The aim of this study was to examine whether Gram-positive cocci isolated from processed poultry in Western Australia provided a potential risk for the transfer of antimicrobial-resistant organisms to humans via commercially prepared ready-to-eat chicken. Research in this study was conducted in three phases: the characterisation of Gram-positive cocci isolated from poultry, an assessment of the isolates' thermal tolerance and the development of a Hazard Analysis Critical Control Points (HACCP) based food-safety program. In the first phase of the study, three specific objectives were investigated. The first determined the presence of Gram-positive cocci on poultry and on processing equipment from poultry-processing plants. The findings confirm the presence of staphylococci and enterococci on incoming live and slaughtered birds and processed carcasses. The data also indicate that carcasses probably become cross-contaminated during processing, when these bacteria are present on the incoming live birds and equipment. The second objective was to characterise staphylococcal isolates by antimicrobial susceptibility testing, and chromosomal and plasmid DNA analysis. The susceptibility of isolates to antimicrobial agents was tested by the disk diffusion method according to the NCCLS (National Committee for Clinical Laboratory Standards) guidelines. Isolates were typed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis of SmaI digested chromosomal DNA, and plasmids were isolated by the cetyltrimethylammonium bromide (CTAB) method. Approximately 37% of Staphylococcus aureus and 16% of coagulase-negative staphylococcal (CNS) isolates were resistant to six or more of the antimicrobial agents tested. Many isolates exhibited resistance to antibiotics that are commonly used in human medicine and registered for veterinary use in Australia. / Among the S. aureus isolates there were twenty-four epidemiologically unrelated SmaI CHEF groups. All staphylococcal isolates, except three CNS, were found to harbour from one to seven plasmids. Some staphylococcal isolates with epidemiologically related CHEF patterns had similar plasmid profiles and resistance patterns. The third objective was to determine the antimicrobial susceptibility of enterococci isolates to the glycopeptide antibiotics. The isolation of two vancomycin-resistant E. faecalis isolates is the first report of VRE outside the health-care setting in Western Australia. Additionally the detection of the vanA gene in an E. gallinarum isolate, a motile enterococcus, has potentially important implications for infection control practices in hospitals. In the second phase of the study, three specific objectives were established to investigate the practical implications of these findings for the chicken industry. The first objective of this phase of the study was to determine the thermal tolerance (D and Z-values) of antimicrobial-resistant, Gram-positive cocci in ground chicken meat. The results indicate that these isolates do not exhibit enhanced thermal-resistance characteristics compared to antimicrobial-susceptible bacteria. The second objective established the internal time-temperature profiles for cooking commercially prepared chicken and estimated the process lethality (F-values). / From three cooking trials, it was confirmed that the internal temperature of at least 70°C was achieved for at least thirty-eight minutes. The third objective of this phase assessed the effectiveness of the thermal process in reducing the risk of the transfer of antimicrobial-resistant cocci via the food chain. The data confirm that the lethal effect (F-values) of the thermal process destroyed these antimicrobial-resistant cocci in commercially prepared ready-to-eat chicken. In the third phase of the study, the data obtained in the earlier parts of the study was incorporated into a model food-safety program for a fast-food chicken chain. The model was based upon the internationally accepted HACCP system, adopted by the Codex Alimentarius Commission. Mindful that the thermal-process step represents only one critical control point in the safe preparation of chicken, this preventative approach ensures that all hazards are controlled at every other step of the process. The data suggest that antimicrobial-resistant, Gram-positive cocci will be present on some ready-to-cook poultry meat processed in Western Australia. This creates opportunities for the potential spread of resistant strains or resistance genes to humans via the food chain. The information from this study will be useful in providing background data and direction for future planning in preventing antimicrobial-resistant bacteria from poultry meat being transmitted through the food chain. The full implementation of the HACCP program would offer substantial benefits and protection to consumers.

Staphylococcus aureus is a Gram positive potentially pathogenic bacterium which has a propensity to gather resistance determinants. Mupirocin is a novel topical antibiotic active against many Gram positive species, including staphylococci and effective in the treatment and prevention of staphylococcal infections. Mupirocin acts by competitively inhibiting the charging of isoleucyl tRNA-synthetase (IRS) with isoleucine. Resistance has been observed in Staphylococcus aureus and coagulase negative staphylococci. Intermediate-level resistance (MIC >8μg ml^-1 >512μg ml^-1) is thought to be due to spontaneous mutations in the native IRS. High-level resistance (>512μg ml^-1) is conferred by a second IRS protein, encoded by mupA which has a much lower affinity for mupirocin than isoleucine. The mupirocin resistance gene (mupA) is usually found on a 4.05kb EcoRI fragment of plasmids of otherwise varied EcoRI restriction pattern which are easily transferred between strains by filter mating. Prior to the onset of these studies, mupirocin resistance had not been found linked to another resistance determinant. Initial investigations intended to identify mechanisms of gene flux resident on mupA plasmids revealed a family of related mupA plasmids, the p3356 family which includes three plasmid types: p3356, p3356D and p3358. p3356 contains a single copy mupA flanked by direct repeats of the staphylococcal insertion sequence IS257. p3356D is identical to p3356 except for the duplication of a "mupA-IS257" cassette in tandem repeat. p3358 is related to p3356D by the insertion of a pT181-like plasmid (tetracycline resistant) accompanied by the duplication of an IS257 in direct repeat to flank the inserted pT181; thus p3358 is the first documented example of linked resistance between mupirocin and another resistance determinant, namely tetracycline. IS257 has been implicated as the recombinogenic site in the gene duplication event involved in the evolution of p3356D from p3356. IS257 co-integrative transposition has been demonstrated to allow the co-integration of pOX7 with p3356 to generate a p3358-type plasmid in which pT181 is replaced by pOX7. Therefore, it is concluded that IS257 transposition and recombination is a mechanism by which staphylococcal replicons can evolve to form multiply resistant replicons.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: E.D. Brown. Includes bibliographical references (leaves 95-104).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 176 p.; also includes graphics (some col.) Includes bibliographical references (p. 158-176). Available online via OhioLINK's ETD Center

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Magister Scientiae - MSc (Biotechnology) / The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a natural barrier against infection. In both healthy and BV infected women Lactobacillus crispatus and Lactobacillus jensennii has been found to be the predominant Lactobacillus species. Many studies have been conducted to assess factors influencing lactobacilli dominance in the vaginal microbiome. However, no study has evaluated the impact of plasmids on the vaginal lactobacilli. In the present study two plasmids, pLc17 and pLc4, isolated from vaginal Lactobacillus species of both healthy and BV infected women were characterized. pLc4 was present in both Lactobacillus crispatus and Lactobacillus jensennii while pLc17 was only present in Lactobacillus crispatus. pLc17 (16663 bp in size) encoded a ribonucleotide diphosphate reductase (RNR), a filamentation induced by cAMP-like (FIC-like) protein and numerous mobile elements.

Master of Science / Department of Plant Pathology / Revathi Govind / Clostridium difficile, a gram-positive, anaerobic bacterium, is a major cause of antibiotic-related diarrhea and pseudomembraneous colitis. In the last decades, C. difficile has emerged as a major threat because of its tendency to cause frequent and severe disease. Because of the severity of the infection and its high rate of recurrence, there is a significant financial burden on healthcare systems. Antibiotic treatments are a primary risk factor for the development of C. difficile infection because they disrupt the normal gut flora in the host, enabling the antibiotic resistant bacterium to colonize the colon. Most of the resistance mechanisms in C. difficile reported to date can be classified as either antibiotic-degrading enzymes or modification of target sites. Another mechanism that can contribute to antibiotic resistance in C. difficile is the extrusion of antimicrobial compounds by efflux pumps. The goal of this project was to provide initial insights into the roles and mechanisms of a putative efflux pump complex. To do this, a number of experiments were designed to provide information about the structures, localization, and functions of this protein complex. It was determined that acidic pH conditions and a small number of antimicrobials, including inorganic compounds, organic compounds, fungicides, and antibiotics, inhibit growth of a C. difficile mutant lacking this pump system. Interestingly, higher NaCl in the medium and alkaline pH seem to promote the growth of a C. difficile mutant lacking this pump or, surprisingly, only inhibit growth of the wild type strain. The experiments performed in this project suggest that this efflux pump might have an essential role in C. difficile physiology, possibly by serving as an efflux pump for toxic metabolites.

Antibiotic resistance is increasing rapidly world wide. Resistance determinants have evolved long before antibiotics were used. Though horizontal gene transfer and mutation play a major role in the dissemination of antibiotic resistance genes their evolutionary origins remain obscure. A model system was used to investigate how they might arise in the first case. Plasmid borne erythromycin resistant clones were selected through marker rescue from genomic libraries of DNA from Gram positive organisms, maintained in E. coli. EryR determinants were recovered from nine libraries of 23 screened. Clone pMP1 (DNA from Mycobacterium parafortuitum) was the most resistant, with an MIC of 400 ìg/ml for erythromycin and 12 ìg/ml for azithromycin. Antibiotic resistance was not expressed in Rhodococcus erythropolis. Restriction maps were constructed for clones pMP1 and pMCX (DNA from Mycobacterium avium). Clone pMP1 was sub-cloned and the fragment carrying the EryR determinant (~2.4 kb) was sequenced. Analysis revealed 2 open reading frames (ORF1 and ORF2). ORF1 showed highest similarity to an FixB/FixA proteins and ORF2 showed similarity to a methyl transferase. Key words: antibiotic resistance,erythromycin, Gram positive DNA / Prof. E.R. Dabbs

Rothia mucilaginosa (Rm) is a gram-positive bacterium residing in the oral cavity. Recent studies in our laboratory have shown that this microorganism is able to cleave gluten, including immunogenic domains implicated in celiac disease. This can be beneficial to patients with celiac disease because exploitation of Rm can provide a novel mode of treatment. The enzymes responsible for this cleavage are as yet unknown. The purpose of this study was to optimize the transformation efficiencies of Rm cells through electroporation, with the ultimate goal to create knock-out mutants for enzyme activity. We have determined various aspects of Rm cells relevant for this project: (1) the growth curve characteristics of Rm; (2) the presence of endogenous restriction enzyme activities; and (3) the conditions facilitating Rm electroporation by varying electroporation voltages. Furthermore, electroporation and transformation of the plasmid pUC18 was conducted in Escherichia coli. The growth curve of Rm cells in BHI growth medium incubated at 37°C while shaking showed a doubling time of approximately 3 hours in the logarithmic growth phase. Using a cell sonicate of Rm cells incubated with Lambda DNA and four different restriction enzyme buffers, we found that there were no apparent endogenous restriction enzyme activities detectable. For the electroporation experiments, we used previously published protocols for the bacterium Staphylococcus aureus, as a standard condition to electroporate Rm cells. Those studies have shown that changing electrical parameters during the electroporation would yield a high efficiency rate of gram-positive bacterial transformation (Lofblom et. al., 2006; Metzler et. al., 1992). Therefore in our study, we increased the field strengths (kV*cm-1) to electroporate Rm cells. Rm cells could not be successfully transformed, and we observed that field strengths exceeding 18 kV*cm-1 destroyed Rm cells. On the other hand, the transformation of E. coli with pUC18 was successful. Our studies have laid the groundwork for investigating the transformation of Rm cells, and future studies can use the results obtained to further investigate ways to optimize transformation of Rm cells for potential utility in celiac patients.

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