Molecular Characterization of ESBL-Producing Escherichia Coli Isolated from Healthy Cattle and Sheep

Abstract The present study aims to characterize ESBL-producing Escherichia coli isolated from healthy cattle and sheep in the Burdur province of Turkey. Fecal samples from a total of 200 cattle and 200 sheep were tested and ESBL-producing E. coli was isolated from 31 (15.5%) cattle and three (1.5%) sheep samples using the Clinical and Laboratory Standards Institute’s combined disk method. Among the ESBL gene classes detected by PCR, blaCTX-M was the most frequent type, followed by the blaTEM and blaSHV families. ESBL-producing E. coli isolates showed co-resistance to multiple classes of antibiotics including aminoglycosides, phenicols, quinolones, folate pathway inhibitors and tetracyclines. The resistance rates were higher in the cattle isolates than in the sheep isolates. Phylogenetic grouping of the E. coli isolates indicated group A (particularly A1) was the predominant phylogenetic group (19/34, 55.9%), followed by groups B1 (9/34, 26.5%) and D (6/34, 17.6%); none of the isolates belonged to group B2. The study shows that ESBL-producing E. coli isolates exist in the intestinal flora of healthy cattle and sheep in the Burdur province of Turkey. This is the first report showing the emergence of CTX-M type ESBL-producing E. coli in sheep farms in Turkey


INTRODUCTION
Extended-spectrum beta-lactamases (ESBLs) are hydrolytic enzymes produced by Gram-negative bacteria, and they confer resistance to many important antibiotics including penicillins, 1 st -4 th generation cephalosporins and monobactams; ESBLs are not active against carbapenems (e.g., imipenem, meropenem and ertapenem) or cephamycins (e.g., cefoxitin). ESBLs are usually inhibited by beta-lactamase inhibitors (e.g., clavulanic acid and tazobactam), which are commonly utilized for laboratory detection and confi rmation of ESBLs [1][2][3]. In the recent years, there has been a steady increase in the emergence of ESBL-producing members of Enterobacteriaceae around the globe, which presents a major challenge for healthcare and is in part a consequence of selective pressure generated by the extensive use of oxyimino-cephalosporins in the treatment of bacterial infections [4]. The most frequently encountered ESBLs in Enterobacteriaceae belong to the TEM, SHV and CTX-M families [1,3]. TEM and SHV variants with ESBL activity have been largely derived from TEM-1/TEM-2 and SHV-1 beta-lactamases respectively [5]. On the other hand, bla CTX-M genes have been captured from the chromosome of Kluyvera spp. onto the conjugative plasmids that mediate their dissemination among Enterobacteriaceae [6]. CTX-M enzymes can be subclassifi ed into clusters 1, 2, 8, 9 and 25, based on similarities in amino acid sequences [7].
The presence of ESBL-producing Escherichia coli has been described in cattle and sheep populations around the world [8][9][10][11][12]. However, very limited information is available on the presence and extent of ESBL-producing bacteria in cattle and sheep populations in Turkey. To date, only a few local studies [13][14][15] have been conducted, and the majority focused only on the phenotypic detection of ESBL-producing E. coli, without detailed characterization of the ESBL types involved. However, in a small-scale study conducted by Kucukbasmaci et al. [15], ESBLs detected in fecal Enterobacteriaceae isolates from cattle and sheep in northwest of Turkey were identifi ed, and none of them were of the CTX-M type. This fi nding was somewhat surprising considering that CTX-M has been increasingly identifi ed in many different sources including humans, animals and the environment and that it has virtually displaced the other ESBLs within Enterobacteriaceae during the last decade [16]. Therefore, the present study was conducted to characterize the ESBL genes found in fecal E. coli isolated from healthy cattle and sheep.

Study population and sampling
The present study was conducted on dairy cattle and sheep populations in Burdur province located in the southwest of Turkey. The study included 16 herds of dairy cattle (Holstein) and 12 fl ocks of sheep (Awassi) selected using the random sampling method. For sample collection, 200 healthy cattle (≥ 12 months of age) and 200 healthy sheep ( ≥ 6 months of age) were selected by random sampling. Fecal samples from each cow and sheep were taken directly from the rectum.

Selective isolation and confi rmation of ESBL-producing isolates
An enrichment procedure was performed to increase the total bacterial population before culturing the fecal samples for ESBL-producing E. coli. A 10% suspension of fecal sample in buffered peptone water (Lab M, UK) was prepared and mixed using a vortex mixer. After incubation of the suspension at 37ºC for 24 hours under aerobic conditions, 50 μl was evenly spread onto Brilliance E. coli/coliform selective agar (Oxoid, UK) supplemented with cefotaxime (CTX, 2 μg/ml) (Sigma-Aldrich, Germany) or ceftazidime (CAZ, 2 μg/ml) (Sigma-Aldrich, Germany) at the same time and incubated for another 24 hours at 37ºC under aerobic conditions. One colony from each plate (one colony from the selective agar supplemented with CTX and one from the selective agar supplemented with CAZ) per positive sample was selected randomly and subcultured on Tryptic Soy agar (Oxoid, UK) for identifi cation. After E. coli identifi cation using conventional methods (Gram staining, acid and gas from glucose, catalase test, citrate utilization, decarboxylation of lysine, hydrogen sulphide production, indole production, methyl red-voges proskauer test, orthonitrophenylbeta-D-galactopyranoside activity, oxidase test and urease production) [17], the isolates were subjected to genetic confi rmation by PCR amplifi cation of a 401 bp fragment of the E.coli 16S rRNA gene [18].
ESBL production by E. coli isolates was confi rmed using the combined disc method recommended by the Clinical and Laboratory Standards Institute (CLSI) [19].
The isolates were classifi ed as resistant, intermediate or susceptible [19][20][21]. E. coli isolates of a single fecal sample cultured on the two selective media containing CTX or CAZ and with the same antibiotic susceptibility profi le were considered to be the same isolate in this study. Multidrug-resistance was defi ned as resistance to at least 3 different classes of antibiotics excluding beta-lactams.

Polymerase chain reaction and sequencing
DNA from E. coli isolates with confi rmed ESBL production was extracted using a genomic DNA purifi cation kit (Thermo Fisher Scientifi c Inc., Massachusetts, USA) and tested by PCR with specifi c primers for the bla TEM , bla SHV and bla CTX-M genes as described elsewhere [22][23][24][25][26][27] with slight modifi cations in cycling conditions. Tag DNA polymerase enzyme, deoxyribonucleotide triphosphates and buffers used in the PCR mixture were obtained from Thermo Fisher Scientifi c Inc. (Massachusetts, USA). The cycling conditions for detection of the bla TEM gene were as follows: initial denaturation at 94 ºC for 5 min, 35 cycles of 94 ºC for 1 min, 48 ºC for 1 min and 72 ºC for 1 min, with a fi nal elongation at 72 ºC for 10 min. The cycling conditions for bla SHV gene detection were initial denaturation at 94 ºC for 5 min, 35  To demonstrate bla CTX-M and bla TEM gene diversity in the cattle population, sequence analysis of the respective genes was performed. E. coli isolates were selected according to their antibiotic susceptibility profi les and phylogenetic groups. Nine E. coli isolates belonging to three phylogenetic groups (A, B1 and D) with nine different antibiotic susceptibility profi les were selected from all of the cattle farms positive for ESBL (n= 8) for further study. For sequence analysis of the bla TEM gene, 10 E. coli isolates (from fi ve farms) belonging to three phylogenetic groups (A, B1 and D) with nine different antibiotic susceptibility profi les were also included in the study. To determine bla SHV gene diversity, we sequenced all of the PCR products (n= 3) that were obtained even if the E. coli isolates were from a single farm and belonged to the same phylogenetic group. All PCR products (3 bla CTX-M and 2 bla TEM ) from E. coli isolates from sheep were sequenced. DNA sequencing of PCR products was performed by Refgen Genetical Research and Biotechnology (Golbasi-Ankara, Turkey). Sequencing was carried out on both strands using the same primer pairs that were used in the PCR. These sequences were then compared to the NCBI GenBank sequences using BLAST to confi rm the subtypes of beta-lactamase genes. Finally, these sequences were submitted to the NCBI GenBank.

Phylogenetic analysis
To reveal whether ESBL-producing E. coli isolates belonging to a particular phylogenetic group were more likely to carry ESBL genes, phylogenetic typing (A, B1, B2 and D) of the isolates was performed according to a triplex PCR protocol as described [28] with modifi ed PCR conditions [29]. To enhance strain discrimination, subgroups (A: A 0 and A 1 ; B2: B2 2 and B2 3 ; D: D 1 and D 2 ) were also identifi ed as previously described [30].

Detection of ESBL-producing E. coli from cattle and sheep feces
E. coli grew on both types of selective media (supplemented with CTX or CAZ) in 47 of the fecal samples (45 cattle and 2 sheep). The number of isolates grown on only medium containing CTX was fi ve cattle and one sheep isolate but with only medium containing CAZ, there was only one cattle isolate. Overall, presumptive ESBL-producing E. coli were isolated from the fecal samples of 51 cattle and 3 sheep. Further characterization using the combined disk method confi rmed that 31 of the 51 E. coli cattle isolates and all of the E. coli sheep isolates produced ESBL. Therefore, 15.5% (31/200) and 1.5% (3/200) of cattle and sheep fecal samples, respectively, were positive for ESBL-producing E. coli. Of the farms tested in this study, ESBLproducing E. coli was obtained from 50% (8/16) of the cattle herds and 25% (3/12) of the fl ocks of sheep.

Antimicrobial susceptibility of ESBL-producing E. coli strains
In antibiotic susceptibility testing for the nine beta-lactams, high resistance rates were detected in the ESBL-producing E. coli isolates from both cattle and sheep ( Table 1). The resistance rates in the cattle isolates against ATM, CPD, CTX, CAZ and CRO, which are used in CLSI initial screening test for ESBL-producing E. coli, were 100%, 96.8%, 100%, 80.6% and 96.8%, respectively (Table 1).

Molecular characterization of ESBL types
PCR screening for the bla TEM , bla SHV and bla CTX-M genes in phenotypically-confi rmed ESBL-producing E. coli isolates of cattle origin indicated that CTX-M was the most common ESBL type, detected in 87.1% (27/31) of the isolates, followed by TEM (77.4%, 24/31) and SHV (9.7%, 3/31). In the ESBL-producing E. coli isolates from sheep, bla CTX-M (100%, 3/3) and bla TEM genes (66.7%, 2/3) were detected, but none of the isolates carried bla SHV genes. Group-specifi c PCR indicated that all of the bla CTX-M genes detected in E. coli isolates of both cattle and sheep belonged to CTX-M group 1.
Multiple beta-lactamase genes were detected in the majority of the E. coli isolates tested in the study. It was determined that 67.7% (21/31) of the isolates from cattle and 66.7% (2/3) of the isolates from sheep were carriers of both bla TEM and bla CTX-M genes. Each of the bla CTX-M + bla SHV and bla TEM + bla SHV gene combinations were found in a single cattle isolate while the sheep isolates did not carry these gene combinations. None of the isolates tested in the study included the bla CTX-M , bla TEM and bla SHV genes together.
Of the bla CTX-M genes detected in the 27 E. coli isolates from cattle, nine were selected for further DNA sequencing. One isolate was CTX-M-3, two isolates were CTX-M-1 and six isolates were CTX-M-15 type ESBL-producers ( Table 2). All PCR products of bla CTX-M genes (n = 3) from the sheep isolates were also sequenced; one isolate was CTX-M-3 and two isolates were CTX-M-15 producers (Table 2). Since all three of these CTX-M types belong to the CTX-M-1 cluster, this fi nding indicates agreement between sequencing and group-specifi c PCR. Among the 24 bla TEM genes detected in cattle isolates, 10 were also selected for sequence analysis, and all were found to encode TEM-1 type beta-lactamase (Table 2). Furthermore, sequence analysis of two bla TEM genes detected from sheep isolates confi rmed to have the TEM-1 genotype ( Table 2). Sequencing of the three bla SHV genes from the cattle isolates indicated the presence SHV-12 type ESBL (Table 2).

Phylogenetic types of ESBL-producing E. coli strains
Of the 31 E. coli isolates of cattle origin that were analyzed, 17 (54.8%) belonged to phylogenetic group A, eight (25.8%) to group B1, and six (19.4%) to group D. Most of the group A isolates of cattle origin (15/17, 88.2%) belonged to subgroup A 1 . Of the three E. coli isolates of sheep origin, two (66.7%) were in group A (subgroup A 1 ) and the third strain (33.3%) was in group B1. None of the cattle and sheep isolates belonged to group B2, the phylogenetic group most likely to be highly virulent. Distribution of the isolates according to phylogenetic groups along with the included ESBL types is given in Table 2.
Of the eight cattle herds which were positive for ESBL-producing E. coli, six farms had more than one isolate. Three isolates which belonged to phylogenetic group D (subgroup D 2 ) and three isolates which belonged to group A (subgroup A 1 ) were identifi ed on farm A and E, respectively. On farms F and G, two isolates from phylogenetic group B1 were identifi ed. Nevertheless on farm D, 12 isolates from three different phylogenetic groups were detected and they belonged to group A (subgroup A 1 , n = 8), group B1 (n = 3) and group D (subgroup D 1 , n = 1). On farm H, seven isolates were distributed in two different phylogenetic groups, A (subgroup A 0 , n = 2; and subgroup A 1 , n = 4) and B1 (n = 1). The SHV-12 type ESBL-producing E. coli isolates were from Farm D, and all isolates belonged to phylogenetic group B1. However, the additional beta-lactamase genes they carried were different; one strain carried only the bla SHV-12 gene, the second carried both the bla SHV-12 and bla TEM gene and the third had the bla SHV-12 and bla CTX-M gene ( Table 2).

DISCUSSION
Emergence and dissemination of ESBL-producing Enterobactericeae of animal and human origin is increasing, which is a cause for considerable concern to both medical and veterinary practitioners around the world. A number of investigations have been conducted in various parts of the world to investigate the presence and types of ESBL in cattle [8][9][10][11][12][31][32][33], but research on ESBL in sheep is limited [8,11,33]. In Turkey, only one study has been conducted so far in which both the presence and types of ESBLs in cattle and sheep were investigated, and this was in the northwest of Turkey [15]. That study reportedly identifi ed only three ESBL-producing E. coli isolates in cattle and none in sheep. Therefore, our study represents the fi rst report of the presence of ESBL-producing E. coli isolates from sheep in Turkey.
The increase in the prevalence of ESBL-producing E. coli may be due to the clonal spread of certain ESBL-producing strains and/or horizontal transfer of ESBLplasmids between strains of different genomic background [5]. Although the types of ESBLs produced by E. coli differ depending on the animal population and geographical areas, detection rates of CTX-M type ESBLs have increased dramatically around the world during the last several years [1,16,34]. In line with this trend, the present study found that the bla CTX-M gene was the most common ESBL type detected in the phenotypically confi rmed ESBL-producing E. coli isolates.
Among CTX-M type ESBLs, CTX-M-1, CTX-M-14 and CTX-M-15 are the most widespread and predominant ones detected in many studies reported from various countries [33,[35][36][37]. In bovine E. coli strains, CTX-M-1, -14, and -15 types in France [36,37], CTX-M-14 and -15 types in the UK [35], and CTX-M-14 and -15 types in Wales [33] have been reported. In sheep, E. coli strains producing CTX-M-1, -14 and -15 types were detected in Switzerland [11]. Similar to the fi ndings of these studies, CTX-M-15 was also found to be the most common ESBL-CTX-M type detected in fecal E. coli isolates from cattle and sheep in our study.
DNA sequencing of the bla TEM genes identifi ed in E. coli isolates from cattle and sheep has shown that all of the isolates are TEM-1 type, which is not considered an ESBL [1]. However, of the 10 E. coli isolates of cattle origin carrying bla TEM-1 , nine also carried the bla CTX-M gene and all of the E. coli isolates (n= 2) of sheep origin with bla TEM-1 also carried the bla CTX-M gene. Only two cattle strains had bla TEM-1 alone, yet exhibited the ESBL phenotype. This is likely due to the production of other ESBL types that were not investigated in the present study.
Intensive use of beta-lactams and other classes of antibiotics in the livestock industry may have contributed to the emergence of multidrug-resistant bacterial phenotypes. In Turkey, beta-lactams, aminoglycosides, phenicols, quinolones, folate pathway inhibitors and tetracyclines are widely used in cattle and sheep production for the treatment of a variety of infections (for example, enteritis, mastitis, pneumonia and septicemia). Studies performed in Turkey show that E. coli isolates of cattle origin are generally more resistant to various antibiotics than isolates of sheep origin [13,38]. Likewise, we found that the overall antibiotic resistance rates of other classes in the ESBL-producing E. coli isolates of cattle origin were higher than those of the sheep isolates. While resistance was observed against CIP, ENR, NAL, FFC, SXT and TET in the cattle isolates, the sheep isolates were not resistant to these antibiotics. Additionally, multidrug-resistant phenotypes were observed in only E. coli isolates of cattle origin in the present study. The higher resistance in the cattle isolates can be attributed to use of these antibiotics more widely in the treatment of a wide variety of infections in the cattle population and co-selection of resistant isolates.
Phylogenetic grouping of E. coli strains shows that most commensal strains generally belong to groups A and B1, whereas group B2, and to lesser extent group D, are generally associated with virulent extraintestinal strains [28,39]. In our study, the predominant phylogenetic group was group A (particularly subgroup A 1 ), followed by group B1 and group D. Even though none of the isolates in our study belonged to the B2 phylogenetic group, which represents the highly virulent extraintestinal E.
coli strains, we found six E. coli isolates in the group D cluster, meaning that some of the isolates may be also pathogenic. On the other hand, Milanov et al. [40] reported E. coli strains from phylogenetic groups A and B1 isolated from bovine mastitis cases, which shows that commensal E. coli strains from group A and B1 can cause various infections in cattle.
The presence of ESBL-producing E. coli isolates from more than one phylogenetic group indicates that there is signifi cant diversity among E. coli isolates carrying ESBL genes in the cattle herds and sheep fl ocks in this region. This is especially supported by the presence of E. coli isolates from three different phylogenetic groups on cattle farm D and two different phylogenetic groups on cattle farm H.
In conclusion, our study shows that bla CTX-M group 1 ESBL genes (especially bla CTX-M-15 ) are predominant in commensal E. coli isolates in cattle and sheep in Burdur province. This is the fi rst report of the presence of this gene in E. coli isolated from sheep in Turkey. However, additional studies using a broader population should be conducted in order to better understand the epidemiology of ESBL genes in animals in Turkey. Furthermore, the veterinary practitioners and farmers should be informed of this important problem and encouraged to be prudent in the use of antimicrobials for animals.