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Journal of Clinical Microbiology, January 1999, p. 248-250, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Systemic Infection of an Immunocompromised
Patient with Methylobacterium zatmanii
B.
Hornei,1,*
E.
Lüneberg,2
H.
Schmidt-Rotte,2
M.
Maaß,1
K.
Weber,3
F.
Heits,3
M.
Frosch,2 and
W.
Solbach1
Institut für Medizinische Mikrobiologie
und Hygiene1 and
Medizinische Klinik
I,3 Medizinische Universität zu
Lübeck, Lübeck, and
Institut für Hygiene
und Mikrobiologie, Universität
Würzburg,2 Würzburg, Germany
Received 9 June 1998/Returned for modification 24 July
1998/Accepted 28 September 1998
 |
ABSTRACT |
We describe the identification of Methylobacterium
zatmanii as the causative agent of bacteremia and fever in an
immunocompromised patient. The patient, a 60-year-old man, had a
5-month history of acute myeloid leukemia and had been on
chemotherapy throughout this period. Seven days after the onset of
neutropenia, the patient developed fever. The combination of
ciprofloxacin, co-trimoxazole, imipenem, amikacin, and vancomycin led
to a complete defervescence. On subculture from six positive blood
cultures, the organism grew only on buffered charcoal yeast extract
agar and not on standard agars. Identification by universal PCR
and subsequent sequence analysis of the amplified 16S rRNA
gene segment was achieved. This identification by molecular
biology techniques was confirmed by conventional biochemical tests. To
our knowledge, this is the first description of M. zatmanii
isolated from patient material.
| |
TEXT |
Advances in the treatment of
leukemia have improved prognosis, but intensive chemotherapy regimens
have increased susceptibility to opportunistic infections. We describe
the isolation and identification of Methylobacterium
zatmanii as the causative agent of bacteremia and fever in an
immunocompromised patient. The genus Methylobacterium is a
group of aerobic, gram-negative, rod-shaped bacteria which were
isolated previously from various environments.
Case report.
A 60-year-old male patient with acute myeloid
leukemia and agranulocytosis had been undergoing chemotherapy for 5 months. A central venous catheter (Hickmann) was implanted at the
beginning of chemotherapy. After complete remission, the patient was
hospitalized and a consolidation chemotherapy was initiated. One week
later, the hospitalized patient developed neutropenia (<500
neutrophils/µl), and an antibiotic prophylaxis with ciprofloxacin and
co-trimoxazole was initiated. Seven days after the onset of
neutropenia, the patient developed fever, which was resistant to
additional ceftazidime and teicoplanin treatment. On day 3 of the fever
period, the antibiotic therapy was changed to a combination of
ciprofloxacin, co-trimoxazole imipenem, amikacin, and vancomycin (Table
1), which led to a complete and durable defervescence.
During the first 3 days of fever, a total of 12 blood specimens of 5 to
10 ml from peripheral blood venipunctures and from the central line
were obtained. After 48 h of incubation at 37°C in a BACTEC 9240 system (Becton Dickinson, Heidelberg, Germany), all aerobically
incubated blood cultures were positive. In contrast, none of the
anaerobic bottles gave a positive signal.
Even though gram-negative vacuolated short rods could be observed by
direct Gram staining of the cultures, several attempts to subcultivate
the organism on standard solid media, such as sheep blood agar (37°C,
aerobic), Mueller-Hinton agar (37°C, aerobic), and chocolate agar
(37°C, 5% CO2), failed. All plates were incubated for 4 days. Media were obtained from Oxoid (Wesel, Germany).
Final plating of the blood cultures on
Legionella-specific agar (
Legionella
charcoal yeast extract agar supplemented with
Legionella
buffered charcoal yeast extract [BCYE] growth supplement
and
Legionella MWY selective supplement [Oxoid]) revealed
faint
white-gray colonies after 48 h of incubation at 37°C in
5% CO
2.
After a further 48-h incubation, colonies turned
slightly red
or pink. Interestingly, for unknown reasons, colonies
grown on
BCYE agar could then be subcultivated on standard media.
Serological
examinations of the colony material with
Legionella
pneumophila specific antisera against serogroups 1, 4, and 6 (Denka Seiken
Ltd., Tokyo, Japan) as well as antisera against
Legionella micdadei and
Legionella dumoffii
(Denka Seiken Ltd.) were negative. The
isolated bacteria were finally
identified by sequence determination
of the 16S rRNA gene. For this
purpose, a universal PCR using
primers that hybridize to the 16S rRNA
of most eubacteria was
performed (
10). The PCR fragments
generated with primers 16Spro27f
(positions 8 to 27 of the 16S rRNA
gene, numbering referring to
Escherichia coli
(
10) and 16Spro342r (positions 342 to 362)
(
9)
were purified and directly sequenced with primer 16Spro342r
(
10). Sequence analysis revealed a 98% identity (229 out of
233 nucleotides were identical) to the 16S ribosomal gene of
M. zatmanii (
7), a bacterium which was first described in
1988
(
6).
This result was confirmed by using conventional biochemical methods.
The isolated gram-negative vacuolated rods showed pink-pigmented
colonies, grew slowly at room temperature, at 30°C, and at 37°C,
and showed weak growth at 42°C. The strain was able to oxidize
methanol (growth on oxidative-fermentative basal medium [Merck,
Darmstadt, Germany]) supplemented with 1% methanol under aerobic
conditions), was oxidative, and gave positive catalase, oxidase,
and urease reactions (API 20 NE, bioMeriéux,
Nürtingen, Germany).
Oxidation of methanol is characteristic of
members of the genus
Methylobacterium and allowed
differentiation from bacteria belonging
to the closely related genus
Roseomonas (
7,
12-14). Further
criteria to
distinguish the strain isolated in this study from
the genus
Roseomonas were Gram-stain morphology and failure to
grow on
MacConkey agar (
12-14). The biochemical characteristics
of
diagnostic importance of the isolated strain are summarized
in Table
2.
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TABLE 2.
Biochemical characteristics of M. zatmanii
isolated in this study and comparison to those of
Methylobacterium spp. and Roseomonas spp.
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Susceptibility testing by microdilution of the isolated
M. zatmanii strain was performed with the Sceptor System (Becton
Dickinson)
at 30°C with a 48-h incubation. The results are
shown in Table
3.
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TABLE 3.
In vitro susceptibilities of the isolated M. zatmanii strain as determined by microdilution procedures at
30°C for 48 h
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|
In spite of sonographic and radiologic examinations, the focus of this
nosocomial bacteremia was not found. The implanted
central line
remained during the infection episode and was removed
1 month later
without further investigations. Microbiological
investigations of the
hospital environment were not performed,
hence the source of the
infection is
unknown.
Discussion.
To our knowledge, this is the first description of
M. zatmanii isolated from patient material. Members of the
genus Methylobacterium (nine species identified hitherto)
are slow-growing, rod-shaped gram-negative bacteria that form pink
colonies (7, 14). They are ubiquitous and are commonly
isolated from water and soil (5). Bacteria isolated from
man-made water distribution systems were found to be highly resistant
to chlorine (7). Some cases of human infections have been
reported to date (1, 3, 8, 9, 11) and in all cases, an
underlying immunosuppression, e.g., leukemia, tuberculosis, AIDS,
or alcohol abuse, existed. The bacteria were isolated from
various patient specimens, such as blood, cerebrospinal fluid, sputum,
bronchoscopic material, and synovial membrane. Since few cases of
infection with Methylobacterium species have been described,
this may reflect the difficulties in cultivation and identification of
the bacteria (13). In general, methylobacteria are
opportunistic pathogens of low virulence. They cause mild clinical
symptoms, such as fever, which can effectively be treated by
appropriate antibiotic therapy. Resistance profiles of a panel of
Methylobacterium strains have been described previously in a
study by Brown et al. (2). In accordance with those results, the M. zatmanii strain isolated here was susceptible to
amikacin and imipenem but resistant to ciprofloxacin, co-trimoxazole,
and ceftazidime. This correlated to the clinical outcome.
Since methylobacteria have been isolated from tap water in hospital
units, and
Methylobacterium mesophilicum have been isolated
from patients who had undergone bronchoscopy (
4), it may be
useful to survey water distribution systems in hospital units
for
immunocompromised patients for the occurrence of methylobacteria.
In
summary, clinicians as well as microbiologists should be aware
of
Methylobacterium species as potentially infectious agents.
We would like to emphasize that subcultivation of the
M. zatmanii strain isolated in this study was successful only after
incubation
on BCYE agar. Therefore, inclusion of BCYE agar for
subcultivation
of bacterial organisms from positive indicated blood
cultures
with gram-negative rods by direct Gram stain appears to be a
valuable
diagnostic tool. Moreover, universal PCR from the pure culture
and subsequent sequence determination of the 16S rRNA gene was
found to
be a practical approach for the identification of uncommon
or
fastidious
organisms.
| |
FOOTNOTES |
*
Corresponding author. Present address:
Hygiene-Institut, Universität Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Phone: 49-228-2875535. Fax: 49-228-2875645. E-mail:
horney{at}mailer.meb.uni-bonn.de.
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Journal of Clinical Microbiology, January 1999, p. 248-250, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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