Diversity of surface protein expression in group B streptococcal colonizing & invasive isolates.

BACKGROUND & OBJECTIVES: The classification of group B streptococcal (GBS) isolates is based on the capsular polysaccharides (Ia-VIII), and antigenic characterization of clinical isolates is augmented by the detection of various surface-localized protein antigens. In our laboratory, all GBS isolates are routinely analysed for the alpha trypsin-resistant and the beta trypsin-sensitive c protein antigens, as well as other trypsin-resistant proteins R1, R3, and R4, as well as BPS. The purpose of this work was to study diversity of protein expression in colonizing isolates (vaginal and rectal sites) from nonpregnant women and from invasive isolates (blood or CSF) from mothers and their less than seven day old newborn infants. METHODS: A total of 289 invasive isolates and 2660 colonizing isolates were collected between 1993-2002. All isolates were tested for polysaccharide serotype and cell surface-expressed protein profile by double immunoprecipation in agarose using monospecific antisera. RESULTS: Among the 289 invasive isolates, 89.6 per cent expressed one or more trypsin-resistant proteins; 93 per cent of the colonizing isolates expressed one or more of these proteins. Overall, the most common surface protein expression profile by GBS serotype was: alpha in type Ia; alpha plus beta in type Ib; alpha and R4 in type II; R4 in type III; and co-expression of R1 plus R4 in isolates of type V. BPS was found in five (1.7%) invasive isolates, alone in two isolates and with other proteins in three isolates. Among 2660 colonizing isolates, BPS was found alone in 15 (0.6%) and in 57 additional isolates with other proteins. Among the total isolates, BPS was found predominantly in serotype Ia isolates, also expressing R1. Uncommon protein profiles of known serotypes included 11 type III isolates expressing alpha plus beta. Among 72 nontypable colonizing isolates, expression of R1 plus R4 was the commonest (33.3%) profile. INTERPRETATION & CONCLUSION: The GBS surface proteins and the common serotypes were distributed comparably in colonizing and invasive isolates. Trypsin-resistant, alpha and alpha-like proteins, R1 and R4 were the most prevalent. The phenotypic diversity of the surface-localized protein antigens of GBS is intriguing, and genotypic analysis will permit consensus in nomenclature from laboratory to laboratory.

Characterization of vaginal & rectal colonization with multiple serotypes of group B streptococci using multiple colony picks.

BACKGROUND & OBJECTIVES: There is paucity of information on vaginal and rectal colonization with multiple serotypes of group B streptococci (GBS). As part of an ongoing cohort study evaluating the natural history of vaginal and rectal colonization by GBS, the colonization with multiple serotypes was studied in 102 non-pregnant women aged 18-30 yr. METHODS: Up to ten separate colony picks of beta-haemolytic streptococci (total 1515 isolates) were selected from vaginal and rectal primary culture plates. The colonies were identified as GBS, and their capsular polysaccharides (CPS) serotypes determined using monospecific rabbit antisera for types Ia-VIII by double immunodiffusion in agarose (DID). A colony dot immunoblot (DB) assay, using monospecific rabbit antisera to purified type polysaccharides conjugated to tetanus toxoid, was developed to serotype efficiently the multiple colony picks of GBS. RESULTS: The CPS serotype distribution, examining only the 177 "a" or first colony picks from the 102 patients, was 30.5 per cent for Ia; 28.2 per cent for type III; 15.3 per cent for type II; and 13.6 per cent for type V. Only 2.8 per cent were nontypeable. Eighty of the 102 patients (78.4%) were colonized with only one serotype; 20 (19.6%) had two serotypes and two patients (2%) had three serotypes in their vaginal and/or rectal paired cultures. Overall, 91.9 per cent of the culture sites colonized with one to three CPS types (from the total number of colonies picked) were identified with a minimum of three colony picks. In 75 patients with vaginal/rectal pairs the GBS serotype concordance of only the "a" colony was 89.3 per cent and concordance decreased to 80 per cent when the serotype concordance of the total colony picks was analyzed. INTERPRETATION & CONCLUSION: In conclusion, there was a relatively high prevalence of serotype nonconcordance in this population, and 21.6 per cent of patients had multiple GBS serotypes.

Gene encoding the group B streptococcal protein R4, its presence in clinical reference laboratory isolates & R4 protein pepsin sensitivity.

BACKGROUND & OBJECTIVES: R proteins were first identified by Lancefield in group B Streptococcus (GBS) as resistant to trypsin at pH8 and sensitive to pepsin at pH2. The R4 protein found predominantly in type III and some type II and V invasive isolates conforms to these criteria. The Rib protein, although structurally and epidemiologically similar to R4, was reported as resistant to both proteases. We report here the gene encoding the R4 protein from a type III group B streptococcal isolate (76-043) well characterized in our laboratory. METHODS: Trypsin extracted GBS proteins were assayed for protease sensitivities by double-diffusion Ouchterlony using varying conditions for the enzyme pepsin. Standard haemoglobin assay was used to examine pepsin enzymatic activity. Thirty clinical isolates of varying protein profiles identified by double-diffusion from our reference strain laboratory were screened by PCR and Southern technique. SDS-PAGE gel purified R4 amino acid sequences were determined and used to design oligonucleotide primers for screening a 76-043 genomic library. RESULTS: R4 was sensitive to pepsin at pH2 but appeared resistant at pH4, the reported pH used for Rib. By standard haemoglobin assay and trypsin extract studies of R4 protein, pepsin was shown to be active at pH2, yet easily inactivated; assays of GBS surface proteins are critical at pH2. Of the amino acids initially sequenced from R4, 88 per cent (61/69) showed identity to Rib; the r4 nucleotide sequence was identical to that of rib. All isolates with strong positive protein reactions for R4 were positive in both PCR and Southern technique, whereas isolates expressing alpha, beta, R1/R4, and R5 (BPS) protein profiles were not. INTERPRETATION & CONCLUSION: Sequenced PCR products aligned with identity to the R4 and Rib nucleotide sequences and confirmed the identity of these proteins and their molecular sequences.