​Isolation and Molecular Characterization of Verotoxin Producing E. coli from Diarrhoeic Goats of Jabalpur Region

Escherichia coli (E. coli) are a group or strains of bacteria found as a normal commensal in the intestinal micro flora of healthy animals and humans and excreted in the feces. Not all the strains are harmless and some strains can cause debilitating and sometimes fatal diseases in both animals and humans, therefore playing a significant role in the epidemiology of human infections (Heller and Chigerwe, 2018). The organisms enter into the food chain via faecal contamination of milk, contamination of meat with intestinal contents during slaughter or contaminated feed and water. Infection can also arise from animal to human contact, as well as human-to-human contact by both direct and indirect ways (Yim et al., 2010).

There are six recognized enteric pathotypes of E. coli, enterotoxigenic (ETEC), enteropathogenic (EPEC), diffusely adherent (DAEC), enteroaggregative (EAEC), enteroinvasive and verotoxigenic (VTEC) are also known as shiga toxigenic, STEC or enterohemorrhagic E. coli (Pal and Ayele, 2017).

Domestic ruminants, including goats, carry genes which produce verotoxins or Shiga-like toxins without having Shiga toxin receptors (Globotriaosylceramide). Therefore, they can act as asymptomatic carrier by STEC bacterium in faeces (Ashraf, 2016). The pathogenicity of VTEC is associated with phage-mediated genes VT1 and VT2 (Parul et al., 2016).

Moreover, verotoxigenic E.coli (VTEC) such as O157: H7 are not pathogenic to ruminants, but they cause serious diseases in humans worldwide, including diarrhoea, hemorrhagic colitis, hemolytic-uremic syndrome, thrombocytopenia purpura and sometimes death (Chileshe and Ateba, 2013).

Keeping in view the above facts in present study, E. coli was isolated from rectal swabs of goats and confirmed by a biochemical test, which was further subjected to molecular confirmation of E. coli along with the presence of VT1 gene in E. coli.

A total of 50 rectal swabs samples were collected aseptically from the diarrhoeic goats (irrespective of age, sex and breeds) of organized and unorganized farms in and around Jabalpur (Table 1). All the samples were processed for bacteriological isolation, molecular confirmation of E. coli and presence of the VT1 gene in E. coli.



Each swab was separately inoculated into nutrient broth and incubated at 37°C for 4 hours. A loopful inoculum was streaked on MacConkey agar plate and incubated at 37ºC for 24 hours. Organisms producing pink coloured colonies (lactose positive) on MacConkey agar plate were further streaked on to Eosin Methylene Blue (EMB) agar and incubated at 37°C for 18-24 hours. The colonies showing characteristic metallic sheen on EMB agar were considered as presumptive of E. coli and subjected to Gram’s staining for identification as per the standard procedure (Quinn et al., 2004). E. coli isolates were further confirmed by biochemical tests viz. Indole, Methyl red, Voges-Proskauer, citrate utilization and carbohydrate utilization in HiIMViC biochemical kit (HiMedia KB001).
 
Detection of verotoxin producing E. coli by slide agglutination test
 
E. coli ‘O’ pool antisera comprising of VTEC serogroup O26, O103, O111, O145, O157 was used for slide agglutination with live cultures to detect verotoxin producing E. coli and the positive reaction was recorded when visible agglutination reaction was seen within 5-10 seconds.
 
Molecular characterization of Escherichia coli by genus-specific primer using 16SrRNA gene
 
The extraction of genomic DNA from broth of overnight grown culture of Escherichia coli was carried out as per the method described by Wilson (1987) with a slight alteration. The Purity of DNA was checked and quantified in a Nanodrop spectrophotometer and stored at -20°C for further use. PCR was performed on extracted DNA samples for amplification 16SrRNA gene fragment specific to genus Escherichia coli by using already published E. coli genus-specific primer (Table 2) as described by Shrivastav (2016). A reaction mixture of 25 μl, was prepared consisting of 12.5 μl, mastermix, forward primer 1 μl, reverse primer 1 μl, 3 μl of template DNA, 7.5 μl nuclease free water. The PCR protocol was performed under the following thermal cyclic conditions. The PCR cycling conditions consisted of an initial denaturation at 95°C for 5 minutes followed by 30 denaturation cycles at 94°C for 30 seconds each, annealing for 30 seconds at 69°C and extension for 2 minutes at 72°C. This was followed for 10 minutes by a final extension at 72°C. PCR amplification was verified on 1.5 per cent agarose horizontal gel electrophoresis.


 
Molecular characterization of VT1 gene specific for verotoxin producing E. coli
 
To investigate the virulence potential of the E. coli, isolates were subjected to PCR methodology as described by Jackson et al., (1987) and Strockbine et al. (1988).

Characterized strain of E. coli was used as positive control and was obtained from Department of Veterinary Microbiology, Jabalpur. In the present study, PCR was optimized with primer targeting gene VT1 (Table 3). Following initial optimization trials, reactions mixtures was standardized in 25 μl  volume containing 12.5 μl master mix, forward primer 1 μl, reverse primer 1 μl, 3 μl of template DNA and 7.5 μl nuclease free water. The standardized amplification reaction began with initial denaturation at 95^oC for 5 minutes, followed by 25 cycles each with denaturation at 94^oC for 1 minute, annealing at 54^oC for 1.5 minute and extension at 72^oC for 1 minute, with final extension for 5 minutes at 72^oC. A nucleic acid blank negative control was taken at each screening along with isolates to check PCR quality control conditions. On completion of PCR amplified products were analysed by 1.5 per cent horizontal agarose gel electrophoresis and the amplified product visualized as a single compact fluorescent band of expected size under U-V light. 

Isolation and identification of E. coli organism
 
In the present study, 52.00 per cent (26/50) samples were positive for E. coli. On MacConkey agar, lactose fermenting E. coli showed smooth bright pink coloured colonies (Fig 1) and dark centered blue black colonies with metallic sheen (Fig 2) were observed on EMB agar. The bacterial smear prepared from EMB agar revealed pink coloured medium sized Gram negative rods with Gram’s staining (Fig 3), which were morphologically identified as E. coli.






 
Characterization of Escherichia coli by various biochemical tests
 
All the isolates (26) were found positive for indole and methyl red tests whereas none were positive for citrate utilization and Voges-Proskauer test as shown in Fig 4.


 
Detection of verotoxin producing E. coli
 
Positive isolates of E. coli obtained from EMB agar was further tested for agglutination reaction with known positive E. coli ‘O’ pooled antisera against verotoxin producing E. coli (VTEC) serogroups (O26, O103, O111, O145, O157). Of them, 38.46 percent (10/26) isolates were positive for VTEC showing agglutination reaction in slide agglutination test (Fig 5). Isolation of caprine VTEC serogroups from rectal contents of diarrhoeic goats was also consistent with the findings of previous studies by Osman et al., (2013), Oluyege and Famurewa (2015) and (Tarabees et al., 2016).


 
Molecular detection of E. coli by genus-specific primer using 16SrRNA gene PCR
 
All positive E. coli isolates were used for molecular detection by targeting the 16SrRNA gene of E. coli. Among them, 21 isolates were found to be positive for E. coli by PCR (amplicon size 1476 bp) (Fig 6). Similar study was done by Begum et al., (2016) who confirmed E. coli by genus specific PCR targeting 16SrRNA gene of E. coli (amplicon size 585 bp). Molecular characterization of E. coli through genus specific PCR targeting 16SrRNA gene of 232 bp were also done by Kumar et al., (2019). Sabat et al., (2000) conducted genus specific PCR for E. coli isolates targeting 16SrRNA gene of 544 bp. Nguyen et al., (2016) confirmed the E. coli isolates by genus specific PCR targeting 16SrRNA gene of 654 bp.


 
Detection of virulence gene VT1 specific for verotoxin producing E. coli by PCR
 
A total of 10 VTEC isolates were used for the detection of VT1 virulence gene by amplifying product of size 256 bp for VT1 gene  in 10.00 per cent (01/10) isolates (Fig 7). The findings are more or less similar with the earlier reports (Oluyege and Famurewa, 2015) and 11.11 per cent per cent from India (Neher et al., 2016). The present findings suggest that, isolates of E. coli which were found negative by PCR may contain another virulence gene except VT1 (VT2, eae and hlyA).



Similarly, higher prevalence of VT1 gene in VTEC isolates was reported in various countries as 69.80 per cent from Spain (Cortes et al., 2005), 63.70 per cent from Spain (Orden et al., 2008), 67.40 per cent from USA (Jacob et al., 2013), 27.50 per cent from Southeast of Iran (Tagadoshi et al., 2018) and 22.20 per cent from turkey (Khider and Buyukcanga, 2018).      

The wide variations in incidence of VT1 gene in goats could be attributed to epidemiological determinants such as herd size, age, sex and season, whereas sampling strategy, sample handling and laboratory methods might also have profound effect on prevalence values reported in different countries.

PCR determined VT1 gene in 08.33 per cent of goats positive for verotoxin producing E. coli indicating the goats as a source for direct transmission of VTEC to humans.