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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Evaluation of Biofilm Production Capability of Shiga Toxin Producing Escherichia coli Isolated from Milk, Milk Products and Hand Swabs 

R
Ravi P. Prajapati1
P
Parul1,*
U
Udit Jain1
B
Barkha Sharma2
R
Raghavendra P. Mishra2
S
Satyendra P. Singh3
G
Gurvinder1
V
Vikram Jeet1
K
Kushaan Seth1
1Department of Veterinary Public Health, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.
2Department of Veterinary Epidemiology, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.
3Department of Animal Genetics and Breeding, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.

ABSTRACT

Background: Food borne pathogens are transmitted to humans through contaminated foods like dairy products, handmade goods and meat. Microbial biofilms are city of microbes in the form of 3D structure and attachment of bacteria with food contact surfaces lead to subsequent development of biofilms that may lead to transmission of disease and food spoilage. This study was designed to evaluate the biofilm production capability of Shiga toxin-producing Escherichia coli (STEC) isolated from milk, milk-based products and hand swabs.

Methods: Samples were collected and processed for isolation of E. coli. STEC were detected on the molecular basis through mPCR. Biofilm formation capability of STEC was screened by methods namely Tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) assay. 

Result: The prevalence of E. coli and STEC was found to be 22.50%, 30.55%, 12.5% and 6.25%, 4.86%, 2.08% in retail raw milk, traditional milk products and hand swabs, respectively. The overall prevalence of E. coli, serotype E. coli O157:H7 and STEC was 24.43%, 1.42% and 5.11%, respectively. The biofilm forming STEC revealed by TCP, TM and CRA were 88.88%, 77.77% and 38.88%, respectively.

INTRODUCTION

Food borne pathogens like Escherichia coli, Shiga Toxin producing Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Listeria monocytogenes can be transmit to people by consumption of contaminated food products of animal and plant origin (Oriekhoe et al., 2024; Rajan et al., 2024). According to the WHO’s report (2015) on the worldwide impact of foodborne illness, in 2010, more than 1.2 million cases of foodborne illness were caused by STEC, resulting in 128 fatalities and almost 13,000 Disability Adjusted Life Years (DALYs) (WHO, 2015).
       
Shiga toxin producing Escherichia coli (STEC) is a subtype of E. coli that causes enteric and systemic diseases in human ranging from diarrhoea to severe haemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and Thrombocytopenic purpura (TPP) (Debbarma et al., 2024; Rosario et al., 2021). Cattle, particularly young ones, have been identified as a primary reservoir of STEC (Abebe et al., 2020). The main sources of STEC-associated foodborne illnesses are milk, meat and its products contaminated with either ruminant feces or other environmental sources (Durairajan et al., 2021; Parul et al., 2021).
       
Bioflm forming food borne pathogens are big issues for food safety and significant challenge in food chains, particularly in less developed countries (Shi et al., 2024). Biofilms are groups of microorganisms in complex extracellular polymer matrix in which cells bind tightly together on various surfaces. Biofilms are extremely resistant to the available antibiotics and disinfectant and is continuous contamination of food due to the detachment of cells from biofilm matrix (Carrascosa et al., 2021). The “top 6” non-O157 serogroups (O26, O45, O103, O111, O121 and O145), along with other STEC serogroups were revealed with biofilm formation attribute and which are frequently implicated with food borne infection (Parul et al., 2023). Thus, the aim of study is evaluation of the biofilm production capability of Shiga toxin-producing Escherichia coli (STEC) isolated from milk, milk-based products and hand swabs in some districts of Uttar Pradesh, India.  

MATERIALS AND METHODS

Sample collection
 
A total of 352 samples, including retail raw milk (160), traditional milk-based products peda (48), burfi/milk cake (48) and laddoo (48) and hand swabs (48) from retail shop workers, were collected from the regions surrounding Mathura, Agra and Aligarh districts in Uttar Pradesh, as well as Bharatpur district in Rajasthan, India from December 2023 to November 2024. The samples were processed at the laboratory or maintained at 4oC until further processing. The work was carried out in the laboratory of Department of Veterinary Public Health, DUVASU, Mathura, India.
 
Isolation and identification of E. coli
 
The isolation and identification of E. coli was done as per the method given by Edwards and Ewing (1972). The samples of milk (1 ml), milk products (25 g) and hand swabs were enriched in 9 ml, 250 ml and 5 ml of Tryptone soya broth at 37oC for 24 hrs, respectively. The loopful culture growth from TSB was streaked on MacConkey lactose agar and incubated at 37oC for 24 hrs further. Lactose fermenting pink-colored colonies were picked and streaked over Eosin methylene blue (EMB) at incubated at 37oC for 24 hrs. The colonies showing green metallic sheen were presumptive E. coli and subjected for biochemical confirmation. The single colony of E. coli was taken from each positive sample.
 
Phenotypic detection of E. coli O157:H7
 
Isolation and identification of E. coli O157:H7 was done as per the procedure given by Jhonson et al. (1996). The EC O157:H7 selective agar media was used for phenotypic detection and this serotype produced dark purple to magenta-coloured colonies while non O157 E. coli produced the greenish blue colour colonies on this agar.
 
Molecular detection of STEC
 
After biochemical confirmation, DNA of STEC was extracted by using kit (Sigma Aldrich) and were further subjected to multiplex PCR for screening of housekeeping Shiga toxin like gene as per the protocol given by Paton and Paton (1998). Primer sequences for stx1F (5'-ATAAATCGCCATTC GTTGACTAC-3') and stx1R (5'-AGAACGCCCACTGAGATCATC-3'), stx2F (5'-GGCACTGTCTGAAACTGCTCC-3') and stx2R (5'-TCGCCAGTTATCTGACATTCTG-3'), eaeAF (5'-GACCCGGC ACAAGCATAA GC-3') and eaeAR (5'-CCACCTGCAGCAACAA GAGG-3'), hlyAF (5'-GCATCATCA AGCGT ACGTTCC-3') and hlyA R (5'-AATGAGCCAAGCTGGTTAAGCT-3'). The amplicon size of stx1, stx2, eaeA and hlyA genes was 180 bp, 255 bp, 384 bp and 534 bp, respectively.  The PCR reaction was performed in a thermal cycler (Cyber lab) using standard cycling condition: an initial denaturation at 95oC for 5 min, followed by 30 cycles of denaturation at 94oC for 1 min, primer annealing at 59oC for 1 min and extension at 72oC for 1 min and a final extension at 72oC for 6 min. DNA quantification was carried out in nanodrop (Eppendorf, Germany) by taking 1 µL of elution buffer used for DNA extraction. The nanodrop was calibrated at 260 nm as well as at 280 nm wavelength, then 1 µL of test sample was taken and concentration was measured at A260/A280 ratio the values measured and this ratio of around 1.9(1.85-1.95) indicated best quality of DNA. The positive culture of Escherichia coli ATCC 25922 was procured from Himedia and used as positive control in entire research.
 
Biofilm production by phenotypic assays
 
STEC isolates were observed for the biofilm forming capacity in vitro by three different assays viz. Congo red agar (CRA) assay, Tube method (TM) and Tissue culture plate (TCP) assay. In CRA assay, black colored colonies with a dry crystalline consistency on CR agar were indicated by biofilm producers, whereas colonies showing red color were considered non-biofilm producers (Panda et al., 2016). In TM, visible film lined in the wall and bottom of the tube were considered as positive and strong biofilm former (Christensen et al., 1985). In TCP assay, OD values were considered as an index of bacteria adhering to the surface and forming biofilms and OD value was measured at 270 nm by the spectrophotometer (Bio RAD, UK). Strains were classified into three categories based on optical density (OD) measurements into weak (<0.120), moderate (0.120-0.240) and strong (>0.240) biofilm producers (Panda et al., 2016).
 
Statistical analysis
 
Chi-square test was used to compare the efficacy of different biofilm forming methods.

RESULTS AND DISCUSSION

Isolation of E. coli
 
Out of 352 samples, eighty-six samples produced pink colored colony on MLA and metallic sheen on EMB and single colonies was taken from each positive sample. The prevalence of E. coli was 22.50%, 30.55% and 12.5% in retail raw milk, traditional milk products and hand swabs, respectively with an overall prevalence of 24.43% (Table 1). Our results of overall prevalence of E. coli are consistent with the study of Madani et al., (2022) who reported a prevalence of 27.0% in milk and milk products from Iran, while Sarba et al., (2023) reported 33.8% in Ethiopia that is higher to this study.

Table 1: Prevalence of E. coli, STEC and virulent gene in dairy products and hand swabs.


       
Prevalence of E. coli in retail raw milk was 22.50%, almost similar prevalence value of E. coli 22.4% was revealed from milk in the work of Awadallah et al., (2016). The overall prevalence of E. coli was 30.55% in traditional milk-based products while Vaidya et al., (2015) reported 31.57% in milk-based products that is similar to this study. Madani et al., (2022) revealed the prevalence of E. coli 43.6% milk products that is quiet higher than this study. The prevalence of E. coli in hand swabs of milk shop workers was 12.5% in present study while Vanitha et al., (2018)  reported 11.11% of E. coli  from hand swabs in India Contamination of E. coli in milk and milk and its products is primarily attributed to inadequate personal hygiene, non-compliance with hygienic practices and suboptimal food processing methods.
 
Phenotypic detection of E. coli O157:H7
 
Out of 86 E. coli five isolates produced purple color colonies over selective agar (Fig 1). The overall prevalence of E. coli O157: H7 and non O157 E. coli was 5.81 (5/86) and 94.18 (81/86).  Sarba et al., (2023) revealed the prevalence of E. coli O157: H7 0.2% from Ethiopia, that is also lower to this study.

Fig 1: Dark purple colony of E. coli O157:H7 on EC O157 Agar.


 
Molecular detection of STEC
 
All the phenotypically detected E. coli strains (n=86) were subjected to mPCR, result showed that housekeeping stx2 gene bearers were 20.93% (18/86) and none of the isolates were found positive for stx1 gene (0/18). Ombarak et al., (2016) revealed 0.9% prevalence of stx1gene from Egypt, consistent to this study. In this study 33.33% (6/18) STEC isolates were positive for eaeA gene and results are consistent with Parul et al., (2021) who reported 33.33% of eaeA in raw milk from India.  Ombarak et al., (2016) revealed 0.0% and 0.45% of eaeA genes from milk and milk-based products, respectively from Egypt. The hlyA gene revealed with percent positivity of 16.66% (3/18) in contrast, this gene was 39.3% from dairy products in the work of Okechukwu et al., (2020).
       
Thus, the prevalence of STEC from retail raw milk, traditional milk products and hand swabs, 6.25%, 4.86% and 2.08%, respectively with an overall prevalence of 5.11% of STEC. In the study of Ranjbar et al., (2018) the prevalence values of retail raw milk 30.0% and from Iran, which is on the higher side to this study whereas Parul et al., (2021) and Madani et al., (2022) reported the prevalence value of 3.7% and 3.7%, of retail raw milk from India and Iran, respectively, which is lower to this study. The prevalence of STEC in traditional milk-based products in the study of Soomro et al., (2002) revealed 51.6% from Pakistan higher to current study. The prevalence of STEC in the hand swabs was found 7.0% while 11.11% reported by Vanitha et al., (2018) from India which is quite higher to our findings. Variations in prevalence values of STEC are largely influenced by differences in hygienic practices during the storage and processing of milk products and by environmental conditions.
 
Biofilm production by phenotypic assays
 
The confirmed STEC isolates were analysed for biofilm formation capability and biofilm forming STEC detected by TCP methods were 88.88% (Fig 4), TM 77.77% (Fig 3) and by CRA 38.88% (Fig 2), respectively (Table 2). By CRA overall 38.88% isolates were biofilm formers and 61.11% were non-biofilm formers and results are in contrast with Ponnusamy et al., (2012) who reported 37.0% biofilm formers and 63% were non-biofilm formers in E. coli isolates. By using the Tube method, 77.77% STEC isolates showed the ability to produce biofilms and categorized strong, moderate and weak was 50.0%, 16.66% and 11.11%, respectively in this assay (Fig 5). Nosrati et al., (2017) revealed 23.0% strong, 59.0% moderate and 18.0% weak biofilm formers and Nachammai et al., (2016), reported 57.0% biofilm forming E. coli from tube method which is lower to current study.  In TCP assay, overall, 88.88% isolates showed the ability to produce biofilms out of which 55.55% of STEC were strong, 22.22% were moderate and 11.11% weak biofilm former. Study of Wang et al., (2016) revealed 25.39% strong, 31.25% moderate and 28.9% weak biofilm formers which is lower to this study.

Fig 2: Black colored colonies of biofilm forming STEC on congo red agar.



Fig 3: STEC biofilm formation by tube method.



Fig 4: STEC biofilm formation by tissue culture plate method.



Fig 5: Overall comparison of biofilm assays (CRA,TM andTCP).


       
A chi -square test showed statistically similar (P>0.05) efficacy of all these three assays (Table 2). The discrepancies in the categorization of biofilm phenotypes could result from differences in the interpretation of results thus standardization of the biofilm method is crucial.

Table 2: Overall Comparison of Biofilm Assays for STEC (CRA, TM and TCP).

CONCLUSION

STEC have ability to form biofilms enables to attach with food contact surfaces like equipment, containers, utensils (milking cane, dippers, measurements) that may cause increase in microbial contamination in dairy products. Resilient nature of biofilms for available antibiotics, disinfectants and sanitizers draws the attention of researchers towards the effective control strategy for biofilm forming food borne pathogen.

ACKNOWLEDGEMENT

The authors wish to express their heartfelt gratitude to DUVASU, Mathura, for providing funds and necessary facilities to carry out part of research work.
 

CONFLICT OF INTEREST

The authors have no conflict of interest.

REFERENCES


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    Full Research Article

    Evaluation of Biofilm Production Capability of Shiga Toxin Producing Escherichia coli Isolated from Milk, Milk Products and Hand Swabs 

    R
    Ravi P. Prajapati1
    P
    Parul1,*
    U
    Udit Jain1
    B
    Barkha Sharma2
    R
    Raghavendra P. Mishra2
    S
    Satyendra P. Singh3
    G
    Gurvinder1
    V
    Vikram Jeet1
    K
    Kushaan Seth1
    1Department of Veterinary Public Health, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.
    2Department of Veterinary Epidemiology, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.
    3Department of Animal Genetics and Breeding, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India.

    ABSTRACT

    Background: Food borne pathogens are transmitted to humans through contaminated foods like dairy products, handmade goods and meat. Microbial biofilms are city of microbes in the form of 3D structure and attachment of bacteria with food contact surfaces lead to subsequent development of biofilms that may lead to transmission of disease and food spoilage. This study was designed to evaluate the biofilm production capability of Shiga toxin-producing Escherichia coli (STEC) isolated from milk, milk-based products and hand swabs.

    Methods: Samples were collected and processed for isolation of E. coli. STEC were detected on the molecular basis through mPCR. Biofilm formation capability of STEC was screened by methods namely Tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) assay. 

    Result: The prevalence of E. coli and STEC was found to be 22.50%, 30.55%, 12.5% and 6.25%, 4.86%, 2.08% in retail raw milk, traditional milk products and hand swabs, respectively. The overall prevalence of E. coli, serotype E. coli O157:H7 and STEC was 24.43%, 1.42% and 5.11%, respectively. The biofilm forming STEC revealed by TCP, TM and CRA were 88.88%, 77.77% and 38.88%, respectively.

    INTRODUCTION

    Food borne pathogens like Escherichia coli, Shiga Toxin producing Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Listeria monocytogenes can be transmit to people by consumption of contaminated food products of animal and plant origin (Oriekhoe et al., 2024; Rajan et al., 2024). According to the WHO’s report (2015) on the worldwide impact of foodborne illness, in 2010, more than 1.2 million cases of foodborne illness were caused by STEC, resulting in 128 fatalities and almost 13,000 Disability Adjusted Life Years (DALYs) (WHO, 2015).
           
    Shiga toxin producing Escherichia coli (STEC) is a subtype of E. coli that causes enteric and systemic diseases in human ranging from diarrhoea to severe haemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and Thrombocytopenic purpura (TPP) (Debbarma et al., 2024; Rosario et al., 2021). Cattle, particularly young ones, have been identified as a primary reservoir of STEC (Abebe et al., 2020). The main sources of STEC-associated foodborne illnesses are milk, meat and its products contaminated with either ruminant feces or other environmental sources (Durairajan et al., 2021; Parul et al., 2021).
           
    Bioflm forming food borne pathogens are big issues for food safety and significant challenge in food chains, particularly in less developed countries (Shi et al., 2024). Biofilms are groups of microorganisms in complex extracellular polymer matrix in which cells bind tightly together on various surfaces. Biofilms are extremely resistant to the available antibiotics and disinfectant and is continuous contamination of food due to the detachment of cells from biofilm matrix (Carrascosa et al., 2021). The “top 6” non-O157 serogroups (O26, O45, O103, O111, O121 and O145), along with other STEC serogroups were revealed with biofilm formation attribute and which are frequently implicated with food borne infection (Parul et al., 2023). Thus, the aim of study is evaluation of the biofilm production capability of Shiga toxin-producing Escherichia coli (STEC) isolated from milk, milk-based products and hand swabs in some districts of Uttar Pradesh, India.  

    MATERIALS AND METHODS

    Sample collection
     
    A total of 352 samples, including retail raw milk (160), traditional milk-based products peda (48), burfi/milk cake (48) and laddoo (48) and hand swabs (48) from retail shop workers, were collected from the regions surrounding Mathura, Agra and Aligarh districts in Uttar Pradesh, as well as Bharatpur district in Rajasthan, India from December 2023 to November 2024. The samples were processed at the laboratory or maintained at 4oC until further processing. The work was carried out in the laboratory of Department of Veterinary Public Health, DUVASU, Mathura, India.
     
    Isolation and identification of E. coli
     
    The isolation and identification of E. coli was done as per the method given by Edwards and Ewing (1972). The samples of milk (1 ml), milk products (25 g) and hand swabs were enriched in 9 ml, 250 ml and 5 ml of Tryptone soya broth at 37oC for 24 hrs, respectively. The loopful culture growth from TSB was streaked on MacConkey lactose agar and incubated at 37oC for 24 hrs further. Lactose fermenting pink-colored colonies were picked and streaked over Eosin methylene blue (EMB) at incubated at 37oC for 24 hrs. The colonies showing green metallic sheen were presumptive E. coli and subjected for biochemical confirmation. The single colony of E. coli was taken from each positive sample.
     
    Phenotypic detection of E. coli O157:H7
     
    Isolation and identification of E. coli O157:H7 was done as per the procedure given by Jhonson et al. (1996). The EC O157:H7 selective agar media was used for phenotypic detection and this serotype produced dark purple to magenta-coloured colonies while non O157 E. coli produced the greenish blue colour colonies on this agar.
     
    Molecular detection of STEC
     
    After biochemical confirmation, DNA of STEC was extracted by using kit (Sigma Aldrich) and were further subjected to multiplex PCR for screening of housekeeping Shiga toxin like gene as per the protocol given by Paton and Paton (1998). Primer sequences for stx1F (5'-ATAAATCGCCATTC GTTGACTAC-3') and stx1R (5'-AGAACGCCCACTGAGATCATC-3'), stx2F (5'-GGCACTGTCTGAAACTGCTCC-3') and stx2R (5'-TCGCCAGTTATCTGACATTCTG-3'), eaeAF (5'-GACCCGGC ACAAGCATAA GC-3') and eaeAR (5'-CCACCTGCAGCAACAA GAGG-3'), hlyAF (5'-GCATCATCA AGCGT ACGTTCC-3') and hlyA R (5'-AATGAGCCAAGCTGGTTAAGCT-3'). The amplicon size of stx1, stx2, eaeA and hlyA genes was 180 bp, 255 bp, 384 bp and 534 bp, respectively.  The PCR reaction was performed in a thermal cycler (Cyber lab) using standard cycling condition: an initial denaturation at 95oC for 5 min, followed by 30 cycles of denaturation at 94oC for 1 min, primer annealing at 59oC for 1 min and extension at 72oC for 1 min and a final extension at 72oC for 6 min. DNA quantification was carried out in nanodrop (Eppendorf, Germany) by taking 1 µL of elution buffer used for DNA extraction. The nanodrop was calibrated at 260 nm as well as at 280 nm wavelength, then 1 µL of test sample was taken and concentration was measured at A260/A280 ratio the values measured and this ratio of around 1.9(1.85-1.95) indicated best quality of DNA. The positive culture of Escherichia coli ATCC 25922 was procured from Himedia and used as positive control in entire research.
     
    Biofilm production by phenotypic assays
     
    STEC isolates were observed for the biofilm forming capacity in vitro by three different assays viz. Congo red agar (CRA) assay, Tube method (TM) and Tissue culture plate (TCP) assay. In CRA assay, black colored colonies with a dry crystalline consistency on CR agar were indicated by biofilm producers, whereas colonies showing red color were considered non-biofilm producers (Panda et al., 2016). In TM, visible film lined in the wall and bottom of the tube were considered as positive and strong biofilm former (Christensen et al., 1985). In TCP assay, OD values were considered as an index of bacteria adhering to the surface and forming biofilms and OD value was measured at 270 nm by the spectrophotometer (Bio RAD, UK). Strains were classified into three categories based on optical density (OD) measurements into weak (<0.120), moderate (0.120-0.240) and strong (>0.240) biofilm producers (Panda et al., 2016).
     
    Statistical analysis
     
    Chi-square test was used to compare the efficacy of different biofilm forming methods.

    RESULTS AND DISCUSSION

    Isolation of E. coli
     
    Out of 352 samples, eighty-six samples produced pink colored colony on MLA and metallic sheen on EMB and single colonies was taken from each positive sample. The prevalence of E. coli was 22.50%, 30.55% and 12.5% in retail raw milk, traditional milk products and hand swabs, respectively with an overall prevalence of 24.43% (Table 1). Our results of overall prevalence of E. coli are consistent with the study of Madani et al., (2022) who reported a prevalence of 27.0% in milk and milk products from Iran, while Sarba et al., (2023) reported 33.8% in Ethiopia that is higher to this study.

    Table 1: Prevalence of E. coli, STEC and virulent gene in dairy products and hand swabs.


           
    Prevalence of E. coli in retail raw milk was 22.50%, almost similar prevalence value of E. coli 22.4% was revealed from milk in the work of Awadallah et al., (2016). The overall prevalence of E. coli was 30.55% in traditional milk-based products while Vaidya et al., (2015) reported 31.57% in milk-based products that is similar to this study. Madani et al., (2022) revealed the prevalence of E. coli 43.6% milk products that is quiet higher than this study. The prevalence of E. coli in hand swabs of milk shop workers was 12.5% in present study while Vanitha et al., (2018)  reported 11.11% of E. coli  from hand swabs in India Contamination of E. coli in milk and milk and its products is primarily attributed to inadequate personal hygiene, non-compliance with hygienic practices and suboptimal food processing methods.
     
    Phenotypic detection of E. coli O157:H7
     
    Out of 86 E. coli five isolates produced purple color colonies over selective agar (Fig 1). The overall prevalence of E. coli O157: H7 and non O157 E. coli was 5.81 (5/86) and 94.18 (81/86).  Sarba et al., (2023) revealed the prevalence of E. coli O157: H7 0.2% from Ethiopia, that is also lower to this study.

    Fig 1: Dark purple colony of E. coli O157:H7 on EC O157 Agar.


     
    Molecular detection of STEC
     
    All the phenotypically detected E. coli strains (n=86) were subjected to mPCR, result showed that housekeeping stx2 gene bearers were 20.93% (18/86) and none of the isolates were found positive for stx1 gene (0/18). Ombarak et al., (2016) revealed 0.9% prevalence of stx1gene from Egypt, consistent to this study. In this study 33.33% (6/18) STEC isolates were positive for eaeA gene and results are consistent with Parul et al., (2021) who reported 33.33% of eaeA in raw milk from India.  Ombarak et al., (2016) revealed 0.0% and 0.45% of eaeA genes from milk and milk-based products, respectively from Egypt. The hlyA gene revealed with percent positivity of 16.66% (3/18) in contrast, this gene was 39.3% from dairy products in the work of Okechukwu et al., (2020).
           
    Thus, the prevalence of STEC from retail raw milk, traditional milk products and hand swabs, 6.25%, 4.86% and 2.08%, respectively with an overall prevalence of 5.11% of STEC. In the study of Ranjbar et al., (2018) the prevalence values of retail raw milk 30.0% and from Iran, which is on the higher side to this study whereas Parul et al., (2021) and Madani et al., (2022) reported the prevalence value of 3.7% and 3.7%, of retail raw milk from India and Iran, respectively, which is lower to this study. The prevalence of STEC in traditional milk-based products in the study of Soomro et al., (2002) revealed 51.6% from Pakistan higher to current study. The prevalence of STEC in the hand swabs was found 7.0% while 11.11% reported by Vanitha et al., (2018) from India which is quite higher to our findings. Variations in prevalence values of STEC are largely influenced by differences in hygienic practices during the storage and processing of milk products and by environmental conditions.
     
    Biofilm production by phenotypic assays
     
    The confirmed STEC isolates were analysed for biofilm formation capability and biofilm forming STEC detected by TCP methods were 88.88% (Fig 4), TM 77.77% (Fig 3) and by CRA 38.88% (Fig 2), respectively (Table 2). By CRA overall 38.88% isolates were biofilm formers and 61.11% were non-biofilm formers and results are in contrast with Ponnusamy et al., (2012) who reported 37.0% biofilm formers and 63% were non-biofilm formers in E. coli isolates. By using the Tube method, 77.77% STEC isolates showed the ability to produce biofilms and categorized strong, moderate and weak was 50.0%, 16.66% and 11.11%, respectively in this assay (Fig 5). Nosrati et al., (2017) revealed 23.0% strong, 59.0% moderate and 18.0% weak biofilm formers and Nachammai et al., (2016), reported 57.0% biofilm forming E. coli from tube method which is lower to current study.  In TCP assay, overall, 88.88% isolates showed the ability to produce biofilms out of which 55.55% of STEC were strong, 22.22% were moderate and 11.11% weak biofilm former. Study of Wang et al., (2016) revealed 25.39% strong, 31.25% moderate and 28.9% weak biofilm formers which is lower to this study.

    Fig 2: Black colored colonies of biofilm forming STEC on congo red agar.



    Fig 3: STEC biofilm formation by tube method.



    Fig 4: STEC biofilm formation by tissue culture plate method.



    Fig 5: Overall comparison of biofilm assays (CRA,TM andTCP).


           
    A chi -square test showed statistically similar (P>0.05) efficacy of all these three assays (Table 2). The discrepancies in the categorization of biofilm phenotypes could result from differences in the interpretation of results thus standardization of the biofilm method is crucial.

    Table 2: Overall Comparison of Biofilm Assays for STEC (CRA, TM and TCP).

    CONCLUSION

    STEC have ability to form biofilms enables to attach with food contact surfaces like equipment, containers, utensils (milking cane, dippers, measurements) that may cause increase in microbial contamination in dairy products. Resilient nature of biofilms for available antibiotics, disinfectants and sanitizers draws the attention of researchers towards the effective control strategy for biofilm forming food borne pathogen.

    ACKNOWLEDGEMENT

    The authors wish to express their heartfelt gratitude to DUVASU, Mathura, for providing funds and necessary facilities to carry out part of research work.
     

    CONFLICT OF INTEREST

    The authors have no conflict of interest.

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