OBJECTIVE: Pseudomonas aeruginosa is a major causative agent of hospital infections. Studies on this subject being rare in Algeria, we determined the antibiotic susceptibility of P. aeruginosa and investigated the mechanisms of beta-lactam resistance and the spread of multidrug resistant strains in the university affiliated Hospital of Tlemcen (Algeria). DESIGN: One hundred and ninety-nine consecutive strains of P. aeruginosa were collected between November 2005 and February 2007. MICs of antibiotics were measured by the agar dilution method. The resistance mechanisms to beta-lactams were identified phenotypically or by molecular methods (isoelectrofocusing, PCR and sequencing). Strains expressing a secondary beta-lactamase were serotyped and genotyped (Random Amplified Polymorphic DNA). RESULTS: The proportion of susceptible isolates were: ticarcillin (56%), piperacillin-tazobactam (81%), ceftazidime (88%), cefepime (80%), aztreonam (64%), imipenem (65%), amikacin (83%), tobramycin (81%) and ciprofloxacin (97%) according to the French CASFM breakpoints. Resistance to beta-lactams was linked to the production of transferable beta-lactamases (16%), overproduction of cephalosporinase AmpC (12%) and/or non-enzymatic mechanisms such as the loss of porin OprD (35%) and overproduction of the active efflux system MexAB-OprM (24%). High level resistance to ticarcillin was due to the expression of beta- lactamase OXA-10 alone or associated with TEM-110. A genotypic analysis revealed the spread of a multidrug resistant epidemic clone expressing these two acquired beta-lactamases in the surgical ICU. CONCLUSIONS: This study shows that resistance to antibiotics, in particular to imipenem of P. aeruginosa, is becoming a cause of concern in the Hospital of Tlemcen.