Veterinary Microbiology 168 (2014) 428–431
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla A. Balbutskaya a, O. Sammra a, S. Nagib a, M. Hijazin a, J. Alber a, C. La¨mmler a,*, G. Foster b, M. Erhard c, P.N. Wragg d, A. Abdulmawjood e, E. Prenger-Berninghoff f a
Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Schubertstr 81, 35392 Gießen, Germany SAC Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UK c RIPAC-LABOR GmbH, Am Mu¨hlenberg 11, 14476 Potsdam-Golm, Germany d Animal Health and Veterinary Laboratories Agency, Penrith Regional Laboratory, Merrythought, Calthwaite, Penrith CA11 9RR, UK e Institut fu¨r Lebensmittelqualita¨t und-sicherheit, Stiftung Tiera¨rztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany f Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t, Frankfurterstr. 85-91, 35392 Gießen, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 September 2013 Received in revised form 6 November 2013 Accepted 12 November 2013
In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Arcanobacterium pluranimalium MALDI-TOF MS 16S rDNA Pluranimaliumlysin pla
1. Introduction According to a proposal of Yassin et al. (2011) genus Arcanobacterium consists of four species namely Arcanobacterium haemolyticum, Arcanobacterium phocae, Arcanobacterium pluranimalium and Arcanobacterium hippocoleae. More recently Arcanobacterium canis and Arcanobacterium phocisimile were described as novel species of genus
* Corresponding author. Tel.: +49 6419938406; fax: +49 6419938409. E-mail address: [email protected] (C. La¨mmler). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.11.015
Arcanobacterium (Hijazin et al., 2012a, 2013). The closely related species Arcanobacterium pyogenes, Arcanobacterium abortisuis, Arcanobacterium bernardiae, Arcanobacterium bialowiezense and Arcanobacterium bonasi were reclassified to the newly described genus Trueperella as Trueperella pyogenes, Trueperella abortisuis, Trueperella bernardiae, Trueperella bialowiezensis and Trueperella bonasi (Yassin et al., 2011). However, both genera belong to the family Actinomycetaceae (http://www.ncbi.nlm. nih.gov/taxonomy). A. pluranimalium was initially described based on two isolates which were recovered from a harbour porpoise and a fallow deer, respectively (Lawson et al., 2001). In the
A. Balbutskaya et al. / Veterinary Microbiology 168 (2014) 428–431
following years this bacterial species was isolated in a ¨ lbegi-Mohyla mixed culture from a dog with pyoderma (U et al., 2010a) and from abortion material, semen, abscesses, viscera, navel ill and peritonitis of sheep and from a milk sample of a cow with mastitis (Foster and Hunt, 2011). The identification of the hitherto described A. pluranimalium was performed with phenotypic methods (Foster and Hunt, 2011) and by sequencing 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR) and 23S rDNA (Lawson et al., ¨ lbegi-Mohyla et al., 2010a). 2001; U The present study was designed to investigate the usefulness of MALDI-TOF MS and the pluranimaliumlysin encoding target gene pla for identification of novel and previously described A. pluranimalium strains of various origins.
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Table 1 Origin of the 13 A. pluranimalium strains investigated in the present study. A. pluranimalium strain designation
Isolated from
S111911-12-1 S214809-11-1 S214961-11-1 S215069-11-1 S420838-12-1 S609650-12-1 12-S0201-04-12 15-S38-11-10 C632524-10-1 24-C359-11-10 D12-0439-1-1-1 D13-0340-5-1-1 1128*
Ovine placenta, S Ovine foetal stomach contents, S Ovine vaginal swab, S Ovine foetal stomach contents, S Ovine foetal stomach contents, S Ovine foetal stomach contents, S Ovine milk, E Ovine brisket abscess, E Bovine foetal stomach contents, S Bovine foetal stomach contents, E Bovine mastitis, G Bovine cervix, G Dog with pyoderma, G
S = Scotland, E = England, G = Germany. ¨ lbegi-Mohyla et al. (2010a). * U
2. Materials and methods The strains used in the present study included reference strain A. pluranimalium DSM 13483, other strains of genus Arcanobacterium (Sammra et al., 2014), A. pluranimalium ¨ lbegi-Mohyla 1128 isolated from a dog with pyoderma (U et al., 2010a) and 12 hitherto not characterized A. pluranimalium strains isolated in Scotland, England and Germany. Further data about the origin of the 13 A. pluranimalium strains are summarized in Table 1. ¨ lbegiThe strains were investigated phenotypically (U Mohyla et al., 2010a; Foster and Hunt, 2011), by 16S rDNA sequencing (Sammra et al., 2014) and by MALDI-TOF MS analysis as described previously (Hijazin et al., 2012b). In addition the previously sequenced pluranimaliumlysin encoding gene pla of A. pluranimalium DSM 13483 (FR745890) was used for the design of pla specific oligonucleotide primer. The oligonucleotide primer had the sequence pla-F 50 -GTT GAT CTA CCA GGA TTG ACG C-30 and pla-R 50 -TTG TCG GGG TGT CCA TTG CC-30 and were used with the following PCR program: one step of 3 min at
94 8C; 30 cycles, with 1 cycle consisting of 45 s at 94 8C, 30 s at 57 8C, and 60 s at 72 8C; and one step of 7 min at 72 8C. Sequencing was performed by Seqlab-Sequence Laboratories, Go¨ttingen, Germany, alignment studies and dendrogram analysis using DNASTAR Lasergene Version 8.0.2 (DNASTAR Inc., Madison, USA) by Clustal W method. 3. Results and discussion All 12 A. pluranimalium strains newly investigated in the present study showed the typical phenotypical properties of this species and could be classified as A. pluranimalium by 16S rDNA sequencing. A typical dendrogram of the 16S rDNA sequencing results is shown in Fig. 1. The phenotypical properties included a moderate hemolysis on sheep blood agar, a slightly enhanced hemolysis on rabbit blood agar, positive CAMP-like reactions with Staphylococcus aureus b-hemolysin, Rhodococcus equi
A. pluranimalium 12-S0201-04-12 (HG423386) A. pluranimalium DSM 13483 (AJ250959) A. pluranimalium 15-S38-11-10 (HG423387) A. pluranimalium S214961-11-1 (HG423378) A. pluranimalium S111911-12-1 (HG423376) A. pluranimalium D12-0439-1-1-1(HG423382) A. pluranimalium D13-0340-5-1-1 (HG423383) A. pluranimalium C632524-10-1 (HG423384) A. pluranimalium 24-C359-11-10 (HG423385) A. pluranimalium 1128 (EU915469) A. pluranimalium S214809-11-1 (HG423377) A. pluranimalium S420838-12-1 (HG423380) A. pluranimalium S215069-11-1 (HG423379) A. pluranimalium S609650-12-1 (HG423381) A. phocisimile (FN562996) A. haemolyticum (AJ234059) A. phocae (X97049) A. canis (FR846134) A. hippocoleae (AJ300767) 2.4 2
0 Nucleotide Substitutions (x100)
Fig. 1. Dendrogram analysis of 16S rDNA sequences of the 13 A. pluranimalium strains of the present study, reference strain A. pluranimalium DSM 13483 and of various other species of genus Arcanobacterium obtained from NCBI GenBank.
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A. pluranimalium S214961-11-1 A. pluranimalium S215069-11-1 A. pluranimalium 1128 A. pluranimalium S111911-12-1 A. pluranimalium S214809-11-1 A. pluranimalium S420838-12-1 A. pluranimalium 15-S38-11-10 A. pluranimalium D12-0439-1-1-1 A. pluranimalium C632524-10-1 A. pluranimalium S609650-12-1 A. pluranimalium 12-S0201-04-12 A. pluranimalium 24-C359-11-10 A. pluranimalium DSM 13483 A. pluranimalium D13-0340-5-1-1 A. phocae DSM 10002 A. phocae DSM 10003 A. haemolyticum DSM 20595 A. canis DSM 25104 A. hippocoleae DSM 15539 Fig. 2. MALDI-TOF MS spectra from 13 A. pluranimalium strains of the present study, reference strain A. pluranimalium DSM 13483 and various other species of the genus Arcanobacterium.
and Arcanobacterium haemolyticum as indicator strains, generally positive reactions for caseinase (n = 10), catalase (n = 8), starch hydrolysis (n = 12), b-glucuronidase (n = 12) and negative reactions for a-glucosidase (n = 12) and Nacetyl-b-glucosaminidase (n = 12). It was of interest that all eight A. pluranimalium strains of ovine origin and the previously described A. pluranimalium of canine origin ¨ lbegi-Mohyla et al., 2010a) were catalase positive (U whereas the four strains of bovine origin were catalase negative (data not shown).
Comparable to previously conducted MALDI-TOF MS analysis of a Trueperella (Arcanobacterium) bernardiae strain of animal origin (Hijazin et al., 2012b), MALDI-TOF MS allowed the identification of all 12 newly described A. pluranimalium strains in the present study and A. pluranimalium 1128 of canine origin to the species level (Fig. 2.), indicating, that MALDI-TOF MS could also be used to reliably identify A. pluranimalium strains isolated from various origins. As shown by numerous authors MALDI-TOF MS appears to be a powerful tool
A. pluranimalium D12-0439-1-1-1 (HG423382) A. pluranimalium D13-0340-5-1-1 (HG423383) A. pluranimalium DSM 13483 (FR745890) A. pluranimalium S420838-12-1 (HG423380) A. pluranimalium S215069-11-1 (HG423379) A. pluranimalium S214961-11-1 (HG423378) A. pluranimalium 12-S0201-04-12 (HG423386) A. pluranimalium 15-S38-11-10 (HG423387) A. pluranimalium 1128 (HG423388) A. pluranimalium S111911-12-1 (HG423376) A. pluranimalium S214809-11-1 (HG423377) A. pluranimalium S609650-12-1 (HG423381) A. pluranimalium C632524-10-1 (HG423384) A. pluranimalium 24-C359-11-10 (HG423385) A. phocae phl (FN999907) A. haemolyticum aln (FJ785427) T. pyogenes plo (U84782) S. pneumoniae ply (GU968219) S. intermedius ily (AB029317) L. monocytogenes hly (EU262920)
71.9 70
60
50 40 30 20 Nucleotide Substitutions (x100)
10
0
Fig. 3. Dendrogram analysis of pla gene sequences of the 13 A. pluranimalium strains of the present study, pla of A. pluranimalium DSM 13483 and the genes plo of T. pyogenes, aln of A. haemolyticum, phl of A. phocae, ply of Streptococcus pneumoniae, ily of Streptococcus intermedius and hly of Listeria monocytogenes obtained from NCBI GenBank.
A. Balbutskaya et al. / Veterinary Microbiology 168 (2014) 428–431
for species classification of a broad spectrum of Grampositive and Gram-negative bacteria (Seng et al., 2009; Murray, 2010; Bizzini et al., 2011). Using the pla specific oligonucleotide primer which were based on the previously published partial gene sequence (FR745890) pla of all 13 A. pluranimalium strains investigated in the present study could be amplified and sequenced yielding 98,9% identity of the pla sequences among each other. Dendrogram analysis of pla of A. pluranimalium, plo of T. pyogenes, phl of A. phocae, aln of A. haemolyticum and of other pore forming toxin encoding genes obtained from NCBI GenBank revealed a close relation of pla, plo, phl and aln (Fig. 3). Comparable to gene plo of T. pyogenes, which appeared to be a constant characteristic of all investigated T. pyogenes (Billington ¨ lbegi-Mohyla et al., 2010b; et al., 1997; Ertas¸ et al., 2005; U Hijazin et al., 2011), pla of A. pluranimalium seems to be also constantly present in all strains of this species and could be used for molecular identification of A. pluranimalium. More recently Moser et al. (2013) also described pla as a novel target for molecular identification of this species. Further studies will give information about the consistent presence and the pathogenic importance of this hitherto unknown virulence factor encoding gene of A. pluranimalium. A. pluranimalium had already been characterized by amplification and sequencing of species specific regions of ¨ lbegi-Mohyla et al., 2010a) and by ISR and 23S rDNA (U sequencing of the target genes rpoB, sodA, cbpA and gap (Hijazin et al., 2011; Sammra et al., 2014). The MALDI-TOF MS analysis of the present study and the target gene pla might improve the identification of A. pluranimalium and might help to elucidate the role this species plays in infections of sheep, bovines, dogs, other animals and possibly in humans. References Billington, S.J., Jost, B.H., Cuevas, W.A., Bright, K.R., Songer, J.G., 1997. The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J. Bacteriol. 179, 6100–6106. Bizzini, A., Jaton, K., Romo, D., Bille, J., Prod’hom, G., Greub, G., 2011. Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains. J. Clin. Microbiol. 49, 693–696.
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¨ zbey, G., Muz, A., 2005. Isolation of Arcanobacterium Ertas¸, H.B., Kilic¸, A., O (Actinomyces) pyogenes from abscessed cattle kidney and identification by PCR. Turk. J. Vet. Anim. Sci. 29, 455–459. Foster, G., Hunt, B., 2011. Distribution of Arcanobacterium pluranimalium in animals examined in veterinary laboratories in the United Kingdom. J. Vet. Diagn. Invest. 23, 962–964. ¨ lbegi-Mohyla, H., Alber, J., La¨mmler, C., Hassan, A.A., AbdulHijazin, M., U mawjood, A., Prenger-Berninghoff, E., Weiss, R., Zscho¨ck, M., 2011. Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. J. Dairy Sci. 94, 1813–1819. Hijazin, M., Prenger-Berninghoff, E., Sammra, O., Alber, J., La¨mmler, C., Ka¨mpfer, P., Glaeser, S.P., Busse, H.J., Hassan, A.A., Abdulmawjood, A., Zscho¨ck, M., 2012a. Arcanobacterium canis sp. nov., isolated from otitis externa of a dog, and emended description of the genus Arcanobacterium Collins et al. 1983 emend. Yassin et al., 2011. Int. J. Syt. Evol. Microbiol. 62, 2201–2205. Hijazin, M., Metzner, M., Erhard, M., Nagib, S., Alber, J., La¨mmler, C., Hassan, A.A., Prenger-Berninghoff, E., Zscho¨ck, M., 2012b. First description of Trueperella (Arcanobacterium) bernardiae of animal origin. Vet. Microbiol. 159, 515–518. ¨ lbegi-Mohyla, H., Nagib, S., Alber, J., La¨mmler, C., Hijazin, M., Sammra, O., U Ka¨mpfer, P., Glaeser, S.P., Busse, H.J., Kassmannhuber, J., PrengerBerninghoff, E., Weiss, R., Siebert, U., Hassan, A.A., Abdulmawjood, A., Zscho¨ck, M., 2013. Arcanobacterium phocisimile sp. nov., isolated from harbour seals. Int. J. Syst. Evol. Microbiol. 63, 2019–2024. Lawson, P.A., Falsen, E., Foster, G., Eriksson, E., Weiss, N., Collins, M.D., 2001. Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. Int. J. Syst. Evol. Microbiol. 51, 55–59. Moser, A., Stephan, R., Sager, J., Corti, S., Lehner, A., 2013. Arcanobacterium pluranimalium leading to a bovine mastitis: species identification by a newly developed pla gene based PCR. Schweiz. Arch. Tierheilkund. 155, 373–375. Murray, P.R., 2010. Matrix-assisted laser desorption ionization time-offlight mass spectrometry: usefulness for taxonomy and epidemiology. Clin. Microbiol. Infect. 16, 1626–1630. Sammra, O., Balbutskaya, A., Nagib, S., Alber, J., La¨mmler, C., Abdulmawjood, A., Timke, M., Kostrzewa, M., Prenger-Berninghoff,., 2014. Properties of an Arcanobacterium haemolyticum strain isolated from a donkey. Berl. Muench. Tieraerztl. Wochenschr. 127, 10–14. Seng, P., Drancourt, M., Gouriet, F., La Scola, B., Fournier, P.E., Rolain, J.M., Raoult, D., 2009. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin. Infect. Dis. 49, 543–551. ¨ lbegi-Mohyla, H., Hassan, A.A., Alber, J., La¨mmler, C., Prenger-BerninghU off, E., Weiss, R., Zscho¨ck, M., 2010a. Identification of Arcanobacterium pluranimalium isolated from a dog by phenotypic properties and by PCR mediated characterization of various molecular targets. Vet. Microbiol. 142, 458–460. ¨ lbegi-Mohyla, H., Hijazin, M., Alber, J., La¨mmler, C., Hassan, A.A., AbdulU mawjood, A., Prenger-Berninghoff, E., Weiss, R., Zscho¨ck, M., 2010b. Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties. J. Vet. Sci. 11, 265–267. Yassin, A.F., Hupfer, H., Siering, C., Schumann, P., 2011. Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al., 1982 emend. Lehnen et al., 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. Int. J. Syst. Evol. Microbiol. 61, 1265–1274.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla A. Balbutskaya a, O. Sammra a, S. Nagib a, M. Hijazin a, J. Alber a, C. La¨mmler a,*, G. Foster b, M. Erhard c, P.N. Wragg d, A. Abdulmawjood e, E. Prenger-Berninghoff f a
Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Schubertstr 81, 35392 Gießen, Germany SAC Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UK c RIPAC-LABOR GmbH, Am Mu¨hlenberg 11, 14476 Potsdam-Golm, Germany d Animal Health and Veterinary Laboratories Agency, Penrith Regional Laboratory, Merrythought, Calthwaite, Penrith CA11 9RR, UK e Institut fu¨r Lebensmittelqualita¨t und-sicherheit, Stiftung Tiera¨rztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany f Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t, Frankfurterstr. 85-91, 35392 Gießen, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 September 2013 Received in revised form 6 November 2013 Accepted 12 November 2013
In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Arcanobacterium pluranimalium MALDI-TOF MS 16S rDNA Pluranimaliumlysin pla
1. Introduction According to a proposal of Yassin et al. (2011) genus Arcanobacterium consists of four species namely Arcanobacterium haemolyticum, Arcanobacterium phocae, Arcanobacterium pluranimalium and Arcanobacterium hippocoleae. More recently Arcanobacterium canis and Arcanobacterium phocisimile were described as novel species of genus
* Corresponding author. Tel.: +49 6419938406; fax: +49 6419938409. E-mail address: [email protected] (C. La¨mmler). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.11.015
Arcanobacterium (Hijazin et al., 2012a, 2013). The closely related species Arcanobacterium pyogenes, Arcanobacterium abortisuis, Arcanobacterium bernardiae, Arcanobacterium bialowiezense and Arcanobacterium bonasi were reclassified to the newly described genus Trueperella as Trueperella pyogenes, Trueperella abortisuis, Trueperella bernardiae, Trueperella bialowiezensis and Trueperella bonasi (Yassin et al., 2011). However, both genera belong to the family Actinomycetaceae (http://www.ncbi.nlm. nih.gov/taxonomy). A. pluranimalium was initially described based on two isolates which were recovered from a harbour porpoise and a fallow deer, respectively (Lawson et al., 2001). In the
A. Balbutskaya et al. / Veterinary Microbiology 168 (2014) 428–431
following years this bacterial species was isolated in a ¨ lbegi-Mohyla mixed culture from a dog with pyoderma (U et al., 2010a) and from abortion material, semen, abscesses, viscera, navel ill and peritonitis of sheep and from a milk sample of a cow with mastitis (Foster and Hunt, 2011). The identification of the hitherto described A. pluranimalium was performed with phenotypic methods (Foster and Hunt, 2011) and by sequencing 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR) and 23S rDNA (Lawson et al., ¨ lbegi-Mohyla et al., 2010a). 2001; U The present study was designed to investigate the usefulness of MALDI-TOF MS and the pluranimaliumlysin encoding target gene pla for identification of novel and previously described A. pluranimalium strains of various origins.
429
Table 1 Origin of the 13 A. pluranimalium strains investigated in the present study. A. pluranimalium strain designation
Isolated from
S111911-12-1 S214809-11-1 S214961-11-1 S215069-11-1 S420838-12-1 S609650-12-1 12-S0201-04-12 15-S38-11-10 C632524-10-1 24-C359-11-10 D12-0439-1-1-1 D13-0340-5-1-1 1128*
Ovine placenta, S Ovine foetal stomach contents, S Ovine vaginal swab, S Ovine foetal stomach contents, S Ovine foetal stomach contents, S Ovine foetal stomach contents, S Ovine milk, E Ovine brisket abscess, E Bovine foetal stomach contents, S Bovine foetal stomach contents, E Bovine mastitis, G Bovine cervix, G Dog with pyoderma, G
S = Scotland, E = England, G = Germany. ¨ lbegi-Mohyla et al. (2010a). * U
2. Materials and methods The strains used in the present study included reference strain A. pluranimalium DSM 13483, other strains of genus Arcanobacterium (Sammra et al., 2014), A. pluranimalium ¨ lbegi-Mohyla 1128 isolated from a dog with pyoderma (U et al., 2010a) and 12 hitherto not characterized A. pluranimalium strains isolated in Scotland, England and Germany. Further data about the origin of the 13 A. pluranimalium strains are summarized in Table 1. ¨ lbegiThe strains were investigated phenotypically (U Mohyla et al., 2010a; Foster and Hunt, 2011), by 16S rDNA sequencing (Sammra et al., 2014) and by MALDI-TOF MS analysis as described previously (Hijazin et al., 2012b). In addition the previously sequenced pluranimaliumlysin encoding gene pla of A. pluranimalium DSM 13483 (FR745890) was used for the design of pla specific oligonucleotide primer. The oligonucleotide primer had the sequence pla-F 50 -GTT GAT CTA CCA GGA TTG ACG C-30 and pla-R 50 -TTG TCG GGG TGT CCA TTG CC-30 and were used with the following PCR program: one step of 3 min at
94 8C; 30 cycles, with 1 cycle consisting of 45 s at 94 8C, 30 s at 57 8C, and 60 s at 72 8C; and one step of 7 min at 72 8C. Sequencing was performed by Seqlab-Sequence Laboratories, Go¨ttingen, Germany, alignment studies and dendrogram analysis using DNASTAR Lasergene Version 8.0.2 (DNASTAR Inc., Madison, USA) by Clustal W method. 3. Results and discussion All 12 A. pluranimalium strains newly investigated in the present study showed the typical phenotypical properties of this species and could be classified as A. pluranimalium by 16S rDNA sequencing. A typical dendrogram of the 16S rDNA sequencing results is shown in Fig. 1. The phenotypical properties included a moderate hemolysis on sheep blood agar, a slightly enhanced hemolysis on rabbit blood agar, positive CAMP-like reactions with Staphylococcus aureus b-hemolysin, Rhodococcus equi
A. pluranimalium 12-S0201-04-12 (HG423386) A. pluranimalium DSM 13483 (AJ250959) A. pluranimalium 15-S38-11-10 (HG423387) A. pluranimalium S214961-11-1 (HG423378) A. pluranimalium S111911-12-1 (HG423376) A. pluranimalium D12-0439-1-1-1(HG423382) A. pluranimalium D13-0340-5-1-1 (HG423383) A. pluranimalium C632524-10-1 (HG423384) A. pluranimalium 24-C359-11-10 (HG423385) A. pluranimalium 1128 (EU915469) A. pluranimalium S214809-11-1 (HG423377) A. pluranimalium S420838-12-1 (HG423380) A. pluranimalium S215069-11-1 (HG423379) A. pluranimalium S609650-12-1 (HG423381) A. phocisimile (FN562996) A. haemolyticum (AJ234059) A. phocae (X97049) A. canis (FR846134) A. hippocoleae (AJ300767) 2.4 2
0 Nucleotide Substitutions (x100)
Fig. 1. Dendrogram analysis of 16S rDNA sequences of the 13 A. pluranimalium strains of the present study, reference strain A. pluranimalium DSM 13483 and of various other species of genus Arcanobacterium obtained from NCBI GenBank.
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A. pluranimalium S214961-11-1 A. pluranimalium S215069-11-1 A. pluranimalium 1128 A. pluranimalium S111911-12-1 A. pluranimalium S214809-11-1 A. pluranimalium S420838-12-1 A. pluranimalium 15-S38-11-10 A. pluranimalium D12-0439-1-1-1 A. pluranimalium C632524-10-1 A. pluranimalium S609650-12-1 A. pluranimalium 12-S0201-04-12 A. pluranimalium 24-C359-11-10 A. pluranimalium DSM 13483 A. pluranimalium D13-0340-5-1-1 A. phocae DSM 10002 A. phocae DSM 10003 A. haemolyticum DSM 20595 A. canis DSM 25104 A. hippocoleae DSM 15539 Fig. 2. MALDI-TOF MS spectra from 13 A. pluranimalium strains of the present study, reference strain A. pluranimalium DSM 13483 and various other species of the genus Arcanobacterium.
and Arcanobacterium haemolyticum as indicator strains, generally positive reactions for caseinase (n = 10), catalase (n = 8), starch hydrolysis (n = 12), b-glucuronidase (n = 12) and negative reactions for a-glucosidase (n = 12) and Nacetyl-b-glucosaminidase (n = 12). It was of interest that all eight A. pluranimalium strains of ovine origin and the previously described A. pluranimalium of canine origin ¨ lbegi-Mohyla et al., 2010a) were catalase positive (U whereas the four strains of bovine origin were catalase negative (data not shown).
Comparable to previously conducted MALDI-TOF MS analysis of a Trueperella (Arcanobacterium) bernardiae strain of animal origin (Hijazin et al., 2012b), MALDI-TOF MS allowed the identification of all 12 newly described A. pluranimalium strains in the present study and A. pluranimalium 1128 of canine origin to the species level (Fig. 2.), indicating, that MALDI-TOF MS could also be used to reliably identify A. pluranimalium strains isolated from various origins. As shown by numerous authors MALDI-TOF MS appears to be a powerful tool
A. pluranimalium D12-0439-1-1-1 (HG423382) A. pluranimalium D13-0340-5-1-1 (HG423383) A. pluranimalium DSM 13483 (FR745890) A. pluranimalium S420838-12-1 (HG423380) A. pluranimalium S215069-11-1 (HG423379) A. pluranimalium S214961-11-1 (HG423378) A. pluranimalium 12-S0201-04-12 (HG423386) A. pluranimalium 15-S38-11-10 (HG423387) A. pluranimalium 1128 (HG423388) A. pluranimalium S111911-12-1 (HG423376) A. pluranimalium S214809-11-1 (HG423377) A. pluranimalium S609650-12-1 (HG423381) A. pluranimalium C632524-10-1 (HG423384) A. pluranimalium 24-C359-11-10 (HG423385) A. phocae phl (FN999907) A. haemolyticum aln (FJ785427) T. pyogenes plo (U84782) S. pneumoniae ply (GU968219) S. intermedius ily (AB029317) L. monocytogenes hly (EU262920)
71.9 70
60
50 40 30 20 Nucleotide Substitutions (x100)
10
0
Fig. 3. Dendrogram analysis of pla gene sequences of the 13 A. pluranimalium strains of the present study, pla of A. pluranimalium DSM 13483 and the genes plo of T. pyogenes, aln of A. haemolyticum, phl of A. phocae, ply of Streptococcus pneumoniae, ily of Streptococcus intermedius and hly of Listeria monocytogenes obtained from NCBI GenBank.
A. Balbutskaya et al. / Veterinary Microbiology 168 (2014) 428–431
for species classification of a broad spectrum of Grampositive and Gram-negative bacteria (Seng et al., 2009; Murray, 2010; Bizzini et al., 2011). Using the pla specific oligonucleotide primer which were based on the previously published partial gene sequence (FR745890) pla of all 13 A. pluranimalium strains investigated in the present study could be amplified and sequenced yielding 98,9% identity of the pla sequences among each other. Dendrogram analysis of pla of A. pluranimalium, plo of T. pyogenes, phl of A. phocae, aln of A. haemolyticum and of other pore forming toxin encoding genes obtained from NCBI GenBank revealed a close relation of pla, plo, phl and aln (Fig. 3). Comparable to gene plo of T. pyogenes, which appeared to be a constant characteristic of all investigated T. pyogenes (Billington ¨ lbegi-Mohyla et al., 2010b; et al., 1997; Ertas¸ et al., 2005; U Hijazin et al., 2011), pla of A. pluranimalium seems to be also constantly present in all strains of this species and could be used for molecular identification of A. pluranimalium. More recently Moser et al. (2013) also described pla as a novel target for molecular identification of this species. Further studies will give information about the consistent presence and the pathogenic importance of this hitherto unknown virulence factor encoding gene of A. pluranimalium. A. pluranimalium had already been characterized by amplification and sequencing of species specific regions of ¨ lbegi-Mohyla et al., 2010a) and by ISR and 23S rDNA (U sequencing of the target genes rpoB, sodA, cbpA and gap (Hijazin et al., 2011; Sammra et al., 2014). The MALDI-TOF MS analysis of the present study and the target gene pla might improve the identification of A. pluranimalium and might help to elucidate the role this species plays in infections of sheep, bovines, dogs, other animals and possibly in humans. References Billington, S.J., Jost, B.H., Cuevas, W.A., Bright, K.R., Songer, J.G., 1997. The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J. Bacteriol. 179, 6100–6106. Bizzini, A., Jaton, K., Romo, D., Bille, J., Prod’hom, G., Greub, G., 2011. Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains. J. Clin. Microbiol. 49, 693–696.
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