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Antimicrobial Agents and Chemotherapy, June 2003, p. 2002-2005, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2002-2005.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evolution of Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Strains Isolated in the Czech Republic between 1984 and 2002

M. Faldynova,1 M. Pravcova,1 F. Sisak,1 H. Havlickova,1 I. Kolackova,2 A. Cizek,3 R. Karpiskova,2 and I. Rychlik1*

Veterinary Research Institute, 621 32 Brno,1 National Institute of Public Health, Center for Food Chain Hygiene,2 University of Veterinary and Pharmaceutical Sciences, 612 42 Brno, Czech Republic3

Received 9 December 2002/ Returned for modification 30 January 2003/ Accepted 26 February 2003


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ABSTRACT
 
In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2002 in the Czech Republic, genes coding for antibiotic resistance were determined by using specific PCRs. We found that the pentadrug-resistant ACSSuT clone first appeared in the Czech Republic in 1990. A new variant of the aadA gene designated aadA21 is described, the 5' end of which was identical to aadA2 and the 3' end of which was identical to aadA1.


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TEXT
 
Multidrug-resistant strains of Salmonella enterica serovar Typhimurium were first isolated in the mid-1980s (22, 24) and are typically identified by pentadrug resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT type of resistance). The genes responsible for this resistance were gradually characterized (2, 6, 7, 11), and recently, the multidrug-resistant genomic island was sequenced completely (5). All the resistance genes are found as a part of class 1 integron (5, 10, 13, 16), and strains with such genetic organization have been described in different parts of the world (4, 8, 17, 22). Besides the genes present in the multidrug-resistant Salmonella serovar Typhimurium strains, other genes related to antibiotic resistance were described in Salmonella serovar Typhimurium or other closely related species. Tetracycline resistance could also be encoded by tetA, tetB, or tetC (1, 20, 23). Resistance to chloramphenicol could be caused by the presence of chloramphenicol transacetylase cat (2, 3), and multiple modes of resistance to aminoglycosides have also been described (14, 19). We were therefore interested in genes present in antibiotic-resistant Salmonella serovar Typhimurium strains isolated in the Czech Republic, with special emphasis on the situation before and after the appearance of the multidrug-resistant strains.

Altogether, 66 randomly selected antibiotic-resistant strains originating from humans, farm animals, and the environment were included in this study. Older strains originated from different laboratories where they were stored either at -70°C in Luria-Bertani with 15% glycerol, Dorset egg medium at 4°C, or even conventional Luria-Bertani agar at room temperature. Therefore, to avoid confusion related to the potential loss of resistance during prolonged storage, in all the strains, when recovered from the storage medium, a single colony was picked up and tested by disk diffusion method for the present antibiotic resistance according to the NCCLS standards (19a). The antibiotics tested were as follows: ampicillin (10 µg), chloramphenicol (30 µg), streptomycin (10 µg), sulfonamide (300 µg), tetracycline (30 µg), ciprofloxacin (5 µg), kanamycin (30 µg), gentamicin (10 µg), sulfamethoxazole-trimethoprim (25 µg), cefotaxime (30 µg), nalidixic acid (30 µg), and amoxicillin-clavulanic acid (30 µg).

Using PCR, all the resistant strains were tested for the presence of the genes typical for the pentadrug-resistant DT104 strains, i.e., blaPSE-1, floR, aadA2, sul1, tetG, and int1, and also for the presence of tetA, tetB, or tetC, cat, strA, sul2, and blaTEM genes. In int1- and sul1-positive strains, but different from ACSSuT or ACSSuTSxt types, the whole integron sequence was amplified by using the 5CS and 3CS primer pair and the resulting PCR products of various sizes were sequenced by using an ABI 310 Genetic Analyser (Applied Biosystems). All the primers used in this study are listed in Table 1.


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  		TABLE 1. Primers used in the study

Although all the strains were tested for the resistance to 12 antibiotics, modes of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline were studied in detail because these resistances occurred by far the most frequently in Salmonella serovar Typhimurium strains isolated in the Czech Republic. We never detected any strains resistant to ciprofloxacin, gentamicin, and cefotaxime. Resistance to other antibiotics, which were detected only sporadically, can be seen in Table 2.


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  		TABLE 2. List of all patterns of resistance (phenotypes) and genotypes identified in this study

All the analyzed strains were clustered into the following 4 groups according to the date of their isolation: those isolated from 1984 to 1985 (12 strains), 1988 to 1991 (7 strains), 1996 to 1999 (18 strains), and 2000 to 2002 (29 strains).

Among the strains isolated from 1984 to 1985, various antibiotic resistance genes were present, including integron-positive strains with the typical structure of 5'- and 3'-end conserved sequences. However, the integron-positive strains did not form the dominant group (Fig. 1) and they also differed from the present isolates in that they contained different genes e.g., aadA1 or blaOXA-1 (Table 2).



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  		FIG. 1. Evolution of types of antibiotic resistances in Salmonella serovar Typhimurium strains from the indicated time periods. Filled bars, integron-positive pentadrug-resistant ACSSuT strains; bars with horizontal lines, integron-positive aadA2 SSu strains; bars with vertical lines, integron-positive, non-ACSSuT, non-aadA2 strains; white bars, integron-independent antibiotic-resistant strains.

The situation changed considerably in the beginning of the 1990s. For the first time, pentadrug-resistant ACSSuT strains appeared in the Czech Republic (Fig. 1). All of these strains were typical by the presence of tetG, blaPSE-1, floR, sul1, and aadA2. Among integron-positive strains resistant to streptomycin and sulfonamides only, clones containing either aadA1 or aadA2 were recovered. In a single SSu strain isolated in 1991, we identified an aadA gene, designated aadA21, the 5' end of which was identical to aadA2 and the 3' end of which was identical to aadA1.

In strains isolated from 1996 to 1999, the integron-positive pentadrug-resistant blaPSE-1, floR, aadA2, and tetG strains became dominant (Fig. 1). Three less usual multidrug-resistant strains were isolated in this period which did not contain int1 and sul1. Instead, these strains contained two high-molecular-weight plasmids that were over 100 kb in size. None of them was, however, the typical serovar-specific plasmid, as these strains were negative by rck-specific PCR (data not shown).

In the very recent isolates (isolated from 2000 to 2002), nearly all isolates belonged to integron-positive, pentadrug-resistant strains. Besides the integron-positive strains, we isolated 4 multidrug-resistant strains which were integron negative, and our preliminary observations suggest that these strains possess a large plasmid similar to that of Salmonella enterica serovar Typhi (21) encoding blaTEM ß-lactamase, chloramphenicol acetylase cat, streptomycin 3" phosphotransferase coded by strA and strB, and either TetB or TetA tetracycline efflux protein.

Throughout the last 15 or more years, different antibiotic-resistant strains with different genetic compositions appeared in the Czech Republic. Our results clearly point to greater variability and different genetic background of antibiotic-resistant Salmonella serovar Typhimurium strains collected in the 1980s and present isolates, although integrons of different genetic structures "were known" also to the oldest Salmonella populations. The increase in proportion of pentadrug-resistant strains in the Czech Republic was observed at the beginning of the 1990s (18), approximately 5 years after these strains were first recorded in cattle in Great Britain and the United States (22, 24). The identification of the aadA21 gene in a strain isolated in 1991 is interesting. This finding further confirms the introduction and mixing of aadA1- and aadA2-positive bacterial populations in the beginning of the 1990s in the Czech Republic. Considering this finding from an evolutionary point of view (12), it is also an example of how new gene variants can be generated by homologous recombination in vivo in the environment.

Nucleotide sequence accession number. The sequence of the aadA21 gene is available in GenBank under accession number AY171244.


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ACKNOWLEDGMENTS
 
We thank Michaela Dekanova for technical assistance.

This study was supported by The Czech Ministry of Health project NH 6083-3 and the grant of the Czech Ministry of Agriculture QC0195.


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FOOTNOTES
 
* Corresponding author. Mailing address: Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic. Phone: 420 541321241. Fax: 420 541211229. E-mail: rychlik{at}vri.cz. Back


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Antimicrobial Agents and Chemotherapy, June 2003, p. 2002-2005, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2002-2005.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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