Dataset Information

PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

ABSTRACT: A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling in blank, positive, and negative DNA controls. Target DNA identification was possible by monitoring changes in the system's charge transfer resistance values (R_ct) before and after PCR treatment through electrochemical impedance spectroscopy (EIS) analysis. Custom-made, flexible gold electrodes were fabricated using chemical etching optical lithography. A PCR cycle study determined the optimum conditions to be at 6 cycles providing fast results while maintaining a good sensitivity. EIS data for the DNA recognition process demonstrated the successful distinction between target interaction resulting in an increase in resistance to charge transfer (R_ct) percentage change of 176% for the positive DNA control vs. 21% and 20% for the negative and non-DNA-containing controls, respectively. Results showed effective fabrication of a fast, PCR-based electrochemical biosensor for the detection of pks genomic island with a calculated limit of detection of 17 ng/μL.

SUBMITTER: Solis-Marcano NE

PROVIDER: S-EPMC8159250 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

Solis-Marcano Nadja E NE Morales-Cruz Myreisa M Vega-Hernández Gabriela G Gómez-Moreno Ramón R Binder Claudia C Baerga-Ortiz Abel A Priest Craig C Cabrera Carlos R CR

Analytical and bioanalytical chemistry 20210527 18

A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling ...[more]

PMID: 34046698

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Project description:Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced 'Helper RNAs' that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes.

Project description:The genotoxin colibactin is a secondary metabolite produced by the polyketide synthase (pks) island harbored by extraintestinal pathogenic E. coli (ExPEC) and other members of the Enterobacteriaceae that has been increasingly reported to have critical implications in human health. The present study entails a high-throughput whole-genome comparison and phylogenetic analysis of such pathogenic E. coli isolates to gain insights into the patterns of distribution, horizontal transmission, and evolution of the island. For the current study, 23 pks-positive ExPEC genomes were newly sequenced, and their virulome and resistome profiles indicated a preponderance of virulence encoding genes and a reduced number of genes for antimicrobial resistance. In addition, 4,090 E. coli genomes from the public domain were also analyzed for large-scale screening for pks-positive genomes, out of which a total of 530 pks-positive genomes were studied to understand the subtype-based distribution pattern(s). The pks island showed a significant association with the B2 phylogroup (82.2%) and a high prevalence in sequence type 73 (ST73; n = 179) and ST95 (n = 110) and the O6:H1 (n = 110) serotype. Maximum-likelihood (ML) phylogeny of the core genome and intergenic regions (IGRs) of the ST95 model data set, which was selected because it had both pks-positive and pks-negative genomes, displayed clustering in relation to their carriage of the pks island. Prevalence patterns of genes encoding RM systems in the pks-positive and pks-negative genomes were also analyzed to determine their potential role in pks island acquisition and the maintenance capability of the genomes. Further, the maximum-likelihood phylogeny based on the core genome and pks island sequences from 247 genomes with an intact pks island demonstrated horizontal gene transfer of the island across sequence types and serotypes, with few exceptions. This study vitally contributes to understanding of the lineages and subtypes that have a higher propensity to harbor the pks island-encoded genotoxin with possible clinical implications.IMPORTANCE Extraintestinal pathologies caused by highly virulent strains of E. coli amount to clinical implications with high morbidity and mortality rates. Pathogenic E. coli strains are evolving with the horizontal acquisition of mobile genetic elements, including pathogenicity islands such as the pks island, which produces the genotoxin colibactin, resulting in severe clinical outcomes, including colorectal cancer progression. The current study encompasses high-throughput comparative genomics and phylogenetic analyses to address the questions pertaining to the acquisition and evolution pattern of the genomic island in different E. coli subtypes. It is crucial to gain insights into the distribution, transfer, and maintenance of pathogenic islands, as they harbor multiple virulence genes involved in pathogenesis and clinical implications of the infection.

Project description:BackgroundMany Escherichia coli strains are considered to be a component of the normal flora found in the human and animal intestinal tracts. While most E. coli strains are commensal, some strains encode virulence factors that enable the bacteria to cause intestinal and extra-intestinal clinically-relevant infections. Colibactin, encoded by a genomic island (pks island), and cytotoxic necrotizing factor (CNF), encoded by the cnf gene, are genotoxic and can modulate cellular differentiation, apoptosis and proliferation. Some commensal and pathogenic pks+ and cnf+ E. coli strains have been associated with inflammation and cancer in humans and animals.ResultsIn the present study, E. coli strains encoding colibactin and CNF were identified in macaque samples. We performed bacterial cultures utilizing rectal swabs and extra-intestinal samples from clinically normal macaques. A total of 239 E. coli strains were isolated from 266 macaques. The strains were identified biochemically and selected isolates were serotyped as O88:H4, O25:H4, O7:H7, OM:H14, and OM:H16. Specific PCR for pks and cnf1 gene amplification, and phylogenetic group identification were performed on all E. coli strains. Among the 239 isolates, 41 (17.2%) were pks+/cnf1-, 19 (7.9%) were pks-/cnf1+, and 31 (13.0%) were pks+/cnf1+. One hundred forty-eight (61.9%) E. coli isolates were negative for both genes (pks-/cnf1-). In total, 72 (30.1%) were positive for pks genes, and 50 (20.9%) were positive for cnf1. No cnf2+ isolates were detected. Both pks+ and cnf1+ E. coli strains belonged mainly to phylogenetic group B2, including B21. Colibactin and CNF cytotoxic activities were observed using a HeLa cell cytotoxicity assay in representative isolates. Whole genome sequencing of 10 representative E. coli strains confirmed the presence of virulence factors and antibiotic resistance genes in rhesus macaque E. coli isolates.ConclusionsOur findings indicate that colibactin- and CNF-encoding E. coli colonize laboratory macaques and can potentially cause clinical and subclinical diseases that impact macaque models.

Dataset Information

PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

Publications

PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

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