No description available.

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, is one of the most and best studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. P. aeruginosa forms biofilms through a series of interactions between the cells and adherence to surfaces, which is mediated by surface appendages such as flagella and type IV pili. A gene cluster, designated htpABCDEFGI, which appears to encode protein products with homology to those encoded by recently described novel pilus biogenesis and assembly systems, has been identified in P. aeruginosa PAO1. Since the pili produced by these systems, designated Flp, are associated with the ability of the bacteria to bind non-specifically to inert surfaces, the aims of this study were to characterize the transcriptional organization of the putative P. aeruginosa PAO1 htp gene cluster and to determine the functional importance of the htp gene cluster in the ability of P. Aeruginosa PAO1 to adhere to surfaces. In silico evidence has suggested that the pilin subunit gene flp is not part of the P. Aeruginosa htp gene cluster thought to encode proteins involved in the synthesis, assembly and export of these pili. To determine the transcriptional organization of this gene cluster, total RNA from P. aeruginosa PAO1 was analyzed by reverse transcriptase-polymerase chain reaction (RTPCR). Primers designed to amplify regions spanning gene junctions yielded amplicons at each individual gene junction from htpA to htpI, as well as an amplicon for flp. Moreover, corresponding sigma 70 (σ70) consensus sequences were identified in the intergenic region between the htpA and flp genes and promoter function of the flp and htpA upstream region was subsequently confirmed using lacZ reporter gene constructs transformed into P.aeruginosa PAO1. The results therefore indicated that the htp gene cluster is an operon transcribed as a polycistronic message, whilst the flp gene is transcribed independently as a monocistronic message. To determine the functional importance of thehtp gene cluster in P. aeruginosa PAO1, the htpD gene, encoding a putative NTPase, was inactivated by in vivo homologous recombination with an appropriately constructed allelic exchange vector to generate the isogenic mutant strain PAOHtpD. Comparative analysis of the wild-type P. aeruginosa PAO1 and mutant PAOHtpD strain revealed that the mutant strain was impaired in its ability to attach to a glass wool substratum and also in its ability to grow as a biofilm. Since the mutant PAOHtpD strain was not growth-impaired, these results indicate that the htp gene cluster plays a role in P. aeruginosa PAO1 biofilm development under the culturing conditions used in this study. Thus, it can be proposed that the flp and htp gene cluster of P. aeruginosa PAO1 may play a role in its ability to successfully colonize abiotic surfaces. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

The Staphylococcus genus comprises a diverse group of Gram-positive bacteria that are opportunistic pathogens of humans and other mammals. S. epidermidis and S. aureus are the most common human pathogens of the staphylococci, causing a variety of infections including biofilm-based medical device infections, skin infections, and pneumonia. Both of these organisms produce proteases whose functions in virulence are not fully characterized. In S. epidermidis, protein-mediated biofilm formation requires a cell wall-anchored adhesin called Aap that must be proteolytically processed in order to allow intercellular adhesion. The S. epidermidis protease(s) responsible for cleaving Aap were unknown. Chapter II describes our findings that the secreted metalloprotease SepA is required for Aap-mediated biofilm formation and cleaves Aap at two different sites. Further, this protease is negatively regulated by the global regulator SarA. Chapter III discusses studies of the S. aureus Spl (serine protease-like) proteases. Although they are produced in vivo, their substrates and role in virulence are unknown. We found that in a rabbit model of pneumonia, a mutant lacking the spl protease operon caused more localized disease compared to wild type S. aureus. Proteomics studies of the secreted and surface proteins in wild type compared to spl mutant S. aureus revealed several changes. We also found that the SplA protease cleaves human Mucin-16, the first identification of a biological substrate of the Spls. Finally, we found that the animal-associated species S. caprae produces an autoinducing peptide (AIP) that is a potent inhibitor of S. aureus quorum sensing. We identified the S. caprae AIP structure as an 8-residue thiolactone ring. A synthetic version of the peptide inhibits S. aureus virulence and quorum sensing induction in a murine skin infection model. This is a novel example of quorum sensing cross talk between staphylococcal quorum sensing systems. These studies are described in Appendix A. On the whole, this work identified two substrates of S. aureus proteases and demonstrated their importance in biofilm formation and infection. We also characterized a novel inhibitor of S. aureus quorum sensing that attenuates virulence. These findings shed light on the importance of staphylococcal secreted proteases and quorum sensing cross talk in the modulation of virulence factor production and the ability to cause disease.

Dispersion is a natural part of a biofilm life cycle in many bacterial species. Dispersion occurs when bacteria revert from a stationary, sessile state to a free-swimming, planktonic state and are freed from a biofilm. Bacterial biofilms consist of proteins, polysaccharides, and extracellular DNA that together make up the extracellular polymeric substances. Surrounded by this mucus-like substance, sessile cells can be extremely difficult to eradicate as compared to the planktonic form of Listeria monocytogenes. Biofilms are robust due to increased surface adherence, inhibition of diffusion of harmful compounds, and increased genetic diversity that exists within a biofilm. As a result, traditional biofilm removal methods are often inadequate; and a novel method for the eradication of Listeria monocytogenes biofilms is needed. Here it is shown that two known biofilm dispersal agents, nitric oxide and cis-2-Decenoic acid, do not induce dispersion in Listeria monocytogenes strain LM23. Nitric oxide and cis-2-Decenoic acid do not influence planktonic cell numbers or biofilm biomass. Ten carbohydrates were screened for their influence on biofilm biomass for use in investigation into natural biofilm dispersion in Listeria monocytogenes strain LM23. Carbohydrate source can significantly increase or decrease biofilm biomass as compared to glucose. Natural biofilm dispersion in Listeria monocytogenes remains inconclusive, yet warrants further investigation. Changes in planktonic cells numbers, sessile cell numbers, and biofilm biomass were tracked under static growth conditions, and suggested a possible dispersion event. However, treatment of biofilms with spent media and observation using scanning electron microscopy did not clarify the results obtained. This research deems the nitric oxide donors, molsidomine (N- (ethoxycarbonyl)-3-(4-morpholinyl)-sydnone imine) and MAHMA NONOate (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), as well as cis-2-Decenoic acid as ineffective in inducing biofilm dispersion. It also brings about new research questions into natural biofilm dispersion in Listeria monocytogenes.

No description available.

archives@tulane.edu / Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current work, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi and exhibit antibiofilm activity against methicillin-resistant S. aureus (MRSA) and Streptococcus mutans. We show that a number of compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid present in B. thailandensis OMVs exert antimicrobial activity. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against various microbes. Rhamnolipid is superior at reducing the integrity of biofilms while HMNQ displays greater bactericidal activity. We attempted to use HMNQ and rhamnolipid to combat MRSA and promote wound healing in a murine full-thickness wound model. However, further optimization of the model and characterization of the molecules in antimicrobial efficacy, wound healing, and host immune responses are required. Overall, this work indicates that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm-forming and multidrug-resistant organisms. / 1 / Yihui Wang

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

The purpose of this thesis is to investigate the use of a single reactor to biologically treat wastewater by simultaneously oxidizing ammonia, and reducing nitrate and nitrite. The Environmental Protection Agency (EPA) places strict discharge restrictions on these compounds due to their inherent toxicity to humans, wildlife, and ecosystems. The use of a simultaneous system can assist the conventional wastewater treatment technology that requires separate systems, by creating a system that needs less time and smaller size to reach effluent requirements. To conduct this research, a bench-scale membrane aerated biofilm reactor was built using silicone tubing for aeration. Batch and continuous-flow experiments were conducted to investigate the reactor’s capability to oxidize ammonia using a defined growth media and monitor nitrate production and reduction. Also, wastewater from a local reclamation facility was used to determine the reactor’s ability to nitrify ammonia and denitrify nitrate concentrations within wastewater. The wastewater was taken from different locations within the reclamation facility, and combinations of primary and nitrified effluent were used to monitor ammonia and nitrate concentration changes. The batch experiments showed the greatest changes, and one batch experiment showed a 79% decrease in ammonium concentrations, and followed a first-order kinetics rate constant of -0.0284 hrs-1. The continuous-flow experiments showed much greater fluctuations in results, but one of the experiments showed an ammonia oxidation efficiency of 86%. The wastewater experiments had even greater fluctuations, and the effluent concentrations of ammonia, nitrate and nitrite showed no changes when compared to the influent.

Biofouling and biocorrosion are due to unwanted deposition of microorganisms on surfaces that are exposed to different types of water. This dissertation focuses on the application of innovative strategies to inhibit biofouling and biocorrosion. Specifically, the strategies examined in this dissertation, namely the use of bacteriophages and quorum quenchers, aim to minimize reliance on the conventional chemical cleaning agents and to reduce chemical-induced hazards on health, safety and environment. First, we analyzed the use of bacteriophages to remove biofoulants on ultrafiltration membrane used in seawater reverse osmosis pretreatment. Our findings revealed that bacteriophages were able to remain active against membrane-associated Pseudomonas aeruginosa at a broad range of temperature, pH and salinity. Bacteriophages were also shown to inhibit biofilm and to reduce transmembrane pressure increment, when applied alone or in combination with chemical agents. Second, this dissertation explores the use of quorum quenchers to inhibit biocorrosion in seawater environment. To do so, we first examined for the presence of quorum sensing system in sulfate reducing bacteria (SRB). Through transcriptomic analysis, we further demonstrate a strong correlation between quorum sensing, biofilm formation and biocorrosion. Therefore, the use of quorum sensing inhibitors was suggested to prevent biofilm formation and biocorrosion caused by SRB in seawater. Through findings from Chapter 2 and 3, we introduced the use of alternative biocidal agents to tackle biofouling and biocorrosion. Compared to quorum quenchers, bacteriophages showed better antibiofilm potential and easier applicability at larger scale. However, bacteriophages alone were insufficient to reduce biofilm formation as phage resistance was observed over long-term experiments. Hence in the last chapter, we further explored the use of bacteriophages to alleviate biofouling that occurred during wastewater treatment process, by combining their infection with UV irradiation. UV was used both for its biocidal effect and to trigger phage infection against bacteria. Our findings indicate that the combined treatment was able to remove mature biofoulants from the membrane. Overall, this dissertation demonstrates the use of bacteriophages and quorum quenchers against biofilm. These two approaches can serve to reduce the amount of chemicals used during cleaning, thus providing a more sustainable way of minimizing biofilm-associated problems.

Extraction of oil and gas in Utah’s Uintah Basin results in large quantities of wastewater, or produced water, with nutrients and residual organic chemical that represent a significant resource for producing energy-related and value-added products. Produced water was obtained as a biomass producing nutrient source from industries operating in Utah’s Uintah Basin. Within the Uintah Basin (defined as Uintah and Duchesne Counties within Utah) approximately 93 million barrels of water were produced in 2013 while only 11% of the water was disposed of through evaporation, with the national average at 2%. The rest is reinjected into the subsurface. The goal of this project was to design a system that utilizes produced water as a nutrient source for growing microalgae biomass in a biofilm form using a Rotating Algal Biofilm Reactor (RABR). The biomass would then be harvested and converted into biocrude oil using hydrothermal liquefaction (HTL). The objectives were to (1) cultivate biomass on produced water, (2) optimize the reactor to reduce energy costs to operate while increasing biomass productivity, and (3) increase feedstock quality for HTL. The RABR was constructed out of polystyrene disks, and experimentation was carried out to optimize rotational speed of the reactor. Two strains of algal biomass were identified as biofilm formers and grown using produced water as the nutrient source. The biomass was then utilized as a HTL feedstock that gave an average yield of 34.5% ash free dry weight.