Genetic diversity, virulence and distribution of antimicrobial resistance among Listeria monocytogenes isolated from milk, beef, and bovine farm environment

Document Type : Full paper (Original article)


1 Ph.D. Student in Veterinary Public Health, Department of Veterinary Public Health and Epidemiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India

2 Department of Veterinary Public Health and Epidemiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India

3 Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Salem-636 112, Tamil Nadu, India

4 Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India

5 Translational Research Platform for Veterinary Biologicals (TRPVB), Madhavaram Milk Colony, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 051, Tamil Nadu, India

6 Division of Animal Health, ICAR Research Complex for North Eastern Hill Region, Meghalaya-793 103, India


Background: Listeria monocytogenes is an opportunistic intracellular foodborne pathogen and is ubiquitous in nature. The occurrence of L. monocytogenes in animal production units coupled with their presence in milk, faeces, feed, water, sewage, and soil is a contributory factor for foodborne listeriosis in humans and animals. Aims: The study was aimed to characterize genotype and serogroup of L. monocytogenes recovered from different types of samples and also to study antimicrobial patterns by phenotypic and genotypic methods. Methods: Multiplex polymerase chain reaction (PCR) was used for the confirmation of L. monocytogenes, the identification of its serogroup and lineage, and the detection of virulence markers. Enterobacterial repetitive intergenic consensus (ERIC), and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize those isolates, and antimicrobial patterns were studied phenotypically by Kirby-Bauer method and genotypically by PCR. Results: Out of the screened 474 samples (274 milk and 50 each of soil, feed, sewage, and beef), ten L. monocytogenes isolates (milk=8, soil=1, and beef=1) were confirmed by PCR targeting the hlyA gene and found to belong to the 1/2a, 3a serogroup and fall under type II lineage. Virulence potential assessment revealed that all the ten isolates harbored the iap gene while the presence of plcA and plcB genes were noticed in seven and eight isolates respectively. Six isolates from milk were found to group in the same cluster by ERIC and RAPD fingerprinting, suggesting both methods to be efficient molecular typing tools for L. monocytogenes. Genotypic characterization of antimicrobial resistance (AMR) genes revealed that seven isolates were positive for tetM, five for mefA, four for msrA, and one for lnuA genes while none of the isolates showed tetK, ermA, ermB, and lnuB genes. Conclusion: The presence of L. monocytogenes in bovine farm environments coupled with virulence markers, and multidrug resistance from the study area suggest a possible transmission from the environment to humans and animals which needs to be monitored regularly to ensure food safety and the well-being of animals and humans.


Main Subjects

Agersborg, A; Dahl, R and Martinez, I (1997). Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods. Int. J. Food Microbiol., 35: 275-280.
Bilung, LM; Chai, LS; Tahar, AS; Ted, CK and Apun, K (2018). Prevalence, genetic heterogeneity, and antibiotic resistance profile of Listeria spp. and Listeria monocytogenes at farm level: A highlight of ERIC- and BOX-PCR to reveal genetic diversity. Biomed. Res. Int., Article ID 3067494, 12 pages. 2018/3067494.
Biswas, BK and Chandra, S (2011). Presence of Listeria spp. in ice cream and sewage water particularly Listeria monocytogenes and its pathogenicity. Int. J. Sci. Tech., 2: 36-39.
Biswas, AK; Kondaiah, N; Bheilegaonkar, KN; Anjaneyulu, AS; Mendiratta, SK; Jana, CH and Singh, RR (2008). Microbial profiles of frozen trimmings and silver sides prepared at Indian buffalo meat packing plants. Meat Sci., 80: 151-154.
Bozdogan, B; Berrezouga, L; Kuo, M; Yurek, D; Farley, K; Stockman, B and Leclercq, R(1999). A new resistance gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025. Antimicrob. Agents Chemother., 43:925-929.
Chen, M; Wu, Q; Zhang, J and Wang, J (2014). Prevalence and characterization of Listeria monocytogenes isolated from retail-level ready-to-eat foods in South China. Food Control. 38: 1-7.
Colaço, D (2011). Characterization of Escherichia coli and Listeria isolated from milk at different levels of collection and processing in Goa. Ph.D. Thesis, Goa University, Goa.
Dhama, K; Karthik, K; Tiwari, R; Shabbir, MZ; Barbuddhe, SB; Malik, SVS and Singh, RK (2015). Listeriosis in animals, its public health significance (food-borne zoonosis) and advances in diagnosis and control: a comprehensive review. Vet. Quart., 35: 211-235. doi: 10.1080/01652176.2015.1063023.
Doijad, SP; Barbuddhe, SB; Garg, S; Poharkar, KV; Kalorey, DR; Kurkure, NV; Rawool, DB and Chakraborty, T (2015). Biofilm-forming abilities of Listeria monocytogenes serotypes isolated from different sources. PLoS One. 10: e0137046. doi: 10.1371/journal pone.0137046.
Doijad, SP; Vaidya, V; Garg, S; Kalekar, S; Rodrigues, J; D’costa, D; Bhosle, S and Barbuddhe, SB (2010). Isolation and characterization of Listeria species from raw and processed meats. J. Vet. Public Health Sci., 8: 83-88.
Doumith, M; Buchrieser, C; Glaser, P; Jacquet, C and Martin, P (2004). Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J. Clin. Microbiol., 42: 3819-3822. doi:10.1128/JCM.42.8.3819-3822.2004.
Enweani, IB; Esumeh, FI; Akpe, RA; Akharume, EE; Aghaiyo, ID; Igbinaduwa, IN and Igbinovia, AE (2003). Isolation of Listeria species from water bodies, sewage and soil samples. J. App. Basic Sci., 1(1-2): 87-90.
Fox, E; O’Mahony, T; Clancy, M; Dempsey, R; O’Brien, M and Jordan, K (2009). Listeria monocytogenes in the Irish dairy farm environment. J. Food Prot., 72: 1450-1456.
Furrer, B; Candrian, U; Hoefelein, C and Luethy, J (1991). Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments. J. Appl. Bacteriol. 70: 372-379. doi:10.1111/j.1365-2672.1991. tb02951.x.
Hunter, PR and Gaston, MA (1988). Numerical index of the discriminatory ability of typing systems: An application of Simpson’s index of diversity. J. Clin. Microbiol., 26: 2456-2466.
Jamali, H; Radmehr, B and Thong, KL (2013). Prevalence, characterisation, and antimicrobial resistance of Listeria species and Listeria monocytogenes isolates from raw milk in farm bulk tanks. Food Control. 34: 121-125.
Krumperman, PH (1983). Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl. Environ. Microbiol., 46: 165-170.
Lina, G; Quaglia, A; Reverdy, ME; Leclercq, R; Vandenesch, F and Etienne, J (1999). Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among Staphylococci. Antimicrob. Agents Chemother., 43: 1062-1066.
Matto, C; Varela, G; Braga, V; Vico, V; Gianneechini, RE and Rivero, R (2018). Detection of Listeria spp. in cattle and environment of pasture-based dairy farms. Pesqui. Vet. Bras., 38: 1736-1741.
Morvan, A; Moubareck, C; Leclerc, A; Hervé-Bazin, M; Bremont, S; Lecuit, M; Courvalin, P and Le Monnier, A (2010). Antimicrobial resistance of Listeria monocytogenes strains isolated from humans in France. Antimicrob. Agents Chemother., 54: 2728-2731.
Nayak, JB; Brahmbhatt, MN; Savalia, CV; Bhong, CD; Roy, A and Kalyani, IH (2010). Detection and characterization of Listeria species from buffalo meat. Buffalo Bull. 29: 83-94.
Nightingale, KK; Schukken, YH; Nightingale, CR; Fortes, ED; Ho, AJ; Her, Z; Grohn, YT; McDonough, PL and Wiedmann, M (2004). Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment. Appl. Environ. Microbiol., 70: 4458-4467.
Nishibori, T; Cooray, K; Xiong, H; Kawamura, I; Fujita, M and Mitsuyama, M (1995). Correlation between the presence of virulence associated genes as determined by PCR and actual virulence to mice in various strains of Listeria spp. Microbiol. Immunol., 39: 343-349. doi:10.1111/j.1348-0421.1995.tb02211.x.
Notermans, SH; Dufrenne, J; Leimeister-Wächter, M; Domann, E and Chakraborty, T (1991). Phophatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl. Environ. Microb., 57: 2666-2670.
Odjadjare, EEO and Okoh, AI (2010). Prevalence and distribution of Listeria pathogens in the final effluents of a rural wastewater treatment facility in the Eastern Cape Province of South Africa. World J. Microbiol. Biotechnol., 26: 297-307.
Orsi, RH; Den Bakker, HC and Wiedmann, M (2011). Listeria monocytogenes lineages: genomics, evolution, ecology, and phenotypic characteristics. Int. J. Med. Microbiol., 301: 79-96.
Paziak-Domanska, B; Boguslawska, E; Wieckowska-Szakiel, M; Kotlowski, R; Rozalska, B; Chmiela, M; Kur, J; Dabrrowski, W and Rudnicka, W (1999). Evaluation of the API test, phosphatidylinositol-specific Phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods. FEMS Microbiol. Lett., 171: 209-214.
Rawool, DB; Doijad, SP; Poharkar, KV; Negi, M; Kale, SB; Malika, SVS; Kurkure, NV; Chakraborty, T and Barbuddhe, SB (2016). A multiplex PCR for detection of Listeria monocytogenes and its lineages. J. Microb. Methods. 130: 144-147.
Sarangi, LN and Panda, HK (2012). Isolation, characterization and antibiotic sensitivity test of pathogenic Listeria species in livestock, poultry and farm environment of Odisha. Indian J. Anim. Res., 46: 242-247.
Seeliger, HPR and Jones, D (1986). Genus Listeria. In: Kandler, O and Weiss, N (Eds.), Regular, non-sporing Gram-positive rods. Bergey’s manual of systematic bacteriology. (2nd Edn.), Baltomore, Williams and Wilkins. PP: 1235-1245.
Shakuntala, I; Das, S; Ghatak, S; Milton, AAP; Rajkumari, S; Puro, K; Pegu, RK; Duarah, A; Barbuddhe, SB and Sen, A (2019). Prevalence, characterization, and genetic diversity of Listeria monocytogenes isolated from foods of animal origin in North East India. Food Biotechnol., 33: 237-250. doi: 10.1080/08905436.2019.1617167.
Shantha, S and Gopal, S (2014). Prevalence of Listeria species in environment and milk samples. Adv. Anim. Vet. Sci., 2: 1-4.
Soni, DK; Singh, RK; Singh, DV and Dubey, SK (2013). Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples at Varanasi, India. Infect. Genet. Evol., 14: 83-91. doi: 10.1016/j.meegid.2012.09.019.
Su, X; Zhang, S; Shi, W; Yang, X; Li, Y; Pan, H; Kuang, D; Xu, X; Shi, X and Meng, J (2016). Molecular characterization and antimicrobial susceptibility of Listeria monocytogenes isolated from foods and humans. Food Control. 70: 96-102.
Swaminathan, B and Gerner-Smidt, P (2007). The epidemiology of human listeriosis. Microb. Infect., 9: 1236-1243.
Taherkhani, A; Attar, HM; Moazzam, MA; Mirzaee, SA and Jalali, M (2013). Prevalence of Listeria monocytogenes in the river receiving the effluent of municipal wastewater treatment plant. Int. J. Environ. Health Eng., 2: 49.
Ward, TJ; Ducey, TF; Usgaard, T; Dunn, KA and Bielawski, JP (2008). Multilocus genotyping assays for single nucleotide polymorphism-based subtyping of Listeria monocytogenes isolates. Appl. Environ. Microbiol., 74: 7629-7642.