De novo acquisition of antibiotic resistance in six species of bacteria

Abstract

Bacteria can become resistant to antibiotics in two ways: by acquiring resistance genes through horizontal gene transfer and by de novo development of resistance upon exposure to non-lethal concentrations. The importance of the second process, de novo build-up, has not been investigated systematically over a range of species and may be underestimated as a result. To investigate the DNA mutation patterns accompanying the de novo antibiotic resistance acquisition process, six bacterial species encountered in the food chain were exposed to step-wise increasing sublethal concentrations of six antibiotics to develop high levels of resistance. Phenotypic and mutational landscapes were constructed based on whole-genome sequencing at two time points of the evolutionary trajectory. In this study, we found that (1) all of the six strains can develop high levels of resistance against most antibiotics; (2) increased resistance is accompanied by different mutations for each bacterium-antibiotic combination; (3) the number of mutations varies widely, with Y. enterocolitica having by far the most; (4) in the case of fluoroquinolone resistance, a mutational pattern of gyrA combined with parC is conserved in five of six species; and (5) mutations in genes coding for efflux pumps are widely encountered in gram-negative species. The overall conclusion is that very similar phenotypic outcomes are instigated by very different genetic changes. The outcome of this study may assist policymakers when formulating practical strategies to prevent development of antimicrobial resistance in human and veterinary health care.IMPORTANCEMost studies on de novo development of antimicrobial resistance have been performed on Escherichia coli. To examine whether the conclusions of this research can be applied to more bacterial species, six species of veterinary importance were made resistant to six antibiotics, each of a different class. The rapid build-up of resistance observed in all six species upon exposure to non-lethal concentrations of antimicrobials indicates a similar ability to adjust to the presence of antibiotics. The large differences in the number of DNA mutations accompanying de novo resistance suggest that the mechanisms and pathways involved may differ. Hence, very similar phenotypes can be the result of various genotypes. The implications of the outcome are to be considered by policymakers in the area of veterinary and human healthcare.

Keywords: antimicrobial resistance; de novo resistance; resistance genes; resistance mutations.

Fig 1

Resistance of six bacterial species evolved against six antibiotics: (A) S. enterica; (B) Y. enterocolitica; (C) A. pittii; (D) E. faecalis; (E) B. subtilis; and (F) S. aureus. The arrows indicate the days that samples for DNA analysis were taken; green indicates replicate 1, and red indicates replicate 2.

Fig 2

The MIC increase at the end of evolution in two replicates of six species of bacteria. Six strains evolved resistance against amoxicillin/cefepime, enrofloxacin, kanamycin, tetracycline, erythromycin, and chloramphenicol. The MIC of six strains at the end of the evolution experiment are compared to the MIC of the wild types. For each antibiotic, two MICs are given, as the duplicates sometimes differed. The MIC increase is expressed in 2^X.

Fig 3

The distribution of point mutations in six species of bacteria. Mutations in early (1) or final (2) time points examined in (A) amoxicillin/cefepime, (B) enrofloxacin, (C) kanamycin, (D) tetracycline, (E) erythromycin, and (F) chloramphenicol. Red color means target mutations; yellow color means efflux pump-related mutations; soft colors represent mutations found in one of the replicates; and bright represents mutation found in both replicates. Even though the mutations differed between replicates, the total number of mutations was more roughly equal for all sets of replicates.

Fig 4

Distribution of allele frequency at the second measurement and analysis of mutation type (A1) S. enterica, (A2) Y. enterocolitica, (A3) A. pittii, (A4) E. faecalis, (A5) B. subtilis, and (A6) S. aureus; panels: the change in acquired mutations from the measurement time point one to two is depicted with transparent green (positive change) and red (negative change) bars. (B) SNP, short insertion, and deletion proportion. (C) Proportion of mutations in the region of exon or non-exon. (D) Ratio of non-synonymous mutations divided by synonymous mutations.

Fig 5

Growth rate as indicator of fitness cost measured in the absence of antibiotics of strains made resistant to the indicated antibiotic: (A) S. enterica, (B) Y. enterocolitica, (C) A. pittii, (D) E. faecalis, (E) B. subtilis, and (F) S. aureus.

Fig 6

The mutational pattern is examined in six species of bacteria against six antibiotics.