The Inoculum Effect in the Era of Multidrug Resistance: Minor Differences in Inoculum Have Dramatic Effect on MIC Determination

Abstract

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 (R² = 0.99) or 1.1-fold (R² = 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.

Keywords: CLSI; MIC; antimicrobial susceptibility testing; broth microdilution; carbapenem-resistant Enterobacteriaceae; extended-spectrum β-lactamase producers; inkjet printing; inoculum; inoculum effect.

FIG 1

Inkjet dispensing precision. (A) Extended-dynamic-range dispensing. Bacteria were dispensed by inkjet printing in a 2-fold dilution series and enumerated by plate count. Each data point is the mean from three independent experiments performed on separate days, with error bars representing 1 standard deviation. There was a strong linear correlation (dashed line) between volume dispensed and CFU per milliliter (R² > 0.99). (B) High-resolution dispensing. Bacteria were dispensed using inkjet technology in a $\sqrt[7]{2}$(≈1.10-fold) dilution series and enumerated by plate count. Data points represent the averages from 3 independent experiments performed on separate days using 4 Enterobacteriaceae strains each, with error bars corresponding to 1 standard deviation from the mean. There was a strong linear correlation (dashed line) between dispense volume and CFU per milliliter delivered to wells (R² = 0.98). Dotted lines indicate the CLSI-recommended inoculum range (2 × 10⁵ to 8 × 10⁵ CFU ml⁻¹).

FIG 2

Extended-range and high-resolution quantification of the inoculum effect. (A to C) Bacteria and antibiotics meropenem (A), cefepime (B), and ceftazidime-avibactam (C) were dispensed in orthogonal 2-fold dilution series. Each data point represents the log₂ difference between the MIC at the indicated inoculum and the corresponding MIC at the CLSI target inoculum averaged across all strains within each category indicated. Error bars represent 1 standard deviation from the mean. Dotted lines demarcate the CLSI-recommended inoculum range. Carbapenem-resistant strains showed a pronounced reduction in meropenem MIC at inocula below the CLSI-recommended inoculum. Cefepime-resistant and -susceptible dose-dependent (SDD) strains showed a rapid increase in cefepime MIC at inocula above the CLSI-recommended inoculum. In contrast, a pronounced inoculum effect was not observed for ceftazidime-avibactam. (D) Bacteria and meropenem were dispensed using inkjet technology in $\sqrt[7]{2}$ (≈1.10-fold) and $\sqrt[4]{2}$ (≈1.19-fold) orthogonal dilution series, respectively. Each data point represents the log₂ difference between the MIC determined at the indicated inoculum and the MIC at the CLSI target inoculum averaged across triplicate experiments performed on separate days using 8 CRE strains. Dotted lines demarcate the CLSI-recommended inoculum range.