Recommended minimal standards for description of new taxa of the genera Bifidobacterium, Lactobacillus and related genera.

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1434–1451

Taxonomic Note

DOI 10.1099/ijs.0.060046-0

Recommended minimal standards for description of new taxa of the genera Bifidobacterium, Lactobacillus and related genera Paola Mattarelli,1 Wilhelm Holzapfel,2 Charles M. A. P. Franz,3 Akihito Endo,4 Giovanna E. Felis,5 Walter Hammes,6 Bruno Pot,7 Leon Dicks8 and Franco Dellaglio5

Correspondence

1

Paola Mattarelli

2

[email protected]

Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy School of Life Sciences, Handong Global University, Pohang, Gyeongbuk 791-798, Republic of Korea

3

Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Department of Safety and Quality of Fruit and Vegetables, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany

4

Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 099-2493 Hokkaido, Japan

5

Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

6

Talstr. 60/1, D-70794 Filderstadt, Germany

7

Institut Pasteur de Lille, Lactic acid Bacteria & Mucosal Immunity, Center for Infection and Immunity of Lille, 1 rue du Pr Calmette, BP 245, F-59019 Lille, France

8

Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, South Africa

Minimal standards for the description of new cultivable strains that represent novel genera and species belonging to the genera Bifidobacterium, Lactobacillus and related genera are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision): the description of novel species should be based on phenotypic, genotypic and ecological characteristics to ensure a rich polyphasic characterization. Concerning genotypic characterization, in addition to DNA G+C content (mol%) data, the description should be based on DNA–DNA hybridization (DDH), 16S rRNA gene sequence similarities and at least two housekeeping gene (e.g. hsp60 and recA) sequence similarities. DDH might not be needed if the 16S rRNA gene sequence similarity to the closest known species is lower than 97 %. This proposal has been endorsed by members of the Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms of the International Committee on the Systematics of Prokaryotes.

Introduction The recommendation 30b of the Bacteriological Code of Nomenclature (1990 Revision) (Lapage et al., 1992) as modified at the 1999 meeting of the International Committee on Systematic Bacteriology (ICSB) and its Judicial Commission (Labeda, 2000) calls for the defining of minimal standards for describing novel bacterial taxa. The aim of this paper is to propose minimal standards for description of novel taxa Abbreviations: DDH, DNA–DNA hybridization; ICSP, International Committee on the Systematics of Prokaryotes; ITS, internally transcribed spacer; LAB, lactic acid bacteria; MLST, multilocus sequence typing; PFGE, pulsed field gel electrophoresis.

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within the genera Bifidobacterium, Lactobacillus and related genera. The number of species of the genera Bifidobacterium, Lactobacillus and related genera has increased considerably during the last 10–15 years. In order to enable taxonomists to correctly identify and allocate a strain to a specific taxon, a clear understanding of the key criteria that need to be applied on a genus and species level is extremely important. It is the intention of this document to provide a firm basis for decision making in the approaches to be taken, for both taxonomic and routine investigation purposes for species and genera considered by the scope of the International Committee on the Systematics of Prokaryotes (ICSP) 060046 G 2014 IUMS Printed in Great Britain

Minimal standards for Bifidobacterium and Lactobacillus

Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms. Species and genera included in the scope of ICSP Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms Gram-staining-positive bacteria group into two of the major bacterial phyla, i.e. the Firmicutes and Actinobacteria. According to the Taxonomic Outline of the Prokaryotes (Garrity et al., 2004) and latest updates, the genus Bifidobacterium belongs to the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Bifidobacteriales and family Bifidobacteriaceae. The other ‘minor genera’ belonging to this family are Aeriscardovia, Alloscardovia, Gardnerella, Metascardovia, Parascardovia and Scardovia. The former genus Falcivibrio has recently been included in the genus Mobiluncus (Hoyles et al., 2004). The phylum Firmicutes include the classes Clostridia (class I), Mollicutes (class II) and Bacilli (Class III). The lactic acid bacteria (LAB) are grouped in class III order, II Lactobacillales (Garrity & Holt, 2001). It was suggested that the common ancestor of the order Lactobacillales (to which the LAB belong) developed from a common ancestor of all members of the class Bacilli (Makarova et al., 2006) by extensive loss of ancestral genes. Beyond gene loss, members of the order Lactobacillales also exhibit clear ancestral adaptations for nutritionally rich and facultatively anaerobic environments, which include acquisition of genes via horizontal gene transfer and duplication of genes for various enzymes including transporters of sugar and amino acid metabolism (Makarova et al., 2006; Makarova & Koonin, 2007). The order Lactobacillales encompasses the families Aerococcaceae (genera Aerococcus, Abiotrophia and Facklamia and the ‘minor genera’ Dolosicoccus, Eremococcus, Globicatella and Ignavigranum), Carnobacteriaceae (genus Carnobacterium and the ‘minor genera’ Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Desemzia, Dolosigranulum, Granulicatella, Isobaculum, Lacticigenium, Marinilactibacillus, Pisciglobus and Trichococcus), Enterococcaceae (genera Enterococcus, Tetragenococcus and Vagococcus, as well as the ‘minor genera’ Bavariicoccus, Catellicoccus, Melissococcus and Pilibacter), Lactobacillaceae (genera Lactobacillus and Pediococcus), Leuconostocaceae (genera Leuconostoc, Fructobacillus, Oenococcus and Weissella) and Streptococcaceae (genera Lactococcus, Lactovum and Streptococcus). On the basis of 16S rRNA gene sequences, three closely related lineages of the LAB were initially described by Woese (1987), i.e. the Leuconostoc group, the Lactobacillus casei/Pediococcus group and the Lactobacillus delbrueckii group. The genera Carnobacterium, Enterococcus, Vagococcus, Aerococcus and Tetragenococcus were considered more closely related to each other than to any other LAB, while the genera Lactococcus and Streptococcus also appeared to be related and were described as forming a separate branch (Schleifer & http://ijs.sgmjournals.org

Ludwig, 1995). The recent availability of complete genomes of representative LAB strains of all major families of the order Lactobacillales enabled a more definitive analysis of their evolutionary relationships (Makarova et al., 2006; Makarova & Koonin, 2007). Accordingly, the streptococci-lactococci branch was considered to be basal in the Lactobacillales tree and the Pediococcus group a sister to the Leuconostoc group within the Lactobacillus clade. Thus, the genus Lactobacillus appeared to be paraphyletic with respect to the Pediococcus– Leuconostoc group and Lactobacillus casei was placed at the base of the Lactobacillus delbrueckii group (Makarova et al., 2006; Makarova & Koonin, 2007), which contradicts the previous classification of the lactobacilli forming a group with the pediococci (Holzapfel et al., 2001). Furthermore, Zhang et al. (2011) confirmed the monophyly of the families Leuconostocaceae, Enterococcaceae and Streptococcaceae in a phylogenetic analysis based on 232 genes from 28 LAB genomes. They divided the LAB species into two groups, where group I included the families Enterococcaceae and Streptococcaceae; within this group the monophyly of the genera Enterococcus, Lactococcus and Streptococcus were strongly supported (Zhang et al., 2011). Group 2 included the families Lactobacillaceae and Leuconostocaceae. An updated representation of the family Lactobacillaceae was published by Salvetti et al. (2012). The ICSP Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms deals with taxonomical matters concerning the genera Bifidobacterium and Lactobacillus and the related genera belonging to the orders Bifidobacteriales and Lactobacillales mentioned above. The genera Streptococcus and Lactococcus, although belonging to order Lactobacillales, are dealt with by the Subcommittee on the Taxonomy of the genera Staphylococcus and Streptococcus. To give a brief overview before the Subcommittee’s recommendations of minimal standards for description of novel species are further elaborated, some general characteristics of the taxa covered are briefly described below. The list of genera and type species at the time of writing (January 2014) are described in Table 1. 1. Family Bifidobacteriaceae Stackebrandt, Rainey & Ward-Rainey 1997VP Type genus: Bifidobacterium Orla-Jensen 1924 (Approved Lists 1980). The family Bifidobacteriaceae contains the Gram-stainingpositive genera Bifidobacterium (42 species), Alloscardovia (2 species), Aeriscardovia (1 species), Metascardovia (1 species), Parascardovia (1 species) and Scardovia (2 species), which are the closest phylogenetic neighbours of members of the family. The Gram-variable genus Gardnerella (1 species) is phylogenetically related to the above genera and possesses the key enzyme fructose-6-phosphate phosphoketolase. All taxa are non-motile, non-endospore forming and usually strictly anaerobic, although sensitivity to oxygen is different among different species and genera. Also, cell morphology 1435

P. Mattarelli and others

Table 1. Families, genera and their type species belonging to the phyla Actinobacteria and Firmicutes comprising the scope of the Subcommittee Taxa

Reference

Phylum Actinobacteria Order Bifidobacteriales Family Bifidobacteriaceae Genus Bifidobacterium (type genus) Bifidobacterium bifidum (type species) Genus Aeriscardovia Aeriscardovia aeriphila (type species) Genus Alloscardovia Alloscardovia omnicolens (type species) Genus Metascardovia Metascardovia criceti (type species) Genus Parascardovia Parascardovia denticolens (type species) Genus Scardovia Scardovia inopinata (type species) Genus Gardnerella Gardnerella vaginalis (type species) Phylum Firmicutes Order Lactobacillales* Family Lactobacillaceae Genus Lactobacillus (type genus) Lactobacillus delbrueckii (type species) Genus Pediococcus Pediococcus damnosus (type species) Family Aerococcaceae Genus Aerococcus (type genus) Aerococcus viridans (type species) Genus Abiotrophia Abiotrophia defectiva (type species) Genus Facklamia Facklamia hominis (type species) Genus Dolosicoccus Dolosicoccus paucivorans (type species), Genus Eremococcus Eremococcus coleocola (type species) Genus Globicatella Globicatella sanguinis (type species) Genus Ignavigranum Ignavigranum ruoffiae (type species) Family Carnobacteriaceae Genus Carnobacterium (type genus) Carnobacterium divergens Genus Alkalibacterium Alkalibacterium olivapovliticus (type species) Genus Allofustis Allofustis seminis (type species) Genus Alloiococcus Alloiococcus otitis (type species) Genus Atopobacter Atopobacter phocae (type species) Genus Atopococcus Atopococcus tabaci (type species) Genus Atopostipes Atopostipes suicloacalis (type species) Genus Desemzia Desemzia incerta (type species) Genus Dolosigranulum Dolosigranulum pigrum (type species) Genus Granulicatella Granulicatella adiacens (type species) Genus Isobaculum Isobaculum melis (type species) Genus Lacticigenium Lacticigenium naphtae (type species) Genus Marinilactibacillus Marinilactibacillus psychrotolerans (type species) Genus Pisciglobus Pisciglobus halotolerans (type species) Genus Trichococcus Trichococcus flocculiformis (type species) Family Enterococcaceae Genus Enterococcus (type genus) Enterococcus faecalis (type species)

Genus Tetragenococcus Tetragenococcus halophilus (type species) Genus Vagococcus Vagococcus fluvialis (type species) Genus Bavariicoccus Bavariicoccus seileri (type species) Genus Catellicoccus Catellicoccus marimammalium (type species) Genus Melissococcus Melissococcus plutonius (type species) Genus Pilibacter Pilibacter termitis (type species) Family Leuconostocaceae Genus Leuconostoc (type genus) Leuconostoc mesenteroides (type species) Genus Fructobacillus Fructobacillus fructosus (type species) Genus Oenococcus Oenococcus oeni (type species) Genus Weissella Weissella viridescens (type species)

Zhi et al. (2009) Orla-Jensen (1924); Skerman et al. (1980) Simpson et al. (2004) Huys et al. (2007) Okamoto et al. (2007a, b) Jian & Dong (2002) Jian & Dong (2002) Greenwood & Pickett (1980)

Winslow et al. (1917); Skerman et al. (1980) Beijerinck (1901); Skerman et al. (1980) Claussen (1903); Skerman et al. (1980) Ludwig et al. (2009d) Williams et al. (1953); Skerman et al. (1980) Kawamura et al. (1995) Collins et al. (1997) Collins et al. (1999a) Collins et al. (1999b) Collins et al. (1992, 1995) Collins et al (1999c) Ludwig et al. (2009b) Collins et al. (1987) Ntougias & Russell (2001) Collins et al. (2003) Aguirre & Collins (1992) Lawson et al. (2000) Collins et al. (2005) Cotta et al. (2004) Stackebrandt et al. (1999) Aguirre et al. (1993, 1994) Bouvet et al. (1989); Collins & Lawson (2000) Collins et al. (2002) Iino et al. (2009) Ishikawa et al. (2003) Tanasupawat et al. (2011) Liu et al. (2002) Ludwig et al. (2009c); Thiercelin & Jouhaud (1903); Andrewes & Horder (1906); Schleifer & Kilpper-Ba¨lz (1984); Ludwig et al. (2009c) Collins et al. (1990a, 1993b) Collins et al. (1989, 1990b) Schmidt et al. (2009) Lawson et al. (2006) White (1912); Bailey & Collins (1982, 1983) Higashiguchi et al. (2006) Schleifer (2009) Van Tieghem (1878); Skerman et al. (1980) Kodama (1956); Endo & Okada (2008) Dicks et al. (1995) Collins et al. (1993a, 1994)

*The order Lactobacillales also contains the family Streptococcaceae, which is not included in the scope of this Subcommittee. 1436

International Journal of Systematic and Evolutionary Microbiology 64


varies from short, regular, thin rods with pointed ends, to coccoidal regular cells, to long cells with a large variety of branching; cells occur singly or in chains. They possess fructose-6-phosphate phosphoketolase (EC 4.1.2.22) as a key characteristic of their obligately saccharoclastic catabolism. Acetic and lactic acid are formed primarily in a 3 : 2 molar ratio. CO2 is not produced (except during the degradation of gluconate). Small amounts of ethanol, formic acid and succinic acid are produced. They do not produce butyric or propionic acids. Optimum growth temperature is 30–42 uC. Growth generally occurs in the range pH 5.0 to 8.0, with optimal growth between pH 6.0 and 7.0. The type genus, Bifidobacterium, comprises 42 species isolated from diverse habitats of the body environment, such as the gastrointestinal tract of most vertebrates and some insects, the human vagina and human dental caries, as well as sewage. Genera of the family Bifidobacteriaceae have DNA G+C contents which fall within the range 42 to 67 mol%, with those of the genus Bifidobacterium ranging from 50 to 67 mol%, and those of other genera as follows: Alloscardovia 47 mol%, Aeriscardovia 54 mol%, Metascardovia 53 mol%, Parascardovia 55 mol% and Scardovia 45 mol%. The genus Gardnerella has the lowest DNA G+C content at only 42 mol%. 2. Family Lactobacillaceae Winslow et al. 1917 Type genus: Lactobacillus Beijerinck 1901 (Approved Lists 1980). The family Lactobacillaceae contains the genera Lactobacillus and Pediococcus, which are phylogenetically intermixed (Felis & Dellaglio, 2007). The former genus Paralactobacillus (Leisner et al., 2000) has recently been included in the genus Lactobacillus (Haakensen et al., 2011), although this inclusion is questionable. More than 150 species are recognized in the genus Lactobacillus (http://www.bacterio.net/lactobacillus.html) and are heterogeneous in numerous properties. In general, cells are Gram-staining-positive, non-endospore-forming rods or coccobacilli, which can also be mobile, catalasenegative when grown without haem in the medium, generally oxygen-tolerant, aciduric or acidophilic, and obligately saccharoclastic with at least 50 % of the carbohydrate end-product being lactate and other fermentation products consisting of acetate, ethanol, CO2, formate and succinate (Hammes & Vogel, 1995; Hammes & Hertel, 2009). Several fermentation types can be recognized: obligately homofermentative, facultatively heterofermentative and obligately heterofermentative metabolisms, based on the type of sugars fermented (hexoses and pentoses) and fermentation products (Hammes & Vogel, 1995). Some exceptions exist (Saier et al., 1996; Biddle & Warner, 1993; Hammes & Hertel, 2009). Although lactobacilli are considered to have a fermentative metabolism (Hammes & Vogel, 1995; Hammes & Hertel 2009), respiration has been http://ijs.sgmjournals.org

reported for Lactobacillus plantarum (Brooijmans et al., 2009). The temperature for growth ranges from 2 to 53 uC (optimal temperature is usually 30–40 uC) and they are able to grow between pH 3 and 8 (optimum usually at pH 5.5–6.2). They are characterized by a low DNA G+C content, although the upper limit of DNA G+C content reaches 59.2 mol% for Lactobacillus nasuensis (Cai et al., 2012). At least six peptidoglycan types have been reported (Schleifer & Kandler, 1972; Hammes & Vogel, 1995) and different isomers of lactic acid are produced by different species; therefore, there is no genus-specificity for these properties. The metabolic groupings suggested by Hammes & Vogel (1995), dividing lactobacilli on the basis of the type of fermented sugars and fermentation products, is not always consistent with the phylogenetic clustering; therefore, taxonomic studies should be comprehensive and include multiple genetic and phenotypic aspects. The pediococci are Gram-staining-positive, chemo-organotrophic, facultatively anaerobic, non-motile and nonendospore-forming lactic-acid-producing bacteria. The cells are typically spherical, occasionally ovoid and divide to form pairs. Moreover, cells can divide alternately in two perpendicular directions to form tetrads (Axelsson, 2004). Chains are never formed. Cells may only occur singly and in pairs, especially during early or mid-exponential growth. Generally oxidase- and catalase-negative, but catalase or pseudocatalase production have been reported for some strains of Pediococcus pentosaceus (Simpson & Taguchi, 1995). Arginine hydrolysis is rare but has been recorded for Pediococcus acidilactici and Pediococcus pentosaceus (Simpson & Taguchi, 1995). Pediococci homofermentatively produce lactic acid from glucose, without CO2. Grow at pH 5 but not at pH 9, except for Pediococcus stilesii (Franz et al., 2006) that can grow at pH 9.6. Optimum growth temperature is 25–35 uC, but is species dependent. All species except Pediococcus claussenii produce DL-lactic acid from glucose. Peptidoglycan type is Lys–D-Asp. The mol% G+C content of the DNA ranges from 35 to 44 mol%. Of note, Lactobacillus dextrinicus includes strains previously assigned to the genus Pediococcus, reclassified on the basis of poor phylogenetic relatedness with this latter genus (Haakensen et al., 2009). 3. Family Aerococcaceae Ludwig et al. 2010 Type genus: Aerococcus Williams et al. 1953 (Approved Lists 1980). The family Aerococcaceae includes the genera Aerococcus, Abiotrophia, Facklamia, Dolosicoccus, Eremococcus, Globicatella and Ignavigranum. The family Aerococcaceae was characterized on the basis of 16S rRNA gene sequences, combining two paraphyletic groups. The majority of the genera form a phylogenetically tight group comprising the genera Abiotrophia, Dolosicoccus, Eremococcus, Facklamia, Globicatella and Ignavigranum, 1437


while the genus Aerococcus represents a separate lineage (Ludwig et al., 2009a). Three genera contain only one species: Dolosicoccus paucivorans, Eremococcus coleocola and Ignavigranum ruoffiae (Collins et al., 1999a, b, c), whereas the genus Globicatella harbours Globicatella sanguinis and Globicatella sulfidifaciens and the genus Facklamia consists of six species, Facklamia hominis, Facklamia ignava, Facklamia languida, Facklamia miroungae, Facklamia sourekii and Facklamia tabacinasalis.

lactate from glucose, some genera (e.g. Alkalibacterium, Atopostipes, Marinilactibacillus, Trichococcus) will also produce acetate, or acetate and formate and/or ethanol in various amounts. Although fermentation is preferred, the bacteria can also respire under aerobic conditions with involvement of cytochromes b and d. No anaerobic types of respiration have been found or reported.

Cells are Gram-staining-positive, catalase-negative or with a weak catalase reaction due to a non-haem pseudocatalase, non-motile, facultatively anaerobic cocci occurring as single cells, in pairs or in short chains and have a strong tendency towards tetrad formation when grown in liquid media. The occurrence of cells in short chains separates the genera Dolosicoccus, Globicatella, Eremococcus, Facklamia and Ignavigranum from the genera Aerococcus, Pediococcus and Tetragenococcus, which have a tetrad cell formation. The fact that the cell walls of members of the genera Aerococcus, Eremococcus, Globicatella and Ignavigranum are of the Lys-direct type (A1a) (Collins et al., 1992, 1999a, b), allows a good distinction of the ‘minor genera’ of the family Aerococcaceae from the pediococci and tetragenococci. However, the cell wall murein composition of Facklamia hominis is of the L-Lys–D-Asp type (type A4a) (Collins et al., 1997), while the composition of the murein type of the genus Dolosicoccus has not yet been reported. All members of the family Aerococcaceae have been associated with human and animal infections, emphasizing their medical importance. The DNA G+C contents range from 37 to 40 mol% for species of the genus Aerococcus and from 35 to 42 mol% for members of the other, ‘minor genera’.

Type genus: Enterococcus (ex Thiercelin & Jouhaud 1903) Schleifer & Kilpper-Ba¨lz 1984.

4. Family Carnobacteriaceae Ludwig et al. 2010

Melissococci are normally found in diseased honeybee larvae (Apis mellifera). Cells are lanceolate cocci (0.5–0.761.0 mm) but pleomorphic and rod-like forms have also been described. Growth is always anaerobic to microaerophilic, with best growth in the presence of 1–5 % (v/v) CO2.

Type genus: Carnobacterium Collins et al. 1987. The family Carnobacteriaceae includes the genera Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Carnobacterium, Desemzia, Dolosigranulum, Granulicatella, Isobaculum, Lacticigenium, Marinilactbacillus and Trichococcus.

5. Family Enterococcaceae Ludwig et al. 2010

The family Enterococcaceae includes the genera Enterococcus, Tetragenococcus and Vagococcus and the ‘minor genera’ Bavariicoccus, Catellicoccus, Melissococcus and Pilibacter. The genus Catellicoccus is phylogenetically related to the family Enterococcaceae and is considered a sister group of the family (Ludwig et al., 2009c). Cells are Gram-staining-positive, catalase-negative, asporogenous and shaped as ovoid cocci. Growth is facultatively anaerobic, anaerobic or microaerophilic. All species are chemo-organotrophic. Some species are carboxyophilic or halophilic and may be resistant to bile. Cells of members of the genus Enterococcus occur singly or in chains and are usually found in the gastro-intestinal tract of animals and humans. Motility and yellow pigmentation are sometimes described (Sˆvec & Devriese, 2009). Glucose is actively fermented and (+)-L-lactic acid is a major end-product. They survive extreme temperatures (5–65 uC), grow at pH 4.5 to pH 10.0, withstand the presence of 6.5 % (w/v) NaCl and grow in the presence and absence of oxygen. The genus is classified as facultatively anaerobic. Some species are haemolytic on trypticase soy agar or Columbia agar with 5 % (v/v) defibrinated sheep blood (Domig et al., 2003).

Cells are Gram-staining-positive, usually catalase-negative, pleomorphic, and coccus- or rod-shaped. Usually facultatively anaerobic, but some species grow aerobically or microaerophilically. May be motile or not, and the cell wall may contain the diamino acids meso-diaminopimelic acid, lysine or ornithine (Ludwig et al., 2009b). Generally Voges–Proskauer-negative, except for species of the genus Carnobacterium. Species may be psychrotolerant, halotolerant, piezophilic, alkalitolerant or alkaliphilic. The DNA G+C contents range from 33 to 49 mol%, with members of the genus Trichococcus showing the highest DNA G+C contents of 45 to 49 mol%. Homo- or hetero-fermentative metabolism with production of lactic- and acetic acid. While most representatives do not produce gas and only

Members of the genus Tetragenococcus are moderately halophilic, slightly alkaliphilic and are usually found in salted food, such as anchovy, soy sauce, pickling brines and fish sauce. The cells are non-motile, spherical, occasionally ovoid (0.5–1.0 mm) and are normally in a tetrad orientation, although they may also occur singly, especially at early or mid-exponential growth. Oxidase is not produced and cytochromes are absent. Growth is facultatively anaerobic. Carbon dioxide is not produced during the fermentation of glucose. Optimum growth occurs between pH 7 and 8 and the optimum temperature for growth is 25–35 uC. Growth is repressed at pH 4.5 and pH 10 and at 45 uC. (+)-Llactic acid is produced from glucose, with occasional traces of (2)-D-lactic acid by some strains. Arginine is not hydrolysed and nitrate is not reduced. Nicotinic acid, pantothenic acid and biotin may be required as growth factors.

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Vagococci have been isolated from human and animal clinical specimens, body fluids, wounds and diseased rainbow trout and include motile, non-pigmented cocci belonging to the serological Lancefield group N (Schmidtke & Carson, 1994). Cells are ovoid, occurring singly, in pairs or in chains and are motile by having peritrichous flagella, although motility is not always observed. Growth is facultatively anaerobic. Gas is not produced from glucose. L-lactic acid is the main end-product of glucose metabolism and some strains produce acetoin (positive Voges– Proskauer test). Most vagococci strains grow at 10 uC but not at 45 uC and generally do not grow with 6.5 % (w/v) NaCl. Some strains grow at pH 9.6. Catellicocci are non-motile, coccoid and arranged in pairs or chains. Growth is facultatively anaerobic and is enhanced by the presence of CO2. The serological group is Lancefield D. Growth in conventional laboratory liquid media is difficult, even in the presence of serum. No haemolytic activity or growth at 10 uC. Grow in 10 % bile. Arginine is hydrolysed and leucine arylamidase is produced. Cell-wall murein: L-Lys–Gly–D-Asp (type A4a). 6. Family Leuconostocaceae Schleifer 2010 Type genus: Leuconostoc van Tieghem 1878 (Approved Lists 1980). Members of the family Leuconostocaceae, including the genera Leuconostoc, Fructobacillus, Oenococcus and Weissella, are Gram-staining-positive, non-endospore-forming and facultatively anaerobic. Motility has only been reported for certain species in the genus Weissella. Species of the genera Leuconostoc and Oenococcus are coccoid, but species of the genus Fructobacillus have rod-shaped cells. The genus Weissella includes both coccoid and rod-shaped cells. Members of the family Leuconostocaceae are obligately heterofermentative and generally produce equimolar amounts of lactic acid, ethanol and carbon dioxide from glucose, except species of the genus Fructobacillus, which produce acetic acid instead of ethanol (Endo & Okada, 2008). Species of the genus Fructobacillus are fructophilic and metabolize glucose in the presence of an external electron acceptor, e.g. oxygen, pyruvate or fructose (Endo & Okada, 2008). Oxygen enhances the growth of species of the genus Fructobacillus and has a significant effect on the growth of some species of the genus Leuconostoc possessing haemdependent respiratory capability. In contrast, oxygen suppresses the growth of species of the genus Oenococcus. The ability to convert L-malic acid to L-lactic acid is an important characteristic, especially for members of the genus Oenococcus. All members of the family Leuconostocaceae are non-thermophilic. Some species of the genus Leuconostoc can grow at 4 uC. The DNA G+C contents range from 37 to 45 mol%. General considerations The description of a new taxon should http://ijs.sgmjournals.org

(i) be based on a polyphasic approach, which integrates genomic and phylogenetic data with phenotypic and chemotaxonomic data; (ii) include a type- and/or reference strain that represents closely related members; (iii) provide a description that not only allows new representatives to be allocated to the taxon based on characteristic features, but also differentiates the new taxon from its phylogenetic or phenotypic neighbours. In this description, (iv) multi-gene encoded characters (e.g. enzyme multiplicity, peptidoglycan type) should be preferentially used as they are of greater significance than a single-gene encoded character (e.g. the splitting of carbohydrates by glycosidases); (v) the analysis procedures for measuring the differential characteristics should either be presented in detail or be readily available from the literature; (vi) novel species descriptions should preferably be based on more than one strain, providing some information of the intra-species phenotypic diversity; (vii) a representative strain of this diversity should be presented as the formal type strain and according to Recommendation 30a of the Bacteriological Code (1990 Revision) (Lapage et al., 1992) and successive revisions (Labeda, 2000), type strains should be deposited, before description, in two different recognized culture collections in different countries (additional changes have been proposed for the use of ‘patent strains’ as type strains; Tindall, 1999). Similarly, (viii) a type species must be designated when a new genus is described. The nature of the species name (a binomial or combination) dictates that it must also be assigned to an already existing or novel genus. The Bacteriological Code also recommends that the placement of a genus in a family should be mentioned and this can be placed within the hierarchical framework as this becomes defined. The ‘Rules and Recommendations of the International Code of Nomenclature of Bacteria’ (Lapage et al., 1992) can provide guidance in naming scientific taxa. For the checking of the Latin grammatical rules, references Tru¨per (2007) and Tru¨per & Euze´by (2009) can be consulted. The descriptions of new taxa should preferably be published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM); when published in another journal, the publication should be submitted to the IJSEM so that the new taxon can be published in one of the Validation Lists that appear periodically in that journal under the procedure described in the Bacteriological Code (1990 Revision) (Lapage et al., 1992). 1439


The sequence of a typical taxonomic analysis should be as follows (i) the investigated strains (type- and reference strains) should be prepared as pure subcultures and characterized by reliable phenotypic procedures; (ii) strains should be shown to exhibit appropriate genus- and family-specific properties; (iii) strains should firstly be located within the 16S rRNA tree (or a tree obtained with alternative markers such as hsp60, recA, etc., for which a sufficiently large number of reference taxa are available), in order to reduce the number of related taxa to be included in further taxonomic investigations; (iv) taxonomic specificity should be demonstrated on the basis of phenotypical and genotypical data, established in relation to all other species of the genus or with all relevant neighbouring genera; (v) a species may be divided into two or more subspecies when a group of strains exhibits consistent phenotypic differences allowing them to be discriminated from the other strains in the species and/or when these strains can be genetically grouped in clusters of strains that still share a high level of DNA–DNA hybridization (DDH) with the remaining strains of the species (Rossello´Mora & Amann, 2001). Once a novel taxon has been confirmed, the characterization leading to its formal description has to be performed, following the differentiation criteria described below and summarized in Table 2. Minimal standard requirements for the description of novel species of the genera Bifidobacterium, Lactobacillus and related genera Here we describe minimal standards related to ecology, phenotype and genotype, which are considered essential for the characterization and differentiation of new taxa within the genera Bifidobacterium and Lactobacillus, and related genera. Additional characteristics are listed which can optionally be used to improve the value of the description but which are not essential for the new taxon description. The criteria considered as well as details on the methods to be used are to be found in the latest edition of Bergey’s Manual of Systematic Bacteriology covering the phyla Firmicutes (De Vos et al., 2008) and Actinobacteria (Goodfellow et al., 2012) or in The Prokaryotes (Dworkin et al., 2006).

Phenotypic criteria General phenotypic criteria, comprising morphological, physiological, biochemical and nutritional characteristics, are summarized here. Because the phenotype may be affected by culturing and test conditions, it is recommended 1440

that authors include in their investigations type strains of relevant reference taxa together with the type strain of the type species of the genus, which is important for comparative purposes at the genus level, rather than simply report data from the literature. This is of particular importance for organisms which appear highly related on the basis of 16S rRNA gene sequences and for species descriptions based on a single strain. The number and significance of differentiating phenotypic characteristics must be sufficiently high so that species boundaries can be reliably delineated. Cell morphology and arrangement Cell shape and form should be adequately described and ideally the description should be supported by an appropriate photomicrograph. Phase-contrast microscopy at 61000 magnification is recommended; electron microscopy may be of value to reveal additional morphological information. As cellular morphology and arrangements are very dependent on growth conditions, a comprehensive description of culture conditions and medium composition should be given. Detailed procedures, i.e. with respect to the differential influences of defined growth conditions, have been suggested by Scardovi (1986). Cell morphology and cell arrangement can be useful in the assignment of an isolate to a specific genus. Bacteria that divide in one plane form pairs and chains when cells remain attached to each other (members of the genera Enterococcus, Lactococcus, Leuconostoc, Streptococcus, Vagococcus and Weissella). Division in two planes at right angles, forming tetrads, is typical of members of the genera Aerococcus, Pediococcus and Tetragenococcus (Facklam & Elliott, 1995). In the genus Bifidobacterium, morphological traits such as the disposition and number of branches of the cells or the cell contours, dimension and arrangements under different culturing conditions can be of great relevance in the recognition of species. Morphologies of cells grown on agar plates may be heterogeneous due to varying oxygen conditions, while cells grown in agar stabs generally show a more homogeneous morphology. Colony (macroscopic) morphology While colony characteristics, including size, shape, colour, edge, elevation, surface, consistency and transparency must be provided, they are generally of very little help to differentiate members of the genera Bifidobacterium, Lactobacillus and related genera, because of the wide variety, both within and between species. Details of colony characteristics may be valuable in the species description, however, if medium and cultural conditions are indicated. Motility Species in the group are generally not motile, but some species belonging to the genera Lactobacillus, Vagococcus, International Journal of Systematic and Evolutionary Microbiology 64


Table 2. Standards for description of new taxa within the families Bifidobacteriaceae, Lactobacillaceae, Aerococcaceae, Carnobacteriaceae, Enterococcaceae and Leuconostocaceae Characteristic REQUIRED Phenotypic criteria Cell morphology and arrangement Colony morphology Motility Gram-staining reaction Formation of endospores Fermentation pattern of carbohydrates Fermentation products from glucose Lactic acid isomer(s) produced from glucose (not specific for the family Bifidobacteriaceae) Fructose-6-phosphate phosphoketolase activity (specific for the family Bifidobacteriaceae) Growth medium and physiological properties, including the relationship to oxygen Presence/absence of catalase Tolerance to NaCl and salt requirement (not essential for the family Bifidobacteriaceae) Nitrate and nitrite reduction Indole production Voges–Proskauer test H2S production Urease production Deamination of arginine Hydrolysis of gelatin Hydrolysis of hippurate (positive for species of the genus Aerococcus) Pyrrolidonylarylamidase production (positive for species of the genus Enterococcus) Bile-aesculin tolerance test (positive for species of the genera Enterococcus, Pediococcus and Tetragenococcus) Haemolysis test Tellurite tolerance (positive for Enterococcus faecalis) Pyruvate utilization (positive for some species of the genus Enterococcus) Chemotaxonomic criteria Peptidoglycan Ecological criteria Source and habitat Genotypic criteria DNA base composition DNA–DNA hybridization (when strains share more than 97 % 16S rRNA gene sequence similarity) 16S rRNA gene sequence similarities Complementary phylogenetic markers ADDITIONAL Phenotypic characteristics Antibiotic susceptibility Bacteriocin typing Chemotaxonomic characteristics Fatty acid analysis Polar lipid analysis Whole-cell protein profiling Electrophoretic mobility of enzymes Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS Genotypic characteristics Plasmid profiling Multilocus sequence typing (MLST) Multiple locus variable number of tandem repeats analysis (MLVA) Genomic fingerprinting [amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), repetitive element palindromic (Rep)-PCR, amplified rDNA restriction analysis (ARDRA), intergenic spacer region (ISR)-PCR]

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Weissella and Enterococcus are known to be motile. Cultures from liquid and solid media should be examined for motility under a phase-contrast microscope. Gram-staining reaction The behaviour of the cells in Gram-staining should be described. The staining reaction may be variable under special circumstances. It is, therefore, recommended that cultures are subjected to the procedure just after growth has become visible and that staining is repeated at late exponential growth phase. All micro-organisms of the group considered should be Gram-staining-positive except for those of the genus Gardnerella, which are Gramstaining variable.

lactate, ethanol or acetic acid and carbon dioxide; moreover, pentoses are fermented via this pathway (Hammes & Vogel, 1995). The theoretical molar ratio of the main fermentation products should be stated. Bifidobacteria are characterized by the fermentation of glucose via the bifidus shunt, with acetic and (+)-L-lactic acid as the main end-products. While the theoretical molar ratio of these acids is 3 : 2, many strains show a considerably higher proportion of acetic acid due to the metabolism of internally formed pyruvic acid to acetic acid, ethanol and formic acid. It is, therefore, recommended to analyse the actual amount of glucose fermented. GC analysis can be used for the determination of the proportions of acetic and (+)-L-lactic acid (Holdeman et al., 1977).

Formation of endospores Species belonging to the genera Bifidobacterium and Lactobacillus and related genera are not endosporeforming. The absence of endospores has to be indicated. Fermentation pattern of carbohydrates The genera Bifidobacterium and Lactobacillus and related genera are characterized by their strictly saccharoclastic activities. Since carbohydrate fermentation data are sensitive to different culture conditions, the use of a specific modified liquid medium containing only half the normal quantity of carbohydrates is recommended, as well as the use of bromocresol purple as a pH indicator. Miniaturized commercial kits such as API (bioMe´rieux) are available for this characterization. For the genus Lactobacillus it is advisable to test the following carbohydrates: cellobiose, melibiose, raffinose, mannitol, amygdalin, sucrose, galactose, lactose, maltose, mannose, salicin, trehalose, arabinose, aesculin, gluconate, melezitose, ribose, sorbitol and xylose. Since the pentoses are of special importance for the genus Bifidobacterium, the following carbohydrates should be tested in addition to those mentioned before: fructose, glycerol, rhamnose and starch. Fermentation products from glucose Lactobacilli are characterized by the fermentation of glucose via different pathways: (1) obligately homofermentative lactobacilli are able to ferment hexoses almost exclusively to lactic acid by the Embden–Meyerhof–Parnas (EMP) pathway while pentoses and gluconate are not fermented, as they lack phosphoketolase;

Lactic acid isomer(s) produced from glucose (specific for all except members of the family Bifidobacteriaceae) The ratio of isomers of lactic acid formed from glucose should preferably be determined enzymically by using a lactic acid dehydrogenase commercial test kit. Fructose-6-phosphate phosphoketolase activity (specific for the family Bifidobacteriaceae) The genera of the family Bifidobacteriaceae are characterized by the key catabolic action on fructose 6-phosphate by fructose-6-phosphate phosphoketolase (EC 4.1.2.22) and subsequent reactions via the pentose phosphate cycle. Evidence of this key enzyme activity should be determined in cell-free extracts, given the importance of the bifidus shunt as the most important genus-specific characteristic (Biavati & Mattarelli, 2006). Growth medium and physiological properties, including the relationship to oxygen The data on growth requirements are part of the characterization of any micro-organism and are essential for the determination of its optimal cultivation conditions. It is therefore important to report (i) the range and optimal values of temperature and pH for growth in suitable media and (ii) whenever possible, the generation time of the bacterium under optimal growth conditions.

(2) facultatively heterofermentative lactobacilli degrade hexoses to lactic acid by the EMP pathway and are also able to degrade pentoses and often gluconate, as they possess both aldolase and phosphoketolase;

Growth at a different pH should be determined up to pH ¢8.5 for carnobacteria and tetragenococci, and also at pH 3.5 for oenococci. Growth at 15 and 45 uC (for presumptive carnobacteria also at 0, 30 and 40 uC) should be determined. While suitable media can be very different, required additives (vitamins and other growth factors, extracts, salt, etc.) should be specified in the species description.

(3) obligately heterofermentative lactobacilli degrade hexoses by the phosphogluconate pathway, producing

Factors affecting growth should be investigated under conditions close to the optimum growth conditions and

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need to be described in detail. Conditions of growth should include the relationship to oxygen, i.e. whether strains are able to grow under aerobic, microaerobic and anaerobic conditions. Qualitatively, this relationship is more easily determined in agar tubes, set up with a suitable anoxic medium, inoculated and carefully mixed with a well-grown culture. Tubes are then exposed to the air at the top to allow oxygen to diffuse into the agar and to establish an oxygen gradient. Presence/absence of catalase While most micro-organisms considered here are generally regarded as anaerobic bacteria that contain neither respiratory chain enzymes nor catalase, most of them can grow aerobically when both haem and a quinone are provided (Yamamoto et al., 2011). In the case of a negative catalase test, it is advisable to characterize other factors that will prevent oxygen damage as these factors might turn out to be interesting differentiating characteristics for the organism. When reporting catalase activity, reagent solution concentration and time of observation should be stated; the standard method employing 3 % H2O2 is recommended (Smibert & Krieg, 1994). Pseudocatalase activity (H2O2 inactivation without haem addition and insensitive to HCN) should be noted. Species of the genus Fructobacillus are an exception in the group and need oxygen as an electron acceptor for growth on glucose. Growth enhancement in the presence of oxygen and other electron acceptors (e.g. fructose and pyruvate) should also be determined. Tolerance to NaCl and salt requirement (not essential for the family Bifidobacteriaceae) The ability to grow at different concentrations of NaCl should be tested for micro-organisms of all new taxa, as some species of the genus Lactobacillus and related genera are differentiated through this characteristic. A liquid growth medium with a minimal content of essential salts is preferable for testing tolerance to NaCl as well as salt requirement. Tolerance towards high concentrations of NaCl has been reported for species of the genus Enterococcus, Tetragenococcus and Vagococcus. Therefore, growth with up to 18 % NaCl should be determined. Other biochemical characteristics Other biochemical activities to be examined for unambiguous delineation of species may vary depending on the group of organisms. The following minimal set of activities is recommended: nitrate and nitrite reduction, indole production, acetylmethyl carbinol-Voges–Proskauer test and H2S production, urease production, deamination of arginine, hydrolysis of gelatin, hydrolysis of hippurate (positive for species of the genus Aerococcus), pyrrolidonylarylamidase production (positive for species of the genus Enterococcus), bile-aesculin tolerance test (positive for species of the genera Enterococcus, Pediococcus and http://ijs.sgmjournals.org

Tetragenococcus), haemolysis test, tellurite tolerance (positive for Enterococcus faecalis), pyruvate utilization (positive for some species of the genus Enterococcus). Miniaturized test combinations and test kits, most of which allow an indicator-based determination of sugar utilization or show reactions based on their specific microbial enzymes, can be utilized in the biochemical phenotypic differentiation.

Chemotaxonomic criteria Type of peptidoglycan The nature of the diamino acid and/or the interpeptide bridge of the cell-wall peptidoglycan is generally considered a valuable taxonomic tool for identification and grouping of members of the genera Bifidobacterium, Lactobacillus and related genera (Schleifer & Kandler, 1972). The determination of these features does not necessarily require the purification of the complete cell wall for all groups. The presence of meso-diaminopimelic acid is characteristic for members of the genera Carnobacterium, Tetragenococcus and Desemzia and certain species of the genus Lactobacillus. The interpeptide bridge is an important taxonomic criterion for the genera Lactobacillus, Leuconostoc, Weissella and Bifidobacterium. Although most lactobacilli possess the L-Lys(Orn)–D-Asp type, within the obligatory heterofermentative lactobacilli interpeptide bridges of the types LLys–L-Ala and L-Lys–Ser–L-Ala2 can be found.

Ecological criteria Source and habitat The source and place of isolation of the new taxon should be detailed, as well as possible pathogenicity and host range. To clarify the possible transfer from a primary habitat (e.g. fermenting food) to a secondary one (e.g. sewage) or to document the deliberate addition to food stuffs of strains originally isolated from animals, a detailed description of the original isolation source is essential, as well as a discussion on the natural habitat(s) and the ecological positioning of the new taxon. This is especially important as representatives of the genera Bifidobacterium and Lactobacillus are often used as supplements (‘culture adjuncts’) or starter cultures in foods or pharmaceutical preparations (Stiles & Holzapfel, 1997; Biavati & Mattarelli, 2006) and, therefore, may be isolated from the (human) host, which is clearly not the natural habitat of the strain. Ecological information, such as, e.g. food type, is important to support the safety status of the organism and can be essential with respect to the application of the ‘Generally Recognized as Safe’ (GRAS) or ‘Qualified Presumption of Safety’ (QPS) status (Gaggıà et al., 2010; Bourdichon et al., 2012), allowing the strains to be used in foods without extensive safety evaluations. 1443


Genotypic criteria The advantage of DNA-based identification techniques is that the methods focus on the nucleic acid composition of the micro-organisms rather than on the expression of phenotypic products encoded by the respective genes, avoiding variation induced by environmentally driven gene regulation. Besides the traditional DDH measurements, other genetic methods such as sequence-based methods, including whole genome sequencing, have become increasingly important as tools for the assessment of the relationships between different taxa. DNA base composition The DNA guanosine and cytosine content (G+C mol%) allows the discrimination between high-G+C and lowG+C Gram-staining-positive bacteria. The indication of the DNA G+C content (mol%) value of a new type strain or the DNA G+C content range of a novel type species should be established using validated methodologies. Different methods can be used for determination of the percentage G+C content such as enzymically hydrolysing DNA and subsequent quantification of the nucleosides by HPLC, e.g. according to the methods of Ezaki et al. (1990) or Tamaoka & Komagata (1984). Also, complete genome sequencing allows determination of the DNA G+C content in a reliable way. The method used should be indicated in parentheses (Tm, HPLC, complete genome, etc.). Since total bacterial genome sequences have become increasingly available (http://www.ncbi.nlm.nih.gov), these sequences represent excellent reference organisms for calibration purposes. Generally, the DNA G+C range observed is not more than 3 % for a well-defined species and not more than 10 % within a well-defined genus (Stackebrandt & Liesack, 1993). However, the genera Lactobacillus and Bifidobacterium are examples of large genera for which the range in DNA G+C content (mol%) can be .10 %. Knowledge of the DNA G+C content is also an important prerequisite for determining the conditions used in DDH. DNA–DNA hybridization Even if there are promising new methods for the definition of bacterial species (Gevers et al., 2005), DDH is still the established gold-standard technique to estimate genetic relationships at the species level in prokaryotes. DDH should be performed in cases where the new taxon contains more than a single strain, in order to show that all members of the taxon share a sufficient degree of similarity to be considered a single species. DDH should also be used to determine that this homogeneous group is genomically different from closely related species. DDH is necessary when strains share more than 97 % 16S rRNA gene sequence similarity. If the new taxon shows this high degree of similarity to more than one species, DDH should be performed with all relevant type strains (usually the closest neighbours based on 16S rRNA gene sequence) to 1444

demonstrate that there is sufficient dissimilarity between the species to support their classification in a new taxon (Tindall et al., 2010). In order to evaluate the stringency of the DDH, it is necessary that experimental conditions (buffer system, ionic strength and reassociation temperature) are properly reported. Several techniques with different advantages and disadvantages can be used in DDH experiments. The filter method using radioactive DNA used to be a highly reproducible technique (Scardovi et al., 1971) that lost its importance with the disappearance of radioactivity from research laboratories. The reliability of the spectrophotometric method is comparable to the filter techniques (Huss et al., 1983). Methods using microtitre plates (Ezaki et al., 1989) offer the advantage of a higher sample throughput. Care should be given though to reproducibility (Goris et al., 2007). 16S rRNA gene sequence similarities The 16S rRNA gene is the most used molecular marker and its full-length sequencing (.1400 nt, ,0.5 % ambiguity) is still the first method to be utilized for the determination of phylogenetic relationships. The public availability of the sequences of most type strains is a great advantage in the identification and phylogenetic positioning of new isolates or taxa. Stackebrandt & Goebel (1994) proposed an upper limit of 97 % 16S rRNA gene similarity as a threshold. While lower sequence similarities suggest different species, similarities of 97 % or higher still require the use of DDH to determine species identity. Stackebrandt & Ebers (2006) proposed a higher cut-off value of 98.7–99 %, but the more recent cut-off proposed was 98.65 % (Kim et al., 2014). Kim et al. (2014) determined this threshold for 16S rRNA gene sequence similarity by performing the pairwise comparison of 6787 genomes of prokaryotes belonging to 22 phyla and showing that this level corresponds to the currently accepted Average Nucleotide Identity (ANI) threshold for species demarcation. For similarity calculation, the correct alignment of the sequences has to be checked using different algorithms. For the reconstruction of phylogenetic trees it is important to verify the reliability of the branching points using a criterion of goodness such as bootstrap analysis. 16S rRNA gene sequences used in any species description should be deposited in a database with public access and the accession numbers need to be included in the species description. To avoid prematurely attributed invalid Latin names in the public database, the sequences should preferably be deposited under the laboratory code and under provisional denominations such as Bifidobacterium sp. isolate no. xxx. Complementary phylogenetic markers 16S rRNA gene sequence similarity alone is not always sufficient to guarantee species discrimination even if it is fundamental for an intial ‘snapshot’. Protein-encoding International Journal of Systematic and Evolutionary Microbiology 64


genes, such as housekeeping genes and non-coding (intergenic) regions, such as 16S–23S internally transcribed spacer (ITS), are more variable and therefore have a greater degree of resolution. They are increasingly being used in the identification and description of novel species (Santos & Ochman, 2004). With the increasing availability of complete bacterial genomes, sets of multiple housekeeping genes have been established that can accurately predict genome relatedness and improve the accuracy of species identification (Coenye et al., 2005). The selection of genes useful for species discrimination should follow some basic rules: (i) genes should be present in one or few copies in most bacterial genomes; (ii) they should possess a higher rate of evolution when compared with rRNA genes; (iii) they should not easily recombine; (iv) they should possess enough variability to allow discrimination of species in a given genus (Zeigler, 2003). Hsp60 (coding for a 60 kDa chaperonin), recA (a protein of the SOS regulon, involved in repairing damaged DNA), pheS (phenylalanyl-tRNA synthase), rpoA (the a-subunit of RNA polymerase), rpoB the b-subunit of RNA polymerase), gyrB (subunit B of DNA gyrase), tuf (elongation factor Tu) and the 16S–23S ITS are examples of complementary phylogenetic markers, useful at the species, subspecies and strain level (Tanigawa & Watanabe, 2011; Naser et al., 2007). The sequencing of a minimum of two well-chosen housekeeping genes, universally distributed, present as single copies and located at distinct chromosomal loci, is recommended. A length covering at least 300 bp from each gene has to be analysed. In 2004, a new database based on chaperonin sequences from bacterial and eukaryotic species was established (http://www.cpndb.ca/cpnDB/search.php) (Hill et al., 2004). Partial housekeeping gene sequence similarity values between species were much lower than those of 16S rRNA genes: for example, similarity of the partial hsp60 gene of the bifidobacteria was 80–96 %, 96.5– 100 % and 95.5–97 % at the inter-species, intra-species and inter-subspecies levels, respectively (Zhu et al., 2003). Similarity values lower than 80 % in fructose-6-phosphate phosphoketolase-positive strains have determined the description of several novel genera of the family Bifidobacteriaceae (Jian & Dong, 2002; Simpson et al., 2004). Although the inter-species diversity of these genes and intergenic regions has been shown to be significantly higher than those of the 16S rRNA genes, sometimes the application of these multiple phylogenetic markers has been hampered by the unavailability of their sequences for many (related) species. In these cases, complementary phylogenetic markers should be chosen on the basis of availability. For example, hsp60 sequences, available for a considerable number of species of the genera Lactobacillus and Bifidobacterium, can be used for the confirmation of new taxa in these genera. http://ijs.sgmjournals.org

In conclusion, in addition to the 16S rRNA gene, we recommend the application of at least two additional genetic markers, such as housekeeping genes or noncoding (intergenic) regions, to improve the accuracy of the genetic identification of isolates, especially when used for the description of novel species. When used for species description, these additional sequences should also be deposited in a public sequence database and accession codes should be provided in the article.

Additional phenotypic characteristics The following phenotypic, chemotaxonomic and genotypic characteristics can be useful for the characterization of a new taxon, although they are not generally considered as part of the ‘minimal standards’. Antibiotic susceptibility Antibiotic resistance due to intrinsic and/or acquired resistance mechanisms can be a useful characteristic of some genera, such as (intrinsic) vancomycin resistance for species of the genera Leuconostoc and Pediococcus. Moreover, the susceptibility of members of the genera Bifidobacterium, Lactobacillus and related genera to antibiotics is of great concern for human and animal safety, especially when used in foods. The presence of transferable antibiotic resistance genes deserves particular attention from a safety point of view. Bacteriocin typing The production of inhibitory substances that are effective against closely related species is a characteristic among certain bacterial groups or strains. As shown for nisin, bacteriocin production can be important for application as a preservative in foods.

Additional chemotaxonomic characteristics Additional chemotaxonomic characteristics may be valuable for novel species or genus descriptions and are recommended as supporting data whenever possible. Fatty acid analysis Gas-chromatographic analysis of the methyl esters of cellular fatty acids is a fast and easy method that has been applied successfully in the taxonomic identification of Gram-staining-positive micro-organisms. The variability in chain length, double-bond position and substituent groups has been shown to be a useful adjunct to phenotypic and genetic data for the characterization of many bacterial taxa. Particular attention must be paid, however, to the influence of culture conditions and growth temperature in particular, which greatly influence bacterial fatty acid 1445


composition. Therefore standardization of media and growth conditions is necessary to obtain highly reproducible fatty acid profiles that may be used as chemotaxonomic markers. Whenever possible, it remains important to test the different chemotaxonomic markers on multiple strains of the same species, allowing the comparison of intra-species with inter-species variability and the detection of possible overlaps. Polar lipids Polar lipids are the major constituents of the lipid bilayer of bacterial membranes (glycolipids, phospholipids, etc.). For comparative taxonomic purposes it is not necessary to structurally characterize lipids but it is sufficient to show their distribution pattern in two-dimensional TLC and identify some properties of lipids on the basis of staining (da Costa et al., 2011).

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS MALDI-TOF MS facilitates the generation of molecular fingerprints for entire micro-organisms producing complex spectra of complex biological macromolecules, through their specific degradation products, which are characteristic for the molecular content of a bacterial cell. When these spectra are compared using appropriate computer software, the bacterial discriminatory power goes to the species level. In some cases, subspecies discrimination might be possible (Zeller-Pe´ronnet et al., 2013). This type of analysis is routinely used in some Microbial Collections for taxonomic control.

Additional genotypic characteristics Additional genotypic characteristics may also be valuable for novel species or genus descriptions and are recommended as supporting data when possible.

Whole-cell protein profiling Whole-cell protein profiles, obtained by a highly standardized method, have proven to be discriminatory at the species and subspecies levels and have been used for the identification and delineation of new taxa (Hertel et al., 1993; Pot et al., 1993; Van Den Berg et al., 1993; Vogel et al., 1994; Vandamme et al., 1996; Morlon-Guyot et al., 1998; Back et al., 1999). In the 1980s and early 1990s, these methods were widely applied to different organisms, but since the 1990s they have been largely surpassed by DNA-based methods. Interestingly, the need for comparative analysis of the complex banding patterns obtained by protein SDS-PAGE was the trigger for the development of dedicated computer software that is now successfully applied to DNA fragment analysis. Within species, strains that have 80 % or greater DDH have identical or nearly identical protein profiles (Biavati et al., 1982; Domig et al., 2003). Electrophoretic mobility of enzymes

Plasmid profiling Plasmid profiles are not a dependable method for strain typing or species identification, because plasmids can be lost during subculturing and technological processes or undergo rearrangements by conjugative transfer. In bifidobacteria, only 8 out of 41 species contain strains with plasmids. Nevertheless, plasmid profiling in association with resistance patterns can be useful for epidemiological typing of strains. Multilocus sequence typing (MLST) MLST is an appropriate technique to establish an unambiguous international database of genetic lineages among species and genera since it is based on identifying alleles from DNA sequences of internal fragments of housekeeping genes (Maiden, 2006). Information should be obtained from the sequences of at least five genes coding for proteins of metabolic function. MLST of six or seven core gene fragments is frequently used to assess evolutionary distances between isolates within a species. Unfortunately, despite the reputation of MLST as being generally applicable and despite a considerable number of gene families being conserved even between the phylum Firmicutes and bifidobacteria (63 gene families), different MLST target gene sets have been proposed for various species and most of these are not conserved between all species (Lukjancenko et al., 2012).

About 50 % of all enzymes investigated so far exist in multiple molecular forms. These isoenzymes are usually directly related to mutations at the gene locus that cause amino acid substitutions in the enzyme coded for by the gene. Differences in the electrostatic charge between the original and substituted amino acid will affect the net charge of the enzyme and hence its electrophoretic mobility. Thus, it is possible to relate mobility differences to different alleles at the gene locus for the enzyme in question. This technique has been shown to offer some potential in the differentiation of enterococci (Tomayko & Murray, 1995). Isozyme patterns such as isozymes of transaldolase and 6-phosphogluconate dehydrogenase can also be used to identify species of bifidobacteria (Biavati & Mattarelli, 2006), and prolyl-aminopeptidase and lactic acid dehydrogenase for identifying species of the genus Lactobacillus (Scolari & Vescovo, 2004).

It is to be expected that MLST schemes will become more widely available through publicly accessible databases. The discriminatory power of an MLST scheme can also be evaluated by electronic tools (Slabbinck et al., 2008) once validation has been performed. Similar to pulsed field gel electrophoresis (PFGE), this sequence-based typing method may be useful for the differentiation of isolates to the subspecies level, in addition to the identification of isolates. Jolley et al. (2012) proposed a universal approach

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for the characterization of bacteria from the domain to the strain by ribosomal MLST, an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrating microbial genealogy and typing. In the original publication a small subset of the genus Lactobacillus was already included, but not discussed in detail. Multiple locus variable number of tandem repeats analysis (MLVA) MLVA is a typing method that has been shown to have good discriminatory power. Tandem repeats are repeated DNA sequences present in the bacterial genome for which the number of units at a given locus may vary among the strains of the same species. The multiple variable number tandem repeat (VNTR) loci are amplified across the genome and the number of repeats in each targeted region is subsequently determined by gel or capillary electrophoresis (van Belkum, 2007). The number of repeats in each locus can be expressed as a numerical code and each unique combination of numbers represents a profile. This effective method can be utilized for the typing of bacterial species. Genomic fingerprinting Rapid DNA-typing methods are considered appropriate tools for the determination of inter- and intra-species relatedness. These methods investigate whole genomes [amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), repetitive element palindromic (Rep)-PCR, PFGE, rrn operons, the 16S rRNA gene (amplified rDNA restriction analysis, ARDRA) or the intergenic 16S–23S rRNA spacer regions (ISR)]. Ribotyping and methods based on whole genomes are especially recommended for examining whether strains belong to the same species. However, ribotyping cannot identify all strains of one species, demonstrating that, as with other fingerprinting techniques, highly similar banding patterns are useful for species identification, whereas different patterns do not necessarily indicate another species. However, further standardization of these techniques is required in order to improve the reproducibility between laboratories before these methods can be proposed as alternative minimal criteria that are equivalent to DDH. A survey of DNA fingerprinting techniques and their application in bacterial systematics is given by Pukall (2006). Though there are currently only a restricted number of complete genome sequences available for members of the genera Bifidobacterium, Lactobacillus and related genera (http://www.genomeonline.org), it can be expected that genome sequences will play an increasing role in understanding the phylogeny of and in the definition of species in the future. Against the background of rapid advances in characterization methodology, novel tools will be included http://ijs.sgmjournals.org

in future minimal standards as soon they have been validated for the identification and characterization within this group of organisms.

Acknowledgements The authors are grateful to all other members of the Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms of the ICSP for their input and careful reading of the manuscript: Bruno Biavati (Bologna University, Bologna, Italy); Johanna Bjo¨rkroth (University of Helsinki, Helsinki, Finland); Christine Bonaparte (University of Veterinary Medicine, Hannover, Germany); Gu¨nter Klein, (University of Veterinary Medicine, Hannover, Germany); Gerhard Reuter (Free University of Berlin, Berlin, Germany); Marc Vancanneyt (Ghent University, Gent, Belgium) and Koichi Watanabe (Yakult, Tokyo, Japan).

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