Plasmid-borne florfenicol resistance in Pasteurella multocida

Corinna Kehrenberg and Stefan Schwarz*

Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany


* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; Email: stefan.schwarz{at}fal.de

Received 24 January 2005; returned 20 February 2005; revised 21 February 2005; accepted 22 February 2005


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Objectives: A florfenicol-resistant Pasteurella multocida isolate from a calf was investigated for the genetic basis of florfenicol resistance and the location of the resistance gene.

Methods: The P. multocida isolate 381 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 10.8 kb florfenicol–chloramphenicol resistance plasmid, designated pCCK381, was identified by transformation into Escherichia coli. The plasmid was mapped with restriction endonucleases, cloned and sequenced completely.

Results: Of the antimicrobials tested, plasmid pCCK381 conferred resistance only to chloramphenicol and florfenicol. It showed extended similarity to the 5.1 kb plasmid pDN1 from Dichelobacter nodosus in the part carrying the mobilization and replication genes. An adjacent 3.2 kb segment was highly homologous to the florfenicol resistance gene region of plasmid pMBSF1 from E. coli. In pCCK381, combined resistance to chloramphenicol and florfenicol was based on the presence of a floR gene that showed 97.2–99.7% identity to so far known floR genes.

Conclusions: The results of this study showed that a plasmid-borne floR gene was responsible for chloramphenicol and florfenicol resistance in the bovine respiratory tract pathogen P. multocida. This is, to the best of our knowledge, the first report of a florfenicol resistance gene in a target bacterium.

Keywords: floR gene , respiratory tract pathogens , antimicrobial resistance , gene transfer


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Florfenicol, a fluorinated chloramphenicol derivative, is one of the few antimicrobial agents that are exclusively licensed for use in veterinary medicine.1 In Europe, it has been licensed in 1995 and 2000 for the control of respiratory pathogens from cattle and pigs, respectively, whereas in the USA, florfenicol is also approved for the treatment of infectious pododermatitis (= interdigital phlegmon) in cattle. In addition, a florfenicol premix is licensed in countries such as the USA, Canada, Chile, Norway, Korea and Japan for the treatment of various fish diseases.1

Since the introduction of florfenicol into clinical veterinary use, continuous monitoring programmes have been conducted to determine MICs of florfenicol of bovine and porcine respiratory tract pathogens. The results of these monitoring programmes indicated that virtually all target bacteria obtained from cattle (Pasteurella multocida, Mannheimia haemolytica and Histophilus somni) and pigs (P. multocida and Actinobacillus pleuropneumoniae) were florfenicol-susceptible and that their MIC50 and MIC90 values had remained stable over the last decade.2,3 To date, no florfenicol resistance genes have been detected in any of these target bacteria. In contrast to the situation in the target bacteria, florfenicol resistance in various Gram-negative enteric bacteria has been detected and related to the gene floR.1 This gene codes for a membrane-associated exporter protein that promotes the efflux of chloramphenicol and florfenicol from the bacterial cell. Closely related floR genes have been detected so far on plasmids of Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica serovar Newport, but also in the chromosomal DNA of E. coli, various Salmonella serovars and Vibrio cholerae.1

In the present study, we analysed a florfenicol-resistant bovine P. multocida isolate for the genetic basis of florfenicol resistance.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
The P. multocida isolate 381 was obtained from the lung of a 4-month-old calf submitted to the Veterinary Laboratories Agency, Thirsk Regional Laboratory, North Yorkshire, UK. The clinical signs were reported as pneumonia, with a 3 day duration prior to death. The treatment history of this calf included the application of florfenicol and a corticosteroid (not further specified) 2 days prior to death and oxytetracycline 1 day prior to death. The species assignment was confirmed biochemically and by a species-specific PCR.4,5 In addition, the capsular type was also determined by PCR.6 In another lung sample from the same calf, an S. enterica subsp. enterica serovar Dublin isolate (no. 336) was identified.4 Both isolates were initially screened for florfenicol resistance by the Veterinary Laboratories Agency, Regional Laboratory Rougham Hill, Bury St Edmunds, Suffolk, UK and then sent to our laboratory. In vitro susceptibility testing was performed by disc diffusion with discs containing ampicillin (10 µg), chloramphenicol (30 µg), florfenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), streptomycin (10 µg), sulfamethoxazole (300 µg), tetracycline (30 µg) or trimethoprim (5 µg).7 MICs of florfenicol were determined by broth macrodilution according to the NCCLS document M31-A2.7 PCR analyses for the florfenicol–chloramphenicol resistance gene floR followed a previously described protocol.8 Plasmid preparation by alkaline lysis and transformation experiments into E. coli recipient strains HB101 and JM101 were conducted as previously described.8 Plasmid DNA obtained from the transformants was subjected to restriction mapping with 18 restriction endonucleases. Overlapping ClaI–PstI fragments of ~5.3 and 2.5 kb, HpaI fragments of 5.5 and 1.1 kb, as well as a HpaI–DraI fragment of 1.5 kb of the plasmid from P. multocida 381 were cloned into either pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands) or pCR-Blunt® II-TOPO (Invitrogen, Groningen, The Netherlands) and transformed into E. coli recipient strains JM109 or TOP10, respectively. Sequence analyses were started with the M13 reverse and forward primers and completed with primers derived from sequences obtained with the aforementioned standard primers. A final segment of 2.5 kb was sequenced by primer walking using DNA from the E. coli JM101:pCCK381 transformant (MWG, Ebersberg, Germany). Sequence comparisons were performed with the Blast programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 14 January 2005) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 14 January 2005). The nucleotide sequence of plasmid pCCK381 has been deposited in the European Molecular Biology Laboratory database under accession number AJ871969.


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
P. multocida 381 proved to be of capsular type A and was resistant to chloramphenicol, florfenicol and sulphonamides, whereas S. Dublin 336 showed the same resistance phenotype in addition to kanamycin resistance. The MICs of florfenicol and chloramphenicol were 16 and 32 mg/L, respectively, for P. multocida 381 and S. Dublin 336. In both cases, plasmid profiling revealed the presence of a single plasmid of ~11 kb. E. coli HB101 and JM101 transformants were obtained on Luria–Bertani agar supplemented with either 15 mg/L chloramphenicol or 10 mg/L florfenicol. Subsequent screening of the transformants for their plasmid content and their in vitro susceptibility revealed that transformants from both donor isolates 381 and 336 carried only the 11 kb plasmid and that this plasmid mediated only resistance to chloramphenicol and florfenicol.

PCR analysis of the transformants indicated the presence of the chloramphenicol–florfenicol resistance gene floR. Since the smallest floR-carrying plasmid known to date was ~35 kb in size,8 the detection of this gene on relatively small plasmids of 11 kb was an interesting new observation. For a better characterization, the 11 kb plasmids from P. multocida 381 and S. Dublin 336 were subjected to extended restriction analysis, and restriction mapping revealed identical results for both plasmids. Based on this observation, the results of PCR analysis and MIC testing, as well as the finding that both strains were from the lung of the same calf, further analysis focused on the floR-carrying plasmid from P. multocida 381. A map of this plasmid, designated pCCK381, is shown in Figure 1. Sequence analysis revealed a total plasmid size of 10874 bp.



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Figure 1. Schematic presentation of plasmid pCCK381 (accession no. AJ871969) in comparison with plasmid pDN1 (accession no. Y19120) as well as parts of plasmids pMBSF1 (accession no. AJ581835) and pRVS1 (accession no. AJ289135). The reading frames are shown as arrows with the arrowhead indicating the direction of transcription (repA, repB, repC: plasmid replication; tnp: transposition; mob: mobilization; sul2: sulphonamide resistance; floR: chloramphenicol-florfenicol resistance; strA, strB: streptomycin resistance). The reading frames marked as A, B and C in the maps of pCCK381 and pDN1 indicate in part overlapping mobilization genes mobA, mobB and mobC. The {Delta} symbol indicates a truncated gene. A distance scale in kb is shown below each map. The grey-shaded areas mark the areas of ≥ 99% nucleotide sequence identity between pCCK381 and the other three plasmids. Restriction sites are abbreviated as follows: B (BamHI), C (ClaI), D (DraI), E (EcoRI), EV (EcoRV), Hp (HpaI), K (KpnI), P (PstI) and Pv (PvuII).

 
The initial 5144 bp of plasmid pCCK381 corresponded closely to the entire 5112 bp plasmid pDN1 from Dichelobacter nodosus.9 This part contained three—in places overlapping—reading frames for the 99 amino acid (aa) protein MobC, the 136 aa protein MobB and the 703 aa protein MobA, all involved in plasmid mobilization. While MobC and MobB from plasmid pCCK381 were identical to the corresponding proteins from plasmid pDN1, MobA from pCCK381 revealed three amino acid exchanges in comparison with MobA from pDN1.9 The 3' end of the mobC reading frame has been reported to constitute a separate reading frame for a 325 aa RepB protein involved in the replication of plasmid pDN1. Such a potential reading frame was also available in pCCK381, and the corresponding RepB protein differed by two amino acids from that of pDN1. Further downstream of mobC and repB in pCCK381, another two reading frames for replication proteins were detected. The gene repA coded for a 279 aa protein that was indistinguishable from RepA of pDN1.9 The repC reading frame overlapped the repA reading frame by 112 bp and coded for a 316 aa protein that differed from the corresponding protein of pDN1 by two amino acids. These comparisons showed that the entire part comprising the genes involved in plasmid replication and mobilization is highly conserved between pCCK381 and pDN1 (Figure 1). Previous studies on pDN1 suggested that its replication and mobilization genes are responsible for a broad host range and that derivatives of pDN1 also replicate in E. coli.9 Our finding, that plasmid pCCK381—which may also be considered as a pDN1 derivative—replicates in Pasteurella, Salmonella, and E. coli hosts, confirms this observation. Moreover, the fact that derivatives of pDN1 were mobilized by plasmid RP4 in E. coli has led to the suggestion that the mobilization determinants of pDN1 are functionally active.9 Since the entire mobrep gene area of plasmid pCCK381 is virtually identical to that of pDN1, we assume that plasmid pCCK381 may also be mobilizable.

Adjacent to the pDN1-homologous region, an ~3.2 kb region was found to exhibit 99% sequence identity to the floR gene regions of the 35 kb plasmid pMBSF1 from porcine E. coli8 (Figure 1) and a 110 kb plasmid from bovine E. coli 10660.10 This region included a truncated transposase gene ({Delta}tnp) and the floR gene coding for an exporter protein of the Major facilitator superfamily that specifically exports phenicol antibiotics. The floR gene of plasmid pCCK381 revealed 97.2–99.7% nucleotide sequence identity to the so far known floR genes.1 The initial 0.8 kb of this floR-homologous region, including part of {Delta}tnp and its downstream region, also showed 99% identity to a part of the Vibrio salmonicida plasmid pRVS1 (accession no. AY171244) (Figure 1). Further downstream of the floR-homologous region, an ~1.6 kb region of pCCK381 exhibited again 99% identity to another part of plasmid pRVS1 (Figure 1). Similarity to pRVS1 ended within a reading frame for a putative Mob-like protein of 269 amino acids whose N-terminal 180 amino acids closely resembled the N-terminal 180 amino acids of the 333 aa Mob protein from Bartonella grahamii (accession no. NP_696963) and the 329 aa Mob protein from Bordetella bronchiseptica (accession no. CAA47269. The C-terminal part between amino acids 144 and 269 was virtually identical to the N-terminal 126 amino acids of the 165 aa Mob protein from plasmid pRVS1.

The structural analysis of plasmid pCCK381, the first florfenicol resistance plasmid of P. multocida, revealed that this plasmid is composed of several segments previously found on other plasmids. All these other plasmids have been found either in bacteria such as E. coli from cattle and pigs, which have previously been shown to carry the floR genes, or in bacteria that cause diseases in fish and ruminants, such as coldwater vibriosis (V. salmonicida) or infectious pododermatitis (D. nodosus), for the control of which florfenicol is used. Although it is not possible to determine in retrospect where and when this plasmid has evolved, the structural analysis suggested that plasmid pCCK381 is most likely the result of interplasmid recombination. The presence of the pDN1-analogous repmob gene region bears the danger of a further dissemination of this plasmid and its floR gene.


    Acknowledgements
 
We thank Vera Nöding for excellent technical assistance as well as Jon Rogers and Paul Todd of the Veterinary Laboratory Agency, Rougham Hill, Bury St Edmunds, Suffolk, UK for providing the two florfenicol-resistant strains and helpful discussions.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Schwarz, S., Kehrenberg, C., Doublet, B. et al. (2004). Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 28, 519–42.[CrossRef][ISI][Medline]

2 . Priebe, S. & Schwarz, S. (2003). In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob Agents Chemother 47, 2703–5.[Abstract/Free Full Text]

3 . Kehrenberg, C., Mumme, J., Wallmann, J. et al. (2004). Monitoring of florfenicol susceptibility among bovine and porcine respiratory tract pathogens collected in Germany during the years 2002 and 2003. J Antimicrob Chemother 54, 572–4.[Free Full Text]

4 . Koneman, E. W., Allen, S. D., Janda, W. M., et al. (1997). Color Atlas and Textbook of Diagnostic Microbiology, 5th edn. Lippincott, Philadelphia.

5 . Townsend, K. M., Frost, A. J., Chiang, W. L. et al. (1998). Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates. J Clin Microbiol 36, 1096–100.[Abstract/Free Full Text]

6 . Townsend, K. M., Boyce, J. D., Chung, J. Y. et al. (2001). Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 39, 924–9.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals—Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA.

8 . Blickwede, M. & Schwarz, S. (2004). Molecular analysis of florfenicol-resistant Escherichia coli from pigs. J Antimicrob Chemother 53, 58–64.[Abstract/Free Full Text]

9 . Whittle, G., Katz, M. E., Clayton, E. H. et al. (2000). Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1. Plasmid 43, 230–4.[CrossRef][ISI][Medline]

10 . Cloeckaert, A., Baucheron, S., Flaujac, G. et al. (2000). Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle. Antimicrob Agents Chemother 44, 2858–60.[Abstract/Free Full Text]