INTRODUCTION

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Microorganisms have been classified and identified on the basis of a variety of characteristics including morphological, growth, tolerance, metabolic, biochemical, and genetic. Recently there has been a tendency to determine definitive classification and taxonomic assignment by nucleic acid hybridization, 16S rRNA sequence analysis, and other molecular genetic techniques. After classification has been established, characteristics are selected for the identification of unknown isolates. Commercial kits based on such a process are available for the identification of clinically important bacteria. It is essential to realize that for routine identification of isolates from human, food, or veterinary specimens, ease of testing and total completion time are critical, since the added value of identification information to the clinical or processing outcome decreases the later it becomes available. Completion times for the identification of bacteria taken from isolated colonies can vary from 2 h to several days. Completion times for tests performed directly on clinical specimens are also variable.

Most growth-dependent tests require at least an overnight incubation; others, based on the ability to utilize a single carbon or nitrogen source, may require as long as 7 days. The molecular genetic techniques are still time-consuming and less amenable to routine application. Moreover, most of the techniques available now are for the specific detection of a limited number of taxa. Alternatively, determinations of the enzymatic activities of isolates with a variety of synthetic substrates (16, 52, 124) can be used for identification and give similar results to those obtained by other characterization methods. The enzymatic characterization of microorganisms by means of synthetic substrates makes use of the fact that many enzymes are constitutively present or easily induced and rapidly detectable, often after incubation times of seconds to 3 h. Thus, identification of bacteria based on enzyme patterns offers simple and rapid results.

The ability to detect specific enzymes rapidly with synthetic chromogenic or fluorogenic substrates has been studied extensively (17, 35, 53, 54, 110, 117, 118, 150). Tests involving some of these substrates have been included in commercial kits for identification or taxonomic studies of bacterial isolates.

The first commercial kit with tests for specific enzymes contained Patho-Tec paper strips (172), a method that evolved to the Micro-ID system (15) for the identification of clinically important gram-negative rods, mainly members of the Enterobacteriaceae. The Micro-ID kit included tests for -galactosidase, cytochrome oxidase, lysine and ornithine decarboxylases, tryptophanase, and urease. Other kits in the form of cards, microtiter trays, or multichamber strips are now available for the identification of certain taxonomic classes of bacteria. The largest number is available for the identification of clinically important aerobic and facultatively anaerobic gram-negative bacteria (e.g., API 20E, MicroScan conventional overnight and MicroScan Rapid GN Identification Systems, and Vitek GNI Card). Fewer kits are available for the identification of gram-positive cocci, staphylococci, streptococci, anaerobic cocci, and yeasts. API 20E, MicroScan conventional overnight, and Vitek GNI Card include growth-dependent tests and a few enzyme tests; in general, they do not use the ability of enzyme tests to provide results rapidly. A specific enzyme test for -galactosidase based on utilization of the synthetic substrate o-nitrophenol--D-galactopyranoside (ONPG) or substrates with other synthetic moieties is included in most kits for identification of the Enterobacteriaceae. Some kits are completely manual, whereas others offer automation with instruments for all or some of the following tasks: inoculation, incubation, determination of test results, and identification. Dade MicroScan Rapid Identification Systems for gram-negative rods and gram-positive cocci are the fastest systems, providing identification in 2 to 2.5 h by measuring enzymatic activities fluorometrically with a high correct identification rate. Thus, overall rates of 98.4 and 92.5% correct identification to the species level, with and without additional tests, respectively, were obtained during the database development phase of the MicroScan gram-negative rapid identification system type 3 (RNID3) for 4,151 isolates comprising 138 fermentative and nonfermentative gram-negative bacteria (1). Clinical studies of the RNID3 system showed 96.8 and 89.5% correct identification for 405 fresh and 247 challenge isolates, respectively (18).

The commercial identification systems also provide databases of expected results, and an unknown isolate is assigned to one of the taxa in the database either by using a code book or by using an automated system and computer-based identification. Most of these kits have been designed for clinically important groups of bacteria, as reflected in the taxa, tests, and expected results included in the databases. Some may be applicable to isolates taken from different environments (143, 240). These kits can also be used for characterization of microorganism groups other than those specified in the database (27). Methods for easy extraction and analysis of enzymatic activities are required for such a purpose.

Commercial identification kits have been optimized for the sets of taxa included in their databases. They require regulatory approval and are expected to provide a high level of accurate identification in comparison with an acceptable reference method. Characterization kits, on the other hand, are designed to provide an easy and reproducible method for testing a variety of unknown isolates. They do not provide expected results and are less rigorously regulated. They are very useful in taxonomic studies, and the results from such studies may be used for the construction of identification kits. Some of these kits have been designed specifically for the characterization of microorganisms with a variety of chromogenic enzyme substrates. These kits are not accompanied by a database, and some may have methods for computer analysis of the data including classification and identification of unknown isolates. They vary in the complement of tests included, the size of the inoculum required, and the incubation period. Generally speaking, tests that require more than 2 h of incubation and/or include a growth-supporting medium in the test compartment allow the detection of inducible enzymes. Tests requiring inoculum densities of 2 McFarland standard units frequently necessitate an additional overnight incubation to achieve a sufficient inoculum.

Studies of the clarification of the taxonomic position of individual taxa have used laboratory-prepared tests (117, 119) as well as commercial characterization and identification kits. Only the latter contain information on expected results for specified taxa in each of the kit tests.

The API ZYM system (API System; bioMérieux, Paris, France) is a semiquantitative micromethod designed for the detection of 19 enzymatic activities (106, 232). The use of API ZYM for taxonomic studies of a variety of taxa of bacteria and other prokaryotic as well as eukaryotic organisms has been tabulated (99). The use of API enzyme research kits detecting 20 glycosidases, 10 esterases, 57 arylamidases, alkaline and acid phosphatases, and phosphoamidase has been reported (151, 153, 164, 226).

In addition, kits based on growth-dependent tests to determine the utilization of amino acids, organic acids, and carbohydrates are available. The API 50CH (12, 123, 164), 50AA, and 50OA (125) tests are strip based; the results are indicated by changes in the color of the pH indicators. The Biolog system (Biolog Inc., Hayward, Calif.) is microtiter tray based and is dependent on the detection of substrate-specific dehydrogenases with tetrazolium salt as an indicator (163). The MAST ID system (Mast Laboratories Ltd., Bootle, England) provides a means of determining the metabolic activities of a number of isolates by agar plate and multipoint inoculator techniques (84, 86, 123, 193). Combinations of these systems permit the detection of over 340 biochemical reactions. A number of studies report the use of commercially available characterization kits alone or in combination with test batteries prepared in their own laboratories (25, 31, 63).

This review will survey the current classification of aerobic and facultative anaerobic gram-positive cocci with emphasis on the role of rapid enzyme tests in characterization and identification.

The aerobic and facultatively anaerobic gram-positive cocci were originally divided into two families, Micrococcaceae and Streptococcaceae, on the basis of catalase activity and cell morphology and aggregation. However, this classification may change, since genetic studies have indicated that Staphylococcus is more closely related to the Bacillus-Lactobacillus-Streptococcus cluster than to Micrococcus or Stomatococcus (129).

The family Micrococcaceae, which includes the aerobic and facultatively anaerobic gram-positive cocci giving a positive reaction in the catalase test, is not a phylogenetically coherent group. Four genera, Micrococcus, Planococcus, Staphylococcus, and Stomatococcus, are recognized (129). Tests useful for the separation of these taxa include oxygen requirement for growth, oxidase test, motility, NaCl tolerance, and susceptibility to bacitracin, furazolidone, and lysostaphin. Genetic studies have suggested that Micrococcus belongs to the actinomycete group, and the isolates originally identified as species of Micrococcus have since been placed in five different genera (see below) (218).

Staphylococcus is the only genus showing susceptibility to lysostaphin, but some exceptions occur among species of Micrococcus and Staphylococcus. The methodology and usefulness of the lysostaphin test for differentiation between Micrococcus and Staphylococcus have been discussed (10, 83, 100, 133, 134, 138, 178, 184, 207, 210, 253). Lysostaphin is a protein preparation derived from culture filtrates of "Staphylococcus staphylolyticus," which contains three enzymes capable of affecting the bacterial cell wall: a glycyl-glycine endopeptidase, an endo--N-acetylglucosaminidase, and an N-acetylmuramoyl-L-alanine amidase (32, 215). The endopeptidase is the component that lyses the cell wall of Staphylococcus aureus by hydrolyzing the polyglycine of the pentapeptide bridge between glycopeptide chains of the staphylococcal cell wall. Sloan et al. (215) have shown that the endopeptidase is capable of catalyzing both hydrolysis and transpeptidation reactions when acting on glycyl peptides. The usefulness of the test depends on the purity of the enzyme preparation, the storage of the enzyme, the lysostaphin concentration (184), and the test procedure. When susceptibility is tested by observation of growth inhibition zones, it also depends on the medium on which the bacteria are grown (144). Susceptibility also depends on the cell wall amino acids of the bacterial species; species that contain serine in the interpeptide bridge, such as S. saprophyticus, S. haemolyticus, and S. hominis, are less susceptible. Interestingly, lytic agents such as lysostaphin are produced by members of the genus Staphylococcus only.

Of the genera included in the family Micrococcaceae, Staphylococcus is the most clinically important and contains 32 species, most of which live on the skin and other external surfaces of animals (129). A comprehensive review of the genera Micrococcus and Staphylococcus and their clinical significance has appeared recently (129). Some staphylococci are human and animal opportunistic pathogens that, under certain circumstances, are major causes of mortality and morbidity. Because of the ability of members of the genus Staphylococcus to acquire resistance to many antimicrobial agents (e.g., strains of methicillin-resistant S. aureus (MRSA), they can cause major clinical and epidemiological problems in hospitals (30), and their presence is monitored carefully. Schemes for the differentiation of members of the family have been published (12, 129, 194). Kits available for the identification of this group include API STAPH-IDENT, Staph-TRAC, API ID 32 Staph, RAPiDEC Staph, and Vitek GPI Card (bioMérieux-Vitek Inc. Hazelwood, Mo., and bioMérieux S.A., Marcy l'Etoile, France); GP Microplate test panel (Biolog); MicroScan Pos ID and Combo, and Rapid Pos ID and Combo panels (Dade International Inc., West Sacramento, Calif.); Pasco Gram-Positive ID (Pasco, Wheat Ridge, Colo.); and Staph-Zym (Rosco, Tastrop, Denmark). The general requirements of the kits and the tests included are listed in Tables 1 to 6, which also contain some published identification schemes for these taxa. In addition to the schemes listed in the tables, Watts et al. (238) have tested the API 20GP (bioMérieux-Vitek) kit, which consists of 20 microcupules containing dehydrated substrates for the identification of staphylococci and group D streptococci. It includes the 10 Staph-Ident tests and 10 tests selected from the API 20S streptococcal identification system, but it has provided only 56.10% correct identification. STAPHYtest (Lachema, Brno, Czech Republic), a microtiter-based system with nine tests and with eight compartments per isolate for identification of Micrococcus, Stomatococcus, and Staphylococcus, has been described (211). A system described by Rhoden et al. (194) is based on 6 screening, 18 primary, and 11 confirmatory tests; the tests used are similar to those described by Kloos and Bannerman (129). Eight enzyme tests are included in this scheme, which reports results and assigns isolates to a taxon by using a six-digit numerical code. The RAPiDEC Staph system is based on detection of the activities of three enzymes: "aurease", -galactosidase, and alkaline phosphatase; it requires a very heavy inoculum and can identify S. aureus, S. epidermidis, S. saprophyticus. Additional tests are required to differentiate between S. intermedius, S. xylosus, and the remaining Staphylococcus species.

The number of taxa identified by these kits varies from 5 by RAPiDEC Staph to 50 by MicroScan Rapid Pos ID panels. Some of the kits are specific to the Micrococcaceae or just to staphylococci, whereas others are intended for identification of both staphylococci and streptococci. Tables 16 show that tests for the formation of acid from a variety of saccharides are used most frequently followed by tests for glycosidases, hydrolases, and peptidases. The number of enzyme tests included in each of these varies from 2 in the Vitek GPI Card to 23 in MicroScan Rapid Pos ID panels. Resistance to certain antimicrobial agents also plays an important role in differentiation of these organisms. The MicroScan Rapid Pos Identification system is the only one relying on enzyme tests and acid formation detected fluorometrically and providing identification of most clinically significant staphylococci, enterococci, and streptococci in 2 h.

Micrococcus. The members of the genus Micrococcus differ from those of Staphylococcus by being obligate aerobes, with a G+C content of 63 to 73 mol%, containing cytochromes a, b, c, and d, lacking teichoic acids in their cell walls, lacking glycine in the interpeptide bridge of their cell walls, being resistant to furazolidone, and being susceptible to bacitracin. Most strains are also resistant to lysostaphin (129); susceptible strains of M. luteus have been described. Lytic activity toward staphylococci, demonstrated by members of the genus Staphylococcus, is not a characteristic of 98.5% of micrococci (205).

Planococcus. Members of the genus Planococcus are cocci arranged in pairs or tetrads. They are positive in the catalase and benzidine tests and have a G+C content of 39 to 52 mol%. They resemble Micrococcus spp. in being strict aerobes, lacking the ability to produce bacterial lytic agents such as lysostaphin, and showing resistance to lysostaphin (205, 228). They can tolerate 12% NaCl and are motile.

Staphylococcus. The members of the genus Staphylococcus differ from those of Micrococcus by being facultative anaerobes with a G+C content of 30 to 39 mol%, containing cytochromes a and b, containing peptidoglycan and teichoic acids in their cell walls with oligoglycine peptides in the interpeptide bridge of their cell walls, and being susceptible to furazolidone and resistant to bacitracin; most strains are susceptible to lysostaphin (129). Lytic activity is produced by 99.5% of staphylococci (205). Lack of lytic activity is confined to a small number of isolates of S. xylosus (205, 228).

Stomatococcus. A description of the genus Stomatococcus, which contains only one species, Stomatococcus mucilaginosus, is available (12, 129, 160, 198, 217). Strains of the species show the morphological characteristics of the family: cocci arranged in pairs, tetrads, and clusters. They exhibit a high G+C content of 56 to 60 mol%, near to that of members of Micrococcus (64 to 75 mol%) and unlike that of staphylococci (30 to 39 mol%). Strains of the species show a low catalase activity and a positive benzidine activity. Responses to metabolic and enzymatic tests resemble those of both staphylococci and micrococci, e.g., production of acid from a number of mono-, di-, and trisaccharides (similar to staphylococci) and hydrolysis of mono-, di-, and triamino acid conjugates (similar to Micrococcus). Enzyme tests used for the identification of S. mucilaginosus include hydrolysis of esculin (-glucosidase), catalase, leucine-aminopeptidases (LAP), and PYR. Identification of members of this taxon has been reported (12, 194, 195). Cross-infection between patients has been studied by using phenotypic characters, acid production from carbohydrates as determined by API 50CH, and MICs (227).

Identification schemes and kits. Reports on the identification of staphylococci with commercial kits are summarized in Table 7; when meaningful, identification of individual taxa is also listed. Not included in the table are early developments of the API systems that have been described (26, 33), studies of Staph-TRAC (11) when used in conjunction with biochemical and susceptibility tests, and STAPH-IDENT (179) for characterization of taxa. In addition, reports on the identification of gram-positive cocci have shown the following percent correct identifications: Pasco Gram-Positive ID (104), 90%; MS GP panels (104), 87.0%; MS Rapid ID panels (19, 38, 92, 216), 95.8, 96.2, 95.7, and 93.0%. MS Rapid ID panels have shown 90.0 and 87.0% correct identification with isolates taken from sheep and horse blood agar, respectively (159).

The family Streptococcaceae and related organisms include the aerobic and facultatively anaerobic gram-positive cocci that generally give a negative result in the catalase test. These organisms are encountered in the mouths and intestinal tracts of humans and animals and play an important role in the food industry as agents of preservation or spoilage of fermentation products (220). Some of the taxa are virulent pathogens, causing pharyngitis, respiratory infections, skin and soft tissue infections, dental caries, infective endocarditis, and septicemia. Others play an important role in fermentation and preservation processes of a variety of food products. Isolates are characterized by homo- or heterofermentative metabolism of carbohydrates. The end product of homofermentative bacteria is lactic acid; the end products of heterofermentative bacteria are lactic and acetic acids as well as CO₂, which can be detected by the production of gas from a glucose medium. Characterization of the streptococci has traditionally been based on cell morphology (cocci or coccobacilli) and aggregation (tetrads or chains), colony size (large or small), appearance on blood agar (alpha-, beta-, or gamma-hemolysis), and antigenic structure (Lancefield grouping A through V). Further classification and identification are obtained by physiological and metabolic tests. In the last two decades, the taxonomy of the group has been actively studied. Applications of genetic methods (24, 41-46, 209, 244, 248), numerical taxonomy (31, 186), and enzymatic techniques (22, 127) have contributed to a further understanding of this complex taxonomic group. In addition, the isolation of vancomycin-resistant bacteria from human infections (37, 48, 115, 200) has necessitated the development of tests and schemes for the identification of clinical isolates of Leuconostoc and Pediococcus. A number of vancomycin-susceptible genera, including Alloiococcus, Globicatella, Helcococcus, and Vagococcus, have also been isolated from clinical specimens (68).

The major changes introduced into the taxonomy include splitting the genus Streptococcus into three genera, Enterococcus, Lactococcus, and Streptococcus; changes to classification of the viridans streptococci; increasing the number of species of Enterococcus; and addition of the genera Alloiococcus, Globicatella, Helcococcus, Tetragenococcus, and Vagococcus. Although the newly described genera resemble members of the family Streptococcaceae, their taxonomic position has not been clearly established.

The taxonomy and clinical significance of Streptococcus (22, 31, 47, 49, 112, 127, 197), Enterococcus (73, 112, 170), and the new genera (68, 198) have been reviewed. Numerical taxonomy of lactic acid bacteria including Streptococcus, Lactobacillus, Leuconostoc, and Pediococcus spp. has been studied by Priest and Barbour (186).

The usefulness of the catalase test for separating micrococci from streptococci has also been studied. The presence of catalase is usually determined by addition of a drop of 3% H₂O₂ to a heavy bacterial suspension and observation of effervescence due to the release of O₂. However, release of O₂ has been observed in Aerococcus and Alloiococcus members of the family Streptococcaceae and is presumed to be mediated by peroxidases (68). Moreover, isolates of Stomatococcus, a member of the family Micrococcaceae, can show a negative or a weak catalase activity. Members of the Micrococcaceae differ from streptococci in their ability to synthesize the iron-porphyrin compounds of the heme group, an essential component of respiratory systems including catalase, cytochromes, and nitrate reductase; streptococci lack this ability. A number of tests have been used to differentiate between members of the Micrococcaceae and Streptococcaceae. The catalase test is the most commonly used, but some members of the Micrococcaceae, e.g., Stomatococcus and some strains of S. aureus, can give weak or even negative reactions. The benzidine test (56) detects iron-porphyrin compounds in catalase- and cytochrome-containing bacteria; it is highly sensitive and is positive for all members of the Micrococcaceae, even those which are catalase negative. Tests originally designed to detect ability of Haemophilus strains to synthesize porphobilinogen and porphyrin from -aminolevulinic acid (126) have been applied to gram-positive cocci (250) for differentiation of micrococci from the streptococci. Production of porphobilinogen correlated 100% with membership in the family Micrococcaceae, including 22 catalase-negative isolates. A modification of the benzidine test has been described (75) for the detection of oxidase-positive members of the Micrococcaceae based on removal of noncovalently linked heme groups prior to reaction with the benzidine reagent making it specific to the covalently protein bound heme of cytochromes c. The modified benzidine test was compared with a modified oxidase test for differentiation within the Micrococcaceae and shown to be positive for all members of the genus Micrococcus and for strains of S. sciuri. By using the modified oxidase test, 2.3% of 302 isolates tested were misclassified mainly because of poor growth; however, the oxidase test was preferred because it is much simpler to perform. Moreover, because benzidine is carcinogenic, its use is not recommended.

The heme-negative coccal group now contains 12 genera. The genera whose cells aggregate in chains include Enterococcus, Globicatella, Lactococcus, Leuconostoc, Streptococcus, and Vagococcus; those arranged in pairs or tetrads include Gemella, Helcococcus, Pediococcus, and Tetragenococcus; while Aerococcus and Alloiococcus, also arranged in pairs and tetrads, are the two genera that may show a weak pseudo-catalase activity.

The genera within these groups are further separated by the production of PYR and LAP, the ability to hydrolyze esculin in the presence of bile salts, the production of gas from glucose, the ability to grow in the presence of 6.5% NaCl, and at 10 and 45°C, motility, susceptibility to vancomycin, and the type of hemolysis.

(i) Enterococcus. The genus Enterococcus, which included E. faecalis and E. faecium, was separated from Streptococcus on the basis of DNA hybridization data by Schleifer and Kilpper-Bälz (208). Collins et al. (44) added E. avium, E. casseliflavus, E. durans, E. gallinarum, and E. malodoratus to the list of Enterococcus species. With the exception of E. malodoratus, all the above species have been isolated from human sources (73). Since then, 12 new species have been described. E. dispar, E. hirae, E. flavescens, E. mundtii, and E. raffinosus have been isolated from humans and other sources (73, 111). E. pseudoavium, and E. saccharolyticus have been isolated from cattle and other mammals. E. columbae and E. cecorum have been isolated from pigeons and chickens, respectively. E. sulfureus has been isolated from plants (111). E. solitarius, which has been isolated from an ear exudate (111), from patients admitted to a public hospital (90), and from the rumens of domestic and wild ruminants (145), is now deemed closer to Tetragenococcus halophilus (248). Similarly, E. seriolicida, which has been isolated from yellow-tail fish (111) and from water buffalo with subclinical mastitis (222), is now deemed closer to Lactococcus garvieae (222). It has been recommended that these two species, which fail to react with the AccuProbe Enterococcus probe, should not be included in the genus Enterococcus at this time (73).

(ii) Globicatella. The genus Globicatella described by Collins et al. (41) contains one species, Globicatella sanguis. Strains of this genus resemble viridans streptococci in colony appearance and many other characteristics, but they are PYR positive and LAP negative, can grow in 6.5% NaCl, and are susceptible to vancomycin. Phylogenetically, the genus is related to Aerococcus; the G+C content of the type strain is 37 mol% (41). Strains have been isolated from patients with bacteremia, urinary tract infections, and meningitis.

(iv) Leuconostoc. The genus Leuconostoc includes heterofermentative coccobacilli that are important in the conservation and preparation of several fermented foods (164), while some are involved in human diseases. The G+C content of members of the genus is 38 to 44 mol%.

(v) Streptococcus. The classification of the genus Streptococcus has been studied and reviewed extensively (5, 31, 47, 111, 116, 186, 197, 212, 232). The genus was previously divided into four groups: enteric, lactic, viridans, and pyogenic streptococci (212). Members of the genus have a G+C content of 34 to 46 mol% and are pathogenic for humans and other animals; some species are found as members of the normal flora of the mouth and gastrointestinal tract. Differentiation within the genus is achieved by colony size, type of hemolysis, and serological, metabolic, and molecular genetic tests. Unfortunately, serological techniques relied upon previously for the identification of pyogenic streptococci identify isolates with group B and F antigens but not individual species of isolates positive for group A, C, and G antigens (197, 224). Isolates carrying the latter antigens may belong to more than one species; they can be subdivided into large- or small-colony-forming strains. Thus, while isolates with the above antigens that grow in large colonies are pyogenic, the small-colony-forming strains may participate in infection but can also be found as commensals. The small-colony-forming strains belong to a number of species which are placed in the "S. milleri" group (197).

(vi) Vagococcus. The genus Vagococcus (42) was created for the motile lactococci, reacting with group N antisera, with a G+C content of 33.6 mol%. Two species have been described, V. fluvialis and V. salmoninarum (40, 42). Isolates of V. fluvialis resemble members of the genus Enterococcus both phenotypically (68) and genetically (42), reacting positively with the AccuProbe Enterococcus test, but unlike Enterococcus spp., they grow poorly at 45°C. Very few isolates have been recovered from human patients. The second species, V. salmoninarum, has been isolated from fish, is nonmotile, does not grow at 40°C, and produces H₂S. Acid formation from glycerol, galactose, sorbitol, and D-tagatose differentiates between the two species; the former is positive in the first three tests, and the latter is positive only in the last (233). Schemes for differentiating the members of this genus from the other gram-positive cocci have appeared (68).

(i) Gemella. The genus Gemella contains two species G. haemolysans, previously classified as Neisseria haemolysans, and G. morbillorum, previously classified as Micrococcus sp., Diplococcus morbillorum, Peptostreptococcus morbillorum, and Streptococcus morbillorum (25, 196). The differences and similarities of the two species were discussed by Berger and Pervanidis (25). The phylogenetic relationship between the two species and other gram-positive bacteria was studied by Stackebrandt et al. (219) and Whitney and O'Connor (246). The clinical significance of the two species has been reviewed (68). The genus contains gram-positive cocci, which may require aerobic (G. haemolysans) or strict anaerobic (G. morbillorum) conditions for isolation and further culturing. It was also shown that 10% CO₂ is optimal for the growth of both species. A G+C content of 30 to 33.5 mol% has been established (149). G. haemolysans may appear gram variable or gram negative. Isolates of both taxa grow poorly with variable hemolysis, which may depend on the source of blood (rabbit, sheep, or horse). Most strains are positive in the LAP and PYR tests but require a heavy inoculum. Discrepancies were observed between the results of these tests when performed with commercial kits or as tube tests (68). The ability to synthesize cytochrome b when grown on media supplemented with hemin was reported (219). Distinguishing these two species from nutritionally variant streptococci is difficult. Schemes for separating the members of this genus from the other gram-positive cocci have appeared (68, 80, 198). On the basis of 16S rRNA studies of two Gemella-like isolates from clinical sources, a new genus, Dolosigranulum, has been proposed (4).

(ii) Helcococcus. Only one Helcococcus species has been described, Helcococcus kunzii (43). The isolates are similar to those of Aerococcus spp. but differ from them with respect to growth on blood agar. Helcococcus isolates grow slowly and form small nonhemolytic viridans-like colonies; the growth is greatly stimulated by serum or Tween 80. Organisms have been isolated from mixed cultures from wounds, commonly foot wounds, leg ulcers, and a purulent breast mass (43). Peel et al. (180) have reported the isolation of H. kunzii in pure culture from an infected sebaceous cyst. Isolates of the species are PYR positive and LAP negative, are susceptible to vancomycin, and have a G+C content of 29.5 to 30 mol%. Members of the genus were phylogentically only remotely related to aerococci, streptococci, and other catalase-negative, facultatively anaerobic gram-positive taxa with a low G+C content (43).

(iii) Pediococcus. The genus Pediococcus, with a G+C content of 34 to 43 mol%, contains five species associated with lactic acid fermentations of vegetables, grain mashes, and cheese (P. acidilactici, P. damnosus, P. dextrinicus, P. parvulus, and P. pentosaceus). Isolates are positive for the LAP and bile-esculin tests but negative for PYR. Some strains are used as starter cultures in silage and various fermented sausages (48). One species (P. damnosus) is an important spoilage agent of beer (186); two species (P. acidilactici and P. pentosaceus) have been isolated from human specimens. The phylogenetic interrelationships within the genus have been discussed by Collins et al. (46). The facts that these organisms are intrinsically resistant to vancomycin and that most strains isolated from human infections have the group D antigen add to the need for their correct identification. Differentiation within the genus is based on tests used in the clinical laboratory, such as formation of acid from carbohydrates and the ability to grow in 6.5% NaCl, and in food microbiology, in particular the ability to grow at low pH, in different beers, and in the presence of hops. Schemes for separating the taxon from others and for differentiation within the genus have been provided (68, 69, 198).

(iv) Tetragenococcus. The genus Tetragenococcus contains one species, Tetragenococcus halophilus (46); this species was previously included in the genus Pediococcus but is susceptible to vancomycin. It resembles Pediococcus in being positive for the LAP and bile-esculin tests but negative for PYR; it has a G+C content of 34 to 36 mol%. The genus represents a distinct line of descent quite separate from aerococci and pediococci (46). It is important in the fermentation of soy moromi to produce soy sauce (220).

(i) Aerococcus. The history, taxonomy, and clinical significance of the genus Aerococcus were summarized by Facklam and Elliott (68). Isolates assigned to this genus show a weak reaction in the catalase test but do not contain cytochrome; they have a G+C content of 35 to 40 mol%.

(ii) Alloiococcus. The genus Alloiococcus contains only one species, Alloiococcus otitis (3), renamed A. otitidis (231). Isolates assigned to this genus are obligately aerobic, slow-growing cocci that give a weak reaction in the catalase test but do not contain cytochrome. Members of the genus have a G+C content of 44 to 45 mol%. Isolates grow very slowly in 6.5% NaCl, are LAP and PYR positive, are susceptible to vancomycin, do not produce gas from glucose, and do not form acid from carbohydrates. Bosley et al. (29) have described 19 isolates of this species from ear fluid samples. Heavy growth occurred in brain heart infusion broth supplemented with 0.07% lecithin and 0.5% Tween 80. Although the species falls within the low-G+C content of gram-positive bacteria, it is genealogically distinct from aerococci and streptococci (3).