JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1983, p. 491-494 Vol. 18, No. 3 0095-1137/83/090491-04$02.00/O Copyright © 1983, American Society for Microbiology
Isolation of Clostridium pseudotetanicum from a Patient with Gas Gangrene NAOKI KATOH,l* KUNITOMO WATANABE,' YOSHIYUKI MIKI,2 TOSHIYUKI MURO,3 AND KAZUE UENO' Institute ofAnaerobic Bacteriology, Gifu University School of Medicine,1 and Clinical Laboratory2 and Department of Orthopedic Surgery,3 Gifu Prefectural Tajimi Hospital, Gifu, Japan Received 22 February 1983/Accepted 14 June 1983 Clostridium pseudotetanicum was isolated along with Serratia marcescens, Proteus mirabilis, and Staphylococcus epidermidis from a patient suffering from gas gangrene who had been injured in the right leg by a power cultivator. Experimental infection of the hind leg of mice with C. pseudotetanicum and the three kinds of aerobic bacteria did not produce any different macroscopic finding in the infection site, compared with aerobic bacterial injection, except for some enlargement of the involved tissue and some slightly altered histolytic findings. Ampicillin, rifampin, and tinidazole were the most active antimicrobial agents against C. pseudotetanicum.
Clostridium pseudotetanicum is an anaerobic the leg revealed accumulation of gas. The diagnosis of bacterium rarely isolated from clinical material. gas gangrene was made. In addition to cephalexin and The etiological role of C. pseudotetanicum in ciclacillin, gentamicin was given intramuscularly in a human infectious disease is still unclear. dose of 40 mg/day for 3 days. The gas gangrene progressed despite these treatments, leading to ampu- We document a case of gas gangrene associat- tation at the proximal end of the femur on hospital day ed with C. pseudotetanicum. In addition, the 8. At surgery, necrosis of subcutaneous tissue of upper pathogenicity of this organism in mice was ex- leg was apparent, and the lower leg showed complete amined, and its susceptibility to various antimi- necrosis with a foul-smelling discharge and copious crobial agents is reported. gas. After surgery, the patient gradually became afe- brile and healed. CASE REPORT A 48-year-old woman was admitted to the Gifu Prefectural Tajimi Hospital, Gifu, Japan, on 29 June MATERIALS AND METHODS 1981 because of injury to the right lower leg by a On hospital day 6, the discharge from the right leg power cultivator 4 days before admission. She was was cultured anaerobically and aerobically. Prere- seen immediately by a private physician who per- duced Gifu anaerobic agar medium (GAM agar; Nissui formed surgical debridement. Seiyaku Ltd., Tokyo, Japan) (11) and hemolysed Physical examination showed an open fracture of sheep blood agar were used for anaerobic culture, the proximal part of the tibia and a contused wound of which was done in an anaerobic glove chamber. To the right leg. On the day after admission, the tempera- detect alcohols and volatile and nonvolatile fatty acids ture was 38.4°C, and the pulse was 114. The erythro- produced by the anaerobic bacterium, a gas chromato- cyte count was 3,220,000. The hematocrit was 28.5%. graphic analysis was performed with a Shimazu GA-7 The leukocyte count was 10,500 with 85% neutrophils, analyzer (Shimazu Co., Ltd., Kyoto, Japan). Identifi- 8% lymphocytes, and 7% monocytes. Arterial blood cation of the anaerobe was performed according to the gas analysis showed a pH of 7.46, PCO2 of 33 mm Hg, Anaerobe Laboratory Manual of the Virginia Poly- P02 of 90 mm Hg, and HCO3 of 24 mmollliter. The technic Institute (7). Sheep blood agar, MacConkey urea nitrogen was 17.8 mg/dl, the uric acid was 1.5 medium (Nissui Seiyaku Ltd.), and heart infusion agar mg/dl, the total bilirubin was 1.1 mg/dl, alkaline phos- (Eiken Chemical Co., Ltd., Tokyo, Japan) were uti- phate was 6.8 KAU, serum glutamic oxaloacetic trans- lized for aerobic culture. Aerobic isolates were identi- aminase was 97 IU, serum glutamic pyruvic transami- fied by conventional procedures (3). nase was 46 IU, and lactic dehydrogenase was 620 IU. The infectivity of the anaerobic bacterium was ex- C-reactive protein was 3+, and the anti-streptolysin 0 amined in mice. The organism was cultured anaerobi- titer was 12. Arterial blood culture yielded no orga- cally in chopped meat medium at 37°C for 1 day. nisms. Medication with cephalexin and ciclacillin was Aerobes isolated (Serratia marcescens, Proteus mira- started at 6 and 1.5 g/day, respectively. On hospital bilis, and Staphylococcus epidermidis) were also cul- day 3, edema of the right leg was noted, followed by tured in GAM broth medium (Nissui Seiyaku Ltd.) at cyanosis of the distal portion of the right foot. On 37°C for 1 day. A 0.02-ml amount of each of the three hospital day 5, a discharge with foul odor appeared in inocula, i.e., the anaerobe alone, the mixture of the the wound of the right lower leg. A roentgenogram of anaerobe and the aerobes, and the mixture of the
491 492 KATOH ET AL. J. CLIN. MICROBIOL. aerobes only, was injected intramuscularly into the benicillin and doxycycline (Taito-Pfizer Co., Ltd., hind leg of each of five mice after mixing with an equal Tokyo, Japan), cephalothin and moxalactam (Shionogi volume of 5% CaCl2. The inocula contained per 0.05 & Co., Ltd., Osaka, Japan), cefazolin (Fujisawa Phar- ml: the anaerobe at 5 x 105, S. marcescens at 6 x 103, maceutical Co., Ltd., Osaka, Japan), cefoxitin (Nip- P. mirabilis at 5 x 102, and S. epidermidis at 7 x 102. pon Merck-Banyu Co., Ltd., Tokyo, Japan), clinda- Animals were examined for swelling and necrosis of mycin (Japan Upjohn Ltd., Tokyo, Japan), the hind leg after 1 week. The toxicity of the anaerobe chloramphenicol (Sankyo Co., Ltd., Tokyo, Japan), was tested by injecting 0.5 ml of a filtrate of a 2-day vancomycin (Eli Lilly International Co., Kobe, Ja- culture of the anaerobe in chopped meat medium pan), rifampin (Japan Ciba-Geigy Co., Tokyo, Japan), intraperitoneally into five mice. Mice were observed and tinidazole (Eizai Co., Ltd., Tokyo, Japan). Disk daily for 8 days. susceptibilities of aerobes to ampicillin, sulbenicillin, Minimal inhibitory concentrations of antimicrobial cefazolin, and gentamicin were performed by conven- agents were examined by an agar dilution method (8) tional methods (3). against the Clostridium sp. isolated and 11 other species of clostridia (C. bifermentans GAI 0010, C. chauvoei GAI 0157, C. fallax GAI 2312, C. limosum RESULTS GAI 0175, C. novyi GAI 0121, C. perfringens GAI 0668, C. septicum GAI 0033, C. sordellii GAI 0155, C. An anaerobic, spore-forming, gram-positive sporogenes GAI 0057, C. subterminale GAI 0037, and rod was isolated from the patient's right lower C. tetani GAI 0019). The antimicrobial agents used leg in addition to S. marcescens, P. mirabilis, were ampicillin (Meiji Seika Co., Tokyo, Japan), car- and S. epidermidis. This spore-forming anaer- obe had the morphological, biochemical, and chromatographic characteristics shown in Table 1. In GAM broth without glucose the anaerobe TABLE 1. Biochemical characteristics of C. grew poorly in contrast to growth in GAM broth pseudotetanicum isolated from a patient with gas with glucose. The anaerobe was identified as C. gangrene pseudotetanicium. Characteristic Presence The virulence of this C. pseudotetanicum iso- Arabinose ...... late was examined in mice. Mice inoculated with Cellobiose ...... C. pseudotetanicum alone showed few patholog- Erythritol ...... + ical changes at the site of injection after 7 days. Esculin pH ...... An abscess measuring 20 to 30 mm in diameter Esculin hydrolysis ...... was seen at the injection site of mice treated Fructose ...... + with the three aerobic bacteria. The abscesses in Glucose ...... mice with Glycogen ...... + injected the four bacteria isolated Inositol ...... were slightly larger than those of mice injected Lactose ...... with the aerobic bacteria only and showed a Maltose ...... slight lysis of muscle. Mannitol...... S. marcescens was susceptible to sulbenicillin Mannose ...... and gentamicin, but not to ampicillin or cefazo- Melezitose ...... lin by the diffusion method. P. mirabilis and S. Melibiose ...... epidermidis were susceptible to ampicillin, sul- Raffinose ...... + benicillin, cefazolin, and gentamicin. The sus- Rhamnose ...... ceptibilities of C. pseudotetanicum and other Salicin ...... Sorbitol ...... clostridia are shown in Table 2. C. pseudotetani- +AL cum was inhibited by 3.13 of Starch pH ...... +b pg cefoxitin and Starch hydrolysis...... moxalactam per ml. Ampicillin, rifampin, and Sucrose ...... tinidazole were the most active (minimal inhibi- Trehalose ...... tory concentration, 0.2 ,ug/ml) against C. pselu- Xylose ...... dotetanicum as well as other clostridia. C. pseu- Ribose ...... dotetanicum was relatively resistant to the Gelatin ...... Milk ...... cephems (cephalothin, cefazolin, cefoxitin, and Indole ...... moxalactam) as compared to other agents. Lecithinase ...... Lipase...... DISCUSSION Acetoin...... C. perfringens is the most frequently isolated GLC profile...... organism from gas gangrene, whereas C. septi- Spore location...... cum and C. novyi are the second in frequency. A Mouse toxicity ...... few other clostridia (C. bifermentans, C. histoly- a GLC, Gas-liquid chromatography; BAL, butyrate, ticum, and C. fallax) have also been isolated (5). acetate, and lactate. In many cases of gas gangrene, one or more b T, Terminal spore. other bacteria are isolated concomitantly with VOL. 18, 1983 C. PSEUDOTETANICUM IN GAS GANGRENE 493 the clostridia (1). C. pseudotetanicum was re- ported to be isolated from one soft tissue infec- -oHO tion and, along with Clostridium ramosum, from a septicemia in a patient with leukemia (6). C. 2. pseudotetanicum bacteremia in a patient with 0. 0 Pasteurella multocida conjunctivitis has also . 010 o . > C) P0 been reported (4). However, there are no reports C)N >.)N0 ~ a o2. of C. pseudotetanicum isolation from gas gan- (Q.. grene. In the present case, C. pseudotetanicum x... 0. was the only anaerobic organism isolated ac- °-j* 00° companied by three aerobic bacteria. We cannot H rule out completely that other clostridia were P1er not present, but no others were Oo r recovered by 0 IA IA IA IA IA IA IA IA IA IA IA > our approach. The patient had a progressive 0 infection; the infected area and free gas in the 0.9 00 n Cs 0) tissues of the right leg increased daily, until a amputation was required. . IA IA cn C. pseudotetanicum displays no particular in- = = = = o oo -X 0. jurious exoenzymes. It is postulated that the lZ 'IC00 00 0\00o'.:h as00 _. CD'F absence of acylneuraminate lyase, present in the la 0 clostridia C. beijerinckii, C. chauvoei, C. per- . IA IA IA IA IA IA IA - N fringens, C. septicum, C. sordellii, and C. terti- r) oS5 Sj w. ili i1i S- >j um, might contribute to the lack of tissue dam- 00000000~,A0'.'ii- i1i i1i 3-~ age by C. pseudotetanicum (4, 12). The 2, .r1 _. experimental infections with C. pseudotetani- '<'71 IA IA IA IA IA IA IA 0 cum of the hind leg of mice suggest that C. zet pseudotetanicum has very limited capability to 0.4 S C) cm initiate myonecrosis in the murine model either S by itself or in the presence of aerobes. (0 IA IA IA IA IA IA IA o o ) The C. pseudotetanicum isolate was most 0.- I C .'01iiii i j i - i - C X o susceptible to ampicillin, tinidazole, and rifam- '<0 CD pin, although certain clostridia may be resistant '
5. Finegold, S. M. 1977. Anaerobic bacteria in human dis- 9. Kesado, T., K. Watanabe, Y. Asahi, M. Isono, and K. ease. Academic Press, Inc., New York. Ueno. 1982. Susceptibilities of anaerobic bacteria to n- 6. Gorbach, S. L., and H. Thadepalli. 1975. Isolation of formimidoyl thienamycin (MK 0787) and to other antibiot- Clostridium in human infections: evaluation of 114 cases. ics. Antimicrob. Agents Chemother. 21:1016-1022. J. Infect. Dis. 131:S81-S85. 10. Rolfe, R. D., and D. M. Finegold. 1982. Comparative in 7. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). vitro activity of ceftriaxone against anaerobic bacteria. 1977. Anaerobe laboratory manual, 4th ed. Virginia Poly- Antimicrob. Agents Chemother. 22:338-341. technic Institute and State University, Blacksburg, Va. 11. Sutter, V. L., D. M. Citron, and S. M. Finegold (ed.). 8. Isono, M., K. Kawashima, N. Yamada, M. Aoki, T. 1980. Wadsworth anaerobic bacteriology manual. The Kobayashi, K. Sawa, K. Watanabe, and K. Ueno. 1982. In C. V. Mosby Co., St. Louis, Mo. vitro and vivo antimicrobial activities of cefmetazole 12. Werner, H., and H. E. Mueller. 1974. Demonstration of against Bacteroides fragilis. Jpn. J. Antibiot. 35:1667- neuraminidase in Clostridium pseudotetanicum. Pathol. 1672. Microbiol. 41:341-347.