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"Title: A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals

URL Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509749/

Markdown Content:
*   [Journal List]
*   [Vet Res]
*   [v.46(1); 2015]
*   PMC4509749

Abstract
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_Erysipelothrix rhusiopathiae_ infections re-emerged as a matter of great concern particularly in the poultry industry.

In contrast to porcine isolates, molecular epidemiological traits of avian _E.

rhusiopathiae_ isolates are less well known.

Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for _E.

rhusiopathiae_, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3).

Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates.

This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together.

PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common.

Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains.

More than 90% of the isolates harboured all 16 putative virulence genes tested and only _intI_, encoding an internalin-like protein, showed infrequent distribution.

MLST data determined _E.

rhusiopathiae_ as weakly clonal species with limited host specificity.

A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen.

### Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0216-x) contains supplementary material, which is available to authorized users.

Introduction
------------

_Erysipelothrix rhusiopathiae_ is a ubiquitous gram-positive bacterial organism which causes erysipelas in mammals and birds, especially in pigs and poultry.

Healthy pigs carrying _E.

rhusiopathiae_ in their lymphoid tissues have been suggested as a reservoir of the pathogen \[[1]\].

The genus _Erysipelothrix_ contains two main species: _E.

rhusiopathiae_ (including serotypes 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21 and N) and _E.

tonsillarum_ (serotypes 3, 7, 10, 14, 20, 22 and 23), the latter being isolated from apparently healthy swine and considered to be non-pathogenic \[[1]\].

_E.

rhusiopathiae_ was first recognized as a human pathogen causing localized cutaneous lesions, called erysipeloid, and sporadic cases of generalized cutaneous forms or septicaemia, often associated with endocarditis \[[2]\].

Erysipelas in swine occurs in different forms characterized by septicaemia often resulting in sudden death (acute form), cutaneous lesions (sub-acute form), polyarthritis and endocarditis (chronic form) \[[1]\].

In recent years, Erysipelas in poultry re-emerged and here, turkeys are most seriously affected and suffer from cyanotic skin to haemorrhages and petechiae in the breast and leg muscles.

Following the change from conventional (battery) cage system to alternative housing systems, problems due to _E.

rhusiopathiae_ also increased in laying hen \[[2]\].

While attenuated live vaccines and bacterins are commercially available to protect pigs and sheep, in Germany there is no licensed vaccine to prevent erysipelas in turkeys and laying hen and this is why the application of autogenous vaccines and/or antimicrobial therapy is the ultimate way to combat the disease \[[1]\].

One of the most promising vaccine candidates is SpaA, a surface protein with high immunogenic properties \[[13]\].

Based on amino acid sequence similarities the Spa proteins can be classified into 3 molecular variants, termed SpaA, SpaB, and SpaC and the occurrence of these variants has been found to be basically associated with the serotype of a strain \[[14]\].

Particularly the N-terminal half of the hyper-variable region of the Spa protein is important for specific immunity, and strains with different _spaA_ gene variants obviously differ in pathogenicity \[[17]\].

_E.

rhusiopathiae_ strains vary considerably in virulence but only little is known about pathogenic mechanisms and the presence and distribution of virulence factors \[[2]\].

Different factors, including neuraminidase, capsule, hemolysin, hyaluronidase, adhesins and the cell wall lipoprotein EwlA have been suggested to be involved in the pathogenesis of the disease \[[2]\].

The availability of the genome sequence of _E.

rhusiopathiae_ strain Fujisawa (Acc.-No.

AP012027.1) offers the opportunity for the _in silico_\-identification of additional genes that have been linked with virulence in other bacterial species before.

The coding sequence ERH\_1472 was for example annotated as internalin gene _intI_, which in case of _Listeria monocytogenes_ contributes to the invasion of the bacteria into epithelial cells \[[18]\].

Various studies have been performed to determine the clonal relatedness of _E.

rhusiopathiae_ strains \[[19]\].

Data from multilocus enzyme electrophoresis (MEE) \[[20]\], restriction fragment length polymorphism (RFLP) analyses \[[19]\], amplified fragment length polymorphism (AFLP) \[[21]\], randomly amplified polymorphic DNA methods (RAPD) \[[23]\], and pulsed-field gel electrophoresis (PFGE) \[[22]\] suggested that _E.

rhusiopathiae_ strains might be genetically diverse even among isolates belonging to the same serotype.

Although the relevance of Erysipelas in poultry increased over the last years, intense epidemiological studies on _E.

rhusiopathiae_ from poultry combining established typing methods such as PFGE, Spa typing and virulence gene typing are limited \[[10]\] Moreover, there is currently no established scheme to study this bacterial species by Multilocus sequence typing (MLST), a widely applied method to determine the phylogeny of a bacterial population based on sequence analysis of representative genes of the bacterial core genome \[[26]\].

The overall objective of this study was to provide a deeper insight into the population structure of _E.

rhusiopathiae_ by developing an MLST scheme and determining sequence types of 165 _E.

rhusiopathiae_ strains predominantly isolated from poultry and also from mammalian hosts, including few human strains.

The congruence of multilocus sequence types of individual strains with their grouping into PFGE clusters and their surface protective antigen (Spa) protein variant was evaluated and the distribution of 16 genes putatively linked with the pathogenic properties of the strains was investigated.

Materials and methods
---------------------

### Bacterial strains and species designation

A total of 165 _E.

rhusiopathiae_ strains primarily from Germany (136) and from a few other countries (29) were included.

The major focus was on isolates from avian sources (chicken, 71; turkey, 43; other birds, 6), while isolates from pigs (_n_ = 36) and other animal species (_n_ = 6) and humans (3) were included as well.

Bacterial strains were isolated from organ and blood samples from diseased animals and from blood samples in case of the human strains.

In case of outbreak situations, only one strain per flock was sampled.

Strains obtained from recurrent disease events in identical flocks were only included if six months had passed since the previous event or if they revealed different banding patterns, as initially determined by pulsed-field gel electrophoresis (data not shown).

_E.

rhusiopathiae_ strains and the non-pathogenic species _E.

tonsillarum_ were differentiated by a species-specific PCR \[[27]\].

Reference strains _E.

rhusiopathiae_ ATCC 19414T, _E.

tonsillarum_ DSM 14972T (ATCC 43339T), and _E.

inopinata_ DSM 15511T as well as two field strains of _E.

tonsillarum_ were also included.

The reference strains were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

All strains were stored at −80 °C in liquid medium (brain heart infusion broth) supplemented with 20% glycerol until used.

### DNA purification

The total DNA of _E.

rhusiopathiae_ was purified using a protocol described previously \[[28]\].

Instead of using a 1.5 mL bacterial culture grown in BHI for 24 h, as recommended in the protocol, we took 20 mL of an overnight culture to obtain a higher amount of DNA.

DNA concentration was determined by UV/VIS spectrophotometry (NanoDrop®ND1000 Spectrophotometer, Thermo Fisher Scientific).

Aliquots (20 ng/μL) were stored at −20 °C until further use.

### Genes for MLST and nucleotide sequencing of gene fragments

To establish an MLST scheme, genes were selected based on published protein sequences of _E.

rhusiopathiae,_ located in the published contigs of the reference strain _E.

rhusiopathiae_ ATCC 19414T (project ID: 38421), sequenced by the National Institute of Animal Health, Japan, in cooperation with the Dragon Genomics Center, Takara Bio Inc., Japan.

Based on reversed translated protein sequences, primers were designed to amplify gene fragments of housekeeping genes _gpsA_ (glycerol-3-phophate-dehydrogenase), _recA_ (recombinase A), _purA_ (adenylosuccinate synthetase), _pta_ (phosphate acetyl-transferase), _prsA_ (ribose-phosphate-pyrophosphokinase), _galK_ (galactokinase), and _ldhA_ (D-lactate dehydrogenase) (Table [1]).

Genes were chosen in line with MLST schemes for other bacteria taking into account (i) a gene size \> 700 bp, (ii) a regular distribution of genes among the _E.

rhusiopathiae_ genome, and (iii) that predicted proteins should be under stabilizing selection.

### Table 1

Primer sets used to amplify and sequence _E.

rhusiopathiae_ housekeeping genes and genetic diversity of gene loci used for MLST analysis

Gene

Primer sequence \[5′-3′\]

Location within genea

Amplicon size PCR (bp)

Allele size MLST (bp)

Mean GC content

No.

of alleles

No.

of variable sites (%)

_d_ _N_ _/d_ _S_ratio

Simpsons index of diversity (_D_)

_gpsA_

fw: AGTTATGATGTGGGGACG

75-745

671

540

38.3%

9

11 (2.0)

0.3610

0.608

rv: TAGCTGTAACGACGAGATCG

_recA_

fw: TTCGGTAGAATAATCTCGCG

38-913

876

640

39.7%

9

13 (1.6)

0.0207

0.775

rv: TGCTATTAGTTCAGGGTCG

_purA_

fw: GATGTTTATGAGGAAGCGC

331-1066

736

611

39.1%

14

11 (1.8)

0.5690

0.764

rv: AACGCATTGATTGTTGCCC

_pta_

fw: TGCTGCAGTACGTTTAGC

90-793

704

544

36.6%

9

12 (2.2)

0.0419

0.579

rv: AGACACGTGCATTACCTG

_prsA_

fw: ACAAGTTCACCAGTAAGTG

190-952

763

575

39.3%

6

4 (0.7)

0.0654

0.657

rv: AGAGTGTACTTACAGGAGT

_galK_

fw: TATTCCTAATGGAGCGGG

357-1041

685

554

36.8%

13

10 (1.8)

0.0766

0.740

rv: AATCGCAATCGCACATCC

_ldhA_

fw: AACGGATATGAAGCTGTTGCC

136-783

648

531

40.3%

11

14 (2.6)

0.3390

0.681

rv: AAGAACATCCAGTCCAACAGC

aAs referred to _E.

rhusiopathiae_ ATCC 19414T (Acc.-No.

NZ\_ACLK02000001 - NZ\_ACLK02000004).

PCRs were performed in a volume of 60 μL containing 60 ng DNA template, 10 pmol of each primer (Sigma Alderich, Munich, Germany), 10 mM dNTP mixture, 10 × PCR buffer including 20 mM MgCl2, and 1 U Taq polymerase (all Rapidozym, Berlin, Germany).

The PCR conditions were initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s (except for _galK_, for which an annealing temperature of 54 °C was used), and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min.

Amplicons were sequenced by LGC genomics (Berlin, Germany).

Sequence analysis was performed with Ridom SeqSphere 1.0.1 \[[29]\].

Each unique sequence of a gene fragment was assigned an allele number.

These allele numbers were consecutively numbered and combined in order to establish an allelic profile, which defined the sequence type (ST).

Using the eBURST program \[[30]\] the MLST data set was divided into groups of related STs (clonal complexes resp.

ST complexes).

We used the most stringent definition of ST complexes, which were defined as groups when six out of seven alleles from the nearest neighbour were the same \[[31]\].

The program START version 2 \[[32]\], was used to determine the number of variable nucleotide sites and their effects on the amino acid sequence changes.

This was done by calculating the ratio of non-synonymous substitutions (_d__N_) to the number of synonymous substitutions (_d__S_) (_d__N_/_d__S_ ratio) \[[33]\].

The _d__N__/d__S_ ratio quantifies the evolutionary pressure on proteins \[[34]\] and thus indicates the presence or absence of a selective force on the locus \[[35]\].

Simpson’s index of Diversity (_D_) was calculated on the basis of the molecular pattern of the seven loci \[[36]\].

A Simpson’s index of diversity with 95% confidence intervals (CI95%) was calculated for a set of 165 strains.

A value close to 1 indicates high diversity, and a value close to 0 reflects little diversity.

### Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) followed a previously published protocol \[[24]\] and was performed to group _E.

rhusiopathiae_ isolates according to their macrorestriction profiles.

PFGE was also applied to discuss the discriminatory power and interpretative value of this band-based method compared with the sequence-based MLST analysis.

The chromosomal DNA was digested with SmaI (Fermentas Life Science, Germany).

Electrophoresis was carried out in a contour-clamped homogeneous electric field (CHEF- DRIII; Biorad, Munich, Germany).

MidRange PFG Marker I (BioLabs, #N3551S) was used as DNA size standard.

Band patterns were analysed with the software BioNumerics (Version 6.6, Applied Maths, Belgium).

Dendrograms were created by an unweighted pair group matching by an arithmetic averages algorithm (UPGMA) and the Dice coefficient.

Optimization was set at 0.9%, band position tolerance at 2.0% \[[39]\].

To compare the discriminatory power of MLST and PFGE the Simpson’s index of Diversity (_D_) was used \[[36]\].

An index of diversity close to 1 indicated a high diversity of the strain collection, whereas a value close to 0 suggested little diversity.

We calculated the Simpson’s index of diversity (_D_) with 95% confidence intervals (CI95%).

### Detection of virulence-associated genes by Multiplex PCR

Two multiplex PCR protocols were established to screen for genes putatively linked with the virulence of _E.

rhusiopathiae_.

The selection of the genes was based on (i) previous studies, linking the gene products with a certain pathogenic mechanism \[[2]\] and (ii) publicly available annotation data of coding sequences of _E.

rhusiopathiae_ (Acc.-No.

AP012027.1), determined by whole genome analysis \[[45]\] (see Table [2]).

The sequences of oligonucleotide primers, either obtained from recent publications \[[14]\] or designed in the present study are given in Table [2].

PCRs were performed in a T300 Thermocycler (Biometra, Göttingen, Germany).

A 25 μL aliquot contained 3 μL of each of purified chromosomal DNA (20 ng), 20 pmol of each primer pair (Sigma Alderich, Munich, Germany), 5 mM of the four deoxynucleoside triphosphates (Rapidozym, Berlin, Germany), 10 × PCR buffer including 20 mM MgCl2, and 0.4 U Dream Green Taq DNA Polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany).

PCR was performed under the following conditions: an initial denaturation step at 94 °C for 10 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 2 min, extension at 72 °C for 7 min, and finally one cycle of extension at 72 °C for 10 min.

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)."


CONCISE SUMMARY: