Isolation of Legionella pneumophila from clinical & environmental ...

Indian J Med Res 131, June 2010, pp 761-764
Isolation of Legionella pneumophila from clinical & environmental
sources in a tertiary care hospital
S. Anbumani, A. Gururajkumar & A. Chaudhury
Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, India
Received May 11, 2009
Background & objectives: Legionella pneumophila and other members of this genus are important
respiratory pathogens but legionellosis often remains a neglected and under reported condition. Hence,
this study was done to find out the presence of this organism in patients admitted to a tertiary care
hospital with community-acquired pneumonia (CAP).
Methods: A total of 470 lower respiratory tract samples and 24 water samples from hospital outlets were
examined. Culture was done on buffered charcoal yeast extract agar with supplements and identification
of the isolates was done by microscopy and biochemical tests.
Results: L. pneumophila could be isolated from 12 (2.55%) patients suffering from community-acquired
lower respiratory tract infection, unassociated with other aetiological agents of bacterial pneumonia. Of
the 24 water samples tested, 8 (33.3%) grew the same organism.
Interpretation & conclusion: Our study has shown that Legionella is present in the hospital environment
and was the aetiological agent of lower respiratory tract infection in 2.55 per cent of patients. A larger
study and reports from other parts of the country may help in determining the true significance of
legionellosis in India.
Key words Legionella culture - legionellosis - pneumonia - water supply
Legionella pneumophila and other members of
genus Legionella are Gram negative bacteria that
are ubiquitous in both natural aquatic and moist soil
environment 1,2 , and in artificial aquatic habitats 3 .
Human infection with Legionella has two distinct
forms- Legionnaires’ disease, which is a severe form
of infection which includes pneumonia; and Pontiac
fever, a milder flu like illness without pneumonia 4 .
Legionellosis occurs both sporadically as well as
outbreaks, in the community and also in the health
761
care setting. It has been implicated as the second most
common cause of community acquired pneumonia 5,6 ,
while nosocomial infection affects less than 1 per cent
of hospitalized patients 7,8 . Though major outbreaks
are most often highlighted in literature, most cases
of legionellosis are likely to be sporadic in nature 9 .
Water is the source of both nosocomial and community
acquired pneumonia. Environmental culturing of water
system for Legionella species has been recommended
for hospitals so that preventive measures can be
initiated 10 .

762 INDIAN J MED RES, JUNE 2010
Legionnaires’ disease remains a grossly under
reported condition as it is difficult to distinguish the
condition from other forms of pneumonia, unless
the organism is specifically looked for. In spite of
the under-reporting, many studies are available from
Europe and North America, but studies regarding
the isolation of Legionella either from clinical or
environmental samples are rare from India 11,12 . We
studied the presence of Legionella infection on the
basis of culture in patients admitted to the hospital with
signs and symptoms of lower respiratory tract infection
acquired in the community. Sampling of the hospital
water was also carried out to detect any Legionella
contamination in the hospital water supply system so
as to alert the hospital infection control committee
about its consequences.
Material & Methods
Patient samples: This study was carried out at Sri
Venkateswara Institute of Medical Sciences, Tirupati,
a post graduate teaching hospital in south India.
Clinical samples from respiratory tract received in
the Department of Microbiology from July 2007 to
December 2008 were included in this study. The samples
were obtained from patients who were hospitalized with
signs and symptoms of lower respiratory tract infection
(LRTI). Apart from sputum, the specimens included
bronchoalveolar lavage (BAL), endotracheal aspirate
and pleural fluid. After careful selection, 470 samples
which did not yield any bacterial respiratory pathogen
on routine culture and which were smear negative for
acid fast bacilli were processed for Legionella.
The samples were decontaminated by acid treatment
(0.2 KCl-HCl) followed by plating on buffered charcoal
yeast extract agar (BCYE) (Oxoid, UK) and BCYE
supplemented with polymyxin(80 u/ml), anisomycin
(40 µg/ml) and cefamandole (4 µg/ml) to make BCYE
selective for respiratory samples 13 . Plates were incubated
at 37 0 C and observed daily for 7 days.
Water samples: A total of 24 water samples were
collected from the taps of various wards and ICUs. Six
samples were obtained at 2 months’ interval from July
2007 to April 2008. The areas included the Medicine
ward, General ward-Male, General ward-Female,
Medicine ICU, Respiratory ICU, and the Emergency
Room. Samples were obtained by introducing a sterile
cotton swab into the opening of the taps and rotating it
along the inner sides of the nozzles. The swab was then
lightly streaked directly onto BCYE and then immersed
in 2.5 ml acid buffer, shaken vigorously and neutralized
with KOH. 0.1ml of the samples was spread with another
swab onto duplicate plates of the same medium 14 .
Identification: Iridescent, frosted glass colonies, 3-4
mm size, typical of Legionella spp. appearing in 3-4
days time were used for identification. Gram stain
was done to show the thin, faintly stained filamentous
Gram-negative morphology. Catalase positive and
oxidase negative colonies showing the typical
morphology were subcultured on plain BCYE and on
blood agar (BA) to determine if L-cysteine is essential
for growth 13 . Colonies growing only on BCYE and not
on BA were presumptively identified as Legionella
spp. The reference strain L. pneumophila Serogroup
1 Paris strain (CIP 107629T) obtained from French
National Legionella Reference Centre, Lyon, France,
was cultured in parallel with the clinical isolates and
the morphology and biochemical characteristics of the
clinical isolates were compared with this reference
strain before definitive identification. Hippurate
hydrolysis test was done by standard method 15 to
identify L. pneumophila species. Briefly, 0.4 ml of
1 per cent sodium hippurate (Hi-Media, Mumbai) was
inoculated with a loopful of organism and incubated
overnight at 37 0 C. Next day, 0.2 ml of 3.5 per cent
ninhydrin (Hi Media, Mumbai) solution in 1:1
acetone:butanol was added to the organism in hippurate
solution, mixed well, and incubated at 37 0 C for 10
min. Blue purple colour developing within 20 min
was considered positive. L. pneumophila CIP 107629T
strain was used as a positive control and Enterobacter
cloacae as the negative control.
Results
A total of 470 respiratory specimens from clinically
suspected cases of LRTI did not yield any respiratory
pathogenic bacterial isolate and were included in this
study. Among the patient population studied, 12 out
of the 470 (2.55%) were found to be culture positive
for L. pneumophila as identified by positive hippurate
hydrolysis test. The profile of these 12 patients,
along with the risk factors and clinical presentation
is shown in the Table. Majority were males, and were
>50 yr of age. Among the risk factors alcohol and /
or smoking habit was found in five patients and one
third of the patients had diabetes mellitus. Three
patients did not have any underlying risk factor. Fever
(100%), dyspnoea (91.7%) and cough (66.7%) were
the most common presenting features. Three quarters
of the patients had some radiological abnormality.
Maximum isolation was from sputum samples
(11/12) and one was from endotracheal aspirate.

Table. Characteristics of patients with legionellosis (n=12)
Characteristics No. (%)
Age (yr)
Range
Age groups (yr)
20-30
31-40
41-50
>50
Males
Females
RISK FACTORS
Smoking status
Current
Ex Smoker
Never
Alcohol consumption
Alcoholic
Non alcoholic
Co-morbid illnesses
None
Diabetes mellitus
Ischaemic heart disease
Asthma
End stage renal disease
Clinical presentation
Fever
Cough
Absent
Productive
Dry
Dyspnoea
Haemoptysis
Chest pain
Vomiting
Diarrhoea
X- ray finding (Consolidation)
ANBUMANI et al: LEGIONELLA ISOLATION FROM PATIENTS & ENVIRONMENT 763
Among 24 samples of water tested, 8 (33%) were
positive for L. pneumophila by culture. Among these
samples, maximum isolations were in the months of
July and October (three each), and one each in January
and April. The colony count was in the range of 180-
356/ plate (swab).
Discussion
24-76
3 (25)
1 (8.3)
0
8 (66.7)
10 (83.3)
2 (16.7)
1 (8.3)
2 (16.7)
9 (75)
2 (16.7)
10 (83.3)
3 (25)
4 (33.3)
3 (25)
3 (25)
1 (8.3)
12 (100)
4 (33.3)
6 (50)
2 (16.7)
11 (91.7)
2 (16.7)
1 (8.3)
4 (33.3)
1 (8.3)
9 (75)
Epidemics were the original presenting scenario
for Legionnaires’ disease in the 1980s, but most cases
are now known to be sporadic. Numerous observational
studies of patients with community-acquired pneumonia
(CAP) requiring hospitalization have documented that
the incidence of Legionnaires’ disease ranges from 2 to
9 per cent 6,16 . A recent Australian study has found that
3.4 per cent of CAP was caused by Legionella 17 , while
another study from UK reported the corresponding
figure to be 3 per cent 18 . Our study showed Legionelle
infection in 2.55 per cent patients. Male population
and age >50 yr are established predisposing factors
which have been found in the present study also. In
these patients, fever was the most common finding
(mean temperature at admission 39 0 C) followed
by respiratory symptom like cough and dyspnoea.
Studies 6,19 have found that a significant population of
patients have mild to moderate disease and do not have
expected co-morbidities like diabetes mellitus which
are also the finding in our study. Apart from systemic
and respiratory symptoms, gastrointestinal symptoms
like nausea, vomiting, and diarrhoea are known to be
common 4 as seen in our patients also. In our study,
majority (75%) had some radiological abnormality,
which is a common feature of Legionella pneumonia
and hence Legionnaires’ disease cannot be distinguished
from other causes of CAP on radiological grounds
alone 4 . For these reasons confining laboratory testing
for this organism specifically to high risk patients will
overlook a notable number of cases and our study also
supports the practice of placing more emphasis on
ascertaining the aetiology of pneumonia 19 .
Centres for Disease Control and Prevention
(CDC) does not support routine environmental culture
for Legionella because of the supposedly ill defined
relationship between the presence of the organism in
water system and risk of acquiring the infection 20 . In our
study, Legionella could be isolated from the hospital
water supply over a nine months period. There is an
increased yield of Legionella from the swab compared
to the filtration concentration technique 21 . This can be
explained by the fact that swab technique results in
direct sampling of the organisms present in the biofilm
consortium, which gives a greater yield than sampling
of water. It has been recommended that swab samples
be collected as part of any environmental Legionella
sampling protocol 21 . Our study has not included
hospital acquired pneumonia patients and the potential
sources of the infections were located outside this
hospital. So no immediate conclusion can be drawn
whether it is causing hospital acquired infections.
However, it has been advocated that if the water
supply is colonized by Legionella, then Legionnaires’
disease should be included in the differential diagnosis
of hospital acquired pneumonia 22 . In a study from
various hospitals, positive environmental sampling
raised the suspicion, and once culture for Legionella of
hospitalized patients started, cases were discovered 23 .

764 INDIAN J MED RES, JUNE 2010
Three important factors need to be mentioned.
Firstly, we have focused on a particular pathogen, but
in literature, concurrent infection particularly with
Streptococcus pneumoniae is well documented 24,25 .
Secondly, the fact that although culture is the most
sensitive method, but still it cannot detect all cases
since its sensitivity is 80 per cent 13 . And lastly, we have
not used other tests like urinary antigen testing or direct
fluorescent antibody stain which might have enhanced
the overall sensitivity of the microbiological diagnostic
workup. Our study highlights the importance of
environmental sampling of water and also the need for
introduction of routine laboratory testing for this not
so uncommon organism. Thus, a greater focus should
be placed on diagnosis of aetiology for early therapy
with appropriate antimicrobial substances. There is an
urgent need to find out the incidence of infection in
other parts of India to establish diagnostic setup for this
organism in referral hospitals.
Acknowledgment
Authors thank the physicians in the department of medicine and
the Emergency department for their co-operation, and acknowledge
Dr Sophie Jarraud of the French National Legionella Reference
Centre, Lyon, France, for providing the reference strain.
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Reprint requests: Dr Abhijit Chaudhury, Additional Professor, Department of Microbiology, Sri Venkateswara Institute of Medical
Sciences, Tirupati 517 507, Andhra Pradesh, India
e-mail: ach1964@rediffmail.com