Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei

Plaque forming, invasion efficiencies and net intracellular survival and replication of <i>B</i>. <i>pseudomallei</i> wild-type and its derivative strains.

<p>(A) Plaque forming and (B) invasion efficiencies of <i>B</i>. <i>pseudomallei</i> K96243 wild-type, 6H4 mutant or 6H4/pME1039 complemented strains in infect HeLa cells. Plaque-forming efficiency was calculated as: number of plaques/bacterial CFU added per well. Percent invasion was determined as: (number of intracellular bacteria post infection/number of CFU added) × 100. <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black bar), 6H4 mutant (white bar) and the 6H4/pME1039 complemented (checked bar) strains were used to infect (C) HeLa and (D) J774A.1 macrophage cells. Intracellular loads of bacteria were enumerated at 4, 6, 8, and 24 h p.i. Asterisks indicate significant differences (<i>P</i> < 0.05, <i>t</i>-test) between wild-type and its derivative strains. Results are presented as standard errors of the means for experiments done in triplicate, with three independent experiments.</p

<i>B</i>. <i>pseudomallei bpsl1039</i> 6H4 mutant is attenuated in mice.

<p>Survival of BALB/c mice inoculated <i>via</i> the intranasal route with <i>B</i>. <i>pseudomallei</i> K96243 wild-type (●), 6H4 (▼) and 6H4/pME1039 complemented (◆) strains were determined (n = 5 per group). The survival curves of mice infected with wild-type and 6H4 were significantly different (<i>P</i> < 0.01). Data were analysed using the Log-rank (Mantel-Cox) test with a Bonferroni correction.</p

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been “harvested” by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6’)-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Effect of nitrate and anaerobic culture conditions on <i>B</i>. <i>pseudomallei</i> growth.

<p><i>B</i>. <i>pseudomallei</i> was inoculated in M9 minimal medium supplemented with (+NaNO₃) or without (-NaNO₃) sodium nitrate, and incubated under aerobic or anaerobic culture conditions. (A) anaerobic and (B) aerobic kinetic growth curves of <i>B</i>. <i>pseudomallei</i> cultured in the presence of nitrate. <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black circle), 6H4 mutant (white circle), 6H4/pME1039 complementation (white square) strains were grown for 72 h. Every 24 h, the numbers of viable bacteria were determined by plating on LB agar for colony count. (C) Growths of <i>B</i>. <i>pseudomallei</i> K96243 wild-type (black bar), 6H4 mutant (white bar) and 6H4/pME1039 (checked bar) strains under aerobic (+O₂) and anaerobic (-O₂) culture conditions at 48 h after bacterial inoculation. Results are presented from at least three replicates with three independent experiments. Asterisks indicate statistically significant differences (<i>P</i> < 0.05, <i>t</i>-test).</p

Nitrate reductase activity of <i>B</i>. <i>pseudomallei</i> wild-type and its derivative strains.

<p><i>B</i>. <i>pseudomallei</i> K96243 wild-type, 6H4 mutant and 6H4/pME1039 complemented strains were cultured in LB medium supplemented with 40 mM sodium nitrate. The nitrate reductase activitiy of each <i>B</i>. <i>pseudomallei</i> strain was determined under permeabilised (dot bar) or unpermeabilised conditions (black bar). The level of nitrate reductase activity was measured at absorbance 420 nm and 540 nm. Results are presented as standard errors of the means for experiments done in quadruplicate with two independent experiments. Asterisks indicate significant differences (<i>P</i> < 0.05, <i>t</i>-test).</p

Gene co-localization of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> and RT-PCR analysis.

<p>(A) Organization of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> genes and the location of primer pairs used in RT-PCR analysis. (B) RT-PCR analysis using primers 1039-F2 and 1040-R showed co-transcription of <i>B</i>. <i>pseudomallei bpsl1039-bpsl1040</i> genes (lane 3). Lanes 1 and 2 represent positive and negative controls, respectively, using wild-type genomic DNA and the extracted RNA, respectively. A negative RT-PCR control (lane 2) confirms that the band observed in the positive reaction is not DNA contamination. (C) RT-PCR analysis of <i>bpsl1040</i> expression, using primers 1040-F and 1040-R, was performed in <i>B</i>. <i>pseudomallei</i> wild-type (K96243) and 6H4 mutant. The 6H4 mutant showed the absence of <i>bpsl1040</i> expression. <i>B</i>. <i>pseudomallei</i> 16S rRNA gene was amplified as control. Lane M represents 1 Kb DNA marker ladder.</p