Microbial Profile and Antibiotic Susceptibility Pattern of Frozen Food in India

This investigation was carried out to examine the safety and microbiological quality of products made from meat, dairy and vegetables. Samples of frozen food were examined for the presence of Gram-negative bacteria. A total of 49 frozen food samples were collected, including chicken nuggets, chicken fillets, chicken sticks, breaded breast chicken, fish, beef burger and minced beef roll, white cheese, camembert cheese, and vegetables (okra and green peas). Standard methods were used to determine the diversity of bacterial isolates in various food samples. About 182 isolates of Escherichia coli , 63 isolates of Salmonella typhi , 51 isolates of Pseudomonas , and 63 isolates of Klebsiella were recovered from the various frozen food samples. Meat samples showed a high prevalence of E. coli and Pseudomonas . The antibiotic susceptibility patterns of the isolated bacterial strains were also examined. Out of 12 antibiotics, only ciprofloxacin and ofloxacin showed a high level of susceptibility. According to the study's findings, the majority of the frozen meat product samples contained a significant number of bacteria and were therefore unsafe for human consumption. These microorganisms can cause infection and are therefore associated with a high risk to the consumers. Therefore, it is important to pay attention to health and education issues in relation to food safety.


INTRODUCTION
Foods that are damaged or tainted because they contain either microorganisms, such as bacteria or parasites, or toxic compounds that render them unsafe for consumption are often referred to as contaminated foods.Biological, chemical, or physical contaminants can affect food; the former two are more frequent.These pollutants can enter a food product through a variety of points along the supply chain (from farm to fork), rendering it unsafe for human consumption. 1Common organisms include Yersinia enterocolitica, Listeria monocytogenes, Salmonella spp., Shigella spp., pathogenic Staphylococcus aureus, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, pathogenic Escherichia coli, Vibrio cholera, V. parahaemolyticus, and V. vulnificus. 2 The food sector constantly deals with numerous regional and worldwide contamination challenges that are both current and emerging. 3cientific and technical advances are being made to address these issues.The nature of the contamination, its origins, consumer dangers, and strategies to reduce or eradicate contamination levels must all be understood by food safety management.To produce food products with a low risk of contamination or those that are free of contamination, excellent scientific understanding is required. 3In numerous documents and reports, the World Health Organization (WHO) has identified food contamination as a global issue. 46][7] Over the course of their lifetimes, the vast majority of people globally acquire a food-or water-borne illness.As a result, millions of people become sick from eating tainted food, and many die as a result.In this situation, "food contamination" becomes a significant issue, and its problems are numerous and constantly expanding.
Microbial standards relating to product safety and quality are required by the food industry globally.Since safety considerations are of utmost importance, they are decided upon in a straightforward manner.Usually, pathogenic bacteria that could cause consumers to have serious health issues are subject to rigorous limits or no tolerance. 8A Foodborne Diseases Active Surveillance Network recently conducted surveillance in the USA for all diseases caused by certain pathogens that are often spread through food.In 2013, 9 it was reported that there were 19,531 infections, 4563 hospitalizations, and 68 fatalities linked to foodborne illnesses.
Every year, foodborne diseases cause a financial burden.1] Even though a wide variety of foods can result in foodborne illnesses, the meat and dairy products sector remains the major safety concern and a focus in many ways.Thus, the goal of the current study was to assess how common Gramnegative bacteria were in frozen foods towards the end of their shelf life, to shed light on the statistics and to indicate where future educational initiatives by food regulatory authorities should be directed.Furthermore, by avoiding the risks associated with such meals and maintaining public health, as well as by identifying defective frozen food products early on, economic losses can be minimized.

Collection of food samples
A total of 49 frozen food samples were randomly collected from 4 different markets in Delhi, India.About 23 meat product samples, 8 dairy samples, and 8 vegetable products were included in the study.These were chicken nuggets, chicken fillets, chicken sticks, breaded breast chicken, fish, beef burger and minced beef roll, white cheese, camembert cheese, and vegetables (okra and green peas).The samples were tagged and analyzed after being collected in sterile plastic bags and transported in ice boxes.

Media and reagents
The media used for bacteriological analysis were MacConkey agar, blood agar, Mueller-Hinton agar and Nutrient broth (NB).The reagents used during the study were crystal violet, Gram's iodine, safranin, 95% ethyl alcohol, 3% hydrogen peroxide, Kovacs indole reagent, normal physiological saline, phosphate buffered saline (PBS), and distilled water.Commercially available antibiotic discs  The commercially available media were prepared according to the manufacturer's instructions, while the non-commercial media were prepared in the laboratory.

Sample processing
To create adequate dilutions for the microbiological examination, 30 g of each sample were combined and homogenized with 225 ml of sterile saline solution.Homogenates were diluted ten-fold before being introduced into the appropriate media.

Isolation of bacterial pathogens
By streaking 0.1 ml of each food sample at a suitable dilution onto blood agar and McConkey agar, bacteria were isolated and counted before being cultured at 37°C for 24 hours.Pure cultures on nutrient agar slants at 37°C for 24 hours were used to isolate typical colonies for subsequent identification. 12The identification of isolated bacteria was carried out based on their microscopic, cultural, and biochemical properties. 13

Antibiotic sensitivity test for bacterial isolates
To test their effectiveness against bacterial isolates, 12 antibiotic discs from different groups that are readily available were selected.The culture broth was transferred aseptically into sterile distilled water overnight and forcefully stirred to produce turbidity that matched the 0.5 McFarland standard (about 108 CFU/ml), as described in Bauer's [14][15] standard Kirby-Bauer disk diffusion procedure.The surface of solidified Mueller-Hinton agar plates was then inoculated with the swab dipped in the culture solution. 16To achieve complete contact with the agar, antibiotic discs were placed on the infected plate surface and gently pressed down with sterile forceps.The plates were then incubated at 37°C for 24 hours in air supplemented with 5% carbon dioxide.Clinical Laboratory Standards "CLS" were used to assess and interpret the inhibition zones surrounding the antibiotic discs.The classification of the results was R (resistant), IS (intermediate sensitive), and S (sensitive). 17
Research by Bezeraa et al., indicates that in 31.4% of samples with positive E. coli results, hamburgers were deemed unfit for human consumption. 18The presence of E. coli in combination with unsanitary conditions, such as a poor waste disposal system, may be a potential source of food contamination.The presence of members of the Enterobacteriaceae family, which have been recognized as significant contributors to foodborne outbreaks, 19 presents a health concern to children and those with underlying illnesses.The overall number of positive samples was 359 and the percentage of E. coli concentration was (182) 50.6% (Table 1 & Figure 1).The use of contaminated water at various stages of processing may be responsible for the occurrence of E. coli.In this regard, the water used for washing raw meat is also used for washing hands and production-related utensils. 20Salmonella typhi and E. coli were both discovered to be contaminants in the beef items. 21Salmonella outbreaks are typically caused by inappropriate food handling and preparation at food service establishments, with infected food handlers not washing their hands.These findings are consistent with those of Zhao et al., 22 who isolated Salmonella in 19-54% of cow carcasses, 1.9% of beef samples purchased at retail, and 4.2% of poultry samples purchased at retail.As of January 16, 2003, Trinidad and Tobago had 49 cases of salmonellosis from weeks 1 to 52 of 2002. 23Gram-negative facultative anaerobes such Klebsiella (K.pneumonia), Salmonella (S. typhi), and Proteus (P.vulgaris) were found in the food samples under investigation, according to Lengeler et al., 24 According to Samson et al., 25 many meat samples under investigation contained Gram-negative aerobes such as Campylobacter (C.jejuni and C. coli), Pseudomonas (P.aeruginosa, P. fluorescens, and P. putida).Since these bacteria are spread by equipment or contact with raw foods, the presence of Enterobacteriaceae and Coliform group is a helpful predictor of cleanliness of post-processing contamination of processed foods.Hassan et al., 26 reported similar findings, demonstrating that the mean value of the Enterobacteriaceae count ranged from 3.9x10 2 to 1x10 3 for each sample of yogurt and feta cheese.Furthermore, Mohammed et al., 27 studies is not consistent with the coliform group's infection % with our investigation results.The pathogenic E. coli O157:H7 was isolated from 19% of the total white cheese samples in the earlier tests conducted in Egypt. 28he bacterial isolates from meat and dairy dietary samples were identified on the basis of their morphological, cultural, and broth consumption manual tests, as described in Bergey's manual. 29Three main types of foodborne bacterial isolates were identified on the basis of biochemical findings (Figure 5).
The findings showed that frozen food samples had high levels of E. coli bacterial contamination.1][32][33][34][35] Alarming multi-resistance frequencies for Salmonella and E. coli isolates from food were also shown by the antibiotic susceptibility tests.7][38][39] Salmonella species isolated from pork and poultry were found to be resistant to one or more antibiotics in 78 to 89% of the cases.

CONCLUSION
The quality of food products in relation to public health standards has attracted the attention of researchers.In this regard, the prevalence of pathogenic bacteria in various frozen food samples was investigated.Strict guidelines for food safety should be observed, along with instructions on safety principles and proper sanitary practices.It is highly recommended that consumers are made aware of the quality standards for frozen food.If these aspects are taken into consideration, infection and toxicity caused by the consumption of these products can be controlled.
test the drug sensitivity pattern.

Figure 1 .Figure 2 .
Figure 1.Total number of bacterial pathogens from different food samples

Figure 3 .
Figure 3. Prevalence of isolated foodborne bacteria from dairy products

Figure 4 .
Figure 4. Prevalence of isolated foodborne bacteria from Vegetable products

Figure 5 .
Figure 5. Diversity of bacteria in different food samples

Figure 6 (Figure 6
Figure 6(a).Antibiotics zone of inhibition against isolated bacterial pathogens

Table 1 .
Distribution of bacterial pathogens in different food samples Bacterial Chicken Chicken Chicken Breaded

Table 2 .
Incidence of Gram-negative bacteria in Meat samples Types of No. of Positive Incidence Average Positive Incidence Average Positive Incidence Average Positive Incidence Average samples percentage log.no samples percentage log.no samples percentage log.no samples percentage log.no samples percentage log.no

Table 3 .
Incidence of Gram-negative bacteria in Dairy samples Types of No. of

Table 4 .
Incidence of Gram-negative bacteria in Vegetable samples Types of No. of

Table 5 .
Antibiogram profile of foodborne pathogens