The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology.
KEYWORDS: Erysipelothrix species, multiplex PCR, serotyping
ABSTRACT
The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.
INTRODUCTION
Erysipelothrix rhusiopathiae is a Gram-positive, non-spore-forming, rod-shaped bacterium and the standard species of the genus Erysipelothrix in the phylum Firmicutes (1). The genus Erysipelothrix is composed of at least five species, E. rhusiopathiae, Erysipelothrix tonsillarum (2), Erysipelothrix inopinata (3), Erysipelothrix larvae (4), and Erysipelothrix piscisicarius sp. nov. (5), the last of which is pathogenic to fish and has been recently proposed as a new member of the genus. Furthermore, the existence of three other species, which are unnamed and tentatively referred to as Erysipelothrix sp. strain 1, Erysipelothrix sp. strain 2, and Erysipelothrix sp. strain 3, has been suggested (6); however, Erysipelothrix sp. strain 2 has now been confirmed to belong to E. piscisicarius sp. nov. (5).
Historically, based on heat-stable peptidoglycan antigens detected in a gel diffusion test, the four species E. rhusiopathiae (serovars 1a, 1b, 2, 4 to 6, 8, 9, 11, 12, 15 to 17, 19, 21, and N [serovar N lacks serovar-specific antigens]), E. tonsillarum (serovars 3, 7, 10, 14, 20, and 22), Erysipelothrix sp. strain 1 (strain Pécs 56; serovar 13), and Erysipelothrix sp. strain 2 (strain 715; serovar 18) have been differentiated by their serovars (2). To date, new serovars have been assigned to E. rhusiopathiae (serovar 23) and E. tonsillarum (serovars 24 to 26). The serovar of the new species E. piscisicarius sp. nov. has been determined; however, the serovars of E. inopinata and E. larvae have not yet been clarified.
Erysipelothrix species are ubiquitous in nature and have been isolated from a variety of animal species. Among the genus Erysipelothrix species, E. rhusiopathiae is the main species that causes swine erysipelas, which may occur as acute septicemia or chronic polyarthritis and endocarditis, septicemic disease in turkeys, chickens, and dolphins, and chronic diseases in other farmed animals (7). In humans, the manifestation of the disease is a skin disease known as erysipeloid, which can lead rarely to septicemia and endocarditis (8). Recently, E. rhusiopathiae infections in pigs and chickens have increased in many countries (9–12) and are also spreading into wildlife in arctic and boreal ecosystems, probably due to climate change (13, 14). Epidemiological and etiological studies have shown that specific E. rhusiopathiae serovars, namely, 1a, 1b, and 2, are isolated mainly from diseased pigs, chickens, and humans (9, 15–19). However, it is common that in pigs, various serovar strains with different virulence can be isolated from an individual animal, and healthy pigs can carry E. tonsillarum in their tonsils (20). Serotyping requires a full set of serovar reference strains and strain-specific rabbit antisera; therefore, only a few studies have examined serovars of isolates from wild animals in which a wide range of serovar strains appear to be prevalent. Thus, serotyping of clinical isolates is important for understanding the epidemiology of infection in domesticated and wild animals and for monitoring serovar prevalence, indicating a clear need for rapid diagnostic methods in low-resource settings.
Recently, the chromosomal region essential for serovar 1a antigenicity and virulence of E. rhusiopathiae was identified, and it was later confirmed that the sequences of the genetic region can be used for the differentiation of serovars 1a, 1b, 2, and 5 (21). In this study, to develop a rapid serotyping system covering all serovars of Erysipelothrix species, we first measured genetic distances between Erysipelothrix species by average nucleotide identity (ANI) and analyzed the serovar-defining chromosomal region of 28 serovar strains of Erysipelothrix species for strain-specific sequences, which can be subsequently targeted by specific primers.
MATERIALS AND METHODS
Bacterial strains.
The Erysipelothrix strains used to test the specificity of the primers are shown in Table S1 in the supplemental material, with the exception of the E. larvae LV19 and E. piscisicarius sp. nov. 15TAL0474 strains. Erysipelothrix strains were grown at 37°C in brain heart infusion (BHI; Becton, Dickinson and Company, Baltimore, MD) supplemented with 0.1% Tween 80 (pH 8.0).
Serotyping.
Serotyping was determined by a double agar-gel precipitation test with autoclaved cell extracts and rabbit antiserum raised against formalin-killed cells of the reference strain, as described previously (22).
Sequence information and analysis.
Draft genome sequences of Erysipelothrix strains were either retrieved from GenBank or generated using the Illumina HiSeq platform, as described previously (12). Trimmed reads were assembled using SPAdes (23), and the assembled contigs were evaluated using QUAST (24). Genetic relatedness among Erysipelothrix strains was analyzed by comparing the ANI using the ANI calculator (JSpecies ver.1.2.1, with default parameters) (25). In E. rhusiopathiae serovar 1a and 2 strains, the chromosomal region between ERH_1438 and ERH_1451 was found to be responsible for serovar 1a and 2 antigenicity and virulence (26). Using the IMCGE (in silico molecular cloning genomics edition) software (27), sequences of the corresponding and neighboring chromosomal regions of each Erysipelothrix serovar strain were analyzed, and primers were designed based on serovar-specific sequences (Table S3 in the supplemental material). For 2 pairs of serovars, the 10 and 11 pair and the 2 (ATCC 19414) and 21 pair, which show similar genetic structures, DNA sequences from each locus were aligned with Clustal W (28) and visually searched for serovar-specific primer sequences.
DNA methods.
The genomic DNA of Erysipelothrix strains was prepared as described previously (12), with the following modifications: after cells were lysed using 10% sodium dodecyl sulfate, the cell lysate samples were mixed with an equal volume of phenol-chloroform solution and centrifuged, and then DNA was recovered by ethanol precipitation. For multiplex PCR assays, DNA was prepared using an alkaline boiling method, as previously described (15). Briefly, a single colony of each strain was suspended in 50 μl of 25 mM NaOH, incubated at 95°C for 5 min, and then neutralized by the addition of 4 μl of 1 M Tris-HCl (pH 8.0). The suspension was centrifuged, and 1.0 μl of the supernatant was used for PCR. The specificity of the PCR was assessed empirically using serovar reference strains, and then validation of the PCR assay was carried out by testing wild-type strains. PCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) and a T-100 thermal cycler (Bio-Rad, CA, USA). Briefly, amplification reaction mixtures were prepared according to the manufacturer’s recommendations but scaled down to 25 μl per reaction. For multiplex PCR assays, the concentration of primers was changed according to the sizes of DNA fragments. The composition of the multiplex PCR mixtures is shown in Table S4 in the supplemental material. The PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by 3 amplification steps (30 cycles) consisting of 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min.
Data availability.
Sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (BioProject accession no. PRJNA607040).
RESULTS AND DISCUSSION
The sequences of the chromosomal region from ERH_1438 through ERH_1451, which were identified as being responsible for virulence and antigenicity of the serovar 1a strain (Fujisawa), and the corresponding genomic regions of other serovar strains are shown in Fig. 1. Sequence analysis revealed that the serovar 13 strain (strain Pécs 56; Erysipelothrix sp. strain 1) lacks the corresponding genetic region. We confirmed that the serovar 13 strain shares 84.16 to 85.20% ANI with the strains of the species E. rhusiopathiae, E. tonsillarum, and Erysipelothrix sp. strain 2 (strain 715; serovar 18) but shares 96.17% ANI with E. inopinata (Table S1), strongly suggesting that Erysipelothrix sp. strain 1 (the serovar 13 strain) and E. inopinata are the same species. In this study, we could not identify the serovar-defining region in these strains, and therefore, the serovar 13 strain and E. inopinata were excluded from the assays.
FIG 1.
Schematic representation of the chromosomal region defining the antigenicity of the respective serovars of the Erysipelothrix species strains. Identical genes with >75% DNA sequence identity are indicated by the same numbers or letters. The genes shown in gray arrows indicate transposons. Note that E. tonsillarum lacks the genes from ERH_1451 to ERH_1453. The small arrows indicate the locations of the primer pairs used for multiplex PCR.
As has been previously observed with the serovar 2/15 strains, which were reactive with both serovar 2 and 15 antisera and positive in a serovar 2-detecting PCR assay (21), it was found that three strain pairs of serovars 10 and 11, serovars 7 and 14, and the E. rhusiopathiae ATCC 19414 type strain, which has been regarded as serovar 2, and serovar 21 possess very similar or the same genetic structure (Fig. 1). It has been reported that antibody cross-reaction occurred between serovars 7 and 14 (29, 30). We observed this cross-reaction and confirmed that anti-serovar 10 serum is partially reactive with the serovar 11 antigen and that anti-serovar 21 serum is strongly reactive with the ATCC 19414 antigen (our unpublished data). Due to the difficulty in finding serovar-specific sequences for differentiation between serovars 10 and 11 and between serovars 7 and 14, primers were designed to detect together the strain pairs of serovars 10 and 11 (10/11) and serovars 7 and 14 (7/14) (Table S3).
For other Erysipelothrix serovar strains, primers were designed to specifically detect each serovar. For convenience, multiplex PCR assays were developed to detect the following four serovar groups: group 1 (serovars 6, 8, 9, 15, and 21), group 2 (serovars 4, 12, 17, 19, and 23), group 3 (serovars 3, 10/11, 16, 24, and 26), and group 4 (serovars 7/14, 18, 20, 22, and 25) (Fig. 2). Each set contained five primer sets that amplified products of correct different sizes for comprehensible differentiation. Validation of the assays with a total of 254 strains using boiled DNA as the template showed no extra bands, confirming the specificity and usability of the assays (Table 1). In the assays, untypeable strains, which are most likely the same as serovar N strains (26), were determined to be either serovar 6, serovar 8, or serovar 21, suggesting that these strains had mutation(s) in the serovar-defining chromosomal region and originated from the respective serovar strains. Thus, together with the multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the described multiplex PCR assays cover all reported serovars of Erysipelothrix species except serovar 13 (Erysipelothrix sp. strain 1), which is most likely the same species as E. inopinata. We believe that the multiplex PCR-based serotyping method should be useful in investigating the disease transmission potential between humans, livestock, and wildlife. However, the number of strains tested was limited for some serovars, and therefore, it is important to verify the system using strains from a variety of different sources.
FIG 2.
Differentiation of serovar reference strains of Erysipelothrix species by multiplex PCR assays. A molecular size marker (1-kb Plus DNA ladder; Invitrogen) is shown to the left (lane M).
TABLE 1.
Validation of PCR with various serovar Erysipelothrix strains from different host origins
| Serovar by species | No. of strains tested | Origin | Serovar(s) by group determined by multiplexed PCR assaya： |
|||
|---|---|---|---|---|---|---|
| Group 1 (serovars 6, 8, 9, 15, and 21) | Group 2 (serovars 4, 12, 17, 19, and 23) | Group 3 (serovars 3, 10/11, 16, 24, and 26) | Group 4 (serovars 7/14, 18, 20, 22, and 25) | |||
| E. rhusiopathiae | ||||||
| 1a | 62 | Pig | − | − | − | − |
| 1a | 1 | Chicken | − | − | − | − |
| 1a | 1 | Unknown | − | − | − | − |
| 1b | 20 | Pig | − | − | − | − |
| 1b | 1 | Human | − | − | − | − |
| 1b | 1 | Chicken | − | − | − | − |
| 1b | 4 | Wild boar | − | − | − | − |
| 2 | 49 | Pig | − | − | − | − |
| 2 | 1 | Human | − | − | − | − |
| 2 | 1 | Dolphin | − | − | − | − |
| 2 | 5 | Wild boar | − | − | − | − |
| 2 | 1 | Raccoon | − | − | − | − |
| 2 | 1 | Unknown | − | − | − | − |
| 2/15b | 1 | Pig | 15 | − | − | − |
| 2/15 | 2 | Wild boar | 15 | − | − | − |
| 2/21c | 3 | Pig | 21 | − | − | − |
| 2/21 | 4 | Crow | 21 | − | − | − |
| 2/21 | 1 | Human | 21 | − | − | − |
| 4 | 1 | Fish | − | 4 | − | − |
| 4 | 6 | Pig | − | 4 | − | − |
| 4 | 1 | Wild boar | − | 4 | − | − |
| 5 | 8 | Pig | − | − | − | − |
| 5 | 1 | Mud of pig farm | − | − | − | − |
| 5 | 2 | Wild boar | − | − | − | − |
| 5 | 1 | Chicken | − | − | − | − |
| 5 | 1 | Crow | − | − | − | − |
| 5 | 1 | Raccoon | − | − | − | − |
| 5 | 1 | Raccoon dog | − | − | − | − |
| 6 | 7 | Pig | 6 | − | − | − |
| 6 | 2 | Dolphin | 6 | − | − | − |
| 6 | 1 | Crow | 6 | − | − | − |
| 6 | 1 | Bustard | 6 | − | − | − |
| 8 | 2 | Wild boar | 8 | − | − | − |
| 8 | 1 | Godwit | 8 | − | − | − |
| 8 | 2 | Pig | 8 | − | − | − |
| 9 | 1 | Fish | 9 | − | − | − |
| 9 | 1 | Pen soil | 9 | − | − | − |
| 9 | 2 | Pig | 9 | − | − | − |
| 11 | 12 | Pig | − | − | 10/11 | − |
| 11 | 1 | Wild boar | − | − | 10/11 | − |
| 12 | 2 | Pig | − | 12 | − | − |
| 15 | 1 | Pig | 15 | − | − | − |
| 16 | 1 | Parrot | − | − | 16 | − |
| 17 | 2 | Pig | − | 17 | − | − |
| 19 | 2 | Pig | − | 19 | − | − |
| 21 | 1 | Sheep dip | 21 | − | − | − |
| 21 | 3 | Pig | 21 | − | − | − |
| 21 | 1 | Wild boar | 21 | − | − | − |
| 23 | 1 | Pig | − | 23 | − | − |
| N | 1 | Pig | 21 | − | − | − |
| Untypeable | 5 | Pig | 21 | − | − | − |
| Untypeable | 2 | Pig | 8 | − | − | − |
| Untypeable | 1 | Penguin | 6 | − | − | − |
| Untypeable | 1 | Wild boar | 6 | − | − | − |
| Untypeable | 1 | Raccoon dog | 6 | − | − | − |
| E. tonsillarum | ||||||
| 3 | 1 | Fish | − | − | 3 | − |
| 7 | 1 | Fish | − | − | − | 7/14 |
| 7 | 1 | Pig | − | − | − | 7/14 |
| 10 | 1 | Squirrel | − | − | 10/11 | |
| 14 | 1 | Mud of zoo pond | − | − | − | 7/14 |
| 20 | 1 | Pig | − | − | − | 20 |
| 22 | 1 | Sheep dip | − | − | − | 22 |
| 24 | 1 | Marine fish | − | − | 24 | − |
| 25 | 1 | Pig slurry | − | − | − | 25 |
| 26 | 1 | Pig slurry | − | − | 26 | − |
| Erysipelothrix sp. strain 2 | ||||||
| 18 | 1 | Pig | − | − | − | 18 |
| 10 | 1 | Pig slurry | − | − | 10/11 | |
| Erysipelothrix sp. strain 1 | ||||||
| 13 | 1 | Pig | − | − | − | − |
| E. inopinata | ||||||
| NTd | 1 | Vegetable broth | − | − | − | − |
Minus signs indicate a negative PCR result.
Reactive with both serovar 2 and 15 antisera.
Reactive with both serovar 2 and 21 antisera.
NT, not tested.
In Erysipelothrix species, serovar switching has been suggested, probably due to the acquisition of foreign genes by horizontal gene transfer, as indicated by G+C contents of the serovar-defining region that were different from those in other parts of the genome (26). As observed in Fig. 1, the hypothesis is further supported by the finding that transposable elements are located within the regions in seven serovar strains, including the ATCC 19414 strain, the type strain of E. rhusiopathiae. Previously, it was confirmed that Erysipelothrix sp. strain 2 (strain 715) (serovar 18) and the new species E. piscisicarius sp. nov. are the same species. However, the E. piscisicarius sp. nov. 15TAL0474 strain has been shown to be a serovar 5 strain (31). Thus, in some strains, the serovar is not strictly associated with whole-genome-based classification of Erysipelothrix species (6). Although serovar 1a, 1b, and 2 strains of E. rhusiopathiae are associated mainly with disease in animals and the role of serovar-defining antigen(s) in virulence in these serovars has been clarified (26), the underlying mechanisms remain largely unknown. It is highly possible that combination(s) of the serovar-defining antigen(s) and other virulence-associated antigen(s) may be necessary to cause the disease. For etiological and epidemiological studies, the use of the described multiplex PCR method in combination with other molecular analyses, including spa typing (32–34), may be important.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by a grant from the National Agriculture and Food Research Organization (to Y.S.).
We declare no conflicts of interest.
Footnotes
Supplemental material is available online only.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (BioProject accession no. PRJNA607040).