Multiantibiotic-resistant Pattern against Pathogenic Bacteria Isolated from...

[Full title: Multiantibiotic-resistant Pattern against Pathogenic Bacteria Isolated from Ready to Eat Food in Ludhiana]

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Asian Journal of Pharmaceutic s • Apr-Jun 2018 (Suppl) • 12 (2) | S 631 Multiantibiotic-resistant Pattern against Pathogenic Bacteria Isolated from Ready to Eat Food in Ludhiana Naresh Kumar, Op Jangir Department of Biotechnology, Maharaj Vinayak, Global University, Jaipur, Rajasthan, India Abstract Objective: The study was conducted to investigate the bacteriological contamination in the most common street food sold in Ludhiana at different places. 90 samples of street food samples were randomly collected from the selected part of Ludhiana. Samples investigated for the occurrence of bacteria by culturing them on selective and differential culture media. Methods: To detect the presence of pathogenic bacteria in food recommended method used like Spread Plate Techniques and identification of bacteria accomplished with the help of differential media and specific media, biochemical testing and molecular characterization also performed to ensure the particular strain of enteropathogenic bacteria. Results: The presence of pathogenic bacteria isolated from all street food were quite high, the percentage of Proteus sp. was 2%. Predominantly staphylococcus sp were found in street food. This was followed by Salmonella (6%) and Staphylococcus aureus (14%). Klebsiella sp. (5%) and Enterococcus sp. (2%) were least predominant in these foods. Conclusions: The study exhibited contagion may be due to poor techniques of food preparation adopted by the street food sellers and its big concern community well-being as well as bad for economy state and India, and there is the loss of 5.2% of national gross domestic product. These bacteria are also known for foodborne illnesses and toxic syndrome Keywords: Coliform count, foodborne illness, pathogenic bacteria, street food INTRODUCTION S treet foods are the food which sold out in streets and roadside, where the commuters or local persons are coming for business or shopping, enjoyment purpose; for instance, these areas are the court, chopati, bus stand, and banking business area. The food sold in streets or roadside, either already prepared or required mild preparation before serving to the person [1] Ingesting of such food may lead to food poisoning and increases the risk of foodborne diseases [2,5] A spread of bacteria causes food poisoning and gastroenteritis basically through the unclean water mixed with fecal material such as food, water, nails, and hand or unhygienic way of handling [20] Especially in developing countries, it is difficult to safeguard the food from cross contamination [6] Street food sellers profited from a direct cash flow and regularly avoid taxation, and second, they can fix their own working hours. Vending snacks, food, and juices or beverages at relatively low prices in comparison of well-maintained hotels and cafeteria, these essential services are easily available for the workers, shoppers, travelers, and people with low wages [5] The individuals who love to have such food are concerned with accessibility instead of its safety, quality, and hygiene. In processing of the street food, clean water to be used but clean water is hardly available specially during the rainy season, sometimes rainy water mixed with municipal corporation water and if this contaminated water used by street vendors may cause health problems [13,14] Even the availability of space for utensils washing readily available, sometimes roadside vendors leave utensils in buckets without washing that attracts the flies and rodents leading to food poisoning. Storing the food at low temperature or under refrigeration protects from direct contact of flies and rodent’s and bacterial multiplication. Keeping these facilities is very difficult for the people having low income that is why the consumers depending on roadside foods, faces the maximum intestinal problem as well as additional food-borne illness after ingesting unlearned food [13] RESEARCH AR TICLE Address for correspondence: Naresh Kumar, Department of Biotechnology, Maharaj Vinayak, Global University, Jaipur, Rajasthan, India. E-mail: naersh 1.kumar@outlook.com Received: 12-03-2018 Revised: 03-06-2018 Accepted: 16-06-2018

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Kumar and Jangir: Multiantibiotic-resistant pattern in street food bacteria Asian Journal of Pharmaceutic s • Apr-Jun 2018 (Suppl) • 12 (2) | S 632 These hawkers or street food sellers’ play an important role in supply food for travelers and urban dwellers at reasonable and easily accessible [8] The demand of ready-to-eat is suitable and growing day by day due to lifestyle change METHODS Location of the study Ludhiana is situated in central part of the district which found Malwa area of Punjab. The population increases considerably during the harvesting season due to the migration of laborers from the eastern states of Uttar Pradesh, Bihar, Odisha, and Delhi. The resources are occupied mainly by small-scale manufacturing units, which manufacture industrial goods, machinery parts, motor vehicle parts, domestic appliances, woolen garments, and another type of garments. Ludhiana is considered as Asia’s largest center for bicycle manufacturing more than 50% of India’s bicycle consumption of approximately 10 million every year, so here office employee and labor mostly depend on the street food. City failed in purity test of most food and water, so researcher has collected Collection of samples Ninety food samples were collected from street vendors at Chandigarh Road, Samrala Chowk, Clock Tower, and Jalandhar bypass. The samples were noodles, burger, bhelpuri, samosa chaat, hulcha chole, masala dosa, chole bhature, chicken tandoori, and pote kaleji, these items are prepared either by frying or boiling, and some ingredients are directly used in serving food; samples were aseptically collected and placed in ice pack contain portable thermocol box and processed in the microbiology laboratory Isolation and identification of bacteria Methodology Ten (10 g) of all food material was taken and made homogenate mixture in mortar and pestle, of that 1 ml of homogenate was added to 9 ml sterile normal saline and prepared the serial dilution of that up to 10 −6 The spread plate technique was used in that, diluted sample were poured on Salmonella-Shigella agar and eosin methylene blue (EMB) agar, and detected blue-black greenish metallic sheen colony of Escherichia coli on EMB, mucoid colonies of Klebsiella on MacConkey agar and black-centred colonies of Salmonella on SS media after a 24 h incubation at 37°C. Proteus sp. was streaked on blood agar to check the swarming growth. All probable isolates were further identified using Kligler Iron Agar [9] Biochemical testing was also performed to confirm the type of bacterial strain after isolation it from street food. To validate the report the quality control also done by using specific bacterial strain purchased from MTCC Molecular characterization of bacteria Pure culture was isolated from food samples and was analyzed by following the standard method, the confirmation isolated bacteria, and the result was matched with the bacteria isolated by the conventional method [8] In polymerase chain reaction protocol 16 S rRNA Universal primers, gene fragment was amplified by means of thermal cycler (MJ Research Peltier make) [11,12] Primer details Primer name Sequence details Number of bases 27 F AGAGTTTGATCMTGGCTCAG 20 1492 R TACGGYTACCTTGTTACGACTT 22 Antibiotic susceptibility testing The sensitivity test by the Kirby–Bauer disc diffusion technique was done against the bacteria isolated from the street food at Ludhiana. 10 different antibiotics with different strength viz: Amikacin, ampicillin cefotaxime, ciprofloxacin, cefazolin, ceftazidime, gentamycin, imipenem, levofloxacin and tetracycline [Graph 2] were used. Bacteria stock solution prepared in normal saline and with the help of sterile swab sticks applied on Muller Hinton (MH) agar. The antibiotic discs were aseptically sited on the MH media using sterile forceps and incubated at 37°C for 24 h after incubation report noted in a format of sensitive or resistant [12] RESULTS AND DISCUSSION The non-vegetarian food samples noted the highest showing almost 50%, whereas in vegetarian food, there was 32% [Graph 3]. The finding of seven different pathogenic bacteria was isolated from street foods in that Proteus sp. were 2% staphylococci was the most predominant. This was followed by Salmonella (6%) and Staphylococcus aureus (14%). Klebsiella sp. (5%) and Enterococcus sp. (2%) were the least predominant [4,10] [Graph 1]. The vendors stay on road or in streets where these foods are contaminated by vehicles passing through. Serving water, direct municipal water, also contributes to several pathogenic bacteria in street food which leads to gastroenteritis or diarrheal diseases, throat infection, etc [6] As a result, access to clean water and health edification to sellers on personal hygiene, food protection, and appropriate disposal improves food quality and reduces foodborne diseases [7] Antimicrobial sensitivity test Drug susceptibility against isolated bacteria was performed by disc diffusion; one well-isolated colony was selected from

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Kumar and Jangir: Multiantibiotic-resistant pattern in street food bacteria Asian Journal of Pharmaceutic s • Apr-Jun 2018 (Suppl) • 12 (2) | S 633 the MS agar plates and EMB agar plate. A colony was touched with a sterile loop and streaked over routinely used nutrient agar and incubated overnight at 37°C. Few well-isolated colonies were inoculated into a tube of sterile physiological saline and mixed well before use [15] A sterile cotton swab sticks were dipped into the bacterial suspension [19] The Graph 1: Number of isolated organisms from food samples Graph 3: Total bacterial isolates in vegetarian and non-vegetarian foods Graph 2: Regarding antibacterial activity against all isolated bacteria

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Kumar and Jangir: Multiantibiotic-resistant pattern in street food bacteria Asian Journal of Pharmaceutic s • Apr-Jun 2018 (Suppl) • 12 (2) | S 634 swab was streaked over the entire surface of Mueller-Hinton agar medium, 3 times, rotating the plate approximately 60° after each application to ensure an even distribution of the inoculums. After incubation the diameter of the zone of complete inhibition, their results are shown in Figure 1 CONCLUSION This study clearly indicated significant levels of contamination in street-vended foods of Ludhiana city. The presence of enteropathogenic or pathogenic bacteria count in all the street samples was high and can be correlated to a number of factors such as inappropriate management and processing, the use of dirty utensils, and rinsing water as probable sources of bacterial contamination in street vendor food [17,18] Most street hawker or vendors did not have basic knowledge of hygiene and how to protect the food from bacterial contamination. However, the bacterias isolated from the food are pathogenic and people are catching the infection after consuming it and even the consumer also hardily bother about it. The presence of S. aureus is an enterotoxins producer and can cause serious health issues. Excess use of antibiotic also the serious issue for world physicians, because some physicians are prescribing antibiotic without antibiotic sensitivity test or diagnosis and consequences of that bacteria becoming multiantibiotic resistant .[3,16] ACKNOWLEDGMENTS The authors would like to acknowledge the support of microbiology laboratory in LPU allowing us to conduct our research work. The authors also wished to state appreciation and sincere thankfulness to their esteemed supervisor Dr. O. P. Jangir Professor, Department of Biotechnology, Maharaj Vinayak, Global University Jaipur, for his priceless help and friendly help in carrying out my research REFERENCES 1. Sultana F, Kamrunnahar, Afroz H, Jahan A, Fakruddin M, Datta S. Multi-antibiotic resistant bacteria in frozen food (ready to cook food) of animal origin sold in Dhaka, Bangladesh. Asian Pac J Trop Biomed 2014;4 Suppl 1: S 268-71 2. Mensah P, Yeboah-Manu D, Owusu-Darko K, Ablordey A. Street foods in Accra, Ghana: How safe are they? Bull World Health Organ 2002;80:546-54 3. Wang H, McEntire JC, Zhang L, Li X, Doyle M. The transfer of antibiotic resistance from food to humans: Facts, implications and future directions. Rev Sci Tech Off Int Epiz 2012;31:249-60 4. Nyenje ME, Odjadjare CE, Tanih NF, Green E, Ndip RN. Foodborne pathogens recovered from ready-to-eat foods from roadside cafeterias and retail outlets in Alice, Eastern Cape province, South Africa: Public health implications Int J Environ Res Public Health 2012;9:2608-19 5. Zige DV, Ohimain EI, Mynepalli SK. Enteric Bacteria from ready to eat food vended in Amassoma community in Niger Delta and its health implication. IOSR J Environ Sci Toxicol Food Technol 2013;6:62-5 6. Wadhai VS, Khobragade KD. Evaluation of microbiological safety of Indian chutneys: A case study of Chandrapur City, India. Sci Res Rep 2012;2:56-8 7. Bai S, Zhao J, Zhang Y, Huang W, Xu S, Chen H, et al . Rapid and reliable detection of 11 foodborne pathogens using thin-film biosensor chips. Appl Microbiol Biotechnol 2010;86:983-90 8. Hafeez Y, Iqbal A, Ahmad M. Biotyping of Bacillus cereus from the street vended foods in Srinagar area of Kashmir, India. Vet World 2012;5:590-3 9. Odu NN, Akano UM. The Microbiological assessment of ready-to-eat-food (shawarma) in port Harcourt City, Nigeria. Nat Sci 2012;10:1-8 10. Kumar A, Bhushan V. Isolation and characterization of microorganisms responsible for different types of food spoilages. Int J Res Pure Appl Microbiol 2011;1:22-31 11. Dias TM, Bricio SM, Almeida DO. Molecular characterization and evaluation of antimicrobial susceptibility of enteropathogenic E. coli (EPEC) isolated from minas soft cheese. Ciência Tecnol Alime 2012;32:747-53 12. Sharma I, Mazumdar JA. Assessment of bacteriological quality of ready to eat food vended in streets of Silchar city, Assam, India. Indian J Med Microbiol 2014;32:169-71 13. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6 14. Tambekar DH, Jaiswal VJ, Dhanorkar DV, Gulhane PB, Dudhane MN. Identification of microbiological hazards and safety of ready-to-eat food vended streets of Amravati City, India. J Appl Biosci 2008;7:195-201 15. Rindhe SN, Zanjad PN, Doifode VK, Siddique A, Mendhe MS. Assessment of microbial contamination of chicken products sold in Parbhani city. Vet World 2008;1:208-10 16. Bhaisare DB, Thyagarajan D, Churchil RR, Punniamurthy N. Bacterial pathogens in chicken meat: Figure 1: Antimicrobial sensitivity test against isolated organism

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Kumar and Jangir: Multiantibiotic-resistant pattern in street food bacteria Asian Journal of Pharmaceutic s • Apr-Jun 2018 (Suppl) • 12 (2) | S 635 Review. Int J Life Sci Res 2014;2:1-7 17. Available from: http://epaper.tribuneindia.com/1687344/ Ludhiana-Tribune/LT-07-June-2018#page/1/1 MostFood, WaterSamplesFail Purity Test–Ludhiana.[Last accessed on 2018 June 07] 18. Gashaw A, Kassu A, Moges F, Tiruneh M, Huruy K. Prevalence of Bacteria and intestinal parasites among food-handlers in Gondar Town, Northwest Ethiopia. J Health Popul Nutr 2008;26:451-5 Source of Support: Nil. Conflict of Interest: None declared 19. Wayne Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing Wayne, PA: Wayne Clinical and Laboratory Standards Institute; 2007. p. 17 20. Stuhlmeier R, Stuhlmeier KM. Fast, simultaneous, and sensitive detection of staphylococci. J Clin Pathol 2003;56:782-5.

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