-
[show abstract]
[hide abstract]
ABSTRACT: Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH2PO4 to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na(+)/H(+) antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.
Applied and environmental microbiology 04/2013; · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Streptococcus suis strains are classified into 35 serotypes on the basis of the antigenicity of their capsular polysaccharides (CPs). CP synthesis genes are known to be clustered on the chromosome (cps gene cluster). The entire cps gene clusters of S. suis have so far been sequenced in 15 serotypes and found to be located between orfZ and aroA. In this study, to provide comprehensive information about S. suis CPs, we sequenced the entire cps gene clusters of the remaining serotypes and analyzed the complete set of S. suis cps gene clusters. Among the 35 cps gene clusters, 22 were located between orfZ and aroA, whereas the other 13 were flanked by other gene(s) on the chromosomes, and the chromosomal locus was classified into five patterns. By clustering analysis, the predicted products of cps genes found in the 35 serotypes were assigned into 291 homology groups, and all serotypes possessed a serotype-specific gene, except for serotypes 1, 2, 1/2 and 14. Because of the presence of genes encoding flippase (wzx) and polymerase (wzy), CPs of all serotypes were thought to be synthesized by the Wzx/Wzy pathway. Our data also implied the possibility of the transfer of the entire or partial cps gene clusters among S. suis strains, as well as the influence of spontaneous mutations in a single or a few genes on the antigenicity of some serotypes. Accumulation of these gene transfers and small-scale mutations may have generated the antigenic diversity of S. suis CPs.
Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
-
The Veterinary record. 12/2012; 171(24):630.
-
[show abstract]
[hide abstract]
ABSTRACT: Bifidobacteria were isolated from the intestinal tract of the Japanese honeybee, Apis cerana japonica, and investigated for potential application as a probiotic agent against Melissococcus plutonius, the causal agent of European foulbrood (EFB), based on the findings of in vitro inhibition assays. A total of 11 bifidobacteria strains (designated as AcjBF1-AcjBF11) were isolated using a culture-dependent method and their 16S rRNA gene sequences were analyzed. The AcjBF isolates belonged to three distinct bifidobacterial phylotypes that were similar to those found in the European honeybee, Apis mellifera. Although the Japanese and European honeybees are distinct species with different traits and habits, the observation that they share highly similar bifidobacterial phylotypes suggests that bifidobacteria are conserved among honeybee species. Despite having extremely high 16S rRNA gene sequence similarities, the AcjBF isolates had markedly different carbohydrate fermentation profiles. In addition, in vitro growth inhibition assays revealed that the cell-free supernatants of all AcjBF isolates exhibited antagonistic effects on M. plutonius growth. These results indicated that the bifidobacteria isolated from the gut of Japanese honeybee could potentially be employed as a new biological agent to control EFB.
Journal of Invertebrate Pathology 10/2012; · 2.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Streptococcus suis is an important pathogen for both swine and humans. In this study, we genotyped 105 S. suis isolates from porcine endocarditis in East Japan on the basis of profiles of capsular serotype-specific, virulence-associated, and pilus-associated genes. The most common genotype was cps2J/mrp+/epf-/sly-/sbp2-/sep1-/sgp1+ (76.19%), followed by nt(non-typeable)/mrp+/epf-/sly-/sbp2-/sep1-/sgp1+ (7.62%) and cps2J/mrp+/epf+/sly+/sbp2+/sep1-/sgp1- (7.62%). The representative isolates of mrp+/epf-/sly-/sbp2-/sep1-/sgp1+ were classified into ST28 complex, a clonal complex previously referred to as ST27 complex, whereas those of mrp+/epf+/sly+/sbp2+/sep1-/sgp1-were classified into ST1 complex by multilocus sequence typing. Because the majority of human clinical isolates were assigned to ST1 and ST28 complexes, most isolates from porcine endocarditis investigated in this study may have the potential to cause S. suis infection in humans.
Journal of Veterinary Medical Science 08/2012; · 0.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report the complete genome sequence of Melissococcus plutonius DAT561, which is a causative agent of European foulbrood. M. plutonius DAT561 is a representative of nonfastidious strains isolated in Japan. The addition of potassium phosphate was not required for normal growth, unlike for typical M. plutonius strain/isolates.
Journal of bacteriology 06/2012; 194(11):3014. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.
Microbes and Infection 04/2012; 14(11):941-50. · 3.10 Impact Factor
-
Rie Arai,
Kiyoshi Tominaga,
Meihua Wu,
Masatoshi Okura,
Kazutomo Ito,
Naomi Okamura,
Hidetaka Onishi,
Makoto Osaki,
Yuya Sugimura,
Mikio Yoshiyama, Daisuke Takamatsu
[show abstract]
[hide abstract]
ABSTRACT: European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.
PLoS ONE 01/2012; 7(3):e33708. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Emerging Infectious Diseases 12/2011; 17(12):2239-44. · 6.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Streptococcus suis, particularly serotype 2, is a pathogen of both pigs and humans associated with a wide range of diseases, including meningitis, septicaemia and endocarditis. Among the genes in the capsular polysaccharide biosynthesis (cps) locus, cps2J exists only in the serotype 2 and 1/2 strains; therefore, cps2J-positive strains are suspected to have capsules of serotype 2 or 1/2. Coagglutination using antiserotype 1 and antiserotype 2 sera and/or transmission electron microscopy analysis of 288 cps2J-positive isolates from pigs showed that 32 (100 %) isolates from meningitis were encapsulated, whereas 86 (34 %) of 256 isolates from endocarditis were unencapsulated, indicating that capsule loss often occurred in the isolates from endocarditis. To investigate the genetic backgrounds, we randomly selected 43 unencapsulated isolates and analysed their cps loci by PCR scanning. Among them, 8 and 10 isolates apparently had deletions and insertions, respectively, in cps loci. In addition, a representative unencapsulated isolate and an unencapsulated strain showed adherence to porcine and human platelets, a major virulence determinant for infective endocarditis, to a significantly greater extent than the encapsulated strains. Although the capsule is considered to be an important virulence factor in S. suis, these results suggest that loss of capsular production is beneficial to S. suis in the course of infective endocarditis.
Journal of Medical Microbiology 07/2011; 60(Pt 11):1669-76. · 2.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report the first completely annotated genome sequence of Melissococcus plutonius ATCC 35311. M. plutonius is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.
Journal of bacteriology 05/2011; 193(15):4029-30. · 3.94 Impact Factor
-
Daisuke Takamatsu
Nippon Saikingaku Zasshi 02/2011; 66(1):7-21.
-
[show abstract]
[hide abstract]
ABSTRACT: Gram-positive pili are composed of covalently bound pilin subunits whose assembly is mediated via a pilus-specific sortase(s). Major subunits constitute the pilus backbone and are therefore essential for pilus formation. Minor subunits are also incorporated into the pilus, but they are considered to be dispensable for backbone formation. The srtG cluster is one of the putative pilus gene clusters identified in the major swine pathogen Streptococcus suis. It consists of one sortase gene (srtG) and two putative pilin subunit genes (sgp1 and sgp2). In this study, by constructing mutants for each of the genes in the cluster and by both immunoblotting and immunogold electron microscopic analysis with antibodies against Sgp1 and Sgp2, we found that the srtG cluster mediates the expression of pilus-like structures in S. suis strain 89/1591. In this pilus, Sgp1 forms the backbone, whereas Sgp2 is incorporated as the minor subunit. In accordance with the current model of pilus assembly by Gram-positive organisms, the major subunit Sgp1 was indispensable for backbone formation and the cognate sortase SrtG mediated the polymerization of both subunits. However, unlike other well-characterized Gram-positive bacterial pili, the minor subunit Sgp2 was required for polymerization of the major subunit Sgp1. Because Sgp2 homologues are encoded in several other Gram-positive bacterial pilus gene clusters, in some types of pili, minor pilin subunits may contribute to backbone formation by a novel mechanism.
Journal of bacteriology 02/2011; 193(4):822-31. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5' end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection.
PLoS ONE 01/2010; 5(1):e8426. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A stillborn calf at 259 days of gestation was examined. The dam showed no clinical signs of disease, and the stillbirth was occurred sporadically and were characterized by focal necrosis surrounded by infiltration of the cells such as macrophages and multinucleated giant cells. The lesions were observed systemically. The hyphae were visible by Grocott's stain and they were positive by immunohistochemical stain using serum of a rabbit immunized with the isolated organism. The isolated bacteria were determined to be Nocardia farcinica by bacteriological and molecular analysis and we confirmed that the stillbirth was caused by infection with and proliferation of Nocardia farcinica. This is the first report of a bovine stillbirth caused by this organism.
Journal of Veterinary Medical Science 12/2009; 71(12):1665-8. · 0.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A-L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.
Veterinary Microbiology 03/2009; 138(1-2):132-9. · 3.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.
Molecular Microbiology 01/2009; 70(5):1120-35. · 5.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We generated by allelic replacement a DeltadltA mutant of a virulent Streptococcus suis serotype 2 field strain and evaluated the contribution of lipoteichoic acid (LTA) d-alanylation to the virulence traits of this swine pathogen and zoonotic agent. The absence of LTA D-alanylation resulted in increased susceptibility to the action of cationic antimicrobial peptides. In addition, and in contrast to the wild-type strain, the DeltadltA mutant was efficiently killed by porcine neutrophils and showed diminished adherence to and invasion of porcine brain microvascular endothelial cells. Finally, the DeltadltA mutant was attenuated in both the CD1 mouse and porcine models of infection, probably reflecting a decreased ability to escape immune clearance mechanisms and an impaired capacity to move across host barriers. The results of this study suggest that LTA D-alanylation is an important factor in S. suis virulence.
Infection and immunity 09/2008; 76(8):3587-94. · 4.21 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: SraP, a platelet-binding surface protein of Staphylococcus aureus, is involved in the pathogenesis of infective endocarditis. In this study, we investigated the importance of SraP in the pathogenesis of bovine mastitis. By means of PCR, sraP was detected in all the isolates tested from bovine bulk milk and humans. However, SraP was not expressed on the cell surface in half of the bovine isolates. Moreover, disruption of sraP did not affect the ability of S. aureus to adhere to cultured bovine mammary epithelial cells. These results suggest that SraP does not seem to be an important factor for S. aureus to adhere to the bovine mammary epithelia.
Journal of Veterinary Medical Science 08/2008; 70(7):735-8. · 0.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Serum opacity factor of Streptococcus suis (OFS) has recently been identified as a virulence determinant of an S. suis strain. In this study, we investigated the prevalence and variations of the ofs gene among 108 S. suis isolates from diseased and healthy pigs, and human patients. PCR screening and sequencing analysis showed that besides the ofs gene reported already (designated type 1), there were three allelic variants of ofs (designated types 2 to 4). Type-1 and type-2 ofs genes were expected to encode functional OFS, and SDS extracts of the isolates with type-1 ofs and type-2 ofs opacified horse serum. Culture supernatants of the isolates with type-2 ofs also showed strong serum opacification activity. In contrast, type-3 ofs was interrupted by a point mutation and type-4 ofs was disrupted by either insertion of an IS element or genetic rearrangement, and therefore the SDS extracts and culture supernatants of the isolates with type-3 ofs and type-4 ofs did not show serum opacification activity. Regardless of their origins, approximately 30 % of the isolates possessed functional OFSs, although type-2 ofs was found only in three isolates from healthy pigs. Multilocus sequence typing analysis showed that most of the isolates with type-1 ofs belonged to the sequence type (ST)1 complex, and most of the isolates with type-3 ofs and type-4 ofs belonged to the ST27 complex. The isolates with type-2 ofs were not assigned to a major ST complex. These results suggest that type-1 OFS contributes to the virulence of a limited number of S. suis isolates, i.e. those of the ST1 complex type, whereas other S. suis may not possess this category of virulence factor; the importance of type-2 OFS is obscure.
Journal of Medical Microbiology 05/2008; 57(Pt 4):488-94. · 2.50 Impact Factor
