Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

Abstract

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ␤-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ␤-lactamase and was previously implicated in resistance to ␤-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

“…Consistent with this prior work, the amrR -mutated strain from CF9 encodes a frameshift in BPSL3085 , and in Pre-DPMS 89, two missense mutations affect this gene (Text S1). Strains from Pre-DPMS 89 and P215 also encode a PenA T147A variant, previously associated with high-level imipenem (32 μg/mL) and amoxicillin-clavulanate (32 μg/mL) resistance in the clinical B. pseudomallei strain, Bp1651 (18); however, despite encoding for this variant, all three strains (MSHR0052, MSHR0664 and MSHR0937) remained sensitive to imipenem (MICs = 0.5μg/mL), indicating that this mutation by itself does not confer imipenem resistance. It is possible that other mutations within penA are required to confer the high-level imipenem resistance phenotype observed previously (18).…”

“…Resistance to ceftazidime can occur by up-regulation or alteration of the β-lactamase PenA (encoded by penA ; (11–16)) or via the loss of penicillin-binding protein 3 (17). Point mutations in penA have recently been shown to lead to imipenem resistance, a carbapenem drug similar to meropenem (18), and missense and promoter mutations affecting penA confer resistance towards amoxicillin-clavulanate (12, 15). Up-regulation of multi-drug efflux pumps can also contribute to antibiotic resistance in B. pseudomallei .…”

Taking the next-gen step: comprehensive antimicrobial resistance detection from Burkholderia pseudomallei genomes

“…All encode a SAM-dependent methyltransferase truncation (BPSL3085 A88fs ) [23] and were predicted by ARDaP to be DOXr. We also observed BPSL3085 A88fs in an unrelated DOXr chronic CF strain, Bp1651 [28] (Table 1). BPSL3085 mutations confer DOXr by altering ribosomal methylation patterns [9,26].…”

Optical microscopy reveals the dynamic nature ofB. pseudomalleimorphology during β-lactam antimicrobial susceptibility testing

“…Then, original cell size, resembling those cells unexposed to antibiotics, was re-established at later time points. The time to morphology restoration was concentration dependent and may be attributed to the degradation of CAZ by the β-lactamase enzyme, PenA, which has acquired mutations correlating to high activity against this antibiotic [14]. In the presence of sub-inhibitory and inhibitory concentrations of cefotaxime, extended spectrum β-lactamase producing E. coli have also been shown to form filaments in lag and early exponential growth phases and revert back to non-elongated cells in exponential and stationary phase [37].…”

“…Drug inactivation and drug target modification are described as mechanisms of resistance. Mutations resulting in amino acid changes in and upstream of the β-lactamase gene penA confer resistance to AMC, CAZ and IPM in strain Bp1651 and to AMC in strain MSHR1655 [14-16]. In addition, a reversible gene duplication and amplification event in a chromosomal region containing penA resulted in acquired CAZ resistance [17].…”