The mechanisms of evolution of antibiotic resistance in Staphylococcus aureus
Final Report Abstract
Staphylococcus aureus is a pathogen that causes nosocomial and community-acquired infections. In recent years S. aureus has acquired resistance to nearly all antibiotics used in clinical practice. As a component of transposons, insertion (IS) elements are involved in the transfer of resistance genes between strains and species. Furthermore, the integration of a single IS element into a gene or its promoter may result in an inactivation or overexpression of the affected gene. IS256 is found frequently in ST239 and other ST8 strains. In the preliminary work we had shown that IS256 transposition activity is affected by low concentrations of antibiotics and that IS256 was involved in generation of intermediate vancomycin resistance in S. aureus in two VISA isolates (VISA: vancomycin intermediate S. aureus). Furthermore, we had observed that the activity of the stress sigma factor B inhibited the transposition of IS256. In this project we identified the 3’ end of the rsbU gene, which encodes a positive regulator of sigma factor B, as a hotspot for IS256 insertion in the clinical isolate S. aureus SA137/93G as well as in the laboratory strain S. aureus HG001 and a further ST239 isolate. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for such rsbU::IS256 insertion mutants. The higher fitness of the cells with low SigB activity might be conferred by their reduced cell wall thickness. In consequence of the inactivation of rsbU, the IS256 transposition frequency was increased 4-fold in the S. aureus HG001 mutant. Furthermore, several antisense IS256 antisense RNAs were characterised and inhibition by SigB could also be shown for IS1181. Sequencing of the genomes of the IS256 containing strains showed that all three ST247 isolates contained the highest number of IS256-insertions compared to all other sequenced strains. Other IS elements (IS1272, IS1181, IS431mec) and transposons (Tn5801, Tn4001 and Tn554) were also present. Several insertion sites of IS256 differed between the two VISA strains. In contrast, there was little evidence of transposition for the other mobile elements and only a few point mutations were detected. In the non-VISA isolate, IS256 was integrated into mutS leading to a mutator phenotype. In addition, it was possible to select a strain with elevated vancomycin resistance which contained an insertion in guaA after passages in the presence of low vancomycin concentrations. Vancomycin resistance was only visible under limiting concentrations of exogenous guanine, which leads to a decrease in intracellular GTP and will upregulate the stringent response. Furthermore, a quick reversion to the wildtype phenotype was observed that corresponded to loss of the insertion in most isolates. Another quick reversion to the wildtype phenotype was demonstrated for a heme dependent clone that harboured an insertion in hemY and showed the geno- and phenotype of a heme dependent SCV. In conclusion, the results of this project show that IS256 is a very active insertion sequence that conveys unanticipated genetic flexibility to S. aureus and – in the presence of subinhibitory concentrations of antibiotics – leads to formation of various mutant genotypes with increased resistance to antibiotics. In conclusion, subinhibitory concentrations of antibiotics during therapy must be avoided in order to slow down resistance development.
Publications
- Antibiotic-induced autoactivation of IS256 in Staphylococcus aureus. Antimicrob Agents Chemother. 2013; 57(12):6381-4
Schreiber F, Szekat C, Josten M, Sahl HG, Bierbaum G
(See online at https://doi.org/10.1128/AAC.01585-13)
