Aavirulence and Antimicrobial Characteristics of Escherichia Coli Isolated from Diseased Chickens in China and Algeria

To reveal and compare the prevalence of pathotypes and virulence genes, as well as antimicrobial resistance and genotyping of poultry E. coli isolates from China and Algeria. Pathotype and seven virulence genes were tested by PCR, susceptibility to antimicrobials was evaluated using broth microdilution method, and genotyping was analyzed by PFGE. Six isolates were identified as pathogenic E. coli. Virulence gene testing showed that the frequency of ompT, iss, fimC, iroN, hlyF, and iutA was high in the isolates from Shandong, Shanxi, Jiangsu, and Xinjiang province, while stx2 was detected in only two isolates from Shandong, and stx2, iss, fimC, hlyF, and iutA could not be detected in Tibet isolates. Importantly, nearly all isolates from Algeria carried seven virulence genes. Drug resistance testing of 141 strains showed that 98.2% (109/111) of the isolates from China and all isolates (30/30) from Algeria resist to more than three classes of antimicrobials. The PFGE genotyping of 157 isolates yielded 134 types, demonstrated a high level of genetic diversity among these isolates. Thus, the poultry E. coli from both China and Algeria exhibited either high frequencies of antimicrobial resistance or high rates of virulence genes carrying.


Introduction
Escherichia coli (E. coli) is a member of the normal flora in human and warm-blooded animal intestinal tracts.
Nevertheless, some E. coli strains are usually pathogenic and have virulence properties that may cause animal diarrhea, hemorrhagic colitis and hemolytic uremic syndrome [1]. Certain strains can even cause public health problems, including life-threatening infections. Up to now, five kinds of E. coli pathotype are widely recognized that may cause colibacillosis both in human and animals according to their specific virulence gene, pathogenic mechanism and genetic features: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC) [2]. The pathogenic E. coli generally possesses rich virulence genes that displayed during disease happening. However, many clinical isolated E. coli strains from diseased or dead chickens may carried abundant virulence genes but cannot attribute to any pathotype.
Pulsed-field gel electrophoresis (PFGE) is a DNA based genetic fingerprinting tool that can preferably differentiate strains by comparing genotypic characteristics. This method has been recently used to estimate the source of Salmonella spp. contamination in poultry products [3], analyze the similarity of E. coli strains isolated from different poultry slaughterhouses [4], as well as identify the route and origin of infection for specifying bacteria that may be responsible for a food-borne illness earlier [5].
Currently, the rising of antimicrobial resistance, especially the emergence of multidrug-resistance (MDR), has become a common public health concern, in particular the abuse and misuse of antibacterials in broiler population. One of the main causes of the MDR strains emerging is bacteria acquired and disseminated exogenous genes by mobile genetic elements [6]. The sustained growth of avian origin E. coli isolates with MDR phenotypes has been published frequently [4,5,7,8].
Though there were some pathogenic or drug resistance characteristic analysis of E. coli isolated from poultry in China or Algeria earlier [4,9,10], few studies have supplied sufficient comparison information of avian origin E. coli strains isolated from representative provinces in China and western African countries. Thus, in this study, we designed to show the prevalence of pathotypes and virulence genes, as well as the antimicrobial characteristics of E. coli isolated from diseased and/or freshly died chickens in different regions of China. Furthermore, we compared them with counterpart of Algeria so as to seek their genetic similarities and differences.

Isolations of E. coli
In this study, a total of 248 strains were ananlyzed, including 30 strains confirmed as E.coli from freshly died broiler chickens in Algeria previously [9]. All Chinese strains were isolated from diseased poultry population or freshly dead chickens in seven representative provinces by local animal hygiene department in 2017 (Fig 1). All isolates were confirmed to be E. coli using API 20E biochemical test strips (bioMérieux, France). Sorbitol fermentation characteristic was examined using sorbitol-MacConkey agar (SM-AC) (Oxoid, UK). The number in red brackets stands for the quantity of E. coli strains obtained.

Pathogenic gene detection
All isolates were tested for the presence of pathogenic genes (eae-EPEC, stx-STEC, est-ETEC, elt-ETEC, ipaH-EIEC, aggR-EAEC) by multiplex PCR assay according to method described earlier [2]. DNA was prepared from overnight grown culture by boiling method. The procedure was advisably modified as follow. Multiplex PCR reaction system was carried out with a 25 μl mixture containing Master Mix, DNA template, and six combined primers. The PCR program was denatured at 95°C, annealed at 52°C and extended at 72°C for 35 cycles. PCR products were then electrophoresed on a agarose gel (AmpliSize; Bio-Rad Laboratories) and visualized by UV transillumination. Quality control E. coli strains of EPEC, STEC, ETEC, EIEC, and EAEC were from Chinese Center for Disease Control and Prevention.

Pulsed-field gel electrophoresis
PFGE of the E. coli strains were performed according to non-O157 STEC subtyping protocol (www.pulsenetinternational.org) with few modifications [4]. The bacteria genomic DNA was digested with 50 U of Xba I (Takara, China) at 37°C for 3h. Xba I-digested Salmonella enterica serovar Braenderup H9812 was used as the controlled DNA marker, PFGE was repeated twice to determine reproducibility. For un-typed isolates, 50 μM thiourea (Sigma, USA) was added to the 0.5×TBE buffer prior to PFGE running as described by Römling and Tümmler [14]. A contour-clamped homogenous electric field apparatus CHEF-Mapper (Bio-Rad, USA) was used for chromosome separation. The pulse time was ramped from 2.16 s to 54.17 s over 19 h at 6.0 V/cm, Gel images were captured with a Gel Documentation 2000 software (Bio-Rad, USA) and converted to Tiff files, then analyzed using BioNumerics software (Applied Maths Belgium).

Statistical analysis
Statistical analysis was conducted by using SPSS 17.0. The difference of drug resistance was analyzed by oneway ANOVA test, P < 0.05, significant difference; P > 0.05, no significant difference.

Pathogenic potentials among 248 E. coli isolates
In order to study the prevalence of pathogenic E. coli, we categorized the E. coli isolates into different pathothypes according to the PCR results for pathogenic marker genes: eae, stx, est, elt, ipaH, and aggR. Six (2.42%, 6/248) isolates were identified as pathogenic E. coli which carried est and/or elt gene, and all these pathogenic strains were classified as ETEC. Of the six pathogenic strains, four strains were from Shandong Province and the other two strains from Shanxi Province and Algeria respectively. No EPEC, STEC, EAEC or EIEC strains were detected in this study.

Prevalence of virulence genes
Seven virulence genes were tested to all the E. coli strains by PCR. These virulence gene frequencies among the E. coli strains were exhibited in Table 2. Gene stx2 was only detected in two isolates (2.7%, 2/75) from Shandong Province. Both ompT and iroN virulence gene were detected from isolates of Tibet, however, stx2, iss, fimC, hlyF, and iutA were all free among Tibet isolates, indicating their lower pathogenic characters.
Importantly, nearly all isolates from Algeria carried seven virulence genes. The frequency of six virulence genes, ompT, iss, fimC, iroN, hlyF, and iutA was high in the isolates from Shanxi, Jiangsu Yunnan, and Xinjiang of China (Table 2).   (Table 3).  It was noteworthy that we found nearly all the E. coli isolates, including the thirty strains from Algeria, were MDR (Table 4, Figure 2). These data therefore reminds us that veterinary clinical irregular use of antimicrobial agents is already a very serious problem not only in China, but also in the African countries, even the world.   shows that E. coli is highly similar in Shanxi Province. An UPGMA dendrogram was constructed in Figure 3. "Xizang" in the right "Source" column also named Tibet.

Discussion
Avian origin E.coli is the mainly etiologic agent of avian colibacillosis. It may cause a large number of chicken diseased and sometimes cause extensive mortality in poultry flocks by primary or secondary infection of E.coli, which lead to huge economic losses in some epidemic areas. Ordinarily, E.coli strains isolated from such tissues as spleen, heart or liver are most potentially pathogenic. However, in this study, of all 218 E.coli isolates from different regions of China, we only confirmed five strains (2.29%, 5/218) as ETEC which carried pathogenic marker gene est or elt.
The prevalence of toxin-encoding virulence genes, namely ompT, stx2, iss, fimC, iroN, hlyF, and iutA, was further evaluated ( Table 2). The similar frequency of virulence genes showed in isolates from Algeria and some provinces of China, it may be illustrate that E. coli strains isolated from poultry population of both Algeria and China has similar evolution process. The positive rate for virulence gene of isolates from Shanxi was 100%, followed by Yunnan (92.3%), Xinjiang (77.8%) and Jiangsu (75.0%). Shandong (34.7%), Jilin (33.3%) and Tibet (20.0%) revealed relatively lower virulence gene carried rate. In addition, except two strains (2.7%, 2/75) from Shandong province hosted stx2 gene, none of other 216 strains harbored it. Shiga toxins play a main role in intense inflammatory reaction and may account for the ability of STEC strains to cause serious illness in both affected human and suffered animals. These toxins were expressed by stx genes which constitute two major subfamilies: stx1 and stx2 [15]. Though we obtained stx2 gene in two isolates from Shandong province (  [19]. MDR rate in 2014 also attained 98.86% among the tested 438 strains of avian origin E.coli in our laboratory [17]. Similarly, the E.coli isolates MDR rate of Algeria also came up to 100% (Table 3). These results suggested that veterinary clinical irregular use or abuse of antimicrobial agents has already become an increasingly serious issue not only in China but also in western African countries.
It is without any surprising that E. coli isolates from Tibet was much more sensitive to most antimicrobial agents.
In conclusion, this study provides the first comparable report of properties of poultry origin E. coli in different representative regions of China and western African country, Algeria. Our findings enrich the knowledge of avian origin E. coli strains virulence gene prevalence and characteristics both in China and Algeria. Both the poultry origin E. coli from China and Algeria exhibited either high frequencies of anti-drug resistance or high rates of virulence genes carrying. These findings indicate that clinical irregular use or abuse of antimicrobials has already been a public concern not only in China but also in western African countries. However, much more isolates should be investigated in western China and western African countries so as to further comprehensive evaluate the prevalence properties of clinical E. coli isolates.
2. The frequency of 7 virulence genes was high in the isolates from Algeria and the other six provinces of China except Tibet.