Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water

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.

Section: Discussionsupporting

confidence: 92%

“…Microbiol., abstr. O-14, 1999) and methods such as PCR (2) and peptide nucleic acid (PNA) probe hybridization followed by signal amplification (18). However, none of these rapid methods provides simultaneous detection, identification, and enumeration.…”

mentioning

confidence: 99%

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

Stender¹,

Broomer²,

Oliveira³

et al. 2001

“…Further work needs to be completed with GFP-labeled bacteria to determine how responsive the fluorescence in the cells is to changes in the physiological state of the bacteria. A second study examined E. coli in distribution water by using a fluorescently labeled peptide nucleic acid oligonucleotide probe targeting the V 1 region of 16S rRNA (33). In this study, E. coli cells were exposed to 1.5 mg of ClOH per liter for 30 min and then stored at room temperature for 14 days before being detected by hybridization, although other measures of cell viability (plate counts, Colilert, and CTC reduction) showed no activity.…”

Section: Vol 69 2003 Growth Of E Coli In Biofilms 5467mentioning

confidence: 99%

Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine

Williams

Braun-Howland

2003

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH 2 Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH 2 Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and ␤ and ␦ proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.Assessment of the microbiological safety of drinking water is based largely on the routine monitoring of water supplies for the presence of total coliforms and Escherichia coli. Detection of E. coli is considered indicative of recent fecal contamination and of the potential presence of enteric pathogens, while the presence of total coliforms is indicative of poor water quality.Whereas routine water quality measurements assess the presence of planktonic bacteria, the vast majority of bacteria indigenous to aquatic environments exist attached to solid particles or surfaces. Within water distribution systems, significant bacterial populations exist as complex, structurally heterogeneous biofilms attached to pipe surfaces. Residence within these complex matrices provides organisms with higher localized nutrient concentrations than are commonly found in drinking waters (10,14,18,22), and recent studies have shown that attached bacteria are more metabolically active than are their free-living counterparts (26, 29). Furthermore, biofilms afford bacteria significant protection from disinfecting agents (3, 31, 35), including hypochlorous acid and monochloramine. Biofilms are dynamic ...

“…However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5,15,17,27,(34)(35)(36).…”

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confidence: 99%

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies ( Enterobacter sakazakii ) in Powdered Infant Formula

Almeida

Azevedo

Iversen

et al. 2009

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.