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.

A cluster of seven human cases of listeriosis occurred in Austria and in Germany between April 2011 and July 2013. The Listeria monocytogenes serovar (SV) 1/2b isolates shared pulsed-field gel electrophoresis (PFGE) and fluorescent amplified fragment length polymorphism (fAFLP) patterns indistinguishable from those from five food producers. The seven human isolates, a control strain with a different PFGE/fAFLP profile and ten food isolates were subjected to whole genome sequencing (WGS) in a blinded fashion. A gene-by-gene comparison (multilocus sequence typing (MLST)+) was performed, and the resulting whole genome allelic profiles were compared using SeqSphere+ software version 1.0. On analysis of 2298 genes, the four human outbreak isolates from 2012 to 2013 had different alleles at ≤6 genes, i.e. differed by ≤6 genes from each other; the dendrogram placed these isolates in between five Austrian unaged soft cheese isolates from producer A (≤19-gene difference from the human cluster) and two Austrian ready-to-eat meat isolates from producer B (≤8-gene difference from the human cluster). Both food products appeared on grocery bills prospectively collected by these outbreak cases after hospital discharge. Epidemiological results on food consumption and MLST+ clearly separated the three cases in 2011 from the four 2012–2013 outbreak cases (≥48 different genes). We showed that WGS is capable of discriminating L. monocytogenes SV1/2b clones not distinguishable by PFGE and fAFLP. The listeriosis outbreak described clearly underlines the potential of sequence-based typing methods to offer enhanced resolution and comparability of typing systems for public health applications.

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.

L isteriosis is a severe, mainly foodborne, human infection associated with higher casefatality and hospitalization rates than other bacterial gastrointestinal pathogens (1). The causative agent, Listeria monocytogenes, occurs ubiquitously in the environment and disseminates into the food production chain. Patients develop either self-limiting noninvasive gastroenteritis or invasive listeriosis (2,3). Listeriosis adversely affects older and immunocompromised persons, as well as pregnant women, causing a severe invasive form of the disease that leads to sepsis, meningitis, and encephalitis, as well as neonatal infections and miscarriage (4). Case-fatality rates of invasive listeriosis are ≈30% for neurolisteriosis and even higher in septic patients (5). In Europe and North America, invasive listeriosis affects 0.3-0.6 persons/100,000 population/year (6,7). L. monocytogenes forms hard-to-remove biofilms in food-processing plants, can acquire tolerance to sanitizers, and multiplies even at temperatures used for refrigeration (8). These properties complicate efficient prevention of L. monocytogenes contaminations in different types of ready-to-eat products, including dairy, meat, and fish, and in fruits and vegetables, all of which have been vehicles for listeriosis outbreaks in the past (9-12).

Our study underlines a dominant role of norovirus and toxigenic C. difficile as etiologic agents of acute gastroenteritis among the patients of general practitioners.