(11). Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR focusing on the operon disclosed large insertions or deletions influencing the gene cluster. A sensitive circulation cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination exposed that approximately one-half of these did communicate capsular polysaccharide. A procedure for easy and reliable capsular gene typing to be included in epidemiological and monitoring studies of is definitely proposed. Intro (group B [GBS]) emerged as an important human being pathogen in the 1960s and is just about the leading cause of neonatal invasive infections (1C3). More recently, has also been recognized as a common cause of infections in immunocompromised individuals and in the elderly (4). In addition, is a notable cause of contagious mastitis in cattle (5). Some evolutionary lineages of the varieties are specifically adapted to either humans or cattle and have spread globally, whereas others are genetically more diverse and may colonize both hosts (6). In addition, may be isolated from additional varieties including additional mammals, amphibians, and fish. Subpopulations of originating from these animal varieties may occasionally cause infections in humans (7, 8). The polysaccharide capsule is definitely a major virulence factor in invasive disease caused by is definitely latex agglutination based on polyclonal antibodies specific for the 10 acknowledged capsular polysaccharides, i.e., serotypes Ia, Ib, and II to Cdh5 IX. Serological methods have limitations, as they may fail to type an isolate due to lack of or low manifestation of capsular polysaccharide under the experimental conditions. Furthermore, these methods are highly dependent on the quality of the antibodies CZC54252 hydrochloride used and on the experience of the laboratory (10). Consequently, capsular gene typing is an attractive alternative. Recently, several methods for typing of based on PCR focusing on genes in the operon were published. In this study, we compared three different PCR-based capsular gene typing methods (11C14) with serotyping by latex agglutination. In addition, the manifestation of capsular polysaccharides in strains that were nontypeable by latex agglutination was evaluated by a sensitive flow cytometric analysis (15) using monoclonal or polyclonal antibodies specific for the five most common serotypes. MATERIALS AND METHODS isolates. A total of CZC54252 hydrochloride 312 isolates were included in the study. Among these, 281 were vaginorectal isolates from pregnant women (= 251) and medical isolates from neonatal infections (= 30) in Belgium, Bulgaria, the Czech Republic, Denmark, Germany, Great Britain, Italy, and Spain. These isolates were collected during the DEVANI project (Design of a Vaccine against Neonatal Infections) supported from the Western Commission Seventh Platform. Among these DEVANI isolates, 77 were received from additional participating laboratories as being problematic in serotyping by latex agglutination. In addition CZC54252 hydrochloride to these human being isolates, 31 bovine strains, nontypeable by serological means, were included from our earlier global study (6). These strains were isolated in Australia (= 5), Europe (= 15), and the United States (= 11). All included strains were confirmed as by a positive Remel Streptex Latex Group B test (Thermo Fisher Scientific), a positive PCR using primers CZC54252 hydrochloride dltS-F and dltS-R as explained previously (14), and a positive CAMP reaction. Isolates were cultured on blood agar plates at 37C in air flow plus 5% CO2 and in Todd-Hewitt broth (Oxoid). Serotyping by latex agglutination. The strains were cultured on blood agar plates. A heavy suspension of the test organism harvested from your blood agar plate was prepared in 250 l phosphate-buffered saline (PBS), pH 7.4. For CZC54252 hydrochloride each of the 10 serotypes, a 20-l aliquot of the bacterial suspension was applied to a Wellcogen disposable reaction cards and mixed with 1 l of latex suspension (reagents Ia, Ib, and II to IX; Strep-B-Latex kit; Statens Serum.