Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates

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.

Correlation between gyrA and CmeR Box Polymorphism and Fluoroquinolone Resistance in Campylobacter jejuni Isolates in China

“…In contrast with previous studies (16, 24), we did not find mutations inactivating CmeR (16). Instead, several nucleotide variations were found in the CmeR-Box of our isolates, making 12 sequence variants (listed in Table 2).…”

“…The cmeABC operon encodes an important drug efflux pump in C. jejuni (22), and it is negatively regulated by CmeR, which binds to a 16-base inverted repeat (IR) sequence (TGTAATA[or T]TTTATTACA; the repeat sequences are underlined) named CmeR-Box located in the promoter region of the cmeABC operon (16,23). In contrast with previous studies (16, 24), we did not find mutations inactivating CmeR (16).…”

“…To investigate the molecular basis of high FQ resistance in the C. jejuni isolates, we analyzed the full gene sequences encoding the DNA gyrase subunits (gyrA and gyrB) (9,10) and the topoisomerase IV subunits (parC and parE) (11,12), which are known targets of FQ, the regulatory protein CmeR, and the sequence of the promoter region of cmeABC, which encodes an efflux pump (13,14), by DNA sequencing as previously described (15,16). The MICs of ciprofloxacin against the 39 isolates which represent different sequence variants were tested according to CLSI guidelines (17,28).…”

Human Campylobacteriosis in Italy: Emergence of Multi-Drug Resistance to Ciprofloxacin, Tetracycline, and Erythromycin

“…High variability in the CmeR-Box region was found, identifying 11 different sequence variants in our strains. Among them, some were previously described as significantly associated with macrolides resistance (Cagliero et al, 2007; Pérez-Boto et al, 2010; Grinnage-Pulley and Zhang, 2015; Zhou et al, 2016; Zhang et al, 2017). In our strains these modifications were not related to ERY resistance, being also identified in ERY-S strains, and some ERY-R strains showed no mutations in this locus.…”

“…The cmeR–cmeA intergenic region and the conserved CmeR-Box (5′- TGTAAT AAAT [or A] ATTACA-3′ ) were analyzed in 72 strains with the aim to identify mutations potentially associated with ERY resistance, as previously described (Cagliero et al, 2007; Pérez-Boto et al, 2010; Grinnage-Pulley and Zhang, 2015; Zhang et al, 2017)…”