PCR-Based Method Using Propidium Monoazide To Distinguish Viable from Nonviable Bacillus subtilis Spores

Abstract

This paper describes a molecular-based method which is able to discriminate between viable and inactivated Bacillus subtilis spores by utilizing the DNA-intercalating dye propidium monoazide. The approach should be valuable in our attempt to employ molecular methods to streamline the evaluation of process validation using bacterial endospores.There is a continued need for the development and application of rapid methods for the detection and enumeration of bacterial endospores, especially as investigators seek to evaluate the efficacy of emerging food-processing technologies. For such thermal-process validation studies, surrogates of Clostridium botulinum, including Bacillus subtilis and Clostridium sporogenes, are commonly used. Currently, standard plating methods remain the "gold standard" for enumeration of survivors of these processes. Molecular amplification approaches, such as quantitative real-time PCR (qPCR), have shown promise but have not been applied to the enumeration of surviving spores because (i) release of nucleic acid from spores is difficult and (ii) the detection of DNA does not necessarily equate with the presence of viable spores.Recently, the DNA-intercalating agents ethidium monoazide and propidium monoazide (PMA) have been used in conjunction with qPCR for the selective detection of live cells of food-borne pathogens (2, 3, 5, 6, 9). These compounds selectively penetrate the membranes of dead cells and form stable DNA monoadducts upon photolysis, resulting in DNA which cannot be amplified by PCR (3). To our knowledge, this technique has not yet been applied to bacterial spores. The purpose of this study was to demonstrate that DNA-intercalating agents could be used in conjunction with qPCR for the selective enumeration of viable, but not inactivated, spores of B. subtilis.All media were supplied by Difco (Franklin Lakes, NJ), and chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Bacillus subtilis ATCC 35021 (American Type Culture Collection, Manassas, VA) was grown overnight in 10 ml of brain heart infusion broth at 37°C. Five-hundred-microliter aliquots of the vegetative cells were spread onto 150-by 15-mm petri dishes containing sporulation agar comprised of 13 g/liter nutrient broth, 15 g/liter agar, 0.51 g/liter MgSO 4 ⅐ 7H 2 O, 0.97 g/liter KCl, 0.2 g/liter CaCl 2 ⅐ 2H 2 O, 3 mg/liter MnSO 4 ⅐ H 2 0, and 0.5 mg/liter FeSO 4 ⅐ 7H 2 O. The plates were incubated aerobically at 37°C for 3 to 5 days until more than 95% of cells had sporulated, as determined by phase-contrast microscopy. Spores were harvested in cold, sterile distilled water (dH 2 O) and washed repeatedly (5 to 10 times). Before final resuspension, the spores were treated with 80 U/ml DNase (SigmaAldrich, St. Louis, MO) at 37°C for 90 min (to degrade residual DNA), with subsequent DNase inactivation by heating at 65°C for 10 min. The final populations were approximately 10 7 spores/ml, and crops were stored at 4°C until use. Using the capillary tube method (8), the D 121°C value (decimal reduction time, i.e., tim...

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

“…Quantitative PCR with PMA modification has been successfully used in a number of studies for different microorganisms (5,8,15,22,27,28,30,37). To our knowledge, the only E. coli strain tested in a PMA-modified quantitative PCR is E. coli O157:H7.…”

Dead or Alive: Molecular Assessment of Microbial Viability

“…The utility of viability PCR has been expanded by modifications to this basic strategy (31). Dithiothreitol cotreatment was reported to facilitate PMA penetration of inactivated Bacillus subtilis spores, thereby improving the ability to discern the viability of spores (35). Deoxycholate (DOC) cotreatment helped PMA penetrate E. coli cells that were inactivated but not disrupted by pasteurization at low temperature (between 52°C and 70°C) (36).…”

Amoeba-Related Health Risk in Drinking Water Systems: Could Monitoring of Amoebae Be a Complementary Approach to Current Quality Control Strategies?

“…The use of qPCR in conjunction with PMA has been effectively evaluated in different bacteria, bacterial viruses, bacterial spores and fungi with excellent results [56][57][58] and is in continuous development. Although viability assessment of human enteric viruses by PCR has not been effectively demonstrated [59], and it is not clear if this technique could be a universal approach for all cases, we bet that this analytical field still has a great potential.…”

“…PMA is a DNA-intercalating dye with the capacity to only penetrate membrane-compromised cells, blocking the amplification cycle. With this approach, cell viability is based on membrane integrity [52][53][54][55][56][57][58][59].…”