D. M. BURAK, Z. ZEYBEK
Turk J Biol
35 (2011) 679-685
© TÜBİTAK
doi:10.3906/biy-0907-93
Investigation of Legionella pneumophila and free living
amoebas in the domestic hot water systems in İstanbul
Dilara Meryem BURAK, Zuhal ZEYBEK
Department of Biology, Fundamental and Industrial Microbiology, Faculty of Science,
İstanbul University, İstanbul - TURKEY
Received: 27.07.2009
Abstract: Legionella pneumophila (L. pneumophila), known to cause legionnaires’ disease, has been found in various
aquatic environments and man-made water systems. Free living amoebas (FLAs) live in the same environments and can
cause infections such as encephalitis, keratitis. Moreover, FLAs play a major role in L. pneumophila transmission. In the
present study, the presence of L. pneumophila and FLAs was examined in water and swab samples obtained from shower
heads (hot water) in 61 houses in İstanbul. Nonnutrient agar (NNA) was used for isolation of FLAs in all samples and
buffered charcoal yeast extract (BCYE) agar with addition of glycine, vancomycine, polymixin, and natamycin (GVPN)
was used for isolation of L. pneumophila. These bacteria have been isolated from 21.3% of the houses examined and
FLAs from 31%. Of the isolated L. pneumophila, 87.5% were named as L. pneumophila serogroup 2-14 and 12.5% as L.
pneumophila serogroup 1. No significant correlation was found between the presence of L. pneumophila and FLAs. A
significant correlation was found between the presence of L. pneumophila and the presence of a central heating system.
Key words: Legionella pneumophila, free living amoeba, domestic hot water, shower heads
İstanbul’daki evlerin sıcak su sistemlerinde Legionella pneumophila ve
özgür yaşayan amiplerin araştırılması
Özet: Lejyoner hastalığı etkeni olarak bilinen Legionella pneumophila (L. pneumophila) çeşitli su ortamlarında ve insan
yapımı su sistemlerinde bulunmaktadır. Aynı ortamlarda özgür yaşayan amipler de (ÖYA) yaşar ve ensefalit, keratit
gibi infeksiyonlara sebep olur. ÖYA, aynı zamanda L. pneumophila’nın yayılmasında büyük bir rol oynar. Bu çalışmada
İstanbul’daki 61 eve ait duş başlıklarından alınan sıcak su ve sürüntü örnekleri L. pneumophila ve ÖYA açısından
incelenmiştir. Örneklerden ÖYA izolasyonu için Nonnutrient agar (NNA) L. pneumophila izolasyonu için glisin,
vankomisin, polimiksin ve natamisin ilave edilmiş buffered charcoal yeast extrakt agar (BCYE-GVPN) kullanılmıştır.
İncelenen evlerin %21,3ünden L. pneumophila, % 31inden ÖYA izole edilmiştir. İzole edilmiş olan L. pneumophila
bakterilerinin %87,5i L. pneumophila serogrup 2-14, %12,5i L. pneumophila serogrup 1 olarak isimlendirilmiştir. L.
pneumophila ve ÖYA ların varlığı arasında anlamlı bir korelasyon bulunmamıştır. L. pneumophila bakterilerinin varlığı
ile merkezi ısıtma sisteminin varlığı arasında anlamlı bir ilişki bulunmuştur.
Anahtar sözcükler: Legionella pneumophila, özgür yaşayan amipler, evsel sıcak su, duş başlığı
Introduction
Legionella pneumophila (L. pneumophila) is
present in low numbers in natural environments such
as fresh water, rivers, and lakes. They pass from these
natural environments to man-made water systems
like cooling towers, houses, hotels, and hospitals (14). When they live both in water and in the biofilm
layers of these environments, they can propagate in
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Investigation of Legionella pneumophila and free living amoebas in the domestic hot water systems in İstanbul
association with the free-living amoebas (FLAs) such
as Acanthamoeba, Naegleria, and Hartmanella (5-9).
It is known that L. pneumophila causes
legionnaires’ disease and Pontiac fever, and FLAs
are a factor for keratitis and encephalitis (1,10,11).
Legionella bacteria present within FLAs can survive
and become resistant to inappropriate environmental
conditions (10,12).
L. pneumophila and FLAs living in the same
aquatic environment can be affected by temperature,
pH, type of heating system and material, age of water
system, content of disinfectants such as chlorine, and
each other. In several legionnaires’ disease epidemics,
FLA and Legionella bacteria have been isolated from
the same infection source and it was found that the
isolated amoebas support the growth of Legionella
bacteria within the cell (13-19).
In the present study, the frequency of the presence
of Legionella bacteria and FLAs was investigated
in hot water and swab samples obtained from the
shower heads of houses in İstanbul. Furthermore,
the relationship between these microorganisms and
the various characteristics of the buildings, waters,
and water systems (temperature, pH, free chlorine
amount, building age, material of water tank, date
and manner of the most recent cleaning, age and
cleanliness of shower head, pipe material and age,
water heating system, etc.) was investigated.
Materials and methods
Sample collection
In the present study, water and swab samples were
obtained from shower heads of 61 houses on the
European and Asian sides of İstanbul; 2000 mL and
500 mL hot water samples were placed into different
sterile containers where sodium thiosulphate (0.05%,
0.1 N sodium thiosulphate) had been added in
advance. During sampling, temperature, pH, and free
chlorine content of water samples were measured.
Swab samples were obtained from the inner
surface of shower heads, by removing the shower
head and using sterile swabs. Then, these swabs were
placed in sterile plastic centrifuge tubes containing 2
mL of hot water obtained from the same point. The
samples were processed for 24 h.
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Isolation of Legionella pneumophila
Hot water samples (2000 mL) obtained from the
shower heads were concentrated using filtration. The
concentrated samples (in 20 mL of distilled water)
were grouped into 2 to be treated with acid (HCl KCl) and heat (50 °C). Then 0.1 mL was taken from
each of them to be inoculated in GVPN - BCYE agar.
Scraping samples taken using swabs were mixed
with the water in their tubes and then they were
removed. After that, HCl-KCl solution was added to
the tubes at the rate of 1/1. These last compositions
were inoculated into the GVPN-BCYE agar after
waiting for 5 min. All petri dishes were kept at 37 °C
for 14 days. Suspicious colonies were identified using
Legionella latex agglutination test kit (Oxoid) (20).
Isolation of FLAs
Hot water samples (500 mL) obtained from the
shower heads were concentrated using a cellulose
nitrate membrane filter (0.45 μm) (Millipore). The
membrane filter was placed inverted onto the nonnutrient agar (NNA) seeded with Escherichia coli
before. Petri dishes were incubated for 10 days at 30
°C. All of them were examined for amoeba cysts and
trophozoites every day under a light microscope at
10× and 40× (11,21).
Characteristics of buildings, water, and water
systems
Information on buildings and water/water
systems was obtained through a questionnaire form.
This form contains information about building age;
materials, most recent cleaning date and cleaning
material for water tanks; age and cleanliness for
showerheads; and age and material for pipes (Table
1).
Results and discussion
The present study covers a total of 122 samples
taken from hot water and swabs from the shower
heads in 61 houses. L. pneumophila was isolated from
13 (21.3%) and FLAs from 19 (31%) of the houses.
L. pneumophila was isolated from 12 (19.6%) of the
water samples and 4 (6.5%) of the swab samples
(Figure). Borella et al. isolated L. pneumophila
from 22.6% of the domestic hot water samples (22).
Zacheus et al. isolated L. pneumophila from 30% of
D. M. BURAK, Z. ZEYBEK
Table 1. Characteristics of buildings and water systems.
Characteristics
L. pneumophila (+)
FLA(+)
Buildings
Age (year)
min/max
8-14
min/max
5-30
Water tanks
Material
Most recent cleaning
Cleaning material
Ceramic
1-6 months ago
Sodium hypochloride
Ceramic
1-6 months ago
Sodium hypochloride
Shower heads
Age (year)
Cleanliness
1-10
3-30 day/detergent
1-10
3-30 day/detergent
Pipes
Age (year)
Material
5-14
plastic and steel
3-15
plastic and steel
FLA: Free living amoeba
5
Swab
Water
L. pneumophila log Cfu/mL
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
D1
D2 D49 D50 D51 D52 D53 D54 D55 D56 D57 D60 D61
Codes of buildings
Figure. L. pneumophila isolated from water and swab samples.
the Finnish domestic hot water samples (23). These
results are similar to those of the present study
(21.3%). However, these bacteria were isolated
at higher rates in the other studies. Pringler et al.
isolated Legionella bacteria from 89% of the hot water
obtained from Danish buildings (24) and Uzel et al.
from 76.2% of the hot water samples from hotels in
İzmir (25).
Although L. pneumophila were isolated in 8
(13%) buildings, no FLAs were isolated in the same
buildings in the present study. Of the 13 houses
where L. pneumophila was isolated, FLAs were also
isolated from 5 houses. FLAs were not isolated
from the remaining 8 houses. On the other hand,
FLAs grew but L. pneumophila did not grow in
14 (23%) of the examined buildings. FLAs and L.
pneumophila grew together in 5 (8%) buildings. It
was not determined whether there was a statistically
significant correlation between the presence of L.
pneumophila and FLAs in the same hot water system
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Investigation of Legionella pneumophila and free living amoebas in the domestic hot water systems in İstanbul
(P > 0.05). Similarly, Sanden et al. have identified
Legionella bacteria in 41% in cases with presence of
amoebas and in 5% in cases with absence of them (26).
Franzin et al. isolated only amoebas from 5.7% and
only Legionella bacteria from 41.6% of the hospital
water systems (27). Although there are many studies
proving that FLAs host the growth of L. pneumophila,
Surman et al. showed that L. pneumophila does not
need protozoons for reproduction (28). New studies
are required showing the interaction between FLAs
and L. pneumophila strains isolated in our samples.
Moreover, isolates of FLAs must be identified at
genus and species level.
L. pneumophila serogroup 1 appears to be
responsible for the majority of legionellosis cases.
In the present study 2 (12.5%) L. pneumophila
serogroup 1 strains and 14 L. pneumophila serogroup
2-14 (87.5%) strains were isolated from water and
swab samples. Uzel et al. have isolated 85.9% L.
pneumophila serogroup 1, 6.25% L. pneumophila
serogroup 2-14, and 7.8% other Legionella types
from the 168 samples obtained from hot water
samples in İzmir (25). Cordes et al. have isolated
L. pneumophila serogroup 1, 4, and 6 from hospital
shower heads (29), while Borella et al. have isolated L.
pneumophila serogroup 1, 3, 6, and 9 from domestic
hot water samples (22). It is known that one of the
factors influencing L. pneumophila infections is the
number of bacteria in water (10). Colony numbers
of L. pneumophila were calculated as 5 × 101-26.6 ×
103 CFU/mL in water samples and 2.9 × 103-28.6 ×
103 CFU/mL in swab samples (about 10 cm2). Borella
et al. have reported the average colony number of L.
pneumophila isolated from Italy in the range 25-8.7
× 104 CFU/mL (22). Alary et al. have calculated the
average colony numbers of L. pneumophila isolated
from domestic hot water in Canada as 16.2 × 103
and 18.3 × 103 CFU/mL (18). In the present study,
the culture method used for investigating both
Legionella bacteria and FLAs is one of the commonly
used methods. In addition to this method, which
is a golden standard, we think necessary molecular
techniques will be used such as polymer chain
reaction and fluorescent in situ hybridisation in the
future, because Legionella bacteria cannot be cultured
when phagocytosed by FLAs and so we cannot know
the actual number of these bacteria in the water
systems.
682
It is known that water temperature, pH, and
free chlorine amounts affect Legionella bacteria and
FLA reproduction. In the water samples in which L.
pneumophila and FLAs were isolated, temperatures
were 45-53 °C, pH values were 6-7, and free chlorine
amounts were 0.3-0.5 ppm. These results are
consistent with other studies (22,30).
Characteristics of the buildings where the
microorganisms under examination were found are
given in Table 1. In our study, we took the specimens
from the biofilm layer with swabs and found out
that the building (from) which L. pneumophila was
isolated from only swab samples was 10 years old and
the shower head was 3 years old. Additionally, all of
the buildings where L. pneumophila were isolated
had water tanks that were suitable for formation of
biofilm layer. A statistically significant correlation
was detected between the presence of a water tank in
the buildings and the isolation of L. pneumophila and
FLAs in the water samples taken from the buildings
in question (χ2 test, P < 0.001). The temperature of
hot water tanks is one of the main factors associated
with the presence of Legionella, which are able to
grow between 25 and 45 °C. Stagnation and biofilm
are other factors. High level of Legionella can also be
explained by the stagnation of water in the storage
tanks, which enhances the formation of biofilm.
Moreover, FLAs frequently live in the biofilm layer
and L. pneumophila can parasitise in these cells.
Therefore, FLAs play an essential role in the ecology
and epidemiology of Legionella. In fact, Stout et al.
have proven that hot water tanks and the microflora
therein support the life and growth of L. pneumophila
(31). For these reasons, we suggest that swab samples
as well as water samples should be examined
especially in the water systems of old buildings.
According to the results of the present study,
although both the shower heads and the water
tanks were cleaned (information derived from the
questionnaire), both L. pneumophila and FLAs grew
from buildings with tanks that had also been cleaned
1 month earlier and the shower heads that had also
been cleaned 3 days ago. In the present study, both
Legionella bacteria and FLA growth in the samples
were obtained from clean shower heads. Similarly,
Cordes and Wiesenthal reported L. pneumophila
was isolated from shower heads. Moreover, they
D. M. BURAK, Z. ZEYBEK
Table 2. The relation between the growth of L. pneumophila and FLAs and the water heating system.
Microorganisms
Central Heating System
(n = 18)
Independent Heating System
(n = 43)
Number
(%)
Number
(%)
L. pneumophila
13
72
0
0
FLAs
5
28
14
33
FLAs: Free living amoebas
found Legionella bacteria again, after 7 days from the
disinfecting of shower heads (29). It can be mentioned
that outer cleaning made in order to prevent
settlement of microorganisms on shower heads is not
very significant in terms of L. pneumophila or FLAs.
The effects of different materials on growth of
L. pneumophila and FLAs in the biofilm have been
shown (32,33). In the present study, it was found that
among the buildings with L. pneumophila growth,
84% had plastic and 15% had steel piping. Among
the buildings with FLA growth, 63% had plastic and
36% had steel piping. However, in a previous study,
L. pneumophila were isolated from 22.8% of water
systems where iron and plastic were used together
but no bacteria were isolated from those made of
plastic only (34).
It was found that of the houses examined 18 were
heated by central system and 43 by independent
system (Table 2). There was a statistically significant
correlation between water being heated centrally and
the presence of L. pneumophila (P < 0.001). Similarly,
Borella et al. have shown that in domestic hot water
systems L. pneumophila contamination mostly
occurs in central heating systems and the distance
between water and heating system (especially in
buildings where the distance is more than 10 m)
largely influences this contamination (22). We did not
isolate L. pneumophila from any independent heating
system houses. Similarly, Bates et al. did not isolate
Legionella bacteria from any of the 100 houses that
were heated by independent heating systems (35).
Contrary to our findings, Alary et al. have isolated
Legionella bacteria 84% from waters that were heated
by independent systems (18).
In conclusion, L. pneumophila and FLAs cohabit
in the water systems of buildings in İstanbul. It
is observed that these systems provide suitable
temperature, pH, and chlorine amount for the survival
of these microorganisms. Moreover, according to
our results, presence of a water tank and central
heating is an advantage for their survival. We do not,
however, know the relationship between our isolates
of L. pneumophila and FLAs. These interactions may
be antagonistic or synergistic in the same aquatic
environment because it is known that some bacteria
strains feed some FLA strains. On the other hand,
sometimes the presence of a mucoid capsule around
bacteria appears to impede phagocytosis by FLAs
and leads to bacterial overgrowth of the amoeba
population. Therefore, further studies are necessary
to show the interaction between the isolates in the
present study.
Acknowledgment
This study has been supported by İstanbul
University Scientific Research Projects Unit with no
T-743/13092005.
Corresponding author:
Zuhal ZEYBEK
Department of Biology,
Fundamental and Industrial Microbiology,
Faculty of Science, İstanbul University,
İstanbul - TURKEY
E-mail: [email protected]
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Investigation of Legionella pneumophila and free living amoebas in the domestic hot water systems in İstanbul
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