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Department of Food Science and Microbiology – Industrial Microbiology Section, University of Milan, Via Celoria 2, 20133 Milan, Italy
Correspondence
M. Grazia Fortina
grazia.fortina{at}unimi.it
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ABSTRACT |
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DNA–DNA relatedness values and the long-fatty-acid composition of Enterococcus italicus sp. nov. and other enterococcal species are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Robredo et al., 2000). However, enterococci are ubiquitous and can be found as free-living micro-organisms in soil, on plants and in large numbers in dairy products, where, in some cases, they predominate with respect to lactobacilli and lactococci (Franz et al., 1999; Giraffa, 2002). The presence of enterococci in dairy products has long been considered as an indicator of inadequate sanitary conditions during the production and processing of milk. In contrast, many authors suggest that enterococci may have a potentially desirable role in some cheeses, because of their proteolytic and lipolytic activities, in the development of typical flavours and for the production of enterocins with anti-Listeria activity (Ennahar & Deschamps, 2000; Giraffa et al., 1997). The important question is whether enterococci originating from food and from community sources possess an equally pathogenic potential, or whether differences in pathogenicity exists, perhaps in relation to the different species. In this context, correct species identification is of great importance to both medical and food microbiologists.
Since the revival of the genus Enterococcus by Schleifer & Kilpper-Bälz (1984), phylogenetic studies have established, on the basis of 16S rRNA gene sequences, the presence of at least four ‘species groups' (Devriese et al., 1993; Devriese & Pot, 1995; Stiles & Holzapfel, 1997), as follows: the ‘faecium group’, comprising Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus mundtii and the newly described Enterococcus villorum (Vancanneyt et al., 2001); the ‘avium group’, comprising Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus and Enterococcus pseudoavium; the ‘gallinarum group’, comprising Enterococcus gallinarum and Enterococcus casseliflavus; and a fourth group, comprising Enterococcus columbae and Enterococcus cecorum. Other described enterococci form individual lines of descent, e.g. Enterococcus sulfureus, Enterococcus saccharolyticus, Enterococcus asini and Enterococcus faecalis. Recently, two novel species, Enterococcus haemoperoxidus and Enterococcus moraviensis (
vec et al., 2001), have been shown to be phylogenetically closely related to Enterococcus faecalis. However, 16S rRNA gene sequence similarities between enterococcal species are mostly high and exceed the generally accepted value of 97 % for species differentiation. For this reason, at the genetic level, only the percentage of DNA–DNA hybridization can clearly indicate their species identity.
Phenotypically, there are no definite characteristics that distinguish the enterococci unequivocally from the other Gram-positive, catalase-negative, coccus-shaped bacteria or within the genus, at the species level. The ability to grow at 10 and 45 °C, in 6·5 % NaCl, at pH 9·6 and on selective media can also be possessed by some non-enterococcal species, while these criteria can exclude several novel enterococcal species, such as Enterococcus columbae, which fails to grow in 6·5 % NaCl (Devriese et al., 1990), or Enterococcus haemoperoxidus (vec et al., 2001), which shows a clear positive catalase reaction.
For these reasons, a polyphasic approach is desirable for correctly identifying ‘unusual’ enterococcal strains. Today, some aspects of the taxonomic and genotypic diversity within the genus Enterococcus can also be further investigated through the use of recently developed molecular techniques, such as intergenic ribosomal PCR, randomly amplified polymorphic DNA and PFGE (Angeletti et al., 2001; Naïmi et al., 1997; Poyart et al., 2000; Quednau et al., 1998).
Recently, during a study on the natural bacterial population obtained from raw cows' milk used in the production of artisanal Italian cheeses (Fortina et al., 2003), we isolated some atypical Enterococcus strains that seemed to represent a novel species of Enterococcus. In the present paper, we describe a polyphasic study of a collection of seven isolates: five originated from Toma piemontese cheese and two were from Robiola piemontese cheese. The results demonstrated that the strains represent a novel species, for which the name Enterococcus italicus sp. nov. is proposed.
Strains TP1.5T, TP1.3, TP2.3, TP1.D and TP3.D were isolated from Toma piemontese cheese, while strains RP1 and RP4 were isolated from Robiola piemontese cheese. They were routinely maintained at 4 °C after growth at 37 °C for 18 h in M17 glucose broth or agar (Difco). On this agar, colonies appeared circular and smooth, with entire margins; after 3 days growth, they were 1 mm in diameter.
Cell morphology was studied using phase-contrast microscopy. Gram staining was done as described by Salle (1961). Growth was checked on kanamycin/aesculin/azide agar (Merck), blood agar and skimmed-milk medium. Sugar-fermentation patterns were determined by using a microtitre plate assay and a basal M17 medium containing chlorophenol red as indicator and the desired filter-sterilized carbohydrate at a final concentration of 0·2 % (w/v). The reproducibility was verified by repeated analyses. A pH-dependent change in the indicator was documented for up to 5 days. Only definitive turnovers were rated as positive. Biochemical tests were also performed using API galleries 50 CH and 20 STREP (bioMérieux) according to the manufacturer's instructions. Susceptibility to antibiotics was evaluated after overnight growth at 37 °C in M17 glucose broth containing different concentrations of antibiotic and using 105 cells ml–1 as the initial inoculum.
DNA was isolated and purified as described previously (Fortina et al., 2001a). The DNA base composition was determined in 0·1x SSC buffer (1x SSC is 0·15 mol NaCl l–1 plus 0·015 mol sodium citrate l–1) by using the thermal denaturation method described by Marmur & Doty (1962) and the equation of Owen & Hill (1979). DNA from Escherichia coli strain B (Sigma) was used as the internal standard. The extent of DNA reassociation was determined as previously described (Fortina et al., 2001b) by using the optical renaturation rates method with a model Response spectrophotometer equipped with an Advance Kinetics Graphic version 1.3 thermoprogrammer (CIBA–Corning Diagnostics). For all samples tested, the reaction was carried out under optimal conditions (25 °C below the melting temperature) in 5x SSC, containing 20 % (v/v) DMSO. The equation of Seidler & Mandel (1971) was used to calculate the extent of DNA–DNA reassociation.
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and sequencing of the PCR products were performed as described previously (Fortina et al., 2003). Pairwise alignment similarities were calculated using the Ribosomal Database Project service (Maidak et al., 2001). Phylogenetic reconstructions were done by using the TREECON software (version 1.3b) (Van de Peer & De Wachter, 1994) and the model of Jukes & Cantor (1969). Amplification of the 16S–23S rRNA gene intergenic transcribed spacer region was performed as previously described (Fortina et al., 2003), employing the primers G1 (5'-GAAGTCGTAACAAGG-3') and L1 (5'-CAAGGCATCCACCGT-3') (Jensen et al., 1993). Following amplification, the reaction mixtures were electrophoresed in 3 % agarose gel in TAE buffer (40 mM Tris/acetate, 1 mM EDTA, pH 8) at 100 V for 2 h, before staining with ethidium bromide. A DNA ladder (50 bp; Amersham Pharmacia Biotech) was used as the molecular size marker.
Analysis of fatty acid methyl esters was performed by GC using the Microbial Identification System (MIDI) at the DSMZ (Braunschweig, Germany).
The strains were Gram-positive with spherical or ovoid cells that occurred in pairs and short chains, catalase-negative, non-motile and non-endospore-forming. The strains did not grow on kanamycin/aesculin/azide agar, showed 
-haemolysis on blood agar and were able to grow in skimmed-milk medium, in which they caused clotting within 48 h incubation. All of the strains were susceptible to vancomycin. Growth was enhanced slightly by cultivation in a 5 % CO2 atmosphere and was more abundant at 37 °C in comparison with 30 or 25 °C. Growth occurred at pH 9·6 and at 42 °C, but was reduced at 10 °C. The ability to grow at 45 °C was variable among the strains tested. The novel isolates failed to grow in M17 broth containing 6·5 % NaCl, as for strains of Enterococcus cecorum (Devriese et al., 1983) and Enterococcus columbae (Devriese et al., 1990). The ability to grow in 6 % NaCl was possessed only by the type strain, TP1.5T, while all isolates grew in 5 % NaCl.
The almost complete 16S rRNA gene sequence (1396 bp) of TP1.5T had the highest similarity to 16S rRNA gene sequences of enterococci: pairwise sequence alignments indicated that the closest relatives of strain TP1.5T are Enterococcus saccharolyticus and Enterococcus sulfureus (96·7 %) and Enterococcus gallinarum (95·4 %). Lower sequence similarities (<95 %) were found with other described species of the genus Enterococcus. The 16S rRNA genes of the species of the genera Melissococcus and Tetragenococcus, for which a close relationship with the enterococcal strains has been previously demonstrated, exhibited 90·4 and 88·8 % sequence identity, respectively, with the 16S rRNA gene sequence of strain TP1.5T. Strain TP1.5T was more distantly related to the streptococcal and lactococcal species tested (<84 % sequence identity). A tree depicting the phylogenetic affinity of strain TP1.5T with other enterococci is shown in Fig. 1. It should be noted that the 16S rRNA gene sequence of strain TP1.5T showed 99 % similarity to those of two unidentified strains (accession nos AF349934 and AY321375) and of a strain deposited as Enterococcus sulfureus (accession no. AY184239). These strains, isolated from an artisanal Sicilian cheese, from human clinical specimens and from a fermented maize beverage, respectively, could belong to Enterococcus italicus sp. nov., indicating the possibility that it occurs in other sources. Whether or not these enterococci belong to Enterococcus italicus remains to be determined.
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PCR amplification of the intergenic transcribed spacer between the 16S and 23S rRNA genes produced an amplification profile characteristic of the novel enterococcal species. This fingerprint pattern showed genetic homogeneity in the cheese isolates and separated them from all enterococcal species included in this study by the presence of three major bands with approximate sizes of 400, 380 and 300 bp (Fig. 2).
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7c (60·6 %); C19 : 0cyclo8c was present in small amounts (2·5 %). Enterococcus sulfureus produced significantly smaller amounts of C18 : 17c and very large amounts of C19 : 0cyclo8c. Enterococcus saccharolyticus showed more balanced amounts of C16 : 0 and C18 : 17c; C19 : 0cyclo8c was not detectable (Supplementary Table B in IJSEM Online).
In complete agreement with the phylogenetic and genotypic examinations described above, the phenotypic studies clearly separated all the novel strains from the other reference species. Indeed, the novel species can be readily distinguished from the other enterococci by using a combination of physiological and biochemical tests (Table 1). In particular, utilization of ribose confirms the separateness of the cheese isolates from all tested enterococcal species. Enterococcus italicus can also be differentiated from its closest relatives, Enterococcus saccharolyticus and Enterococcus sulfureus, by its inability to acidify L-arabinose, melezitose, melibiose and raffinose.
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Description of Enterococcus italicus sp. nov.
Enterococcus italicus (i.ta'li.cus. L. masc. adj. italicus from Italy, where the bacterium was first isolated).
Cells are facultatively anaerobic, non-motile, non-spore-forming, catalase-negative, non-pigmented, Gram-positive cocci that occur in pairs or short chains. Colonies are whitish-grey, circular and smooth, with entire margins; 1 mm in diameter after 3 days growth on M17 glucose agar. Growth is enhanced slightly by cultivation in a 5 % CO2 atmosphere and is optimal at 37 °C. Growth occurs at 42 °C, but is weak at 10 °C. The ability to grow at 45 °C is variable among the strains. Strains grow at pH 9·6 and in 5 % NaCl. A few strains are positive for growth in 6 % NaCl; no growth is seen in 6·5 % NaCl. The strains do not grow on kanamycin/aesculin/azide agar, show -haemolysis on blood agar and are able to grow in skimmed-milk medium, where they cause clotting within 48 h incubation. All strains are susceptible to vancomycin. Homofermentative, producing lactic acid and no gas. Acid is produced from galactose, D-glucose, D-fructose, D-mannose, methyl -D-glucoside (often delayed), N-acetylglucosamine, aesculin, salicin, maltose, lactose, sucrose, trehalose and starch. Weak reactions can occur on cellobiose. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl 
-xyloside, L-sorbose, rhamnose, dulcitol, inositol, methyl -D-mannoside, amygdalin, melibiose, inulin, melezitose, D-raffinose, glycogen, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Strain-dependent reactions are observed with mannitol (4/7 strains), -gentiobiose (3/7 strains), D-tagatose (3/7 strains), arbutin (3/7 strains) and sorbitol (5/7 strains). Positive results are obtained in API tests for acetoin production, aesculin hydrolysis, pyrrolidonyl arylamidase and leucine arylamidase. Strains are negative for hippurate hydrolysis, -galactosidase, -glucuronidase, -galactosidase, alkaline phosphatase and arginine dihydrolase. The DNA G+C content ranges from 39·9 to 41·1 mol%, as determined by melting temperature. The strains form a distinct relatedness group, as determined by DNA–DNA hybridization. The strains can also be differentiated from the other species of Enterococcus by their unique intergenic transcribed spacer profile and the sequence of their 16S rRNA genes.
Strains were isolated from dairy products in Piedmont, Italy. The type strain is DSM 15952T (=LMG 22039T)
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ACKNOWLEDGEMENTS |
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