Med. Microbiol. Immunol. 161,263--271 (1975) (~) by Springer-Verlag 1975
Studies on Bacteriophages of Propionibacterium aches E. C. Jong, H. L. Ko, and G. Pulverer Hygiene-Institut der Universit/it K51n Received May 20, 1975 Abstract. With the help of adaptation experiments, 61 phage preparations out of 36 Propionibaeterium acnes bacteriophages (32 isolated by us and 4 sent from abroad) were established. On the basis of their stability, spectrum of activity, and virulence, 13 phages were selected for phagetyping. 58 well-classified P. aeries strains were grouped into 7 phage-types. 7 strains of P. granulosum, two strains of P. avidum, and one yet ungroupable microaerophilic propionibacterium strain were resistant to all 61 phages, even when tested in i00 • I~TD. A combination of phagetyping with biotyping resulted in data especially useful for the differentiation of P. aches.
There is no doubt that microaerophilie coryneforms are of medical interest. According to Johnson and Cummins [2], these eoryneforms can be grouped in the following species: P. acnes, P. granulosum, and P. avidum. Hereby P. acnes corresponds to group I of Voss [9]. P. granulosum to group II. Our own fermentative and serological studies [6] confirmed these proposals, as did the recent investigations of Marples and McGinley [5]. Recently Cummins and Johnson [1] published a list of characters for identifying the abovementioned propionibacteria. We started investigations on bacteriophages of propionibacteria because of a lack of knowledge of this subject. The following study presents a set of 13 bacteriophages for phagetyping of P. acnes. Some data have appeared in a preliminary paper [7]. Materials and Methods Bacterial ~trains. 68 strains of mieroaerophilie propionibacteria were studied. Of these, 5 strains were obtained from the American Type Culture Collection: ATCC 6919, 6921, 6922, 6923, 11827; 5 strains were sent by V. R. Dowell, Center for Disease Control, Atlanta, Georgia: CDC 5159, 6949, 6981, 6994, 7010; 12 strains came from J. G. Voss, Proctor & Gamble & Co., Cincinnati, Ohio: go C 51, V-l, D 34, UCLA-18, MC, 0391, A-3, A C 45, 174 V, C 55, D 21; 5 strains were obtained from C. S. Cummins, Anaerobe Lab., Virginia Polytectmio Institute, Blacksburg, Virginia: Cu 3706, 0162, 0575, 0589, 0507. An additional 41 strains were isolated in our laboratory. Most of the strains had been examined biochemieally and serelogically during our first study [6]. Bacterial Cultivation. Stock cultures of bacteria were subeultured once a month on A-medium agar plates prepared by the Fortner method [6], and incubated at 37 ~ C for 3 - - 4 days. Broth cultures were prepared in test tubes containing N-bouillon with a thick vaseline overlay added after inoculation of the broth. Broth cultures were incubated for 48 hrs at 37 ~ C. Media. A-medlum agar was used for routine bacterial cultures, for the basal agar layer when the soft agar overlay technique was employed in phage studies, and in phage "dropping" experiments. The medium consisted of: Difco Casitone, 12 g; Difco yeast extract, 12 g; glucose, 4 g; KHzPOd, 4 g; MgSOa 9 7H~O, I g; Difco agar. 28 g; and distilled water, 1000 ml. The medium was adjusted to p H 7.2 after autoclaving. Bolt Agar. A composition like t h a t of the A-medium was used, but with I)ifco agar in a concentration of 0.750]0. 19 Med. Microbiol, Immunol., Vol. 161
264
E.C. Jong etal.
N-bouillon was used to prepare bacterial and phage stock suspensions. N-bouillon consisted of: Difco Tryptone, 15 g; Difco yeast extract, 4 g; NaC1, 8 g; and distilled water, 1000 ml. The broth was adjusted to pH 7.5 after autoclaving. Phosphate Bu[/er. 612 g of 1/15 M Na2HPO 4 . 2H20 and 388 g of 1/15 M KttzPO4 were mixed carefully, and pH was adjusted to 7.2. 2'ermentative studies were done according to the media and methods published eralier [6]. Bacteriophages. Phage strains 26, 156, and 20 A were obtained from Puhvel, University of California, Los Angeles; phage strain 174 of Zierdt etaL [11] was sent by Voss, Procter & Gamble Co., Cincinnati, Ohio. 32 additional phage strains were isolated in our laboratory by spontaneous lysogeny and lysogeny induction with the use of Mitemycin C [7]. Phages ATCC 6922, H12, and H22 were released spontaneously; the other phages were induced by Mitomyein C. Phage strains were named according to donor strains; propagation strains are cited in parentheses. All phages were first indicated and propagated on P. aches strains KB, ATCC 6923, and 173. Thereafter adaption experiments were performed using other propagation strains. A total of 61 phages were propagated, adapted, purified, and tested for their activity spectra. Spontaneous Lysogeny Test. Supernatents obtained after centrifugation of 48 hrs broth cultures of all P. syncs strains were dropped (0.1 ml) onto the surface of A-medium agar plates, which had been previously flooded with a suspension of the indicator strains used (P. aches) strains KB, ATCC 6923, and 173). The indicator strain suspension was made by suspending bacterial growth from solid medium in broth to a concentration of 106 to 107cfu/ml (equal to the density of a Mc:Farland nephelometer tube :No. 5). After the supernatent drops had dried, the plates were sealed with use of a modification of the :Fortner method: sterile fiat glass plates (9 • 9 cm) were prepared by placing 1--2 ml of agar medium in the center of each plate. After the agar had hardened, each agar "island" was heavily inoculated with Serratia marcescens. The experimental plates were inverted onto the Serratia plates, and the edges were completely sealed by a border of plastiline. After 48 hrs incubation at 37~ C, the plates were observed for plaques. Zysoffeny Induction by Mitomycin C. A suspension of indicator strain bacteria (P. aches strains KB and ATCC 6923, separately) and test-strain bacteria was prepared in broth in a ratio of 5:1, respectively. Aliquots of 0.1 ml of each experimental combination were dropped onto A-medium agar plates, and were evenly spread with sterile glass spatulas. A sterile filter paper disk (Schleicher & Schiill, 9 mm diameter) was placed on the surface of each plate, and 0.05 ml of 50 ~/ml mitomycin C solution (Schuehardt) was applied to each disc. The plates were sealed as previously described and were incubated for 3 days at 37~ C. Isolation and Propagation o/Phages. Plaques in the bacterial lawn were cut out of the agar and homogenized in N-bouillon. After centrffugation, the supernatents were dropped (0.05 ml) onto the surfaces of indicator bacterial plates. The above procedure was begun once more, after which propagation was performed using the soft agar overlay technique. Phage titration was also done with this method. Phage titers of 10~ to 109 pfu/ml were thus obtainable. Adaptation el Phages. Each on strain K_B, ATCC 6923, and 173 propagated phage stock was tested against all P. ashes strains until lysis by a given phage with clear, sharp plaques appeared (characteristic of virulent phages; temperated phages produce only turbid plaques). Such P. aches strains were then designated as new propagation strains for a given phage. Purl/ication o/ Phages. Before stock phage preparations were begun, each phage was cloned three times by single plaque isolation on a given propagating P. syncs strain. Each stock phage preparation was checked for purity (virulent plaques only, identical plaque morphology), pfu]ml, and RTD (routine test dilution = confluent lysis on the propagating strain). Host ~pectrum 1)etermination. F~ach phage was tested in RTD and in 100• on all propionibacterial strains with use of the simple "dropping" test. All experiments were repeated at least once. Results Clzrnrnlns a n d J o h n s o n [1] r e c e n t l y p u b l i s h e d a v e r y practicable a n d useful differentiation m o d e l for P . acne.s, P. granulosum, a n d P . avidum. W e used this schedule for the g r o u p i n g of our 68 strains. T a b l e 1 shows t h a t , with one e x c e p t i o n
Unknown 1 strain
2 strains
P. avidum
7 strains
P. granulosum
1
Vo A
Ca 0575, Cu 0589
s 140
1
2
95, VoC51, VoV-1, VoD34, Cu 0507, VoD21
3714
6
1
6994,172, 147, 89, 90, KB, Gerath, 173, 902, 225, H~, Hz3, ATCC 11827, 122, CaO 162, 330, VoA-3, 567, 372
H,, H~,H~,H6,H~,Hs, Hm HIz,H,5, H~, H~, Hao,Haa,CDG6981,CDC
33
+
+
+
-~
-~
+
H~6,1~, ATCC 6922, CDG 6949, CDC -b 7010, CDC 5159, 65, 259, 73, 286, ATGC 6921, ATGC 6919, ATCC 6923, 3045,112, Cu 3706, Vo 174 V, VoMG, Vo 0391, VOC45, Vo UCLA-18, S16, 125
24
.
+
+
%
.
.
--
.
.
.
+
+
-~
--
.
.
.
.
--
.
.
.
-}-
.
.
.
+
.
--
.
.
.
.
Glucose Sucrose Maltose Sorbitol Aesculin hydrolysis
P. acnes 58 strains
Strain no.
No. of strains
Species
.
--
~-
.
-}-
-{-
.
Indole
--
-}-
-{-
~
+
§
--
-{-
-t-
Nitrate Gelatin
Table 1. Differentiation of strains used in species according to the recommendations of Cummins and Johnson [1]
['0
3"
266
E . C . J o n g e~ al.
Table 2. Biotypes of strains used according to recommendations of Pulverer and Ko [6] Species
Biotype No. of strains
Strain no.
P . aches 58 strains
A
29
CDC 6994, CDC 6981, -H4, Hs, Hs, HT, Hg, Hn, H15, H ~ , IL~, It3 o, tt33, Hz~, H 1, 89, 172, 90, 902, 225, 147, 173, Gerath, KB, 330, 567, 372, 3714, Cu 0162
--
--
--
B
1
H~4
+
--
--
--
D
3
Hz3,122, ATCC 11827
--
--
-{-
--
E
10
ATCC 6922, CDC 6949, -CDC 7010, CDC 5159, Hz6, H~, 65, 259, Cu 3706, Vo 0391
--
--
-4-
F
2
73, 286
-[-
--
--
-[-
G
9
ATCC 6919, ATCC -6921, ATCC 6923, 112, 3045, VoMC, $16, 125, Vo UCL A-18
--
-}-
-f-
I
3
Vo 174 V, VoC 55, Vo C45
+
--
~-
-~
K
1
Vo A-3
+
--
+
--
P . granulosum C 7 strains
5
95, Vo C51, Vo V-l, S140, Cu 507
--
-t-
--
--
H
2
Vo D34, Vo D21
--
+
-}-
--
C
1
Cu 0575
--
A-
--
--
L
1
Cu 0589
+
+
--
--
A
1
Vo A
.
P. avidum 2 strains
Unknown 1 strain
Inositol maltose Mannitol
.
.
Sorbitol
.
( s t r a i n V o A), i t w a s p o s s i b l e t o c l a s s i f y all o u r s t r a i n s . T h e d a t a o f V o s s a n d C u m m i n s for t h e i r s t r a i n s c o u l d be c o n f i r m e d . A l t h o u g h r e c o m m e n d e d b y J o h n s o n a n d C u m m i n s [2] d i f f e r e n t i a t i o n o f P . a c n e s s t r a i n s i n t o t y p e s I a n d I I w a s n o t d o n e . T h e i r m e t h o d o f s u b d i v i s i o n d o e s n o t s e e m t o be v e r y u s e f u l f o r p r a c t i c a l p u r p o s e s b e c a u s e o f t e c h n i c a l difficulties. I n 1973 we p u b l i s h e d t h e r e s u l t s o f f e r m e n t a t i v e a n d s e r o l o g i c a l s t u d i e s o n m i e r o a e r o p h i l i c p r o p i o n i b a c t e r i a [6]. W e classified t h e s t r a i n s t e s t e d i n t o 7 b i o t y p e s a n d 11 s e r o t y p e s w h i c h p r o v e d t o b e u n r e l a t e d . T h e r e s u l t s o f t h e b i o t y p i n g o f o u r p r e s e n t s t r a i n s a r e l i s t e d in T a b l e 2. S e r o t y p i n g w a s d o n e in all s t r a i n s , b u t t h e r e s u l t s will n o t b e p r e s e n t e d h e r e i n t h e i n t e r e s t o f c l a r i t y .
Bacteriophages of Propionibacterium aches
267
The 7 biotypes established earlier [6] were named A - - H and 2 more biotypes (I and K) were found in our study. As seen in Table 2, all propionibacteria tested could be classified into our proposed biotypes. Altogether 32 phages were isolated, the result of either spontaneous lysogeny or mitomycin C induction. Four further phages (phage strains 174, 26, 156, and 20 A) were sent from abroad. Each of these phages was at first isolated and propagated on one or all of the P. aches-indicator strains used (strains KB, ATCC 6923, and 173). The preliminary results were published in 1973 [7], but the data from phagetyping were inconclusive, and most of the phage-stocks later proved to be mixtures of virulent and temperate phages. Further, the same indicator and propagation strains were for all phages used. We therefore continued to isolate new phages and adapt phages to different propagation strains. I n sum, we acquired 61 different phage preparations. Following the aim of our study, we wished to have in hand pure virulent phages. Our experience with phages of different bacterial species showed, that especially temperate phages cause trouble in phagetyping. Important, too, were certain difficulties of propagation and the stability of phage stocks. Finally, the spectrum of activity had to be taken into consideration. The result of our experiments was the selection of 13 phages for the phagetyping of P. acnes. These 13 phages were named K5 1--13. The origin and propagating strains of these phages are given in Table 3 : the first name belongs to the original donor strain (phage 174 was originally released b y P. aches strain ATCC 11827 [11 ]; the origin of phage 26 is not known), and the name in parentheses marks the propagation strain used. Phage K5 11 was first adapted to P. acnes strain CDC 7010, and then adapted to P. acnes strain 172 because of difficulties in yielding pure virulent phage stocks. As shown in Table 3, our 58 strains of P. acnes could be classified into 7 phagetypes. Since it was difficult to find P. acnes phages with real and stable differences in their activity spectra, it is not surprising to find that 46 out of the 58 P. aches strains belonged to one phage-type. Only one strain (P. acnes strain 330 of biotype A) was absolutely resistant to all 61 phages examined, even, when using phages in 100 • RTD. Nearly all negative results in R T D were found to be negative also in 100 X RTD. As expected, all examined strains of P. granulosum and P. avidum, as well as strain Vo A, were not lysed by any of the 61 phages tested, even when applied in 1 0 0 x R T D . However, our data indicate that it m a y be possible to adapt some of our P. aches phages onto some P. granulosum strains. Discussion
The taxonomic position of microaerophilie coryncforms was until recently very confused and unclear [6]. I t is the merit of Voss [9] to have given new impulses to clear up the problems of differentiation of these bacteria. Johnson and Cummins [2] established the basis of further taxonomy in proving that nearly all microaerophilic coryneforms belong to three species; P. acnes, P. granulosum, and P. avidum. Our own studies [6] completed the spectrum of fermentative activities of P. aches and P. granulosum (known as group I I of Voss). Furthermore, we
1
1
VII
Un~'nown
1
VI
2
3
V
P. avidum
Cu 0162
4
IV
7
259, 173, 3741
2
HI
0
0
d-
+
0
+
-~
-}-
K6 1 174/Vo0391 9
VoA
0
Cu 0575, Cu 0589 0
95, VoC 51, VoV-1, VoD 34, Cu 0507, S 140, VoD 2~
330
CDC 5159, VoO 45, VoC 55, 286
VoMc, Vo 194 V
1
II
CDC 7010
46
I
Phage- No. of Strain no. type strains
P. granu~osum
P. acne~ 58 strains
Species
0
0
0
0
0
-[-
0
+
d-
-}-
r
0 0
0
0
-1-
+
0
0
0
+
0
0
0
0
q-
0
0
-~
~-
+
0
0
0
0
0
0
0
d-
-~
+
0
0
0
0
0
0
0
0
0
+
K6 2 K6 3 K8 4 K5 5 K6 6 H2,/CDC 6994 8/ATCC6919 H~s/Vo 0391 CDC6949/CDC6994 H1JATCC 6922
Table 3. Activitsrspeetrumofl3bact~riophagesofP. aches
0o
1
1
VI
VII 0
0
0
0
0
+
+
+
K5 7 CDC6981[172
YeA
0
Ou 0575, Ou 0589 0
95, VoC 51, VoV-1, VoD 34, Cu 0507, S 140, VoD 21
330
Cu 0162
259, 173, 3741
CDC 5159, VoC 45, VoC 55, 286
VoMC, Vo 174V
CDC 7010
Strain no.
+ = Lysis (CL, SCL, ~ 20 pfu/drop). a 174 = donor strain, Vo 0391 = propagation strain.
1
3
V
"Unknown
4
IV
2
2
III
P. avldum
1
II
7
46
No. of strains
I
Phage type
P. granuloaum
P. o~nes 58 strains
Species
0
0
0
0
O
+
+
+
+
+
I{6 8 95/VOC45
Table 3 (continued)
0
0
0
0
0
+
+
+
+
+
K6 9 26/H21
0
0
0
0
0
+
0
+
+
+
K6 10 2741/His
0
0
0
0
0
0
0
+
+
+
K6 11 ATCC6921/172
0
0
0
0
+
+
0
+
0
+
I{6 12 156/H~e
0
0
0
0
0
0
0
+
+
+
K5 13 20A/H n
O
o~
~~ Q
E. C. Jong et al.
270
Table 4. Phagetyping and biotyping of P . aches Phage-type I
III
No. of strains
Biotype
No. of strains
46
A B D E F G K
25 1 3 7 l 8 1
2
G
1
I
1
IV
4
E F I
1 1 2
V
3
A E
2 1
demonstrated possibilities for biotyping and serotyping microaerophilicpropionibacteria. Recently Marples and McGinley [5] and Cummins and Johnson [1] published lists of characters for differentiation of the abovementioned species. The proposal of Cummins and Johnson [1] seems to us to be especially useful in routine laboratory work (see Table 1). All in all, confusion over the taxonomy of microacrophilie propionibaeteria can be largely dispelled. However, the authors recommend not to leave the well-founded basis of Johnson and Cummins [2]. Although much is known now about the taxonomic position of microaerophflie propionibaeteria, little is known about the bacteriophages of these bacteria. Zierdt et al. [11] in 1968 demonstrated t h a t phage 174 out of spontaneous plaques of P . acnes strain ATCC 11827 could be isolated. This phage was able to lyse 147 of 167 strains tested ( = 88~ Voss [9] stated that 75 of 106 strains of his group I ( ~ 70~ were lysed by this pjage 174, although only 1 of 15 group I I strains was thus lysed. Voss mentioned that suspectibility to phage 174 was closely paralleled b y susceptibility of Puhvel's phage 26. These phages were used by others for differentiation [8, 10]. Marples et al. [4] stated that they observed spontaneous plaques in 30~ of P . acnes strains examined. They isolated 8 phages (A--G), some of which were used for classification of anaerobic coryneforms, us well [3--5]. I n 1973 we published the preliminary results of our studies on bacteriophages of P . acnes [7]. We then pointed out our experience with spontaneous lysogeny tests and lysogeny induction experiments with the use of UV rays, Novobiocin and mitomycin C. Only mitomyein C proved to have inducing effects. In addition, we demonstrated the possibility of differentiating P . aches phages according to their activity spectra and their resistance to UV rays, hydroxylamine, and acridine orange in the dark. In our paper we present a set of 13 virulent P. acnes phages, selected out of 61 phage preparations tested. These 13 phages proved to be stable and to cause no
Bacteriophages of Propionibacterium aches
271
g r e a t difficulties concerning p r o p a g a t i o n a n d storage. T a b l e 3 d e m o n s t r a t e s t h e p o s s i b i l i t y o f phage~yping w i t h this set. W h e r e a s all t e s t e d s t r a i n s o f P . granulosum a n d of P. avidum were r e s i s t a n t t o all p h a g e s in R T D a n d in 1 0 0 • o n l y one o f 58 P . acnes s t r a i n s was p h a g e - r e s i s t a n t . A l t h o u g h t h e p h a g e - t y p i n g d a t a a r e n o t y e t optimal, 46 o u t o f 58 s t r a i n s b e l o n g e d t o o n l y one p h a g e t y p e , t h i s set o f p h a g e s can nevertheless be useful for d i f f e r e n t i a t i o n purposes. U n t i l p h a g e a r e f o u n d which can m o r e s a t i s f a c t o r i l y s p l i t u p t h e species P . aches, which seems us t o be r a t h e r difficult, p h a g e t y p i n g can be c o m b i n e d w i t h o t h e r m a r k e r s y s t e m s such as b i o t y p i n g (see Tables 2 a n d 4). As seen in T a b l e 4, 46 P . acne8 s t r a i n s o f p h a g e t y p e I can t h u s be classified i n t o 7 b i o t y p e s .
References 1. Cummins, C. S., Johnson, J. L.: Corynebacterium parvum: a synonym for Propionibacterium aches ? J. gen. Microbiol. 80, 433--442 (1974) 2. Johnson, J. L., Cummins, C. S. : Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria and strains of Arachnia propionica. J. Bact. 109, 1047--1066 (1972) 3. Marplcs, R. R., Leyden, J . J . , Stewart, R. N., Mills, O. It., Kligman, A.M.: The skin microflora in Acne vulgaris. J. invest. Derm. 62, 37--41 (1974) 4. Marplcs, R. R., !~IcGinley, K. J., Mills, O. H. : Microbiology of comedoncs in Acne vulgaris. J. invest. Derm. 60, 80--83 (1973) 5. ~Iarples, R.R., McGinley, K . J . : Corynebacterium aches and other anaerobic diphtheroids from human skin. J. mcd. Microbiol. 7, 349--357 (1974) 6. Pulverer, G., Ko, It. L.: Fermentative and serological studies on Propionibacterium acnes. Appl. Microbiol. 25, 222--229 (1973) 7. Pulverer, G., Sorgo, W., Ko, H. L.: Bakteriophagen yon Propionibacterium aches. Zbl. Bakt., I. Abt. Orig. A 225, 353--363 (1973) 8. Reid, J. D., Joya, M. A. : A study of the morphologie and biochemical characteristics of certain anaerobic corynebacteria. Int. J. syst. Baet. 19, 273--280 (1969) 9. Voss, J. G.: Differentiation of two groups of Corynebacterium acnes. J. Bact. 101, 392 to 397 (1970) 10. Whiteside, J . A . , Voss, J. G.: Incidence and lipolytic activity of Propionibacterium aches (Corynebacterium aches group I) and P. granulosum (C. acnes group II) in Acne and in normal skin. J. invest. Derm. 60, 94--97 (1973) 11. Zierdt, C. H., Webster, C., Rude, W. S.: Study of the anaerobic corynebacteria. Int. J. syst. Bact. 18, 33--47 (1968) Prof. Dr. Gerhard Pulverer Direktor des Hygiene-Instituts D-5000 K61n 41, F~irst PQckler-Str. 56 Federal Republic of Germany
Studies on Bacteriophages of Propionibacterium aches E. C. Jong, H. L. Ko, and G. Pulverer Hygiene-Institut der Universit/it K51n Received May 20, 1975 Abstract. With the help of adaptation experiments, 61 phage preparations out of 36 Propionibaeterium acnes bacteriophages (32 isolated by us and 4 sent from abroad) were established. On the basis of their stability, spectrum of activity, and virulence, 13 phages were selected for phagetyping. 58 well-classified P. aeries strains were grouped into 7 phage-types. 7 strains of P. granulosum, two strains of P. avidum, and one yet ungroupable microaerophilic propionibacterium strain were resistant to all 61 phages, even when tested in i00 • I~TD. A combination of phagetyping with biotyping resulted in data especially useful for the differentiation of P. aches.
There is no doubt that microaerophilie coryneforms are of medical interest. According to Johnson and Cummins [2], these eoryneforms can be grouped in the following species: P. acnes, P. granulosum, and P. avidum. Hereby P. acnes corresponds to group I of Voss [9]. P. granulosum to group II. Our own fermentative and serological studies [6] confirmed these proposals, as did the recent investigations of Marples and McGinley [5]. Recently Cummins and Johnson [1] published a list of characters for identifying the abovementioned propionibacteria. We started investigations on bacteriophages of propionibacteria because of a lack of knowledge of this subject. The following study presents a set of 13 bacteriophages for phagetyping of P. acnes. Some data have appeared in a preliminary paper [7]. Materials and Methods Bacterial ~trains. 68 strains of mieroaerophilie propionibacteria were studied. Of these, 5 strains were obtained from the American Type Culture Collection: ATCC 6919, 6921, 6922, 6923, 11827; 5 strains were sent by V. R. Dowell, Center for Disease Control, Atlanta, Georgia: CDC 5159, 6949, 6981, 6994, 7010; 12 strains came from J. G. Voss, Proctor & Gamble & Co., Cincinnati, Ohio: go C 51, V-l, D 34, UCLA-18, MC, 0391, A-3, A C 45, 174 V, C 55, D 21; 5 strains were obtained from C. S. Cummins, Anaerobe Lab., Virginia Polytectmio Institute, Blacksburg, Virginia: Cu 3706, 0162, 0575, 0589, 0507. An additional 41 strains were isolated in our laboratory. Most of the strains had been examined biochemieally and serelogically during our first study [6]. Bacterial Cultivation. Stock cultures of bacteria were subeultured once a month on A-medium agar plates prepared by the Fortner method [6], and incubated at 37 ~ C for 3 - - 4 days. Broth cultures were prepared in test tubes containing N-bouillon with a thick vaseline overlay added after inoculation of the broth. Broth cultures were incubated for 48 hrs at 37 ~ C. Media. A-medlum agar was used for routine bacterial cultures, for the basal agar layer when the soft agar overlay technique was employed in phage studies, and in phage "dropping" experiments. The medium consisted of: Difco Casitone, 12 g; Difco yeast extract, 12 g; glucose, 4 g; KHzPOd, 4 g; MgSOa 9 7H~O, I g; Difco agar. 28 g; and distilled water, 1000 ml. The medium was adjusted to p H 7.2 after autoclaving. Bolt Agar. A composition like t h a t of the A-medium was used, but with I)ifco agar in a concentration of 0.750]0. 19 Med. Microbiol, Immunol., Vol. 161
264
E.C. Jong etal.
N-bouillon was used to prepare bacterial and phage stock suspensions. N-bouillon consisted of: Difco Tryptone, 15 g; Difco yeast extract, 4 g; NaC1, 8 g; and distilled water, 1000 ml. The broth was adjusted to pH 7.5 after autoclaving. Phosphate Bu[/er. 612 g of 1/15 M Na2HPO 4 . 2H20 and 388 g of 1/15 M KttzPO4 were mixed carefully, and pH was adjusted to 7.2. 2'ermentative studies were done according to the media and methods published eralier [6]. Bacteriophages. Phage strains 26, 156, and 20 A were obtained from Puhvel, University of California, Los Angeles; phage strain 174 of Zierdt etaL [11] was sent by Voss, Procter & Gamble Co., Cincinnati, Ohio. 32 additional phage strains were isolated in our laboratory by spontaneous lysogeny and lysogeny induction with the use of Mitemycin C [7]. Phages ATCC 6922, H12, and H22 were released spontaneously; the other phages were induced by Mitomyein C. Phage strains were named according to donor strains; propagation strains are cited in parentheses. All phages were first indicated and propagated on P. aches strains KB, ATCC 6923, and 173. Thereafter adaption experiments were performed using other propagation strains. A total of 61 phages were propagated, adapted, purified, and tested for their activity spectra. Spontaneous Lysogeny Test. Supernatents obtained after centrifugation of 48 hrs broth cultures of all P. syncs strains were dropped (0.1 ml) onto the surface of A-medium agar plates, which had been previously flooded with a suspension of the indicator strains used (P. aches) strains KB, ATCC 6923, and 173). The indicator strain suspension was made by suspending bacterial growth from solid medium in broth to a concentration of 106 to 107cfu/ml (equal to the density of a Mc:Farland nephelometer tube :No. 5). After the supernatent drops had dried, the plates were sealed with use of a modification of the :Fortner method: sterile fiat glass plates (9 • 9 cm) were prepared by placing 1--2 ml of agar medium in the center of each plate. After the agar had hardened, each agar "island" was heavily inoculated with Serratia marcescens. The experimental plates were inverted onto the Serratia plates, and the edges were completely sealed by a border of plastiline. After 48 hrs incubation at 37~ C, the plates were observed for plaques. Zysoffeny Induction by Mitomycin C. A suspension of indicator strain bacteria (P. aches strains KB and ATCC 6923, separately) and test-strain bacteria was prepared in broth in a ratio of 5:1, respectively. Aliquots of 0.1 ml of each experimental combination were dropped onto A-medium agar plates, and were evenly spread with sterile glass spatulas. A sterile filter paper disk (Schleicher & Schiill, 9 mm diameter) was placed on the surface of each plate, and 0.05 ml of 50 ~/ml mitomycin C solution (Schuehardt) was applied to each disc. The plates were sealed as previously described and were incubated for 3 days at 37~ C. Isolation and Propagation o/Phages. Plaques in the bacterial lawn were cut out of the agar and homogenized in N-bouillon. After centrffugation, the supernatents were dropped (0.05 ml) onto the surfaces of indicator bacterial plates. The above procedure was begun once more, after which propagation was performed using the soft agar overlay technique. Phage titration was also done with this method. Phage titers of 10~ to 109 pfu/ml were thus obtainable. Adaptation el Phages. Each on strain K_B, ATCC 6923, and 173 propagated phage stock was tested against all P. ashes strains until lysis by a given phage with clear, sharp plaques appeared (characteristic of virulent phages; temperated phages produce only turbid plaques). Such P. aches strains were then designated as new propagation strains for a given phage. Purl/ication o/ Phages. Before stock phage preparations were begun, each phage was cloned three times by single plaque isolation on a given propagating P. syncs strain. Each stock phage preparation was checked for purity (virulent plaques only, identical plaque morphology), pfu]ml, and RTD (routine test dilution = confluent lysis on the propagating strain). Host ~pectrum 1)etermination. F~ach phage was tested in RTD and in 100• on all propionibacterial strains with use of the simple "dropping" test. All experiments were repeated at least once. Results Clzrnrnlns a n d J o h n s o n [1] r e c e n t l y p u b l i s h e d a v e r y practicable a n d useful differentiation m o d e l for P . acne.s, P. granulosum, a n d P . avidum. W e used this schedule for the g r o u p i n g of our 68 strains. T a b l e 1 shows t h a t , with one e x c e p t i o n
Unknown 1 strain
2 strains
P. avidum
7 strains
P. granulosum
1
Vo A
Ca 0575, Cu 0589
s 140
1
2
95, VoC51, VoV-1, VoD34, Cu 0507, VoD21
3714
6
1
6994,172, 147, 89, 90, KB, Gerath, 173, 902, 225, H~, Hz3, ATCC 11827, 122, CaO 162, 330, VoA-3, 567, 372
H,, H~,H~,H6,H~,Hs, Hm HIz,H,5, H~, H~, Hao,Haa,CDG6981,CDC
33
+
+
+
-~
-~
+
H~6,1~, ATCC 6922, CDG 6949, CDC -b 7010, CDC 5159, 65, 259, 73, 286, ATGC 6921, ATGC 6919, ATCC 6923, 3045,112, Cu 3706, Vo 174 V, VoMG, Vo 0391, VOC45, Vo UCLA-18, S16, 125
24
.
+
+
%
.
.
--
.
.
.
+
+
-~
--
.
.
.
.
--
.
.
.
-}-
.
.
.
+
.
--
.
.
.
.
Glucose Sucrose Maltose Sorbitol Aesculin hydrolysis
P. acnes 58 strains
Strain no.
No. of strains
Species
.
--
~-
.
-}-
-{-
.
Indole
--
-}-
-{-
~
+
§
--
-{-
-t-
Nitrate Gelatin
Table 1. Differentiation of strains used in species according to the recommendations of Cummins and Johnson [1]
['0
3"
266
E . C . J o n g e~ al.
Table 2. Biotypes of strains used according to recommendations of Pulverer and Ko [6] Species
Biotype No. of strains
Strain no.
P . aches 58 strains
A
29
CDC 6994, CDC 6981, -H4, Hs, Hs, HT, Hg, Hn, H15, H ~ , IL~, It3 o, tt33, Hz~, H 1, 89, 172, 90, 902, 225, 147, 173, Gerath, KB, 330, 567, 372, 3714, Cu 0162
--
--
--
B
1
H~4
+
--
--
--
D
3
Hz3,122, ATCC 11827
--
--
-{-
--
E
10
ATCC 6922, CDC 6949, -CDC 7010, CDC 5159, Hz6, H~, 65, 259, Cu 3706, Vo 0391
--
--
-4-
F
2
73, 286
-[-
--
--
-[-
G
9
ATCC 6919, ATCC -6921, ATCC 6923, 112, 3045, VoMC, $16, 125, Vo UCL A-18
--
-}-
-f-
I
3
Vo 174 V, VoC 55, Vo C45
+
--
~-
-~
K
1
Vo A-3
+
--
+
--
P . granulosum C 7 strains
5
95, Vo C51, Vo V-l, S140, Cu 507
--
-t-
--
--
H
2
Vo D34, Vo D21
--
+
-}-
--
C
1
Cu 0575
--
A-
--
--
L
1
Cu 0589
+
+
--
--
A
1
Vo A
.
P. avidum 2 strains
Unknown 1 strain
Inositol maltose Mannitol
.
.
Sorbitol
.
( s t r a i n V o A), i t w a s p o s s i b l e t o c l a s s i f y all o u r s t r a i n s . T h e d a t a o f V o s s a n d C u m m i n s for t h e i r s t r a i n s c o u l d be c o n f i r m e d . A l t h o u g h r e c o m m e n d e d b y J o h n s o n a n d C u m m i n s [2] d i f f e r e n t i a t i o n o f P . a c n e s s t r a i n s i n t o t y p e s I a n d I I w a s n o t d o n e . T h e i r m e t h o d o f s u b d i v i s i o n d o e s n o t s e e m t o be v e r y u s e f u l f o r p r a c t i c a l p u r p o s e s b e c a u s e o f t e c h n i c a l difficulties. I n 1973 we p u b l i s h e d t h e r e s u l t s o f f e r m e n t a t i v e a n d s e r o l o g i c a l s t u d i e s o n m i e r o a e r o p h i l i c p r o p i o n i b a c t e r i a [6]. W e classified t h e s t r a i n s t e s t e d i n t o 7 b i o t y p e s a n d 11 s e r o t y p e s w h i c h p r o v e d t o b e u n r e l a t e d . T h e r e s u l t s o f t h e b i o t y p i n g o f o u r p r e s e n t s t r a i n s a r e l i s t e d in T a b l e 2. S e r o t y p i n g w a s d o n e in all s t r a i n s , b u t t h e r e s u l t s will n o t b e p r e s e n t e d h e r e i n t h e i n t e r e s t o f c l a r i t y .
Bacteriophages of Propionibacterium aches
267
The 7 biotypes established earlier [6] were named A - - H and 2 more biotypes (I and K) were found in our study. As seen in Table 2, all propionibacteria tested could be classified into our proposed biotypes. Altogether 32 phages were isolated, the result of either spontaneous lysogeny or mitomycin C induction. Four further phages (phage strains 174, 26, 156, and 20 A) were sent from abroad. Each of these phages was at first isolated and propagated on one or all of the P. aches-indicator strains used (strains KB, ATCC 6923, and 173). The preliminary results were published in 1973 [7], but the data from phagetyping were inconclusive, and most of the phage-stocks later proved to be mixtures of virulent and temperate phages. Further, the same indicator and propagation strains were for all phages used. We therefore continued to isolate new phages and adapt phages to different propagation strains. I n sum, we acquired 61 different phage preparations. Following the aim of our study, we wished to have in hand pure virulent phages. Our experience with phages of different bacterial species showed, that especially temperate phages cause trouble in phagetyping. Important, too, were certain difficulties of propagation and the stability of phage stocks. Finally, the spectrum of activity had to be taken into consideration. The result of our experiments was the selection of 13 phages for the phagetyping of P. acnes. These 13 phages were named K5 1--13. The origin and propagating strains of these phages are given in Table 3 : the first name belongs to the original donor strain (phage 174 was originally released b y P. aches strain ATCC 11827 [11 ]; the origin of phage 26 is not known), and the name in parentheses marks the propagation strain used. Phage K5 11 was first adapted to P. acnes strain CDC 7010, and then adapted to P. acnes strain 172 because of difficulties in yielding pure virulent phage stocks. As shown in Table 3, our 58 strains of P. acnes could be classified into 7 phagetypes. Since it was difficult to find P. acnes phages with real and stable differences in their activity spectra, it is not surprising to find that 46 out of the 58 P. aches strains belonged to one phage-type. Only one strain (P. acnes strain 330 of biotype A) was absolutely resistant to all 61 phages examined, even, when using phages in 100 • RTD. Nearly all negative results in R T D were found to be negative also in 100 X RTD. As expected, all examined strains of P. granulosum and P. avidum, as well as strain Vo A, were not lysed by any of the 61 phages tested, even when applied in 1 0 0 x R T D . However, our data indicate that it m a y be possible to adapt some of our P. aches phages onto some P. granulosum strains. Discussion
The taxonomic position of microaerophilie coryncforms was until recently very confused and unclear [6]. I t is the merit of Voss [9] to have given new impulses to clear up the problems of differentiation of these bacteria. Johnson and Cummins [2] established the basis of further taxonomy in proving that nearly all microaerophilic coryneforms belong to three species; P. acnes, P. granulosum, and P. avidum. Our own studies [6] completed the spectrum of fermentative activities of P. aches and P. granulosum (known as group I I of Voss). Furthermore, we
1
1
VII
Un~'nown
1
VI
2
3
V
P. avidum
Cu 0162
4
IV
7
259, 173, 3741
2
HI
0
0
d-
+
0
+
-~
-}-
K6 1 174/Vo0391 9
VoA
0
Cu 0575, Cu 0589 0
95, VoC 51, VoV-1, VoD 34, Cu 0507, S 140, VoD 2~
330
CDC 5159, VoO 45, VoC 55, 286
VoMc, Vo 194 V
1
II
CDC 7010
46
I
Phage- No. of Strain no. type strains
P. granu~osum
P. acne~ 58 strains
Species
0
0
0
0
0
-[-
0
+
d-
-}-
r
0 0
0
0
-1-
+
0
0
0
+
0
0
0
0
q-
0
0
-~
~-
+
0
0
0
0
0
0
0
d-
-~
+
0
0
0
0
0
0
0
0
0
+
K6 2 K6 3 K8 4 K5 5 K6 6 H2,/CDC 6994 8/ATCC6919 H~s/Vo 0391 CDC6949/CDC6994 H1JATCC 6922
Table 3. Activitsrspeetrumofl3bact~riophagesofP. aches
0o
1
1
VI
VII 0
0
0
0
0
+
+
+
K5 7 CDC6981[172
YeA
0
Ou 0575, Ou 0589 0
95, VoC 51, VoV-1, VoD 34, Cu 0507, S 140, VoD 21
330
Cu 0162
259, 173, 3741
CDC 5159, VoC 45, VoC 55, 286
VoMC, Vo 174V
CDC 7010
Strain no.
+ = Lysis (CL, SCL, ~ 20 pfu/drop). a 174 = donor strain, Vo 0391 = propagation strain.
1
3
V
"Unknown
4
IV
2
2
III
P. avldum
1
II
7
46
No. of strains
I
Phage type
P. granuloaum
P. o~nes 58 strains
Species
0
0
0
0
O
+
+
+
+
+
I{6 8 95/VOC45
Table 3 (continued)
0
0
0
0
0
+
+
+
+
+
K6 9 26/H21
0
0
0
0
0
+
0
+
+
+
K6 10 2741/His
0
0
0
0
0
0
0
+
+
+
K6 11 ATCC6921/172
0
0
0
0
+
+
0
+
0
+
I{6 12 156/H~e
0
0
0
0
0
0
0
+
+
+
K5 13 20A/H n
O
o~
~~ Q
E. C. Jong et al.
270
Table 4. Phagetyping and biotyping of P . aches Phage-type I
III
No. of strains
Biotype
No. of strains
46
A B D E F G K
25 1 3 7 l 8 1
2
G
1
I
1
IV
4
E F I
1 1 2
V
3
A E
2 1
demonstrated possibilities for biotyping and serotyping microaerophilicpropionibacteria. Recently Marples and McGinley [5] and Cummins and Johnson [1] published lists of characters for differentiation of the abovementioned species. The proposal of Cummins and Johnson [1] seems to us to be especially useful in routine laboratory work (see Table 1). All in all, confusion over the taxonomy of microacrophilie propionibaeteria can be largely dispelled. However, the authors recommend not to leave the well-founded basis of Johnson and Cummins [2]. Although much is known now about the taxonomic position of microaerophflie propionibaeteria, little is known about the bacteriophages of these bacteria. Zierdt et al. [11] in 1968 demonstrated t h a t phage 174 out of spontaneous plaques of P . acnes strain ATCC 11827 could be isolated. This phage was able to lyse 147 of 167 strains tested ( = 88~ Voss [9] stated that 75 of 106 strains of his group I ( ~ 70~ were lysed by this pjage 174, although only 1 of 15 group I I strains was thus lysed. Voss mentioned that suspectibility to phage 174 was closely paralleled b y susceptibility of Puhvel's phage 26. These phages were used by others for differentiation [8, 10]. Marples et al. [4] stated that they observed spontaneous plaques in 30~ of P . acnes strains examined. They isolated 8 phages (A--G), some of which were used for classification of anaerobic coryneforms, us well [3--5]. I n 1973 we published the preliminary results of our studies on bacteriophages of P . acnes [7]. We then pointed out our experience with spontaneous lysogeny tests and lysogeny induction experiments with the use of UV rays, Novobiocin and mitomycin C. Only mitomyein C proved to have inducing effects. In addition, we demonstrated the possibility of differentiating P . aches phages according to their activity spectra and their resistance to UV rays, hydroxylamine, and acridine orange in the dark. In our paper we present a set of 13 virulent P. acnes phages, selected out of 61 phage preparations tested. These 13 phages proved to be stable and to cause no
Bacteriophages of Propionibacterium aches
271
g r e a t difficulties concerning p r o p a g a t i o n a n d storage. T a b l e 3 d e m o n s t r a t e s t h e p o s s i b i l i t y o f phage~yping w i t h this set. W h e r e a s all t e s t e d s t r a i n s o f P . granulosum a n d of P. avidum were r e s i s t a n t t o all p h a g e s in R T D a n d in 1 0 0 • o n l y one o f 58 P . acnes s t r a i n s was p h a g e - r e s i s t a n t . A l t h o u g h t h e p h a g e - t y p i n g d a t a a r e n o t y e t optimal, 46 o u t o f 58 s t r a i n s b e l o n g e d t o o n l y one p h a g e t y p e , t h i s set o f p h a g e s can nevertheless be useful for d i f f e r e n t i a t i o n purposes. U n t i l p h a g e a r e f o u n d which can m o r e s a t i s f a c t o r i l y s p l i t u p t h e species P . aches, which seems us t o be r a t h e r difficult, p h a g e t y p i n g can be c o m b i n e d w i t h o t h e r m a r k e r s y s t e m s such as b i o t y p i n g (see Tables 2 a n d 4). As seen in T a b l e 4, 46 P . acne8 s t r a i n s o f p h a g e t y p e I can t h u s be classified i n t o 7 b i o t y p e s .
References 1. Cummins, C. S., Johnson, J. L.: Corynebacterium parvum: a synonym for Propionibacterium aches ? J. gen. Microbiol. 80, 433--442 (1974) 2. Johnson, J. L., Cummins, C. S. : Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria and strains of Arachnia propionica. J. Bact. 109, 1047--1066 (1972) 3. Marplcs, R. R., Leyden, J . J . , Stewart, R. N., Mills, O. It., Kligman, A.M.: The skin microflora in Acne vulgaris. J. invest. Derm. 62, 37--41 (1974) 4. Marplcs, R. R., !~IcGinley, K. J., Mills, O. H. : Microbiology of comedoncs in Acne vulgaris. J. invest. Derm. 60, 80--83 (1973) 5. ~Iarples, R.R., McGinley, K . J . : Corynebacterium aches and other anaerobic diphtheroids from human skin. J. mcd. Microbiol. 7, 349--357 (1974) 6. Pulverer, G., Ko, It. L.: Fermentative and serological studies on Propionibacterium acnes. Appl. Microbiol. 25, 222--229 (1973) 7. Pulverer, G., Sorgo, W., Ko, H. L.: Bakteriophagen yon Propionibacterium aches. Zbl. Bakt., I. Abt. Orig. A 225, 353--363 (1973) 8. Reid, J. D., Joya, M. A. : A study of the morphologie and biochemical characteristics of certain anaerobic corynebacteria. Int. J. syst. Baet. 19, 273--280 (1969) 9. Voss, J. G.: Differentiation of two groups of Corynebacterium acnes. J. Bact. 101, 392 to 397 (1970) 10. Whiteside, J . A . , Voss, J. G.: Incidence and lipolytic activity of Propionibacterium aches (Corynebacterium aches group I) and P. granulosum (C. acnes group II) in Acne and in normal skin. J. invest. Derm. 60, 94--97 (1973) 11. Zierdt, C. H., Webster, C., Rude, W. S.: Study of the anaerobic corynebacteria. Int. J. syst. Bact. 18, 33--47 (1968) Prof. Dr. Gerhard Pulverer Direktor des Hygiene-Instituts D-5000 K61n 41, F~irst PQckler-Str. 56 Federal Republic of Germany
