a Department of Microbiology, IDIBAPS, Hospital Clinic, School of Medicine, University of Barcelona, Villarroel 170, 08036 Barcelona; b Department of Microbiology, Faculty of Sciences, University of Málaga, 29071 Málaga, Spain; c Choice Canning Company, Narayana Reddy Pet, Nellore, India
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Abstract |
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Introduction |
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Isolation of drug-resistant micro-organisms from food is an increasing public health problem. Being easily spread among the population, they can cause an epidemic outbreak, especially if sanitary conditions are not optimum.
This report concerns the various mechanisms of antibiotic resistance of a multiresistant clone of Salmonella typhimuriumisolated from food.
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Materials and methods |
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Five strains of S. typhimurium were isolated from finfish in Coimbatore, India.
Determination of MICs
Susceptibility to antimicrobial agents was assessed by the Etest method (AB Biodisk, Solna, Sweden).
Epidemiological analysis
Epidemiological relationships among the five strains were studied by plasmid analysis and REP-PCR. Plasmids were isolated using Wizard Plus Maxipreps (Promega, Madison, WI, USA), following the procedure described by the manufacturers. Recovered products were resolved by electrophoresis in 0.7% agarose gels. REP-PCR was performed as previously described. 4
Detection of mutations in the gyrA and parC genes
Amplification and sequencing of the fragments of gyrA and parC genes containing the respective quinolone-resistance-determining regions were performed as previously described. 5
Detection of chloramphenicol acetyl-transferase activity
The presence of chloramphenicol acetyl-transferase activity was determined using the spectrophotometric method described by Azemun et al. 6
Detection of ß-lactamases
Extraction of ß-lactamases was performed by ultrasonication of exponentially growing cultures. Their presence was determined by isoelectrofocusing (IEF) gel electrophoresis in a PhastSystem (Pharmacia AB, Uppsala, Sweden) and detection with nitrocefin. TEM-1, PSE-2, OXA-2 and OXA-3 were used as controls of pI.
Detection of ß-lactamase by PCR was performed using the primers and conditions for the amplification of the bla OXA gene described by Gallardo et al. 2
Detection of the ß-lactamase gene in an integron
The presence of the ß-lactamase gene in an integron was investigated by two different PCRs. The first was performed with the upper primer for bla OXA, 2 and the lower primer for the integron described by Levesque & Roy. 7 The second was performed with the lower primer for bla OXA, 2 and the upper primer for the integron. 7 Both PCR amplifications were performed following the procedure described previously. 5 The PCR products were then sequenced.
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Results |
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The IEF showed the presence of a ß-lactamase with a pI of c. 7.3 in the four isolates resistant to ß-lactam antibiotics. PCR results showed the presence of a bla OXA gene located in an integron element with a molecular weight of c. 1900 bp. The integron was sequenced, showing the presence of a bla OXA1 and an aadA gene.
Resistance to chloramphenicol in the five isolates was associated with the presence of chloramphenicol acetyl-transferase activity.
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Discussion |
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In previous studies on the molecular basis of quinolone resistance in S. typhimurium the presence of mutations in the gyrA gene has been associated with the development of quinolone resistance. 5,8 These mutations are located in the positions equivalent to the Ser-83 and/or Asp-87 of Escherichia coli. Sequencing studies showed a mutation in position 87, resulting in a substitution of Asp by Gly. This mutation has been previously described in Indian clinical isolates of S. typhimurium. 8 No mutations were observed in the parC gene of our strains.
Chloramphenicol resistance was associated in all the strains with the presence of chloramphenicol acetyl-transferase activity, as previously reported in S. typhimurium. 2 In Gram-negative bacteria, resistance to ß-lactam antibiotics is mainly associated with the production of ß-lactamases. In our study this resistance was explained by the presence of a ß-lactamase of pI c. 7.3, identified by PCR as an OXA-1 type ß-lactamase. The susceptibility of the S-9 strain, which could be considered the parenteral strain, to co-trimoxazole and ß-lactam antibiotics suggests the possibility that the genes encoding enzymes responsible for these resistances are included in mobile genetic elements, such as integrons or transposons. Genes codifying both OXA-type ß-lactamases and trimethoprim resistance have been described in these elements. 7 In our study the ampicillin-resistant S. typhimuriumstrains had an integron containing a bla OXA-1 and an aadA gene. An integron containing a bla OXA-1 and an aadA gene has been described by Colonna et al. 9 in Salmonella wien, suggesting that this could be a frequent mechanism of resistance transference in these micro-organisms.
Our results indicate the potential human health hazard of multiresistant S. typhimurium isolated from food and corroborate the increasing levels of antibiotic resistance in this micro-organism.
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Acknowledgments |
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Notes |
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References |
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2 . Gallardo, F., Ruiz, J., Marco, F., Towner, K. J. & Vila, J. (1999). Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance. Journal of Medical Microbiology (In press).
3 . Piddock, L. J. V., Griggs, D. J., Hall, M. C. & Jin, Y. F. (1993). Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients. Antimicrobial Agents and Chemotherapy 37 ,6626.[Abstract]
4 . Vila J., Marcos, M. A. & Jiménez de Anta, M. T. (1996). A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticus- A. baumannii complex. Journal of Medical Microbiology 44 ,482 9.
5 . Ruiz, J., Castro, D., Goñi, P., Santamaria, J. A., Borrego, J. J. & Vila, J. (1997). Analysis of the mechanisms of quinolone resistance in nalidixic acid-resistant clinical isolates of Salmonella serotype Typhimurium. Journal of Medical Microbiology 46, 6238.[Abstract]
6 . Azemun, P., Stull, T., Roberts, M. & Smith, A. L. (1981). Rapid detection of chloramphenicol resistance in Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 20, 16870.[ISI][Medline]
7 . Levesque, C. & Roy, P. H. (1993). PCR analysis of integrons. In Diagnostic Molecular Microbiology: Principles and Applications, (Persing, D. H., Smith, T. F., Tenover, F. C. & White, T. J., Eds), pp. 5904. American Society for Microbiology, Washington, DC.
8 . Brown, J. C., Thomson, C. J. & Amyes, S. G. B. (1996). Mutations of the gyrA gene of clinical isolates of Salmonella typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quinolone drugs. Journal of Antimicrobial Chemotherapy 37, 3516.[Abstract]
9 . Colonna, B., Bernardini, M., Micheli, G., Malmone, F., Nicoletti, M. & Casalino, M. (1988). The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936 -kanamycin resistance element. Plasmid 20, 22131.[ISI][Medline]
Received 15 July 1998; returned 15 September 1998; revised 24 November 1998; accepted 5 January 1999