ASSESSMENT OF METRONIDAZOLE SUSCEPTIBILITY甲硝唑敏感性评价.doc

EL-MINIA MED., BULL., VOL. 16, NO. 1, JAN., 2005 Mahmoud et al _ASSESSMENT OF METRONIDAZOLE SUSCEPTIBILITY IN CLOSTRIDIUM DIFFICILE ISOLATES RECOVERED FROM CASES WITH ANTIBIOTIC DIARRHEAL DISEASESByMahmoud Shokry Mahmoud, Hazem A. Rezk, Wafaa K. M. Mahdi* and Enas Mamdouh HefzyDepartment of Microbiology and Immunology, El-Minia Faculty of MedicineABSTRACT:Background and Purpose: Clostridium difficile (C. difficile) is the most common cause of nosocomial acquired, antibiotic-associated diarrhea (AAD), thats called Clostridium difficile associated disease (CDAD). This organism causes about 20% of the cases of AAD, up to 75% of the cases of antibiotic-associated colitis, and virtually all the cases of pseudomembranous colitis (PMC). Patients and Methods: Sixty isolates were collected; all had been obtained from hospitalized patients under antibiotic therapy. Clostridium difficile associated diarrhea was defined as the presence of diarrhea 6 watery stools in a 36 hours period in a patient with a positive stool culture or a positive cytotoxin assay. Primary cultures were done on selective medium for C. difficile, cefoxitin-cycloserine fructose agar (CCFA). C. difficile isolates were presumptively identified by their colony morphology. Additional biochemical tests were also used. API ATB 32A Rapid ID Anaerobe Identification Kit was also used for confirmation of the biological identification. Gas liquid chromatography (GLC) was used to determine the volatile fatty acids produced by the growth of the organisms. Production of C. difficile toxin A (TcdA) is determined by the ToxA TEST immuno assay. The presence of C. difficile toxin B was determined by demonstrating a specific cytopathic effect on MRC-5 cells. Using HindIII restriction endonuclease did DNA extraction, PCR amplification and restriction endonuclease analysis. The pattern of each isolate was visually compared with the patterns of the previously identified REA groups in a well-characterized REA patterns library. Most patients were treated for respiratory tract infections (48% of cases) either lower (17 = 28%) or upper (12 = 20%). Results: Patients treated by a combination of antibiotics formed 35% (25/60) of cases, 2 or 3 or 4 antibiotics. Cephalexin was associated with the highest incidence of CDAD (12=20%). Clindamycin and amoxicillin formed 11 cases ( = 18%). The majority of CDAD cases developed after 6-9 days use of antibiotics (45 patients = 75%). Screening the isolates for sensitivity to metronidazole using E-test showed that, MIC50 (= 0.125 g/mL) and MIC90 (= 1 g/mL). Only 2 isolates (= 3.033%) were inhibited at MIC = 16 g/mL and these isolates are identified as resistant strains (MIC 16 g/mL).Conclusion: From this study we can ensure that CDAD is a serious problem caused by the use of broad-spectrum antibiotics for long periods especially in old age group of patients. Also we can conclude the high sensitivity of C. difficile to metronidazole; in spite of the recently emerging and rapidly expanding problem of resistance, which is still in need for further research work to be justified.KEY WORDS: Broad-spectrum antibioticsC. difficileMetronidazole261INTRODUCTION: Clostridium difficile (C. difficile) is the most common cause of nosocomial acquired, antibiotic-associated diarrhea (AAD), thats called Clostridium difficile associated disease (CDAD)1. This organism causes about 20% of the cases of AAD, up to 75% of the cases of antibiotic-associated colitis, and virtually all the cases of pseudo-membranous colitis (PMC)2. Pseudomembranous colitis (PMC) are of a common pathological lesion of the disease; sigmoidoscopy shows characteristic patchy, yellow pseudo-membranes surrounded by normal looking mucosa3. CDAD is generally thought to occur after disturbance of the balance of patients normal protective bowel flora following use of broad-spectrum antibiotics4. This state leaves the patient sensitive to overgrowth by C. difficile that is already in the patient in small numbers (endogenous) or, more commonly, from another patient or the environment (exogenous)5. The third generation cephalosporins, clinda-mycin and amoxicillin are associated with the greatest risk for developing AAD because of their widespread use in the hospital as well as the community, but almost all antibiotics can cause the disease6. Two toxins, enterotoxin A and cytotoxin B, have been recognized as the main virulence factors of C. difficile. These toxins belong to the group of large clostridial cytotoxins (LCT) and are characterized by a high molecular mass (250kDa)7. The enterotoxin and cytotoxin genes, tcdA and tcdB, and three additional genes (tcdC, tcdD and tcdE) are arranged within a well-defined chromosomal region termed the pathogenicity locus (PaLoc)8. Other virulence factors include; adhesion, chemotaxis, capsule, hydro-lytic enzymes. The typical manifes-tations of CDAD are cramping abdo-minal pain, profuse diarrhea, watery stools, low-grade fever, and leuko-cytosis. These can start a few days after start of antibiotic therapy or up to 8 weeks after its discontinuation9. The gold standard for diagnosis of CDAD is detection of toxin B by cytotoxin assay. Other methods include: clinical assessment, endoscopy, detec-tion of C. difficile products, detection of C. difficile genes and Isolation and typing of C. difficile10. The most successful single control measure to reduce symptoms is the immediate stoppage of the predisposing anti-biotic11. The drugs most commonly used to treat CDAD are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile isolates are susceptible to metroni-dazole and vancomycin12. Metroni-dazole is the drug of choice due to its in vitro activity, its efficacy by either the oral or the intravenous route of administration, its presumed lower potential for selection of Vancomycin Resistant Enterococcus (VRE) and the low cost of treatment with the drug13. The aim of the present study is to asses the rates of in- vitro susceptibility to metronidazole in C. difficile isolates recovered from patients with AAD over 12-months period of study.MATERIALS AND METHODS: Sixty isolates were collected from El-Minia University Hospital all over 12-months study, all had been obtained from hospitalized patients under antibiotic therapy. Full demographical & clinical details including: antibiotic usage and health status were included in the individual clinical reports. Clostridium difficile associated diarrhea was defined as the presence of diarrhea 6 watery stool in a 36 hours period in a patient with a positive stool culture or a positive cytotoxin assay. Stool samples were collected in our labs before the start of antibiotic treatment for AAD. Primary cultures were done on selective medium for C. difficile, cefoxitin-cycloserine fructose agar (CCFA). Suspected C. difficile isolates were detected, then confir-mation of diagnosis and the other steps of the study were done. Suspected isolates were grown on Fastidious Anaerobe Agar (FAA) supplemented with 6% horse blood in an anaerobic atmosphere at 37C. The C. difficile isolates were presumptively identified by their colony morphology, yellow color, ground-glass texture, characteristic horse-dung smell, Gram staining and fluorescent properties. Additional biochemical tests were also used. These tests included tests for the fermentation of glucose, maltose, mannitol, lactose, sucrose, xylose, salicin, arabinose, rhamnose, and trehalose. Isolates were tested for the production of catalase, indole, urease, lecithinase, and lipase. The isolates were also tested for gelatin liquefaction, nitrate reduction, esculin hydrolysis, and growth in bile. API ATB 32A Rapid ID Anaerobe Identification Kit was also used for confirmation of the biological identification. This is a 32 test strip used as a rapid identification system for anaerobes. Gas liquid chromatography (GLC) was used to determine the volatile fatty acids produced by the growth of the organisms(fatty acids as acetic acid butyric acid isobutyric valeric isovalyric and isocaproic acids). Production of C. difficile toxin A (TcdA) is determined by the ToxA TEST immunoassay The presence of C. difficile toxin B was determined by demonstrating a specific cytopathic effect on MRC-5 cells14.Restriction Endonuclease Analysis (REA): Crude template DNA was prepared by suspension of cells harvested from FAA in a 5% solution of Chelex-100 (Bio-Rad) and boiling for 12 min. Cell debris was separated by centrifugation (15000c/m for 10 min) and the supernatant fluid was used for polymerase chain reaction (PCR). PCR products were then digested by adding HindIII (restriction endonu-clease enzyme). Tubes were centrifuged briefly and incubated at 37C for 5 h. The fragments were separated on a 0.7% agarose gel, producing a charac-teristic banding pattern for each isolate after photographing gel under UV light. The pattern of each isolate was visually compared with the patterns of the previously identified REA groups in a well-characterized REA patterns libraryV.Antimicrobial susceptibility testing by E-test: A sterile cotton swab was used to apply a suspension of the organism to a fresh FAA plate. E-test strip impregnated with metronidazole was placed at the median plane of the plate. The plates were then incubated in an anaerobic chamber at 37C for 24 h.MIC determination: The MIC value read from the E test strip at the intercept of the inhibition ellipse with the MIC scale on the E-test strip. MIC50 and MIC90 were calculated by pasting all the MICs for each strain into an Excel spreadsheet and sorting it into ascending order. The MIC50 was taken as the MIC that was the median value.RESULTS: The mean age was 50.7 years (range, 1989 years). The highest incidence was between the age group 60 years old (36=60%). The biochemical reactions of the anaerobic culture of the oragnisms showed: Glucose: positive (+ve), Lactose: negative (ve), Mannitol: positive (+ve), Mannose: positive (+ve), Melibiose: negative (-ve), Maltose: negative (-ve), sucrose: negative (-ve), Rhamnose: negative (-ve), Xylose: negative (-ve). Growth on bile is equal growth to control without bile, lecithinase: negative, lipase: negative, indole: nega-tive, catalase: negative, nitrate: nega-tive, urease: negative, starch hydrolysis: negative, gelatin hydrolysis: positive, esculin hydrolysis: positive. GLc results showing production of volatile fatty acids as acetic acid, butyric, isobutyric, valeric, isovaleric and isocaproic acids. Restriction endonuclease analysis of Clostridium difficile DNA digested by Hind III restriction endonuclease (Figure 1). Lane 1 (left hand lane) shows the sizing markers in kilobases, Lane 2 to 9, show digestion pattern of REA group, CF, AL,Z, B, CF, RF and BN respectively. According to National Committee for Clinical Laboratory Standards (NCCLS) recommendations, (NCCLS, 2000), resistance of C. difficile to metronidazole was defined as MIC 16 g/mL. The MIC (Minimal Inhibitory Concentration) was defined as the lowest concentration of the agent that inhibited growth of 60 isolates, this was cell 30. Similarly, the MIC90 was the value found in row 54 (90 % of 60) and represented the concentration of antibiotic that would inhibit 90% of the isolates tested.(see Figure 2). The mean age was 50.7 years (range, 1989 years). The highest incidence was between the age group 60 years old (36 = 60%), secondly in the age group 41-60 years old (12 = 20%), third in the age group 60 years old) (Figure 3). Most patients were treated for respiratory tract infections either lower respiratory infections; bronchopneu-monia (17 = 28%) or upper respiratory tract infections, ear, nose and throat infections (12 = 20%). So we can find that respiratory tract infections formed 48% of cases and this high value means that antibiotics used for treatment of these infections precipitates acquisition or C. difficile infections. Gastroin-testinal tract infections formed (10 = 17%). Lower incidence was among patients with urinary tract infection (UTI) (9 = 15%). Other miscellaneous infections were 12 cases (12 = 20%) (Figure 4). Patients treated by a combination of antibiotics formed 42% (25/60) of cases, 2 or 3 or 4 antibiotics. But most of patients included in the study (35 = 58%) were treated with one broad spectrum antibiotic (Figure 5). Figure 5 shows the different antibiotics associated with the onset of diarrhea; treatment with cephalexin was associated with the highest incidence of CDAD (12 = 20%). Clindamycin and amoxicillin formed 11cases (=18%) beta-lactam agents other than amoxi-cillin showed high, penicillin (10 = 17%), and erythromycin (9 = 15%). Gentamycin (6 = 10%), tetracycline (6 = 10%), cotrimoxazole forms (7 = 12%) and other miscellaneous non-beta-lactam antibiotics (22 = 37%) Cephalexin was associated with the highest incidence of CDAD (12 =20%). Clindamycin and amoxicillin formed 11 cases (=18%), beta-lactam agents other than amoxicillin showed high incidence, penicillin (10 =17%), and erythromycin (9 =15%). Gentamycin was (6 =10%), tetracycline (6 =10%), cotrimoxazole forms (7 =12%) and other miscellaneous non-beta-lactam antibiotics (22 = 37%) as detected in Figure 6. Figure 7 show the actual duration of antibiotic treatment followed with diarrhea. From these charts we conclude that the use of antibiotic for more than 5 days increases the incidence of diarrhea as (45=75%), significant value for patients developed diarrhea after 6-9 days of antibiotic treatment while (7 =12%), of patients used antibiotics for 4-5 days and whom were treated with antibiotics for 10-14 days formed (8=13%). The majority of CDAD cases developed after 6-9 days use of antibiotics (45 patients = 75%) as shown in Figure 7. Table 1 shows the values of MIC50, MIC90, range of MICs and geometric mean for MIC of metroni-dazole against C. difficile. Screening the isolates for sensitivity to metronidazole using E-test showed that most of the isolates were inhibited at low MICs, and this was reflected by a low MIC50 (0.125 g/mL) and MIC90 (= 1 g/mL). Only 2 isolates (= 3.033%) were inhibited at MIC =16 g/ml and these isolates are identified as resistant strains (MIC 16 g/mL) which are mentioned in Figure 8.Figure 1. Restriction endonuclease analysis of Clostridium difficile DNA digested by HindIII restriction endonuclease Figure 2. Susceptibility testing of C. difficile to metronidazole using E-test showing MIC of 0.016 g/mL Figure 3. Relationship between infection increases and ageFigure 4. Type of infection associated with CDADFigure 5. Number of antibiotics given per patient under studyFigure 6. The different antibiotics associated with the onset of diarrheaFigure 7. Actual duration of antibiotic treatment followed with diarrheaTable 1: MIC50, MIC90 mean and rangeMIC50MIC90RangeGeometric Mean0.125g/mL1g/mL0.016- 16g/mL0.9g/mL. Figure 8. Screening the isolates for sensitivity to metronidazole using E-testDISCUSSION: The increasing problem of antibiotic associated diarrhea had directed the worldwide study of C. difficile as the first causative organism of diarrhea. C. difficile is considered susceptible to metronidazole in most institutions15. Despite high clinical efficacy, a small number of patients with Clostridium difficile-associated disease (CDAD) do not respond to treatment with metronidazole, so that studies on the emergence of resistant strains of C. difficile against metronidazole is increasing although reports of metronidazole resistance in C. difficile are rare. Also it is well known, however, that CDAD may develop in patients during metronidazole therapy 16. In the current study the mean age was 50.7, range was (19-89) (Figure 3), male to female ratio was 1:1.9 (66% females). Pneumonia and upper repiratory tract infection (URTI) were the most commonly underlying diseases and amoxicillin, clindamycin, penicillins and other B-lactam antibiotics were the commonest antibiotics used. Using a combination of antibiotics formed 35% (25/60) of cases. All these observations coped with those obtained by Frenz and McIntyre, 200318. We found in this work that amoxicillin, clindamycin, penicillins and other beta-lactam agents are the antibiotics most commonly associated with CDAD. And these results agreed with results obtained by Climo 199819 and Pear 199420. The study done by Sanchez and colleagues, 199921, detected 13 different REA groups and revealed that no particular C. difficile strain was associated with metronidazole resistance, and this goes with our results in which 11 REA groups were isolated and no correlation