Clonal distribution of an atypical MRP⁺, EF^*, and suilysin⁺ phenotype of virulent Streptococcus suis serotype 2 strains in Brazil

Abstract

Streptococcus suis is considered one of the most important bacterial swine pathogens worldwide. The distribution of the 35 described serotypes in diseased animals may vary in different regions. Data regarding S. suis isolation from pigs in South America is not available. In the present study, 51 isolates of S. suis recovered in pure culture or as the predominant species from diseased animals in Brazil, were analyzed. These isolates were classified as serotypes 2 (58.8%), 3 (21.5%), 7 (13.7%), 1 (3.9%), and 14 (2%). Serotype 2 isolates were further studied for their production of virulence-related proteins muramidase-released protein (MRP), extracellular factor (EF), and suilysin. In addition, the genetic diversity was studied by randomly amplified polymorphic DNA. All but 1 of the serotype 2 isolates showed a clonal distribution of an atypical phenotype (MRP⁺, EF^*, suilysin⁺), different from the known European (MRP⁺, EF⁺, suilysin⁺), and North American (MRP^v, EF⁻, suilysin⁻), phenotypes.

Streptococcus suis infection has been considered a major worldwide problem in the swine industry, particularly over the past 10 y. This pathogen is responsible for pathological conditions such as meningitis, endocarditis, arthritis, pneumonia, and septicemia followed by sudden death. Pigs can also be clinically healthy carriers and S. suis is usually isolated from the upper respiratory tract, nasal cavities, and palatine tonsils of these animals (1). Thirty-five capsular types have been described for S. suis, with type 2 being the most prevalent serotype recovered from diseased animals in most countries (2,3,4). However, the distribution of predominant serotypes may vary in different regions. For example, in Australia and The Netherlands, S. suis serotype 9 is frequently isolated from diseased pigs, whereas serotypes 7 and 14 are the most prevalent serotypes in Finland and Scotland, respectively (5,6). Streptococcus suis serotypes 2 and 14 are also recognized as zoonotic agents since they have been identified to cause meningitis, septicemia, and endocarditis in humans, particularly those occupationally exposed to pigs or pig products (7).

Phenotypic markers used to distinguish virulent from avirulent isolates of S. suis serotype 2 include the expression of muramidase-released protein (MRP), extracellular factor (EF), and suilysin (8,9). In several countries (specifically in Europe), MRP⁺EF⁺ serotype 2 strains are mainly isolated from diseased pigs and are associated with severe clinical signs of disease after experimental infection. In contrast, MRP⁻EF⁻ serotype 2 strains are frequently isolated from healthy pigs and are non-pathogenic after experimental inoculation (3,9). The MRP⁺EF* serotype 2 strains, which produce high molecular weight variants of EF, have been reported to be weakly virulent for young pigs (9). In addition, larger and smaller forms of MRP have been described (MRP* or MRP^s, respectively), but the virulence of these phenotypes is still unknown (10,11). Despite the fact that MRP and EF are strongly associated with highly virulent isolates in Europe, they do not appear to confer virulence directly, as demonstrated by the use of gene knockout technology (12). Moreover, most S. suis serotype 2 strains isolated from diseased pigs in Canada do not produce these virulence markers (13).

A similar situation has been described for the suilysin. This toxin has been described to play a certain role in the pathogenesis of the infection, since it is toxic to endothelial, epithelial, and phagocytic cells (14). However, it does not seem to be a critical virulence factor, since a nonhemolytic knock-out mutant was shown to be only slightly attenuated in a porcine model of systemic infection (15). As it is the case for the MRP and EF proteins, the suilysin is absent in most North American field isolates (13).

Different molecular typing methods; such as, multilocus enzyme electrophoresis (16), restriction endonuclease analysis (17), ribotyping (18), and randomly amplified polymorphic DNA (RAPD) (19), have been used to evaluate the diversity of S. suis serotype 2 isolates originating from Europe, North America, and Australia. However, data about the epidemiology and phenotypical characteristics of this organism isolated from diseased pigs in South America are not available. Therefore, the objective of this work was to study the serotype distribution of S. suis isolates originating from diseased pigs in Brazil. In addition, phenotypic and genotypic characterization of serotype 2 isolates was carried out.

Reference strains of all serotypes of S. suis originated from a collection from the University of Montréal. Strains 31533 and TD10 of S. suis serotype 2 were used as positive and negative controls, respectively, for EF, MRP, and suilysin detection. Strain 31533 (MRP⁺, EF⁺, suilysin⁺) is a virulent strain isolated from an acute case of meningitis in France (20). The non-virulent strain TD10 (MRP⁻, EF⁻, suilysin⁻) (21) was kindly provided by Dr. T. Alexander (University of Cambridge, England). The reference strain of S. suis serotype 2 (S-735) was used as an EF* control. All S. suis were grown on broth (THB) or agar (THA) (Todd-Hewitt broth, Todd-Hewitt agar; Difco Laboratories, Detroit, Michigan, USA) at 37°C under aerobic conditions. For long term storage, cultures were maintained in THB plus 50% glycerol at −70°C.

Samples were collected with sterile swabs from different tissues of dead or diseased pigs, mainly with clinical manifestations of septicemia and meningitis. Isolates incorporated in this study were recovered in pure culture, or as predominant bacteria from different tissues (cases of septicemia), lung (cases of pneumonia), or brain (cases of meningitis). Most pigs were between 25- to 45-days old and originated from unrelated different farms (from totally independent production systems) located in 3 different states of Brazil (Minas Gerais, Sao Paulo, Parana). The study was conducted from January to December, 1999. Samples were plated on a blood agar base (Columbia Blood Agar base; Oxoid Limited, Basingstone, Hants, England), containing 5% bovine sterile blood with the supplement SR-126 (Oxoid Limited), and were incubated at 37°C for 24 h. The presumptive bacteriologic diagnosis was based on the following characteristics: presence α-hemolysis on blood agar (5% bovine), negative Voges-Proskauer test, and production of amylase (22). Strains that had this profile were submitted for further tests; such as, absence of growth in THB plus NaCl 6.5%, fermentation of trehalose, and inulin and the lack of fermentation of mannitol and sorbitol. All streptococal isolates with the standard pattern of S. suis were confirmed by the API 20 STREP system (bioMérieux sa, Marcy l'Etoile, France).

Serotyping was carried out by the coagglutination test using rabbit hyperimmune sera against reference strains of all serotypes of S. suis, as previously described (23). A quick reaction (1 min) was recorded as positive. Conversely, no reaction or a weak one after several minutes was recorded as negative.

Production of MRP and EF were detected by immunoblotting using methodology and antisera, as previously described (13). In order to detect the expression of suilysin by immunoblot, a specific antibody was produced, as previously described (24). The activity and specificity of the anti-suilysin immunoglobulin G were confirmed by inhibition of the hemolytic activity and immunoblotting with purified suilysin and with supernatant of the suilysin-positive strain 31533. A perfect correlation of positive immunoblot reactions with suilysin production had previously been confirmed in our laboratory (unpublished results).

Genomic DNA was extracted by guanidine thiocyanate method according to Pitcher et al (25). The RAPD test was performed as previously described (19). Amplified products were separated by electrophoresis in a 1.2% agarose gel (Sigma-Aldrich, Oakville, Ontario) and were visualized as white bands on a black background by ultraviolet (UV) transillumination following ethidium bromide staining. A 1 kb DNA ladder (Invitrogene, Burlington, Ontario) was used in each gel as a molecular size standard. A negative control consisting of the same reaction mixture, but with water instead of template DNA, was included in each run. In addition, a positive control was also included, containing the same reaction mixture with a template of DNA from a well characterized reference strain (S. suis strain S-735). Each isolate was tested under the same conditions at least 3 times.

A total of 51 isolates were collected in pure culture or as the predominant specie from diseased or dead pigs; 26 isolates from pigs with meningitis, 17 from pigs with septicemia, and 8 from pigs with pneumonia. All isolates from cases of pneumonia were serotype 2. The majority of isolates recovered in this study belonged to serotype 2 (58.8%) (Table I). This result is in agreement with others, in which serotype 2 is the predominant S. suis serotype in most European countries, Japan, USA, and Canada (2,3,4,10,26). In addition, serotypes 3 (21.6%) and 7 were also frequently isolated in the Brazilian S. suis population examined in this study (Table I). Interestingly, serotypes reported to cause serious infections in humans (2 and 14) (7) are present in Brazil.

Table I.

European studies have shown that the MRP and EF proteins, as well as the suilysin (3,27), are strongly associated with the virulence of S. suis serotype 2 isolates. On the other hand, these proteins are not frequently associated with virulent North American strains (13). Taking into account this major difference in the phenotype of virulent S. suis serotype 2 strains, we analyzed the Brazilian isolates of S. suis for all currently known putative virulence traits. The analysis of the 3 virulence markers showed that all but 1 isolate of S. suis serotype 2 were MRP⁺, EF*, and suilysin⁺. The remaining isolate expressed the 3 proteins MRP, EF, and suilysin. The EF* protein is a higher molecular weight variant of EF and it has been proposed that strains producing this variant protein are weakly virulent (9). However, isolates evaluated in this study were all collected from animals with typical acute manifestations of S. suis infection. In addition, all Brazilian isolates studied also expressed the suilysin, which was previously reported as toxic for different host cells (14). Interestingly, the expression of suilysin had not been investigated in previous studies where the low virulence of strains presenting the phenotype MRP⁺ and EF* was reported (9).

The diversity of S. suis serotype 2 isolates was examined by RAPD, a technique with high resolution power, which has successfully been used to study S. suis populations in the past (19). Although S. suis serotype 2 isolates were recovered from geographically very distant farms (more than 400 km apart in 3 different States), all but 1 of these isolates presented the same RAPD pattern (Figure 1). This finding is in disagreement with previous reports, which demonstrated a relative genetic diversity for this serotype (16,17,18). However, Chatellier et al (19) found that RAPD clusters are more related to the phenotype defined by the suilysin, MRP, and EF profiles than to the geographic or host origin of the strains. This seems to be the case for the present study, since the RAPD technique was able to discriminate the only isolate which had a different phenotype (MRP⁺, EF⁺, and suilysin⁺) from the rest of the population (MRP⁺, EF*, and suilysin⁺) (Figure 1).

Figure 1. Illustration of the randomly amplified polymerase DNA (RAPD) patterns generated with primers OPB07, OPB10, and OPB17, as described by Chetellier et al (19). Lane 1, negative control; lanes 2–4 and 6, representative isolates from different States of Brazil, with a MRP⁺, EF*, and suilysin⁺ phenotype; lane 5, the only isolate with a MRP⁺, EF⁺, and suilysin⁺ phenotype.

M — 1kb DNA ladder (DNA molecular size marker).

In conclusion, we reported that serotype 2 is an important S. suis serotype isolated from diseased pigs in Brazil. In addition, this serotype seems to have a clonal distribution as determined by the RAPD technique, with predominance of the phenotype MRP⁺, EF*, and suilysin⁺, which is different from European (MRP⁺, EF⁺, and suilysin⁺) and North American (MRP^v, EF⁻, and suilysin⁻) virulent strains. It would be interesting, in the near future, to investigate the distribution of different serotypes, phenotypes, and genotypes of S. suis isolates recovered from clinically healthy animals.