A multiplex PCR method was developed for the detection of Clostridium difficile toxin genes tcdA, tcdB, ctdA, and cdtB and the major in-frame deletion types (18, 39, and 54 bp) of tcdC. The method has high specificity for PCR ribotype 027 and may identify other C. difficile strains of clinical and epidemiological importance.Toxigenic and epidemic Clostridium difficile is a well-established health threat in the nosocomial environment. Additionally, several reports have shown C. difficile acquisition in the community and C. difficile has been isolated from various food, environmental, and animal sources, often with genetic profiles similar to those observed in human infections (13). Epidemic and clinically important types of C. difficile are evolving and include several PCR ribotypes (5). Therefore, sufficient diagnostic methods need to be continuously updated in order to comply with this bacterium's changing epidemiology. C. difficile toxins include toxin A, toxin B, and a binary toxin. Toxins A and B are encoded by the genes tcdA and tcdB, which are located on the pathogenicity island PaLoc, which also includes the negative and positive regulators tcdC and tcdR. The binary toxin is encoded by the genes cdtA and cdtB, which constitute another operon together with the positive regulator cdtR. A number of different genetic alterations in the tcdC gene have been observed. Most prominent are the in-frame deletion of 18, 39, or 54 bp and the truncating mutation at position 117 (1-bp deletion) or 184 (C3T transition). In this respect, PCR ribotype 027 (North America pulsed-field type 1), which has been responsible for the most severe outbreaks around the world, is often reported to have an 18-bp in-frame deletion and ⌬117. Evidently, such genetic changes in the negative regulator have been proposed to be responsible for increased toxin production and hence hypervirulence (9, 15), but a clear association with clinical manifestations has not been determined (8,14,16,20). No matter what role tcdC deletions and mutations may have in relation to clinical outcome, the internal in-frame deletion of 18 bp and ⌬117 have been targets for real-time PCR screening for PCR ribotype 027 (4,11,18,21). This allows clinicians to introduce patient quarantine and hygienic precautions much earlier than when only laborious PCR ribotyping is performed.The present method is based on a conventional multiplex PCR (mPCR) applied to bacterial colonies derived from selectively cultured stool samples. This method allows the simultaneous identification of the toxin genes tcdA, tcdB, cdtA, and cdtB combined with an approximate determination of the inframe tcdC deletion size, all features that may assist in the evaluation of pathogenic potential and relatedness to clinically important strains. The C. difficile isolates used in the present study originated from local clinical microbiology laboratories and general practitioners and were sent to the National Reference Laboratory at the Statens Serum Institut as part of a national surveillance program dir...

Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.A eromonas spp. are highly adapted to aquatic environments and have been described as pathogenic to humans and animals. The genus Aeromonas consists of more than 20 valid species, of which A. hydrophila, A. caviae (synonymous with A. punctata), A. media, A. veronii bv. sobria, and A. veronii bv. veronii are of particular clinical significance, because they can cause gastroenteritis, wound and soft tissue infections, and septicemia (1). Aeromonas spp. may produce an array of virulence factors (e.g., cytolytic toxins with hemolytic activity and enterotoxins). Recent reviews suggested that only a subset of Aeromonas spp. are truly pathogenic and may be transmitted by hitherto unknown routes, and they proposed that further epidemiological and molecular studies are needed (2, 3). Aeromonas species identification has traditionally been performed by a combination of different biochemical tests. However, these are not always conclusive, since some Aeromonas species display heterogeneous biochemical properties; compared to molecular methods, the correct identification rate with biochemical tests has been shown to be very low (4, 5). Molecular species identification has been exploited by the 16S rRNA gene, either by restriction fragment length polymorphism (RFLP) (6-9) or direct sequencing (10, 11). However, due to insufficient interspecies sequence variation and heterogeneity among copies of ribosomal RNA operons in the same bacteria (10, 12, 13), this gene may not be an optimal target. A number of studies have shown that the sequences of several different housekeeping genes are able to differentiate this tight taxonomic group of organisms. These genes include an RNA polymerase B subunit (rpoB), an RNA polymerase D subunit (rpoD), and a DNA gyrase B subunit (gyrB) (10, 14-16). The objective of the present study was to identify species of Aeromonas by partial gyrB and rpoB sequencing in order to develop a multiplex PCR (mPCR) that targets the four most prevalent and clinically relevant species identified by sequence analysis on a Danish strain collection.The strain collection used in this study was composed of 51 Aeromonas spp. collected from diarrheagenic patients during the period of 2005 to 2010. Each stool specimen was grown on enteric medium (Statens Serum Institut, Hillerød, Denmark) (17), and Aeromonas was identified by its distinct colony morphology, while further delineation of clinically relevant species was performed manually according to their biochemical characteristics, including Voges-Proskauer test results and lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, glucose (gas), esculin, and acid production fr...