Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

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.

Keywords: ISO 11731:2017; Legionella pneumophila; culture method; molecular detection; transport media.

Figure 1

Scheme for the preparation of bacterial suspensions in SRK™ medium with final Lp concentrations of approximately 10⁴, 10³ and 10² CFU/mL. Bacteria recovery using culture (tube A) and molecular methods (tube B) was evaluated at time 0, 24 and 48 h (T0, T24, T48) following storage at 20–25 °C and at time 0, 24, 48 and 72 (T0, T24, T48, T72) hours at 2–8 °C, respectively.

Figure 2

Genomic units/sample detected from 10⁴, 10³ and 10² CFU/mL dilutions stored at (a) 2–8 °C and (b) 20–25 °C using iQ-Check^® Quanti Legionella spp. Real-Time assay.

Figure 3

Genomic units/sample detected from 10⁴, 10³ and 10² CFU/mL dilutions stored at (a) 2–8 °C (b) and 20–25 °C using qualyfast^® Legionella qPCR detection Kit.