Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate)

Abstract

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H 2O 2, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate). © 2012 The Societies and Blackwell Publishing Asia Pty Ltd.