Detection of Legionella pneumophila in a biological treatment plant by co-cultivatiobn with Acanthamoeba castellanii

Abstract

Legionella pneumophila was identified in the aeration ponds of a biological wastewater treatment plant at the pulp and paper industry Borregaard, Sarpsborg, Norway. After 3 outbreaks of Legionaires’ disease reported in this area in 2005 and 2008, the aeration ponds were shut down by the Norwegian authorities in September 2008. During the shutdown of these ponds, September to December 2008, the viable counts of L. pneumophila decreased from 107 to < 10 CFU/mL measured using the International Standard growth (ISO11731) method. The aim of this work was to use amoebal coculture with Achantamoebae castellanii to recover and detect L. pneumophila from the complex microbial community in the pond during the shutdown period. This work shows that the viable counts of the environmental L. pneumophila ST 462 outbreak strain present in the pond samples during shutdown, was increased from 0-10 CFU/mL (no amoebae added) to 107 -108 CFU/mL in co-culture with A. castellanii. This indicates that pathogenic L. pneumophila isolates present in the environment may not be detected using standard culture methods. As a consequence, methodological improvements are needed to ensure more reliable detection and isolation of Legionella. By using amoebal co-culture, the concentration of L. pneumophila increased by 5-7 log units, allowing low concentrations and bacteria not detected using standard growth methods (according to the ISO11731), to be detected. Cells in the viable but non-culturable (VBNC) form will not be detected using the ISO 11731 standard culture method, and growth on agar media may be inhibited by other organisms and inhibitors present in complex environmental samples. The methodological procedure described in this paper may assist in providing a general more robust and sensitive approach to detect L. pneumophila in more complex environmental samples and may assist in providing improved hazard assessments.