Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

Abstract

Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of Legionella.

Therapeutic and diagnostic applications of antisense peptide nucleic acids

“… 76 Eubacterial 16S rRNA 15-mer PNA Blood plasma Fluorogenic (Alexaflour 488) Detection of E. coli, K. pneumoniae, and P. aeruginosa within water samples Time to detection: 5–6 h for E. coli and K. pneumoniae, and 10 h for P. aeruginosa Nácher-Vázquez et al. 77 …”

“…Legionalla contamination of natural and manmade water systems contributes to the development of Legionnaires’ disease and Pontiac fever. 77 Current methods of contamination detection rely on guidelines outlined within the International Organization for Standardization 11731:2017, which recommends culturing techniques that are time-consuming and of low sensitivity. 77 …”

“… 77 Current methods of contamination detection rely on guidelines outlined within the International Organization for Standardization 11731:2017, which recommends culturing techniques that are time-consuming and of low sensitivity. 77 …”

“…characterized a novel, culture-independent PNA-FISH method designed for the detection of the genus Legionella within contaminated tap water samples. 77 The PNA probe was developed by aligning the highly conserved 16S rRNA gene sequences of Legionella and comparing with other non-target strains of bacteria that may also be present in water. The selected FISH-PNA probe, named LEG22, specifically targets the 16S rRNA between positions 634 and 648 of the Legionella pneumonophila subspecies, which is estimated to cause 97% of all infectious outbreaks.…”

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

“…They developed a parallel sequential FISH (par-seqFISH) to capture cells gene expression profiles, maintaining biofilm structure integrity to study cellular physical and biogeographic context. For L. pneumophila , gene expression using FISH is a promising area yet to explore, but until the present day, this technique was only applied for Legionella detection in water samples [ 189 , 190 ], industrial settings [ 191 , 192 ] and biofilms (species detection) [ 31 , 193 ].…”