BACKGROUND: Antimicrobial susceptibility testing (AST) of Clostridium difficile is increasingly important because of the rise in resistant strains. The standard medium for the AST of C. difficile is supplemented Brucella agar (sBA), but we found that the growth of C. difficile on sBA was not optimal. Because active growth is critical for reliable AST, we developed a new, modified C. difficile (mCD) agar. C. difficile grew better on mCD agar than on sBA. METHODS: C. difficile isolates were collected from patients with healthcare-associated diarrhea. sBA medium was prepared according to the CLSI guidelines. Homemade mCD agar containing taurocholate, L-cysteine hydrochloride, and 7% horse blood was used. For 171 C. difficile isolates, we compared the agar dilution AST results from mCD agar with those from sBA. RESULTS: No significant differences were observed in the 50% minimal inhibitory concentration (MIC50) and 90% minimal inhibitory concentration (MIC90) of clindamycin (CLI), metronidazole (MTZ), moxifloxacin (MXF), piperacillin-tazobactam (PTZ), and rifaximin (RIX), but the values for vancomycin (VAN) were two-fold higher on mCD agar than on sBA. The MICs of CLI, MXF, and RIX were in 100% agreement within two-fold dilutions, but for MTZ, VAN, and PTZ, 13.7%, 0.6%, and 3.1% of the isolates, respectively, were outside the acceptable range. CONCLUSIONS: The MIC ranges, MIC50 and MIC90, were acceptable when AST was performed on mCD agar. Thus, mCD agar could be used as a substitute medium for the AST of C. difficile.
Anti-Infective Agents/*pharmacology ; Clostridium Infections/microbiology ; Clostridium difficile/*drug effects/isolation & purification ; Diarrhea/*microbiology ; Humans ; Microbial Sensitivity Tests/*methods

PURPOSE: Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI. MATERIALS AND METHODS: In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection. RESULTS: ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h. CONCLUSION: Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.
Chromogenic Compounds/chemistry ; Clostridium difficile/chemistry/*isolation & purification ; Culture Media/*chemistry

To evaluate the ability of microbial mix-culture fermenting syngas into ethanol, we studied the microbial mix-cultures A-fm 4, G-fm 4, Lp-fm 4 and B-fm 4 obtained by enrichment and compared with Clostridium autoethanogenum DSM10061 with 10% and 25% inoculation size. The results show that, with 10% inoculation size, the ethanol production of A-fm 4, G-fm 4, Lp-fm 4, B-fm 4 and C. autoethanogenum were 349.15, 232.16, 104.25, 79.90 and 26.99 mg/L respectively. With 25% inoculation size, the ethanol production were 485.81, 472.73, 348.58, 272.52 and 242.15 mg/L respectively. Higher inoculation size will increase the production of ethanol. The tested mix-culture exhibited a significant yield advantage compared with the maximum production of C. autoethanogenum reported in the literature (259.64 mg/L). This research provided a practical method to improve ethanol production from syngas.
Bacteria ; classification ; metabolism ; Clostridium acetobutylicum ; metabolism ; Ethanol ; isolation & purification ; metabolism ; Fermentation ; Gases ; metabolism ; Hydrogen ; metabolism

Clostridioides ; Clostridioides difficile/isolation & purification* ; Clostridium Infections/epidemiology* ; Guidelines as Topic ; Humans

Infection with Clostridium difficile is a growing concern because of the increasing prevalence and spread of nosocomial infections. Emergence of the hypervirulent 027/NAP1/BI strain is also notable. Existing diagnostic methods have low sensitivity or are time-consuming. Therefore, establishing a rapid and accurate microbiological diagnostic assay is needed. We evaluated the Xpert C. difficile assay (Xpert CD assay; Cepheid, USA) to detect toxigenic C. difficile. This assay is a real-time multiplex PCR assay that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive 027/NAP1/BI strain. A total of 253 loose stool specimens were collected and toxigenic cultures, VIDAS C. difficile A & B assays (VIDAS CDAB assay; bioMerieux, France), and the Xpert CD assay were performed. In comparison to toxigenic cultures, the sensitivity, specificity, and positive and negative predictive values were 100%, 94.6%, 83.1%, and 100%, respectively, for the Xpert CD assay and 40.8%, 98.0%, 100%, and 88.9%, respectively, for VIDAS CDAB assay. Because of the low prevalence of the PCR ribotype 027 in Korea, the evaluation of the usefulness of the Xpert CD assay for screening for the 027 strain was limited. The Xpert CD assay provides great sensitivity in diagnosing toxigenic C. difficile infection. In addition, this method has excellent usability because it is simple and fast.
Clostridium Infections/*diagnosis/epidemiology/microbiology ; Clostridium difficile/genetics/*isolation & purification/metabolism ; Face/microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Prevalence ; Reagent Kits, Diagnostic/*standards ; Sensitivity and Specificity

BACKGROUND: Clostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility. METHODS: We analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods. RESULTS: Among 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A-B+) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin. CONCLUSIONS: This is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.
Clostridium Infections/microbiology ; Clostridium difficile/classification/*genetics/isolation & purification ; Diarrhea/microbiology ; *Drug Resistance, Bacterial ; Enterotoxins/*genetics ; Genetic Variation ; Genotype ; Hospitals, University ; Humans ; Microbial Sensitivity Tests ; Republic of Korea ; Ribotyping

Clostridial septicemia usually occurrs in patients with immunocompromised diseases such as diabetes and malignancy. Clostridial liver abscess is very rare but highly fatal. We experienced a case of Clostridial septicemia due to liver abscess in a 73-year-old man. He was presented with fever and chills. On admission, abdominal CT scan showed about 35 mm sized hypoattenuated lesion with multiple central air-bubbles. After the diagnosis of liver abscess, the patient underwent prompt empirical antimicrobial therapy and percutaneous drainage. In spite of early therapy, the patient had gone into shock and death.
Aged ; Clostridium/*isolation & purification ; Clostridium Infections/diagnosis/*microbiology ; Drainage ; Humans ; Liver/radiography ; Liver Abscess/complications/*diagnosis/microbiology ; Male ; Sepsis/complications/*diagnosis ; Tomography, X-Ray Computed

OBJECTIVETo develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin.

METHODSTaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi) of C. difficile strains and the toxins A(TcdA), B(TcdB) and truncated toxin A(TcdAT). Sensitivity, specificity and anti-interference ability of these methods were estimated, as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay.

RESULTSThe detection limits of tpi were 6×10⁻² CFU/µl and 6 × 10⁻¹ CFU/µl in the non-oxin producing and toxin producing strains, respectively. The coefficients of variability(CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3% . The CVs of intra-assay and inter-assay for the detection limit of tpi, tcdA, tcdB and tcdAT in the toxin producing strain were 3.0% and 3.4%, 2.9% and 3.2%, 5.3% and 5.7%, 2.7% and 2.8%, respectively. No interference was detected from other genus or species in clostridium. From 50 clinical samples, thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique, in which 3 were dubious and 2 were negative under VIDAS.

CONCLUSIONThe newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis, clinically.

Bacterial Proteins ; isolation & purification ; Bacterial Toxins ; isolation & purification ; Clostridium difficile ; isolation & purification ; Enterotoxins ; isolation & purification ; Real-Time Polymerase Chain Reaction ; methods

Laboratory-based pathogen isolation, identification, and toxicity determination were performed on samples from a suspected case of infant botulism. Mice injected with cultures generated from the enema sample and ingested Powered infant formula (PIF) presented typical signs of botulism. Antitoxins to polyvalent botulinum neurotoxins (BoNTs) and monovalent BoNT type B antitoxin had protective effects. Clostridium botulinum isolated from the enema and residual PIF samples were positive for type B toxin. Pulsed-field gel electrophoresis (PFGE) revealed that the two strains of C. botulinum isolated from the two samples produced indistinguishable pulsotypes. These findings confirmed this case of type B infant botulism associated with the ingestion of PIF contaminated by type B C. botulinum spores.
Animals ; Beijing ; epidemiology ; Botulinum Toxins ; isolation & purification ; toxicity ; Botulism ; diagnosis ; epidemiology ; Clostridium botulinum ; isolation & purification ; Gastrointestinal Tract ; microbiology ; Humans ; Infant ; Mice ; Toxicity Tests

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.
Bacterial Proteins/*analysis ; Bacterial Toxins/*analysis ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/enzymology/*isolation & purification/metabolism ; Enterotoxins/*analysis ; Feces/microbiology ; Glutamate Dehydrogenase/*analysis ; Humans ; *Immunoenzyme Techniques ; Reagent Kits, Diagnostic ; Sensitivity and Specificity