Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens

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1 Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens Microbiol. Immunol., 40(10), , 1996 Akihito Wada*,', Yoshishige Masuda', Makiko Fukayama2, Tsutomu Hatakeyama2, Yoshitoki Yanagawa3, Haruo Watanabe', and Takashi Inamatsu2 'Department of Bacteriology, National Institute of Health, Shinjuku-ku, Tokyo 162, Japan, 'Division of Infectious Diseases, Tokyo Metropolitan Geriatric Hospital, Itabashi-ku, Tokyo 173, Japan, and 'Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo 169, Japan Received January 12, 1996; in revised form, July 15, Accepted July 16, 1996 Abstract: To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C. perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C. perfringens isolates from those samples were grouped by slide agglutination and by pulsed-field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C. perfringens, and not by a food-borne outbreak. Key words- Nosocomial diarrhoea, Enterotoxigenic Clostridium perfringens, Reverse passive latex agglutination assay, Pulsed-field gel electrophoresis Clostridium perfringens, a Gram-positive spore-forming anaerobe, causes various clinical problems ranging from a self-limiting enteric disease to life-threatening gas gangrene. Enterotoxin-producing strains of types A and less frequently C cause enteric disease characterized by diarrhoea and abdominal cramps with minimal systemic toxicity. It has been thought that the majority of the enteric disease associated with enterotoxigenic C. perfringens is food-borne. Recently, the epidemiological spectrum of the disease has been expanded to include sporadic cases of diarrhoea (10), antibiotic-associated diarrhoea (3) and diarrhoea in geriatric wards (4, 9, 13). We present here epidemiological evidence for the nosocomial spread of C. perfringens in our geriatric hospital; spread which was not thought to be associated with a food-borne outbreak. From January to August 1993 in Tokyo Metropolitan Geriatric Hospital, 69 faecal specimens from 58 elderly patients having symptoms of anorexia, diarrhoea and/or vomiting were cultured to isolate C. perfringens. Multiple specimens were presented from ten patients to reexamine the etiological organism(s) causing diarrhoea persisting more than five days, or to confirm that the number of C. perfringens was reduced to less than a significant level. As described in a manual (1), faecal *Address correspondence to Dr. Akihito Wada, Department of Bacteriology, National Institute of Health, Toyama , Shinjuku-ku, Tokyo 162, Japan. specimens were heated at 80 C for 10 min without dilution to select spores. One loopful of each specimen (less than 10 E.tl) was then streaked on a GAM agar plate (Nissui, Tokyo) with egg yolk, and was cultured for one or two days under anaerobic conditions in a chamber and/or jar. If more than 100 colonies were observed, one colony from each specimen was subjected to further analysis. All specimens were cultured for other enteropathogenic organisms as well. Environmental C. perfringens in ward E09 was examined at 84 points as well as from two staff members employing a stamp method (7), and was cultured using GAM agar (Nissui) with egg yolk. No spore selection was performed in this method. Identification of C. perfringens was performed using a Sceptor (BBL, Md., U.S.A.). The enterotoxin production of C. perfringens isolates was examined using a reverse passive latex agglutination assay kit (Mitsubishi Chemical Corporation, Tokyo). Each isolate was cultured for 48 hr in a modified Duncan-Strong medium (11), and then the supernatant was tested for the enterotoxin. The enterotoxin in faecal specimens was also examined using the same kit. A 0.5 ml volume of specimen was mixed with an equal amount of the buffer supplied in the kit and centrifuged at 2,000 X g for 15 min. Then, the supernatant was examined for the enterotoxin by the same method. Abbreviation: PFGE, pulsed-field gel electrophoresis. 767

2 768 A. WADA ET AL Serogrouping of the isolates was performed using a slide agglutination procedure with rabbit antisera (8, 12). Antisera TW18 and TW40 were previously raised in our laboratory from C. perfringens strains which do not react with Hobbs' antisera (8). Antiserum WX2 was newly raised from a C. perfringens isolate of food poisoning using a conventional method. Briefly, the isolate was cultured using a GAM agar plate (Nissui). The cells were fixed in saline with 0.6% formaldehyde, washed three times with saline and then used to immunize rabbits intravenously. The resulting serum was used without absorption by other C. perfringens strains. Pulsed-field gel electrophoresis (PFGE) was performed according to the description by Canard et al (5). About 1 µg genomic DNA of each isolate was digested using 20 units of restriction endonuclease Smal (Toyobo, Tokyo) and was subjected to PFGE using CHEF DRII (Bio-Rad, Calif., U.S.A.). The conditions of PFGE were as follows: 1% agarose in 0.5 X TBE (Trisborate 45 mm, EDTA ph mm), with an electric field strength of 6.2 V per cm and a 25 sec switching interval which continued for 20 hr running time. After PFGE, DNAs were stained with ethidiumbromide, and photographs were taken under 254 nm UV transillumination. Sixty C. perfringens isolates were obtained from 49 of the 58 patients; three isolates from one patient, two each from nine patients and one each from 39 patients. Fifty two isolates from 43 patients tested positive for enterotoxin. As to the three isolates from the single patient, two were positive for enterotoxin and the other was negative, which was isolated from a specimen presented after the patient had recovered from diarrhoea. Eight of the nine patients, each from whom two isolates were obtained, had persistent diarrhoea, and all 16 isolates were positive for enterotoxin. The ninth patient had recurrent diarrhoea, but no enterotoxin was detected from the two isolates obtained. All of the 34 isolates, each of which was obtained from one of the 34 patients, were enterotoxin-positive. Enteropathogenic organisms other than C. perfringens were not isolated from any specimen. Consequently, the diarrhoea of 43 of the total 58 patients could be diagnosed as due to enterotoxigenic C. perfringens infection. Fifty six faecal specimens from 46 patients were directly examined for C. perfringens enterotoxin using latex agglutination assay, and 43 specimens from '35' patients were positive for enterotoxin. Enterotoxin-positive isolates were obtained from 42 of the 43 enterotoxin-positive faeces. But enterotoxin-positive isolates were also obtained from six of the 13 enterotoxin-negative faeces. The sensitivity and specificity of the direct detection of enterotoxin from faeces compared to the isolation of enterotoxin-positive C. perfringens were 87.5% (42 positive faeces out of 48 positive isolates) and 87.5 % (7 negative faeces out of 8 negative isolates), respectively. Fifty of the 60 isolates reacted with the WX2 serum, four isolates with TW18, one isolate with TW40 and five isolates did not react with any antisera tested. Fifty seven of the 60 isolates could be grouped by PFGE; three isolates could not be characterized due to degradation of the genomic DNAs despite multiple repeated experiments. Figure la shows a representative result of genomic comparison by PFGE among C. perfringens isolates from three wards E09 (lane 1 to 11), Ell (lane 12) and W09 (lane 13). Nine isolates from ward Fig. 1. PFGE analysis of C. perfringens isolates with restriction enzyme Smal. (a) Isolates on lanes 1 to 11 were from ward E09, lane 12 from ward Ell and lane 13 from ward W09. Serogroups were all WX2 except for those on lanes 6 (TW18) and 10 (TW40). All isolates except for these two isolates shared the same PFGE pattern, and were grouped to PFGE-group I. The sizes of the DNA marker are indicated in kilobases. (b) The PFGE patterns which are not shown in (a). An isolate on lane 8 showed a single band according to the conditions used for this PFGE.

3 NOTES 769 Table 1. Serogrouping and PFGE typing of Clostridium perfringens isolates from various wards.) Not determined by PFGE due to DNA degradation; b) enterotoxin was not detected (one each from wards E08 and Ell). E09 on lanes 1-5, 7-9 and 11, and two isolates from the other wards on lanes 12 and 13 shared the same genomic pattern, designated as PFGE-group I. Isolates on lanes 6 and 10 were serogrouped as TW18 and TW40, respectively. Thirty six of the 50 serogroup-wx2 isolates and two untypable isolates belonged to PFGEgroup I. Thirteen PFGE patterns, which are not shown in Fig. la, are shown in Fig. 1b. The results of serogrouping, enterotoxin production and PFGE analysis are summarized in Table 1 and Fig. 2. Small self-limiting epidemics of diarrhoea were repeatedly observed in multiple wards such as E09, E08, W09 and Ell. Thirty one of 42 isolates from these wards reacted with antiserum WX2 and also belonged to PFGE-group I. All of these isolates were enterotoxin-positive except one from ward Ell, which was isolated from the patient which had recovered from diarrhoea (vide supra). The C. perfringens isolated from 19 of 84 environmental points (including two staff members) reacted with WX2 (not included in Table 1). An examination for enterotoxin production and PFGE analysis were not performed on the environmental isolates. Serogrouping is a major method for the epidemiological study of C. perfringens (8, 10). In addition to this, Fig. 2. C. perfringens isolation from multiple wards. A closed triangle shows an isolate which reacted with WX2 and belonged to PFGE-group I. An open triangle shows an isolate which belonged to serogroups other than WX2, or PFGE groups other than PFGE-group I. Enterotoxin was not detected from an isolate shown with an asterisk. PFGE has come to be used to compare C. perfringens isolates to each other. Recently, physical genomic maps of ten C. perfringens strains were constructed and com-

4 770 A. WADA ET AL pared to each other (6). For analyzing epidemics of diarrhoea in our hospital, we used both serogrouping and PFGE because the cross-reactivity of WX2 was not yet characterized. Indeed 36 of the 50 serogroup-wx2 isolates belonged to PFGE-group I, but the other 14 isolates showed ten different PFGE patterns (lanes 1, 3-9, 12 and 13 in Fig. 1b) or could not be grouped by PFGE due to degradation of the genomic DNA. In addition, in spite of the documented good specificity of TW18 (8), this serum reacted with four isolates, one of which showed a different PFGE pattern (lane 6 in Fig. 1a) from the others (lane 11 in Fig. 1b). The observed epidemics of diarrhoea might have been due to a small amount of food contamination by C. perfringens strains. We think, however, this possibility is very low because: 1) the times of the emergence of symptoms, and also of isolation of C. perfringens were not coincident but consecutive over a period of several days in each ward; and 2) only a small number of patients were affected at one time, while all food in the hospital was served from the same source. Although isolates which did not belong to PFGE-group I and serogroup-wx2 were obtained, a major part of the isolation was sporadic and might not be a result of nosocomial infection. After all, the epidemic diarrhoea in the four wards was thought to be caused by the nosocomial infection of enterotoxigenic C. perfringens, which reacted with WX2 and belonged to PFGE-group I. The minimum dose to cause enteritis upon ingestion of C. perfringens is thought to be 108 living vegetative cells, which sporulate in the small intestine and release enterotoxin (2). Therefore, foods heavily contaminated with this organism are the most common source of an outbreak. In addition to this, other situations resulting in enteritis have also been proposed (3, 4, 9, 10, 13). Several reports described recurring diarrhoea among elderly patients due to enterotoxigenic C. perfringens strains, which were not related to a food-borne outbreak (4, 9, 13). In our cases, the majority of patients had diminished physical activity and required staff members to maintain their sanitary conditions. Direct patient-to-patientransfer of a sufficient amount of stool due to low personal hygiene was one possible mechanism for the spread of the C. perfringens strain. This explanation could not, however, account for all of the cases. Another possibility was that orally-ingested spores from the environment and/or staff members would distend the vegetative cells in the intestine, and then proliferate and release enough enterotoxin to cause diarrhoea during re-sporulation. C. perfringens has been reported to readily overgrow in the intestine of elderly patients (13, 14). The preliminary monitoring of environmental C. perfringens showed that all isolates were serogrouped as WX2, supporting the latter hypothesis. To confirm it, extended monitoring of C. perfringens spores in various hospital environments, and an epidemiological study of their relationship to the onset of diarrhoea in elderly patients are required. This study was partially supported by a grant from the Ministry of Health and Welfare, Japan. References 1) Allen, S.D Clostridium, p In Lennette, E.H., Balows, A., Hausler, W.J., Jr., and Shadomy, H.J. (eds), Manual of clinical microbiology, 4th ed, American Society for Microbiology, Washington, D.C. 2) Bartlett, J.G Gas gangrene (other clostridium-associated diseases), p In Mandell, G., Douglas,. R.G., and Bennett, J.E. (eds), Infectious diseases, 3rd ed, Churchill Livingstone, New York. 3) Borriello, S.P., Larson, H.E., Welch, A.R., and Barclay, F Enterotoxigenic Clostridium perfringens: a possible cause of antibiotic-associatediarrhoea. Lancet is ) Borriello, S.P., Barclay, F.E., Welch, A.R., Stringer, M.F., Watson, G.N., Williams, R.K.T., Seal, D.V., and -Sullen, K Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens. J. Med. Microbiol. 20: ) Canard, B., and Cole, S.T Genome organization of the anaerobic pathogen Clostridium perfringens. Proc. Natl. Acad. Sci. U.S.A. 86: ) Canard, B., Saint-Joanis, B., and Cole, T Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens. Mol. Microbiol. 6: ) Clabots, C.R., Bettin, K.M., Peterson, L.R., and Gerding, D.N Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium di, f jtcile from environmental sites. J. Clin. 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5 NOTES ) Williams, R., Piper, M., Borriello, P., Barclay, F., Welch, A., Seal, D., and Sullens, K Diarrhoea due to enterotoxigenic Clostridium perfringens: clinical features and management of a cluster of ten cases. Age Ageing 14: ) Yamagishi, T., Serikawa, T., Morita, R., Nakamura, S., and Nishida, S Persistent high numbers of Clostridium perfringens in the intestine of Japanese aged adults. Jpn. J. Microbiol. 20: