Dissertation (Ph.D.)--University of South Florida, 2006. / Title from PDF of title page. Document formatted into pages; contains 90 pages. Includes vita. Includes bibliographical references.

Source: Masters Abstracts International, Volume: 47-01, page: 0341. Adviser: Luis Ostrosky. Includes bibliographical references.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen associated with significant morbidity and mortality. Previous colonisation with this pathogen is a risk factor for the development of subsequent infection. Tolllike receptors (TLRs) are a family of transmembrane receptors of the innate immune system that recognize pathogen-associated molecular patterns. The role of nasal colonisation of S. aureus has started to receive more attention. In spite of this, there are not enough studies looking at its effects on human primary nasal epithelial cells and their response to TLR ligands. The respiratory tract itself seems to pose a contradiction given by the clinical observation that its upper portion (nasal compartment) allows the growth of bacteria, acting like a reservoir, whereas the lower portion (lung compartment) reacts with an exuberant inflammatory response to the same organisms, as noted during pneumonia. The mechanism related with this phenomenon remains to be elucidated. A negative regulator of the TLR signalling cascade called toll-interacting protein (tollip) has been demonstrated to induce hyporesponsiveness in the gastrointestinal tract in the presence of bacteria. So far, tollip has not been demonstrated in the respiratory tract. Aims: To compare the responses of the upper and lower respiratory tract to TLR ligands, to characterise the role of tollip in the respiratory tract and its effects in the induction of tolerance, and to determine the cellular response to nasal carriage of S. aureus. Materials and Methods: The cell line RPMI 2650 (representative of nasal epithelium) and the cell line A549 (representative of type II alveolar epithelium) were used to establish the cytokine response to stimulation with TLR ligands and to demonstrate the presence of tollip protein by immunocytochemistry and enzymelinked immunosorbent assay (ELISA). Primary human nasal epithelial and type II alveolar epithelial cells were isolated and cultured from consented subjects. The cytokine response to stimulation was measured using cytokine bead array and the presence of tollip was determined by immunofluorescence and quantitative polymerase chain reaction. The presence of TLRs was assessed by immunocytochemistry in primary nasal and type II alveolar epithelial cells and the response to stimulation with the TLR9 agonist CpG-C ODN was assessed in these cells as well as in primary human type II alveolar epithelial cells. Subjects were also assessed for nasal carriage of S. aureus and their associated cytokine responses. Results: The RPMI 2650 cell line, despite retaining phenotypic characteristics of the nasal epithelium, appears unresponsive to stimulation with TLR ligands. In contrast, the A549 cell line responded significantly to stimulation with TLR ligands. Primary human nasal epithelial cells responded by secreting higher amounts of interleukin (IL)-8 and IL-6 in response to stimulation with S. aureus peptidoglycan (PGN) and tumour necrosis factor alpha (TNF-α) with a strong trend toward statistical significance. These cells did not respond to stimulation with Pseudomonas aeruginosa LPS. Primary type II alveolar epithelial cells responded significantly to stimulation with S. aureus PGN by increasing the secretion of IL-8, IL-6, IL-1β, TNF-α and IL-10 into cultured supernatant. Cells from the upper respiratory tract displayed a more tolerant phenotype given by the lower levels in cytokine production in response to stimulation with S. aureus PGN, in contrast to alveolar epithelial cells. TLRs were identified in primary nasal epithelial cells. The negative regulator tollip was identified in cell lines as well as primary cells of the respiratory tract in its three segments: nasal, bronchial and type II alveolar. It was not possible to demonstrate an up-regulation of tollip after stimulation with TLR ligands in any of the cell types studied, although, it was possible to observe a significantly higher constitutive level in tollip mRNA transcripts from primary nasal epithelial cells in comparison to type II alveolar epithelial cells. TLR9 was identified in human primary nasal epithelial cells, although it was not possible to observe an increase in cytokine production after stimulation with a TLR9 agonist. TLR9 was expressed strongly in primary type II alveolar epithelial cells which responded by significantly increasing IL-8 production after stimulation with CpG-C ODN. Primary nasal epithelial cells from individuals who carry S. aureus exhibit a proinflammatory profile, as evidenced by higher basal levels of IL-8 and IL-6 in comparison to non-colonised controls. Conclusion: The upper respiratory tract epithelium displays a tolerant phenotype in response to stimulation with TLR ligands in comparison to the lower respiratory epithelium, potentially favouring nasal colonisation by S. aureus. Tollip m-RNA transcripts appear to be up-regulated constitutively in the nasal epithelium which might favour this response. Staphylococcus aureus colonisation is however associated with a local pro-inflammatory state in the nasal epithelium of carrier individuals.

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Hung Ming Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 146-169). / Abstracts in English and Chinese. / Abstract --- p.I / Abstract (Chinese version) --- p.III / Acknowledgments --- p.V / Contents --- p.VI / List of Tables --- p.XII / List of Figures --- p.XIV / List of Abbreviations & Symbols --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Methicillin-resistant Staphylococcus aureus --- p.1 / Chapter 1.1.1 --- Definition and Identification --- p.1 / Chapter 1.1.2 --- Antibiotic resistance --- p.2 / Chapter 1.1.2.1 --- Methicillin/oxacillin resistance --- p.2 / Chapter 1.1.2.2 --- Other antibiotic resistance --- p.2 / Chapter 1.2 --- Evolution of MRSA --- p.3 / Chapter 1.2.1 --- Staphylococcal Chromosome Cassette mec (SCCmec) --- p.3 / Chapter 1.2.2 --- The origin of mec element --- p.7 / Chapter 1.2.3 --- The acquisition of SCCmec: from MSSA to MRSA --- p.7 / Chapter 1.2.4 --- Five pandemic clones --- p.8 / Chapter 1.3 --- Epidemiology of MRSA --- p.10 / Chapter 1.3.1 --- MRSA Infections --- p.12 / Chapter 1.3.2 --- Community-acquired MRSA --- p.13 / Chapter 1.4 --- Staphylococcal Gene Regulators --- p.14 / Chapter 1.4.1 --- Accessory gene regulator (agr) --- p.14 / Chapter 1.4.1.1 --- Structure and functions of accessory gene regulator (agr) --- p.15 / Chapter 1.4.1.2 --- Autoinducing Peptide (AIP) --- p.16 / Chapter 1.4.1.3 --- Implication of agr I-IV --- p.18 / Chapter 1.4.2 --- Staphylococcal accessory regulator (sar) --- p.19 / Chapter 1.4.2.1 --- Structure and function of sar --- p.19 / Chapter 1.4.3 --- Difference in Functions of sar and agr --- p.20 / Chapter 1.4.4 --- "Other sar A homologues, SarS, R，T, U, and other Gene Regulators" --- p.22 / Chapter 1.5 --- Adhesin and toxin genes --- p.25 / Chapter 1.5.1 --- Adhesins --- p.25 / Chapter 1.5.2 --- Exotoxins --- p.26 / Chapter 1.6 --- Typing Methods --- p.29 / Chapter 1.6.1 --- Phenotypic typing method --- p.31 / Chapter 1.6.1.1 --- Antibiotic-susceptibility Test --- p.31 / Chapter 1.6.1.2 --- Bacteriophage Typing --- p.31 / Chapter 1.6.1.3 --- Serotyping --- p.32 / Chapter 1.6.1.4 --- Electrophoretic Protein Typing and Immunoblotting --- p.33 / Chapter 1.6.1.5 --- Multilocus Enzyme Electrophoresis --- p.34 / Chapter 1.6.1.6 --- Limitations of phenotyping --- p.34 / Chapter 1.6.2 --- Genotyping methods --- p.35 / Chapter 1.6.2.1 --- PCR Assays --- p.35 / Chapter 1.6.2.2 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.36 / Chapter 1.6.2.3 --- Multi-Locus Sequence Typing (MLST) --- p.37 / Chapter 1.6.2.4 --- Amplified Fragment Length Polymorphism (AFLP) --- p.38 / Chapter 1.6.2.5 --- Ribotyping --- p.38 / Chapter 1.6.2.6 --- Plasmid Typing --- p.39 / Chapter 1.6.2.7 --- Restriction Fragment Length Polymorphism --- p.39 / Chapter 1.6.2.8 --- Nucleic Acid Hybridization --- p.40 / Chapter 1.7 --- Objectives of the project --- p.41 / Chapter Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Bacterial Isolates & Culture conditions --- p.42 / Chapter 2.1.1 --- Bacterial Isolates --- p.43 / Chapter 2.1.2 --- Reference Strains --- p.43 / Chapter 2.1.3 --- Identification of MRSA --- p.44 / Chapter 2.2 --- Antibiotic Susceptibility Test --- p.45 / Chapter 2.3 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.47 / Chapter 2.3.1 --- DNA preparation --- p.47 / Chapter 2.3.2 --- Restriction digestion --- p.48 / Chapter 2.3.3 --- PFGE --- p.48 / Chapter 2.3.4 --- Analysis of band patterns --- p.48 / Chapter 2.4 --- Phage Typing --- p.50 / Chapter 2.4.1 --- Source of Phages & Propagating Strains --- p.50 / Chapter 2.4.2 --- Procedures of phage typing --- p.50 / Chapter 2.4.3 --- Routine check of the lytic strength of phages --- p.51 / Chapter 2.4.4 --- Evaluation of the reproducibility of phage typing --- p.51 / Chapter 2.5 --- Detection of SCCmec and mec Genes by Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.5.1 --- DNA preparation for PCR --- p.55 / Chapter 2.5.2 --- Master mix preparation and PCR conditions --- p.55 / Chapter 2.5.3 --- Electrophoresis of DNA Amplicors (PCR products) --- p.55 / Chapter 2.5.4 --- Detection of mecA gene by PCR --- p.56 / Chapter 2.5.5 --- Detection of SCCmec I-IV by PCR --- p.58 / Chapter 2.6 --- Multiplex Polymerase Chain Reaction --- p.60 / Chapter 2.6.1 --- Detection of agr I-IV --- p.60 / Chapter 2.6.2 --- Detection of Pyrogenic Toxin Genes --- p.62 / Chapter 2.6.2.1 --- Primer Mix Preparation for Multiplex PCR --- p.66 / Chapter 2.6.2.1.1 --- Sea-see Multiplex PCR --- p.66 / Chapter 2.6.2.1.2 --- Seg-sej Multiplex PCR --- p.66 / Chapter 2.6.2.1.3 --- "Eta, etb, tsst-1 for multiplex PCR" --- p.66 / Chapter 2.6.2.2 --- Multiplex Master Mix Preparation --- p.67 / Chapter 2.6.2.3 --- "PCR controls for pyrogenic toxins (sea-sej, eta-b, tsst-1)" --- p.67 / Chapter 2.6.2.4 --- Sequencing of PCR products --- p.68 / Chapter 2.6.3 --- PCR for adhesin and toxin genes --- p.69 / Chapter 2.6.3.1 --- "Multiplex Master mix preparation for lukE, fib, cna, icaD" --- p.71 / Chapter 2.6.3.2 --- "Multiplex Master mix preparation for fnbA,fnbB, hla, hlb, icaA" --- p.71 / Chapter 2.6.3.3 --- PCR controls for adhesin and toxin genes --- p.71 / Chapter 2.6.3.4 --- Size of PCR products resolved by gel electrophoresis --- p.72 / Chapter 2.6.3.5 --- Sequencing of PCR products --- p.72 / Chapter 2.7 --- Multi-locus sequence typing --- p.73 / Chapter 2.7.1 --- Bacterial Isolates --- p.73 / Chapter 2.7.2 --- Primer design --- p.73 / Chapter 2.7.3 --- PCR Master mix preparation --- p.73 / Chapter 2.7.4 --- PCR for MLST gene --- p.75 / Chapter 2.7.5 --- Sequencing of PCR products --- p.75 / Chapter 2.7.6 --- Data Analysis --- p.75 / Chapter 2.8 --- Sequence typing of Coa gene --- p.76 / Chapter 2.8.1 --- Bacterial Isolates --- p.76 / Chapter 2.8.2 --- Amplification of Coa Gene by PCR --- p.76 / Chapter 2.8.3 --- coa sequencing --- p.77 / Chapter 2.8.4 --- Sequence Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.79 / Chapter 3.1 --- Bacterial Isolates --- p.79 / Chapter 3.2 --- PCR for mecA gene --- p.80 / Chapter 3.3 --- Antibiotics Susceptibility Test --- p.81 / Chapter 3.3.1 --- Antibiotic Resistance Profiles of MRSA --- p.82 / Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.85 / Chapter 3.4.1 --- Optimization of PFGE and Inter-gel Variation --- p.85 / Chapter 3.4.2 --- Analysis of PFGE profiles --- p.87 / Chapter 3.4.3 --- PFGE patterns and antibiotic profiles --- p.93 / Chapter 3.5 --- Phage Typing --- p.96 / Chapter 3.6 --- SCCmec Typing --- p.98 / Chapter 3.6.1 --- PCR Optimization --- p.98 / Chapter 3.6.2 --- PCR and distribution of SCCmec types --- p.101 / Chapter 3.6.3 --- PFGE patterns and SCCmec types --- p.103 / Chapter 3.7 --- agr Typing --- p.104 / Chapter 3.7.1 --- Optimization of PCR --- p.104 / Chapter 3.7.2 --- PCR and Distribution of agr groups --- p.106 / Chapter 3.7.3 --- PFGE patterns and agr groups --- p.108 / Chapter 3.7.4 --- SCCmec types and agr groups --- p.108 / Chapter 3.8 --- "PCR for adhesin, pyrogenie and other toxin genes" --- p.110 / Chapter 3.8.1 --- Optimization of PCR --- p.110 / Chapter 3.8.2 --- "Distribution of adhesin, pyrogenic and other toxin genes" --- p.120 / Chapter 3.9 --- Multi-Locus Sequence Typing --- p.124 / Chapter 3.10 --- coa Sequence Typing --- p.126 / Chapter Chapter 4 --- Discussion --- p.128 / Chapter 4.1 --- Evaluation of the Typing methods for MRSA --- p.128 / Chapter 4.1.1 --- Antibiotic Susceptibility Test --- p.128 / Chapter 4.1.2 --- PFGE --- p.128 / Chapter 4.1.3 --- Phage Typing --- p.130 / Chapter 4.1.4 --- "PFGE, SCCmec typing and agr typing" --- p.131 / Chapter 4.1.5 --- MLST --- p.132 / Chapter 4.1.6 --- Sequencing typing of coa gene --- p.133 / Chapter 4.2 --- Characteristics of MRSA --- p.134 / Chapter 4.2.1 --- PCR for adhesin and toxin genes --- p.134 / Chapter 4.3 --- Key Characteristics of Hong Kong isolates --- p.137 / Chapter 4.4 --- Conclusion --- p.144 / Chapter 4 5 --- Future Research --- p.145 / Reference List --- p.146 / Appendix I - Materials/Reagents for Methods --- p.170 / Appendix II - Reagents Formula --- p.178 / Appendix III - coa sequence alignment --- p.180 / Appendix IV - Typing patterns and Characteristics of reference isolates --- p.193 / Appendix V - Miscellaneous --- p.195 / Appendix VI - Typing Patterns and Characteristics of PFGE type isolates --- p.204

Thesis (M.S.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 160-171). Also available via World Wide Web.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background Methicillin resistant Staphylococcus aureus (MRSA) is endemic in healthcare settings in many countries of the world. Patients who have acquired MRSA serve as a source of transmission by contamination of their surrounding environments. Numerous studies illustrate that many different inanimate surfaces in hospitals can become a reservoir for MRSA. Objectives The objective of this study is to examine the presence of MRSA on environmental surfaces and its relationship between patients’ acquisition of MRSA by studying their molecular characteristics. Methodology The near-patient surfaces of 30 MRSA positive patients, 30 control patients and the ward environments were sampled from June 2011 to September 2011. The swabs were enriched and cultured for the presence of MRSA. The MRSA isolates obtained from environmental samples and from the clinical samples of the patients were then characterized by Spa typing. Results The MRSA found in case patients and control patients’ environmental surfaces was 97% (29/30) and 40% (12/30) respectively. Environmental surfaces that were highly contaminated by MRSA positive patients were bed sheets (70%), followed by pillows (55%), patient bed frames (52%) and patient lockers (52%). On the environmental surfaces other than the near-patient areas, ambulatory chair armrests had the highest amount of MRSA (21%), followed by fax machines which accounted for 14%. Among the 216 MRSA isolates (30 clinical isolates and 151 environmental isolates), eight spa types were found and the most predominant spa type was t1081 (63.3%) followed by t032 (17.6%) and t037 (7.4%). 27 patients were found to have the MRSA isolates with same spa type in the clinical samples and their surrounding environments. The agreement between the MRSA isolated from the clinical sample of patients and their surrounding environment was 93.1%. Conclusion Identical isolates were recovered from the patient and their environment (93.1%) which suggests possible environmental contamination of the ward cubicles, possibly contributing to endemic MRSA. More effective and rigorous use of current approaches to cleaning and decontamination is required and consideration of newer technologies to eradicate MRSA. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Methicillin-resistant Staphylococcus aureus (MRSA) is an important and common pathogen causing community- and healthcare-associated infection. Culture methods were used for identification of MRSA for a long period of time, however it spends a lot of time on incubation and 1 to 2 days is needed to obtain the identification and antibiogram. Molecular tests were developed in the past decades and different genes were used. In this study a Staphylococcus aureus-specific gene, sau gene was designed and accompanied with mecA gene to detect the presence of MRSA in 322 nasal swabs from Tuen Mun Hospital. To evaluate the performance of in-house RT-PCR, samples were run in parallel with LightCycler? MRSA Advanced test and BBLTM CHROMagar? MRSA. 75 (23%) of samples were MRSA positive. The sensitivities and specificities of in-house RT-PCR and LightCycler? MRSA Advanced test were 76.7%/ 89.2% and 87.8%/ 96.6% respectively. The mean processing time for a batch of 32 samples by CHROMagar, in-house RT-PCR and LightCycler? MRSA Advanced test were 48.9 hours, 134.4 mins and 149.8 mins. In-house RT-PCR showed comparable performance and short turnaround time. sau gene can be used with mecA gene for the detection of MRSA in nasal swab. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Thesis (Ph. D.)--Illinois State University, 1999. / Title from title page screen, viewed July 20, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Radheshyam K. Jayaswal, Alan J. Katz, Anthony J. Otsuka, David L. Williams. Includes bibliographical references and abstract. Also available in print.