Identification, Typing, and Drug Resistance Analysis of Escherichia coli in Two Different Types of Broiler Farms in Hebei Province

Simple Summary Antibiotic resistance is a global health crisis faced by humanity. In response to this crisis, in 2018, China released the National Pilot Work Program for Action to Reduce the Use of Veterinary Antimicrobial Drugs. Pilot work to reduce the use of antimicrobial drugs was carried out on livestock and poultry scale farms across the country, and the farms implementing the reduction in antimicrobial drug use were accepted as meeting the standards by the “Evaluation Standards and Methods for Reducing the Use of Veterinary Antimicrobial Drugs on Livestock and Poultry Farms”. To evaluate the effect of antimicrobial drug reduction on antibiotic resistance, we selected six broiler farms in Hebei Province for the first time, four of which implemented antimicrobial drug reduction and passed national acceptance (SFs), and the other two did not implement antimicrobial drug reduction (NSFs). By collecting cloacal swabs from healthy broilers, isolating and characterizing Escherichia coli (E. coli), and determining their resistance phenotypes, resistance profiles, and genotypes to 16 antimicrobial drugs, as well as ST typing of the isolates, we compared the differences in resistance and the genetic evolutionary relationships of two different types of broiler farms in Hebei Province, China. Abstract Hebei Province is an important area for breeding broiler chickens in China, but the antimicrobial resistance and prevalence of Escherichia coli (E. coli) are still unclear. A total of 180 cloacal samples from broiler farms in Hebei Province were collected and used for the isolation and identification of E. coli. The isolates were subjected to resistance phenotyping, resistance profiling, and genotyping, and some multiresistant strains were subjected to multilocus sequence typing (MLST). The results showed that 175 strains were isolated. Among both types of broiler farms, the ampicillin resistance rate was the highest, and the meropenem resistance rate was the lowest. Serious multiresistance was present in both types of broiler farms. Thirty strains of multidrug-resistant E. coli were typed by MLST to obtain a total of 18 ST types, with ST10 being the most prevalent. This study was to simply analyze the antimicrobial resistance and prevalence of E. coli in broiler chickens in Hebei Province after the implementation of the pilot work program of action to reduce the use of veterinary antimicrobials in standard farms (SFs) and nonstandard farms (NSFs). This study will provide a research basis and data support for the prevention and control of E. coli in Hebei.


Introduction
Escherichia coli (E.coli) is a gram-negative bacterium in the Enterobacteriaceae family of bacteria that is widely present throughout the entire feeding chain of chicken farms, in chicken houses, in the air, and even in the food chain, that can cause significant economic loss to the livestock industry and seriously affect global public health security [1][2][3][4].Currently, there is still no effective vaccine for the prevention of E. coli infection, and the use of antibiotics remains the primary choice.Antibiotics are extensively used in livestock and samples were collected from healthy broilers, and 30 chickens were randomly selected from each broiler farm.The samples were kept in an ice box and sent to the laboratory in a timely manner.Information on antibiotic treatment for the six sampled farms during the study period is shown in Table S2.

Sample Source
A total of 180 cloacal swabs were collected from six broiler farms located in the Baoding, Chengde, Tangshan, and Cangzhou regions in Hebei Province, including four SFs and two NSFs (Figure 1).The six broiler farms' details are shown in Table S1.All cloacal samples were collected from healthy broilers, and 30 chickens were randomly selected from each broiler farm.The samples were kept in an ice box and sent to the laboratory in a timely manner.Information on antibiotic treatment for the six sampled farms during the study period is shown in Table S2.

Isolation and Identification of E. coli
The collected cloacal swabs were streaked onto MacConkey (MAC) and eosin methylene blue agar (EMB) and cultured at 37 • C for 15 h.Following three rounds of purification, purified single colonies were selected for Gram staining, and the morphological characteristics of the isolated strains were observed under a microscope.The E. coli strains were confirmed by PCR detection of the 16S rRNA gene using the following primers: 16S rRNA-F, 5 -ATCATGGCTCAGATTGAACG-3 , 16S rRNA-R, 5 -CCAGTAATTCCGATTAACGC-3 , 552 bp.PCR conditions were as follows: initial denaturation at 94 • C for 5 min, 30 denaturation cycles at 94 • C for 40 s, annealing at 54 • C for 30 s, amplification at 72 • C for 1 min, and a final extension at 72 • C for 10 min.PCR amplification products were identified by 1% agarose gel electrophoresis, and positive products were sequenced by Sangon Bioengineering (Shanghai, China) Co., Ltd.

Drug Susceptibility Testing
The Kirby-Bauer disk diffusion method was used to test the susceptibility of the isolated strains to 16 kinds of antibacterial drugs.According to the Clinical and Laboratory Standards Institute (CLSI) standards, the drug susceptibility test results were judged in three forms: sensitivity (S), intermediate (I), and resistance (R).Escherichia coli ATCC 25922 was used as the quality control strain.

Multilocus Sequence Typing
To investigate the prevalence of multidrug-resistant strains, 30 multidrug-resistant E. coli strains (SF: n = 20, NSF: n = 10) were selected from 175 E. coli strains for multilocus sequence typing.Primer sequences of the seven housekeeping genes of E. coli (adk, fumC, icd, purA, gyrB, recA, and mdh) were obtained from http://enterobase.warwick.ac.uk/species/ ecoli/allele_st_search, accessed on 13 August 2023 [19,20].The primer sequences, fragment sizes, and annealing temperatures are shown in Table S4.The sequencing results were spliced by SeqMan and uploaded to the database, generating an allelic map to determine the STs of E. coli.A phylogenetic tree was constructed and analyzed using Mega 7 software to analyze the genetic evolution.

Statistical Analysis
Statistical differences in AMR rates in E. coli isolates from two types of broiler farms were analyzed with the chi-square test and Fisher's test by SPSS 25.0 (IBM Corporation, Somers, NY, USA).A value of probability (p) <0.05 was considered statistically significant.

Isolation and Identification
A total of 175 strains (SF 115; NSF 60) were isolated and identified from 180 cloacal swab samples after separation and purification by MacConkey medium and eosin methylene blue medium, Gram staining morphological characteristics identification, and 16S rRNA PCR detection (Figure 2).

Isolation and Identification
A total of 175 strains (SF 115; NSF 60) were isolated and identified from 180 cloacal swab samples after separation and purification by MacConkey medium and eosin methylene blue medium, Gram staining morphological characteristics identification, and 16S rRNA PCR detection (Figure 2).

Results of Drug Resistance Phenotype Detection
The antibiotic sensitivity test results of 175 of the E. coli strains to 16 antibiotics are shown in Table 1.Both types of broiler farms had the highest rate of resistance to ampicillin, which was above 90.0%,and the lowest rate of resistance to meropenem, which was below 10.0%.
Based on the analysis, it was found that the E. coli strains in the SFs had the highest resistance rate to ampicillin (93.9%), followed by florfenicol, ceftiofur, enrofloxacin, sulfisoxazole, and cotrimoxazole, which were all over 70.0%.However, the resistance rates to ofloxacin, gentamicin, colistin, acetylmethaquine, ceftazidime, and meropenem were lower at 31.3%, 26.1%, 22.6%, 22.6%, 16.5%, and 7.0%, respectively.On the other hand, in the NSFs, the E. coli strains had a high resistance rate to florfenicol, ampicillin, and ceftiofur, with the resistance rate being over 90.0%.Additionally, the resistance rates for spectinomycin, apramycin, enrofloxacin, sulfisoxazole, and cotrimoxazole were above 70%.However, the resistance rates for colistin (28.3%), acetylmethaquine (13.3%), ceftazidime (16.7%), and meropenem (1.7%) were lower.These results indicated that the tested E. coli strains of the SFs and NSFs showed a high resistance rate to ampicillin and were sensitive to meropenem.
The analysis revealed that there were significant differences in the resistance rates of E. coli in the SFs and NSFs.Specifically, the resistance rates of gentamicin, spectinomycin, and ceftiofur were significantly lower in the SFs than in the NSFs (p < 0.05).Conversely, the resistance rate of cotrimoxazole was significantly lower in the NSFs than in the SFs (p < 0.05).This may be due to the extensive usage of cotrimoxazole before, resulting in relatively high drug resistance levels.Furthermore, the resistance to apramycin, tetracycline, florfenicol, ampicillin, ceftazidime, enrofloxacin, and colistin was lessened in the SFs than in the NSFs, although the difference was not statistically significant (p > 0.05).According to the study's findings, the National Action Program for the Reduction of Veterinary Antimicrobial Drug Use has successfully reduced antibiotic resistance to a certain extent in Hebei Province, China.However, the resistance situation is still relatively serious, and

Results of Drug Resistance Phenotype Detection
The antibiotic sensitivity test results of 175 of the E. coli strains to 16 antibiotics are shown in Table 1.Both types of broiler farms had the highest rate of resistance to ampicillin, which was above 90.0%,and the lowest rate of resistance to meropenem, which was below 10.0%.Based on the analysis, it was found that the E. coli strains in the SFs had the highest resistance rate to ampicillin (93.9%), followed by florfenicol, ceftiofur, enrofloxacin, sulfisoxazole, and cotrimoxazole, which were all over 70.0%.However, the resistance rates to ofloxacin, gentamicin, colistin, acetylmethaquine, ceftazidime, and meropenem were lower at 31.3%, 26.1%, 22.6%, 22.6%, 16.5%, and 7.0%, respectively.On the other hand, in the NSFs, the E. coli strains had a high resistance rate to florfenicol, ampicillin, and ceftiofur, with the resistance rate being over 90.0%.Additionally, the resistance rates for spectinomycin, apramycin, enrofloxacin, sulfisoxazole, and cotrimoxazole were above 70%.
However, the resistance rates for colistin (28.3%), acetylmethaquine (13.3%), ceftazidime (16.7%), and meropenem (1.7%) were lower.These results indicated that the tested E. coli strains of the SFs and NSFs showed a high resistance rate to ampicillin and were sensitive to meropenem.
The analysis revealed that there were significant differences in the resistance rates of E. coli in the SFs and NSFs.Specifically, the resistance rates of gentamicin, spectinomycin, and ceftiofur were significantly lower in the SFs than in the NSFs (p < 0.05).Conversely, the resistance rate of cotrimoxazole was significantly lower in the NSFs than in the SFs (p < 0.05).This may be due to the extensive usage of cotrimoxazole before, resulting in relatively high drug resistance levels.Furthermore, the resistance to apramycin, tetracycline, florfenicol, ampicillin, ceftazidime, enrofloxacin, and colistin was lessened in the SFs than in the NSFs, although the difference was not statistically significant (p > 0.05).According to the study's findings, the National Action Program for the Reduction of Veterinary Antimicrobial Drug Use has successfully reduced antibiotic resistance to a certain extent in Hebei Province, China.However, the resistance situation is still relatively serious, and there is still a need to continue long-term monitoring of antibiotic resistance in broiler farms.

Results of Multidrug Resistance of Isolated Strains
Most E. coli strains were MDR strains (96.57%, 169/175) resistant to three or more different types of antimicrobials.The MDR rates were different between the E. coli strains of the SFs (95.65%, 110/115) and NSFs (98.33%, 59/115).The 175 E. coli strains showed a broad multidrug-resistance pattern with 151 AMR patterns.The MDR strains of SFs (74.8%, 84/115) and NSFs (90.0%, 54/60) were both the majority resistant to 6-12 classes of antimicrobials (Figure 3).The E. coli strains of NSF have a more severe multidrug resistance situation, with the majority of E. coli strains being resistant to more types of antibiotics.Overall, the pilot work slightly alleviated the multidrug resistance of chicken-derived E. coli strains in Hebei Province, China.

Results of Multidrug Resistance of Isolated Strains
Most E. coli strains were MDR strains (96.57%, 169/175) resistant to three or more different types of antimicrobials.The MDR rates were different between the E. coli strains of the SFs (95.65%, 110/115) and NSFs (98.33%, 59/115).The 175 E. coli strains showed a broad multidrug-resistance pattern with 151 AMR patterns.The MDR strains of SFs (74.8%, 84/115) and NSFs (90.0%, 54/60) were both the majority resistant to 6-12 classes of antimicrobials (Figure 3).The E. coli strains of NSF have a more severe multidrug resistance situation, with the majority of E. coli strains being resistant to more types of antibiotics.Overall, the pilot work slightly alleviated the multidrug resistance of chicken-derived E. coli strains in Hebei Province, China.
The resistance rates to 16 antibiotics were correlated with the detection rates of related resistance genes in two different types of broiler farms.Overall, there is a similar prevalence and diversity of 23 resistance genes in the E. coli strains of the two types of broiler farms in Hebei Province, China.

MLST Typing Results
Multilocus sequence typing (MLST) analysis showed that the 30 multidrug-resistant E. coli strains belonged to 18 ST types and one novel ST (Table 3).ST10 of E. coli strains was the dominant genotype (13.3%), and the percentages of ST156 and ST2847 were both 10.0%, followed by ST93, ST69, ST6843, ST457, and ST1011, which were all 6.67%, and the other 10 ST types were all 3.33%.There were differences in the distribution of E. coli ST types in the two types of broiler farms.In the SFs, the dominant STs were ST10 and ST2847, while in the NSFs, ST156 was the most common (Figure 4).
The cluster analysis results of 30 multidrug-resistant E. coli strains of two types of broiler farms using Mega 7.0 software showed (Figure 5) that there was a certain genetic relationship between the SFs and NSFs.Out of the 30 multidrug-resistant E. coli strains, there were 18 STs with 28 multidrug-resistant patterns.Notably, strains with the same ST may not share the same multidrug-resistance pattern; for instance, the ST10 strain YX4, ST43 strain YX8, ST2847 strain YM7, and ST69 strain YC23.There was no obvious correlation between the ST types of the strain and the MDR patterns.
ST10 of E. coli strains was the dominant genotype (13.3%), and the percentages of ST156 and ST2847 were both 10.0%, followed by ST93, ST69, ST6843, ST457, and ST1011, which were all 6.67%, and the other 10 ST types were all 3.33%.There were differences in the distribution of E. coli ST types in the two types of broiler farms.In the SFs, the dominant STs were ST10 and ST2847, while in the NSFs, ST156 was the most common (Figure 4).The cluster analysis results of 30 multidrug-resistant E. coli strains of two types of broiler farms using Mega 7.0 software showed (Figure 5) that there was a certain genetic relationship between the SFs and NSFs.Out of the 30 multidrug-resistant E. coli strains, there were 18 STs with 28 multidrug-resistant patterns.Notably, strains with the same ST may not share the same multidrug-resistance pattern; for instance, the ST10 strain YX4, ST43 strain YX8, ST2847 strain YM7, and ST69 strain YC23.There was no obvious correlation between the ST types of the strain and the MDR patterns.

Discussion
The increased use of antimicrobial medications in livestock and poultry production has resulted in a number of issues with antibiotic resistance [21].A governmental initiative

Discussion
The increased use of antimicrobial medications in livestock and poultry production has resulted in a number of issues with antibiotic resistance [21].A governmental initiative to lower the usage of veterinary antimicrobial medicines has been in place in China since 2018.In this study, we examined and analyzed the resistance to a total of 16 antimicrobial drugs in seven classes and the carriage of resistance genes from the two types of broiler farms (SF and NSF) in Hebei Province following the implementation of the National Veterinary Antimicrobial Drug Use Reduction Action Pilot Work Program.A differential analysis of the drug resistance in E. coli strains from the two types of broiler farms was performed.
Ampicillin resistance rates were found to be highest in E. coli strains in two types of broiler farms, exceeding 90.0%.Additionally, resistance rates to florfenicol, ceftiofur, enrofloxacin, and sulfisoxazole were over 70.0%.This is consistent with the results of domestic and foreign surveys in recent years.Similarly, high resistance to ampicillin, tetracycline, and sulfisoxazole was observed in chicken-derived E. coli strains of Shandong Province, China [22,23].Likewise, most E. coli isolate strains displayed serious resistance to tetracycline, sulfamethoxazole, florfenicol, and ampicillin in Eastern China [24].According to a report by Osman et al. [25], resistance to antibiotics such as ampicillin, tetracycline, erythromycin, sulfamethoxazole, and doxycycline and streptomycin is the most common.However, most of chicken-derived E. coli were strains showed highly sensitive to the ceftazidime and meropenem of β-lactams in this study.Similar results were observed in Zhejiang Province, China, where most isolates showed highly sensitive to ceftazidime (12.81%) and meropenem (0.75%), but serious resistance to tetracycline (92.92%), sulfisoxazole (93.05%), florfenicol (83.11%), and ampicillin (78.27%) [26], which is also similar to the previous reports in Jiangxi Province, China [27].It has been observed that in France, Germany, Italy, and the United Kingdom, E. coli strains have a high resistance to ampicillin and cotrimoxazole, while resistance to ceftazidime and meropenem is very low [28].Antibiotic resistance is closely linked to the use of antibiotics, and it has been found that food animals [29], especially chickens [30], are given high levels of antimicrobial drugs such as tetracyclines, fluoroquinolones, β-lactams, and aminoglycosides.Among β-lactams, amoxicillin, ampicillin, and ceftiofur are legally and extensively used, followed by fluoroquinolones such as norfloxacin and ofloxacin.This explains the high resistance levels to these antibiotics on broiler farms.Moreover, a study conducted between 2013 and 2019 revealed that the resistance and multiresistance of E. coli strains to important antibiotics for human medicine, such as third-generation cephalosporins and meropenem, have increased over time compared to 2013 [31].This trend of antibiotic resistance is concerning.It is necessary to strictly control antibiotic use and monitor resistance to protect human and public health.
Bacterial resistance genotypes are determinants of the resistance phenotype.By identifying carriers of resistance genes in multidrug-resistant strains is crucial to support clinical rational antibiotic use and gain insight into antibiotic resistance gene epidemic characteristics [32].Among the E. coli strains in this study, aph(3 )-II, aadA, TEM, gyrA, gyrB, parC, and flor resistance genes were highly prevalent with the detection rates all above 80%.The primary resistance mechanism of aminoglycosides is inactivated by aminoglycosidemodifying enzymes.Our study found that E. coli isolates were mainly resistant to aminoglycosides due to the presence of aph(3 )-II encoding aminoglycoside phosphotransferases (APHs) and aadA encoding aminoglycoside nucleotidyltransferase (ANTs).According to Liu et al., the highest detection rate for aadA (80%) was observed in chicken-derived E. coli strains in Shandong Province, China [33], while Song et al. noted that the detection frequencies of the aphA3 resistance genes in chicken-derived E. coli strains were above 50% [34].The CTX-M and TEM family of extended-spectrum beta-lactamases (ESBLs) has long been considered the most widespread globally, among the 262 ESBL-positive E. coli isolates in central China, CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) [35,36].Similarly, our study found that CTX-M and TEM primarily mediate β-lactam antibiotic resistance.In terms of quinolone resistance, gyrA, gyrB, and parC genes are the main contributors, followed by qnrA, qnrB, and qnrS genes.However, fluoroquinolone-modified acetyltransferase genes (aac(6 )-Ib-cr) are less common [37,38].Flor genes encoding mainly mediate the florfenicol resistance, and it has been reported that among 106 florfenicol resistant E. coli strains, approximately 91.51% (97/106) of E. coli strains carry flor genes [39].The key to antibiotic resistance is the effective expression of resistance genes [32,40].We observed a correlation between the detection rates of related resistance genes and the resistance rates to 16 antibiotics in two different types of broiler farms.The resistance to a particular class of antibiotics increased with the detection rate of its related resistance genes.Specifically, the resistance rates of gentamicin, spectinomycin, and ceftiofur were significantly lower in the SFs than in the NSFs (p < 0.05).This may be because the NSF uses more β-lactams antibiotics in the early fattening stage and more aminoglycosides antibiotics in the terminal fattening stage than that in the SF, in addition, the overall level of antibiotic use in the SF is lower than that in the NSF.The results of significant difference analysis showed that the detection rates of the aadA, aph(3 )-II, aac2, and aac4 genes of E. coli in the SFs were lower than those of the NSFs, especially the detection rate of aac2, which significantly lower than that of the NSFs (p < 0.05).This may be related to the lower use of aminoglycoside antibiotics in the SF.
In this study, the antibiotic resistance in SFs decreased to a certain extent compared with NSFs in Hebei Province, China, but the difference in some antimicrobial drugs was not significant.On the one hand, this may be due to the short period of antimicrobial drug reduction in SFs (3-4 years), and the fact that antibiotics have been administered in poultry farming for more than 50 years, and the resistance genes remain at high levels for a long time, regardless of the antibiotic pressure; on the other hand, there are different medication habits in different regions [41].Overall, serious antibiotic resistance problems still exist in the two types of broiler farms in Hebei Province, China, and the farms should further strengthen the rational use of antibiotics to effectively curb antibiotic resistance.
In recent years, multilocus sequence typing (MLST) technology has been widely used in bacterial typing, which can clarify the affinity and evolutionary relationship of strains and provide a reference for the epidemiological investigation, control, and prevention of E. coli [42,43].It was found that 30 multidrug-resistant E. coli strains from two types of broiler farms were categorized into a total of 18 ST types, with ST10 as the dominant ST type, followed by ST156, ST2847, ST93, ST69, ST6843, ST457, and ST1011.In the SFs, the dominant STs were ST10 and ST2847, while in the NSFs, ST156 was the most common ST type.Zhou et al. [11] reported that among strains of porcine and avian origin, ST10 was the dominant ST type, followed by ST48, ST58, and ST162.A study of the MLST data showed that the E. coli strains of ST10, ST48, ST95, and ST117 were dominant in poultry [36,40,44], of which ST10 is predominant in human, poultry, and swine EXPEC at home and abroad, and it has been shown that the strains of ST10, ST95, ST23, ST117, and ST131 may cause zoonotic diseases [45].Notably, strains with the same ST may not share the same multidrug-resistance pattern.Out of all the E. coli strains that were isolated, only the ST10 type showed a crossover between the SFs and NSFs.This suggests that the ST types have a complex and diverse genetic makeup.However, due to the limited number of samples and the short implementation period for antimicrobial reduction, it is unclear whether the implementation had a significant impact.Therefore, more monitoring and research are needed to fully understand the effects.
The limitation of this study is that only six farms were analyzed, which were further divided into two categories, with monitoring of antibiotic reduction (four farms) and without monitoring (two farms), and only 30 cloacal samples were analyzed per farm.In the future long-term studies, we will expand the sampling range and increase the number of samples, so as to make the conclusions of our study more abundant and reliable.

Conclusions
In this study, we investigated and analyzed the antibiotic resistance of E. coli strains and ST types of some multidrug-resistant E. coli strains from two types of broiler farms in

Figure 1 .
Figure 1.The distribution of broiler farms in Hebei Province, China.

Figure 1 .
Figure 1.The distribution of broiler farms in Hebei Province, China.

Figure 3 .
Figure 3. Multidrug resistance of isolated strains in the two types of broiler farms.Figure 3. Multidrug resistance of isolated strains in the two types of broiler farms.

Figure 3 .
Figure 3. Multidrug resistance of isolated strains in the two types of broiler farms.Figure 3. Multidrug resistance of isolated strains in the two types of broiler farms.

Figure 4 .
Figure 4. Cluster analysis was performed by geoBURST software v1.1.5.The black number represents ST types; The red number represents the difference in allele numbers between different ST types.

Figure 4 . 15 Figure 5 .
Figure 4. Cluster analysis was performed by geoBURST software v1.1.5.The black number represents ST types; The red number represents the difference in allele numbers between different ST types.Animals 2023, 13, x FOR PEER REVIEW 10 of 15

Figure 5 .
Figure 5. Phylogenetic tree of E. coli strains based on STs and multiresistance patterns.The red dots represent E. coli strains of SFs, and the blue dots represent E. coli strains of NSFs.

Table 1 .
Resistance rates of isolates from two types of broiler farms to 16 antimicrobials.

Table 1 .
Resistance rates of isolates from two types of broiler farms to 16 antimicrobials.Note: different uppercase letters on the shoulders of peer data indicate extremely significant differences (p < 0.01); different uppercase and lowercase letters on the shoulders indicate significant differences (p < 0.05); the same letters or no letters on the shoulders indicate no significant differences (p > 0.05).

Table 2 .
The detection rate of resistance genes of the two types of broiler farms.
Note: different uppercase letters on the shoulders of peer data indicate extremely significant differences (p < 0.01); different uppercase and lowercase letters on the shoulders indicate significant differences (p < 0.05); same letters or no letters on the shoulders indicate no significant differences (p > 0.05).

Table 3 .
Distribution of the ST type of the 30 isolates and multidrug resistance profile.