First Isolation and Molecular Characterization of blaCTX-M-121-producting Escherichia coli O157:H7 from China Cattle

Background: To study antibiotic resistance and the molecular epidemiology of bovine Escherichia coli ( E.coli ) O157:H7 and to explore the intrinsic relationship among different isolates, we collected 27 strains of bovine E. coli O157:H7 in Xinjiang from 2012 to 2017 and assessed virulence genes, antibiotic resistance and pulsed-field gel electrophoresis (PFGE) molecular typing. Results: In this study, 21 strains carried at least one virulence gene, and 19 strains carried eae gene (70.4%) including 8 carrying stx1 + stx2 + eae + hly + tccP . Most strains were sensitive to all antibiotics tested, 4 strains were bacteria-resistant, and 2 strains possessed multi-drug resistance, including one ESBL-producing strain. This is the first report of a bla CTX-M-121 gene in bovine E. coli O157:H7. Moreover, bla CTX-M-121 gene can be transmitted horizontally by plasmid between strains. The PFGE spectral similarity of the 27 strains was between 65.8% and 100%. Two PFGE types including clusters Ⅰ and Ⅱ were obtained through cluster analysis. Conclusions: E. coli O157:H7 may have undergone clonal propagation in cattle farms as well as cross-regional transmission. A horizontal transmission path with E. coli O157:H7 appeared in different areas.

States, Canada, Japan and China [3][4][5][6]. As reported, cattle are a major reservoir and source of infection with E. coli O157:H7. E. coli O157:H7 from healthy cattle have been reported worldwide [7]. Cattle irregularly expel E. coli O157:H7 out of the body without suffering any pathological symptom and transmit strains to humans through food, water, direct contact with animals, or the environment [8]. Shiga toxins (stx), one of the major virulence factors involved in the pathogenesis of E. coli O157:H7, is encoded by the stx1 or stx2 genes [9]. Intimin and enterohemolysin (encoded by the eae gene and the hly gene respectively) are another two markers that play a major role in the pathogenesis [9]. The tccP protein encoded by tccP gene is a pathogenic molecule of E. coli O157:H7 and is transduced into host cells by the type III secretion system to exert its pathogenic effect [10]. These genetic virulence characteristics are often used in epidemiological studies on strains from various sources.
Undoubtedly, antimicrobia are a main tool for the prevention and treatment of bacterial diseases in animals, but the antibiotic resistance of bacteria has caused great concern. Antibiotic resistance has become a serious problem worldwide, especially in developing countries where the quality, distribution and use of antibiotics in human medicine and veterinary medicine are under limited control [11,12]. The E.coli-caused diseases often require antimicrobial therapy, but antibiotic-resistant strains of this bacterium cause more chronic and more severe illnesses than their antibiotic-susceptible counterparts [11]. Although antibiotics are not recommended for patients with suspected E. coli O157:H7 infection, E. coli O157:H7 isolated from infected humans or animals is resistant against multiple antibiotics [13]. The emergence of multi-drug resistant (MDR) E. coli O157:H7 is a public health problem.
Xinjiang is a big province of cattle raising industry in China. Effective prevention and control of bovine pathogenic microorganisms is the premise to ensure the healthy and sustainable development of cattle raising and the safety of consumers.
To further assess the potential impacts of these isolates on public health, we investigated the pathogenicity and antibiotic resistance of E. coli O157:H7 in these farm-and abattoir-sourced isolates, studied the intrinsic relationship among different isolates and assessed the potential dissemination of MDR profiles in vitro.

Isolation of E. coli O157:H7
A total of 27 E. coli O157:H7 strains were isolated from 2,657 bovine samples in Xinjiang, including 2 collected from 1 carcass swab sample, 4 from 3 feed samples, 8 from 8 feces samples and 13 from 5 rectal swab samples (Table 1).

Antimicrobial susceptibility
Of the 27 E. coli O157:H7 strains tested, the majority (92.6%) were sensitive to all antibiotics. Only 4 out of the 27 tested strains (14.8%) showed resistance against one, eight or ten antibiotics by three different patterns ( Table 2). Two strains were multi-resistant; whereas one of them was an Extended Spectrum Beta-Lactamases (ESBLs)-producing strain. These three strains were isolated from the same cattle farm in Yili.

Prevalence of antibiotic resistance genes in E. coli O157:H7 isolates
The resistance genes identified in all of the 4 drug-resistant isolates were summarized in Table 2. One bla CTX-M-121 gene screened was detected. Four tetracycline-resistant isolates were detected, with only one isolate carrying the tetA gene encoding the tetracycline efflux pump.

Conjugal transfer of resistance
Resistance transfer by conjugation was observed in 1 bla CTX-M -producing isolate (Y4-A109) that was subjected to conjugation assays. The resistance against ampicillin, cefotaxime, ceftazidime, trimethoprim-sulfamethylisoxazole and tetracycline, and the bla CTX-M gene from the bla CTX-M -producing O157:H7 isolate can be transferred to the recipient.

Plasmid incompatibility
The bla CTX-M-121 genes were carried by untypeable plasmid.

Epidemiological typing
The chromosomal DNA of 27 isolates was available for PFGE typing and the isolates displayed 14 different PFGE profiles (Fig. 1). The similarity among the types was above 65.80%, with two dominant clusters I and II, each accounting for 41%, and other types accounted for 18%. Cluster Ⅰ mainly included type p4, and cluster Ⅱ mainly consisted of types p11 and p12. The 9 strains in type p4 and the 5 strains in type p12 were highly consistent in sampling time and location, which was identified as clonal propagation. Obvious differences were found between p4 and p12 strains, and the isolates were separated in different regions and years. Four strains of drugresistant bacteria belonged to four different types. Discussion E. coli O157:H7 is an important foodborne pathogen and is mainly associated with cattle as the reservoir [1]. The presence of E. coli O157:H7 in cattle indirectly reflects its potential harm to humans. In this study, a total of 2,657 cattle sourced samples were collected from 18 cattle farms (8/18, 44.4%) and 1 slaughterhouse in Tacheng, Bole, Yili, Wujiaqu, Changji, Wulumuqi and Aksu, and 25 strains (25/2609, 0.1%) were isolated from 8 cattle farms. The positive rate of cattle farms was high and that of samples was low. E. coli O157:H7 was isolated from samples from Yili, Wulumuqi and Aksu, but not from Tacheng, Bole, Wujiaqu or Changji, which indicated the presence of regional differences in bacterial distribution. The distribution of E. coli O157:H7 is affected by many factors, including the influence factors on bacterial excretion and transmission [14]. Seasonal and geographical characteristics affect the excretion of E. coli O157:H7 in cattle, and sampling frequency also impacts the bacteria detection rate [15,16]. Generally, the seasonal shedding of E. coli O157:H7 in cattle is well documented and characterized by higher prevalence in summer [15]. In this study, the seven regions differed not only in geographical distribution, but also in climate, environment, temperature and other aspects. These reasons may lead to the differences in the separation rate of E. coli O157:H7 in cattle.
The pathogenicity of E. coli O157:H7 is associated with several virulence factors, including production of at least Shiga toxins (stx1 and/or stx2), intimin (eae), enterohemolysin (hly) and tir couple cytoskeleton protein (tccP). Results showed 37.0% and 59.3% of the E. coli O157:H7 isolates were positive for stx1 and stx2 genes respectively. Epidemiological research shows stx2-producing strains are more virulent than stx1 producers [17]. The eae gene, which is necessary for the attaching and effacing activity, encodes the intimin protein that is essential for pathogenesis [18]. In our study, this important virulence gene was detected in 70% of the E. coli O157:H7 isolates. We identified the tccP gene in 56% of the E. coli O157:H7 strains. Noticeably, tccP gene is highly correlated with both eae gene and hly gene, but not with stx gene.
Despite the small sample size in the slaughterhouse, the isolation rate of carcass swab samples was higher than others in cattle farms, and one of them was the MDR bacteria, which showed evidence of coselection for antibiotic resistance and virulence. In this study, E. coli O157:H7 was found to be resistant against newer and more clinically important antimicrobial compounds, such as fluoroquinolone and cephalosporins. Production of β-lactamases is the main mechanism underlying the cephalosporin resistance in Gram-negative bacteria [19]. Extended-spectrum cephalosporins are an important class of drugs in both human and veterinary medicine. We investigated various narrow-spectrum (bla TEM and bla SHV ) and extended-spectrum (bla CTX− M ) β-lactamase-encoding genes, but only identified one --bla CTX− M . This is the first report of a bla CTX− M− 121 gene in bovine E. coli O157:H7.
The tetA, which is one of the most widespread tet genes found in Enterobacteria [20], was the only tetracycline-resistant gene identified in four tetracyclineresistant strains. However, to the best of our knowledge, this is the first report about the presence of tetA in bovine E. coli O157:H7 in Xinjiang. Conjugative transfer of untypeable plasmid was observed. Conjugation experiments produced successful transconjugation with MDR to β-lactamases, sulfonamides and tetracycline. This study highlights the importance of encouraging the appropriate use of antibiotics.
Dendrogram analysis of PFGE results showed the two E. coli O157:H7 strains separated from the same carcass swab samples from the slaughterhouse belonged to clusters I and II respectively, suggesting cross-contamination may occur during slaughter. The Y4-A20-1, Y4-A20-3, Y4-A20-4 from cluster I, and Y4-A20-5 from cluster II were isolated from the same rectal swab, indicating different E. coli O157:H7 strains were colonized in cattle. Cluster I W1-E51-5 and cluster II W1-E51-3F were isolated from the same feed sample, suggesting the cattle farm feed was contaminated with different E. coli O157:H7 strains. Cluster II Y1-166 and Y3-F328 were isolated from different cattle farms in the same region at the same time, which further proved horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. Cluster I Y4-A20-1, W2-A61-2 and W1-E51-5, and cluster II Y2-F25, A1-F13 and A2-F14 were isolated at different time points and from different regions, and these cattle farms were far apart. The trans-regional spread of bacteria may be caused by cross-regional trading of live animals. Based on analysis of virulence genes and drug resistance of E. coli O157:H7, we speculated that the virulence and drug resistance may be acquired or lost during the evolution and transfer of the same cluster of strains.

Conclusions
In this study E. coli O157:H7 contamination was found in cattle farms and the slaughterhouse in Xinjiang, and most isolates carried at least one virulence gene. E. coli O157:H7 had horizontal transmission pathways in the two regions, during which the virulence and drug resistance of the bacterium were constantly evolving.

Bacterial isolates
Samples (feces/feed/water) each either 1 g or 1 ml were aseptically added to 9 ml of a trypticase soya broth (TSB) containing 20 mg/l of novobiocin and were incubated for 6-8 h at 37 °C. One rectal swab was transferred into a separate tube containing 2 ml of a nutrient broth and cultured at 37 °C for 24 h [21]. One carcass swab was put into a stomacher bag and added with 500 ml of a modified trypticase soya broth containing 8 mg/l novobiocin. Each sponge was mixed in the stomacher bag for 2 min and incubated for 20 h at 37 °C [22]. This was streaked out onto sorbitol MacConkey agar supplemented with 1 mg/l potassium tellurite and incubated for one day at 37 °C. One or more pale colonies were each picked as presumptive E.
coli O157 per sample. The prevalence of E. coli O157:H7 was assessed via PCR (rfbE and fliC genes [23]) ( Table 4). The positive isolates were each inoculated into separate TSB and incubated for one day at 37 °C, from which glycerol stock was made and then stored at -80 °C for further analyses.

Antimicrobial susceptibility tests
The isolates were tested for the susceptibility to antibiotics using the Kirby-Bauer  [26]. E. coli ATCC25922 was used as a quality control strain in the susceptibility tests. The ESBLs-producing isolates were determined by double-disk synergy tests according to CLSI [26].

Conjugation experiments and plasmid analysis
Conjugation was conducted with bla CTX-M -producing isolates by the filter method, using a sodium azide-resistant E. coli J53 as the recipient. Transconjugants were selected on Mac Conkey agar containing cefotaxime or ceftazidime (4 μg/ml) and sodium azide (200 μg/ml). ESBLs and antibiotic susceptibility were also tested in selected transconjugants, and the presence of bla genes was determined using PCR as described above. The resistance plasmids carried by transconjugants were typed by using PCR-based replicon typing [34].

Epidemiological typing
All available isolates were referred for PFGE and analyzed according to some existing criteria [35]. XbaI-digested chromosomal DNA of isolates was characterized by PFGE using the CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA, USA).
A Salmonella serotype Braenderup H9812 (ATCC BAA-664) was chosen as the molecular weight marker. Electrophoresis was run at 6.0 V/cm for 18.5 h with an angle of 120° at 14°C.

Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.    Figure 1 Dendrogram of XbaI pulsed-field gel electrophoresis profiles of E.coli O157:H7 isolates