The performance of the Liofilchem omadacycline MIC Test Strip (MTS) was evaluated in a multi-site study. Three testing sites collected/tested clinical isolates and one site tested challenge isolates that totaled 175
S. aureus
, 70
S. lugdunensis
, 121
E. faecalis
, 100
E. faecium
, 578 Enterobacterales, 142
Haemophilus
spp., 181
S. pneumoniae
, 45
S. anginosus
group, 35
S. pyogenes
and 20
S. agalactiae
. MIC testing was performed by CLSI broth microdilution (BMD) and MTS. Fastidious isolates testing included BMD and MTS testing with both CLSI and EUCAST Mueller Hinton Fastidious (MH-F). In addition, each site performed reproducibility for non-fastidious and fastidious isolates and QC by MTS and BMD. All BMD and MTS results for the QC strains were within expected ranges, with exception of one MTS HTM result for
H. influenzae
ATCC 49247. Among reproducibility isolates, omadacycline MTS results were within one dilution of the modal MIC for 95.2% of non-fastidious Gram-positive, 100% of Gram-negative, 99.3% and 98.5% of fastidious isolates tested on CLSI and EUCAST media, respectively. MTS results for all study isolates were within one doubling dilution of the CLSI BMD MIC for 98.9% of
S. aureus
, 100% of
S. lugdunensis
, 98.3% of
E. faecalis
, 100% of
E. faecium
, and 99.6% of Enterobacterales. Essential agreement rates for CLSI and EUCAST MH-F agar compared to CLSI BMD were 98.2% and 98.2%, for
H. influenzae
, 91.1% and 73.6%, for
S. pneumoniae
and 100% and 85-91.7% for other
streptococcus
species, respectively. Based on CLSI media, all categorical errors were minor errors and categorical agreement rates were >90% with exception of
C. freundii
, S. lugdunensis,
E. faecalis
, S. anginosus
and S. constellatus.