International Journal of Medical Microbiology 300 (2010) 387–395

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International Journal of Medical Microbiology

journal homepage: www.elsevier.de/ijmm

Mini Review

The emergence of ‘hypervirulence’ in Clostridium difficile

Stephen T. Cartman, John T. Heap, Sarah A. Kuehne, Alan Cockayne, Nigel P. Minton ∗
Centre for Biomolecular Sciences, School of Molecular Medical Sciences, Nottingham Digestive Diseases Centre NIHR Biomedical Research Unit, University of Nottingham,
University Park, Nottingham, NG7 2RD, UK

a r t i c l e i n f o a b s t r a c t

Keywords: The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the devel-
Clostridium difficile oped world is considerable in terms of mortality, morbidity, and disease management. The incidence
Hypervirulence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-
Virulence factors
called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and
ClosTron
increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027,
Mutant generation
which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe,
it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiolog-
ical factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is
known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to
fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative
strains have been suggested to produce more toxin A and B in the ‘laboratory flask’ (most likely due to a
frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also
exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However,
the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile vari-
ants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means
that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse
genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence
factors using this approach should help lead to the rational development of therapeutic countermeasures
against CDAD.
© 2010 Elsevier GmbH. All rights reserved.

1. Introduction
All members of the genus Clostridium are obligate anaerobic bac-
teria that are unified by their ability to form endospores, the most
highly resistant life forms on earth. They are one of the largest bac-
terial genera. More than 300 species have been described, although
currently less than 100 are recognised at the species level. The vast
majority of the genus is entirely benign, undertaking all manner
of useful biotransformations, including the production of biofuels.
Thus, concerns over global warming and escalating crude oil costs
dictate the reintroduction of the commercial production of bio-
fuels such as butanol by solventogenic clostridia from renewable
biomass, e.g., C. acetobutylicum. Moreover, experimental advances
have now demonstrated that clostridia have a significant role in
the treatment of cancer, by capitalising on the unique ability of
spores to selectively germinate in tumours (e.g., C. sporogenes
and C. novyi). However, despite the beneficial properties of many
clostridial species, it is the antics of a few that give the genus a
∗ Corresponding author. Tel.: +44 115 846 7458; fax: +44 115 823 2120.
E-mail address: nigel.minton@nottingham.ac.uk (N.P. Minton).
bad name. Over 90% of clinical disease is caused by just 12 species,
and it is these organisms that tend to make the headlines. Thus,
necrotic enteritis due to C. perfringens is causing devastation to
intensively reared fowl following the EU-directed withdrawal of
antibiotic growth promoters from feedstuffs. Post 9/11, there are
heightened public concerns over the use of C. botulinum by bioter-
rorists, but the largest and most strident headlines are currently
reserved for C. difficile.
2. Clostridium difficile-associated disease (CDAD)
Toxinogenic strains of C. difficile are carried by 7–11% of hospi-
talised patients (Poutanen and Simor, 2004). The reservoirs of C.
difficile include contaminated environments and surfaces within
healthcare facilities and infected patients. Colonized individuals
may become asymptomatic carriers or manifest symptoms, depen-
dent on their status. Healthy adults are generally resistant, a
consequence of the protective effects of their colonic flora. Per-
turbation of this flora, most often following exposure to broad
spectrum antibiotics (such as penicillins, cephalosporins, and clin-
damycin), leads to loss of colonisation resistance, enabling C. difficile
to proliferate and cause disease symptoms. Other risk factors

1438-4221/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2010.04.008
388 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
include various physical interventions (surgery, nasogastric tubes,
and enemas), antiperistaltic drugs and antacids, and exposure to
chemotherapy or immunosuppressive agents (Poutanen and Simor,
2004). The symptoms of those individuals that develop the disease
range from a mild, self-limiting diarrhoea to the potentially fatal
pseudomembranous colitis (PMC) (Mylonakis et al., 2001). Mortal-
ity rates can be as high as 30%.
Elderly patients are especially at risk of C. difficile-associated
disease (CDAD). Although the carriage rate in neonates is higher
(over 60%), they rarely develop disease, most likely a conse-
quence either of an inability of toxins to attach to the neonatal
mucosa or through protection from maternally-acquired anti-
bodies against the toxins. Neonatal carriage rates fall to levels
equivalent to adults by the age of 3 years and upwards (Kelly et
al., 1994; Poutanen and Simor, 2004). Although generally regarded
as a healthcare-associated disease, the occurrence of community-
associated disease is increasingly being reported, e.g., 9.3% of 703
patients with diarrhoea visiting German general practitioners over
a 5-month period were diagnosed with CDAD (Weil et al., 2007). C.
difficile is now also recognised as an important cause of enteric dis-
ease in animals (Songer and Anderson, 2005; Clooten et al., 2008).
Until recently, it had been generally regarded that strains present
in animals were distinct from those causing human infections. This
notion has now been modified somewhat by Debast et al. (2009)
who found that strains of PCR ribotype 078 (toxinotype V) isolated
from pigs with diarrhoea were identical to strains isolated from
cases of human CDAD.
The impact of CDAD in healthcare settings is considerable.
Patients require isolation, revised supportive therapy for under-
lying disease and for CDAD, specific therapy to eliminate C. difficile,
scrupulous hygiene in nursing, environmental decontamination,
and (in outbreaks) ward closure. The financial impact of CDAD
on the healthcare system is substantial and has recently been
estimated to cost at least $2 billion dollars in Europe (European
Centre for Disease Prevention and Control. ECDC Summary of CDAD
infection, 2005–2007) and $3.2 billion dollars per year in the US
(O’Brien et al., 2007). As the disease is principally a disease of the
elderly, these costs can be expected to almost double over the next
four decades in line with an increasing elderly population.
3. Current therapeutic and management options
Given the recognised role of antibiotic therapy in precipitating
CDAD, mild disease may be treated by withdrawing antibiotics and
providing additional supportive therapy (Hedge et al., 2008). The
organism has long been recognised as multiply antibiotic resistant
(e.g., erythromycin, clindamycin, lincomycin, tetracycline, chlor-
amphenicol), principally due to the presence of a large proportion
of mobile elements in the genome carrying antibiotic resistance
genes (Sebaihia et al., 2006). As a consequence, just 2 antibiotics are
available for treatment, metronidazole and vancomycin, which in
combination with improved hygiene and patient isolation currently
remain the main therapeutic option.
Whilst the latter has fewer side-effects and is more effective,
metronidazole has until now been preferred due to its lower price
and concerns over using a last resort antibiotic such as vancomycin.
However, metronidazole is becoming increasingly ineffective and
reports on the isolation of less susceptible strains are beginning
to appear (Brazier et al., 2001, 2008; Peláez et al., 2005; Baines
et al., 2008). This has led to moves towards adopting vancomycin
as the front-line antibiotic. This is extremely worrying, as there
are numerous examples of gene transfer between Enterococcus and
Clostridium, either demonstrated or inferred by isolation of con-
jugative transposons common to both genera (Roberts et al., 2001;
Stinear et al., 2001; Agersø et al., 2006; Launay et al., 2006). As the
major source of the vancomycin resistance gene vanA is Enterococ-
cus faecium, and it lives in the same niche as C. difficile, transfer of
vanA to C. difficile would appear to be just a matter of time. The
emergence of metronidazole-resistant strains carrying vanA would
have catastrophic consequences.
Other approaches are being investigated (Critchley et al., 2009;
Hedge et al., 2008; Rohde et al., 2009), including the use of
alternative antimicrobial agents, toxin-binding agents, immune-
modifying agents, probiotics, phage-based approaches, and faecal
replacement therapy. As yet, none have achieved wide acceptance.
As such, vaccination still remains an option. Current vaccine devel-
opment approaches have been based on a limited understanding
of the detailed mechanisms of disease causation and consequently
have focussed primarily on the generation of antibody responses
to neutralise the effects of toxins A and B which have histori-
cally been considered key virulence determinants in C. difficile. A
vaccine consisting of toxoids prepared from toxins A and B has
already undergone Phase 1 clinical trials (Acambis, 2007). Evidence
to support the potential of this approach comes from early exper-
imental vaccination studies using purified toxoids of toxins A and
B or crude culture filtrates which showed that systemic anti-toxin
IgG responses are important in protecting hamsters from lethal C.
difficile challenge and in preventing gut pathology (Giannasca et
al., 1999). Anti-TcdA responses have also been shown to protect
hamsters from C. difficile challenge (Kim et al., 1987), although a
recent study with isogenic tcdA mutants showed that TcdA is not
an essential virulence determinant (Lyras et al., 2009). This obser-
vation supports findings that clinical isolates of C. difficile which
are unable to produce TcdA still cause disease in humans (Drudy
et al., 2007). It is clear that the exact role played by TcdA in clinical
disease requires further clarification. Other hamster immunisation
studies have shown varying levels of protection from lethality and
diarrhoea following vaccination with TcdB toxoids (Giannasca et
al., 1999; Torres et al., 1995). However, anti-toxin responses alone
do not appear to result in elimination of C. difficile from the gut, and
vaccinated animals still remain colonised (Kim et al., 1987; Torres
et al., 1995) and potentially infectious. In addition, observed varia-
tion in the sequence of tcdB among C. difficile isolates (Stabler et al.,
2008) raises the possibility that a vaccine based on TcdB alone may
ultimately encounter problems with the breadth of cover elicited.
4. Increased incidence of CDAD
Once regarded as relatively uncommon, there has been a
steadily increasing upward trend in the incidence of CDAD. Analy-
sis of infection rates in US hospitals from 1986 to 2001 found that
the incidence had significantly increased (incidence rate approxi-
mately 5.5 per 10,000 patient-days) in hospital intensive care units
(ICU) with >500 beds (Archibald et al., 2004). The upward trend was
confirmed in 2 subsequent studies, where rates were perceived to
have doubled. Thus, McDonald et al. (2006) reported a doubling
of the rate (as any diagnosis) between 1996 and 2003 (from 31
to 61 per 100,000 population) and Elixhauser and Jhung (2008)
found 76.9 episodes of CDAD per 10,000 discharges in 2005, a rate
more than twice of that in 1999 (37.6 per 10,000 discharges). More
recently, a survey of 12.5% of all US acute-care facilities indicated
that there had been no decrease in rates reported by 82% of respon-
dents over the preceding 3 years; the overall C difficile prevalence
rate was 13.1 per 1000 inpatients (Jarvis et al., 2009). Broadly sim-
ilar infection rates were also noted in Canada (Gravel et al., 2009),
where the overall incidence rate for adult patients admitted to the
hospitals surveyed was 4.6 cases per 1000 patient admissions and
65 per 100,000 patient-days.
Nowhere is the rise in CDAD rates more visible than in the
UK. Accordingly, there has been a significant documented increase
through the 1990s and 2000s, rising from 1100 cases in 1990
peaking at 55,635 in 2006 (equivalent to 2.45 infections per 1000
 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
Fig. 1. C. difficile incidence figures for England and Wales. Figures from The Health
Protection Agency.
bed-days) (Fig. 1). Not unexpectedly, there has been a concomi-
tant increase in the number of deaths attributable to CDAD. This
trend has continued into 2007, where despite the decrease in
reported incidence (from 55,635 to 50,392), there was a 28%
increase in C. difficile-associated deaths in 2007 compared to 2006.
This equates to over 5 times as many deaths (8324) in England
and Wales than MRSA (1593). Similar trends have been noted
elsewhere in Europe (Borgmann et al., 2008; Suetens, 2008). For
example, in Spain (Asensio et al., 2008), the prevalence rates of
CDAD increased from 3.9 to 12.2 cases per 10,000 hospitalised
patients and showed a significantly increasing secular trend from
1999 through 2007 (prevalence rate ratio per each year increment
1.09; 95% CI 1.05–1.14).
In the UK at least, various reasons have been suggested for
this extraordinary rise in incidence and mortality. These include
the introduction of mandatory reporting in the UK from 2004,
the increasing age of the population and therefore the number at
risk, increased antibiotic resistance, lower standards of hygiene in
healthcare facilities, overcrowding in hospitals, and the emergence
of so-called hypervirulent strains.
5. Hypervirulent strains
In the view of many, the emergence of more highly virulent
strains has contributed to the problem of increased CDAD inci-
dence (Kuijper et al., 2007; Pépin et al., 2004). The most prominent
of these are strains which belong to ribotype 027, but are also
characterised as toxinotype III, North American pulsed field gel
electrophoresis type 1 (NAP1) and restriction endonuclease anal-
ysis group BI. The occurrence of these strains is associated with
an excessive use of quinolone antibiotics and in North Amer-
ica has been responsible for a 5-fold increase in the historical
average of CDAD, more severe disease (complications increased
from 7.1% to 18.2%), higher relapse rates (from 20.8% to 47.2%),
increased mortality (from 4.7% to 13.8%), and greater resistance
to fluoroquinolone antibiotics (Kuijper et al., 2006). Since 2005,
individual countries have developed surveillance studies to mon-
itor the spread of ribotype 027. In the United States, cases of C.
difficile ribotype 027 infection have been reported from at least
38 states (http://www.cdc.gov/ncidod/dhqp/id Cdiff.html). It has
been reported in 16 European countries (Kuijper et al., 2008). Ribo-
type 027 isolates have been responsible for outbreaks in Belgium,
Germany, Finland, France, Ireland, Luxembourg, The Netherlands,
Switzerland, and the United Kingdom (England, Wales, Northern
Ireland, and Scotland). They have also been detected in Austria,
Denmark, Sweden, Norway, Hungary, Poland, and Spain. Three
countries experienced imported patients with CDAD due to ribo-
type 027 who acquired the infection abroad (Kuijper et al., 2007).
During May and June of 2005, a prospective CDAD study was con-
ducted in 38 hospitals from 14 different European countries. A total
389
of 66 different PCR ribotypes were characterised, with ribotype 027
accounting for only 6.2% of isolates (Barbut et al., 2007).
Whilst the proportion of 027 isolates in the Barbut et al. (2007)
study is low, it is an average taken across 14 countries and may
not accurately reflect the present day situation. The speed with
which ribotype 027 can become predominant is amply demon-
strated by events in the UK. Laboratory reports amassed over the
period 1990–2003 had indicated that the predominant ribotype
was 001 (55%), with ribotype 106 a distant second (Brazier, 2005).
Over the period 2005–2007, 3 ribotypes had come to predominate
in approximately equal proportions (Brazier et al., 2007). These
were ribotypes 106 (26.2%), 027 (25.9%), and ribotype 001 (25.1%).
More recently, 677 isolates were obtained from 186 hospitals in
the 9 geographical regions of England over the period April 2007 to
the end of March 2008. Typing revealed that PCR ribotype 027 was
now the most common strain isolated from symptomatic patients,
accounting for over 41.3% of all isolates. Ribotype 106 was the sec-
ond most common strain (20.2%), and Ribotype 001 had reduced to
only 7.8% of the total.
Typically, 027 strains possess a binary toxin gene and encode
a variant tcdC repressor gene (with an 18-bp deletion and a
frameshift mutation due to a single base pair deletion at position
117 relative to the ATG start codon). The alterations have been
suggested to account for the observed increase in toxin produc-
tion when cultured in the laboratory (Warny et al., 2005). Detailed
molecular analysis has shown that this variant TcdC repressor is
indeed defective, but as a consequence of the frameshift and not the
18-bp deletion (Dupuy et al., 2008). Other data, presented at the 2nd
International Meeting on C. difficile (June 2007), has demonstrated
that 027 strains possess altered surface proteins and increased
adherence to human intestinal epithelial cells which may also con-
tribute to enhanced virulence. A further contributing factor to the
emergence of 027 strains may have been acquisition of resistance
to fluoroquinolones and erythromycin antibiotics due to chromoso-
mal mutations in gyrA/B and the 23s RNA genes, respectively (Pépin
et al., 2005; Spigaglia et al., 2008; Schmidt et al., 2007). Thus, the
first 027 strain to be isolated in France during 1988 was susceptible
to both antibiotics.
It should be noted that whilst ribotype 027 strains have received
all the attention, other strains may present an equivalent threat
in terms of disease severity. In many countries, a different ribo-
type can predominate, but be extremely rare elsewhere. Thus,
the second most common ribotype in the UK, ribotype 106, was
entirely absent from the European study of Barbut et al. (2007). In
The Netherlands, ribotype 078 has significantly increased over the
period February 2005 to February 2008, from 3% to 13%. Patients
infected with ribotype 078 were younger (67.4 vs. 73.5 years;
p < 0.01) than those infected with 027 and more frequently had
community-associated disease (17.5% vs. 6.7%; odds ratio 2.98; 95%
CI 2.11–8.02). The rates of severe diarrhoea and attributable mor-
tality were essentially the same for both ribotypes. Ribotype 078
isolates possess a number of features in common with ribotype
027 strains. They both harbour genes encoding toxin A and toxin
B and the genes for binary toxin. They also possess a variant tcdC
gene, although the deletion and frameshift are different for the 2
ribotypes (a 39-bp deletion and a single base pair deletion at posi-
tion 184 in the case of ribotype 078). Significantly, the ribotype
078 strains isolated from humans have recently been shown to be
genetically related to isolates from pigs (Goorhuis et al., 2008).
6. Virulence factors of C. difficile
The emergence of dominant strains that cause more severe dis-
ease is suggestive of one or more changes in bacterial encoded
determinants that contribute to the disease process. Despite
390 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
its medical importance, the pathogenesis of C. difficile infection
remains poorly understood. Attention has largely focussed on the
2 large toxins, A and B. Initially, toxin A was widely acknowledged
as the principle factor responsible for disease. However, the sub-
sequent recognition that a significant number of strains do not
produce toxin A, yet remain highly pathogenic suggested that toxin
A could have little if any role to play in disease. This interpretation
has been supported by the study of Lyras et al. (2009) which demon-
strated that inactivation of the tcdA gene through the creation of an
unstable, single cross-over mutant, had little effect on the virulence
of C. difficile. However, it should be noted that a proportionately
stable mutant created in our laboratory using the ClosTron yield
contradictory results. Thus, a tcdB mutant producing only TcdA
proved lethal in the hamster model (Sarah Kuehne, Steve Cart-
man, John Heap, Michelle Kelly, Alan Cockayne, and Nigel P. Minton,
unpublished results).
Certain C. difficile strains, and in particular strains of ribotype
027, also produce an actin-specific ADP-ribosyltransferase CDT, a
binary toxin encoded by cdtA-cdtB. Whilst not absolutely required
for virulence, CDT may enhance virulence. Other factors undoubt-
edly contribute to virulence, particularly the initial colonisation
process. Some putative virulence factors that could play a role in
adherence and intestinal colonization have already been identi-
fied. These include the S-layer proteins (Cerquetti et al., 2000), the
cell wall protein Cwp66 (Waligora et al., 2001), GroEL (Hennequin
et al., 2001), Fbp68 fibronectin-binding protein (Hennequin et al.,
2003), and FliC-FliD, components of flagella (Tasteyre et al., 2001).
In addition to mediating the attachment of bacteria to host tis-
sues, adhesins may be biological effectors in vivo that influence
the outcome of the host–pathogen interactions. Thus, C. diffi-
cile S-layer proteins may contribute to the pathogenicity of the
microorganism by perturbing the fine balance of inflammatory and
regulatory cytokines in human monocytes and monocyte-derived
dendritic cells (Ausiello et al., 2006). Cwp84, a cysteine protease,
may contribute to the degradation of host tissue integrity, to the
dissemination of the infection and to Slp maturation (Janoir et al.,
2007).
Surface antigens frequently elicit a host immune response dur-
ing colonisation and infection. Indeed, levels of antibodies directed
against the flagellar proteins and Cwp84 were significantly higher
in a control group than in a patient group with CDAD (Péchiné et al.,
2005a). Similarly, the S-layer proteins have shown to be immun-
odominant in the host. This suggests that these proteins are able
to induce an immune response that could play a role in the host
defence mechanisms.
Blocking of the initial colonisation process, which involves adhe-
sion to the colonic mucosa, has been considered as an alternative
target for vaccination approaches. Analysis of serum antibody
responses in control and C. difficile-infected groups identified fla-
gellar antigens FliC and FliD and the surface protease Cwp84 as
potential vaccine candidates (Péchiné et al., 2005a,b). Subsequent
immunisation studies showed that recombinant FliD in combi-
nation with a crude flagella extract or Cwp84 and a cell surface
extract all reduced colonisation in the human flora-associated
mouse model though colonisation was not totally eliminated in
any of the vaccinated animals (Péchiné et al., 2007). However, in a
second study, immunisation with a crude surface layer extract and
adjuvant failed to elicit significant protective immune responses
against lethal challenge in the hamster infection model (Ní Eidhin
et al., 2008).
7. Mutant generation
Other potential virulence factors which present possible ther-
apeutic targets are apparent in both the annotated genome
of strain 630 and the various partial sequences available
(http://www.sanger.ac.uk). However, definitive proof of their
involvement in disease has yet to be obtained. It is only through
mutation that the function of any particular gene can be definitively
ascribed. However, despite the best efforts of numerous laborato-
ries around the globe, in the preceding decades there have been
relatively few directed mutants generated in the genus Clostrid-
ium. Those that have been generated have almost exclusively been
generated using classical recombination-based methods by select-
ing for the integration of either replication defective or deficient
plasmid on the basis of acquisition of an antibiotic resistance gene
(Heap et al., 2008). The larger proportion of these mutants has been
based on the mutagenic integration of the entire plasmid by a single
cross-over event and has therefore been unstable. In C. difficile, the
handful of mutants generated to date have all been made through
integration of either a deficient (Liyanage et al., 2001) or defec-
tive (O’Connor et al., 2006; Dineen et al., 2007; Lyras et al., 2009)
plasmid. They are, therefore, unstable.
8. Group II introns
An alternative approach to recombination is to utilise group
II introns. These interrupt protein coding or RNA genes and are
widespread in both eukaryotic organelle and prokaryotic genomes.
Group II introns rely on the activity of an element-encoded
multifunctional intron-encoded protein (IEP) to bring about the
self-catalytic splicing of the intron from RNA. One of the best stud-
ied elements is the ‘Ll.LtrB intron’ found in the ltrB gene (and
its cognate IEP, LtrA) of selected Lactococcus lactis plasmids. The
details of the process by which this intron mediates its splicing
and subsequent insertion (retro-homing) into intron-free copies
of ltrB have been thoroughly characterised. By determining those
factors important in ltrB target specificity, the Lambowitz labora-
tory have been able to devise procedures whereby defined changes
to the intron sequence may be employed to target Ll.LtrB-derived
introns to almost any gene of interest (Mohr et al., 2000; Perutka
et al., 2004). In the continued presence of LtrA, dependent on the
orientation of insertion, the intron is capable of secondary retro-
transposition events. To prevent this, in the system devised, ltrA
was moved from within Ll.LtrB to a distal plasmid-borne location.
It follows that the selection of cells in which the plasmid-carrying
LtrA is lost, prevents any subsequent mobility of the intron element.
The insertions generated are therefore stable. The retargeted intron
elements created have been termed ‘Targetrons’.
Basic Targetron technology was first used to generate a plc
mutant of C. perfringens (Chen et al., 2005) using a simple phe-
notypic plate assay to identify the desired mutant. It is, however,
disadvantaged by an inability to positively select cells in which the
intron element has inserted into the desired location. As integra-
tion frequencies can vary widely between target sites, if a simple
phenotypic screen is unavailable, then screening for the desired
mutant can be prohibitively laborious. To overcome this deficiency,
the Lambowitz laboratory constructed a modified Ll.LtrB intron in
which an antibiotic resistance gene was inserted that had been
inactivated by insertion of a region of DNA encoding a group I
intron. In the ‘twintron’ element made, the constitutive elements
were orientated relative to one another such that the group I intron
is lost (through self-catalytic splicing) during group II intron retro-
transposition. As a consequence, the antibiotic gene in the inserted
group II intron becomes reactivated, allowing positive selection of
the desired mutant (Karberg et al., 2001; Zhong et al., 2003). This
new marker was termed a retrotransposition-activated selectable
marker (RAM). A widely used example is based on the kan gene,
and a plasmid carrying this element, pACD4K-C, is available from
Sigma Aldrich (http://www.sigma-genosys.com/targetron/.
 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395 391

9. The ClosTron

As kanamycin cannot be used as a selectable marker in Clostrid-

Fig. 2. The second generation ClosTron vector pMTL007C-E2 (Heap et al., 2010).
The second generation ClosTron plasmid pMTL007C-E2 uses the strong fdx pro-
moter from C. sporogenes to direct expression of the Gp II intron, and contains
FRT sites flanking the RAM to facilitate FLP-mediated marker removal. The plas-
mid also features a lacZ’ stuffer sequence that is replaced during intron retargeting,
allowing clones containing successfully retargeted plasmids to be identified by
blue/white screening, restriction analysis, or PCR. Further details may be found at
www.clostron.com.
ium spp., we elected to construct a new element incorporating a
clostridial RAM based on the ermB gene of the Enterococcus fae-
calis plasmid pAM␤1. This new element, the ClosTron (Heap et al.,
2007), enables the group II intron integration event to be positively
selected (using erythromycin or lincomycin). Therefore, it is pos-
sible to inactivate any non-essential gene, without the need for a
phenotypic screen appropriate to the gene of interest.
Insertion of the group I intron into the ermB gene to generate an
ermB-based RAM was achieved by extending the 5 end of the ermB
gene by 12 codons. In addition, the weak native promoter of ermB
was replaced with the stronger promoter of the thlA gene of C. ace-
tobutylicum ATCC 824 (Girbal et al., 2003) to increase expression
of the new ermB element. The complete ermB RAM was incorpo-
rated into a group II intron cassette within an E. coli/clostridial
shuttle vector, giving rise to the plasmid pMTL007 (Heap et al.,
2007). This novel targeted intron system was termed the ‘ClosTron’
to denote that it is functionally analogous to Targetron systems,
but optimised for use in clostridia. Superfluous sequence has now
been removed from the original pMTL007 vector, giving rise to the
second-generation vector pMTL007C-E2 (Fig. 2). Further details on
this improved system may be found at www.clostron.com.

Fig. 3. ClosTron mutagenesis using plasmid pMTL007C-E2. (A) The plasmid is transferred into C. difficile by conjugation. (B) Expression from the C. acetobutylicum thiolase
promoter (PthlA ) does not confer erythromycin resistance, as the Grp I intron is transcribed in the wrong orientation to splice out of the ermB transcript. (C) Expression of
the Grp II intron yields a transcript which binds to the LtrA protein to form a ribonuclear protein complex (RNP). In this case, the Grp I intron is transcribed in the correct
orientation and thus splices out of the transcript. (D) The RNP locates the desired genomic target, the Grp II intron RNA is inserted, and LtrA reverse transcribes the cDNA
strand. Host enzymes then displace the RNA strand and repair the insertion site to give a permanent dsDNA insertion. The plasmid pMTL007C-E2 is subsequently lost due to
segregational instability (not shown). (E) The ermB marker can be removed from the insertion site by introducing a plasmid expressing the FLP recombinase enzyme. (F) This
catalyses recombination between the FRT sites flanking ermB, resulting in ‘flip-out’ of the ermB gene. The plasmid expressing FLP is also lost due to segregational instability
and the ClosTron can then be used again to make further mutations (Heap et al., 2010).
392 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395

Fig. 4. PCR screening of ClosTron mutants. (A) PCR primers P1 and P2 are designed to anneal either side of the ClosTron insertion site. Primer P3 (EBS universal primer) anneals
to Grp II intron sequence. PCR screening is carried out using primer pairs P1 and P2 (B), and P2 and P3 (C). Lane M, 1 kb plus DNA ladder (Invitrogen); lane 1, H2 O-negative
control; lane 2, genomic DNA from parent C. difficile strain; lane 3, plasmid pMTL007C-E2; lane 4, erythromycin-resistant C. difficile mutant.

In ‘proof-of-principle’ studies, the ClosTron was used to inser-
tionally inactivate a total of 19 clostridial genes (Heap et al., 2007),
including 5 in the C. difficile strain 630erm (Hussain et al., 2005).
Typically, 1000s of erythromycin-resistant colonies were obtained
in each individual experiment with C. difficile, with the majority
of the integration events taking place at the intended target site
(Heap et al., 2007). Predictably, no evidence was obtained for mul-
tiple insertion of the element, and the mutations generated were
extremely stable. The latter conclusion was based on the observa-
tion that when late exponential cultures of the 3 clostridial pyrF
mutants were obtained (in C. acetobutylicum, C. sporogenes, and C.
difficile) and plated out without dilution onto minimal media lack-
ing uracil, no growth (reverted colonies) was obtained (Heap et al.,
2007).
10. Use of the ClosTron
The practicalities of gene inactivation using the ClosTron are
extremely straightforward. The group II intron is retargeted by
substituting a ∼350-bp fragment in plasmid pMTL007 with a
proportionately-sized fragment carrying the desired nucleotide
changes. The fragment is generated in a one-step ‘Splicing by
Overlap Extension’ (SOE)-PCR using primers which are auto-
matically designed by a computer algorithm (http://www.sigma-
genosys.com/targetron/), and template DNA supplied in the Sigma
Aldrich Targetron Gene Knockout System kit. The modified plas-
mid is transferred from an E. coli donor into C. difficile 630erm
by conjugation (Purdy et al., 2002). C. difficile transconjugants are
selected based on thiamphenicol resistance, which is encoded by
the catP gene of pMTL007 vectors. In order to isolate ClosTron
integrant colonies, transconjugant colonies are simply transferred
from growth medium supplemented with thiamphenicol onto
growth medium supplemented with erythromycin (but lacking thi-
amphenicol). A detailed account of events occurring in the host
cell during this time is illustrated in Fig. 3. Erythromycin-resistant
clones are screened for insertion of the ClosTron by PCR (Fig. 4).
Typically, 25–100% of the clones are found to have inserted in the
correct gene. Clones confirmed to be correct by PCR are screened
for thiamphenicol sensitivity to confirm loss of the ClosTron plas-
mid (and thus loss of the ltrA gene). Both plasmids, pMTL007
and pMTL007C-E2, are based on the replicon of pCB102 (Minton
and Morris, 1981), which is segregationally unstable in C. diffi-
cile. Therefore, they are easily lost from this host in the absence of
selection. Indeed, simple restreaking of colonies in the absence of
thiamphenicol is sufficient for the loss of pMTL007 and pMTL007C-
E2 from C. difficile.
The ClosTron is an insertional mutagen. There are practical
issues to be borne in mind in its use. Not all insertions may be muta-
genic. A proximal insertion site may result in a novel ORF encoding
a fore-shortened protein with potential activity. Distal insertions
can result in the production of a truncated protein which retains
activity. Insertion sites should be chosen appropriately. The poten-
tial for polar effects should also be considered. Depending upon
the orientation of intron insertion, downstream genes or upstream
genes (via antisense and transcriptional interference effects) may
be affected. To alleviate polar effects, second-generation versions of
pMTL007 have been constructed (pMTL007C-E2) in which the ermB
can be subsequently removed using FLP:FRT technology (Heap et
al., 2010). The use of this system has the added benefit that it
provides the facility to make multiple mutations. Thus, following
the removal of ermB from a ClosTron-generated mutant, a second
gene can be targeted, and its inactivation selected on the basis of
acquisition of erythromycin resistance. More significantly, we have
now provided, free-of-charge, at www.clostron.com, access to an
automated intron design bioinformatics tool, which, in combina-
tion with outsourced construction of retargeted intron plasmids,
has significantly simplified the process. Using the resource at
www.clostron.com, the application of this technology is more rapid
 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
and cost-effective, and more fully exploits the potential of group II
introns.
11. Conclusions
The occurrence of CDAD has now risen to epidemic propor-
tions in healthcare facilities throughout the developed world. The
evolution of C. difficile strains which are better adapted to the mod-
ern healthcare environment is almost certainly the reason for this,
although the specific factors involved remain unclear at present.
The apparent dominance of ribotype 027 strains and the recent
emergence of ribotype 078 strains are particularly interesting.
Traits which are common to strains of these ribotypes, includ-
ing variant tcdC genes and the presence of binary toxin, may be
important. However, it is not clear whether they are sufficient to
explain the increased incidence of CDAD, and other factors may
well be involved. Fluoroquinolone resistance may be important,
although this is also common in less prevalent strains of C. diffi-
cile (Spigaglia et al., 2008). Nonetheless, the emergence of more
antibiotic-resistant profiles amongst strains of C. difficile would
have profound implications for how the disease is treated. Of par-
ticular concern is the prospect of lateral transfer of van genes from
enterococcal species which may give rise to vancomycin-resistant
strains of C. difficile.
It is clear that genetic studies will play a vital role in identifying
the unrecognized virulence factors of C. difficile and thus, mutage-
nesis tools will play a vital role. Indeed, the value of reverse genetic
studies has recently been demonstrated by Lyras et al. (2009) in
elucidating the role of toxin B in C. difficile virulence. Historically,
genetic manipulation of C. difficile has been extremely difficult.
However, the advent of ClosTron technology now provides a facile
means of generating genetically stable mutants of C. difficile (Heap
et al., 2007). Since publication of the ClosTron, we have generated
over 150 different mutants between ourselves and collaborators.
Moreover, we have made numerous mutants in C. difficile strain
R20291, the ribotype 027 isolate responsible for the outbreaks of
CDAD in the UK’s Stoke Mandeville hospital in 2003–2004 and
2004–2005. It is worth noting that the ClosTron procedure is
extremely rapid (taking as few as 10–14 days per mutant), highly
efficient (100s of retargeted clones obtained per experiment), and
reproducible (100% success rate among non-essential genes), and
is revolutionizing functional genomic studies in clostridia based on
reverse genetics (Bradshaw et al., 2010; Burns et al., 2010; Emerson
et al., 2009; Kirby et al., 2009; Twine et al., 2009; Underwood et al.,
2009). In contrast, forward genetic studies have until now been
prohibited due to the absence of an effective random mutagen.
The situation has now changed with the recent development of a
mariner-based transposon system (Cartman and Minton, 2010). Its
utility has been exemplified in C. difficile R20291, where transposi-
tion occurred at random genomic loci, as determined by sequencing
the insertion site of 60 randomly picked transposition events. The
only requirement for insertion is the sequence ‘TA’.
The deployment of mutational tools will facilitate studies aimed
at providing fundamental aspects of clostridial biology. One such
area is spore formation and germination, an as yet little stud-
ied phenomenon in Clostridium. Spore longevity is as remarkable
as their ability to withstand inimical agents, such as extremes of
temperature, desiccation, radiation, and disinfectants. Spores are
thought to play a pivotal role in the spread of CDAD. It has been
suggested that epidemic strains of C. difficile appear to be more
proficient in their ability to form spores than standard laboratory
strains (Akerlund et al., 2008; Fawley et al., 2007). If this is gener-
ally the case, infection control measures which specifically target
spores and their formation offer hope as a means of controlling
the spread of CDAD throughout healthcare settings. However, it
393
should be noted that data in our laboratory does not support this
observation (Burns et al., 2010). Further analysis is clearly required.
Acknowledgements
The authors wish to acknowledge the financial support of the
supported by SysMO (project number 2; http://www.sysmo.net),
the BBSRC (grants BB/D522797/1, BD/D522289/1, BB/F003390/1),
the MRC (G0601176), and the EU (grants LSHE-CT-2006-037870
and HEALTH-F3-2008-223585).
References
Acambis, 2007. Acambis Concludes C. difficile Vaccine Formulation Work
On Schedule and Will Commence Proof-of-concept Trial in 2008,
http://www.acambis.co.uk/default.asp?id=2033 [online], accessed March
30, 2009.
Asensio, A., Vaque-Rafart, J., Calbo-Torrecillas, F., Gestal-Otero, J.J., López-Fernández,
F., Trilla-Garcia, A., Canton, R., EPINE Working Group, 2008. Increasing rates
in Clostridium difficile infection (CDI) among hospitalised patients, Spain
1999–2007. Euro Surveill. 13 (July (31)), 18943.
Agersø, Y., Pedersen, A.G., Aarestrup, F.M., 2006. Identification of Tn5397-like and
Tn916-like transposons and diversity of the tetracycline resistance gene tet(M)
in enterococci from humans, pigs and poultry. J. Antimicrob. Chemother. 57,
832–839.
Akerlund, T., Persson, I., Unemo, M., Noren, T., Svenungsson, B., Wullt, M., Bur-
man, L.G., 2008. Increased sporulation rate of epidemic Clostridium difficile type
027/NAP1. J. Clin. Microbiol. 46, 1530–1533.
Archibald, L.K., Banerjee, S.N., Jarvis, W.R., 2004. Secular trends in hospital-acquired
Clostridium difficile disease in the United States. J. Infect. Dis. 189, 1585–1589.
Ausiello, C.M., Cerquetti, M., Fedele, G., Spensieri, F., Palazzo, R., Nasso, M., Frezza, S.,
Mastrantonio, P., 2006. Surface layer proteins from Clostridium difficile induce
inflammatory and regulatory cytokines in human monocytes and dendritic cells.
Microbes Infect. 8, 2640–2646.
Baines, S.D., O’Connor, R., Freeman, J., Fawley, W.N., Harmanus, C., Mastrantonio, P.,
Kuijper, E.J., Wilcox, M.H., 2008. Emergence of reduced susceptibility to metron-
idazole in Clostridium difficile. J. Antimicrob. Chemother. 62, 1046–1052.
Barbut, F., Mastrantonio, P., Delmée, M., Brazier, J., Kuijper, E., Poxton, I., European
Study Group on Clostridium difficile (ESGCD), 2007. Prospective study of Clostrid-
ium difficile infections in Europe with phenotypic and genotypic characterisation
of the isolates. Clin. Microbiol. Infect. 13, 1048–1057.
Borgmann, S., Kist, M., Jakobiak, T., Reil, M., Scholz, E., von Eichel-Streiber, C., Gru-
ber, H., Brazier, J.S., Schulte, B., 2008. Increased number of Clostridium difficile
infections and prevalence of Clostridium difficile PCR ribotype 001 in southern
Germany. Euro Surveill. 13, pii: 19057.
Bradshaw, M., Marshall, K.M., Heap, J.T., Tepp, W.H., Minton, N.P., Johnson, E.A., 2010.
Construction of a nontoxigenic Clostridium botulinum strain for food challenge
studies. Appl. Environ. Microbiol. 76, 387–393.
Brazier, J.S., Fawley, W., Freeman, J., Wilcox, M.H., 2001. Reduced susceptibility of
Clostridium difficile to metronidazole. J. Antimicrob. Chemother. 48, 741–742.
Brazier, J.S., 2005. Antimicrobial susceptibility of polymerase chain reaction ribo-
types of Clostridium difficile commonly isolated from symptomatic hospital
patients in the UK. J. Hosp. Infect. 61, 11–14.
Brazier, J.S., Patel, B., Pearson, A., 2007. Distribution of Clostridium difficile PCR ribo-
type 027 in British hospitals. Euro Surveill. 12, 3182.
Brazier, J.S., Raybould, R., Patel, B., Duckworth, G., Pearson, A., Charlett, A., Duerden,
B.I., 2008. HPA Regional Microbiology Network, 2008. Distribution and antimi-
crobial susceptibility patterns of Clostridium difficile PCR ribotypes in English
hospitals, 2007–2008. Euro Surveill. 13 (41), pii: 19000.
Burns, D.A., Heap, J.T., Minton, N.P., 2010. SleC is essential for germination of Clostrid-
ium difficile spores in nutrient-rich medium supplemented with the bile salt
taurocholate. J. Bacteriol. 192, 657–664.
Cartman, S.T., Minton, N.P., 2010. A mariner-based transposon system for in
vivo random mutagenesis of Clostridium difficile. Appl. Environ. Microbiol. 76,
1103–1109.
Cerquetti, M., Molinari, A., Sebastianelli, A., Diociaiuti, M., Petruzzelli, R., Capo, C.,
Mastrantonio, P., 2000. Characterization of surface layer proteins from different
Clostridium difficile clinical isolates. Microb. Pathog. 28, 363–372.
Chen, Y., McClane, B.A., Fisher, D.J., Rood, J.I., Gupta, P., 2005. Construction of an alpha
toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile
group II intron. Appl. Environ. Microbiol. 71, 7542–7547.
Clooten, J., Kruth, S., Arroyo, L., Weese, J.S., 2008. Prevalence and risk factors for
Clostridium difficile colonization in dogs and cats hospitalized in an intensive
care unit. Vet. Microbiol. 129, 209–214.
Critchley, I.A., Green, L.S., Young, C.L., Bullard, J.M., Evans, R.J., Price, M., Jarvis, T.C.,
Guiles, J.W., Janjic, N., Ochsner, U.A., 2009. Spectrum of activity and mode of
action of REP3123, a new antibiotic to treat Clostridium difficile infections. J.
Antimicrob. Chemother. 63, 954–963.
Debast, S.B., van Leengoed, L.A., Goorhuis, A., Harmanus, C., Kuijper, E.J., Bergwerff,
A.A., 2009. Clostridium difficile PCR ribotype 078 toxinotype V found in diarrhoeal
pigs identical to isolates from affected humans. Environ. Microbiol. 11, 505–511.
394 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
Dineen, S.S., Villapakkam, A.C., Nordman, J.T., Sonenshein, A.L., 2007. Repression of
Clostridium difficile toxin gene expression by CodY. Mol. Microbiol. 66, 206–219.
Drudy, D., Harnedy, N., Fanning, S., Hannan, M., Kyne, L., 2007. Emergence and con-
trol of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium
difficile. Infect. Control. Hosp. Epidemiol. 28, 932–940.
Dupuy, B., Govind, R., Antunes, A., Matamouros, S., 2008. Clostridium difficile toxin
synthesis is negatively regulated by TcdC. J. Med. Microbiol. 57, 685–689.
Emerson, J.E., Reynolds, C.B., Fagan, R.P., Shaw, H.A., Goulding, D., Fairweather, N.F.,
2009. A novel genetic switch controls phase variable expression of CwpV, a
Clostridium difficile cell wall protein. Mol. Microbiol. 74, 541–556.
Elixhauser, A., Jhung, M., 2008. Clostridium difficile-associated disease in US hos-
pitals, 1993–2005. In: HCUP Statistical Brief No. 50, April 2008. US Agency for
Healthcare Research and Quality, Rockville, MD, Available at: http://www.hcup-
us.ahrq.gov/reports/statbriefs/sb50.pdf, accessed March 23, 2009.
European Centre for Disease Prevention and Control, 2005. ECDC Summary of CDAD
Infection, 2005–2007.
Fawley, W.N., Underwood, S., Freeman, J., Baines, S.D., Saxton, K., Stephenson, K.,
Owens Jr., R.C., Wilcox, M.H., 2007. Efficacy of hospital cleaning agents and
germicides against epidemic Clostridium difficile strains. Infect. Control Hosp.
Epidemiol. 28, 920–925.
Giannasca, P.J., Zhang, Z.X., Lei, W.D., Boden, J.A., Giel, M.A., Monath, T.P., Thomas Jr.,
W.D., 1999. Serum antitoxin antibodies mediate systemic and mucosal protec-
tion from Clostridium difficile disease in hamsters. Infect. Immun. 67, 527–538.
Girbal, L., Mortier-Barriere, I., Raynaud, F., Rouanet, C., Croux, C., Soucaille, P.,
2003. Development of a sensitive gene expression reporter system and an
inducible promoter-repressor system for Clostridium acetobutylicum. Appl. Env-
iron. Microbiol. 69, 4985–4988.
Goorhuis, A., Bakker, D., Corver, J., Debast, S.B., Harmanus, C., Notermans, D.W., Berg-
werff, A.A., Dekker, F.W., Kuijper, E.J., 2008. Emergence of Clostridium difficile
infection due to a new hypervirulent strain, polymerase chain reaction ribotype
078. Clin. Infect. Dis. 47, 1162–1170.
Gravel, D., Miller, M., Simor, A., Taylor, G., Gardam, M., McGeer, A., Hutchinson,
J., Moore, D., Kelly, S., Boyd, D., Mulvey, M., Canadian Nosocomial Infection
Surveillance Program, 2009. Health care-associated Clostridium difficile infec-
tion in adults admitted to acute care hospitals in Canada: a Canadian Nosocomial
Infection Surveillance Program study. Clin. Infect. Dis. 48, 568–576.
Heap, J.T., Pennington, O.J., Cartman, S.T., Carter, G.P., Minton, N.P., 2007. The
ClosTron: a universal gene knock-out system for the genus Clostridium. J. Micro-
biol. Methods 70, 452–464.
Heap, J.T., Cartman, S.T., Pennington, O.J., Cooksley, C.M., Scott, J.C., Blount, B., Burns,
D., Minton, N.P., 2008. Development of genetic knock-out systems for clostridia.
In: Bruggermann, H., Gottschalk, G. (Eds.), Clostridia: Molecular Biology in the
Post-Genomic Era. Caister Academic Press.
Heap, J.T., Kuehne, S.A., Ehsaan, M., Cartman, S.T., Cooksley, C.M., Scott, J.C., Minton,
N.P., 2010. The ClosTron: mutagenesis in Clostridium refined and streamlined. J.
Microbiol. Methods 80, 49–55.
Hedge, D.D., Strain, J.D., Heins, J.R., Farver, D.K., 2008. New advances in the treatment
of Clostridium difficile infection (CDI). Ther. Clin. Risk. Manage. 4, 949–964.
Hennequin, C., Porcheray, F., Waligora-Dupriet, A., Collignon, A., Barc, M., Bourlioux,
P., Karjalainen, T., 2001. GroEL (Hsp60) of Clostridium difficile is involved in cell
adherence. Microbiology 147, 87–96.
Hennequin, C., Janoir, C., Barc, M.C., Collignon, A., Karjalainen, T., 2003. Identification
and characterization of a fibronectin-binding protein from Clostridium difficile.
Microbiology 149, 2779–2787.
Hussain, H.A., Roberts, A.P., Mullany, P., 2005. Generation of an erythromycin-
sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and
demonstration that the conjugative transposon Tn916DeltaE enters the genome
of this strain at multiple sites. J. Med. Microbiol. 54, 137–141.
Jarvis, W.R., Schlosser, J., Jarvis, A.A., Chinn, R.Y., 2009. National point prevalence of
Clostridium difficile in US health care facility inpatients. Am. J. Infect. Control 37,
263–270.
Janoir, C., Péchiné, S., Grosdidier, C., Collignon, A., 2007. Cwp84, a surface-associated
protein of Clostridium difficile, is a cysteine protease with degrading activity on
extracellular matrix proteins. J. Bacteriol. 189, 7174–7180.
Karberg, M., Guo, H., Zhong, J., Coon, R., Perutka, J., Lambowitz, A.M., 2001. Group
II introns as controllable gene targeting vectors for genetic manipulation of
bacteria. Nat. Biotechnol. 19, 1162–1167.
Kelly, C.P., Pothoulakis, C., LaMont, J.T., 1994. Clostridium difficile colitis. N. Engl. J.
Med. 330, 257–262.
Kim, P.H., Iaconis, J.P., Rolfe, R.D., 1987. Immunization of adult hamsters against
Clostridium difficile-associated ileocecitis and transfer of protection to infant
hamsters. Infect. Immun. 55, 2984–2992.
Kirby, J.M., Ahern, H., Roberts, A.K., Kumar, V., Freeman, Z., Acharya, K.R., Shone, C.C.,
2009. Cwp84, a surface-associated, cysteine protease, plays a role in the matura-
tion of the surface layer of Clostridium difficile, J. Biol. Chem. 284, 34666–34673.
Kuijper, E.J., Barbut, F., Brazier, J.S., Kleinkauf, N., Eckmanns, T., Lambert, M.L., Drudy,
D., Fitzpatrick, F., Wiuff, C., Brown, D.J., Coia, J.E., Pituch, H., Reichert, P., Even, J.,
Mossong, J., Widmer, A.F., Olsen, K.E., Allerberger, F., Notermans, D.W., Delmée,
M., Coignard, B., Wilcox, M., Patel, B., Frei, R., Nagy, E., Bouza, E., Marin, M.,
Akerlund, T., Virolainen-Julkunen, A., Lyytikäinen, O., Kotila, S., Ingebretsen, A.,
Smyth, B., Rooney, P., Poxton, I.R., Monnet, D.L., 2008. Update of Clostridium
difficile infection due to PCR ribotype 027 in Europe. Euro Surveill. 31 (13), 31.
Kuijper, E.J., Coignard, B., Tüll, P., ESCMID Study Group for Clostridium difficile;
EU Member States; European Centre for Disease Prevention and Control, 2006.
Emergence of Clostridium difficile-associated disease in North America and
Europe. Clin. Microbiol. Infect. 12 (Suppl. 6), 2–18.
Kuijper, E.J., van Dissel, J.T., Wilcox, M.H., 2007. Clostridium difficile: changing epi-
demiology and new treatment options. Curr. Opin. Infect. Dis. 20, 376–383.
Launay, A., Ballard, S.A., Johnson, P.D., Grayson, M.L., Lambert, T., 2006. Transfer
of vancomycin resistance transposon Tn1549 from Clostridium symbiosum to
Enterococcus spp. in the gut of gnotobiotic mice. Antimicrob. Agents Chemother.
50, 1054–1062.
Liyanage, H., Kashket, S., Young, M., Kashket, E.R., 2001. Clostridium beijerinckii and
Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glyc-
erol dehydrogenase. Appl. Environ. Microbiol. 67, 2004–2010.
Lyras, D., O’Connor, J.R., Howarth, P.M., Sambol, S.P., Carter, G.P., Phumoonna, T.,
Poon, R., Adams, V., Vedantamm, G., Johnson, S., Gerding, D.N., Rood, J.I., 2009.
Toxin B is essential for virulence of Clostridium difficile. Nature 458, 1176–1179.
McDonald, L.C., Owings, M., Jernigan, J.B., 2006. Clostridium difficile infection
in patients discharged from US short-stay hospitals. Emerg. Infect. Dis. 12,
409–415.
Minton, N.P., Morris, J.G., 1981. Isolation and partial characterisation of three cryp-
tic plasmids from strains of Clostridium butyricum. J. Gen. Microbiol. 127, 325–
331.
Mohr, G., Smith, D., Belfort, M., Lambowitz, A.M., 2000. Rules for DNA target-site
recognition by a lactococcal group II intron enable retargeting of the intron to
specific DNA sequences. Genes Dev. 14, 559–573.
Mylonakis, E., Ryan, E.T., Calderwood, S.B., 2001. Clostridium difficile-associated diar-
rhea. Arch. Intern. Med. 161, 525–533.
Ní Eidhin, D.B., O’Brien, J.B., McCabe, M.S., Athié-Morales, V., Kelleher, D.P., 2008.
Active immunization of hamsters against Clostridium difficile infection using
surface-layer protein. FEMS Immunol. Med. Microbiol. 52, 207–218.
O’Brien, J.A., Lahue, B.J., Caro, J.J., Davidson, D.M., 2007. The emerging infectious
challenge of Clostridium difficile-associated disease in Massachusetts hospi-
tals: clinical and economic consequences. Infect. Control Hosp. Epidemiol. 28,
1219–1227.
O’Connor, J.R., Lyras, D., Farrow, K.A., Adams, V., Powell, D.R., Hinds, J., Cheung, J.K.,
Rood, J.I., 2006. Construction and analysis of chromosomal Clostridium difficile
mutants. Mol. Microbiol. 61, 1335–1351.
Péchiné, S., Janoir, C., Boureau, H., Gleizes, A., Tsapis, N., Hoys, S., Fattal, E., Col-
lignon, A., 2007. Diminished intestinal colonization by Clostridium difficile and
immune response in mice after mucosal immunization with surface proteins of
Clostridium difficile. Vaccine 25, 3946–3954.
Péchiné, S., Gleizes, A., Janoir, C., Gorges-Kergot, R., Barc, M.C., Delmée, M., Collignon,
A., 2005b. Immunological properties of surface proteins of Clostridium difficile.
J. Med. Microbiol. 54, 193–196.
Pechine, S., Janoir, C., Collignon, A., 2005a. Variability of Clostridium difficile sur-
face proteins and specific serum antibody response in patients with Clostridium
difficile-associated disease. J. Clin. Microbiol. 43, 5018–5025.
Peláez, T., Alcala, L., Alonso, R., Martín-Lopez, A., Garcia-Arias, V., Marín, M., Bouza, E.,
2005. In vitro activity of ramoplanin against Clostridium difficile, including strains
with reduced susceptibility to vancomycin or with resistance to metronidazole.
Antimicrob. Agents Chemother. 49, 1157–1159.
Pépin, J., Valiquette, L., Alary, M.E., Villemure, P., Pelletier, A., Forget, K., Pépin, K.,
Chouinard, D., 2004. Clostridium difficile-associated diarrhea in a region of Que-
bec from 1991 to 2003: a changing pattern of disease severity. Can. Med. Assoc.
J. 171, 466–472.
Pépin, J., Saheb, N., Coulombe, M.A., Alary, M.E., Corriveau, M.P., Authier, S., Leblanc,
M., Rivard, G., Bettez, M., Primeau, V., Nguyen, M., Jacob, C.E., Lanthier, L., 2005.
Emergence of fluoroquinolones as the predominant risk factor for Clostridium
difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin.
Infect. Dis. 41, 1254–1260.
Perutka, J., Wang, W., Goerlitz, D., Lambowitz, A.M., 2004. Use of computer-designed
group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase
genes. J. Mol. Biol. 336, 421–439.
Poutanen, S.M., Simor, A.E., 2004. Clostridium difficile-associated diarrhea in adults.
Can. Med. Assoc. J. 171, 51–58.
Purdy, D., O’Keeffe, T.A., Elmore, M., Herbert, M., McLeod, A., Bokori-Brown, M.,
Ostrowski, A., Minton, N.P., 2002. Conjugative transfer of clostridial shuttle vec-
tors from Escherichia coli to Clostridium difficile through circumvention of the
restriction barrier. Mol. Microbiol. 46, 439–452.
Roberts, A.P., Johanesen, P.A., Lyras, D., Mullany, P., Rood, J.I., 2001. Comparison
of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the
CW459tet(M) element from Clostridium perfringens shows that they have similar
conjugation regions but different insertion and excision modules. Microbiology
147, 1243–1251.
Rohde, C.L., Bartolini, V., Jones, N., 2009. The use of probiotics in the prevention and
treatment of antibiotic-associated diarrhea with special interest in Clostridium
difficile-associated diarrhea. Nutr. Clin. Pract. 24, 33–40.
Sebaihia, M., Wren, B.W., Mullany, P., Fairweather, N.F., Minton, N., Stabler, R., Thom-
son, N.R., Roberts, A.P., Cerdeno-Tarraga, A.M., Wang, H., Holden, M.T., Wright,
A., Churcher, C., Quail, M.A., Baker, S., Bason, N., Brooks, K., Chillingworth, T.,
Cronin, A., Davis, P., Dowd, L., Fraser, A., Feltwell, T., Hance, Z., Holroyd, S., Jagels,
K., Moule, S., Mungall, K., Price, C., Rabbinowitsch, E., Sharp, S., Simmonds, M.,
Stevens, K., Unwin, L., Whithead, S., Dupuy, B., Dougan, G., Barrell, B., Parkhill, J.,
2006. The multidrug-resistant human pathogen Clostridium difficile has a highly
mobile, mosaic genome. Nat. Genet. 38, 779–786.
Schmidt, C., Löffler, B., Ackermann, G., 2007. Antimicrobial phenotypes and molec-
ular basis in clinical strains of Clostridium difficile. Diagn. Microbiol. Infect. Dis.
59, 1–5.
Songer, J.G., Anderson, M.A., 2005. Clostridium difficile: an important pathogen of
food animals. Anaerobe 12, 1–4.
 S.T. Cartman et al. / International Journal of Medical Microbiology 300 (2010) 387–395
Spigaglia, P., Barbanti, F., Mastrantonio, P., Brazier, J.S., Barbut, F., Delmée, M., Kuijper,
E., Poxton, I.R., European Study Group on Clostridium difficile (ESGCD), 2008.
Fluoroquinolone resistance in Clostridium difficile isolates from a prospective
study of C. difficile infections in Europe. J. Med. Microbiol. 57, 784–789.
Stabler, R.A., Dawson, L.F., Phua, L.T., Wren, B.W., 2008. Comparative analysis of
BI/NAP1/027 hypervirulent strains reveals novel toxin B-encoding gene (tcdB)
sequences. J. Med. Microbiol. 57, 771–775.
Stinear, T.P., Olden, D.C., Johnson, P.D., Davies, J.K., Grayson, M.L., 2001. Enterococ-
cal vanB resistance locus in anaerobic bacteria in human faeces. Lancet 357,
855–856.
Suetens, C., 2008. Clostridium difficile: summary of actions in the European
Union. Euro Surveill. 13 (31), pii: 18944, Available online: http://www.
eurosurveillance.org/ViewArticle.aspx?ArticleId=18944.
Tasteyre, A., Barc, M.C., Collignon, A., Boureau, H., Karjalainen, T., 2001. Role of FliC
and FliD flagellar proteins of Clostridium difficile in adherence and gut coloniza-
tion. Infect. Immun. 69, 7937–7940.
Torres, J.F., Lyerly, D.M., Hill, J.E., Monath, T.P., 1995. Evaluation of formalin-
inactivated Clostridium difficile vaccines administered by parenteral and mucosal
routes of immunization in hamsters. Infect. Immun. 63, 4619–4627.
Twine, S.M., Reid, C.W., Aubry, A., McMullin, D.R., Fulton, K.M., Austin, J., Logan, S.M.,
2009. Motility and flagellar glycosylation in Clostridium difficile. J. Bacteriol. 191,
7050–7062.
395
Underwood, S., Guan, S., Vijayasubhash, V., Baines, S.D., Graham, L., Lewis, R.J.,
Wilcox, M.H., Stephenson, K., 2009. Characterisation of the sporulation initia-
tion pathway of Clostridium difficile and the role in toxin production. J. Bacteriol.
191, 7296–7305.
Waligora, A.J., Hennequin, C., Mullany, P., Bourlioux, P., Collignon, A., Karjalainen,
T., 2001. Characterization of a cell surface protein of Clostridium difficile with
adhesive properties. Infect. Immun. 69, 2144–2153.
Warny, M., Pépin, J., Fang, A., Killgore, G., Thompson, A., Brazier, J., Frost, E., McDon-
ald, L.C., 2005. Toxin production by an emerging strain of Clostridium difficile
associated with outbreaks of severe disease in North America and Europe. Lancet
366, 1079–1084.
Weil, H.P., Fischer-Brügge, U., Harmanus, C., Mattner, F., Gastmeier, P., Kuijper, E.J.,
2007. High incidence of Clostridium difficile-associated diarrhoea with a com-
munity onset in a hyperendemic region in Germany. In: Abstracts of the 17th
Europ. Congr. Clin. Microbiol. Infect. Dis., ICC, Munich, Germany, March 31–April
4, 2007.
Zhong, J., Karberg, M., Lambowitz, A.M., 2003. Targeted and random bacte-
rial gene disruption using a group II intron (targetron) vector containing
a retrotransposition-activated selectable marker. Nucleic Acids Res. 31,
1656–1664.