Beta-lactam antimicrobials are first line anti-bacterial infection drugs for humans due to their high potency, broad anti-bacterial spectrum, and minimal side effects. They are widely used in the treatment of various infections, such as those of the lungs, urinary tract, and the bloodstream. However, widespread use of antibiotics has intensified the problem of antibiotic resistance in bacteria (Paterson and Bonomo, 2005; Biedenbach et al., 2014; D'Angelo et al., 2016). The production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of antimicrobial resistance in Enterobacteriaceae, especially Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), and the enzyme can hydrolyze penicillin, cephalosporin, and monocyclic amide antibiotics, but its activity is usually inhibited by beta-lactamase inhibitors, such as sulbactam, clavulanic acid, and tazobactam (Bush et al., 1995; Bradford, 2001).

Currently, over hundreds of ESBLs have been identified; the most prevalent genotypes are bla_TEM, bla_SHV, and bla_CTX−M. Within the past decade, the genotype bla_CTX−M has rapidly increased and is now widely found in clinically isolated E. coli across the world (Paterson and Bonomo, 2005; Livermore et al., 2007). In practice, bla_CTX−M genes have already become the major ESBL genotype in American, European, and Asian countries (Pitout et al., 2005; Livermore et al., 2007; Ben-Ami et al., 2009; Zhang et al., 2014). Emergence of community-associated infections caused by ESBL-producing E. coli has been reported in Europe and the United States (Ben-Ami et al., 2009). Moreover, relevant studies from Oceania, Asia, and South America have also reported that ESBL-positive E. coli are the key pathogens in community-onset infections (Baas and Ahmad, 2001; Bell et al., 2002; Munday et al., 2004; da Silva Dias et al., 2008; Baurin et al., 2009; Rawat et al., 2013).

Numerous studies in China have already demonstrated that ESBL-producing E. coli in tertiary and county hospitals is becoming an epidemic (Xiao et al., 2011, 2012, 2013; Zhang et al., 2014; Liu et al., 2015). Previous studies that monitored infections in tertiary hospitals of China indicated that the prevalence of ESBL-producing E. coli was rapidly on the rise, increasing from an ESBL-positive rate of <20% in 2000 to 72.2% in 2011 (Xiao et al., 2011, 2012, 2013). A similar study that examined infections in county hospitals across China also reported an ESBL-positive rate of up to 46.5% in E. coli (Zhang et al., 2014). However, these studies were focused on city hospitals, and there are very few reports that have examined ESBL-producing E. coli in town hospitals of rural areas in China. Therefore, this study was undertaken to investigate drug-resistance and molecular epidemiology of ESBL-producing E. coli isolated from outpatients in town hospitals of Shandong province, in order to provide comprehensive and reliable epidemiological information for preventing dissemination of resistance genes.

To our best knowledge, this study was the first time to investigate drug resistance and molecular characteristics of ESBL-producing E. coli from outpatients in town hospitals of Shandong province, China. The isolation rate of ESBL-producing E. coli in our study was 62.8%, which was similar with Zhao's (62.5%) and Wang's (67.8%) results conducted in Shanghai, China (Zhao et al., 2015; Wang et al., 2016), but higher than those reported in Argentina (18.1%), Mexico (48.4%), Chile (23.8%), and Brazil (12.8%; Gales et al., 2012). All ESBL-producing E. coli from these three regions carried bla_CTX-M genes, which was composed of 10 genotypes including bla_{CTX-M-1, -3, -14, -15, -24, -27, -55, -65, -79, and-101}. This indicates that ESBL-producing E. coli from Shandong province have diverse CTX-M genotypes, and similar results were also found in the tertiary and county hospitals of China (Zhang et al., 2014; Zhao et al., 2015; Wang et al., 2016).

E. coli isolated in this study were found to be highly resistant to penicillin derivatives, fluoroquinolone, folate pathway inhibitors, and third generation cephalosporins, but were 100% susceptible to imipenem and meropenem. In addition, these isolates displayed low resistance to amikacin, piperacillin/tazobactam, ceftazidime, and cefepime. The antibiotics to which the E. coli was found to be highly resistant are common medications used in Shandong town hospitals, and therefore our findings should caution clinicians for the rational use of antibiotics.

We found that bla_CTX-M-14 was the most prevalent genotype of ESBL-producing E. coli in Shandong town hospitals, followed by bla_CTX-M-55 and bla_CTX-M-15, which is consistent with findings reported in Chinese county hospitals between 2010 and 2011 (Zhang et al., 2014), as well as in 3 Shanghai hospital studies between 2011 and 2013 (Zhao et al., 2015). CTX-M-55 genotype, which only has 1 amino acid site mutation (Ala-77-Val) compared to CTX-M-15 genotype, was first discovered in clinically isolated E. coli and K. pneumoniae from Thailand in 2007 (Kiratisin et al., 2007), and was subsequently detected in Salmonella in China, US, Korea, and Switzerland (Shi et al., 2009; Sjölund-Karlsson et al., 2011). At present, CTX-M-55 genotype is frequently detected in ESBL-producing E. coli that originates from animals (Dinubile et al., 2005; Ma et al., 2012; Zheng et al., 2012; Zurfluh et al., 2013; Li et al., 2016). In China, two nationwide breeding farm studies have shown that CTX-M-55 was, respectively, the second (26.1%, 29/111) and third (18.5%, 10/54) most frequently detected ESBL gene (Li et al., 2010; Zheng et al., 2012). These findings demonstrated that bla_CTX-M-55 gene may have already been passed from animals to humans through the food chain. The subjects of this study were outpatients from rural town hospitals. Since residents from these regions have greater exposure to food animals and breeding farms, compared to those living in the cities, the chance of transmission of drug-resistant bacteria from animals to humans is therefore increased. However, the transmission mechanism of drug-resistant bacteria from animals to humans is currently unclear, and further studies are required to elucidate this process. Additionally, it is an interesting finding that the resistance to cefotaxime and ceftriaxone is not 100% while only CTX-M-type ESBLs were found in this study, which is needed to be further studied.

Although ST131 was the most common ST among the 201 ESBL-producing E. coli strains, it only accounted for 13.4% of the total ST. Similarly, some recent nationwide studies in tertiary and county hospitals have also shown that ST131 was found in 9.6% and 12.7% of ESBL-producing E. coli, respectively, indicating that no predominant ESBL-producing E. coli ST epidemic was found in China (Cao et al., 2011; Zhang et al., 2014). In contrast, the percentage of ST131 ESBL-producing E. coli in many European and American countries is far greater than that in China. For example, a community infection study in U.S showed that 53% of ESBL gene CTX-M-carrying E. coli were ST131 (Pitout et al., 2005). Another community infection study in the U.K also reported that ST131 comprised 64% of the cephalosporin-resistant E. coli (Lau et al., 2008b). Furthermore, a similar community infection study in Belgium between 2006 and 2007 showed that 64% of CTX-M-15-carrying E. coli was also ST131 (Smet et al., 2010). Of note, some sequence types found in this work belong to known international clonal complexes, such as ST131, ST393, and ST405 (Wirth et al., 2006; Hrabák et al., 2009). These international clonal complexes have been described as E. coli clones disseminating on a global scale (Coque et al., 2008; Nicolas-Chanoine et al., 2008; Literacka et al., 2009; Lee et al., 2010).

Taken together, These findings demonstrated the high isolation rate of ESBL-producing E. coli (62.8%) detected in outpatients in town hospitals, China, and the bla_CTX-M gene was most dominant and was composed of a variety of subtypes. More importantly, this study spotlights the necessity to carry out long-term surveillance of ESBL-producing E. coli in hospital environments, especially in underdeveloped areas.

ZM and SL conceived and designed the experiment. LW, YZ, and SL collected these isolates. SL, LW, YZ, and WS performed the experiments. ZM and SL analyzed the data and wrote the paper.

This study was supported by the National Natural Science Foundations of China (81501357) and Science and Technology Development Project of Shandong Province (2014GSF118044).