Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

Abstract

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Occurrence of Salmonella spp. in samples from pigs slaughtered for consumption: A comparison between ISO 6579:2002 and 23S rRNA Fluorescent In Situ Hybridization method

“…FISH was also responsible for false-negative results (2.2%), which is in accordance with the work by Fang et al (2003). These results could be due to the presence of small numbers of the target molecules (Fang et al, 2003;Stender et al, 2001) or to the insufficient accessibility of the target molecules (Amann et al, 1995). Although some authors suggest that a pre-enrichment step should be included to improve method sensitivity (Blasco, Ferrer, & Pardo, 2003;Fang et al, 2003;Moreno et al, 2003), in our study this step didn't eliminate the false-positive results.…”

GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND SYNECHOCOCCUS (MARINE CYANOBACTERIA) MEASURED BY WHOLE‐CELL HYBRIDIZATION OF rRNA‐TARGETED PEPTIDE NUCLEIC ACIDS1

“…PNA probes have been used to probe coastal (non–PE‐containing) Synechococcus strains (WH8007 and WH8101) and Prochlorococcus strains (Worden et al 2000) as well as a number of other microbes (Padilla et al 2000, Perry‐O'Keefe et al 2001, Stender et al 2001). In situ hybridization of PE‐containing strains with oligonucleotide probes has been hampered by the high background fluorescence engendered by this pigment.…”

PNA2/DNA Triplexes: Stability and Specificity

“…Based on this method, a test system was developed for express detection of Staphylococcus aureus in blood [48]. Another promising field of microbiological application of PNA is identification of microorganisms such as bacteria, algae, and yeasts in industry (e.g., food processing [49] and wine production [50]) and ecological monitoring [51]. Methods of microbiological analysis with the use of PNA are continuously improving [52,53].…”