Kim H., Fittipaldi B., Hoque F., Wang C., Zefi O., Li W., Goldman Z., Peter Y., Basu P., Fittipaldi B.
Keywords:
Antibiotic tolerance,
d-Alanyl-d-alanine carboxypeptidases Pbp4, Group B streptococcus, Mechanisms of tolerance
Group B streptococci (GBS) bacteria are the leading cause of neonatal infections in the US. GBS strains may exhibit prolonged resilience to clinical treatments, becoming antibiotic tolerant. Penicillin tolerance (PT) in many Gram-positive bacteria is associated with the highly conserved penicillin-binding protein 4 (pbp4). To determine potential amino acid polymorphisms (AAP) associated with PT in GBS, we characterized the pbp4 gene of 48 clinical isolates. Defining PT as a detectable growth of ≥ 1× MIC90 in the penicillin killing and penicillinase regrowth assay, 7/48 (14.6%) of the strains were PT, and 11/48 (22.9%) were characterized as paradoxical-PT (PE–PT). Sequence alignment of pbp4, identified two AAPs, previously found to be in linkage disequilibrium, G168D, and V289I, that were associated with the PT strains. Within the isolates, the mutant pbp4 allele (pbp4mu) was found in 0/30 (0%) of susceptible, as opposed to 5/7 (71.4%) of PT and 7/11 (63.7%) of PE–PT samples (χ2 = 40.1; df = 2; P < 0.001). To confirm the effect of the G168D and V289I substitutions in GBS, we replaced the wild-type pbp4 (pbp4wt) in a susceptible strain (A909) with pbp4mu and substituted the pbp4mu with pbp4wt in a PT strain (090R). Prevalence of pbp4mu in A909 stabilized cell wall peptidoglycan cross-linkage, decreased membrane damage, and penicillin-mediated killing increasing regrowth tolerance. The reverse was seen upon expression of pbp4wt in 090R. Our findings indicate that AAPs in the penicillin-binding protein 4 of GBS generated by two novel single nucleotide polymorphisms (SNPs) in the pbp4 gene are associated with PT and that the occurrence of one or both mutations can serve as biomarkers used in real-time PCR-based screening assays for detection of PT in GBS and consequently improve treatment guidelines.
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