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Era A. Izano
B.A. 2004, Rutgers University
Thesis Advisor: Jeffrey B. Kaplan, Ph.D.
Department of Oral Biology
Thursday, November 6, 2008
Background: Chronic infections are often caused by biofilms, which are defined as surface-associated communities of bacteria encased in a self-synthesized extracellular polymeric matrix. The biofilm matrix holds the bacterial cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In this thesis we investigated the roles of two biofilm matrix polymers, poly-N-acetylglucosamine (PGA) and extracellular DNA (eDNA), in biofilm cohesion and biocide resistance in the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis, and in the Gram-negative bacteria Aggregatibacter actinomycetemcomitans, Actinobacillus pleuropneumoniae and Haemophilus influenzae.
Methods: Two different approaches were taken. In the first approach, transposon mutagenesis and genetic complementation were used to investigate the biosynthesis and function of PGA in A. actinomycetemcomitans and A. pleuropneumoniae. In the second approach, various matrix-degrading enzymes including DNase I, micrococcal nuclease, restriction endonucleases, and the PGA-degrading enzyme dispersin B were used to investigate the role PGA and eDNA in biofilm formation by S. aureus, S. epidermidis, A. pleuropneumoniae, A. actinomycetemcomitans and H. influenzae. Biofilms formation was assessed in vitro by using crystal violet staining, CFU enumeration and microscopy. The resistance of biofilms to killing by the anionic detergent sodium dodecyl sulfate and the cationic detergent cetylpyridinium chloride was also measured in vitro.
Results: Results from genetic studies showed that pgaC, which encodes a putative glycosyltransferase, is required for the biosynthesis of PGA in A. pleuropneumoniae and A. actinomycetemcomitans. Results from studies with matrix-degrading enzymes showed that PGA and eDNA perform different functions in different bacterial species. PGA appears to be a major intercellular adhesin in A. pleuropneumoniae and S. epidermidis biofilms, whereas eDNA plays a more important role in biofilm cohesion in S. aureus and H. influenzae. In A. actinomycetemcomitans, both PGA and eDNA contribute to biofilm cohesion. Both PGA and eDNA were shown to play an important role in biofilm-mediated detergent resistance.
Conclusions: Taken together, these studies show a structural and functional role of matrix adhesins in pathogenic bacterial biofilms. They highlight the complexity and diversity of biofilms among different microorganisms even when they are closely related at the genetic level. Furthermore, the use of biocides and matrix-degrading enzymes offers an innovative way of treating highly resistant biofilm infections.