Legionella Detection in Water Networks as per ISO 11731:2017: Can Different Filter Pore Sizes and Direct Placement on Culture Media Influence Laboratory Results?

Abstract

Determination of Legionella concentrations in water networks is useful for predicting legionellosis risks. The standard culture technique using concentration with membranes filters is the most commonly used method for environmental surveillance of Legionella. The aim of this study was to verify whether filtration with different filter pore sizes (0.2 and 0.45 µm) according to (ISO) 11731:2017, followed by directly placing them on culture media, can influence Legionella detection. Three laboratories participated in an experimental study that tested a known suspension of Legionella pneumophila (Lpn) serogroup 1 (ATCC 33152) (approximate final cell density of 15 CFU/mL). E. coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 25668) were included as control tests. The average (95% CI) percentage of recovery of Lpn was 65% using 0.45-µm filters and 15% using 0.2-µm filters (p < 0.0001). For control tests, the average (95% CI) percentage of recovery was higher with 0.45 vs. 0.2 µm filters: 97% vs. 64% for Escherichia coli (p < 0.00001) and 105% vs. 97% (p = 0.0244) for P. aeruginosa. Our results showed that the 0.45-µm filters provided the greatest detection of Legionella. Because the current national guidelines leave the choice of membrane porosity to the operator, experimental studies are important for directing operators towards a conscious choice to standardize Legionella environmental surveillance methods.

Water Sampling Module for Collecting and Concentrating Legionella pneumophila from Low-to-Medium Contaminated Environment

“…In comparison to the filtration with 0.45 µm membranes, the lower values of RR and CF obtained with 0.22 µm membranes are likely related to the reduced efficiency of eluting bacteria from a small pore size structure of membranes. These results are consistent with the results of De Giglio et al [ 24 ] who reported recovery rates of L. pneumophila at 65% and 15% for 0.45 and 0.2 µm membranes, respectively.…”

“…To suppress the concentration of L. pneumophila to below the critical level, it would be advantageous to perform routine quasi-continuous collection of water samples to be analyzed on the spot, well ahead of the potential outbreak. A possible method of detecting low numbers of bacteria consists of taking the filters from filtration stations and placing them directly on culture media [ 24 ]. However, to efficiently fight disease outbreaks, it is important to analyze samples at the source of water [ 25 ], especially in view that no scientific evidence has been established for a safe level of L. pneumophila contamination [ 26 , 27 , 28 , 29 ] and infectious doses were not well defined [ 30 ].…”

Assessing the Risk of Legionella Infection through Showering with Untreated Rain Cistern Water in a Tropical Environment

“…Significant progress has been made in recent years on improving the method for culture detection of Legionella in drinking water and the water distribution network [35][36][37]. The Legionella monitoring methods by ISO 11731 and the U.S. CDC also provide a reliable framework for culturing Legionella in drinking water [38,39].…”

“…Moreover, identification of Legionella to the species level could improve our understanding of the pathogenic vs. non-pathogenic species in water. Additionally, our model does not account for additional Legionella that may be growing in the plumbing and showerhead due to biofilm release or the presence of amoeba [36]. There have also been reported differences in Legionella concentrations between the cistern and the in-home faucet due to fluctuations in water age and in uncertain chlorine residuals from chlorinated cisterns, causing pipes to act as Legionella reservoirs [31].…”

Assessment of Viability of Legionella Pneumophila in Environmental Samples: On Filter Application of Ethidium Monoazide Bromide

“…Using this method, however, the results, expressed in Colony Forming Units (CFU), can be reported after long time, usually 11 days after the beginning of the analysis. In addition, an underestimation of Legionella species may occur because culture method detects only living bacteria and can be in uenced by the presence of interfering bacterial ora (Leoni et al 2001, Collins et al 2015, Kirschner et al 2016, Collins et al 2017, De Giglio et al 2020. Otherwise, qPCR strengths regard i) the ability to amplify and simultaneously quantify the DNA of the species of Legionella, expressing the results in Genomic Units (GU), ii) the rapid response times from the beginning of the analysis (hours instead of days) and iii) the capacity to also detect VBNC bacteria (Fittipaldi et al 2011 (EMA), a uorescent dye capable of crossing damaged cell membrane of dead bacteria and covalently binding to their DNA.…”