Florfenicol resistance in Salmonella enterica serovar Newport mediated by a plasmid related to R55 from Klebsiella pneumoniae

Danièle Meunier1, Sylvie Baucheron1, Elisabeth Chaslus-Dancla1, Jean-Louis Martel2 and Axel Cloeckaert1,*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Florfenicol resistance has emerged over the past few years in multidrug-resistant Salmonella enterica serovars Typhimurium, Agona and Paratyphi B. The floR gene encoding florfenicol resistance is chromosomally located in these serovars within a genomic island of 43 kb called SGI1 (Salmonella genomic island 1). In the present study, we characterized florfenicol resistance in a strain of S. enterica serovar Newport isolated from a turkey in 1990 and that lacked SGI1. Florfenicol resistance was mediated by a conjugative plasmid related to R55 from Klebsiella pneumoniae, which was characterized initially in the 1970s and harbours a gene 95% identical to floR.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Florfenicol is a fluorinated analogue of chloramphenicol, approved in Europe for use against respiratory infections in cattle since January 1995. Previous studies have shown that florfenicol is active against chloramphenicol-resistant strains that produce chloramphenicol acetyl transferases.1 Florfenicol resistance was first described in 1996 in the fish pathogen Pasteurella piscicida,2 recently renamed Photobacterium damselae subsp. piscicida. The pp-flo resistance gene identified in this pathogen, conferring cross-resistance to chloramphenicol, was located on a transferable plasmid. According to amino acid homologies, the pp-flo gene product is predicted to belong to the 12 transmembrane segment family of the major facilitator superfamily of export proteins reviewed by Paulsen et al.3

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
S. enterica serovar Newport strain 5823 was isolated from a turkey in France in 1990. Antibiotic susceptibility was evaluated by the disc diffusion method. All antibiotic discs except florfenicol discs were purchased from Bio-Rad (Marnes-La-Coquette, France). Florfenicol discs and the drug itself were obtained from Schering-Plough Animal Health (Kenilworth, NJ, USA). MICs of florfenicol and chloramphenicol were determined by a standard agar dilution method. The MIC breakpoints for chloramphenicol and florfenicol were defined by the Comité de l’Antibiogramme de la Société Française de Microbiologie (CASFM) or by the manufacturer, and were susceptible (MIC <= 8 mg/L), intermediate (MIC 16 mg/L) or resistant (MIC >= 32 mg/L).

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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Figure 1. Structural organization of the 6179 bp floR locus of plasmid R55.11 PCR fragments A and B and their sizes are indicated. BglI restriction sites are abbreviated as B. The distance scale below the map of the floR locus is given in base pairs. DR, direct repeat.

 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
S. enterica serovar Newport strain 5823 showed a multidrug resistance profile, with resistances to ampicillin, chloramphenicol/florfenicol, streptomycin, sulphonamides and trimethoprim. This strain was as resistant to florfenicol as strains of S. enterica serovars Typhimurium DT 104, Agona and Paratyphi B harbouring SGI1 (MIC 32 mg/L).5,6 However, strain 5823 lacked SGI1, as shown by negative results in PCR mapping and Southern blot analysis with appropriate probes.46 The floR gene was detected by PCR, and partial nucleotide sequencing revealed 100% nucleotide identity to the recently reported floR gene variant of plasmid R55.11 This plasmid is known to confer non-enzymic resistance to chloramphenicol.

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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Figure 2. (a) SacI (lanes 1 and 2) and BglI (lanes 3 and 4) restriction profiles of plasmid R55 (lanes 1 and 3) and the S. enterica serovar Newport strain 5823 plasmid (lanes 2 and 4). (b) Southern-blot hybridization of above restriction profiles with a probe consisting of the 12 kb SacI floR-containing fragment of plasmid R55.

 
In conclusion, these results suggest that plasmids related to R55 may have been circulating for a long time among Salmonella serovars, and also that this represents another means to acquire florfenicol resistance besides SGI1. Other conjugative plasmids carrying floR, previously shown in E. coli animal isolates,7 might also occur in Salmonella serovars. This is currently under investigation.


    Acknowledgements
 
We thank C. Mouline for expert technical assistance. This study was funded by the Direction Générale de l’Alimentation (Ministère de l’Agriculture), projet AQS 1999/S13 and partly by INRA, projet Transversalité.


    Footnotes
 
* Corresponding author. Tel: +33-2-47-42-77-50; Fax: +33-2-47-42-77-74; E-mail: cloeckae{at}tours.inra.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cannon, M., Harford, S. & Davies, J. (1990). A comparative study on the inhibitory actions of chloramphenicol, thiamphenicol and some fluorinated derivatives. Journal of Antimicrobial Chemotherapy 26, 307–17.[Abstract]

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, 665–9.[ISI][Medline]

3 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiological Reviews 60, 575–608.[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, 5725–32.[Abstract/Free Full Text]

5 . Cloeckaert, A., Sidi Boumedine, K., Flaujac, G., Imberechts, H., D’Hooghe, 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, 1359–61.[Abstract/Free Full Text]

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, 430–3.[ISI][Medline]

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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, 49–54.[Abstract/Free Full Text]

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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, 4593–8.[Abstract/Free Full Text]

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