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The Inoculum Effect in the Era of Multidrug Resistance: Minor Differences in Inoculum Have Dramatic Effect on MIC Determination.
Antimicrobial Agents and Chemotherapy 2018 August
The observed MIC may depend on the number of bacteria initially inoculated into the assay. This phenomenon is termed the inoculum effect (IE) and is often most pronounced for β-lactams in strains expressing β-lactamase enzymes. The Clinical and Laboratory Standards Institute (CLSI)-recommended inoculum is 5 × 105 CFU ml-1 with an acceptable range of 2 × 105 to 8 × 105 CFU ml-1 IE testing is typically performed using an inoculum 100-fold greater than the CLSI-recommended inoculum. Therefore, it remains unknown whether the IE influences MICs during testing performed according to CLSI guidelines. Here, we utilized inkjet printing technology to test the IE on cefepime, meropenem, and ceftazidime-avibactam. First, we determined that the inkjet dispense volume correlated well with the number of bacteria delivered to microwells in 2-fold ( R 2 = 0.99) or 1.1-fold ( R 2 = 0.98) serial dilutions. We then quantified the IE by dispensing orthogonal titrations of bacterial cells and antibiotics. For cefepime-resistant and susceptible dose-dependent strains, a 2-fold increase in inoculum resulted in a 1.6 log2 -fold increase in MIC. For carbapenemase-producing strains, each 2-fold reduction in inoculum resulted in a 1.26 log2 -fold reduction in meropenem MIC. At the lower end of the CLSI-allowable inoculum range, minor error rates of 34.8% were observed for meropenem when testing a resistant-strain set. Ceftazidime-avibactam was not subject to an appreciable IE. Our results suggest that IE is sufficiently pronounced for meropenem and cefepime in multidrug-resistant Gram-negative pathogens to affect categorical interpretations during standard laboratory testing.
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