JOURNAL OF BACTERIOLOGY, Feb. 1969, p. 561-570Copyright © 1969 American Society for Microbiology

Vol. 97, No. 2Printed In U.S.A.

Cultural Characteristics and Fatty AcidComposition of Propionibacteria

C. WAYNE MOSS, V. R. DOWELL, JR., D. FARSHTCHI, L. J. RAINES, AND W. B. CHERRY

National Communicable Disease Center, Atlanta, Georgia 30333

Received for publication 2 November 1968

The cultural characteristics and cellular fatty acid composition of 40 strains rep-

resenting 7 species of Propionibacterium and of 9 cultures of anaerobic coryne-bacteria were studied. The cultures were characterized by means of 23 separatecultural and biochemical tests. Cultures of the two genera differed consistently inonly two reactions; the propionibacteria did not produce indole or liquefy gelatin,whereas the anaerobic corynebacteria were consistently positive with these tests.The fatty acids were extracted from whole cells and examined as methyl esters bygas-liquid chromatography. The most abundant acid in the seven Propionibacteriumspecies was a C15-saturated branched-chain acid which was present in both the iso-and anteiso-form. Based on a comparison of the relative abundance of these iso-mers (i-C15 and a-C15), the species were separated into two groups. P. freudenreichiiand P. shermanii (group one) were similar and contained the a-C15 isomer as thepredominant acid. The i-C15 isomer was the most abundant acid in the second group(P. arabinosum, P. jensenii, P. pentosaceum, P. thoenii, and P. zeae). The fatty acidprofiles of the anaerobic corynebacteria were somewhat similar to those of the secondgroup of propionibacteria, but were distinct from the profiles of P. freudenreichiiand P. shermanii. The addition of branched-chain amino acids (L-leucine and L-isoleucine) to the growth medium increased the synthesis of the specific fatty acid(s)structurally related to the added amino acid.

Members of the genus Propionibacterium aretaxonomically very similar to anaerobic species ofCorynebacterium. C. acnes, one of the most fre-quently encountered anaerobic species in clinicalmaterials (5, 13), has been tentatively assigned toPropionibacterium by some workers on the basisof similar biochemical and morphological charac-teristics (3, 7). Others, however, have reportedthat C. acnes is sufficiently different in morpho-logical and physiological features, and in deoxy-ribonucleic acid composition, to warrant itsseparation from the genus Propionibacterium (14,16, 17).

Previous work from this laboratory has shownthat the relationship of C. acnes to the genusPropionibacterium extends beyond morphologicaland cultural similarities to include common cellu-lar constituents such as fatty acids. We found thatthe fatty acid profiles of C. acnes were essentiallyidentical to those of P. freudenreichii and P.shermanii, and that the three species were charac-terized by the presence of large amounts of asaturated C15 branched-chain fatty acid (10).Further study (9), however, indicated that C.acnes could be distinguished from P. freudenreichii

and P. shermanii on the basis of the point ofbranching of the methyl group in the C15 fattyacid chain; C. acnes contained 13-methyltetra-decanoic acid (i-C15) as the most abundant acid,whereas 12-methyltetradecanoic acid (a-C,5) wasthe most abundant acid in the two Propionibac-terium species.Data on the cultural characteristics and fatty

acid composition of additional cultures of P.shermanli, P. freudenreichii, and other Propioni-bacterium species are presented. These cultureswere compared with several strains of anaerobiccorynebacteria.

MATERIALS AND METHODSOrganisms. Cultures used in this study were gra-

ciously supplied by the following: G. W. Reinbold,Iowa State University, Ames; C. H. Zierdt, NationalInstitutes of Health, Bethesda, Md.; R. Latta, Na-tional Research Council of Canada, Ottawa; R. E.Hargrove and J. A. Alford, U.S. Department of Agri-culture, Washington, D.C.; P. A. Hansen, Universityof Maryland, College Park; and E. McCoy, Univer-sity of Wisconsin, Madison. The source of the strainsand the identifying number assigned by the supplyinglaboratory are listed in Table 1.

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MOSS ET AL.

Maintenance of cultures. All cultures were main-tained in Fluid Thioglycollate Medium (BBL) andweretransferred weekly. As standard procedure, all liquidmedia were boiled for 10 min and cooled rapidly in awater bath just prior to inoculation. Cultures weregrown in an electrically heated, catalyst-type Breweranaerobe jar which was evacuated and filled succes-sively three times with a mixture of 80% Ng, 10%H2, and 10% CO2 (Matheson and Co., East Ruther-ford, N.J.) and catalyzed 10 min prior to incubation at35 C. Smith's modified methylene blue indicator mix-ture was used to test for anaerobic conditions (2).

Fermentation studies and differential biochemicaltests. The media, reagents, and procedures used todetermine cultural and biochemical characteristicswere described previously (4, 9).

Growth media and cultural procedures for fatty acidstudies. Cultures for fatty acid studies were routinelygrown in thioglycolate medium (pH 7.4) which con-tained 1.7% Trypticase (BBL), 0.3% Phytone (BBL),0.6% dextrose (Difco), 0.05% Sodium Thioglycollate(Difco), 0.25% NaCl (Fisher Scientific Co., Pittsburgh,Pa.), 0.01% Na2SO3 (Fisher), and 0.025% L-cystine(Nutritional Biochemicals Corp., Cleveland, Ohio).The same lot of each ingredient of this medium wasavailable in bulk supply and was used throughout thestudy. Where indicated, comparative studies to deter-mine the effects of growth medium were made inAlternate Thioglycollate Medium (ATM, Fisher), andin ATM supplemented with 10 mmoles of L-isoleucine(Calbiochem, Los Angeles, Calif.) or L-leucine(Fisher). Cell crops were produced by two successivetransfers of culture in the respective growth mediumprior to final transfer into 250 ml of medium. Cultureswere routinely incubated for 24 to 48 hr in a Breweranaerobe jar. Cells from the third transfer (250 ml ofmedium) were collected by centrifugation at 4 C andanalyzed without washing. Preliminary studies indi-cated the presence of trace amounts of certain fattyacids in the growth medium but not in amounts suffi-cient to affect the results reported here. The proce-dures used for saponification of whole cells and forextraction and methylation of acidic components weredescribed earlier (11). The methylated bacterial fattyacid samples were analyzed immediately or werestored at -20 C.

Gas-liquid chromatography (GLC) analysis. Themethyl esters were analyzed with a Barber-ColmanModel 5000 Gas Chromatograph (Barber-ColmanCompany, Rockford, Ill.) equipped with a hydrogen-flame ionization detector and a disc integrator recorder(series 8000). Samples were analyzed on an 8-ft (2.4-meter) U-tube glass column containing 15% ethyleneglycol succinate (EGS) coated on 80/100 meshChromosorb W (Applied Science Laboratories, StateCollege, Pa.), and also on a 6-ft (1.83-meter) columnof 2% SE-30 methyl silicone rubber gum coated on80/100 mesh Chromosorb P (Applied Science). TheEGS column was used to resolve branched-chain fattyacids (9). Operating parameters of the instrumentwhen employing the SE-30 column were: injectiontemperature, 230 C; detector temperature, 250 C;column temperature, 110 to 215 C at 3 C per min;carrier gas, nitrogen.

For routine analysis, 3 pliters of the methylatedbacterial fatty acid samples was analyzed for 45 minafter injection onto the SE-30 column under the above-stated conditions. This time interval was sufficient todetect normal saturated fatty acid methyl esters ofcarbon chains from 8 to 23 carbons in length. Thefatty acid methyl ester peaks tentatively were identi-fied by comparison of retention times on both col-umns (EGS and SE-30) with retention times of highlypurified methyl ester standards (Applied Science andNational Institutes of Health). Peak areas were de-termined by disc integrator, and the percentage ofeach acid was calculated from the ratio of the area ofits peak to the total area of all peaks. In this study,data are presented on those acids which were presentat concentrations greater than 1%.

RESULTSCultural and biochemical characteristics of

Propionibacterium. The cultures when receivedwere activated by growth in thioglycolate brothand checked for purity with Gram stained smearsand blood-agar streak plates incubated aerobi-cally and anaerobically. Single-colony isolateswere studied in detail as described previously (10).The cultural and biochemical characteristics ofthe 40 Propionibacterium cultures examined areshown in Table 1. All of the strains fermentedglucose and glycerol and were negative for he-molysis of rabbit blood, indole production, gela-tin hydrolysis, motility, and urease activity. Amajority of the cultures hydrolyzed esculin andproduced catalase. In general, the results obtainedwith the various Propionibacterium species con-formed to those differential reactions outlined inBergey's Manual of Determinative Bacteriology(Table 2).

Cellular fatty acids of Propionibacterium.Table 3 shows the principal fatty acid compositionof cells after growth in thioglycolate medium. Allstrains of the genus were uniform in that thesingle most abundant acid was a C15 branched-chain acid. Two isomeric forms, 12-methyltetra-decanoic acid (a-C15) and 13-methyltetradecanoicacid (i-C15), were present in each strain, but indifferent proportions. Comparisons of the relativeamounts of these two acids allowed the strains tobe placed into two distinct groups.The overall fatty acid profiles of P. freuden-

reichii were essentially identical to those of P.shermanii. The characteristic and predominantfatty acid in each strain of these two species wasthe a-C15 acid which was generally present atconcentrations two to three times that of anyother single acid. The i-C15 acid also was presentin each strain, but in concentrations 4 to 25 timesless than the a-C15 acid. The next most abundantacids in most strains of these two species werepentadecanoic, hexadecanoic, and a Cu7 saturated

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CHARACTERISTICS OF PROPIONIBACrERIA

TABLE 2. Comparison of biochemical characteristics of Propionibacterium species as determined In the present study to reactionslisted In Bergey's Manual of Determinative Bacteriologya

P. freudenrweichii P. skermaiii P. arabinosum P. jeuseuii P. peniosaceum P. ikoenii P. aeeeReaction _

Pres- Ber- Pres- Ber- Pres- Ber- Pres- Ber- Pres- Ber- Pres- Ber- Pres- Ber-ent gey's ent gey's ent gey's ent gey's ent gey's ent gey's ent gey's

Gelatin li-quid. -

Indole pro-duction. - -

Milk coagu-lation. - - V + _+ __ + + + + - V - +

NOs --

NO2 _ .(+) - V NR - _ V + - _ -_Glucose.....A A A(A) A A A A A A A A A A AMannitol... _ _ _ - A A A V A A V - A ALactose.... A A V ? V A A A A A - ASucrose .... (A) - A A A A A A A(A) A A AMaltose.... (A) ..(A) - A A A- A A A A A A ASalicin. ... (A) NR (A) - A- (A) - V A A (A) AGlycerol .... A A A A A A A A A A A A A AFructose.... V A V A A A A A A A A A A AXylose....-(A) - V - _-(A) - - - A A _(A) - _ -(A) -

Arabinose.. -(A) - V V A A - - A A A - _(A) AStarch hy-

drolysis... - NR - - _ r) ? - - + NR V - VEsculin hy-

drolysis... V + V + + NR +_ + + + + + + NRCatalase ....+ + + + V +sl++ - +s V + + +

a Abbreviations and symbols: + = positive test;- = no reaction; A = acid; () = delayed reaction; V = variable reaction;sl = slight reaction; ? = doubtful reaction; NR = not recorded. Superscripts denote delayed or occasional reactions.

branched-chain acid. In addition, some culturescontained relatively large amounts of acids withgreater than 19 carbon atoms (C2o, C21, C22, C2s).Other acids detected were generally present in lowconcentration.

Although the large percentage of the a-C15 acidwas characteristic of P. shermanii and P. freuden-reichii, differences in the relative amounts of thisacid among strains were observed. Most strains(13) contained between 41 and 50% of this acid,but 9 strains contained between 31 and 40%, and1 strain contained 57%.The i-C15 acid was the principal fatty acid in

the other five Propionibacterium species tested(P. arabinosum, P. jensenii, P. pentosaceum,P. thoenii, and P. zeae; Table 3). Although thesespecies were similar to each other, each wasreadily distinguishable from the P. shermanii-P.freudenreichii group which, as noted, containedrelatively small amounts of i-C1s acid and largeamounts of the anteiso-isomer (a-C1s). The rela-tive abundance of the i-C15 acid was the onlyconsistent and distinguishing characteristic ofthese five species; other cellular fatty acids weresimilar to those of one or more strains of P.shermanii and P. freudenreichii (Table 3).

Cultural and biochemical characteristics ofanaerobic corynebacteria. Physiological charac-teristics of the corynebacteria studied are listed in

Table 4. The results show a close relationshipamong the various cultures in their culturalandbiochemical activity. Moreover, the data showthat the cultures labeled as various anaerobicspecies of Corynebacterium differ no more fromC. acnes than C. acnes strains differ among them-selves. All strains fermented glucose, glycerol, andfructose, but not lactose, sucrose, maltose, xylose,or arabinose. They were all indole-positive, non-motile, urease-negative, failed to hydrolyze escu-lin or starch, and produced traces of H2S whentested with lead acetate paper strips. Variablereactions were noted for hemolysis of rabbit bloodand mannitol fermentation. All strains werecatalase-positive, reduced nitrate to nitrite, andliquefied gelatin. These results are consistent withthose of Zierdt, Webster, and Rude (17), andwith those from an earlier study (10).Fatty adds of anaerobic corynebacteria. Table 5

shows the principal cellular fatty acid compositionof the corynebacteria after growth in fluid thio-glycolate medium. Each of the cultures testedpossessed similar fatty acid profiles, thus limitingtheir grouping into species by fatty acid analyses.The fatty acid composition of the four C. acnescultures was consistent with our earlier findings(9, 10). The results indicate apparent homogeneitynot only among strains of C. acnes, but alsoamong the other anaerobic corynebacteria. Each

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culture of the anaerobic corynebacteria contained are less easily distinguished from other Propioni-the i-C15 acid as the single most abundant acid bacterium species. As noted above, strains of P.with relatively small amounts of the a-Cis acid. arabinosum, P. jensenii, P. pentosaceum, P. thoenii,On this basis, they are clearly distinguishable from and P. zeae each contained the i-C15 acid as theP. shermanii and P. freudenreichii; however, they major component, but, unlike the anerobic co-

TABLE 3. Percentage ofprincipal cellular fatty acids of Propionibacterium species

Fatty acid

Organism NCDC--__ __-__no. 1712:01 Un | 14:0 |iCio|aC o 15.0@ 161 160917: 17:0 18:1 18:0 19:0 20:0 21:0 22:0 23:012:u-~niOBr

P.freudenreichii

P. shermanii

P. arabinosum

P. jensenii

P. pentosaceum

P. thoenii

P. zeae

30963103343534403442345134523441

3087308930903094309831043106310831093100343834473446B34483450

309130993431344434453449

308630923101-131023443

30883433

30953434

30973432

tPbtr111111

tr111tr1trtrtr112111

trtr1121

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tr1

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tr2

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trtrtrtr11

trtrtrtrtr

tr1

tr1

tr2

11112315

222112113512242

tr13121

12

22

35

32

23

33532322

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39

410

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36

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11

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61

22

102

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73

31

131

107242134

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b Numbers refer to percentages of total acids; tr = less than 1%.

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568 MOSS ET AL. J. BACTERIOL.

rynebacteria, these species also contained rela- relative abundance of the major acids, i-C15 intively large amounts of the a-C15 acid. The ratio C. acnes and a-C18 acid in the two Propionibac-of i-C15 to a-C15 acid was generally 2:1 in the terium species, was comparable in each medium.Propionibacterium species, but 6:1 or greater in the Studies with various bacterial species haveanaerobic corynebacteria (Tables 3 and 5). shown that the relative abundance of cellular

Effect of culture media on fatty acid composi- branched-chain fatty acids is markedly affected bytion. The fatty acid composition of C. acnes, P. the availability in the growth medium of pre-shermanii, and P. freudenreichii after growth in cursors of the terminal portion of the acids (1, 6).Alternate Thioglycollate Medium is presented in Two potential amino acid substrate precursors ofTable 6. The fatty acid profiles in this medium branched-chain acids, L-isoleucine and L-leucine,were not markedly different from those observed were added to ATM to determine their effect onin thioglycolate medium (Table 3). Moreover, the fatty acid composition of several strains of C.

TABLE 5. Percentage ofprincipal cellular fatty acids of anaerobic corynebacteria

Fatty acid

Organismn NCDC --____ ______no.17012:0° Un 14:0 iC1s:oaCis:o 15:0 16:1 16:0 | lrj 17:0 18:1 18:0 19:0 20:0 21:0 22:0 23:0

C. liquefaciens . 3420 lb 1 2 55 3 4 2 10 8 2 2 2 tr 2 6 tr trC. granulosum. . 3421 2 1 1 37 7 10 1 11 6 1 2 2 tr 7 5 7 trC. anaerobium 3422 tr tr 1 62 2 A 1 10 14 2 1 1 tr 1 tr 1 trC. diph-

theroides . ... 3423 tr tr 2 41 2 13 1 7 17 2 1 2 2 1 1 7 1C. acnes........ 3425 tr tr 3 32 2 16 1 15 24 3 1 1 1 1 tr tr tr

3426 1 tr 1 43 2 4 1 10 11 1 2 2 tr 1 12 9 tr3427 1 1 3 58 3 3 1 6 8 1 1 1 1 1 6 5 tr3428 1 2 5 25 1 9 1 14 9 1 3 3 2 4 7 13 tr

C. pyogenes..... 3429 1 tr 1 33 5 2 1 3 2 1 2 3 7 16 17 4 2

a See footnote a, Table 3.b See footnote b, Table 3.

TABLE 6. Percentages of principal celluar fatty acids of C. acnes and Proplonibacterlum species grown In ATM and In ATM with10 mmoles of leucine (leu) or isoleucine (fleu)

Fatty acidOrganism Growth medium _ ' aC

12.Oa Un 14:0 iC aC 15:0 16:1 16:0 17: 17:0 18:1 18:019:0 20:0 21:0 22:023:01s:o0 1a:o0 OBr

C. acnes1925 ATM trb tr 1 44 2 3 3 10 10 3 3 3 1 3 6 4 4

ATM + ileu 1 tr 2 27 14 4 3 12 4 3 2 4 4 5 7 5 31934 ATM 1 1 2 44 2 2 1 4 9 5 2 1 2 4 5 7 8

ATM + ileu 2 2 1 32 13 6 1 3 3 5 4 1 3 5 6 7 62000 ATM 1 tr 3 48 2 6 2 5 19 5 1 1 tr 2 3 2 tr

ATM + ileu 1 tr 2 33 18 7 2 7 16 5 1 1 tr 2 2 3 tr2022 ATM 2 tr 2 43 2 4 2 5 18 2 2 2 1 4 2 8 1

ATM + ileu 1 tr 1 34 14 5 3 6 14 3 1 3 2 3 2 7 1P.shermanl3094 ATM 2 2 4 2 41 9 1 4 20 2 1 2 tr 3 2 4 1

ATM + leu 1 1 5 21 33 8 1 5 15 1 tr 2 tr 1 2 3 13104 ATM 2 1 2 4 45 10 1 5 16 1 3 1 tr 2 3 4 tr

ATM + leu 3 1 3 21 32 8 tr 5 14 3 1 2 tr 1 3 2 1P. freundenrekchff3096 ATM 3 1 4 3 49 10 1 10 8 tr 4 1 1 tr 2 2 1

ATM + leu 2 2 5 18 33 12 tr 11 7 tr 2 3 tr 1 3 1 tr3103 ATM 5 2 7 2 38 8 2 12 13 2 1 4 tr 1 2 1 tr

ATM + leu 4 tr 6 19 30 10 2 12 6 1 1 2 1 2 1 2 1

a See footnote a, Table 3.b See footnote b, Table 3.

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CHARACTERISTICS OF PROPIONIBACTERIA

acnes and of Propionibacterium. The results inTable 6 show that L-isoleucine, which serves as aprecursor of anteiso-acids, markedly increasedthe relative amount of a-C15 acid in each C. acnesculture. L-Leucine, a precursor of iso-acids,effected analogous changes in the Propionibac-terium cultures by increasing the relative amountsof i-C15 acid. The increase in i-C15 acid was ac-companied by a corresponding decrease in a-C15and possibly in C17:0 Br acids, whereas the in-crease in a-C15 acid in C. acnes was consistentwith corresponding decreases in i-C15 and 17:OBracids (Table 5). These data indicate that theaddition of branched-chain amino acid sub-strates increases the synthesis of the specific fattyacid(s) structumlly related to the added sub-strate and decreases the synthesis of otherbranched-chain acid(s).

DISCUSSIONThe cultural characteristics and biochemical

activities of C. acnes have previously been shownto be similar to those of P. shermanii and P.freudenreichii (10). The present results, obtainedfrom studying 23 additional strains of the twoPropionibacterium species and 4 additional C.acnes cultures, support the earlier findings. Thedata reemphasize the difficulty in assigning exactgeneric and familial status to these organisms onthe basis of present cultural and biochemicalcriteria. As a group, the propionibacteria showedonly a limited number of homogeneous reactions;they were uniformly positive for production ofacid from glucose and glycerol and uniformlynegative for indole production, gelatin hydrolysis,motility, and urease activity (Table 1). Similarreactions were noted for C. acnes (Table 4), withthe exception that all strains of this species wereconsistently indole-positive and liquefied gelatin(10). Separation of C. acnes from propionibac-teria by these two reactions is debatable, especiallysince the reactions have been reported to bevariable for C. acnes (3, 12, 17).The fatty acid analyses of P. shermanii and P.

freudenreichil reported here are consistent withearlier findings (9, 10). The present study, whichwas expanded to include fatty acid determinationsof other propionibacteria, shows heterogeneityamong some species. The single most abundantacid in the seven species examined was a C15saturated branched-chain acid which was presentin each culture in both the iso- and anteiso-form.Based on the relative abundance of these isomers(i-C15 and a-C1s), the species were separated intotwo groups. The first group consisted of P. sher-manii and P. freudenreichii, which were indistin-guishable from each other. All cultures in thisgroup contained a-C15 acid as the predominant

acid and only relatively small amounts of i-C15acid. In contrast, the i-C15 isomer was the mostabundant acid in the second group, which con-tained P. arabinosum, P. jensenli, P. pentosaceum,P. thoenil, and P. azeae. The fatty acid profiles ofthese latter species, although clearly distinguish-able from those of the first group, were notmarkedly different from each other; Repeatedfatty acid analyses on representative strains ofthese two groups showed essentially identicalfatty acid profiles. The results, therefore, reflecttrue differences between the two groups in respectto their fatty acid metabolism.There are clear differences in the cellular fatty

acid composition of C. acnes and certain Propioni-bacterium species (Tables 3 and 5). Without ex-ception, each of 36 C. acnes cultures examined inthis and in earlier studies contained i-C15 acid asthe single most abundant acid with only minoramounts of a-C15 acid. Hence, these cultures canbe distinguished from P. shermanli and P. freuden-reichii, which contained relatively small amountsof i-C15 acid but large amounts of a-C15 acid. Theuse of these differences to distinguish betweenthese species seems valid since the data were ob-tained from a relatively large number of strainsfrom several different sources.The fatty acid profiles of P. arabinosum, P.

jensenii, P. pentosaceum, P. thoenii, and P. zeaeresembled those of C. acnes more closely thanthose of the P. shermanii-P. freudenreichii group.However, they could be distinguished from C.acnes by comparison of the ratios of i-C15 toa-C15 acid. Separation on this basis must, how-ever, be approached cautiously in view of themarked changes in fatty acid composition whichresult from varying the concentration ofbranched-chain fatty acid precursors in thegrowth medium (1, 6,15; Table 6).

In a recent study, Zierdt, Webster, and Rude(17) concluded that the anaerobic species ofCorynebacterium were so closely related bio-chemically, serologically, and morphologicallythat a reduction in the number of species wasfeasible. They designated C. acnes as the onlylegitimate anaerobic species and considered allother species to be synonyms of C. acnes. Thedata from the present study of cultures obtainedfrom Zierdt and labeled as C. liquefaciens, C.diphtheroides, C. granulosum, C. anaerobium, andC. pyogenes show that these cultures are essen-tially identical to cultures of C. acnes, both inbiochemical characteristics and in cellular fattyacids. Our results and those of Zierdt, Webster,and Rude (17), however, are based on the exami-nation of only one culture of each of thedesignated Corynebacterium species, none ofwhich represented the type culture of the species

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(17). Moreover, the biochemical results do notagree with the original descriptions of thesespecies (3, 12). Consequently, designation of C.acnes as the only anaerobic species of the genusdoes not seem justified at present. This viewpointis shared by others (8). The classification of theanaerobic corynebacteria should not be changeduntil studies are performed with large numbers ofcultures of anaerobic, nonsporulating, gram-positive rods.

LITERATURE CITED

1. Allision, M. J., M. P. Bryant, I. Katz, and M. Keeney. 1962.Metabolic function of branched-chain volatile fatty acids,growth factors for runinococci. II. Biosynthesis of higherbranched-chain fatty acids and aldehydes. J. Bacteriol.83:1084-1093.

2. Bailey, W. R., and E. G. Scott. 1966. Diagnostic microbiology,2nd ed. The C. V. Mosby Co., St. Louis.

3. Douglas, H. C., and S. E. Gunter. 1946. The taxonomic posi-tion of Corynebacterium acnes. J. Bacteriol. 52:15-23.

4. DoweUl, V. R., Jr., and T. M. Hawkins. 1968. Laboratorymethods in anaerobic bacteriology. U.S. Public HealthServ. Publ. No. 1803.

5. Evans, C. A., W. M. Smith, E. A. Johnston, and E. R. Giblett.1950. Bacterial flora of the normal human skin. J. Invest.Dermatol. 15:305-324.

6. Kaneda, T. 1966. Biosynthesis of branched-chain fatty acids.IV. Factors affecting relative abundance of fatty acids pro-

duced by Bacillus subtfls. Can. J. Microbiol. 12:501-514.7. Moore, W. E. C., and E. P. Cato. 1963. Validity of Proploni-

bacterium acnes (Gilchrist) Douglas and Gunter comb. nov.J. Bacteriol. 85:870-874.

8. Moore, W. E. C., L. V. Holdenian, and C. S. Cummings.1968. Objection: Corynebacterlum acnes is not the solelegitimate anaerobic species of Corynebacterlum. Intern. J.System. Bacteriol. 18:273-274.

9. Moss, C. W., and W. B. Cherry. 1968. Characterization of theCu branched-chain fatty acids of Corynebacterium acnes bygas chromatography. J. Bacteriol. 95:241-242.

10. Moss, C. W., V. R. Dowell, Jr., V. J. Lewis, and M.A. Schekter. 1967. Cultural characteristics and fatty acidcomposition of Corynebacterlum acnes. J. Bacteriol. 94:1300-1305.

11. Moss, C. W., and V. J. Lewis. 1967. Characterization ofclostridia by gas chromatography. I. Differentiation ofspecies by cellular fatty acids. Appl. Microbiol. 15:390-397.

12. Prevot, A. R., and V. Fredette. 1966. Manual for the clas-sification and determination of the anaerobic bacteria. Leaand Febiger, Philadelphia.

13. Puhvel, S. M. 1968. Characterization of Corynebacteriumacnes. J. Gen. Microbiol. 50:313-320.

14. SeBald, M., F. Gasser, and H. Werner. 1965. DNA base com-position of bifidobacteria and related genera. Ann. Inst.Pasteur 109:251-269.

15. Tornabene, T. G., E. 0. Bennett, and J. Or6. 1967. Fatty acidand aliphatic hydrocarbon composition of Sarcina luteagrown in three different media. J. Bacteriol. 94:344-348.

16. Werner, H. 1967. Untersuchungen tiber die Lipase und Leci-thinase-Aktivitat von Aeroben und Anaeroben Coryne-bacterium-und von Propionibacterium. Zentr. Bakteriol.Parasitenk. Abt. I Orig. 204:127-138.

17. Zierdt, C. H., C. Webster, and W. S. Rude. 1968. Study of theanaerobic corynebacteria. Intern. J. System. Bacteriol. 18:33-47.

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