A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR

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.

Keywords: Mycobacterium avium subsp. paratuberculosis; live-dead discrimination; mycobacteria; platinum; propidium monoazide; qPCR; viability.

FIGURE 1

Schematic overview of the optimization of Pt compound treatment.

FIGURE 2

Direct effect of Pt compound solutions at various concentrations on amplification of MAP DNA diluted with TE buffer or ultrapure water. The ΔCq values express the mean difference of individual Cq values of Pt-treated and mean Cq value of untreated control samples calculated from four replicates (biological and technical). The significance of differences between ΔCq was calculated by two-way ANOVA with factors of concentration and dilution medium and Tukey’s HSD test. The same letters indicate statistically insignificant differences (P > 0.05) between ΔCq values, while different letters indicate significant differences (P < 0.05). Error bars represent standard deviations calculated from four replicates. The bar above value of 18 in ΔCq on the y-axis signifies that no amplification occurred.

FIGURE 3

Effect of treatment of M. smegmatis cells with cis-dichlorodiammine platinum(II) at different concentrations and for different times. The aim was to find the treatment condition providing the highest possible value for ΔCq _{dead with Pt – live with Pt} and ΔCq _{dead with Pt – dead without Pt} and the lowest possible value for ΔCq _{live with Pt – live without Pt}. The ΔCq values express the individual mean differences of Cq values calculated from four replicates (biological and technical). The significance of differences between ΔCq was calculated by one-way ANOVA and Tukey’s HSD test. The same letters indicate statistically insignificant differences (P > 0.05) between ΔCq values, while different letters indicate significant differences (P < 0.05). The values of ΔCq _{dead with Pt – live with Pt} were marked with lowercase letters, and the values of ΔCq _{live with Pt – live without Pt} were marked with capital letters, as it was evaluated separately. Error bars represent standard deviations calculated from four replicates.

FIGURE 4

Effect of different incubation temperatures (37 and 5°C) on the treatment of M. smegmatis cells with 100 μM cis-dichlorodiammine platinum(II) for 60 min. The aim was to find the incubation temperature providing the highest possible value for ΔCq _{dead with Pt – live with Pt} and ΔCq _{dead with Pt – dead without Pt} and the lowest possible value for ΔCq _{live with Pt – live without Pt}. The ΔCq values express the individual mean differences of Cq values calculated from four replicates (biological and technical). The significance of differences between ΔCq was calculated by Welch’s t-test. The same letters indicate statistically insignificant differences (P > 0.05) between ΔCq values, while different letters indicate significant differences (P < 0.05). The values of ΔCq _{dead with Pt – live with Pt} were marked with lowercase letters, and the values of ΔCq _{live with Pt – live without Pt} were marked with capital letters, as it was evaluated separately. Error bars represent standard deviations calculated from four replicates.

FIGURE 5

Comparison of Pt and PMA treatment of MAP cells and the effect of different resuspension media (ultrapure water or TE buffer with carrier DNA solution) used to resuspend the cells after the treatment. The aim was to determine the agent showing the highest possible value for ΔCq _{dead with Pt/PMA – live with Pt/PMA} and ΔCq _{dead with Pt/PMA – dead without Pt/PMA} and the lowest possible value for ΔCq _{live with Pt/PMA – live without Pt/PMA}. The ΔCq values express the individual mean differences of Cq values calculated from four replicates (biological and technical). The significance of differences between ΔCq was calculated by two-way ANOVA with factors of resuspension medium and agent used, and Tukey’s HSD test. The same letters indicate statistically insignificant differences (P > 0.05) between ΔCq values, while different letters indicate significant differences (P < 0.05). The values of ΔCq _{dead with Pt – live with Pt} were marked with lowercase letters, and the values of ΔCq _{live with Pt – live without Pt} were marked with capital letters, as it was evaluated separately. Error bars represent standard deviations calculated from four replicates.