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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. An extensive virulence gene profiling showed that over 90% of isolates carried 16 putative virulence genes, indicating a weakly clonal nature of the species and its limited host specificity. The study also characterized the PCR amplification methods used to detect virulence genes, identified the spaA gene variants, and categorized the strains based on their Spa protein sequences, which revealed no association with host type. These findings suggest shared reservoirs and pathogenic pathways among diverse hosts and highlight the need for comprehensive epidemiological studies to understand *E. rhusiopathiae* strains' pathogenic mechanisms and virulence factors better.
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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).

The number of C-terminal tandem repeats of similar 20-amino-acid sequences in the SpaA proteins of field isolates ranged from 7 to 13, corresponding to the different _spaA_ nucleotide sequence lengths observed.

The majority of strains (89.7%) harboured a domain with 9 repeats similar to that present in strain Fujisawa and in all SpaA-positive serotype reference strains, except for serotype 9 strain Kaparek (Table [4]).

The remaining field isolates showed either higher or lower number of repeats.

One strain from a chicken with septicaemia was truncated by 40 amino acids at positions 502 and 534 and another seven strains lacked a 20 amino acids repeat either at position 534 or 558.

Seven strains revealed 10 repeats and two porcine strains even showed 11 and 13 repetitions of similar 20-amino-acid sequences in the C-terminal part of their SpaA protein, respectively.

A host-specific distribution of SpaA types was not observed.

Portraying the different SpaA alleles against the phylogenetic background of the strains reveals a strong linkage of group I (amino acid changes Ile-55, Asn-70, Asp-178, Asn-195, Ile-257 and Gln-303 compared with Fujisawa strain) with ST complex 9 (Figure [2B]).

Group II (Ser-101, Ile-257) and group III isolates (Ile-257) occur in several STs which are not related to this major ST complex.

Only in one case, different SpaA types occur in the same sequence type, namely ST48, which is made up by porcine isolates (Figure [2A]).

### Nucleotide sequence accession numbers

Nucleotide sequence of _spaA_ genes of 165 _E.

rhusiopathiae_ strains have been submitted to the GenBank database under accession nos.

KR606141 – KR606305.

Discussion
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Although there is an increasing notion of severe outbreaks of Erysipelas in poultry \[[10]\], possibly related to the ban of conventional cages within EU from 2012 and the large changes of housing systems due to welfare demands for laying hens \[[9]\], only little is known about the molecular epidemiology of avian _E.

rhusiopathiae_ isolates.

To overcome this, we investigated isolates of _E.

rhusiopathiae_, mainly from poultry but also from mammalian hosts by a newly developed species-specific MLST scheme and in addition by more established methods such as PFGE, virulence genotyping and Spa typing.

The novel MLST scheme for _E.

rhusiopathiae_ is based on the identification of nucleotide polymorphisms of internal fragments from seven housekeeping genes involved in metabolic pathways of the bacterial pathogen.

The target genes were chosen with a dN/dS ratio of <1 to guarantee a very low degree of evolutionary pressure on the proteins and absence of a selective force on the respective gene locus.

This ratio was calculated for other MLST schemes, recently developed e.g.

for the bovine pathogen _Mannheimia haemolytica_ \[[48]\] or for _Enterococcus faecalis_, which has been associated with amyloid arthropathy in chickens \[[49]\] as well.

The lower number of alleles in the _E.

rhusiopathiae_ MLST scheme, which varies between 6 (_pta_) and 14 (_purA_) among 165 _E.

rhusiopathiae_ strains together with a diversity of index _D_ of 0.69 (95% CI, 0.579 to 0.775), which is also lower than that observed for other schemes \[[48]\] might indicate a lower diversity as compared e.g.

to the Gram negative pathogen _E.

coli_ or to the Gram positive species _Streptococcus gallolyticus_.

However, this assumption should be treated cautiously as the MLST databases of these two species contain significantly more isolates, i.e.

7.500 isolates in case of _Escherichia coli_ \[[52]\] and 292 isolates in case of _Sc.

gallolyticus_ \[[53]\].

Whether _E.

rhusiopathiae_ would still appear as a bacterial species with low diversity after including more isolates from different sources remains to be determined.

The distribution of the STs as presented in a minimum spanning tree (MST) shows accumulations of _E.

rhusiopathiae_ strains isolated from various animals in certain STs (Figure [2A]).

Even if there was a bias towards avian isolates and towards isolates from Germany in our sample material, there was no indication for host or geographic specificity.

Strains from poultry and swine randomly scattered throughout the MSTree and commonly shared identical STs, such as ST9, ST8 and ST5.

This might indicate that birds and pigs may be infected by phylogenetically similar strains either by a common reservoir or by direct or indirect transmission from one animal species to the other.

A limited host specificity has also been suggested in another recent study, where marine _E.

rhusiopathiae_ isolates were shown to be capable of inducing classical skin lesions in pigs in a skin inoculation model \[[54]\].

However, looking deeper into the clonal nature of our strains by PFGE, those from poultry and pigs were commonly found in same PFGE clusters, but no identical pairs of isolates were observed between the two species.

This supports a previous finding by Eriksson et al.

who also didn’t observe a 100% band profile identity among any of the pig and poultry isolates albeit the strains were not separated into two distinct populations by PFGE analysis \[[22]\].

Notably, the most prominent ST observed in the present study – ST9 - includes human and animal strains.

The grouping of porcine strain ATCC 19414T and human strain IMT9137 in an identical sequence type and in the same PFGE cluster (89.9% similarity in banding pattern) indicates that these strains may be closely related and that a zoonotic potential of animals strains should be taken into consideration.

Sporadic diseases in humans have been reported and these often occurred in farmers and in other persons whose work is closely related with contaminated animals, underlining that _E.

rhusiopathiae_ infection in humans is occupationally-related \[[2]\].

Overall, our MLST analysis distinguished 165 isolates into 72 sequence types (STs) and only partial congruence was observed to the grouping of strains into 58 PFGE clusters.

MLST intends to portray the long term epidemiology and the evolutionary history of a bacterial pathogen based on a representative set of the bacterial core genome rather than to unravel disease outbreaks, at least not without additional typing methods \[[26]\].

In contrast, PFGE is a suitable tool to monitor short-term local outbreaks mainly by identifying clones \[[57]\].

In studies comparing MLST to PFGE e.g.

among _Enterococcus faecalis_, _Staphylococcus aureus_, and _Vibrio cholerae_ isolates, MLST has been found to have similar or greater discriminatory ability than PFGE \[[58]\].

In contrast, a study comparing MLST and PFGE data in _Escherichia coli_ O157:H7, found that PFGE revealed a greater discriminatory ability than MLST did, which may be due to the highly clonal nature of this _E.

coli_ serotype \[[62]\].

In case of the Gram-negative pathogen _Pseudomonas aeruginosa_ Johnson et al.

found that MLST was better for detecting genetic relatedness, while PFGE was more discriminatory than MLST for determining genetic differences in this bacterial species and to confirm the a patient-to-patient transmission \[[63]\].

Nemoy et al.

observed a greater discriminatory ability and reproducibility of MLST than PFGE for ESBL-producing _E.

coli_ \[[58]\].

They suggested MLST is suitable to a priori define genetically related bacterial strains, which might be very helpful for surveillance.

Our MLST data for the first time allow determining _E.

rhusiopathiae_ as a weakly clonal species as 165 isolates are dispersed among 72 different sequence types.

The accumulation of a number of strains in ST complex 9 may indicate that this group of genetically related strains represents a particularly successful subpopulation.

If this holds true after including more strains from different countries is uncertain.

Likewise, possible reasons for this success or at least high prevalence of STC9 strains (e.g.

high virulence, environmental survival) also need to be studied.

Interestingly, although serotypes for only 32 of our isolates were available (serotype 1a and 1b (_n_ = 20); serotype 2 (_n_ = 9); serotype N (_n_ = 3)), these serotypes were not strictly linked with a certain sequence type or ST complex (data not shown).

In addition, different serotypes were observed in identical STs, indicating that the O antigen encoding genetic structure may have, at least partly, evolved independently from the phylogenetic background of the strains, which has likewise been reported for other bacterial species, such as _E.

coli_ and _Bordetella_ spp.

\[[64]\].

No correlation between serotype and PFGE pattern could also be observed in a recent study including _E.

rhusiopathiae_ isolates from poultry, pigs, emus, and the poultry red mite _Dermanyssus gallinae_ \[[22]\].

Here, the authors found that serotypes were randomly scattered throughout the dendrogram based on PFGE patterns and that isolates with the same PFGE pattern were of different serotypes in some cases.

They concluded that serotyping may be less suitable for studies on epidemiological relationships between flocks.

Concerning the putative virulence genes investigated in our study, there was no congruence between one of these genes with a certain sequence type or PFGE cluster, which is primarily attributable to the frequent detection of the genes.

Almost all 16 markers selected and putatively involved in pathogenesis, except for _intI_ (85.5%), the Alginate-O-acetyltransferase gene _algI_ (97.6%), and the attachment protein gene _dnaB_ (99.4%) were regularly present in our isolates.

According to the annotation of whole genome sequenced _E.

rhusiopathiae_ strains Fujisawa (GenBank AP012027) and SY1027 (GenBank CP005079) _intI_ encodes an internalin-like protein.

We observed 15% _intI_\-negative isolates that occurred in 12 different STs, mostly from poultry and two isolates from sheep.

But, as the function of this internalin-like protein has been deduced from amino acid sequence comparisons only, further molecular genetic studies and in vitro assays would have to be performed to verify its putative role in _E.

rhusiopathiae_ pathogenesis.

Although most of the other genes were regularly present in our isolates, their expression might vary due to different body compartments or environmental factors.

For some of these factors, such as the neuraminidase, a correlation between the produced amount of this enzyme, which may serve the nutritional requirements and play a role in adherence and antiphagocytic activity of the bacteria, and the virulence of strains has been reported \[[2]\].

It was not unexpected to detect the capsule biosynthesis locus in all our isolates.

In previous studies capsule-negative mutants or the loss of capsule in general resulted in avirulent strains which did not resist phagocytosis by murine polymorphonuclear leukocytes and lost the capability of intracellular survival \[[2]\].

Thus, such strains would not have been able to cause severe disease in the various hosts.

This study was mainly focussed on poultry isolates, as we intended to provide a rational typing method to assess the clonal relatedness of these strains, particularly with respect to the selection of epidemiological relevant strains to be included in future vaccines.

In this respect we also aimed to determine, whether the N-terminal hyper-variable region of the surface protein SpaA, which apart from SpaC, represents one of the most promising vaccine candidates \[[13]\] might show a co-evolution with the genomic backbone of _E.

rhusiopathiae_.

The exclusive finding of SpaA and the absence of SpaB and SpaC in our sample collection might be due the prevalence of certain serotypes among our strains.

Our partial serotype data confirm what has been reported in previous studies, namely that the _spaA_ gene usually, but not exclusively, occurs in strains belonging to serotype 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17 and type N, while _spaB_ and _spaC_ are mainly observed in _E.

rhusiopathiae_ serotypes 4, 6, 11, 19, and 21 and in serotype 18, respectively \[[3]\].

Ingebritson et al.

reported that one strain may contain more than one Spa type and that these were not always confined to a certain serotype \[[3]\].

It is not likely that single isolates of our study harboured different Spa types as this would have become apparent due to ambiguous sequencing data.

Different authors have explored the variations in the N-terminal half of the _spaA_ gene, particularly in the protective domain which is located at amino acid positions 30 to 413, and in the repeat domain which is located at amino acid positions 448 to 626 (in case of 8 to 10 tandem repeats) \[[14]\].

A broad collection of avian isolates and their _spa_ gene has not been investigated so far, limiting our knowledge about the diversity of this major antigen considerably.

By sequence analysis of the _spaA_ gene of 165 clinical _E.

rhusiopathiae_ isolates, thereof 120 from birds, we found high sequence identity within the signal sequence region, located at the most N-terminal part of the Spa protein, corroborating the findings of To and Nagai and supporting the idea that Spa proteins may cross bacterial cell membranes and eventually be secreted from the bacterial cell by using a secretion machinery commonly present in _E.

rhusiopathiae_ strains \[[14]\].

Although most of our isolates (89.7%) revealed nine C-terminal tandem repeats in their SpaA protein, which is also common to most of the serotype reference strains investigated by To and Nagai and listed in Table [4] \[[14]\], the diversity in number was quite high with 7 to 13 tandem repeats and has not been reported before.

As this repeat domain has been suggested to function as an anchor for binding Spa proteins tightly to the bacterial surface it remains to be determined, e.g.

by in vitro adhesion experiments, if this binding might perhaps be influenced by the number of repeats \[[13]\].

Regarding the amino acid substitutions in the N-terminal protective part of the Spa protein we detected five different variants (group I-V) that occurred independently of the original host species and have been described at least in part in previous publications on porcine isolates as well.

Among swine isolates from Japan, Uchiyama et al.

observed three groups, looking at substitutions at amino acid positions 195, 203 and 257 \[[17]\].

These positions were also part of our investigations, while we additionally included another 6 amino acid substitutions resulting in a finer resolution of the groups.

Thus, their group 2 resembles our groups II-IV; group 1 matches with our group V; and group 3 was not detected among our isolates at all.

In that study, more than half of the 34 porcine strains showed the Met-203 variant in combination with Ile-257 (group 1 according to Uchiyama et al.

\[[17]\]), corresponding to group 2 recently determined among another collection of isolates from Japan \[[69]\].

To et al.

\[[68]\] found that 95% of their overall 80 pig isolates carried the Met-203 variant of the _spaA_, while the same variant accounted for 55.6% of the isolates included by Uchiyama et al.

\[[17]\].

Notably, we observed this variant only very infrequently (2.4%) among our isolates.

In the present strain collection we rather detected SpaA variants (group I, 52.7%; group II, 21.8%; group III, 22.4%) that have been described for different serotype reference strains before (Table [4]) \[[14]\].

Of note, the distribution of _spaA_ variants demonstrated to be irrespective of the original host of the strains, i.e.

avian or mammalian.

Whether the current geographic linkage of the Met-203 variant to Japan remains after studying more isolates from different countries is uncertain \[[17]\].

Uchiyama et al.

could show that isolates with Met-203 were highly pathogenic in mice with LD50 values between 0.45 and 1.45 CFU/mouse, but comparative in vivo data with isolates representing other _spaA_ groups were not provided \[[17]\].
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