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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 2  |  Page : 198-199
Clostridium perfringens enterotoxin in antibiotic-associated diarrhea


Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012, India

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   Abstract 

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research

Keywords: Antibiotic-associated diarrhea, Clostridium perfringens

How to cite this article:
Vaishnavi C, Kaur S. Clostridium perfringens enterotoxin in antibiotic-associated diarrhea. Indian J Pathol Microbiol 2008;51:198-9

How to cite this URL:
Vaishnavi C, Kaur S. Clostridium perfringens enterotoxin in antibiotic-associated diarrhea. Indian J Pathol Microbiol [serial online] 2008 [cited 2019 Oct 24];51:198-9. Available from: http://www.ijpmonline.org/text.asp?2008/51/2/198/41681



   Introduction Top


Antibiotic-associated diarrhea (AAD) is precipitated by various microorganisms even though Clostridium difficile is implicated in the most severe cases. However, C. difficile is much less often found in diarrhea or colitis where pseudomembranes are absent and interestingly, some C. difficile -negative cases of AAD present with bloody diarrhea.

Heat-resistant forms of Clostridium perfringens type A (CPA) are also associated with pathogenic outcome in humans and 2-5% of all CPA produce enterotoxin. It has been reported that about 5-20% of the AAD cases and sporadic non-food borne diarrhea may be due to CPA. [1] It is not known whether antibiotics permit infection by enterotoxigenic C. perfringens or allow overgrowth of small numbers of the organisms that may normally be resident in the gut of these patients. In an earlier study, [2] we found that CPA was present in 8.77% of patients with AAD, but no C. perfringens enterotoxin (CPE) detection was done. So in the present study, the stool samples of patients with AAD negative for C. difficile toxin were examined for CPE. Correlation of the incidence with the clinical profile of the patients was done so that optimum control and treatment measures could be defined in the future.


   Materials And Methods Top


Patients and samples

One hundred fecal samples negative for C. difficile toxin were investigated in the microbiology section of the Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh between March and July, 2005. These samples came from hospitalized patients who developed diarrhea regardless of antibiotic intake. These patients were undergoing treatment for various ailments such as sepsis, renal diseases, liver diseases, pancreatitis, ulcerative colitis and necrotizing enterocolitis. Ten normal healthy volunteers without antibiotic exposure for at least 6 weeks prior to testing served as controls. Patients with infective diarrhea caused by other enteric pathogens were not part of the study.

Clinical profile

Of the 100 samples tested, 65 were male patients and 35 were female patients. The age of the patients ranged from 6 days to 75 years with an average of 26.4 years. Seventy-eight of the patients were on various antibiotics, the predominant ones being metronidazole, cephalosporins, ciprofloxacin and vancomycin. During the time of sampling, diarrhea was present in 93, fever in 45 and abdominal pain in 31 of the patients.

Clostridium perfringens ELISA

An enzyme-linked immunoassay (ELISA) was carried out for the detection of CPE as per the manufacturer's instructions (RIDASCREEN, OSB Agencies, India). Briefly, break away microtiter wells were coated with polyclonal antibodies directed against epitopes of CPE. An aliquot of fecal suspension was added to the wells and incubated simultaneously with an anti-CPE monoclonal antibody conjugated to horse radish peroxidase. After the wells were washed thrice with washing buffer, the substrate was added and the plate was incubated in the dark for 15 min at 25C. After the incubation was completed, stop solution was added and the intensity of the developed colour was measured at 450 nm in an ELISA reader (Bio Rad Model 550, Japan).

Culture for other isolates

Routine cultures were set up to look for other possible etiological factors.


   Results Top


The cut-off value was calculated by extinction for the negative control + 0.150 and was 0.199. Samples were considered positive if the absorbance value was higher than 10% over the determined cut-off value as mentioned by the manufacturers. Thus, samples above 10% of 0.199 (i.e. >0.218) were considered positive. CPE was positive in 2/100 (2%) of the patients receiving antibiotics but was negative in the control samples. Both the samples were also positive for C. perfringens in culture. One of the patients (60 years, female) positive for CPE was diagnosed as having AAD and was on multiple antibiotics viz. ciprofloxacin, vancomycin and metronidazole. The second patient was a 30-year-old female, diagnosed as having acute myeloid leukemia and was also on multiple antibiotics. Her stool sample also grew Candida sp. in pure culture.

Other organisms isolated from the patients were non-toxigenic C. difficile (4%), Staphylococcus aureus Scientific Name Search  (6%), Candida sp. (18%) and Klebsiella pneumoniae (1%). The samples from the 10 healthy controls did not yield any growth of significance.


   Discussion Top


Borriello et al, [3] were the first to suggest that the association of C. perfringens with antibiotic treatment is causal rather than fortuitous. They reported the prevalence of C. perfringens and its enterotoxin in 1.6% patients with C. difficile toxin positivity in 14.4% of their patients and 2/11 of them excreting both the organisms. Samuel et al, [4] tested 721 diarrheal specimens, of which 25 were shown to be positive for enterotoxigenic C. perfringens with most of them being hospital inpatients. Pituch et al, [5] isolated both C. difficile and C. perfringens from the same stool samples of 4/158 patients suspected of AAD. Joshy et al, [6] reported 10% of C. perfringens in C. difficile -negative samples in patients with AAD and suggested that it may result in extended hospitalization, particularly, in frail elderly patients. Clostridium perfringens was also detected in 8.77% of the patients with AAD in our previous study [2] without looking for CPE. So the finding of CPE in 2% of our patients in the present study is quite interesting.

Apart from this, 4% samples grew C. difficile and they probably represented non-toxigenic isolates as only samples negative for C. difficile toxin were included in the assay. Klebsiella pneumoniae was isolated in pure culture from one of the samples; and during analysis, the sample was found to belong to a 4-year-old male having nosocomial diarrhea without any history of antibiotic intake. Here it may be pointed out that Klebsiella oxytoca has earlier been reported to be associated with AAD. [7]

Evidence supporting a causal role for C. perfringens in non-food borne diarrheal disease includes the presence of enterotoxin in the feces of patients with AAD. Clostridium perfringens AAD is a distinct entity [8] though it is unclear if antibiotic exposure primarily permits the proliferation of small numbers of resident C. perfringens strains or allows their acquisition. Clostridium difficile may account for only approximately 20% of all cases of AAD [8] and C. perfringens up to 15%. [9] Sparks et al, [10] in their study with North American isolates provide important evidence, regardless of geographic origin or date of isolation, that plasmid cpe isolates cause most C. perfringens -associated AAD and chromosomal cpe isolates cause most CPA food poisoning.

The incidence of C. perfringens diarrhea is expected to increase with the growing population of immunocompromised individuals and the increased use of antibiotic intake. As detection of C. perfringens is not part of the routine laboratory investigation due to resource implications, one tends to miss out on the diagnosis of AAD due to this organism. The criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens -associated AAD by further research.

 
   References Top

1.Carman RJ. Clostridium perfringens in spontaneous and antibiotic associated diarrhoea of man and other animals. Rev Med Microbiol 1997;8:S43-5.  Back to cited text no. 1    
2.Vaishnavi C, Kaur S, Singh K. Clostridium perfringens type A and antibiotic associated diarrhoea. Indian J Med Res 2005;122:52-6.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Borriello SP, Larson HE, Welch AR, Barday F, Stringer MF, Bartholomew BA. Enterotoxigenic Clostridium perfringens : A possible cause of antibiotic associated diarrhoea. Lancet 1984;1:305-7.  Back to cited text no. 3    
4.Samuel SC, Hancock P, Leigh DA. An investigation into Clostridium perfringens enterotoxin associated diarrhoea. J Hosp Infect 1991;18:219-30.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Pituch H, Martirosian G, Obuch-Woszezatynski P, Meisel Mikolajezyk F, Luezak M. Intestinal flora of patients with suspected antibiotic associated diarrhoea (AAD). I. Clostridium perfringens . Med Dosw Mikrobiol 2000;52:375-82.  Back to cited text no. 5    
6.Joshy L, Dhawan B, Chaudhary R. Antibiotic associated diarrhea: A possible role of clostridium species. Abstract book of XXVII National Conference of Indian Association of Medical Microbiologists, Mumbai: 2003. p. 148.  Back to cited text no. 6    
7.Minami J, Katayama S, Matsushita O, Sakamoto H, Okabe A. Enterotoxic activity of Klebsiella oxytoca cytotoxin in rabbit intestinal loops. Infect Immun 1994;62:172-7.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Modi N, Wilcox MH. Evidence for antibiotic induced Clostridium perfringens diarrhoea. J Clin Pathol 2001;54:748-51.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Asha NJ, Wilcox MH. Laboratory diagnosis of Clostridium perfringens antibiotic associated diarrhoea. J Med Microbiol 2002;51:891-4.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Sparks SG, Carman RJ, Sarker MR, McClane BA. Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhoea and food poisoning in North America. J Clin Microbiol 2001;39:883-8  Back to cited text no. 10  [PUBMED]  [FULLTEXT]

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Correspondence Address:
Chetana Vaishnavi
Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.41681

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