Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain.

Streptococcus suis serotype 2 is an important swine bacterial pathogen causing sudden death, septic shock, and meningitis. However, serotype 2 strains are phenotypically and genotypically heterogeneous and composed of a multitude of sequence types (STs) whose distributions greatly vary worldwide. It has been previously shown that the lipoprotein (LPP) maturation enzymes diacylglyceryl transferase (Lgt) and signal peptidase (Lsp) significantly modulate the inflammatory host response and play a differential role in virulence depending on the genetic background of the strain. Differently from Eurasian ST1/ST7 strains, the capsular polysaccharide of a North American S. suis serotype 2 ST25 representative strain only partially masks sub-capsular domains and bacterial wall components. Thus, our hypothesis is that since LPPs would be more surface exposed in ST25 strains than in their ST1 or ST7 counterparts, the maturation enzymes would play a more important role in the pathogenesis of the infection caused by the North American strain. Using isogenic Δlgt and Δlsp mutants derived from the wild-type ST25 strain, our studies suggest that these enzymes do not seem to play a role in the interaction between S. suis and epithelial and endothelial cells, regardless of the genetics background of the strain used. However, a role in the formation of biofilms (also independently of the STs) has been demonstrated. Moreover, the involvement of LPP dendritic cell activation in vitro seems to be somehow more pronounced with the ST25 strain. Finally, the Lgt enzyme seems to play a more important role in the virulence of the ST25 strain. Although some differences between STs could be observed, our original hypothesis that LPPs would be significantly more important in ST25 strains due to a better bacterial surface exposition could not be confirmed.

Improvement of the mismatch amplification mutation assay-PCR for discrimination between Streptococcus suis serotypes 2 and 1/2.

A mismatch amplification mutation assay (MAMA)-PCR, which detects a single-nucleotide polymorphism contributed to serological difference between Streptococcus suis serotypes 2 and 1/2, is used to discriminate between these serotypes. The present study reports unusual serotype 1/2 isolates untypable by the MAMA-PCR and improvement of the MAMA-PCR for typing such isolates.

Streptococcus suis surface-antigen recognition by antibodies and bacterial elimination is influenced by capsular polysaccharide structure.

Streptococcus suis is an encapsulated bacterium causing severe diseases in swine. Here, we compared the protective properties of the capsular polysaccharide (CPS) of different S. suis serotypes by using serotype-switched mutants in a mouse model of infection. CPS structure influenced bacterial survival in mice, antibody binding, and antibody-mediated bacterial killing. The CPS of serotypes 3, 4 and 14 allowed more antibody binding and bacterial elimination than the CPS of serotypes 2, 7 and 9. Results suggest that the different CPS structures of S. suis provide varying levels of protection by influencing antigen availability and elimination by the host immune system.

Development of a Streptococcus pluranimalium-specific PCR assay.

Streptococcus pluranimalium, an emerging zoonotic pathogen associated with infections in various animal species and humans, cannot be reliably identified by phenotypic characterization using the commercial kits routinely used in laboratories. We herein developed the first S. pluranimalium-specific PCR assay useful for the easy and reliable identification of this species.

Suppurative meningoencephalitis and perineuritis caused by Streptococcus gallolyticus in a Japanese Black calf.

A 179-day-old calf, which was weak and stunted, showed neurological signs and was euthanized. Postmortem examination revealed extensive and severe cloudy area in the meninges, and pleural pneumonia. Gram-positive cocci were isolated from systemic organs. Biochemical and 16S rRNA gene sequence analyses identified the isolate as Streptococcus gallolyticus, and its subspecies was suggested to be gallolyticus (SGG). The isolate was classified as a novel sequence type (ST115) by the multilocus sequence typing scheme for SGG and showed susceptibility to penicillin, ampicillin, amoxicillin, florfenicol, sulfamethoxazole-trimethoprim, and chloramphenicol. Histopathologically, suppurative meningoencephalitis and perineuritis were detected. As SGG has been isolated solely from a cow with mastitis in Japan, this is the first SGG infection in a calf with suppurative meningoencephalitis and perineuritis in this country.

Role of Maturation of Lipoproteins in the Pathogenesis of the Infection Caused by Streptococcus suis Serotype 2.

Streptococcus suis serotype 2 is an important porcine bacterial pathogen associated with multiple pathologies in piglets. Bacterial lipoproteins (LPPs) have been described as playing important roles in the pathogenesis of the infection of other Gram-positive bacteria as adhesins, pro-inflammatory cell activators and/or virulence factors. In the current study, we aimed to evaluate the role of the prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) enzymes, which are responsible for LPP maturation, on the pathogenesis of the infection caused by two different sequence types (STs) of S. suis serotype 2 strains (virulent ST1 and highly virulent ST7). Through the use of isogenic Δlgt, Δlsp and double Δlgt/Δlsp mutants, it was shown that lack of these enzymes did not influence S. suis adhesion/invasion to porcine respiratory epithelial cells. However, in the absence of the Lsp and/or Lgt, a significant reduction in the capacity of S. suis to activate phagocytic cells and induce pro-inflammatory mediators (in vitro and in vivo) was observed. In general, results obtained with the double mutant did not differ in comparison to single mutants, indicating lack of an additive effect. Finally, our data suggest that these enzymes play a differential role in virulence, depending on the genetic background of the strain and being more important for the highly virulent ST7 strain.

Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain.

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70-80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

Capsular polysaccharide switching in Streptococcus suis modulates host cell interactions and virulence.

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Comparative Study of Immunogenic Properties of Purified Capsular Polysaccharides from Streptococcus suis Serotypes 3, 7, 8, and 9: the Serotype 3 Polysaccharide Induces an Opsonizing IgG Response.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C-C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly.

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

Update on Streptococcus suis Research and Prevention in the Era of Antimicrobial Restriction: 4th International Workshop on S. suis.

Streptococcus suis is a swine pathogen and a zoonotic agent afflicting people in close contact with infected pigs or pork meat. Sporadic cases of human infections have been reported worldwide. In addition, S. suis outbreaks emerged in Asia, making this bacterium a primary health concern in this part of the globe. In pigs, S. suis disease results in decreased performance and increased mortality, which have a significant economic impact on swine production worldwide. Facing the new regulations in preventive use of antimicrobials in livestock and lack of effective vaccines, control of S. suis infections is worrisome. Increasing and sharing of knowledge on this pathogen is of utmost importance. As such, the pathogenesis and epidemiology of the infection, antimicrobial resistance, progress on diagnosis, prevention, and control were among the topics discussed during the 4th International Workshop on Streptococcus suis (held in Montreal, Canada, June 2019). This review gathers together recent findings on this important pathogen from lectures performed by lead researchers from several countries including Australia, Canada, France, Germany, Japan, Spain, Thailand, The Netherlands, UK, and USA. Finally, policies and recommendations for the manufacture, quality control, and use of inactivated autogenous vaccines are addressed to advance this important field in veterinary medicine.

Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.

Different impacts of pMP19 on the virulence of Melissococcus plutonius strains with different genetic backgrounds.

Virulence factors responsible for bacterial pathogenicity are often encoded by plasmids. In Melissococcus plutonius, the causative agent of European foulbrood of honey bees, a putative virulence plasmid (pMP19) possessing mtxA, which encodes a putative insecticidal toxin, was found by comparative genome analyses. However, as the role of pMP19 in the pathogenesis of European foulbrood remains to be elucidated, we generated pMP19 cured-M. plutonius from representative strains of the three genetically distinct groups (CC3, CC12 and CC13) and compared their virulence against Apis mellifera larvae using our in vitro infection model. Under the conditions tested, the loss of pMP19 abrogated the pathogenicity in CC3 strains, and > 94% of pMP19-cured CC3 strain-infected larvae became adult bees, suggesting that pMP19 is a virulence determinant of CC3 strains. However, introduction of mtxA on its own did not increase the virulence of pMP19-cured strains. In contrast to CC3 strains, the representative CC12 strain remained virulent even in the absence of pMP19, whereas the representative CC13 strain was avirulent even in the presence of the plasmid. Thus, pMP19 plays a role in the virulence of M. plutonius; however, its impact on the virulence varies among strains with different genetic backgrounds.

Development of a novel Trueperella pyogenes-specific PCR assay.

Trueperella pyogenes is an opportunistic pathogen that causes a wide variety of purulent infections. We recently isolated a T. pyogenes strain unable to be identified by the previously reported T. pyogenes pyolysin gene (plo)-specific PCR from the lung of a sheep with astasia. Sequence comparison of plo among representative strains revealed several nucleotide substitutions in the primer-annealing regions. As such substitutions were considered to be a reason for the low PCR specificity, we designed novel primers in conserved regions of plo. Under optimized conditions, the novel primers precisely identified all T. pyogenes strains tested, and no products were generated from any other bacterial strains, suggesting the usefulness of the novel PCR assay for the diagnosis of T. pyogenes infections.

Genotypic diversity of Streptococcus suis and the S. suis-like bacterium Streptococcus ruminantium in ruminants.

Although Streptococcus suis has attracted public attention as a major swine and human pathogen, this bacterium has also been isolated from other animals, including ruminants. However, recent taxonomic studies revealed the existence of other species that were previously identified as S. suis, and some of these isolates were reclassified as the novel species Streptococcus ruminantium. In Japan, biochemically identified S. suis is frequently isolated from diseased ruminants; however, such isolates have not yet been identified accurately, and their aetiological importance in ruminants is unclear. Therefore, to understand the importance of S. suis and S. suis-like bacteria in ruminants, we reclassified S. suis isolates from ruminants according to the updated classification and investigated their genetic diversity. Although both S. suis and S. ruminantium were isolated from healthy and diseased ruminants, most of the isolates from diseased animals were S. ruminantium, implying that S. ruminantium is more likely to be associated with ruminant disease than S. suis. However, the ruminant S. suis and S. ruminantium isolates from diseased animals were classified into diverse genotypes rather than belonging to certain clonal groups. Genome sequence analysis of 20 S. ruminantium isolates provided information about the antibiotic resistance, potential virulence, and serological diversity of this species. We further developed an S. ruminantium-specific PCR assay to aid in the identification of this bacterium. The information obtained and the method established in this study will contribute to the accurate diagnosis of ruminant streptococcal infections.

Complete Genome Sequences of Two Melissococcus plutonius Strains with Different Virulence Profiles, Obtained by PacBio Sequencing.

Melissococcus plutonius attacks honeybee larvae, causing European foulbrood. Based on their virulence toward larvae, M. plutonius isolates were classified into three types, highly virulent, moderately virulent, and avirulent. We herein performed whole-genome sequencing of M. plutonius isolates with different virulence levels to promote an understanding of the pathogenesis of this disease.

High rate misidentification of biochemically determined Streptococcus isolates from swine clinical specimens.

In this study, 22 bacterial isolates from swine necropsy specimens, which were biochemically identified as Streptococcus suis and other Streptococcus species, were re-examined using species-specific PCR for authentic S. suis and 16S rRNA gene sequencing for the verification of the former judge. Identification of S. suis on the basis of biochemical characteristics showed high false-positive (70.6%) and false-negative (60%) rates. The authentic S. suis showed various capsular polysaccharide synthesis gene types, including type 2 that often isolated from human cases. Five of 22 isolates did not even belong to the genus Streptococcus. These results suggested that the misidentification of the causative pathogen in routine veterinary diagnosis could be a substantial obstacle for the control of emerging infectious diseases.

Structure determination of Streptococcus suis serotypes 7 and 8 capsular polysaccharides and assignment of functions of the cps locus genes involved in their biosynthesis.

Streptococcus suis serotypes 7 and 8 are counted among the top six S. suis serotypes causing clinical disease in pigs. Yet, limited information is available on these serotypes. Since S. suis serotyping system is based upon capsular polysaccharide (CPS) antigenicity and the CPS is considered a major virulence factor for encapsulated pathogens, here we determined for the first time the chemical compositions and structures of serotypes 7 and 8 CPSs. Chemical and spectroscopic data gave the following repeating unit sequences: [3)L-Rha(α1-P-2)D-Gal(α1-4)D-GlcA(ß1-3)D-FucNAc4N(α1-]n for serotype 7 and [2)L-Rha(α1-P-4)D-ManNAc(ß1-4)D-Glc(α1-]n for serotype 8. As serotype 8 CPS is identical to Streptococcus pneumoniae type 19F CPS, dot-blot analyses showed a strong reaction of the 19F polysaccharide with reference anti-S. suis serotype 8 rabbit serum. A correlation between S. suis serotypes 7 and 8 sequences and genes of those serotypes' loci encoding putative glycosyltransferases and polymerases responsible for the biosynthesis of the repeating units was tentatively established. Knowledge of CPS structure and composition will contribute to better dissect the role of this bacterial component in the pathogenesis of the disease caused by S. suis serotypes 7 and 8.

Bovine vegetative endocarditis caused by Streptococcus suis.

A 5-month-old crossbred beef steer died after exhibiting astasia. A postmortem examination revealed verrucous endocarditis and numerous renal hemorrhages. Gram-positive bacteria were identified in the necrotic lesions of the verruca and mitral valve via histopathological analysis. Multifocal necrosis and hemorrhage were detected in the renal cortex. Gram-positive cocci isolated from the verruca were identified via biochemical tests and 16S rRNA gene sequence analysis as Streptococcus suis. Serotyping indicated that the S. suis isolates were untypable, following which these isolates were classified as a new sequence type (ST1000) via multi-locus sequence typing. S. suis is an important pathogen of pigs. However, clinical cases in cattle are rare. This report is intended to provide information that may be useful in the diagnosis of streptococcal disease in cattle.