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1 Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
2 Alimentary Pharmabiotic Centre, Cork, Ireland
3 Department of Microbiology, University College Cork, Cork, Ireland
Correspondence
Catherine Stanton
cstanton{at}moorepark.teagasc.ie
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ABSTRACT |
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Published online ahead of print on 12 September 2003 as DOI 10.1099/ijs.0.02667-0.
The GenBank/EMBL/DDBJ accession numbers for the partial HSP60 gene sequences of LMG 21775T (=NCIMB 13940T), LMG 21773T (=NCIMB 13939T) and LMG 21774 are AY339132, AY339131 and AY339130, respectively.
Phase-contrast and whole-cell FISH images of strains studied in this paper are available as supplementary material in IJSEM Online.
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; Sghir et al., 2000; Harmsen et al., 2002). At the time of writing, the genus Bifidobacterium is represented by over 30 species and subspecies, which were mostly recovered from faecal, GIT and sewage samples (Biavati et al., 2000; Hoyles et al., 2002; Jian & Dong, 2002; Sakata et al., 2002). In a previous study within our laboratory (Simpson et al., 2003), 160 pig caecal isolates were assigned provisionally to the genus Bifidobacterium through detection of fructose-6-phosphate phosphoketolase activity (Scardovi, 1986). The isolates were considered to represent 15 strains, based on distinct PFGE macrorestriction patterns; four species were identified, based primarily on 16S rDNA sequencing. Two were considered to be novel species and were provisionally termed ‘Bifidobacterium pyschroaerophilum’ (which consisted of 23 isolates with the same PFGE macrorestriction pattern, termed PFGE type F) and ‘Bifidobacterium aerophilum’ (which consisted of 77 isolates with three distinct PFGE macrorestriction patterns, termed PFGE types B, E and Ea) (Simpson et al., 2003). ‘B. psychroaerophilum’ segregated in subcluster I of the 16S rDNA phylogenetic tree, with the majority of Bifidobacterium species. However, ‘B. aerophilum’ grouped in subcluster II, which was considered previously to represent an undefined genus that was related closely to Bifidobacterium, but with a lower DNA G+C content (Miyake et al., 1998). Indeed, based on heat-shock protein 60 (HSP60) gene sequencing, the two existing species within subcluster II, Bifidobacterium inopinatum and Bifidobacterium denticolens, were transferred to Scardovia gen. nov. and Parascardovia gen. nov., respectively (Jian & Dong, 2002).
The DNA G+C content of the isolates was determined by HPLC (BCCM/LMG, Belgium) as described by Mesbah et al. (1989). Values reported are means of three measurements on the same DNA. Partial HSP60 gene sequences were obtained by PCR as described by Jian et al. (2001) with the following modifications: the PCR included each primer at 10 pM and MgCl2 at 1 mM and was extended to 35 cycles. The amplicon (
590 bp) was extracted from an agarose gel and sequenced directly by using primers H60F and H60R. Analysis of HSP60 gene sequences and phylogenetic tree construction were done as described previously (Simpson et al., 2003). Biochemical profiles for strains from both proposed species were obtained from triplicate tests with the API Rapid ID32A and 20A test strips (bioMérieux). Strains were cultured under anaerobic conditions in modified MRS medium (mMRS), which comprised Lactobacilli MRS medium (Difco) supplemented with 0·05 % (w/v) cysteine/HCl, at 37 °C. Growth at low pH was determined by using pH-adjusted mMRS (with HCl). Growth on mMRS agar under aerobic conditions was determined by streaking cultures and/or spread-plating serial dilutions of overnight anaerobic mMRS broth cultures onto mMRS agar. Selected isolates were deposited with the LMG (BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent, Belgium) and NCIMB (National Collection of Industrial and Marine Bacteria, Aberdeen, UK) culture collections. High-molecular-mass DNA preparations and PFGE analysis were done as described previously (Simpson et al., 2003), except that cells were removed from the surface of mMRS agar plates. Whole-cell fluorescent in situ hybridization (FISH) was performed on cells from colonies by using an FITC-labelled 16S rRNA probe that was specific for either the genus Bifidobacterium or all bacteria (Microscreen; Ribotechnologies). Images were viewed with an Olympus BX51 epifluorescence microscope and captured by using an Olympus DP50 digital camera (15 ms exposure for fluorescent images). Cell-size determinations were done as described previously (Simpson et al., 2003).
Isolates T4, T6T, T8 and T16T were taken as representatives of PFGE types B, E, Ea and F, respectively. Isolate T16T (=LMG 21775T) had a DNA G+C content of 59·2 mol%, which was consistent with the reported range for the genus Bifidobacterium (55–67 mol%) (Crociani et al., 1996). This concurs with the general grouping of the strain with other Bifidobacterium species within the 16S rDNA (Simpson et al., 2003) and partial HSP60 gene (Fig. 1) phylogenetic trees. In both cases, inclusion of the strain resulted in separation of Bifidobacterium minimum from the three insect-derived species, Bifidobacterium asteroides, Bifidobacterium indicum and Bifidobacterium coryneforme. The highest similarity value for the partial HSP60 gene sequence was 83·58 %, which was shared with B. minimum, B. asteroides and B. indicum. These data support the previous assignment of the strain to the genus Bifidobacterium (Simpson et al., 2003). In the current study, the specific epithet has been corrected to pyschraerophilum. Isolates T4 (=LMG 21774) and T6T (=LMG 21773T) had the same DNA G+C content (54·7 mol%); this relatively low value is consistent with their grouping with Scardovia inopinata and Parascardovia denticolens in subcluster II of the 16S rDNA phylogenetic tree (Simpson et al., 2003). Based on partial HSP60 gene sequencing, both strains were again clearly related, as they shared 98·44 % sequence similarity. However, isolate T6T was distinct from the genera Scardovia, Parascardovia and Bifidobacterium, having 71·41, 71·97 and 73·81 % (mean value of 70·85–76·82 %) similarity, respectively (Fig. 1). The latter value is comparable to those reported previously for S. inopinata and P. denticolens (Jian et al., 2001) and we propose that ‘B. aerophilum’ should be transferred to a novel genus. Given the grouping of the novel species in II of the 16S rDNA phylogenetic tree, we have termed the novel genus Aeriscardovia gen. nov., in keeping with the taxonomy proposed by Jian & Dong (2002) and to highlight the aerotolerance of the strains. In accordance with the gender of the proposed genus name, the specific epithet was changed to aeriphila.
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. For many Bifidobacterium species, carbohydrate profiles are known to vary between strains (Scardovi, 1986; Gómez Zavaglia et al., 1998); this was also apparent for Aeriscardovia aeriphila gen. nov., sp. nov. In addition, given the relatively low number of strains (based on the number of distinct macrorestriction patterns) that are evident within the proposed species, the ability to identify the groups by biochemical profiling can not be stated. No Bifidobacterium species appear to grow at <20 °C (Scardovi, 1986; Biavati et al., 2000). However, at 4 and 8 °C, isolate T16T reduced the culture pH from 6·2 to 5·91 and 5·46, respectively, after 16 days incubation, compared to 4·6 after 24 h at 37 °C. Cells cultured at 8 °C yielded a PFGE macrorestriction pattern that was characteristic for PFGE type F (data not shown). Although sensitivity to oxygen is known to vary between Bifidobacterium species (Scardovi, 1986; Shimura et al., 1992; Meile et al., 1997), no existing species have been reported to grow on agar media under aerobic conditions. However, isolates T4, T6T and T16T all had comparable colony counts following spread-plating on mMRS agar and incubation under aerobic or anaerobic conditions. Based on colony size, growth was clearly reduced in the presence of air. When the type strains for B. minimum, S. inopinata and P. denticolens, the nearest neighbours to the novel species (Fig. 1
and Simpson et al., 2003) were streaked onto mMRS agar and incubated aerobically, only B. minimum showed signs of growth. However, growth was limited to the initial inoculation and no individual colonies were observed. Therefore, both novel species appear to be readily distinguished. It is not clear whether the proposed species represent food-associated bacteria that are passing through the GIT or are actual residents of the caecum. Given the relatively high concentration of dissolved oxygen in the pig caecum (Hillman et al., 1993), either habitat could explain their distinctive aerotolerance. Indeed, 25 % of anaerobes recovered from a human caecum were considered to be facultative anaerobes, compared to only 0·1 % for faecal isolates (Marteau et al., 2001). Similarly, bifidobacteria isolated from the chicken crop were considered to have aerotolerant properties (Petr & Rada, 2001). S. inopinata and P. denticolens have an atypical bifidobacterial morphology that is similar to that of isolates T4, T6T and T8 (see Supplementary Figure in IJSEM Online, panels 2a and 4a). In addition, for each species, cell morphology changed to an elongated rod with enlarged ends when exposed to oxygen (Crociani et al., 1996; Supplementary Figure, panel 5a). The morphology change was also evident when isolates T4, T6T and T8 were cultured at 30 or 46 °C (data not shown). From whole-cell FISH with a Bifidobacterium genus-specific 16S rRNA probe (Ventura et al., 2001), elongation appeared to be caused by incomplete cell division, resulting in compartmental distribution of the probe (Supplementary Figure, panel 5b). Incomplete cell division was previously considered to be the cause of elongation of cells of Bifidobacterium longum following exposure to oxygen (Ahn et al., 2001). The weak signal observed with strains LMG 21773T and LMG 21774 (Supplementary Figure, panel 2b) may relate to their non-Bifidobacterium status, although the signal was greater than that observed for the negative control culture, Lactobacillus rhamnosus (Supplementary Figure, panel 3b). It is not clear why, when elongated, cells of isolates T4, T6T and T8 had a strong signal (Supplementary Figure, panel 5b). By using a universal 16S rRNA probe, both morphology types yielded equivalent signals (Supplementary Figure, panels 4b and 6b), indicating that the weak signal was not related to poor probe penetration into the cells. PFGE patterns obtained from colonies grown under aerobic and anaerobic conditions were identical and characteristic for each strain, indicating that elongation represented an adaptation to stress. No change in cell morphology was seen when strain LMG 21775T was cultured aerobically and an equally strong signal was observed with the Bifidobacterium-specific probe for both aerobically and anaerobically cultured cells. This is consistent with the assignment of the novel species to the genus Bifidobacterium.
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and 3, respectively.
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r air; N.L. adj. philus from Gr. adj. philos loving; N.L. neut. adj. psychraerophilum cold- and air-loving).
From PFGE analysis, 23 pig caecal isolates appeared to represent a single strain, termed PFGE type F. The following description is based on a single isolate of this strain. Cells are Gram-positive, catalase- and oxidase-negative, non-motile, non-spore-forming, short and irregularly shaped rods, approximately 0·7–1·0 µm wide and 0·8–1·5 µm long with occasional bifurcations, arranged singly or in pairs. Colonies on mMRS agar under anaerobic conditions are white, circular and convex with smooth edges and reach a diameter of up to 3 mm after 3 days incubation at 37 °C. Colonies are also formed under aerobic conditions, but attain a reduced diameter of 1 mm after 3 days incubation. Optimal temperature for growth is 37 °C; maximum temperature for growth is 42 °C, with no growth at 46 °C. Growth occurs at 4 °C, although it is considerably reduced. Lowest pH attained is 4·0, with a minimum initial pH for growth of 4·5. DNA G+C content is 59·2 mol%. Biochemical characteristics are shown in Table 1.
Type strain is T16T (PFGE type F) (=LMG 21775T=NCIMB 13940T). Isolated from a pig caecum (contents and epithelium) in Fermoy, Ireland. Previously termed ‘Bifidobacterium pyschroaerophilum’.
Description of Aeriscardovia gen. nov.
Aeriscardovia (Aer'i.scar.do'vi.a. L. masc. n. aer, aeris air; N.L. fem. n. Scardovia a bacterial generic name to honour Vittorio Scardovi, an Italian microbiologist; N.L. fem. n. Aeriscardovia cells similar to the genus Scardovia that can grow in air).
Gram-positive, catalase- and oxidase-negative, non-motile, non-spore-forming, short and irregularly shaped rods. Optimal growth occurs under anaerobic conditions, with aerobic growth yielding elongated cells. DNA G+C content of the type species is 54·7 mol%. Isolated from a porcine caecum. According to 16S rDNA and HSP60 gene sequence comparisons, Aeriscardovia belongs to the family Bifidobacteriaceae. Only one species, the type species Aeriscardovia aeriphila, has thus far been described.
Description of Aeriscardovia aeriphila sp. nov.
Aeriscardovia aeriphila (L. masc. n. aer, aeris air; Gr. adj. philos loving; N.L. fem. adj. aeriphila air-loving).
From PFGE analysis, 77 pig caecal isolates appeared to represent three strains, termed PFGE types B, E and Ea. The following phenotypic description is based on a representative isolate for each strain. Cells are Gram-positive, catalase- and oxidase-negative, non-motile, non-spore-forming, short and irregularly shaped rods, approximately 0·6–0·9 µm wide and 0·8–1·5 µm long, that are arranged singly or in pairs, but not in chains. Colonies on mMRS agar under anaerobic conditions are grey–white, circular and flat to convex with entire edges and reach a diameter of up to 3 mm after 3 days incubation at 37 °C. Colonies are also formed under aerobic conditions, but a diameter of only 1 mm is attained after 5 days incubation. Under aerobic conditions, cell morphology includes elongated cells of approximately 4–6 µm in length. Optimum temperature for growth is 37 °C, with a maximum of 46 °C and minimum of 30 °C. No growth occurs at 48 or 25 °C. In mMRS, the lowest pH value attained is 4·2; minimum initial pH for growth is 4·5. DNA G+C content of strains of PFGE types B and E is 54·7 mol%. Biochemical characteristics are shown in Table 1. A single plasmid of 30 kbp may be present in isolates of PFGE type Ea, based on macrorestriction pattern data.
Type strain is T6T (PFGE type E) (=LMG 21773T=NCIMB 13939T). Isolated from a pig caecum (contents and epithelium) in Fermoy, Ireland. Previously termed ‘Bifidobacterium aerophilum’.
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ACKNOWLEDGEMENTS |
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