Antimicrobial Susceptibility Testing and ESBL Production in Clinical Isolates of Proteus Mirabilis: An Evaluation with the Phoenix ™ Automated Microbiology System F. LUZZARO, 1 G. LOMBARDI, 1 M. PERILLI, 2 R. BELLONI, 1 G. AMICOSANTE, 2 AND A. TONIOLO 1 Laboratory of Microbiology, Ospedale di Circolo and University of Insubria, Varese, Italy and Department of Sciences and Biomedical Technology University of L’Aquila, L’Aquila, Italy Clinical isolates of ESBL-producing Proteus mirabilis have been obtained at an increasing frequency over the last few years. Expression of ESBL is often associated with multidrug resistance. At our Institution, the majority of these strains caused UTIs, but also respiratory infections and septicemia. We evaluated the ability of the Phoenix ™ Automated Microbiology System (BD Diagnostic Systems, Sparks, MD) to measure drug susceptibility and detect ESBL-mediated resistance in clinical isolates of P. mirabilis. Sixty-four ESBL-producing isolates were studied in comparison to 54 ESBL-negative isolates (susceptible or resistant to ampicillin). ESBL production was assessed by the double-disk synergy test and by Etest strips (AB Biodisk, Solna, Sweden) containing ceftazidime/ceftazidime plus clavulanate or cefotaxime/cefotaxime plus clavulanate. ESBL-coding genes were investigated by colony hybridization with bla TEM and bla SHV probes. MIC values obtained by the Phoenix System (NMIC/ID-5 panels) were compared to those obtained by the Etest. ESBL-positive isolates were shown to produce TEM-type enzymes and were characterized by: a) reduced susceptibility or resistance to 3rd- and 4th-generation cephalosporins (MIC ≥ 2mg/L); b) MICs for meropenem ≤ 0.5 mg/L; c) resistance to gentamicin, and 100% sensitivity to amikacin; d) over 75% resistance to ciprofloxacin; e) resistance to piperacillin completely abolished by tazobactam (MIC ≥ 256 vs. ≤ 2 mg/L). Overall, the essential agreement of MIC values between the Phoenix System and the Etest method was 96.8%, with no very major errors and 0.5% major errors. Production of ESBL was correctly assessed in 64/64 positive isolates by the ESBL screening test integrated in Phoenix panels. No false positive results were obtained for ESBL-negative isolates. The average time for ID plus AST was 12.4 ± 2.3 hr for ESBL-positive and 9.3 ± 1.6 hr for ESBL-negative strains. Thus, the automated Phoenix System appears to perform properly in measuring MIC and ESBL production both in susceptible and multidrug-resistant isolates of P. mirabilis. INTRODUCTION Proteus mirabilis isolates are commonly susceptible to most antibiotics, including beta-lactams. However, acquired resistance to the above mentioned drugs has increased over the last few years, due to the production of extended-spectrum beta- lactamases (ESBL). A 3-year survey conducted in France from 1988 to 1990 showed that only 0.8% of P. mirabilis strains produced ESBL [Sirot, 1992]. Subsequently, the prevalence of ESBL-positive strains has increased to 6.9% in France and 9.5% in the U.S. [De Champs, 2000; Saurina, 2000]. A nationwide survey showed that, among ESBL-producing Enterobacteriaceae, P. mirabilis was the second most frequent species in Italy [Spanu, 2000]. At our Institution, 8.8% of P. mirabilis isolates was shown to produce ESBL [Luzzaro, 2001]. Due to the therapeutic relevance of ESBL production for the management of infections caused by gram-negative pathogens, automated diagnostic methods capable of detecting these enzymes would be useful. Elevated MIC values for broad- spectrum cephalosporins usually suggest ESBL production. However, manual confirmatory methods based on the synergic effect between clavulanate and oxyimino-cephalosporins or monobactams are still required [NCCLS, 2000]. Moreover, in the case of P. mirabilis and Providencia stuartii, ESBL are often expressed at low levels. This makes the detection of beta-lactam hydrolyzing enzymes particularly difficult and usually requires a modified synergy test [Sirot, 1996]. The aim of this study was to evaluate the ability of the Phoenix ™ System (BD Diagnostic Systems, Sparks, MD) to automatically measure drug susceptibility of clinical isolates of P. mirabilis and to detect the production of ESBL by members of this species. ABSTRACT Presented at the 101st General Meeting of the American Society for Microbiology, Orlando, Florida, 2001. Phoenix Automated Microbiology System