Development of a duplex PCR genotyping assay for distinguishing Clostridium perfringens type A isolates carrying chromosomal enterotoxin (cpe) genes from those carrying plasmid-borne enterotoxin (cpe) genes

About 5% of Clostridium perfringens type A isolates carry the cpe gene encoding the C. perfringens enterotoxin. Those cpe-positive type A isolates are important causes of food-poisoning and non-food-borne cases of diarrheas in humans, as well as certain veterinary cases of diarrhea. Previous studies have determined that the enterotoxigenic type A isolates causing both non-food-borne human gastrointestinal disease and veterinary disease carry their cpe genes on plasmids, while the type A isolates causing human food poisoning carry a chromosomal cpe gene. The present study reports on the successful development of a duplex PCR assay that can rapidly genotype enterotoxigenic type A isolates (i.e., determine whether those cpe-positive isolates carry a chromosomal or a plasmid-borne cpe gene). The availability of this rapid cpe genotyping assay capable of handling large numbers of samples provides a powerful new investigative tool for diagnostic, epidemiologic, and basic research purposes.     

Synthetic DNA probes for detection of enterotoxigenic Clostridium perfringens strains isolated from outbreaks of food poisoning.

Four synthetic oligonucleotides encoding different parts of the Clostridium perfringens enterotoxin gene were used to test the enterotoxigenicity of C. perfringens strains isolated from confirmed outbreaks of food poisoning. Of the 245 strains isolated from food and feces originating from 186 separate outbreaks, 145 (59%) gave hybridization reactions with each of the four DNA probes used, while 104 strains did not hybridize with any of the probes. There was no correlation between serotype and the presence of the enterotoxin gene, although the C. perfringens enterotoxin gene was rarely detected among nontypable strains (17%). Results show that DNA hybridization is a suitable method for the identification of C. perfringens strains which have the potential to produce enterotoxin.     

Clostridium perfringens type A strains carrying a plasmid-borne enterotoxin gene (genotype IS1151-cpe or IS1470-like-cpe) as a common cause of food poisoning

The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). These results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning.     

Analysis of protection afforded by a Clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases C

The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the alpha-toxin. Previously, mice have been protected against challenge with heterologous alpha-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa(247-370) or alpha-toxoid) of the alpha-toxin. In this study, we have determined the ability of the alpha-toxoid to protect against the lethal effects of a divergent C. perfringens alpha-toxin and against the PLCs of C. absonum or C. bifermentans, species which have been isolated from cases of clostridial myonecrosis. Protection against the C. perfringens alpha-toxin variant, the C. absonum alpha-toxin or the C. bifermentans PLC was elicited by immunisation with the alpha-toxoid in vivo.     

Use of a Clostridium perfringens vector to express high levels of SIV p27 protein for the development of an oral SIV vaccine

Clostridium perfringens is a normal bacterial flora of the small and large intestines of humans and other animals. The current study investigates the potential use of a noncytotoxic C. perfringens as an oral vaccine vehicle for expression and intestinal delivery of a large amount of SIV antigens. Here we report the construction of a recombinant C. perfringens vaccine vector expressing high levels of SIV p27 during sporulation. Following oral administration of this recombinant C. perfringens vaccine vector to mice, large amounts of intact p27 protein were detected in the terminal ileum where the majority of Peyer's Patches (PPs) are located. Furthermore, dendritic cells (DCs) beneath the mucosal surface in the PPs were able to capture SIV p27 antigen, when PPs were exposed to C. perfringens expressing SIV p27 antigen. In addition, uptake of C. perfringens was able to induce maturation of mouse DCs. These results support the potential use of C. perfringens as an oral SIV/HIV vaccine vector.     

The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice

Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine.     

Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).     

Cytotoxic effects by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence ofClostridium perfringens iota toxin

The ADP-ribosylating toxinsClostridium botulinum C2 toxin andC. perfringens iota toxin, which ADP-ribosylate monomeric G-actin at Arg-177 but not the polymeric F-actin, induce depolymerization of the actin cytoskeleton in cultured cells. Since ADP-ribosylated G-actin has properties of a barbed-end-capping protein, we studied whether the ADP-ribosylated actin affects the actin cytoskeleton of PtK2 cells even in the absence of ADP-ribosylating toxin. Skeletal muscle actin was ADP-ribosylated byC. perfringens iota toxin and the toxin was removed using an anti-iota toxin antibody. Microinjection of ADP-ribosylated actin caused retraction of the cell body, redistribution and depolymerization of the actin cytoskeleton in a concentration-and time-dependent manner. The finding that ADP-ribosylated actin affects per se the actin cytoskeleton explains the cytopathic effects of ADP-ribosylating toxins on microfilaments, although F-actin is not directly modified by the toxins.     

Dihydropteroate synthase gene mutations in Pneumocystis jiroveci strains isolated from immunocompromised patients

The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase (DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.     

Molecular properties of each subcomponent in Clostridium botulinum type B haemagglutinin complex

The role of each subcomponent of Clostridium botulinum serotype B haemagglutinin (HA), which is one component of 16S toxin, and consists of four subcomponents (HA1, 2, 3a, and 3b), was investigated. In order to identify the subcomponent contributing to the stability of a neurotoxin in the gastro-intestinal tract, each recombinant HA (rHA) subcomponent was incubated with gastro-intestinal proteases. Although rHA1 and rHA3 were stable to these proteases except for specific cleavage, rHA2 was not. Anti-free whole HA serum reacted with neither rHA2 nor HA2 in 16S toxin on both Western blot and ELISA, while anti-rHA2 serum reacted with both rHA2 and HA2 in 16S toxin on Western blots, although it did not react with 16S toxin in ELISA. Binding or haemagglutination activity against erythrocytes was found in rHA1 and rHA3, but not in rHA2. In addition, only HA1 bound to the intestinal section. These results indicate that the HA (and 16S toxin) complex is assembled in the way that HA1 and HA3 (HA3a plus HA3b) encase HA2, followed by modification with trypsin-like bacterial protease, leading to the conclusion that HA1 and HA3 act as protective factors for the neurotoxin and as attachment factors to host cells.     

Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence

Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans. In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene. In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence. When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C. perfringens food poisoning in Japan. The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C. perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.     

The First Case of Antibiotic-associated Colitis by Clostridium difficile PCR Ribotype 027 in Korea

Clostridium difficile (C. difficile) is a common causative agent of pseudomembranous colitis (PMC). C. difficile-associated diarrhea (CDAD) ranges from mild diarrhea to life threatening PMC. Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan. A 52-yr-old woman with anti-tuberculosis medication and neurogenic bladder due to traffic accident experienced five episodes of C. difficile PMC after taking antibiotics for pneumonia along with septic shock and acute renal failure. She was readmitted to the intensive care unit and treated with oral vancomycin with refractory of oral metronidazole, inotropics and probiotics for over 60 days. C. difficile isolated both at the first and the last admission was identified as C. difficile ribotype 027 by ribotyping, toxinotyping, and tcdC gene sequencing, which turned out the same pathogen as the epidemic hypervirulent B1/NAP1 strain. This is the first case of C. difficile PCR ribotype 027 in Korea. After discharge, she was maintained on probiotics and rifaximin for 3 weeks. She had no relapse for 6 months.     

Rapid identification of myxoma virus variants by long-range PCR and restriction fragment length polymorphism analysis

A long-range PCR method directed at the Myxoma virus (MV) left hand and right hand terminal inverted repeats (TIRs) for rapid amplification of genomic DNA and MV isolate differentiation by restriction fragment length polymorphism (RFLP) analysis is described. The efficacy of this method was tested by comparing the results from full genome RFLPs with those from TIRs amplified separately using reference strain Lausanne (Lu) and a field MV strain characterised previously for its virulence in rabbits. The usefulness of this method was also demonstrated by amplifying MV DNA directly from the eyelid tissue of an infected rabbit and comparative RFLP analysis with respect to Lu. The results proved the long-range PCR technique to be a simple highly efficient method for identifying mutations between MV genomes by RFLP analyses of the amplified TIRs and may be used in future studies to identify variable regions for phylogenetic studies.     

Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains

Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease was investigated. The gene of strain E86 from a diseased eel (type B vvp) was 95.2% identical with that of strain L-180 from human blood (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the gene showed that eel avirulent strains (9 isolates) commonly carry type A vvp, whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes from 12 strains including strain E86 were placed on type B while those from 3 strains were on type A. Other strains were found to be vvp-negative, but PAGE and amino acid sequencing analysis showed that they secreted a serine protease (VVA0302) instead of the metalloprotease. This protease is an orthologue of a toxic protease from Vibrio parahaemolyticus, a human pathogen causing wound infection as well as gastroenteritis. These findings suggest that, in addition to metalloprotease, the extracellular serine protease may contribute to pathogenicity of V. vulnificus.     

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.     

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10³ cfu mL⁻¹ for Campylobacter jejuni, 10⁴ cfu mL⁻¹ for Salmonella, and 10⁵ cfu mL⁻¹ for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples (n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.     

Five novel acid-tolerant oligotrophic thiosulfate-metabolizing chemolithotrophic acid mine drainage strains affiliated with the genus Burkholderia of Betaproteobacteria and identification of two novel soxB gene homologues

Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958^T (96-98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8^T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417^T (99.6%) and Burkholderia vietnamiensis LMG 10929^T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958^T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.     

Interval based fuzzy systems for identification of important genes from microarray gene expression data: Application to carcinogenic development

In the present article, we develop two interval based fuzzy systems for identification of some possible genes mediating the carcinogenic development in various tissues. The methodology involves dimensionality reduction, classifying the genes through incorporation of the notion of linguistic fuzzy sets low, medium and high, and finally selection of some possible genes mediating a particular disease, obtained by a rule generation/grouping technique. The effectiveness of the proposed methodology, is demonstrated using five microarray gene expression datasets dealing with human lung, colon, sarcoma, breast cancer and leukemia. Moreover, the superior capability of the methodology in selecting important genes, over five other existing gene selection methods, viz., Significance Analysis of Microarrays (SAM), Signal-to-Noise Ratio (SNR), Neighborhood analysis (NA), Bayesian Regularization (BR) and Data-adaptive (DA) is demonstrated, in terms of the enrichment of each GO category of the important genes based on P-values. The results are appropriately validated by earlier investigations, gene expression profiles and t-test. The proposed methodology has been able to select genes that are more biologically significant in mediating the development of a disease than those obtained by the others.