Isolation of Escherichia coli from raw milk and detection of antibiotic resistance genes by blaTEM PCR
244 / 116
Authors
-
Varsha Patange
-
Varsha Devidas Thorat
Keywords:
Raw milk, E.coli, blaTEM PCR assay, Total coliform countAbstract
Escherichia coli is an important pathogen that causes urinary, gastrointestinal infections and septicaemia. The present study aimed to identify the prevalence of E. coli in raw milk samples and relate the results with public health implications of the infection. A total of 106 raw milk samples of cattle and buffalo were collected from the local vendors and organized as well as unorganized dairy farms in and around the Mumbai region. Total coliform count and isolation of E. coli from milk samples was done on MacConkey and Eosin Methylene blue agar. The recovered isolates were subjected to a blaTEM PCR assay. The results showed that the total coliform count range was from 3.7x105 to 1.2x106, 2.6x105 to 1.8x106, 3.6x105 to 1.48x107, 4.4x105 to 6.2x106, and 4.9x105. The rate of isolation of E. coli from raw milk samples in our study was 39.62 %. In vitro antimicrobial sensitivity testing of E. coli showed that 92.85% of the isolates were sensitive to Ciprofloxacin, followed by 69.04% to Imipenem, 66.66% to Amikacin, 59.52% to Ceftriaxone, 57.14% to Tetracycline and 11.90% to Amoxicillin + clavulanic acid. Whereas 88.09% of the isolates were resistant to Amoxicillin + clavulanic acid followed by 42.85% to Tetracycline, 40.47% to Ceftriaxone, 33.33% to Amikacin, 30.95% to Imipenem and 7.14% to Ciprofloxacin. Out of 42 isolates, ten isolates (23.8%) showed a blaTEM gene-specific amplicon of 1215 bp. There is a need to reduce the contaminants by implementing appropriate measures as resistant E. coli contaminated raw milk samples were found in the Mumbai region.
References
Akarsh KL, Prejit, Asha K, Vergis J, Vinod VK, Andrews HP (2019) Occurrence and Characterization of Extended Spectrum β-lactamase Producing E. coli and Salmonella spp. from Raw Milk Samples in Wayanad District, Kerala, India. Int j curr microbiol 8: 2089-2098.
Altalhi AD, Hassan SA (2009) Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food control 20: 913-917. doi:org/10.1016/j.foodcont.2009.01.005.
Ansharieta R, Effendi, MH, Plumeriastuti H (2020) Detection of multidrug-resistant (MDR) Escherichia coli isolated from raw milk in East Java Province, Indonesia. Indian J Forensic Med Toxicol 14: 3403-3407.
Bajpai T, Pandey M, Varma M, Bhatambare G S (2017) Prevalence of TEM, SHV, and CTX-M Beta-Lactamase genes in the urinary isolates of a tertiary care hospital. Avicenna J Clin Med 7: 12-16.
Bauer AW, Kirby WM, Sherris JC and Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45(4): 149-158
Bindayna KM, Senok AC, Jamsheer AE (2009) Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bahrain. J. Infect. Public Health vol. 2:129–135.
Boor KJ, Brown DP, Murphy SC, Kozlowski SM, Bandler DK (1998) Microbiological and chemical quality of raw milk in New York State. J Dairy Sci 81: 1743-1748. doi:org/10.3168/jds.S0022-0302(98)75742-X.
Clinical and Laboratory Standards Institute (2018) Performance standards for antimicrobial susceptibility testing. M100 28th edn., Wayne, PA
Cruickshank R (1975) Medical Microbiology: A Guide Molecular detection of Staphylococcus aureus from raw milk in Mumbai 269 to Diagnosis and Control of Infection. E and S Livingston Ltd. Edinburgh, London, pp 888
De Silva SASD, Kanugala KANP, Weerakkody NS (2016) Microbiological quality of raw milk and effect on quality by implementing good management practices. Procedia Food Sci 6: 92-96. doi:org/10.1016/j.profoo.2016.02.019.
Dewangan P, Panigrahi M, Kumar A, Saravanan BC, Ghosh S, Asaf VN, Parida S, Gaur GK, Sharma D, Bhushan B (2015) The mRNA expression of immune-related genes in crossbred and Tharparkar cattle in response to in vitro infection with Theileria annulata. Mol Biol Rep 42: 1247-1255. doi:org/10.1007/s11033-015-3865-y.
Douglas JR (2003) Bulk tank cultures are the dairy man best friend. University of Wisconsin Milking Res Inst Lab (Report) No, 2223.
Edwards PR, Ewing WH (1972) Identification of Enterobacteriaceae, 3rd edn., Burgess Publishing Co., Minneapolis, USA
FAO and WHO (2004) Code of hygienic practice for milk and milk products, AC/RCP 57, Codex Alimentarius, Rome, Italy
Gran HM, Mutukumira AN, Wetlesen A, Narvhus JA (2002) Smallholder dairy processing in Zimbabwe: the production of fermented milk products with particular emphasis on sanitation and microbiological quality. Food control,13: 161-168. doi:org10.1016/S0956-7135(01)00094-9
Kaper JB, Nataro JP, Mobley HL (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 2: 123-140. doi:org/10.1038/nrmicro818.
Kumar R, Prasad A (2010) Detection of E. coli and Staphylococcus in milk and milk products in and around Pantnagar. Vet. World 3: 495.
Lanz R, Kuhnert P, Boerlin P (2003) Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet Microbiol 91: 73-84. doi:org/10.1016/S0378-1135(02)00263-8.
Lee JT, Park SY (1983) Quality of raw milk in Korea. Korean J Dairy Sci 5: 22-28.
Megersa R, Mathewos M, Fesseha H (2019) Isolation and Identification of Escherichia coli from dairy cow raw milk in Bishoftu Town, Central Ethiopia. Arch Vet Anim Sci, 1(1).
Murphy SC, Boor KJ (2000) Trouble-shooting sources and causes of high bacteria counts in raw milk. Dairy food environ sanit 20: 606-611.
Ogot H A, Ochuodho HO, Machoka R (2015) Microbial analysis of raw and boiled milk sold at Baraton center in Nandi County, Kenya. In Proceedings of the Second Annual Baraton International Interdisciplinary Research Conference Proceedings 2015 Emerging Issues in Globalization, Baraton Interdisciplinary Research Journal (pp. 1-8).
OIE (2004) Verocytotoxigenic Escherichia coli. In. Manual of Diagnostic Test and Vaccines for Terrestrial Animals. World Organisation for Animal Health (OIE), 2004; Available at: http:// www.oie.int
OIE (2009) World Animal Health Information Database - Version: 1.4. Paris, France: World Organization for Animal Health. Available at http://www.oie.int In: Bovine genital Campylobacteriosis, (2009) (last modified 2013). Animal Disease Data sheet
Ombarak RA, Hinenoya A, Elbagory ARM, Yamasaki S (2018) Prevalence and molecular characterization of antimicrobial resistance in Escherichia coli isolated from raw milk and raw milk cheese in Egypt. J Food Prot 81: 226-232.
Paghdar D, Nayak J, Mathakiya RA, Parmar BC, Gida HK, Bhavsar PP (2020) Isolation and Molecular Characterization of Extended Spectrum Beta Lactamase Producing Escherichia coli from Milk. J Anim Res 10: 143-148.
Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA (2007) Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 51: 3471-3484. doi:org/10.1128/AAC.01464-06.
Quinn PJ (1994) Clinical Veterinary Microbiology. Edinburgh, Mosby
Ryser ET (1998) Public Health Concerns. In: Applied Dairy Microbiology Marth EH and JL Steele (Eds.) Marcel Dekker Inc. New York pp: 263-403.
Saeed M O, Hassan MN, Mujeebu MA (2009) Assessment of municipal solid waste generation and recyclable materials potential in Kuala Lumpur, Malaysia. Waste Manage, 29: 2209-2213.
Sambrook J, Russell DW(2006) The condensed protocols from molecular cloning: a laboratory manual.
Shojaei ZA, Yadollahi A (2008) Physicochemical and microbiological quality of raw, pasteurized and UHT milks in shops. Asian J Sci Res 1: 532-538
Soomro AH, Arain MA, Khaskheli M, Bhutto B (2002) Isolation of Escherichia coli from raw milk and milk products in relation to public health sold under market conditions at Tandojam, Pakistan. Pakistan J Nutr, 1: 151-152, doi: 10.3923/ pjn.2002.151.152
Thaker HC, Brahmbhatt MN, Nayak JB (2012) Study on occurrence and antibiogram pattern of Escherichia coli from raw milk samples in Anand, Gujarat, India. Vet World 5: 556
Wanjala GW, Mathooko FM, Kutima PM, Mathara J M (2017) Microbiological quality and safety of raw and pasteurized milk marketed in and around Nairobi region. African J Food Agric Nutr Dev 17: 11518-11532. doi:org/10.18697/ajfand.77.15320
Yohannes G (2018) Isolation, identification and antimicrobial susceptibility testing of Escherichia coli isolated from selected dairy farms in and around Mekelle, Tigray, Ethiopia. J Vet Sci Technol 9: 518.
