Macrolide resistance trajectories across three Bordetella pertussis genetic backgrounds under stepwise erythromycin exposure

Background Macrolide resistance in Bordetella pertussis remains a clinical concern, yet how broader genomic background may be associated with resistance-associated trajectories under antibiotic exposure is not fully understood. Methods We performed stepwise erythromycin (ERY) induction using three clinical macrolide-susceptible parental isolates representing clinically relevant genetic backgrounds. Resistance-associated variants were tracked by whole-genome sequencing together with phenotypic readouts, including antimicrobial susceptibility testing, growth and biofilm assays, and ELISA-based measurements of selected virulence- and metabolism-associated factors. Results ERY minimum inhibitory concentration (MIC) trajectories diverged across genetic backgrounds. B19068 and B181 reached high-level resistance and exhibited a 23S rRNA G2046A substitution in the consensus sequence, whereas B197 followed a lower-MIC trajectory and accumulated variants in efflux-associated loci. Whole-genome sequencing further revealed genomic alterations across genetic backgrounds, including structural variation in a subset of isolates, indicating that serial passaging under antibiotic exposure can be accompanied by genomic alterations beyond candidate resistance loci. Antibiotic-exposed lineages also displayed differences in growth and biofilm formation across genetic backgrounds, and ELISA readouts (PT, FHA, LPS, DHFS, and DHFR) differed across backgrounds under the in vitro conditions used. Conclusion Under a controlled induction regimen, three representative clinical genomic backgrounds exhibited divergent macrolide-resistance trajectories and accompanying phenotypic differences. These observations are consistent with background-linked differences in resistance evolution and provide a basis for future validation in replicated evolution experiments.