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1 Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA
2 School of Food Biosciences, University of Reading, Reading, RG6 6AP, UK
Correspondence
Terence R. Whitehead
whitehtr{at}ncaur.usda.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 1 August 2003 as DOI 10.1099/ijs.0.02719-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains NRRL B-23456T and NRRL B-23458T are AF445264 and AF445239, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Spoelstra, 1980; Yasuhara et al., 1984; Zahn et al., 1997). In addition, production of gaseous emissions (such as ammonia) within confined facilities, such as those used with swine, can pose potential health problems to both animals and human workers. Although production of these chemicals is the result of microbiological activity, little is known about the types of micro-organisms that are responsible for their production. During the course of an ongoing study into the microbial diversity that is present within manure storage pits (Whitehead & Cotta, 2000; Cotta et al., 2003), we characterized four strictly anaerobic, Gram-positive, asporogenous, rod-shaped organisms of uncertain taxonomic position. Based on the results of a polyphasic taxonomic study, it is proposed that the isolates should be assigned to a novel genus, Hespellia gen. nov., as Hespellia stercorisuis sp. nov. and Hespellia porcina sp. nov. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Leedle & Hespell, 1980) or 80 % (v/v) clarified swine manure slurry (Slurry medium; 8000 g, 20 min, 4 °C) (Cotta et al., 2003). Media used in these experiments were prepared anaerobically by using the method of Hungate, as modified by Bryant (1972). Basic media contained macro- and microminerals, buffers, reducing agents and other components, as in RGM medium described by Hespell et al. (1987) or anaerobic BHI medium as described by Whitehead & Flint (1995). No additional volatile fatty acids (FAs) were added to slurry-containing media. Glucose, xylose, cellobiose, maltose, starch (each at 0·05 %, w/v) and peptone (0·3 %, w/v) were provided as complex carbon, nitrogen and energy sources. In the case of isolates PC80T and PC82, agar medium contained 10 µg erythromycin ml-1 (Cotta et al., 2003). Plates were incubated anaerobically in a Coy anaerobic chamber in a carbon dioxide/hydrogen (96 : 4) atmosphere. Plates were incubated initially at room temperature (approx. 24 °C) for manure slurry samples, to simulate the pit environment (Cotta et al., 2003). The new isolates were found subsequently to grow equally well at 37 °C. Single colonies were picked and streaked out repeatedly until pure cultures were obtained. For morphological and physiological studies, strains were grown on RGM medium with 0·2 % carbohydrate or BHI medium. Presence of spores was determined by visual examination, as well as by incubation of cultures in 95 % ethanol followed by plating onto anaerobic agar medium. Fermentation end products were determined by using GC and HPLC; GC, including hydrogen production analyses, was performed on a Hewlett Packard model 5890A gas chromatograph with a HP Innowax capillary column (30 mx0·32 mm, 0·5 µm film thickness; Agilent Technologies) (Miller, 2001). HPLC detection of organic acids was performed by using a Bio-Rad Aminex HPX-87H column at 65 °C with 5 mM H2SO4 as solvent. Peaks were detected by a Waters model 410 differential refractometer and identified by comparison with retention times of authentic standards (Miller, 2001). For long-chain cellular FA analyses, isolates were grown in brain heart infusion broth. FAs were identified by using the MIDI system according to the manufacturer's instructions. Determination of DNA G+C content was carried out by thermal denaturation of chromosomal DNA, using a Beckman model DU 640 spectrophotometer equipped with a high-performance temperature controller and Tm analysis software (Johnson, 1994). 16S rRNA gene fragments were generated by PCR with universal primers pA (positions 8–28, Escherichia coli numbering) and pH* (1542–1522). Amplified products were purified by using a QIAquick PCR Purification kit (Qiagen) and sequenced directly by using primers to conserved regions of the 16S rRNA gene. Sequencing was performed by using a PRISM Taq Dyedeoxy Terminator Cycle Sequencing kit and a model 373A automatic DNA sequencer (both from Applied Biosystems). To establish the closest relatives of the isolates, preliminary searches in GenBank/EMBL were performed with the program FASTA. Closely related sequences were retrieved and aligned with the newly determined sequences by using the program DNATools (Rasmussen, 1995). Approximately 100 bases at the 5' end of the resulting multiple sequence alignment were omitted from further analysis because of alignment uncertainties (due to the highly variable region V1) by using the program GeneDoc (Nicholas et al., 1997). A phylogenetic tree was reconstructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs DNATools and TREEVIEW (Page, 1996); stability of the groupings was estimated by bootstrap analysis (1000 replications). |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The DNA G+C contents of isolates PC18T and PC80T were found to be 43·7 and 43·8 mol%, respectively. Both sets of chemical data reinforced the affinity between the isolates.
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), a large suprageneric cluster that embraces taxa with a variety of generic names, such as Anaerostipes, Butyrivibrio, Catonella, Clostridium, Coprococcus, Dorea, Eubacterium, Johnsonella, Lachnospira, Roseburia, Ruminococcus and Sporobacterium. Within this suprageneric rDNA group, the two pairs of isolates formed a distinct and robust subcluster that was supported by a bootstrap value of 100 % (Fig. 1
). Based on both sequence divergence and tree topology considerations, it is clear that the unidentified isolates do not possess a particularly close association with any described species. The nearest apparent relative to the new isolates corresponds to an rDNA clone (designated OTU-95; 1453 bases) that was derived from an uncultured organism from the gastrointestinal tract of pigs (Leser et al., 2002). However, 6·1–6·4 % sequence divergence between the pig manure isolates and this uncultured organism and the low bootstrap resampling value (Fig. 1) indicate that this association is not particularly close. Leser et al. (2002) published a comprehensive rDNA inventory of the pig gastrointestinal tract and described over 300 novel phylotypes, which may correspond to as-yet-uncharacterized bacterial genera or species. Surprisingly, none of these novel lines corresponded to either of the two groups of isolates reported here. Taxonomically, it is evident that the two groups of unknown isolates represent closely related, albeit different, species. 16S rRNA gene sequence divergence of 3 % is consistent with separate species (Stackebrandt & Goebel, 1994) and the two groups show major phenotypic differences. Before the advent of 16S rRNA gene sequencing, the two swine manure species would have been considered taxonomically to conform to the genus Eubacterium, which has in the past acted as a repository for strictly anaerobic, Gram-positive, asporogenous, rod-shaped organisms. However, it is now acknowledged universally that the eubacteria represent a phylogenetically very heterogeneous group of organisms and there is a growing consensus that the genus Eubacterium should be restricted to the type species Eubacterium limosum and its close phylogenetic relatives (Eubacterium barkeri, Eubacterium callanderi and Eubacterium aggregans) (Willems & Collins, 1996; Kageyama et al., 1999). E. limosum and related species are far removed phylogenetically (approx. 20 % 16S rRNA gene sequence divergence) from the unidentified isolates reported here and belong to a different phylogenetic group (Collins et al., 1994; Willems & Collins, 1996). Furthermore, the unknown isolates are incompatible phenotypically with eubacteria, as they do not produce butyrate as a major fermentation product. Based on phylogenetic findings, it is evident that the unidentified isolates form a distinct group and do not display a close affinity to any recognized genus within the C. coccoides rDNA cluster. The unknown organisms also differ phenotypically from all other genera within this suprageneric grouping. For example, they can be distinguished from clostridial species and other spore-forming taxa (e.g. Sporobacterium) in not producing endospores, from Lachnospira by the absence of curved cellular shapes, from Dorea by hydrolysis of starch and the end products of glucose metabolism, from Coprococcus and Ruminococcus by cellular morphology and the end products of glucose fermentation and from Roseburia and Butyrivibrio in the end products of glucose metabolism (i.e. not producing butyric acid) and in being non-motile. Based on their overall phenotypic and phylogenetic resemblance, combined with their distinctiveness from other members of the C. coccoides group, we consider that the two unidentified species merit classification in a novel genus, Hespellia gen. nov. We propose that isolates PC17 and PC18T should be classified as Hespellia stercorisuis sp. nov. and isolates PC80T and PC82 should be designated Hespellia porcina sp. nov.
Description of Hespellia gen. nov.
Hespellia (Hes.pel'li.a. N.L. fem. n. Hespellia to honour the late American microbiologist Robert B. Hespell, in recognition of his many contributions to anaerobic microbiology).
Gram-positive-staining, non-spore-forming, non-motile, rod-shaped cells. Strictly anaerobic and catalase- and oxidase-negative. Glucose and some other sugars are fermented. Major end products of glucose metabolism are formic, acetic, lactic and propionic acids; hydrogen is produced. Butyric acid is not formed. Aesculin and starch are hydrolysed, but gelatin is not. Indole-negative. Nitrate is not reduced to nitrite. Long-chain cellular FAs consist of complex mixtures of FAs and DMAs, together with small amounts of ALDs. DNA G+C content is 43·7–43·8 mol%. The type species of the genus is Hespellia stercorisuis.
Description of Hespellia stercorisuis sp. nov.
Hespellia stercorisuis (ster.co.ri.su'is. L. masc. n. stercus, -oris faeces, manure; L. gen. n. suis of a pig, N.L. gen. n. stercorisuis from pig faeces/manure).
Cells consist of Gram-positive-staining rods that are obligately anaerobic, non-motile and non-spore-forming. Cells are 0·5–1·0 µm wide by 1·5–5·0 µm long and occur singly, in pairs or in short chains. After 48 h anaerobic incubation at 37 °C under a gas phase of N2 and CO2 (80 : 20, v/v) on RGM/glucose agar, colonies are grey, convex, smooth, shiny and translucent. Catalase- and oxidase-negative. Hydrogen is formed after growth on glucose. End products of metabolism from BHI broth are formic, acetic, lactic and propionic acids. Glucose, lactose, cellobiose, trehalose, amygdalin, sorbitol, maltose, mannose, sucrose, fructose and xylose are utilized as energy sources, but arabinose, inositol, raffinose, rhamnose and inulin are not. Aesculin and starch are hydrolysed, but gelatin is not. Nitrate is not reduced. Indole is not produced. Long-chain cellular FAs consist of complex mixtures of FAs and DMAs, together with small amounts of ALDs; predominant components are C14 : 0 FA, C16 : 0 FA, C16 : 1cis9 DMA and C18 : 1cis11 DMA. DNA G+C content of the type strain is 43·7 mol%.
The type strain is PC18T=NRRL B-23456T=CCUG 46279T=ATCC BAA-677T. Isolated from pig manure.
Description of Hespellia porcina sp. nov.
Hespellia porcina (por.ci'na. L. fem. adj. porcina of pigs, pertaining to pigs).
Cells are Gram-positive-staining, non-motile, non-spore-forming rods. Obligately anaerobic. Cells are 0·5–1·0 µm wide by 1·5–4·0 µm long and occur singly, in pairs or in short chains. After 48 h anaerobic incubation at 37 °C under a gas phase of N2 and CO2 (80 : 20, v/v) on RGM/glucose agar, colonies are grey, convex, smooth, shiny and translucent. Catalase- and oxidase-negative. Hydrogen is formed after growth on glucose. End products of metabolism from BHI broth are formic, acetic, lactic and propionic acids. Glucose, arabinose, inositol, maltose, mannose, sucrose, fructose and xylose are utilized as energy sources, but amygdalin, cellobiose, lactose, raffinose, rhamnose, sorbitol, trehalose and inulin are not. Nitrate is not reduced. Aesculin and starch are hydrolysed, but gelatin is not. Indole is not produced. Long-chain cellular FAs consist of complex mixtures of FAs and DMAs, together with small amounts of ALDs; predominant components are C14 : 0 FA, C14 : 0 DMA, C16 : 0 FA and C16 : 1cis9 DMA. DNA G+C content of the type strain is 43·8 mol%.
The type strain is PC80T=NRRL B-23458T=ATCC BAA-674T. Isolated from pig manure.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.[CrossRef][Medline]
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