Biofilms are bacterial communities that adhere to biotic and abiotc surfaces, and are embedded in a polymeric matrix composed mainly of polysaccharides and proteins. Not only are biofilms a public health problem, but they are also a hindrance in industrial practices. Due to their intractability by conventional cleaning agents, a number of alternative agents, including enzymes, have been investigated as potential biofilm detachment-promoting agents. Two major types of enzymes, i.e. proteases and polysaccharases, have been used for biofilm removal and their use is aimed at degrading or promoting the collapse of the biofilm matrix. Consequently, the aim of this investigation was primarily to assess the use of enzymes originating from a wastewater biofilm to remove biofilms from three Pseudomonas species, viz. P. aeruginosa PAO1, P. fluorescens and P. putida. To investigate, biofilms were sampled from an aerobic reactor at an industrial wastewater treatment plant. Dissolution of the biofilm, as evidenced by reductions in the soluble chemical oxygen demand (COD) and total suspended solids (TSS), coincided with detectable protease and carbohydrate-degrading enzyme activities. Crude extracellular enzyme extracts prepared from the wastewater biofilm were subsequently shown to remove P. aeruginosa PAO1 biofilms from a glass surface, suggesting that the wastewater biofilms expressed enzymes that may be used towards the removal of detrimental biofilms. Consequently, representative bacteria were isolated from the wastewater biofilm and, based on 16S rRNA gene sequencing and analyses, were found to represent four major phylogenetic divisions of bacteria, i.e. Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Screening of the bacterial isolates for different enzyme activities indicated that nine isolates produced proteases, while ten isolates produced polysaccharide-degrading enzymes that comprised amylase, xylanase, cellulase, á-glucosidase and â-glucosidase. The ability of these enzymes to degrade proteins and polysaccharides present in purified EPS from P. aeruginosa PAO1, P. putida and P. fluorescens was confirmed by SDS-polyacrylamide gel electrophoresis and an increase in the amount of reducing sugar, respectively, while their efficacy to remove single-and multi-species biofilms cultured in microtiter plates was evaluated using a quantitative spectrophotometric assay. Proteases produced by four of the strains were effective in degrading the EPS proteins of all three Pseudomonas spp., while all bacterial strains that produced polysaccharide-degrading enzymes were capable of degrading the EPS polysaccharides, albeit with different efficiencies. Efficient removal of P. aeruginosa PAO1 biofilms was only achieved when mixtures of enzyme extracts, containing protease and different types of polysaccharase activities, were used. Biofilms of P. putida and P. fluorescens were readily removed with single enzyme extracts prepared from B. subtilis and B. pumilus. Enzyme combinations showing high biofilm removal for all three Pseudomonas species were tested against a mixed species biofilm. These enzyme extracts yielded lower biofilm removal efficiencies than those obtained for mono-species pseudomonad biofilms, possibly due to the heterogenous nature of the EPS. Nevertheless, it may be possible that the enzymes identified in this study could be used in combination with other treatments to increase the biofilm removal effectiveness or in combination with other enzymes to degrade the mixture of proteins and polysaccharides present in the EPS of multi-species biofilms. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Microorganisms, particularly bacteria, can associate together to form complex communities called biofilms. These communities are embedded in extracellular polymeric substances and can form on numerous surfaces such as implanted devices (catheters, central lines, joint replacement) in patients. These biofilms cause bloodstream and systemic infections that are difficult to treat and increase the chances of sepsis. Previously, our laboratory has identified a protein secreted by Klebsiella that has inhibitory effects on other members of the Enterobacteriacea bacterial family, namely Citrobacter and Enterobacter. Our current interest lies in the ability of the protein to potentially inhibit this bacterial family from establishing biofilms. In the present study, we wanted to explore: (1) if it is possible to form Citrobacter and Enterobacter biofilms in 6-well plates and on microscope coverslips; (2) if treating these biofilms with the secreted protein shows inhibition similar to previous planktonic cultures; (3) if these biofilms and inhibition could be visualized by a variety of staining techniques. To determine if it is possible to create Citrobacter and Enterobacter biofilms, bacteria were inoculated into 6-well plates, grown under static conditions in a 37°C incubator for 24 hours, and stained with crystal violet. Images showed that robust biofilms grew in the 6-well control plates while wells treated with the Klebsiella protein displayed reduced biofilms. To determine if it was possible to see Citrobacter and Enterobacter biofilms at a microscopic level, microscope slides were placed into 6-well plates, treated as above, and the slides were Gram stained. Images show thick biofilms consisting of Gram negative rods on control slides, while slides treated with the Klebsiella molecule become sparse and poorly grown. To determine if Citrobacter and Enterobacter biofilms could be fluorescently labeled for visualization, the same process was employed as above, but stained with a LIVE/DEAD cell viability kit where live cells fluoresce green and dead cells fluoresce red. Control slides showed bright thick green fluorescing biofilms while slides treated with the Klebsiella molecule had fewer green fluorescing cells and some red cells. From these observations, it is our conclusion that: (1) it is possible to grow Citrobacter and Enterobacter biofilms on both 6-well plates and microscope slides; (2) Citrobacter and Enterobacter biofilms can be visualized both by simple staining and fluorescent staining; (3) Citrobacter and Enterobacter biofilms are inhibited by Klebsiella secreted proteins. Currently, the identity of this protein is unknown. However, it is possible that this unknown protein could be of future use in the treatment of bacterial biofilms one identified.

Escherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain.

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Even though it has been decades since antibiotics were put into widespread use, bacterial infections are a worsening source of morbidity and mortality worldwide. This is partially due to the formation of biofilms. Biofilms are populations of microbial cells embedded in self-produced matrices and their formation can enhance survival of the pathogen in the host. Pseudomonas aeruginosa is a major cause of acute and chronic infections and an excellent model for the study of opportunistic, biofilm-based infections. It produces a plethora of virulence factors and we do not fully understand how it harms the host. This thesis investigates the synthesis and characteristics of the Refractile-body (R-body), a newly identified P. aeruginosa virulence factor and potential roles of this virulence factor during host colonization. R-bodies are large proteinaceous polymers that are produced as a coiled ribbon but can extend to form a spear-like structure that is longer than a bacterial cell. Further, the R-body is produced stochastically and the producing minority is thought to contribute to success of the population through altruistic suicide. The purpose of this thesis is to characterize yet another virulence factor in the arsenal of the notorious pathogen P. aeruginosa. Further, the capacity for R-body production is present in diverse bacteria, and characterization of its function could be pertinent for our understanding of other bacteria with roles in medicine, agriculture, and industry. In Chapter 1, I introduce concepts from the fields of bacterial infectious disease, population biology and gene expression to provide context for my research findings on the R-body. In Chapter 2, I describe the discovery of R-body polymers in the P. aeruginosa PA14 biofilm. Using mass spectrometry analysis, I identified a novel P. aeruginosa R-body protein absent in the Caedibacter taeniospiralis and Azorhizobium caulinodans genomes, two bacteria for which R-body production had previously been described. Further, results in the chapter elucidate the role of R-bodies in P. aeruginosa PA14 colonization in the plant and virulence in the nematode hosts. The work described in Chapter 3 focuses on the transcription factor RcgA, which is required for R-body production. The gene encoding RcgA lies in a cluster and is co-expressed with R-body structural genes. Using established genetic tools, I asked the question, “what signal does RcgA sense?” I found that RcgA binding to a cyclic nucleotide is necessary for its function in turning on R-body genes. I present data in Chapter 3 and 4 that sheds light on the regulatory logic of R-body production in P. aeruginosa. Specifically, using single-cell resolution methods, I have been able to characterize the impact of various genes on stochasticity of R-body production in the population. Data presented in these chapters are another example of the importance of studying heterogeneity and stochasticity of virulence factor expression in the population. Taken together, the work in this thesis provides an expanded and multifaceted understanding of a fascinating virulence factor found across bacterial phylogeny. The R-body produced by P. aeruginosa, a notorious human pathogen, is unique in its makeup and should be further characterized. This work also underscores the necessity of studying bacterial pathogenicity in the context of the biofilm lifestyle.

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative seleniumdependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.

Antibiotics are small molecules that kill bacteria by inhibiting essential processes. However, the concentrations used to kill bacteria in a clinical setting are typically much higher than the concentrations generated in nature, where most antibiotics are secreted by microbes. This discrepancy in concentrations, combined with a recognition that the human use of antibiotics bears little resemblance to the role of antibiotics in nature, prompted questions about whether growth inhibition was the primary function of antibiotics. Studying the effects of antibiotics at sub-lethal concentrations on bacteria could provide new insights into the natural role of antibiotics. One striking effect of bacterial encounters with sub-lethal antibiotics is the stimulation of biofilm formation. Biofilms are surface-adhered communities of bacteria. The biofilm lifestyle confers many benefits for bacteria and is a major mode of bacterial growth. Therefore, the ability of sub-lethal antibiotics to cause a transition from planktonic to biofilm growth indicates that antibiotics could be a driving force behind the assembly and abundance of bacterial communities in nature. Chapters Two and Three investigate the underlying mechanisms of this response in Escherichia coli and Pseudomonas aeruginosa, and suggest that sub-lethal antibiotics perturb central metabolism and respiration, changes that are sensed and relayed into increased biofilm formation to provide population-level protection. Chapters Four and Five investigate the effects of sub-lethal antibiotics on peptidoglycan metabolism in P. aeruginosa and E. coli. Peptidoglycan is an essential macromolecule for bacterial survival and is deeply integrated into their physiology. Furthermore, peptidoglycan synthesis is among the most favoured targets of antibiotics. Chapter Four investigates interactions between peptidoglycan-targeting antibiotics and folate metabolism-targeting antibiotics, and characterizes an overlooked connection between folate and peptidoglycan metabolism. Based on this work, we rationally designed a new inhibitor that potentiates folate and peptidoglycan-targeting antibiotics. Chapter Five sheds new light on peptidoglycan recycling by leveraging a pathway in P. aeruginosa for sensing and responding to sub-lethal doses of PG-targeting antibiotics. Finally, Chapter Six summarizes the understanding gained from Chapters Two through Five and synthesizes this information for broader insights on the possible roles of antibiotics in nature. / Thesis / Doctor of Philosophy (PhD) / The ability to cure infections with antibiotics revolutionized modern medicine and kick-started decades of research into the growth inhibitory properties of antibiotics. Although the therapeutic role of antibiotics as anti-bacterials is clear, the natural role of antibiotics is not. In nature, microbes export antibiotics, allowing them to interact with surrounding microbes that import those antibiotics, changing the physiology of the recipient. Understanding how antibiotics affect bacterial physiology at concentrations below the lethal dose can provide information about the natural role of antibiotics, which in turn can inform future antibiotic discovery. This thesis investigates two major effects of sub-lethal antibiotics on pathogenic bacteria. The first is the ability of antibiotics to induce the formation of surface-attached clusters of bacteria called biofilms. We showed that sub-lethal antibiotics have a common effect of disrupting cell metabolism, and this effect is translated into a signal for increased biofilm formation. The second is the effects of antibiotics on bacterial cell wall metabolism. We discovered that sub-lethal antifolate antibiotics impact cell wall metabolism in at least two different ways, and used this information to rationally design an inhibitor that overcomes antibiotic resistance. Further, sub-lethal antibiotics were used to identify new features of a cell wall recycling pathway. Overall, this work furthers our understanding of bacterial physiology at scales ranging from sub-cellular to multi-cellular, and reveals new impacts of sub-lethal antibiotics.

The transition from unicellular to multicellular life is captivating because free-living individuals become complex, coordinated assemblages that display unique properties and behaviors. It is a transformative step in biology that optimizes survival and resource utilization, especially in fluctuating environments. In microbiology, this multicellular organization assumes an intriguing form known as biofilms. Bacterial biofilms, assemblages of cells encased in a self-produced matrix, are sophisticated structures that provide protection from environmental challenges. The emerging understanding of biofilms reveals that bacteria within them do not exist as passive, isolated entities. Instead, they display spatial organization, physiological differentiation, and even metabolic interactions such as cross-feeding. The pathogenic bacterium Pseudomonas aeruginosa, which is a common cause of biofilm-based infections and a popular model organism, has been shown to form metabolic subpopulations and differentially regulate gene expression across depth in biofilms. However, one open question is the nature of this cellular arrangement in P. aeruginosa biofilms, the mechanisms governing it, and its physiological ramifications. My thesis addresses the overarching question: Does cellular arrangement in P. aeruginosa biofilms influence nutrient distribution, metabolic activity, antibiotic tolerance, and metabolic cross feeding? Through the use of paraffin embedding, thin-sectioning, and confocal microscopy, I delve deep into the biofilm, particularly in the z-direction, byproducing high-resolution images that provide insights into the three-dimensional structure and dynamics of these bacterial communities. The first chapter, serving as the foundation of this exploration, provides an introduction of the principles of multicellularity. It draws attention to the hallmarks of multicellularity, encompassing metabolic cross-feeding, protective advantages, and labor specialization while also shedding light on its challenges. In the context of multicellularity, biofilms are introduced, emphasizing the formation of bacterial biofilms, their environmental and medical implications, and specifically highlighting the importance of P. aeruginosa biofilms for understanding microanatomy and physiology. Chapter 2 presents the crux of our exploration, underlining how cellular arrangement directly impacts metabolic activity and antibiotic tolerance in P. aeruginosa biofilms. A striking observation was the presence of vertical, clonal striations, suggesting the presence of an organized architecture within mature biofilms. Mutants with disordered cell arrangements, particularly in O-antigen attachment, showed altered patterns of nutrient distribution and metabolic activity in addition to distinct patterns of antibiotic- induced cell death. Such findings build on prior knowledge by illuminating the intricate relationships between biofilm anatomy, metabolic differentiation, and drug tolerance. Chapter 3 introduces the use of light-sheet microscopy for live imaging of pellicle biofilms, which offers a real-time window into biofilm development and cellular dynamics. In Chapter 4, the narrative takes a broader perspective, focusing on the influence of various carbon sources on cellular arrangement. It introduces the presence of metabolic cross-feeding among different biofilm subpopulations and hints at the potential relationship between cell arrangement and heterogeneous metabolic activity patterns. The work in this thesis reveals that the arrangement of cells within P. aeruginosa biofilms determines metabolic outcomes, antibiotic responses, and potential cross- feeding interactions. In a world where biofilm-related infections account for an alarming 80% of persistent bacterial infections, understanding biofilm microanatomy has implications for therapeutic strategies and possibly reshaping our battle against antibiotic tolerance. A more detailed picture of the relationship between cell arrangement, physiological differentiation, and metabolic cooperation within biofilms has the potential to provide inroads toward new approaches to combating these recalcitrant structures.

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