Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention22172538331117111-060910.3201/eid1712.110609ResearchLineage and Virulence of Streptococcus suis Serotype 2 Isolates from North AmericaLineage and Virulence of S. suis Serotype 2FittipaldiNahuelXuJianguLacoutureSoniaTharavichitkulPrasitOsakiMakotoSekizakiTsutomuTakamatsuDaisukeGottschalkMarceloUniversité de Montréal, St-Hyacinthe, Quebec, Canada (N. Fittipaldi, S. Lacouture, M. Gottschalk);National Institute for Communicable Disease Control and Prevention, Beijing, People’s Republic of China (J. Xu);Chiang Mai University, Chiang Mai, Thailand (P. Tharavichitkul);Agriculture, Forestry and Fisheries Research Council, Tokyo, Japan (M. Osaki);The University of Tokyo, Tokyo (T. Sekizaki);National Institute of Animal Health, Tsukuba, Japan (D. Takamatsu)Address for correspondence: Marcelo Gottschalk, Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, CP5000, St-Hyacinthe, Quebec J2S 7C6, Canada; email: marcelo.gottschalk@umontreal.ca122011171222392244

Two sequence types predominate and have lower virulence than other types.

We performed multilocus sequence typing of 64 North American Streptococcus suis serotype 2 porcine isolates. Strains were sequence type (ST) 28 (51%), ST25 (44%), and ST1 (5%). We identified nonrandom associations between STs and expression of the virulence markers suilysin (SLY), muramidase-relased protein (MRP), and extracellular factor (EF). Expression of pili encoded by the srtF and srtG pilus clusters was also nonrandomly associated with STs. ST1 strains were SLY+ EF+ MRP+ srtF pilus+ srtG pilus−. ST25 strains were SLY− EF− MRP− srtF pilus− srtG pilus+, and most ST28 strains were SLY− MRP+ EF− srtF pilus+ srtG pilus+. ST28 isolates proved essentially nonvirulent in a mouse infection model; ST25 strains showed moderate virulence and ST1 isolates were highly virulent. ST1 is responsible for a high proportion of S. suis disease in humans worldwide. Its presence in North America indicates that potential zoonotic S. suis outbreaks in this continent cannot be disregarded.

Keywords: Streptococcus suisbacteriaserotype 2multilocus sequencing typingMLSTNorth Americazoonosesswinestreptococcilineagevirulence

Streptococcus suis causes meningitis and septicemia in pigs and is a zoonotic agent (1). In the Western hemisphere, human S. suis disease is infrequent and usually affects workers in the swine industry. However, S. suis is the most commonly reported cause of streptococcal meningitis in adults in Vietnam and the second in Thailand (2,3). Two outbreaks of human S. suis disease have occurred in People’s Republic of China, affecting hundreds of persons and causing 39 deaths (4). Most cases of animal and human S. suis infection have been caused by serotype 2 strains (5). The percentage of S. suis serotype 2 strains recovered from diseased pigs and the number of cases of human disease is lower in North America than in other parts of the world (6,7).

Multilocus sequence typing (MLST) has shown that S. suis serotype 2 strains can be divided into at least 16 sequence types (STs). Closely related STs are grouped in the so-called ST complexes. Although ST complexes 1, 27, and 87 dominate the S. suis population, most invasive isolates belong to the ST1 complex (8). For example, most strains isolated from human patients in Japan were ST1 (9), whereas those causing the human outbreaks in People’s Republic of China were ST7, included in the ST1 complex (10,11). However, Takamatsu et al. showed that 80% of the isolates recovered from blood or cerebrospinal fluid of humans in Thailand belonged to STs grouped in the ST27 complex (12).

Most of the S. suis serotype 2 strains genotyped so far by MLST originated in Europe and Asia (812). Isolates from Canada and the United States have received less attention. In this study, we used MLST to genotype a relatively large collection of US and Canadian S. suis serotype 2 strains.

Materials and Methods<italic>S</italic>. <italic>suis</italic> Field Strains

Sixty-four strains of S. suis serotype 2 isolated from pigs with clinical disease in different and nonrelated farms in major swine production areas of Canada and the United States were used. For comparison purposes, 19 porcine and 1 human S. suis serotype 2 strains isolated in Japan and 12 human S. suis serotype 2 strains isolated in Thailand were included (12,13). All strains are listed in Table A1.

MLST and Phylogenetic Analysis

S. suis genomic DNA was prepared from overnight cultures by using the QIAamp DNA Minikit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. MLST was performed by PCR amplification and DNA sequencing of the cpn60, dpr, recA, aroA, thrA, gki, and mutS genes as described (8). For each isolate, the alleles at each of the 7 loci defined the ST. MLST information in the S. suis database (http://ssuis.mlst.net) identified the phylogenetic position of strains. eBURST software (14) was used to identify S. suis clonal complexes and to display the overall structure of the population.

PCRs for Virulence Markers and Pili Cluster Genes

Amplification of sly, mrp, and epf genes was performed by PCR as described (6). Genes in the srtF and srtG pilus clusters were amplified by PCR by using the primers and conditions described by Takamatsu et al. (13).

MRP, EF, and Pili Expression and Hemolysis Assays

S. suis strains were grown in Todd-Hewitt broth at 37°C (at 28°C for detection of the srtG pilus). Bacteria were harvested by centrifugation during the late exponential phase of growth, and supernatants were concentrated 10-fold by using Ultrafree-MC centrifugal filters (Millipore Corp., Bedford, MA, USA). Expression of extracellular factor (EF) and muramidase-released protein (MRP) was determined by Western blotting of the concentrated supernatant fraction by using monoclonal antibodies against MRP or EF, as described (15). Mutanolysin extracts were prepared from pelleted bacteria as described (16,17) and used to detect pili encoded by the srtF and srtG pilus clusters by Western blotting with antibodies directed against the major subunit of each pilus (16,17). The ability of strains to lyse horse erythrocytes (an indication of the production by the strains of the hemolysin known as suilysin, SLY) was determined as described (6).

Experimental Infection of Mice

All animal experiments followed the guidelines of the Canadian Council on Animal Care and were approved by the Ethics Committee, Université de Montréal. We used a validated CD1 mouse infection model (18). In a first experiment, 60 female 6-week-old mice (Charles River Laboratories, Wilmington, MA, USA) were divided in 4 groups. Group 1 was inoculated with ST1 strain P1/7; groups 2 and 3 received ST25 strains 89-1591 and 1085543, respectively. Group 4 received ST28 strain 1088563. The inocula (5 × 107 CFU/animal) were delivered intraperitoneally. Mice were monitored 3×/d for 10 days for clinical signs and assigned clinical scores as described (18). Blood was collected daily from the tail vein (5 μL) and at necropsy by cardiac puncture and used to evaluate bacterial load by plating onto sheep blood agar plates and enumeration. Colonization of the liver and spleen of infected animals was evaluated at necropsy as described (18). A second experiment was performed essentially as described above, but the mice received a 10-fold higher dose of ST28 strains 1088563, 1054471, and 1097205. In this second experiment, groups contained 5 mice.

Results

Most of the 64 strains from North America were ST28 (n = 33) or ST25 (n = 28). Together, these 2 STs accounted for 95% of all S. suis serotype 2 strains from North America that were investigated (Table 1). However, a higher ST28 prevalence was true only for the United States; most strains from Canada were ST25. The remaining 3 strains belonged to ST1, which is commonly found in Europe and Southeast Asia.

STs identified among the <italic>Streptococcus suis</italic> serotype 2 isolates from North America*
CountryNo. strainsST1ST25ST28
Canada4402618
United States203215
Total6432833

*ST, sequence type.

Nonrandom Association between STs and Expression of Virulence Markers

SLY (encoded by the sly gene), MRP (mrp gene), and EF (epf gene) are virulence markers that have been used in elaborated genotypic and phenotypic schemes to try to predict the virulence of a given S. suis strain (1,19). For example, Silva et al. designed a multiplex PCR test that can discriminate between at least 6 naturally occurring genetic variants of mrp, named mrps, mrp, mrp*, mrp**, mrp***, and mrp**** (20). We investigated possible associations between STs and these widely used markers in our collection of S. suis serotype 2 strains from North America. To assess whether associations found are independent of the geographic origin of the strains, we included 32 described (12,13) S. suis serotype 2 strains of STs 28, 25, and 1 isolated in Japan and Thailand (Table A1).

Independently of geographic origin, we found clear, nonrandom associations between STs and expression of virulence markers. All but 2 ST1 strains had the phenotype SLY+MRP+ EF+. All ST25 strains were SLY−MRP−EF− and all ST28 strains were SLY−MRP (or its variants)+ EF− (Table 2). Most ST1 strains had an sly+mrp+epf+ genotype, in agreement with results of previous reports (1113). ST25 and ST28 strains had an sly− genotype and, with the exception of 3 ST28 strains, an epf− genotype. No clear relationships were found between ST25 strains and a particular mrp gene variant genotype. All but 3 ST25 strains were positive by PCR for 1 mrp gene variant, yet none of these strains expressed the protein (Table 2). In a recent report, all mrp+/MRP− strains that were investigated (of various S. suis serotypes) had truncations or point mutations in the mrp gene that prevented expression of MRP (6). Although we have not sequenced the mrp gene in our collection of strains, we hypothesize that similar genetic rearrangements are likely to explain the mrp+/MRP− results we observed in ST25 strains in this study. Three mrp gene variants were associated with ST28, although variant mrp was the most prevalent (85%) among this ST.

Association of <italic>Streptococcus suis</italic> serotype 2 STs and commonly used virulence markers in isolates from North America†
STNo. strainsPresence of factor-encoding gene
Phenotype
slymrp variant‡
epf
mrpmrpsmrp*mrp**mrp***NDHemolysis§MRP¶EF
111119000021111911
2536001182330000
28490426100030490

†ST, sequence type; SLY, suilysin; MRP, muraminidase-released protein; ND, no amplification of the mrp gene was detected by PCR under the conditions used; EF, extracellular factor.
‡Variants of the mrp gene are those described by Silva et al. (20).
§Hemolysis of horse erythrocytes by the strains was considered to be an indication of the expression of SLY.
¶Molecular mass MRP variants identified by Western blotting were in agreement with those expected on the basis of the mrp gene variant identified by PCR.

Nonrandom Association between STs and Expression of Pili

Takamatsu et al. reported associations between particular STs and the presence or absence of putative pilus gene clusters, designated srtBCD, srtE, srtF, and srtG clusters (13). All ST25 and ST28 strains investigated by these authors were positive by PCR for all genes in the srtF and srtG pilus clusters (13). Consistently, we found that all ST25 and ST28 strains in our collection were positive for all genes in these 2 pilus clusters (Table 3). Furthermore, by using specific antibodies directed against the major pilin subunits (16,17), we identified a clear, nonrandom association between ST28 strains and expression of both pili (Table 3). However, although all ST25 strains expressed the srtG pilus, none produced the srtF pilus (Table 3).

Association of <italic>Streptococcus suis</italic> serotype 2 STs and <italic>srtF</italic> and <italic>srtG</italic> pilus clusters in isolates from North America*
STNo. strainssrtF pilus cluster
srtG pilus cluster
Gene†
Pili expression‡, Sfp1Gene†
Pili expression, Sgp1‡
srtFsfp1sfp2sipFsrtGsgp1sgp2
11111111111113330
253636363636036363636
2849494949494649494947

*ST, sequence type.
†The presence of the genes was detected by PCR by using primers and conditions described by Takamatsu et al. (13).
‡Expression of pili encoded by the srtF and srtG pilus clusters was performed by Western blotting by using described antibodies directed against the major subunits of these structures (16,17).

It has been shown that one ST25 isolate from Canada, which does not have a discrete srtF pilus cluster and is unable to express the srtF pilus, is nonetheless PCR positive for each of the individual srtF genes because PCR amplicons can be generated from homologs of these genes found at various genome locations (13,16). We hypothesized that the ST25 strains analyzed in our study have a genetic organization similar to that ST25 isolate. Consistent with our hypothesis, our attempts to amplify the srtF pilus cluster in ST25 strains by using a primer pair annealing to the first and last gene of the srtF cluster were unsuccessful (data not shown). All the ST1 strains had the srtF cluster genes but, with the exception of 3 strains, not the srtG cluster genes. When we assessed the pilus phenotype by Western blotting, all ST1 strains expressed the srtF pilus but none expressed the srtG pilus (Table 3). The reason(s) the 3 ST1 strains that have the srtG cluster genes do not express the corresponding pilus are currently under investigation.

Mouse Infection Model

Inasmuch as the MLST data showed that more than half of the strains from North America analyzed were ST28 and the second most represented ST was ST25, we performed a comparison of the virulence of representative ST25 and ST28 strains by using a standardized mouse infection model (18). For comparison, we included the well-characterized and highly virulent ST1 strain P1/7. Most mice in the ST1 group showed severe clinical signs of septicemia, such as depression, swollen eyes, weakness, and prostration during the first 24 hours postinoculation. Several mice died of septicemia during the first 2 days of the trial, and the remaining animals were humanely killed for ethical reasons at day 3 postinoculation (Figure 1). S. suis was isolated in pure cultures at high titers (>1 × 107 CFU/mL) from blood samples and organs, such as the liver and spleen, of septicemic animals in the ST1 group (>1 × 107 CFU/0.5 g of tissue in most animals).

Survival of CD1 mice inoculated with Streptococcus suis strains of different sequence types (STs). Most animals that received the ST1 strain P1/7 died from septicemia during the first 3 days of the trial. Several animals in this group died from meningitis from day 6 postinfection. Two groups of mice received ST25 strains 89–1591 and 1085543, respectively. Survival of mice in these 2 groups was higher than in the group that received the ST1 strain. However, >40% of the animals in the 89–1591 group and 60% of the animals in the 1085543 group died or were killed for ethical reasons before the end of the trial. In strong contrast, all 15 mice in the ST28 strain group survived the trial. Significant differences in survival were noted between groups (log-rank test, p values indicated in the figure body).

The virulence of ST25 strains was intermediate. They caused moderate clinical signs and relatively low mortality among inoculated mice (Figure 1). Statistical analysis demonstrated that ST25 strains were significantly less virulent than ST1 strains. However, ST25 strains were significantly more virulent than ST28 strains. In fact, no mice in the ST28 group died (Figure 1) or showed clinical signs associated with S. suis infection, with the exception of slight depression immediately after inoculation, which subsided after 24 hours postinoculation. Bacteria could not be isolated from the blood of most mice in this group >48 hours postinoculation, and we could not isolate S. suis from different organs at necropsy (results not shown). Given this surprising absence of clinical signs, we repeated the experiment by inoculating 3 additional groups of 5 mice each with the previously used and 2 other ST28 strains by using an infective dose that was 10-fold higher than the one previously used. Despite this increased infective dose, similar low virulence was observed for ST28 strains (Figure 2).

Survival of CD1 mice inoculated with the different Streptococcus suis sequence type 28 strains from North America. In this experiment, the infectious dose was 1 × 108 CFU/animal, 10-fold higher than in the previous experimental inoculation. Doses were intraperitoneally injected into the animals. No significant differences were found between groups.

Discussion

In this article, we show that most S. suis isolates from North America belong to ST28 and ST25 and that strains of these STs are significantly less virulent than ST1 strains. Although ST28 strains were essentially nonvirulent for mice, ST25 strains were of intermediate virulence and able to induce severe disease.

With a population of ≈115 million pigs, Canada and the United States combined are second only to the People’s Republic of China in terms of swine production. Although S. suis infections are a main cause of postweaned piglet deaths in North America, the prevalence of S. suis serotype 2 strains is much lower on this continent than in other regions of the world (6,7). We show here that in North America the most common STs among S. suis serotype 2 strains are ST28 and ST25. By using a mouse infection model, we also show that S. suis serotype 2 ST28 and ST25 strains are of lower virulence than ST1 strains. In contrast to Europe and Asia, where >60% of virulent serotype 2 isolates are ST1 (2123), in North America only a small percentage (5%) of strains belonged to this more virulent ST.

Only 3 cases of S. suis serotype 2 in locally infected humans have been reported in North America (5). Our results suggest that this low prevalence of human infections might be connected to the lower virulence of the circulating serotype 2 strains among the swine population in North America. In addition to a low prevalence of ST1 strains, we did not find any strains in our collection from North America belonging to STs 101, 102, 103, and 104, which are agents of human disease in Thailand (12). On the basis of its low frequency of isolation, we speculate that the ST1 strains we identified were introduced in North America by importation of animals. Human travel might also contribute to dissemination of ST1 strains, as exemplified by a reported case of human S. suis meningitis caused by an ST1 strain involving a patient who contracted S. suis in the Philippines but in whom clinical signs appeared only after he returned to the United States (24). The deadly human outbreaks in Asia caused by ST1 complex strains (25) and the fact that ST1 strains are replacing at a fast pace STs of lower virulence and causing human disease in countries such as Vietnam and Thailand (21,25,26) highlight that maintaining a low prevalence of ST1 strains among the swine population in North America is crucial for animal and human health. Of note, the only locally acquired human infection in the United States described so far (27) was caused by an ST1 strain (M. Gottschalk, unpub. data).

Another concern for the swine industry and for public health authorities is the presence in North America of S. suis ST25 strains. Many human cases reported in Thailand and 2 cases in Canada of S. suis serotype 2 disease were caused by ST25 strains (5,12). On the other hand, we found that strains of the most prevalent ST28 are of low virulence. Two strains shown here to be ST28 (1330 and 0891; see Table A1) had been reported as nonvirulent S. suis serotype 2 (8). No human S. suis cases attributable to ST28 strains have been reported in North America. However, all ST28 strains included in this study were isolated from diseased pigs, and 1 human case in Japan and 1 human case in Thailand were caused by ST28 strains (9,12). Nonvirulent S. suis strains have been hypothesized to cause disease in immunocompromised animals or humans who have a concurrent infection with another bacterial or viral pathogen(s) (5). Porcine S. suis infections in North America are usually associated with a concomitant infection with the porcine respiratory and reproductive virus (1). We do not know the immunologic status of the animals from which the ST28 strains were isolated to test the aforementioned hypothesis. Toward this goal, however, we are developing a co-infection model of S. suis and porcine respiratory and reproductive virus.

Our results provide evidence that genotyping schemes based on sly, mrp, epf, and pilus cluster genes, although useful in discriminating highly virulent ST1 strains from other groups (8,13,20), are of limited use in differentiating between ST25 and ST28 strains. Although not ideal because protein expression levels may be affected by many factors, typing methods based on protein expression of these markers might a priori differentiate these STs of different virulence. The fact that ST25 strains do not express MRP or the srtF pili, yet they are more virulent than ST28 strains, further demonstrates the dispensability of these factors for the full virulence of S. suis (16,28). Our results also highlight that subunit vaccines based on purified MRP or srtF pilus subunits might be of little use to counter S. suis infections caused by ST25 strains.

Our work provides more support to the longstanding hypothesis that S. suis serotype 2 strains in North America are of lower virulence than strains from Eurasia. However, we do not yet understand the reasons for this lower virulence. The genome sequences of several S. suis serotype 2 ST1 and an ST25 strains have been published or made available (25,29,30). Genome sequencing of a larger number of additional S. suis strains of these and other STs could help elucidate the genetic basis of virulence differences among strains of this swine pathogen and zoonotic agent.

Suggested citation for this article: Fittipaldi N, Xu J, Lacouture S, Tharavichitkul P, Osaki M, Sekizaki T, et al. Lineage and virulence of Streptococcus suis serotype 2 isolates from North America. Emerg Infect Dis [serial on the Internet]. 2011 Dec [date cited]. http://dx.doi.org/10.3201/eid1712.110609

This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada to M.G. (no. 154280 and Discovery Accelerator Supplement 380299) and Minister of Economic Development, Innovation and Export Trade, China-Quebec Collaboration grants to M.G and J.X. (no. 2008FA31830 and 2008DFA31830, respectively). N.F. is partially supported by the Canadian Institutes of Health Research.

<italic>Streptococcus suis</italic> serotype 2 strains used in study of North American isolates and summary of results†
Strain nameCountryProvince/state/ prefectureIsolation dateSTGenotype
Phenotype
HostTissue/ disease
gdhmrpslyepfsipFsfp1sfp2srtfsgp1sgp2srtGMRPSLYEFSfp1Sgp1
P1/7United KingdomNo data19811++++++++++++PigMeningitis
1043248CanadaQuebec2007 Jan25+***++++++++PigBrain
1043629CanadaQuebec2007 Feb25+***++++++++PigLung
1044423CanadaOntario2007 Jun25+***++++++++PigNo data
1053253CanadaManitoba2008 Jan25+***++++++++PigBronchious
1085543CanadaQuebec2008 Jan25+***++++++++PigMeninges
1054470CanadaQuebec2007 Jun25+***++++++++PigLung
1058691CanadaManitoba2007 Aug25+***++++++++PigMeninges
1063930CanadaOntario2007 Mar25+***++++++++PigNo data
1064496CanadaQuebec2007 Jun25+***++++++++PigBrain
1055923CanadaSaskatchewan2007 Aug25+***++++++++PigBronchious
1072913CanadaQuebec2007 Jan25+***++++++++PigPleura
1074055CanadaQuebec2008 Feb25+++++++++PigMeningitis
1078217CanadaOntario2008 Feb25+***++++++++PigBrain
1078679CanadaOntario2008 Feb25+***++++++++PigLung
1084568CanadaQuebec2008 Mar25+*++++++++PigBrain
1086117CanadaManitoba2008 Apr25+s++++++++PigEndocardium
1087028CanadaManitoba2008 May25+***++++++++PigBrain
1088904CanadaQuebec2008 May25+***++++++++PigMeningitis
1091168CanadaQuebec2008 Jun25+***++++++++PigMeningitis
1093400CanadaOntario2008 Jun25+***++++++++PigBrain
1097204CanadaQuebec2008 Jun25+***++++++++PigLung
1098986CanadaQuebec2008 Jul25+***++++++++PigLung
1102864CanadaQuebec2008 Aug25+***++++++++PigMultiple
1102337CanadaQuebec2008 Jul25+***++++++++PigMeningitis
1111483CanadaQuebec2008 Sep25+***++++++++PigBrain
1084708CanadaOntario2008 Apr28++++++++++++PigPleura
1085273CanadaQuebec2008 Apr28+s++++++++++PigNasal sample
1054471CanadaManitoba2007 Jul28++++++++++++PigBrain
1064089CanadaSaskatchewan2007 Sep28+s++++++++++PigTonsil
1057906CanadaSaskatchewan2007 Jul28+s++++++++++PigBrain
1064773CanadaQuebec2007 Oct28+++++++++++PigLung
1077008CanadaQuebec2008 May28+*++++++++PigEndocardium
1082563CanadaQuebec2008 Apr28++++++++++++PigEndocardium
1088563CanadaQuebec2008 May28++++++++++++PigBrain
1089976CanadaQuebec2008 May28+s++++++++++PigEndocardium
1090152CanadaOntario2008 Jun28++++++++++++PigLung
1090686CanadaSaskatchewan2008 Jun28++++++++++++PigNo data
1097205CanadaOntario2008 Jun28++++++++++++PigBrain
1097811CanadaOntario2008 Jun28+s++++++++++PigLiver/lung/kidney
1110359CanadaQuebec2008 Sep28+s++++++++++PigPleura
1111490CanadaManitoba2008 Sep28++++++++++++PigLung
89-1591CanadaManitoba198925+***++++++++PigSepticemia, meningitis
1330CanadaQuebec199528++++++++++++PigLung
0891CanadaQuebec199528++++++++++++PigLung
NIAH11433JapanNo data19891++++++++++++PigMeningitis
DAT261JapanGunma1993 Jan1++++++++++++PigMultiple serositis, pneumonia
DAT264JapanGunma1994 Jun1++++++++++++PigMeningitis
DAT229JapanAichi2006 Jan1++++++++++++PigEndocarditis
DAT273JapanNagasaki2002 Feb1++++++++++HumanMeningitis
DAT292JapanOkinawa2005 Dec28++++++++++++PigHealthy carrier
DAT242JapanIbaraki1990 Jan28++++++++++++PigMeningitis
DAT245JapanIbaraki1995 Jan28+++++++++++PigMeningitis
DAT246JapanIbaraki1996 Apr28++++++++++++PigSepticemia
DAT251JapanIshikawa1991 Jun28++++++++++++PigLiver
DAT253JapanIshikawa1993 Aug28++++++++++++PigBrain
DAT254JapanIshikawa1996 Jul28++++++++++++PigBrain
DAT255JapanNiigataNo data28++++++++++++PigSepticemia
DAT256JapanNiigataNo data28++++++++++++PigMeningitis
DAT259JapanGunma1992 Nov28++++++++++++PigMultiple serositis, pneumonia
DAT260JapanGunma1992 Dec28++++++++++++PigMeningitis
DAT272JapanYamagataNo data28++++++++++++PigBrain
DAT274JapanKumamoto1994 Mar28++++++++++++PigEndocarditis
DAT281JapanKumamotoNo data28+++++++++++PigMeningitis
DAT285JapanIbarakiNo data28++++++++++++PigEndocarditis
MNCM01ThailandChiang Mai2000 Jun1++++++++++++HumanEndocarditis
MNCM06ThailandChiang Mai2000 Aug1++++++++++++HumanMeningitis
MNCM16ThailandChiang Mai2000 Nov1++++++++++++HumanMeningitis
MNCM04ThailandChiang Mai2000 Aug25+**++++++++HumanMeningitis
MNCM10ThailandChiang Mai2000 Sep25+**++++++++HumanSepticemia
MNCM24ThailandChiang Mai2001 Aug25+**++++++++HumanEndocarditis
MNCM26ThailandChiang Mai2001 Nov25+**++++++++HumanMeningitis, endocarditis
MNCM51ThailandChiang Mai2002 Oct25+**++++++++HumanSepticemia
MNCM55ThailandChiang Mai2002 Dec25+**++++++++HumanSeptic shock
LPH4ThailandLamphun2001 May25+**++++++++HumanSepticemia
LPH12ThailandLamphun2002 Mar25+**++++++++HumanSeptic shock
MNCM43ThailandChiang Mai2002 Jun28++++++++++++HumanEndocarditis
MGGUS1United StatesMinnesota2003 Mar1+++++++++++++PigNo data
MGGUS2United StatesWisconsin2003 Feb1+++++++++++++++PigBrain
MGGUS3United StatesIowa2003 May1+++++++++++++++PigBrain
MGGUS4United StatesIowa2005 May25+++++++++PigSepticemia
MGGUS5United StatesNebraskaNo data25+++++++++PigSepticemia
MGGUS6United StatesMinnesotaNo data28+++++++++++++PigNo data
MGGUS7United StatesKansas199528+++++++++++++PigNo data
MGGUS8United StatesKansas199528+++++++++++++PigNo data
MGGUS9United StatesOklahoma2003 Dec28+++++++++++PigHeart
MGGUS10United StatesIllinois2005 May28++++++++++++PigLung
MGGUS11United StatesVirginia2005 May28++++++++++++PigLung
MGGUS12United StatesIowa2005 May28++++++++++++PigLung
MGGUS13United StatesOklahoma2004 Oct28++++++++++++PigLiver/lung/ kidney
MGGUS14United StatesOklahomaNo data28++++++++++++PigLung
MGGUS15United StatesOklahomaNo data28++++++++++++PigLiver/lung/ kidney
MGGUS16United StatesNebraska2004 Jun28++++++++++++PigLung
MGGUS17United StatesOklahoma2004 Dec28++++++++++++PigSpleen
MGGUS18United StatesNorth CarolinaNo data28++++++++++++PigLiver/lung/ kidney
MGGUS19United StatesNebraskaNo data28++++++++++++PigLiver/lung/ kidney
MGGUS20United StatesKentucky2004 Jan28++++++++++++PigSpleen

†ST, sequence type; MRP, muramidase-released protein; SLY, suilysin; EF, extracellular protein factor; +, positive; −, negative.
‡The identified mrp variant (mrps, mrp, mrp*, mrp**, mrp***, mrp****) is indicated.

Dr Fittipaldi is a postdoctoral fellow in the Center for Molecular and Translational Human Infectious Diseases Research of the Methodist Hospital Research Institute in Houston, Texas, USA. His primary research interest is the molecular basis of streptococcal pathogen–host interactions.

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