Bovine mastitis, inflammation of the mammary gland, is the most prevalent and economically important disease of dairy animals (Halasa et al., 2007). Mastitis can be caused by a variety of bacterial pathogens, but Escherichia coli is one of the leading causes (Dahmen et al., 2013). Antimicrobial agents are used for therapeutic as well as preventive measures against bacterial infections including bovine mastitis in farm animals. Beta-lactams, such as ampicillin and amoxicillin, remain the first-line treatment in veterinary medicine but an increase in drug-resistance to these antibiotics has been observed. Therefore, extended-spectrum cephalosporins (ESC) such as ceftiofur have been approved in China for the treatment of animal diseases (MAO, 2010). Unfortunately, several recent studies have reported the increasing occurrence of highly resistant extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, mainly E. coli, isolated from food-producing animals from various countries including China (Rao et al., 2014; Xu et al., 2015; Seni et al., 2016).

Bacterial resistance to β-lactams, popular antibiotics due to their proven safety and efficiency, is increasing at an alarming rate. This resistance is mainly achieved through β-lactamases that can hydrolyse most β-lactam antibiotics including the third and fourth generation ESCs and monobactams (Bush and Jacoby, 2010). ESBLs are predominantly produced in gram negative bacteria, particularly in E. coli, and are considered a key mechanism conferring resistance to cephalosporins (Perez et al., 2007). Multi-drug resistance (MDR) has been commonly observed in most ESBL-producers and more alarmingly, co-resistance to other commonly used antibiotics like aminoglycosides, fluoroquinolones, tetracycline has been often reported (Chen et al., 2010; Timofte et al., 2014; Xu et al., 2015). This renders these organisms resistant to a wide range of antibiotics with limited therapeutic options. ESBL encoding genes have been categorized into three main types: bla_CTX-M, bla_SHV, and bla_TEM. The bla_CTX-M has been further categorized into five sub-groups (bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-8, bla_CTX-M-9, bla_CTX-M-25) and more than 150 variants have been documented (http://www.lahey.org/studies). In the past few years, CTX-M, especially CTX-M-15, has emerged as the most dominant type of ESBLs globally (D'Andrea et al., 2013). Recently, CTX-M-15 producing E. coli have been frequently documented from various sources including humans and food producing animals (Timofte et al., 2014; Liu et al., 2015; Xu et al., 2015), showing the broad spectrum of reservoirs carrying and spreading these genes. Food-animals are well established reservoirs of ESBL-producing E. coli, which can be transmitted from animals to humans by various direct and indirect means (Dahmen et al., 2013; Geser et al., 2015). This is also verified by Madec et al. (2012), they reported that the plasmids carrying CTX-M-15 genes in E. coli isolated from cattle were highly similar to those found in ESBL-producing E. coli isolates from human beings.

Integrons are genetic elements that play a vital role in the development and dissemination of MDR in clinical isolates due to their ability to capture, integrate and express gene cassettes (Vinue et al., 2008; Chen et al., 2010). Three main classes of integrons (1–3), carrying the gene cassettes encoding for antimicrobial resistance genes, are generally found to be associated with antibiotic resistance genes in pathogenic E. coli, Class 1 integrons are the most common in clinical E. coli, followed by less frequent class 2 integrons (Vinue et al., 2008; Xu et al., 2015). Class 1 integrons contain a 5′ conserved segment (CS) and 3′CS, followed by a variable region that contains one or more gene cassettes. The 5′CS consists of an integrase gene (intI1), a recombination-site (attI1), and the Pc promoter(s), and the 3′CS includes qacEΔ1 and sul1 genes which encode for quaternary ammonium compound and sulphonamide resistance (Hall and Stokes, 1993). Moreover, insertion sequences like ISCR1 (insertion sequence common region 1) as part of the complex class 1 integrons are found to be associated with ESBL and other resistance encoding genes and are probably involved in their mobilization and transposition. ISCR1 may mobilize the truncated 3′CS and nearby sequences from one integron to the 3′CS of another integron utilizing rolling circle transposition, thus facilitating dissemination of resistance elements (Eckert et al., 2006; Toleman et al., 2006).

Limited studies, particularly from China, have characterized ESBL-producing E. coli isolated from diseased food producing animals, mainly from mastitic cows (Lu et al., 2010; Timofte et al., 2014). Thus, we designed the current study to investigate the prevalence of pathogenic ESBL-producing E. coli and to characterize the ESBL genes and genetic elements which are likely to be responsible for their mobility and dissemination. To the best of our knowledge, this is the first comprehensive study into the molecular characterization of ESBL genes in E. coli isolated from dairy cows in China.

In the past few years, ESBL-producing E. coli have been increasingly isolated from food-producing animals raising global concerns for veterinary and public health (Seiffert et al., 2013). The current study reports on the higher occurrence (23.53%) of ESBL-producing E. coli (n = 36) among 153 E. coli isolates from mastitic cows, as compared to previous reports from China (Yu et al., 2015) and other countries (Dahmen et al., 2013; Geser et al., 2015; Freitag et al., 2016). Interestingly, the majority of ESBL-producing E. coli (23 isolates) were recovered from herds in Inner Mongolia province possibly linked to dense farming (2.37 million dairy cows) with the largest dairy herds in this province (Dou, 2014). Dense farming is significantly correlated with the incidence of bovine mastitis (Ali et al., 2014), and mastitis is the main reason for frequent and prolonged use of antibiotics that exert selective pressure for emergence and dissemination of resistant isolates (Berge et al., 2005). Our results revealed that CTX-M, mainly the CTX-M-15, was the most prevalent genotype, followed by TEM and SHV. These findings agree with other contemporary studies from China and around the world that also reported CTX-M as the dominant ESBL genotype (Locatelli et al., 2009; Dahmen et al., 2013; Geser et al., 2015; Kar et al., 2015). This goes along with the recent detection of CTX-M-15 producing E. coli from cattle and other food-animals in east Asia (Ohnishi et al., 2013; Yu et al., 2015), India (Upadhyay et al., 2015), the United Kingdom (Timofte et al., 2014), Germany (Freitag et al., 2016) and Tanzania (Seni et al., 2016). A national resistance surveillance study in China reported that the prevalence of ESBL-producing E. coli in humans has persisted above 50% since 2000 (Xiao et al., 2011), and recently Liu et al. reported even higher prevalence (68.2%) of ESBLs in clinical E. coli isolates, mainly the bla_CTX-M-15 (Liu et al., 2015). Similarly, in animals the prevalence of ESBL-producing E. coli in China has considerably increased in recent years with CTX-M being the major prevailing gene encoding for ESBLs (Rao et al., 2014). It is known that, generally, ESBL genes are located on plasmids that could spread easily among commensal and pathogenic bacteria in the herd and the environment. Due to limited resources, in the present study, we could not investigate the prevalence of ESBL genes in other bacteria, in other healthy cows, or in cows with subclinical mastitis which is usually 30–40% higher than clinical mastitis (Halasa et al., 2007). We presume that the actual prevalence of ESBL-producers, particularly E. coli, may be much higher than the reported.

In ESBL-positive E. coli, phylogenetic group A represented the most prevalent group followed by virulent extra-intestinal group D; however, this was in contrast to our previous study (Liu et al., 2014), which reported that the pathogenic E. coli associated with mastitis mainly belonged to phylo-group B1 (58.6%) rather than group A (35.7%). Nevertheless, similar phylogenetic distributions were observed in some ESBL-producing E. coli isolated from animals (Abraham et al., 2014; Xu et al., 2015). Our results indicated that all the ESBL-producing E. coli isolates were MDR. The majority of these isolates (54–100%) were found resistant to cephalosporins. In addition, low susceptibility was also observed against the common β-lactam and non-β-lactam antibiotics such as ampicillin, aminoglycosides, tetracycline and fluoroquinolones. Recently, many studies have reported MDR ESBL-producing E. coli isolated from cattle (Timofte et al., 2014), poultry (Kar et al., 2015), pigs (Xu et al., 2015), and humans (Gu et al., 2008). Fluoroquinolones, following ciprofloxacin, are the second important antimicrobial drug in veterinary and human medicine (Coque et al., 2008). Quinolone resistance is traditionally caused by chromosomal mutations in gyrase or topoisomerase encoding genes or efflux pump expression regulating genes (Hopkins et al., 2005); nonetheless, plasmid mediated quinolone resistance is also increasingly reported in ESBL-producing E. coli (Xu et al., 2015).

Integrons play an important role in the emergence of MDR bacteria and in the dissemination of resistance genes. Published reports on the characterization of integrons in ESBLs-positive E. coli from dairy cows are scarce, but previous studies have been conducted in other food-animals, humans and the environment (Gu et al., 2008; Chen et al., 2010; Xu et al., 2015). In accordance to these studies, clinical class 1 integrons were found in the majority of ESBL-positive E. coli (83.33%). The gene cassette arrays of the class 1 integron variable regions contained five different gene combinations that likely impart additional resistance features to our isolates (see Table 3). Six of the intI1 positive amplicons were failed to generate gene cassettes which may be related to the absence of 3′CS in these integrons (Lu et al., 2010). The dfrA17-aadA5 was the predominant gene array that corroborates with the previous studies in China (Gu et al., 2008; Xu et al., 2015). Strikingly, we determined that majority of bla_CTX-M genes were associated with ISCR1 elements, but no ESBL genes were found in the class 1 integron cassettes. It agrees with other published reports that also did not detect ESBL genes in the cassettes (Kiiru et al., 2013; Kar et al., 2015; Xu et al., 2015). Notably, our findings of the most pre-dominant CTX-M type (bla_CTXM-15) and its association with the ISCR1 elements rather than gene cassette arrays indicated that they are more likely mobilized by ISCR1 elements. Conversely, bla_TEM, when found alone or not associated with bla_CTXM-15, was not often found linked to ISCR1, and therefore, was comparatively less prevalent. It has been proposed that antibiotic resistance gene elements are added to the 3′-CS of class 1 integrons by co-mobilization with the nearby ISCR1 from the neighbor integron, implying rolling circle transposition and homologous recombination mechanisms, thus facilitating the formation of complex class 1 integrons (Toleman et al., 2006). Taken together, the current high occurrence of multi-resistant ESBL-producing E. coli carrying clinical class 1 integrons and its association with ISCR1 is worrisome. This may suggest these bacteria are armouring against the antibiotics by devising various tools to render antibiotics useless. Fear exists that this co-existence of ESBL genes along with class 1 integrons as gene cassettes and ISCR1 mobile elements would more robustly disseminate resistance elements within bacterial populations. This calls for an efficient control policy with restriction on the consumption of extended spectrum cephalosporins for long term use.

Here, we report on the high occurrence of ESBL-producing E. coli from bovine mastitis. Genotypic characterization indicated a dominance of bla_CTX-M-15 genes harboring clinical class 1 integrons associated with ISCR1 elements, indicative rapid and wider dissemination potential and posing threats to veterinary and public health. To the best of our knowledge, this is the first comprehensive study to report on the alarming high prevalence of bla_CTX-M-15 and class 1 integron resistance conferring elements in ESBL-producing E. coli from mastitic cows in China.

BH, TA, and SR, conceived and designed the experiment. TA, MS, and SZ, performed the research. JG, GL, and LZ, contributed in reagents/materials/analysis. BH, TA, and SR, wrote the manuscript.

This research was supported by the Chinese Twelfth “Five-year” National Science and Technology Support Project (No. 2012BAD12B03), Ministry of Education in China major project (No. 313054), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) State Education Ministry (No. 20120008110042), and the National Natural Science Foundation of China (No. 3151101034) and (NO. 31572587).