International Journal of Systematic and Evolutionary Microbiology (2010), 60, 000–000
Chryseobacterium palustre sp. nov. andChryseobacterium humi sp. nov., isolated fromindustrially contaminated sediments
Carlos Pires,1,2 Maria F. Carvalho,1 Paolo De Marco,3,4 Naresh Magan2and Paula M. L. Castro1
1Escola Superior de Biotecnologia, Universidade Cato´lica Portuguesa, Rua Dr Anto´nio Bernardino
2Cranfield Health, Cranfield University, Building 52A, Cranfield, Bedford MK43 0AL, UK
3IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Campo Alegre, 823,
4CICS – Centro de Investigac¸a˜o em Cieˆncias da Sau´de, Institudo Superior de Cieˆncias da Sau´de –
Norte, CESPU, 4585-116 Gandra PRD, Portugal
Two Gram-staining-negative bacterial strains, designated 3A10T and ECP37T, were isolated fromsediment samples collected from an industrially contaminated site in northern Portugal. These twoorganisms were rod-shaped, non-motile, aerobic, catalase- and oxidase-positive and formedyellow colonies. The predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 1v9c andiso-C17 : 0 3-OH. The G+C content of the DNA of strains 3A10T and ECP37T was 43 and34 mol%, respectively. The major isoprenoid quinone of the two strains was MK-6. 16S rRNAgene sequence analysis revealed that strains 3A10T and ECP37T were members of the familyFlavobacteriaceae and were related phylogenetically to the genus Chryseobacterium. Strain3A10T showed 16S rRNA gene sequence similarity values of 97.2 and 96.6 % to the type strainsof Chryseobacterium antarcticum and Chryseobacterium jeonii, respectively; strain ECP37Tshowed 97.3 % similarity to the type strain of Chryseobacterium marinum. DNA–DNAhybridization experiments revealed levels of genomic relatedness of ,70 % between strains3A10T and ECP37T and between these two strains and the type strains of C. marinum, C. antarcticum and C. jeonii, justifying their classification as representing two novel species of thegenus Chryseobacterium. The names proposed for these organisms are Chryseobacteriumpalustre sp. nov. (type strain 3A10T 5LMG 24685T 5NBRC 104928T) and Chryseobacteriumhumi sp. nov. (type strain ECP37T 5LMG 24684T 5NBRC 104927T).
The genus Chryseobacterium was proposed by Vandamme
levels of contamination, especially by heavy metals (Costa
et al. (1994). It belongs to the family Flavobacteriaceae and,
& Jesus-Rydin, 2001; Oliveira et al., 2001; Carvalho et al.,
at the time of writing, comprises 37 recognized species
2002). Two different sampling occasions yielded two
(Ka¨mpfer et al., 2009). Chryseobacterium species may be
unidentified organisms that were related phylogenetically
found in soil and water environments and in clinical and
to the family Flavobacteriaceae (Bernardet et al., 2002). In
dairy sources (Bernardet et al., 2006). Three flavobacteria
the present study, a detailed classification of the two strains
previously classified as Sejongia antarctica, Sejongia jeonii
is provided on the basis of a polyphasic study, including
(Yi et al., 2005) and Sejongia marina (Lee et al., 2007)
analysis of morphological and physiological characteristics,
cellular fatty acid profiling, DNA–DNA hybridization
Chryseobacterium (Ka¨mpfer et al., 2009).
experiments and phylogenetic analysis of 16S rRNA genesequences. On the basis of the results obtained, strains
Recently, the bacterial diversity of sediments collected from
3A10T and ECP37T are considered to represent two novel
an industrially polluted site at Estarreja in northern
species of the genus Chryseobacterium.
Portugal was investigated. Sediments at the site have high
Sediment samples were serially diluted in saline solution
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA genesequences of strains 3A10T and ECP37T are EU360967 and
(0.85 %, w/v, NaCl) and were inoculated on trypticase soy
agar (TSA; Oxoid), adjusted to pH 7.0, at 30 uC. Strains
3A10T and ECP37T, selected on the basis of colony
after 24, 48, 72 and 120 h incubation at 25 uC. Hydrolysis of
morphology and colour, were purified by subculturing
arbutin, gelatin, casein, Tweens 20 and 80 and starch was
and were preserved at 280 uC in modified Luria–Bertani
tested on TSA as described by Hudson et al. (1986) and
broth (Tiago et al., 2004), supplemented with 15 % (v/v)
Smibert & Krieg (1994). Antibiotic susceptibility was
glycerol. Unless stated otherwise, all morphological and
examined on TSA at 25 uC for 72 h with Oxoid discs (see
tolerance tests were performed by using TSA.
species descriptions for details) following the interpretationcriteria proposed by the Comite´ de l’Antibiogramme de la
Cell morphology and gliding motility were examined by
Socie´te´ Franc¸aise de Microbiologie (1998). The phenotypic
phase-contrast microscopy. Flagellar motility was tested as
characteristics of strains 3A10T and ECP37T are given in
described by Alexander & Strete (2001). Gram staining and
Table 1 and in the species descriptions.
catalase and cytochrome oxidase tests were performed asdescribed by Murray et al. (1994) and Smibert & Krieg
Genomic DNA for determination of the G+C content was
(1994). Cell size was determined by using a Leica
obtained as described by Cashion et al. (1977). G+C ratios
DM4000B light microscope equipped with Leica Applied
were estimated by the Identification Service of the
Suite software. Presence of flexirubin pigments was
investigated by using the bathochromatic test with a 20 %
Zellkulturen (DSMZ; Braunschweig, Germany) by HPLC
(w/v) KOH solution as described by Bernardet et al.
as described by Mesbah et al. (1989). Fatty acid methyl
esters were obtained from fresh wet biomass, grown on
Phenotypic tests listed below were performed on strains
TSA at 28 uC for 24 h, by saponification, methylation and
3A10T and ECP37T and on their closest phylogenetic
extraction as described by Kuykendall et al. (1988), and the
neighbours, namely Chryseobacterium marinum NBRC
fatty acids were separated, identified and quantified
103143T, Chryseobacterium antarcticum AT1013T and
according to the protocol of the Microbial Identification
Chryseobacterium jeonii AT1047T. The pH range for growth
System, Sherlock version 4.6 (MIS-MIDI). The closest
was determined in buffered trypticase soy broth (TSB)
phylogenetic neighbours of strains 3A10T and ECP37T were
adjusted to pH 3–10 (at 1 pH unit intervals). Turbidity of
not included in the fatty acid analysis because they could
the cultures grown in an orbital shaker at 25 uC was
not be grown under the same conditions as the new
measured at 610 nm. All buffer solutions used to adjust the
isolates. Analysis of respiratory quinones was also carried
pH of TSB were prepared from 1 M stock solutions
according to Gomori (1990). Citrate buffer was used for
The major respiratory lipoquinone of strains 3A10T and
pH 3–6, phosphate buffer for pH 7, Tris buffer for pH 8,
ECP37T was menaquinone 6 (MK-6), which is in
and a carbonate–bicarbonate buffer for pH 9 and 10.
accordance with all recognized members of the family
Growth temperature ranges were determined on TSA
Flavobacteriaceae (Bernardet et al., 2006). The fatty acid
incubated at 4, 10, 15, 20, 25, 30, 37 and 50 uC.
profile of strains 3A10T and ECP37T, like those of
Growth in the presence of 0–20 % (w/v) NaCl (at 1 %
recognized species of the genus Chryseobacterium, was
intervals up to 10 %, then at 12, 15 and 20 %) was
dominated by branched components, namely iso-C15 : 0,
examined in TSB adjusted to pH 7 and incubated at 25 uC.
iso-C17 : 1v9c, anteiso-C15 : 0 and iso-C17 : 0 3-OH. The
The ability to grow under anaerobic conditions was
detailed fatty acid profiles of strain 3A10T and ECP37T
evaluated by incubating TSA plates in an anaerobic jar
are compared with those reported in the literature for
related Chryseobacterium species (Yi et al., 2005; Lee et al.,
using an AnaeroGen (Oxoid) at 25 uC for 7 days.
2007) in Table 2. The G+C content of the DNA of strains3A10T and ECP37T was 43 and 34 mol%, respectively. The
Acid production from carbohydrates was examined in API
DNA G+C content of strain ECP37T is in line with those
50 CH test strips by using API 50 CHB/E medium
reported for recognized Chryseobacterium species (Yi et al.,
(bioMe´rieux) according to the manufacturer’s instructions.
2005; Bernardet et al., 2006; Lee et al., 2007), but that of
Single carbon source assimilation was also determined in
strain 3A10T is significantly higher (Bernardet et al., 2006),
API 50 CH test strips (bioMe´rieux) by using cells
being similar to that of Kaistella koreensis (Kim et al.,
suspended in 0.1 M phosphate buffer (pH 7) supplemen-
ted with 0.7 % yeast nitrogen base (Difco) and 0.05 %NH
Extraction of genomic DNA, PCR amplification of the 16S
4Cl (Tiago et al., 2005, 2006). Bacterial cells were
suspended in sterilized water to reach a turbidity
rRNA gene and sequencing of the purified PCR products
corresponding to McFarland No. 6 standard. Cell suspen-
were carried out as described by Rainey et al. (1996).
sions (3 ml) were then added to 60 ml medium and
Cloning of the amplicons into pGEM T-Easy vector
inoculated in the API 50 CH test strip wells, according to
(Promega) and cycle sequencing were performed at
Macrogen Inc. (Seoul, Republic of Korea) by using
Nitrate reduction, indole production and the presence of
universal bacterial 16S rRNA primers (f27, f357, f519,
b-galactosidase, L-arginine dihydrolase and urease activities
f1114, r519, r800, r1056, r1492) (Lane, 1991). For
were determined by using API 20 NE strips and API AUX
phylogenetic analyses, the sequences were aligned by using
medium (bioMe´rieux). All the above results were recorded
the BioEdit program (version 220.127.116.11) (Hall, 1999) and
International Journal of Systematic and Evolutionary Microbiology 60
Two novel Chryseobacterium species from sediment
Table 1. Differential characteristics between strains 3A10T and ECP37T and related species of the genus Chryseobacterium
Strains: 1, 3A10T; 2, ECP37T; 3, C. marinum NBRC 103143T; 4, C. antarcticum AT1013T; 5, C. jeonii AT1047T. All data are from the present study. +, Positive; 2, negative; W, weakly positive. Data in parentheses were reported in the original descriptions of the species.
were analysed via the DNAML, SEQBOOT, DNAPARS, DNADIST
formed a monophyletic group with C. antarcticum
(Kimura’s two-parameter correction), NEIGHBOR, FITCH and
AT1013T, C. jeonii AT1047T and C. marinum NBRC
CONSENSE programs of the PHYLIP package (Felsenstein,
103143T supported by high bootstrap values (82–99 %).
1995). 16S rRNA gene sequences of other members of the
The other phylogenetic trees showed essentially the same
family Flavobacteriaceae were obtained from the NCBI
topology (not shown). These results show that the two
database (Benson et al., 2007). A manually corrected and
novel strains belong to the genus Chryseobacterium. Strain
degapped alignment of 31 sequences of 1249 nt was used.
3A10T showed 16S rRNA gene sequence similarities of 97.2
The robustness of the phylogenetic tree was confirmed by
and 96.6 % to the type strains of C. antarcticum and C.
using bootstrap analysis based on 100 resamplings of the
jeonii, respectively, while strain ECP37T showed 97.3 %
sequences (1000 for the neighbour-joining analysis). Non-
similarity to the type strain of C. marinum.
homologous and ambiguous nucleotide positions wereexcluded from the calculations. In the neighbour-joining
For DNA–DNA hybridization experiments, the genomic
phylogenetic tree (Fig. 1), strains 3A10T and ECP37T
DNA of strains 3A10T and ECP37T was hybridized and
Table 2. Fatty acid contents of strains 3A10T and ECP37T
strain 3A10T and C. marinum NBRC 103143T, C.
and the type strains of related Chryseobacterium species
antarcticum AT1013T and C. jeonii AT1047T, respectively. Levels of DNA–DNA relatedness between strain ECP37T
Strains: 1, 3A10T; 2, ECP37T; 3, C. marinum NBRC 103143T (data
and C. marinum NBRC 103143T, C. antarcticum AT1013T
from Lee et al., 2007); 4, C. antarcticum AT1013T (Yi et al., 2005);
and C. jeonii AT1047T were 10.1, 11.0 and 7.4 %,
5, C. jeonii AT1047T (Yi et al., 2005). Not all strains were cultivated
under the same conditions. Values are percentages of the total fatty
acids. Cellular fatty acids that amount to ,1 % of the total fatty acidcontent in all strains are not shown. ”, Not detected; tr, trace
The data presented herein demonstrate that strains 3A10T
and ECP37T represent two novel species of the genusChryseobacterium, for which the names Chryseobacterium
palustre sp. nov. and Chryseobacterium humi sp. nov.,respectively, are proposed. The new isolates can be
differentiated from closely related species based on a
combination of phenotypic features (Tables 1 and 2).
Chryseobacterium palustre (pa.lus9tre. L. neut. adj. palustre
Cells are Gram-staining-negative, aerobic, chemohetero-
trophic rods (1.5–2.1 mm long and 0.6 mm in diameter).
No flagellar or gliding motility. Colonies grown on TSA for
3 days are 0.3–1.2 mm in diameter, circular with regular
edges and yellow. Flexirubin-type pigments are absent.
Oxidase- and catalase-positive. Growth occurs at 10–37 uC
(optimum, about 30 uC), at pH 6.0–9.0 (optimum, pH 7)
and in the presence of 0–6.0 % NaCl (optimum, 2–3 %).
Nitrite and nitrate are not reduced. Casein, gelatin, arbutin,
aesculin and starch are hydrolysed. Positive for the Voges–
Proskauer reaction (API 20NE). Urease and b-galactosidase
activities are absent. Assimilates D-glucose, L-arabinose, D-
mannose, N-acetylglucosamine, maltose, adipic acid, malic
acid, trisodium citrate, L-xylose, D-fructose, L-sorbose, L-rhamnose, inositol, D-mannitol, D-sorbitol, lactose, suc-
*Unknown fatty acid; numbers indicate equivalent chain-length.
rose, glycogen, potassium gluconate, potassium 2-ketoglu-
DSummed features are groups of two or three fatty acids that cannot
conate and potassium 5-ketogluconate. Acid is produced
be reliably separated by GLC with the MIDI system. Summed feature
from galactose, D-glucose, D-mannose, maltose and starch.
3 comprised iso-C15 : 0 2-OH and/or C16 : 1v7c; summed feature 4
Assimilation or acid production is negative for the other
comprised iso-C17 : 1 I and/or anteiso-C17 : 1 B.
carbon sources in the API 50CH and API 20NE strips. Thetype strain is resistant to discs containing penicillin G(10 mg), polymyxin B (300 mg), gentamicin (10 mg),sulfamethoxazole (25 mg), colistin sulfate (50 mg) and
each was hybridized with the DNA of the type strains of
their closest phylogenetic neighbours, namely C. antarc-
(15 mg), ceftazidime (30 mg), cephalothin (30 mg), tetra-
ticum AT1013T, C. jeonii AT1047T and C. marinum NBRC
cycline (30 mg), amoxicillin (25 mg), ciprofloxacin (5 mg),
103143T. DNA–DNA hybridization was performed at the
ticarcillin (75 mg), sulfamethoxazole/trimethoprim (1.25/
DSMZ, as described by De Ley et al. (1970) with the
23.75 mg), vancomycin (30 mg), meropenem (10 mg),
modifications described by Huß et al. (1983), by using a
streptomycin (10 mg), lincomycin (2 mg), rifampicin
model Cary 100 Bio UV/visual spectrometer equipped with
(30 mg), cefoxitin (30 mg), cephalothin (30 mg), amoxicil-
a Peltier-thermostatted 666 multicell changer and a
lin/clavulanic acid (20/10 mg), chloramphenicol (30 mg)
temperature controller with in-situ temperature probe
and ampicillin (10 mg). The major respiratory lipoquinone
(Varian). DNA was isolated by using a French pressure cell
is MK-6. The major cellular fatty acids (¢6 % of the total)
(Thermo Spectronic) and was purified by chromatography
on hydroxyapatite as described by Cashion et al. (1977).
15 : 0, anteiso-C15 : 0, iso-C17 : 1v9c and iso-C17 : 0 3-
OH. Other cellular fatty acids are listed in Table 2. The
DNA–DNA hybridization experiments revealed levels of
G+C content of the genomic DNA of the type strain is
genomic relatedness of 15.2, 29.5 and 21.8 % between
International Journal of Systematic and Evolutionary Microbiology 60
Two novel Chryseobacterium species from sediment
Fig. 1. Neighbour-joining phylogenetic tree,based on 16S rRNA gene sequences, show-ing the relationships between strains 3A10Tand ECP37T and representative members ofthe family Flavobacteriaceae. Bootstrap values.80 % are shown at nodes. The sequence ofFlavobacterium aquatile ATCC 11947T wasused as an outgroup. Bar, 0.01 substitutionsper nucleotide position.
The type strain, 3A10T (5LMG 24685T 5NBRC 104928T),
floxacin (5 mg), colistin sulfate (50 mg), ticarcillin (75 mg),
was isolated from a rhizosphere sediment sample collected
sulfamethoxazole/trimethoprim (1.25/23.75 mg), merope-
near a stream at a polluted site located in the industrial
nem (10 mg), streptomycin (10 mg), lincomycin (2 mg),
complex of Estarreja, northern Portugal.
rifampicin (30 mg), cefoxitin (30 mg), cephalothin (30 mg),amoxicillin/clavulanic acid (20/10 mg), chloramphenicol(30 mg) and ampicillin (10 mg). The major respiratory
Description of Chryseobacterium humi sp. nov.
lipoquinone is MK-6. The major cellular fatty acids (¢8 %
Chryseobacterium humi (hu9mi. L. gen. n. humi of earth,
of the total) are iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1v9c
and anteiso-C15 : 0. Other cellular fatty acids are listed inTable 2. The G+C content of the genomic DNA of the
Cells are Gram-staining-negative, aerobic, chemohetero-
trophic rods (1.6–2.5 mm long and 0.5–0.6 mm in dia-meter). No flagellar or gliding motility. Colonies grown on
The type strain, ECP37T (5LMG 24684T 5NBRC 104927 T),
TSA for 3 days are 0.2–1.4 mm in diameter, circular with
was isolated from a soil sample collected at a polluted site
regular edges and yellow. Flexirubin-type pigments are
located in the industrial complex of Estarreja, northern
absent. Oxidase- and catalase-positive. Growth occurs at 4–
37 uC (optimum, 25–30 uC), at pH 6.0–9.0 (optimum,pH 7.0–8.0) and in the presence of 0–7.0 % NaCl
(optimum, 2 %). Nitrite and nitrate are not reduced. Casein, gelatin, arbutin, aesculin and starch are hydrolysed.
C. P. and M. F. C. acknowledge research grants from Fundac¸a˜o para a
Positive for the Voges–Proskauer reaction (API 20NE).
Cieˆncia e a Tecnologia (FCT), Portugal (SFRH/BD/25493/2005 andSFRH/BPD/44670/2008, respectively), and FCT projects POCI/AMB/
Urease and b-galactosidase activities are absent. Assimilates
60131/2004 and POCI/V.5/A0105/2005. We are indebted to Milton da
D-glucose, L-arabinose, D-mannose, maltose, potassium
Costa (University of Coimbra, Coimbra, Portugal) and to Fernanda
gluconate, amygdalin, cellobiose, maltose, glycogen and b-
Nobre for help with fatty acid analysis. We also thank the Unit of
gentiobiose. Acid is produced from lactose, D-glucose,
Research and Development of Nephrology (Faculty of Medicine,
cellobiose, starch, glycogen, amygdalin, arbutin, salicin,
University of Porto, Porto, Portugal) for help with cell measurements.
xylitol and gentiobiose. Assimilation or acid production isnegative for the other carbon sources in the API 50CH and
API 20NE strips. The type strain is resistant to discscontaining sulfamethoxazole (25 mg), vancomycin (30 mg),
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International Journal of Systematic and Evolutionary Microbiology 60
IJCCM October-December 2003 Vol 7 Issue 4 Indian J Crit Care Med July-September 2007 Vol 11 Issue 3 Review Article TNF-alpha inhibitors: Current indications Rashmi Sharma, Chaman Lal Sharma* Advances in the DNA hybrid technology led to the development of various biologicals that specifi cally target TNF-α. There are currently three anti- TNF-α drugs available- etanercept, infl ix
Accidents aigus des nouvelles toxicomanies Consensus d'actualisation SFAR - Médecine d'urgence 1999 P. Mols, N. Dedecker, G. Taton, M. Amuli Itegwa Service des urgences adultes, CHU Saint-Pierre, rue Haute 322, B -1000 Bruxelles, Belgique POINTS ESSENTIELS · Les amphétamines autorisées légalement sont soumises à prescription. Elles ont pour indication la narcolepsie, les troub