Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli

Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome, and currently only supportive therapy is available. Lack of suitable animal models has hindered study of this disease. Induced human intestinal organoids (iHIOs), generated by in vitro differentiation of pluripotent stem cells, represent differentiated human intestinal tissue. We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses. Commensal and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high numbers. Commensal E. coli did not cause damage, and were completely contained within the lumen, suggesting defenses, such as mucus production, can constrain non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew as filaments, consistent with activation of the bacterial SOS stress response. SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased ROS production. Transcriptional profiling (RNAseq) demonstrated that both commensal and O157:H7 upregulated genes associated with gastrointestinal maturation, while infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment, and infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO tissue

Cognitive and Microbiome Impacts of Experimental Ancylostoma ceylanicum Hookworm Infections in Hamsters

Hookworms are one of the most prevalent and important parasites, infecting ~500 million people worldwide. Hookworm disease is among the leading causes of iron-deficiency anemia in the developing world and is associated with significant growth stunting and malnutrition. In humans, hookworms appear to impair memory and other forms of cognition, although definitive data are hard to come by. Here we study the impact of a human hookworm parasite, Ancylostoma ceylanicum, on cognition in hamsters in a controlled laboratory setting. We developed tests that measure long-term memory in hamsters. We find that hookworm-infected hamsters were fully capable of detecting a novel object. However, hookworm-infected hamsters were impaired in detecting a displaced object. Defects could be discerned at even at low levels of infection, whereas at higher levels of infection, hamsters were statistically unable to distinguish between displaced and non-displaced objects. These spatial memory deficiencies could not be attributed to defects in infected hamster mobility or to lack of interest. We also found that hookworm infection resulted in reproducible reductions in diversity and changes in specific taxanomic groups in the hamster gut microbiome. These data demonstrate that human hookworm infection in a laboratory mammal results in a specific, rapid, acute, and measurable deficit in spatial memory, and we speculate that gut alterations could play some role in these cognitive deficits. Our findings highlight the importance of hookworm elimination and suggest that finer tuned spatial memory studies be carried out in humans

Dysbiosis in a canine model of human fistulizing Crohn\u27s disease

Crohn\u27s disease (CD) is a chronic immune-mediated inflammatory condition caused by the loss of mucosal tolerance toward the commensal microbiota. On average, 29.5% and 42.7% CD patients experience perianal complications at 10 and 20 y after diagnosis, respectively. Perianal CD (pCD) result in high disease burden, diminished quality of life, and elevated health-care costs. Overall pCD are predictors of poor long-term outcomes. Animal models of gut inflammation have failed to fully recapitulate the human manifestations of fistulizing CD. Here, we evaluated dogs with spontaneous canine anal furunculosis (CAF), a disease with clinical similarities to pCD, as a surrogate model for understanding the microbial contribution of human pCD pathophysiology. By comparing the gut microbiomes between dogs suffering from CAF (CAF dogs) and healthy dogs, we show CAF-dog microbiomes are either very dissimilar (dysbiotic) or similar (healthy-like), yet unique, to healthy dog\u27s microbiomes. Compared to healthy or healthy-like CAF microbiomes, dysbiotic CAF microbiomes showed an increased abundance of Bacteroides vulgatus and Escherichia coli and a decreased abundance of Megamonas species and Prevotella copri. Our results mirror what have been reported in previous microbiome studies of patients with CD; particularly, CAF dogs exhibited two distinct microbiome composition: dysbiotic and healthy-like, with determinant bacterial taxa such as E. coli and P. copri that overlap what it has been found on their human counterpart. Thus, our results support the use of CAF dogs as a surrogate model to advance our understanding of microbial dynamics in pCD

Alzheimer\u27s Disease Microbiome Is Associated with Dysregulation of the Anti-Inflammatory P-Glycoprotein Pathway

The microbiota-gut-brain axis is a bidirectional communication system that is poorly understood. Alzheimer\u27s disease (AD), the most common cause of dementia, has long been associated with bacterial infections and inflammation-causing immunosenescence. Recent studies examining the intestinal microbiota of AD patients revealed that their microbiome differs from that of subjects without dementia. In this work, we prospectively enrolled 108 nursing home elders and followed each for up to 5 months, collecting longitudinal stool samples from which we performed metagenomic sequencing and in vitro T84 intestinal epithelial cell functional assays for P-glycoprotein (P-gp) expression, a critical mediator of intestinal homeostasis. Our analysis identified clinical parameters as well as numerous microbial taxa and functional genes that act as predictors of AD dementia in comparison to elders without dementia or with other dementia types. We further demonstrate that stool samples from elders with AD can induce lower P-gp expression levels in vitro those samples from elders without dementia or with other dementia types. We also paired functional studies with machine learning approaches to identify bacterial species differentiating the microbiome of AD elders from that of elders without dementia, which in turn are accurate predictors of the loss of dysregulation of the P-gp pathway. We observed that the microbiome of AD elders shows a lower proportion and prevalence of bacteria with the potential to synthesize butyrate, as well as higher abundances of taxa that are known to cause proinflammatory states. Therefore, a potential nexus between the intestinal microbiome and AD is the modulation of intestinal homeostasis by increases in inflammatory, and decreases in anti-inflammatory, microbial metabolism.IMPORTANCE Studies of the intestinal microbiome and AD have demonstrated associations with microbiome composition at the genus level among matched cohorts. We move this body of literature forward by more deeply investigating microbiome composition via metagenomics and by comparing AD patients against those without dementia and with other dementia types. We also exploit machine learning approaches that combine both metagenomic and clinical data. Finally, our functional studies using stool samples from elders demonstrate how the c microbiome of AD elders can affect intestinal health via dysregulation of the P-glycoprotein pathway. P-glycoprotein dysregulation contributes directly to inflammatory disorders of the intestine. Since AD has been long thought to be linked to chronic bacterial infections as a possible etiology, our findings therefore fill a gap in knowledge in the field of AD research by identifying a nexus between the microbiome, loss of intestinal homeostasis, and inflammation that may underlie this neurodegenerative disorder

The high prevalence of Clostridioides difficile among nursing home elders associates with a dysbiotic microbiome

Clostridioides difficile disproportionally affects the elderly living in nursing homes (NHs). Our objective was to explore the prevalence of C. difficile in NH elders, over time and to determine whether the microbiome or other clinical factors are associated with C. difficile colonization. We collected serial stool samples from NH residents. C. difficile prevalence was determined by quantitative polymerase-chain reaction detection of Toxin genes tcdA and tcdB; microbiome composition was determined by shotgun metagenomic sequencing. We used mixed-effect random forest modeling machine to determine bacterial taxa whose abundance is associated with C. difficile prevalence while controlling for clinical covariates including demographics, medications, and past medical history. We enrolled 167 NH elders who contributed 506 stool samples. Of the 123 elders providing multiple samples, 30 (24.4%) elders yielded multiple samples in which C. difficile was detected and 78 (46.7%) had at least one C. difficile positive sample. Elders with C. difficile positive samples were characterized by increased abundances of pathogenic or inflammatory-associated bacterial taxa and by lower abundances of taxa with anti-inflammatory or symbiotic properties. Proton pump inhibitor (PPI) use is associated with lower prevalence of C. difficile (Odds Ratio 0.46; 95%CI, 0.22-0.99) and the abundance of bacterial species with known beneficial effects was higher in PPI users and markedly lower in elders with high C. difficile prevalence.C. difficile is prevalent among NH elders and a dysbiotic gut microbiome associates with C. difficile colonization status. Manipulating the gut microbiome may prove to be a key strategy in the reduction of C. difficile in the NH

Differential effects of Akkermansia-enriched fecal microbiota transplant on energy balance in female mice on high-fat diet

Estrogens protect against weight gain and metabolic disruption in women and female rodents. Aberrations in the gut microbiota composition are linked to obesity and metabolic disorders. Furthermore, estrogen-mediated protection against diet-induced metabolic disruption is associated with modifications in gut microbiota. In this study, we tested if estradiol (E2)-mediated protection against obesity and metabolic disorders in female mice is dependent on gut microbiota. Specifically, we tested if fecal microbiota transplantation (FMT) from E2-treated lean female mice, supplemented with or without Akkermansia muciniphila, prevented high fat diet (HFD)-induced body weight gain, fat mass gain, and hyperglycemia in female recipients. FMT from, and cohousing with, E2-treated lean donors was not sufficient to transfer the metabolic benefits to the E2-deficient female recipients. Moreover, FMT from lean donors supplemented with A. muciniphila exacerbated HFD-induced hyperglycemia in E2-deficient recipients, suggesting its detrimental effect on the metabolic health of E2-deficient female rodents fed a HFD. Given that A. muciniphila attenuates HFD-induced metabolic insults in males, the present findings suggest a sex difference in the impact of this microbe on metabolic health.Peer reviewe

Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA

Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis

BACKGROUND: P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function. RESULTS: We have identified a functional core microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression. CONCLUSION: Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation. Video abstract

Early microbial and metabolomic signatures predict later onset of necrotizing enterocolitis in preterm infants

Background: Necrotizing enterocolitis (NEC) is a devastating intestinal disease that afflicts 10% of extremely preterm infants. The contribution of early intestinal colonization to NEC onset is not understood, and predictive biomarkers to guide prevention are lacking. We analyzed banked stool and urine samples collected prior to disease onset from infants 99% versus 99% versus 38% in the other NEC cases and 84% in controls, P = 0.01). NEC preceded by Firmicutes dysbiosis occurred earlier (onset, days 7 to 21) than NEC preceded by Proteobacteria dysbiosis (onset, days 19 to 39). All NEC cases lacked Propionibacterium and were preceded by either Firmicutes (≥98% relative abundance, days 4 to 9) or Proteobacteria (≥90% relative abundance, days 10 to 16) dysbiosis, while only 25% of controls had this phenotype (predictive value 88%, P = 0.001). Analysis of days 4 to 9 urine samples found no metabolites associated with all NEC cases, but alanine was positively associated with NEC cases that were preceded by Firmicutes dysbiosis (P < 0.001) and histidine was inversely associated with NEC cases preceded by Proteobacteria dysbiosis (P = 0.013). A high urinary alanine:histidine ratio was associated with microbial characteristics (P < 0.001) and provided good prediction of overall NEC (predictive value 78%, P = 0.007). Conclusions: Early dysbiosis is strongly involved in the pathobiology of NEC. These striking findings require validation in larger studies but indicate that early microbial and metabolomic signatures may provide highly predictive biomarkers of NEC

Oropharyngeal Microbiome Profiled at Admission is Predictive of the Need for Respiratory Support Among COVID-19 Patients [preprint]

The clinical course of infection due to respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the causative agent of Coronavirus Disease 2019 (COVID-19) is thought to be influenced by the community of organisms that colonizes the upper respiratory tract, the oropharyngeal microbiome. In this study, we examined the oropharyngeal microbiome of suspected COVID-19 patients presenting to the Emergency Department and an inpatient COVID-19 unit with symptoms of acute COVID-19. Of 115 enrolled patients, 74 were confirmed COVID-19+ and 50 had symptom duration of 14 days or less; 38 acute COVID-19+ patients (76%) went on to require respiratory support. Although no microbiome features were found to be significantly different between COVID-19+ and COVID-19-patients, when we conducted random forest classification modeling (RFC) to predict the need of respiratory support for the COVID-19+ patients our analysis identified a subset of organisms and metabolic pathways whose relative abundance, when combined with clinical factors (such as age and Body Mass Index), was highly predictive of the need for respiratory support (F1 score 0.857). Microbiome Multivariable Association with Linear Models (MaAsLin2) analysis was then applied to the features identified as predicative of the need for respiratory support by the RFC. This analysis revealed reduced abundance of Prevotella salivae and metabolic pathways associated with lipopolysaccharide and mycolic acid biosynthesis to be the strongest predictors of patients requiring respiratory support. These findings suggest that composition of the oropharyngeal microbiome in COVID-19 may play a role in determining who will suffer from severe disease manifestations. Importance: The microbial community that colonizes the upper airway, the oropharyngeal microbiome, has the potential to affect how patients respond to respiratory viruses such as SARS-CoV2, the causative agent of COVID-19. In this study, we investigated the oropharyngeal microbiome of COVID-19 patients using high throughput DNA sequencing performed on oral swabs. We combined patient characteristics available at intake such as medical comorbidities and age, with measured abundance of bacterial species and metabolic pathways and then trained a machine learning model to determine what features are predicative of patients needing respiratory support in the form of supplemental oxygen or mechanical ventilation. We found that decreased abundance of some bacterial species and increased abundance of pathways associated bacterial products biosynthesis was highly predictive of needing respiratory support. This suggests that the oropharyngeal microbiome affects disease course in COVID-19 and could be targeted for diagnostic purposes to determine who may need oxygen, or therapeutic purposes such as probiotics to prevent severe COVID-19 disease manifestations