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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1999, p. 3778–3779 0095-1137/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 37, No. 11

CASE REPORT Septicemia in Neutropenic Patients Infected with Clostridium tertium Resistant to Cefepime and Other Expanded-Spectrum Cephalosporins SOPHIA STEYAERT,1* RENAAT PELEMAN,2 MARIO VANEECHOUTTE,1 THIERRY DE BAERE,1 GEERT CLAEYS,1 AND GERDA VERSCHRAEGEN1 Department of Clinical Chemistry, Microbiology and Immunology1 and Department of Internal Medicine, Division of Infectious Diseases,2 University Hospital, 9000 Ghent, Belgium Received 8 March 1999/Returned for modification 25 May 1999/Accepted 9 August 1999

Clostridium tertium was isolated from two immunocompromised patients with septicemia, fever, and gastrointestinal symptoms. The strains were resistant to ceftazidime, cefepime, and clindamycin; intermediately resistant to penicillin; and susceptible to metronidazole, quinolones, and vancomycin. of 99.9% for the second. MICs (Table 1) were determined by using the E test (AB Biodisk, Solna, Sweden) on brucella blood agar supplemented with hemin and vitamin K1 (Becton Dickinson, Erembodegem, Belgium) under anaerobic conditions for 48 h (6, 12). Quality control was performed by the Stokes method with Bacteroides fragilis ATCC 25285. No ␤-lactamase production was detected. The identification as C. tertium was confirmed for both strains by means of amplified ribosomal DNA restriction analysis (11). The AluI pattern consisted of fragments of approximately 60, 110, 190, 210, 230, and 610 bp, and the CfoI pattern consisted of fragments of approximately 210, 410, and 840 bp. The patterns were completely identical for reference strain NIH 4522 and both clinical strains. Sequence determination of the amplified 16S rRNA gene (first 800 bp) of the first clinical strain revealed 98% identity with C. chauvoei (GenBank U51843) and 97% identity with C. septicum (GenBank U59278) as the closest matches. However, the database contained no C. tertium sequences. Therefore, the type strain of C. tertium (ATCC 14573) was sequenced (GenBank AJ245413, Thierry De Baere). Comparison of the first 800 bp of 16S rRNA of the first clinical strain (GenBank AJ132605, Mario Vaneechoutte) with that of the type strain revealed 100% sequence identity. C. tertium has been isolated from soil (1), the oral cavity (4), feces of healthy neonates and infants (1), appendices and feces of healthy adults (1), and 5 to 26% of fecal specimens of patients (8). This species has been isolated in relation to a brain abscess (1), meningitis (5), neutropenic enterocolitis (2), soft tissue infections (1), and war wounds (1). C. tertium is usually regarded as nonpathogenic, and no toxin production has been described (1). Clostridium species account for 0.5 to 2% of all clinically significant bacteria in blood cultures, with C. perfringens as the most common isolate (7). The significance of C. tertium in blood cultures is unclear, particularly in polymicrobial cultures (3). Our patients presented with fever and gastrointestinal symptoms with C. tertium as the only isolate. Therefore, we considered both isolates to be clinically significant. In previous reports, C. tertium septicemia in neutropenic patients has been associated with abdominal discomfort, diarrhea, colonic bleeding, or perianal cellulitis (8, 9). The intestinal mucosa is a rapidly proliferating epithelium which can be damaged by treatment with antineoplastic drugs. This mucosal damage is the most likely portal of entry for C. tertium. The

CASE REPORTS Case 1. A 65-year-old male was diagnosed with acute myeloid leukemia and treated with chemotherapy. During neutropenia, he became febrile and developed abdominal pain and watery diarrhea after the institution of ceftazidime and amikacin. Six sets of blood cultures revealed Clostridium tertium. Vancomycin was administered with good therapeutic response. Case 2. A 55-year-old male was treated for acute lymphoblastic leukemia with chemotherapy. The patient developed severe abdominal pain and watery diarrhea with chills and fever up to 40°C. Cefepime and amikacin were started with good clinical response. After a few days, the patient developed a new episode with fever in conjunction with bloody diarrhea. Blood culture revealed C. tertium. The antibiotherapy was switched to vancomycin with improvement of the patient’s condition. Blood was inoculated in BacT/Alert anaerobic and FAN aerobic bottles and incubated at 37°C in the BacT/Alert3D instrument (Organon Teknika, Turnhout, Belgium). Gram staining revealed gram-negative rods with a “squarish” morphology and intracellular gram-positive granules. Subculture on blood agar (Becton Dickinson, Erembodegem, Belgium) revealed small colonies after 24 h at 37°C in a 5% CO2-enriched atmosphere. More luxuriant growth of gray, translucent colonies with an irregular, nonspreading edge could be seen on the plates incubated anaerobically at 37°C in a jar by using GasPak Plus (Becton Dickinson). Terminal oval spores could be demonstrated in organisms incubated anaerobically. The catalase test was negative. Both strains fermented carbohydrates, but were nonproteolytic. No lipase or lecithinase activity was present. The first strain reduced nitrate, while the second did not. The Api20A (bio-Me´rieux, Marcy-l’Etoile, France) strip produced a profile number, 4 774 602 3, giving an identification as C. tertium with a probability of 98.9% for the first strain and a profile number, 4 774 612 3, with a probability * Corresponding author. Mailing address: Department of Clinical Chemistry, Microbiology and Immunology, University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Phone: 32 (0)9 240 34 22. Fax: 32 (0)9 240 49 85. E-mail: [email protected]. 3778

VOL. 37, 1999

CASE REPORT

TABLE 1. MICs of 11 antimicrobial agents tested for both C. tertium isolates Antimicrobial agent

Amoxicillin-clavulanate Cefoxitin Ceftazidime Cefepime Ciprofloxacin Clindamycinc Gentamicin Imipenem Metronidazole Penicillin Vancomycin

Isolate 1

Isolate 2

Result by NCCLS interpretive standarda

0.5 1 ⬎256 ⬎256 0.5 12 ⬎256 0.125 2 1 2

0.25 0.38 ⬎256 ⬎256 0.19 8 ⬎256 0.125 0.38 0.75 1.5

Susceptible Susceptible NAb NA NA Resistant NA Susceptible Susceptible Intermediate NA

MIC (␮g/ml) for:

a National Committee for Clinical Laboratory Standards (NCCLS) approved standard M11-A4. b NA, not available. c Tested on Fastidious Anaerobe blood agar supplemented with hemin and vitamin K (LAB M, Bury, Lancashire, United Kingdom) by D. Pierard, Academic Hospital, Free University of Brussels, Brussels, Belgium.

chance of fecal carriage of C. tertium in the setting of neutropenia may be related to selection by previously administered antimicrobial agents (2, 3, 8–10). This could explain the emergence of C. tertium as a pathogen in neutropenic patients, because this organism is resistant to standard empirical antibiotic regimens, such as ceftazidime (2a), and was shown here to be resistant to expanded-spectrum cephalosporins with enhanced activity against gram-positive organisms, such as cefepime. C. tertium is a gram-positive organism that is easily decolorized in gram-stained smears and can be mistaken for a gramnegative organism. However, C. tertium does not grow on selective media for gram-negative organisms. The aerotolerance of C. tertium can result in the misidentification of this organism as Bacillus spp. or Lactobacillus spp. A negative catalase test is an easy tool to differentiate C. tertium from Bacillus spp., which are catalase positive. C. tertium will only form spores under anaerobic conditions, in contrast to Bacillus spp., which will sporulate aerobically. Lactobacilli are catalase negative, but do not form spores and grow on a tomato juice agar. Other aerotolerant Clostridium species are C. carnis and C. histolyticum (Table 2). Although penicillin and clindamycin show excellent activity against most of the clostridial species, resistance can occur for C. tertium (1, 8–10). In general, metronidazole (3, 8–10), fluoroquinolones (8), and imipenem (8, 10) are active against C. tertium, while aminoglycosides (4), ceftazidime, and other expanded-spectrum ␤-lactam drugs are not (4, 8, 9). Although cefepime is an expanded-spectrum cephalosporin with enhanced activity against gram-positive organisms, both strains reported here were resistant to cefepime. Vancomycin appears to be efficacious (8, 9). In conclusion, C. tertium is not common and is not very virulent, but appears to be an emerging pathogen in neutropenic patients. The recognition of C. tertium is hampered by its

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TABLE 2. Differential characteristics of aerotolerant Clostridium species Characteristic

Lactose Mannitol Melibiose Gelatinase NO3 reduction Spores

Result fora: C. tertium

C. carnis

C. histolyticum

⫹ ⫹w ⫹w ⫺ ⫹/⫺ OT

d ⫺ ⫺ ⫺ ⫺ OS/T

⫺ ⫺ ⫺ ⫹ ⫺ OS

a Compiled from reference 1. ⫹, 90 to 100% of strains positive; ⫹/⫺, 61 to 89% of strains positive; d, 40 to 60% of strains positive; ⫺, 90 to 100% of strains negative; w, weak reaction; O, oval; T, terminal; S, subterminal.

aerotolerance and variable gram-staining properties, and this organism may be refractory to standard antimicrobial agents such as ceftazidime and cefepime. We thank Denis Pierard, Department of Microbiology, Academic Hospital, Free University of Brussels, Brussels, Belgium, for performing the clindamycin E test on both isolates. REFERENCES 1. Cato, E. P., W. L. George, and S. M. Finegold. 1986. Clostridium, p. 1141– 1200. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 2. The Williams and Wilkins Co., Baltimore, Md. 2. Coleman, N., G. Speirs, J. Khan, V. Broadbent, D. G. D. Wight, and R. E. Warren. 1993. Neutropenic enterocolitis associated with Clostridium tertium. J. Clin. Pathol. 46:180–183. 2a.EORTC International Antimicrobial Therapy Cooperation Group. 1987. Ceftazidime combined with a short or long course of amikacin for empirical therapy of Gram-negative bacteremia in cancer patients with granulocytopenia. N. Engl. J. Med. 317:1692–1698. 3. Gosbell, I. B., C. G. Johnson, P. J. Newton, and J. Jelfs. 1996. Clostridium tertium bacteremia: 2 cases and review. Pathology 28:70–73. 4. Johnson, J. R., and F. C. Tenover. 1988. Clostridium tertium bacteremia in a patient with aspiration pneumonia: an elusive diagnosis. J. Infect. Dis. 157: 854–855. 5. Kourtis, A. P., R. Weiner, K. Belson, and F. O. Richards. 1997. Clostridium tertium meningitis as the presenting sign of a meningocele in a twelve-yearold child. Pediatr. Infect. Dis. J. 16:527–529. 6. National Committee for Clinical Laboratory Standards. 1997. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard M11-A4. National Committee for Clinical Laboratory Standards, Villanova, Pa. 7. Onderdonk, A. B., and S. D. Allen. 1995. Clostridium, p. 574–586. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C. 8. Speirs, G., R. E. Warren, and A. Rampling. 1988. Clostridium tertium septicemia in patients with neutropenia. J. Infect. Dis. 158:1336–1340. 9. Thaler, M., V. Gill, and P. A. Pizzo. 1986. Emergence of Clostridium tertium as a pathogen in neutropenic patients. Am. J. Med. 81:596–600. 10. Valtonen, M., A. Sivonen, and E. Elonen. 1990. A cluster of seven cases of Clostridium tertium septicemia in neutropenic patients. Eur. J. Clin. Microbiol. Infect. Dis. 1:40–42. 11. Vaneechoutte, M., C. P. Cartwright, E. C. Williams, B. Ja ¨ger, H.-V. Tichy, T. De Baere, A. De Rouck, and G. Verschraegen. 1996. Evaluation of 16S rRNA gene restriction analysis for the identification of cultured organisms of clinically important Clostridium species. Anaerobe 2:249–256. 12. Wu ¨st, J., and U. Hardegger. 1992. Comparison of the E-test and a reference agar dilution method for susceptibility testing of anaerobic bacteria. Eur. J. Clin. Microbiol. Infect. Dis. 11:1169–1173.