Emergence of a Potent Multidrug Efflux Pump Variant That Enhances Campylobacter Resistance to Multiple Antibiotics

Abstract

Bacterial antibiotic efflux pumps are key players in antibiotic resistance. Although their role in conferring multidrug resistance is well documented, the emergence of "super" efflux pump variants that enhance bacterial resistance to multiple drugs has not been reported. Here, we describe the emergence of a resistance-enhancing variant (named RE-CmeABC) of the predominant efflux pump CmeABC in Campylobacter, a major zoonotic pathogen whose resistance to antibiotics is considered a serious antibiotic resistance threat in the United States. Compared to the previously characterized CmeABC transporters, RE-CmeABC is much more potent in conferring Campylobacter resistance to antibiotics, which was shown by increased MICs and reduced intracellular accumulation of antibiotics. Structural modeling suggests that sequence variations in the drug-binding pocket of CmeB possibly contribute to the enhanced efflux function. Additionally, RE-CmeABC expands the mutant selection window of ciprofloxacin, enhances the emergence of antibiotic-resistant mutants, and confers exceedingly high-level resistance to fluoroquinolones, an important class of antibiotics for clinical therapy of campylobacteriosis. Furthermore, RE-CmeABC is horizontally transferable, shifts antibiotic MIC distribution among clinical isolates, and is increasingly prevalent in Campylobacter jejuni isolates, suggesting that it confers a fitness advantage under antimicrobial selection. These findings reveal a new mechanism for enhanced multidrug resistance and an effective strategy utilized by bacteria for adaptation to selection from multiple antibiotics.

Importance: Bacterial antibiotic efflux pumps are ubiquitously present in bacterial organisms and protect bacteria from the antibacterial effects of antimicrobials and other toxic compounds by extruding them out of cells. Thus, these efflux transporters represent an important mechanism for antibiotic resistance. In this study, we discovered the emergence and increasing prevalence of a unique efflux pump variant that is much more powerful in the efflux of antibiotics and confers multidrug resistance in Campylobacter, which is a major foodborne pathogen transmitted to humans via the food chain. Unlike other specific resistance determinants that only allow bacteria to resist a particular antimicrobial, the acquisition of a functionally enhanced efflux pump will empower bacteria with simultaneous resistance to multiple classes of antibiotics. These findings reveal a previously undescribed mechanism for enhanced multidrug resistance and open a new direction for us to understand how bacteria adapt to antibiotic treatment.

FIG 1

Accumulation of ciprofloxacin in NCTC 11168, 11168-V1, and 11168-V2. Each bar represents the mean and standard deviation of triplicate samples. A t test was used to perform the statistical analysis. *, P < 0.05; **, P < 0.01.

FIG 2

Sequence analysis and structure prediction of CmeB. (a) Phylogenetic analysis of Campylobacter CmeB sequences identified in this study and deposited in the GenBank database. Different Campylobacter species are shown in different colors as follows: red, C. coli; blue, C. jejuni; green, other Campylobacter species. The purple square indicates CmeB of isolate DH161. The unique subtree of RE-CmeB and its homologs from C. jejuni and C. coli is shaded gray. The purple dot indicates multidrug efflux RND transporter AcrB (accession number WP_044694729.1) from E. coli, which was used as the outgroup. (b) The CmeB structures predicted by the Modeller program. For clarity, only the predicted periplasmic domain structures of NCTC 11168 CmeB (pink) and RE-CmeB (DH161, light green) are shown. The 22 mutated residues located in the periplasmic drug-binding cavity of the mutant transporter are represented by green sticks. The corresponding residues in wild-type CmeB are represented by cyan sticks. (c) The predicted bound ciprofloxacin molecules are magenta. The residues involved in binding are represented by sticks. (d) The predicted bound florfenicol molecules are orange. The residues involved in binding are represented by sticks.

FIG 3

Distribution of ciprofloxacin and florfenicol MICs for Campylobacter isolates with RE-cmeABC or other CmeABC types. (a) Distribution of ciprofloxacin MICs for C. jejuni. (b) Distribution of ciprofloxacin MICs for C. coli. (c) Distribution of florfenicol MICs for C. jejuni. (d) Distribution of florfenicol MICs for C. coli. In all panels, “Variant” indicates strains that contain RE-cmeABC while “Wildtype” depicts isolates containing a cmeABC operon that is not RE-cmeABC.

FIG 4

PFGE analysis of representative RE-cmeABC-positive Campylobacter isolates. SmaI was used for digestion. The regions of isolation (provinces and cities) include Guangdong (GD), Shandong (SD), Ningxia (NX), Henan (HN), and Shanghai (SH). The host species include chickens (C), swine (S), and ducks (D). (I) Representative RE-cmeABC-positive C. jejuni (CJ) isolates (n = 36). With 80% genetic similarity as a cutoff, the C. jejuni isolates were grouped into 17 clusters (PFGE patterns represented by multiple strains) and 24 unique PFGE patterns. (II) Representative RE-cmeABC-positive C. coli (CC) isolates (n = 15). C. coli isolates were grouped into 11 clusters and 15 unique PFGE patterns. (III) Representative RE-cmeABC-positive C. jejuni isolates from Henan Province in 2014 (n = 25). The isolates were grouped into two clusters and three unique PFGE patterns.