Acidic pH Strongly Enhances In Vitro Biofilm Formation by a Subset of Hypervirulent ST-17 Streptococcus agalactiae Strains

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.     

Molecular pathogenesis of neonatal group B streptococcal infection: no longer in its infancy

The process of human infection by group B Streptococcus (GBS) is complex and multifactorial. While this bacterium has adapted well to asymptomatic colonization of adult humans, it remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Sequencing of two complete GBS genomes has provided additional context for interpretation of experimental data and comparison to other well-studied pathogens. Here we review recent discoveries regarding GBS virulence mechanisms, many of which are revealed or magnified by the unique circumstances of the birthing process and the deficiencies of neonatal immune defence. Appreciation of the formidable array of GBS virulence factors underscores why this bacterium remains at the forefront of neonatal pathogens.     

A practical method for subtyping of Streptococcus agalactiae serotype III, of human origin, using rolling circle amplification

Group B streptococcus (GBS; Streptococcus agalactiae) serotype III is one of the most common and virulent serotypes of the species. It can be divided into several subtypes, which vary in their distribution among invasive isolates from different patient groups. In this study, we used 91 well-characterized GBS serotype III isolates to compare three subtyping methods, and developed a novel padlock probe and rolling circle amplification (RCA) method to identify informative single nucleotide polymorphisms (SNPs) that define the major subtypes. There was good agreement between partial sequencing of the capsule polysaccharide synthesis (cps) gene cluster, a 3-set genotyping system and multilocus sequence typing (MLST). Serosubtype III-2/multilocus sequence type (ST)-17 represents a virulent clone which is significantly associated with late onset GBS neonatal infections. RCA provides a simple, reproducible method for rapid identification of the two most common GBS serotype III subtypes (III-1/ST-19 and III-2/ST-17).     

Group B streptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells

Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.     

Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.     

晚期孕妇生殖道感染无乳链球菌与新生儿感染的相关性研究

目的探讨妊娠晚期妇女生殖道感染无乳链球菌（GBS）与新生儿感染的关系。方法在慈溪市妇幼保健院1192例胎膜早破妊娠晚期妇女进行宫颈分泌物培养,同时对其分娩的1196个新生儿进行鼻咽分泌物培养作为观察组,同时选择同期无胎膜早破的妊娠晚期妇女500例作为对照组进行比较。观察胎膜早破合并GBS阳性妊娠晚期妇女与新生儿感染的相关性。结果观察组分离无乳链球菌比例为16．1％（192／1192）,对照组分离无乳链球菌比例为6．0％（30／500）,两组比较无乳链球菌感染与胎膜早破差异有统计学意义（P〈0．05）。早破合并GBS阳性的妊娠晚期妇女新生儿感染率与破膜时间和分娩产程有关,破膜时间〈24h与〉24h的妊娠晚期妇女,其新生儿感染率与破膜时间成正比,产程〈24h的妊娠晚期妇女新生儿感染率明显低于24h以上的妊娠晚期妇女。结论妊娠晚期妇女无乳链球菌感染与胎膜早破有关,从而引起新生儿感染,为了早期预防新生儿感染,应对GBS阳性的妊娠晚期妇女及时治疗,并严格控制早破时间和缩短分娩产程。     

A streptococcal NRAMP homologue is crucial for the survival of Streptococcus agalactiae under low pH conditions

Streptococcus agalactiae or Group B Streptococcus (GBS) is a commensal bacterium of the human gastrointestinal and urogenital tracts as well as a leading cause of neonatal sepsis, pneumonia and meningitis. Maternal vaginal carriage is the main source for GBS transmission and thus the most important risk factor for neonatal disease. Several studies in eukaryotes identified a group of proteins natural resistance‐associated macrophage protein (NRAMP) that function as divalent cation transporters for Fe²⁺ and Mn²⁺ and confer on macrophages the ability to control replication of bacterial pathogens. Genome sequencing predicted potential NRAMP homologues in several prokaryotes. Here we describe for the first time, a pH‐regulated NRAMP Mn²⁺/Fe²⁺ transporter in GBS, designated MntH, which confers resistance to reactive oxygen species (ROS) and is crucial for bacterial growth and survival under low pH conditions. Our investigation implicates MntH as an important colonization determinant for GBS in the maternal vagina as it helps bacteria to adapt to the harsh acidic environment, facilitates bacterial adherence, contributes to the coexistence with the vaginal microbiota and plays a role in GBS intracellular survival inside macrophages.     

产前B族链球菌筛查对妊娠结局及新生儿的影响

目的研究妊娠期B族链球菌(GBS)感染对妊娠结局和新生儿的影响,探讨GBS筛查的临床意义。方法选取2016年1月至2017年6月在我院进行GBS筛查的800例孕妇为受试对象,实时荧光定量PCR技术检测GBS携带情况,根据GBS筛查结果及是否愿意接受干预治疗分为治疗组、非治疗组和GBS阴性组,对比三组妊娠结局、新生儿情况。结果 800例受试对象中,GBS阳性136例(17.0%);治疗组胎膜早破、早产、宫内感染、胎儿窘迫及新生儿肺炎、窒息、败血症、病理性黄疸发生率与GBS阴性组相比,差异均无统计学意义(Ps0.05);而与非治疗组相比,除新生儿脑膜炎差异无统计学意义(P0.05)外,其他指标差异均有统计学意义(Ps0.05)。结论妊娠期进行GBS感染的筛查具有重要意义,及时采取干预措施可有效减少不良妊娠结局及新生儿感染的发生率。     

Epidemiologically and clinically relevant Group B Streptococcus isolates do not bind collagen but display enhanced binding to human fibrinogen

Group B Streptococcus (GBS) is the leading cause of neonatal septicemia and meningitis. Pili appendages were shown to play a critical role in bacterial adhesion and colonization of human tissues. Recently it was claimed that binding of the pilus-associated adhesin PilA to collagen is a critical, initial step in promoting interactions with the α2β1 integrin expressed on brain endothelial cells. Here we show that strain NCTC10/84 used in this study is not representative for GBS isolates and question the importance of collagen as a critical extracellular matrix component for GBS infections of the central nervous system.     

Survival and biofilm formation by Group B streptococci in simulated vaginal fluid at different pHs

The Group B Streptococcus (GBS, Streptococcus agalactiae) is an important cause of neonatal and maternal infection. GBS is a commensal organism of the lower gastrointestinal and vaginal tract. A frequent mode of neonatal infection is vertical transmission from pregnant women to their foetus or neonate. The aim of this study was to evaluate the survival and biofilm production of 10 GBS strains in simulated vaginal fluid at pH 4.2, 5.5 and 6.5. GBS survived longer at higher pH than at normal vaginal pH. At pH 4.2, with the exception of two isolates that were recovered up to 48 and 72 h, viable GBS numbers declined below the limit of detection by 24 h. At higher pH, GBS survived between 3 and 15 days. All isolates investigated were biofilm producers but biofilm production was greater in tryptone soy broth compared to simulated vaginal fluid. The quantity of biofilm produced increased with the rise in the pH. This study suggests that high vaginal pH may influence both GBS survival and biofilm production and thus could be a risk factor for GBS infection.     

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.     

Srr2, a multifaceted adhesin expressed by ST‐17 hypervirulent Group B Streptococcus involved in binding to both fibrinogen and plasminogen

The Group B Streptococcus (GBS) ‘hypervirulent’ ST‐17 clone is strongly associated with invasive neonatal meningitis. Comparative genome analyses revealed that the serine‐rich repeat (Srr) glycoprotein Srr2 is a cell wall‐anchored protein specific for ST‐17 strains, the non‐ST‐17 isolates expressing Srr1. Here, we unravel the binding capacity of GBS Srr proteins to relevant components of the host fibrinolysis pathway. We demonstrate that: (i) Srr2 binds plasminogen and plasmin whereas Srr1 does not; (ii) the ability of ST‐17 strains to bind fibrinogen reflects a high level surface display of Srr2 combined with a higher affinity of Srr2 than Srr1 to bind this ligand; and (iii) Srr2 binding to host plasma proteins results in the formation of bacterial aggregates that are efficiently endocytosed by phagocytes. Importantly, we show that Srr2 increased bacterial survival to phagocytic killing and bacterial persistence in a murine model of meningitis. We conclude that Srr2 is a multifaceted adhesin used by the ST‐17 clone to hijack ligands of the host coagulation system, thereby contributing to bacterial dissemination and invasiveness, and ultimately to meningitis.     

B族链球菌感染妊娠妇女产时 抗生素治疗临床效果评价

目的探讨沈阳地区孕晚期妊娠妇女B族链球菌(group B Streptococcus,GBS)的定植率及其耐药性,评估GBS感染妊娠妇女分娩期给予产时抗生素预防(intrapartum antibiotic prophylaxis,IAP)的临床效果。方法选择2017年9月至2017年12月在沈阳市妇婴医院行GBS筛查的691例怀孕35～37周的妊娠妇女为研究对象,将GBS筛查阳性妊娠妇女中采用顺产方式分娩的38例妊娠妇女作为研究组,638例GBS筛查阴性妊娠妇女作为对照组。研究组妊娠妇女给予IAP,对照组妊娠妇女不作处理。分析两组妊娠妇女GBS定植情况、GBS菌株耐药情况及给予IAP后新生儿不良事件发生率。结果 691例妊娠妇女中有53例GBS培养阳性,GBS定植率为7.67%。全部GBS菌株对青霉素、头孢唑林及万古霉素的敏感率均为100.00%;对红霉素、克林霉素耐药率分别为81.48%和73.95%。研究组中新生儿黄疸发生率为7.89%,新生儿窒息发生率为2.63%,脑膜炎、肺炎、败血症的发生率均为0.00%。对照组中新生儿黄疸发生率为3.13%,新生儿窒息发生率为0.63%,肺炎发生率为0.16%,新生儿脑膜炎及败血症发生率均为0.00%。两组新生儿在新生儿黄疸、新生儿窒息、脑膜炎、肺炎和败血症发生率方面比较差异无统计学意义(均P0.05)。结论沈阳地区妊娠妇女GBS带菌率较高,青霉素可作为治疗的首选药物。预防性使用抗生素治疗可以改善新生儿结局。     

Genetic analysis of Streptococcus agalactiae strains isolated from neonates and their mothers

Streptococcus agalactiae or group B streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis in neonates. One of the major questions is whether the GBS strains able to cause neonatal invasive disease have peculiar genetic features. A collection of S. agalactiae strains, isolated from cervix, vagina and rectum of 10 mothers and from throat, ear and umbilicus of their newborns was genetically characterized by pulsed-field gel electrophoresis (PFGE). This study demonstrated that the strains isolated from each mother and her child were all genetically identical but that the strains from the 10 mother/child pairs mutually were genetically heterogeneous and 10 different PFGE patterns were found. Although it has been suggested that PFGE would be able to identify virulence traits to direct decisions in antibiotic management, the heterogeneous feature of GBS strains does not support broad application.     

Occurrence of surface protein genes from alpha-like protein (Alp) family in Streptococcus agalactiae isolates

Distribution of serotypes and alpha-like surface protein (Alp) of Streptococcus agalactiae (Group B Streptococci - GBS) vary with geographical region, ethnic origin and the virulence of clinical isolates. Demonstration of different genotypes based on surface protein genes improves the potential of GBS subtyping, is essential in research on new vaccines against invasive neonatal infections and may be useful in epidemiological studies. The molecular characterization of protein gene profile of GBS isolates was the main aim of this study. We evaluated the applicability of multiplex PCR for the identification of GBS protein genes from Alp family, such as: epsilon, bca, rib, alp2, alp3, alp4 and evaluated presence of these genes in the group of GBS isolates originating from vaginal or rectal carriage in pregnant women. For statistical analysis the G2 (Likelihood ratio) test was used. P values of < 0.05 were considered significant. The surface protein genes were found in all investigated strains. The epsilon gene dominated (27%) in GBS isolates originating from healthy pregnant women. The other genes were detected with the following frequency: rib (21%), alp2 (21%), bca (17%) and alp3 (14%). In the analyzed population, GBS strains with alp4 gene were not found. A statistically significant relationship between surface protein genes and capsular polysaccharides was demonstrated (p < 0.0001). The results of our study show immense diagnostic usefulness of multiplex PCR for identification of genes encoding GBS surface proteins from Alp family.     

The Optimisation of the Expression of Recombinant Surface Immunogenic Protein of <Emphasis Type="Italic">Group B Streptococcus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Response Surface Methodology Improves Humoral Immunity

Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity. The response surface methodology (RSM) and Box–Behnken design can optimise the performance in the expression of recombinant proteins. However, the biological effect in mice immunised with an immunogenic protein that is optimised by RSM and purified by low-affinity chromatography is unknown. In this study, we used RSM for the optimisation of the expression of the rSIP, and we evaluated the SIP-specific humoral response and the property to decrease the GBS colonisation in the vaginal tract in female mice. It was observed by NI–NTA chromatography that the RSM increases the yield in the expression of rSIP, generating a better purification process. This improvement in rSIP purification suggests a better induction of IgG anti-SIP immune response and a positive effect in the decreased GBS intravaginal colonisation. The RSM applied to optimise the expression of recombinant proteins with immunogenic capacity is an interesting alternative in the evaluation of vaccines in preclinical phase, which could improve their immune response.     

Respiration metabolism of Group B Streptococcus is activated by environmental haem and quinone and contributes to virulence

Group B Streptococcus (GBS) is a common constituent of the vaginal microflora, but its transmission to newborns can cause life-threatening sepsis, pneumonia and meningitis. Energy metabolism of this opportunist pathogen has been deduced to be strictly fermentative. We discovered that GBS undergoes respiration metabolism if its environment supplies two essential respiratory components: quinone and haem. Respiration metabolism led to significant changes in growth characteristics, including a doubling of biomass and an altered metabolite profile under the tested conditions. The GBS respiratory chain is inactivated by: (i) withdrawing haem and/or quinone, (ii) treating cultures with a respiration inhibitor or (iii) inactivating the cydA gene product, a subunit of cytochrome bd quinol oxidase, in all cases resulting in exclusively fermentative growth. cydA inactivation reduced GBS growth in human blood and strongly attenuated virulence in a neonatal rat sepsis model, suggesting that the animal host may supply the components that activate GBS respiration. These results suggest a role of respiration metabolism in GBS dissemination. Our findings show that environmental factors can increase the flexibility of GBS metabolism by activating a newly identified respiration chain. The need for two environmental factors may explain why GBS respiration metabolism was not found in previous studies.