In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens

Research in Veterinary Science /988. 45, 337-340

In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens F. KONDO, Department oj Veterinary Public Health, Faculty oj Agriculture, Miyazaki University, 7710 Kumano, Miyazaki-shi, Japan 889-21

Viable Clostridium perfringens ranging from 1~ to few strains of C perjringens originating from necrotic lOS g-t was detected in all of 88 intestinal content enteritis in broiler chickens. The purpose of the present study was to assay the in specimens of necrotic enteritis in broiler chickens. In vitro lecithinase activity and sensitivity to 22 anti- vitro lecithinase activity of such C perfringens strains microbial agents were determined for the 88 isolates. and the minimum inhibitory concentration of 22 The activities of lecithinase in the culture filtrate of drugs currently in medicinal and veterinary use and to isolates were estimated to be 0·5 to 4·0 AE ml" t as a- compare the results with those of earlier workers. antitoxin equivalent. With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used Materials and methods as growth promoters, showed that all except baci- Cultures tracin had low minimum inhibitory concentration levels (1' 6 lAg ml- t or less) against this organism; A large number of broiler chickens, aged 10days to polyethers of monensin, salinomycin and lasalocid six weeks, died from necrotic enteritis on a farm in were generally adequate in low concentrations while Miyazaki Prefecture, during the period May to July there was a high level of resistance to three tetra- 1987. The intestinal contents, mainly jejunum and cyclines in 90 per cent of the strains and all isolates ileum, from 88 broilers were collected in anaerobic were insusceptible to streptomycin of the aminoglyco- transport vials. Each specimen was inoculated on to side antibiotics. cw agar medium containing kanamycin (Nissui Pharmaceutical, Japan) to detect C perjringens and CLOSTRIDIUM perfringens is one of the normal to count the viable n'l.tmber of organisms in the bacterial flora of the gastrointestinal tract in both specimen. All plates were incubated anaerobically in humans and animals. It has also been shown to cause Gas-Pak jars for 24 hours at 37°C. Organisms were identified to be C perjringens by a number of diseases in humans and animals and reported to be a causal agent of necrotic enteritis, an the three procedures of lecithinase production on an important avian disease throughout the world. The egg-yolk agar plate (Willis and Hobbs 1958), routine types of C perfringens in this disease are type A and biochemical tests and the identification of metabolic type C (Parish 1961, Long 1973). The lecithinase end-products by gas-liquid chromatography in activity (a toxin) of these isolates of necrotic enteritis cultures (Holdeman and Moore 1972). Stock cultures of these strains were maintained in cooked meat broth has not so far been examined in detail. There are many antibiotics available for medicinal and C perfringens type A PB6K, kindly provided by and veterinary use and some are used as growth- Dr S. Nishida of the Department of Microbiology, promoting agents. For broiler chickens under typical , School of Medicine, Kanazawa University, Japan, commercial husbandry conditions in Japan, feed was used as a control. additive antibiotics including penicillin, tetracycline, bacitracin, enramycin and thiopeptin have 'been used. The in vitro susceptibility of C perfringens to anti- Typing test biotics has been described in a numberof papers All isolates" were examined for toxigenic typing by (Traub 1971, Traub and Raymond 1971, Sapico et al the toxin-antitoxin neutralisation test, using mice, 1972, Tally et al 1975, Bryan et al1979, Dutta and according to the method of Oakley (1955). Antitoxins Devriese 1980). However, most of these reports were of each type except type A of C perjringens were limited mainly to a few major antibiotics and to only a purchased from Wellcome Research Laboratories,

338

F. Kondo

i.

50

~

40

'E

30

.,

41·1

t:l>

.

.5:

Cl.

27·5

(.)

'0

.. ] ~

20 12·6 10 1·1 1·1

1·1

0'050·1 0·2 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0 a-antitoxin equivalent ml-' FIG 1: Distribution of a toxin activities of isolates

Beckenham, England. Type A antitoxin (6ooiu ml- I ) was obtained from Chiba Serum Institute, Japan.

centrations (MICs) for 22 antimicrobial agents were determined by an agar dilution method. The plates were inoculated with a Steers replicator to yield a final inoculum of 106 colony forming units mlr ". The medium used was GAM agar medium (Nissui Pharmaceutical, Tokyo) . After I ml of each respective antibiotic dilution had been incorporated with 9 ml of GAM agar at 50°C, plates were poured in 90 mm Petri dishes, allowed to solidify, dried for 30 minutes at 37°C, and used immediately. Inoculated plates were incubated in anaerobic jars at 37°C for 24 hours. Plates without antimicrobial agents were incubated as controls. After the incubation period, the MIC for each strain was recorded as the lowest concentration of antimicrobial agent which completely inhibited the growth of C perfringens.

Lecithinase activity

Results

Lecithinase production was detected on an eggyolk plate according to the method of Willis and Hobbs (1958). Cooked meat broth containing I per cent (w/v) fructose (Nishida et al 1962) was used for production of lecithinase (a toxin). The in vitro activity of lecithinase in culture filtrates was estimated as a-antitoxin equivalent (AE) ml" ' by the method of Evans (1945). C perfringens type A antitoxin from Chiba Serum Institute was used.

Isolation and typing

Antimicrobial agents

A total of 22 antimicrobial agents were supplied by the manufacturers as powders suitable for susceptibility tests. They were tylosin, enrarnycin, avoparcin and monensin (Takeda Chemical Industries, Osaka), cephazolin, thiopeptin and lasalocid (Fujisawa Pharmaceutical, Osaka), penicillin-G, ampicillin, chloramphenicol and chloropolysporin-C (Sankyo, Tokyo), tetracycline (Lederle Japan, Tokyo), oxytetracycline (Taito Pfizer, Tokyo), and streptomycin (Meiji Seika, Tokyo). Chlortetracycline, erythromycin, virginiamycin, bacitracin and salinomycin were purchased from the National Veterinary Assay Laboratory, Tokyo. Vancomycin, nalidixic acid and rifampicin were purchased from Sigma Chemical, St Louis, USA. The antimicrobial agents were dissolved in appropriate solvents, such as distilled water, methanol, dimethylformamide and 0'1 N sodium hydroxide. Minimum inhibitory concentrations

All drugs were tested at a concentration of 100 J.lg ml- I and in twofold serial dilutions to 0·025 J.lg ml- I in sterile distilled water. Minimum inhibitory con-

C perfringens was detected in all the 88 specimens from necrotic enteritis. The actual number of the organisms ranged from lOS to I()8 g- I of the specimen and they all produced type A toxin. Lecithinase activity

The distribution of in vitro lecithinase activities (AE ml' I) of the 88 isolates of C perfringens is shown in Fig .1. The a toxin activity of C perfringens type A PB6K was 6' 0 AE ml- I. The lecithinase activities for the isolates ranged from O·5 to 4'0 AE ml- 1, and 41. I per cent of all strains showed activity of 3.0 AE ml- I. Susceptibility (MICs)

The MICs of antimicrobial ~ents against C perfringens type A PB6I.< and the ranges of MICs against the isolates and the values that inhibited 50 per cent and 90 per cent of the isolates are summarised in Table I. Penicillins and cephalosporins. No penicillinresistant strains were isolated. The inhibition concentration for 90 per cent of C perfringens isolates was less than I . 3 jAg ml- I. The majority of strains of C perfringens were somewhat less susceptible to penicillin-G than to ampicillin. The MIC of cephazolin against 90 per cent of strains was I· 205 J.lg ml- 1 or less. Tetracyclines. The susceptibility patterns to the three tetracyclines were similar and there was a high level of resistance to them in 90 per cent of the isolates.

339

C perfringens sensitivity to antimicrobial agents TABLE 1: Susceptibility of C perfringens to 22 antimicrobial agents

C perfringens Antimicrobial agent Penicillins Penicillin-G Ampicillin Cephalosporins Cephazolin Tetracyclines Tetracycline Oxytetracycline Chlortetracycline Macrolides Erythromycin Tylosin Aminoglycosides Streptomycin Chloramphenicol Chloramphenicol Peptides Enramycin Thiopeptin Virginiamycin Bacitracin Vancomycin Avoparcin Chloropolysporin-C Polyethers Monensin Salinomycin Lasalocid Others Rifampicin Nalidixic acid

type A PB6K 0·025 0·025

0·025-6·25 0·025-12·5

0·056 0·075

0·650 0·300

0·20

0·025-25

0·33

1·205

0·39 0·39 0·20 0·78 0·20

O·lO-over 100 O·2o-over 100 O·:zo.:over 100 0·39-50 0·05-50

9·375 18·250 4·553

"22·917

0·631 0·160

1·393 0·330

38·333 10·294

over 100

over 100

over 100

over 100

1-56

1-56·6,25

1'334

2-713

0·20 0·10 0·39 0·39 1·56 0·39 0·10

0·025-1·56 0·025-3·13 0·10·6·25 0·20·50 0·78-6·25 0·10-6·25 0·025-3·13

0·134 0·05 0·200 2·340 0·834 0·235 0·058

0·493 0·150 0·379 32·692 1·587 0·387 0·150

3·13 0·39 0·78

1·56-12·5 0,10-12,5 0·20-12·5

2·563 0·343 0·529

5·448 0·731 1·226

under 0·025 6·25

under 0·025-3·13 3,13·25

0·019 4·997

0·024 9·926

Macrolides. Tylosin was active against most of the isolates at O:33 Jlg rnl: 1. The MIC values of erythromycin against 96 per cent of strains were less than . I, 56 Jlg ml- I • Amlnoglycosides. All isolates were insusceptible, showing MIC values over 100Jlgml- I to streptomycin. Chloramphenicol. The MIC of chloramphenicol against all strains was 6· 25 Jlg ml- I or less and 90 per cent were inhibited at a concentration of 2·713 Jlg ml- I or less. Peptides. All except bacitracin had Jlg ml- I or less.

MIC (fig ml- 1) 88 isolates of C perfringens Range 50% 90%

MIC levelsof

I .6

Polyethers. Monensin, salinomycin and lasalocid, which are used as anti protozoan drugs, generally required low concentrations (0,025 to 6'25/Ag ml- I ) to produce 90 per cent inhibition. Others. Rifampicin was most active, inhibiting 90 per cent of all isolates at a concentration of 0·024 Jlg rnl "! and the MIC for nalidixic acid was 3 '13 to 25 Jlg mlr ',

Discussion Large numbers of C perfringens were recovered from the small intestine of chickens which had died of necrotic enteritis and a'llisolates were type A although type A and C have been reported by others (Parish 1961, Long 1973). The activity of lecithinase which is a major component of the a toxin of C perfringens was found to range from 0'5 to 4,0 AE ml- I for the isolates studied and these are generally higher than for those of isolates from normal humans and animals (Kondo and Ogata 1975). It is not known whether outbreaks of necrotic enteritis associated with C perfringens result from strains found in the gut of normal birds or strains from the environment or both. . The MICs for the penicillins are in agreement with those of others (Traub 1971, Traub and Raymond 1971, Tally et al 1975) and ampicillin was among the more effective antimicrobials while the tetracyclines and the streptomycins were among the least effective. Of the peptides (frequently used as food additives) all had low MICs except for bacitracin to which the isolates showed high resistance as also demonstrated by Dutta et al (1980). Chloropolysporin-C, which is a relatively new glycopeptide, is not active against

340

F. Kondo

Gram-negative bacteria (Takatsu et al 1987) but shows excellent in vitro activity against C perfringens and is worthy of use in field trials. References BRYAN, L. E., KOWAND, S. K. & VAN DEN ELZEN, H. M. (1979) Antimicrobial Agents and Chemotherapy 15.7-13 DUTTA, D. G. & DEVRIESE, L. A. (1980) Journal 0/ Veterinary Pharmacology and Therapeutics 3. 227-236 EVANS, D. G. (1945) Journal 0/ Pathological Bacteriology 57, 75-85 HOLDEMAN, L. V. & MOORE, W. E. C. (eds) (1972) Anaerobe Laboratory Manual. 2nd edn. Blacksburg, Virginia Polytechnic Institute KONDO, F. & OGATA, M. (1975) Japanese Journal 0/ Bacteriology 30,477-485 LONG, J. R. (1973) Canadian Journal ofComparative Medicine 37. 302-308 NISHIDA, S., MURAKAMI," M. & YAMAGISHI, T. (1962) Japanese Journal 0/ Microbiology 6. 33-40

OAKLEY, C. L. (1955) Journal 0/ General Microbiology 12, 344-347 PARISH, W. E. (1961) Journal 0/ Comparative Pathology 71 377-393 • SAPICO, F. L., KWOK, U. Y., UTTER. V. L. & FINGOLD, S. M. (1972) Antimicrobial Agents and Chemotherapy 2. 320-325 TAKATSU, T., KAKAYAMA, T., NAKAJIMA, M., TAKAHASHI, S., HANEISHI, T., MAGARIBUCHI, T. & TAJIMA, M. (1987) Journal 0/ Antibiotics 40. 946-952 TALLY, F. P., JACOBUS, N. V., BARTLETT, J. G. & GORBACH, S. L. (1975) Antimicrobial Agents and Chemotherapy 7,128-132 TRAUB, W. H. (1971) Chemotherapy 16. 11-17 TRAUB, W. H. & RAYMOND, E. A. (1971) Chemotherapy 16. 162-165 WILLIS, A. T. & HOBBS, G. (1958) Journal 0/ Pathological Bacteriology 75. 299-305

Received October 13, 1987 Accepted December 18, 1987

..