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E-Book 2nd Congress

  • Identification and diagnosis of pathogenic Clostridium species in medical microbiology
  • Mojtaba Alimolaei,1,*
    1. Research and Development Department, Kerman branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Kerman, Iran


  • Introduction: Clostridium genus is a phylogenetically heterogeneous group of prokaryotic bacteria which currently consists of 204 validly described species (http://www.bacterio.net). Clostridium species are Gram-positive (mostly), pleomorphic spore-forming rods with a considerable variety in their oxygen tolerance, from obligate to aerotolerant. They are widely distributed in the environment as well as the microbiota in humans and animals. They produce a variety of potent exotoxins/enzymes, which lead the clinical features of disease in their hosts. Tetanus, gas gangrene, botulism, pseudomembranous colitis, antibiotic-associated diarrhoea, food-borne illness, cholecystitis, pneumonia, bacteremia, empyema, abscesses, and etc. are the most important clostridial diseases in humans. So, the Clostridium spp. can be isolated from various clinical specimens and thus their reliable identification is important. This study summarizes the standard identification of Clostridium species involved in medical microbiology.
  • Methods: Primary isolation of Clostridium spp. can be performed by the culture of specimens on agar plates containing blood and incubation anaerobically at 35-37 °C for 40-48h or Egg Yolk agar incubated anaerobically at 35-37 °C for 16-24h. The selective culture media are also available for some species as tryptose sulfite cycloserine (TSC) agar for C. perfringens, and cycloserine cefoxitin fructose agar (CCFA), C. difficile moxalactam norfloxacin (CDMN), and cefoxitin cycloserine egg yolk agar (CCEY) for Clostridioides difficile. Colonial appearance on agar plates varies with each clostridia species. Gram and spore staining techniques are useful to determine the gram-positive rods and the shape and position of spores, respectively. The specific biochemical tests (Lecithinase, Lipase, Indole, Urease, …) as described in Bergey’s manual can be carried out to differentiate the Clostridium species. The Nagler and reverse CAMP tests can be used for differentiation of C. perfringens from other Clostridium species, too. The specialized methods as enzyme immunoassay (EIA), ELISA, Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS), and molecular assays as PCR, real-time PCR, Pulsed- Field Gel Electrophoresis (PFGE), Fluorescent Amplified Fragment Length Polymorphism (AFLP),16S rDNA gene sequencing, PCR- restriction fragment length Polymorphism (PCR-RFLP), Microarray analysis, Multiple-Locus Variable-Number Tandem-Repeat Analysis (MVLA), and even whole-genome sequencing (WGS) were also can be carried out. To confirm the clostridia isolates, they can be referred to the anaerobe reference laboratory for further identification as the clostridia laboratory in Razi Vaccine and Serum Research Institute-Kerman branch, Iran.
  • Results: Different Clostridium species can be isolated as the commonly (C. perfringens, C. septicum, C. tertium, and C. difficile), rarely (C. novyii type A and C. sordellii), very rarely (C. tetani, C. histolyticum, and C. botulinum) and the commonly “non-pathogenic” clostridia (C. sporogenes, C. ramosum. C. innocuum, C. paraputrificum, C. cadaveris, C. bifermentans, C. fallax, and C. clostridioforme) from human clinical specimens. They can be differentiated by the identification methods that described above.
  • Conclusion: Despite the clinical significance of clostridia in medical microbiology, reliable, practical, and fast identification methods are few. Although simple tests can serve to identify most commonly isolated Clostridium species, the identification of other clostridia by conventional biochemical testing is still laborious, expensive, and time-consuming. Due to these evident drawbacks of conventional methods, there is a growing trend toward molecular diagnostics of clostridia that are difficult to identify by phenotypic characters.
  • Keywords: Identification; Diagnosis; Clostridium; medical microbiology