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Previous Article | Next Article 
Journal of Clinical Microbiology, December 2001, p. 4588-4590, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4588-4590.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
One Case Each of Recurrent Meningitis and
Hemoperitoneum Infection with Ralstonia
mannitolilytica
Mario
Vaneechoutte,1,*
Thierry
De
Baere,1
Georges
Wauters,2
Sophia
Steyaert,1
Geert
Claeys,1
Dirk
Vogelaers,3 and
Gerda
Verschraegen1
Department of Chemistry, Microbiology and
Immunology1 and Department of Infectious
Diseases,3 Ghent University Hospital, Ghent, and
Department of Microbiology, UZ St. Luc,
Brussels,2 Belgium
Received 30 July 2001/Returned for modification 21 August
2001/Accepted 1 October 2001
 |
ABSTRACT |
Two clinical cases of infection with Ralstonia
mannitolilytica are described: a recurrent meningitis on an
implanted intraventricular catheter and an infected hemoperitoneum as a
complication of a cholangiocarcinoma. The strains were first
misidentified as Pseudomonas fluorescens and
Burkholderia cepacia. Further testing lead to the
identification as Ralstonia pickettii biovar
3/"thomasii," which was recently shown to represent
a separate species, R. mannitolilytica (List editor N. Weiss, Int. J. Syst. Evol. Microbiol. 51:795-796, 2001),
originally described as R. mannitolytica (De Baere et al., Int. J. Syst. Evol. Microbiol. 51:547-558,
2001). R. mannitolilytica can be distinguished
from all described Ralstonia species by its acidification
of D-arabitol and mannitol and by its lack of
nitrate reduction and of alkalinization of tartrate. In order to
determine the true prevalence of infections with this species,
colistin-resistant "P. fluorescens" strains and strains
growing on B. cepacia selective medium deserve further attention.
| |
CASE REPORTS |
Case report 1. In 1997, a
Caucasian woman, 38 years old, presented with fever of unknown origin.
At the age of 17 she had received a ventriculoatrial draining for
hydrocephalia after an intracerebral hematoma. Twenty years later,
after a localized epileptic insult, further neurological testing and
imaging pointed to a diagnosis of cavernous hemangiomas, for which she
was treated surgically. The nonfunctional ventricular drain was
partially removed, leaving the intrathoracic part in place.
Postoperatively, the patient developed meningitis. Culture remained
negative, and the patient was treated with 2 g of ceftriaxone
intravenously (i.v.) twice daily during 2 weeks. Two days after
antibiotic therapy was stopped, the meningitis recurred. Culture of the
liquor yielded a gram-negative bacillus that could grow on
Burkholderia cepacia selective medium (Mast Diagnostics,
Merseyside, United Kingdom) and therefore was first identified as
B. cepacia. The same organism was cultured from the removed
intracerebral catheter segment. Ceftriaxone (2 g i.v. twice per day)
was started, and ciprofloxacin (400 mg i.v. thrice per day) was added.
Fever subsided and liquor cultures remained negative.
In February 1998, the patient presented in a private hospital with a
generalized epileptic insult and high fever (39.5°C) and was treated
with amoxicillin and clavulanic acid. The fever subsided, and the
single blood culture, positive for "Pseudomonas fluorescens," was considered contaminated. From March 1998 onwards, the patient had repeatedly febrile episodes and lost 10 kg of weight. Blood cultures were not performed. In November 1998, the patient was admitted in the same private hospital for high fever and
tonic-clonic insults. Seven blood cultures were found positive for
"P. fluorescens," and the patient was referred to the
Ghent University Hospital for removal of the endovascular catheter
segment. Cultures of the removed catheter were positive and were
identified as Ralstonia pickettii biovar
3/"thomasii." Retrospectively, it was shown that this
was also the correct identification for the "P.
fluorescens" isolates that had been obtained from the private hospital. The strain was resistant to ampicillin, gentamicin, temocillin, and aztreonam but was susceptible to cotrimoxazole, piperacilin, cefuroxime, cefotaxime, ceftazidime, imipenem, and quinolones. The patient was treated, according to the susceptibility testing results, with cotrimoxazole and doxycycline. Since then, the
patient has been doing well.
Case report 2.
In December 1997, primary cholangiocarcinoma
with extensive hepatic involvement was diagnosed in a 32-year-old
woman. Chemotherapy with cis-platinum and 5-fluoroacil was
started with good clinical response and improvement of the hepatic
lesions. Six months later, the patient was seen in the surgery
department for partial resection of the liver in order to reduce the
tumor mass and to improve the effect of chemotherapy. One week after
the resection, computerized axial tomography (CAT) scanning revealed
that the Kehr drain was leaking into the abdomen. A hemoperitoneum was
diagnosed, and a review of the abdomen revealed bleeding of the right
vena subhepatica. Intraoperatively, a specimen was taken from
the hematoma for culture. After enrichment in thioglycolate broth,
Enterococcus sp. and a gram-negative nonfermenting bacillus
were isolated. Three days after review, the patient developed fever
with peaks up to 39°C and antibiotherapy with cefuroxime was started.
Abdominal drainage fluid culture yielded the same gram-negative
nonfermenter. This strain was resistant to ampicillin, gentamicin,
colimycin, and temocillin but susceptible to cotrimoxazole, cefuroxime,
and quinolones. Despite cefuroxime treatment, fever persisted and
metronidazole was added. A CAT scan of the abdomen showed an excessive
amount of free abdominal fluid. One week later, small numbers of the nonfermenter and of Enterococcus sp. could still be isolated
from the abdominal drainage fluid. Antibiotherapy was switched to
piperacillin and tazobactam, and a new abdominal drain was placed. As
the patient remained febrile, drug fever was suspected and antibiotics
were stopped. A subsequent fluid specimen again grew
Enterococcus sp. and a gram-negative, nonfermenting
bacillus, later identified as R. mannitolilytica. The
patient's condition improved very slowly, and finally the patient was
discharged from the hospital knowing that new intrahepatic lesions were
detected on CAT scan.
Strain LMG 19090, obtained from patient 1, was isolated on conventional
media and could grow on Burkholderia cepacia selective medium, containing 100 mg of ticarcillin/liter and 300 U of polymyxin B/ml. API 20NE (BioMérieux, Marcy l'Etoile, France) testing
identified the strain as P. fluorescens (profile code 0 054 555). Because of colistin resistance, this strain was studied in more
detail (3, 7, 13, 14), which led to an identification as
R. pickettii biovar 3/"thomasii."
The further data gathered by means of polyphasic taxonomy led to the
description of this biovar as a separate species, named R. mannitolilytica, referring to its characteristic acidification of
mannitol, unlike all other described Ralstonia species
(5). The original spelling of the specific epithet
"mannitolytica" (5) was corrected to
"mannitolilytica" (8).
In retrospect, strain LMG 19091 from patient 2, which had been
identified previously as P. fluorescens (API 20NE profile
code 0 045 555), and strain LMG 6866T, isolated
at St. Thomas' Hospital (London, United Kingdom) during an outbreak
and deposited as "Pseudomonas thomasii" in 1972 (NCIB 10805) (11, 12), could both be identified as R. mannitolilytica.
The G+C content for all three R. mannitolilytica strains
tested was 66.2 mol%, which is higher than the values for R. pickettii (64.0 to 64.1%) (5). The 16S rDNA
sequences of the clinical strains (GenBank accession numbers AJ270256
and AJ270257) were identical and clustered at more than 99.5% sequence
similarity with the R. mannitolilytica type strain LMG
6866T (GenBank accession number AJ270258)
(5). The 16S rDNA sequences for the R. pickettii biovar Va-1 and Va-2 strains clustered at 96%
similarity versus R. mannitolilytica. DNA hybridization
confirmed that the two clinical strains and the type strain belonged to a separate species (5). When tRNA PCR was performed
(1, 9), all three strains had a PCR fragment of 108.4 bp
(standard deviation, 0.06 bp), in combination with one or two other
variably present fragments. The obtained tRNA PCR fingerprints were
sufficiently discriminative for us to recognize each strain as being
R. mannitolilytica.
The two clinical R. mannitolilytica strains were motile by a
single polar flagellum, while motility was not observed for the culture
collection R. mannitolilytica type strain LMG
6866T. It was observed that freshly isolated
strains were very motile and that motility decreased upon prolonged
preservation and subculture, which could explain the nonmotility of the
type strain. All three strains grew at 30, 37, and 42°C and were
viable for less than 6 days on tryptic soy agar (Becton Dickinson,
Cockeysville, Md.) at 25°C. Oxidase and catalase were positive. They
were resistant to desferrioxamine, O:129, and colistin. No acid was
produced from ethylene glycol. Urease, pyrrolidonyl arylamidase (Rosco, Taastrup, Denmark), Tween esterase, and phenylalanine deaminase were
positive. Acid was oxidatively produced from glucose,
L-arabinose, lactose, maltose, mannitol,
D-arabitol, and D-xylose.
Alkalinization occurred on minimal mineral agar with acetate, serine,
malonate, 
-alanine, 4-aminobutyrate, azelate, succinate, fumarate,
butyrate, formate, malate, mucate, galacturonate, citrate, histidine,
and lactate but not with acetamide, adipate, alginate, allantoin, amygdalin, L-arginine, benzoate,
L-ornithine, maleate, and tartrate (5).
In the routine clinical laboratory, R. mannitolilytica can
be differentiated from P. fluorescens and Pseudomonas
aeruginosa by a negative pyoverdin test, by its inability to grow
on salmonella-shigella agar, and by a negative arginine dihydrolase
test (Table 1). Growth on B. cepacia selective medium pointed to an identification as B. cepacia. Differentiation from B. cepacia, especially
from the genomovar II strains (i.e., Burkholderia
multivorans), which do not decarboxylate lysine or acidify sucrose
(15), is difficult and is only possible by means of a
positive pyrrolidonyl arylamidase test for R. mannitolilytica.
Finally, several clear phenotypic differences exist between
R. mannitolilytica and the other Ralstonia
species (Table 1). R. mannitolilytica can be differentiated
from the other described Ralstonia species through its
assimilation and acidification of mannitol and
D-arabitol. R. mannitolilytica strains
differ from R. pickettii and Ralstonia
solanacearum by their resistance towards desferrioxamine
and from R. pickettii because of their lack of alkalinization of tartrate and of nitrate reductase. Strains of R. mannitolilytica were previously reported to be adonitol
and ethanol acidification negative, like the R. pickettii
biovars Va-1 and Va-2, and cellobiose positive, like R. pickettii biovar Va-1 (13).
A limited number of cases of hospital outbreaks with "P.
thomasii" and R. pickettii biovar
3/"thomasii" isolates have been reported in the
literature (2, 6, 10, 11). The first report
(12) dealt with bacteremia and bacteriuria in 25 patients due to parenteral fluids prepared with deionized water contaminated with "P. thomasii" (11, 12). Pan et al.
(10) reported that 23 of 39 R. pickettii
isolates of an epidemic involving 24 patients that was caused by
contaminated saline solution (prepared by the hospital pharmacy)
belonged to "P. thomasii." A pseudo-outbreak has been
described as well (4). Although no serious
non-outbreak-related infections have been described thus far, the
clinical importance of R. mannitolilytica may have been
overlooked, possibly due to misidentification as P. fluorescens, B. multivorans, and/or R. pickettii.
We reported two cases of infection with R. mannitolilytica,
first identified as P. fluorescens and/or B. cepacia. Colistin-resistant "P. fluorescens"
isolates and strains growing on B. cepacia selective medium
should be considered to be possibly R. mannitolilytica, a
species that was formerly known as R. pickettii biovar
3/"thomasii" and that can be differentiated from
P. fluorescens by its colistin resistance and its absence of
arginine dihydrolase activity, from B. cepacia and B. multivorans by its pyrrolidonyl peptidase activity, and from
other Ralstonia species by the acidification of mannitol. Correct identification of this organism may be of importance, since
appropriate treatment was postponed in at least case 1, due to
misidentification as P. fluorescens and B. cepacia, pointing to the presence of a contaminant and also
obscuring the long-term presence of the same bacterial organism.
| |
ACKNOWLEDGMENTS |
We thank Leen Van Simaey and Catharine De Ganck for excellent
technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory
Bacteriology & Virology, Blok A, Ghent University Hospital, De
Pintelaan 185, B9000 Gent, Belgium. Phone: 32 9 240 36 92. Fax: 32 9 240 36 59. E-mail:
Mario.Vaneechoutte{at}rug.ac.be.
| |
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Journal of Clinical Microbiology, December 2001, p. 4588-4590, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4588-4590.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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