Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Multifaceted genome control by Set1 Dependent and Independent of H3K4 methylation and the Set1C/COMPASS complex.

Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress Tf2 retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (mat) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the mat locus and subtelomeres while dispensing Set1C in repressing Tf2s and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of Saccharomyces cerevisiae orthologs. Moreover, unlike S. cerevisiae, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

Gut Microbial Species and Endotypes Associate with Remission in Ulcerative Colitis Patients Treated with Anti-TNF or Anti-integrin Therapy.

BACKGROUND AND AIMS: The gut microbiota contributes to aberrant inflammation in inflammatory bowel disease, but the bacterial factors causing or exacerbating inflammation are not fully understood. Further, the predictive or prognostic value of gut microbial biomarkers for remission in response to biologic therapy is unclear. METHODS: We perform whole metagenomic sequencing of 550 stool samples from 287 ulcerative colitis patients from a large, phase 3, head-to-head study of infliximab and etrolizumab. RESULTS: We identify several bacterial species in baseline and/or post-treatment samples that associate with clinical remission. These include previously described associations [Faecalibacterium prausnitzii_F] as well as new associations with remission to biologic therapy [Flavonifractor plautii]. We build multivariate models and find that gut microbial species are better predictors for remission than clinical variables alone. Finally, we describe patient groups that differ in microbiome composition and remission rate after induction therapy, suggesting the potential utility of microbiome-based endotyping. CONCLUSIONS: In this large study of ulcerative colitis patients, we show that few individual species associate strongly with clinical remission, but multivariate models including microbiome can predict clinical remission and have better predictive power compared with clinical data alone.

Toxigenic Clostridium perfringens Isolated from At-Risk Paediatric Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Short- and long-read metagenomics of urban and rural South African gut microbiomes reveal a transitional composition and undescribed taxa.

Human gut microbiome research focuses on populations living in high-income countries and to a lesser extent, non-urban agriculturalist and hunter-gatherer societies. The scarcity of research between these extremes limits our understanding of how the gut microbiota relates to health and disease in the majority of the world's population. Here, we evaluate gut microbiome composition in transitioning South African populations using short- and long-read sequencing. We analyze stool from adult females living in rural Bushbuckridge (n = 118) or urban Soweto (n = 51) and find that these microbiomes are taxonomically intermediate between those of individuals living in high-income countries and traditional communities. We demonstrate that reference collections are incomplete for characterizing microbiomes of individuals living outside high-income countries, yielding artificially low beta diversity measurements, and generate complete genomes of undescribed taxa, including Treponema, Lentisphaerae, and Succinatimonas. Our results suggest that the gut microbiome of South Africans does not conform to a simple "western-nonwestern" axis and contains undescribed microbial diversity.

Comparative genomics of Enterobacter cloacae complex before and after acquired clinical resistance to Ceftazidime-Avibactam.

Resistance to Ceftazidime-Avibactam in Enterobacter cloacae is poorly understood. Whole genome sequencing identified 6 variants in isolates collected from a patient before and after acquiring Ceftazidime-Avibactam resistance. This included a Phe396Leu mutation in acrB, a component of the AcrAB-TolC efflux pump, possibly mediating enhanced efflux of Ceftazidime and/ or Avibactam.

A Fructo-Oligosaccharide Prebiotic Is Well Tolerated in Adults Undergoing Allogeneic Hematopoietic Stem Cell Transplantation: A Phase I Dose-Escalation Trial.

Alterations of the gut microbiota after allogeneic hematopoietic cell transplantation (allo-HCT) are a key factor in the development of transplant-related complications such as graft-versus-host disease (GVHD). Interventions that preserve the gut microbiome hold promise to improve HCT-associated morbidity and mortality. Murine models demonstrate that prebiotics such as fructo-oligosaccharides (FOSs) may increase gut levels of short-chain fatty acids (SCFAs) such as butyrate and consequently induce proliferation of immunomodulatory FOXP3+CD4+ regulatory T cells (Tregs), which impact GVHD risk. We conducted a pilot phase I trial to investigate the maximum tolerated dose of FOS in patients undergoing reduced-intensity allo-HCT (n = 15) compared with concurrent controls (n = 16). We administered the FOS starting at pretransplant conditioning and continuing for a total of 21 days. We characterized the gut microbiome using shotgun metagenomic sequencing, measured stool short-chain fatty acids (SCFAs) using liquid chromatography-mass spectrometry, and determined peripheral T cell concentrations using cytometry by time-of-flight. We found that FOS was safe and well-tolerated at 10 g/d without significant adverse effects in patients undergoing allo-HCT. Community-level gut microbiota composition differed significantly on the day of transplant (day 0) between patients receiving FOS and concurrent controls; however, FOS-associated alterations of the gut microbiota were not sustained after transplant. Although the impact of FOS was fleeting, transplantation itself impacted a substantial number of taxa over time. In our small pilot trial, no significant differences were observed in gut microbial metabolic pathways, stool SCFAs, or peripheral Tregs, although Tregs trended higher in those patients who received FOS. A marker of CD4+ T cell activation (namely, CTLA4+) was significantly higher in patients receiving FOS, whereas a non-significant trend existed for FOP3+CD4+ Treg cells, which were higher in those receiving FOS compared with controls. FOS is well tolerated at 10 g/d in patients undergoing reduced-intensity allo-HCT. Although the alterations in gut microbiota and peripheral immune cell composition in those receiving FOS are intriguing, additional studies are required to investigate the use of prebiotics in HCT recipients.

Author Correction: Acquisition, transmission and strain diversity of human gut-colonizing crAss-like phages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Acquisition, transmission and strain diversity of human gut-colonizing crAss-like phages.

CrAss-like phages are double-stranded DNA viruses that are prevalent in human gut microbiomes. Here, we analyze gut metagenomic data from mother-infant pairs and patients undergoing fecal microbiota transplantation to evaluate the patterns of acquisition, transmission and strain diversity of crAss-like phages. We find that crAss-like phages are rarely detected at birth but are increasingly prevalent in the infant microbiome after one month of life. We observe nearly identical genomes in 50% of cases where the same crAss-like clade is detected in both the mother and the infant, suggesting vertical transmission. In cases of putative transmission of prototypical crAssphage (p-crAssphage), we find that a subset of strains present in the mother are detected in the infant, and that strain diversity in infants increases with time. Putative tail fiber proteins are enriched for nonsynonymous strain variation compared to other genes, suggesting a potential evolutionary benefit to maintaining strain diversity in specific genes. Finally, we show that p-crAssphage can be acquired through fecal microbiota transplantation.

Surveying Gut Microbiome Research in Africans: Toward Improved Diversity and Representation.

Descriptive and translational investigations into the human gut microbiome (GM) are rapidly expanding; however, studies are largely restricted to industrialized populations in the USA and Europe. Little is known about microbial variability and its implications for health and disease in other parts of the world. Populations in Africa are particularly underrepresented. What limited research has been performed has focused on a few subject domains, including the impact of long-term lifestyle and dietary factors on GM ecology, its maturation during infancy, and the interrelationships between the microbiome, infectious disease, and undernutrition. Recently, international consortia have laid the groundwork for large-scale genomics and microbiome studies on the continent, with a particular interest in the epidemiologic transition to noncommunicable disease. Here, we survey the current landscape of GM scholarship in Africa and propose actionable recommendations to improve research capacity and output.

Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016.

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.

Precision identification of diverse bloodstream pathogens in the gut microbiome.

A comprehensive evaluation of every patient with a bloodstream infection includes an attempt to identify the infectious source. Pathogens can originate from various places, such as the gut microbiota, skin and the external environment. Identifying the definitive origin of an infection would enable precise interventions focused on management of the source1,2. Unfortunately, hospital infection control practices are often informed by assumptions about the source of various specific pathogens; if these assumptions are incorrect, they lead to interventions that do not decrease pathogen exposure3. Here, we develop and apply a streamlined bioinformatic tool, named StrainSifter, to match bloodstream pathogens precisely to a candidate source. We then leverage this approach to interrogate the gut microbiota as a potential reservoir of bloodstream pathogens in a cohort of hematopoietic cell transplantation recipients. We find that patients with Escherichia coli and Klebsiella pneumoniae bloodstream infections have concomitant gut colonization with these organisms, suggesting that the gut may be a source of these infections. We also find cases where typically nonenteric pathogens, such as Pseudomonas aeruginosa and Staphylococcus epidermidis, are found in the gut microbiota, thereby challenging the existing informal dogma of these infections originating from environmental or skin sources. Thus, we present an approach to distinguish the source of various bloodstream infections, which may facilitate more accurate tracking and prevention of hospital-acquired infections.

Epidemiologic Investigation of a Cluster of Neuroinvasive Bacillus cereus Infections in 5 Patients With Acute Myelogenous Leukemia.

Background. Five neuroinvasive Bacillus cereus infections (4 fatal) occurred in hospitalized patients with acute myelogenous leukemia (AML) during a 9-month period, prompting an investigation by infection control and public health officials. Methods. Medical records of case-patients were reviewed and a matched case-control study was performed. Infection control practices were observed. Multiple environmental, food, and medication samples common to AML patients were cultured. Multilocus sequence typing was performed for case and environmental B cereus isolates. Results. All 5 case-patients received chemotherapy and had early-onset neutropenic fevers that resolved with empiric antibiotics. Fever recurred at a median of 17 days (range, 9-20) with headaches and abrupt neurological deterioration. Case-patients had B cereus identified in central nervous system (CNS) samples by (1) polymerase chain reaction or culture or (2) bacilli seen on CNS pathology stains with high-grade B cereus bacteremia. Two case-patients also had colonic ulcers with abundant bacilli on autopsy. No infection control breaches were observed. On case-control analysis, bananas were the only significant exposure shared by all 5 case-patients (odds ratio, 9.3; P = .04). Five environmental or food isolates tested positive for B cereus, including a homogenized banana peel isolate and the shelf of a kitchen cart where bananas were stored. Multilocus sequence typing confirmed that all case and environmental strains were genetically distinct. Multilocus sequence typing-based phylogenetic analysis revealed that the organisms clustered in 2 separate clades. Conclusions. The investigation of this neuroinvasive B cereus cluster did not identify a single point source but was suggestive of a possible dietary exposure. Our experience underscores the potential virulence of B cereus in immunocompromised hosts.