Group B Streptococcus Growth Research Articles

PhenoMATRIX™ for the screening of Group B Streptococcus (GBS) carriage in pregnant women: ready to get rid of the LIM broth?

AbstractThere is a constant need to reduce turn-around times and keep costs as low as possible for the carriage screening of GBS in pregnant patients. Laboratory automation might provide an edge in this field. The objectives of the present study were: i) to compare the performance of the direct chromID™ Strepto B agar (CA) plating against LIM-broth enriched plating on CA for the detection of GBS from vagino-rectal screening-swabs; and ii) to assess the usage of PhenoMATRIX™ for the automated screening of GBS. Between January 2021 and December 2023, 9′107 vagino-rectal specimens were collected from pregnant women at Geneva University Hospitals and were used to address the first objective. There was a small difference in the GBS detection rates between direct CA plating (13.2%; 1′202/9′107) and LIM-broth enriched plating on CA (13.2%; 1′198/9′107). Based on the LIM-broth enrichment results, the sensitivity and specificity of the direct CA plating were 98.3% (95% CI, 97.3%—98.9%) and 99.7% (95% CI, 99.5%—99.8%), respectively. Importantly, for 25 specimens, GBS growth was only detected by direct CA plating. We used a random set of 8′768 CA plate pictures for the machine learning of PhenoMATRIX™. The validation was carried out on an additional set of 830 CA plate pictures. The sensitivity and specificity of PhenoMATRIX™ were 100% (95% CI, 96.6%—100.0%) and 90.2% (95% CI, 87.8%—92.1%), respectively. We established that for GBS screening, the performance of direct CA plating is not inferior to the LIM-broth enriched approach. By relying on PhenoMATRIX™, the negative predictive value for GBS screening reaches 100% (95% CI, 99.4%—100.0%), enabling the automatic release of GBS-negative cases within 24 h.

Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus.

Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.

Prevalence and Risk Factors of Group B Streptococcus Colonization in Pregnant Women: A Pilot Study in Palestine.

Background Maternal Streptococcus agalactiae (Group B Streptococcus (GBS)) colonization is an important cause of complications in mothers and neonates during gestation and after delivery. The data regarding GBS colonization among pregnant women in Palestine is scarce. The aim of this study is to determine the prevalence of GBS colonization, its associated risk factors, and the antibiotic sensitivity patterns in Nablus, West Bank, Palestine. Methods A cross-sectional, single center study conducted at Rafidia Governmental Hospital in Nablus, West Bank, Palestine. Samples were collected between November 2019 and January 2020. Vaginal swabs from 200 pregnant women (≥35 weeks of gestation) attending the labor and delivery department were plated directly on CHROMagarTM StrepB (CHROM agar, France) and placed in an incubator at 35–37°C. After 24 and 48 hours, the plates were checked for growth and classified into three categories: growth of GBS with mauve colonies on chromogenic media, no growth, or other growth. The identification of the mauve colonies was confirmed by the CAMP test. Identified GBS isolates were tested for susceptibility to vancomycin, ampicillin, clindamycin, cefotaxime, erythromycin, and levofloxacin using the disc diffusion method. Clinical and demographic information were collected using a questionnaire. Result The overall prevalence of GBS colonization was 12%. The median age of the study population was 27 years. GBS colonization was significantly associated with age (p=0.013), history of previous preterm delivery (p=0.013), and parity (p=0.015). No association was noted with smoking, previous abortion, previous history of fetal demise, vaginitis, or urinary tract infection. Resistance to ampicillin, vancomycin, cefotaxime, erythromycin, clindamycin, and levofloxacin was found to be 91.7%, 54.2%, 45.8%, 29.2%, 25%, and 8.3%, respectively. Conclusion The prevalence of vaginal GBS in this study was 12% from Nablus, West Bank. Further research is needed to determine the GBS serotypes common in West Bank and the burden they cause on the health system. Moreover, this study also highlights the need to establish a screening program suited to a developing country with low control on the antibiotic's prescription protocols.

Use of Recombinant Endolysin to Improve Accuracy of Group B Streptococcus Tests.

ABSTRACTGroup B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test.IMPORTANCE Endolysin is a bacteriophage-derived enzyme that degrades the peptidoglycan in the cell wall of host bacteria; it could be used as an antimicrobial agent for selectively eliminating specific bacterial genera/species. Group B Streptococcus (GBS) causes neonatal infection via vertical transmission; prenatal GBS screening test, in which enrichment culture is followed by bacterial identification, is used to detect the presence of GBS in pregnant women. However, the presence of commensal bacteria such as Enterococcus faecalis in clinical specimens can inhibit GBS growth in the selective enrichment culture, resulting in false-negative result. Here, we demonstrated that the application of originally isolated endolysin in the enrichment culture improved the test accuracy by inhibiting unwanted E. faecalis growth and therefore avoiding false-negative results, not only in experimental settings, but also in tests using vaginal swabs.

Downregulation of Ribosomal Contents and Kinase Activities Is Associated with the Inhibitive Effect on the Growth of Group B Streptococcus Induced by Placental Extracellular Vesicles.

Simple SummaryThe bioactive properties of extracellular vesicles (EVs) in the physiological and pathophysiological conditions of pregnancy and cancers have been well investigated. However, the interaction of EVs and bacterial cells has not been studied yet. Transferring and releasing of cargos in EVs intracellularly impact the function of target cells. In this study, we investigated the effect of placental EVs on the growth of opportunistic pathogens such as Group B Streptococcus (GBS), which is a major cause of some complications of pregnancy. We found that placental micro-EVs or nano-EVs attenuated the growth of a Gram-positive bacterium, GBS. This attenuative effect at least partially required an interaction of placental EVs with GBS. Proteomic analysis showed that changes in protein synthesis or cellular energy in GBS may contribute to this inhibitory effect.Background: Like many other cell types, the human placenta produces large amounts of extracellular vesicles (EVs). Increasing evidence has shown that placental EVs contribute to the regulation of maternal immune and vascular systems during pregnancy via the transfer of their cargos. In this study, we investigated the effect of placental EVs on the growth of opportunistic pathogens that commonly colonise the female reproductive tract. Methods: Gram-positive bacterium Group B Streptococcus (GBS) and Gram-negative bacterium Escherichia coli (E. coli) were treated with placental EVs that were collected from placental explant cultures, and the growth, susceptibility, and resistance to antibiotics of the bacteria were measured. In addition, comparative proteomics analysis was also performed for the GBS with or without exposure to placental EVs. Results: When treated with placental micro-EVs or nano-EVs, the GBS growth curve entered the stationary phase earlier, compared to untreated GBS. Treatment with placental EVs also inhibited the growth of GBS on solid medium, compared to untreated GBS. However, these biological activities were not seen in E. coli. This attenuative effect required interaction of placental EVs with GBS but not phagocytosis. In addition, the susceptibility or resistance to antibiotics of GBS or E. coli was not directly affected by treatment with placental EVs. The proteomic and Western blotting analysis of GBS that had been treated with placental EVs suggested that the downregulation of cellular components and proteins associated with phosphorylation and cell energy in GBS may contribute to these attenuative effects. Conclusion: We demonstrated the attenuative effect of the growth of GBS treated with placental EVs. Downregulation of cellular components and proteins associated with phosphorylation and cell energy may contribute to the physiological changes in GBS treated with placental EVs.

Front Cover: Antibacterial and Anti‐biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus (ChemBioChem 12/2021)

The cover feature picture is a composite of a high-resolution scanning electron microscopy (SEM) of Streptococcus agalactiae biofilms at high (10 000×) magnification pseudocolored purple with ribbon diagram structures of the lactoferrin molecule between cells. S. agalactiae, also known as Group B Streptococcus (GBS) is a bacterial pathogen that causes invasive infections during pregnancy that can lead to preterm birth and neonatal sepsis. The work demonstrates that the glycoprotein lactoferrin, which is a dominant component of human breast milk, can inhibit GBS growth, viability, biofilm formation, and adherence to reproductive tissues, such as gestational membranes. More information can be found in the Full Paper by S. D. Townsend, J. A. Gaddy et al.

The impact of Lactobacillus on group B streptococcal interactions with cells of the extraplacental membranes

Group B Streptococcus (GBS) causes adverse pregnancy outcomes and neonatal disease. The recommended preventative measure is intrapartum antibiotic prophylaxis, which can prevent early onset neonatal disease but not chorioamnionitis, preterm labor, stillbirth, or late-onset disease. Novel prevention methods are therefore needed. Use of probiotics including Lactobacillus spp., has been suggested given that they are dominant members of the lower reproductive tract microbiome. Although Lactobacillus was shown to reduce recto-vaginal colonization of GBS, no studies have examined how Lactobacillus impacts GBS in the extraplacental membranes. Since Lactobacillus has been detected in the placental membranes, we sought to characterize GBS-Lactobacillus interactions in vitro using a colonizing and invasive GBS strain. While live Lactobacillus did not affect growth or biofilms in GBS, co-culture with L. gasseri led to a 224-fold increase in GBS association with decidualized human endometrial stromal cells for both GBS strains (p < 0.005). Increased association did not result in increased invasion (p > 0.05) or host cell death, though some GBS and Lactobacillus combinations contributed to a significant reduction in host cell death (p < 0.05). Since Lactobacillus secretes many inhibitory compounds, the effect of Lactobacillus supernatants on GBS was also examined. The supernatants inhibited GBS growth, biofilm formation and invasion of host cells, though strain dependent effects were observed. Notably, supernatant from L. reuteri 6475 broadly inhibited growth in 36 distinct GBS strains and inhibited GBS growth to an average of 46.6% of each GBS strain alone. Together, these data show that specific Lactobacillus strains and their secreted products have varying effects on GBS interactions with cells of the extraplacental membranes that could impact pathogenesis. Understanding these interactions could help guide new treatment options aimed at reducing GBS-associated maternal complications and disease.

The importance of porphyrins in blue light suppression of Streptococcus agalactiae

It is well documented that blue light absorption by bacterial chromophores triggers downstream production of reactive oxygen species (ROS), which in turn results in bacterial cell death. To elucidate the importance of chromophores in the bactericidal effect of blue light, and to determine whether blue light absorption per se or the presence of porphyrins known to engender ROS is crucial in blue light treatment, we studied the effect of 450 nm pulsed light on Streptococcus agalactiae, also known as Group B Streptococcus (GBS) strain COH1. GBS does not synthesize porphyrins but has a blue light-absorbing chromophore, granadaene. We irradiated planktonic cultures of GBS with or without exogenous chromophore supplementation using either protoporphyrin IX (PPIX), coproporphyrin III (CPIII), Nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide (FAD), or Flavin mononucleotide (FMN). Quantification of surviving bacterial colonies, presented as percent survival and CFU/mL (log10), showed that (1) 450 nm blue light does not suppress the growth of GBS, even though its endogenous chromophore, granadaene, absorbs light in the 450 nm spectrum. (2) The addition of either of the two exogenous porphyrins, PPIX or CPIII, significantly suppressed GBS, indicating the importance of porphyrins in the antimicrobial action of blue light. (3) Adding exogenous FMN or FAD, two known absorbers of 450 nm light, minimally potentiated the bactericidal effect of blue light, again confirming that mere absorption of blue light by chromophores does not necessarily result in bacterial suppression. (4) Irradiation of GBS with or without NAD+ or NADH supplementation—two weak absorbers of 450 nm light—minimally suppressed GBS, indicating that a blue light-absorbing chromophore is essential for the bactericidal action of blue light. (5) Collectively, these findings show that in addition to the presence of a blue light-absorbing chromophore in bacteria, a chromophore with the right metabolic machinery and biochemical structure, capable of producing ROS, is necessary for 450 nm blue light to suppress GBS.

Are Mikvehs Responsible for GBS Carriers among Orthodox Jewish Women?—A Prospective Observational Study

Background: Maternal colonization with group B streptococcus (GBS) is an important risk factor for neonatal sepsis. A “mikveh” is a pool of water for ritual immersion by Jewish women. It had been reported to be a risk factor for GBS colonization. Objective: To determine whether there is an association between ritual immersion in the mikveh pools and GBS carriership. Methods: Water samples and bacterial swabs were taken from eight mikveh pools centers at two separate occasions and tested for GBS growth. For determination of the total number of live, aerobic bacteria, each sample was grown on strep selective agar for 48 hours at 37°C and CO2 5%. Results: All the samples were tested. No trace of GBS was found in any of the samples. Conclusions: The findings of this study refute earlier findings and suggest that there is no association between ritual immersion in mikvehs and GBS carriership.

In vitro synergistic activity of erythromycin and nisin against clinical Group B Streptococcus isolates.

This study investigated the potential synergy between erythromycin and nisin against clinical Group B Streptococcus (GBS) strains. The combination of erythromycin and nisin was examined for synergistic activity using checkerboard and time-kill assays against invasive and colonizing GBS strains. Additionally, the immunological effect of the antibiotic combination was investigated in vitro using human U937 cells and ELISA analysis. Checkerboard assays confirmed an additive effect when the antimicrobials were combined, while time-kill assays demonstrated a synergistic effect when antimicrobials were combined for invasive GBS isolates. Furthermore, a significantly lower TNF-alpha response (P<0·05) was observed in U937 cells challenged with GBS when erythromycin and nisin were used in combination. The results suggest that erythromycin and nisin can act synergistically to inhibit the growth of GBS. Group B Streptococcus is the leading cause of invasive neonatal disease worldwide and is becoming increasingly more prevalent in adults. Resistance to some conventionally used antibiotics, such as erythromycin and clindamycin, continue to rise among GBS, indicating a need for alternative treatments. This study demonstrates the potential of an erythromycin-nisin combination for treatment of GBS infections and encourages further investigation of this treatment option.

Adaptive response of neonatal sepsis-derived Group B Streptococcus to bilirubin

Hyperbilirubinemia is so common in newborns as to be termed physiological. The most common bacteria involved in early-onset neonatal sepsis are Streptococcus agalactiae, commonly called Group B Streptococcus (GBS). Whilst previous studies show bilirubin has antioxidant properties and is beneficial in endotoxic shock, little thought has been given to whether bilirubin might have antibacterial properties. In this study, we performed a transcriptomic and proteomic assessment of GBS cultured in the presence/absence of bilirubin. Our analysis revealed that increasing levels of bilirubin (>100 µmol/L) negatively correlated with GBS growth (18% reduction from 0–400 µmol/L on plate model, p < 0.001; 33% reduction from 0–100 µmol/L in liquid model, p = 0.02). Transcriptome analysis demonstrated 19 differentially expressed genes, almost exclusively up-regulated in the presence of bilirubin. Proteomic analysis identified 12 differentially expressed proteins, half over-expressed in the presence of bilirubin. Functional analysis using Gene Ontology and KEGG pathways18 revealed a differential expression of genes involved in transport and carbohydrate metabolism, suggesting bilirubin may impact on substrate utilisation. The data improve our understanding of the mechanisms modulating GBS survival in neonatal hyperbilirubinemia and suggest physiological jaundice may have an evolutionary role in protection against early-onset neonatal sepsis.

Group B Streptococcus Induces Neutrophil Recruitment to Gestational Tissues and Elaboration of Extracellular Traps and Nutritional Immunity.

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a gram-positive bacterial pathogen associated with infection during pregnancy and is a major cause of morbidity and mortality in neonates. Infection of the extraplacental membranes surrounding the developing fetus, a condition known as chorioamnionitis, is characterized histopathologically by profound infiltration of polymorphonuclear cells (PMNs, neutrophils) and greatly increases the risk for preterm labor, stillbirth, or neonatal GBS infection. The advent of animal models of chorioamnionitis provides a powerful tool to study host-pathogen relationships in vivo and ex vivo. The purpose of this study was to evaluate the innate immune response elicited by GBS and evaluate how antimicrobial strategies elaborated by these innate immune cells affect bacteria. Our work using a mouse model of GBS ascending vaginal infection during pregnancy reveals that clinically isolated GBS has the capacity to invade reproductive tissues and elicit host immune responses including infiltration of PMNs within the choriodecidua and placenta during infection, mirroring the human condition. Upon interacting with GBS, murine neutrophils elaborate DNA-containing extracellular traps, which immobilize GBS and are studded with antimicrobial molecules including lactoferrin. Exposure of GBS to holo- or apo-forms of lactoferrin reveals that the iron-sequestration activity of lactoferrin represses GBS growth and viability in a dose-dependent manner. Together, these data indicate that the mouse model of ascending infection is a useful tool to recapitulate human models of GBS infection during pregnancy. Furthermore, this work reveals that neutrophil extracellular traps ensnare GBS and repress bacterial growth via deposition of antimicrobial molecules, which drive nutritional immunity via metal sequestration strategies.

Role of human milk oligosaccharides in Group B Streptococcus colonisation.

Group B Streptococcus (GBS) infection is a major cause of morbidity and mortality in infants. The major risk factor for GBS disease is maternal and subsequent infant colonisation. It is unknown whether human milk oligosaccharides (HMOs) protect against GBS colonisation. HMO production is genetically determined and linked to the Lewis antigen system. We aimed to investigate the association between HMOs and infant GBS colonisation between birth and postnatal day 90. Rectovaginal swabs were collected at delivery, as well as colostrum/breast milk, infant nasopharyngeal and rectal swabs at birth, 6 days and days 60–89 postpartum from 183 Gambian mother/infant pairs. GBS colonisation and serotypes were determined using culture and PCR. 1H nuclear magnetic resonance spectroscopy was used to characterise the mother's Lewis status and HMO profile in breast milk. Mothers who were Lewis-positive were significantly less likely to be colonised by GBS (X2=12.50, P<0.001). Infants of Lewis-positive mothers were less likely GBS colonised at birth (X2=4.88 P=0.03) and more likely to clear colonisation between birth and days 60–89 than infants born to Lewis-negative women (P=0.05). There was no association between Secretor status and GBS colonisation. In vitro work revealed that lacto-N-difucohexaose I (LNDFHI) correlated with a reduction in the growth of GBS. Our results suggest that HMO such as LNDFHI may be a useful adjunct in reducing maternal and infant colonisation and hence invasive GBS disease. Secretor status offers utility as a stratification variable in GBS clinical trials.

Activity of Genital Tract Secretions and Synthetic Antimicrobial Peptides against Group B Streptococcus.

Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. None of the CVL samples inhibited GBS. The human neutrophil peptide-1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 μg/mL and ≥50 μg/mL, respectively, while human beta-defensin 2 and LL-37 did not inhibit at highest concentration tested (100 μg/mL). In contrast, all AMP inhibited E. coli. Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms.

Avaliação da atividade antimicrobiana do alho (Allium sativum Liliaceae) e de seu extrato aquoso

Objetivou-se avaliar a atividade antimicrobiana do alho (Allium sativum Liliaceae), in natura, e do extrato aquoso, frente à Candida albicans (Ca) e a Estreptococos do grupo B (EGB). O alho in natura e os extratos aquosos 30% e 170%, foram submetidos à avaliação da atividade antimicrobiana usando os métodos de Difusão em Ágar pela técnica do disco e do poço. Os resultados mostraram que o alho, in natura, apresentou halo de inibição de 55,3 ± 2,6 milímetros (mm) frente a Ca e de 27,1 ± 2,6 mm frente à EGB, enquanto o halo de inibição do miconazol foi de 24 ± 0,5 mm e o da Penicilina G de 29,8 ± 0,3 mm. O extrato aquoso de alho a 30% não apresentou atividade antimicrobiana frente à Ca e à EGB Já o extrato aquoso de alho a 170% apresentou halo de inibição frente a Ca, de 11,3 ± 0,7 mm na técnica do disco e de 14,5 ± 0,9 mm na técnica do poço, porém não inibiu o crescimento da EGB Conclui-se que o alho in natura apresentou melhor efeito inibitório frente às cepas de Ca e de EGB

Selection of reference genes for real-time expression studies in Streptococcus agalactiae

Streptococcus agalactiae, group B streptococci (GBS) is the leading cause of severe bacterial infections in newborns. GBS expression studies allowed the identification and characterization of virulence factors and a better understanding of the host–pathogen–environment interactions. The measurement of transcript levels by quantitative real‐time PCR (qRT-PCR) is a widely used technique in GBS; however, a systematic evaluation and validation of reference gene stability for normalization purposes in GBS expression studies is currently lacking. Therefore, we analyzed the stability of 10 candidate reference genes (16SrRNA, glcK, glnA, groEL, gyrA, recA, rpoB, rpsL, sdhA and tkt) in three GBS prototype strains (O90R, NEM316 and 2603V/R) grown at different temperature conditions (37°C and 40°C). Our approach was based on the calibration of transcript levels from each gene against the number of bacteria from the same sample (ratio messenger RNA/genomic DNA). As a complementary analysis, reference gene stability was also investigated through the bioinformatic applications, geNorm and NormFinder. Considering the whole GBS development cycle, only a minority of genes were stable under both growth conditions, but this number increased when restricting the analysis to the logarithmic time-points. The range of stable genes was higher at 37°C, where recA and sdhA were stable simultaneously for the three strains, and six out of 10 genes were stable for at least two strains. At 40°C, recA showed up again as one of the best options, suggesting its potential use as reference gene in future qRT-PCR studies. The results generated with geNorm and NormFinder were consistent with those obtained experimentally and evidenced minor variations either among strains or temperature conditions.In conclusion, the fluctuation of expression of reference genes observed among different GBS strains and growth conditions highlights the importance of carefully validating, for each experimental scenario, the use of reference genes for qRT-PCR normalization purposes. Nevertheless, recA seems to be a good candidate for such optimizations.

Synergistic Effect Between Two Bacteriocin-like Inhibitory Substances Produced by Lactobacilli Strains with Inhibitory Activity for Streptococcus agalactiae

Group B streptococci (GBS) are bacterial species that colonize the vagina in pregnant women and as such may cause serious infections in neonates that passed through the birth channel. The objective of this work was to study the inhibitory activities produced by each bacteriocin-like inhibitory substance (BLIS) of Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23, and the effects of the combined BLIS-es of these lactobacilli on GBS. The interactions between the BLIS-es were assessed by qualitative and quantitative methods on agar plates. The minimum inhibitory concentrations (MICs) and fractional inhibitory concentrations (FICs) were determined by a modification of the broth microdilution and checkerboard methods, respectively. Antibiotic susceptibilities of all S. agalactiae strains were assayed and the results of these tests were evaluated for statistical significance. A 7.5% of GBS isolates were recovered from 760 pregnant women and 91% of those strains were susceptible to each BLIS produced by L. fermentum, L. rhamnosus, and also to a mixture of them. The comparisons among the BLIS-es showed statistically significant differences, with a combination of the BLIS-es from the two Lactobacillus species being better than the BLIS of each one alone (P < 0.05) as GBS growth inhibitors. Synergistic activities between the BLIS-es were found on 100% of susceptible GBS strains, MICs ranges of BLIS of L23 and L60 were 80-160 and 160-320 UA ml(-1), respectively. By the checkerboard method, the BLIS-es combination showed synergistic effect on all sensitive strains tested, with values of FICs ranging from 0.131 to 0.218. The BLIS-es produced by these lactobacilli of vaginal origin were able to inhibit S. agalactiae isolates. The results indicate that these strains may have probiotic potential for the control of GBS in women and may consequently prevent GBS infections in newborns.

IL-4 Deficiency Decreases Mortality but Increases Severity of Arthritis in Experimental Group BStreptococcusInfection

IL-4 is an anti-inflammatory cytokine that inhibits the onset and severity in different experimental arthritis models. Group B streptococci (GBS) have been recognized as an ever-growing cause of serious invasive infections in nonpregnant adults. Septic arthritis is a clinical manifestation of GBS infection. To investigate the role of IL-4 in experimental GBS infection, IL-4 deficient or competent mice were inoculated with 1 × 107 GBS/mouse. Mortality, appearance of arthritis, GBS growth in the organs, and local and systemic cytokine and chemokine production were examined. IL-4−/− mice showed lower mortality rates but increased severity of arthritis and exhibited a lower microbial load in blood, kidneys, and joints than wt mice. Increased local levels of IL-1 β, IL-6, TNF-α, MIP-1α, and MIP-2 accompanied the more severe arthritis in IL-4−/− mice. Our results suggest a detrimental role of IL-4 in GBS sepsis, whereas it plays a beneficial effect on GBS-induced arthritis.

Metallopeptidases produced by group B Streptococcus: Influence of proteolytic inhibitors on growth and on interaction with human cell lineages

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.

Activité in vitro de la moxifloxacine (8-méthoxyquinolone) seule ou en association avec le céfotaxime sur les streptocoques du groupe B

Our objective is to determine in vitro efficiency of moxifloxacin (MXF) alone or in combination with cefotaxime (CTX) on Group B streptococcus (GBS). For 21 strains of GBS isolated from newborn invasive infections (6 meningitis and 15 bacteraemia), the bacterial growth in Mueller Hinton broth with MXF and/or CTX leaded to the determination of MIC and MBC, the determination of tolerance for CTX and the evaluation of the bacteriostatic action of these antibiotics combination by calculating the FIC index. Time-kill studies were conducted for MXF and CTX alone or in combination for the first four hours, with concentrations likely reached in CSF. Study of GBS growth with crossed concentrations of MXF and CTX showed no resistant strains, no tolerant strains, and no antagonism between MXF and CTX. Killing curves demonstrated that MXF is ten-fold more active than CTX in the first four hours. MXF is an interesting antibiotic for its good activity on the GBS, suggesting that MXF is a good candidate for further evaluation in GBS meningitis in animal model.