Emergence of CTX-M-2-producing Escherichia coli in diseased horses: evidence of genetic exchanges of blaCTX-M-2 linked to ISCR1

Sir, The impact of CTX-M-producing bacteria on animal health remains unclear.1 The CTX-M-2 enzyme was first identified in a Salmonella Mbandaka strain isolated from faeces of a 4-month-old child.2 Later on, CTX-M-2-producing Enterobacteriaceae were also reported in faeces from healthy poultry and horses.1,3–5 In this study, we describe the molecular characterization of different blaCTX-M-2-carrying plasmids in multidrug-resistant Escherichia coli isolates recovered from cases of diseased horses. From November 2008 through June 2010, five cephalosporin-resistant E. coli isolates were obtained from diseased horses hospitalized at the Faculty of Veterinary Medicine, Ghent University, Belgium. The isolates were recovered from horses with an abdominal fistula (E. coli 2657), abdominal wound infection (E. coli 3744), peritonitis (E. coli 1443), funiculitis (E. coli 1454) and arthritis (E. coli 4410) (Table S1, available as Supplementary data at JAC Online). Most horses received a prolonged empirical therapy with several antimicrobial agents, such as β-lactams, aminoglycosides and fluoroquinolones. Antimicrobial susceptibility testing of the E. coli isolates was assessed by the disc diffusion method and interpreted according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (Supplementary Data).6 PCRs to screen for extended-spectrum β-lactamase (ESBL) genes were performed.5 All isolates harboured the plasmid-encoded blaCTX-M-2 gene. E. coli 4410 and 3744 also carried a narrow-spectrum blaTEM-1 gene. The epidemiological relationship among the isolates was studied by PFGE.3 XbaI PFGE revealed no genetic relatedness between the isolates (data not shown). Plasmid transfer experiments were then carried out.5 Briefly, E. coli J5, resistant to rifampicin, was used as the recipient strain.3 Conjugation experiments were performed overnight in Luria–Bertani broth at 37°C with a donor/recipient ratio of 1 : 5. Transconjugants were selected on MacConkey agar plates (Oxoid Ltd, Basingstoke, UK) supplemented with ceftiofur (8 mg/L) and rifampicin (250 mg/L). The antimicrobial susceptibility of the transconjugants was also determined3 to identify the co-transferred non-β-lactam resistance (Table 1). The blaCTX-M-2-carrying plasmids were extracted from the transconjugants and were further characterized by EcoRI restriction fragment length polymorphism (RFLP) analysis and Southern blot hybridization.3 The plasmids pB4-25,5 p1684Sa023 and p04-92751 were included in this analysis. The incompatibility (Inc) group of each blaCTX-M-2-carrying plasmid was defined by the PCR-based replicon typing method.7 The genetic environment of blaCTX-M-2 was investigated as described previously.8 The different genetic regions (integron cassette array, IntI1 to ISCR1, ISCR1 to blaCTX-M-2, and blaCTX-M-2 to 3′-CS) were defined by PCR, and the sizes were compared with known genetic regions present on the blaCTX-M-2-carrying plasmids pB4-25,5 p1684Sa02 (accession number EF592570)3 and p04-9275 (accession number EF592571).1 Integron cassette arrays were also further characterized by sequencing (Table 1).8 Characteristics of the blaCTX-M-2-carrying plasmids (>100 kb) analysed in this study aAntimicrobial susceptibility testing of the E. coli isolates was assessed by the disc diffusion method (Kirby–Bauer) and interpreted according to the guidelines of the Antibiogram Committee of the French Society for Microbiology. The antimicrobial drugs used were tetracycline (TET), sulphonamides (SUL), trimethoprim (TMP), streptomycin (STR), kanamycin (KAN) and fourth-generation cephalosporins (4GC). bpB4-25, blaCTX-M-2-carrying plasmid isolated from E. coli, poultry, Belgium, 2008;5 p1684Sa02, blaCTX-M-2-carrying plasmid isolated from Salmonella enterica serovar Virchow (GenBank accession number EF592570), poultry, Belgium, 2002;3 and p04-9275, blaCTX-M-2-carrying plasmid isolated from S. enterica serovar Typhimurium (GenBank accession number EF592571), human (gastroenteritis), French Guiana, 2004.1 cdfrA, gene encoding resistance to trimethoprim; aadA, gene encoding resistance to streptomycin. Characteristics of the blaCTX-M-2-carrying plasmids (>100 kb) analysed in this study aAntimicrobial susceptibility testing of the E. coli isolates was assessed by the disc diffusion method (Kirby–Bauer) and interpreted according to the guidelines of the Antibiogram Committee of the French Society for Microbiology. The antimicrobial drugs used were tetracycline (TET), sulphonamides (SUL), trimethoprim (TMP), streptomycin (STR), kanamycin (KAN) and fourth-generation cephalosporins (4GC). bpB4-25, blaCTX-M-2-carrying plasmid isolated from E. coli, poultry, Belgium, 2008;5 p1684Sa02, blaCTX-M-2-carrying plasmid isolated from Salmonella enterica serovar Virchow (GenBank accession number EF592570), poultry, Belgium, 2002;3 and p04-9275, blaCTX-M-2-carrying plasmid isolated from S. enterica serovar Typhimurium (GenBank accession number EF592571), human (gastroenteritis), French Guiana, 2004.1 cdfrA, gene encoding resistance to trimethoprim; aadA, gene encoding resistance to streptomycin. All isolates contained a high-molecular-weight conjugative blaCTX-M-2-carrying plasmid (>100 kb). Plasmids p1443, p1454, p2657 and p3744 belonged to the IncHI1 group, and only plasmid p4410 belonged to IncFIB (Table 1). To our knowledge, this is the first time that blaCTX-M-2 has been identified on IncHI1 and IncFIB plasmids. RFLP analysis of plasmid DNA revealed the same fingerprint pattern for three out of four IncHI1 plasmids (Figure S1, available as Supplementary data at JAC Online). The differences seen in the RFLP patterns for the IncHI1 plasmids may possibly reflect the rapid evolution of these plasmids. Southern blot hybridization with a blaCTX-M-2 probe showed a >10 kb EcoRI fragment for all plasmids (Supplementary Data). Characterization of the genetic environment showed that blaCTX-M-2 was part of novel complex class 1 integrons (Table 1). The gene cassette array of the complex class 1 integron located on the IncHI1 plasmids differed in size from known gene cassette arrays of blaCTX-M-2-carrying complex class 1 integrons (Table 1). Sequencing of the 1664 bp cassette array revealed the association of the trimethoprim resistance gene cassette dfrA17 and the streptomycin/spectinomycin resistance gene cassette aadA5. The IncFIB plasmid p4410 showed the same gene cassette array, dfrA1-aadA1, as pB4-25 and p1684Sa02, but the size of the IntI1-ISCR1 region of plasmid p4410 was ∼15–20 kb, in comparison with the known size of 5000 bp (Table 1). The common region 1 (ISCR1) was found to be linked to the blaCTX-M-2 gene, as reported.1 All these results may indicate that ISCR1 mobilized the blaCTX-M-2 gene from one integron to another through rolling circle transposition, a phenomenon that has been reported.1 Together, these findings show the recent emergence of blaCTX-M-2-producing E. coli in diseased horses. Previous reports have shown that blaCTX-M-2 is consistently linked with ISCR1 and located on IncHI2 plasmids from bacteria of human and animal origin.1,3–5 The fact that blaCTX-M-2 was here found on IncHI1 and IncFIB plasmids suggests the mobility of blaCTX-M-2 via ISCR1 between plasmids of different incompatibility groups. The potential for the transposition of blaCTX-M-2 has health implications, since extended-spectrum cephalosporins are extensively used in human and veterinary medicine. Indeed, the horses from this study received a prolonged treatment with different antimicrobial agents to recover from an infection with an ESBL-producing bacterium. The flexibility of blaCTX-M-2 also highlights the need to develop appropriate means to control the dissemination of this gene and associated resistance genes. Therefore, comparative genomic analysis of different plasmids carrying blaCTX-M-2 might be useful to fully understand their evolution, plasticity and spread. While blaCTX-M-2 has frequently been detected in human pathogens,1 this is the first report of blaCTX-M-2-producing E. coli isolated from diseased animals and, more specifically, horses. The emergence of blaCTX-M-2 is therefore a clear cause for concern. This work was supported by internal funding. None to declare. We thank Mrs Nathalie Van Ryselberghe and Isabelle Monchaux for their skilled technical assistance.