Plasmid-borne florfenicol and ceftiofur resistance encoded by the floR and blaCMY-2 genes in Escherichia coli isolates from diseased cattle in France

Introduction

In numerous reported cases, resistance to extended-spectrum cephalosporins (ESCs), such as ceftriaxone and ceftiofur, in Salmonella enterica and Escherichia coli is conferred by chromosomal or plasmid-borne AmpC β-lactamases. The β-lactamase CMY-2 has been described worldwide as the most prevalent plasmid-borne AmpC β-lactamase in S. enterica and E. coli (Alvarez et al., 2004; Li et al., 2007).

The association of the bla_CMY-2 gene and genes conferring resistance to other antimicrobials, such as aminoglycosides, sulphonamides, tetracyclines and phenicols, on the same plasmid could also contribute to selecting multidrug resistance phenotypes (Doublet et al., 2004).

Florfenicol is a fluorinated structural analogue of chloramphenicol and thiamphenicol approved exclusively for veterinary use. The florfenicol-resistance gene floR has been characterized on different mobile genetic elements, on the Salmonella genomic island 1 (SGI1) in various S. enterica serovars and Proteus mirabilis, on conjugative and non-conjugative plasmids in E. coli and S. enterica serovars Typhimurium and Newport, as well as on the chromosome in E. coli (Boyd et al., 2008; Mulvey et al., 2006). The floR gene has also been reported as part of a transposon on a plasmid of a bovine E. coli strain, suggesting that this insertion sequence contributes to the diffusion of the floR gene between different plasmids and also the chromosome (Doublet et al., 2005). This transposition process has been shown to occur via a circular intermediate form. Characteristics of this TnfloR are very similar to those associated with site-specific integrating transposons such as Tn554, Tn5406 and Tn558, i.e. production of circular form, no inverted repeat at their termini and presence of 6–7 bp sequences at their left and right junctions. However, it has recently been demonstrated that this circular intermediate form may be the result of an homologous recombination between insertion sequence ISCR2 and its truncated form located on both sides of the floR gene (Toleman et al., 2006). The presence of these insertion sequences would play a role in the transposition of floR but would not constitute a transposable element.

Data have been reported on S. enterica isolates in the United States and France which harboured both bla_CMY-2 and floR on the same plasmid (Alcaine et al., 2005; Doublet et al., 2004; Egorova et al., 2008). Here, we characterized plasmids carrying floR and bla_CMY-2 in bovine E. coli isolates recovered in 2003 and 2004. We also investigated the mobile genetic elements potentially involved in the capture and dissemination of these genes by analysing the genetic environments of floR and bla_CMY-2 on the plasmids.

Methods

Bacterial isolates.. The bovine E. coli isolate 13688 was collected in 2003 from a lung of a calf with clinical signs reported as respiratory disease. Two other bovine E. coli isolates, 13954 and 13956, were obtained from faeces of cattle suffering from digestive disease (diarrhoea) in 2003 and 2004, respectively (Table 1). These isolates were collected through the RESAPATH network and initial screening of ESC-resistant isolates was done using ceftiofur as an indicator.

Table 1. Characteristics of the blaCMY-2-carrying Escherichia coli strains analysed in this study

E. coli J53 (pro met azi) was used as a control for mating experiments.

Antimicrobial susceptibility testing.. Antimicrobial susceptibility testing was determined by the disc diffusion method on Mueller–Hinton agar according to the guidelines of the Antibiogram Committee of the French Society for Microbiology () with 32 antimicrobial drugs (Sirot et al., 1996).

E. coli ATCC 25922 was used as a control in antimicrobial susceptibility testing. The MICs for chloramphenicol and florfenicol were determined by the standard agar doubling dilution method (Sirot et al., 1996). The MIC breakpoints for chloramphenicol and florfenicol were defined by the Antibiogram Committee of the French Society for Microbiology.

Characterization of β-lactam- and florfenicol-resistance genes.. PCRs were performed to detect the bla_TEM, bla_SHV, bla_OXA and bla_CMY β-lactam-resistance genes and the florfenicol-resistance gene floR using primers previously described (Arcangioli et al., 1999; Eckert et al., 2004). The PCR products of bla_TEM, bla_CMY and floR were sequenced on both strands by Cogenics (Meylan, France). The nucleotide sequences were analysed with the BLAST program (Altschul et al., 1997).

Phylogenetic grouping.. Phylogenetic grouping of the E. coli isolates was performed by amplification by PCR of two genes, chuA and yjaA, and an anonymous DNA fragment, TspE4.C2 (Clermont et al., 2000).

Conjugation and plasmid analysis.. Mating experiments were carried out in liquid media with E. coli K-12 J53 (pro met azi) used as recipient strain and E. coli isolates 13688, 13954 and 13956 used as donor strains. Transconjugants were selected on brain heart infusion agar supplemented with ceftiofur (2 mg l^–1) and sodium azide (500 mg l^–1). Plasmid DNA of the E. coli transconjugants was obtained as described by Takahashi & Nagano (1984) and was then subjected to restriction mapping with two restriction endonucleases, BglI and EcoRI, to evaluate the relatedness of plasmids.

A PCR-based replicon typing method was performed on each bla_CMY-2-carrying plasmid to determine plasmid incompatibility groups (Carattoli et al., 2005).

Analysis of the genetic environments of bla_CMY-2- and floR-resistance genes.. The presence of floR and bla_CMY-2 on the same plasmid and their genetic environment were studied by Southern blotting of BglI-digested plasmidic DNA using as probes floR and bla_CMY-2 genes and the ΔtnpA-floR-tnpA gene cluster (hereafter described as ΔISCR2-floR-ISCR2), obtained from the floR plasmid pEF03 (Doublet et al., 2002). The genetic environment of bla_CMY-2 and floR was assessed by PCR mapping with primers Tn-F/AmpC-R, AmpC-F/SugE-R, AmpC-F/AmpC-R and Tn-F/SugE-R to amplify the different regions of the element carrying bla_CMY-2 (Su et al., 2006) and with primers floRcirc1 and floRcirc2 to detect the circular intermediate of the floR-carrying element (Doublet et al., 2005).

Results And Discussion

E. coli isolates 13688, 13954 and 13956 were resistant to amoxicillin, amoxicillin–clavulanic acid, ceftiofur, cefoxitin, ceftazidime, kanamycin, gentamicin, streptomycin, apramycin, tetracycline, chloramphenicol, nalidixic acid, enrofloxacin, trimethoprim and sulphonamides and displayed intermediate resistance to cefotaxime and aztreonam by the disc diffusion method. Resistance to florfenicol was also observed in E. coli strains 13688 and 13956. All three strains showed susceptibility to cefepime, cefquinome and amikacin (Table 1). Phylogenetic grouping assigned the E. coli isolates 13688 and 13956 to groups A and B1, respectively, which most commensal strains belong to. E. coli 13954 belonged to group D, which is a source of extraintestinal pathogenic E. coli.

Identification of resistance genes by PCR and DNA sequencing in the three isolates showed that resistance to ESCs was conferred by the bla_CMY-2 gene. The narrow-spectrum β-lactamase gene bla_TEM-1 was also detected in these three E. coli strains.

bla_CMY-2 has been shown to be the most prevalent type of plasmid AmpC β-lactamase in members of the Enterobacteriaceae of animal origin (Li et al., 2007). The dissemination of this resistance gene throughout bacterial populations can occur by the spread of an epidemic clone or by plasmid transfer. In this study, macrorestriction profiles of the three E. coli strains DNA were distinct. The three isolates were probably not epidemiologically linked according to epidemiological data (data not shown).

Then, to assess the transfer of plasmid-borne bla_CMY-2, mating experiments were carried out with the three strains. Transconjugants were obtained on plates supplemented with ceftiofur (2 mg l^–1) and were subjected to antimicrobial susceptibility testing. All transconjugants (13688-1, 13954-1 and 13956-1) showed a phenotypic β-lactam-resistance profile (resistance to amoxicillin, amoxicillin plus clavulanic acid, cephalotin, ceftazidime, cefoxitin, cefotaxime and ceftiofur) related to the presence of bla_CMY-2 on conjugative plasmids. In addition, two of the three transconjugants tested (13688-1 and 13956-1) were also resistant to sulphonamides, trimethoprim and tetracycline. Moreover, the E. coli transconjugants 13688-1 and 13956-1 were also resistant to chloramphenicol and florfenicol, as were their parental strains, both with MICs of 256 mg l^–1 (Table 1). Analysis of EcoRI (data not shown) and BglI restriction profiles of the bla_CMY-2-carrying plasmids in strains 13688-1, 13954-1 and 13956-1 showed that the three plasmids were different from each other and also different from the previously described bla_CMY-2- and floR-carrying plasmid isolated from the human S. enterica serovar Typhimurium strain 2039 and from the floR-carrying plasmid recovered from the bovine E. coli strain BN10660 (Fig. 1a) (Doublet et al., 2002, 2004), suggesting an independent acquisition of the floR and bla_CMY-2 genes on these plasmids. This result differs from others recently reported in France in food animals where identical bla_CTX-M-1-carrying plasmids were encountered in E. coli strains recovered from poultry, cattle and swine (Girlich et al., 2007; Meunier et al., 2006).

(32K): Fig. 1. BglI restriction patterns of plasmids (a), Southern blot hybridization with the ΔISCR2-floR-ISCR2 probe of BglI-digested plasmids (b) and Southern blot hybridization with the blaCMY-2 probe of BglI-digested plasmids (c) from transconjugant E. coli 13688-1 (lanes 1), transconjugant E. coli 13954-1 (lanes 2), transconjugant E. coli 13956-1 (lanes 3), transconjugant E. coli 2039 TC1 (lanes 4) and transconjugant E. coli BN10660-1 (lanes 5). Molecular mass marker: Smart Ladder (Eurogentec).

In members of the Enterobacteriaceae, the spread of bla_CMY-2 seems to be ensured by the acquisition of the bla_CMY-2-carrying structure ISEcp1-bla_CMY-2-blc-sugE (Su et al., 2006). Overlapping PCR mapping confirmed that an identical structure to that described in S. enterica serovar Choleraesuis strain SC-B67 (Su et al., 2006) was detected in transconjugants 13688-1 and 13956-1, in contrast to transconjugant 13954-1, in which only a truncated form of ISEcp1 was located upstream of bla_CMY-2 (Fig. 2a). The presence of bla_CMY-2 on these plasmids was also confirmed by Southern blotting using a bla_CMY-2 probe, which yielded a single BglI fragment of around 5 kb (Fig. 1c). Thus, even if the plasmids carrying bla_CMY-2 are different in the three E. coli isolates, the similarity of the regions surrounding the gene strongly suggests a transfer of bla_CMY-2 among different plasmids in E. coli strains. Further to the acquisition of the bla_CMY-2 gene, plasmids can be transmitted within the same species or between different genera of bacteria as it has been reported between E. coli and S. enterica (Winokur et al., 2001).

(17K): Fig. 2. (a) Genetic organization of the regions surrounding the blaCMY-2 gene in transconjugant E. coli strains 13688-1 and 13956-1 (i) and in transconjugant E. coli strain 13954-1 (ii). (b) Genetic organization of regions surrounding the floR gene in transconjugant E. coli strains 13688-1 and 13956-1.

The florfenicol resistance of transconjugants 13688-1 and 13956-1 was conferred by the presence of the floR gene. The regions adjacent to floR were studied by Southern blotting of plasmid DNA digested by BglI with the ΔISCR2-floR-ISCR2 gene cluster used as a probe (Fig. 1b). Results indicated that floR and the flanking regions were well-conserved in the two transconjugants 13688-1 and 13956-1 and were similar to the structure initially described in E. coli strain BN10660 (Fig. 2b) (Doublet et al., 2005).

A PCR-based replicon typing method showed that two of the bla_CMY-2 plasmids isolated from E. coli strains 13688-1 and 13956-1 were of the replicon type A/C. IncA/C plasmids harbouring bla_CMY-2 have been reported worldwide in both humans and animals, suggesting that this IncA/C plasmid could be a successful and widely distributed plasmid circulating on different continents (Hopkins et al., 2006). In E. coli 13954-1, the bla_CMY-2 plasmid was of the IncI1 replicon type.

Previous reports described the presence on IncA/C plasmids harbouring bla_CMY-2 of genes conferring resistance to antimicrobials other than β-lactams, suggesting that these types of plasmids are involved in dissemination of multidrug-resistant strains (Lindsey et al., 2009; Mataseje et al., 2009). Interestingly, in transconjugant 13954-1, the IncI1 plasmid seemed not to confer additional antimicrobial drug resistance. This result was also reported by Hopkins et al. (2006), suggesting an evolution of plasmids and an ability to gain or to lose resistance determinants.

The association of floR and bla_CMY-2 on the same IncA/C plasmid significantly increases the risk of co-selection and then horizontal dissemination and persistence of ceftiofur and florfenicol co-resistant E. coli in animals. Recently, a plasmid with both floR and bla_CMY-2 was recovered from a catfish pathogen, Edwardsiella ictaluri, in the United States whereas no cephalosporins are currently approved for aquaculture, suggesting that the use of florfenicol in aquaculture could contribute to the dissemination of such a multidrug resistance plasmid (Welch et al., 2009). This is of particular concern as, based on phenotypic data, it has recently been demonstrated that healthy calves are rapidly colonized after birth by ceftiofur and florfenicol co-resistant E. coli (Donaldson et al., 2006).

Identification of insertion sequences and transposons argues in favour of the important role played by these mobile genetic elements in the acquisition of resistance genes by chromosomes or plasmids. The presence of several antimicrobial resistance genes on the same plasmid highlights the role of co-selection in horizontal transfer of multiple antimicrobial resistance genes among bacterial pathogens.

Acknowledgements

The authors would like to acknowledge the veterinary laboratories which participate in the RESAPATH network. This work was supported by a grant from the French Ministry of Agriculture (Direction Générale de l'Alimentation).