Identification of indicator microorganisms using a standardized PNA FISH method

A standardized fluorescent in situ hybridization (FISH) method using Peptid e Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas a eruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and I yeast species, representing both phyloge netically closely related species, as well as species important to both cli nical and industrial settings. The S. aureus and P. aeruginosa PNA probes d id not cross react with any of the organisms tested., whereas the E. coli P NA probe, as expected from sequence data, also detected Shigella species. T he Salmonella PNA probe reacted with all of the 13 Salmonella strains, repr esenting the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identifi cation of multiple species, both gram-negative and gram-positive, in a mixe d population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereb y demonstrating the potential of multiplex FISH for various diagnostic appl ications within both clinical and industrial microbiology. (C) 2001 Elsevie r Science B.V. All rights reserved.