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1 Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul 135-710, Korea
2 Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
3 Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
4 Dong-A University Hospital, Busan 602-715, Korea
Correspondence
Won Sup Oh
wsoh{at}smc.samsung.co.kr
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ABSTRACT |
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Fatty acid compositions of strains SMC 4352-1T and SMC 4352-2T are detailed in a supplementary table in IJSEM Online.
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MAIN TEXT |
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). Most are widely distributed saprophytes, but some are pathogenic, including Bacillus alvei, B. anthracis, B. brevis, B. cereus, B. circulans, B. coagulans, B. licheniformis, B. macerans, B. pumilus, B. sphaericus, B. subtilis and B. thuringiensis (Logan & Turnbull, 2003). In this paper, we report on the isolation of two bacterial strains, SMC 4352-1T and SMC 4352-2T, from a newborn child. 16S rRNA gene sequencing, DNA–DNA hybridization and biochemical testing showed that these two bacterial isolates constitute two novel species of the genus Bacillus. A 5-day-old female newborn child was admitted to our neonatal intensive-care unit because of cyanosis after birth. Transthoracic echocardiography showed the presence of a large (35 mm diameter) patent ductus arteriosus. On day 5 of hospitalization, fever and hypotension developed, and two sets of blood cultures were found to harbour Gram-positive bacilli. However, these isolates could not be identified by using conventional methods, such as VITEK (bioMérieux) and Microscan (Dade-Microscan), in the clinical microbiology laboratory.
When the bacterial isolates were tested repeatedly with API 50 CH kits (bioMérieux) to characterize their biochemical traits, they were both found to be positive for D-xylose, galactose, glucose, fructose, mannitol, sorbitol, methyl 
-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, maltose, melibiose, sucrose, trehalose, raffinose, starch, glycogen and gluconate. Cellobiose, lactose and inulin were positive only in the case of strain SMC 4352-1T, whereas glycerol, ribose, mannose, inositol, xylitol, gentiobiose and 5-ketogluconate were positive only for strain SMC 4352-2T. Because conventional biochemical tests failed to identify these isolates to given species in the clinical microbiology laboratory, we subjected them to 16S rRNA 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, b). Colonies on blood agar plates were suspended in lysis buffer (100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA and 1 % Triton X-100) and then incubated at 90 °C for 10 min. The mixture was then briefly centrifuged and the aqueous phase was used as template for a PCR. The 16S rRNA gene was amplified using the universal primers 16S-F0 (5'-GATCCTGGCTCAGGACGAAC-3') and 16S-R0 (5'-CTTGTTACGACTTCACCCCA-3') (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 °C for denaturation, 30 s at 60 °C, and 1 min at 72 °C for extension, followed by a final extension at 72 °C 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 (5'-TATTGGGCGTAAAGCGAGCGC-3') (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).
The 16S rRNA gene sequences of strains SMC 4352-1T (1367 bp) and SMC 4352-1T (1437 bp) showed a pairwise similarity of 95.2 %, indicating that they belong to the same genus but to different species. Comparisons with the GenBank database revealed that their sequences did not match those of any known bacterium. The bacterium with the greatest pairwise similarity to strain SMC 4352-1T was B. 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 included the 16S rRNA gene sequences of nearly all Bacillus species with validly published names. Of these, 19 Bacillus species found to have a close relationship to the two novel bacterial strains in the initial analysis were selected and analysed (Fig. 1). A phylogenetic tree constructed using the neighbour-joining method suggested that strains SMC 4352-1T and SMC 4352-2T are members of the genus Bacillus but they represent distinct species. Strain SMC 4352-1T clustered with B. firmus and Bacillus siralis with a low bootstrap value (<50 %), whereas strain SMC 4352-2T clustered with B. cibi – a relationship supported by bootstrap value of 100 %. Other methods of phylogenetic reconstruction, such as maximum parsimony and maximum likelihood, showed relationships similar to those presented in Fig. 1.
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DNA–DNA reassociation was measured fluorometrically by using the microplate hybridization method described by Ezaki et al. (1989). Strain SMC 4352-1T showed 36 % DNA–DNA reassociation with B. firmus ATCC 8247T, while strain SMC 4352-2T showed 23 % DNA–DNA reassociation with B. cibi KCTC 3880T. The DNA G+C contents of strains SMC 4352-1T and SMC 4352-2T were determined spectrophotometrically using the thermal denaturation method (Marmur & Doty, 1962) and found to be 40.8 and 41.2 mol%, respectively.
Although these two strains were isolated from a neonate, their association with neonatal sepsis could not be determined. In addition to these two isolates, several other bacterial organisms, such as vancomycin-susceptible enterococci, Acinetobacter lwoffii, Alcaligenes xylosoxidans and methicillin-resistant, coagulase-negative staphylococci, were also isolated from the patient. Since these strains were isolated from blood obtained via a central venous catheter, which is prone to contamination, they may not be pathogens but rather contaminants or colonizers. B. firmus and B. cibi, the closest relatives of these two strains, also do not cause human disease (Logan & Turnbull, 2003; Yoon et al., 2005), although their pathogenicity remains to be investigated.
On the basis of their biochemical characteristics and cellular fatty acid profiles, the two isolates were demonstrated to represent two different species (Table 1 and Supplementary 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 the highest level of pairwise similarity to that of the type strain of B. firmus (98.2 %), while that of strain SMC 4352-2T showed the highest level of pairwise similarity to that of the type strain of B. cibi (97.1 %). These pairwise similarities are sufficient to consider strains SMC 4352-1T and SMC 4352-2T as belonging to novel species, as many Bacillus species having sequence pairwise similarities >98.5 % are considered as different species. For example, the 16S rRNA gene sequences of the type strains of Bacillus bataviensis, B. soli, B. dretensis, B. novalis and B. vireti, which are distinct species, show 98.7–99.6 % pairwise similarity, whereas the type strains of B. mojavensis, B. subtilis, B. amyloliquefaciens, B. vallismortis and B. atrophaeus show 98.5–99.6 % pairwise similarity and yet they are regarded as distinct species. In addition, DNA–DNA hybridization results supported species differentiation of the two strains, because their values for DNA–DNA reassociation with the closest species were below 40 %. Phenotypically, strain SMC 4352-1T could be differentiated from B. firmus in that it was positive for utilization of glycogen, inulin and salicin (Table 1). Strain SMC 4352-2T was positive for utilization of mannitol and salicin, while B. cibi was negative for both (Table 1). Thus, our data suggest that the two isolates represent novel Bacillus species.
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Description of Bacillus infantis sp. nov.
Bacillus infantis (in.fan'tis. L. gen. n. infantis of an infant, baby, the putative source of the type strain).
Aerobic, Gram-positive, catalase-positive, oxidase-negative bacillus. Grows well on blood agar at 37 °C. When assayed with the API 50 CH system, it is positive for utilization of D-xylose, galactose, glucose, fructose, mannitol, sorbitol, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, maltose, melibiose, sucrose, trehalose, raffinose, starch, glycogen, gluconate, cellobiose, lactose and inulin and is negative for utilization of glycerol, ribose, mannose, inositol, xylitol, gentiobiose and 5-ketogluconate. The major fatty acid is iso-C15 : 0 (44.0 %), followed by anteiso-C15 : 0 (30.9 %) and anteiso-C17 : 0 (7.4 %), and its 16S rRNA gene sequence shows most similarity (98.2 %) to that of the type strain of B. firmus. DNA–DNA reassociation with B. firmus ATCC 8247T is 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.
Description of Bacillus idriensis sp. nov.
Bacillus idriensis (id.ri.en'sis. N.L. masc. adj. idriensis arbitrary specific epithet pertaining to IDRI, the Infectious Disease Research Institute, where this study was performed).
Aerobic, Gram-positive, catalase-positive, oxidase-negative bacillus. Grows well on blood agar at 37 °C. When assayed with the API 50 CH system, it is positive for utilization of D-xylose, galactose, glucose, fructose, mannitol, sorbitol, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, maltose, melibiose, sucrose, trehalose, raffinose, starch, glycogen, gluconate, glycerol, ribose, mannose, inositol, xylitol, gentiobiose and 5-ketogluconate, but negative for utilization of cellobiose, lactose and inulin. The predominant fatty acid is anteiso-C15 : 0 (26.0 %), followed by iso-C15 : 0 (18.0 %) and anteiso-C17 : 0 (6.9 %), and its 16S rRNA gene sequence shows most similarity (97.1 %) to that of the type strain of B. cibi. DNA–DNA reassociation with B. cibi KCTC 3880T is 23 %. The DNA G+C content is 41.2 mol%.
The type strain, SMC 4352-2T (=KCCM 90024T=JCM 13437T), was isolated from a newborn child with sepsis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Hong, T., Heibler, N. & Tang, Y.-W. (2003). "Bacillus hackensackii" sp. nov., a novel carbon dioxide sensitive bacterium isolated from blood culture. Diagn Microbiol Infect Dis 45, 143–147.[CrossRef][Medline]
Ko, K. S., Lee, N. Y., Oh, W. S., Lee, J. H., Ki, H. K., Peck, K. R. & Song, J. H. (2005a). Tepidimonas arfidensis sp. nov., a novel Gram-negative and thermophilic bacterium isolated from the bone marrow of a patient with leukemia in Korea. Microbiol Immunol 49, 785–788.[Medline]
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Logan, N. A. & Turnbull, P. C. B. (2003). Bacillus and other aerobic 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.
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Yoon, J. H., Lee, C. H. & Oh, T. K. (2005). Bacillus cibi sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 55, 733–756.
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