit, High fidelity DNA polymeraseare. QIAquick PCR purification kit.

5

Qiagen Kit for yeast plasmid DNA prep

Qiagen (USA)

6

Qiagen Kit for plasmid DNA prep from Bacteria

Qiagen (USA)

7

RNeasy Mini kit for purification total RNA from bacteria

Qiagen (USA)

۱ ۲ ۳

Qiagene (USA)

Qiagen (USA)

2.1.5. Standard strains quality control bacteria All standard strain and quality control bacteria used throughout this study are listed in table 2-5:

Table 2-5: The standard strains and quality control bacteria used in the present study . Relevant Genotype, description, or sequence MATa/MATα leu2/leu2 trp1Saccharomyces 289/trp1-289 ura3-52/ura3-52 his3cerevisiae (InvSc1) Δ1/his3-Δ1 thi pro hsdR-hsdM+ ΔrecA RP4Escherichia coli S172::TcMu-Km::Tn7 1(λpir) Name

lˉ f80dlacZDM15D (lacZYA-argF) U169 recA1 endA hsdR17(rKˉ mKˉ ) supE44 thi-1 gyrA relA1

DH5α

Source Invitrogen/USA Simon et al., 1983 Life Technologies/ USA

Table 2-5 continued

Name Pseudomonas aeruginosa PAO1 oprD::isphoA/hah Escherichia coli Pseudomonas aeruginosa

Relevant Genotype, description, or sequence Wild type PAO1 with isphoA/hah insertion in oprD;Tcʳ ATCC 25922/ Sensitive for all antibiotic ATCC 27853/ Sensitive for all antibiotic

Source Jacobs et al.,2003 Jacobs et al.,2003 ATCC/USA ATCC/USA

2.1.6 Primers 2.1.6.1. Primers used in oprD genetic complementation and sequencing All primers used in oprD complementation and sequencing were designed according to http://www.idtdna.com website listed in table 2-6.

Table 2-6: oprD gene primer used in Genetic complementation and sequencing Primers Name oprD comp 5′ oprD comp 3′

Primer sequence (5′ – 3′)

Origin

ttctccatacccgtttttttggggaaggagatatacatATGAAAGTG ATGAAGTGGAG taatctgtatcaggctgaaaatcttctctcatccgccTCACAGGATC GACAGCGGATAG

oprD seq a-f

ATGAAAGTGATGAAGTGGAGC

oprD seq a-r

AGGGAGGCGCTGAGGTT

oprD seq b-f

AACCTCAGCGCCTCCCT

P730

GCAACTCTCTACTGTTTCTCC

Integrated DNA Technology (IDT)/USA

Note: In primer sequences, lowercase letters indicate sequence homology to the cloning vector, uppercase letters indicate a Pseudomonas gene-specific sequence, boldface and lower case letters indicates a His tag sequence.

2.1.6.2. Quantification real time PCR primers All primer used in oprD complementation and sequencing were designed according to http://www.idtdna.com website are listed in table 2-9. Table 2-9: oprD primers used in q RT-PCR Primer name

Primer sequence (5′ – 3′)

oprD-RT Forward

CCGCAGGTAGCACTCAGTTCG

Origin

IDT/USA oprD-RT Reverse

GTAGTTGCGGAGCAGCAGGTC

2.1.6.3. Primer used in Arbitrary PCR All primers used in Arbitrary PCR are listed in table 2-7. Table 2-7: Primer used in Arbitrary PCR Primer name

Primer sequence (5′ – 3′)

P235

GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT

P236

GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC

P237

GGCCACGCGTCGACTAGTACNNNNNNNNNNAGAG

P238 P239 P240 P241

TATAATGTGTGGAATTGTGAGCGG GGCCACGCGTCGACTAGTAC ACAGGAAACAGGACTCTAGAGG CACCCAGCTTTCTTGTACAC

Origin

IDT/USA

2.1.6.4. Primers used in pilY1 genetic complementation and sequencing All primer used in pilY1Genetic complementation and sequencing were designed according to http://www.idtdna.com web site are listed in table 28. Table 2-8: pilY1 primers used in Genetic complementation and sequencing Primer sequence (5′ – 3′) tctccatacccgtttttttgggctagcgaattcgaaggagatataca tATGAAATCGGTACTCCACCAG pilY1His comp 3′ tcttctctcatccgccaaaacagccaagcttgcatgcctTCAgt ggtgatggtggtggtgGTTCTTTCCGATGGGGC pilY1 seq Rev 2 TGAACGGACAGGTACAGATCC

Origin

pilY1 seq 3

GGATCTGTACCTGTCCGTTC

IDT/USA

pilY1 seq 4

GGCGAGTTTCTCAAGAAGACC

pilY1 seq 5

CTTCCAGGACATCCTCAACCG

Primer name pilY1 comp 5′

pilY1 seq 6

AGCCCAGCGGTAACTACTCC

pilY1 seq 7 P730

CAAGGTCAACCAGGACGATC GCAACTCTCTACTGTTTCTCC

Note: In primer sequences, lowercase letters indicate sequence homology to the cloning vector, uppercase letters indicate a Pseudomonas gene-specific sequence, boldface and lower case letters indicates a His tag sequence.

2.1.7. Plasmids and vectors used in this study All the plasmids and vector were used in this study were summarized in table 2-10. Table 2-10: Plasmids and vectors Plasmids

description, or sequence

Origin

pMQ72

poprD

Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; GmR Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; ApR Vector carrying mariner transposon; ApR GmR * (marker on transposon) oprD gene cloned in pMQ72; GmR

Shanks et al., 2006 Shanks et al., 2006 Kulasekara et al., 2005 This study

pPilY1His

His-tagged pilY1 gene cloned in pMQ70; ApR

This study

PMQ70 pBT20

*GmR: Gentamicin resistance, ApR: Ampicillin resistance 2.1.8. Buffers and solutions All buffers and solutions were prepared according to British Pharmacopeia (2012) unless it is mentioned elsewhere. 2.1.8.1. Magnesium sulfate MgSO4 1 M MgSO4 (24.65) g was dissolve in ~75 ml distilled water, then brought up to 100 ml volume and then sterilized with autoclave (15 minutes, 121°C).

2.1.8.2. Glucose 20% Glucose (200) g was added to one litter of water and then autoclaved for 5 min at 121°C. 2.1.8.3. Lazy bones solution Polyethylene glycol (40%) and Lithium acetate (0.1 M), 10mM TrisHCL (pH 7.5) and 1mM EDTA were mixed 2.1.8.4. Sodium borate (SB) buffer 20X This buffer was prepared as follows: 8 g NaOH and 40 g boric acid were added to 1 liter of distilled water. For routine, SB buffer was diluted to 1X concentration. 2.1. 8.5. Ethylendiaminetetraacetic Acid (EDTA), 0.5 and 0.05 M This buffer was prepared as follows: 18.61 g EDTA in 100 ml distilled water to achieve 0.5 M concentration followed by pH adjustment to 8.0 and sterilization was done by filtration. This solution was used for the preparation of TE buffer. The 0.5 M EDTA solution was diluted by adding 10 ml of this solution to 90 ml distilled water in order to attain the 0.05 M EDTA solution. 2.1. 8.6. Tris buffer (1M) pH 8 Tris base (122.2) g was added to one liter of distilled water. 2.1. 8.7. Tri- EDTA (TE) buffer Tris stock (1 M ) 10 ml was add to 2 ml of 0.5 M of EDTA, final concentration should be 10mM Tris pH8, 1mM EDTA pH8. 2.1. 8.8. loadeing buffer 6X dye

Mixture of 30% sucrose and 0.25% bromophenol blue was made. The mixture was completed to 100 ml and autoclaved and then kept at 4°C until used. 2.1. 8.9. Ethanol (70%) Absolute ethanol (70 ml) was completed to 100 ml with distilled water. 2.1. 8.10. Glycial acetic acid 30% Absolute glycial acetic acid (70 ml) was completed to 100 ml with distilled water. 2.1.9.11. Casamino acids 20% (CAA) 200 g of CAA was added to one litter of water and then autoclaved for 15 min at 121°C. 2.2. Methods 2.2.1. Sterilization Sterilization was achieved by heating; wet heat sterilization by autoclave at 121˚C/15 psi for 15 min. One more method used is filtration using 0.22 μm filter unit. All equipment and materials used in this study were sterilized through these two methods unless mentioned elsewhere. 2.2.2. Laboratory prepared culture media 2.2.2.1. Yeast peptone dextrose (YPD) broth media The broth was prepared as follows: 10 g yeast extract, 20 g peptone and 20 g dextrose were dissolve in 1L of distilled water, pH was adjusted to 7.5. 2.2.2. 2. Yeast peptone dextrose (YPD) agar plate

The agar plate was prepared as follows: 10 g yeast extract, 20 g peptone, 20 g dextrose and 15 g agar were dissolved in one liter of distilled water, pH was adjusted to 7.5. 2.2.2. 3. Lysogeny broth (LB) broth The broth was prepared according to Sambrook and Russell (2001) and was done as follows: 10 g tryptone, 5 g Yeast extract, 5 g NaCl, these chemicals were dissolved in 1L of distilled water, the pH was adjusted to 7, autoclaved, then kept at 4°C until used. 2.2.2. 4. Lysogeny broth (LB) agar plates The agar plate was prepared according to Sambrook and Russell (2001). In brief, 10 g tryptone, 5 g Yeast extract, 5 g NaCl, and 15 g Agar these chemicals were dissolved in 1L of distilled water, pH was adjusted to 7, autoclaved, then poured in petri plates. 2.2.2. 5. Minimal salts medium 5X M63 The minimal media was prepared according to Sambrook and Russell (2001). Briefly, 60g KH2PO4, 140g K2HPO4 and 40g (NH4) 2SO4, the reagent were mixed well in 4L of distilled water and autoclaved, pH was adjusted to 7. 2.2.2. 6 Minimal salts medium 5X M8 The minimal media was prepared according to the following method: 64 g of Na2HPO4.7H2O (or 30 g NaHPO4), 15 g KH2PO4 and 2.5 g of NaCl, were dissolved in four liters of distilled water and autoclaved, pH was adjusted to 7. 2.2.2. 7 Minus uracil medium

Synthetic medium was prepared by dissolving Yeast Nitrogen Base 6.7 g, 0.76 g supplement mixture minus uracil (CSM-URA), 15g Dextrose, 20 g Agar and 0.77 g of DoB or DoBA were dissolved in one liter of distilled water. Afterward, the mixture was autoclave at 121 °C for 15 min. 2.2.2. 8. Glycerol for -80ºC LB powder no agar ( 20) g was added to 800 ml distilled water and 200 ml glycerol (100%) and mix well. 2.2.2. 9. Stabs nutrient broth (2.4) g, 3 g agar and 15 g thymine were mixed and melted in a microwave. Later, 2 ml was added to vial by syringe and needle, caps were left loosely and autoclaved. Thereafter, caps were tightened when it became cool. 2.2.3. McFarland standard (no. 0.5) preparation The 0.5 McFarland standard was prepared in accordance with the British Society for Antimicrobial Chemotherapy (Vandepitte et al. 2003), by adding 0.۰٥ ml of 0.048 M barium chloride (1.17% w/v BaCl2. 2H2O) to 99.۹5 ml of 0.18 M sulphuric acid (1% w/v H2SO4) with constant stirring. The suspension was distributed to five glass tubes of the same size and volume as those used in growing the broth cultures, the absorbance was measured in a spectrophotometer at a wavelength of 625 nm and the acceptable absorbance range for the standard is 0.08-0.13. Afterward, each tube was thoroughly mixed on a vortex mixer to ensure that it is even. These turbidity standard tubes were sealed tightly to prevent loss by evaporation. Stored protected from light at room temperature. Before use, the tubes were

vigorously agitated by hand. It was used for the turbidity standardization for antibiotic susceptibility test. 2.2.4. Single Stranded Carrier DNA Single stranded carrier DNA prepared in accordance with (Burke et al., 2000), high-molecular weight DNA (Deoxyribonucleic acid sodium salt type III from Salmon testes; sigma), TE buffer (pH 8.0) (10 mM Tris-Hcl pH 8.0, 1mM EDTA). ·

DNA (200) mg was weighed and placed into 100 µl TE buffer. This mixture was mixed vigorously on a magnetic stirrer for 2-3 hours or until fully dissolve.

·

the DNA was aliqouted and stored at -20ºC

·

Prior to use, the DNA was boiled an aliquot in water bath for 10 minutes and quickly placed in ice.

2.2.5. Preservation of bacterial strains Two methods were used for the preservation of bacterial strains: A) Glycerol method: A loopful of overnight growth pure culture was added to LB broth and incubated at 37 ºC. After 18 hr., 0.5 ml of culture was added to 0.5 ml Glycerol (Vandepitte et al., 2003). B) The strains of bacteria were subcultured in stabs, kept in cool incubator 4-8 °C and resubcultured every 3 months in a new slant. 2.2.6. DNA agarose gel electrophoresis Standard method of the (Sambrook and Russell, 2001) was followed to prepare horizontal agarose gel electrophoresis for genomic DNA, plasmid and PCR product:

·

Agarose at concentrations of 1% was prepared for PCR product for plasmid and chromosomal DNA electrophoresis. Agarose was dissolve in 100 ml of 1X SB buffer and solubilized by heating with stirring .The agarose was left to cool at 60°C before adding Syper safe stain and poured into the taped plate.

·

A comb was placed near one edge of the gel.

·

Syper safe was added with the pouring of the agarose

·

The gel was left to harden until it became opaque; gently the comb and tape were removed.

·

SB (1X) buffer was poured into gel tank and the slab was placed horizontally in electrophoresis tank.

·

About 2µl of loading buffer was applied to each 5 ml of plasmid, the well was fill with the mixture. The power supply was set, 1kp molecular ladder served as marker. Five

microliters of the DNA ladder were mixed with one microliter of blue 6X loading dye and subjected to electrophoresis in single lane. After electrophoresis, the gel was exposed to UV using UV transilliuminator and then photographed. 2.2.7. Imipenem stock preparation The potency of Imipenem contains is 1000 µg per mg (Wiegand et al., 2007). The stock solutions were prepared using the formula: 1000 ×V×C W= P Where P = potency given by the manufacturer (µg/mg), V =volume required (ml), C = final concentration of solution (multiples of 1000)

(mg/L), and W = weight of antibiotic (mg) to be dissolved in volume V (mL). Further stock solutions were prepared from the initial 10 000 mg/L solution: 1 ml of 10 000 mg/L solution was added to 9 ml diluent = 1000 mg/L, 100 µl of 10 000 mg/L solution was added to 9.9 ml diluent = 100 mg/L. 2.2.8. Antibiotic stocks used in this study Antibiotic stocks used in this study represent 1000X. Gentamicin (GM), Nalidixic acid (NA), Ampicillin (Ap) and Carbincillin (CA) were prepare in concentration of 10, 20, 150, 50 mg/ml respectively, distilled water represent diluent. 2.2.9. Biofilm Media M63 (minimal salts medium, 200 ml) 5X was supplemented with 1 M MgSO4 (1 ml ), 20% glucose ( 10 ml) and 20% CAA ( 25 ml), all the reagents were mixed and autoclaved at 121 °C for 15 min and then stored at room temperature 25±2 ºC. 2.2.10. Swarming motility media M8 (minimal salts medium 200 ml) 5X was supplemented with 1 M MgSO4 (1 ml), 20% glucose (10 ml) and 20% CAA (25 ml), afterward, they were mixed and solidified with agar (0.5% final concentration), finally they were autoclaved at 121 °C for 15 min and stored at room temperature 25±2 ºC. 2.2.11. Twitching motility media 5X M63 (200 ml) was supplemented with 1 M MgSO4 (1 ml), 20% glucose (10 ml) and 20% CAA (25 ml), thereafter they were mixed and

solidified with agar (1.5% final concentration) , finally they were autoclaved at 121 °C for 15 min and stored at room temperature 25±2 ºC. 2.2.12. Standard and clinical strains culture Clinical strains and E. coli DH5α and S17-1 λpir strains carrying the plasmid pBt20 were routinely cultured on lysogeny broth (LB) medium, which was solidified with 1.5% agar when necessary. Gentamicin (Gm) was used at from 25 to 50 µg/ml for P. aeruginosa and at 10 µg/ml for E. coli. Carbincillin (CA) was used at 50 µg/ml and 10 µg/ml for E. coli. Nalidixic acid (NA) at 20 µg/ml for E. coli. Saccharomyces cerevisiae strain InvSc1, used for plasmid construction via in vivo homologous recombination, was grown with yeast extractpeptone-dextrose (1% Bacto yeast extract, 2% Bacto peptone, and 2% dextrose) (Shanks etal., 2006). Selections with InvSc1 were performed using synthetic defined agar-uracil. 2.2.13. Determination of MIC of Imipenem for strains 2.2.13. 1. The E-test method All strains of P. aeruginosa were subjected to determine minimum inhibitory concentrations (MICs) against Imipenem. The E-test method has been used for MIC determination according to the manufacturer’s instructions. In brief, bacterial suspensions were prepared from fresh colonies and the concentration has been adjusted to 1.5 to 2 ×108 cfu/ml McFarland turbidity. Each strain was inoculated by streaking the bacteria all over a Mueller Hinton agar (MHA) plate. An E-test strip of Imipenem (from 0.002 to 32 μg/ml) was placed on the surface of cultured media, after overnight incubation at 37°C, MIC has been determined and the sensitive, intermediate and resistant phenotypes were tested according to Clinical and

Laboratory Standards Institute (CLSI, 2013). MIC was also determined by microdilution on microtiter plates to check the similarity of results. Escherichia coli ATCC 25922 was used as quality control strains for E test.

2.2.13. 2. Microdilution method The minimum inhibitory concentration (MIC) of P. aeruginosa strains was determined, using the twofold serial microdilution method (Wiegand et al., 2007) and using Muller Hinton broth (MHB). A serial dilutions ranging from 0.5-32 μg/ml for Imipenem were prepared in microtiter plate wells. Bacterial culture of 1.5 to 2 ×108 cell/ml was prepared using MHB and 1.5 to 2 ×108 cfu/ml McFarland’s standard tube, 10 μl of bacterial culture was added to each microtiter plate well. The MIC values were taken as the lowest concentration of the antibiotic in the well that showed no turbidity after 24 hours of incubation at 37°C. The turbidity of the wells in the microtiter plate was interpreted as visible growth of the microorganisms, P. aeruginosa ATCC 27853 was used as quality control strains for microdilution assay. Microdilution assay was used to confirm the results of E test in regard to the resistance strains. 2.2.14. Modified Hodge Test (MHT) Modified Hodge test is a phenotypic method for detection of carbapenemases (Anderson et al., 2007; Noyal et al., 2009). 0.5 McFarland dilution of the E. coli ATCC 25922 was prepared in 5 ml of saline. 1:10 dilution was prepared by adding 0.5 ml of overnight culture to 4.5 ml of saline. Thereafter, a lawn of the 1:10 dilution of E. coli ATCC 25922 was streaked to a Mueller Hinton agar. Afterward, 10 µg

Imipenem susceptibility disc was placed in the center of the test area. In a straight line, the test organism was streaked from the edge of the disc to the edge of the plate. Up to four organisms can be tested on the same plate with one drug, and then the plate was incubated overnight at 37ºC in ambient air for 16–24 hours. After incubation period, the plate was examined for a clover leaf-type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disk. MHT Positive test has a clover leaf-like indentation of the E. coli 25922 growing along the test organism growth streak within the disk diffusion zone. MHT Negative test has no growth of the E. coli 25922 along the test organism growth streak within the disc diffusion. 2.2.15. Biofilm formation assay Biofilm formation in 96-well microtiter plates was assayed and quantified as previously described by Caiazza and O’Toole (2004). All biofilm assays were performed using M63 minimal medium supplemented with glucose, MgSO4, and CAA. Strains were grown overnight (~16 hours) in LB broth at 37°C, the 96well plate(s) were prepared for the assay (label strains). Each strain suspension was diluted (1:50) into an aliquot of the Biofilm media and mixed well by swirling and pipetting up and down.

The wells were

inoculated (at least 4 wells per strain) of the 96-well plate (100 µl/well) from the strain mixture using a multi-channel pipet. The 96-well plate was covered with a lid and placed in the warm room (37°C) for up to 24 hours. After the incubation period, the wells were shaken out to remove the unattached bacteria and then were rinsed twice in the water container and shaken out the excess water by tapping plate on paper towels. Subsequently,

125 µl of Crystal violate (CV) stain (at 0.1% concentration) was added to each well and the control un-inoculated well, and then the plate was let sit to 10-15 min. The excess stain was shaken out into the waste container and the plate was rinsed twice. The plate was taped on paper towels to dry. In order to quantify the biofilms, 125 µl of 30% glacial acetic acid was added to biofilm wells and to the control well (no bacterial cells, just stained with crystal violet). This step was done with multichannel pipette. Plates were allowed to sit at room temp for at least 15 minutes. Later, the solubilized crystal violet was pipetted up and down gently to evenly mix just prior to transferring 100 µl from each well to a 96-well flat-bottomed plate (non-sterile). Finally, the plate was read by a spectrophotometer at an absorbance of 550 nm. 2.2.16. Extraction of genomic DNA Extraction of Genomic DNA was accomplished using the Gentra Puregen Yeast/Bacteria Kit. Phusion enzyme was used to amplify oprD gene to reduce the chance that errors will be introduced during the PCR reaction (since we are specifically looking for mutations that have arisen by growth in the presence of imipenem). The purity was checked by Nanodrop at OD 260/280.

2.2.17. Amplification of oprD gene by polymerase chain reaction technique (PCR) The PCR technique was employed for amplifying whole oprD gene in P. aeruginosa strains. Mastermix reaction and reaction conditions are summarized in table 2-11 and table 2-12, respectively.

Table 2-11: Mastermix reaction for single reaction Reagents

Volume (µl)

5X HF Buffer*

10

dNTP mix (10 mM)

1

DMSO

1.5

For Primer (25 µM)

1

Rev Primer (25 µM)

1

Template Genomic DNA

1

Phusion enzyme (U/ 50 µl)

0.5

Distilled water

34

*High Fidelity (HF) buffer was used that comes with Phusion enzyme kit, the primers were listed in table 2-6

Table 2-12: The reaction conditions of PCR Stage Initial denaturation Denaturation Annealing Extension Final extension Hold

Temperature (time) 98°C (1min) 98°C (10sec) 69°C (30sec) 30 cycles 72°C (40sec) 72°C (10min) 4°C

Annealing temperature was determined by adding 3°C to lowest Tm of primer pair, extension times was determined, (1 kb ) was added in each 30 second (when using complex templates like genomic DNA), the primers were used in oprD amplification are listed in table 2-6.

2.2.18. Sequencing After PCR, gel was run if the band of expected size was seen, then Qiagen PCR clean-up kit was used to clean up the PCR product for the reactions of sequencing. The mix of reaction summarized in table 2-13. Table 2-13: The sequence reaction mix Reagent PCR product Primer (100 µM) Distelled water total volume

Volume(µl) 1 µl 1 µl 18 µl 20 µl

The primers were listed in table 2-6

After reaction, samples were sent to the Core Facility in Dartmouth College. Sequence analysis was performed according to www.ncbi.org using, Gene construction kit software and finch TV. The results DNA sequences were aligned to the PAO1 oprD genomic sequence using the NCBI BLAST. 2.2.19. Quantitative reverse transcription-PCR (qRT-PCR) (Kuchma et al., 2005) The qRT-PCR was used for measuring the oprD expression in clinical strains 2.2.19.1. Bacterial Harvest Bacterial suspension were diluted from LB-grown overnight cultures 1:100 into M63 minimal medium (2.2.2.5) and grown for 8 hours to an

optical density of 0.4 at 600 nm (OD600) (Normalized to a volume of 10 ml). Samples were span down in a centrifuge (37°C at 5000 rpm for 2 minutes). Thereafter, supernatant was discarded and the tubes were placed in a dry ice ethanol bath for 10 minutes. The tubes were stored in -80°C freezer, until use. 2.2.19.2. RNA extraction and cDNA RNA preparation by Qiagen Rneasy Kit, RNA was run in 1% gel to assess purity. RNA was quantified by Nanodrop at OD

260/280.

2 to 3 µg of

RNA was taken to make cDNA using the Invitrogen Superscript III cDNA Kit. Primers were designed according to http://www.idtdna.com. The 96 well plates were set with appropriate number of strains three replica for each one. 2 µl of 1000 pmol/µl cDNA (read by Nanodrop) was added for each well and 82.5 µl of Syper green mix to that tube, negative control was prepared as well. When loading is completed the pressure sensitive sealing film was added on the plate. The real time plate was sent to core facility Quantitative reverse transcription-PCR (qRT-PCR) was performed using an ABI 7500 Fast System and analyzed using ABI Fast System software version 1.4. Expression levels were quantified in picograms of input cDNA using a standard curve method for absolute quantification, and these values were normalized to rplU expression. Results shown are based on the average from two independent experiments with three replicates per sample. The primers used were listed in table 2-9. 2.2.20. Genetic complementation steps of oprD mutant strain 2.2.20. 1. Digestion of PMQ72

PMQ72 was purified from E. coli and 20 µl PMQ72 was digested with enzyme Sac1 for overnight at 37ºC. Plasmids for complementation and overexpression were generated using vectors via homologous recombination in yeast. PMQ72 vector was used for the complementation of oprD mutant (shanks et al., 2006). The poprD complementation constructs was generated by PCR amplification using the high-fidelity DNA polymerase. 2.2.20. 2. Yeast transformation (Burke et al., 2000) S. cerevisiae (InvSc1) was grown in YPD overnight. After that, couple of large colonies were picked up with a sterile toothpick and transferred to 5 ml LB broth and incubated for overnight. Then 0.5 ml from overnight culture was applied in 1.5 ml microfuge tube and spun down pellet at 10000 rpm for 10 second. To the pellet, 0.5 ml of lazy bones solution, 20 µl of carrier DNA (salmon sperm DNA), 20 µl PCR product and of 5 µl vector were mixed and then vortexed hard for 1 minute.

The mixture was

incubated overnight to 4 days at room temperature (after 1- 4 days the plasmid efficiency goes down in the yeast). The mixture was exposed to heat shock for 10-12 minutes at 42ºC. Cell was pelleted, optimally was washed with T.E., as PEG inhibits growth, and cultured onto selective media. At the end of the incubation period (5-7 days), the growth was harvested by spreader from plate and the suspension was taken for plasmid preparation. Qiagene kit was used for DNA plasmid prep from yeast. Then amount of plasmid was transferred into E. coli to amplify the plasmid. 2.2.20. 3. Electroporation (Oldenburg et al., 1997) A. Preparation of E.coli DH5α

A liter of LB was inoculated with 1/100 of fresh overnight E.coli DH5α, cells grown with vigorous shaking to OD

600

of 0.5 at 1 hour. The

culture was chilled on ice for 15 to 30 min, and then centrifuged in cold centrifuge at 4000X g for 15 min. Successively, the pellets was resuspended in a total of 1litter cold water and centrifuged as before, and then the pellet was resuspended in 0.5 L cold water and centrifuged. The pellet was resuspended in 10% glycerol and centrifuged. The pellet was resuspended in a final volume of 2 to 3 ml in 10% glycerol. The cells were aliquoted and frozen on dry ice. Finally the cell was storaged at -80ºC; the cells are good for at least six months. B. Electroporation of E. coli The cells were thawed at room temperature and placed on ice, 40 µl of cells was mixed with 1 µl to 2 µl of DNA in low ionic strength buffer and put in pre-chilled cuvette. The cells were let to sit on ice for a minute. Thereafter, the gene pulser apparatus was set at 25 µF and 2.5 KV, the cuvette was put in the slide and the slid was push into the chamber, the pulsing was once pulsed (time constant should be 4.5 to 5 sec), 500 µl was added after pulsing immediately of LB and the cells were resuspended. Subsequently, the cells was transferred to appendroff tube and incubated at 37ºC for 1 hr, the cells were plated on selective media by spreader. QIAprep Spin Miniprep kit was used for plasmid preparation from E. coli and run on agarose gel to confirm the presence of the expected band. Then the sequence analysis was achieved to ensure there is no mutation in interesting gene that may happen during PCR cycle. C. Electroporation of P. aeruginosa

From overnight culture, 1 ml of cell were spun to pellet (maximum speed, 1min), the supernatant was remove and resuspended in 1 ml of 300 mM sucrose, the cell was spun to pellet, the supernatant was removed and this wash was repeated in sucrose two more times. After the finally spin, the supernatant was remove carefully and the cells was resuspended in 80 µl of 300mM sucrose, the( DNA no more than 1.5) was pipetted onto the side of electroporation cuvette to allow cell mixture to go in to the well of the cuvette (no bubbles), electroporate using the E. coli setting (the putton was push for the time constant setting so you can see the puls time), immediately, 0.5 ml of LB was added to recover the cells and transfered to fresh microfuge tube, the cells was recovered at the 37ºC on the wheel for 1.5 – 2 hours, 10 µl of the mixture plus 50 µl was plated on the appropriate selective media. After that, the phenotypic restoration was screened. 2.2.21. mariner transposon mutagenesis of the highest biofilm producers; 576 and 214 (Kulasekara et al., 2005) 2.2.21.1. Conjugation and selection of mutants The donor strain E. coli S-17 l pir (strain carrying the plasmid pBT20 – carries Mariner transposon) and the recipient P. aeruginosa were grown for overnight with appropriate antibiotic selection (antibiotic markers were ampicillin marker on plasmid backbone – ampicillin was used for growth of E. coli; gentamicin marker on Mariner was used for P. aeruginosa). The transposon carries an outward-directed Ptac promoter so Tn insertions can lead to overexpression of downstream genes. 1 ml of each fresh strain overnight culture was pelleted. Enough samples were included to set up single plating of each strain as negative controls for the conjugations, i.e.

another 1 ml of each P. aeruginosa and E. coli (that was using for the conjugation) strain was spun. Each strain was washed twice by resuspending in 1 ml of fresh LB and pelleted. Then cells were resuspended in a final volume of 100 µl, the suspensions of each strain were mixed in a tube and 60 µl of this mixture was spotted onto 2 sterile LB plates (without antibiotics), additional plates were plated for conjugation were spots to scale this up if necessary. The conjugation was allowed to proceed for at least 1 hour at 30°C (up to 3 hours). Afterward, the cells were recovered from plate by scraping up the pellet with a sterile stick and resuspended in a small volume of LB in a 1.5 ml microfuge tube. The cells were vortexed to break up clumps. The aliquots (10, 20 and 40 µl) of the conjugation mixture were plated to test for appropriate yield of colonies onto selective LB agar plates (Gentamicin to select for Mariner; include nalidixic acid to select against E. coli. The plating was spreaded evenly over entire plate with a sterile glass spreader; the conjugation plate was stored at 4°C until results of initial plating are evaluated. Gentamicin was used to select against P. aeruginosa 25 µg/ml (2.5X), Nalidixic Acid was used to select against the E. coli strain 20 µg/ml (1X). The plates were incubated at 37°C overnight, then additional time at room temperature.

2.2.21.2. Storing/screening the library The library plates (96-well dishes) were filled with sterile LB plus antibiotic (Gm), 100 µl per well was added for each well except control wells for (576 and 214) comparison control wells (e.g. A1, A2) were filled with LB alone. Colonies were picked into wells using sterile toothpicks, with

taking care to maintain a consistent inoculums, the control strains were pick into appropriate wells, the plates were allowed to grow at 37°C 12-18 hours. When substantial uniform growth has been achieved, plates can be screened right away or stored for later screening. 2.2.21.3. Screening for biofilm deficient Frog (multipronged device) was used to transfer samples from 96well sterile plate into wells of 96-well biofilm plate filled with appropriate media and incubate for desired time at 37ºC. 2.2.21.4. To store the library 100 µl of sterile 20% glycerol was added to the library (to give final of 10%) to all of the wells, and mixed carefully. Thereafter, the plates were frozen at -80°C with 2 layers of protection (e.g. parafilm or aluminum foil, Ziploc bag). 2.2.21.5. Mapping Mariner transposons by Arbitrary PCR (ARB PCR) Mapping of Mariner transposon was done according to CaetanoAnolles, (1993) and O’Toole and Kolter (1998), when mutants strains were isolated and confirmed, the arbitrary PCR was used to identify the insertion point of the transposon in gene. In arbitrary PCR there are 2 rounds. Before round1, 5 µl of the genomic template was extracted by the Gentra kit (2.1.4) and then was digested with 5 µl of EcoR1 enzyme for overnight at 37ºC. Then use 5µl of that as template for round 1, the mastermix reaction and ARB PCR program condition for round 1 are summarized in table 2-14 and table 2-15, respectively.

Table 2-14: The mastermix reaction for ARB PCR round 1 Reagent P235 (1µg/µl) P236 (1µg/µl) P237 (1µg/µl) P238 (1µg/µl) Taq Buf. 10X dNTP mix (10mM each) MgCl2 (50mM) DMSO H2O Taq DNA polymerase (U/ 50 µl) Digested gDNA Total Volume

Volume 1µl 1 µl 1 µl 1 µl 5 µl 2 µl 0.5 µl 2.5 µl 30 µl 1 µl 5 µl 50µl

Table 2-15: PCR1 program for round 1 – The annealing temperature was very low. Stage Temperature (time) Temperature (time) Initial denaturation 94°C (2min) 94°C (2min) Denaturation 94°C (30sec) 94°C (30sec) Annealing 42°C (30sec)* 5 cycles 65°C (30sec) 25 cycles Extension 72°C (3min) 72°C (3min) 4°C (∞) 1cycle Hold None *-1 °C for each cycle The PCR product was cleaned up using Qiagen kit and eluted with 50 µl distilled water. In regard to round 2, the mastermix reaction and ARB PCR program conditions are summarized in table 2-16 and table 2-17, respectively.

Table 2-16: The mastermix reaction for ARB PCR round 2 Reagent P239 (1µg/µl) P240 (1µg/µl) Taq Buf. 10X dNTP mix (10mM each)

Volume 1 µl 1 µl 5 µl 2 µl

MgCl2 (50mM)

1 µl

DMSO H2O TaqDNA polymerase (U/ 50 µl) PCR 1produt Total Vol.

2.5 µl 35.5 µl 1 µl 1 µl 50µl

Table 2-17: PCR1 program for round 2 –Annealing Temperature was increased Stage Initial denaturation Denaturation Annealing Extension Final extension Hold

Temperature (time) 94°C (2min) 94°C (30sec) 57°C (30sec) 30 cycles 72°C (3min) 72°C (5min) 4°C(∞)

An agarose gel was run to check the PCR. Several bands were seen, PCR product sample was purified using the Qiagen kit and eluted in 40µl distilled water. The primer P241 was used for sequencing. PCR products were sequenced at the Molecular Biology and Proteomics Core at Dartmouth

College. The resulting DNA sequences were aligned to genomic sequence using the NCBI BLAST program, the primers was used in ARB PCR are listed in Table (2-7) 2.2.22. Construction of mutant strains and plasmids PMQ70 was purified from E. coli and digest 20 µl with 4 µl of enzyme Sac1 for overnight in 37 ºC. Then use 5 µl of was used as template. PMQ70 vector was used for complementation of PilY1 mutant (shanks et al, 2006), pPilY1 complementation construct was generated by PCR amplification of pilY1 gene using the high-fidelity Phusion DNA polymerase. The reaction conditions for the amplification pilY1 gene are sumerized in table 2-18.

Table 2-18: The condition reaction for amplification pilY1 gene Stage Initial denaturation Denaturation Annealing Extension Final extension Hold

Temperature (time) 98°C (30min) 98°C (10sec) 72°C (20sec) 30 cycles 72°C (2min) 72°C (10min) 4°C(∞)

The primers that used for the pilY1 amplification are listed in table 218. 2.2.23. Twitching assays Twitch motility plates consisted of M63 medium supplemented with MgSO4, 20% glucose and 20% CAA and solidified with 1.5% agar. Twitch

assays were performed as previously reported (Whitchurch, 1990; O’Toole & Kolter 1998). Briefly, cells were stab inoculated with a toothpick through a thin (approximately 3 mm) LB agar layer to the bottom of the Petri dish. After overnight growth at 37ºC, the zone of twitching motility between the agar and Petri dish interface was visualized by staining with crystal violet. 2.2.24. Swarming motility Swarm motility plates were comprised of M8 medium supplemented with MgSO4, 20% glucose, and 20% CAA and solidified with 0.5% agar. Swarming assays were performed as previously reported (Köhler et al., 2000). Agar was allowed to cool slightly and then was poured thick plate approximately 25 ml/ plate; these plates were set at room temperature for few hours. For testing individual strains, 2 µl of grown overnight cultures was inoculated onto the surface of the swarm plates and incubated for 16 h at 37°C. Wild type was included as apositive control. 2.2.25. Estimation of polysaccharide extracts We quantified Psl production via ELISA with some modifications from an existing protocol (Honko et al., 2006). Flat-bottom 96-well MaxiSorp plates (Nunc) were coated in triplicate overnight at 4°C with 100 µl /well. Plate was washed thrice with PBS plus 0.1% Tween-20 for 3 minutes each and tapped to mix block plate with 300µL/well of PBS plus 1% Bovine serum albumin (BSA), 60 min at 4°C. Primary antibody diluted in PBS plus 0.1% BSA was added for 1-2 hr at room temp (or 4°C). Thereafter, Horseradish peroxidase (HRP)-conjugate secondary antibody diluted in PBS plus 0.1% BSA was added. 1:5,000 dilutions from reconstituted stock were made and finally the plate was

incubated for 1 hr at room temp or 4°C. Subsequently, plate was washed and 100 µl/well TMB SureBlue development reagent to half of the plate at a time was added as well. 100 µl /well 0.2 N H2SO4 was added to terminate development in the hood. Measurement was achieved at 450 nm. 3.3. Statistical analysis The data were analyzed by GraphPad Prism software was originally designed for experimental biologists in medical schools and drug companies, which was applied for the comparison among different values when the enumerative data are qualitative using ANOVA One way Analysis (http://www.graphpad.com). difference.

P 8 µg/l).

Figure 3-1: Modified Hodge test, the negative strain shows an undistorted zone of inhibition by p. aeruginosa.

3.2. Assessing biofilm formation and imipenem resistance in clinical strains To explore the relationship between biofilm formation and imipenem resistance, these phenotypes were assessed for an imipenem sensitive clinical strain SMC631, a number of imipenem resistant variants of SMC631, and several additional P. aeruginosa clinical strains. As shown in table 3-1, all these variants were tested for their ability to form a biofilm and compared to the parent SMC631.

Table 3-1: Biofilm and imipenem resistance phenotypes of clinical strains Strains

Imipenem MIC (µg/ml)a

Biofilm (A550 ± SDb)

SMC4973

0.5

0.96 ± 0.0509

SMC576

1

0.84 ± 0. 2305

SMC4972

1

0.77 ± 0.0257

SMC631

1.5

0.19 ± 0.0226

PAO1

4

0.88 ± 0.0134

SMC214

4

0.70 ± 0.1634

SMC4979

4

0.53 ± 0.0626

SMC631C

16

0.12 ± 0.0153

SMC631F

16

0.06 ± 0.0161

SMC631H

16

0.10 ± 0.0188

SMC631J

16

0.11 ± 0.0151

SMC5810

23

0.02 ± 0.0121

SMC631K

24

0.12 ± 0.0344

SMC5811

24

0.01 ± 0.0325

SMC4974

32

0.04 ± 0.0051

Sensitive (≤2 µg/ml), intermediate (4 =µg/ml) or resistant (≥8 µg/ml). bLSD= standard deviation. LSD= 0.13, P= 1.755 *10-18. a

Markedly, an inverse relationship (r= -0.83) was found between biofilm formation and imipenem resistance in P. aeruginosa. The imipenem resistant strains SMC4974, SMC5810, and SMC5811 showed significantly low (P < 0.01) biofilm formation compared to the imipenem sensitive or intermediate P. aeruginosa clinical strains SMC576, SMC214, SMC4972, SMC4973, and SMC4979, which all formed relatively robust biofilms (Table 3-1).

Noticeably, table 3-1 demonstrates nonuniformity in clinical strains biofilm results. Even though the imipenem sensitive SMC631 has low biofilm value (0.19 ± 0.0226) in comparison to its spouses of MIC category ≤ 4 µg/ml (SMC576, SMC214, SMC4972, SMC4973, and SMC4979), all its imipenem resistance variants (SMC631C, SMC631H, SMC631F and SMC631J) formed low biofilm by comparison to the parent SMC631. These data confirmed the possibility of an inverse relationship between biofilm formation and P. aeruginosa imipenem resistance. 3.3. Analysis of Sequencing of oprD gene Loss of oprD is one of the most important mechanisms of resistance to imipenem in P. aeruginosa. Multiple studies have evaluated the importance of oprD mutation in clinical strains of P. aeruginosa resistant to carbapenems. Always authors demonstrated a correlation between the levels of expression of oprD and the degrees of susceptibility to imipenem (Dib et al., 1995; Ocampo-Sosa et al., 2012; Lee et al., 2012). The relationship between oprD mutation type and imipenem susceptibility profiles in imipenem-susceptible ,intermediate, and resistance clinical strains of P. aeruginosa was investigated. The impact of oprD mutations in 19 clinical strains, 11 were metallo-ß-lactamase- negative P. aeruginosa and 8 sensitive for imipenem was analyzed (Table 3-2). Selection of the strains was based on their imipenem susceptibility profiles, including organisms with a broad range of susceptibility: susceptible (MICs ≤ 2 µg/ml), intermediately susceptible (MIC = 4 µg/ml) or resistance (MICs ≥ 8 µg/ml) to imipenem. The evaluation of the

mutations found in oprD of clinical strains was compared with the analysis of the oprD of PAO1. Sequencing of the entire oprD gene regions (Appendix A) permitted analysis of the mutation in oprD gene in such strains. Most of the strains presented point mutations consisting of single nucleotide insertion or nucleotide deletion in positions −167 and −177, respectively (relative to the ATG start codon). Such mutations were observed in both susceptible and resistant strains; therefore, it considered the involvement in decreased imipenem susceptibility not relevant. It is likely there is another passage for the imipenem or this mutation does not affect the OprD function as a porin. Wolter et al. (2009) suggested that carbapenems can enter through an alternative route. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility. Patterns of mutations found in oprD (Tables 3-2) were assembled into three groups: group I showed full length comprising PAO1, SMC 4963, SMC 4972, SMC 4986 and clinical strains (SMC 631, SMC 631 K, SMC 4974). Witch were included several oprD allelic variants, due to amino acid substitutions. Group II substitution of a nucleotide in the oprD gene resulted in a premature termination of translation whereas the last pattern group III comprises those oprD genes harboring a frameshift mutation due to nucleotide insertions or deletions. Group II and group III called deficient types, as their mutations resulted in loss of oprD porin (Ocampo-Sosa et al., 2012). Following the usual pattern, most of the oprD full-length type strains were susceptible strains. Several oprD allelic variants compared to PAO1 oprD were found (Table 3- 2). The most frequent amino acid substitutions were T103S, K115T, and F170L, found in the parent SMC631 susceptible

strains and the five resistance derivatives of 631 (SMC631C, F, H, J, K) and SMC4974 resistance strains, oprD variants showing these amino acid substitutions, were described before in clinical strains of P. aeruginosa (El Amin et al., 2005; Gutiérrez et al., 2007; Rodríguez-Martínez et al., 2009; Ocampo-Sosa et al., 2012). Other frequently found amino acid substitutions are shown in (Table 3-2); some of them were previously described in strains with different susceptibility profiles to imipenem (El Amin et al., 2005). The OprD “deficient types” included one strain, SMC631F, showing an earlier termination of translation due to premature stop codons. Most strains have frameshift mutations caused by nucleotide insertions or deletions in the oprD structural gene. The nt 152Gnt 153 mutation in SMC631 H, SMC 631J and SMC631C in variants strains from clinical strain SMC631. The remaining mutations (D

43

E,

nt 167Gnt 168, nt 177ΔC)

were found in

susceptible, intermediately susceptible and resistance (Table 3-2). The majority of mutations found within this oprD type consisted of single nucleotide insertions at different positions in the oprD gene. The insertion of a G at 167 the most frequent mutation, observed in susceptible and resistance strains and intermediately susceptible. Most of the strains harboring these mutations remained susceptible to imipenem (Table 3-2). 3.4. Analysis of oprD expression The relationship between biofilm formation, imipenem resistance, and oprD expression (Appendix B) was assessed in some imipenem resistant clinical strains (SMC631C, SMC631F, SMC631J, SMC631H, SMC631K and SMC 4974). As it is illustrated in table 3-3, all resistant strains showed low oprD expression and low biofilm values as comparison with the

sensitive strains. Remarkably, the correlation coefficient (r=0.5) appreciates the possibility of a direct relationship between oprD expression and biofilm formation. Table 3-2: Different oprD mutation types found in clinical strains of P. aeruginosa shown different Imipenem susceptibility. Name of strain

Imipenem MIC (µg/ml)*

mutation(s)

Type of mutation

PAO1 SMC 631 SMC 631 K SMC 4974

2 1.5 24 32

None T 103 S, K 115 T, F 170 L T103 S, K 115 T, F 170 L T 103 S, K 115 T, F 170 L

Full-length Wild type

SMC 631 F

16

T103 S, K 115 T, F 170 L, W 417 opa

SMC 631 H

16

T103 S, K 115 T,

SMC 631 J

16

T 103 S, K 115 T, nt 152Gnt 53

SMC 631 C

16

T 103 S, K 115 T, nt 152Gnt 153

nt 152Gnt 153

Amino acid substitutions Premature stop codon Amino acid substitutions, Frame shift mutations due to insertions

nt 167Gnt 168

SMC 5810

23

, S 56 K, G 57 V, nt 177ΔC, I 210 V, E 230 K, S 240 T, N 262 T, A 267 S, A 280 G, K 295 Q , Q 300 E, R 310 G, V 359 L

Amino acid substitutions

SMC 5811

24

D 43 N, nt 167Gnt 168, S 56 K, G 57 V, nt 177ΔC, nt 567ΔC, E 102 Q, I 210 V, E 230 K, S 240 T, N 262 T, A 267 S, A 280 G, K 295 Q, Q 300 E, R 310 G , V 359 L

SMC 4980

32

nt 167Gnt 168, nt 177ΔC, nt861ΔG

SMC 4963 SMC 4972 SMC 4986 SMC 4970 SMC 4973 SMC 4966 SMC 4964 SMC 4979

1 1 1 0.5 0.5 0.5 0.75 4

None None None D 43 E, nt 167Gnt 168, nt 177ΔC D 43 E, nt 167Gnt 168, nt 177ΔC D 43 E, nt 167Gnt 168, nt 177ΔC D 43 E, nt 167Gnt 168, nt 177ΔC D 43 E, nt 167Gnt 168, nt 177ΔC

Amino acid substitutions, Frame shift mutations due to insertions and deletions

Frame shift mutations due to insertions and deletions Full-length type

Amino acid substitutions, Frame shift mutations due to insertions and deletions,

*

Sensitive (≤ 2 µg/ml), intermediate (4 µg/ml) or resistant (≥ 8 µg/ml)

The imipenem sensitive or intermediate P. aeruginosa clinical strains (SMC576, SMC4972, SMC4973, and SMC4979) formed relatively robust biofilms; however, the oprD expression in SMC576 and SMC4972 was high. Such finding is compatible with our theory (there is an inverse correlation between oprD expression in respect to imipenem resistance and biofilm formation). Decrease in oprD expression that appeared to be relevant was also detected in imipenem-susceptible and intermediate susceptible in two strains SMC4973, SMC4979 respectively, indicated these low expressions of oprD related to other mechanism. Less commonly there is a mutation or deletions within mexT convert inactive MexT into an active form. Somehow mutations occur in mexS located upstream of mexT, lead to accumulate various metabolites that serve as effector molecules for MexT, which, in turn or in both cases, the expression of mexEF-oprN occurs at high level, alongside with a decline in the expression of oprD which is inadequate to elaborate quantities of OprD in the outer membrane sufficient for normal cellular function (Fukuda et al., 1995; Köhler et al., 1997). Wolter et al. (2009) also showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. These strains had decreased expression of the mexAB–oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD–oprJ and mexEF–oprN efflux systems normally associated with acquired resistance. Once again this might means there are other routes for carbapenems entrance.

It can be concluded that oprD inactivating mutations in clinical strains of P. aeruginosa are not restricted only to imipenem-resistant strains but are

also found in strains with imipenem MICs ≤ 2 µg/ml during sequence analysis, and from oprD expression experiment we appreciated may be there is a link between oprD gene and biofilm formation.

Table 3-3: correlation between oprD expression and biofilm formation of P. aeruginosa strains. Imipenem susceptibility

Resistance

Sensitive

Intermediate

Name of strains

oprD expression values ± SDb

Biofilm values (A550 ± SD)

SMC631 C

0.060 ± 0.034

0.12 ± 0.0153

SMC 631F

0.136 ± 0.007

0.06 ± 0.0161

SMC 631J SMC 631H SMC 631K SMC 4974

0.066 ± 0.003 0.063 ± 0.028 0.101 ± 0.033 0.015 ± 0.004

0.10 ± 0.0188 0.11 ± 0.0151 0.12 ± 0.0344 0.04 ± 0.0051

SMC 4972

0.202 ± 0.094

0.77 ± 0.0257

SMC 4973 SMC 576

0.072 ± 0.007 1.231 ± 0.227

0.96 ± 0.0509 0.84 ± 0.2305

SMC 4979

0.079 ± 0.014

0.53 ± 0.0626

Sensitive (≤ 2 µg/ml), intermediate (4 µg/ml) or resistant (≥ 8 µg/ml). bSD= standard deviation. a

There is little literature available that correlates mechanisms of planktonic antimicrobial resistance and biofilm production. Dheepa et al. (2011) indicated the resistance to antibiotics such as ceftazidime, cefepime and pipercillin in Acinetobacter baumannii was comparatively higher among biofilm producers than non-biofilm producers. Similarly, Rao et al. (2008) and Ibrahim et al. (2012) investigated clinical strains of A. baumannii, that had a significant of biofilm associated with multiple drug resistance. These

data indicate the possibility that there may be an under-appreciated link between planktonic resistance mechanisms and biofilm formation. 3.5. OprD participates in biofilm formation. In the present study the critical role of OprD in imipenem resistance well-established, the defected in the oprD gene that may display an altered biofilm phenotype using the 96 well microtiter assay. To test this hypothesis, the ability of an oprD transposon mutant was investigated for biofilm and compared to the PAO1 strain. Results revealed that oprD mutant showed a significant reduction in biofilm formation compared to P. aeruginosa PAO1 (Figure 3-2A).

The biofilm formation defect of the oprD mutant was

complemented by introducing a wild-type copy of the oprD gene on plasmid, but the vector control (pMQ72) was not able to rescue this defect (Figure 3-2A). To confirm the role of oprD mutant with imipenem resistance, we performed an E-test assay on the strains described in figure 3-2A. The oprD transposon mutant and the mutant carrying the vector control (pMQ72) showed significantly higher resistance to imipenem compared to the parental P. aeruginosa PAO1 and the oprD mutant complemented with a plasmid carrying the wild-type copy of this gene (Figure 3-2B). To evaluate whether these phenotypes might be observed in a clinical strain as well as the PAO1 laboratory strain, we investigated the biofilm formation and imipenem resistance phenotypes of a clinical strain of P. aeruginosa isolated from the sputum of a cystic fibrosis patient. Non-mucoid strain, designated SMC631, could form a biofilm in the 96 well microtiter plate and was sensitive to imipenem (Table 3-1).

Figure 3-2: OprD participates in biofilm formation as well as imipenem resistance. (A) Quantification of biofilm formation for the indicated strains, including P. aeruginosa PAO1, the oprD::IsphoA/hah transposon mutant and the oprD mutant complemented with the vector control (pMQ72) or a plasmid carrying a wild-type copy of oprD (poprD+). In this and all figures, each strain was tested in four wells per experiment. Error bars represent standard deviations of means from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test comparison. ns, not significantly different; **, P < 0.01, compared to P. aeruginosa PAO1. (B) Imipenem

susceptibility was investigated by Etest strips for the same strains described in panel A. Error bars represent standard deviations of averages from three independent experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test comparison. ns, not significantly different; ***, P