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, 3 (9), e519

Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia Pseudomallei Clinical Isolates From Thailand

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Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia Pseudomallei Clinical Isolates From Thailand

Lily A Trunck et al. PLoS Negl Trop Dis.

Abstract

Background: Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a.

Methodology/principal findings: qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s). Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively). This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model.

Conclusions/significance: Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Delta(amrAB-oprA) mutants in such experiments.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. amrB transcript levels in gentamicin susceptible and resistant strains.
mRNA levels in LSLB without antibiotics-grown late-log cultures of the indicated strains were determined with an amrB-specific primer set. Data were normalized using the 23S rRNA gene as the housekeeping control. amrB transcript levels were determined A in strain 2188a and its gentamicin resistant derivative Bp35 and B in strain 3799a and its gentamicin resistant derivative Bp24. Relative quantifications were performed using 2188a and 3799a, respectively.
Figure 2
Figure 2. Extent of chromosome 1 deletion in strain 708a compared to K96243.
708a contains a deletion fusing the bold sequences of BPSL1717 and BPSL1807, respectively. Some notable genes and gene clusters present in K96243 but missing from 708a are: 1 amrR-amrAB-oprA; 2 a three gene operon (BPSL1801-BPSL1800-BPSL1799) encoding a putative type-1 fimbrial protein along with its outer membrane usher protein and chaperone; 3 the 13 gene malleobactin biosynthetic gene cluster and its extracytoplasmic sigma factor MbaS defined by mbaF-fmtA-mbaA-mbaI-mbaJ-mbaE-BPSL1781-BPSL1782-BPSL1783-BPSL1784-BPSL1785-BPSL1786-mbaS; 4 a cluster of 18 genes (BPSL1755-BPSL1773) encoding a putative aerobic (or late cobalt insertion) vitamin B12 biosynthetic pathway with an embedded gene (BPSL1763) encoding a putative exported chitinase; 5 arcD (BPSL1742) and arcABC (BPSL1743-BPSL1744-BPSL1745) coding for the arginine deiminase pathway; and 6 a two gene cluster (BPSL1732-BPSL1731) coding for a putative methyl-accepting chemotaxis citrate transducer and chemotaxis protein CheW2, respectively. Strain 1710b contains an additional 10 kb of DNA in this region.
Figure 3
Figure 3. Large deletion verification in chromosome 1 of strain 708a by whole genome sequencing.
Genomic sequencing data from strain 708a were aligned against the K96243 reference genome. Panel A shows the read density near positions 2,024,621 and 2,155,359 on chromosome 1. Panel B shows the 708a read density across the ∼4.5 Kb flanking the deletion in chromosome 1 of strain K96243. The yellow highlighted region in panel B marks a ∼130.7 Kb region with a near-zero read coverage, which correspond to the panel A coordinates. This lack of reads is strong evidence for deletion of the entire region in strain 708a.
Figure 4
Figure 4. Strain 708a is fully virulent in an acute murine melioidosis infection model.
BALB/c mice (n = 4–5 mice) were infected intranasally with 5×103 CFUs of 1026b ▪, 5×103 CFUs of strain 708a ▴, and 5×103 • or 5×104 ○ colony forming units of the isogenetic Δ(amrRAB-oprA) 1026b derivative Bp50. Statistical differences in survival times were determined by Kaplan-Meier curves followed by log-rank test. The Bonferroni corrected threshold was applied and comparisons with p <0.017 were considered significant. (**, p <0.01 for strain 1026b vs. Bp50 (5,000 CFU) and 708a vs. Bp50 (5,000 CFU). Data are representative of 2 independent experiments.
Figure 5
Figure 5. Deleted genes are absent from the 708a genome.
PCR was performed with genomic DNA isolated from K strain K96243 or A strain 708a with gene-specific primers. These included 2037 & 2038 for BPSL1801, 2035 & 2036 for BPSL1774, 2033 & 2034 for BPSL1755, 1954 & 1955 for BPSL1743, 2031 & 2032 for BPSL1732, and 1742 & 1743 for BPSL1810. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. Sizes of the expected PCR fragments (in bp and based on K96243 genomic sequence) are indicated above the respective bands. Gene annotations are according to K96243 and gene names, where known, are in parentheses. Lanes M contained the Hi-Lo DNA ladder (Minnesota Molecular, Minneapolis, MN) and the sizes of pertinent fragments are indicated on the left.

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References

    1. Cheng AC, Currie BJ. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev. 2005;18:383–416. - PMC - PubMed
    1. Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ. Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol. 2006;4:272–282. - PubMed
    1. Currie BJ, Dance DAB, Cheng AC. The global distribution of Burkholderia pseudomallei and melioidosis: an update. Trans R Soc Trop Med Hyg. 2008;102/S1:S1–S4. - PubMed
    1. Stone R. Racing to defuse a bacterial time bomb. Science. 2007;317:1022–1024. - PubMed
    1. Ciervo A, Mattei R, Cassone A. Melioidosis in an Italian tourist injured by the tsunami in Thailand. J Chemother. 2006;18:443–444. - PubMed
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