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

Abstract

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.

Keywords: Gut Inflammation; Microbial Biomarkers; Microbiota; PCR; Stool Samples.

Figure 1

PCR amplification of the pks island genes was visualized on a 2% agarose gel in which the first four lanes contain the DNA size marker, a positive control (+), a negative control (−) and a blank (0), respectively. The DNA size markers used for samples 1 – 24 is 1kb Opti-DNA Marker from ABM band for sample 25 – 41 the 100 bp Opti-DNA Marker by ABM. The presence of the pks island gene can be established by the presence of a band at 733 bp (black arrows). The data for the 41 samples is shown in two different gels with eight positives (3, 7, 8, 13, 14, 26, 36 and 41).

Figure 2

The DNA sequences for all the fragments generated in this study were confirmed by the Sanger method. Interestingly, three distinct variants of the pks island sequence were identified in three different samples (Hu033, Hu037 and Hu038). The sequencing chromatograms corresponding to regions 8300 – 8322 and 8372 – 8392 of the pks island gene sequence (Ge-neBank Accession No. AM229678) are shown for all three samples. Sequence variability was observed in positions 8312 and 8383 of this amplicon.

Figure 3

For the quantification of DNA copy number, a standard curve was generated for each of the pro-inflammatory genes as described in the methods section. Standards containing 393 ng/μl (77,058,825 copies per μl), 100 ng/μl (19,607,843 copies per μl), 10 ng/μl (1,960,784 copies per μl), 1 ng/μl (196,078 copies per μl) and 0.1 ng/μl (19,607 copies per μl) were used to make the calibration curve. Typical copy number estimates for tcpC-positive samples ranged from 100,000 – 800,000 copies per μl. In this figure we show (a) the agarose gel electrophoresis for the PCR amplification of tcpC gene using different known amounts of E. coli DNA in the reaction mixture, (b) the qPCR reaction for the standards monitored in real time and (c) the calibration curve for the quantification of tcpC copies.