Browsing by Subject "Staphylococcus aureus"
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Item Comparative genomics, antimicrobial resistance determinants, and pathogenicity of community-associated Staphylococcus aureus(2016-05) Lee, Grace Choi; Frei, Christopher R.; Lawson, Kenneth A; Wilson, James P; Wang, Yufeng; Olsen, RandallStaphylococcus aureus is a major human pathogen and a global public health issue. It is considered an opportunistic pathogen as it asymptomatically colonizes its host, but can occasionally cause diseases that range in severity from relatively minor skin and soft tissue infections (SSTI) to life-threatening cases of pneumonia and endocarditis. There is a critical need to better understand mechanisms that lead to the evolution, resistance, and severity of S. aureus infections. Bacterial whole genome sequencing (WGS) techniques have offered new insights into S. aureus genomic populations and have the potential to predict antimicrobial resistance and infection severity. This study applied WGS 1) to describe the diversity and distribution of resistance mechanisms among community-associated S. aureus isolates, and 2) to identify S. aureus genetic signatures associated with SSTI isolates and derive a predictive risk model. WGS was performed on S. aureus isolates from patients within 14 primary care clinics in the South Texas Ambulatory Research Network from 2007 to 2015. The bacterial genomes were compared to a reference genome, FPR3757 (USA300 strain) to identify single nucleotide polymorphisms (SNPs). Phylogenetic analyses were conducted using concatenated SNP nucleotides in the core genomes. In the first study, the resistome was assembled by identifying antimicrobial resistance determinants related to the phenotypically derived antibiogram. The findings of this study identified that multidrug-resistant S. aureus isolates have emerged in the South Texas community; approximately one-third were multidrug-resistant. There was an increasing resistance pattern to fluoroquinolones. Furthermore, the genotype demonstrated to be highly predictive of antimicrobial resistance (very major error rate=0% and major error rate=1.4%). These findings highlight the genomic diversity of S. aureus strains in the South Texas community and demonstrate the utility of next generation sequencing to define the diversity and distribution of resistance mechanisms within S. aureus. Further work to explore antimicrobial selective pressures is needed. The second study utilized a bacterial genome-wide association study to identify specific variants associated with S. aureus pathogenicity. This study revealed the heterogeneity of S. aureus SSTI and nasal colonization isolates and identified potentially novel pathogenic mechanisms.Item Comparison of Three Techniques for Detecting Enterotoxin A (SEA) in Clinically Relevant Staphylococcus aureus StrainsHarrison, Cari Riccay; Jones, Crosby W; Ammerman, Loren K; Guardiola, Amaris R; Keith, Susan EThirty-three clinical strains of Staphylococcus aureus were screened for the production of staphylococcal enterotoxin A (SEA) protein and/or its gene using three techniques, reversed passive latex agglutination (RPLA), Ouchterlony double diffusion (ODD), and polymerase chain reaction (PCR). Of the isolates, two produced detectable levels of SEA (6%), while 22 (64.7%) harbored the gene for SEA. These results indicate that clinical S. aureus isolates commonly have the potential of producing SEA. ODD testing revealed promise for using it to detect prolific producers of enterotoxins. RPLA was shown to detect enterotoxins with specificity and accuracy. PCR revealed that although clinical strains frequently have the sea gene, they often do not produce SEA. Further studies should examine the presence of other staphylococcal enterotoxin genes and products. Growth conditions should also be evaluated to determine the ideal environments for enterotoxin production.Item The dimerization of Staphylococcus aureus sortase A on cell membrane(2010-05) Zhu, Jie, 1980-; Zhang, Zhiwen JonathanStaphylococcus aureus sortase A (SrtA) transpeptidase is a prominent membrane bound virulence factor in gram-positive bacteria, which organizes the peptidoglycan cell wall of the organism. Here, we report the first direct observation of the self-association behavior of SrtA. Formation of a SrtA dimer is highly selective in vitro in E. coli and in vivo on the S. aureus cell membrane. Quantitative analysis of protein binding affinity indicated a moderate association between two SrtA molecules with an apparent K[subscript d] of about 55 [micrometres] in vitro. Furthermore, to address the importance of dimerization for enzyme function, site-directed mutagenesis on potential target residues was performed to generate monomer only SrtA mutant proteins to completely disrupt dimer formation both in vitro and in vivo. Finally, an in vivo activity assay was performed to evaluate the function of SrtA wild type protein as well as its monomer only mutants. Our data demonstrated that S. aureus cells expressing mutant SrtA in a monomer only form are more successful at invading human epithelial cells than those expressing wild type SrtA in dimer-monomer equilibrium. It suggested that the monomeric form of SrtA is more active than the dimeric enzyme. We also demonstrated the uniqueness of SrtA dimerization by identifying that at least one other sortase family protein, SrtB only exists in monomer form. SrtA dimerization may have significant implications for understanding its biological function at both the cellular and molecular levels, which will lead to the development of new anti-infective therapies against gram-positive pathogens.Item Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university medical center day care facility(2009-05-02) Angela Lois Hewlett; C. Glen Mayhall M.D.; Norbert J. Roberts Jr. M.D.; Christine M. Arcari PhDBACKGROUND: Few data are available on MRSA colonization in day care. We performed a study in a medical university child care center to determine the epidemiology of MRSA in this population. \r\n\r\nMETHODS: A cross sectional study was done involving 104 day care attendees and 32 adult employees. Swab samples were taken from children, employees, the environment, and household contacts of participants found to be colonized with MRSA. Parents and employees completed questionnaires. Swabs were incubated in broth, then plated on agar and identified as MRSA by routine methods. Isolates were analyzed for relatedness using molecular typing. Statistical analysis was performed. \r\n\r\nRESULTS: The prevalence of MRSA in the children was 6.73%. One employee (3.13%) was colonized with MRSA. Cultures of 6 of 17 (35.3%) family members of participants positive for MRSA yielded MRSA. MRSA was recovered from 4 environmental samples. On molecular typing, many of the MRSA isolates were indistinguishable. Univariate analysis identified macrolide antibiotics (p=0.004), asthma medications (p=0.036), other medications (p=0.036), and previous surgery (p=0.022) as risk factors. On multivariable analysis, receipt of macrolide antibiotics (p=0.002; OR 39.6; 95% CI 3.4-651.4), and receipt of asthma medications (p=0.024; OR 26.9; 95% CI 1.5-500.7) remained related to MRSA colonization. \r\n\r\nCONCLUSIONS: There was a low prevalence of MRSA colonization in children and employees in the child care center. A higher prevalence of colonization was found among household contacts of children and employees colonized with MRSA. Molecular typing showed that transmission of MRSA likely occurred in the child care center. Macrolide antibiotics may increase the risk of MRSA colonization in this population.\r\nItem Isolation of bacteriophages specific to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in livestock from west-central Texas(2013-05-24) Kang, Jonathan; Kang, Jonathan; Jones, Crosby W.; Negovetich, Nicholas J.; Strenth, Ned E.; Bechtol, Bruce; Angelo State University. Department of Biology.Cross-species infections of methicillin-resistant Staphylococcus aureus (MRSA) from livestock to humans have been reported. Prior studies have isolated S. aureus and associated bacteriophages from dairy cattle. This research is undertaken to isolate bacteriophages specific for Escherichia coli, S. aureus and Pseudomonas aeruginosa from 13 beef cattle, 15 sheep and 12 goat fecal samples from a ranch in Tom Green Co., Texas. Phage enrichment was carried out using E. coli American Type Culture Collection (ATCC) strain 23848, S. aureus ATCC strain 13565 and P. aeruginosa ATCC strain PA01. Phage presence was detected using lawn spotting and reconfirmed using plaque assay. Bacteriophages specific to E. coli, S. aureus and P. aeruginosa were isolated from fecal samples of sheep and goats, while only S. aureus phages were isolated from cattle feces. Statistical analysis using Fisher's exact test and nested set modeling showed significant differences in phage isolation success between livestock types.Item Quinolone trafficking via outer membrane vesicles in Pseudomonas aeruginosa(2008-08) Warren, Lauren Mashburn, 1981-; Whiteley, MarvinPseudomonas aeruginosa is a Gram-negative opportunistic pathogen often infecting the lungs of individuals with the heritable genetic disease cystic fibrosis and the peritoneum of those undergoing continuous peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this dissertation I used a rat dialysis membrane peritoneal model to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo. Included in this analysis are genes important for iron acquisition and growth in lowoxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in the environment. This lysis was shown to be dependent on antimicrobial quinolones produced by P. aeruginosa. I demonstrate that these quinolones are present in outer membrane vesicles (MVs). Not only were these quinolones present in MVs, but the quorum sensing molecule; 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal; PQS) was also packaged into MVs and was necessary for MV formation. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviors in P. aeruginosa. Although MVs are involved in important processes besides signaling, the molecular mechanism is unknown. To provide insight into the molecular mechanism of MV formation, I examined the interaction of PQS with bacterial lipids. In this work, I demonstrated that PQS interacts strongly with the acyl chains and 4’-phosphate of bacterial lipopolysaccharide. The results of my studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signaling functions. Finally, I propose a model of PQSmediated MV formation where PQS interacts with specific outer membrane components to allow the necessary curvature for MV formation.Item Staphylococcal growth patterns in chicken tetrazzini prepared for a cook/chill foodservice system(Texas Tech University, 1978-12) Carlton, Edythe SueChicken tetrazzini was prepared and stored for a simulated cook/chill foodservice operation. Immediately following preparation half of the product was inoculated with Staphylococcus aureus at a concentration of 200 org/gm of product. Samples were stored at 5°C for 1, 3, 5, and 10 days. Microbial analysis for total plate count and S. aureus was determined at the following intervals: prior to reconstitution, after heating in a conventional oven until an internal temperature of 160°F C71°C) had been reached, and after 15, 45, and 90 minutes holding on a simulated steam table. The numbers of viable cells in the product prior to heating were influenced by the excessive growth of organisms during the first 8 hours of refrigeration, when the temperature of chicken tetrazzini was in the temperature range that permitted rapid growth of S. aureus (6.5°-46°C). Conventional heating of the product prior to the holding produced inconsistent temperatures throughout the food mass. A decrease in number of organisms was apparent after the product had been adequately heated. Conditions for increase in microorganisms were optimal with lengthening holding time under simulated steam table conditions for Day 1 product. On subsequent days of research, microbial count decreased as the product was held on the steam table. S. aureus count and total plate count decreased to a low point at 5 days of refrigerated storage. Viable cells of S. aureus were detected at low levels at each phase of analysis throughout the research project. Utilization of appropriate time-temperature relationships, sanitation and quality control, and effective employee training are important aspects of successful cook/chill foodservice production.Item Staphylococcal growth patterns in chicken tetrazzini prepared for a cook/freeze foodservice system(Texas Tech University, 1978-05) Stewardson, Patti JoNot availableItem Systems Biology of Staphylococcus Aureus Infection Ex Vivo and in Vitro(2012-07-09) Banchereau, Romain; Ramilo, OctavioStaphylococcus aureus has emerged as one of the most common community-acquired bacterial infections, with significant morbidity and mortality. Emergence of multidrug resistant strains worldwide, combined with limited treatment options demand novel approaches to further elucidate host-pathogen interactions, and especially host responses to infection. To this end, we leveraged systems biology approaches to better characterize the status of the host immune system during S. aureus infection ex vivo and in vitro. The transcriptional profiles of PBMC and whole blood from patients with community-acquired S. aureus infection were characterized by microarray analysis, and leukocyte population frequencies were measured by polychromatic flow cytometry. To refine our understanding of inflammatory networks involved, an in vitro system of antigen-presenting cell stimulation with various pathogens, including S. aureus as well as other bacteria and viruses, and their components, was used to identify early inflammatory programs induced in innate immune cells. To reduce the dimension and complexity of the data generated, we developed modular frameworks to analyze and interpret the fingerprints obtained from both the ex vivo and in vitro studies. // Overall, the blood transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis transcriptional programs, and under-expression of adaptive immunity programs. Flow cytometry and standard cell blood count (CBC) revealed an increase in absolute numbers of circulating monocytes, neutrophils and antigen-presenting cells, including dendritic cells and B cells, combined with a decrease in central memory T cells. To identify transcriptional correlates of clinical heterogeneity, we obtained individual fingerprints and derived the molecular distance to health, a numerical score of transcriptional perturbation for each patient. Patient-by-patient analysis without a priori knowledge of clinical diagnoses identified four major transcriptional clusters based on inflammation, erythropoiesis and interferon-induced profiles. Clinical presentation, bacterial dissemination and time between hospitalization and blood sampling were identified as major factors influencing the signature. The framework obtained from in vitro stimulation of monocyte-derived DC helped us refine the characterization of inflammatory programs activated during S. aureus infection. In addition to inflammatory antibacterial programs, S. aureus induced a subset of interferon response modules, also observed in viral infections and autoimmunity, as well as a specific set of modules linked to cell compartmentalization and lipid biosynthesis. Systems biology approaches provide a global and comprehensive assessment of host responses to acute bacterial infections, bringing a new understanding of disease pathogenesis and underlying patient heterogeneity. [Keywords; Staphylococus aureus, microarray, systems biology, module framework, transcription]