Can the Etest Correctly Determine the MICs of β-Lactam and Cephalosporin Antibiotics for Beta-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae ?

Abstract

A recent study reporting surprisingly high beta-lactam MICs against beta-lactamase-negative ampicillin-resistant (BLNAR) strains when tested by the Etest prompted us to compare results of the Etest with those of the standard broth microdilution concerning BLNAR strains (5). In that study, the MIC90s of BLNAR and beta-lactamase-positive amoxicillin-clavulanate-resistant (BLPACR) strains in response to ampicillin, amoxicillin-clavulanate, cefuroxime, cefaclor, cefixime, and meropenem were 256 and 256, 256 and 256, 8 and 256, 256 and 256, 32 and 256, and 32 and 256 μg/ml, respectively. In previous studies, BLNAR isolates almost always showed low ranges of MICs. For instance, Dabernat et al. reported MIC ranges of the ftsI mutant gene strains as 0.5 to 8, 0.5 to 8, 4 to 16, and 0.03 to 0.25 μg/ml for amoxicillin-clavulanate, cefuroxime, cefaclor, and cefixime, respectively (2). Similarly in a recent study, mean MICs of meropenem were <0.3 μg/ml, although BLNAR strains showed increasing MIC trends (8). For the antibiotic susceptibility method, the author performed only the Etest; however, both of the previous studies with lower MICs used the broth microdilution method (2, 6). Concerning the reliability of the Etest, the authors referred to two studies; however, the numbers of Haemophilus influenzae isolates with relatively low MICs was small, and the mutation status of the ftsI gene was not evaluated in either study (4, 7). In the current study, our main concern was whether remarkably high MIC patterns of BLNAR strains in response to beta-lactam antibiotics (beta-lactams) and meropenem arise from the test method or their actual status. To confirm the high MIC values, we compared results of the Etest with those of broth microdilution using H. influenzae isolates with known mechanisms of beta-lactam resistance. We examined MICs of 153 H. influenzae strains by using the Etest and the broth microdilution method simultaneously (Table ​(Table1).1). MICs of all strains were determined by broth microdilution using the standard method (1) and by the Etest according to the manufacturer's instructions. Two persons independently took readings of MICs, and the agreement was almost ±log21. H. influenzae ATCC 49247 was included in each batch as a control. MICs of the Etest were rounded up to the next log2 concentration, and agreement was defined as the results of broth microdilution and those of Etest in the range of ±log21 of each other. The mutation status of the ftsI gene in all strains was checked by using primers described previously (3, 8). MICs of BLNAS and beta-lactamase-positive ampicillin-resistant (BLPAR) strains showed consistent results by both tests, and the agreement was 92.59 to 100% for beta-lactams and 90 to 92.59% for meropenem, respectively. However, BLNAR and BLPACR strains showed diverse results, and the agreement for beta-lactam and meropenem decreased to a range of 48.27% to 100% and 42.10% to 44.82%, respectively (Table ​(Table1).1). Furthermore, 9.2% of the intermediate BLNAR strains determined by the broth microdilution method were highly resistant to ampicillin (256 μg/ml) by the Etest method. Similarly, all strains were susceptible to ceftriaxone by broth microdilution, but 42.5 to 68.42% of BLNAR and BLPACR strains showed high MICs to ceftriaxone (32 μg/ml). The fluoroquinolone levofloxacin showed high agreement (98.85% to 100%) between microbroth and Etest results (Table ​(Table11). TABLE 1. Comparison of Etest and broth microdilution MICs determined for BLNAS and BLNAR H. influenzae isolatesa From these results, we considered that determining MICs to beta-lactams by the Etest is not a reliable method for strains with the fstI mutant gene but is acceptable for BLNAS and BLPAR strains. High MICs to beta-lactams and meropenem may be due to the presence of small colonies within the inhibition ellipses of the Etest and the variable expression of ftsI during bacterial growth between agar and broth media. Matic et al. reported low MICs for BLPACR strains to amoxicillin-clavulanate and excluded the possibilities of extended-spectrum beta-lactamase associated with amoxicillin-clavulanate resistance in BLPACR strains (6). We considered that remarkably high MICs of BLNAR strains might arise from an antibiotic susceptibility method such as the Etest rather than an actual elevation of MICs. In conclusion, we considered that Etest results alone might not represent the actual MIC status of BLNAR and BLPACR strains for beta-lactams and meropenem. Considering the essential roles of beta-lactams in the treatment of invasive diseases, utilizing only the Etest may misguide a community about the susceptibility of H. influenzae and would enhance the development of BLNAR strains by selection of an inappropriate antibiotic. Although the Etest is not a national or international reference method, it is a convenient commercial product for generating MICs and can be applicable in regions with a low prevalence of BLNAR strains. Finally, we suggest that MICs of H. influenzae isolates should be measured by the broth microdilution method in conjunction with the PCR technique.