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We studied the occurrence of Streptococcus group G in throat cultures obtained from school children of poor socioeconomic status and in sick children brought to our hospital. The results revealed a high occurrence of group G Streptococci (44% and 91.5% respectively) in cases positive for beta-hemolytic Streptococci.
The content of protein and carbohydrate polymers was estimated in the cell wall of Streptococcus, group A, type 29. A method was developed for analysing peptidoglycane in a polysaccharide-peptidoglycane complex after the prior oxidation by sodium periodate. It was found that the cell wall peptidoglycane bears two carbohydrate and three amino acid residues, i. e. N-acetylglucosamin, muramic acid, glutamic acid, alanine and lysine, in the ratio 1:1:1:4:1, respectively. The data on the cell wall composition prior to and after its oxidation with sodium periodate are given, and the ratio between the main structural components is determined: proteins (60% mol), polysaccharide (23% mol), peptidoglycane (17% mol).
PURPOSE: To report the clinical findings, treatment, and outcomes of four cases of beta-streptococcus Group G (BHS-G) ocular infection. METHODS: The medical and microbiologic records of four cases of BHS-G ocular infection were retrospectively reviewed. RESULTS: Two cases of BHS-G endophthalmitis and two cases of BHS-G keratitis were recorded. Three patients developed fulminant infection within 12 hours of the onset of symptoms. One patient's history was incomplete. One patient developed endophthalmitis from a contaminated donor button; another following cataract surgery. One developed keratitis in a keratoplasty suture tract; and another patient developed a corneal abscess after being struck with a tree branch. The patient with the contaminated donor button developed overwhelming endophthalmitis resulting in no light perception vision, severe pain, and evisceration. The postoperative cataract patient developed a purulent endophthalmitis and is still hypotonus with light perception vision. The second keratitis patient developed a significant suture abscess with marked stromal loss but eventually healed. The traumatic keratitis patient developed a large ulcer with hypopyon and descemetocele but was lost to follow-up. CONCLUSIONS: This is the first report of a series of BHS-G ocular infections. The ocular infections were characterized by rapid onset, extreme inflammation, and--despite in vitro antibiotic sensitivity--a poor or sluggish response to antibiotic therapy.
The allyl glycosides of a tri-, penta- and hexasaccharide corresponding to the Streptococcus Group A cell-wall polysaccharide were coupled to solid or soluble supports to give immunoaffinity columns and neoglycoproteins, respectively. Cysteamine hydrochloride was added to the allyl glycosides and the resulting cysteamine adducts were used for subsequent coupling to linkers via the amine functionality. The tri- and penta-saccharide cysteamine adducts were coupled directly to the azalactone-derivatized 3M Emphase Biosupport Medium AB 1 to yield two affinity columns. The penta- and hexa- saccharides were coupled to bovine serum albumin or ovalbumin via the conjugate addition of the epsilon-amino groups of lysines on the proteins with the N-acryloylated sugars or the oligosaccharide-squarate adducts, derived in turn from the cysteamine adducts. The efficiency of the above methods is compared.
In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.
A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococcus Group A is described. The strategy relies on the preparation of a key linear trisaccharide unit beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap which has previously resisted our efforts. The trisaccharide functions both as a glycosyl acceptor and donor to give an elusive hexasaccharide. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hitherto unknown hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high-resolution 1H and 13C NMR spectroscopy is also described.
Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.
Our investigation concerns 14 streptococcus D sepsis neonates, thirteen of which were collected over a six year period during which eighty-two neonate infections were recorded. This rate slightly higher than generally recorded and may be due to the initial prescription of third generation cephalosporin to the mothers. The clinical, hematological and biological data are not specifically those of group D streptococcus. The issue was unfavourable in 15% of our cases and in 8 to 33% of the recorded cases. The sensitivity to antibiotics varies depending on the species of group D streptococcus. The effectiveness of Amoxicillin, of Mezlocillin and of Ampicillin justifies the initial prescription in association with an aminoglycoside because of the possibility of synergy; cephalosporins are contraindicated as they are inactive on this germ.
This paper describes the use of a protocol for conformational analysis of oligosaccharide structures related to the cell-wall polysaccharide of Streptococcus group A. The polysaccharide features a branched structure with an L-rhamnopyranose (Rhap) backbone consisting of alternating alpha-(1-->2) and alpha-(1-->3) links and D-N-acetylglucosamine (GlcpNAc) residues beta-(1-->3)-connected to alternating rhamnose rings: [formula: see text] Oligomers consisting of three to six residues have been synthesized and nuclear magnetic resonance (NMR) assignments have been made. The protocol for conformational analysis of the solution structure of these oligosaccharides involves experimental and theoretical methods. Two-dimensional NMR spectroscopy methods (TOCSY, ROESY and NOESY) are utilized to obtain chemical shift data and proton-proton distances. These distances are used as constraints in 100 ps molecular dynamics simulations in water using QUANTA and CHARMm. In addition, the dynamics simulations are performed without constraints. ROE build-up curves are computed from the averaged structures of the molecular dynamics simulations using the CROSREL program and compared with the experimental curves. Thus, a refinement of the initial structure may be obtained. The alpha-(1-->2) and the beta-(1-->3) links are unambiguously defined by the observed ROE cross peaks between the A-B',A'-B and C-B,C'-B' residues, respectively. The branch-point of the trisaccharide CBA' is conformationally well-defined. Assignment of the conformation of the B-A linkage (alpha-(1-->3)) was problematic due to TOCSY relay, but could be solved by NOESY and T-ROESY techniques. A conformational model for the polysaccharide is proposed.
Group B streptococcus was isolated from the blood, penile exudate and needle aspirate of cellulitis in a severely ill neonate with a rapidly progressive infection of the scrotum, penis and lower abdominal wall. This case demonstrates the need to evaluate and to manage newborns with scrotal cellulitis differently than older children and adults. A different group of organisms and a greater likelihood for systemic involvement are to be anticipated in newborns.
Convergent syntheses of di-, tri, tetra-, penta-, and hexa-saccharide allyl glycosides corresponding to the beta-hemolytic Streptococcus Group A cell-wall polysaccharide are described. The strategy relies on the preparation of related di- and tri-saccharide building blocks: beta-D-Glc pNAc-(1-3)-alpha-L-Rhap and alpha-L-Rhap-(1-2)-[(beta-D-Glc p NAc-(1-3)]-alpha-L-Rhap, which could be used either as glycosyl donors or acceptors in subsequent glycosylation reactions. The protecting groups were chosen to allow the selective removal of the allyl aglycon to access the intermediate glycosyl donors but also to allow their own removal without affecting the allyl group. The allyl group was intended for use in conjugation of the oligosaccharides to soluble protein carriers or solid supports for the preparation of antigens and immunoadsorbents, respectively.
Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.
Group B Streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-h td) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at a td of 1. 4 h than at a td of 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at a td of 1. 4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.
Group B streptococcus (GBS) is the major cause of bacterial sepsis and meningitis in neonates and poses a significant threat to parturient women. Recently, we identified in GBS the polypeptide PcsB, which is a protein required for cell separation of GBS, and which is also involved in the antibiotic sensitivity of these bacteria. In the present study, the introduction of the pcsB-carrying plasmid pATpcsB into the PcsB-deficient GBS mutant Sep1 restored the phenotype and the antibiotic susceptibility of this strain to that of the GBS wild-type. Although Northern blots revealed a four- to five-fold increased transcription of pcsB in pATpcsB-carrying GBS strains, overexpression of pcsB did not result in higher amounts of PcsB in the cell wall and in the culture supernatant of GBS, indicating regulatory mechanisms that control the translation or secretion of PcsB in these bacteria. In the culture supernatant of mutant Sep1 significant amounts of enolase were identified. As this protein was also present in extracts of cell wall-bound proteins from the GBS wild-type, it can be speculated that GBS can translocate enolase across the cytoplasmic membrane. Northern blot analysis exhibited similar expression of the enolase gene in the GBS strains 6313 and Sep1, indicating that mutant Sep1 is impaired in the anchoring of this protein to its cell wall.
Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal alpha 2-3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of the O-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase gene neuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.
Group B streptococcus (GBS) induces apoptosis in macrophages. Growth conditions minimizing beta-hemolysin expression, such as high glucose, reduce apoptosis. We constructed an isogenic mutant strain of GBS 874391 lacking the beta-hemolysin structural gene cylE and investigated the role that beta-hemolysin plays in apoptosis of J774 macrophages. Viability of macrophages infected with wild-type or cylE GBS was similar and significantly less than that of macrophages infected with GBS grown in high-glucose media. Thus, apoptosis in GBS-infected macrophages is dependent not on beta-hemolysin but on a factor coregulated with beta-hemolysin by glucose.
Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lens-shaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.
Group B Streptococcus (GBS) is an important pathogen responsible for a variety of diseases in newborns and the elderly. A clinical GBS isolate is considered nontypable (NT) when serological methods fail to identify it as one of nine known GBS serotypes. Eight clinical isolates (designated A1-A4, B1-B4) showed PFGE profiles similar to that of a GBS serotype V strain expressing R1, R4 surface proteins. These unique isolates were further characterized by immunologic and genetic methods. Rabbit sera to isolates A1 and A2 reacted weakly with concentrated HCl extracts of A1-A4 isolates, but not with those of B1-B4 isolates. In addition, a type V capsular polysaccharide (CPS) inhibition ELISA revealed that cell wall extracts from isolates A1-A4, but not from B1-B4, expressed low but measurable amounts of type V CPS. Molecular serotyping with PCR analysis showed that all eight isolates contained a type V-specific CPS gene (cpsO) and harboured the gene encoding the surface protein Alp3. Multilocus sequence typing identified isolate A1 as belonging to a new sequence type (ST) designated ST-173, whereas the other seven isolates keyed to ST-1. Sequencing of the 18 genes (17 736 bp) in the cps locus showed that each NT isolate harboured one to three unique polymorphisms, and also identified an IS1381 element in cpsE of the B4 isolate. Collectively, genetic and immunologic analyses revealed that these NT isolates expressing R1, R4 proteins have a genetic profile consistent with that of type V, an emergent, antigenically diverse and increasingly prevalent GBS serotype.