JAC Advance Access originally published online on May 2, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):13-17; doi:10.1093/jac/dkl174
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Plasmid-mediated florfenicol resistance in Pasteurella trehalosi
1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL) Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Agence Française de Sécurité Sanitaire des Aliments (AFSSA) 69007 Lyon, France 3 Institut National de la Recherche Agronomique (INRA), Plasticité Génomique Biodiversité, Antibiorésistance UR1282, 37380 Nouzilly, France
*Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de
Received 7 February 2006; returned 22 March 2006; revised 4 April 2006; accepted 7 April 2006
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Abstract |
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Objectives: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR.
Methods: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization.
Results: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events.
Conclusions: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.
Keywords: floR gene , respiratory tract pathogens , antimicrobial resistance , gene transfer , recombination , insertion sequences
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Introduction |
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Relatively few novel antimicrobial agents have been approved for veterinary use during recent years. One of them, the fluorinated chloramphenicol derivative florfenicol, was licensed in 1995 and 2000 for the control of respiratory pathogens from cattle and pigs, respectively.1 A drug-specific monitoring programme in Germany that aimed at determining MICs of florfenicol among bovine (Pasteurella multocida, Mannheimia haemolytica) and porcine respiratory tract pathogens (P. multocida and Actinobacillus pleuropneumoniae) revealed that virtually all target bacteria were florfenicol-susceptible and that their MIC50 and MIC90 values have remained stable over the past decade.2 Recently, the first florfenicol-resistant bovine P. multocida isolate from the UK carrying the florfenicol resistance gene floR on a plasmid has been described.3
The gene floR codes for a membrane-associated efflux protein of the major facilitator superfamily and specifically exports phenicols from the bacterial cell.4 This gene was previously detected as part of the Salmonella genomic island 1 associated multiresistance gene cluster, but was also detected in various Gram-negative enteric bacteria on plasmids or in the chromosomal DNA.1 In 2005, the floR gene was identified to be part of the small non-conjugative transposon TnfloR.5
In contrast to P. multocida, little is known about antimicrobial resistance in other Pasteurella species.6 This applies in particular to Pasteurella trehalosi. Originally described as biotype T of Pasteurella haemolytica, P. trehalosi was recognized as a separate species in 1990.7 P. trehalosi is mainly a pathogen of sheep where it causes septicaemia in older lambs.6 It has also been isolated from other ruminants including goats,8 bisons9 and cattle.10 In the present study, we identified the first florfenicol-resistant bovine P. trehalosi isolate and investigated the genetic basis of this resistance trait and the localization of the corresponding resistance gene.
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Bacterial strain and species identification
The P. trehalosi isolate 13698 was obtained from the lung of a calf. The clinical signs were reported as respiratory disease. The strain was collected in France through the RESAPATH network. For species assignment, biochemical tests11 were backed up by 16S rDNA sequencing. The latter approach was performed at the DSMZ, the German National Resource Centre for Biological Material in Braunschweig, Germany.
Antimicrobial susceptibility testing
In vitro susceptibility testing was performed by disc diffusion with discs containing ampicillin (10 µg), chloramphenicol (30 µg), enrofloxacin (5 µg), florfenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), spectinomycin (100 µg), streptomycin (10 µg), sulfamethoxazole (300 µg), tetracycline (30 µg) or trimethoprim (5 µg). MICs of florfenicol, chloramphenicol and sulphonamides were determined by broth macrodilution according to the NCCLS document M31-A2. The transformant P. multocida P4000::pCCK13698 was investigated for chloramphenicol acetyltransferase (CAT) activity by a colorimetric CAT assay.12 The chloramphenicol-susceptible recipient strain P. multocida P4000 served as a negative control.
DNA techniques
PCR detection of florfenicol, chloramphenicol and sulphonamide resistance genes followed previously described protocols and primers.4,13 Plasmid preparation by alkaline lysis and transformation experiments (heat-shock transformation into CaCl2-treated competent cells or electrotransformation) into Escherichia coli recipient strains JM109, JM110, JM101, HB101 and C600 as well as P. multocida strain P4000 were conducted as described previously.13,14 Transformants were selected on Luria–Bertani agar or sheep blood agar supplemented with either chloramphenicol (15 mg/L) or florfenicol (10 mg/L). Plasmid DNA obtained from the P. multocida transformants was subjected to restriction mapping; SspI fragments were cloned into pCR-Blunt® II-TOPO (Invitrogen, Groningen, The Netherlands) and transformed into E. coli recipient strain TOP10. Sequence analysis by the dideoxy chain termination method (MWG Biotech, Martinsried, Germany) was started with the M13 reverse and forward primers and completed with primers derived from sequences obtained with the aforementioned standard primers. Sequence comparisons were performed with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 29 January 2006) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 29 January 2006).15 The nucleotide sequence of plasmid pCCK13698 has been deposited in the European Molecular Biology Laboratory (EMBL) database under accession number AM183225.
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Results and discussion |
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Identification of plasmid pCCK13698
P. trehalosi 13698 carried a plasmid of 
15 kb, designated pCCK13698, which mediated resistance to chloramphenicol, florfenicol and sulphonamides when transferred into the P. multocida recipient strain P4000. No transformants were obtained in repeated transformation experiments with any of the E. coli recipient strains used, suggesting that this plasmid does not replicate in E. coli, but in Pasteurella hosts. The MICs for the original P. trehalosi strain and the P. multocida P4000::pCCK13698 transformant of florfenicol, chloramphenicol and sulphonamides were 16, 32 and 512 mg/L, respectively. PCR analysis of the transformant indicated the presence of not only the chloramphenicol-florfenicol resistance gene floR, but also the chloramphenicol acetyltransferase gene catA3 and the sulphonamide resistance gene sul2. Because of the comparatively low MIC of chloramphenicol, we performed a CAT assay that showed a >20-fold higher deacetylation rate of acetyl-CoA in the cell-free lysates of P. multocida P4000::pCCK13698 as compared with the plasmid-free recipient strain P. multocida P4000 and hence confirmed pCCK13698-based CAT activity. The reason for the low MIC of chloramphenicol in the presence of two functionally active chloramphenicol resistance genes, catA3 and floR, remains to be answered.
For a better characterization of the florfenicol resistance plasmid from P. trehalosi and a structural comparison with the floR-carrying plasmid pCCK381, previously identified in P. multocida,3 plasmid pCCK13698 was sequenced completely. Sequence analysis revealed a total plasmid size of 14 969 bp. A map of plasmid pCCK13698 is shown in Figure 1.
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Structure and organization of plasmid pCCK13698
The initial 635 bp of plasmid pCCK13698 as presented in Figure 1 consists of small segments that exhibited similarity to plasmid pCCK3813 (positions 1–225) and to the mobC gene region of plasmid RSF101016 (positions 169–635). The RSF1010-like part ended at a 96 bp sequence that was identical to an internal segment of the transposase reading frames of IS1592 and IS1593, the latter of which is an insertion sequence from M. haemolytica (EMBL database accession no. AJ439064). A large part of plasmid pCCK13698, comprising 6770 bp (positions 731–7500), corresponded closely (99.5% identity) to plasmid pHS-Rec from Haemophilus parasuis.17 This segment included the genes parA coding for a 207-amino-acid partition protein, rec coding for a 255-amino-acid recombinase protein, orfX coding for a 168-amino-acid hypothetical protein of not further specified function, repB coding for a 372-amino-acid plasmid replication protein and int coding for a 261-amino-acid integrase protein (Figure 1). Immediately after the pHS-Rec homologous part, a complete copy of an IS1593-like insertion sequence was detected (positions 7497–8527). This insertion sequence, designated IS1592, was 1031 bp in size, had perfect 20 bp inverted repeated sequences at its termini and coded for a transposase protein of 294 amino acids that differed by three amino acid exchanges from that of IS1593 from M. haemolytica. Further downstream of the IS1592 element, a 2812 bp RSF1010-like segment (positions 8526–11 337) was detected. It included a truncated 
repA gene and a complete repC gene, which represent part of the rep gene area of the broad-host-range plasmid RSF1010, and an RSF1010-associated sul2 gene.16 The sul2 gene, which codes for a 272-amino-acid sulphonamide-resistant dihydropteroate synthase, was followed by a catA3 gene coding for a 213-amino-acid CAT and a largely truncated, functionally inactive strA gene coding for a streptomycin phosphotransferase. Only the initial 73 codons of the strA reading frame were present in plasmid pCCK13698. The 1831 bp segment (positions 10 475–12 305), comprising the genes sul2, catA3 and strA, showed 99.7% identity to the multiresistance gene cluster previously identified on plasmid pMVSCS1 from Mannheimia varigena.18 The strA gene was followed by a 1801 bp segment (positions 12 306–14 149) that exhibited 99.7% identity to the sequence of TnfloR.5 It contained the complete gene floR coding for a 404-amino-acid exporter protein, the reading frame for a 101-amino-acid LysR-like transcriptional regulator and the initial 51 bp of the TnfloR-associated transposase reading frame. Immediately thereafter, a complete copy of the insertion sequence IS26 was detected (positions 14 150–14 969). This IS26 element was 820 bp in size, had perfect 14 bp terminal inverted repeats and coded for a transposase protein of 234 amino acids. It was identical to that associated with the kanamycin resistance transposon Tn268019 and to a large number of IS26 sequences from various enterobacterial species deposited in the databases, such as E. coli (AF550679), Salmonella enterica (AY333434), Klebsiella pneumoniae (AY123253), Citrobacter freundii (AF550415), Enterobacter cloacae (AY532647), Serratia marcescens (BX664015) and Proteus vulgaris (AP004237). It should also be noted that an IS26-flanked region encompassing the genes repA, repC, sul2, strA and strB has been described recently on a plasmid from S. enterica subsp. enterica serovar Enteritidis.20
Integration and recombination sites in plasmid pCCK13698
The presence of a 96 bp IS1592 relic, which was in the opposite orientation to the complete IS1592 sequence, suggested that originally a not further specifiable part of pHS-Rec or of a pHS-Rec-related plasmid was flanked by two inverted copies of IS1592. While the right-hand copy remained complete, the left-hand copy was found to be largely truncated. A comparison of the pCCK13698 sequence with the corresponding sequences of the two plasmids RSF1010 and pHS-Rec revealed the presence of two potential recombination sites (see Figure 2). Recombination site 1 is 17 bp in size with 16/17 bases matching the IS sequence and 12/17 bases matching the RSF1010 sequence. Recombination site 2 consists of 15 bp with 15/15 bases matching the IS sequence and 9/15 bases matching the pHS-Rec sequence. Recombination involving these two sites might explain the deletion of the left-hand IS1592 copy. A third recombination site was detected in the strA sequence and might have been used for recombination with part of the floR upstream sequence of TnfloR. This recombination site 3 comprised 16 bp with 12/16 bases matching the strA sequence of pMVSCS1 and 14/16 bases matching the TnfloR sequence.
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A closer look at the sequences flanking the two complete insertion sequences revealed no direct repeats. For IS1592, it is not known whether it produces directly repeated sequences at the integration site. However, the Tn5706-associated elements IS1596 and IS1597, which represent close derivatives of IS1592, are known to produce a 7 bp direct repeat at the integration site.21 For IS26, it is known that it generates an 8 bp direct repeat.12 The lack of direct repeats might suggest that besides the integration of these IS elements, further processes have occurred by which the directly repeated sequences at one or both ends were deleted or modified. The observation that the sequences up- and downstream of both IS elements were different might support this assumption.
In conclusion, this is the first report of antimicrobial resistance genes in P. trehalosi. The structural analysis of plasmid pCCK13698 revealed that it is almost exclusively composed of segments previously associated with other plasmids, transposons or insertion sequences and that interplasmid recombination processes might have played a role in the formation of this plasmid. Most of these plasmid and transposon segments have already been found in members of the family Pasteurellaceae. However, the occurrence of IS26—an insertion sequence widely distributed among Enterobacteriaceae—in the genus Pasteurella is a novel observation that supports the hypothesis of a gene flow between Pasteurellaceae and Enterobacteriaceae.
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None to declare.
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Footnotes |
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These authors contributed equally to this work. 
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Acknowledgements |
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We thank Vera Nöding for excellent technical assistance. We acknowledge the veterinary laboratories participating in RESAPATH. Part of this work was supported by a grant of the French Ministry of Agriculture (Direction Générale de l'Alimentation).
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