Evaluation of Legiolert for Quantification of <i>Legionella pneumophila</i> from Bath Water Samples

Abstract

Monitoring of Legionella pneumophila populations in environmental water is very important for public health, because the bacterium is the primary cause of Legionnaires' disease. The conventional plate culture method, similar to ISO 11731 (2017) , is generally used to enumerate Legionella spp. in water samples. The method, however, is highly complicated and requires sample handling and colony identification skills due to the growth of non-target bacteria and fungi on the agar plates. Thus, the development of a simpler method that could deliver accurate enumeration results is urgently needed.The Legiolert ® / Quanti-Tray ® most probable number (MPN) method (IDEXX Laboratories, USA) is a novel method to enumerate L. pneumophila in potable (e.g., tap water) and non-potable (e.g., cooling tower water) water samples. According to the manufacturer's instructions, the principle of this method is based on a bacterial enzyme detection technology that signals the presence of L. pneumophila through utilization of a substrate present in the Legiolert reagent. L. pneumophila cells grow rapidly and reproduce using the rich supply of nutrients present in the Legiolert reagent. Actively growing strains of L. pneumophila use the added substrate to produce a brown color indicator. There are four different protocols in the manufacturer's instructions, two for potable water and the other two for non-potable water. Recent papers have reported method comparison studies for the enumeration of L. pneumophila between Legiolert and the conventional plate culture method from potable water, such as hot water taps, showers, and ice machines (Sartory et al., 2017;Petrisek and Hall, 2017;Spies et al., 2018) and non-potable water, such as cooling tower water (Petrisek and Hall, 2017;Rech et al., 2018) . In this paper, we report the first comparison of the enumeration of L. pneumophila from bath water samples using the two Legiolert potable water methods and the conventional plate culture method.All water samples were collected from natural hot springs (53 samples) and public baths using tap water (79 samples) between May 2018 and March 2019. These samples were collected in sterile 500-mL polypropylene bottles with sodium thiosulfate, and were examined within 3 d after sample collection.The Legiolert/Quanti-Tray MPN method was carried out according to manufacturer's instructions. We compared the two potable water protocols (10 mL and 100 mL protocols) described in the instructions. In the 10 mL protocol (Legiolert-10 mL) , 10 mL of each water sample was transferred into a sterile 120 mL vessel and diluted with 90 mL of sterile deionized water. In the 100

Comparison of two culture methods for the enumeration of Legionella pneumophila from potable water samples

“…Particularly, all 9 false positive samples were taken from small wells. Relatively high specificity of Legiolert is comparable to other published studies ( Sartory et al 2017 ; Petrisek & Hall 2018 ; Spies et al 2018 ; Inoue et al 2020 ; Scaturro et al 2020 ). It should be noted that those studies performed secondary confirmations by sub-culture.…”

“…The manufacturer claims that there is no need for further verification for L. pneumophila due to the high selectivity against non-pneumophila species. More recently, research groups from Europe and the United States have reported that Legiolert yielded a higher sensitivity (i.e., higher counts of correctly identified positive samples) and had a higher specificity (i.e., higher counts of correctly identified negative samples) for L. pneumophila compared with the conventional BCYE agar method ( Sartory et al 2017 ; Petrisek & Hall 2018 ; Spies et al 2018 ; Inoue et al 2020 ; Scaturro et al 2020 ). While this method shows promising practical applications (i.e., simplistic and less laborious) over the traditional culture method, further testing against various types of environmental water samples is necessary to ensure its reliability.…”

Legionella pneumophila recovery using Legiolert and a traditional culture method

“…This study confirms results reported from 10 previous studies using Legiolert, indicating this method works as well or better than BCYE‐based methods for recovery and detection of L. pneumophila in water samples: cooling towers (Barrette, 2019); drinking water in distribution systems (LeChevallier, 2019a, 2019b); public bathing water and hot springs (Inoue et al, 2020), potable and nonpotable (Monteiro et al, 2021; Petrisik & Hall, 2018); nonpotable, cooling towers (Rech et al, 2018); building potable and nonpotable (Sartory et al, 2017); potable water (Scaturro et al, 2020); and building potable and nonpotable (Spies et al, 2018).…”

Cross‐reactivity of the IDEXX Legiolert method with other Gram‐negative bacteria and waterborne pathogens leads to false‐positive assay results

“…One study using Legiolert in water distribution systems reported false positives due to Brevundimonas vesicularis, B. diminuta, Sphingomonas koreensis, Ochrobactrum intermedium and Elizabethkingia anophelis (LeChevalier 2019). In public bath water samples, false positivity was noted for Legiolert with two Pseudomonas species (P. otitidis and P. mendocina) and S. marcescens (Inoue et al 2020). These organisms, and other species of these genera, can be regularly found in drinking water and also be opportunistic human pathogens of potential concern to immunocompromised individuals (Koskinen et al 2000;Szymanska 2007;Ryan and Adley 2010;Lee et al 2011;Lau et al 2016;Bharucha et al 2017;Kyritsi et al 2018).…”

“…otitidis and P. mendocina ) and S. marcescens (Inoue et al . 2020). These organisms, and other species of these genera, can be regularly found in drinking water and also be opportunistic human pathogens of potential concern to immunocompromised individuals (Koskinen et al .…”