Microbiology of Legionnaires' Disease Bacterium HENRY D. ISENBERG, Ph.D.; New Hyde Park, New York
Legionnaires' disease bacterium in tissue does not readily react with the Gram stain but can be seen by other stains and direct immunofluorescence. It is a slow-growing, aerobic, gram-negative rod that can be cultivated over a narrow temperature range on Mueller-Hinton agar supplemented either with complex biological mixtures or certain ferric salts and cysteine. The bacterium produces unique, branched-chain fatty acids, catalase, oxidase (weakly), and gelatinase and uses starch while ignoring other carbohydrates. Pigment production is related to tyrosine in the medium. In-vitro studies suggest susceptibility to all antibiotics except vancomycin, but a class 1 beta-lactamase has been demonstrated. Analysis of DNA confirmed the unrelatedness of this bacterium to previously recognized prokaryotes. Diagnosis of the disease has depended largely on serologic test findings and the demonstration of the bacterium in tissue and, occasionally, on isolation. Additional, simpler, and more rapid diagnostic tests should soon be available.
T H E ISOLATION of a hitherto unknown bacterium from the lungs of patients with Legionnaires' disease (1) surprised the scientific community. Since World War II, new environmental prokaryotes have entered the intimate human biosphere, largely as a result of various medical and pharmacologic manipulations of patients compromised by disease or as part of treatment for life-threatening ailments. The discovery of the Legionnaires' disease (LD) bacterium marks a significant moment in the history of microbiology and infection because it underlines the inadequacy of our collective understanding of infectious • F r o m the Long Island Jewish-Hillside Medical Center; New Hyde Park, New York.
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disease, the mechanisms of the host-parasite equilibrium, and the role of our environment as a reservoir for microorganisms with the potential to challenge a segment of the population. Subsequent to the initial characterization of L D bacterium, it has been demonstrated that the bacterium is and has been involved in other outbreaks and sporadic cases of pneumonia, adding to the mystery of how it has escaped laboratory definition for so long. Tinctorial Properties
The best excuse for the microbiologist for this failure is the inability to demonstrate L D bacterium in lungs or other organs by the usual tissue-adopted modifications of the Gram stain (2). One might suggest that the significance of the successful isolation of L D bacterium from pleural fluid by Dumoff and his group (3) would not have been recognized had it preceded the Philadelphia outbreak and the availability of some appropriate laboratory tools. Numerous L D bacteria have been demonstrated by the Dieterle impregnation method (2) and direct immunofluorescent staining (4) in the very tissues that failed to yield L D bacteria by G r a m stain. Such a consistent failure of large numbers of gram-negative bacteria in tissue to react properly has not been observed previously. All available theoretical explanations of this incompletely understood stain do not account for the inability of L D bacterium to react (5). One could postulate that the fixation process altered the ability to the dyes to react properly, which would imply a singular intracellular pH for L D bacterium. An alternative explanation would require ©1979 American College of Physicians
the organism to be coated with high molecular-weight substances, usually implicated in a false-positive reaction of gram-negative bacteria. In this instance, a high molecular-weight bacterial outer layer may interact with host cell materials to prevent contact between the bacterium and the dyes. Thus, the inability of L D bacterium to react with the Gram stain in tissue may provide scientists with an opportunity to investigate the basic mechanisms of this important tinctorial procedure. In tissue sections stained with the Dieterle method (2), L D bacteria appear as short pleomorphic rods measuring 2 to 4 jam in length and up to 1 jum in diameter. Some have beading or appear bipolar. Direct immunofluorescent staining, especially when used on scrapings of formalin-fixed lung tissue, shows numerous L D bacteria intracellular^ and extracellularly from severely ill or deceased patients (4), leading to a set of criteria applicable to fresh tissue or fluid specimens from the lower respiratory tract and to the scrapings or slides of formalin-fixed preparations. However, L D bacterium in fresh tissue was not as readily discernible as in formalin-fixed preparations, possibly because of "envelopes" that obscure their outline. The Gimenez stain can be applied to fresh but not fixed tissues and to preparations from eggs. The great specificity of the fluorescent antibody reagent, which did not react with 374 bacteria representing 59 known species, 25 known genera, and 54 unidentified cultures, makes this procedure the most diagnostic of the tinctorial modalities applied to date. Recently a culture of Pseudomonas fluorescens has been shown to react with a conjugate (5). While morphologic differences between the two bacteria prevail, this observation must serve as a caveat and a reminder that other confirmatory examinations must be applied for correct diagnosis. With direct fluorescent antibodies, the usually numerous bacteria in tissue sections have varying structures due to the thickness of the sections and the orientation of the individual bacterium within host structures. A more uniform appearance is seen in scraping preparations and especially in smears from cultures. The structure of the bacteria as pleomorphic rods that vary in size and occasionally form filaments can be quite readily seen. In lung tissue, Cherry (6) pointed out that extracellular material of bacterial origin reacts with the fluorescent antibodies. This material may play a role when future tests for rapid diagnosis are devised. Fine Structure of LD Bacterium
Fine structure studies of L D bacterium in human and animal tissues show typical prokaryotic morphology. The cell wall appears thin and occasionally wavy. Many of the L D bacteria have inclusions identified as intracellular fat that probably correspond to the sudanophilic vacuoles seen with light microscopy (7). Unfortunately, one group of observers (8, 9) has described certain structures visualized in electron micrographs as spores. These structures were seen frequently in one preparation obtained from yolksac cultures, rarely in a second, and not at all in the three remaining eggs included in the study. Since many degenerating bacteria or shadow forms were seen in these
preparations, one might explain the sporelike structures as fusions of shadow forms. No spore forms have been observed by any other investigators, not even in old cultures where such forms would be expected and readily detected by light microscopy. Since no bacteriologic quality control data accompanied the report of spores, contamination by a spore-forming bacterium cannot be ruled out. These authors alone have also reported L D bacterium as gram-variable. Growth Requirement
Once the prokaryotic nature of L D bacterium was established, attempts to define its in-vitro growth requirements were initiated (10), aided by the isolation of the bacterium in a clinical laboratory on an IsoVitaleX®-hemoglobin enriched G C agar base (Baltimore Biological Laboratories [BBL], Cockeysville, Maryland) (3). Using yolk-sac grown L D bacteria representing Philadelphia isolates 1 and 2, Flint isolate 1, and Pontiac isolate 1, Mueller-Hinton agar (BBL), supplemented with IsoVitaleX and hemoglobin (MH-IH) supported growth. Careful analysis of all constituents of this combination led to the formulation of a new medium designated F-G agar (10), consisting of acid hydrolyzed casein, beef extracts, L-cysteine hydrochloride, soluble ferric pyrophosphate, starch, and agar. The L-cysteine hydrochloride and ferric pyrophosphate substituted for IsoVitaleX and hemoglobin, respectively. None of the other IsoVitaleX constituents (adenine, p-aminobenzoic acid, cocarboxylase, L-cystine, glucose, nicotinamide adenine dinucleotide, ferric nitrate, glutamine hydrochloride, guanine hydrochloride, thiamine hydrochloride, and vitamin B12) were essential for the growth of L D bacterium when used individually to supplement Mueller-Hinton agar enriched with hemoglobin or when each chemical was deleted from the combinations tested. Ferric pyrophosphate met the increased iron requirements of L D bacterium better than hemoglobin, ferric nitrate, or ferric phosphate; the latter two compounds supported better yields than did hemoglobin or other iron salts. The requirement for the proper peptone was quite narrow. Only Bacto-tryptone® (Difco Laboratories, Detroit, Michigan) and biosate (BBL) or acidicase (BBL) combined with beef extracts (BBL) supported growth of the bacterium adequately. A 2 . 5 % C 0 2 concentration was optimal for growth. Reduction of 0 2 decreased the growth of the organism while anaerobic conditions abolished proliferation. Tolerance of L D bacterium for pH variations was confined to p H 6.9 and 7.0. The optimal growth temperature was 35 °C; slight growth occurred at 29 °C, but none was observed at 25 °C or 42 °C. In broth, better results were achieved with ferric pyrophosphate levels reduced from 0.025% to 0 . 0 1 % . Legionnaires' disease bacterium grew better and faster on FG agar than on M H - I H , requiring only 4 days for macroscopically discernible pinpoint colonies that increased in size with age. The colonies had a characteristic cut-glass appearance when observed microscopically. In areas of heavy growth, a brown pigment was formed. In addition, L D bacterium produced a fluorescent substance on F-G Isenberg
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agar that involved the colonies and the surrounding agar, detectable with long-wave (366 nm) ultraviolet light. A simpler growth medium has also been described (11) consisting of yeast extract, soluble starch, NaCl, L-cysteine hydrochloride, ferric pyrophosphate, and agar at pH 6.9. Discernible growth of LD bacterium is manifest in 3 days, but no pigment is produced on this agar even after 1 week's incubation. The tyrosine content of the yeast extract in the concentration used is only 100 mg/L. Increasing the amino acid concentration to 550 m g / L resulted in comparable pigment production. Thus, LD bacteria may join microbial genera such as Mycobacterium, Pseudomonas, and Streptomyces that produce melanin by enzymatic oxidation of tyrosine. Gas chromatographic analysis of several representatives (12) revealed close correspondence between LD bacterium isolates from different sources. Results indicated an inordinately high content of branched-chain fatty acids (81% to 90%), whereas the usual bacterial fatty acids were not detected. Therefore, the fatty acid profile could be an important tool for the recognition of LD bacterium. Only two other gram-negative bacteria are known to contain branched-chain fatty acids in appreciable quantity—Therm us aquaticus and a thermophilic Flavobacterium—but the profile of LD bacterium is unique. Physiologic Activity
Biochemical and physiologic studies indicate that LD bacterium is a somewhat fastidious, gram-negative bacterium that produces catalase, is weakly oxidase positive, uses starch, and elaborates gelatinase. No nitratase or urease activities have been found, while the use of carbohydrates thus far is confined to starch (13). Molecular Biology
In an attempt to find a proper taxonomic niche for L D bacterium, Brenner and associates (13) applied the critical technology of D N A hybridization to the problem, characterizing the organism by genome size and guaninecytosine content as well as hybridization studies to establish the extent of relatedness between strains of LD bacteria and other bacteria. They included four isolates from Philadelphia and those from Pontiac, Flint, Vermont, Knoxville, Bellingham, Albuquerque, and Berkeley. Other bacteria included Escherichia coli, Proteus mirabilis, Yersinia enterocolitica, and Edwardsiella tarda, the representatives of Enterobacteriaceae that cover the relatedness within the family. In addition, Vibrio cholerae, Aeromonas hydrophila, Staphylococcus epidermidis, Bordetella pertussis, Rochalimaea quintana, Flavobacterium species, and Pasteurella multocida were used. These investigators established that the guanine-cytosine content of LD bacterium D N A is 39%, restricting D N A relatedness tests to bacteria with similar ratios. The studies revealed LD bacterium D N A has a molecular weight of about 2.5 X 109 daltons, sufficient genetic information to specify some 3000 genes corresponding in magnitude to the potential of E. coli K-12 D N A . It has been accepted by molecular biologists that members of the given species are at least 70% related in hybridization studies, 504
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25% to 60% if they belong to the same genus, while the level of total lack of relatedness is at 5% or less. Under the most stringent conditions, all strains of LD bacteria behave as a single species. The most divergent strain was Pontiac 1, which, understandably, led these investigators to speculate on the relation between severity of disease and the relatedness of the strains. Hybridization studies with other bacteria indicated total lack of relatedness between LD bacteria and the bacteria studied. Guided by the restrictions gained by D N A analysis, the isolation of LD bacteria from the environment, and the demonstration of bacteria reactive with direct fluorescent antibodies in homogenates of soil invertebrates and lower vertebrates (CHERRY WB: Personal communication), the search for taxonomic connections will include Cytophaga, Flexithrix, Flexibacter, and Microcyclus, gram-negative bacteria from soil with suitable guanine-cytosine content. Response to Antibiotics
The LD bacterium response to antibiotic andchemotherapeutic agents was studied in guinea pigs (14), eggs (15), and in vitro by agar dilution method (16). The laboratory profile shows the various strains susceptible to most agents and resistant only to vancomycin, a potentially helpful observation in attempts to formulate selective media. Thornsberry and Kirven (17) have demonstrated the presence of a class 1 beta-lactamase, that is, a cephalosporinase with penicillinase activity, in nine isolates of LD bacteria, an observation significant in therapeutic considerations. The favorable susceptibility established by the laboratory studies is not reflected in the in-vivo models. Thus in guinea pigs (14), only erythromycin and rifampin prevented death of the animals infected intraperitoneal^. The superiority of these two agents was confirmed in chick-embryo experiments (15), useful information for the treatment of patients. Diagnostic Procedures
At present, the only readily applicable diagnostic laboratory procedure for the diagnosis of epidemic or sporadic Legionnaires' disease is the indirect fluorescent-antibody test (1), a technically difficult procedure (18). With paired sera (acute and convalescent), a fourfold rise in titer to 1:128 or greater is considered diagnostic. When only convalescent sera are available, a titer equal to or greater than 1:256 is required. Recently, a microagglutination method with heat-killed LD bacteria and a microenzyme-linked immunosorbent assay using buffer extracted antigen have been described (18). Although only a limited number of sera have been tested, both tests hold promise in diagnosis of the disease, especially the microagglutination method, which represents technology widely used in clinical microbiology laboratories. Suggestions
The present state of knowledge on this bacterium can form the basis for the development of rapid diagnostic tools. While such efforts are under way with serodiagnostic tests, media selective for LD bacteria are needed to
help detect the bacterium in polymicrobic specimens. This may mean the search for organic and inorganic inhibitory or enrichment-type compounds useful in sequestering L D bacterium from companion microorganisms. Although the D N A studies prove that LD bacterium is a single species, the question of the degree of antigenic relatedness has not been answered. The suggestion that the severity of disease may be related to a particular strain underlines the need for some biotyping mechanism. Such an effort may make use of antigenic differences, fatty acid analysis, or biochemical and physiologic properties as yet undetermined. Information on the experience of populations vis-a-vis LD bacterium should lead to the identification of susceptible persons in a given group and an appreciation of the host factors that permit overt clinical disease. Along these lines, the considerable iron requirement of L D bacterium may be a clue to understanding the pathologic process of LD bacterium infection in susceptible patients (19). The thin cell wall and the presence of branched-chain fatty acids may play a role not only in the tissue tinctorial problems but also in the production of capsular or extracellular materials that may be extremely chemotactic in addition to inflicting intracellular injury to macrophages and other cell lines. While the chemistry and physics of the host-parasite interactions in pneumococcal, mycoplasmal, or any other pneumonia have not been elucidated, L D bacterium offers the rare opportunity to understand disease production at the cellular level by investigating the various host-cell receptor sites and their reaction with LD bacterium. The distribution of LD bacterium in the environment and its role in natural hosts must be assessed to determine if it exists in nature in a more infective form than is encountered in human tissues. The lack of person-fo-person contagion observed thus far suggests the possibility of a primitive cycle in which the infective, environmental bacteria differ in some way from the seemingly nontransmissible forms in the diseased host. Finally, the discovery of LD bacterium reinforces the need for a re-examination of established definitions of pathogenicity, the mechanisms of host-parasite interactions, and the role of the autochthonous and environmental microbiota in the complex relationship of health and disease (20). • Requests for reprints should be addressed to Henry D. Isenberg, Ph.D.;
Department of Laboratories, Long Island Jewish-Hillside Medical Center; New Hyde Park, NY 11040. Received 15 November
1978; revision accepted 8 January 1979.
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