Antibacterial activity of chitosan against Burkholderia pseudomallei

Abstract The ability of Burkholderia pseudomallei to persist and survive in the environment is a health problem worldwide. Therefore, the antibacterial activities of chitosan against four environmental isolates of B. pseudomallei from soil in Khon Kaen, Thailand, were investigated. Antibacterial activities were assessed by a plate count technique after treatment with 0.2, 0.5, 1, 2 or 5 mg ml−1 chitosan for 0, 24 and 48 hr. Chitosan at 5 mg ml−1 completely killed all four B. pseudomallei isolates within 24 hr, whilst 2 mg ml−1 chitosan lowered the viability of B. pseudomallei by 20% within the same time span. Chitosan may act by disruption of the cell membrane, releasing intracellular components that can be detected spectrophotometrically at 260 and 280 nm. Transmission electron microscopy inspection of chitosan‐treated B. pseudomallei revealed damage to the bacterial membranes. This study demonstrated the effective antibacterial activity by chitosan against B. pseudomallei. Chitosan causes disruption of the bacterial cell membrane, release of intracellular constituents and cell death. This study revealed the inhibitory potential of chitosan for mitigating B. pseudomallei occurrences.

We know of three reports on antibacterial activity and mechanism of action of chitosan solutions against members of the genus Burkholderia. One study concerned B. seminalis, the apricot fruit-rot pathogen, a member of the B. cepacia complex (Lou et al., 2011).
Another study on members of the same complex was conducted in sputum from cystic fibrosis (CF) patients in China (Fang et al., 2010).
The third report focused on the multidrug-resistant B. cenocepacia (Ibrahim et al., 2014). Chitosan can lethally damage bacterial cell membranes leading to the leakage of proteins, nucleic acids and other intracellular components. To date, there has been no research on the antibacterial activity of chitosan against B. pseudomallei. This, therefore, was the aim of our study. Assays of the integrity of the bacterial cell membrane were done and transmission electron microscopy observations used to elucidate the mechanism of the antibacterial activity of chitosan against B. pseudomallei.

| Materials
Chitosan with degrees of N-deacetylation not less than 85%, practical grade, from crab shells, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan was dissolved in 1% (v/v) acetic acid to produce stock solutions of 10 mg ml −1 . The pH was adjusted to 5.6 using NaOH and continuous stirring at 160 rpm for 24 hr at room temperature. This was followed by autoclaving at 121°C for 20 min. The stock chitosan solution was diluted to the desired concentrations in sterile deionized water of pH 5.6. In the control treatment, sterile deionized water of pH 5.6 was substituted for chitosan stock. water. Thereafter, the bacterial cells were resuspended in sterile distilled water (pH 5.6) and density adjusted to achieve OD 600 of 0.6 (approximately 10 8 colony forming unit (cfu) ml −1 ) for the antibacterial activity assay.

| Antibacterial activity of chitosan against B. pseudomallei
Final concentrations of chitosan used in our experiments were 0.2, 0.5, 1, 2, and 5 mg ml −1 . Bacterial suspension was adjusted to approximately 10 8 cfu ml −1 . One hundred microliter of bacterial suspension was added to chitosan solutions in a 96-well plate and incubated for 0, 24 or 48 hr at 37°C with agitation at 180 rpm as previously described by Lou and others (Lou et al., 2011). The chitosan-treated bacteria were thereafter serially diluted and 10 μl of each dilution was plated on LB agar in 10 replicate (Herigstad, Hamilton, & Heersink, 2001). After incubation at 37°C for 24 hr, the viable bacteria were enumerated based on numbers of colonyforming units. The percentage of killing was calculated using the formula [1-(log10 sample/log10 inoculum)]× 100 (Fang et al., 2010).
Each experiment was carried out in duplicate in three independent experiments. Each experiment was carried out in duplicate in three independent experiments.

| Transmission electron microscopy
One milliliter of B. pseudomallei ST-39 suspension of approximately 10 8 cfu ml −1 was added into sublethal chitosan solutions with final concentrations of 1 and 2 mg ml −1 . After incubation at 37°C, 180 rpm for 24 hr, the suspension was centrifuged, washed twice with sterile distilled water and fixed with 2.5% (v/v) glutaraldehyde (EM grade; EMS, Electron microscopy Sciences, USA) in 0.1 mol L -1 phosphate buffer (PBS, pH 7.4) at 4°C for 2 hr. Subsequently, the samples were washed three times with 0.1 mol L -1 PBS followed by postfixing with 1% (w/v) OsO 4 in 0.1 mol L -1 PBS for 2 hr at room temperature. After three washes with the same buffer, the samples were dehydrated by a graded series of ethanol solutions (70%, 80%, 90% and 100%) as previously described by Lou and others (Lou et al., 2011). The samples were then embedded in Spurr's resin and sectioned with an ultramicrotome (Leica EM UC7 ultramicrotome, Germany) at room temperature. Thereafter, the sections were double-stained with saturated uranyl acetate and lead citrate. The grids were examined with a transmission electron microscope (Hitachi HT7700, Japan) at an operating voltage of 80 kV.

| Antibacterial activity of chitosan against B. pseudomallei
The

| Integrity of B. pseudomallei cell membranes
To illustrate the antimicrobial mechanism of chitosan against B. pseudomallei, the integrity of cell membranes in the treated B. pseudomallei was assessed using the release of intracellular materials as an indicator. The A260 and A280 values, estimating nucleic acid and protein released from treated B. pseudomallei treated with 0-5 mg ml −1 chitosan for 24 hr, were determined. The positive control used 0.01% Triton X-100 to lyse all cells. Release of intracellular components was concentration-dependent up to the highest concentration used in this study (5 mg ml −1 ) ( Figure 2). Notably, the damage to bacterial cell membranes by chitosan is in agreement with the results of the bactericidal activity investigation.

| Transmission electron microscopy
The

| DISCUSSION
Burkholderia pseudomallei is a soil-dwelling saprophyte that can be acquired from the environment, leading to a fatal infection termed melioidosis (Cheng & Currie, 2005;Limmathurotsakul & Peacock, 2011;Wiersinga et al., 2012). The remarkable ability of B. pseudomallei to survive for months to years in the environment may increase the risk of transmission to humans (Inglis & Sagripanti, 2006;Kamjumphol, Chareonsudjai, Taweechaisupapong, & Chareonsudjai, 2015). In light of this, attempts have been made to lower the bacterial population to decrease the risk of their transmission to humans (Na-ngam et al., 2004;Wang-Ngarm et al., 2014;Boottanun et al., 2017).
In this study, we demonstrated that the greatest killing activity of all four environmental B. pseudomallei strains occurred when bacteria were exposed to 5 mg ml −1 of chitosan. Chitosan at a concentration of 2 mg ml −1 inhibited bacterial growth by about 20% within 24 hr. The inhibitory activity of chitosan against B. pseudomallei was evidently a function of the concentration of chitosan since the control treatment (sterile deionized water of pH 5.6) had almost no effect on the survival of B. pseudomallei. This result is consistent with the optimum pH of 5-8 determined for the survival of B. pseudomallei by Tong et al. (1996). Our results are in line with those of Fang and colleagues who demonstrated that chitosan was a potential bactericidal agent against cells of the B. cepacia complex isolated from cystic fibrosis patients.
In those cases, effective concentrations of chitosan were 10-100 μg ml −1 (Fang et al., 2010). Activity of chitosan against B. cenocepacia, a multidrug-resistant pathogen that is difficult to eradicate, has also been demonstrated (Ibrahim et al., 2014). Leaving aside human pathogens, the apricot fruit-rot pathogen, B. seminalis, could be eradicated by treatment with chitosan at 2 mg ml −1 (Lou et al., 2011). However, our findings demonstrated that B. pseudomallei can withstand higher chitosan concentrations than do other Burkholderia species. Chitosan is known to act against bacteria by damaging the bacterial cell integrity and causing cell membrane permeabilization. The positive charge of chitosan is assumed to interact with anionic components such as lipopolysaccharides, phospholipids and bacterial cell-surface proteins (Chung et al., 2004;Kong et al., 2010). Given that B. pseudomallei bears highly species-specific antigens that can be exploited for diagnosis (Pitt, Aucken, & Dance, 1992;Perry, MacLean, Schollaardt, Bryan, & Ho, 1995;Ho et al., 1997;Dharakul, Songsivilai, Smithikarn, Thepthai, & Leelaporn, 1999), its cell surface structure is presumably different from that of other Burkholderia species. Nevertheless, like other members of the genus, B. pseudomallei is susceptible to chitosan.
We showed here that chitosan disrupts B. pseudomallei cell membranes with the release of intracellular contents resulting in a drastic increase in absorbance at A260 and A280 compared to untreated controls. Damage to the cell membrane was directly observed by transmission electron microscopy in chitosan-treated B. pseudomallei.
We demonstrated disruption of the bacterial cell membrane and confirmed the antibacterial activity of chitosan against B. pseudomallei. To extend the applicability of our findings, further studies are required to demonstrate that chitosan can control B. pseudomallei in soil or, combined with other antimicrobial agents, that chitosan may improve outcomes for melioidosis patients.
In summary, our work has added more information concerning the antibacterial activity of a natural and nontoxic biopolymer, chitosan.
At a concentration of 5 mg ml −1 , chitosan can kill B. pseudomallei, the causative agent of melioidosis. The mechanism by which chitosan exerts this effect is damage to the bacterial cell membrane leading to leakage of intracellular components. Chitosan has the potential to limit the numbers of B. pseudomallei cells.