1 Departamento de Biología Funcional, Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, C/Julián Clavería 6, 33006 Oviedo; 2 Laboratorio de Salud Pública, Consejería de Sanidad, Principado de Asturias, Carretera del Rubín s/n, 33001 Oviedo, Spain
Received 2 January 2002; returned 25 May 2002, revised 22 August 2002; accepted 4 February 2003
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Abstract |
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Introduction |
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The aim of the present work was to ascertain the situation regarding antimicrobial resistance in Salmonella Enteritidis isolated in the Principality of Asturias (PA), Spain, in 2002. Asturias is a Northern Spanish Region with 1 million inhabitants and covering an area of
10 565 km2. In the past, antimicrobial resistance has been rare in this serotype. However, in the early and late 1990s the occurrence of resistance to ampicillin and nalidixic acid has increased, whereas resistance to other antimicrobials remained infrequent.
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Materials and methods |
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PCR-based procedures, using previously described protocols and primers, were used to detect mutations in DNA gyrase genes by amplification/restriction of the gyrA gene,8 by AS-PCR-RFLP of gyrA and parC genes,9 by bla, aadA, qacE1, sul1 genes,6,13 and the plasmid virulence gene spvC.13 RAPD typing was performed with the C-primer.14 PCR products, representing different amplicons, were collected from the gels, purified and directly analysed by partial sequencing in the Servicio de Secuenciacion de DNA, CIB-CSIC, Madrid, Spain. Sequences obtained were compared with those registered in the GenBank database. Plasmid analysis (DNA isolation, restriction, hybridization, conjugation in liquid medium and transformation by electroporation) was performed as described by Guerra et al.13 and Sambrook et al.15
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Results |
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When NALr isolates were tested for mutations in the gyrA and parC genes, the gyrA amplicons of 97 and 15 isolates generated HinfI profiles compatible with mutations within codons Asp-87 and Ser-83.8,9 For parC amplicons, no isolates generated HaeII profiles compatible with a mutation at codon Ser-80.9 Amplicons containing gyrA, gyrB and parC genes from eight isolates (three with a mutation in Asp-87 and five with a Ser-83 mutation) were sequenced. In all cases, the expected mutations in gyrA were confirmed (Asp-87: GACTAC and Asp
Tyr; and Ser-83: AGC
ATC and Ser
Phe); no other functional mutation was detected. AMPr isolates were grouped into three ß-lactam resistance patterns: blaR1 (ampicillin), blaR2 (ampicillin/piperacillin) and blaR3 [ampicillin/cefalothin/cefotaxime], comprising 78, seven and two isolates. IEF revealed the presence of ß-lactamases with a pI of 5.4 in 18 isolates (nine, seven and two showing blaR1, blaR2 and blaR3 patterns, data corresponding to TEM 1-like ß-lactamases). These isolates generated amplification products with generic blaTEM primers. Nucleotide sequencing of the products, from isolates representing each bla pattern, confirmed that all contained blaTEM1 genes (accession no. AF126482). No other bla genes were detected.
When the drug-resistant isolates were tested for the presence of plasmids and integrons, all were found to carry plasmids but only two possessed Class 1 sul1 integrons with the aadA1a gene cassette. Eleven plasmid profiles were identified (Figure 1a). With the exception of three isolates, all possessed a plasmid of 60 kb, typical of the serovar-specific Enteritidis virulence plasmid. Representative plasmid profiles were tested by hybridization using spvC, blaTEM1, aadA1 and qacE
1-sul1 probes. Results are shown in Figure 1(b). The capacity for the transfer, by transformation or conjugation of AMPr plasmids from 10 isolates representing different plasmid profiles to E. coli strains, was also tested. Plasmids from the 10 isolates were transferred by transformation and from nine by conjugation. The self-transferable plasmids showed the blaR1 pattern and were of three molecular masses: 40, 90 and 100 kb. The single non-self-transferable plasmid had the blaR2 pattern and a molecular mass of
7 kb. The genetic relationship between AMPr plasmids collected from transconjugants (derived from representative Enteritidis isolates: eight with 40, six with 90 and one a 100 kb plasmid) was tested by restriction followed by hybridization. All 40 kb plasmids generated identical restriction profile, being identified as a single type (pUO-SeR1); the 90 kb plasmids generated two types of restriction profile (pUO-SeR2 and pUO-SeR3), and the 100 kb plasmid another profile (pUO-SeR4). Hybridization revealed that the blaTEM1 probe mapped to different fragments in each restriction profile. In contrast, the qacE
1-sulI and aadA1a probes mapped only to fragments in pUO-SeR4 profiles and in the chromosome in the profiles from the control strain LSP 473/98 (Figure 1cf).
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Discussion |
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In this investigation, RAPD analysis was applied to differentiate Enteritidis isolates, together with R pattern and plasmid analysis. RAPD is simple, reproducible, screens stable traits and shows good discriminatory power within and between Salmonella serotypes.14 The results have demonstrated that, in PA, ampicillin resistance in Salmonella Enteritidis is mediated mostly by pUO-SeR1 plasmids and associated mainly with a type identified as PT 6a, RAPD-C6. In a minority of strains, other types of AMPr plasmid (pUO-SeR2-R5) have also been identified, but for the most part in isolates obtained after 1997.
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Acknowledgements |
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Footnotes |
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References |
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