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Title
Phenotypic and genotypic determinants of colonization and pathogenesis in group B Streptococcus
Creator
Parker, Robert Edward
Date
2016
Collection
Electronic Theses & Dissertations
Description
Group B Streptococcus (GBS) is a leading cause of sepsis and meningitis in neonates, an important factor in premature and still births worldwide, and a threat to immunocompromised and elderly adults. The prevention strategy for neonatal invasive disease targets maternal colonization, the primary risk factor, through late-gestational screening and intrapartum antibiotics. This approach has resulted in a significant decline in disease rates; however, this decrease has stagnated in the 2000s,...
Show moreGroup B Streptococcus (GBS) is a leading cause of sepsis and meningitis in neonates, an important factor in premature and still births worldwide, and a threat to immunocompromised and elderly adults. The prevention strategy for neonatal invasive disease targets maternal colonization, the primary risk factor, through late-gestational screening and intrapartum antibiotics. This approach has resulted in a significant decline in disease rates; however, this decrease has stagnated in the 2000s, and maternal re-colonization following treatment is common. For my doctoral work, I examined phenotypic and genotypic factors which facilitate colonization, antibiotic tolerance, and persistence of GBS including biofilm production, quorum sensing, and phenotypic heterogeneity. The assessment of biofilm formation across a diverse set of isolates, including colonizing and invasive clinical human strains, found that weak biofilm production correlated with genotype, pilus profile, and invasive disease. Furthermore, asymptomatic colonization was associated with strong biofilm production suggesting a colonization advantage for strong biofilm producers. The role of a putative quorum-sensing auto-inducing peptide, RgfD, was investigated through the creation of a deletion mutant through homologous recombination. In this work, rgfD was found to drive adherence to decidualized human endometrial cells through the upregulation of the regulator of fibrinogen-binding two-component system suggesting quorum-sensing in GBS is important for colonization. Lastly, the importance of phenotypic diversification in a single strain of GBS was assessed through the identification and characterization of a locked mutant small colony variant (SCV) derived from a clinical isolate. This is the first demonstration of SCV formation in GBS. The mutant SCV displayed increased penicillin tolerance and biofilm production, but reduced phagocytic uptake by THP-1 macrophages. Furthermore, the SCV phenotype was inducible when treated with antibiotics or exposed to acidic pH, which, alongside whole transcriptome analysis, suggests variant-formation to be driven by stress response in GBS. The work contained herein furthers the understanding of GBS colonization and identifies key phenotypic and genotypic characteristics driving colonization and persistence that must be considered in the development of future therapeutics.
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Title
The impact of lactobacillus and bacteriophage on group b streptococcus and the placental membranes
Creator
Shiroda, Megan
Date
2019
Collection
Electronic Theses & Dissertations
Description
The human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in...
Show moreThe human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in this site, but few studies have been conducted to characterize their interactions in vitro or to understand their impact on the placental membranes. Further, our knowledge of the viral component of the microbiome in human health remains incomplete.In this dissertation, Lactobacillus, a well-studied probiotic in other body sites, was evaluated for its effect in the placental membranes. As these membranes were previously considered sterile, we sought to assess the ability of Lactobacillus to colonize these cells and evaluate its impact on them. A cell line model of the outermost layer of these membranes, the decidual cells, was used to establish that Lactobacillus can associate and impact a known cell signaling pathway, the Mitogen Activated Protein Kinase (MAPK) pathway, which is associated with inflammation and host cell death. Total protein level of p38, an important upstream protein in this pathway, was found to be significantly lower with Lactobacillus than in mock infection. These data suggest that Lactobacillus could maintain a commensal interaction in the placental membranes as described in other body sites. Lactobacillus is also known to inhibit pathogen invasion. Group B Streptococcus (GBS) can ascend from the vaginal tract to infect placental membranes, triggering premature birth or neonatal infection. Four Lactobacillus strains representing three species were characterized to assess their impact on two GBS strains (colonizing and invasive). We found live Lactobacillus does not affect GBS growth or biofilm production. L. gasseri increased association of both strains of GBS to the decidual cells but did not result in increased invasion of the cells. Instead, co-culture with Lactobacillus reduced host cell death. Secreted products of Lactobacillus drastically reduced growth in 35 GBS strains that broadly represent GBS diversity and could prevent biofilm formation; this inhibition was strain dependent. Unfortunately, increased GBS-induced host cell death with Lactobacillus supernatants was observed. Collectively, these data suggest that both live Lactobacillus and its supernatant could impact GBS interactions with the placental membranes.Bacteriophage are one of the most abundant members of the microbiome but their impact on opportunistic pathogens such as GBS remains unknown. As GBS can be isolated from gastrointestinal tract, we hypothesized fecal phage communities would inhibit the growth of GBS in vitro. Approximately 6% of the tested communities inhibited the growth of 35 GBS strains. To better understand differences in GBS strain inhibition, we examined capsule, sequence and clinical types of the strains. As no significant differences were found with these traits, we next examined Clustered Regularly Interspaced Palindromic Repeats (CRISPR), which serve as an adaptive immune system against invading foreign DNA by the acquisition of spacer sequences. GBS strains with fewer than nine spacer sequences were less likely to be lysed by a phage community than strains with more than sixteen spacers. We further hypothesized that presence of GBS in the corresponding bacterial component of each phage community would correlate to its ability to inhibit GBS growth. While this correlation did not exist across all GBS strains tested, sensitive strains of GBS were significantly more likely to be inhibited by phage communities with a lower abundance of GBS. Collectively, these data suggest that the phage component of the intestinal microbiome could impact GBS colonization. To further characterize these interactions, an individual bacteriophage should be isolated from these communities.
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Title
Variation in host-pathogen interactions among genetically diverse strains of Group B Streptococcus
Creator
Korir, Michelle Lynn
Date
2016
Collection
Electronic Theses & Dissertations
Description
"Streptococcus agalactiae, or Group B Streptococcus (GBS), is a highly diverse species that can be found asymptomatically colonizing the gastrointestinal and genital tracts of healthy adults, but is also capable of causing severe invasive disease. GBS is a leading cause of sepsis and meningitis in neonates and the only preventative measure is antibiotic therapy given to pregnant mothers during labor to prevent transmission. Although this method was effective at reducing case rates upon...
Show more"Streptococcus agalactiae, or Group B Streptococcus (GBS), is a highly diverse species that can be found asymptomatically colonizing the gastrointestinal and genital tracts of healthy adults, but is also capable of causing severe invasive disease. GBS is a leading cause of sepsis and meningitis in neonates and the only preventative measure is antibiotic therapy given to pregnant mothers during labor to prevent transmission. Although this method was effective at reducing case rates upon implementation, case rates have remained unchanged since the initial decline and some mothers remain persistently colonized by GBS. Due to the high level of diversity among strains it is important to understand how strains differ at the various stages of disease progression in order to have a more complete understanding of GBS pathogenesis. Here, I examined how genotypically diverse strains differ in their interactions with human cells. The examination of strains of the same serotype in ability to associate with decidual cells and lung epithelial cells revealed that strains within the same serotype, and even the same sequence type (ST) differed in attachment and invasion, but this variation was dependent on host cell type. More specifically, strains of the hypervirulent lineage, ST-17 associated with decidual cells significantly more than the other STs, but the opposite was true for lung epithelial cell attachment. Mechanisms of persistent colonization was explored by comparing antibiotic tolerance and macrophage survival between ST-17 and ST-12 strains, which persisted and was eradicated after antibiotic prophylaxis, respectively. This study revealed that although the ST-17 strain was not tolerant to antibiotics, subinhibitory antibiotics enhanced phagocytic uptake of this strain where it was able to survive for an extended period of time. Additionally, intracellular survival of the ST-17 strain was dependent on acidification of the phagosome, whereas altered pH had no effect on survival of the ST12 strain, suggesting GBS can use different mechanisms of survival. Moreover, serotype III GBS strains were better able to survive phagosomal stress compared to other serotypes. Lastly, transcriptome analysis of the ST-17 strain during intracellular survival revealed temporal gene expression responses to long term survival and identified a large number of factors important for intracellular survival. Through mutagenesis studies, the roles of NADH peroxidase (Npx) and cadmium resistance protein (CadD) in GBS intracellular survival was examined. These studies demonstrated that Npx promotes resistance to reactive oxygen stress through detoxification of hydrogen peroxide and CadD serves as a heavy metal efflux pump to confer resistance to intoxication by certain divalent metal cations. The work described here reveals new insights in GBS pathogenesis and helps identify key virulence factors that can serve as targets for alternative therapeutics and vaccine development."--Pages ii-iii.
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Title
Genetic diversity and virulence gene characterization of Group B Streptococcus
Creator
Springman, Amber Cody
Date
2012
Collection
Electronic Theses & Dissertations
Description
Group B Streptococcus (GBS, Streptococcus agalactiae) is a Gram-positive, beta-hemolytic bacterium that was first described as a causative agent in bovine mastitis. GBS has since become a leading cause of pneumonia, sepsis, and meningitis newborns and has also emerged as an opportunistic pathogen in the elderly and adults with underlying medical conditions. The accessibility to a collection of GBS strains from colonized women, newborns, adults, elderly, and bovines has provided an excellent...
Show moreGroup B Streptococcus (GBS, Streptococcus agalactiae) is a Gram-positive, beta-hemolytic bacterium that was first described as a causative agent in bovine mastitis. GBS has since become a leading cause of pneumonia, sepsis, and meningitis newborns and has also emerged as an opportunistic pathogen in the elderly and adults with underlying medical conditions. The accessibility to a collection of GBS strains from colonized women, newborns, adults, elderly, and bovines has provided an excellent basis for conducting molecular epidemiological studies. The use of molecular subtyping methods including multilocus sequence typing (MLST), PCR-based RFLP, and mulitiplex PCR has allowed for inferences into the phylogenetic relationships among strains and the identification of specific virulence determinants associated with different clonal lineages. During the first study of this dissertation, the extent of genetic diversity and allelic variation within key virulence genes were analyzed in a panel of 94 GBS strains from diverse sources (e.g. pregnant women, newborns, elderly adults, and bovines). In the second study, a more inclusive set of 295 human-derived and bovine-derived strains representing 73 sequence types were evaluated for the presence of and variation in horizontally acquired pilus islands (PIs). Extensive genetic analyses conducted in these studies revealed that recombination and horizontal gene transfer have played a major role in the diversification of GBS clonal lineages. Furthermore, evolutionary pressures have favored the selection of specific allelic determinants that has led to the emergence of highly specialized lineages that have become successful at causing disease in humans. Overall, this research has strong implications for vaccine development since many of the virulence genes examined herein have been proposed as vaccine candidates. The high degree of sequence variability observed in many of the potential vaccine targets underscores the difficulty of developing a broadly protective, universal vaccine against GBS. Continued efforts to assess the genetic diversity of GBS populations and the mechanisms involved in pathogenesis will prove critical for establishing new prevention measures to combat GBS-associated disease.
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Title
A study of experimental streptococcic mastitis in dairy cattle
Creator
Bryan, Claude Stever, 1908-
Date
1937
Collection
Electronic Theses & Dissertations