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Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.
Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase [EC 3.2.1.18] and an intracellular acylneuraminate pyruvate lyase [EC 4.1.3.3]. The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature optimum of 50 degreesC and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2 mM Hg2+). The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Escherichia coli. The structural gene is represented by 2,319 bp encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids possess the character of a signal peptide. The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 bp downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases. For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.
INTRODUCTION: Clostridium tertium septicaemia, although usually described in neutropenic patients, can also occur in the absence of neutropenia, as in our case report. OBSERVATION: A 61 year-old woman presented with pain in the right hypochondrium and epigastrium, constipation and vomiting that led to the diagnosis of acute pancreatitis of biliary origin. During septic shock, analysis of the liquid of an intra-abdominal mass and the results of the blood cultures revealed a Gram positive bacillus identified as C. tertium. During laparotomy, necrotic-hemorrhagic acute pancreatitis lesions were found with subtotal colic necrosis. Analysis of the peritoneal fluid revealed C. tertium. The patient died on D 46 after several nosocomial pneumopathies without identification of C. tertium. DISCUSSION: Three factors appear related to the onset of C. tertium septicaemia: neutropenia, lesions of the intestinal mucosa and previous treatment with 3rd generation, broad spectrum, cephalosporins. Such septicaemias are rare in immunocompetent patients and do not appear to have been reported before in a context of acute pancreatitis.
Clostridium tertium is infrequently isolated from blood in patients with underlying diseases. Laboratory diagnosis is often delayed because Clostridium tertium is aerotolerant and resistant to metronidazole. Clinically it is a problem because it is commonly resistant to metronidazole, clindamycin and cephalosporins. We present 3 cases illustrating these characteristics.
A cluster of seven febrile and severely neutropenic patients who developed Clostridium tertium septicemia during a 13-month period is described. The patients had received third generation cephalosporins for 7 to 13 days (mean 9 days) at the time Clostridium tertium was isolated from blood cultures. Two patients had perirectal and one patient pericaecal cellulitis. The organism was also isolated from bronchial secretions in one patient. No patient had diarrhea. Five of six strains tested were resistant to clindamycin (MIC 2-8 micrograms/ml), and six of seven strains moderately resistant to penicillin (MIC 1-4 micrograms/ml). In one patient Clostridium tertium grew from blood cultures although metronidazole had been administered for two days. Six patients recovered on antibiotic therapy. In view of the unusual susceptibility pattern of Clostridium tertium, an accurate diagnosis of infection with this organism is important for the choice of an appropriate antimicrobial treatment.
Clostridium tertium septicemia is a rare condition that predominantly occurs in neutropenic patients with concomitant abdominal disease. We report the fatal case of a nonneutropenic, 51-year-old patient with mechanical ileus and post-operative C. tertium septicemia, resulting in widespread pathology with multi-organ failure. As C. tertium is aerotolerant, often gram-variable and mostly resistant to broad-spectrum cephalosporins, differentiation is difficult and empirical therapeutic strategies may fail.
Clostridium tertium bacteremia is unusual, seen most often with gastrointestinal disease and/or neutropenia. Two cases are described. The first was a 19-yr-old female with acute leukemia, who developed gastrointestinal symptoms and C. tertium bacteremia while neutropenic. The second was a 57-yr-old female with quiescent ulcerative colitis, who presented with fever, rigors and epigastric pain. Four organisms including C. tertium were isolated from blood cultures. This patient responded to broad spectrum antimicrobial therapy, whereas the first patient required the addition of specific agents to recover. C. tertium is aerotolerant and thus can be misidentified as a Bacillus or Corynebacterium spp. Our isolates had a distinctive Gram stain morphology, were catalase negative and failed to sporulate aerobically--this aided in the recognition of this significant Gram-positive bacillus.
Bacteriologic examination of an abscess found between blubber and musculature of a striped dolphin (Stenella coeruleoalba) found dead on 30 March 2002 in the Adriatic Sea, Croatia, was performed and an aerotolerant, nontoxogenic Clostridium tertium was isolated in pure culture. National Collections of Industrial Food and Marine Bacteria (NCIMB Ltd., Aberdeen, Scotland, UK) confirmed the results. Sequencing results showed it to be C. tertium with 100% similarity. The strain was named Clostridium tertium strain Zagreb, acceded to the culture collection and assigned the accession number NCIMB 13970. This is the first record of C. tertium in marine mammals.
In the nonneutropenic host, bacteremia due to Clostridium tertium is rare and of unclear significance. We describe a patient in whom presentation with Clostridium tertium bacteremia was the harbinger of Crohn's disease. In order to understand the significance of C. tertium bacteremia in neutropenic and nonneutropenic hosts, we review all 32 cases of C. tertium bacteremia that occurred at Duke University Medical Center from 1992 to 1999.
Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium. Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation. The effect was even more dramatic in C. tertium than in C. perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter. Unlike C. perfringens, C. tertium responded to the presence of nitrate in the medium with an increased growth rate. A slight increase in the Y ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of Stouthammer and Bettenhaussen that Y ATP will vary with the growth rate. Thus, C. tertium, like C. perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this may be widespread among the nitrate-reducing anaerobes.
Eighteen adult patients with hematologic malignancy developed bacteremia due to Clostridium tertium while neutropenic. Fifteen had accompanying abdominal pain, colonic bleeding, or diarrhea, and three had perianal cellulitis. Fourteen recovered with antibiotic therapy alone; no patient was treated by surgery. C. tertium is an unusual Clostridium because it is resistant to many beta-lactam antibiotics and to metronidazole but is susceptible to vancomycin, trimethoprim-sulfamethoxazole, and ciprofloxacin. It is possible that use of third-generation cephalosporins (cefotaxime, ceftizoxime, ceftazidime) for treating febrile episodes in the absence of any selective intestinal decontamination with trimethoprim-sulfamethoxazole or ciprofloxacin may have resulted in selection for C. tertium in our patients.
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.
A method was described for the partial purification of beta galactosidase and beta glucosaminidase from Clostridium tertium culture supernatants. Treatment of erythrocytes with preparations containing both enzymes decreases their ability to react with anti-I cold agglutinins, and with Type XIV antipneumococcal horse serum. Erythrocytes of blood group A(1) are altered more rapidly and extensively than are group O cells. The enzymatic treatment of stroma results in a decrease in ability to absorb anti-I agglutinins and the release of galactose and N-acetylglucosamine as monosaccharides. The data suggest that these two sugars may be structural units of the erythrocyte I determinant, but no direct evidence is available.
Although usually considered a non-pathogen, Clostridium tertium was isolated from 10 immunosuppressed patients including seven patients with bacteremia. This organism can grow aerobically and can be easily disregarded as a contaminant. It also has a somewhat unusual susceptibility pattern, with significant resistance to the penicillins, cephalosporins, and clindamycin, possibly explaining its emergence in immunocompromised patients already receiving multiple antibiotics.
A procedure was developed for the formation and regeneration of protoplasts in Clostridium tertium. Although in the parent strain most of the protoplasts gave rise to L-form colonies, derivatives could be isolated which exhibited high regeneration frequencies.
Of 10 strains of mannanase-producing anaerobic bacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A, which was isolated from lotus soil, produced high amounts of extracellular beta-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growth cultivated with no addition of reducing agents was also stable. High yields of mannanase were obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation end products on galactomannan fermentation were formate, acetate, lactate, butyrate, carbon dioxide and hydrogen. The extracellular mannanase displayed high activity on galactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spino gum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an active mannanase-producing anaerobic bacterium from natural environments.
A 15 year old boy being treated for relapsed acute lymphoblastic leukaemia developed severe diarrhoea and abdominal pain which worsened despite empirical antibiotic treatment. A right hemicolectomy was performed. The caecum and ascending colon showed changes typical of neutropenic enterocolitis. Clostridium tertium was isolated from faeces, blood cultures, and from the resected gut wall, with no evidence of other organisms capable of causing such a condition. As far as is known, this is the first reported case in which neutropenic enterocolitis has been associated with well documented C tertium infection, an organism previously described as a cause of bacteraemia in neutropenic patients.
A young girl developed an intracranial abscess and necrotizing cellulitis following penetrating injury from a lawn dart. Initial identification of a gram-positive rod growing aerobically from clinical specimens was as a Bacillus organism, but the observation that the isolate grew poorly in subcultures for susceptibility testing but quite well under standard anaerobic culture techniques led to the identification of the organism as an aerotolerant Clostridium tertium. Early management of penetrating head trauma should include cranial imaging studies to detect fractures and intracranial pathology. Clinical microbiologists and clinicians should be aware of the phenomenon of aerotolerance in anaerobic bacteria to avoid errors in choice of antibiotic therapy.