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Contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of healthcare-associated C. difficile infection. The aim of this study was to test the effect of an automated room disinfection system that provides an aerosol of 7.5 % hydrogen peroxide (H2O2) disinfectant, on spores of two different strains of C. difficile, and to evaluate the impact of biological soiling on the efficacy of H2O2 disinfection. The strains used were a C. difficile PCR ribotype 027 and a C. difficile ATCC 9689. Spore suspensions of each strain were applied to ceramic tiles and exposed to aerosolized H2O2 at different locations in a test room. Biological soiling was simulated by bovine serum albumin and sheep erythrocytes. At set time points spores were recovered, plated onto Columbia 5 % sheep blood agar, and surviving bacteria were counted as colony-forming units (cfu). No viable spores of either strain were recovered after a 3 h exposure to gaseous H2O2. Spores located inside a drawer showed recovery of approximately 1E5 cfu/ml for C. difficile ribotype 027 after 1 h. In the presence of organic matter, a more than fivefold log reduction compared with not exposed controls could be observed for spores of either strain tested. Appropriate decontamination of surfaces exposed to spores of C. difficile is challenging for conventional cleaning methods. Aerosolized H2O2 delivered by automated room disinfection systems could possibly improve surface decontamination and thereby reduce transmission of healthcare-associated C. difficile infection. Also in the presence of organic matter H2O2 disinfection appears to be an effective adjunct for decontamination of environmental surfaces.

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Clostridium difficile is mainly considered a nosocomial pathogen associated with diarrhea and pseudomembranous colitis in hospitalized patients. Austrian hospitals reported 2761 cases of C. difficile infection (including 277 lethal outcomes) in 2007, compared with 777 cases (including 54 lethal outcomes) in 2003. The occurrence of community-acquired C. difficile infection is also increasingly reported. Recent studies have shown the occurrence of C. difficile in food and animals. The aim of the present study was to determine the occurrence of C. difficile in food and animals in Austria. Between March and July 2008, gut or fecal samples from 67 cows, 61 pigs and 59 broiler chickens were collected at Austrian abattoirs. Between February and April 2008 meat samples (51 beef [25 ground], 27 pork [17 ground] and 6 samples of chicken meat) were purchased at retail outlets. Of the 187 samples tested, eight yielded C. difficile: in cows 3/67 samples (4.5%) were positive, in pigs 2/61 (3.3%), in broiler chickens 3/59 (5%). Six of the eight isolates yielded toxigenic C. difficile (toxins A and B): 2/67 (3%) cow samples, 2/61 (3.3%) pig samples, 2/59 (3.4%) chicken samples. Genes for the binary toxin were detected in one of the two pig isolates, a PCR ribotype 126 strain. None of the 84 meat samples yielded C. difficile. The results of this Austrian study suggest that animal reservoirs are possible sources, via food, of human C. difficile infection.

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006–2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.

From November 2008 to 15 April 2009, 36 isolates of CD027 identified in Austria, all originating from four hospitals in Vienna. All isolates were positive for toxin A, toxin B and the binary toxin, and showed a characteristic 18 bp deletion in the tcdC gene. Clostridium difficile is an anaerobic spore-forming bacterium. Some strains may cause diarrhoea due to formation of toxins. Symptomatic C. difficile infection (CDI) is primarily linked with hospital admission and antibiotic treatment, although antibiotic exposure is neither necessary nor sufficient for CDI [1,2]. In Belgium, for instance, one third of CDI cases reported in the hospital surveillance system are not hospital-associated [3]. Symptoms range from mild diarrhoea to serious manifestations such as pseudomembranous colitis, toxic megacolon or perforation of the colon. C. difficile challenges hygiene standards as it is forms spores. The risk of infection rises with increasing age, underlying disease and immunodeficiency [4]. In recent years, a particularly virulent strain, ribotype 027 (CD027), has emerged in a number of countries, particularly in connection with hospital outbreaks, but also in community-acquired diarrhoea cases [5]. The risk of serious disease and death associated with CD027 exceeds that of other C. difficile strains. The classical CD027 is characterised - among other things - by an increased production of toxins A and B, production of a binary toxin and resistance to newer fluoroquinolones such as moxifloxacin. The first three Austrian cases of CD027 occurred in 2006 and in March 2008 [6,7]. Since August 2006, the Austrian National Reference Centre for C. difficile has ribotyped approximately 2,700 human C. difficile isolates received from all nine Austrian provinces. In recent months, a drastic increase in CD027 cases has been noted, all originating from four hospitals in Vienna. From November 2008 to 15 April 2009, 36 isolates of CD027 were received at the National Reference Centre. The Figure summarises these C. difficile 027 cases by month of reception of the sample at the reference centre.

In order to assess the lethality of Clostridium difficile-associated disease (CDAD) and the PCR ribotypes prevalent in Austria, the Austrian Agency for Health and Food Safety requested isolates of C. difficile from patients in a structured but arbitrary sampling scheme. In the allocated period from February 2006 to January 2007, local hospital laboratories within each of the nine provinces were asked to submit C. difficile isolates from at least ten cases of CDAD. Confirmation of species identification, toxin detection, susceptibility testing against four antimicrobial agents and typing using a PCR ribotyping method were performed at the reference laboratory. In total, 149 isolates of putative C. difficile were submitted, from which 142 were included for study. Antimicrobial susceptibility patterns revealed resistance to clindamycin in 57 % and high-level resistance to moxifloxacin in 38 % of isolates tested. CDAD manifested as diarrhoea (including eight cases of bloody diarrhoea) in 126 cases (88.7 %), as pseudomembranous colitis in 15 cases (10.6 %) and as toxic megacolon in one case. Twelve of the 142 patients died within 30 days of specimen collection (8.45 % lethality). A lethal outcome occurred in 2/15 cases (13.3 %) when pseudomembranous colitis was present and in 10/126 cases (7.9 %) in the absence of pseudomembranous colitis or toxic megacolon. Among the 142 isolates from 25 health-care facilities, 41 PCR ribotype patterns were found. The most frequent ribotypes were AI-5 (including six lethal cases out of 26 patients), 014 (two out of 24) and 053 (one out of 24). The typing patterns demonstrated the occurrence of clusters in hospitals.