Autecology of Legionella pneumophila

Autecology of Legionella pneumophila

Zbl. Bakt. H yg., I. Ab t. Orig. A 255, 58- 63 (1983) Autecology of Legionella pneumophila CARL B. FLIERMANS Savannah River Laboratory, E.I. du Pont ...

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Zbl. Bakt. H yg., I. Ab t. Orig. A 255, 58- 63 (1983)

Autecology of Legionella pneumophila CARL B. FLIERMANS Savannah River Laboratory, E.I. du Pont de Nemours & Co., Aiken, South Carolina, USA

Abstract Species of Legionella are readily isolated from freshwater aquatic habitats throughout the United States,which are diverse in their physical and chemical parameters. Temperature studies indicate that Legionel/a is not present in habitat s with temperatures above 63 °C, and isolates have been obtained from frozen rivers. The presence of the organism in natural hot springs and man-made thermal habitats suggest that Legionel/a may have been present for a long period of time, but has only recently had man-made thermal habitats suitable to establish niches common to man. Keywords: Autecology, ecological niche, environmental habitats, in situ activity, thermophile. " Is there any thing where of it may be said, See, this is new ? It has already been, in the vast ages of time which were before us." Ecclesiastes 1: 10 Introduction

Legionella is a fascina ting bacteri um th at will tell us man y things abo ut th e world in which we live, provided we have th e ears to hear an d th e eyes to see. The scope of th is contribution is to exp lore th e ecolog ical niche and th e autecology of Legionella pneum ophila with und erstanding sight. Not until recent year s has such a successful marriage betw een microbial ecology and medical microbiology been con sumated. The format for such a marriage was esta blished with Robert Ko ch's work in 1876 on anthrax, a disease of cattle caused by Bacillus anthracis. The use of Koch's postulates develop ed in th e study of medical microbiology is equally applicable to the study of micr obi al ecology . Ko ch's postulates ada pted to microbial eco logy wo uld be defined as the follow ing : 1. The selected microorganism sho uld always be found in th e habitat which is r esponding in a cha rac teristic mann er, and shou ld not be present in high densities in habitats th at do not respond in such a manner. 2. The microorganism must be iso lated and cultivat ed in pure cultu re away from the environment. 3. The isolated culture wh en in ocul ated back into th e enviro nment or a su bsample of th at environment should produce the characte ristics of th e hab itat containing th e microor gani sm. 4. The microo rga nism mu st th en be reisolated from the in oculat ed habitat , cultur ed in the lab or atory, and be th e same microorganism as th e original.

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Koch's contributions set the stage for subsequent successful ecological studies over the past century, and it is from this heritage that the ecological research on Legionella draws its breath.

Overview of Ecological Techniques Although within the aquatic environment a large and diverse number of microorganisms exist, specific bacterial components such as Legionella constitute a very small portion of the total population. In order to determine the presence and density of selected bacteria in a water sample, the sample must be concentrated in such a manner that the bacteria of choice are readily separated and identified from the surrounding milieu. Initially, Fliermans, Cherry, Orrison et al. (1979) and Fliermans, Cherry, Tison et al. (1981) used continuous flow centrifugation to concentrate 500 fold the total microbial population in 22 liters of sample collected from selected aquatic habitats. The advantage of the technique is clearly its ability to concentrate large volumes of water in a relatively short period of time. Continuous flow centrifugation has been used in field sampling in conjunction with the use of a mobile laboratory as well as bringing the samples to a centrally located laboratory for processing (Christensen, Tyndall, Fliermans et al., 1982). An overriding ecological problem with any culturing procedures is that they are basically indirect. Indirect techniques attempt to obtain information about the density and the in situ physiology of Legionella in its natural habitat via indirect means. Only under very rare circumstances, i. e., in extreme habitats, can such indirect procedures be used with any confidence, and then the data are not always clear (Brock, 1978; Tison, Pope, Cherry, and Fliermans, 1980). The growth of Legionella adapted to an aquatic habitat with a myriad of interactions may not have any relation to an agar plate. The use of laboratory media to selectively enrich and differentiate Legionella for the purpose of evaluating the habitat from which the sample came, misses the point of ecological studies. Ecological techniques should be designed to study the organism in its natural habitat, in situ, with the minimal disturbance so that the maximum can be learned both about the habitat and the organism. Thus, learning Legionella ecology through indirect laboratory manipulations is tenuous at best. From the available data it appears that a selective, differential enrichment medium is still to be produced that allows Legionella to express all of its characteristics of growth and survival at elevated temperatures, low oxygen tensions, and wide ranges of pH values. The organism is certainly capable of existing in a wide variety of habitats (Fliermans, Cherry, Tison et al., 1981) as well as growing under experimental conditions in the laboratory that are not produced by any of the presently available media. The direct fluorescent antibody technique (DFA) in conjunction with guinea pig infectivity and subsequent isolation is the most widely used technique for assessing Legionella in natural aquatic systems (Fliermans, Bettinger and Fynsk, 1982; Fliermans, Saracco and Pope, 1981 j Tison, Baross and Seidler, 1982). As with any technique, particularly in the realm of microbial ecology, none are panacea, but the disadvantages are far outweighed, as we have previously discussed (Fliermans, Cherry, Tison et al., 1981). The DFA technique provides the basis for studying

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selected microorganisms in a habit at that is exceedingly complex in its microbial diversity. The ecological study of a particular organi sm in relationship to its habitat is termed aut ecology (Fliermans and Schmidt, 1975). Because Legionella or any other bacterium that remains int act will be detected by the direct fluorescent anti body technique regardle ss of whether it is alive or dead , it is necessary to establish techniques whereby the difference between living and dead bacteria can be assessed and thu s the aute cology determined. The technique using the tetrazolium dye, INT, (1-p-iodophenyl-2-p-nitro-phenyl tetr azolium chloride) in combination with the DFA was developed to answer just this question (Fliermans, Soracco and Pope, 1981). The technique makes use of the fact th at Legionella has an electron transport system as do most all known respiring bacteria (Zimmerman, Iturriaga and Becker-Brick, 1978). The technique of DFA and INT (FAINT) has been used with a number of organisms and has been used in the field extensively with Legionella. Monthly samples taken from the Par Pond System were evaluated for INT activity levels of Legionella with regard to water column position and seasonality. As there appears to be a seasonality to the infection levels of Legionella for guinea pigs (Fliermans, Cherry, Tison et al., 1981), there appe ars to be a greater activity of Legionella in the surface waters. Likewise, a seasonality of electron transport activity may occur with Legionella. Th e technique has provided physiological inform ation in situ that would not have been obtained by any oth er technique. Studies in the field as well as in pure culture using temperature tr ansfer experiments have demon strated th at Legionella doe s indeed have an electron transport system that is active over a wide range of temperatur es (Fliermans, Soracco and Pope, 1981). The INT studies support and help confirm the fact th at Legionella is related to thermophilic bacteria in its ability to survive high temper atures and to remain active over a wide temperature range. It is significant that th e highest temperature hab itat from which Legionella has been isolated is 63 DC, and corresponds very nicely to the upper temperature limit at which th e electron tr ansport system of Legionella functions as measured by the INT technique. Secondly, the isolates obtained fro m the field and those from clinical specimens have th e same response with regard to th eir electron transport activity under temperature tr ansfer experiments. Orrison, Cherry, Fliermans et al. (1981) have demon strated th at the environmental isolates of Legionella pneumophila are essentially identical with the clinical isolates in all regards. Thus there is no a priori reason at present to separate the Legionella from natural habitats as opposed to those from clinical specimens. They are the same organism, capable of the same metabolic and physiological processes, and have the capability of causing a serious infection in the host. Th e use of the INT technique is not an elixir, but does pro vide a techn ique that allows the gath ering of in situ information that would otherwise not be available. Ecological Niche Much of the research th at has been conducted on the ecological distribution of Legionella has been done with the first four serogroups of L. pneumophia. Part of the reasoning behind such a study is histori cal but additi onally, these serogroups have composed a significant fraction of the Legionellosis cases in th e United States

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(Cordes and Fraser, 1981). Our laboratory has been involved with the question of geographic distribution of Legionella since 1978. We have a 35 ft mobile laboratory that affords us the versatility of moving into a variety of environments in order to process samples in the field within minutes of collection so that changes due to storage or shipping are kept at a minimum. Thus, physiological ecology can be performed with the minimum of sample alteration. Water samples have been collected from all parts of the country and processed for the presence and density of Legionella. These samples have been primarily from surface waters, including major rivers and large lakes both natural and man-made, both ambient and thermally altered. The story appears to be consistent with other aquatic bacteria (Hazen, 1979), in that wherever one finds water, one finds Legionella. The initial ecological studies (Fliermans, Cherry, Tison et al., 1981) demonstrated that Legionella was somewhat unusual at least in its temperature relationships, and that non ambient habitats should be considered. The temperature phenomenon was initially studied because of my research and acquaintance with other thermal habitats, particularly those in Yellowstone National Park. Those years of investigation had led the thermophilic community of scientitsts to recognize characteristics of bacteria associated with natural thermal environments. One of these characteristics was the general pattern that thermophilic bacteria possessed a large percentage of branched-chained fatty acids such as Thermus aquaticus, Bacillus caldolyticus and B. caldotenax. Not only is Legionella associated with thermal habitats but we have also isolated the organism from ice covered rivers (Tyndall and Fliermans, unpublished results). Although one of the aims of the ecological research has been to establish a surrogate for Legionella, such an indicator has not been established. Although our studies have centered around man-made thermal habitats, we have sampled some natural warm and hot springs habitats across the United States (Table 1). The data indicate that Legionella is indeed part and parcel of the habitats that are naturally thermal. Many of these natural habitats are alleged to be quite old (Hague, Iddings, Weed et al., 1899) and certainly they are when compared to the man-made habitats of power production facilities. Generally, the samples contained densities of Legionella comparable to those observed in the man-made thermal habitats. Selected naturally thermal habitats have densities two to three orders of magnitude above those observed in the natural nonthermal habitats. Isolations from these natural thermal habitats are more difficult to obtain than from man-made thermal habitats. The data seem to indicate that the man-made thermal habitats are more likely to contain infectious Legionella than the natural thermal habitats. The reason for such findings is unclear but may be related to the cycling of warm water in the man-made aquatic habitats and the stability of natural hot springs (Brock, 1978). Density estimates of Legionella spp. have been made in the Savannah River and its estuary. The data indicate that the waters flowing into the coastal area do contain species of Legionella, but that the densities are reduced once the levels of salinity increase. Samples collected in the open ocean were not even DFA positive for any of the species of Legionella tested. The ecological data are preliminary indications that Legionella may not be tolerant of marine or estuarine environments. The eruption of Mt. St. Helens in Washington State has provided lake conditions in which Legionella may grow. Initially, in 1980, we processed water samples from the various lakes formed or significantly altered during the eruption and demonstrated substantial densities of Legionella in these lakes. Tison, Baross and Seidler

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T abl e 1. Densities of L. pneum ophila in Selected Natural Wa rm and Hot Springs in the United Srates H abitat

Cyanid ium Creek Cyan idium Creek Cyan idium Creek Cyanidium Creek Bath Lake Octopus Lake Oct opus Lake Octo pus Lake Octopu s Lake M ineral Hot Spr ings M adison River T err ace Spring Old Bath O bsidia n Cr eek Springs Amphith eatre Springs

L. pneumcphila /l iter

Loca tion

T emperatur e °C

Knoxville

To gus

YNP YNP YNP YNP YNP YNP YNP YNP YNP CO

42 45 50 55 36 45 50 55 60 54

BD BD BD 2.3 1.5 BD BD 6.8 9.6 5.8

BD BD BD BD BD BD BD BD BD BD

YNP YNP YNP YNP

13 48.5 36.5 35.5

6.5 x lOG 1.1 x 105 BD 8.4 x 104

YNP

31.3

BD

x 108 x 108 x 107 x 107 x 105

Bloom ington BD BD BD BD BD BD BD BD BD BD

Los Angeles

Isolation

BD BD BD BD 4.1 x 107 2.7 x 107 BD BD BD 4.1 x 104

NT NT NT K K,LA Neg Neg K K Neg

1.1 x 105 BD BD BD 2.8 x 10· 1.7 x io7.1 X 104 BD

9.1 X 103 6.6 x 10 8 1.7 x to8.2 x l ot

Neg LA Neg NT

BD

BD

NT

BD

BD = Below Detecti on « 9. 1 X 103 ) . YNP = Yellowstone N atio nal Park.

(1982) have extended these studies and have show n that these habitats cont ain densities of Legionella that are pr obably greater than the levels observed in many natural habitats. Likewise, the distribution of Legionella pneumophila serogroups in Mt. St. Helens habitats are prim arily four and six while other habitats show a preponderance for serogro ups one and four. T errestrial habitats have remained a potential source of Legionella but have not been confirmed as a reservoir for the organism. Numerous researchers have attem pted to isolate Legionella from the terrestrial habitat without success (Cherry, Fliermans and Tiedie, unpubli shed results). It is indeed true that within soil organisms (such as earthworms, arthropods, and the like), a large number of Legionella-like bacteria can be observed by the direct fluorescent antibody technique but the organisms have not been isolated and the necessary confirmat ion is lacking (Cherry, Pittman, Harris et al., 1978). Thus , one is left with the hypothesis that Legionella may be a member of the terre stri al commun ity, but it is in fact not culturable from this "source". The data remain that Legionella is a part of the aqu atic community and has thu s far not proven itself to be part of terrestrial habitats. Finally, one wonders whether th e author of Ecclesiastes had insight into the ecology of Legionella. The ecological data on Legionella from natural hot springs certainly suggests that this bacterium has been associated with aquatic habitats for

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a considerable period of time. Only recently when man has provided extensive man-made habitats suitable for the amplification, growth and dissemination of the bacterium have the niches of man and Legionella had extensive overlap. Therein lies part of the story of Legionnaires' Disease and autecology of Legionella. Acknowledgements: I continue to express my appreciation to W. B. Cherry and R. L. Tyndall for their encouragement and insights into the ecological fascinations of Legionella. These colleagues have had eyes to see and ears to hear the tale Legionella weaves.

References Brock, T.D.: Thermophilic Microorganisms and Life at High Temperatures, pp.465. Springer-Verlag, New York (1978) Cherry, W. B., B.Pittman, P. P. Harris, G. A. Herbert, B. M. Thomason, L. Thacker, and R. E. Weaver: Detection of Legionnaires' Disease bacterium by direct immunofluorescent staining. J. Clin, Microbio!' 8 (1978) 329-338 Christensen, S. W., R. L. Tyndall, C. B. Fliermans, [, A. Soloman, and S. B. Gough: In: Electrical Power Research Institute (Ed.), Legionnaires' Disease bacterium in power plant cooling systems, Interim Report, p. 97 (1982) Cordes, L. G. and D. W. Fraser: Legionellosis, Legionnaires' Disease; Pontial Fever. Med. Clin, N. Amer. 64 (1980) 395 Fliermans, C. B., G. E. Bettinger, and A. W. Fynsk: Treatment of cooling systems containing high levels of Legionella pneumopbila. Water Res. 16 (1982) 903-909 Flierrnans, C. B., W. B. Cherry, L. H. Orrison, and L. Thacker: Isolation of Legionella pneumophila from nonepidemic-related aquatic habitats. Appl, Environ. Microbio!. 37 (1979) 1239-1242 Fliermans, C. B., W. B. Cherry, D. L. Tison, R. B. Smith, and D. H. Pope: Ecological distribution of Legionella pneumophila. Appl. Environ. Microbio!. 41 (1981) 9-16 Fliermans, C. B. and E. L. Schmidt: Autoradiography and immunofluorescence combined for autoecological study of single cell activity with Nitrobacter as a model system. Appl, Microbio!' 30 (1975) 676-684 Pliermans, C. B., R.]. Saracco, and D. H. Pope: Measure of Legionella pneumophila activity in situ. Curr. Microbio!' 6 (1981) 89-94 Hague, A., ].P.Iddings, and W.H. Weed: Geology of Yellowstone National Park. U.S. Geo!' Survey Monograph. 32, pt 2 (1899) 893 Hazen, T. c.: Ecology of Aeromonas hydrophila is a South Carolina cooling cooling reservoir. Microbial Eco!' 5 (1979) 179-195 Orrison, L. H., W. B. Cherry, C. B. Fliermans, S. B. Dees, L. K. McDougal, and D.]. Dodd: Characteristics of environmental isolates of Legionella pneumophila. Appl. Environ. Microbio!' 42 (1981) 109 Tison, D. W., [, A. Baross, and R.]. Seidler: Legionella in aquatic habitats in the Mt. St. Helens blast zone. Abst. Amer. Soc. Microbio!' 82 (1982) 212 Tison, D. L., D. H. Pope, W. B. Cherry, and C. B. Fliermans: Growth of Legionella pneumophila in association with blue-green algae (Cyanobacteria). Appl, Environ. Microbio!. 39 (1980) 456-459 Zimmerman, R., R.Iturriaga, and ].Becker-Brick: Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. Environ. Microbio!' 36 (1978) 926-935