The Korean Journal of Internal Medicine : 23:9-15, 2008 Antibiotic-Associated Diarrhea: Candidate Organisms other than Clostridium Difficile Hyun Joo Song, M.D.1, Ki-Nam Shim, M.D.1, Sung-Ae Jung, M.D.1, Hee Jung Choi, M.D.1, Mi Ae Lee, M.D.2, Kum Hei Ryu, M.D.1, Seong-Eun Kim, M.D.1 and Kwon Yoo, M.D.1 Department of Internal Medicine1 and Laboratory Medicine2, Ewha Medical Research Institute, College of Medicine, Ewha Womans University, Seoul, Korea Background/Aims : The direct toxic effects of antibiotics on the intestine can alter digestive functions and cause pathogenic bacterial overgrowth leading to antibiotic-associated diarrhea (AAD). Clostridium difficile (C. difficile) is widely known to be responsible for 10～20% of AAD cases. However, Klebsiella oxytoca, Clostridium perfringens, Staphylococcus aureus, and Candida species might also contribute to AAD. Methods : We prospectively analyzed the organisms in stool and colon tissue cultures with a C. difficile toxin A assay in patients with AAD between May and December 2005. In addition, we performed the C. difficile toxin A assays using an enzyme-linked fluorescent assay technique. Patients were enrolled who had diarrhea with more than three stools per day for at least 2 days after the initiation of antibiotic treatment for up to 6～8 weeks after antibiotic discontinuation. Results : Among 38 patients (mean age 59±18 years, M:F=18:20), the organism isolation rates were 28.9% (11/38) for stool culture, 18.4% (7/38) for colon tissue cultures and 13.2% (5/38) for the C. difficile toxin A assay. The overall rate of identification of organisms was 50.0% (19/38). Of the five patients that had a positive result by the C. difficile toxin A assay, two had no organism isolated by the stool or colon tissue culture. The organisms isolated from the stool cultures were C. difficile (4), Klebsiella pneumoniae (K. pneumoniae) (3), Candida species (3), and Staphylococcus aureus (1). C. difficile (4) and K. pneumoniae (3) were isolated from the colon tissue culture. Conclusions : For C. difficile negative AAD patients, K. pneumoniae, Candida species, and Staphylococcus aureus were found to be potential causative organisms. Key Words : Antibiotic-associated; Diarrhea; Organism INTRODUCTION medical cost. Clostridium difficile (C. difficile) is widely known to be responsible for approximately 10～20% of cases of AAD and Antibiotic-associated diarrhea (AAD) is defined as diarrhea that almost all cases of pseudomembranous colitis4-7). However, occurs in association with the administration of antibiotics1). The Klebsiella oxytoca8), enterotoxin-producing Clostridium perfrin- direct toxic effects of antibiotics on the intestine can alter gens9-11), Staphylococcus aureus12), Candida species13, 14), digestive functions secondary to reduced concentrations of the Salmonella species, and Pseudomonas aeruginosa15) might also normal gut bacteria, or cause pathogenic bacterial overgrowth2, 3). contribute to the development of AAD2, 5). Understanding the different mechanisms that cause AAD may However, little is known about other candidate organisms in help to prevent this condition, improve medical care and reduce this context and the clinical correlation between endoscopic ∙Received : May 4, 2007 ∙Accepted : December 21, 2007 ∙Correspondence to : Ki-Nam Shim, M.D., Department of Internal Medicine, Ewha Womans University Mokdong Hospital, 911-1 Mok-dong, Yangcheon-gu, Seoul 158-710, Korea Tel : 82-2-2650-2632, Fax : 82-2-2655-2076, E-mail : [email protected] 10 The Korean Journal of Internal Medicine : Vol. 23, No. 1, March 2008 AB CD EF Figure 1. Three groups of antibiotic-associated diarrhea detected by endoscopy. Normal (A), non-specific colitis (B, C, D), and pseudomembranous colitis (E, F). Hyun Joo Song, et al : Candidate Organisms in Antibiotic-Associated Diarrhea 11 findings and the organisms isolated in patients with AAD. Table 1. Demographic characteristics of the AAD patients Thus, a better understanding of the causes of AAD is needed. Variables No. (%) In this study, we prospectively analyzed the organisms Sex (male : female) 18 : 20 associated with AAD using stool and colon tissue cultures in Mean age±SD (range) (years) 59±18 (22-86) combination with the C. difficile toxin A assay in patients with Underlying disease Total =38 AAD. In addition, we evaluated the clinical correlation between Diabetes 14 (36.8) endoscopic findings and the organisms isolated from stool and Cancer 9 (23.7) Cerebrovascular disease 9 (23.7) colon tissue cultures. Chronic renal failure 8 (21.1) Trauma 6 (15.8) Chronic liver disease 4 (10.5) MATERIALS AND METHODS Chronic obstructive pulmonary disease 3 (7.9) Congestive heart failure 3 (7.9) Patient enrollment No underlying disease 1 (2.6) Data on the stool and colon tissue cultures from 38 patients Cause of antibiotic uses with AAD who were treated at the Ewha Womans Mokdong Surgical prophylaxis 9 (23.7) Pneumonia 8 (21.2) Hospital from May to December in 2005 were collected Infection prophylaxis 6 (15.8) prospectively. In addition, we tested for the presence of C. CAPD peritonitis 4 (10.5) difficile toxin A using the enzyme-linked fluorescent assay (ELFA) Urinary tract infection 3 (7.9) technique (VIDASⓇ C. difficile Toxin A II, bioMérieuxⓇSa, Marcy Neutropenic fever 4 (10.5) Sepsis 1 (2.6) I'Etoile, France), an automated test for the qualitative detection Cellulitis 1 (2.6) of C. difficile toxin A in stool specimens. The relative sensitivity Cathter associated infection 1 (2.6) Ⓡ and specificity of VIDAS for C. difficile toxin A assay compared Unknown 1 (2.6) with the commercial enzyme immunoassay are 87.6%, and AAD, antibiotic‐associated diarrhea; CAPD, continous ambula- 98.7%, respectively. We enrolled patients who had three or more tory peritoneal dialysis; SD, standard deviation stools a day for at least two days from the start of antibiotic administration to 6～8 weeks after antibiotic discontinuation. Candida, MacConkey media for Klebsiella oxytoca and Klebsiella We excluded patients without a history of antibiotic use, those pneumoniae, and Salmonella-Shigella agar for Salmonella. Insult who discontinued using antibiotics 2 months before the diarrhea kits (VITEK IIⓇ GN, bioMérieuxⓇ Sa, Marcy I'Etoile, France) were started, and those with a diagnosis of acute gastroenteritis, used to identify gram-negative species. The endoscopic findings radiation colitis, inflammatory bowel disease, ischemic colitis, or associated with AAD were classified into three groups as follows: diarrhea due to a carcinoid tumor. The clinical characteristics of 1) normal, no inflammation, 2) nonspecific colitis, which showed the patients with AAD were evaluated. Written informed consent erythema, edema and shallow aphthous ulcers, and 3) was obtained from each patient for the protocol, which was pseudomembranous colitis (PMC) with whitish exudates, approved by the ethics committee of the Ewha Womans hemorrhage or deep ulcers (Figure 1). University Mokdong Hospital. Statistical analysis Endoscopic and microbiologic investigations Statistical analysis was performed using the Fisher's exact test For all patients who satisfied the above criteria, stool and the linear-by-linear association in SPSS for Windows (Ver. specimens and colon tissue specimens were obtained and 11.0.1. 2001. Chicago, USA, SPSS Inc.). Throughout the study, cultured. The stool specimens were placed in transport medium Ⓡ p values less than 0.05 were considered statistically significant. (Amines , w/o Charcol, Asan Pharmaceuticals Co. Ltd., Hwasung, Korea). Four tissue specimens from severely inflamed colon mucosa or normal mucosa were obtained during RESULTS endoscopy using sterile biopsy forceps. The tissue specimens were collected into sterile eppendorf tubes (Safe lock test tube, Demographic characteristics of the AAD patients Handok Pharmaceuticals Co. Hwasung, Korea). Thirty-eight patients who were treated between May and All stool and tissue specimens were cultured in selective December 2005 were enrolled; there were 18 men and 20 medium containing cycloserine, cefoxitin and fructose in egg-yolk women. The mean age was 59 (range, 22-86) years. Eight agar (CCFA) for C. difficile, Brucella medium for Clostridium (21.1%) patients were evaluated by colonoscopy and 30 (78.9%) perfringens, blood agar plate for Staphlyococcus aureus and by sigmoidoscopy. All but one patient had co-morbid disease such as diabetes (14 cases), cerebrovascular disease (9), cancer 12 The Korean Journal of Internal Medicine : Vol. 23, No. 1, March 2008 Table 2. Comparison of isolated organism between stool and colon tissue cultures Stool culture Total Isolated organism No organism Isolated organism 1 (2.6%) 6 (15.8%) 7 (18.4%) Tissue culture No organism 10 (26.3%) 21 (55.3%) 31 (81.6%) Total 11 (28.9%) 27 (71.1%) 38 (100.0%) p=0.648, tested by Fisher’s exact test Table 3. Isolated organisms according to tissue and stool cultures with Clostridium difficile toxin A assays C. difficile Organisms Tissue culture Stool culture Total toxin A assay Clostridium difficile 4 (10.5%) 4 (10.5%) 5 (13.2%) 9 (23.7%) Klebsiella pneumoniae 3 (7.9%) 3 (7.9%) - 6 (15.8%) Candida albicans 0 (0.0%) 2 (5.3%) - 2 (5.3%) Candida tropicalis 0 (0.0%) 1 (2.6%) - 1 (2.6%) Staphylococcus aureus 0 (0.0%) 1 (2.6%) - 1 (2.6%) No organism 31 (81.6%) 27 (71.0%) 33 (86.8%) 19 (50.0%) C. difficile, Clostridium difficile Table 4. Summary of Clostridium difficile-positive cases by tissue cultures and stool cultures with Clostridium difficile toxin A assays C. difficile Patients Sex/Age Tissue culture Stool culture toxin A assay 1 M/80 + + + 2 M/66 - - + 3 M/80 - - + 4 F/46 + - - 5 F/71 + - + 6 M/73 + - - 7 M/71 - + + 8 M/57 - + - 9 F/76 - + - C. difficile, Clostridium difficile (9), chronic renal failure (8), trauma (6), chronic liver disease (4), (%) Culture positive p =0.008 chronic obstructive pulmonary disease (3), and congestive heart 60 p<0.001 19 (50.0%) failure (3).