Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

The complete nucleotide sequence of a 7.7 kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. ã2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


Introduction
Actinobacillus pleuropneumoniae, one of the main pathogens contributing to swine respiratory disease throughout the world, has been used as an indicator organism in surveillance studies of antimicrobial resistance in bacteria of animal origin (de Jong et al., 2014;Hendriksen et al., 2008). Tetracyclines, beta-lactams, and trimethoprim/sulphonamides are the commonest frontline treatments for porcine pleuropnuemonia in Europe (European Medicines Agency, 2012), however, levels of resistance are increasing (de Jong et al., 2014;Vanni et al., 2012). To a lesser extent, florfenicol, a fluorinated thiamphenicol derivative licensed to treat respiratory diseases of pigs and cattle in Europe since 2000 and 1995, respectively , has been used. Most surveys of A. pleuropneumoniae antimicrobial susceptibility have shown that nearly all isolates are susceptible to florfenicol, except in Korea where resistance levels of 34% were recently reported (Gutiérrez-Martín et al., 2006;Kucerova et al., 2011;Priebe and Schwarz, 2003;Shin et al., 2005;Vanni et al., 2012;Yoo et al., 2014).
Here we describe the isolation and characterisation of a 7.7 kb florfenicol resistance plasmid from a clinical isolate of A. pleuropneumoniae. Comparative sequence analysis of this plasmid with others reported as mediating florfenicol resistance in bovine isolates of Pasteurella multocida Kehrenberg et al., 2008) and Mannheimia haemolytica (Katsuda et al., 2012) is also described.

Plasmid isolation and confirmation of floR by PCR
Plasmid DNA was isolated using a Qiaprep Spin Miniprep kit (Qiagen) according to the manufacturer's protocol. The presence of the floR gene was confirmed by PCR using primers floR_for (CGACGCCCGCTATGATCCAACTC) and floR_rev (CCCAAAAAGCCG-GACTCGCGAAG). Initially, florfenicol resistance was transferred to E. coli Stellar cells (Clontech) by heat shock with the MIDG3446 plasmid extract, according to the manufacturer's protocol, with selection of transformants on LB agar supplemented with 10 mg/ml florfenicol. Subsequently, mobilisation into selected Pasteurellaceae strains was assessed using a mating protocol previously described (Bossé et al., 2009;Bossé et al., 2015). Recipient strains included nalidixic acid resistant derivatives of M. haemolytica (MIDG1579Nal R ), P. multocida (MIDG1570 Nal R ), and Haemophilus parasuis (MIDG3176Nal R ), as well as the NADindependent A. pleuropneumoniae isolate, MIDG2331DureC::nadV, previously described (Bossé et al., 2009;Bossé et al., 2014). Transconjugants were selected on Brain Heart Infusion agar (with or without 0.01% NAD or 20 mg/ml nalidixic acid, as appropriate) supplemented with 2 mg/ml florfenicol. Transformants and transconjugants were tested by PCR for the presence of floR, as above, and for the nadV gene, as previously described (Bossé et al., 2015), where appropriate. MICs for florfenicol and chloramphenicol were determined, as above, for recipient strains +/À plasmid.

Plasmid sequence
A plasmid carrying the floR gene was isolated from a selected E. coli transformant (above) and the complete nucleotide sequence was determined using a primer walking strategy. Sequence analysis was carried out using BLASTn and BLASTx. Alignments with other floR plasmids were done using ClustalW. The sequence of the A. pleuropneumoniae floR plasmid (pM3446F) has been deposited to Genbank (accession number KP696484).

Results and discussion
A serovar 2 isolate of A. pleuropneumoniae, MIDG3446, was found to be resistant to florfenicol and chloramphenicol ( Table 1). Analysis of DNA from MIDG3446 revealed multiple small plasmids, one of which when transformed into E. coli Stellar cells (Clontech), conferred resistance to florfenicol and chloramphenicol ( Fig. 1; Table 1). Additionally, mobilisation from MIDG3446 was successful into A. pleuropneumoniae MIDG2331DureC::nadV and M. haemolytica MIDG1579Nal R , but not P. multocida MIDG1570Nal R nor H. parasuis MIDG3176Nal R . The location of the genes encoding the conjugation machinery was not determined, and no attempts were made to optimise mating conditions, nor were other isolates of these species tested. PCR confirmed the presence of floR in transformants and transconjugants (Fig. 1), and MICs for florfenicol and chloramphenicol, determined as above, for recipient strains +/À plasmid indicated transfer of resistance with the plasmid (Table 1).
The complete 7,709-bp sequence of pM3446F, isolated from a selected E. coli transformant, was determined using a primer walking strategy. Sequence analysis using BLASTn and BLASTx (http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed extended similarity to pMh1405 (AB621552), a 7,674-bp plasmid isolated in 2009 from an M. haemolytica strain recovered from a calf in Japan (Katsuda et al., 2012), and to a lesser extent to pCCK381 (AJ871969), a 10,874-bp plasmid isolated in 2005 from a P. multocida strain recovered from a calf in Thirsk, England , and subsequently from bovine and porcine isolates of P. multocida in Germany (Kehrenberg et al., 2008). All three Pasteurellaceae-derived plasmids (Fig. 2) have virtually identical replication (repA-C) and mobilisation genes (mobA-C), as well as oriV and oriT sequences, as those comprising pDN1 (Fig. 2), a 5,112-bp RSF1010-like IncQ broad-host-range plasmid (Y19120) isolated from Dichelobacter nodosus, the causative agent of ovine footrot Rawlings and Tietze, 2001;Whittle et al., 2000). However, whereas pDN1 does not encode resistance genes, these Pasteurellaceae plasmids contain differing fragments of what was initially described as TnfloR, a transposable element encoding floR, a lysR transcriptional regulator, and the tnpA transposase (Doublet et al., 2005), and subsequently shown to be an ISCR2-floR element (Toleman et al., 2006). Similar sequences have been found in various plasmids and chromosomes of different bacterial species, suggesting recombination and/or deletion events following integration have given rise to different truncated forms of the genes flanking floR (Doublet et al., 2005;Schwarz et al., 2004;Toleman et al., 2006).
The sequences flanking floR in these Pasteurellaceae plasmids show greatest similarity to those found in pAQU1 (AB571865), a 200 kb conjugative plasmid isolated from Photobacterium damselae, subsp. damselae in Japan (Nonaka et al., 2012). In Figure 2, only the sequence around the ISCR2-floR element of pAQU1 is shown (from bases 138014 to 143017 in the Genbank annotated sequence), with the Dtnp gene upstream of floR almost identical to the last 720 bases of the tnp gene downstream. In pCCK381, in addition to the partial ISCR2-floR (99% identity with pAQU1 bases 138160-141381), the 2062 bases comprising the end of the lysR gene to the start of the oriV are 99% identical to bases 146509-148591 of pAQU1. Whereas, upstream of the Dtnp gene in pCCK381, there are sequences 99-100% identity with other regions (bases 148616-148778 and bases 148992-149190) of pAQU1. In pMh1405, the 2.5 kb upstream of oriV shares 99% identity with These data suggest that these Pasteurellaceae plasmids share a common origin, with sequences from pAQU1 (or a related plasmid) having integrated into pDN1, and subsequent deletions/rearrangements of the inserted sequences. Two further Pasteurellaceae floR plasmids have been identified: pCCK1900 (NC_011378), a 10,226-bp plasmid from a porcine  ) and transfer (oriT; ) are shown. Grey blocks between sequences indicate ! 98% nucleotide sequence identity. A distance scale in kb is shown.

Conclusion
In summary, to our knowledge, this is the first report of a complete sequence of a mobilisable florfenicol resistance plasmid from A. pleuropneumoniae. Structural analysis of pM3446F revealed extensive similarity to two florfenicol resistance plasmids found in other members of the Pasteurellaceae, and mating experiments confirmed the ability to mobilise between species. This highlights the importance of continued surveillance of florfenicol susceptibility in A. pleuropneumoniae and other Gram-negative pathogens that may co-exist within the respiratory tract of pigs.

Funding
This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. MTGH was supported by Wellcome Trust grant 98051. The MIC work was funded from the former AHVLA's Research and Development Internal Investment Fund RD0030c . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.