Alternate Germinants of C. Difficile, a Leading Hospital Pathogen

Clostridium difficile infections (CDI) are the leading nosocomial infections worldwide. Humans are asymptomatic carriers of C. difficile spores in the intestinal tract. The process known as germination occurs when otherwise harmless C. difficile spores are converted to toxin-producing cells upon recognition of bile salts in humans. This distinctive transition ultimately leads to the onset of disease and recurrent CDI. Germination profiles will be characterized in response to peptidoglycan (PG) fragments isolated from various bacterial species. These specific peptidoglycan fragments contain different amino acid residues that may induce different germination responses. Purification and structural determination of the peptidoglycan fragments will be carried out by HPLC-MS. In this study, C. difficile germination will be tested against exhausted media containing cellular debris, as well as with solutions obtained from post-germination assays. This will reveal if germination of C. difficile induces other spores to germinate as well. If it is shown that there are alternant germinants of C. difficile, further characterization and modeling of C. difficile can be made, and further inhibitors can be tested to ensure complete inactivation of spores, ultimately preventing CDI

Kinetics of Bacillus anthracis and Bacillus cereus spore germination in soil and the C. elegans intestine

Bacillus anthracis and Bacillus cereus are both described as soil bacteria, but are almost exclusively found as spores within the soil. Soil is generally not a nutrient-rich environment and may lack the amino acids and nucleosides necessary for spore germination and vegetative reproduction. We aim to determine if soil alone can cause germination in these two species in order to produce vegetative cells that can reproduce. In addition, nematodes, decaying meat, maggots, and plant roots will be tested for their ability to cause germination in these species

Synthesis of chimeric receptors essential for spore germination

Various species of bacteria have been reported to form an endospore, a metabolically dormant cell, during times of nutrient deficiencies and extreme stress. These said structures are outstandingly resistant to harsh chemicals, extreme temperatures, and can revert back to a metabolically active cell, through a process known as germination, when the necessary conditions are met. The rigid membrane of the endospore contains various germination (Ger) receptors which sense the external environment for necessary metabolites and germinants. Ger receptors are encoded by tricistronic operons that produce three distinct membrane proteins, the A, B, and C subunits. Although the function of the Ger receptor has been established by genetics, no information is currently available for germinant binding site. Bioinformatic and genetic approaches has predicted that the C-terminus of the B subunit is the most likely candidate to contain the germinant binding site. B. Subtilis and B. Megaterium, two species of the Bacilli genus, germinate in response to different germinants; B. Subtilis germinates in response to L-alanine by activation of the GerA receptor, while B. Megaterium germinates in response to L-leucine by the activation of its GerU receptor. The focus of this study is to construct chimeric genes in which fragments of B. Subtilis GerA receptors and B. Megaterium receptors are fused together. These B. Subtilis::B. Megaterium chimeric receptors will be introduced into the B. Subtilis genome and the mutant B. Subtilis spores will then be tested for the ability to germinate with leucine in order to establish the leucine binding site of GerUB. During the initial pilot studies, the regions coding for the Nterminus of the GerA receptor from B. Subtilis and the C-terminus of the GerU receptor from B. Megaterium were amplified using polymerase chain reaction with primer ends complementary to each other in order to further produce the desired hybrid genes without the use of restriction enzymes

Examination of germination receptors of B. subtilis and B. megaterium

Many bacterial species including those in the Bacilli group form spores as a mechanism to survive harsh conditions such as extreme temperature, radiation, chemicals, and nutrient starvation. By forming spores, they can remain metabolically dormant for an extended period and revert to their vegetative form when environment becomes favorable. This resumption of metabolism and growth is marked by a process called germination that is triggered by exogenous nutrients such as amino acids, sugars, and nucleotides. The (Ger) germination receptors that are postulated to respond to these germinants, in the case of B. subtilis and B. megaterium, are a complex of at least three different proteins (the A-, B-, and C- subunits) transcribed from the same operon. While similar in gene arrangement and protein complex formation, these two Bacilli sp. respond to different germinants. This experiment investigates the GerA receptor of B. subtilis and the GerU receptor from B. megaterium. GerA of B. subtilis is activated with L-alanine, while GerU of B. megaterium is activated with L-proline. In order to determine the location of the binding site, different fragments of the GerAB gene and the GerUB genes encoding for protein A and B from each operon were amplified and fused together in frame to make a chimeric gene product. recombination. Spores from B. subtilis mutant strains expressing chimeric protein complexes will be tested for germination in the presence of L-proline and/or L-alanine. These studies will provide insights into how bacteria sense their environment and possible strategies to control and prevent growth

The Release of calcium in Bacillus anthracis pathogenicity methods

Anthrax infection starts with germination of Bacillus anthracis spores in macrophages. Some bacteria, including B. anthracis, can sporulate in response to environmental stress, such as starvation. During germination, large concentrations of calcium ions are released from the B. anthracis spore. Calcium ions are hydrophilic secondary messengers, and may therefore interfere with detection of the spore by confusing the cell signaling pathways. We investigated calcium release on infected macrophage viability by replacing the calcium stored in B. anthracis spores for other cations via demineralization/remineralization. It was discovered that calcium ions typically out-performed other cations in germination of B. anthracis

Germination and characterization of Bacillus anthracis and Bacillus cereus

Bacillus cereus and Bacillus anthracis are micro organisms found in soil. Normally, only their spores are found in soil. We recently showed that, B. anthracis and B. cereus do not germinate in soil. Thus, how does B. cereus and B. anthracis continue their life cycle if they can not replicate in soil? We hypothesize that B. cereus and B. anthracis spores may germinate in the gut of nematodes. Caenorhabditis elegans was used as our model nematode to investigate this possibility. Subsequently, the goal of our research is to determine the effect of C. elegans on the life cycle of B. anthracis and B. cereus. Three sets of experiments were attempted. Synchronized cultures of C. elegans strain N2 and BA1 was used to determine if B. anthracis and B. cereus have a significant effect on the lifespan of nematodes. Co-plating B. anthracis/B. cereus, B. cereus/E.coli and B. anthracis/E.coli enabled us to find out which food source was preferred over the other and in what conditions. Finally, B. anthracis spore germination was monitored in real time by feeding C. elegans with a B. anthracis strain that fluorescence\u27s upon germination

Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA

A High-Fat/High-Protein, Atkins-Type Diet Exacerbates Clostridioides (Clostridium) difficile Infection in Mice, whereas a High-Carbohydrate Diet Protects

Clostridioides difficile (formerly Clostridium difficile) infection (CDI) can result from the disruption of the resident gut microbiota. Western diets and popular weight-loss diets drive large changes in the gut microbiome; however, the literature is conflicted with regard to the effect of diet on CDI. Using the hypervirulent strain C. difficile R20291 (RT027) in a mouse model of antibiotic-induced CDI, we assessed disease outcome and microbial community dynamics in mice fed two high-fat diets in comparison with a high-carbohydrate diet and a standard rodent diet. The two high-fat diets exacerbated CDI, with a high-fat/high-protein, Atkins-like diet leading to severe CDI and 100% mortality and a high-fat/low-protein, medium-chain-triglyceride (MCT)-like diet inducing highly variable CDI outcomes. In contrast, mice fed a high-carbohydrate diet were protected from CDI, despite the high levels of refined carbohydrate and low levels of fiber in the diet. A total of 28 members of the Lachnospiraceae and Ruminococcaceae decreased in abundance due to diet and/or antibiotic treatment; these organisms may compete with C. difficile for amino acids and protect healthy animals from CDI in the absence of antibiotics. Together, these data suggest that antibiotic treatment might lead to loss of C. difficile competitors and create a favorable environment for C. difficile proliferation and virulence with effects that are intensified by high-fat/high-protein diets; in contrast, high-carbohydrate diets might be protective regardless of the source of carbohydrate or of antibiotic-driven loss of C. difficile competitors. IMPORTANCE: The role of Western and weight-loss diets with extreme macronutrient composition in the risk and progression of CDI is poorly understood. In a longitudinal study, we showed that a high-fat/high-protein, Atkins-type diet greatly exacerbated antibiotic-induced CDI, whereas a high-carbohydrate diet protected, despite the high monosaccharide and starch content. Our study results, therefore, suggest that popular high-fat/high-protein weight-loss diets may enhance CDI risk during antibiotic treatment, possibly due to the synergistic effects of a loss of the microorganisms that normally inhibit C. difficile overgrowth and an abundance of amino acids that promote C. difficile overgrowth. In contrast, a high-carbohydrate diet might be protective, despite reports on the recent evolution of enhanced carbohydrate metabolism in C. difficile

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

Análise da parametrização nacional do Sistema de Apoio à Prática de Enfermagem - SAPE

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