Occurrence of OXA-48 Carbapenemase and Other β-Lactamase Genes in ESBL-Producing Multidrug Resistant Escherichia coli from Dogs and Cats in the United States, 2009–2013

Objective: The aim of this study was to explore the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemases among ESBL-producing multidrug resistant (MDR) Escherichia coli from dogs and cats in the United States. Methods: Of 2443 E.coli isolated from dogs and cats collected between August 2009 and January 2013, 68 isolates were confirmed as ESBL-producing MDR ones. PCR and sequencing were performed to identify β-lactamases and plasmid-mediated quinolone resistance (PMQR) genes, and shed light on the virulence gene profiles, phylogenetic groups and ST types. Results: Phylogenic group D and B2 accounted for 69.1% of the isolates. 50 (73.5%) isolates carried CTX-M ESBL gene, and the most predominant specific CTX-M subtype identified was blaCTX−M−15 (n = 33), followed by blaCTX−M−1 (n = 32), blaCTX−M−123 (n = 27), blaCTX−M−9 (n = 19) and blaCTX−M−14 (n = 19), and blaCTX−M−123 was firstly reported in E. coli isolates in the United States alone or in association. Other β-lactamase genes blaTEM, blaSHV, blaOXA−48, and blaCMY−2 were detected in 41.2, 29.4, 19.1, and 17.6% of 68 ESBL-producing MDR isolates, respectively. The blaTEM and blaSHV genes were classfied as ESBLs with the exception of the blaTEM−1 gene. Additionally, 42.6% (29/68) of isolates co-expressed blaCTX−M−15 and PMQR gene aac(6′)-Ib-c. The overall occurrence of virulence genes ranged from 11.8 (ireA) to 88.2% (malX), and most of virulence genes were less frequent among CTX-M-producing isolates than non-CTX-M isolates with the exception of malX and iutA. The 68 isolates analyzed were assigned to 31 STs with six being novel. Three pandemic clonal lineages ST131 (n = 10), ST648 (n = 9), and ST405 (n = 9) accounted for more than 41% of the investigated isolates, and ST648 and ST405 of phylogenetic D were firstly reported in E. coli from dogs and cats in the United States. Conclusion: blaCTX−M−123 of ESBLs and carbapenemase blaOXA−48 were firstly reported in ESBL-producing MDR E.coli from dogs and cats in the United States, and ST131, ST648, and ST405 were the predominant clonal groups.


INTRODUCTION
Extraintestinal pathogenic strains of Escherichia coli (ExPEC) are the most important dogs and cats bacterial pathogens associated with extraintestinal infections (Beutin, 1999). However, extended-spectrum β-lactamase (ESBL)-producing ExPEC are isolated worldwide with increasing frequency from human and animal clinical isolates (Pitout, 2012). The occurrence of β-lactamases, including ESBLs, plasmidmediated AmpC β-lactamases (pAmpC) and carbapenemases among E. coli pose serious challenges to the use of penicillins, extended-spectrum cephalosporins (3rd and 4th generation cephalosporins), monobactams, and carbapenems (Karisik et al., 2008;Geser et al., 2012). Furthermore, ESBL-producing isolates are often cross-resistant to fluoroquinolones and other antimicrobial agents, thus expressed multidrug resistance (MDR). This combination of properties can significantly affect the course and outcomes of infections. β-lactamase genes commonly located on mobile genetic elements, such as plasmids, transposons, or integrons, and the resistance plasmids can easily be transferred between bacterial isolates by conjugation mechanism. Accordingly, transmission of βlactamase genes between companion animals and owner has become a subject of active discussion as companion animals could be potential sources of ESBL-producing E. coli isolates causing community-acquired infections (Schmiedel et al., 2014).
Although the ESBLs, pAmpCs and carbapenemases in E. coli isolates from humans and animals have been characterized in various studies around the world, knowledge about the βlactamases and population structure in MDR E. coli isolates from companion animals in the United States is limited. Prior to the current study only two studies have described the occurrence and the diversity of ESBLs in E. coli from dogs and cats in the United States (O'Keefe et al., 2010;Shaheen et al., 2011), and the isolates were collected from September 2004 to December 2007, and May 2008to May 2009. However, the β-lactamases, particularly CTX-M-type ESBLs, are characterized by ongoing and complex evolution. Currently, greater than 150 variants have been identified, and several chimeras, e.g., bla CTX−M−64 and bla CTX−M−123 have been reported since 2009 (He et al., 2013). Moreover, several novel β-lactamases, e.g., bla KPC , bla NDM−1 , and bla OXA−48 are emerging worldwide in E. coli isolated from humans or animals.
The aim of the present study was to (i) investigate the occurrence and molecular characterization of ESBL-producing MDR E. coli recovered from clinical cases of infection in dogs and cats in the United States, over a period of time ranging from August 2009 to January 2013, and (ii) characterize the association between β-lactamases, phylogenetic groups, virulence genes and the ST types.

Bacterial Isolates
Between August 2009 and January 2013, a total of 2443 E. coli isolates from urine, wound, ear, genital tract, anal sac, nasal structure, and soft tissue samples of dogs and cats with presumed naturally-occurring infection in six geographical regions of the United States: West (California), South (North Carolina), Central (Missouri), Midwest (Ohio and Illinois), and Southeast (Alabama), Northeast (Massachusetts) were received from a nationally recognized veterinary diagnostic laboratory. Isolates were reconfirmed to be E. coli upon receipt by the Clinical Pharmacology Laboratory (CPL) at Auburn University based on reculture overnight on CHROMagar Orientation (BD Diagnostics, Franklin Lakes, NJ) at 37 • C, and then the isolates were harvested and stored in tryptic soy broth containing 30% glycerol at −80 • C until studied.

Susceptibility Testing and Initial ESBL Identification
Antimicrobial susceptibility testing was performed for all 2443 isolates using 96 well custom microdilution susceptibility plates according to the manufacturer's protocol (Trek Diagnostic Systems, Inc., Cleveland, OH). Susceptibility testing was performed using 16 antimicrobials representing six antimicrobial classes and classified into 12 antimicrobial categories: penicillins: ampicillin; penicillins + β-lactam inhibitor: amoxicillin-clavulanic acid; anti-pseudomonal + β-lactam inhibitor: ticarcillin-clavulanic acid; non-extended spectrum cephalosporins (1st generation cephalosporins): cephalothin; extended-spectrum cephalosporins (3rd and 4th generation cephalosporins): cefotaxime, ceftazidime, and cefpodoxime; cephamycins: cefoxitin; carbapenems: meropenem; tetracyclines: doxycycline; phenicols: chloramphenicol; fluoroquinolones: enrofloxacin and ciprofloxacin; aminoglycosides: gentamicin and amikacin; and folate pathway inhibitor: sulfamethoxazoletrimethoprim (Magiorakos et al., 2012;Thungrat et al., 2015). All MIC determinations were performed in triplicates and E. coli ATCC 25922 was used for quality control. The results were interpreted according to the guidelines of Clinical Laboratories Standards Institute (CLSI; CLSI, 2013). The MICs were recorded using the Sensititre Vizion system (Trek Diagnostic Systems), and each isolate was categorized in terms of its resistant phenotype as to: susceptible (S), non-multidrug resistance (DR) or MDR. DR was defined as resistance to 1 or 2 antimicrobial classes, and MDR was defined as resistance to three or more antimicrobial classes.
Additionally, all the 2443 E. coli isolates were screened for ESBL production using microdilution-based Sensititre (TREK diagnostic systems, Cleveland, Ohio) with ESBL Confirmatory MIC plates (ESB1F) as described previouly . Finally, the ESBL-producing isolates expressed MDR phenotype were used in the current study.

Phylogenetic Grouping and Virulence Genotyping
The distribution of phylogenetic groups amongest the ESBLproducing MDR isolates was determined by the new quadruplex PCR as recently described by Clermont et al. (Clermont et al., 2013). Genomic DNA were extracted from bacterial preparations using the PreMan R Ultra Preparation Reagent according to the manufacturer's protocol. The presence of 17 virulence genes [fimH, sfa/focDE, afa/draBC, papA, papC, papG alleles (I, II, III), hlyA, cnf1, kpsM II, fyuA, iutA, ireA, iroN, traT, and malX] known for their association with pathogenicity ExPEC isolates was ascertained in each isolate by use of established PCR assay as reported previously (Johnson and Stell, 2000;Liu et al., 2015).

Transfer of Resistance Genes by Conjugation
We tested whether the ESBL-producing E. coli isolates harboring bla CTX−Ms , bla TEM , bla SHV , or bla OXA−48 enzymes were transferable. Conjugation was performed by broth mating at 37 • C on 10 ESBL-producing MDR isolates using plasmid-free sodium azide resistant E. coli J53 (J53 AZ r ) as recipient as described previously (Shaheen et al., 2011). Transconjugants were selected on tryptic soy agar plates supplemented with sodium azide (150 µg/ml) and cefotaxime (2 µg/ml). Antimicrobial susceptibility, confirmatory tests for ESBL production, and PCR detection of ESBL genes were performed on all transconjugants as described to confirm transfer of ESBL genes.

Multilocus Sequence Typing (MLST)
MLST was performed using seven conserved housekeeping genes of E. coli (adk, fumC, gyrB, icd, mdh, purA, and recA). A detailed scheme of the MLST procedure, including the primers, PCR conditions, allelic type and sequence type assignment methods, is available at MLST databases at the at the Warwick University website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli).

Statistical Analysis
Significance was determined by Pearson's Chi-squared test with Yates continuity correction using "R" software (version 3.0.1), and the level of significance was set at P < 0.05.

Conjugation Experiments
We tested whether bla CTX−M genes or other β-lactamase genes in 10 selected isolates were transferable by conjugation experiments, and seven out of the 10 ESBL-producing isolates successfully transferred the β-lactamase genes to the recipient E. coli. PCR analysis showed the presence of respective bla CTX−M genes and other β-lactamase genes, including two bla OXA−48 -carrying plasmids from all the transconjugants (Table 3). Meanwhile, PMQR gene aac-(6')-Ib-cr was co-transferred with β-lactamase genes. Generally, all donors and their transconjugants were resistant to amoxicillin-clavulanic acid, ampicillin, cefotaxime, cefoxitin, cefpodoxime, cephalothin, and ticarcillin-clavulanic acid, and all transconjugants exhibited an increase of at least eight-fold in MICs compared to the recipient, E. coli J53 AZ r . The ciprofloxacin MICs for four transconjugants harboring aac-(6 ′ )-Ib-cr ranged from 0.06 to 0.125 mg/L, representing an increase of two-fold to four-fold compared with the recipient (Table 3). Additionally, the transconjugants remained susceptible to meropenem, ciprofloxacin, gentamicin, chloramphenicol, and doxycycline, whereas one transconjugant harboring bla OXA−48 was resistant to sulfamethoxazole-trimethoprim and reduced the susceptibility to meropenem.
Frontiers in Microbiology | www.frontiersin.org pAmpC genes bla CMY−2 were distributed in nine STs. Notably, all ST131, ST405, and ST648 isolates expressed resistance to ciprofloxacin and 3rd generation cephalosporins, whereas all ST131 isolates remained susceptible to cefoxitin. A strong correlation was revealed between the virulence gene profiles and STs, and the same STs showed the similar virulence gene profiles. Among the three most common STs, ST405 isolates harbored more virulence genes (mean 4.6), followed by ST131 (mean 4.4), and virulence genes were less abundant in ST648 isolates (mean 3.4). Almost all of the ST131 and ST405 isolates were positive for afa/draBC, traT, and malX genes, ST648 isolates were significantly associated with fimH, malX and traT, but negative for afa/draBC.

DISCUSSION
ESBLs, pAmpC and carbapenemases are mostly responsible for the emerging resistance to the β-lactam antibiotics, especially the 3rd generation cephalosporins and carbapenems in E. coli (Pitout, 2012). In the present study, we conducted a molecular detection and characterization of the β-lactamase genes in ESBLproducing MDR E. coli isolates from dogs and cats in the United States over a period of time ranging from August 2009 to January 2013, and also revealed the association between the phylogenetic groups, virulence gene profiles, genetic backbones and β-lactamase types. The prevalence of 3.8% ESBL-producing E. coli found in this study is similar to that recorded in a recent study (3%; Shaheen et al., 2011) but higher than the first survey (1%; O'Keefe et al., 2010) among E. coli from dogs and cats in the United States. Surprisingly, 73.9% (68/92) of the ESBL-producing E. coli exhibited MDR phenotype, and 75% of MDR isolates were resistant to more than 10 antimicrobial agents tested. Phylogenetic groups D and B2 were the main phylogenetic groups in this study, and it was similar to the phylogenetic subtype distribution of the ESBL-producing isolates from human patients , which further demonstrated that isolates in phylogenetic groups D and B2 were associated with extraintestinal infections. Among the 68 ESBL-producing MDR isolates, bla CTX−M was prominent and detected in 73.5% (50/68) of isolates, whereas two previous similar surveys carried out in different states in the United States showed that the corresponding prevalence of bla CTX−M were 16.7% and 89.7%, respectively (O'Keefe et al., 2010;Shaheen et al., 2011). It is indicated that the geographical regions, time, resistant phenotype and the history of antimicrobial treatment of the animals can affect the prevalence of bla CTX−M gene. The high prevalence of bla CTX−M strongly suggests a significant role for E. coli isolates from companion animals as ESBL gene reservoirs, which poses an additional risk to humans. Therefore, monitoring of the spread of bla CTX−M genes in E. coli isolates in dogs and cats is urgently needed. Althouth bla CTX−M−15 was still the most frequently encountered gene, the specific genotype of bla CTX−M is undergoing changes, which was supported by available evidence from the occurrence of CTX-M-9 group as well as the occurrence of a novel hybrid β-lactamase gene bla CTX−M−123 . bla CTX−M−123 was firstly discovered in E. coli from pig feces in China in 2013 (He et al., 2013), and afterward in human specimen . It is interesting to note that bla CTX−M−15 is also the most widely distributed ESBL gene among human-associated Enterobacteriaceae (Cantón and Coque, 2006). These finding revealed the possibility of crosstransmission between animals and humans. Moreover, several isolates appear only with bla TEM−1 , bla CMY−2 , or bla OXA−48 , suggesting that these isolates perhaps carry other ESBL genes, which will require further studies.
bla CMY−2 was the most prevalent pAmpC, and it not only confer resistance to a wide range of extended-spectrum cephalosporins but also are not affected by β-lactamase inhibitors. bla CMY−2 was dected in 17.6% of the isolates in our study, and it was significantly lower than the occurance of bla CMY−2 (89%) in E. coli from companion animals in a previous study in the United States (Shaheen et al., 2011). We supposed that the occurance of bla CMY−2 might be underestimated since only the ESBL-producing MDR isolates were characterized in this study. Meanwhile, our results showed that majority (58.3%) of CMY-2-producing isolates belonged to phylogenetic group D, consistent with a previous study in E. coli from human in Australia (Sidjabat et al., 2014). This similar distribution of phylogenetic group further certified that bla CMY−2 can also be transferred between different bacterial species and between animals and humans (Li et al., 2007;Shaheen et al., 2011). bla OXA−48 was initially reported in Klebsiella pneumoniae isolates in Turkey in 2001 (Poirel et al., 2004) and afterward in other Mediterranean countries (Spain, France, Italy, Egypt, and Lebanon Turkey) (Girlich et al., 2014). In 2013, it was firstly discovered in E. coli from dogs in Germany (Stolle et al., 2013). bla OXA−48 can hydrolyze carbapenems and βlactamase inhibitors but has no activity toward broad-spectrum cephalosporins (Mathers et al., 2013). Our data showed that about 19% of the isolates carried the bla OXA−48 , and they were mostly associated with meropenem resistance, sequence types ST648, ST405, and ST131 as well as different combinations of β-lactamase genes. To our knowledge, bla OXA−48 was firstly reported in the United States in 2012 (Poirel et al., 2012), and the present study is the first report of bla OXA−48 in E. coli from dogs and cats in the United States. Moreover, bla OXA−48 can transfer with other β-lactamases and aac(6 ′ )-Ib-cr. This finding also revealed possibility of the transfer between humans and companion animals appears highly probable through multiple potential pathways although bla OXA−48 is still sporadic occurrence in animals.
aac(6 ′ )-Ib-cr was the exclusive PMQR gene in this study, and CTX-M-producing isolates (particularly bla CTX−M−15 positive isolates) showed significantly higher occurrence of aac(6 ′ )-Ibcr compared to non-CTX-M or non-ESBL isolates (62 vs. 11.1 vs. 10%, P < 0.001). The frequent combination of bla CTX−M−15 and aac(6 ′ )-Ib-cr in this study further supported the previous studies that coproduction of β-lactamases and PMQR genes could conduce to the dissemination of MDR isolates, and also reflect the fact that genes encoding resistance to β-lactams and quinolones are located on the same plasmid. Although it was not the primary focus of this study, our results coincided  with a previous study (Qin et al., 2013) that ESBL-producing isolates presented a lower occurrence of studied virulence genes compared with non-ESBL isolates (the data from another study in our laboratory) with the exception of malX gene, and CTX-M-producing E. coli harbored fewer virulence genes than non-CTX-M isolates (P<0.0001). A possible reason why individual virulence gene increased among ESBL-producing is that might be a fitness trade-off for the ESBL to survive antibiotics exposure (Qin et al., 2013) and the difference source of E. coli. The exact explanation needs additional study in the future.
A previous review suggested that attention should be paid to the rising of E. coli ST131, ST648, ST405, and ST38 isolates as they can play an important role in the worldwide distribution of CTX-M-producing E. coli (Pitout, 2012). It was further confirmed by our results since ST131, ST648, and ST405 accounted for 54% of the CTX-M-producing MDR isolates. ST131 was the predominant clone in this study, and all ST131 isolates remained susceptible to cefoxitin, which has been recently suggested as an alternative carbapenems for the treatment of infections by ESBLproducing E. coli (Guet-Revillet et al., 2014). It is noteworthy that nine ST648 isolates were strongly associated with bla CTX−M−15 (88.9%, 8/9), bla OXA−48 (55.6%, 5/9), and severe clinical signs. The zoonotic potential of ST648 ESBL-producing isolates has been indicated in the isolates from humans, domestic and wild animals in previous studies (Nicolas-Chanoine et al., 2008;Cortes et al., 2010), and two recent studies in Europe further suggested that ST648 clone may represent a novel genotype that combines MDR phenotype, extraintestinal virulence and zoonotic potential in companion animals (Huber et al., 2013;Ewers et al., 2014). Furthermore, ST131, ST648, and ST405 isolates have the similar β-lactamase gene combinations and resistance profiles, respectively. While it is alarming that other STs have various β-lactamase gene combinations, especially one ST38 isolate, which was associated with the highest frequency of β-lactamases and aac(6 ′ )-Ib-cr, high level cephalosporins resistance (MICs ≥ 32 µg/ml), lowest frequency of virulence genes and severe clinical signs. Nevertheless, constant attention and further investigations for ST648 and ST38 isolates in companion animals are necessary as they are now rapidly and globally disseminated as well as the companion animals are more and more considered an important source of human infections as the physical closeness. were the predominant clonal groups among the ESBLproducing E. coli, and all ST131 isolates remained susceptible to the cefoxitin. This information will be useful for assessing the epidemiological risk factors and appropriate use of antimicrobials for ESBL-producing E. coli infections of dogs and cats.