Multiple factors drive the progression from healthy mucosa towards sporadic colorectal carcinomas and accumulating evidence associates intestinal bacteria with disease initiation and progression. Therefore, the aim of this study was to provide a first high-resolution map of colonic dysbiosis that is associated with human colorectal cancer (CRC). To this purpose, the microbiomes colonizing colon tumor tissue and adjacent non-malignant mucosa were compared by deep rRNA sequencing. The results revealed striking differences in microbial colonization patterns between these two sites. Although inter-individual colonization in CRC patients was variable, tumors consistently formed a niche for Coriobacteria and other proposed probiotic bacterial species, while potentially pathogenic Enterobacteria were underrepresented in tumor tissue. As the intestinal microbiota is generally stable during adult life, these findings suggest that CRC-associated physiological and metabolic changes recruit tumor-foraging commensal-like bacteria. These microbes thus have an apparent competitive advantage in the tumor microenvironment and thereby seem to replace pathogenic bacteria that may be implicated in CRC etiology. This first glimpse of the CRC microbiome provides an important step towards full understanding of the dynamic interplay between intestinal microbial ecology and sporadic CRC, which may provide important leads towards novel microbiome-related diagnostic tools and therapeutic interventions.

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, b-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, b-2-microglobin, b-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal-iron, or high-iron diets and after two weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron-deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high-iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status and gut microbiota composition.

BACKGROUND:Intestinal bacteria have long been implicated in colorectal cancer pathology, and many reports point to a close linkage between Streptococcus bovis biotype I (recently renamed Streptococcus gallolyticus) infections and tumors of the human colon. This work aims to investigate the humoral immune response to this bacterium during different stages of colorectal cancer. METHODS: The presence of serum antibodies against S. bovis antigen RpL7/L12, previously assigned as a potential diagnostic antigen, was evaluated in Dutch (n ¼ 209) and American (n ¼ 112) populations using a newly developed enzyme-linked immunosorbent assay. RESULTS: The analyses consistently showed that an immune response against this bacterial antigen was increased in polyp patients and stage I/II colorectal cancer patients as compared with asymptomatic individuals. This was not paralleled by increased antibody production to endotoxin, an intrinsic cell wall component of the majority of intestinal bacteria, which implies that the humoral immune response against RpL7/L12 is not a general phenomenon induced by the loss of colonic barrier function. Notably, increased anti-RpL7/L12 levels were not or were only mildly detected in late stage colorectal cancer patients having lymph node or distant metastasis. CONCLUSIONS: These findings are indicative of an increased exposure to antigen RpL7/L12 during early stages of colon carcinogenesis and suggest that intestinal bacteria such as S. bovis constitute a risk factor for the progression of premalignant lesions into early stage carcinomas. Clearly, the current findings emphasize the necessity for further studies on the possible etiologic relationship between intestinal bacteria and human colorectal cancer. Cancer 2010;116:4014-22.

Streptococcus gallolyticus subsp. gallolyticus (previously called Streptococcus bovis biotype I) infections have long been associated with colorectal cancer (CRC). This work aimed to investigate the CRC-associated humoral immune response to four pilus proteins of this bacterium by newly developed ELISAs. Pilus proteins are interesting diagnostic targets as they are the building blocks of pilin-like structures that mediate bacterial virulence and are readily exposed to the host immune system upon infection. The presence of serum antibodies against these pilus proteins was evaluated in Dutch and American populations. These analyses showed that an immune response to these antigens was specific for clinical S. gallolyticus subsp. gallolyticus infections, but that increased serum antibody titers to multiple pilus proteins in single individuals were rarely observed. However, a multiplex approach based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis of S. gallolyticus subsp. gallolyticus–associated CRC.

Smoking and high red meat intake have been associated with colorectal cancer (CRC) risk. Increased iron exposure may be a common factor, favoring the colonization of certain bacterial pathogens that preferentially grow in an iron-rich luminal environment. We analyzed the data from a population-based case-control study of CRC and measured antibody levels against flagelin of Salmonella (FliC), one of the irontrophic bacteria, in two independent blood collections. The risk of CRC synergistically increased by combined exposures to heme iron intake and pack-years (PY) of cigarette smoking (P-value for the interaction = 0.039 on the continuous scale). There was a marginally significant interaction between heme iron intake and PY in increasing FliC antibody in the US control subjects (P=0.055), although no iron or smoking data were available for Dutch samples. Furthermore, FliC antibody levels were significantly higher in patients with colorectal polyps and cancer than in controls in both Dutch (3.93 vs. 2.23) (P=0.014) and US samples (6.65 vs. 4.37) (P<0.001). Potential roles of iron from cigarette smoking and dietary heme in CRC through altering irontrophic luminal bacterial population may warrant further investigation.

One of the few bacteria that have been consistently linked to colorectal cancer (CRC) is the opportunistic pathogen Streptococcus gallolyticus. Infections with this bacterium are generally regarded as an indicator for colonic malignancy, while the carriage rate of this bacterium in the healthy large intestine is relatively low. We speculated that the physiological changes accompanying the development of CRC might favor the colonization of this bacterium. To investigate whether colon tumor cells can support the survival of S. gallolyticus, this bacterium was grown in spent medium of malignant colonocytes to simulate the altered metabolic conditions in the CRC microenvironment. These in vitro simulations indicated that S. gallolyticus had a significant growth advantage in these spent media, which was not observed for other intestinal bacteria. Under these conditions, bacterial responses were profiled by proteome analysis and metabolic shifts were analyzed by 1 H-NMR-spectroscopy. In silico pathway analysis of the differentially expressed proteins and metabolite analysis indicated that this advantage resulted from the increased utilization of glucose, glucose derivates, and alanine. Together, these data suggest that tumor cell metabolites facilitate the survival of S. gallolyticus, favoring its local outgrowth and providing a possible explanation for the specific association of S. gallolyticus with colonic malignancy.

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