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

**Ming Zou 1, Ping-Ping Ma 1, Wen-Shuang Liu 1, Xiao Liang 1, Xu-Yong Li 2, You-Zhi Li <sup>3</sup> and Bao-Tao Liu 1,\***


**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 *bla*CTX-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 *bla*NDM, 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%). *bla*CTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring

**Citation:** Zou, M.; Ma, P.-P.; Liu, W.-S.; Liang, X.; Li, X.-Y.; Li, Y.-Z.; Liu, B.-T. Prevalence and Antibiotic Resistance Characteristics of Extraintestinal Pathogenic *Escherichia coli* among Healthy Chickens from Farms and Live Poultry Markets in China. *Animals* **2021**, *11*, 1112. https://doi.org/10.3390/ani11041112

Academic Editors: Paola Roncada and Bruno Tilocca

Received: 8 February 2021 Accepted: 7 April 2021 Published: 13 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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 *bla*NDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying *mcr-1* and/or *bla*NDM, 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 *bla*NDM from healthy chickens.

**Keywords:** characteristics; extraintestinal pathogenic *E. coli*; healthy chickens; multidrug-resistant

#### **1. 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.

#### **2. Materials and Methods**

#### *2.1. 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 (BectonDickinson 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.

#### *2.2. 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).

#### *2.3. Detection of Antibiotic Resistance Genes*

Mobilized colistin resistance genes (*mcr-1* to *mcr-9*) among all *E. coli* isolates in this study were identified using multiplex PCRs as previously described [30,31]. Carbapenemase-encoding genes including *bla*IMP, *bla*VIM, *bla*NDM, *bla*SPM, *bla*AIM, *bla*DIM, *bla*GIM, *bla*SIM, *bla*KPC, *bla*BIC and *bla*OXA−<sup>48</sup> were detected by PCRs [32]. Plasmid-mediated tigecycline resistance determinant *tet*(X4) was amplified as previously described [33]. All *E. coli* isolates were also screened for the presence of plasmid-mediated quinolone resistance (PMQR) genes (*qnrA*, *qnrB*, *qnrS*, *qnrC*, *qnrD*, *qepA*, and *oqxAB*) [34–38]. The presence of extended-spectrum β-lactamases (ESBLs) (*bla*CTX-M-1G, *bla*CTX-M-9G, *bla*CTX-M-2G, and *bla*CTX-M-25G) and plasmid-mediated AmpC β-lactamases (pAmpC) (*bla*CMY-2 and *bla*DHA-1) both conferring resistance to cephalosporins was also analyzed as previously described [39–42]. Resistance genes *rmtB*, *fosA* and *fosA3* were also screened as previously reported [43,44].

#### *2.4. 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.

After PCR identification of ExPEC, the 30 most prevalent serogroups including O1, O2, O4, O6, O7, O8, O9, O15, O18, O21, O22, O25, O26, O45, O55, O78, O83, O86, O101, O103, O111, O113, O117, O121, O138, O145, O149, O157, O158 and O165, were screened among these ExPEC isolates by PCRs as previously described [45].

#### *2.5. 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−<sup>1</sup> 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 *Xba*I (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−<sup>1</sup> 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.

The ExPEC isolates were also subtyped by the multilocus sequence typing (MLST) method using seven house-keeping genes (*adk*, *fumC*, *gyrB*, *icd*, *mdh*, *recA* and *purA*) of *E. coli* as previously described [47]. All the PCR amplicons were sequenced and imported into the *E. coli* MLST database website (https://pubmlst.org/bigsdb?db=pubmlst\_ escherichia\_seqdef&page=sequenceQuery (accessed on 29 July 2020)).

#### *2.6. Statistical Analysis*

Differences in proportions were compared using the χ<sup>2</sup> 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.

#### **3. Results**

#### *3.1. 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).

**Table 1.** Comparison of the resistance rates of *E. coli* isolates from chickens of different origins.


\* The different lowercase letters in the same line were considered significantly different (*p* ≤ 0.05) between two groups using a χ2 test with SPSS software version 19.0. OLA, olaquindox; COL, colistin; FFC, florfenicol; DOX, doxycycline; AMP, ampicillin; CTX, cefotaxime; CTF, ceftiofur; CIP, ciprofloxacin; LEV, levofloxacin; FOS, fosfomycin; MEM, meropenem; NAL, nalidixic acid; GEN, gentamicin; ENR, enrofloxacin; KAN, kanamycin; STR, streptomycin; AMK, amikacin; TET, tetracycline. TIG, tigecycline.

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).

#### *3.2. Detection of Resistance Genes*

Among the 926 *E. coli* isolates, *bla*NDM was found in 45 (4.9%) isolates, and no other carbapenemase-encoding genes was found in this study (Table 2). For the ESBLs-encoding genes, *bla*CTX-M-9G found in 222 (24.0%) of the total *E. coli* isolates was the most prevalent gene, consisting of 52 (14.0%) from 371 brown-egg layers, 54 (69.2%) from 78 white-feather broilers and 116 (24.3%) from 477 yellow-feather broilers of live poultry markets. There were 130 isolates (14.0%) carrying *bla*CTX-M-1G, including 43, 32 and 55 isolates from layer farms, broiler farm and live poultry markets, respectively. A total of 22 isolates harbored both *bla*CTX-M-1G and *bla*CTX-M-9G and no other ESBLs-encoding genes was found in this study. pAmpC-encoding genes *bla*CMY-2 and *bla*DHA-1 were found in 53 (5.7%) and 3 (0.3%) of the 926 isolates. Among the PMQR determinants, *qnrS* and *oqxAB* found in 311 (33.6%) and 181 (19.5%) isolates, respectively, were the two most prevalent genes, followed by *qnrB* (34, 3.7%) and *qnrD* (21, 2.3%) (Table 2). There was no *qnrA* and *qnrC* found in this study. Of the 926 *E. coli* isolates, plasmid-borne fosfomycin resistance (PFR) genes were found in 191 isolates, and the number of isolates harboring *fosA3* and *fosA* was 189 and 2, respectively. Notably, 157 (17.0%) of the isolates were found to harbor *mcr-1*, consisting of 22 (5.9%) from 371 brown-egg layers, 53 (67.9%) from 78 white-feather broilers and 82 (17.2%) from 477 yellow-feather broilers, and no other *mcr* genes were found in this study. In addition, *rmtB* was present in 35 isolates (2.8%). Luckily, none of the 926 isolates carried the plasmid-mediated tigecycline-resistance determinant *tet*(X4).

The detection rate of *bla*NDM, *bla*CTX-M-9G, *bla*CTX-M-1G, *mcr-1*, *qnrS*, *fosA3* and *rmtB* in *E. coli* from white-feather broilers, respectively, was significantly higher than that from layer farms and that from live poultry markets (*p* < 0.05) (Table 2). For *bla*CTX-M-9G, *bla*CMY-2, *mcr-1*, *oqxAB*, *qnrB*, *qnrD*, *fosA3* and *rmtB*, respectively, the yellow-feather broilers from live poultry markets possessed significantly higher detection rate than that from brown-egg layers (*p* < 0.05) (Table 2).

#### *3.3. 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).


**Table 2.** Prevalence of resistance genes among the 926 *E. coli* isolates from chickens.

\* The different lowercase letters in the same line were considered significantly different (*p* ≤ 0.05) between two groups using a χ2 test with SPSS software version 19.0.


**Table 3.** Prevalence and origins of the 22 ExPEC isolates in this study.

**Figure 1.** Characteristics and PFGE dendrogram patterns of the 22 ExPEC isolates from healthy chickens in this study.

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).

#### *3.4. 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.** Resistance phenotypes and genotypes of the 22 ExPEC isolates in this study.


**Table 4.** *Cont.*
