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Journal of Clinical Microbiology, August 2000, p. 3128-3130, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Isolation of Legionella oakridgensis
from Two Patients with Pleural Effusion Living in the Same
Geographical Area
François
Lo
Presti,1
Serge
Riffard,1
Sophie
Jarraud,1
Florence
Le
Gallou,2
Hervé
Richet,2
François
Vandenesch,1 and
Jerome
Etienne1,*
Centre National de Référence des
Légionelles EA1655, Faculté de Médecine R. T. H. Laennec, Rue Guillaume Paradin, 69372 Lyon Cedex
08,1 and Laboratoire de
Bactériologie-Virologie, Institut de Biologie des Hôpitaux
de Nantes, Centre Hospitalier Universitaire de Nantes, Quai Moncousu,
44035 Nantes Cedex 01,2 France
Received 4 January 2000/Returned for modification 25 February
2000/Accepted 1 May 2000
 |
ABSTRACT |
Two cases of Legionnaire's disease caused by Legionella
oakridgensis were diagnosed at the university hospital in Nantes, France. The two patients' isolates were identified by means of phenotyping and genotyping methods. Epidemiological investigations concluded that the first case was hospital acquired while the second
case was considered community acquired.
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CASE REPORTS |
Case 1.
A 69-year-old man with a
1-year history of rheumatoid arthritis was admitted to Hotel Dieu
hospital on 20 September 1993 with idiopathic thrombopenic purpura
requiring splenectomy. He was discharged 9 days after surgery and
returned home to a town located 50 km from Nantes. Fifteen days later
he was readmitted to the hospital for fever, dyspnea, and chest pain.
Bilateral ronchi and crackles were heard on auscultation of the lungs;
the chest X-ray film showed left pleural effusion, and computed
tomography revealed an encysted pleural pocket. Bronchial brushing
performed on 26 October with culture on buffered charcoal yeast extract (BCYE) agar yielded a gram-negative Legionella-like
bacterium. Cultures of pleural fluid were sterile. Serologic tests for
Legionella pneumophila were negative. He recovered after
receiving intravenous (i.v.) cefotaxim and oral ciprofloxacin for 6 days, followed by oral roxithromycin for 2 weeks.
Case 2.
A 79-year-old woman living in Nantes who had a 2-year
history of rheumatoid arthritis and Parkinson's disease was admitted to Hotel Dieu hospital on 21 November 1993 for severe pneumonia of the
left lung with chest pain. Chest radiography revealed bilateral lower
lobe pneumonia with interstitial opacities and left pleural effusion.
Bronchial brush culture performed on 22 November yielded gram-negative
bacilli on BCYE agar after 9 days of incubation. The patient was first
treated with oral ciprofloxacin and oral amoxicillin-clavulanate for 10 days and then with oral roxithromycin for 5 days. She recovered from
the pneumonia and was discharged.
Microbiological investigations.
On primary culture, the
two isolates grew on BCYE agar supplemented with 0.1%
-ketoglutarate (BCYE) and BCYE agar supplemented with glycine,
vancomycin, and colistin, but not on blood agar or on BCYE agar
without L-cysteine. Each isolate yielded positive catalase
and oxidase tests, negative glucose fermentation, and no urease or
nitrate reduction. Neither isolate was autofluorescent under UV
light (365 nm). These biochemical characteristics are shared by
Legionella hackeliae serogroup 1, Legionella
jordanis, Legionella londiniensis, Legionella
moravica, Legionella sainthelensi serogroup 1, Legionella santicrucis, Legionella spiritensis,
and Legionella oakridgensis (although L. oakridgensis reference strain OR-10/ATCC 33761 is described as
oxidase negative [7]). The two isolates were tested by
direct immunofluorescent assay (DFA) with unadsorbed antisera as
previously described (1) and reacted strongly with antisera
against L. oakridgensis, L. sainthelensi serogroup 1, and L. hackeliae serogroup 1.
Random amplified polymorphic DNA (RAPD) typing was performed on the two
isolates by using primer SK2 (5'-CGGCGGCGGCGG-3') (6), and infrequent-restriction-site PCR (IRS-PCR) was
carried out using primers FGPS1490-72 (5'-TGCGGCTGGATCCCCTCCTT-3')
and FGPL132'-38 (5'-CCGGGTTTCCCCATTCGG-3')
(8). RAPD profiles were consistently reproducible in
several independent determinations, and the two profiles obtained were
more than 91% related to that of the L. oakridgensis type
strain (Fig. 1) and to those of six L. oakridgensis isolates cultured from four French thermal
spas and two hospitals (one in Lille, France, and one in Portugal), all
of which had previously been characterized by RAPD (5) (not
shown). These profiles were less than 80% related to those of all
other Legionella species (Fig. 1). Similarly, the 16S-23S intergenic spacer region PCR profiles of the clinical isolates were
identical to that of the L. oakridgensis type strain
OR-10/ATCC 33761 (data not shown). The two clinical isolates were
therefore identified as L. oakridgensis (deposited in the
American Type Culture Collection as strains ATCC 700515 and ATCC
700516), and the diagnosis of L. oakridgensis pneumonia with
pleural effusion was established in both patients.
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FIG. 1.
RAPD profiles of L. oakridgensis strains and
other Legionella reference strains. Lanes M, molecular
weight marker Ready-load 100 bp (Gibco BRL); lane 1, L. oakridgensis ATCC 33761; lane 2, strain 930101868/ATCC 700515 (patient 1, Nantes); lane 3, strain 930101937/ATCC 700516 (patient 2, Nantes); lane 4, strain 88020285; lane 5, strain 88020400; lane 6, strain 88020413; lane 7, strain 88020417 (lanes 4 to 7 are
environmental isolates from Nantes hospital); lane 8, L. oakridgensis strain 91020991 (thermal spa Bourbon-Lancy); lane 9, L. oakridgensis strain ATCC 33761; lane 10, L. hackeliae ATCC 35250; lane 11, L. jordanis ATCC 33263;
lane 12, L. londiniensis ATCC 49505; lane 13, L. moravica ATCC 43877; lane 14, L. sainthelensi ATCC
35248; lane 15, L. santicrucis ATCC 35301; lane 16, L. spiritensis ATCC 35249.
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As patient 1 was considered to have hospital-acquired legionellosis,
the source was investigated by analyzing samples of the
hospital's hot
water distribution system. Cultures yielded
Legionella isolates which were identified as
L. oakridgensis by using
the
same molecular methods as above (phenotypically, the isolates
were
oxidase positive, like the patients' isolates) (Fig.
1, lanes
4 to
7).
The clinical and environmental isolates were compared by means of
IRS-PCR with
PstI and
XbaI restriction enzymes.
The IRS-PCR
profiles of our clinical and environmental isolates were
compared
to those of the six
L. oakridgensis control
environmental isolates
mentioned above. The reproducibility of the
method was 100% when
each isolate was tested three times. Nine
different IRS-PCR patterns
were obtained, each composed of three to
nine bands ranging from
200 to 1,000 bp, confirming the discriminatory
power of the method
(Fig.
2). The two
patients' isolates and two of the four Nantes
hospital isolates each
had a unique IRS-PCR profile (Fig.
2, lanes
1, 3, 6, 9), and the third
and fourth Nantes hospital isolates
each had a distinct profile (Fig.
2, lanes 2 and 4).
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FIG. 2.
IRS-PCR profiles of L. oakridgensis reference
strains and isolates. Lanes M, molecular weight marker VI (Boehringer
Mannheim); lane 1, strain 89020417; lane 2, strain 88020285; lane 3, 89020400; lane 4, strain 89020413 (lanes 1 to 4 contain isolates from
the water distribution system of the Nantes hospital); lane 5, strain
92203624 (from the water distribution system of a Lille hospital); lane
6, strain 930101868/ATCC 700515 (patient 1, Nantes); lane 7, L. oakridgensis strain OR-10/ATCC 33761; lane 8, strain 9020498 (thermal spa, Aix-les Bains); lane 9, strain 930101937/ATCC 700516 (patient 2, Nantes); lane 10, strain 90020551 (from the water
distribution system of a Portuguese hospital).
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L. oakridgensis was originally isolated from water in the
environment (
7,
13) and was subsequently found to be
responsible
for several human cases of pleurisy (
4,
10,
11).
However,
these human infections were diagnosed by DFA on clinical
specimens
and were not confirmed by culture of the organism (
4,
10,
11). Our two cases of
L. oakridgensis
legionellosis were diagnosed
on the basis of strain isolation. Isolates
were recovered from
bronchial brush specimens of patients with pleural
effusion and/or
pneumonia. Both patients had a previous history of
immunological
disorders treated with steroids (idiopathic
thrombocytopenic purpura
and rheumatoid arthritis, respectively),
possibly rendering them
more susceptible to infection. Pleural effusion
was also present
in the three previously reported cases of human
L. oakridgensis infection (
4,
10,
11), suggesting
that this species might
produce specific virulence factors (adhesins
and/or extracellular
products) favoring tropism for pleural
tissue.
Biochemical tests poorly distinguish
L. oakridgensis from
other
Legionella species (
2,
12), and
cross-reactions occur
with
L. sainthelensi in DFA
(
3). Definite
L. oakridgensis identification
is
more easily achieved with molecular techniques, such as RAPD
and
ISR-PCR (
5,
8), and we have successfully applied these
methods in the present report. If pulsed-field gel electrophoresis
and
IRS-PCR have proven useful for epidemiological comparison
of
L. pneumophila isolates (
9), we show here that IRS-PCR can
be applied to
L. oakridgensis for microbiological
investigation
of clinical cases. Case 1 was considered hospital
acquired, with
probable infection directly from the hospital water:
L. oakridgensis isolates from the patient and from hospital
sources had the same
IRS-PCR profiles. Case 2 was considered community
acquired, as
the patient had not been in contact with the hospital for
several
months before onset, even though the isolate had an IRS-PCR
profile
identical to that of the first patient's isolate. As patient 2
lived in Nantes, it is conceivable that the town water distribution
system contained the same
L. oakridgensis strain, but
cultures
were not done at the time. Hence, patients from the same
geographical
area can be infected by an unusual
Legionella
strain that has
only rarely been associated with human
infections.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centre National de
Référence des Légionelles EA 1655, Faculté de
Médecine R. T. H. Laennec, Rue Guillaume Paradin, 69372 Lyon
Cedex 08, France, Phone: 33 (0) 478-77-86-57. Fax: 33 (0) 478-77-86-57. E-mail: jetienne{at}univ-lyon1.fr.
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Journal of Clinical Microbiology, August 2000, p. 3128-3130, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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