Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis

Abstract

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.

Keywords: Bartonella; antibiotic resistance; evolution; rifampicin; slow-growing bacteria.

Fig. 1.

Rifampicin resistant mutants are generated within the inhibition zone of antibiotic diffusion tests after prolonged incubation. (A) Bartonella krasnovii wild-type strain showed the emergence of colonies (indicated by yellow arrows) within the inhibition zone produced by rifampicin (RIF) Etest after prolonged incubation. (B) The colonies that grew within the inhibition zone presented higher resistance phenotype after isolation and re-evaluation of their minimal inhibition concentration (MIC) on subsequent Etests. (C) RIF resistant colonies emerged on chocolate and/or blood agar plates containing in house RIF disks (RD, 25 µg RIF). (D) Resistant colonies are not preexisting and only emerge on RIF diffusion disk plates (RD), whereas plates with homogeneous RIF concentrations inhibited the emergence of resistant colonies.

Fig. 2.

Bartonella krasnovii phenotypic response to RIF exposure. (A) Bartonella krasnovii population analysis profile (PAP) for RIF, indicating the minimal inhibition concentration for the 99% of the population (MIC₉₉) and the heteroresistance window from the lowest antibiotic concentration giving maximum growth inhibition (0.005 µg/ml) to the highest noninhibitory concentration (0.04 µg/ml). (B) Dye-dilution test of B. krasnovii liquid cultures marked with eFluor450. After 5 days of incubation with different RIF concentrations at 37 °C (control at 4 °C), the bacteria were labelled with live/dead markers, Syto9 and propidium iodine (PI), and analyzed on ImageStream. eFluor450 intensities correspond to the survival population (Syto9+, PI−). (C) Death curve on RIF (0.2 µg/ml; 10X-MIC), indicating the minimal duration for killing the 99% of the population (MDK₉₉=8 days). Analysis based on liquid cultures of B. krasnovii monitored for live bacteria every 24 h on antibiotic-free plates. Error bars indicate SD from the mean.

Fig. 3.

RIF degradation test during incubation. (A) Sterile chocolate agar plates with RIF diffusion disks (RD) were incubated at 37 °C + 5% CO₂. At every time point (from 0.5 to 10 days), three plates were recovered, and three agar plugs were cut and processed per plate. RIF was extracted with acetonitrile and measured by HPLC. (B) RIF concentrations surpassed the MIC_original (0.02 µg/ml, indicated by a blue-dash line) after 24 h, and maintained at supra-MIC for the rest of the experiment. (C) Confirmation assay of accumulation of RIF during time. Five sterile RD plates were incubated, recovered at different time points, and inoculated with fresh Bartonella krasnovii liquid cultures. Fresh RD plates were prepared in parallel and used as controls (N = 5). (D) After reincubation of aged and fresh inoculated RD, the inhibition zones were measured and compared. Fold change of the radius measured showed that aged plates always produced higher inhibition zones (above the FC = 1, red dash line). (E) Representative RD plates from the previous experiment (C and D) showing the increment of the inhibition zone in aged plates.

Fig. 4.

A mathematical model for microbial growth and adaptation in the presence of a drug due to heteroresistence and resistance. (A) Illustration of the ordinary differential equation system of the model, logistic growth is assumed but not shown. The model was run at experimentally defined parameters (i.e., normal rates) versus hypothetical rates representing increments of µ, mutation, and v, sensitive-to-heteroresistance switching rates, and inoculum size (all N = 20 runs per group). (B) Resulting number of hypothetical resistant (top panel) and heteroresistant colonies (bottom panel) obtained in the model (altering µ and/or and v) versus the experimental data. Model parameters that best fitted the experimental results (purple) are highlighted in yellow. (C) Resulting number of hypothetical resistant colonies obtained by increasing the initial population size (N_{0 alt}) and the heteroresistant mutation rates (µ_H). (D) Resulting number of mutated cells during a 24 h period obtained per parameter set, altering heteroresistant, µH, or general population mutation rates, µS, and allowing maximum growth rate of sensitive cells during incubation (r_S=r_R).

Fig. 5.

The emergence of resistant colonies within the inhibition zone of RD is inhibited at microaerophilic conditions. (A) Comparison of parallel cultures seeded on RD plates and incubated at either capnophilic or microaerophilic conditions (N = 30 per group). Right panel illustrating representative RD plates incubated at the different oxygen atmospheres (yellow arrows highlighted the emergence of CFUs on the inhibition zone of capnophilic incubated plates). ****P < 0.00001. (B) Mutation spectra obtained from colonies isolated from the inhibition zone of RD plates versus colonies recovered from typical fluctuation test RIF plates. χ² test for homogeneity is indicated.

Fig. 6.

Differential RNA expression of Bartonella krasnovii in response to RIF stress. (A) Heatmap of the RNAseq results from the seeding cultures (0h) and the cultures incubated for 18h with or without RIF (0.02 µg/ml). (B) Volcano plots of the pairwise comparison between the treatments.

Fig. 7.

Major differences in the RNA expression of Bartonella in response to RIF stress. Fold change (log2) of the RNA expression between 18h RIF versus 18h free cultures of: (A) Repair genes; (B) Phage genes (prophage nomenclature based on phaster.ca); (C) Plasmid genes; (D) Transcriptional regulators. Red dash lines indicate the threshold of FC of 1.5 applied. Abbreviations: GTA, gene transfer agent loci; ROR, run-off replication loci; BER, base excision repair; DSBR, double-strand break repair; HR, homologous recombination; MMR, DNA mismatch repair; NER, nucleotide excision repair; T4SS, type IV secretion system. Error bars indicated SD from the mean.