Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

Marianna Martinelli,Sergio Frugoni,Rosario Musumeci,Federica Perdoni,Maria Scaturro,Enrico Calaresu,Maria Luisa Ricci,Santina Castriciano,Chiara Giubbi,Clementina E Cocuzza

doi:10.3390/ijerph18168551

Abstract

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Highlights

Legionella spp. are aquatic bacteria that are ubiquitously found in nature, in both anthropogenic structures and in environmental waters [1]
Molecular methods allowed the quantification of all three bacterial suspensions in SRKTM medium (104, 103 and 102 CFU/mL) as genomic units (GU)
The results of this study demonstrated the high sensitivity and reproducibility of both molecular methods investigated in this study, indicating that the composition of the SRKTM medium does not affect the stability and conservation of bacterial nucleic acids and does not interfere with the amplification reagents used in the assays

Summary

Introduction

Legionella spp. are aquatic bacteria that are ubiquitously found in nature, in both anthropogenic structures and in environmental waters [1]. The most common pathogenic species is Legionella pneumophila (Lp) serogroup 1 and is responsible for up to 80% of Legionnaires disease (LD) cases [2]. The exact incidence of LD worldwide is unknown because countries differ greatly in the methods used to ascertain whether someone has the infection and in reporting known cases [3]. Environmental sampling for the detection of Lp represents an important tool to obtain data of epidemic risk assessment and to provide remedial interventions. Monitoring water systems involves choosing sampling sites, and the number and type of samples to be obtained (water and/or biofilm), as well as the sampling method to be used [5]

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Conclusion