Comparison of agar dilution, broth dilution, and disk diffusion testing of ampicillin against Haemophilus species by using in-house and commercially prepared media

Abstract

An evaluation to determine the optimal method for the in vitro susceptibility testing of Haemophilus strains to ampicillin was undertaken. In our hands, in-house-prepared Haemophilus Test Medium used by either the broth macrodilution or agar dilution method produced the most consistent results, especially for I-lactamasenegative, ampicillin-resistant H. influenzae strains.In 1985, the National Committee for Clinical Laboratory Standards (NCCLS) published disk diffusion (DD) and dilution susceptibility testing criteria for the in vitro susceptibility testing of Haemophilus influenzae (M2-A3 [12] and M7-A [13], respectively). These standards applied to the testing of ampicillin, amoxicillin-clavulanate, ampicillin-sulbactam, and chloramphenicol when H. infiuenzae was tested with chocolatized Mueller-Hinton agar for DD testing and Mueller-Hinton broth supplemented with lysed horse blood for dilution susceptibility testing.In January 1990, NCCLS published revised guidelines for H.influenzae DD and dilution susceptibility testing with Haemophilus Test Medium (HTM) (M2-A4 [14] and M7-A2 [15], respectively). HTM was adopted for DD testing because it had certain advantages over chocolatized Mueller-Hinton agar, including transparency and the ability to support DD testing of trimethoprim-sulfamethoxazole (chocolatized Mueller-Hinton agar contained large quantities of thymidine and thymidine analogs). HTM was also advantageous for both DD and broth dilution testing because of its low cost and its ability to adequately support the growth of H. influenzae (2,9). Nevertheless, problems with HTM have been reported, including its inability to support the growth of H. infiuenzae and provide consistency in susceptibility testing results (11). Interand intramanufacturer variabilities with batches of HTM (1, 3, 6, 7) as well as lot-to-lot and within-lot variabilities of HTM have also been reported (1, 3, 7). In June 1992, NCCLS realized that the HTM DD method could falsely categorize ,3-lactamase-negative, ampicillin-susceptible (BLNAS) H. influenzae strains as 3-lactamase-negative, ampicillin-resistant (BLNAR) strains. New interpretive criteria for zone diameters for DD testing were developed to alleviate this problem (2).We undertook a study to determine the optimal method for the in vitro susceptibility testing of H. influenzae strains to ampicillin. Specifically, we were interested in comparing the performance characteristics of agar dilution (AD) methods with HTM in comparison with those of our standard Mueller-Hinton agar supplemented with 1% hemoglobin and 1% IsoVitaleX (SMH). Furthermore, we wished to evaluate the * Corresponding author. broth dilution method using HTM and the DD method using HTM from two commercial sources (Remel and BBL). Finally, although current NCCLS guidelines do not specifically address other Haemophilus species, we included BLNAS Haemophilus parainfluenzae strains in our evaluation. These strains were previously characterized as ampicillin susceptible by our conventional SMH agar dilution technique...

Comparison of in-house and commercially prepared haemophilus test media for disk diffusion testing of ampicillin against Haemophilus species

“…Characterization of each strain as H. influenzae or H. parainfluenzae was performed by screening for Xand V-factor requirements. The reference or ''gold standard'' susceptibility (interpretive category) for each Haemophilus strain was derived from a consensus among the majority of testing methods used in a prior study (5). These testing methods included agar dilution with HTM prepared in-house or supplemented Mueller-Hinton medium, broth dilution with HTM prepared in-house, and disk diffusion with HTM from BBL or Remel.…”

“…We previously determined that Haemophilus Test Medium (HTM) prepared in-house and by the broth or agar dilution method produced the most consistent results for the susceptibility testing of ampicillin against Haemophilus strains including ␤-lactamase-positive (BLP); ␤-lactamase-negative, ampicillinsusceptible (BLNAS); and ␤-lactamase-negative, ampicillinresistant (BLNAR) strains (5). By these methods, no very major interpretive errors were observed and major interpretive errors were infrequent.…”

Antibacterial Activity of Environmentally Sustainable Polyurethane Based Composites from Castor Oil

“…Then the small pieces of sheets were placed on the surface of Mullar-Hinton medium and the plates were kept for incubation at 37°C for 24 hours. At the end of incubation, inhibition zones were examined around the disc and measured with transparent ruler in millimetres (Kohner et al, 1994;Mathabe et al, 2006). here, standard streptomycin disc used as positive control and sterile distilled water used as negative control.…”

Antimicrobial and Phytochemical Properties of Crude Extracts of Some Selected Plants Against Some Microbial Isolates

“…Each assay was carried out in triplicates under strict aseptic conditions. The absence of zone inhibition was interpreted as the absence of activity (Kohner et al, 1994;Mathabe et al, 2006).The activities are expressed as resistant, if the zone of inhibition was less than 7 mm, intermediate or mildly sensitive (8-10 mm) and sensitive if more than 11 mm (Assam et al, 2010).Commercially prepared antibiotics discs (Tetracycline, Ampicillin, Chloramphenicol and Ofloxacin) were used to determine the drug sensitivity and resistance pattern of bacteria. These discs were placed on the plates inoculated with different strains.…”