Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.
Abstract. Hospital-based and community-based studies were conducted to understand the prevalence and mode of transmission of Cryptosporidium parvum infection in Surabaya, Indonesia. In both studies people with and without diarrhea were examined for oocysts. A community-based survey included questionnaires to a community and stool examination of cats. Questionnaires covered demographic information, health status, and hygienic indicators. In the hospital, C. parvum oocysts were found in 26 (2.8%) of 917 patients with diarrhea and 15 (1.4%) of 1,043 control patients. The most susceptible age was less than two years old. The prevalence was higher during the rainy season. A community-based study again showed that C. parvum oocysts were frequently detected in diarrhea samples (8.2%), exclusively during rainy season. Thirteen (2.4%) of 532 cats passed C. parvum oocysts. A multiple logistic regression model indicated that contact with cats, rain, flood, and crowded living conditions are significant risk factors for Cryptosporidium infection.The protozoan Cryptosporidium parvum is now recognized as an important cause of diarrhea in both immunocompetent and immunosuppressed patients. A number of surveys carried out in recent years have described the worldwide distribution of Cryptosporidium. [1][2][3][4][5][6][7][8][9][10] The prevalence of Cryptosporidium infection is higher among children than among adults and is more common in developing countries than in developed countries. 1-10 Infection occurs by ingestion of oocysts either through human-to-human or animal-to-human contact, or via contaminated water. 5,6,[11][12][13][14][15][16] Despite its wide distribution and obvious relevance to public health, Cryptosporidium remains a little-studied protozoan in Indonesia. The present studies were undertaken to 1) assess the importance of Cryptosporidium in the causation of acute diarrhea among patients of all age groups who visited a hospital, and 2) understand the community prevalence and the mode of transmission of Cryptosporidium infection in Surabaya, Indonesia. MATERIALS AND METHODSStudy population and sample collection. Hospital-based study. Observations were made over a period of one year from August 1992 to July 1993 at the Dr. Soetomo Hospital, which is part of the University of Airlanaga in Surabaya. The hospital has 1,466 beds. During the study period, 92,061 patients visited the hospital and 41,622 patients were admitted. At total of 3,892 of 41,622 in-patients and 1,982 of 50,439 out-patients had diarrhea. The study population consisted of 917 patients with acute diarrhea: 715 in-patients and 202 out-patients. A group of 1,043 in-patients selected during the study period served as controls; none of these patients had any gastrointestinal problems. A stool specimen was collected from each patient within 24 hr of visiting the hospital.Community-based study. The studies were conducted in eight communities in Surabaya during the rainy season (December 1992 to March 1993) and dry season (June to July 1993). The total ...
The gene encoding a thermostable β-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-β-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.
SUMMARY: Diarrheagenic Escherichia coli (DEC) is a major etiologic agent of childhood diarrhea in developing countries. We investigated the frequency of DEC in stool samples from 125 diarrheal children (age, 1-10 years) and 92 non-diarrheal children in Surabaya, Indonesia. The non-diarrheal children served as healthy controls. DEC was detected in 23 of 125 (18.4z) and 47 of 92 (51.1z) samples in the diarrheal and non-diarrheal children, respectively. Enteropathogenic E. coli was the most prevalent in the non-diarrheal children (25.0z), and its prevalence was significantly higher than that in the diarrheal children (0.8z) (P º 0.0001). Interestingly, Shiga toxin-producing E. coli (4.3z) was detected only in the non-diarrheal children ( P = 0.031). This is the first study comparing between diarrheal children with non-diarrheal or healthy children to investigate the role of DEC in pediatric diarrheal diseases in Indonesia.
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