Characterization of the Inoculum Effect with Haemophilus influenzae and beta-lactams

An inoculum effect is defined as a four-fold or greater increase in MIC wit h an increase in bacterial inocula. Haemophilus influenzae was tested for a n inoculum effect with ampicillin, cefuroxime, and amoxicillin/clavulanate using the standard initial inocula (5 x 10(5) CFU/mL) and a higher initial inocula (1 x 10(7) CFU/mL). An inoculum effect was observed with both beta- lactamase (TEM-1, ROB-1) positive and beta-lactamase negative strains of H. influenzae when MICs were determined based on turbidity. MICs based on via ble cell counts however, demonstrated that only p-lactamase positive strain s of H. influenzae produced an inoculum effect. These observations suggest that MICs determined based on turbidity, using high initial inocula, are no t reliable when examining the inoculum effect in H. influenzae. The magnitu de of the inoculum effect with beta-lactamase positive strains was beta-lac tam dependent (ampicillin > amoxicillin/clavulanate > cefuroxime). beta-lac tam kill-curves confirmed the aforementioned results. Addition of the beta- lactamase inhibitor clavulanate completely reversed the inoculum effect in beta-lactamase (TEM-1 and ROB-1) positive strains of H. influenzae with all beta-lactams tested. Introduction of the beta-lactamase gene TEM-1 on plas mid vector pLS88 into a beta-lactamase negative strain of H. influenzae (Rd ) produced an inoculum effect based on viable cell counts. In conclusion, o ur results suggest that the beta-lactam inoculum effect demonstrated by H. influenzae is the result of p-lactamase production and is poorly assessed b y turbidity. (C) 1999 Elsevier Science Inc.