Clostridium difficile is the etiologic agent of pseudomembranous colitis (CPM); it is also responsible for many cases of diarrhea or colitis consecutive antibiotic therapy. These diseases are caused by the production and action of both toxins in the colon: an enterotoxin and cytotoxin.
C. difficile was isolated in 1935 by Hall and O’Toole from newborn stool. In 1971 George and Symonds showed the presence in the intestine, in the pseudo-membranous colitis, toxin neutralized by a serum Clostridium sordellii UNA. Bartiett et al. 1978 isolate C. difficile in this disease and make the connection between this species and the toxin produced. In 1981, this same team, and that of Wilkins, shows that some strains of C. difficile secrete two toxins: a cytotoxin (or toxin B) and enterotoxin (or toxin).
I – CLASSIFICATION:
C. difficile belongs to the group of Clostridium III.
C. difficile GC 28%, similar to that of the main species of Clostridium.
II – HABITAT:
The intestine of man as well as many species of animals can harbor C. difficile in humans colonizing the rate varies according to age. 20-70% of healthy children younger than 1 year, only 3 % of healthy adults. But in the latter, the frequency of porting can be influenced by various factors: antibiotics, digestive disease, hospitalization. In the stool of patients, C. difficult may be present in high numbers: 106-108 / gram stool.
C. difficile can survive several months in the environment through its spores that are resistant to disinfectants.
III – PATHOPHYSIOLOGY:
Diarrhea, colitis and pseudomembranous colitis associated with C. difficult are conditions that are usually observed in hospital.
The occurrence of diarrhea or colitis in adults follows:
– An exogenous contamination with C. difficult
– An imbalance in the intestinal flora by antibiotic therapy.
All antibiotics given orally or parenterally can induce this condition, particularly those incompletely absorbed orally and / or having an enterohepatic cycle. The most frequently implicated antibiotics are aminopenicillins, cephalosporins and clindamycin.
It is not a secondary infection; C. difficile remains, in most cases, sensitive in vitro to the responsible antibiotic. C.sporulerait difficult in the colon with regeneration of the vegetative form when the antibiotic levels in the colonic lumen is relatively low. The intestinal flora of the colon, exercising its barrier effect, normally inhibit the growth of C.difficult, and it is only thanks to an imbalance, with the waning of antibiotics that C. difficile to grow and produce toxins.
Observation of CPM in use of certain cancer (methotrexate, 5-fluorouracil, …) is possible (reached flora or digestive toxicity?).
Despite the presence of C. difficult in their intestinal flora, children under 2 years never CPM except predisposing circumstances. In infants of non-toxigenic strains are especially isolated.
The two toxins produced by C. difficult provoke an inflammatory lesion of the colonic wall. When the lesions and inflammation are not too severe, the only symptom is diarrhea. If the lesions were larger, a colon biopsy highlights mucosa congestive covered by a
pseudo-membrane, ie from isolated or confluent plaques cream-colored or yellow-green; they consist of a layer of fibrin and mucin including leukocytes.
For unknown reasons, these toxins have no effect on the colonic mucosa of young children, as well as that of cystic fibrosis patients (absence of receptors on enterocytes?).
IV – PATHOGENICITY:
C. difficile causes various intestinal diseases ranging from asymptomatic carriage to pseudomembranous colitis.
Digestive disorders appear on average less than a week after the start of treatment, or even 4 or 6 weeks after discontinuation.
It would be responsible for 15-25% of diarrhea associated with antibiotic therapy.
The clinical forms are diverse: mundane diarrhea or severe diarrhea, or not associated colitis.
The CPM is suspected in the following symptoms: loose stools, mucous membranes, abundant, abdominal cramps, fever, leukocytosis, Hypocholesterolemia. The clinical diagnosis of pseudomembranous colitis is evoked during endoscopic observation pseudomembranes on the colon wall.
The CPM can progress to the rare complications, but severe: toxic mégacôlons, perforations, peritonitis. Extra-digestive symptoms are unusual.
V – BACTERIOLOGICAL CHARACTERS:
A – morphological characters:
C. difficile is a Gram-positive bacterium, strictly anaerobic, giving oval subterminal spores deforming. The majority of bacteria isolated from pathological products are mobile through peritrichous ciliature.
In some strains, fimbriae or a capsule have been demonstrated, but their role in the pathogenesis is still controversial. Their adhesion properties to be confirmed.
B – Cropping characters:
Incubation of all culture is at 37 ° C in an anaerobic atmosphere.
In a broth consisting of peptones, yeast extracts and glucose (PGY or TGY broth), a homogeneous disorder is achieved in 24 h with a plentiful sediment.
On blood agar, colonies are circular, flat or slightly curved, opaque, grayish or whitish without hemolysis zone.
Sporulation is obtained by culture after more than 48 hours and it is favored by the presence of sodium taurocholate 0.1%.
C – biochemical characters:
– Protein Metabolism: not digest milk, gelatin hydrolysis
– Lack of nitrate reductase and urease.
– Carbohydrate metabolism: fermentation of glucose, fructose, mannitol and mannose, esculin hydrolysis.
Broth PGY, production of the following volatile fatty acids: acetic acid, isobutyric, butyric, isovaleric, valeric, isocaproic, formic, lactic.
– Lipid metabolism: no lipase no phospholipase C.
D – Toxins:
C. difficile produces two toxins:
– Toxin A (308 kDa) called enterotoxin is about 1000 times less cytopathic on cell cultures that toxin B, toxin gives a positive test in the rabbit ileal loop, it gives a severe hemorrhagic necrosis and fluid secretion ;
– The B toxin or cytotoxin (270 kDa) causes cell rounding on cells in culture, a test ileal loop negative but hemorrhagic ulceration hamster cecum.
The cytotoxic effect on the cells appear identical for both toxins with disorganization and condensation of the actin cytoskeleton, suggesting a common mechanism of action. Fixated on different receptors explain the diversity of effects. The destruction of the brush border of intestines by toxin A precede the action of toxin B.
Production of two toxins is not related to sporulation and appears to be co-regulated, the two corresponding genes were cloned which are contiguous on the bacterial chromosome and possess the same promoter.
VI – BACTERIOLOGICAL DIAGNOSIS:
It is based on the isolation of C. difficile in stool and demonstration of a toxic activity of the strain or the stool filtrate.
A – Sample:
The stool should be freshly issued or stored at -20 ° C.
B – Culture:
Feces are diluted or directly seeded on CCFA medium (this medium is the selective and specific medium George based cefoxitin, cycloserine, fructose and agar). The plates are incubated anaerobically at 37 ° C for 24 to 48 hours.Colonies ferment fructose and give a yellow color, they have a typical appearance with irregular filamentous margination.
The identification uses the biochemical characteristics described above. Some marketed galleries can help diagnosis (API An-ident System APIZYM Micro-system, Rapid ID System … ANA).
The diagnosis can be completed by a study of glucose fermentation products analyzed by gas chromatography (the peak isocaproic acid is typical).
Epidemiological markers have been proposed (antibiotic resistance phenotypes, sensitivity to bacteriophages and bacteriocins, analysis of protein profiles or DNA after digestion with restriction enzymes, serotyping).
C – Search toxins:
Toxins are searched, or in the culture supernatant or in the supernatant centrifuged stool after sterilizing filtration.
Cytotoxicity assay in cell culture (reference method)
Toxin B is highlighted with its powerful cytotoxic effect on many cell lines (McCoy, Vero, MRC-5 …). Stool filtrates may have a non-specific toxic effect and the cytotoxic effect must be neutralized with antitoxin serum or C. sordellii anti-toxin B.
– Counter-immunoelectrophoresis and sensitized latex particles: these methods are discussed (cross-reactions with other bacterial species, no distinction between toxigenic and non-toxigenic)
– Several ELISA methods have been described but their use is still part of the same, a quick test “Dot immunobinding assay” (C. diff-CUBE, Difco) is proposed.
– The use of the polymerase chain reaction (PCR) technique is still in its beginning.
VII – TREATMENT:
The treatment of diarrhea or colitis C. difficult begins with the interruption of antibiotic therapy and correction of electrolyte disturbances.
In severe cases of diarrhea and colitis, must be added to oral vancomycin or metronidazole: antibiotics whose high concentrations in the stool inhibit the growth of C. difficult. All the symptoms disappear in 5 or 7 days.
Note the high frequency of relapses in the weeks after stopping digestive disorders (25 to 50% depending on the study). Therapeutic relay processing have been proposed: cholestyramine (chelating resin 2 toxins) or takingSaccharomyces boulardii (restaurant the barrier effect).
Prophylaxis seems simple in appearance, but in hospital C. difficult can behave like a true opportunist could lead to outbreaks of contamination between patients.