The Streptococcus Anginosus/Milleri group (SMG) colonize mucosal surfaces in humans but are also associated with numerous respiratory and invasive infections. These infections are often polymicrobial in nature, with obligate anaerobes often being isolated. The group consists of three species, S. anginosus, S. constellatus and S. intermedius. SMG are considered to be lactic acid bacteria, producing acids such as lactate, formate and acetate as byproducts of their metabolism. Their genomes have been recently sequenced but little is known about their metabolism. Understanding the basis of their metabolism is beneficial in determining optimal growth conditions and mechanisms associated with their pathogenicity. The isolation of obligate anaerobes from SMG polymicrobial infections suggests that they have anoxic microenvironments. There is also some evidence for synergy between SMG species and anaerobes. While cooperation might be occurring with certain anaerobes, streptococci also produce inhibitors such as hydrogen peroxide and short peptides called bacteriocins. These give streptococci a competitive advantage in polymicrobial commensal communities such as the oral cavity. The Streptococcus invasion locus controls bacteriocin production in Group A streptococci and has been identified in SMG species as well. It is unknown if SMG have mechanisms to compete with closely related streptococci. The goal of my thesis is to characterize the cooperative and competitive interactions of S. intermedius with other species. In chapter 2, we characterized the in vitro metabolism of S. intermedius under aerobic (5% CO2) and anaerobic conditions. Using a transcriptomic and metabolomic approach, we mapped the pathways involved in S. intermedius B196 metabolism. We found that there was a minimal upregulation of core pathways including carbohydrate metabolism under anaerobic conditions. Under aerobic conditions, oxidative stress genes were induced. An increased growth rate was also observed anaerobically. In chapter 3, I demonstrated that Streptococcus strains, including S. intermedius, can deplete oxygen and create an anaerobic environment. Certain strains could support the viability of the obligate anaerobe Prevotella melaninogenica in broth cultures under hypoxic conditions, while others inhibited Prevotella by producing hydrogen peroxide. S. intermedius B196 has an alkylhydroperoxidase system (ahpCF), which is thought to endogenously detoxify peroxides. An S. intermedius ahpCF mutant produced hydrogen peroxide and inhibited P. melaninogenica in coculture. Complementation in S. intermedius restored P. melaninogenica viability in coculture. I demonstrated that the ahpCF peroxide detoxification system directly protects S. intermedius from peroxides and indirectly affects a polymicrobial community. In chapter 4, we used a subcutaneous abscess model in BALB/c mice to demonstrate that S. intermedius promotes P. melaninogenica survival during co-infection in comparison to a P. melaninogenica mono-infection. S. intermedius induced abscesses appeared to induce apoptosis, necrosis and NETosis in neutrophils that infiltrated the site of infection. Our results demonstrate the complexity of SMG infections. In chapter 5, I demonstrated that S. intermedius B196 produces inhibitors of other SMG in response to stimulation with the pheromone peptide SilCR. This is the first case of S. intermedius inhibiting a closely related SMG strain. A bioinformatic analysis was done on the sil system in SMG. The system is associated with a genetically heterogeneous bacteriocin cluster which can carry any combination of sixteen putative open reading frames, six of which are putative bacteriocins. Together, my thesis outlines that S. intermedius has specific mechanisms of cooperation and competition. These allow it to cooperate with obligate anaerobes such as P. melaninogenica and inhibit other SMG species. Oxygen depletion, hydrogen peroxide production and bacteriocin production are only three factors addressed in this thesis. However, there are many factors involved in shaping a polymicrobial environment with SMG species. More research in SMG polymicrobial interactions is required to fully understand SMG pathogenicity. / Thesis / Doctor of Philosophy (PhD)

The rise of antibiotic resistance in common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition against the competing microbes. Four of these A. faecalis mutant strains were successfully sequenced and compared to NCBI’s genomic database. The proteins of the interrupted genetic elements were identified as a FAD-binding oxidoreductase, MFS transporter, and mechanosensitive ion channel. Further analysis of these mutants is needed to determine their role in 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, C. albicans, and Bacillus species treatments.

Antimicrobial-resistant bacteria are a major source of ailment in modern-day nosocomial settings, with numerous risks including leading to possible further drug resistance or spreading to those who cannot fight off the infection due to immune suppression or dysfunction. Previous work in our laboratory has determined that Klebsiella pneumoniae possesses inhibitory effects on the growth of a variety of bacteria that contain antimicrobial-resistant properties in the Enterobacteriaceae family, a major source of nosocomial antimicrobial-resistance. This novel property of K. pneumoniae inhibits the growth of Citrobacter freundii, Enterobacter aerogenes, and Enterobacter cloacae through an anti-microbial protein. The antimicrobial protein secreted from K. pneumoniae has been shown to reduce the density and growth of C. freundii, E. aerogenes, and E. cloacae in both biofilm and planktonic forms. The work performed in this thesis has shown that the antimicrobial protein is plasmid mediated by introducing a transposon (Tn5) to the plasmid to provide resistant selection and a possible way to create a mutant knockout to find the exact location of the gene in the plasmid. Upon transposon mutagenesis of the plasmid, it was electroporated into Rec- E. coli. The E. coli were then able to produce the antimicrobial protein allowing the formation of zones of inhibition when screened on C. freundii, E. aerogenes, and E. cloacae lawns. Upon confirmation that the plasmid mediates the anti- microbial protein, the plasmid was sent for sequencing to further characterize the gene responsible for coding the anti-microbial protein. This novel antimicrobial protein has high sequence similarity to bacteriocins and, thus far, is a novel and uncharacterized protein of plasmid origin found in only in this particular strain of K. pneumoniae. Further research involving this new bacteriocin could aid in the development of treatments for the highly drug resistant Enterobacteriaceae family members.

The rise of antibiotic resistance of common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition of the competing microbes. In A. faecalis mutant strains P2-9 and P1-42, the interrupted gene was identified as a FAD-binding oxidoreductase with a 94% and 90% match of nucleotide sequence. Mutant strain P2-25’s interrupted gene was identified as an MFS transporter with a 100% match and P2-30’s interrupted gene was identified as a mechanosensitive ion channel with a 100% match. Further analysis of these mutants is needed to determine their role in 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, C. albicans, and Bacillus treatments.

Antibiotic resistance occurs when bacteria change in response to selective pressures induced by antibiotics, which has become a major concern worldwide and one of the biggest threats to global health. Antibiotic resistance can occur naturally, but the misuse and overuse of antibiotics is accelerating the process. One way to combat this process is to understand the different relationships between microbes, also known as polymicrobial interactions. Bacteria can interact with one another synergistically or antagonistically and understanding the mechanisms behind these interactions can lead to the discovery of new therapeutics or targets to fight and kill pathogenic microbes. The rarely pathogenic Gram-negative bacterium, Alcaligenes faecalis, has previously been shown in our lab as playing an important role in potentially fighting antibiotic and antifungal resistance due to its competitiveness during polymicrobial interaction. Our research has found that A. faecalis kills Bacillus cereus, Staphylococcus aureus, and Candida albicans. This is a unique characteristic as these targets encompass both prokaryotic (bacteria) and eukaryotic (fungi) microbes. These three species are known to cause numerous infections in humans and have increased cases of antibiotic and antifungal resistance. In the present study, we investigated the genetic elements A. faecalis utilizes to inhibit growth when interacting with B. cereus, S. aureus, and C. albicans. Transposon mutagenesis was performed to create a genetic library of A. faecalis loss-of-function mutants. These strains were then screened against all three microorganisms to determine which mutants no longer inhibited growth. The mutants that lacked zones-of-inhibition were sequenced to determine the gene that had been interrupted. BLAST analysis of these sequences identified a MFS transporter, a 2FE-2S iron sulfur binding protein, a mechanosensitive ion channel, and a glucose-6-phosphate isomerase as instrumental in this inhibitory mechanism. Results from this research study can be used to further study polymicrobial interactions and potentially discover new therapeutics to combat antimicrobial resistance.

Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection. / Master of Science

Throughout existence, fungi and bacteria have long shared ecological niches and thus engage in numerous interactions to mutually enhance survival or antagonistically gain competitive advantages. Of importance to human health are those interactions that involve bacteria with the opportunistic fungi, Candida albicans. An important virulence factor of C. albicans is the ability to control morphology, which allows the transition between yeast, pseudohyphal, and hyphal phenotypes. Morphological control in C. albicans is governed by quorum sensing and the secreted autoregulatory molecule farnesol. Quorum sensing allows individual cells to sense the environment and respond as a group. Bacteria also use quorum sensing to communicate and control virulence. Despite their abundance in nature, very little is known about the interactions of C. albicans with bacteria on a genetic and molecular level. The objective of our research is to identify the genetic elements involved in C. albicans-bacterial interactions and characterize the genes that may participate in these relationships. To accomplish this, we screened a C. albicans mutant library for the ability to filament in the presence of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, where 3 typically, these three bacterial species inhibit C. albicans filamentation. We identified 836 C. albicans mutants that displayed a filamentous phenotype in the presence of bacteria. Collectively, 295 of these mutants filamented in the presence of all 3 bacterial species. Candidates were subsequently sequenced to identify the location of the mutation and the affected genetic element. CDR4, a putative ABC transporter, and ALS6, a putative adhesion, were further characterized for their specific involvement in Candida-bacterial interactions. Using a filamentation assay, cdr4 and als6 deletion strains demonstrated a decreased response to the inhibitory effects of farnesol, as well as bacterial molecules known to inhibit the production of hyphal-filaments. Additionally, the ability of cdr4 and als6 deletion strains to attach and form biofilms was significantly enhanced even in the presence of farnesol and bacterial inhibitors. The results of this study contribute to the body of knowledge involving polymicrobial interactions and these findings may lead to new antifungal targets for therapeutic interventions.

Pseudomonas aeruginosa is a ubiquitous bacterium that is commonly isolated from soil and water. Additionally, this bacterium can cause infections in individuals with compromised immune systems and in those with underlying debilitating conditions. Individuals with cystic fibrosis, burn wounds, AIDS and diabetes are more likely to being infected by P. aeruginosa than healthy individuals. In individuals with CF, there is a marked increase in the accumulation of lung mucus that serves as a source of nutrition for P. aeruginosa and other bacterial species resulting in chronic and often fatal infections. While CF lung infections are initially caused by more than one species of bacteria, over time P. aeruginosa emerges as the dominant species. P. aeruginosa also causes chronic infections in association with other bacteria in wounds. Microbes within these infections are engaged in complex interactions with each other. Often, these interactions are synergistic resulting in infections that are recalcitrant to antimicrobial therapy. While many studies have documented the occurrence of synergistic polymicrobial infections, little is known about the molecular mechanisms prevailing in these infections. Interestingly, production of virulence factors by P. aeruginosa has been shown to correlate with the presence of specific nutrients in their growth environment. Expanding on the idea of available nutrients affecting virulence, I demonstrate the ability of N-Acetylglucosamine (GlcNAc) and GlcNAc-containing polymers such as peptidoglycan to induce production of virulence factors in P. aeruginosa. Peptidoglycan shed by Gram-positive bacteria acts as a cue for P. aeruginosa in polymicrobial environments, to enhance production of virulence factors. In the context of a polymicrobial infection, this results in enhanced pathogenesis. Here, I provide insights into mechanisms influencing such interspecies interactions between the opportunistic pathogen Pseudomonas aeruginosa and S.aureus. / text

In this dissertation I describe a model system to characterize the response of an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans to the metabolic byproducts of a representative member of the oral flora, Streptococcus gordonii. A. actinomycetemcomitans is a causative agent of periodontal infections in humans. To cause infection, A. actinomycetemcomitans must overcome numerous challenges, including the host immune system and toxic metabolite production from other microbes. The most numerically dominant flora in the oral cavity are oral streptococci, which are well known for their ability to produce copious amounts of lactic acid and H₂O₂. By studying the response to H₂O₂ and lactic acid in pure and co-cultures, I have demonstrated that A. actinomycetemcomitans responds to these metabolites by several novel mechanisms that both enhance its survival in the presence of the host immune system and in the presence of the model oral streptococci S. gordonii. These studies have demonstrated that metabolites produced by normal flora can impact the survival of a single species in vivo as much as previously known virulence factors have done. In addition, I present a new method for measuring metabolite production in an attached cell population. This method is a novel application of scanning electrochemical microscopy (SECM) and I used this technique to study H₂O₂ production in the three dimensional space surrounding a multispecies biofilm in real time. In a related study I present the use of SECM to discover a novel redox chemistry phenomenon in the opportunistic pathogen Pseudomonas aeruginosa. / text

OBJECTIVE:: To examine peripheral leukocyte Dectin-1 regulation in clinically relevant models of fungal and polymicrobial sepsis. DESIGN:: Prospective animal study. SETTING:: University medical school research laboratory. SUBJECTS:: Age, weight, and sex matched ICR/HSD mice. INTERVENTIONS:: Mice were infected with Candida albicans (1 × 10, intravenously) or were subjected to cecal ligation and puncture to induce polymicrobial sepsis. MEASUREMENTS:: Blood, spleen, and peritoneal exudate were harvested and leukocytes were isolated. Leukocytes were evaluated for membrane-associated Dectin-1 expression and cell phenotype by flow cytometry. MAIN RESULTS:: In C. albicans infection, Dectin-1-positive blood and splenic leukocytes were increased from 23.5% to 58.9% over the course of infection. The increased percentage of Dectin-1-expressing cells was primarily attributable to neutrophilia. However, the amount of Dectin-1 expressed by blood and splenic neutrophils in C. albicans-infected mice was decreased by a range of 49.0% to 53.3%. C. albicans infection also resulted in an infiltration of Dectin-1-positive macrophages and neutrophils into the kidney. In contrast, polymicrobial sepsis decreased blood leukocyte Dectin-1-expressing cells by up to 51.4%. This reduction was due to a decrease in Dectin-1-positive neutrophils in the periphery. However, the percentage of Dectin-1-expressing cells in the peritoneal cavity increased by 774% with cecal ligation and puncture. Treatment of isolated neutrophils with three soluble glucans, mannan, lipopolysaccharide, or a variety of cytokines revealed that glucans, alone or in combination, were the only treatment that resulted in a decrease in Dectin-1-positive neutrophils. CONCLUSIONS:: We conclude that peripheral leukocyte Dectin-1 expression is differentially regulated in fungal vs. polymicrobial sepsis. These data demonstrate that leukocyte Dectin-1 levels are modulated in response to infections of fungal and nonfungal origin.