Human and bovine group B streptococci: two distinct populations

Group B streptococci ( Streptococcus agalactiae ) from humans and animals were compared for cultural, biochemical, serological and bacteriocin sensitivity properties. Each isolate possessed the group B carbohydrate antigen, hydrolysed hippurate, and was CAMP test positive. Most human isolates were characterized as bacitracin resistant, pigment producing, haemolytic, and salicin but not lactose utilizing. In contrast bovine isolates were usually bacitracin sensitive, non-pigment producing, non-haemolytic, salicin and lactose utilizing. Isolates from other animals behaved similarly to those from humans. Whereas human isolates belonged to a variety of serotypes and were uniformly sensitive to bacteriocins, bovine isolates showed varying sensitivity to bacteriocins and most belonged to serotype II or were non-typable. We believe these results support the belief that Strep. agalactiae from humans and cattle are separate populations sharing the same group B carbohydrate antigen.     

Vaginal microbiota in healthy pregnant women and prenatal screening of group B streptococci (GBS)

The microbiota of the lower female genital tract was evaluated from vaginal swabs obtained from 623 healthy pregnant women at gestation periods of 35–40 weeks. Isolated and identified microorganisms were expressed as percentages of total samples. As expected, lactobacilli made up the dominant vaginal microbiota (70%). Enterobacteriaceae, mainly Escherichia coli, Klebsiella spp. and Proteus, were present in 38% of the samples, which might reflect the possible contamination of vaginal tract with rectal microorganisms. Candida albicans was present in 10% of healthy pregnant woman assayed. Streptoccocci (Streptococcus sp. and Enterococcus faecalis with 3% and 4%, respectively) and other gram-positive cocci (Staphylococcus sp., 5%), along with other microorgansisms such as Gardnerella vaginalis (5%) and Pseudomonas aeruginosa (2%) may represent a potential infection risk. Streptococcus agalactiae (group B streptococci β-hemolytic, GBS) was detected in 7% of the samples. GBS infection is a leading cause of neonatal morbidity and mortality in the developed world. Furthermore, GBS was often co-isolated with C. albicans (54.5%) in the samples. A complete and detailed evaluation of the vaginal biota swab, with particular attention to the presence of potential pathogens such as GBS, is a preventive strategy that can provide useful information to obstetricians and gynecologist in managing the last days of pregnancy and delivery. Electronic Publication     

Morphological stabilization of capsules of group B streptococci, types Ia, Ib, II, and III, with specific antibody.

Antibody prepared to the type-specific capsular polysaccharides of group B streptococci was used to demonstrate a stabilizing effect on the capsular glycocalyx. This permitted visualization by electron microscopy of the size of the capsule relative to the rest of the bacterial cell, and clear differences in the dimensions of untreated and antibody-treated capsular material were noted. Antibodies produced against group B streptococci types Ia, Ib, II, and III were used to demonstrate morphologically that the organization and extent of the capsular glycocalyx more closely resembles its natural state on stabilization by reaction with specific antibody.     

Genetic heterogeneity of the pathogenic potentials of human and bovine group B streptococci

One-hundred seventy-two B-streptococcal strains of human and bovine origin were analyzed for the presence of 9 genes potentially involved in virulence. Some of genes (glnA, cyl, hylB, scaA andcfb) were revealed in all the strains. However, the presence of others (bca, bac, scpB, lmb) varied from strain to strain. Taken together, 3 and 5 different types of pathogenic potential were found among human and bovine group B streptococci (GBS) strains, respectively, and only one type (bca ⁺ bac scpB⁺ glnA⁺ cyl⁺ hylB⁺ lmb⁺ scaA⁺ cfb⁺) was common for both kinds of strains. We propose that different virulence genes can be involved in the development of infectious processes in humans and animals. A reliable PCR protocol with 3 pairs of primers (for the genesbca, bac andscpB) in the same reaction mixture was developed for the fast identification of the pathogenic potential of GBS. In comparison with the classical immunological methods this procedure displayed higher specificity and sensitivity as well as a shorter time of analysis. It can be recommended for use in the clinical and veterinary practice for studying the epidemiological relationship between the isolates and the ready identification of the clone causing the infection.     

Findings of serotypes of group B streptococci in human and bovine sources.

Five hundred and fifty-five strains of S. agalactiae of human or bovine origin were serologically typed. In human strains, serotype Ia was the most frequent irrespective of the source and kind of cultivation material, but serotype R was very frequent in urine. In bovine strains, one serotype was found as a rule in one stable both in small private and large socialist farms. The reason for such uniformity of serotypes is not known. Monocolonisation is one of the alternatives, but it seems more reasonable to assume that, the most resistant or more invasive strain will predominate in the herd in the course of time.     

Purification and partial characterization of neuraminidase from type III group B streptococci

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.     

Identification of cpsD, a gene essential for type III capsule expression in group B streptococci

We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916ΔE. The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916ΔE insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis. A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor. The galactose–acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.     

Adherence of group B streptococci isolated from man and cattle to human and bovine epithelial cells

Proof of adherence of group B streptococci (GBS) to human and bovine vaginal epithelial cells and to bovine cells of milk cisternae of the mammary gland was employed as a criterion determining the possibility of colonization of these organs with GBS, or as another method of testing the transfer of GBS between man and cattle GBS of both human and animal origin adhered to human epithelial cells in a similar way On the other hand, a significantly stronger adherence of bovine GBS to vaginal epithelial cells and cells of milk cisternae of cattle was found than of human GBS Thus the direction of colonization man—animal is more prob able than the opposite way Neither in animal nor in human strains a correlation between the equipment of strains with type antigens and intensity of adherence could be found     

Correlation between the virulence of streptococci for mice and their IgG FcR activity in in vivo experiments

The immunological analysis of 24 spontaneous Strr, Rifr and Kanr mutants of streptococcal strain IP, highly virulent for mice and capable of binding polyclonal human IgG (IgG FcR+), was made. The characteristic feature of all these mutants was decreased virulence, restored after their passage in vivo. 23 mutants were capable of binding polyclonal IgG; one Strr mutant had no such capacity, but acquired it, together with an increase in virulence, after its passage in vivo. When stored in meat-peptone agar without antibiotics, 5 out of 10 Strr mutants lost their capacity for binding polyclonal human IgG. After passage in vivo they regained this property simultaneously with virulence.     

Role of L-ficolin/mannose-binding lectin-associated serine protease complexes in the opsonophagocytosis of type III group B streptococci

Serotype III group B streptococci (GBS) are a common cause of neonatal sepsis and meningitis. Although deficiency in maternal capsular polysaccharide (CPS)-specific IgG correlates with susceptibility of neonates to the GBS infection, serum deficient in CPS-specific IgG mediates significant opsonophagocytosis. This IgG-independent opsonophagocytosis requires activation of the complement pathway, a process requiring the presence of both Ca(2+) and Mg(2+), and is significantly reduced by chelating Ca(2+) with EGTA. In these studies, we defined a role of L-ficolin/mannose-binding lectin-associated serine protease (MASP) complexes in Ca(2+)-dependent, Ab-independent opsonophagocytosis of serotype III GBS. Incubation of GBS with affinity-purified L-ficolin/MASP complexes and C1q-depleted serum deficient in CPS-specific Ab supported opsonophagocytic killing, and this killing was inhibited by fluid-phase N-acetylglucosamine, the ligand for L-ficolin. Binding of L-ficolin was proportional to the CPS content of individual strains, and opsonophagocytic killing and C4 activation were inhibited by fluid-phase CPS, suggesting that L-ficolin binds to CPS. Sialic acid is known to inhibit alternative complement pathway activation, and, as expected, the bactericidal index (percentage of bacteria killed) for individual strains was inversely proportional to the sialic acid content of the CPS, and L-ficolin-initiated opsonophagocytic killing was significantly increased by addition of CPS-specific IgG2, which increased activation of the alternative pathway. We conclude that binding of L-ficolin/MASP complexes to the CPS generates C3 convertase C4b2a, which deposits C3b on GBS. C3b deposited by this lectin pathway forms alternative pathway C3 convertase C3bBb whose activity is enhanced by CPS-specific IgG2, leading to increased opsonophagocytic killing by further deposition of C3b on the GBS.     

Rapid method for the detection of group B streptococci from human sources

A modification of the classical CAMP test has been devised for the rapid detection of streptococci of serological Group B from human sources. The method was compared with detection based on the development of orange pigmented colonies on a starch-based medium and with detection by conventional methods. In a survey of vaginal carriage of Group B streptococci in parturient women, the modified CAMP test detected a carriage rate of 13.09%, the starch-based, pigment enhancing medium, 5.76% and the conventional methods, 8.38%. It proved to be particularly useful for detecting the organisms in the presence of other bacteria.     

Three different types of organization of the vir regulon in group A streptococci

Summary The DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation vir regulon for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated large vir regulon (LVR), consists of virR -fcrA(-like) emm - ennX(-like) - scpA. The second, designated small vir regulon (SVR), contains virR - emm- scpA, and the last, designated unusual vir regulon (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3 ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.     

贺兰山岩羊（Pseudois nayaur）集群特征的季节变化

2004年11月～2005年10月,在贺兰山对岩羊(Pseudois nayaur)的集群行为进行了研究,将其集群类型划分为雌性群、雄性群、雌雄群、母仔群、混合群和独羊6种类型.共观察到岩羊1 023群次,计4 866只次,平均群大小为(4.86±2.54)只,最大的群为51只,最小的为独羊.其中,母仔群459群(44.87%)为最多的集群类型,其余为混合群(20.72%)、雄性群(14.86%)、独羊(9.09%)、雌雄群(5.57%)、雌性群(4.89%).母仔群出现的频次在4个季节均最高,除母仔群外,春季雄性群出现的频次最高,而夏、秋、冬季都是混合群出现的频次最高,不同类型集群出现频次的季节间差异极显著.在4个季节中都以2～5只的群居多,其出现的频次占各季节群数50%以上,不同季节群大小差异极显著,而不同集群类型群大小季节间不存在显著差异.除独羊外,不同季节混合群大小差异极显著,母仔群、雌性群大小差异显著,而雄性群、雌雄群大小无显著差异.研究结果显示,贺兰山岩羊集小群是其显著特点,随着季节的变化,其集群类型、集群大小均会发生一定的变化.     

Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America)

Isolates of Toxoplasma gondii, which is responsible for a wide range of clinical manifestations are grouped into three clonal lineages of different virulence in mice. However, it is not clear whether this genotypic pattern is associated with the clinical profile of the disease in humans nor is the geographical distribution of the genotypes known. This is mainly due to difficulties in obtaining parasitic DNA from patients. The available data are therefore limited and originate from acute or congenital infections or from animals. A non-invasive assay is needed to address issues of strain type, geographical distribution and severity of clinical toxoplasmosis. To serotype T. gondii strains, we have developed an enzyme-linked immunosorbent assay (ELISA) that uses polymorphic polypeptides specific to the three clonal lineages and derived from two dense granule antigens, GRA5 and GRA6. Two hundred and fifty-two sera from chronically infected pregnant women from three different European countries and Colombia were investigated. The analysis of genotype-specific antibody response showed a homogeneous type II distribution in the European samples compared with types I and III but no type II in the Colombian population. Our data concord with those obtained from the genotyping of other isolates from Europe and South America. We demonstrated that, despite some limitation due to antigen and/or antibody specificity, serotyping is a promising assay to investigate the relationship between type of strain and severity of the disease.     

A comparison of four methods for the serotyping of group B streptococci.

Group B streptococci are implicated in a wide range of clinical conditions in human adults and neonates. The Group is subdivided into five serotypes Ia, Ib, Ic, II and III, which are differentiated on the basis of capsular polysaccharides. In the interests of epidemiology and efficiency a cheap, rapid method which is easily interpreted would be advantageous. In this study four methods of serotyping, namely, counter-immunoelectrophoresis (CIEP), microimmunodiffusion (MID), coagglutination (COA), and the Lancefield capillary precipitin (CP) test were compared in terms of ease of operation and interpretation, accuracy and rapidity. Todd Hewitt Broth (THB) cultures and acid extracts of the group B streptococcal strains were used as antigens for these methods. It was concluded that COA using THB cultures allows cheap and rapid screening for presumptive serotyping, having a 93-96% correlation with the CP test. MID gives an accurate (100% correlation with the CP test) and unambiguous confirmatory diagnosis of serotype.     

The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.