Advertisement\nResearch Article\n26 May 2020\nShare on\nDevelopment of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species\nAuthors: Yoshihiro Shimoji, Kazumasa Shiraiwa, Haruka Tominaga, Sayaka Nishikawa, Masahiro Eguchi, Hirokazu Hikono, Yohsuke OgawaAuthors Info & Affiliations\nDOI: https://doi.org/10.1128/jcm.00315-20\n00\nMetrics\nTotal Citations10\nTotal Downloads1,367\nView all metrics\nCite\nPDF/EPUB\nABSTRACT\nThe Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species.\n\nHistorically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping.\n\nAmong 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.\n\nThe 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.\n\nTo 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.\n\nWe 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.\n\nWe created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars.\n\nTogether 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.\n\nINTRODUCTION\nErysipelothrix rhusiopathiae is a Gram-positive, non-spore-forming, rod-shaped bacterium and the standard species of the genus Erysipelothrix in the phylum Firmicutes (1).\n\nThe genus Erysipelothrix is composed of at least five species, E. rhusiopathiae, Erysipelothrix tonsillarum (2), Erysipelothrix inopinata (3), Erysipelothrix larvae (4), and Erysipelothrix piscisicarius sp.\n\nnov. (5), the last of which is pathogenic to fish and has been recently proposed as a new member of the genus.\n\nFurthermore, the existence of three other species, which are unnamed and tentatively referred to as Erysipelothrix sp.\n\nstrain 1, Erysipelothrix sp.\n\nstrain 2, and Erysipelothrix sp.\n\nstrain 3, has been suggested (6); however, Erysipelothrix sp.\n\nstrain 2 has now been confirmed to belong to E. piscisicarius sp.\n\nnov. (5).\n\nHistorically, 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.\n\nstrain 1 (strain Pécs 56; serovar 13), and Erysipelothrix sp.\n\nstrain 2 (strain 715; serovar 18) have been differentiated by their serovars (2).\n\nTo date, new serovars have been assigned to E. rhusiopathiae (serovar 23) and E. tonsillarum (serovars 24 to 26).\n\nThe serovar of the new species E. piscisicarius sp.\n\nnov. has been determined; however, the serovars of E. inopinata and E. larvae have not yet been clarified.\n\nErysipelothrix species are ubiquitous in nature and have been isolated from a variety of animal species.\n\nAmong 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).\n\nIn humans, the manifestation of the disease is a skin disease known as erysipeloid, which can lead rarely to septicemia and endocarditis (8).\n\nRecently, 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).\n\nEpidemiological 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).\n\nHowever, 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).\n\nSerotyping 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.\n\nThus, 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.\n\nRecently, 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).\n\nIn 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.\n\nMATERIALS AND METHODS\nBacterial strains.\n\nThe 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.\n\nnov. 15TAL0474 strains.\n\nErysipelothrix 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).\n\nSerotyping.\n\nSerotyping 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).\n\nSequence information and analysis.\n\nDraft genome sequences of Erysipelothrix strains were either retrieved from GenBank or generated using the Illumina HiSeq platform, as described previously (12).\n\nTrimmed reads were assembled using SPAdes (23), and the assembled contigs were evaluated using QUAST (24).\n\nGenetic relatedness among Erysipelothrix strains was analyzed by comparing the ANI using the ANI calculator (JSpecies ver.1.2.1, with default parameters) (25).\n\nIn 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).\n\nUsing 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).\n\nFor 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.\n\nDNA methods.\n\nThe 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.\n\nFor multiplex PCR assays, DNA was prepared using an alkaline boiling method, as previously described (15).\n\nBriefly, 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).\n\nThe suspension was centrifuged, and 1.0 μl of the supernatant was used for PCR.\n\nThe 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.\n\nPCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) and a T-100 thermal cycler (Bio-Rad, CA, USA).\n\nBriefly, amplification reaction mixtures were prepared according to the manufacturer’s recommendations but scaled down to 25 μl per reaction.\n\nFor multiplex PCR assays, the concentration of primers was changed according to the sizes of DNA fragments.\n\nThe composition of the multiplex PCR mixtures is shown in Table S4 in the supplemental material.\n\nThe 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.\n\nData availability.\n\nSequence data generated in this study have been deposited in the NCBI Sequence Read Archive (BioProject accession no.\n\nPRJNA607040).\n\nRESULTS AND DISCUSSION\nThe 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.\n\n1.\n\nSequence analysis revealed that the serovar 13 strain (strain Pécs 56; Erysipelothrix sp.\n\nstrain 1) lacks the corresponding genetic region.\n\nWe 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.\n\nstrain 2 (strain 715; serovar 18) but shares 96.17% ANI with E. inopinata (Table S1), strongly suggesting that Erysipelothrix sp.\n\nstrain 1 (the serovar 13 strain) and E. inopinata are the same species.\n\nIn 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.\n\nFIG 1\nAs 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.\n\n1).\n\nIt has been reported that antibody cross-reaction occurred between serovars 7 and 14 (29, 30).\n\nWe 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).\n\nDue 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).\n\nFor other Erysipelothrix serovar strains, primers were designed to specifically detect each serovar.\n\nFor 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.\n\n2).\n\nEach set contained five primer sets that amplified products of correct different sizes for comprehensible differentiation.\n\nValidation 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).\n\nIn 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.\n\nThus, 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.\n\nstrain 1), which is most likely the same species as E. inopinata.\n\nWe believe that the multiplex PCR-based serotyping method should be useful in investigating the disease transmission potential between humans, livestock, and wildlife.\n\nHowever, 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.\n\nFIG 2\nTABLE 1\nIn 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).\n\nAs observed in Fig.\n\n1, 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.\n\nPreviously, it was confirmed that Erysipelothrix sp.\n\nstrain 2 (strain 715) (serovar 18) and the new species E. piscisicarius sp.\n\nnov. are the same species.\n\nHowever, the E. piscisicarius sp.\n\nnov. 15TAL0474 strain has been shown to be a serovar 5 strain (31).\n\nThus, in some strains, the serovar is not strictly associated with whole-genome-based classification of Erysipelothrix species (6).\n\nAlthough 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.\n\nIt 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.\n\nFor 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.\n\nACKNOWLEDGMENTS\nThis work was supported by a grant from the National Agriculture and Food Research Organization (to Y.S.).\n\nWe declare no conflicts of interest.\n\nSupplemental Material\nFile (jcm.00315-20-s0001.xlsx)\nASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article.\n\nThe authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files.\n\nPlease contact the corresponding author directly for reuse.\n\n----------\nAs a professional microbiologist, create a concise and comprehensive Chinese scientific report on Erysipelothrix Spp. research based on the provided text, whether it's an article, post, conversation, or passage. Follow these guidelines:\n\n 1. Produce a detailed and in-depth report that remains clear and concise.\n 2. Focus on main ideas and essential information, removing any extraneous language.\n 3. Strictly rely on the provided text without adding external information.\n 4. Format the report in paragraphs for easy understanding.\n 5. Conclude with “[End of Notes, Message #X],” where “X” represents the total number of messages I’ve sent. Increment the message counter with each new message.\n 6. Use English for technical terms, industry terminology, proper nouns, and species names. After you finish, add the following words: {{{ I_finished_papa!!! }}} to the end of you answer