Characterization of the inoculum effect with Haemophilus influenzae and B-lactams

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Balko, Tamara
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An inoculum effect is defined as a four-fold or greater increase in minimum inhibitory concentration (MIC) with an increase in bacterial inocula. Using the National Committee for Clinical Laboratory Standards (NCCLS) MIC determination protocol (a turbidimetric method) Haemophilus influenzae demonstrated an inoculum effect with ampicillin, cefaclor, loracarbef, cefuroxime, and amoxicillin/clavulanate when initial inocula were increased from $5\times 10\sp5$ CFU/ml (low inocula) to $1 \times 10\sp7$ CFU/ml (high inocula). Using this method an inoculum effect was observed with both $\beta$-lactamase (TEM-1, ROB-1) positive and $\beta$-lactamase negative strains of H. influenzae. A viable cell count MIC determination method however, demonstrated that only $\beta$-lactamase positive strains of H. influenzae produced an inoculum effect suggesting that turbidimetrically determined MICs using high initial inocula are not reliable when examining the inoculum effect in H. influenzae. The magnitude of the inoculum effect with $\beta$-lactamase positive strains was $\beta$-lactase dependent (ampicillin $>$ cefaclor = loracarbef $>$ amoxicillin/clavulanate $>$ cefuroxime). $\beta$-lactam 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. The absence of an inoculum effect in $\beta$-lactamase negative strains of H. influenzae and the elimination of the inoculum effect with the addition of clavulanate suggested that the inoculum effect of $\beta$-lactams with H. influenzae was due to the activity of $\beta$-lactamase. To test this supposition directly the $\beta$-lactamase gene TEM-1 was inserted into pLS88 and introduced into a $\beta$-lactamase negative strain, H. influenzae Rd. This transformation produced an inoculum effect suggesting that the inoculum effect resulted directly from $\beta$-lactamase production.