Synthesis and evaluation of antibacterial activity of quaternized biopolymer from Klebsiella terrigena

Authors


Abstract

Aims

Microbial exopolymer with antimicrobial properties, in particular, has gathered considerable interest due to their enormous scope of modification and wide gamut of application. The purpose of present study was to evaluate the antibacterial spectrum of a chemically modified biopolymeric flocculant produced by Klebsiella terrigena.

Methods and Results

N,N,N trimethyl biopolymer (TMB) was synthesized using dimethyl sulfate as methylating agent and was characterized by nuclear magnetic resonance and mass spectroscopic analysis, which confirmed the presence of quaternary ammonium groups on the TMB structure. The antibacterial activity of TMB was investigated against three selected bacterial pathogens viz. Aeromonas hydrophila ATCC 35654, Listeria monocytogenes ATCC 19111 and Escherichia coli O157:H7 ATCC 32150. An inactivation of 3 log CFU ml−1 of all pathogens was noticed for TMB when compared to native polymer over a short contact time (60 min) and low dosage (60–80 μg ml−1) at ambient temperature. A marked increase in glucose level, protein content and lactate dehydrogenase (LDH) activity was observed concurrently in the cell supernatant suggesting damage of the cell membranes to be a possible reason for inactivation.

Conclusions

The quaternization of amino rich biopolymer isolated from a bacterium led to a water-soluble bioactive agent with enhanced inhibitive capability against all the selected bacterial pathogens.

Significance and Impact of Study

The results of this study suggest a potential application of TMB as an effective disinfectant in water treatment.

Introduction

Infections by microbial pathogens are of great concern in many fields, including in healthcare products, food packaging and water purification systems. (Munoz-Bonilla and Fernandez-Garcia 2012). Bacterial pathogens are becoming resistant faster than the rate at which new microcides are being made available, leaving bacterial disease as major cause of concern in many parts of the world (Cabral 2010). The antibacterial agents used as disinfectants are able to keep bacteria, viruses and fungi in check, but the chemicals found in these products may pose environmental and health risks (Werschkun et al. 2012). Hence, there is an urgent need to develop strong, economically viable and ecofriendly replacements of conventional disinfectants.

Distinctive nature of microbial extracellular polymers containing amino groups can be explored in hope to obtain effective, economically viable and safe substitute to chemical biocides. Microbial exopolymers are produced by micro-organisms during growth, including glycoprotein, polysaccharide, cellulose and DNA, and have the advantage of being safe, nontoxic, biodegradable and environment-friendly (Kurane et al. 1986; Yokoi et al. 1997). Cationic biopolymers containing quaternary ammonium groups have gained wide attention in the recent years (Carmona-Ribeiro et al. 2013; Tan et al. 2013). It has been known for many years that quaternary ammonium compounds (QACs) are membrane-active agents with a target site, predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane (McDonnell and Russell 1999). Well-known biopolymers such as chitosan have been modified through various techniques. Despite the extensive research over the last decade, the exact mode of action of quaternized biopolymers is not yet fully understood (Siedenbiedel and Tiller 2012).

The biopolymer produced by Klebsiella terrigena has already been reported to be effective in removal through flocculation of Cryptosporidium oocysts and Salmonella (Ghosh et al. 2009a,b). In the present study, the biopolymer has been chemically modified to develop antibacterial property in the polymer. The amino group of the biopolymer was quaternized using dimethyl sulfate as methylating agent and interestingly, the quaternized biopolymer possessed excellent antibacterial activity against all the three selected pathogens. Our research has basically focused on the possibility of developing biopolymer as a natural antibacterial agent. It can be used as a disinfectant for water treatment.

Materials and methods

All the chemicals were purchased from Sigma chemicals company (Sigma, St. Louis, MO) and were of the highest grade available commercially. Standard media components were purchased from Hi-Media (Mumbai, India).

Bacterial cultures and growth conditions

Biopolymer producing strain

Klebsiella terrigena (Accession number EU082029) was used in this study (Ghosh et al. 2009a). The strain was preserved in glycerol stock solutions at −80°C. Bacterial cells were grown in 1 l of medium (Polypeptone 5 g l−1, diammonium sulfate 2 g l−1, yeast extract 1 g l−1, CaCl2 0·7 g l−1, NaCl 0·1 g l−1, MgSO4 2 g l−1, K2HPO4 1 g l−1, glucose 1 g l−1, agar 3 g l−1) on a rotary shaker (120 rpm min−1) at 30°C for 48 h.

Indicator strains

Aeromonas hydrophila ATCC 35654, Listeria monocytogenes ATCC 19111 and Escherichia coli O157:H7 ATCC 32150 were grown in brain heart infusion (BHI) broth by incubating at 37°C, with agitation for 6–8 h at 120–150 rpm.

Biopolymer production

The culture was centrifuged at 12 000 g at 4°C for 20 min; biopolymer was separated by the addition of two volumes of ethanol (99·5%) to 500 ml of concentrated supernatant, and allowed to precipitate at 40°C for 24 h. The precipitated polymer was further recovered and purified as described by Ghosh et al. (2009a,b).

Preparation of antibacterial compound

The biopolymer was dissolved in dimethylsulfate (2·4 ml) and deionized water (0·6 ml). The solution was then filtered to eliminate the impurities. Sodium hydroxide (0·18 mg) and sodium chloride (0·132 mg) were added to the resulting suspension, and solution was stirred at ambient temperature for 6 h. The product was precipitated using acetone, filtered and vacuum-dried. White precipitates obtained were redissolved in deionized water (20 ml) and purified through dialysis method using a dialysis tubing (Cellulose, MWCO 12 000 Da) for 1 day. Dialyzed solution was subjected to lyophilization to get white fluffy powder (4·5 mg) (Belalia et al. 2008).

NMR Analysis

1H NMR spectra of biopolymer and N, N, N-trimethyl derivative (TMB) were recorded using Bruker Avance II (400 MHz) spectrometer. For this analysis, samples were dissolved in D2O.

Molecular weight determination

Molecular weight determination of biopolymer and TMB was performed with a Bruker Daltonics MALDI-TOF mass spectrometer. The MALDI matrix used was 2, 5-dihydroxybenzoic acid (DHB).

Compositional analysis

The total sugars, neutral sugar, uronic acids, amino sugar content and pyruvic acid of the quaternized biopolymer were performed as described by Yokoi et al. (1997). Elemental analysis was carried out with a 2400 II elemental analyser (Perkin Elmer Company, Bedford, MA). Fractionation and purification were achieved using gel chromatography on a Sepharose 4B column followed by elution with a 0·4 mol l−1 NaCl buffer. The ultrastructure of purified TMB was observed using a scanning electron microscopy (JSM 541-V, JEOL, Tokyo, Japan) (results not shown).

Evaluation of antibacterial activity

Agar disc diffusion assay

The uniform sized discs (6 mm) prepared from Whatman filter paper no. 1 were soaked in sterile solution of 1 mg ml−1 of each native and N, N, N trimethyl biopolymer. These soaked discs were then placed on the assay plates containing 100 μl of indicator bacterial cultures on BHI agar. The plates were incubated at 37°C for 12 h, after which the diameters of inhibition zones were observed. The experiments were performed in triplicates.

Inhibition kinetics

Antibacterial activity of TMB was assayed by microdilution method, using a sterile 96-well-microtiter plate reader (Bioscreen C, Thermo labsystems, Helsinki, Finland). Briefly, serial twofold dilutions of TMB solutions were prepared in the appropriate culture medium in sterile 96-well round bottom polystyrene microtiter plates (Raafat et al. 2008). Final TMB concentrations used were 1–100 μg ml−1. The indicator strains were grown in the respective broth at 37°C to an optical density of 1 at 600 nm and subsequently diluted in the same medium to about 10CFU ml−1. The solution of biopolymer and TMB was added, and minimal inhibitory concentration (MIC) was determined (Andrews 2001) for 3 h.

Minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound required to completely inhibit microbial growth after incubation. The MIC determinations were carried out in triplicates, with two independent experiments performed. The surviving log10 CFU ml−1 was plotted against time for each of the different quaternized biopolymer concentrations.

Thin agar layer method

The thin agar layer (TAL) method as described by Wu et al. (2001) was used to enumerate injured pathogens. This method involves overlaying 14 ml of nonselective medium (tryptic soy agar [TSA]) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish. The indicator micro-organisms (Aerhydrophila, Lmonocytogenes and Ecoli) were exposed to various concentrations of TMB as noted above and the recovery with the TAL method was compared with that of TSA and respective pathogen-specific, selective media.

Leakage of glucose, lactate dehydrogenase and protein from treated bacterial cells

To examine the effect of water-soluble biopolymeric derivative on cell leakage and the viability of the indicator strain, inoculum of the test organism (1 ml) was inoculated into sterile deionized water (10 ml) with or without TMB in a culture tube. The mixture, containing 50–80 μg ml−1 of water-soluble biopolymeric derivative, and indicator cultures were incubated at 37°C with shaking (120 rpm) for 12 h. During the predetermined incubation period; aliquots were withdrawn for determination of protein and glucose contents and lactate dehydrogenase (LDH) activity. The cell suspension was centrifuged at 8 000 g for 15 min, and the supernatant was measured for LDH activity, protein and glucose contents. The glucose content was analysed by a glucose assay kit (DiaSys Diagnostic Systems GmbH, Holzheim, Germany). A sample or glucose standard (0·0–3·0 mg dl−1, 200 μl) was added to reagent (1 ml) containing glucose dehydrogenase. After incubation at 25°C for 15 min, the absorbance at 334 nm was recorded. The LDH activity was analysed by an LDH assay kit (Clontech, Mountain View, CA). A 200 μl sample was added to 1 ml reagent containing NADH and incubated at 25°C. LDH activity was then determined by measuring the rate of decrease in the NADH concentration, which was monitored by recording the change of absorbance at 334 nm. The protein concentration was measured by absorbance at 280 nm.

Electron microscopy

Bacterial cells treated with TMB as were used to visualize structural damage at different time intervals (supporting information) by scanning electron microscopy (SEM).

Statistical analysis

The mean values and the standard deviation were from the data of triplicate trials. Mean values were compared by analysis of variance (anova) with Duncan's multiple range method for comparing groups (SAS 1989). A significance level of 5% was adopted for all comparisons of triplicate trials.

Results

Synthesis and characterization of antibacterial compound

The 1H-NMR spectrum of the biopolymer and its quaternized derivative is compared in Fig. 1. The spectra revealed an intense signal at 3·66 corresponding to the trimethylammonium group, which was in accordance with the results of a study by Sieval et al. (1998). The peak at 3·6 ppm is assigned to the trimethyl amino group; the peak at 3·1 ppm is assigned to dimethyl amino groups; and the peaks between 4·7 and 5·7 ppm are assigned to 1H protons.

Figure 1.

1H NMR spectrum of biopolymer (a) and N,N,N trimethyl biopolymer (TMB) (b) dissolved in D2O.

Figure 2 depicts the MALDI-Mass spectra of TMB compared with that of native biopolymer. Results were also in agreement with 1H NMR spectra showing an increase in molecular weight after quaternization.

Figure 2.

MALDI-TOF spectrum of biopolymer (a) and TMB (b) dissolved in D2O.

Both physical and chemical characteristics of the quaternized biopolymer were compared with its native counterpart. The total sugar and total protein content of the biopolymer were 66·8 and 2·45% (w/w), respectively, indicating a primarily polysaccharide structure of TMB similar to the native biopolymer. The amino sugars (5·8%), acidic polysaccharides including uronic acid (2·83%) and pyruvic acid (7·4%) did not differ significantly than native biopolymer (Table 1).

Table 1. Compositional analysis of biopolymer and N,N,N trimethyl biopolymer (TMB)
S.no.ComponentsBiopolymer composition (%)TMB composition (%)
1.Total sugar69·866·8
2.Amino sugar6·875·8
3.Protein6·732·45
4.Pyruvic acid0·67·4
5.Uronic acid1·122·83
6.Carbon14·7312·73
7.Hydrogen1·231·01
8.Nitrogen0·640·44

Evaluation of antibacterial activity

Results obtained by the agar disc diffusion assay of the quaternized biopolymer against three water-borne pathogens showed that the inhibitory effect of TMB was higher than that of native biopolymer as indicated by their zone of inhibitions (Table 2).

Table 2. Inhibition zone diameter (mm) against pathogens (Values represent the diameter (mm) of zone produced around each disc and are average of three separate experiments) ND, not determined; NZ, no zone observed; Diameters includes the diameter of disc (6 mm)
BacteriaConcentration of compounds (μg ml−1) Diameter of zone of inhibition (mm)
BiopolymerTMB
255075100255075100
Listeria monocytogenes NZ10131410131616
Escherichia coli NZND121410141820
Aeromonas hydrophilla NZNZ141913182023

Based on the above analysis, the antibacterial activity of quaternized biopolymer has been shown to be higher than that of unmodified polymer.

The inhibition kinetics of biopolymer and TMB against the indicator bacterial strains is presented in Fig. 3a–c. The inhibitory effect of TMB was higher than that of native biopolymer on different water-borne pathogens. Inactivation of the indicator bacteria with the quaternized derivative could be effectively achieved with a low derivative dose of 60–80 μg ml−1, within a contact time of 60 min at ambient temperature (Table 3). The analysis of bacterial growth in the presence of various concentrations of quaternized biopolymer over time indicated no significant change (P > 0·05) in growth behaviour at a concentration of 10 μg ml−1, with respect to the control (cells without TMB). For concentrations of 10 and 45 μg ml−1, the growth profile was different from the control (P < 0·05), whereas at concentrations of ≥50 μg ml−1, the quaternized biopolymer inhibited cell growth. The killing efficacy of 60 μg ml−1 of TMB when challenged upon c. 10cells per ml of pathogens is illustrated in Fig. 3, 3 log reduction was achieved by 60 min at ambient temperature.

Table 3. Minimal inhibitory concentration (MIC) of N,N,N trimethyl biopolymer (TMB) and biopolymer against selected bacterial pathogens
BacteriaMIC (μg ml−1)
Quaternized biopolymerBiopolymer
Listeria monocytogenes 80>100
Escherichia coli 73>100
Aeromonas hydrophila 65>100
Figure 3.

Inhibition kinetics for biopolymer (●) and N,N,N-trimethyl biopolymeric derivative, N,N,N trimethyl biopolymer (TMB) (■) against (a) Aeromonas hydrophila ATCC 35654 (65 μg ml−1), (b) Escherichia coli O157:H7 ATCC35150 (73 μg ml−1) and (c) Listeria monocytogenes ATCC 1911 (80 μg ml−1). Each value is expressed as mean ± SD (n = 3).

In the thin agar layer (TAL) plate injured cells resuscitate and grow on TSA during the first few hours of incubation; then, the selective agents from the selective medium diffuse to the top layer, interact with the recovered micro-organisms and start to produce typical reactions. The injured cells thereby are identified onto their respective selective medium. The method is simple and allows one-step, convenient procedure for recovery and enumeration of injured bacterial cells from environment. Lack of bacterial growth following treatment with TMB on TAL plates, as well as on specific selective media plates indicated complete inactivation of the spiked pathogens (results not shown).

To elucidate the possible mechanism of the bactericidal action of TMB, Glucose, LDH and protein leakage from treated pathogens were studied. In the present study, treatment for pathogens with a concentration of 76 μg ml−1 of TMB led to rapid leakage of proteins, glucose and LDH within a period of 3 h (Table 4), although a minor increase in glucose, protein and LDH levels in the extracellular media was observed after 6 h (results not shown), no further increase in the levels of glucose, protein or LDH occurred thereafter.

Table 4. Effect of biopolymeric derivative [N,N,N trimethyl biopolymer (TMB)] (75 μg ml−1) on glucose concentration, lactate dehydrogenase activity and protein level in the extracellular media of bacterial pathogens cultured at 37°C for up to 3 h. Control is bacterial cells in distilled water without TMB treatment. Each value is expressed as mean ± SD (n = 3)
Bacterial strainsLDH activity (U l−1)Glucose (mg dl−1)Protein absorbance (280 nm)
0 h3 h0 h3 h0 h3 h
Aeromonas hydrophila 016·37 ± 0·0700·40 ± 0·0500·520 ± 0·06
Control00·02 ± 0·00100·03 ± 0·00100·018 ± 0·004
Escherichia coli O157:H7015·63 ± 0·0600·36 ± 0·0800·46 ± 0·08
Control00·01 ± 0·00100·025 ± 0·00500·020 ± 0·004
Listeria monocytogenes 014·0 ± 0·7000·34 ± 0·0400·42 ± 0·02
Control00·005 ± 0·00300·020 ± 0·00100·025 ± 0·001

Discussion

Chitosan is a well-known amino rich biopolymer and has been widely used for biomedical applications due to its biodegradability, nontoxicity and antimicrobial activity. However, its insolubility in most solvents at neutral or high pH, except in organic acids substantially limits its usefulness (Tan et al. 2013). Although, chitosan have been extensively modified by quaternization and have resulted in several derivatives with distinct properties and applications (Sajomsang et al. 2009; Tan et al. 2013), but the dual property of these amino rich biopolymers of being flocculant and disinfectant has not yet been exploited. Thus, for practical purposes, studies with superior quaternized biopolymeric derivatives against a range of water-borne pathogens are desired. A complete characterization of the biopolymer structure is important for its chemical modification; to produce ‘customized’ biopolymers with a unique combination of mechanical, chemical and biological properties. To this end, very few biologically produced polymers have been thoroughly characterized limiting the generation of chemically modified biopolymers. In the current past, we have extensively characterized an exocellular biopolymer produced by a strain of Kl. terrigena. The biopolymer has very good stability under environmental conditions and can flocculate a wide range of colloidal particles rapidly in water; the polymer has been purified and structurally elucidated (Ghosh et al. 2009a,b). Dimethyl sulfate (DMS) was used as methylating agent as it is less toxic and less expensive in comparison with commonly used alkylating agents (iodomethane), and no solvent is required for the reaction due to high boiling point of DMS (Britto and Assis 2007). Synthesis of TMB leads to methylation of the amino groups in the C-2 position of biopolymer to form quaternary amino groups with fixed positive charges on the repeating units of the quaternized polymer chain.

In our study, it was found that biopolymer has small diameters of inhibition zones attributable to lower natural antibacterial activity due to positively charged amino groups. The quaternized biopolymer in comparison shows higher zone of inhibition against the gram-negative and gram-positive pathogens as the antibacterial activity was found to increase with an increase in ammonium salt moieties. The higher antibacterial activity of quaternized biopolymeric derivative could be due to the permanent positive charges on the biopolymeric flocculant chain, as a consequence of the quaternization of the amino groups. The activity resulted from the interaction between the positively charged amino groups of the biopolymer and negatively charged cell surface of gram-negative bacteria. In our study, the inhibitory effect of the TMB on the growth of Aerhydrophila and Escherichia coli was significantly different from its effect on L. monocytogenes. This could be attributed to differences in cell wall structure or due to the inhibition of the growth of Listeria by high concentrations of the TMB. We examined antibacterial activity of the synthesized biopolymeric derivative only at low concentrations and after short-exposure times with the intention of investigating its potential practical use as an antibacterial agent against bacterial pathogens.

Several authors have proposed that the antimicrobial action of chitosan could be explained by a more direct disturbance of membrane functions (Tsai and Su 1999). The reactive amino groups in chitosan could interact with a multitude of anionic groups on the surface of the cell to alter its permeability. This causes the leakage of intracellular components such as glucose, LDH and protein, resulting in a destabilized cell membrane beyond repair and subsequent cell death.

Two gram-negative and one gram-positive bacterial strain were used in the present study. The micro-organism may also affect the antimicrobial activity of the quaternized biopolymers (Tan et al. 2013). Electrostatic interaction between the polycationic structure and the anionic components of the micro-organisms play a fundamental role in antibacterial activity. The antibacterial modes in both gram-negative and gram-positive bacteria, begin with interactions at the cell surface and compromise the cell wall or outer membrane first. Lipoteichoic acids in gram-positive bacteria may provide a molecular linkage for biopolymeric derivatives at the cell surface, allowing it to disturb membrane functions (Raafat et al. 2008). For gram-negative bacteria, lipopolysaccharide and proteins in the outer membrane are held together by electrostatic interactions with divalent cations that are required to stabilize the membrane. Polycations may compete with divalent metals present in the cell wall, which will disrupt the integrity of the cell wall or influence the activity of degradative enzymes (Tan et al. 2013). Contact between biopolymeric derivative and the cell membrane, which is essentially a negatively charged phospholipid bilayer, may slightly change the membrane permeability. The molecular size of the antimicrobial compounds is one of the determining factors in their interactions with bacteria. The relatively large sizes of the TMB hinder their ability to penetrate into the cell wall and membrane, and as a result, they may only cause damages to the cell surfaces and cause cell aggregation.

The ultra structural change caused by TMB treatment on bacterial cells, scanning electron microscopic (SEM) data, was collected for treated bacterial cells at various time intervals (supporting information). The intracellular changes were observed in TMB-treated gram-negative bacteria (Escherichia coli O157:H7 ATCC 32150) when compared to the nontreated cells. Besides, remarkable modifications of cell membrane and disruption of cell membranes occurred after only a short period of exposure. The ultra structural changes were observed in the TMB-treated gram-positive bacterial cells (L. monocytogenes ATCC 19111), where modification on the cell membrane was observed after 90 min of exposure.

In contrast to the bioactivity of biopolymer, the inhibitory activity of TMB was maintained during the incubation time. In view of the vast number of bacteria, which reside in the water environment, pathogen-specific inactivation would be more desirable than general disinfection. Although the antibacterial activity of bioflocculants has been poorly emphasized, our results demonstrate for the first time the antibacterial activity of the quaternized biopolymer against one of the major bacterial pathogens. The results suggest that inactivation of these bacterial pathogens using TMB as disinfectants may be practically achievable. However, prior to actual application, the interactive effects of other water-borne contaminants with the quaternized biopolymeric derivative need to be established. The resistance to exposure of quaternary ammonium compounds to pathogens might arise (Li et al. 2009); however, in this context, it may be emphasized that the exposure time for pathogen inactivation is very less for the biopolymeric derivative. However, an attempt to investigate whether bacterial resistance arises will be worthwhile proposition in the future. Further, the toxicity studies carried out using swiss albino mice did not indicate any toxic effect of TMB for upto a dose of 280 mg kg−1. These results confirmed the safety of the quaternized derivative (unpublished results).

The quaternization of amino rich biopolymer isolated from a bacterium led to a water-soluble bioactive agent with enhanced inhibitive capability against all the selected bacterial pathogens. TMB exhibited high water solubility and enhanced bactericidal action against gram-positive and gram-negative pathogens as compared to native biopolymer. Results revealed rapid loss of intracellular glucose, LDH and proteins from treated cells. Mechanistically, an electrostatic interaction of non-pH-dependent positive charge of the quaternized derivative with the negatively charged cell wall of pathogenic cells is suggested as a cause of antibacterial action. Nevertheless, studies will be pursued to determine the impact of TMB on field samples and mode of action of the quaternized biopolymer will be investigated in the future. In conclusion, these polymers may be good candidates as disinfectant against bacterial pathogens. Further studies are required to characterize these antibacterial polymers in detail.

Acknowledgements

We are grateful to Department of Science and Technology (DST) New Delhi for their financial support.

Conflict of interest

The authors have declared that no conflict of interest exists.

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