Summary

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 (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 108 cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 106 cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 102 (cfu/ml) over the same period. After incubation for 1.5 years at 15°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 BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, 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 BCYEα. 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.

References

[1]
Anand
C.M.
Skinner
A.R.
Malic
A.
Kurtz
J.B.
(
1983
)
Interaction of Legionella pneumophila and a free-living amoeba (Acanthamoeba palestinensis)
J. Hyg.
 ,
91
,
167
168
.
[2]
Benson
R.F.
Thacker
W.L.
Waters
R.P.
Quinlivan
P.A.
Mayberry
W.R.
Brenner
D.J.
Wilkinson
H.W.
(
1989
)
Legionella quinlivanii sp. nov. isolated from water
Curr. Microbiol.
 ,
18
,
195
197
.
[3]
Fliermans
C.B.
Cherry
W.B.
Orrison
L.H.
Smith
S.J.
Tison
D.L.
Pope
D.H.
(
1981
)
Ecological Distribution of Legionella pneumophila
Appl. Environ. Microbiol.
 ,
41
,
9
16
.
[4]
Shahamat
M.
Paszko-Kolva
C.
Keiser
J.
Colwell
R.R.
(
1991
)
Sequential cultural method improves recovery of Legionella spp. from contaminated environmental samples
Zbl. Bakt.
 ,
275
,
312
319
.
[5]
Tison
D.L.
Pope
D.H.
Cherry
W.B.
Fliermans
C.B.
(
1980
)
Growth of Legionella pneumophiila in association with blue green algae (cyanobacteria)
Appl. Environ. Microbiol.
 ,
39
,
456
459
.
[6]
Berg
J.D.
Hoff
J.C.
Roberts
P.V.
Matin
A.
(
1985
)
Growth of Legionella pneumophila in aontinous culture
Appl. Environ. Micro.
 ,
49
,
1534
1537
.
[7]
Hussong
D.
Colwell
R.R.
O'Brien
M.
Weiss
A.D.
Pearson
A.D.
Weiner
R.M.
Burge
W.D.
(
1987
)
Viable Legionella pneumophila not detectable by culture on agar media
Biotechnology
 ,
5
,
947
950
.
[8]
Csonka
C.N.
(
1989
)
Physiological and genetic responses of bacteria to osmotic stress
Microbiol. Rev.
 ,
53
,
121
147
.
[9]
LeChevallier
M.W.
Cawthon
C.D.
Lee
R.G.
(
1988
)
Factors promoting survival of bacteria in chlorinated water supplies
Appl. Environ. Microbiol.
 ,
54
,
649
654
.
[10]
Warren
W.J.
Miller
R.D.
(
1979
)
Growth of Legionnaires' disease bacterium in a chemically defined medium
J. Clin. Microbiol.
 ,
10
,
50
55
.
[11]
Pine
L.
George
J.R.
Reeves
M.W.
Harrell
W.H.
(
1979
)
Development of a chemically defined medium for growth of Legionella pneumophila
J. Clin. Microbiol.
 ,
9
,
615
626
.
[12]
Bornstein
N.
Nowicki
M.
Fleurette
J.
(
1984
)
Loss of virulence of Legionella pneumophila serogroup 1 with conversion of cells to long filamentous rods
In
Legionella Proceedings of the 2nd International Symposium
 
Thornsberry
C.
Ballows
A.
Feeley
J.C.
Jakubowski
W.
, Eds) Atlanta, Georgia, pp
70
71
American Society for Microbiology
,
Washington, DC
.
[13]
McDade
J.E.
Shepard
C.C.
(
1979
)
Virulent to avirulent conversion of Legionnaires' disease bacterium (Legionella pneumophila). Its effect on isolation technique
J. Infect. Dis.
 ,
139
,
707
711
.
[14]
Paszko-Kolva
C.
Yamamoto
H.
Shahamat
M.
Sawyer
T.K.
Morris
G.
Colwell
R.R.
(
1991
)
Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations
Appl. Environ. Microbiol.
 ,
57
,
163
167
.
[15]
Feely
J.C.
Gibson
R.J.
Gorman
G.W.
Langford
N.C.
Rasheed
K.
Mackel
D.
Baine
W.B.
(
1979
)
Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila
J. Clin. Microbiol.
 ,
10
,
437
441
.
[16]
Hobbie
J.E.
Daley
R.
Jasper
S.
(
1977
)
Use of Nucleopore filters for counting bacterial by fluorescent microscopy
Appl. Environ. Microbiol.
 ,
33
,
1225
1228
.
[17]
Cherry
W.B.
Pittman
P.P.
Harris
G.A.
Herbert
B.M.
Thomason
L.
Thacker
W.L.
Weaver
R.E.
(
1978
)
Detection of Legionnaires' disease bacteria by direct immunofluorescent staining
J. Clin. Microbiol.
 ,
8
,
329
338
.
[18]
Armon
R.
Payment
P.
(
1990
)
A transparent medium for isolation of Legionella pneumophila from environmental of Legionella pneumophila from environmetal water sources
J. Microbiol. Methods
 ,
11
,
65
71
.
[19]
Laemmli
U.K.
(
1970
)
Cleavage of structural proteins during the assembly of the head of the bacteriophage T4
Nature
 ,
227
,
680
685
.
[20]
Yee
R.B.
Wadowsky
R.M.
(
1982
)
Multiplication of Legionella pneumophila in unsterilized tap water
Appl. Environ. Microbiol.
 ,
43
,
1330
1334
.
[21]
Fliermans
C.B.
Cherry
C.B.
Orrison
L.H.
Thacker
L.
(
1979
)
Isolation of Legionella pneumophila from nonepidemic related aquatic habitats
Appl. Environ. Microbiol.
 ,
37
,
1239
1242
.
[22]
Wright
J.B.
Ruseska
I.
Athar
M.A.
Cobert
S.
Costerton
J.W.
(
1989
)
Legionella pneumophila grows adherent to surfaces in vitro and in situ
Infect. Contol. Hosp. Epidemiol.
 ,
10
,
408
415
.
[23]
Paszko-Kolva
C.
Shahamat
M.
Yamamoto
H.
Sawyer
T.K.
Colwell
R.R.
(
1991
)
Survival of Legionella pneumophila in the Aquatic environment
Microb. Ecol.
 ,
22
,
75
83
.
[24]
Saito
A.
Rolfe
R.D.
Edelstein
P.
Finegold
S.
(
1981
)
Comparison of liquid growth media for Legionella pneumophila
J. Clin. Microbiol.
 ,
14
,
623
627
.
[25]
Flesher
A.R.
Ito
S.
Mansheim
B.J.
Kasper
D.L.
(
1979
)
The cell envelop of the Legionnaires' disease bacterium. Morphologic and biochemical characteristics
Ann. Intern. Med.
 ,
90
,
628
630
.
[26]
Surgot
M.
Barioz
C.M.
Nowicki
M.
Bornstein
N.
Fleurette
J.
(
1988
)
An electron microscopy study of Legionella pneumophila after in vitro and in vivo culture
Zbl. Bakt. Hyg. A
 ,
269
,
26
33
.
[27]
Kogure
K.
Simidu
U.
Taga
N.
(
1979
)
A tentative direct microscopic method for counting living marine bacteria
Can. J. Microbiol.
 ,
25
,
415
420
.
[28]
Ishida
Y.
Imai
I.
Miyagaki
T.
Kadota
H.
(
1982
)
Growth and uptake kinetics of facultatively oligotrophic bacterium at low nutrient concentrations
Microbiol. Ecol.
 ,
8
,
23
32
.
[29]
Kuznetsov
S.I.
Dubinina
G.A.
Lapteva
N.A.
(
1979
)
Biology of oligotropic bacteria
Ann. Rev. Microbiol.
 ,
33
,
377
387
.
[30]
Herbert
D.
(
1958
)
Some principles of continuous culture
 , In
Recent Progress in Microbiology, VIIth International Congress of Microbiology
  pp
381
396
.
[31]
Reichardt
W.
Morita
R.Y.
(
1982
)
Survival stages of a psychotrophic Cytophaga johnsonae strain
Can. J. Microbiol.
 ,
28
,
841
850
.
[32]
Rozak
D.B.
Colwell
R.R.
(
1987
)
Survival strategies of bacteria in the natural environment
Microbiol. Rev.
 ,
51
,
365
379
.
[33]
Barbaree
J.M.
Gorman
G.W.
Martin
W.T.
Fields
B.S.
Morrill
W.E.
(
1987
)
Protocol for sampling environmental sites for Legionellae
Appl. Environ. Micro.
 ,
51
,
422
424
.

Author notes

1
Present address: Advanced Technology Laboratories, Alta Loma, CA 91701, USA and Metropolitan Water District of Southern California, Water Quality Division, LaVerne, CA 91750, USA.