Characterization of CTX-M-14-producing Escherichia coli from food-producing animals

Bacterial resistance to the third-generation cephalosporin antibiotics has become a major concern for public health. This study was aimed to determine the characteristics and distribution of blaCTX-M-14, which encodes an extended-spectrum β-lactamase, in Escherichia coli isolated from Guangdong Province, China. A total of 979 E. coli isolates isolated from healthy or diseased food-producing animals including swine and avian were examined for blaCTX-M-14 and then the blaCTX-M-14 -positive isolates were detected by other resistance determinants [extended-spectrum β-lactamase genes, plasmid-mediated quinolone resistance, rmtB, and floR] and analyzed by phylogenetic grouping analysis, PCR-based plasmid replicon typing, multilocus sequence typing, and plasmid analysis. The genetic environments of blaCTX-M-14 were also determined by PCR. The results showed that fourteen CTX-M-14-producing E. coli were identified, belonging to groups A (7/14), B1 (4/14), and D (3/14). The most predominant resistance gene was blaTEM (n = 8), followed by floR (n = 7), oqxA (n = 3), aac(6′)-1b-cr (n = 2), and rmtB (n = 1). Plasmids carrying blaCTX-M-14 were classified to IncK, IncHI2, IncHI1, IncN, IncFIB, IncF or IncI1, ranged from about 30 to 200 kb, and with insertion sequence of ISEcp1, IS26, or ORF513 located upstream and IS903 downstream of blaCTX-M-14. The result of multilocus sequence typing showed that 14 isolates had 11 STs, and the 11 STs belonged to five groups. Many of the identified sequence types are reported to be common in E. coli isolates associated with extraintestinal infections in humans, suggesting possible transmission of blaCTX-M-14 between animals and humans. The difference in the flanking sequences of blaCTX-M-14 between the 2009 isolates and the early ones suggests that the resistance gene context continues to evolve in E. coli of food producing animals.


INTRODUCTION
Enterobacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing extended-spectrum β-lactamase genes (ESBLs), have been detected in a wide range of food-producing animals. In 1989, CTX-M-type β-lactamases, whose carriers are highly resistant to cefotaxime but sometimes susceptible to ceftazidime, were considered as a novel group of class-A β-lactamases with extended-spectrum properties. This family of enzymes are well inhibited by clavulanate and tazobactam (Tzouvelekis et al., 2000). Since then, the CTX-M family of ESBLs has become prominent and is common in E. coli with many infections occurring in human patients in the community (Livermore and Hawkey, 2005). In addition, the occurrence and prevalence of CTX-M in food-producing animals were also reported frequently (Hammerum et al., 2012;Reich et al., 2013). Rapid dissemination of bla CTX−M genes involved plasmids and mobile genetic elements as well as epidemic spread of particular strains (Eckert et al., 2006). ISEcp1-like insertion sequences (ISs) have repeatedly been observed upstream of open reading frames (ORFs) encoding members belonging to the CTX-M-1, CTX-M-2, and CTX-M-9 clusters. ISs such as IS10, IS26, and IS903 have also been observed flanking the ORF region of bla CTX−M genes (Arduino et al., 2002).
Of the CTX-M enzymes, the CTX-M-1 and CTX-M-9 clusters have been the most frequently reported worldwide, and although in some places CTX-M-2 group remains endemic, the emergence of new CTX-M groups (mainly CTX-M-1 and CTX-M-9) has been documented (D' Andrea et al., 2013). In addition, the CTX-M-14 enzyme is, besides CTX-M-9, the most widespread enzyme of the CTX-M-9 group (Valverde et al., 2009), especially in China (Li et al., 2010;Zheng et al., 2012). CTX-M-14 was first isolated from hospital in China in 1997 (Chanawong et al., 2002). It is a member of the CTX-M-9 cluster and differs from bla CTX−M−9 only by the substitution Ala 231−→Val (Ma et al., 2002), and it has spread almost all over the world (Canton and Coque, 2006). Reports showed that ISEcp1, IS26, ORF513, IS903, and ORF1005 located upstream and downstream of bla CTX−M−14 , respectively, which might have contributed to its widespread transmission (Izumiya et al., 2005;Eckert et al., 2006;Bae et al., 2007;Navarro et al., 2007). Moreover, most of the literature indicates that novel CTX-M enzyme genes were often derived from or recombinated with CTX-M-14-like β-lactamase (Djamdjian et al., 2011;He et al., 2013;Tian et al., 2014), indicating that bla CTX−M genes evolve by homologous recombination between members of different groups, especially with bla CTX−M−14 (Tian et al., 2014).
Due to the importance of bla CTX−M−14 in antimicrobial resistance and its limited information in food producing animals, we examined the characteristics and distribution of bla CTX−M−14 in E. coli of food-producing animals in China in this study.

Bacterial Isolates and CTX-M-14 Detection
From 2002 to 2009, a total of 979 E. coli isolates, including 455 isolates in 2002, 119 isolates in 2003-2004, 76 isolates in 2007 and 329 isolates in 2009 were isolated from healthy or diseased foodproducing animals from Guangdong Province in China. Samples were collected from rectal swabs of healthy animals, or the liver, heart, lung, or muscle samples of diseased or dead animals. Samples were seeded on MacConkey agar at 37 • C, and one colony with typical E. coli morphology was selected from each sample. Each isolate was from an individual animal. The bacterial strains were identified by classical biochemical methods and confirmed using the API-20E system (bioMérieux). All confirmed E. coli isolates were stored at −80 • C in the Luria-Bertani broth medium containing 30% glycerol.
Cefotaxime-resistant E. coli isolates were selected on MacConkey agar containing cefotaxime at 2 μg/mL and then the bla CTX−M−14 gene was detected by PCR analysis with the primer described previously (Yu et al., 2007), the primers and PCR programs were listed in Supplementary Table S1. PCR products were directly sequenced, and then made comparison in the GenBank nucleotide database.

Clonal Relatedness
A multiplex PCR methodology was employed to assign isolates harboring bla CTX−M−14 to one of the four phylogenetic groups (A, B1, B2, or D). Primers and methodology have been described previously (Doumith et al., 2012). For isolates carrying bla CTX−M−14 , multilocus sequence typing (MLST) was performed using seven conserved housekeeping genes (adk, purA, recA, mdh, icd, gyrB, and fumC;Wirth et al., 2006). The internal fragments of all loci were sequenced. Allelic profiles and sequence type (ST) determinations were performed according to the E. coli MLST website 1 scheme. MLST data were analyzed by using the eBURST program (version 3 2 ), which assesses the relationship within clonal complexes. The set minimum group allele number was five. The UPGMA method of START program (version 2) was used to construct phylogenetic grouping tree. The genetic distance is 0.1.
Frontiers in Microbiology | www.frontiersin.org  (Carattoli et al., 2005). PFGE with S1 nuclease (TakaRa Biotechnology, Dalian, China) digestion of whole genomic DNA was performed for all 14 transconjugants and transformants as described previously (Barton et al., 1995). After Southern transfer to a Hybond-N + membrane (GE Healthcare, Little Chalfont, UK), the plasmids were probed with the bla CTX−M−9G gene (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany).

Clonal Relatedness and Transfer of bla CTX−M−14
Phylogenetic group analysis showed that group A (7/14) was dominant amongst the isolates that produced the CTX-M-14 enzymes, followed by group B1 (4/14) and group D (3/14). None of them belonged to group B2 ( Table 1). The result of MLST showed that the 14 isolates have 11 ST, among which ST2929 and ST2962 were newly discovered (Supplementary Table S2). The MLST results belonged to five groups. ST10, ST206, ST2929, ST2930, and ST2962 belonged to Group 1 (Figure 1A), while ST155, ST224, and ST602 were classified into Group 2 ( Figure 1B). Furthermore, ST648, ST359, and ST405 belonged to Group14, Group16, and Group17, respectively. Phylogenetic grouping tree suggested that ST10, ST2929, ST206, and ST2930 were close in one branch, while ST224, ST602, ST155, ST359, and ST2962 were clustered in another branch. ST648 and ST405 were separated from others (Figure 2). Eight transconjugants and six transformants were successfully obtained by conjugation/transformation experiments. Co-transfer of bla TEM−1 or rmtB or floR genes were also detected. The bla CTX−M−14 -positive strain isolated in 2005 co-transferred with bla TEM−1 . Among the 2009 isolates, one had co-transfer of rmtB, two had co-transfer of floR, and another one had co-transfer of both bla TEM−1 and rmtB. MICs of cefotaxime, ceftiofur, and ceftriaxone increased two-fourfold compared with the recipients.

Plasmids and Genetic Environment of bla CTX−M−14
Plasmids containing bla CTX−M−14 were predominately belonging to IncF (n = 5), IncK (n = 3), and lncI1 (n = 2). Additionally, 2 of the 14 were positive for two replicons (IncHI1 and IncN), another one was positive for IncFIB and IncF, and the In this study, phylogenetic group analysis showed that group A (7/14) was dominant amongst the isolates that produced the CTX-M-14 enzymes, followed by group B1 (4/14) and group D (3/14), which was consistent with the reports in Portugal, Spain, and China (Valverde et al., 2009;Zheng et al., 2012). Previous studies showed that most E. coli strains responsible for urinary tract infections and other extraintestinal infections in humans belong to group B2 or, to a lesser extent, to group D (Johnson and Stell, 2000;Yang et al., 2012). Investigation of urinary E. coli isolates from 20 widely dispersed tertiary Chinese hospitals revealed although phylogroups D and B2 were most frequently observed, phylogroups A and B1 were also found in bla CTX−M−14 -producing E. coli isolates (Cao et al., 2011).
According to recent reports, replicon types of bla CTX−M−14positive plasmids belonged to lncF, lncFIB, lncI1, lncA/C, lncN, lncFII, and lncI1-Iγ (Millan et al., 2011;Song et al., 2011;Tamang et al., 2011). In this study, bla CTX−M−14 -carrying plasmids predominately belonged to IncF and IncK. The spread of bla CTX−M−14 in E. coli in Spain is reported to be mediated by IncK plasmids (Valverde et al., 2009), while in Korea and France bla CTX−M−14 is mostly carried on IncF plasmids (Marcade et al., 2009). IncF plasmids were found frequently to be associated with CTX-M enzyme genes of E. coli (Matsumura et al., 2013;Mnif et al., 2013). IncK plasmids may facilitate the ability of E. coli to colonize the intestine and, consequently, enhance the pathogenic profile of specific clones or clonal groups (Oshima et al., 2008). Besides, reports showed that the acquisition of IncK plasmids containing bla CTX−M−14 by group A and B1 E. coli clones could have enhanced their ability to colonize the urinary tract in patients exposed to antibiotics (Valverde et al., 2009). IncHI1, IncHI2 and IncN plasmids were rarely reported in bla CTX−M−14 -producing E. coli.
In this study, 11 different STs (including two new STs) were detected among 14 bla CTX−M−14 -producing E. coli isolates.
The findings indicate that no ST predominates in CTX-M-14producing E. coli from food-producing animals of Guangdong. ST10 and ST648 were common in E. coli isolated from human and animals (Shabana et al., 2013;Maluta et al., 2014;Xia et al., 2014;Jamborova et al., 2015). ST155 was once found in human, duck, and bovine (Ben Sallem et al., 2012;Sváb et al., 2013;Maluta et al., 2014). ST359 was once reported in human and duck (Maluta et al., 2014). ST405, a global clonal group associated with the global increase of ESBLs, was mainly reported in human origin as well as once reported in rooks and food origins (Jouini et al., 2013;Matsumura et al., 2014;Jamborova et al., 2015). ST602 in E. coli of cats was once reported (Nebbia et al., 2014). ST224 was detected in E. coli of human, dogs and buffalo origin (Mshana et al., 2011;Dahmen et al., 2013;Aizawa et al., 2014), while ST224 was found in duck origin in 2009 in this study 3 . Moreover, recent reports revealed that E. coli of human origin, especially E. coli-producing ESBLs associated with urinary tract infection, mainly belonged to the ST10 complex. In Portugal, Spain, and Brazil, ST155 and ST359 were found rising in patients suffering from urinary tract infection (Canton and Coque, 2006). In this study, the STs we have found were mainly reported in human, suggesting that bla CTX−M−14 could transfer between human and food-producing animals.
Insertion sequences played an important role in the transfer of bla CTX−M−14 . In this study, ISEcp1 was detected 42 nucleotides upstream of both bla CTX−M−14a and bla CTX−M−14b . It is of interest to note that an identical 42-bp region has also been detected upstream of different genes encoding ESBLs of the CTX-M-9 cluster, such as CTX-M-9, −14, −16, and −17 (Barlow et al., 2008), which means this subtype may have the same origin as bla CTX−M−14 . From 2002 to 2007, the genetic environment of bla CTX−M−14 -positive isolates was the same, with ISEcp1 and IS903 found upstream and downstream, respectively, while bla CTX−M−14 -positive isolates in 2009 showed diversity of the genetic platform. IS26 and ORF513 were both found in 2009. It is important to note that ORF513 located upstream of bla CTX−M−14a in strain 16-D was the same as bla CTX−M−14b of strain 103-D. This showed that resistant genes of incompatible plasmids have the possibility to transfer and then recombine.
Extended-spectrum β-lactamase genes genes were often found to be strongly associated with PMQR or 16S rRNA methyltransferase (16S-RMTase) genes, and some were often found to be located on the same plasmid, both in human and animals (Carattoli, 2009;Liu et al., 2013). In this study, bla CTX−M−14 of the isolates isolated from 2002 to 2007 tended to conjugate alone, while co-transfer with bla TEM−1 , rmtB, or floR on the same plasmid were common in the 2009 isolates. Co-existence or co-spread of ESBLs with PMQR, rmtB or floR suggests that the resistant isolates could be selected by different classes of antibiotics. The fourteen isolates carrying bla CTX−M−14 were found to be multidrug resistant and showed resistance to more than two non-β-lactam antimicrobial agents, including kanamycin, tetracycline, doxycycline, nalidixic acid, ciprofloxacin, enrofloxacin, and florfenicol. Some of them were also resistant to other cephalosporins, including ceftiofur, cefoxitin, and ceftriaxone, but remained susceptible to ceftazidime fortunately. In addition, although the bla CTX−M−14positive isolates showed resistance to kanamycin and gentamycin, most of them (13/14) remain susceptible to amikacin (data not shown), which indicated amikacin might be effective for treating bla CTX−M−14 -positive E. coli infection.

CONCLUSION
The evolution of bla CTX−M−14 gradually became diversified in food-producing animals of Guangdong, China, from 2002 to 2009. Findings from this study and previous publications by others suggest that antibiotics, especially the third-and fourthgeneration cephalosporins, should be used more prudently in food-producing animals.