Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages.

Gut phages have an important impact on human health. Methylation plays key roles in DNA recognition, gene expression regulation and replication for phages. However, the DNA methylation landscape of gut phages is largely unknown. Here, with PacBio sequencing (2120×, 4785 Gb), we detected gut phage methylation landscape based on 22 673 gut phage genomes, and presented diverse methylation motifs and methylation differences in genomic elements. Moreover, the methylation rate of phages was associated with taxonomy and host, and N6-methyladenine methylation rate was higher in temperate phages than in virulent phages, suggesting an important role for methylation in phage-host interaction. In particular, 3543 (15.63%) phage genomes contained restriction-modification system, which could aid in evading clearance by the host. This study revealed the DNA methylation landscape of gut phage and its potential roles, which will advance the understanding of gut phage survival and human health.

Squalene epoxidase drives cancer cell proliferation and promotes gut dysbiosis to accelerate colorectal carcinogenesis.

OBJECTIVE: Aberrant lipid metabolism is a hallmark of colorectal cancer (CRC). Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is upregulated in CRC. Here, we aim to determine oncogenic function of SQLE and its interplay with gut microbiota in promoting colorectal tumourigenesis. DESIGN: Paired adjacent normal tissues and CRC from two cohorts were analysed (n=202). Colon-specific Sqle transgenic (Sqle tg) mice were generated by crossing Rosa26-lsl-Sqle mice to Cdx2-Cre mice. Stools were collected for metagenomic and metabolomic analyses. RESULTS: SQLE messenger RNA and protein expression was upregulated in CRC (p<0.01) and predict poor survival of patients with CRC. SQLE promoted CRC cell proliferation by inducing cell cycle progression and suppressing apoptosis. In azoxymethane-induced CRC model, Sqle tg mice showed increased tumourigenesis compared with wild-type mice (p<0.01). Integrative metagenomic and metabolomic analyses unveiled gut dysbiosis in Sqle tg mice with enriched pathogenic bacteria, which was correlated to increased secondary bile acids. Consistent with detrimental effect of secondary bile acids, gut barrier function was impaired in Sqle tg mice, with reduced tight junction proteins Jam-c and occludin. Transplantation of Sqle tg mice stool to germ-free mice impaired gut barrier function and stimulated cell proliferation compared with control mice stool. Finally, we demonstrated that terbinafine, a SQLE inhibitor, could be repurposed for CRC by synergising with oxaliplatin and 5-fluorouracil to inhibit CRC growth. CONCLUSION: This study demonstrates that SQLE mediates oncogenesis via cell intrinsic effects and modulation of gut microbiota-metabolite axis. SQLE represents a therapeutic target and prognostic marker in CRC.

Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites.

OBJECTIVE: Cigarette smoking is a major risk factor for colorectal cancer (CRC). We aimed to investigate whether cigarette smoke promotes CRC by altering the gut microbiota and related metabolites. DESIGN: Azoxymethane-treated C57BL/6 mice were exposed to cigarette smoke or clean air 2 hours per day for 28 weeks. Shotgun metagenomic sequencing and liquid chromatography mass spectrometry were parallelly performed on mice stools to investigate alterations in microbiota and metabolites. Germ-free mice were transplanted with stools from smoke-exposed and smoke-free control mice. RESULTS: Mice exposed to cigarette smoke had significantly increased tumour incidence and cellular proliferation compared with smoke-free control mice. Gut microbial dysbiosis was observed in smoke-exposed mice with significant differential abundance of bacterial species including the enrichment of Eggerthella lenta and depletion of Parabacteroides distasonis and Lactobacillus spp. Metabolomic analysis showed increased bile acid metabolites, especially taurodeoxycholic acid (TDCA) in the colon of smoke-exposed mice. We found that E. lenta had the most positive correlation with TDCA in smoke-exposed mice. Moreover, smoke-exposed mice manifested enhanced oncogenic MAPK/ERK (mitogen-activated protein kinase/extracellular signal­regulated protein kinase 1/2) signalling (a downstream target of TDCA) and impaired gut barrier function. Furthermore, germ-free mice transplanted with stools from smoke-exposed mice (GF-AOMS) had increased colonocyte proliferation. Similarly, GF-AOMS showed increased abundances of gut E. lenta and TDCA, activated MAPK/ERK pathway and impaired gut barrier in colonic epithelium. CONCLUSION: The gut microbiota dysbiosis induced by cigarette smoke plays a protumourigenic role in CRC. The smoke-induced gut microbiota dysbiosis altered gut metabolites and impaired gut barrier function, which could activate oncogenic MAPK/ERK signalling in colonic epithelium.

Streptococcus thermophilus Inhibits Colorectal Tumorigenesis Through Secreting ß-Galactosidase.

BACKGROUND & AIMS: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention. METHODS: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by ß-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing. RESULTS: Oral gavage of S thermophilus significantly reduced tumor formation in both Apcmin/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. ß-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and ß-galactosidase activity assay. ß-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional ß-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via ß-galactosidase. ß-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus. CONCLUSION: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of ß-galactosidase.

Non-bacteria microbiome (virus, fungi, and archaea) in gastrointestinal cancer.

The gastrointestinal tract houses millions of microbes collectively referred to as the gut microbiome. The gut microbes comprise of bacteria, viruses, fungi, archaea, and microscopic eukaryotes, which co-evolved or colonize the gut forming complex symbiotic and mutualistic relationships. A state of homeostasis is required between host and gut microbiome relationship to maintain several host beneficial processes. Alterations in the taxonomic and functional composition of the gut microbes are associated with several human diseases including gastrointestinal cancers. Owed to their overwhelming abundance and ease of characterization, several studies focus on the role of bacteria in gastrointestinal cancers. There is however growing evidence that non-bacteria gut microbes are associated with the pathogenesis of gastrointestinal cancers. This review details the association of non-bacteria gut microbes including fungi, viruses, and archaea and their potential manipulation in the prevention and treatment of human gastrointestinal cancers.

Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is an increasing healthcare burden worldwide. We examined the role of dietary cholesterol in driving NAFLD-HCC through modulating gut microbiota and its metabolites. DESIGN: High-fat/high-cholesterol (HFHC), high-fat/low-cholesterol or normal chow diet was fed to C57BL/6 male littermates for 14 months. Cholesterol-lowering drug atorvastatin was administered to HFHC-fed mice. Germ-free mice were transplanted with stools from mice fed different diets to determine the direct role of cholesterol modulated-microbiota in NAFLD-HCC. Gut microbiota was analysed by 16S rRNA sequencing and serum metabolites by liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. Faecal microbial compositions were examined in 59 hypercholesterolemia patients and 39 healthy controls. RESULTS: High dietary cholesterol led to the sequential progression of steatosis, steatohepatitis, fibrosis and eventually HCC in mice, concomitant with insulin resistance. Cholesterol-induced NAFLD-HCC formation was associated with gut microbiota dysbiosis. The microbiota composition clustered distinctly along stages of steatosis, steatohepatitis and HCC. Mucispirillum, Desulfovibrio, Anaerotruncus and Desulfovibrionaceae increased sequentially; while Bifidobacterium and Bacteroides were depleted in HFHC-fed mice, which was corroborated in human hypercholesteremia patients. Dietary cholesterol induced gut bacterial metabolites alteration including increased taurocholic acid and decreased 3-indolepropionic acid. Germ-free mice gavaged with stools from mice fed HFHC manifested hepatic lipid accumulation, inflammation and cell proliferation. Moreover, atorvastatin restored cholesterol-induced gut microbiota dysbiosis and completely prevented NAFLD-HCC development. CONCLUSIONS: Dietary cholesterol drives NAFLD-HCC formation by inducing alteration of gut microbiota and metabolites in mice. Cholesterol inhibitory therapy and gut microbiota manipulation may be effective strategies for NAFLD-HCC prevention.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis.

OBJECTIVE: Using faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis. DESIGN: The tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry. RESULTS: L. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo. CONCLUSION: L. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

Altered Gut Archaea Composition and Interaction With Bacteria Are Associated With Colorectal Cancer.

BACKGROUND & AIMS: Changes in the intestinal microbiota have been associated with development and progression of colorectal cancer (CRC). Archaea are stable components of the microbiota, but little is known about their composition or contribution to colorectal carcinogenesis. We analyzed archaea in fecal microbiomes of 2 large cohorts of patients with CRC. METHODS: We performed shotgun metagenomic analyses of fecal samples from 585 participants (184 patients with CRC, 197 patients with adenomas, and 204 healthy individuals) from discovery (165 individuals) and validation (420 individuals) cohorts. Assignment of taxonomies was performed by exact k-mer alignment against an integrated microbial reference genome database. RESULTS: Principal component analysis of archaeomes showed distinct clusters in fecal samples from patients with CRC, patients with adenomas, and control individuals (P < .001), indicating an alteration in the composition of enteric archaea during tumorigenesis. Fecal samples from patients with CRC had significant enrichment of halophilic and depletion of methanogenic archaea. The halophilic Natrinema sp. J7-2 increased progressively in samples from control individuals, to patients with adenomas, to patients with CRC. Abundances of 9 archaea species that were enriched in fecal samples from patients with CRC distinguished them from control individuals with areas under the receiver operating characteristic curve of 0.82 in the discovery cohort and 0.83 in the validation cohort. An association between archaea and bacteria diversities was observed in fecal samples from control individuals but not from patients with CRC. Archaea that were enriched in fecal samples from patients with CRC had an extensive mutual association with bacteria that were enriched in the same samples and exclusivity with bacteria that were lost from these samples. CONCLUSIONS: Archaeomes of fecal samples from patients with CRC are characterized by enrichment of halophiles and depletion of methanogens. Studies are needed to determine whether associations between specific archaea and bacteria species in samples from patients with CRC contribute to or are a response to colorectal tumorigenesis.

Aspirin Reduces Colorectal Tumor Development in Mice and Gut Microbes Reduce its Bioavailability and Chemopreventive Effects.

BACKGROUND & AIMS: Alterations in the intestinal microbiota affect development of colorectal cancer and drug metabolism. We studied whether the intestinal microbiota affect the ability of aspirin to reduce colon tumor development in mice. METHODS: We performed studies with APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium to induce colorectal carcinogenesis. Some mice were given antibiotics to deplete intestinal microbes, with or without aspirin, throughout the entire experiment. Germ-free mice were studied in validation experiments. Colon tissues were collected and analyzed by histopathology, quantitative reverse-transcription polymerase chain reaction, and immunoblots. Blood samples and gut luminal contents were analyzed by liquid chromatography/mass spectrometry and an arylesterase activity assay. Fecal samples were analyzed by 16S ribosomal RNA gene and shotgun metagenome sequencing. RESULTS: Administration of aspirin to mice reduced colorectal tumor number and load in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium that had been given antibiotics (depleted gut microbiota), but not in mice with intact microbiota. Germ-free mice given aspirin developed fewer colorectal tumors than conventionalized germ-free mice given aspirin. Plasma levels of aspirin were higher in mice given antibiotics than in mice with intact gut microbiota. Analyses of luminal contents revealed that aerobic gut microbes, including Lysinibacillus sphaericus, degrade aspirin. Germ-free mice fed L sphaericus had lower plasma levels of aspirin than germ-free mice that were not fed this bacterium. There was an inverse correlation between aspirin dose and colorectal tumor development in conventional mice, but this correlation was lost with increased abundance of L sphaericus. Fecal samples from mice fed aspirin were enriched in Bifidobacterium and Lactobacillus genera, which are considered beneficial, and had reductions in Alistipes finegoldii and Bacteroides fragili, which are considered pathogenic. CONCLUSIONS: Aspirin reduces development of colorectal tumors in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium, depending on the presence of intestinal microbes. L sphaericus in the gut degrades aspirin and reduced its chemopreventive effects in mice. Fecal samples from mice fed aspirin were enriched in beneficial bacteria, with reductions in pathogenic bacteria.

Gastric microbes associated with gastric inflammation, atrophy and intestinal metaplasia 1 year after Helicobacter pylori eradication.

OBJECTIVE: Helicobacter pylori is associated with gastric inflammation, precancerous gastric atrophy (GA) and intestinal metaplasia (IM). We aimed to identify microbes that are associated with progressive inflammation, GA and IM 1 year after H. pylori eradication. DESIGN: A total of 587 H. pylori-positive patients were randomised to receive H. pylori eradication therapy (295 patients) or placebo (292 patients). Bacterial taxonomy was analysed on 404 gastric biopsy samples comprising 102 pairs before and after 1 year H. pylori eradication and 100 pairs before and after 1 year placebo by 16S rRNA sequencing. RESULTS: Analysis of microbial sequences confirmed the eradication of H. pylori in treated group after 1 year. Principal component analysis revealed distinct microbial clusters reflected by increase in bacterial diversity (p<0.00001) after H. pylori eradication. While microbial interactions remained largely unchanged after placebo treatment, microbial co-occurrence was less in treated group. Acinetobacter lwoffii, Streptococcus anginosus and Ralstonia were enriched while Roseburia and Sphingomonas were depleted in patients with persistent inflammation 1 year after H. pylori eradication. A distinct cluster of oral bacteria comprising Peptostreptococcus, Streptococcus, Parvimonas, Prevotella, Rothia and Granulicatella were associated with emergence and persistence of GA and IM. Probiotic Faecalibacterium praustznii was depleted in subjects who developed GA following H. pylori eradication. Functional pathways including amino acid metabolism and inositol phosphate metabolism were enriched while folate biosynthesis and NOD-like receptor signalling decreased in atrophy/IM-associated gastric microbiota. CONCLUSION: This study identified that gastric microbes contribute to the progression of gastric carcinogenesis after H. pylori eradication.

Enteric fungal microbiota dysbiosis and ecological alterations in colorectal cancer.

OBJECTIVES: Bacteriome and virome alterations are associated with colorectal cancer (CRC). Nevertheless, the gut fungal microbiota in CRC remains largely unexplored. We aimed to characterise enteric mycobiome in CRC. DESIGN: Faecal shotgun metagenomic sequences of 184 patients with CRC, 197 patients with adenoma and 204 control subjects from Hong Kong were analysed (discovery cohort: 73 patients with CRC and 92 control subjects; validation cohort: 111 patients with CRC, 197 patients with adenoma and 112 controls from Hong Kong). CRC-associated fungal markers and ecological changes were also validated in additional independent cohorts of 90 patients with CRC, 42 patients with adenoma and 66 control subjects of published repository sequences from Germany and France. Assignment of taxonomies was performed by exact k-mer alignment against an integrated microbial reference genome database. RESULTS: Principal component analysis revealed separate clusters for CRC and control (p<0.0001), with distinct mycobiomes in early-stage and late-stage CRC (p=0.0048). Basidiomycota:Ascomycota ratio was higher in CRC (p=0.0042), with increase in Malasseziomycetes (p<0.0001) and decrease in Saccharomycetes (p<0.0001) and Pneumocystidomycetes (p=0.0017). Abundances of 14 fungal biomarkers distinguished CRC from controls with an area under the receiver-operating characteristic curve (AUC) of 0.93 and validated AUCs of 0.82 and 0.74 in independent Chinese cohort V1 and European cohort V2, respectively. Further ecological analysis revealed higher numbers of co-occurring fungal intrakingdom and co-exclusive bacterial-fungal correlations in CRC (p<0.0001). Moreover, co-occurrence interactions between fungi and bacteria, mostly contributed by fungal Ascomycota and bacterial Proteobacteria in control, were reverted to co-exclusive interplay in CRC (p=0.00045). CONCLUSIONS: This study revealed CRC-associated mycobiome dysbiosis characterised by altered fungal composition and ecology, signifying that the gut mycobiome might play a role in CRC.

Alterations in Enteric Virome Are Associated With Colorectal Cancer and Survival Outcomes.

BACKGROUND & AIMS: Patients with colorectal cancer (CRC) have a different gut microbiome signature than individuals without CRC. Little is known about the viral component of CRC-associated microbiome. We aimed to identify and validate viral taxonomic markers of CRC that might be used in detection of the disease or predicting outcome. METHODS: We performed shotgun metagenomic analyses of viromes of fecal samples from 74 patients with CRC (cases) and 92 individuals without CRC (controls) in Hong Kong (discovery cohort). Viral sequences were classified by taxonomic alignment against an integrated microbial reference genome database. Viral markers associated with CRC were validated using fecal samples from 3 separate cohorts: 111 patients with CRC and 112 controls in Hong Kong, 46 patients with CRC and 63 controls in Austria, and 91 patients with CRC and 66 controls in France and Germany. Using abundance profiles of CRC-associated virome genera, we constructed random survival forest models to identify those associated with patient survival times. RESULTS: The diversity of the gut bacteriophage community was significantly increased in patients with CRC compared with controls. Twenty-two viral taxa discriminated cases from controls with an area under the receiver operating characteristic curve of 0.802 in the discovery cohort. The viral markers were validated in 3 cohorts, with area under the receiver operating characteristic curves of 0.763, 0.736, and 0.715, respectively. Clinical subgroup analysis showed that dysbiosis of the gut virome was associated with early- and late-stage CRC. A combination of 4 taxonomic markers associated with reduced survival of patients with CRC (log-rank test, P = 8.1 × 10-6) independently of tumor stage, lymph node metastases, or clinical parameters. We found altered interactions between bacteriophages and oral bacterial commensals in fecal samples from patients with CRC compared with controls. CONCLUSIONS: In a metagenomic analysis of fecal samples from patients and controls, we identified virome signatures associated with CRC. These data might be used to develop tools to identify individuals with CRC or predict outcomes.

Mucosal microbiome dysbiosis in gastric carcinogenesis.

OBJECTIVES: We aimed to characterise the microbial changes associated with histological stages of gastric tumourigenesis. DESIGN: We performed 16S rRNA gene analysis of gastric mucosal samples from 81 cases including superficial gastritis (SG), atrophic gastritis (AG), intestinal metaplasia (IM) and gastric cancer (GC) from Xi'an, China, to determine mucosal microbiome dysbiosis across stages of GC. We validated the results in mucosal samples of 126 cases from Inner Mongolia, China. RESULTS: We observed significant mucosa microbial dysbiosis in IM and GC subjects, with significant enrichment of 21 and depletion of 10 bacterial taxa in GC compared with SG (q<0.05). Microbial network analysis showed increasing correlation strengths among them with disease progression (p<0.001). Five GC-enriched bacterial taxa whose species identifications correspond to Peptostreptococcus stomatis, Streptococcus anginosus, Parvimonas micra, Slackia exigua and Dialister pneumosintes had significant centralities in the GC ecological network (p<0.05) and classified GC from SG with an area under the receiver-operating curve (AUC) of 0.82. Moreover, stronger interactions among gastric microbes were observed in Helicobacter pylori-negative samples compared with H. pylori-positive samples in SG and IM. The fold changes of selected bacteria, and strengths of their interactions were successfully validated in the Inner Mongolian cohort, in which the five bacterial markers distinguished GC from SG with an AUC of 0.81. CONCLUSIONS: In addition to microbial compositional changes, we identified differences in bacterial interactions across stages of gastric carcinogenesis. The significant enrichments and network centralities suggest potentially important roles of P. stomatis, D. pneumosintes, S. exigua, P. micra and S. anginosus in GC progression.

Whole genome sequence analysis of multidrug-resistant Mycobacterium tuberculosis Beijing isolates from an outbreak in Thailand.

The Mycobacterium tuberculosis Beijing family is often associated with multidrug resistance and large outbreaks. Conventional genotyping study of a community outbreak of multidrug-resistant tuberculosis (MDR-TB) that occurred in Kanchanaburi Province, Thailand was carried out. The study revealed that the outbreak was clonal and the strain was identified as a member of Beijing family. Although, the outbreak isolates showed identical spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeats patterns, a discrepancy regarding ethambutol resistance was observed. In-depth characterization of the isolates through whole genome sequencing of the first and the last three isolates from our culture collection showed them to belong to principal genetic group 1, single nucleotide polymorphism (SNP) cluster group 2, sequence type 10. Compared with the M. tuberculosis H37Rv reference genome, 1242 SNPs were commonly found in all isolates. The genomes of these isolates were shown to be clonal and highly stable over a 5-year period and two or three unique SNPs were identified in each of the last three isolates. Genes known to be associated with drug resistance and their promoter regions, where applicable, were analyzed. The presence of low or no fitness cost mutations for drug resistance and an additional L731P SNP in the rpoB gene was observed in all isolates. These findings might account for the successful transmission of this MDR-TB strain.

Blood microbial signatures associated with mortality in patients with sepsis: A pilot study.

Sepsis is a life-threatening illness caused by the dysregulated host response to infection. Nevertheless, our current knowledge of the microbial landscape in the blood of septic patients is still limited. Next-generation sequencing (NGS) is a sensitive method to quantitatively characterize microbiomes at various sites of the human body. In this study, we analyzed the blood microbial DNA of 22 adult patients with sepsis and 3 healthy subjects. The presence of non-human DNA was identified in both healthy and septic subjects. Septic patients had a markedly altered microbial DNA profile compared to healthy subjects over α- and ß-diversity. Unexpectedly, the patients could be further divided into two subgroups (C1 and C2) based on ß-diversity analysis. C1 patients showed much higher bacteria, viruses, fungi, and archaea abundance, and a higher level of α-diversity (Chao1, Observed and Shannon index) than both C2 patients and healthy subjects. The most striking difference was seen in the case of Streptomyces violaceusniger, Phenylobacterium sp. HYN0004, Caulobacter flavus, Streptomyces sp. 11-1-2, and Phenylobacterium zucineum, the abundance of which was the highest in the C1 group. Notably, C1 patients had a significantly poorer outcome than C2 patients. Moreover, by analyzing the patterns of microbe-microbe interactions in healthy and septic subjects, we revealed that C1 and C2 patients exhibited distinct co-occurrence and co-exclusion relationships. Together, our study uncovered two distinct microbial signatures in the blood of septic patients. Compositional and ecological analysis of blood microbial DNA may thus be useful in predicting mortality of septic patients.

Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis.

BACKGROUND: Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is 'RHA' unlike 'RHG' found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. RESULTS: Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. CONCLUSION: Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.

Altered gut microbiome composition by appendectomy contributes to colorectal cancer.

Appendectomy impacts the homeostasis of gut microbiome in patients. We aimed to study the role of appendectomy in colorectal cancer (CRC) risk through causing gut microbial dysbiosis. Population-based longitudinal study (cohort 1, n = 129,155) showed a 73.0% increase in CRC risk among appendectomy cases throughout 20 years follow-up (Adjusted sub-distribution hazard ratio (SHR) 1.73, 95% CI 1.49-2.01, P < 0.001). Shotgun metagenomic sequencing was performed on fecal samples from cohort 2 (n = 314). Gut microbial dysbiosis in appendectomy subjects was observed with significant enrichment of 7 CRC-promoting bacteria (Bacteroides vulgatus, Bacteroides fragilis, Veillonella dispar, Prevotella ruminicola, Prevotella fucsa, Prevotella dentalis, Prevotella denticola) and depletion of 5 beneficial commensals (Blautia sp YL58, Enterococcus hirae, Lachnospiraceae bacterium Choco86, Collinsella aerofaciens, Blautia sp SC05B48). Microbial network analysis showed increased correlation strengths among enriched bacteria and their enriched oncogenic pathways in appendectomy subjects compared to controls. Of which, B. fragilis was the centrality in the network of the enriched bacteria. We further confirmed that appendectomy promoted colorectal tumorigenesis in mice by causing gut microbial dysbiosis and impaired intestinal barrier function. Collectively, this study revealed appendectomy-induced microbial dysbiosis characterized by enriched CRC-promoting bacteria and depleted beneficial commensals, signifying that the gut microbiome may play a crucial role in CRC development induced by appendectomy.

Altered gut metabolites and microbiota interactions are implicated in colorectal carcinogenesis and can be non-invasive diagnostic biomarkers.

BACKGROUND: Gut microbiota contributes to colorectal cancer (CRC) pathogenesis through microbes and their metabolites. The importance of microbiota-associated metabolites in colorectal carcinogenesis highlights the need to investigate the gut metabolome along the adenoma-carcinoma sequence to determine their mechanistic implications in the pathogenesis of CRC. To date, how and which microbes and metabolites interactively promote early events of CRC development are still largely unclear. We aim to determine gut microbiota-associated metabolites and their linkage to colorectal carcinogenesis. RESULTS: We performed metabolomics and metagenomics profiling on fecal samples from 386 subjects including 118 CRC patients, 140 colorectal adenomas (CRA) patients and 128 healthy subjects as normal controls (NC). We identified differences in the gut metabolite profiles among NC, CRA and CRC groups by partial least squares-discriminant and principal component analyses. Among the altered metabolites, norvaline and myristic acid showed increasing trends from NC, through CRA, to CRC. CRC-associated metabolites were enriched in branched-chain amino acids, aromatic amino acids and aminoacyl-tRNA biosynthesis pathways. Moreover, metabolites marker signature (twenty metabolites) classified CRC from NC subjects with an area under the curve (AUC) of 0.80, and CRC from CRA with an AUC of 0.79. Integrative analyses of metabolomics and metagenomics profiles demonstrated that the relationships among CRC-associated metabolites and bacteria were altered across CRC stages; certain associations exhibited increasing or decreasing strengths while some were reversed from negative to positive or vice versa. Combinations of gut bacteria with the metabolite markers improved their diagnostic performances; CRC vs NC, AUC: 0.94; CRC vs CRA, AUC 0.92; and CRA vs NC, AUC: 0.86, indicating a potential for early diagnosis of colorectal neoplasia. CONCLUSIONS: This study underscores potential early-driver metabolites in stages of colorectal tumorigenesis. The Integrated metabolite and microbiome analysis demonstrates that gut metabolites and their association with gut microbiota are perturbed along colorectal carcinogenesis. Fecal metabolites can be utilized, in addition to bacteria, for non-invasive diagnosis of colorectal neoplasia. Video Abstract.

Parvimonas micra promotes colorectal tumorigenesis and is associated with prognosis of colorectal cancer patients.

Large-scale fecal shotgun metagenomic sequencing revealed the high abundance of Parvimonas micra in colorectal cancer (CRC) patients. We investigated the role and clinical significance of P. micra in colorectal tumorigenesis. The abundance of P. micra was examined in 309 fecal samples and 165 colon biopsy tissues of CRC patients and healthy subjects. P. micra was significantly enriched in fecal samples from 128 CRC patients compared to 181 healthy subjects (P < 0.0001); and in colon tissue biopsies from 52 CRC patients compared to 61 healthy subjects (P < 0.0001). Multivariate analysis showed that P. micra is an independent risk factor of poor survival in CRC patients (Hazard Ratio: 1.93). P. micra strain was isolated from feces of a CRC patient. Apcmin/+ mice gavaged with P. micra showed significantly higher tumor burden and tumor load (both P < 0.01). Consistently, gavage of P. micra significantly promoted colonocyte proliferation in conventional mice, which was further confirmed by germ-free mice. P. micra colonization up-regulated genes involved in cell proliferation, stemness, angiogenesis and invasiveness/metastasis; and enhanced Th17 cells infiltration and expression of Th17 cells-secreted cytokines (Il-17, Il-22, and Il-23) in the colon of Apcmin/+, conventional and germ-free mice. P. micra-conditioned medium significantly promoted the differentiation of CD4+ T cells to Th17 cells (IL-17+CD4+ phenotype) and enhanced the oncogenic Wnt signaling pathway. In conclusion, P. micra promoted colorectal tumorigenesis in mice by inducing colonocyte proliferation and altering Th17 immune response. P. micra may act as a prognostic biomarker for poor survival of CRC patients.