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Can BactoBox® be used for Mycoplasma viable cell counting?

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Gautier-Bouchardon, Anne, V. | Ferré, Séverine | Tocqueville, Véronique | Jaÿ, Maryne | Tardy, Florence

Edité par CCSD -

International audience. Optical density measurements at a wavelength of 600 nm (OD600) are often used as a proxy to estimate the number of cells in bacterial suspensions. However, it is not applicable to Mycoplasma spp. because of the small size and pleomorphic nature of their cells. In consequence, mycoplasma cell counts rely on long and tedious methods such as counting colony forming units (CFU/mL) on agar plates or estimating colony changing units (CCU/mL) by following up the extinction of growth by serial broth dilutions in microtiter plates. Count results are then only available within 2-7 days (agar) and up to 1-3 weeks (broth) depending on mycoplasma species and strains. Impedance-based flow cytometry offers a sensitive methodology to detect and count bacterial cells. The BactoBox® (SBT instruments) claims to provide a simple portable solution for bacterial cell count (https://sbtinstruments.com/bactobox/resources). Whether this technique applies also to mycoplasmas has yet to be assessed. SBT contacted us within the framework of the MyMIC network to propose BactoBox® as a potential help in inoculum calibration for antimicrobial susceptibility testing (AST). To determine whether this technology could be used to quickly and reliably enumerate mycoplasma suspensions, tests were carried out in parallel with traditional cell counting techniques. Strains of different mycoplasma species isolated from ruminants (Mycoplasma (M.) bovis, M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum, M. putrefaciens), pigs (M. hyopneumoniae, M. hyorhinis, M. flocculare) and poultry (M. gallisepticum, M. synoviae, M. meleagridis, M. iowae, M. pullorum, M. glycophilum, M. iners, M. gallinarum) were tested. For most of the species tested, the results in CFU/mL obtained in a few minutes with the BactoBox® were comparable to those obtained on agar medium in several days. However, cell counts obtained during late growth phase or with lysed mycoplasma cells showed that the BactoBox® was not able to discriminate living from deadmycoplasmas. Due to conductivity issues for low dilutions, the lowest count determined by BactoBox® was at 10^5 CFU/mL. Despite these limitations, and provided that BactoBox® counts are standardized per species, it could really allow a gain of time in the preparation of strains’ suspensions for AST.

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