Caseous lymphadenitis (CLA) is a worldwide disease of small ruminants caused by Corynebacterium pseudotuberculosis, a facultative intracellular pathogen that is able to survive and multiply in certain white blood cells of the host. In this study, 33 strains of C. pseudotuberculosis were isolated from sheep and goats suffering from CLA on nine farms in the Czech Republic. All these strains were tested for their antibiotic susceptibility, ability to form a biofilm and resistance to the effects of commonly used disinfectant agents. To better understand the virulence of C. pseudotuberculosis, the genomes of strains were sequenced and comparative genomic analysis was performed with another 123 genomes of the same species, including ovis and equi biovars, downloaded from the NCBI. The genetic determinants for the virulence factors responsible for adherence and virulence factors specialized for iron uptake and exotoxin phospholipase D were revealed in every analyzed genome. Carbohydrate-Active Enzymes were compared, revealing the presence of genetic determinants encoding exo-α-sialidase (GH33) and the CP40 protein in most of the analyzed genomes. Thirty-three Czech strains of C. pseudotuberculosis were identified as the biovar ovis on the basis of comparative genome analysis. All the compared genomes of the biovar ovis strains were highly similar regardless of their country of origin or host, reflecting their clonal behavior.

Department of Microbiology and Antimicrobial Resistance Veterinary Research Institute 62100 Brno Czech Republic

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Burkovski A. The Role of Corynomycolic Acids in Corynebacterium-Host Interaction. Antonie Van Leeuwenhoek. 2018;111:717–725. doi: 10.1007/s10482-018-1036-6. PubMed DOI

Soares S.C., Silva A., Trost E., Blom J., Ramos R., Carneiro A., Ali A., Santos A.R., Pinto A.C., Diniz C., et al. The Pan-Genome of the Animal Pathogen Corynebacterium pseudotuberculosis Reveals Differences in Genome Plasticity between the Biovar Ovis and Equi Strains. PLoS ONE. 2013;8:e53818. doi: 10.1371/journal.pone.0053818. PubMed DOI PMC

Biberstein E., Knight H., Jang S. Two Biotypes of Corynebacterium pseudotuberculosis. Vet. Rec. 1971;89:691–692. doi: 10.1136/vr.89.26.691. PubMed DOI

Britz E., Spier S.J., Kass P.H., Edman J.M., Foley J.E. The Relationship between Corynebacterium pseudotuberculosis Biovar Equi Phenotype with Location and Extent of Lesions in Horses. Vet. J. 2014;200:282–286. doi: 10.1016/j.tvjl.2014.03.009. PubMed DOI

Schlicher J., Schmitt S., Stevens M.J.A., Stephan R., Ghielmetti G. Molecular Characterization of Corynebacterium pseudotuberculosis Isolated over a 15-Year Period in Switzerland. Vet. Sci. 2021;8:151. doi: 10.3390/vetsci8080151. PubMed DOI PMC

Baird G.J., Fontaine M.C. Corynebacterium pseudotuberculosis and Its Role in Ovine Caseous Lymphadenitis. J. Comp. Pathol. 2007;137:179–210. doi: 10.1016/j.jcpa.2007.07.002. PubMed DOI

Literák I., Horváthová A., Jahnová M., Rychlík I., Skalka B. Phenotype and Genotype Characteristics of the Slovak and Czech Corynebacterium pseudotuberculosis Strains Isolated from Sheep and Goats. Small Rumin. Res. 1999;32:107–111. doi: 10.1016/S0921-4488(98)00174-6. DOI

Langova D., Slana I., Okunkova J., Moravkova M., Florianova M., Markova J. First Evidence of the Presence of the Causative Agent of Caseous Lymphadenitis—Corynebacterium pseudotuberculosis in Dairy Products Produced from the Milk of Small Ruminants. Pathogens. 2022;11:1425. doi: 10.3390/pathogens11121425. PubMed DOI PMC

Olson M.E., Ceri H., Morck D.W., Buret A.G., Read R.R. Biofilm Bacteria: Formation and Comparative Susceptibility to Antibiotics. Can. J. Vet. Res. 2002;66:86–92. PubMed PMC

Sá M.d.C.A., Veschi J.L.A., Santos G.B., Amanso E.S., Oliveira S.A.S., Mota R.A., Veneroni-Gouveia G., Costa M.M. Activity of Disinfectants and Biofilm Production of Corynebacterium pseudotuberculosis. Pesqui. Veterinária Bras. 2013;33:1319–1324. doi: 10.1590/S0100-736X2013001100006. DOI

Yaacob M.F., Murata A., Nor N.H.M., Jesse F.F.A., Raja Yahya M.F.Z. Biochemical Composition, Morphology and Antimicrobial Susceptibility Pattern of Corynebacterium pseudotuberculosis Biofilm. J. King Saud. Univ. Sci. 2021;33:101225. doi: 10.1016/j.jksus.2020.10.022. DOI

de Sá M.C.A., da Silva W.M., Rodrigues C.C.S., Rezende C.P., Marchioro S.B., Rocha Filho J.T.R., Sousa T.d.J., de Oliveira H.P., da Costa M.M., Figueiredo H.C.P., et al. Comparative Proteomic Analyses Between Biofilm-Forming and Non-Biofilm-Forming Strains of Corynebacterium pseudotuberculosis Isolated From Goats. Front. Vet. Sci. 2021;8:614011. doi: 10.3389/fvets.2021.614011. PubMed DOI PMC

Merino N., Toledo-Arana A., Vergara-Irigaray M., Valle J., Solano C., Calvo E., Lopez J.A., Foster T.J., Penadés J.R., Lasa I. Protein A-Mediated Multicellular Behavior in Staphylococcus aureus. J. Bacteriol. 2009;191:832–843. doi: 10.1128/JB.01222-08. PubMed DOI PMC

Christensen G.D., Simpson W.A., Younger J.J., Baddour L.M., Barrett F.F., Melton D.M., Beachey E.H. Adherence of Coagulase-Negative Staphylococci to Plastic Tissue Culture Plates: A Quantitative Model for the Adherence of Staphylococci to Medical Devices. J. Clin. Microbiol. 1985;22:996–1006. doi: 10.1128/jcm.22.6.996-1006.1985. PubMed DOI PMC

Stepanović S., Vuković D., Dakić I., Savić B., Švabić-Vlahović M. A Modified Microtiter-Plate Test for Quantification of Staphylococcal Biofilm Formation. J. Microbiol. Methods. 2000;40:175–179. doi: 10.1016/S0167-7012(00)00122-6. PubMed DOI

Chemical Disinfectants and Antiseptics—Quantitative Suspension Test for the Evaluation of Bactericidal Activity of Chemical Disinfectants and Antiseptics Used in the Veterinary Area—Test Method and Requirements (Phase 2, Step 1) iTeh Standards; Portland, OR, USA: 2019.

European Committee on Antimicrobial Susceptibility Testing Media Preparation for EUCAST Disk Diffusion Testing and for Determination of MIC Values by the Broth Microdilution Method. EUCAST Version 6.0. 2020. [(accessed on 1 January 2020)]. Available online: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/2020_manuals/Media_preparation_v_6.0_EUCAST_AST.pdf.

European Committee on Antimicrobial Susceptibility Testing EUCAST Reading Guide for Broth Microdilution, EUCAST Version 4.0. 2022. [(accessed on 1 January 2022)]. Available online: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/2022_manuals/Reading_guide_BMD_v_4.0_2022.pdf.

European Committee on Antimicrobial Susceptibility Testing EUCAST Clinical Break-Point Tables, Version 12.0. 2022. [(accessed on 1 January 2022)]. Available online: https://www.eucast.org/clinical_breakpoints.

Marosevic D.V., Berger A., Kahlmeter G., Payer S.K., Hörmansdorfer S., Sing A. Antimicrobial Susceptibility of Corynebacterium diphtheriae and Corynebacterium ulcerans in Germany 2011–17. J. Antimicrob. Chemother. 2020;75:2885–2893. doi: 10.1093/jac/dkaa280. PubMed DOI

Ewels P., Magnusson M., Lundin S., Käller M. MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report. Bioinformatics. 2016;32:3047–3048. doi: 10.1093/bioinformatics/btw354. PubMed DOI PMC

Wick R.R., Judd L.M., Gorrie C.L., Holt K.E. Unicycler: Resolving Bacterial Genome Assemblies from Short and Long Sequencing Reads. PLoS Comput. Biol. 2017;13:e1005595. doi: 10.1371/journal.pcbi.1005595. PubMed DOI PMC

Jain C., Rodriguez-R L.M., Phillippy A.M., Konstantinidis K.T., Aluru S. High Throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries. Nat. Commun. 2018;9:5114. doi: 10.1038/s41467-018-07641-9. PubMed DOI PMC

Seemann T. Prokka: Rapid Prokaryotic Genome Annotation. Bioinformatics. 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Zhang H., Yohe T., Huang L., Entwistle S., Wu P., Yang Z., Busk P.K., Xu Y., Yin Y. DbCAN2: A Meta Server for Automated Carbohydrate-Active Enzyme Annotation. Nucleic Acids Res. 2018;46:W95–W101. doi: 10.1093/nar/gky418. PubMed DOI PMC

Huerta-Cepas J., Szklarczyk D., Heller D., Hernández-Plaza A., Forslund S.K., Cook H., Mende D.R., Letunic I., Rattei T., Jensen L.J., et al. EggNOG 5.0: A Hierarchical, Functionally and Phylogenetically Annotated Orthology Resource Based on 5090 Organisms and 2502 Viruses. Nucleic Acids Res. 2019;47:D309–D314. doi: 10.1093/nar/gky1085. PubMed DOI PMC

Buchfink B., Reuter K., Drost H.-G. Sensitive Protein Alignments at Tree-of-Life Scale Using DIAMOND. Nat. Methods. 2021;18:366–368. doi: 10.1038/s41592-021-01101-x. PubMed DOI PMC

Page A.J., Cummins C.A., Hunt M., Wong V.K., Reuter S., Holden M.T.G., Fookes M., Falush D., Keane J.A., Parkhill J. Roary: Rapid Large-Scale Prokaryote Pan Genome Analysis. Bioinformatics. 2015;31:3691–3693. doi: 10.1093/bioinformatics/btv421. PubMed DOI PMC

Kozlov A.M., Darriba D., Flouri T., Morel B., Stamatakis A. RAxML-NG: A Fast, Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic Inference. Bioinformatics. 2019;35:4453–4455. doi: 10.1093/bioinformatics/btz305. PubMed DOI PMC

Letunic I., Bork P. Interactive Tree Of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation. Nucleic Acids Res. 2021;49:W293–W296. doi: 10.1093/nar/gkab301. PubMed DOI PMC

Jia B., Raphenya A.R., Alcock B., Waglechner N., Guo P., Tsang K.K., Lago B.A., Dave B.M., Pereira S., Sharma A.N., et al. CARD 2017: Expansion and Model-Centric Curation of the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2017;45:D566–D573. doi: 10.1093/nar/gkw1004. PubMed DOI PMC

Zankari E., Hasman H., Cosentino S., Vestergaard M., Rasmussen S., Lund O., Aarestrup F.M., Larsen M.V. Identification of Acquired Antimicrobial Resistance Genes. J. Antimicrob. Chemother. 2012;67:2640–2644. doi: 10.1093/jac/dks261. PubMed DOI PMC

Gupta S.K., Padmanabhan B.R., Diene S.M., Lopez-Rojas R., Kempf M., Landraud L., Rolain J.-M. ARG-ANNOT, a New Bioinformatic Tool To Discover Antibiotic Resistance Genes in Bacterial Genomes. Antimicrob. Agents Chemother. 2014;58:212–220. doi: 10.1128/AAC.01310-13. PubMed DOI PMC

Doster E., Lakin S.M., Dean C.J., Wolfe C., Young J.G., Boucher C., Belk K.E., Noyes N.R., Morley P.S. MEGARes 2.0: A Database for Classification of Antimicrobial Drug, Biocide and Metal Resistance Determinants in Metagenomic Sequence Data. Nucleic Acids Res. 2020;48:D561–D569. doi: 10.1093/nar/gkz1010. PubMed DOI PMC

Feldgarden M., Brover V., Haft D.H., Prasad A.B., Slotta D.J., Tolstoy I., Tyson G.H., Zhao S., Hsu C.-H., McDermott P.F., et al. Validating the AMRFinder Tool and Resistance Gene Database by Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates. Antimicrob. Agents Chemother. 2019;63:e00483-19. doi: 10.1128/AAC.00483-19. PubMed DOI PMC

Viana M.V.C., Figueiredo H., Ramos R., Guimarães L.C., Pereira F.L., Dorella F.A., Selim S.A.K., Salaheldean M., Silva A., Wattam A.R., et al. Comparative Genomic Analysis between Corynebacterium pseudotuberculosis Strains Isolated from Buffalo. PLoS ONE. 2017;12:e0176347. doi: 10.1371/journal.pone.0176347. PubMed DOI PMC

Join-Lambert O.F., Ouache M., Canioni D., Beretti J.-L., Blanche S., Berche P., Kayal S. Corynebacterium pseudotuberculosis Necrotizing Lymphadenitis in a Twelve-Year-Old Patient. Pediatr. Infect. Dis. J. 2006;25:848–851. doi: 10.1097/01.inf.0000234071.93044.77. PubMed DOI

Koliwer-Brandl H., Syson K., van de Weerd R., Chandra G., Appelmelk B., Alber M., Ioerger T.R., Jacobs W.R., Geurtsen J., Bornemann S., et al. Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis. PLoS Pathog. 2016;12:e1005768. doi: 10.1371/journal.ppat.1005768. PubMed DOI PMC

Vanaporn M., Titball R.W. Trehalose and Bacterial Virulence. Virulence. 2020;11:1192–1202. doi: 10.1080/21505594.2020.1809326. PubMed DOI PMC

Naumthong W., Ito K., Pongsawasdi P. Acceptor Specificity of Amylomaltase from Corynebacterium glutamicum and Transglucosylation Reaction to Synthesize Palatinose Glucosides. Process Biochem. 2015;50:1825–1834. doi: 10.1016/j.procbio.2015.07.003. DOI

Wesener D.A., Levengood M.R., Kiessling L.L. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae. J. Biol. Chem. 2017;292:2944–2955. doi: 10.1074/jbc.M116.759340. PubMed DOI PMC

Dietrich C., Li de la Sierra-Gallay I., Masi M., Girard E., Dautin N., Constantinesco-Becker F., Tropis M., Daffé M., van Tilbeurgh H., Bayan N. The C-terminal Domain of Corynebacterium glutamicum Mycoloyltransferase A Is Composed of Five Repeated Motifs Involved in Cell Wall Binding and Stability. Mol. Microbiol. 2020;114:1–16. doi: 10.1111/mmi.14492. PubMed DOI

Shadnezhad A., Naegeli A., Collin M. CP40 from Corynebacterium pseudotuberculosis Is an Endo-β-N-Acetylglucosaminidase. BMC Microbiol. 2016;16:261. doi: 10.1186/s12866-016-0884-3. PubMed DOI PMC

Park S.-C., Lee K., Kim Y.O., Won S., Chun J. Large-Scale Genomics Reveals the Genetic Characteristics of Seven Species and Importance of Phylogenetic Distance for Estimating Pan-Genome Size. Front. Microbiol. 2019;10:834. doi: 10.3389/fmicb.2019.00834. PubMed DOI PMC

Costa S.S., Guimarães L.C., Silva A., Soares S.C., Baraúna R.A. First Steps in the Analysis of Prokaryotic Pan-Genomes. Bioinform. Biol. Insights. 2020;14:1177932220938064. doi: 10.1177/1177932220938064. PubMed DOI PMC

Oliveira A., Teixeira P., Azevedo M., Jamal S.B., Tiwari S., Almeida S., Silva A., Barh D., Dorneles E.M.S., Haas D.J., et al. Corynebacterium pseudotuberculosis May Be under Anagenesis and Biovar Equi Forms Biovar Ovis: A Phylogenic Inference from Sequence and Structural Analysis. BMC Microbiol. 2016;16:100. doi: 10.1186/s12866-016-0717-4. PubMed DOI PMC

Viana M.V.C., Sahm A., Góes Neto A., Figueiredo H.C.P., Wattam A.R., Azevedo V. Rapidly Evolving Changes and Gene Loss Associated with Host Switching in Corynebacterium pseudotuberculosis. PLoS ONE. 2018;13:e0207304. doi: 10.1371/journal.pone.0207304. PubMed DOI PMC

Gallardo A.A., Toledo R.A., González Pasayo R.A., Azevedo V., Robles C., Paolicchi F.A., Estevao Belchior S.G. Corynebacterium pseudotuberculosis Biovar Ovis: Evaluación de La Sensibilidad Antibiótica in Vitro. Rev. Argent. Microbiol. 2019;51:334–338. doi: 10.1016/j.ram.2018.12.001. PubMed DOI

Robaj A., Hamidi A., Bytyqi H., Sylejmani D. Frequency and Antimicrobial Susceptibility of Bacterial Isolates from Caseous Lymphadenitis in Sheep in Kosovo. Bulg. J. Agric. Sci. 2017;23:1033–1036.

El Damaty H.M., El-Demerdash A.S., Abd El-Aziz N.K., Yousef S.G., Hefny A.A., Abo Remela E.M., Shaker A., Elsohaby I. Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats. Animals. 2023;13:2337. doi: 10.3390/ani13142337. PubMed DOI PMC

Jagielski T., Bakuła Z., Brzostek A., Minias A., Stachowiak R., Kalita J., Napiórkowska A., Augustynowicz-Kopeć E., Żaczek A., Vasiliauskiene E., et al. Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains. Antimicrob. Agents Chemother. 2018;62:e01093-18. doi: 10.1128/AAC.01093-18. PubMed DOI PMC

Zou J., Chorlton S.D., Romney M.G., Payne M., Lawson T., Wong A., Champagne S., Ritchie G., Lowe C.F. Phenotypic and Genotypic Correlates of Penicillin Susceptibility in Nontoxigenic Corynebacterium diphtheriae, British Columbia, Canada, 2015–2018. Emerg. Infect. Dis. 2020;26:97–103. doi: 10.3201/eid2601.191241. PubMed DOI PMC

Sá M.d.C.A.d., Gouveia G.V., Krewer C.d.C., Veschi J.L.A., Mattos-Guaraldi A.L.d., Costa M.M.d. Distribution of PLD and FagA, B, C and D Genes in Corynebacterium pseudotuberculosis Isolates from Sheep and Goats with Caseus Lymphadenitis. Genet. Mol. Biol. 2013;36:265–268. doi: 10.1590/S1415-47572013005000013. PubMed DOI PMC

Trost E., Ott L., Schneider J., Schröder J., Jaenicke S., Goesmann A., Husemann P., Stoye J., Dorella F.A., Rocha F.S., et al. The Complete Genome Sequence of Corynebacterium pseudotuberculosis FRC41 Isolated from a 12-Year-Old Girl with Necrotizing Lymphadenitis Reveals Insights into Gene-Regulatory Networks Contributing to Virulence. BMC Genom. 2010;11:728. doi: 10.1186/1471-2164-11-728. PubMed DOI PMC

Ibraim I.C., Parise M.T.D., Parise D., Sfeir M.Z.T., de Paula Castro T.L., Wattam A.R., Ghosh P., Barh D., Souza E.M., Góes-Neto A., et al. Transcriptome Profile of Corynebacterium pseudotuberculosis in Response to Iron Limitation. BMC Genom. 2019;20:663. doi: 10.1186/s12864-019-6018-1. PubMed DOI PMC

Corrêa J.I., Stocker A., Trindade S.C., Vale V., Brito T., Bastos B., Raynal J.T., de Miranda P.M., de Alcantara A.C., Freire S.M., et al. In Vivo and in Vitro Expression of Five Genes Involved in Corynebacterium pseudotuberculosis Virulence. AMB Express. 2018;8:89. doi: 10.1186/s13568-018-0598-z. PubMed DOI PMC

Qiu J., Shi Y., Zhao F., Xu Y., Xu H., Dai Y., Cao Y. The Pan-Genomic Analysis of Corynebacterium striatum Revealed Its Genetic Characteristics as an Emerging Multidrug-Resistant Pathogen. Evol. Bioinform. 2023;19:11769343231191481. doi: 10.1177/11769343231191481. PubMed DOI PMC

Ott L., Hacker E., Kunert T., Karrington I., Etschel P., Lang R., Wiesmann V., Wittenberg T., Singh A., Varela C., et al. Analysis of Corynebacterium diphtheriae Macrophage Interaction: Dispensability of Corynomycolic Acids for Inhibition of Phagolysosome Maturation and Identification of a New Gene Involved in Synthesis of the Corynomycolic Acid Layer. PLoS ONE. 2017;12:e0180105. doi: 10.1371/journal.pone.0180105. PubMed DOI PMC

Seidel M., Alderwick L.J., Birch H.L., Sahm H., Eggeling L., Besra G.S. Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, Such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J. Biol. Chem. 2007;282:14729–14740. doi: 10.1074/jbc.M700271200. PubMed DOI

Gaskell A., Crennell S., Taylor G. The Three Domains of a Bacterial Sialidase: A β-Propeller, an Immunoglobulin Module and a Galactose-Binding Jelly-Roll. Structure. 1995;3:1197–1205. doi: 10.1016/S0969-2126(01)00255-6. PubMed DOI

Silva W.M., Dorella F.A., Soares S.C., Souza G.H.M.F., Castro T.L.P., Seyffert N., Figueiredo H., Miyoshi A., Le Loir Y., Silva A., et al. A Shift in the Virulence Potential of Corynebacterium pseudotuberculosis Biovar Ovis after Passage in a Murine Host Demonstrated through Comparative Proteomics. BMC Microbiol. 2017;17:55. doi: 10.1186/s12866-017-0925-6. PubMed DOI PMC

de Pinho R.B., de Oliveira Silva M.T., Bezerra F.S.B., Borsuk S. Vaccines for Caseous Lymphadenitis: Up-to-Date and Forward-Looking Strategies. Appl. Microbiol. Biotechnol. 2021;105:2287–2296. doi: 10.1007/s00253-021-11191-4. PubMed DOI PMC

Rodríguez Domínguez M.C., Montes de Oca Jiménez R., Varela Guerrero J.A. Linfadenitis Caseosa: Factores de Virulencia, Patogénesis y Vacunas. Revisión. Rev. Mex. Cienc. Pecu. 2022;12:1221–1249. doi: 10.22319/rmcp.v12i4.5699. DOI