Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates

Abstract

A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.

FIG. 1

PCR amplicons of STEC integrons. Lanes 2 to 5 were generated with the CS primers specific for class 1 integron. Lanes 6 to 9 were obtained using the sul primers specific for the sul gene that confers sulfonamide resistance. Lanes 10 to 13 were obtained using the primers specific for integrase. Specifically, lanes 2, 6, and 10 show positive controls (serovar Typhimurium DT104); lanes 3, 7, and 11 show E. coli O111:H8 strain CVM1877; lanes 4, 8, and 12 show E. coli O157:H7 strain CVM990; and lanes 5, 9, and 13 show negative controls (E. coli K-12).

FIG. 2

PFGE profiles with XbaI digestion of donor, recipient, and transconjugant strains. Lanes 1 to 5 show, respectively, results obtained with donor strains CVM990 (E. coli O157:H7) and CVM1884, (E. coli O111:NM) and recipient strains JM263 (E. coli O157:H7), H. alvei CVM1202, and H. alvei CVM1203 before the conjugations. Lanes 6 to 8 show results with the transconjugants CVM990-JM263, CVM990-CVM1202, and CVM990-CVM1203, respectively, that have gained a 90-kb band. Lanes 9 to 11 show results with the transconjugants CVM1884-JM263, CVM1884-CVM1202, and CVM1884-CVM1203, respectively, that have gained a 127-kb band.