Failure to detect ampicillin-resistant, non-beta-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing

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Abstract
We questioned whether the apparent rarity of ampicillin-resistant, non-beta-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillin-susceptible H. influenzae by the agar dilution technique, using 10(5)- and 10(3)-CFU inocula. We compared the disk susceptibility of 18 ampicillin-resistant NBLP strains, 13 ampicillin-resistant, beta-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10- and 2-micrograms ampicillin disks on two different media. We also investigated the possibility that disks containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 micrograms of ampicillin, we correctly differentiated all the ampicillin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of less than or equal to 15 mm was considered resistant). In contrast, the 10-micrograms ampicillin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of less than or equal to 19 mm was considered resistant). The MIC of ampicillin with the 10(3)-CFU inoculum correlated better with zone diameters than with the 10(5)-CFU inoculum. A difference in zone diameters of greater than or equal to 8 mm between the disk containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin and the disk containing only 2 micrograms of ampicillin correctly identified all beta-lactamase-producing strains. We conclude that the 2-micrograms ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-micrograms ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.