Antibacterial Susceptibility of Clostridium sordellii Strains

Zbl. Bakt. Hyg. A 261, 345-349 (1986)

Antibacterial Susceptibility of Clostridium sordellii Strains SHINICHI NAKAMURA, KIYOTAKA YAMAKAWA, and SHOKI NISHIDA Department of Bacteriology, School of Medicine, Kanazawa University, Kanazawa 920, Japan Received September 9, 1985 . Accepted November 4, 1985

Summary A total of 24 Clostridium sordellii strains was tested for susceptibility to 24 antibacterial agents. All strains exhibited a high degree of resistance to streptom ycin, kanamycin, sulfamethoxazole , and trimethoprim. Zusammenfassung 24 Clostridium sordel/ii-Stiimme wurden auf ihre Empfindlichkeit gegen 24 amibakterielIe Verbindungen untersucht. Aile Stamrne zeigten hohe Resistenz gegen Streptomycin, Kanamycin, Sulfamethoxazole , und Trimethoprim.

Introduction Toxigenic Clostridium difficile has been found to be the etiologic agent of most cases of antibiotic-associated pseudomembranous colitis and of some cases of antibioticassociated diarrhea (2). Two toxins, enterotoxin (toxin A) and a cytotoxin (to xin B), have been described as the potential virulence factors of C. difficile (15). Although the detailed relationship between the two toxins of C. difficile and the antitoxin serum of C. sordellii has not been elucidated yet, it is known that cytotoxicity in culture filtrates of C. difficile is neutralized by C. sordellii antitoxin serum (3, 12). In some cases of antibiotic-associated diarrhea, the cytotoxicity neutralizable with C. sordellii antitoxin serum was detectable but C. difficile was not recovered from fecal samples (1, 6, 16,

17).

Recently it was found that culture filtrates of lethal toxin-positive C. sordellii strains were cytotoxic at dilutions between 10- 2 and 10- 4 against baby hamster kidneys cells, and the cytotoxicity could be neutralized by C. difficile antitoxin serum (11 ). These findings imply a possibility that C. sordellii could play an etiological role in the diarrhea without an association between the presence of cytotoxicity and the recovery of C. difficile (8). Development of a selective medium for C. sordellii would presum-

346

S. Nakamura, K. Yamakaw a, and S. Nishida

abl y be useful to in vestigate the etio lo gic role of C. sordel/ii a nd th e d istribution of C. sordel/ii in normal intestinal flora which is little known (14). In th e present study we examined the antibacterial suscep ti bility of C. sordellii strains , in order to find a nti bacteria l agents effective for th e select ive isolation o f C. sordel/ii. Materials and Methods

Strains. A total of 24 C. sordellii strains, 19 cyto to xigenic and 5 non -cytot oxigenic, were tested. Twel ve strains were obtained from L. DS. Smith, Montan a State Universit y, Montana, U.S.A.; 7 strains from I. Batty, Wellcome Research Laboratories, Beckenham , England; 4 strai ns from C. T. Huang, Hong Kong University, Hong Kong; and one strain from A . R. Preuot, Pasteur Institute, Par is, France. All str ains ha ve been sto cked in our lab oratory over 10 years. Antibacterial agents. A total of 24 anti bacterial agents was tested. The y were as follows: penicillin G (M eiji Seika, Tokyo, j ap an ); ampicillin (Meiji Seika ); carbenicillin (Pfizer Taito, Tokyo ); cefoxitin (Merck Sharpe and Dohme Research Laboratori es, West Point, U.S.A.); cephalexin (Tor ii, Tokyo); ceph aloridine (Japan Glaxo, Tokyo); cephalothin (Torii); streptomycin (M eiji Seika); kanamycin (Meiji Seika ); neomycin (Takeda , O sak a, japan); gentami cin (Shionogi, Osaka); linc omy cin (Japa n Upjohn, Tokyo ); clind amycin (Japan Upjoh n ); er ythromycin (Abott Laboratorie s, N orth Chicago, U.S.A.); tetracycline (Japan Lederle, Tok yo); chloramphenico l (Sankyo, Tokyo ); vanco mycin (Eli Lilly, Indi an apolis, U.S.A.); metronidazole (Shionogi), cyclose rine (Sigma, St. Lou is, U.S.A); rifampicin (Japan Ciba Geigy, Tokyo); fusidic acid (Sankyo); nalid ix ic aci d (Sigma ); sulfamethoxazo le (Shio nogi); and trimethoprim (Shio no gi). All drugs except tetrac ycline, rifa mpici n, fusidic acid , chloramphenicol, nalid ixic acid, sulfamethox azole, and tr imeth oprim were dissol ved in distilled water, steril ized by membrane filtra tio n, and diluted in sterile d istilled water to th e desired final concentration. Tetracycline, rifampicin, fusid ic acid, and chlor amphenicol wer e dissol ved in ethanol ; nal idixic acid in 0.1 N N aO H, and sulfame thoxazo le and trimethoprim in distilled water, by th e respe ctive addition of 10 % NaO H and 0.05 N HC!. Determination of minimum inhibit ory concentration (MIC). Th e aga r dilut ion method of the j ap an ese Association for Infectiou s Diseases (9) was used. GAM -broth and -agar (N issui, T ok yo) were used; GAM-b roth consists o f peptone 23 g, digested ser um powder 13.5 g, yeast extract 5 g, meat ext ra ct 2.2 g, liver extrac t 1.2 g, glucose 3 g, KH 2P 0 4 2.5 g, NaCl 3 g, so luble sta rch 5 g, L-cysteine . H Cl 0.3 g, sod ium thio glycolate 0.3 g, and distill ed water 1,000 ml (pH 7.2). Two ml o f the serial twofold diluti on of each drug we re added to 18 ml of melted GAMagar. The agar was mixed , poured into 9 cm Petri dishe s and allow ed to set. C. sordellii strai ns were grown anaerobically in GAM-broth overnight under hydr ogen . The bacterial suspension for inoculation wa s prepared by diluting the broth cultu re with 0.05% yeast extra ct solut ion to give a concentrat ion of about 109 colony forming units/ml, A loopful (1 mm in diameter) of the bacterial suspension was streaked 2 cm in length on the plates containing drugs. The plates inocul ated were incubated anaerobically fo r 24 h at 3rc under hydrogen. The MIC was defined as the lowest concentration of the drug inhibiting growth completely as judged by th e nak ed eye.

Results The MICs of 24 antibacterial agents for 24 strai ns of C. sordel/ii a re presented in Table 1. Erythromycin, penicill in G , rifampicin , tetra cycl ine , a m pici llin, clinda my cin, fu sidi c ac id, cephaloridine, cefox itin , cephalothin, metron id a zol e, a nd ca r beni cillin

Eryth rom ycin, penicillin G, rifampicin, tetracycline, ampicillin, clindamycin, fusidic acid, ceph aloridine, cefoxitin, cepha lothin M et ron idazole Ca rbenicillin Vancom ycin Lincom ycin Chlora mphenicol Cepha lexin Genta micin Neomy cin Nalid ixic acid Cyclose rine Strepto myc in Kan amy cin Sulfamethox azo le Trimethoprim

Ant iba cterial agent

24 21 14 1 2

3

10 4 1

3

< 0.78 0,78

Table 1. Antibacterial susceptibility of C. sorde/lii stra ins

18 2 16

1.56

1 13 2

3.13

6 3 24

6.25

I

12.5

2

25

14 1 4 1

50

7 15 12 1 1 1

100

Number of strains showing MIC (ug/ml ) of

1 22 14 1 1 1 1

400

8 7

200

8 22 24

22

> 400

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348

S.Nakamura, K. Yamakawa, and S.Nishida

were the most active antibacterial agents; they inhibited all strains at a concentration of 0.78 ug/ml, Vancomycin, lincomycin, chloramphenicol, and cephalexin were only slightly less active and inhibitory at a concentration of 6.25 ug/rnl. Moderately high MICs to gentamicin, neomycin, and nalidixic acid were observed; MICs ranged between 50 and 400 ug/rnl except for 3 strains in gentamicin. All strains were inhibited at concentrations of 100 ug/rnl or more in cycloserine, streptomycin, kanamycin, sulfamethoxazole, and trimethoprim except for one strain in cycloserine. Discussion In the present study we used GAM-agar as the test medium. GAM-agar as well as Brucella base blood agar or brain heart infusion base blood agar were recommended as test media for the determination of MIC of antibacterial agents for anaerobic bacteria by the Japanese Association for Infectious Diseases (9). We had previously tested the antibacterial susceptibility of C. difficile by using GAM-agar (10). Results obtained had resembled those described by other investigators (4, 5, 13), in which brain heart infusion broth, Brucella base blood agar, or lysed blood DTS agar were used .. CCFA (7), which contains cefoxitin and cycloserine at concentrations of 16 and 500 ug/ml, respectively, was developed for the isolation of C. difficile on the basis of resistance of this organism to these drugs, and has been widely used for the isolation of C. difficile (1, 6, 17). The MIC of cefoxitin for C. sordellii strains tested in this study was less than 0.78 ug/ml, i.e. distinctively lower than that for C. difficile (32 ug/ml or more) (4,5,10,13). Therefore, it is supposed that C. sordellii in fecal specimens could not grow on CCFA. In C. sordellii, the high level of resistance to gentamicin, neomycin, nalidixic acid, streptomycin, kanamycin, sulfamethoxazole, and trimethoprim as found in this study may be useful for the development of a selective medium for isolation of the organism from a mixed microbial population, although this requires further investigations on the composition of the medium. Acknowledgment. We wish to thank Miss J. Izumi for her technical assistance and Dr. K. Baczko, Insitute of Virology and Immunology, University of Wiirzburg, FRG, for his help in

the preparation of the manuscript. References

1. Aronson, B., R. Mollby, and C.-E. Nord: Antimicrobial agents and Clostridium difficile in acute enteric disease: epidemiological data from Sweden, 1980-1982. J. infect. Dis. 151 (1985) 476-481 2. Bartlett, J. G., T. W. Chang, N. S. Taylor, and A. B. Onderdonk: Colitis induced by Clostridium difficile. Rev. infect. Dis. 1 (1979) 370-373 3. Chang, T. W., S. 1. Gorbach, and J. G. Bartlett: Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxin. Infect. Immun. 22 (1978) 418-422 4. Fekety, R., J. Silva, R. Toshniwal, M. AlIo, J. Armstrong, R. Browne, J. Ebright, and G. Rifkin: Antibiotic-associated colitis: effects of antibiotics on Clostridium difficile and the disease in hamsters. Rev. infect. Dis. 1 (1979) 386- 397 5. George, W. 1., B. D. Kibby, V. 1. Sutter, and S. M. Finegold: Antimicrobial susceptibility of Clostridium difficile. In: D. Schlessinger (ed.), Microbiology 1979, pp. 267-271. American Society for Microbiology, Washington D.C. (1979)

Antibacterial Susceptibility of C. sordellii

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6. George, W. L., R. Rolfe, and S. M. Finegold: Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions. J. Clin. Microbiol. 15 (1982) 1049-1053 7. George, W. L., V. L. Sutter, D. Citron, and S. M. Finegold: Selective and differential medium for isolation of Clostridium difficile. J. Clin. Microbiol. 9 (1979) 214-219 8. Kobayashi, T., H. Kono, M. Aoki, and K. Ueno: Diagnosis of antimicrobial associated diarrhea by latex immuno reagent. Proc. 13 Ann. Meet. japn, Assoc. Anaerobic. Res. 13 (1983) 83-88 (in Japanese) 9. Kozakai, N., K. Ueno, S. Goto, S. Mitsuhashi, S. Nakayama, K. Shimada, K. Tamai, and T. Koguri: Method for examination of minimum inhibitory concentration of anaerobes. Chemotherapy 27 (1979) 559-560 (in Japanese) 10. Nakamura, S., S. Nakashio, M. Mikawa, K. Yamakawa, S. Okumura, and S. Nishida: Antimicrobial susceptibility of Clostridium difficile from different sources. Microbiol. Immunol. 26 (1982) 25-30 11. Nakamura, S., N. Tanabe, K. Yamakawa, and S. Nishida: Cytotoxin production by Clostridium sordellii strains. Microbiol. Immunol. 27 (1983) 495-502 12. Rifkin, G. D., R. Fekety, and]. Silva [r.: Neutralization by Clostridium sordellii antitoxin of toxins implicated in clindamycin-induced cecitis in the hamsrer. Gastroenterology 75 (1978) 422-424 13. Shuttleworth, R., M. Taylor, and D. Jones: Antimicrobial suscepribilities of Clostridium difficile. J. Clin. Path. 33 (1980) 1002-1005 14. Smith, L. DS.: The pathogenic anaerobic bacteria (2nd ed.). Charles C Thomas, Springfield/Illinois (1975) 15. Taylor, N. S., G. Thorne, and]. G. Bartlett: Comparison of two toxins produced by Clostridium difficile. Infect. Immun. 34 (1981) 1036-1043 16. Willey, S. H. and]. G. Bartlett: Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin. J. Clin. Microbiol. 10 (1979) 880-884 17. Wu, T. C. and]. C. Fung: Evaluation of the usefulness of counterirnmunoelectrophoresis for diagnosis of Clostridium difficile-associated colitis in clinical specimens. J. Clin. Microbiol. 17 (1983) 610-613 Dr. Shinichi Nakamura, Dept. of Bacteriology, School of Medicine, Kanazawa University, Kanazawa 920, Japan