Rampant Parasexuality Evolves in a Hospital Pathogen during Antibiotic Selection

Abstract

Horizontal gene transfer threatens the therapeutic success of antibiotics by facilitating the rapid dissemination of resistance alleles among bacterial species. The conjugative mobile element Tn916 provides an excellent context for examining the role of adaptive parasexuality as it carries the tetracycline-resistance allele tetM and has been identified in a wide range of pathogens. We have used a combination of experimental evolution and allelic frequency measurements to gain insights into the adaptive trajectories leading to tigecycline resistance in a hospital strain of Enterococcus faecalis and predict what mechanisms of resistance are most likely to appear in the clinical setting. Here, we show that antibiotic selection led to the near fixation of adaptive alleles that simultaneously altered TetM expression and produced remarkably increased levels of Tn916 horizontal gene transfer. In the absence of drug, approximately 1 in 120,000 of the nonadapted E. faecalis S613 cells had an excised copy of Tn916, whereas nearly 1 in 50 cells had an excised copy of Tn916 upon selection for resistance resulting in a more than 1,000-fold increase in conjugation rates. We also show that tigecycline, a translation inhibitor, selected for a mutation in the ribosomal S10 protein. Our results show the first example of mutations that concurrently confer resistance to an antibiotic and lead to constitutive conjugal-transfer of the resistance allele. Selection created a highly parasexual phenotype and high frequency of Tn916 jumping demonstrating how the use of antibiotics can lead directly to the proliferation of resistance in, and potentially among, pathogens.

The Ribosomal S10 Protein Is a General Target for Decreased Tigecycline Susceptibility

“…Interestingly, all colonies from one population had a twelve-nucleotide deletion in rpsJ (S10 R53Q-⌬54-57ATHK ) that is identical to a deletion identified in strains of E. faecalis S613 that were also adapted in vitro to TGC resistance in a previous study ( Fig. 1) (10). The net effect of the deletion is to remove residues 54 to 57 but restore the reading frame of the S10 protein and change arginine 53 to glutamine.…”

“…To date, clinical isolates expressing tetM have not been shown to confer resistance to TGC, although we showed recently that overexpression of tetM or amplification of gene copy number can produce TGC resistance in E. faecalis during experimental evolution (10). Using PCR, we established that E. faecium R499 does not carry any tetracycline resistance genes.…”

“…The clinically isolated strains Enterococcus faecalis S613 (GCA_ 000163795.1) (11), E. faecalis R712 (GCA_000163815.1) (11), E. faecalis S613(S10 R53Q-⌬54-57ATHK ) (SAMN03164156) (10), Enterococcus faecium 105 (12), E. faecium R499 (GCA_000294875.1) (13), methicillin-resistant S. aureus MRSA131 (GCA_000187145.1), and A. baumannii AB210 (GCA_000189655.2) (14) and the lab strains E. faecalis OG1RF (GCA_000172575.2) and E. coli BW25113 (GCA_000750555.1) were used for this study. Enterococcal strains were cultured in 80% lysogeny broth-20% brain heart infusion (LBHI) medium at 37°C with or without agitation.…”

“…This could be due to the essential role of S10 in translation and transcription; S10 is both a component of the 30S ribosomal subunit and an important transcription factor involved in lambda N-mediated antitermination (20). In the absence of successful allelic replacement, we tested the effect of rpsJ mutations directly using an evolved strain with only a single mutation in rpsJ, as determined by whole-genome sequencing and showed that it had an elevated MIC of 0.5 g/ml, compared to 0.125 g/ml for the susceptible ancestral strain (Table 1) (10). This shows that a mutation in S10 alone can reduce susceptibility to TGC.…”

“…The averages for three replicates run in parallel were plotted; error bars show standard deviations. (10). Therefore, we adapted two strains of E. faecium to TGC: one that carries tetM (E. faecium 105) and one without tetM (E. faecium R499).…”