Microbiology and Infectious Disease / NOVEL SPECIES OF ROSEOMONAS
Bacteriologic Characterization of 36 Strains
of Roseomonas Species and Proposal of Roseomonas
mucosa sp nov and Roseomonas gilardii subsp
rosea subsp nov
Xiang Y. Han, MD, PhD,1 Audrey S. Pham, PhD,1 Jeffrey J. Tarrand, MD,1
Kenneth V. Rolston, MD,2 Leta O. Helsel,3 and Paul N. Levett, PhD3
Key Words: Bacterial taxonomy; Sequencing of 16S rDNA; Roseomonas
DOI: 10.1309/731VVGVCKK351Y4J
Abstract
We used a polyphasic approach (sequencing
analysis of the 16S ribosomal RNA gene and phenotypic
analyses) to characterize 36 strains of Roseomonas
species isolated from blood. Five strains, represented by
strain MDA5176 (M.D. Anderson Cancer Center), were
identified as Roseomonas gilardii. One strain belonged
to Roseomonas genomospecies 4. The 22 strains
represented by strain MDA5527 showed significant
differences genotypically and phenotypically with R
gilardii and other Roseomonas species and represented
a new Roseomonas species; Roseomonas mucosa sp
nov was proposed to denote its prominent mucoid,
almost runny colonies. Eight strains, represented by
strain MDA5605, had minor differences with R gilardii
and displayed obvious pink to red colonies;
Roseomonas gilardii subsp rosea subsp nov was
proposed. For subspecies differentiation, R gilardii was
proposed to be R gilardii subsp gilardii subsp nov.
Unique patterns of biochemical reactions were
established for these Roseomonas species, which may
assist routine identification of these organisms. All 36
strains and 2 American Type Culture Collection strains
were susceptible to amikacin and ciprofloxacin but
resistant to cefepime and ceftazidime. They also were
frequently susceptible to imipenem and ticarcillinclavulanate but far less susceptible to ceftriaxone,
trimethoprim-sulfamethoxazole, and ampicillin. R
mucosa strains were most resistant, whereas R gilardii
subsp gilardii strains were most susceptible.
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DOI: 10.1309/731VVGVCKK351Y4J
The genus Roseomonas was established in 1993 by
Rihs et al1 based on studies of the morphologic features,
phenotypes, and genome similarity of 42 strains of pinkpigmented, aerobic, and slow-growing gram-negative
bacteria. This genus includes 6 species: Roseomonas
gilardii (or genomospecies 1, the type species),
Roseomonas cervicalis (genomospecies 2), Roseomonas
fauriae (genomospecies 3), and 3 unnamed Roseomonas
genomospecies 4, 5, and 6. These organisms have been
isolated from the aquatic environment and various clinical
samples, such as blood and wound.
Since then, many clinical studies of various Roseomonas
infections have been reported, from several single case
reports2-9 to 2 series of multiple cases.10,11 However, most
clinical microbiology laboratories are unable to confidently
identify these organisms owing to lack of expertise and lack
of commercially available kits that offer positive identification. In addition, further microbiologic characterizations of
these organisms are few, and the 16S ribosomal DNA
(rDNA) of these organisms have not been sequenced, which
hampers research activity and makes identification impossible by the increasingly popular sequencing methods. Therefore, a Canadian group and we independently sequenced the
16S rDNA of all 6 Roseomonas genomospecies (GenBank
accession numbers AF533352-56 and AY150045-50, respectively). These unpublished results suggest that while
Roseomonas genomospecies 1, 2, 4, and 5 are valid taxa, the
genomospecies 3 and 6 are not. These invalid Roseomonas
species belong to the genus Azospirillum, a nitrogen-fixing
plant symbiont that is in a different order of bacteria.12 A
report describing the taxonomic revision of the genus
Roseomonas is being prepared.
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
In this report, we characterized 36 strains of
Roseomonas species that were isolated from patient blood
samples and initially were unidentified or misidentified.
Sequencing analysis of the 16S rDNA and routine phenotypic methods were used for the study. This study led to
proposals of novel species and modification of current
species, which may prove useful clinically and academically.
Materials and Methods
Bacteria and Cultures
The 36 bacterial strains were isolated from blood
cultures performed from January 1991 to December 2002
at the University of Texas M.D. Anderson Cancer Center,
Houston, a 500-bed tertiary cancer center. These pink,
mucoid, and gram-negative but occasionally gram-variable
bacteria had been banked, along with 13 strains of other
“pink bacteria” that later were identified by sequencing
analysis to be Gordonia terrae (5 strains),13 Rhodococcus
equi (7 strains), and Methylobacterium extorquens (1
strain) (X.Y.H., unpublished data, 2002). All 36 strains
were previously unidentified or misidentified by routine
biochemical tests as Methylobacterium mesophilicum
(another pink, slow-growing, gram-negative coccobacillus)
or Rhodococcus (a pink, gram-positive coccobacillus). Of the
36 strains, 27 (75%) were from the 1998-2002 period, during
which there was better recognition of these organisms. Two
Roseomonas strains from the American Type Culture Collection (ATCC) were included in the study for comparison:
ATCC 49956T, the type species of Roseomonas and type
strain of R gilardii; and ATCC 49959T, the type strain of
Roseomonas genomosp 4. Two nontype strains, CDC5465
and E9464 that previously were included in the R gilardii
group,1 also were used for the study.
Blood cultures were performed using the Bactec automated culturing system (BD Diagnostic Systems, Sparks,
MD) and Isolator tubes (Wampole Laboratories, Princeton,
NJ). Approximately 30,000 blood cultures were performed
yearly in the institution. All subcultures were plated on blood
agar or chocolate agar (BBL, Becton Dickinson Microbiology
Systems, Cockeysville, MD) and incubated at 35°C aerobically with 5% carbon dioxide. The organisms were fastidious
and generally required an incubation period of 2 to 3 days.
Sequencing of 16S rDNA and Phylogenetic Analysis
Extraction of the bacterial genomic DNA, amplification
of the 16S rDNA by polymerase chain reactions, and subsequent sequencing of the amplicons were performed as
described previously.14,15 All strains were amplified and
sequenced for a 593-base-pair fragment using a set of
universal bacterial primers15: 5'-TGCCAGCAGCCGCGGTAATAC-3' and 5'-CGCTCGTTGCGGGACTTAACC-3'
(corresponding to positions 515-1107 of Escherichia coli
J01859). For the groups with multiple identical strains, ie,
the MDA5176 (M.D. Anderson Cancer Center), MDA5605,
and MDA5527 groups, at least 2 representative strains of
each group were further amplified and sequenced for 1,480
base pairs (near full length of the 16S rDNA). Two additional sets of primers: 5'-GTTTGATCCTGGCTCAGAGCG3' and 5'-AGGAATTTCACGCCTGACTTGG-3' (11-613),
and 5'-GCACAAGCGGTGGAGCATGTG-3' and 5'AGGAGGTGATCCAACCGCA-3' (932-1539) (E coli
J01859) were used for this purpose. Single strain,
MDA1060, was sequenced only to 593 base pairs because
further sequencing was deemed unnecessary. Sequencing
was performed using the dye-terminator method in an ABI
377 sequencer (Applied Biosystems, Foster City, CA).
Sequences were queried to the GenBank for best matches.
Multiple alignments of sequences were performed using the
ClustalW (http://www.ebi.ac.uk/clustalw/index.html).
Other Characterizations
The biochemical reactions were performed primarily
with API 20NE (bioMérieux, Marcy l’Etoile, France), and
results were read after a 48-hour incubation. Scanning electron microscopy was performed at our institutional core
facility according to a standard procedure described previously.14 Cell wall fatty acids were analyzed in a commercial
laboratory (Microbial ID, Newark, DE) using high pressure
liquid chromatography and Sherlock Version 4.0 software
(Microbial ID). The antibiotic susceptibility tests were
performed using Etest (Biodisk, Solna, Sweden) and interpreted according to the National Committee for Clinical
Laboratory Standards.16
Results
Sequencing of 16S rDNA and Phylogenetic Analysis
The 16S rDNA of all 36 strains initially was sequenced
to 593 base pairs. These sequences were queried to the
GenBank for the best matches. All strains matched best,
from 94% to 100%, with R gilardii ATCC 49956T, the type
species of Roseomonas and type strain of the species. By the
match percentages, these analyses further placed the 36
Roseomonas strains into 4 homogeneous groups (100%
matches within group), represented by MDA1060 (1 strain),
MDA5176 (5 strains), MDA5605 (8 strains), and MDA5527
(22 strains). It is noteworthy that the sequences of R gilardii
ATCC 49956T recently were determined independently by
Han et al (AY150045) and by Bernard et al (AF533352)
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DOI: 10.1309/731VVGVCKK351Y4J
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Han et al / NOVEL SPECIES OF ROSEOMONAS
(both unpublished), and both sequences matched 99.86%
(1,431/1,433) with 1 pseudogap and a nucleotide mismatch,
thereby demonstrating reliability of the sequences.
The strain MDA1060 matched 93.8% with R gilardii
but 100% with Roseomonas genomosp 4 (AY150048). Thus,
it was positively identified as Roseomonas genomosp 4, and
further sequencing of its 16S rDNA was deemed unnecessary. For the other groups, at least 2 strains from each group
were sequenced further to 1,480 base pairs (nearly the full
length of the gene) for detailed analysis. All 5 strains in the
MDA5176 group matched 100% with R gilardii (up to 1,480
base pairs analyzed); thus, these organisms also were positively identified as R gilardii. The MDA5605 group matched
99.39% (1,468/1,477) with R gilardii, and 6 of the 9
mismatches were within the hypervariable regions ❚Table 1❚.
Therefore, the MDA5605 group probably was a subspecies
of R gilardii. The MDA5527 group matched 98.24%
(1,452/1,478) with R gilardii, suggesting a significant difference with R gilardii and the possibility of a novel species of
Roseomonas.
We noted that in the initial characterization of 23 strains
of the R gilardii group, there were considerable genotypic
and phenotypic variations,1 suggesting the possible presence
of other species and subspecies within the group. Therefore,
2 nontype R gilardii strains, CDC5465 and E9464 from the
initial study, 1 were sequenced to 1,480 base pairs for
analysis. The CDC5465 matched 100% with MDA5527, and
morphologically, the 2 strains also were identical (data not
shown). Since previous DNA hybridization experiments had
shown that CDC5465 hybridized 66% (at 85°C) and 75% (at
70°C) with the R gilardii type strain (ATCC49956T),1 we
deemed it unnecessary to perform hybridization experiments
between MDA5527 and R gilardii (ATCC49956T). Strain
E9464 (GenBank AY150051) matched 99.19%
(1,469/1,481) with MDA5527 (and also CDC5465), 98.72%
(1,466/1,485) with MDA5605, and 98.24% (1,451/1,477)
with R gilardii (ATCC49956T). At the genome level,
previous data have shown that E9464 and CDC5465
hybridized 47% (at 85°C) and 58% (at 70°C).1 Therefore,
E9464 and MDA5527 were most closely related.
The preceding results are summarized in Table 1 and
❚Table 2❚ , in which multiple alignments of the variable
nucleotides and the matched percentages are shown. Within
the genus, considerable phylogenetic distance was evident
between Roseomonas genomosp 4 and the other groups that
are more closely related. The data also suggest heterogeneity
among the groups, and further classification is needed.
Morphologic Features
The strains within each of the 4 groups exhibited consistent and uniform colonial morphologic features; however,
among the groups, discernible differences were observed
❚Image 1❚. Pink pigmentation, as the genus name implies, was
demonstrated more readily on a chocolate agar plate or by a
cotton swab. The color was more pronounced for the
MDA5605 group (Image 1A) and the Roseomonas genomosp
4 group (MDA1060 and ATCC 49959T) (Image 1B), in
which the pink pigment also turned to red after several days,
which was not observed in MDA5176 and MDA5527 groups.
The most noticeable feature of the MDA5527 group was the
mucoid, almost runny, sometimes teardrop-shaped colonies
(Image 1C), which began autolysis after several days. The
pink Roseomonas genomosp 4 organisms were least mucoid,
and the colonies were flat and the smallest (Image 1B). In
contrast, the colonies of MDA5176 group (R gilardii) were
round and elevated (Image 1D). Both the Roseomonas
genomosp 4 and MDA5176 groups had the slowest growth
rate (usually 3 days) among all. On Gram stain, all 36 strains
were gram-negative, coccoid to short rods, and sometimes
chaining, and they occasionally retained some of the purple
stain (thus, they had been confused with rhodococci in the
past). An electron photomicrograph of MDA5527 is shown in
❚Image 2❚, in which the organism was estimated to be 0.9 × 11.9 µm. Some cells had a single fragile flagellum (Image 2
and other photomicrographs not shown).
Biochemical Characterization
The biochemical reactions were performed primarily
with API 20NE for convenience and consistency and for its
frequent use in clinical microbiology laboratories. The
❚Table 1❚
Phylogenetic Analyses of Roseomonas Strains: Multiple Alignments Showing Representative Variable Nucleotides, Positions
According to Roseomonas gilardii (AY150045)
Position
61
Roseomonas gilardii
MDA5605
MDA5527
Roseomonas
genomosp 4
258
258
74
GTGGTTTCGGCCAT
GTGGTTTCGGCCAT
GCAGCAATG---------T
GCAGCAATG---------T
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951
956
961
G
A
A
A
C
T
T
T
C
T
T
C
1375
1406
GTCG TTGCGCTAACC--GCAA--GGGGGCAGGCGAC
GTCGTTGCGCTAACCAGCGATGGGGGCAGGCGAC
GTCGTTGCGCTAACCAGCGATGGGGGCAGGCGAC
GCAGGTGCGCGAACCCGCAAGGGATGTAGCTTGC
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
❚Table 2❚
Phylogenetic Analyses of Roseomonas Strains: NucleotideMatched Percentages for the Strains
Roseomonas
gilardii
MDA5605
R gilardii
MDA5605
MDA5527
Roseomonas
genomosp 4
100.0
99.4
98.2
93.8
—
100.0
98.7
93.9
MDA, M.D. Anderson Cancer Center.
MDA5527
Roseomonas
genomosp 4
—
—
100.0
94.3
—
—
—
100.0
results were read after a 48-hour incubation owing to the
fastidious nature of the organisms ❚Table 3❚, ❚Table 4❚, and
❚Table 5❚. The MDA5176 group and MDA1060 essentially
matched with ATCC49956T and ATCC49959T, respectively,
in these reactions. All 38 strains (36 MDA strains and 2
ATCC strains) were positive in the catalase test and for
malate assimilation. In addition, utilization of urea was
another consistent feature: positive for 31 strains (82%),
weakly positive for 5 strains (13%), and negative for only 2
strains (5%) in the MDA5605 group.
The 2 Roseomonas genomosp 4 strains reduced nitrate
to nitrite but did not utilize arabinose, citrate, or glucose.
These reactions differed from those of the other 3 groups.
The latter strains (35 MDA strains and ATCC49956T) all
A
B
C
D
❚Image 1❚ Colonial morphology of Roseomonas species on 4-day culture on chocolate agar. A, MDA5605 (Roseomonas gilardii
subsp rosea subsp nov). B, Roseomonas genomosp 4 (ATCC49959T) (note smallest colony size). C, MDA5527 (Roseomonas
mucosa sp nov). D, MDA5176 (R gilardii subsp gilardii subsp nov).
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Han et al / NOVEL SPECIES OF ROSEOMONAS
were negative for nitrate reduction except 1 strain (1/36
[3%]), but they utilized arabinose (36/36 [100%]), citrate
(35/36 [97%]), and also frequently glucose (31/36 [86%]).
These biochemical differences correlated with the phylogenetic distance noted (Table 2).
The R gilardii strains exhibited minor differences with
the MDA5605 and MDA5527 groups (Tables 3-5). For
instance, none of the 6 R gilardii strains assimilated adipate,
whereas 22 (73%) of 30 strains in the MDA5605 and
MDA5527 groups weakly assimilated this sugar. Five of the
6 R gilardii strains weakly assimilated mannitol, whereas
other strains were mostly negative. The MDA5605 and
MDA5527 groups overlapped frequently for the biochemical
reactions, although they probably were different species as
determined by the 16S rDNA sequencing analysis (98.7%
match; Table 2).
The API 20NE code of each strain was given for
comparison and convenient identification in most clinical
microbiology laboratories (Tables 3-5). The codes were more
uniform when not accounting for the weakly positive reactions, and the 2 most common codes were 0241041 (13
strains) and 0201041 (7 strains). Except for 1 code
(0241064) that also matched with M mesophilicum, all other
codes (75/76 [99%]) were unique, whether the weakly positive reactions were included or not. The oxidase reactions
were more variable: in our hands, 27 strains of the 38 were
negative, although in the previous study,1 all 23 R gilardii
strains were positive. We observed that young cultures on
❚Image 2❚ Scanning electron microscopy of MDA5527
(Roseomonas mucosa sp nov) (×15,000). Bar indicates 1 µm.
blood agar tended to be positive, whereas cultures on chocolate agar or older than 3 days tended to be negative.
Fatty Acid Analysis
Two representative strains (100.00% match of 1,480
base pairs) of each of the MDA5176, MDA5605, and
MDA5527 groups were analyzed for cell wall fatty acids
❚Table 3❚
Number of Positive and Weakly Positive* Biochemical Reactions of 36 Strains of Roseomonas Species
Roseomonas gilardii
Reaction
Nitrate reduction
To nitrite
To nitrogen
Indole production
Glucose acidification
Arginine hydrolysis
Urea utilization
Esculin hydrolysis
β-galactosidase activity
Assimilation of
Glucose
Arabinose
Mannose
Mannitol
N-acetylglucosamine
Maltose
Gluconate
Caprate
Adipate
Malate
Citrate
Phenylacetate
Oxidase
Catalase
Roseomonas genomosp 4
ATCC49956T
MDA5176
Group (n = 5)
MDA5605
Group (n = 8)
MDA5527
Group (n = 22)
ATCC49959T
MDA1060
0
0
0
0
0
1
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
3 (3)
0
0
1
0
0
0
0
21 (1)
0
0
1
0
0
0
0
(1)
0
0
1
0
0
0
0
1
0
0
0
1
0
(1)
0
0
0
0
0
1
1
0
0
1
2 (3)
5
0
1 (3)
0
0
0 (1)
0
0
5
5
1 (1)
0
5
3 (2)
5 (3)
0
0 (1)
0
0
0 (2)
0
3 (2)
8
8
0 (1)
1
8
15 (6)
22
0
0 (6)
0
0
0 (1)
0
3 (14)
22
20 (1)
0
6
22
0
0
0
0
0
0
0
0
(1)
1
0
0
1
1
0
0
0
0
0
0
(1)
0
1
1
0
0
1
1
ATCC, American Type Culture Collection; MDA, M.D. Anderson Cancer Center.
* In parentheses.
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© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
❚Table 4❚
API 20NE Reaction Codes Without Weakly Positive Reactions*
MDA5176
Group (n = 5)
MDA5605
Group (n = 8)
MDA5527
Group (n = 22)
—
0201041 (3)
0241041 (1)
0241043 (1)
—
—
0041061 (3)
0000041 (2)
0201041 (1)
0201045 (2)
0200041 (1)
—
—
—
0241041 (12)
0201041 (2)
0201045 (2)
0201044 (2)
0241061 (1)
0201061 (1)
0241065 (1)
0241045 (1)
ATCC49956T
0201041 (1)
ATCC49959T
MDA1060
0200064 (1)
0200044 (1)
—
—
—
—
—
—
ATCC, American Type Culture Collection; MDA, M.D. Anderson Cancer Center.
* The number of strains is given in parentheses.
❚Table 5❚
API 20NE Reaction Codes With Weakly Positive Reactions*
MDA5176
Group (n = 5)
MDA5605
Group (n = 8)
MDA5527
Group (n = 22)
—
0245041 (2)
0245043 (1)
0241041 (1)
0245443 (1)
—
—
—
0241041 (2)
0041061 (2)
0245061 (1)
0201461 (1)
0201043 (1)
0201465 (1)
—
—
—
—
0241061 (6)
0241041 (5)
0245065 (3)
0241065 (2)
0245061 (2)
0241064 (1)
0245461 (1)
0201061 (1)
1241061 (1)
ATCC49956T
0205041 (1)
—
ATCC49959T
MDA1060
1200064 (1)
1200464 (1)
—
—
—
—
—
—
—
—
ATCC, American Type Culture Collection; MDA, M.D. Anderson Cancer Center.
* The number of strains is given in parentheses.
❚Table 6❚. Results showed that the MDA5176 group was
identical to R gilardii (similarity index, 0.858 of 1.000),
whereas the MDA5605 group exhibited minor differences
(similarity index, 0.722), suggestive of a variant or
subspecies of R gilardii. In contrast, the MDA5527 group
differed significantly (similarity index, 0.593) from the
others in a number of fatty acid peaks, especially 19:0
CYCLO w8c and 18:1 2OH, suggestive of a species distinct
from R gilardii. These results were consistent with the findings from the 16S rDNA sequencing data (Tables 1 and 2).
Antibiotic Susceptibility
Results of antibiotic susceptibility testing by the Etest
are given in ❚Table 7❚. The MDA5176 group and MDA1060
also matched with ATCC49956T and ATCC49959T, respectively. All 38 strains were susceptible to amikacin and
ciprofloxacin but resistant to cefepime and ceftazidime. Most
strains also were susceptible to imipenem (36/38 [95%) and
ticarcillin-clavulanate (31/38 [82%]).
The overall susceptibilities to ceftriaxone, trimethoprimsulfamethoxazole, and ampicillin were lower (47% to 18%),
and there were differences among the groups (Table 7). The
R gilardii strains were susceptible to ceftriaxone (6/6 [100%])
but variably susceptible to trimethoprim-sulfamethoxazole
(3/6 [50%]) and ampicillin (1/6 [17%]). The MDA5605
group organisms frequently were susceptible to ampicillin
(6/8 [75%]) and ceftriaxone (5/8 [63%]) but barely susceptible to trimethoprim-sulfamethoxazole (1/8 [13%]). The
strains in the MDA5527 group all were resistant to ampicillin and infrequently susceptible to ceftriaxone (7/22
[32%]) and trimethoprim-sulfamethoxazole (5/22 [23%]).
Both Roseomonas genomosp 4 strains were resistant to
ceftriaxone and ampicillin but susceptible to trimethoprimsulfamethoxazole.
Overall, these results suggest that R gilardii strains were
most susceptible, whereas the organisms in the MDA5527
group were most resistant. The third-generation cephalosporins
cefepime, ceftazidime, and ceftriaxone would be poor
choices for the treatment of Roseomonas infections. These
results also demonstrate the uniformity of antibiotic susceptibility within a phylogenetically homogeneous species.
Discussion
The preceding polyphasic characterizations suggest that
the MDA5527 group differs sufficiently in the genotype and
phenotype from R gilardii to warrant species status for these
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organisms. Therefore, we propose MDA5527 be named
Roseomonas mucosa sp nov, descriptive of the prominent
mucoid nature of the colonies. In addition, minor genotypic
and phenotypic differences between the MDA5605 group
and R gilardii were demonstrated, which suggest subspecies
status for the MDA5605 group organisms. Therefore, we
propose MDA5605 be named Roseomonas gilardii subsp
rosea subsp nov, descriptive of the apparent pink colonies of
these organisms. For subspecies differentiation, the R gilardii
group is proposed to be R gilardii subsp gilardii subsp nov.
Description of Roseomonas mucosa sp nov
R mucosa (mu co' sa, L. fem. adj. mucosa, mucous, slimy)
is slightly fastidious and forms very mucoid, almost runny
colonies. It is coccoid to bacillary, measures 0.9 × 1.0-1.9 µm,
and has a single poorly formed flagellum. This organism splits
urea and assimilates arabinose, malate, citrate, and glucose.
Strains of this species are consistently resistant to cefepime,
ceftazidime, and ampicillin and frequently resistant to
trimethoprim-sulfamethoxazole and ceftriaxone. However, they
are consistently susceptible to amikacin and ciprofloxacin and
frequently susceptible to imipenem and ticarcillin-clavulanate.
The type strain of R mucosa is MDA5527 (ATCC BAA-692 or
NCTC 13291). It was isolated from a patient’s blood in 2000.
The 16S rDNA sequence is GenBank AF538712.
Description of Roseomonas gilardii subsp rosea subsp nov
R gilardii subsp rosea (roz ea, L. fem. adj. rosea, rosy,
rose-colored, or pink) forms mucoid and pink colonies. The
color intensifies after several days. It assimilates malate,
citrate, and arabinose. Strains of this species variably utilize
urea, glucose, and adipate. Biochemically, it differs from
❚Table 6❚
Fatty Acid Analyses of the MDA5176, MDA5605, and
MDA5527 Groups*
Fatty Acid
14:0 3OH/16:1 ISO I
16:1 w7c/15 iso 2OH
16:1 w5c
16:0
16:0 2OH
16:0 3OH
18:2 w6,9c/18:0 ANTE
18:1 w7c
18:1 w5c
18:0
19:0 CYCLO w8c
18:1 2OH
Total
MDA5176
Group
MDA5605
Group
1.00
0.93
0.00
20.12
0.00
3.39
0.00
53.55
0.26
1.00
9.29
10.44
99.98
MDA5527
Group
0.65
1.30
0.23
16.93
0.00
2.86
0.73
60.64
0.27
1.15
4.19
11.26
100.21
0.89
1.23
0.00
19.53
1.24
4.13
0.40
46.55
0.00
1.16
21.55
3.82
100.50
MDA, M.D. Anderson Cancer Center.
* Data are given as percentages and represent the average value of 2 strains from each
group. The similarity index to Roseomonas gilardii (of 1.000) and interpretation
were as follows: MDA5176 group, 0.858, same; MDA5605 group, 0.722, variant;
MDA5527 group, 0.593, different.
Roseomonas genomosp 4, another species with a prominent
red pigment, because the latter reduces nitrate to nitrite but
does not assimilate citrate or arabinose, as shown in this
study and previously.1 Strains of R gilardii subsp rosea are
consistently susceptible to amikacin and ciprofloxacin and
frequently susceptible to imipenem, ticarcillin-clavulanate,
ampicillin, and ceftriaxone. However, they are consistently
resistant to cefepime and ceftazidime and often resistant to
trimethoprim-sulfamethoxazole. The type strain of R gilardii
subsp rosea is MDA5605 (ATCC BAA-691 or NCTC
13290). It was isolated from a patient’s blood in 1999. The
16S rDNA sequence is GenBank AY220740.
❚Table 7❚
Antibiotic Susceptibility of 36 Strains of Roseomonas Species*
Roseomonas gilardii
Antibiotic
All susceptible
Amikacin
Ciprofloxacin
Mostly susceptible
Imipenem
Ticarcillin-clavulanate
Variably susceptible
Ceftriaxone
Trimethoprimsulfamethoxazole
Ampicillin
All resistant
Cefepime
Ceftazidime
ATCC49956T
(n = 1)
MDA5176
Group (n = 5)
Roseomonas genomosp 4
MDA5605
Group (n = 8)
MDA5527
Group (n = 22)
ATCC49959T
(n = 1)
MDA1060
(n = 1)
All
(n = 38)
1 (1.5)
1 (0.25)
5 (0.75-1.5)
5 (0.38-0.5)
8 (1.0-3.0)
8 (0.19-1.5)
22 (0.5-2.0)
22 (0.13-2.0)
1 (0.75)
1 (0.047)
1 (3.0)
1 (0.1)
38 (100)
38 (100)
1 (0.5)
1 (6.0)
5 (0.38-0.75)
5 (0.38-6.0)
7 (0.38 to >32)
6 (0.5 to >256)
21 (0.5 to >32)
17 (1.5 to >256)
1 (1.0)
1 (1.0)
1 (0.19)
1 (0.38)
36 (95)
31 (82)
1 (2.0)
0 (>32)
5 (0.5-1.0)
3 (0.19 to >32)
5 (0.5 to >32)
1 (2.0 to >32)
7 (1 to >32)
5 (0.13 to >32)
0 (>32)
1 (0.008)
0 (>32)
1 (0.004)
18 (47)
11 (29)
0 (>256)
1 (0.75 to >256)
6 (0.25 to >256)
0 (>256)
0 (>256)
0 (>256)
7 (18)
0 (>32)
0 (>256)
0 (>32)
0 (64 to >256)
0 (>32)
0 (>256)
0 (>32)
0 (>256)
0 (>32)
0 (64)
0 (>32)
0 (24)
0 (0)
0 (0)
ATCC, American Type Culture Collection; MDA, M.D. Anderson Cancer Center.
* Data are given as number of susceptible strains and minimum inhibitory concentration range (µg/mL), except in the “All” column, in which they are given as number
(percentage).
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Am J Clin Pathol 2003;120:256-264
DOI: 10.1309/731VVGVCKK351Y4J
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
Description of Roseomonas gilardii subsp gilardii subsp nov
While the majority of the initial descriptions of R
gilardii1 still hold true, the present study defines R gilardii
subsp gilardii through studies of 6 genotypically homogeneous strains. This organism is fastidious and generally
requires an incubation period of 3 days. It forms round,
elevated, and mucoid colonies. The organism splits urea and
assimilates arabinose, malate, and citrate. Most strains also
assimilate glucose or weakly do so. Strains of R gilardii
subsp gilardii are susceptible to amikacin, ciprofloxacin,
imipenem, ticarcillin-clavulanate, and ceftriaxone but resistant to cefepime and ceftazidime and variably susceptible to
trimethoprim-sulfamethoxazole and ampicillin. These
features, along with cellular morphologic features on Gram
stain and many negative biochemical reactions, positively
identify this organism. The type strain of R gilardii subsp
gilardii remains ATCC49956T. The 16S rDNA sequence is
GenBank AY150045.
In the present study, R mucosa was the most common
Roseomonas species (22/36 [61%]), followed by R gilardii
subsp rosea (8/36 [22%]), R gilardii subsp gilardii (5/36
[14%]), and Roseomonas genomosp 4 (1/36 [3%]). All 36
organisms were isolated from blood, none from other
sources. Other valid Roseomonas species, ie, R cervicalis and
Roseomonas genomosp 5, were not encountered. Therefore,
even considering the possibility of selection bias for patients,
the overwhelming number of R mucosa suggests that this
organism probably is the most clinically significant species in
the genus. This study has led to better recognition of these
organisms in our laboratory, and as such, they were isolated
approximately once per month. The unique biochemical
reactions, shown by API 20NE codes (Tables 3-5), should
provide a solid basis for routine biochemical identification of
these organisms, along with the morphologic features illustrated in Images 1 and 2. Distinction between Roseomonas
and M mesophilicum should be confident, because the
colonies of M mesophilicum are flat and dry, in addition to
the biochemical differences. At the 16S rDNA level, the 2
genera match at 83.27% (1,170/1,405; AY150045 vs
D3222517). Methylobacteria were rare in our laboratory:
from 1998 to 2002, only 1—M extorquens, identified by 16S
rDNA sequencing analysis—was isolated, and no M
mesophilicum were isolated. In contrast, 27 Roseomonas
species were isolated during the same period. In retrospect,
the confusion between Roseomonas and Rhodococcus was
largely attributed to technologists’ experience.
Roseomonas species have been isolated elsewhere in the
world, such as the United Kingdom,7,8 Spain,4,5 Canada
(AF533357-60; Bernard et al, unpublished data, 2002), and
France (AF227831 and AF227855 for 2 previously unknown
strains, and AF531769 for “candidatus Roseomonas
marsilliae”; Raoult et al, unpublished data, 2002). Complete
sequence match between candidatus Roseomonas marsilliae
and R mucosa suggests that they probably are identical.
However, the name R mucosa is preferable in our view
because of its descriptive nature and, more important, the large
number of strains that were characterized in the present study.
All of our 36 strains and the 2 ATCC strains were
susceptible to amikacin and ciprofloxacin but resistant to
cefepime and ceftazidime. Frequent susceptibilities to
imipenem and ticarcillin-clavulanate also were shown.
Results with ceftriaxone, trimethoprim-sulfamethoxazole,
and ampicillin were variable, depending on the species. All
22 strains of R mucosa also were resistant to ampicillin and
frequently were resistant to trimethoprim-sulfamethoxazole
and ceftriaxone. Therefore, R mucosa seems most resistant,
which is in contrast with the R gilardii subsp gilardii strains
that are most susceptible. Distinguishing these species may
prove useful in guiding empiric antibiotic treatment. Clinically, most organisms caused catheter-related bacteremia,
resulting in fever and, in some cases, disseminated infections
and prolonged colonization of the catheter.18 A report of the
detailed analysis of the 36 cases is being prepared.
From the Sections of 1Clinical Microbiology and 2Infectious
Diseases, the University of Texas M.D. Anderson Cancer Center,
Houston; and 3Meningitis and Special Pathogens Branch,
National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, GA.
Supported by a Startup Fund (to X.Y.H.) from the University
of Texas M.D. Anderson Cancer Center and by institutional core
research grant CA16672 from the National Institutes of Health,
Bethesda, MD, for the Sequencing Core Facility and Electron
Microscopy Core Facility, M.D. Anderson Cancer Center.
Address reprint requests to Dr Han: Section of Clinical
Microbiology, the University of Texas M.D. Anderson Cancer
Center, Unit 84, 1515 Holcombe Blvd, Houston, TX 77030.
Acknowledgment: We thank the staff in our laboratory for
isolating the organisms, Barbara LeBlanc for banking some of
the strains, Sary Joudah for assisting with some of the
experiments, the staff in our Sequencing Core Facility for
sequencing analysis, and Hans G. Trueper, PhD, for assistance
with Latin language.
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© American Society for Clinical Pathology