Disinfectant-like activity of lipopeptide biosurfactant produced by Bacillus tequilensis strain SDS21

Colloids and Surfaces B: Biointerfaces 185 (2020) 110514

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Disinfectant-like activity of lipopeptide biosurfactant produced by Bacillus tequilensis strain SDS21

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Anil Kumar Singha, , Prakriti Sharmab a b

Department of Botany, Sant Baba Bhag Singh University, Jalandhar, Punjab, 144030, India College of Animal Biotechnology, Guru Angad Dev Veterinary And Animal Sciences University, Ludhiana, Punjab, 141004, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Antibacterial Biofilm Bactericidal Surfactin

Antiseptics and disinfectants are widely applied for eliminating microorganisms. However, microorganisms dwelling in the biofilm are less susceptible and in some cases resistant to biocide treatment. The present study describes isolation and characterization of lipopeptide biosurfactant exhibiting disinfectant-like activity. Biosurfactant was produced by an endo-rhizospheric bacterium Bacillus tequilensis strain SDS21. Biosurfactant reduced the surface tension of water from 72 to 30 mN/m with CMC of 40 mg/l. The Liquid Chromatography–Mass Spectrometry analysis of biosurfactant suggested it to be a mixture of C14, C15, C16 and C17 surfactin homologues. The lipopeptide biosurfactant exhibited bactericidal activity against planktonic cells and biofilm residing sessile cells. The biosurfactant treatment eradicated more than 99% of bacterial biofilm present on polystyrene, glass and stainless steel surface. The biosurfactant retained its bactericidal and biofilm eradicating activities even after exposure to extreme conditions like high temperate and extreme pH. Unlike some of the commonly used disinfectant, biosurfactant retained its bactericidal and biofilm removing activity even in the hard water containing Mg2+ and Ca2+ ions. Thus, suggesting that biosurfactant produced by strain SDS21 can be used as a disinfectant or in disinfectant-like formulations effective against both planktonic and biofilm residing bacteria.

1. Introduction Antiseptics and disinfectants are widely used in hospital and health care setting, food industries and households to kill or to limit the growth of microorganisms. Antiseptics are biocides that inhibit or destroy growth of microorganisms in or on living tissue [1] while disinfectants can be best described as chemical agents applied on inanimate objects to inactivate virtually all recognized pathogenic microorganisms [2]. Although biocide treatment eliminates most of the surface contamination, some microorganisms may survive and give rise to substantial problems. There are numerous reports exhibiting the survival of microorganisms even after cleaning and disinfectant treatment [3–5]. Most of these disinfectant resistant microorganisms remain associated with biofilm [3–5]. Cells residing within the biofilm differ from those of their planktonic counterparts, and display an increased resistance to biocide treatments [5,6]. An efficient disinfectant formulation must have biocide activity against both planktonic and biofilm dwelling cells [6]. Biofilms are known to cause bio-corrosion, decrease water quality, increase chances of recurrence of infection and major source of contamination for hygienic products [1]. Literatures suggest

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that apart from biofilm menace, efficacy of disinfectant is also influenced by several abiotic factors like pH, temperature, water hardness, concentration and duration of exposure to hostile conditions [7]. Most of the disinfectants currently being used are synthetic chemical with long half life or non-biodegradable in nature [1,6]. The release of biocides in the environment is a cause of concern as it can damage or disturb microbial ecology [8] and also provides a suitable environment for the microbes to develop resistance or tolerance against common biocides [6]. Thus, there is a need for new biocides with efficient biofilm eradication ability and easy degradation in nature for developing environment friendly disinfectants [6]. Biosurfactant with antimicrobial and biofilm dislodging activity have potential to be used in disinfectant formulation. OppenheimerShaanan et al. postulated that small molecules like biosurfactants can be used as natural agents for dispersion and disassembly of biofilm by acting as cell envelope-modifying or anti-matrix molecules [9]. Biosurfactants are better than synthetic surfactants as they have excellent surface active properties, biodegradability and activity even at adverse environmental conditions [10]. The aim of this work was to evaluate the heat stable lipopeptide

Corresponding author. E-mail address: [email protected] (A.K. Singh).

https://doi.org/10.1016/j.colsurfb.2019.110514 Received 4 May 2019; Received in revised form 25 August 2019; Accepted 17 September 2019 Available online 03 October 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

Colloids and Surfaces B: Biointerfaces 185 (2020) 110514

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incubated overnight at 4 °C. The precipitated biosurfactant was collected by centrifugation (15,000×g; 20 min) and dissolved in methanol. After evaporation of methanol using rotary evaporator, biosurfactant was lyophilised to obtain the off-white powder. The HPLC analysis of CFS and purified biosurfactant (dissolved in methanol) was performed using HPLC system equipped with a Phenomax-C18 column (5 μ, 250 mm x 4.6 mm).The mobile phase consisted of 90% methanol and 10% water (0.1% TFA). An aliquot of the sample (40 μl) was injected and analyzed using UV detector (Prominence UV–vis detector, Shimadzu, Japan) at 210 nm. Surfactin purchased from Sigma-Aldrich Co. USA, served as the standard (98% purity). Biosurfactant was subjected to LC–MS analysis by Agilent 6550 ifunnel Q-TOF LC/MS system (Santa Clara, CA, USA) following the earlier mentioned LC condition. Mass spectrometry conditions were 200–1800 m/z mass range, Dual AJS ESI as ion source, 9 l/min gas flow, 45 psi nebulizer pressure, 250 °C as drying gas temperature, and 3500 Vas nozzle voltage. Amino acid constituent of biosurfactant was determined by Waters Pico Tag method [15].

biosurfactant produced by an endo-rhizospheric bacterium for bactericidal and biofilm eradication activity so that it can be used as environment friendly disinfectant or in disinfectant-like formulations. Since the effect of a surfactant on biofilm may differ depending on both the type of microorganism and the type of surface, the present study considered three different surfaces namely polystyrene, glass and stainless steel for biofilm formation. Further, influence of abiotic factors on bactericidal and biofilm eradication activity of biosurfactant was studied to look into the potential application under realistic conditions. 2. Materials and methods 2.1. Microorganisms The biosurfactant producing microorganism used in the present study was isolated from endo-rhizosphere of wild plant Parthenium hysterophorus following the serial dilution plating method on Tryptone Soya Agar (TSA) plates. Ability of bacteria to reduce the surface tension of growth medium while growing on minimal salt medium (MSM) was taken as a criterion for selecting biosurfactant producer. The MSM (pH = 7.0 ± 0.5) used for screening and biosurfactant production was composed of 4 g/l NH4NO3, 4 g/l KH2PO4, 5.68 g/l Na2HPO4, 0.78 mg/l CaCl2, 197.18 mg/l MgSO4, 1.112 mg/l FeSO4 and 30 g/l sucrose. Biosurfactant producer was identified by morphological, biochemical and physiological method. Identity of biosurfactant producer was also confirmed by 16S rRNA gene sequencing [11]. The 16S rRNA gene sequence was compared with the sequences of cultured bacteria present in the EzTaxon Server [12] and NCBI GenBank database. The sequence obtained was further utilized for constructing phylogenetic tree by neighbor-joining algorithm of MEGA software version 6 [13]. The antimicrobial and biofilm eradicating activity of biosurfactant was tested against Gram-negative and Gram-positive bacteria obtained from Microbial Type Culture Collection (MTCC IMTECH, Chandigarh, India). All the bacteria were grown on TSA or TSB at 37 °C as per requirement.

2.3. Surface activity and critical micelle concentration (CMC) determination The surface, interfacial tension (against n-hexadecane) and CMC were measured at 25 °C using a duNouy tensiometer (CSC Scientific Company Inc., USA) based on ring detachment method [15]. 2.4. Antimicrobial and bactericidal activity of biosurfactant Minimum inhibitory concentration (MIC) of biosurfactant was estimated by micro-dilution method in 96-well flat bottom polystyrene tissue culture plates [16]. The MIC was defined as the lowest biosurfactant concentration that inhibited visible bacterial growth (OD at 600 nm) after 24 h of incubation at 37 °C. Minimum bactericidal concentration (MBC) of biosurfactant was determined following broth micro-dilution assay as per recommendation of Centers for Disease Control and Prevention, USA (2000). Minimum biosurfactant concentration that kills more than 99.0% of bacterial culture after 24 h of treatment was reported as MBC.

2.2. Production, purification and characterization of biosurfactant Biosurfactant production was carried out in Erlenmeyer flask (2 l) containing 500 ml aliquots of MSM at 30 °C with agitation of 200 rpm for 48 h. MSM was inoculated with 5 ml of seed culture (OD = 0.5 at 600 nm). Samples were withdrawn from flasks at regular interval. Withdrawn samples were used for determining bacterial growth in MSM, bacterial biomass, carbohydrate utilization and biosurfactant production. Bacterial growth in MSM was determined by measuring the colony forming unit per ml (cfu/ml). The samples were centrifuged (8000×g; 10 min; 4 °C) to obtain bacterial cell mass and cell free supernatant (CFS). Bacterial cell mass was washed with phosphate buffered saline (PBS; pH = 7.4) and dried (65 °C; 12 h) to obtain dry bacterial biomass. The biomass was weighed to obtain bacterial cell mass per liter of MSM. Carbohydrate utilization by the strain SDS21 was determined by estimating sucrose concentration in CFS. The CFS (1 ml) was mixed with Anthrone reagent (4 ml) and heated in boiling water bath for 10 min. After cooling, the absorbance was taken at 630 nm. Decrease in sucrose concentration was estimated by comparing absorbance of uninoculated MSM with the absorbance of CFS. Anthrone reagent was prepared by dissolving of 200 mg of anthrone in 100 ml ice cold concentrated H2SO4. Biosurfactant production was determined by measuring the surface tension of growth medium and High-performance liquid chromatography (HPLC) analysis of CFS. Various fermentation parameters were determined following earlier reported equations [14]. Earlier reported procedure and conditions were followed for purification of biosurfactant produced by strain SDS21 [15]. In brief, CFS obtained by centrifugation was acidified with 6 N HCl to pH 2 and

2.5. Time kill assay The bactericidal activity of biosurfactant at MBC was examined by challenging the cells (∼1 × 10 6) with biosurfactant and monitoring colony forming unit (cfu/ml) at different time intervals. Bacterial cells collected from log-phase and stationary-phase of growth was used for studying bactericidal activity. 2.6. Removal of biofilm present on polystyrene, stainless steel and glass surface Biofilm formation and quantification was performed on polystyrene flat-bottomed 96-well microtiter plate (BD, Falcon, USA) following earlier described method [17]. In brief, 100 μl of overnight grown bacterial cell suspension (1 × 106 cfu/ml) prepared in TSB were added into the wells and the cells were allowed to adhere for 6 h. The wells were decanted, washed with PBS and 200 μl of fresh TSB was added. The plates were incubated at 37 °C for 48 h. After incubation, the planktonic cells and weakly adherent cells were removed by PBS washing. Further, the wells with biofilm were treated with biosurfactant solution at different concentrations (0 mg/ ml, 0.250 mg/ml, 0.5 mg/ml, 2 mg/ml, 4 mg/ml and 6 mg/ml) and time intervals. After decanting biosurfactant solution, the plates were air dried and each well was stained with 200 μl of 1% crystal violet aqueous solution for 45 min. After staining, excess of dye was removed and washed with sterile water. After drying of wells, adherent dye was solubilized by 2

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200 μl of 95% ethanol. An aliquot of 100 μl from each well was transferred to a new microtiter plate and absorbance was taken at 595 nm for quantification of biofilm (Synergy 2 Multi-Mode Microplate Reader, BioTek, USA). Viability of dislodged and biofilm residing cells present on polystyrene surface was examined by cfu/ml determination. Biofilm formation and quantification on stainless steel coupons (type 304, with no.4 finish and surface area = 3 × 3 × 0.1 cm) was performed following earlier described method with certain modifications [5]. In brief, sterile stainless steel coupons were placed in the Petri dishes containing 25 ml of bacterial cell suspension (1 × 108 cfu/ml) in TSB. After incubation at 37 °C for 48 h, the samples were withdrawn aseptically and fresh TSB was added. Further Petri dishes were incubated for another 48 h. The coupons with biofilm were removed from TSB and treated with biosurfactant at different concentrations (0 mg/ ml, 0.250 mg/ml, 0.5 mg/ml, 2 mg/ml, 4 mg/ml and 6 mg/ml) and time intervals by dipping the coupons in biosurfactant solution. For quantifying biofilm after treatment, biofilm was removed from the coupons by swabbing it with sterile cotton and transferring it into 10 ml of 0.85% NaCl solution. After vigorous shaking, standard spread plating was performed on TSA plates. Plates were incubated at 37 °C for 24 h and cfu/ml was determined. Confocal Scanning Laser Microscopy (CSLM) and cfu/ml determination were applied to visualize the effect of biosurfactant treatment on bacterial biofilm present on the microscopic cover glass surface. Bacterial biofilm was formed on the glass surface by allowing bacterial cells to adhere to the surface. For adherence cover glasses were incubated in the bacterial cell suspension (1 × 108) prepared in TSB for 24 h at 37 °C. After incubation, glasses with adhering bacteria were dipped in fresh TSB and incubated at 37 °C for another 48 h for developing biofilm. Glasses were then treated with biosurfactant solution at different concentrations (0 mg/ml, 0.250 mg/ml, 0.5 mg/ml, 2 mg/ml, 4 mg/ml and 6 mg/ml) and time intervals. After treatment, cells present on cover glasses were fixed with 2% (v/v) glutaraldehyde in PBS for 10 min. Excess fixative was removed by washing it with PBS. The bacterial cells were stained with SYTO 9 (Excitation /Emission – 485 nm/500 nm) and Propidium iodide (Excitation /Emission –535/ 617 nm) purchased from Invitrogen, CA, USA. SYTO 9 stain was used to stain live as well as dead cells while Propidium iodide was used to visualize dead cells. Nikon A1R confocal microscope system (Nikon Instuments Inc. NY, USA) was used for visualization. Multiple (5) images were scanned and analyzed using the image processing software NIS-Element AR. The representative images are presented here. Biofilm residing cells present on the glass surface after biosurfactant treatment was also examined by cfu/ml determination.

Hydrophobicity (CSH) by determining percentage adherence to hexadecane [18]. 2.9. Statistical analysis All statistical parameters were estimated using the Microsoft Excel 2007. The variation in the observation was recorded as means ± SD. The statistical significance of the results were calculated using Twoway ANOVA, of treated samples in comparison to untreated samples, significances values being annotated as ***= < 0.001. 3. Results and discussion 3.1. Identification of biosurfactant producer A total of 4 morphologically distinct endo-rhizospheric bacteria were isolated from the roots of P. hysterophorus. Screening of endorhizospheric bacterial isolates for biosurfactant production resulted in isolation of strain SDS21. Strain was able to reduce the surface tension of sucrose supplemented MSM from 64 mN/m to 30 mN/m within 24 h of growth. Thus, indicating strain SDS21 as a biosurfactant producer. The strain SDS21 is a Gram positive, rod shaped, facultative anaerobic, spore-forming and flagellated bacterium. The strain SDS21 exhibited growth in the range of temperature 15 °C–45 °C (optimum 30 °C), pH values 5–9 (optimum pH 7.0) and salt concentration (NaCl %) up to 5%. Biochemical analysis demonstrated that strain SDS21 is positive for catalase, oxidase, hydrolysis of gelatin, starch, casein, indole and citrate utilization. Strain SDS21 is negative for methyl red, Voges-Proskauer, nitrate reduction and urea hydrolysis. Acid production was observed from carbohydrates namely arabinose, ribose, adonitol, galactose, dextrose, fructose, maltose, dulcitol, sorbitol, mannose, rhamnose, sucrose and trehalose. Results of biochemical and physiological tests of strain SDS21 were similar to strain 10bT thus suggesting the strain SDS21 to be Bacillus tequilensis [19]. Further, 16S rRNA gene sequence comparison with the type strains available in EzTaxon Database and NCBI GenBank Database confirmed the bacterium to be Bacillus tequilensis (Gen Bank accession number: KJ559528). Phylogenetic 16S rRNA gene sequence comparison using neighbor joining tree method suggested a close relationship between SDS21 and Bacillus tequilensis 10bT (HQ223107) (Fig. 1A). 3.2. Biosurfactant production and characterization Strain SDS21 exhibited luxuriant growth in MSM which was accompanied by decrease in surface tension of growth medium and sucrose concentration (Fig. 1B). Maximum decrease in surface tension was observed during early exponential growth phase. Table 1 shows main kinetic results of biosurfactant production by B. teqelensis SDS21. Strain SDS21 was able to utilize 30.67 ± 1.87% of available carbon source and produce 1879.43 ± 30.4 mg/l of biosurfactant after 40 h of growth. After 40 h of incubation, growth and biosurfactant production was inhibited even though the carbon source was available in MSM. This may be due to limitation of nitrogen source needed for bacterial metabolism. Appropriate carbon to nitrogen ratio is important for bacterial growth and production of bioactive molecules. Also accumulation of toxic waste product at the end of fermentation may inhibit growth and biosurfactant production [14]. Bacillus sp. produces lipopeptides biosurfactant as a mixture of different species or isoforms [15]. Surfactin is well studied lipopeptide type biosurfactant [14,15]. In the present study, HPLC chromatogram of purified biosurfactant and standard surfactin was compared. The chromatogram of purified biosurfactant and standard surfactin was almost similar (Supplementary Fig. 1). Thus, suggesting microbial surfactant produced by strain SDS21 to be surfactin-like biosurfactant. As determined by HPLC, the purity of biosurfactant obtained after purification steps was 88.0 ± 4.0% with respect to standard surfactin

2.7. Effect of abiotic factors on bactericidal and biofilm eradication activity of biosurfactant Effect of heat on bactericidal and biofilm eradication activity of biosurfactant was studied by boiling biosurfactant solution at 100 °C for 3 h and then testing its various activities following the procedure performed for untreated biosurfactant solution. Effect of pH was studied by challenging biosurfactant solution to extreme pH range of 5–12 for 3 h. After incubation at 25 °C, pH was neutralized to 7.0 ± 1.0 and scrutinized for its activity. The 6 N NaOH and 6 N HCl was used for changing the pH of biosurfactant solution. For studying the effect of hard water on biosurfactant, aqueous solution was prepared in hard water (pH = 8.0 ± 0.5) containing Mg2+ (108 ppm) and Ca2+ (114 ppm). Further, this solution was used for studying bactericidal and biofilm removing activity. 2.8. Bacterial cell surface hydrophobicity Biofilm formation was carried out in 6 well polystyrene plates following above mentioned conditions. Cells removed by biosurfactant or PBS treatment were collected and examined for its Cell Surface 3

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Fig. 1. (a) The evolutionary relationships of Bacillus tequilensis strain SDS21. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the association taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of evolutionary distance used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum composite Likelihood method and are in the units of the number of bases substitutions per site. Evolutionary analyses were conducted in MEGA 6 [13]. (b): Time course of growth, surface tension reduction and total carbohydrate utilization by strain SDS21. Growth was carried out at 30 °C with agitation of 200 rpm. Values given are mean ± SD of three independent experiments.

Mass peaks differ by interval of 14 Da, thus suggesting that biosurfactant has different number of methylene groups in fatty acid chain of lipid moiety [20]. Comparison of molecular weight with those available in literature suggests that biosurfactant is a mixture of surfactin-like homologues namely C14-surfactin, C15-surfactin, C16-surfactin, and C17surfactin [20]. Biosurfactant produced by strain SDS21 has C14-surfactin as the most abundant homologue while C15-surfactin was the least abundant homologue. Amino acid analysis of biosurfactant exhibited presence of asparitic acid, glutamic acid, valine and leucine in ratio 1:1:1:4. These amino acids are known to be present in surfactin [15]. B. teqelensis has been reported to produce cyclic lipopeptide type biosurfactant [21–23]. However, production of C17-surfactin like homologue has been rarely reported [23]. Surfactin is regarded as a most efficient biosurfactant [14,15]. Efficient biosurfactant reduces the surface tension of water to minimum with minimum amount. Thus, amount of surfactant required to reduce surface tension would be less for efficient biosurfactant. The purified biosurfactant from strain SDS21 reduced the surface tension of water from 72 to 30 mN/m with CMC of 40 mg/l. The interfacial tension of 1 mN/m with n-hexadecane was observed for biosurfactant. Surfactants that can reduce the surface tension of water below 35 mN/m are generally regarded as efficient biosurfactant [15].

Table 1 Main kinetic parameters of biosurfactant production by strain SDS21. Fermentation Parameters

Equation

Calculated Value

Substrate conversion

ΔS (%) = (S0-S/S0) X 100 X max P max

30.67 ± 1.87 1124.04 ± 15.0 mg/l 1879.43 ± 30.4 mg/l

Pp = P max /tpmax PX = X max /txmax Yp/x = Pf-P0/Xf - X0

46.98 ± 0.76 mg/l/h 28.1 ± 0.3 mg/l/h 1.70 ± 0.05

Maximum biomass concentration Maximum biosurfactant concentration Volumetric Surfactin productivity Volumetric biomass productivity Specific biosurfactant yield

Initial substrates concentration (S0); Final Substrate concentration (S); Maximum biosurfactant concentration (P max); Maximum biomass (X max); Time for attaining maximum biosurfactant yield (tpmax); Time for attaining maximum biomass (txmax); Product concentration at particular time (Pf); Biomass concentration at particular time (Xf); Product concentration at time zero (P0); Biomass concentration at time zero (X0).

(≥98%, Sigma). The LC–MS analysis of biosurfactant produced by strain SDS21 exhibited four prominent peaks at m/z ratio of 1022.67, 1036.69, 1050.70 and 1064.72 (Fig. 2). These peaks may be attributed to [M+H] + ions.

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Table 2 Minimum inhibitory concentration and Minimum bactericidal concentration of biosurfactant produced by Bacillus tequilensisstrain SDS21 (Three independent experiments with three replicates were performed to determine MIC and MBC) Bacterial Strains

Escherichia coli V517 MTCC 131 Pseudomonas aeruginosa MTCC 4306 Staphylococcus aureus MTCC 3160 Staphylococcus epidermidis MTCC 435 Salmonella typhi MTCC 733 Salmonella typhimurium MTCC 98

Biosurfactant concentration (mg/ml) MIC

MBC

2.0 2.0 0.5 4.0 4.0 4.0

4.0 4.0 1.0 6.0 6.0 6.0

MIC: Minimum inhibition concentration. MBC: Minimum bactericidal concentration.

However, concentrations above MIC become lethal and microorganisms cannot be revived even if transferred to an antimicrobial free growth supporting medium [16]. Biosurfactant at lower concentrations inhibited bacterial growth while higher concentrations proved lethal to the target bacteria. Biosurfactant concentration of 6 mg/ml demonstrated bactericidal activity against all the tested bacteria. Biosurfactant with longer fatty acid chain are more effective in interacting and damaging cell membranes than shorter chain homologues. Biosurfactant from strain SDS21 has longer fatty acid homologues hence exhibited efficient bactericidal activity. Challenging bacterial cells with MBC of biosurfactant for 1 h reduce the cfu/ml by more than 90% (Fig. 3). Contrary, bacterial cells suspended in PBS (without biosurfactant) did not exhibit any significant decrease in cfu/ ml even after 1 h of incubation (n = 15; P > 0.1). Interestingly, no significant difference was observed in the bactericidal activity of surfactin against log and stationary phase bacterial cells (n = 15; P > 0.1). Usually stationary phase cells are less susceptible to biocides than log phase cells since most of the biocides target enzymes that are highly expressed during cell division, including those involved in cell-wall synthesis, DNA synthesis, metabolism and protein synthesis [26]. Surfactin being amphiphile molecule targets cellular membrane causing cell lysis and this mechanism is not significantly influenced by the bacterial growth phase [27]. Biocides with membrane damaging properties have lesser chance of resistance development in microbes and hence, offers promising alternative against emergence of biocide resistance. Antimicrobial lipopeptide from Bacillus species has been reported to be effective against both Gram-positive and Gramnegative bacteria [28–30]. Biosurfactant produced by B. circulans inhibited growth of multi-drug resistant pathogenic bacteria like E. coli and Klebsiella pneumoniae at concentration of 200 μg/ml and 60 μg/ml,

Fig. 2. Liquid Chromatography–Mass Spectrometry (LC–MS) chromatogram analysis of purified biosurfactant obtained from Bacillus tequilensis strain SDS21. (A) C15-surfactin (B) C14-surfactin (C) C16-surfactin (D) C17-surfactin.

3.3. Antibacterial and bactericidal activity of biosurfactant Bacteria (Escherichia coli V517 MTCC 131, Pseudomonas aeruginosa MTCC 4306, Staphylococcus aureus MTCC 3160, Staphylococcus epidermidis MTCC 435, Salmonella typhi MTCC 733 and Salmonella typhimurium MTCC 98 selected for the study are either opportunistic or conditional pathogen very often associated with biofilm related nuisance that affect industry and public health [24,25]. Therefore, removing these bacteria from food processing plants, medical implants and devices, public drinking water system and many other places are important issues that deserve full consideration for public well being. As evident from Table 2 purified biosurfactant from strain SDS21 exhibited growth inhibition and bactericidal activity against both Gram-positive and Gram-negative bacteria. The MIC value was observed in the range of 0.5 mg/ml to 4 mg/ml depending upon the bacteria. The antimicrobials at MIC may act as a bacteriostatic agent.

Fig. 3. Bactericidal activity of biosurfactant at MBC. Log phase bacterial cells were challenged with MBC of biosurfactant at 37 °C with agitation of 150 rpm. (a) E. coli V517 MTCC 131, (b) P. aeruginosa MTCC 4306, (c) S. aureus MTCC 3160, (d) S. epidermidis MTCC 435, (e) S. typhi MTCC 733 and (f) S. typhimurium MTCC 98. Values given are mean ± SD of three independent experiments (n = 15). The Two- way ANOVA, was used to determine if there was a statistically significant difference between the treated and untreated samples (*** = P < 0.001). 5

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respectively [28]. Das et al. observed that 200 μg/ml and 500 μg/ml of biosurfactant executed growth inhibition and bactericidal activity against Methicillin-resistant Staphylococcus aureus (MRSA) [28]. Liu et al. postulated use of surfactin in combination to other antibiotic as an efficient means to combat pathogen [27]. Recently, Garg et al. reported that biosurfactant produced by Candida parapsilosis exhibited antibacterial activity against E.coli and S. aureus at concentration of 10 mg/ ml and 5 mg/ml, respectively [31]. 3.4. Biofilm eradication by biosurfactant Three different methods (namely crystal violet staining method, confocal scanning laser microscopy and cfu/ml estimation) commonly used in biofilm dislodging study were used in the present study [5,17,28,32]. Crystal violet staining method is handy and simple method of estimating anti-adhesive and anti-biofilm activity of a substance on polystyrene surface [17]. Biofilm quantification by colony forming unit is relatively laborious but superior method as it also indicates viability of bacterial cells [5,32]. Confocal scanning laser microscopy provided visual evidence of biofilm eradication activity of a substance [32]. Disrupting the multi-cellular structure of bacterial biofilm has been proposed as the most promising strategy for increasing the sensitivity of pathogens in biofilm to bactericidal agents and host immune systems [9]. Presence of extracellular polymeric substances (EPS) in the biofilm provides resistance to bactericidal agent by limiting the diffusion of compounds. Efficient biofilm eradicating agent must diffuse through EPS and penetrate between the sessile cells and adhering surface, and modify cell surface properties to induce disruption or dislodging of biofilm [9]. Biofilm eradication efficiency of a compound depends on several biotic and abiotic factors like chemical nature of the compound, target microorganism and surface on which biofilm has been formed. In the present study extent of biofilm developed by each bacterium was observed to be surface and microorganism dependent. Among all the bacteria used in the present study E. coli V517 MTCC 131 and P. aeruginosa MTCC 4306 exhibited good biofilm development on all the surfaces i.e glass, stainless steel and polystyrene. Glass and stainless steel are common materials used in the hospital, households and food industries. While polystyrene is the most common material applied in laboratory for biofilm studies. Biosurfactant treatment was able to remove biofilm from glass, stainless steel and polystyrene surfaces. Removal of biofilm by biosurfactant was observed to be concentration, time and microorganism dependent. Treating polystyrene surface with biosurfactant (4 mg/ml) for 3 h removed more than 99% of the bacterial biofilm in comparison to PBS treated biofilm (Fig. 4A). Most efficient biofilm removal was observed in S. aureus MTCC 3160 where 0.5 mg/ml of biosurfactant removed more than 90% of biofilm and also dislodged cells were not viable. Cell removed by biosurfactant at lower concentrations (less than 4 mg/ml) were viable unlike the cells dislodged at higher biosurfactant concentration (6 mg/ml). More than 99.0% of the cells removed by 6 mg/ ml of biosurfactant solution were not viable. Thus, suggesting that 6 mg/ml of biosurfactant has bactericidal effect on both the sessile cells of biofilm and the planktonic cells. Rivardo et al. have reported biofilm inhibition by biosurfactant produced by Bacillus spp [32]. Lipopeptide biosurfactant produced by Bacillus spp has been reported to dislodge biofilm present on polystyrene surface [29,30]. Polystyrene surface pretreated with lipopeptide biosurfactant reduced bacterial adherence to the surface and thus acted as an anti-biofilm agent [30]. Biosurfactant produced by Paenibacillus polymyxa reduced the biofilm in single and mixed species biofilm [33]. As evident from cfu/ ml count, challenging preformed biofilm present on stainless steel with biosurfactant (4 mg/ml) removed significant (n = 15; P < 0.001) amount of bacteria (except S. aureus MTCC 3160 where 0.5 mg/ml of biosurfactant was observed to be effective) (Fig. 4B). Interestingly, more than 99.0% of biofilm cells removed by

Fig. 4. The effect of biosurfactant treatment on biofilm present on the (A) polystyrene surface (B) stainless steel coupons and (C) glass surface. Biosurfactant treatment was carried out for 3 h at 25 °C. (a) E. coli V517 MTCC 131, (b) P. aeruginosa MTCC 4306, (c) S. aureus MTCC 3160, (d) S. epidermidis MTCC 435, (e) S. typhi MTCC 733 and (f) S. typhimurium MTCC 98. Values given are mean ± SD of three independent (n = 15) experiments. The Two- way ANOVA, was used to determine if there was a statistically significant difference between the treated and untreated samples (*** = P < 0.001).

6 mg/ml of biosurfactant were not viable indicating bactericidal activity against biofilm. Simoes et al. exhibited that the synergistic action of synthetic cationic surfactant and application of high shear stress is required to eradicate the biofilm from stainless steel surface [34]. However, in the present study, simple biosurfactant treatment was sufficient to eradicate biofilm highlighting biosurfactant effectiveness as an anti-biofilm agent. As evident from Fig. 4C, treating biofilm present on glass surface with biosurfactant removed significant amount of biofilm. This was also confirmed and visualized by CLSM (Fig. 5). Additional the CLSM images suggested that the cell adhering to the glass surfaces were viable as no Propidium iodide stained bacterial cells were observed. Most efficient biofilm eradication was observed in S. aureus MTCC 3160 where 0.5 mg/ml of biosurfactant treatment for 3 h removed considerable amount of biofilm whilst for other bacterial biofilm 2 mg/ml of surfactin was observed to be effective. Challenging bacterial biofilm with 6 mg/ml of biosurfactant for 3 h not only removed biofilm but also proved lethal to the biofilm dwelling cells as more than 99.0% of cells lost its viability. Dusane et al. observed that biosurfactant is more efficient in eradicating Yarrowia lipolytica biofilm from glass surface as compared to chemical surfactants [35]. Biosurfactant, cetyl-trimethyl ammonium bromide and sodium dodecyl sulfate disrupted 76%, 38%

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Fig. 5. Confocal microscopic images of bacterial biofilm present on cover glass slides. Control were washed with PBS (pH = 7.2) while treated were challenged with 2 mg/ml biosurfactant solution (except S. aureus MTCC 3160 that was treated with 0.5 mg/ml surfactin solution) for 3 h at 25 °C (Scale bar 10 μm).

biofilm dislodging efficiency of biosurfactant. Both the activities were very similar to the biosurfactant solution prepared in the normal water (n = 15; P < 0.1). Though change in pH and presence of mono/divalent cations is known to modify the lipopeptides structures in bulk and interfacial phase [37]. Thimon et al. have reported that presence of aspartate and glutamate residues in surfactin facilitates binding of divalent metals ions like magnesium, manganese, calcium, barium, lithium and rubidium [38]. However, observation made in the present study suggests that conformational changes induced by divalent metal ions do not have any significant influence on bactericidal and biofilm eradication efficiency of biosurfactant.

and 53% of Y. lipolytica biofilm at sub-MIC concentration, respectively [35]. 3.5. Effect of pH, temperature and hard water on bactericidal and biofilm eradication activity of biosurfactant Generally, bactericidal agents are heat liable and often lose it activity upon exposure to high temperature during storage or transportation [7]. Very often heat degraded disinfectant produces potential health hazards for human. On the contrary, biosurfactant from strain SDS21 was found to retain its bactericidal and biofilm eradicating activity even after prolong heat exposure, thus making it an interesting heat stable antimicrobial. The activity difference observed between the heat treated and un-treated biosurfactant solution was not significant (n = 15; P < 0.1). This observation may be attributed to strong bond present between heptapeptide and β-hydroxy fatty acid chain to form cyclic lactone ring structure. Bactericidal and biofilm eradicating activity of biosurfactant was also not influenced by exposing it to extreme pH range of 5–12. No significant change in the bactericidal and biofilm dislodging activity was observed between treated and untreated biosurfactant solution (n = 15; P < 0.1). Some commonly used disinfectants (like phenols, hypochlorites) are known to loss it antimicrobial activity upon exposure to higher pH [7]. Hard water is known to reduce efficacy of several disinfectants like Quaternary ammonium compounds, phenols. Divalent cations (like Mg2+ and Ca 2+) present in the hard water interact with the disinfectant to form insoluble precipitates, thus reducing its bactericidal activity [36]. In the present study, use of hard water for solution preparation did not have any considerable effect on bactericidal and

3.6. Effect of biosurfactant on cell surface hydrophobicity (CSH) Biosurfactants can influence surface properties of bacterial cell and substrate thus influencing structural integrity of biofilm [39]. In the present study, cell removed by biosurfactant from polystyrene surface had decreased CSH as compared to the cells removed by PBS (Fig. 6). This suggests that biosurfactant mediated cell surface modification may have contributed extensively in removing bacterial cells from biofilm. Bacterial cells removed from biofilm may sometime again re-adhere to the surface. However, this may not be possible after biosurfactant treatment as cell surface property of dislodged bacteria were not suitable for adherence. Bacterial cells with high CSH favors adherence to the polystyrene surface. According to Rodrigues et al. biosurfactant treatment reduces hydrophobic interactions between bacterial cell and solid surfaces, consequently microbial adhesion [39]. Hydrophobic interaction favors microbial colonization of surfaces as it facilitate the close approach between microorganism and solid substratum, favoring the elimination of interfacial water present in the interacting surfaces. 7

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Fig. 6. Bacterial cell surface hydrophobicity of cells dislodged from biofilm. (a) E. coli V517 MTCC 131, (b) P. aeruginosa MTCC 4306, (c) S. aureus MTCC 3160, (d) S. epidermidis MTCC 435, (e) S. typhi MTCC 733 and (f) S. typhimurium MTCC 98. Values given are mean ± SD of three independent experiments (n = 15). The Two- way ANOVA, was used to determine if there was a statistically significant difference between the treated and untreated samples (*** = P < 0.001).

Orientation of biosurfactant during adsorption on the cell surface decides modification of hydrophobic region into hydrophilic or hydrophilic into hydrophobic region [39]. During biosurfactant mediated eradication lipopeptide may have adhered to the bacterial cell with its hydrophobic end embedded into the cell surface and hydrophilic end protruding free into the surface thus making the dislodged cells hydrophilic. 4. Conclusions The present work demonstrated that Bacillus tequilensis strain SDS21 is an efficient lipopetide type biosurfactant producer. Biosurfactant from strain SDS21 exhibited heat and pH stable bactericidal and biofilm dislodging activity against several opportunistic pathogenic bacteria. Biocide activity of biosurfactant was retained even in the hard water. Biofilm eradication ability of biosurfactant was equally effective against the biofilm present on polystyrene, glass and stainless steel surface. Thus, suggesting that biosurfactant from strain SDS21 can be used as an environment friendly microbial product in preparing heat and pH stable disinfectant or disinfectant formulations efficient even in the hard water against bacterial colonization. Acknowledgements The authors thank the SBBSU, Punjab for laboratory facilities. Authors also acknowledge Director CSIR-IMTECH, Chandigarh for CLSM and LC–MS. References [1] G. McDonnell, A.D. Russell, Antiseptics and disinfectants: activity, action, and resistance, Clin. Microbiol. Rev. 12 (1999) 147–179. [2] Centers for Disease Control and Prevention, USA, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved standard M7-A5. Wayne, PA (2000). [3] H. Wang, L. Cai, Y. Li, X.Xu G. Zhou, Biofilm formation by meat-borne Pseudomonas fluorescens on stainless steel and its resistance to disinfectants, Food Control 91 (2018) 397–403. [4] A. Charlebois, M. Jacques, M. Boulianne, M. Archambault, Tolerance of Clostridium perfringens biofilms to disinfectants commonly used in the food industry, Food Microbiol. 62 (2017) 32–38. [5] J. Krolasik, Z. Zakowska, M. Krepska, L. Klimek, Resistance of bacterial biofilms formed on stainless steel surface to disinfecting agent, Pol. J. Microbiol. 59 (2010) 281–287. [6] A. Bridier, R. Briandet, V. Thomas, F. Dubois-Brissonnet, Resistance of bacterial biofilms to disinfectants: a review, Biofouling 27 (2011) 1017–1032. [7] W.A. Rutala, D.J. Weber, Current principles and practices; new research; and new technologies in disinfection, sterilization, and antisepsis, Am. J. Infect. Control 41 (2013) S1.

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4427–4435. [38] L. Thimon, F. Peypoux, G. Michel, Interactions of surfactin, a biosurfactant from Bacillus subtilis, with inorganic cations, Biotechnol. Lett. 14 (1992) 713–718. [39] L.R. Rodrigues, J.A. Teixeira, Biomedical and therapeutic applications of biosurfactants, Adv. Exp. Med. Biol. 672 (2010) 75–87.

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