UV-light fluorescence as a confirmation method for presumptive Legionella colonies isolated from water samples

Legionnaires' disease (LD) is a severe and potentially fatal form of bacterial pneumonia caused by Legionella spp. We evaluated the use of UV-light for detecting Legionella non-pneumophila in water samples according to the NEN-EN-ISO 11731:2017 methodology (reference method) in a collaborative effort involving 10 laboratories. First, a test panel was constructed of 298 strains: 157 Legionella strains and 141 non-Legionella strains were cultured on buffered charcoal yeast extract (BCYE)-medium and confirmed according to ISO 11731: 2017 (cultured on BCYE agar plates with and without l-cysteine), and by matrix-assisted laser desorption-ionization time of flight or next generation sequencing. All strains were additionally exposed to an UV-light to assess if they showed a bright blue fluorescence effect (UV-positive) or not (UV-negative). Second, in an interlaboratory study, 10 laboratories analyzed a blinded set of 16 Legionella strains and 8 non-Legionella strains using both methods. The test panel analyses showed 100% accordance between the UV-light and reference method. In addition, the interlaboratory study results showed full agreement between both methods. Our results support the implementation of UV-light detection to confirm Legionella presumptive colonies during analyses of water samples according to the NEN-EN-ISO 11731:2017 methodology. Implementation of UV-light confirmation could reduce workload, time-to-result and costs for the analyses of water samples for the presence of Legionella.