Effect of upstream regulatory regions and growth conditions on erm(B)-mediated constitutive MLS resistance in Group A streptococci

Objective: The upstream sequence of the gene is considered a key factor in the translational control of erm expression, which can lead to either a constitutive (cMLS) or an inducible (iMLS) macrolide, lincosamide, and streptogramin B resistance phenotype. We observed that some GAS expressing a cMLS phenotype and harbouring erm(B) did not show a constant phenotype. The present work sought to study the relationship occurring between this phenotypic instability and the sequence context of the erm(B). Methods: cMLS phenotype of five GAS clinical isolates was studied by growth in liquid medium using cells induced by preincubation with ERY (0.1 mg/L) followed by challenge with either josamycin (JOS) or clindamycin (CLI) (both at 50 mg/L) in supplemented Muller-Hinton II (MH). All cultures were incubated at 35–37ºC in 5% CO₂ for a maximum of 24 h. The effect of inoculum preparation (broth culture or direct colony suspension) was also evaluated. The sequence of the erm(B) regulatory regions were determined following standard procedures and the general characteristics of the genetic elements harbouring erm(B) were determined using PCR. Results: When inoculum was prepared by direct colony suspension, both CLI and JOS resistance of 3 strains was induced during the logarithmic phase of growth (i-cMLS). When the inoculum was prepared by the growth method, the same set of strains were insensible to the induction of CLI and JOS resistance but were susceptible to both antibiotics until the end of the logarithmic phase. In these strains, the upstream regulatory sequences and the genetic context surrounding erm(B) were identical. A fourth strain had the same genetic characteristics of the i-cMLS strains but showed a constitutive MLS resistance in all the tested growth conditions, as well as a cMLS strain used as reference, the latter having a consistent deletion of the upstream regulatory region of the erm(B). Conclusions: In GAS, the expression of erm(B)-mediated MLS resistance strongly depends on growth conditions. The contribution of erm(B) specific sequences seems to play a secondary role.