A Molecular Investigation of Extended Spectrum Beta-Lactamase genes in Escherichia coli and Klebsiella spp. in raw cow Milk

Abstract

Objective: Raw milk is an important source of nutrients. Therefore, today, there is a great demand for raw milk consumption. The positive side of milk consumption on growth and development cannot be ignored, but unfortunately, pathogens in raw milk are always potential public health risks for transmission pathogens. Bacteria such as Enterobacteriaceae in normal flora can cause serious problems due to their extended-spectrum beta-lactamase (ESBL) production. These bacteria and their resistance genes have been reported in raw milk. In this matter, the aim of the study is to determine the status of blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes responsible for the production of ESBL enzyme in Escherichia coli and Klebsiella spp. strains to identify risk factors in raw milk consumption and to gain an understanding of the epidemiology of these resistant strains. Materials and methods: A total of different 50 raw milk samples were collected and subjected to phenotypic microbiological analysis and Real-time PCR targeting blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes. In the phenotypic analyses, suspicious isolates were identified by classical microbiological methods and antibiotic resistance profiles were revealed. Results: These results indicated that raw milk is a potential reservoir for ESBL producing E. Coli, Klebsiella spp. strains are obviously significant. And It was determined that CTX-M based ESBL genes are predominant in ESBL production. The present study revealed that raw milk is epidemiologically involved in the transmission of ESBL genes. Raw milk could be distributed to ESBL genes widely with which is consumed in Şanlı Urfa.

Keywords

• e coli
• esbl genes
• klebsiella spp
• raw cow milk
• qPCR

References

1. Altun B, Besler T and Ünal S, (2002): Ankara’da satılan sütlerin değerlendirilmesi. Sted. 11(2):51.
2. Ansharieta R, Ramandinianto SC, Effendi MH, et al., (2021): Molecular identification of blaCTX-M and blaTEM genes encoding extended-spectrum ß-lactamase (ESBL) producing Escherichia coli isolated from raw cow’s milk in East Java, Indonesia. Biodiversitas Journal of Biological Diversity, 2021: 22(4).
3. Athanasakopoulou Z, Reinicke M, Diezel Y, et al., (2021): Antimicrobial Resistance Genes in ESBL-Producing Escherichia coli Isolates from Animals in Greece. Antibiotics, 10(4), 389.
4. Badri AM, Ibrahim IT, Mohamed SG, et al., (2017): Prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, and Klebsiella pneumoniae isolated from raw milk samples in Al Jazirah state, Sudan. Mol. Biol, 7(1): 201.
5. Baran A, Adigüzel MC, and Yüksel M, (2020): Prevalence of Antibiotic-Resistant and Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Chicken Meat from Eastern Turkey. Pak. Vet. J, 40, 355-359.
6. Casti D, Scarano C, Pala C, et al. (2016): Evolution of the Microbiological Profile of Vacuum-Packed Ricotta Salata Cheese During Shelf-Life. Ital J Food Saf. 5(2):5501.
7. Demirci M, Celepler Y, Dincer S, et al., (2020): Should we leave the paper currency? A microbiological examination. Rev Esp Quimioter. 2020;33(2):94-102.
8. Diassa N, Sibhat B, Mengistu S, et al., (2017): Prevalence and Antimicrobial Susceptibility Pattern of E. coli O157:H7 Isolated from Traditionally Marketed Raw Cow Milk in and around Asosa Town, Western Ethiopia. Veterinary Medicine International. 7581531.

9. EUCAST, The European Committee on Antimicrobial Susceptibility Testing. Routine and extended internal quality control for MIC determination and disk diffusion as recommended by EUCAST. Version 6.1, 2016. [cited 11 November 2021]. Available from: http://www.eucast.org.
10. Gaffer W, Gwida M, Samra RA, Al-Ashmawy M, (2019): Occurrence and molecular characterization of extended-spectrum beta-lactamase-producing Enterobacteriaceae in milk and some dairy products. Slovenian Veterinary Research, 56.
11. Jena J, Sahoo RK, Debata NK, Subudhi E, (2017): Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum β-Lactamase-producing Escherichia colistrain isolated from urinary tract infections in adults.3 Biotech, 7 (4): 244.
12. Kim IS, Hur YK, Kim EJ, et al., (2017): Comparative analysis of the microbial communities in raw milk produced in different regions of Korea. Asian-Australas J Anim Sci. 30(11):1643-1650.
13. Odenthal S, Akineden Ö, Usleber E, (2016): Extended-spectrum β-lactamase producing Enterobacteriaceae in bulk tank milk from German dairy farms. Int J Food Microbiol, 238: 72-78.
14. Oliver SP, Boor KJ, Murphy SC, et al. (2009): Food safety hazards associated with consumption of raw milk. Foodborne Pathog Dis. 6(7):793-806.
15. Paghdar D, Nayak J, Mathakiya RA, Parmar, et al., (2020): Isolation and Molecular Characterization of Extended-Spectrum Beta-Lactamase Producing Escherichia coli from Milk. Journal of Animal Research, 10(1): 143-148.
16. Plassard V, Gisbert P, Granier SA, et al., (2021): Surveillance of Extended-Spectrum β-Lactamase-, Cephalosporinase- and Carbapenemase-Producing Gram-Negative Bacteria in Raw Milk Filters and Healthy Dairy Cattle in Three Farms in Île-de-France, France. Front Vet Sci. 8:633598.
17. Ramos, GLDPA and Santos Nascimento J. (2021): Antibiotic resistance profile and detection of degradative enzymes by Enterobacteriaceae isolated from raw goat milk. Germs, 11(2), 211.
18. Salinas L, Loayza F, Cárdenas et al., (2021): Environmental Spread of Extended-Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli and ESBL Genes among Children and Domestic Animals in Ecuador. Environmental health perspectives, 129(2): 027007.
19. Shi Y, Chen P, Huo W, Chen X, Barkema HW, De Buck J, Gao J, (2020): ESBL-Producing Escherichia coli from Bovine Mastitis Induced Apoptosis of Bovine Mammary Epithelial Cells Via Alteration of ROS/MMP/bax/bcl-2 Signaling Pathway. Pakistan Veterinary Journal, 40(3).
20. Tekiner İH, Özpınar H, (2016): Occurrence and characteristics of extended-spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin. Braz J Microbiol. 47(2):444-51.
21. Tóth AG, Csabai I, Krikó E, et al., (2020): Antimicrobial resistance genes in raw milk for human consumption. Sci Rep. 10(1):7464. doi:10.1038/s41598-020-63675-4.
22. Uraz G , Arslan S, (1998): Beyaz Peynir, Çiğ ve Pastörize Süt Örneklerinden İzole Edilen Bakterilerde İodometrik Test ve Kromojenik Sefalosporin Test (Nitrocefin) Yöntemleriyle Beta Laktamaz Araştırması. GIDA. 23(2):147-155.
23. Vendramin T, Kich DM, Moline RD et al ., (2014): Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms. Food Sci. Technol (Campinas), 34 (3):604-608.
24. Waade J, Seibt U, Honscha W, et al., (2021): Multidrug-resistant enterobacteria in newborn dairy calves in Germany. PLoS One. 16(3):e0248291.
25. Widodo A, Effendi MH, Khairullah AR, (2020): Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli from livestock.Sys Rev Pharm, 11(7): 382-392.
26. Yao F, Qian Y, Chen S, et al., (2007): Incidence of extended-spectrum beta-lactamases and characterization of integrons in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolated in Shantou, China. Acta Biochim Biophys Sin (Shanghai) 39 (7): 527-532.
27. Yun MJ. and Lee YJ, (2020): Characteristics of Escherichia coli from bulk tank milk of dairy company. Journal of Prventive Veterinary Medicine, 2020: 44(4), 236-239.