MOLECULAR DETECTION OF SHIGA TOXIN ( stx1 and stx2 ) AND INTIMIN ( eaeA ) GENES IN ESCHERICHIA COLI ISOLATED FROM FECAL SAMPLES OF CATTLE, SHEEP, AND HUMAN IN BASRAH GOVERNORATE

The present study aims to isolate and identify Escherichia coli from fecal samples of farm animals and human, also, it aims to molecular detection of shigatoxin and intimin genes in isolates. A total of (264) fecal samples and swabs were collected from different parts of Basrah in the period extending from September 2018 to January 2019. These samples were composed of (85) samples from cows, (94) samples from humanand (85) samples from sheep. Different techniques were used in this study to detect the presence of E. coli ; these techniques included conventional microbiological assays and molecular techniques (amplification of uidA gene by using polymerase chain reaction). The results of these techniques indicated 50 (18.9%) were E. coli from the tested samples. These isolates were subjected to PCR to detect Shiga toxins and intimin genes (stx 1 , stx2 , and eaeA ). The results of PCR confirmed all (50) isolates were harbor at least one virulence gene. Out of 50 isolates 20 (40%) carried stx2 gene alone, the percentages of the carrier were (66.7 %, 41.7% and 23.5%) from human, sheep and cattle samples, respectively. The genes (stx 1 and stx2) were detected together in 9/50 (18%), represent (52.9%) of cattle isolates. The intimin gene ( eaeA ) alone was detected in 2/50 (4%),


INTRODUCTION
Escherichia coli is a Gram-negative, facultative anaerobe, non-sporulating rod within the family Enterobacteriaceae. It can ferment different sugars, but lactose fermentation (with the production of acid and gas) is a characteristic of the species (1).
Shiga toxin-producing E. coli (STEC) appears to be widespread in the gastrointestinal tracts of wild and domestic animals and, not surprisingly, meat and other animal products are the significant sources of human infections (2). STEC has emerged as a group of foodborne pathogens that can cause severe human disease, such as hemolytic uremic syndrome (HUS) (3).
The STEC strains are characterized by their ability to produce either one or both of these cytotoxins, referred to as Stx1 [first described as Shiga-like toxin I], Stx2 or [first described as Shiga-like toxin II] (4).
The STEC strains were isolated from a variety of animals. However, cattle are considered the main reservoir (5). Other studies have indicated that small domestic ruminants, including sheep and goats, are also key reservoirs of STEC (6 and 7). Moreover (8), stated that, the STEC remains a significant cause of foodborne-related disease in humans.
This study aimed to isolate and identify Escherichia coli form fecal samples and swabs from Humans and domestic animals (cattle and sheep) also, molecular detection of E. coli isolates that carrying gene (stx1, stx2, and eaeA).

MATERIALS AND METHODS -Samples collection
The fecal samples and swabs were collected from different parts of Basrah province. A total of 264 fecal samples and swabs were collected from Cattle, Sheep, and Human in the period extending from September 2018 to January 2019. Table (1)

Isolation and identification of bacteria
The samples were treated according to (9). Briefly, all the collected samples were transported immediately to the laboratory of Veterinary college by using an icebox. In the laboratory, the samples were inoculated in nutrient broth and incubate at 37°C for overnight. The preincubated samples were subcultured on MacConkey's agar (Micromedia / Iran) and again incubated at 37° C for overnight. Next day 2-3 rose pink colonies randomly picked and were subcultured on to EMB agar (Himedia / India) followed by overnight incubation at 37° C. The colonies which observed as a metallic sheen, single colony were subject to Gram's stain (10), oxidase test; indole test and methyl red ( M.R) and voges -proskauer (V.P) Tests (11), and Citrate utilization test using Simmons citrate agar (12).

Molecular techniques
The suspected isolates of E. coli by using conventional microbiological techniques were submitted to conformation by amplification of uidA gene by using PCR technique. Bacterial DNA was extracted according to the manufacturer of bacterial extraction kit (Genaid, Korea).

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Oligonucleotide primers for PCR amplification: Primers used for detection of virulence genes (stx1,stx2, and eae A) in E. coli isolated from fecal samples of farm animals and human were adopted from (14), Table (4).

The Amplification conditions
Amplification conditions of the genes (stx1 and stx2) were optimized and illustrated in the table (5). However, the optimized amplification conditions of eae A gene listed in the table (6).

RESULTS
The total number of collected samples was 264; the samples included fecal samples and swabs from farm animals (cows, sheep) and human. The isolation rate of E. coli identified by using conventional microbiological and molecular techniques, as shows in Table (   The mixture was run on1.5% agarose gel, stained with ethidium bromide. M: marker, and ds. uidA product size (203 bp).
Note: The sample in well No. 1 was negative control, and (2-11) were positive samples.

Molecular detection of stx1, stx2 and eaeA genes
All isolates of E. coli were submitted to molecular detection of genes (stx1, stx2, and eaeA) by using primers. Table (

Isolation rates of E. coli
The conventional microbiological methods used for E. coli detection relies on the enrichment of the sample on nutrient broth, then differentiating on MacConkey agar followed by culturing on selective media Eosin methylene blue and submitting to biochemical confirmation and molecular method according to (9).
The net isolation rate E. coli from total number of samples was (18.9%). The isolation rate of E.
coli of human samples was 9.6 %. This result was lower than that, reported by (`17), who found E. coli in (21.4%) of stool culture. The isolation rate of E. coli from cattle in this study was (20%), this rate was higher than (10.9%) which reported by (18) in Basrah province. On the other hand, the isolation rate of E. coli from sheep was (28.2%).

Antimicrobial susceptibility
Antimicrobial agents have primarily been used to cure infectious diseases caused by bacteria.
The use of antibiotics is a significant risk factor for extension of resistance to these agents (19).
Multidrug resistance in Escherichia coli has become a worrying issue that is increasingly observed in human but also in veterinary medicine worldwide (20).
By using the disc diffusion method, 50 isolates of Escherichia coli were submitted to antimicrobial susceptibility test toward 7 antimicrobial agents. The results of the current study clarified that E. coli isolate resistant to Ampicillin (92%), and 100% susceptible to imipenem.
Table (8), these results are in agreement with (21) who found that the resistance to ampicillin was 100% and all of E. coli isolates were susceptible to imipenem. The reported susceptibility to gentamycin in this study was 92 %; this result is higher than 66.9% recording by (22). From the obtained result, the rate of resistance to tetracycline was 74%; this result is lower than 84.76% reported by (23) and the rate of resistance for chloramphenicol in this study was 22 %, this result is lower than 33.3%, found by (24). Furthermore, in this study, the resistance against cefotaxime and ciprofloxacin were found (36%, 22%) respectively. These results are similar to (22), who found that the resistance against Cefotaxime and ciprofloxacin were (36.6% and 18.3%), respectively.
The slight differences in resistance may result from the source of isolates; also, the reason behind continuous increasing in resistance to these antibiotics may attribute to over usage of these antibiotics. Consuming antibiotics without prescription has been assumed as one of the causes of reducing bacterial sensitivity to the antibiotics (25), moreover, in Iraq, antibiotics can be easily obtained without a prescription. All of the previous reasons might be responsible for such a high prevalence of resistance.

Molecular detection of stx1, stx2, and eaeA genes
Shiga toxin-producing Escherichia coli (STEC) are globally known pathogens that cause diarrhea, hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC) in humans. STEC are predominately shed within the feces of healthy meat-producing animal species and aren't considered to be pathogens of ruminant species except once infections occur in young (preweaned) animals (26).
For 50 E. coli, isolates were submitted to PCR targeting the characteristics of virulence genes (stx1, stx2, and eaeA). All isolates were potentially pathogen hence harbor at least one specific virulence trait. This study revealed that the detection rate of stx2 gene in cattle was 23.5%. This result is lower than 35% who found by (27), while the percentage of stx2 gene in human was 66.7 %, this result was higher than 24% reported by (28). Moreover, the stx2 ratio in sheep was 41.7%; this result was much higher than 14% found by (29), Table ( is 100 times more potent than Stx1 (32).
Regarding eaeA gene, it was detected in 4 % of isolates. This result is in agreement with (33), who reported that eae A gene was 4.1% isolates. Moreover, the distribution of the stx2 and eaeA genes of human in this study was 33.3 %; this result was higher than 14.3% reported by (34).
While in sheep, the distribution of the stx2 and eaeA genes was 45.8 %, this result disagreed with (35) who found 8.7% of fecal samples were positive for both genes.
In the present study, the presence of the three genes (stx1, stx2, and eaeA) in both cattle and sheep was similar in percentage 11.8%,12.5 %, respectively, this result was comparable to (36), who found the presence of three genes in cattle and sheep was (6.52% and 11.42), respectively,

Conclusions
All E. coli isolates were potentially pathogen; hence, harboring specific virulence genes. The plurality of isolates were resistant to ampicillin followed by tetracycline, whereas, all the isolates were susceptible to imipenem followed by gentamycin.