Antimicrobial Resistance and Molecular Epidemiology of ESBL-Producing Escherichia coli Isolated from Outpatients in Town Hospitals of Shandong Province, China

ORIGINAL RESEARCH published: 24 January 2017 doi: 10.3389/fmicb.2017.00063 Antimicrobial Resistance and Molecular Epidemiology of ESBL-Producing Escherichia coli Isolated from Outpatients in Town Hospitals of Shandong Province, China Zengmin Miao 1* † , Song Li 2 † , Lei Wang 3 , Wengang Song 2 and Yufa Zhou 4 1 Department of Life Sciences, Taishan Medical University, Tai’an, China, 2 Department of Basic Medicine, Taishan Medical University, Tai’an, China, 3 Department of Pediatrics, Maternal and Child Health hospital of Laiwu, Laiwu, China, 4 Disease Controlling Center, Veterinary Bureau of Daiyue, Tai’an, China Edited by: Fatah Kashanchi, George Mason University, USA Reviewed by: Ákos Tóth, National Center for Epidemiology, Hungary Jozsef Soki, University of Szeged, Hungary *Correspondence: Zengmin Miao † These authors have contributed equally to this work. Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Received: 21 November 2016 Accepted: 10 January 2017 Published: 24 January 2017 Citation: Miao Z, Li S, Wang L, Song W and Zhou Y (2017) Antimicrobial Resistance and Molecular Epidemiology of ESBL-Producing Escherichia coli Isolated from Outpatients in Town Hospitals of Shandong Province, China. Front. Microbiol. 8:63. doi: 10.3389/fmicb.2017.00063 This study aimed to investigate antimicrobial resistance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) isolated from outpatients in town hospitals of Shandong province, China. Antimicrobial susceptibility of ESBL-producing E. coli was tested using the disk diffusion and resistance genes encoding for β-lactamases (blaTEM , blaCTXM , and blaSHV ) were detected by polymerase chain reaction (PCR). Multilocus sequence typing (ST) of ESBL-producing E. coli was analyzed in this study. Our results showed that of 320 E. coli isolates, 201 carried ESBL genes (201/320, 62.8%), and these isolates all carried blaCTX-M genes, the most common being blaCTX-M-14 (116/201, 57.7%), followed by blaCTX-M-55 (47/201, 23.4%) and blaCTX-M-15 (31/201, 15.4%). ESBL-producing E. coli exhibited highly resistant to penicillin derivatives, fluoroquinolones, folate pathway inhibitors, and third-generation cephalosporins, but no carbapenem-resistant isolates were found in this study. Forty-two STs were found among the 201 ESBL-producing E. coli, and the most common ST was ST131 (27/201, 13.4%), followed by ST405 (19/201, 9.5%) and ST69 (15/201, 7.5%). Taken together, a high isolation rate of ESBL-producing E. coli (62.8%) was found among outpatients in town hospitals. blaCTX-M gene was most dominant and was composed of a variety of subtypes. No dominant ST was detected among ESBL-producing E. coli, indicating that these ESBL-producing E. coli isolates derive from different clones. Keywords: outpatient, antimicrobial resistance, ESBL, ST, town hospital INTRODUCTION 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 Frontiers in Microbiology | www.frontiersin.org 1 January 2017 | Volume 8 | Article 63 ESBL-Producing E. coli within Town Hospitals Miao et al. for E. coli isolation (Figure 1). The outpatients were selected according to the following three conditions: (1) they had not stayed at the hospital within the past 3 months, (2) they had no long-term intubation, and (3) they had not taken antimicrobial medication for over 72 h before treatment. 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 blaTEM , blaSHV , and blaCTX−M . Within the past decade, the genotype blaCTX−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, blaCTX−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 communityassociated 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. Isolation and Identification of E. coli Samples were transported back to the lab on ice within 6–10 h of collection, for E. coli isolation and identification. Samples were inoculated onto MacConkey agar plates using sterile cotton swabs and were incubated overnight at 37◦ C in aerobic conditions. Five single red colonies from each patient sample were selected for further colony purification, and the colonies were subsequently identified using conventional biochemical methods and API20 assays (bioMérieux, Durham, NC, USA). All positively identified E. coli strains (...truncated)