THE GROUP D STREPTOCOCCI'

Transcription

1 BACTERIOLOGICAL REVIEWS Vol. 28, No. 3, p September, American Society for Microbiology Printed in U.S.A. THE GROUP D STREPTOCOCCI' R. H. DEIBEL2 American Meat Institute Foundation, The University of Chicago, Chicago, Illinois INTRODUCTION EARLY TAXONOMY AND NOMENCLATURE PHYSIOLOGY AND METABOLISM Proteolytic and Peptidase Activity Amino Acid Catabolism Arginine Agmatine Canavanine Allantoin Serine Tyrosine Phenylalanine Carbohydrate Catabolism Pyruvate Citrate Malate Succinoxidase system Aerobic diversion of glucose metabolism Glycerol Gluconate Pentose Galactose Hydrogen Transport Catalase Slime Formation Dextran formation by S. bovis Slime production by S. faecalis Unusual Characteristics of Enterococci NUTRITION Vitamins and Cofactors Lipoate requirements of enterococci Amino Acids Growth requirements Transaminase activity and D-amino acid utilization SEROLOGY Group Antigen Type Antigens S. faecalis S. faecium S. bovis CELL STRUCTURE Cell-Wall Chemistry Cytoplasmic Membrane Chemistry Nutritional Aspects of Wall Synthesis Neuraminidase Sensitivity Bacteriophage CURRENT CONCEPTS OF GROUP D STREPTOCOCCUS TAXONOMY Publication no. 266, American Meat Institute Foundation. 2 Present address: Division of Bacteriology, College of Agriculture, Cornell University, Ithaca, N.Y. 330

2 VOL. 28, 1964 GROUP D STREPTOCOCCI 331 Enterococci S. bovis and S. equinus COMMENTS REGARDING ISOLATION ECOLOGY Species Distribution in the Human Host Species Distribution in Domestic Animals Occurrence of Enterococci in Wild Animals Incidence of Enterococci in Insects, Plants, and Soil PUBLIC HEALTH SIGNIFICANCE Food Poisoning Pathogenicity Group D Streptococci as Indicators of Fecal Contamination LITERATURE CITED INTRODUCTION The keen interest in the metabolism, nutrition, and taxonomy of the group D streptococci is reflected in the plethora of literature associated with these subjects. These microorganisms have been involved in numerous studies dealing with clinical and food microbiology, and their utility as biochemical tools in studying fundamental metabolic processes cannot be overlooked. Within the past decade, an improved taxonomic scheme has emerged which facilitates a more definitive speciation within the enterococcus division as defined by Sherman (170). Previously, this division consisted of Streptococcus durans, and S. faecalis and its varieties liquefaciens and zymogenes. However, recent studies indicate that two metabolically distinct species exist within the previously considered S. faecalis species, and the revised scheme now recognizes S. faecalis and S. faecium. This speciation has evolved as a result of a spectrum of differential characteristics including nutritional patterns, serological properties, metabolic activities, and possibly cell structure as reflected by neuraminidase (lysozyme) sensitivity. S. durans is physiologically similar to S. faecium, and a varietal status (S. faecium var. durans) has been suggested (44). The observation that enterococcal proteolytic ability varies in degree among different strains places doubt on the establishment of S. faecalis varieties. These taxonomic considerations pervade both fundamental and applied disciplines with which these microorganisms are associated. In the immediate past, the recent nature of this improved classification scheme has not allowed its widespread utilization. The possible utility of enterococci as indices of fecal contamination or as a means of determining the sanitary history of food products, their association with food-poisoning outbreaks, and their occurrence in human pathological conditions attest to their importance in public health considerations. In each of these areas, isolation, quantitation, and precise identification are requisites for proper interpretation of results. Moreover, the relative incidence of the respective species, as well as the influence of a spectrum of different selective media upon their incidence, have not been realized and merit continued study. The employment of enterococci as indicator organisms of fecal contamination has received renewed impetus recently. However, the use of these bacteria in this capacity has not been accepted generally, because their significance and the environmental factors relating to their occurrence remain to be elucidated fully. A factor which tends to discount their employment as fecal indicators in some food products is their ability to grow in environments far removed from the original source of fecal contamination. Thus, once introduced into a food-processing plant, these bacteria can become established, and their subsequent introduction and growth in a food product does not infer necessarily fecal contamination. In the past, enterococci have been implicated as the etiological agent of food-poisoning incidents. This association has been questioned (46), and definitive evidence affirming or negating their involvement in food-borne gastroenteritis is lacking. Despite the stigma associated with their fecal origin and doubtful food-poisoning propensity, these bacteria possess potential value in some food fermentations. Indeed, one strain has been proposed as a starter culture for the rapid ripening of Cheddar cheese (36), and this process has enjoyed some commercial success. There is no reason to doubt that enterococci may

3 332 DEIBEL BACTERIOL. REV. be employed similarly in other food fermentations. In the study of fundamental metabolic processes, the use of enterococci without regard to their exact taxonomic position may lead to confusion. Although S. faecalis and S. faecium possess a number of common characteristics, their metabolic dichotomy is apparent. Establishment of the precise taxonomic position of the strain(s) under study will not only avoid conflicting results, but it may ultimately aid in a more definitive classification of these streptococci. It is the aim of this communication to relate some bacteriological and biochemical characteristics of the group D streptococci with their taxonomy, and to discuss certain aspects of metabolism which are peculiar to these microorganisms. Finally, a consideration of their ecology and public health significance will be presented. EARLY TAXONOMY AND NOMENCLATURE The first definitive streptococcal classification scheme dates back to 1906, when Andrewes and Horder (2) suggested seven groups of streptococci based primarily on morphology, fermentative ability, and growth characteristics in milk. The predominant Streptococcus, isolated from human feces, was termed S. faecalis, and it was defined by its active fermentation of several carbohydrates and its abundant growth at 20 C. In contrast to S. faecalis, isolates from horse feces did not ferment lactose, and required relatively high temperatures for growth. These strains were named S. equinus. The next significant contribution to streptococcal classification was made by Orla-Jensen (139), who based his divisions on fermentation characteristics, tolerance to heat and sodium chloride, and temperature limits of growth. The heat-resistant streptococci of fecal origin that initiated growth at 10 and 45 C were divided into two physiological types: (i) S. faecium and (ii) S. glycerinaceus and S. liquefaciens. S. liquefaciens differed from S. glycerinaceus only in its ability to liquify gelatin. In contrast to the S. glycerinaceus type, S. faecium fermented arabinose and seldom fermented glycerol or sorbitol. In addition, Orla-Jensen described the occurrence of streptococci from bovine feces that possessed unique physiological characteristics which afforded their differentiation from other fecal streptococci. For these strains, he proposed the name S. bovis. It was not until 1937 when Sherman (170) published his classification scheme that a truly definitive separation of the genus Streptococcus was made. This scheme divided the genus into four divisions based on the ability to initiate growth at 10 and 45 C. Some of the fecal streptococci were included in the enterococcus group which was characterized by growth at both 10 and 45 C. The term enterococcus was employed to commemorate its use by Thiercelin (193), who first employed it in his early description of a fecal Streptococcus. Sherman's enterococcus division excluded S. bovis and S. equinus, as these organisms did not initiate growth at 10 C or grow in media containing 6.5% sodium chloride. These streptococci were grouped in his "viridans" division. Aside from the ability to grow at 10 and 45 C and sodium chloride tolerance, Sherman's enterococcus division was characterized further by its resistance to heat (60 C for 30 min) and its initiation of growth at ph 9.6. At first, four species were included in this division; however, in a later study (171), the close relationship of S. faecalis, S. liquefaciens, and S. zymogenes was noted, and a varietal status for S. liquefaciens and S. zymogenes was suggested because these organisms differed only in their proteolytic and hemolytic abilities. S. durans possessed a number of differing physiological characteristics, and it was considered that this Streptococcus was distinctive at the species level (172, 173). In the following years, Sherman (174, 185) endeavored to relate Orla- Jensen's S. glycerinaceus species to Andrewes and Horder's S. faecalis, and discounted the S. faecium species because all enterococci fermented glycerol. Subsequently, Orla-Jensen (140) substantiated and extended the validity of S. faecium by associating the fermentation of melibiose and the inability to ferment melezitose and inositol with this species. In addition, S. faecium grew characteristically at 50 C. Both S. glycerinaceus and S. liquefaciens evidenced opposite fermentation reactions and seldom initiated growth above 45 C. In 1943 Gunsalus and Sherman (74) observed that all enterococci tested fermented glycerol aerobically. However, two types of enterococci could be discerned by the ability to ferment this polyol under anaerobic conditions. Later,

4 VOL. 28, 1964 GROUP D STREPTOCOCCI 333 Gunsalus (69) reported that those strains fermenting glycerol anaerobically required an exogenous hydrogen acceptor which occurred naturally in yeast extract. This hydrogen acceptor could be replaced by fumarate. In view of the differential activity with glycerol under anaerobic conditions, Gunsalus suggested a reconsideration of the speciation proposed by Orla-Jensen. Skadhauge (181) further observed that strains corresponding to S. glycerinaceus and S. liquefaciens (both correspond to Sherman's S. faecalis) grew in media containing 0.04% tellurite in contrast to S. faecium and S. durans. In addition, he confirmed Orla-Jensen's speciation based on fermentative characteristics and temperature limits of growth. Shattock (167) substantiated the distinction between S. faecium and S. faecalis in her study of physiological characteristics of 350 strains of fecal streptococci, and Barnes (9) added another differentiating characteristic by noting the ability of S. faecalis to reduce tetrazolium salts, in contrast to the inability of S. faecium to reduce these compounds. Deibel, Lake, and Niven (44) further reported that S. faecalis was distinctive in that it utilized citrate, gluconate, and glycerol (anaerobically) as sources of energy, and its growth in a semisynthetic medium did not require supplementation with folic acid. S. faecium strains gave opposite reactions. In addition, it was noted that S. durans was similar physiologically to S. faecium, and a varietal status for this organism was suggested. Serologically, all streptococci adhering to Sherman's enterococcus criteria contain the group D antigen (171, 184), and the employment of special techniques has afforded the demonstration of this antigen in S. bovis and S. equinus (92, 166, 183). Group D serological characteristics will be discussed in detail in another section. However, for clarity of presentation, it is deemed necessary to point out that all of the enterococci as defined by Sherman, as well as S. bovis and S. equinus, possess the group D antigen. With the advantage of time, it would appear that another step toward a rational and systematic scheme for the classification of fecal streptococci presents itself. The group D streptococci may now be considered to embrace S. bovis, S. equinus, and Sherman's enterococcus division, which consists of S. faecalis and S. faecium (and its variety durans). In this review, the term enterococcus will be employed to connote specifically those group D streptococci which follow the Sherman criteria (i.e., S. faecalis and S. faecium). In contrast to the enterococci, S. bovis and S. equinus have not been studied to the extent that a definitive speciation exists; further studies with these organisms may result in a somewhat altered speciation. PHYSIOLOGY AND METABOLISM Proteolytic and Peptidase Activity Proteolytic activity has been associated chiefly with S. faecalis; S. bovis, S. equinus, and S. faecium are generally considered to be devoid of this activity. Classically, streptococcal proteolytic activity has been detected in test-tube cultures. However, it has been observed (44) that this method is less sensitive than the agar plate method described by Burnett, Pelezar, and Conn (20). Some strains of S. faecalis and S. faecium that were previously considered to be nonproteolytic were found to possess relatively weak activity when tested by the plate method. As a result of these studies and the frequent loss of hemolytic activity observed in enterococcus strains, it is suggested that the designation of S. faecalis varieties (i.e., liquefaciens and zymogenes) be discontinued. Unlike the proteolytic activity of the group A streptococci (40), the activity of enterococci is not enhanced by anaerobiosis. Moreover, incubation under increased CO2 tension does not enhance proteolysis. Heat-denatured pepsin, casein, gelatin, protamine, and wheat gluten are usually hydrolyzed by the actively proteolytic strains. However, only activity with gelatin is demonstrable in plate cultures with the less active strains (Deibel, unpublished data). An optimal ph of 7.4 has been reported for enterococcal proteolytic activity (51), but Rabin and Zimmerman (153) demonstrated an optimal ph of 6.3 for enzyme synthesis. The amino acid requirements for proteinase synthesis parallel those necessary for growth. In contrast, purines and pyrimidines are not required for growth but appear to be necessary for optimal proteinase biosynthesis. The vitamin requirements also differ, in that only riboflavine and pyridoxal are necessary for proteinase synthesis (77). Dudani (51) observed the peculiar requirement of vitamin B 12 for enzyme production in one of nine proteolytic strains investigated.

5 334 DEIBEL BACTERIOL. REV. None of the enterococci has been reported to require this cofactor for growth. The relationship of proteolytic enterococci to food poisoning and certain disease conditions has received attention in the past. Guthof (76) associated increased numbers of proteolytic enterococci in the feces of individuals with various pathological conditions, but Deibel and Silliker (46) discounted the possible association of gelatin-hydrolyzing enterococci or the end products of their activity with food-poisoning propensity. Peptidase activity of the enterococci has received little attention, and aside from the work of Dudani (51) the literature is sparse in this area. The latter investigation demonstrated peptidase activity with glycyl-l-leucine and D,L-alanylglycine, and both alkaline and acidic ph optima were reported for these enzymes. Depending upon the substrate involved and the ph value of the test system, magnesium or manganese was required for activation of the enzyme. The majority of the enterococci hydrolyze hippurate at the peptide bond, forming glycine and benzoic acid. The characteristics of this reaction have not been studied, and the relationship to peptidase activity remains to be elucidated. Hokoma and Salle (84) described the hydrolysis of glycocholic acid by S. faecalis, and related this activity to the peptidases characteristic of the mammalian gastrointestinal tract. In each instance in which peptidase activity was associated with the enterococci, the amino acid glycine was involved in the formation of the peptide. However, definitive studies regarding the specificity of the enzyme(s) are lacking. Amino Acid Catabolism The degradation of amino acids by group D streptpcocci is limited, although recent observations indicate that this activity is somewhat greater than considered previously. It has been demonstrated that degradative activity is not confined to the amino acids arginine and tyrosine but includes serine, agmatine, phenylalanine, and canavanine. S. bovis and S. equinus do not hydrolyze arginine (134) or decarboxylate tyrosine (119), and their degradative activity with other amino acids has not been investigated extensively. Arginine. Since Hills (82) first described the hydrolysis of arginine by streptococci, the distribution of this characteristic among the streptococci and the hydrolytic sequence have been the subject of numerous investigations. Niven, Smiley, and Sherman (134) reported the deamination of arginine by all enterococci. Gale (64) observed the ability of this amino acid to serve as an energy source in the active uptake of certain amino acids into the amino acid pool. Subsequently, the mechanistic aspects of degradation were investigated [see reference material in (41)], and it was demonstrated that the arginine dihydrolase enzyme of Hills consisted, in reality, of a series of enzymes. The first step involves the hydrolytic deamination of arginine to citrulline. In the hydrolysis of citrulline to ornithine, adenosine triphosphate (ATP) is formed. This reaction involves the generation of carbamyl phosphate, as first described by Jones, Spector, and Lipmann (95) in work with S. faecalis R (S. faecium in the current classification scheme). The characteristics and reaction spectrum of carbamyl phosphate were reviewed recently (94). Bauchop and Elsden (14) related the ATP generated to the total cell crop produced, and demonstrated the utilization of the resultant energy in growth processes. A molar relationship was established between the arginine hydrolyzed and the ATP generated. In our laboratory (41), it has been observed that, although all enterococci hydrolyze arginine, only S. faecalis can couple the energy liberated with growth processes. This observation has taxonomic utility and aids in the differentiation of S. faecalis from S. faecium (and var. durans). The inability of S. faecium to utilize the energy released in the hydrolytic process is an enigma, and certainly represents a loss in the overall economy of the cell. Whether or not the detailed mechanism of hydrolysis differs in these two enterococcal species, or factors other than those associated with its degradation are operative, will remain a subject for further study. Agmatine. The utilization of agmatine as a source of energy for growth processes parallels arginine utilization (41). Again, S. faecium cannot utilize this compound as an energy source, and only approximately 50% of the strains are capable of hydrolyzing it with the production of ammonia (41). Moller (124) first observed agmatine hydrolysis in a strain of S. faecalis, and by analogy formu-

6 VOL. 28, 1964 GROUP D STREPTOCOCCI 335 lated an agmatine dihydrolase system. In this sequential reaction, monocarbinylputrescine and putrescine correspond to citrulline and ornithine, respectively, in the arginine dihydrolase system. The observation that S. faecalis utilizes agmatine as an energy source and the structural similarity of this compound to arginine lead to the supposition that the energy-yielding mechanism in both systems is similar, if not identical. Canavanine. Canavanine is a competitive inhibitor of arginine desimidation, and several pathways have been reported by which it is metabolized and thus detoxified by enterococci. In a reaction requiring glucose as an energy source, canavanine is cleaved to form guanidine and homoserine (98). The various strains of enterococci employed in the latter study evidenced differential degradative activity. Canavanine can also be hydrolyzed to O-ureido-Lhomoserine and ammonia (97). Although the cell suspensions employed in this investigation were considerably more active in arginine desimidation, these reactions may reflect the activity of separate and distinct enzymes. Growth of the strain used in these studies (S. faecalis R) was not inhibited by canavanine (198). Moreover, canavanine could not replace the growth requirement for arginine. Recently, Himmel and Zimmerman (83) observed a stimulation of S. faecalis growth in the presence of canavanine and arginine. It was assumed that the arginine dihydrolase system was inhibited by canavanine, thus affording a greater concentration of arginine for synthetic reactions. Additional studies with this arginine analogue in relation to its inhibition of growth, inhibition of arginine hydrolysis, and possible differential degradation by S. faecalis and S. faecium may furnish an insight to the previously noted difference in arginine metabolism by the two species. Allantoin. The analogy between degradation of the cyclic ureide allantoin by S. allantoicus and decomposition of the ureido group of citrulline by enterococci deserves comment. S. allantoicus was isolated and described by Barker (8). The taxonomic position of this organism has not been established specifically, although it does resemble the enterococci in its overall physiological characteristics. This organism ferments allantoin, and the primary attack has been identified as a hydrolytic cleavage of the ring to yield allantoic acid. The subsequent steps are unknown, but the major end products are urea, ammonia, oxamic acid, and carbon dioxide. Lesser amounts of lactic, acetic, formic, and glycolic acids are produced. Valentine and Wolfe (195, 196) demonstrated the phosphorolytic cleavage of the ureido group in carbamyl oxamate, resulting in the formation of carbamyl phosphate and oxamic acid. Apparently, this is the energy-yielding reaction in the allantoin fermentation. It is not known whether or not the enterococci can utilize allantoin or, more specifically, carbamyl oxamate as an energy source. Further studies as well as a more complete description of S. allantoicus (including its serological relationships) are indicated. Serine. This amino acid is degraded by S. faecalis but not by S. faecium (42). The observation has taxonomic utility in that S. faecalis can utilize serine as a source of energy for growth purposes, in contrast to S. faecium. A molar relationship between the serine utilized and ammonia produced has been established for S. faecalis. The most probable pathway for this degradative process is the deamination to pyruvate, which may be mediated by a serine dehydrase. This enzyme has been reported to occur in certain strains of lactobacilli (102, 103). In a subsequent section, the utilization of pyruvate as an energy source for the growth of S. faecalis (in contrast to S. faecium) will be presented. As will be discussed, this fermentation requires an exogenous source of the cofactor lipoic acid for growth in a semisynthetic medium. Similarly, when S. faecalis is cultured in the semisynthetic medium containing serine as the energy source, lipoic acid is required. Thus, it would appear that serine is deaminated to pyruvate, and the energy-yielding step occurs in the lipoate-linked metabolism of this compound. Only L-serine is utilized by S. faecalis; D-serine as well as D, L-homoserine and L-seramine are neither utilized as an energy source nor deaminated extensively. A large number of other amino acids, including both optical forms of alanine and some of its structural analogues, were tested. However, none was found to support growth or give rise to ammonia production (Deibel and Niven, unpublished data). Tyrosine. The majority of the enterococci decarboxylate tyrosine to form the correspond-

7 336 DEIBEL BACTERIOL. REV. ing amine tyramine; this reaction was the subject of a previous review (63). This activity is somewhat variable among the enterococcus species, particularly the S. faecium strains (10). A possible role of tyramine-producing enterococci in neonatal diarrhea was suggested (62). However, a subsequent study failed to relate these bacteria to the disease condition (162). In addition, the involvement of tyramine-producing strains in outbreaks of food poisoning was discounted, since large amounts of the amine failed to produce illness when consumed by human volunteers (35). Phenylalanine. Although Gale (61) associated a high degree of substrate specificity with his tyrosine decarboxylase preparations, it has been demonstrated that phenylalanine is also decarboxylated (117). With acetone powders of S. fascalis R (S. faecium), sufficient decarboxylase activity with phenylalanine was observed such that the assay for tyrosine gave somewhat higher values than the actual amount of tyrosine present. The end product of phenylalanine decarboxylation was determined and identified as phenylethylamine by the formation of its derivatives. In another study (4), phenylalanine decarboxylase activity was observed with S. faecalis, and the inhibition of this activity by tetracycline was considerably higher than that obtained with the tyrosine decarboxylase. Phenylethylamine was also identified as the end product of the reaction in this study. More recently, Lestrovaya and Mardashev (108) demonstrated phenylalanine decarboxylase activity with S. faecalis, and noted its inhibition by halogen-substituted arylamines. In addition, these investigators observed the decarboxylation of dihydroxyphenylalanine-a reaction not previously associated with the enterococci. In this laboratory, the decarboxylase activity of phenylalanine was compared with tyrosine decarboxylase activity in growing cultures. Ten representative enterococcus strains were cultured in two complex media: one containing 1.0% L-tyrosine; the other, 1.0% L-phenylalanine. The modified Eldrige technique of Williams and Campbell (207) was used to detect the evolved carbon dioxide. After 1 week of incubation, tyrosine decarboxylase activity was detected visually in nine of the cultures; however, none of the strains evidenced demonstrable activity with phenylalanine. Thus, it would appear that decarboxylase activity with phenylalanine is limited, and relatively sensitive methods must be employed to detect this activity. Carbohydrate Catabolism The enterococci are generally considered to be catalase-negative microorganisms, not containing cytochrome and lacking a tricarboxylic acid cycle. The anaerobic fermentation of glucose is a homofermentative process resulting in the formation of lactic acid, and the dextrorotatory isomer of lactate is formed. Until recently, the hexose diphosphate pathway was considered the primary, if not the sole, mechanism for carbohydrate degradation and energy production. However, the fermentation of gluconate, arabinose, pyruvate, citrate, and malate serves to illustrate the metabolic diversity of carbohydrate degradation by the enterococci, and suggests a certain degree of independence from the classical hexose diphosphate pathway. Pyruvate. In relation to the hexose diphosphate system, pyruvate serves as a "hydrogen sink," allowing the regeneration of oxidized nicotinamide adenine dinucleotide (NAD) through the agency of lactic dehydrogenase. Thus, the addition of pyruvate in substrate quantities would not be expected to alter radically the total cell crop, because energy generation is not associated with the reduction of pyruvate to lactate. However, the observation that pyruvate can serve as an energy source for the growth of S. faecalis requires a departure from concepts associated with its role in the hexose diphosphate pathway, and opens a heretofore unexpected avenue of enterococcal carbohydrate metabolism with a spectrum of ramifications (43). Pyruvate is utilized as an energy source only by S. faecalis (43). The fermentation is adaptive (38) and occurs under anaerobic and aerobic conditions of growth. The utilization of pyruvate as an energy source has taxonomic value, and may be employed as an aid in the differentiation of S. faecalis from S. faecium. Only a few strains of S. bovis have been tested for this characteristic (with negative results), but whether or not this species or S. equinus has the ability to ferment pyruvate is unknown. When S. faecalis is cultured in a semisynthetic, casein-hydrolysate medium with pyruvate as the energy source, the cofactor lipoic acid is required for growth. Culture of the organism

8 V:OL. 28, 1964 GROUP D STREPTOCOCCI 337 with other carbohydrate-energy sources such as hexoses, ribose, glycerol, and gluconic acid does not manifest a lipoate requirement. As previously discussed, growth with serine also evidenced a lipoate requirement, thus implicating the metabolism of pyruvate in the degradation of the amino acid. Although pyruvate could be metabolized by a number of mechanisms, only the phosphoroclastic and dismutation pathways require consideration in relation to anaerobic energy metabolism. The dismutation pathway has received considerable attention relative to the phosphoroclastic pathway, and the overall mechanisms involved in dismutative activity have been described and reviewed by Gunsalus (71). The key oxidation associated with energy production has not been identified, but is believed to involve an aldehyde level of oxidation which ultimately results in the formation of acetyl-coenzyme A (CoA). In this system, lipoate serves as an acyl carrier and undergoes a cyclic oxidation and reduction. Hydroxyethyl thiamine diphosphate has been identified as the primary reaction product resulting from the initial decarboxylation of pyruvate, and the involvement of this compound in the oxidation and generation of energy in the dismutation pathway has been suggested (101). The requirement for lipoate in the fermentation of pyruvate by S. faecalis suggests a significant role of dismutative activity in pyruvate catabolism. However, fermentation balances indicate that the phosphoroclastic pathway is also operative, as evidenced by the production of significant quantities of formate (43). The mechanistic aspects of the phosphoroclastic reaction have not been elucidated, and the involvement of lipoate has not been demonstrated. Although good carbon recoveries and O/R balances were not obtained with pyruvate as the energy source (due to the formation of a viscous slime), formate was consistently found as an end product, and its concentration varied from 0.31 to 0.42 mmole per mmole of pyruvate fermented. The absolute requirement for lipoate in growth studies afforded the speculation that lipoate is involved also in the phosphoroclastic reaction. However, studies with cell-free systems failed to demonstrate the involvement of this cofactor. An absolute requirement for thiamine diphosphate was observed (43), and Wood and O'Kane (211) demonstrated significant stimulation with tetrahydrofolic acid and yeast extract. The demonstration of energy generation in the phosphoroclastic reaction (109) and the apparent lipoate-nonlinked nature of the reaction would suggest the ability of S. faecalis to utilize pyruvate in the absence of exogenous lipoate. The relationship of this reaction to the overall fermentation of pyruvate by S. faecalis is not understood, and presents an interesting and presently paradoxical accumulation of data. Citrate. A detailed study of citrate fermentation by the enterococci finds its origin in the investigations of Campbell and Gunsalus (23, 73). At alkaline ph values, resting-cell suspensions accumulated pyruvate, acetate, and carbon dioxide. From the data presented with growing cultures, a phosphoroclastic breakdown of the pyruvate occurred at alkaline ph values, a mixed phosphoroclastic-dismutation breakdown occurred at neutrality, and an almost complete dismutative metabolism took place under acidic conditions. A variation in the end products was noted also as a function of the strain employed. These results indicate that the ph value of the medium, as well as the genetic potential of the strain, determine the relative activity of phosphoroclastic and dismutation reactions. Previously, Gunsalus and Niven (72) noted the radical effect of ph on the enterococcal glucose fermentation. At ph 9.0, the yield of lactate substantially decreased, and large amounts of acetate, formate, and ethanol accumulated. Only 88% of the carbon was recovered, owing to the formation of a viscous polysaccharide under these conditions. The results indicated an altered pyruvate metabolism and an enhancement of phosphoroclastic activity, as judged by the increased formate concentrations. The primary reaction involved in enterococcal citrate metabolism is a C2-C4 cleavage, and these products have been identified as acetate and oxalacetate, respectively (65). Acetyl-CoA was not formed in the reaction, and the enzyme associated with this activity was termed citridesmolase. If the extracts were freed from oxalacetate carboxylase, acetate and oxalacetate accumulated. Thus, the overall degradation of citrite may be summarized as follows: citrate is cleaved to acetate and oxalacetate, the latter is decarboxylated to pyruvate, and the metabolic fate of this compound is ph- and strain-dependent.

9 338 DEIBEL BACTERIOL. REV. The utilization of citrate as an energy source is characteristic of S. faecalis; S. faecium is unable to grow with this compound as the energy source (44). The related compounds, isocitrate and cis-aconitic acid, are not utilized as energy sources by either species. In addition, when S. faecalis is grown with citrate as the energy source in a casein-hydrolysate medium, lipoate is required (43). Consequently, it was inferred that the energy-yielding step in citrate fermentation by the enterococei is a lipoate-linked system, and most probably occurs in the fermentation of pyruvate. Malate. The fermentation of malate by enterococci was reported in thesis form by Whittenbury (204) and Jones (90). While a large number of various compounds were screened for their ability to serve as energy substrates, it was also observed in this laboratory that malate is utilized by S. faecalis but not by S. faecium (42). Cultures incubated under anaerobic conditions on complex media with malate as the energy source evidenced a slow rate of growth as well as a submaximal growth response. These results were comparable to those obtained with glycerol or mannitol as the energy source. Generally, malate degradation in other biological systems involves an initial dehydrogenation. These considerations prompted experiments utilizing fumarate as an external hydrogen acceptor in the system. As noted in Table 1, growth was stimulated with fumarate under anaerobic conditions of incubation. Aerobic incubation also afforded enhanced growth, and it was surmised that oxygen can also serve as a hydrogen acceptor in malate degradation. Pyruvate was implicated in the fermentation of serine and citrate by demonstrating a lipoate requirement in cultures grown with these substrates. However, when this method was extended to the malate fermentation, it was observed that growth took place in the absence of exogenous lipoate, and repeated attempts failed to demonstrate a lipoate requirement. The addition of lipoate stimulated growth, and a combination of lipoate and fumarate produced a maximal cell crop (Table 2). These results indicate that another and heretofore unsuspected mechanism of energy generation is associated with malate degradation. Apparently, pyruvate is not involved in this energy-generating step, and further studies with this reaction merit consideration. Succinoxidase system. Gunsalus (69) demonstrated the ability of fumarate to serve as a hydrogen acceptor in the glycerol fermentation. In this laboratory, it was observed that cultures containing glucose and fumarate evidenced carbon dioxide formation and an increased production of volatile acids. When growth responses were compared in a glucose versus a glucosefumarate medium, a superior response was noted in cultures of the latter. These characteristics were peculiar to the majority of the S. faecalis strains in the collection and were not demonstrable with S. faecium. TABLE 1. Effect of fumarate on the growth response of Streptococcus faecalis strain FB82 to malatea Condition of incubation Addition to basal mediunmb Anaer- Atmos- Aerobicd obic phericc None Fumarate (1.0%) Fumarate + malate Malate (1.0%) a Growth is expressed as optical density times 100 ḃtryptone-yeast extract medium, 24-hr incubation. c Incubated with 10 ml of medium in 18-mm test tubes. d Cultured in 125-ml flasks and incubated on a reciprocating shaking apparatus. The increased growth response and production of carbon dioxide and volatile acids in the cultures grown in glucose-fumarate medium led to the hypothesis that fumarate diverted the normal glucose fermentation, and the association of this activity with S. faecalis implicated pyruvate metabolism. It was postulated that fumarate reacted with the reduced NAD generated in the oxidation of glyceraldehyde-3- phosphate, and thus altered the role of pyruvate in the regeneration of oxidized NAD. The metabolism of pyruvate via the dismutation and phosphoroclastic pathways would then account for the increased growth response and the volatile acid and carbon dioxide production. Alternatively, the hydration of fumarate by a fumarase, leading to malate formation and its metabolism, would also account for the observed results. To test this hypothesis, S. faecalis was cultured in a medium containing glucose and fumarate. Each

10 VOL. 28, ml of the complex medium contained 4.4 mmoles of fumarate and 2.8 mmoles of glucose; 5 jxc of 1,4-C'4-fumarate were added to one flask, and another received 5 isc of 2,3-C'4- fumarate. After overnight incubation, the evolved carbon dioxide was trapped in barium hydroxide tubes, and, after zinc sulfate clarification by the method of Neish (129), samples were steam distilled for volatile acid content. No significant amounts of radioactivity were detected in either the volatile acid fractions or the precipitated barium carbonate samples TABLE 2. Stimulatory effect of lipoate and fumarate on malate fermentation by Streptococcus faecalis FB82a Addition to basal mediumb GROUP D STREPTOCOCCI Per cent malate in medium None Lipoatec Fumarated Lipoate + fumarate a Results are expressed as optical density times 100 ḃsemisynthetic medium (44) incubated 20 hr under atmospheric conditions in 18-mm test tube cultures., Amount added was 20 mug per 10 ml of medium. d Amount added was 0.1 g per 10 ml of medium. (Table 3), indicating the inability of the organism to degrade extensively the added fumarate. To substantiate this conclusion, a carbon balance was performed. If fumarate were further metabolized to carbon dioxide and volatile acids, then a significant increase in recovered carbon would be manifested if the added fumarate had not been taken into account in the carbon balance. However, if fumarate were reduced to succinate and not further metabolized, then a carbon recovery of approximately 100% would be expected. The experiment was performed with glucose and fumarate as substrates and, for comparative purposes, with glucose alone. As seen in Table 4, the recoveries approached 100%. Perhaps a higher value could have been obtained if ethanol had been quantitated, since small amounts of this alcohol are normally produced (149). These results tend to negate the possible involvement of a fumarase and strengthen the role of fumarate as a hydrogen acceptor in the fermentation reactions of S. faecalis. The recent studies of Sanadi and Fluharty (159) indicate that an energy-generating reaction occurs in beef heart mitochondria in the TABLE 3. Fate of radioactive fumarate in the glucose-fumarate fermentation effected by Streptococcus faecalis FB82 Sample Radioactivity (counts per min)* in culture containing 1,4-C14- fumarate 2,3-C'4- fumarate Culture medium Before inoculation 6,450,000 6,210,00 After incubation, clarified... 6,380,000 6,180,000 CO2... 1, Volatile acids.. 65,000 55,000 Volatile acid residue.. 6,610,000 6,390,000 * All values are corrected for background count. TABLE 4. Carbon recoveries with cultures of Streptococcus faecalis FB82 grown in glucose and glucose-fumarate Substrate or product Glucose. Lactate... Acetoin... CO2... Formate... Acetate... Carbon recovery (%)... C6 (mmoles/ 100 mmoles) Glucose Condition of culture c (mmoles) Glucose and fumarate C6 (mmoles/ 100 mmoles) c (mmoles) oxidation of reduced NAD and concurrent reduction of fumarate. Conceivably, the reduction of fumarate could also be coupled to highenergy phosphate generation in the fermentation reactions of the enterococci, thus indicating that further studies with enterococcal preparations are desirable. Aerobic diversion of glucose metabolism. Seeley and VanDemark (161) observed a 30% increase

11 340 DEIBEL BACTERIOL. REV. in cell crop when S. faecalis B33A was incubated aerobically, as compared with anaerobic incubation, with glucose as the substrate. Previously, O'Kane (137) demonstrated the effect of lipoate (pyruvate oxidation factor) on the aerobic oxidation of glucose in cell suspensions of S. faecalis loci. Glucose was degraded to pyruvate both aerobically and anaerobically. Aerobically, in the absence of lipoate, pyruvate accumulated, or it was metabolized to acetoin. However, in the presence of lipoate, pyruvate was oxidized to acetate and CO2. In toto, these results suggest a diversion of aerobic glucose metabolism in analogy to that effected by fumarate. In this instance, oxygen rather than fumarate is serving as the final hydrogen acceptor, and the additional growth response under aerobic conditions of incubation may reflect the additional energy generated by pyruvate oxidation. More recently, London and Appleman (110) reported significant differences in the aerobic metabolism of glucose by S. faecium and S. faecalis. The acetateacetoin ratio for S. faecalis was 1:1 in contrast to a 35:1 ratio for S. faecium. In addition, a 40% increase in aerobic growth was observed with S. faecalis, and a 12% increase was noted with S. faecium. These investigators ascribed the superior aerobic growth response of S. faecalis to a higher ph value, resulting from decreased acid formation. This concept is diametric to the diversion scheme, and additional studies are indicated prior to resolving the effect of aerobiosis on the increased growth response. Glycerol. Glycerol fermentation has occupied a central role in the taxonomy of the enterococci, and the details of glycerol degradation have been elucidated. Gunsalus and Sherman (74) noted the "fermentation" of glycerol by all enterococci under aerobic conditions and the anaerobic utilization by only certain strains. End-product analyses (69) indicated that the metabolism of glycerol proceeded through the hexose diphosphate pathway, and lactate accumulated. As previously mentioned, fumarate or other unidentified substances in the complex medium could serve as hydrogen acceptors in the fermentation of this reduced substrate. The strains that did not ferment glycerol anaerobically were dependent upon a primary phosphorylation to a-glycerophosphate. This phosphorylated intermediate was oxidized to lactate, and hydrogen peroxide accumulated (75). Jacobs and VanDemark (89) demonstrated a fundamental difference in aerobic and anaerobic glycerol metabolism by S. faecalis loc1. Cell crops were grown aerobically and anaerobically, and the hydrogen transport was followed in the two systems. Aerobically, glycerol was first phosphorylated (glycerol kinase), forming a-glycerophosphate. a-glycerophosphate oxidase mediated the oxidation of this compound (ultimately forming lactate), and this enzyme was flavinlinked. Hydrogen peroxide accumulated, and the absence of a NAD linkage was observed. In sharp contrast to the aerobic system, glycerol is first dehydrogenated anaerobically, probably forming dihydroxyacetone, which is subsequently phosphorylated and metabolized to lactate (89). The glycerol dehydrogenase was NAD-linked, and could be coupled either to reduce fumarate directly or with the intermediate reduction of exogenous flavin as demonstrated with crude cell-free preparations. The reduction of fumarate with NAD (which may involve a flavin intermediate) merits stressing, as definitive evidence was obtained in this study relating the regeneration of oxidized NAD through the agency of fumarate. Recently, it was demonstrated that the anaerobic utilization of glycerol is characteristic of S. faecalis (44). In general, S. faecium does not grow under these conditions, and a differential test utilizing a glycerol-fumarate medium was proposed. Gluconate. Sokatch and Gunsalus (189) demonstrated an inducible fermentation of gluconate in S. faecalis locl. End-product analyses and the metabolism of labeled substrates indicated that the degradation occurred by the combined action of the pentose phosphate and Entner- Doudoroff pathways. The distribution of this characteristic was investigated (44), and only S. faecalis utilized gluconate as an energy source. The test has differential value, and may be employed in distinguishing among the enterococcus species. Pentose. S. faecium ferments L-arabinose in contrast to S. faecalis, and only an occasional enterococcus strain ferments D(+)xylose. None of ten representative enterococci tested in our laboratory fermented L(-)xylose, D(-)arabinose, D(+)arabitol, L(-)arabitol, D(-)xylitol, or D(-)lyxose. All enterococcus strains ferment D (-)ribose. Thus, aside from ribose, pentose

12 VOL. 28, 1964 GROUP D STREPTOCOCCI 341 fermentation by the enterococci appears to be rather limited, and confined to arabinose fermentation by S. faecium. A reinvestigation of arabinose fermentation by S. faecalis (most probably S. faecium) gave unexpected results (58). Resting-cell suspensions converted 95% of the substrate to lactate, and growing cultures converted 80 to 85% to lactate. No carbon dioxide or acetate was produced, and only small amounts of formate were detected. These yields of lactate are far in excess of the 60% theoretical yield if the pentose were cleaved to lactate and acetate, as observed in other lactic acid bacteria. The ability to ferment sedoheptulose led to the conclusion that a new mechanism of pentose fermentation was operative, involving the pentose phosphate pathway. Confirmation and extension of these results appear to be in order and indicate, as the authors suggest, a new mechanism of pentose degradation. Galactose. The end products of galactose metabolism have been shown to differ radically from those of glucose (148, 190, 202). In S. pyogenes cultures, approximately 50%O of the substrate is converted to formate, acetate, and ethanol in a 2:1:1 ratio. Although the same end products and ratios were observed by Gunsalus and Niven (72) in the alkaline fermentation of glucose, a similar effect of ph on the galactose fermentation by S. pyogenes could not be demonstrated. S. faecalis converted 67c% of the galactose fermented to lactic acid. Subsequently, Fukuyama and O'Kane (59) extended this observation, and in experiments with S. faecalis loc1 the fermentation of labeled substrates gave results indicating galactose was dissimilated via the hexose diphosphate pathway. The alteration of end products presumably occurs at the pyruvate level (59, 148). However, the exact mechanism is yet to be resolved. The fermentation of galactose by both S. faecalis and S. faecium and the inability of S. faecium to ferment pyruvate afford a speculation regarding a difference in end products of galactose catabolism by these enterococcus species. Hydrogen Transport The group D streptococci lack cytochrome pigments, and the flavin coenzymes occupy a central role in aerobic hydrogen transport. Generally, these enzymes are NAD-linked to the specific substrate dehydrogenases and mediate hydrogen transfer to oxygen, artificial hydrogen acceptors, and possibly fumarate (89). Dolin (50) reviewed the characteristics of five distinct flavoprotein enzymes that are associated with reduced NAD oxidation in S. faecalis. These activities include a reduced NAD oxidase and peroxidase, a cytochrome c reductase (which is perhaps of vestigial significance), a diaphorase, and a menadione reductase. The enterococcal fumarate reductase has not been studied. The evidence obtained to date indicates the absence of an energy-yielding reaction that is coupled with the oxidation of reduced NAD (49). Seeley and VanDemark (161) described the formation of a peroxidase in S. faecalis B33A. Anaerobically grown cells accumulated hydrogen peroxide when exposed to air, in contrast to aerobically grown cells. The enzyme was formed adaptively and differed from the classical peroxidase in that it lacked hemin. As yet, the distribution of the enzyme among the group D streptococci is not known. A concomitant 30% increase in cell crop was observed in the aerobically grown, peroxidase-producing cultures with glucose as the energy source. Thus, an additional energy-yielding reaction presumably takes place when the organism is incubated aerobically. The slow growth rate and submaximal growth response of the enterococei, when cultured anaerobically with glycerol or malate, and the stimulation of growth activity under these conditions by the addition of fumarate, reflect the difficulty of the organism in regenerating oxidized NAD. Similarly, fumarate may serve to regenerate oxidized NAD in the glucose fermentation and, thus, afford the generation of additional energy in the subsequent metabolism of pyruvate. In an analogous manner, the oxidation of glucose under aerobic conditions of growth may be diverted by the regeneration of oxidized NAD, with oxygen as the terminal hydrogen acceptor. The ability of S. faecalis to couple the energyyielding reactions of pyruvate dissimilation to growth processes would suggest the restriction of this activity to S. faecalis strains, and a difference in end products as well as growth response may be manifested in a comparison of the S. faecalis and S. faecium activities. These considerations merit further study, and may aid in explaining the observed increase of cell crop under aerobic conditions of growth. Catalase. The lack of catalase activity is one

13 342 DEIBEL BACTERIOL. REV. of the classical physiological tests employed to differentiate group D streptococci from morphologically similar staphylococci. Only a rare enterococcus strain possesses this activity, and these strains, like the typical strains without catalase activity, also lack iron-porphyrin compounds (45). The only enterococci which have been reported to possess catalase activity were isolated from silage (106). A study of two of these strains (both S. faecalis) in this laboratory by Jones (91) revealed a gradual loss of activity when the strains were maintained by daily transfer in broth media. The activity could not be reactivated by growth under a variety of conditions in the strains which had lost the characteristic, although high activity could be maintained by daily transfer on agar slopes. Cationic supplementation of the growth medium with ferric, manganese, and, to a lesser extent, zinc ions, coupled with aerobiosis, enhanced significantly the catalase activity. However, the activity in cell suspensions and cell-free extracts (all cell crops grown in the absence of added cations) failed to respond to added cations and indicated that an active metabolism was a perrequisite for cationic stimulation. The exact role of the cations in catalase activity is not understood. As is demonstrable with the classical enzyme, quantitative oxygen evolution was observed; however, the enterococcal activity was not inhibited by cyanide or azide. In addition, iron-porphyrin compounds were not detected either chemically or spectrophotometrically. In the lactic acid bacteria, catalase activity is not restricted to the enterococci, and a large number of pediococci and lactobacilli have been reported to possess this enzyme. [See references (45)]. Some properties of the Lactobacillus and Pediococcus enzymes were described by Delwiche (47, 48), and iron-porphyrin compounds are also lacking in these bacteria (45). The enterococcal activity is not inhibited by Atebrin (91), and, as yet, the functional group of the enzyme has not been identified in any of the lactic acid bacteria possessing the activity. Slime Formation Slime formation is not a constant characteristic of any group D Streptococcus species. However, when cultured under appropriate conditions, the majority of S. bovis strains elaborate a polysaccharide when cultured with sucrose. This substance has been characterized as a dextran. The enterococci do not form a polysaccharide under these conditions, but some S. faecalis strains produce a viscous slime in broth cultures when the ph of the medium is maintained at neutrality or at slightly alkaline conditions. The major component of this material has been identified tentatively as a ribonucleic acid (RNA) nucleoprotein. Dextran formation by S. bovis. While studying the ability of S. salivarius to form slime from sucrose, it was observed that an occasional strain of S. bovis also formed a polysaccharide under these cultural conditions (133). Unlike the water-soluble levan produced by S. salivarius, S. bovis elaborated an insoluble dextran. Later, carbon dioxide was demonstrated to have a profound effect on dextran production by S. bovis (37). Only 5 of 87 strains formed grossly detectable polysaccharide when sucrose-gelatin, agar-plate cultures were incubated aerobically. On the other hand, incubation under increased carbon dioxide tension resulted in dextran production by 71 of the strains. In another study (25), this effect of carbon dioxide was not demonstrable. The agar stab method employed to detect slime formation, however, is open to criticism. The enhancement of slime production by carbon dioxide was substantiated and investigated further (5, 6, 7, 142, 212). Under anaerobic conditions of incubation, Tween 80 replaced the carbon dioxide enhancement of dextran synthesis (37). Studies on the quantitative production of slime stressed the adequacy of the buffering system employed in the medium (142). Acetate was more effective than phosphate in this respect, as higher acetate concentrations could be used. Although bicarbonate could replace carbon dioxide, maximal dextran synthesis was shown to involve the dissolved gas and not the bicarbonate ion. The most probable function of carbon dioxide in the production of dextran by S. bovis centers on its relationship to enzyme synthesis, rather than actual incorporation in the polysaccharide molecule. Growth of the organism with C1402 results primarily in the labeling of aspartic acid and, to a lesser extent, threonine, glutamate, and several purines and pyrimidines (212). Tween 80 may function by altering the cell's permeability, thus facilitating a higher intracellular aspartate

14 VOL. 28, 1964 GROUP D STREPTOCOCCI 343 concentration (212). In this respect, the Tween has a similar effect to that noted in the nutrition of the "minute" streptococci (115). The observation that dextransucrase is a constitutive enzyme in S. bovis (7), coupled with the nutritional and radioactive carbon dioxide studies, indicates that an overall enhancement of enzyme synthesis, including dextransucrase, is effected by increased carbon dioxide tensions (12). The properties of the dextransucrase isolated from S. bovis are sufficiently similar to the Leuconostoc enzyme to suggest identity (5). The chief differences lie in the temperature required for optimal activity (37 to 44 C for the S. bovis enzyme and 25 to 29 C for the Leuconostoc enzyme) and the degree of branching in the dextran polymer produced. Usually, the Leuconostoc polymers are highly branched, in contrast to the unbranched dextran elaborated by S. bovis (5). Slime production by S. faecalis. When cultured with pyruvate as the energy source in a complex medium (Tryptone, 1.0%; yeast extract, 0.5%; NaCl, 0.5%; K2HPO4, 0.5%; sodium pyruvate, 1.0%), 9 of 22 S. faecalis strains tested formed a viscous substance which was easily detected by twirling the culture tube (39). Some strains produced a large quantity of the viscous substance, whereas cultures of other strains never formed detectable quantities. Staphylococcus epidermidis has also been shown to produce slime under these conditions (93). The streptococcal slime was detected only when the cultures were incubated under anaerobic conditions or when stationary test-tube cultures were employed. Maximal slime production was observed usually after 18 to 24 hr of incubation and, upon continued incubation, the viscosity of the culture would decrease rapidly until there was no evidence of slime. Capsule formation was not detected when preparations were stained by the amended method of Hiss (32) or the India ink method. A tendency to form slime was noted when some of the strains were cultured with arginine as the energy source. When either pyruvate or arginine is fermented by S. faecalis, the final ph of the medium is always significantly higher (pyruvate, 5.4 to 5.8; arginine, 8.2 to 8.6) than that observed with glucose (4.0 to 4.2). These considerations prompted an experiment in which the glucose fermentation was neutralized continuously, and the amount of slime formed was quantitated. For comparative purposes, cultures with arginine and pyruvate were included. Previous experiments demonstrated that a solubilization of the slime could be effected by adjusting the ph to 8.5 and, thus, this procedure facilitated the centrifugation and removal of the cells. The slime could then be precipitated by adjusting the ph to 4.5 by the addition of mineral acids; the resulting precipitate was harvested by centrifugation, and its dry weight was determined. When a neutral ph value was maintained during the fermentation of glucose, a significant quantity of slime was formed (Table 5). Consequently, the specific energy source is of minor import in slime produc- TABLE 5. Effect of neutralization during fermentation on the production of slime by Streptococcus faecalis strain 2 Neutralization Dry wt of slime Substrate during (mg/200 ml fermentation of culture) Arginine... No Arginine... Yes Pyruvate... No Pyruvate... Yes Glucose... No 0.0 Glucose... Yes tion, and the major factor appears to be the maintenance of a neutral or slightly acidic ph value. In an experiment designed to quantitate the major components of the slime, 500 ml of the complex medium containing 1.0% pyruvate were inoculated with S. faecalis strain 2. After a 20-hr incubation period, the supernatant was decanted (the heavy slime tends to settle on the bottom of the culture flask), and the slime was resuspended in 1 M sodium chloride and adjusted to ph 8.5. Sodium chloride appeared to facilitate solubilization of the slime. After centrifugation to remove the cells, samples were removed for chemical analyses (crude preparation). The remaining supernatant was dialyzed against tap water for 28 hr, during which a slight haze formed. After removing the haze by centrifugation, samples of the clear supernatant solution were removed for subsequent analyses. The slime was then precipitated by adjusting the ph to 4.5, and the precipitate was harvested by centrifugation and redissolved by ph adjustment to 8.5. The chemical analyses of the slime at the

15 344 DEIBEL BACTERIOL. REV. three stages of purification are presented in Table 6. Acid precipitation of the slime altered significantly its chemical composition. From the analyses of the preparation obtained after dialysis, it appears that the slime consists predominantly of protein (54%) and RNA (35%). Samples taken after dialysis and after acid precipitation (the latter sample was dissolved at an alkaline ph) were placed on a Sephadex column and eluted with 1 M NaCl; the fractions obtained were analyzed spectrophotometrically. Acid precipitation of the dialyzed preparation altered its fractional-elution diagram, as com- TABLE 6. Comparison of analyses of Streptococcus faecalis slime in different stages of purification Analysis* Crudet Prepn after Acid dialysis precipitated mg/mi mg/mi mg/mi Carbohydrate DNA... o (6.9)t 0.06 (9.5) RNA (34.5) 0.15 (23.8) Protein (54.2) 0.40 (63.5) Dry weight * The following references state the method employed in the respective analyses: total carbohydrate (26); DNA (199); RNA (122); and protein (112). t Total volume of crude preparation was 140 ml. t Figures in parentheses represent per cent of dry weight. pared with the dialyzed preparation's pattern (Fig. 1). The acid precipitation not only decreased the elution range, but it also increased the concentration of the fractions. These results, coupled with the altered chemical composition of the acid-precipitated preparation, tend to negate this step in the purification procedure. The behavior of the slime material in Sephadex columns, its precipitability at acidic ph values and solubility at an alkaline ph (123), as well as its chemical composition, favor the probability that the major slime component consists of a RNA nucleoprotein. A review of the literature has yielded no information on the production of slime, as distinct from capsule formation, by the enterococci. Reports on the formation of an RNA slime by any microorganism are few, and the elaboration of a RNA nucleoprotein slime has not been reported. Three theories have been advanced to account for the production of an extracellular slime of nucleic acid nature. The formation of DNA (deoxyribonucleic acid) slime by staphylococci may result from an inhibition of deoxyribonuclease production (27). A deranged metabolism, involving the accumulation of slime, has also been suggested (24). Another possibility involves an altered permeability of the cells, such as that observed in DNA slime production by Micrococcus halodenitrificans; however, the same was not true with another halophile, Vibrio costicolus (188). Dialyzed Acid-Precipitated Sample Sample 1.4- E 1U0-1.0 X Protein C E~ 0.6 Acid 0.2 L Fraction Number FIG. 1. Comparison of Sephadex G-50 elution diagrams of two slime preparations. Each fraction contained 4.2 ml. Nucleic acid and protein were estimated spectrophotometrically by optical-density determinations at 260 and 280,, respectively. The chemical composition of the slime isolated from the enterococci, namely, an RNA-protein complex with small concentrations of DNA, does not favor a concept of lysis with the production of slime. With the meager evidence obtained, it would appear that alkaline culture of S. faecalis may result in a deranged metabolism with, perhaps, an attending altered permeability. Unusual Characteristics of Enterococci In recent years, a number of reports have appeared in which physiological characteristics not associated with classical descriptions of enterococci are related to specific strains. This, perhaps, is to be expected as more detailed investigations are undertaken and additional studies on large culture collections are performed to detect these variants. The observations do not detract from the classical descriptions of the

16 VOL. 28, 1964 GROUP D STREPTOCOCCI 345 more commonly occurring strains, but serve to illustrate the flexibility that is incorporated in all bacterial taxa. Motility in streptococci other than the group D organisms is a rarity. The nonmotile nature is often extended to include all streptococci, but this generalization is contrary to past and recent studies with the enterococci. Accounts of motility in enterococcus strains published prior to 1957 were considered by Graudal (66, 67). This investigator studied the characteristics of 129 motile strains, predominantly of fecal origin, and found them all to be enterococci, including both S. faecalis and S. faecium. All but one strain possessed the same flagellar antigen. Subsequent studies of the motile variety indicated that these organisms are widely distributed and not characteristic of any given environment (87, 105). Aside from their motility, these strains are typical enterococci, and the establishment of a varietal status (i.e., mobilis) has little taxonomic value and does not appear to be warranted. S. faecalis has been reported to clot citrated rabbit plasma (56). Only citrate-fermenting strains evidenced this property, and cultures not adapted to citrate required a 7- to 8-hr incubation period in the test system, in contrast to the 3- to 4-hr period required by adapted strains. None of the strains clotted plasma containing other anticoagulants, such as fluoride, heparin, or oxalate, whereas staphylococci clotted plasma regardless of the anticoagulants employed. It was concluded that plasma coagulation was due to metabolic decomposition of citrate rather than the possession of a coagulase enzyme. In another study (17), Aerobacter aerogenes demonstrated similar activity, and 4 of 13 enterococcus strains also clotted citrated plasma. The activity of both the Aerobacter and enterococcus strains was inhibited by sodium fluoroacetate. Peculiarly, 0.1 M potassium cyanide hastened clotting by the enterococci. In a subsequent study, Wood (210) refuted the correlation between citrate utilization and clotting activity. A nutrient broth was prepared in which the culture had grown within a cellophane container. It was reasoned that neither bacteria nor extracellular enzymes would diffuse into the test broth. After incubation, the sterile broth coagulated plasma in less than 5 hr. The diametric results obtained in these two studies (56, 210) have not been resolved, and the "coagulase activity" of the enterococci cannot be stated with certainty. Langston and Williams (107) described the reduction of nitrate by two strains of S. faecium. The reduction was inhibited by oxygen and cyanide, and the optimal ph for activity was 8.0. Although a number of reports have appeared associating this activity with lactobacilli (156), this is the only study in which nitrate reductase activity has been implicated in enterococcal physiology. The production of L( +)lactic acid is generally considered to be characteristic of all streptococci. However, in two studies, the production of inactive lactic acid by enterococci has been reported (104, 111). Evidently, these strains, unlike the typical enterococci, possess a lactic acid racemase. NUTRITION In the following discussion, no attempt will be made to cover exhaustively the nutritional aspects of the group D streptococci, as much of this material has been incorporated in previous reviews (68, 125). Some general aspects will be presented and specific topics which are peculiar to this group of streptococci will be entertained. Vitamins and Cofactors The uniquely simple requirements of S. bovis for exogenous amino acids and vitamins serve to differentiate this organism from all other streptococci. Most S. bovis strains require biotin and are stimulated by thiamine, pantothenate, and nicotinate when the cultures are incubated under atmospheric conditions (135). When cultivated under anaerobic conditions with an increased carbon dioxide tension in a medium containing Tween 80 (thus obviating a biotin requirement), no vitamin requirements were demonstrable (57). Strain variation exists; some require pantothenate, and others are stimulated by thiamin (12). Generally, S. bovis does not require exogenous purines or pyrimidines; however, here again, strain variations do occur (12). Unlike S. bovis, the nutritional requirements of the enterococci are complex. The majority of the strains require biotin, nicotinate, pantothenate, riboflavine, and pyridoxine (132). Characteristically, S. faecium requires folic acid in contrast to S. faecalis, and this requirement has taxonomic

17 346 DEIBEL BACTERIOL. REV. utility in differentiating these species (44). Folate can be replaced by thymine in the nutrition of S. faecium (68). This observation, coupled with the known role of folate in purine synthesis, is indicative of its function in the metabolism of S. faecium. The absence of a folate requirement by S. faecalis suggests not only its synthesis but that of thymine as well. Lipoate requirements of enterococci. O'Kane and Gunsalus (138) first observed that cell suspensions of S. faecalis required an unknown factor present in yeast extract (pyruvate oxidation factor, POF) which was necessary for the aerobic oxidation of pyruvate. In subsequent studies, the factor was purified, crystallized, and finally synthesized, and it is commonly referred to as lipoic acid. Several reviews have appeared covering its chemistry, function, assay, and biological spectrum of activity (70, 71, 191). S. faecalis requires lipoate when grown in a semidefined, casein-hydrolysate medium containing pyruvate as the energy source. This requirement appears to be linked specifically with pyruvate metabolism, as it is not demonstrable when hexoses, hexitols, ribose, gluconate, or glycerol is supplied as the energy source. The adaptive nature of the pyruvate fermentation necessitates growth of the inoculum culture in a complex medium with pyruvate as the energy source prior to its growth in the casein-hydrolysate medium. Glucose-grown inocula give erratic and variable responses when tested in the semidefined medium (energy source, pyruvate) with various concentrations of lipoate (38). When a pyruvate-adapted inoculum is used, a dose-response curve is obtained with 0.8 to 4.0 m/ug of D, ilipoate per 10 ml of medium. It has been demonstrated previously that only the L form of lipoate is active for S. faecalis (86). Thus, these concentrations represent one-half of the utilizable lipoate. In our laboratory, a testtube bioassay method (similar to that employed in the B-vitamin assays with other lactic acid bacteria) has been used to quantitate lipoate with S. faecalis strain FB82. The assay is performed with 10 ml of the casein-hydrolysate medium (energy source, pyruvate) in 18-mm test tubes and varying concentrations of lipoate (0.8 to 4.0 mjug per 10 ml of medium). The pyruvateadapted inoculum is washed once with water and diluted tenfold, and one drop is used to inoculate the assay tubes. After 18 to 20 hr of incubation at 37 C, the turbidity is measured, and a doseresponse curve is plotted. The method compares favorably with other assay methods and offers the advantage of simplicity. Moreover, the requirement is specific in that it cannot be replaced by acetate, nor does acetate exert a sparing effect. Initial attempts to grow S. faecalis in a lipoatesupplemented, casein-hydrolysate medium with pyruvate as the energy source under conditions of strict aerobiosis (vigorous shaking in air) met with failure. Regardless of the method employed to grow the inoculum (aerobically or anaerobically), no growth was manifested in TABLE 7. Effect of oxygen on the lipoate requirement of Streptococcus faecalis strain FB82* Condition of incubation Concn of DL-lipoate necessary for half-maximal growth (myg/10 ml of medium) Anaerobic Static-atmospherict Aerobict * Casein-hydrolysate medium; 20 hr. Energy source, 1.0% pyruvate. t Cultured in 18-mm test tubes (10 ml). t Cultured in 125-ml flasks (10 ml) and incubated on a reciprocating shaking apparatus. aerobically incubated cultures. Subsequent experiments revealed that the optimal concentration of lipoate under aerobic conditions was significantly higher than that under anaerobic conditions, and thereby offered an explanation for the failures encountered in the initial experiments. Thus, the amount of lipoate required for growth may be expressed as a function of the oxygen tension under which the culture is incubated (Table 7). No explanation is available to account for this increased lipoate requirement, although the results may indicate either an oxidation or partial destruction of the lipoate or an increased requirement by the organism when cultured aerobically. The addition of reducing compounds, such as thioglycolate and ascorbate, decreased the lipoate requirement, when cultures were incubated under static-atmospheric conditions, to a level approximating that observed when the cultures were incubated anaerobically. However, when cultures were incubated aerobically with these compounds in concentrations as high as

18 VOL. 28, 1964 GROUP D STREPTOCOCCI 347 TABLE %, no sparing effect on the lipoate requirement was demonstrable. When S. faeium is cultured with any fermentable carbohydrate under anaerobic or staticatmospheric conditions of incubation, lipoate is not required. In contrast, 11 of 20 strains tested required lipoate when cultured aerobically with glucose as the energy source. Additional experiments with these 11 strains indicated a lipoate requirement for aerobic growth with all fermentable substrates. Included in the 52 compounds tested were pentoses, hexoses, polyols, di- and tri-saccharides, and organic acids. Conversely, lipoate was not required for growth of the redentified factor were required (197). Recently, a reinvestigation of the process established the identity of the unknown factor as lipoic acid (31). However, the requirement of this strain for lipoate, regardless of the substrate employed under aerobic conditions, may indicate its involvement not only in glycerol oxidation but, more generally, in aerobic metabolism. In view of the varied reports regarding lipoate requirements of the enterococci, an attempt will be made to collate the independent studies and perhaps clarify the results so that orientation for further study may be enhanced. To recapitulate, studies in our laboratory have demonstrated an Comparison of the effect of energy source and oxygen on the lipoate requirement of Streptococcus faecalis and S. faecium Condition of incubation* Organism Energy source Aerobic Anaerobic + Lipoate - Lipoate + Lipoate - Lipoate S. faecalis Glucose FB82 Pyruvate S. faecium Glucose F24 Ribose S. faecium Glucose Igau Ribose * Optical density X 100; 48-hr incubation period. maining nine S. faecium strains, regardless of the energy source or condition of incubation. Growth responses of S. faecalis, and lipoate-requiring and nonrequiring strains of S. faecium, are compared in Table 8. Neither S. faecalis FB82 nor nine other representative S. faecalis strains require lipoate when cultured aerobically with glucose as the energy source. The lipoate requirement for one-half maximal growth of S. faecium (energy source, glucose) under aerobic conditions is approximately 0.45 mjug per 10 ml of medium. Some strain variation was encountered, and growth with concentrations of lipoate producing submaximal responses was erratic. As noted in Table 7, this concentration is significantly lower than that required by S. faecalis under aerobic conditions, and approximates the level required by S. faecalis under anaerobic conditions. In a study of the cofactor requirements for glycerol oxidation by cell suspensions of S. faecalis F24 (S. faecium), it was observed that thiamine, nicotinate, riboflavine, and an uniabsolute requirement for lipoate when S. faecalis is grown with pyruvate as the energy source. This requirement is specific and cannot be replaced with acetate. The amount of lipoate required for growth may be expressed as a function of the oxygen tension under which the culture is incubated, and significantly higher concentrations are required under highly aerobic conditions. S. faecium, on the other hand, does not utilize pyruvate as an energy source and does not require lipoate or acetate when cultured anaerobically with any energy source. However, when cultured aerobically, approximately 50% of the strains require lipoate regardless of the energy source. In contrast, S. faecalis does not require lipoate when grown aerobically with glucose as the energy source. In a study employing S. faecalis 734, the organism was incubated under static-atmospheric conditions with glucose as the energy source, and it was observed that acetate or a preparation of lipoate (POF) was not required for growth (113). However, cultures incubated with glu-

19 348 DEIBEL BACTERIOL. REV. conate as the energy source manifested an acetate or POF requirement. Carbon dioxide (bicarbonate), or compounds whose metabolism resulted in carbon dioxide production, replaced the acetate or POF requirement in gluconategrown cultures. The fermentation of gluconate by this strain suggests its classification (in the current scheme) as S. faecalis. However, studies in our laboratory have failed to associate a lipoate requirement with the gluconate fermentation by other S. faecalis strains. The relationship of the lipoate requirement (as well as the acetate and carbon dioxide effect) with the gluconate fermentation merits further study and may involve strain variation. Shockman (175) observed that acetate is required only under aerobic conditions when S. faecalis R (S. faecium) and another strain (S. faecalis ATCC 9790, not yet identified in terms of current classification) are cultured with glucose as the energy source. A combination of lipoate and thiamine replaced the acetate requirement. Under anaerobic conditions of incubation, neither acetate nor lipoate was required. Thus, the results obtained in our laboratory with the lipoaterequiring S. faecium strains are identical to those observed previously by Shockman. These studies suggest that the aerobic requirement of S. faecium for lipoate can be replaced by acetate. It has been demonstrated (192) that relatively large amounts of propionate (1 to 3 mg per ml of medium) will inhibit the growth of S. faecalis R (S. faecium) and that this inhibition can be reversed competitively with acetate or protogen (lipoate). Oleate reversed the inhibition noncompetitively. An assay method was proposed subsequently (191) for acetate or lipoate with propionate-inhibited cultures of S. faecalis ATCC 8043 (S. faecium). Hill (81) first observed an inhibition of growth with propionate and S. faecalis 8043 (S. faecium) and postulated that the inhibition resulted from the combination of propionate with CoA. Collectively, the results obtained with the S. faecium strains indicate that acetate is an essential metabolite regardless of the oxygen tension. Anaerobically, this requirement can be demonstrated by propionate inhibition, and the inference can be made that a sufficient quantity of acetate is metabolically produced to satisfy growth requirements. The aerobic requirement for exogenous acetate or lipoate may reflect either an inhibition of acetate generation or an increased physiological requirement under aerobic growth conditions. Although these considerations may aid in an explanation of the results obtained in growth studies, the lipoate requirement in glycerol oxidation by cell suspensions may involve other functions, such as electron transfer. This role of lipoate has been established in the catabolism of lactate by Butyribacterium rettgeri (100). The possible inhibition effected by propionate either aerobically or anaerobically with glucose or pyruvate as the energy source has not been investigated in the metabolism of S. faecalis. In summation, it would appear that progress has been made in elucidating the role of lipoate in enterococcus nutrition; however, complete resolution of the problem must await further study. Amino Acids Growth requirements. In the first report describing the amino acid requirements for the growth of S. bovis, the absence of a requirement for any specific amino acid was noted (135). In a medium containing only arginine and glutamate, satisfactory serial growth occurred with the majority of the strains studied. Supplementa, tion of a medium containing arginine with leucine, isoleucine, threonine, and equal amounts of phenylalanine and tyrosine produces an antagonistic effect presumbly by blocking the synthesis of other amino acids (200). Subsequently, an enhanced growth response was observed in the arginine-containing medium when bicarbonate was added (150). Bicarbonate also reversed the inhibitory effect of isoleucine, leucine, and other amino acids, and the stimulatory effect was associated with growth in the lag phase (151). As discussed previously, Wright (212) studied the incorporation of carbon dioxide, associated its fixation in asparate, and noted the generalized stimulation of growth which was ascribed to an increased concentration of intermediates necessary for protein and nucleic acid synthesis. Paul (143) observed a requirement for numerous amino acids and a peptide factor for satisfactory growth of strains isolated from sheep and bovine sources. These results indicate a more complex nutrition than that described by previous investigators. Included in the study were strains of S. equinus, and their requirements paralleled those of S. bovis. In this study, no attempt to grow S. bovis

20 VOL. 28, 1964 GROUP D STREPTOCOCCI 349 in a simple medium with arginine and glutamate was reported. The complex requirements observed may reflect amino acid antagonisms, as demonstrated previously. Almost simultaneously, reports from two laboratories indicated that S. bovis was capable of utilizing ammonium salts as a sole source of nitrogen (152, 208). Growth with ammonium salts was stimulated by acetate (152) or bicarbonate, and glutamine also served as a sole source of nitrogen (208). Growth with ammonium salts, but not with glutamine, required an unknown substance formed by autoclaving phosphate with glucose (209). The addition of potential electron acceptors or a lowering of the ph value of the medium replaced this requirement. Asparagine also satisfies the nitrogen requirement (18), and enzymatic studies indicate that glutamine and asparagine are the most likely primary products of ammonia incorporation (18). The unique ability of S. bovis to utilize ammonium salts as a sole source of nitrogen is not shared by other Streptococcus species. In comparison, the enterococci possess relatively complex requirements in that at least ten amino acids are necessary for growth, and still others are significantly stimulatory (116, 132). Variations in the specific requirements occur and may represent strain differences. Also, other factors such as the composition of the medium and the method of sterilization employed may be implicated (116). As yet, the requirements of S. faecium and S. faecalis have not been compared. In our laboratory, it has been observed that replacement of an acid and enzymatic hydrolysate of casein with amino acids results in an inferior growth response. The possible involvement of peptide stimulation was considered. However, Langston (personal communication) demonstrated a doubling of the growth response of S. faecalis with 0.25% sodium carbonate in an adequately buffered synthetic medium. These results parallel the carbon dioxide stimulation obtained with S. bovis, and indicate the desirability of additional studies. Transaminase activity and D-amino acid utilization. In a study of the transaminase activity of S. faecalis R (S. faecium), the a-keto acids corresponding to histidine, leucine, isoleucine, tryptophan, tyrosine, and valine were transaminated, and the activity was pyridoxal-linked (68). The inactivity of a-ketoglutarate and oxalacetate was noted, and deemed peculiar in view of the known glutamic and aspartic transaminase activity. The impermeable nature of these compounds was suggested to account for their inactivity. Only the a-hydroxy acids corresponding to leucine and valine were utilized, and a dehydrogenation to the a-keto acid and subsequent transamination were shown to be involved. The pyridoxal-linked, alanine racemase activity of the enterococci, and the reciprocity of D-alanine and pyridoxal have been considered previously (68). A methionine racemase, which is not pyridoxal-linked, has been reported (176). This racemase is active in the post-log phase of growth and affords the utilization of D-methionine but only in the presence of the L isomer. Unlike other amino acids, L-methionine is not assimilated quantitatively by S. faecalis 9790, and approximately one-fifth of it is metabolically converted to extracellular D-methionine which is subsequently oxidized nonbiologically to the sulfoxide (177). SEROLOGY Group Antigen The early literature concerning the serology of the group D streptococci was considered extensively by Skadhauge (181). As mentioned previously, all enterococci fulfilling the Sherman criteria possess the group D antigen. In addition, the presence of this antigen has been demonstrated in S. boyts and S. equinus. The intracellular location of the antigen in all group D streptococci, as well as its chemical identity, merit detailed consideration. Initial work pointed out the variability of response and the difficulty of producing a potent group D antiserum in rabbits (166). Strain variation was observed, and repeated immunizing doses were necessary over protracted periods of time. Quite often, commercially produced, group antisera are encountered that possess extremely low levels of group-specific antibody (168). In recent years, studies by Shattock and others have offered an explanation for these peculiar results. In contrast to the cell-wall location of the group antigen in other streptococci, the group D antigen occurs intracellularly (54, 92). Previously, Shattock (166) observed the ease with which the antigen could be demonstrated in strains of S. bovis when the cell preparation employed for immunization

21 350 DEIBEL BACTERIOL. REV. was first mechanically shaken to disrupt the cells. Another factor enhancing serological demonstration of the antigen, and also associated with its intracellular nature, is its concentration by alcoholic precipitation prior to use in the precipitin test (166). The intracellular location of the antigen in all group D streptococci (92) offers an explanation for the refractory nature of most strains to the serological demonstration of the group antigen. Shattock (168) surmised that the antigen is not readily available at the site of antibody formation, and that disruption of the cell afforded liberation of the antigen and thus indirectly contributed to its antigenicity. Likewise, extraction and precipitation effects concentration and detection of the antigen in extracts. The procedures employed to prepare the antigen for immunization and the extracts for serological testing were considered by Shattock (166). Medrek and Barnes (118) demonstrated high yields of the antigen when the culture medium contained 0.5 to 1.0% glucose and poor yields in media containing low glucose concentrations. Characteristically, growth of S. bovis, S. equinus, and S. faecium in the Todd-Hewitt medium resulted in an inferior group reaction in contrast to the results obtained with S. faecalis. No explanation was available for this difference, although growth in glucose-containing media resolved these differences, and group reactions were enhanced in all species. The possible involvement of nutritional conditions affecting the synthesis of the group antigen has been suggested (92), since media which support excellent growth of the organism do not always produce cells with substantial quantities of antigen. Equivocal results obtained in cross-reactions of S. bovis extracts with enterococcal group D sera have been noted (130, 165, 171), and Niven (130) observed the failure of absorption tests to resolve these cross-reactions. These results led to Shattock's demonstration of the group D antigen in S. bovis (166). Subsequently, Smith and Shattock (183) observed variable results with intact cells of both S. bovis and S. equinus. However, in disintegrated preparations the antigen was demonstrable in both species. The results with S. equinus were confirmed in another laboratory (60), and Jones and Shattock established the identity of the group D antigen in the enterococci and S. bovis (92). The peculiar location of the group antigen in the group D organisms serves to differentiate them from all other streptococci. The exact location of the antigen has been investigated, and Hijmans (79) observed the absence of the antigen in L forms of enterococci, thus obviating its possible cytoplasmic location. Shattock and Smith (169) further reported the occurrence of the antigen in cultures of one L form after continual subculture in a medium containing an increased glucose concentration. However, approximately 97% of the antigen was found in the medium and not within the L form, and 85% of the group antigen was released during digestion of intact cells with a phage-associated, cell-wall lysin. These results indicated the absence of the antigen in the cytoplasm and, when associated with previous studies regarding its absence in the cell wall, led to the conclusion that the antigen is located between the cytoplasmic membrane and the cell wall. These conclusions have been confirmed in another study (206). Recently, the chemical nature of the group D antigen has been elucidated (205, 206) and found to be a peculiar type of glycerol-teichoic acid. Unlike other teichoic acids in which the D-alanine is linked to the polyol, the group D antigen contains D-alanine in ester linkage with the hydroxyl groups of glucose, and the glucose is esterified to the C2 group of glycerol. The glycerol molecules are joined through phosphodiester linkages in the 1, 3 positions to form the polymer. The high sugar content and amino acid attachment to the glucose residues were considered peculiar to the group D antigen (205). Some variation was noted in the teichoic acids from S. faecalis 39 and S. faecium 8191 (205). In S. faecalis 39, the side chain consisted of a glucose disaccharide (kojibiose) in contrast with the glucose trisaccharide (kojitriose) of S. faecium In further contrast, the S. faecium polymer contained ilysine as well as D-alanine, whereas the S. faecalis complex contained only D-alanine. Evidence was presented indicating that the immunological specificity resided in the carbohydrate side chains, since no reaction occurred with a poly-glycerol phosphate preparation obtained from a group A Streptococcus. Indirect results and the analogous lack of amino acid participation in the immunological specificity of Staphylococcus teichoic acids led to the conclusion that amino acids were not involved as immunological determinants in the group D polymer.

22 VOL. 28, 1964 In the isolation of the group D teichoic acid, the formation of complexes with cellular RNA was noted. It was concluded, however, that teichoic acid did not occur in the cell as an RNA-teichoic acid complex and that, in the course of cellular disruption and purification, the complex was formed (205). In the traditional method of preparing extracts for serological studies, the antigen is acid-extracted from the cells with heating. The lability of glucosyl residues on the side chain of the polymer was suggested as a possible source of error in this procedure (205), which may account for the serological identity of the group D antigen in the enterococci and S. bovis, as reported previously (92). The possibility of subtle differences in the composition of the antigen as a function of species exists; however, further studies are necessary for affirmation or negation of this speculation. GROUP D STREPTOCOCCI Type Antigens S. faecalis. Skadhauge (181) observed both thermostable 0 antigens and thermolabile K antigens in S. faecalis strains. The K antigens were associated with the surface of the cell (envelope antigen), and were deemed unsuitable for serological studies because of variability incurred by storage and repeated subculture of the strains. Six types were established on the basis of the 0 antigens. In his study of 350 enterococcus strains, Nyman (136) reported that 90% of the typable strains belonged to three types, and one of the three types was associated principally with strains from pathological material. The unpublished studies of Sharpe, as quoted by Shattock (168), indicate that approximately 20 types of S. faecalis exist, and that these antigens are distinctive and not shared by other members of the group D streptococci. Elliott (54) examined the chemical composition of the type-specific antigens in three different serological types of S. faecalis and one S. faecium var. durans type. In each instance, the antigen was composed primarily of glucosamine, rhamnose, and glucose. The antigens formed an integral part of the cell wall, and this situation is analogous to the group antigens of other streptococci. It is assumed that these type antigens are identical with the 0 antigens of Skadhauge. S. faecium. The cell-wall, type-specific antigens of S. faecium and its variety durans are also distinctive within this physiological group (no crossreactions with S. faecalis), and a sharing of common type antigens exists between strains of S. faecium and its variety durans (164). A common type-specific antigen has been reported to occur in two strains of S. faecium (163) and a strain of S. lactis (group N). Previously, Niven (130) also noted the existence of a common type antigen in certain enterococcus and S. lactis strains. S. bovis. In an extensive study of the type specificity of S. bovis, Medrek and Barnes (119) observed that 75 of 152 strains were typable, and these 75 strains fell into 12 types. In one type, represented by one strain, the antigen was located in the cell wall; in the remaining types, it occurred in the capsule. Heterogeneity was indicated by the inability of a number of S. bovis strains, isolated in other studies, to react with the 12 type-specific antisera prepared in this study. In addition, none of the 12 type sera reacted with other group D organisms, indicating serological distinction from these streptococci. However, in other studies it was observed that S. bovis shares a common type antigen with streptococci of group E (145) and group N (146). Clearly, the type antigens of S. bovis are somewhat heterogeneous. They may differ in their location, but, unlike the enterococci, they occur generally in the capsular material. The type-specific antigens of S. equinus have not been studied. CELL STRUCTURE Cell-Wall Chemistry 351 Since a comprehensive review of cell-wall metabolism and composition appeared recently (144), only specific topics related to the group D streptococci will be included in this discussion. As stated previously, the type-specific antigens, located in the cell wall, are species specific in the enterococci, and on this basis one might expect chemical variation in the wall composition of these two species. However, in the limited studies conducted to date, no significant differences have been observed. This may reflect either subtle differences in composition or variance in chemical structure within the realm of similar composition. Further, the taxonomic dichotomy of S. faecalis and S. faecium is not, as yet, recognized widely, and minor variations in cell walls associated with individual species may have been overlooked. The overall composition of the cell walls of the

23 352 DEIBEL BACTERIOL. REV. group D streptococci is similar. In the only comparative study of group D wall composition (three S. faecalis strains and one each of S. faecium, S. bovis, and S. faecium var. durans were examined), lysine, glutamate, alanine, rhamnose, glucose, and glucosamine were the principal compounds detected, and each was common to all species (92). Galactosamine was also a principal component in all species except S. bovis. Mannose (a minor component) has been reported to occur only in S. faecalis; however, in one strain (S. faecalis 370) its presence was detected in two laboratories (34, 92) and its absence was reported from another laboratory (182). Also, Cummins and Harris (34) detected mannose in S. faecalis 6782, in contrast to the results obtained by Salton (157). Considerable variation has been observed in the occurrence of galactose. Two of three S. faecalis strains contained this sugar, as did a strain of S. faecium var. durans and S. bovis; the absence of galactose was noted in S. faecium 4MEC (92). As in other lactic acid bacteria, four major fractions of polymeric compounds (mucopeptide, "protein," and ribitol and glycerol teichoic acids) have been identified (88) in S. faecalis R (S. faecium). D-Alanine occurs in the mucopeptide, and both rhamnose and D-alanine are common to the two types of teichoic acid. Unlike the ribitol teichoic acid, the glycerol teichoic acid does not contain an amino sugar. The occurrence of both types of teichoic acid in the wall of S. faecalis was observed in another study (3). As yet, the relationship between these teichoic acids and type specificity has not been studied. Cytoplasmic Membrane Chemistry The chemical composition of the membrane in S. faecalis 9790 was found to differ in preparations of cells taken from the log and stationary phases of the growth cycle (178). Log-phase membranes contained more protein (49 to 55 %) and less lipid (28 %) than did stationary phase membranes (42% protein, 36% lipid). A large number of various amino acids occurred in the protein fraction, and the relative absence of D-amino acids was noted. The lipid-phosphorus concentration (3%), coupled with the low nitrogen content, indicated that the lipid consists primarily of phosphatidic acids. In another study (15), the relative absence of rhamnose in membrane preparations of S. faecalis R (S. faecium), as compared with its occurrence in the cell wall, was noted and could be employed to indicate the degree of wall contamination in membrane preparations. Nutritional Aspects of Wall Synthesis Studies in Shockman's laboratory, concerned primarily with S. faecalis 9790, divorced cell-wall synthesis from active growth (179, 180). Wall synthesis continued after growth cessation in the absence of amino acids required for protoplasmic protein synthesis but in the presence of amino acids necessary for wall synthesis (i.e., lysine, alanine). Depletion of wall-required amino acids in the medium resulted in rapid lysis. Hydroxylysine does not replace the lysine growth requirement for S. faecalis R (S. faecium), but it does exert a sparing effect (147) and prevents lysine-depletion lysis (186). The absence of hydroxylysine conversion from lysine has been demonstrated, and cells grown with lysine contain no hydroxylysine (194). The marked stimulation of growth by hydroxylysine observed in lysinelimiting media has been associated with active hydroxylysine incorporation in the cell wall. Cells containing hydroxylysine in their walls were more resistant to autolysis, the lytic effects of neuraminidase and penicillin, and the disruption by sonic oscillation, in comparison with cells grown solely with lysine (187). Neuraminidase Sensitivity The differential ability of neuraminidase to lyse various living enterococcal species has not been studied extensively. Generally, some strains are refractory to the lytic effect of this enzyme, and others are lysed rapidly. Chesbro (28) observed some indication of species variation within the enterococci. S. faecalis appeared to be relatively resistant in comparison with most S. faecium strains. Lysis of S. faecium was enhanced by growth in a carbonate-containing medium and accentuated by growth at ph 9.4. In contrast, supplementation of the medium with potassium or sodium ions decreased the neuraminidase sensitivity. Abrams (1), working with S. faecalis 9790, associated the neuraminidase sensitivity of his strain with the absence of wall galactose, since Salton's neuraminidase-resistant strain contained this sugar in the cell wall (158). This relationship has been questioned by Perkins (144). Utilizing the action of neuraminidase, Bibb and Straughn (15) prepared protoplasts with S. faecalis 9790

24 VOL. 28, 1964 GROUP D STREPTOCOCCI 353 (178), S. faecium HF8AG (28), and S. faecalis F24 (S. faecium). Bleiweis and Zimmerman (16) observed that spheroplast formation could be invoked by the action of neuraminidase on cells grown previously in the presence of penicillin. The employment of penicillin in the production of enterococcal L forms was studied by Hijmans and Kastelein (80). Using heat-treated cells (70 C for 15 min) and the synergistic action of neuraminidase and trypsin, Hartsell and Caldwell (78) proposed a lytic scheme for the differentiation of the group D streptococci. The excellent agreement of this scheme with the physiological classification suggested its employment as a rapid method for the identification of these streptococci. In the procedure, S. bovis can be separated from the enterococci because it is resistant to the combined enzymes (neuraminidase and trypsin) while S. faecalis and S. faecium are lysed. S. equinus was not included in the study. When the procedure was repeated with another sample of heated cells, an increase in the neuraminidase content, the omission of trypsin, and the addition of base at the end of the reaction period resulted in the lysis of S. faecium and its variety durans but not of S. faecalis. S. faecium could be differentiated from its variety durans in that the latter agglutinated when acidified cell suspensions were heated. Confirmation and extension of these results to include S. equinus are lacking, and additional study is desirable, especially in view of the possible ease and rapidity of species identification within the group D streptococci. Bacteriophage Publications prior to 1947 dealing with the relationship between bacteriophage and group D streptococci were considered by Evans and Chinn (55). These investigators observed the lysis of most enterococci by a single phage strain; less than 50% of the strains were lysed by another type. No correlation between phage-induced lysis and the classification scheme employed at that time could be made. Recently, the possible specificity of phages for the various group D species has been investigated and two serologically distinct phages either separately or in combination lysed 91% of the S. faecalis strains (30). A third serologically distinct phage lysed S. faecium, S. bovis, and atypical S. faecalis strains. As yet, a definite separation of group D species by lysotyping has not been accomplished. Some specific characteristics of group D bacteriophages have been reported (155). Bleiweis and Zimmerman (16) employed a lytic enzyme, obtained from a phage-infected culture of S. faccalis, to induce protoplast formation, and this enzyme was used by Shattock and Smith (169) in their study on the location of the group D antigen. CURRENT CONCEPTS OF GROUP D STREPTOcoccUs TAXONOMY Two characteristics, common to all group D streptococci, are the ability to grow at 45 C and in media containing 40% bile. S. bovis and S. equinus do not initiate growth at 10 C, at ph 9.6, or in media containing 6.5% NaCl, and these species usually fail to hydrolyze arginine or decarboxylate tyrosine. Generally, the enterococci give opposite reactions in these tests; thus, the primary differentiation of the two physiological entities is afforded. As mentioned previously, use of the term enterococcus or enterococci to connote specifically S. faecalis or S. faecium, or both, has been employed in this review. The author does not desire to stress or suggest the employment of this terminology; however, its facility and convenience in this restricted sense prompted its utilization. Enterococci To differentiate the enterococci from other streptococei, Sherman (170) suggested the use of certain tolerance tests-specifically, the ability of these organisms to grow in the presence of 6.5% sodium chloride, at ph 9.6, and at 10 and 45 C, and to withstand heating at 60 C for 30 min. These tests are marginal in nature, and the occurrence of strains that fail to give a positive result under one or more of these conditions is to be expected. Some indication of the critical nature of factors associated with the performance of these tests has been demonstrated. White (203) reported that tolerance to 60 C for 30 min was correlated closely with the ph value of the medium. At ph 6.8, the highest heat tolerance occurred, and the ability to withstand this temperature dropped precipitously on either side of this ph value. In addition, Chesbro and Evans (29) showed a marked effect of medium composition and buffering system on the ability of enterococci to grow at ph 9.6. The inclusion of oleate in the medium and the replacement of

25 354 DEIBEL BACTERIOL. REV. glycine buffer with carbonate facilitated growth at ph values up to Still another consideration is the physiological state of the organism. Recently isolated strains are encountered which fail to grow under one or more of the tolerance conditions. However, after transfer in laboratory media, these strains may evidence growth under the test condition that previously gave a negative result (114). In all identification procedures, dependence must be placed on a spectrum of characteristics possessed by the strain in question, and its failure to comply in a few specific tests does not constitute sufficient grounds to negate speciation if it conforms with the overall species description, As in any taxonomic scheme, the occurrence of some transitional types is to be expected. Caution must be exercised, however, to define these types and not to avoid their species establishment on the premise of a few differing characteristics. In the past, speciation of the enterococci has been confusing, although an indication of some order is emerging. Orla-Jensen (139, 140) and, subsequently, Gunsalus (69) recognized the two grossly different, physiological types of enterococci, and the studies of Shattock (167), Skadhauge (181), and Barnes (9) provided sufficient characterization to effect a species definition of S. faecium and S. faecalis. Subsequent investigations in a number of laboratories have substantiated the validity of this speciation. Thus, on the basis of tellurite sensitivity, tetrazolium reduction, differential susceptibility to neuraminidase, fermentation pattern, various energy-utilization tests, nutritional differences, and serological type specificity, S. faecalis can be differentiated from S. faecium (Table 9). Accessory tests, although not as conclusive as those mentioned previously, include growth at 50 C by S. faecium, rapid reduction of litmus (8 hr) in milk cultures by S. faecalis, and reactions in blood-agar (Table 9). From the array of differentiating characteristics, it would appear that this speciation rests on a firm foundation. Some generalizations regarding S. faecalis and S. faecium may be drawn from the information gained to date. S. faecium grows at a higher temperature and tolerates heat to a greater extent than does S. faecalis. This explains the common occurrence of S. faecium in marginally processed, canned hams. Also, there are indications that this species is more tolerant to highly alkaline condi- TABLE 9. Physiological characteristics employed to differentiate group D streptococci Physiological characteristic S. fae- S.feaeS- bovis S ui calis cium S. "vṡcus- Fermentation (acid production) Arabinose... Sorbitol... Melibiose.. Melezitose... Mannitol... Glycerol (anaerobic)... Energy utilization Pyruvate... Arginine... Citrate... Serine... Agmatine... Gluconate... Malate... Hydrolysis Arginine... Agmatine... Starch... Growth ph % NaCl C C... loc... 40% bile % tellurite... Folic acid requirement... Tyrosine decarboxylation... Tetrazolium reduction... Rapid reduction of litmus (8 hr)... Blood-agar reaction... Slime production from sucrose. Species _* V + ± + fl,a vt V + V 0t * The symbols + and - represent a positive or negative test, respectively. t V denotes strain differences and a variable result. I Zero indicates insufficient data available. tions of growth (29). In contrast to S. faecium, S. faecalis enjoys a metabolic diversity reflected by its ability to obtain energy from a wide variety of organic compounds. ± + V 0 av a

26 VOL. 28, 1964 GROUP D STREPTOCOCCI 355 S. bovis and S. equinus Serological studies have established definitively the presence of the group D antigen in S. bovis and S. equinus (92, 166), and consequently these organisms must be included in the group D streptococci in spite of the physiological differences that separate them from the enterococci. However, the distinctiveness of these two species has not been resolved fully, and their overall physiological characteristics are similar. Slime production from sucrose by S. bovis and the inability of S. equinus to ferment lactose are the chief differentiating characteristics. In a collection of 87 S. bovis strains, a correlation was noted between slime production, mannitol fermentation, and a-hemolysis (37). Seventy-one strains formed slime, produced an a reaction in blood-agar, and failed to ferment mannitol. The remaining strains did not produce slime, but 12 fermented mannitol. On the basis of the fermentation pattern of the two groups, Smith and Shattock (183) observed sufficient variation to suggest separation into two species. In addition, S. equinus does not hydrolyze starch to the level of reducing sugars. S. bovis hydrolyzes starch to the monosaccharide level, and produces acid in contrast to the partial hydrolysis and absence of acid formation effected by S. equinus (52). From the studies completed to date, it would appear that the polysaceharide-forming, mannitol-nonfermenting strains of S. bovis form a taxonomic entity. However, the polysaccharidenonforming, mannitol-fermenting types, as well as the S. equinus strains, are not c4early defined. Undoubtedly, further study is indicated prior to a definitive assessment of speciation among these streptococci. In summary, the group D streptococci may be separated into two physiological groups: the enterococci and the S. bovis-s. equinus group. A large number of characteristics can be employed in the separation of S. faecalis and S. faecium, and these streptococei appear to be distinctive at the species level. The S. bovis-s. equinus group is not separated as readily, and additional study is indicated prior to definitive speciation. COMMENTS REGARDING ISOLATION The increasing attention associated with the occurrence of group D streptococci in foods and water necessitates an improved methodology regarding their selective isolation, quantitation, and species identification. In the past decade, a multitude of various selective media have been suggested for facilitating isolation and quantitation of these bacteria, and confusion exists regarding the efficacy of over 30 different media that have been proposed for this purpose. Moreover, disagreement is encountered as to what constitutes a "fecal Streptococcus," in that some investigators include S. salivarius and S. mitis, as well as other streptococci, in this category. Compounding this rather fluid situation is the lack of agreement in speciation and the inadequate testing of the various media to ascertain their specificity as well as the possible differential inhibition among specific group D species. Some indication of species selectivity in different media was reported by Chesbro and Evans (29). It was observed that cultivation in a carbonate-containing medium at ph 10.5 favored the isolation of S. faecium. In contrast, use of an azide-dextrose medium favored the isolation of S. faecalis. The variety of media available to isolate group D streptococci is similar to the situation encountered in the detection of Salmonella and in the quantitation of Staphylococcus aureus. Obviously, as is the case with the latter microorganisms, there is no single medium that is equally satisfactory for the isolation and quantitation of group D streptococci encountered in diverse environments. In addition, factors such as type of sample, numbers of group D streptococci and their proportion to other bacteria, and the species of streptococci to be quantitated are important factors when choosing a specific medium. As yet, these considerations have not been resolved. Recently, a medium designed for the selection and enumeration of enterococci was proposed which contains only citrate as the energy source (160). It would be expected that this medium does not afford quantitation of the enterococci, since S. faecium does not utilize this substrate. However, the medium may have merit in the isolation of S. faecalis to the exclusion of other group D streptococci. It would appear that a critical assessment of a small number of different selective media is in order to determine their species specificity and quantitative ability. Such a study would aid in the resolution of the overall problem. Future studies regarding the design of selective media may well center on isolation and quantitation of specific species rather than the group as a whole. In this respect, employment of tellurite or certain energy sources such as pyruvate, citrate, or ar-

27 356 DEIBEL BACTERIOL. REV. ginine may aid in the quantitation of S. faecalis. Methods for the isolation of S. faecium may center on this species' tolerance to higher ph values in the medium or the ability to grow at 50 C, or both. In addition, the design of a medium capitalizing on the simple nutrition of S. bovis may afford its quantitation. A factor which offers promise in the speciation of a large number of isolates is the differential scheme for identification based on neuraminidase sensitivity (78). This procedure, in addition to some key physiological tests, may result in a more rapid identification of isolates in studies involving floral characterizations. ECOLOGY Studies involving the distribution of group D streptococci have employed media designed to isolate the group as a whole rather than specific species. As mentioned previously, these media may exert an influence on recovery and quantitation of these streptococci. To obviate the pitfalls of a single medium, some investigators have employed a number of media. Variation in the group D streptococcal flora of animals has been associated with geographical location, diet, age, and species of the host animal. In some instances, seasonal effects have been observed. Also, differences in the predominant flora of the same host examined at different time intervals have been reported. Thus, in ecological studies relating the numbers of group D streptococci in the alimentary canal, attention must be focused not only on the methodology employed but also on a number of factors which may influence their occurrence in the host species. In some studies, a direct comparison of the results obtained is difficult due to the method(s) used in isolation or the taxonomic criteria employed, or both. Detailed aspects of the overall problem have been discussed previously (99, 154, 168). In spite of the potential for variation, significant differences in the distribution of group D Streptococcus species have been observed in host animals, and the predominating species have been identified in the more common hosts. In general, the enterococci predominate in the human alimentary canal while the S. bovis-s. equinus species are found in superior numbers in cattle, swine, sheep, and horses. Species Distribution in the Human Host In Great Britain, Cooper and Ramadan (33) reported the predominance of S. faecalis (69%) in human feces. S. faecium var. durans comprised 4% of the isolates, and 25% were classified as "atypical S. faecalis" (S. faecium?). Only one strain of S. bovis was encountered in the study. In the United States, Bartley and Slanetz (13) observed a greater frequency of S. faecalis over S. faecium; however, in this study an organism resembling S. inulinaceus (now considered S. bovis) characteristically outnumbered both S. faecalis and S. faecium. An extension of this observation merits consideration. Generally, the S. bovis-s. equinus species are not found in significant numbers in the human host. Also in the United States, Kenner, Clark, and Kabler (96) observed the predominance of enterococci over other streptococcal species in human feces. In contrast to the higher frequency of S. faecalis as discussed above, approximately equal or superior numbers of S. faecium are found in human hosts of continental Europe. In France, Buttiaux (21) observed a higher frequency of S. faecium over S. faecalis in human feces. In Denmark, Kjellander (99) observed a similar frequency. In adult subjects in Germany, Guthof (76) reported the dominance of S. faecium; however, in children S. faecalis was encountered more frequently, but its numbers decreased with age. Shattock (168) suggested the possible influence of diet on the geographical variation observed. Species Distribution in Domestic Animals The incidence of group D streptococci in cattle, swine, and sheep was reported by Kjellander (99), and the results of his study were compared with those of previous investigators. In general, S. bois constituted the predominant Streptococcus in the feces of these animals. The incidence of S. faecium and S. faecalis was decidedly lower, and, in Kjellander's study, these species constituted approximately 10% of the total streptococcal flora. A preponderance of S. faecium over S. faecalis in the feces of sheep, cattle, and swine has been observed in most investigations conducted to date. In Great Britain, the superiority of S. faecalis in human subjects and its rare occurrence in swine prompted an association of these species

28 VOL. 28, 1964 with fecal contamination originating from the respective hosts in the processing of bacon (11). Bartley and Slanetz (13) noted also the rare occurrence of S. faecalis in domestic animals, with the exception of chickens. In the latter host, S. faecalis was found in larger numbers, although S. faecium and "S. inulinaceus" were encountered frequently. Occurrence of Enterococci in Wild Animals Mundt (126) surveyed the incidence of enterococci in wild mammals, reptiles, and birds in a native state where the influence of man was negligible. Of 216 various mammalian species, 71 % harbored enterococci. In wild boars, a higher incidence of S. faecium over S. faecalis was noted. These results parallel those observed in domestic swine, and indicate a selective effect of this host upon the enterococcal types occurring in its alimentary canal. With the exception of the results obtained from wild boars in Mundt's survey, S. faecalis occurred most frequently in the feces of wild mammals. The sporadic incidence of enterococci in rodents, coupled with the low numbers when present (141), casts doubt regarding the possibility that these animals are natural hosts. Mundt (126) noted that a large number (85%) of reptiles contained enterococci, with a superior incidence of S. faecalis over S. faecium. No extensive survey of avian species was attempted; however, only 7 of 22 specimens tested yielded enterococci. The feeding habits of all animals, regardless of diet, provides ample opportunity for implantation of enterococci in the alimentary canal. However, the total absence or extremely low numbers in some animals suggests a selective action of the digestive tract upon these bacteria (121). As yet, the mechanism of this action is unknown. Incidence of Enterococci in Insects, Plants, and Soil The identity and distribution of enterococci associated with insects was studied and reviewed by Eaves and Mundt (53). No pattern of species distribution was noted, and the random occurrence of these streptococci coupled with a high incidence of other streptococci (pyogenic and lactic streptococci) led these investigators to conclude that the association was circumstantial. In the insect digestive tract, the enterococci are transient residents, and their occurrence on the GROUP D STREPTOCOCCI 357 insect exterior is due most probably to mechanical transfer. As early as 1937, Sherman (170) noted the common occurrence of enterococci on plants. The absence of hemolytic varieties, common in the intestinal tract of humans, led Sherman to suggest that some growth took place on plants, thus negating an accidental occurrence or mere survival on the plant. More recent and extensive studies in Mundt's laboratory (127, 128) verified the common occurrence of enterococci, particularly on domestic plants. An epiphytic relationship was suggested, since the bacteria could establish a cycle in plants with transmission in the plant seed (128). In addition, the enterococci may contaminate plants through the agency of insects and wind. There is a general agreement that enterococci are not native to the soil, and their presence in soil samples represents contamination from either animal or plant sources. In 369 undisturbed soil samples, Medreck and Litsky (120) found only 8 samples containing enterococci. In cultivated soils and adjacent areas, Mundt (127) observed a significantly higher incidence, and associated this finding with enterococcal growth on the domestic plant host. In this environment, the enterococci are disseminated most probably via wind, rain, and insects. PUBLIC HEALTH SIGNIFICANCE Food Poisoning A number of reports have appeared implicating enterococci in food-poisoning outbreaks, and the literature associated with this subject has been reviewed (46, 168). In most instances, the mere finding of enterococci in the suspect food has constituted grounds for their implication in the food-poisoning outbreak. However, definitive studies relating enterococci with food-borne gastroenteritis are lacking, and their association with this condition has not been substantiated. The reported clinical symptoms vary widely, but involve supposedly a longer incubation period than that observed in Staphylococcus food poisoning. As yet, a toxic substance has not been identified, and the feeding of contaminated foods, pure cultures, or culture filtrates has led to equivocal or negative results. A collection of strains implicated in various food-poisoning outbreaks indicated an approximate equal distribution of the S. faecalis and S. faecium species (46).

29 358 DEIBEL BACTERIOL. REV. If these streptococci are capable of causing food-poisoning, it is indeed surprising that a high incidence of this condition is not noted, in light of their common occurrence in various food products. From the evidence obtained to date, it must be concluded that, if enterococci are at all capable of producing food poisoning, then this ability is peculiar to a truly rare strain, or else to an extremely unusual set of environmental conditions. Pathogenicity In the normal habitat of the alimentary canal, the group D streptococci, like Escherichia coli and other gram-negative commensals, constitute a part of the normal flora. All of these bacteria, however, are potential pathogens in the animal body outside of the digestive system. E. coli is encountered commonly in suppurative processes, and this organism is frequently the etiological agent of human urinary-tract infections. Enterococci are involved also in urinary-tract infections; however, the comparative incidence is lower than that of E. coli. S. bovis and enterococci are associated occasionally with subacute, bacterial endocarditis, and the occurrence of enterococci in suppurative conditions, including peritonitis and meningitis, has been observed (201). In terms of the current classification system of enterococci, no data are available regarding the species incidence in pathological conditions. Whether or not one enterococcal species possesses pathogenic potential over the other deserves study. Group D Streptococci as Indicators of Fecal Contamination The employment of group D streptococci in the analysis of water is receiving increased attention due to questions which arise when complete reliance is placed upon the use of E. coli as the indicator organism. In the routine procedures employed, E. coli may be confused with physiologically similar bacteria. In addition, its relatively rapid death in water, in comparison with pathogenic microorganisms including enteric viruses, had led some public health officials to question its utility as the sole index microorganism for fecal contamination. Although the group D streptococci have been proposed as index organisms for this purpose, their acceptance in the past has met with disfavor owing to the ease of detecting E. coli in polluted water, the lack of quantitative recovery and methods of rapid indentification, and the absence of information regarding their source, survival, and significance in water supplies. More recent studies indicate that some of these objections can be resolved. From the ecological studies conducted to date, it may be surmised that the chief source of group D streptococci is the alimentary canal of animals. Thus, the occurrence of these bacteria in water infers either direct or indirect fecal contamination. Recent studies associated with the ecology of the group D streptococci indicate the necessity for quantitating both the enterococci and the S. bovis-s. equinus species in the water analysis. The latter species could be included to detect possible animal contamination. Although the isolation of enterococci does not indicate the origin of contamination, the presence of S. bovis or S. equinus may be associated with animal contamination (13). Thus, the methodology employed should quantitate all the group D Streptococcus species. Generally, the coliform-streptococcus ratio in polluted water is significantly greater than unity; however, the streptococci occasionally may outnumber the coliforms. After initial contamination, both groups may exhibit a slight increase in numbers (presumably as a function of the organic material available in the water and the temperature) followed by a pronounced decrease (13). A study of the coliform-streptococcus ratio in various water sources indicated a more rapid decrease in coliforms, and a marked tendency was noted for the ratio to approach unity (19). Characteristically, group D streptococci were not detected in waters known to be free from fecal contamination. Buttiaux and Mossel (22) related a higher resistance of group D streptococci over the coliforms to chlorination, and these investigators stated that relatively low numbers of streptococci are to be expected in water. Under laboratory conditions, Kjellander (99) also noted the greater resistance of streptococci to chlorination. Burman (19) challenged the tolerance of these bacteria to chlorination, and his study indicated that the occurrence of streptococci represents postchlorination contamination. Thus, under actual conditions of water purification, where the amount of organic and microbial contamination is relatively low, the differential susceptibility to chlorination

30 VOL. 28, 1964 GROUP D STREPTOCOCCI 359 is not manifested. It is entirely possible that Burman's results reflect efficacy of treatment rather than differential susceptibility. As Kjellander (99) noted, the difference in sensitivity to chlorination of E. coli and the gram-negative pathogens occurring in water is insignificant. Ideally, the indicator organism should possess superior resistance to chlorination, and herein lies a possible strong point for the use of group D streptococci as indicator organisms. The employment of both E. coli and the group D streptococci as index organisms for the most accurate appraisal of fecal contamination in water is receiving increased attention. Certainly, the group D streptococci cannot replace E. coli as an index for pollution. It would appear that continued study with the streptococci is necessary, in regard to the development of specific media for species isolation, and their incidence under field conditions in various water supplies. The use of enterococci as an index microorganism to reflect the sanitary history of various frozen foods was reviewed by Niven (131). Most, but not all, of these food products are precooked or undergo some thermal processing prior to freezing. Thus, the bacteria found in these products represent recontamination after thermal processing and prior to freezing. E. coli is particularly susceptible to freezing and the subsequent holding of the product in the frozen state. In contrast, the enterococci are relatively resistant and provide a reliable index as to the sanitary history of the food product. Enterococci may occur in comminuted, curedmeat products either as a result of survival of thermal processing or from postprocessing contamination. These products are heated to kill most vegetative bacteria [155 F (68.3 C) and above]; however, the processing schedules are based on the premise that the raw sausage mix does not contain excessive bacterial numbers. The high heat and salt resistance of enterococci as well as a high initial population in the sausage mix are factors contributing to their survival in these marginally processed products. Even under ideal conditions, the product may be recontaminated in the subsequent slicing and prepackaging operations. In these instances, the occurrence of enterococci does not necessarily suggest direct fecal contamination. Quite often, these bacteria become established in food plants and grow in areas far removed from the original source of fecal contamination. Complete avoidance of enterococci in these products is difficult, and recourse to control of numbers rather than avoidance of occurrence must be practiced. Generally, good sanitary procedures including proper temperature control will restrict the growth of these contaminants. ACKNOWLEDGMENTS The author's research was supported in part by grant AI-2499 from the National Institutes of Health. The author is grateful to N. J. Jacobs, J. M. Moulder, C. F. Niven, Jr., H. W. Seeley, J. H. Silliker, and J. A. Johnson for their critical reading of this manuscript. LITERATURE CITED 1. ABRAMS, A acetyl groups in the cell wall of Streptococcus faecalis. J. Biol. Chem. 230: ANDREWES, F. W., AND T. J. HORDER A study of the streptococci pathogenic for man. Lancet 2: ARMSTRONG, J. J., J. BADDILEY, J. G. Bu- CHANAN, A. L. DAVISON, M. V. KELEMAN, AND F. C. NEUHAUS Teichoic acids from bacterial cells. Nature 184: BACHRACH, U., M. SEGAL, AND R. ROZANSKY Effect of tetracyclines on formation of amines by bacteria. Proc. Soc. Exptl. Biol. Med. 97: BAILEY, R. W Transglucosidase activity of rumen strains of Streptococcus bovis. 2. Isolation and properties of dextransucrase. Biochem. J. 72: BAILEY, R. W., AND A. E. OXFORD A quantitative study of the production of dextran from sucrose by rumen strains of Streptococcus bovis. J. Gen. Microbiol. 19: BAILEY, R. W., AND A. E. OXFORD Prerequisites for dextran production by Streptococcus bovis. Nature 182: BARKER, H. A Streptococcus allantoicus and the fermentation of allantoin. J. Bacteriol. 46: BARNES, E. M Tetrazolium reduction as a means of differentiating S. faecalis from S. faecium. J. Gen. Microbiol. 14: BARNES, E. M., AND M. INGRAM The identity and origin of faecal streptococci in canned hams. Ann. Inst. Pasteur Lille 7: BARNES, E. M., M. INGRAM, AND G. C. IN-

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