Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany
Received 24 January 2005; returned 20 February 2005; revised 21 February 2005; accepted 22 February 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: The P. multocida isolate 381 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 10.8 kb florfenicolchloramphenicol 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 97.299.7% 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR analysis of the transformants indicated the presence of the chloramphenicolflorfenicol 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.
|
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 (
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 97.299.7% nucleotide sequence identity to the so far known floR genes.1
The initial 0.8 kb of this floR-homologous region, including part of
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Priebe, S. & Schwarz, S. (2003). In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob Agents Chemother 47, 27035.
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, 5724.
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, 1096100.
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, 9249.
7 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From AnimalsSecond 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, 5864.
9 . Whittle, G., Katz, M. E., Clayton, E. H. et al. (2000). Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1. Plasmid 43, 2304.[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, 285860.