Gene Factors and Serotypes Related to Polysaccharide and Protein-Based Candidate Vaccines Among Streptococcus agalactiae Isolates.

Background: Acute otitis media (AOM) is one of the most common pediatric infections. We aimed to investigate the bacterial profile of AOM in children, the serotype distribution, and the main genetic virulence factors involved in adhesion, immune evasion, and tissue spread. Methods: In total, 121 AOM cases involving children aged 0 to 14 years were studied. Middle ear fluids (MEF) (n = 42) and nasopharyngeal samples (n = 79) were collected. All strains were identified using routine microbiological tests, conventional PCRs and real-time PCR methods. Molecular serotyping was performed for S. pneumoniae and H. influenzae isolates. An immunofluorescence serotyping technique was employed for M. catarrhalis. Target genetic factors were determined for all involved bacterial agents using singleplex or multiplex PCRs. Results: We analyzed 148 nasopharyngeal and MEF. Among 121 AOM cases, a total of 127 bacterial agents were identified, including S. aureus (n = 41), S. pneumoniae (n = 28), H. influenzae (n = 23), M. catarrhalis (n = 19), and S. pyogenes (n = 16). The leading three serotypes among S. pneumoniae were: 19A (18.0%), 6A (14.3%), and 15B (14.3%). 91.3% of H. influenzae isolates were non-typeable (lacking a capsule-NTHi). The M. catarrhalis isolates were distributed in serotypes A (57.9%), B (26.3%), and C (15.8%). Presence of pili type 1 was detected in 21.4% pneumococci, and the fimbrial gene hifA was found in 34.8% of the H. influenzae strains. In 73.6% of the M. catarrhalis strains, ompCD was identified, while 84.2% contained ompE. 62.5% of the S. pyogenes isolates harbored the sdc gene, and 56.2% possessed the sdaD gene, predominantly in the MEF isolates. The cna adhesin was found in 28.0% of the S. aureus strains. Conclusions: The monitoring of bacterial pathogens responsible for otitis media, along with their serotype distribution and the prevalence of genetic factors involved in disease pathogenesis, is essential for public health and can help predict disease severity and treatment options.

Prevention of Neonatal Group B Streptococcal Disease

Background21 million pregnant women worldwide (18%) are estimated to carry Group B Streptococcus (GBS), which is a risk for invasive disease in newborns, pregnant women, and stillbirths. Adults ≥ 60 years or with underlying health conditions are also vulnerable to invasive GBS disease. We undertook systematic reviews on GBS organism characteristics including: capsular polysaccharide (serotype), sequence type (multi-locus sequence types (MLST)), and virulence proteins. We synthesised data by at-risk populations, to inform vaccine development. MethodsWe conducted systematic reviews and meta-analyses to estimate proportions of GBS serotypes for at risk populations: maternal colonisation, invasive disease in pregnant women, stillbirths, infants 0–90 days age, and older adults (≥60 years). We considered regional variation and time trends (2001–2018). For these at-risk population groups, we summarised reported MLST and surface proteins. ResultsBased on 198 studies (29247isolates), 93–99% of GBS isolates were serotypes Ia, Ib, II, III, IV and V. Regional variation is likely, but data gaps are apparent, even for maternal colonisation which has most data. Serotype III dominates for infant invasive disease (60%) and GBS-associated stillbirths (41%). ST17 accounted for a high proportion of infant invasive disease (41%; 95%CI: 35–47) and was found almost exclusively in serotype III strains, less present in maternal colonisation (9%; 95%CI:6–13),(4%; 95%CI:0–11) infant colonisation, and adult invasive disease (4%, 95%CI:2–6). Percentages of strains with at least one of alp 1, alp2/3, alpha C or Rib surface protein targets were 87% of maternal colonisation, 97% infant colonisation, 93% infant disease and 99% adult invasive disease. At least one of three pilus islands proteins were reported in all strains. DiscussionA hexavalent vaccine (serotypes Ia, Ib, II, III, IV and V) might provide comprehensive cover for all at-risk populations. Surveillance of circulating, disease-causing target proteins is useful to inform vaccines not targeting capsular polysaccharide. Addressing data gaps especially by world region and some at-risk populations (notably stillbirths) is fundamental to evidence-based decision-making during vaccine design.

INTRODUCTION: Group B Streptococcus (GBS) has been recognized as an important pathogen in neonatal infectious disease. However, there are few data on the prevalence of neonatal GBS infection in China. OBJECTIVE: Our aim was to estimate the infection rate of GBS in neonatal pneumonia in China and identify distribution of the GBS serotype. METHODS: We retrospectively studied 200 children with fatal neonatal pneumonia who died between 1953 and 2004; 34 fatal neonatal cases without any infectious disease were used as a control group. Paraffin-embedded lung tissues were collected for total genomic DNA extraction. Polymerase chain reaction (PCR) and Southern blotting were used for GBS detection and molecular serotyping. RESULTS: (1) The positive rate of GBS in the pneumonia group was significantly higher than that in the control group (PCR: 26% vs 3% [P < .01]; Southern blot: 65% vs 18% [P < .01]). (2) The positive rate in neonates younger than 7 days was significantly higher than that in neonates older than 7 days (PCR: 37% vs 13% [P < .01]; Southern blot: 72% vs 52% [P < .05]). (3) Risk factors were identifiable for most GBS-positive cases. (4) In the pneumonia group, 22 GBS-positive cases were serotypeable: 7 cases were identified as serotype Ia, 6 cases were serotype III, 5 cases were serotype II, and 1 case was serotype Ib. CONCLUSIONS: In China, GBS is an important pathogen in fatal neonatal pneumonia, especially in early-onset cases. Serotypes Ia, III, and II were the most common serotypes identified.

1. Laura Sass, MD* 1. *Division of Infectious Diseases, Children's Specialty Group, Children's Hospital of The King's Daughters, Norfolk, VA; Pediatrics, Eastern Virginia Medical School, Norfolk, VA. * Abbreviations: CDC: : Centers for Disease Control and Prevention CPS: : capsular polysaccharides CSF: : cerebrospinal fluid EOD: : early onset disease GBS: : group B Streptococcus IAP: : intrapartum antibiotic prophylaxis NAAT: : nucleic acid amplification test UTI: : urinary tract infection In 2010, revised guidelines were published with updated algorithms for group B Streptococcus screening, intrapartum prophylaxis, antibiotic doses, and revised management for newborns. After completing this article, readers should be able to: 1. Know the mode of transmission of group B Streptococcus (GBS) and the peripartum risk factors associated with a high risk of infection. 2. Know the major clinical manifestations of GBS and be able to differentiate the epidemiology and clinical presentation of early onset GBS disease from that of late-onset disease. 3. Know the laboratory testing for GBS: isolation, antigen detection, and susceptibility tests. 4. Know the treatment of GBS infections: drugs of choice, alternative drugs, and ineffective drugs. 5. Review the most recent guidelines for the prevention of perinatal GBS disease. 1. Understand the importance of maternal screening tests for GBS. 2. Know the criteria for the treatment of mothers known to be GBS carriers at the time of delivery. 3. Differentiate the prevention of early onset disease from that of late onset. Group B Streptococcus (GBS), or Streptococcus agalactiae , is an encapsulated gram-positive diplococcus that produces a narrow zone of β-hemolysis on blood agar and generally is resistant to bacitracin. GBS can be further differentiated by type-specific capsular polysaccharides (CPS) and protein antigens. Current circulating serotypes include Ia, Ib, Ia/c, II, III, IV, V, VI, VII, VIII, and IX. GBS initially was believed to be a bovine mastitis pathogen, but cases of puerperal sepsis in humans were described in the 1930s. The organism then became more prominent in the 1970s as a cause of maternal and neonatal disease. The most common maternal manifestations are asymptomatic bacteriuria, urinary tract infection (UTI), bacteremia, chorioamnionitis, and endometritis. In …

Distribution of genotypes and antibiotic resistance genes among invasive Streptococcus agalactiae (group B streptococcus) isolates from Australasian patients belonging to different age groups

The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain terminal galactose and sialic acid, being α-2,6 for S. suis but α-2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. Here, we investigated the role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis using inter-species allelic exchange mutagenesis. To this aim, a new molecular biogenetic approach to express CPS with modified sialic acid linkage was developed. We showed that sialic acid (and its α-2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α-2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). It was demonstrated that the observed differences in sialyltransferase activity and specificity between S. suis and GBS were serotype unrestricted. This is the first time that a study investigates the interspecies exchange of capsular sialyltransferase genes in Gram-positive bacteria. The obtained mutants represent novel tools that could be used to further investigate the immunomodulatory properties of sialylated CPSs. Finally, in spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS.

To define factors influencing vertical transmission of and neonatal colonization with group B streptococci (GBS) in neonates representing ethnically and economically diverse populations, and to determine the serotype distribution of isolates, especially new types IV-VIII. Prospective, cross-sectional study of neonates born to women evaluated for GBS colonization at admission for delivery to one of four hospitals between January 1994 and February 1995. Cultures of throat, umbilicus, and rectum were obtained from 24- to 48-hour-old infants for isolation of GBS. Isolates were classified by capsular polysaccharide (I-VIII) and C protein (alpha and beta) antigen components. Colonization was detected in 28% of 546 mothers, was higher in blacks than whites (40.6% vs 20.3%) and Hispanics (26. 9%), and was not influenced by socioeconomic status. Overall, ethnic origin did not seem to be related to GBS serotype, but whites were more likely to carry the new type V strain than blacks (6 out of 24 [25%] vs 1 out of 43 [2%]). Vertical transmission of GBS to neonates was significantly diminished when their mothers had intrapartum antibiotics (0% vs 52%), rupture of membranes <12 hours before delivery (38.4% vs 73.3%), or delivery by cesarean section (25.9% vs 45.2%). Colonization with GBS was found in 13.8% of 549 neonates, was acquired vertically in 97%, and was less frequent in neonates at the private hospitals (4% vs 20%) where intrapartum antibiotics were given more frequently (34.7% vs 17.3%). Among isolates from neonates, serotype Ia predominated (31.6%) followed by types II (25%), III (22.4%), and V (11.8%); approximately 40% of strains contained C protein antigen. Changes in the epidemiology of GBS colonization included diminished rates in some populations associated with use of maternal intrapartum antibiotics, and a shift in serotype prevalence, with Ia as predominant and V, in addition to II and III, as common.

Invasive Streptococcus agalactiae (GBS) infections are increasingly common among neonates and the elderly. Therefore, GBS surveillance for better antibiotic treatment and prophylaxis strategies are needed. We retrospectively evaluated the clinical aspects of invasive infections and the phenotypic and genetic diversity of infectious isolates from Nara, Japan, collected between 2007 and 2016, by using information from hospital records. GBS strains collected from the blood and cerebrospinal fluid cultures were evaluated for capsular types, multi-locus sequence typing (MLST), antibiotic susceptibility, antibiotics resistance gene, and pulsed-field gel electrophoresis. Forty GBS isolates (10 from children and 30 from adults) were analyzed, and the distribution of molecular serotype and allelic profiles varied between children and adults. We found the rates of early-onset disease in neonates with birth complications to be higher than that of previous reports, indicating that there could be relevance between complications at birth and early-onset disease. Standard antibiotic prophylaxis strategies may need to be reconsidered in patients with birth complications. In adults, the mean age of the patients was 68 years (male: 63%). Primary bacteremia was the most common source of infection. In the neonates, six had early-onset diseases and four had late-onset diseases. The most frequently identified strains were molecular serotype Ia ST23 (40%) and molecular serotype Ib ST10 (20%) in children and molecular serotype Ib ST10 (17%), molecular serotype VI ST1 (13%), and molecular serotype V ST1 (13%) in adults. Levofloxacin-resistant molecular serotype Ib strains and molecular serotypes V and VI ST1 were common causes of GBS infection in adults but were rarely found in children. Furthermore, pulsed-field gel electrophoresis in our study showed that specific clone isolates, that tend to have antibiotics resistance were widespread horizontally for a decade. Continuous surveillance and molecular investigation are warranted to identify the transmission route and improve antibiotic treatment strategies.

This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae.

Group B streptococci (GBS) colonizing the female genital tract will often infect newborn infants during delivery. In 200 pregnant women studied, 14% were colonized with GBS in the cervix, 12% in the rectum, and 9% in both cervix and rectum. We have previously reported that antibody levels to GBS serotypes Ia, II, and III in sera and cervical secretions were increased in women colonized in the rectum and/or cervix, when analyzed by a whole-cell ELISA. Here, we report the levels of antibodies to GBS serotype III capsular polysaccharide antigen (CPS III) and to protein antigen R4, which are present in most GBS III strains. Compared to culture-negative women, the group of women colonized rectally had markedly elevated levels of immunoglobulin (Ig)A and IgG antibodies in cervical secretions to both CPS III and protein R4 (P < 0.01 and P < 0.001, respectively). In sera, the corresponding differences between culture-negative and culture-positive women were less pronounced, or not present. In contrast to antibody levels to whole-cell GBS, antibody levels to CPS III and protein R4 in cervical secretions were not significantly increased in women colonized only in the cervix, except that IgA antibodies to protein R4 were slightly elevated (P < 0.05). These findings suggest that capsular type-specific polysaccharides and protein R4 in a mucosal vaccine might induce protective antibodies against GBS colonization of the uterine cervix.

All roads lead to Rome: N-acetylation of GBS Capsular Polysaccharides is linked to conjugate vaccine immunogenicity.

Group B Streptococcus (GBS) are frequent colonisers of the gastrointestinal tract of healthy adults with worldwide rates of 18% colonisation; however, they are also opportunistic pathogens capable of causing invasive disease, particularly in neonates and adults with underlying conditions. Indeed, GBS is the leading cause of invasive neonatal disease worldwide with a global incidence of 0.49 per 1000 live births and GBS disease in adults is increasing globally, with estimated rates of 10.9 cases per 100,000 people. While GBS remain largely susceptible to penicillin, there have been reports of reduced beta-lactam susceptibility in certain countries and resistance to other antibiotic classes continues to rise. GBS strains are distinguished by their serotype, defined by an expressed capsular polysaccharide, and vaccine efforts have primarily focused on this capsule as a target; however, as of yet, there is no vaccine licensed for use. The aims of this thesis were to identify the predominant serotypes and antimicrobial resistance properties of circulating GBS isolates in Ireland, to investigate pathogenic traits of these isolates and to examine novel therapeutic options for GBS. The molecular epidemiology of n = 253 clinical GBS strains was investigated by determining the serotype and antimicrobial resistance traits in a collection of both colonising (n = 202) and invasive (n = 51) isolates. High levels of erythromycin (22.5%) and clindamycin (21.3%) resistance were observed and the emergence of the unusual L phenotype was detected, which had not been previously reported in Ireland. Resistance to both erythromycin and clindamycin was higher among colonising isolates than colonising isolates (P &lt; 0.05), with mean zones of X mm and X mm for erythromycin and clindamycin respectively. The serotype distribution revealed that overall serotypes Ia (28.1%), III (26.88%), II (13.44%), V (11.86%) and Ib (11.46%) were the most common among GBS strains. Serotype distribution was differed among invasive and colonising isolates, whereby serotype III (31.37%) predominated among invasive isolates while serotypes Ia (29.7%) and III (25.745) were the most common in colonising strains. Serotype III predominated among resistant isolates (33.3%). To examine novel antimicrobials for GBS, the bacteriocin nisin was explored, and some level of susceptibility characterised by zones of inhibition was detected in 91% of isolates. Investigation of the genetic basis for resistance to nisin in GBS revealed the presence of a nisin resistance gene (nsr) in 98.4% of isolates, as well as the presence of an ABC transporter, nsrFP, in 84.4% of isolates. A bioengineered derivative of nisin, nisin PV, designed to resist cleavage by the nisin resistance protein (NSR), displayed significantly enhanced activity against isolates (P &lt; 0.05) compared to the wild-type peptide, with an MIC50 of 20.1 μg/ml and 6.3 μg/ml for nisin A and nisin PV respectively. Investigating the potential of novel alternative antimicrobial strategies determined that erythromycin and nisin used in combination exerted a synergistic effect in a proportion of isolates by time-kill curve assays. This combination also reduced TNF-α production (P &lt; 0.05) in U937 macrophages challenged with GBS. The capacity of GBS to form biofilms and the underlying genetic determinants associated with biofilm formation were investigated within the collection. Reducing gastrointestinal colonisation could help limit GBS infections and anti-biofilm agents are an interesting approach to help achieve this. Consuming food products with antimicrobial activity could prevent biofilm formation and therefore the potential for certain food products to inhibit and reduce GBS biofilms was assessed. Biofilm formation was significantly inhibited and reduced by natural food products garlic, honey, feta and goats cheese by the XTT assay for cell viability (P &lt; 0.05). This thesis provides a current assessment of the characteristics of clinical GBS strains circulating in the South of Ireland and has highlighted novel antimicrobial strategies for the control of GBS.

Invasive group B streptococcal (GBS) infections are common in neonates but are rare after the 1st month of life. It is not known why GBS infections have this age distribution which differs from that of invasive infections caused by other encapsulated bacteria. The aim of this study was to test the possibility that serum antibodies against the GBS capsular polysaccharides (CPS) are acquired during the first months of life thereby preventing infections after the neonatal period. Cord sera were collected from 321 healthy term newborns. A second blood sample was collected at 2, 4, 8, 13 or 26 weeks of age. IgG CPS antibodies (measured by ELISA) against serotypes Ia, II and III were present in 98%-100% of all cord sera and decreased continuously during the first 6 months of life. No IgM antibodies against serotype III CPS were present in cord sera. Only 16%-17% of the children acquired IgM antibodies against serotype III CPS at 3 and 6 months of age. Early acquisition of IgG or IgM antibodies against CPS of the most common GBS serotypes was not demonstrated and cannot explain the rare occurrence of invasive GBS infections in children after the 1st month of life.

Introduction: Maternal genitalia colonising bacteria i.e., Group B Streptococcus (GBS) well known as Streptococcus agalactiaeis responsible for serious health complications in newborns like sepsis, meningitis and pneumonitis. Centre for Disease Control and Prevention (CDC) recommends a thorough culture based screening in 35-37 weeks gestation mothers for further therapeutic steps. Previously no study was carried out on the occurrence of GBS among pregnant mothers in Assam with phylogenetic analysis. Aim: To bring the scenario of GBS infection in antepartum women by targeting the capsular serotypes with their antibiogram profile and to bring the phylogenetic relationship of the prevailing GBS isolates found in Dibrugarh district, Assam, India. Materials and Methods: This was a hospital based observational study which was carried out in District Urban Health Centre, Assam, India. Lower vaginal swabs without using speculum were collected from the enrolled late trimester pregnant women. Socio-demographic data were collected with their consent. Out of the total enrolled participants (n=345), GBS was isolated in 52 samples. These were inspected by culture techniques and further confirmed using molecular methods. Serotyping was carried out by employing multiplex PCR. Antibiotic susceptibility test was conducted as per Clinical and Laboratory Standards Institute (CLSI) guidelines and phylogenetic tree was reconstructed. Data analysis was done using Statistical Package for the Social Sciences (SPSS) version 16.0. Results: This study showed a GBS carriage rate of 15.1% in the colonising participating women. No significant association with any of the demographic and clinical factors was found. Serotype Ia (42.1%) was the ruling one followed by VI (31.6%), II (15.8%) and VII (10.5%). While 36.7% of the GBS isolates were grouped into not typeable. Serotypes Ia showed resistant towards cefotaxime, erythromycin and clindamycin and serotype II towards vancomycin. Phylogenetic evaluation showed the presence of four distinct clusters viz., I, II, III and IV with unique evolutionary trends in human GBS population in the study site. Conclusion: So far in Assam, this study reports for the first time on GBS prevailing rate in late trimester mothers which may be helpful in declining the rate of adverse neonatal outcomes with on time maternal therapeutic administration by real time monitoring of antibiogram profiling. This study also paves a way of designing Capsular Polysaccharide (CPS) based vaccines for immunising the expectant mothers to prevent adverse outcomes of the newborns.