Abstract
Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.
Highlights
The detection of mycobacteria in recent years relied mainly on cultivation-based methods
The other three Pt compounds [tetrakis(triphenylphosphine)platinum(0), chloroplatinic acid hexahydrate and platinum(IV) chloride] showed a limited effect on Mycobacterium avium subsp. paratuberculosis (MAP) DNA amplification ( Cq < 5) even at the highest concentration tested (1,000 μM), for which reason the treatment with these compounds was excluded from the following optimization on mycobacterial cells
The viability assay combining the sample treatment with Pt compounds and quantitative PCR (qPCR) is able to detect the viable mycobacterial cells within 1 day and is clearly faster than cultivation, a gold standard for viability evaluation, which is very challenging for slow-growing bacteria such as MAP
Summary
The detection of mycobacteria in recent years relied mainly on cultivation-based methods. These are only able to detect viable mycobacteria if they are cultivable, which may represent a substantial deficiency of these conventional methods. Paratuberculosis (MAP), which is the causative agent of paratuberculosis, in domestic and wild ruminants is very time consuming (several weeks to months). Another disadvantage of MAP cultivation is the need for chemical decontamination of the sample to suppress the growth of competitive microorganisms, which can result in a decrease in the sensitivity of detection (Slana et al, 2008). The number of MAP cells may be underestimated due to the tendency of MAP cells to form clumps (Pickup et al, 2005)
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