CLINICAL GA STROENTEROLOGY

Yersinia infection with Clostridium difficile colitis

M.J. FISHMAN. MD, M.A. NOBLE. MD, FRCPC, H.J. FREEMAN. MD, FRCPC, FACP

ABSTRACT: Antibimic associated diarrhea appears to be largely due to Closcrid­ium difficile and may have, at least in part. a toxin mediated pathogenesis. Because a poor correlation exists between measurable toxin titres and symptoms, additional copathogenic factors may be important. All patients seen during a two year period with diarrhea within four weeks of antibiotic therapy, a positive $tool culture for C difficile and a positive stool cytotoxin assay specific for C difficile were investigated. All patients had stools cultured for cnteric bacterial pathogens including Salmonella , Shigella. Campylobacter, Aeromonas and Yersinia species. Seven patients had Yersinia species isolated during the course of their illness; no other entcric pathogens were identified . In four patients, Y enrerocolitica was cultured simultaneously with C difficile prior to treatment, and in one of these, Y fredriksenii was also isolated. Of six patients with persistent or recurrent sympcoms after treatment for C difficile ( ie, vancomycin in five and mecronidazole in one patient), four had positive yersinia cultures at the conclusion of therapy (Y enrerocolicica in three and Y fredriksenii in two patients). All but one of these patients had been yersinia culture negative prior to therapy for C difficile Patients with and without yersinia isolates were then compared with respect to age, sex, clinical symptoms and sigmoldoscopic as well as rectal biopsy findings. The presence of yersinia was associated with male sex, younger age and abdominal pain; other features including hematochezia, fever, arthralgia , cytotoxin titre , sig­moidoscopy and rectal biopsy could not distinguish patients with and without Yc.-rsinia species. Thus, ycrsinia may be associated with an antibiotic related diarrheal illness usually attributed to C difficile alone and may be observed in the setting of persistent or recurrent symptoms following treatment for C difficile diarrhea. Can J Gastro• enterol 1989;3( 1 )121-25

Key Words: Anribiocic diarrhea and colitis, Clostridium difficile , Clostridium difficile cywwxin. Pse.udomembrnnous colitis, Yeninia diarrht!a, Yersinia entercolitica

L'infection a yersinia et la colite attribuable a Clostridium difficile

RESUME: Les diarrhces associees aux antibiothcrapies semblent surtout attribuables a Closcridium difficile et pourraient presenter, en par tie tout du moins, une pathogenesc lice aux toxincs. Paree qu'il existe une faible correlation entre Les titres de coxines mcsurables et leb symptomes, les factcurs co-pathogenes supplcmentaires sont peut­ecrc importants. Ont etc examines: tousles patients·( l4 J vus sur une periode de deux ans ct souffrant de diarrhees moins de quatre semaines a pres une annbiotherapie, et ayant subi un coproculture d estinee a rcpcrer C difficile avec rcsultats positifs. Pour

Departments uf Medicine (Gasrroenterology) and Pathology ( Medical Microbiology). Uni1iersi1y of Briti.1h Columbia and che Un1t•ersicy Hospira I. Vancouver. Bricish Columbia

Correspondence and repnnlS Dr Hugh Freeman. Head. Gastroenr.erology, ACU F-137. Uni1oersir:y Hu~pical ( UBC Sni!I. 2211 Wesbrook Mall. Vancout•cr, British Columbia V6T I W5. Telephone (6(4J 228-7216

Received for /mbl1curion lune 17. 1988. Acccpced Ocwber 7. 1988

Vol. 3 No. I, Feb ruary 1989

C OLITIS MAY COMPLICATE ANTIMl­

crobial therapy and, in the vast majority of patie nts, appears to be due co Clos1ridium difficile ( l ). In most cases, treatment wi th oral vancomycin is effec­tive although one or more symptomatic rela pses may occur ( 1). Failure to e radi­cate the organism, possibly due to spor­ularion, or acquisi tion of a new strain of C difficile, may be responsible (2). Alter­natively, coinfection with another organ­ism or acqu isition of a second organism could occu r.

In the present stud y, all patien ts seen in the hospi tal d uring a two year period with C diffici le associated colitis were eval­uated prior to and at the conclusion of therapy fo r a second bacte rial pathogen or parasite. In this specific setting, a sig­nifican t propo rtion of patients wi th dis­ease initially attributed to C diffici le were observed co have Yersinia species infec­tion , but no o the r, bacte ria l pathogens.

MATERIALS AND METHODS Specimen source: Fecal specime ns were deri ved fro m in pati ents b e in g investigated for diarrhea or from patients referred to an ou tpatient clinic special­izing in gastrointestina l diseases; a small numbe r of specimens were submitted from a university student health service. During the two year period of this study, 40 3 stool specime ns from 269 patients were cultured for C difficile and its cyto­tox in; a ll stool specime ns were routinely cultured for Yersmia species. A total of 14 patients were identified with diarrhea beginning within fou r weeks of antibi­otic administration associated with a pos­itive stool culture and cytotoxin assay for

21

FISHMAN ec a/

tous !es patients, la mise en culture bactcriologique a porre sur !es agents pathogenes enteriqucs - Salmonella, Shigella, Campylobacrer, Aeromonas et Yeninia species y compris. Durant le cours de !cur maladic, sept des patients etaient porteurs des especes Yersinia ct aucun autre agent pathogene n'a cte identifie. Pour quatre patients, Y enrerocolicica et C difficile ont ete cu ltivcs sim ultancment avant le traitement. et chez l'un deux, y fredriksenii a etc isole. Chez six patients Souffrant de symptomcs persis­tcnts OU recurrents apres avoir ete traites pour C difficile ( vancomycin chez cinq des patients et metronidazole pour l'autre), quatrc ont donne des cultures positives pour la detection de Yersinia au terme de la therapie (Y en11..>rocolitica chez trois d'cntre cux et Y fredriksenii chez !cs deux autres) A !'exception d 'un seul, tous ccs patients avaient donne des resultats ncgatifs avant d 'etre traites contre C d1ffic1/e. Les patients chez qui Yersinia avait ete isole OU non, ont ensuite ete compares selon !'age, le sexe, les symptomcs cliniques et sigmoidoscopiques, ainsi que d'aprcs !cs resultats des biop­sies reccales. La presence de Yersinia est associee aux patients de sexe masculin, d'un age relativement plus jcune et souffrant de douleurs abdominalcs. Les autrcs caracteristiques inclucnt !'emission de selles sanglantcs, la fievre . l'arthralg1e. Les titres de cytotoxines, !es sigmoidoscopies et biopsies rectales ne sont parvenues a ecablir aucune distinction entrc !cs malades atteints de divers types de Ycrsinia et le:, autres. Ainsi, Yersinia peut etre associc aux diarrhces consccutives aux annbiotherapics que !'on a coutume d 'attribuer a C difficile seulcmcnt et pcut ctrc detccte lors des symptomes persistants et recurrents qui sc manifestent a pres le traitement des diarrhees provoquces par C difficile. La colite qui compliquc parfois les therapies anti ­microbiennes semble attribuable, clans la grande maiorite des cas, a C difficile Le plus sou vent, un traitemcnt a base de vancomycine par voie orale suffit, bien qu'une rcchute symptomatique ou plusieurs puissent se produire. Si !'on ne parvient pas a eliminer l'organisme, ii est possible qu'il y ait sporulation ou qu'une nouvelle souche de C difficile soit rcsponsable de l'echec; ou encore, qu'il y ait co-infection par un autre organisme ou acquisition d'un second agent. Dans l'etude presente, qui porte sur une pcriodc de dcux ans, rous les patients attcints d 'une colitc associee a C difficile ont ete evalues avant et au terme d 'une thcrapie destince a combattre un second agent pathogene ou parasite. Dans ce cadre particulier, un nombre significatif des patients atteints de maladies tout d'abord attribuces a C difficile sc sont averes porteurs de Yersinia, mais d'aucun autre agent bacterien pathogene

C diffici le. O f these, no patient developed diarrhea in hospital and all were referred fro m within British Columbia. Srool specimens were obtained fro m all pa­tients at the onset and conclusion of ther­apy for diarrhea presumed to be caused by C difficile infec tion. Specimens were submitted in contai ners with Cary-Blair transport medium or fresh withou t trans­port med ium .

companied by addi tional fecal samples to examine for the presence of in testinal parasites. No fecal samples were exam­ined for enteric viruses. Yersinia culture and de tection me• thods: Fecal mate ria l was streaked on yersinia selective {Schiemann CIN) me­dium (Gibso, Madison. Wisconsin) and incubated at 35°C for 18 to 24 h afte r cold enrichment in te rvals o f 24 h , three days and again after one and two weeks.

Yersinia species were fi rst detected by their characte ristic colonial morphology appearance on ye rsinia selection CIN medium . As described e lsewhere (3),

TABLE 1

typical colonies have a 'bullseye' appear­ance with a deep cherry red centre and transparent margins. Colon ies also have a ground glass appearance under the ste­reo microscope and have a distinctive odour. Suspect yersinia isolates were fur­the r screened using a routine triple sugar iron agar slant for de tection of glucose, lactose and sucrose fe rmen tation or hydrogen gas production, as well as an urea slant for urease production. In addi­tion , lysine and indo lc test reactions as well as motil ity after incubation at 28°C were assessed . Fur ther confirmation of Yersmra series was done using an AP! 20E enterobacteriaccae system (API Lab­oratory Products Inc, St Laurent, Que­bec), incubated at 28°C. A typical group o f sugar fermentation reactions for yer­sin ia permitting initial distinction of the diffe rent species is tabulated in Table I. All positive isolates identi fied to species were subm itted to an independent ref­erence laboratory for confirmation as well as definition of biotype and serotype of the organism Clostridium difficile: C difficile was cul­tured anaerobically on CD and Colum­bia CNA media for 48 h . {Capco Anaer­obic Environmental System, Santa Clara, California). Iden tification was based on colonial appcarnnce , typical 'barnyard odour', Gram sta in morphology, aerobic and anaerobic subcultu re and anaero­bic A PI sugar fermentation profile. Cyto­toxin assay followed the method of Allen (4) using filte red srool su per natant (10,000 g; 0 .45 µm fil ter pore size), human foreskin fib roblast cultures and C difficile antitoxin (TD Wilkins, Vi rginia Polytechnic Institute, Blacksburg, Vir­ginia). Ti tres were determined using serial 10-fold dilutio ns of the super­natant.

RESULTS Clinical features: Table 2 shows the age and sex of the 14 patients with C difficile

Culture media and processing: All specimens were ro utinely cul tured on sheep blood agar {Prepared Media Lab­oratories, Tualatin. O regon), McConkey agar (Difeo, Detroit, Michigan), deoxy­cholate agar (Difeo), thiosulphate-citrate­bile-sucrose agar (Oxoid, Basingstoke, UK), campylobacter agar {PML, Tualatin, Oregon), aeromonas agar (Difeo) and sel­enite bro th (Oxoid) . Isolates of Esche­richia coli were tested for their ability to

ferment sorbitol as a screen for hemor­rhagic colitis associated strains. Approx­imately 90% of the specimens were ac-

Typical sugar fermentation reactions of Yersinio species

Species Suc rose Rhamnose Roffinose fvleliblose

Y enterocotit,co + Y fred riksenii + + Y 1ntermed10 + + + Y krtstensenii

22 CAN J G ASTROENTEROL

TABLE 2 Clinical features

Clinical feature Negative

Age(years) Range 24 89

Mean 61

Sex Male 2 Female 5

Diarrhea (bloody) 7 (1)

Abdominal pain 2

Fever 2

Arthrolgias

diarrhea. Yernnia species tended to occur in younger patients compared to the group with negative cultures In addi­tion, males were more often likely to have

a positive culture. Table 2 also shows symptoms elicited

in the 14 patients with C Jifficilc diar­rhea. Inclusion criteria required the pres­ence of diarrhea within four weeks of antibiotic therapy; one patient with bloody diarrhea had a negative yersinia culture , one had a positive initial cul­ture and cine with initially negative cul­tures. whose diarrhea became bloody after failed treatment, subsequently haJ yersin ia isolated .

Abdominal pain or tenderness at the time of initial culture was more common when yersin ia was present ( three of four patients. 75",;,) than when yersinia was absent (two of seven patients. 29'~[,). With fa iled therapy for C difficile, one patient had persistent abdominal pain and per­sistence of Y enteroc.:olitica in stool. In one patient with persistent d iarrhea and ini­tially negative cultures for yersinia, abdominal pain and the presence ofboth Y enterocolitica anJ Y Jrcdriksenii devel­oped after vancomycin therapy for C difficile. Duration of symptoms did not differ in yersinia positive patients com­pared co yersinia negative patients.

TABLE 3

Yersinia cultures Positive Positive

(before therapy) (after therapy)

31 77 27-77

48 52

2 2

2 3 4 (1) 5 (1)

3 2

1 0

Neither fever nor arthralgia could pre­dict the presence lH absence of Yersi11ia species. Fever was present m two patients with negative yersinia cultures, in one patient with a positive Y enterocoliuca iso­late who responded well t<) vancomycin therapy and in one patient who had recu rrent diarrhea with Y entcrocoltttca and Y fredriksenii isolates after being ini­ti ally yersinia culture negative. Stool cultures: Results of yersinia cul­tures arc shown in Table 3. All 14 patients had a positive stool cu lture for C difficile simultaneously with positive assay for C difficile cyroroxin. Four patients (29"{,) also had confirmed isolation of Y enterocolittca frum the same initial stool specimen while Y /redriksenii was concomitantly identified in <)ne of these patients. ln all patients, positive cultures for Yersinia spe­cies were not confirmed for 14 days, therefore. therapy initiated to eradicate C difficile diarrhea was not altered be­cause of subsequent detection of Yersinia species.

Six of 14 patients 14 3%) had persis­tent or recurrent diarrhea following treat­ment with vancomycin ( five patients) or mctronidazole (one patient). Further stool cultures after this therapy resulted in isolation of Yersinia species in four of these six patien ts (6r{,): Y enreroco/1cica

Yersinia cultures in Clostridium difffc ile diarrhea

Species

Yenterocolitica Biotype 1. Serotype 0·41 Biotype 1. Serotype 0:6.30 Nontypeable

Y tredrikseni, Yintermedia

Vol. 3 No. I, February 1989

Culture positive before treatment

4 1 2 2 1 0

Culture positive after treatment

3 0 1 2 2

Yersinia and C/ostrldium dlfflc/lecohtls

in three patients and Y Jrednksenu in two (isolated concurrently with Y enterocol1t 1ca in one patient) Only one pmient with Y encerocol1ticu had been yersmta culture positive prior m treatment One further patient with a positive Y CTlk.rrocolittca cul­ture at the on~et of treatment for diar­rhea hecame asymptomatic after vanco­mycin therapy However, a repeat stool culture after 16 days of tn:atment re­vealed Y intermedw. in chis paucnt nei­ther C difficile nor Y encerocolmca could he identified following treatment No o ther enteric pathogens were isolated at

any time during the illnesses of these 14 patients. Sigmoidoscopic and biopsy findings: Flexible fibreoptic sigmoidoscopy with biopsy was done in all 14 patients at chc omet of symptoms and was repeated in all pauents with failed therapy (Table 4 ). The endoscopic and histologic observa­tions did not differentiate patients with ycr~inia 1wlated in stool cultures com­pared to patients wtthout positive cul­tu res . The patient with persisting Y entcrocolwca bdore and after treatment has a 'nonspecrfrc' cnlnis histologically confirmed at hoth times wtth positive

cultures. Clostridium difficile cytotoxin titres: Tahle 5 shows C Jrj/ICtlc cytotoxin titres in patients wtth and without positive yersinra isolates. No differences m toxm titres were observed All four patients with pcrsbient diarrhea and positive ye rsinia cultures after therapy had C difficile cytotoxin titres measured; all of these patil'nts had negative cytotoxin assays after therapy. Two other patients with persistent symptoms fo llowing treat­ment had negauve ycrsmia cultures; one of these patients had a persistently pos­itive C difficile stool cytotoxin assay ( titres of 1/ 10.000 before and after a course of vancomycin therapy).

DISCUSSION Several organisms have been etiolog­

ically implicated in post antibiotic coli­tis. Staphylococcus aureu.,1 received much attention initially ( 5) and. more recently. C perfnngcns has bt·en suggested as a pos­sible cause (6) However. the majority of cases are presently ascnhed to C difficile C difficile produces an entemtoxin (ie , toxin A) and a potent cytomxin ( re, cox in

Fl~HMAN er al

TABLE 4 Sigmoldoscopic a nd biopsy findings

Pathology features

Performed Pseudomembronous

colitis Aphthoid ulceration Nonspecific diffuse colitis Normal

TABLE 5

Negative

7

3 1 2

Clostridium difficile cytotoxin titres

Titre

1 100 111000 1110.000

Negative

3 3

B) (7-9). The cytotoxin may induce typi­cal changes in the colonic mucosa (8) and its detection has been used to establish the diagnosis (I). However, there appears m be a poor correlation between cyro­toxin titre (toxin B) and the severity of the clinical illness ( 10). Although toxin A may correlate more strongly with symptoms, other factors may also be involved ( 11)

Of 108 patients with post antibiotic C diffiale colitis in Sweden, none had other enteric pathogens isolated; some patients, however, had detectable cymtoxin but negative cultures for C difficile ( 12). This raised the possibility of C difficile cyto­toxin production by undetected organ­isms, perhaps other than C difficile ( 12, 13) Bartlett (14) has reported that five of 144 patients with significant titres of C difficile cytotoxin had negative cultures; attempts to identify other organisms in such circumstances were unsuccessful. In contrast, other studies have indicated that more 'traditional' pathogens such as Salmonella and Camtrylobacter species may be isolated from patients with C difficde. Culture techniques and/or, pos­sibly, geographic factors may have ac­counted for the unusually high fre­quency of yersinia in seven (or 50%) of the present patients with C difficile diar­rhea. Post antibiotic colitis due to Y e11terocolitica in the absence of C difficile has also been reported ( 17). Finally, three

24

Yersinio cultures Positive

(before therapy)

4

1

0 2

Yersinio cultures Positive

(before therapy)

2 0 2

Positive (ofter the rapy)

4

2

1

0

Positive (ofter therapy)

4 0

patients with Y enrerocolinca associated diarrhea have been described with C difficile cyrotoxin also detected (3 ).

Administration of antibiotics in the month prior to positive stool culture was previously noted in 29 of 122 patients with Y enterocolirica associated diarrhea (3 ). The role of antibiotics in predispos­ing to coinfection with Yersinia species and C difficile is uncertain but compel­ling Of particular interest is the role of vancomycin or metronidazole therapy in the development of new isolates of Yer­sinia species in four patients reported here. This seemed to be especially asso­ciated with persisting or recurrent diarrhea.

Clinical differentiation of patients with and without yersinia in the presence of C difficile appears to be difficult. Abdom­inal pain was more common with yersinia in the present study, but this symptom is well recognized in patients with C diffiale colitis ( 18). Fever anJ bloody diar­rhea were also present in both groups. Arthralgias, reported in up to 30% of patients with Y enterocolirica infection ( 19,20) also occurs in C difficile colitis ( 21-24) and could not predict the pres­ence of Yersinia species in the present patient:;.

Yersinia enterocolitis has been associ­ated with normal mucosa, non:;pecific colitis and colonic mucosa! aphrhoid ulcerations (25), but in this study, sig-

moidoscopic examination and rectal mucosa! biopsy of patients with C difficile diarrhea could not differentiate between patients with and without yersinia co­infection. Pseudomembranes were seen in a minority of patients wrth and with­out yersinia. Two of the present patients, both having isolates of C difficile and Y encerocolicica, presented as an exacerba­tion of previously diagnosed inflamma­tory bowel disease; both C difficile (26-28) and yersinia ( 3,29) have been implicated

in this setting. Under these circum­stances, morphologic diagnosis may be very difficult.

In summary, an inordinately high prev­alence of Yersinia species exists in the stools of patients referred to the authors' institution with post antibiotic C difficile colitis. Failed therapy of C difficile colitis may be associated with yersinia coinfec· tion. Yersmia species, particularly Y enterocolitica, must be considered poten­tial enteric pathogens in the post antibi­otic state and the confirmation of C difficile does not obviate the need to care­fuily seek their presence in this situation

ACKNOWLEDGEMENTS: The author, thank Dr Sandu Toma of the National Ref­erence Centre of Yersinia. Ontano Ministry of Health, Public Health Lahoramries. To­ronto, Ontario.

REFERENCES l. Freeman HJ Antih1otic-inducecl

rseudomembranous colitis. New approaches. Drug Therapy 1985; 15:117-30.

z. Walters BAJ, Roberts R, Stafford R, Seneviratone E. Relapse of ant1biouc associated colitis. Endogenous pcrs1s• tence of Clostrrdium diffic,le during vancomycin therapy Gur 1981;24:206-12

3. Simmonds SD, Noble MA. Freeman HJ Gastrointestinal features of culture· rositivc Ycrsmia cnterocoli11ca infection Gastroenrerology 1987 ,9 2: 11 Z-7.

4. Allen SD. Clos1nd11m1 In. Lcnnerte EH, ed. Manual of Clinical Microbiology, 4th cdn . Washington, DC: American Society of Microbiology, 1985-4 34-44

5. Hummel RP, Altemeier WA, Htll E. Iatrogenic staphylococcal enterocolius. Ann Surg 1964:138:551-60.

6. Borrielo SP. Larson HE. Enterotoxigcnic Clostridi11m perJrmgens. A possible cause of amibiotic-.1ssociated diarrhea Lanc<"I 1984;i: 305-7

CAN J GAS1 ROENT EROl

7 T.1ylor NS. Thorne GM. Bartlett JG Comp:mson of 1wo toxins rroduced hy CloicraJ1um d1ffirilc lnfcu lrnrnun 1981. 34· 1036-4 3

q Lyerly DM. Lockwood Dt, R1chard,011 SH. Wilkins TD B1olog1cal acuv1t1cs of toxins A and Bo( Clo.1md1um difficile lnfw Irnmun 1982. 35 1147· 'iO

9 Sullivan NM. Pcllt•t S. Wilkins TD Purification and charactem:ltlon of toxins A and B of Closcmlnm1 J1fficile. Infect lmmun 1982.35 IOU-4l).

IO. Burdon DW. George RH, Mogg G Fat•cal mxin and severity of anubiotic·associated pscudornembra nouscohtis.J Clin Pathol 1981, 34548-51

11 Justus PG, Martin JL. Goldht·rg DA. et al Myoelecmc effects of ClmcnJ1um difficile Motil it\ altering factors distinct from llS cyttlfOXII) and entcmtoxm m rahh1cs Gastrot·n1ert1logy 1982 ;8 3 :8 36-4 3

12. Awn,son B. M1,llhv R Nord CE Ant1m1crob1al agents and Closcnd111m difficile m acute entcric diseast· Ep1dcrn1olt,g1c data from "weden. 1980-82 J lnfw D1s 1985, 15147(d'll

13 Nakamura S Cy1otoxin rmducucm hy CIMtridnmt ~orJcllii strain, Microhiol lmmunol 198 3.17 495-502 .

14 Bartlett JC, Anubmuc associated colitis. Dis Mon 1984;30 I 54

15 Falsen E. Ka11ser B. Nehls L, Nygren 8, Svedham A Clo.11rnl1um difficile in rdation to entcm bacterial pathogens J C lin Milmbiol 1980, 12:297-300.

16. Brittle RP Wallace E. Closmd111m d1/ficile assonatl'd d1arrhl'a J lnfccuon 1984;!-t IZ 3-8

17 Brown R. Tedesw rJ, Assad RT. Rao R )ernn1<1 cohus masquerading as pscudomt•mhran11us colttts. Dig Dis Sri 1986;31: 548-51

18 Mogg GM. Keighley M An11hi1,11c associatt·d wlitis A review 1l 66 cases. Br J Surg 1979;66 7 '38-42

19. Ahvoncn P, Sievers K, Aho K Arthritis associated with Yerm11u en!t!rocol111cu iniectton Acta Rheum .;,,and 1%9.15.23Z-'i3

Z0 Ahvo1wn P Human yersin 111s1s m Finland 11 Clinical features Ann Clin Res 197 Z.4 39-48

Z I Rollin DE. Moeller D Acute migratory polyarthrttis associated with anubiotic· induced rseudomembranous colt tis Am J Gastroenteml 1976,65 • '35 3-6

Z2 Rothchild BM. Masi AT.June PL Arthmis associated with ampicillin colitts Arch Intern Mt·d 1977 135 1605-7

Yersinia and C/ostrfd/um difficl/e colihs

Z 3 Bolton RP. Wood GM. Lo'<,wsky MS. Acute arthniis assornttl•d w11h Cl,MraJium J1/fiole rol1tl\ Br McJ J 1981,Z8l 102 H

24. Lofgren RP, T..idh k 1 M. Sol 11, RD Acute oligoarchmis associated w11h Clo.11r1dium difficile pscudomcmhranous coitus. Arch Intern Mt·d 1%4.144 617-9

25 Vantrappcn G. A1-ig HO, Ponl'tlt' E, Gehoes K, Bertrand PH Yt·rnnw enteritis and cntcrocolttis · Gasrm1•n1cm• logical aspects. Ga,troenterology 1980;72:220-7.

26 La MontJT, Trnka Y Therapcutlt' imrlicati11ns of Closrndium J1/ftnl1· t<lxm during rclarsc of chrome inflammatory howcl disease Lancet 1980;i· '381 Z.

l.7 Trnka YM. La Mont JT As,ociatton of Clomidium difficile toxin with symptomauc rel,1rse of rhronK mflamm,itory b1,wcl dist•ase Ga,trt>· entcmlogy 1981 ;80:69 >·6.

28. Mey1•rs S, Mayer L. &,none E. Desmond E, Janowm HD Occurrence of Closmd111m difficile toxin durtnl! the course of mflammamry bowel disease Gastroentcwlogy 1981 ,!:\0 697-700.

Z9 Trcacher OF, Jerwell DP Ycrsmw colitis associated with Crohn', disease Postgrad Mt·d J 1985.61.17 H

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Disease Markers

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com