JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1977, p. 555-558Copyright i) 1977 American Society for Microbiology

Vol. 6, No, 6Printed in U.S.A.

Enzymatic and Hemolytic Properties of Propionibacteriumacnes and Related Bacteria

ULRICH HOEFFLER

Institute ofHygiene, University of Cologne, Cologne, Germany

Received for publication 10 June 1977

The production of chondroitin sulfatase, hyaluronidase, deoxyribonuclease,gelatinase, phosphatase, lecithinase, and hemolysins was examined in 95 strainsof Propionibacterium acnes and four related species of anaerobic, respectively,microaerophilic coryneform bacteria (P. avidum, P. lymphophilum, P. granu-losum, and Corynebacterium minutissimum). All enzymes could be demonstratedin at least one representative of the species tested. Those Propionibacteriumspecies most frequently found in acne vulgaris lesions, i.e., P. acnes and P.granulosum, proved to be the most active organisms concerning the productionof the enzymes tested. P. avidum, on the other hand, showed the highest rate ofhemolytic activity.

There has been little information about theproduction of toxins and enzymes by anaerobicand microaerophilic coryneform bacteria. Exclu-sively, the production of lipases by Propionibac-terium acnes as an etiological factor in acnevulgaris has been widely discussed (17). Somereports have appeared about further enzymesof some few strains of P. acnes (7-10, 15, 18).Concerning representatives of the new Propi-onibacterium species, proposed by Johnson andCummins in 1972 (2, 4), no data have yet ap-peared. The aim of this study was to determinewhether there are differences in the enzymeproduction of P. acnes and four related species,as a mean of identification and, possibly, as anindicator of their virulence.

MATERLALS AND MErHODSBacterial strains. A total of95 well-defined strains

of anaerobic, respectively, microaerophilic propioni-bacteria and corynebacteria were examined. Thesestrains belonged to the following species.

(i) P. acneu (40 strains). Twenty-one strains ofP. acnes were received from abroad: American TypeCulture Collection (ATCC) strains 6919, 6921, 6922,6923, 11827, and 11828; V. R. Dowell, Jr., Center forDisease Control, Atlanta, Ga.: strains 5159, 6949, 6981,6994, and 7010; J. G. Voss, The Proctor & GambleCo., Cincinnati, Ohio: strains MC, A, M3, C45, 174V,0391, and UCLA18; C. S. Cummins, Anaerobe Labo-ratory, Virginia Polytechnic Institute and State Uni-versity, Blacksburg: strains 0162 and 3706. Nineteenstrains of P. acnes were isolated in our laboratoryfrom human hair and from acne vulgaris lesions, acti-nomycosis (11), and nonspecific anaerobic infectionsof the cervicofacial region (14).

(ii) P. avidum (33 strains). Thirty strains of P.avidum were isolated in Cologne from normal humanskin. Three additional strains were supplied by ATCC

(strain 25577) or by C. S. Cummins (strains 0575 and0589).

(iii) P. lymphophilum (one strain). The only P.lymphophilum strain tested was the ATCC strain27520.

(iv) P. granulosum (18 strains). Twelve strainsof P. granulosum were found in our institute in acnevulgaris lesions and on normal human skin. Six strainswere received from ATCC (strain 25564), J. G. Voss(strains C51, D21, D34, and V1), or C. S. Cummins(strain 0507).

(v) Corynebacterium minutisimum (3 strains).Strains 22347, 22348, and 22349 were obtained fromATCC.

All strains were differentiated according to Bergey'sManual of Deterninative Bacteriology (2); most ofthem have been used in our previous studies (3, 5, 12,13).

Media. Subcultures were done on A-agar medium,the composition of which has been described by Yonget al. (5). Bacterial suspensions were prepared in N-broth (1.5 g of trypton [Difco]-0.4 g of yeast extract[Difco]-0.8 g of NaCl-100 ml of distilled water [pH7.4]).

Preparation of inocula. One loop of a fresh cul-ture on A-agar medium was transferred to N-broth.After 24 h of cultivation at 37°C, 1 drop (Pasteurpipette) of each suspension was used as the inoculum.

Test methods. The bacteria were tested for theproduction of chondroitin sulfatase (15), hyaluroni-dase (15), deoxyribonuclease (8), and phosphatase (1)according to the procedures described by previousinvestigators. Production of lecithinase was detectedby incorporating egg yolk emulsion (7%; Oxoid) inplates with blood agar base (Oxoid) containing 1%NaCl. The medium for the gelatinase tube test was 3ml of nutrient gelatin (BBL; 16 g; distilled water wasadded to 100 ml). The hemolytic activity of the strainswas investigated by adding different concentrationsof blood (washed erythrocytes) obtained from rabbits,sheep, and humans to blood agar base (Oxoid). All

555

556 HOEFFLER

substrate plates were freshly prepared. After solidifi-cation, they were dried for 3 h at 37°C and theninoculated immediately. Plates were incubated for 48h at 37°C by using the Heraeus incubator VT/N2 (W.C. Heraeus GmbH, 6450 Hanau, Germany; 95% N2and 5% CO2 at normal atmospheric pressure), and theresults were read immediately thereafter. Each assay

was performed in duplicate. All reactions were devel-oped and interpreted as described by the originalinvestigators, with the exception of the hemolytic ac-

tivity of the strains, which was recorded as "positive"when a clear, colorless zone around the colonies ap-

peared or "weak" when slight and incomplete hemol-ysis under the colonies was found.

RESULTS

The results of the enzyme tests on 95 strainsof propionibacteria and corynebacteria are sum-

marized in Table 1. All enzymes could be dem-onstrated in at least one representative of thespecies tested.

Chondroitin sulfatase was found to be pro-

duced by 68% of the P. acnes strains and 28%of the P. granulosum strains. Hyaluronidaseproduction was found in 73% of the P. acnesstrains, 18% of the P. avidum strains, and 33%of the P. granulosum strains. All other speciesshowed no or only weak activities regarding bothenzymes.Concerning deoxyribonuclease production, all

strains tested showed positive or weak reactions,with the striking exception of 70% of the P.acnes strains. Gelatinase was produced bynearly all strains of P. acnes and P. avidum butnone of the P. granulosum and the C. minutis-simum strains.

All the propionibacteria examined showedonly weak or no phosphatase activity, whereasall three strains of C. minutissimum gave clearpositive reactions. In our study, lecithinase couldbe demonstrated in only a few strains of P.avidum and in a great number of the P. granu-losum strains (30 and 89%, respectively).Summarizing, hemolytic activity was common

in tested propionibacteria and corynebacteria

(Tables 2 and 3). Obviously, it was more clearlyvisible in agar plates with lower concentrationsof blood (Table 3). Some differences betweenhemolysis of sheep, rabbit, and human erythro-cytes were observed. Rabbit erythrocytes were

more susceptible to the lytic action of P. acnes

and P. granulosum strains than human ones.

Sheep erythrocytes seemed to be the most re-

sistant. All strains with hemolytic activityagainst sheep blood were also active againstrabbit and human erythrocytes. P. avidumstrains proved to be some of the most activeamong the species tested.

DISCUSSIONLittle information about the production of

enzymes by microaerophilic coryneform bacteriawas found in previous publications. The produc-tion of chondroitin sulfatase and hyaluronidaseby three strains of P. acnes has been reportedin a brief technical note by Smith and Willet(15). Puhvel and Reisner (10) found hy-aluronidase activity in seven strains of P. acnes,

using the turbidity reducing method. Deoxyri-bonuclease and phosphatase production havebeen examined in four strains of P. acnes byPorschen and Sonntag (8) and Porschen andSpaulding (9). No activity was found. Werner(18) examined 15 P. acnes strains for their leci-thinase activity at different anaerobic condi-tions. He reported practically the same numberof active strains as we do under conditions sim-ilar to those in our study. Only gelatinase activ-ity has been demonstrated by a lot of investiga-tors (2, 6, 12, 16). The possible pathogenic roleof neuraminidase and acylneuraminic acid lyasehas been discussed by Muller (7). Hemolyticactivity against sheep erythrocytes was foundby Marples and McGinley (6). Our data confirmand extend these observations with regard tofive further species of microaerophilic coryne-form bacteria.The results of our study with 95 strains of

microaerophilic coryneform bacteria ind cate

TABLE 1. Enzymes of P. acnes and related bacteriaa

Enzyme"

SpeciesNo. of Chondroitin Hyaluronidase Deoxyribonu- Gelatinase Phosphatase LecithinaseSeis strains sulfatase Hauoise clease

- w + - w + - w + - w + - w + - w +

P. acnes 40 13 5 22 11 0 29 28 7 5 2 0 38 19 21 0 40 0 0P. avidum 33 29 4 0 27 0 6 0 4 29 0 0 33 12 21 0 23 1 9P.lymphophilum 1 1 0 0 1 0 0 0 1 0 0 1 0 1 0 0 1 0 0P.granulosum 18 13 3 2 12 3 3 0 3 15 18 0 0 5 13 0 2 2 14C. minutissimum 3 3 0 0 3 0 0 0 0 3 3 0 0 0 0 3 3 0 0

Absolute numbers were used.-, No reaction; w, weak reaction; +, moderate to strong reaction.

J. CLIN. MICROBIOL.

ENZYMATIC AND HEMOLYTIC PROPERTIES OF P. ACNES 557

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TABLE 3. Hemolytic action of Propionibacteriumacnes and related bacteria on sheep, human, and

rabbit bloodaHemolysis of:

Species No. of Sheep Human Rabbitstrains blood (% blood (% blood (%

positive) positive) positive)

P. acnes 40 22.5 42.5 47.5P. avidum 33 69.6 75.7 69.6P. lymphophilum 1 0.0 0.0 0.0P. granulosum 18 0.0 5.6 100.0C. minutissimum 3 0.0 0.0 0.0

a Positive reactions obtained with a concentration of 3%blood. Weak reactions (Table 2) are not included.

that P. acnes, the Propionibacterium most fre-quently found in acne vulgaris lesions, is themost active species concerning the productionof chondroitin sulfatase and hyaluronidase. P.avidum and P. granulosum produce deoxyribo-nuclease much more frequently than the otherspecies examined. Differences in susceptibilityof erythrocytes of different mammalian speciesto the lytic action of some products of propion-ibacteria tested may indicate an existence ofseveral substances with a distinct hemolyticspecificity.

LITERATURE CITED

1. Barber, M., and S. W. H. Kuper. 1951. Identificationof Staphylococcus pyogenes by the phosphatase reac-tion. J. Pathol. Bacteriol. 63:65-68.

2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-gey's manual of determinative bacteriology, 8th ed.Williams & Wilkins Co., Baltimore.

3. Hoeffler, U., H. L. Ko, and G. Pulverer. 1976. Antimi-crobial susceptibility of Propionibacterium acnes andrelated microbial species. Antimicrob. Agents Chemo-ther. 10:387-394.

4. Johnson, J. L., and C. S. Cummins. 1972. Cell wallcomposition and deoxyribonucleic acid similaritiesamong the anaerobic coryneforms, classical propioni-bacteria, and strains of Arachnia propionica. J. Bac-teriol. 109:1047-1066.

5. Jong, E. C., H. L. Ko., and G. Pulverer. 1975. Studieson bacteriophages of Propionibacterium acnes. Med.Microbiol. Immunol. 161:263-271.

6. Marples, R. R., and K. J. McGinley. 1974. Corynebac-terium acnes and other anaerobic diphtheroids fromhuman skin. J. Med. Microbiol. 7:349-357.

7. Miller, H. E. 1971. Uber das Vorkommen von Neuramin-idase bei Corynebacterium acnes. Z. Med. Mikrobiol.Immunol. 156:240-249.

8. Porschen, R. K., and S. Sonntag. 1974. Extracellulardeoxyribonuclease production by anaerobic bacteria.Appl. Microbiol. 27:1031-1033.

9. Porschen, R. K., and E. H. Spaulding. 1974. Phospha-tase activity of anaerobic organisms. Appl. Microbiol.27:744-747.

10. Puhvel, S. M., and R. M. Reisner. 1972. The productionof hyaluronidase (hyaluronate lyase) by Corynebacte-rium acnes. J. Invest. Dermatol. 58:66-70.

11. Pulverer, G. 1974. Problems of human Actinomygosis.Postepy Hig. Med. Dosw. 28:253-260.

12. Pulverer, G., and H. L. Ko. 1973. Fermentative andserological studies on Propionibacterium acnes. Appl.

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Microbiol. 25:222-229.13. Pulverer, G., W. Sorgo, and H. L. Ko. 1973. Bakterio-

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14. Schaal, K. P. 1974. Naturliches Vorkomxnen und klin-ische Bedeutung grampositiver, nicht sporenbildenderAnaerobier. Zentralbl. Bakteriol. Parasitenkd. Infek-tionskr. Hyg. Abt. 1 Orig. Reihe A 228:56-62.

15. Smith, R. F., and N. P. Willett. 1968. Rapid platemethod for screening hyaluronidase and chrondroitin

sulfatase-producing microorganisms. Appl. Microbiol.16:1434-1436.

16. Voss, J. G. 1970. Differentiation of two groups of Cory-nebacterium acnes. J. Bacteriol. 101:392-397.

17. Voss, J. G. 1974. Acne vulgaris and free fatty acids. Areview and criticism. Arch. Dermatol. 109:894-898.

18. Werner, H. 1967. Untersuchungen uber die Lipase- undLecithinase-Aktivitat von aeroben und anaeroben Cor-ynebacterium- und von Propionibacterium-Arten. Zen-tralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1 Orig. Reihe A 204:127-138.