Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia.
Appl Microbiol Biotechnol
; 107(12): 4069-4077, 2023 Jun.
Article
em En
| MEDLINE
| ID: mdl-37148337
ABSTRACT
Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland-Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. KEY POINTS ⢠Enumeration of live biotherapeutics is essential for product quality and safety. ⢠Conventional CFU counting may not differentiate between strains in microbial products. ⢠This approach was developed for direct enumeration of mixed bacterial strains simultaneously.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bactérias
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos