Precision editing of the gut microbiota ameliorates colitis.

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

The Antibiotic Efflux Protein TolC Is a Highly Evolvable Target under Colicin E1 or TLS Phage Selection.

Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug-resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria (Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, et al. 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 61(10); Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. 2018. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018(1):60-66; Petrovic Fabijan A, Lin RCY, Ho J, Maddocks S, Ben Zakour NL, Iredell JR, Westmead Bacteriophage Therapy Team. 2020. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat Microbiol. 5(3):465-472). One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS phage (Pagie L, Hogeweg P. 1999. Colicin diversity: a result of eco-evolutionary dynamics. J Theor Biol. 196(2):251-261; Andersen C, Hughes C, Koronakis V. 2000. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep. 1(4):313-318; Koronakis V, Andersen C, Hughes C. 2001. Channel-tunnels. Curr Opin Struct Biol. 11(4):403-407; Czaran TL, Hoekstra RF, Pagie L. 2002. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. 99(2):786-790; Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev. 71(1):158-229). By systematically assessing the distribution of fitness effects of ∼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.

A Bayesian zero-inflated negative binomial regression model for the integrative analysis of microbiome data.

Microbiome omics approaches can reveal intriguing relationships between the human microbiome and certain disease states. Along with identification of specific bacteria taxa associated with diseases, recent scientific advancements provide mounting evidence that metabolism, genetics, and environmental factors can all modulate these microbial effects. However, the current methods for integrating microbiome data and other covariates are severely lacking. Hence, we present an integrative Bayesian zero-inflated negative binomial regression model that can both distinguish differentially abundant taxa with distinct phenotypes and quantify covariate-taxa effects. Our model demonstrates good performance using simulated data. Furthermore, we successfully integrated microbiome taxonomies and metabolomics in two real microbiome datasets to provide biologically interpretable findings. In all, we proposed a novel integrative Bayesian regression model that features bacterial differential abundance analysis and microbiome-covariate effects quantifications, which makes it suitable for general microbiome studies.

Rapid ultrasensitive detection platform for antimicrobial susceptibility testing.

Rapid detection and phenotyping of pathogenic microbes is critical for administration of effective antibiotic therapies and for impeding the spread of antibiotic resistance. Here, we present a novel platform, rapid ultrasensitive detector (RUSD), that utilizes the high reflectance coefficient at high incidence angles when light travels from low- to high-refractive-index media. RUSD leverages a principle that does not require complex manufacturing, labeling, or processing steps. Utilizing RUSD, we can detect extremely low cell densities (optical density [OD] ≥ 5 × 10-7) that correspond to approximately 20 bacterial cells or a single fungal cell in the detection volume, which is nearly 4 orders of magnitude more sensitive than standard OD methods. RUSD can measure minimum inhibitory concentrations (MICs) of commonly used antibiotics against gram-negative and gram-positive bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, within 2 to 4 h. Here, we demonstrate that antibiotic susceptibility tests for several pathogens can rapidly be performed with RUSD using both small inoculum sizes (500 cells/mL) and larger inoculum sizes (5 × 105 cells/mL) used in standard antibiotic susceptibility tests. We anticipate that the RUSD system will be particularly useful for the cases in which antibiotic susceptibility tests have to be done with a limited number of bacterial cells that are available. Its compatibility with standard antibiotic susceptibility tests, simplicity, and low cost can make RUSD a viable and rapidly deployed diagnostic tool.

The Impact of Lactobacillus Probiotics on the Gut Microbiota in Children With Short Bowel Syndrome.

BACKGROUND: Children with short bowel syndrome (SBS) frequently struggle with malabsorption and poor growth. The intestinal microbiota plays an important role in gut function, and children with SBS have known deficiencies in some commensal gut microbes. One strategy to enhance the gut microbiota is by taking probiotics. However, the efficacy of this approach is not well established. We hypothesized that probiotic supplementation would result in increased levels of the supplemented bacteria and improved growth. MATERIALS AND METHODS: Children with SBS who had weaned from parenteral nutrition but with suboptimal growth were randomized to receive probiotics (Lactobacillus rhamnosus and Lactobacillus johnsonii) or placebo daily for 2 mo. The gut microbiota from monthly stool samples were compared between groups using 16S ribosomal ribonucleic acid sequencing and quantitative polymerase chain reaction. Growth between groups was also compared. Statistical analysis was completed using Mann-Whitney, Kruskal-Wallis, and chi-square tests as appropriate. RESULTS: Eighteen children with SBS completed the study (n = 9 per group). There were no significant changes to the major bacterial families in either group. Median relative abundance of Lactobacillus did not differ between groups at baseline or at the end of the study (7.67 versus 13.23, P = 0.523 and 1.93 versus 15.8, P = 0.161). Median z scores for weight and length did not differ between groups at the beginning or end of the study. CONCLUSIONS: The efficacy of daily probiotic use in children with intestinal failure is unknown. In this study, Lactobacillus probiotics did not result in a predictable change to the fecal microbiota or overall growth compared with placebo in these patients.

Antibiotic-Induced Depletion of Anti-inflammatory Clostridia Is Associated with the Development of Graft-versus-Host Disease in Pediatric Stem Cell Transplantation Patients.

Adult stem cell transplantation (SCT) patients with graft-versus-host-disease (GVHD) exhibit significant disruptions in gut microbial communities. These changes are associated with higher overall mortality and appear to be driven by specific antibiotic therapies. It is unclear whether pediatric SCT patients who develop GVHD exhibit similar antibiotic-induced gut microbiota community changes. Here, we show that pediatric SCT patients (from Children's Medical Center Dallas, n = 8, and Cincinnati Children's Hospital, n = 7) who developed GVHD showed a significant decline, up to 10-log fold, in gut anti-inflammatory Clostridia (AIC) compared with those without GVHD. In fact, the development of GVHD is significantly associated with this AIC decline and with cumulative antibiotic exposure, particularly antibiotics effective against anaerobic bacteria (P = .003, Firth logistic regression analysis). Using metagenomic shotgun sequencing analysis, we were able to identify specific commensal bacterial species, including AIC, that were significantly depleted in GVHD patients. We then used a preclinical GVHD model to verify our clinical observations. Clindamycin depleted AIC and exacerbated GVHD in mice, whereas oral AIC supplementation increased gut AIC levels and mitigated GVHD in mice. Together, these data suggest that an antibiotic-induced AIC depletion in the gut microbiota is associated with the development of GVHD in pediatric SCT patients.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Potential for Monitoring Gut Microbiota for Diagnosing Infections and Graft-versus-Host Disease in Cancer and Stem Cell Transplant Patients.

BACKGROUND: Gut microbiota, the collective community of microorganisms inhabiting the intestine, have been shown to provide many beneficial functions for the host. Recent advances in next-generation sequencing and advanced molecular biology approaches have allowed researchers to identify gut microbiota signatures associated with disease processes and, in some cases, establish causality and elucidate underlying mechanisms. CONTENT: This report reviews 3 commonly used methods for studying the gut microbiota and microbiome (the collective genomes of the gut microorganisms): 16S rRNA gene sequencing, bacterial group or species-specific quantitative polymerase chain reaction (qPCR), and metagenomic shotgun sequencing (MSS). The technical approaches and resources needed for each approach are outlined, and advantages and disadvantages for each approach are summarized. The findings regarding the role of the gut microbiota in the health of patients with cancer and stem cell transplant (SCT) patients (specifically in modulating the development of gut-derived bacterial infections and a posttransplant immune-mediated complication known as graft-vs-host-disease) are reviewed. Finally, there is discussion of the potential viability of these approaches in the actual clinical treatment of cancer and SCT patients. SUMMARY: Advances in next-generation sequencing have revolutionized our understanding of the importance of the gut microbiome to human health. Both 16S rRNA gene sequencing and MSS are currently too labor-intensive or computationally burdensome to incorporate into real-time clinical monitoring of gut microbiomes. Yet, the lessons learned from these technologies could be adapted to currently used methods (e.g., qPCR) that could then be rigorously tested in the clinical care of these patients.

FMAP: Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies.

BACKGROUND: Given the lack of a complete and comprehensive library of microbial reference genomes, determining the functional profile of diverse microbial communities is challenging. The available functional analysis pipelines lack several key features: (i) an integrated alignment tool, (ii) operon-level analysis, and (iii) the ability to process large datasets. RESULTS: Here we introduce our open-sourced, stand-alone functional analysis pipeline for analyzing whole metagenomic and metatranscriptomic sequencing data, FMAP (Functional Mapping and Analysis Pipeline). FMAP performs alignment, gene family abundance calculations, and statistical analysis (three levels of analyses are provided: differentially-abundant genes, operons and pathways). The resulting output can be easily visualized with heatmaps and functional pathway diagrams. FMAP functional predictions are consistent with currently available functional analysis pipelines. CONCLUSION: FMAP is a comprehensive tool for providing functional analysis of metagenomic/metatranscriptomic sequencing data. With the added features of integrated alignment, operon-level analysis, and the ability to process large datasets, FMAP will be a valuable addition to the currently available functional analysis toolbox. We believe that this software will be of great value to the wider biology and bioinformatics communities.

Murine models of Candida gastrointestinal colonization and dissemination.

Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory and gastrointestinal (GI) tracts. The human GI tract is colonized with trillions of commensal microbes, including numerous Candida spp. Some commensal microbes in the GI tract can cause serious human infections under specific circumstances, typically involving changes in the gut environment and/or host immune conditions. Therefore, utilizing animal models of fungal GI colonization and dissemination can lead to significant insights into the complex pathophysiology of transformation from a commensal organism to a pathogen and host-pathogen interactions. This paper will review the methodologic approaches used for modeling GI colonization versus dissemination, the insights learned from these models, and finally, possible future directions using these animal modeling systems.

Collaboration between macrophages and vaccine-induced CD4+ T cells confers protection against lethal Pseudomonas aeruginosa pneumonia during neutropenia.

The usefulness of vaccine-based strategies to prevent lethal bacterial infection in a host with neutropenia is not well-defined. Here, we show in a neutropenic mouse model that immunity induced by mucosal vaccination with a live-attenuated Pseudomonas aeruginosa vaccine is protective against lethal P. aeruginosa pneumonia caused by both vaccine-homologous and vaccine-heterologous strains, whereas passive immunization confers only vaccine-homologous protection. Cells in the macrophage lineage served as crucial innate cellular effectors in the neutropenic host after active immunization. Vaccine efficacy was CD4(+) T-cell dependent and associated with accumulation of macrophage-lineage cells in the alveolar space after infection, as well as with enhanced P. aeruginosa clearance from the lung. Adaptive CD4(+) T cells produced granulocyte-macrophage colony-stimulating factor (GM-CSF) on restimulation in vitro, and local GM-CSF was critical for vaccine efficacy. Thus, collaboration between the innate and adaptive effectors induced by mucosal vaccination can overcome neutropenia and confer protection against lethal bacterial infection in the profoundly neutropenic host.

Breathe and bloom: Gut hypoxia limits C. albicans growth.

Multiple host and microbial factors dictate whether Candida albicans can colonize the mammalian gastrointestinal tract. In this issue of Cell Host & Microbe, Savage et al. demonstrate that restoration of intestinal epithelial hypoxia is sufficient to restore Candida albicans colonization resistance, even when other Candida inhibitory effectors remain depleted.

Susceptible bacteria can survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance.

Antibiotic resistance and evasion are incompletely understood and complicated by the fact that murine interval dosing models do not fully recapitulate antibiotic pharmacokinetics in humans. To better understand how gastrointestinal bacteria respond to antibiotics, we colonized germ-free mice with a pan-susceptible genetically barcoded Escherichia coli clinical isolate and administered the antibiotic cefepime via programmable subcutaneous pumps, allowing closer emulation of human parenteral antibiotic dynamics. E. coli was only recovered from intestinal tissue, where cefepime concentrations were still inhibitory. Strikingly, "some" E. coli isolates were not cefepime resistant but acquired mutations in genes involved in polysaccharide capsular synthesis increasing their invasion and survival within human intestinal cells. Deleting wbaP involved in capsular polysaccharide synthesis mimicked this phenotype, allowing increased invasion of colonocytes where cefepime concentrations were reduced. Additionally, "some" mutant strains exhibited a persister phenotype upon further cefepime exposure. This work uncovers a mechanism allowing "select" gastrointestinal bacteria to evade antibiotic treatment.

IsoAnalytics: A Single-cell Proteomics Web Server.

Motivation: Single-cell proteomics provide unprecedented resolution to examine biological processes. Customized data analysis and facile data visualization are crucial for scientific discovery. Further, userfriendly data analysis and visualization software that is easily accessible for the general scientific community is essential. Results: We have created a web server, IsoAnalytics , that gives users without computational or bioinformatics background the ability to directly analyze and interactively visualize data obtained from the Isoplexis single cell technology platform. We envision this open-sourced web server will increase research productivity and serve as a free, competitive alternative for single-cell proteomics research. Contact: Andrew.Koh@utsouthwestern.edu and Xiaowei.Zhan@utsouthwestern.edu. Availability: IsoAnalytics is free and available at: https://cdc.biohpc.swmed.edu/isoplexis/ and is implemented in Python, with all major browsers supported. Code for IsoAnalytics is free and available at: https://github.com/zhanxw/Isoplexis_Data_Analysis . Supplementary Information: Supplementary data are available at Bioinformatics online.

IsoAnalytics: a single-cell proteomics web server.

Motivation: Single-cell proteomics provide unprecedented resolution to examine biological processes. Customized data analysis and facile data visualization are crucial for scientific discovery. Further, user-friendly data analysis and visualization software that is easily accessible for the general scientific community is essential. Results: We have created a web server, IsoAnalytics, that gives users without computational or bioinformatics background the ability to directly analyze and interactively visualize data obtained from the Isoplexis single cell technology platform. We envision this open-sourced web server will increase research productivity and serve as a free, competitive alternative for single-cell proteomics research. Availability and implementation: IsoAnalytics is free and available at: https://cdc.biohpc.swmed.edu/isoplexis/ and is implemented in Python, with all major browsers supported. Code for IsoAnalytics is free and available at: https://github.com/zhanxw/Isoplexis_Data_Analysis. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Susceptible bacteria survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance.

In vitro systems have provided great insight into the mechanisms of antibiotic resistance. Yet, in vitro approaches cannot reflect the full complexity of what transpires within a host. As the mammalian gut is host to trillions of resident bacteria and thus a potential breeding ground for antibiotic resistance, we sought to better understand how gut bacteria respond to antibiotic treatment in vivo . Here, we colonized germ-free mice with a genetically barcoded antibiotic pan-susceptible Escherichia coli clinical isolate and then administered the antibiotic cefepime via programmable subcutaneous pumps which allowed for closer emulation of human parenteral antibiotic pharmacokinetics/dynamics. After seven days of antibiotics, we were unable to culture E. coli from feces. We were, however, able to recover barcoded E. coli from harvested gastrointestinal (GI) tissue, despite high GI tract and plasma cefepime concentrations. Strikingly, these E. coli isolates were not resistant to cefepime but had acquired mutations â€" most notably in the wbaP gene, which encodes an enzyme required for the initiation of the synthesis of the polysaccharide capsule and lipopolysaccharide O antigen - that increased their ability to invade and survive within intestinal cells, including cultured human colonocytes. Further, these E. coli mutants exhibited a persister phenotype when exposed to cefepime, allowing for greater survival to pulses of cefepime treatment when compared to the wildtype strain. Our findings highlight a mechanism by which bacteria in the gastrointestinal tract can adapt to antibiotic treatment by increasing their ability to persist during antibiotic treatment and invade intestinal epithelial cells where antibiotic concentrations are substantially reduced.

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

Precision modulation of dysbiotic adult microbiomes with a human-milk-derived synbiotic reshapes gut microbial composition and metabolites.

Manipulation of the gut microbiome using live biotherapeutic products shows promise for clinical applications but remains challenging to achieve. Here, we induced dysbiosis in 56 healthy volunteers using antibiotics to test a synbiotic comprising the infant gut microbe, Bifidobacterium longum subspecies infantis (B. infantis), and human milk oligosaccharides (HMOs). B. infantis engrafted in 76% of subjects in an HMO-dependent manner, reaching a relative abundance of up to 81%. Changes in microbiome composition and gut metabolites reflect altered recovery of engrafted subjects compared with controls. Engraftment associates with increases in lactate-consuming Veillonella, faster acetate recovery, and changes in indolelactate and p-cresol sulfate, metabolites that impact host inflammatory status. Furthermore, Veillonella co-cultured in vitro and in vivo with B. infantis and HMO converts lactate produced by B. infantis to propionate, an important mediator of host physiology. These results suggest that the synbiotic reproducibly and predictably modulates recovery of a dysbiotic microbiome.