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

Abstract

We questioned whether the apparent rarity of ampicillin-resistant, non-4-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillinsusceptible H. influenzae by the agar dilution technique, using 105-and 103-CFU inocula. We compared the disk susceptibility of 18 ampicihlin-resistant NBLP strains, 13 ampicillin-resistant, (8-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10-and 2-,ug ampicillin disks on two different media. We also investigated the possibility that disks containing 10 ,ug of clavulanic acid and 2 ,ug of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 ,ug of ampicillin, we correctly differentiated all the ampiciflin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of s15 mm was considered resistant). In contrast, the 10-,ug ampiciflin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of sl9 mm was considered resistant). The MIC of ampicillin with the 103-CFU inoculum correlated better with zone diameters than with the 105-CFU inoculum. A difference in zone diameters of .8 mm between the disk containing 10 ,g of clavulanic acid and 2 ,ug of ampicillin and the disk containing only 2 ,ug of ampicillin correctly identified all j3-lactamase-producing strains. We conclude that the 2-,ug ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-,ug ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.Although the most common mechanism of ampicillin resistance (Amp) in Haemophilus influenzae is plasmidmediated production of TEM P-lactamase (4,7,17, 32,37), pathogenic Ampr type b H. influenzae lacking detectable P-lactamase activity has been reported (12,14,24,26).Clinical isolates with this phenotype appear to be even more common among nontypable respiratory tract H. influenzae (1,29,37). Nontypable H. influenzae has been increasingly implicated in community-acquired and nosocomial pneumonia in adults (3,9,19,20,22,40,41)

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae

“…Table 1 shows the amoxicillin and amoxicillin-clavulanate MICs for the three phenotypic groups tested. Although no overlap was observed between Amps and Amp'NBLP strains, MICs for two of the latter strains were 1 ,ug/ml with HTM media, whereas previously MICs for all strains were .1.5 ,ug/ml at the lower inoculum of 103 CFU when tested on sBHI agar (15). AmprBLP strains with the other antibiotics tested (amoxicillin-clavulanate, cefaclor, cefuroxime, ceftriaxone, and cefotaxime).…”

“…P-Lactamase production was determined by incubating suspensions of each strain with 3-lactamase substrate (chromogenic cephalosporin; Calbiochem-Behring, La Jolla, Calif.) at room temperature for 30 min and visually assessing color development. The Ampr NBLP strains were identified previously (15,16), with ampicillin MICs of .1.5 jig/ml in comparison to Amps isolates, for which MICs were -0.5 ,ug/ml. All 18 susceptible strains were nontypable, with the site of isolation as follows: 10 respiratory tract, 5 eye, 1 ear, 1 ATCC 33391, and 1 site unknown.…”

Haemophilus influenzae: antibiotic susceptibility

“…However, little has been documented regarding beta-lactamase-negative, ampicillin-resistant isolates. The limited evidence which exists suggests that these isolates are less influenced by inoculum than are the beta-lactamase-producing strains (68). Results may vary sufficiently to warrant the testing of a higher inoculum.…”

“…Mendelman et al (68) reported that 8 of 18 ampicillinresistant strains which lacked beta-lactamase were classified as falsely susceptible by the NCCLS guidelines. They proposed the use of a 2-itg ampicillin and 2-pug ampicillin/10-pug clavulanic acid disk as an alternative method for classifying resistant and susceptible strains.…”

“…Those with zone diameters of >19 mm should be considered susceptible (74). Although several investigators have validated this breakpoint for ampicillin-susceptible and betalactamase-producing resistant strains (28,48), the categorization of ampicillin resistance in non-beta-lactamaseproducing strains is less satisfactory (49,68,80).…”