Original Paper Digestion 1992;53:121-128

Public Health Laboratory, Odstock Hospital, Salisbury, UK; PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK; General Infirmary, Salisbury, UK; Royal and New Cross Hospitals. Wolverhampton, UK; Princess Margaret Hospital. Swindon, UK

KeyWords Ulcerative colitis Inflammatory bowel disease Enteric infection Aetiology Faeces Microflora Colonisation resistance Cytotoxin

Search for Enteric Microbial Pathogens in Patients with Ulcerative Colitis

Abstract Microbial pathogens were sought in faeces of patients with active ulcerative colitis and again after 3 months treatment. 64 patients were examined during their first episode of ulcerative colitis and 30 with relapse of chronic disease. At presentation, bacterial pathogens were not found; 1 patient had cryptosporidiosis. In 10 patients treatment appeared to result in some loss of colonisation resistance as evidenced by colonisation with (3-haemolytic streptococci, Staphylococcus aureus, Can­ dida and Clostridium difficile. Unidentified cytotoxic activity was present in the faeces of 4 patients at presentation and 2 patients during or after treatment. We conclude that enteric infection is an uncommon finding in patients with active ulcerative colitis.

The role of infection in the aetiology and pathogenesis of ulcerative colitis is obscure. The pathological features of several infective enteropathies can resemble ulcerative colitis. Many studies have claimed to have discov­ ered a relationship between microbial patho­ gens and colitis; those incriminated include mycobacteria, treponemes, clostridial toxin, cell-wall-deficient bacteria and virus. How­ ever, subsequent studies have failed to con­ firm such findings, and no aetiological agent

Received: February 24. 1992 Received in revised form: July 27,1992

of ulcerative colitis has been identified [1,2]. Although enteric infection was once thought to predispose to ulcerative colitis [3-5], this is seldom observed today [1, 2], However, spo­ radic reports continue to implicate infection as an initiating factor; recent examples in­ clude salmonella [6] and aeromonas [7] infec­ tions. Concurrent infection in colitic patients can complicate differential diagnosis, and pa­ tient management can be problematic [8],

M.J. Hudson Division of Pathology PHLS Centre for Applied Microbiology & Research Porton Down, Salisbury. Wilts. SP4 0JG (UK)

© 1992 S. Kargcr AG, Basel 0012-2823/92/ 0534-0121$2.75/0

Downloaded by: Stockholm University Library 130.237.165.40 - 12/5/2017 5:15:45 AM

W.J. Brown3, M.J. Hudsonb S. Patricka S. C. W. Matthewsa M.J. Hillb, A.E. Gentc R.H. Graced, M D. Hellierc E.T. Swarbrickd

Patients and Methods Patients anil Samples The patients presented for diagnosis and treatment of active colitis at primary referral clinics in hospitals collaborating in a research programme on the diagno­ sis and management of idiopathic inflammatory' bowel disease (the IBD Study Group). Study protocols were approved by local ethics committees, and patients gave informed consent. 64 patients (68%) had active ulcer­ ative colitis but no previous history of inflammatory bowel disease, and 30 patients (32%) had active ulcer­ ative colitis in relapse. Routine clinical investigations included sigmoidoscopy and rectal mucosal biopsy, and diagnosis was confirmed by characteristic clinical history and typical biopsy histology supported by radi­ ology and endoscopy when these were indicated. The anatomical extent of disease on presentation was de­ termined accurately for 73 patients: 38 (52%) had dis­ tal disease or proctitis only, 13 (18%) had left-sided disease, 8 (11%) had colitis extending to the hepatic

122

flexure, and 14 (19%) had total (entire) colitis. The patients were treated with steroids and/or sulphasalazine. Diagnosis of ulcerative colitis was confirmed by follow-up examinations and supported by the response to treatment: after remission was achieved, withdrawal of maintenance treatment resulted in worsening of symptoms or frank relapse of active colitis. Patients who had taken antimicrobials in the preceding 2 weeks or those with either Crohn’s disease or indeterminate colitis were excluded from the analysis. Samples of faeces, obtained at presentation and at a follow-up examination after 3 months, were snapfrozen in liquid Ni or on dry' ice and stored frozen at -70 °C. Matched serum samples were stored frozen. Microscopy and Culture Paired faecal samples from each patient were thawed rapidly and examined for pathogens by stan­ dard diagnostic laboratory procedures. Wet films stained with dilute iodine were examined for ova, cysts and parasites. Fixed smears were stained by a modified Ziehl-Neelsen procedure [ 14] for Cryptosporidium sp. oocysts and confirmed using a safranin/methylcneblue stain [15]. Faecal samples were inoculated onto a wide range of non-selective, selective and enrichment media (ta­ ble 1) and incubated for the time and under the condi­ tions indicated. Each batch of media was quality-con­ trolled for selectivity and growth characteristics. Mi­ crobial growth over the four quadrants of streaked plates was estimated. Colonies were purified by sub­ culture, identified presumptively and confirmed by specific biochemical assays and serology. Validation Study Microbiological methods were validated using fae­ cal samples from the routine diagnostic laboratory or faeces seeded artificially with pathogens. Estimates of pathogens were made by microscopy or culture before and then I week and 6 months after freezing at-20°C . Faeces containing Cryptosporidium oocysts, G. lam­ bda cysts or Campylobacter jejuni were selected from routine clinical specimens. Normal faeces were seeded with various enteric pathogens using neat or a 10-4 dilution of 18-hour cultures of Bacillus cereus, Staphy­ lococcus aureus, Escherichia coli 0111, Salmonella typhimurium, Shigella Jlexneri, Aeromonas hydrophila. Yersinia enterocolitica, Vibrio parahaemolyticus, Clostridium perfringens type A and Candida albicans.

Brown/Hudson/Palrick/Matlhews/Hill/ Gcnt/Grace/Hell ier/Swarbrick

Enteric Pathogens in Ulcerative Colitis

Downloaded by: Stockholm University Library 130.237.165.40 - 12/5/2017 5:15:45 AM

Many factors have been implicated in pre­ cipitating relapse of chronic ulcerative colitis although conclusions from different studies are often conflicting. Of particular interest in a microbiological context is the not infre­ quent observation that a relatively trivial in­ fection immediately preceeds relapse, most commonly an upper respiratory tract infec­ tion [9-13] or gastroenteritis [II]. These cor­ relations are supported by other, associated, risk factors such as recent foreign travel [11]. treatment with antibiotics [11] and use of analgesics [13]. This paper describes a study designed to re-examine the role of enteric infection in ulcerative colitis by retrospective study of an unselected patient group present­ ing at primary referral clinics with active dis­ ease. two thirds of whom presented with no previous history of inflammatory bowel dis­ ease. Faeces were screened for a comprehen­ sive range of enteric pathogens and potential pathogens, both before and after treatment, together with relevant serological assays.

Table 1. Microbiological culture media and conditions Medium

Incubation conditions

Organisms sought

Blood agar (BA (Oxoid CM271)

aerobic + 5% CO2

Staphylococci E. coli Streptococci B. ceretts

NeoTTC agar

aerobic + 5% CO2

C. albicans

[BA base with neomycin 500 mg/l & TTC (Oxoid SR 148)]

BA neomycin ( 100 mg/l)

anaerobic

C. perfringens

MacConkey agar (Oxoid CM7)

aerobic

E. coli Plesiomonas shigelloides

DCA (Oxoid CM227) aerobic [and after Selenite F broth (Oxoid R39) enrichment]

Salmonella sp. Shigella sp.

TCBS (Oxoid CM 333)

Vibrio sp.

aerobic

[and after alkaline peptone water (Oxoid CM9) enrichment]

BA ampicillin ( 10 mg/l)

aerobic, 24 h, 30 °C

Aeromonas sp.

[and after alkaline peptone water (Oxoid CM9) enrichment at 25 °C]

CIN agar (Oxoid CM653)

aerobic, 24-48 h, 30°C

Yersinia sp.

[and after enrichment in buffered peptone (CM509I water at 4 0.5). One patient had slightly elevated IgG but high lgM in both scrum samples (IgM: 9.18 g/1 on day 0. 9.99 g/1 at 3 months); there was no obvious correlation with micro­ biological or clinical findings. A patient with unidentified cytotoxin had a raised IgM con­ centration at 3 months, but this had not

Discussion We chose to conduct this survey retrospec­ tively in order that batches of samples could be processed using standardised media and assays. This necessitated storage of the faeces frozen at -70 °C for up to 18 months before analysis. A validation survey confirmed that frozen storage even at - 2 0 °C did not alter markedly the sensitivity of detection of the majority of the microbial pathogens sought. We did note that whereas Cryptosporidium oocysts were found in 1 patient sample frozen at -7 0 °C for 18 months, oocysts in the con­ trol sample frozen at -2 0 °C for 6 months decolourize in the modified Ziehl-Neelscn procedure. This suggests that storage of faeces at -2 0 °C is inappropriate for Cryptosporidia investigation. Our observation of loss of via­ bility of the aeromonad, vibrio and yersinia after freezing requires further study. It is pos­ sible that these organisms might survive freez­ ing better in naturally infected faeces, and our observed loss of viability in seeded stool sam­ ples is artefactual. In the patient survey we did not isolate any enteric pathogens at onset of active ulcerative colitis, other than to identify a single case of cryptosporidiosis. The results of the valida­ tion study suggest that, if present in the pa­ tient samples, most pathogens would have been recovered even after frozen storage. Our negative culture results are supported by a recent comparable microbiological survey in

126

which Ljungh and Wadstrom [16] failed to culture any enteropathogen or find C. difficile cytotoxin in fresh faeces from 68 patients with active relapse of ulcerative colitis. In view of the poor recovery of aeromonas, yer­ sinia and vibrio from our frozen faecal sam­ ples. it is of particular interest to note that Ljungh and Wadstrom [16] also did not find Y. enterocolilica. At follow-up examination after 3 months of treatment, several of our patients were colonised by Candida, staphylo­ cocci or toxigenic C. difficile, although there was no evidence that this markedly affected clinical outcome. A patient who failed to im­ prove with treatment was thought to have been colonised with Y. ruckeri, but we ques­ tioned this finding since this is very rarely iso­ lated from man [17], It was subsequently identified as H. alvei, an organism of uncer­ tain pathogenicity [17], The contribution of this organism to the disease activity in this patient is unknown; his colitis eventually sett­ led on steroid enemas. We were unable to detect consistent alter­ ations in the normal flora in our patients, either at onset of active disease or after treat­ ment. The characterisation of the predomi­ nant obligately anaerobic faecal flora was beyond the scope of this study. The predomi­ nant facultative flora comprised faecal strep­ tococci (enterococci). We noted the numbers and varieties of E. coli, streptococci and C. perfringens type A in an attempt to detect alterations in the balance of the flora in active disease. In patients with large numbers in active but not treated disease, we sought a rise in specific serum antibodies but found none. There was similarly no marked changes in total serum immunoglobulin concentrations. The carriage rates of E. coli of 33 and 43% (acute and treated samples, respectively) were rather low in comparison with rates in excess of 80% in healthy adults [18] or colitics [19. 20], and we can advance no explanation for

Brown/Hudson/Patrick/Matthews/Hill/ Gent/Gracc/Hellier/Swarbrick

Enteric Pathogens in Ulcerative Colitis

Downloaded by: Stockholm University Library 130.237.165.40 - 12/5/2017 5:15:45 AM

specimens with this cytotoxic activity had no E. coli detected and 1 had only very sparse E. coli. We did not attempt serial passage of the cytotoxic activity nor did we examine affected cells by electron microscopy for vi­ rus; the rapid development of the cytotoxic effect, usually evident after 12h, argued against virus.

these would not have been missed in the Swedish study [ 16]. The nature and role of the faecal cytotoxin found in some patients re­ mains to be determined. The balance of the normal intestinal flora appears to be some­ what disturbed by the disease or by treatment and this may result in loss of colonisation resistance; this may be reflected in the cases of concurrent C. difficile, salmonella and Campy­ lobacter infection which have all been well documented in the literature. That microbes play some part in the pathogenesis of ulcer­ ative colitis is a widely held belief [1], but their precise role remains obscure. Recent reports of the efficacy of antimicrobials such as vancomycin [28] and tobramycin [29] in the treatment of active ulcerative colitis jus­ tify further investigation of this relationship.

Note Added in Proof A recent survey of concurrent infection in IBD by Weber and colleagues (Tübingen) found evidence of infection in two of 15 colitis patients. One had S. typhimurium and another had C. difficile. In neither case did the infection influence clinical course. (.1 Clin Gastroent 1992;14:302-308.)

Acknowledgements Our thanks to Mrs. Cherry Sell and Mrs. Veronica Brown (Royal Hospital. Wolverhampton, UK) for sample collection and collation of patient data. Dr. Howard Tranter. PHLS Centre for Applied Microbiol­ ogy and Research kindly provided antiserum to C. per­ fringens enterotoxin. We thank Dr. H. Malnick, Cen­ tral Public Health Laboratory, and Dr. Robert Davies, University of Glasgow. UK, for helpful advice. We are most grateful to Dr. G.F. Batstonc. Salisbury General Infirmary, for the serum immunoglobulin estimations and to Pharmacia (UK) for a grant to cover the cost of these analyses.

127

Downloaded by: Stockholm University Library 130.237.165.40 - 12/5/2017 5:15:45 AM

this observation; recovery of enteropathogenic E. coli used in the validation study was unaffected by freezing. In contrast, carriage rates of 50 and 48%. respectively, for C. perfringens fell within the normal range [ 18. 21 ]. There was no evidence of faecal enterotoxin in those patients with high counts of C. pcrfringens type A. Unidentified cytotoxic activity was de­ tected in samples from 6 patients. We did not attempt serial passage of the Vero-cell cyto­ toxic factor nor did we examine cells or super­ natant for virus by electron microscopy. It was due to neither C. perfringens type A nor C. difficile: we lacked an antiserum specific to the Shiga-like verotoxin produced by some strains of E. coli with which to perform neu­ tralisation studies. Others have observed a specific cytotoxic effect of ulcerative colitis faecal supernatants in tissue culture cell lines [22. 23], and several recent reports describe the presence of verotoxic E. coli in both active and quiescent phases of the disease [ 16, 2426], Infection with verotoxic E. coli can both mimic ulcerative colitis and also obscure its diagnosis [24], Verotoxic E. coli are quickly lost from faeces [27], and it is notable that 1 of the 6 patients had scanty E. coli and the remaining 5 had none. We can summarise that enteric infection appears to be an uncommon finding at onset of ulcerative colitis as seen at primary referral clinics in the UK. This is an agreement with a recent Swedish survey [16] but differed in the important respect that the majority of our patients had no previous history of ulcerative colitis. Thus we can conclude that infection with enteropathogens is unlikely to be the trig­ ger event either in the initiation of the disease or in precipitating relapse. Our findings are qualified with the proviso that our methodol­ ogy might have caused us to miss some cases of infection with yersinia, aeromonas and vibrio, although this does seem improbable as

1 Kirsner JB. Shorter RG: Recent de­ velopments in 'nonspecific' inflam­ matory bowel disease. N Engl J Med 1982;306:837-848. 2 Thayer WR Jr: Infectious agents in inflammatory bowel disease; in Jarncrot G (ed): Inflammatory Bowel Disease. New York. Raven Press. 1987. pp 101-107. 3 Felsen J: The relationship o f bacil­ lary dysentery to distal ileitis, chronic ulcerative colitis and non­ specific intestinal granuloma. Ann Intern Med 1936;10:645-669. 4 Banks BM. Korelitz Bl. Zetzel L: The cause o f non-specific ulcerative colitis: Review o f twenty years' ex­ perience and late results. Gastroen­ terology 1957;32:983-1012. 5 Lindeman RJ. Weinstein L. Levitan R. Patterson JF: Ulcerative colitis and intestinal salmonellosis. Am J M edSci 1967:254:855-861. 6 Taylor-Robinson S. Miles R. Whitehead A. el al: Salmonella infection and ulcerative colitis. Lancet I989:i: 1145. 7 Willoughby JMT, Rahman AFMS, Gregory MM: Chronic colitis after Aeromonas infection. Gut 1989:30: 686-690. 8 Dronficld MW. Fletcher J. Langman MJS: Coincident salmonella infections and ulcerative colitis: Problems o f recognition and man­ agement. BrM edJ 1974:i:99-100. 9 Jewell DP: Factors precipitating re­ lapse o f ulcerative colitis and Crohn's disease: in Jârnerot G (ed): Inflammatory Bowel Disease. New York. Raven Press, 1987. pp 141 — 145. 10 Mee AS. Jewell DP: Factors induc­ ing relapse in ulcerative colitis. Br Med J 1978;iii:801—802. 11 Isgar B, Harman M, Whorwell PJ: Factors preceding relapse in ulcer­ ative colitis. Digestion 1983:26: 236-238.

12 Kangro HO. Chong SKF. Hardiman A. el al: A prospective study of viral and mycoplasma infections in chronic inflammatory bowel dis­ ease. Gastroenterology 1990:98: 549-553. 13 Rampton DS. McNeil N l. SamerM: Analgesic ingestion and other fac­ tors preceding relapse in ulcerative colitis. Gut 1983:24:187-189 14 Casemore DP, Armstrong M. Jackson B: Screening for Cryptospori­ dium in stools. Lancet 1984:i:734— 735. 15 Baxby D. Blundell N: Sensitise, rap­ id, simple methods for detecting Cryptosporidium in faeces. Lancet 1983;ii: M49. 16 Ljungh A. Wadstrom T: Subepithelial connective tissue protein bind­ ing o f Escherichia coli from patients with ulcerative colitis; in MacDermott RP (ed): Inflammatory Bowel Disease: Current Status and Future Approach. Amsterdam. Excerpta Medica, 1988. pp 571-575. 17 Farmer JJ III. Davis BR. HickmanBrenner FW. et al: Biochemical identification o f new species and biogroups o f Enterobacteriaccae iso­ lated from clinical specimens. J Clin Microbiol 1985:21:46-76. 18 Fincgold SM. Sutter VL, Malhisen GE: Normal indigenous intestinal flora: in Hentges DJ (ed): Human Intestinal Flora in Health and Dis­ ease. London. Academic Press. 1983. pp 3-31. 19 Cooke EM: A quantitative compari­ son o f the faecal flora o f patients with ulcerative colitis and that o f normal persons. J Pathol Bactcriol 1967;9:439-444. 20 West B. Lendrum R. Hill MJ. Walker G: Effects o f sulphasalazine (Salazopyrin) on faecal flora in pa­ tients with inflammatory bowel dis­ ease. Gut 1974;15:960-965.

21 Stringer MF: Clostridium perfringens type A food poisoning: in Borriello SP (ed): Clostridia in Human Disease. Boca Raton. CRC Press. 1985. pp 117-143. 22 O'Mora in C. Prestage H. Harrison P. et al: Cytopathic effects in cul­ tures inoculated with material from Crohn's disease. Gut 1981:22:823826. 23 McClaren L, Gilnick G: Ulcerative colitis and Crohn's disease tissue cytotoxins. Gastroenterology 1982:82: 1381-1388. 24 Hunt CM. Harvey JA. Youngs ER. et al: Clinical and pathological vari­ ability o f infection by enterohaemorrhagic (Vcro cytotoxin produc­ ing) Escherichia coli. J Clin Pathol 1989:42:847—852. 25 Ljungh A. Eriksson M. Eriksson O. et al: Shiga-like toxin production and connective-tissue protein bind­ ing o f Escherichia coli isolated from a patient with ulcerative colitis. Scand J Infect Dis 1988:20:443446. 26 von WullTen, Russmann H, Karch H. et al: Verocytotoxin-producing Escherichia coli 02:H 5 isolated from patients with ulcerative colitis. Lancet 1989:i: 1 149-1150. 27 Pai CH. Gordon R. Sims HV. Bryan LE: Sporadic cases o f hemorrhagic colitis associated with Escherichia coli OI57:H7. Ann Intern Med 1984;101:738-742. 28 Dickinson RJ. O'Connor HJ. Pinder I. et al: Double blind controlled trial o f oral vancomycin as adjunctive treatment in acute exacerbations o f idiopathic colitis. Gut 1985:26: 1380-1384. 29 Burke DA, Axon ATR, Clavden SA. et al: The efficacy o f tobramycin in the treatment o f ulcerative colitis. Aliment Pharmacol Ther 1990:4: 123-129.

128

Brown/Hudson/Patrick/Matthews/Hill/ Gent/Grace/Hellier/Swarbrick

Enteric Pathogens in Ulcerative Colitis

Downloaded by: Stockholm University Library 130.237.165.40 - 12/5/2017 5:15:45 AM

References

Search for enteric microbial pathogens in patients with ulcerative colitis.

Microbial pathogens were sought in faeces of patients with active ulcerative colitis and again after 3 months treatment. 64 patients were examined dur...
1MB Sizes 0 Downloads 0 Views