the enterococci: their '.d l. maryland · the recognition of the enterococci hasincreased in...

THE ENTEROCOCCI: WITH SPECIAL REFERENCE TO THEIRASSOCIATION WITH HUMAN DISEASE

ALICE C. EVANS '.D ALICE L. CHINNNational Institute of Health, Bethesda, Maryland

Received for publication July 1, 1947

During the past 15 years, when beta hemolytic streptococci of Lancefield's(1933) groups A and C were subjects of investigation in this laboratory, strainsof group D were acquired from time to time and were included in the compara-tive studies. In this paper they will be called enterococci, a term first used byThiercelin (1899).Because the enterococci have been found to be resistant to the sulfonamides

and to penicillin, and because the doctor wants to know as early as possible inthe course of a disease what kind of treatment is indicated, the importance ofthe recognition of the enterococci has increased in recent years.Sherman and his collaborators (1931, 1937, 1943) studied the enterococci

extensively, and Sherman (1937) published a review of the literature. Theirstudies included few pathogenic strains, and the review did not include a fullconsideration of the incidence of enterococci in human disease.

THE HARDINESS OF ENTEROCOCCI

Sherman (1938) reported other distinguishing characteristics of the enterococciin addition to precipitation in serum of group D-namely, the ability to growat 10 C and 45 C; survival at 60 C for 30 minutes; and tolerance for inhibitorysubstances, as shown by ability to grow in the presence of 6.5 per cent sodiumchloride, 0.1 per cent methylene blue, or 40 per cent bile.The tolerance of the enterococci for inhibitory substances is of practical

importance. A high degree of resistance to the sulfonamides was reported byBliss and Long (1937), Long and Bliss (1938), Neter (1938, 1940), Helmholz(1937, 1941), Francis (1941), Rantz and Kirby (1943), and MacNeal and Blevins(1945).In his original paper on the inhibitory action of penicillin on many species of

bacteria, Fleming (1929) noted that enterococci were resistant to it. Thisfinding was confirmed by later investigators (Bornstein, 1940; Heilman andHerrell, 1942; McKee and Rake, 1942; Watson, 1944; White and Sherman,1944; MacNeal and Blevins, 1945).

THE SOURCES OF THE STRAINS OF OUR COLLECTION

Our collection contained 23 strains from human pathologic sources and 11strains from other sources, included for comparison. Three of the latter werefrom the stools of normal human subjects; one was from a normal human throat;two were from different pooled lots of human plasma; one was from an unknownhuman source; one was isolated from milk powder and one from pasteurized

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milk; one was from the diseased lung of a dog; one, received from the AmericanType Culture Collection, had a history of having been isolated from pus from ahorse with strangles. The authenticity of this origin is questionable, becausethe strain came indirectly from the original investigator and was labeled "S.equi."Some of the strains listed in table 1 are duplicates from the same patient.

It appears that more than one of the strains (nos. 1188, 1308, 1355, and 1357)that were received from Dr. Sherman may have been isolated at different timesfrom the same patient, because, according to Sherman, Stark, and Mauer(1937), S. zymogenes was isolated on various occasions from one subject. Strains1332 and 1333 were originally the same strain, one of the branches having had ahistory of undergoing variation in its ability to hemolyze blood.

THE CHARACTERISTICS OF THE ENTEROCOCCI

The strains included in table 1 belonged to group D, according to Lance-field's precipitin test; all grew at 10 C and 45 C; all grew in media containing6.5 per cent NaCl and in media having an initial pH value of 9.6; all testedstrains (31) grew in media containing 40 per cent bile; all hydrolyzed esculin;none hydrolyzed starch; all attained a final pH of 4.4 or lower in glucose broth,except one (no. 945) which produced a final pH of 4.6; all strains fermentedmaltose; of 27 strains tested, all fermented salicin; of 29 strains tested, all fer-mented trehalose. None of 25 tested strains fermented dulcitol or inulin.The characteristics which were common to all strains are omitted from table

2, which includes those reactions which showed interesting differences betweenstrains. Omitted from the table are the following reactions: hydrolysis of sodiumhippurate, production of ammonia in 4 per cent peptone, and virulence for mice.These reactions appeared not to be correlated with significant characteristics.Though not considered in table 2, a general statement in regard to the viru-

lence of the enterococci for mice may be of interest. Mice were injected intra-peritoneally with broth cultures, which had been inoculated very lightly byplatinum needle and incubated for about 11 hours. Two strains killed mice in10-2 dilution; 22 killed in 101 dilution but not in higher dilutions; 9 strainsfailed to kill in the 101 dilution. According to these results, the virulence ofenterococci for mice is low as compared with the virulence of many strains ofstreptococci of groups A and C.

Uncorrelated with characteristics which appeared to be significant for classi-fication purposes are the following, listed in table 2: type of hemolysis; liquefac-tion of gelatin; sensitivity to bacteriophage D2-1188; production of acid fromlactose, arabinose, and raffinose. The characteristics of distinction were foundto be agglutinative reactions, sensitivity to phage D-693, growth in milk con-taining 0.1 per cent methylene blue, survival at 60 C for 30 minutes, and pro-duction of acid from sucrose, mannitol, and sorbitol. In the study of a largerseries of strains, the production of acid from glycerol might be found to be ofsome significance.

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THE ENTEROCOCCI

TABLE 1Histories of streptococcal strains of group D

NIH ~~~~~~~~~~~~~~~~~SoURCENIO PREVIOUS INVESTIGATORS PREVIOUS DESIGNATION |

Host | Material Disease~~~~~~l I~~~~~~~~~~Inetalhmr

Kendrick and Hol-lon; Abbott Lab-oratories

The Lilly ResearchLaboratories

Allen-Sandlin Lab-oratories

Graham

Thompson and Me-grail

Dr. John S. Buckley;Am. Type CultureCollection, Cata-logue of Cultures,1927

Torrey and Montu

Lederle Labora-tories

FarrellSherman, Stark, andMauer

Saunders; Torreyand Montu

S. fecalis no. 311900.89

S. hemolyticusno. 1527

18297S56S 112S 115A.M.S 6; 827

Intestinal strep-tococcus no. 11




L-14L-5032

1198

291

Hilligest

R-36

66

83

140

Human |Feces

Human IPus

Human

CanineHumanHumanHumanEquine?

LungToothFecesFecesPus?

Human Feces

Human Lesion

Human Feces

Human IFeces

HumanHumanHuman Pus

Human Heart blood

Human Pus

Human

HumanHuman

Human

Human

Human

Feces

Intestinal hemor-rhages

Empyema

Sore throat

InfectedNoneNoneStrangles?*

Chronic ulcera-tive colitis



None

Osteomyelitis

Peritoneal ab-scess

Septicemia fol-lowing small-pox vaccina-tion

Ear infection fol-lowing measles

Meningitis fol-lowing mas-toiditis

Normal throatDiarrhea

Gastric ulcer car-cinoma

Gastric ulcer

Gastric ulcer car-cinoma

693

696

702

894912913914945

977

978

979

980

112111221130

1131

1132

1181

11871188

1275

1276

1278 I

19471 A97


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ALICE C. EVANS AND ALICE L. CHINN

TABLE 1-Continued

___ ~~~~~~~~~~souscN PREVIOUS IVSTIGATORS PREVIOUS DESIGNATIONNO.

Host Material Disease.- - .1 .~.-

1308 Sherman, Stark, and S. zymogees no. 1 Human Feces Intestinal dis-Mauer order

1309 Sherman and Wing S. durans Wing 3 Milk powder1332 }Dr. E. C. Rosenow 257.21} Human Endocarditis1333 ISh7rman1355 26CC Human1357 Sherman, Stark, and 36Cs Human Intestinal dis-

Mauer order1359 132B Pasteurized

milk1531 Varn Human Peritonitis1574 Rigens Human Pus Otitis media1588 3365 Human Pooled

plasma1600 31A Human Pooled

plasma

The authenticity of the source of strain 945 is questioned (see the text).t Strains 1332 and 1333 were from the same patient.

TESTS FOR THE DIFFERENTIATION OF ENTEROCOCCAL SPECIES

According to the key in the fifth edition of Bergey's Manucl (1939) liquefac-tion of gelatin and hemolysis are considered to be distinctive characters, onwhich the differentiation of enterococcal species is based. Sheiman, Stark, andMauer (1937) mentioned the "thin and shaky boundaries" which separate the"supposed" enterococcal species, but Sherman (1938) recognized 3 species whichhe differentiated on the basis of the two characteristics mentioned above.Durand and Dufourt (1923) reported that they found a precise correlation

between liquefaction of gelatin and agglutinative reactions. According toother investigators, however, liquefaction of gelatin is an unstable property ofno significance in classification. Houston (1934) noted that the action ofbacteriophage may alter the gelatin-liquefying property. Elser and Thomas(1936) found gelatin-liquefying strains of enterococci which agreed well withnonliquefying strains in cultural and biochemical properties. Wheeler andFoley (1943) stated that biologic characteristics of enterococci could not becorrelated with serologic type.Lack of correlation between liquefaction of gelatin and significant char-

acteristics may be noted in table 2. For example, strains 1278 and 1600 gavealmost identical reactions in all tests excepting that for liquefaction of gelatin.Gordon (1922) found that hemolytic and nonhemolytic strains of enterococci

behaved alike in agglutinin absorption tests. Frobisher and Denny (1928),Elser and Thomas (1936), and Sherman and Stark (1931) found that hemolyticand nonhemolytic strains resembled each other in every respect excepting thereaction on blood. The literature records many instances of the loss of hemo-

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TABLE 2Characteristics of the enterococci

SENSIIY-

AGOUTINATIONIN SERUM I TO ACID PRODUCTION FROMPHAGE OZ

NO. __ _ _ _ _ _ _ __ _ _ _ _

1188 1130894

. 8n8Streptococcus zymogenes (MacCallum and Hastings)1188 500t 100 1,000 + + + - + + - + + _ + + +1308 500 100 1,000 + + + - + + - + + + + +914 50000 1,000 + ++ - + + - + + + + +1275 500 1000 1,000 + + - _ + + _ + + + + +1276 500 1,000 1,000 + + - _ + + - + + + +1278 500 - 1,000+,-+ + -++ ++ +1600 100 100 500 + + - + + + - + + + +1355 10 100 10 + + + + + + - + + _ + + +693 100 50 1 + + + + + + - + + + + +702 500 100 1,000 + - + - + + + + + + + + +912 500 1,000 + + - + + + + - + + - + + +1357 500- 1,000 + - + _ + + - + + - + + +1588 100 _ 100 + - _ + + + - + + - + + +1359 5- 100 - + - + - + + - + + - + + +980 100 10 100 + - + + + + + + + - + + +1531 100 10000-+ - + - + + - + + - + + +913 100 100 100 + -+ - - + + - + + +

Group 2

1130- 10 100 + __- + + --+-++-1122 -10 100 + + - + + --+ -+ +1121 10 50 10 + + - + +-- + -+ +1131 - 10 _ + _ + - +- +ILI +Lmz+r+nI977 10 10 _ + +

+ + + + - + + +978 - 1,000 _ _ + _+ + 1- - + - + + +1187 - 100 _ + + -_ + ++ + +6961 - 100l 1,000 + _+ -- + 1-+ --+ + +

Group 3

1132 - - 100 + - + + ++-1+1+9791 - | - 1001Z1+l + } - + -+ + - + ++8941 - - 11,0001+-+ I - I+I + 1--+1-1+1+1+

945$1 - - 10 . - - - + + + + + - + _ +

Group 4

15784 - - | +1+ + + + + 1- 1+1+1-+1+1+Group 5

13321 - - -- - I+ I- 1+1+1+1+1+1 1+13331 - - -- - I+ I- 1+ + + + +-+

S. durans (Sherman and Wing)

1xv1-I- -- + I +I +1+-+----* Strong reduction in milk containing 0.1 per cent methylene blue.t The titers are expressed as reciprocals of the highest serum dilution which aggluti-

nated.t Strain 945 was peculiar in that the final pH was only 4.6.


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ALICE C. EVANS AND ALICE L.. CHINN

lytic power in streptococci (Grinnell, 1928; Todd, 1928; Fry, 1933; Lancefield,1934). That this phenomenon may occur in enterococci was observed byseveral investigators (Gordon, 1922; Stein, 1933; Noel, 1934). Our strain 1333was originally a hemolytic strain which, when it was received, had a history ofhaving produced a green variant from a pure line culture. In our laboratoryneither strain 1333 nor the substrain 1332 produced beta hemolysis. Thereverse change, the acquisition of hemolytic power by enterococci of the alphatype, was reported by Meyer (1926).

In our laboratory repeated changes in type of hemolysis were observed instrain 693. This strain had been isolated by Kendrick and Hollon (1931) fromfeces in a case of intestinal hemorrhages. They noted that when first isolatedit was strongly hemolytic, but that it soon lost its hemolytic power and becamealpha hemolytic. After transmission to our laboratory, however, it producedbeta hemolysis. Because it did not behave in accordance with its previoushistory, another subculture of the strain was requested. It also produced betahemolysis in our laboratory when first received, and it has done so consistently.However, a subculture of our beta hemolytic 693 was given to another laboratoryof the National Institute of Health, and it was reported as having changed toan alpha hemolytic strain. A subculture of this alpha strain was returned toour laboratory. It was tested for type of hemolysis on agar containing rabbitblood, which was in general use in our laboratory, and on sheep blood, whichwas in use in the other laboratory. On rabbit blood agar, beta hemolysisoccurred, but on sheep blood, alpha hemolysis occurred. This observation ofdifferences in the type of hemolysis dependent on the source of blood was madealso by Kobayashi (1940), who reported that the enterococcus does not hemolyzethe blood corpuscles of the goat or sheep, although it may hemolyze the cor-puscles of man, horse, cow, or rabbit.The inconstancy of hemolysis in strain 693 illustrates the unreliability of the

hemolytic property as a character of specific significance. The lack of correla-tion between type of hemolysis and other characteristics is illustrated in table 2by strains 914 and 1275, which gave almost identical reactions in all tests ex-cepting that for hemolysis.

Enterococcal bacteriophage. That enterococcal bacteriophage may be morewidespread than phages attacking other streptococci is suggested by the morefrequent mention of it in the literature. Beckerich and Hauduroy (1922), andalso Hadley and Dabney (1926), studied this phage. Bagger (1926) reportedthe sudden appearance of bacteriophage in a plate culture of a strain of enter-ococcus which had been under cultivation for a long time, many similar platecultures having been made previously. According to Houston (1936), an activeenterococcal phage can often be isolated from the stools in cases of ulcerativecolitis. This author also reported (1934) that in a septic focus the enterococcususually occurs in a phage-infected form. He believes that the action of thephage results in variation in the characteristics of the organism. Graham andBartley (1S39) found that 34 of 36 strains of enterococci were sensitive to allthree phages which they studied. In our experience, enterococcal phage couldbe readily obtained from sewage.

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Kendrick and Hollon (1931) noted the parallelism between serologic and bac-teriophagic relationships in a group of fecal streptococci. One of us (A. C. E.)studied their phage, and reported (1934) that sensitivity to this phage, desig-nated D, differentiated the enterococci from other streptococci, and that on thebasis of bacteriophagic reactions the grouping of enterococci corresponded withthe grouping recognized on the basis of other characteristics.During the course of our studies on bacteriophage, a race designated D2 was

found in a sample of sewage taken in Washington, D. C. It lysed enterococci,but differed from phage D in that the antilysins prepared against the two phagesbehaved differently. Antilysin D2 neutralized phage D as well as phage D2whereas antilysin D neutralized the homologous phage but not phage D2.Each phage was prepared by propagation on a strain of enterococcus found to

be highly sensitive. Phage D was propagated on strain 693, and phage D2 waspropagated on strain 1188. The techniques of isolating the phage, preparingthe filtrates and the antilysins, making the serologic tests, and determining thesensitivity of the streptococci to the phages were described in previous publications(Evans, 1934, 1942; Evans and Sockrider, 1942). All strains of enterococciwere tested for sensitivity to filtrates of both phages, D and D2, with results asshown in table 2.

Serologic relatio8nships. A number of investigators (Gordon, 1922; Durandand Dufourt, 1923; Meyer and L6wenstein, 1926; Takeda, 1935; Meyer, 1937)reported that the majority of enterococci from various sources fall into a fewwell-defined groups according to agglutinative reactions, which were confirmedby agglutinin absorption tests in the studies of some of the investigators.

Saunders (1930) reported that in a large series of cases enterococci from thetissues of resected gastric and intestinal ulcers and from certain types of ulcersin other parts of the body exhibited similar serologic characteristics. Torreyand Montu (1936) found that enterococci serologically related to Saunders'strains occurred more frequently in patients showing intestinal lesions than innormal adults. A few of Saunders' strains (nos. 1275, 1276, and 1277 of tables1 and 2) and a few of Torrey and Montu's strains (nos. 977, 978, 979, and 980)were available for the present study. It was found that the strains from casesof ulcer, received from Saunders and from Torrey and Montu, resembled strainsfrom other pathologic as well as nonpathologic sources in serologic behavior aswell as in physiologic and biochemic reactions.That the commonest serologic types of enterococci are widely distributed is

suggested by the studies of the following authors: Houston (1936) reportedthat type 1 of the "Belfast classification" was identical with one of Meyer'stypes of continental European strains, and Meyer (1937) reported that histype 1 agreed with Takeda's (1935) type 1 of strains isolated in Japan.

Agglutinating serums were prepared against three strains, 894, 1130, and 1188,strain 1188 having been selected because it was the strain previously utilizedfor the propagation of phage D2. Strain 1130 was selected to represent strainswhich failed to agglutinate in antiserum 1188; strain 894 was selected to repre-sent strains which failed to agglutinate in either serum.

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Rabbits were injected with antigen prepared from fresh broth culture heatedat 56 C for 1 hour. The cultures were centrifuged and the sediments weresuspended in saline solution containing 0.2 per cent tricresol. The first 3 doses,injected subcutaneously on successive days, each consisted of 0.5 ml of antigenof a turbidity equivalent to 2,000 ppm of the silica standard. They werefollowed by 2 intraperitoneal doses of 1 ml, 400 ppm, on successive days. Intra-peritoneal injections were made with increasing doses 3, 4, or 5 times duringthe following 2 weeks, the largest being 1.0 ml, 1,000 ppm. Trial bleedingswere then made 6 days after the last injection. If the serum did not show agood titer of agglutinins, further injections were made.

In the case of strain 1188, a satisfactory serum was obtained after treatmentof the rabbit for 3 weeks; strain 894 required 4 weeks. In the case of strain1130, the serum was unsatisfactory, when tested against the homologous strain,after treatment for 6 weeks. It agglutinated in 1:10 but not in higher dilutions.However, it agglutinated many heterologous strains in a titer of 1:100 or 1:1,000(table 2). Torrey and Montu (1936) reported that some of their strains lackedagglutinogenic properties.

Distinguishing characteristics of the enterococci. In table 2 the 34 strains ofenterococci are arranged in 6 groups, primarily according to agglutinativereactions. It may be noted that certain other characteristics are more or lesscorrelated with agglutinative reactions.The first group includes 17 strains, one of which, no. 1308, was labeled Strep-

tococcus zynwgenes when received from Dr. Sherman. All strains of this groupagglutinated in serum 1188; all but two agglutinated in serum 1130, and all butanother two agglutinated in serum 894. All strains were sensitive to phageD-693; all grew in milk containing 0.1 per cent methylene blue; all survived60 C for 30 minutes; all produced acid from sucrose, lactose, mannitol, and sor-bitol. The strains varied in sensitivity to phage D2-1188, hemolysis, liquefac-tion of gelatin, and production of acid from glycerol. Only one strain producedacid from arabinose, and it was also the only one which produced acid fromraffinose. The table shows that liquefaction of gelatin and type of hemolysis areuncorrelated with other characteristics. According to all available evidence the17 strains of the group belong to one species. In agreement with priority ofnomenclature the group should be designated Streptococcus zymogenes (Mac-Callum and Hastings, 1899), and the names faecalis and liquefaciens should beeliminated.The last group in table 2 includes only one strain, no. 1309, which was re-

ceived from Dr. Sherman with the designation "S. thermodurans." AfterwardsSherman and Wing (1937) changed the specific name to Streptococcus durans.These authors reported that S. durans does not have as strong a reducing actionas other strains of group D; it lacks the ability to produce acid from glyceroland sorbitol; usually it does not attack mannitol or sucrose. These distin-guishing characteristics of S. durans were confirmed in our studies (table 2),which show, further, that strain 1309 does not agglutinate in the serums which

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agglutinate the strains of S. zymogenes, and that it is resistant to phages D-693and D2-1188.Between the widely different species S. zymogenes and S. durans are strains of

intermediate characteristics (see table 2). These intervening groups show agradation of divergence from the characteristics of S. zymogenes toward thecharacteristics of S. durans. Group 2 differs from S. zymogenes chiefly in thefailure to agglutinate in antiserum 1188, and in the failure of most of the strainsto produce acid from sucrose and sorbitol. Group 3 diverges further in that thestrains fail to agglutinate in antiserum 1130. The strains of group 4 agglutinatein none of the serums, but in other characteristics they resemble those of S.zymogenes.The data in table 2 are insufficient to determine whether any one or more of

the groups 2, 3, and 4 or any combination of them should be regarded as aseparate species. However, the failure of production of acid from sucrose andsorbitol appears to have some significance. Possibly a new species should berecognized largely on the basis of those characteristics.The strains of group 5, one of which was derived from the other, are clearly

differentiated from the two recognized species, S. zymogenes and S. durans.They differ from S. zymogenes in their failure to react in the three agglutinatingserums, in their resistance to phage D-693, and in their failure to survive 60 Cfor 30 minutes. They differ from S. durans in their strong reduction of meth-ylene blue, in their failure to survive 60 C for 30 minutes, and in their productionof acid from sucrose, mannitol, and sorbitol.Graham and Bartley (1939) mentioned a variety of enterococcus which lacked

the property of resistance to heat. Another author mentioned strains whichresiLt-d 60 C for 10 minutes, but not for 30 minutes. This report was seen byone of us (A. C. E.), but the reference was lost. If group 5 is found to be ofnumerical significance, it should be given a specific name.

Enterococci in various animal hosts. The hardiness of the enterococci enablesthem to multiply under a wide variety of conditions. They are found in healthand disease, not only in various species of mammals, but also in lower forms ofanimal life. Steinhaus (1941) isolated enterococci from 5 species of insects;Sherman (1937) quoted several authors who considered the so-called Strep-tococcus apes, which is associated with European foul brood of bees, to be anenterococcus. Plummer (1941) isolated an enterococcus from the eye of a ferret;Sylvester and Benedict (1941) isolated it from the viscera of foxes and minks;and Elser and Thomas (1936) isolated it from the cervix of guinea pigs. Orcutt(1926) found enterococci in the digestive tract of normal calves and in calvessuffering from diarrhea or scours. Torrey and Montu (1936), and also Plummer(1941), studied enterococci which had been isolated from milk in cases of bovinemastitis. Two of our strains, nos. 1309 and 1359, were probably of bovineorigin, not associated with disease.Hont and Banks (1944) cultivated enterococci from a pig which died of

endocarditis, and they produced disease in a young pig from a healthy herd by

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inoculating the culture intravenously. Thomson and Thomson (1927) men-tioned the isolation of enterococci from the respiratory tract of dogs sufferingfrom distemper. One of the hemolytic straiins of our collection, no. 894, wasisolated from the diseased lung of a dog, where it was associated with a non-hemolytic streptococcus which the writers did not have an opportunity to study.

The sources of the enterococci found in human infections. The data in tables1 and 2 indicate that strains of enterococci similar to those found in pathologiclesions of man may be derived from the flora of the normal human intestines, orfrom animal sources. Takeda (1935) also was unable to find differences be-tween the enterococci of the healthy and thcse of diseased intestines.Among the 17 strains designated S. rymogenes, one was apparently of bovine

origin, having been isolated from pasteurized milk; 2 were apparently fromnonpathologic human sources, pooled normal plasma; 3 were from normalstools; and the remaining 11 were from human pathologic sources. Among the8 strains of group 2, one was from a normal throat, one was from an unknownhuman source, and the remaining 6 were from human pathologic sources. Amongthe 4 strains of group 3, 2 were from animal sources, and 2 were from humanpathologic sources. It may be noted that strain 894, from the diseased lung ofa dog, behaved in every reaction like strain 979, from a human case of chroniculcerative colitis.No evidence that S. durans is pathogenic for man has yet appeared. None

of our 23 strains from human pathologic sources showed the characteristics ofthis species. Brown and Schaub (1945) reported finding one strain of S. duransamong 386 strains of group D isolated from autopsy material, but they did notstate that they found evidence that it had been involved in any disease process.

The enterococci in human disease. The literature on enterococci in humandisease contains many extensive reviews (Schmitz, 1912; Meyer and Schbnfeld,1926; Thomson and Thomson, 1927; Dible, 1929). The German literature onthe pathologic significance of the enterococci was reviewed by Ehrismann(1935). In the following review reference will be made to Ehrismann but notto the authors whom he quotes. This review will omit references to papers inwhich the description of strains leaves doubt as to whether they were enterococci.That enterococci are more frequently associated with human disease than they

were formerly believed to be is indicated by the report of Brown and Schaub(1945), who stated that nearly 50 per cent of strains of streptococci isolatedfrom autopsy material belonged to group D. These investigators were of theopinion that many of the strains were derived from post-mortem enterococcalinvasion, but that many were associated with disease. Rantz and Kirby (1943)reported the finding of many enterococcal infections.

Enterococci in the human digestive tract. That the enterococci are commoninhabitants of the normal human intestines was demonstrated by Andrewesand Horder (1906) and by many subsequent investigators. Rantz and Kirby(1943) reported that they found enterococci to be constantly present in thenormal bowel. On the other hand, Schmitz (1912) failed to find them in normalstools.

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Thiercelin (1899) found enterococci involved in enteritis and concluded thatthey are important in most affections of the. digestive tract. Sherman, Stark,and Mauer (1937) investigated the occurrence of S. zymogenes in a subject whoharbored the organism. It could not always be isolated from the stools, but itcould be isolated frequently, and usually with ease, during periods; of intestinaldisturbance (strain 1188 of tables 1 and 2 was from this case). Ross and Peck-ham (1920) reported finding enterococci in the stools during an outbreak of 12cases of severe dysentery, 5 of which were fatal.

Linden, Turner, and Thom (1926) were the first to report outbreaks of foodpoisoning due to streptococci in cheese. Others have been reported morerecently. The streptococcus from one of the early outbreaks was identifiedwith Lancefield's group D by Sherman, Smiley, and Niven (1943). Accordingto Sherman (1945), symptoms similar to those in man may be produced in catsby feeding them with milk in which enterococci have grown.

Saunders (1930) reviewed the literature on the aEsociation of streptococciwith ulcers of the digestive tract, and he reported finding the constant occurrenceof enterococci in the tissues of resected gastric, duodenal, and gastrojejunalulcers. His observations were confirmed by Torrey and Montu (1936).

According to Felsen (1936), after an acute infection with "Bacterium dysen-teriae" a secondary infection with enterococci and "B. coli" sometimes takesplace at the site of the mucosal ulcerations, presumably originally produced bythe toxin of the dysentery organism.

Strain 693 of our collection (see the tables) was isolated from feces in a caseof intestinal hemorrhage.

Purulent abdominal infections with enterococci. Thiercelin's findings (1899)led him to believe that enterococci are important in the production of appen-dicitis. A number of more recent investigators have confirmed this association.(Meyer and L6wenstein, 1926; Ehrismann, 1935; Elser and Thomas, 1936;Lodenk&mper, 1937; Muroi, 1938).

Enterococci are sometimes associated with purulent infections resulting froma damaged intestine or bladder (Schmitz, 1912; Rantz and Kirby, 1943; Wheelerand Foley, 1943; Brown and Schaub, 1945). Strain 1130 of our collection wasfrom a case of peritonitis.

The streptococci of wounds. Fleming (1915) was of the opinion that thestreptococci as well as other bacteria of wounds inflicted during war are generallyof intestinal origin. Burnet and Weissenbach (1918) reported finding enter-ococci in 30 per cent of osteomuscular war wounds of less than 7 days' duration.Dible (1921) quotes a number of authors who noted the presence of entero-cocci in wounds studied during the First World War, and other more recentinvestigators have reported enterococcal infection of wounds (Morin, Caudibre,and Certonciny, 1924; Takeya, 1938; Francis, 1941).

Infections of the urinary tract. Enterococcal infections of the urinary tractwere reported by Andrewes and Horder (1906), Meyer and L6wenstein (1926),Ehrismann (1935), and ELser and Thomas (1936). That enterococci occurfrequently in genitourinary infections was shown by Porch (1941), who found

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that 73 out of 100 strains of streptococci isolated from specimens of urine be-longed to group D. Hollander (1942) found that 22 out of 40 (55 per cent) ofstreptococci from infections of the genitourinary tract belonged to group D.Rantz and Kirby (1943) found 27 per cent of streptococci isolated from speci-mens of urine belonged to group D. From 5 of their cases which presentedsigns of pyelonephritis they isolated enterococci in pure culture.

Puerperal 8epsis. A good many authors have reported finding enterococciassociated with puerperal sepsis (Meyer and Lowenstein, 1936; Ehrismann,1935). Gordon (1922) found them in 8 cases, Ramsay and Gillespie (1941) in6, and Rantz and Kirby (1943) in 2. Witebsky and his coworkers (1939)isolated a strain of enterococcus from the blood in a case of septicemia followingabortion.Takezawa (1937) found 50 strains of enterococci among 216 strains of strep-

tococci from female genital organs in various diseases. Hare and Colebrook(1934) isolated streptococci with the characteristics of enterococci from 7 of 34women who had low-grade fever during the puerperium, but in only a few in-stances were the organisms isolated in pure culture. Lancefield and Hare(1935) found no streptococci of group D among 46 strains from cases of severeinfection of the uterus, but they found 8 strains of group D among 18 strainsfrom "minor infections." Brown and Schaub (1945) found that 9 per cent of232 strains of streptococci from the uteri of patients with febrile puerpera wereenterococci.

Otitis media, mastoiditis, and meningiti. Thiercelin (1899) isolated enter-ococci from cases of meningitis, and Andrewes and Horder (1906) found themin cases of otitis media, mastoiditis, and meningitis. Subsequent investigatorshave confirmed those early reports. Ehrismann (1935) quoted two authorswho reported cases of meningitis due to enterococci; Lang, Lode, and Schutter-mayer (1937) reported 2 cases; Wheeler and Foley (1943), 1 case. Rantz (1942)reported 1 case in which meningitis followed a prolonged ear infection. Rantzand Kirby (1943) found that about 10 per cent of streptococcal infections of themiddle ear were caused by enterococci.Among the strains of our collection, no. 1132 was from a case of ear infection

following measles; no. 1181 was from a case of meningitis following mastoiditis;and no. 1574 was from a case of otitis media.

Endocarditis. MacCallum and Hastings (1899) obtained an organism whichthey called Micrococcu zymogenes from the blood in a case of endocarditis, andSherman (1937) was convinced that their organism was an enterococcus. Sub-sequently many authors reported the isolation of enterococci in cases of endo-carditis (Andrewes and Horder, 1906; Hicks, 1912; Gordon, 1922; Meyer andLowenstein, 1926; Dible, 1929; Wallach, 1934; Houston, 1934; Baum, 1935;Ehrismann, 1935; Elser and Thomas, 1936; Reiners, 1936; Fox, 1936; Waaler,1937; Clements, 1937; Otto, 1938; Moran, 1938; Rohleder, 1938; Williams, 1939;Lederle, 1940; Skinner and Edwards, 1942; Rantz and Kirby, 1943; Wheeler andFoley, 1943; MacNeal and Blevins, 1945; Brown and Schaub, 1945). Twostrains of our collection, nos. 1332 and 1333, were from one case of malignantendocarditis.

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Some of the investigators mentioned reported on the frequency of occurrenceof enterococci in their cases of endocarditis. Elser and Thomas encounteredenterococci "not infrequently" in the blood of patients suffering from a subacuteform of endocarditis. Andrewes and Horder found 4 strains of enterococciamong the streptococci from 24 cases of malignant endocarditis; Dible reportedthat 1 strain out of 6 isolated from the blood in cases of ulcerative endocarditiswas an enterococcus; Moran found it in 5 out of 20 cases; Lederle in 8 out of10 cases; Rantz and Kirby in 3 out of 16 cases; and MacNeal and Blevins in6 out of 36 cases.The portal of entry was determined in a number of cases of endocarditis

revieved by Skinner and Edwards (1942). Enterococci obtained from theblood stream were derived from an infected finger in 1 case, infected tonsils in1, the gall bladder in 2, the urinary tract in 2, septic abortion in 3, and thegastrointestmial tract in 5.

Miscellaneous diseases. Rantz and Kirby (1943) called attention to thefact that the enterococci are rarely found associated with infections of therespiratory tract. They reported 8 cases; Wheeler and Foley (1943) reported1 case. Brown and Schaub (1945) found enterococci in mixed cultures in casesof pneumonia. One strain of our collection (no. 702) was from a case of sorethroat.The enterococcus is occasionally reported to be associated with various

diseases not mentioned above. Meyer and L6wenstein (1926) found it incholecystitis, osteomyelitis, and pancreatitis; Houston (1934) found it in septictonsils, the root canal of septic teeth, postnasal catarrh, septic antra, excisedgall bladders, abscesses in various parts of the body, certain forms of acne andother skin lesions, and invariably in chronic onychia; Wheeler and Foley (1943)found it in dermatomyositis and in emphysema. Our collection includes strainsfrom empyema (no. 696), an infected tooth (no. 912), osteomyelitis (no.1121), and septicemia following smallpox vaccination (no. 1131).There is an extensive literature on the association of enterococci with rheu-

matic diseases. It is omitted here because, if the ansociation should be provedto be significant, this literature should be treated separately.

SUMMARY

The literature on human infections with enterococci is reviewed, and theresults of a study of 34 strains, 23 from human pathologic sources and 11 fromother sources, are reported.

Enterococci have been found in a great variety of human ailments. Theyappear to be important causal agents in some cases of endocarditis, intestinaldisorders, abdominal infections due to injury of the intestinal tract, infectionsof wounds inflicted during war, and infections of the urinary tract.The following characteristics distinguish the enterococci from other strep-

tococci: reaction in serum of group D according to Lancefield's precipitin test,growth at 10 and 45 C, growth in media containing 6.5 per cent sodium chloride,growth in media having an initial pH value of 9.6, and growth in media con-taining 40 per cent bile.

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The hardiness of the enterococci is of practical significance in that they areresistant to the sulfonamides and penicillin.The following characteristics are useful in distinguishing subgroups or species

of enterococci: agglutinative reactions; sensitivity to bacteriophage D-693;growth in milk containing 0.1 per cent methylene blue; survival at 60 C for 30minutes; and the production of acid from sucrose, mannitol, and sorbitol.The type of hemolysis and liquefaction of gelatin, characteristics on which the

differentiation of species hitherto have been based, were found to be uncorrelatedwith other significant characteristics.The data indicate that enterococci pathogenic for man may be derived from

the flora of normal human intestines or from the tissues or intestines of variousspecies of animals.

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J. Path. Bact., 37, 337-340.GORDON, M. H. 1922 The classification of streptococci. J. State Med., 30, 932-935.GRAHAM, N. C., AND BARTLEY, EILEEN 0. 1939 Some observations on the classification

of enterococci. J. Hyg., 39, 538-552.HADLEY, PHILIP, AND DABNEY, EUGENIA 1926 The bacteriophagic relationships between

B. coli, S.fecalis, and S. lacticus. Proc. Soc. Exptl. Biol. Med., 24. 13-18.GRINNELL, FRANCIs B. 1928 Streptococcus studies. I. Streptococcus viridans derived

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HEILMAN, DOROTHY, AND HERRELL, WALLACE E. 1942 Comparative bacteriostatic activ-ity of penicillin and gramicidin. J. Bact., 43, 12.

HELMHOLZ, HENRY F. 1937 A comparison of mandelic acid and sulfanilamide as urinaryantiseptics. J. Am. Med. Assoc., 109, 1039-1041.

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HOUsTON, THOMAS 1934 A theory that may account for the bacteriology of rheumatism.Ulster Med. J., 3, 224-258.

HOUSTON, THOMAS 1936 Enterococci. Rept. Proc., Second International Congress forMicrobiology.

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KOBAYASHI, Roxuzo 1940 Studies concerning hemolytic streptococci. Kitasato Arch.Exptl. Med., 17, 218-241.

LANCEFIELD, REBECCA C. 1933 Serological differentiation of human and other groups ofhemolytic streptococci. J. Exptl. Med., 57, 571-595.

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LANCEFIELD, REBECCA C., AND HARE, RONALD 1935 Serologicsl differentiation of patho-genic and non-pathogenic strains of hemolytic streptococci from parturient women.J. Exptl. Med., 61, 335-349.

LANG, F. J., LODE, A., AND SCHUTTERMAYER, F. 1937 Enterococcic infections of thecentral nervous system. Wien klin. Wochschr., 50, 29-35.

Lederle Laboratories 1940 Recent %dvances in the classification of streptococci. Bull.Lederle Lab., 8, 15-18.

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LINDEN, B. A., TURNER, W. R., ANi THom, CIRuzs 1926 Food poisoning from a strepto-coccus in cheese. U. S. Pub. Health Service, Pub. Health Repts., 41, 1647-1652.

LODENEiMPER, HANs 1937 Studien tiber die Streptokokkengruppe, insbesondere tiberdie Enterokokken. Zentr. Bakt. Parasitenk., I, Orig., 139, 287-297, 371-381.

LONG, PERRIN H., AND BLISS, ELEANOR A. 1938 Observations upon the mode of actionand the clinical use of sulfanilamide in urinary tract infections. Southern Med. J.,31, 308-315.

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MEYER, KURT 1926 Ueber hamolytische Enterokokken. Zentr. Bakt. Parasitenk., I,Orig., 99, 416-419.

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Bact., 11, 129-139.OTTo, EDWARD 1938 Beitrag zur Kenntnis der Enterokokken-endokarditis. Klin.

Wochschr., 17, 1847-1850.PLUMMER, HELEN 1941 A serological and biochemical study of hemolytic streptococci.

J. Immunol., 42, 91-107.PORCH, MARY L. 1941 A bacteriological study of streptococci isolated from the genito-

urinary tract. J. Bact., 41, 485-494.RAMSAY, A. M., AND GILLESPIE, M. 1941 Puerperal infection associated with hemolytic

streptococci other than Lancefield's group A. J. Obstet. Gynaecol. Brit. Empire, 48,569-585.

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ROHLEDER, THEzLA 1938 Ein Beitrag zur Frage der Enterokokken-endokarditis.Monatsschr. Kinderheilk., 73, 361-366.

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SAUNDERS, EDWARD WATT8 1930 The serologic and etiologic specificity of the alphastreptococcus of gastric ulcer. Arch. Internal Med., 46, 347-382.

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from subacute bacterial endocarditis. Address before the Washington Branch of theSociety of American Bacteriologists, Feb. 27, 1945.

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Tomz, JOHN C., AND MONTU, ELIZABETH 1934 The cultural and agglutinative relation-ships of intestinal streptococci. J. Infectious Diseases, 55, 340-355.

TORREy, JOHN C., AND MoNTu, ELIZABETH 1936 Comparative observations on strepto-cocci from human gastrointestinal ulcerations and from bovine mastitis. J. InfectiousDiseases, 58,105-114.

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WH1TE, J. C., AND SHERMAN, J. M. 1944 Occurrence of enterococci in milk. J. Bact.,48, 262.

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WITEBSKY, E., MACCALLUM, J., NETER, E., AND RICHBART, C. 1939 A strain of entero-coccus (Streptococcus fecalis) giving cross-reactions with pneumococcus type IIIantiserum, isolated from the blood stream of a patient. J. Bact., 87, 225.


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the enterococci: their '.d l. maryland · the recognition of the enterococci hasincreased in...

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