Prevalence and characteristics of extended spectrum β-lactamaseproducing Escherichia coli from bovine mastitis cases in China

Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050,P.R.China

1. lntroduction

Bovine mastitis is one of the most prevalent and costly diseases of dairy industry worldwide. Mastitis can be caused by 137 different microorganisms, butEscherichiacoliis one of the primary pathogens causing bovine mastitis(Kempfet al. 2016; Yanget al. 2016). Antibiotic therapy is the mainstay of treatment for this disease. However, the results of this therapy have been disappointing in large part due to the resistance to most of the antimicrobial agents,especially the β-lactams and their derivatives (Metzger and Hogan 2013).

Resistance to β-lactams inE.coliis mainly based on β-lactamases-mediated antibiotics hydrolysis. Bacteria harboring extended-spectrum β-lactamase (ESBL)-encoding genes can hydrolyze virtually all penicillins and cephalosporins (Hijaziet al. 2016). ESBL producers are usually multidrug-resistant (MDR) against non-β-lactam antibiotics, including fluoroquinolones, aminoglycosides,tetracyclines, sulfamethoxazole and chloramphenicol, which makes the treatment difficult (Hoet al. 2007). The great majority of ESBLs belong to 3 types, including TEM, CTX-M,and SHV types (Liet al. 2014). The predominant genotype varied across different geographic regions. CTX-M β-lactamases was the most prevalent ESBLs in China,especially CTX-M-14 and CTX-M-15 (Zhao and Hu 2013;Liuet al. 2015; Aliet al. 2016). Recently, lines of studies have reported that the prevalence of ESBL-producingE.coliwas increasing in food producing animals from many parts of the world (Liet al. 2014; Raoet al. 2014; Liuet al.2015; Xuet al. 2015). The increasing ESBL-producingE.coliisolated from animal sources may pose a potential risk for public health because the isolates and resistance genes can be transferred to humans through the food chain(Kilaniet al. 2015).

The aim of this study was to investigate the prevalence and characterization of ESBL-producingE.colifrom bovine mastitis cases in China. To the best of our knowledge, this study reports the largest screening of ESBL-producingE.colicausing bovine mastitis so far in China.

2. Materials and methods

2.1. Sample collection and bacterial isolates

Mastitic milk samples of cows (n=2 897) were collected from 98 commercial dairy herds located in 19 regions of China during August 2013 to April 2017 (Table 1). Mastitis cases were confirmed by the California mastitis test(CMT). Samples were collected aseptically for the further bacteriological assay as previously described (Pitk?l?et al.2004). Isolation and identification ofE.coliwere performed by morphological characterization and biochemical testing(Cressierand Bissonnette 2011).

2.2. ESBL-producing E. coli identification

ESBL-producingE.coliisolates were identified by ChromID ESBL agar (bioMerieux, France) according to the manufacturer’s recommendation. EachE.coliisolate was aerobically incubated on ChromID ESBL agar at 37°C for 18 to 24 h. Bacterial colonies show pink to burgundy color on the agar were confirmed as ESBL-producingE.colistrains.

2.3. DNA amplification and sequencing

Bacterial DNA was extracted by the Bacterial DNA Kit(Omega Bio-Tek, USA) according to the manufacturer’s recommendation. ESBL-encoding genesblaTEM,blaSHVandblaCTX-Mwere detected by PCR with the gene-specific primers as listed in Table 2. All of the PCR products were confirmed by bi-directionally sequencing after purified through a QIAquick PCR Purification Kit (Qiagen, Hilden,Germany). The DNA sequence obtained was compared with those in GenBank using the BLAST Program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.4. Antimicrobial susceptibility testing

ESBL-producingE.coliisolates were subjected to susceptibility testing against 17 antimicrobial agents by disc diffusion method according to the recommendations of CLSI (2015). The antibiotics (Oxoid, United Kingdom)tested were: ampicillin (10 μg), amoxicillin-clavulanic acid(20 μg/10 μg), cefotaxime (30 μg), cefuroxime (30 μg),ceftazidime (30 μg), cefaclor (30 μg), cefpodoxime (10 μg),aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg),streptomycin (10 μg), gentamicin (10 μg), tetracycline(30 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg),trimethoprim-sulfamethoxazol (1.25 μg/23.75 μg) and chloramphenicol (30 μg).E.coliATCC 25922 was used as a quality control strain. Isolates resistant to three or more antimicrobial categories were classified as multidrugresistant (MDR) (Magiorakoset al. 2012).

3. Results

3.1. Bacterial isolates

In this study, a total of 318E.coliisolates (10.98%) were recovered from 2 897 mastitic milk samples from 19 regions in China. Out of these isolates, 73 isolates (22.96%) were confirmed as ESBL-positive based on ChromID ESBL agar in 8 regions. Gansu Province showed the highest prevalence of ESBL producers (27.85%), followed by Shaanxi Province (27.27%), and Ningxia Hui Autonomous Region (26.87%) (Table 1).

3.2. Characterization of ESBL-producing E. coli

According to the PCR and sequencing results (Fig. 1 and Appendix A), 97.26% ESBL-producingE.coliisolates(71/73) wereblaCTX-Mpositive, includingblaCTX-M-15(65.75%,48/73),blaCTX-M-14(10.96%, 8/73),blaCTX-M-55(9.59%, 7/73),blaCTX-M-64(5.48%, 4/73),blaCTX-M-65(4.11%, 3/73), andblaCTX-M-3(1.37%, 1/73). Fifty-two isolates (71.23%) were positive forblaTEM-1. Furthermore, theblaTEM-1andblaCTX-Mwere observed together in 68.49%E.coliisolates (50/73).GeneblaSHVwas not detected in any of the isolates.

3.3. Antimicrobial susceptibility

The β-lactam susceptibility profiles and the associated antimicrobial resistance of the ESBL-producing isolates were summarized in Table 3 and Appendix A. Overall, 72ESBL-producingE.coliisolates (98.63%) were found to be MDR. All isolates were resistant to ampicillin. The majority of isolates were resistant to cefotaxime (98.63%), cefaclor(98.63%), cefpodoxime (97.26%), cefuroxime (95.89%),amoxicillin-clavulanic (79.45), aztreonam (63.01%) and ceftazidime (52.05%). None of the tested isolates was resistant to carbapenems. Notably, 35 isolates (47.95%)were resistant to all tested penicillins, cephalosporins and monobactams. Besides the β-lactam resistance, the isolates were also tested for resistance to other antimicrobial categories. Resistance was most frequently observed against tetracycline (94.52%), followed by streptomycin(86.30%), nalidixic acid (83.56%), chloramphenicol(79.45%), trimethoprim-sulfamethoxazole (76.71%),gentamicin (75.34%), and ciprofloxacin (67.12%).

Table 1 Prevalence of extended spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from 19 regions (cities,provinces, and autonomous regions) in China

4. Discussion

The prevalence of ESBL-producingE.coliamong clinical isolates has increased dramatically in the past several years (Chandramohan and Revell 2012). In this study, 318E.coliisolates were collected from bovine mastitis cases in China during August 2013 to April 2017, the detection rate of ESBL producer was 22.96%, which was similar to previously study of bovine mastitisE.coliin China (Aliet al.2016), but much higher than the data from other countries(Geseret al. 2012; Dahmenet al. 2013; Ohnishiet al. 2013;Freitaget al. 2016). Interestingly, the majority of ESBL producers were distributed in underdeveloped regions in northern China. In these regions, antibiotics abuse may be unavoidable under the situation of high mastitis incidences due to poorly planned dairy infrastructures, sub-optimal housing hygiene and lacking knowledge and skills among the producers (Kivariaet al. 2007). Besides, the use of extended-spectrum cephalosporins in animals should be the primary driving force for the spread of ESBLs (Kilaniet al. 2015). Therefore, we should pay great attention to the problem and try to make appropriate prevention and treatment strategies.

Recently, studies have reported that CTX-M-type ESBLs replaced TEM- and SHV-type ESBLs in Asia, Europe,and Canada as the most frequent ESBL type amongEnterobacteriaceae(Chandramohan and Revell 2012). Our results also confirmed that theblaCTX-Mwas the predominant ESBL gene, followed byblaTEM. This is consistent with other reports from China and other countries that revealed CTX-M was the most prevalent ESBL inE.colifrom bovine mastitis (Freitaget al. 2016; Pehlivanogluet al. 2016).Besides, our results are in accord with previous studies and confirm that CTX-M-15 is the most common CTX-M subtype among bovine mastitisE.coliin China (Yuet al. 2015; Aliet al. 2016). The same situation is also observed in Japan(Ohnishiet al. 2013) and Korea (Tarket al. 2017). These results illustrate the large spread of CTX-M-15-producingE.colifrom bovine mastitis in the east Asia countries.Moreover, the CTX-M-64 and CTX-M-65 producers also detected in our study, which had never been reported to date amongE.coliisolates from bovine mastitis.

Although antibiotics like imipenem, meropenem,ciprofloxacin, and chloramphenicol are banned to use in food animals, some farmers still use these antibiotics to treat or prevent infection, especially to promote animalgrowth in practice. It is necessary to test their antimicrobial susceptibility which can provide scientific data for the government and guide the veterinarian to select the most appropriate antimicrobial agent (Hilaryet al. 2016).Nowadays, resistance to β-lactam among Gram-negative bacteria from food producing animals is increasing at an alarming rate (Geseret al. 2012). The susceptibility test data in our study showed that 98.63% of ESBL-producingE.coliisolates were MDR. ESBL producers which were resistant to most β-lactams were high frequently resistant to the non-β-lactam antibiotics such as aminoglycosides and tetracycline. This is consistent with other reports from China that most ESBLs producers from food animals were MDR(Liet al. 2014; Yuet al. 2015; Aliet al. 2016). MDR is on the rise and pose a serious threat to society because theseisolates might enter the food chain. Therefore, we should avoid antibiotics abuse and misuse in clinical treatment,reducing opportunities for emergence of ESBLs.

Table 2 Primers used in this study

Fig. 1 Distribution of extended-spectrum β-lactamases(ESBLs)-encoding genes and CTX-M subtypes from ESBL-producing Escherichia coli isolates (73) from bovine mastitis.

Table 3 Resistance patterns of extended spectrum β-lactamase (ESBL)-producing Escherichia coli isolates (73) from bovine mastitis

5. Conclusion

This study revealed high prevalence and multidrug-resistant rate of ESBL-producingE.coliisolates from bovine mastitis cases in China. CTX-M type β-lactamases were the predominant ESBLs produced byE.coli, particularly CTX-M-15. To the best of our knowledge, this is the first report of CTX-M-64 and CTX-M-65 ESBLs inE.colicausing bovine mastitis.

Acknowledgements

This work was funded by the National Key R&D Program of China (2017YFD0502200) and the Central Public-Interest Scientific Institution Basal Research Fund, China(1610322017013).

Appendixassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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