A multi-faceted diagnostic approach to lung infections in patients with cystic fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

An ethnopharmacological approach to multidrug -resistant Staphylococcus aureus: Evaluation of Italian plants used in the traditional healing of skin disease

One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infections (SST's). Methicillin-resistant Staphylococcus aureus (MRSA), a common cause of SSTIs, is responsible for increased morbidity and mortality from infections. Therapeutic options are limited by antibiotic resistance. Many plants possess potent antimicrobial compounds for these disorders. Validation of traditional medical practices is important for the people who rely on medicinal plants. Moreover, identification of novel antibiotics and anti-pathogenic agents for MRSA is important to global healthcare.^ I took an ethnopharmacological approach to understand how Italian medicinal plants used for the treatment of SSTIs affect MRSA growth and virulence. My hypothesis was that plants used in folk remedies for SSTI would exhibit lower cytotoxicity and greater inhibition of bacterial growth, biofilm formation and toxin production in MRSA than plants used for remedies unrelated to the skin or for plants with no ethnomedical application. The field portion of my research was conducted in the Vulture-Alto Bradano area of southern Italy. I collected 104 plant species and created 168 crude extracts. In the lab, I screened samples for activity against MRSA in a battery of bioassays. Growth inhibition was analyzed using broth microtiter assays for determination of the minimum inhibitory concentration. Interference with quorum-sensing (QS) processes, which mediate pathogenicity, was quantified through RP-HPLC of δ-toxin production. Interference with biofilm formation and adherence was assessed using staining methods. The mammalian cytotoxicity of natural products was analyzed using MTT cell proliferation assay techniques.^ Although bacteriostatic activity was limited, extracts from six plants used in Italian folk medicine (Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius ) significantly inhibited biofilm formation and adherence. Moreover, plants used to treat SSTI demonstrated significantly greater anti-biofilm activity when compared to plants with no ethnomedical application. QSI activity was evident in 90% of the extracts tested and extracts from four plants ( Ballota nigra, Castanea saliva, Rosmarinus officinalis, and Sambucus ebulus) exhibited a significant dose-dependent response. Some of the plant remedies for SSTI identified in this study can be validated due to anti-MRSA activity.^

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.