Extensive Adaptive Changes Occur in the Transcriptome of Streptococcus agalactiae (Group B Streptococcus) in Response to Incubation with Human Blood

To enhance understanding of how Streptococcus agalactiae (group B streptococcus, GBS) adapts during invasive infection, we performed a whole-genome transcriptome analysis after incubation with whole human blood. Global changes occurred in the GBS transcriptome rapidly in response to blood contact following shift from growth in a rich laboratory medium. Most (83%) of the significantly altered transcripts were down-regulated after 30 minutes of incubation in blood, and all functional categories of genes were abundantly represented. We observed complex dynamic changes in the expression of transcriptional regulators and stress response genes that allow GBS to rapidly adapt to blood. The transcripts of relatively few proven virulence genes were up-regulated during the first 90 minutes. However, a key discovery was that genes encoding proteins involved in interaction with the host coagulation/fibrinolysis system and bacterial-host interactions were rapidly up-regulated. Extensive transcript changes also occurred for genes involved in carbohydrate metabolism, including multi-functional proteins and regulators putatively involved in pathogenesis. Finally, we discovered that an incubation temperature closer to that occurring in patients with severe infection and high fever (40°C) induced additional differences in the GBS transcriptome relative to normal body temperature (37°C). Taken together, the data provide extensive new information about transcriptional adaptation of GBS exposed to human blood, a crucial step during GBS pathogenesis in invasive diseases, and identify many new leads for molecular pathogenesis research

The application of multiplex PCR to detect seven different DNA targets in group B streptococci

Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection

Increased risk of miscarriage among women experiencing physical or sexual intimate partner violence during pregnancy in Guatemala City, Guatemala: cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Violence against women by their male intimate partners (IPV) during pregnancy may lead to negative pregnancy outcomes. We examined the role of IPV as a potential risk factor for miscarriage in Guatemala. Our objectives were: (1) To describe the magnitude and pattern of verbal, physical and sexual violence by male intimate partners in the last 12 months (IPV) in a sample of pregnant Guatemalans; (2) To evaluate the influence of physical or sexual IPV on miscarriage as a pregnancy outcome.</p> <p>Methods</p> <p>All pregnant women reporting to the maternity of a major tertiary care public hospital in Guatemala City from June 1st to September 30th, 2006 were invited to participate in this cross-sectional study. The admitting physician assessed occurrence of miscarriage, defined as involuntary pregnancy loss up to and including 28 weeks gestation. Data on IPV, social and demographic characteristics, risk behaviours, and medical history were collected by interviewer-administered questionnaire. Laboratory testing was performed for HIV and syphilis. The relationship between IPV and miscarriage was assessed through multivariable logistic regression.</p> <p>Results</p> <p>IPV affected 18% of the 1897 pregnant Guatemalan women aged 15-47 in this sample. Verbal IPV was most common (16%), followed by physical (10%) and sexual (3%) victimisation. Different forms of IPV were often co-prevalent. Miscarriage was experienced by 10% of the sample (<it>n </it>= 190). After adjustment for potentially confounding factors, physical or sexual victimisation by a male intimate partner in the last 12 months was significantly associated with miscarriage (ORadj 1.1 to 2.8). Results were robust under a range of analytic assumptions.</p> <p>Conclusions</p> <p>Physical and sexual IPV is associated with miscarriage in this Guatemalan facility-based sample. Results cohere well with findings from population-based surveys. IPV should be recognised as a potential cause of miscarriage. Reproductive health services should be used to screen for spousal violence and link to assistance.</p

Maternal colonization with Streptococcus agalactiae and associated stillbirth and neonatal disease in coastal Kenya

Streptococcus agalactiae (group B streptococcus, GBS) causes neonatal disease and stillbirth, but its burden in sub-Saharan Africa is uncertain. We assessed maternal recto-vaginal GBS colonization (7,967 women), stillbirth and neonatal disease. Whole-genome sequencing was used to determine serotypes, sequence types and phylogeny. We found low maternal GBS colonization prevalence (934/7,967, 12%), but comparatively high incidence of GBS-associated stillbirth and early onset neonatal disease (EOD) in hospital (0.91 (0.25-2.3)/1,000 births and 0.76 (0.25-1.77)/1,000 live births, respectively). However, using a population denominator, EOD incidence was considerably reduced (0.13 (0.07-0.21)/1,000 live births). Treated cases of EOD had very high case fatality (17/36, 47%), especially within 24 h of birth, making under-ascertainment of community-born cases highly likely, both here and in similar facility-based studies. Maternal GBS colonization was less common in women with low socio-economic status, HIV infection and undernutrition, but when GBS-colonized, they were more probably colonized by the most virulent clone, CC17. CC17 accounted for 267/915 (29%) of maternal colonizing (265/267 (99%) serotype III; 2/267 (0.7%) serotype IV) and 51/73 (70%) of neonatal disease cases (all serotype III). Trivalent (Ia/II/III) and pentavalent (Ia/Ib/II/III/V) vaccines would cover 71/73 (97%) and 72/73 (99%) of disease-causing serotypes, respectively. Serotype IV should be considered for inclusion, with evidence of capsular switching in CC17 strains

Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of Group B Streptococcus

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response

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

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution

<p>Abstract</p> <p>Background</p> <p>Group B <it>Streptococcus </it>(GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations.</p> <p>Methods</p> <p>To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence.</p> <p>Results</p> <p>Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005).</p> <p>Conclusion</p> <p>This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.</p

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress