Mechanisms of penicillin-resistance in streptococcus uberis
Résumé
Background: S. uberis is a member of the streptococcal pyogenic group and a causative agent of bovine mastitis, which are frequently treated with penicillin. Despite the report of decreased susceptibility in certain environmental isolates, the ability of S. uberis to develop penicillin-resistance has not been documented yet. Thus, since multimodular PBPs are commonly considered as the major resistance determinants in penicillin-resistance, we looked for PBP mutations occurring during penicillin-resistance selection in the S. uberis reference strain ATCC 19436 and 9 susceptible environmental isolates. In parallel, we determined the PBP modifications accumulated in environmental bacteria naturally presenting a decreased susceptibility to penicillin. Methods: PBPs were identified by standard in silico analyses. Liquid cultures of S. uberis were cycled with increasing concentrations of penicillin and the increase in MIC was followed. Mutations were determined by sequencing the transpeptidase domains of PBP1A, PBP2B and PBP2X genes. Results: S. uberis carries 5 multimodular PBPs, which share homologies with other streptococcal PBPs and were named accordingly (class A PBP1A, 1B and 2A; class B PBP2B and 2X). By culture cycling, resistance increased stepwise from an MIC of about 0.016 μg/ml to 1-2 μg/ml in ca. 30 cycles. And the same trend was observed for each cycled isolate. PBP mutations were detected in PBP2X (including the characteristic streptococcal Q554E substitution), PBP2B and PBP1A, and accumulated concomitantly to the increase of resistance. Moreover, a high number of silent mutations was identified in these genes. Point mutations, but no mosaic-like PBPs, were also detected in the environmental isolates. Conclusion: In S. uberis, resistance development was readily obtained by culture cycling, contrarily to S. pyogenes. In the cycled strains as well as in the environmental isolates, decreased susceptibility was associated with point mutations in pbp genes, and no mosaic PBPs were detected in the natural strains. Consequently, while fully able to develop strongly decreased susceptibilities under laboratory conditions, S. uberis isolated from cattle mastitis might only present limited levels of penicillin-resistance probably because of the absence of a long-lasting bacterial community on the site of infection, contrarily to what is occurring in the buccal cavity for oral streptococci.