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

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 (Rct) 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 (Rct) 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.

Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

The Presence of Gut Microbial Genes Encoding Bacterial Genotoxins or Pro-Inflammatory Factors in Stool Samples from Individuals with Colorectal Neoplasia.

Gut bacterial toxins are thought to contribute to the development of colorectal cancer (CRC). This study examines the presence of specific gut bacterial toxin genes in stool samples from individuals with colorectal neoplasia (adenomas and/or CRC). The presence of bacterial genes encoding genotoxic or pro-inflammatory factors (pks, tcpC, gelE, cnf-1, AMmurB, and usp) was established by PCR of stool samples from individuals from mainland US (n = 30; controls = 10, adenoma = 10, CRC = 10) and from Puerto Rico (PR) (n = 33; controls = 13; adenomas = 8; CRC = 12). Logistic regression models and multinomial logistic regression models were used to estimate the magnitude of association. Distinct bacterial gene profiles were observed in each sample cohort. In individuals with CRC, AMmurB was detected more frequently in samples from the US and gelE in samples from PR. In samples from PR, individuals with ≥2 gut bacterial toxin genes in stool had higher odds of having colorectal neoplasia (OR = 11.0, 95%: CI 1.0⁻637.1): however, no significant association between bacterial genes and colorectal neoplasia was observed in the US cohort. Further analyses are warranted in a larger cohort to validate these preliminary findings, but these encouraging results highlight the importance of developing bacterial markers as tools for CRC diagnosis or risk stratification.

The Presence of Genotoxic and/or Pro-inflammatory Bacterial Genes in Gut Metagenomic Databases and Their Possible Link With Inflammatory Bowel Diseases.

Background: The human gut microbiota is a dynamic community of microorganisms that mediate important biochemical processes. Differences in the gut microbial composition have been associated with inflammatory bowel diseases (IBD) and other intestinal disorders. In this study, we quantified and compared the frequencies of eight genotoxic and/or pro-inflammatory bacterial genes found in metagenomic Whole Genome Sequences (mWGSs) of samples from individuals with IBD vs. a cohort of healthy human subjects. Methods: The eight selected gene sequences were clbN, clbB, cif, cnf-1, usp, tcpC from Escherichia coli, gelE from Enterococcus faecalis and murB from Akkermansia muciniphila. We also included the sequences for the conserved murB genes from E. coli and E. faecalis as markers for the presence of Enterobacteriaceae or Enterococci in the samples. The gene sequences were chosen based on their previously reported ability to disrupt normal cellular processes to either promote inflammation or to cause DNA damage in cultured cells or animal models, which could be linked to a role in IBD. The selected sequences were searched in three different mWGS datasets accessed through the Human Microbiome Project (HMP): a healthy cohort (N = 251), a Crohn's disease cohort (N = 60) and an ulcerative colitis cohort (N = 17). Results: Firstly, the sequences for the murB housekeeping genes from Enterobacteriaceae and Enterococci were more frequently found in the IBD cohorts (32% E. coli in IBD vs. 12% in healthy; 13% E. faecalis in IBD vs. 3% in healthy) than in the healthy cohort, confirming earlier reports of a higher presence of both of these taxa in IBD. For some of the sequences in our study, especially usp and gelE, their frequency was even more sharply increased in the IBD cohorts than in the healthy cohort, suggesting an association with IBD that is not easily explained by the increased presence of E. coli or E. faecalis in those samples. Conclusion: Our results suggest a significant association between the presence of some of these genotoxic or pro-inflammatory gene sequences and IBDs. In addition, these results illustrate the power and limitations of the HMP database in the detection of possible clinical correlations for individual bacterial genes.

Real-Time Detection of Telomerase Activity in Cancer Cells using a Label-Free Electrochemical Impedimetric Biosensing Microchip.

The enzyme telomerase is present in about 85% of human cancers which makes it not only a good target for cancer treatment but also an excellent marker for cancer detection. Using a single stranded DNA probe specific for telomerase binding and reverse transcription tethered to an interdigital gold electrode array surface, the chromosome protection provided by the telomerase was replicated and followed by Electrochemical Impedance Spectroscopy as an unlabeled biosensor. Using this system designed in-house, easy and affordable, impedance measurements were taken while incubating at 37 °C and promoting the probe elongation. This resulted in up to 14-fold increase in the charge transfer resistance when testing a telomerase-positive nuclear extract from Jurkat cells compared to the heat-inactivated telomerase-negative nuclear extract. The electron transfer process at the Au electrodes was studied before the elongation, at different times after the elongation, and after desorption of non-specific binding.

Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool.

Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.