This method has applications in the pharmaceutical, food microbiology, semiconductor, and other industries requiring surface sterilization.
The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores.
Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.
While this PMA-based selective chemistry has been applied to several polymerase chain reaction (PCR)-based molecular protocols, it has never been coupled with fluorescence in situ hybridization (FISH)-based microscopic methods. FISH microscopy is a powerful technique for visualizing and enumerating microorganisms present in a given sample, which relies on the ability of fluorescently labeled oligonucleotide probes to gain access to, and hybridize with, specific nucleic acid sequences within cells. Dogmatic principles suggest that by first treating a sample with PMA and covalently modifying the DNA originating from dead cells, downstream FISH-based microscopy should then enable the direct, specific visualization and enumeration of only living, viable microorganisms. An effective and efficient coupling of PMAbased chemistry with downstream FISHmicroscopic methods would significantly empower the current ability to discern viable from dead microbes by direct visualization.
The basic principle of this method is that PMA penetrates only the dead cells and/or spores, due to their compromised membrane structures. Once inside the cell, PMA strongly intercalates with DNA. PMA has a photoactive azide group that allows covalent cross-linkage to DNA upon exposure to bright white light. This photoactivation results in the formation of PMA-DNA complex that renders DNA inaccessible for hybridization reaction during FISH assay. To avoid the difficulties and problems associated with current methods for determining the actual numbers of living versus dead cellular entities examined, and biases associated therewith, a novel molecular-biological protocol was developed for selective detection and enumeration of viable microbial cells. After having been subjected to the procedures described herein, the viability (live vs. dead) of bacterial cells and spores could be discerned. Following treatment with PMA, living, viable cells and spores were shown to be receptive to fluorescently labeled oligonucleotide probes, as hybridization and FISH-based mi cros copy was successful. Dead cells and spores, however, were not detected, as the pretreatment with PMA rendered their DNA unavailable to hybridization with the FISH-probes.
The true novelty of the technology is the coupling of a downstream, highly specific means of visualizing microbial cells and spores with a chemical pretreatment that precludes the portion of the microbial consortium that is not living (non-viable) from being detected. This results in the ability to selectively visualize and enumerate only the living cells and spores present in a given sample, in a molecular biological fashion, without the need for heavily biased cultivationbased methodologies. This novel study demonstrates that PMA penetrates only the heat-killed spores, which precludes downstream hybridization reactions in the FISH assay. This novel PMA-FISH method is an attractive tool to detect viable endospores in spacecraft-associated environments, which is of crucial importance and benefit to planetary protection practices aimed at reducing the abundance of spacecraft-borne microbial contaminants.
In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to:
Innovative Technology Assets Management
Mail Stop 202-233
4800 Oak Grove Drive
Pasadena, CA 91109-8099