Monoclonal antibody to streptococcal group B carbohydrate: applications in latex agglutination and immunoprecipitin assays

Two monoclonal mouse antibodies with specificities for group B streptococcal capsular antigens were evaluated in assays for the identification of group B streptococci (GBS). One of these antibodies (A9) was shown to precipitate group B carbohydrate antigen in reactions with both purified group B antigen and antigen present in autoclave or enzyme extracts of GBS. A9 antibody was also specific for group B antigen in gel diffusion reactions with extracts of Lancefield group A, B, C, D, F, and G streptococci and was a highly sensitive reagent in detecting soluble group B antigen by counterimmunoelectrophoresis. Antigen extracted from all five serotypes of GBS was shown to be precipitated by A9 antibody. A second monoclonal antibody (C8) was reactive with intact GBS but did not precipitate soluble antigen in bacterial extracts. In contrast with what has been shown for polyclonal rabbit anti-group B antiserum, neither antibody was significantly inhibited in binding or precipitation assays by high concentrations of free rhamnose or other monosaccharides of carbohydrates found in group B antigen. Rhamnose, the most abundant carbohydrate of the group B antigen, does not appear therefore to be an immunodominant determinant in the binding of A9 or C8 antibody. The epitopes of both monoclonal antibodies are exposed on the surface of live as well as heat-fixed GBS cells. A9 antibody-coated latex particles were compared with a commercially available polyclonal latex agglutination reagent and shown to be equally sensitive and specific in the detection of soluble group B antigen in urine and cerebrospinal fluid from patients with GBS infections. Because of its uniformity and defined antigen specificity, which includes reactivity with all five serotypes of GBS, A9 antibody offers the potential of an improved immunodiagnostic reagent for the identification of GBS.     

MATERNAL AND NEONATAL SCREENING FOR GROUP B STREPTOCOCCI BY SCP B GENE BASED PCR: A PRELIMINARY STUDY

Objective: To detect the magnitude of group B streptococcal (GBS) colonization and disease among a sample of pregnant women and their infants in Egypt. Study Design: Prospective observational study. Participants: The study included 95 pregnant females, 35–37 weeks of gestational age, attending the antenatal outpatient clinic at AlFayom University Hospital between September 2006 and June 2007. All participants were screened with vaginorectal swabs by a conventional GBS PCR assay. Participants were grouped into group A (GBS present, 17 patients) and group B (GBS absent, 78 patients). Details with regard to labor and delivery were recorded and placental pathology was examined to detect histological chorioamnionitis. Ninety-five infant data were also recorded. All neonates of group A (17 out of 95 with known positive maternal GBS) underwent collection of simultaneous specimens from surface sites for PCR before their first bath and within four hours of birth. Results: GBS carriage rate in the study sample was 17.89%. Chorioamnionitis confirmed in three patients by placental pathology (one was in group A and two in group B) was statistically not significant. Twenty-two women had rupture of membranes (<12 hours) before delivery (four from group A and 18 from group B) that was not statistically significant. There were three infants out of 17 in group A who had GBS colonized at one or more sites by PCR which was statistically significant. However, only one infant was admitted to neonatal intensive care unit (NICU) that was not statistically significant. Conclusion: Maternal GBS carriage is associated with a significant increase in neonatal infection rate but is not associated with an increase in neonatal intensive care admission. An accurate evaluation of colonization rate (using a larger sample) is desired to evaluate neonatal invasive disease and determine the cost effectiveness of PCR to select an appropriate preventive strategy in Egypt.     

Group B streptococcal colonization patterns in mothers and their infants.

Maternity patients and their newborn infants were cultured for group B streptococci (GBS) at Tampa General Hospital, Tampa, Fla., from September 1982 to May 1983. Culture swabs were placed into Lim Group B Strep Broth (GIBCO Laboratories, Madison, Wis.) and quantitated for GBS. A strong correlation was found between the numbers of GBS in the maternal vagina and the infant rectum. Infants symptomatic for early-onset GBS disease were delivered by mothers heavily colonized (greater than or equal to 3 X 10(4) GBS per swab) at the vagina. Such mothers were identified as GBS carriers by slide coagglutination and latex agglutination after their broth cultures had been incubated for 5 h. These data indicate that maternity patients at high risk of delivering infants heavily colonized with GBS and potentially symptomatic for early-onset GBS disease can be rapidly and selectively identified.     

Endocarditis caused by nonhemolytic group B streptococcus.

We report a case of bacterial endocarditis caused by nonhemolytic group B streptococcus (GBS) in a 67-year-old man with no predisposing risk factors. Nonhemolytic GBS strains rarely cause illness and are usually detected in perinatal infections. We believe this to be the first reported case of endocarditis caused by a nonhemolytic strain of GBS.     

Prevention of perinatal group B streptococcal infections: A review with an Indian perspective

Group B Streptococcus (GBS) is an important cause of maternal and neonatal morbidity and mortality in many parts of the world. Asymptomatic colonisation of the vagina and rectum with Group B streptococci is common in pregnancy. Maternal colonisation of GBS can vary depending on ethnicity and geographical distribution. Vertical transmission of this organism from mother to foetus may lead to neonatal GBS disease. Intra-partum use of antibiotics in these women has led to a decrease in the rate of early onset but not late onset GBS disease. Identification of women with GBS is the key factor in the prevention of perinatal GBS disease. There are different screening strategies available to identify women at risk of perinatal GBS disease. Clinicians continue to face the challenge of choosing between preventive strategies to reduce the impact of perinatal GBS disease. Controversy exists regarding the ideal preventive strategy. In India, the mortality and morbidity associated with the GBS disease remains largely a under-recognised problem. This comprehensive review summarises the salient features of GBS disease and discusses the epidemiology, risk factors, screening strategies, intra-partum antibiotic prophylaxis with an Indian perspective and how it compares with the Western nations.     

Cell-associated collagenolytic activity by group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.     

Group B streptococcus induces trophoblast death

Group B streptococcus (GBS) is one of the leading causes of neonatal infection; however the molecular mechanisms involved are not clearly known. Here we used high and low hemolytic GBS isolates and mutant GBS that lacks beta-hemolysin expression and showed that GBS infection or exposure to GBS hemolysin extract induces primary human trophoblast, placental fibroblast and JEG3 trophoblast cell line death, and that GBS-induced trophoblast death was beta-hemolysin dependent. The fibroblasts and trophoblasts provide an innate immune barrier between fetal and maternal circulation in the placenta. These data suggest that GBS may disrupt this barrier to invade fetal circulation.     

Group B streptococci--gastrointestinal organisms?

Matched perianal swabs, rectal swabs, and faecal samples from a group of male homosexual patients attending a clinic for sexually transmitted disease were examined for the presence of group B streptococci (GBS). GBS recovery rates were as follows: perianal skin 31/115 (27%), rectal mucosa 18/72 (25%) and faeces 7/115 (6%). The recovery of GBS from faeces was similar to that obtained from faecal samples sent to the laboratory for routine investigation (5%). Although there was no difference in GBS recovery rates from rectal and perianal swabs, the latter did show heavier colonisation. These results suggest that gastrointestinal GBS carriage is mainly limited to the rectum and anal canal and that this may represent contamination from perianal skin.     

Functional activity of antibodies to the group B polysaccharide of group B streptococci elicited by a polysaccharide-protein conjugate vaccine.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in infants. While antibodies directed to the type-specific GBS capsule have been shown to be protective, it is less clear whether antibodies to the group B polysaccharide, a noncapsular, cell wall-associated antigen, may play a role in immunity. To investigate the functional activity of group B polysaccharide-specific antibodies, we tested sera from rabbits vaccinated with group B polysaccharide coupled to tetanus toxoid (B-TT). Anti-B-TT was weakly opsonic in vitro for a highly encapsulated type III strain, while antiserum elicited by vaccination with type III capsular polysaccharide linked to tetanus toxoid (III-TT) was a very effective opsonin. In contrast to anti-III-TT, anti-B-TT given before or after bacterial challenge was only marginally effective in protecting newborn mice against lethal infection with type III GBS. The number of C3 molecules bound to type III GBS was augmented by anti-III-TT but not by high antibody concentrations of anti-B-TT. These results suggest that the difference in opsonic activity between anti-B-TT and anti-III-TT may be due to a difference in their ability to deposit C3. In addition, the maximum number of antibody molecules bound to the bacterial surface was greater for anti-III-TT than for anti-B-TT. That anti-B-TT binds to fewer sites than anti-III-TT may explain the differences in complement activation and in opsonic and protective efficacy of antibodies to group B polysaccharide compared with antibodies to the type-specific capsular polysaccharide.     

Characterization of polymorphonuclear leukocyte aggregation in vitro induced by heat-inactivated group B streptococcus

We studied the aggregatory characteristics of human polymorphonuclear leukocytes (PMNs) in response to heat-inactivated group B streptococcus. PMNs suspended in physiologic salt solution do not aggregate to heat-inactivated group B streptococcus (GBS) unless the GBS is previously opsonized in autologous plasma. The aggregating activity of both opsonized GBS and activated plasma are reduced if the plasma is decomplemented before incubation with GBS. Pretreatment of PMNs with pronase inhibited opsonized GBS-induced aggregation, suggesting aggregation via cell membrane receptors foropsonic fragments of C3. Pronase pretreatment had no significant effect on aggregation induced by activated plasma or arachidonic acid. Unlike PMNs in physiologic salt solution, PMNs suspended in plasma aggregate when stimulated by unopsonized GBS. GBS aggregates PMNs via complement cascade activation, opsonization, and interaction with cell membrane receptors to stimulate cellular mechanisms resulting in PMN aggregation.     

Rapid detection of group B streptococcal antigen in human amniotic fluid.

Infants exposed in utero to group B streptococcus (GBS)-infected human amniotic fluid (HAF) are at high risk for serious infection. Latex particle agglutination (LPA) tests are not approved for detection of GBS in HAF. Two LPA systems, Patho-Dx Strep B and Wellcogen Strep B, were used to test unfiltered sterile HAF and filtered HAF containing concentrations of GBS carbohydrate from 0.2 to 100 micrograms/ml. Four different processing techniques were used to prevent nonspecific LPA: EDTA, nitrous acid, enzyme, and nitrous acid-heat. GBS (10(2) CFU/ml) was inoculated into filtered HAF, incubated, sampled serially, processed with enzyme, and tested by LPA. Unprocessed, unfiltered HAF showed 33% nonspecific agglutination when tested by LPA. Processing of HAF removed nonspecific agglutination and improved GBS antigen detection. Without processing, LPA could not detect less than 100 micrograms of GBS carbohydrate per ml. With nitrous acid or enzyme processing, as little as 0.2 microgram/ml could be detected. Results were easier to read after enzyme processing than after nitrous acid processing. Although both LPA systems were equally efficient, testing was easier with the Patho-Dx system. After enzyme processing, LPA could detect as few as 10(4) CFU/ml when agglutination was read with a 4 X hand lens. Substances in HAF induce false-positive reactions during LPA testing. Processing removes the interference and improves the detection of GBS. LPA testing of HAF may allow earlier identification and treatment of infants at risk for serious GBS infection.     

Latex assay for serotyping of group B Streptococcus isolates

We developed a group B streptococcus (GBS) latex serotyping kit that reduces the numbers of GBS nontypeable isolates by nearly 50%. A total of 232 isolates were tested, and 203 isolates were serotyped by the GBS latex test, while the capillary precipitation test serotyped 184 isolates.     

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.     

Xpert GBS Assay for Rapid Detection of Group B Streptococcus in Gastric Fluid Samples from Newborns

The Xpert GBS real-time PCR assay was applied to gastric fluid samples from 143 newborns, and it detected group B streptococcus (GBS) within 1 h for 16 (11.2%) cases, while microscopic examination detected only 2 cases. The sensitivity and specificity of the Xpert GBS were 80% and 100%, respectively, with regard to 20 cases of GBS colonization or infection. Concordance of Xpert GBS results versus culture was 92.3%. This test detects in a timely manner newborns at risk for invasive GBS disease.     

Reduction of morbidity and mortality rates for neonatal group B streptococcal disease through early diagnosis and chemoprophylaxis.

Pregnant women, part of the term service population at Orlando Regional Medical Center, were screened for group B streptococci (GBS), using Lim Group B Strep Broth (GIBCO Laboratories, Madison, Wis.) and the Phadebact Strep B Test (Pharmacia Diagnostics, Piscataway, N.J.). Of the 803 women screened, 173 were confirmed as colonized with GBS at the time of admission in labor. Eighty of these women were treated with ampicillin at least 6 h prior to delivery. The remaining 93 women received no ampicillin. None of the infants born to the treated women was colonized with GBS at surface culture sites. Forty-three of the infants born to untreated women were colonized. Rapid identification of GBS colonization in women, combined with ampicillin chemoprophylaxis, significantly reduced vertical transmission of GBS.     

Optimisation of Prenatal Group B Streptococcal Screening

The purpose of the study presented here was to confirm the high yield of group B streptococci (GBS) on Granada medium for the detection of pregnant GBS carriers and to compare the results with those obtained using standard Columbia blood agar at two participating centers in Belgium. Culture results of the vaginorectal swabs obtained at the two centers were also compared. A total of 1,142 samples (838 in Leuven and 304 in Bonheiden) obtained from consecutive pregnant women were cultured onto both media. Of all GBS carriers 84.7% were detected on Columbia blood agar and 93.4% on Granada agar (P<0.01, McNemar test). The addition of Granada agar was responsible for a 15% higher rate of detection of GBS carriers. As a result of this study, both participating hospitals will use a combination of Granada agar with Columbia blood agar for optimal GBS screening in the future.     

Group B streptococcal transmission via a prolonged colonizer in a neonatal intensive care unit

This article reports five invasive Group B streptococcal (GBS) infections that occurred in a neonatal intensive care unit for about 3 months. This outbreak might have been associated with a prolonged GBS colonized infant and adjacent environmental contaminations. Infection control interventions prevented the additional spread of GBS infections.     

Phosphoglycerate kinase inhibits epithelial cell invasion by group B streptococci

Group B streptococci (GBS) are opportunistic human pathogens that cause infection and invasive disease in newborns, pregnant women and non-pregnant adults. The internalization of GBS into eukaryotic cells occurs in an actin-microfilament dependent process. The objective of our study was to understand what host cell and/or bacterial factors may be involved in this process. We focused on alpha-actinin, an actin binding protein closely associated with cytoplasmic F-actin in the eukaryotic cell, to determine if it is involved in actin recruitment upon GBS internalization. Initial work revealed that GBS does not recruit alpha-actinin. However, it was found that alpha-actinin antibodies bound to the surface of the GBS, suggesting GBS possess surface-exposed actin binding protein(s). Slide agglutination experiments revealed that when the bacteria were emulsified with F-actin, visible agglutination occurred, further suggesting the presence of an actin binding protein on the GBS cell. Western blot analysis found that anti-alpha-actinin antibodies bound to a 42 kDa protein; mass spectra analysis identified this protein as GBS phosphoglycerate kinase (PGK). Competitive binding assays suggest that the PGK-actin interaction is not a factor in the initial binding of GBS to epithelial cells, however, treating epithelial cells with PGK prior to performing an invasion assay inhibited GBS internalization. This occurred in a dose dependent manner with 10 microg/mL of PGK inhibiting invasion by over 70%, and 50 microg/mL PGK inhibits GBS invasion completely.     

Oxygen regulates invasiveness and virulence of group B streptococcus

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (t(d)) of 1.8 h than at a slower t(d) of 11 h. Invasion was optimal when GBS was cultured at a t(d) of 1.8 h in the presence of >or=5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t(d) of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t(d) of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.     

Induction of cyclooxygenase-2 by human monocytes exposed to group B streptococci

Group B streptococcal (GBS) infections are associated with high morbidity and mortality. The molecular pathways mediating the pathophysiological events in GBS infection are not fully delineated. Cyclooxygenases (COX) are the enzymes that convert arachidonate to active eicosanoids. To identify the effects of GBS on eicosanoid metabolism and regulatory mechanisms, we exposed human monocytes to GBS and found that they secreted prostaglandin E2, prostacyclin, and thromboxane A2. Exposure to GBS caused monocytes to express COX-2 mRNA and protein in both a time- and concentration-dependent manner that correlated with eicosanoid production. COX-1 protein was unchanged. Addition of the anti-inflammatory cytokines interleukin (IL)-4 or IL-10 markedly attenuated GBS-induced COX-2 protein accumulation after GBS exposure, as did inhibition of p38 MAPK. Our experiments are the first to show that exposure of monocytes to a gram-positive bacterium (GBS) results in induction of functional COX-2, suggesting that eicosanoids may play important roles in the pathogenesis of GBS infections.