Biofilm formation and genetic profiles of Escherichia coli isolated from milk and flavoured milk

Introduction

The World Health Organization has reported that hundreds of millions of people worldwide suffer from foodborne diseases each year in both developed and developing countries. More than 100 million cases of food-related illnesses are anticipated to occur each year in India by 2030 (Bisht et al., 2021) [2] . The increasing frequency of foodborne disease outbreaks across various states in India further underscores this burden National Health Portal. (2019). Bacterial biofilms as complex microbial communities encased in extracellular polymeric substances, emphasizing the matrix components such as polysaccharides, proteins, and extracellular DNA and their role in biofilm formation and resilience (Zhao, Sun and Liu, 2023) [16] . Microbes embedded in biofilms exhibit far greater resistance to cleaning agents than their free-floating (planktonic) counterparts. Several human pathogens are involved in biofilm formation, including Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus (Galié et al., 2018) [6] . Biofilms consist of a collection of microbial cells that are permanently attached to a surface and are surrounded by a matrix mainly composed of polysaccharide substances, offering certain benefits to the bacteria and making them resistant to cleaning and disinfection (Flemming et al., 2016) [4] . Biofilms are a persistent issue on food contact surfaces such as stainless steel, plastic, and rubber in processing environments. These biofilms not only threaten food safety but can also cause equipment degradation and reduce processing efficiency (Liu et al., 2023) [16] . Escherichia coli is a gram-negative commensal gastrointestinal microflora in humans and animals. They may be difficult to detect and treat owing to their short doubling time, ability to adhere to surfaces, and biofilm formation (Bhardwaj et al., 2021) [1] .

Biofilm formation in E. coli is a coordinated process that requires multiple genes for surface sensing and attachment (fimH, csgA), (Foroogh et al., 2021; Bu, Dee and Liu, 2024) [5, 3] , motility, quorum sensing (luxS), (Laganenka and Sourjik, 2018) [7] and maturation. These gene products interact to allow the bacteria to colonise surfaces, resist environmental stress, and disperse when conditions are favourable. This study investigated the biofilm-forming capacity of Escherichia coli isolated from raw and flavoured milk collected from local markets. Subsequently, the genes associated with biofilm formation were assessed in the field isolates

Materials and Methods

Food samples

Raw milk (20), flavoured milk (10) samples were collected in separate containers from the retail outlet in and around Chennai, Tamil Nadu.

Isolation, identification and characterization of foodborne microorganisms

A total of 25 g of the sample was mixed with 225 mL of nutrient broth for enrichment and incubated at 37 °C for 18-24 hours. After enrichment, 100 µL of the inoculum was plated onto MacConkey agar (MCA) plates, which were then incubated at 37 °C for 18-24 hours. Pink bacterial colonies on the MCA were identified as E. coli. The E. coli isolates were inoculated into BHI broth and incubated at 37 °C for 24 hours. The cultures were stored at-20 °C in glycerol for future use.

Identification of biofilm forming organism and quantification of biofilm:

Positive isolates were grown in 10 mL of BHI broth, and the bacterial culture turbidity was adjusted to an optical density (OD) of 0.1 (range: 0.08-0.1) at 600 nm. In microtiter plate wells, 180 µL of BHI broth and 20 µL of the inoculum were added in triplicate, with broth-only wells serving as negative controls. The plates were incubated at 37 °C for 48 h to allow biofilm formation. After incubation, the contents were removed, and the wells were washed three times with sterile distilled water to remove planktonic cells, then air-dried for 15 min. The attached cells were stained with 0.1% crystal violet solution for 30 min, washed, and air-dried. A mixture of 75% ethanol and 25% acetone was added to solubilize the bound dye. Biofilm formation was quantified by measuring absorbance at 597 nm, and isolates were classified as weak (< 0.3), moderate (0.3-0.6), or strong (> 0.6) biofilm producers (Obe et al., 2021) [11] .

Molecular identification and amplification of adhesion genes

Genomic DNA was extracted from E. coli isolates using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions. All positive colonies were confirmed by PCR using 16S rRNA primers specific for E. coli. Adhesion-associated genes involved in biofilm formation by E. coli were identified using primers targeting various genes (Table 1). The PCR protocol consisted of 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1.5 min, following an initial denaturation at 95 °C for 3 min and ending with a final extension at 72 °C for 7 min. The amplified products were analysed by electrophoresis on a 1.8% agarose gel using a 100 bp molecular weight marker and were documented with a UV light transilluminator. [15] 3. Results and Discussion

Isolation and identification

In this study, 20 raw milk and 10 flavoured milk samples were collected for the isolation of foodborne microorganisms using MacConkey agar for the isolation of E. coli. In the present study, Out of 20 raw milk samples, 12 formed pink colonies on MacConkey agar (60 %) and in flavoured milk samples out of 10, three formed pink colonies on MacConkey agar (30 %). The results of the present study are consistent with Ravi et al. (2025), who reported that the prevalence of E. coli was 22.50%, 30.55%, and 12.5% in retail raw milk and traditional milk products, respectively. The prevalence of E. coli isolates was higher in traditional dairy products than in pasteurized dairy products (Madani et al., 2022) [9] .

Identification of biofilm forming organism

The biofilm forming ability of E. coli isolates from raw milk and flavoured milk are given in Table 2. In raw milk out of seven isolates screened, three were strong biofilm producers (42.8%), two were moderate biofilm producers (28.5%), and two were weak biofilm producers (28.5%). Out of three isolates from flavoured milk, two were weak producers and one was a strong biofilm producer. Madani et al. 2022 [9] reported that 28.9% isolates classified as strong biofilm producers, 10.5% isolates showed moderate biofilm formation, 28.9% isolates showed weak biofilm formation, and 12 (31.6%) isolates did not form any biofilms. The present study revealed a higher rate of strong biofilm formation in E. coli isolates from raw milk samples.

Molecular identification and amplification of adhesion genes

The PCR amplification and the agarose gel electrophoresis results are presented in Figure 1, 2, 3, and 4. Molecular characterisation of seven isolates from raw milk and three isolates from flavoured milk were screened for 16S rRNA specific primer to E. coli. All the samples were positive for E. coli. The amplification of adhesion genes in E. coli associated with biofilm production were luxS (100%), csgA (71%), and fimH (57%). All the isolates from raw milk were negative for fimA and papC in the milk samples. The PCR results are presented in Table 3

Conclusion

The prevalence of E. coli in raw and flavoured milk samples was high, and the majority of E. coli isolates were strong biofilm producers. The prevalence of biofilm-associated genes in this study confirmed that luxS, csgA, and fimH are responsible for biofilm formation by E. coli isolates. This highlights the importance of hygienic practices during the production and processing of milk and milk products to minimise contamination associated with biofilm-forming E. coli in the dairy industry.

Acknowledgement

AcknowledgementThe authors thank the Department of Animal Biotechnology, Madras Veterinary College, Chennai-600 007, and College of Food and Dairy Technology, Chennai-600 052 for providing laboratory facilities to carry out the research work.

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