Abstract
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.
Highlights
Multidrug efflux pumps play key roles in bacterial physiology, conferring intrinsic and acquired resistance to diverse toxic compounds
We investigated the expression of cmeABC in C. jejuni isolates from turkeys, chickens, and humans, examined the mechanisms associated with the differential expression, and measured the functional consequences associated with the differential expression
The region spanning from cmeR to cmeA was sequenced for all overexpression level (OEL) isolates, 4 of the wild typelevel (WEL) isolates, and C. jejuni strain 81–176 to identify genetic mutations that were potentially associated with the differential cmeABC expression (S1 Table)
Summary
Multidrug efflux pumps play key roles in bacterial physiology, conferring intrinsic and acquired resistance to diverse toxic compounds. ArcAB-TolC, MexAB-OprM, and MexXY-Z in Escherichia coli and Pseudomonas aeruginosa are examples of well-characterized RND efflux pumps that extrude bile salts, organic cations, detergents, and various classes of antimicrobials [2,3,4,5]. These efflux pumps have been associated with intrinsic and acquired resistance to antimicrobial compounds. CmeABC, a member of the RND family, is the predominant efflux pump in Campylobacter jejuni [6] and plays a key role in the resistance to structurally diverse compounds, such as bile salts, ciprofloxacin, erythromycin, ethidium bromide, and various detergents [6, 7]. Due to its significant role in bile resistance, CmeABC is essential for Campylobacter colonization and adaptation in the intestinal tract of animals [7]
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