Prevalence and molecular detection of shiga toxin producing Escherichia coli from diarrheic cattle

Shiga toxin-producing Escherichia coli (STEC) are zoonotically important pathogen which causes hemorrhagic colitis, diarrhea, and hemolytic uremic syndrome in animals and humans. The present study was designed to isolate and identify the STEC from fecal samples of diarrheic cattle. A total of 35 diarrheic fecal samples were collected from Bangladesh Agricultural University (BAU) Veterinary Teaching Hospital. The samples were primarily examined for the detection of E. coli by cultural, morphological and biochemical characteristics, followed by confirmation of the isolates by Polymerase Chain Reaction (PCR) using gene specific primers. Later, the STEC were identified among the isolated E. coli through detection of Stx-1 and Stx-2 genes using duplex PCR. Out of 35 samples, 25 (71.43%) isolates were confirmed to be associated with E. coli, of which only 7 (28%) isolates were shiga toxin producers, and all of them were positive for Stx-1. However, no Stx-2 positive isolate could be detected. From this study, it may be concluded that cattle can act as a reservoir of STEC which may transmit to human or other animals.


Introduction
Escherichia coli (E.coli) is a Gram-negative, rod-shaped, flagellated, motile, oxidase negative, facultative anerobic organism, and is classified under the family Enterobacterioceae (Riley et al., 1983).E. coli produces septicemia and diarrhea in cattle and other animals such as piglets, kids, foals and lambs.Cats and dogs are susceptible to cystitis and other urogenital infections caused by E. coli (Riley et al., 1983).Pathogenic E. coli are mainly divided into two types namely Enteropathogenic E. coli and uropathogenic E. coli.Further, pathogenic E. coli are grouped into enterotoxic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC ) and enterohaemorrhagic E. coli (EHEC) (Riley et al., 1983).E. coli O157:H7 is an enterohemorrhagic strain of the bacterium E. coli and a cause of illness through food.While most strains are harmless and normally found in the intestines of mammals, this strain may produce shiga-like toxins which is a member of a class of pathogenic E. coli known as EHEC.Often they are referred to depending on their toxin producing capabilities, known as verocytotoxin producing E. coli (VTEC) or shiga-like toxin producing E. coli (STEC).The etiologic agent of the illness was identified as a rare O157:H7 serotype of E. coli in 1983.This serotype had only been isolated once before, from a sick patient in 1975 (Riley et al., 1983).
STEC is an important group of E. coli that can cause severe diarrhea and food-borne illness worldwide.The serotype of a STEC is based on the 'O' antigen determined by the polysaccharide portion of cell wall lipopolysaccharide and the 'H' antigen by the flagella protein.STEC can grow in temperatures ranging from 7°C to 50°C, with an optimum temperature of 37°C (Rehman et al., 2014).Healthy dairy and beef cattle are a major reservoir of STEC (Rehman et al., 2014).Contamination of meat with the bacterium can occur during slaughtering.Ground beef possesses higher risk than intact meat because it can be contaminated during the grinding process (Rehman et al., 2014).Besides, food and water can be contaminated by cattle manure.In many countries, STEC was isolated from diarrheic cattle as one of the major causal agents of diarrhea.In Bangladesh, the incidence of diarrheal diseases in calf is high.However, so far no report has been observed on the detection or isolation of STEC from diarrheic cattle in Bangladesh.The prevalence of E. coli infection was higher in diarrheic calves (13.71%) as compared to that of non-diarrheic calves (9.1%), as reported by Nazir et al. (2005).Considering the above points, the present study was conducted to isolate and detect shiga toxin producing E. coli from diarrheic cattle.

Materials and Methods
Collection of samples A total of 35 fecal samples were collected from diarrheic cattle that were presented to the Bangladesh Agricultural University (BAU) Veterinary Teaching Hospital, Mymensingh during the period from January 2015 to March 2015.After collection, each sample was inoculated into nutrient broth (NB) and incubated at 37 0 C overnight.

Isolation of E. coli in culture media
After primary culture of the organism, a 10 fold dilution was made to reduce overgrowth of the organisms.Then 100 µL was inoculated onto Mac-Conkey agar.Single well defined pink colony was further subcultured onto the Eosin Methylene Blue (EMB) agar media.

Gram staining
The E. coli colonies were characterized morphologically using Gram stain according to the method described by Nazir et al. (2005).

Identification by biochemical test
The sugar fermentation test was performed by inoculating a loop full of NB culture of the organisms into each tube containing five basic sugars (e.g., dextrose, sucrose, lactose, maltose, and mannitol) separately and incubated for 24 h at 37 0 C. Indole test, MR-VP test were performed accordingly to the method described by Nazir et al. (2005).

DNA extraction by boiling method
A pure bacterial colony was mixed with 200 uL of distilled water which were boiled for 10 min, then immediately kept on ice for cold shock followed by centrifugation at 10,000 rpm for 10 min.The supernatant was collected and used as DNA template in PCR.

PCR Assay
A total volume of 25 µL of PCR reaction mixture was prepared; the mixture contained PCR master mixture (Promega, USA): 12.5 µL, Forward primer (10 pmol/µL): 1 µL, Reverse primer (10 pmol/µL): 1 µL, DNA template: 5 µL, Nucleus free water: up to 25 µL.Thermal profiles for the amplification of 16SrRNA gene were 95 Agarose gel electrophoresis of PCR products PCR products were analyzed by 1.5% agarose gel electrophoresis.In brief, gel containing tray was assembled with gel comb of appropriate teeth size and number.1.5% Agarose gel was prepared in TAE buffer by boiling in a microwave over followed by solidification and preparing gel cast.The PCR product of each sample was electrophoresed at 100 V for 45 min and stained with ethidium bromide (EtBr) for 10 min.After that the PCR products were analyzed by trans-illuminator (biometra, Germany) and photograph was taken.

Results and Discussion
Cultural properties of E. coli on Mac-Conkey and EMB agar E. coli was detected in 25 out of 35 samples.They were gram-negative, pink colored, small rod shaped, single or paired organisms but produce bright pink colonies on Mac-Conkey agar and greenish black colonies with metallic sheen on EMB agar.The media used in this study were selected considering the experience of the past researchers worked in various fields relevant to the present study (Nazir et al., 2005;Nazir, 2007).Colony growth character on different media exhibited characteristics reaction.In this study, colony characteristics of E. coli observed on EMB and MC agars were similar to the findings of other authors (Rehman et al., 2014;Hassan et al., 2014;Mamun et al., 2016;Tanzin et al., 2016;Elafify et al., 2016;Islam et al., 2016).

Identification of E. coli by Gram staining
The microscopic examination of isolated bacteria with Gram stained smears from Mac-Conkey and EMB agar revealed that the isolated bacteria were Gram negative, pink colored, small rod shaped organisms arranged in single, pairs or short chain.These findings were in support of the findings of Hassan et al. (2014), Tanzin et al. (2016), Elafify et al. (2016) and Islam et al. (2016).

Identification of E. coli by Biochemical tests
In fermentation test, all the isolates fermented the five basic sugars (dextrose, sucrose, lactose, maltose and manitol) and sorbitol producing both acid and gas.Acid production was indicated by the color change from reddish to yellow and the gas production was noted by the appearance of gas bubbles in the inverted Durham's tubes.This result was also reported by Nazir et al. (2005) and Rehman et al. (2014).Fecal isolates revealed a complete fermentation of basic sugars as stated by Beutin et al. (1993), Sandhu et al. (1996) and Mckec et al. (1995).E. coli isolates were able to ferment the five basic sugars producing both acid and gas; however, differentiation of E. coli into species level was difficult as showed similar reaction in various sugars (Elafify et al., 2016).All the isolates fermented dextrose, sucrose, fructose, maltose and mannitol with the production of acid and gas within 24 h of incubation.Results of E. coli isolates were positive as reported by Nazir et al. (2005).Each isolate produced a dark red color in the reagent and bright red colony after incubation of methyl-red (MR) broth positive and no color was produced in Voges-Proskauer (VP) broth, indole positive.This indicated the isolates were E. coli.The isolates also revealed positive reaction in MR test and Indole test but negative reaction in VP test, which was supported by several authors (Hassan et al., 2014;Mamun et al., 2016;Tanzin et al., 2016;Elafify et al., 2016;Islam et al., 2016).

Confirmation of E. coli by PCR
PCR was performed to detect shiga toxin (Stx-1 and Stx-2) of E. coli.First of all, EC16SrRNA primer was used for the confirmation of E. coli sample.All 25 E. coli isolates were identified as E. coli sample and band of EC16SrRNA (585-bp) was formed in agarose gel.Primers targeting Stx-1 gene of E. coli amplified 606-bp fragments of DNA confirmed the identity of Stx-1 from EC16SrRNA positive E. coli.Finally, among 25 of E. coli isolates, only 7 isolates were found positive for Stx-1 confirmed by amplification of Stx-1 gene and band of Stx-1 (606-bp) was formed in agarose gel, and none of them was EC Stx-2 positive.The result of PCR presented in Fig. 1 (EC16SrRNA) and Fig. 2 (ECStx-1).

Prevalence of shiga-toxin producing E. coli
The prevalence of STEC in the isolated E. coli was 71.43% (n=25/35).Out of 25 E. coli isolates 7 samples were confirmed as STEC due to presence of shiga-toxin producing gene Stx-1, no Stx-2 positive E. coli was detected in this study.In this study, out of 35 samples, 25 were confirmed to be associated with E. coli and out of these 25 isolates, only 7 were shiga-toxin producers, and all of them were positive for Stx-1.No Stx-2 positive isolates was detected in duplex PCR.This result suggested that 28% (p<0.01)isolates were bearingStx-1 gene.From this study, it is concluded that 71.43% (p<0.01)cattle were positive for E. coli and 28% (p<0.01) were positive for STEC.
In present study, the results of PCR showed that 28% (p<0.01)stains carried Stx-1 gene and all the strains were Stx-2 negative, 7 (28%) were positive for Stx-1 genes.Higher frequency of Stx-1 gene (28%) than Stx-2 gene (0%) in diarrheic cattle observed in the present study is contrary to the observations of Dastmalchi et al. (2012) who reported predominance of Stx-2 over Stx-1 in diarrheic calves in Iran.This variation might be due the difference in geographical location, management practices and immune status of the animals.
The prevalence of STEC, including STEC O157, was significantly higher in buffalo feces than in cow or goat feces (p<0.05).Within similar geographical locations, a few studies of the occurrence of STEC have been done, and most of the studies were done in India and Thailand (Leelaporn et al., 2003;Manna et al., 2006).Recently, a study was done in central Vietnam that found a prevalence of STEC of 27% in buffaloes, 23% in cattle, and 38.5% in goats.In India, Manna et al., (2006) reported that the prevalence of STEC O157 in fecal samples from slaughtered cattle and diarrheic calves was 2.0% and 7.6%, respectively (Manna et al., 2006).
STEC O157 has also been isolated in India from foods of cattle origin namely raw minced beef samples (9%; n=22) (Dutta et al., 2000), beef surface swabs (3.7%; n=27), and milk samples (2.4%; n=81) (Manna et al., 2006).In China, STEC O157:H7 was isolated from 10 to 20% of the animals in the villages, including pigs, cattle, goats, and chickens (Zheng et al., 2005;Fernández et al., 2009).When buffalo samples were tested for the presence of Stx1 genes by PCR, around 80% of the samples were positive.Of the cows and goats, around 73% and 12%, respectively, were positive.By observing that prevalence rate of different animals we collected fecal sample from diarrheic cattle for the detection of Stx-1 and Stx-2 genes and only Stx-1 gene was amplified from 7 samples.
It is therefore difficult to determine whether the results reported reflect true differences in isolation rates or are the consequence of the different methodologies adopted.The relative occurrence of STEC virulence factors changed as the calves became older; as a result, Stx-1 positive isolates might be replaced by Stx-2 positive isolates.In the current study, however, Stx-1 sequences were present in a higher number (28%) of isolates from diarrheic cattle.This may be due to differences in age of calves, season of collection, ruminal development, immune response, diet and other aspects of calves' management.

Conclusion
It is concluded that cattle are an important reservoir of virulent E. coli carrying toxigenic gene, which may cause illness in cattle as well as human.
min in final extension and holding temperature was 4 0 C until use.The total cycling was 30 times.Similarly, thermal profile to amplify Stx-1 and Stx-2 genes of E. coli were 95 0 C for 5 min, followed by denaturation at 94 0 C for 30 sec, 56 0 C for 1 min in annealing, 72 0 C for 1 min in extension, and 72 0 C for 10 min for final extension and holding temperature was 4 0 C until use.The total cycling was 30 times.

Table . 1
Prevalence of shiga toxin producing E. coli