Bacillus licheniformis ATCC 21415 cells were immobilized on different carriers using different methods of immobilization including physical adsorption, covalent binding, ionic binding and entrapment. The immobilized cells were prepared by covalent binding on wool (as a new carrier) through 1% glutaraldehyde had the highest enzyme activity (9.0 U/mL) with the highest specific productivity (6.17 U/g wet cells/h). Alkaline protease production and the stability of biocatalyst were investigated in both free and immobilized cells. The results showed that the immobilized cells were more efficient for enzyme production by repeated batch fermentation (5 cycles, 480 h) with 57% residual activity whereas the free cells retained 35% after 2 cycles. In continuous production the highest enzyme activity (9.9 U/mL) was obtained at a dilution rate of 0.1/h while the highest enzyme yield (763.6 U/h) and the highest reactor productivity (3.32 U/mL/h) were attained at a dilution rate of 0.4/h. Packed-bed bioreactor was a successful method for continuous production of alkaline protease for a long time (168 h) with 53% relative activity. The bioreactor affected the highest specific productivity (118.2 U/g wet cells/h) which was 12-24 times higher than other systems of enzyme production.

Article InfoDevelopment of carbapenem resistance is common in Pseudomonas aeruginosa. This resistance, which is due mainly to alteration of the OprD porin, the specific uptake pathway of carbapenems, may also result from acquisition of foreign genes encoding Ambler class A, class B, or class D B-lactamases. These enzymes able to hydrolyze carbapenems at various degrees. Thus, detection of carbapenemase producers in clinical laboratories is of utmost importance for the determination of appropriate therapeutic schemes and the implementation of infection control measures. In order to improve the detection of carbapenemase producers, various inhibitor-based tests and enzymatic assays (e.g., NP-Carba) have been proposed as a first screening step prior to the use of confirmatory molecular techniques. However, because most of imipenem-non susceptible strains are just OprD-deficient mutants, these phenotypic or enzymatic tests usually yield low rates of positivity.A new test is developed in order to screen for OprD-deficient mutants thus discriminating carbapenemase producingP. Aeruginosa strains from nonproducers. This test combines imipenem and cloxacillin, a strong inhibitor of intrinsic cephalosporinase AmpC. It is based on the observation that imipenem resistance resulting from OprD deficiency requires constitutive and/or carbapenem-induced overproduction of AmpC. Therefore, inhibition of AmpC by cloxacillin is expected to restore partial or complete sensitivity to imipenem in OprD-deficient strains but not in carbapenemase positive strains. The aim of this study was to evaluate the performance of a simple, inexpensive detection method applicable in medical laboratories that used combined disk testing of Imipenem and cloxacillin, in order to discriminate carbapenemase producing P. aeruginosastrains from non-producers(OprD-deficient mutants).Fifty clinical isolates of imipenem resistant P. aeruginosa, which were well characterized by MHT for carbapenamse production and by real time PCR for (bla IMP ) gene and (bla KPC ) gene from previous studies, were used. Combined test testing (CDT) using a carbapenem disk (imipenem) supplemented with various loads of cloxacillin was assessed. Out of 50 P.aeruginosa isolates,15 (30%)isolates were positive by Imipenem-Cloxacillin CDT at conc. 4000.This CDT detected OprD-deficient mutants which were non carbapenamse producer strains. In comparison to (bla IMP) gene positivity and (bla KPC ) gene positivity, and MHT results, sensitivity and specificity of imipenem-cloxacillin at conc. 4000ug were 93.33% and 75% respectively. In conclusion, the Imipenem-Cloxacillin at conc. 4000 test is a simple and inexpensive presumptive method that can be added to the standard antibiogram for routine screening of OprD-deficient mutants thus discriminating carbapenemase producing P. aeruginosa strains from non-producers.