its name) but can be grown on highly selective CCFA (cefoxitin, cycloserine, and fructose agar)
media (19). The bacteria can exist in spore and vegetative forms. Outside the colon it survives
in the spore form. The spores are resistant to heat, acid, and antibiotics. In the colon, the spores
convert to their vegetative, toxin-producing form and become susceptible to killing by
antimicrobial agents.
C. difficileproduces two potent protein exotoxins, toxin A and B, the largest bacterial
toxins known (33) and the B1/NAP1 strain also produces a binary toxin. The toxins mediate
colitis and diarrhea. Both toxin A and B are optimally expressed at body temperature (19).
Purified toxins are capable of causing the full spectrum of disease (17).
Toxin A is a 308-kDa enterotoxin that produces acute inflammation, leading to intestinal
fluid secretion and mucosal injury (33). Toxin B is a 270-kDa cytotoxin that is 10 times more
potent than toxin A in mediating mucosal damage in vitro. The toxins appear to act
synergistically (17). Both toxins act intracellularly by inactivating proteins in the Rho
subfamily, which regulate the F-actin cytoskeleton. This results in disaggregation of actin,
opening the tight junctions between cells, and resulting in cell retraction and apoptosis
manifested as characteristic cell rounding in tissue culture assays and shallow ulceration on
the intestine mucosal surface (17,34).
Both toxins are also proinflammatory, inducing release of cytokines, phospholipase A2,
platelet-activating factor (33), tumor necrosis factor-a, and substance P. This results in the
activation of the enteric nervous system, leading to neutrophil chemotaxis and fluid secretion.
C. difficilealso produces tissue degradation enzymes such as collagenase and hyaluronidase,
(3) promoting the development of PMC.
Toxigenic strains ofC. difficileare not equally virulent; some strains that clearly possess
toxin genes demonstrate low levels of gene transcription, resulting in minimal toxin
production (35). While most strains produce both toxins, some produce toxin B only but can
be equally virulent as strains with both toxins. Rare cases of CDI caused by strains producing
neither toxin A nor B have been reported, (34) but nontoxigenic strains are generally
considered nonpathogenic.
Microbiology of the Epidemic Strain, B1/NAP1
The epidemic strain B1/NAP1 is emerging as an important contributor to the current epidemic
of CID, but it has been isolated only rarely in the past (6). This strain has had several names,
based on the biologic properties tested; NAP1 by pulse filed gel electrophoresis, B1 on
restriction endonuclease analysis, toxinotype III and ribotype 027 by polymerase chain
reaction. Currently, the name B1/NAP1 is favored.
There are several unique features with B1/NAP1, the following five factors have been
found in nearly all of the strains (6):
- The epidemic strain B1/NAP1 produces substantially more toxins A and B in vitro (36).
- All B1/NAP1 strains are toxinotype III. Toxinotyping is based on analysis of the
region of theC. difficilegenome known as the pathogenicity locus (PaLoc) that
includes genes that encode for toxin A(tcdA) and toxin B(tcdB) and neighboring
regulatory genes. More than 80% of non-B1/NAP1 strains are toxinotype 0 (36,37). - The epidemic strain B1/NAP1 has a deletion oftcdC, which is a gene in the PaLoc
responsible for downregulation of toxin production (37). - The epidemic strain B1/NAP1 produces a binary toxin in addition to toxin A and B.
The binary toxin is an iota-like toxin similar to that produced byC. perfringenstype E
(38). Its role in the pathogenesis of CDI is unclear. - The epidemic strain B1/NAP1 is resistant in vitro to fluoroquinolones, which is
infrequently observed in strains collected before 2001 (11,37,39).
CLINICAL PRESENTATION
Most patients exposed toC. difficile, even after antibiotic exposure, do not develop clinical
disease. Colonization rates of 25% to 80% are seen in healthy infants and neonates but clinical
illness is rare (3). For unclear reasons, colonization appears to wane with advancing age, and
Clostridium difficileInfection in Critical Care 275