Protease activity of Clostridium difficile strains.
Poilane I. Karjalainen T. Barc MC. Bourlioux P. Collignon A.
Departement de microbiologie, Faculte de pharmacie, Universite Paris-Sud, Chatenay-Malabry, France.
The production of proteolytic enzymes by 10 Clostridium difficile isolates of varying toxigenicity and clinical origin was studied to determine if all isolates secreted proteases. Different protease substrates were studied: gelatin, collagen, phenylazobenzyloxycarbonyl-leucyl-glycyl-L-prolyl-D-arginine (Pz-peptide), casein, azocasein, and azocoll. All isolates degraded gelatin, collagen, and azocoll. The supernatants of all isolates contained an enzyme capable of attacking gelatin incorporated in a polyacrylamide gel (zymograms) and forming two closely spaced lytic bands with an estimated molecular mass of 35-40 kDa. Polyclonal antibodies, produced against the C. difficile gelatinase, revealed in Western blots a 35-kDa protein in the culture supernatants of all C. difficile isolates. In the same manner, Clostridium perfringens collagenase polyclonal antibodies detected a 120-kDa protein in the culture supernatants of all isolates; this suggests that at least two proteases may exist in C. difficile. The protease activities of the 10 strains examined did not seem strikingly different quantitatively but were in general weak and their role in pathogenicity is suspect.
Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus.
Arnal C. Ferre-Aubineau V. Besse B. Billaudel S.
Laboratoire de virologie, Institut de biologie, Centre Hospitalier Regional Universitaire de Nantes, France. email@example.com
Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.