Pharyngeal carriage of group B streptococci: detection by three methods.

The prevalence of pharyngeal carriage of group B streptococci was evaluated in patients with and without the complaint of a sore throat by three methods (blood agar plates, Columbia CNA agar plates, and a selective enrichment broth containing gentamicin and nalidixic acid). The overall carriage rate of group B streptococci was 12%, and there was no significant difference between the two groups of patients. The selective broth medium was more sensitive than the two solid agar plate methods in detecting carriage, and 37% of all group B streptococci were recovered solely from the broth. Use of the broth alone would have permitted a 94% detection of the group B streptococcal carriers.     

Grouping of streptococci by Streptex.

Streptex was compared to routine laboratory identification methods available. The results from Streptex sometimes required several attempts before final identification could be achieved. In the main, group D streptococci other than Strep. faecalis failed to group with the Streptex antisera, and this method cannot therefore be used exclusively as a means of identifying this group of streptococci.     

New Granada Medium for detection and identification of group B streptococci.

A methotrexate-containing medium for the detection of beta-hemolytic group B streptococci from clinical specimens on the basis of detection of pigment is described. The medium contained peptone, starch, serum, MgSO4, glucose, pyruvate, methotrexate (as pigment enhancer), phosphate-morpholine-propanesulfonic acid buffer, and selective agents. The recovery of beta-hemolytic group B streptococci was comparable to that obtained with selective broth.     

Rapid detection of group B streptococci directly from vaginal swabs.

Duplicate vaginal swabs were obtained from patients who attended obstetric or gynecologic clinics affiliated with the Magee Womens Hospital in Pittsburgh. One swab was cultured semiquantitatively on 5% sheep blood agar to detect group B streptococci (GBS). The other swab was subjected to a rapid method (25 min) for antigen detection and micronitrous acid exposure to extract the GBS antigen, followed by latex particle agglutination. A total of 464 swabs were evaluated by direct plating. Fifty-two swabs (11.2%) were found to contain GBS. Overall, the rapid method detected 21 of 52, or 40.4%, positive specimens. The sensitivity of the rapid method for identifying the most heavily colonized samples was 85.7%. This method can be used to identify maternity patients who are heavily colonized with GBS and are at high risk of delivering septic infants.     

Grouping of beta-haemolytic streptococci by agglutination.

Streptococcal grouping sera, diluted and absorbed to remove cross-reactions, were bound to staphylococci and used to group trypsinised beta-haemolytic streptococci by coagglutination. The results compared well with those obtained using the Phadebact streptococcal grouping kit. The same sera bound to staphylococci without prior dilution and absorption could be used to group enzyme extracts of haemolytic streptococci by slide agglutination, the results again comparing favourably with those of the Phadebact kit.     

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.     

Extracellular neuraminidase production by group B streptococci.

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-1 acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.     

Granada medium for detection and identification of group B streptococci

A new starch serum medium, Granada medium, for isolation and identification of group B streptococci (GBS) anaerobically as red colonies is described. The medium contains 3.8% Proteose Peptone no. 3 (Difco), 15% soluble starch 1252 (Merck), 10% coagulated horse serum, 15 micrograms of trimethoprim per ml, and 0.06 M phosphate buffer (pH 7.8; medium pH 7.4). This medium inhibited fecal flora and at the same time supported growth of GBS. A new pigment-enhancing effect of folate inhibitors on GBS is reported and used in the formulation of the medium. The good selective and differential properties of the Granada medium favor quicker and easier detection of GBS in heavily contaminated specimens. Since the medium is convenient to use and requires only 18 h of incubation to detect and identify GBS, it should be useful in any clinical microbiology laboratory and would assist in the early detection of GBS in clinical specimens.     

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.     

Specimen storage in transport medium and detection of group B streptococci by culture

Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21 degrees C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4 degrees C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21 degrees C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4 degrees C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration.     

Respiratory epithelial cell invasion by group B streptococci.

Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period; however, the pathogenesis of this infection is poorly understood. We investigated the ability of GBS to enter epithelial cells in culture. Two strains of GBS were capable of invading immortalized respiratory epithelial cell lines in vitro at different levels, suggesting strain differences in invasiveness. Intracellular replication was not observed. Invasion required actin microfilaments but not microtubular cytoskeletal elements. Active bacterial protein, DNA, and RNA syntheses were required for invasion. These findings are consistent with our previous observation of intracellular GBS in the lungs of infected primates. We hypothesize that this organism may access the bloodstream by direct invasion of the epithelial cell barrier.     

Faecal carriage of group B streptococci.

Consecutive stool samples from 116 female and 98 male patients (both adults and children), and rectal and vaginal swabs from 28 and 53 cases respectively, were quantitatively cultured for group B streptococci using Islam's medium. Group B streptococcus was recovered from 5% and 2% of faeces in female and male patients respectively, and the colony counts ranged from 10(2) to 10(3)/g. In women, the faecal carriage rate was 6%, which was significantly lower than the rectal carriage rate (p 0.02), suggesting that the higher recovery rate (27%) from rectal specimens may be due to contamination of swabs by perianal skin flora. Type II group B streptococcus was the only faecal isolate in adults (numbers involved are small for statistical significance), and we suspect that this type strain may be the only resident gut flora in adults, and the gastrointestinal tract is unlikely to serve as the main reservoir of all group B streptococci.     

Lysis and protoplast formation of group B streptococci by mutanolysin.

Immunoprecipitation methods were used to analyze sera and cerebrospinal fluids from patients with multiple sclerosis for measles virus-specific antibodies. The sera contained antibodies against all virus antigens except the matrix polypeptide. Of the nine cerebrospinal fluids investigated, two contained undetectable levels of measles-specific antibodies, four contained antibodies against the nucleocapsid and another internal antigen, and three contained antibodies against the surface antigens as well as the internal antigens.     

Identification of streptococci: serogrouping by immunofluorescence.

This paper deals with the fluorescent antibody (FA) method for identifying six commonly occuring and two rare groups of streptococci by using commercially prepared (Difco) conjugates. We have shown that group-specific FA produced frequent cross-reactions with heterologous groups of organisms. These reactions varied with different strains of the same serogroup. Nonetheless, there was distinct overall patterns in the intensity and appearance of the homologous and heterologous reactions. When monitored by the precipitin test with Rantz and Randall extracts, these patterns led to the correct identification of 90 to 100% of specimens of serogroup A, B, C, and G streptococci. Many members of groups D and F also showed distinctive reaction patterns. However, there was a significant number of strains of both groups D and F that either failed to strain or stained poorly with the homologous conjugate. As a result, the identification of these serogroups by FA was less reliable.     

Rapid recognition of group B streptococci by pigment production and counterimmunoelectrophoresis.

Streptococci from clinical isolates were studied for their ability to produce pigment in stab cultures in Columbia agar. Serological grouping of these organisms was done by counterimmunoelectrophoresis using Burroughs-Wellcome antisera. In this group of isolates, 66 of the 68 organisms grouped as B by serological testing produced pigment in the Columbia agar stab cultures. Pigment was not produced by any of the other 36 streptococci studied (11 group A, 9 group C, 4 group D, and 12 nongroupable). The use of the Columbia agar stab culture is recommended as a rapid and simple test for recognition of group B streptococci. The counterimmunoelectrophoresis test is also suggested as a convenient, rapid, and sensitive method for grouping the streptococci.     

Rapid detection of group B streptococci in pregnant women at delivery

BACKGROUND: Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. METHODS: We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. RESULTS: Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. CONCLUSIONS: Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.     

Invasion of brain microvascular endothelial cells by group B streptococci.

Group B streptococci (GBS) are the leading cause of meningitis in newborns. Although meningitis develops following bacteremia, the precise mechanism or mechanisms whereby GBS leave the bloodstream and gain access to the central nervous system (CNS) are not known. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells (BMEC), the single-cell layer which constitutes the blood-brain barrier. In order to test this hypothesis, we developed an in vitro model with BMEC isolated from a human, immortalized by simian virus 40 transformation, and propagated in tissue culture monolayers. GBS invasion of BMEC monolayers was demonstrated by electron microscopy. Intracellular GBS were found within membrane-bound vacuoles, suggesting the organism induced its own endocytic uptake. GBS invasion of BMEC was quantified with a gentamicin protection assay. Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently than strains from other common GBS serotypes. GBS survived within BMEC for up to 20 h without significant intracellular replication. GBS invasion of BMEC required active bacterial DNA, RNA, and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. The polysaccharide capsule of GBS attenuated the invasive ability of the organism. At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury; this cytotoxicity was correlated to beta-hemolysin production by the bacterium. Finally, GBS demonstrated transcytosis across intact, polar BMEC monolayers grown on Transwell membranes. GBS invasion of BMEC may be a primary step in the pathogenesis of meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial blood-brain barrier.     

Direct detection of group B streptococci from vaginal specimens compared with quantitative culture.

Determination of prenatal vaginal carriage of group B streptococci (GBS) is important in the management of newborns. A pronase extraction-latex particle agglutination method (Streptex; Wellcome Diagnostics, Dartford, England) was used to rapidly detect GBS species-specific antigen directly from vaginal specimens. It was compared with quantitative and broth enrichment cultures. A total of 434 vaginal swab specimens were obtained before delivery. GBS cultures were positive for 14.7% of the specimens (64 of 434). Colony counts ranged from 2 to greater than 10(6) CFU per swab. The sensitivities of the direct antigen analysis were 19% (12 of 64) for all cultures and 63% (12 of 19) for specimens heavily colonized with GBS (greater than 10(4) CFU per swab). The specificity of the antigen test was 99.7%, with only one false-positive. There were three false-negative tests with colony counts of greater than 10(6) CFU per swab. The predictive values were 92% for a positive antigen test and 88% for a negative antigen test. The direct immunochemical detection of GBS antigen can be useful in a population of heavily colonized women. Direct latex particle agglutination does not appear to be salutary for a lightly colonized population and does not appear to be able to replace either culture or antigen detection after growth amplification at this time.