Effects of sodium chloride on heat resistance, oxidative susceptibility, motility, biofilm and plaque formation of Burkholderia pseudomallei

Abstract Burkholderia pseudomallei is an environmental saprophyte and the causative agent of melioidosis, a severe infectious disease prevalent in tropical areas, including southeast Asia and northern Australia. In Thailand, the highest incidence of melioidosis is in the northeast region, where saline soil and water are abundant. We hypothesized that B. pseudomallei develops an ability to thrive in saline conditions and gains a selective ecological advantage over other soil‐dwelling microorganisms. However, little is known about how an elevated NaCl concentration affects survival and adaptive changes in this pathogen. In this study, we examined the adaptive changes in six isolates of B. pseudomallei after growth in Luria‐Bertani medium containing different concentrations of NaCl at 37°C for 6 hr. The bacteria were then investigated for resistance to heat at 50°C and killing by hydrogen peroxide (H2O2). In addition, flagellar production, biofilm formation, and the plaque formation efficiency of B. pseudomallei after culture in saline conditions were observed. In response to exposure to 150 and 300 mmol L−1 NaCl, all B. pseudomallei isolates showed significantly increased thermal tolerance, oxidative resistance, and plaque‐forming efficiency. However, NaCl exposure notably decreased the number of B. pseudomallei flagella. Taken together, these results provide insight into the adaptations of B. pseudomallei that might be crucial for survival and persistence in the host and/or endemic environments with high salinity.

melioidosis vary considerably, ranging from acute fulminant septicemia to chronic localized infection. In its acute form, death can occur within days of the onset of symptoms. However, the longest reported incubation period between initial acquisition of the organism and subsequent infection is a remarkable 62 years. Furthermore, a high rate of relapse has been recognized (Ngauy, Lemeshev, Sadkowski, & Crawford, 2005). Unfortunately, there is currently no effective vaccine available for the prevention of melioidosis. The treatment of melioidosis generally involves the antibiotics ceftazidime or carbapenem as B. pseudomallei exhibits resistance to several empiric antimicrobial therapies.
In Thailand, the highest prevalence of B. pseudomallei and the highest incidence of melioidosis are in the northeast region, where saline soil and water are plentiful. The electrical conductivity of soil samples from northeast Thailand ranges from 4 to 100 dS/m, which is higher than that of normal soil from other regions (approximately 2 dS/m) (Development Department of Thailand). We hypothesized that B. pseudomallei may develop an ability to adapt to saline conditions and gain cross-protection to other stress conditions. There is evidence of a link between high NaCl concentrations and an ability to survive in saline conditions in other closely related organisms, namely, the Burkholderia cepacia complex (BCC). These organisms are opportunistic pathogens of cystic fibrosis (CF) sufferers (Mahenthiralingam, Baldwin, & Vandamme, 2002;Vandamme et al., 1997) whose lung airways have an increased concentration of NaCl in the surface liquid (Widdicombe, 2001), approximately twofold higher than that of healthy lungs (Joris, Dab, & Quinton, 1993). The potential pathogenic role of B. pseudomallei in CF lung disease has also been reported (O'Carroll et al., 2003).
Several studies have shown that exposure to NaCl can influence the adaptive survival and virulence of pathogenic bacteria.
The relevance of this has been shown in Salmonella enterica serovar Typhimurium (12), Staphylococcus aureus (Park et al., 2012), and Listeria monocytogenes (Garner, James, Callahan, Wiedmann, & Boor, 2006), whereby bacteria cultured in medium-containing high NaCl show increased heat tolerance (Park et al., 2012;Yoon, Park, Oh, Choi, & Yoon, 2013), antibiotic resistance (Yoon et al., 2013), and invasion ability into host cells (Garner et al., 2006;Yoon et al., 2013). Our previous study also showed that B. pseudomallei grown under salt stress displayed significantly greater resistance to the antibiotic ceftazidime (Pumirat et al., 2009). Salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 (Pumirat et al., 2010). However, only one B. pseudomallei isolate was used in our previous study and adaptive responses of B. pseudomallei to high NaCl concentrations remain largely unknown.
In this study, we further investigated the adaptive response of six B. pseudomallei isolates grown in Luria-Bertani (LB) medium with different concentrations of NaCl for 6 hr at 37°C. The concentrations of NaCl used were 0, 150, and 300 mmol L −1 which are equivalent to 0, 15, and 30 dS/m, respectively. The bacteria under salt stress were then tested for heat resistance, oxidative susceptibility, swarm motility, flagellar production, and biofilm and plaque formation.

| Bacterial strains, growth, and salt treatment
Experiments were performed using six clinical isolates of B. pseudomallei: strains 153, 576, 1026b, 1530, 1634, and the reference strain K96243. All strains were obtained from clinical specimens of six patients presenting with melioidosis in northeast Thailand. The bacteria were generally maintained on LB agar at 37°C. To examine the effect of NaCl, B. pseudomallei was subcultured in NaCl-free LB broth and incubated at 37°C with shaking at 200 rpm overnight. The bacteria were then inoculated at a dilution of 1:10 into 10 ml of LB broth containing 0, 150, and 300 mmol L −1 NaCl and incubated at 37°C for 6 hr with shaking. The salt-treated and untreated B. pseudomallei were adjusted to an OD 600 of 0.15. A serial dilution was performed to determine the number of colony-forming units (CFU) to obtain the starting number of bacteria.

| Heat resistance assay
A heat stress resistance assay was performed as described previously (Vanaporn, Vattanaviboon, Thongboonkerd, & Korbsrisate, 2008) with some modifications. Briefly, B. pseudomallei cultured in LB medium containing different salt concentrations (0, 150, and 300 mmol L −1 NaCl) at 37°C for 6 hr were washed with phosphate-buffered saline (PBS) and resuspended in PBS to an OD 600 of 0.15. One milliliter of the bacterial suspension was then added into a prewarmed tube and incubated at 50°C for 15 min. Before and after heat challenge, bacterial survival was enumerated on LB agar plates after incubating at 37°C for 24 hr. The number of surviving bacteria was expressed as a percentage of the viable cells.

| Oxidative stress assay
The survival of B. pseudomallei under oxidative conditions was determined by observing the number of viable bacteria after exposure to an oxidative agent. After 6 hr of culturing in LB medium containing different salt concentrations (0, 150, and 300 mmol L −1 NaCl), B. pseudomallei cells were harvested, washed, and resuspended in PBS. The bacterial concentration was adjusted to an OD 600 of 0.15. Then, 100 μl of bacterial suspension was treated with H 2 O 2 (at a final concentration of 1 μmol L −1 ) or left untreated at room temperature for 15 min. A 10-fold dilution of treated and untreated bacteria was performed and plated on LB agar.
After incubation at 37°C for 24 hr, colonies were counted. The number of colonies of treated bacteria was compared with that of untreated bacteria (without oxidant) and presented as the % bacterial survival.

| Motility assay
A motility assay was undertaken using the swarm plate method as previously described (Deziel, Comeau, & Villemur, 2001). Briefly, B. pseudomallei were grown in LB broth with 0, 150, or 300 mmol L −1 NaCl for 6 hr at 37°C. Bacterial pellets were collected, washed, and adjusted in PBS to approximately 10 8 CFU/ml. Swarm plates were inoculated by placing 2 μl of the prepared inoculum onto the agar surface at the center of the plate. The diameter of the swarming motility zone was measured from the point of inoculation after incubation at 37°C for 24 hr.

| Electron microscopic examination
The presence of B. pseudomallei flagella was examined using a transmission electron microscope. Fifty microliters of B. pseudomallei grown in LB broth with different salt concentrations was harvested and dropped onto parafilm. Formvar-coated carbon grids were placed on top of the parafilm for 10 min to transfer the bacterial cells. The liquid was then carefully removed with filter paper. The samples were stained with 1% uranyl acetate for 10 min, then the liquid was removed again. The grid was dried at room temperature overnight.

Bacteria were observed under a Hitachi Electron Microscope H-7000
(Japan). The presence of bacterial flagella was recorded for 100 bacteria per condition.

| RNA preparation and real-time RT-PCR
RNA was isolated from 6 hr culture of B. pseudomallei grown at 37°C by adding 10 ml of RNAprotect bacterial reagent (QIAGEN) to 5 ml of bacteria culture and incubating for 5 min at room temperature.
Subsequently, total RNA was extracted from bacterial pellets using

| Biofilm formation assay
Quantification of biofilm formation was performed using a microtiter plate assay as previously described (Leriche & Carpentier, 2000;Stepanovic, Vukovic, Dakic, Savic, & Svabic-Vlahovic, 2000). Briefly, biofilm formation of B. pseudomallei was induced in trypticase soy broth at 37°C for 24 hr. After incubation, the adherent bacteria were washed using deionized water three times and fixed with 99% methanol for 15 min at room temperature. The bacteria were stained for 15 min with 1% crystal violet and solubilized with 33% (v/v) glacial acetic acid. The quantity of biofilm was measured at 630 nm using a microplate reader (Bio-Rad). Each B. pseudomallei isolate was assayed in duplicate, using eight wells per experiment.

| Plaque formation assay
Plaque-forming efficiency was assessed as previously described (Pumirat et al., 2014). HeLa cells were infected with B. pseudomallei at a multiplicity of infection of 20 and incubated at 37°C with 5% CO 2 for 2 hr. Thereafter, the infected cell monolayers were washed and replaced with medium-containing kanamycin (250 μg/ml). The plates were incubated at 37°C in a humidified 5% CO 2 atmosphere for 20 hr.
Plaques were stained with 1% (w/v) crystal violet in 20% (v/v) methanol and counted by microscopy. Plaque-forming efficiency was calculated by determining the number of plaques per CFU of bacteria added per well.

| Statistical analysis
All assays were conducted in triplicate, and an unpaired t-test of independent experiments was performed using the GraphPad Prism 6 program (STATCON). Results were considered significant at a p ≤ .05.

| NaCl stress induces cross-protection against heat and oxidative agents
Different growth rates may affect the number of viable bacteria under NaCl stress conditions. Therefore, prior to observing the effect of NaCl stress on cross-protection against heat and oxidative agents, the T A B L E 1 Oligonucleotide primers used in this study  (Pumirat et al., 2010). In this study, we investigated the growth kinetics of six B. pseudomallei isolates in LB media containing 0, 150, or 300 mmol L −1 NaCl for 6 hr after incubation at 37°C. Similar growth curves were observed for the six isolates under conditions of 0, 150, and 300 mmol L −1 NaCl ( Figure S1). Therefore, salt concentrations ranging from 0 to 300 mmol L −1 and a culture time of 6 hr were chosen for further investigations.
To evaluate the effect of NaCl on heat resistance in B. pseudomallei, six B. pseudomallei isolates were cultured in LB broth with different concentrations of NaCl for 6 hr to reach the log phase of bacterial growth, followed by heating at 50°C for 15 min. Figure 1 shows the percentage of surviving bacteria and demonstrates a significant difference in heat resistance between B. pseudomallei isolates cultured in NaCl-free medium and those cultured in LB with 150 mmol L −1 NaCl (p = .014 for K96243, p = .011 for 153, p = .028 for 576, p = .027 for 1026b, p = .011 for 1530, and p = .040 for 1634) or those cultured in LB with 300 mmol L −1 NaCl (p = .020 for K96243, p = .004 for 153, p < .001 for 576, p < .001 for 1026b, p < .001 for 1530, and p = .002 for 1634). In addition, the data also showed a significant difference in the percentage of bacterial survival between B. pseudomallei isolates cultured in LB supplemented with 150 and 300 mmol L −1 NaCl (p = .038 for K96243, p = .002 for 153, p = .001 for 576, p < .001 for 1026b, p = .002 for 1530, and p = .008 for 1634). The mean and standard deviation (SD) of bacterial survival in NaCl-free medium of the six B. pseudomallei isolates after heat treatment were 2.2 ± 0.5%. By contrast, the mean and SDs of bacterial survival of the six isolates in medium containing 150 mmol L −1 and 300 mmol L −1 NaCl were 18.2 ± 2.9% and 67.9 ± 8.9%, respectively. These data clearly revealed that salinity is associated with increased resistance of B. pseudomallei to heat stress. In the presence of H 2 O 2, the mean survival rate of untreated B. pseudomallei isolates was 1.7 ± 0.6%, compared with 5.6 ± 1.2% for those exposed to 150 mmol L −1 NaCl and 12.7 ± 2.3% for those exposed to 300 mmol L −1 NaCl.  (Jitprasutwit et al., 2014;Korbsrisate et al., 2005;Vanaporn et al., 2008). We therefore investigated whether NaCl affects the expression of the rpoE, groEL, htpG, bopA, bopE, and bipD. The rpoE, groEL, and htpG genes were selected because they code transcription factors or heat shock proteins that have previously been reported to be involved in heat and oxidative stress (Jitprasutwit et al., 2014;Korbsrisate et al., 2005;Vanaporn et al., 2008). The bopA, bopE, and bipD were T3SS genes which may be important for cell invasion (Gong et al., 2011;Muangsombut et al., 2008;Stevens et al., 2003). Realtime RT-PCR results showed that B. pseudomallei K96243 when exposed to NaCl (150 and 300 mmol L −1 ) exhibited increased expression of all tested genes, compared with bacteria grown under NaCl-free conditions ( Figure 3). These data suggested that NaCl is involved in increasing the expression of stress response proteins, which might be responsible for the enhanced resistance of B. pseudomallei to heat and oxidative stress.

| NaCl decreases the expression of B. pseudomallei flagella
Motility is a crucial factor for bacterial pathogenesis. diameter of the swarming zone was measured ( Figure S2). The mean and SDs of the swarming zone diameters of the six B. pseudomallei isolates were 23.7 ± 0.9, 21.8 ± 1.2, and 17.4 ± 1.6 mmol L −1 for bacteria exposed to 0, 150, and 300 mmol L −1 NaCl, respectively (Table 2).
To determine whether altered expression of the fliA gene affects bacterial flagella, we examined the number of flagella on the six B.
pseudomallei isolates during growth under different salt conditions using an electron microscope. The results showed that the number of flagella decreased with increasing concentrations of NaCl ( Figure   S3). The number of flagella counted on 100 bacteria for each of the six isolates is shown in Table 3. The majority of B. pseudomallei isolates (70.7 ± 3.5%) grown in LB with 300 mmol L −1 NaCl showed no flagella. By contrast, only 38.0 ± 3.8% and 49.3 ± 4.3% of B. pseudomallei cultured in NaCl-free and 150 mmol L −1 NaCl-supplemented media, respectively, had no flagella. The number of unflagellated bacteria among the B. pseudomallei isolates grown in 300 mmol L −1 NaCl-supplemented medium was therefore significantly higher than among those grown in salt-free (p < .001) or 150 mmol L −1 NaClsupplemented medium (p = .003, respectively). This phenomenon indicated that salinity affects flagella production in B. pseudomallei. pseudomallei isolates tended to show increased biofilm formation when grown in the presence of NaCl compared with those grown in 0 mmol L −1 NaCl, we could not detect a significant difference in biofilm formation when comparing bacteria grown in the presence of 0, 150, and 300 mmol L −1 NaCl. Rattanachetkul, Wanun, Utaisincharoen, & Sirisinha, 2000), which is an important characteristic for pathogenesis. Previously, NaCl was found to increase expression of the Burkholderia secretion apparatus (Bsa) type III secretion system (T3SS), which involved a virulence-associated interaction with the host cell (Pumirat et al., 2010). In particular, the translocon "BipB" and the secreted effector protein "Cif" homolog in B. pseudomallei were reported to induce cell-to-cell dissemination (Pumirat et al., 2014;Suparak et al., 2005). Hence, we investigated whether salt stress affects cell-to-

| DISCUSSION
B. pseudomallei is a saprophyte that can survive and multiply under different environmental conditions (Cheng & Currie, 2005;Dharakul & Songsivilai, 1999;White, 2003). It is a difficult microorganism to kill. It can inhabit harsh environments for many years, especially in endemic areas, including northeast Thailand (Wuthiekanun et al., 1995) where saline soil and water are abundant. B. pseudomallei was reported as potential opportunist pathogens of CF patients (Mahenthiralingam et al., 2002;O'Carroll et al., 2003;O'Sullivan et al., 2011;Vandamme et al., 1997),   (Volker, Mach, Schmid, & Hecker, 1992), and Escherichia coli (Gunasekera, Csonka, & Paliy, 2008), have also reported that activation of the salt stress response conferred cross-protection against other stresses, that is, increased resistance to heat and H 2 O 2 . Recently, Yuan, Agoston, Lee, Lee, & Yuk, (2012) and Yoon et al., (2013) also showed that the heat resistance of Salmonella enterica was increased after exposure to NaCl. Moreover, it is evident that growing Vibrio harveyi in LB broth supplemented with 2% NaCl (34.2 mmol L −1 ) resulted in increased resistance to menadione killing compared with the same organism grown in normal LB broth (Vattanaviboon, Panmanee, & Mongkolsuk, 2003).
It is possible that the salt stress adaptation may reflect the ability of these bacteria, including B. pseudomallei, to survive under hostile environmental conditions, such as high temperature and oxidative stress.
As B. pseudomallei is an intracellular organism, it has the capability to survive in phagocytic cells (Allwood, Devenish, Prescott, Adler, & Boyce, 2011). While trafficking within macrophages, B. pseudomallei may be exposed to oxidative stress. Interestingly, Scott & Gruenberg (2011) reported that chloride and sodium ion channels play important roles in regulating the phagosomal environment through counter ion regulation and charge compensation of macrophages. Therefore, the salt content in the phagosome may promote bacterial resistance to oxidative stress and allow B. pseudomallei to survive within the host cell.
These oxidative and heat protective effects of NaCl could be a result of the increased expression of stress response cellular components. The increased expression of the rpoE and groEL genes detected in this study was in agreement with previous reports for the B. pseudomallei transcriptome (Pumirat et al., 2010) and secretome (Pumirat et al., 2009) under high salinity conditions. The expression of groEL (bpss0477) and rpoE (bpsl2434) was upregulated in B. pseudomallei cultured in LB containing 320 mmol L −1 NaCl, by approximately 1.2-and 1.4-fold, respectively, compared with B. pseudomallei cultured in 170 mmol L −1 NaCl at the 6-hr time point (Pumirat et al., 2010). Indeed, the secretomic profile confirmed the presence of GroEL in the culture supernatant only after exposure to 320 mmol L −1 NaCl (Pumirat et al., 2009). Moreover, our results were consistent with the observation that inactivation of the rpoE operon increased susceptibility of B. pseudomallei to killing by menadione and H 2 O 2 and high osmolarity . Furthermore, it has been demonstrated that rpoE regulated a heat-inducible promoter of the rpoH gene in B.
pseudomallei (Vanaporn et al., 2008). These data implied that RpoE plays an important role in the increased resistance of B. pseudomallei in response to heat and oxidative stress.   (Fida et al., 2012) and B. subtilis (Hoper, Bernhardt, & Hecker, 2006;Steil, Hoffmann, Budde, Volker, & Bremer, 2003). All six B. pseudomallei isolates exhibited a smaller mean diameter for their motility zone when cultured under high salt conditions (300 mmol L −1 NaCl), compared with culturing under salt-free or low salt conditions (0 and 150 mmol L −1 NaCl). This observation implied that salt stress plays an important role in regulating the production of bacterial flagella. One possible explanation for this is that in order to cope with stressful environmental conditions the bacteria conserve energy by diminishing nonvital activities, such as motility, by reducing the production of flagella by decreasing the expression of the motility regulator gene.
The ability to form a biofilm is important for B. pseudomallei to gain resistance to numerous environmental factors, including certain antibiotics and stresses (Cheng & Currie, 2005;Inglis & Sagripanti, 2006;Kamjumphol et al., 2013). Our study detected the increased ability of B. pseudomallei to form a biofilm when bacterial isolates were grown in medium supplemented with NaCl, compared with salt-free medium (Table 4). This was consistent with the findings of Kamjumphol et al. who demonstrated that biofilm formation was increased when B. pseudomallei was grown in modified Vogel and Bonner's medium containing 0.85-1.7 mol L −1 NaCl (Kamjumphol et al., 2013). This indicated that B. pseudomallei responds to salt stress by producing a biofilm that could confer cross-protection against other environmental stresses.
Exposure to high salinity is likely to be associated with pathogenesis in B. pseudomallei. Previously, invasion of A549 cells was enhanced by culturing of B. pseudomallei K96243 in salt-supplemented LB medium (Pumirat et al., 2010). Our results showed that when grown in the presence of NaCl, all six B. pseudomallei isolates exhibited significantly increased plague formation in HeLa cells (Figure 4). The elevated rate of cellular invasion in response to NaCl may increase the load of intracellular bacteria, contributing to cell-to-cell spread or enhance cell cytotoxicity. Several studies have demonstrated the requirement of the Bsa T3SS and type VI secretion system (T6SS) for the intracellular pathogenicity of B. pseudomallei (Burtnick et al., 2008(Burtnick et al., , 2011Lim et al., 2015;Shalom, Shaw, & Thomas, 2007;Stevens et al., 2002;Warawa & Woods, 2005). We postulate that these systems may participate in the enhanced plaque formation of B. pseudomallei observed after exposure to NaCl. However, further experiments are required to investigate this possibility.

| CONCLUSIONS
In conclusion, our results demonstrated that high salt conditions modulate adaptive responses in B. pseudomallei isolates. These adaptive responses include increased thermal resistance, plaque formation, and decreased flagella and oxidative susceptibility. Similar results were observed in all six isolates tested; suggesting that salt stress induces a general, conserved response in B. pseudomallei. Our findings provide insight into how these bacteria persist in endemic environments abundant in saline soil and water, and may indicate the link between the establishment and pathogenesis of B. pseudomallei infection in CF patients.