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.

Burkholderia pseudomalleiAdaptation for Survival in Stressful Conditions

“…Like other bacteria, B. pseudomallei possesses various mechanisms to modulate its gene expression for survival under stress ( Table 1 ). The adaptation of B. pseudomallei to stress includes modulation of the expression of genes encoding important proteins, such as short-chain dehydrogenase/oxidoreductase (SDO) [ 31 ], acyl-CoA dehydrogenase [ 27 ], Burkholderia secretion apparatus (Bsa) T3SS [ 27 ], beta-lactamase-like protein [ 28 ], sigma factor E (RpoE) [ 27 , 32 ], and heat-shock proteins [ 27 , 32 ] for salt stress; KatG and KatE catalase enzymes [ 33 ], sigma factor S (RpoS) [ 34 ], succinyl-CoA: 3-ketoacid CoA transferase (SCOT) [ 34 ], and DpsA [ 35 ] for oxidative stress; Fur [ 36 ], pyochelin [ 37 ], pyoverdine [ 37 ], ornibactin [ 37 ], cepabactin [ 37 ], and biofilm formation-associated regulator [ 38 ] for iron stress; and ATP synthases [ 25 ], polyhydroxybutyrate synthase [ 25 ], pyruvate dehydrogenase [ 25 ], acetate kinase [ 25 ], alcohol dehydrogenase [ 25 ], motility-mediated proteins [ 25 ], stress-related proteins [ 25 ], and virulence factors [ 25 ] for oxygen stress. Most of these stress-response proteins have been observed to react with sera from melioidosis patients [ 39 ], potentially indicating their important roles in the adaptation of bacteria to survive under ecologically stressful conditions.…”

Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance

“…Real time RT-PCR was performed using the Brilliant II SYBR R Green QPCR Master Mix, one step (Agilent Technologies, Santa Clara, CA, USA). Amplifications of five genes (kex, wzt, qor, proS and hsp) were performed under the following conditions: reverse transcription at 50 • C for 30 min, enzyme activation at 95 • C for 10 min, then 40 cycles of denaturation at 95 • C for 30 s, annealing at 55 • C for 1 min, and melting curve analysis at 72 • C for 1 min in a CFX96 Touch TM Real-Time PCR Detection System (Bio-Rad, Singapore) as previously described (Pumirat et al, 2017). The primer sequences are shown in Table 1.…”

“…A heat stress resistance assay was performed as described previously (Pumirat et al, 2017) with some modifications. Briefly, B. pseudomallei cultured in LB medium at 37 • C for 6 h were washed with phosphate-buffered saline (PBS) and resuspended in PBS to an OD 600 of 0.15.…”

“…Analysis of B. pseudomallei biofilm formation was performed by a microtiter-plate assay as previously described (Pumirat et al, 2017). Each B. pseudomallei strain was assayed with at least eight replicates per experiment, along with positive and negative controls.…”

Peer Review #1 of "Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance (v0.1)"

“…DataAnalysis™ software version 3.4 (Bruker) was used to convert raw data 145 format (.d) files to mascot generic files (.mgf), which were further searched by Mascot software Standard PCR for the 23S RNA gene was used to verify that there was no gDNA contamination 155 in the DNase-treated RNA samples. Real time RT-PCR was performed using the Brilliant II 156 SYBR® Green QPCR Master Mix, one step (Agilent Technologies, Santa Clara, CA, USA).Manuscript to be reviewed 177 Analysis of B. pseudomallei biofilm formation was performed by a microtiter-plate assay 178 as previously described(Pumirat et al, 2017). Each B. pseudomallei strain was assayed with at 179 least eight replicates per experiment, along with positive and negative controls.…”