Taxonomic Classification of Strain PO100/5 Shows a Broader Geographic Distribution and Genetic Markers

Home , Corynebacterium ulcerans

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Taxonomic classification of strain PO100/5 shows a broader geographic distribution and genetic markers

2 of the recently described Corynebacterium silvaticum

4 Marcus Vinicius Canário Viana1,2, Rodrigo Profeta1, Alessandra Lima da Silva1, Raquel Hurtado1, Janaína

5 Canário Cerqueira1, Bruna Ferreira Sampaio Ribeiro1, Marcelle Oliveira Almeida1, Francielly Morais-

6 Rodrigues1, Manuela Oliveira3, Luís Tavares3, Henrique Figueiredo4, Alice Rebecca Wattam5, Debmalya

7 Barh6, Preetam Ghosh7, Artur Silva2, Vasco Azevedo1*

9 1 Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal University of

10 Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

11 2 Department of Genetics, Institute of Biological Sciences, Federal University of Pará, Belém, Pará, Brazil

12 3 Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of

13 Lisbon, Lisboa, Portugal

14 4 National Reference Laboratory of Aquatic Animal Disease, Federal University of Minas Gerais, Belo

15 Horizonte, Minas Gerais, Brazil

16 5 Biocomplexity Institute, University of Virginia, Charlottesville, Virginia, USA

17 6 Institute of Integrative Omics and Applied Biotechnology, Purba Medinipur, West Bengal, India

18 7 Department of Computer Science, Virginia Commonwealth University, Richmond, Virginia, USA

20 * Corresponding author: Dr. Vasco Azevedo

21 Email: [email protected] (VA)

22 Abstract

23 The bacterial strain PO100/5 was isolated from a skin abscess of a pig (Sus scrofa domesticus) in

24 the Alentejo region of southern Portugal. It was identified as Corynebacterium pseudotuberculosis using

25 biochemical tests, multiplex PCR and Pulsed Field Gel Electrophoresis. After genome sequencing and

26 rpoB phylogeny, the strain was classified as C. ulcerans. To better understand the taxonomy of this strain

27 and improve identification methods, we compared strain PO100/5 to other publicly available genomes

28 from the C. diphtheriae group. Taxonomic analysis reclassified it and three others strains as belonging to

29 the recently described C. silvaticum, which have been isolated from wild boar and roe deer in Germany

30 and Austria. The results showed that PO100/5 is the first sequenced genome of a C. silvaticum strain from

31 a domestic animal and a different geographical region, is a putative producer of the diphtheriae toxin, and

32 has a unique sequence type. Genomic analysis of PO100/5 showed four prophages and eight conserved

33 genomic islands when compared to C. ulcerans. Pangenome analysis of 38 C. silvaticum and 76 C.

34 ulcerans samples suggest that C. silvaticum is a clonal species, with 73.6% of conserved genes and a

35 pangenome near to being closed (α > 0.952). 172 conserved genes are unique to C. silvaticum when

36 compared to C. ulcerans, with most related to nutrient uptake and metabolism, prophages or immune

37 evasion. These unique genes could be used as genetic markers for species identification. This information

38 can be useful for identification and surveillance of this pathogen, especially in regard to the possibility of

39 zoonotic transmission.

41 Introduction

42 The genus Corynebacterium from phylum Actinobacteria has Gram-positive bacteria of

43 biotechnological, veterinary and medical relevance with free, commensal and pathogenic lifestyles.

44 Within its pathogenic members, the most prominent species are the nearly exclusively human pathogen C.

45 diphtheriae and the zoonotic species, C. pseudotuberculosis and C. ulcerans. These species compose the

46 C. diphtheriae group, a clade of species that can be lysogenized by phages harboring the diphtheria toxin

47 (DT) gene (tox) [1]. In this group, three new species were recently described. C. belfantii is a

48 reclassification of C. diphtheriae biovar Belfanti [2] and a synonym of C. diphtheriae subspecies

49 lausannense [3], C. rouxii [3] is reclassifications of C. diphtheriae biovar Belfanti, and C. silvaticum [4]

50 is a reclassification of atypical C. ulcerans strains. Strains of C. silvaticum were previously described as

51 atypical non-toxigenic but tox-gene-bearing (NTTB) strains of C. ulcerans, isolated from wild boars and

52 roe deer in Germany, which caused caseous lymphadenitis similar to C. pseudotuberculosis infections [5–

53 7]. This variant, examined using genomics and proteomics, was initially named as a “wild boar cluster”

54 (WBC) of C. ulcerans [5–7] and later reclassified as C. silvaticum [4].

55 The strain PO100/5 was isolated from caseous lymphadenitis lesions in a Black Alentejano pig

56 (Sus scrofa domesticus) from a swine farm in the Alentejo region of Portugal. It was identified as

57 Corynebacterium pseudotuberculosis by both biochemical tests (Api Coryne® kit) and by multiplex PCR

58 and Pulsed Field Gel Electrophoresis [8]. Genome sequencing and rpoB phylogeny showed that this strain

59 was closer to C. ulcerans and the genome was deposited in GenBank as a strain from this species

60 (accession number CP021417.1). During the submission and review of this manuscript, the description of

61 C. silvaticum was published and suggested PO100/5 as a strain of this species by rpoB phylogeny [4],

62 while a genomic analysis of 28 C. ulcerans strains suggested that PO100/5, W25 and KL1196 could

63 represent a new species [9], although KL1196 had already been classified as C. silvaticum [4].

64 Pigs and boars are reservoirs of C. silvaticum [4–7] and C. ulcerans [10–12] and are known to

65 transmit pathogens to humans and domestic animals [10,11,13]. Rapid, simple and reliable identification

66 of species is essential for diagnosis, treatment and surveillance [14,15]. To better understand the

67 taxonomy of PO100/5, the genome diversity of C. silvaticum and to identify molecular markers of this

68 species, we performed a comparative analysis of 34 C. silvaticum and 80 C. ulcerans genomes, as well as

69 other publicly available genomes from the C. diphtheriae group. We reclassified PO100/5 and other three

70 strains recently deposited as C. silvaticum and found a unique sequence type and genes that can be useful

71 for species classification.

73 Materials and Methods

74 Genomes, assembly and annotation

75 For the taxonomic, phylogenetic and genome plasticity analyses, a total of 120 genomes (S1 File)

76 were selected, including 80 C. ulcerans and 34 C. silvaticum strains and six type strains of C. diphtheriae

77 group. Assembled genomes were retrieved from Pathosystems Resource Integration Center (PATRIC)

78 [16], while genomes available as sequencing reads were assembled in PATRIC using SPAdes [17]

79 strategy. All genomes were annotated using the Rapid Annotation using Subsystems Technology

80 (RASTtk) pipeline [18] that is available in PATRIC.

82 Taxonomic analysis

83 Average Nucleotide Identity (ANI) was estimated using FastANI v1.3 [19]. An automatic

84 genome-based taxonomic analysis was performed using Type (Strain) Genome Server (TYGS)

85 (https://tygs.dsmz.de) [20]. First, TYGS pipeline identifies the closest type strains using MASH [21] for

86 genomic sequences and BLAST [22] for 16S rRNA sequences. Second, it identifies the 10 closest type

87 strains using Genome Blast Distance Phylogeny (GBDP) [23]. Third, it uses digital DNA:DNA

88 hybridization (dDDH) [23] to cluster species and subspecies using a threshold of 70 and 79%,

89 respectively [24].

90 Phylogenetic trees of rpoB and tox were built using the Maximum Likelihood method [25]

91 implemented in MEGA v10.1.6 [26]. The tox tree included all sequences in genomes of C. ulcerans and

92 outgroups from C. silvaticum, C. pseudotuberculosis and C. belfantii. C. rouxii was not included due to

93 all sequenced genomes being tox- [3]. All trees were visualized using iTOL [27].

94 Multi Locus Sequence Typing (MLST) was performed using MLSTcheck [28], using the scheme

95 for C. diphtheriae and C. ulcerans (genes atpA, dnaE, dnaK, fusA, leuA, odhA and rpoB) [12]. The

96 Minimmum Spanning Tree (MST) generated using goeBURST algorithm was built using PHYLOViZ

97 [29].

99 Genome plasticity analysis

100 Prophages of PO100/5 were predicted using PHASTER [30]. Genomic islands were predicted

101 using GIPSy v1.1.2 [31], using C. ulcerans NCTC7910T and C. pseudotuberculosis ATCC19410T as

102 references. A circular map was generated using BRIG v0.95 [32]. The presence of true or candidate

103 virulence factors of Corynebacterium [33,34] were verified using PATRIC’s Protein Family Sorter tool,

104 Proteome Comparison tool, and by comparing the gene neighborhood with other strains using Artemis

105 Comparison Tool 17.0.1 [35]. Signal peptide and conserved protein domains were verified using

106 InterProScan [36], while cell wall sorting signal (CWSS) was verified using CW-PRED [37]. The

107 identification of homologous genes (orthogroups) was performed using OrthoFinder v2.3.12 [38]. In

108 house scripts were used to extract all orthogroups (pangenome) and subsets representing orthogroups

109 conserved across all genomes (core genome), orthogroups shared by more than one but not all genomes

110 (shared genome), orthogroups exclusive from a genome (singletons), orthogroups shared by more than

111 one but not all genomes or exclusive of a genome (accessory genome) [39] and orthogroups conserved

112 and exclusive to a group of genomes (exclusive core); and to calculate the development of pangenome,

113 core genome and singletons [39]. The development of the pangenome was calculated according to Heaps’

114 law fit formula n = κ*Nγ, in which n is the number of genes, N is the number of genomes, and κ and γ (α =

115 γ -1) are free parameters determined empirically. The pangenome is estimated as closed when α > 1 (γ <

116 0), which means no significant increase with the addition of new genomes. The pangenome is estimated

117 to be open when α ≤ 1 (0 < γ < 1). The development of core genome and singletons were calculated using

118 least-squares fit of the exponential regression decay n = κ*exp[-N/τ] + tg(θ), in which n is the number of

119 genes, N is the number of genomes, and κ, τ, and tg(θ) are free parameters determined empirically [39]. A

120 functional annotation of genes was performed using eggNOG-mapper v2 [40].

121

122 Results

123 Taxonomic analysis

124 ANI results showed that C. ulcerans strains PO100/5, 04-13, 05-13 and W25 had identity values

125 ≥ 99.74% with C. silvaticum KL0182T and ≤ 91.03% with C. ulcerans NCTC7910T (S2 File). The

126 taxonomic classification using TYGS classified those strains as C. silvaticum, due to genome and 16S

127 rRNA GBDP trees, dDDh > 70% in the formula d4 and GC content difference > 1% with C. ulcerans

128 genomes (S3 File). In the rpoB phylogeny, the C. ulcerans strains PO100/5, 04-13, 05-13 and W25

129 clustered with C. silvaticum KL0182T, while other C. ulcerans strains formed two clades (Fig 1). In the

130 phylogenetic tree of tox gene, the same four strains (PO100/5, 04-13, 05-13 and W25) also cluster with C.

131 silvaticum, separated from C. ulcerans, C. diphtheriae and C. pseudotuberculosis clusters (Fig 2). MLST

132 analysis classified strains 04-13, 05-13 and W25 as ST578 (53-60-121-70-76-66-57) and PO100/5 as a

133 new ST, differing from ST578 by a new allele in locus odhA (66~) (S4 File, Fig 3). Due to those results

134 those four strains were reclassified for the next analyses, changing the number of C. silvaticum genomes

135 from 34 to 38 and C. ulcerans genomes from 80 to 76.

136

137 Fig 1. Phylogenetic tree of rpoB gene from Corynebacterium species. The phylogeny was inferred

138 using the Maximum Likelihood method and the Tamura-Nei (TN93 + G) model implemented in the Mega

139 v10.1.6. The Corynebacterium ulcerans strains PO100/5, 04-13, W25 and 05-13 cluster with the

140 Corynebacterium silvaticum KL0182T (yellow). C. ulcerans strains form lineage 1 (red, colapsed) and

141 lineage 2 (orange).

142

143 Fig 2. Phylogenetic tree of the tox gene from Corynebacterium species.

144 The phylogeny was inferred using the Maximum Likelihood method and the Tamura-Nei (T92 + G)

145 model implemented in Mega v10.1.6. The strains PO100/5, 04-13, W25 and 05-13 cluster with

146 Corynebacterium silvaticum. Clade colors represent rpoB clades of C. silvaticum (yellow) and C.

147 ulcerans (red and orange).

148

149 Fig 3. goeBURST diagram for the MLST data set of 38 Corynebacterium silvaticum and 76 C.

150 ulcerans strains generated using PHILOViZ. The red numbers on the links indicate the number of

151 divergent alleles between STs.

152

153 The taxonomic analysis led to additional insights. TYGS classified nine C. ulcerans strains as a

154 potential new species: 03-8664, 04-7514, 131002, FRC11, KL0349, LSPQ-04227, LSPQ-04228,

155 NCTC8666 and NCTC12077. These genomes had dDDH values greater than 70% (99.8 – 75.9%) within

156 them and less than 70% (63 to 67.2%) with other C. ulcerans genomes, although the GC content

157 difference was less than 1% (S3 File). In ANI analysis, those nine genomes were more similar to each

158 other than to the other genomes. They had values between 95.52 and 96.57% with C. ulcerans

159 NCTC7910T, and ≥ 97.82% when one of them (NCTC12077) was used as reference for the other eight

160 (S2 File). MLST analysis classified them as having the unique sequence types ST345, ST341, ST344 and

161 a new ST derived from ST345 (S4 File). The ANI analysis showed 99.3% identity between C. diphtheriae

162 lausannense strains CHUV2995 and C. belfantii FRC0043T (S2 File). A further analysis using TYGS

163 classified C. diphtheriae lausannense strains CHUV2995T and CMCNS703 as C. belfantii, and the nine

164 C. belfantii genomes, besides the type strain FRC0043T, as C. diphtheriae (S3 File).

165

166 Genome plasticity analysis

167 The presence of genes encoding 16 known true or candidate virulence factors of

168 Corynebacterium [33,34] in C. silvaticum is shown in Table 1. The genes rhuM, rpb and tspA are absent,

169 while all the pilus genes except spaB are pseudogenized, lacking the signal peptide or CWSS. C.

170 silvaticum has the two pilus gene clusters structured as srtA, spaBC, and srtB, spaD, srtC and spaEF,

171 despite fragmentation of pilin genes. Only eight genomes had the tox gene (04-13, 05-13, KL0182,

172 KL0884, KL0957, KL1196, PO100/5 and W25), and only PO100/5 does not have a two bases insertion

173 (GG) in position 48 that introduces a frameshift. In C. ulcerans, tspA is present in all strains, rpb is

174 present only in strain 809, and rhuM is present in 16 strains from Austria, France and Germany isolated

175 from humans, cats and dogs (02-13, FRC58, KL0195, KL0246-cb3, KL0251-cb4, KL0252-cb5, KL0349,

176 KL0387-cb8, KL0475, KL0497, KL0541, KL0547, KL0796, KL0867, KL0880, NCTC12077).

177 Table 1. Presence of 16 known true or candidate virulence factors of Corynebacterium in C. silvaticum

Gene Product Reference locus Reference protein C. silvaticum protein Manual curation

tag family family

endoE Endoglycosidase E (former CULC809_01974 PLF_1716_00006954 PLF_1716_00006954 Present

corynebacterial protease

CP40)

cwlH Cell wall-associated CULC809_01521 PLF_1716_00062893 PLF_1716_00062893 Present

hydrolase

nanH Sialidase (neuraminidase H) CULC809_00434 PLF_1716_00002393 PLF_1716_00002393 Present

pld Phospholipase D CULC809_00040 PLF_1716_00029465 PLF_1716_00029465 Present

rbp Shiga-like ribosome-binding CULC809_00177 PLF_1716_00033486 - Absent

protein

rhuM RhuM-like protein CulFRC58_0285 PLF_1716_00026137 - Absent

rpfI Resuscitation-promoting CULC809_01133 PLF_1716_00001449 PLF_1716_00001449 Present

factor-interacting protein

9 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

spaB Surface-anchored protein CULC809_01980 PLF_1716_00010184 PLF_1716_00010184 Present

(minor pilus subunit)

spaC Surface-anchored protein CULC809_01979 PLF_1716_00004783 PLF_1716_00004783 Pseudogene, no cell wall sorting

(pilus tip protein) signal

spaD Surface-anchored protein CULC809_01952 PLF_1716_00090862 PLF_1716_00102654 Pseudogene, no cell wall sorting

(major pilus subunit) signal

spaE Surface-anchored protein CULC809_01950 PLF_1716_00007274 PLF_1716_00079271 Pseudogene, no signal peptide

(minor pilus subunit)

spaF Surface-anchored protein CULC809_01949 PLF_1716_00006760 PLF_1716_00006760 Pseudogene, no signal peptide

(pilus tip protein)

tox Diphtheria toxin CULC0102_0213 PLF_1716_00005191 PLF_1716_00005191 Present in 8 out of 38 genomes

tspA Trypsin-like serine protease CULC809_01848 PLF_1716_00007827 - Absent

vsp1 Venom serine protease CULC809_00509 PLF_1716_00104602 PLF_1716_00104343 64% identity with C. ulcerans 809

vsp2 Venom serine protease CULC809_01964 PLF_1716_00015799 PLF_1716_00116381 74% identity with C. ulcerans 809

- C. diphtheriae DIP0733 CULC22_00609 PLF_1716_00030114 PLF_1716_00030114 Present

homolog

178

179 Sixteen and eight islands were predicted by comparing PO100/5 with the reference strains C.

180 pseudotuberculosis ATCC19410T and C. ulcerans NCTC7910T, respectively, and no island was detected

181 in comparison to C. silvaticum KL0182T (Table 2, Fig 4). The content of the islands is described in S5

182 File. Within them, four incomplete prophages were predicted, one of them harboring the tox gene (Table

183 3, Fig 4). The number of orthogroups in pangenome, core genome, accessory genome and singletons of C.

184 silvaticum and C. ulcerans is shown in Table 4 and S6 File. The core genome represented 73.6 and 40%

185 for C. silvaticum and C. ulcerans, respectively. The pangenome, core genome and singletons development

186 graphs and formulas are shown in Fig 5.

187

188 Table 2. Genomic islands in strain PO100/5 in comparison to Corynebacterium pseudotuberculosis

189 ATCC19410T and C. ulcerans NCTC7910T.

n Position compared Size Position Size Type Prophage

to Cp compared to Cul content

1 29362-38109 8.75 kb - - - -

2 55224-60448 5.22 kb 55224-60448 5.22 kb PA -

3 69256-74863 5.6 kb - - PA, RE, SY -

4 97842-103378 5.53 kb - - PA, RE -

5 167859-206438 38.58 kb 167859-206209 38.35 kb - Prophage I

6 311533-318567 7.03 kb 311533-318567 70.34 kb - -

7 422293-430839 85.46 kb - - RE, SY -

8 694806-746966 52.16 kb 694806-746966 52.16 kb RE Prophage II

9 925047-938225 13.18 kb 925047-938225 13.18 kb - -

10 1235769-1244625 8.86 kb 1235769-1244625 8.86 kb - -

11 1613625-1644953 31.33 kb 1614013-1639302 25.29 kb - Prophage III

12 1793740-1802246 8.5 kb - - PA, ME -

13 2029016-2035120 6.1 kb - - - -

14 2109299-2139505 30.2 kb 2110317-2139505 29.19 kb - Prophage IV

15 2255307-2261370 6.06 kb - - - -

16 2517632-2529863 12.23 kb - - ME -

190 Cp – C. pseudotuberculosis, Cul – C. ulcerans, PA – pathogenicity island, RE – resistance island, ME –

191 metabolic island, SY – symbiotic island.

192

193 Fig 4. Circular map of Corynebacterium silvaticum genomes generated using BRIG v0.95.

194 From inner to outer circle: strain PO100/5 (reference); CG content; CG Skew; strains KL0182T, KL0183,

195 KL0259, KL0260, KL0374, KL0382, KL0386, KL0394, KL0395, KL0396, KL400, KL0401, KL0581,

196 KL0598, KL0615, KL0707, KL0709, KL0773, KL0774, KL0882, KL0883, KL0884, KL0886, KL0887,

197 KL0938, KL0957, KL0968, KL1003, KL1006, KL1007, KL1008, KL1009, KL1010, KL1196, 05-13, 04-

198 13, W25; genomic islands compared to C. pseudotuberculosis ATCC19410T (blue) and C. ulcerans strain

199 NCTC7910T (grey); and prophages (black). Genomic islands (GI) and prophage detection were performed

200 using GIPSy and PHASTER, respectively.

201

202 Table 3. Incomplete prophages in strain PO100/5.

Prophage Length Position

Prophage I (tox+) 44.5 kb 161483-206048

Prophage II 23.5 kb 719365-742927

Prophage III 44.3 kb 1594987-1639315

Prophage IV 9.9 kb 2129289-2139193

203

204 Table 4. Pangenomics of Corynebacterium silvaticum and C. ulcerans

Species Genomes Pangenome Core Shared Singletons α

genome genome

C. silvaticum 38 3,002 2,209 603 190 0.9520

C. ulcerans 76 4,351 1,747 1,706 898 0.8142

C. silvaticum 114 4,916 1,618 2,349 949 _

and C. ulcerans

205

206

207

208 Fig 5. Pangenome, core genome and singletons development graphs and formulas for 38 genomes

209 of Corynebacterium silvaticum and 76 C. ulcerans genomes.

210

211

212 Both species had genes conserved in all strains that were absent in the other species, or the

213 exclusive core. In C. silvaticum, 172 orthogroups were detected in this subset. They are represented in

214 strain PO100/5 by 174 proteins, 81 of them located across genomic islands 1, 2, 5, 6, 8, 9, 10, 11, 12 and

215 14. C. ulcerans lineage 2 had a hypothetical protein with 37 amino acids (S6 File). A graph comparing the

216 distribution of Cluster of Homologous Groups (COG) categories of the exclusive core genome of both

217 species is shown in Fig 6.

218

219

220 Fig 6. Clusters of Orthologous Groups (COGs) in the accessory and exclusive core genomes of

221 Corynebacteirum silvaticum and C. ulcerans annotated using eggNOG-mapper v2. COG categories 14

222 are sorted from most abundant to less abundant in C. silvaticum. In C. silvaticum, 356 out of 793 proteins

223 from the accessory genome and 93 out of 174 proteins of the exclusive core genome had a COG category.

224 In C. ulcerans, 1,065 out of 2,064 proteins from the accessory genome and six out of nine proteins of the

225 exclusive core genome had a COG category

226

227 Discussion

228 Strain PO100/5 was isolated from a caseous lymphadenitis lesion of a pig from a swine farm in

229 the Alentejo region of Portugal. It was classified as C. pseudotuberculosis by biochemical tests (Api

230 Coryne® kit), multiplex PCR and Pulsed Field Gel Electrophoresis [8]. After genome sequencing, a rpoB

231 phylogeny clustered it close to C. ulcerans and the genome was deposited in GenBank as this species in

232 2017 (accession number CP021417.1). During the submission and review of this manuscript, PO100/5

233 was suggested as C. silvaticum by rpoB phylogeny, while another study of 28 C. ulcerans genomes

234 suggested strains PO100/5, W25 and KL1196 [4] as a new species [9]. Here, we confirmed the taxonomy

235 of PO100/5 as C. silvaticum and analyzed the genome diversity of this species, using 34 C. silvaticum and

236 80 C. ulcerans genomes, four of the C. ulcerans reclassified, as well as other publicly available genomes

237 from the C. diphtheriae group (S1 File).

238 Taxonomic analysis showed that PO100/5, W25, 04-13 and 05-13 are strains that belong to the

239 recently described C. silvaticum [4]. This is supported by ANI values above 95% [19] (S2 File), genome

240 and 16S rRNA GBDP clustering [23], dDDH > 70% in the formula d4, GC content difference > 1% with

241 C. ulcerans genomes [20,23,41] (S3 File), rpoB phylogenetic clustering (Fig 1) and the unique sequence

242 type ST578 from C. silvaticum [4] (Fig 3). Strain PO100/5 has a new allele in locus odhA (S4 File, Fig 4),

243 which results in a new ST derived from ST578. The original misclassification of those strains was

244 understandable, as prior to the development of methods to identify C. silvaticum [4,5], the use of

245 biochemistry tests (API Coryne and VITEK2-compact) and the clinical picture would classify this strains

246 as C. pseudotuberculosis [4,42], while DNA sequence analysis and fourier-transformed infrared

247 spectroscopy would classify it as C. ulcerans [6,7,42].

248 Analysis of genome plasticity identified unique characteristics of C. silvaticum. The analysis of

249 16 known true or candidate virulence factors showed the absence of rpb, rhuM, and tsA, and spaB as the

250 only non-fragmented pili gene in C. silvaticum (Table 1). The Shiga-like ribosome-binding protein (rpb)

251 has a ribosome inactivating protein domain and was reported only in C. ulcerans 809 [33,43]. The RhuM-

252 like protein (rhuM) was reported only in C. ulcerans strain KL0387 from a human, probably transmitted

253 by a cat [44]. A RhuM mutant of Salmonella enterica had a significant decrease in epithelial cell invasion

254 [45]. Here, we identified this protein in 15 other strains from humans, dogs and cats form Austria, France

255 and Germany. Serine proteases can promote the survival and dissemination of pathogens in the host [46].

256 Venom serine proteases (vsp1 and vsp2) and Trypsin-like serine protease (tspA) are secreted proteases

257 that could have multiple potential pathogenic functions [47]. The effect of the absence of tspA in C.

258 silvaticum is unknown, but it can be used as a marker to differentiate it from C. ulcerans. Bacterial pili are

259 adhesion structures required for colonization of host tissues. The Corynebacterium pili are SpaA-type, a

260 heterotrimeric structure composed by major (pilus shaft), minor and tip pilins, the last two required for

261 adhesion. The pilus is assembled and anchored to the cell wall by the housekeeping sortase SrtA and pili

262 sortases SrtB and SrtC [48]. As with C. ulcerans [33], C. silvaticum has the two pili gene clusters spaBC

263 and spaDEF, although only spaB appears to be functional due to the presence of a signal peptide and a

264 CWSS. The SpaB is a minor pilin that in C. diphtheriae has a role in adhesion on pharyngeal epithelial

265 cells and could be functional when linked to the cell wall [49] as shown for the heterodimeric structure

266 SpaB-SpaC in C. diphtheriae [50] and suggested for C. ulcerans [33]. Intriguingly, only eight of the 38

267 genomes had the tox gene (04-13, 05-13, KL0182, KL0884, KL0957, KL1196, PO100/5 and W25),

268 although the strains lacking it was reported to be tox+ [6]. The absence of tox could be the result of an

269 assembly artifact, due to a repetitive region prior to this gene. This can be seen in the circular map as a

270 blank space in the tox gene region of the other strains (Fig 3). In a previous study, the tox gene from C.

271 silvaticum strains W25 and KL1196 was shown to have an insertion of two bases (GG) in position 48,

272 turning it into a pseudogene, while PO100/5 did not has this insertion [9]. This suggest PO100/5 could be

273 a toxigenic C. silvaticum.

274 In PO100/5, four incomplete prophages (Table 2, Fig 3) were found, one harboring the tox gene.

275 Sixteen and eight genomic islands were found when compared to C. pseudotuberculosis ATCC 19410T

276 and C. ulcerans NCTC7910T (Table 3, S5 File, Fig 3), with four of them containing the incomplete

277 prophages. No island was found when compared to C. silvaticum KL0182T. Genomic islands are regions

278 acquired by horizontal gene transfer that can provide adaptive traits [31]. In previous studies, the

279 phylogenetic analysis of prophages and other mobile elements, tox and DtxR gene sequences showed

280 species-specific clades, including the atypical C. ulcerans clade that now represents C. silvaticum [6].

281 This implies different or independent events of acquisition of virulence factors that could be related to

282 different disease manifestations, progression, as well as an expanded host reservoir that can result in

283 zoonotic transmission [6,43].

284 C. silvaticum was estimated to be a more clonal species than C. ulcerans and to have a

285 pangenome near to being closed, and bigger core genome, with higher values of core genome

286 development (Fig 5) and α closer to 1 (Table 4). This result could be influenced by the samples of C.

287 silvaticum being from two countries, Germany (n = 37) and Portugal (n = 1), and two species of host (Sus

288 scrofa and Capreolus capreolus). A total of 172 and 8 orthogroups were uniquely shared by all C.

289 silvaticum and C. ulcerans, respectively, some in genomic islands (S6 File, Fig 6). For C. silvaticum, the

290 most abundant functions are involved in nutrient acquisition such as transport and metabolism of

291 inorganic ions, carbohydrates and amino acids (COG categories E, G and P), or are related to phages or

292 immunity against them (COG category L). For example, two of them are a Type I restriction-modification

293 system [51] in genomic island 11 and an “ABC-type dipeptide oligopeptide nickel transport system”. The

294 function of those genes in the phenotype and infection must be investigated, but they are candidates for

295 genetic markers for a rapid and cost-effective diagnostic using multiplex polymerase chain reaction

296 (PCR) [52–54], along with the MLST analysis, including the new ST of PO100/5, and other established

297 methods [4,5].

298 Additionally, the TYGS results suggest that the nine C. ulcerans corresponding to lineage 2 [43]

299 represent a potential new species, with dDDH of less than 70% with lineage 1 genomes. MLST classified

300 these genomes with the unique sequence types ST345, ST341, ST344 and a new one derived from ST345

301 (S3 and S4 Files). In a previous analysis, ST335 and ST344 were the only known STs found in lineage 2

302 [43]. Further investigation is required to verify whether this lineage could be classified as a new species.

303 Recently, C. belfantii and C. diphtheriae lausannense were suggested as synonyms [3]. Our analysis

304 using TYGS corroborated the suggestion and showed that other genomes of C. belfantii besides the type

305 strain (FRC0043T) are C. diphtheriae (S3 File), showing the difficulty in classifying this recently

306 described species [2].

307 We showed that strains PO100/5 (domestic pig), W25 (wild boar), 04-13 and 05-13 are C.

308 silvaticum. This species was previously described as atypical NTTB C. ulcerans or “wild boar cluster”

309 (WBC) that infects wild boars and roe deer in Germany and Austria and causes lymphadenomegaly

310 similar to C. pseudotuberculosis infections [5–7]. As other strains of this group are found in Germany and

311 Austria [5,7], PO100/5 is the first genome of this species isolated from a different geographical area

312 (Portugal) and from a domesticated animal (farm pig) [8] and to have a unique ST. The production on DT

313 was not tested in strain PO100/5 [8], but the absence of a two bases insertion in the tox gene, that occurs

314 in the other strains, suggest that it may be the first toxigenic strain described for this species [6,7].

315 In addition to the veterinary concerns posed by this pathogen, C. silvaticum could also have a

316 medical relevance. Its known host range is limited to wild boars, domestic pig and roe deer [4,7,8]. Wild

317 boars are reservoirs for viruses, bacteria and other parasites that can be transmitted to domestic animals

318 and humans, during opportunities provided by deforestation and use of lands for agricultural purposes,

319 hunting activities and consumption of wild boar meat [13]. Besides other hosts, pigs and boars are a

320 reservoir of C. ulcerans that can cause zoonotic transmission to humans [10–12]. By the same route, C.

321 silvaticum could be transmitted to humans and cause infection. In addition, it could be misidentified as C.

322 ulcerans or C. pseudotuberculosis due to limitations of the standard methods [4,5]. The data generated in

323 this study along with the available literature can be used in the identification, treatment and surveillance

324 of this pathogen.

325 Conclusions

326 The taxonomic analysis shows PO100/5 and four other genomes deposited as C. ulcerans should

327 be included in the recently described species C. silvaticum. The comparative genomic analysis showed

328 this species is more clonal than C. ulcerans, has SpaB as the only probably functional pilin subunit, and

329 has conserved genomic islands and 172 genes that could be used as molecular makers for PCR

330 identification. In contrast to the other strains from the same species, PO100/5 is the first one to be isolated

331 from a domestic animal, the first isolation from Germany, and also has a unique ST and a non-

332 pseudogenized tox gene. This data can be used in the identification, treatment and surveillance of this

333 pathogen.

334

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495 Supporting information

496 S1 File. Genomes of Corynebacterium species used for taxonomic analysis of the strain PO100/5.

497 S2 File. Average Nucleotide Identity and digital DNA-DNA hybridization among strains of

498 Corynebacterium.

499 S3 File. Taxonomic classification of Corynebacterium ulcerans and C. silvaticum. The analysis was

500 performed using Type Strain Genome Server.

501 S4 File. Multilocus Sequence Typing data of Corynebacterium silvaticum and C. ulcerans genomes.

502 The analysis was performed using MLSTchecker.

503 S5 File. Genomic islands content in strain PO100/5.

504 S6 File. Pangenome analysis of 38 Corynebacterium silvaticum and 76 C. ulcerans samples. Gene

505 homology groups were predicted using OrthoFinder v2.12.2 and functional annotation was performed

506 using eggNOG-mapper v2.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.206599; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.