Characterization of invasive isolates of Streptococcus pneumoniae among Taiwanese children

Accurate molecular surveillance is important in monitoring the dynamics of Streptococcus pneumoniae . A prospective study was conducted to collect invasive isolates of S. pneumoniae from children for genetic analysis from January 2004 to December 2006 in Taiwan. PCRs were performed to detect the zmpC and zmpD genes, both encoding a metalloprotease virulence factor in pneumococci, among these invasive isolates. During the study period, 68 invasive isolates of S. pneumoniae were obtained for analysis. Serotype 14 was the most common type isolated from children with invasive disease and was significantly associated with pneumonia (OR 3.1; 95% CI] 1.1–8.8; p 0.035). Serotype 23F was significantly associated with bacteraemia (OR 7.5; 95% CI  1.8–31.3; p 0.006). The seven-valent conjugate vaccine covered 83.8% of invasive isolates, but non-vaccine serotypes were more frequently isolated from patients with underlying diseases than from patients without underlying diseases (p 0.007 by Fisher's exact test). Clonal complexes related to international clones Spain23F ST81, Spain6B ST95, England14 ST9, Taiwan19F ST236, Taiwan23F ST242 and Colombia23F ST338 accounted for 52.9% of invasive isolates. Dissemination of the penicillin-resistant clones ST876, ST46, ST76 and ST2889, which were first identified in Taiwan, was also found; 1.5% of these invasive isolates carried the zmpC gene, and 47.1% of these invasive isolates carried the zmpD gene. In conclusion, the spread of certain international clones and some domestic antibiotic-resistant clones resulted in invasive diseases among Taiwanese children.     

Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis

Streptococcus agalactiae is considered one of the major causes of bovine intramammary infections. It is also found in the vaginas of women without any apparent clinical symptoms, but reports of neonatal infections, causing significant morbidity, are relatively frequent. The aim of this study was to evaluate the genetic diversity of S. agalactiae strains isolated from bovine milk and from asymptomatic women in Québec, Canada, by randomly amplified polymorphic DNA (RAPD) analysis. A total of 185 bovine isolates and 38 human isolates were first serotyped for capsular polysaccharide by double diffusion in agarose gel (bovine isolates) and coagglutination (human isolates). Strains were then studied by RAPD using 3 primers, designated OPS11, OPB17, and OPB18, which were selected from 12 primers. Thirty-eight percent of bovine isolates and 82% of human isolates could be serotyped. Prevalent serotypes were type III (28%) for bovine isolates and types V (26%) and III (24%) for human isolates. RAPD results showed that, taken together, all isolates (of bovine and human origin) shared 58% similarity. Ninety-four percent of these isolates were clustered in four groups (I, II, III, and IV) with 70% similarity among them. Three clusters, A (48 isolates), B (14 isolates), and C (32 isolates), with 79 to 80% similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. Relatively high heterogeneity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.     

Molecular subtyping and characterization of bovine and human Streptococcus agalactiae isolates

Streptococcus agalactiae causes severe invasive disease in humans and mastitis in cattle. Temporally matched bovine milk isolates and clinical human invasive isolates (52 each) collected in New York State over 18 months were characterized by molecular subtyping and phenotypic methods to probe the interspecies transmission potential of this species. EcoRI ribotyping differentiated 17 ribotypes, and DNA sequencing of the housekeeping gene sodA and the putative virulence gene hylB differentiated 7 and 17 allelic types, respectively. Human and bovine isolates were not randomly distributed between ribotypes or hylB and sodA clusters. The combined analysis of all subtyping data allowed the differentiation of 39 clonal groups; 26 groups contained only bovine isolates, and 2 groups contained both human and bovine isolates. The EcoRI ribotype diversity among bovine isolates (Simpson's numerical index of discrimination [mean +/- standard deviation], 0.90 +/- 0.05) being significantly higher than that among human isolates (0.42 +/- 0.15) further supports that these isolates represent distinct populations. Eight human isolates, but no bovine isolates, showed an IS1548 transposon insertion in hylB, which encodes a hyaluronidase. Based on data for 43 representative isolates, human isolates, on average, showed lower hyaluronidase activities than bovine isolates. Isolates with the IS1548 insertion in hylB showed no hyaluronidase activity. Human and bovine isolates did not differ in their abilities to invade HeLa human epithelial cells. Our data show that (i) EcoRI ribotyping, combined with hylB and sodA sequencing, provides a discriminatory subtype analysis of S. agalactiae; (ii) most human invasive and bovine S. agalactiae isolates represent distinct subtypes, suggesting limited interspecies transmission; and (iii) hyaluronidase activity is not required for all human infections.     

Prevalence of erythromycin and clindamycin resistance among Streptococcus agalactiae isolates in Germany

The antimicrobial susceptibilities of 338 clinical Streptococcus agalactiae isolates from two geographical regions in Germany were determined by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. The overall frequencies of erythromycin and clindamycin resistance were 11% and 4.7%, respectively. Determination of resistance phenotypes among the 37 erythromycin-resistant isolates revealed constitutive and inducible MLS(B) resistance in 40.6% and 37.8% of isolates, respectively, and susceptibility to clindamycin in 21.6% of isolates. Only 14.3% of isolates with inducible MLS(B) resistance were identified as clindamycin-resistant by determination of clindamycin MICs. Pulsed-field gel electrophoresis suggested a clonal distribution pattern among the erythromycin-resistant isolates.     

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.     

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.     

Genomic characterization of emerging invasive Streptococcus agalactiae serotype VIII in Alberta,Canada

European Journal of Clinical Microbiology & Infectious Diseases - Invasive Group B Streptococcus (GBS) can infect pregnant women, neonates, and older adults. Invasive GBS serotype VIII is...     

Antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Ireland

Between January 1999 and June 2002, 646 invasive isolates of Streptococcus pneumoniae were collected in Ireland. MICs of penicillin, ciprofloxacin, cefotaxime, moxifloxacin and linezolid were determined by Etest methodology. Eighty-seven (13.5%) isolates showed intermediate resistance to penicillin, while seven (1.1%) showed high-level resistance. Eighty-seven (13.5%) isolates were resistant to erythromycin, but all isolates were susceptible to cefotaxime, moxifloxacin and linezolid. The prevalence of pneumococcal isolates non-susceptible to penicillin in Ireland is worryingly high, but currently there are alternative agents available to treat invasive infection.     

Identification of a Streptococcus agalactiae protein antigen associated with bovine mastitis isolates.

Immunoblotting was used to analyze the immune response of cows to Streptococcus agalactiae. Antibody from the milk of cows immunized (via the superficial inguinal lymph node) with formalinized S. agalactiae cells or from the milk of cows with S. agalactiae mastitis reacted strongly with a group of high-molecular-weight proteinaceous antigens. The two most predominant antigenic polypeptides in this group had apparent molecular weights of 97,000 and 104,000. Because the data indicated that these two antigens, as well as several minor antigens sometimes observed in the 70- to 100-kilodalton size range, seemed to be different forms of the same protein, we refer to the entire group as Sas97/104. A monoclonal antibody that was reactive with Sas97/104 was derived and was used to purify the antigen by affinity chromatography. Whole-cell and colony blot enzyme-linked immunoassays with either the monoclonal antibody or a polyclonal serum sample raised against the affinity-purified antigen indicated that this antigen (or cross-reactive proteins with higher molecular weights) is present on the S. agalactiae strains that were freshly isolated from mastitic cows. However, the antigen was not detected in S. agalactiae of human origin, bovine strains of S. agalactiae maintained for a prolonged period in the laboratory, or other streptococci. The data are consistent with the notion that Sas97/104 is a surface antigen and does not correspond to previously described type-specific antigens of group B streptococci.     

Phenotypic and molecular characteristics of Streptococcus agalactiae isolates recovered from milk of dairy cows in Brazil

Information on the characteristics of Streptococcus agalactiae obtained from bovine sources in Brazil is still very limited. The aim of this study was to assess the phenotypic and genotypic diversity among S. agalactiae isolates from milk of dairy cows presenting clinical or subclinical mastitis in the southeast region of Brazil. Phenotypic characterization was based on physiological and serological tests. Antimicrobial susceptibility tests were carried out by the disk method. Genetic diversity was evaluated by using random amplified polymorphic DNA-PCR (RAPD-PCR) (by using the primer 1254) and pulsed-field gel electrophoresis (PFGE) (by using SmaI as the restriction enzyme) and by PCRs for detection of genes associated with resistance to erythromycin and tetracycline as well as PCRs for detection of genes coding for cell surface-associated proteins. According to the results of physiologic tests, 45 (52.9%) isolates showed beta-hemolysis and 44 (51.7%) were susceptible to bacitracin. Fourteen different biotypes were detected. The two most frequent biotypes comprised strains that were non-beta-hemolytic; fermented galactose, lactose, and salicin; produced protease; and were negative for DNase production. Serotype III was predominant (66 isolates [77.6%]), followed by serotypes II, Ia, Ib, and VI. Resistance to tetracycline and erythromycin was found in 38 (44.7%) and 9 (10.5%) isolates, respectively, with tet(O) (31.7%) and erm(B) (100%) being the most frequently occurring resistance genes. Three genes coding for surface proteins, bca, lmb, and scpB, were detected in 55 (64.7%), 7 (8.2%), and 43 (50.5%) isolates, respectively. In most cases, isolates from animals in the same herd presented closely related genetic profiles (determined by either RAPD-PCR or PFGE), which were distinct from those of isolates from different herds.     

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.     

Clinical aspects of invasive infections with Streptococcus dysgalactiae ssp. equisimilis in Japan: differences with respect to Streptococcus pyogenes and Streptococcus agalactiae infections

Streptococcus dysgalactiae ssp. equisimilis (SDSE) is increasingly being identified as a pathogen responsible for invasive and non-invasive infections. We compared the clinical features of invasive SDSE infections with those of invasive infections caused by Streptococcus pyogenes (group A streptococcus (GAS)) and Streptococcus agalactiae (group B streptococcus (GBS)). Active surveillance for invasive SDSE, GAS and GBS was maintained over 1 year at 142 medical institutions throughout Japan. Clinical information was collected together with isolates, which were characterized microbiologically. Two hundred and thirty-one invasive SDSE infections were identified, 97 other patients had infections with GAS, and 151 had infections with GBS. The median age of the SDSE patients was 75 years; 51% were male and 79% had underlying diseases. Forty-two SDSE patients (19%) presented to the emergency department. Among the 150 patients (65%) for whom follow-up was completed, 19 (13%) died and eight (5%) had post-infective sequelae (poor outcome). Insufficient white blood cell responses (<5000 cells/µL) and thrombocytopenia on admission each suggested significantly higher risk of poor outcome (ORs 3.6 and 4.5, respectively). Of 229 isolates, 55 (24%) showed an stG6792 emm type, which was significantly associated with poor outcome (OR 2.4). Clinical manifestations of invasive SDSE infections were distinct from those of invasive GBS infections. Primary-care doctors should consider invasive SDSE infections when treating elderly patients.     

Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis.

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).     

Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts

To better understand the emergence and transmission of antibiotic-resistant Streptococcus agalactiae, we compared phenotypic and genotypic characteristics of 52 human and 83 bovine S. agalactiae isolates. Serotypes found among isolates from human hosts included V (48.1%), III (19.2%), Ia and Ib (13.5% each), and II (5.8%). Among isolates from bovine hosts, molecular serotypes III and II were predominant (53 and 14.5%, respectively). Four and 21 different ribotypes were found among human and bovine isolates, respectively. A combination of ribotyping and serotyping showed that two bovine isolates were indistinguishable from human isolates. Resistance to tetracycline and erythromycin was more common among human (84.6% and 26.9%, respectively) than bovine (14.5% and 3.6%, respectively) isolates. tetM was found in all tetracycline-resistant human isolates, while tetO was the predominant resistance gene among bovine isolates. tet genes were found among various ribotypes. ermB, ermTR, and mefA were detected among erythromycin-resistant human isolates, while ermB was the only erythromycin resistance determinant among isolates from bovine hosts. For isolates from human hosts, erythromycin resistance genes appeared to be associated with specific ribotypes. We conclude that (i) human and bovine S. agalactiae isolates represent distinct populations; (ii) human host-associated S. agalactiae subtypes may occasionally be transmitted to bovines; (iii) while emergence of erythromycin and tetracycline resistance appears to largely occur independently among human and bovine isolates, occasional cross-species transfer of resistant strains or transmission of resistance genes between human- and bovine-associated subtypes may occur; and (iv) dissemination of antibiotic-resistant S. agalactiae appears to include both clonal spread of resistant strains as well as horizontal gene transfer.     

In vitro method to differentiate isolates of type III Streptococcus agalactiae from symptomatic and asymptomatic patients

Streptococcus agalactiae (group B streptococci) isolates from infected infants have been demonstrated to have three- to fourfold or higher levels of cell-associated lipoteichoic acid than isolates from asymptomatically colonized infants, suggesting a role for this cell surface polymer in the relative virulence of these organisms. The present study indicates that symptomatic isolates of type III group B streptococci can be readily differentiated from asymptomatic strains by their response to various levels of phosphate in a chemically defined medium (FMC). Both classes of isolates had the same doubling time (TD of 30 to 35 min) in FMC containing 65 mM sodium phosphate. However, levels of phosphate greater than 125 mM distinguished the two classes of strains. Asymptomatic strains pregrown in 65 mM phosphate to the stationary phase rapidly initiated growth at elevated phosphate levels, while symptomatic strains initiated growth only after a prolonged incubation period (greater than 400 min). These results suggest that the physiological growth response of clinical isolates of group B streptococci to phosphate can serve as a diagnostic aid in screening potentially virulent strains in pregnant women and newborn infants.     

Epidemiology of Streptococcus agalactiae colonization in Germany

Streptococcus agalactiae can cause severe pneumonia, sepsis and meningitis in neonates and remains one of the most prevalent causes of invasive neonatal infections. Maternal transmission of S. agalactiae during delivery can be prevented by prenatal screening and peripartal antibiotic prophylaxis. Implementation of CDC guidelines for group B streptococci (GBS) disease prevention resulted in a significant decline of invasive neonatal S. agalactiae infections in the USA. Similar national guidelines were issued in 2000 for Germany. However, the epidemiology of S. agalactiae colonization in Germany has not been investigated for more than 15 years and the impact these guidelines will have is therefore unknown. To assess colonization rates in Germany, we cultured vaginal and rectal swabs for S. agalactiae from pregnant and non-pregnant adult patients in the region of Aachen and Munich. Swabs were cultivated in selective broth medium for 24h and subsequently plated on blood agar plates according to the CDC recommendations. Colonies negative for catalase and pyrrolidonyl aminopeptidase were further differentiated by the CAMP test and a DNA probe specific for S. agalactiae. Rectal or vaginal colonization of S. agalactiae was found in 34 (16%) of 210 pregnant patients and in 41 (16%) of 250 non-pregnant women. S. agalactiae was found only in rectal swabs in 4% of pregnant and non-pregnant patients. For further characterization of the strains capsular serotypes and major surface protein antigens were determined by Ouchterlony immunodiffusion and PCR. Among the 75 different patient isolates serotype III was the most prevalent with 21 (28%) isolates, followed by 16 (21%) isolates of serotype II, 13 (17%) isolates of serotype Ia, 12 (16%) of serotype V, 11 (15%) of serotype Ib and only 2 (3%) isolates of serotype IV. The vast majority of all strains harbored genes for the major surface protein antigens, the alpha-C-protein or alpha-C-protein like antigens like Alp2-4, epsilon and Rib. These data show that S. agalactiae colonization is common in Germany and strict adherence to the guidelines for the preventions of GBS disease will result in peripartal antibiotic prophylaxis in up to 20% of all deliveries.