LONG-TERM SURVIVAL OF LEGIONELLA-PNEUMOPHILA SEROGROUP-1 UNDER LOW-NUTRIENT CONDITIONS AND ASSOCIATED MORPHOLOGICAL-CHANGES

Extended survival of Legionella pneumophila, using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEalpha). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 x 10(8) cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 x 10(6) cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10(2) (cfu/ml) over the same period. After incubation for 1.5 years at 15-degrees-C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEalpha. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEalpha, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEalpha. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.