The point mutation A1387G in the 16S rRNA gene confers aminoglycoside resistance in and .

Thermotolerant spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient and was ~2.5 log lower compared to the control -A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177-1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA_A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among . spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures.