lecithinase-negative variants of clostridium perfringens ; the identity of c . plagarum with c ....

Lecithinase-negative variants of Clostridium perfringens; the identity of C. plagarum with C . perfringens

A N D

T. TATSUKI, Y. YANAGASE, Y. HI GASH^, A N D T. AMANO D(,pnr.tt~retrt oJ'Bncto.iology. Sclrool ofMcdicit~e, Oscrktr Utrir~et.sity. Oscrkcr. Jnpcrt~

Accepted June 29, 1976

N A K A M U R A , S , M . SAKURAI, S. NISHIDA. T. TATSUKI, Y . Y H N A G A S E , Y . HIGASHI. and T. AMANO. 1976. Lecithinase-negative variants of Clos~t~iclirrt~r pc~t:/i.itrgc,rr.s; the identity of C. l~ltrgcrrrtt~r with C. /ICI:/~.~II,~~,IIS, Can. J . Microbial. 22: 1497-1501,

Identity of Closrt.idirrt~r p/ogot.ro)r (PrCvot) com. nov. (1938) with C. pc,~:/i.i~~grtrs was demonstrated in the tests for the cultural and biochemical properties, DNA-DNA homology, susceptibility to C. prr:fi.i~~gi~~~s-specific lysin. comp1emental.y synthesis of C. per:fi.it~gc,~l.s 0-toxin between C. p1ogar111~1 and 0-toxin-negative mutants of C. pc,rji.i~rgr~rs, as well as the tests for gas-chromatographic patterns.

N A K A M U R A , S.. M. SAKUHAI, S. NISHIDA, T. TATSUKI. Y. YANAGASE, Y . HIGASHI et T . AMANO. 1976. Lecithinase-negative variants of Clo.striditr~,l /~~t j i i i~rgi ,~~.s ; the identity of C. plogtrrtt~?~ with C. pc.rji-i~lgc,~r.s. Can. J . Micl'obiol. 22: 1497-1501,

L'identite de Clo.s/ri(lirt~?r plcrgorri~?~ (Prevot) com. nov. (1938) avec C. ~ P I ~ ~ . ~ I I ~ ~ I I S a ete demontree par les caracttres culturaux, les proprietes biochimiques, I'homologie du DNA-DNA, la sensibilite vis-5-vis d'une lysine specifique de C. ~ c I : ~ ~ . ~ / I ~ P I I s , la synthese complement;lire de 121 toxine H de C. pet:fi.i~rgp~~.s entre C. pItrgtrt-rr~?r et des mutants de C. podi.i~rgc.~rs deficients en toxine 0. ainsi que par les profils obtenus en chromatographie gazeuse.

[Traduit par le journal]

Introduction Clostridial strains having all the characteristics

of a particular species except a fact that they did not produce the characteristic toxin or soluble antigen are liable to be rejected from the species (1 1 , 15). This standpoint seetns to give rise to taxonomic confusions because non-toxigenic variants of originally toxigetiic strains are coni- mon enough. Recently, we encountered several strains of lecithinase- and 0-haemolysin-negative Clostridiln~i perfiingons-like strains which were identified, by Miwa (6), as Clostridii1177 ~ I ~ ~ c I ~ L I I ~ I , according to the manuals of Holdeman and Moore (4) and Smith and Hobbs (14). In the present study, we reinvestigated these strains, suspecting that they might be a- and B-toxin- negative variants of C. perfijt~gens in the tests for the cultural and biochemical properties and DNA-DNA homology, as well as in the tests by gas chromatography.

Since we recently demonstrated that mitomycin C-induced lysate of a C. perfiitlgens strain

'Received April 2, 1976.

KZ 2 19 included a C. perjkingens-specific lysin (71, we used the lysin for the present taxonomic purpose. Also, since Higashi pt (11. (2) demonstrated that 0-toxin-negative mutants of a C. perfiit7gen.r strain BP6K produced the characteristic 0-toxin in association with another 0- toxin-negative strain of this species, we, in this study, attempt to carry the complementation test between C. p l rgm~i t l~ and the 0-toxin-negative mutants of C. perfringens for the same ob- ject.

Materials and Methods Sltnitls

Four strains of C. p l r ~ ~ l o r r ~ ~ , ~ KZ 1340, K Z 1341, KZ 1342, and KZ 1343 were kindly provided by Prof. S. Suzuki, Department of Bacteriology, Gifu University, Gifu, Japan. Clos1ridilo17plr1got-~r11t ATCC 25769 was ~lsed as the referencc strain in the tests ror biochemical and cultural properties and DNA-DNA homology. Clos11.i- dilrt~~ perf,.itrgc~tts BPBK, K Z 215, KZ 216, K Z 2 17, KZ 21 8, K Z 21 9, KZ 220, K Z 221, KZ 222, KZ 223, KZ 224, K Z 225, KZ 226, KZ 227, K Z 228, KZ 229, K Z 230, KZ 231, K Z 232, KZ 233, and K Z 234 were used as referencc strains ror the cultu~~al and bioche~nical pr,operties. Clos- IriClirt~n pc,rJfi.irr,yet~s BP6K was also used as the rererence strain for DNA-DNA ho~i~ology.

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1498 CAN. J. MICROBIOL. VOL. 22. 1976

Clostr~itii~rr~~ perfibrg~rrs K Z 219 was used for prepara- tion of the C. po./j.irr~errs-specific lysin (7).

Clostriclirrrr~ obsorrrrrr~ ATCC 27555, ATCC 27635, ATCC 27636, ATCC 27637, K Z 1221, K Z 1222, and K Z 1223 and C. pol-rrperfiirrger2s ATCC 27638, ATCC 27639, ATCC 27640, K Z 1469, K Z 1470, K Z 1471, and KZ 1472 were used to examine susceptibility to the C. po.fiirlgerrs- specific lysin.

Bioc/rerrricol a r d Crrltrrrol Properties Fermentation of carbohydrates, liquefaction of gelatin,

and digestion of coagulated albumen were examined according to the description of Sterne and van Heyningen (16). The production of indole was tested with 21- and 48-h cultures in cooked meat broth (10). The test for nitrate reduction was performed according to the method of Hauschild and Hilsheimer (3). The digestion of milk was examined in skim milk nlediun~. The test for digestion of casein was as follows: cultures were streaked on brain heart infusion agar (Nissan Co., Tokyo, Japan) containing 7% (w/v) casein and after incubation for 48 h the plates were flooded with 10% HCI. Cultures showing a clear area around the streaks were recorded as casein- digestion-positive. Ten percent blood agar with 2% glucose and brain heart infusion agar were used to observe colonial properties.

and a strain M2-I 1 as the b-type mutant. The two @-toxin- negative mutants showing complementary production of 0-toxin were obtained originally from a C. perfiirlgens strain BP6K. The reference species and strains of clostridia are as follows: C. brrtyricrrrrz K Z 589, C. oroticrrrrz ATCC 25750, C. recturn ATCC 2575 1, C. paraperfringens ATCC 27639, C. rlrbrrrrrz ATCC 14949, C. fa1la.x ATCC 25754, C. pnste~u~ior~rrrrl ATCC 601 3, C. sticklor~dii ATCC 12662, C. tyrobrrtyricrrrt~ ATCC 25755, C. propiorziclrrrl ATCC 25522, C. gl~orli ATCC 25757, C. rrlangcrlotii ATCC 25761, C. ocetobrrtyric~rtn K Z 586, C. rlficile ATCC 9689, C. spllenoides ATCC 19403, C. irldolis ATCC 25771, C. scotologenes ATCC 25775, C. rr~aler~ornirmtrr~n ATCC 25776, C. tertirrrn ATCC 14573, C. sartagoforrrlrrrr~ ATCC 25778, C. cellobio/)arrrrr~ ATCC 15832, C. pseu- htetorlicrrrr~ ATCC 25779, C. poroprrtrificrrrrl ATCC 25780, C. orrrir~ounlericrrrt~ ATCC 13725, C. glycolicrrrr~ ATCC 14880, C. sporospl~er~oidrs ATCC 25781, C. coclz- leorirrrr~ ATCC 17787, C. rorr~osrrrrr ATCC 25582, C. bar- keri ATCC 25849, C. pererlrle ATCC 25782, C. corlooeris ATCC 25783, and C. ler~toptrtrescerzs ATCC 17794.

Clostr~idirrr?~ perfiirrger~s type A antiserum was pur- chased from Wellcome Research Laboratories (Becken- ham, England) and the 0-toxin-specific antibody (20 units/ml) was supplied by the courtesy of Dr. R. Murata, NIH. Tokvo. Jaoan: the unit of @-antitoxin was cali-

Arrolysis of Ferrrrarrtotior~ Prodrrcts brat& according to'0-antitoxin unit in Wellcome Re-

Strains tested were incubated for 48 h at 37 "C in modi- search Laboratories.

fied PY medium (4) containing 1% glucose (PYG); the DNA-DNA ~ o r n o ~ o g y modified PY medium consists of Bacto-peptone (Difco) DNA-DNA homology was performed according to 1.0% (w/\'), Yeast extract (Difco) 1.0% (w/v), NaCl 0.5% the method previously described (9). and cysteine-HCI-H20 0.05% (pH 7.2). Fatty acids and alcoholic fermentation products were extracted from the Results cultures and detected by gas chronlatography according to the description of Holdeman and Moore (4) but using Biochetnical and Cultural Pro~erties a Nihon Denshi gas chromatograph model JGC-1100 (Nihon Denshi, Tokyo, Japan). A glass column (2 mm inside diameter, 2 m long) was packed with chromosorb W (AW) coated with 10% Reoplex 400 (Nihon Chromato Co., Tokyo, Japan). The chromatograph was operated at 120 "C and with N 2 carrier gas at 4.0 kg/cm3 pressure.

Pj~rrtoate Utilizotior~ T o determine pyruvate utilization, products from a

48-h culture in modified PY medium containing pyruvate 0.9% (v/v) (PYP) were compared with those from a 48-h culture in modified PY medium by gas chromatography according to the above-mentioned procedure.

Lecit/ritmse Renctiorl This was examined on modified Nagler's agar (17).

C. p e r f i i g e ~ s - p e c c Lysirl This was induced by mitomycin C from the culture of

a C. perfiirgerls strain K Z 219 by the method previously described (7).

Preporntiorl of Cells to be Tested for Lysirl orlrl Estirr~atiorl of Tlreir Srrsceptibility

These were performed according to the method previously described (7).

Cornplerner~tory Prorlrrctior~ of C. perfiirrgetis 0-toxin by C. plagnr~rnz

This was examined according to the method of Higashi et 01. (2). A strain M4-1 was used as the a-type mutant

To examine the identity of C. plagarum with C. perfringens we examined five strains of C. plaganmz including four wild strains and a type- strain ATCC 25768 and 21 reference strains of C. petfiingens stocked in our laboratory for the cultural and biochemical properties. All of the C. plagar~mz and C. perfringens strains agreed in the following properties: they attacked fructose, galactose, glucose, lactose, maltose, mannose, melibiose, raffinose, ribose, and sucrose, and did not attack arabinose, dulcitol, inulin, mannitol, melezitose, rhamnose, sorbitol, and xylose. They digested gelatin but not albumen. They were negative in the test for indole production. Nitrate was reduced by all of them with formation of nitrite. They produced slightly greenish colonies on 10z blood agar supplemented with 2% glucose and their cultures on brain heart infusion (BHI) agar, 10% blood agar with 2% glucose, and in liver broth consistently showed capsule formation around the thick rod form, although the two characters, i.e., greenish colonies and capsule formation, are not stated in the present manuals for taxonomy.

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N A K A M U R A ET AL. 1499

Some strains of C. perpingens and C. p/crgarut~ showed a positive reaction in fermentation of cellobiose, inositol, starch, salicin, and trehalose. Some strains of C. perflingens and all of C. plagarlrm strains digested casein. Stormy fermentation more slowly occurred in the cultures of C. plagarum than in those of C. perfrit~gens in milk medium. Distinctly different properties between C. perpingens and C. plagarlrm were lecithinase reaction on Nagler's agar and haemolysis on blood agar.

Fern7entation Products On gas-chromatographic analysis for cultures

of five C.plagarum and 1 1 C. pe19ingens strains in PYG medium, the two species exhibited the same patterns of fatty acids. The major peaks were acetic, propionic, and butyric acids as volatile acids as well as lactic and succinic acids as non- volatile acids (Figs. 1, 2).

MINUTES

FIG. 1. Gas-chromatographic patterns of volatile acids. A, C. perfrit~getzs BP6K; B, C. plagaruttz ATCC 25768. 1 , acetic a c ~ d ; 2, propionic acid; 3, butyric acid.

L

0 5 0 5 MINUTES

FIG. 2. Gas-chromatographic patterns of non-volatile acids. A, C. perf ib~get~s BP6K; B, C. pl(~garl~ttz ATCC 25768. 1, lactic acid; 2, succinic acid.

Utilizatioiz of Pyruuate Pyruvate in the P Y P medium was dernon-

strated to be utilized with increase of acetic and butyric acids in both species.

Taxonotnical App/ication of C. perfiit~gens-specific Lysin

Five strains of C. plcrgarlrnf as well as 35 reference strains of 7 C. pclrcrpetj2tzget7s, 7 C. ab- sonum, and 21 C. perflingens strains were examined for the susceptibility to the C . perfiitl- geiw-specific lysin induced from a C. perpitzgetls strain K Z 219. All of five C. plagarlrtn and 21 C. petfiitlgens strains tested were sensitive, whilst none of 7 C.parapctfringens and of 7 C . absonutn strains tested were sensitive to the lysin. The representative lysis curves are shown in Fig. 3.

Complenzentary Production of C. perpingens @-toxin by C. plcigarum

Five C. plagarutn strains including a type- strain ATCC 25768 together with a @-toxin- negative a-type mutant of C. perfrit~gens BP6K

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1500 CAN. J . MICROB IOL. VOL. 22. 1976

M l NUTES

FIG. 3. Susceptibility to C. /)c,~ji.i~~,:.e~ls-specific lys~n . A, C. pc~1fii11gc.11~ K Z 219; B, C. l ) l r / ~ ~ r r r l ~ ~ ~ r ATCC 25768; C, C. /)or~/)crJi.i~cgr,rs ATCC 27639; D, C. rrbsoll/rt,l ATCC 27555. Lysin-added (O), control (a).

were cultured in cross culture with O-toxin-negative a- and 6-type mutants, M4-1 and M2-l I . All of C. plngar~riii strains as well as the n-type mutant M4-1 yielded narrow P-haemolysis sur- rounded by the wide zone of incomplete haemolysis on 10% sheep blood agar by cross culture with the 6-type mutant M2-11 but not with the n-type mutant M4-1 (Fig. 4). Botli of the complete and incomplete haemolysis caused by these strains were suppressed on the blood agar supplemented with 1% (vh) C. pei$.iilgeils type A antitoxin and when 1 % 0-toxin-specific antiserum was used, only the complete haemolysis was neu- tralized, leaving the wide incomplete haemolysis zone intact. Furthermore, one strain each of 32 species of clostridia were tested for complementary production of C. peifiiiigens 0-toxin in cross culture with a- and 6-type mutants of C. per- friilgeils. Thirty-one of the 32 species tested were negative; the only exceptional case was caused by C. ghoni.

Meas~ct~en7ent of DNA- DNA Hoi71010gy We examined wild strains of C. plagaiuin, the

type-strain ATCC 25768, and the reference strain of C. perfiingeiu for their DNA-DNA homology. The results shown in Table 1 indi- cate that the four strains named as C. plngai~ui7i as well as the type-strain ATCC 25768 should be grouped into C. perfiiiigei2s.

' I ' r llgc'l~s FIG. 4. Complenlentary production of C. pc f i @-toxin. C. P / O ~ N ~ I I I ) I strains ( I ) ATCC 25768, ( 2 ) K Z 1340, (3) KZ 1341, (4) K Z 1342, (5) K Z 1343. First, rr (M4-1)- and b (MZ-11)-type mutants were streaked in parallel from t o p to bottom and then C./)lcrgorrrr?l strains and both of the two type nlutants were streaked in cross culture from right to left. Wide a n d incomplete haernol- ysis (white arrow) is due t o o-toxin produced by two mutants. Narrow and complete haen~olysis (black arrow) is due to complementarily produced 0-toxin.

Discussion

It is stated in the manuals for taxonomy (13, 14) that C. plagailriii is a name designated for Bacillus L of Adamson (I). He described that Bacillus L morphologically resembled C. per- fiiilgens. PrCvot renamed the Bacillus L as Iii-

j?abilis plagarui2 with amended characters (12). This species has newly been adopted in the 8th edition of Bergey's Manual (14). Recently, however, Johnson and Francis (5) demonstrated in the tests for ribosomal RNA homology that C. peifiingeiis had a similarity of 100% with the nucleotide sequence for C. plngaivi~i. The present studv revealed in the tests for cultural and biochemical properties, gas chromatography, and DNA-DNA homology that C. plagai~un~ is a lecithinase- or haemolysin-negative variant of C. perfiingens. It seems, however, to be a matter of controversy to regard the C. pefiingeils-like

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NAKAMURA ET AL. 1501

TABLE 1. DNA-DNA homology between C. perfrit~getls and C. p l a g n r u t ~ ~

% hon~ology t o D N A from :

Organisms BP6K A T C C 25768

C. perfiirlgerls

New isolates of C. plngrrrrrrrl K Z 1341 86 99 K Z 1343 84 98 K Z 1340 8 3 86 K Z 1342 8 1 89

C. plngarrit?~ ATCC 15768

but lecithinase-negative stralns as C. perfringens (11, 15), because a-toxin or lecithinase is regarded as the most important criterion for identification of C. perfiingens. We, therefore, used further additional tests for susceptibility to C. perfiit~gens-specific lysin (7) and for complementary production of C. perfii~lgens 8-haemolysin (2). It was already demonstrated by us that the lysin susceptibility was confined to C. pe~fiitlgeils and C. p/rgnt.wu and. furthermore, the present study confirmed this co~lclusion using increased numbers of strains of C. plngm~tin and the other species that are taxonomically close to C. per- fri;7ge11s (8). The test for complementary production of C. perj.i~lgens 8-haemolysin seems to be taxonomically useful since 31 of the 32 nonhaemolytic species of clostridia tested were negative in the test. Although thecomplementary haemolysis was caused by a C. ghoni strain, we did not carry out further investigation on this species. After all, considering the agreements in the tests for cultural and biochemical properties, gas chromatography, and DNA-DNA homology as well as in the two above-mentioned tests, we claim that C. p/rtga~.~/~?z should be regarded as a variant of C. petfiingetls and that the species name should be cancelled.

Acknowledgement We thank Dr. L. DS. Smith, Anaerobe Labo-

ratory, Virginia Polytechnic Institute and State University, Va., for his instructive advice; Dr. S. Suzuki, Gifu University, Gifu, Japan, for providing C. plrgarw17 isolates; and Dr. R. Murata, NTH, Tokyo, Japan, for C. perfiingsls 0-toxin-specific antiserum.

1. ADAMSON, R. S . 1919. On the cultu~.al characters of certain anaerobic bacteria isolated from war wounds. J. Pathol. Bacteriol. 2: 345-400.

2. HIGASHI, Y., M. CHAZONO. K. INOUE, Y. YANA- GASE, and T. AMANO. 1973. Complementation of 6-toxigenicity between mutants of two groups of Closrridirrrn per:fiirzgerl.s. Biken J . 16: 1-9.

3. HAUSCHILD, A. H. W., and R. HILSHEIMER. 1974. Evaluation of media for enumeration of Closrridirrr~~ pf,rJiirrger7s. Appl. Microbiol. 27: 78-82.

4 . HOLDEMAN. L. V., and W. E. C. MOORE. 1972. Anaerobe Laboratory Manual. Virginia Polytechnic Institute and State University. Blacksburg, Va.

5. JOHNSON. J . L .? and B. S. FRANCIS. 1975. Taxonomy of the clostridia: ribosomal ribonucleic acid ho- mologies among the species. J . Gen. Microbiol. 88: 229-244.

6. M I W A , T . 1975. Clostridia in the Antarctic. Jpn. J. Med. Sci. Biol. 28: 201-213.

7. NAKAMURA. S . , J . MIZUKO. and T . ABE. 1976. MC- or UV-induced lysins and their applicability for taxonomy of clostridia. 111 Abstracts of the 49th Annual Meeting of the Jpn. Soc. Microbiol. Jpn. J . Bacteriol. 31: 126. (in Japanese).

8. NAKAMURA. S., T. S H I M A M U R A . M. HAYASE, and S. NISHIDA. 1973. Numer-ical taxonomy of saccharolytic clostridia, pa~.ticularly Closrridir~rr~ pc~rfiingeris-like strains: description of Clostriclirrrn n b ~ o r ~ r i m sp.n. and Clo.stridirrrrr prrrrrpe~iir1g~r1.s. Int. J . Syst . Bacteriol. 23: 4 19-429.

9. N A K A M U R A , S.. T. SHIMAMURA. H. HAYASHI, and S. N I S H I D A . 1975. Reinvestigation of the laxonomy of Closrrirlir~rn l)$rrrner7rnrr.s and Closrrirlirrrn .sor~clellii. J. Med. Microbiol. 8: 299-309.

10. NAKAMURA, S.. K. TAKEMATSU, and S . NISHIDA. 1975. Susceptibility to mitomycin C and lecithinase activities of Closrridirrrn oerlrrr~crrierrs (C. rloisvi) types B a n d D. J . Med. Microbiol. 8: 289-297.

11. OAKLEY, C. L. 1962. Soluble bacterial antigens as discriminants in classification. 112 Microbial classification. Erlired by G. C. Ainsworth and P. H . A. Sneath. Cambridge Univ. Press, London. pp. 242- 248.

12. PnivoT, A. R. 1938. gtudes de systimatique bacterienne IV. Critique de la conception actuelle du genre Closrrirlirrnr. Ann. Inst. Pasteur. Paris, 61: 72-9 1.

13. PREVOT, A. R., A. T U R P I K . and P. K A I S E R . 1967. Les batteries anaerobies. Dunod, Paris. p. 603.

14. S M I T H , L. DS., and G . H o ~ s s . 1974. Clo.s/ridirrrf~. Irf Bergey's Manual of determinative bacteriology. 8th ed. Editcd by R. E. Buchanan and N. E. Gibbons. Willir~ms & Wilkins Conlpany. Baltimore, Md. pp. . - 551-572.

15, S M I T H . L. DS.. and L. V HOLDEMAN. 1968. The pathogenic anaerobic bacteria. 1st ed. Charles C. Thomas, Publishers, Springfield, 111.

16. STERNE, M., and W. E. VAN H E Y N I N G E N . 1958. The clostridia. In Bacterial and mycotic infections of man. 3rd cd. Edired by R. J. Dubos. J . B. Lippincott Com- pany. Philadelphia. pp. 343-35 1.

17. WILLIS, A. T.. and G. HOBRS. 1958. A medium for the identification of clostridia producing opalescence in egg-yolk emulsion. J . Pathol. Bacteriol. 75: 299-305.

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