Characterisation of invasive group B streptococci based on investigation of surface proteins and genes encoding surface proteins.

The joint distributions of the six genes bca, bac, epsilon/alp1, alp2, alp3 and rib (encoding alpha-C-protein, beta-C-protein, epsilon/Alp1, Alp2, Alp3, and Rib, respectively) and the proteins alpha-C-protein, beta-C-protein and Rib were investigated in invasive isolates of group B streptococcus (GBS). In total, 297 invasive isolates (123 from neonates, 174 from adults) from south-west Sweden were collected during a 13-year period. Genes were detected using multiplex and specific PCRs, and expression of the surface proteins was demonstrated using monoclonal antibodies. The genes studied were found alone or in combinations in 294 (99%) of the invasive isolates. The most common genes were rib (n = 127 isolates, 43%), alp3 (n = 78, 26%) and epsilon/alp1 (n = 42, 14%). The bac gene was never found alone, but was found in combination with one other gene in 36 isolates. The surface proteins studied were detected alone or in combinations in 152 (51%) isolates, with the most common being Rib (n = 80, 27%), alpha-C-protein (n = 68, 23%) and beta-C-protein (n = 24, 8%). Several genes were associated significantly with particular serotypes (e.g., epsilon/alp1 with serotype Ia; bca and bac with serotypes Ib and II; rib with serotype III; alp3 with serotype V). Overall, it was concluded that demonstration of different genes and surface proteins of GBS strains can be useful in epidemiological studies and in formulation of vaccines, but disappointingly, no single gene or surface protein included in the study was sufficiently common for it to be considered as the basis for a successful GBS vaccine.

Invasive group B streptococcus (GBS) disease in Norway 1996-2006.

The aim of this study was to survey the occurrence of invasive group B streptococcus (GBS) disease in Norway and detect possible trends in characteristics of invasive GBS strains from 1996 to 2006. Data from national monitoring systems for infectious diseases in Norway were analysed. Of 638,452 live births in the period, 434 cases of invasive GBS disease in infants were reported. In adults and children older than 1 year of age, 969 cases were reported. The incidence of invasive GBS disease increased significantly in the elderly, while the incidence of neonatal early-onset disease was stable with 0.46 cases per 1,000 live births. The incidence of late-onset disease increased in 2005 and 2006. The lethality of GBS in infants increased from an average of 6.5% in 1996-2005 to 20% in 2006. Serotypes III and V were predominant in 839 invasive GBS strains characterized-type III in infants and type V in the elderly. The distribution of serotypes did not change throughout the period. The distribution of detected surface proteins was stable from 1996 to 2005, but the detection rates in types III and V were low. Molecular methods for GBS typing introduced in 2006 made characterization of nearly all strains possible and appear more applicable to epidemiological studies of GBS than conventional methods. Resistance to erythromycin and clindamycin increased significantly in 2006. The increased incidence in the elderly, the increased lethality in infants in 2006, and the increased resistance to erythromycin and clindamycin the same year might indicate changing characteristics of invasive GBS strains.

Streptococcus agalactiae (group B streptococcus (GBS)) colonisation and persistence, in pregnancy; a comparison of two diverse communities (rural and urban).

OBJECTIVE: To establish the extent of GBS colonisation, persistence of colonisation in pregnancy and influence of obstetric history in two diverse communities (rural and urban) in Zimbabwe. DESIGN: Cross sectional survey. SETTING: Rutsanana Clinic in Highfield, Harare (representing the urban area) and Chitsungo Mission Hospital in Lower Guruve, (representing the rural area). SUBJECTS: 300 and 100 pregnant women from the urban and rural areas respectively. MAIN OUTCOME MEASURES: GBS colonisation and persistence rates for both urban and rural areas were established, together with pregnancy outcome. RESULTS: Mother colonisation rate was significantly higher in the rural areas (60%) as compared to the urban areas (46%). GBS colonisation persistence was evidently more in rural (48%) that in urban women (12%). Baby colonisation was also more in the rural (23%) that in urban area (5%). In both the rural and urban areas, flu-like illness was a common feature and was equally reported by the subjects. Vaginal discharge requiring treatment, previous stillbirths and previous miscarriages were equally reported in both communities.

A Streptococcus agalactiae R protein analysed by polyclonal and monoclonal antibodies.

Unexpected cross-reactivity between two Streptococcus agalactiae (GBS) isolates formed the basis for purification of a GBS protein called the Ra antigen, and raising of murine monoclonal antibody (MAb) against Ra. The Ra protein was resistant to trypsin digestion, susceptible to pepsin digestion, formed a ladder-like pattern of lines with a periodicity of approximately 8 kD on immunoblotting, was surface-localized in GBS strains, and was variably expressed by GBS. These characteristics provided evidence that the Ra antigen belonged to the R proteins of GBS. By testing of reference GBS isolates and antiserum, including an anti-R4 protein serum, cross-reactivity was recorded consistent with the assumption that Ra is a R4 protein. The Ra/R4 protein also showed cross-reactivity with a previously described GBS protein called protein Rib (J. Exp. Med. 177: 1593-1603, 1993). Several characteristics of the Ra/R4 protein were similar to those of the GBS protein c alpha, but the two proteins showed no cross-reactivity. The anti-Ra/R4 MAb has proved useful in serosubtype determination of GBS of known serotype and should be a valuable tool for studying the immunobiological function of antibodies targetting the surface-localized Ra/R4 protein.

Properties and distribution of the putative R3 protein of Streptococcus agalactiae.

Strain-variable Streptococcus agalactiae (group B streptococci; GBS) proteins exposed at the bacterial cell surface are important markers in GBS serotyping. These proteins include the c proteins c(alpha) and c(beta) and the R proteins R1 through R4, of which R1 and R4 have been studied most extensively. This study presents the characteristics of a protein which was expressed by a capsular antigen type V GBS strain shown by means of polyclonal and monoclonal antibody testing. Examination of a number of reference and prototype strains by fluorescent antibody testing and Western blotting provided evidence that the serotype V-derived protein was the R3 protein of GBS, previously defined on the basis of immunoprecipitation assays. The putative R3 protein formed ladder-like banding patterns on Western blotting with polypeptides in the 30 kDa to > or = 140 kDa range, was destroyed by pepsin digestion, and partially degraded by trypsin digestion. The protein was expressed by 10 (6.5%) of 153 clinical GBS strains tested, the expression being restricted to isolates of the capsular antigen types II, III, and V. Some isolates expressed both the c(beta) and the R3 protein. Expression in combination with c(alpha) or R4 protein synthesis was not detected. Inclusion of the anti-R3 monoclonal antibody among antibody reagents for GBS serotyping will enhance the discriminatory power of this typing method.

Comparison of three different methods in monoclonal antibody-based detection of Streptococcus agalactiae protein serotype markers.

Surface-exposed proteins are important serotype markers in Streptococcus agalactiae (group B streptococci; GBS). The proteins include the c proteins c(alpha) and c(beta), the R4 protein and a protein provisionally called P. For all of these markers, protein-specific monoclonal antibodies (MAbs) have been generated. We have compared whole-cell-based fluorescent antibody testing (FAT), ELISA, and dot blotting for MAb-based detection of these proteins by testing a panel of 52 GBS isolates of different capsular antigen types. Of a total of 208 observations with each of the tests, positive signalling in the dot assay was observed in 32.2%, with ELISA in 27.8%, and with FAT in 26.4% of the recordings. Discordant results were noted most frequently with the c(beta) and c(alpha) MAbs. In the case of c(alpha) the reason for the discordant test results was further examined and it appeared that this could be attributed to low level expression of the c(alpha) protein, although structural variations of c(alpha) proteins cannot be excluded. Our findings favour dot blotting as the method of choice although we consider all three methods acceptable for serotyping of GBS.

Binding of human IgA to HCl-extracted c protein from group B streptococci (GBS).

The c beta protein of group B streptococci obtained by HCl extraction appears as a ladder-like pattern in SDS-PAGE when detected by a rabbit anti-c beta serum, and a similar picture is seen when the crude extract is incubated with human IgA and an anti-human IgA conjugate. Affinity-purified c beta antigen and IgA receptors from GBS gave identical pictures in Western blots using rabbit anti-c beta serum. Both the c beta antigen and the IgA receptor are exposed on the surface of GBS as demonstrated by immunofluorescence.

Low prevalence of Bartonella henselae infections in Norwegian domestic and feral cats.

Bartonella henselae is the causative agent of cat scratch disease (CSD). This clinical entity is very rarely encountered in human medical practice in Norway. B. henselae infections including bacteraemia in cats have been frequently reported. The objective of the present study was to investigate the seroprevalence rate and the degree of B. henselae bacteraemia in Norwegian domestic and feral cats. One hundred cats investigated at a small animal veterinary practice in the middle of Norway were included in the study. Blood collected in Isolator blood-lysis tubes and lysates of erythrocytes after freezing and thawing were cultured. PCR analysis of whole blood was also performed. Serology was performed by indirect fluorescence assay (IFA) and enzyme immunoassay (EIA) using immobilised B. henselae Houston-1 strain as antigen. None of the 100 cats investigated was found to be bacteraemic. All 100 cats were seronegative when analysed by IFA; one cat was positive by EIA. The discrepancy between IFA and EIA of this particular cat is probably due to cross-reactive antibodies. Contrary to findings reported from several geographic regions, B. henselae infections in Norwegian cats appear to be virtually absent. This in turn may explain why CSD has not been reported in human medical practice in Norway.

Characterization of the alpha antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody.

A murine monoclonal antibody raised against the alpha antigen of the group B streptococcal c proteins was analysed by immunofluorescence and whole-cell ELISA against a collection of 22 c protein-producing GBS. All the strains showing fluorescence and reactivity in ELISA turned out to be alpha antigen-carrying strains as defined by polyclonal rabbit antisera, while none of the strains producing only the beta antigen was positive. Western blot analyses of the alpha antigen released into the culture medium of growing bacteria suggest that the alpha antigen is present as distinct proteins of variable molecular weights. The upper limit of the molecular weights varies considerably from one strain to another, from approximately 200 kD to 70 kD. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD throughout the gel. Some strains gave rise to 15-16 bands, while others gave rise to only one or two bands. The present investigation suggests that alpha antigens include several, probably identical, repeating subunits of approximately 10 kD. The epitope recognized by the MAb seems to be located on a 10-kD fragment, and in addition, it appears to be surface located, making the MAb a suitable tool in serodiagnostic work.

Serological testing for campylobacteriosis with sera forwarded for Salmonella and Yersinia serology.

Single serum specimens forwarded for Salmonella and Yersinia serology, from a total of 250 patients, were tested for anti-C. jejuni antibodies of the IgG class. Anti-Salmonella and anti-Y. enterocolitica O3 antibodies were examined by a microagglutination test and anti-C. jejuni antibodies by ELISA against a C. jejuni ultrasonicate before (ELISA) and after neutralization of antibodies which cross-reacted with Helicobacter pylori antigens (ELISA-Abs). Blood donor sera (n = 50) and sera (n = 40) from patients with various infectious diseases served as controls. A positive test for anti-Salmonella antibodies was recorded in 4/250 (1.6%) of the patients, for anti-Yersinia antibodies in 7/250 (2.8%), and for anti-C. jejuni antibodies in 7/250 (2.8%) in the ELISA; in 25/250 (10%) in the ELISA-Abs. No mixed infection was recorded by the serological testing. The ELISA-Abs showed a diagnostic specificity of 97.7%. Our results support the inference that diagnostic serology for enteropathogenic bacteria should include serology for C. jejuni, preferably by tests which exclude participation by antibodies which cross-react with H. pylori antigens.

Evaluation of monoclonal antibodies in serovar classification of group B streptococci (GBS). Brief report.

We have produced murine MAbs against the c protein from GBS and selected one anti-c alpha and one anti-c beta MAb for serovar classification of c-protein-producing strains. A total of 61 GBS isolates, classified by serotype-specific rabbit antisera, were tested in an immunofluorescent assay and whole-cell ELISA. The MAbs permitted classification of the isolates into 13 serovars. A total of 34 strains contained the c alpha antigen, seven strains the c beta antigen, and 20 strains both the c alpha and the c beta antigen. The distribution of GBS with regard to c-protein serovars was identical to that recorded when rabbit anti-c alpha and -c beta sera were used. The results show that the MAbs are directed against common epitopes on c alpha and beta, respectively. The MAbs will be useful in the serovar determination of clinical GBS isolates and in the study of the structure and immunobiology of c proteins.

Streptococcus agalactiae beta gene and gene product variations.

Streptococcus agalactiae (group B streptococci; GBS) are serotyped on the basis of the capsular polysaccharide antigens and subtyped on the basis of the strain-variable and surface-localised c proteins c alpha, c beta, and R proteins. This study compared c beta protein detection and the polymerase chain reaction (PCR) for beta gene detection, by examining 50 clinical GBS strains. The c beta protein was detected by antibody-based immunofluorescence in a GBS whole-cell assay and Western blotting by probing with the anti-c beta antibody or human IgA. Absorption experiments were performed to test for surface-anchoring of c beta; and bacterial supernates were examined to test for c beta production. Primers for the PCR target regions resulted in a 620-bp product that included beta gene-encoding IgA-binding domains. The results demonstrated four categories of GBS with respect to the beta gene and the c beta protein: (1) strains (16 of 50) that harboured the beta gene and regularly expressed normal surface-localised c beta with a M(r) of 120 kDa; (2) strains (5 of 50) that harboured the gene but did not express the protein; (3) strains (2 of 50) that harboured the gene but expressed a c beta that was not surface-localised and had reduced M(r); (4) strains (27 of 50) without beta gene and c beta expression. One strain amongst the third group generated a PCR product of 1330 bp. These results demonstrate considerable strain variability of the beta gene of GBS and of its product the c beta protein.

Distribution and expression of bca, the gene encoding the c alpha protein, by Streptococcus agalactiae.

A total of 52 clinical isolates of group B streptococci (GBS) was tested for expression of the c protein c(alpha) by a fluorescent antibody test (FAT) and by PCR amplification of a 202-bp stretch within the repeat unit of the bca gene encoding the c(alpha) protein. The strains were categorised as follows: c(alpha) FAT positive and PCR positive with amplification products of multiple sizes (category A, n = 12); FAT negative and with PCR products of multiple sizes (category B, n = 11); FAT negative and with a single PCR product of c. 200 bp (category C, n = 5); negative in both tests (category D, n = 24). A single amplification product of minimum size and additional products of larger sizes corresponded to one and more bca repeats, respectively. Five of the 11 category B strains showed expression of low Mr c(alpha) in whole cell-based Western blotting. The results showed that a proportion of the GBS isolates harboured bca gene elements that either were not expressed or they expressed c(alpha) molecular variants which could not be detected by the whole cell-based FAT. This genotype/phenotype discrepancy should be considered in relation to GBS typing, including the selection of antibody reagents and the technical approach to c(alpha) protein detection.

Identification of nasopharyngeal carriage of an outbreak strain of Neisseria meningitidis by pulsed-field gel electrophoresis versus phenotypic methods.

The clustering of four cases of meningococcal disease during a 3-month period in a small community with 2233 inhabitants prompted an interventional carrier survey in persons < 19 years old and in family members of the patients. The aims of the survey were to identify the nasopharyngeal carriers and the carriage rate of the outbreak strain, to offer chemoprophylaxis to those carrying the outbreak strain, and to study the discriminatory power of phenotypic methods versus pulsed-field gel electrophoresis (PFGE) on carrier isolates during an outbreak. A high percentage of the population in the age group 0-19 years (73.7%) participated in the study. Among the 469 samples collected in this age group, meningococci were grown from 43 (9.2%). The highest carriage rates were in the age group 18-19 years (36.4%). With a provisional definition of the outbreak strain (group B or non-groupable Neisseria meningitidis with reduced sulphonamide sensitivity), six carriers were identified. All were treated with a single dose of ofloxacin. Four of these persons (0.76% of all tested) were later shown to have harboured the outbreak strain when analysed by PFGE. Three of them were epidemiologically closely related to one of the index cases. Serogrouping alone is not sufficient for the identification of an epidemic strain of N. meningitidis. Complete concordance of type and subtype antigens correctly identified the outbreak strain in this study. PFGE is well suited for the identification of an outbreak strain of N. meningitidis versus non-epidemic strains in tonsillo-pharyngeal specimens.

Distribution of serovariants of group B streptococci in isolates from England and Norway.

The distribution of capsular polysaccharide antigen (CHO) types, surface-exposed c proteins alpha (c alpha) and beta (c beta) and an R-protein antigen was examined in 334 group B streptococci (GBS) isolates from three groups of patients hospitalised in England and Wales or Norway. The isolates were from 108 carriers, 67 cases of neonatal infection and 154 cases of adult infection. Each group contained all CHO types (Ia, Ib, II, III, IV, V and NT); type III strains predominated except in the adult infected group. Strains within each CHO type could be further subdivided by the protein markers into five subtypes by a combined typing system. The proportion of type Ib and type III strains in the neonatal infection cases and of type Ib strains in the adult infection cases significantly outnumbered isolates of these serotypes among the carrier strains. Twenty-nine different serovariants were identified; 24, 13 and 23 serovariants among the carrier, neonatal infection and adult infection isolates, respectively. Certain CHO antigen-protein associations were identified, notably those between Ia/c alpha, Ib/c alpha beta and III/R. The proportion of invasive isolates that expressed protein was not higher than in the carrier isolates. All CHO-type Ib isolates contained a c protein, but 7% of the Ib isolates did not contain any of these proteins. These findings indicate that this combined typing approach may be useful in examining epidemiological problems associated with GBS.

Antibiotic sensitivity still prevails in Norwegian blood culture isolates.

We describe the antimicrobial susceptibility of bacteraemia isolates from Norway. From March 1998 to February 1999, four university hospitals covering all parts of Norway collected their first 10 isolates each month. Minimal inhibitory concentrations were determined for: Enterobacteriaceae (n=192), staphylococci (n=89) and Streptococcus pneumoniae (n=69) using the Etest. NCCLS breakpoints were used. About 20% of all blood culture isolates in Norway in this period were investigated. Compared with countries outside Scandinavia antibiotic sensitivity still prevails. Only minor differences in resistance were found between participating hospitals, between hospital departments and between hospital- and community-acquired pathogens. The prudent use of antibiotics in Norway may contribute to the fact that antibiotic resistance still remains low in the most common bacterial pathogens causing bloodstream infections.

Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR with TaqMan probe hybridization.

BACKGROUND & OBJECTIVES: Implementation of a screening based strategy for the prevention of perinatal group B streptococcus (GBS) disease is anticipated to increase the demand of fast laboratory techniques. The aim of the present study was to develop a real-time PCR method for the specific detection of GBS in vaginal swabs. METHODS: Based on partial sequencing of the sip gene, primers and a TaqMan hybridization probe were constructed for a real-time PCR assay. The performance of the assay was tested on 100 consecutive vaginal specimens submitted to the laboratory for culture. Lysis of bacteria and DNA extraction was performed by lysozyme, mutanolysin, proteinase K and Quiagen spin columns. PCR was performed using LightCycler. Highly purified DNA from GBS was used as positive control. RESULTS: Of the 100 samples investigated, 25 were positive by culture (16 abundant/moderate growth, 6 sparse growth and 3 after enrichment only). At a cut-off of 12 fg per reaction (corresponding to 4 bacterial cells), PCR was positive in 32 samples. A complete concordance was noted between PCR positivity and abundant/moderate and sparse growth by culture. One of 3 samples that were positive by culture only after enrichment was negative in PCR. On the other hand, 8 samples were positive by PCR and negative by culture. INTERPRETATION & CONCLUSION: The real-time PCR assay was sensitive and rapid and enabled detection of GBS in less than 2 h after collection. Due to the rapidity of the assay by which results could be obtained, the assay harbors the potential for intrapartum detection of GBS.

The Ibc proteins of group B streptococci: isolation of the alpha and beta antigens by immunosorbent chromatography and test for human serum antibodies against the two antigens.

The Ibc protein fraction from group B streptococci contains at least two antigens, named alpha and beta. Rabbit antisera to each of these antigens were used to prepare affinity-purified alpha and beta antigens. Analysis of the purified antigens by SDS-PAGE showed at least three weakly stained proteins in the alpha antigen - and multiple protein bands in the beta antigen preparation. Conditions for optimalization of ELISA for testing of human serum antibodies against these antigens were investigated. When tested at a dilution of 1 in 200, ELISA activity corresponding to an OD405 greater than 0.2 was shown by 11.2 per cent of 157 blood donor sera in tests with the alpha antigen, and 77.7 per cent with the beta antigen. Increasing antibody activity against the alpha antigen was shown by a patient with septicaemia caused by an alpha antigen-producing strain. The antigens were immunoblotted and tested against human and rabbit antibodies. With the alpha antigen, antibodies from both species produced a diffuse stained area, and with the beta antigen multiple stained bands, some of which were colinear with the human and rabbit antibody. Thus, antibodies to the beta antigen of group B streptococci are present in a large proportion of the healthy population. These antibodies may be important in the protection against group B streptococcal disease.

Ibc proteins as serotype markers of group B streptococci.

A collection of 106 clinical isolates of group B streptococci were type classified by a direct fluorescent antibody test (FAT). In all, 99 of the isolates were typable and belonged to the serotypes Ia (8 strains), Ib (9 strains), Ic (30 strains), II (15 strains), or III (37 strains). The presence of Ibc proteins was demonstrated in 60 of the strains, including 6 of the non-typable isolates. Indirect FAT was performed to test whether the bacteria produced the alpha and beta antigens of the Ibc protein fraction, using specific anti-alpha and anti-beta sera. Both alpha and beta were produced by 22 strains, only alpha by 33 strains, and only beta by 5 strains. The results show that the alpha and beta antigens can be used as markers to detect serovars among group B strains which carry identical serotype-specific carbohydrate antigens, or strains listed as nontypable according to the classification currently used.