Bacillus Infantis Sp. Nov. And Bacillus Idriensis Sp. Nov ...
International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2541–2544
DOI 10.1099/ijs.0.64213-0
Bacillus infantis sp. nov. and Bacillus idriensis sp.
nov., isolated from a patient with neonatal sepsis
Kwan Soo Ko,1,2 Won Sup Oh,2 Mi Young Lee,1 Jang Ho Lee,3
Hyuck Lee,4 Kyong Ran Peck,2 Nam Yong Lee3 and Jae-Hoon Song1,2
Correspondence
1Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul 135-710, Korea
Won Sup Oh
2,3Division of Infectious Diseases2 and Department of Laboratory Medicine3, Samsung Medical
wsoh@smc.samsung.co.kr
Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
4Dong-A University Hospital, Busan 602-715, Korea
Two Gram-positive bacilli, designated as strains SMC 4352-1T and SMC 4352-2T, were isolated
sequentially from the blood of a newborn child with sepsis. They could not be identified by using
conventional clinical microbiological methods. 16S rRNA gene sequencing and phylogenetic
analysis revealed that both strains belonged to the genus Bacillus but clearly diverged from known
Bacillus species. Strain SMC 4352-1T and strain SMC 4352-2T were found to be closely related to
Bacillus firmus NCIMB 9366T (98?2 % sequence similarity) and Bacillus cibi JG-30T (97?1 %
sequence similarity), respectively. They also displayed low DNA–DNA reassociation values (less
than 40 %) with respect to the most closely related Bacillus species. On the basis of their
polyphasic characteristics, strain SMC 4352-1T and strain SMC 4352-2T represent two novel
species of the genus Bacillus, for which the names Bacillus infantis sp. nov. (type strain SMC
4352-1T=KCCM 90025T=JCM 13438T) and Bacillus idriensis sp. nov. (type strain SMC
4352-2T=KCCM 90024T=JCM 13437T) are proposed.
The genus Bacillus comprises aerobic or anaerobic,
of hospitalization, fever and hypotension developed, and
endospore-forming, Gram-positive bacteria. More than
two sets of blood cultures were found to harbour Gram-
100 bacterial species are included in the genus Bacillus,
positive bacilli. However, these isolates could not be
although many species have been transferred into other
identified by using conventional methods, such as VITEK
genera, e.g. Paenibacillus, Brevibacillus and Alicycloba-
(bioMe´rieux) and Microscan (Dade-Microscan), in the
cillus (http://www.bacterio.cict.fr/b/bacillus.html; Logan &
clinical microbiology laboratory.
Turnbull, 2003). Most are widely distributed saprophytes,
but some are pathogenic, including Bacillus alvei, B.
When the bacterial isolates were tested repeatedly with API
anthracis, B. brevis, B. cereus, B. circulans, B. coagulans, B.
50 CH kits (bioMe´rieux) to characterize their biochemical
licheniformis, B. macerans, B. pumilus, B. sphaericus,
traits, they were both found to be positive for D-xylose,
B. subtilis and B. thuringiensis (Logan & Turnbull, 2003).
galactose, glucose, fructose, mannitol, sorbitol, methyl a-D-
glucoside, N-acetylglucosamine, amygdalin, arbutin, aescu-
In this paper, we report on the isolation of two bacterial
lin, salicin, maltose, melibiose, sucrose, trehalose, raffinose,
strains, SMC 4352-1T and SMC 4352-2T, from a newborn
starch, glycogen and gluconate. Cellobiose, lactose and
child. 16S rRNA gene sequencing, DNA–DNA hybridization
inulin were positive only in the case of strain SMC 4352-1T,
and biochemical testing showed that these two bacterial
whereas glycerol, ribose, mannose, inositol, xylitol, gentio-
isolates constitute two novel species of the genus Bacillus.
biose and 5-ketogluconate were positive only for strain SMC
A 5-day-old female newborn child was admitted to our
4352-2T. Because conventional biochemical tests failed to
neonatal intensive-care unit because of cyanosis after birth.
identify these isolates to given species in the clinical
Transthoracic echocardiography showed the presence of a
microbiology laboratory, we subjected them to 16S rRNA
large (35 mm diameter) patent ductus arteriosus. On day 5
gene sequence analysis in order to identify them.
Bacterial DNA for the amplification of the 16S rRNA gene
was extracted using a boiling lysis method (Ko et al., 2005a,
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
gene sequences of strains SMC 4352-1T and SMC 4352-2T are
b). Colonies on blood agar plates were suspended in lysis
AY904032 and AY904033, respectively.
buffer (100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA and
Fatty acid compositions of strains SMC 4352-1T and SMC 4352-2T are
1 % Triton X-100) and then incubated at 90 uC for 10 min.
detailed in a supplementary table in IJSEM Online.
The mixture was then briefly centrifuged and the aqueous
64213 G 2006 IUMS
Printed in Great Britain
2541
K. S. Ko and others
phase was used as template for a PCR. The 16S rRNA gene
was amplified using the universal primers 16S-F0 (59-GA-
TCCTGGCTCAGGACGAAC-39) and 16S-R0 (59-CTTG-
TTACGACTTCACCCCA-39) (Hong et al., 2003; Zhu et al.,
2002). Template DNA and 20 pmol each primer were
added to the PCR mixture tubes (AccuPower PCR PreMix;
Bioneer), and the reaction mixtures were then subjected to
35 amplification cycles. Each cycle consisted of 30 s at 95 uC
for denaturation, 30 s at 60 uC, and 1 min at 72 uC for
extension, followed by a final extension at 72 uC for 5 min.
Amplified PCR products were purified for sequencing using
a PCR purification kit (CoreOne). Purified PCR products
were sequenced directly using the PCR amplification
primers and primer 16S-F5 (59-TATTGGGCGTAAAGCG-
AGCGC-39) (Ko et al., 2005a, b). The 16S rRNA gene
sequences of the two novel bacterial strains and those of
other Bacillus species retrieved from GenBank were aligned
using the CLUSTAL X program (Thompson et al., 1997). The
phylogenetic relationships among the two novel strains and
other Bacillus species were determined by using the
neighbour-joining, maximum-parsimony and maximum-
likelihood methods within PAUP, version 4.0 (Swofford,
1999).
Fig. 1. Neighbour-joining phylogenetic tree, based on almost-
The 16S rRNA gene sequences of strains SMC 4352-1T
complete sequences of 16S rRNA genes, showing the relation-
(1367 bp) and SMC 4352-1T (1437 bp) showed a pairwise
ships among the two novel isolates and species of the genus
similarity of 95?2 %, indicating that they belong to the same
Bacillus. Bootstrap values are shown as percentages from
genus but to different species. Comparisons with the
1000 replications at branch points. Species names and acces-
GenBank database revealed that their sequences did not
sion numbers are given as cited in the GenBank database.
Bacillus niacini S147 (GenBank accession no. AY509227)
match those of any known bacterium. The bacterium with
was used as an outgroup (not shown). Bar, 5 substitutions per
the greatest pairwise similarity to strain SMC 4352-1T was B.
100 nucleotides.
firmus NCIMB 9366T (98?2 %), whereas Bacillus cibi JG-30T
most closely matched strain SMC 4352-2T, showing a
pairwise similarity of 97?1 %. Initially, we retrieved and
SMC 4352-2T, the results of which are shown in
included the 16S rRNA gene sequences of nearly all Bacillus
Supplementary Table S1 available in IJSEM Online. The
species with validly published names. Of these, 19 Bacillus
profiles of the two strains differed: in strain SMC 4352-1T,
species found to have a close relationship to the two novel
the predominant fatty acid was iso-C
bacterial strains in the initial analysis were selected and
15 : 0 (44?0 %), followed
by anteiso-C
analysed (Fig. 1). A phylogenetic tree constructed using the
15 : 0 (30?9 %) and anteiso-C17 : 0 (7?4 %); in
strain SMC 4352-1T, however, the predominant fatty acid
neighbour-joining method suggested that strains SMC
was anteiso-C
4352-1T and SMC 4352-2T are members of the genus
15 : 0 (26?0 %), followed by iso-C15 : 0 (18?0 %)
and anteiso-C
Bacillus but they represent distinct species. Strain SMC
17 : 0 (6?9 %).
4352-1T clustered with B. firmus and Bacillus siralis with a
DNA–DNA reassociation was measured fluorometrically by
low bootstrap value (<50 %), whereas strain SMC 4352-2T
using the microplate hybridization method described by
clustered with B. cibi – a relationship supported by bootstrap
Ezaki et al. (1989). Strain SMC 4352-1T showed 36 % DNA–
value of 100 %. Other methods of phylogenetic reconstruc-
DNA reassociation with B. firmus ATCC 8247T, while strain
tion, such as maximum parsimony and maximum like-
SMC 4352-2T showed 23 % DNA–DNA reassociation with
lihood, showed relationships similar to those presented in
B. cibi KCTC 3880T. The DNA G+C contents of strains
Fig. 1.
SMC 4352-1T and SMC 4352-2T were determined spectro-
photometrically using the thermal denaturation method
The cellular fatty acid compositions of the two novel strains
(Marmur & Doty, 1962) and found to be 40?8 and
were examined using GC (6890A; Hewlett Packard) and a
41?2 mol%, respectively.
MIDI aerobe method (Chem Station, version 4.02) at
MicroID (Seoul, Korea). For fatty acid analysis, the strains
Although these two strains were isolated from a neonate,
were grown at 35 uC on blood agar for about 24 h. The
their association with neonatal sepsis could not be
cellular fatty acid profiles determined were compared with
determined. In addition to these two isolates, several
published profiles. Analysis of the cellular fatty acid
other bacterial organisms, such as vancomycin-susceptible
composition was performed for strains SMC 4352-1T and
enterococci, Acinetobacter lwoffii, Alcaligenes xylosoxidans
2542
International Journal of Systematic and Evolutionary Microbiology 56
Two novel Bacillus species from a patient
and methicillin-resistant, coagulase-negative staphylococci,
be differentiated from B. firmus in that it was positive for
were also isolated from the patient. Since these strains were
utilization of glycogen, inulin and salicin (Table 1). Strain
isolated from blood obtained via a central venous catheter,
SMC 4352-2T was positive for utilization of mannitol and
which is prone to contamination, they may not be pathogens
salicin, while B. cibi was negative for both (Table 1). Thus,
but rather contaminants or colonizers. B. firmus and B. cibi,
our data suggest that the two isolates represent novel Bacillus
the closest relatives of these two strains, also do not cause
species.
human disease (Logan & Turnbull, 2003; Yoon et al., 2005),
although their pathogenicity remains to be investigated.
On the basis of the above biochemical data, cellular fatty acid
compositions and molecular phylogenetic results, strains
On the basis of their biochemical characteristics and cellular
SMC 4352-1T and SMC 4352-2T represent two novel species
fatty acid profiles, the two isolates were demonstrated to
of Bacillus, for which we propose the names Bacillus infantis
represent two different species (Table 1 and Supplementary
sp. nov. and Bacillus idriensis sp. nov.
Table S1); this was confirmed by the results of 16S rRNA
gene sequence comparisons and DNA–DNA hybridizations.
The 16S rRNA gene sequence of strain SMC 4352-1T showed
Description of Bacillus infantis sp. nov.
the highest level of pairwise similarity to that of the type
Bacillus infantis (in.fan9tis. L. gen. n. infantis of an infant,
strain of B. firmus (98?2 %), while that of strain SMC 4352-
baby, the putative source of the type strain).
2T showed the highest level of pairwise similarity to that of
the type strain of B. cibi (97?1 %). These pairwise similarities
Aerobic, Gram-positive, catalase-positive, oxidase-negative
are sufficient to consider strains SMC 4352-1T and SMC
bacillus. Grows well on blood agar at 37 uC. When assayed
4352-2T as belonging to novel species, as many Bacillus
with the API 50 CH system, it is positive for utilization of D-
species having sequence pairwise similarities >98?5 % are
xylose, galactose, glucose, fructose, mannitol, sorbitol,
considered as different species. For example, the 16S rRNA
methyl a-D-glucoside, N-acetylglucosamine, amygdalin,
gene sequences of the type strains of Bacillus bataviensis, B.
arbutin, aesculin, salicin, maltose, melibiose, sucrose,
soli, B. dretensis, B. novalis and B. vireti, which are distinct
trehalose, raffinose, starch, glycogen, gluconate, cellobiose,
species, show 98?7–99?6 % pairwise similarity, whereas the
lactose and inulin and is negative for utilization of glycerol,
type strains of B. mojavensis, B. subtilis, B. amyloliquefaciens,
ribose, mannose, inositol, xylitol, gentiobiose and 5-
B. vallismortis and B. atrophaeus show 98?5–99?6 % pairwise
ketogluconate. The major fatty acid is iso-C15 : 0 (44?0 %),
similarity and yet they are regarded as distinct species. In
followed by anteiso-C15 : 0 (30?9 %) and anteiso-C17 : 0
addition, DNA–DNA hybridization results supported
(7?4 %), and its 16S rRNA gene sequence shows most
species differentiation of the two strains, because their
similarity (98?2 %) to that of the type strain of B. firmus.
values for DNA–DNA reassociation with the closest species
DNA–DNA reassociation with B. firmus ATCC 8247T is
were below 40 %. Phenotypically, strain SMC 4352-1T could
36 %. The DNA G+C content is 40?8 mol%.
The type strain, SMC 4352-1T (=KCCM 90025T=JCM
13438T), was isolated from a newborn child with sepsis.
Table 1. Comparison of the biochemical characteristics
of strains SMC 4352-1T and SMC 4352-2T and related
species
Description of Bacillus idriensis sp. nov.
Taxa: 1, strain SMC 4352-1T; 2, strain SMC 4352-2T; 3, B. cibi KCTC
Bacillus idriensis (id.ri.en9sis. N.L. masc. adj. idriensis
3880T; 4, B. firmus; 5, B. siralis; 6, B. nealsonii; 7, B. megaterium.
arbitrary specific epithet pertaining to IDRI, the Infectious
Biochemical profiles of strains SMC 4352-1T, SMC 4352-2T and B.
Disease Research Institute, where this study was performed).
cibi KCTC 3880T were determined using API 50 CH, and those of
related species were retrieved from Logan & Turnbull (2003),
Aerobic, Gram-positive, catalase-positive, oxidase-negative
Pettersson et al. (2000) and Venkateswaran et al. (2003). Symbols:
bacillus. Grows well on blood agar at 37 uC. When assayed
+, positive; 2, negative; V, variable; ND, no data available. All strains
with the API 50 CH system, it is positive for utilization of D-
are negative for utilization of D-arabinose.
xylose, galactose, glucose, fructose, mannitol, sorbitol,
methyl a-D-glucoside, N-acetylglucosamine, amygdalin,
Biochemical reaction
1
2
3
4
5
6
7
arbutin, aesculin, salicin, maltose, melibiose, sucrose,
Utilization of:
trehalose, raffinose, starch, glycogen, gluconate, glycerol,
Glycerol
2
ribose, mannose, inositol, xylitol, gentiobiose and 5-
+
V
2
2
+
+
Glycogen
ketogluconate, but negative for utilization of cellobiose,
+
+
+
2
2
2
+
Inulin
+
2
2
2
ND
2
+
lactose and inulin. The predominant fatty acid is anteiso-
Mannitol
+
+
2
V
2
2
+
C15 : 0 (26?0 %), followed by iso-C15 : 0 (18?0 %) and anteiso-
Salicin
+
+
2
2
2
+
+
C17 : 0 (6?9 %), and its 16S rRNA gene sequence shows most
Trehalose
+
+
+
V
2
+
+
similarity (97?1 %) to that of the type strain of B. cibi. DNA–
Starch
+
+
+
+
2
ND
+
DNA reassociation with B. cibi KCTC 3880T is 23 %. The
DNA G+C content is 41?2 mol%.
http://ijs.sgmjournals.org
2543
K. S. Ko and others
The type strain, SMC 4352-2T (=KCCM 90024T=JCM
Logan, N. A. & Turnbull, P. C. B. (2003). Bacillus and other aerobic
13437T), was isolated from a newborn child with sepsis.
endospore-forming bacteria. In Manual of Clinical Microbiology, 8th
edn, pp. 445–460. Edited by P. R. Murray, E. J. Baron, J. H.
Jorgensen, M. A. Pfaller & R. H. Yolken. Washington, DC: American
Society for Microbiology.
Acknowledgements
Marmur, J. & Doty, P. (1962). Determination of the base composition
This work was partly supported by the Asian-Pacific Research
of deoxyribonucleic acid from its thermal denaturation temperature.
Foundation for Infectious Diseases (ARFID) and the Samsung
J Mol Biol 5, 109–118.
Biomedical Research Institute (SBRI, C-A5-319-1).
Pettersson, B., de Silva, S. K., Uhle´n, M. & Priest, F. G. (2000).
Bacillus siralis sp. nov., a novel species from silage with a higher
order structural attribute in the 16S rRNA genes. Int J Syst Evol
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International Journal of Systematic and Evolutionary Microbiology 56
