Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation.

BACKGROUND: Fecal microbiota transplantation (FMT) aims to cure Clostridioides difficile infection (CDI) through reestablishing a healthy microbiome and restoring colonization resistance. Although often effective after one infusion, patients with continued microbiome disruptions may require multiple FMTs. In this N-of-1 study, we use a systems biology approach to evaluate CDI in a patient receiving chronic suppressive antibiotics with four failed FMTs over two years. METHODS: Seven stool samples were obtained between 2016-18 while the patient underwent five FMTs. Stool samples were cultured for C. difficile and underwent microbial characterization and functional gene analysis using shotgun metagenomics. C. difficile isolates were characterized through ribotyping, whole genome sequencing, metabolic pathway analysis, and minimum inhibitory concentration (MIC) determinations. RESULTS: Growing ten strains from each sample, the index and first four recurrent cultures were single strain ribotype F078-126, the fifth was a mixed culture of ribotypes F002 and F054, and the final culture was ribotype F002. One single nucleotide polymorphism (SNP) variant was identified in the RNA polymerase (RNAP) ß-subunit RpoB in the final isolated F078-126 strain when compared to previous F078-126 isolates. This SNV was associated with metabolic shifts but phenotypic differences in fidaxomicin MIC were not observed. Microbiome differences were observed over time during vancomycin therapy and after failed FMTs. CONCLUSION: This study highlights the importance of antimicrobial stewardship in patients receiving FMT. Continued antibiotics play a destructive role on a transplanted microbiome and applies selection pressure for resistance to the few antibiotics available to treat CDI.

Gut Microbiome Signatures Are Predictive of Infectious Risk Following Induction Therapy for Acute Myeloid Leukemia.

BACKGROUND: The majority of studies that provide insights into the influence of the microbiome on the health of hematologic malignancy patients have concentrated on the transplant setting. Here, we sought to assess the predictive capacity of the gastrointestinal microbiome and its relationship to infectious outcomes in patients with acute myeloid leukemia (AML). METHODS: 16s rRNA-based analysis was performed on oral swabs and stool samples obtained biweekly from baseline until neutrophil recovery following induction chemotherapy (IC) in 97 AML patients. Microbiome characteristics were correlated with clinical outcomes both during and after IC completion. RESULTS: At the start of IC, higher stool Shannon diversity (hazard ratio [HR], 0.36; 95% confidence interval [CI], .18-.74) and higher relative abundance of Porphyromonadaceae (HR, 0.36; 95% CI, .18-.73) were associated with increased probability of remaining infection-free during neutropenia. A baseline stool Shannon diversity cutoff of <2 had optimal operating characteristics for predicting infectious complications during neutropenia. Although 56 patients received therapy >72 hours with a carbapenem, none of the patients had an infection with an extended spectrum ß-lactamase-producing organism. Patients who received carbapenems for >72 hours had significantly lower α-diversity at neutrophil recovery (P = .001) and were approximately 4 times more likely to have infection in the 90 days following neutrophil recovery (HR, 4.55; 95% CI, 1.73-11.93). CONCLUSIONS: Our results suggest that gut microbiome evaluation could assist with infectious risk stratification and that improved targeting of antibiotic administration during IC could decrease subsequent infectious complications in AML patients.Baseline microbiome diversity is a strong independent predictor of infection during acute myeloid leukemia induction chemotherapy (IC) among clinical and microbiome covariates. Higher baseline levels of Porphyromonadaceae appear protective against infection, while carbapenem use is associated with consequences to the microbiome and infection susceptibility post-IC.

Gut Microbiome Modulates Response to Cancer Immunotherapy.

With the advent of next-generation sequencing approaches, there has been a renaissance in the microbiome field. Microbial taxonomy and function can now be characterized relatively easily and rapidly-no longer mandating complex culturing approaches. With this renaissance, there is now a strong and growing appreciation for the role of the microbiome (referring to microbes and their genomes) in modulating many facets of physiology-including overall immunity. This is particularly true of the gut microbiome, and there is now an evolving body of the literature demonstrating a role for gut microbes in modulating responses to cancer treatment-particularly immunotherapy. Gut microbes can modulate immunity and anti-tumor responses via a number of different interactions, and these will be discussed herein. Additionally, data regarding the impact of gut microbes on cancer immunotherapy response will be discussed, as will strategies to manipulate the microbiome to enhance therapeutic responses. These efforts to date are not completely optimized; however, there is evidence of efficacy though much additional work is needed in this space. Nonetheless, it is clear that the microbiome plays a central role in health and disease, and strategies to manipulate it in cancer and overall precision health are being explored.

Microbial diversity changes with rhizosphere and hydrocarbons in contrasting soils.

In the ecotoxicological assessment of petroleum hydrocarbon-contaminated soil, microbial community profile is important aspect due to their involvement in soil functions. However, soil physicochemical properties and the inhabiting plants could dictate the microbial composition. A question remains unanswered is, how an integrated approach may be utilized to account for various contrasting soil properties, plant types (reference vs. native) and the nature of the hydrocarbon contamination. In this study, we utilized bacterial DNA profiling techniques to investigate the relationship between soil properties, contaminant and plant species. Results identified that Proteobacteria and Actinobacteria were the most abundant bacteria of the 45 phyla identified in the hydrocarbon-contaminated soil. The bulk and rhizosphere microbiome showed that the contaminated soil originally had quite distinct bacterial communities compared to the artificially contaminated soil (mine soil = 95 genera vs. other soils = 2-29 genera). In these cases, not significantly but the native plant slightly increased bacterial diversity and relative abundance in the same soils. Also, within each site, the bacterial community was significantly altered with the hydrocarbon concentration. In this instance, the influence of the contaminant was strong and also with the soil pH and organic matter. These results would significantly contribute to the novel insights on the molecular technique-based hydrocarbon toxicity assessment and the development of the further integrative approach with other microbial community and their metabolic profile in the contaminated sites.

Nodal immune flare mimics nodal disease progression following neoadjuvant immune checkpoint inhibitors in non-small cell lung cancer.

Radiographic imaging is the standard approach for evaluating the disease involvement of lymph nodes in patients with operable NSCLC although the impact of neoadjuvant immune checkpoint inhibitors (ICIs) on lymph nodes has not yet been characterized. Herein, we present an ad hoc analysis of the NEOSTAR trial (NCT03158129) where we observed a phenomenon we refer to as "nodal immune flare" (NIF) in which patients treated with neoadjuvant ICIs demonstrate radiologically abnormal nodes post-therapy that upon pathological evaluation are devoid of cancer and demonstrate de novo non-caseating granulomas. Abnormal lymph nodes are analyzed by computed tomography and 18F-fluorodeoxyglucose positron emission tomography/computer tomography to evaluate the size and the maximum standard uptake value post- and pre-therapy in NEOSTAR and an independent neoadjuvant chemotherapy cohort. NIF occurs in 16% (7/44) of patients treated with ICIs but in 0% (0/28) of patients after neoadjuvant chemotherapy. NIF is associated with an inflamed nodal immune microenvironment and with fecal abundance of genera belonging to the family Coriobacteriaceae of phylum Actinobacteria, but not with tumor responses or treatment-related toxicity. Our findings suggest that this apparent radiological cancer progression in lymph nodes may occur due to an inflammatory response after neoadjuvant immunotherapy, and such cases should be evaluated by pathological examination to distinguish NIF from true nodal progression and to ensure appropriate clinical treatment planning.

Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.

The gut microbiome has been shown to influence the response of tumors to anti-PD-1 (programmed cell death-1) immunotherapy in preclinical mouse models and observational patient cohorts. However, modulation of gut microbiota in cancer patients has not been investigated in clinical trials. In this study, we performed a phase 1 clinical trial to assess the safety and feasibility of fecal microbiota transplantation (FMT) and reinduction of anti-PD-1 immunotherapy in 10 patients with anti-PD-1-refractory metastatic melanoma. We observed clinical responses in three patients, including two partial responses and one complete response. Notably, treatment with FMT was associated with favorable changes in immune cell infiltrates and gene expression profiles in both the gut lamina propria and the tumor microenvironment. These early findings have implications for modulating the gut microbiota in cancer treatment.

Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Neoadjuvant nivolumab or nivolumab plus ipilimumab in operable non-small cell lung cancer: the phase 2 randomized NEOSTAR trial.

Ipilimumab improves clinical outcomes when combined with nivolumab in metastatic non-small cell lung cancer (NSCLC), but its efficacy and impact on the immune microenvironment in operable NSCLC remain unclear. We report the results of the phase 2 randomized NEOSTAR trial (NCT03158129) of neoadjuvant nivolumab or nivolumab + ipilimumab followed by surgery in 44 patients with operable NSCLC, using major pathologic response (MPR) as the primary endpoint. The MPR rate for each treatment arm was tested against historical controls of neoadjuvant chemotherapy. The nivolumab + ipilimumab arm met the prespecified primary endpoint threshold of 6 MPRs in 21 patients, achieving a 38% MPR rate (8/21). We observed a 22% MPR rate (5/23) in the nivolumab arm. In 37 patients resected on trial, nivolumab and nivolumab + ipilimumab produced MPR rates of 24% (5/21) and 50% (8/16), respectively. Compared with nivolumab, nivolumab + ipilimumab resulted in higher pathologic complete response rates (10% versus 38%), less viable tumor (median 50% versus 9%), and greater frequencies of effector, tissue-resident memory and effector memory T cells. Increased abundance of gut Ruminococcus and Akkermansia spp. was associated with MPR to dual therapy. Our data indicate that neoadjuvant nivolumab + ipilimumab-based therapy enhances pathologic responses, tumor immune infiltrates and immunologic memory, and merits further investigation in operable NSCLC.

Dietary simple sugars alter microbial ecology in the gut and promote colitis in mice.

The higher prevalence of inflammatory bowel disease (IBD) in Western countries points to Western diet as a possible IBD risk factor. High sugar, which is linked to many noncommunicable diseases, is a hallmark of the Western diet, but its role in IBD remains unknown. Here, we studied the effects of simple sugars such as glucose and fructose on colitis pathogenesis in wild-type and Il10-/- mice. Wild-type mice fed 10% glucose in drinking water or high-glucose diet developed severe colitis induced by dextran sulfate sodium. High-glucose-fed Il10-/- mice also developed a worsened colitis compared to glucose-untreated Il10-/- mice. Short-term intake of high glucose or fructose did not trigger inflammatory responses in healthy gut but markedly altered gut microbiota composition. In particular, the abundance of the mucus-degrading bacteria Akkermansia muciniphila and Bacteroides fragilis was increased. Consistently, bacteria-derived mucolytic enzymes were enriched leading to erosion of the colonic mucus layer of sugar-fed wild-type and Il10-/- mice. Sugar-induced exacerbation of colitis was not observed when mice were treated with antibiotics or maintained in a germ-free environment, suggesting that altered microbiota played a critical role in sugar-induced colitis pathogenesis. Furthermore, germ-free mice colonized with microbiota from sugar-treated mice showed increased colitis susceptibility. Together, these data suggest that intake of simple sugars predisposes to colitis and enhances its pathogenesis via modulation of gut microbiota in mice.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

The Tumor Microbiome in Pancreatic Cancer: Bacteria and Beyond.

There is increasing appreciation of the microbial influence on cancer, with emerging evidence about the tumor microbiome and crosstalk with the gut microbiome. A great example of this is pancreatic cancer, in which intra-tumoral microbes such as bacteria and fungi have been shown to contribute to carcinogenesis and therapeutic responses, both positively and negatively.

NLRP12 suppresses hepatocellular carcinoma via downregulation of cJun N-terminal kinase activation in the hepatocyte.

Hepatocellular carcinoma (HCC) is a deadly human cancer associated with chronic inflammation. The cytosolic pathogen sensor NLRP12 has emerged as a negative regulator of inflammation, but its role in HCC is unknown. Here we investigated the role of NLRP12 in HCC using mouse models of HCC induced by carcinogen diethylnitrosamine (DEN). Nlrp12-/- mice were highly susceptible to DEN-induced HCC with increased inflammation, hepatocyte proliferation, and tumor burden. Consistently, Nlrp12-/- tumors showed higher expression of proto-oncogenes cJun and cMyc and downregulation of tumor suppressor p21. Interestingly, antibiotics treatment dramatically diminished tumorigenesis in Nlrp12-/- mouse livers. Signaling analyses demonstrated higher JNK activation in Nlrp12-/- HCC and cultured hepatocytes during stimulation with microbial pattern molecules. JNK inhibition or NLRP12 overexpression reduced proliferative and inflammatory responses of Nlrp12-/- hepatocytes. In summary, NLRP12 negatively regulates HCC pathogenesis via downregulation of JNK-dependent inflammation and proliferation of hepatocytes.

Burkholderia pseudomallei-absent soil bacterial community results in secondary metabolites that kill this pathogen.

Burkholderia pseudomallei is a Gram-negative bacterium found in soil and the causative agent of a severe disease in humans and animals known as melioidosis. It is intrinsically resistant to many antibiotics and has been reported resistant to the drugs of choice; ceftazidime. Microbial communities in soil in the presence and absence of B. pseudomallei were investigated using metagenomics approach. The variation in bacterial species diversity was significantly higher in soil samples without B. pseudomallei. Abundances of phyla Actinobacteria and Firmicutes were found significantly higher in B. pseudomallei-negative soils. Bacillus amyloliquefaciens KKU1 in phylum Firmicutes was discovered from negative soil and its secondary metabolites could inhibit clinical, environmental and drug resistant isolates of B. pseudomallei, together with some pathogenic Gram-negative but not Gram-positive bacteria. The antimicrobial activity from KKU 1 against B. pseudomallei was abolished when treated with proteinase K, stable in a wide range of pH and remained active after heating at 100 °C for 15 min. Precipitated proteins from KKU1 were demonstrated to cause lysis and corrugated surfaces of B. pseudomallei. The minimum inhibitory concentrations and minimum bactericidal concentrations of the precipitated proteins from KKU1 against B. pseudomallei were 0.97 µg/ml and 3.9 µg/ml. Interestingly, Native SDS-PAGE showed small active compounds of less than 6 kDa, along with other information collectively suggesting the properties of antimicrobial peptides. For the first time, culture-independent information in melioidosis endemic area could lead to a suspected source of metabolites that may help defense against B. pseudomallei and other pathogenic Gram-negative bacteria.