Jpn. J. Infect. Dis., 55, 135-138, 2002

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Laboratory and Epidemiological Communications

Identification and Characterization of Transferable Integron-Mediated Antibiotic Resistance among Salmonella serovar Typhimurium and Salmonella serovar Infantis Isolates from 1991 to 2002

Kokichi Hamada*, Hidetaka Tsuji and Kahori Oshima

Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032

Communicated by Takashi Kawamura

(Accepted October 4, 2002)

Salmonella serovar Typhimurium and Salmonella serovar Infantis have been major causes of Salmonella infections in Japan during the past decades, though in 1989 Salmonella serovar Enteritidis suddenly emerged and its epidemic still prevails (1). But the drug-resistant S. Enteritidis has been rare (2). We report here transferable class 1 integrons which conferred the drug resistance on non-DT104 S. Typhimurium and S. Infantis. Class 1 integrons are predominant among Enterobacteriaceae (3), Vibrionaceae (4,5), and some non-fermenting gram-negative bacteria (3). Integrons contain gene cassettes that can be mobilized via their association with transposons or conjugative plasmids (3). They are important sources for the spread of antibiotic resistance among bacteria (3).

We analyzed a total of 30 S. Typhimurium isolates obtained in 1991-2002 in Hyogo Prefecture, 29 from humans and one from chicken, and 11 S. Infantis isolates, which were all multidrug resistant (MDR) (submitted for publication). The 29 isolates of S. Typhimurium from humans were obtained from six outbreaks and 23 sporadic cases. One S. Typhimurium isolate was judged as DT104 from the drug resistance pattern, but others were not.

Antibiotic sensitivity was assayed by means of the disk diffusion method by using commercial antibiotic disks (Becton Dickinson Microbiology Systems, Cocksville, Md., USA) or by the agar dilution method on Mueller-Hinton agar plates following the recommendation by the National Committee for Clinical Laboratory Standards (6). Antibiotic disks were those of ampicillin (Am), cefotaxime, kanamycin (Km), gentamicin, streptomycin (Sm), tetracycline (Tc), trimethoprim (Tm), ciplofloxacin, fosfomycin, chloramphenicol (Cm), Sulfamethoxazole-trimethoprim (Su-Tm), and nalidixic acid (Na). The plates for agar dillution method contained Tc (25 mg/ml), Am (30 mg/ml), Km (25 mg/ml), Sm (25 or 50 mg/ml), Cm (25 mg/ml), Tm (25 mg/ml), or Su (100 mg/ml). MaConkey agar plates containing 0.5% lactose (Lac-MAC) were also used for the agar dilution method.

Class 1 integrons were detected by a consensus sequence for PCR primer pair, int I-F (5'-GGCATCCAAGCAGCAAGC-3') and int I-R (5'-AAGCAGACTTGACCTGAT-3'), and PCR amplification was carried out as reported previously (6). Pulsed-field gel electrophoresis (PFGE) patterns of DNA from S. Typhimurium isolates and trasconjugants were analyzed by using a Gene Path Typing System (Program No. 5/No. 10; Bio-Rad Laboratories, Hercules, Calif., USA). Bacterial chromosomal DNAs were digested with XbaI or BlnI (Takara Shuzo Co., Ltd., Kyoto) and the method for isolation of plasmid DNA and its detection on PFGE were the same as reported (submitted for publication). The molecular size markers were 100 bp DNA ladder and lambda (l) - HindIII digest (Takara Shuzo), pGEM^TM marker (Promega Corporation, Madison, Wis., USA), 48.5 kb (l) DNA 1adder (Roche Diagnostics GbmH, Mannheim, Germany).

PFGE patterns of XbaI-digested chromosomal DNA of 30 S. Typhimurium strains are shown in Fig. 1 (Figs. 1A and B and lanes 1-6 in Fig. 1C). The overall patterns were similar among the isolates. There were four mutually identical pairs, Tyml3 and Tym15 (lanes 1, 3 in Fig. 1B), Tym21 and Tym22 (lanes 9, 10 in Fig. 1B), Tym23 and Tym24 (lanes 11, 12 in Fig. 1B), and Tym26 and Tym30 (lanes 2, 6 in Fig. 1C). However, others were dissimilar, i.e., the S. Typhimuriun isolates used had different genetic constitutions. PFGE patterns of BlnI-digests appeared less variable among the isolates (lanes 7-12 in Fig. 1C).

Among the 30 S. Typhimurium isolates, 22 strains were drug resistant (Table 1) and the remaining eight were sensitive to all the drugs tested (see the legend to Fig. 1). Out of the 22 drug-resistant S. Typhimurium strains, 19 strains exhibited MDR patterns while three strains (Tym02 , Tym09, and Tym11) were resistant to Tc only (Table 1). The MDR patterns exhibited by the 19 strains varied widely while those in S. Infantis were relatively homogeneous (submitted for publication). Class I integrons were present in the MDR strains of S. Typhimurium and S. Infantis (Table 1 and Fig. 2). Twelve strains among 21 drug-resistant S. Typhimurium and all of the 11 MDR strains of S. Infantis had class 1 integrons. All the 11 S. Infantis had the same sized integrons (1.0 kb long, C type) (Fig. 2). On the other hand, S. Typhimurium had two kinds of integrons different in size; 7 strains had 1.0- and 1.2-kb integrons (A type), while five strains had a 2.0-kb integron only (B type) (Fig. 2). All the strains that harbored integrons were Tc-, Am-, and Cm-resistant (Tc^r, Am^r, Cm^r). All the strains with type A integron were Sm-resistant (Sm^r), while all the strains that harbored type B integron were Sm-sensitive (Sm^s). All the isolates with type A integron were Km-sensitive (Km^s), while all the strains with B-type integrons except Tym13 were Km-resistant (Km^r) (Table 1).

The 11 MDR S. Infantis strains were known to have antibiotics resistance genes, along with a 1.0-kb integron, on a 300-kb R plasmid (submitted for publication). We examined here 21 strains of S. Typhymurium for their capacity to transfer drug resistance by conjugation to S. Litchfield strain AO Lac+Nal^r-01 (Lac+Nal^r)(7). When the donor strain was Na-resistant, the rifampicin-resistant (Rif^r) derivative recipient was used in place of the parental strain. The mating time was 4 h. The transconjugants were selected on Lac-MAC plates containing either Tc, Am, Km, Sm, or Cm (concentrations were the same as above). The donor strains were eliminated by Na (25 mg/ml) or by Rif (25 mg/ml) depending upon the combination of donors and recipients.

We found that, among 21 strains, Tym04, Tym13, Tym15, and Tym20 had an ability of conjugal transfer (Tables 1 and 2). Among them, Tym04 strain had no integrons, while Tym13, Tym15, and Tym20 had B type integrons. Transfer of antibiotic resistance markers and integrons by conjugation are shown in Table 2.

Mating experiments using Tym04 as a donor gave a result which suggested that Tc^r, Km^r and Sm^r were linked but Am^r and Tc^rKm^rSm^r were not (Table 2), i.e., it was possible that Am^r and Tc^rKm^rSm^r were on separate plasmids or a plasmid and chromosome. To test the possibility, the competent Escherichia coli K-12 DH5 a cells (Takara Shuzo) were transfected with plasmid DNA fraction prepared from Tym04. The transfectants were selected on Lac-MAC plates containing Tc, Am, Km, or Sm. Seventy-four transfectants were obtained. Eight colonies selected on Tc plates, 25 colonies selected on Km plates and 18 colonies selected on Sm plates were all Tc^r Km^r Sm^r Am^s, while 23 colonies selected on Am plates were all Tc^s Km^s Sm^s Am^r. No Tc^r Km^r Sm^r Am^r transfectants were obtained. The data supported the hypothesis that Tym04 starin had two plasmids, one with Tc^r Km^r Sm^r and the other with Am^r.

When Tym13, Tym15, and Tym20 as donors were used as donors, the order of transfer of drug resistance genes was suggested to be Cm^r, Tm^r, and Km^r/Am^r. This result was best explained by postulating that these drug resistance genes were present on the bacterial chromosomes in that order, because, if the drug resistance genes were on a plasmid, all the genes should have been transferred together at a time.

In this study we demonstrated here transferability of integron-mediated drug resistance in non-DT104 S. Typhimurium and S. Infantis. The gene cassette structures of the integrons of these bacteria remains to be clarified in comparison with those of the well characterized DT104 (8).

The authors are grateful to Drs. Hidemasa Izumiya and Haruo Watanabe, National Institute of Infectious Diseases, for the phage typing of our isolates and for a critical reading of the manuscript.

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*Corresponding author: Tel: +81-78-511-6787, Fax: +81-78-531-7080

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