Purpose: Analyses of 34 water samples from 13 healthcare structures revealed how culture method and quantitative PCR (qPCR) often differ in the detection of Legionella pneumophila (Lp). With these considerations in hand, culture method, PCR and Ethidium Monoazide Bromide (EMA) qPCR have all been compared in order to detect Lp in water samples, identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude non-viable bacteria using a commercial kit for extraction and amplification as well as modification of the protocol. Methods: Pure water samples artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 were analyzed using a commercial kit for both qPCR and EMA-qPCR, while ISO 11731-2-2004 was used for culture method. Results: Only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA-qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the non-viable ones. In both viable and VBNC strains, a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration. Conclusions: Discrepancies between culture method and EMA-qPCR were observed and may be due to different causes such as membrane-dye interactions, presence of interfering compounds and the sensitivity of the kit used. Study significance and impact: In the presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, seeing that in our experience EMA pretreatment on the filter membrane did not come up with the expected results, it would be necessary to proceed with other experiments and/or different dyes.

Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide / M. Consonni, A. Grassi, S. Scuri, M. Gori, E. Tanzi, M. Tesauro. - In: ANNALS OF MICROBIOLOGY. - ISSN 1590-4261. - 71:1(2021 Dec), pp. 43.1-43.7. [10.1186/s13213-021-01653-5]

Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

M. Consonni
Primo
Investigation
;
M. Gori
Methodology
;
E. Tanzi
Penultimo
Methodology
;
M. Tesauro
Ultimo
Conceptualization
2021

Abstract

Purpose: Analyses of 34 water samples from 13 healthcare structures revealed how culture method and quantitative PCR (qPCR) often differ in the detection of Legionella pneumophila (Lp). With these considerations in hand, culture method, PCR and Ethidium Monoazide Bromide (EMA) qPCR have all been compared in order to detect Lp in water samples, identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude non-viable bacteria using a commercial kit for extraction and amplification as well as modification of the protocol. Methods: Pure water samples artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 were analyzed using a commercial kit for both qPCR and EMA-qPCR, while ISO 11731-2-2004 was used for culture method. Results: Only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA-qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the non-viable ones. In both viable and VBNC strains, a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration. Conclusions: Discrepancies between culture method and EMA-qPCR were observed and may be due to different causes such as membrane-dye interactions, presence of interfering compounds and the sensitivity of the kit used. Study significance and impact: In the presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, seeing that in our experience EMA pretreatment on the filter membrane did not come up with the expected results, it would be necessary to proceed with other experiments and/or different dyes.
Legionella, Detection, Rapid methods, Water, Environmental health;
Settore MED/42 - Igiene Generale e Applicata
Settore MED/07 - Microbiologia e Microbiologia Clinica
12-nov-2021
Article (author)
File in questo prodotto:
File Dimensione Formato  
Consonni_et_al-2021-Annals_of_Microbiology.pdf

accesso aperto

Tipologia: Publisher's version/PDF
Dimensione 847.47 kB
Formato Adobe PDF
847.47 kB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/883624
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 1
  • ???jsp.display-item.citation.isi??? 0
social impact