Title page for ETD etd-08052008-111424

Type of Document Dissertation
Author Mastropaolo, Matthew David
Author's Email Address mmastrop@vt.edu
URN etd-08052008-111424
Title Studies of Three Human Intestinal Opportunistic Pathogens
Degree PhD
Department Biology
Advisory Committee
Advisor Name Title
Melville, Stephen B. Committee Co-Chair
Stevens, Ann M. Committee Co-Chair
Inzana, Thomas J. Committee Member
Larson, Timothy J. Committee Member
  • diabetes
  • mouse experiments
  • cytotoxic necrotizing factor 1
  • α-hemolysin
  • Escherichia coli
  • systemic infection
  • cell culture
  • polymicrobial infections
  • lux reporter
  • Bacteroides
Date of Defense 2008-07-30
Availability unrestricted
Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain.

We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice.

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