Evaluation of the Osiris Expert System for Identiﬁcation of (cid:2) -Lactam Phenotypes in Isolates of Pseudomonas aeruginosa

Osiris is a video zone size reader for disk diffusion tests featuring a built-in extended expert system (EES). The efﬁcacy of the EES for the identiﬁcation of the (cid:1) -lactam susceptibility phenotypes of Pseudomonas aeruginosa isolates was evaluated. Thirteen (cid:1) -lactams were tested in four laboratories by the disk diffusion test with 53 strains with well-characterized resistance mechanisms, including the production of 12 extended- spectrum (cid:1) -lactamases (ESBLs). The plates were read with the Osiris system and the results were interpreted with the ESS, and then the phenotype identiﬁed by the EES was compared to the resistance mechanism. The strains were also screened for the presence of ESBL production by a double-disk synergy test by placing the strains between an extended-spectrum cephalosporin-containing disk and a clavulanic acid-containing disk at distances of 30, 20, 15, and 10 mm from each other. Overall, the EES accurately identiﬁed the phenotypes of 88.2% of the strains and indicated an association with several mechanisms for 3.8% of the strains. No phenotype was identiﬁed in four strains with low levels of penicillinase production. Misidentiﬁcations were observed for two penicillinase-producing strains: one strain with partially derepressed cephalosporinase production and one strain overexpressing the MexA-MexB-OprM efﬂux system. The production of only four ESBLs was detected by the standard synergy test with a 30-mm distance between the disks. The production of ﬁve further ESBLs was identiﬁed by reducing the distance to 20 mm, and the production of the last three ESBLs was detected only at a distance of 15 or 10 mm. Our results indicate that the Osiris EES is an effective tool for the identiﬁcation of P. aeruginosa (cid:1) -lactam phenotypes. A speciﬁc double-disk synergy test with reduced disk distances is necessary for the detection of ESBL production by this organism. Brieﬂy, the strains were subcultured onto tryptic soy agar. A bacterial cell suspension in Mueller-Hinton broth adjusted to (cid:1) 10 6 CFU/ml was used to inoculate a square petri plate containing Mueller-Hinton agar (Bio-Rad) by ﬂooding. Disks containing the antibiotics were distributed onto the plates according to a predeﬁned scheme by using a disk dispenser. After incu-bation at 37°C for 18 h, the plates were read with the Osiris video instrument and the results were interpreted with the EES according to the instructions of the manufacturer. The phenotype identiﬁed by the EES was compared to the (cid:2) -lac- tam resistance mechanism determined by biochemical and molecular techniques. Detection of ESBLs. Strains were screened for the presence of ESBLs by the double-disk synergy test, as described by Jarlier et al. (8). On the square petri plate inoculated for susceptibility testing, the ticarcillin-clavulanic acid disk was placed next to the ceftazidime, cefepime, and aztreonam disks at a distance of 30 mm (center to center). Moreover, an additional test was done in order to detect OXA-derived ESBLs, which are poorly susceptible to clavulanic acid (14). A round Mueller-Hinton agar plate was inoculated with the bacterial suspension in the same way described above for the disk diffusion procedure. Three ceftazidime disks were placed at distances of 20, 15, and 10 mm, respectively, from a central amoxicillin-clavulanic acid disk. The test result was considered positive when an enhancement of the inhibition zone around at least one of the ceftazi- dime, cefepime, or aztreonam disks toward the clavulanic acid disk was observed.