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The oral microbiome represents an extremely diverse environment that harbors many species of bacteria; over 700 different species have been identified overall. These organisms may be either commensal or pathogenic, and reside in multi-species communities of bacterial biofilms. As such, these bacteria may be 100-1000 times less susceptible to antibiotic treatment than their planktonic counterparts. One pathogenic organism that exists as a biofilm in the oral cavity is Streptococcus mutans, the main etiologic agent contributing to dental caries. Recently, the myxobacterial natural product carolacton was isolated and shown to be lethal to S. mutans cells in a biofilm at low (10 nM) concentration. As part of an endeavor to take inspiration from natural products to develop new therapeutics to combat biofilms, our group became interested in carolacton. This dissertation describes research conducted into the synthesis and biological evaluation of carolacton. Total synthesis enabled the biological evaluation of carolacton as well as several analogs. A novel compound was identified that was shown to elicit a phenotypic response from S. mutans that was different from that elicited by carolacton. In an effort to uncover novel simplified carolacton derivatives that maintain bioactivity and/or act via a different mechanism, we have exploited the power of diverted total synthesis in order to obtain a 1st-generation library of carolacton analogs. By leveraging a common intermediate that we were then able to diversify, we have obtained a library of simplified aryl analogs. Preliminary testing of these analogs has revealed a compound that inhibits growth and formation of S. mutans biofilms. This research has enabled us to obtain compounds that will serve to guide future drug discovery efforts, as well as act as tool compounds to help identify novel drug targets in S. mutans biofilms. / Chemistry

Streptococcus mutans is a member of the dental plaque and is the principal causative agent of dental caries. It can metabolize a wide array of sugars which results in the production of acid that causes demineralization of the tooth surface. S. mutans can also persist for extended periods of starvation, which may occur in different niches within the oral cavity. Previous studies have shown that mucin prolonged the survival of S. mutans in batch cultures and biofilms. Our laboratory has shown that the pyruvate dehydrogenase (pdh) operon is upregulated in stationary phase and is important for prolonged survival during nutrient starvation in chemically defined medium (CDM). In this study, I found that mucin enabled S. mutans to grow in a minimal chemically defined medium (MCDM) containing glucose and lacking all amino acids. A pdh mutant was found to be impaired in survival in CDM or MCDM supplemented with mucin. Additionally, I have shown that a galactose utilization mutant was impaired in survival in CDM with mucin and no exogenous glucose; galactose is the most abundant utilizable sugar residue in mucin. Mucin, supplemented in minimal medium with sucrose, also enabled S. mutans to form biofilms. Survival of the biofilms was pdh independent. Although biofilm formation was not seen in the absence of sucrose, cells did adhere to the glass coverslip; their survival was found to be pdh dependent. Using a previously constructed reporter strain, pdh expression was observed in a majority of cells in this condition. The expression of the pdh operon was also monitored in batch cultures. The starting inoculum size affected the percentage of fluorescent cells and the outcome of survival in media (CDM or MCDM) supplemented with mucin and no sugar. Greater than 50% of the stationary phase population expressed pdh in CDM with galactose. Less than 1% expressed pdh in CDM with glucose. However, galactose was unable to prolong survival of S. mutans in batch cultures in contrast to the effect of mucin. These results show that mucin may have a metabolic role in promoting long term survival of S. mutans. However it is possible that different components of mucin can be utilized in different conditions. / Microbiology and Immunology

Long-term Stationary Phase Behavior of Streptococcus pyogenes Biofilms Department of Microbiology and Immunology Streptococcus pyogenes is the etiological agent of many human diseases ranging from mild superficial skin infections and pharyngitis to life-threatening necrotizing fasciitis. There can be several complications as a result of S. pyogenes infection including post-streptococcal glomerulonephritis and rheumatic fever, which leads to rheumatic heart disease. Despite the significant virulence associated with the pathogen, the bacteria can also persist asymptomatically in human host carriers. S. pyogenes is characterized by significant strain-to-strain variation with many single nucleotide polymorphisms and differences in genetic content of up to 33% of the genome. Active infection is associated with the rapid growth of the pathogen, whereas survival or carriage is associated with slow growth. Our laboratory has demonstrated that during survival in long-term stationary phase cultures and in eukaryotic cells, S. pyogenes diversifies into a mixed population. Isolates from this population show diversification in their proteome, in metabolism, and in virulence factor transcription patterns. These are stable, heritable changes with unique mutations in global gene regulators in some isolates, suggesting that an accumulation of genetic mutations leads to diversification. There are two proposed modes of survival in the human host; by taking residence intracellularly in host cells and as biofilms. Previous studies showed that isolates surviving within eukaryotic cells acquire heritable changes in metabolism and virulence factor expression. Biofilms are highly organized structures formed by many bacteria, which provide resiliency to harsh environmental conditions. It has been demonstrated that S. pyogenes form biofilms in vivo and in vitro, and up to 90% of clinical isolates can form biofilms. Considering the resiliency of biofilms, and the organized roles played by individual cells in biofilms, we hypothesized that biofilms may provide S. pyogenes with a niche for persistence and diversification. Despite the capacity for survival of planktonic cells, we have found that viable cells could not be isolated from static biofilms after 10 days. No metabolic variants were found among biofilm isolates prior to loss of biofilm viability. Biofilm structure was examined using confocal microscopy to image cells after LiveDead® staining. These experiments revealed that the biofilms lost viability rapidly, and also appeared to disperse. Dispersion of 2-day old biofilms could be induced with culture supernatants collected from 7-day old planktonic cells. Overall, the results of these studies suggest that secreted factors from late stationary phase cultures induce biofilm dispersion and biofilms do not serve as a niche for long-term survival and diversification of S. pyogenes. Therefore, S. pyogenes biofilms may be more critical for initial colonization of the oropharynx. These studies may provide a valuable insight to the role of biofilms in S. pyogenes infections. / Microbiology and Immunology

Strains of pathogens are typically described as virulent or non-virulent. However, in the majority of pathogens, strains often vary continuously and quantitatively in their virulence and pathogencity. Biofilm formation is one of the recently recognized virulence factors in many human pathogens and little is known about the variation and evolution of biofilms among natural strains. In this study, I examined quantitative variation of biofilms among natural strains of the human pathogenic yeast Candida albicans. A total of 115 natural strains of C. albicans from three sources (vaginal, oral and environmental) were quantified by two mebods: (i) the XTT tetrazolium reduction assay, and (ii) optical density following staining by crystal violet dye. Mature biofilm was confirmed by observation using confocal laser scanning microscopy. My analyses indicated that strains from each of the three scurces varied widely in biofilm formation abilities and that biofilm formation ability was positively correlated to cell surface hydrophobicity (CSH). For each strain, multilocus genotypes were determined by PCR-RFLP, my comparative genotype and biofilm analyses denonstrated that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variation for biofilm formation. I also examined potential interactions among strains within C. albicans and between different Candida species. My preliminary results suggest significant variation and complex patterns of strains or species interaction during Candida biofilm development. / Thesis / Master of Science (MS)

The hybrid activated sludge-biofilm system called Integrated Fixed Film Activated Sludge (IFAS) has recently become popular for enhanced nitrification and denitrification in aerobic zones because it is an alternative to increasing the volume of treatment plant units to accomplish year round nitrification and nitrogen removal. Biomass is retained on the fixed-film media and remains in the aerobic reactor, thus increasing the effective mean cell resident time (MCRT) of the biomass and providing the temperature sensitive, slow growing nitrifiers a means of staying in the system when they otherwise would washout. While the utilization of media in aerobic zones to enhance nitrification and denitrification has been the subject of several studies and full-scale experiments, the effects and performances of fixed film media integrated into the anoxic zones of biological nutrient removal (BNR) systems have not adequately been evaluated as well as the impacts of integrated media upon enhanced biological phosphorus removal (EBPR). Also, user-friendly software designed specifically to simulate the complex mixture of biological processes that occur in IFAS systems are not available. The purpose of this research was to more fully investigate the effects of integrated fixed film media on EBPR, to evaluate the impacts of media integrated into the anoxic zone on system performance, and to develop a software program that could be used to simulate the effects of integrating the various types of media into suspended growth biological nutrient removal (BNR) systems. The UCT type configuration was chosen for the BNR system, and Accuweb rope-like media was selected for integration into the anoxic zones of two IFAS systems. The media also was integrated into the aerobic reactors of one of the systems for comparison and for further investigation of the performance of the Accuweb media on enhanced nitrification and denitrification in the aerobic zones. The experiments were conducted at 10 day total MCRT during the initial phase, and then at 6 days MCRT for the experimental temperature of 10 oC. A13 hour hydraulic retention time (HRT) was used throughout the study. A high and a low COD/TP ratio were used during the investigation to further study the effects of integrated media on EBPR. The PC Windows based IFAS program began with the concepts of IAWQ model No. 2 and a zero-dimensional biofilm model was developed and added to predict the IFAS processes. Experimental data from the initial study and existing data from similar studies performed at high temperatures (>10oC) indicated that there were no significant differences in BNR performances between IFAS systems with media integrated into the anoxic and aerobic or only aerobic zones and a suspended growth control system maintained at the same relative high MCRT and temperature values. Even though greater biological nitrogen removal could not be achieved for the experimental conditions used, the experimental results indicated that the IFAS systems with fixed film media installed in the anoxic zone have a greater potential for denitrification than conventional BNR systems. As much as 30 percent of the total denitrification was observed to occur in the aerobic zones of the system installed the media only anoxic zones and 37% in the system with integrated media in both anoxic and aerobic zones where as no denitrification was observed in the aerobic zones of the control system when the systems were operated at 6 days MCRT and COD/TP of 52. It is statistically confirmed EBPR can be maintained in IFAS systems as well as Control systems, but the IFAS processes tend to have more phosphorus release in the anoxic zones with integrated fixed film installed. Further, the combination of split flow to the anoxic zone and fixed film media in the anoxic zone resulted in the decreased EBPR performances in the IFAS system relative to the control system. / Ph. D.