Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

Metaorganismal choline metabolism shapes olfactory perception.

Microbes living in the intestine can regulate key signaling processes in the central nervous system that directly impact brain health. This gut-brain signaling axis is partially mediated by microbe-host-dependent immune regulation, gut-innervating neuronal communication, and endocrine-like small molecule metabolites that originate from bacteria to ultimately cross the blood-brain barrier. Given the mounting evidence of gut-brain crosstalk, a new therapeutic approach of "psychobiotics" has emerged, whereby strategies designed to primarily modify the gut microbiome have been shown to improve mental health or slow neurodegenerative diseases. Diet is one of the most powerful determinants of gut microbiome community structure, and dietary habits are associated with brain health and disease. Recently, the metaorganismal (i.e., diet-microbe-host) trimethylamine N-oxide (TMAO) pathway has been linked to the development of several brain diseases including Alzheimer's, Parkinson's, and ischemic stroke. However, it is poorly understood how metaorganismal TMAO production influences brain function under normal physiological conditions. To address this, here we have reduced TMAO levels by inhibiting gut microbe-driven choline conversion to trimethylamine (TMA), and then performed comprehensive behavioral phenotyping in mice. Unexpectedly, we find that TMAO is particularly enriched in the murine olfactory bulb, and when TMAO production is blunted at the level of bacterial choline TMA lyase (CutC/D), olfactory perception is altered. Taken together, our studies demonstrate a previously underappreciated role for the TMAO pathway in olfactory-related behaviors.

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome.

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting.

BACKGROUND: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus. OBJECTIVES: To evaluate the clinical utility of SARS-CoV-2 sgRNA RT-PCR testing in a pediatric population. STUDY DESIGN: Retrospective analysis was performed on inpatients from February-September 2022 positive for SARS-CoV-2 by RT-PCR with a concomitant order for sgRNA RT-PCR. Chart abstractions were conducted to determine clinical outcomes, management, and infection prevention and control (IPC) practices. RESULTS: Of 95 SARS-CoV-2 positive samples from 75 unique patients, 27 (28.4%) were positive by sgRNA RT-PCR. A negative sgRNA RT-PCR test allowed for de-isolation in 68 (71.6%) patient episodes. Regardless of age or sex, a positive sgRNA RT-PCR result significantly correlated with disease severity (P = 0.007), generalized COVID-19 symptoms (P = 0.012), hospitalization for COVID-19 (P = 0.019), and immune status (P = 0.024). Moreover, sgRNA RT-PCR results prompted changes in management in 28 patients (37.3%); specifically, therapeutic escalation in 13/27 (48.1%) positives and de-escalation in 15/68 (22.1%) negatives. CONCLUSIONS: Taken together, these findings underscore the clinical utility of sgRNA RT-PCR testing in a pediatric population as we report significant associations between sgRNA RT-PCR results and clinical parameters related to COVID-19. These findings align with the proposed use of sgRNA RT-PCR testing to guide patient management and IPC practices in the hospital setting.

A gut microbial metabolite of dietary polyphenols reverses obesity-driven hepatic steatosis.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Monkeypox: Clinical Considerations, Epidemiology, and Laboratory Diagnostics.

Monkeypox virus (MPXV) has garnered recent attention as outbreaks are continually reported outside historic regions of endemicity in Africa. Consequently, MPXV is becoming routinely included in the differential diagnosis of rash illnesses, requiring clinicians and laboratorians alike to quickly adapt to a new public health emergency. This review discusses the epidemiology, clinical presentation, and laboratory testing of MPXV in the context of recent outbreaks.

Flavin-Containing Monooxygenase 3 (FMO3) Is Critical for Dioxin-Induced Reorganization of the Gut Microbiome and Host Insulin Sensitivity.

Exposure to some environmental pollutants can have potent endocrine-disrupting effects, thereby promoting hormone imbalance and cardiometabolic diseases such as non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiorenal diseases. Recent evidence also suggests that many environmental pollutants can reorganize the gut microbiome to potentially impact these diverse human diseases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent endocrine-disrupting dioxin pollutants, yet our understanding of how TCDD impacts the gut microbiome and systemic metabolism is incompletely understood. Here, we show that TCDD exposure in mice profoundly stimulates the hepatic expression of flavin-containing monooxygenase 3 (Fmo3), which is a hepatic xenobiotic metabolizing enzyme that is also responsible for the production of the gut microbiome-associated metabolite trimethylamine N-oxide (TMAO). Interestingly, an enzymatic product of FMO3 (TMAO) has been associated with the same cardiometabolic diseases that these environmental pollutants promote. Therefore, here, we examined TCDD-induced alterations in the gut microbiome, host liver transcriptome, and glucose tolerance in Fmo3+/+ and Fmo3-/- mice. Our results show that Fmo3 is a critical component of the transcriptional response to TCDD, impacting the gut microbiome, host liver transcriptome, and systemic glucose tolerance. Collectively, this work uncovers a previously underappreciated role for Fmo3 in integrating diet-pollutant-microbe-host interactions.

A 3D-printable device allowing fast and reproducible longitudinal preparation of mouse intestines.

Accurate and reproducible analysis of murine small and large intestinal tissue is key for preclinical models involving intestinal pathology. Currently, there is no easily accessible, standardized method that allows researchers of different skill levels to consistently dissect intestines in a time-efficient manner. Here, we describe the design and use of the 3D-printed "Mouse Intestinal Slicing Tool" (MIST), which can be used to longitudinally dissect murine intestines for further analysis. We benchmarked the MIST against a commonly used procedure involving scissors to make a longitudinal cut along the intestines. Use of the MIST halved the time per mouse to prepare the intestines and outperformed alternative methods in smoothness of the cutting edge and overall reproducibility. By sharing the plans for printing the MIST, we hope to contribute a uniformly applicable method for saving time and increasing consistency in studies of the mouse gastrointestinal tract.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

Helicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization. IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

Gut microbial trimethylamine is elevated in alcohol-associated hepatitis and contributes to ethanol-induced liver injury in mice.

There is mounting evidence that microbes residing in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite TMA is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis.

L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe-/- mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

A Single Human-Relevant Fast Food Meal Rapidly Reorganizes Metabolomic and Transcriptomic Signatures in a Gut Microbiota-Dependent Manner.

BACKGROUND: A major contributor to cardiometabolic disease is caloric excess, often a result of consuming low cost, high calorie fast food. Studies have demonstrated the pivotal role of gut microbes contributing to cardiovascular disease in a diet-dependent manner. Given the central contributions of diet and gut microbiota to cardiometabolic disease, we hypothesized that microbial metabolites originating after fast food consumption can elicit acute metabolic responses in the liver. METHODS: We gave conventionally raised mice or mice that had their microbiomes depleted with antibiotics a single oral gavage of a liquified fast food meal or liquified control rodent chow meal. After four hours, mice were sacrificed and we used untargeted metabolomics of portal and peripheral blood, 16S rRNA gene sequencing, targeted liver metabolomics, and host liver RNA sequencing to identify novel fast food-derived microbial metabolites and their acute effects on liver function. RESULTS: Several candidate microbial metabolites were enriched in portal blood upon fast food feeding, and were essentially absent in antibiotic-treated mice. Strikingly, at four hours post-gavage, fast food consumption resulted in rapid reorganization of the gut microbial community and drastically altered hepatic gene expression. Importantly, diet-driven reshaping of the microbiome and liver transcriptome was dependent on an intact microbial community and not observed in antibiotic ablated animals. CONCLUSIONS: Collectively, these data suggest a single fast food meal is sufficient to reshape the gut microbial community in mice, yielding a unique signature of food-derived microbial metabolites. Future studies are in progress to determine the contribution of select metabolites to cardiometabolic disease progression and the translational relevance of these animal studies.

Microbial Flavonoid Metabolism: A Cardiometabolic Disease Perspective.

Cardiometabolic disease (CMD) is a leading cause of death worldwide and encompasses the inflammatory metabolic disorders of obesity, type 2 diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. Flavonoids are polyphenolic plant metabolites that are abundantly present in fruits and vegetables and have biologically relevant protective effects in a number of cardiometabolic disorders. Several epidemiological studies underscored a negative association between dietary flavonoid consumption and the propensity to develop CMD. Recent studies elucidated the contribution of the gut microbiota in metabolizing dietary intake as it relates to CMD. Importantly, the biological efficacy of flavonoids in humans and animal models alike is linked to the gut microbial community. Herein, we discuss the opportunities and challenges of leveraging flavonoid intake as a potential strategy to prevent and treat CMD in a gut microbe-dependent manner, with special emphasis on flavonoid-derived microbial metabolites.

Identification of essential genes for Escherichia coli aryl polyene biosynthesis and function in biofilm formation.

Aryl polyenes (APEs) are specialized polyunsaturated carboxylic acids that were identified in silico as the product of the most widespread family of bacterial biosynthetic gene clusters (BGCs). They are present in several Gram-negative host-associated bacteria, including multidrug-resistant human pathogens. Here, we characterize a biological function of APEs, focusing on the BGC from a uropathogenic Escherichia coli (UPEC) strain. We first perform a genetic deletion analysis to identify the essential genes required for APE biosynthesis. Next, we show that APEs function as fitness factors that increase protection from oxidative stress and contribute to biofilm formation. Together, our study highlights key steps in the APE biosynthesis pathway that can be explored as potential drug targets for complementary strategies to reduce fitness and prevent biofilm formation of multi-drug resistant pathogens.

A surgical method for continuous intraportal infusion of gut microbial metabolites in mice.

Gut microbe-derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe-derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

Simultaneous Measurement of Urinary Trimethylamine (TMA) and Trimethylamine N-Oxide (TMAO) by Liquid Chromatography-Mass Spectrometry.

Trimethylamine (TMA) is a gut microbial metabolite-rendered by the enzymatic cleavage of nutrients containing a TMA moiety in their chemical structure. TMA can be oxidized as trimethylamine N-oxide (TMAO) catalyzed by hepatic flavin monooxygenases. Circulating TMAO has been demonstrated to portend a pro-inflammatory state, contributing to chronic diseases such as cardiovascular disease and chronic kidney disease. Consequently, TMAO serves as an excellent candidate biomarker for a variety of chronic inflammatory disorders. The highly positive correlation between plasma TMAO and urine TMAO suggests that urine TMAO has the potential to serve as a less invasive biomarker for chronic disease compared to plasma TMAO. In this study, we validated a method to simultaneously measure urine TMA and TMAO concentrations by liquid chromatography-mass spectrometry (LC/MS). Urine TMA and TMAO can be extracted by hexane/butanol under alkaline pH and transferred to the aqueous phase following acidification for LC/MS quantitation. Importantly, during sample processing, none of the nutrients with a chemical structure containing a TMA moiety were spontaneously cleaved to yield TMA. Moreover, we demonstrated that the acidification of urine prevents an increase of TMA after prolonged storage as was observed in non-acidified urine. Finally, here we demonstrated that TMAO can spontaneously degrade to TMA at a very slow rate.

Obesity-linked suppression of membrane-bound O-acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease.

Recent studies have identified a genetic variant rs641738 near two genes encoding membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4) that associate with increased risk of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol-related cirrhosis, and liver fibrosis in those infected with viral hepatitis (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017). Based on hepatic expression quantitative trait loci analysis, it has been suggested that MBOAT7 loss of function promotes liver disease progression (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017), but this has never been formally tested. Here we show that Mboat7 loss, but not Tmc4, in mice is sufficient to promote the progression of NAFLD in the setting of high fat diet. Mboat7 loss of function is associated with accumulation of its substrate lysophosphatidylinositol (LPI) lipids, and direct administration of LPI promotes hepatic inflammatory and fibrotic transcriptional changes in an Mboat7-dependent manner. These studies reveal a novel role for MBOAT7-driven acylation of LPI lipids in suppressing the progression of NAFLD.