Asian Journal of Dairy and Food Research, volume 42 issue 2 (june 2023) : 247-254

Enumeration, Serotypes and Virulence Genes Associated with Shigatoxigenic (STEC) and Enterotoxigenic (ETEC) Escherichia coli from Beef and Chicken of Mizoram, India

M. Debbarma1, D. Deka1,*, P. Roychoudhury2
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
2Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
Cite article:- Debbarma M., Deka D., Roychoudhury P. (2023). Enumeration, Serotypes and Virulence Genes Associated with Shigatoxigenic (STEC) and Enterotoxigenic (ETEC) Escherichia coli from Beef and Chicken of Mizoram, India . Asian Journal of Dairy and Food Research. 42(2): 247-254. doi: 10.18805/ajdfr.DR-1950.
Background: E. coli is one of the most important zoonotic pathogen and also an indicator of faecal contamination of food and water. There is paucity of data regarding the level of contamination of raw meat and characterization of pathogenic E. coli from slaughtered cattle and chicken in un-organized sector from Mizoram, India.

Methods: Raw meat samples from traditionally slaughtered cattle and chicken were collected from Aizawl, Kolasib and Champhai districts of Mizoram and analyzed for E. coli count (ECC), serotypes and virulence genes of STEC and ETEC.

Result: A proportion of 65.55 per cent beef and 58.89 per cent chicken had unacceptable level of ECC. The most predominant serotypes of E. coli were O118 (13.33%) in beef and O8 (13.89%) in chicken. Both STEC (8.00% and 6.94%) and ETEC (12.00% and 26.38%) pathotypes were detected in beef and chicken, respectively. Detection of serotype O121 in chicken and O26 and O111 in both beef and chicken along with virulence genes of STEC indicated the contamination of raw meat with highly pathogenic STEC strains.
Food-borne infections and zoonoses are major causes of illness and death worldwide with subsequent economic losses and thus constitute one of the threat multiplier for food safety and security costing billions of dollars in morbidity and mortality. Diarrhoeal diseases are the most common illnesses resulting from the consumption of contaminated food, causing 550 million people to fall ill and 230 000 deaths every year (Brooks et al., 2021).

Amongst the approximately 200 different types of food borne diseases, bacterial contamination is the most common cause; some are even capable of producing heat-resistant toxins, accounting for more than 70 per cent of deaths associated with food borne transmission (Thomas, 2017). India is prone to food borne problems because of climatic conditions, food habits, poverty, inadequate basic hygienic and sanitary facilities and low public awareness of food safety and lack of a risk-based food control system to reduce food borne diseases in conventional food production settings (World Health Organization, 2020). Food can be contaminated at any point of production and distribution and the primary responsibility lies with food producers and handlers. 

A large proportion of the world’s population relies on meat as a source of protein (Bradeeba and Sivakumaar, 2013). North-East region of India is inhabited by different ethnic tribes, where majority of the populations consume meat as an important component of their diet. There is ever increasing demand of meat in Mizoram. Beef and chicken are predominantly consumed meat besides pork which is produced in unorganized sector and usually sold in the retail markets. The meat available at retail outlets comes through a long chain of slaughtering and transportation where each step may pose a risk of microbial contamination.

Escherichia coli is a member of coliform group of bacteria and its presence in food is indicative of faecal contamination of food and water, poor hygienic conditions or existence of enteric pathogens and E. coli itself is the most important zoonotic food borne pathogen (Gozde and Emec, 2019). Some of the E. coli strains are highly pathogenic in human and animals and people with low immunity (Akbar et al., 2014).

The unhygienic production, processing and selling environment of meat bear high chances of bacterial contamination of meat. Rapid and simultaneous detection of food-borne pathogens is a key step towards ensuring food safety and can be easily facilitated by PCR (Radji et al., 2010). The present paper describes the enumeration of E. coli, its serotypes and STEC and ETEC pathotypes in raw meat from conventionally slaughtered cattle and chicken of Mizoram, India.
The present study was carried out in the Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and Animal Husbandry, Aizawl, Mizoram during 2019-20.

A total of 180 raw meat samples, 90 each of beef and chicken, were collected randomly from conventionally slaughtered cattle and chicken on weekly Saturday market days from three districts, Aizawl, Kolasib and Champhai, of Mizoram state of India. The E. coli count (ECC) was determined on Hicrome E. coli agar using spread plate technique and the number of colonies in appropriate dilution was recorded as the total number of bacteria (cfu/ g) = Average number E. coli colony count on the plates x 10 x dilution factor. The E. coli isolates were presumptively identified based on biochemical characteristics (Quinn et al., 2004).

All the presumptive E. coli isolates were serotyped based on their somatic (O) antigen at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh (India) and were also screened for STEC and ETEC pathotypes by PCR assay by detection of virulence genes of STEC (stx1, stx2, eaeA and hlyA) and ETEC (LTA and ST1) as described by Paton and Paton (1998) and Phipps et al., (1995), respectively (Table 1 and 2). The bacterial DNA lysate was prepared by boiling and snap chilling method.

Table 1: Oligonucleotide primers used for determination of virulence genes of E. coli.

Table 2: PCR thermal cycling conditions for virulence genes of STEC (stx1, stx2, eae A and hly A) and ETEC (LT1 and STA).

Escherichia coli count in retail meat
A proportion of 65.56 per cent beef and 58.88 per cent chicken samples exceeded the maximum permissible limit of ECC as per food safety and standards regulations (FSSAI, 2010) with the mean value of 3.07±0.2 log10 (cfu/g) and 2.98 ±0.03 log10 (cfu/g), respectively indicating high microbial contamination of raw meat. The finding was in close agreement with Ahmed et al., (2013) (2.94 log10 cfu/g) from Lahore, Pakistan. Heetun et al., (2015) and Huges et al., (2015) reported 5.1 log10 cfu/g and 4.80 log10 cfu/g E. coli from beef in Mauritus and Accra region, Ghana, respectively.
Prevalence of E. coli in retail meat
Overall 83.33 per cent and 80.00 per cent prevalence of E. coli strains in retail beef and, respectively were detected by 16S-r RNA gene analysis from 3 districts of Mizoram. The prevalence of E. coli was significantly (P£0.01) higher in chicken from Aizawl and Kolasib than Champhai district (Table 3). Saikia and Joshi (2012), Ahmed et al., (2013) and Ramya et al., (2015) also reported high prevalence of E. coli with 98, 54 and 76 per cent in chicken meat from retail market of North- East region (India), Lahore (Pakistan) and Bangalore (India), respectively. It indicated the contamination of retail beef and chicken might be due to unhygienic handling of raw meats during slaughtering, butchering and processing, improper storage, long exposure in the market environment and contamination from the handlers itself.

Table 3: Prevalence of E. coli in retail meats from 3 districts of Mizoram.

Serotypes of E. coli strains
The distribution of serotypes in 75 E. coli strains of beef showed that O118 (13.33%) was the most pre dominant serotype whereas the serotype O8 (13.89%) was most common in 72 E. coli strains from chicken followed by other serotypes (Fig 1 and 2). The serotypes O118 and O8 are frequently isolated from the potentially pathogenic STEC and ETEC pathotypes. The sero-groups O26, O45, O103, O111, O121 and O145 are the most commonly found non-O157 STEC strains (Gonzalez and Cerqueira, 2019). Detection of O118, O26, O111, O121 and O128 indicates the possible chance of transmitting the zoonotic STEC from retail meat to human. The most common ETEC serotypes associated with diarrhoea are O6, O8, O25, O78, O148 and O153 (Croxen et al., 2013). Rathore et al., (2010) reported O8 and O9 as the most frequent serotypes from raw meat and meat products collected from Uttar Pradesh, India. Hazarika et al., (2007) had reported O145 as the predominant serotype in raw beef from retail shops of Assam, India. Jana and Mondal (2013) reported O120 as the most predominant serotype of E. coli isolated from raw poultry meat in West Bengal. E. coli serotypes O2, O20, O22 and O102 were reported from chicken meat in Mumbai (Zende et al., 2013). Pratik et al., (2020) reported the serotypes O8, O35, O83, O88, O119 and O149 in chicken from Gujarat, India.

Fig 1: Distribution of E. coli serogroups in beef.

Fig 2: Distribution of E. coli serogroups in chicken meat.

Detection of STEC genes
The prevalence of STEC in beef and chicken was recorded as 8.00% and 6.94 per cent, respectively with over all prevalence of 7.48 per cent (Fig 3). The prevalence of STEC in beef was higher than that of chicken meat which might be due to the fact that cattle are the most important reservoir of STEC contaminating environment, animal origin food and vegetables besides sheep and goat (Gonzalez and Cerqueira, 2019). Rashid et al., (2013) and Rasheed et al., (2014) had reported 25 per cent and 10.70 per cent of STEC from raw beef in Hyderabad and Jammu, India, respectively. Dutta et al., (2011) recorded 14.00 per cent STEC in chicken faeces from Mizoram, India.

Fig 3: Distribution of STEC in beef and chicken meat from 3 districts of Mizoram.

The prevalence of stx2 (6.67% and 4.16%) gene in E. coli strains was higher than the stx1 (1.33% and 2.78%) in both beef and chicken, respectively (Fig 4, 5 and 6). However, 2.04 per cent E. coli strains from beef and chicken possessed both stx1 and stx2 genes. Hazarika et al., (2007), Aradhye et al., (2014) and Mohammed et al., (2014) also reported higher prevalence of stx2 gene than stx1 from beef in Assam, Mumbai and Pune, India, respectively. Zende et al., (2013) had reported 27.27 per cent stx2 gene with 4.54 per cent prevalence of STEC in chicken from Mumbai but Saikia and Joshi (2012), Kiranmayi et al., (2011) and Rasheed et al., (2014) recorded higher prevalence of stx1 gene than stx2 in E. coli strains from chicken of North-East region, Hyderabad and Jammu region, India, respectively. Shiga toxin producing stx2 gene is considered to be the most important virulence factor associated with E. coli infection in human. The predominance of stx2 gene either alone or in combination with stx1 has been found to be highly associated with HUS. The presence of STEC genes in E. coli in retail meat indicated the possibility of transmission of such organisms to human beings through food chain.

Fig 4: Distribution of stx1, stx2 of STEC and associated eaeA and hlyA genes in E. coli isolates from beef and chicken meat.

Fig 5: PCR amplification products of stx1 (180 bp) and stx2 (255 bp) genes of E. coli. (L1: 100 bp DNA ladder, L2: Positive control of stx1 and stx2, L3: No template control, L 4, 5: Positive isolate for stx2, L6: Positive isolate for stx1 and stx2).

Fig 6: PCR amplification products of eaeA (384 bp) and hlyA (534 bp) genes of E. coli. (L1: 100 bp DNA ladder, L2: Positive control of eaeA and hlyA, L3: No template control, L4 and 7: Positive isolates for eaeA, L5: Positive isolate for hlyA, L6 and 8: Positive isolates for both eaeA and hlyA).

Higher prevalence of eae A gene in E. coli strains was recorded in beef (14.66%) than chicken (9.72%). Saikia and Joshi (2012) reported much higher prevalence (50%) of eae A gene in E. coli strains from retail chicken from North-East region, India. The eae A gene is an important factor for attachment and effacing lesion in human intestinal epithelial cells. Although eae A gene simultaneously occurs with stx gene, it is not essential required for STEC pathogenicity (Gonzalez and Cerqueira, 2019) and eae A positive STEC  occurs in both diarrhoeic and non-diarrheic  animals (Coura et al., 2017) reinforcing that only some strains are able to cause disease. The occurrence of hly A gene was low in beef E. coli strains, 2.36 per cent singly and 2.04 per cent in combination of eaeA and hlyA and absent in chicken E. coli strains. Kiranmayi et al., (2011) and Rasheed et al., (2014) observed 10.50 per cent and 2 per cent hlyA gene in E. coli from retail meat of Hyderabad and Jammu region, respectively.

One beef E. coli strain was found to carry both the stx2 and hlyA genes which belonged to the serotype O111. A virulent E. coli strains harboring both stx and hlyA genes may pose a higher risk to human health (Manna et al., 2006). Serotype O26 and O111, one each from beef and chicken harbouring stx2 gene has been frequently encountered with HC and HUS in human.
Detection of ETEC genes
The prevalence of ETEC strains was higher in chicken (26.38%) than beef (12.00%) with highest occurrence from Champhai district (31.57%) (Fig 7 and 8). The prevalence of ST1 and LTA genes in chicken (15. 27% and 11.11%) was higher than beef (6.67% and 5.33%), respectively (Fig 9). Although ETEC infections are well studied in human, there is paucity of information on the occurrence of ETEC in meat from India. Zende et al., (2013) observed 18.28 per cent ST1 gene and absence of LTA gene in E. coli from chicken in Mumbai. Yadav et al., (2007) detected 6.67 per cent ST1 and 26.67 per cent LTA gene in E. coli strains from mutton in Madhya Pradesh. Few reports are also available on detection of ETEC in mithun (Rajkhowa et al., 2009) and diarrhoeic lambs (Bandyopadhyay et al., 2011) from North East India. Similarly, Mahanti et al., (2014) detected ETEC with ST1 gene from healthy water buffalo in West Bengal.

Fig 7: Distribution of ETEC in E. coli isolates of beef and chicken meat from 3 districts of Mizoram.

Fig 8: Distribution of ST1 and LTA virulence genes in E. coli isolates from beef and chicken.

Fig 9: PCR amplification products of ST1(190 bp) and LTA(450 bp) genes of E. coli. (L1: 100 bp DNA ladder, L2: Positive control of ST1 and LTA, L3: No template control, L4: Positive isolate for ST1, L5: Positive isolate for LTA, L6: Positive isolate for both ST1 and LTA).

The prevalence of pathogenic E. coli in meat may vary in different geographical locations. However, the paucity of studies makes it difficult to evaluate the actual role of retail meat as a STEC and ETEC vehicle in Mizoram. Detection of these two potentially pathogenic E. coli pathotypes in retailed beef and chicken bears public health significance owing to the fact that contaminated meat might act as vehicle for transferring pathogenic E. coli to human. Further, detection of potentially pathogenic E. coli serotype O26 and O111 harbouring stx2 in retail meat of Mizoram are common serotypes associated with HC and HUS in human.

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