REVIEWS

Clostridium difficile infection:

new developments in epidemiology
and pathogenesis
Maja Rupnik*, Mark H. Wilcox and Dale N. Gerding
Abstract | Clostridium difficile is now considered to be one of the most important causes of
health care-associated infections. C. difficile infections are also emerging in the community
and in animals used for food, and are no longer viewed simply as unpleasant complications
that follow antibiotic therapy. Since 2001, the prevalence and severity of C. difficile infection
has increased significantly, which has led to increased research interest and the discovery
of new virulence factors, and has expanded and focused the development of new treatment
and prevention regimens. This Review summarizes the recent epidemiological changes in
C. difficile infection, our current knowledge of C. difficile virulence factors and the clinical
outcomes of C. difficile infection.

Pseudomembranous colitis
found in some (generally the
more severe cases) but not all
patients with Clostridium
difficile infection, and refers to
changes on the inner surface of
the lining of the large intestine
(colon). Characteristically, the
colon is inflamed and has
visible patches caused by an
inflammatory membrane that
consists of red and white blood
cells, fibrin and bacteria.
*Institute of Public Health
Maribor, Centre for
Microbiology, Prvomajska 1,
2000 Maribor, Slovenia, and
University of Maribor, Faculty
of Medicine, Slomaskov trg
15, 2000, Maribor, Slovenia.
Clostridium difficile was first described in 1935 as part of
the intestinal microflora in neonates. Although the severe
form of C. difficile disease (pseudomembranous colitis; PMC)
was first described in 1893, C. difficile was not identified as
the causative agent of human disease until 1978 (Ref. 1).
C. difficile infection (CDI) is a toxin-mediated intes-
tinal disease, and extraintestinal manifestations are rare.
The clinical outcomes of CDI can range from asympto-
matic colonization to mild diarrhoea and more severe
disease syndromes, including abdominal pain, fever
and leukocytosis. Fulminant or severe complicated CDI
is characterized by inflammatory lesions and the forma-
tion of pseudomembranes in the colon (which is typical
for PMC), toxic megacolon or bowel perforation, sepsis,
shock and death.
C. difficile is recognized as the main cause of infec-
tious diarrhoea that develops in patients after hospitali-
zation and antibiotic treatment (fIG. 1). The association
The changing epidemiology of CDI
In 2002, the University of Pittsburgh Medical Center in
the United States reported an increase in severe CDI,
which signalled the beginning of a continuous rise in
the rate of CDI in Canada46, the United States7 and
Europe8,9.
In 2006, the CDI discharge diagnosis rates in US
hospitals exceeded 300,000 cases per year, an increase
from <150,000 cases in 2000. It is currently esti-
mated that there are ~500,000 cases of CDI per year
in US hospitals and long-term care facilities, based
on annual data from the state of Ohio in 2006 (Ohio
Department of Health; see Further information). An
estimated 15,000 to 20,000 patients die from CDI in
the United States each year. Few countries in Europe
have the necessary systems in place to measure system-
atically the incidence of CDI and the associated death
rates and, in some cases, even to test for C. difficile. In

Leeds General Infirmary,

Leeds, LS1 3EX, UK.
between antimicrobial therapy and CDI has been almost Saxony, Germany, the incidence of CDI increased from

Hines Veterans Affairs
Hospital and Loyola
University Chicago Stritch
School of Medicine, ACOS
Research and Development,
5th Avenue and Roosevelt
Road, Building 1, Hines,
Illinois 60141, USA.
e-mails:
maja.rupnik@uni-mb.si;
mark.wilcox@leedsth.nhs.uk;
dale.gerding2@med.va.gov
doi:10.1038/nrmicro2164
universal, as C. difficile can only colonize the gut if the
normal intestinal microbiota is disturbed or absent.
Although elderly hospitalized patients receiving anti-
biotics are still the main group at risk of infection (fIG. 1),
an increase in CDI in younger populations with no pre-
vious contact either with the hospital environment or
with antibiotics is emerging. Furthermore, CDI in spe-
cific populations that were previously at low risk, such as
children2 and pregnant women3, is increasing. Therefore,
CDI should be considered when diagnosing any patient
with persistent diarrhoea.
1.7 to 3.8 cases per 100,000 people in 2002 to 14.8 cases
per 100,000 people in 2006 (Ref. 10). In Spain, the inci-
dence of hospital discharges in patients aged >65 years
with a diagnosis of CDI increased threefold between
1997 and 2005 (Ref. 11), although the epidemic C. difficile
strain (C. difficile BI/NAP1/027; discussed below) has
not been reported in this country. In England, all cases
of CDI must be reported, and data for each health care
facility are made publicly available every 3 months.
The marked decrease in the number of CDI cases in
England after years of steady increases (although more

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 REVIEWS

Asymptomatically
colonized

Non-toxigenic C. difficile

Asymptomatically
colonized

Toxigenic C. difficile;
C. difficile IgG response to ToxA
negative

Symptomatic
CDI
Toxigenic C. difficile;
no IgG response to
ToxA

Figure 1 | Model for the acquisition of Clostridium difficile infection (CDI). Patients are exposed to C. difficile spores
through contact with the hospital environment or health care workers. After taking an antibiotic, Nature
theyReviews | Microbiology
develop CDI if they
acquire a toxigenic C. difficile strain and fail to mount an anamnestic serum immunoglobulin G (IgG) antibody response to
toxin A (ToxA; also known as TcdA)64; if they can mount an antibody response they become asymptomatically colonized
with C. difficile. If they acquire a non-toxigenic C. difficile strain, they also become asymptomatically colonized. Colonized
patients have been shown to be protected from CDI126.

Leukocytosis
A term used to refer to an
individual with an increased
number of white blood cells.
A common explanation for
leukocytosis is infection, and
in general the higher the
number of white blood cells
(particularly neutrophils) in the
blood the greater the severity
of the infection.
Toxic megacolon
An uncommon condition that
occurs in only the most severe
cases of Clostridium difficile
infection. The large bowel
(colon) becomes dangerously
inflamed and dilated, and can
eventually perforate.
Ribotype
Characterized by the pattern
of amplified intergenic regions
in the ribosomal RNA operons
present in Clostridium difficile
in multiple copies.
than 10,000 cases still occur each quarter 12) might
reflect the recent national mandatory target to reduce
the incidence of CDI by 30% by 2011.
In 2005, molecular analysis led to the identification
of the C. difficile strain that was responsible for a large
number of infections during an increase in CDI rates in
hospitals across North America. This type was charac-
terized as group BI by restriction endonuclease analy-
sis (REA), as North American pulse-field type NAP1
by pulse-field gel electrophoresis (PFGE) (BOX 1) and as
ribotype 027; the differing terminology reflects the pre-
dominant techniques that were used for epidemiological
typing and in this Review we refer to this strain as C. dif-
ficile BI/NAP1/027 (Ref. 13). Since then, C. difficile BI/
NAP1/027 has been documented in hospitals in 40 states
in the United States, in all the provinces of Canada and
in most European countries4,5,8,1315. The C. difficile BI/
NAP1/027 strains in North America and Europe seem
to be highly related by some typing approaches16, but in
a collection of 91 isolates of C. difficile BI/NAP1/027 from
9 hospitals in England, PFGE discriminated 5 pulsovars,
whereas multiple loci variable number tandem repeat
analysis detected 23 types, and identified enlarged and
additional hospital clusters of CDI cases17.
The rising rates of CDI have largely been attributed
to the presence of BI/NAP1/027 but are not limited to
the spread of this strain. Depending on the country, other
strains (including PCR ribotypes 001, 053 and 106) can
be often associated with outbreaks and severe cases18.
The prevalence of C. difficile ribotype 078 has increased
recently from 3% to 13% in several countries in Europe19,20.
In the Netherlands, patients infected with ribotype 078
were younger (67.4 versus 73.5 years) and had community-
associated disease more frequently (17.5% versus 6.7%;
odds ratio = 2.98; 95% confidence interval = 2.118.02)
than patients infected with ribotype 027.
Community-associated infections
Patients in the community are also at risk for CDI, albeit at
a considerably lower rate than those who are hospitalized.
The community CDI rates in the United States have been
reported as 7.7 cases per 100,000 person years, of which
35% received no antibiotics within 42 days of C. difficile
detection21. More recent studies by the Centers for Disease
Control and Prevention found similar community rates,
but an increased severity of the disease22,23. Wilcox et al.24
found that in urban and semi-rural areas of the United
Kingdom the infection rates were higher (29.5 and 20.2
cases per 100,000 individuals) in 1999, and only 52%
of patients received antibiotics within 4 weeks of C. difficile
detection.
Community-associated CDI without previous direct
or indirect contact with a hospital environment remains
rare compared with hospital-acquired CDI. Nevertheless,
it has been reported in populations that were previously
thought to be at low risk, such as young individuals and
pregnant women22.
In North America, most cases of CDI that have
been attributed to C. difficile BI/NAP1/027 have been
described in hospitals, particularly hospitals asso-
ciated with outbreaks or epidemics. The C. difficile
BI/NAP1/027 strain has only recently been described
in community-associated cases, and studies indicate
that there is a correlation between the presence of this
epidemic strain in a hospital and the detection of
this strain in the surrounding community 25,26.
Possible community sources for CDI include soil,
water, pets, animals used for food, meats and vegeta-
bles27. There is no conclusive evidence that C. difficile
contamination of food has led to clinical CDI in humans.
However, as we lack clear explanations for why the rates
of CDI have risen so rapidly in recent years, a common
vehicle, such as food, cannot be ruled out. The C. difficile

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Box 1 | Summary of typing methods for Clostridium difficile

PCr ribotyping
PCR ribotyping exploits differences in the spacer regions of 16S and 23S ribosomal RNA. Specific primers are used
for PCR-mediated amplification of the DNA that encodes these RNA regions. This method generates a few DNA bands
as visualized by gel electrophoresis; the DNA band patterns are referred to as ribotypes.
Pulsed field gel electrophoresis (PFgE)
PFGE involves using an enzyme that cuts the bacterial genome infrequently, resulting in large DNA fragments. The
fragments are then slowly separated in a polyacrylamide gel that is submitted to an electrical field in which the voltage
repeatedly switches. This enables the large DNA fragments to migrate varying distances through the gel according to
their size. The fragments are then visualized by DNA staining to reveal differences in banding patterns that are
sometimes referred to as pulsovars.
Multilocus variable number tandem repeat analysis (MlVA)
MLVA is a method of counting the numbers of repeat alleles in the genome for a series of predefined, conserved loci that
are amplified by PCR. This method requires expensive equipment but is highly discriminatory, and produces a consistent
numerical result (code) for each strain that should be comparable between different laboratories. This method is well
known in forensic science, as it is the basis of DNA fingerprinting in humans.
restriction endonuclease analysis (rEA)
REA relies on more frequent cutting of the bacterial genome than PFGE, resulting in large numbers of DNA fragments.
These fragments are separated by electrophoresis in an agarose gel. This method is usually highly discriminatory, but
produces complex DNA banding patterns that can be difficult to interpret and reproduce.
other methods
Other methods that are used for typing C. difficile include toxinotyping (BOX 2); multilocus sequence typing (MLST),
which is similar in principle to MLVA; and amplified fragment length polymorphism (AFLP), which uses restriction
enzymes to cut genomic DNA, followed by ligation of adaptors to the ends of the restriction fragments. A subset of the
restriction fragments are then amplified using primers that are complementary to the adaptor and part of the restriction
site fragments, with the DNA visualized following gel electrophoresis.

Toxinotype
A group of C. difficile strains
with identical changes in the
toxin-coding region known as
the pathogenicity locus
(PaLoc).
BI/NAP1/027 strain has been detected in meat 28,29, but
the strains that are most commonly found in animals
and meat are toxinotype V, PCR ribotype 078 and REA
group BK28,29; these strains are now also emerging in
hospital- and community-associated CDI19,20,30.
C. difficile veterinary disease
In animals, C. difficile was mainly known as an impor-
tant pathogen in horses, although it has been reported
to infect numerous wild and domestic animals31 and,
more recently, piglets and calves32,33. As with infection
of humans, disease in animals is associated with non-
protective normal gut flora, owing to either antibiotics
or young age3234. The heterogeneity of the genotypes that
have been isolated from animals (approximately 3050
different PCR ribotypes) is lower than the heterogeneity
of human isolates (approximately 190 PCR ribotypes).
This could be caused by the limited number of animal
typing studies that have been performed in comparison to
human typing studies. The most prevalent PCR ribotypes
differ between animal and human populations, but a
substantial number of PCR ribotypes have been isolated
from both populations19,33,35.
Antibiotics and CDI
CDI occurs when the natural flora in the gut is
disrupted by antibiotics. It is becoming evident from
16S ribosomal RNA sequencing that disruption of the
gut microbiota following antibiotic treatment is much
greater than was previously realized36 and is a contrib-
uting factor to recurrent CDI treated with antibiotics37.
The fact that C. difficile is resistant to a wide range
of antibiotics enables the bacterium to colonize and
infect in the presence of antibiotics (fIG. 2). In studies
that used a hamster model of CDI, susceptible organ-
isms were not able to colonize the gut until several
days after the last dose of clindamycin, presumably
when the level of clindamycin in faeces had fallen
below the minimum inhibitory concentration (MIC)
of the organism, whereas clindamycin-resistant strains
colonized readily in the presence of daily administra-
tion of the drug. Susceptibility to CDI persisted for a
variable period after administration of the last dose
that depended on the drug being administered; colo-
nization resistance recovered rapidly following treat-
ment with cephalosporins, but in the hamster model
clindamycin treatment led to a much longer period of
susceptibility to infection38,39. Restoration of coloniza-
tion resistance of the normal flora is therefore a key
factor in the prevention of CDI in patients.
The antibiotic susceptibility of C. difficile strains,
including epidemic clones, is changing. Historically
most of the prevalent types in human populations were
clindamycin resistant 6,40. The emergence and spread of
C. difficile BI/NAP1/027 correlates with acquired resist-
ance to the fluoroquinolone antibiotics gatifloxacin and
moxifloxacin, a trait that was not present in historic
strains of the same genotype13. However, outbreaks
caused by additionally clindamycin-resistant ermB-
positive C. difficile 027 strains have occurred in three
European countries8.
Resistance to the antibiotics that are currently used
to treat CDI (metronidazole and vancomycin) has thus
far not posed a significant threat. The reduced clini-
cal response to treatment with metronidazole has not
been attributed to resistance to the drug in C. difficile,

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Box 2 | Toxinotyping
Toxinotyping is a PCR-based method in which Clostridium difficile strains are assigned to a toxinotype according to the
lengths and restriction patterns of two (the B1 and A3 fragments) of the ten fragments that make up the pathogenicity
locus (PaLoc)124. Comparison with the reference strain C. difficile VPI 10463 generates 27 variant toxinotypes (I to XXVII)
(Clostridium difficile toxinotypes web page; see Further information). The tcdC gene is another highly variable region of
the PaLoc125.
All strains in a given toxinotype have identical changes in the PaLoc. In some toxinotypes, these changes are minor
and are usually deletions in repetitive sequences of the tcdA gene (the A3 region). In a large proportion of toxinotypes,
however, the changes are spread over the entire PaLoc. These are the major toxinotypes, and they correlate well with
ribotyping and other molecular typing methods.
The variant toxin genes encode variant toxins with altered properties or can even result in the absence of one or
both toxins. Because of their unusual pattern of toxin production, the TcdATcdB+ strains were the first variant strains
to be discovered.
At the clinical level, given toxinotypes can be linked to specific disease characteristics or patient populations in epidemic
settings, but in general, toxinotype is not predictive of clinical diseas e expression. In the past, variant toxinotypes
represented a large proportion (40100%) of the strains of animal origin, but only up to 10% of isolates from humans.
However, the proportion of variant strains in humans is increasing, and some variant strains (toxinotype III (also known
as ribotype 027 and REA BI), toxinotype VIII (also known as ribotype 017 and REA CF) and toxinotype V (also known as
ribotype 078 and REA BK)) have been associated with outbreaks worldwide.

Heteroresistance
A type of resistance in which
some but not all of the cells
in a population are resistant
to an antibiotic; the remainder
retain their susceptibility to
the antibiotic.
Negative predictive value
A measurement (usually
expressed as a percentage)
of all negative test results
that are truly negative.
Positive predictive value
A measurement (usually
expressed as a percentage)
of all positive test results that
are truly positive.
although subtle increases in MIC (MIC creep)41 and
heteroresistance have been observed 42 ; however,
these observations have not yet been correlated with
clinical treatment failure. Patients with CDI are occa-
sionally treated with rifaximin, a poorly absorbed oral
rifamycin, and increasing resistance in C. difficile to
this drug has been reported, particularly in C. difficile
BI/NAP1/027 strains43.
Clinical aspects of CDI
Diagnosis. Suspicion of CDI, which is often based on
diarrhoea that has a typical foul-smelling odour, is
not sufficiently accurate for a definitive diagnosis44.
The high negative predictive value (NPV) (8292%)
of odour assessment for the likelihood of CDI might
be useful when assessing patients with diarrhoea to
determine priority for isolation in settings where
this capacity is limited, especially if rapid laboratory
diagnosis is not available4547. Clearly, however, all
patients with diarrhoea who are at risk of CDI should
be tested for CDI.
The traditional gold standard for C. difficile diagno-
sis is a cytotoxin assay that detects the cell cytotoxicity
of toxin B (ToxB; also known as TcdB) (and to some
extent toxin A (ToxA; also known as TcdA) depend-
ing on the cell line used) in faecal eluate. That C. dif-
ficile toxin is the cause is confirmed by neutralization
of the cytotoxic effect by anti-toxin antibodies (TABLe 1).
An alternative reference standard test is to culture
C. difficile then carry out a cytotoxin assay (known as
cytotoxigenic culture), which detects C. difficile strains
that have the capacity to produce toxin (or toxins) as
opposed to detecting the presence of toxins in a stool
sample per se. Several commercial toxin detection kits
(enzyme immunoassays and membrane assays) are also
available, but kits that only detect TcdA should not be
used for routine microbiological diagnosis of CDI, as
they cannot detect pathogenic strains that are negative
for TcdA. A recent systematic review of studies on the
accuracy of six toxin detection kits found that the positive
predictive value (PPV) of these assays is unacceptably low
(<50% in some circumstances)48. Although the NPVs
are high (typically >95%), crucially, the PPV reduces
markedly as the prevalence of CDI decreases. For exam-
ple, in the community setting only 2% of diarrhoeal
samples tested might be positive for C. difficile toxin24.
New molecular methods that detect the gene encod-
ing C. difficile TcdB could offer a rapid and sensitive
alternative49. However, similar to cytotoxigenic culture,
a positive result is not synonymous with the presence of
the toxin, and therefore this methodology might identify
C. difficile strains that carry the gene but do not produce
the toxin. Toxigenic strains can be carried asymptomati-
cally by up to 50% of elderly patients who are residents of
a long-term care or nursing home facilty 50. Studies that
take into account the clinical diagnosis and outcome
of treatment of patients will be required to assess the
accuracy and value of tests that do not detect C. difficile
toxin activity.
False-positive and false-negative results have
major implications for patient care; for example, false-
positive results lead to unnecessary treatment and iso-
lation and false-negative results lead to increased risk of
delay in treatment and cross-infection. Some research-
ers have recommended using a two-step process to
improve diagnostic accuracy, for example using an ini-
tial test that has a high NPV to identify those individu-
als who require a second definitive toxin test 51. The
screening test could involve the detection of glutamate
dehydrogenase, which is produced by most C. difficile
strains. However, a recent study found that glutamate
dehydrogenase detection can have a low level of sensi-
tivity, and it remains uncertain whether this reflects an
inherent problem with glutamate dehydrogenase or is
related to the insensitivity of the assay used.
Treatment of CDI
Until recently, the treatment of CDI had not changed
appreciably since oral vancomycin was found to be
highly effective in 1981 (Ref. 52). Metronidazole was
shown to be as effective as clindamycin a few years
later 53. Recently, two studies have shown that for

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a b c d
No antibiotic Antibiotic No antibiotic No antibiotic
Normal flora Normal flora
CDI
risk
Flora disrupted Flora disrupted
No CDI risk C. difficile that is C. difficile that is No CDI risk
resistant to the resistant to the
antibiotic has a antibiotic has
selective advantage no advantage
Figure 2 | The effect of antibiotics on the normal gut flora and the risk of
Clostridium difficile infection (CDI). Patients are resistantNature to CDIReviews
if their normal gut
| Microbiology
flora is not disrupted by antibiotics (a). Once antibiotic treatment starts, infection with
a C. difficile strain that is resistant to the antibiotic is more likely while the antibiotic is
being administered owing to the presence of the antibiotic in the gut (b). When the
antibiotic treatment stops, the levels of the antibiotic in the gut diminish rapidly, but the
microflora remains disturbed for a variable period of time (indicated by the break in the
graph), depending on the antibiotic given (c). During this time, patients can be infected
with either resistant or susceptible C. difficile. Finally, after the microflora recovers,
colonization resistance to C. difficile is restored (d).
severe, but not mild to moderate, CDI, vancomycin
provides a superior response rate to metronidazole
(p = 0.02)54,55; it should be noted that severe CDI was
defined differently in these studies.
Two of the most difficult challenges for CDI treat-
ment are the management of multiple recurrence and
the management of fulminant or severe complicated
CDI. Patients with multiple recurrences of CDI typically
respond to treatment with vancomycin or metronida-
zole, but then diarrhoea symptoms resume within days
to weeks after treatment is stopped. Between 20% and
50% of these recurrences are caused by new C. difficile
organisms, indicating reinfection rather than a relapse
of the original infection56,57. No highly effective means to
treat these multiple recurrences have been devised, and
most are treated with prolonged tapering and pulse dos-
ing of vancomycin given every other or every third day
in the hope of keeping C. difficile from regrowing while
the normal flora recovers. The most effective treatment
for these patients has been replenishment of the normal
bacterial flora with a faecal transplant delivered either by
nasogastric tube or by enema58.
No highly effective treatment has been found for
severe complicated CDI, and if medical management
with intravenous fluids, vasopressors, oral vancomycin,
intravenous metronidazole and vancomycin enemas is
not effective, surgical removal of the colon can be the
only remaining life-saving measure59. Newer agents for
treatment of multiple relapsing and fulminant CDI are
desperately needed, and several approaches are under
530 | jUly 2009 | VOlUME 7
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investigation, including the use of flora-sparing anti-
biotics, vaccines, toxin-binding agents and passive
antibodies, including monoclonal antibodies.
Patient risk factors for CDI
The main risk factor for CDI is exposure to specific anti-
biotics (fIGS 1,2). There have been some recent reports of
patients with community-onset CDI who had not been
exposed to antibiotics. However, these cases are infre-
quent compared with the number of patients with CDI
in hospital who have been exposed to an antibiotic in
the 23 months before infection60. Every antibiotic has
been associated with subsequent CDI, but some carry a
higher risk than others, including clindamycin, cepha-
losporins and, more recently, fluoroquinolones5,14,40,61,62.
All fluoro quinolones have been implicated, includ-
ing levofloxacin, moxifloxacin, gatifloxacin and cip-
rofloxacin. The rise in the fluoroquinolone-associated
risk has been concomitant with the rising incidence of
C. difficile BI/NAP1/027 and other strains that carry
high-level fluoroquinolone resistance. The use of cepha-
losporins, to which virtually all C. difficile strains are resist-
ant, has been implicated as a CDI risk factor in hospitals
for several decades5,6. Exposure to stomach acid-reducing
agents, such as H2 blockers and proton pump inhibitors,
remains a controversial risk factor, and has been associated
with CDI in some hospital studies but not in others5,62,63.
Hospitalization is a risk because it brings together
multiple major CDI risks, including exposure to anti-
biotics, a spore-contaminated environment, inad-
equate hand hygiene by health care workers and a
highly susceptible elderly population of patients. The
high incidence and mortality rates of CDI in older
patients have been attributed to the failure of these
individuals to mount an anti-TcdA serum immu-
noglobulin G (IgG) immune response when first
exposed to the toxins (fIG. 1) ; this lack of immune
response has also been associated with a higher rate of
recurrent disease64,65. However, in a subsequent valida-
tion study, the observation of higher recurrence rates
in the absence of the anti-TcdA serum IgG antibody
response did not enhance the ability of researchers to
predict a recurrence over the use of clinical predictors
(such as an age of >65, the presence of severe or fulmi-
nant underlying disease and the receipt of additional
antibiotics)66.
C. difficile virulence factors
The main C. difficile virulence factors are TcdA and
TcdB. The genome of C. difficile strain 630 has been
sequenced67, and comparative genomic analysis of this
genome with the genomes of several other C. difficile
strains that are currently being sequenced will hopefully
reveal additional virulence factors.
Toxins. C. difficile produces the enterotoxin TcdA, the cyto-
toxin TcdB and the binary toxin CDT (fIG. 3). Biochemical
and molecular studies have shown that the main clini-
cal symptoms and signs of CDI (secretory diarrhoea and
inflammation of the colonic mucosa (fIG. 4)) can largely
be explained by the actions of TcdA and TcdB6870 (fIG. 5).
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Table 1 | Diagnosing Clostridium difficile infection

Question to Detection Advantages Disadvantages
be answered method
Is C. difficile Culture Sensitive, but presence does not Slow detection (days)
present? equate with infection as many Suboptimal sensitivity in inexperienced
C. difficile strains are non-toxigenic hands
Useful for epidemiological Requires anaerobic culturing capability
investigation and surveillance
Antigen High negative predictive value* Not specific for C. difficile and therefore
(glutamate Rapid detection (hours) requires supplementary testing
dehydrogenase)
detection
Is C. difficile Cytotoxin assay Sensitive Slow (minimum 12 days)
toxin High specificity for infection Requires access to and/or experience of
present? cell culture methods
Enzyme Familiar methodology that can be Variable sensitivity and specificity resulting
immunoassay used widely in low positive predictive values, especially
Rapid (hours) in populations with low prevalence of
C. difficile infection
Requires laboratory facilities
Membrane assays Does not necessarily require Variable sensitivity and specificity resulting
laboratory facilities in low positive predictive values, especially
Rapid (minutes to hours) in populations with low prevalence of
C. difficile infection
Does the C. Cytotoxigenic High sensitivity Uncertain specificity for infection
difficile have culture Slow (days)
the capacity
to produce
toxin? Detection of High sensitivity Uncertain specificity for infection
toxin B gene Rapid (hours) Requires laboratory and molecular
expertise
High cost
*There are recent contradictory data regarding assay sensitivity.

Both toxins are cytotoxic, causing disruption of the
actin cytoskeleton and tight junctions, and resulting in
decreased transepithelial resistance, fluid accumulation
and destruction of the intestinal epithelium68,69,71.
C. difficile toxins also cause the release of various
inflammatory mediators from intestinal epithelial
cells, mast cells and macrophages. Such cytokines
affect enteric nerves and sensory neurons, and pro-
mote an influx of inflammatory cells, thereby add-
ing to the fluid secretion, intestinal inflammation
and transmigration of neutrophils 72. Interestingly,
TcdB caused damage and oedema in cardiac tissue in
zebrafish embryos73 and lung damage in hamsters74.
The recent development of two systems for the
genetic manipulation of C. difficile has allowed fur-
ther investigation of the role of these toxins in C. dif-
ficile pathogenesis75,76. Comparison of mutants that
lacked one of the toxins revealed that TcdATcdB+
mutants retained the ability to kill hamsters, whereas
TcdA+TcdB mutants were not virulent in hamsters76.
These results are consistent with the clinical findings
that TcdATcdB+ strains cause the entire spectrum of
symptoms of CDI, and with experiments on human
colonic tissue in which TcdB was shown to be more
potent than TcdA in causing mucosal necrosis and
decreasing barrier function71. These data contradicted
early experiments which showed that TcdA by itself had
enterotoxic effects and caused haemorrhagic fluid secre-
tion, inflammation and necrosis of intestinal tissue after
intragastric challenge of hamsters whereas TcdB by itself
had no effect and caused oedema in the small intestine,
haemorrhage in the lungs and death only when applied
with a sub-active concentration of TcdA74.
TcdA and TcdB belong to a group of large clostrid-
ial toxins (lCTs) that includes Tcsl and TcsH from
Clostridium sordellii, TcnA from Clostridium novyi and
Tcpl from Clostridium perfringens types B and C77,78.
lCTs are single-chain proteins with three main functional
domains: an amino-terminal binding domain with charac-
teristic repeats, a carboxy-terminal catalytic domain and a
putative translocation domain77. The role of several other
functional motifs has been further elucidated by struc-
tural studies70 (fIG. 3). lCTs glycosylate small GTPases of
the Rho and Ras families in the host cell, rendering them
inactive and leading to the cytoskeletal changes described
above68,70,79. The trisaccharide Gal1(13)Gal(14)
GlcNac is part of the receptor for TcdA in animals, but is
probably not present in humans. Instead, in human cells,
the glycoprotein gp96 was shown to be a co-receptor for
TcdA80. The toxins have different tropisms for the host cell
membrane; TcdA binds more effectively on the apical side
of the host cell and TcdB binds to an unknown receptor
on the basolateral side of the host cell68. After endocytosis,
the proteolytic activity of TcdA and TcdB leads to cleavage
of the catalytic domain from the holotoxin, which is then
transferred into the cytoplasm through a toxin-mediated
pore. This cleavage requires only inositol phosphate from
the host cell as a co-substrate81,82.

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REVIEWS

a PaLoc (19 kb)

tcdR tcdB tcdE tcdA tcdC

Catalytic domain Translocation domain Binding domain

102 286 288 365 516 544 767 956 1128 1652 1678
N C
Trp DXD motif Cysteine Hydrophobic Aspartate
enzymatic protease region protease
activity
Subtrate
specificity
b CDT locus (4.3 kb)

cdtR cdtA cdtB

18 1383 1 2631
Catalytic domain Translocation and binding domain

N 50 kDa C N 100 kDa C

Figure 3 | Toxins produced by Clostridium difficile. a | Two large toxins, toxin A and toxin B (TcdA and TcdB),
are encoded on the pathogenicity locus (PaLoc), which comprises five genes. In non-toxigenic strains,
Nature this| Microbiology
Reviews region
is replaced by a short 115 bp sequence. Both toxins are single-chain proteins, and several functional domains and
motifs have been identitifed. TcdB is shown in detail below the PaLoc. b | A third toxin, the binary toxin or CDT, is
encoded on a separate region of the chromosome (CdtLoc) and comprises three genes. The binary toxin is composed
of two unlinked proteins, CdtB and CdtA. CdtB has a binding function and CdtA is the enzymatic component.

TcdA and TcdB are encoded together with tcdR (for-
merly known as tcdD), which encodes an alternative
sigma factor that is involved in positive transcriptional
regulation83, tcdC, which encodes a negative regulator 84,
and tcdE, which encodes a protein that has similarity
to phage holins85, in a well defined genetic element, the
pathogenicity locus (Paloc)86,87. The Paloc is present at
the same chromosomal integration site in all toxigenic
C. difficile strains that have been analysed to date. In non-
toxinogenic (TcdATcdB) strains, the Paloc is replaced
by 115 bp of non-coding sequence. The DNA sequence
of the Paloc is variable, and strains with changes in this
region are defined as different toxinotypes88 (BOX 2).
TcdA and TcdB are produced during the late log and
stationary phases89, and their production depends on
the strain and environmental factors, such as nutrient
levels (for example, levels of glucose, amino acids and
biotin), temperature and the presence of sub-inhibi-
tory levels of antibiotics9092. In addition to TcdR and
TcdC, factors outside the Paloc, including Cody93,
also participate in the regulation of toxin synthesis.
The binary toxin, CDT, belongs to the clostridial
binary toxins, a group of toxins that are unrelated
to TcdA and TcdB, and is composed of two pro-
teins, CdtA and CdtB. CdtB binds to host cells and
translocates CdtA, the catalytic component, into the
cytosol where it ADP-ribosylates actin molecules.
Interestingly, binary toxin is usually produced by
C. difficile strains with variant (but still functional)
tcdA and tcdB genes 88,94 . The genes that encode
CdtA and CdtB are located in the binary toxin locus
(Cdtloc), together with one other gene, the regulatory
cdtR gene 95. The role of CDT in disease is not well
understood. It is cytotoxic for Vero cells in vitro 96 and
enterotoxic in a rabbit ileal loop assay. However, wild-
type strains that produce CDT but do not produce
TcdA or TcdB colonize but do not kill hamsters97.
Surface layer proteins and adherence. The surface layer
proteins of vegetative C. difficile are additional potential
virulence factors. These proteins are integral to the adher-
ence of the organism to the gut mucosa and can induce
both inflammatory and antibody responses in the host98102.
There is considerable variability between the surface pro-
teins of different strains, particularly in surface layer pro-
tein A (SlpA). Investigation of the relationship between
surface proteins and the virulence of epidemic C. difficile
BI/NAP1/027 strains is preliminary, but the results
indicate that SlpA is altered in these strains and is asso-
ciated with increased adherence to human intestinal
epithelial cells103.
Sporulation. C. difficile forms spores that are highly
resistant to desiccation, chemicals and extreme tem-
peratures. Spores frequently contaminate the environ-
ment around patients with CDI, potentially persisting
for months and even years. Interestingly, C. difficile
epidemic strains have a greater sporulation capacity
in vitro than non-outbreak strains104.
C. difficile spores were recently shown to survive the
temperatures and disinfectant treatment of typical hos-
pital laundering cycles and to cross-contaminate bed lin-
ens during a wash cycle105. In vitro, exposure of epidemic
C. difficile strains to sub-inhibitory concentrations

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2009 Macmillan Publishers Limited. All rights reserved


a b
Figure 4 | The clinical outcome of Clostridium difficile infection. The local
inflammatory effects of C. difficile infection result in the formation
NatureofReviews
volcano-like
| Microbiology
lesions (a); later, pseudomembranes are formed that are composed of the destroyed
intestinal cells and leukocytes (b).
of non-chlorine-based cleaning agents (detergent or
hydrogen peroxide) significantly increased sporula-
tion capacity, an effect that is not generally seen with
chlorine-based cleaning agents104,106. Working-strength
concentrations of five different cleaning agents inhibited
the growth of C. difficile in vitro but only chlorine-based
cleaning agents inactivated C. difficile spores104.
There is no simple relationship between antibiotic-
mediated depletion of the colonic microbiota and the
induction of C. difficile spore germination with sub-
sequent toxin production. Rather, antibiotic exposure
might directly stimulate C. difficile proliferation (that
is, cause the germination of spores, which are the usual
type of cells that are acquired and can remain quies-
cent in the gut) and toxin production, which occurs
in late log phase91,92. The bacteriological response to
vancomycin varies among strains that cause CDI, and
possibly correlates with the germination capacity 107.
Vancomycin was not active against spores in the gut
model, but oritavancin, an investigational lipoglyc-
opeptide, prevented the outgrowth of C. difficile
spores108. Further investigation of the factors that affect
both sporulation and germination could provide insights
into the risk factors and treatment options for CDI.
Prevention of CDI
Barrier methods and environmental hygiene. Infection
control measures to prevent CDI in hospitals are
of two main types: those that attempt to prevent
C. difficile spores from reaching patients and those that
reduce the risk of CDI should the patient ingest the
organism. Isolation of patients with CDI (in private
rooms if possible), and the use of gowns and gloves by
health care workers are effective barrier methods 109.
Hand hygiene is also an important barrier method,
although the commonly used alcohol hand rubs or
gels that are effective against other organisms do not
remove C. difficile spores, and therefore traditional
hand washing is preferred. In addition, the handles
of electronic thermometers become readily contami-
nated and can spread C. difficile and other pathogens,
such as vancomycin-resistant enterococci, to other
patients. Removal of these thermometers and replace-
ment with disposable thermometers has resulted in
marked reductions in CDI rates110,111.
NATURE REVIEWS | MICrobIology
2009 Macmillan Publishers Limited. All rights reserved
REVIEWS
The hospital environment is contaminated with C. dif-
ficile spores in proportion to the type of patient in the
room, and the hands of health care workers become con-
taminated in proportion to the contamination of the room
environment 112. The rooms of patients with CDI and
active diarrhoea have the highest contamination rates (up
to 50%), followed by the rooms of patients who are colo-
nized with C. difficile but do not have diarrhoea (~25%)
and the rooms of patients who do not harbour C. difficile
(<10%)113. Efforts to reduce environmental contamina-
tion by C. difficile spores is difficult because traditional
detergents and quaternary ammonium cleaning agents are
not sporicidal and might actually enhance sporulation in
some cases106. A sporicidal hypochlorite solution (at least
1,000 parts per million available chlorine) can significantly
reduce spore contamination and CDI rates114,115. Because
of its corrosive effect on the environment, pungent odour,
and cutaneous and respiratory irritation to those who
use it, bleach cleaning is usually reserved for use during
outbreaks or settings where CDI is epidemic.
Altered antibiotic prescribing. Some antibiotics are
less likely to induce CDI than others. These include
penicillin and vancomycin, and some broad-spectrum
agents, such as gentamicin and anti-pseudomonal
penicillins, with or without a -lactamase inhibitor.
However, prescribing low-risk antibiotics must be
combined with restricted prescribing of high-risk anti-
biotics, as shown during a 5-year surveillance study
of the effects of introducing piperacillintazobactam
and restricting cefotaxime as a control intervention to
reduce the incidence of CDI116.
Studies in which the use of broad-spectrum antibiotics,
specifically cephalosporins or clindamycin, was restricted
provided the most robust evidence that altering antibiotic
prescribing can control CDI117,118. Despite the association
of fluoroquinolones with an increased incidence of CDI,
which has been notable since the emergence of ribotype
027, intervention studies that provide convincing evi-
dence of cause and effect are lacking. Fluoroquinolones
were identified retrospectively as a significant risk fac-
tor during a large outbreak of CDI62. CDI rates subse-
quently decreased in association with the restriction of
first- (21%), second- (93%) and third-generation (79%)
cephalosporins, clindamycin (87%), macrolides (78%) and
ciprofloxacin (29%). However, use of respiratory fluoro-
quinolones (predominantly moxifloxacin) and piperacil-
lintazobactam increased by 79% and 114%, respectively,
as the outbreak was controlled119. Following one outbreak
of CDI, multiple infection control measures were used,
including an increase in the prescription of moxifloxacin
and ciprofloxacin (levofloxacin was removed from the
formulary), to reduce the incidence of infection from
peak levels to baseline rates120. These reports emphasize
why efforts to manipulate antibiotic prescribing must not
displace optimal infection prevention and control prac-
tice, which aims to reduce to a minimum the chance that
pathogens such as C. difficile can spread in health care set-
tings. Clearly, even the use of low-risk antibiotics can lead
to CDI if it is undermined by frequent patient acquisition
of C. difficile.
VOlUME 7 | jUly 2009 | 533
REVIEWS

TcdA
Bacterial cells
TcdB
Pseudomembrane

Mediators

Blood vessel

Figure 5 | Clostridium difficile pathogenesis. C. difficile colonizes the intestine (colon) after disruption of the normal
intestinal flora. To what extent adhesion and biofilm production are involved in the pathogenesis of C. difficile is unknown;
Nature
in the schematic, bacterial cells are shown as free cells and attached to host cells. Toxigenic strains Reviewstoxin
produce | Microbiology
A and
toxin B (TcdA and TcdB). TcdA binds to the apical side of the cell and, after internalization, causes cytoskeletal changes
that result in disruption of tight junctions and loosening of the epithelial barrier, in cell death or in the production of
inflammatory mediators that attract neutrophils. Disruption of tight junctions enables both TcdA and TcdB to cross the
epithelium. TcdB binds preferentially to the basolateral cell membrane. Both toxins are cytotoxic and induce the release of
various immunomodulatory mediators from epithelial cells, phagocytes and mast cells, resulting in inflammation and the
accumulation of neutrophils. In an animal model, TcdB was shown to have a tropism for cardiac tissue, which would require
that TcdB enter the bloodstream.

Probiotics. A meta-analysis provided insufficient evi-
dence to support the use of probiotics as a preventive
measure for CDI121; some caution is needed in inter-
preting such reviews, given the diversity of probiotics
and end points used in various studies122,123. lack of
standardization of preparations, including quality con-
trol to minimize variations in bacterial counts during
storage, and the possibility of inducing bacteraemia or
fungaemia remain drawbacks of probiotic use.
Summary
The rising incidence and severity of infections with C. dif-
ficile BI/NAP1/027 has refocused clinical and research
efforts on CDI. Past and current epidemiological trends
in the United Kingdom and Canada indicate that other
types will eventually supplant this type in most hospitals.
With the availability of molecular typing methods, we are
now better prepared to recognize these changes, but it will
be of utmost importance to maintain a practice of stool
culturing to make these organisms available for molecu-
lar tracking. Clinical diagnostic testing remains prob-
lematic, as no single test is sensitive, specific and rapid.
The possibility of an increase in community-associated
CDI must be clarified and addressed to avoid replicat-
ing the experience with community-associated meti-
cillin-resistant Staphylococcus aureus infection. Clearly,
non-hospital-associated reservoirs are emerging and
C. difficile is capable of spreading in animal hosts. The
development of more tools for the genetic manipulation
of C. difficile will be crucial to identifying additional
virulence factors, especially in the toxin variant strains.
Understanding the relationship between C. difficile
spores and vegetative cells with the mucosal interface in
the host, and the subsequent host immune response, is
also crucial. The results of these studies should in turn
lead to an understanding of bacterial interference strate-
gies and, ultimately, the development of vaccines for the
prevention of CDI.

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Acknowledgements
M.R. was supported by EU grant 223585, ERA NET
PathoGenoMics grant and ARRS grant J3-0194-0377-08.
Competing financial interests
The authors declare competing financial interests: see web
version for details.
DATABASES
Entrez Genome Project:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprj
Clostridium difficile | Clostridium novyi | Clostridium
perfringens | Staphylococcus aureus
UniProtKB: http://www.uniprot.org
CDT | CdtA | CdtB | SlpA | TcdA | TcdB
FURTHER INFORMATION
Clostridium difficile toxinotypes:
http://www.mf.uni-mb.si/mikro/tox/
Ohio Department of Health:
http://www.odh.ohio.gov/alerts/cdiff1.aspx
All lInks ArE ACTIVE In ThE onlInE PDF
www.nature.com/reviews/micro