Respiratory pathogens in the oral cavity: Molecular epidemiology and interaction with salivary components
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The oral cavity may serve as a reservoir for respiratory pathogens. Staphylococcus aureus has been isolated from oral microbial biofilms and saliva of healthy individuals and, more frequently, from hospitalized patients. The function of saliva is frequently impaired in hospitalized patients, and proteins in human saliva either promote the colonization or foster the clearance of pathogens from the oral cavity. The purpose of our first study ( Chapter 2 ) was to determine the genetic relationship between strains of respiratory pathogens initially isolated from the oral cavity and later isolated from the lung of patients with suspected ventilator-associated pneumonia (VAP). Plaque and tracheal secretion samples were obtained on the day of hospital admission and every other day thereafter until discharge from the intensive care unit from 100 patients who underwent mechanical ventilation. Bronchoalveolar lavage (BAL) was performed for 30 patients with suspected VAP. Pulse-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relationships of strains obtained from oral, tracheal, and bronchoalveolar lavage samples. In most patients, isolates of S. aureus, Pseudomonas aeruginosa, Acinetobacter species, and enteric species recovered from plaque were genetically indistinguishable from isolates recovered from BAL fluid. Nearly one-half of the Pseudomonas strains showed identical genetic profiles between patients, which suggested a common environmental source of infection. Thus, dental plaque serves as an important reservoir for respiratory pathogens in VAP patients. The goals of the next projects ( Chapters 3 and 4 ) were to identify the salivary components that interact with S. aureus , and to elucidate the influence of biofilm formation on the binding of salivary proteins. Bacteria were exposed to unlabeled or fluorescence (Cy3)-labeled saliva, and after washing, bound proteins were released by SDS-containing buffer and separated by SDS-PAGE. Alternatively, bound salivary proteins were released from the bacteria by limited trypsin treatment. Protein bands excised from gels or limited trypsin digests were subjected to complete trypsin digestion, analyzed by LC-MS/MS, and identified by matching peptide profiles to the Swiss-Prot database. Protein identities were additionally confirmed by Western blotting. Components bound to S. aureus were different from those bound to the oral commensal bacterium Streptococcus gordonii, in that salivary &agr;-amylase bound strongly to S. gordonii, but did not bind to S. aureus. Binding of salivary components to S. aureus occurred rapidly and was not solely mediated by electrostatic or hydrophobic interactions. Major salivary components bound to S. aureus included DMBT1/gp-340/salivary agglutinin (DMBT1 gp-340 ), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9 and lysozyme. Additionally, carbonic anhydrase (CA) VI, SPLUNC2/BPIFA2/parotid secretory protein (SPLUNC2) and zymogen granule protein 16 homolog B (ZG16B) could be detected by LC/MS-MS analysis. Biofilm-grown S. aureus and a biofilm-forming strain of S. epidermidis bound fewer salivary components identified as salivary immunoglobulins IgG and IgA. The corresponding adhesive component on the S. aureus surface was identified as staphylococcal protein A (SpA) and confirmed using a SpA-deficient mutant strain of S. aureus and SpA-coated Sepharose beads in combination with Western blotting. However, SpA did not mediate the binding of non-immunoglobulin components, including mucin-7, to the staphylococcal surface. For the binding of these molecules, other surface adhesive components on S. aureus must be responsible. These results shed new light on the role of saliva and oral biofilm formation in colonization by S. aureus .