Lessons learned from a one-year study of Legionella spp. cultivation from activated sludge samples

Risk assessment and management of Legionella spp. contamination in activated sludge in wastewater treatment plants is carried out using the culture method. Underestimation of Legionella spp. is frequently reported in the literature, but a comprehensive long-term study of the performance of the method under comparable conditions is still lacking. The aim of this study is to evaluate the recovery rate and limit of detection of the culture method for Legionella spp. from activated sludge samples collected during the different seasons of the year. Activated sludge samples spiked with Legionella pneumophila subsp. pneumophila strain Philadelphia-1 (mean concentration 5.2 ± 0.35 logCFU/mL) were analysed monthly for one year using the culture method. Three different sample pre-treatments were compared, namely filtration, acid treatment and thermal treatment, and the recovery rate and limit of detection were assessed for each. The recovery rate of the culture method for Legionella spp. depended on the type of sample pre-treatment and the season of activated sludge sampling, while the limit of detection depended only on the sample pre-treatment. The best performance of the culture method, defined as the combination of the highest recovery rate and lowest limit of detection, was obtained for the filtered acid pre-treated samples (recovery rate: 89 ± 4 %; limit of detection: 1.3 logCFU/mL in 83 % of the samples). The lowest limit of detection was observed for the filtered thermally pre-treated samples (1.0 logCFU/mL in 93 % of the samples). Simultaneously, both thermally pre-treated samples showed up to a third lower recovery rates than the other pre-treatments in winter, while untreated and acid pre-treated samples showed consistently high recovery rates (>80%, logCFU/mL). The recovery rates of the unfiltered and filtered thermally pre-treated samples showed significant weak to strong positive correlations with the organic and phosphorus load in the influent as well as with the water and atmospheric temperatures, indicating that the recovery rate depends on the seasonal variation of the wastewater composition. This study presents new insights into the detection and quantification of Legionella spp. in activated sludge samples and considers seasonal dependencies in analytical results.