The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.
The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
We grew liquid co-cultures of P. aeruginosa and S. aureus in LB Lennox media and examined their absolute and relative cell densities by plating the co-cultures on selective media. We evaluated the influence of oxygen concentration and co-inoculation vs. staggered inoculation on the ability to achieve a co-cultures with two P. aeruginosa (PA) and two S. aureus (SA) strains. The method that consistently produced PA:SA ratios in the range of 1:1 to 1:100 was to allow a SA mono-culture to reach stationary phase, and then re-suspend the SA cells in fresh media before inoculating with PA. With this method, it is possible to grow both PA and SA to stationary phase, a necessity for studying how PA and SA alter phenotypes in the presence of one another.
P. aeruginosa was found to produce less pyocyanin in the presence of S. aureus, but reduction in pyocyanin expression was depended on the strain of S. aureus. Elastase production differed between the two P. aeruginosa strains as well as between the two S. aureus strains, one increasing and one decreasing in expression. This data indicates that the responses of P. aeruginosa to S. aureus differ depending on both the P. aeruginosa and S. aureus strain present.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
An investigation of academic achievement and achievement motivation in children with cystic fibrosis
Sheldon Clark Reed helped establish the profession of genetic counseling in the US during the twentieth century. In 1947 Reed coined the term genetic counseling to describe the interaction of a doctor explaining to a patient the likelihood of passing a certain trait to their offspring. With physicians being able to test for genetic abnormalities like cystic fibrosis, Reed helped trained individuals give patients the tools to make informed decisions. In 1955 Reed published the book Counseling in Medical Genetics. Reed educated patients about how certain genetically transmitted traits could adversely affect their offspring and provided options for remedying those effects.
Cystic fibrosis (CF) is a fatal, inherited disease found in humans and characterized by buildup of thick, sticky mucus, particularly in the respiratory and digestive tracts. The abnormally thick mucus prevents the pancreas from functioning normally; it often leads to digestive problems and chronic lung infections. Cystic fibrosis is most prevalent in Caucasian individuals, and approximately 1 in every 29 individuals in the US is a carrier for the mutated CF gene. There are an estimated 30,000 reported cystic fibrosis cases in the US, and 70,000 reported cases worldwide, although the international number is undoubtedly low due to underreporting or early deaths.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene was identified in 1989 by geneticist Lap-Chee Tsui and his research team as the gene associated with cystic fibrosis (CF). Tsui's research pinpointed the gene, some mutations to which cause CF, and it revealed the underlying disease mechanism. The CFTR gene encodes a protein in the cell membrane in epithelial tissues and affects multiple organ systems in the human body. Mutations in the CFTR gene cause dysfunctional regulation of cell electrolytes and water content. Research on the CFTR mutation has shed light on the ways in which this gene is vital to normal human development.