Prevalence and Antibiotic Resistance Characteristics of Extraintestinal Pathogenic Escherichia coli among Healthy Chickens from Farms and Live Poultry Markets in China

Simple Summary Chicken meat has been proved to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC), causing several diseases in humans, and bacteria in healthy chickens can contaminate chicken carcasses at the slaughter; however, reports about the prevalence and molecular characteristics of ExPEC in healthy chickens are still rare. In this study, among 926 E. coli isolates from healthy chickens in China, 22 (2.4%) were qualified as ExPEC and these ExPEC isolates were clonally unrelated. A total of six serogroups were identified in this study, with O78 being the most predominant type, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant and most isolates carried both blaCTX-M and fosA3 resistance genes. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates, among which two carried additional carbapenemase gene blaNDM, compromising both the efficacies of the two critically important drugs for humans, carbapenems and colistin. These results highlight that healthy chickens can serve as a potential reservoir for multidrug resistant ExPEC isolates, including mcr-1-containing ExPEC. Abstract Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). blaCTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and blaNDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or blaNDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring blaNDM from healthy chickens.


Introduction
Escherichia coli is a commensal member of the intestinal tract of warm-blooded animals and most E. coli strains are harmless; however, a subgroup has possessed the ability to cause diseases, especially extraintestinal infections caused by the extraintestinal pathogenic Escherichia coli (ExPEC) [1]. ExPEC strains could colonize the human gastrointestinal tract, not causing disease; however, diverse infections occur when they enter a normally sterile body site [2]. For example, ExPEC strains have been the leading cause of urinary tract infections primarily affecting women [3] and been also the most common cause of bloodstream infections in humans [4]. Importantly, ExPEC infections would impose a large economic burden due to both medical costs and lost productivity, besides their association with morbidity and mortality [5].
A molecular definition of ExPEC is E. coli isolate harboring at least two of five virulence markers: papA and/or papC, sfa/foc, afa/dra, kpsM II and iutA [6], and this molecular criteria has been widely applied in epidemiological studies. ExPEC strains have been found in various water sources, including environmental water [7], wastewater [8] and drinking water [9]. Retail meats proved to be classic vehicles for several foodborne pathogens, are also commonly contaminated with ExPEC strains [10,11], posing a potential risk to consumers. In recent years, researchers found that human and animal-source ExPEC shared highly similar virulence genes and clonal backgrounds [12,13] and animal-source ExPEC were capable to adhere or invade human intestinal epithelial [14], suggesting that foodproducing animals have been a potential source of human ExPEC. Investigations of the ExPEC within poultry were mainly focused on the avian pathogenic Escherichia coli (APEC), a subset of ExPEC, from poultry with colibacillosis [15,16] and APEC mainly caused respiratory and systemic disease in poultry; however, the molecular definition criteria of APEC in those studies was different from that of ExPEC [6,17]. Recently, ExPEC isolates within diseased chickens were also reported [18,19]. Notably, the feces of healthy chickens also carried ExPEC isolates [14,20], and the fecal ExPEC isolates could contaminate chicken carcasses at slaughter, including from rupture of the digestive system during processing, and then transmitted to humans by the food chain or direct human-animal contact [21]. However, studies focusing on ExPEC isolates in healthy chickens are still rare, especially in China, which has huge chicken production.
The presence of antibiotic resistance, one of the ten threats to global health for 2019 as determined by the World Health Organization, among ExPEC isolates has been another big concern. Antibiotic resistance genes, such as extended-spectrum β-lactamases (ESBLs)encoding genes have been reported in ExPEC isolates [22] and ExPEC including those from poultry can also acquire different resistance genes [18], which would inevitably reduce the therapeutic options, increase morbidity and mortality of ExPEC infections, and eventually bring an increased risk to public health [23,24]. Although antibiotics do not select virulent strains such as ExPEC intrinsically [25], the heavy use of antibiotics in foodproducing animals could facilitate the dissemination of ExPEC because such pathogens from environment and diseased animals have been often reported to be multidrug resistant (MDR) [7,14]. However, reports focusing on the antibiotic resistance among ExPEC isolates in healthy chickens remain rare, especially in China which has the largest consumption of antibiotics in the world [26].
Therefore, intense research efforts are warranted to fully understand the characteristics of ExPEC isolates from healthy animals to devise new strategies to prevent their dissemination. In this study, we investigated the prevalence of ExPEC isolates among healthy chickens from farms and live poultry markets in 10 cities in China, and the phenotypic and genotypic characteristics of antimicrobial resistance in these ExPEC isolates were also analyzed.

Sampling and Bacterial Isolation
From May 2015 to February 2017, a total of 926 fecal samples were collected from healthy chickens of seven layer farms (371 samples), one white-feather broiler farm (78 samples) and 17 live poultry markets (477 samples) in 10 cities of three provinces (Shandong, Anhui and Shanxi) in China, and 813 samples used in this study were from Shandong province because Shandong has the first largest broiler and layer production in China (Table S1). The white-feather broilers were five-weeks old when the 78 fecal samples were collected, and all the 371 layer fecal samples were from 70-weeks old brown-egg layers before being rejected. All the chickens in the eight farms would be slaughtered within one week after sampling. The 477 fecal samples collected from 17 live poultry markets were from yellow-feather broilers about 12 weeks old sold for consumption and all the yellow-feather broilers from each market were sampled. The chickens in these farms and live poultry markets had been fed with non-medicated feed for at least two weeks before we collected these samples.
A total of 2 g of fecal sample was suspended in 18 ml of trypticase soy broth (Bec-tonDickinson Co., Cockeysville, MD, USA) and incubated aerobically overnight at 37 • C. The broth was then diluted in series of 1:10 and streaked onto MacConkey agar (Qingdao Haibo Microorganism Reagent Co., Ltd., Qingdao, China) followed by incubation for 18 h at 37 • C. Suspected colonies were streaked onto eosin methylene blue (EMB) agar (Qingdao Haibo Microorganism Reagent Co., Ltd., Qingdao, China), and one colony with typical E. coli morphology was selected from each sample. The E. coli isolates were identified by classical biochemical methods as previously described [27] and confirmed by API 20E system (bioMérieux, Marcy l'Étoile, France). Each farm or live poultry market was sampled only one time when we surveyed the prevalence of antibiotic resistance of E. coli and the sampling period covered the four seasons.

Antimicrobial Susceptibility Tests
The minimal inhibitory concentrations (MICs) of 17 antimicrobials-namely, meropenem, cefotaxime, ceftiofur, ampicillin, ciprofloxacin, enrofloxacin, levofloxacin, nalidixic acid, amikacin, gentamicin, kanamycin, streptomycin, tigecycline, doxycycline, tetracycline, florfenicol and fosfomycin-for these isolates were determined by the agar dilution method following the guidelines of the Clinical and Laboratory Standards Institute [28]. The MIC of colistin was determined according to the method of 2017 EUCAST (available at http://www.eucast.org/clinical_breakpoints/ (accessed on 29 June 2018)). The resistant breakpoints for colistin and tigecycline were recommended by the 2017 EU-CAST (http://www.eucast.org/clinical_breakpoints/ (accessed on 28 July 2018)), while the breakpoints for the remaining antimicrobials were recommended by the CLSI [28,29]. E. coli ATCC 25,922 was used as the control strain. Ceftiofur, florfenicol and tigecycline were from Solarbio Life Sciences Co. (Beijing, China) and the remain antimicrobials used in this study were purchased from China National Institutes for Drug Control (Beijing, China).

Detection and Serotyping of ExPEC Isolates
All isolates were investigated for the following five key virulence markers: papA and/or papC (P fimbriae; counted as 1), sfa and/or foc (S and F1C fimbriae, respectively), afa and/or dra (Afimbrial and Dr-binding adhesion, respectively), kpsM II (group 2 capsule), and iutA (aerobactin system). The isolates carrying ≥ 2 of the above 5 ExPEC-defining markers were classified as ExPEC [6]. All PCR amplicons were sequenced to confirm these virulence genes.

Pulse-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST)
To determine the genetic relationship between ExPEC isolates, PFGE was carried out as previously described [46]. Briefly, the ExPEC isolates were grown on Luria-Bertani agar (Qingdao Haibo Microorganism Reagent Co., Ltd., Qingdao, China) overnight at 37 • C and diluted to an optical density of 0.5. Subsequently, the bacteria dilutions were embedded in SeaKem Gold agarose (Lonza, Rockland, ME, United States) and culture plugs were lysed with 100 µg mL −1 protease K (Solarbio, Beijing, China) by incubation in a shaking water bath at 55 • C for 2 h. Then, the lysed plugs were washed using sterilized water and Tris-EDTA buffer, respectively. The plugs were then digested with XbaI (TaKaRa, Dalian, China) and subjected to PFGE analysis using Chef Mapper electrophoresis system (Bio-Rad Laboratories). The gels were run at 6.0 V cm −1 with an initial/final switch time of 2.16 s/54.17 s for 19 h. PFGE patterns were analyzed with BioNumerics software version 7.0 (Applied Maths, Kortrijk, Belgium) by using Dice coefficients and the unweightedpair group method to achieve dendrograms with a 1.5% band position tolerance. Salmonella enterica serotype Braenderup H9812 standards served as size markers.

Statistical Analysis
Differences in proportions were compared using the χ 2 test implemented in SPSS software (Version 17.0; SPSS Inc., Chicago, IL, USA). All tests of significance were two-tailed, and a value of p ≤ 0.05 was considered statistically significant.

Antimicrobial Susceptibilities
A total of 926 E. coli isolates were obtained including 78 isolates from white-feather broilers, 371 isolates from brown-egg layers and 477 isolates from yellow-feather broilers in 17 live poultry markets ( Table 1). As shown in Table 1, resistances to tetracyclines were observed most often among the total 926 E. coli isolates in this study, and 89.3% and 83.8% of the isolates were resistant to tetracycline and doxycycline, respectively, although none of the isolates were resistant to the newly tetracyclines drug, tigecycline, a last-resort treatment for infections caused by MDR Gram-negative bacteria in humans. For the β-lactam drugs, the rate of resistance to ampicillin was the highest (87.1%), followed by ceftiofur (44.7%), cefotaxime (41.8%), and meropenem (4.9%). Among aminoglycosides, resistance to streptomycin was the greatest (60.7%), followed by kanamycin (50.5%), gentamicin (31.9%), and amikacin (8.9%). For quinolones, the old quinolone drug nalidixic acid possessed the highest resistance rate (77.1%), and the rates of resistance to the three fluoroquinolones (enrofloxacin, ciprofloxacin, and levofloxacin) varied from 36.3% to 58.4%. Moreover, 20.6% and 69.1% of these isolates were resistant to fosfomycin and florfenicol, respectively. Worryingly, 17.0% of the isolates were resistant to colistin, a critically important antimicrobial for humans (Table 1). For isolates from white-feather broilers, brown-egg layers and yellow-feather broilers (live poultry markets), respectively, the rates of resistance to ampicillin, tetracycline and doxycycline were all above 80.0% (Table 1). Notably, except levofloxacin and tigecycline, the resistance rate for each of the remaining 15 antimicrobials tested among E. coli from whitefeather broilers, was significantly higher than that from brown-egg layers and that from yellow-feather broilers (p < 0.05) ( Table 1). The rate of resistance to ampicillin, cefotaxime, colistin, and fosfomycin among E. coli from yellow-feather broilers was significantly higher than that from brown-egg layers, respectively (p < 0.05). For meropenem, levofloxacin and streptomycin, respectively; however, the E. coli isolates from brown-egg layers possessed significantly higher resistance rate than that from yellow-feather broilers (p < 0.05) ( Table 1).

Prevalence of ExPEC Isolates and Their Serogroups
In the present study, 22 (2.4%) of the 926 chicken isolates were qualified as ExPEC. As shown in Table 3, six ExPEC isolates were found in the white-feather broiler farm, and a total of six ExPEC isolates were also detected in four of the seven layer farms. The detection rates of ExPEC ranged from 0.7% to 6.3% among the four layer farms. Chickens harboring ExPEC in live poultry markets were found in three of the six cities we collected samples from and the detection rates of ExPEC among the yellow-feather broilers from poultry markets were 4.4% (3/68) in city Linyi, 3.0% (6/199) in city Qingdao and 1.8% (1/55) in city Yantai ( Table 3). The six ExPEC isolates in Qingdao were from three live poultry markets (Table 3 and Figure 1). Notably, the detection rate (7.7%, 6/78) of ExPEC among whitefeather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). There was no significant difference between the detection rate (1.6%, 6/371) of ExPEC among brown-egg layers and that among yellow-feather broilers (2.1%, 10/477) (p = 0.611) ( Table 3).   Among the 22 ExPEC isolates, iutA was the most prevalent ExPEC-defining marker, followed by kpsM II (18 isolates) and papA (4 isolates) ( Figure 1). All ExPEC isolates carried two of the five ExPEC-defining markers, and no other markers were found in our study. After the PCR-based serotyping method was applied to the 22 ExPEC isolates, the serogroups of 16 isolates were successfully identified and they belonged to six serogroups (O78, O26, O86, O18, O45 and O83). O78 detected in nine of the 22 ExPEC isolates (40.9%) was the most prevalent serogroup, followed by O26 (9.1%, 2/22) and O86 (9.1%, 2/22) (Figure 1).

Antimicrobial Resistance Phenotypes and Genotypes of the ExPEC Isolates
An antimicrobial susceptibility test showed that all 22 ExPEC isolates were resistant to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%), followed by resistance to tetracycline (95.5%), and doxycycline (90.9%) ( Table 4 and Figure 2). All the rates of resistance to florfenicol, streptomycin, kanamycin and nalidixic acid among these isolates were 81.8%. For the third-generation cephalosporins, the rates of resistance to cefotaxime and ceftiofur were both 72.7% (16/22), and the resistance rates to fluoroquinolones ranged from 45.5% to 59.1% (Figure 2). The number of isolates resistant to amikacin and fosfomycin were six (27.3%) and ten (45.5%), respectively. A total of six (27.3%) and two (9.1%) ExPEC isolates were resistant to the two critically important antibiotics colistin and meropenem, respectively. Notably, two ExPEC isolates WF1-5-13 and WF1-5-40 were resistant to both colistin and meropenem (Table 4). Luckily, no isolate was resistant to tigecycline. Detailed results of the antibiotic resistance profiles for the 22 ExPEC isolates were presented in Table 4. Interestingly, the rate of resistance to cefotaxime, ceftiofur, fosfomycin, amikacin, kanamycin and streptomycin in ExPEC isolates was significantly higher than that of non-ExPEC isolates in this study, respectively (p < 0.05) (Figure 2). Worryingly, all the ExPEC isolates in this study were MDR (resistance ≥ 3 three classes of antibiotics) in nature (Table 4).     (Table 4). bla CTX-M-9G and bla CMY-2 -type pAmpC-encoding gene were present in isolate AH234 from layer farm B, accounting for the resistances to the thirdgeneration cephalosporins. fosA3 was found in ten ExPEC isolates resistant to fosfomycin and this gene was only distributed among bla CTX-M -positive isolates. For the six colistin and two meropenem resistant ExPEC isolates, the presence of mcr-1 and bla NDM could account for their corresponding resistance, respectively. Notably, the two ExPEC isolates (WF1-5-13 and WF1-5-40) harboring both mcr-1 and bla NDM , carried additional bla CTX-M , fosA3 and floR genes, and isolate WF1-5-40 also possessed rmtB (Table 4).

Genetic Relationships of the ExPEC Isolates
All 22 ExPEC isolates could be successfully analyzed by PFGE and 20 different PFGE profiles were obtained indicating that clonal dissemination of these ExPEC isolates was unlikely ( Figure 1). As shown in Figure 1, 12 types were identified by the MLST subtyping method along with one new ST (6-6-804-10-9-1-6 in isolate G-1-5) not previously registered in the E. coli MLST database. The most prevalent ST types were ST117 (four) and ST93 (four), followed by ST569 (three), ST1485 (two) and ST2944 (two). Most isolates sharing the same ST types had different PFGE profiles and were from different farms or markets of different cities (Figure 1). For example, the four isolates (AH234, L2-JC-34, WF1-5-10 and LS-A-3) belonging to ST93 were from four chicken markets/farms in four cities and had different PFGE patterns. Notably, the two ExPEC isolates WF1-5-13 and WF1-5-21 from the same chicken farm in city Weifang shared identical PFGE pattern and ST type; however, different resistance genotypes and phenotypes were found in these two isolates ( Figure 1 and Table 4). Different resistance phenotypes were also found in isolates JS-JC-4 and JS-JC-7, which were from the same market and possessed identical PFGE pattern and ST type ( Figure 1 and Table 4).

Discussion
The presence of ExPEC colonizing healthy chickens could be a huge threat to both animal and human health. For China, having the largest consumption of antibiotics in the world, the prevalence and antibiotic resistance of ExPEC among healthy chickens urgently need to be studied. In this study, we investigated the resistance of E. coli isolates from healthy chickens of seven layer farms, one white-feather broiler farm and 17 live poultry markets in China, and the ExPEC among these commensal isolates were also characterized.
Both the rates of resistance to tetracycline and doxycycline among isolates in this study were above 80.0%, consistent with that from chickens in China after 2012 [48]. The high resistance rates to these drugs could be attributed to the heavy usage of tetracyclines in poultry, because oxytetracycline, tetracycline, chlortetracycline and doxycycline have been heavily used for decades in animal production including poultry [49]. Tigecycline, a last-resort treatment for human infections caused by MDR Gram-negative bacteria, has never been used in animal husbandry. Luckily, isolate resistant to tigecycline was not found in our E. coli isolated during 2015-2017; however, the heavy usage of tetracyclines in animals could increase the prevalence of newly mobile tigecycline-resistance gene tet(X4) in E. coli and this should be paid more attention [33]. The resistance rates to meropenem (4.9%) and colistin (17.0%), two critically important antimicrobials in human medicine, among the 926 isolates in our study could be well accounted for the presence of bla NDM (4.9%) and mcr-1 (17.0%), respectively (Tables 1 and 2). qnrS was the most prevalent PMQR gene in this study, differing from that in humans [50] and animals [51], in which the most prevalent PMQR gene was oqxAB. In some countries, qnrB was the most prevalent type [52]. The prevalence of CTX-M-type ESBLs (35.6%) in healthy chickens in this study was similar to that (38.5%) in E. coli isolates from chickens in China [48], but lower than that in chicken production of India [53]. Notably, except levofloxacin and tigecycline, the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers, was significantly higher than that from brown-egg layers and that from yellow-feather broilers (p < 0.05) ( Table 1). Such phenomena could be attributed to that consumption of antibiotics in white-feather broilers is the largest among the three types of chickens. The rate of resistance to ampicillin, cefotaxime, colistin, and fosfomycin among E. coli from yellowfeather broilers was significantly higher than that from brown-egg layers, respectively (p < 0.05). This might because that β-lactams and colistin are often used in early feeding period of the yellow-feather broilers while almost all antimicrobials are forbidden in layer farms during the laying period.
Based on the molecular criteria of Johnson et al. [6], 2.4% (22/926) of the healthy chicken fecal E. coli isolates were qualified as ExPEC in this study. The ExPEC could asymptomatically colonize the gut of a fraction of healthy animal population and survive in extra-intestinal environments, causing diseases in animals and humans through the food chain [54]. The threat to human health posed by the healthy chicken ExPEC isolates in this study could been further proved by that healthy poultry ExPEC were capable to adhere or invade human intestinal epithelial [14]. In this study, virulence markers iutA and KpsM II were the two most prevalent genes among the ExPEC isolates from healthy chickens, consistent with the finding about MDR E. coli in healthy chickens in Brazil [14]. Besides isolation methods, geographic locations and management practices, different classification criteria for ExPEC has been the main factor contributing to differences in frequency of ExPEC between studies. We will focus the studies using the same PCR-based screening method for ExPEC as that in our study. The detection rate of ExPEC in our samples was 2.4%, similar to that (4.7%, 5/108) among chicken egg E. coli isolates (p > 0.05), but lower than that (21.5%, 130/606) in chicken meat isolates reported in the USA (p < 0.05) [55]. Notably, the prevalence of ExPEC isolates (2.4%, 22/926) in our study was also significantly lower than that (13.2%, 40/304) from farmed healthy chickens in Quebec, Canada [20]. This might be explained by that boiled DNA extracts from total cultures of samples were initially screened for all possible ExPEC strains in the previous study, contributing to a high recovery rate of ExPEC. In this study, the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brownegg layers and that (2.1%, 10/477) from yellow-feather broilers in live poultry markets (p < 0.05). The phenomenon could be attributed to the selective pressure on the dissemination of ExPEC posed by antimicrobials frequently used in white-feather broilers. This was proved by that highly similar PFGE patterns were found in the two ExPEC isolates WF1-5-13 and WF1-5-21 from the same Farm ( Figure 1).
In this study, the most two prevalent ST types of the 22 ExPEC isolates were ST117 and ST93 (Figure 1). Since ST117 and ST93 types of ExPEC had been found to be associated with meningitis of humans in Brazil in 1999 (http://enterobase.warwick.ac.uk/species/ecoli/ search_strains?query=st_search (accessed on 7 October 2020)), both ST types of E. coli have caused sepsis among humans [56,57] and disseminated among humans around the world including the European countries and China (http://enterobase.warwick.ac.uk/species/ ecoli/search_strains?query=st_search (accessed on 7 October 2020)). Besides ST2944, all other STs in this study have been also found in human isolates (http://enterobase.warwick.ac.uk/species/ecoli/search_strains?query=st_search (accessed on 7 October 2020)). Serogroups of the ExPEC isolates from animals were rarely studied, although the APEC isolates from diseased poultry were often serotyped. In this study, O78 was the most predominant serogroup among ExPEC isolates from healthy chickens, followed by O26 and O86. This was slightly different from that of a previous study about APEC from Korea in which O78 was the most prevalent serogroup followed by O2 and O53 [15], both of which were not identified in our study. Notably, O78 was also a common serogroup among human ExPEC isolates from neonatal meningitis in Europe [58]. The distribution rate of each serogroup in this study was also different from that of APEC obtained between 2005 and 2008 in Guangdong, China [59]. The presence of serogroup O86 in two ExPEC from healthy chickens in this study are of interest since this serogroup was only identified in human ExPEC strains in Brazil rather than in strains from poultry in a previous report [57]. In the present study, one ExPEC isolate from healthy chicken belonged to O18 serogroup, which was frequently found in APEC from diseased avian in the United States and also the main serogroup of ExPEC isolates causing newborn meningitis in the Europe [58,60,61]. O83 and O45, which were prevalent in neonates with E. coli meningitis from the Netherlands [61] and France [62], respectively, were also found in our ExPEC isolates. All these results show that the ExPEC isolates from healthy chickens in this study might transmit to humans and the prevalence of ExPEC isolates in healthy animals should be monitored in the future.
Notably, all the ExPEC isolates in this study were MDR isolates. Although the use of antibiotics in animals does not select ExPEC strains intrinsically [25], it will favor the dissemination of ExPEC with MDR phenotypes among healthy animals, contributing to the emergence of MDR ExPEC in human infections [22]. Almost all ExPEC isolates in this study were resistant to ampicillin (100%), sulfamethoxazole-trimethoprim (100%), tetracycline (95.5%), and doxycycline (90.9%), which were all used extensively in animal husbandry in China, further favoring the dissemination of ExPEC among animals and humans by co-selection. In recent years, ExPEC isolates producing ESBLs or AmpC in human infections have been increasing [63], and such pathogens have been also found in healthy poultry in Brazil recently [14]. In our study, CTX-M-type ESBLs were found in 15 of the 22 ExPEC isolates from healthy chickens. The increase in ESBLs or AmpC among ExPEC from poultry will inevitably reduce the therapeutic options of ExPEC infections in humans, because cephalosporins are important to human medicine. Fosfomycin has been widely recommended for treating uncomplicated urinary tract infection especially caused by ESBLs-producing or fluoroquinolone-resistant ExPEC isolates [64]. However, ten ExPEC isolates in this study harbored the fosfomycin resistance gene fosA3 and all ten isolates also carried bla CTX-M , among which six were resistant to fluoroquinolones (Table 4). Such pathogens in healthy chickens will pose a great threat to human health because they will compromise the efficacies of fosfomycin, fluoroquinolones and cephalosporins. For ExPEC from different markets/farms, three isolates (YJ-JC-8, WF1-5-21 and LS-A-7) carrying virulence genes papA and iutA belonged to serogroup O78 and they were all ST117 type, however, different PFGE and resistance profiles were present in the three isolates ( Figure 1 and Table 4). This indicates that the ExPEC isolates have undergone a complex evolutionary process resulting in genetically diverse isolates although they share identical ST type and serogroup at the beginning. Even for ExPEC isolates with identical PFGE pattern and ST type from the same farm, such as WF1-5-13 and WF1-5-21, different resistance genotypes and phenotypes were also observed, further proving the complex evolutionary process within ExPEC.
Worryingly, besides mcr-1 and bla NDM , the two ExPEC isolates WF1-5-13 and WF1-5-40 carried additional bla CTX-M , fosA3 and floR genes, with isolate WF1-5-40 also harboring rmtB ( Table 4). The presence of such ExPEC isolates co-harboring bla NDM and mcr-1 in healthy chickens in this study will threaten the health of consumers because such pathogens will not only compromise the efficacies of cephalosporins, fosfomycin and aminoglycosides, but also threaten the usage of carbapenems and colistin, two critically important antimicrobials used for serious infections caused by MDR ExPEC [65]. mcr has been also found in two ExPEC isolates from diseased poultry in Brazil [18] and two ExPEC isolates from healthy ducks in China [66]. All four ExPEC isolates carrying mcr from animals in the two previous reports were susceptible to carbapenems, although NDM-producing ExPEC isolates susceptible to colistin have been reported in humans [67]. To the best of our knowledge, this is the first report about mcr-1-positive ExPEC isolates harboring bla NDM from healthy chickens.

Conclusions
In conclusion, we observed that the resistances in E. coli from white-feather broilers were more serious than those from layer farms and those from live-poultry markets in China, respectively. This study also reported that 2.4% of these E. coli isolates from healthy chickens were qualified as ExPEC using a molecular detection method. The most predominant serogroup of these ExPEC isolates was O78, followed by O26 and O86, and almost all serogroups identified in our study were frequently reported in human ExPEC isolates in many countries, suggesting that ExPEC isolates from healthy poultry could be a source of potentially virulent ExPEC causing multiple diseases in humans. Notably, all the ExPEC isolates in this study possessed MDR phenotypes and most showed resistances to cephalosporins and fosfomycin, which made co-selection of these ExPEC possible when corresponding drugs were used. More worryingly, six ExPEC isolates in this study carried mcr-1, including two harboring both bla NDM and mcr-1, which could compromise both the efficacies of carbapenems and colistin. The presence of MDR ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or bla NDM , is alarming and will pose a serious health threat to consumers. Interventions need to be taken to reduce these pathogens in the chicken intestine and prevent clinical ExPEC infections in humans by reducing transmission via poultry products. Further studies are required for monitoring the prevalence of MDR ExPEC in healthy chickens in China and other countries. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring bla NDM from healthy chickens.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ani11041112/s1, Table S1: Origins of the samples collected in this study.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.