Occurrence of multidrug-resistant and ESBL-producing atypical enteropathogenic Escherichia coli in China

Background Atypical enteropathogenic Escherichia coli (aEPEC) is regarded as a globally emerging enteropathogen. aEPECs exhibit various level of resistance to a range of antibiotics, which is increasing alarmingly. The present study investigated the antimicrobial resistance of aEPEC isolates recovered from diarrheal patients, healthy carriers, animals, and raw meats. Results Among 267 aEPEC isolates, 146 (54.7%) were resistant to tetracycline, followed by ampicillin (49.4%), streptomycin (46.1%), and piperacillin (41.2%). Multidrug resistance (MDR) was detected in 128 (47.9%) isolates, and 40 MDR isolates were resistant to ≥ 10 antimicrobial agents. A total of 47 (17.6%) aEPEC isolates were identified as extended-spectrum β-lactamase (ESBL)-producers. The blaCTX-M-14 and blaCTX-M-15 genes were predominant among ESBL-producing isolates. Conclusions This investigation depicted the occurrence of multidrug-resistant and ESBL-producing aEPEC isolates in China. The results suggested that it is necessary to continuously monitor the emergence and spread of MDR aEPEC. Electronic supplementary material The online version of this article (10.1186/s13099-018-0234-0) contains supplementary material, which is available to authorized users.


Background
Escherichia coli remains one of the most common etiological agents of diarrheal illness among children under 5 years old in developing countries [1,2]. Six major diarrheagenic E. coli are well-characterized: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) [3]. EPEC are the primary cause of summer diarrhea in infants in developing countries [4]. It was estimated that about 79,000 deaths every year are linked with EPEC, which was the first to be identified and is the most prevalent pathotype of diarrheagenic E. coli [5].
EPEC isolates carry the locus of enterocyte effacement (LEE) island, which can induce the hallmark histopathology on the surfaces of intestinal epithelial cells, known as the attaching and effacing (A/E) lesion. A/E results in electrolyte disruption and eventual diarrhea [3,6,7]. Some EPEC isolates possess the adherence factor (EAF) plasmid, which carries the bundle-forming pilus genes, the plasmid-encoded regulator genes, and other virulence-related factors [3]. Depending on the presence or absence of the EAF plasmid, EPEC strains are divided into two subgroups: typical EPEC (tEPEC) and atypical EPEC (aEPEC) [8]. In developing countries, tEPEC was considered to be the main cause of infantile diarrhea for decades [6]. However, further studies have shown an apparent increase in the involvement of aEPEC strains in endemic childhood diarrhea and outbreaks in adults in recent years [9][10][11][12][13][14]. Thus, aEPEC strains have been regarded as emerging enteropathogens and have caused a number of infections [15][16][17]. Humans and animals, including food-production animals and pet animals, can be reservoirs of aEPEC, while the major reservoirs of tEPEC are humans [6].
Multidrug resistance (MDR), which was designated as resistance to one agent in three or more antibiotic classes [18], has been increasing alarmingly in E. coli (http://www.ecdc.europa.eu/en/healthtopics/antimicro-bial_resistance/database/Pages/map_reports.aspx) [19]. The establishment of MDR is mediated by many diverse and interactive mechanisms, e.g., drug efflux, enzymatic inactivation, and target protection [20]. The determinants responsible for MDR are widely distributed among E. coli isolates, irrespective of their resources [20]. Production of extended-spectrum β-lactamase (ESBL) is one of the main mechanisms conferring the spread of MDR [21], because most ESBL-producing isolates show extensive resistance to other antimicrobial agents [22]. The genes encoding ESBLs are usually located on plasmids and different types of ESBLs have been identified globally [23]. According to their amino acid sequences, ESBLs are classified into several types, such as TEM, SHV, CTX, OXA, PER, and GES [24]. Currently, the most frequently detected genetic type of ESBL is CTX-M [25]. There are five major sublineages of CTX-M: 1, 2, 8, 9, and 25 [26].
The spread of antibiotic resistance among pathogens has become an emerging public health concern [21]. In China, aEPEC appeared to be one of the most common pathogens associated with infectious diarrhea [27]. However, there are few data available regarding the resistance of aEPEC. The present study aimed to determine the overall antimicrobial resistance profiles, the current prevalence of MDR, the ESBL genotype distribution, and the determinants of resistance in aEPEC isolates recovered from diarrheal patients, healthy carriers, animals, and raw meat in China. The results will fill in large knowledge gaps concerning this pathogen in China, and provide further information and guidance for the application antimicrobials in farm animals and in clinical treatment.

Isolation and identification of aEPEC isolates
Samples from different sources (diarrheal patients, healthy carriers, animals, and raw meat) were collected during 2006-2015 in ten geographical regions (Henan, Shanxi, Heilongjiang, Beijing, Qinghai, Guangdong, Sichuan, Shanghai, Guizhou, and Anhui) of China. Fecal samples of diarrheal patients were collected when patients were admitted to sentinel hospitals; stools from healthy carriers were sampled during routine physical examinations; while stool samples of animals and raw meat samples were collected during routine surveys.
The samples were processed as previously described [28]. In brief, the overnight enrichment culture of each sample was centrifuged and the cells were lysed in lysis buffer (10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 1 mM EDTA [pH 9.0], 1% Triton X-100). The released DNA was then examined for eae gene by polymerase chain reaction (PCR) assays. The enrichment culture with eae + were streaked on CHROMagar ™ ECC plate (CHROMagar Co., Paris, France) and incubated at 37 °C for 18-24 h. Ten E. coli-like colonies from each culture were selected to detect the presence of the eae gene. The eae + colonies were then subcultured on Luria-Bertani (LB) (Oxoid, Basingstoke, UK) plates, incubated for another 18-24 h, and subjected to PCR assays for the eae, stx 1 , stx 2 , and bfpA genes. Isolates that were eae positive, but bfpA and stx 1 /stx 2 negative, were defined as aEPEC [6].
A total of 267 aEPEC isolates were identified and included in this study (Additional file 1). Among them, 151, 32, and 51 isolates were recovered from the stools of diarrheal patients, healthy carriers, and animals (cattle, pig, chicken, bird, pika, and marmot), respectively. The remaining 33 strains were isolated from raw meat (beef, pork, mutton, and chicken meat).

Screening and confirmation of ESBL producing isolates
ESBL production was screened phenotypically using cefotaxime (30 μg). The presumptive isolates were confirmed by combination disk tests with cefotaxime and ceftazidime (30 μg), with and without clavulanic acid (10 μg), as described by the CLSI guidelines [29]. A ≥ 5 mm increase in the zone diameter for cefotaxime or ceftazidime in combination with clavulanic acid versus the zone diameter of the corresponding antimicrobial agent alone defined an ESBL producer [29]. Klebsiella pneumoniae ATCC ® 700603 was used as a positive control.

Identification of β-lactamase genes
DNA templates were prepared by crude extraction, as previously described [30]. All isolates were screened for the presence of the bla CTX-M [26], bla TEM , and bla SHV [31] gene using PCR. Four sets of group-specific primers were further used to identify five subgroups (bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-8/25/26 , and bla CTX-M-9 ) of bla CTX-M [26]. The PCR products were resolved on a 1% agarose gel and then subjected to sequencing using an ABI 3730 Automated DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were compared against the sequences in GenBank (https://blast.ncbi. nlm.nih.gov/Blast.cgi).

Whole genome sequencing and identification of antimicrobial resistance genes
Based on their serotypes, pulse-field gel electrophoresis (PFGE) patterns and multi-locus sequence typing (MLST), 96 isolates (69 from diarrheal patients, 16 from healthy carriers, and 11 from raw meat) were selected from among the 267 aEPEC strains for whole genome sequencing. Bacterial genomic DNA was extracted using a Wizard ® Genomic DNA Purification Kit (Promega Co., Madison, WI, USA) according to the manufacturer's instructions. Genomic DNA was sequenced using an Illumina HiSeq 2500 PE125 instrument (Illumina, Santiago, CA, USA) with 500-bp libraries at the Beijing Novogene Bioinformatics Technology Co., Ltd. Coverage greater than 100× was obtained. The sequence read data was filtered by quality control using the Illumina data pipeline. High-quality filtered reads were assembled into contigs and scaffolds using SOAP de novo (http://soap.genomics.org.cn/soapdenovo.html). Based on the N90, N50, minimum contig size, maximum contig size, and number of contigs, the optimum genome assembly was chosen. Contigs with length > 500 bp were used for further analysis. Assembled draft genomes of all 96 isolates were then used to predict coding genes using the GeneMarkS program [32]. tRNAs and rRNAs were identified using tRNAscan-SE [33] and the rRNAmmer [34], respectively. Seven databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups, Non-Redundant Protein Database, Transporter Classification Database, Swiss-Prot, and TrEMBL) were used to predict gene functions. The Antibiotic Resistance Genes Database (http://ardb.cbcb.umd.edu/) was used to search for antimicrobial resistance genes [35]. The raw data of these genomes have been submitted to the GenBank under accession numbers listed in Additional file 2.

Statistical analysis
Differences in the antimicrobial resistance patterns among aEPEC origins were assessed by a two-tailed Chi square test or Fisher's exact test, with a level of significance of P < 0.05. All statistical analyses were performed using Epi Info software, version 3.5.3 [36].

Molecular characteristics of ESBL genes
The presence of bla CTX-M , bla TEM , and bla SHV genes in 47 ESBL-producing isolates was screened using PCR. The bla CTX-M-1 subgroup was identified in 20 (42.6%) ESBLproducing isolates, with 17 from diarrheal patients and three from healthy carriers. The bla CTX-M-9 subgroup was found in 30 (63.8%) isolates, with 24 from diarrheal patients, three from raw meat, two from healthy carriers, and one from animals. A total of 26 isolates recovered from diarrheal patients possessed the bla TEM subgroup ( Table 3). None of the 47 isolates examined in this study was positive for the genes belonging to subgroups bla CTX-M-2 , bla CTX-M-8/25/26 , or bla SHV . DNA sequencing showed that bla CTX-M-14 gene was the most prevalent, and was present in 28 (59.6%) ESBLproducing isolates, with 22 from diarrheal patients, three from raw meat, two from healthy carriers, and one from animal. The bla CTX-M-15 gene was identified in 11 (23.4%) isolates, with nine from diarrheal patients and two from healthy carriers. The bla CTX-M-55 and bla CTX-M-3 genes were found in four and five isolates, respectively. The two genes, bla CTX-M-13 and bla CTX-M-65 , belonging to the subgroup bla CTX-M-9 , were only found in two separate diarrheal patient-derived isolates. In addition, all of the 26 bla TEM genes were identified as bla TEM-1 . The coexistence of subgroup bla CTX-M-1 and bla CTX-M-9 genes was identified in three diarrheal patient isolates, including one that harbored bla CTX-M-14 and bla CTX-M-55 , and two that harbored bla CTX-M-14 and bla CTX-M-15 ( Table 3).

Discussion
Globally, EPECs displaying different levels of resistance to a range of antibiotics are increasing alarmingly [37]. The antimicrobial resistance of EPEC has been reported in many countries, including Brazil [38,39], India [40], Iran [41], Ireland [42], the United Kingdom [43], and Singapore [44]. In China, only two studies are available: one characterizing 39 EPEC isolates in ready-to-eat foods [45] and another examining 58 EPEC isolates recovered from pediatric diarrheal patients [46]. These EPEC strains were either restricted to being from foods or were regionally restricted. In the present study, the 267 aEPEC isolates were recovered from different sources (diarrheal patients, healthy carriers, animals and raw meat) from ten provinces/cities of China. This was the first study to reveal the comprehensive antimicrobial resistance of aEPEC in China and to provide further insight into the current situation of this specific diarrheagenic E. coli.
Of the 151 diarrheal patient-derived aEPEC isolates, the highest resistance rate was to tetracycline, followed by ampicillin and streptomycin, which was different from reports in Iran [47], Brazil [39], and India [40]. Physicians in China should pay attention to the antimicrobial resistance of clinical aEPEC isolates, because EPEC is still one of the most common pathogens associated with infectious diarrhea. Domestic animals, such as sheep, cattle, poultry, and pigs, have been considered as the main reservoirs of aEPEC [14]. In Europe, the predominant Table 3 continued antimicrobial agents administered to animals are sulphonamides and/or trimethoprim, tetracyclines and β-lactams [48]. However, there is little antimicrobials consumption data available in this field in China. It was reported that high doses and multiple types of veterinary antimicrobial products were used routinely in livestock husbandry [49]. The agents mentioned above are also included in the antimicrobials that can be used in the treatment and prevention of animal diseases. The high prevalence of antimicrobial-resistant aEPEC in raw meat and animals could be explained by the possible overuse and misuse of tetracyclines, ampicillin, and trimethoprim/sulphonamides in veterinary practice and agriculture. Poor sanitary conditions or practices might also play a role in the spread of resistant aEPEC.

Origin (no. of isolates) Isolates Antimicrobial resistance pattern
The emergence of multidrug resistance, especially among Enterobacteriaceae, i.e., E. coli, has become a critical public concern [18]. In this study, nearly half of the 267 aEPEC strains were multidrug resistant. These MDR strains showed high resistance to tetracycline (92.2%) and ampicillin (89.8%), and 31.3% of that showed resistance to ≥ 10 antimicrobial agents. In addition, in this study, significantly more aEPEC strains from diarrheal patients showed multidrug resistance than did strains from healthy carriers and animals. Thus, diarrheal patients may be the main source of MDR aEPEC strains in China and clinicians should be careful when using antibiotics as therapy for EPEC infections. A recent study showed that wild birds could also act as carriers of MDR EPEC [50]. Consistent with this, we found that 19 (37.3%) aEPEC strains from animals, including birds, pika, and marmot, were MDR. In this sense, MDR aEPEC could emerge in the natural environment and then pose potential risk to public health.
Most multidrug resistances in Enterobacteriaceae are associated with ESBLs [51]. E. coli has become one of main producers of ESBL and has posed a major challenge in the treatment of bacterial infection [19]. A previous study showed that occurrence of ESBL-producing E. coli in patients in China varied from 30.2 to 57.0% [52]. In our study, 47 (17.6%) aEPEC isolates were identified as ESBLproducing strains, with 38 the isolates coming from diarrheal patients. Most ESBL-producing isolates showed co-resistance to other antimicrobial agents, such as aminoglycosides, tetracyclines, and sulfonamides, and even to fluoroquinolones [22]. The present results showed that ESBL-producing aEPEC isolates displayed co-resistance to aminoglycosides, tetracycline, nalidixic acid, trimethoprim-sulfamethoxazole, and nitrofurantoin, but not to fluoroquinolones. It is worth noting that MDR E. coli usually implies significant increase of resistance and pathogenic potential, such as the emergence of ESBLproducing clone ST131 [53] and another clinically relevant ESBL-producing clone ST410 [54]. The multi-locus sequence typing (MLST) analysis in our previous study indicated that these aEPEC isolates showed high clonal diversity, but none of them were identified as ST131 or ST410 [28].
TEM, SHV, and CTX-M are the three main genetic types of ESBLs [19]. Currently, the CTX-M-type ESBLs have dramatically increased and largely outnumber other types of ESBLs [25]. However, there are extensive geographical variations in the distribution of dominant CTX-M types across different countries, such as CTX-M-2 in Japan, CTX-M-1 in Italy, and CTX-M-2 and CTX-M-15 in Brazil. By contrast, CTX-M-15 widespread throughout the world [22,55,56]. In the present study, all 47 ESBL-producing aEPEC isolates possessed CTX-M  [57] and clinical patients in China [52]. CTX-M-55 was observed only in four aEPEC strains from diarrheal patients, although it was demonstrated to be widespread in E. coli isolates from food-producing animals and environmental samples in China [58,59]. These findings suggested that humans might acquire these strains from animals, as well as from the food chain. High levels of resistance to tetracycline, ampicillin, and streptomycin were identified among 96 genome sequenced aEPEC isolates. More than half of the ampicillin resistant strains harbored the bla TEM-1 gene in this study. It has been reported that bla TEM was the most frequent β-lactamase gene involved in ampicillin resistance in E. coli [60]. Of the known tetracycline resistance genes, only tetA, tetB, and tetC (alone or in combination) were detected, indicating that the major mechanism involved in tetracycline resistance in aEPEC isolates is active efflux. This is consistent with the investigation of EPEC from diarrheic rabbits in Portugal [60]. Among the aEPEC resistant to aminoglycosides, 69.7% of the isolates resistant to gentamicin carried aac3iia; 54.5% isolates resistant to streptomycin possessed genes aph33ib and aph6id; and most isolates resistant to kanamycin harbored aph3ia. These results suggested that aminoglycoside acetyltransferases are the main mechanism of resistance to gentamicin, while aminoglycoside phosphotransferases are the predominant mechanism mediating streptomycin and kanamycin resistance. With respect to determinants responsible for resistance to trimethoprim-sulfamethoxazole, our results demonstrated that sul1, sul2, dfra12, and/or dfra17 were the predominant genes, as revealed by a previous study [60].
Some limitations exist in this study. Compared with the number of strains from diarrheal patients, fewer isolates from healthy carriers, animals, and raw meat were included. Further investigations are needed to clarify the association between virulence and antimicrobial resistance.
In conclusion, our investigation revealed the occurrence of multidrug-resistant and ESBL-producing aEPEC isolates in China. These results suggest that it is necessary to continuously monitor the emergence and spread of MDR aEPEC to guide the application of antimicrobials in farm animals and in clinical treatment.