Transformed Legionella for application in engineering process validation in the built environment

Legionella pneumophila is a waterborne pathogen that causes Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. In a culture-based technique, Legionella is detected using buffered charcoal-yeast extract (BCYE) agar supplemented with L-cysteine, Iron salt and antibiotics. These supplements provide essential and complex nutrient requirements and help in the suppression of non-target bacteria in Legionella analysis. Legionella occurs naturally in freshwater environments and for their detection; a sample is plated on solid agar media and then incubated for several days. There are many challenges in the detection of Legionella in environmental waters and the built environments. A common challenge is that a variety of environmental bacteria can be presumptively identified as Legionella using the culture-based method. In addition, proper identification of Legionella requires long incubation period (3-9 days) while antibiotics used in BCYE agar have relatively short half-life time. In order to overcome some of the challenges, Legionella has been genetically modified to express reporter genes such Green Fluorescent Protein (GFP) that can facilitate its detection in process validation studies under controlled laboratory conditions. However, such studies had limited success due to the instability of genetically modified Legionella strains. The development of a genetically modified Legionella with a much rapid growth rate (1-2 days) in simulated environmental systems (tightly-controlled water distribution system) is achieved. The mutant Legionella is engineered by transforming with a specific plasmid encoding CymR, LacZ and TetR genes. The newly engineered Legionella can grow on conventional BCYE agar media without L-Cysteine, Iron salt and only require one antibiotic (Tetracycline) to suppress the growth of other microorganisms in media. To the best of our knowledge, this is the first report of L. pneumophila strain capable of growing without L-Cysteine. We believe that this discovery would not only facilitate the study of the fate and transport of this pathogen in environmental systems, but also further our understanding of the genetics and metabolic pathways of Legionella.