The gut microbiota contains between 5 and 8 million active genes distributed over a great diversity of mostly uncultivable and novel species [1] - [3] . This complex community of bacteria has been shown to promote a healthy gut homeostasis as it is tasked with housekeeping functions like catabolism of complex polysaccharides, biosynthesis of vitamins and other essential nutrients, degradation of xenobiotics and the maintenance of serotonin levels in plasma [4] - [6] . Additionally, it is believed that a healthy commensal gut microbiota aids in the clearance of pathogenic bacteria and in the prevention of intestinal inflammation [7] [8] . Shifts in the composition of the resident microbiota, or microbial dysbioses, have been shown to lead to a variety of intestinal disorders [9] [10] . With the advent of powerful tools for parallel DNA sequencing, there has been a renewed interest in exploring the possible involvement of the gut microbiota in the development of intestinal disorders such as inflammatory bowel Disease (IBD), colorectal cancer (CRC) and gut-brain associated diseases [11] - [13] .

Clinical correlations have been established that link chronic bacterial infections and intestinal disorders. For instance, the human pathogen Helicobacter pylori is widely recognized as a strong cofactor for the development of gastric adenocarcinoma [14] . Similarly, gut colonization with Clostridium difficile is followed by development of colitis, toxic megacolon and perforation [15] .

In addition to the known gastrointestinal pathogens, there are the typically non-pathogenic members of the microbiota, which may harbor genes associated with inflammation. For instance, certain strains of commensal Escherichia coli have been found to harbor a genomic island encoding a colibactin polyketide synthase multienzyme (pks island) that induces DNA damage followed by the activation of DNA repair pathways [16] . The presence of the pks island induces mammalian chromosome instability in mouse and human colon cells in culture [17] . Strains of E. coli harboring the pks island were found in higher frequency in mucosal samples from individuals suffering from CRC and IBD compared with the control group [18] .

Other pro-inflammatory gene products found in certain strains of E. coli include tcpC, which has been found to impair toll-like receptor (TLR) function and the cytotoxic necrotizing factor-1 (CNF-1), an activator of onco- genic Rho GTPases [19] - [21] . Additionally, there are certain strains of commensal Enterococcus species that are not pathogenic but contain a subset of the virulence genes including gelE—a gene encoding a collagen-degrading metalloprotease which has been found to induce inflammation in a mouse model [22] .

It has been established that these pro-inflammatory genes promote the colonization of pathogenic strains of bacteria. Substantially less clear is the effect of these pro-inflammatory genes when harbored by non-pathogenic commensal bacteria in the microbiota over a period of time [23] . Additionally, the distribution and frequency of most of these pro-inflammatory genes in the human population are not known and methods have not been implemented for the quick and direct detection of these genes in easily obtained stool samples.

In this work, we have developed a rapid assay for the detection of pro-inflammatory genes directly in a small stool sample (0.5 g). The assay can be performed in diagnostic format (classical PCR) or a quantitative format (rtPCR). Our results show that pro-inflammatory genes are highly prevalent (up to 20% of the samples tested) and in high enough copy number (up to 1.3 million copies per mg stool) for direct detection in a small stool sample. The assays described in this report could prove useful in future explorations of the relationship between the presence of these pro-inflammatory genes and intestinal diseases.

The elucidation of the role of the gut microbiota in human health is an active area of research [26] - [28] . A number of efforts worldwide have served to elucidate important associations between the microbiota and health [29] - [31] . For instance, an unequivocal link has been established between the gut microbiota and obesity [32] [33] . Additionally, the presence of pathogens such as Helicobacter pylori and Clostridium difficile has been found to correlate with inflammation and ultimately with the development of gastric cancer [14] [15] . More recently, there have been reports that the gram-negative bacterium of the genus Fusobacterium is more highly represented in colorectal tumor tissue than in normal mucosal tissue from the same patients [34] [35] .

In this report, instead of focusing on the presence of bacterial species, we have developed a quantitative assay to detect specific bacterial genes that have been associated with inflammation, directly in a small stool sample. Some of these genes in this report were found to be present in as many as 20% of the stool samples and in copy numbers as high as 1.3 million copies per mg of stool. The assay described in this report provides a quick method for the screening of stool samples to ascertain the presence of pro-inflammatory genes in case-control studies involving individuals with colorectal cancer or inflammatory bowel diseases. The genes in this report, namely pks island, tcpC, cnf-1, gelE and cdt, have all been the subject of scrutiny for their possible crosstalk with inflammatory processes. Here we discuss each of them separately:

pks Island Of the genes tested, the pks island was the most prevalent and was found in high copy numbers (>10⁶ copies per mg stool). The pks genomic island encodes a number of multidomain enzymes termed polyketide synthases that typically synthesize secondary metabolites in numerous microbial species, although the actual compound made by this multienzyme has not been chemically characterized [16] . The pks island has been found in pathogenic and in non-pathogenic strains of E. coli [23] . Although the pks island is not pro-inflamma- tory per se, the presence of this genomic island in some strains of E. coli has been implicated in the formation of colorectal tumors in inflammation-prone mice, which lack the inteleukin-10 gene [18] . That study also reported that the pks island was more highly represented in the intestinal mucosa from colorectal cancer (CRC) patients than in non-CRC controls [18] . In the present study, the pks island genes were found in 20% of the stool samples analyzed, a frequency consistent with previous findings reporting the pks island to be present in 20% of strains isolated from human mucosa samples from non-cancer donors [18] and in 32% of E. coli strains isolated from the feces of hospitalized veterans [23] . Although our assay is carried out on total DNA isolated from a stool sample, without strain isolation or cultivation, our results are similar to those obtained using the more conventional culture-dependent method.

tcpC Similarly, the tcpC gene has been found in strains of E. coli associated with severe kidney infection [36] . tcpC subverts toll-like receptor-dependent inflammation by binding directly to the receptor, resulting in the formation of kidney abscesses in mice infected with strains of uropathogenic E. coli [19] . However, there are no reports of the tcpC gene in the commensal E. coli strains that inhabit the GI tract. Much to our surprise, seven (7) of our fecal samples were found to harbor the tcpC gene in high enough copy number to detect by PCR.

cnf-1 The cnf-1 gene encodes an enzyme, the cytotoxic necrotizing factor-1, which is found in strains of E. coli commonly associated with urinary tract infections [37] . The Cnf enzyme deaminates Rho GTPase in glutamine 63 causing it to be locked in the active GTP-bound state. Although the cnf-1 gene has been found in bacterial isolates from feces of children with diarrhea, this is the first report of this gene in high enough copies to be detected directly in stool [38] . Only one (1) sample in the present study was found to contain the cnf-1 gene.

gelE GelE gene encodes a metallo-collagenase from the genus Enterococcus. The GelE collagenase has been found to promote chronic inflammation in mice lacking the interleukin-10 gene [22] . More recently, gelE gene was found in higher frequency in bacterial isolates from the feces of children with IBD vs. children without IBD [39] . Here we report 3 stool samples with detectable levels of gelE gene directly in stool.

With the growth of affordable parallel sequencing methods, scientists have gained a whole new view of the diversity of bacterial species (or taxa) in a complex community such as the gut microbiota [40] [41] . One important line of experimentation centers on building a comprehensive list of bacterial species present in the community for comparison between disease cases and controls. However, most community profiling efforts rely on molecular markers (such as the 16S rRNA gene) and do not necessarily detect additional genes that may have been acquired in time. For instance, the bacterial community profile for any stool sample would likely reveal the presence of E. coli, but it would not necessarily show whether the E. coli strains in the sample contain pro-in- flammatory genes (tcpC, pks island, etc.). In addition, E. coli is not the only species that harbor the pks island, as the genes can also be carried by other bacteria, such as Klebsiella pneumoniae, Enterobacter aerogenes and Citrobacter koseri [42] .

The assay described in this report is meant to complement ongoing efforts, whether by bacterial community profiling or by shotgun metagenomics, to ascertain the presence of harmful or protective genes, in fecal samples regardless of which species carries the genes. Clearly, other complementary assays will have to be developed to determine whether the pro-inflammatory bacterial genes are being transcribed in the gut, or whether the gene products are being synthesized in active form. But the development of a quick and quantitative assay for the detection of pro-inflammatory genes in stool samples is a first step toward the quick interrogation of stool samples in large-scale case-control studies involving IBD and CRC patients.

We report the detection, directly in DNA extracted from a fecal sample, of several genes of bacterial origin which had been previously associated with inflammation. Although the presence of these genes had been ascertained in strains of bacteria known to colonize other tissues, this is the first report of these genes being found in the normal commensal flora. The assay described in this report provides a quick and inexpensive method for the analysis of a large number of samples from case-control studies involving patients of IBD or CRC.

This publication was made possible by NSF grant CHE0953254 to AB-O and NIGMS grant R25GM061838 to RG-M. The laboratory also received support from an institutional grant to the Puerto Rico Clinical and Translational Consortium (PRCTRC: U54MD007587). Shared instrumentation was purchased with NIH Grant G12RR03051 (RCMI Program).

The authors also wish to thank Rosa I. Martínez, MT from Laboratorio Clínico El Monte in San Juan, Puerto Rico for the kind donation of stool sample remnants used in this study, and Dr. Luis Ríos-Hernández from the University of Puerto Rico in Mayagüez for providing the E. faecalis strains used as positive controls. The authors express gratitude to Carmelo Orengo, Vilmarie Mercado and Caleb Fernández for their technical support.