INTRODUCTION

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Gram-positive anaerobic cocci (GPAC) are better known to most bacteriologists as peptococci or peptostreptococci; most clinical isolates are identified to species in the genus Peptostreptococcus. GPAC are a major part of the normal human flora and are frequently recovered from human clinical material (35, 84, 136, 251); they constituted 24 to 31% of all isolates in four surveys of anaerobic pathogens (28, 138, 196, 295). However, they have been little studied (186, 196, 262, 273) and their importance as human pathogens is insufficiently recognized. Many factors have contributed to this lack of interest. The classification is unsound and has been confused by many loosely defined taxa (136, 166, 167, 190); of the five species most frequently reported from clinical infections, two are known to be genetically heterogeneous (136, 167, 190, 202). Routine diagnostic laboratories can rarely give adequate resources to the isolation of slowly growing anaerobes; until recently, identification schemes relied on the availability of gas-liquid chromatography (GLC) (136, 251, 254) and therefore were impractical for most laboratories. From the clinician's standpoint, most GPAC are cultured from polymicrobial infections (35, 129, 196, 273), often with well-recognized pathogens such as microaerophilic streptococci or fusobacteria, and their isolation is relatively unimportantthe patient has usually been given therapy that is effective against anaerobes long before the GPAC are cultured. Therefore, there has been little laboratory or clinical interest in the field.

Several recent advances have made the subject somewhat less impenetrable. Taxonomic studies involving nucleic acid techniques (67, 142, 166, 167, 188) should soon lead to a revision of the classification, better identification schemes and, eventually, a closer association between taxon and clinical context. With the introduction of preformed enzyme kits (59, 192-194), a simple, relatively rapid method of identification is now available for use in routine laboratories. Surveys of the clinical importance of GPAC have defined more clearly the pathogenic potential of different species (14, 33, 35, 196). The importance of Peptostreptococcus micros in intraoral infections is now recognized (110, 185, 221, 267). Studies of the pathogenicity of P. magnus have led to the description of virulence factors, some of which may have industrial applications (149, 158, 205, 286). This review discusses what is known of the biology of GPAC, describes recent advances, and defines areas in particular need of further study.

The study of GPAC has suffered from a proliferation of synonyms; at various stages, the terms "anaerobic streptococcus," "anaerobic coccus," "Peptococcus and Peptostreptococcus," and "anaerobic gram-positive coccus" have been used to describe them. As the classification has evolved, the precise meaning of some of these terms has changed; for instance, until 1974 both Peptococcus and Peptostreptococcus included species of microaerophilic streptococci (134). At present, most species of clinical importance are classified in the genus Peptostreptococcus (35, 84, 136). However, this review will use the acronym GPAC; not only is it shorter, but the genus Peptostreptococcus is a phylogenetically heterogeneous taxon and will undergo radical revision (67, 167, 188). Many studies have preferred to use the term GPAC (85, 190, 192) or AGPC (anaerobic gram-positive coccus) (82, 84, 117, 142, 261). Furthermore, the term GPAC is useful in the routine diagnostic laboratory, because it gives a broad morphological description of organisms isolated under specified atmospheric conditions; it is a term of convenience, nothing more. Watt and Jack (272) defined anaerobic cocci as "cocci that grow well under satisfactory conditions of anaerobiosis and do not grow on suitable solid media in 10% CO₂ in air even after incubation for 7 days at 37°C." This is a valuable working definition, which will be used in this review.

These organisms have been fair game for amateur taxonomists. Hare (114)

An awareness of the many changes in nomenclature (Table 1) makes it easier to assess previous identification schemes and old clinical reports. The classification has always been very unsatisfactory; at least 40 species have been described (114), but many were poorly defined. Some species, for instance Peptococcus activus, were not included in the 1980 Approved Lists of Bacterial Names because no strains were available (136, 237). Others are now recognized as synonyms, e.g., Peptococcus aerogenes for Peptostreptococcus asaccharolyticus (136, 237), or have been placed on the list of nomina rejicienda, for instance, Peptococcus anaerobius, now known as P. magnus (136).

The genera Peptococcus and Peptostreptococcus were described by Kluyver and van Niel in 1936 (154). They were separated on the basis of morphological characteristics: peptococci, the anaerobic equivalent of staphylococci, were arranged in clumps and were assigned to the tribe Micrococcaceae, whereas peptostreptococci, as anaerobic streptococci, were arranged in chains and were placed in the tribe Streptococcaceae. This distinction is influenced by so many variables, for instance the composition of the medium, that it is unreliable (84, 136); however, the scheme lasted until the application of DNA hybridization techniques in 1983 (87). A later scheme by Prevot in 1948 (218), based on microscopic appearance, divided GPAC into eight genera; the identification to the species level was based on criteria such as production of indole, liquefaction of gelatin, and growth in litmus milk. Most of these tests are now disregarded. A different approach was taken by Hare and coworkers (114, 116, 263). Their classification of anaerobic cocci used fermentation and gas production from five carbohydrates and five organic acids, combined with serological tests, from which they distinguished 10 groups of anaerobic cocci. Since they found that the results of carbohydrate fermentation reactions varied with the quantity of fatty acids in the medium, the tests were standardized by careful definition of their basal media. These authors were able to place 90% of nearly 400 human strains in well-defined groups (114). Commendably, they did not add more Latin names to a classification already burdened by loosely defined species. Hare's scheme unfortunately included both microaerophilic and overtly aerobic groups, but its merits have been demonstrated by the identification of Hare group III with P. hydrogenalis (190) and the recent proposal of Hare group VIII as "P. octavius" (188).

In 1971, Rogosa (224) united three genera, Peptococcus, Peptostreptococcus, and Ruminococcus, in a new family of strictly anaerobic gram-positive cocci or coccobacilli, the Peptococcaceae; he added Sarcina in 1974 (225). He noted that peptococci and most peptostreptococci could use the products of protein decomposition as their sole energy source whereas ruminococci and sarcinae required the presence of carbohydrates for fermentation. He characterized ruminococci by the digestion of cellulose and the fermentation of cellobiose and sarcinae by the conspicuous arrangement of cells in packets. He noted the heterofermentative ability of most peptostreptococci and proposed that this property was of sufficient taxonomic importance that it should be used to distinguish them from the streptococci, which were characterized by the homofermentation of carbohydrates to form lactic acid. Holdeman and Moore (134) therefore transferred Peptococcus morbillorum, Peptococcus constellatus, and Peptostreptococcus intermedius to the genus Streptococcus, thus removing all microaerophilic species from the Peptococcaceae; this important revision was later supported by analysis of cell wall structure (276) and nucleic acid studies (142). Holdeman and Moore also described a new genus from human feces, Coprococcus, which they assigned to the family Peptococcaceae; coprococci, like ruminococci, used fermentable carbohydrates for growth, but they formed different metabolic end products, notably butyric acid. When the Approved Lists of Bacterial Names were published in 1980 (237), the present classification was beginning to take shape; seven species were recognized in the genus Peptococcus, and four were recognized in the genus Peptostreptococcus (Table 1).

The introduction of nucleic acid techniques in the 1980s soon led to a major revision of the classification (87) but regrettably little consensus, because the two investigations employing DNA-DNA hybridization techniques (87, 142) came to conflicting conclusions. Ezaki et al. (87) studied 65 strains, mainly from human clinical specimens, which they characterized by standard biochemical methods (carbohydrate fermentation reactions, volatile fatty acid [VFA] patterns detected by GLC, and tests for enzyme activity with the API ZYM commercial kit) and taxonomic techniques (cellular fatty acid analysis, determination of guanine-plus-cytosine content of DNA, and DNA-DNA homology experiments). They noted that the G+C content of Peptococcus niger, the type strain of the genus Peptococcus, was 51 mol%, but for other species in the genus Peptococcus, they recorded a G+C content of 29 to 34 mol%, a range they considered to be unacceptably large for one genus. Comparison of DNA-DNA homology values and cellular fatty acid profiles led them to reclassify four species of Peptococcus in the genus Peptostreptococcus, retaining only P. niger in the genus Peptococcus.

The data from the study of Ezaki et al. (87) finally disposed of the spurious distinction between peptococci and peptostreptococci based on cellular arrangement, and the new scheme was adopted in Bergey's Manual of Systematic Bacteriology in 1986 (136). However, this revision was almost immediately challenged by a study of Huss et al. (142), which used similar techniques. The results of G+C contents and DNA-DNA hybridization values often differed between the two studies; for instance, Ezaki et al. recorded a binding value of 46% between ATCC 14963^T and ATCC 9321^T (the type strains of P. asaccharolyticus and P. prevotii, respectively), which indicated a distinct relationship, whereas Huss et al. detected no DNA homology between these strains. These studies also recorded significantly different values for the G+C contents of certain key strains, for instance, ATCC 14963^T and ATCC 29427^T, the type strains of P. asaccharolyticus and P. indolicus, respectively. Huss et al. assessed the degree of relatedness at the genus level by use of DNA-rRNA cistron similarity studies; they concluded that P. anaerobius DSM 20357 (not the type strain) was more closely related to the type strains of Eubacterium tenue and Clostridium lituseburense than to other species of GPAC. The conclusions of Huss et al. were not generally accepted at the time but they have been confirmed by later studies (67, 82, 211).

The application of nucleic acid techniques has led to the recognition that several species are only very remotely related to most species of GPAC. Analysis of nucleic acid relatedness data and cell wall peptidoglycan structure revealed that Peptococcus saccharolyticus should be transferred to the genus Staphylococcus (150). Most strains of Staphylococcus saccharolyticus will grow only under anaerobic conditions on primary isolation but on subculture will grow in an aerobic atmosphere including 10% CO₂ (81); S. saccharolyticus is therefore not an anaerobic coccus by the definition of Watt and Jack (272), but it is a potential source of confusion. Peptostreptococcus parvulus was reclassified in the genus Streptococcus by Cato (57), but recent comparisons of 16S rRNA sequence data have led to its reassignment to a new genus, Atopobium, with two species that were previously placed in the genus Lactobacillus (68). Peptostreptococcus heliotrinreducens is also only very remotely related to other GPAC (167, 190) and should probably be classified with oral, asaccharolytic species at present in the genus Eubacterium (102, 216, 268).

Within the Peptococcaceae, P. magnus, P. micros, and P. anaerobius are generally accepted as being phylogenetically valid species. Peptococcus glycinophilus was shown to be a synonym of P. micros by Cato et al. (58), who compared the soluble cell protein patterns of the type strains by polyacrylamide gel electrophoresis (PAGE). The classification of Peptostreptococcus productus is under active review. P. productus is a strongly saccharolytic species with a distinctive oval cell morphology and a G+C content of 44 to 45 mol%, much higher than that of other peptostreptococci. Recently it was reclassified in the genus Ruminococcus (82). However, this study did not examine the type species of the genus Ruminococcus; other workers (219, 289) have demonstrated at least two unrelated groups in the genus. Until there is a definitive revision of the classification of ruminococci, Willems and Collins (289) have recommended that P. productus be retained in its present taxonomic position.

Several species of butyrate-producing GPAC have recently been described (83, 166, 188); they have all been assigned to the genus Peptostreptococcus as a placement of convenience. P. asaccharolyticus and P. prevotii, the two butyrate-producing species commonly reported from human pathological material, have long been recognized as genetically heterogeneous (87, 117, 166, 202). Until recently, standard identification schemes (128, 133, 254) assigned all indole-positive strains of GPAC to the species P. asaccharolyticus or P. indolicus; however, the type strains of both species are asaccharolytic. P. hydrogenalis was described by Ezaki et al. (83) from saccharolytic, indole-positive strains previously identified as P. asaccharolyticus; Hare group III (114, 116, 192, 263) is a synonym of P. hydrogenalis (190, 193). Another group of indole-positive, saccharolytic strains, the `trisimilis' group, differs from P. hydrogenalis in whole-cell composition (190) and biochemical activity (186, 192) and probably merits species status. A group of strains biochemically similar to P. asaccharolyticus but differing in 16S rRNA sequence and whole-cell composition has recently been proposed as "Peptostreptococcus harei" (188, 190).

The species P. prevotii is still often used as a loose description for all strains of indole-negative, butyrate-producing GPAC (203), although Rogosa recognized that the group was heterogeneous and recommended that P. prevotii be placed on the list of nomina rejicienda in 1974 (225). Other species in this group have now been described: P. tetradius in 1983 (87) and P. vaginalis, P. lactolyticus, and P. lacrimalis in 1992 (166). However, there is strong evidence (166, 167, 190, 192) that related species of clinical importance still await formal description.

The present situation is that the genus Peptostreptococcus contains 13 recognized species, with a G+C range from 28 to 37 mol%, except for P. productus (44 to 45 mol%); there is now but one species in the genus Peptococcus, Peptococcus niger, which has a G+C content of 50 to 51 mol% (84). A recent study (190) compared conventional techniques with whole-cell composition by pyrolysis mass spectrometry (PyMS) (Fig. 1); it has led to the proposal of three new species: "P. harei," "P. ivorii," and "P. octavius" (Fig. 2) (188). Several further undescribed groups of strains formed distinct clusters by both techniques; these included GAL (-galactosidase) strains (192) and the `trisimilis' group, both of which probably merit species status.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Clustering of 26 reference and 101 clinical strains of GPAC on the basis of whole-cell composition as assessed by PyMS. Numbered but unnamed clusters did not contain type strains of recognized or proposed species. Adapted from reference 190 with permission of the publisher.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Phylogenetic tree constructed by the neighbor-joining method, showing the position of Peptostreptococcus species within Clostridium rRNA clusters XI, XIII, and XIVa (67). Significant bootstrap values (90% and higher), expressed as a percentage of 500 replications, are indicated at the branching points. Adapted from reference 188 with permission of the publisher.

Recent studies based on comparisons of 16S rRNA sequence data have confirmed that Peptococcus niger (the type species of the family Peptococcaceae), is not related to other GPAC (67, 167), and they agree with those of Huss et al. (142) that P. anaerobius, the type species of the genus Peptostreptococcus, is more closely related to some members of the genus Clostridium than to other GPAC (67, 82, 163, 211). Li et al. (167) have recommended that the genus Peptostreptococcus be divided into at least seven genera. The most comprehensive survey, by Collins et al. (67), compared over 100 reference strains of low-G+C content gram-positive anaerobes, mainly type strains of the genus Clostridium. Collins et al. proposed splitting clostridia and related groups into 19 clusters based on 16S rRNA gene analysis. Of the 11 species in the genus Peptostreptococcus in this survey, 9 were assigned to cluster XIII, which also contained Helcococcus kunzii but no recognized species of Clostridium. Cluster XI contained one species of GPAC, P. anaerobius, with E. tenue and 18 species of Clostridium. P. productus was assigned to cluster XIVa with Streptococcus hansenii and species of Clostridium, Coprococcus, and Ruminococcus. Sarcina ventriculi and Sarcina maxima fell into a redefined genus Clostridium (cluster I) and were found to be closely related to Clostridium perfringens (288). Atopobium parvulum was placed in the Actinomycetales. A possible revised structure for GPAC based on these data is presented in Table 2.

It is evident that the genus Peptostreptococcus requires radical revision (67, 163, 167). The continual changes in nomenclature are extremely confusing, and the constant addition of new species, although essential, makes the situation even more complicated for those not familiar with the field. Furthermore, several clinically important species still await formal description (166, 190, 192). It is difficult to escape the conclusion that the lack of a sound and stable classification has substantially contributed to the neglect of GPAC.

Proper collection and transport of specimens is crucial for recovery of anaerobes in the laboratory. Finegold (90)

Most species in the genus Peptostreptococcus have been isolated from human clinical material and are not extremely oxygen sensitive; they are strict anaerobes in that they need an anaerobic atmosphere for multiplication, but the very limited data available suggest that many clinical strains are moderately aerotolerant. Fourteen clinical strains studied by Tally et al. (258) all survived exposure to atmospheric oxygen for 8 h; nine (63%) survived more than 72 h. Investigations (187) of four fresh clinical isolates of P. magnus and P. micros revealed that 1% of cells were still viable after exposure to air for 48 h.

Aspirates or tissue are generally considered to be the best specimens for culture of obligate anaerobes; swabs are less satisfactory (172). Specimens should be delivered to the laboratory as quickly as possible and must not be allowed to dry out, because moisture is important in maintaining viability (20). Anaerobic transport systems are necessary to optimize recovery rates (6, 20, 84, 243) and are essential for swabs; Mangels (172) presents an excellent overview of the subject. According to Watt and Smith (273), growth is best in the temperature range from 35 to 37°C and is enhanced by the presence of 10% CO₂ in the atmosphere; a palladium catalyst should be present to remove traces of oxygen.

The nutritional requirements of Peptostreptococcus spp. have been very little studied and would repay investigation; given the heterogeneity of the genus, it is difficult to make generalizations. Commercial nonselective solid media vary in their ability to support the growth of fresh clinical isolates; in one study (119), Fastidious Anaerobe Agar (Lab M, Bury, United Kingdom) consistently gave the best growth. Sodium oleate (Tween 80; final concentration, 0.02%) enhances the growth of some species (114) but is not essential; neither are vitamin K nor hemin supplements (136). Satellitism of some fresh clinical isolates of the GAL group has been noted around colonies of P. magnus on unenriched blood-containing media (187). Solid media that do not contain blood, such as Gifu anaerobic medium (Nissui, Tokyo, Japan) can support growth well (84). Ezaki et al. (84) stated that prereduced media are necessary for growth, but this is not my experience (187). Viability in chopped-meat broth depends on the commercial source; some preparations appear not to support growth at all, but most strains will survive in high-quality products for several months, if not years, at room temperature on the open bench, providing a very convenient method of short-term storage (187). For long-term storage, Holdeman Moore et al. (136) recommend lyophilization of cultures in the early stationary phase of growth in a medium containing less than 0.2% fermentable carbohydrate. GPAC retain viability well at 80°C or in liquid nitrogen (136).

Ruminococci, coprococci, and sarcinae are more sensitive to oxygen and will grow only under well-maintained anaerobic conditions (84). Carbohydrates are essential for the growth of ruminococci and sarcinae and stimulate the growth of coprococci (84). Most strains will not grow under the conditions encountered in routine laboratories; Bryant (53) and Ezaki et al. (84) give excellent guidelines for their isolation and culture.

Unfortunately, there have been few attempts to develop selective media for GPAC. This is a subject that deserves investigation; because most clinical isolates of GPAC grow relatively slowly on standard media and are usually present in mixed culture, they are frequently overgrown by other organisms, and many clinical studies will therefore give a falsely low estimate of their frequency. However, because GPAC are heterogeneous, a single medium is unlikely to support the growth of all representatives yet be reasonably selective. GPAC are very susceptible to most antibiotics, including, quite often, neomycin and polymyxin (187); they are usually resistant to bicozamycin (241, 270), but this broad-spectrum agent may no longer be available. Wren (292) showed that nalidixic acid-Tween blood agar (10 mg of nalidixic acid per liter, 0.1% Tween 80) gave better isolation than neomycin blood agar (75 mg of neomycin per liter) but recommended that a combination of different media be used to maximize recovery rates. Petts et al. (213) reported that oxolinic acid was superior to nalidixic acid for suppression of staphylococci but permitted the growth of nonsporing anaerobes, including GPAC. Ezaki et al. (84) recommended phenylethylalcohol agar (Difco, Detroit, Mich.), but Turng et al. (265) reported that 79% of strains of P. micros did not grow on PEA. Recently, Turng et al. (265) have described P. micros medium (PMM), a selective and differential medium for P. micros, which contains colistin-nalidixic acid agar (Difco), a selective base for gram-positive cocci, supplemented with glutathione and lead acetate. Strains of P. micros can use the reduced form of glutathione to form hydrogen sulfide, which reacts with lead acetate to form a black precipitate under the colony.

The absence of clear guidelines for characterisation [of GPAC] is a constant source of discouragement to the clinical microbiologist and this has held back studies on the role of these organisms in health and disease. Watt and Jack (272)

These comments were written in 1977; the situation has improved since but is still far from satisfactory; since there is no simple, accessible, and generally accepted identification scheme, few routine laboratories identify clinical isolates to the species level.

Grouping a clinical isolate can be surprisingly difficult; the microbiologist must decide whether an organism is a strict anaerobe and whether it is a coccus or a rod. In addition, many strains retain Gram's stain poorly and therefore can appear gram negative. GPAC must be distinguished from microaerophilic organisms, most of which, from human clinical specimens, will be streptococci. Other species which can be misidentified as GPAC include Staphylococcus saccharolyticus and Gemella morbillorum. Microaerophilic strains of these species may appear only on anaerobic plates on primary incubation but will grow in 10% CO₂ on repeated subculture. However, routine laboratories cannot wait 7 days to detect growth in an aerobic environment; a simple, reliable test is needed to distinguish strict anaerobes from microaerophiles. A cheap but effective technique is to apply a 5-µg metronidazole disc to the edge of the inoculum; two studies (192, 272) have reported that all strains of GPAC examined after incubation for 48 h showed zones of inhibition of 15 mm or greater whereas microaerophilic strains showed no zones. Metronidazole-resistant strains of GPAC have been reported (78, 230, 236, 280), but they appear to be rare.

Streptococci and gemellae are strongly saccharolytic, unlike most GPAC, and produce large quantities of lactic acid, which can be detected by GLC. S. saccharolyticus is a normal member of the skin flora; strains ferment several carbohydrates, produce urease and catalase, and reduce nitrate (81, 84). Some GPAC, particularly strains of P. asaccharolyticus, decolorize rapidly with Gram's stain and can be confused with gram-negative anaerobes such as veillonellae; testing for vancomycin susceptibility with a 5-µg disc appears to be a simple and reliable method of separation (129, 192). The cell morphology of older cultures of GPAC can be very irregular, with many coccobacillary and rod-like forms, and can lead to possible confusion with lactobacilli or clostridia; P. anaerobius is particularly prone to pleomorphism, but its close relationship to some species of clostridia (67) shows that morphological distinctions are often arbitrary. The problem is similar for the facultative catalase-negative, gram-positive cocci and is well covered by Facklam and Elliott (88); the distinction relies considerably on the microbiologist's judgement.

Early identification schemes depended on microscopic appearance, colonial morphology, ability to form gas from carbohydrates and organic acids, and carbohydrate fermentation reactions (114, 116, 218, 225). However, these tests proved to be of limited value for a group of bacteria that are often pleomorphic and usually asaccharolytic.

Gas-liquid chromatography. In the 1970s, GLC was introduced for the detection of fatty acid by-products of metabolism (133, 254). Unfortunately, GPAC produce a very limited range of VFAs, but GLC can be used to classify them into three groups (Table 3): an acetate group containing several species, notably P. magnus and P. micros, which produce acetic acid or no VFAs at all; a large butyrate group, containing eight recognized species and a large number of unrecognized groups, which product butyric acid as their major terminal VFA; and a caproate group, whose members produce large quantities of longer-chain VFAs. The most important species in this last group is P. anaerobius, the only species of GPAC to produce a major terminal peak of isocaproic acid; GLC is a reliable method for its identification (133, 192). GLC is also useful for identifying the rarely isolated Peptococcus niger and the recently proposed "P. octavius" (188), which produce n-caproic acid, and "P. ivorii," also recently proposed (188), which produces a terminal peak of isovaleric acid. Few workers except Ezaki et al. (87) have studied production of nonvolatile fatty acids (NVFAs), but it appears to be of little value except to exclude strains of streptococci, which, by definition, form large quantities of lactic acid (113). The results of GLC correlate very closely with biochemical tests (192) and whole-cell composition (190). Both liquid and solid media can be used for the detection of VFAs (285). Unfortunately, GLC is time-consuming, and the capital equipment is costly; although it is beyond the capabilities of most diagnostic laboratories, most identification manuals (133, 136, 251, 254) have recommended schemes in which GLC is an essential step.

Standard identification schemes. Further identification has relied mainly on the fermentation of carbohydrates and other standard bacteriological tests such as nitrate reduction and urease production (129, 254), which may be valuable for other groups of bacteria but are of little use for GPAC. Most schemes (84, 129, 133, 251, 254) include testing for indole production, but its reliability is doubtful (190, 192). Since relatively few species of GPAC produce acid from carbohydrates (85, 136), many strains have been characterized on the basis of negative reactions (129, 254). Some recent schemes (129, 251) still base differentiation of P. magnus and P. micros on cell size, a highly unsatisfactory method of discrimination; its use requires the prior application of GLC and may lead to confusion with P. heliotrinreducens and asaccharolytic eubacteria (187). Colony morphology is a useful guide, particularly for many strains of P. micros, which exhibit a characteristic halo (195, 254), but this feature is not completely reliable (192). To quote a review from 1987 (262), "simple and reliable methods for the differentiation and taxonomy of these organisms have not yet been described."

Preformed enzyme kits. Identification methods should be appropriate to the metabolism of the organisms concerned. It has long been accepted that most species of GPAC are strongly proteolytic and use the products of protein decomposition as a major energy source (154, 225). In 1985, Ezaki and Yabuuchi (85) examined 77 strains of GPAC, comparing their amidase and oligopeptidase activities with VFA production and carbohydrate fermentation reactions. These tests were performed in an aerobic atmosphere at 37°C with a 4-h incubation period, conditions which are much more convenient for the routine diagnostic laboratory than those for carbohydrate fermentation tests. They found that the carbohydrate fermentation reactions were almost always negative but that most groups studied produced numerous positive peptidase reactions; in particular, P. magnus and P. micros were reliably differentiated by several tests. Peptidase activity correlated well with VFA production. These authors also examined 21 strains of microaerophilic streptococci and S. saccharolyticus, which they could easily distinguish by their strong saccharolytic activity and generally weak proteolytic activity. Furthermore, they were able to differentiate "classic" strains of P. asaccharolyticus from a group of saccharolytic indole-positive organisms, which they later described as P. hydrogenalis (83). This study showed that proteolytic enzyme profiles (PEPs) could distinguish clearly and reproducibly among recognized species of GPAC; it also revealed the value of PEPs for distinguishing undescribed groups of GPAC from recognized taxa.

Other techniques. Few other identification tests are of value. Graves et al. (104) described a simple but useful method for the provisional identification of P. anaerobius; they showed that strains of P. anaerobius are susceptible to sodium polyanethol sulfonate (SPS; Liquoid) but that other species of GPAC are resistant. Later evaluations (192, 284) have confirmed that this test is both sensitive and specific. SPS paper discs are commercially available. Other antibiotic susceptibility tests, for susceptibility to novobiocin (296) or as used for gram-negative anaerobic rods (255), do not appear to be helpful.

Virtually the only source of anaerobes participating in infection is the indigenous flora of mucosal surfaces and, to a much lesser extent, the skin. Finegold (90)

GPAC are part of the normal flora of the mouth, upper respiratory and gastrointestinal tracts, female genitourinary system, and skin (7, 84, 126, 201, 251). Since many commensal groups are rarely isolated from clinical specimens (84, 91, 183), there has been little stimulus to study them. Consequently, many commensal organisms cannot be assigned to recognized species and few reports of the normal flora have attempted identification to the species level. Analysis is complicated by conflicting data; for example, reports disagree about the presence of P. anaerobius in the oral and vaginal flora (123, 126, 136, 201, 241).

GPAC constitute 1 to 15% of the normal oral flora (201, 251, 253); they are found mainly in plaque and the gingival sulcus. P. micros is usually considered to be the predominant species of GPAC (136, 221), although, in a recent study (168) it was not isolated from gingival samples of 25 individuals with healthy sulci. Most workers would agree that P. anaerobius is part of the gingival flora (201, 235), and P. magnus has been reported to be a member (241), but Holdeman Moore et al. (136) stated that neither are members of the normal flora. It is possible that these species are regularly present but in undetectable numbers; the threshold for detection of GPAC is relatively high because of the lack of a selective medium.

The gastrointestinal tract hosts a wide variety of GPAC, including most recognized species of Peptostreptococcus and poorly studied groups such as Coprococcus, Ruminococcus, and Sarcina (7, 71, 91, 136, 183). Using optimal anaerobic techniques, Finegold et al. (91) and Moore and Holdeman (183) showed that P. productus is one of the commonest organisms in the gastrointestinal flora. Fecal composition is related to age (201, 247) and diet (71, 91). Finegold et al. associated high counts of P. micros with American rather than Japanese diets, while Crowther (71) reported that Sarcina ventriculi was common in the gastrointestinal tracts of vegetarians, in counts of up to 10⁸/g of feces, but very infrequent in persons whose diets contained animal products.

Large numbers of GPAC can be found in the female genitourinary tract; counts vary with physiological processes such as the stage of the menstrual cycle and pregnancy (177), making assessment of the normal flora extremely complicated. P. tetradius, P. lactolyticus, and P. vaginalis were first described from vaginal discharges (87, 166); P. anaerobius, P. asaccharolyticus, P. hydrogenalis, P. magnus, P. micros, P. prevotii, and Peptococcus niger have also been isolated (11, 136, 201). Bartlett et al. (11) reported carriage rates of 40% and mean counts of 10^8.7/g in normal vaginal secretions; the most common species were P. magnus, P. asaccharolyticus, and P. prevotii. In a valuable recent study, Hillier et al. (126) reported carriage rates in pregnant women of 92% and mean counts of 10^4.2 CFU/ml; the most common species, in terms of both carriage rates and counts, were P. asaccharolyticus followed by P. magnus. Hill et al. (123) noted higher counts of GPAC in the vaginal flora of prepubertal girls than in the flora of normal adult women; the major species were P. tetradius and P. anaerobius. Unfortunately, neither Hill nor Hillier reported finding strains of P. vaginalis because satisfactory methods for its identification were not available at the time.

The skin flora contains GPAC (7, 90, 251), but its composition at the species level appears to have been little studied. Murdoch et al. (196) suggested that P. magnus, P. asaccharolyticus, and P. vaginalis are probably components because they can often be isolated from superficial wound infections.

It is clear that few investigations have been carried out on this difficult subject. Further studies would be very welcome; they should aim to characterize as many isolates as possible to the species level and to carry out quantitative counts when appropriate. The ability to identify recently described species, rather than to place strains in poorly defined species such as P. prevotii is essential.

GPAC are commonly present in human clinical specimens; data from four surveys of anaerobic infections (28, 138, 196, 295) are consistent that they account for about 25 to 30% of all anaerobic isolates (Table 5). They can be cultured from a wide variety of sites, particularly abscesses and infections of the mouth, skin and soft tissues, bone and joints, and upper respiratory and female genital tracts (Fig. 3) (35, 90, 92).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Sites of pathology for species of GPAC. Species of major importance are underlined, ? denotes uncertain pathology.

Most infections involving GPAC are polymicrobial (35, 90), particularly abscesses and those developing from mucocutaneous surfaces. However, there are many instances of their isolation in pure culture; most relate to P. magnus (14, 32, 38, 51, 64, 72, 77, 94, 141, 173, 196, 207, 214, 217, 231), but there are also reports of P. anaerobius (181), P. asaccharolyticus (196), P. indolicus (13), P. micros (210, 278), P. vaginalis (189, 196), and "P. harei" (188).

When a report employs the term "anaerobic streptococcus," it will be used in the following discussion, because this term is likely to include microaerophilic streptococci such as Streptococcus intermedius and Streptococcus constellatus, which were removed from the genus Peptostreptococcus in 1974 (134). Otherwise the term GPAC is used, with the caveat that some organisms formerly included in the group would not be described as such nowadays. Data from mucocutaneous sites will be presented first, followed by data from sites that are usually sterile.

Most dental infections are polymicrobial but are dominated by anaerobic organisms (260). The crevicular fluid is probably the major source of nutrients in the subgingival ecosystem; it contains relatively high concentrations of glycoproteins and peptides but low concentrations of carbohydrates; consequently, the flora is largely asaccharolytic (235). P. micros would appear well adapted to this serum-rich, oxygen-depleted environment, and many studies have associated it with periodontitis, but it is not always detected at active sites. Haffajee et al. (109) isolated it from only 5 of 33 adults with active periodontal disease, but in these patients it constituted 5 to 10% of the mean bacterial count. Moore et al. (184) reported P. micros counts of >1% in the supragingival flora of 21 young adults with severe periodontitis; it was present in 48% of subgingival samples, more often than at supragingival sites, and raised subgingival counts correlated with gingival inflammation. Likewise, Socransky et al. (245) isolated P. micros from 24% of subgingival plaque samples and linked it to gingival redness, deeper pockets, and loss of periodontal attachment. However, in a related study, Haffajee et al. (110) associated P. micros with deep progressing pockets but not raised subgingival temperature. It has been suggested that periodontal disease is episodic rather than a continual progression; a longitudinal study by Rams et al. (221) found a higher prevalence in patients with active disease (47%) than in patients with inactive disease (14%). In a prospective study of adults with chronic periodontitis by Moore et al. (185), P. micros, Campylobacter rectus, and Fusobacterium nucleatum were the only species isolated from all patients, but no quantitative difference was detected between the flora of active and inactive sites. A recent comparison of the subgingival microflora of mobile and nonmobile teeth (103) reported higher counts of C. rectus and P. micros around mobile teeth; the authors suggested that tooth mobility might create a subgingival environment conducive to overgrowth by periodontal pathogens.

One approach has been to identify organisms, termed risk markers, which occur more frequently than others in active disease or whose presence is linked to a less favorable outcome. Dzink et al. (76) compared the cultivable subgingival flora from sites with active periodontal disease and inactive sites; they reported that a site was more likely to be active if Bacteroides forsythus, Porphyromonas gingivalis, P. micros, C. rectus, or Prevotella intermedia was isolated. Another approach has been to associate groups of potential pathogens, termed species clusters, with dental disease. Socransky et al. (244) associated P. micros with the presence of several species, including Prevotella melaninogenica and P. gingivalis, but found a very strong negative correlation with another putative periodontal pathogen, Bacteroides forsythus. This observation may help to explain why P. micros is not always present in patients with active periodontitis.

Periodontitis can dramatically worsen in patients with human immunodeficiency virus (HIV). Rams et al. (220) reported that although the subgingival flora of 14 patients with HIV was usually qualitatively unchanged, counts of P. micros from normal sites were raised and those from infected sites were very high; other putative pathogens such as C. rectus were also more common. The authors suggested that P. micros can act as an opportunist pathogen in HIV-positive patients.

Dental implants may be susceptible to plaque-induced gingivitis, which can progress to peri-implantitis. Alcoforado et al. (1) cultured a wide range of organisms from failing implants; P. micros was the most common isolate, accounting for 24% of the cultivable flora. Rosenberg et al. (226) reported major differences between the flora of implant failures attributed to trauma and those due to infection. Implant sites with traumatic failure exhibited a microflora consistent with periodontal health and did not yield P. micros. In contrast, implant sites with infectious failure yielded a wide variety of periodontal pathogens; P. micros was isolated from 75% of sites and made up more than 10% of the cultivable flora.

Thus, there is good evidence that P. micros is associated with periodontal infections, but it is extremely difficult to prove causation in a microbial flora as complex as that of the subgingival plaque. The development of techniques such as DNA probes (108, 297, 298) may help to resolve this problem. The evidence implicating other GPAC is much weaker. In a cross-sectional study by Moore et al. (184), P. anaerobius was a major component (>1%) of the supragingival flora but was less common in the subgingival flora. However, in a later report, the same workers (182) associated P. anaerobius with both gingivitis and periodontitis. Wade et al. (269) isolated P. micros, P. anaerobius, P. asaccharolyticus, and rarely P. productus from subgingival plaque in patients with chronic periodontitis.

P. micros is often implicated in other oral infections (260). In a quantitative study of 10 endodontic abscesses by Williams et al. (290), P. micros was the most common organism (accounting for 18% of all isolates) and was the predominant organism in five abscesses. A quantitative study of 50 acute dentoalveolar abscesses by Lewis et al. (164) yielded a mainly anaerobic flora; along with "Streptococcus milleri," Prevotella oralis, and Prevotella melaninogenica, GPAC (30%) were the most common organisms; unfortunately, they were not identified to the species level. Pericoronitis is an acute condition in which the soft tissues investing the crown of a partially erupted tooth become inflamed; Wade et al. (267) reported that in a group of 20 patients with pericoronitis, P. micros was the second most common organism. Recent investigations have shown that anaerobes, including GPAC, are able to invade cementum via periapical periodontal tissue (152) and nonexposed dental pulps (140), possibly via dentinal tubules. Dental infections can extend to cause brain abscesses (92), either directly or by hematogenous spread.

Two recent studies (147, 179) of peritonsillar abscesses have distinguished a group of patients with mainly aerobic infections, in which Streptococcus pyogenes predominated, from a larger group with mixed, primarily anaerobic infections. Both studies reported that the most frequent isolates in the second group were P. micros, "Streptococcus milleri," and Prevotella and Fusobacterium species. Surface swabs do not reliably reflect the organisms present in the tonsillar core (180); P. micros is significantly more common in the core.

The complications of peritonsillar abscess can be serious and occasionally fatal. A recent case report (63) described a peritonsillar abscess that yielded at least eight organisms, including P. anaerobius and P. asaccharolyticus. The case was complicated by a retropharyngeal abscess, which yielded a similar range of organisms, and anaerobic myonecrosis, in which seven organisms including P. anaerobius were cultured from neck muscle. This case illustrates the extraordinary range of organisms that can be isolated from intraoral abscesses.

There have been relatively few reports of GPAC from the upper respiratory tract in health or disease. Thomas and Hare (263) isolated strains of several Hare groups from the nasal flora; the predominant group, Hare group VIII, has recently been recognized as "P. octavius." Brook (24) compared the flora of 25 children with purulent nasopharyngitis with that of 25 controls and showed a statistically significant increase in counts of GPAC as well as Streptococcus pneumoniae, Haemophilus influenzae, and Fusobacterium and Prevotella spp. A more recent report (50) noted that anaerobes, including GPAC, predominated in chronic but not acute sinusitis. Data from surveys by Brook (34) and Murdoch et al. (196) (Table 6) indicate that the major species are P. magnus and P. micros. Other reports have shown the importance of GPAC in retropharyngeal abscesses (23), wound infections after head and neck surgery for cancer (46), chronic suppurative otitis media (49), and tracheostomy site wounds (21). Brook (34) isolated a wide range of GPAC from children with otitis media; 16 of 33 GPAC (48%) isolated from patients with acute otitis were recovered in pure culture, compared with 4 of 71 strains (6%) isolated from patients with chronic otitis.

Anaerobic infections of the lower respiratory tract are relatively common but underdiagnosed; GPAC form one of the major pathogenic groups (8). The same species as in intraoral disease are found, and it is likely that the gingival crevice is the site of origin of many of these organisms (8). However, since transtracheal aspiration is now rarely used, it is difficult to obtain uncontaminated specimens to determine the pathogens responsible. The most common clinical syndromes are aspiration pneumonia, lung abscess, and empyema (8); the course of infection is usually indolent with abscess formation, but a more rapid presentation resembling pneumococcal pneumonia can occur. Early studies in laboratory animals by Smith (238, 239) demonstrated that combinations of anaerobes were essential to induce lung abscess formation. In a classic study of 83 cases of empyema by Bartlett et al. (9), anaerobes were cultured in 76% of patients and aerobes were cultured in only 65%. A similar study by the same workers (10) of 54 patients with aspiration pneumonia documented anaerobes in 93% of patients and aerobes in 54%. In both series, the predominant organisms were Prevotella spp., Fusobacterium nucleatum, Peptostreptococcus spp., and microaerophilic streptococci. However, the peptostreptococci were not fully identified in these studies, and it is likely that some strains would now be classified in the genus Streptococcus (134). Four cases of empyema reported by Murdoch et al. (195) all yielded a similar flora; the GPAC were all identified as P. micros. Recent investigations (62, 173, 196) support the predominance of P. micros but have also reported isolation of P. anaerobius, P. magnus, P. vaginalis, and P. heliotrinreducens. However, an analysis of 197 cases of empyema in a younger age group (39) revealed that aerobes (particularly S. aureus and S. pneumoniae) were more common than anaerobes; the predominant species of GPAC was P. magnus. Several factors, including the age of the patient and the cause of the empyema, could account for these differences; further reports would be valuable. P. magnus has been isolated in pure culture from a patient with necrotizing pneumonia (207).

Anaerobes constitute more than 99% of the fecal flora, and counts of GPAC often exceed 10¹⁰/g (90, 91). However, to quote Moore et al. (183), "it is most significant that many of the more numerous fecal organisms have not been reported from human infections." As an example, P. productus is probably the commonest GPAC in the normal gut flora (91, 183), but it is very rarely cultured from properly obtained clinical specimens (136, 196). In a summary of five studies of intra-abdominal sepsis (92), GPAC accounted for 5% of strains, a much lower percentage than that of gram-negative anaerobes. Few reports have attempted to identify intraabdominal GPAC to the species level. In two surveys of anaerobic infections, Brook (28, 34) isolated strains of most species of GPAC, but unidentified strains comprised the largest group in both reports. In a more recent survey (196), intra-abdominal sites yielded 38 strains (18% of all isolates), 23 of which were from abscesses; the most common species was P. anaerobius (11 isolates), but almost all species of GPAC were recovered. P. anaerobius was also the commonest anaerobe in a recent series of infected hemorrhoids (43).

In 1972, Sabbaj et al. (229) reported a series of 47 liver abscesses, including 15 that were purely anaerobic; "anaerobic streptococci" or anaerobic cocci were the most common isolates after microaerophilic streptococci. Brook and Frazier (40) described 14 pediatric liver abscesses that yielded 29 organisms, mostly anaerobes; GPAC (5 isolates) were the most common organisms, but only one strain (P. magnus) was identified.

Infection of the urinary tract by anaerobes appears to be an uncommon event (92). GPAC have been isolated from several sites, most frequently superficial abscesses (29, 30, 196), but rarely from infections of the upper tract (34). Brook (30) documented 103 localized suppurative genitourinary infections, mostly skin related, in which mixed infections of GPAC and "Bacteroides" group spp. were the most common combination of organisms; P. anaerobius was the most common species of GPAC.

GPAC are commonly involved in gynecological and obstetric sepsis (28, 35, 92), but they have received much less study than gram-negative anaerobic rods, probably because the classification and identification methods are less well established. The association between bacterial vaginosis (BV) and a range of gynecological and obstetric infections is at present a very active area of research.

Gynecological sepsis. The normal adult vaginal flora contains about 10⁵ to 10⁷ bacteria/ml of fluid and is dominated (>95%) by lactobacilli (79, 126); the low pH (<4.5) and the ability of many lactobacilli to form hydrogen peroxide (126) appear to be important factors in its maintenance. BV represents a condition of bacterial overgrowth with replacement of the lactobacilli by a more anaerobic flora; bacterial counts are 100 to 1,000 times higher than normal (79). The classical indicator organism is Gardnerella vaginalis (79), but BV is now clearly associated with increased carriage of Mycoplasma hominis and raised counts of anaerobes, particularly gram-negative rods (126, 139, 256); these include several Prevotella species, notably Prevotella bivia and Prevotella disiens, Porphyromonas and Mobiluncus species, and F. nucleatum. The role of GPAC in BV is less clear.

Obstetric infections. "Anaerobic streptococci" were associated with puerperal infection by Schottmüller in 1910 (233); later reports by Schwarz and Dieckmann (234), Brown (52), and Colebrook and Hare (65) clearly established their importance. Infection classically spread from the placental site via the uterine veins to the broad ligaments, often causing thrombophlebitis, abscess formation, and infertility (114); in contrast to S. pyogenes infection, septicemia and metastatic infection were rare. Other pathogens such as gram-negative anaerobes and aerobic streptococci were usually present. Fortunately, improved obstetric practices and the ready availability of antibiotics have made puerperal fever a rare disease in the developed world. However, it is still common in the Third World, although it has been very little investigated. A unique study by Hare and Polunin (115) suggests that GPAC may play an important role. They compared the Murut, a tribe in the interior of British North Borneo (now Sarawak), whose high rates of infertility were leading to depopulation, with the Dusun, a neighboring, unaffected tribe. Gynecological abnormalities consistent with postabortion fever or postpartum fever were common in the Murut but not the Dusun. The authors reported that the cervical carriage rate of the predominant organism, Hare group I (now P. hydrogenalis [190]), was 33% in infertile members of the Murut but 3% in members of the Dusun.