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ABSTRACTClostridium difficileis the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficileis closely related to the production of toxins A and B. Toxigenic C. difficiledetection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficiletoxin genes tcdAand tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficilestrains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile(n= 14), including Clostridium sordelliifor which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdAand tcdBwere efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficiledirectly from feces samples.

A 4.4‐kb DNA fragment was cloned from Actinobacillus pleuropneumoniae(strain 4074, serotype 1) by genetic complementation with Escherichia coli groES‐groELmutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)‐encoding gene homolog (deoD), heat‐shock response‐encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino‐acid sequence of groESand groELgenes showed extensive sequence identity (80–95%) with other Pasteurellaceae. The gene organization surrounding the groElocus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES‐groELgenes were capable of complementing the growth of a λ lytic phage, indicating a structural as well as functional conservation.

Several letters to the editor are presented in response to previous articles including the marathon bombing in the July 2013 issue, "Just Imagine" in the July 2013 issue, and "Standing Ovation" in the July 2013 issue.

A 4.4-kb DNA fragment was cloned from Actinobacillus pleuropneumoniae (strain 4074, serotype 1) by genetic complementation with Escherichia coli groES-groEL mutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)-encoding gene homolog (deoD), heat-shock response-encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino-acid sequence of groES and groEL genes showed extensive sequence identity (80–95%) with other Pasteurellaceae. The gene organization surrounding the groE locus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES-groEL genes were capable of complementing the growth of a λ lytic phage, indicating a structural as well as functional conservation.