Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.     

Capsular Gene Typing of Streptococcus agalactiae Compared to Serotyping by Latex Agglutination

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes 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 targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.     

Multilocus sequence typing system for group B streptococcus

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.     

Serotype Identification of Group B Streptococci by PCR and Sequencing

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.     

Phenotypic and genotypic characterization of group B streptococcal isolates in southern Brazil

One-hundred sixty-eight group B streptococcal (GBS) isolates from a Brazilian hospital were phenotypically and genotypically characterized. Isolates were recovered from human sources from April 2006 to May 2008 and classified as either invasive, noninvasive, or colonizing isolates. Classical methods for serotyping and antibiotic resistance profiling were employed. Clonal groups were also defined by pulsed-field gel electrophoresis (PFGE). Results showed that susceptibility to beta-lactam antimicrobials was predominant among the isolates. Only 4.7% were resistant to erythromycin and clindamycin. The erm(B) gene was widely detected in our GBS isolates, according to our phenotypic results (constitutive macrolide-lincosamide-streptogramin B [cMLSB] resistance phenotype), and the erm(A) gene was also detected in some isolates. MLSB resistance was restricted to strains isolated from patients with noninvasive infections and carriers. Serotype Ia was predominant (38.1%), serotype IV isolates were found at a high frequency (13.1%), and few isolates of serotype III were identified (3%). Pulsed-field gel electrophoresis results revealed a variety of types, reflecting the substantial genetic diversity among GBS strains, although a great number of isolates could be clustered into two major groups with a high degree of genetic relatedness. Three main PFGE clonal groups were found, and isolates sharing the same PFGE type were grouped into different serotypes. Furthermore, in a few cases, isolates from the same patients and possessing the same PFGE type were of different serotypes. These findings could be related to the occurrence of capsular switching by horizontal transfer of capsular genes.     

Population structure of invasive and colonizing strains of Streptococcus agalactiae from neonates of six U.S. Academic Centers from 1995 to 1999

The purpose of this study was to describe the population structure of group B streptococci (GBS) isolated from infected and colonized neonates during a prospective active-surveillance study of early-onset disease in six centers in the United States from July 1995 to June 1999 and to examine its relationship to bovine strains of GBS. The phylogenetic lineage of each GBS isolate was determined by multilocus sequence typing, and isolates were clustered into clonal complexes (CCs) using the eBURST software program. A total of 899 neonatal GBS isolates were studied, of which 129 were associated with invasive disease. Serotype Ia, Ib, and V isolates were highly clonal, with 92% to 96% of serotype Ia, Ib, and V isolates being confined to single clonal clusters. In contrast, serotype II and III isolates were each comprised of two major clones, with 39% of serotype II and 41% of serotype III isolates in CC 17 and 41% of serotype II and 54% of serotype III isolates in CC 19. Further analysis demonstrates that the CC 17 serotype II and III GBS are closely related to a previously described "ancestral" lineage of bovine GBS. While 120 (93%) of invasive GBS were confined to the same lineages that colonized neonates, 9 (7%) of the invasive GBS isolates were from rare lineages that comprised only 2.7% of colonizing lineages. These results are consistent with those for other geographic regions that demonstrate the highly clonal nature of GBS infecting and colonizing human neonates.     

Evaluation of methodology for serotyping invasive and nasopharyngeal isolates of Haemophilus influenzae in the ongoing surveillance in Brazil

To assess the magnitude of discrepant results obtained by routine Haemophilus influenzae serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (n = 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (n = 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of H. influenzae serotypes in the H. influenzae type b vaccine era.     

Adherence to, invasion by, and cytokine production in response to serotype VIII group B Streptococci

The adherence to and invasion of the human epithelial cell line A549 by group B streptococcus (GBS) serotype VIII strains were compared with those of serotype III strains by a conventional method and the dynamic in vitro attachment and invasion system. Twenty GBS strains, including nine vaginal isolates and one invasive isolate each of serotypes III and VIII, were used in the conventional attachment and invasion assay. Adherence to and invasion of A549 cells by serotype VIII GBS strains were significantly greater (P < 0.0001) than those by serotype III strains for both the invasive strain and vaginal isolates. Cytokine production by A549 cells following stimulation with GBS serotypes III and VIII or their purified capsular polysaccharides (CPS) was measured. Serotype III strains stimulated significantly greater tumor necrosis factor alpha (TNF-alpha) (P < 0.0001) and interleukin-10 (IL-10) (P < 0.05) production than did serotype VIII strains. IL-8 production in response to serotype VIII was significantly higher (P < 0.001) than that in response to serotype III. TNF-alpha, IL-8, and IL-10 production was greater in A549 cells infected with GBS than in the untreated control cells. TNF-alpha production was significantly greater (P < 0.005) after stimulation with purified GBS serotype III CPS than after stimulation with serotype VIII CPS, a result similar to that after stimulation with whole GBS. IL-12 production by A549 cells was observed only in response to infection with GBS serotype III, resulting in the possibility of a greater TH1 response in serotype III GBS. These results suggest differences in immune responses to infection with GBS serotypes III and VIII.     

Epidemiology of and prenatal molecular distinction between invasive and colonizing group B streptococci in The Netherlands and Taiwan

The identification of markers for virulent group B streptococci (GBS) could guide prenatal prevention and intervention strategies. We compared the distribution of serotypes and potential pathogenicity islands (PPIs) between invasive and colonizing GBS. Colonizing and invasive strains from The Netherlands and Taiwan were serotyped. We used polymerase chain reaction (PCR) for the amplification of several new PPI markers. Several combinations of PPI-specific markers and serotypes were associated with invasiveness. For Dutch neonatal strains, a receiver operating characteristic (ROC) curve with serotype and five PPI markers showed an area under the curve (AUC) of 0.963 (95% confidence interval [CI] 0.935–0.99). For Taiwanese neonatal strains, serotype and four different PPI markers resulted in an ROC curve with an AUC of 0.894 (95% CI 0.826–0.963). PPI-specific and serological markers can distinguish local neonatal invasive GBS strains from colonizing ones. Apparently, there are clear regional differences in the GBS epidemiology and infection potential of clones.     

Phenotypical and genotypical characteristics of invasive group B Streptococcus isolates in Western Sydney 2000-2005

AIMS: To analyse antimicrobial susceptibility and serotypes of group B streptococcus (GBS) bloodstream isolates from different patient groups. METHODS: Susceptibility to penicillin, erythromycin and clindamycin was measured for 99 bloodstream GBS isolates collected between October 2000 and July 2005. Multiplex PCR-based reverse line blot (mPCR/RLB) assays were used to identify macrolide resistance genes and capsular serotype for each isolate. Clinical correlation was obtained from chart review. RESULTS: Adult bacteraemia accounted for 84 of 99 (85%) isolates, and were usually associated with underlying diseases such as diabetes, malignancy and renal failure. Overall mortality was 10%. Known macrolide resistance genes [ermB (2), ermA/TR (3) and mefA/E (2)] were detected in seven of eight erythromycin resistance isolates. Four of these isolates expressed MLSB phenotype, two with constitutive (ermB) and two with inducible (ermA/TR) clindamycin resistance. Of four M phenotype isolates, two had mefA/E, one had ermA/TR and one had no detectable macrolide resistance genes. Serotype III was significantly more common in neonatal isolates; serotype V was more common among adult isolates and was associated with increased mortality. CONCLUSIONS: mPCR/RLB is a rapid molecular method to identify GBS serotype and macrolide resistance genes. This is the first major study correlating these characteristics with demographic data for invasive isolates.     

Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types.     

Application of capsular sequence typing (CST) to serotype non-viable <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> isolates from an old collection

Serotyping of Streptococcus pneumoniae is essential for monitoring changes in the pneumococcal population and the impact of vaccines. Recently, various DNA-based methods have become available and are increasingly used because they are cheaper and easier to perform than the Quellung reaction. Our aim was to apply a DNA-based method, capsular sequence typing (CST), to a collection of non-viable lyophilized pneumococcal isolates dating from the 1980s to elucidate the serotypes circulating in Italy 30 years ago. As a preliminary evaluation of the method, CST was applied to 68 recent pneumococcal isolates representative of the most common serotypes circulating in Italy in invasive pneumococcal disease (IPD) previously serotyped by the Quellung reaction. CST was then applied to 132 lyophilized non-viable isolates. A serotype-specific polymerase chain reaction (PCR), using primers suggested by the Centers for Disease Control and Prevention (CDC), was performed when CST did not yield a univocal serotype. Considering the control isolates, CST concordance with the Quellung reaction was 95.6 %. For the non-viable lyophilized isolates, CST identified a univocal serotype for 59.4 % of the isolates. This percentage increased to 78.1 % if CST was combined with serotype-specific PCR. The most frequent serotypes in the collection of non-viable strains were: 3 (15.6 %), 14 (11.7 %), 35B (5.5 %), 19A (5.5 %), and 8 (4.7 %). CST proved to be a valid method for serotyping pneumococcal strains and provided information about pneumococcal serotypes present in Italy 30 years ago. The combination of CST with serotype-specific PCR was an effective strategy to identify pneumococcal serotypes that can be suggested also for routine laboratories.     

Phylogenetic lineages of invasive and colonizing strains of serotype III group B Streptococci from neonates: a multicenter prospective study

This study compares the phylogenetic lineages of invasive serotype III group B streptococci (GBS) to those of colonizing strains in order to determine lineages associated with invasive disease. Isolates from 29 infants with early-onset disease (EOD) and from 196 colonized infants, collected in a prospective, multicenter study, were assigned a sequence type (ST) by multilocus sequence typing. Overall, 54.5% of the isolates were in the ST-19 complex, and 40.4% were in the ST-17 complex. Invasive strains were more likely to be in the ST-17 complex than were colonizing strains (59% versus 38%, P = 0.03). After we adjusted for potential confounders, the ST-17 complex was more likely to be associated with EOD than were other lineages (odds ratio = 2.51, 95% confidence interval = 1.02 to 6.20). These data support the hypothesis that ST-17 complex GBS are more virulent than other serotype III GBS.     

Serotype IX, a Proposed New Streptococcus agalactiae Serotype

We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Calpha and Cbeta proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3' end of cpsE-cpsF and 5' end of cpsG, approximately 700 bp; 3' end of cpsH and 5' end of cpsM, approximately 560 bp) were sequenced. All eight isolates expressed Calpha, and seven expressing the Cbeta protein and the corresponding genes, bca and bac, respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.     

Serotype distribution and clinical correlation of Streptococcus agalactiae causing invasive disease in infants and children in Taiwan

BackgroundStreptococcus agalactiae, or group B Streptococcus (GBS), remains to be one of the leading pathogens causing invasive infections in infants.MethodsThe clinical GBS isolates from sterile sites of patients younger than 18 years old were collected from October 1998 to December 2014 in two hospitals in Taiwan. Medical records were retrospectively reviewed. Every isolate was serotyped with a multiplex PCR assay. Multilocus sequence typing (MLST) was performed in representative isolates of different serotypes. A total of 205 GBS isolates were collected from 181 patients with 182 infection episodes.ResultsSerotype Ia was the most common in patients less than 72 h old, whereas III the most common in patients older than 72 h. In early-onset disease (0–6 days), Ia and III each caused 27.5% of the infection, followed by Ib (14.5%). In late-onset disease (7–89 days), serotype III predominated (75.3%), followed by Ia (10.1%) and Ib (6.8%). Thirty-one episodes (17%) were complicated with culture-confirmed meningitis. We compared serotype Ia and III patients, and found that serotype Ia patients were significantly younger (median age, 3 days), had more perinatal maternal fever and higher mortality. ST17 and ST19 were exclusively found in serotype III, while ST23 and ST24 comprised of 85% of serotype Ia.ConclusionIn Taiwan, serotypes Ia and III are the most common cause for early-onset and late-onset neonatal GBS infections, respectively. Some differences in the clinical features of invasive GBS infections caused by serotype Ia and III were observed.     

Rapid detection of group B streptococcal antigen by monoclonal antibody sandwich enzyme assay.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Infants at greatest risk to develop invasive disease are delivered to women colonized with GBS in their birth canals and lacking immunity to the colonizing serotype. We have investigated the sensitivity and specificity of a recently developed monoclonal antibody sandwich enzyme immunoassay for detection of GBS antigen. The sandwich enzyme immunoassay detected types II and III GBS at a concentration of 5 X 10(4) CFU/ml and types Ia and Ib GBS at 5 X 10(5) CFU/ml. No cross-reactions were noted when each of the GBS serotypes was reacted with antibodies of differing serotypes specificities. Type III GBS native antigen was detected at a concentration of 1 ng/ml. The sandwich enzyme assay is more sensitive than other methods currently in use for rapid detection of GBS and is serotype specific. This assay system should prove useful for the detection of GBS colonization during labor and for identification of neonates with invasive disease.     

Decreased capacity for type-specific-antigen synthesis accounts for high prevalence of nontypeable strains of group B streptococci in Mexico.

The low incidence of group B streptococcal (GBS) invasive neonatal disease in Mexico has been attributed to the low prevalence of serotype III strains, a major serotype in developed countries. In addition, nontypeable strains account for 12% of the isolates in Mexico and < 1% of the isolates in the United States. In this study, 57 GBS isolates (28 nontypeable by the Lancefield procedure) from carrier and infected neonates and women from Mexico were also examined for the presence of type-specific antigen by an enzymatic procedure using N-acetylmuramidase digestion of the cell wall to release soluble type-specific antigen. Of the 28 nontypeable strains from Mexico, 23 were typeable by the enzyme extraction procedure, with serotype III being the predominant serotype in invasive disease. These results suggest that nontypeable isolates of GBS should be further examined by the enzymatic extraction procedure to determine the presence of type-specific antigen. Furthermore, these limited results suggest that serotype III is likely a major serotype in invasive disease also in Mexico.     

Characterisation of invasive group B streptococci from adults in Denmark 1999 to 2004

The aim of this study was to characterise the group B streptococci (GBS) isolates causing severe invasive infections in patients >15 years of age in Denmark from 1999 to 2004. A total of 411 invasive GBS isolates were phenotypically characterised by the capsular polysaccharide (CPS) serotype and protein Cα, Cβ and R4. The incidence of invasive GBS disease ranged from 2.2 to 3.2 per 100,000 adults in the study period, being highest among adults over 65 years of age. Diabetes was observed in 15% of the cases, 12% had alcohol abuse and 7% had cancer. Of all isolates, 77% were CPS serotypes Ia, Ib, III or V. The surface proteins Cα or R4 were detected as the only protein in 57% of the GBS isolates. Cβ was detected in 12% of the isolates, but always in combination with either Cα or both Cα and R4. The incidence of invasive GBS infections continued to increase in Denmark from 1999 to 2004. In that period, the overall case fatality was 14%. The most prevalent CPS serotypes were serotypes III, Ia, V and Ib. The most prevalent surface protein was R4 when testing for R4, Cα and Cβ. There was no clear relation between the GBS phenotype and infections with fatal outcome.     

Multilocus sequence typing of Swedish invasive group B streptococcus isolates indicates a neonatally associated genetic lineage and capsule switching

Streptococcus agalactiae, also designated group B streptococcus (GBS), is an important pathogen in neonates, pregnant women, and nonpregnant adults with predisposing conditions. We used multilocus sequence typing (MLST) to characterize 158 GBS isolates that were associated with neonatal and adult invasive disease and that were collected in northern and western Sweden from 1988 to 1997. Five major genetic lineages (sequence type [ST] 19, ST-17, ST-1, ST-23, and ST-9 complexes) were identified among the isolates, including serotype Ia, Ib, and II to V isolates, indicating a highly clonal population structure among invasive GBS isolates. A number of STs were found to contain isolates of different serotypes, which indicates that capsule switching occurred rather frequently. Two distantly related genetic lineages were identified among isolates of serotype III, namely, clonal complex 19 (CC19), and CC17. CC19 was equally common among isolates from adult and neonatal disease (accounting for 10.3% of GBS isolates from adult disease and 18.7% from neonatal disease), whereas CC17 significantly appeared to be associated with neonatal invasive disease (isolated from 21.9% of neonatal isolates but only 2.6% of adult isolates). The distribution of the mobile elements GBSi1 and IS1548 reveals that they can act as genetic markers for lineages CC17 and CC19, respectively.     

Penicillin susceptibility and epidemiological typing of invasive pneumococcal isolates in the Republic of Ireland

A national study was undertaken to investigate the incidence of invasive pneumococcal disease in the Republic of Ireland and to examine the associated isolates. In 1999, 144 S. pneumoniae isolates, all recovered from cases of invasive disease, were received from 12 microbiology laboratories. The incidence of invasive pneumococcal disease was estimated to be 6.6/100000 population. All isolates were analyzed for serotype, penicillin susceptibility, chromosomal relatedness (by using pulsed-field gel electrophoresis [PFGE]), and penicillin-binding protein (pbp) fingerprinting. Several findings of note were observed regarding the pneumococcal population in Ireland. First, isolates of 25 different serotypes were represented, with serotypes 14, 9V, 8, 5, 4, and 3 being the most common. This finding, together with the pbp fingerprinting and PFGE typing results, indicated the clonal spread of strains of these serotypes in Ireland. Second, 27 (18.7%) isolates had reduced susceptibility to penicillin, and 74% of these were serotype 9V. Of these, 80% appeared to belong to the same clone. This could suggest the spread of the international Spanish/French 9V penicillin-resistant clone into Ireland. Third, nine different pbp genotypes were identified, four of which were new. Two pbp genotypes accounted for the majority of isolates dividing them according to their penicillin susceptibility status but irrespective of serotype and PFGE type. This is strong evidence for the occurrence of horizontal transfer of pbp genes between strains, observed with both penicillin-susceptible and penicillin-nonsusceptible isolates. Fourth, there was evidence of serotype transformation since isolates, indistinguishable by pbp fingerprinting and PFGE typing, expressed different capsular types.