Biology of Clostridium difficile: Implications for Epidemiology and Diagnosis

MI65CH25-Carroll ARI 27 July 2011 9:11

Biology of Clostridium difficile:Implications for Epidemiologyand DiagnosisKaren C. Carroll1 and John G. Bartlett2

1Division of Medical Microbiology and 2Division of Infectious Diseases, The JohnsHopkins Medical Institutions, Baltimore, Maryland 21205; email: [email protected]

Annu. Rev. Microbiol. 2011. 65:501–21

First published online as a Review in Advance onJune 16, 2011

The Annual Review of Microbiology is online atmicro.annualreviews.org

This article’s doi:10.1146/annurev-micro-090110-102824

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4227/11/1013-0501$20.00

Keywords

toxins, toxinotypes, hypervirulent, microbiota, risk factors

Abstract

Clostridium difficile is an anaerobic, spore-forming, gram-positive rodthat causes a spectrum of antibiotic-associated colitis through the elab-oration of two large clostridial toxins and other virulence factors. Sinceits discovery in 1978 as the agent responsible for pseudomembranouscolitis, the organism has continued to evolve into an adaptable, aggres-sive, hypervirulent strain. Advances in molecular methods and improvedanimal models have facilitated an understanding of how this organ-ism survives in the environment, adapts to the gastrointestinal tract ofanimals and humans, and accomplishes its unique pathogenesis. Theadvances in microbiology have been accompanied by some importantclinical observations including increased rates of C. difficile infection, in-creased virulence, and multiple outbreaks. The major new risk is fluoro-quinolone use; there is also an association with proton pump inhibitorsand increased recognition of cases in outpatients, pediatric patients,and patients without recent antibiotic use. The combination of moreaggressive strains with mobile genomes in a setting of an expanded poolof individuals at risk has refocused attention on and challenged assump-tions regarding diagnostic gold standards. Future research is likely tobuild upon the advancements in phylogenetics to create novel strategiesfor diagnosis, treatment, and prevention.

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Phylogenetics:whole-genomecomparisons ofbacteria using a varietyof moleculartechniques to developa model of bacterialevolution

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 502THE BIOLOGY OF

CLOSTRIDIUM DIFFICILE . . . . . . . 502Microbiology . . . . . . . . . . . . . . . . . . . . . . 502Summary of the Clostridium difficile

Strain 630 Genome Sequence . . . 503The Pathogenicity Locus . . . . . . . . . . . 504Toxins A and B . . . . . . . . . . . . . . . . . . . . 504C. difficile Transferase . . . . . . . . . . . . . . 506Other Virulence Factors . . . . . . . . . . . . 507Toxin Variant Strains . . . . . . . . . . . . . . 507Pathophysiology . . . . . . . . . . . . . . . . . . . 508

EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . 508People and Patients as Reservoirs . . . 508Risks for C. difficile Infection . . . . . . . . 509Changing Epidemiology . . . . . . . . . . . 510Emergence of Ribotype 027 . . . . . . . . 510

DIAGNOSIS . . . . . . . . . . . . . . . . . . . . . . . . . 510Nucleic Acid Amplification

Methods. . . . . . . . . . . . . . . . . . . . . . . . 513

INTRODUCTION

Major contributions to the current knowledgeon Clostridium difficile and the disease it causesare the discovery of C. difficile in 1935 (63, 125),studies of antibiotic-associated enterocolitis inguinea pigs as reported by Hambra et al. (64)in 1943 and later by De Somer and colleagues(37), the recognition of a cytopathic toxin in theguinea pig model by Green in 1974 (62), thehighly successful clinical experience with oralvancomycin for Staphylococcus aureus enterocol-itis as reported by Kahn & Hall in 1966 (71),and the prospective study of clindamycin col-itis by Tedesco et al. reported in 1974 (132).These observations set the stage to challengeS. aureus as the major pathogen of antibiotic-associated colitis using rodent models and clin-ical observations (18, 19). These studies con-verged with reports published in the late 1970sdefining the role of C. difficile as the major agentof antibiotic-associated colitis in people and an-imals (14–17, 19, 54).

Understanding the biology of C. difficilecolonization as it relates to the human in-testinal microbiota has been facilitated bythe development of numerous mouse modelssuch as gnotoxenic (germ-free animals) andmonoxenic (single-species inoculation) mice(32). Human fecal flora remains stable and re-tains bacterial and enzymatic properties wheninoculated into the gnotobiotic mice (32). Suchhuman microbiota-associated (HMA) animalsfacilitate research on the effects of antimicro-bial treatment on the human microbiota andon the protective effects of probiotics (32).Data from the human microbiome project arelikely to extend the observations provided bythe HMA mouse model.

In addition to better animal models, molecu-lar advances including whole-genome sequenc-ing have enabled sophisticated phylogeneticsstudies (5, 66, 121, 127). This review summa-rizes to date our knowledge of the biology ofC. difficile and the application of that knowl-edge to the understanding of important facetsof C. difficile epidemiology and diagnosis.

THE BIOLOGY OFCLOSTRIDIUM DIFFICILE

Microbiology

C. difficile is an obligate anaerobic, gram-positive, spore-forming rod that is bothsaccharolytic and proteolytic. Its name isderived from observations by Hall & O’Toole(63) on the difficulty in isolating the organismbecause of its slow growth (doubling time40 to 70 min) compared with that of otherClostridium spp. (35, 63). During logarithmicgrowth, when vegetative cells predominate,the organism is very aerointolerant. C. difficilespores are the transmissible form, contributeto survival of the organism in the host, andare responsible for recurrence of disease whentherapy is stopped. Like other bacterial spores,they are metabolically dormant, survive forlong periods, and are resistant to harsh physicalor chemical treatments.

Lawley et al. (80) developed protocols toeffectively purify spores of C. difficile strain

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Toxinotype: refers toa particular strain ofC. difficile based onPCR-restrictionfragment analysis ofthe PaLoc

Pathogenicity locus(PaLoc): the sectionof the C. difficilegenome that encodesthe genes that regulatetoxin production

630. Transmission electron microscopy of thesehighly purified spores showed that they arecomposed of an electron-dense outer surface,with the spore coat composed of concentric in-ner rings (80). The outer surface of the sporecoat contains an exosporium that is smooth inthe dormant state but develops numerous fila-mentous projections that attach to the colonicmicrovilli during germination (101). Beyondthe exosporium are the spore coat, a clearer cor-tex region, and then an inner membrane adja-cent to the core (80). Densely packed ribosomesand nucleoproteins are seen in the core (80).

Highly purified spores permitted the studyof their biology and infectivity (80). Cholate,taurocholate, or glycocholate supplementationof brain heart infusion (BHI) increased germi-nation rates 100- to 1000-fold more than BHIagar plates alone, an observation that has im-plications for recovery of C. difficile from clin-ical and environmental samples (80). Purifiedspores demonstrated resistance to high temper-atures and 70% ethanol but were inactivated bysporicidal agents. Environmental spores infectmice in a dose-dependent manner; the dose re-quired to infect 50% of the mice (ICD50) is∼7 spores per cm2.

Genes encoding the spore proteins aredispersed throughout the bacterial genome.Proteases and stress response proteins thatputatively protect the spore from oxidativestress during germination are abundant, asare metabolic proteins (80). Surface-exposedproteins may interact with the extracellularenvironment and may be important in cellularadhesion (e.g., S-layer protein, SlpA) andgermination (80). These surface-exposedproteins are potential candidates for vaccinetargets and novel diagnostic tests. Regulationof sporulation is intimately associated withtoxin regulation (137).

Summary of the Clostridium difficileStrain 630 Genome Sequence

The complete genome sequence of C. difficilestrain 630, an epidemic, toxinotype X clinicalstrain that is virulent and multi-drug resistant,

was determined by Sebaihia et al. (121). Thiswork has provided insight into the geneticfactors responsible for organism survival, re-sistance, and virulence. Some of the importantobservations are highlighted here. The genomeof this organism has a circular chromosome(4,290,252 bp) and a plasmid (7,881 bp).When compared with the genomes of fourother pathogenic Clostridium spp. includingC. botulinum and C. tetani, C. difficile shares only15% of its coding sequences with these speciesand 50% of the coding sequences are unique toC. difficile (121). Moreover, 11% of the genomeconsists of mobile genetic elements such asconjugative transposons capable of integratinginto and excising from the host genome (121).

One of the most important regions of thegenome is the pathogenicity locus (PaLoc),which contains five genes, tcdA, tcdB, tcdC, tcdR,and tcdE, responsible for the synthesis and reg-ulation of toxins A (TcdA) and B (TcdB). Thegenes (cdtA and cdtB) encoding the binary toxinare not found on the PaLoc. Other importantgenetic loci important in virulence include slpA,a single gene that encodes the S-layer; genes en-coding extracellular matrix-binding domains; acollagen protease gene, suggesting that diges-tion of collagen may occur in vivo; surface-anchored proteins important for covalent at-tachment to peptidoglycan; a putative TypeIV pilus biosynthesis locus involved in fimbrialbiosynthesis; and a cluster of genes involved inextracellular polysaccharide synthesis (121).

C. difficile has a large number of genes im-portant for survival in the gastrointestinal tract.Several of these coding sequences are dedicatedto carbohydrate transport and metabolism. Onedistinguishing feature is the presence of genesthat encode an enzyme p-hydroxyphenylacetatedecarboxylase that allows C. difficile to produceand tolerate high concentrations of p-cresol, abacteriostatic compound. The ability to toler-ate this compound may provide a competitiveadvantage over other intestinal microbes and isresponsible for the horse manure odor charac-teristic of this organism in culture. A 19-genecluster exists that is involved in ethanolaminedegradation, an important constituent of

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Hypervirulent: refersto toxin variant strainsof C. difficile that areassociated withincreased toxinproduction and severeclinical disease

Clade: a group ofgenetically relatedorganisms

phospholipids and a source of carbon and nitro-gen for C. difficile found in the host’s diet. Fi-nally, C. difficile encodes an enzyme (missing inother sequenced clostridia) important for sec-ondary bile acid synthesis, as well as other genesthat allow it to tolerate bile acids.

Stabler et al. (127) carried out whole-genome microarray analysis based on thesequenced genome of C. difficile strain 630.They applied Bayesian phylogeny after arrayhybridization. Their observations confirmthose of Sebaihia et al. and provide someinsight into the phylogenetic evolution ofC. difficile. Fifty-five human isolates, repre-senting several hypervirulent strains, toxinvariant isolates, and toxin-negative isolates,and 20 animal isolates were analyzed. Thisdetailed analysis revealed four major clades:a hypervirulent clade that contained 20 of 21hypervirulent isolates; a defective toxin cladewith all 14 TcdA−, TcdB+ isolates; and twoclades, HA1 and HA2, in which animal isolateswere intermixed with human isolates (127).

The genomes of the hypervirulent strainshad a number of deletions compared withstrain 630. Some fragments of conjugativetransposons (CTn2 and Ctn5) present in theepidemic and hypervirulent strains are not seenin nonepidemic older isolates. The function ofthe loci is not clear, but they may participate indrug transport (127). The toxin-defective cladeconsisted of TcdA−, TcdB+ isolates fromthe United States, the United Kingdom, andIreland, confirming widespread geographicaldistribution of this clone. HA1 had mostlyhuman strains including toxinotype 0 and ref-erence strain VPI 10463, a toxinotype 0 isolatethat hyperproduces toxins A and B. The HA2clade contains mostly animal isolates. Stableret al. (127) also made several important observa-tions, not detailed here, regarding deleted lociand genetic diversity that may be important forunderstanding host adaptation and virulence.

The Pathogenicity Locus

The PaLoc of C. difficile is approximately19.6 kb in size and is stable and conserved in

toxigenic strains (30, 138). Nontoxigenic strainslack the PaLoc; however, isolates with a defec-tive PaLoc can still cause disease (30, 34). Fivegenes are present on the PaLoc, tcdA, tcdB, tcdC–tcdE, and tcdR (Figure 1). The two toxin genes,tcdA and tcdB, are closely aligned, separated byan intervening sequence (tcdE), and both aretranscribed in the same direction (138). tcdEencodes a holin, a protein whose pore-formingactivity allows the release of TcdA and TcdBfrom the cell (40, 138). tcdR, found upstreamof tcdB, is a major positive regulator of tcdAand tcdB expression (40, 138). It is responsiveto environmental conditions and is increasedduring stationary phase (40, 138). tcdC is founddownstream of tcdA (Figure 1) and is a nega-tive regulator of toxin production, an antisigmafactor that destabilizes the TcdR holoenzymeto prevent transcription of the PaLoc (30, 36,40, 42, 90, 138). tcdC is expressed during the ex-ponential phase of growth; all other genes areexpressed during stationary phase (30, 36, 42,90, 138).

Dineen et al. (42) have also shown that toxingene expression is repressed by another globalgene regulator called CodY. This regulator actsby monitoring environmental nutrient factors.The authors demonstrated that in the presenceof sufficient nutrients, such as certain aminoacids and GTP, CodY binds to the promoterregion of tcdR and represses toxin gene expres-sion (42). When nutrients in the environmentare lacking, toxin gene expression is derepressed(42). Identification of CodY as a toxin gene reg-ulator may provide a potential target for the de-velopment of compounds that are useful in thetreatment of C. difficile.

Toxins A and B

TcdA and TcdB are part of the large clostridialtoxin family along with Clostridium sordelliilethal toxin and hemorrhagic toxin and Clostrid-ium novyi alpha toxin (36, 138). Large clostridialtoxins are so named because of their large size(205 to 308 kDa), cytotoxicity, and mechanismof action. Both toxins are glucosyltrans-ferases. They transfer a UDP-glucose to small


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tcdR tcdB tcdE

PaLoc (19 kb)

tcdA tcdC

a

b

N C

1,6781,6521,128956544516365288286102 767

Aspartateprotease

Hydrophobic region

Substratespecificity

Trp DXD motifenzymatic activity

Cysteineprotease

CDT locus (4.3 kb)

18

cdtR cdtA cdtB

1,383 1 2,631

CC N 100 kDaN

Catalytic domain Translocation and binding domain

50 kDa

Binding domainTranslocation domainCatalytic domain

Figure 1(a) Two large toxins, toxin A and toxin B (TcdA and TcdB), are encoded on the pathogenicity locus (PaLoc),which comprises five genes. Both toxins are single-chain proteins, and several functional domains and motifshave been identified (see text for an explanation). TcdB is shown in detail below the PaLoc. (b) A third toxin,the binary toxin or CDT (C. difficile transferase), is encoded on a separate region of the chromosome(CdtLoc) and comprises three genes. The binary toxin is composed of two unlinked proteins, CdtB andCdtA. CdtB has a binding function and CdtA is the enzymatic component. Adapted by permission fromMacmillan Publishers Ltd: Nature Reviews Microbiology, Rupnik M, et al. 7:532 copyright 2009 (116).

GTPases, such as Rho, Rac, and Cdc42, inthe cell. These small proteins are important inregulating signaling pathways. Glycosylationdisrupts these pathways, which results in mor-phological changes, inhibition of cell divisionand membrane trafficking, and eventual celldeath (36, 138).

TcdA (308 kDa), an enterotoxin, was orig-inally believed to be the toxin associated withdisease, and therefore necessary for virulence,until TcdA−, TcdB+ strains were associatedwith outbreaks of severe C. difficile infection(52). TcdB (270 kDa), a cytotoxin, is 100- to1000-fold more toxic to culture cells than TcdAis. Lyras et al. (88) reported that TcdB, notTcdA, is essential for virulence. Hamsters in-fected with toxin B mutants (active toxin A,no toxin B) were less likely to die, and thedegree of in vitro cytotoxicity was less thanthat of wild-type animals (88). TcdA mutant

hamsters died as rapidly as wild-type animals.However, a recent paper by Kuehne et al. (74)refutes the assertion that only toxin B is essentialfor virulence and re-establishes the observationthat both toxins can cause significant disease.In that study, TcdA+, TcdB− isolates were aslikely to cause disease as the wild-type strains(74). The authors hypothesize that differencesin the hamster models or in the C. difficile iso-lates themselves used in the respective studiesmay account for the differences in their obser-vations (74, 88).

Both toxins are large single-stranded pro-teins, and recent X-ray crystallography andsmall angle X-ray scattering models (SAXS)of TcdB suggest four structural domains (5).These domains include (a) a biologically ac-tive N-terminal glucosyltransferase protrudingfrom the core of the protein; (b) a cysteine pro-tease domain; (c) a middle translocation section


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that contains a hydrophobic region implicatedin toxin delivery; and (d ) a C-terminal receptor-binding domain (5).

Toxin activity is located in the N-terminaldomain, and the first 543 amino acids (aa) aresufficient to elicit full glucosyltransferase activ-ity. This portion is delivered into the cytosolof host cells (68, 119). Cleavage of the bio-logically active 543-aa segment occurs betweenLeu543 and Gly544 in a highly conserved re-gion (115). Separation occurs by autoproteoly-sis via the cysteine protease domain (5, 58, 68,111, 115) and is dependent on host cell inosi-tolphosphate (111).

The C-terminal domain has short combinedrepetitive oligopeptides (CROPs) for receptorbinding. In animal models of TcdA, carbohy-drate structures play a role in toxin binding (5,116, 138). These carbohydrates are not presentin humans and the glycoprotein gp96 present inthe human colon is the receptor for TcdA (116).The central translocation domain is large, tak-ing up approximately 50% of the total size ofthe holotoxin. There is a hydrophobic stretchon the protein that likely facilitates membranepenetration during translocation (58).

An understanding of the genetics, structure,and function of each of these domains has led tothe following model of toxinogenesis. Toxinsenter the host cells by receptor-mediated endo-cytosis, and they require an acidified endosomefor translocation (36, 58, 111, 119, 138). Acidifi-cation induces a conformational change in bothtoxins, exposing the hydrophobic regions of thetranslocation domain and leading to subsequentpore formation through which the uncleavedcatalytic domain, followed by the cysteineprotease domain, translocates through theendosomal membrane into the cytosol (12, 58,108, 111, 115, 119). Host cell inositol hexaphos-phate induces autocleavage of the toxin by aC. difficile aspartate protease, resulting inbiologically active toxin (3, 58, 111, 116).

Once inside the cell, the toxins targetthe Ras superfamily of small GTPases (Rho,Rac, and Cdc) (42), modifying them throughglycosylation (138). Glycosylation preventsthe structural changes required for active

conformation of Rho GTPases, significantlyaltering the function of these proteins. Cellsshrink and become rounded owing to disaggre-gation of the actin cytoskeleton, eventually dy-ing (119, 138). Tight junctions between epithe-lial cells are disrupted. This allows neutrophilsto migrate to the intestines, contributing to theinflammatory response, typical of colitis (138).Biological inactivation of the GTPases resultsin serious physiological consequences such asinhibition of secretion, transcriptional regula-tion, and eventual apoptosis (119, 138). In addi-tion to direct cytotoxic effects, TcdA stimulatesrelease of tumor necrosis factor from activatedmacrophages as well as cytokine production(138). These activities cause fluid accumulationand further the inflammatory responses (138).

C. difficile Transferase

C. difficile transferase (CDTa), also known as bi-nary toxin (120, 130), is encoded by the Cdt lo-cus (CdtLoc). It is found in approximately 6%–12.5% of strains overall. Strains without binarytoxin CDT have a conserved 68-bp sequencein place of the CdtLoc (36). CDT is an ADP-ribosylating toxin that disrupts the cytoskeletonof the cell, leading to excessive fluid loss, round-ing of the cell, and eventual cell death (130).

CDT is composed of two subunits, CDTaand CDTb. Each component alone is notcytotoxic, together they cause cytotoxicity invitro (57, 130). C. difficile CDT is similar to thebinary toxins of other clostridia. The bindingsite for CDTa in the cell is unknown and itsrole in pathogenesis is unclear. The fact thatthe incidence of CDT is higher in some of theepidemic strains suggests that it contributesto the severity of disease. This is evidenced inthe work by Geric et al. (57). The investigatorschallenged hamsters and inoculated rabbit ilealloops with a TcdA−, TcdB−, CDT+ strain.Marked nonhemorrhagic fluid responses wereseen in the rabbit ileal loops. Of the hamsters,70%–80% became colonized, but showed nosigns of disease and no histological changes,and they did not die, compared with animals


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inoculated with TcdA+, TcdB+, CDT−strains that did die (57).

New findings by Schwan et al. (120) revealthat CDT induces the formation of novelthin, dynamic, microtubules on the surface ofepithelial cells, leading to increased adherenceof Clostridia in vitro and in vivo. Electronmicroscopy showed that these protrusionsincreased the adherence of Clostridia to the ep-ithelial cell surface by approximately fivefold invitro and fourfold in the mouse large intestine,thereby possibly playing an important role inintestinal colonization (120).

Other Virulence Factors

Although TcdA and TcdB are the major vir-ulence mechanisms in C. difficile disease, otherfactors are likely important to disease. C. difficilehas a crystalline or paracrystalline S-layer con-sisting of two proteins on the outer cell surfacethat coats the entire surface of the vegetativeorganism (26, 45, 50). These proteins are inte-gral to adherence and stimulate inflammationand induce antibody responses in the host (26,45, 50, 116). Several other C. difficile proteinsplay a role in attachment to intestinal epithelialcells. Ampicillin and clindamycin stimulate theincreased expression of these surface adhesins(36).

Toxin Variant Strains

Many strains of C. difficile have alterations in thesequences of one or more genes in the PaLoccompared with the sequences of the referencestrain VPI 10463. Rupnik et al. (114) devel-oped a polymerase chain reaction-restrictionfragment length polymorphisms (PCR-RFLP)method for toxinotyping C. difficile based onmutations in these genes (114). Currently thereare approximately 28 known toxinotypes (46).Despite the known role of TcdA in disease,the number of strains lacking TcdA recoveredfrom patients with clinical disease has increased.These strains have TcdB but contain deletionsin tcdA, resulting in a truncated form of thetoxin. The first A−, B+ strain to be character-ized was strain 8864 (toxinotype X) (46, 138).

It contained a 5.9-kb deletion in the 3′ end oftcdA as well as an insertion in tcdE (138). Thisstrain, which caused tissue damage in rabbitileal loops, is uncommon in humans (46). MostTcdA− strains have a 1.8-kb deletion in the 3′

CROP region of tcdA, the region responsiblefor attachment to host cell receptors (see ThePathogenicity Locus, above). The absence ofthis region also prevents detection by diagnos-tic assays that use antibodies directed againstthis area of the toxin. These isolates have beenrare causes of disease in the United States butconstitute anywhere from 3% of all isolates inthe United Kingdom and France to as high as39% in Japan (46). The A−, B+ strains de-scribed to date belong to toxinotypes VIII, X,XVI, and XVII. The last two also have CDT. Ofthese, toxinotype VIII seems to be most clini-cally significant (46). Severe disease has beenreported with these strains (ribotype 017) as hasemergence of resistance to both the macrolide-lincosamide-streptogramin antibiotics and flu-oroquinolones (36, 46).

In addition to changes in tcdA and tcdB,changes in the other genes of the PaLoc mayalso alter virulence. In 2003, outbreaks ofsevere disease in the United States and Canadawere caused by a clone of C. difficile designatedNorth American pulsed-field type 1 (NAP1),toxinotype III, ribotype 027, and restrictionendonuclease analysis (REA) type B1 (84, 91).These isolates possessed binary toxin CDT andhad an 18-bp deletion in the PaLoc tcdC (91).Quinolone resistance was also an important fea-ture of the clone. Since the original descriptionof the increased and widespread disseminationof this clone, several investigations into thegenotypic and phenotypic characteristics ofthese isolates have been published (118, 126,127, 139). In brief, Warny et al. (139) showedthat 027 strains produced toxins A and Bfaster and in greater quantities than control(non-027) strains. In addition, others haveshown that the duration of toxin productionby 027 strains markedly exceeds that of otherclones (118) and that these effects may be re-lated partly to stimulation of germination andtoxin production by quinolones (118). Toxin


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B from both historical and modern 027 strainsis more potent in vitro against multiple celllines than toxinotype 0 is (127). Spigaglia et al.(126) studied modifications to the accessorygenes of the PaLoc. They detected 39- and18-bp deletions in tcdC and they showed thatnonsense mutations in tcdC reduced the TcdCprotein from 232 to 61 aa (126). The 18-bpdeletion has no effect on toxin production (48,90). Rather, there is a correlation betweentruncation of TcdC, due to the single base-pairdeletion at position 117 that results in theformation of a stop codon, and increased toxinproduction due to derepression of the PaLoc(34, 90, 126, 127). At least one study failed todemonstrate a consistent correlation betweena truncated tcdC alone and increased toxinactivity, and the authors caution against usingmutations to diagnose patients as having morevirulent strains and using this information todetermine treatment (95).

Aside from having altered TcdC, 027 strainshave five unique genetic regions not presentin historical 027 strains (127). These genes in-clude mutations that explain not only enhancedtoxicity as described above, but motility, sur-vival, increased sporulation (2), and antibioticresistance (127). These factors combined mayexplain the increased severity of disease andmortality associated with these hypervirulentclones.

In addition to emergence of 027 and the A−,B+ 017 clone, strains of two other hyperviru-lent clones have recently been recognized. PCRribotypes 053 and 078 (toxinotypes V) cause se-vere disease in humans. Strain 078 is interestingfor a number of reasons. It causes disease in bothanimals, particularly calves and pigs, and hu-mans. Studies to date have shown a high degreeof genetic relatedness in the animal and humanstrains (38, 60). Both 027 and 078 strains haveincreased in frequency in the Netherlands, andclinical illness caused by both strains has similarpresentations.

Pathophysiology

As described above, the C. difficile genome en-ables the organism to express a variety of factors

that ensure its survival in the gastrointestinaltract of humans and animals once introduced.Physiological factors that allow colonizationinclude a disturbed or absent microbiota, an im-portant barrier. The HMA model showed thatamoxicillin–clavulanic acid treatment of micedid not modify the total numbers of microbesbut rather modified the type of microbiota.The authors showed that the Bacteroides-Porphyromonas-Prevotella group increased andthe Clostridium coccoides–Eubacterium rectalegroup decreased dramatically (11). Othershave assessed the impact of fluoroquinolonetreatment on gut microbiota, which decreasesthe quantities of enterococci and lactobacilli,Bacteroides spp., and bifidobacteria (118). In thissetting C. difficile endogenous or exogenousspores germinate and vegetative cells multiply.The organism adheres to the mucus layer bymeans of its multiple adhesins and penetratesthe mucus with aid of flagella and proteases.Once it penetrates mucus, the organism adheresto enterocytes and the first phase of patho-genesis, namely colonization, begins. Putativevirulence factors that promote colonization in-clude the proteolytic enzyme cysteine proteaseCwp84, adhesins such as S-layer proteins, a 66-kDa cell wall protein Cwp66, the GroEL heatshock protein, a 68-kDa fibronectin-bindingprotein, and the flagella components FliC(flagellin) and FliD (flagellar cap protein) (40).Genes encoding these proteins are in closeproximity on the genome. The second phase ofthe pathogenic process is toxin production (seeThe Pathogenicity Locus, above). As describedabove, alterations in genes that encode all thesevirulence factors, and not just those encoded inthe PaLoc, are important for and explain strainvariation in C. difficile disease.

EPIDEMIOLOGY

People and Patients as Reservoirs

C. difficile is widely distributed in the environ-ment and is often detected in the colon withoutpathologic consequences. C. difficile infection(CDI) is restricted to patients with C. difficile


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Table 1 Rates of recovery of C. difficile and frequency of positive cytotoxicity tests in culturepositive specimensa

Population tested Positive stool cultures Positive toxin testPatients with pseudomembranous colitis 99%–100% 96%Patients with antibiotic-associated diarrhea 15%–25% 10%–20%Antibiotic treatment without diarrhea 10%–20% <0%–5%Hospitalized patients 10%–25% 2%–8%Healthy adults 2%–3% <0.1%Healthy neonates 5%–70% 5%–63%

aAdapted from Reference 13.

replication and toxin production. Rates of col-onization and positive stool toxin tests basedon multiple reports are summarized in Table 1(13, 39, 53, 136).

C. difficile has been detected in stool from10% to 40% of most animal species, includ-ing cattle, horses, camels, donkeys, snakes,seals, dogs, and cats (82, 141). It is not clearthat it causes disease in most of these animalsexcept in antibiotic-treated horses (21) and ro-dent models. The organism has also been re-ported from diverse environmental sources; onereport based on 2,580 samples showed positivecultures in 184 samples (7%) including 47% ofsamples from river water, 21% of soil samples,and 2% of home environment samples (4).

Foodborne C. difficile infection. FoodborneCDI is an unproven but biologically plausi-ble mechanism of CDI based on detection ofC. difficile in an aggregate total of 90 of 592(15%) retail meat supplies from diverse animalsources and geographic locations in the UnitedStates and Canada (61, 117, 140); the dominantstrains in these reports are ribotypes 027 and078.

Risks for C. difficile Infection

Major risks for CDI are antibiotic exposure, ad-vanced age, and exposure to acute or chroniccare facilities. Antibiotic treatment is alwaysthe leading risk for CDI in people and an-imals. The initial studies focused on clin-damycin (132), which was highest in incidence,and cephalosporins, which were highest in

prevalence. In a review of 503 cases in Swedenfrom 1980 to 1982, the relative risk for theseagents compared with other antibiotics was 10to 70 times higher (6). The fluoroquinolonesemerged as major inducing agents and were im-plicated in several outbreaks that could be con-trolled only by restraining or prohibiting use ofthe entire class (55, 69). The prominent role ofthis class since 2000 presumably reflects highuse rates combined with the emergence of re-sistance by ribotype 027 (29, 55, 91).

Advanced age is a risk, with most reportsshowing sharp increases in incidence in personsover 65 years and a direct correlation with ageabove that threshold (6, 102). A possible con-tributing factor is immunosenescence, based onreports showing humoral response to toxins Aand/or B determines clinical expression (77) andprotection afforded by monoclonal antibodiesto toxins A and B (86). It is unclear that de-fective cell-mediated immunity is an importantrisk factor.

Another important risk is contact with thehealthcare system, which is heavily contami-nated by C. difficile, especially case-associatedareas (72). The potential sources of C. difficileacquisition include surface contamination, hos-pital personnel, patient cases and asymptomaticcarriers, and hospital air (22, 31, 44, 52,72). This risk is well verified by reports ofC. difficile acquisition and CDI in hospitals andchronic care facilities. A prospective study of428 admissions to Harborview Hospital in Seat-tle showed 6 (1.5%) patients were colonized onadmission from home and 83 (21%) patients be-came colonized during their hospital stay (92).


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Risks associated with C. difficile acquisition andinfection include age over 65 years, ICU (inten-sive care unit) admission, antibiotic treatment,length of hospital stay, gastrointestinal pro-cedures, and hospitalization in case-associatedareas (10).

There is increasing evidence that protonpump inhibitors also promote CDI (41). A sys-tematic review of the literature from 1988 to2005, based on 12 relevant publications and2,948 patients with CDI, showed a relative riskof 1.94 (95% CI 1.4–2.8) for proton pump in-hibitors (81, 83).

Changing Epidemiology

Since 2000, there has been a dramatic increasein rates and severity of CDI noted in NorthAmerica and much of Europe. The first reportswere from Sherbrooke in Quebec, Canada, in2003 showing a fourfold increase in CDI rates,from 35.6 to 156.3/100,000. Subsequent re-ports indicated this experience affected multiplehospitals in Quebec and included reviews show-ing the following: an attributable mortality of6.9%, fluoroquinolones as the dominant induc-ing agents, 10-fold-higher rates in persons over65 years, and the “hypervirulent” ribotype 027as the cause of the epidemic (84, 85, 103). Inthe United States an analysis by ICD-9 dis-charge data showed that the CDI rate doubled,the case mortality increased 80%, and the rateof colectomies for CDI tripled in 2003 com-pared with 1993 (47, 110, 113, 143). In addi-tion, the U.S. experience suggested increases inselected patient populations including pediatricpatients, persons without recent antibiotic ex-posure, community-acquired cases, and severecases associated with pregnancy. European dataalso showed increased CDI rates and multiplehospital-associated outbreaks (52).

Emergence of Ribotype 027

The increasing incidence and severity of CDI inNorth America and much of Europe has beenattributed to the emergence of ribotype 027.This strain accounted for only 14 of over 6,000

(<0.02%) typed strains collected from U.S.cases from 1984 to 1993, but was implicatedin 96 of 187 (51%) of strains tested in eightU.S. outbreaks from 2000 to 2003 (91, 100).This strain has been associated with large out-breaks of severe CDI in hospitals in the UnitedStates, Canada, and Europe (75, 84, 96, 100).The factor thought to contribute to incidence isthe strain’s resistance to fluoroquinolones, andenhanced virulence may reflect increased toxinproduction in vitro (139).

In 2008, the European Center for DiseaseControl established a surveillance system to de-fine CDI incidence and implicated strains in 34European countries. This showed a mean in-cidence of 5.5 cases per 10,000 patient days,and ribotype 027 accounted for just 5% of allcases (52). Unfortunately, there is no naturalsurveillance system in the United States, but alarge clinical trial of a new therapeutic agent in2005–2007 showed that ribotype 027 accountedfor 36% of U.S. strains and 8% of Europeanstrains (56).

The data reviewed provide strong supportfor the role of ribotype 027 in some devastat-ing outbreaks, the important new role of flu-oroquinolones for driving incidence, and thecritical role of fluoroquinolone restriction incontrolling some outbreaks (69, 75). However,this conclusion may be overly simplified be-cause ribotype 027 accounted for only 5% of 89typed strains in the 2008 European surveillancestudy (20); a case control study found “no evi-dence to support assertions that ribotype 027 ismore virulent than other PCR ribotypes” (93).Some institutions report that beta-lactams arestill much more important inducing agents thanfluoroquinolones (20) and that ribotype 078 hasplayed an important part in some outbreaks(75).

DIAGNOSIS

Guidelines for diagnosis and treatment ofC. difficile disease have been developed bythe Society for Healthcare Epidemiology ofAmerica and the Infectious Diseases Society ofAmerica (SHEA/IDSA) in the United States


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Table 2 Performance characteristics of various test methods

Performance characteristics

Methods/assays Sensitivity (%, range) Specificity (%, range)Toxigenic anaerobic culture N/A N/A

Enzyme immunoassaysa 31–99 84–100

Cell culture neutralization assaysb 67–86 97–100

Glutamate dehydrogenasec 71–100 76–98

Nucleic acid amplification testsd

BD-GeneOhmTM 84–96 94–100

Prodesse ProGastroTM 77–92 95–99

GeneXpertTM 94–100 93–99

illumigeneTM 99 98

aDerived from References 33, 49, and 107 and includes both solid-phase and membrane assays combined. Data are notstratified by comparative method.bCompiled from References 9, 49, 105, and 129.cCompiled from References 33, 51, 124, 135, and 142.dCompiled from References 8, 9, 49, 59, 67, 70, 73, 76, 98, 99, 128, 129, 131, and 134.

and the European Society of Clinical Microbi-ology and Infectious Diseases (ESCMID) (31,33). Both groups recommend that testing beperformed only on diarrheal (unformed) stoolexcept in rare instances (31, 33). Asymptomaticpatients should not be tested even for test ofcure.

Table 2 lists the variety of test methodsavailable for the diagnosis of CDI and a sum-mary of their performances based on publishedstudies. Cell culture cytotoxicity neutralizationassays (CCCNAs) are performed by inoculatinga stool filtrate onto a monolayer of a particularcell line (e.g., human foreskin fibroblasts) andthen observing for toxin-induced cytopathiceffect (CPE). Once CPE is observed, neutral-ization with an antiserum, either C. sordelliiantitoxin or C. difficile antitoxin, is performed.For many years, CCCNAs were consideredthe diagnostic gold standard. However, theirperformance has recently been re-evaluated incomparisons with toxigenic culture and nucleicacid amplification tests (NAATs) (9, 49, 105,128) (Table 2). In general, CCCNAs are tooinsensitive (<90%) to be considered acceptablereference methods (31).

Enzyme immunoassays (EIAs), the methodmost frequently used in U.S. laboratories, use

polyclonal or monoclonal antibodies targetingTcdA alone or both TcdA and TcdB. Becauseof the emergence of virulent TcdA−, TcdB+isolates, clinical laboratories should use a testthat detects both toxins. About two dozensolid-phase, well-type, and rapid membraneassays are available. Several recent compre-hensive reviews evaluating the performance ofthese assays have been published (33, 49, 107).Planche et al. (107) performed a comprehensivereview of 28 studies assessing six Toxin A/Bassays commonly used in the United Kingdom.The authors noted significant heterogeneityamong the assays, but the diagnostic oddsratio implied no difference in performanceamong them. Most of the assays had higherspecificities than sensitivities, but none of theassays met the criteria for an acceptable test,defined as having a sensitivity of 90% and afalse-positive rate of ≤3% (107). Eastwoodet al. (49) evaluated six EIAs and three lateralflow assays on the same set of 600 diarrhealsamples. Sensitivities ranged from 60% to 81%and specificities from 91% to 99.4% comparedwith toxigenic culture (49). None of the assaysmet the criteria as defined by Planche et al. (49).

Because EIAs lack sensitivity, it has be-come common practice to perform “stools for


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C. difficile x 3.” However, recent reports demon-strate the lack of utility of repeat testingregardless of the method used (1, 24, 27, 87,97, 106). Given the poor sensitivity and positivepredictive value in low-prevalence populations,both practice guidelines consider EIAs subop-timal for the diagnosis of C. difficile infections(31, 33).

Glutamate dehydrogenase (GDH) is ametabolic enzyme expressed at high levels byall strains of C. difficile. GDH is present in bothtoxigenic and nontoxigenic strains, so a positiveGDH test must be combined with an assay thatalso detects toxin. The C. DIFF CHEKTM-60solid-phase microtiter plate GDH assay (Tech-Lab, Blacksburg, Virginia) demonstrated highnegative predictive values (98.5% to 99.7%) inseveral studies (51, 124, 135, 142). Therefore,many laboratories have adopted a two-step al-gorithm in which the GDH is performed first,and if negative, no further testing is required.If the GDH test is positive, a toxin test shouldfollow. If the GDH is positive but the toxin testis negative, either the patient is colonized withnontoxigenic C. difficile or (depending upon theassay) the toxin test is falsely negative. If boththe GDH and the toxin test are positive, thenthe symptomatic patient likely has CDI. Al-though some investigators have recently notedsuboptimal performance of the GDH assaycompared with culture and/or molecular meth-ods (76, 99, 133), a recent meta-analysis reportsthat GDH has a greater than 90% sensitivitywith a negligible false-positive rate when com-pared with culture (123). This two-step methodis the approach that is currently recommendedby both ESCMID and SHEA/IDSA (31, 33).

A newer immunochromatogenic membraneversion of the test combines both GDH andtoxin testing into a single device (C. DIFF QuikChek CompleteTM, TechLab) and takes about30 min to perform. In two published studieswhen this device was compared to toxigenicculture, the sensitivity of the GDH componentwas 100% (109, 131). However, the toxin com-ponent performed poorly; its sensitivity rang-ing from 61% to 78% (109, 131). Given theinsensitivity of the toxin component of the

assay, some laboratories are using the devicein an expanded three-step approach, testing theGDH-positive, toxin-negative samples with an-other method such as a NAAT.

The increase in the incidence of C. difficileinfections, the emergence of hypervirulentstrains causing more severe disease, and morerecently, the need for a better method againstwhich to assess evolving technologies, such asNAATs, have compelled laboratories to revisitanaerobic toxigenic culture. Toxigenic culturerequires inoculating stool specimens to selec-tive anaerobic media that contain substancesinhibitory to normal stool microbiota whilepromoting the germination and growth ofC. difficile. Both toxigenic and nontoxigenic iso-lates are recovered, so isolates must be tested fortoxin production by EIA, CCCNA, or NAAT.Toxigenic culture performed after negativedirect toxin testing by EIA or CCNA increasesthe yield of positives by 15% to 23% (25, 112).

Although culture methods have not beenstandardized, there are some important caveatswith respect to its performance. Spore enrich-ment significantly enhances recovery and isuseful when culturing is done for epidemi-ological purposes. There are two ways toenrich for spores: heat shock and treatment ofthe specimen with ethanol. The methods aredetailed in References 28, 65, 78, and 89.

A variety of media exist for culturingC. difficile. The original cycloserine, cefoxitin,fructose agar (CCFA), as described by Georgeet al. (53) contained an egg yolk fructose agarbase with 500 μg ml−1 of cycloserine and16 μg ml−1 of cefoxitin. CCFA takes advantageof the ability of C. difficile to ferment fructose.CCFA variants with reduced concentrations ofthe antimicrobial agents are less sensitive thanthe formulation by George et al. (94, 104). Ad-ditives such as horse blood, taurocholate, andlysozyme improve recovery by enhancing vege-tation (23; K. Carroll, personal observations). Arecent study demonstrated improved recoverywith CCF broth supplemented with 0.1% tau-rocholate compared with plating on solid agar(7). Whichever method is chosen, it is impor-tant to use prereduced media, as the failure to


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do so can affect the sensitivity of the culturemethod (104). Despite its sensitivity, toxigenicculture is not practical for use in most clinicallaboratories. However, it is particularly usefulin the setting of an outbreak, when evaluating anew test method, for surveillance of antimicro-bial resistance, and for management of patientswith recurrent/refractory disease.

Nucleic Acid Amplification Methods

In the United States there are currently fourFDA-cleared NAATs available for diagnosis ofCDI (Table 2): (a) the BD-GeneOhmTM Cdiffassay (BD, Franklin Lakes, New Jersey), (b) theProdesse ProGastroTM Cd assay (Gen-Probe,Inc. San Diego, California), (c) the CepheidGeneXpert R© C difficile (Cepheid, Sunnyvale,California), and (d ) the illumigeneTM C. difficileassay (Meridian Diagnostics, Inc., Cincinnati,Ohio). The first three assays target conservedregions of tcdB, and the last uses loop-mediatedisothermal amplification to detect the PaLoc ata conserved region of tcdA that is present evenin TcdA−, TcdB+ strains. The assays vary con-siderably in terms of ease of use, instrumenta-tion, and cost. The performance characteristicsof these assays are well published in the litera-ture and are highlighted in Table 2. In general,they are superior to all methods except toxi-genic culture. To date, there is no publishedstudy that compares them all.

An important observation came from theanalysis of the data from the GeneXpert clin-ical trials. The authors noted that the perfor-mance of all the assays evaluated appeared tobe affected by the ribotype of the strain causingthe infection. Compared with toxigenic culture,the GDH assay and qPCR assay had compa-rable sensitivity for detection of ribotype 027(91%) (133). However, for non-027 infections,the overall sensitivity for the GDH assay was70% compared with 92% for the GeneXpert as-say (133). These results were statistically signif-icant (133). Likewise, the performance of twoToxin A/B EIAs revealed extensive variability,with sensitivities as low as 16% for some ri-botypes (133). Although these observations can

Diarrheal stool specimen (N = 150)

C. DIFF Quik Chek CompleteTM

GDH positive (N = 22) GDH negative (N = 114)

C. difficile negative

Toxin negative (N = 11)Toxin positive (N = 11)

Presumed CDI

XpertTM C. difficile PCR

PCR positive (N = 8) PCR negative (N = 3)

CDI (N = 4)or toxigenic carriage (N = 4)

Clinical assessment essential

Nontoxigenic carriage (N = 2)or false-positive GDH (N = 1)

No CDI

Figure 2Proposed testing algorithm for detection of C. difficile in stool samples usingthe C. DIFF Quick Chek CompleteTM test followed by testing GDH-positive,EIA-negative specimens with a commercial real-time PCR method. Thenumbers in parentheses reflect the numbers of specimens that required testingby each method in the authors’ study. Modified and reprinted with permissionfrom Swindells J, et al. 2010. J. Clin. Microbiol. 48:606–8 (131).

certainly explain the broad variability in testperformance, especially for the EIA methods,it would be useful to see additional data fromother sites and other platforms with respect tosuch observations.

NAAT testing is rapidly replacing othermethods in clinical microbiology laboratories,particularly in laboratories that already havethe required instrumentation for testing. Thesenew technologies have yet to be endorsed byprofessional society guidelines (31, 33). Thereare practical and theoretical concerns that, ifclarified, could reassure healthcare practition-ers regarding their use. One of these concernsis whether genetic drift of tcdB or other genetargets in regions of primer/probe binding islikely to suddenly affect assay performance. Asdiscussed above, there is a great deal of het-erogeneity in the genome of various C. difficile


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strains. However, the full and partial genomesequencing of several strains of C. difficile haselucidated regions that are conserved, includ-ing regions of genes on the PaLoc that havebeen targeted for these assays. At least one studyshowed performance variability based on ribo-type (133). Information about the impact ofNAATs on C. difficile transmission and theircost-effectiveness is also needed.

Given the costs of NAATs, some laborato-ries have explored using them on a limited basisin three-step algorithms as outlined in Fig-ure 2. The algorithm begins with the C. DIFFQuik CHEK CompleteTM test, and NAATtesting is performed only on GDH-positive,EIA-negative specimens. Several studies havedemonstrated the cost-effectiveness of such anapproach (43, 79, 122).

SUMMARY POINTS

1. The complete genome of a clinical strain of C. difficile and partial genomes of toxin variantstrains have been sequenced.

2. C. difficile causes disease by elaborating one or both large clostridial toxins encoded on aPaLoc with three additional genes that regulate toxin production.

3. Both toxins are large single-stranded proteins, and recent X-ray crystallography and smallangle X-ray scattering models of TcdB suggest four structural domains are necessary fortoxin entry and cytotoxicity.

4. Binary toxin, which is not encoded on the PaLoc, may contribute to disease severity byits cytotoxic activity and by induction of novel thin, dynamic microtubules on the surfaceof epithelial cells, leading to increased adherence of Clostridia.

5. Mutations in the PaLoc have resulted in the evolution of four clades of C. difficile inclusiveof hypervirulent toxinotypes responsible for aggressive disease.

6. Risk factors such as quinolone antibiotics, proton pump inhibitors, and advanced age, incombination with more pathogenic strains, have created the “perfect storm” for recentepidemics.

7. The recognition of toxin variant strains in domestic animals and food sources may explainthe onset of community disease. This needs further study.

8. Increased incidence and severity of disease has refocused attention on the inadequacy ofEIAs and CCCNAs and has sparked development of diagnostic molecular assays.

DISCLOSURE STATEMENT

Dr. Karen C. Carroll has received research funding from BD Diagnostics (Sparks, MD) andProdesse, now Gen-Probe, Inc.(San Diego, CA). Dr. John G. Bartlett has no relevant disclosures.

ACKNOWLEDGMENTS

The authors wish to thank Ms. Janet Landay for her secretarial assistance.

LITERATURE CITED

1. Aichinger E, Schleck CD, Harmsen WS, Nyre LM, Patel R. 2008. Nonutility of repeat laboratory testingfor detection of Clostridium difficile by use of PCR or enzyme immunoassay. J. Clin. Microbiol. 46:3795–97


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

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f T

exas

- A

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07/1

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. For

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2. Akerlund T, Persson I, Unemo M, Noren T, Svenungsson B, et al. 2008. Increased sporulation rate ofepidemic Clostridium difficile type 027/NAP1. J. Clin. Microbiol. 46:1530–33

3. Aktories K. 2007. Self-cutting to kill: new insights into the processing of Clostridium difficile toxins. ACSChem. Biol. 2:228–30

4. al Saif N, Brazier JS. 1996. The distribution of Clostridium difficile in the environment of South Wales.J. Med. Microbiol. 45:133–37

5. Albesa-Jove D, Bertrand T, Carpenter EP, Swain GV, Lim J, et al. 2010. Four distinct structural domainsin Clostridium difficile toxin B visualized using SAXS. J. Mol. Biol. 396:1260–70

6. Aronsson B, Mollby R, Nord CE. 1985. Antimicrobial agents and Clostridium difficile in acute entericdisease: epidemiological data from Sweden. J. Infect. Dis. 151:476–81

7. Arroyo LG, Rousseau J, Willey BM, Low DE, Staempfli H, et al. 2005. Use of a selective enrichmentbroth to recover Clostridium difficile from stool swabs stored under different conditions. J. Clin. Microbiol.43: 5341–43

8. Babady NE, Stiles J, Ruggiero P, Khosa P, Huang D, et al. 2010. Evaluation of the Cepheid XpertClostridium difficile EPI assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strainat a cancer hospital. J. Clin. Microbiol. 48:4519–24

9. Barbut F, Braun M, Burghoffer B, Lalande V, Eckert C. 2009. Rapid detection of toxigenic strains ofClostridium difficile in diarrheal stools by real-time PCR. J. Clin. Microbiol. 47:1276–77

10. Barbut F, Petit JC. 2001. Epidemiology of Clostridium difficile–associated infections. Clin. Microbiol. Infect.7:405–10

11. Barc MC, Bourlioux F, Janoir C, Charrin-Sarnel C, Janoir C, et al. 2004. Effect of amoxicillin-clavulanateon human fecal flora in a gnotobiotic mouse model measured with group specific 16SrRNA targetedoligonucleotide probes in combination with flow cytometry. Antimicrob. Agents Chemother. 48:1365–68

12. Barth H, Pfeifer G, Hofmann F, Maier E, Benz R, Aktories K. 2001. Low pH-induced formation of ionchannels by Clostridium difficile toxin B in target cells. J. Biol. Chem. 276:10670–76

13. Bartlett JG. 1994. Clostridium difficile: history of its role as an enteric pathogen and the current state ofknowledge about the organism. Clin. Infect. Dis. 18(Suppl. 4):S265–72

14. Bartlett JG, Chang TW, Gurwith M, Gorbach SL, Onderdonk AB. 1978. Antibiotic-associated pseu-domembranous colitis due to toxin-producing clostridia. N. Engl. J. Med. 298:531–34

15. Bartlett JG. 1980. Experimental studies of antibiotic associated colitis. Scand. J. Infect. Dis. (Suppl.22):11–15

16. Bartlett JG, Chang TW, Onderdonk AB. 1978. Will the real Clostridium species responsible for antibiotic-associated colitis please step forward? Lancet 1:338

17. Bartlett JG, Moon N, Chang TW, Taylor N, Onderdonk AB. 1978. Role of Clostridium difficile inantibiotic-associated pseudomembranous colitis. Gastroenterology 75:778–82

18. Bartlett JG, Onderdonk AB, Cisneros RL. 1977. Clindamycin-associated colitis in hamsters: protectionwith vancomycin. Gastroenterology 73:772–76

19. Bartlett JG, Onderdonk AB, Cisneros RL, Kasper DL. 1977. Clindamycin-associated colitis due to atoxin-producing species of Clostridium in hamsters. J. Infect. Dis. 136:701–5

20. Bauer MP, Notermans DW, Van Benthem BH, Brazier JS, Wilcox MH, et al. 2011. Clostridium difficileinfection in Europe: a hospital-based survey. Lancet 377:63–73

21. Baverud V. 2002. Clostridium difficile infections in animals with special reference to the horse. A review.Vet. Q. 24:203–19

22. Best EL, Fawley WN, Parnell P, Wilcox MH. 2010. The potential for airborne dispersal of Clostridiumdifficile from symptomatic patients. Clin. Infect. Dis. 50:1450–57

23. Bliss DZ, Johnson S, Clabots CR, Savik K, Gerding DN. 1997. Comparison of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance ofhospitalized patients. Diagn. Microbiol. Infect. Dis. 29:1–4

24. Borek AP, Aird DZ, Carroll KC. 2005. Frequency of sample submission for optimal utilization of thecell culture cytotoxicity assay for detection of Clostridium difficile toxin. J. Clin. Microbiol. 43:2994–95

25. Bouza E, Pelaez, T, Alonso R, Catalan P, Munoz P, Creixems MR. 2001. “Second-look” cytotoxicity:an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis ofCDAD. J. Hosp. Infect. 48:233–37


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1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


26. Calabi E, Ward S, Wren B, Paxton T, Panico M, et al. Molecular characterization of the surface layerproteins from Clostridium difficile. Mol. Microbiol. 40:1187–99

27. Cardona DM, Rand KH. 2008. Evaluation of repeat Clostridium difficile enzyme immunoassay testing.J. Clin. Microbiol. 46:3686–89

28. Clabots CR, Gerding SJ, Olson MM, Peterson LR, Gerding DN. 1989. Detection of asymptomaticClostridium difficile carriage by an alcohol shock procedure. J. Clin. Microbiol. 27:2386–87

29. Clements AC, Magalhaes RJ, Tatem AJ, Paterson DL, Riley TV. 2010. Clostridium difficile PCR ribotype027: assessing the risks of further worldwide spread. Lancet Infect. Dis. 10:395–404

30. Cohen SH, Tang YJ, Silva J Jr. 2000. Analysis of the pathogenicity locus in Clostridium difficile strains.J. Infect. Dis. 181:659–63

31. An authoritativeguideline on currentconcepts in testing,treatment, and infectioncontrol.

31. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, et al. 2010. Clinical practice guidelinesfor Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemi-ology of America (SHEA) and the infectious diseases society of America. Infect. Control Hosp.

Epidemiol. 31:431–5532. Collignon A. 2010. Methods for working with the mouse model. In Clostridium difficile: Methods in

Molecular Biology, ed. P Mullany, AP Roberts, pp. 229–37. New York: Springer. 646 pp.33. Crobach MJT, Dekkers OM, Wilcox MH, Kuijper EJ. 2009. European Society of Clinical Microbiology

and Infectious Diseases (ESCMID): data review and recommendations for diagnosing Clostridium difficile-infection (CDI). Clin. Microbiol. Infect. 15:1053–66

34. Curry SR, Marsh JW, Muto CA, O’Leary MM, Pasculle AW, Harrison LH. 2007. tcdC genotypesassociated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile.J. Clin. Microbiol. 45:215–21

35. Curry S. 2010. Clostridium difficile. Clin. Lab. Med. 30:329–4236. Dawson LF, Valiente E, Wren BW. 2009. Clostridium difficile—a continually evolving and problematic

pathogen. Infect. Genet. Evol. 9:1410–1737. De Somer P, Van de Voorde H, Eyssen H, Van Dijck P. 1955. A study on penicillin toxicity in guinea

pigs. Antibiot. Chemother. 5:463–6938. Debast SB, Vaessen N, Choudry A, Wiegeers-Ligtvoet EA, van den Berg RJ, et al. 2009. Successful

combat of an outbreak due to Clostridium difficile PCR ribotype 027 and recognition of specific riskfactors. Clin. Microbiol. Infect. 15:427–34

39. Delmee M, Verellen G, Avesani V, Francois G. 1988. Clostridium difficile in neonates: serogrouping andepidemiology. Eur. J. Pediatr. 147:36–40

40. Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A. 2009. New trends in Clostridium difficilevirulence and pathogenesis. Intern. J. Antimicrob. Agents 33:S24–28

41. Dial S, Delaney JA, Barkun AN, Suissa S. 2005. Use of gastric acid-suppressive agents and risk ofcommunity-acquired Clostridium difficile–associated disease. JAMA 294:2989–95

42. Dineen SS, Villapakkam AC, Nordman JT, Sonenshein AL. 2007. Repression of Clostridium difficile toxingene expression by CodY. Mol. Microbiol. 66:206–19

43. Doing KM, Hintz MS, Keefe C, Horne S, LeVasseur S, Kulikowski ML. 2010. Reevaluation of thePremier Clostridium difficile toxin A and B immunoassay with comparison to glutamate dehydrogenasecommon antigen testing evaluating Bartels cytotoxin and Prodesse ProGastro Cd polymerase chainreaction as confirmatory procedures. Diagn. Microbiol. Infect. Dis. 66:129–34

44. Donskey CJ. 2010. Preventing transmission of Clostridium difficile: Is the answer blowing in the wind?Clin. Infect. Dis. 50:1458–61

45. Drudy D, Calabi E, Kyne L, Sougioultzis S, Kelly E, et al. 2004. Human antibody response to surfacelayer proteins in Clostridium difficile infection. FEMS Immunol. Med. Microbiol. 41:237–42

46. Drudy D, Fanning S, Kyne L. 2007. Toxin A-negative, toxin B-positive Clostridium difficile. Int. J. Infect.Dis. 11:5–10

47. Dubberke ER, Butler AM, Yokoe DS, Mayer J, Hota B, et al. 2010. Multicenter study of Clostridiumdifficile infection rates from 2000 to 2006. Infect. Control Hosp. Epidemiol. 31:1030–37

48. Dupuy B, Govind R, Antunes A, Matamouros S. 2008. Clostridium difficile toxin synthesis is negativelyregulated by TcdC. J. Med. Microbiol. 57:685–89


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


49. Eastwood K, Else P, Charlett A, Wilcox M. 2009. Comparison of nine commercially available Clostridiumdifficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenasedetection assay to cytotoxin testing and cytotoxigenic culture methods. J. Clin. Microbiol. 47:3211–17

50. Fagan RP, Albesa-Jove D, Qazi O, Svergun DI, Brown KA, Fairweather NF. 2009. Structural insightsinto the molecular organization of the S-layer from Clostridium difficile. Mol. Microbiol. 71:1308–22

51. Fenner L, Widmer AF, Goy G, Rudin S, Frei R. 2008. Rapid and reliable diagnostic algorithm fordetection of Clostridium difficile. J. Clin. Microbiol. 46:328–30

52. Provides acomprehensive reviewof the epidemiology ofC. difficile infectionsincluding devastatingoutbreaks in hospitals,regional trends in rates,and distribution ofribotypes.

52. Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, et al. 2010. The changing epidemiologyof Clostridium difficile infections. Clin. Microbiol. Rev. 23:529–49

53. George WL, Sutter VL, Citron D, Finegold SM. 1979. Selective and differential medium for isolationof Clostridium difficile. J. Clin. Microbiol. 9:214–19

54. George RH, Symonds JM, Dimock F, Brown JD, Arabi Y, et al. 1978. Identification of Clostridiumdifficile as a cause of pseudomembranous colitis. Br. Med J. 1:695

55. Gerding DN. 2004. Clindamycin, cephalosporins, fluoroquinolones, and Clostridium difficile–associateddiarrhea: This is an antimicrobial resistance problem. Clin. Infect. Dis. 38:646–48

56. Gerding DN. 2010. Global epidemiology of Clostridium difficile infection in 2010. Infect. Control Hosp.Epidemiol. 31(Suppl. 1):S32–34

57. Geric B, Carman RJ, Rupnik M, Genheimer CW, Sambol SP, et al. 2006. Binary toxin-producing largeclostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters.J. Infect. Dis. 193:1143–50

58. Giesemann T, Egerer M, Jank T, Aktories K. 2008. Processing of Clostridium difficile toxins. J. Med.Microbiol. 57:690–96

59. Goldenberg SD, Dieringer T, French GL. 2010. Detection of toxigenic Clostridium difficile in diarrhealstools by real-time polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 67:304–7

60. Goorhuis A, Bakker D, Corver J, Debast, Harmanus C, et al. 2008. Emergence of Clostridium difficileinfection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin. Infect. Dis.47:1162–70

61. Gould LH, Limbago B. 2010. Clostridium difficile in food and domestic animals: a new foodbornepathogen? Clin. Infect. Dis. 51:577–82

62. Green RH. 1974. The association of viral activation with penicillin toxicity in guinea pigs and hamsters.Yale J. Biol. Med. 47:166–81

63. Hall IC, O’Toole E. 1935. Intestinal flora in newborn infants: with a description of a new pathogenicanaerobe, Bacillus difficilis. Am. J. Dis. Child. 49:390

64. Hambre DM, Rake G, McKee CM, MacPhillamy HB. 1943. The toxicity of penicillin as prepared forclinical use. Am. J. Med. Sci. 206:642

65. Hanff PA, Zaleznik DF, Kent KC, Rubin MS, Kelly E, et al. 1993. Use of heat shock for culturingClostridium difficile from rectal swabs. Clin. Infect. Dis. 16(Suppl. 14):S245–47

66. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal geneknock-out system for the genus Clostridium. J. Microbiol. Methods 70:452–64

67. Huang H, Weintraub A, Fang H, Nord CE. 2009. Comparison of a commercial multiplex real-timePCR to the cell cytotoxicity neutralization assay for diagnosis of Clostridium difficile infections. J. Clin.Microbiol. 47:3729–31

68. Jank T, Aktories K. 2008. Structure and mode of action of clostridial glucosylating toxins: the ABCDmodel. Trends Microbiol. 16:222–29

69. Kallen AJ, Thompson A, Ristaino P, Chapman L, Nicholson A, et al. 2009. Complete restriction offluoroquinolone use to control an outbreak of Clostridium difficile infection at a community hospital.Infect. Control Hosp. Epidemiol. 30:264–72

70. Karre T, Sloan L, Patel R, Mandrekar J, Rosenblatt J. 2011. Comparison of two commercial molecularassays to a laboratory-developed molecular assay for diagnosis of Clostridium difficile infection. J. Clin.Microbiol. 49:725–27

71. Khan MY, Hall WH. 1966. Staphylococcal enterocolitis: treatment with oral vancomycin. Ann. Intern.Med. 65:1–8


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


72. Kim KH, Fekety R, Batts DH, Brown D, Cudmore M, et al. 1981. Isolation of Clostridium difficile fromthe environment and contacts of patients with antibiotic-associated colitis. J. Infect. Dis. 143:42–50

73. Knetsch CW, Bakker D, de Boer RF, Sanders I, Hofs S, et al. 2011. Comparison of real-time PCRtechniques to cytogenic culture methods for diagnosing Clostridium difficile infection. J. Clin. Microbiol.49:227–31

74. Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. 2009. The role of toxin A andtoxin B in Clostridium difficile infection. Nature 467:711–14

75. Kuijper EJ, van den Berg RJ, Debast S, Visser CE, Veenendaal P, et al. 2006. Clostridium difficile ribotype027, toxinotype III, The Netherlands. Emerg. Infect. Dis. 12:827–30

76. Kvach EJ, Ferguson D, Riska PF, Landry ML. 2010. Comparison of BD GeneOhm Cdiff real-time PCRassay with a two-step algorithm and a toxin A/B enzyme-linked immunosorbent assay for diagnosis oftoxigenic Clostridium difficile infection. J. Clin. Microbiol. 48:109–14

77. Kyne L. 2010. Clostridium difficile—beyond antibiotics. N. Engl. J. Med. 362:264–6578. Lahn M, Tyler G, Daubener W, Hadding U. 1993. Improvement of Clostridium difficile isolation by

heat-shock and typing of the isolated strains by SDS-PAGE. Eur. J. Epidemiol. 9:327–3479. Larson AM, Fung AM, Fang FC. 2010. Evaluation of tcdB real-time PCR in a three-step diagnostic

algorithm for detection of toxigenic Clostridium difficile. J. Clin. Microbiol. 48:124–3080. Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, et al. 2009. Proteomic and genomic characteri-

zation of highly infectious Clostridium difficile 630 spores. J. Bacteriol. 191:5377–8681. Leonard J, Marshall JK, Moayyedi P. 2007. Systematic review of the risk of enteric infection in patients

taking acid suppression. Am. J. Gastroenterol. 102:2047–5682. Levett PN. 1986. Clostridium difficile in habitats other than the human gastrointestinal tract. J. Infect.

12:253–6383. Linsky A, Gupta K, Lawler EV, Fonda JR, Hermos JA. 2010. Proton pump inhibitors and risk for

recurrent Clostridium difficile infection. Arch. Intern. Med. 170:772–7884. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, et al. 2005. A predominantly clonal multi-

institutional outbreak of Clostridium difficile–associated diarrhea with high morbidity and mortality.N. Engl. J. Med. 353:2442–49

85. Loo VG, Libman MD, Miller MA, Bourgault AM, Frenette CH, et al. 2004. Clostridium difficile: aformidable foe. Can. Med. Assoc. J. 171:47–48

86. Lowy I, Molrine DC, Leav BA, Blair BM, Baxter R, et al. 2010. Treatment with monoclonal antibodiesagainst Clostridium difficile toxins. N. Engl. J. Med. 362:197–205

87. Luo RF, Banaei N. 2010. Is repeat PCR needed for diagnosis of Clostridium difficile infection? J. Clin.Microbiol. 48:3738–41

88. Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, et al. 2009. Toxin B is essential forvirulence of Clostridium difficile. Nature 458:1176–79

89. Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. 1992. Comparison of five culturalprocedures for isolation of Clostridium difficile from stools. J. Clin. Microbiol. 30:514–16

90. Matamouros S, England P, Dupuy B. 2007. Clostridium difficile toxin expression is inhibited by the novelregulator TcdC. Mol. Microbiol. 64:1274–88

91. McDonald LC, Kilgore GE, Thompson A, Owens RC Jr, Kazakova SV, et al. 2005. An epidemic, toxingene-variant strain of Clostridium difficile. N. Engl. J. Med. 353:2433–41

92. McFarland LV, Mulligan ME, Kwok RY, Stamm WE. 1989. Nosocomial acquisition of Clostridiumdifficile infection. N. Engl. J. Med. 320:204–10

93. Morgan OW, Rodrigues B, Elston T, Verlander NQ, Brown DF, et al. 2008. Clinical severity of Clostrid-ium difficile PCR ribotype 027: a case-case study. PLoS One 3:e1812

94. Mundy LS, Shanholtzer CJ, Willard KE, Gerding DN, Peterson LR. 1995. Laboratory detection ofClostridium difficile. A comparison of media and incubation systems. Am. J. Clin. Pathol. 103:52–56

95. Murray R, Boyd D, Levett PN, Mulvey MR, Alfa MJ. 2009. Truncation in the tcdC region of Clostridiumdifficile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A.BMC Infect. Dis. 9:103


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


96. Muto CA, Pokrywka M, Shutt K, Mendelsohn AB, Nouri K, et al. 2005. A large outbreak of Clostridiumdifficile–associated disease with an unexpected proportion of deaths and colectomies at a teaching hospitalfollowing increased fluoroquinolone use. Infect. Control Hosp. Epidemiol. 26:273–80

97. Nemat H, Khan R, Ashraf MS, Matta M, Ahmed S, et al. 2009. Diagnostic value of repeated enzymeimmunoassays in Clostridium difficile infection. Am. J. Gastroenterol. 104:2035–41

98. Noren T, Alriksson I, Andersson J, Akerlund T, Unemo M. 2011. Rapid and sensitive loop-mediatedisothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and cultureas gold standard. J. Clin. Microbiol. 49:710–11

99. Novak-Weekley SM, Marlowe EM, Miller JM, Cumpio J, Nomura JH, et al. 2010. Clostridium difficiletesting in the clinical laboratory by use of multiple testing algorithms. J. Clin. Microbiol. 48:889–93

100. O’Connor JR, Johnson S, Gerding DN. 2009. Clostridium difficile infection caused by the epidemicBI/BNAP1/027 strain. Gastroenterology 136:1913–24

101. Panessa-Warren BJ, Tortora GT, Warren JB. 2007. High resolution FESEM and TEM reveal bacterialspore attachment. Microsc. Microanal. 13:251–66

102. Represents the firstmajor report in NorthAmerica on the regionaloutbreak of cases due toribotype 027.

102. Pepin J, Valiquette L, Alary ME, Villemure P, Pelletier A, et al. 2004. Clostridium difficile–associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of diseaseseverity. Can. Med. Assoc. J. 171:466–72

103. Pepin J, Valiquette L, Cossette B. 2005. Mortality attributable to nosocomial Clostridium difficile–associated disease during an epidemic caused by a hypervirulent strain in Quebec. Can. Med. Assoc.J. 173:1037–42

104. Peterson LR, Kelly PJ, Nordbrock HA. 1996. Role of culture and toxin detection in laboratory test-ing for diagnosis of Clostridium difficile–associated diarrhea. Eur. J. Clin. Microbiol. Infect. Dis. 15:330–36

105. Peterson LR, Manson RU, Paule SM, Hacek DM, Robicsek A, et al. 2007. Detection of toxigenicClostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficile–associated diarrhea. Clin. Infect. Dis. 45:1152–60

106. Peterson LR, Robicsek A. 2009. Does my patient have Clostridium difficile infection? Ann. Int. Med.151:176–79

107. A useful review of28 papers on EIAs forC difficile diagnosis thatsupports the view thatthese tests aresuboptimal.

107. Planche T, Aghaizu A, Holliman R, Riley P, Poloniecki J, et al. 2008. Diagnosis of Clostridium

difficile infection by toxin detection kits: a systematic review. Lancet Infect. Dis. 8:777–84108. Qa’Dan M, Spyres LM, Ballard JD. 2000. pH-enhanced cytopathic conformational changes in Clostridium

difficile toxin B. Infect. Immun. 68:2470–74109. Quinn CD, Sefers SE, Babiker W, He Y, Alcabasa R, et al. 2010. C. Diff Quik Chek complete enzyme

immunoassay provides a reliable first-line method for detection of Clostridium difficile in stool specimens.J. Clin. Microbiol. 48:603–5

110. Redelings MD, Sorvillo F, Mascola L. 2007. Increase in Clostridium difficile–related mortality rates,United States, 1999–2004. Emerg. Infect. Dis. 13:1417–19

111. Reineke J, Tenzer S, Rupnik M, Koschinski A, Hasselmayer O, et al. 2007. Autocatalytic cleavage ofClostridium difficile toxin B. Nature 446:415–19

112. Reller ME, Lema CA, Perl TM, Cai M, Ross TL, et al. 2007. Yield of stool culture with isolate toxintesting versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridiumdifficile. J. Clin. Microbiol. 45:3601–5

113. Ricciardi R, Rothenberger DA, Madoff RD, Baxter NN. 2007. Increasing prevalence and severity ofClostridium difficile colitis in hospitalized patients in the United States. Arch. Surg. 142:624–31

114. Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmee M. 1998. A novel toxinotyping schemeand correlation of toxinotypes with serogroups of Clostridium difficile isolates. J. Clin. Microbiol. 36:2240–47

115. Rupnik M, Pabst S, Rupnik M, von Eichel-Streiber C, Urlaub H, Soling HD. 2005. Characterizationof the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridiumdifficile toxin B (TcdB) by host cells. Microbiology 151:199–208

116. Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difficile infection: new developments in epi-demiology and pathogenesis. Nat. Rev. Microbiol. 7:526–36


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


117. Rupnik M. 2010. Clostridium difficile: (re)emergence of zoonotic potential. Clin. Infect. Dis. 51:583–84118. Saxton K, Baines SD, Freeman J, O’Connor R, Wilcox MH. 2009. Effects of exposure of Clostridium diffi-

cile PCR ribotypes 027 and 001 to fluoroquinolones in a human gut model. Antimicrob. Agents Chemother.53:412–20

119. Schirmer J, Aktories K. 2004. Large clostridial cytotoxins: cellular biology of Rho/Ras-glucosylatingtoxins. Biochim. Biophys. Acta 1673:66–74

120. Explains howC. difficile binary toxinmay contribute to theseverity of disease.

120. Schwan C, Stecher B, Tzivelekidis T, van Hamm M, Rohde M, et al. 2009. Clostridium difficile

toxin CDT induces formation of microtubule-based protrusions and increases adherence ofbacteria. PLoS Pathog. 5:1–14

121. Summarizes thecomplete genomesequence of a clinicalisolate of C. difficile.

121. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, et al. 2006. The multi-drugresistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet.

38:779–86122. Sharp SE, Ruden LO, Pohl JC, Hatcher PA, Jayne LM, Ivie WM. 2010. Evaluation of the C. Diff Quik

Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay foruse in rapid, simple diagnosis of Clostridium difficile disease. J. Clin. Microbiol. 48:2082–86

123. Shetty N, Wren MWD, Coen PG. 2011. The role of glutamate dehydrogenase for the detection ofClostridium difficile in faecal samples: a meta-analysis. J. Hosp. Infect. 77:1–6

124. Snell H, Ramos M, Longo S, John M, Hussain Z. 2004. Performance of the TechLab C DIFF CHEK-60enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficilepanel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile–associated diarrhea. J. Clin.Microbiol. 42:4863–65

125. Snyder MD. 1937. Further studies on Bacillus difficilis. J. Infect. Dis. 60:223126. Spigaglia P, Mastrantonio P. 2002. Molecular analysis of the pathogenicity locus and polymorphism in

the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates.J. Clin. Microbiol. 40:3470–75

127. Discusses andclassifies toxin variantstrains and their clinicaland epidemiologicalassociations.

127. Stabler RA, Gerding DN, Songer JG, Drudy D, Brazier JS, et al. 2006. Comparative phy-logenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulentstrains. J. Bacteriol. 188:7297–305

128. Stamper PD, Alcabasa A, Aird D, Babiker W, Wehrlin J, et al. 2009. Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for directdetection of toxin-producing Clostridium difficile in clinical samples. J. Clin. Microbiol. 47:373–78

129. Stamper PD, Babiker W, Alcabasa R, Aird D, Wehrlin J, et al. 2009. Evaluation of a new commercialTaqMan PCR assay for direct detection of the Clostridium difficile toxin B gene in clinical stool specimens.J. Clin. Microbiol. 47:3846–50

130. Sundriyal A, Roberts AK, Ling R, McGlashan J, Shone CC, Acharya KR. 2010. Expression, purificationand cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile. Protein Exp. Purif. 74:42–48

131. Swindells J, Brenwald N, Reading N, Oppenheim B. 2010. Evaluation of diagnostic tests for Clostridiumdifficile infection. J. Clin. Microbiol. 48:606–8

132. Tedesco FJ, Barton RW, Alpers DH. 1974. Clindamycin-associated colitis: a prospective study. Ann.Intern. Med. 81:429–33

133. Tenover FC, Novak-Weekley S, Woods CW, Peterson LR, Davis T, et al. 2010. Impact of strain type ondetection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassayapproaches. J. Clin. Microbiol. 48:3719–24

134. Terhes G, Urban E, Soki J, Nacsa E, Nagy E. 2009. Comparison of a rapid molecular method, the BDGeneOhm Cdiff assay, to the most frequently used laboratory tests for detection of toxin-producingClostridium difficile in diarrheal feces. J. Clin. Microbiol. 47:3478–81

135. Ticehurst JR, Aird DZ, Dam LM, Borek AP, Hargrove JT, Carroll KC. 2006. Effective detection oftoxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin. J. Clin.Microbiol. 44:1145–49

136. Torres JF, Cedillo R, Sanchez J, Dillman C, Giono S, Munoz O. 1984. Prevalence of Clostridium difficileand its cytotoxin in infants in Mexico. J. Clin. Microbiol. 20:274–75


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f T

exas

- A

rlin

gton

on

07/1

9/12

. For

per

sona

l use

onl

y.


137. Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, et al. 2009. Characterization ofthe sporulation initiation pathway of Clostridium difficile and its role in toxin production. J. Bacteriol.191:7296–305

138. Discusses thepathogenesis ofC. difficile toxins.

138. Voth DE, Ballard JD. 2005. Clostridium difficile toxins: mechanism of action and role in disease.Clin. Microbiol. Rev. 18:247–63

139. Warny M, Pepin J, Fang A, Killgore G, Thompson A, et al. 2005. Toxin production by an emergingstrain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.Lancet 366:1079–84

140. Weese JS, Avery BP, Rousseau J, Reid-Smith RJ. 2009. Detection and enumeration of Clostridium difficilespores in retail beef and pork. Appl. Environ. Microbiol. 75:5009–11

141. Weese JS, Finley R, Reid-Smith RR, Janecko W, Rousseau J. 2010. Evaluation of Clostridium difficile indogs and the household environment. Epidemiol. Infect. 138:1100–4

142. Zheng L, Keller SF, Lyerly DM, Carman RJ, Genheimer CW, et al. 2004. Multicenter evaluation of anew screening test that detects Clostridium difficile in fecal specimens. J. Clin. Microbiol. 42:3837–40

143. Zilberberg MD. 2009. Clostridium difficile-related hospitalization among US adults, 2006. Emerg. Infect.Dis. 15:122–24


Ann

u. R

ev. M

icro

biol

. 201

1.65

:501

-521

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

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Biology of Clostridium difficile: Implications for Epidemiology and Diagnosis

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Transcript of Biology of Clostridium difficile: Implications for Epidemiology and Diagnosis