The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

No description available.

Shigellae are pathogenic bacteria that cause the disease shigellosis. Two methods for studying secreted effectors encoded by this pathogen’s virulence plasmid are described. First, protein microarrays were used to identify substrates of an E3 ubiquitin ligase called IpaH7.8. Second, a deletion collection containing mutants for every gene on the virulence plasmid was used in two screens: one to identify mutants that elicit atypical levels of Interleukin-8 (IL-8) from U937 cells, and one to identify mutants that bind the dye Congo red abnormally. Although protein microarrays were an ineffective tool, the deletion collection proved valuable. Most mutants were less effective at sequestering Congo red than wild-type S. flexneri, although this ability was enhanced in several mutants. Four mutants, ?ospB, ?orf186, ?mxiH and ?mxiK, elicited higher levels of IL-8 from U937 cells than wild type S. flexneri. These results validate the use of the deletion collection as a tool for studying bacterial pathogenesis.

The sources of gentamicin resistant pseudomonads and enterobacteria were studied in detail. A total of 1703 gentamicin resistant gram negative bacilli (GRGNB) isolated from patients, staff and their immediate environment were studied over a 6 month period . Of these 954 were isolated from clinical specimens obtained from patients and 540 from their immediate environment. A furthur 209 stains were isolated from the staff members who were responsible for the care of these patients. Pseudomonas aeruginosa; pyocin type 1 phage type F7 and .serotype 11 was the commonest isolate. It constituted 24,9% of all isolates in this study. This organism was distributed in all the wards investigated and was isolated throughout the 6 month study period. This strain, therefore, appears to be part of the "resident'' flora of King Edward Vlll Hospital for it was found on patients, staff and their immediate environment. Among the Enterobacteriaceae, Klebsiella pneumoniae was the commonest isolate and made up 13,6 % of all isolates. All the isolates obtained in this study were resistant to five of more antibiotics tested (gentamicin, tobramycin, kanamycin, streptomycin, carberricillin, polymyxin B amikacin and sisomicin). Of 310 staff members screened 25,2% harboured GRGNB on their hands. Among patients the commonest source of GRGNB was stool which yielded 141 (14,8 %) of the clinical isolates. Of the environmental sources studied, sinks harboured 87 (14%) GRGNB. The isolates from the environment and staff members were identical to patient strains. The significance of these findings is discussed. / Thesis (Fellowship of the Society of Medical Laboratory Technologists of South Africa)-University of Natal, Durban, 1985.

The susceptibility of 80 StaphyIococcus aureus isolated to oxacillin was investigated using microtitre, agar dilution and Stokes' disc diffusion methods. There was a bimodal distribution of the isolates according to the oxacillin minimum inhibitory concentration (MIC) values. For the sensitive isolates, the agar dilution method generally gave lower MIC values than the microtitre method, while for the resistant isolates the agar dilution method gave comparable to slightly lower MIC values than the microtitre method. The Stokes disc diffusion method yielding the best results when performed on Mueller-Hinton agar incubated at 30°C for 18 hours; however local strains grew poorly when incubated at 30 C for 18 hours. The next best medium which provided clear disc diffusion results plus good growth was Mueller-Hinton agar incubated at 35°C for 18 hours, on which 10 % of the sensitive isolates appeared intermediate in susceptibility, and none resistant, while all the resistant isolates (microtitre MIC >8mg/1) appeared resistant. Oxacillin resistance among strains of Staphylococcus aureus tested by Stokes' disc diffusion method correlated best with gentamicin resistance, and less often with tetracycline resistance. Therefore gentamicin- or tetracycline-resistance may indicate oxacillin resistance in Staphylococcus aureus. / Thesis (M.Med.)-University of Natal, Durban, 1988.

A survey was conducted to answer two basic questions: 1) What are the medical bacteriology laboratories of Indiana using as criteria for identifying microorganisms? and 2) What is the basis for these criteria? The author developed a questionnaire used as the survey instrument. One hundred fifty questionnaires were mailed to medical laboratories throughout Indiana. Sixty percent of the laboratories responded. This study lists all responses and gives a description and/or evaluation of each procedure.In addition, the researcher discusses some of the factors influencing the type of procedures that are being used. Data collected by this research will be submitted to the biology department at Ball State University to help establish more concrete guidelines that can be used to update course content.

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

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No description available.

Thesis equivalent (M.P.H.)--University of Michigan, 1948.