Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems

Abstract

A HindII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficik isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. dfficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies. During the 1980s, several antibiotic-associated diarrhea

U.S.-Based National Surveillance for Fidaxomicin Susceptibility of Clostridioides difficile-Associated Diarrheal Isolates from 2013 to 2016

“…A randomly selected sample of isolates, stratified by center (ϳ30 isolates from each center per year), were referred to the Microbiology Reference Laboratory at the Hines Veterans Administration Hospital (Hines, IL) for restriction endonuclease analysis (REA) strain typing. Following purification, total cellular DNA was cut with HindIII restriction enzyme, and fragments were separated by electrophoresis on a 0.7% agarose gel as previously described (32,33). HindIII restriction patterns with a 90% similarity index were included in the same REA group (letter designation).…”

Use of in-house studies of molecular epidemiology and full species identification for controlling spread of vancomycin-resistant Enterococcus faecalis isolates

“…Isolates with Ն90% similarity (or five or fewer band differences) were considered to be subtypes within the same type letter designation. Isolates with more than five band differences were considered to be sufficiently different to represent a new type letter designation (2). Determination of relatedness had to be the same by both enzyme analyses in order for isolates to be identified as clonal.…”

Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium : Assessment Using Clinical Epidemiologic Data

“…We have used REA of total genomic DNA with success in epidemiologic study of other organisms (6) and have applied this technique to type enterococcal isolates (2). REA typing.…”

“…The DNA band patterns for each new isolate digested with a common restric-tion enzyme were systematically compared according to the method described first by Clabots and colleagues (6). The first isolate in this analysis with a new DNA band pattern was arbitrarily designated a reference REA type.…”