Cronic otitis media is a common problem associated with a nonintact tympanic membrane frequently involving Staphylococcus aureus and Pseudomonas aeruginosa. The virulence of Pseudomonas bacteria is related to the production of two matrix metalloproteinases, elastase and alkaline protease. Serine proteases, such as neutrophil elastase, are produced by the host inflammatory response. These proteases are thought to contribute to tissue destruction and assist bacterial invasion during infection. This preliminary study was done to identify protease activity in otorrhea samples from patients with otitis media and a nonintact tympanic membrane and to examine the ability of selective protease inhibitors to decrease protease activity. Ilomostat (galardin) is a synthetic, specific inhibitor of matrix metalloproteinases including P. aeruginosa elastase and alkaline protease, where-as α1-antitrypsin inhibits serine proteases including neutrophil elastase. Samples were collected and cultured from 20 patients with otorrhea resulting from tympanic membrane perforations or pressure-equalization tubes. A protease assay that used azocasein as the substrate was used to quantify protease activity, with and without addition of selective protease inhibitors. Cultures revealed P. aeruginosa alone in 7 samples, P. aeruginosa plus other organisms in 10, and S. aureus alone in 3. Protease activity was detected in 15 (75%) of the samples. A statistically significant ( p < 0.05) decrease in protease activity was seen with the addition of α1-antitrypsin or Ilomostat plus α1-antitrypsin, but not with Ilomostat alone. Analyzing the 10 samples with the highest protease activity, a statistically significant decrease in activity was seen with Ilomostat or αα1-antitrypsin alone and with both Ilomostat and α1-antitrypsin together. Bacteriologic type, source of sample, age and gender of the subject, and duration of infection were not significantly related to protease activity. This is the first study to quantify protease activity and inhibition by selective protease inhibitors in human otorrhea. Protease inhibitors effectively decrease protease activity in most cases and in addition to standard antibiotic therapy might prove beneficial in the treatment of otitis media with a nonintact tympanic membrane. This study supports future clinical investigations into the role of proteases and inhibition of protease activity in the treatment of otitis media.
Sporulation and Extracellular Alkaline Protease Activity of Bacillus subtilis 168 Grown in the Presence of Tween
