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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.

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.

BackgroundAlterations 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 (FOS) may increase gut levels of short-chain fatty acids (SCFAs) such as butyrate, and consequently induce proliferation of immunomodulatory CD4+ FOXP3+ T-regulatory cells (Tregs), that impact GVHD risk.MethodsWe conducted a pilot Phase I trial to assess the investigate the maximum tolerated dose of FOS in patients undergoing reduced-intensity (RIC) allo-HCT (n=15) compared to concurrent controls (n=16). We administered FOS starting at pretransplant conditioning and continuing for a total of 21 days. We characterized the gut microbiome using shotgun metagenomic sequencing, measured stool SCFAs using LC-MS, and determined peripheral T-cell concentrations using CyTOF.ResultsWe found that FOS was safe and well-tolerated at 10g per day without significant adverse effects in patients undergoing reduced-intensity conditioning allo-HCT. Community-level gut microbiota composition was significantly different on the day of transplant (day 0) between patients receiving FOS compared to 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 in peripheral Tregs although Tregs trended higher in those patients who received FOS. Early alterations in gut microbiota composition in those receiving FOS are especially intriguing although it remains unclear what impact this has on outcomes following transplantation and larger studies are required to investigate the use of prebiotics in HCT recipients.ConclusionsFOS is well-tolerated at 10g per day in patients undergoing RIC allo-HCT.

Background Comparisons of traditional hunter-gatherers and pre-agricultural communities in Africa with urban and suburban Western North American and European cohorts have clearly shown that diet, lifestyle and environment are associated with gut microbiome composition. Yet, little is known about the gut microbiome composition of most communities in the very diverse African continent. South Africa comprises a richly diverse ethnolinguistic population that is experiencing an ongoing epidemiological transition and concurrent spike in the prevalence of obesity, largely attributed to a shift towards more Westernized diets and increasingly inactive lifestyle practices. To characterize the microbiome of African adults living in more mainstream lifestyle settings and investigate associations between the microbiome and obesity, we conducted a pilot study, designed collaboratively with community leaders, in two South African cohorts representative of urban and transitioning rural populations. As the rate of overweight and obesity is particularly high in women, we collected single time-point stool samples from 170 HIV-negative women (51 at Soweto; 119 at Bushbuckridge), performed 16S rRNA gene sequencing on these samples and compared the data to concurrently collected anthropometric data. Results We found the overall gut microbiome of our cohorts to be reflective of their ongoing epidemiological transition. Specifically, we find that geographical location was more important for sample clustering than lean/obese status and observed a relatively higher abundance of the Melainabacteria, Vampirovibrio, a predatory bacterium, in Bushbuckridge. Also, Prevotella, despite its generally high prevalence in the cohorts, showed an association with obesity. In comparisons with benchmarked datasets representative of non-Western populations, relatively higher abundance values were observed in our dataset for Barnesiella (log2fold change (FC) = 4.5), Alistipes (log2FC = 3.9), Bacteroides (log2FC = 4.2), Parabacteroides (log2FC = 3.1) and Treponema (log2FC = 1.6), with the exception of Prevotella (log2FC = − 4.7). Conclusions Altogether, this work identifies putative microbial features associated with host health in a historically understudied community undergoing an epidemiological transition. Furthermore, we note the crucial role of community engagement to the success of a study in an African setting, the importance of more population-specific studies to inform targeted interventions as well as present a basic foundation for future research.

Human gut microbiome research focuses on populations living in high-income countries or on the other end of the spectrum, namely 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. We present the first study evaluating gut microbiome composition in transitioning South African populations using short- and long-read sequencing. We analyzed stool samples from adult females (age 40 - 72) living in rural Bushbuckridge municipality (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 characterization of microbiomes of individuals living outside high-income countries, resulting in artificially low species-level beta diversity measurements. To improve reference databases, we generated complete genomes of undescribed taxa, including Treponema, Lentisphaerae, and Succinatimonas species. Our results suggest that the gut microbiome in South African populations do not exist along a simple “western-nonwestern” axis and that these populations contain microbial diversity that remains to be described.

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.

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., CrAss-like phages are bacterial viruses often found in the human gut. Here, Siranosian et al. analyze gut metagenomic data to evaluate the patterns of acquisition, transmission and strain diversity of these phages in mother-infant pairs and in patients undergoing fecal microbiota transplantation.

Summary The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

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) bla(OXA-48 like), 20.8% (5/24) bla(KPC), 20.8% (5/24) bla(NDM), 20.8% (5/24) bla(SME), 8.3% (2/24) bla(IMP), and 4.2% (1/24) bla(VIM). Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing bla(KPC) were 100% susceptible to ceftazidime–avibactam, meropenem–vaborbactam, and imipenem–relebactam; bla(OXA-48 like)–positive isolates were 100% susceptible to ceftazidime–avibactam; and metallo β-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 β-lactamase inhibitors.

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.

Bloodstream infection is the most common infectious complication in hematopoietic cell transplantation recipients. Culture-based sequencing approaches have been previously applied to interrogate the intestine as a reservoir of these bloodstream pathogens. To evaluate the genomic concordance of bloodstream pathogens and bacterial strains within the intestinal microbiome using whole genome sequencing (WGS), we developed StrainSifter, a bioinformatic pipeline to compare nucleotide variation between bacterial isolate strains and stool metagenomes. We applied StrainSifter to bloodstream isolates and stool metagenome samples from a cohort of 30 hematopoietic stem cell transplant recipients with bloodstream infections at a single academic center. StrainSifter is designed to 1) identify single nucleotide variants (SNVs) between isolate and metagenomic short reads aligned to assembled draft genomes using stringent alignment, coverage, and variant frequency criteria, and 2) output both phylogenetic trees and SNV counts for clinical and epidemiologic strain comparison. Using StrainSifter, we identified enteric BSI isolates - including Enterococcus faecium, Klebsiella pneumoniae, Escherichia coli, and Streptococcus mitis - that were highly concordant with those in the gut microbiota. We also identified highly concordant strains of Staphylococcus epidermidis and Pseudomonas aeruginosa between the bloodstream and gut microbiome, an unexpected finding for typically non-enteric organisms. In contrast to previous studies demonstrating intestinal domination by pathogenic bacteria preceding BSI, we found significant heterogeneity in relative pathogen abundance prior to infection. These findings demonstrate not only the utility of StrainSifter in strain matching between isolate and metagenomic sequencing data, but also provide a more precise and comprehensive investigation of the intestine as a reservoir of diverse pathogens capable of causing bloodstream infections in hematopoietic stem cell transplant patients.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging as a major health threat in North America. The mechanism of resistance to carbapenems has therapeutic and public health implications. We comprehensively characterized the underlying mechanisms of carbapenem resistance in CRE isolates recovered between 2013 and 2016 at a health system in Northern California. Genotypic methods were used to detect carbapenemases and plasmid-encoded cephalosporinases, and mass spectrometry was used to quantify relative porin levels for OmpC and OmpF and their analogs. MICs for imipenem-relebactam, meropenem-vaborbactam, ceftazidime-avibactam, and ceftolozane-tazobactam were measured. Whole genome sequencing was used for strain typing. A carbapenemase gene encoding blaOXA-48 like, blaNDM, blaKPC, blaSME, blaIMP, and blaVIM was detected in 38.7% (24/62) of CRE isolates. Porin levels was down at least 2-fold in 91.9% (57/62) of isolates. Including carbapenemase genes and porin loss, the mechanism of resistance was identified in 95.2% (59/62) of CRE isolates. Of the carbapenemase gene-positive isolates, blaKPC -positive isolates were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and blaSME-positive isolates were 100% susceptible to meropenem-vaborbactam and ceftolozane-tazobactam. 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) of carbapenemase gene-negative CRE isolates were susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were genetically identical. In conclusion, at this health system in Silicon Valley, carbapenemase-producing CRE occurred sporadically and were mediated by diverse mechanisms. Nucleic acid testing for blaOXA-48 like, blaNDM, blaKPC, blaIMP, and blaVIM was sufficient to distinguish between carbapenemase-producing and non-producing CRE and accurately predicted susceptibility to ceftazidime-avibactam, meropenem-vaborbactam and imipenem-relebactam.

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.

Paper to Plastics (P2P) is an interdisciplinary program that combines chemistry and biology in a research setting. The goal of this project is 2-fold: to engage students in scientific research and to educate them about sustainability and biodegradable materials. The scientific aim of the project is to recycle unwanted office paper to the useful biodegradable polymer poly(lactic acid) (PLA). Through this program, students learn firsthand how chemistry and biology interact to form useful materials from waste. Students combine biological techniques, such as enzymatic digestion and fermentation, with chemical techniques, such as distillation and catalysis, to accomplish the conversion of waste paper into PLA. Through this summer program, students ultimately become familiar with diverse laboratory techniques, while learning how their scientific interests can be used to address important social problems.

Recipients of hematopoietic stem cell transplantion (HCT) are at high risk for bloodstream infections (BSI); culture-based and marker gene sequencing approaches have been previously applied to interrogate the intestine as a reservoir of these pathogens. Identifying the source of bloodstream infections can impact infection control and prevention practices such as removing central venous catheters and placing patients in isolation. To evaluate the genomic concordance of bloodstream pathogens and bacterial strains within the intestinal microbiome, we developed StrainSifter (Figure 1), a streamlined bioinformatic pipeline using whole genome shotgun sequencing to compare nucleotide variation between bacterial isolate strains and metagenomes. We applied StrainSifter to bloodstream isolates and stool metagenome samples from a cohort of 30 HCT recipients with bloodstream infections at a single academic center. StrainSifter is designed to 1) identify single nucleotide variants (SNVs) between isolate and metagenomic short reads aligned to assembled draft genomes using stringent alignment, coverage, and variant frequency criteria, and 2) output both phylogenetic trees and SNV counts for clinical and epidemiologic strain typing and comparison. Using StrainSifter, we identified enteric BSI isolates - including Enterococcus faecium, Klebsiella pneumoniae, Escherichia coli, and Streptococcus mitis - that were identical or nearly identical with those in the gut microbiota. We also identified highly concordant strains of Staphylococcus epidermidis and Pseudomonas aeruginosa between the bloodstream and gut microbiome, an unexpected finding for typically non-enteric organisms. Although intestinal domination by pathogenic bacteria preceded BSI with E. faecium and E. coli as previous studies have demonstrated, we found significant heterogeneity in the relative abundance of pathogens prior to infection for other bacteria. These findings demonstrate the utility of StrainSifter in strain matching between isolate and metagenomic sequencing data, and provide a more precise and comprehensive investigation of the intestine as a potential reservoir of diverse pathogens capable of causing bloodstream infections in HCT recipients. These findings may have implications for the tracking and prevention of BSI previously classified as central-line associated bloodstream infections. Disclosures No relevant conflicts of interest to declare.

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.

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.

Background Infection is a major preventable cause of transplant-related morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HCT). Bacteremia is the most common infectious complication in HCT, often occurring during periods of mucositis when the risk for microbial translocation from the intestine is increased. Prior research in HCT patients using 16S rRNA sequencing demonstrated that gut microbiota dominance by either Enterococcus spp. or Proteobacteria was associated with the development of bacteremia with Enterococcus spp. and Gram-negative organisms, respectively. No studies to date, however, have compared bacteremia isolates and gut microbiota samples at a strain-specific level using next-generation shotgun metagenomic sequencing (NGS). Methods In order to assess the degree of genetic similarity between bacteremia isolates and the gut microbiota, we identified patients who had undergone HCT at Stanford and developed a bacteremia between October 2015 and September 2016 for whom we had both saved blood culture isolates and stool samples within 30 days preceding bacteremia. We identified 15 patients from whom we had 17 bacteremia isolates, and performed NGS (Illumina HiSeq 4000) on stool and isolate DNA. We generated draft assemblies of isolate genomes using the SPAdes assembler, and aligned stool metagenomic reads to the draft isolate genomes using Bowtie2, filtering reads for perfect end-to-end alignment. Results Enteric gram-negative bacteremia isolates were identical to those in the gut microbiota, as has been demonstrated in prior studies using older strain-typing Methods. Surprisingly, we also identified gram-positive organisms that were identical in both the blood and stool prior to bacteremia, which challenges existing dogma regarding sources of gram-positive bacteremia-causing organisms. Conclusion Using a highly sensitive and accurate NGS-based strain typing method, we provide evidence of translocation of organisms from the gut microbiota and subsequent bacteremia. The gut was confirmed as a source for both classic enteric gram-negative and classically non-enteric Gram-positive bacteremia in HCT patients. These findings may have implications for the origins of bacteremia in HCT patients previously classified as CLABSIs. Disclosures A. Bhatt, Janssen Human Microbiome Institute / Johnson and Johnson: Consultant, Consulting fee