Journal of Antimicrobial Chemotherapy (1999) 43, 699-702
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
Mechanisms of resistance to ampicillin, chloramphenicol and quinolones in multiresistant Salmonella typhimurium strains isolated from fish
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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Mechanisms of antibiotic resistance and epidemiological relationships were investigated for five multiresistant strains of Salmonella typhimurium isolated from fish in India. Four strains showed resistance to nalidixic acid, chloramphenicol, tetracycline, co-trimoxazole, gentamicin and ß -lactam antibiotics. The remaining strain was susceptible to all ß -lactam antibiotics tested and to co-trimoxazole but resistant to the other antibiotics tested. Epidemiological analysis performed by REP-PCR showed that the five isolates belonged to the same clone. Resistance to nalidixic acid was related to a single mutation in the gyrA gene. Chloramphenicol resistance was related to the production of chloramphenicol acetyl-transferase. An OXA-1 ß -lactamase, located in an integron, was responsible for resistance to ampicillin. These results indicate the health hazard posed by the fact that S. typhimurium may acquire or develop several mechanisms of resistance to a variety of antibiotics, including quinolones, and can thus cause disease in humans which may be difficult to treat.
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Introduction |
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Salmonella spp. are recognized as some of the most common pathogens causing enteritis worldwide. 1 Diarrhoea is an important problem in developing countries and is normally related to unhealthy sanitary conditions. Furthermore, the development of antibiotic resistance in enteropathogens, including Salmonellaspp., has increased the problem. Resistance to some ß-lactam antibiotics, tetracyclines, chloramphenicol, or trimethoprim is reported with increasing frequency. 2 Quinolones are an alternative therapy. However, the development of quinolone resistance in Salmonellaspp. during therapy of patients with enterocolitis has been reported. 3
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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Bacterial strains
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 epidemiological relationships of the five S. typhimurium strains were established by plasmid profiles and REP-PCR. Three of the strains (S-6, S-9 and S-10) had a plasmid of 3.0 kb that was not present in the other two. REP-PCR results showed that all strains belonged to the same clone (Figure).
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The resistance pattern of these strains is shown in the Table. All the strains were resistant to nalidixic acid, tobramycin, gentamicin, chloramphenicol, tetracycline and nitrofurantoin. Only the S-9 strain was susceptible to all the ß-lactam antibiotics tested and to co-trimoxazole.
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In order to explain the reduced susceptibility to quinolones, a 343 bp fragment of the gyrA gene was amplified and sequenced, showing that all the strains presented an Asp-87
RGly
substitution. A 291 bp fragment of the parC gene was amplified and sequenced, but no
mutations were found. 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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Five multiresistant strains of S. typhimurium isolated from food were analysed. Plasmid profile analysis showed that three of the strains contained a plasmid of 3.0 kb (S-6, S-9 and S-10), whereas in the other two strains (S-7 and S-8) no plasmid was detected. However, the gain or loss of plasmids is well established, therefore the analysis of the plasmid profile is not a definitive typing method. Epidemiological studies performed by REP-PCR showed that these five strains were the same clone. A similar power of discrimination was observed between REP-PCR and low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis. 2
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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This work was supported in part by grant SAF97/0091. We thank Servicios Cientifico Técnicos of the University of Barcelona for their help in DNA sequencing.
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Notes |
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* Corresponding author. Tel: +34-3-2275522; Fax: +34-3-2275454; E-mail: vila{at}medicina.ub.es
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References |
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Received 15 July 1998; returned 15 September 1998; revised 24 November 1998; accepted 5 January 1999
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