CC BY
0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
ASSESSMENT OF BACTERIAL CONTAMINATION AND ANTIBIOTIC RESISTANCEGENE (TETM) IN VEGETABLE AND MEAT SAMPLES FROM A LOCAL MARKET
1Fahmi Naznine, 2Raj Kumar, 3Mohd Ikram Ansari
Research scholar, Department of Biosciences, Integral University, Lucknow (UP), India Student, Department of Biosciences, Integral University, Lucknow (UP), India Assistant Professor, Department of Biosciences, Integral University, Lucknow (UP), India
https://doi.org/10.5281/zenodo.13846134
Abstract. Food samples were collected in February 2022-2023 from three separate locations of each local marketfood samples. A total of 90 Bacteria were isolatedfrom local market for food sample, Lucknow. All the isolates were tested for their resistance to various antibiotics by disc diffusion method. Antibiotic-resistant bacteria were found in water samples from the local market of food samples. The percent of antibiotic-resistant bacteria for Buffalo meat and Goat meat, were resistant for ampicillin (80, 40%), sulphadiazene (60, 30 %), kanamycin (20, 10%). The percent of antibiotic-resistant bacteria for cauliflower samples and spinach, were highest for ampicillin (0, 80%), sulphadiazene (50%, 20%) and kanamycin (20, 20%). Further, antibiotic resistance genes in local market food samples disclose distinctive patterns, including resistance to tetM (25%) for buffalo. Results revealed significant contamination in vegetables due to poor handling and unhygienic conditions, while meat samples showed minimal contamination. The presence of antibiotic resistance genes in isolated bacteria underscores the need for continuous monitoring and responsible antibiotic use. The study highlights the importance of proper hygiene practices and preventive measures to reduce bacterial contamination and the spread of antibiotic resistance, ensuring food safety and public health.
Keywords: antibiotic resistance genes, Environments, Ampicillin, Sulphadiazne, Vegetable.
Introduction
Food contamination is a critical global issue highlighted by the World Health Organization. It affects millions worldwide, leading to numerous illnesses and fatalities. The WHO emphasizes that food contamination can impact individuals far from its origin, making it a significant concern. Key challenges include the contamination of fresh produce, the presence of antibiotics in food products, and deliberate acts of contamination [1]. In recent years, global production of fresh vegetables and fruits has increased by 30%, from 30 million to 60 million metric tonnes [2]. This growth has been uneven, with Asian countries nearly doubling their production and exports, while Europe sees a decline in export value. Fresh produce is essential for human nutrition, providing vital vitamins, minerals, and dietary fiber. The rise in global consumption of fresh fruits and vegetables has been accompanied by increased microbial contamination, posing a significant threat to food safety [3].
Fresh vegetables and fruits are essential for human nutrition, providing vital vitamins such as B, C, and K, minerals like calcium, potassium, and magnesium, and dietary fiber [4]. These nutrients help maintain a healthy diet and prevent chronic diseases like heart disease, cancer, diabetes, and obesity, while addressing micronutrient deficiencies, especially in developing nations [5]. However, raw vegetables can carry human pathogens. Consumed raw or lightly cooked
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
to preserve their nutritional value, they can cause food-borne infections and disease outbreaks in countries like China, India, Iran, Vietnam, Turkey, Russia, Nigeria, Egypt, Mexico, and the USA. The global rise in fresh fruit and vegetable consumption has led to increased microbial contamination, threatening food safety. Vegetable production is concentrated among the top ten producing countries globally, with China, India, and the USA leading in both vegetable and meat production. Between 1986 and 1995, per capita vegetable consumption was around 0.95%, with China consuming the highest proportion of fruits at 6.4% [6]. Sub-Saharan Africa had the lowest consumption at 0.19%. From 1980 to 2004, fresh produce production increased by 94% due to population growth. Recently, Asia has the highest vegetable consumption, followed by Europe, Northern America, Oceania, and Africa, with Africa showing slower but steady growth.
This study highlights the urgent need to address antibiotic-resistant bacteria, understand gene transmission pathways, and elucidate disease mechanisms. Emphasizing horizontal gene transfer's role, we stress the importance of studying microorganism interactions and the critical impact on public health, aiming to inspire effective measures to combat this global threat.
Materials and Method
Sampling and Study Site Description
The research was conducted at a local market on Kuris Road, near Integral University in Lucknow, Uttar Pradesh. This market features various food vendors selling meat, spinach, and cauliflower. Lucknow experiences a summer subtropical climate, with temperatures reaching up to 43°C from February 2022-2023. Rapid urbanization has led to poor handling and unhygienic conditions, promoting microorganism growth in food. The increasing population has strained natural resources, affecting food quality. Meat and vegetable samples were collected using sterile tubes to ensure sample integrity.
Collection of sample
Three 100-gram samples of meat or vegetables were collected from a local market. Samples were placed in labeled polybags with GPS locations and stored in a cooler bag to maintain temperature control. They were then transferred to a storage facility at 4°C to preserve freshness and quality until analysis.
Isolation and Sub culturing of Bacteria
The spread plate method was used to isolate bacteria and determine the total plate count. Samples were mixed with saline, serially diluted, and inoculated on agar plates. After 16-18 hours of incubation at 37°C, pure cultures were prepared using aseptic techniques and streaking. Plates were incubated for 24 hours.
Gram-Staining
For Gram-staining, slides were labeled and a drop of deionized water was added. Pure cultures were transferred using a sterilized loop, dried, and heat-fixed. Slides were stained with crystal violet, rinsed, treated with iodine, decolorized with ethanol, and counterstained with safranin. After drying, they were examined under a microscope.
Antibiotic sensitivity testing
Antibiotic sensitivity testing was conducted using the Disc Diffusion Method. Antibiotics used included Ampicillin (30 |g), Tetracycline (30 |g), Leomycin (5 |g), Sulphadiazine (100 |g), and Kanamycin (30 |g), sourced from Hi-media, Mumbai. Sensitivity was interpreted per CLSI guidelines (2020).
PCR based Detection of Antibiotics Resistance Genes
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
Template DNA was prepared by boiling lysates of 3-5 colonies in TE buffer, boiling at 95°C for 15 minutes, and centrifuging. DNA was stored at 20°C and diluted 1:10 for PCR.
PCR Detection of Target Genes
PCR was used to detect the tetM gene in environmental samples and bacterial strains. The reaction mixture included PCR Taq Master Mix, primers, template DNA, and nuclease-free water. Thermocycling involved denaturation, annealing, and extension steps. Products were separated on agarose gel and visualized using a gel documentation system.
Discussion. This study investigated bacterial contamination and antibiotic resistance genes in vegetable and meat samples from a local market. Findings revealed significant contamination in vegetables due to poor handling and environmental conditions, while meat showed minimal contamination. The study emphasizes the importance of proper hygiene practices and continuous food safety monitoring. It also highlights the need for awareness and preventive measures to reduce bacterial transmission and antibiotic resistance. Limitations include a small sample size and focus on a specific market. Further research with larger samples and broader geographical representation is needed for comprehensive insights.
Conclusion. In conclusion, our study investigated bacterial contamination and antibiotic resistance genes in vegetable and meat samples from a local market. Handling practices significantly influenced contamination, with vegetables showing higher contamination due to unhygienic conditions. Meat samples had minimal contamination. The study underscores the importance of proper hygiene and preventive measures to reduce bacterial spread and antibiotic resistance. Continuous monitoring and responsible antibiotic use are crucial. Despite limitations like small sample size and limited geographic scope, the findings highlight the need for improved food safety practices to ensure safe, uncontaminated food for public health.
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