IncA/C Plasmid-Mediated Spread of CMY-2 in Multidrug-Resistant Escherichia coli from Food Animals in China

Objectives To obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China. Methods A total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the bla CMY-2 genotype was determined using PCR and sequencing, characterization of the bla CMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE), PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST). Results All 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (bla CTX-M-14 or bla CTX-M-55), plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6′)-Ib-cr), floR and rmtB. The co-transferring of bla CMY-2 with qnrS1 and floR (alone and together) was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12) and A (n = 11), and virulent group D (n = 8). There was a good correlation between phylogenetic groups and sequence types (ST). Twenty-four STs were identified, of which the ST complexes (STC) 101/B1 (n = 6), STC10/A (n = 5), and STC155/B1 (n = 3) were dominant. Conclusions CMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.


Introduction
The prevalence of plasmid-encoded AmpC (pAmpC) blactamases, which confer resistance to extended-spectrum cephalosporins in Gram-negative bacilli, has increased in both humans and livestock isolates worldwide, and is a global problem [1]. CMY-2 is the most common pAmpC in E. coli from different geographical areas including Asia, North America, and Europe [2][3][4][5], and has now been reported in Salmonella and Escherichia coli isolates from a variety of food animals and products on all continents except Australia [6]. Some studies reported that the increased incidence of infections in humans with S. enterica serovar Newport possessing CMY-2 in North America was associated with exposure to dairy cattle as well as the consumption of raw milk, raw or improperly cooked beef mince, and the cross-contamination of raw meat with other foods [7,8]. CMY-2-bearing plasmids, predominantly the A/C, I1, or K/B replicon types [9,10], were readily transferable between Salmonella and E. coli from food animals and humans [10,11]. Therefore, livestock-associated plasmid-encoded CMY-2 has posed increasing concerns to public health worldwide 6 .
E. coli is one of most common pathogens of nosocomial, healthcare-associated, and community infections [12]. According to CHINET (antimicrobial resistance surveillance networks in China), bacteria isolated from various samples from 14 hospitals in 10 regions or Provinces were predominantly E. coli, comprising 61.8% of the total isolates [13]. CMY-2 AmpC in Chinese pediatric patients was detected first in E.coli between 2003 and 2005; the occurrence of AmpC b-lactamase in E. coli and K. pneumoniae had the highest prevalence [13]. The resistance to multiple drugs, including third-generation cephalosporins, and the carriage of both pAmpC and extended spectrum b-lactamase (ESBL) genes in E. coli isolates from food animals has increased rapidly [14][15][16][17]. The detection rate of bla CMY-2 in isolates from chickens, which emerged between 2000 and 2003, increased rapidly from 2004-2007 [16]. However, limited information is available regarding the characteristics of the CMY-2 plasmid type or the clonal dissemination of CMY-2 in E.coli of food animal origin in China. The aim of this study was to assess the molecular epidemiology and characteristics of CMY-2-bearing plasmidproducing E. coli in food animals including pigs, chickens, ducks, and geese.

Bacterial isolates and antimicrobial susceptibility
A total of 1083 unique E. coli isolates from a range of food animal species (pigs, n = 424; chickens, n = 306; ducks, n = 175; geese, n = 178) were recovered between October 2010 and January 2012 from 58 fixed farms described previously [15]. Of the 1083 E. coli isolates, 587 were cultured from feces, 456 from the viscera, 14 from blood samples, and 26 from drinking water and subsurface soil of duck farms. These isolates were collected partially from the Guangdong Province Surveillance Program on Antibiotic Resistance in bacteria isolated from animals. The program was carried out by the Laboratory of Clinical Microbiology, Veterinary Research Institute, Guangdong Academy of Agricultural Sciences. Methods for sample collection and bacterial isolation were described previously [18]. Susceptibility testing was determined for all isolates using the standard agar dilution method on Mueller-Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [19,20]. The 13 antimicrobials tested were ampicillin (Amp), ceftiofur (Cft), cefotaxime (Ctx), ceftazidime (Caz), cefoxitin (Cxt), ceftriaxone (Ctr), gentamicin (Gen), kanamycin (Kan), amikacin (Ami), florfenicol (Flf), tetracycline (Tet), ciprofloxacin (Cip), and olaquindox (Oqx). Cefoxitinresistant strains (MIC $8 mg/L) were used for selecting CMY-2producing strains via PCR amplification of the bla CMY gene and DNA sequencing, molecular characterization of drug resistance, and epidemiology of bla CMY-2 -harboring strains. E. coli ATCC 25922 was used as the control strain.

Genetic environment of bla CMY-2 gene and detection of integrons
The genetic environment of the bla CMY-2 genes was investigated using PCR and sequencing. The ISEcp1 forward primer and CMY-2 reverse primer were used to investigate regions upstream of the bla CMY-2 genes. The CMY-2 forward primer and reverse primers for IS903, IS26, orf447, mucA, or orf513 were used to characterize regions downstream of the bla CMY-2 genes. Integrons of class 1 or class 2 as well inserted gene cassettes were detected. Sequences of the above primers are listed in Tables S2 and S3.

Conjugation and plasmid analysis
Conjugation experiments were performed on bla CMY-2 -containing strains using streptomycin-resistant E. coli C600 as the recipient [4]. Transconjugants were selected on MacConkey agar plates supplemented with streptomycin (1000 mg/L) and cefoxitin (8 mg/L). Transconjugants were tested for the presence of drug resistance genes and antimicrobial susceptibility, as described above.
Plasmids were typed using PCR-based replicon typing (PBRT) [21]. PFGE with S1 nuclease (TakaRa Biotechnology, Dalian, China) digestion of whole genomic DNA was performed for all 15 donor strains and transconjugants, as described previously [22]. After Southern transfer to a Hybond-N + membrane (GE Healthcare, Little Chalfont, United Kingdom), the plasmids were probed with the bla CMY-2 gene and respective replicons (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany).

Antimicrobial susceptibility
A total of 233 (21.5%) out of 1083 E. coli isolates were resistant to cefoxitin with MIC values ranging from 8 mg/L to .512 mg/ L. Only 33 of the cefoxitin-resistant isolates produced CMY, including 19 from pigs, 10 from chickens, and two each from ducks and geese. Of the 33 bla CMY bearing isolates, 31 carried bla CMY-2 from feces (Table 1), and two from the feces and viscera of geese carried bla CMY-41 and bla CMY-64 , respectively (data not shown). All 33 CMY-producing isolates exhibited multi-drug resistance profiles, and showed resistance to both b-lactam drugs and more than two non-b-lactam drugs. The most common resistance pattern was Amp-Ctx-Caz-Ctr-Flf-Gen-Kan-Tet-Cip-Oqx (20/33, 61%). The presence of resistance to olaquindox (a growth promoter used extensively in pig and poultry farms) was detected in the majority of bla CMY-2 -bearing strains (28/33, 85%). Twenty-three bla CMY-2 -bearing isolates were resistant to ceftiofur, a newly approved b-lactam for veterinary use in China. Resistance to amikacin was relatively low (8/32, 25%).

Characterization of bla CMY-2 harboring isolates
All except one CMY-2-producing isolate harbored more than one resistance gene conferring resistance to different antimicrobial drugs (Table 1). Of the detected ESBLs genes, bla CTX-M type alone (bla CTX-M-14 and bla CTX-M-55 ) was identified in two strains; no isolate harbored the bla SHV gene. The narrow b-lactamaseencoding genes bla TEM-1 and bla OXA-1 were found in 22 and three strains, respectively. Among the detected PMQR determinants, oqxA, qnr, and aac-(69)-Ib-cr were detected in 17, 15, and five strains, respectively (Table 1). Of the 15 qnr genes, 14 were qnrS1, and one was qnrB6. No qnrA, qnrD, qnrC, or qepA genes were detected. The floR and rmtB genes were identified in 18 and four strains, respectively. Both ESBL producers also carried the qnrS1, oqxA, and floR genes. Remarkably, one strain (L461) contained five genes conferring resistance to five antimicrobial drug classes Table 1. Overall results of co-resistance, phylogenetic grouping, MLST and plasmid replicon analysis of CMY-2producing E. coli isolates of food producing animals in China.

Population structure analysis
All 31 bla CMY-2 strains were distributed into groups B1 (n = 12) and A (n = 11) of the commensal strains, strains associated with enterotoxigenic and enterohemorrhagic infections, and the potentially virulent phylogenetic group D (n = 7). The strain carrying bla CMY-41 from the liver of a diseased goose belonged to group D, and the strain containing bla CMY-64 from a goose intestine belonged to group A.
A total of 18 different PFGE-types were detected among 22 typeable CMY isolates (20 CMY-2, one CMY-41, and one CMY-64), including 19 single types, and two clusters (.90% similarity) containing two (Cluster 2) and four isolates each (Cluster1) (Fig. 1). Interestingly, the four isolates in Cluster1 were obtained from two different locations and further divided into two phylogenetic groups, each containing two isolates. The Cluster 2 isolates belonged to the same phylogenetic group and originated from the same place.

Transferability of bla CMY-2 and plasmid analysis
The transferability of bla CMY-2 was observed in 15 out of 31 isolates at transfer frequencies ranging from 10 23 to 10 27 transconjugants per recipient. Of the 15 transferable bla CMY-2 , 10 were located on the IncA/C type plasmid with sizes of 160 to 200 kb, two 40 kb IncFIB plasmids, two IncK plasmids sized 160 kb and 200 kb, and one 220 kb HI2 plasmid. Genes encoding resistance to other antimicrobials also co-transferred in some strains. Remarkably, qnrS1 and floR co-transferred with bla CMY-2 in eight of 15 isolates. The co-transfer of bla CMY-2 , bla CTX-M-55 , and qnrS1 was observed in one isolate from a duck.

Discussion
In the present study, we performed a broad molecular epidemiological characterization of CMY-2-producing E. coli collected from the Guangdong Province Surveillance Program on antibiotic resistance in bacteria isolated from animals between 2010 and 2012. Compared with earlier surveillance on E. coli antibiotic resistance from fixed food animal farms, resistance to a panel of cephalosporins was increased significantly from ,7% to between 20 and 60% (data not shown) for all tested drugs in 2003 to 2005 [14,15]; resistance to cefotaxime, ceftriaxone, cefoxitin, ceftiofur, and ceftazidime was 55.8, 59.7, 21.5, 60, and 20.8%, respectively. The rapid increase in resistance to third-generation cephalosporins, particularly for ceftiofur, which is newly approved for use in veterinary clinical settings, was consistent with previous reports from other Provinces in China [16,17].
Cefoxitin/ceftiofur resistant isolates from E. coli and Salmonella occur frequently worldwide, which is likely to be associated with the production of CMY enzyme(s) [24]. The use of ceftiofur in the veterinary clinical setting has encouraged selection of CMY-2 AmpC in both Salmonella and E. coli [6]. In China, the occurrence of CMY-2 in E. coli originating in chickens increased rapidly [16]; the detection rate of bla CMY-2 in chicken isolates was higher than in those from pigs [25], which is consistent with reports from Japan [26]. In the present study, all CMY-2 producers were isolated from feces, consistent with a previous study [14], suggesting that the gastro-intestinal tract of animals is a reservoir for CMY-2 producers. Only half of the samples were collected from animal feces in the present study, which might explain the lower occurrence of CMY-2. Of note, many CMY-2 producers were isolated from pigs, which was an increased prevalence compared with previous reports [14,25]. The use of ceftiofur was approved for pigs in 2005 in China, which may have contributed to this rapid increase.
Salmonella and E. coli isolates carrying bla CMY-2 have been associated with community-acquired infections [1,6,27]. Plasmids carrying AmpC genes often carry other genes that confer resistance to non-b-lactams, but rarely ESBL genes [28]. Previous studies demonstrated that CMY-2-producing E. coli easily acquired other resistance genes, resulting in a multidrug resistant profile. The floR gene was the most common gene acquired, which conferred resistance to florfenicol. Some studies have shown that the increasing occurrence of CMY-2 was associated with the use of florfenicol in the veterinary clinic [29]. In the present study, most CMY-2 producers harbored not only genes encoding b-lactamases (including ESBLs), but also many diverse genes encoding resistance to other antimicrobials. In addition to floR, variants of qnr, a plasmid-mediated quinolone resistant gene, were detected frequently. The presence of qnr or floR, either alone or in combination, was detected frequently in CMY-2 isolates. Transconjugation experiments confirmed that the bla CMY-2 gene could co-transfer with multiple antibiotic resistance genes, frequently with qnrS1+floR or qnrS1, driven by 160 and 200 kb-sized IncA/C plasmids. Previous studies demonstrated that plasmids carrying CMY-2 could self-transfer between different strains alone, but rarely co-transferred with multiple genes [4,27,30]. The spread of bla CMY-2 was driven mainly by the IncA/C, IncI1, or IncK plasmids [31].
Co-localization of bla CMY-2 and floR on an IncA/C plasmid was detected commonly. A close relationship between the IncA/C plasmid and the multidrug resistance (MDR) of plasmid bearing isolates from human, animal, and environmental origins has been reported [9]. Considering the high occurrence of MDR genes (such as floR and qnr) in IncA/C plasmids and the co-transfer of these genes within the same plasmids in the present study, we suggest that there should be a stable association of these resistance genes with A/C plasmids. In addition, bla CMY-2 co-transferred with qnrS1 located on the IncK plasmid, with sizes of 160 and 200 kb. These results highlight the potential risk for co-selection of isolates carrying bla CMY-2 via the use of florfenicol or fluoroquinolones in the raising of food animals [32,33]. Interestingly, the cotransfer of bla CMY-2 with qnrS1+floR located on the IncHI2 plasmid was first reported in a chicken isolate. The IncHI2 plasmid was most prevalent in Salmonella isolates of food animal origin, and could readily capture PMQR [34]. Our results suggest that the dissemination of IncHI2 plasmids carrying CMY-2, PMQR, and floR between E. coli and Salmonella might have occurred.
Although the occurrence of OqxAB was surprisingly high in China (39%) [35] compared with Denmark (1.8%), and Korea (0.4%) [36,37], the encoded genes rarely co-transferred with bla CMY-2 on the same plasmid. Previous findings demonstrated that rmtB was the most prevalent 16S rRNA methylase gene in the Enterobacteriaceae isolates that produced ESBLs in China [23,38]. Similarly, only the rmtB gene was detected in five strains in this study. However, in contrast with the previously described cotransfer of rmtB and ESBL-encoding genes, the co-transfer of rmtB with bla CMY-2 was not observed in the current study, as demonstrated by a conjugation experiment.
Plasmids and integrons can contain or capture a variety of resistance genes that are beneficial for survival of the bacterial host and help them adapt to changing environments [39,40]. Integrons are genetic platforms involved in the spread of different previously captured gene cassettes that encode determinants of antimicrobialresistance and represent a fundamental resource for bacterial evolution [41]. Integrons are divided into five classes based on integrase gene sequence; class 1 integrons are by far the most common in clinical isolates of Gram-negative bacteria [41]. In this study, we found five types of integrons encompassing eight different genes: aadA1, aadA2, aadA5, aadA22, dfrA1, dfrA17, sat1, and orfF. The most common integron profile (dfrA17-aadA5) was found in Salmonella and other Enterobacteriaceae, and recently in two Staphylococcus species isolated in China, suggesting the successful spread of this integron around the world and across bacterial phyla [40]. Different types of integrons contained by CMY-2 producers Figure 1. Dendrogram of XbaI-PFGE patterns of CMY-producing E. coli strains recovered from food-producing animals. All the strains were CMY-2 producers, except for T19 (CMY-41) and T129 (CMY-64). Similarity analysis was performed using the Dice coefficient, and clustering was performed by following the unweighted-pair group method using average linkages (UGPMA). A total of 16 PFGE patterns were identified, and the two clusters with highly similar PFGE patterns were labeled C1 and C2. represented a diverse trend in strain evolution. DNA fingerprints also revealed that most CMY-2 producers were unlikely to be derived from a single E. coli clone. However, the clonal dissemination of bla CMY-2 between different farms at the same and different geographical locations was also found.
There was strong correlation between phylogenetic groups and STs in this study. One of the most common ST lineages, STC101, was isolated from pigs, and belonged to the avirulent phylogroup B1. Most of these harbored class 1 integron-containing drfA17+ aadA5 cassette arrays. The global spread of bla CTX-M-9G ESBLs and bla NDM-1 was driven by ST101/B1 E. coli, and could be explained by the accumulation of a large number of virulence genes [42]. Similarly ST101C/B1 CMY-2, which is prevalent dominantly and consistently at pig farms from different geographical regions, could be explained by its ability for acquiring class 1 integrons with the most common cassettes arrays and antibiotic resistant genes. Capturing and acquiring these genes could help establish the dissemination of STC101/B1 CMY-2 isolates between pig farms. The other most common ST, STC10, belonged to the avirulent phylogroup A, and was distributed widely between pigs, chickens, and geese. The significant contribution of ST10C to the spread of resistance in humans was reported in Europe and Canada [43]. Interestingly, a chicken origin ST48 (belonging to ST10C) contained qnrS1+floR, as well a class 2 integron carrying a sat1+aadA1 cassette array. Compared with ST10, its SLV or DLV contained more antibiotic resistant genes, which helps the host bacteria adapt to their surroundings under antibiotic selective pressures. Three strains isolated from a diverse range of animal species corresponded to ST648, which belongs to virulent phylogroup D. It was worthy noting that two of these three strains contained class 1 integrons with different cassette arrays, which might allow the potential persistence between animals. E.coli strains of ST648/D clones were reported to cause most cases of ESBL-producing E. coli bacteremia in the Netherlands [44]. Additional concerns arise from this ST belonging to phylogroup D, due to its ability to produce New Delhi metallo (NDM)-type carbapenemases in hospitalized patients in Pakistan and the United Kingdom [45,46]. In a recent study, ST648/D clones were the main vectors that allowed the spread of bla CMY-2 between dogs in the Republic of Korea [47].
This study demonstrated the presence of bla CMY-2 in broad hostrange conjugative plasmids. To our knowledge, this represents the first comprehensive analysis of CMY-2 plasmids in E. coli isolated from food animals in China. bla CMY-2 was co-transferred with qnrS1 and/or floR, linked to diverse lineages of E. coli STCs (including 101, 10, and 155), and disseminated among different food producing animals in China. The acquisition of multiple antimicrobial resistant genes and integrons might have allowed CMY-2-positive isolates to persist in the environment and evolve under antibiotic selective pressure. Continued surveillance of CMY-2 in animal reservoirs is necessary to curb the spread of multidrug resistant pathogens from animals to humans.

Supporting Information
Table S1 Primers used for the PCR amplification of antimicrobial resistance genes. (DOC)