Structural characterization of Bacillus licheniformis

Download PDF
1 downloads 0 Views 9MB Size Report
Comment
constituents of the cell wall, providing physical and chemical protection from the ...... (2015) Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide ...
Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-2002-6

RESEARCH ARTICLE

Structural characterization of Bacillus licheniformis Dahb1 exopolysaccharide—antimicrobial potential and larvicidal activity on malaria and Zika virus mosquito vectors Muthukumar Abinaya 1 & Baskaralingam Vaseeharan 1 & Mani Divya 1 & Sekar Vijayakumar 1 & Marimuthu Govindarajan 2,3 & Naiyf S. Alharbi 4 & Jamal M. Khaled 4 & Mohammed N. Al-anbr 4 & Giovanni Benelli 5,6 Received: 14 February 2018 / Accepted: 10 April 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Microbial polysaccharides produced by marine species play a key role in food and cosmetic industry, as they are nontoxic and biodegradable polymers. This investigation reports the isolation of exopolysaccharide from Bacillus licheniformis Dahb1 and its biomedical applications. Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) was extracted using the ethanol precipitation method and structurally characterized. FTIR and 1H-NMR pointed out the presence of various functional groups and primary aromatic compounds, respectively. Bl-EPS exhibited strong antioxidant potential confirmed via DPPH radical, reducing power and superoxide anion scavenging assays. Microscopic analysis revealed that the antibiofilm activity of Bl-EPS (75 μg/ml) was higher against Gramnegative (Pseudomonas aeruginosa and Proteus vulgaris) bacteria over Gram-positive species (Bacillus subtilis and Bacillus pumilus). Bl-EPS led to biofilm inhibition against Candida albicans when tested at 75 μg/ml. The hemolytic assay showed low cytotoxicity of Bl-EPS at 5 mg/ml. Besides, Bl-EPS achieved LC50 values < 80 μg/ml against larvae of mosquito vectors Anopheles stephensi and Aedes aegypti. Overall, our findings pointed out the multipurpose bioactivity of Bl-EPS, which deserves further consideration for pharmaceutical, environmental and entomological applications. Keywords Exopolysaccharide . 1H-NMR . Dengue fever . Insecticide . Drug development . Microbial pathogens . Pesticide . Yellow fever

Introduction Microbial exopolysaccharides (EPS) are macromolecules of high molecular weight polymers with glycosidic linkages. Recently, natural polymers have been increasingly

requested for numerous industrial applications. EPS can be secreted by several microorganisms, including bacteria, fungi, and algae during their growth (Wang et al. 2010a, b; Ismail and Nampoothiri 2010; Poli et al. 2010; Zhang et al. 2016). As a general trend, it has been highlighted

Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-018-2002-6) contains supplementary material, which is available to authorized users. * Baskaralingam Vaseeharan [email protected] 1

2

Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Block, 6th floor, Burma Colony, Karaikudi, Tamil Nadu 630004, India Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalai Nagar, Tamil Nadu 608 002, India

3

Department of Zoology, Government College for Women, Kumbakonam, Tamil Nadu 612 001, India

4

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

5

Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy

6

The BioRobotics Institute, Sant’Anna School of Advanced Studies, viale Rinaldo Piaggio 34, 56025 Pisa, Pontedera, Italy

Environ Sci Pollut Res

that bacteria have a better EPS excreting ability if compared to fungi and algae (Sutherland 1972; Lee et al. 1997). In particular, Gram-positive Bacillus spp. are great exopolysaccharides producing species, which are now recognized as potentially benign probiotics (Patel et al. 2009; Rani et al. 2017). Polysaccharides of microbial derivation are the integral constituents of the cell wall, providing physical and chemical protection from the environment, ensuring nutrition storage, repressing defense during infection, and helping in molecular recognition (biofilm formation/quorum sensing), affecting protein folding and cell adhesion (Barbara et al. 2009). They also play a crucial role in defense mechanisms triggered by environmental factors, bacteriophage attacks, as well as attachment to surfaces, nutrient gathering, and antigenicity (Kanmani et al. 2011; Sajna et al. 2013). Therefore, they have multipurpose applications in biomedical science (Arena et al. 2006; Abdhul et al. 2014). Earlier, EPS produced by marine bacteria have been recognized as important resource. Unfortunately, only few of these bacteria have been commercially exploited in the Breal world^ due to the low yields and lack of cost-effective routes for EPS production. To utilize these bacterial EPS, a key requisite is investigating the production, structural characterization, and potential applications of EPS from marine microorganisms. Recent reports elucidated that the microbial biopolymers can be exploited for a number of biological and biomedical applications of EPS, with special reference to the development of new antimicrobial drugs, to face the growing resistance level of microbial pathogens to commonly used antibiotics (Lima et al. 2008; Lin 2010). Hence, the present study aims to isolate microbial biopolymers from a probiotic B. licheniformis Dahb1 strain isolated from shrimp intestine. Accordingly, BlEPS was extracted from B. licheniformis Dahb1 and structurally characterized it using FTIR spectroscopy and 1HNMR. Emulsification assays with different hydrocarbons (xylene, n-hexane, and toluene) were carried out. Then, the antioxidant activity was assessed via DPPH, reducing power and superoxide anion scavenging assays. The growth inhibition activity against four bacterial species was evaluated relying to minimum inhibitory concentration (MIC) estimation. Light and confocal laser scanning microscopy (CLSM) were used to study the antibiofilm activity of Bl-EPS against Gram-negative (P. aeruginosa and P. vulgaris) and Gram-positive (B. subtilis and B. pumilus), as well as on the fungal pathogen C. albicans. Lastly, the larvicidal activity of Bl-EPS against the malaria vector Anopheles stephensi (Benelli and Beier 2017) and the Zika virus vector Aedes aegypti (Benelli and Romano 2017) was also investigated. Histological damages in mosquito larvae triggered by the exposure to this bacterial product were investigated.

Materials and methods Bacterial culture and maintenance The probiotic strain B. licheniformis Dahb1 was isolated from a shrimp culture. Bacillus licheniformis Dahb1 molecular identification was done by 16S rRNA gene sequencing (NCBI GenBank accession no HM235407.1). The isolation, identification, probiotic efficacy, and maintenance (Luria-Bertani broth, sterile glycerol − 20 °C, 20% v/v) of B. licheniformis Dahb1 were reported in our earlier studies (Jayaseelan et al. 2013; Vinoj et al. 2014, 2015). The culture was grown at 37 °C under continuous agitation in nutrient broth (200 ml) plus 3.5% NaCl (w/v) and 0.02% (w/v) glucose in an Erlenmeyer flask (500 ml) and incubated at 150×g for 24–72 h. The pH of the medium ranged from 5.0 to 7.0 and was maintained using 1 N NaOH. At regular intervals, aliquots (5 ml) of the bacterial culture were drawn for turbidity measurement at OD600nm, and culture medium was stored at 4 °C for further analysis.

Autoaggregation assay Autoaggregation assay was determined as reported by Del Re et al. (2000). Briefly, overnight, B. licheniformis culture was centrifuged at 2000×g for 10 min at 4 °C. Cell suspensions (3 ml) were vortexed for 5 min and incubated for 1 h at 37 °C. Absorbance was measured by spectrophotometer at OD600 nm. Autoaggregation (%) was estimated as 1 − (At/A0) × 100, where A0 is the absorbance at 0 h and At is the absorbance at after 1 h incubation, respectively.

Hydrophobicity Hydrophobicity analysis was conducted by the method of Rosenberg (2006). Briefly, overnight bacterial suspension (3 ml) was transferred into a fresh tube with xylene (0.4 ml), briefly vortexed and incubated at 30 °C for 20 min. Post-incubation, the supernatant was removed and absorbance measured at OD600 nm. The hydrophobicity (%) was calculated as (A0 − A)/ A0 × 100, where A0 represents the absorbance before xylene extraction and A represents the absorbance after xylene extraction.

Production of Bl-EPS The B. licheniformis Dahb1 was inoculated into 500 ml of optimized fermentation medium and grown at 37 °C for 72 h. The cell wall containing Bl-EPS was dissolved by heating the culture at 100 °C for 30 min and then centrifuged at 8000×g for 10 min to separate the cells. Supernatant fluid containing Bl-EPS was precipitated by adding three volume of ice-cold 95% ethanol (20 °C) and placed at 4 °C for 36 h. The sample was then centrifuged at 8000×g for 10 min, and the pellet was re-dissolved in 5 ml of d.H2O and dialyzed using

Environ Sci Pollut Res

d.H2O. After overnight dialysis at 4 °C, the sample was dried in desiccator and stored at 30 °C (Fang et al. 2010). Total BlEPS concentration was determined by phenol-sulfuric method (Dubois et al. 1956) with glucose (Glc) as a standard. Protein content was analyzed following the technique of Lowry (Bensadoun and Weinstein 1976). Uronic acid content was measured by carbazole-sulfuric acid method (Bitter and Muir 1962) relying to glucuronic acid as standard.

Characterization of Bl-EPS

The percentage of DPPH radicals scavenging ability of BlEPS was calculated as follows:   Scavenging activity ð%Þ ¼ 1− Asample −Ablank =Acontrol  100% where Asample is the presence of the sample, Ablank is the absence of the DPPH solution, and Acontrol is the absence of the sample. Control was ascorbic acid.

FTIR spectral analysis Bl-EPS was analyzed by FTIR spectroscopy (PerkinElmer infrared spectrum GX). Finely ground 10 mg of Bl-EPS and potassium bromide (KBr) 100 mg were pressed into a disc with hydraulic press. The pellets were subjected to FTIR spectroscopy, 4000–400cm−1, resolution 4cm−1 (Wang et al. 2011). NMR spectra analysis Noise-decoupled 1H-nuclear magnetic resonance (1H-NMR) spectrum of Bl-EPS was carried out using Bruker Avance-II 200 Spectrometer, at 400 MHz with 5 mm inverse probe. Briefly, 100 mg of Bl-EPS was dissolved in deuterium oxide (D2O, 0.5 ml) in a NMR tube. The proton NMR spectra were studied at 27 °C, and chemical shifts were expressed as parts per million (Singh et al. 2011). Emulsifying activity of Bl-EPS The emulsification of Bl-EPS was performed as in Bramhachari et al. (2007) with minor modifications. In brief, the dried Bl-EPS (0.5 mg/ml) was suspended in dd.H2O and prepared with PBS (2 ml; pH 7.2). The reaction mixture was then shaken for 5 min behind the addition of 3 ml of hydrocarbons (xylene, n-hexane and toluene) and incubated at 30 °C for 30 and 60 min. Control was 2 ml of PBS without Bl-EPS. The absorbance was recorded at OD540 nm. The proportion confinement of emulsifying agent during incubation time was estimated as (t) = At/A0 × 100, where A0 is the absorbance at 0 h and At is the absorbance at 30 and 60 min after incubation respectively. In vitro antioxidant assay of Bl-EPS DPPH radical (DPPH) scavenging activity The scavenging activity of Bl-EPS on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) was determined following the DPPH standard method (Molyneux 2003; Tadhani et al. 2007; Prior et al. 2005) with slight modifications. Briefly, the mixture containing 0.5 mg/ml of Bl-EPS at 20, 40, 60, 80, and 100 μg/ml was added to 4 ml DPPH solution (0.2 mM, suspend in methanol 95%, v/v).

Reducing power The reducing power of Bl-EPS was studied following a standard method (Kumar et al. 2004) with little modifications. Briefly, the Bl-EPS sample (0.5 mg/ml) solutions at various concentrations (20, 40, 60, 80, and 100 μg/ml) were mixed with phosphate-buffered saline (2.5 ml of 0.2 M PBS; pH − 6.6), potassium ferricyanide (2.5 ml; 1% (w/v)), and then incubated at 50 °C for 20 min. After incubation, trichloroacetic acid (2.5 ml; 10% (w/v)) was added and centrifuged at 2000×g for 10 min. The supernatant was mixed with d.H2O (2.5 ml) and (1 ml; 0.1% (w/v)) FeCl3, and then the OD was read at 700 nm. Control was ascorbic acid. Superoxide radical scavenging activity The superoxide radical scavenging activity of Bl-EPS was studied as in Zhang et al. (2016) with slight modifications. Briefly, 4.5 ml of 0.5 mM Tris buffer (pH 8.0) solution was mixed into sample (0.5 mg/ ml) with concentrations of 20–100 μg/ml and then incubated at 37 °C for 10 min. After incubation, preheated pyrogallol (0.2 ml; 7 mM) was added and read at OD320 nm. Control was ascorbic acid. Superoxide radical scavenging activity (%) = (1 − Asample/A) × 100.

Minimum inhibitory concentrations (MIC) assay The minimum inhibitory concentration (MIC) of Bl-EPS was assessed against Gram-positive (B. subtilis and B. pumilus) and Gram-negative (P. aeruginosa and P. vulgaris) bacteria as well as on the fungus C. albicans, by 96-well micro-titer assay using resazurin as an indicator of cell growth (Elshikh et al. 2016). One hundred microliters of nutrient broth plus with Bl-EPS (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/ ml) was added. Then, resazurin indicator (10 μl) solution was added to each well. Finally, 10 μl microbial suspension (5 × 106 CFU/ml) was added to each well. PBS (pH 7.4) was used as a control and plates were incubated for 24 h at 37 °C. After that, the color change was assessed visually.

Live and dead cell assay The bacterial cell viability was assessed by following the methods of Velusamy et al. (2015). Briefly, overnight cultures

Environ Sci Pollut Res

of bacteria (1.0 × 105 CFU/ml) were inoculated to each well of a 24-well plate. The plates containing bacterial cells were treated with 50 and 75 μg/ml concentrations of Bl-EPS and incubated at 37 °C for 1 h. After, the cells were transferred into 1.5-ml Eppendorf tubes and centrifuged at 7000×g for 10 min. The resulting pellet was washed with PBS buffer following the cells that were stained with 10 μl SYTO®9 (3 mM) and 20 μl propidium iodide (20 mM) for 15 min under dark conditions. The stained bacterial cells were examined under CLSM (Carl Zeiss LSM 710) using a 488-nm argon laser and BP 500–640 band pass emission filter and Zen 2009 software (Carl Zeiss, Germany).

Antibacterial activity of Bl-EPS Antibacterial activity was tested on Gram-positive Bacillus s u b ti li s ( K T 7 6 3 0 78 . 1 ) , B a c i l l u s p u m i l u s D a h b 3 (HQ693273.1) and Gram-negative Pseudomonas aeruginosa Dahp1 (HQ400663.1), and Proteus vulgaris Dahp1 (HQ116441.1) bacteria, as well as on the fungus C. albicans, by agar well diffusion method (Mahendran et al. 2013). All the isolates were maintained in nutrient broth, and overnight culture of 100 μl of each microbial species was spread onto LB agar plates using sterile cotton swab. Then, LB agar was punched with the help of sterile cork borer to create 6 mm well. Bl-EPS concentrations of (25, 50, 75, 100 μg/ml) were loaded on marked wells with micropipette. Deionized water without Bl-EPS was considered as control; bacteria plates were kept for incubation at 37 °C for 24 h, while fungus plates were kept at 37 °C for 48 h. After incubation, the zone of inhibition was measured in terms of millimeters (mm).

Antibiofilm activity of Bl-EPS Antibiofilm assays were carried out using the microtiter plate technique (Junter et al. 2016). Briefly, overnight grown cultures of Gram-positive (B. subtilis and B. pumilus) and Gramnegative (P. aeruginosa and P. vulgaris) bacteria were grown on sterile glass pieces (1 × 1 cm) placed in 24-well plates containing nutrient broth (1.5 ml) with 75 μg/ml of Bl-EPS and incubated for 3 days at 37 °C. After incubation, the liquid suspension was removed and the glass pieces were recovered, washed twice with 1× PBS, stained with 0.4% of crystal violet, and examined under inverted light microscopy (Nikon, ECLIPSE Ti ×100, Japan) at 40x magnification. Independently, additional set of glass pieces with biofilm grown as above were washed with 1× PBS, stained with 0.1% of acridine orange, and the biofilm was assessed under CLSM. The thickness of biofilm was evaluated using the COMSAT software. The same procedure was followed to assess the antibiofilm activity of Bl-EPS on C. albicans (Peralta et al. 2015).

Cytotoxicity of Bl-EPS by hemolytic potentiality test Hemolytic activity of Bl-EPS was assessed by Abinaya et al. (2018). Briefly, the blood (9 ml) was collected from 8 weekold female chicken. 1 ml of 3.8% sodium citrate that was mixed to it inhibiting the blood coagulation. The sample was centrifuged at 3000×g for 10 min. The pellet containing RBCs was washed three times with 10 ml of PBS (pH 7.4) to remove the buffy coat of RBCs. Finally, the cells were uniformly suspended in PBS. The mixture containing 2 ml of erythrocyte suspension with Bl-EPS at different concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 mg/ml) was added to each test tube and gently inverted. The mixtures were incubated at 37 °C for 1 h and PBS was used as a control. After incubation, the samples were centrifuged at 3000×g for 10 min to pellet out the RBC cells. The supernatant was separated out and used for absorption studies at OD540 nm using a UV-vis spectrophotometer. The percentage of hemolysis was calculated as follows:  Hemolysis ð%Þ ¼ Asample −Ablank =Acontrol  100 where Asample is the absorbance in the presence of the sample, Ablank is the absorbance in the absence of the sample with the same amount of erythrocyte suspension to PBS, and Acontrol is the absorbance of PBS, respectively.

Mosquito larvicidal activity of Bl-EPS Third instars larvae of An. stephensi and Ae. aegypti were reared as illustrated by Govindarajan and Benelli (2016). The larvicidal activity was assessed by the standard procedure of WHO (2005). In this assay, third instar larvae were separated into five sets. Each set was provided with 20 larvae plus 200 ml of water. Different concentrations of Bl-EPS (30, 60, 90, 120, and 150 μg/ml) were evaluated. The control was dechlorinated tap water. Dead larvae were counted after 24 h of exposure, and mortality (%) was reported as the average of five replicates.

Morphological and histological observations post-exposure to Bl-EPS The morphological changes in An. stephensi and Ae. aegypti third instar larvae were examined as described by Yu et al. (2015) post-exposure to Bl-EPS at LC50 and LC90 for 24 h, then results were compared with control larvae. Before the morphological observations, mosquito larvae were washed with distilled water, then morphological changes in body segments were observed under stereo microscopy (Nikon SMZ 745T Japan). The histological observations on An. stephensi and Ae. aegypti post-exposure to Bl-EPS were carried out following the procedure by Sundararajan and Ranjitha Kumari (2017)

Environ Sci Pollut Res

with minor changes. Briefly, larvae were embedded in a block with melted paraffin. The paraffin blocks were sectioned at 8 μm thickness using rotary microtome and stained with hemotoxylin and eosin. The glass slides were observed under stereomicroscope (Nikon SMZ 745T Japan).

Statistical analysis The experiments were carried out in randomized block design with three replications, and the final data were presented the mean ± standard deviation (SD). The larvae mortality data were subjected to probit analysis and LC50, and LC90 values were calculated. Chi squares were not significant (Benelli 2017). Scavenging activity data and microbial pathogen growth inhibition data were evaluated using ANOVA followed automatically by post hoc Tukey’s HSD test. We used the SPSS version 21 software. In all data analyses, P < 0.05 was considered as statistically significant to assess the differences among the control and treated groups.

Results Autoaggregation and hydrophobicity The autoaggregation ability of the probiotic strain B. licheniformis Dahb1 was studied based on sedimentation rates. The B. licheniformis Dahb1 exhibited strong autoaggregation (97.9%) after 1 h of incubation. Hydrophobicity was measured based on their adhesion to a hydrophobic substratum. It revealed to be hydrophobic with 59.1% adhesion to xylene.

Fig. 1 Growth curve of Bacillus licheniformis Dahb1: influence of pH changes and profile of B. licheniformis exopolysaccharide (Bl-EPS) production

Production of Bl-EPS The EPS content from probiotic strain B. licheniformis Dahb1 varied from time to time; the maximum production was achieved until the late log phase of bacterial growth (pH 7.0) with 620mg−1 dry cell weight after 3 days at 37 °C (Fig. 1). The quantity of carbohydrates, protein, and uronic acid in BlEPS was 680.43, 386.15, and 56.72mg−1, respectively.

Characterization of Bl-EPS FTIR spectral analysis The FTIR spectrum of Bl-EPS is shown in Fig. 2a. Several peaks were detected from 3798.7 to 486.2 cm−1. Broadly stretched intense peaks were noted at 3798.7 and 3441.5 cm−1 due to the presence of the hydroxyl groups (O-H) containing sugar residues. The peaks at 2923.6, 2853.3, and 2358.9 cm−1 were assigned to the weak CH stretching peak of methyl groups. The strong absorption peak at 1650.2 cm−1 indicated the presence of carboxylate groups and confirmed the polysaccharide characteristic of Bl-EPS. Further, the bands at 1566.2, 1456.5, and 1403.7 cm −1 were as attributed to asymmetrical stretching vibrations of carboxyl groups revealing the complexity of Bl-EPS. The broad stretch of C-O-C, C-O at1077.7 cm−1 confirmed the polymer presence. An intense peak at 486.2 cm−1 indicated the presence of carbohydrates, with glycosidic linkage bonds between the glycosyl groups present in the Bl-EPS. NMR spectral analysis The 1H-NMR spectral analysis of Bl-EPS is shown in Fig. 2b. Alkyl substituted anomeric proton chemical shifts appeared as broad signals, they were formed in downfield confirming the presence of polysaccharides. The signals at δ 0.75–δ 1.3981 ppm was linked to the 6-deoxy sugars methyl protons. The signals at δ 1.23 and δ 2.087 ppm were assigned to the CH3 and were predicted as acetylenic proton with triple bond, and CC H stretching and δ 2.379 ppm indicated the benzylic protons (Ar C H). The signal around 2 ppm was characteristic N-acetyl glucosamine residues; δ 2.4852 ppm was exclusively detected in Bl-EPS indicating the presence of succinyl group. The peak δ 3.070–δ 3.2980 ppm indicated the presence of carbohydrate derivatives and alcohols (H C OH), while δ 3.318 and δ 3.964 ppm showed that the presence of esters (RCOO C H). The peak at 4.7 ppm is due to hydrogendeuterium oxide (HDO), while signals between δ 5.719 and δ 5.733 ppm were compatible with the presence of α-anomers. The anomeric peaks at δ 6.758–δ 7.4692 ppm were assigned as α − (1 → 6) linked glucan and indicate

Environ Sci Pollut Res

Fig. 2 a FTIR spectrum and b 1H-NMR analysis of Bacillus licheniformis Dahb1 exopolysaccharide

the existence of as α − (1 → 3) linked glycosyl moieties. Overall, it was depicted the heterogeneous properties of

Bl-EPS due to the convergence of most sites of the signals in the region at 400 MHz (Table 1).

Environ Sci Pollut Res Table 1 Characteristic proton 1H NMR chemical shifts of Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS)

Compound

Hydrogen type

Chemical shifts (δ) value (ppm)

Alkanes

RCH3 (1° aliphatic)

0.75–1.3 1.2–1.5 1.5–2.0 1.6–1.9 2.2–2.5

RCH2R (2° aliphatic) R3CH (3° aliphatic) R2C=C(R)–CH3

Allylic Benzylic

ArCH3

Alkyl chloride

RCH2Cl RCH2Br ROCH2R HOCH2R RCOCH3 R2C=CH2 R2C=CRH

3.6–3.8 3.4–3.6 3.3–3.9 3.3–4.0 2.1–2.6 4.6–5.0 5.2–5.7

Aromatic Acetylinic

ArH RC=CH

6.5–8.5 2.5–3.1

Alchol hydroxyl Phenolic Amino Amine Amides

ROH ArOH R-NH2 HC-NHR RNHC(=O)R′

0.5–6.0 4.5–7.7 1.0–5.0 1.5–2.0 5–8.5

Alkyl bromide Ether Alcohol Ketone Vinylic

Emulsifying activity of Bl-EPS The emulsifying activity of Bl-EPS was determined against different hydrocarbons and Tween 20 (Table 2). The highest emulsification was obtained with n-hexane (81.6 ± 1.43) at 30 min. In addition, toluene (77.6 ± 0.95) and xylene (58.7 ± 0.64) were considerably emulsified. The emulsion was found to be more stable after incubation at 30 min over 60 min. In vitro antioxidant assay of Bl-EPS The antioxidant activity of Bl-EPS was investigated by DPPH radicals, reducing power and superoxide anion scavenging assays (Supplementary Online Material Fig. S1). The scavenging capability of Bl-EPS was observed

Table 2 The emulsifying activity of Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) tested against different hydrocarbons Tested compound

Xylene Toluene n-Hexane

Emulsifying activity of Bl-EPS (0.5 mg/ml) Control

After 30 min

After 60 min

51.2 ± 0.95a 63.9 ± 0.76b 72.1 ± 0.67c

58.7 ± 0.64a 77.6 ± 0.95b 81.6 ± 1.43b

43.0 ± 0.55a 55.5 ± 1.29b 66.2 ± 1.15c

Within each column, different superscript letters show significant differences (ANOVA, Tukey’s HSD, P < 0.05)

as 17 ± 2.1% testing 20 μg/ml, and 82 ± 6.7% testing 100 μg/ml. Positive control ascorbic acid expressed its scavenging capacity from 22 ± 0.6 to 89 ± 3.8% testing 2–10 μg/ml, respectively. However, the scavenging activity of Bl-EPS was relatively lower if compared to that of the control. In addition, the tested samples were changed from yellow color to various shades of green and blue color indicating the reducing power of Bl-EPS. Higher absorbance indicated better reductive potential compared with control ascorbic acid. In superoxide radical scavenging activity assays, we noted a maximum of 22 ± 5.5% testing 20 μg/ml and 58 ± 6.1% testing 100 μg/ml (Fig. 3).

Minimum inhibitory concentration (MIC) assay The MIC of Bl-EPS against Gram-positive and negative bacteria and the fungus C. albicans was tested by using r e s a z u r i n m i c r o t i t e r p l a t e a s s a y, a s s h o w n i n Supplementary Online Material Fig. S2. After incubation, complete color change from blue to pink was observed in all control wells, whereas no color change was observed in the wells containing Bl-EPS, indicating no growth and confirming the microbial pathogen sensitivity to the BlEPS. The MIC estimated for Gram-positive bacteria B. subtilis and B. pumilus was 40 μg/ml. Concerning Gram-negative bacteria, P. aeruginosa and P. vulgaris, as well as the fungus C. albicans MIC was 30 μg/ml.

Environ Sci Pollut Res Fig. 3 Live (green) and dead (red) bacterial cell assay testing Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) against Gram-positive (Bacillus subtilis and Bacillus pumilus) and Gram-negative (Pseudomonas aeruginosa and Proteus vulgaris) bacteria

Live and dead cell assay The live and dead bacterial cells were examined by confocal microscopy using BacLight fluorescent staining, as shown in Fig. 3. The green fluorescence indicates live bacterial cells, while the red depicts dead ones. After treatment with Bl-EPS against Gram-positive (B. subtilis and B. pumilus) and Gramnegative (P. aeruginosa and P. vulgaris) bacteria, it was observed that Bl-EPS had highly effective bactericidal properties at 75 μg/ml, if compared to the control samples, which showed a large number of live cells (Fig. 3). Antibacterial activity of Bl-EPS Results showed significant antibacterial activity of Bl-EPS against Gram-positive (B. subtilis and B. pumilus) and Gram-negative (P. aeruginosa and P. vulgaris) bacteria and the fungus C. albicans, using the agar well diffusion method

(Table 3). The inhibition zone of Gram-positive bacteria B. subtilis ranged from 8.23 ± 0.87 mm testing 75 μg/ml, while B. pumilus inhibition zones ranged from 9.27 ± 0.25 mm testing 100 μg/ml. Gram-negative P. aeruginosa ranged from 10.4 ± 0.75 mm testing 75 μg/ml, while P. vulgaris and C. albicans inhibition zones ranged from 11.7 ± 0.85 and 8.93 ± 0.64 mm testing 100 μg/ml. Overall, Bl-EPS showed greater inhibition on Gram-negative bacteria at 100 μg/ml if compared to Gram-positive bacteria. Antibiofilm activity of Bl-EPS The antibiofilm activity of Bl-EPS against Gram-positive (B . s u b t i l i s a nd B . p u m i l u s) and Gr am-ne gativ e (P. aeruginosa and P. vulgaris) bacteria and the fungus C. albicans was assessed and visualized by CLSM (Fig. 4a) and light microscopy (Fig. 4b). Control biofilm (without BlEPS) showed higher surface colonization in control for all

Environ Sci Pollut Res Table 3 Growth inhibition activity triggered by Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) against Gram-positive (Bacillus subtilis and Bacillus pumilus) and Gram-negative (Pseudomonas aeruginosa and Proteus vulgaris) bacteria and the fungus Candida albicans

Species

Zone of inhibition (mm) 25 μg/ml

Bacillus subtilis Bacillus pumilus Pseudomonas aeruginosa Proteus vulgaris Candida albicans

50 μg/ml

75 μg/ml

100 μg/ml

3.97 ± 0.21a

5.23 ± 1.17a

8.27 ± 0.47b

8.23 ± 0.87b

a

b

b

9.27 ± 0.25c 9.93 ± 0.42c 11.7 ± 0.85c 8.93 ± 0.64d

6.17 ± 0.25 5.17 ± 0.32a 7.17 ± 0.42a 3.07 ± 0.15a

8.03 ± 0.31 7.27 ± 0.25b 8.17 ± 0.35ab 4.8 ± 0.36b

8.23 ± 0.38 10.4 ± 0.75c 9.97 ± 0.55bc 6.57 ± 1.17c

Within each row, different superscript letters show significant differences (ANOVA, Tukey’s HSD, P < 0.05)

bacterial strains and fungus C. albicans, whereas in the presence of Bl-EPS, Gram-positive, Gram-negative bacteria and fungus showed weak adherence and disintegrated biofilm testing 75 μg/ml (Fig. 5), respectively. In addition, after the exposure to Bl-EPS, the thickness (12 μm) of bacterial biofilm was significantly reduced (P < 0.05) as confirmed through COMSTAT analysis. Cytotoxicity of Bl-EPS by hemolytic potential assay The initial assessment of the biocompatibility of Bl-EPS was determined by hemolytic potential activity assay (Table 4). The interaction of Bl-EPS with RBCs revealed low hemolytic activity. It showed a significant increase in hemolysis activity increasing in concentration, since 5% hemolysis is considered as permissible limit for biomaterials, till a concentration of 5.0 mg/ml of Bl-EPS can be taken for hemolytic activity (Table 4). Mosquito larvicidal activity of Bl-EPS The larvicidal activity of Bl-EPS against third instars larvae of An. stephensi and Ae. aegypti were studied. Mortality (%) increased by increasing the tested concentration of Bl-EPS. The maximum mortality was 98.2% against An. stephensi and 94.6% against Ae. aegypti larvae, when Bl-EPS were tested at 150 μg/ml (Table 5). The larvicidal LC50 values and related 95% CL of Bl-EPS against the two mosquito vectors were provided in Table 5. The LC50 and LC90 values of BlEPS against An. stephensi were 61.31 and 127.45 μg/ml. The LC50 and LC90 values of Bl-EPS against Ae. aegypti were 79.28 and 150.68 μg/ml, respectively (Table 5). Morphological and histological observations post-exposure to Bl-EPS The stereomicroscopic visualization of third instar mosquito larvae of An. stephensi and Ae. aegypti treated with Bl-EPS is reported in Fig. 6a, revealing the loss of lower and upper head hairs, antennae, caudal, and lateral hairs in An. stephensi, while darkened and large body structural damage at the

stigmal plate on the siphon apex, and disintegration of the epithelial layer and outer cuticle was observed in Ae. aegypti, 24 h post-exposure to 150 μg/ml of Bl-EPS. Further, great shrinkage in the abdominal region of Ae. aegypti was examined in treated Bl-EPS. The histological results on mosquito larvae exposed to Bl-EPS are shown in Fig. 6b. Compared to control, larvae treated with Bl-EPS showed various histological changes. From our results, we noted that the shrinkage in the abdominal region and damages to the midgut and muscles are the most common changes in An. stephensi and Ae. aegypti larvae treated with Bl-EPS.

Discussion Developing eco-friendly antimicrobials and insecticides is a crucial challenge nowadays, in order to face the growing resistance of targeted organisms to drugs and pesticides (Banumathi et al. 2017; Benelli 2018a,b; Benelli and Duggan 2018). The EPS from marine bacteria are receiving high research attention for preparing novel combination of natural polymers with potential application in various industrial sectors. As indicated by earlier literature, EPS producing marine bacteria from Bacillus species include various strains of B. licheniformis, e.g., UD061 from squid (Loligo chinensis spp.) (Fanga et al. 2013a), B. licheniformis OSTK95 from mud samples (Fang et al. 2013b), B. licheniformis from thermal sources (Dahech et al. 2013), B. licheniformis from seaweeds (Singh et al. 2011), B. licheniformis 8-37-0-1from soil samples (Liu et al. 2010a), and B. licheniformis from ropy cider (Larpin 2002), were isolated and their antioxidant properties were also reported. However, to the best of our knowledge, the isolation and characterization of EPS from probiotic B. licheniformis Dahb1 has not been studied. Moreover, reports on the antibiofilm activity of these bacteria are very scarce. Hence, the present study focused on the isolation of B. licheniformis Dahb1 EPS, its structural characterization, and biomedical properties. The probiotic B. licheniformis Dahb1 efficiency was studied through its ability to aggregate and adhere (hydrophobicity) to epithelial cells because both represent

Environ Sci Pollut Res

Fig. 4 Antibiofilm activity of Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) against Gram-positive (Bacillus subtilis and Bacillus pumilus) and Gram-negative (Pseudomonas aeruginosa and Proteus vulgaris) bacteria. a Confocal laser scanning microscopy. b Light microscopy

prerequisites for successful colonization and protection of gastrointestinal tract (Del Re et al. 2000). The sedimentation rate of B. licheniformis Dahb1 strain exhibited strong autoaggregation of 97.9% and hydrophobic with 59.1% adhesion to xylene. Differences in the aggregation and hydrophobicity abilities have been reported among different Bifidobacterium spp. (Rahman et al. 2008; Wang et al. 2010b). In this study, the percentage of autoaggregation and hydrophobicity was higher over the previously reported values for probiotic Enterococcus faecium BDU7, which was 72.7 and 54.8% (Abdhul et al. 2014). Thus, our findings

revealed that B. licheniformis Dahb1 can be further considered as a potential probiotic strain (see also Khaled et al. 2018). The yield of EPS content from B. licheniformisDahb1 showed maximum production after 3 days, with 620 mg/l dry cell weight. The Bl-EPS quantity in the present study was higher if compared to the previously reported value for the endophytic seaweed-associated strain of B. licheniformis, which produced 576 mg/l of EPS (Singh et al. 2011). The content of carbohydrates, proteins, and uronic acid in BlEPS was 680.43, 386.15, and 56.72/mg, respectively. Ordinarily, sugar contents of EPS were higher over proteins,

Environ Sci Pollut Res Fig. 5 Antifungal activity of Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) against the fungus Candida albicans. a Confocal laser scanning microscopy. b Light microscopy

a feature that has also been detected for other bacterial EPS (Kumar et al. 2007). The FTIR spectrum of Bl-EPS exhibited many peaks from 3798.7 to 486.2 cm−1. It showed a broadly stretched intense peak that indicated the presence of Bl-EPS (see also Liu et al. 2010a, b). The existence of both carboxyl groups and hydroxyl could be indicators of elevated polysaccharide presence (Kanmani et al. 2011). The 1H-NMR data showed alkyl substituted aromatic rings, the aromatic hydrogen chemical shifts appeared as a broad signal designed for EPS production by B. licheniformis Dahb1 in the present study, in a comparable manner to 6-deoxy sugars methyl protons (Singh et al. 2011). Bl-EPS showed stability in retaining the emulsion breaks during incubation. Bl-EPS was able to emulsify hydrocarbons at a significant proportion over the control. Based on our findings, both xylene and toluene were effectively emulsified by Bl-EPS, with higher levels, if compared to earlier reports Table 4 Hemolysis of red blood cells by Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) Treatment

Optical density at 540 nm

Hemolysis (%)

Saline (control) 0.25 mg/ml

1.207 ± 0.0010 0.0023 ± 0.0015

– 0.19 ± 0.13a

0.007 ± 0.0010 0.010 ± 0.0021 0.011 ± 0.0015 0.014 ± 0.0020

0.58 ± 0.09ab 0.80 ± 0.17bc 0.94 ± 0.13cd 1.16 ± 0.17d

0.50 mg/ml 1.0 mg/ml 2.5 mg/ml 5.0 mg/ml

Within the column, different superscript letters show significant differences (ANOVA, Tukey’s HSD, P < 0.05)

(Bermudez et al. 2004). The Bl-EPS emulsions were stable, potentially leading to lower toxicity, higher biodegradability, and better environmental compatibility (Sun et al. 2009). This overall result suggests that Bl-EPS may have potential application in food industry as reliable emulsifiers. In vitro antioxidant activity of Bl-EPS was studied through DPPH scavenging assay, a stable free radical trial depend on the absorbance reduction of methanolic DPPH solution at OD517 nm in existence of DPPH free radicals that can receive hydrogen radical to convert a stable diamagnetic molecule (Liu et al. 2010a, b). The maximum scavenging activity of Bl-EPS at 100 μg/ml was 82 ± 6.7%. This observation was encouraging in comparison with an earlier study on E. faecium reporting a maximum EPS activity of 63.5% at 10 mg/ml (Abdhul et al. 2014). The reducing capacity of BlEPS was confirmed through the change of yellow color test solution into green and blue color that indicated the reducing power of Bl-EPS. Greater absorbance indicated higher reductive potential, which was compared with control ascorbic acid. Similarly, study on Pseudomonas PF-6 high activity was reported (Ye et al. 2012). The capacity of antioxidant compounds to reduce Fe3+/Fe2+ complex was also considered as an important antioxidant action (Liu et al. 2010a, b). Based on our knowledge, this is the first investigation on the antibacterial activity of Bl-EPS revealing maximum growth inhibition in Gram-negative over Gram-positive bacteria, at 100 μg/ml concentration. Mahendran et al. (2013) reported that EPS can effectively interact with both bacteria categories depending to their cell membrane permeability, then attacking respiratory chain, cell division machinery, and leading to cell death.

Environ Sci Pollut Res Table 5 aegypti

Larvicidal activity of Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) against third instar larvae of Anopheles stephensi and Aedes

Mosquito species

Concentration (μg/ml)

Mortality (%) ± SD

LC50 (95% LCL-UCL) (μg/ml)

LC90 (95% LCL-UCL) (μg/ml)

Regression equation

χ2 (df = 4)

Anopheles stephensi

30 60 90 120 150 30 60 90 120 150

31.7 ± 0.8 45.6 ± 0.6 67.3 ± 1.2 86.5 ± 1.6 98.2 ± 1.2 23.3 ± 1.2 32.5 ± 0.8 56.7 ± 1.4 71.3 ± 1.2 94.6 ± 1.6

61.31 (53.50–68.09)

127.45 (117.71–140.45)

y = 13.69 + 0.5797x

3.731n.s.

79.28 (72.11–86.07)

150.68 (139.08–166.41)

y = 1.260 + 0.6047x

6.225n.s.

Aedes aegypti

No mortality was observed in control n.s. chi squares were not significant (P > 0.05)

In addition, the antibiofilm activity assays conducted with Bl-EPS showed weak adherence and disintegrated biofilm formation Gram-negative P. aeruginosa and P. vulgaris over Gram-positive bacteria post-exposure to Bl-EPS at 75 μg/ml. This agrees with earlier research on antibiofilm activity (Kanmani et al. 2011; Vijayabaskar et al. 2011). Moreover, Bl-EPS had high impact on C. albicans biofilms at 75 μg/ ml. Therefore, close-related antibiofilm effects have been also documented (Peralta et al. 2015), they may be due to the stimulation of bacterial cell detachment during biofilm

formation. Overall, the Gram-negative bacteria were highly inhibited by Bl-EPS if compared to Gram-positive bacteria, and this can be due to thick cell walls. Indeed, Gram-positive bacteria are generally composed of a three-dimensional thick peptidoglycan layer, if compared to those of Gram-negative bacteria (Nazzaro et al. 2013). The cytotoxicity test of Bl-EPS revealed low hemolytic activity at 5.0 mg/ml. Besides, the assay shows an increase in hemolysis when increasing the tested dosage. Notably, 5% hemolysis is considered as permissible limit for biomaterials.

Fig. 6 Stereomicroscopic images of third instar larvae of Anopheles stephensi and Aedes aegypti post-exposure to Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS). a Morphological changes. b Histological observations. Arrows indicate damages in mosquito larvae

Environ Sci Pollut Res

Consequently, it can be indicated that Bl-EPS is biocompatible in nature at the tested concentration (Surendra et al. 2016). Furthermore, the effective control of mosquito young instar populations is crucial to prevent and manage vector borne diseases (Benelli 2015, 2018c; Roni et al. 2015; Benelli and Mehlhorn 2016; Govindarajan et al. 2016; Murugan et al. 2018). Notably, there are no reports on microbial biopolymer Bl-EPS larvicidal activity, while more information is available on plant extracts and nano-formulated pesticides (Benelli 2016a,b; Benelli et al. 2017, 2018, Khater 2012, 2013, 2017; Khater et al. 2016, 2018; Benelli and Pavela 2018a,b; Pavela et al. 2018). Here, larvicidal activity of Bl-EPS against was assessed on mosquito larvae of An. stephensi and Ae. aegypti, two important vectors of malaria and Zika virus, respectively. Bl-EPS exhibited maximum mortality against both vectors at 150 μg/ml. LC50 and LC90 values of Bl-EPS against An. stephensi were 61.31 and 127.45 μg/ml, while LC50 and LC90 values of Bl-EPS against Ae. aegypti were 79.28 and 150.68 μg/ml, respectively. The mechanisms triggered by Bl-EPS causing the death of mosquito larvae are still unknown. Therefore, we carried out preliminary histological and morphological observations, to shed light on the kind of damage and the most affected mosquito tissues. The stereomicroscopic studies revealed the loss of upper and lower head hairs, antennae, and lateral hair, in An. stephensi, while disintegration of epithelial layer and outer cuticle, darken body and destruction of the structure at the stigmal plate on the siphon apex were outlined in Ae. aegypti at 24 h post-exposure to BlEPS. In other words, the histological assays of Bl-EPS towards the larvae of both species allowed us to highlight that midgut and muscles are the most affected tissues in larvae. In agreement with our findings, Ishwarya et al. (2017) showed that Ae. aegypti exposed to Pedalium murex seed extract experienced damages to hindgut and muscles, and the P. amboinicus leaf extract tested on larvae of An. stephensi and Culex quinquefasciatus led to damages on midgut, hindgut, muscles, and nerve ganglia (Vijayakumar et al. 2015).

Conclusions The Bl-EPS obtained from probiotic B. licheniformis Dahb1 was structurally characterized by FTIR and 1H-NMR. The chemical properties of Bl-EPS showed the higher amount of carbohydrates over proteins and uronic acid. The emulsification activity analyzed testing different hydrocarbons revealed that Bl-EPS can be exploited for environmental bioremediation targeting hydrocarbon pollutants. Three in vitro assays studying the Bl-EPS antioxidant activity showed that it had strong reducing power and limited scavenging action on DPPH. Bl-EPS exhibited greater antibacterial and antibiofilm activity against Gram-negative bacteria compared to Grampositive bacteria. The Bl-EPS also successfully inhibited the

biofilm growth of C. albicans. In addition, Bl-EPS revealed permissible limit of hemolysis activity at its lower concentration, allowing us to consider it as biocompatible. Moreover, it exhibited effective larvicidal properties against malaria and Zika virus mosquito vectors. Overall, our findings shed light on the interesting properties of Bl-EPS, which may be used to develop effective control tools against mosquito larval populations, and which deserves further research for potential applications in pharmaceutical and biomedical fields. Acknowledgments The authors gratefully acknowledge the financial support of the Department of Biotechnology (DBT), Government of India, New Delhi, India, under the project grant code BT/PR7903/ AAQ/3/638/2013. M.A. thanks the support of DST-INSPIRE fellowship [IF160623], New Delhi, India. The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RG-1438-091.

References Abdhul K, Ganesh M, Shanmughapriya S, Kanagavel M, Anbarasu K, NatarajaSeenivasan K (2014) Antioxidant activity of exopolysaccharide from probiotic strain Enterococcus faecium (BDU7) from Ngari. Int J Biol Macromol 70:450–454. https://doi. org/10.1016/j.intimp.2005.07.004 Abinaya M, Vaseeharan B, Divya M, Sharmili A, Govindarajan M, Alharbi NS, Kadaikunnan S, Khaled JM, Benelli G (2018) Bacterial exopolysaccharide (EPS)-coated ZnO nanoparticles showed high antibiofilm activity and larvicidal toxicity against malaria and Zika virus vectors. J Trace Elem Med Biol 45:93–103. https://doi.org/10.1016/j.jtemb.2017.10.002 Arena A, Maugeri TL, Pavone B, Iannello D, Gugliandolo C, Bisignano G (2006) Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis. Int J Immunopharmacol l6:8–13 Banumathi B, Vaseeharan B, Periyannan R, Prabhu NM, Ramasamy P, Murugan K, Canale A, Benelli G (2017) Exploitation of chemical, herbal and nanoformulated acaricides to control the cattle tick, Rhipicephalus (Boophilus) microplus—a review. Vet Parasitol 244: 102–110 Barbara V, Miao C, Rusell JC, Elena PI (2009) Bacterial extracellular polysaccharides involved in biofilm formation. Molecules 14: 2535–2554 Benelli G (2015) Research in mosquito control: current challenges for a brighter future. Parasitol Res 114:2801–2805 Benelli G (2016a) Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol Res 115:23–34. https://doi.org/10.1007/ s00436-015-4800-9 Benelli G (2016b) Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer—a brief review. Enzym Microb Technol 95:58–68. https://doi.org/10.1016/j.enzmictec.2016.08.022 Benelli G (2017) Commentary: data analysis in bionanoscience—issues to watch for. J Clust Sci 28:11–14 Benelli G, Maggi F, Romano D, Stefanini C, Vaseeharan B, Kumar S, Higuchi A, Alarfaj AA, Mehlhorn H, Canale A (2017) Nanoparticles as effective acaricides against ticks – a review. Ticks Tick-borne Dis 8: 821-826, https://doi.org/10.1016/j.ttbdis.2017.08.004 Benelli G, Duggan MF (2018) Management of arthropod vector data –Social and ecological dynamics facing the One Health perspective. Acta Tropica, 182:80-91, https://doi.org/10.1016/j.actatropica.2018.02.015

Environ Sci Pollut Res Benelli G (2018a) Gold nanoparticles—against parasites and insect vectors. Acta Trop 178:73–80. https://doi.org/10.1016/j.actatropica. 2017.10.021 Benelli G (2018b) Plant-borne compounds and nanoparticles: challenges for medicine, parasitology and entomology—GREEN-NANOPEST&DRUGS. Environ Sci Poll Res 25:10149–10150. https:// doi.org/10.1007/s11356-017-9960-y Benelli G (2018c) Mode of action of nanoparticles against insects. Environ Sci Poll Res, doi: https://doi.org/10.1007/s11356-018-1850-4 Benelli G, Beier J (2017) Current vector control challenges in the fight against malaria. Acta Trop 174:91–96. https://doi.org/10.1016/j. actatropica.2017.06.028 Benelli G, Mehlhorn H (2016) Declining malaria, rising dengue and Zika virus: insights for mosquito vector control. Parasitol Res 115(5): 1747–1754. https://doi.org/10.1007/s00436-016-4971-z Benelli G, Pavela R (2018a) Repellence of essential oils and selected compounds against ticks—a systematic review. Acta Trop 179:47–54 Benelli G, Pavela R (2018b) Beyond mosquitoes—essential oil toxicity and repellency against bloodsucking insects. Ind Crop Prod 117: 382–392 Benelli G, Romano D (2017) Mosquito vectors of Zika virus. Entomol Gen 36:309–318. https://doi.org/10.1127/entomologia/2017/0496 Benelli G, Maggi F, Pavela R, Murugan K, Govindarajan M, Vaseeharan B, Petrelli R, Cappellacci L, Kumar S, Hofer A, Youssefi MR, Alarfaj AA, Hwang JS, Higuchi A (2018) Mosquito control with green nanopesticides: towards the One Health approach? A review of non-target effects. Environ Sci Pollut Res 5(11):10184–10206. https://doi.org/10.1007/s11356-017-9752-4 Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70(1):241–250. https://doi.org/ 10.1016/S0003-2697(76)80064-4 Bermudez J, Rosales N, Loreto C, Briceño B, Morales E (2004) Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures. World J Microbiol Biotechnol 20(2):179–183. https://doi.org/10.1023/B: WIBI.0000021754.59894.4a Bitter T, Muir HM (1962) A modified uronic acid carbazolereaction. Anal Biochem 4(4):330–334. https://doi.org/10.1016/0003-2697(62) 90095-7 Bramhachari PV, Kishor PBK, Devi RR, Kumar R, Rao BR, Dubey SK (2007) Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3. J Microbiol Biotechnol 17(1):44–51 Dahech I, Fakhfakh J, Damak M, Belghith H, Mejdoub H, Belghith KS (2013) Structural determination and NMR characterization of a bacterial exopolysaccharide. Int J Biol Macromol 59:417–422. https:// doi.org/10.1016/j.ijbiomac.2013.04.036 Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacteriumlongum. Appl Microbiol 31(6):438–442 Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–356. https://doi.org/10.1021/ ac60111a017 Elshikh M, Ahmed S, Funston S, Dunlop P, McGaw M, Marchant R, Banat IM (2016) Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants. Biotechnol Lett 38(6):1015–1019. https://doi.org/ 10.1007/s10529-016-2079-2 Fang Y, Lu M, Xu W, Liu S, Jiao Y, Wang S (2010) Screening and optimization of fermentation medium of an antioxidant exopolysaccharide producing marine strain. Food Sci 31(23):276–280 Fang Y, Liu S, Lu M, Jiao Y, Wang S (2013a) A novel method for promoting antioxidant exopolysaccharides production of Bacillus licheniformis. Carbohydr Polym 92(2):1172–1176. https://doi.org/ 10.1016/j.carbpol.2012.10.016

Fang Y, Ahmed S, Liu S, Wang S, Lu M, Jiao Y (2013b) Optimization of antioxidant exopolysaccharides production by Bacillus licheniformis in solid state fermentation. Carbohydr Polym 98(2): 1377–1382. https://doi.org/10.1016/j.carbpol.2013.07.076 Govindarajan M, Benelli G (2016) Artemisia absinthium-borne compounds as novel larvicides: effectiveness against six mosquito vectors and acute toxicity on non-target aquatic organisms. Parasitol Res 115(12):4649–4661. https://doi.org/10.1007/s00436-0165257-1 Govindarajan M, Khater HF, Panneerselvam C, Benelli G (2016) One-pot fabrication of silver nanocrystals using Nicandra physalodes: a novel route for mosquito vector control with moderate toxicity on nontarget water bugs. Res Vet Sci 107:95–101 Ishwarya R, Vaseeharan B, Anuradha R, Rekha R, Govindarajan M, Alharbi NS, Kadaikunnan S, Khaled JM, Benelli G (2017) Ecofriendly fabrication of Ag nanostructures using the seed extract of Pedalium murex, an ancient Indian medicinal plant: histopathological effects on the Zika virus vector Aedes aegypti and inhibition of biofilm-forming pathogenic bacteria. J Photochem Photobiol 174: 133–143. https://doi.org/10.1016/j.jphotobiol.2017.07.026 Ismail B, Nampoothiri KM (2010) Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510. Arch Microbiol 192(12): 1049–1057. https://doi.org/10.1007/s00203-010-0636-y Jayaseelan BD, Vaseeharan B, Maharajan A, Shanthi S, Vinoj G (2013) Vibrostatistic effects of probiotic B. licheniformis Dahb1 and its molecular phylogeny resolved through RAPD markers. Ann Microbiol 63(4):1601–1609. https://doi.org/10.1007/s13213-0130623-z Junter G, Thébault P, Lebrun L (2016) Polysaccharide-based antibiofilm surfaces. Acta Biomater 30:13–25. https://doi.org/10.1016/j.actbio. 2015.11.010 Kanmani P, Satishkumar R, Yuvaraj N, Paari KA, Pattukumar V, Arul V (2011) Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional characteristics activity in vitro. Bioresour Technol 102(7): 4827–4833. https://doi.org/10.1016/j.biortech.2010.12.118 Khaled JM, Al-Mekhlafi FA, Mothana RA, Alharbi NS, Alzaharni KE, Sharafaddin AH, Kadaikunnan S, Alobaidi AS, Bayaqoob NI, Govindarajan M, Benelli G (2018) Brevibacillus laterosporus isolated from the digestive tract of honeybees has high antimicrobial activity and promotes growth and productivity of honeybee’s colonies. Environ Sci Poll Res 25:10447–10455. https://doi.org/10. 1007/s11356-017-0071-6 Khater HF (2012) Prospects of botanical biopesticides in insect pest management. Pharmacologia 3:641–656 Khater HF (2013) Bioactivity of essential oils as green biopesticides: recent global scenario. In: Govil JN, Bhattacharya S, eds, vol 37. Essentials oils II. Recent progress in medicinal plants. Houston: Studium, 151–218 Khater HF (2017) Introductory Chapter: Back to the future: solutions for parasitic problems as old as pyramids. In: Khater H (ed) Natural remedies in the fight against parasites. InTech, Rijeka, pp 4–19 23 Khater H, Hendawy N, Govindarajan M, Murugan K, Benelli G (2016) Photosensitizers in the fight against ticks: safranin as a novel photodynamic acaricide to control the camel tick Hyalomma dromedarii (Ixodidae). Parasitol Res 115(10):3747–3758 Khater HF, Ali AM, Abouelella GA, Marawan MA, Govindarajan M, Murugan K, Abbas RZ, Vaz NP, Benelli G (2018) Toxicity and growth inhibition potential of vetiver, cinnamon, and lavender essential oils and their blends against larvae of the sheep blowfly, Lucilia sericata. Int J Dermatol 57(4):449–457 Kumar CG, Joo HS, Choi JW, Koo YM, Changa CS (2004) Purification and characterization of an extracellular polysaccharide from halo alkalophilic Bacillus sp. I-450. Enzym Microb Technol 34(7):673– 681. https://doi.org/10.1016/j.enzmictec.2004.03.001

Environ Sci Pollut Res Kumar A, Mody K, Jha B (2007) Evaluation of biosurfactant/ bioemulsifier production by a marine bacterium. Bull Environ Contam Toxicol 79(6):617–621. https://doi.org/10.1007/s00128007-9283-7 Larpin S (2002) Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider. Int J Food Microbiol 77(1–2):1–9 Lee YI, Seo TW, Kim JG, Kim KM, Ahn GS, Kwon SG, Park HY (1997) Optimization of fermentation conditions for production of exopolysaccharide by Bacillus polymyxa. Bioprocess Biosyst Eng 16(2):71–75. https://doi.org/10.1007/s004490050290 Lima LF, Habu S, Gern JC, Nascimento BM, Parada JL, Noseda MD (2008) Production and characterization of the exopolysaccharides produced by Agaricus brasiliensis in submerged fermentation. Appl Biochem Biotechnol 151(2–3):283–294. https://doi.org/10. 1007/s12010-008-8187-2 Lin ES (2010) Production of exopolysaccharides by submerged mycelial culture of Grifola frondosa TFRI1073 and their antioxidant and antiproliferative activities. World J Microbiol Biotechnol 27(3): 555–561. https://doi.org/10.1007/s11274-010-0489-1 Liu C, Lu J, Lu L, Liu Y, Wang F, Xiao M (2010a) Isolation, structural characterization and immunological activity of an exopolysaccharide produced by Bacillus licheniformis 8-37-0-1. Bioresour Technol 101(14):5528–5533. https://doi.org/10.1016/j. biortech.2010.01.151 Liu J, Luo J, Ye H, Sun Y, Lu Z, Zeng X (2010b) In vitro and in vivo antioxidant activity of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3. Carbohydr Polym 82(4):1278– 1283. https://doi.org/10.1016/j.carbpol.2010.07.008 Mahendran S, Saravanan S, Vijayabaskar P, Anandapandian KTK, Shankar T (2013) Antibacterial potential of microbial exopolysaccharide from Ganoderm alucidum and lysine Bacillus fusiformis. Int J Recent Sci Res 4:501–505 Molyneux P (2003) The use of the stable free radical diphenylpicrylhydrazy (DPPH) for estimating antioxidant activity. J Sci Technol 26(2):211–219 Murugan K, Priyanka V, Dinesh D, Madhiyazhagan P, Panneerselvam C, Subramaniam J, Suresh U, Chandramohan B, Roni M, Nicoletti M, Alarfaj AA, Higuchi A, Munusamy MA, Khater HF, Messing RH, Benelli G (2018) Predation by Asian bullfrog tadpoles, Hoplobatrachus tigerinus, against the dengue vector, Aedes aegypti, in an aquatic environment treated with mosquitocidal nanoparticles. Parasitol Res 114:3601–3610 Nazzaro F, Fratianni F, De Martino L, Coppola R, De Feo V (2013) Effect of essential oils on pathogenic bacteria. Pharmaceuticals 6(12): 1451–1474. https://doi.org/10.3390/ph6121451 Patel AK, Ahire JJ, Pawar SP, Chaudhari BL, Chincholkar SB (2009) Comparative accounts of probiotic characteristics of Bacillus spp. isolated from food wastes. Food Res Int 42(4):505–510. https://doi. org/10.1016/j.foodres.2009.01.013 Pavela R, Maggi F, Lupidi G, Mbuntcha H, Woguem V, Womeni HM, Barboni L, Tapondjou LA, Benelli G (2018) Clausena anisata and Dysphania ambrosioides essential oils: from ethno-medicine to modern uses as effective insecticides. Environ Sci Poll Res 25: 10493–10503. https://doi.org/10.1007/s11356-017-0267-9 Peralta MA, da Silva MA, Ortega MG, Cabrera JL, Paraje MG (2015) Antifungal activity of a prenylated flavonoid from Dalea elegans against Candida albicans biofilms. Phytomedicine 22(11):975–980. https://doi.org/10.1016/j.phymed.2015.07.003 Poli A, Anzelmo G, Nicolaus B (2010) Bacterial exopolysaccharides from extreme marine habitats: production, characterization and biological activities. Mar Drugs 8(6):1779–1802. https://doi.org/10. 3390/md8061779 Prior RL, Wu X, Schaich K (2005) Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary

supplement. Food Chem 53(10):4290–4302. https://doi.org/10. 1021/jf0502698 Rahman MM, Kim WS, Kumura H, Shimazaki KI (2008) Autoaggregation and surface hydrophobicity of bifidobacteria. World J Microbiol Biotechnol 24(8):1593–1598. https://doi.org/10. 1007/s11274-007-9650-x Rani RP, Anandharaj M, Sabhapathy P, Ravindran AD (2017) Physiochemical and biological characterization of novel exopolysaccharide produced by Bacillus tequilensis FR9 isolated from chicken. Int J Biol Macromol 96:1–10. https://doi.org/10. 1016/j.ijbiomac.2016.11.122 Roni M, Murugan K, Panneerselvam C, Nicoletti M, Madhiyazhagan P, Dinesh D, Suresh U, Khater HF, Wei H, Canale A, Alarfaj AA, Munusamy MA, Higuchi A, Benelli G (2015) Characterization and biotoxicity of Hypnea musciformis-synthesized silver nanoparticles as potential eco-friendly control tool against Aedes aegypti and Plutella xylostella. Ecotoxicol Environ Saf 121:31–38 Rosenberg M (2006) Microbial adhesion to hydrocarbons: twenty-five years of doing MATH. FEMS Microbiol Lett 262(2):129–134. https://doi.org/10.1111/j.1574-6968.2006.00291.x Sajna KV, Sukumaran RK, Gottumukkala LD, Jayamurthy H, Dhar KS, Pandey A (2013) Studies on structural and physical characteristics of a novel exopolysaccharide from Pseudozyma sp. NII 08165. Int J Biol Macromol 59:84–89. https://doi.org/10.1016/j.ijbiomac.2013. 04.025 Singh RP, Shukla MK, Mishra A, Kumari P, Reddy CRK, Jha B (2011) Isolation and characterization of exopolysaccharides from seaweed associated bacteria Bacillus licheniformis. Carbohydr Polym 84(3): 1019–1026. https://doi.org/10.1016/j.carbpol.2010.12.061 Sun HH, Mao WJ, Chen Y, Guo SD, Li HY, Qi XH, Chen YL, Xu J (2009) Isolation, chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicilliumsp.F23-2. Carbohydr Polym 78(1):117–124. https://doi.org/10.1016/j. carbpol.2009.04.017 Sundararajan LB, RanjithaKumari BD (2017) Novel synthesis of gold nanoparticles using Artemisia vulgaris L. leaf extract and their efficacy of larvicidal activity against dengue fever vector Aedes aegypti. J Trace Elem Med Biol 43:187–196. https://doi.org/10.1016/j.jtemb. 2017.03.008 Surendra TV, Roopan SM, Arasu MV, Al-Dhabi NA, Sridharan M (2016) Phenolic compounds in drumstick peel for the evaluation of antibacterial, hemolytic and photocatalytic activities. J Photochem Photobiol 161:463–471. https://doi.org/10.1016/j.jphotobiol.2016. 06.013 Sutherland IW (1972) Bacterial exopolysaccharides. Adv Microb Physiol 8:143–213 Tadhani MB, Patel VH, Subhash R (2007) In vitro antioxidant activities of Stevia rebaudiana leaves. J Food Compos Anal 20(3–4):323– 329. https://doi.org/10.1016/j.jfca.2006.08.004 Velusamy P, Das J, Pachaiappan R, Vaseeharan B, Pandian K (2015) Greener approach for synthesis of antibacterial silver nanoparticles using aqueous solution of neem gum (Azadirachta indica L.). Ind Crop Prod 66:103–109. https://doi.org/10.1016/j.indcrop.2014.12. 042 Vijayabaskar P, Babinastarlin S, Shankar T, Sivakumar T, Anandapandian K T K ( 2 0 11 ) Q u a n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f exopolysaccharides from Bacillus subtilis (MTCC 121). Adv Biol Res 5(2):71–76 Vijayakumar S, Vinoj G, Malaikozhundan B, Shanthi S, Vaseeharan B (2015) Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae. Spectrochim Acta A Mol Biomol Spectrosc 137:886–891. https:// doi.org/10.1016/j.saa.2014.08.064 Vinoj G, Vaseeharan B, Thomas S, Spiers AJ, Shanthi S (2014) Quorumquenching activity of the AHL-Lactonase from Bacillus

Environ Sci Pollut Res licheniformisDAHB1 inhibits Vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality. Mar Biotechnol 16(6):707–715. https://doi.org/10.1007/s10126-014-9585-9 Vinoj G, Rashmirekha P, Avinash S, Vaseeharan B (2015) In vitro cytotoxic effects of gold nanoparticles coated with functional AHL Lactonase protein from Bacillus licheniformis and its antibiofilm activity against Proteus species. Antimicrob Agents Chemother 59(2):763–771. https://doi.org/10.1128/AAC.03047-14. Wang Y, Li C, Liu P, Ahmed Z, Xiao P, Bai X (2010a) Physical characterization of exopolysaccharide produced by Lactobacillus plantarum KF5 isolated from Tibet Kefir. Carbohydr Polym 82(3): 895–903. https://doi.org/10.1016/j.carbpol.2010.06.013 Wang LQ, Meng XC, Zhang BR, Wang Y, Shang YL (2010b) Influence of cell surface properties on adhesion ability of bifidobacteria. World J Microbiol Biotechnol 26(11):1999–2007. https://doi.org/10.1007/ s11274-010-0384-9 Wang CL, Huang TH, Liang TW, Fang CY, Wang SL (2011) Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023, N. Biotechnol 28(6):559–565. https:// doi.org/10.1016/j.nbt.2011.03.003

World Health Organization (2005) Guidelines for laboratory and field testing of mosquito larvicides. Communicable disease control, prevention and eradication, WHO pesticide evaluation scheme, WHO, Geneva. WHO/CDS/WHOPES/GCDPP/1 3:2005 Ye S, Liu F, Jihui W, Wang H, Zhang M (2012) Antioxidant activities of an exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Carbohydr Polym 80(1):764–770. https://doi. org/10.1016/j.carbpol.2011.08.057 Yu KX, Wong CL, Ahmad R, Jantan I (2015) Larvicidal activity, inhibition effect on development, histopathological alteration and morphological aberration induced by seaweed extracts in Aedes aegypti (Diptera: Culicidae). Asian Pac J Trop Dis 8(12):1006–1012. https:// doi.org/10.1016/j.apjtm.2015.11.011 Zhang J, Cao Y, Wang J, Guo X, Zheng Y, Zhao W, Mei X, Guo T, Yang Z (2016) Physicochemical characteristics and bioactivities of the exopolysaccharide and its sulphated polymer from Streptococcus thermophilus GST-6. Carbohydr Polym 146(1):368–375. https:// doi.org/10.1016/j.carbpol.2016.03.063