Staphylococcus aureus has developed resistance to several antibiotics including vancomycin, which is often used as a “last resort” treatment. There is an ever-increasing need to develop novel antimicrobial treatments to combat S. aureus and other drug resistant bacteria. Microorganisms are most often found in polymicrobial communities where they either exhibit synergistic or antagonistic relationships. Competition between microorganisms can lead to the discovery of new antimicrobial targets as the specific mechanisms of resistance are elucidated. In addition, synergistic treatments are being evaluated for their combined effect and potential to decrease the concentration of drugs needed, and thus the side effects also. Alcaligenes faecalis is a microorganism that our lab has previously shown to inhibit S. aureus and other various bacterial species. In this study, we found that A. faecalis reduces the planktonic growth of S. aureus by 94.5% and biofilm growth by 76.6%. A. faecalis also has a synergistic effect when paired with bacitracin to reduce the planktonic growth by 99.9% and biofilm growth by 99.7%. Transposon mutagenesis was successfully performed on A. faecalis, and loss of function mutations were attained. Two mutants were no longer able to inhibit the growth of Staphylococcus aureus, Candida albicans, or Bacillus megaterium. Further analysis and genomic sequencing of these mutants is needed to determine the gene(s) that were interrupted and the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus treatments.

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells (ECs) and has been reported to protect against cardiac dysfunction from endotoxemia or myocardial infarction. This study investigated the mechanisms by which endothelial HSPA12B protects polymicrobial sepsis–induced cardiomyopathy. Wild-type (WT) and endothelial HSPA12B knockout (HSPA12B–/–) mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP). Cecal ligation and puncture sepsis accelerated mortality and caused severe cardiac dysfunction in HSPA12B–/– mice compared with WT septic mice. The levels of adhesion molecules and the infiltrated immune cells in the myocardium of HSPA12B–/– septic mice were markedly greater than in WT septic mice. The levels of microRNA-126 (miR-126), which targets adhesion molecules, in serum exosomes from HSPA12B–/– septic mice were significantly lower than in WT septic mice. Transfection of ECs with adenovirus expressing HSPA12B significantly increased miR-126 levels. Increased miR-126 levels in ECs prevented LPS-stimulated expression of adhesion molecules. In vivo delivery of miR-126 carried by exosomes into the myocardium of HSPA12B–/– mice significantly attenuated CLP sepsis increased levels of adhesion molecules, and improved CLP sepsis–induced cardiac dysfunction. The data suggest that HSPA12B protects against sepsis-induced severe cardiomyopathy via regulating miR-126 expression which targets adhesion molecules, thus decreasing the accumulation of immune cells in the myocardium.

Candida albicans is the most common human fungal pathogen. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. The hypothesis of this study is that C. albicans and both E. aerogenes and E. cloacae have a positive relationship and work together to infect the host. In this study, the physical cell-to-cell interaction, molecular components of said interaction, and the impact of the interaction on a live organism were explored. Results indicate that Enterobacter adheres to C. albicans and inhibits growth with unidentified secreted molecules. Als1p has potential involvement in the attachment of E. cloacae to C. albicans. Out of 480 E. cloacae mutants, 6 showed reductions in attachment to C. albicans. The presence of C. albicans in C. elegans may lead to less Enterobacter colonization. Future work involving this interaction should strive to identify the Enterobacter secreted molecules and genes necessary for their production.

Candida albicans is a fungal microorganism found on the human body and in the environment. An opportunistic pathogen causing local and systemic infection, this fungus is one of the leading causes of nosocomial infections. More alarming is its growing resistance against the limited number of antifungals we have for treatment Candida infections. An area of current research, termed polymicrobial interactions, focuses on how different microorganisms interact with each other for limited space, nutrients, and survival. The current study focuses on attempting to inhibit planktonic and biofilm growth stages by using the benign bacterium Alcaligenes faecalis, previously shown in our lab to kill C. albicans. Under a variety of growth conditions and phases, co-cultures of A. faecalis and C. albicans have demonstrated that the bacterium drastically inhibits all forms of Candida growth. The results of this study may provide information on potential new therapeutic targets that Alcaligenes may employ in inhibition of Candida.

<p>Despite the advanced knowledge of the mechanisms of influenza infection and improved vaccines, Influenza A Virus still causes a life-threatening respiratory disease, especially during pandemics. Past investigations have proposed a synergism between Influenza A virus and a simultaneous or subsequent bacterial superinfection as the predominant cause of death. The recent development of animal models to study these heterologous infections has shed light onto the diverse mechanisms by which Influenza A Virus may increase the susceptibility to contract a secondary bacterial infection. These studies suggested an important role for the innate immune system in mediating such disease. We developed a model of heterologous infection combining Influenza A Virus and <em>Bordetella parapertussis</em> that demonstrated a critical role for MIP-2 to drive pulmonary neutrophilia in the pathology associated with bacterial superinfection of influenza. However, the origin of this increased MIP-2 production and the mechanisms underlying the immunopathology remained to be elucidated. The present studies proposed IL-1β overproduction as the upstream cause of the increased MIP-2 production observed in heterologous infection. This exaggerated IL-1β production was likely related to the increased bacterial burden observed in heterologously infected mice. This study also demonstrated that reduction in IL-1β production by blockade of the inflammasome seemed to provide an improvement in the clinical symptoms and the immunopathology of the disease. Thus, interventions to attenuate the exacerbated bacterial burden and the inflammatory responses derived from the subsequent IL-1β overproduction should be further investigate as possible therapeutic approaches to treat bacterial superinfections.</p> / Master of Science (MSc)

Bats are crucial to ecological function and provide key ecosystem services to people but face a variety of significant threats. One current threat to North American bats is white-nose syndrome (WNS), a disease caused by the invasive fungal pathogen Pseudogymnoascus destructans (Pd) that has killed millions of hibernating bats across the continent. Remnant populations of affected bat species persist but are so depleted that they may now be highly vulnerable to new threats, or to the synergistic effects of multiple existing threats. The emergence of novel or opportunistic pathogens in bat hosts is a particular concern for the survival of these small, isolated colonies. Apart from studies of WNS and zoonotic pathogens of humans, however, bat diseases remain poorly understood. In this paper, I describe the pathology of a new, highly aggressive bat disease affecting hibernating little brown myotis (Myotis lucifugus) and identify candidate microbes as possible causative agents. The pathological signs that were observed diverged from those of WNS, and included blue fluorescence in the wings when trans-illuminated with ultraviolet light, and the rapid development of wing necroses and mortality within weeks of the onset of hibernation. Pathology, wing swab cultures, post-mortem analyses, and hemolysis testing identified an array of candidate species, but suggest that a possible cause is a polymicrobial infection involving two etiological agents – Trichosporon yeast and Serratia bacteria. Both species have been documented as part of the mycobiota and microbiota of healthy bats, and cave environments. They are also opportunistic pathogens, known to cause infection in other wild animals and immunocompromised humans. Opportunistic pathogens have been increasingly implicated as a cause of mass mortality events in wildlife. The disease identified here has, to my knowledge, not previously been described, and could represent a new threat to North American bats, compounding concerns for populations facing an already precarious situation. / Biology

The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to assess cell surface Dectin-1 expression. C. albicans sepsis resulted in an increase in the percentage of Dectin-1 positive (Dectin+) blood and splenic leukocytes by increasing the percentage of neutrophils. C. albicans infection increased the percentage of Dectin+ splenic T cells. CLP decreased the percentage of highly Dectin-1 positive leukocytes in the blood by decreasing the percentage of Dectin+ neutrophils. GP treatment in sepsis further decreased the percentages of Dectinhigh blood leukocytes and Dectin+ neutrophils. CLP decreased the percentage of Dectin+ splenic leukocytes by decreasing the percentage of splenic macrophages. GP administration to CLP mice further decreased the percentage of Dectin+ splenocytes by decreasing the percentage of Dectin+ macrophages. Administration of GP resulted in a prolonged decrease in the percentage of Dectinhigh blood leukocytes. The changes in Dectin-1 expression with GP were because of decreases in the percentage of Dectin+ neutrophils and monocytes. In the trafficking studies, macrophages were incubated with fluorescent labeled glucans and then stained for intracellular organelles and signal transduction molecules. Cells were imaged using confocal microscopy. GP is internalized by clathrin and trafficked to the Golgi apparatus. GP internalization is regulated but not dependent on caveolin-1. GP co-localized with SRA, TLR2, and PI3K/p85. The trafficking of laminarin and particulate glucan is similar. We speculate that loss of cell surface Dectin-1 may be important in the protection conferred by glucans in sepsis. Additionally, intracellular trafficking and interaction with signaling components may be important steps in modulation of cellular function by glucan-pattern recognition receptor complexes.

Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.

In the first study, three different transport systems for bacteria were evaluated. The CLSI M40-A guideline was used to monitor the maintenance of both mono- and polymicrobial samples during a simulated transportation at room temperature that lasted 0-48 h. All systems were able to maintain the viability of all organisms for 24 h, but none of them could support all tested species after 48 h.  The most difficult species to recover was Neisseria gonorrhoeae, and in polymicrobial samples overgrowth was an observed problem. The aim of the second study was to study the presence of TSST-1 and three other important toxin genes in invasive isolates of Staphylococcus aureus collected during the years 2000-2012 at two tertiary hospitals. The genes encoding the staphylococcal toxins were detected by PCR, and whole-genome sequencing was used for analyzing the genetic relatedness between isolates. The results showed that the most common toxin was TSST-1, and isolates positive for this toxin exhibited a clear clonality independent of year and hospital. The typical patient was a male aged 55-74 years and with a bone or a joint infection. The third study was a clinical study of the effect of silver-based wound dressings on the bacterial flora in chronic leg ulcers. Phenotypic and genetic silver-resistance were investigated before and after topical silver treatment, by determining the silver nitrate MICs and by detecting sil genes with PCR. The silver-based dressings had a limited effect on primary wound pathogens, and the activity of silver nitrate on S. aureus was mainly bacteriostatic. A silver-resistant Enterobacter cloacae strain was identified after only three weeks of treatment, and cephalosporin-resistant members of the Enterobacteriaceae family were relatively prone to developed silver-resistance after silver exposure in vitro. The last study was undertaken in order to develop an easy-to-use method for simulating the laundering process of hospital textiles, and apply the method when evaluating the decontaminating efficacy of two different washing temperatures. The laundering process took place at professional laundries, and Enterococcus faecium was used as a bioindicator. The results showed that a lowering of the washing temperature from 70°C to 60°C did not affect the decontamination efficacy; the washing cycle alone reduced the number of bacteria with 3-5 log10 CFU, whereas the following tumble drying reduced the bacterial numbers with another 3-4 log10 CFU, yielding the same final result independent of the washing temperature. To ensure that sufficient textile hygiene is maintained, the whole laundering process needs to be monitored. The general conclusion is that all developmental work in the bacterial field requires time and a large strain collection.