The clostridia are the endospore-forming, obligately anaerobic (or aerotolerant), catalase-negative, gram-positive bacilli (Figure 1). The rods are pleomorphic and may be arranged in pairs or short chains. If spores are not present on Gram stain, the ethanol shock spore or heat shock spore test can separate this group from the non–spore-forming anaerobic bacilli on the Evolve site). Some strains of C. perfringens, C. ramosum, and C. clostridioforme may not produce spores or survive a spore test, so it is important to recognize these organisms using other characteristics. Some clostridia typically stain gram negative, although they are susceptible to vancomycin on the disk test. Several species of clostridia grow aerobically (C. tertium, C. carnis, C. histolyticum, and occasional strains of C. perfringens), but they produce spores only under anaerobic conditions. C. perfringens may appear weakly catalase positive.

Fig1. Gram stain of Clostridium perfringens. 

Clostridium species are widespread in nature because of their ability to form spores, referred to as endospores, in the mother cell (Table 1). In addition, they are present in large numbers as normal flora in the gastro intestinal tract of humans and animals, the female genital tract, and the oral mucosa.

Table1. Characteristics of Clinically Significant Clostridium species

C. botulinum is listed by the Centers for Disease Control and Prevention (CDC) as a potential agent of bioterror ism. A diagnosis of botulism is made by the demonstration of botulinum neurotoxin in serum, feces, gastric contents, vomitus, or suspect food (food poisoning) or environmental specimen (potential bioterrorism incident). This means that most hospital laboratories must know how to package and ship such a specimen to the State Health Department or CDC. Isolation of C. botulinum is rarely seen in the clinical microbiology laboratory.

Laboratory Diagnosis and Specimen Collection

 As stated in Chapter 41, the proper collection and trans port of specimens for anaerobic culture cannot be over emphasized. General considerations are included in the discussion in Chapter 41. However, special collection instructions must be followed for some clostridial illnesses, specifically foodborne C. perfringens and C. botulinum, C. difficile pseudomembranous enterocolitis, and C. septicum neutropenic enterocolitis (NEC). Food and freshly passed fecal specimens must be sent to a public health laboratory for confirmation of C. perfringens food poisoning; these should be transported at 4° C. The specimens should be processed within 24 hours of collection. The clinical diagnosis of botulism is confirmed by demonstration of botulinum toxin in serum, feces, vomitus, or gastric contents, as well as by recovery of the organism from the stool of patients (Figure 2). Several methods are available, including cell culture assays, enzyme-linked immunosorbent assay (ELISA), and latex agglutination.

Fig2. Clostridium difficile on cycloserine cefoxitin fructose  agar (CCFA). (Courtesy Anaerobe Systems, Morgan Hill, Calif.)

C. perfringens–associated enteritis necroticans infection requires the collection of three blood cultures, stool and bowel contents, or bowel tissue. Specimens should be Gram-stained and cultured. Follow-up tests to identify the organism are determined by the interpretation of the initial Gram stain. The isolate should be serologically typed. In addition, polymerase chain reaction (PCR) testing is available for C. perfringens.

Suspected C. difficile infection (CDI) indicates collection of a freshly passed stool specimen for culture and toxin assays for both toxin A and toxin B. Only liquid or unformed stools should be processed for CDI to prevent the treatment of patients colonized with the bacterium. Formed stools or rectal swabs are adequate to detect carriers. Specimens should be cultured within 2 hours after collection. Figure 3 demonstrates the isolation of C. difficile on cycloserine cefoxitin fructose agar (CCFA) and on anaerobic blood agar. Specimens may be stored in anaerobic transport bags at 4° C for up to 48 hours; however, this reduces the recovery rate of viable organ isms in culture. Specimens for toxin assays may be stored at 4° for 72 hours or frozen at –70° C if a longer delay is expected.

Fig3. Clostridium perfringens on anaerobic blood agar. Note  double zone of beta-hemolysis. 1, First zone; 2, second zone.  (Courtesy Anaerobe Systems, Morgan Hill, Calif.)

A variety of immunoassays are commercially available for the identification of C. difficile enterotoxin. In addition, a variety of molecular-based assays have been developed for the amplification of the toxin A (tcdA) and toxin B (tcdB) genes. Stool samples may be submitted for PCR amplification. The assays include amplification of the glutamate dehydrogenase (GDH) gene or 16srRNA as internal control housekeeping genes. Detection of the GDH and 16s rRNA genes without the presence of a toxin gene would indicate a nonpathogenic strain or carrier state. A new molecular assay is currently available: Illumigene C. difficile, (Meridian Bioscience, Inc., Memphis, TN), using LAMP isothermal amplification. See Chapter 8 for information on LAMP methodology. Cell culture cultivation is still recommended for molecular stain typing and epidemiologic studies.

The CDC maintains a 24-hour/day, 365-day/year hotline to provide emergency assistance in cases of botulism. Botulinum toxin is a potential bioweapon. Accept able specimens for the diagnosis of C. botulinum or C. tetani infection include feces, enema fluid, gastric aspirates, vomitus, tissue, exudates, or postmortem specimens. Specimens for infant botulism should include serum and stool; those for wound botulism should include serum, stool, and tissue biopsy. Serum specimens should be collected immediately after the onset of symptoms. All specimens should be stored and shipped at 4° C. Detection of the toxin BoNT is diagnostic for C. botulinum infection. The mouse bioassay remains the recommended method of analysis for the identification of BoNT. The bioassay requires that the specimen be split into two samples. One sample is boiled at 80° C for 10 minutes, inactivating the toxins. The two samples are each injected intraperitoneally into a mouse. One mouse serves as the negative control (inactivated specimen), and the other serves as the “test” sample. The mice are then observed for neurologic symptoms. The presence of toxin is presumptively indicated with the development of symptoms and death in the test animal but not the control animal. The toxins associated with C. botulinum and C. tetani (BoNT and tetanus neurotoxin) are considered extremely dangerous. The CDC recommends the use of Biosafety Level 3 practices and precautions, including immunization for the toxins.

The specimens of choice for neutropenic enterocolitis involving C. septicum are three different blood cultures, stool, and lumen contents or tissue from the involved ileocecal area; a muscle biopsy sample should also be collected if myonecrosis (death of muscle tissue) is suspected. Table 2 provides an identification scheme for representative anaerobic organisms.

Table2. Differentiation of Representative Gram-Negative Bacilli and Gram-Positive Cocci

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المجمع العلمي يجري اختباره للمتقدمين على قناة حفظة القرآن الكريم في النجف الأشرف

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مواضيع ذات صلة

Gram-Negative Rods

Gram-Positive, non–Spore- Forming Bacilli

Morphology and Identification of Enterobacteriaceae

Clinical Findings of Enterococci

Streptococcus pneumoniae

Anaerobic Media

Specimen Processing of Anaerobic Bacteriology

Specimen Collection and Transport of Anaerobic Bacteriology

Gonococcal Arthritis

Nongonococcal Bacterial Arthritis

Acute Bacterial Arthritis

Streptococcus pyogenes