Keywords
sequence analysis
bacterial genomes
livestock
MLVA
How to Cite
Abstract
Clostridium perfringens is a component of the normal gut microbiota in humans and animals. However, it can become pathogenic under certain conditions, causing serious intestinal and systemic diseases. Enterotoxaemia is one of these diseases causing significant mortality in livestock annually. Analysis of genetic diversity or relatedness of isolates with molecular typing methods can be helpful for understanding the epidemiology of these infections and observing population structures of these bacteria. The aim of this study was to perform molecular typing of Clostridium perfringens isolates obtained from both healthy and clinically suspected enterotoxemic livestock in Fars Province using Multiple Locus Variable Tandem Repeat Analysis (MLVA) method. Collection of previously isolated and preserved Clostridium perfringens along with new collected isolates from healthy and diseased livestock were evaluated for toxin type and some virulence genes including cpe, tpeL, netB, and β2. Genetic typing of isolates was done using specific primers for amplification of eight Variable Number Tandem Repeat (VNTR) loci. PCR was done and electrophoresis banding patterns of fragments were analyzed by BioNumerics software. The results of typing revealed 71 distinct MLVA profiles from 74 strains. The strains analyzed included 27 clinical isolates, 43 non-clinical isolates, three vaccine strains, and the reference strain 13 obtained from the GenBank database. Clostridium perfringens strain 13 sequence was used as a reference strain and was evaluated for comparison of sequences for each locus. Clustering of isolates according to the minimum spanning tree showed 5 different clusters, excluding three orphan isolates. Clinical and non-clinical isolates were scattered in all clusters and no defined cluster related to health status, date of isolation, kind of host and place of isolation was detected. The total diversity index of our typing method was estimated to be 0.99. Overall MLVA molecular typing method shows high genetic diversity among Clostridium perfringens isolates.
References
Afshari, A., Jamshidi, A., Razmyar, J. Rad, M. 2016. Genomic diversity of Clostridium perfringens strains isolated from food and human sources. Iranian Journal of Veterinary Research, 17, 3, 160-164.
Alimolaei, M., Shamsaddini Bafti, M. 2023. Isolation and Molecular Characterization of Clostridium perfringens Toxinotypes F & G in Diarrhoeic Sheep (Ovis aries) Flocks in Southeast of Iran. Archives of Razi Institute, 78, 3, 1159–1168. https://doi.org/10.22092/ARI.2023.360450.2582
Bailey, M.A., Macklin, K.S., Krehling, J.T. 2013. Use of a Multiplex PCR for the Detection of Toxin-Encoding Genes netB and tpe Lin Strains of Clostridium perfringens. ISRN Veterinary Science, 11, 865702. https://doi.org/10.1155/2013/865702
Benson, G. 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Research, 27, 2, 573–580. https://doi.org/ 10.1093/nar/27.2.573
Bueschel, D.M., Jost, B.H., Billington, S.J., Trinh, H.T., Songer, J.G. 2003. Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: Correlation of genotype with phenotype. Veterinary Microbiology, 94, 2, 121-129. https://doi.org/10.1016/s0378-1135(03)00081-6
Camargo, A., Guerrero-Araya, E., Castañeda, S., Vega, L., Cardenas-Alvarez, M.S., Rodríguez, C., Paredes-Sabja, D., Ramírez J.D., Muñoz, M. 2022. Intra-species diversity of Clostridium perfringens: A diverse genetic repertoire reveals its pathogenic potential. Frontiers in Microbiology, 22 13, 952081. https://doi.org/10.3389/fmicb.2022.952081
Chalmers, G., Martin, S.W., Prescott, J.F., Boerlin, P. 2008. Typing of Clostridium perfringens by multiple-locus variable number of tandem repeats analysis. Veterinary Microbiology, 128, 1-2, 126–136. https://doi.org/ 10.1016/j.vetmic.2007.09.018
Chan, G., Farzan, A., Soltes, G., Nicholson, V.M., Pei, Y., Friendship, R., Prescott, J.F. 2012. The epidemiology of Clostridium perfringens type A on Ontario swine farms, with special reference to cpb2-positive isolates. BMC veterinary research, 8, 156. https://doi.org/10.1186/1746-6148-8-156
Feng, Y., Fan, X., Zhu, L., Yang, X., Liu, Y., Gao, S., Jin, X., Liu, D., Ding, J., Guo, Y., Hu, Y. 2020. Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens. Microbial Genomics, 6, 10, mgen000441. https://doi.org/10.1099/mgen.0.000441
Fisher, D.J., Miyamoto, K., Harrison, B., Akimoto, S., Sarker, M.R., McClane, B.A. 2005. Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene. Molecular Microbiology, 56, 3, 747-762. https://doi.org/10.1111/j.1365-2958.2005.04573.x.
Freedman, J.C., Theoret, J.R., Wisniewski, J.A., Uzal, F.A., Rood, J.F., McClane, B.A. 2015. Clostridium perfringens type A-E toxin plasmids. Research in microbiology, 166, 4, 264-279. https://doi.org/ 10.1016/j.resmic.2014.09.004
Gelfand, Y., Rodriguez, A., Benson, G. 2007. TRDB—The Tandem Repeats Database. Nucleic Acid Research, 35, Database issue, 80–87. https://doi.org/10.1093/nar/gkl1013
Gulliver, E.L., Adams, V., Marcelino, V.R., Gould, J., Rutten, E.L., Powell, D.R., Young, R.B., D'Adamo, G.L., Hemphill, J., Solari, S.M., Revitt-Mills, S.A., Munn, S., Jirapanjawat, T., Greening, C., Boer, J.C., Flanagan, K.L., Kaldhusdal, M., Plebanski, M., Gibney, K.B., Moore, R.J., Rood, J.I., Forster, S.C. 2023. Extensive genome analysis identifies novel plasmid families in Clostridium perfringens. Microbial Genomics, 9, 4, 000995. https://doi.org/10.1099/mgen.0.000995
Hassan, W.M.M., Abd El Tawab, A., El-Shannat, S.M. 2020. Current advances in molecular subtyping using multilocus variable number of tandem repeat analysis of Salmonella entritidis and Salmonella typhimurium in Egyptian chickens. Veterinary Worlds, 13, 10, 2252-2259. https://doi.org/10.14202/vetworld.2020.2252-2259
Hayati, M., Tahamtan, Y. 2021. Toxin typing of Clostridium perfringens associated with enterotoxaemia in sheep in Fars province. Archives of Razi Institute, 76, 3, 691-697. https://doi.org/ 10.22092/ari.2020.343573.1509
Hunter, P.R., Gaston, M.A. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. Journal of Clinical Microbiology, 26, 11, 2465–2466. https://doi.org/10.1128/jcm.26.11.2465-2466.1988
Johansson, A., Aspan, A., Bagge, E., Båverud, V., Engström, B.E., Johansson, K.E. 2006. Genetic diversity of Clostridium Perfringens type A isolates from animals, food poisoning outbreaks and sludge, BMC Microbiology, 6, 47. https://doi.org/10.1186/1471-2180-6-47
Keyburn, A.L., Boyce, J.D., Vaz, P., Bannam, T.L., Ford, M.E., Parker, D., DiRubbo, A., Rood, J.I., Moore, R.J. 2008. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS pathogens, 4, 2, e26. https://doi.org/10.1371/journal.ppat.0040026
Li, J., Adams, V., Bannam, T.L., Miyamoto, K., Garcia, J.P., Uzal, F.A., Rood, J.I., McClane, B.A. 2013. Toxin Plasmids of Clostridium perfringens. Microbiology and Molecular Biology Reviews, 77, 2, 208-233. https://doi.org/10.1128/MMBR.00062-12
Lindstedt, B.A. 2005. Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis, 26, 13, 2567-82. https://doi.org/10.1002/elps.200500096
Lukinamaa, S., Nakari, U.M., Ekland, M., Sitonen, A. 2004. Application of molecular genetic methods in diagnostics and epidemiology of food‐borne bacterial pathogen. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 112, 11-12, 908-929. https://doi.org/10.1111/j.1600-0463.2004.apm11211-1213.x.
MacFaddin, J.F. 2000. Biochemical Tests for Identification of Medical Bacteria, 3rd еdition, Lippincott Williams& Wilkins, Baltimore.
Manteca, C., Daube, G., Jauniaux, T., Linden, A., Pirson, V., Detilleux, J., Ginter, A., Coppe, P., Kaeckenbeeck, A., Mainil, J.G. 2002. A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia. Veterinary Microbiology, 86, 3, 191-202. https://doi.org/10.1016/s0378-1135(02)00008-1
Meer, R., Songer, J.G. 1997. Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. American Journal of Veterinary Research, 58, 7, 702-705.
Myers, G.S.A., Rasko, D.A., Cheun, J.K., Ravel, J., Seshadri, R., DeBoy, R.T. Ren, Q., Varga, J., Awad, M.M., Brinkac, L.M., Daugherty, S.C., Haft, D.H., Dodson, R.J., Madupu, R., Nelson, W.C., Rosovitz, M.J., Sullivan, S,A., Khouri, H., Dimitrov, G.I., Watkins, K.L., Mulligan, S., Benton, J., Radune, D., Fisher, D.J., Atkins, H.S., Hiscox, T., Jost, B.H., Billington, S.J., Songer, J.G., McClane, B.A., Titball, R.W., Rood, J.I., Melville, S.B., Paulsen, I.T. 2006. Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome research, 16, 8, 1031-1040. https://doi.org/10.1101/gr.5238106
Park, M., Khan, S.A., Folley, S.L., Nayak, R., Songer, J.G., Seibel, J.R., Khan, S.A., Rooney, A.P., Hecht, D.W., Rafii, F. 2016. Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids. Anaerobe, 38, 25-35. https://doi.org/10.1016/j.anaerobe.2015.11.003
Razmyar, J., Kalidari, G.H., Tolooe, A., Rad, M., Movassaghi, A.R. 2014. Genotyping of Clostridium perfringens isolated from healthy and diseased ostriches. Iranian Journal of Microbiology, 6, 1, 31-36.
Rood, J.I., Adams, V., Lacey, J., Lyras, D., McClane, B.A., Melville, S.B., Moore, R.J., Popoff, M.R., Sarker, M.R., Songer, J.G., Uzal, F.A., Van Immerseel, F. 2018. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe, 53, 5-10. https://doi.org/10.1016/j.anaerobe.2018.04.011
Sawires, Y.S., Songer, J.G. 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe, 11, 5, 262–272. https://doi.org/10.1016/j.anaerobe.2005.03.004
Sawires, Y.S., Songer, J.G. 2006. Clostridium perfringens: insight into virulence evolution and population structure. Anaerobe, 12, 1, 23-43. https://doi.org/ 10.1016/j.anaerobe.2005.10.002
Sievers, F., Barton, G.J., Higgins, D.G. 2020. Multiple Sequence Alignment. In Bioinformatics. Eds. A.D. Baxevanis, G.D. Bader, D.S. Wishart, 4th edition, Wiley, 227-250.
Smyth, J.A., Mishra, N., Shivaprasad, H.L. 2022. Toxinotyping of Clostridium perfringens Strains Recovered from U.S. Turkeys with Necrotic Enteritis. Avian diseases, 66, 1, 1–4. https://doi.org/10.1637/aviandiseases-D-22-00003
Songer, G.J. 2010. Clostridia as agents of zoonotic disease. Veterinary Microbiology, 140, 3-4, 399–404. https://doi.org/ 10.1016/j.vetmic.2009.07.003
Sting, R. 2009. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia. Berliner und Munchener tierarztliche Wochenschrift, 122, 9-10, 341–347.
Suzuki, H., Donaka, B.K., Hashimoto, A. 2021. Evaluation of Clostridium perfringens cpe-positive strains as a source tracking indicator of human contamination in freshwater environments. Journal of Water and Environmental Technology, 19, 1, 1-12. https://doi.org/10.2965/jwet.20-089
Uzal, F.A., Freedman, J.C., Shrestha, A., Theoret, J.R., Garcia, J., Awad, M.M., Adams, V., Moore, R.J., Rood, J.I., McClane, B.A. 2014. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiology, 9, 3, 361-377. https://doi.org/10.2217/fmb.13.168
Van Asten, A.J., Nikolaou, G.N., Gröne, A. 2010. The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the beta2-toxin in enteric disease of domestic animals, wild animals and humans. Veterinary journal, 183, 2, 135–140. https://doi.org/10.1016/j.tvjl.2008.11.005
Van Belkum, A. 2007. Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA). FEMS Immunology and Medical Microbiology, 49, 1, 22-7. https://doi.org/10.1111/j.1574-695X.2006.00173.x.
Wang, B., Dong, W., Ma, L., Dong, Y., Wang, S., Yuan, Y., Ma, Q., Xu, J., Yan, W., Nan, J., Zhang, Q., Xu, W., Ma, B., Chu, Y., Zhang, .J, Li, L., Li, Y. 2021. Prevalence and genetic diversity of Clostridium perfringens isolates in hospitalized diarrheal patients from central China. Infection and Drug Resistance, 14, 4783-4793. https://doi.org/10.2147/IDR.S338593
Wasinski, B. 2007. Contribution of Clostridium perfringens type A with β2 toxin gene in etiology of porcine enteric diseases. The Bulletin of the Veterinary Institute in Pulawy, 51, 4, 509-513.
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