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

Gómez-Moreno R, Robledo IE, Baerga-Ortiz A - Adv Microbiol (2014)

Bottom Line: Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation.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.

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Affiliation: Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico ; Molecular Sciences Building, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: A total of 41 anonymous human stool samples were analyzed for the presence of the genes for pks island, tcpC, gelE, and cnf-1. The presence of bacteria was confirmed in all of the samples by amplification of the 16S-rRNA gene (data not shown). The presence of the pks island genes was established by the amplification of a DNA fragment of 733 bp (Figure 1(a)). A total of 8 samples out of 41 were found to contain the gene for pks island (20%). The other pro-inflammatory gene, tcpC, was detected in a total of 7 samples (17%) as evidenced by the presence of a band of 216 bp (Figure 1(b)) and the enterococcal gelE was found in 3 samples easily detected by a band of 213 bp (7%). The gene for cytotoxic necrotizing factor-1 (cnf-1) was only found in a single sample while the gene for the cytolethal distending toxin (cdt) was not found in this group of samples. The results from the PCR screens are summarized in Table 2.


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

Gómez-Moreno R, Robledo IE, Baerga-Ortiz A - Adv Microbiol (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307837&req=5

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).
Mentions: A total of 41 anonymous human stool samples were analyzed for the presence of the genes for pks island, tcpC, gelE, and cnf-1. The presence of bacteria was confirmed in all of the samples by amplification of the 16S-rRNA gene (data not shown). The presence of the pks island genes was established by the amplification of a DNA fragment of 733 bp (Figure 1(a)). A total of 8 samples out of 41 were found to contain the gene for pks island (20%). The other pro-inflammatory gene, tcpC, was detected in a total of 7 samples (17%) as evidenced by the presence of a band of 216 bp (Figure 1(b)) and the enterococcal gelE was found in 3 samples easily detected by a band of 213 bp (7%). The gene for cytotoxic necrotizing factor-1 (cnf-1) was only found in a single sample while the gene for the cytolethal distending toxin (cdt) was not found in this group of samples. The results from the PCR screens are summarized in Table 2.

Bottom Line: Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico ; Molecular Sciences Building, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico.

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.

No MeSH data available.


Related in: MedlinePlus