clostridium perfringens — specific lysin

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Page 1: Clostridium perfringens               — specific lysin

Clostridium perfringens - specific lysin

S. NAKAMURA, I . OKADA, J . M I Z U N O , A N D S. NISHIDA Depcrrtn~enr of Bacteriology, School of Medicine, Kcincrzciwa Utzi~ersity, Kot~nzcr~va, Japntz

Accepted January 26, 1977

NAKAMURA, S., I . OKADO, J. MIZUNO, and S. NISHIDA. 1977. Clostridiiimperfiit~get~s-specific lysin. Can. J. Microbiol. 23: 601406.

A mitomycin C induced lysate of Clos~ridiiin~petfringens strain KZ219 was lytic to 50strains of C . perfiitzgens of types A-E, and three strains of C . plcigariim. The lysin was active against only 2 out of 87 strains of 51 other clostridial species. The optimum pH of the lytic agent was 5.5. The activity was largely inactivated by proteolytic enzymes, and nearly completely inactivated by heating at 60°C for 5 min.

NAKAMURA, S., I . OKADO, J. MIZUNO et S. NISHIDA. 1977. Clostridiritnperfiingetls - specific lysin. Can. J. Microbiol. 23: 601406.

Une lysate de Clos~ridiritnpe~frit~gens souche KZ 219 induite par la mitomycine C est lytique envers 50 souches de C . perfritlgetls des types A-E et de trois souches de C. plcigarrim. La iysine est active envers seulement 2 des 87 souches de 51 autres espkces de clostridies. Le pH optimum de la substance lytique est de 5.5. L'activite est grandement inactivee par les enzymes proteolytiques et presque complitement inactivee par un chauffage a60°C durant 5 min.

[Traduit par le journal]

Introduction

In a study on the susceptibility of Clostridilrm perfringens to mitomycin C , we encountered a mitomycin-induced lysin with considerable spe- cies-specificity in the lysate of a C . perflingens strain. Susceptibility to this lysin was one of the pertinent criteria which allowed us to demon- strate identity of the newly adopted species of C . plagarunl ( 1 2) with C. perflingens (7). In the present study, we have examined further the taxonomic applicability of the lysin and investi- gated some of its biochemical properties.

Materials and Method Strains

Closrridiin,~ perfiinge~is, strain KZ 219, was used for production of the lytic agent and C . perfibrgens, strain BP6K, as the indicator strain to examine the character of the 1ytic agent. Strains used for examining the spectrum of the lytic agent are listed in Table 1. Most of the clostridial-type strains listed in the 8th edition of Bergey's Manual (12) were used.

Mediimi PYG medium consists of proteose peptone No. 2

(Difco), 2%; yeast extract (Difco), 0.5%; glucose, 1%; NaCI, 0.5%; and cysteine HCI-HZO, 0.05% (pH 7.2). Bacteria grown overnight in liver broth were used as the preculture.

Preparation of It~dicaror Cells To prepare freeze-thawed cells (FT-cells), bacteria

grown for 16 h in the PYG medium at 37°C were harvested by centrifugation, washed once with 0.05 M phosphate buffer (pH 5.5) by centrifugation, resuspended in the same buffer, and immediately stored at -20°C. Acetone-, chloroform-, and heat-treated bacteria were prepared

according to the methods described by Mitsui el al. (6). Formalin-treated organisms were prepared as described by Ogata and Hongo (S), except that 0.05 M phosphate buffer (pH 5.5) was used.

Preparariotz of Cell Walls Walls of C . perfiinget~s, strain BP6K, were prepared

according to the method described by Takumi and Kawata (13).

Preparalio~z of Lysale To induce production of the lytic agent, cells growing

exponentially at 37°C in PYG medium were treated with mitomycin C (MC) (Sankyo, Tokyo, Japan). M C (final concentration 2 wg/ml) was added to the culture when the optical density at 560 nm reached 0.14-0.18. The culture was incubated a further 3.0 to 3.5 h until bacterial lysis was complete; the lyzed culture was used as the lysate. Untreated bacteria grown for the same period and then sonicated for 5 min using a Tomy ultrasonic vibrator model UR-ZOOP, 20 kcycles (Tomy, Tokyo, Japan), to obtain an extract were used as the control. Each lysate and extract was cleared of bacterial debris by centrifuga- tion (3000 x g, 15 min), and the supernatants were used as cell-free lysate and cell-free extract, respectively. The cell-free lysate was fractionated further into a super- natant fraction (phage-free lysate) and a pellet fraction by ultracentrifugation (100 000 x g, 90 min); the pellet was resuspended in volume of the culture with PYG medium. Both cell-free and phage-free lysates could be stored at 4°C for at least 2 weeks without measurable loss of their lytic activities.

Esritnation of Siiscep~ibili~y to Lysin Susceptibility of the indicator cells to the lysin was

estimated turbidimetrically using a Shimazu Bausch & Lomb Spectronic 20 calorimeter. An amount of 0.5 ml of the cell-free lysate was added to 3.0 ml of indicator cells suspended in 0.05 M phosphate buffer (pH 5.5) in a tube (1 x 10 cm) to give an optical density at 535 mm of 0.45 to 0.50; the mixture was incubated at 37°C. OD,,,

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Page 2: Clostridium perfringens               — specific lysin

602 CAN. J . MICROBIOL. VOL. 23, 1977

TABLE 1. Strains used for lytic spectrum Results

ATCC" No. strains C . absorrum 10984, 10985, 10986; C. nr~~inoualericu~~~ 13725; C. nurnt~/ibrrtyricrr,n 17777; C . harkeri 25849; C . beijerinckii 25752; C. cadaueris 25783; C. cantis 25777; C . cellobioparrrr~r 15832; C. cocl~learirrm 17787; C . dlficile 9689; C . fallax 25754; C . ghorii 25757; C . glycolicrrnr 14880; C . I~is/olyticuni 25770; C . indolis 25771 ; C. irrnocurrm 14501 ; C . lentoputrescerzs 17794; C . lir~rosr~rrr 25760; C. litlrsebrrrerrse 25759; C . rnalerro- rr~inntum 25776; C. nrangenolii 25761 ; C . oceanicrrrn 25647; C. oroticrrm 25750; C . paraperfiitigens 25753, 27639, 27640; C . parap~rtrifcrrrr~ 17864, 25780; C . pastelrrianrmr 6013; C. perenne 25782; C . plagnrurn 25768; C. propior~icrmr 25522; C . pserrdo/etat~icrrm 25779; C . prrtrefacierls 25786; C . putrifcurn 25784; C . rrrbrrrrn 14949; C . sartngoforr~~~rn~ 25778; C. scatolo- genes 25775 ; C . sphenoides 19403 ; C . sporosplienoides 25781 ; C . sticklor~c/ii 12662; C. s~rhtern~inale 25774; C . tertirrrlr 14573 ; C . tetarli 8033 ; C. tyrobrrtyricrm~ 25755.

KZb No. strains C. ocetobrrtyric~rn~ 586; C. biferrner~tarrs 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 1012, 1013, 1015, 1033, 1035 ; C . botrrlir~rrm (type A) 532, (type B) 533, 534, (type C) 536, (type D) 537, (type E) 582, (type F) 539, 562; C . brrtyricrrrn 589, 591; C. charruoei 382; C. cochlearirrnl 61 1, 612, 1176, 1202; C . l~aernoly- ticlrr?~ 415; C . nouyi (type A) 671, (type B) 3690: C . perf,irg>gens (type-A) ~ I ~ ( B P ~ K ) , 215; -216, 21 7, 21 8; 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, (type B) 301, 438, 439, (type C) 111, 112, 113, 114, 116, 117, 119, 120, 302, 303, 732, 738, 750, (type D) 305, 442, 443, 444, 445, 446, 447,449,450,451, (type E) 316, 339, 452; C . plagarum 1340, 1341 ; C . septicrrm 993, 1009; C. sordellii 1018, 1023; C . sporogerres 1072, 1073.

"American Type Culture Collection Rockville, MD, U.S.A. bKanazawa University, Kanazawa japan.

readings were taken every 2.5 min up to 10 min. FT-cells without lysate served as the controls. Cells were con- sidered to be susceptible to the lysin when the difference in OD was more than 0.1 per 10 minutes.

Assay of Lytic Actiuity The assay of lytic activity was performed according to

the method of Mitsui et a / . (6). The extent of cell lysis during the first 5 min of incubation was measured and defined as the lytic activity of the lysate. One unit of lytic activity is defined as the decrease in optical density at 535 nm of 1.0 per hour under the conditions of test.

Detectiorr of Pl~age Particles by Electron Microscopy Preparation of samples and the procedures for electron

microscopy were as described by Shirnaniura a/. (1 1).

Production of Lytic Agent Of several C. perfringens strains tested for the

susceptibility to MC, strain KZ 219 exhibited the highest sensitivity. The MC-lysate of this strain was demonstrated to include a lytic agent highly active against FT-cell suspensions of C. perfringens (Fig. 1). Lytic activity appeared in the culture 90 min after the addition of MC, when the bacterial lysis occurred. The lytic activity of sonic extracts prepared from cultured cells grown for 30, 60, 90, and 120 min after addition of M C were 0, trace, 9.5 units/ml, and 0, respectively. No lytic activity was detected in the culture without MC.

The induction process is sensitive to chloram- phenicol (Fig. 2, Table 2). To MC-added bac- terial cultures, chloramphenicol at a concentra- tion of 50 pg/ml was added at different intervals. Cellular lysis and production of the lytic agent

Clienlicals FIG. 1. Effect of MC on bacterial growth and the pro- Pronase (Kaken Co., Tokyo, Japan), trypsin (Difco), duction of a lytic agent. Growth of the bacteria: MC

nagarse (Nagase Co., Tokyo, Japan), and chloramphen- treated (@), untreated (0). Lytic activity in cell-free icol (Sankyo Co., Tokyo, Japan) were purchased from lysate (A). Arrow indicates the time of MC addition in the respective companies. culture.

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Page 3: Clostridium perfringens               — specific lysin

N A K A M U R A ET AL.

col. dLytic activity was measured 180 min

after MC addition.

20-

-I z '3i. I- - z 3 -

60 120 180 240 g 1 0 - MINUTES 2

I- U FIG. 2. Effect of chloramphenicol on growth of MC-

treated cultures. Time of chloramphenicol addition after MC addition: 30 min (A), 60 min (*), 75 min (O) , 90 min (A), 105 min (O) , control without chloramphenicol > (0). Arrow indicates the time of M C addition in cultures.

TABLE 2. Effect of chloramphen- ico14 on production of lysin

Time, Lytic act.d minb unitslml

were completely inhibited by chloramphenicol if added within 60 rnin of MC addition.

4 )

A - I I

Optitnum Conditions for Susceptibility Testing The optimum pH of the lysin was 5.5 (Fig. 3).

The effect of lysin on differently treated cell suspensions is shown in Fig. 4. The cells used were from a 4 h culture of C. perfringens, but further investigation on FT-cells indicated that 16-h FT-cells were equally suitable (Fig. 5). The susceptibility of FT-cells decreased as the treat- ment temperature increased. Since the FT-cells were most sensitive, cells from 16-h cultures were used throughout the following study.

0 4 5 6 7 8 9

30 60 0 P H 75 5.8 FIG. 3. Effect of pH on the activity of the lytic agent: 90 14.0 FT-cells suspended in 3 ml of 0.05 M phosphate buffer

105 20.0 (0) or 0.1 N acetate buffer ( 0 ) . Controlc 39.0

Lytic Spectrum The lytic spectrum of MC-lysate from C.

petfiingens, strain KZ 219, was as follows: all of 50 strains of C. petfiingens types A, B , C, D, and E were highly sensitive (Table 3). Tests against three strains each of C , perfringens - related species, C. plngnrlmi, C. parnperfiingens, and C. absotam~, and 8 1 reference strains including one or more of each of 49 clostridial species showed that none of the strains, except C. bifermentnns, strain K Z 1012, and C. cochlearium strain ATCC 17787, were sensitive. The remaining 15 strains of C. bifern~entar~s and 4 strains of C. coch- learium were insensitive.

Additional Clzaracteristics of Lysirz When the MC-lysate of C. perfiit~gens K Z 2 19

was centrifuged at 100 000 x g for 90 min, the total activity remained in the supernatant; the pellet showed no lytic activity (Fig. 6), although phage-like particles were observed in the pellet (Fig. 7). The lytic activity was completely inac- tivated by heating the lysate at 60°C for 10 rnin (Fig. 8). It was partially inactivated by proteo-

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Page 4: Clostridium perfringens               — specific lysin

604 CAN. J. MICROBIl 3 L . VOL. 23, 1977

I I I I

5 10 15 M 1 NUTES

FIG. 4. Susceptibility of differently treated indicator cells to the lytic agent. Treatment of cells: FT (O), acetone-powdered (A) , chloroform-treated ( n ) , Forma- lin-treated (a), untreated cells (A). The lysin concentra- tion used was 17.2 unitslml against FT-cells.

lytic enzymes: pronase, trypsin, and nagarse at a concentration of 100 pg/ml (Fig. 9). The lysate was also active against cell walls, although the lysis of the cell wall occurred more slowly (Fig. 10) than did that of FT-cells.

Discussion The present study indicates that the lytic

spectrum of the MC-induced lysin of C. per- fiingens, strain K Z 219, was nearly species- specific, with the exception of C. plagarum. Clostridium plagarum has been adopted in the 8th edition of Bergey's Manual (12) as a new species although it has all the characteristics of C. perfringens, except lecithinase. Identity of C. plagarurn with C. perfringens was demonstrated by us in the preceding paper (7); we used the MC-induced lytic agent of strain K Z 219 as one of the pertinent criteria.

A lytic agent, welchicin, was already demon- strated by Sasarman and Antohi (9, lo), but welchicin is so widely active against aerobic and anaerobic bacteria that it is not applicable for the identification of C. perfiingens. Mahony ( 5 ) and Uchiyama (14) demonstrated a bacteriocin-like substance in the culture filtrates of C. perfringens

MINUTES

FIG. 5. Heat susceptibility of FT-cells prepared from 4-h and 16-h cultures. Open and closed symbols are 16- and 4-h FT-cells, respectively. Treatment of cells: non-heated (O,.), heated at 50°C (A,A), 60°C (UiD), IOOoC, (0,*) for 10 min. The lysin concentration was 17.2 units/ml against FT-cells.

TABLE 3. Lytic spectrum of MC-lysate KZ 219

No. of No. of strains strains sensitive t o

Species Type tested MC-lysate

C. perfiitigens A 2 1 2 1 B 3 3 C 13 13 D 10 10 E 3 3

C. perfringens - related species C. plagarrrnz 3 3 C. paraperfiinger~s 3 0 C. absonum 3 0

Other clostridia C. bifermenrans 16 1 C. cockleariurn 5 1 47 species 60 0

strains. However, these bacteriocins were far from being active against all the C. perfringens strains and hence could not be used to differen- tiate C. perfringens from other clostridial species. None of the above lytic agents was induced by

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Page 5: Clostridium perfringens               — specific lysin

N A K A M U R A ET A L

- 5 10 15

MINUTES

FIG. 6. Activity of lysate fractions against FT-cell suspensions. Cell-free lysate (O), phage-free lysate (O), pellet fraction (A).

FIG. 7. Phage-like particles in M C lysate. x 100 000.

ultraviolet light or MC, while the lysin described in the present study was MC-induced. It still remains to be determined whether the distinct specificity of our MC-induced lysin is due to the character of strain KZ 219 or to our method of testing, and to elucidate the manner in which the specificity of the lysin occurs. The present study, however, suggests that the lysin is useful for the identification of C. perfiingens as well as other criteria (1, 2, 3, 15).

Since autolysin had no effect on Formalin- treated cells, while endolysin did (S), the C. peflringens - specific lysin was tested against the formalin-treated cells and was active against these cells as well as cell walls.

The rate of lysis of FT-cells by C. perfringens - specific lysin was decreased when heated indi- cator cells were used. This suggests that autolytic

M I NUTES

FIG. 8. Inactivation of the lytic agent by heating. Phage-free lysate was heated at 37°C (O), 40°C (U), 45°C (A), 50°C (e) , 5S0C (A), or 60°C (U). The lysin concentration was 18.4 unitslml.

u 30 6 0

M I N U T E S

FIG. 9. Inactivation of the lytic agent by proteolytic enzymes. Pronase (A), trypsin, (O), nagarse (O), control (A). The lysin concentration was 18.4 units/rnl.

M I N U T E S

FIG. 10. Activity of the lytic agent against cell walls. MC-lysate added (O), control ( e ) . The lysin concentra- tion was 50.4 unitslrnl.

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606 CAN. 1. MICROBIOL. VOL. 23, 1977

enzymes of the indicator cells might contribute to the lytic process. The C. perfiingens - specific lvsin has a number of characteristics of an enzyme: the activity was heat-sensitive, depen- dent upon pH, and inactivated by trypsin, pronase, and nagarse. Similar lysins were demonstrated in other clostridia (4, 1 l), although these were less species-specific than the C. per- jiingens - specific lysin described in the present study.

Acknowledgment We thank Dr. L. DS. Smith, Anaerobe

Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, VA, for providing type strains described in the 8th edition of Bergey's Manual, and Dr. K. Kiritani for his instructive advice.

1. HARMON, S. M. 1976. Collaborative study of an im- proved method for the enumeration and confirmation of Clostridirrm perfringens in foods. J . Assoc. Off. Agrlc. Chem. 59: 606412.

2. HAUSCHILD, A. H. W. 1975. Criteria and procedures for implicating Clostridirrm perfringens in food-borne outbreaks. Can. J. Public Health, 66: 388-392.

3. HAYASE, M., N. MITSUI, K . T A M A I , S. NAKAMURA, and S. NISHIDA. 1974. Isolation of Closrridirrtn rib- sonrrm and its cultural and biochemical properties. Infect. Immun. 9: 15-19.

4. KIRITANI, K. , N. MITSUI, S. NAKAMURA, and S . NISHIDA. 1973. Numerical taxonomy of Closrridirrm botulitlrrm and Clostridi~rm sporogenes strains, and their susceptibilities to induced lysins and to mitomy- cin C . Jpn. J. Microbiol. 17: 361-372.

5. MAHONY, D. E. 1974. Bacteriocin susceptibility of Clostridilrm petfringens: aprovisional typing schema. Appl. Microbiol. 28: 172-176.

6. MITSUI, N., K. KIRITANI, and S . NISHIDA. 1973. A lysin(s) in lysates of Closfridi~rm borrrli~~rrm A 190 induced by ultraviolet ray or mitomycin C. Jpn. J . Microbiol. 17: 353-360.

7. NAKAMURA, S. , M. S A K U R A I , S. NISHIDA, T . TAT- SUKI, Y. YANAGASE, Y. HICASHI, and T. AMANO. 1976. Lecithinase-negative variants of Clostridium

perfringens; the identity of C . plagnrrrm with C . per- fringens. Can. J. Microbiol. 22: 1497-1501.

8. OGATA, S., and M. HONGO. 1974. Lysis induced by sodium ion and its relation to lytic enzyme systems in Clostridi~rm saccharoperbrr~lacerm~icrit~~. J. Gen. Microbiol. 81: 315-323.

9. SASARMAN, A., and M. ANTOHI. 1963. Presence d e s bacteriocines chez Clostridirrm petfiit~gens. Arch. Roum. Pathol. Exp. Microbiol. 22: 377-381.

10. SASARMAN, A., and M. ANTOHI. 1967. Sensibilitt a la welchicine A , comme test d'identification de Welchia petfritrblgens. In The anaerobic bacteria. Edited by V. Fredette. T h e Institute of Microbiology and Hygiene of Montreal University, Canada. pp. 125-13 1.

11. S H I M A M U R A , T. , S. NAKAMURA, M. HAYASE, and S . NISHIDA. 1973. Mitomycin-induced lysis of Clos- friditrm sordellii. J. Med. Microbiol. 7: 277-283.

12. SMITH, L . DS., and G. HOBBS. 1974. Closfridirrm. It1 Bergey's Manual of determinative bacteriology. 8th ed. Edited by R. E. Buchanan and N. E. Gibbons. Williams & Wilkins Compamy, Baltimore, MD. pp. 551-572.

13. TAKUMI, K . , and T. KAWATA. 1970. Chemical corn- position of the cell walls of Closrridiurn botrrlin~rrn type A. Jpn. J . Microbiol. 14: 57-63.

14. UCHIYAMA, K. 1966. Studies on bacteriocin-like sub- stances produced by Closrridirrm perfringens. I . Ac- tivity spectrum of the inhibitory substances produced by CI. perfringens. Med. J . Kagoshima Univ. 18: 131-144.

15. WILLIS,A. T. , and G. HOBBS. 1958.A mediurnfor the identification of clostridia producing opalescence in egg-yolk emulsions. J. Pathol. Bacterial. 75: 299-305.

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