Development of fluorescent in situ hybridization for detection of L. pneumophila using solid phase laser cytometry

Abstract

Legionella pneumophila was first identified and documented after a pneumonia outbreak during an American Legion convention held in Philadelphia in 1976, with 25 casualties. L. pneumophila is a gram negative bacillus found mostly in fresh water environments. It is known as a facultative intracellular parasite of protozoa in its natural habitat, and can also infect macrophages in humans when contaminated aerosols are inhaled. The current detection procedure for L. pneumophila is a culture based method, which is slow and may not detect viable but non-culturable cells; presence of L. pneumophila usually takes 3 to 10 days to confirm by this method, due to its slow growth. New more reliable, rapid and sensitive methods are needed. In this research, a method coupling fluorescent in situ hybridization (FISH) and ScanRDI solid phase laser cytometry was developed to detect L. pneumophila relying on growth with extended incubation to obtain cultures. Published FISH methods were optimized by addition of HCl and Tween 20 incubation, dextran sulfate, helper probes and tyramide signal amplification to increase fluorescence intensity of probed L. pneumophila. ScanRDI settings were also adjusted to be able to detect TSA-FISH probed L. pneumophila. The resulting TSA-FISH method coupled with ScanRDI was able to detect almost 90% of L. pneumophila cell compared to plate counts. This optimized TSA-FISH method was also used on Escherichia coli and Aeromonas hydrophila; the ScanRDI detected 79.5% and 39.7% of these organisms respectively compared to plate counts. The results suggested that L. pneumophila can be detected and enumerated using the method developed in this research. It can be completed within 12 hours, which is a significant improvement compared to at least 3 days for current detection methods. The TSA-FISH method can be used for detection and enumeration of at least 2 microorganisms, L. pneumophila and E. coli. For reliable detection and enumeration of other organisms such as A. hydrophila, procedures need to be further optimized and ScanRDI settings adjusted for each organism. It is anticipated that several microbes could be detected on one membrane filter using appropriate probes, procedures and ScanRDI settings.