OBJECTIVE: The in vitro activity of apalcillin plus Ro 48-1220, a novel penam sulfone beta-lactamase inhibitor, was compared with apalcillin alone, piperacillin/tazobactam, ticarcillin/clavulanic acid, amoxicillin/clavulanic acid, imipenem, ceftazidime and cefepime. METHODS: Agar dilution and broth microdilution testing of 854 bacterial strains, subcultured from frozen stocks incubated for 24 h in 5% carbon dioxide, was carried out to determine the minimum bactericidal (MBC) and minimum inhibitory concentrations (MIC) of each of the study drugs in accordance with the NCCLS M26-T method. RESULTS: Apalcillin/Ro 48-1220 was active against all gram-negative aerobic and anaerobic isolates except Klebsiella oxytoca (MIC90 32 microg/mL). Among the Enterobacteriaceae, synergy for apalcillin/Ro 48-1220 (4 microg/mL fixed concentration) vs apalcillin alone was demonstrated for nearly all species when comparing MIC90 results. Apalcillin/Ro 48-1220 was highly potent against beta-lactamase-producing Moraxella catarrhalis, Haemophilus influenzae and Neisseria gonorrhoeae (MICs less-than-or-equal 1 microg/mL). However, much of this activity was due to the direct antimicrobial action of Ro 48-1220 alone (MICs less-than-or-equal 4 microg/mL). All Pseudomonas aeruginosa, Stenotrophomonas (Xanthomonas) maltophilia and Acinetobacter species were inhibited by apalcillin/Ro 48-1220 (MIC90 0.25 to 4 microg/mL). For the aerobic gram-positive organisms, none of the drugs tested were consistently effective against oxacillin-resistant staphylococci, Corynebacterium jeikeium and Enterococcus species other than E. faecalis. Apalcillin/Ro 48-1220 was as effective as piperacillin/tazobactam against Escherichia coli producing extended-spectrum TEM enzymes, but less active against isolates producing SHV-type beta-lactamases. When tested against 204 ceftazidime-, gentamicin- or fluoroquinolone-resistant organisms, 78%, 91% and 66% of strains, respectively, were susceptible to apalcillin/Ro 48-1220 (less-than-or-equal 16 microg/mL). CONCLUSIONS: Apalcillin/Ro 48-1220 is bactericidal with a modest inoculum effect; its wide spectrum of activity favors continued studies of spectrum, pharmacokinetics and in vivo efficacy.