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We have provided an existing summary up to a certain point: The study investigates the molecular epidemiology of *Erysipelothrix rhusiopathiae* strains affecting poultry and mammals, focusing on the development of a multilocus sequence typing (MLST) scheme. It analyzed 165 isolates, revealing high genetic diversity with 72 sequence types identified. Notably, 34.5% of isolates clustered into a predominant sequence type (STC9) comprising strains from both avian and mammalian origins, including humans. The congruence of strain clustering from pulsed-field gel electrophoresis (PFGE) with MLST was minimal, and five SpaA protein variants were identified based on strain phylogeny. Over 90% of isolates carried 16 putative virulence genes, highlighting the weakly clonal nature of the species and its limited host specificity. These findings suggest shared reservoirs and pathogenic pathways among diverse hosts. The study emphasizes the need for comprehensive epidemiological studies to understand *E. rhusiopathiae* strains' pathogenic mechanisms and virulence factors better.
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PCR products were separated by gel electrophoresis in a 1.5% agarose gel containing ethidiumbromid for 90 min at 120 V and analysed by gel documentation system (Easy doc, Herolab, Wiesloch, Germany).

### Table 2

Oligonucleotide primers used for multiplex PCRs to detect 16 genes putatively linked with virulence of _E.

rhusiopathiae_

Gene / locus tag

Gene product / predicted function \[reference\]

Primer sequence (5′ – 3′) (forward/reverse)a

GenBank Acc.

No

Localization within gene

Size (bp)

Gene pre-valence (%)

ERH\_1356

ABC transporter metal-binding protein / adhesion of host cells \[[45]\]

CATGAAGGGTAACACCTTGG/ GGGCGATAAAGTTGCGGTAGAA

NC\_015601.1

579 – 787

209

100

_intI-like_

Internalin / invasion of epithelial cells \[[18]\]

ACAGTTTCGGATACTTCCGG/ ACCCTCGTCATATTTACCAGC

AP012027.1

386 – 714

329

85.5

_rspB_

Rhusiopathiae surface protein /biofilm formation \[[41]\]

ATCTTTACCCAATTCGACGT/ ATGAACCCAGTCCAAGATTGG

AB052682.1

6882 – 7287

406

100

_rspA_

Rhusiopathiae surface protein /biofilm formation \[[41]\]

ATCGACTGGTATTCAGTTGG/ ATCACGAGACATACCGCCAA

AB052682.1

597 – 1131

535

100

_cap locus_

Capsule / resistance to phagocytosis, intracellular survival \[[40]\]

TATCTTTGTAGCGTAGTTGG/ CAATAAAAGGAAATACCAGTGC

D64177

1333 – 1987

645

100

_algI_

Alginate-O-acetyltransferase / resistance to phagocytosis \[[45]\]

AGTTATCTTGGACTTGGTCC/ AGATAAGTGCGCATTGATCC

AP012027.1

121 – 887

767

97.6

_ewlA_

Lipoprotein / adhesion to host cells \[[45]\]

TAATATTAGATAGCGAGGAAT/ AAGAAAAGGGAGTGTGAATAT

U52850.1

226 –1185

960

100

_nanH_

Neuraminidase / spreading factor, nutritive \[[44]\]

ATGAAGCGCTTACATTTGAAT/ TACATAAGGTTGACCAAAGTC

AB019122

295 –1401

1.107

100

_sub_

S8-subtilisin / peptidase \[[45]\]

AAGCCTGAGATATCTGCACC/ TTGTACAATTGGATGAGCCG

AP012027.1

1360 –1585

226

100

_sodA_

Superoxide dismutase / antioxidans \[[45]\]

AGAAGACATCCGCACAGCAGT/ GCATGTTCCCAAACATCAAGA

AP012027.1

195 – 509

315

100

_mviN1_

Integral membrane protein / cell adhesion, transport protein \[[45]\]

AAATCATGCTTGTAATGGCGG/ ATTCGACGTTAAAACAACCGC

AP012027.1

565 –1032

378

100

_hep_

Heparinase / inactivation of heparin \[[45]\]

ATGGAAGTACCGATCTCACT/ TCATTGTAGCAACATGGCTTC

AP012027.1

997 –1480

484

100

_hlyIII_

Hemolysin / lytic activity on red blood cells \[[42]\]

TACGATTGCGACAAAGTGTGCG/ ATGGAAACATAGGGAAGGCTG

AP012027.1

16 – 559

544

100

_fbpA_

Fibronectin-binding protein / adhesion \[[41]\]

ATCTCGCCGCTTTTAGAACG/ GCGTCTTCAACTGTTGCTTG

AP012027.1

565 –1166

602

100

_hlyA_

Hyaluronidase / spreading factor \[[2]\]

AGGATCACTTACCGCTATGG/ CAGCACTCAGCATGTTCTC

AP012027.1

598 –1538

941

100

_dnaB_

Membrane-, attachment protein / proliferation, adhesion \[[45]\]

AATAGCCCCTGATCAAATGG/ CTCTCCTTTACTTAACATCCC

ACLK02000002.1

39 –1155

1.117

99.4

### Sequencing of spaA genes

In order to distinguish the three different molecular variants of the Spa protein \[[14]\] we performed restriction length polymorphism (RFLP) analysis and single locus sequence typing (SLST) of the _spa_ gene.

The Spa type variant genes _spaA_, _spaB_ and _spaC_ were PCR amplified under standard PCR conditions (60 ng template DNA, 10 pmol each primer (Sigma Alderich, Munich, Germany), 10 × PCR buffer including 20 mM MgCl2, 5 mM dNTP mixture, and 0.5 U Dream Green Taq DNA Polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany), using primers Spa-fw (5′- ATGAAAAAGAAAAAACACCTA – 3′), located at position 1–21, and Spa-rv (5′- CTATTTTAAACTTCCATCGTT – 3′), located at position 1881–1862 of serotype 1a reference strain Fujisawa (Acc.-No.

AP012027.1).

The cycling conditions were 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 52 °C and 130 s at 72 °C.

Final extension was done at 72 °C for 10 min.

Alignment of primers with publicly available _spaB_ (Acc.-No.

AB238211 - AB238215) and _spaC_ (Acc.-No.

AB238210) gene sequences of serotype 4, 6, 11, 19, 21, and 18 strains, respectively, revealed that they could amplify all known gene variants correctly.

Amplicons were then digested with restriction enzyme _HhaI_ overnight at 37 °C.

Spa genes revealing different RFLP patterns were double-strand sequenced using the same primers as above and data were analyzed with RidomSeqSphere 1.0.1 \[[29]\] and the EditSeq and MegAlign programs of the DNAStar software package (DNASTAR Inc. Madison, Wisconsin, USA).

Alignments of nucleotide and amino acid sequences were performed with RidomSeqSphere.

Results
-------

### Multilocus sequence typing

Allele sizes for the genes included for MLST analysis of 165 _E.

rhusiopathiae_ strains ranged between 531 bp for _ldhA_ to 640 bp for _recA_ (Table [1]).

The mean GC content varied from 36.6% (_pta_) to 40.3% (_ldhA_).

Among the 165 investigated isolates with the corresponding 3.995 bp of the concatenated sequences of seven loci, 75 (1.9%) variable sites were confirmed.

The number of alleles ranged from six for _prsA_ to 14 for _purA_.

The nucleotide diversity within the single loci varied between four (0.7%) for _prsA_ and 14 (2.6%) for _ldhA,_ the diversity index (_D_) for each allele between 0.579 (_pta_) and 0.775 (_recA_).

The _d__N__/d__S_ ratio for all seven loci demonstrated to be < 1 (Table [1]).

The combination of allele numbers and the corresponding sequence types are shown in Table [3].

A list of all strains with their sequence types is available as additional file (see Additional file [1]).

Of 72 sequence types (STs) assigned to 165 _E.

rhusiopathiae_ isolates, more than half (58.3%) were singletons.

The remaining 29 STs were represented by two or more strains.

ST9 was by far the most common sequence type, which grouped 18 isolates (10.9%) from poultry (_n_ = 12), pigs (_n_ = 3), sheep (_n_ = 1), and also from a case of wound infection and a case of endocarditis in humans together.

Sequence type 4 (_n_ = 11 poultry isolates) and ST5 (_n_ = 10 poultry and one pig isolate) were the second most frequent STs.

Overall, eBURST analysis based on all allelic profiles revealed a highly divergent population structure among the _E.

rhusiopathiae_ strains under study (Figure [1]).

### Table 3

Sequence types (ST) with corresponding allele numbers demonstrated for 165 _E.

rhusiopathiae_ and number of each ST

ST

_gpsA_

_recA_

_purA_

_pta_

_prsA_

_galK_

_ldhA_

n

1

1

3

1

1

4

4

4

2

2

1

3

1

3

1

1

1

4

3

2

4

3

2

2

2

2

6

4

2

4

2

2

2

5

2

11

5

2

4

4

2

2

2

3

10

6

2

2

2

2

2

2

2

3

7

1

3

1

1

1

1

1

6

8

1

1

1

1

1

4

1

3

9

1

1

1

1

1

1

1

18

10

1

1

6

2

2

2

2

4

11

1

1

1

1

1

1

3

1

12

1

2

1

1

1

4

1

2

13

2

4

2

2

4

6

2

2

14

3

2

1

1

1

3

1

1

15

2

4

7

2

4

6

2

1

16

2

5

8

2

4

2

2

1

17

2

1

6

1

2

5

2

1

18

2

5

5

2

2

2

3

1

19

2

4

9

1

1

1

2

5

20

2

4

4

2

2

2

4

2

21

1

1

1

1

1

7

1

1

22

3

2

1

4

1

3

1

1

23

1

1

1

1

3

4

1

1

24

4

2

6

2

2

2

2

1

25

5

3

2

5

4

1

1

1

26

1

4

1

1

1

5

1

2

27

2

5

3

2

2

2

3

3

28

2

5

6

2

2

2

2

1

29

1

6

1

1

1

4

1

1

30

1

3

11

1

1

1

1

1

31

1

1

1

1

1

1

6

1

32

5

1

1

1

1

1

1

5

33

2

1

3

2

1

1

2

1

34

2

2

6

2

2

5

2

1

35

1

7

12

6

1

5

7

1

36

1

2

6

2

2

5

2

2

37

6

1

1

2

1

4

1

2

38

2

5

10

2

4

5

3

1

39

2

5

3

2

2

5

2

2

40

2

4

1

7

4

2

3

4

41

2

2

2

2

4

2

2

1

42

2

5

3

2

2

8

2

1

ST

_gpsA_

_recA_

_purA_

_pta_

_prsA_

_galK_

_ldhA_

n

43

1

1

1

1

3

1

1

2

44

1

1

1

1

2

1

1

1

45

1

3

1

1

1

1

9

1

46

1

3

13

1

1

1

1

1

47

2

4

2

8

2

6

3

2

48

2

2

2

2

5

1

2

7

49

2

2

14

2

2

2

8

2

50

2

5

2

2

4

5

2

1

51

1

2

2

1

4

10

10

1

52

5

8

1

1

1

1

9

1

53

6

2

1

1

1

9

1

1

54

7

1

1

1

1

1

1

2

55

2

2

3

2

4

2

3

4

56

1

1

1

1

6

4

1

1

57

1

2

1

1

1

2

1

1

58

1

4

1

9

1

5

1

1

59

2

1

3

2

4

4

2

1

60

2

1

3

2

2

5

11

2

61

2

5

1

2

2

11

2

1

62

2

4

9

1

1

12

2

1

63

1

9

1

1

1

4

1

1

64

2

2

6

2

2

2

3

1

65

8

3

2

1

1

1

1

2

66

1

3

13

1

4

1

1

1

67

5

1

2

1

2

1

1

1

68

1

2

6

1

2

2

2

1

69

5

2

6

1

1

4

1

1

70

9

3

3

2

2

13

2

1

71

1

1

1

1

1

2

1

2

72

2

4

3

2

4

2

2

1

]

**Population snapshot of 165** **_E.

rhusiopathiae_** **isolates assigned to 72 sequence types (STs).

** STs connected by a line share six out of seven alleles.

The dominant ST complex (STC9) which is named after its predicted founder is highlighted by a dotted circle.

Only one sequence type complex, i.e.

a group of phylogenetic related strains was identified.

ST complex 9 (STC9), named after its predicted founder, included 22 STs and accounted for 34.5% of the total collection of MLST-analysed strains.

As depicted in Figure [2A], 40 avian and 17 mammalian _E.

rhusiopathiae_ strains were among the STC9 strains.

In general, no host-specific or host-restricted sequence type was observed, as isolates from different hosts scattered around the various STs identified (Figure [2A]).

In addition, different countries shared isolates with identical STs, e.g.

ST3 (isolates from Austria, Germany, Sweden), ST9 (Austria, Germany, USA), and ST19 (Estonia, Germany, USA), not suggesting any association between certain STs with distinct countries.

However, due to a sample bias towards isolates from Germany, this should be verified by including a larger number of isolates from various countries.

]

**Minimum spanning tree based on allele profiles of 165** **_E.

rhusiopathiae_** **isolates and distribution of** **(A)** **host and (** **B** **) SpaA type** (for Groups I-V see Table [4]).

Sequence types (STs) are indicated by numbers and sequence type complexes are highlighted by grey shade.

### Pulsed-field gel electrophoresis and comparison of sequence types with PFGE clusters

Based on an 85% cut-off- value we identified 54 different PFGE clusters containing one (_n_ = 17 singletons) to 13 strains among 165 _E.

rhusiopathiae_ isolates (Additional file [2]).

Identical macrorestriction profiles were only rarely observed which is consistent with our criteria for strain selection, i.e.

exclusion of copy strains from the analysis.

In contrast to PFGE, MLST revealed 72 different sequence types and, as expected, the grouping achieved by both methods revealed only partial congruence.

The most homogenous macrorestriction profile was observed for strains assigned to ST4.

All eleven ST4 strains, isolated from 1999 to 2011 from chickens and turkeys in Germany clustered exclusively together at a similarity value of 90.9% (Figure [3A]).

In contrast, strains of the most prominent sequence type ST9 were distributed among 12 different PFGE clusters, including three singletons, with an overall similarity of 64.5% (Figure [3B]).

These PFGE clusters frequently harboured strains of other sequence types as well, which is also the case for most of the other non-singleton PFGE clusters.

]

**Dendrogram for** **_E.

rhusiopathiae_** **isolates based on PFGE banding patterns produced by SmaI restriction.

** Illustrated are dendrograms for (**A**) 45 _E.

rhusiopathiae_ isolates of sequence type complex (STC) 9 and isolates of other STs clustering with ST9 strains and (**B**) 11 ST4 isolates that do not cluster with strains of other STs.

Additional information on isolation year and country, host, SpaA-groups (based on amino acid substitutions) and the number of SpaA C-terminal repeats are provided in separate rows.

PFGE clusters (85% cut-off) are indicated by random colours; singletons are left black.

A calculation of the Simpsons index of discriminatory (_D_) with 95% confidence interval (CI95%) revealed that the ability of MLST to discriminate _E.

rhusiopathiae_ isolates (_D_ = 0.973; CL95% \[0.963 to 0.983\]) was similar to that of PFGE (_D_ = 0.976; CL95% \[0.970 to 0.982\]).

However, as exemplified by the dendrograms shown in Figure [3] and in the Additional file [2], strain groups established by the band-based method PFGE and the sequence-based method MLST frequently differ in their composition.

### Virulence gene typing

Highly homogenous patterns of putative virulence genes were observed among the 165 _E.

rhusiopathiae_ isolates with 136 (82.4%) of them possessing all 16 investigated genes (Table [2]).

Another 24 (14.5%) isolates differed only by the absence of internalin gene _intI_.

Five isolates lacked either _algI_ (_n_ = 4), or _dnaB_ (_n_ = 1), which might be associated with colonization and resistance to phagocytosis and with bacterial proliferation and adhesion, respectively \[[45]\].

None of the 16 genes investigated were present in the _E.

inopinata_ reference strain DSM 15511T, whereas _nanH_ and _ewlA_ were detected in the _E.

tonsillarum_ strain DSM 14972T as well as in both field strains of _E.

tonsillarum_.

### Spa typing

Sequence analysis of the amplified DNA fragments of _spa_ genes revealed the consistent presence of _spaA_ and the absence of _spaB_ and _spaC_, respectively, among all 165 _E.

rhusiopathiae_ isolates.

The size of _spaA_ open reading frames ranged from 1761 to 2181 bp (encoding 587 to 727 amino acids).

The signal sequences of 29 amino acids were conserved among the Spa proteins of 164 isolates (99.4%).

Only one isolate from a duck (IMT28342; Acc.-No.

KR606269) showed a mutation at nucleotide position 27 (A to G) and at amino acid position 10 (Lysin to Glutamic acid), respectively.

Sequences of the N-terminal hypervariable region of the _spaA_ gene of field isolates and of _E.

rhusiopathiae_ serotype reference strains were compared with that of the virulent wild-type reference Fujisawa serotype 1a strain (see Table [4]).

Based on patterns of amino acid changes at eight different positions, the field isolates were divided into five groups as follows: (i) 87 isolates with isoleucine at position 55 (Ile-55), Asn-70, Asp-178, Asn-195, Ile-257 and Gln-303 classified as group I, (ii) 36 isolates with Ser-101 and Ile-257 classified as group II, (iii) 37 isolates with Ile-257 termed group III, (iv) one isolate with Ile-257 and Gln-303 classified as group IV, and (v) 4 isolates with Met-203 and Ile-257 termed group V (group I-V as specified in Table [4]).

### Table 4

Substitutions in amino acids in the N-terminal hypervariable region of the _spaA_ gene and number of C-terminal tandem repeats in 165 _E.

rhusiopathiae_ field isolates and serotype reference strains compared with the corresponding sequence of _E.

rhusiopathiae_ Fujisawa strainc

_E.

rhusiophatiae_ strain or group (no.

of isolates)

Substitutions in N-terminal nucleotides (amino acid position)a,b

No.

of C-terminal tandem repeats

Nucleotide (aa 55)

Nucleotide (aa 70)

Nucleotide (aa 101)

Nucleotide (aa 178)

Nucleotide (aa 195)

Nucleotide (aa 203)

Nucleotide (aa 257)

Nucleotide (aa 303)

Fujisawa serotype 1a

GTA (Val)

AAA (Lys)

AAC (Asn)

GGT (Gly)

GAT (Asp)

ATT (Ile)

CTT Leu

GGG (Gly)

9

Field isolates (No.)

Group I (87)

ATA (Ile)

AAT (Asn)

GAT (Asp)

AAT (Asn)

ATT (Ile)

GAG (Gln)

8-11, 13

Group II (36)

AGC (Ser)

ATT (Ile)

7-10

Group III (37)

ATT (Ile)

8, 9

Group IV (1)

ATT (Ile)

GAG (Gln)

8

Group V (4)

ATG (Met)

ATT (Ile)

9

Serotype reference strains

Serotype 1a (Koganei) 5, 15

ATA (Ile)

AAT (Asn)

GAT (Asp)

AAT (Asn)

ATT (Ile)

GAG (Gln)

9

Serotype 8, 17

AGC (Ser)

ATT (Ile)

9

Serotype 1b, 9, 12, 16, N

ATT (Ile)

8, 9

Serotype 2

ATA (Ile)

AAT (Asn)

ATT (Ile)

9

Except for serotype 2 reference strain ATCC19414T, which revealed a unique pattern (Ile-55, Asn-70 and Ile-257) all other reference strains included (see Table [4]) corresponded to one of the SpaA groups observed among the field isolates.

None of the SpaA groups correlated with a certain host type (mixed avian and mammalian).

As far as serotypes were available (_n_ = 32; serotype 1a and 1b (_n_ = 20); serotype 2 (_n_ = 9), serotype N (_n_ = 3), they also did not correlate with certain SpaA groups.

In group I, we observed both serotype 1a/b and serotype 2 strains and within group III, strains of serotypes 1a/b, 2 and N were present (data not shown).
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