Annemarie Boleij

Towards the human colorectal cancer microbiome

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.

A bacterial driver–passenger model for colorectal cancer: beyond the usual suspects

Cancer has long been considered a genetic disease. However, accumulating evidence supports the involvement of infectious agents in the development of cancer, especially in those organs that are continuously exposed to microorganisms, such as the large intestine. Recent next-generation sequencing studies of the intestinal microbiota now offer an unprecedented view of the aetiology of sporadic colorectal cancer and have revealed that the microbiota associated with colorectal cancer contains bacterial species that differ in their temporal associations with developing tumours. Here, we propose a bacterial driver–passenger model for microbial involvement in the development of colorectal cancer and suggest that this model be incorporated into the genetic paradigm of cancer progression.

Clinical Importance of Streptococcus gallolyticus Infection Among Colorectal Cancer Patients: Systematic Review and Meta-analysis

BACKGROUND Streptococcus bovis has long been associated with colorectal cancer (CRC). However, not all genospecies are as closely related to CRC. With this systematic review, we aim to increase the awareness of the association between S. bovis biotype I (Streptococcus gallolyticus) and CRC and urge for uniform molecular microbiological classification. METHODS In January 2011, the PubMed database was searched for all studies that investigated the association between S. bovis, infective endocarditis (IE), and CRC. A total of 191 studies were screened for eligibility and yielded 52 case reports and 31 case series, of which 11 were used for meta-analysis on the association between S. bovis biotype, IE, and adenomas/carcinomas (CRC). RESULTS Among the S. bovis-infected patients who underwent colonic evaluation, the median percentage of patients who had concomitant adenomas/carcinomas was 60% (interquartile range, 22%), which largely exceeds the disease rate reported in the general asymptomatic population. Meta-analysis showed that patients with S. bovis biotype I infection had a strongly increased risk of having CRC (pooled odds ratio [OR], 7.26; 95% confidence interval [CI], 3.94-13.36) and IE (pooled OR, 16.61; 95% CI, 8.85-31.16), compared with S. bovis biotype II-infected patients. Notably, CRC occurred more often among patients with S. bovis IE than among patients with S. bovis infection at other sites (pooled OR, 3.72; 95% CI, 2.03-6.81). CONCLUSIONS Our meta-analysis clearly indicates that S. bovis should no longer be regarded as a single species in clinical practice, because S. gallolyticus (S. bovis biotype I) infection, in particular, has an unambiguous association with CRC.

The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients

BACKGROUND Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. METHODS We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. RESULTS The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). CONCLUSIONS The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Novel Clues on the Specific Association of Streptococcus gallolyticus subsp gallolyticus With Colorectal Cancer

BACKGROUND The prevalence of Streptococcus gallolyticus subsp gallolyticus ( Streptococcus bovis biotype I) endocarditis is in general low but very often linked to colorectal cancer. Therefore, this study aimed to reveal the virulence characteristics that distinguish this opportunistic pathogen from a panel of (closely related) intestinal bacteria. METHODS The route of infection was reconstructed in vitro with adhesion, invasion, and translocation assays on differentiated Caco-2 cells. Furthermore, cellular immune responses upon infection and bacterial biofilm formation were analyzed in a comparative manner. RESULTS S. gallolyticus subsp gallolyticus strains were demonstrated to have a relative low adhesiveness and could not internalize epithelial cells. However, these bacteria were uniquely able to paracellularly cross a differentiated epithelium without inducing epithelial interleukin 8 or 1β responses. Importantly, they had an outstanding ability to form biofilms on collagen-rich surfaces, which in vivo are found at damaged heart valves and (pre)cancerous sites with a displaced epithelium. CONCLUSIONS Together, these data show that S. gallolyticus subsp gallolyticus has a unique repertoire of virulence factors that facilitate infection through (pre)malignant colonic lesions and subsequently can provide this bacterium with a competitive advantage in (1) evading the innate immune system and (2) forming resistant vegetations at collagen-rich sites in susceptible patients with colorectal cancer.

Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface.

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.

Gut bacteria in health and disease: a survey on the interface between intestinal microbiology and colorectal cancer

A healthy human body contains at least tenfold more bacterial cells than human cells and the most abundant and diverse microbial community resides in the intestinal tract. Intestinal health is not only maintained by the human intestine itself and by dietary factors, but is also largely supported by this resident microbial community. Conversely, however, a large body of evidence supports a relationship between bacteria, bacterial activities and human colorectal cancer. Symbiosis in this multifaceted organ is thus crucial to maintain a healthy balance within the host‐diet‐microbiota triangle and accordingly, changes in any of these three factors may drive a healthy situation into a state of disease. In this review, the factors that sustain health or drive this complex intestinal system into dysbiosis are discussed. Emphasis is on the role of the intestinal microbiota and related mechanisms that can drive the initiation and progression of sporadic colorectal cancer (CRC). These mechanisms comprise the induction of pro‐inflammatory and pro‐carcinogenic pathways in epithelial cells as well as the production of (geno)toxins and the conversion of pro‐carcinogenic dietary factors into carcinogens. A thorough understanding of these processes will provide leads for future research and may ultimately aid in development of new strategies for CRC diagnosis and prevention.

The itinerary of Streptococcus gallolyticus infection in patients with colonic malignant disease

Bacteria constitute about 90% of all cells in the human body. The densest and most complex bacterial community is in the large intestine. This population is quite stable in healthy intestines, but intestinal disease distorts the ecological balance and induces dysbiosis. Results of studies have indicated that the epithelial and metabolic changes that occur with colorectal cancer provide a competitive advantage to a subset of intestinal bacteria. Strikingly, however, Streptococcus gallolyticus gallolyticus (previously known as Streptococcus bovis biotype I) is one of the very few opportunistic pathogens that has been clinically linked to colonic malignant diseases. In this Personal View we describe how S. gallolyticus gallolyticus exploits its unique range of virulence features to cause infections in patients with colorectal cancer. We postulate that distinct virulence factors on one hand enable this bacterium to establish a symptomatic infection in susceptible individuals, and on the other hand make its ability to do this dependent on pre-existing colonic abnormalities. We believe that our current reconstruction of this route of infection aids understanding of how S. gallolyticus gallolyticus infections can be best exploited for early detection of colorectal cancer.

Association between Streptococcus bovis and Colon Cancer

We read with great interest the article by Marcella Beck and colleagues (1). We fully support their recommendation that proper distinction between the “Streptococcus bovis” strains belonging to Streptococcus gallolyticus and Streptococcus infantarius (previously biotypes I and II/2, respectively) should be made in future studies to obtain a clear picture of the disease associations of these opportunistic pathogens; above all, because proper bacterial classification and subsequent recognition of their association with colon cancer can be a life-saving event for S. bovis-infected individuals with undiagnosed colon cancer (4, 5, 8). We were therefore somewhat puzzled by the authors’ conclusion that the association between S. bovis bacteremia and colon cancer (7%) may not be as strong as previously thought. Beck and colleagues based their conclusion on the finding that 3 out of 46 individuals with S. bovis bacteremia presented with a coincidental colon carcinoma. The authors recognize, however, that only 15 of these 46 patients underwent full bowel examination and that in the other patients asymptomatic colon tumors could be missed. In two cited papers, Ruoff et al. (6) and Corredoira et al. (2) report respective associations of 100% and 57% for the association of S. bovis biotype I and colon cancer in patients that underwent colonoscopy. Importantly, these associations also took into account the presence of premalignant adenomas that are generally regarded as (earlystage) precursors of carcinomas. In fact, Corredoira et al. (3)

Pharmacomicrobiomics: The Impact of Human Microbiome Variations on Systems Pharmacology and Personalized Therapeutics

The Human Microbiome Project (HMP) is a global initiative undertaken to identify and characterize the collection of human-associated microorganisms at multiple anatomic sites (skin, mouth, nose, colon, vagina), and to determine how intra-individual and inter-individual alterations in the microbiome influence human health, immunity, and different disease states. In this review article, we summarize the key findings and applications of the HMP that may impact pharmacology and personalized therapeutics. We propose a microbiome cloud model, reflecting the temporal and spatial uncertainty of defining an individuals microbiome composition, with examples of how intra-individual variations (such as age and mode of delivery) shape the microbiome structure. Additionally, we discuss how this microbiome cloud concept explains the difficulty to define a core human microbiome and to classify individuals according to their biome types. Detailed examples are presented on microbiome changes related to colorectal cancer, antibiotic administration, and pharmacomicrobiomics, or drug-microbiome interactions, highlighting how an improved understanding of the human microbiome, and alterations thereof, may lead to the development of novel therapeutic agents, the modification of antibiotic policies and implementation, and improved health outcomes. Finally, the prospects of a collaborative computational microbiome research initiative in Africa are discussed.