A study was conducted to find the effect of magnesium, calcium, manganese and zinc ions on the infection of Psudomonas aeruginosa strain 1C by RNA bacteriophage 7s. When an 18 hour progeny experiment was performed, it was found that magnesium, calcium and manganese had different effects on bacteriophage production and was dependent on the bacterial growth conditions. RNA bacteriophage 7s progeny production was significantly enhanced by the addition of magnesium to cultures of Psudomonas aeruginosa 1C grown in a magnesium deficient medium. Under these environmental conditions there was a slight increase in progeny in the presence of calcium. When Psudomonas aeruginosa 1c was grown in a complete medium, the infection of cells by bacteriophage 7s was enhanced by magnesium and calcium but not manganese or zinc, as demonstrated by the One Step Growth Curve.

Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control. / Ph. D.

No description available.

Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and is of particular concern due to its intrinsic resistance to many frontline antibiotics. To aid in infection, Pseudomonas encodes an arsenal of virulence factors, including type IV pili (T4P), hair-like adhesins involved in many processes, such as twitching motility and surface attachment. T4P are primarily composed of the major pilin, PilA, whose expression is tightly regulated by the PilS-PilR two-component system. The sensor kinase, PilS, monitors the inner membrane PilA inventory and modifies activity of the response regulator, PilR, to regulate pilA transcription. Here, we demonstrate that P. aeruginosa virulence in a roundworm infection model is reduced when the amount of T4P expressed at the cell surface increases, regardless of the ability of the bacteria to twitch. We propose that inappropriate increases in surface T4P expression may impair pathogenicity-associated systems which require intimate host-cell contact. New genes in the regulon of the PilS-PilR two-component system were also identified. A tool to fluorescently label and image T4P in real-time using microscopy was established in the lab. This work highlights the consequences of increased surface T4P expression, providing potential new targets for antipseudomonal therapeutics which act on components involved in T4P expression and function.

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The problem of finding an efficient antibiotic against Pseudomonas aeruginosa which can be used in the clinical treatment of genito-urinary tract infections resistant to treatment by streptomycin has not yet been solved. Therefore, this problem has consisted of first, the acquisition of possible inhibitors of the streptomycin resistant strain of Pseudomonas aeruginosa; second, the selection and identification of those which show a marked antagonism toward this organism; third, the determination of the antibiotic spectra of the inhibitors; fourth, the determination of whether the streptomycin resistant strain could also acquire a resistance to the antibiotic produced by its inhibitors; and last, an attempt to evaluate the therapeutic possibilities of the antibiotics demonstrated.

Thesis (D.P.H.)--University of Michigan.