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Enterococcus phoeniculicola sp. nov., a novel member of the enterococci isolated from the uropygial gland of the Red-billed Woodhoopoe, Phoeniculus purpureus

¹ Percy FitzPatrick Institute of African Ornithology, University of Cape Town, Private Bag 1, Rondebosch, 7701, Cape Town, South Africa
² Department of Molecular and Cell Biology, University of Cape Town, Private Bag 1, Rondebosch, 7701, Cape Town, South Africa

Correspondence
Paul R. Meyers
pmeyers{at}science.uct.ac.za

	ABSTRACT

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A facultatively anaerobic, Gram-positive, coccoid, non-spore-forming, non-motile, catalase-negative bacterium was isolated from the uropygial (preen) gland of wild Red-billed Woodhoopoes (Phoeniculus purpureus) and designated strain JLB-1^T. Physiological and biochemical testing suggested that this homofermentative, lactic-acid-producing bacterium could belong to the genus Enterococcus or the genus Streptococcus. Comparison of the 16S rRNA gene sequence of strain JLB-1^T with other 16S rDNA sequences in the GenBank database by BLAST analysis showed that its closest relatives are Enterococcus faecium, Enterococcus avium and Enterococcus asini. Strain JLB-1^T may be differentiated from these species by the fact that it does not produce acid from lactose, D-mannitol, D(+)-melezitose or D-sorbitol. Furthermore, it does not hydrolyse arginine or hippurate and cannot grow in the presence of 6·5 % NaCl or 40 % bile. It differs from the streptococci in that it does not lyse erythrocytes. Strain JLB-1^T is a novel member of the enterococci, for which the name Enterococcus phoeniculicola sp. nov. is proposed. The type strain is JLB-1^T (=ATCC BAA-412^T=DSM 14726^T).

The GenBank accession number for the 16S rRNA gene sequence of Enterococcus phoeniculicola JLB-1^T is AY028437.

A phylogenetic tree showing the relationship between E. phoeniculicola and related species is available as supplementary data in IJSEM Online.

	MAIN TEXT

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Prior to the description of the genus Enterococcus by Schleifer & Kilpper-Bälz (1984), this group of organisms was a subdivision of the streptococci (Hardie, 1986). At the time of writing, the genus Enterococcus comprises 27 species (http://www.bacterio.cict.fr/e/enterococcus.html) that are divided into four groups, based upon 16S rDNA sequence similarity (Vancanneyt et al., 2001). Phylogenetically, the enterococci belong to the clostridial subdivision of the Gram-positive bacteria and cluster with the genera Vagococcus, Tetragenococcus and Carnobacterium (Vancanneyt et al., 2001). Physiologically, the enterococci are related to the streptococci by their abilities to grow at 10 and 45 °C, at pH 9·6, and in the presence of 6·5 % NaCl and 40 % bile (Schleifer & Kilpper-Bälz, 1984). However, not all enterococci are able to grow at such a high pH or at such high bile and salt concentrations (Devriese et al., 1983, 1990). Analysis of the 16S rRNA gene sequence of enterococci has played a large part in their differentiation from streptococci (Devriese et al., 1990).

Many species of bacteria and fungi have been isolated from birds and their nests (Hubálek, 1976; Cooper et al., 1986; Cooper, 1990, 1993; Cornelissen et al., 1991; Hubálek et al., 1995; Singleton & Given Harper, 1998). In most of these cases, the micro-organisms were pathogenic.

We have been investigating the hypothesis that the odour and taste of the uropygial (preen) gland secretion of Red-billed Woodhoopoes is offensive to certain predators of these birds, and that this secretion may thus play a role in preventing predation on Red-billed Woodhoopoes. Recently, we isolated a Gram-positive, non-motile, non-sporing coccus from the uropygial secretion of the Red-billed Woodhoopoe, Phoeniculus purpureus. The bacterium was found to modify the colour, viscosity and smell of the uropygial secretion produced by the birds. After direct treatment of the uropygial glands of three adult birds with enrofloxacin, no bacteria could be cultured from the uropygial secretion of the treated birds. Furthermore, the uropygial secretion had become lighter in colour, less viscous and less malodorous than that of the control group of three adult birds (Law-Brown, 2001).

Samples (2–4 µl) were gently collected from the uropygial glands of six individual adult Red-billed Woodhoopoes (P. purpureus) using an automatic pipette. The samples were pipetted into sterile nutrient broth (Biolab Diagnostics) in sterile universal bottles and incubated at 37 °C with agitation for 16 h. The resulting cultures were examined microscopically and inoculated onto nutrient agar (Biolab Diagnostics). Based on microscopic examination and colony morphology, only one type of bacterium was cultured by this isolation method. A representative culture was chosen for further investigation. This organism was designated strain JLB-1^T and was maintained on nutrient agar. No attempt was made to isolate bacteria by direct plating of the uropygial secretion.

Strain JLB-1^T was subjected to physiological tests and the sequence of its 16S rRNA gene was determined. Physiological tests were carried out according to established methods (Gordon et al., 1974; Palleroni, 1984). All tests were carried out at 37 °C unless indicated otherwise.

To test whether strain JLB-1^T is a lactic acid bacterium, it was cultured in nutrient broth at 37 °C with agitation for 24 h and the bacteria were harvested by centrifugation. The culture supernatant was filter-sterilized and prepared for volatile and non-volatile fatty acid analysis according to the method of Summanen et al. (1993). Fatty acids were assayed using a Chrompak model 437A gas chromatograph.

PCR amplification of the 16S rRNA gene was performed with universal eubacterial primers (forward, 5'-AGAGTTTGATCITGGCTCAG-3'; reverse, 5'-ACGGITACCTTGTTACGACTT-3', where I=inosine) and Super-Therm thermostable polymerase (JMR Holdings). The forward and reverse primers were modified from primers fD1 and rP2, respectively, of Weisburg et al. (1991). Both strands of the PCR-amplified DNA were sequenced by the chain-termination technique, using an ALFexpress DNA automated sequencer and a Thermo Sequenase Cy 5.0 Dye Terminator Cycle Sequencing kit (both from Amersham Biosciences).

Strain JLB-1^T is a Gram-positive bacterium with spherical or ovoid cells that occur in pairs and short chains. The organism is facultatively anaerobic, non-motile, catalase- and oxidase-negative and does not form endospores. Optimum growth occurred between 30 and 37 °C and at pH 8. Strain JLB-1^T grew in the presence of 3 % NaCl, but not in the presence of 6 % NaCl, 40 % bile or 10 % ethanol.

Strain JLB-1^T is a homofermentative lactic acid bacterium and is thus precluded from belonging to the heterofermentative genera of lactic acid bacteria, such as Lactobacillus, Carnobacterium and Desemzia. Its characterization suggested that it might belong to the genus Enterococcus or the genus Streptococcus. BLAST analysis (Altschul et al., 1997) indicated that the almost-complete 16S rDNA sequence (1472 bp) of JLB-1^T has highest similarity to 16S rDNA sequences of enterococci: pairwise sequence alignments of the 16S rDNA sequence of JLB-1^T with Enterococcus species, using the DNAMAN software (version 4.13; Lynnon BioSoft), showed high sequence similarity to Enterococcus avium (97·53 %) and Enterococcus faecium (97·34 %). Strain JLB-1^T differs from these species in not producing acid (within 24 h) from lactose, D-mannitol, D(+)-melezitose or D-sorbitol. JLB-1^T differs further from these species by not hydrolysing arginine, and by being unable to grow in the presence of 40 % bile or 6·5 % NaCl. The ability to grow in the presence of 6·5 % NaCl and 40 % bile was included in the original definition of the genus Enterococcus (Schleifer & Kilpper-Bälz, 1984), but not all validly described Enterococcus species exhibit this phenotype (Devriese et al., 1983, 1990).

Strain JLB-1^T is differentiated from other genera of homofermentative lactic acid bacteria by certain phenotypic differences and in low 16S rRNA gene sequence similarities. A phylogenetic tree is available as supplementary data in IJSEM Online. The tree shows that strain JLB-1^T groups with the Enterococcus cluster.

We are unaware of any reports of bacteria being cultured from the uropygial secretion of Red-billed Woodhoopoes. The results of the physiological tests and the 16S rDNA sequence comparisons indicate that strain JLB-1^T is a homofermentative lactic acid bacterium that belongs to the genus Enterococcus. However, the characteristics of this bacterium are sufficiently different from previously described species of Enterococcus to suggest that it represents a novel species, for which we propose the name Enterococcus phoeniculicola sp. nov.

Description of Enterococcus phoeniculicola sp. nov.
Enterococcus phoeniculicola (phoe.ni.cu.li'co.la. L. suff. n. -cola inhabitant; N.L. masc. adj. phoeniculicola growing in Phoeniculus, the genus of the Woodhoopoe).

Cells are facultatively anaerobic, non-motile, non-spore-forming, catalase- and oxidase-negative, Gram-positive cocci that occur in pairs or short chains. Colonies are whitish-grey, circular and 0·5 mm in diameter when grown on nutrient agar at 37 °C for 16 h. Optimum growth occurs between 30 and 37 °C and at pH 8. Growth occurs in the presence of 3 % NaCl, but not in the presence of 6 % NaCl, 40 % bile or 10 % ethanol. Homofermentative, producing lactic acid and no gas (minor quantities of acetic acid were detected by GC). Acid is produced from L(+)-arabinose, D(+)-cellobiose, cyclodextrin, D(-)-fructose, D(+)-glucose, maltose, D(+)-mannose, L(+)-rhamnose, D(-)-ribose, salicin, sucrose, trehalose and D(+)-xylose. Acid production is delayed (takes 48 h) from dulcitol, glycerol, meso-inositol, lactose, D-mannitol, D-sorbitol and L(-)-sorbose. No acid is produced from D(+)-galactose, inulin, D(+)-melezitose, D(+)-melibiose, raffinose or sodium succinate. Does not liquefy gelatin, reduce nitrate, or produce H₂S, acetoin or indole. Does not hydrolyse casein, Tween 80, starch, hippurate, DNA or arginine. No haemolysis on blood agar. Does not grow on M9 minimal medium (Sambrook et al., 1989) or M9 medium enriched with Casitone (Difco), but grows on M9 medium enriched with 0·1 % yeast extract.

The type strain, Enterococcus phoeniculicola JLB-1^T (=ATCC BAA-412^T=DSM 14726^T), was isolated from the uropygial gland of a Red-billed Woodhoopoe (Phoeniculus purpureus) captured in the Morgan Bay region of the Eastern Cape province, South Africa (32° 43' S, 28° 47' E).

	ACKNOWLEDGEMENTS

 
We thank Giselle Murison, Patrick Brackley and Dr Andrew Jenkins for help with uropygial sample collection, Di James for DNA sequencing, John Clausen for help with GC and Professor Morné du Plessis for guidance in matters ornithological. J. L.-B. holds a bursary from the South Africa National Research Foundation. P. M. holds research grants from the University of Cape Town Research Committee and the Medical Research Council of South Africa.

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