The Pic Protease of Enteroaggregative \u3cem\u3eEscherichia coli\u3c/em\u3e Promotes Intestinal Colonization and Growth in the Presence of Mucin

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 108 CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log10 difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization

Further development of crank mechanism through PLC / Wan Hasidi Wan Muhamad, Sheikh Rizuan Jalaluddin dan Muhd Hanif Hasan

Effisiensi pengeluaran selalunya adalah merupakan kunci dalam mencapai sesuatu kejayaan dalam mengharungi arus pembangunan industri pengeluaran yang semakin hebat.Effisiensi pengeluaran meliputi bidang-bidang seperti; • Kelajuan di mana peralatan pengeluaran dan juga line pengeluaran dapat diselaraskan untuk sesuatu proses pengeluaran. • Mengurangkan kos bahan dan kos buruh untuk sesuatu produk. • Meningkatkan kualiti dan mengurangkan bahan buangan. • Meminimakan susutmasa sesuatu peralatan pengeluaran. • Kos peralatan pengeluaran yang rendah. Programmable logic Controllers memenuhi sebahagian daripada keperluan di atas dan merupakan kunci yang dapat membawa effisiensi pengeluaran lebih laju dalam proses industri. Secara tradisionalnya, automasi hanya dapat diaplikasikan untuk satu barangan dalam suatu proses pengeluaran yang tinggi.Tetapi pada masa kini automasi pengeluaran dapat digandakan untuk melakukan pelbagai jenis barangan, dalam kuantiti yang banyak, mencapai overall pengeluaran yang tinggi dan yang memerlukan pelaburan yang rendah dalam peralatan dan juga logi. Sistem pengeluaran Flexible memenuhi kriteria-kriteria di atas. Sistem ini terdiri daripada peralatan automatik seperti mesin NC, robot industri, pengangkutan automatik dan pengawalan berkomputer untuk sesuatu pengeluaran. Oleh itu Programmable Logic Controller selalunya digunakan dalam peralatan pengeluaran automatik

Regulation of the Overlapping pic/set Locus in Shigella flexneri and Enteroaggregative Escherichia coli

Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37°C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus

Molecular Epidemiological Study of Nosocomial Enterobacter aerogenes Isolates in a Belgian Hospital

In 1995, the rate of isolation of Enterobacter aerogenes in the Saint-Pierre University Hospital in Brussels, Belgium, was higher than that in the preceding years. A total of 45 nosocomial E. aerogenes strains were collected from 33 patients of different units during that year, and they were isolated from 19 respiratory specimens, 13 pus specimens, 7 blood specimens, 4 urinary specimens, 1 catheter specimen, and 1 heparin vial. The strains were analyzed to determine their epidemiological relatedness and were characterized by their antibiotic resistance pattern determination, plasmid profiling, and genomic fingerprinting by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The majority of the strains (82%) were multiply resistant to different commonly used antibiotics. Two major plasmid profiles were found: most strains (64%) harbored two plasmids of different sizes, whereas the others (20%) contained a single plasmid. PFGE with SpeI and/or XbaI restriction enzymes revealed that a single clone (80%) was responsible for causing infections or colonizations throughout the year, and this result was concordant with those obtained by plasmid profiling, with slight variations. By comparing the results of these three methods, PFGE and plasmid profiling were found to be the techniques best suited for investigating the epidemiological relatedness of E. aerogenes strains, and they are therefore proposed as useful tools for the investigation of nosocomial outbreaks caused by this organism

Use of a Continuous-Flow Anaerobic Culture To Characterize Enteric Virulence Gene Expression

We developed an in vitro culture method to characterize the expression of bacterial genes under conditions mimicking the colonic environment. Our culture system (the intestinal simulator) comprised a continuous-flow anaerobic culture which was inoculated with fecal samples from healthy volunteers. As a test organism, we employed enteroaggregative Escherichia coli (EAEC), an emerging diarrheal pathogen that is thought to cause infection in both the small and large intestines. After the simulator culture achieved equilibrium conditions, we inoculated the system with prototype EAEC strain 042 and assessed the expression of three EAEC virulence-related genes. We focused particularly on expression of aggR, which encodes a global transcriptional regulator of EAEC virulence factors, and two AggR-regulated genes. By using real-time quantitative reverse transcription-PCR, we showed that aggR expression in the simulator is increased 3- to 10-fold when 042 is grown under low-pH (5.5 to 6.0) conditions, compared with results with neutral pH (7.0). Interestingly, however, this effect was seen only when the strain was grown in the presence of commensal bacteria. We also found that expression of aggR is 10- to 20-fold higher at low NaCl concentrations, and this effect was also observed only in the presence of commensal bacteria. Using coculture and conditioned-media experiments, we identified specific strains of Enterococcus and Clostridium that upregulated aggR expression; in contrast, strains of Lactobacillus and Veillonella downregulated aggR expression. Our data provide new insights into regulation of virulence genes in EAEC and suggest the utility of intestinal simulation cultures in characterizing enteric gene regulation

The Pic Protease of Enteroaggregative Escherichia coli Promotes Intestinal Colonization and Growth in the Presence of Mucin▿

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 108 CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log10 difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization

Isolation and characterization of enteroaggregative Escherichia coli (EAggEC) by genotypic and phenotypic markers, isolated from diarrheal children in Congo

Objective: To determine the prevalence of enteroaggregative Escherichia coli (EAggEC) in African diarrheal children in Lwiro, Congo, to characterize EAggEC isolates by possible genotypic and phenotypic markers, and to evaluate the EAggEC probe pCVD432 in identifying EAggEC. Methods: The Hep-2 cell adhesion assay and colony-blot hybridization assays were carried out for the identification of EAggEC. O:H serotyping, biotyping, antibiogram and plasmid-profile analysis were done. To detect the E. coli LT and ST, ELISA tests were used and, for VT, a vero cell assay was used. Results: EAggEC strains were isolated from 56 out of 115 diarrheal children (48.7%): the organism was present alone and presumed to cause diarrhea in 22 (19.1%) cases. The rest of the cases were associated with two or more diarrheagenic E. coli strains. EAggEC strains were isolated from 25% of total diarrheal children (first day of isolation) and 8.86% of age-matched healthy individuals (p < 0.03). This isolation rate was significantly higher than the one found for other diarrheagenic E. coli strains. In parallel, we evaluated the sensitivity and specificity of EAggEC probe pCVD432, and found that it had 56% sensitivity with 100% specificity compared with the Hep-2 cell test. EAggEC isolates were characterized by serotyping, biotyping, antibiotic resistance pattern, plasmid profiling and toxin production analysis. They did not produce any one of these classical toxins and nor did they relate to any particular serotypes. Plasmid analysis of the 79 EAggEC isolates (n = 315) showed seven different profiles. Ten resistance patterns were identified and 34 strains were sensitive to all drugs. There was no association between plasmid profiles and antibiotic resistance patterns. All 16 classical E. coli biotypes were found in this small EAggEC population. Conclusions: EAggEC has been emerging as a cause of childhood diarrhea in African children in Congo. From the accumulated data it was found that there is a great heterogeneity in EAggEC populations.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Typical Enteroaggregative Escherichia coli Is the Most Prevalent Pathotype among E. coli Strains Causing Diarrhea in Mongolian Children

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic Escherichia coli. In Mongolia, the epidemiology of diarrheagenic E. coli has not been well studied. A total of 238 E. coli strains from children with sporadic diarrhea and 278 E. coli strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic E. coli (EPEC) eae, tir, and bfpA; enterotoxigenic E. coli (ETEC) lt and st; enteroinvasive E. coli (EIEC) ipaH; enterohemorragic E. coli stx1 and stx2; and enteroaggregative E. coli (EAEC) aggR and astA. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%; P = 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes—aggA, 2 (5.5%); aafA, 4 (11.1%); agg-3a, 5 (13.8%); aap, 8 (22.2%); aatA, 11 (30.5%); capU, 9 (25.0%); pet, 6 (16.6%); and set, 3 (8.3%)—and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains

A novel dispersin protein in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product “dispersin,” and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen