1

Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis

Peter Kuhnert1,*, Isabelle Brodard1, Maher Alsaaod1,2, Adrian Steiner2, Michael H. Stoffel3 and Joerg Jores1

TAXONOMIC DESCRIPTIONKuhnert et al., Int. J. Syst. Evol. Microbiol.

DOI 10.1099/ijsem.0.004027

Author affiliations: 1Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; 2Clinic for Ruminants, Vetsuisse Faculty, University of Bern, Bern, Switzerland; 3Division of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, Bern, Switzerland.*Correspondence: Peter Kuhnert, peter. kuhnert@ vetsuisse. unibe. chKeywords: cattle; lameness; digital dermatitis; hoof disease; Treponema.Abbreviations: ANI, average nucleotide identity; DD, digital dermatitis; MALDI- TOF, matrix assisted laser desorption ionization- time of flight; MLST, multi locus sequence typing; MSP, MALDI- TOF reference spectra; TSA, tryptic soy agar.The GenBank accession number for the 16S rRNA gene of T. phagedenis B43.1T is MN396624. The genome sequences of T. phagedenis strains have been deposited under accession numbers CP042818 (B43.1T), CP042817 (B36.5), CP042816 (B31.4), CP042815 (S2.3), CP042814 (S8.5), CP042813 (S11.1), and VOQA00000000 (ATCC 27087).One supplementary table is available with the online version of this article.

004027 © 2020 The Authors

Abstract

‘Treponema phagedenis’ was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomen-clature. Six Treponema strains positive in a ‘T. phagedenis’ phylogroup- specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain ‘T. phagedenis’ ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI- TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with ‘T. phagedenis’-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human ‘T. phagedenis’ were observed. Slight genomic as well as phenotypic vari-ations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Species of the genus Treponema are spiral- shaped, strictly anaerobic or microaerophilic Gram- negative bacteria. They are often associated with specific hosts and a fraction of them are well- recognized pathogens. The genus currently comprises 28 validated species names according to the List of Prokary-otic Names with Standing in Nomenclature [1]. However, although on this list, type strains and their corresponding 16S rRNA gene sequences are not available for Treponema minutum, Treponema paraluiscuniculi, Treponema pertenue and Treponema pallidum, even though the latter is the type species of the genus. The list does not include ‘Treponema phagedenis’ and this name has not been validly published until today. To be validly published, a bacterial name must be (i) contained in the Approved List of Bacterial Names [2] or after 1980 (ii) be published in the IJSB/IJSEM or (iii) if published outside IJSEM be included in a validation list published in there and, finally, (iv) the type strain of the species must be

designated and deposited in at least two culture collections in different countries [3]. None of these criteria have been met for ‘T. phagedenis’.

The fact that ‘T. phagedenis’ till this day has not been validly published is somehow surprising. The species has been known for more than 100 years and the isolation and partial charac-terization of ‘T. phagedenis’ or ‘T. phagedenis’-like bacteria from bovine digital dermatitis (DD), a globally leading form of foot disease- related lameness in cattle [4], has been reported many times over the years [5–14]. A possible reason might be the fact, that ‘T. phagedenis’ was originally isolated from a woman [15] and that the only ‘T. phagedenis’ strain deposited at a culture collection, strain ATCC 27087, is also of human origin and was isolated from a case of syphilis [16]. This strain has originally been deposited as T. pallidum Kazan 8.

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Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

In 1912, Noguchi [15] first described 'Spirochaeta phagedenis' isolated from a phagedenic lesion on the external genitalia of a woman for which later the name ‘T. phagedenis’ was used [17, 18]. The first report of bovine isolates similar to ‘T. phagedenis’ was in 1997 when Choi et al. [12] described a strain which was isolated from a bovine DD lesion and which was related to human ‘T. phagedenis’ based as on 16S rRNA gene sequences. This was later confirmed by other studies using biochemical and genetic comparisons [10, 19]. Bovine DD has emerged worldwide since its first descrip-tion in 1974 [20] and has become the most common and most important infectious foot disease causing lameness in cattle. Three different Treponema phylogroups were isolated and characterized from DD lesions, i.e. T. medium phylo-group, T. pedis phylogroup and ‘T. phagedenis’ phylogroup [19, 21]. While the former two are recognized species, the latter still awaits taxonomic appraisal [22]. Moreover, several authors have shown that human and bovine ‘T. phagedenis’ strains showed a low level of diversity and should actually be described as the same species [10, 19]. A complete taxo-nomic description would help to investigate the role of this species in pathogenicity and host–pathogen interaction. It is of utmost importance to decipher the causative pathogens of DD, to develop diagnostic assays and to conduct epide-miological studies related to DD. We, therefore, propose the validation of the taxon name Treponema phagedenis sp. nov., nom. rev.

For this purpose, we analysed six Swiss strains isolated from bovine DD lesions in the framework of a prevalence study [23] as well as the human strain ATCC 27087 (Table 1). These strains were first compared to each other using 16SrRNA gene analysis. Afterwards they were phenotypically characterized by using the API ZYM and VITEK2 microbiological identification systems, MALDI- TOF and scanning electron microscopy. Afterwards, next generation sequencing was used to characterize and compare the genome sequences. Results of the phenotypic and genotypic characterization were compared to published data on human T. phagedenis and to bovine T. phagedenis phylogroup isolates from various countries as well as to T. pedis and T. medium, the other two species or phylogroups associated with DD in cattle [22].

ISOlATION AND STRAINSFeet from animals with DD lesions were collected at the slaughterhouse in Langnau (Canton of Bern) and St. Gallen (Canton of St. Gallen) and further processed in the labora-tory on the same day. Lesions were cleared mechanically from dirt and a swab was taken for nested- PCR analyses [13]. Strain isolation from samples being PCR- positive for the T. phagedenis phylogroup was done according to Evans et al. [13] from a punch biopsy cut into pieces in an anaerobic work station (Don Whitley Scientific). Six isolates from different animals were obtained and preserved at −80 %. Three isolates were obtained from Canton of Bern (B43.1T, B36.5 and B31.4) and another three from the Canton of St. Gallen (S2.3, S8.5 and S11.1). If needed, strains were grown from stocks in oteb (Anaerob Systems) supplemented with 10 % rabbit serum (Sigma- Aldrich) or on tripticase soy agar (TSA)–blood plates (Oxoid) under anaerobic conditions at 37 °C. The human T. phagedenis ATCC 27087 as well as the type strains of T. medium ATCC 700293T and T. pedis DSM 18691T were obtained from their corresponding culture collections.

PhENOTyPIC AND ChEMOTAXONOMIC ChARACTERIzATIONThe API ZYM strips (bioMérieux) were used to determine enzyme profiles of isolates according to the manufacturer's recommendations. Table  2 shows the enzyme profiles of T. phagedenis isolates in comparison to other Treponema species. Treponema phagedenis showed consistent results for all reactions except for leucine arylamidase, naphthol- AS- BI- phosphohydrolase and β-glucuronidase. Therefore, T. phagedenis can be separated from all other Treponema species by two or more characters used on the API ZYM system. Conflicting results compared to published data were repeatedly obtained with the type strains of T. pedis (acid phosphatase) and T. medium (alkaline phosphatase, C8 esterase lipase and acid phosphatase) [24]. This may be due to the subjective interpretation of reactions on a scale from 0 to 5, whereby 0, 1, 2 are considered negative while 3, 4 and 5 are positive according to the supplier.

In order to gain objective and extended enzyme profiles, we generated data on the automated system VITEK2 using ANC cards (bioMérieux). The T. phagedenis isolates were thereby compared to type strains of T. pedis and T. medium (Table 3). This way, it was possible to unambiguously identify and separate T. phagedenis by five and four stable characters from T. pedis and T. medium, respectively. Interestingly, the human T. phagedenis ATCC 27087 showed different reactions for LeuA, TyrA, PheA and OPS compared to all six bovine isolates (Table 3). These markers allowed to phenotypically differentiate the human strain from bovine T. phagedenis isolates.

Reference spectra (MSP) of type strains of T. pedis and T. medium as well as from the T. phagedenis field isolates B43.1T and B31.4 were generated on a Microflex LT (Bruker) from liquid cultures by the extraction method according to standard procedures [25]. Newly generated spectra and

Table 1. Treponema phagedenis strains characterized in this study

Designation Year Origin

B43.1T 2018 Bovine DD lesion, Switzerland

B36.5 2018 Bovine DD lesion, Switzerland

B31.4 2018 Bovine DD lesion, Switzerland

S2.3 2019 Bovine DD lesion, Switzerland

S8.5 2019 Bovine DD lesion, Switzerland

S11.1 2019 Bovine DD lesion, Switzerland

ATCC 27087 1965 Human syphilis, Kazan, Russia

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Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

Tabl

e 2.

Com

pari

son

of A

PI Z

YM p

rofil

es o

f Tre

pone

ma

phag

eden

is s

trai

ns w

ith ty

pe s

trai

ns o

f oth

er T

repo

nem

a sp

ecie

s

Enzy

mes

tes

ted:

1, a

lkal

ine

phos

phat

ase;

2, e

ster

ase

(C4)

; 3, e

ster

ase

lipas

e (C

8); 4

, lip

ase

(C14

); 5,

leuc

ine

aryl

amid

ase;

6, v

alin

e ar

ylam

idas

e; 7

, cys

tine

aryl

amid

ase;

8, t

ryps

in; 9

, α-c

hym

otry

psin

; 10

, aci

d ph

osph

atas

e; 1

1, n

apht

hol-

AS

- BI-

phos

phoh

ydro

lase

; 12,

α-g

alac

tosi

dase

; 13,

β-g

alac

tosi

dase

; 14,

β-g

lucu

roni

dase

; 15,

α-g

luco

sida

se; 1

6, β

-glu

cosi

dase

; 17,

N- a

cety

l-β-

gluc

osam

inid

ase;

18

, α-m

anno

sida

se; 1

9, α

-fuc

osid

ase.

+, P

ositi

ve; –

, neg

ativ

e.

Spec

ies

Stra

in1

23

45

67

89

1011

1213

1415

1617

1819

Trep

onem

a ph

aged

enis

B43.

1T+

+–

––

––

––

++

–+

––

–+

––

Trep

onem

a ph

aged

enis

B36.

5+

+–

–+

––

––

++

–+

––

–+

––

Trep

onem

a ph

aged

enis

B31.

4+

+–

––

––

––

+–

–+

––

–+

––

Trep

onem

a ph

aged

enis

S2.3

++

––

+–

––

–+

+–

+–

––

+–

–

Trep

onem

a ph

aged

enis

S8.5

++

––

+–

––

–+

+–

++

––

+–

–

Trep

onem

a ph

aged

enis

S11.

1+

+–

–+

––

––

++

–+

+–

–+

––

Trep

onem

a ph

aged

enis

ATC

C 2

7087

++

––

+–

––

–+

+–

++

––

+–

–

Trep

onem

a pe

dis

DSM

186

91T

–+

+–

––

–+

++

––

––

––

––

–

Trep

onem

a m

ediu

mAT

CC

700

293T

–+

––

+–

––

–+

––

+–

––

––

–

Trep

onem

a re

ctal

eaC

HPA

T–

+–

––

––

––

––

++

––

––

––

Trep

onem

a ru

min

isaD

SM 1

0346

2T–

–+

–+

––

––

––

–+

––

+–

––

Trep

onem

a pa

rvum

aAT

CC

700

770T

++

+–

––

––

–+

+–

–+

––

––

–

Trep

onem

a be

rline

nsea

ATC

C B

AA-

909T

––

––

––

––

–+

+–

––

––

––

–

Trep

onem

a po

rcin

uma

ATC

C B

AA-

908T

–+

––

––

––

–+

+–

––

+–

––

–

Trep

onem

a br

enna

bore

nsea

DSM

121

68T

++

+–

––

––

–+

+–

+–

+–

+–

–

Trep

onem

a pe

ctin

ovor

uma

ATC

C 3

3768

T–

++

––

––

––

++

––

––

––

––

Trep

onem

a so

cran

skiia

ATC

C 3

5536

T+

+–

––

––

––

++

––

––

––

––

subs

p. b

ucca

leAT

CC

355

34+

++

––

––

––

++

––

+–

––

––

subs

p. p

ared

isAT

CC

355

35+

++

––

––

––

++

––

––

––

––

Trep

onem

a m

alto

philu

ma

ATC

C 5

1939

T+

++

––

––

––

++

+–

–+

––

–+

Trep

onem

a am

ylov

orum

aAT

CC

700

288T

++

––

––

––

–+

+–

––

––

––

+

Trep

onem

a de

ntico

laa

ATC

C 3

5405

T–

+–

––

––

++

––

––

––

––

––

Trep

onem

a pu

tidum

aAT

CC

700

334T

++

+–

+–

–+

++

++

+–

++

––

–

Trep

onem

a lec

ithin

olyt

icum

aAT

CC

700

332T

++

+–

––

––

–+

+–

++

––

+–

+

a, D

ata

take

n fr

om [2

4].

4

Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

Table 3. Comparison of VITEK2 reactions of Treponema phagedenis, Treponema pedis and Treponema medium

Strains: 1, Treponema phagedenis B43.1T; 2, Treponema phagedenis B36.5; 3, Treponema phagedenis B31.4; 4, Treponema phagedenis S2.3; 5, Treponema phagedenis S8.5; 6, Treponema phagedenis S11.1; 7, Treponema phagedenis ATCC 27087; 8, Treponema medium ATCC 700293T; 9, Treponema pedis DSM 18691T.

Test (VITEK2 abbreviation) Strain

1 2 3 4 5 6 7 8 9

d- Galactose (dGAL) − − − − − − − − −

Cellobiose (dCEL) − − − − − − − − −

Sucrose (SAC) − − − − − − − − −

β-Galactopyranosidase indoxyl (BGALi) + + + + + + + − −

Maltotriose (MTE) − − − − − − − − −

Phosphatase (PHOS) + (+) − + − + + − −

Leucine–arylamidase (LeuA) + + + + + + − + +

Tyrosine–arylamidase (TyrA) + + + + + + − + +

Arbutin (ARB) − − − − − − − − −

α-Arabinosidase (AARA) (+) (+) − + − + + − −

Aesculin hydrolysis (ESC) − − − − − − − − −

l- Arabinose (IARA) − − − − − − − − −

Ellman (ELLM) + + + + + + + − +

Ala–Phe–Pro–arylamidase (APPA) − − − − − − − − +

N- acetyl- d- glucosamide(NAG) − − − − − − − − −

5- brom-4- chlor-3- indoxyl-α-Galactoside (AGALi) − − − − − − − − −

β- d- Fucosidase(BdFUC) + + (-) + + + + − +

d- Ribose 2 (dRIB2) − − − − − − − − −

Phenylalanine–arylamidase (PheA) + + + + + + − + +

d- Glucose (dGLU) − − − − − − − − −

5- brom-4- chlor-3- indoxyl-β-Glucoside (BGLUi) − − − − − − − − −

β-Mannosidase (BMAN) − − − − − − − − −

5- brom-4- chlor-3- indoxyl-β- N- Acetyl- glucosamide (BNAGi) + + − + − + − − −

phenylphosphonate (OPS) − − − − − − + − −

l- Proline–arylamidase (ProA) − − − − − − − − +

d- Mannose (dMNE) − − − − − − − − −

Urease (URE) − − − − − − − − −

Arginine–GP (ARG) − − − − − − − − −

5- brom-4- chlor-3- indoxyl-α-Mannoside (AMANi) − + − (+) − + + − −

α-Arabinfuranoside (AARAF) − − − − − − − − −

l- Pyrrolidonyl–arylamidase (PyrA) + − − − − − + − +

Maltose (dMAL) − − − − − − − − −

5- brom-4- chlor-3- indoxyl-β-Glucuronide (BGURi) + + + + + + + − −

Pyruvate (PVATE) − − − − − − − − −

Continued

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Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

available spectra of spirochaetes from the Bruker database were used to generate a tree in MALDI Biotyper Compass Explorer 4.1 (Bruker) using standard parameters. The two T. phagedenis strains matched well and separated clearly from the other DD associated species as well as from other spirochaetes (Fig. 1). Furthermore, the other field isolates as well as the human strain ATCC 27087 were tested against the new project database and all were clearly identified by score values ≥2.5 with best score values to other species being far below 2.0 (data not shown). Thereby MALDI- TOF is a highly suitable and straightforward method for the identification of T. phagedenis. Best results were obtained by the extraction method from liquid culture, while identification was still possible when using colony material directly from agar plates (score values >2.0).

Scanning electron microscopy was performed with T. phagedenis strains B43.1T and ATCC 27087 as well as with type strains of T. medium and T. pedis (Fig. 2) as reported previ-ously [26] with a field emission scanning electron microscope

DSM 982 Gemini (Zeiss) at an acceleration voltage of 5 kV and at a working distance of 4 mm. The T. phagedenis strains are generally straighter and less spiral- shaped than the other two species. This is in astonishing accordance to the original description by Noguchi [15] who, using a dark- field micro-scope, reported that ‘The number of waves also varied greatly … and some were almost straight’. Cells of T. phagedenis are of variable length from 4 to 14 µm and about 0.25 µm wide.

GENOMIC AND PhylOGENETIC ChARACTERIzATIONDNA was isolated from T. phagedenis cultures using phenol–chlorophorm extraction and sent for PacBio sequencing to the Lausanne Genomic Technologies Facility, located at the Centre for Integrative Genomics of the University of Lausanne, Switzerland. Closed genomes were obtained for the bovine isolates B43.1T (accession no. CP042818), B36.5 (CP042817), B31.4 (CP042816), S2.3 (CP042815), S8.5

Test (VITEK2 abbreviation) Strain

1 2 3 4 5 6 7 8 9

α- l- Fucosidase (AIFUC) + + + + + + + − −

d- Xylose (dXYL) − − − − − − − − −

Table 3. Continued

01002003004005006007008009001,000

Brachyspira intermedia AN621_97 AHVLA

Brachyspira intermedia AN885_94 AHVLA

Brachyspira intermedia AN519_97 AHVLA

Brachyspira intermedia AN1707_96 AHVLA

Brachyspira innocensAN3706_90 AHVLA

Brachyspira innocensC109 AHVLA

Brachyspira murdochii DSM 12563T

Brachyspira pilosicoli AN652_02 AHVLA

Brachyspira pilosicoli C162 AHVLA

Brachyspira pilosicoli GD82 GDD

Brachyspira pilosicoli GD83 GDD

Borrelia burgdorferi OE TWF

Borrelia garinii AE TWF

Borrelia spielmanii IE TWF

Treponema mediumATCC700293T

Treponema pedis DSM1869T

Treponema phagedenis B31.4

Treponema phagedenisB43.1T

MSP Dendrogram

Distance Level

Fig. 1. Dendrogram derived from similarity matrices based on MSP profiles. The distance level is normalized to a maximum value of 1000. The type strain of T. phagedenis is indicated in bold.

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Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

(CP042814) and S11.1 (CP042813), while four contigs were gained for the human strain ATCC 27087 (VOQA00000000). Genome sizes varied from 3.2 to 3.7 Mbp.

Average nucleotide identity (ANI) values were calculated based on genome sequences including the three published ones using the OrthoANIu algorithm [27]. Comparison of T. phagedenis genomes showed ANI values around 99 % or higher and dropped drastically to <70 % when compared to T. pedis and T. medium, thus confirming the species identity of the T. phagedenis strains (Table S1, available in the online version of this article). The human T. phagedenis showed slightly higher ANI values to each other than to the bovine isolates. However, the ANI values were always far above the species boundary of 95–96 % [28]. Phylogenetic analysis of 16S rRNA gene sequences corroborates the results from the genome comparisons, showing similarities of >99 % between available T. phagedenis 16S rRNA gene sequences of bovine and animal isolates (Fig. 3). In the study of Clegg et al. [19] 16S rRNA gene analysis of 70 T. phagedenis phylogroup isolates also showed a high sequence conservation of >99 % within this gene, in accordance with our findings.

Whole genome- based phylogenetic analysis was done using realphy [29]. A high quality merged tree from the 10 trees was built on individual alignments using each genome sequence as reference genome (Fig. 4). The merged tree as well as all 10 individual trees clearly showed phylogenetic separation of the two human and the six bovine isolates.

No spatiotemporal clustering of bovine T. phagedenis was observed. This actually substantiates the findings based on MLST analysis of the 70 T. phagedenis strains by Clegg et al. [19] showing human strains to be as diverse as animal isolates but being separated from the animal isolates in a minimum spanning tree.

PROPOSAl Of Treponema phagedenis SP. NOv., NOM. REv.In summary, our analyses together with published data show that T. phagedenis isolated from bovine DD form a genetically and phenotypically homogenous group with human isolates. Data also indicate some genetic as well as phenotypic variation allowing us to differentiate between commensal human and putative pathogenic bovine strains. Elucidating those differences could help define potential virulence factors of T. phagedenis involved in the pathogen-esis of bovine DD. To investigate the role of T. phagedenis in bovine DD further, we recommend to keep the long standing name in literature and propose this taxon under its commonly used species name that has been revived as Treponema phagedenis sp. nov., nom. rev. The type strain is B43.1T (=DSM 110455T=NCTC 14362T) isolated from bovine DD in Switzerland.

Fig. 2. Scanning electron micropgraphs of Treponema species at various magnification (×1000, ×5000 and ×10000). (a) Bovine Treponema phagedenis B43.1T. (b) Human Treponema phagedenis strain ATCC 27087. (c) Treponema pedis DSM 18691T. (d) Treponema medium ATCC 700293T.

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Treponema maltophilum

Treponema lecithinolyticum

Treponema saccharophilum

Treponema succinifaciens

Treponema ruminis

Treponema brennaborense

Treponema pectinovorum

Treponema berlinense

Treponema bryantii

Treponema socranskii subsp. paredis

Treponema socranskii subsp. socranskii

Treponema socranskii subsp. buccale

Treponema rectale

Treponema parvum

Treponema amylovorum

Treponema porcinum

Treponema isoptericolens

Treponema azotonutricium

Treponema primitia

Treponema stenostreptum

Treponema caldarium

Treponema zuelzerae

Treponema medium

Treponema pallidum subsp. pertenue

Treponema pedis

Treponema denticola

Treponema putidum

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Treponema phagedenis

Spirochaeta psychrophila

DSM 27366T

OMZ 684T

DSM 2985T

DSM 2489T

DSM 103462T

DSM 12168T

ATCC 33768T

ATCC BAA-909T

DSM 1788T

ATCC 35535T

ATCC 35536T

ATCC 35534T

DSM 103679T

DSM 16260T

ATCC 700288T

14V28T

SPIT5T

ATCC BAA-888T

ATCC BAA-887T

DSM 2028T

DSM 7334T

DSM 1903T

ATCC 700293T

str. Gauthier

DSM 18691T

DSM 14222T

ATCC 700334T

Kazan 5

ATCC 27087

F0421

B43.1T

B31.4

Reiter

Kazan 8

CIP 62.29

T257

Mayo-A

4A

YG3903R

V1

S11.1

S8.5

B36.5

S2.3

DSM 23951T

X87140

X87139

M71238

M57738

GU566698

Y16568

GU562449

AY230217

FR749895

AF033307

AF033306

AF033305

GU566699

AF302937

Y09959

AY518274

AM182455

AF320287

AF093252

FR733664

EU580141

FR749929

D85437

AF426102

EF061268

AF139203

AJ543428

M57739

CP042818

CP042816

KR025824

KR025835

EF645248

EF061257

FM210038

AF546875

FJ004921

DQ470655

CP042813

CP042814

CP042817

CP042815

AB598279

0.1

VOQA00000000

AEFH01000000100

100

100

100

100

100

100

100

100

100

100

83

48 32

56

22

74

29

30

50

68

96

91

37

98

65

46

69

Fig. 3. Phylogenetic tree based on 16S rRNA gene sequences of Treponema phagedenis strains and all currently recognized species of the genus Treponema. Spirochaeta psychrophila was included as an outgroup for rooting the tree that was built in Bionumerics 7.6.3 by using Jukes–Cantor correction and the neighbour- joining method for cluster analysis. Bootstrap values from 500 iterations are given at branches. Bar, 10 % sequence divergence. The strain designation and accession number is given besides the species name. The type strain of T. phagedenis is indicated in bold.

8

Kuhnert et al., Int. J. Syst. Evol. Microbiol. 2020

DESCRIPTION Of Treponema phagedenis (ex NOGuChI 1912) BRuMPT 1922 SP. NOv., NOM. REv.Treponema phagedenis ( pha. ge. de'nis. Gr. fem. n. phagedaina a cancerous sore; N.L. gen. n. phagedenis of a cancerous sore).

Cells are Gram- negative, motile, obligatory anaerobic spiro-chaetes. Indole is positive and catalase negative. Cells only show few helical coils, can even be straight and also show differences in length from 4 to 14 µm and about 0.25 µm wide. Optimal growth is obtained at 37 °C in liquid oteb medium supplemented with 10 % rabbit serum, but once isolated, cells also grow on TSA plates containing 5 % sheep blood. Colony morphology is variable, fuzzy, and often star- like with a dense centre. No haemolysis is observed. API ZYM reactions are positive for alkaline phosphatase, esterase (C4), acid phos-phatase, β-galactosidase and N- acetyl-β-glucosaminidase. Negative for esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucosidase, α-mannosidase and α-fucosidase. Variable reactions are observed for leucine arylamidase (type strain negative), naphthol- AS- BI- phosphohydrolase (type strain positive) and β-glucuronidase (type strain negative). Reaction on the VITEK2 ANC card is positive for BGALi, ELLM, BGURi and AIFUC, while nega-tive for dGAL, dCEL, MTE, ARB, ESC, lARA, APPA, NAG, AGALi, dRIB2, dGLU, BGLUi, BMAN, ProA, dMNE, URE, ARG, AARAF, dMAL, PVAT and dXYL. Variable reactions were observed for PHOS (type strain positive), LeuA (bovine isolates positive), TyrA (bovine isolates positive), AARA (type strain positive), BdFUC (type strain positive), PheA (bovine isolates positive), BNAGi (type strain positive), OPS (bovine isolates negative), AMANi (type strain negative) and PyrA (type strain positive).

DNA G+C content is 39.9–40.1 mol% as determined on whole genome sequences.

The type strain is B43.1T (=DSM 110455T=NCTC 14362T), isolated in Switzerland from a bovine DD lesion. The GenBank accession numbers for the 16S rRNA gene and whole genome sequence of T. phagedenis B43.1T are MN396624 and CP042818, respectively. The genome sequences of the other T. phagedenis strains have been deposited under accession numbers CP042817 (B36.5), CP042816 (B31.4), CP042815 (S2.3), CP042814 (S8.5), CP042813 (S11.1) and VOQA00000000 (ATCC 27087).

Funding informationThe authors received no specific grant from any funding agency.

AcknowledgementsWe thank Helga Mogel for preparing the scanning electron micrographs.

Author contributionsP. K. contributed to conceptualization, supervision, methodology, investigation, validation, visualization and writing – original draft. I. B. contributed to investigation, methodology, data curation and writing – review and editing. M. A. contributed to resources and writing – review and editing. A. S. contributed to supervision and writing – review and editing. M. H. S. contributed to resources and writing – review and editing. J. J. was involved in conceptualization, supervision, funding and writing – original draft.

Conflicts of interestThe authors declare that there are no conflicts of interest.

References 1. Parte AC. LPSN - List of prokaryotic names with standing in

Nomenclature ( bacterio. net), 20 years on. Int J Syst Evol Microbiol 2018;68:1825–1829.

2. Skerman VBD, Sneath PHA, McGowan V. Approved Lists of bacte-rial names. Int J Syst Evol Microbiol 1980;30:196–.

3. Parker CT, Tindall BJ, Garrity GM. International Code of Nomencla-ture of prokaryotes. Int J Syst Evol Microbiol 2019;69:S1–s111.

4. Wilson- Welder JH, Alt DP, Nally JE. Digital dermatitis in cattle: current bacterial and immunological findings. Animals 2015;5:1114–1135.

5. Pringle M, Bergsten C, Fernström L- L, Höök H, Johansson K- E. Isolation and characterization of Treponema phagedenis- like spiro-chetes from digital dermatitis lesions in Swedish dairy cattle. Acta Vet Scand 2008;50:40.

6. Trott DJ, Moeller MR, Zuerner RL, Goff JP, Waters WR et al. Charac-terization of Treponema phagedenis- like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle. J Clin Micro-biol 2003;41:2522–2529.

7. Mushtaq M, Manzoor S, Pringle M, Rosander A, Bongcam- Rudloff E. Draft genome sequence of 'Treponema phagedenis' strain V1, isolated from bovine digital dermatitis. Stand Genomic Sci 2015;10:67.

8. Yano T, Moe KK, Chuma T, Misawa N. Antimicrobial susceptibility of Treponema phagedenis- like spirochetes isolated from dairy cattle with papillomatous digital dermatitis lesions in Japan. J Vet Med Sci 2010;72:379–382.

9. Yano T, Yamagami R, Misumi K, Kubota C, Moe KK et al. Genetic heterogeneity among strains of Treponema phagedenis- like spiro-chetes isolated from dairy cattle with papillomatous digital derma-titis in Japan. J Clin Microbiol 2009;47:727–733.

10. Wilson- Welder JH, Elliott MK, Zuerner RL, Bayles DO, Alt DP et  al. Biochemical and molecular characterization of Treponema phagedenis- like spirochetes isolated from a bovine digital derma-titis lesion. BMC Microbiol 2013;13:280.

11. Stamm LV, Bergen HL, Walker RL. Molecular typing of papilloma-tous digital dermatitis- associated Treponema isolates based on analysis of 16S- 23S ribosomal DNA intergenic spacer regions. J Clin Microbiol 2002;40:3463–3469.

ATCC 27087 (human, Kazan, < 1965, VOQA00000000)

F0421 (human, USA, < 2011, AEFH01000000)

B43.1T (bovine, Switzerland, 2018, CP042818)

B36.5 (bovine, Switzerland, 2018, CP042817)

B31.4 (bovine, Switzerland, 2018, CP042816)

S8.5 (bovine, Switzerland, 2019, CP042815)

S2.3 (bovine, Switzerland, 2019, CP042814)

S11.4 (bovine, Switzerland, 2019, CP042813)

4A (bovine, USA, < 2003, AQCF00000000)

V1 (bovine, Sweden, < 2014, CDNC00000000)

Fig. 4. Phylogenetic tree derived from available genome sequences of bovine and human T. phagedenis isolates. A merged tree from the 10 trees was built based on individual alignments using each genome sequence as reference genome using REALPHY 1.12 [18]. The host, country of isolation, most recent year of isolation and genome accession number is given in brackets.

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12. Choi BK, Nattermann H, Grund S, Haider W, Göbel UB. Spirochetes from digital dermatitis lesions in cattle are closely related to trep-onemes associated with human periodontitis. Int J Syst Bacteriol 1997;47:175–181.

13. Evans NJ, Brown JM, Demirkan I, Murray RD, Vink WD et al. Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle. Vet Microbiol 2008;130:141–150.

14. Nally JE, Hornsby RL, Alt DP, Whitelegge JP. Phenotypic and prot-eomic characterization of treponemes associated with bovine digital dermatitis. Vet Microbiol 2019;235:35–42.

15. Noguchi H. Pure cultivation of Spirochæta phagedenis (new species), a spiral organism found in phagedenic lesions on human external genitalia. J Exp Med 1912;16:8–.

16. Ovcinnikov NM, Delektorskij VV. Morphology of Treponema pallidum. Bull World Health Organ 1966;35:223–229.

17. Huffman OV. Spirochaeta pallida or Treponema pallidum? JAMA 1916:LXVII(25).

18. Schaudinn F. Korrespondenzen. Dtsch med Wochenschr 1905;31:1:1728.

19. Clegg SR, Carter SD, Birtles RJ, Brown JM, Hart CA et al. Multilocus sequence typing of pathogenic treponemes isolated from Cloven- Hoofed animals and comparison to treponemes isolated from humans. Appl Environ Microbiol 2016;82:4523–4536.

20. Cheli R, Mortellaro C. La dermatite digitale del bovino. Proceedings of the 8th International Conference on Diseases in Cattle. Milan, Italy: Piacenza; 1974. pp. 208–213.

21. Clegg SR, Crosby- Durrani HE, Bell J, Blundell R, Blowey RW et al. Detection and isolation of digital dermatitis treponemes from bovine pressure sores. J Comp Pathol 2016;154:273–282.

22. Evans NJ, Murray RD, Carter SD. Bovine digital dermatitis: current concepts from laboratory to farm. Vet J 2016;211:3–13.

23. Alsaaod M, Locher I, Jores J, Grimm P, Brodard I et al. Detection of specific Treponema species and Dichelobacter nodosus from digital dermatitis (Mortellaro's disease) lesions in Swiss cattle. Schweiz Arch Tierheilkd 2019;161:207–215.

24. Staton GJ, Newbrook K, Clegg SR, Birtles RJ, Evans NJ et  al. Treponema rectale sp. nov., a spirochete isolated from the bovine rectum. Int J Syst Evol Microbiol 2017;67:2470–2475.

25. Kuhnert P, Bisgaard M, Korczak BM, Schwendener S, Chris-tensen H et al. Identification of animal Pasteurellaceae by MALDI- TOF mass spectrometry. J Microbiol Methods 2012;89:1–7.

26. Thiel A, Mogel H, Bruggisser J, Baumann A, Wyder M et al. Effect of Clostridium perfringens β-Toxin on platelets. Toxins 2017;9:336.

27. Yoon S- H, Ha S- M, Lim J, Kwon S, Chun J. A large- scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 2017;110:1281–1286.

28. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018;68:461–466.

29. Bertels F, Silander OK, Pachkov M, Rainey PB, van Nimwegen E. Auto-mated reconstruction of whole- genome phylogenies from short- sequence reads. Mol Biol Evol 2014;31:1077–1088.

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