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1 School of Food Biosciences, University of Reading, Whiteknights, Reading RG6 6AP, UK
2 Université de Montréal, Faculté de Médecine Vétérinaire, St Hyacinthe, Quebec, Canada
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Sweden
Correspondence
Matthew D. Collins
M.D.Collins{at}reading.ac.uk
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ABSTRACT |
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Strain 01-570-1T (=CCUG 45438T=CIP 107425T) was isolated from stored porcine semen. The unidentified bacterium was cultured on Columbia blood-agar base supplemented with 5 % horse blood at 37 °C, under anaerobic conditions. The organism was characterized biochemically using the API Rapid ID 32Strep, API Rapid ID 32A and API ZYM systems according to the manufacturer's instructions (API bioMérieux). Cell-wall murein was prepared by mechanical disruption of cells and acid hydrolysates were analysed as described by Schleifer & Kandler (1972)
, except that ascending TLC with cellulose sheets was used. Long-chain cellular fatty acids were analysed as described by Kämpfer & Kroppenstedt (1996). The G+C content of DNA was determined by HPLC according to Mesbah et al. (1989). The 16S rRNA gene of the isolate was amplified by PCR and sequenced directly using a Taq Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the novel isolate were determined by performing database searches. These sequences and those of other known related strains were retrieved from GenBank and aligned with the newly determined sequence using the program DNATools (Rasmussen, 1995). The resulting multiple sequence alignment was corrected manually and a distance matrix was calculated with the programs PRETTY and DNADIST (using the Kimura 2-parameter correction) (Felsenstein, 1989). A phylogenetic tree was constructed according to the neighbour-joining method with the program NEIGHBOR and the stability of the groupings was estimated by bootstrap analysis (500 replications) using the programs DNABOOT, DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1989).
The unidentified organism recovered from porcine semen consisted of short, Gram-positive rods. The organism was facultatively anaerobic but grew better under anaerobic conditions and was catalase-negative. Using the API Rapid ID 32Strep system, the unknown organism did not produce acid from any of the carbohydrates tested. Activity was observed for arginine dihydrolase, alkaline phosphatase, alanine-phenylalanine-proline arylamidase, 
-glucosidase, glycyl tryptophan arylamidase, N-acetyl--glucosaminidase, pyroglutamic acid arylamidase and -mannosidase. All other enzyme tests were negative with this kit and the organism did not hydrolyse hippurate and did not produce acetoin. Using the API Rapid ID 32A system, the organism failed to produce acid from mannose and raffinose, did not reduce nitrate and did not produce indole. The organism displayed activity for alanine arylamidase, alkaline phosphatase, arginine arylamidase, arginine dihydrolase, 
-fucosidase, -glucosidase, glycine arylamidase, histidine arylamidase, proline arylamidase, leucyl glycine arylamidase, leucine arylamidase, N-acetyl--glucosaminidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, serine arylamidase and tyrosine arylamidase. All other enzyme tests in this kit gave negative results. With the API ZYM system, acid phosphatase, alkaline phosphatase, phosphoamidase, leucine arylamidase and valine arylamidase were detected. All other tests in the API ZYM kit were negative. The long-chain cellular fatty acids of the organism were found to be of the straight-chain saturated and monounsaturated types, with C16 : 0 (32·7 %), C16 : 1 (4·3 %), C18 : 0 (19·3 %) and C18 : 1
9c (40·2 %) predominating. Cell wall murein analysis showed the presence of an A1 murein type: L-Lys-direct. The G+C content of DNA of the porcine bacterium was 39 mol%.
The cell wall murein structure and low G+C content of DNA of the isolate were incompatible with its provisional identification as belonging to one of the genera Actinomyces or Arcanobacterium. Therefore, to investigate the phylogenetic position of the unidentified bacterium, its almost complete 16S rRNA gene sequence was determined. Sequence database searches revealed that the unknown organism was most closely related to Alkalibacterium olivapovliticus, Alloiococcus otitis, Carnobacterium species, Dolosigranulum pigrum, Enterococcus species, Granulicatella species and related catalase-negative organisms. Actinomyces and other high-G+C-content bacteria revealed very low levels of similarity to the isolate (data not shown). A tree constructed using the neighbour-joining method showing the phylogenetic relationships of the unidentified bacterium is illustrated in Fig. 1 and demonstrates that the isolate possesses an affinity with Alkalibacterium olivapovliticus, Alloiococcus otitis and Dolosigranulum pigrum. This association was confirmed by maximum-parsimony analysis.
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). In terms of sequence similarities, the unknown organism displayed 88·5 % sequence similarity to Dolosigranulum pigrum NCFB 2975T, a coccus-shaped organism associated with human clinical sources (Aguirre et al., 1993). A comparable level of sequence similarity (88·4 %) was also displayed between the porcine bacterium and some uncultured bacteria (16S rDNA accession numbers AF124034 and AF124041) associated with sheep scab mites (Hogg & Lehane, 1999). Marginally lower levels of similarity were exhibited to Alloiococcus otitis NCFB 2890T (87·9 %), an ovoid-shaped organism associated with human ear infections (Aguirre & Collins, 1992), and Alkalibacterium olivapovliticus WW2-SN4aT (87·9 %), a rod-shaped alkaliphile recovered from wash waters of edible olives (Ntougias & Russell, 2001). From its branching position in the tree and sequence divergence values of approximately 11–12 % from these genera, it is clear that the porcine bacterium merits classification at a similar taxonomic rank (i.e. genus). It is pertinent to note that the separateness of the novel bacterium is strongly supported by phenotypic considerations. The isolate does not match any known Gram-positive organism phenotypically. The unidentified organism differs from Alloiococcus otitis by forming rod-shaped cells, growing anaerobically and by numerous biochemical reactions. Cells of Alloiococcus otitis are ovoid in shape and the species is nutritionally very fastidious and does not grow under anaerobic conditions (Aguirre & Collins, 1992; Miller et al., 1996). Similarly, the isolate is incompatible with belonging to the genus Dolosigranulum in that it forms rod-shaped cells, synthesizes an A1-type L-Lys-direct cell wall murein and fails to produce acid from a variety of carbohydrates. By contrast, cells of Dolosigranulum pigrum are ovoid in shape, possess an L-lys–D-Asp-type murein and ferment a variety of carbohydrates (Aguirre et al., 1993). The isolate also differs from Dolosigranulum pigrum in numerous other biochemical traits (Table 1) and differs markedly from Alkalibacterium olivapovliticus in not being alkaliphilic or psychrotolerant. Alkalibacterium olivapovliticus is an obligate alkaliphile, growing within a pH range of 
8·5–10·8, with an optimum at pH 9–10 (Ntougias & Russell, 2001). Based on sequence divergence values of >11 % from its nearest phylogenetic relatives and the distinct subline formed by the novel bacterium, in concert with its quite distinct phenotypic characteristics, we are of the opinion that the unknown bacterium from porcine semen merits assignment to a new genus and species, for which the name Allofustis seminis gen. nov., sp. nov. is proposed.
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Cells are Gram-positive, non-spore-forming rods. Facultatively anaerobic and catalase-negative. Arginine dihydrolase, leucine arylamidase and pyroglutamic acid arylamidase are produced. Indole-negative. Nitrate is not reduced. Voges–Proskauer-negative. The long-chain cellular fatty acids are of the straight-chain saturated and monounsaturated types. Cell wall murein is based on L-lys variation a1 (type L-Lys-direct). The type species is Allofustis seminis. The G+C content of genomic DNA of the type species is 39 mol%.
Description of Allofustis seminis sp. nov.
Allofustis seminis (sem.in'is. L. n. semen seed; N.L. gen. n. seminis of semen).
Cells stain Gram-positive and are rod-shaped. Non-spore-forming. -Haemolytic reaction on sheep blood. Facultatively anaerobic; grows well on chocolate agar anaerobically or in CO2, at 37 °C. Catalase-negative. Acid is not produced from L-arabinose, D-arabitol, cyclodextrin, glycogen, lactose, maltose, mannitol, mannose, melibiose, melezitose, pullulan, raffinose, D-ribose, sorbitol, sucrose, D-tagatose or trehalose. Arginine dihydrolase, arginine arylamidase, acid phosphatase, alkaline phosphatase, alanine arylamidase, alanine-phenylalanine-proline arylamidase, glycine arylamidase, glycyl tryptophan arylamidase, histidine arylamidase, leucyl glycine arylamidase, leucine arylamidase, phosphoamidase, proline arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, -mannosidase, serine arylamidase, tyrosine arylamidase and valine arylamidase are produced. Activity for -arabinosidase, esterase C-4, ester lipase C8, -galactosidase, -galactosidase, -galactosidase-6-phosphate, -glucosidase, -glucuronidase, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, lipase C14, -mannosidase, chymotrypsin, trypsin and urease is not detected. -Fucosidase, -glucosidase and N-acetyl--glucosaminidase may or may not be detected. Hippurate and aesculin are not hydrolysed. Indole and acetoin are not produced and nitrate is not reduced. The major long-chain cellular fatty acids are C16 : 0, C16 : 1, C18 : 0 and C18 : 1
9c. Other chemotaxonomic properties are as described for the genus.
The type strain is strain 01-570-1T (=CCUG 45438T=CIP 107425T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Aguirre, M., Morrison, D., Cookson, B. D., Gay, F. W. & Collins, M. D. (1993). Phenotypic and phylogenetic characterization of some Gemella-like organisms from human infections: description of Dolosigranulum pigrum gen. nov., sp. nov. J Appl Bacteriol 75, 608–612.[Medline]
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Hogg, J. C. & Lehane, M. J. (1999). Identification of bacterial species associated with the sheep scab mite (Psoroptes ovis) by using amplified genes coding for 16S rRNA. Appl Environ Microbiol 65, 4227–4229.
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Miller, P. H., Facklam, R. R. & Miller, J. M. (1996). Atmospheric growth requirements for Alloiococcus species and related gram-positive cocci. J Clin Microbiol 34, 1027–1028.[Abstract]
Ntougias, S. & Russell, N. J. (2001). Alkalibacterium olivoapovliticus gen. nov., sp. nov., a new obligately alkaliphilic bacterium isolated from edible-olive wash-waters. Int J Syst Evol Microbiol 51, 1161–1170.[Abstract]
Rasmussen, S. W. (1995). DNATools, a software package for DNA sequence analysis. Copenhagen: Carlsberg Laboratory.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
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