Jpn. J. Infect. Dis., 56, 123-126, 2003

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Laboratory and Epidemiology Communications

Drug Resistance Genes Encoded in Integrons and in Extra-Integrons: Their Distribution and Lateral Transfer among Pathogenic Enterobacteriaceae including Enterohemorrhagic Escherichia coli and Salmonella enterica Serovars Typhimurium and Infantis

Kokichi Hamada*, Kahori Oshima and Hidetaka Tsuji

Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe 652-0032

Communicated by Kazue Tabita

(Accepted July 2, 2003)

*Corresponding author: Mailing address: Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032, Japan. Fax: +81-78-531-7080

Salmonella enterica serovars Typhimurium and Infantis have been major causes of Salmonella infections in Japan during the past decades, though S. Enteritidis suddenly emerged in 1989 and continues to prevail (1). While rare in S. Enteritidis, multidrug resistance (MDR) is frequent among S. Typhimurium and S. Infantis (1). The drug resistance genes are transferred among these Salmonella spp. along with the class 1 integrons (1) present in transposons and conjugative plasmids (1,2). Class 1 integrons are predominant within (2,3) and outside (4,5) the family Enterobacteriaceae. In Japan, enterohemorrhagic Escherichia coli (EHEC), particularly O157, O26, and O111 serotypes, has prevailed since 1996. Though well documented for O157 and O111 serotypes, integron-mediated antibiotic resistance among O26 serotype has remained relatively unknown (3). Here, we present a systematic investigation of drug resistance genes carried by integrons (2-5) or extra-integrons (2,4,6) in S. Typhimurium, S. Infantis, and EHEC with special reference to their transferability.

The strains used in this study were collected by authors. Salmonella strains were those used previously (1) and one additional S. Infantis strain (Inf32). EHEC strains were 29 serotype O26 strains (including two verotoxin non-producers), one O111, and three O157 strains, all collected in 1996-2003. The O111 and O157 EHEC strains were chosen randomly from a small number of MDR strains (resistant to more than three drugs) found in approximately 550 strains collected in 1996 - 2002.

The strains were tested for sensitivities to ampicillin (Am), cefotaxime, kanamycin (Km), gentamicin, streptomycin (Sm), tetracycline (Tc), trimethoprim (Tm), ciplofloxacin, fosfomycin (Fm), chloramphenicol (Cm), sulphamethoxazole (Su), and nalidixic acid (Na). We used antibiotic disks (Becton Dickinson Microbiology Systems, Cockeysville, Md., USA) on Mueller-Hinton agar (MH) plates and agar dilutions on MacConkey (MAC) and/or MH agar plates (1). Table 1 shows the antibiograms of 14 MDR S. Typhimurium strains (among 22 strains tested), all the tested 12 MDR S. Infantis strains, and all the tested 11 MDR E. coli strains. A susceptible E. coli ('02-S.031) and a susceptible Salmonella (Inf01) were included for comparison. By means of polymerase chain reaction (PCR) (1), we searched for class 1 integrons, for ant (3")-1a and qac△E1sul1 in close association with the 3'-conserved segment (3'-CS) of integron (2-5), and for drug resistance genes often located in integrons (2-5) or outside of integrons (2,4,6) . The primer pairs used for PCR and their PCR products are shown in Fig. 1. Primer concentration was 0.2 mM and Taq polymerase concentration 0.25 units/50 ml for all the genes except for tet genes (tetA, B, C, D, and E [4]). For PCR of tet, the primer concentration was 0.4 mM, and Taq polymerase concentration 0.5 units/50 ml.

Table 1 summarizes the characteristics of bacterial strains examined in the present and previous (1) reports.

  1. When the int l primer pair was used, various sizes of PCR products were obtained. We conveniently classified them in terms of the size of PCR product. Among 22 drug-resistant S. Typhimurium strains, seven had 1.0-kb and 1.2-kb integrons (such strains are called A type) and five strains had a 2.0 kb-integron only (they are called B type). All the 12 MDR S. infantis strains had a 1.0 kb-integron only (they are called C type). One non-EHEC E. coli strain ('96-E.094) was C type.
  2. All the pathogenic species of E. coli and Salmonella harboring integrons possessed an ant(3")-1a gene (0.75 or 2.0 kb in PCR product size) encoding an aminoglycoside-modifying enzyme.
  3. aadA2 encoding aminoglycoside-adenyltransferase was detected in one half of S. Typhimurium and in one EHEC ('00-E.051).
  4. qacE△1sul1 is responsible for insecticide and sulfonamide resistances. All the Su-resistant S. Typhimurium except Tym04 or Infantis had the gene, while all the Su-resistant EHEC strains except '96-E.094 were negative for the gene.
  5. TetA and TetB are known to be responsible for Tcr. Among Tc-resistant E. coli strains, a half of the strains had tetA or tetB. The remaining half was negative for tetA or tetB. All the MDR S. Infantis had tetA. Most Tc-resistant S. Typhimurium had tetB. S. Typhimurium strains of the B integron type had tetB and those of A integron type had cmlAtetR (Cmr and a regulatory gene for Tcr). Tym22 of the A integron type (1) having cmlAtetR and tetA (2) is an exception.
  6. All the Km-resistant strains had aphA1-LAB gene.
  7. All the Am-resistant E. coli harbored TEM (blaTEM) genes. PSE-1 (blaPSE-1) genes were found only among Am-resistant Salmonella of the A integron type. An exception was integron-negative Tym04 with both the bla genes. Am-resistant S. Typhimuriun of the B integron type having a 2.0 kb ant (3")-la casette was devoid of both PSE and TEM-1 genes.

We examined 11 MDR strains of E. coli (Table 1) for transferability of drug resistance genes. Two protocols were used. Conjugal transfer I (CT-I): Each of the E. coli strains was conjugated with a rifampicin (Rf)-resistant derivative of S. Litchfield AOLac+Nalr-01 (lac+Nar) (1,8). The mating time was 4 h in liquid cultures at 37C. The transconjugants were selected on sucrose (0.5%)-MAC plates containing Am (30 mg/ml), Km (25 mg/ml), Sm (25 and 50 mg/ml), or Tc (25 mg/ml). The donor strains were eliminated by Na (25 mg/ml) and/or Rf (25 mg/ml). Conjugal transfer II (CT- II): In order to know whether the resistance genes were present on transferable plasmids or not, we conducted transformation followed by conjugal transfer. For transformation, competent E. coli K12 DH5a (lac gylA) cells (Takara Shuzo, Co., Ltd., Kyoto) were transfected with plasmid DNA fractions prepared from the MDR strains. The transfectants were selected on the lactose (0.5%)-MAC plates containing Am, Km, Sm, or Tc. DH5a transfectants were crossed with a lac+ revertant of E. coli K12 WA921-3 (lacNarRfr) (8). The MAC contained Rf in order to eliminate the DH5a transfectants used as donors. Table 2 shows CT-I and CT-II data. The results are summarized as follows:
(1) Among 11 MDR EHEC, conjugal transfer (CT-1) was successful for four strains, '96-E.152, '98-E.001, '98-E.002, and '99-E.003. Segregation of drug resistance genes, especially Amr from other resistant genes, was observed. This suggests Amr and the other genes are located on a plasmid and the choromosome, respectively (see also TF data in the table).
(2) CT-II was positive only for '96-E.152 and '99-E.003. In '96-E.152, there was a rare segregation of resistance genes, AmsSmrSur from AmrSmrSur, during CT-II. For '99-E.003 no such segregation was observed.

Thus, four of 11 EHEC were able to transfer drug resistance genes through conjugation, and at least two ('96-E.152 and '99-E.003) of them harbored all the drug resistance genes also on the same plasmids.

REFERENCES

  1. Hamada.K., Tsuji, H. and Oshima, K. (2002) : Identification and characterization of transferable integron-mediated antibiotic resistance among Salmonella serovar Typhimurium and Salmonella serovar Infantis isolates from 1991 to 2002. Jpn. J. Infect. Dis., 55, 135-138.
  2. Briggs, C. E. and Fratamico, P.N. (1999): Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104. Antimicrob. Agents Chemother., 43, 846-849.
  3. Zhao, S., White, D.G., Ge, B., Ayers, S., Friedman, S., English, L., Wagner, D., Gaines, S. and Meng, J. (2001): Identification and characterization of integron-mediated antibiotic resistance among shiga toxin-producing Escherichia coli isolates. Appl. Environ. Microbiol., 67, 1558-1564.
  4. Schmidt, A. S., Bruun, M. S., Dalsgaard, I. and Larsen, J. L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl. Environ. Micribiol., 67, 5675-5682.
  5. Dalsgaard, A., Forslund, A., Tam, N.V., Vinh, D.X. and Cam, P.D. (1999): Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in Vibrio cholerae O1 strains isolated from 1979 to 1996. J. Clin. Microbiol., 37, 734-741.
  6. Frana, T.S., Carlson, S.A. and Griffith, R.W. (2001): Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype Typhimurium phage type DT104. Appl. Environ. Microbiol., 67, 445-448.
  7. Carlson, S.A., Bolton, L.F., Briggs, C.E., Hurd, H.S., Sharma, V.K., Fedorka-Cray, P.J. and Jones, B.D. (1999): Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR. Mol. Cell. Probes, 13, 213-222.
  8. Hamada, K. and Nakayama, Y. (1989): Location on the chromosome of the lac gene in a lactose-fermenting Salmonella litchfield strain. Microbiol. Immunol., 33, 87-97. 

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