The production of extended-spectrum β-lactamases (ESBLs) is the main mechanism of resistance to β-lactam antibiotics in E. coli. ESBLs hydrolyse penicillins, cephalosporins, and aztreonam; however, they are inhibited by clavulanic acid, sulbactam, and tazobactam [1],[2]. The enzymes TEM and SHV were previously recognised as the main ESBLs. However, CTX-Ms have recently become more prominent, and are now considered to be the most prevalent β-lactamases found in clinical isolates of E. coli globally [3],[4]. A nationwide survey of the United States during 2009–2010 revealed that 91% of ESBL-producing E. coli strains carried CTX-M-type genes [5]. CTX-M-type ESBLs are also dominant in European and Asian countries [4],[6], and in China they are the most common ESBLs found in Gram-negative bacteria isolated from tertiary hospitals [7]-[9]. ESBL-producing E. coli strains are a major cause of community-associated infections [5],[10], and their spread has been well-documented in Europe and North America [5],[11]-[15]. Strains of ESBL-producing E. coli have been recorded in other regions of the world, including Oceania, Asia, and South America [10],[16]-[18]. Several studies have characterised ESBL-producing Enterobacteriaceae in Chinese tertiary hospitals [7],[19],[20]. A national monitoring program of antimicrobial resistance in E. coli caused bacteriaemia (mostly nosocomial infections) in Chinese tertiary hospitals reported an increase in ESBL-producing E. coli from less than 20% to 72.2% between 2000 and 2011 [21]-[23]. Another survey of Chinese tertiary hospitals reported that the incidence of ESBL-producing E. coli among community-associated intra-abdominal infections increased from 19.1% in 2002–2003 to 61.6% in 2010–2011 [20]. None of these studies focused on bacteria isolated from outpatients in primary and non-central city hospitals. In light of the increasing importance of ESBL-producing E. coli within communities, it is important to understand the pattern of antibiotic resistance and the prevalence of ESBLs in bacterial isolates from primary and non-central city healthcare institutions that provide medical services to patients in China.

Most Chinese people live in rural areas or small to medium-sized cities, and seek their healthcare at county-level hospitals, which play an important role in China. The high global prevalence of ESBL-producing E. coli strains among community-onset infections warrants an urgent need to investigate the characteristics of antimicrobial resistance in China. This study focuses on community-onset infections in patients at selected hospitals in seven regions of China. The purpose was to investigate the antimicrobial resistance, the incidence of ESBLs and their genetic types, and the molecular characteristics of E. coli strains isolated from patients, with the intention of developing strategies to control and prevent antimicrobial resistance.

The findings of this study reveal, for the first time, the high prevalence of ESBL-producing E. coli associated with community-onset infections in county hospitals across China. The results indicate that 46.5% of clinical E. coli isolates carried genes for ESBLs, and that this figure varied significantly across the different regions, ranging 30.2% to 57.0%. In particular, the proportion of isolates that carried ESBL genes was 50% or greater in the North, Northwest, and Southwest regions of China. PCR amplification revealed a high diversity of CTX-M-type ESBLs in China, with 12 subtypes detected. The results showed that CTX-M-14 was the most abundant of those CTX-M enzymes detected, and that it is prevalent across China. This is supported by previous studies that have reported the prevalence of ESBLs in isolates from both community-associated and hospital-acquired infections in Chinese tertiary hospitals, and also in healthy people from large cities [7],[8],[32].

A developing characteristic of ESBL epidemiology in several hospitals is the emergence of CTX-M-55 as a dominant genotype. This is the first study to report that the abundance of clinical isolates in China containing CTX-M-55 have exceeded those with CTX-M-15 as the second most common genotype. In 2009, CTX-M-55 was detected in only two of 49 CTX-M-1 group E. coli strains in Guangzhou Province in South China [33]. Following this report, there were limited investigations into the presence of CTX-M-55 in Chinese clinical isolates. However, Hu et al. recently reported that eight CTX-M-55 strains were detected from 17 CTX-M-1 group E. coli isolates from the faeces of healthy humans in Hangzhou (Zhejiang Province) [32].

CTX-M-15 differs from CTX-M-55 by a single amino acid substitution of Ala-77-Val. CTX-M-55 was first detected in clinical isolates of E. coli and K. pneumonia from Thailand in 2005 [34], and was subsequently found in Salmonella spp. in China, the United States, Korea, and Switzerland [35]-[37]. Although CTX-M-55 is widely distributed in E. coli strains isolated from pets and food-producing animals, its range currently appears restricted to specific geographic regions, mainly in Asia [15],[34],[38]-[41]. Two previous studies of the epidemiology of ESBL-producing E. coli strains in food-producing animals in China reported that CTX-M-55 was the second (26.1%, 29/111) and third (18.5%, 10/54) most common genotype, both following CTX-M-14 [39],[42]. A further study showed that CTX-M-55 was the second most common (25%, 27/108 strains) genotype detected among ESBL-producing E. coli strains isolated from healthy ducks and environmental samples from a duck farm in South China [40]. These findings suggest that CTX-M-55 has already transferred from animals to humans, and spread among both healthy individuals and patients in China, and that this particular β-lactamase may be displacing bla _CTX-M-14 as the most common genotype in some regions.

Drug resistance in E. coli can be transferred horizontally between strains that are common to food-producing animals and humans. E. coli can also transfer resistance genes to bacteria that are part of the commensal intestinal flora, and it may serve as an important reservoir for these transmissible traits [43]-[47]. Winokur et al. reported plasmid transfer of β-lactamase genes between Salmonella and E. coli, and suggested that they may have been transmitted between food-producing animals and humans [44]. Fey et al. reported the acquisition of ceftriaxone-resistant Salmonella enterica serotype typhimurium by a 12-year-old boy from cattle [45]. This finding highlights the potential threat to public health through the transfer of highly resistant bacterial strains from livestock to humans, particularly farmers, ranchers, and animal handlers [45]. Findings from this study, and others, suggest that CTX-M-55 can be transmitted from animals to humans. Most outpatients in county hospitals live in rural areas and are likely to have contact with infected food-producing animals and farm sewage. Further investigations are needed to test this hypothesis and resolve the mechanism by which blaCTX-M-55 transfers to, and exists in, humans.

In this study, 64 different STs (including nine new STs) were detected among 213 ESBL-producing E. coli isolates, and ST131 was the most abundant type (12.7%). These findings are similar to those from a tertiary hospital investigation, which reported that 9.6% of 94 bla _CTX-M-14- and bla _CTX-M-15-containing E. coli strains belonged to ST131 [48]. An investigation of ESBL-producing E. coli isolated from river samples and the faeces of healthy humans found that 14.4% (20/139) of isolates belonged to ST131 [32]. The findings indicate that no particular ST of E. coli is predominant in China, unlike European and North American countries where there is a higher prevalence of ST131 [6]. For example, 53% of ESBL-producing E. coli isolates belonged to ST131 in the United States [5]. A study from Calgary for the period 2000–2007 revealed that E. coli clone ST131, which produces CTX-M-15, was an important cause of community-onset bacteraemia, with its incidence increasing from 6% (1/18) in 2000–2003 to 41% (20/49) in 2004–2007 [49]. In one region of the United Kingdom, 64% of community-acquired cefpodoxime-resistant E. coli infections were caused by ST131 [50]. Similarly, data from Belgium covering the period 2006–2007 shows that 62% of CTX-M-15-carrying E. coli causing community-acquired infections belonged to ST131.[51] In France, 25% of community-onset ESBL-producing E. coli infections belonged to ST131 [13].

No dominant ST type was detected in any region; however, in Northwest China, 19.5% (8/41) of isolates belonged to ST38 (detected in three of four hospitals). These four hospitals are all located in Xinjiang province, and are far away from each other. Results indicated that 164 bla _CTX-M-14-containing strains clustered into 40 different STs, and 47 bla _CTX-M-55-containing strains were scattered between 22 different STs. There was no concentrated distribution of STs in these two, or any other, ESBL-producing genotypes (Figure 2). The results indicate no evolutionary convergent relationships among ESBL-producing E. coli in Chinese hospitals, and suggest that the clonal dissemination of ESBL-producing strains is complicated in China and requires further investigation.

This study provides the first report of an OXA-10-like β-lactamase sequence detected in a member of the Enterobacteriaceae in China, although it was previously reported in Pseudomonas aeruginosa [52]. OXA-10 has also been recorded in multi-drug-resistant clinical isolates of E. coli, K. pneumonia, and Proteus. mirabilis [53]-[55]. OXA-10 is a class D broad-spectrum β-lactamase that hydrolyses oxacillin, methicillin, and cloxacillin, and is inhibited by sodium chloride and clavulanic acid [56]. This β-lactamase was first detected in P. aeruginosa in 1988 [56], and has a similar function to OXA-2 as the parent of OXA-type ESBL, the extended-spectrum variant was continually found in P. aeruginosa during the past two decades, although it has not been detected in Enterobacteriaceae [57],[58]. The detection of OXA-10 in the Enterobacteriaceae reported here, and in other studies, suggests further investigations are needed into the origin of this P. aeruginosa-derived β-lactamase and its transmission.

In summary, this study reports high rates of ESBL-producing isolates of E. coli community-associated infections in county hospitals in China; although the prevalence was lower than in tertiary hospitals. The CTX-M ESBL was the dominant enzyme, and CTX-M-14 was the major molecular type detected in community-onset infections. A diverse range of STs was detected, and no evolutionary convergent relationship among ESBL-producing E. coli was found. We also report the detection of the OXA-10 type of ESBL in clinical isolates of E. coli. These evolutionary characteristics of ESBL epidemiology in clinical strains of E. coli may complicate the future management of antibiotic resistance in China if no effective measures are taken. Further investigations are needed to resolve the mechanisms underlying the increasing incidences of CTX-M-55 and the emergence of OXA-10 in strains of E. coli in county hospitals.