Legionella pneumophila sp. nov.: 

 The Legionnaires' Disease Bacterium 



Don J. Brenner. Arnold G. Steigenvalt. 

 G. Ann Hebert, and Joseph E. McDade 



In the 2 years since the outbreak in Philadelphia (7) that brought Legionnaires" disease 

 worldwide attention and led to the isolation of the Legionnaires" disease bacterium (LDB) at the 

 Center for Disease Control (7/). intense effort has been focused on understanding the "new" 

 disease and the "new"' bacterium. We have learned that the LDB is a gram-negative, weakly 

 oxidase-positive. catalase-positive, fastidious organism that has narrow optimal pH and tempera- 

 ture ranges and will not grow anaerobically. It does not reduce nitrates, utilize carbohydrates, 

 degrade urea, or appear to possess decarboxylases for lysine and ornithine or an arginine dihydro- 

 lase (79). It has a unique fatty acid profile and no major differences in cellular fatty acids among 

 strains (75). Four different serogroups have been identified (6, 12). 



Deoxyribonucleic acid (DNA) hybridization has proved extremely useful in the classifi- 

 cation of bacteria (7. 8. 76. 7 7). We have characterized the DNA of LDB by genome size, guanine 

 plus cytosine (GC) content, and DNA hybridization in order to determine the extent of related- 

 ness between strains of LDB and possible relatedness of the LDB to other microorganisms. 



DNA relatedness studies were first done before biochemical data were available for LDB and 

 before its GC composition had been detemiined. Thus the initial experiments were designed to 

 rule on the possibility that the LDB was a fastidious Klebsiella or another fastidious member of 

 Enterobacteriaceae. Since DNA relatedness studies have previously been done on all described 

 species within Enterobacteriaceae (7, 2. 3). we were able to test possible relatedness between the 

 LDB and Enterobacteriaceae. DNAs from Philadelphia 1 and Flint 1 were reacted with labeled 

 DNA from Escherichia coli K-12, Proteus mirabilis 1. Yersinia enterocolitica 498-70, and Ed- 

 wardsiella tarda 3889-64 (Table 1). Relatedness was between 0% and 3% (5). All members of 

 Enterobacteriaceae are at least 15% related to one or more of these four species (7. 2, 3). 

 Therefore, the LDB is not a member of Enterobacteriaceae. Relatedness values below 5% are not 

 considered to be significant. Similar experiments proved that LDB had no relatedness to Staphy- 

 lococcus epidermidis, Bordetella pertussis, Aeromonas hydrophila, and Vibrio cholerae (Table I). 

 Although Vibrio parahaemolyticus and Vibrio alginolyticus were not tested directly (Table 1), 

 these and some other vibrios were ruled out on the basis of their known DNA relatedness to V. 

 cholerae. 



Preliminary approximations, using optical thermal denaturation, indicated that the GC con- 

 tent of LDB DNA was slightly lower than that o( Proteus mirabilis. GC content in strains of P. 

 mirabilis is 39% to 42%. Quantitative determinations of GC content by optical thermal de- 

 naturation (9) and by CsCI buoyant density ultracentrifugation (70) showed that both Phila- 

 delphia 1 and Flint 1 DNA contained 39% GC. With this knowledge we were able to restrict DNA 

 relatedness tests mainly to those organisms with known GC values between 35% and 43%. 



Highest priority was given to detemiining relatedness between LDB and the organisms 

 shown in Table 2. Rochalimaea (formerly Rickettsia) qui)itana, which has a GC content of 37%, 

 was given high priority because its pattern of growth on artificial media is comparable to that of 



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