International Biodeterioration & Biodegradation 31 (1993) 55--63

Environmental Factors Affecting the Colonization of Cool-

ing Towers by Legionella spp. in South Australia
R. H. Bent ham
Repatriation General Hospital, Daw Park, South Australia 5041
(Received 26 June 1992; accepted 27 November 1992)
A B S T R A CT
Legionella colonization in 31 cooling towers in or near Adelaide in South
Australia was investigated. Al l towers were on identical cleaning and
chemical t reat ment programmes, including t reat ment with a monomeri c
quaternary ammoni um biocide. Tower construction varied, wood, f i bregl ass
and stainless steel models being included. Over a t wo-year period, water
samples were taken f r om towers twice weekl y and records kept of tower
water pH, conductivity, total bacterial count, basin-water temperature,
ambient temperature and hours usage o f each tower at the t i me o f collection.
Temperat ure and tower usage showed direct correlation with Legionella
counts. Bet ween 60 and 75% of cooling tower waters were colonized by
Legionella spp. during the summer months. This f i gure f el l to 20- 30%
during the winter. Basin-water temperatures were shown to be a maj or
determinant in Legionella colon&ation; regardless of ambient temperature,
towers with elevated basin-water temperatures were f requent l y colonized.
Anal ysi s showed that Legionella counts in towers used continuously
throughout the year were not influenced by ambient temperature.
I NTRODUCTI ON
The presence and multiplication of Legi onel l a spp. and particularly L.
pne umophi l a, the causative organism of Legionnaires' disease, in cooling
water systems is well established (Dondero et al . , 1980; Fliermans et al. ,
1982; Ager & Tickner, 1983). Although studies have been carried out to
55
International Biodeterioration & Biodegradation 0964-8305/93/$06-00 1993 Elsevier
Science Publishers Ltd, England. Printed in Great Britain.
56 R. H. Bentham
investigate the factors i nfl uenci ng col oni zat i on of cool i ng wat er systems
(Kurt z e t al. , 1982; Chri st ensen et al . , 1983; Chri st ensen e t al . , 1985;
Schofield & Locci, 1985; I kedo & Yabuuchi , 1986), t he maj ori t y have been
conduct ed over relatively short t i me peri ods wi t h small number s of towers.
Frequent l y, a desire for mor e compr ehensi ve field dat a has been expressed
( Howl and & Pope, 1983; Edelstein, 1985a; States e t al . , 1987). One of t he
l engt hi er studies ( Howl and & Pope, 1983) has shown t hat there is seasonal
vari at i on in L. p n e u mo p h i l a col oni zat i on of cool i ng towers, with highest
concent r at i ons being det ect ed in s ummer mont hs. It was also not ed in these
studies t hat towers oper at i ng all year r ound showed relatively const ant
numbers.
The present st udy was under t aken to investigate t he influence of a range
of envi r onment al fact ors on Le gi one l l a col oni zat i on of towers. The
exami nat i on of waters f r om a large number of towers over an ext ended
peri od was i nt ended to achieve a mor e generalized and widely appl i cabl e
view of fact ors i nfl uenci ng col oni zat i on.
MATERI ALS AND METHODS
Sampling of tower water
Over a t hree-year peri od, 70 ml wat er sampl es were t aken f r om 31 cool i ng
t ower basins, and basi n-wat er t emper at ur es recorded. Sampl es were
collected twice weekly if Le g i o n e l l a were det ect ed in previ ous sampl es
and once weekly if not. Hour s- r un met ers installed on t he towers were read
and r un t i me recorded at each sampl i ng.
The pH and conduct i vi t y (gS) of all wat er sampl es were recorded in t he
l abor at or y pr i or to processing, using cal i brat ed pH and conduct i vi t y
meters. Daily mean ambi ent t emper at ur e records for t he dur at i on of t he
i nvest i gat i on were obt ai ned f r om t he met eorol ogi cal office.
Tot al bacterial count s were recorded usi ng doubl e-si ded pl at e count agar
dipslides ( PC- PC, Di sposabl e Pr oduct s PTY. ) i ncubat ed at 37C for 3
days. These dipslides gave a sensitivity range of 103-107 col ony f or mi ng
units (cfu) ml "1.
Culture
The sampl es were pre-t reat ed at 50C for 30 mi n (Dennis, 1988) wi t hout
concent r at i on. Inocul a ( 0. 025 ml) were pl at ed on buffered charcoal yeast
ext ract agar (Oxoid, BCYE; Denni s, 1988) and 0. 25ml on Modi fi ed
Colonization of cooling towers by Legionella spp. 57
Wadowsky and Yee agar (Oxoid, MWY; Wadowsky & Yee, 1981). Plates
were i ncubat ed at 37C in an at mosphere cont ai ni ng 5% CO2 for 7 days,
and exami ned using a plate microscope and under UV light to detect
aut ofl uorescent species.
This procedure has a theoretical limit of detection of 4000cf uL -~ on
MWY and 40000cf uL -~ on BCYE plates, assuming 100% recovery of
Legionella spp. on the selective media, but previous investigation of
recovery of Legionella on selective medi a has indicated t hat these
pret reat ment met hods may cause a reduced viable Legionella cell count
from samples dependent on the species (Roberts et al., 1987). Time
constraints and the number of samples t aken precluded concent rat i on of
samples by centrifugation or filtration, which woul d have given greater
sensitivity. This limit of detection was sufficient to allow mai nt enance of
the cooling towers accordi ng to Aust ral i an published guidelines and
st andards (Depart ment of Housi ng and Const ruct i on, Australia, 1987;
Aust ral i an St andard, AS3666-1989, 1989).
Presumptive identification of suspect Legionella colonies was by
subculture ont o BCYE and bl ood agar plates. Latex aggl ut i nat i on tests
for L. pneumophila SG 1-14 (Disposable Product s Pty. Ltd) were carried
out on single colonies of presumptively identified Legionella. Species not
identified by latex aggl ut i nat i on were sent to a reference l aborat ory for
identification (Edelstein, 1985b; Dennis, 1988).
Statistical analysis
Possible correlations between the different envi ronment al factors and
Legionella count s were investigated by Spearman rank analysis.
RESULTS
Species isolated
Of approxi mat el y 1500 positive cultures, the maj ori t y of organisms isolated
were L. pneurnophila SG 1 (70%), with L. anisa (23%), L. rubrilucens
(6. 9%) and L. pneumophila SG 2-14 (0.1%) also being isolated in lesser
degrees. Because of the poor recovery of L. anisa on MWY agar in quality
cont rol , only the BCYE cultures were used to enumerat e this species. This
reduced the maxi mum limit of det ect i on for L. anisa to approxi mat el y
40 000 cfu L -1.
58 R. H. Bentharn
pH
Results from the study showed that Legionella were isolated from towers
anywhere within the pH range 7.0-10.0. When the pH rose above 10.0,
Legionella numbers were below detectable limits; pH values below 7.0 were
not encountered. Apart from this, there was no indication of any
correlation between pH and colonization.
Conductivity
Conductivity (a measurement of dissolved solids) was typically in the range
0.5-3.0/~S. Higher conductivities were recorded on occasion, but as these
are undesirable for efficient operation of the towers they were corrected by
adjustment of the bleed rate from the tower. No correlation was found
between Legionella concentrations and conductivity.
Total bacterial counts
Cooling towers commonly gave total bacterial counts of 103- 107 cfu ml 1.
Legionella spp. were isolated from towers with total bacterial counts over
this entire range. No correlation was found between total bacterial counts
and Legionella concentrations.
Temperature effects
Results of the monitoring of the cooling towers are presented in Fig. 1.
During the summer period (December-March), an average of 65% showed
lOO
.~ 8 0
60
c
0
5 2O
u
o
Fig. 1. Average mont hl y percent age of Legionella-colonized cool i ng towers* in Sout h
Aust r al i a from December 1988 to May 1991.
* N= 31.
Colonization of cooling towers by Legionella spp. 59
colonization of the circulating water at levels > 4000cfu Legionella L -t.
Duri ng the winter mont hs (June-September), this fell to 21%, on average.
Cont i nuousl y operating systems made up the greater proport i on of
Legionella colonized systems duri ng the winter mont hs (6 of 31).
Non-paramet ri c statistical analysis (Table 1) showed a significant
correlation between ambi ent air t emperat ure and Legionella colonization
(p < 0-05) in 44% of cooling towers; in 48% of towers there was significant
correlation between Legionella concent rat i ons and basin-water tempera-
ture (Table 1). Furt her analysis revealed basin-water t emperat ure to be the
maj or predictor of Legionella (Table 2). It was also noted t hat there was a
mi ni mum basin-water temperature of approxi mat el y 16.5C below which
Legionella was not detected.
T A B L E 1
Spearman Rank Correlation Coefficient (p) for Correlation
Between Legionella Count and Ambient and Basin-Water
Temperatures
Tower Ambient t e mp . Basi n-wat er temp.
C C
1 0-15 0-16
2 0.38* 0.50*
3 0.30* 0.60*
4 0.13 0.30*
5 0-57* 0.54
6 -0-005 -0-16
7 0-59* 0.54*
8 0.22* 0.007
9 0.10 0.40*
10 0.13 0.04
11 0.13 0.00
12 0.20* 0.19
13 0.27* 0.31"
14 0.05 -0.23
15 0-32* 0.28*
16 0-48* 0-57*
17 -0.17 0.00
18 -0.009 0.60*
19 0.03 0.00
20 0.50 0.60*
21 0.008 0.00
22 0.44* 0.45*
23 -0-09 -0.27
*Indicates significant correlation (p > 0-05)
60 R. H. Bentham
TABLE 2
Statistical Analysis of the Relationship Between Legionella Concentration
and Environmental Factors
Predictor p Probability N
Air temperature 0-18 0-0001 2730
Basin temperature 0-42 0.0001 1418
Operation 0.26 0.0001 573
p = Spearman correlation coefficient.
N = Number of observations in each correlation.
Tower usage
Analysis gave a significant correlation between tower usage and Legi onel l a
count (Table 2). Fur t her analysis of Legi onel l a counts (not shown) in terms
of average summer and winter counts with tower usage showed correl at i on
duri ng the winter to be greater t han duri ng summer.
DI SCUSSI ON
As in ot her studies, there was a lack of correl at i on between bot h the pH and
conductivity/dissolved solids of tower wat er and Legi onel l a col oni zat i on
(Miller & Bott, 1982; Christensen et al., 1983; Gr ow et al., 1984;
Christensen et al., 1985). An upper limit of pH 10.0 for Legi onel l a
colonization was not ed and, al t hough use of high pH to cont rol Legi onel l a
has been suggested by States et al. (1987), operat i on of towers at this pH
woul d be mechanically destructive in Sout h Aust ral i an cooling water-
systems.
The lack of correl at i on between total bacterial count s and Legi onel l a
concent rat i ons agrees with ot her studies (Ikedo & Yabuuchi , 1986; Gar net t
et al., 1990). Dipslides are current l y routinely used to assess biological
cont ami nat i on of cooling towers in Australia. An investigation of the value
of dipslides in assessing the likelihood of Legi onel l a cont ami nat i on seemed
pertinent and the possible i naccuracy of dipslide values in assessing total
bacterial count s was considered less critical.
Gi ven the absolute requirement of Legi onel l a for ami no acids, especially
L-cysteine, it woul d be reasonable to assume t hat the absence of ot her
micro-organisms or ot her sources of these nutrients from the system may
preclude colonization. The study revealed no direct evidence of synergism
between Legi onel l a and ot her bacteria, al t hough relationships with ot her
micro-organisms, including bacteria, algae and amoebae have been
document ed (Pope et al., 1982; Wadowsky & Yee, 1985; Fields et al., 1989).
Colonization of cooling towers by Legionella spp. 61
This study has shown that, of the factors investigated, basin-water
t emperat ure was the maj or det ermi nant in colonization of the circulating
water. This colonization is dependent on ambi ent t emperat ure and tower
usage, but may also be affected by heat load placed on the tower, and also
tower situation. Towers located inside buildings coul d be expected to have
higher basin-water temperatures duri ng the winter period regardless of
ambi ent t emperat ure, t ower usage or heat load.
The seasonal vari at i on in Legionella colonization of cooling towers and
the correl at i on between Legionella colonization and tower usage has been
document ed ( Howl and & Pope, 1983). The low sensitivity of the detection
met hod woul d suggest that the number of towers with circulating water
colonized by Legionella may be much higher. The low limit of detection for
L. anisa suggests t hat this organi sm may be present in a higher percentage
of the cooling towers t han is indicated in the present study. Previous
investigations and compl ement ary studies to the present investigation have
shown t hat Legionella may be present in biofilms and sediments when
pl ankt oni c Legionella are undet ect ed (Schapira, 1988; FaUon & Rowbo-
t ham, 1990; Bent ham et al., 1992).
The lower limit of 16.5C for Legionella detection in the present study is
somewhat lower t han not ed in ot her studies (Col bourne & Dennis, 1989).
In an operat i ng cooling tower system, the basin t emperat ure will typically
be approxi mat el y 5C lower t han t hat of the condenser and post-condenser
pipework. Taki ng these facts into consideration, this lower t emperat ure
limit is not inconsistent with previous findings. This suggests t hat the tower
basin-water may not necessarily be the pri mary site of Legionella
multiplication.
The correl at i on between usage and concent rat i ons of Legionella (heavy
col oni zat i on correlates with high usage) may not be simply attributable to
heating of the water. Ot her ecological factors must be considered, such as
increased oxygen supply, increased flow of wat er t hrough pi pework duri ng
cont i nuous operat i on possibly enhanci ng biofilm format i on or condi t i ons
favouring sloughing of biofilm (Miller & Bott, 1982), the di st urbance of
sediments which are known reservoirs of Legionella col oni zat i on in cooling
towers mi ght also be involved ( Howl and & Pope, 1983). Nevertheless, the
correl at i on between tower usage and wat er t emperat ure demonst rat ed in
this study suggests t hat heating of the water is an i mport ant factor in
Legionella multiplication.
REFERENCES
Ager, B. P. & Tickner, J. A. (1983). Control of microbiological hazards associated
with air conditioning and ventilation systems. Annals of Occupational
Hygiene, 27, 341-58.
62 R. H. Bentham
Australian Standard AS 3666-1989. (1989). Air-handling and Water Systems of
Buildings--Microbial Control. Standards Australia, Nort h Sydney, New
South Wales.
Bentham, R. H., Broadbent, C. R. & Marwood, L. N. (1992). The influence of the
sessile population in the Legionella colonization of cooling towers.
Proceedings of the 1992 International Legionella Symposium. American
Society for Microbiology, Washington, D.C. (in press).
Christensen, S. W., Solomon, J. A., Gough, S. B., Tyndall, R. L. & Fliermans, C. B.
(1983). Legionnaires' disease bacterium in power plant cooling systems:
Phase I. Final report. Electric Power Research Institute Research Project
1909-1, Report EA-3153.
Christensen, S. W., Solomon, J. A., Gough, S. B., Tyndall, R. L. & Fliermans, C.
B. (1985). Legionnaires' disease bacterium in power plant cooling systems:
Phase 2. Electric Power Research Institute Research Project 1909-1, Report
EA-4017.
Colbourne, J. S. & Dennis, P. J. (1989). The ecology and survival of L.
pneumophila. Journal of the Institution of Water and Environmental
Management, 3, 345-50.
Dennis, P. J. (1988). Isolation of legionellae from environmental specimens. In A
Laboratory Manual for Legionella, ed. T. G. Harrison & A. G. Taylor. John
Wiley, New York, pp. 31-171.
Department of Housing Construction, Australia. (1987). Measures to control
Legionnaires' disease hazards in buildings. Technical Information TI 178
ME.
Dondero, T. J., Rendtorff, R. C., Mallison, G. F., Weeks, R. M., Levy, J. S.,
Wong, E. W. & Schaffner, W. (1980). An outbreak of Legionnaires' disease
associated with a contaminated air-conditioning cooling tower. New England
Journal of Medicine, 320, 365-70.
Edelstein, P. H. (1985a). Environmental aspects of Legionella. American Society
for Microbiology News, 51, 460-5.
Edelstein, P. H. (1985b). Legionella. In Manual of Clinical Microbiology (4th edn)
ed. E. H. Lennette, A. Balows, W. J. Hauster & H. J. Shadonny. American
Society for Microbiology, Washington, D.C., pp. 373-9.
Fallon, R. J. & Rowbot ham, T. J. (1990). Microbiological investigations into an
outbreak of Pontiac fever due to Legionella micdadei associated with use of a
whirlpool. Journal of Clinical Pathology, 43, 479-83.
Fields, B. S., Sanden, G. N., Barbaree, J. M., Morrill, W. E., Wadowsky, R. M.,
White, E. H. & Feeley, J. C. (1989). Intracellular multiplication of Legionella
pneumophila in amoebae isolated from hospital hot water tanks. Current
Microbiology, 18, 131-7.
Fliermans, C. B., Bettinger, G. E., & Fynsk, A. W. (1982). Treatment of cooling
systems containing high levels of Legionella pneumophila. Water Research,
16, 903-6.
Garnett, H. M., Gilmore, K. & Liu, J. (1990). Legionella: an unwelcome pollutant.
Environmental Technology, 11,393-400.
Grow, K. M., Wood, D. O., Coggin, J. H. & Leinbach, E. D. (1984).
Environmental factors influencing growth of Legionella pneumophila in
operating, biocide-treated cooling towers. In Proceedings of the Second
International Legionella Symposium, ed. C. Thornsberry, A. Balows, J. C.
Colonization of cooling towers by Legionella spp. 63
Feeley & W. Jakubowski. American Society for Microbiology, Washington,
D.C. pp. 316-18.
Howland, E. B. & Pope, D. H. (1983). Distribution and seasonality of Legionella
pneumophila in cooling towers. Current Microbiology, 9, 319-23.
Ikedo, M. & Yabuuchi, E. (1986). Ecological studies of Legionella species. I.
Viable counts of Legionella pneumophila in cooling tower water. Micro-
biology and Immunology, 30, 413-23.
Kurtz, J. B., Bartlett, C., Tillet, H. & Newton, U. (1982). Field trial of biocides in
control of Legionellapneumophila in cooling water systems. In Proceedings of
the Second International Legionella Symposium, ed. C. Thornsberry, A.
Balows, J. C. Feeley & W. Jakubowski. American Society for Microbiology,
Washington, D.C., pp. 340-2.
Miller, P. C. & Bott, T. R. (1982). Effects of biocide and nutrient availability on
microbial contamination of surfaces in cooling-water systems. Journal of
Chemical Technology and Biotechnology, 32, 538-46.
Pope, D. H., Soracco, R. J., Gill, H. K. & Fliermans, C. B. (1982). Growt h of L.
pneumophila in two membered cultures with algae and cyanobacteria.
Current Microbiology, 7, 319-22.
Roberts, K. P., August, C. M. & Nelson, J. D. (1987). Relative sensitivities of
environmental legionellae to selective isolation procedures, Applied Environ-
mental Microbiology, 53, 2704-7.
Schapira, S. (1988). The formation, implications and control of biofilms in
industrial cooling water systems. Journal of Hospital Engineering, 42, 22-4.
Schofield, G. M. & Locci, R. (1985). Colonization of components of a model hot
water system by Legionella pneumophila. Journal of Applied Bacteriology, 58,
151-62.
States, S. J., Conley, L. F., Towner, S. G., Wolford, R. S., Stephenson, T. E.,
McNamara, A. M., Wadowsky, R. M. & Yee, R. B. (1987). An alkaline
approach to treating cooling towers for control of Legionella pneumophila.
Applied and Environmental Microbiology, 53, 1775-9.
Wadowsky, R. M. & Yee, R. B. (1981). Glycine-containing selective medium for
isolation of Legionellaceae from environmental specimens. Applied and
Environmental Microbiology, 42, 768-72.
Wadowsky, R. M. & Yee, R. B. (1985). Effect of non-legionellaceae bacteria on
the multiplication of L. pneumophila in potable water. Applied and
Environmental Microbiology, 49, 1206-10.