1 Unité de Pathologie Aviaire et Parasitologie, Institut National de la Recherche Agronomique, 37380 Nouzilly; 2 Agence Française de Sécurité Sanitaire des Aliments, 69007 Lyon, France
Received 11 March 2002; returned 6 June 2002; revised 26 June 2002; accepted 7 January 2003
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Abstract |
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Introduction |
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Florfenicol resistance has emerged over the past few years in multidrug-resistant Salmonella enterica serovars Typhimurium, mainly of definitive phage type (DT) 104, Agona and Paratyphi B.46 The floR gene, responsible for florfenicol resistance, showed 97% identity to pp-flo, and was located in these serovars within a chromosomal cluster of antibiotic resistance genes as part of a genomic island of 43 kb called SGI1 (Salmonella genomic island 1).4 Other antibiotic resistance genes accounted for the remaining resistances of the multidrug-resistance pattern, i.e. resistance to ampicillin, streptomycin/spectinomycin, tetracyclines and sulphonamides. Florfenicol resistance conferred by floR, located either on plasmids or on the chromosome, has also been reported in Escherichia coli strains isolated from cattle and poultry.710 A floR gene variant (95% nucleotide identity) was recently identified on plasmid R55 from Klebsiella pneumoniae, initially described in the 1970s as conferring non-enzymic chloramphenicol resistance, thus suggesting that spread of the floR gene may have occurred a long time before the introduction of florfenicol.11 Spread of florfenicol-resistant strains may have occurred prior to the introduction of florfenicol, through the use of chloramphenicol or other unrelated antibiotics, and selection of strains where floR is associated with other antibiotic resistance genes, either on plasmids or on the chromosome, as in the case of multidrug-resistant S. enterica serovar Typhimurium DT 104.4
In the present study, we characterized florfenicol resistance in an S. enterica serovar Newport strain isolated from a turkey in 1990.
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Materials and methods |
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The presence of SGI1 and antibiotic resistance genes was determined by PCR and Southern blotting, as described previously.46 The floR gene was detected by PCR using primers cml01 and cml15, as described previously.5 Its nucleotide sequence was determined. Florfenicol resistance plasmids were detected by conjugation and plasmid extraction, as described previously.7 PCR mapping of floR and downstream open reading frames, as found on plasmid R55, was performed using primers A1 (5'-ACTGCTGCTGATGGCTCCTT-3') and A2 (5'-GAACAATGAAAGTCTCGCCG-3') for fragment A (Figure 1) and B1 (5'-GACTGCCATACCCGACCGAT-3') and B2 (5'-GATGAACGCCCGCCTCGCCG-3') for fragment B (Figure 1). The flanking regions of floR were also assessed by Southern blotting BglI-digested plasmid, using a 12 kb SacI fragment of plasmid R55 cloned in pGEM-7Zf (Promega, Charbonnieres, France) and that included the previously sequenced 6 kb BamHI region containing floR.11
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Results and discussion |
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We investigated the presence of a plasmid related to R55 in the serovar Newport strain. Florfenicol resistance was transferred from strain 5823 by conjugation to E. coli strain BM14 (pro met azi). The antibiotic resistance pattern conferred by the conjugative plasmid transferred was similar to that of plasmid R55, i.e. ampicillin, chloramphenicol/florfenicol, sulphonamides and gentamicin, but with additional resistance to trimethoprim. Restriction analysis of this plasmid showed SacI and BglI restriction profiles similar to those of plasmid R55 with some differences (Figure 2a). PCR mapping using primers A1, A2, B1 and B2 (fragments A and B in Figure 1) indicated that the flanking regions of floR in the serovar Newport plasmid were the same as those of floR on plasmid R55 (data not shown). Southern-blot analysis, using a probe consisting of a 12 kb SacI fragment of R55 containing the floR gene variant and its flanking regions, revealed identical patterns with either BglI- or SacI-digested R55 or the serovar Newport plasmids (Figure 2b), indicating that this region at least is conserved on both plasmids.
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Acknowledgements |
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Footnotes |
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References |
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2 . Kim, E. H. & Aoki, T. (1996). Sequence analysis of the florfenicol resistance gene encoded in the transferable R-plasmid of a fish pathogen, Pasteurella piscicida. Microbiology and Immunology 40, 6659.[ISI][Medline]
3 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiological Reviews 60, 575608.[Abstract]
4
.
Boyd, D., Peters, G. A., Cloeckaert, A., Sidi Boumedine, K., Chaslus-Dancla, E., Imberechts, H. et al. (2001). Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. Journal of Bacteriology 183, 572532.
5
.
Cloeckaert, A., Sidi Boumedine, K., Flaujac, G., Imberechts, H., DHooghe, I. & Chaslus-Dancla, E. (2000). Occurrence of a Salmonella enterica serovar Typhimurium DT104-like antibiotic resistance gene cluster including the floR gene in S. enterica serovar Agona. Antimicrobial Agents and Chemotherapy 44, 135961.
6 . Meunier, D., Boyd, D., Mulvey, M. R., Baucheron, S., Mammina, C., Nastasi, A. et al. (2002). Salmonella enterica serovar Typhimurium DT104 antibiotic resistance genomic island I in serotype paratyphi B. Emerging Infectious Diseases 8, 4303.[ISI][Medline]
7
.
Cloeckaert, A., Baucheron, S., Flaujac, G., Schwarz, S., Kehrenberg, C., Martel, J. L. et al. (2000). Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle. Antimicrobial Agents and Chemotherapy 44, 285860.
8
.
Doublet, B., Schwarz, S., Nußbeck, E., Baucheron, S., Martel, J. L., Chaslus-Dancla, E. et al. (2002). Molecular analysis of chromosomally florfenicol-resistant Escherichia coli isolates from France and Germany. Journal of Antimicrobial Chemotherapy 49, 4954.
9
.
Keyes, K., Hudson, C., Maurer, J. J., Thayer, S., White, D. G. & Lee, M. D. (2000). Detection of florfenicol resistance genes in Escherichia coli isolated from sick chickens. Antimicrobial Agents and Chemotherapy 44, 4214.
10
.
White, D. G., Hudson C., Maurer J. J., Ayers S., Zhao S., Lee M. D. et al. (2000). Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea. Journal of Clinical Microbiology 38, 45938.
11
.
Cloeckaert, A., Baucheron, S. & Chaslus-Dancla, E. (2001). Nonenzymatic chloramphenicol resistance mediated by IncC plasmid R55 is encoded by a floR gene variant. Antimicrobial Agents and Chemotherapy 45, 23812.