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Copyright 0 1976 American SocietyforMicrobiology Printed in U.S.A.

Long-Chain

Fatty Acids of Peptococci and Peptostreptococci

CAROL L. WELLS* ANDCHARLES R. FIELD

Wisconsin StateLaboratory of HygieneandDepartment of Medical Microbiology,* University of Wisconsin, Madison, Wisconsin 53706

Received for publication23July 1976

The long-chain fatty acids extracted from the whole cells of 12 clinically

significant species of peptococci and peptostreptococci werecharacterizedby

gas-liquid chromatography. The resulting methylated fatty acid profiles (andsome

unidentified compounds) of 82 strains allowed the 12 species to be separated into four groups. Fifteen strains of Peptostreptococcus anaerobius were placed in

group Ibecause they had aunique, prominent compoundthat occurred inthe

area where a 0, to

C01

fatty acid would be expected. Group II, consisting of

Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus parvulus, Peptococcus morbillorum, and Peptococcus constellatus, produced

C14,

C16:1, C18:1

,andC18fatty acids. Peptococcus prevotii, Peptococcus variabilus,

Peptococcus magnus, Peptococcus asaccharolyticus, and Peptostreptococcus

productus wereplaced in group III becausethey contained three to six

addi-tional, unidentified compounds that strikingly differentiated them fromgroup

II. Peptococcus saccharolyticus was the single species assigned to group IV

because it yielded C14, C16,

C18:1,

C18, and C20 fatty acids and a prominent

unidentified peak that occurred between C14 and C16 fatty acids. This study

indicated that cellular long-chain fatty acids may be an important tool in

clarifying thetaxonomy of the peptococci and peptostreptococci.

Peptococci and peptostreptococci arefoundas

normal flora of the skin, upper respiratory

tract, oral cavity, large intestine, and female

genitalia (3); they are frequently associated withinfectiousdiseases(13, 18),and their

path-ogenicityformanis now widely accepted.

Accurate laboratory speciation of the medi-cally important anaerobic gram-positivecocciis difficult. Different species of peptococci and

peptostreptococci have similar biochemical

re-actions, andgaschromatography of short-chain acidmetabolites (C1to C8)often reveals identi-calfermentation products. Thespeciationof the peptococciandpeptostreptococci isfurther

com-plicated by theexistence ofmany strains that

donotfitthe already describedspeciesof

anaer-obiccocci (3). In addition, four of the foremost

authorities in clinical anaerobic bacteriology

differ markedly in theirguidelines for

specia-tion ofpeptococci and peptostreptococci (5, 7,

14-16).

This study presents an analysisof the

long-chain fatty acids (LCFAs) extracted from the

whole cells of 12 clinically significant species

(82 strains) of peptococciand peptostreptococci.

The LCFAs (C8 to C20) of the anaerobic cocci

havenotyet beencharacterized,and such

anal-yses have been useful in

clarifying

the

taxon-omy of other organisms (10, 12).

MATERIALS AND METHODS

Organisms and media. The organisms studied wereobtained from stock cultures maintained at the Wisconsin State Laboratory of Hygiene, Madison, Wis. All cultures were clinical isolates sent to the State Laboratory for identification over a 4-year period from 1972 to 1975. The following identifica-tion tests were routinely performed: Gram stain from agarandpeptone-yeast-glucose broth; fermen-tation tests oncellobiose, esculin, fructose, glucose, lactose, maltose, mannitol, and sucrose; esculin

hy-drolysis;gelatinliquefaction; indole production;

ni-tratereduction; gas-liquid chromatography of short-chainacid metabolites. In addition, other tests, such as stimulation by Tween 80, reactions in milk and meat, salicin and starch fermentation, starch

hy-drolysis,andhemolysis, were occasionally done. All

tests were performedaccording to, and with results compatible with, the Virginia Polytechnic Institute (VPI) Anaerobe Laboratory Manual, 2nd ed. (7). Twelve species were studied: Peptostreptococcus an-aerobius, Peptostreptococcus intermedius,

Pepto-streptococcus micros,Peptostreptococcus productus,

Peptostreptococcusparvulus, Peptococcus

asaccharo-lyticus, Peptococcus constellatus, Peptococcus mag-nus,Peptococcusmorbillorum, Peptococcus prevotii, Peptococcus variabilus, and Peptococcus

saccharo-lyticus.Afteridentification, the cultureswerestored

at -70°C in chopped-meat-glucose broth. Before

analysis, each culturewasthawed and transferred

tochopped-meat-glucose broth for24to48 hat370C. 515

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The purity of each culture was checked by Gram stain. Allcultures were then transferred to 7 ml of peptone-yeast-glucose broth prepared according to the formula given in the VPI Anaerobe Laboratory Manual (7) (peptone made by GIBCO Laboratory, Madison, Wis.; yeast extract and glucose made by DifcoLaboratories, Detroit, Mich.) for 48 h at370C.

The cultures were centrifuged, the supernatant was pouredoff, and the cell pellet was stored at -70°C until the fatty acids were extracted.

LCFA extraction. Fatty acids were extracted from the wholecells according to the method of Moss

etal. (11) with some modifications. The cells were thawed, suspended in 5 ml of 5% NaOH in 50% aqueous methanol and heated at 100°C for 15 min. The saponified material was cooled and acidified with 6 NHClto apHbelow2.One milliliter of boron trifluoride methanol (BF3-CH30H, Applied Science Laboratories, State College, Pa.) was added to the saponified mixture and it was heated at 100°C for 5 min. The methylated solution was then added to 10 ml of saturated NaCl and the fatty acid methyl esterswereextracted twice with an equal volume of 1:1 ether-hexane. The combined ether-hexane ex-tracts were evaporated at room temperature to about5mlunder agentlestreamofdrynitrogen and anhydrous sodium sulfate was then added to remove any water. Aprecipitatefrequently formed during the evaporationprocedure and was removed by cen-trifugation at 1,800 rpm for5 min. Evaporationof the extract withdry nitrogen was continued until a final volume of 0.1 to 0.2 ml was achieved. The extractwasstoredat-70°Cbefore fatty acid analy-sis.

Gas chromatography. A Hewlett-Packard gas

chromatograph (model 5710A) with automatic

sam-pler(model 7671A) and reporting integrator(model

3380A) was used throughout this study. The gas

chromatograph wasequipped with aflame

ioniza-tiondetector. The flamewasmaintainedbya

mix-tureofhydrogenand air with flowratesof 30ml/min

and 250 ml/min, respectively. The reporting inte-grator was set for aslope sensitivity of1 mV, an

attenuationof16 or32, andachartspeedof0.5cm/ min. The integrator printed the retention time of eachpeak, calculated theareaundereachpeak, or

identified each peak relative to anexternal

stan-dard. The external standard contained caprylate

(C8), caprate (C10), laurate (C12), myristate (C14),

palmitoleate (C16:), palmitate (C16), oleate

(C8:1),

stearate (C18), and arachidate (C20) methyl esters

(Applied ScienceLaboratories, State College, Pa.). Themethylestersof LCFAswereseparatedon a

coiled-glasscolumn6feet(ca. 183cm)longwitha

4-mmIDandaone-fourth-inch(ca. 0.64cm) OD. The carrier gaswasprepurifiednitrogen(Badger Weld-ingSupplies, Madison, Wis.), withaflowrateof50

ml/min.Methylatedfattyacidextractswere run on

bothapolar andanonpolarcolumn. Thenonpolar

columnwas packed with3% SE-30on100/120 Gas-Chrom Q(Hewlett-Packard, Skokie,Ill.). The

tem-perature of the nonpolarcolumn was

programmed

from110 to240°C at4°C/min, withatotalanalysis

timeof45min.Foranalysisonthenonpolarcolumn,

thesamplesize was1,ul, the injection port

tempera-turewas 200°C, and thedetector temperature was

350'C.Thepolar column, used primarily for confir-mation of identified peaks, was packed with 15% EGSS-X on 80/100 Gas-Chrom P(AppliedScience,

State College, Pa.). The temperature of the polar column wasmaintained at172°C for a totalanalysis timeof50 min. Foranalyses on the polarcolumn,

thesample size was 10

gl,

theinjection port

temper-ature was200°C, and the detector temperaturewas

4000C.

All samples werefirst analyzed on the nonpolar column. Theremaining extract was diluted 1:3 in

ether-hexanetoobtainasufficient volume for anal-ysis on the polar column. Consequently, a 10-,ul sample size was needed to obtain adequate peak size fromthe polar column, whereas a 1-,ul sample size gaveadequate peak size from the nonpolar column.

RESULTS

Results of this study demonstrated that the

peptococciand peptostreptococci,onthe basisof

gas chromatographic profiles of their

methyl-atedLCFAs, could bedividedintofourgroups.

Group I (Fig. 1) contained onlyone species, Peptostreptococcus anaerobius. Fifteen strains

of thisorganismallyieldedasimilar

chromato-graphic profile which containeda

characteris-tic, prominent, unidentified peak with a

reten-tion time between C8 and C10. The average

percent fatty acid composition of this

unidenti-fied compoundwas 20% (witharangeof 2.4to

29.0%).This compound was always present and

could be usedasamarker forgroupI anaerobic

cocci since it was not produced by the other

anaerobic cocciassayed. P.anaerobius was also

unique among the other peptococci and

pepto-streptococci because each of 15 P. anaerobius

strainsanalyzed contained from 14 to 25

differ-ent peaks (with an average of 19 peaks). All

other species of peptococci and

peptostrepto-cocci studied never produced over 12

differ-ent peaks. After analysis of 15 P. anaerobius

strains, thefollowing statements could be made

about group I: (i) a prominent, unidentified

peak is located betweenC8andC01;(ii) a total of

14to 25differentpeaks areproduced; (iii)

C14,

C16,

C18:1,

and

C18

areusually present; the latter

fatty acidswerepresent in13 outof15strains

studied.

On thebasis of theircellular LCFAcontent,

Peptostreptococcus intermedius,

Peptostrepto-coccus micros, Peptostreptococcus parvulus,

Peptococcus morbillorum, and Peptococcus

constellatus were all placed in group II. The

average percentages ofthe LCFA composition

oftheseorganismsaresummarizedinTable 1.

All strains within group II had C16 or C18:1 as

the mostprominent LCFAs on the

chromato-gram. Group II alsoproduced smaller, but

de-tectable amounts ofC14,

C16:1,

andC18. A

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llJ (I) z 0 a. Uf) LLI

w

a:

a:

0

wL

a:

GROUP I

18:1

ULAJ

GROUPmf

18

14

16

18:1

GROUP EI

181

16

161

14

8

14

16:1

18 16

18:1

l

0 7 14 21 28 35 0 7 14 21 28 35

MINUTES

FIG. 1. Representativechromatograms ofeachofthefourgroupsofpeptococciandpeptostreptococcibased onlong-chain fattyacidanalysis ofwhole cells.

sentative

chromatogram

for this group is

showninFig. 1.

Table2shows theaverage percentagesof the fattyacid composition of group III organisms.

GroupmincludedPeptococcus variabilus,

Pep-tococcus magnus, Peptococcus

asaccharolyti-cus,Peptococcusprevotii, and

Peptostreptococ-cusproductus. Themostprominentfatty acids

ingroup III wereidenticaltothefatty acidsin

group 11

(C16:1,

C16,

C18:1,

and C18). Again, as in

group II, the highest peak on the

chromato-gram was consistently C16 or

Cj8:l.

Group

Ill

differed fromgroupII,

however, by

thepresence

of three to six additional

compounds (Fig.

1). Thesepeaks wereallunidentified and occurred

between C14 and

C16:1,

between C16 and C18:1,

and between C18 and C20.

GroupIVcontainedoneorganism,

Peptococ-cussaccharolyticus. The LCFAs extracted from

P.saccharolyticusareshown inTable3.

Promi-nentpeaksofC16,

C08:1,

and C18weresimilarto

those seen in groups II andIII;however, C16:1

was conspicuously absent from group IV. P.

saccharolyticus

contained significant amounts

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TABLE 1. Average percentages of the long-chain fatty acids extracted from group II cocci

Peptostreptococcus sp. Peptococcus sp. Fatty acids P.

intermedius

P.

micros

P. parvulus P. morbillorum P. constellatus

(15 strains) (8strains) (1strain) (5strains) (5strains)

C12 0.7 1.4

(0-3.7)a (0.6-2.6)

Unidentified 1.2 0.3

(0-6.2) (0-1.0)

C14 4.6 3.2 4.2 3.1 4.8

(2.3-7.3) (0-6.8) (1.9-4.0) (3.4-6.1)

C16:1 8.4 5.0 9.6 10.2 7.3

(4.0-28.3) (2.3-6.7) (2.9-30.1) (6.4-8.9)

C16 35.1 22.5 18.4 20.1 32.5

(13.7-44.7) (19.6-35.2) (14.2-31.5) (19.5-40.0)

C18:1

39.8 53.7 54.8 53.5 43.1

(27.3-50.2) (41.5-63.2) (23.2-69.1) (34.9-58.3)

C08 11.2 15.8 13.0 12.1 10.7

(9.4-16.1) (11.6-20.7) (5.1-18.0) (8.9-12.1)

aThe numbers in parentheses are the range (in percent) for all organisms tested.

of C20 and an unidentified compound with a

retention timebetween

C14

and

C16,

which

ap-pearedtobe unique characteristics of this

spe-cies in group IV. Fig. 1presents a

representa-tivechromatogram ofgroup IV(P.

saccharoly-ticus).

DISCUSSION

Thetaxonomy of the peptococci and the

pep-tostreptococci is unclear. Four of the most

nota-ble publications in this area differ greatly in

thespeciationof theseorganisms. Publications

ofRogosa (14, 15),Sutteretal. (16),Dowell and

Hawkins (5), and Holdeman and Moore (7) de-scribe 11species ofpeptococci and six species of peptostreptococci, and they differon the

num-berand identity of species within eachgenus.

Sinceall of the peptococci and peptostreptococci

(exceptPeptococcus niger)areconsideredtobe

associated with infectious disease in humans, thereshould be reliable methods for identifying theminthe clinicallaboratory. Rogosa (14, 15)

listssixspeciesofpeptococciand fivespeciesof peptostreptococci. Of thelatter 11species, five species (Peptococcus niger,

Peptococcus

aero-genes, Peptococcus activus, Peptococcus

an-aerobius, and Peptostreptococcus lanceolatus)

are not

recognized

by either Sutteret al. (16),

Dowell and Hawkins (5), or Holdeman and

Moore (7). Table4 presents a summaryofthe

current classification of the 12 most

generally

recognized species of peptococci and

peptostrep-tococci and includes the grouping based on

LCFAprofiles. Of the 12 specieslistedinTable

4,thereisgeneralagreement on the taxonomic

status ofonly two species, Peptostreptococcus

anaerobius and Peptostreptococcus

interme-dius. It is evident that much work needs to be

doneonthetaxonomyof thepeptococciandthe

peptostreptococci.

This study, for the first time, characterized

the LCFAs of the12mostuniformly recognized species of peptococci and peptostreptococci. On the basis of their LCFA chromatographic

pat-terns, each species of peptococcus and

pepto-streptococcus assayedcould be placedinto one

offourdifferent groups.

Asaresult ofLCFAprofiles,

Peptostreptococ-cusanaerobiuswasplacedina groupbyitself. One distinguishing feature ofP. anaerobius

wasthe large number (14 to 25)of peaksonthe chromatograms. No other species studied

pro-duced over 12 peaks. The most characteristic feature of the profile ofP. anaerobius was a

unique, prominent peak between C5 and C00.

Unfortunately, the latter peak hasnotyetbeen identified andmaybe an iso-acid.

Group II cocci containedPeptostreptococcus intermedius, Peptostreptococcus micros,

Pep-tostreptococcusparvulus, Peptococcus

morbil-lorum, and Peptococcus constellatus. The

spe-cies in group II produced characteristic

amounts ofC14,

C16:i,

C16,

Cl8:1,

and

Cl5

fatty

acids. The latterprofileissimilartothe

profile

reported for fatty acids in some ofthe

faculta-tivestreptococci (2, 9). Some of the organisms

inthisgroup are now considered tobe members

of the genus

Streptococcus

since

they produce

lacticacidas amajor metabolicproduct. Onthe

latter basis, P. intermedius is now

generally

recognized as belongingto the genus

Strepto-coccus (4, 8, 14-16). Similarly,P.morbillorum

and P. constellatus are now

widely

considered

to be members of the

streptococci (4, 8, 16).

Peptostreptococcus micros, which

produces

lac-tic acid as a minor product, is still

generally

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TABLE 2. Average percentagesofthelong-chain fattyacids extractedfromgroupIII cocci

Peptococcus sp.

Peptostreptococcus

Fatty acids P. variabilus P.magnus P.asaccharolyti- P. prevotii productus(1 strain) (5 strains) (8strains) cus(9 strains) (6 strains)

C12 0.4 2.9

(0-1.8)a

Unidentified 1.8 0.8

(1.0-2.5) (0-3.4)

C14 2.4 5.7 2.5 6.9

(0-3.3) (2.9-8.5) (0-9.0)

Unidentified 0.2 0.6 0.8 0.8 3.0

(0-1.0) (0-1.6) (0-4.1) (0-2.9)

Unidentified 2.3 0.7

(0-3.6) (0-2.8)

C16:1 5.2 4.7 10.5 6.2 2.3

(2.9-6.6) (3.4-6.4) (3.1-15.3) (2)2-13.0)

C16

11.6 11.1 24.7 13.0 26.8

(6.8-17.3) (9.3-13.4) (17.7-31.1) (2.6-27.1)

Unidentified 17.2 13.3 5.0 9.0 15.8

(13.2-20.0) (10.3-21.1) (2.0-9.5) (2.9-16.7)

Unidentified 1.9 4.2 5.5

(0-5.6) (0-12.2) (0.5-8.8)

C18:1

43.6 36.6 31.3 49.1 13.4

(38.0-48.9) (30.4-44.4) (18.7-49.2) (30.0-77.6)

C18

6.6 7.6 5.8 8.4 7.5

(4.4-8.6) (6.8-8.8) (3.9-10.4) (5.6-11.4)

Unidentified 10.2 18.8 8.3 4.6 14.2

(2.9-21.4) (17.0-21.7) (3.7-12.0) (2.3-8.1)

Unidentified 1.4 4.6 0.9 0.3 7.3

(0-4.7) (2.8-6.5) (0-2.2) (0-1.1)

aThe numbersinparentheses are the range (in percent)for all organisms tested.

TABLE 3. Average percentages ofthelong-chain fatty

acids extractedfromgroup IV cocci

FattyFattyacidsacids Peptococcus saccharolyticus

~~(4

strains)

C14

Unidentified

C16

Unidentified

C18:1 C.8

Unidentified

Unidentified

C20

2.9

(1.4-5.0)a

26.5 (16.9-32.5)

12.8 (5.6-23.9)

2.5 (0-9.8) 14.3 (9.5-22.7)

18.3 (13.3-22.7)

2.5 (2.0-2.7)

5.7 (0-8.8) 13.2 (8.5-15.7)

aThe numbersinparentheses aretherange (in percent)forallorganisms tested.

consideredtobeamember of the

peptostrepto-cocci.Thefatty acidsinP.parvulus also placed

this organism in group II; however, only one

strain was analyzed and this should not be

considered a definitive characterization. The

VPI Anaerobe Laboratory Manual also de-scribes P. parvulus on the basis of a single isolate and the Manual also notes that this

organismproduceslacticacidas amajor

meta-bolic product (7). Thus, the most significant

interpretationsof the LCFA profiles ofgroupII

organismsare: (i) theseLCFAprofilesare

con-sistent with theprofilesof the facultative

strep-tococci;and(ii)allspeciesnowbeingconsidered

for classification with thestreptococci have all

beenplacedingroup II(Table 4) on the basis of

LCFAanalysis.

Group

IIIcocci, consisting ofPeptococcus

var-iabilus, Peptococcus magnus, Peptococcus

asaccharolyticus, Peptococcus prevotii, and Peptostreptococcusproductus, appeared to be a

miscellaneous assortment of organisms that produce similar LCFA profiles that consist of

C16:1, C16,

C08:1,

and

C1l

fatty acidsin amounts

similartothoseseeningroupII. The

chromato-grams oftheorganisms in group m contained

from threeto sixadditional, unidentified peaks

notseen in group H organisms, and thus

war-rantedthe formation of a separate group. The

organisms in group III possessed some

associa-tions that were consistent with the published

literature on anaerobic cocci. Holdeman and

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TABLE 4. Current classification of the species of peptococci andpeptostreptococcia

Organism Rogosa (14,15)8 Sutter et al. Dowell and Holdeman and Long-chain fatty

(16) Hawkins (4, 5) Moore (6-8) acidgrouping

Ps. anaerobius R R Rc R 1

Ps. intermedius S S S S 2

Pc. morbillorum U S S S 2

Pc. constellatus R R S S 2

Ps. micros R R Q R 2

Ps. parvulus R R Q R 2

Pc. magnus Ud Rd Q Re 3

Pc. variabilus Ud Rd Q Re 3

Pc. asaccharolyticus R R Rf R 3

Pc. prevotii Ug R Rh R 3

Ps. productus R R Q R 3

Pc. saccharolyticus U U RI R 4

a Abbreviations: Pc., Peptococcus; Ps., Peptostreptococcus; R, recognized species; U, unrecognized species;

Q, questionable status; S, Streptococcus species.

b Numbers in parentheses indicate literature reference.

c Designated Peptostreptococcus Center for Disease Control (CDC) group 3.

dConsidered the equivalent of Peptococcus anaerobius.

eConsidered equivalent organisms.

' Designated Peptostreptococcus CDC group 1.

9Considered the equivalent of Peptococcus asaccharolyticus.

hDesignated Peptostreptococcus CDC group 2. iDesignated Peptococcus CDC group 2.

Moore(6)considerP.magnusandP. variabilus tobe thesame organism sincetheir

biochemi-calprofilesareidenticalifoneadds Tween 80 to

their growth medium. Rogosa (14) claims that

P.variabilus and P. magnus are the equivalent

of aPeptococcus anaerobius. As a solution to

this obvious confusion, West and Holdeman

(17) propose that Peptococcus anaerobius

should be

rejected

sinceitisa nomenconfusum

due to the existence ofPeptostreptococcus

an-aerobius. An example oftwo other organisms

thatmay notbeseparatespecies are P.prevotii

and P.

asaccharolyticus.

Rogosa (14)statesthat

P.prevotii and P.asaccharolyticusare

indistin-guishable since they differ only by an indole

reaction out ofover 100 other characteristics.

The placement ofP. productus in group III

must notbeconsidereda reliableclassification

sinceonlyonestrain wasavailable foranalysis.

Thus,as seen inTable4, group

Ill

containsfive

organisms and any two organisms considered

equivalent by any taxonomist were always

placedinthe same LCFAgroup.

On the basis of the LCFAprofiles ofwhole cells, asingle species, P. saccharolyticus, was

placed in group IV. P. saccharolyticus

con-tained

fatty

acids C14, C16,

Ci8:1,

and C18 that appeared to be typical of the other peptococci and peptostreptococci.Thisorganism,however,

had twounique characteristics not seen inthe

other peptococci and peptostreptococci: (i) sig-nificantamountsof

C20

wereproduced;and (ii)

a prominent, unidentified peak with a

reten-tion time between C14 and

C16

was present.

This peak may be a branched-chain 15-carbon

fatty acid similar to that reported in cell

ex-tractsofthe genus Ruminococcus (1). Some of

theliterature on the peptococci and

peptostrep-tococci support placing P. saccharolyticus in a

separate group. Dowell and Hawkins(5),inthe

Center for Disease ControlLaboratoryManual,

listed P. saccharolyticus astheonly speciesin

the peptococci. Rogosa (14) claimed thatP.

sac-charolyticus was frankly saccharoclastic and

should be excluded from thegenusPeptococcus,

without, however, mentioningwhere the

orga-nism should be placed. On the basis of LCFA

analysis, these studies agree with Rogosa (14,

15) and with Dowell and Hawkins (5), thatP.

saccharolyticus is significantly different from

the other peptococci andpeptostreptococci and

should beplacedinagroup by itself.

This study showed that LCFA analysis can

be used as an aid intaxonomy,but cannot be

relied on as a definitive test forspecies

designa-tion. Further work must be doneifthe

specia-tion of thepeptococci andthepeptostreptococci

is tobe clarified. Additional biochemical tests,

serological studies, guanine-cytosine ratios, or

deoxyribonucleic acid homologystudies might

prove helpful for speciation. The grouping of

speciesobtained from the LCFAprofilesmaybe

an indicationthat thesespeciesdooverlapeach

other agreat deal.Perhapsourgoalshould not

be to describenew species ofthese organisms,

but to consolidate the species known to exist.

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LCFAs OF PEPTOCOCCI AND PEPTOSTREPTOCOCCI 521

Thisapproachseems morelogical,atleast until

definitivepathogenicitystudieshave been

com-pleted.

ACKNOWLEDGMENTS

Thisstudywassupported by the Wisconsin Alumni

Re-search Federation. Wearegrateful forthecooperationof the members of the GeneralBacteriology Laboratoryatthe WisconsinStateLaboratory of Hygiene. Weespecially ap-preciateassistance givenby A. Helstad. We also wishto acknowledge E. Balish for his advice in thepreparation of this manuscript.

LITERATURE CITED

1. Allison, M. J., M. P. Bryant, I. Katz, and M. Ke_a_y. 1962. Metabolic function of branched-chain volatile fatty acids,growth factors for ruminococci. II.

Bio-synthesis of higher branched-chain fattyacids and aldehydes.J. Bacteriol. 83:1084-1093.

2. Amatein,C. F., and P. A. Hartman.1973. Differentia-tionofsomeenterococci bygaschromatography. J.

Bacteriol. 113:38-41.

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