F33:A−:B− and F2:A−:B− Plasmids Mediate Dissemination of rmtB-blaCTX-M-9 Group Genes and rmtB-qepA in Enterobacteriaceae Isolates from Pets in China

ABSTRACT This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the blaCTX-M-9 group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A−:B−, carrying rmtB-qepA, and F33:A−:B−, carrying the rmtB-blaCTX-M-9 group genes (and especially blaCTX-M-65), shared restriction patterns within each incompatibility group.

The gene type of bla CTX-M was confirmed by PCR and DNA sequencing (25). In addition, this study also included 132 Enterobacteriaceae isolates previously confirmed to be CTX-M negative that were collected from pets in Guangdong province of China during 2006 and 2008 (18,25). The presence of 16S rRNA methylase genes was identified by PCR using previously designed primers (5,6,9). Of the 135 CTX-M producers, 60 (ϳ44%) were positive for rmtB and 5 (ϳ4%) were positive for armA (Table 1). No isolate was positive for the rmtA, rmtC, rmtD, rmtE, or npmA genes. The rmtB gene was also detected in 9 (ϳ7%) of the 132 CTX-M-negative isolates. Therefore, of the 69 rmtB-positive isolates, 60 were CTX-M producers, and most of the enzymes produced belonged to the CTX-M-9 group. Since the plasmid-mediated fluoroquinolone efflux pump gene qepA is frequently associated with rmtB (1,2,17,22,23,28,32), RmtB-producing isolates were screened for the qepA gene (17). Our results showed that 31 (44.9%) RmtBproducing isolates were positive for qepA. The pulsed-field gel electrophoresis analysis revealed that most of the rmtB-positive isolates were clonally unrelated ( Table 1).
The transferability of the rmtB genes was studied by conjugation experiments as previously described (7). When plasmid cotransfer occurred, the transformation experiment was carried out. The presence of rmtB, qepA, and bla CTX-M in the transconjugants and transformants was confirmed by PCR as previously described (5,17). Of the 69 rmtB-positive isolates, 73 transconjugants/transformants containing rmtB were obtained from 66 isolates, with 6 donors generating two or three transconjugants carrying different plasmids and resistance genes. rmtB was cotransferred with bla CTX-M or qepA genes in 33 and 25 transconjugants/transformants, respectively ( Table  2). In addition, 8 transconjugants carried rmtB, qepA, and bla CTX-M-9G simultaneously.
PCR-based plasmid replicon typing was performed to characterize the conjugative plasmids carrying rmtB (4). The IncFII, IncFIB, and IncN replicon types were detected in 58 (79.5%), 5 (6.8%), and 4 (5.5%) of the plasmids from the 73 transconjugants, respectively, with 7 other transconjugants carrying two replicons (FII in combination with FIB or N) ( Table  2). To better clarify the IncF plasmids, a replicon sequence typing scheme discriminating IncF plasmid variants, described by Villa et al. (26), was used to characterize the IncFII and IncFIB replicons. Among these transconjugants, 10 and 3 different alleles were identified for the FII and FIB replicons, respectively ( Table 2). The F2 allele was detected in 24 out of the 34 transconjugants that carried both rmtB and qepA, and the F33 allele, a new F allele identified in this study, was detected in 12 out of the 22 transconjugants that carried both rmtB and bla CTX-M-9G . Three transconjugants obtained from one E. coli donor (0113DDF) carried different IncF plasmids (p0113J, p0113-1, and p0113-2T) encoded by different resistance genes (Table 3), indicating the coexistence of three unrelated plasmids in one bacterium. It has been demonstrated that the IncF plasmids possess great versatility in intracellular adaptation due to the rapid evolution of the regulatory sequences of the replicons (21,26). The coexistence and maintenance of three IncF plasmids that belong to different subgroups, F2:AϪ:BϪ, F35:AϪ:BϪ, and F33:AϪ:BϪ, in the same bacterial strain indicated preliminarily that these differences in the F alleles could result in the emergence of compatible plasmids.
Since most of the rmtB-qepA and rmtB-bla CTX-M-9G gene combinations were associated with the F2:AϪ:BϪ and F33: AϪ:BϪ plasmids, respectively (Table 3), these plasmids were subjected to restriction enzyme digestion analysis to clarify whether a specific plasmid had been disseminated among the isolates. Plasmids extracted from the transconjugants or transformants containing only a single plasmid were digested with the endonucleases EcoRI and BamHI (TaKaRa Biotechnology, Dalian, China). Twenty-one F2:AϪ:BϪ plasmids carrying both rmtB and qepA were obtained, 18 of which, including one plasmid bearing rmtB, qepA, and bla CTX-M-14 , showed identical plasmid restriction patterns. Interestingly, these patterns are the same as or highly similar to the patterns obtained from F2:AϪ:BϪ plasmids carrying both rmtB and qepA in E. coli isolates obtained from pigs, the environment, and farmers in 2002 (Table 3) (7). It is suggested that this F2:AϪ:BϪ plasmid is a rather stable plasmid circulating in different members of Enterobacteriaceae and is present in different animal and human reservoirs in China. The F2:AϪ:BϪ plasmids are also associated with bla CTX-M genes in Enterobacteriaceae isolates from China, Hong Kong, South Korea, Vietnam, Italy, Canada, the United Kingdom, and Belgium (12,14,20,24,26,30,34). Further studies are needed to address the mechanisms underlying the worldwide dissemination of these plasmid types.
Twelve F33:AϪ:BϪ plasmids carrying both rmtB and bla CTX-M-9G (including seven carrying bla CTX-M-65 ) and one F33:AϪ:BϪ plasmid carrying only rmtB showed the same BamHI digestion profiles and highly similar EcoRI digestion    (Table 3). bla CTX-M-65 is one of the dominant CTX-M types in animal isolates obtained in China after 2005 (15,16,33) and has also been identified as colocalized with rmtB in pRB1 in an E. coli isolate from a patient in the United States (8). This suggests that the increasing prevalence of CTX-M-65 in E. coli isolates may be due to the dissemination of plasmids carrying both rmtB and bla CTX-M-65 .
In conclusion, the dissemination of rmtB, qepA, and bla CTX-M genes in Enterobacteriaceae isolates from pets is mediated mainly by the F2:AϪ:BϪ and F33:AϪ:BϪ plasmids. The coexistence of these resistance determinants in a single plasmid increases the selection by one or more of the antimicrobials used in clinical practice. Therefore, prudent use of antimicrobial agents in pets is urgently needed.
Nucleotide sequence accession numbers. New replicon sequences described in this work were deposited in the GenBank database with assigned accession numbers GU477621, HQ706665, HQ706666, HQ706667, HQ882837, HQ882838, and HQ882839.