Multilocus Sequencing of<i> Corynebacterium pseudotuberculosis</i> Biotype Ovis Strains

Sellyei, Boglárka; Bányai, Krisztián; Bartha, Dániel; Hajtós, István; Fodor, László; Makrai, László

doi:https://doi.org/10.1155/2017/1762162

BioMed Research International

On this page

Abstract Introduction Materials and Methods Results Discussion Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2017 | Article ID 1762162 | https://doi.org/10.1155/2017/1762162

Multilocus Sequencing of Corynebacterium pseudotuberculosis Biotype Ovis Strains

Boglárka Sellyei,¹Krisztián Bányai,¹Dániel Bartha,¹István Hajtós,²László Fodor,³and László Makrai³

Academic Editor: Chiu-Chung Young

Received04 Mar 2017

Revised26 Jul 2017

Accepted08 Aug 2017

Published11 Oct 2017

Abstract

Thirteen Corynebacterium pseudotuberculosis biotype ovis strains isolated from clinical cases of caseous lymphadenitis in Hungary were characterised using multilocus sequencing and their phylogenetic comparison was carried out on the basis of four housekeeping genes (groEL1, B, dnaK, and leuA). The in silico analysis of the 16 frequently studied housekeeping genes showed that C. pseudotuberculosis strains could be readily distinguished from C. diphtheriae and C. ulcerans strains; however, sequences of the same genes in the two biotypes of the C. pseudotuberculosis were highly similar; the heterogeneity values were low. Genes dnaK, B, groEL1, and leuA showed marked genetic variation within C. pseudotuberculosis, and strains of the two biotypes of C. pseudotuberculosis could be differentiated. Analysis of the individual genes showed a fairly conservative nature of C. pseudotuberculosis biotype ovis strains. The greatest genetic differentiation was seen in the dnaK and infB genes and concatenations of these two genes were very useful in the genetic separation of the studied strains.

1. Introduction

Corynebacterium pseudotuberculosis was isolated from a sheep and described by Preisz [1] as a Gram-positive, facultative intracellular pathogen. Two major biotypes can be distinguished based on the ability of reduction of nitrate; the nitrate negative biotype (C. pseudotuberculosis biotype ovis) causes caseous lymphadenitis (CLA) in small ruminants [2–4], whereas the nitrate positive biotype (C. pseudotuberculosis biotype equi) is the causative agent of ulcerative lymphangitis in horses, cows, camels, buffaloes, and occasionally humans [5, 6]. CLA is a chronic, contagious disease that is characterised by abscess formation in or near major peripheral lymph nodes (external form) or within the internal organs and lymph nodes (internal form). CLA occurs worldwide; however, it is more frequent in the tropical areas and causes important economic losses in ovine and caprine herds by reducing wool, meat, and milk production [4, 7–10].

Classification of C. pseudotuberculosis was originally based on cultural, morphological, and biochemical characteristics [11]. In addition to nitrate reduction various molecular methods, restriction fragment length polymorphism (RFLP) of chromosomal DNA, ribotyping, and whole genome sequence analysis were used for the differentiation of C. pseudotuberculosis biotypes [12–14]. C. pseudotuberculosis biotype equi strains showed greater genetic divergence than C. pseudotuberculosis biotype ovis strains when examined with pulsed field gel electrophoresis (PFGE), BOX-PCR, random amplified polymorphic DNA (RAPD), and amplification of DNA surrounding rare restriction sites (ADSRRS) [3, 15, 16].

Multilocus sequence typing has become popular in bacterial genotyping technique over the past decade, including genotyping Corynebacterium spp.; it allows the identification of potential molecular genetic marker(s) in conservative genes for the differentiation of closely related bacterial strains [17, 18].

The objective of this study was characterisation of Hungarian field strains of C. pseudotuberculosis biotype ovis using multilocus sequencing and their phylogenetic comparison on the basis of selected housekeeping genes.

2. Materials and Methods

2.1. Bacterial Strains and Culture Condition

Thirteen C. pseudotuberculosis biotype ovis field strains isolated between 1994 and 2014 were randomly selected from the strain collection of the Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary. After identification using standard methods [20], they had been stored at −80°C till the examinations. Details about the strains used in this study are described in Table 1.

Reference strains of C. pseudotuberculosis biotype ovis (DSM-7180) and C. pseudotuberculosis biotype equi (DSM-7177) from the Leibniz Institute, German Collection of Microorganisms, and Cell Cultures (Braunschweig, Germany) were also included in the examinations.

The strains were cultured on Columbia blood agar (LabM, Lancashire, UK) with the addition of 5% (vol/vol) sterile defibrinated sheep blood and incubated at 37°C for 48 h.

2.2. In Silico Sequence Analysis and Primer Design

In order to distinguish C. pseudotuberculosis biotypes from the closely related species and to find potential markers, the nucleotide sequences of 16 housekeeping genes of 15 C. pseudotuberculosis, 13 C. diphtheriae strains, and one C. ulcerans strain (Table 2) were compared. The nucleotide sequences of 7 genes including the ATP synthase alpha chain (atpA), the DNA polymerase III alpha subunit (dnaE), the chaperone Hsp70 (dnaK), the elongation factor G (fusA), the 2-isopropylmalate synthase (leuA), the 2-oxoglutarate dehydrogenase E1 and E2 components (odhI), and the DNA-directed RNA polymerase beta chain (rpoB) were taken from [22, 23], while sequences of 9 genes involving ATP synthase beta chain (atpD), the heat shock proteins GroEL and GroES (groES, groEL1, and groEL2), the translation initiation factor IF2 (B), the DNA recombinational repair system (recA, recN), the alpha subunit of DNA dependent RNA polymerase (rpoA), and the manganese-dependent superoxide dismutase (sodA) were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). In silico analysis of the selected genes was performed with the ClustalW multiple alignment algorithm [24] in MegAlign software of the Lasergene 7 program suite (DNAStar, Madison, WI, USA) to find potentially useful markers for genetic differentiation and molecular epidemiological investigations.

Four genes (groEL1, B, dnaK, and leuA) were selected for primer design; they are located at different genomic regions of bacteria (Figure 1). Primer sets were designed to amplify 580 bp, 654 bp, 641 bp, and 647 bp long fragments from the respective genomic regions; pending on the genes, the number of SNPs varied from 7 to 31 within the amplified fragments (Table 3). PCR primers were designed using Oligo Primer Analysis software 7 [25].

2.3. PCR and Sequencing

A loopful of 48 h culture of C. pseudotuberculosis was suspended in 6% Chelex-solution (Bio-Rad Laboratories, Hercules, CA, USA) and then heated to 65°C for 30 min and to 100°C for 8 min; afterwards, it was centrifuged. The supernatant was collected in a new tube and stored at −20°C until use. Fragments of the selected housekeeping genes were amplified by PCR in an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, Carlsbad, CA, USA). The 25 μl amplification reaction mixture contained 1x Dream Taq Buffer, 200 nM dNTP-mix, 1 μM of each primer, 1 U Dream Taq (Thermo Fisher Scientific, Carlsbad, CA, USA), template DNA, and water. The PCR program included 3 min of initial denaturation at 95°C and then 30 cycles at 95°C for 30 s, at 60°C for 30, and at 72°C for 30 s and 7 min of the final elongation. PCR products were purified for direct sequencing using the Geneaid PCR purification Kit (Geneaid Biotech, New Taipei, Taiwan). Templates were sequenced with the PCR primers using the BigDye Terminator Ready Reaction Mix v3.1. Nucleotide sequences were run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

2.4. Phylogenetic Analysis

Fragments of four protein-coding highly diverse housekeeping genes (groEL1, B, dnaK, and leuA) were sequenced for all strains.

Sequences were aligned using the ClustalW algorithm in the MEGA 6 software [26] and trimmed manually at the same position before being used for comparison with sequences of other C. pseudotuberculosis strains deposited in the GenBank. The same gene set from C. diphtheriae HC02 was used as an outgroup.

After the single gene alignments, the sequences were joined to make a concatemer of loci at head-to-tail in-frame. The phylogenetic trees were constructed by using the neighbour joining (NJ) method. The bootstrap technique [27] was employed to evaluate the reliability of tree topologies by resampling the sequence alignment 1000 times.

2.5. Nucleotide Sequence Accession Numbers

The determined partial gene sequences were deposited in GenBank under accession numbers: from MF491615 to MF491629 (dnaK); from MF464070 to MF464084 (B); MF476990 and from MF476992 to MF477005 (groEL1); MF446654 and from MF446656 to MF446669 (leuA).

3. Results

As the use of MLST protocol of Corynebacterium diphtheriae (https://pubmlst.org/cdiphtheriae/) proved to be inadequate for analysis of C. pseudotuberculosis strains, developing of new scheme has been required.

The in silico analysis of the 16 frequently studied housekeeping genes showed that C. pseudotuberculosis strains could be readily distinguished by sequence analysis from C. diphtheriae and C. ulcerans strains; however, sequences of the same genes of the C. pseudotuberculosis strains belonging to the two biotypes were highly similar, and the heterogeneity values were low (Table 4). Genes dnaK, B, groEL1, and leuA showed marked genetic variation within C. pseudotuberculosis biotypes and they were selected for further genetic differentiation and molecular epidemiological investigations. The sequence diversity in these regions exceeded 5%.

C. pseudotuberculosis biotype ovis and C. pseudotuberculosis biotype equi field strains were differentiated with sequence analysis of the target gene fragments. Analysis of the individual genes showed a fairly conservative nature of C. pseudotuberculosis biotype ovis strains, although segregation of some Hungarian strains (e.g., C1 and C19, C3, C23, and C27) together with strains from Australia, Israel, USA, South Africa, Scotland, France, and Brazil was also evident but they were clearly located in the cluster of C. pseudotuberculosis biotype ovis. The greatest genetic differentiation was seen in the dnaK and infB genes and concatenations of these two genes were very useful in the genetic separation of the studied strains (Figure 2).

4. Discussion

C. pseudotuberculosis biotype ovis is the causative agent of caseous lymphadenitis, a contagious, chronic disease of sheep and goats [3].

Several previous studies showed high sequential homology of the C. pseudotuberculosis biovar ovis strains confirming their clonal origin; however, diversity of the clinical signs in sheep and goats and different efficacy of the vaccines suggest that more than one pathogen clone can exist [14].

The analysis of sequence data of 16 frequently studied housekeeping genes (atpA, dnaE, dnaK, fusA, leuA, odhI, rpoB, atpD, groES, groEL1, groEL2, B, recA, recN, rpoA, and sodA) of 15 C. pseudotuberculosis, 13 C. diphtheriae, and C. ulcerans genomes from the GenBank proved to be related and all studied genes could be used to differentiate C. diphtheriae, C. ulcerans, and C. pseudotuberculosis strains. All these genes allow the differentiation of biotype ovis and biotype equi strains of C. pseudotuberculosis but for detailed examination of C. pseudotuberculosis biovar ovis field isolates four genes (dnaK, groEL1, B, and leuA) seemed to have higher differentiation power. The partial sequence analysis of adequate genes delineated phylogenetic trees with various topology and depth of branches. The highest resolution and topology of the tree were obtained with the dnaK sequences.

The clusters could be explained by movement of breeding animals, but no epidemiological connection was found between the examined C. pseudotuberculosis strains. The distance between the geographical place of isolation and the year of the isolation do not support any epidemiological connection between the strains, so circulation of several clones of C. pseudotuberculosis biotype ovis was verified.

According to our data, multilocus sequence typing can differentiate C. pseudotuberculosis and the most important Corynebacterium species, and examination of selected genes helps to differentiate the two biotypes of C. pseudotuberculosis, and it can be used for epidemiological follow-up of these strains.

Conflicts of Interest

None of the authors have any conflicts of interest to declare.

Authors’ Contributions

All authors have been involved in designing the study, analysing the data, and drafting of the manuscript.

Acknowledgments

This project was supported by the Hungarian Scientific Research Fund (OTKA PD 101091) and by the János Bolyai Research Scholarship of the Hungarian Academy of Science to B. Sellyei. This research was supported by the Hungarian Ministry of Human Capacities (12190-4/2017/FEKUTSTRAT Grant).

References

H. Preisz, About a case of pseudotuberculosis in sheep and about pseudotuberculosis in general, 1891 (Hungarian).
E. L. Biberstein, H. D. Knight, and S. Jang, “Two biotypes of Corynebacterium pseudotuberculosis.,” Veterinary Record, vol. 89, no. 26, pp. 691-692, 1971.
View at: Publisher Site | Google Scholar
K. M. Connor, M. M. Quirie, G. Baird, and W. Donachie, “Characterization of United Kingdom isolates of Corynebacterium pseudotuberculosis using pulsed-field gel electrophoresis,” Journal of Clinical Microbiology, vol. 38, no. 7, pp. 2633–2637, 2000.
View at: Google Scholar
L. H. Williamson, “Caseous lymphadenitis in small ruminants.,” The Veterinary clinics of North America. Food animal practice, vol. 17, no. 2, pp. 359–371, 2001.
View at: Publisher Site | Google Scholar
M. M. Peel, G. G. Palmer, A. M. Stacpoole, and T. G. Kerr, “Human lymphadenitis due to Corynebacterium pseudotuberculosis: report of ten cases from Australia and review,” Clinical Infectious Diseases, vol. 24, no. 2, pp. 185–191, 1997.
View at: Publisher Site | Google Scholar
D. T. L. Liu, W.-M. Chan, D. S. P. Fan, and D. S. C. Lam, “An infected hydrogel buckle with Corynebacterium pseudotuberculosis,” British Journal of Ophthalmology, vol. 89, no. 2, pp. 245-246, 2005.
View at: Publisher Site | Google Scholar
I. Hajtós, G. Malik, and J. Varga, “Occurrence of caseous lymphadenitis caused by Corynebacterium pseudotuberculosis in goats in Hungary , (In Hungarian: A kecskék Corynebacteriumpseudotuuberculosis okozta sajtos nyirokcsomó-gyulladásának elofordulása hazánkban,” MagyarÁllatorvosokLapja, vol. 40, no. 1, pp. 25–29, 1985.
View at: Google Scholar
I. Hajtós, I. Makkai, L. Fodor et al., “Occurrence of ovine caseous lymphadenitis (pseudotuberculosis) in Northern Hungary , (In Hungarian: Sajtos nyirokcsomó-gyulladás (pseudotuberculosis) észak-magyarországi juhállományban,” Magyar Állatorvosok Lapja, vol. 127, no. 6, pp. 339–347, 2005.
View at: Google Scholar
F. A. Dorella, L. G. C. Pacheco, S. C. Oliveira, A. Miyoshi, and V. Azevedo, “Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence,” Veterinary Research, vol. 37, no. 2, pp. 201–218, 2006.
View at: Publisher Site | Google Scholar
A. de Sá Guimarães, F. Borges do Carmo, R. Barbosa Pauletti et al., “Review: veterinary microbiology: Caseous lymphadenitis: epidemiology, diagnosis, and control,” The IIOAB Journal, vol. 2, no. 2, pp. 33–43, 2011.
View at: Google Scholar
C. A. Muckle and C. L. Gyles, “Characterization of strains of Corynebacterium pseudotuberculosis,” Canadian Journal of Comparative Medicine, vol. 46, no. 2, pp. 206–208, 1982.
View at: Google Scholar
S. S. Sutherland, R. A. Hart, and N. B. Buller, “Genetic differences between nitrate-negative and nitrate-positive C. pseudotuberculosis strains using restriction fragment length polymorphisms,” Veterinary Microbiology, vol. 49, no. 1-2, pp. 1–9, 1996.
View at: Publisher Site | Google Scholar
J. C. Ruiz, V. D'Afonseca, A. Silva et al., “Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains,” PLoS ONE, vol. 6, no. 4, Article ID e18551, 2011.
View at: Google Scholar
S. C. Soares, A. Silva, E. Trost 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, vol. 8, no. 1, Article ID e53818, 2013.
View at: Publisher Site | Google Scholar
K. M. Connor, M. C. Fontaine, K. Rudge, G. J. Baird, and W. Donache, “Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis,” Veterinary Research, vol. 38, no. 4, pp. 613–623, 2007.
View at: Publisher Site | Google Scholar
I. Stefańska, M. Rzewuska, and M. Binek, “Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland,” Polish Journal of Microbiology, vol. 57, no. 2, pp. 105–112, 2008.
View at: Google Scholar
A. Deshpande, T. Inkster, K. Hamilton et al., “Colonisation with toxigenic corynebacterium diphtheriae in a scottish burns patient, june 2015,” Eurosurveillance, vol. 20, no. 49, pp. 1–4, 2015.
View at: Publisher Site | Google Scholar
T. Eisenberg, N. Mauder, M. Contzen et al., “Outbreak with clonally related isolates of Corynebacterium ulcerans in a group of water rats,” BMC Microbiology, vol. 15, no. 1, article 384, 2015.
View at: Publisher Site | Google Scholar
E. Trost, J. Blom, S. C. de Soares et al., “Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia,” Journal of Bacteriology, vol. 194, no. 12, pp. 3199–3215, 2012.
View at: Publisher Site | Google Scholar
B. Markey, F. Leonard, and M. Archambault, Clinical veterinary microbiology, Mosby/Elsevier, Ltd, Edinburgh, UK, 2nd edition, 2013.
A. A. S. O. Dias, F. C. Silva Jr., G. A. Pereira et al., “Corynebacterium ulcerans isolated from an asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil.,” Vector borne and zoonotic diseases (Larchmont, N.Y.), vol. 10, no. 8, pp. 743–748, 2010.
View at: Publisher Site | Google Scholar
A. Khamis, D. Raoult, and B. La Scola, “rpoB gene sequencing for identification of Corynebacterium species,” Journal of Clinical Microbiology, vol. 42, no. 9, pp. 3925–3931, 2004.
View at: Publisher Site | Google Scholar
F. Bolt, The population structure of the Corynebacteriumdiphtheriae group [Ph.D. thesis], University of Warwick, 2009.
J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research, vol. 22, no. 22, pp. 4673–4680, 1994.
View at: Publisher Site | Google Scholar
W. Rychlik, “OLIGO 7 Primer Analysis Software,” in PCR Primer Design, A. A. Yuryev, Ed., pp. 35–59, Humana Press Inc, Totowa, NJ, USA, 2007.
View at: Google Scholar
K. Tamura, G. Stecher, D. Peterson, A. Filipski, and S. Kumar, “MEGA6: Molecular Evolutionary Genetics Analysis version 6.0,” Molecular Biology and Evolution, vol. 30, no. 12, pp. 2725–2729, 2013.
View at: Publisher Site | Google Scholar
J. Felsenstein, “Confidence limits on phylogenies: an approach using the bootstrap,” Evolution, vol. 39, pp. 783–791, 1985.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2017 Boglárka Sellyei et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

1585

Downloads

840

Citations