Genotypic Diversity and Antimicrobial Resistance of Escherichia Coli Isolated From Pigs in Hubei, China

Intestinal infections with Escherichia coli (E. coli) are mostly occur in piglets between 1~10 days old, which major lead to diarrhea and edema in newborn piglets. These diseases caused by E. coli can increase mortality, morbidity and growth delay of piglets, which are responsible for economic losses. In this study was to investigate the prevalence of antibiotic resistance, transmission mechanisms, and molecular epidemiology of E. coli strains isolated from pig farms in Hubei province. Furthermore, clonal and genetic diversity of isolates were identied. Results A total of 29 E. coli isolates were obtained from fecal of weaned piglets from Hubei province. The E. coli isolates in different regions demonstrate different genetic diversity. Multilocus sequence typing (MLST) presented that ST165 was the common sequence type, accounting for 27.6% of all E. coli isolates, followed by ST744, ST1081, ST101 and ST10. All of the isolates were resistant to the tested antibiotics to vary degrees, and more than 80% of E. coli isolates presented high resistance rates to ampicillin, lincomycin, doxycycline, tetracycline, sulfaisoxazole and ampicillin. There was one E. coli strain that was resistant to the fteen antimicrobial agents tested. Overall, most of the isolates were conferring resistance to 5-7 antimicrobial agents tested. Our study reported E. coli isolates with high antimicrobial resistance and explores the genetic diversity of E. coli isolated from swine-origin. From the results obtained it can be concluded that these isolates present high prevalent multi-drug resistance. These data provide a greater understanding of the genetic diversity and antimicrobial resistance of E. coli. sulsoxazole and tetracycline of several old drugs with a high resistance rate (100%). The E. coli isolates in different regions demonstrate different genetic diversity In this study, a high antimicrobial resistance and the genotypic diversity of E. coli were observed isolated from swine-origin in Hubei province. From the results obtained it can be concluded that these isolates present high prevalent multi-drug resistance. These data provide a greater understanding of the genetic diversity and antimicrobial resistance of E. coli.


Introduction
Escherichia coli (E. coli) is one of the major commensal bacteria that normally operates in the digestive tract of normal humans and warmblooded animals [1,2,3] . Intestinal infections with E. coli principally occur in piglets between and 1 ~ 10 days old, which major lead to diarrhea and edema in newborn piglets [4] . E. coli can be classi ed as commensal E. coli and pathogenic E. coli based on the differences in pathogenicity [5] . Pathogenic E. coli includes at six major categories on the basis of the virulence mechanisms and prospective progression to infection: enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), adherent invasive E. coli (AIEC) [6] . Furthermore, several types of E. coli have been causing infections in humans, as a public health problem, which has been in uencing all aspects of life, causing severe economic losses to the world [7] .
Several studies of epidemiological studies on E. coli have been initiated in parts of China. In Northeastern China, a survey showed that the separation rate of E. coli isolated from pig fecal samples reached 88% [8] . An investigation on pig farms in Henan province found that the positive rate of E. coli was 70.74%, of which the commonly sequenced types (STs) 10 and 101 were identi ed [9] . However, data on genetic diversity and antimicrobial resistance of E. coli is still restricted in the Hubei province of central China.
In the last few decades, antimicrobial resistance has emerged as one of the considerable global threats to human health [10] . The enhancing antimicrobial resistance of Gram-negative bacteria isolated from animals, which can be transmission from animal to human via the food chain and the environment [11] . In subsequent years, antibiotic resistance has expanded with the application of antibiotics in piglets. In Great Britain, the antimicrobial resistance of E. coli isolates from pigs seems higher than that of E. coli isolates from cattle and sheep [12] . Therefore, it is important to comprehend the antibiotic resistance of pathogenic E. coli in pig farms in China. From 2011 to 2012, a survey of drug resistance in pigs demonstrated that the great majority of swine-origin E. coli isolates resistance to tetracycline (79.57%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin (55.91%) [13] . A survey in Guangdong Province showed that a total of 333 E. coli isolates were acquired from pig farms from 2013 to 2016, which were all multidrug-resistant strains [14] . Multidrug-resistance Gram-negative bacterial clinical isolates are responsible for high rates globally [15] and comprise a challenge for animal treatment.
Multilocus sequence typing (MLST) has been used to study the evolution and epidemiology of a number of bacterial pathogens. It has become the method of choice for typing epidemiologically important strains. MLST is a standard molecular subtyping technique that identi es the genetic relatedness of strains and determines the strains with high discriminatory power [16] . Investigating the trend characterization of epidemic strains will help us to better understand epidemiology.
This present study was carried out to investigate the prevalence and characteristics of E. coli, the MLST genotypes and antibiotic resistance of E. coli isolates were examined, collected from 2018 to 2019 in Hubei province of China. These ndings provide information and implication for safeguarding and commanding the occurrence of diseases in future studies.

Isolation of Escherichia coli
From 2018 to 2019, a total of 29 E. coli isolates were collected from pig fecal samples in Hubei province. These strains isolated by MacConkey agar incubated at 37℃ and further identi ed by PCR test as previously described [17] with some slight modi cations. Total DNA was available by boiling the lysis of isolated colonies for 10 min in distilled water. The samples were then centrifuged at 12,000×rpm/min for 5 min and the supernatant was transferred into a new 1.5 mL tube to be used as a template and applied for the following reactions.

Antibiotic resistance pro les
According to the guidelines of the Clinical and Laboratory Standards Institute [18] , the con rmed E. coli was identi ed for antimicrobial susceptibility. E. coli isolates were examination for susceptibility to antimicrobial drugs utilizing a disk diffusion assay. All samples were analyzed for the presence of resistant bacteria. A total of 18 antimicrobials were tested, comprising cefuroxime (CXM), ceftriaxone (CRO), cephalothin (CEP), cefotaxime (CTX), ampicillin (AMP), amoxicillin (AMX), lincomycin (MY), doxycycline (DOC), tetracycline (TEC), kanamycin (KMC), gentamicin (GEN), amikacin (AMK), cipro oxacin (CIP), enoxacin (ENO), lome oxacin (LOM), azithromycin (AZM), sulfafurazole (SFN). Inoculated plates were incubated at 37 ℃ for 24 h, subsequently the diameters (in mm) of the inhibition zone were measured. Based on the minimal inhibitory concentration determined for each drug, the isolates were classi ed as "susceptible", "intermediate", or "resistant". The E. coli strain ATCC 25922 was utilized for quality control. Multidrug-resistant (MDR) of an isolate was designated as clinical resistance to at least one agent in three or more antimicrobial categories [19] .

MLST and Phylogenetic tree
The multilocus sequence typing (MLST) was executed on 29 E. coli isolates according to the E. coli MLST database guidelines (http://enterobase.warwick.ac.uk/ species/ecoli/allele st search), accreting to the protocols published on the web site. Brie y, the seven house-keeping genes adk, fumC, gyrB, icd, mdh, purA and recA were ampli ed employing a PCR protocol, and the amplicons sequenced utilizing the ampli cation primers. Investigated individual gene sequences and allocated an allelic pro le number in line with the MLST database. Sequence type (STs) and clone complexes (CCs) designations of each strain were comprised of seven alleles. The calculated tree of the E. coli resistant isolates was constructed by applying the UPGMA cluster analysis based on seven housekeeping gene sequences.

Isolation of Escherichia coli
As shown in Table 1, E. coli isolates were obtained from fecal of weaned piglets from Hubei province. 29 strains of E. coli were separated from positive samples and further identi ed by PCR tests as E. coli. There are 3 strains of E. coli isolated from Suizhou city, 6 strains of E. coli isolated from Xiangyang city, 15 strains of E. coli isolated from Wuhan city and 5 strains of E. coli isolated from Yichang city. Antimicrobial Susceptibility pro le of Escherichia coli isolates As shown in Table 2, E. coli in different regions has separate antibiotic resistance. All of the E. coli isolates showed resistance to lincosamides, tetracyclines, and sulfonamide. As shown in the Fig. 1   As shown in the Fig. 2, all of the isolates were resistant to test antibiotics to vary degrees, and 100% of the isolates were resistant to more than six drug classes. There was one E. coli strain that was resistant to the fteen antimicrobial agents tested. Overall, most of the isolates were conferring resistance to 5-7 antimicrobial agents tested. The most frequent multidrug resistance pattern was resistance to ampicillin, amoxicillin, lincomycin, doxycycline, tetracycline, gentamicin and sulfaisoxazole, which covered 8 isolates.
The identi cation of 12 ST types belonged to 5 clone complexes and unassigned clone complexes. To further analyzing STs utilized the UPGMA cluster analysis (Fig. 3). 29 identi ed STs were classi ed into three major groups. Group 1 covered a great majority of STs, containing 24 isolates, belonging to the CC-10, CC-46, CC-165 and unassigned clone complexes. CC-10 contained 2 of our STs (ST10 and ST617). Group 2 included 3 isolates that only belonged to one CC-101. There are 2 isolates included CC-23 and unassigned clone complexes constitute Group 3.

Discussion
Escherichia coli is one of the main pathogenic bacteria that impact the production and growth of pigs in pig farms. It is associated with gastrointestinal diseases such as diarrhea, edema disease, and systemic infections such as septicemia and polyserositis [2] . These diseases caused by E. coli can increase mortality, morbidity and growth delays of piglets, which are responsible for economic losses. This study analyzed the prevalence, genetic diversity and antibiotic resistance of disease, which may help us to improve methods of prevention and treatment.
The distribution of swine-origin E. coli in pigs differs between countries and regions. From 2002 to 2008, the prevalence of E. coli isolated from pork chop samples was 44% in the United States [20] . However, various incidence rates have also been reported in different regions of China. From 2003 to 2009, the prevalence of E. coli isolates from pig farms was 77.78% in China [21] . From 2013 to 2016, the positive rates of E. coli between farm 1 and farm 2 were 40.25% and 59.75% in Guangdong province [14] . Between 2016 and 2017, a survey indicated that the separation rate of E.
coli isolated from pig fecal swabs reached 88% in northeastern China, including Heilongjiang, Jilin and Liaoning [8] . In this study, we collected samples from the fecal of weaned piglets, and a total of 29 strains of E. coli were isolated from 4 cities in Hubei province.
Diarrhea in weaned piglets driven by E. coli remains a principal cause of economic losses for the pig industry. This commonly seeks antimicrobial drug treatment, which is considerable to cure pathogen animals. In 2011, Danish scholar Agersө et al. study found that 32% of isolates have multidrug resistance, mainly concentrated on ampicillin (27%) and tetracycline (29%) [24] . In 2012, Tadesse et al. tested 1729 isolates of E. coli antibiotic susceptibility varied from different sources, the resistance rate of E. coli increased from 7.2-63.6% but the most common resistance to tetracycline, streptomycin and sulfonamides [25] . Total of 131 E. coli isolates were obtained from the pigs presenting from diarrhea in Switzerland from 2014 to 2015, isolates exhibited resistance to tetracycline (50%), sulfamethoxazole (49%), ampicillin (26%), gentamicin (17%), cipro oxacin (8%) [26] . However, this caused a rise in the employment of various antimicrobial agents, such as lincosamides, tetracyclines and sulfonamides, which may expand antimicrobial resistance.
In this study, E. coli isolates results of the antimicrobial susceptibility tests presented that the most prevalent antibiotic resistance was to lincosamides, tetracyclines, sulfonamides. More than 80% of E. coli isolates presented high resistance rates to ampicillin, lincomycin, doxycycline, tetracycline, sulfaisoxazole and ampicillin. All of the isolates were resistant to test antibiotics to vary degrees, and Most of E. coli isolates advent high prevalent multi-drug resistance. There was one E. coli strain that was resistant to the fteen antimicrobial agents tested. Overall, more than half of the isolates were conferring resistance to 8-15 antimicrobial agents tested. The most frequent multidrug resistance pattern was resistance to ampicillin, amoxicillin, lincomycin, doxycycline, tetracycline, gentamicin and sulfaisoxazole, which covered 8 isolates. In some other studies in China, Jiang et al. revealed that E. coli isolates had high rates of resistance to ampicillin (99.5%), tetracycline (93.4%) and amoxicillin (65.1%).
Resistance to cephalosporins, quinolones, and aminoglycosides was also quite prevalent [27] . Meng et al. study results showed that the great majority of E. coli isolates resistance to tetracycline (79.57%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin in China (55.91%) [13] . However, E. coli isolates showed the highest resistance to sulfamethoxazole (61.6%), followed by tetracycline (61.2%), ampicillin (48.2%) and kanamycin (22.4%) in Sichuan province between 2012 and 2013 [28] . E. coli were isolated from pig farms from seven provinces that the resistance rate to ampicillin was 81.44%, 94.37% to tetracycline and 88.36% to sulfaisoxazole [29] . These ndings provide important information and implications for the application of antibiotics in future studies.

Conclusions
In this study, a high antimicrobial resistance and the genotypic diversity of E. coli were observed isolated from swine-origin in Hubei province. From the results obtained it can be concluded that these isolates present high prevalent multi-drug resistance. These data provide a greater understanding of the genetic diversity and antimicrobial resistance of E. coli.

Abbreviations
MLST multilocus sequence typing STs sequence types CCs clonal complexes.

Declarations Author′ contribution
The experiments were performed mainly by ZL, XL, NW and WL, and some experiments were performed with the help of TG and DZ, XL, ZL, RG, WL and WB performed the data analysis. The study was designed by FY and YT. All authors read and approved the nal manuscript.