Legionella species colonization

in cooling towers: Risk factors

and assessment of control measures
Varvara A. Mouchtouri, BSc, PhD,
a,b
Georgia Goutziana, MD,
a
Jenny Kremastinou, MD, PhD,
b
and Christos Hadjichristodoulou, MD, PhD
a
Larissa and Athens, Greece
Background: Cooling towers can be colonized by Legionella spp, and inhalation of aerosols generated by their operation may cause
Legionnaires disease in susceptible hosts. Environmental investigations of Legionnaires disease outbreaks linked with cooling
towers have revealedpoorlymaintainedsystems, lackof control measures, andfailure of systemequipment. The purposeof this study
was toidentify Legionella-contaminatedcooling towers, identify risk factors for contamination, and assess the effectiveness of control
measures.
Methods: A total of 96 cooling towers of public buildings were registered and inspected, and 130 samples were collected and mi-
crobiologically tested. Microbiological test results were associated with characteristics of cooling towers, water samples, inspection
results, and maintenance practices.
Results: Of the total 96 cooling towers examined, 47(48.9%) were colonizedby Legionella spp, and22 (22.9%) requiredremedial action.
Atotal of 65 samples (50.0%) were positive ($500 cfu L
21
), and 30 (23%) were heavily contaminated ($10
4
cfu L
21
). Of the 69 isolates
identied, 55 strains (79.7.%) were L pneumophila. Legionella colonization was positively associated with the absence of training on
Legionella control (relative risk [RR] 51.66; P 5.02), absence of regular Legionella testing (RR52.07: P 5.002), absence of sunlight
protection (RR51.63: P 5.02), with samples in which the free residual chlorine level in the water sample was ,0.5 mg/L
(RR52.23; P 5.01), and with total plate count (P 5.001). Colonization was negatively associated with chemical disinfection
(RR50.2; P 5.0003) andwiththe presence of a risk assessment andmanagement plan (RR50.12; P 5.0005). Astatistically signicant
higher age (P 5.01) was found in legionellae-positive cooling towers (median, 17 years; interquartile range [IQR] 55.0 to 26.0 years)
compared with noncolonized cooling towers (median age, 6 years; IQR51.0 to 13.5 years). After the 22 legionellae-positive cooling
towers were disinfected with chlorine, 2 (9%) of them remained positive for Legionella spp with a concentration $1000 cfu L
21
.
Conclusions: Cooling towers can be heavily colonized by Legionella spp and thus present a potential risk for infection. This study
demonstrates the importance of a risk assessment and management plan. Water chlorination effectively reduces legionellae
contamination. Proper training of cooling tower operators is paramount.
Key Words: Legionella; cooling tower; factor; disinfection; risk assessment.
Copyright 2010 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights
reserved. (Am J Infect Control 2010;38:50-5.)
Cooling towers, devices used to cool warm water by
evaporation in atmospheric air, can be found in air-con-
ditioning systems of buildings such as hotels and hospi-
tals.
1
Legionella spp colonization in cooling towers is
well documented.
2-5
Aerosols generated by the
operation of cooling towers can transmit legionellae to
susceptible hosts.
1
Many epidemiologic and environ-
mental investigations have linked outbreaks of Legion-
naires disease with contaminated water in cooling
towers by matching the molecular subtyping ndings
betweenwater andpatient isolates.
6-8
Environmental in-
vestigations of Legionnaires disease outbreaks linked
with cooling towers have revealed poorly maintained
systems,
9
lack of control measures and failure of system
equipment;
10
consequently, investigators recommend
regular maintenance of cooling towers.
7,11-13
Public health authorities in some countries have
introduced recommendations, mandatory guidelines,
and even legislation for the registration of cooling
towers and application of control measures.
9,10,14
The
European Surveillance Scheme for Travel-Associated
Legionnaires Disease and the European Working
Group for Legionella Infections (EWGLI) have published
guidelines aimed at controlling and preventing travel-
associated Legionnaires disease. These guidelines
From the Department of Hygiene and Epidemiology, Medical School,
University of Thessaly, Larissa, Greece
a
; and Department of Public
and Administrative Health, National School of Public Health, Athens,
Greece.
b
Address correspondence to Christos Hadjichristodoulou, MD, PhD,
Department of Hygiene and Epidemiology, Medical School, University
of Thessaly, Papakiriazi Str, 22, 41222, Larissa, Greece. E-mail: xhatzi@
med.uth.gr.
Conicts of interest: none to report.
0196-6553/$36.00
Copyright 2010 by the Association for Professionals in Infection
Control and Epidemiology, Inc. Published by Elsevier Inc. All rights
reserved.
doi:10.1016/j.ajic.2009.04.285
50
include design, operational, and management mea-
sures for controlling the risk of legionellae proliferation
in cooling towers.
15
The World Health Organization
(WHO) suggests a hazard analysis approach by imple-
menting water safety plans to control the risk of infec-
tion from cooling towers.
1
Such a water safety plan
species measures for the water source, heat
exchanger, distribution system, and the cooling tower,
including disinfection, monitoring, and maintenance.
But although maintenance of cooling towers and appli-
cation of control measures is reasonable and many
guidelines make such recommendations, no data exist
to support the claim that maintenance minimizes colo-
nization by Legionella.
16
Accordingly, the present study
was conducted to identify Legionella-contaminated
cooling towers, identify risk factors for contamination,
and assess the effectiveness of control measures.
METHODS
Cooling towers of public buildings (eg, hotels, hospi-
tals, athletic venues, shopping centers) located inAthens,
Thessaloniki, Patra, Volos, and Irakliowere registered, in-
spected, and microbiologically tested. Legionella-
contaminated cooling towers were disinfected using
chlorine compounds according to the EWGLI technical
guidelines.
15
Prefecture Department of Public Health
personnel conducted the registrations, inspections, and
sampling. Microbiological test results were examined to-
gether with characteristics of cooling towers, water sam-
ples, inspection results, and maintenance practices.
Registration and inspection
Cooling towers were registered by completing a stan-
dardized registry form, including information on name
of building, unique registration code, address and typical
characteristics such as age, location in the building (eg,
rooftop, courtyard), responsible person, methods and
means for disinfection and chemical control of fouling,
presence of drift eliminators, record keeping, existence
of a riskassessment plan, water capacity, and daily water
loss. The cooling tower inspections were performed us-
ing a standardized inspection report that included 39
scored items. These inspection items were classied
into 6 categories: construction, operation, cleaning-dis-
infection, personnel, record keeping and onsite manu-
ally conducted tests.
17
Data included in the inspection
and the registration forms were used to evaluate risk fac-
tors possibly associated with Legionella colonization.
Sample collection and microbiological analysis
methods
Methods used for sample collection, storage condi-
tions, and microbiological analysis have been
described elsewhere.
17
Microbiological analyses for Le-
gionella spp were performed by the National Legionella
Reference Laboratory of Southern Greece in Athens
and the National Legionella Reference Laboratory of
Northern Greece in Thessaloniki.
The lower limit of detection of the microbiological
analysis was 500 cfu L
21
. Remedial actions were un-
dertaken in any cooling tower with a Legionella bacte-
ria count $10,000 cfu L
21
in one sample or and
aerobic count .100,000 cfu mL
21
in one sample.
Disinfection procedure
Chlorine disinfection was applied to all Legionella-
postitive cooling towers according to the following pro-
cedure. First, the cooling tower water was chlorinated.
The chlorinated water containing 5 mg/L of free chlo-
rine, was circulated through the system for 5 hours
with the fan off. If the system pH value was .8.0,
then the recommended measured residual was in the
range of 15 to 20 mg/L of free chlorine to achieve the
required disinfection level. All areas of the tower
were then cleaned. Cleaning staff wore suitable respira-
tory protective equipment. After the cleaning opera-
tions, the system was relled and chlorinated to
maintain a minimum level of 5 mg/L of free chlorine
for a period of 5 hours with the fan off and checked
hourly. An alternative cleaning method involved
increasing the free chlorine level to, for example,
50 mg/L for 1 hour or 25 mg/L for 2 hours, followed
by dechlorination, draining, ushing, relling with
fresh water, and adding appropriate startup doses of
treatment chemicals, including biocides.
15
Between 2 and 7 days after disinfection, 1 sample
was collected from each cooling tower. Disinfection
was considered successful when the sample exhibited
a Legionella spp concentration ,1000 cfu L
21
and an
aerobic count ,100,000 cfu mL
21
.
The standardized disinfection guidelines were
printed and distributed by the public health personnel
in the Prefecture Departments to the persons responsi-
ble for the various buildings. Hoteliers, hospital staff, or
other responsible persons applied the disinfection
procedure, supervised by public health personnel
when feasible.
Data analysis
Data were analyzed with Epi-Info 2000 (Centers for
Disease Control and Prevention, Atlanta, GA) and SPSS
for Windows release 15.0 (SPSS Inc, Chicago, IL) by
the t-test or Mann-Whitney test for quantitative data
and by the x
2
test or Fishers exact test for qualitative
data. The median and the interquartile range (IQR)
were used to delineate the dispersion of quantitative
data. Relative risk (RR) and 95% condence interval
www.ajicjournal.org
Vol. 38 No. 1
Mouchtouri et al. 51
(CI) were calculated to assess categorical risk variables
associated with legionellae-positive test results. Varia-
bles that were signicant inunivariate analysis were en-
tered into a backward conditional logistic regression
model. By using conditional logistic regression models,
independent predictors of colonization were estab-
lished. Variables were retained in the model if the likeli-
hood ratio test result was signicant (P # .05).
RESULTS
A total of 96 cooling towers were examined, and 130
samples were collected. Forty-seven (48.9%) of the
cooling towers were Legionella-postitive, and 22
(22.9%) of these required remedial action. Table 1 pre-
sents a descriptive analysis of the cooling towers and
maintenance and water treatment practices applied
during the study period.
Of the 130 samples evaluated, 65 (50.0%) were from
systems colonized by Legionella spp ($ 500 cfu L
21
).
Thirty samples (23%) had a Legionella concen-
tration $10
4
cfu L
21
, and 10 samples (7.7%) had a con-
centration .10
5
cfu L
21
. Of the 69 isolates, 55 strains
(79.7.%) were L pneumophila, including 52 serogroup
1 (75.4% of total strains) and 3 serogroup 2 to
14 (4.3%), and 14 strains (20.3%) were nonL pneumo-
phila (Table 2).
Table 3 characterizes the association between cool-
ing tower system and operational characteristics with
Legionella spp contamination using univariate analysis.
Legionella colonization was positively associated with
cooling towers in which the responsible person was
not trained on Legionella control and Legionella testing
was not done regularly (ie, every 3 months).
Characteristics of cooling towers that were treated
with chlorine or not treated were associated with the
concentration of free chlorine measured in the sam-
ples. A positive association was found with cooling
towers in which the free-residual chlorine of the water
sample was ,0.5 mg/L and that were not protected
fromsunlight. There was negative association with col-
onization and chemical disinfection of cooling towers.
In addition, cooling towers with a risk assessment and
management plan were negatively associated with
colonization.
Tables 4 and 5 present the median and IQR of char-
acteristics of cooling towers and water samples in asso-
ciation with contamination with Legionella spp.
A statistically signicant higher age (P 5.01) was found
in the legionellae-positive cooling towers (median, 17
years; IQR 55.0 to 26.0 years) comparison with the
noncolonized cooling towers (median, 6 years;
IQR 51.0 to 13.5 years). The median total plate count
was signicantly higher in the legionellae-positive
samples than in the legionellae-negative samples
(4200 cfu/mL vs 422 cfu/mL; P 5.001). The statistically
signicant association between legionellae-positive
samples and cooling tower age (odds ratio
[OR] 53.00; 95% CI 51.04 to 8.62; P 5.04), system
chlorination (OR 52.27; 95% CI 50.08 to 0.89;
P 5.03), and microbiological monitoring (OR 54.88;
95% CI 51.33 to 17.86; P 5.01) persisted after strati-
ed and logistic regression analysis. After chlorine dis-
infection of the cooling towers, legionellae-positive
samples with a concentration $1000 cfu L
21
were
found in 2 of the 22 cooling towers resampled (9%).
DISCUSSION
Our results indicate that about half of the cooling
towers examined were contaminated with Legionella
spp, and 23% of the collected samples were highly
contaminated with a concentration $10
4
cfu L
21
. In
another study conducted in 15 cooling towers in Spain,
about 15% of the collected samples were positive with
a concentration .10
4
cfu L
21
.
18,19
The great majority
of the systems examined were contaminated with L
pneumophila serogroup 1, whereas the primary legion-
ellae associated with outbreaks of Legionnaires dis-
ease from cooling towers appear to be L pneumophila
serogroup 1 MAb2-reactive strains.
1
The fact that only 11.4% of the cooling towers had a
water system operational manual available, 24.1% had
a risk assessment and management plan in place, and
41.8% had a cleaning and maintenance log indicates
that cooling tower operation is not systematically mon-
itored in the majority of the devices. No methodical
Table 1. Results of descriptive analysis: cooling tower
system characteristics, maintenance, and water treatment
practices applied
Characteristics
Number of cooling
towers/total (%)
Presence of water system operational manual 9/79 (11.4)
Presence of risk assessment and management plan 14/58 (24.1)
Presence of CT cleaning and maintenance log 33/79 (41.8)
Microbiological results record keeping 38/59 (64.4)
Application of chemical disinfection 24/60 (40.0)
Application of chlorine disinfection 21/94 (22.3)
CT located near windows or air inlet conduit 18/79 (22.8)
Protection of CT from sunlight 28/79 (35.4)
Water source: municipality 48/94 (51.0)
Water source: well 1/94 (1.0)
Presence of drift eliminators 53/68 (77.9)
Presence of automatic chlorinator 13/60 (21.7)
System cleaning at least every 6 months 10/79 (12.6)
Application of disinfection before CT cleaning 30/79 (37.9)
Evidence of system water leakage 8/79 (10.1)
Responsible person is trained 24/79 (30.4)
Water examination for Legionella 38/79 (48.1)
CT, cooling tower.
52 Mouchtouri et al. American Journal of Infection Control
February 2010
hazard analysis has been conducted to assess the risks
and dene control measures. Only a small number of
cooling towers were cleaned (12.6%) and disinfected
(22.3%) at least every 6 months. About 30% of the re-
sponding cooling tower operators had received training
regarding the application of control measures and
maintenance of the system. Awareness and instruc-
tions regarding prevention of Legionnaires disease
are necessary not only for operators, but also for con-
tractors and persons who install and clean the devices.
This is crucial, considering that many cooling towers
are located near windows or air inlet conduits and
that 60% of them were not disinfected before cleaning,
to limit the risk of inhaling contaminated water.
The Hellenic Ministry of Health has enacted manda-
tory guidelines for the prevention and control of
Legionnaires disease, which include provisions for
cooling tower maintenance and application of control
measures. Our ndings show that only a small propor-
tion of cooling tower operators adhere to these guide-
lines, however. An ofcial registry of cooling towers
would help identify target groups, provide training,
and conduct routine inspections. Another study of
175 cooling towers in Glasgow, Scotland yielded simi-
lar ndings, with inadequate drift control, failure to
use chemicals, and absence of a log book and guide-
lines.
20
Cooling tower maintenance standards should
be published and information campaigns directed at
those responsible for cooling tower maintenance
established.
20
Cooling towers have not yet been linked
to incidents of legionellosis in Greece, but the disease is
likely underdiagnosed and underreported. No pub-
lished data on investigations of outbreaks or incidents
in Greece have implicated cooling towers, however.
WHO
1
and EWGLI
15
guidelines recommend regular
maintenance of cooling tower systems based on the
control of fouling due to scale, salt, and microbial
growth and maintenance of mechanical components
of the system. In addition, regular inspection and appli-
cation of corrective measures to eliminate parts of the
system that facilitate stagnant water are recommen-
ded. Further measures include cleaning all internal
parts, including the heat exchanger, to remove dirt,
dust, dissolved solids, and organic material. Finally,
Table 2. Legionella spp. contamination in cooling towers water samples examined
Number of samples/total (%)
Organisms Positive
With 500 to
999 cfu L
21
With 10
3
to
9999 cfu L
21
With 10
4
to
10
5
cfu L
21
With .10
5
cfu L
21
Legionella spp. 65/130 (50.0) 7/130 (5.4) 28/130 (21.5) 20/130 (15.4) 10/130 (7.7)
L. pneumophila serogroup 1 52/65 (80.0) 6/7 (85.7) 23/28 (82.1) 15/20 (75.0) 8/10 (80.0)
L. pneumophila serogroups 2 to 14 3/65 (4.6) 1/7 (14.3) 2/20 (10.0)
Legionella spp. other
than L. pneumophila
14/65 (21.5) 9/28 (32.1) 3/20 (15.0) 2/10 (20.0)
Table 3. Association of cooling towers system and operational characteristics with Legionella spp contamination (univariate
analysis)
Number of positive cooling towers for Legionella spp
Characteristics With characteristic (%) Without characteristic (%) RR (95% CI) P
Algaecide water treatment 3 (60.0) 14 (58.3) 1.02 (0.46-2.26) .60
Antiscale water treatment 19 (51.4) 8 (57.1) 0.89 (0.51-1.55) .40
Chemical disinfection 4 (16.7) 23 (63.9) 0.2 (0.10-0.65) .0003
Cleaning every .6 months 3 (75.0) 35 (46.7) 1.60 (0.86-2.97) .20
Heterotrophic colony count tests not conducted 26 (60.5) 32 (42.1) 1.43 (1.00-2.05) .04
Legionella spp. tests every 3 months not conducted 25 (65.8) 13 (31.7) 2.07 (1.25-3.43) .002
Sprinklers not properly operated 0 (0.0) 38 (48.1)
System components damaged 2 (50.0) 36 (48.0) 1.04 (0.38-2.85) .60
Responsible person not trained 16 (66.7) 22 (40.0) 1.66 (1.08-2.56) .02
Free-residual chlorine ,0.5 mg/L 33 (55.9) 5 (25.0) 2.23 (1.01-4.94) .01
Presence of risk assessment and management plan 1 (7.1) 26 (59.1) 0.12 (0.01-0.81) .0005
Chlorine disinfection 6 (28.6) 41 (56.2) 0.50 (0.25-1.03) .02
Cooling tower not protected from sunlight 18 (64.3) 20 (39.2) 1.63 (1.05-2.54) .02
www.ajicjournal.org
Vol. 38 No. 1
Mouchtouri et al. 53
water treatment with corrosion inhibitors to minimize
scaling and corrosion and with disinfectants is
suggested. In this study, we associated legionellae col-
onization with practices applied in cooling towers to
identify the factors that can effectively contribute to re-
duced legionellae proliferation. Our results demon-
strate less colonization in cooling towers with a risk
assessment and management plan. This plan includes
an integrated approach based on hazard assessment,
identication and monitoring of control measures,
application of management procedures, and establish-
ment of a verication program.
1,15
Biocidal treatment of cooling tower water reduces the
density of legionellae.
21,22
According to our results,
chemical water disinfection, including chlorination
seems to be more effective than other measures imple-
mented to maintain the system in good condition and
operation, suchas cleaning, repair of constructiondam-
ages, and antiscale treatment. These measures did not
inhibit contamination signicantly. As indicated in
another study, L pneumophila occurs even in well-main-
tained cooling towers.
23
However, ndings of other
studies are contradictory, because the presence of
deposits or aggregates, biolm, and algae seemed to
be related to viable counts of legionellae.
18,24
Our ndings suggest that old cooling towers are at
greater risk for colonization. Thus, control measures
should be applied more rigorously as towers age. The
risk assessment and management plan should be re-
viewed regularly, and special measures should be taken
as necessary, such as frequent application of control
measures and monitoring of old devices.
The lower colonization in cooling towers that were
periodically monitored for the presence of heterotro-
phic colony counts or Legionella spp may be attributed
to the fact that possible changes to the microbiological
quality of the water were identied and corrective
actions were taken when necessary. Contrarily, cooling
towers with no verication plan in place could not
track trends and changes in the water microbial load,
which is necessary to implementation of corrective ac-
tions and validation of implemented prevention and
control measures. Microbiological monitoring is part
of the verication and surveillance system of a water
safety plan, which can be applied to assess and manage
the risks associated with legionellae in cooling towers.
1
In a small proportion of the legionellae-positive cool-
ing towers (9%), the disinfection procedure was not ef-
fective in reducing the bacteria to acceptable levels. In
those cases, the chlorine disinfection procedure proba-
bly was not done properly. Measurement of pH and
free chlorine concentration and proper timing are es-
sential to ensure an efcient disinfection procedure.
The cooling tower system also should be reassessed
Table 4. Median (IQR) of cooling tower characteristics in association with Legionella spp contamination
Characteristics Legionella-positive cooling tower Legionella-negative cooling tower P
Water temperature,
o
C 26.8 (23.4-29.1) 25.1 (20.2-29.2) .30
Cooling tower age, years 17 (5.0-26.0) 6 (1.0-13.5) .01
Water loss, L 100 (0.0-6,000) 29 (0.25-80) .40
Total plate count 4,200 (450-22,600) 422 (14-9,600) .001
pH 8.1 (7.9-8.4) 8.0 (7.6-8.5) .40
Table 5. Association of Legionella spp colonization of cooling tower water samples with pH values, total plate counts, and
free chlorine concentrations
Legionella contamination of samples and water characteristics, cfu L
21
, 500 500 to 999 10
3
to 9999 10
4
to 10
5
. 10
5
P
pH
Number of samples 59 4 23 16 8
Median (IQR) 8 (7.6-8.5) 7.8 (7.5-7.9) 8.1 (8.0-8.5) 8.1 (7.9-8.4) 8.2 (7.7-8.3) .40
Total plate count, cfu mL
21
Number of samples 61 7 26 19 9
Median (IQR) 422 (14-9600) 170 (10-40,900) 7,006 (1700-20,000) 2,279 (679-11,580) 30,160 (402-137,840) .005
Free chlorine, mg L
21
Number of samples 49 3 22 16 7
Median (IQR) 0.22 (0.10-0.57) 2.52 (0.33-3.85) 0.12 (0.02-0.15) 0.09 (0.01-0.16) 0.16 (0.06-0.72) .002
54 Mouchtouri et al. American Journal of Infection Control
February 2010
for hazards, such as areas of standing water, with correc-
tive actions taken as necessary. Continuous water treat-
ment with chlorine will limit the risk of recolonization.
Cooling towers can be heavily colonized by Legion-
ella spp and thus present a potential infection risk.
Our study demonstrates the importance of a risk
assessment and management plan aimed at reducing
legionellae populations in cooling towers. Water chlori-
nation effectively reduces legionellae contamination.
Training of cooling tower operators is paramount.
Older cooling towers are at greater risk for legionellae
proliferation and thus require frequent, rigorous
preventive and control measures.
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