Gene Of Clostridium Difficile Research Articles

Rapid, label-free genetic detection of enteropathogens in stool without genetic isolation or amplification

Current genetic detection methods require gene isolation, gene amplification and detection with a fluorescent-tagged probe. They typically require sophisticated equipment and expensive fluorescent probes, rendering them not widely available for rapid acute infection diagnoses at the point of care to ensure timely treatment of the diseases. Here we report a rapid genetic detection method that can detect the bacterial gene directly from patient stools using a piezoelectric plate sensor (PEPS) in conjunction with a continuous flow system with two temperature zones. With stools spiked with sodium dodecyl sulfate (SDS) in situ bacteria lysing and DNA denaturation occurred in the high-temperature zone whereas in situ specific detection of the denatured DNA by the PEPS occurred in the lower-temperature zone. The outcome was a rapid genetic detection method that directly detected bacterial genes from stool in < 40 min without the need of gene isolation, gene amplification, or expensive fluorescent tag but with polymerase chain reaction (PCR) sensitivity. In 40 blinded patient stools, it detected the toxin B gene of Clostridium difficile with 95% sensitivity and 95% specificity. The all-electrical, label-free nature of the detection further supports its potential as a low-cost genetic test that can be used at the point of care.

Simultaneous detection and characterization of toxigenic Clostridium difficile directly from clinical stool specimens.

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x2 = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.

Mo1868 Role of Rpon (σ54) in the Regulation of Clostridium difficile Gene Expression and Growth

Mo1868 Role of Rpon (σ54) in the Regulation of Clostridium difficile Gene Expression and Growth

Evaluation of the Diagnostic Performance of the XpertClostridium difficileAssay and Its Comparison With the Toxin A/B Enzyme-Linked Fluorescent Assay and In-House Real-Time PCR Assay Used for the Detection of ToxigenicC. difficile

Clostridium difficile genes or toxin can be detected using several laboratory techniques. In this study, we compared the performance of the Xpert C. difficile assay with that of a toxin A/B enzyme-linked fluorescent immunoassay (ELFA) and an in-house real-time PCR assay for the tcdB gene. From April 2011 through January 2012, 138 soft or liquid stool samples from 138 adult patients at Paik Hospital were tested using the toxin A/B ELFA, in-house real-time PCR assay, and Xpert C. difficile assay to detect toxigenic C. difficile. Specimens were considered true positives if results were positive in both the in-house real-time PCR for tcdB gene and Xpert C. difficile assays. Sensitivity of the toxin A/B ELFA, in-house tcdB gene real-time PCR, and Xpert C. difficile assay were 67.6%, 97.3%, and 100.0%, respectively. The specificity of the in-house tcdB gene real-time PCR assay was 100%, while the specificity was 98.0% for the other two methods. The turnaround time (TAT) was 50 min for the Xpert C. difficile assay, 75 min for the toxin A/B ELFA, and 160 min for the in-house real-time PCR assay. The Xpert C. difficile assay and the in-house real-time PCR assay had higher sensitivity than the toxin A/B ELFA; however, the specificities of the three assays were similar. Considering its rapid TAT and high sensitivity, use of the Xpert C. difficile assay is highly recommended for rapid and accurate diagnosis of C. difficile infection.

Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR

In this study, a total of 650 stool samples were tested to show that our method is capable of detecting four Clostridium difficile genes; tcdA, tcdB, encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin C. difficile transferase genes (cdtA and/or cdtB) encoding CDT toxin. Besides detecting the targeted C. difficile genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID™ C. difficile, can be applied directly for screening C. difficile-associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing C. difficile infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent C. difficile strains.

Evaluation of the BD MAX Cdiff assay for the detection of the toxin B gene of Clostridium difficile out of faecal specimens

In this evaluation study, 180 faecal specimens were tested in parallel with the new BD MAX™ Cdiff assay, the Premier™ Toxins A&B, and toxigenic culture as reference method. BD MAX™ Cdiff presented a high sensitivity (96.0%) and specificity (99.4%) with a negative predictive value of 99.4%.

Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile.

We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Nucleotide and peptide sequences of the open reading frame encoding a truncated toxin A gene of Clostridium difficile strain CCUG 20309.

The open reading frame encoding a putative truncated toxin A gene of Clostridium difficile in strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, was sequenced by cycle sequencing method. The coding region contains 2097 base pairs and has a GC content of 26.4%. The deduced polypeptide is 50 kDa and is generally hydrophilic. Although strain CCUG 20309 of C. difficile was reported not to produce toxin A, it is enterotoxic, an inherent property of toxin A in pathogenic strains of C. difficile. It therefore remains to be determined whether a truncated form of toxin A could actually be expressed from the open reading frame by this strain of C. difficile.

Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile

We have used six independent polymerase chain reactions (A1–A3 and B1–B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5′ third of tcdB (B1 fragment) and/or the 3′ third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Macrofragment localization of the toxin A and toxin B genes of Clostridium difficile.

We report the physical mapping of the toxin A and B genes to the bacterial chromosome of Clostridium difficile ATCC 43594 by pulsed-field gel electrophoresis. Single and double digestions with restriction endonucleases NruI and SacII allowed localization of the toxin genes to a specific 577-kb fragment and estimation of genome size to be approximately 3.8 megabases. This effort represents the initial step in the construction of a physical map of the whole genome.

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.

Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli

Cloned fragments constituting the 8.1-kb toxin A gene of Clostridium difficile were used to reconstruct the intact gene. The recombinant toxin expressed in Escherichia coli was cytotoxic, enterotoxic, and lethal. In addition, toxic lysate caused hemagglutination of rabbit erythrocytes. The toxic activities were inhibited by antibody specific for toxin A. Our findings demonstrate that the biological activities exhibited by native toxin A are functions of a single protein encoded by the 8.1-kb toxin A gene, independent of any other C. difficile gene products.

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

Nucleotide sequence ofClostridium difficiletoxin A gene fragment and detection of toxigenic strains by polymerase chain reaction

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

Cloning and Characterization of Overlapping DNA Fragments of the Toxin A Gene of Clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.

Cloning of the carbohydrate-binding portion of the toxin A gene ofClostridium difficile

A portion of the toxin A gene ofClostridium difficile was cloned into pBR322 withEscherichia coli Chi 1776 as the host. Five identical clones, each containing a 4.7-kbPstI restriction endonuclease fragment and producing toxin A antigens, were detected with affinity-purified, monospecific antibodies against toxin A. Digestion of the cloned DNA withPstI revealed as internal restriction site that resulted in two fragments (2.1 and 2.6 kb in size). Probe DNA from either of these fragments hybridized with DNA in the 4.7 kb region ofPstI-digested, high-molecular-weight DNA from the sourceC. difficile strain, indicating that the internalPstI site is protected. The probe DNA also hybridized with restriction-digested DNA from five additional toxigenic strains, but it did not hybridize with DNA from four nontoxigenic strains. In addition, a DNA fragment from a toxigenic strain ofClostridium sordellii, whose toxin cross-reacts with antibody toC. difficile toxin A, hybridized with the clonedC. difficile DNA. Unlike native toxin A, the cell lysate from the recombinant clone was not cytotoxic to Chinese hamster ovary cells or enterotoxic in hamsters. It did agglutinate rabbit red blood cells, a characteristic of toxin A. The cell lysate also exhibited a line of partial identity when compared with purified toxin A in Ouchterlony assays, and it reacted with monoclonal antibody to toxin A in an enzyme-linked immunosorbent assay. The cloned DNA appears to code for a nontoxic binding portion of toxin A, which is responsible for binding to galactose-α1-3-galactose-β1-4-N-acetylglucosamine.