Investigation of ampicillin-intermediate strains of Haemophilus influenzae by using the disk diffusion procedure and current National Committee for Clinical Laboratory Standards guidelines

Abstract

It was noted in our laboratory that certain strains of Haemophilus influenzae yielded zone sizes interpreted as resistant to the ampicillin (AMP) disk on chocolate-Mueller-Hinton agar (CMH) but showed no evidence of 13-lactamase (13-Lac) activity. Although it is known that a second mechanism of AMP resistance exists, strains with this mechanism are uncommon. To investigate this apparent discrepancy, a study of 100 consecutive clinical isolates of H. influenzae collected over a 6-month period was performed. Isolates were simultaneously tested against five antibiotics (AMP, chloramphenicol, cefotaxime, ciprofloxacin, and AMP-sulbactam) on CMH and on two brands of Haemophilus test medium (HTM) by using the disk diffusion procedure and National Committee for Clinical Laboratory Standards (NCCLS) standards. By using CMH and NCCLS standard M2-A3-S2, strains of H. influenzae showing zone sizes of .20 mm with AMP were considered sensitive. By using HTM and NCCLS standard M2-A4, strains showing zone sizes of .25 mm to AMP on HTM were considered sensitive. Intermediate strains had zone sizes of 22 to 24 mm. The majority of isolates (68%) were sensitive to all antibiotics. Two percent of the isolates were resistant to chloramphenicol. Seventeen percent of the isolates were AMP-resistant, 13-Lac-producing strains of H. influenzae. Thirteen percent of the isolates gave at least one intermediate or resistant zone for AMP but were 13-Lac negative. MIC determinations with NCCLS standard M7-A2 were performed with resistant and intermediate strains. MICs for 13-Lacproducing strains of H. influenzae were .8.0 ,ug/ml. MICs for 13-Lac-negative strains were '1.0 ,ug/ml and were highly reproducible. If one uses the current NCCLS zone diameter interpretive criteria, results should be viewed with caution. Further investigation of zone size interpretive criteria is warranted. It is suggested that in the case of serious infections with H. influenzae, 13-Lac-negative, AMP-resistant or -intermediate strains be confirmed by the MIC procedure.

In vitro susceptibility testing of Haemophilus influenzae: review of new National Committee for Clinical Laboratory Standards recommendations

“…In June 1992, the following zone diameter criteria were adopted for HTM ampicillin disk diffusion tests: .18 (R) and .22 (S). This change is consistent with the observations of one recent investigation (11).…”

“…Extensive anecdotal experience indicates frequent episodes of poor growth or actual growth failures with HTM. Two published studies have alluded to this problem (1,11). By contrast, the six-center study cited above, which was conducted with the intent of reassessing zone diameter interpretive criteria with antimicrobial agents such as cefaclor and cefprozil, did not experience major problems with growth failures.…”

“…A further difficulty pertains to inter-and intramanufacturer variability with batches of HTM (2,7,11,12). Such variability is perhaps not surprising, as the individual ingredients used to make HTM have profound effects on media performance.…”

Molecular Epidemiology of Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae

“…According to the accepted limit of the MIC, their incidence varies. The frequencies of occurrence of resistant strains are Ͻ1 to 2.5% (MICs, Ն2 mg/liter) in Canada and the United States (1, 2, 5, 10, 11, 34), 5 to 7% (MICs, Ն1 mg/liter) in the United Kingdom (29-31), Ͻ1 to 7% (MICs, Ն1 or Ն2 mg/ liter) in Australia (3, 6), 13% (MICs, Ն1 mg/liter) in Greece (14), 1% (MICs, Ն2 mg/liter) in France (7), and 0.3% (MICs, Ն4 mg/liter) in an international study (15). This variability in the incidence of AMP r ␤Ϫ strains illustrates the difficulties encountered in highlighting this kind of resistance.…”

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

“…These minor errors (susceptible or resistant called intermediate) ranged in frequency from 10.1 to 24.4% for all strains tested and resulted from calling susceptible (BLNAS) or resistant (BLNAR but not BLP) strains intermediate (Tables 2 and 4). For these strains, then, the clinical decision remains whether to accept the disk diffusion result as indicative of probable ampicillin resistance and to use an alternative treatment which could be more expensive, toxic, or unnecessary or to perform other, more definitive susceptibility testing for any intermediate result, as recommended by Scriver and colleagues (11,12) and Heelan and colleagues (4). Also in the present study, unacceptably high frequencies of very major errors (resistant strains called susceptible) were observed for HTM prepared in-house or by Remel (range, 6.6 to 10.5%) when 1993 NCCLS guidelines were followed (Table 1); however, no very major interpretive errors were observed when 1990 NCCLS guidelines were followed (Table 1).…”