Killing Of Group B Streptococci Research Articles

Erythrocyte-Coated Nanoparticles Block Cytotoxic Effects of Group B Streptococcus β-Hemolysin/Cytolysin.

Group B Streptococcus (GBS) emerged as a leading cause of invasive infectious disease in neonates in the 1970s, but has recently been identified as an escalating public health threat in non-pregnant adults, particularly those of advanced aged or underlying medical conditions. GBS infection can rapidly develop into life-threatening disease despite prompt administration of effective antibiotics and initiation of state-of-the-art intensive care protocols and technologies due to deleterious bacterial virulence factors, such as the GBS pore-forming toxin β-hemolysin/cytolysin (β-H/C). β-H/C is known to have noxious effects on a wide range of host cells and tissues, including lung epithelial cell injury, blood brain barrier weakening, and immune cell apoptosis. Neonatal and adult survivors of GBS infection are at a high risk for substantial long-term health issues and neurologic disabilities due to perturbations in organ systems caused by bacterial- and host- mediated inflammatory stressors. Previously engineered anti-virulence inhibitors, such as monoclonal antibodies and small molecular inhibitors, generally require customized design for each different pathogenic toxin and do not target deleterious host pro-inflammatory responses that may cause organ injury, septic shock, or death. By simply wrapping donor red blood cells (RBCs) around polymeric cores, we have created biomimetic “nanosponges.” Because nanoparticles retain the same repertoire of cell membrane receptors as their host cell, they offer non-specific and all-purpose toxin decoy strategies with a broad ability to sequester and neutralize various bacterial toxins and host pro-inflammatory chemokines and cytokines to attenuate the course of infectious disease. This proof-of-concept study successfully demonstrated that intervention with nanosponges reduced the hemolytic activity of live GBS and stabilized β-H/C in a dose-dependent manner. Nanosponge treatment also decreased lung epithelial and macrophage cell death following exposure to live GBS bacteria and stabilized β-H/C, improved neutrophil killing of GBS, and diminished GBS-induced macrophage IL-1β production. Our results, therefore, suggest biomimetic nanosponges provide a titratable detoxification therapy that may provide a first-in-class treatment option for GBS infection by sequestering and inhibiting β-H/C activity.

Regulation and Function of Pilus Island 1 in Group B Streptococcus

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.

BibA Induces Opsonizing Antibodies Conferring In Vivo Protection against Group BStreptococcus

We recently characterized the function of BibA, an immunogenic surface-associated antigen expressed by group B Streptococcus (GBS) that is involved in virulence. In this study, we performed a bibA gene variability analysis on a panel of 72 clinical isolate strains. The bibA gene was present in all the strains analyzed, and 4 allelic variants, correlating with serotype, were identified. Moreover, although the BibA protein was expressed in all strains tested, only 54% of strains demonstrated surface exposure of the antigen in vitro. Importantly, expression of BibA on the bacterial surface was correlated with protection, because mice immunized with BibA were protected against challenge by a GBS strain with high levels of surface exposure of the antigen. In agreement with these findings, serum samples from mice immunized with recombinant BibA induced neutrophil-mediated in vitro opsonophagocytic killing of GBS. In conclusion, we propose BibA as a novel GBS vaccine candidate.

CsrRS Regulates Group BStreptococcusVirulence Gene Expression in Response to Environmental pH: a New Perspective on Vaccine Development

To identify factors involved in the response of group B streptococci (GBS) to environmental pH, we performed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that the transcription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregulated. The global response to acid stress included the differential expression of genes involved in transport, metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components, were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are known to be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/R with that of a csrRS deletion mutant strain revealed that the pH-dependent regulation of 90% of the downregulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that many virulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inactivation of csrS, suggesting the implication of the CsrS protein in pH sensing. These results imply that the translocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals the up-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition, the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killing of GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specific antibodies to surface-exposed GBS proteins.

Dexamethasone Affects the Neutrophil-Mediated Bacterial Killing of Group B Streptococcus

Dexamethasone use is sometimes associated with an increased risk of infection. Group B streptococcus (GBS) is a significant cause of infection in immunocompromised adults, pregnant woman, and neonates. Neutrophil-mediated bacterial killing is an important defense against GBS infection. We hypothesized that dexamethasone would affect neutrophil-mediated bacterial killing of GBS and studied this in vitro. Neutrophils isolated from healthy adults and neonates were either 1) incubated with dexamethasone concentrations from 0 to 100 μg/mL in the presence of GBS, complement, and antibody; or 2) incubated only with similar concentrations of dexamethasone for 1 hour, then incubated with GBS, complement, and antibody. Colony counts were performed, bacterial killing calculated, and results expressed as the log antibody-1 level at which the largest reciprocal dilution promoted ≥ 90% bacterial killing. Adult neutrophil-mediated bacterial killing was not affected by dexamethasone exposure during the bacterial killing assay but neutrophil exposure to dexamethasone before the assay resulted in improved killing (p < 0.05). Neonatal neutrophil-mediated bacterial killing was significantly decreased with dexamethasone exposure during the bacterial killing assay (p < 0.05), whereas neutrophil exposure before the assay suggested an increase in bacterial killing. All these effects were concentration dependent. Dexamethasone affects the adult and neonatal neutrophil-mediated bacterial killing of GBS in vitro. The interaction of corticosteroids, neutrophils, and bacteria appears complex and dependent on the timing of these interactions, population of neutrophils, and dexamethasone concentration. These factors may account for the variable susceptibility and response to infection associated with corticosteroids.

Role of C5a-ase in group B streptococcal resistance to opsonophagocytic killing

Type III group B streptococci (GBS) can be subdivided into three subtypes, RDP III-1, III-2, and III-3, on the basis of numerical analysis of HindIII restriction endonuclease digestion patterns (HindIII RDP) with their chromosomal DNAs. In the present study, the effect of C5a on opsonophagocytic killing of a representative strain from each RDP type was investigated by using a novel optical method for determining opsonophagocytic killing, and the effect of C5a-ase treatment of C5a on opsonophagocytic killing was also investigated. Pre-stimulation of polymorphonuclear leukocytes (PMNs) with C5a significantly increased opsonophagocytic killing of all three strains. The increase in killing was abolished by pretreating the C5a with GBS that express C5a-ase, a treatment that also destroyed the chemoattractant activity of the C5a. The kinetics of killing of the RDP III-2 strain differed from those of the other two strains. The survival of the RDP III-2 bacteria continued to decline over the entire 60-min incubation of the opsonophagocytic assay when PMNs were prestimulated with C5a or with C5a that had been inactivated with GBS C5a-ase (dC5a). In contrast, killing of the RDP III-1 and III-3 strains almost ceased after 20 or 60 min when PMNs were prestimulated with dC5a or C5a, respectively. A difference in bacterial killing between the III-2 strain and the III-1 and III-3 strains therefore became increasingly apparent with prolonged incubation time. The percentage of bacteria surviving in the extracellular fluid was approximately the same as the percentages of bacteria surviving in both intracellular and extracellular locations when PMNs were prestimulated with either C5a or dC5a. These data imply that the majority of bacterial killing occurred following phagocytosis and suggest that the enhanced killing of GBS following prestimulation of PMNs with C5a resulted from increased ingestion of the bacteria.

A monoclonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci

Group B streptococci (GBS) are the leading causes of neonatal sepsis and meningitis in the United States, with a high rate of fatality and serious morbidity despite appropriate therapy. The C-protein antigens of GBS appear to be important in immunity to experimental infection, yet these antigens remain incompletely characterized with respect to their number, structure, and function. None of these proteins has yet been purified to homogeneity. We have developed a novel method for extraction of surface proteins from the A909 (Ia/c) strain of GBS by using mutanolysin. Antibodies raised in rabbits against these partially purified proteins conferred passive protection to lethal GBS infection in mice challenged with a GBS strain expressing C proteins with a heterologous capsule type. In addition, mouse monoclonal antibodies were produced and identified by reactivity with the mutanolysin-extracted proteins. One of these monoclonal antibodies (4G8) identifies an epitope on the alpha-antigen of the GBS C proteins (identified by protease susceptibility and mouse protection). On sodium dodecyl sulfate-polyacrylamide gels, this epitope appears as a series of regularly spaced bands ranging in apparent molecular mass from 160,000 to 30,000 Da. The monoclonal antibody 4G8 induces opsonic killing of GBS and protects mice from lethal challenge with GBS. Thus, the 4G8 monoclonal antibody identifies a fully protective epitope on the C-protein alpha-antigen of GBS.

Surfactant therapy of newborn rabbits impairs lung macrophage bactericidal activity.

Because in vitro studies indicate that pulmonary alveolar macrophages (PAM's) filled with phospholipid vesicles have depressed microbicidal capacity, we tested the intrapulmonary bactericidal activity of newborn PAM's after surfactant treatment. Term newborn rabbits received intratracheally either homologous surfactant or one of two artificial phospholipid vesicle preparations followed by pulmonary aerosol infection with group B streptococci (GBS). Four hours after lung infection, phagocytic killing of GBS was reduced by 70-90% in animals treated with the homologous and one of the artificial surfactants compared with untreated animals or animals that received intrapulmonary injections of the surfactant vehicle (P less than 0.02). The other artificial phospholipid preparation decreased intrapulmonary inactivation of GBS by 30-40% compared with the controls. The phospholipid vesicles in the three preparations were avidly ingested and processed by newborn PAM's. The diminished in vivo killing of GBS was not attributed to decreased viability or phagocytic behavior of the PAM's toward GBS. The bactericidal defect that was evident in the newborn PAM's appeared related to the uptake of large phospholipid vesicles in the preparations rather than to the phospholipid content of the surfactants themselves. When in vitro conditions that stimulated the alveolar environment were used, the natural surfactant preparation promoted GBS proliferation, whereas the artificial preparations did not. Our findings indicate that surfactant administration reduces the bactericidal activity of neonatal PAM's. We conclude that additional investigations are needed to ascertain the effect of surfactant replacement therapy on lost defenses of the lung.

Immunoglobulin therapy of neonatal Group B streptococcal infections

Group B streptococci (GBS) are a major cause of sepsis and meningitis in newborn babies. Neonatal GBS infections are often rapidly progressive, suggesting that the immunity to GBS is deficient. Studies have shown that opsonic antibody is required for efficient phagocytosis and killing of GBS, and neonatal GBS infections have been associated with diminished levels of anti-GBS antibody. Intravenous immunoglobulin (IVIG) has been shown to provide protective immunity in experimental GBS infection models. However, lot to lot variation in opsonic antibody levels occurs in standard IVIG preparations. Recently hyperimmune anti-GBS IVIG has been prepared with high levels of opsonic and protective antibody to GBS. Hyperimmune IVIG preparations will allow physicians to give higher quantities of specific anti-GBS antibody without having to administer large fluid volumes or large amounts of nonspecific immunoglobulin. In addition specific immunoglobulin preparations will ensure that the IVIG contains reliable antibacterial activity. Although human studies are limited they suggest that IVIG therapy in neonates may be safe and effective in treating neonatal sepsis. However, further studies are necessary to determine the role of IVIG in preventing or treating neonatal infections.

Assessment by a fluorochrome microassay of phagocytic killing of group B streptococci adherent to glass

An in vitro adhrence assay was developed to study the interaction between bovine polymorphonuclear cells (PMN) and group B streptococci (GBS) on glass. The kinetics of uptake and killing of GBS in the presence and absence of opsonins were compared by staining bacteria with a fluorochrome (acridine orange). Opsonization was not required for the PMN to ingest and kill glass-adherent GBS. Nevertheless, the presence of serum during the phagocytosis incubation, or preopsonization of bacteria, both increased the rate of ingestion and the number of bacteria per PMN at the end of the reaction (90 min). Precolostral calf serum (PCS), almost devoid of antibodies, was as effective as normal bovine serum in this respect. By contrast, PCS was not an effective opsonin source when phagocytosis took place in suspension, demonstrating further that different underlying mechanisms operate depending on the physical presentation of bacteria to phagocytes. It was noted that PMN monolayers were able to ingest unopsonized bacteria that merely settled on the glass surface during phagocytosis incubation. The method should permit investigations on the mechanisms involved in surface phagocytosis, a defense mechanism of potential importance in local infections.

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells.

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.

AMPICILLIN-GENTAMICIN SYNERGY IN TREATMENT OF GROUP B STREPTOCOCCAL MENINGITIS

Ampicillin and gentamicin in combination accelerate killing of group B streptococci (GBS) in vitro. Penicillin-amlnoglycoside combinations have been recommended for the treatment of GBS meningitis despite poor penetration of the aminogiycosides into the cerebrospinal fluid. Previous studies have demonstrated accelerated killing of GBS by combinations of penicillin or ampicillin and gentamicin. These studies utilized concentrations of amlnoglycosides not consistently attainable in the CSF following systemic administration. We examined 20 strains of GBS for accelerated killing with gentamicin concentrations attainable in the CSF by systemic administration. All strains (Ia, Ib, Ic, II, III) were Isolated from infants with sepsis or meningitis. Preliminary studies revealed that mean MIC's by tube dilution were: penicillin 0.02, ampicillin 0.16, and gentamicin 4.5ugm/ml. No synergy was detected by the checkerboard titration method. Killing kinetics were determined using 10ugm/ml of ampicillin or penicillin alone or in combination with 0.1 or 0.5ugm/ml of gentamicin.Fifteen of the 20 strains demonstrated acceierated killing with gentamicin concentrations comparable to those attained in the CSF. These findings provide a rationale for the use of a combination of a penicillin and gentamicin in treating GBS meningitis in spite of "subtherapeutic" levels of gentamicin in the CSF.

Functional characteristics of a modified immunoglobulin preparation for intravenous administration: Summary of studies of opsonic and protective activity against group B streptococci

Group B streptococci (GBS) remains a major cause of morbidity and mortality in newborn babies, despite antibiotic therapy, Recent studies suggest that phagocytosis and killing of GBS is ineffective due to a deficiency in anti-GBS antibody. Using an opsonophagocytic bacterial assay and a suckling rat model of GBS sepsis, we analyzed a modified human immunoglobulin for opsonic and protective antibody. Immune globulin (IGIV) prepared for intravenous use (Gamimune, Cutter Laboratories, Inc.) was highly protective in this experimental GBS model. Using the opsonophagocytic assay, antibody activity to several strains of types Ia, II, and III GBS were also demonstrated with IGIV. To ensure that the IGIV activity was in fact antibody, globulin from IGIV was isolated and analyzed. Purified IgG retained opsonic activity in vitro and also provided protection in experimental GBS disease. Variation in the quantity of IgG necessary for protection was observed in different GBS strains. Since IGIV has opsonic and protective activity against several strains and serotypes of GBS, its intravenous administration may provide a valuable adjunct to standard antibiotic therapy for neonatal GBS infections.