JAC Advance Access originally published online on December 12, 2002
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Journal of Antimicrobial Chemotherapy (2003) 51, 180-182
© 2003 The British Society for Antimicrobial Chemotherapy
Correspondence |
Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources
Institute for Animal Science, Federal Agricultural Research Centre (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee, Germany
Keywords: Salmonella Typhimurium var. Copenhagen, resistance phenotype, resistance genotype
Sir,
Salmonella enterica subsp. enterica (S.) serovar Typhimurium is of major zoonotic importance, whereas its O:5-negative variant, designated variant Copenhagen (v.c.), has rarely been detected in connection with diseases in humans. In pigeons however, S. Typhimurium v.c. causes various clinical symptoms depending on the age of the infected animals, such as fatal septicaemia or meningoencephalitis in young pigeons or arthritis affecting joints in wings and legs in older pigeons. Although S. Typhimurium v.c. infections are mainly seen in pigeons, these bacteria have also been isolated from salmonellosis cases in other animals, such as cattle and swine, at high frequencies. Occasionally S. Typhimurium v.c. isolates have also been detected in dogs and cats.
In the present study, 19 isolates of S. Typhimurium v.c. obtained from epidemiologically unrelated food-producing animals and pets (Table 1) in different parts of Germany were investigated for their clonal relationships and their antimicrobial resistance. Studies of the resistance patterns as determined by agar disc diffusion according to the NCCLS1 were extended to PCR of resistance genes to compare resistance phenotypes with genotypes (Table 1).
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Serotyping was carried out using commercially available antisera (Behring Werke, Marburg, Germany) and phage types were determined at the National Reference Center for Salmonellae and other Enteric Pathogens (Wernigerode, Germany). All isolates were investigated for their clonal relationships using IS200 typing and macrorestriction analysis with BlnI, SpeI and XbaI. Specific PCR assays were used for the detection of the resistance genes blaTEM, blaPSE (ampicillin resistance), tet(A-E), tet(G) and tet(H) (tetracycline resistance), sul1 and sul2 (sulphonamide resistance), catA1-catA3 and floR (chloramphenicol or chloramphenicol/florfenicol resistance), strA, ant(3'')-Ia, aadA2 (streptomycin resistance), aph(3')-Ia (kanamycin resistance), ant(2'')-Ia (kanamycin and gentamicin resistance), as well as aac(3)-IVa (gentamicin resistance). PCR analyses followed previously described protocols.2–5 Four PCR assays that enabled the simultaneous detection of closely related dfr genes (trimethoprim resistance) were developed: dfrB1/dfrB2/dfrB3 [forward, 5'-CAAAGTAGCGATGAAGCCA-3'; reverse, 5'-CAGGATAAATTTGCACTGAGC-3'; amplicon size 205 bp, annealing temperature (TA) 53°C], dfrA5/dfrA14 [forward, 5'-GATTGGTTGCGGTCCA-3'; reverse, 5'-CTCAAAAACAACTTCGAAGG-3'; amplicon size 379 bp; TA 48°C], dfrA7/dfrA17 [forward, 5'-CAGAAAATGGCGTAATCG-3'; reverse, 5'-TCACCTTCAACCTCAACG-3'; amplicon size 345 bp; TA 50°C] and dfrA1/dfrA15/dfrA16 [forward, 5'-GATATTCCATGGAGTGCCA-3'; reverse, 5'-ACCCTTTTGCCAGATTTG-3'; amplicon size 414 bp; TA 50°C]. The different PCR amplicons were verified by sequence and/or restriction analysis.
Five different phage types were identified among the 19 isolates, with DT104 being the predominant phage type (Table 1). Macrorestriction analysis with BlnI, SpeI and XbaI revealed seven, six and five major types of pattern with more than six bands difference, respectively. Within isolates of a major pattern, variations of up to five bands difference were seen. The DT104 isolates yielded identical XbaI patterns, but BlnI and SpeI macrorestriction analysis showed minor variations in eight of the 11 DT104 isolates. IS200 typing revealed five different hybridization patterns each consisting of five to nine hybridizing bands.
All 19 S. Typhimurium v.c. isolates exhibited at least resistance to ampicillin, streptomycin, sulfamethoxazole and tetracycline. Additional resistances to chloramphenicol, florfenicol, kanamycin, gentamicin and/or trimethoprim were seen in the majority of the isolates (Table 1). The resistance genes detected in the DT104 strains were mainly those previously identified as part of the chromosomal multiresistance cluster [aadA2, floR, tet(G), blaPSE, sul1].6 However, a second type of ampicillin resistance gene, blaTEM, a second type of chloramphenicol resistance gene, catA1, and in one case a second type of tetracycline resistance gene, tet(A), were detected. These resistance genes are commonly located on transposons, such as Tn3 (blaTEM), Tn9 (catA1) or Tn1721 [tet(A)], all of which are widespread among Gram-negative bacteria.7 Independent acquisition of these transposons before the development/acquisition of the chromosomal multiresistance gene cluster might provide an explanation for the presence of more than one gene coding for the same resistance property in these strains. In addition, genes coding for resistance to kanamycin [aph(3')-Ia], kanamycin and gentamicin [ant(2'')-Ia] or trimethoprim (dfrA1/dfrA15/dfrA16) were identified in several DT104 isolates (Table 1). In contrast, the DT204c isolates carried only a single ß-lactamase gene, blaTEM, but a second type of trimethoprim resistance gene, dfrA5/dfrA14. Moreover, chloramphenicol resistance was exclusively based on the expression of the catA1 gene and kanamycin resistance was the result of the expression of the gene aph(3'')-Ia. Finally, gentamicin resistance in a single DT204c strain was associated with the gene aac(3)-IVa. The five isolates resistant to nalidixic acid by disc diffusion were investigated for resistance to nalidixic acid (Nal), enrofloxacin (En) and ciprofloxacin (Cip) by agar dilution. All five isolates showed MICNal values of 
512 mg/L, but fluoroquinolone resistance (MICEn 32 mg/L, MICCip 32 mg/L) was exclusively detected in the four DT204c strains; the DT104 isolate 1 showed MICEn 0.5 mg/L and MICCip 0.25 mg/L.
This study confirmed (i) that multiresistant S. Typhimurium v.c. isolates carry a wide variety of resistance genes and also (ii) that isolates with the same resistance phenotype often have different resistance genotypes.
Footnotes
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de 
References
1 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA, USA.
2 . Frech, G. & Schwarz, S. (2000). Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar, Saintpaul: construction and application of specific gene probes. Journal of Applied Microbiology 89, 633–41.[CrossRef][Medline]
3 . Kehrenberg, C. & Schwarz, S. (2001). Occurrence and linkage of genes coding for resistance to sulfonamides, streptomycin and chloramphenicol in bacteria of the genera Pasteurella and Mannheimia. FEMS Microbiology Letters 205, 283–90.[CrossRef][Web of Science][Medline]
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Ng, L.-K., Mulvey, M. R., Martin, I., Peters, G. A. & Johnson, W. (1999). Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrobial Agents and Chemotherapy 43, 3018–21.
5 . Sandvang, D. (2001). Aminoglycoside resistance genes and their mobility in gramnegative bacteria from production animals. PhD thesis, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
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Briggs, C. E. & Fratamico, P. M. (1999). Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104. Antimicrobial Agents and Chemotherapy 43, 846–9.
7 . Berg, D. E. (1989). Transposable elements in prokaryotes. In Gene Transfer in the Environment (Levy, S. B. & Miller, R. V., Eds), pp. 99–138. McGraw-Hill Publishing Company, New York, NY, USA.
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