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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
A study of the lactobacilli in tomato whey medium and soy bean milk
Creator
Grover, Donald Alonzo
Date
1934
Collection
Electronic Theses & Dissertations
Title
Effects of lactic acid bacteria on the immune system
Creator
Simon, Maria Victoria Tejada
Date
1998
Collection
Electronic Theses & Dissertations
Title
Inhibition of Neisseria gonorrhoeae epithelial cell interactions by vaginal Lactobacillus species
Creator
Spurbeck, Rachel R.
Date
2010
Collection
Electronic Theses & Dissertations
Title
Investigating the role of Lactobacillus reuteri and the microbiota in bone health
Creator
Quach, Darin
Date
2016
Collection
Electronic Theses & Dissertations
Description
Osteoporosis is a disease characterized by low bone mass, which can subsequently lead to an increased risk of sustaining a bone fracture. With the advancements made in research and medicine in the past century, the average lifespan has increased substantially. However, this has also lead to an increase in the elderly population that is susceptible to age-related diseases such as osteoporosis. It is currently estimated that over 300 million people worldwide are impacted by osteoporosis. Taking...
Show moreOsteoporosis is a disease characterized by low bone mass, which can subsequently lead to an increased risk of sustaining a bone fracture. With the advancements made in research and medicine in the past century, the average lifespan has increased substantially. However, this has also lead to an increase in the elderly population that is susceptible to age-related diseases such as osteoporosis. It is currently estimated that over 300 million people worldwide are impacted by osteoporosis. Taking into consideration the side effects stemming from medications used to treat this illness, there has been an increase in research efforts to develop novel therapeutics for osteoporosis. One area of research that has garnered recent interest involves investigating the therapeutic potential of the gut microbiota in bone health. As a result, an overarching goal in this area of research revolves around identifying microbes that impact bone health and understanding the mechanisms mediating these responses. In this thesis, I present the beneficial use of the probiotic bacterium Lactobacillus reuteri (L. reuteri) in an in vivo murine model of osteoporosis mediated by estrogen deficiency. Using female Balb/c mice that are rendered estrogen deficient following ovariectomy, we demonstrated that supplementation with L. reuteri was capable of preventing bone loss. In addition to this, we identified that the process of osteoclastogenesis was down-regulated following L. reuteri treatment in these mice suggesting that this could be the mechanism by which L. reuteri confers its benefit on bone health. Osteoclasts are the main cell type responsible for bone resorption. Using an in vitro model of osteoclastogenesis, we demonstrated that cell-free conditioned medium (CCM) from L. reuteri inhibited the maturation of osteoclasts from macrophages. We further characterized this by demonstrating that L. reuteri CCM halted osteoclastogenesis at an intermediate stage characterized by fused polykaryons. Using an antagonist to the G protein coupled receptor GPR120, we decreased the ability of L. reuteri CCM to suppress osteoclastogenesis from 70% to 38% suggesting that L. reuteri is partially signaling through GPR120 to suppress osteoclastogenesis. Taking into account that GPR120 was a receptor for long chain fatty acids, we investigated the impact of lactobacillic acid (LA), a long chain fatty acid produced by L. reuteri, and observed that the suppression of osteoclastogenesis by L. reuteri involved the production of LA. Moreover, purified LA could suppress osteoclast formation in a dose dependent manner. To elucidate the effect of L. reuteri treatment on host cell physiology, we performed RNA sequencing at multiple time points during osteoclastogenesis. An analysis of the transcriptome data identified several pathways that were modulated following L. reuteri treatment. Further investigations indicated that NF-κB and p38 activation were impacted by L. reuteri in RAW264.7 cells. These sets of experiments have led to the identification of a possible effector molecule produced by L. reuteri and mechanism by which it acts to benefit bone health. In the last part of this thesis, I present an in vivo murine model using germ free (GF) mice to study the impact of the gut microbiota on bone health. By colonizing GF mice with microbiotas with different compositions, the goal was to identify specific microbes that could be impacting bone health in either a positive or negative manner. Interestingly enough, we discovered that the microbial communities that were introduced into the different groups of mice in our studies did not impact bone health in comparison to the GF control group. This was in stark contrast to existing literature that reported a deleterious effect in bone health following the introduction of a gut microbiota. This reinforced the fact that our knowledge remains limited in terms of understanding how the gut microbiota impacts bone health. Nevertheless, the discoveries stemming from these studies contribute to the growing body of work in this discipline and will guide future research that aims to uncover novel therapeutic options to combat osteoporosis.
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Title
Construction of a secretome mutant library of Lactobacillus reuteri
Creator
Ortiz Villalobos, Javiera
Date
2013
Collection
Electronic Theses & Dissertations
Description
During the last 10 years increased scientific attention has been given to probiotics due to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri 6475 has emerged as a potential probiotic strain that has demonstrated various probiotic therapeutic features. These characteristics include the ability of this strain to produce and secrete the antimicrobial compound reuterin and anti-inflammatory factors. Because the main probiotic effects...
Show moreDuring the last 10 years increased scientific attention has been given to probiotics due to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri 6475 has emerged as a potential probiotic strain that has demonstrated various probiotic therapeutic features. These characteristics include the ability of this strain to produce and secrete the antimicrobial compound reuterin and anti-inflammatory factors. Because the main probiotic effects of L. reuteri 6475 that have been identified thus far are secreted, we were interested in developing a mutant library consisting of disruptions of genes that encode for secreted or cell-wall proteins. To complete this task we employed two different strategies: the identification of secretome protein sequences by predictive mathematical methods and the disruption of the protein coding sequences by single-stranded DNA recombineering. As a result, we have developed a mutant library of 127 secretome genes that would drive the elucidation of important L. reuteri probiotic mechanisms of action. To demonstrate the utility of this library, I screened all 127 mutants for the ability to produce reuterin and found 11 genes that increase reuterin secretion and 3 that eliminate reuterin secretion when disrupted. Future characterization of these genes will further elucidate the bacterial pathways that are critical for reuterin production.
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