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)

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.

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

Candida albicans is the most common human fungal pathogen. Its relationship with various bacterial species has been documented, showing an increase in host mortality in some cases and a decrease in others. The need for new antibiotics and antifungal treatments have led to studies on polymicrobial interactions and how those interactions impact host health. Interactions between microbes within the human body are inevitable, and exploring these relationships can aid in the development of novel antimicrobials and can deepen our understanding of the complex human microbiome. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. Both are commensal organisms, living asymptomatically in immunocompetent individuals. 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 was analyzed by utilizing planktonic (free-floating) and biofilm co-cultures, performing live/dead staining, observing the effect of morphology on the interaction, and examining if Enterobacter alters C. albicans morphology. C. albicans was cultured with Enterobacter lysate to determine if Enterobacter can inhibit C. albicans without physical contact. Enzyme-linked immune assays (ELISAs) were performed on C. albicans ALS deficient mutants to ascertain their potential involvement in cell-to-cell adherence. ELISAs were also utilized to screen E. cloacae mutants for deficiency in attachment to C. albicans. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to compare expression of the HWP1 gene in C. albicans when mono-cultured versus co-cultured. Caenorhabditis elegans were used as a host model to examine the effect that co-culture has on survival and microbial burden. It was found that Enterobacter can inhibit C. albicans growth, no matter its morphology, and it is not necessary for Enterobacter cells to be present for inhibition to occur. C. albicans appears to have more hyphae when incubated with Enterobacter for 24 hours than incubated alone. Research involving ELISAs, RT-PCR, and C. elegans is ongoing.

Buruli ulcer disease (BUD) remains a ‘mysterious disease’ due to the unknown mode of M. ulcerans transmission and pathogenesis. To understand these, it is important to determine the reservoir of the organism in its natural environments, and stress response and interactions of M. ulcerans in its natural niche and during infection of a host. The major virulence factor of M. ulcerans is mycolactone, a lipid cytotoxin that is encoded on a giant plasmid pMUM001. Genetic analysis suggests that plasmid pMUM001 was acquired by M. ulcerans during evolution from its progenitor, M. marinum. Coincidental evolution of virulence hypothesis suggests that many microbes evolve to acquire traits to outcompete or overcome biotic and abiotic forces during their normal life cycle in the outside-host environment, which can confer virulence during infection of a human host. Hence in this study, we exposed M. ulcerans to selective abiotic forces such as UV, and dynamic oxygen and temperature conditions to determine their effect on M. ulcerans growth, and mycolactone and global gene expression. We also studied the role of mycolactone in determining polymicrobial interaction of M. ulcerans in its natural aquatic habitat by exposing mycolactone coated and uncoated slides in M. ulcerans endemic and non-endemic aquatic locations and determining differences in microbial community composition between them. Further, we studied quorum quenching ability of mycolactone against an opportunistic pathogen, S. aureus. The results obtained showed that exposure of M. ulcerans to abiotic stresses such as higher temperature and lower than optimal oxygen conditions modulate its global and mycolactone gene expression. Further, we also showed that mycolactone can impact overall microbial community structure in a polymicrobial environment in its natural, aquatic habitat. Mycolactone also effected virulence and quorum sensing in an opportunistic pathogen, S. aureus, without inhibiting its growth. These findings are important as they provide insight toward potential reservoirs or environmental niches which may harbor M. ulcerans and inform new potential mechanisms of pathogenesis. Further, our novel research of synergistic or antagonistic interactions within the complex polymicrobial communities colonizing skin and aquatic habitats is a powerful approach in determining M. ulcerans colonization efficiency, resiliency, and transmission mechanisms.

Polymicrobial interactions are an important, yet understudied area of research. Candida albicans is the most common human fungal pathogen. The bacterial genus, Enterobacter, is a source of nosocomial acquired infections and increased drug resistance. Our lab has previously discovered that Enterobacter preferentially adheres to C. albicans hyphae. From an E. cloacae transposon library screen, six candidates displayed reduction in C. albicans attachment. These candidates were identified genetically and characterized for involvement in attachment to C. albicans. A fluorescent plasmid was introduced into E. cloacae to measure and observe adherence to C. albicans in planktonic and biofilm growth. In vivo experiments using Caenorhabditis elegans showed no significant differences in microbial burden or nematode survivability exposed to Candida and Enterobacter. Candida-Enterobacter co-infections were observed microscopically within C. elegans. This study highlights the complex dynamics of C. albicans-E. cloacae interactions, underscoring the importance of understanding polymicrobial relationships in research and clinical settings.

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.

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.