Rapid detection of group B streptococcal antigen by monoclonal antibody sandwich enzyme assay.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Infants at greatest risk to develop invasive disease are delivered to women colonized with GBS in their birth canals and lacking immunity to the colonizing serotype. We have investigated the sensitivity and specificity of a recently developed monoclonal antibody sandwich enzyme immunoassay for detection of GBS antigen. The sandwich enzyme immunoassay detected types II and III GBS at a concentration of 5 X 10(4) CFU/ml and types Ia and Ib GBS at 5 X 10(5) CFU/ml. No cross-reactions were noted when each of the GBS serotypes was reacted with antibodies of differing serotypes specificities. Type III GBS native antigen was detected at a concentration of 1 ng/ml. The sandwich enzyme assay is more sensitive than other methods currently in use for rapid detection of GBS and is serotype specific. This assay system should prove useful for the detection of GBS colonization during labor and for identification of neonates with invasive disease.     

Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and alpha.

Encapsulated bacteria cause some of the most common diseases in humans. Although the polysaccharide capsules of these pathogens have attracted the most attention with regard to vaccine development, recent evidence suggests that bacterial surface proteins may also be used to confer protective immunity. We have analyzed this possibility in group B streptococcus (GBS), an encapsulated bacterium that is the major cause of invasive bacterial disease in the neonatal period. Previous work has shown that the majority of GBS strains causing invasive infections express the Rib protein, and that most strains lacking Rib express a protein designated alpha. Here we report that active immunization with highly purified preparations of Rib or alpha protected mice against lethal infection with strains expressing the corresponding protein. Vaccination with the Rib protein protected against two strains of capsular type III and two strains of type II, and vaccination with the alpha protein protected against one strain of type II and one strain of type Ib. The mice vaccinated with Rib or alpha showed a good immunoglobulin G response to the immunogen. These data suggest that a vaccine against GBS disease may be based on cell surface proteins and support the notion that proteins may be used for immunization against encapsulated bacteria.     

Low Prevalence of the Immunoglobulin-A-Binding β Antigen of the C Protein Among Streptococcus agalactiae Isolates Causing Neonatal Sepsis

 Streptococcus agalactiae (Group B streptococcus, GBS) is the most important pathogen causing neonatal sepsis. The role of bacterial proteins contributing to pathogenicity in GBS infections has not yet been clearly determined, but the C protein complex has been suggested to be involved in both virulence and protective immunity. The aim of this study was to assess the prevalence of GBS strains bearing the gene encoding for the β antigen of the C protein among clinical isolates from 68 neonates with sepsis, 45 newborns colonized without clinical signs of infection, and 50 isolates from pregnant women. The prevalence of the β antigen gene in all three groups was low (22% vs. 19% vs. 22%), and the differences between groups were not statistically significant. Clinical characteristics and cytokine plasma levels did not differ between septic patients with β antigen-positive and -negative strains. The β-antigen gene was not found among serotype III isolates, which accounted for roughly half of all the strains isolated. Thus, polymerase chain reaction (PCR) analysis based on the β antigen gene seems not helpful for distinguishing invasive from colonizing GBS strains. A vaccine based on peptide antigens from the β antigen of the C protein would most probably not provide protection against the majority of GBS isolates. When analyzing the PCR products of the C protein β antigen gene by DNA sequencing, a genetic heterogeneity was observed, revealing small repetitive genetic elements within the amplified fragment, an observation that should be studied further.     

Genetic relatedness among extended-spectrum beta-lactamase-producing Klebsiella pneumoniae outbreak isolates associated with colonization and invasive disease in a neonatal intensive care unit

Pulsed-field gel electrophoresis typing of 60 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBLKp) isolates obtained in a neonatal intensive care unit during an outbreak indicated the dissemination of two major bacterial genotypes associated with colonization and invasive disease: one composed by aminoglycoside-resistant isolates and the other by aminoglycoside-susceptible isolates. A urease-negative phenotype was observed among aminoglycoside-resistant ESBLKp. Six pairs of isolates from gastrointestinal (GI) colonization and isolates from invasive disease that occurred 3-23 days later were shown to belong to the same genotype, reinforcing a direct association between colonization and subsequent disease. These data indicate that screening for ESBLKp GI colonization in an outbreak setting may be useful to detect neonates at a higher risk of invasive disease.     

Type III group B streptococcal polysaccharide induces antibodies that cross-react with Streptococcus pneumoniae type 14

Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that of Streptococcus pneumoniae type 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.     

Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.     

Correlation of epithelial cell invasiveness of group B streptococci with clinical source of isolation

Neonatal infections caused by Group B streptococci (GBS) may lead to pneumonia, sepsis, or meningitis indicating that GBS are able to invade tissues and enter the bloodstream from infected sites. In this study, we showed that the tissue invasiveness of GBS may be related to their ability to invade epithelial cellsin vitroby correlating the degree of GBS invasion of cultured human respiratory epithelial cells with the clinical source of isolation. Among 77 isolates tested, those from invasive infections of neonates and adults were significantly (P<0.001) more invasive than those from vaginal carriers and colonised neonates without clinical symptoms. Furthermore, isolates from the blood were more invasive (P<0.05) than those from other sites. GBS invasion seemed to be mediated by bacterial surface proteins since trypsin treatments of streptococci significantly reduced their invasion into epithelial cells and invasiveness was not limited to a certain capsular serotype. The two major GBS surface protein antigens c and R, however, were not involved in the invasion process. These results indicate thatin vitroinvasion of cultured human cells reflects thein vivoinvasive property of GBS and involves bacterial surface components different from known virulence factors such as capsule or protein antigens c and R.     

Decreased capacity for type-specific-antigen synthesis accounts for high prevalence of nontypeable strains of group B streptococci in Mexico.

The low incidence of group B streptococcal (GBS) invasive neonatal disease in Mexico has been attributed to the low prevalence of serotype III strains, a major serotype in developed countries. In addition, nontypeable strains account for 12% of the isolates in Mexico and < 1% of the isolates in the United States. In this study, 57 GBS isolates (28 nontypeable by the Lancefield procedure) from carrier and infected neonates and women from Mexico were also examined for the presence of type-specific antigen by an enzymatic procedure using N-acetylmuramidase digestion of the cell wall to release soluble type-specific antigen. Of the 28 nontypeable strains from Mexico, 23 were typeable by the enzyme extraction procedure, with serotype III being the predominant serotype in invasive disease. These results suggest that nontypeable isolates of GBS should be further examined by the enzymatic extraction procedure to determine the presence of type-specific antigen. Furthermore, these limited results suggest that serotype III is likely a major serotype in invasive disease also in Mexico.     

Laboratory detection of group B <Emphasis Type="Italic">Streptococcus</Emphasis> for prevention of perinatal disease

Group B Streptococcus (GBS) or Streptococcus agalactiae emerged in the 1970s as the leading cause of neonatal morbidity and mortality. Today, GBS remains one of the leading causes of sepsis and meningitis in newborns despite important prevention efforts, including the issuance of recommendations for prevention of perinatal GBS disease by the American College of Obstetricians and Gynecologists, the Centers for Disease Control and Prevention, and the American Academy of Pediatrics in 1996/1997. The gastrointestinal tract is the natural human reservoir for GBS and is the likely source of vaginal colonization. GBS disease in newborns usually results from ascending spread of GBS into the amniotic fluid, which leads to neonatal colonization and to invasive disease in some infants. This review analyzes the various laboratory methods available for the detection of GBS from clinical samples collected from pregnant women and will discuss their impact in the prevention of neonatal GBS infections and in the rationalization of antibiotic use. The recent commercial availability of a rapid and highly sensitive real-time polymerase chain reaction assay suitable for the specific detection of GBS from vagino-rectal samples obtained from pregnant women during delivery, which is approved by the US Food and Drug Administration, provides improvements in the accuracy and rapidity of GBS colonization screening compared to the standard culture-based method using the recommended selective enrichment broth.     

Prevention of group B streptococcal neonatal disease revisited. The DEVANI European project

The purpose of this paper was to present the current knowledge on the prevention of group B streptococcus (GBS) neonatal infections and the status of prevention policies in European countries and to present the DEVANI pan-European program, launched in 2008. The aim of this program was to assess the GBS neonatal infection burden in Europe, to design a new vaccine to immunize neonates against GBS infections, to improve the laboratory performance for the diagnosis of GBS colonization and infection, and to improve the methods for the typing of GBS strains. The current guidelines for GBS prevention in different countries were ascertained and a picture of the burden before and after the instauration of prevention policies has been drawn. After the issue of the Centers for Disease Control and Prevention (CDC) guidelines, many European countries have adopted universal screening for the GBS colonization of pregnant women and intrapartum prophylaxis to colonized mothers. Nevertheless, some European countries continue advocating the risk factor approach to GBS prevention. Most European countries have implemented policies to prevent GBS neonatal infections and the burden of the disease has decreased during the last several years. Nevertheless, further steps are necessary in order to develop new strategies of prevention, to improve microbiological techniques to detect GBS colonization and infection, and to coordinate the prevention policies in the EU.     

In vitro systems for investigating group B streptococcal: host cell and extracellular matrix interactions

Streptococcus agalactiae or group B streptococci (GBS) are gram-positive diplococci and are the leading bacterial cause of pneumoniae, sepsis, and meningitis in neonates. Neonatal GBS infections may occur prior to or during birth. GBS have been cultured from the chorioamnionic membrane of pregnant women and have therefore been associated with chorioamnionitis and premature labor. A potential route for GBS to establish infection of a neonate would be to penetrate the placental membrane of colonized pregnant women. In our laboratory, we have constructed in vitro systems to emulate certain events during the colonization and invasion of host epithelial cell tissues by GBS. By utilizing techniques to grow primary cultures of both chorion cells and amnion cells isolated from human C-section placentas, we have established a relevant model to investigate certain aspects of GBS adherence and invasion into the placental membrane. To identify relevant molecules required for GBS to colonize the multiple tissues it encounters during an infection, we have applied a variety of biochemical approaches with host cell membrane preparations as well as purified extracellular matrix proteins. These techniques are enabling us to further characterize the pathogenic mechanisms utilized by GBS.     

Improved methods for typing nontypeable isolates of group B streptococci

Group B streptococci (GBS) are classified by capsular polysaccharide (CPS) type and by cell surface-expressed proteins (c and R). Isolates lacking detectable CPS are considered nontypeable (NT) although they frequently express surface proteins. Immunological and genetic methods were used to study 91 NT GBS isolates collected during surveillance studies for invasive disease or colonization in pregnant or non-pregnant women and neonates less than seven days of age. CPS production was upregulated by the addition of glucose and sodium phosphate to Todd-Hewitt broth (THB) and cells were extracted using hot HCl or mutanolysin. Extracts were tested with antisera for specific CPS types Ia, Ib, and II - VIII by double immunodiffusion (DD) in agarose. By mutanolysin extraction, 12 (13.2%) of the 91 isolates were typeable. In contrast, only four of these 12 newly typeable isolates tested positive for CPS with the HCl extracts of cells grown in modified THB. DNA was analyzed by pulsed-field gel electrophoresis (PFGE) using SmaI restriction with NT isolates grouped by protein profile to facilitate analysis. PFGE results of the NT isolates were compared to DNA profiles of typeable isolates and were correlated with the DD results. The DNA profiles of the newly typeable isolates were similar to profiles of isolates with corresponding defined CPS type. Of the remaining 78 NT isolates digested by SmaI, 63 (80.8%) had DNA profiles that resembled those of specific types of GBS. These approaches will be useful for classification of NT isolates in continued epidemiological surveillance associated with GBS vaccine trials.     

Cervical Secretions in Pregnant Women Colonized Rectally with Group B Streptococci have High Levels of Antibodies to Serotype III Polysaccharide Capsular Antigen and Protein R

Group B streptococci (GBS) colonizing the female genital tract will often infect newborn infants during delivery. In 200 pregnant women studied, 14% were colonized with GBS in the cervix, 12% in the rectum, and 9% in both cervix and rectum. We have previously reported that antibody levels to GBS serotypes Ia, II, and III in sera and cervical secretions were increased in women colonized in the rectum and/or cervix, when analyzed by a whole-cell ELISA. Here, we report the levels of antibodies to GBS serotype III capsular polysaccharide antigen (CPS III) and to protein antigen R₄, which are present in most GBS III strains. Compared to culture-negative women, the group of women colonized rectally had markedly elevated levels of immunoglobulin (Ig)A and IgG antibodies in cervical secretions to both CPS III and protein R₄ ( P  < 0.01 and P  < 0.001, respectively). In sera, the corresponding differences between culture-negative and culture-positive women were less pronounced, or not present. In contrast to antibody levels to whole-cell GBS, antibody levels to CPS III and protein R₄ in cervical secretions were not significantly increased in women colonized only in the cervix, except that IgA antibodies to protein R₄ were slightly elevated ( P  < 0.05). These findings suggest that capsular type-specific polysaccharides and protein R₄ in a mucosal vaccine might induce protective antibodies against GBS colonization of the uterine cervix.     

Group B Streptococcus (GBS) colonization is dynamic over time,whilst GBS capsular polysaccharides-specific antibody remains stable

Group B Streptococcus (GBS) is a leading cause of adverse pregnancy outcomes due to invasive infection. This study investigated longitudinal variation in GBS rectovaginal colonization, serum and vaginal GBS capsular polysaccharide (CPS)-specific antibody levels. Non-pregnant women were recruited in the UK and were sampled every 2 weeks over a 12-week period. GBS isolates were taken from recto-vaginal swabs and serotyped by polymerase chain reaction. Serum and vaginal immunoglobulin G (IgG) and nasal immunoglobulin A (IgA) specific to CPS were measured by Luminex, and total IgG/A by ELISA. Seventy women were enrolled, of median age 26. Out of the 66 participants who completed at least three visits: 14/47 (29.8%) women that were GBS negative at screening became positive in follow-up visits and 16/19 (84.2%) women who were GBS positive at screening became negative. There was 50% probability of becoming negative 36 days after the first positive swab. The rate of detectable GBS carriage fluctuated over time, although serum, vaginal, and nasal CPS-specific antibody levels remained constant. Levels of CPS-specific antibodies were higher in the serum of individuals colonized with GBS than in non-colonized, but similar in the vaginal and nasal mucosa. We found correlations between antibody levels in serum and the vaginal and nasal mucosa. Our study demonstrates the feasibility of elution methods to retrieve vaginal and nasal antibodies, and the optimization of immunoassays to measure GBS-CPS-specific antibodies. The difference between the dynamics of colonization and antibody response is interesting and further investigation is required for vaccine development.     

Transcriptional and proteomic profiles of group B Streptococcus type V reveal potential adherence proteins associated with high-level invasion

Group B Streptococcus (GBS) is an opportunistic organism that can harmlessly colonize the human gut, vagina, and rectum but can also cause pneumonia, sepsis, and meningitis in neonates born to colonized mothers. We have shown previously that growth rate and oxygen level regulate the ability of GBS to invade eukaryotic cells in vitro. Herein we extend and expand on these observations to show that GBS type V, an emergent serotype, grown in a chemostat at a cell mass-doubling time (t(d)) of 1.8 h with oxygen invaded human ME-180 cervical epithelial cells in large numbers compared with those grown at the same t(d) without oxygen or at a slower t(d) of 11.0 h. The fact that several GBS type V cell wall-associated and membrane proteins were expressed exclusively under the invasive growth condition prompted an investigation, using genomics and proteomics, of all upregulated genes and proteins. Several proteins with potential roles in adherence were identified, including an undefined surface antigen (SAG1350), a lipoprotein (SAG0971), penicillin-binding protein 2b (SAG0765), glyceraldehyde-3-phosphate dehydrogenase (SAG0823), and an iron-binding protein (SAG1007). Mouse antisera to these five proteins inhibited binding of GBS type V to ME-180 cells by > or =85%. Recombinant undefined surface antigen (SAG1350), lipoprotein (SAG0971), and penicillin-binding protein 2b (SAG0765) each bound to ME-180 cells in a dose-dependent fashion, confirming their ability to act as ligands. Collectively, these data increase the number of potential GBS adherence factors and also suggest a role for these surface-associated proteins in initial pathogenic events.     

BsaB,a Novel Adherence Factor of Group B Streptococcus

Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of neonatal sepsis and meningitis, peripartum infections in women, and invasive infections in chronically ill or elderly individuals. GBS can be isolated from the gastrointestinal or genital tracts of up to 30% of healthy adults, and infection is thought to arise from invasion from a colonized mucosal site. Accordingly, bacterial surface components that mediate attachment of GBS to host cells or the extracellular matrix represent key factors in the colonization and infection of the human host. We identified a conserved GBS gene of unknown function that was predicted to encode a cell wall-anchored surface protein. Deletion of the gene and a cotranscribed upstream open reading frame (ORF) in GBS strain 515 reduced bacterial adherence to VK2 vaginal epithelial cells in vitro and reduced GBS binding to fibronectin-coated microtiter wells. Expression of the gene product in Lactococcus lactis conferred the ability to adhere to VK2 cells, to fibronectin and laminin, and to fibronectin-coated ME-180 cervical epithelial cells. Expression of the recombinant protein in L. lactis also markedly increased biofilm formation. The adherence function of the protein, named bacterial surface adhesin of GBS (BsaB), depended both on a central BID1 domain found in bacterial intimin-like proteins and on the C-terminal portion of the BsaB protein. Expression of BsaB in GBS, like that of several other adhesins, was regulated by the CsrRS two-component system. We conclude that BsaB represents a newly identified adhesin that participates in GBS attachment to epithelial cells and the extracellular matrix.     

Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy.     

Population structure of invasive and colonizing strains of Streptococcus agalactiae from neonates of six U.S. Academic Centers from 1995 to 1999

The purpose of this study was to describe the population structure of group B streptococci (GBS) isolated from infected and colonized neonates during a prospective active-surveillance study of early-onset disease in six centers in the United States from July 1995 to June 1999 and to examine its relationship to bovine strains of GBS. The phylogenetic lineage of each GBS isolate was determined by multilocus sequence typing, and isolates were clustered into clonal complexes (CCs) using the eBURST software program. A total of 899 neonatal GBS isolates were studied, of which 129 were associated with invasive disease. Serotype Ia, Ib, and V isolates were highly clonal, with 92% to 96% of serotype Ia, Ib, and V isolates being confined to single clonal clusters. In contrast, serotype II and III isolates were each comprised of two major clones, with 39% of serotype II and 41% of serotype III isolates in CC 17 and 41% of serotype II and 54% of serotype III isolates in CC 19. Further analysis demonstrates that the CC 17 serotype II and III GBS are closely related to a previously described "ancestral" lineage of bovine GBS. While 120 (93%) of invasive GBS were confined to the same lineages that colonized neonates, 9 (7%) of the invasive GBS isolates were from rare lineages that comprised only 2.7% of colonizing lineages. These results are consistent with those for other geographic regions that demonstrate the highly clonal nature of GBS infecting and colonizing human neonates.