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Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles.

ABSTRACT: PCR detection of enterotoxigenic Escherichia-coli (ETEC) can be used directly on stool sample. However, it still has limitations due to presence of PCR inhibitors and interferences. This study, oligonucleotide primer specific to ETEC was immobilized onto MNPs and applied for separation and enrichment of ETEC-DNA from contaminants in stool after boiling. DNA separation efficiency was evaluated using conventional PCR and magneto-PCR-enzyme linked-gene-assay (MELGA). Due to high specificity of primer and efficiency of nanoparticles to bring down PCR inhibitors, DNA separation using primer-immobilized-MNPs exhibited 100-fold increase of sensitivity compared to that using simple boiling. Moreover, the sensitivities in stool were increased from 10⁸ to 10⁶ CFU/mL and 10⁴ to 10² CFU/mL when PCR products were detected by gel electrophoresis and MELGA, respectively. Results suggested that oligonucleotide-immobilized-MNPs combined with boiling DNA extraction method was successfully used to separate the DNA of ETEC in stool with high sensitivity using MELGA.

SUBMITTER: Jangpatarapongsa K

PROVIDER: S-EPMC8487978 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Increased sensitivity of enterotoxigenic <i>Escherichia coli</i> detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles.

Jangpatarapongsa Kulachart K Saimuang Kween K Polpanich Duangporn D Thiramanas Raweewan R Techakasikornpanich Mongkol M Yudech Papichaya P Paripurana Venusrin V Leepiyasakulchai Chaniya C Tangboriboonrat Pramuan P

Biotechnology reports (Amsterdam, Netherlands) 20210924

PCR detection of enterotoxigenic <i>Escherichia-coli</i> (ETEC) can be used directly on stool sample. However, it still has limitations due to presence of PCR inhibitors and interferences. This study, oligonucleotide primer specific to ETEC was immobilized onto MNPs and applied for separation and enrichment of ETEC-DNA from contaminants in stool after boiling. DNA separation efficiency was evaluated using conventional PCR and magneto-PCR-enzyme linked-gene-assay (MELGA). Due to high specificity ...[more]

PMID: 34631437

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Project description:Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species-E. coli.

Project description:Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for TibA modification. These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process.

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Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles.

Publications

Increased sensitivity of enterotoxigenic <i>Escherichia coli</i> detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles.

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