Genome-wide association studies reveal distinct genetic correlates and increased heritability of antimicrobial resistance in Vibrio cholerae under anaerobic conditions

Abstract

The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria are exposed to anaerobic conditions within the gastrointestinal tract, yet little is known about how oxygen exposure influences AMR. The facultative anaerobe Vibrio cholerae was chosen as a model to address this knowledge gap. We obtained V. cholerae isolates from 66 cholera patients, sequenced their genomes, and grew them under anaerobic and aerobic conditions with and without three clinically relevant antibiotics (ciprofloxacin, azithromycin, doxycycline). For ciprofloxacin and azithromycin, the minimum inhibitory concentration (MIC) increased under anaerobic conditions compared to aerobic conditions. Using standard resistance breakpoints, the odds of classifying isolates as resistant increased over 10 times for ciprofloxacin and 100 times for azithromycin under anaerobic conditions compared to aerobic conditions. For doxycycline, nearly all isolates were sensitive under both conditions. Using genome-wide association studies, we found associations between genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations. These AMR phenotypes were more heritable, and the AMR-associated genetic elements were more often discovered, under anaerobic conditions. These AMR-associated genetic elements are promising targets for future mechanistic research. Our findings provide a rationale to determine whether increased MICs under anaerobic conditions are associated with therapeutic failures and/or microbial escape in cholera patients. If so, there may be a need to determine new AMR breakpoints for anaerobic conditions.

Keywords: Vibrio cholerae; anaerobic; antibiotics; antimicrobial resistance; cholera; enteropathogens.

Fig. 1.

Distribution of MICs under aerobic and anaerobic conditions among clinical isolates from the primary collection. Ciprofloxacin (CIP) (a), azithromycin (AZI) (b) and doxycycline (DOX) (c). Data are from 67 human-shed V. cholerae isolates. The MICs for each isolate under aerobic (green) and anaerobic (blue) conditions were enumerated, and the number of isolates with a given MIC (µg ml⁻¹) are represented as bars; values in grey text represent concentrations not tested (Table S4). Dotted lines are the breakpoints for resistance as per CLSI standards, which are based on assays under aerobic conditions (CIP=2 µg ml⁻¹; AZI=8 µg ml⁻¹; DOX=8 µg ml⁻¹). S, Sensitive; R, resistant. * indicates a significant difference in the frequency of isolates identified as resistant to ciprofloxacin and azithromycin by McNemar’s test (both P<0.001).

Fig. 2.

AMR phenotypes and known AMR genetic elements in human-shed V. cholerae isolates from the primary collection. Pink, yellow and blue colour coding is used to indicate AMR phenotypes and genotypes to azithromycin (AZI), doxycycline (DOX) and ciprofloxacin (CIP), respectively; colours blend when overlapped. Isolates with no resistance (sensitive) are shown in white circles. (a) Proportional Venn diagram (Euler) of AMR phenotypes to AZI, DOX and/or CIP under aerobic (left) and anaerobic conditions (right). Counts indicate the number of isolates with the corresponding phenotype. (b) AMR genotypes from known resistance genes in whole-genome sequences. The distribution of known AMR genetic elements is shown by proportional Venn diagram (Euler; left) and bar chart (right). On the right, the x-axis of the bar chart depicts the presence (black points) of known AMR genes [mphA, gyrA, parC, tet(59), qnr_Vc ] in a given genome and the y-axis depicts the number of isolates that share the given combination of AMR genes. Coloured bars to the left indicate the number of isolate genomes encoding resistance genes to CIP (blue), AZI (pink) or DOX (yellow). AMR genetic elements to other antibiotics are not shown.

Fig. 3.

Correlation analysis of growth phenotypes at different concentrations of antibiotics under aerobic and anaerobic conditions among V. cholerae clinical isolates from the primary collection. Antibiotic exposures were to ciprofloxacin (a), azithromycin (b) and doxycycline (c). AUC was analysed as the growth parameter. Aerobic/anaerobic conditions are labelled horizontally and vertically with the antibiotic concentration in µg ml⁻¹ (e.g. anaerobic-0.06). Analyses are grouped: aerobic versus aerobic, red boxes; anaerobic versus aerobic, purple boxes; anaerobic versus anaerobic, blue boxes. Heatmaps show correlation coefficients (the keys are on the right) for similar (yellow) versus dissimilar (purple) growth at two given conditions.

Fig. 4.

Distribution of AMR genes associated with AMR growth phenotypes at different concentrations of antibiotics under aerobic and anaerobic conditions among isolates from the primary collection. Venn diagrams show the overlap between genes associated with (a) ciprofloxacin resistance under aerobic versus anaerobic conditions, (b) ciprofloxacin at different concentrations (µg ml⁻¹) under aerobic conditions, (c) ciprofloxacin at different concentrations (µg ml⁻¹) under anaerobic conditions, and (d) doxycycline at different concentrations (µg ml⁻¹) under anaerobic conditions. Genes shown in boxes had statistically significant associations.

Fig. 5.

Antibiotic detection in stool supernatants by LC-MS/MS among cholera samples from the primary collection. Green with +, detected; white with −, not detected. CIP, Ciprofloxacin; TET/DOX, tetracycline and/or doxycycline; NAL, nalidixic acid; MET, metronidazole; BAC, sulfamethoxazole and/or trimethoprim; AMO, amoxicillin; ERY, erythromycin; CEF, ceftriaxone. Stool supernatants were not available for EN80, 86, 88, 92, 100, 103, 109, 116–120, 126, 124, 130 and 131.