Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ

To determine the structure of the resistance region in an IncHI1 plasmid conferring resistance to multiple antibiotics, including gentamicin, recovered from a Salmonella enterica serovar Typhimurium isolate from a horse. Plasmids were recovered by conjugation. The plasmid type, resistance genes and their context were identified by PCR, cloning, hybridization and DNA sequencing. The sequence was compared using bioinformatic tools with available resistance region sequences. In isolate SRC27, an IncI1 plasmid, pSRC27-I, conferred streptomycin resistance via the strA and strB genes contained within Tn5393a. An IncHI1 plasmid, pSRC27-H, was found to carry the aacC2 gentamicin resistance gene within a 34.6 kb multiple antibiotic resistance region that included nine further antibiotic resistance genes, aadA2, aphA1, bla(TEM), catA1, dfrA12, strA and strB, sul2 and tetA(B), conferring resistance to streptomycin and spectinomycin, kanamycin and neomycin, ampicillin, chloramphenicol, trimethoprim, streptomycin, sulfamethoxazole and tetracycline, respectively. This complex resistance region has evolved from Tn2670 and Tn10 via loss and gain of DNA segments. It includes Tn6029 and Tn4352, and a new transposon carrying the aacC2 gene. It also contains five copies of IS26, two of IS1 and one each of IS10 and ISCfr1. This region of pSRC27-H is related to ones present at the same position in three sequenced IncHI1 plasmids, pHCM1, pO111_1 and pMAK1, but has acquired new segments carrying antibiotic resistance genes. Evolution via loss and gain of resistance genes has occurred within the large resistance region of pHCM1-type IncHI1 plasmids leading to different resistance phenotypes.