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Cloning and characterization of a dextranase gene (dexS) from Streptococcus suis.

A gene (dexS) coding for a Streptococcus suis capsular type 2 dextranase (DexS) was detected in a recombinant gene library constructed in phage lambda ZapII, and its nucleotide sequence was determined. Sequence comparison showed that the dexS gene product had significant similarities with enzymes which hydrolyze glucose polymers. Moreover, conserved amino acids that are suggested to be part of the active site of the glucosidases are also found in DexS. The dexS gene, adjacent to the gene encoding a S. suis IgG-binding protein, encoded a protein of approximately 62 kDa which exhibited DexS activity.

Amino Acid Sequence↗

Fatal Streptococcus suis meningitis in man.

A case of Streptococcus suis meningitis is observed in a 39-year-old and previously healthy meat factory worker. Neurological recovery was incomplete despite adequate and sustained antimicrobial therapy. Early deafness was demonstrated by brainstem auditory evoked potentials. Death due to aspiration pneumonia, cardiac arrest and subsequent cerebral anoxia occurred late in the course of the illness.

Adult↗

Virulence-associated gene profiling of Streptococcus suis isolates by PCR.

Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.

Animals↗

Comparison of experimental models for Streptococcus suis infection of conventional pigs.

Four different experimental models for Streptococcus suis-induced disease were compared to find a model that closely mimics naturally occurring disease in conventional pigs. Fourteen, 2-week old pigs free of S. suis type 2 were used in 2 experiments. In experiment 1, 3 pigs were inoculated intravenously (IV) and 3 pigs intranasally (IN) with S. suis. Two out of 3 of the IV-inoculated pigs exhibited signs of severe central nervous system disease (CNS) and were euthanized. Streptococcus suis type 2 was isolated from whole blood, joints, and serosal surfaces of both pigs. No clinical signs and no growth of S. suis were detected in the IN-inoculated pigs. In experiment 2, 4 pigs were inoculated IV and another 4 were inoculated IN with the same isolate as in experiment 1. One hour before inoculation the IN-inoculated pigs were given 5 mL of 1% acetic acid intranasally (IN-AA). All the IV-inoculated pigs showed CNS disease and lameness, and 2 of the pigs became severely affected and were euthanized. All the IN-AA inoculated pigs exhibited roughened hair coats and 2 pigs developed severe CNS disease and were euthanized. Streptococcus suis was isolated from the joints and blood of 3 pigs in the IV-inoculated group. Streptococcus suis was isolated from blood of 2 pigs, meninges of 3 pigs, and joints of 1 pig in the IN-AA inoculated group. Natural exposure to S. suis most likely occurs by the intranasal route. The IN-AA model should serve as a good model for S. suis-induced disease, because the natural route of exposure is intranasal and the IN-AA model was effective in inducing disease that mimics what is observed in the field.

Administration, Intranasal↗

[Chemical modification of Streptococcus suis type 2 haemolysin].

The haemolysin of Streptococcus suis type 2 Jiangsu isolate was purified. Effects of protein modification reagents on the haemolysin activity were detected. The haemolysin was not affected by PCMB, succinic anhydride (SA), EDC, and N-AI modification, and it indicated that sulfhudryl groups, amino groups, carboxyl groups and tyrosine residues were not essential to haemolysin activity. The haemolysin activity was significantly decreased after NBS, DEPC, 2, 3-Diacetyl, H2O2 modification and was greatly increased after DTT modification. The results indicated that tryptophane residues, histidine residues, arginine residues and disulfides groups seemed to be essential to the haemolysin activity.

Bacterial Proteins↗

Conjugation of antibiotic resistance in Streptococcus suis.

Forty-eight clinical isolates of Streptococcus suis were examined for antibiotic sensitivity and the presence of plasmid DNA. It was determined that isolates from this study showed a substantial increase in resistance to erythromycin (ery), clindamycin, and tetracycline (tet) compared to a similar study conducted five years earlier. Eleven of the 48 isolates contained plasmid DNA as revealed by DNA isolation and gel electrophoresis. Plasmid DNA from four strains resistant to the above three antibiotics was tested for the ability to transform an antibiotic sensitive recipient. No transformation of antibiotic resistance could be demonstrated. In other experiments, the above four strains, along with four plasmid-negative triply resistant strains were tested for the ability to transfer tet or ery resistance to tet and ery sensitive recipients by conjugation. In each mating, antibiotic resistance was transferred at frequencies averaging 2.4 x 10(-6) recombinants/recipient for ery and 3.4 x 10(-6) recombinants/recipient for tet resistance. DNA from each clinical specimen, as well as the recombinants mentioned above was probed with tn916. Autoradiographs revealed that several clinical isolates and recombinants bound the probe. It is concluded that conjugation of antibiotic resistance in these clinical strains is possibly mediated by a transposon similar to tn916.

Animals↗

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes.

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.

Amino Acid Sequence↗

Pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to Streptococcus suis infection.

Eighty 3-week-old crossbred pigs were randomly assigned to six groups (13-14 pigs/group). Group 1 pigs served as uninoculated controls, group 2 pigs were inoculated intranasally (i.n.) with Streptococcus suis serotype 2, group 3 pigs were inoculated i.n. with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine, group 4 pigs were inoculated i.n. with the same vaccine and with S. suis, group 5 pigs were inoculated i.n. with VR-2385 (a high-virulence strain of PRRSV), and group 6 pigs were inoculated i.n. with VR-2385 and S. suis. Pigs exposed to both PRRSV and S. suis were inoculated with PRRSV 7 days prior to S. suis inoculation. The pigs were 26 days old when inoculated with S. suis. Respiratory disease was significantly more severe in groups 5 and 6. Mortality rate was the highest in group 6 (87.5%). This rate was significantly higher than that observed in all other groups except group 4 (37.5%). The mortality rate in group 2, inoculated with S. suis alone, was 14.3%. No pigs from groups 1, 3, or 5 died prior to the scheduled necropsies at 10 and 28 days postinoculation with PRRSV (DPI). To study the effect of PRRSV and/or S. suis on pulmonary clearance by pulmonary intravascular macrophages, six pigs from each group were intravenously infused with 3% copper phthalocyanine tetrasulfonic acid in saline prior to necropsy at 10 DPI. Mean copper levels in the lungs of pigs in groups 2, 5, and 6 were significantly lower than those in control pigs. The mean percentage of lung tissue grossly affected by pneumonia at 10 DPI was 0%, 1%, 0%, 3%, 64%, and 62% for groups 1-6, respectively. Both gross and microscopic interstitial pneumonia lesions were significantly more severe in the VR2385-inoculated groups (5 and 6). PRRSV was isolated from bronchoalveolar lavage fluid collected at necropsy from 100% of the pigs in groups 5 and 6, 71.4% of pigs in group 4, 38.5% of pigs in group 3, and none of the pigs in groups 1 or 2. Streptococcus suis serotype 2 was cultured from the internal tissues of 7.7%, 28.6%, and 78.6% of the pigs in groups 2, 4, and 6, respectively. Streptococcus suis serotype 2 was isolated from whole blood at necropsy from 7.7%, 35.7%, and 78.6% of pigs in groups 2, 4, and 6, respectively. Significantly more pigs in group 6 had S. suis isolated from whole blood and internal tissues. In summary, both high-virulence PRRSV and S. suis decreased copper clearance, and the incidence of isolation of S. suis and PRRSV was higher in dually inoculated pigs. PRRSV-induced suppression of pulmonary intravascular macrophage function may in part explain PRRSV-associated increased susceptibility to S. suis infection.

Administration, Intranasal↗

Streptococcus suis meningitis with ventriculoperitoneal shunt infection and spondylodiscitis.

Streptococcus suis is a zoonotic pathogen which causes meningitis, bacteremia, and endocarditis in pigs. Human infection is rare and often presents as meningitis with the sequela of permanent deafness and endocarditis. Previous cases were reported from pig-rearing countries such as Holland and Hong Kong. We report a 55-year-old bedridden man with S. suis meningitis complicated with ventriculoperitoneal shunt infection and lumbar spine spondylodiscitis. He presented with fever, delirium, neck stiffness, lower leg weakness and sudden onset hearing loss for several days. He was successfully treated with intravenous antibiotics, ventriculoperitoneal shunt replacement, lumbar spinal laminotomy and discectomy. Cerebrospinal fluid culture initially misidentified the organism as Streptococcus acidominimus, and S. suis was later identified by 16S rRNA sequencing. Misidentification of the microbiological findings may lead to a failure to correctly diagnose this disease. S. suis meningitis should be included in the differential diagnosis of patients with meningitis and sudden hearing loss.

Anti-Bacterial Agents↗

Description of 14 new capsular types of Streptococcus suis.

Fourteen new capsular types of Streptococcus suis (types 9 to 22) are described. All reference strains are morphologically and biochemically similar to types previously described. Reference strain types 9 to 13, 15, 16, and 22 were isolated from diseased pigs, whereas types 17 to 19 and 21 came from clinically healthy pigs; type 14 was isolated from a human case of meningitis, and type 20 was isolated from a diseased calf. The group T streptococcus of de Moor has been included in the typing system as type 15. Two-way cross-reactions between types 6 and 16 and a one-way cross-reaction between types 2 and 22 have been demonstrated. In addition, several cross-reactions probably not due to capsular material were detected among different types by using the coagglutination test. This test should not be used alone; weak or multiple positive reactions must be confirmed by the capsular reaction test or the capillary precipitation test.

Agglutination Tests↗

Occupational exposure to Streptococcus suis type 2.

Antibody titres to Streptococcus suis type 2 were measured with an enzyme linked immunosorbent assay (ELISA) in four occupational groups in New Zealand. No veterinary students, 9% of dairy farmers, 10% of meat inspectors and 21% of pig farmers were seropositive to S. suis type 2. The development of antibody to S. suis type 2 was associated with occupational contact with pigs or their meat products. Subclinical infection with S. suis type 2 appears to occur in humans and the antibody produced is of only short duration. The annual incidence of subclinical infection and seroconversion in pig farmers may approach 28%. Thus S. suis type 2 may be one of the most infectious potentially zoonotic agents present in New Zealand, although very rarely resulting in clinical disease.

Animal Husbandry↗

Factors affecting the survival of Streptococcus suis type 2.

The survival of Streptococcus suis type 2 was assessed in experimentally inoculated faeces and dust stored at 0, 9 and 22 to 25 degrees C. The organism survived in faeces for 104 days at 0 degrees C, up to 10 days at 9 degrees C and up to eight days at 22 to 25 degrees C. It survived in dust for up to 54 days at 0 degrees C and up to 25 days at 9 degrees C but could not be isolated from dust stored at room temperature for 24 hours. The organism survived at 4 degrees C in nutrient medium for up to nine months but in distilled water for only one to two weeks. At 50 degrees C it survived in water or broth for up to two hours but at 60 degrees C it only survived for 10 minutes. The organism was rapidly inactivated by disinfectants and cleansers, commonly used on farms and in laboratories, at concentrations less than those recommended for use by the manufacturers.

Animals↗

Experimental infection of specific pathogen free piglets with French strains of Streptococcus suis capsular type 2.

A standardized model of Streptococcus suis type 2 infection in specific-pathogen-free piglets, housed in high-security barns, was used to compare the virulence of 3 French field strains of S. suis serotype 2 isolated from tonsils of a healthy pig (strain 65) or from diseased pigs (meningitis, strain 166', or septicemia, strain 24). In one of the 2 trials, 7-week-old pigs, in 3 groups of 8, were inoculated intravenously with 2 x 10(8) colony-forming units of S. suis type 2. In each group, 1 uninfected animal was a sentinel. Eight animals were also used as negative control group. The experiment was repeated under similar conditions with strains 65 and 166'. Virulence differed markedly among these S. suis strains when clinical signs, zootechnical performances, lesions, and bacteriological data were analyzed. Strain 65 did not induce clinical signs in inoculated pigs. In contrast, pigs infected with the other 2 strains exhibited clinical signs and typical lesions of S. suis type 2 infections. Differences in virulence were also observed between the 2 virulent strains. Sentinel animals exhibited the same manifestations as those recorded in inoculated piglets. Results were similar in the second trial, indicating that under the present experimental conditions, results were reproducible. The standardized conditions described in this study could be a useful tool to further study about the S. suis infection.

Animals↗

Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system.

Streptococcus suis is an important swine and human pathogen. Inflammation, a hallmark of S. suis infection, is thought to be responsible for most clinical signs of meningitis, septicaemia and sudden death. In this work, using a porcine whole blood model, S. suis serotype 2 was shown to trigger the release of several pro-inflammatory cytokines as evaluated by reverse transcriptase-PCR and enzyme-linked immunosorbent assay. Although individual variations were observed among different S. suis strains, no correlations were observed between the strain origin/phenotype and cytokine levels. Live bacteria induced higher tumour necrosis factor alpha, interleukin-1 beta (IL-1beta) and IL-6 levels than did heat-killed bacteria. In contrast, heat-killed bacteria stimulated higher levels of IL-8 and monocyte chemotactic protein one (MCP-1). The bacterial cell wall was observed to be the major cytokine-inducting components, whereas capsule expression was important for MCP-1 activation. The presence of specific antibodies suppressed bacterial growth resulting in significantly reduced levels of cytokine production. Thus, antibody-mediated bacterial phagocytosis combined with suppressed inflammation may be beneficial for infection control strategies. We provide first evidence of S.suis-induction of pro-inflammatory swine cytokines and demonstrate the strength and relevance of the whole blood culture systems in the investigation of S. suis modulation of cytokine production.

Animals↗

Human infective endocarditis caused by Streptococcus suis serotype 2.

Human infective endocarditis due to Streptococcus suis occurs infrequently. The case of a 43-year-old male who sustained life-threatening infective endocarditis is reported. Since growth of S. suis on commonly used sheep blood agar plates resembles viridans streptococci, diagnosis of S. suis constitutes a diagnostic pitfall. Identification was accomplished by using molecular methods.

Adult↗

Detection and molecular typing of Streptococcus suis in tonsils from live pigs in France.

Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.

Animals↗

Streptococcus suis serotype 2 binding to extracellular matrix proteins.

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.

Animals↗