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The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An  Escherichia coli  mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly - ). Generation of Hly -  clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the  hha  allele ( hha ::Tn 5 ). Detailed analysis of different Hly -  clones evidenced that recombination between partial IS 91  sequences that flank the  hly  operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly -  derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the  hha ydgT  mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS 91  flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type  E. coli  strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly -  clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS 91  is flanking several virulence determinants in different enteric bacterial pathogenic strains from  E. coli  and  Shigella . The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Antibiotics Shaping Bacterial Genome: Deletion of an IS91 Flanked Virulence Determinant upon Exposure to Subinhibitory Antibiotic Concentrations