Broth microdilution plate-based minimal inhibitory concentration testing using MGIT-positive cultures

The current WHO-endorsed 96-well-plate-based broth microdilution testing (BMD) for minimum inhibitory concentration (MIC) testing of Mycobacterium tuberculosis complex (MTBc) relies on inoculum prepared from a solid-medium culture, which requires long incubation, limiting its use in clinical settings. We aimed to develop a protocol for BMD-MIC testing from freshly positive, actively growing MGIT cultures.

Bacterial suspensions were prepared by resuspending well-dispersed pellets obtained by centrifuging the contents of purity-confirmed, 3-5 days old positive MGIT cultures in sterile distilled water (SDW). The bacterial suspensions with optical density (OD) ≤McF 0.5, were diluted 1:100 in supplemented 7H9 broth to be used as the inoculum for BMD-MIC testing while those with OD >McF 0.5 were diluted in SDW to obtain an OD of Mcf 0.5 before diluting 1:100 in 7H9. MICs obtained for moxifloxacin, levofloxacin, clofazimine, bedaquiline, linezolid, and delamanid using an MGIT inoculum were compared to the MICs obtained using a solid medium inoculum.

We tested 16 MTBc isolates, yielding 96 MICs: 10/16 MGIT inocula achieved an OD of McF 0.5, the OD of the remaining six ranged between McF 0.38-0.47. All 16 isolates had interpretable MICs to all drugs tested with an average turnaround time of 16.5 days since MGIT positivity. The difference between the MICs obtained by the two methods was within the acceptable range for 90/96 MICs: zero for 33 MICs and +/-1 dilution for 67. Few (6/96 MICs) differed by 2 drug dilutions.

Our results suggest that standardized MGIT inocula yield comparable BMD-MICs to that of solid medium inoculum while reducing the turn-around time.