persistence of Clostridium difficile during vancomycin

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1 Giat Relapse of antibioti assoiated olitis: endogenous persistene of lostridium diffiile during vanomyin therapy B A J WALTERS. R ROBERTS. R STAFFORD AND E SENEVIRATNE From tlie Departmtietnt of Mirobiology Repatriation General Hospital Brisbane Australia and the Departmrlenit of' Mediinte niversitv of Quieenslanid Queenslandl Alustralia SMMARY This study reports 24 patients with antibioti assoiated olitis due to lostridiunt diffiile. Fifteen patients were treated with vanomyin due to the severity of the olitis and in eight of these a linial relapse of the olitis ourred after vanomyin therapy was stopped. Bateriologial investigations of these patients indiated that diffiile was able to persist in stool samples during vanomyin therapy in the absene of detetable ytotoxin. This was in ontrast with the seven patients suessfully treated with vanomyin without relapse and those not treated with vanomyin where both stool ultures and ytotoxin assays beame negative. These results suggest that patients being treated with vanomyin for antibioti assoiated olitis due to diffiile should have stool ultures done during and after treatment. Persistene of the organism in the absene of detetable ytotoxin may identify those patients who relapse and lead to either reommenement of vanomyin or alternative therapeuti approahes. Oral vanomyin has been shown to be effetive in the treatment olitis aused by diffiile. 13 In some patients relapses have been reported after vanomyin therapy was disontinued46 and alternative treatment has been used or onsidered. These inlude baitrain orally7 metronidazolei and the use of holestyramine alone or in sequene with an antibioti.9 Possible reasons for relapse after vanomyin treatment inlude a failure to totally eradiate the organism from the bowel reinfetion from a ontaminated environment or a ombination of these. Resistane to vanomyin has not been desribed for diffiile and strains isolated during relapse have all been sensitive.46 Spore formation by diffiile has been observed in the stool of animals being treated with vanomyin and these return to the vegetative form when vanomyin is terminated. ") Vanomyin is not ative against baterial spores. This provides one possible mehanism whereby diffiile may persist in the presene of the high onentration of vanomyin ahieved in the stool by standard oral doses.2 In vitro studies have shown that subinhibitory onen Address for orrespondene and reprint requests: Dr E Seneviratne. Department of Mirobiology. Repatriation General Hospital. Newdegate Street. Greenslopes Australia. Reeived for publiation 14 June 1982 trations of vanomyin an inrease toxin prodution by diffiile" and normal gastrointestinal flora an inhibit its growth.12 Thus the time taken for some patients to reestablish their normal gastrointestinal flora after vanomyin therapy together with the persistene of diffiile as spores may predispose them to relapses as toxin prodution reommenes with falling vanomyin levels. Reinfetion from environmental soures has been suggested as another possible ause of relapse.4 13 Several studies have reported environmental ontamination by diffiile during and after the presene of known to be exreting the organism.' 5 These studies also found that diffiile was able to persist in the environment for several months thus providing an exogenous soure of reinfetion. There does not appear to be any predisposing fators that allow patients who relapse after vanomyin therapy to be identified. Bateriologial assessment of patients with antibioti assoiated diffiile olitis has usually been onfined to the initial diagnosis and onfirmation of the relapse. No studies have been onerned with bateriologial assessments during vanomyin therapy. This paper desribes our observations on serial stool sampling of patients 26 Gut: first published as /gut on 1 Marh Downloaded from on 11 November 218 by guest. Proteted by opyright.

2 Relapse of antibioti assoiated olitis with antibioti assoiated diffiile olitis who were treated either with vanomyin or by stopping antibiotis. Methods PATIENTS Initial stool speimens were submitted to the laboratory from inpatients and outpatients when the linial history and/or sigmoidosopy suggested antibioti assoiated olitis. Diagnosis of antibioti assoiated olitis was suspeted when there was diarrhoea starting on or soon after a ourse of antibiotis the absene of past or subsequent evidene of noninfetive inflammatory bowel disease the presene of stool leuoytes and the absene of other enteri pathogens in stool ulture. onfirmation of the diagnosis was indiated by the isolation of diffiile in the absene of other enteri pathogens and the demonstration of a faeal ytotoxin neutralised by sordellii antitoxin. Patients treated with vanomyin were all inpatients and followup speimens were obtained while the patients were being treated and after disharge from hospital. Patients not treated had less severe symptoms at the time of submission of the first speimen and after disharge followup speimens were obtained from four out of nine patients. Patients with relapses of olitis had reurrene of diarrhoea and were doumented by sigmoidosopy reisolation of diffiile detetion of the ytotoxin and the absene of other enteri pathogens. None of the patients had been on antimirobial agents in the interval between the end of vanomyin therapy and the relapse. Other relevant linial information appears in the tables. TEHNIQES Stools olleted from patients being investigated for antibioti assoiated diarrhoea were assayed for the presene of ytotoxin neutralised by sordellii antitoxin in a W138 ell line.16 ultural tehniques inluded plating of the speimen emulsified in reinfored lostridial broth RB (Oxoid Ltd London England) on to efoxitin yloserine frutose agar FA'7 and a ommerially available modifiation of this diffiile agar DA (Oxoid Ltd London England). Approximately 2 ml of the emulsified stool was inoulated diretly into RB ontaining.2% p resol.18 Subulture of this broth to seletive media (FA DA) was routinely done at two and seven days. ultures were inubated anaerobially at 37 and olonies having the gross morphology of diffiile were identified using Virginia Polytehni Institute methods. '9 Sensitivity of the isolates to vanomyin was determined in broth by standard methods.2" Toxin prodution by the isolates was determined on the supernates of 48 h ooked meat medium. Other baterial pathogens inluding Salmonella Shigella ampylobater and pathogeni Vibrio were exluded as well as parasites ova and ysts. All the ultures for baterial pathogens were performed on stools within one hour of olletion and ytotoxin assays were set up on the same day or on these speimens stored overnight at 2(. Results 2)7 The linial and laboratory findings for the eight patients infeted with diffiile who were treated with vanomyin and had relapses are shown in Table 1. The strains of diffiile isolated from these patients at the time they were being treated with vanomyin were all toxigeni in the tissue ulture assay although ytotoxin was not deteted in serial spool speimens obtained from these patients at that time. The number of speimens obtained during therapy from eah individual patient varied; two patients (D and RF) were inpatients and speimens were obtained daily; two patients (F and GH) supplied four speimens and the remaining patients supplied two speimens eah. All these speimens without exeption were shown to ontain diffiile in the absene of detetable ytotoxin. Relapses ourred as early as three days (patient D) and as late as 28 days (patient GH) after the essation of vanomyin. In the eight patients who relapsed vanomyin retreatment resulted in a ure in six and diffiile was not isolated nor the ytotoxin deteted in serial stool speimens olleted during the seond treatment. With one patient in this group (patient D) retreatment was not onsidered and in another (patient RF) retreatment did not ure the diarrhoea. These two patients ontinued to have diarrhoea and exrete the organism up to the time of their death beause of unrelated auses. In Table 2 are listed the patients treated with vanomyin who were ured without relapse. Serial stool speimens from these patients while being treated with vanomyin were both ulture and ytotoxin negative for diffiile. All the patients treated with vanomyin had severe olitis determined by sigmoidosopy and in 14/15 there was evidene of a pseudomembrane. Initial vanomyin therapy was terminated for both groups when the linial symptoms resolved although in four patients who had relapses (patients GH AM D and RF) there was not a omplete return to prediagnosis bowel frequeny. Patients reeiving no treatment (Table 3) had less severe symptoms and stool Gut: first published as /gut on 1 Marh Downloaded from on 11 November 218 by guest. Proteted by opyright.

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5 21() speimens were diffiult to obtain from this group after diarrhoea terminated. In this group four patients were ooperative and the persistene of diffiile as well as the ytotoxin in these patients ranged from three days to 22 days. diffiile was readily isolated from the seletive agar media (FA. DA) and reovered from the broth for all the initial speimens submitted. In five out of eight patients however who had relapses isolation of diffiile during treatment was only suessful from the p resol broth. There was an abrupt end to the suessful ultivation of the organism from the seven patients suessfully treated with vanomyin often after the first day of treatment. In two out of seven patients (RB and L) the last speimen from whih diffiile was isolated was via the p resol broth. This also ourred with two patients (EO and MM) who were not treated. Resistane to vanomyin (MI >2 ug/ml) was never enountered with any of the strains isolated and all the strains were toxigeni. Disussion These results onfirm the high inidene of relapse of antibioti assoiated olitis after treatment with vanomyin. For the patients treated with vanomyin in this report relapse was not apparently related to sex age ytotoxin onentration severity of the disease or previous exposure to any partiular antibioti. Patients treated with vanomyin with no relapse however were exposed to fewer antibiotis than the patients who relapsed. Four of the eight patients who relapsed reeived three antibiotis and one reeived two. Their ourse of vanomyin was at a slightly higher dose (1.9 g vs 13 g/day) and for a shorter period (9.8 days vs 13.3 days) than patients treated with vanomyin but who did not relapse. Relapse in patients treated with vanomyin was assoiated with the ontinued presene of diffiile in the absene of detetable ytotoxin in the stool. This suggests that the relapses observed in this report may be the result of the endogenous presene of the organism despite the high levels of vanomyin shown to exist in the stool by the treatment protools used.2 3 All strains of diffiile isolated were sensitive to vanomyin although in vitro sensitivity to a given antibioti does not guarantee its suess in eradiating diffiile. There have been several reports of antibioti assoiated olitis aused by isolates highly suseptible to the antibioti impliated as the ause Further work is required to understand the mehanism by whih diffiile is able to survive antibioti therapy in vivo. Relapse was not enountered in any of the patients Walters Roberts Stafford anid Seneviratne treated by withdrawal of antibiotis; none of these patients required antibiotis in the ensuing six months. The nature of the hanges in suseptibility of the bowel to olonisation by diffiile are not known. learly the soure of diffiile in an individual who develops pseudomembranous olitis is ritial to an understanding of the epidemiology of this disease in the ommunity as a whole. If the soure of diffiile is initially endogenous antibioti therapy may allow the organism to proliferate although antibioti assoiated olitis is not simply due to overgrowth of toxin produing diffiile.2' Alternatively it may be that the major soure of diffiile is exogenous as is suggested by several reports of lusters of pseudomembranous olitis and also our own report of the epidemiology of the disease in an intensive are unit Reinfetion from exogenous soures may be potential ause of relapse. The bateriologial data presented here would suggest that one of the reasons for relapse is the persistene of the organism presumably as spores within the affeted olon. The fat that faeal toxin disappears quikly implies that the number of vegetative forms of diffiile are rapidly redued by therapy with vanomyin or vanomyin inhibits toxin prodution in vivo. On essation of vanomyin therapy reversion to vegetative forms with rerudesene of the disease may potentially our if the appropriate onditions are present. Little is understood of the interations between normal faeal flora and diffiile but the observation that ertain strains of Staphyloous Pseudomonas Bateroides and Latobaillus inhibit the growth of diffiile and that the growth of ertain strains of the genera Peptoous Peptostreptoous and Bateroides were inhibited by diffiile suggests a fine balane exists between the various organisms olonising the normal large bowel.'2 Presumably if this balane is not restored before vegetation of spores ours a relapse of the illness may be possible. Thus failure to demonstrate a ytotoxin may be beause of its nonprodution by diffiile in the spore form its low onentration by redued numbers of the vegetative form or loss of the ability of diffiile to produe toxin. Some evidene for all these mehanisms was observed in the eight patients who relapsed. diffiile was isolated during vanomyin treatment from the broth in only five patients suggesting the presene of spores. Two patients (D and RF) had severely redued normal faeal flora but ontinued to exrete the organism in large numbers during treatment although a ytotoxin was not deteted. These strains whih grew on all the media used were subsequently shown Gut: first published as /gut on 1 Marh Downloaded from on 11 November 218 by guest. Proteted by opyright.

6 Relapse of antibioti assoiated olitis 211 to produe ytotoxin after growth in ooked meat medium for 48 hours. The reason why these strains did not produe ytotoxin during vanomyin therapy is not known. Patient AM had normal faeal flora present and there were only a few olonies of diffiile on the FA media (a tehnique apable of deteting as few as 1 fu of diffiilelg of stool). 17 The absene of ytotoxin from the speimens obtained from this patient during vanomyin therapy may have been the result of ytotoxin levels below detetable limits by the tissue ulture method. There is some evidene that the initial dilution neessary in the tissue ulture method may prevent the detetion of low levels of the toxin.26 The results reported in this series of patients with antibioti assoiated olitis may be of value in prognosis and treatment. Whereas mild ases of antibioti assoiated olitis respond well to the withdrawal of antibioti therapy a signifiant number of the more serious ases treated with vanomyin may require more prolonged and intensive treatment to prevent relapse. This is presumably beause diffiile is able to reestablish infetion after vanomyin therapy whih is ineffetual when diffiile is present in spores. ultural methods that inlude seletive enrihment tehniques have the advantage of deteting spores or low numbers of the organism. We have evidene that the preliminary enrihment in nonseletive broth before plating to FA is more sensitive than diret plating alone for the reovery of diffiile from stool (unpublished observations). The presene of diffiile in the stool of patients being treated with vanomyin for antibioti assoiated olitis may potentially identify those patients predisposed to relapse. Finally if the rapid reestablishment of the normal bowel flora is essential for the prevention of relapse then a more speifi antibioti for diffiile should be used. As no suh antibioti is available at the present time lower or intermittent doses of vanomyin may have less effet on the normal bowel flora. This study was supported by a grant from the Department of Veterans' Affairs of the Australian Government. We wish to thank Mrs W Gemmell and Miss J Thursby for seretarial assistane. We are grateful to the various loal liniians who referred patients and provided speimens for this study. Mr R Grie State Virology Laboratory Brisbane kindly performed the toxin assays. Referenes 1 Modigliani R Delhien J. Vanomyin for antibiotiindued olitis. Lanet 1978; 1: Tedeso F. Markham R Gurwith M hristie D Bartlett JG. Oral vanomyin for antibiotiassoiated pstid(omembrainous olitis. Lanet 1978; 2: Keighley MRB. Burdon DW Arabi Y ei al. Randomised ontrolled trial of vanomyin for pseudomembranous olitis and postoperative diarrhoea. Br Med J 1978; 2: (iorge WL Volpielli NA Stiner OB et al. Relapse of pseudomembranous olitis after vanomyin therapy. N Engl J Med 1979; 31: Roberts RK Seneviratne E. Vanomyin therapy (losiridium diffiile. Med J Aust 198; 2: Bartiett JG Tedeso FJ Shull S Lowe B hang T. Symptomati relapse after oral vanomyin therapy of antibiotiassoiated pseudomembranous olitis. Gastroenterology 198; 78: George WL Kirby BD. Sutter VL et al. Antimirobial suseptibility of lostridium diffiile. In: Shlessinger D ed. Mirobiology Washington D: Amerian Soiety for Mirobiology 1979: Trinh Dinh H Kernbaum S Frottier J. Treatment of antibiotiindued olitis by metronidazole. Lanet 1978; 1: Keutzer EW Milligan FD. Treatment of antibiotiassoiated pseudomembranous olitis with holestyramine resin. Johns Hopkins Med J 1978; 143: Onderdonk AB isneros R Bartlett JG. Study of lostridium diffiile gnotobioti mie. Infet Iminunol 198; 28(1): Onderdonk AB Lowe BR Bartlett JG. Effet of environmental stress on lostridium diffiile toxin levels during ontinuous ultivation. Appl Environ Mirobiol 1979; 38: Rolfe RD Finegold SM. Inhibitory interations between normal feal flora and lostridium diffiile. Am J lin Nutr 198; 33: Mulligan ME George WL Rolfe RD Finegold SM. Epidemiologial aspets of lostridium diffiileindued diarrhoea and olitis. Am J lin Nutr 198; 33: Walters BAJ Stafford R Roberts RK Seneviratne E. ontamination and rossinfetion with lostridium diffiile in an intensive are unit. Aust NZ J Med 1982; 12: (3) Kim KH Fekety R Batts DH et al. Isolation of lostridium diffiile from the environment and ontats of patients with antibiotiassoiated olitis. J Infet Dis 1981; 143: hang T Lavermann M Bartlett JG. ytotoxiity assay in antibioti assoiated olitis. J Infet Dis 1979; 14: George WL Sutter VL itron D Finegold SM. A seletive and differential medium for the isolation of lostridium diffiile. J lin Mirobiol 1979; 9: Hafiz S Oakley L. lostridium diffiile: isolation and harateristis. J Med Mirobiol 1976; 9: Holdeman K ato EP Moore WE. Anaerobi laboratory manual 4th ed. Bloksburg Virginia: Virginia Polytehni Institute and State niversity Gut: first published as /gut on 1 Marh Downloaded from on 11 November 218 by guest. Proteted by opyright.

7 212 Walters Roberts Stafford and Seneviratne 2 Wilkins TD. Thiel T. Moditied brothdis method for testing the antibioti suseptibility of anaerobi bateria. Anitimirob Agents (heinother 1973; 3: Saginur R. Hawev R. Bartlett JR. olitis assoiated with metronidazole therap'y. J l Dfl'lis 198: 141: George WL. Sutter VL. Finegold SM. Toxigeniity and antimirobial suseptibility of' lostriditimn diffiile as a ause of antimirobiail agentassoiated olitis. urr Mirobiol 1978; 1: Onderdonk AB. Bartlett JG. The biologial and linial signifiane of lostridium diffiile. rit Rev lin Lab Si 1981; 13: (3): Keighley MRB. Burdon DW. Mogg RH et al. Pseudomembranous olitis. Lanet 1979; 1: Kabins SA Spira TJ. Outbreak of lindamyinassoiated olitis. Ann Intern Med 1975; 83: Ryan RW. Kwasnik I Tilton R. Rapid detetion of lostridium diffiile toxin in human faees. J lin Mirobiol 198; 12: Gut: first published as /gut on 1 Marh Downloaded from on 11 November 218 by guest. Proteted by opyright.