Protocol for Metatranscriptomic analysis of Intestinal Microbiota

The objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70×75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples

Stool consistency as a major confounding factor affecting microbiota composition : an ignored variable?

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of BMJ Publishing Group for personal use, not for redistribution. The definitive version was published in Gut 65 (2016): 1-2, doi:10.1136/gutjnl-2015-310043.2016-07-1

Agent-based dynamic knowledge representation of Pseudomonas aeruginosa virulence activation in the stressed gut: Towards characterizing host-pathogen interactions in gut-derived sepsis

<p>Abstract</p> <p>Background</p> <p>There is a growing realization that alterations in host-pathogen interactions (HPI) can generate disease phenotypes without pathogen invasion. The gut represents a prime region where such HPI can arise and manifest. Under normal conditions intestinal microbial communities maintain a stable, mutually beneficial ecosystem. However, host stress can lead to changes in environmental conditions that shift the nature of the host-microbe dialogue, resulting in escalation of virulence expression, immune activation and ultimately systemic disease. Effective modulation of these dynamics requires the ability to characterize the complexity of the HPI, and dynamic computational modeling can aid in this task. Agent-based modeling is a computational method that is suited to representing spatially diverse, dynamical systems. We propose that dynamic knowledge representation of gut HPI with agent-based modeling will aid in the investigation of the pathogenesis of gut-derived sepsis.</p> <p>Methodology/Principal Findings</p> <p>An agent-based model (ABM) of virulence regulation in <it>Pseudomonas aeruginosa </it>was developed by translating bacterial and host cell sense-and-response mechanisms into behavioral rules for computational agents and integrated into a virtual environment representing the host-microbe interface in the gut. The resulting gut milieu ABM (GMABM) was used to: 1) investigate a potential clinically relevant laboratory experimental condition not yet developed - i.e. non-lethal transient segmental intestinal ischemia, 2) examine the sufficiency of existing hypotheses to explain experimental data - i.e. lethality in a model of major surgical insult and stress, and 3) produce behavior to potentially guide future experimental design - i.e. suggested sample points for a potential laboratory model of non-lethal transient intestinal ischemia. Furthermore, hypotheses were generated to explain certain discrepancies between the behaviors of the GMABM and biological experiments, and new investigatory avenues proposed to test those hypotheses.</p> <p>Conclusions/Significance</p> <p>Agent-based modeling can account for the spatio-temporal dynamics of an HPI, and, even when carried out with a relatively high degree of abstraction, can be useful in the investigation of system-level consequences of putative mechanisms operating at the individual agent level. We suggest that an integrated and iterative heuristic relationship between computational modeling and more traditional laboratory and clinical investigations, with a focus on identifying useful and sufficient degrees of abstraction, will enhance the efficiency and translational productivity of biomedical research.</p

Membership and behavior of ultra-low-diversity pathogen communities present in the gut of humans during prolonged critical illness.

UnlabelledWe analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a "commensal lifestyle." However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style.ImportanceDuring critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis.

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown

Media from macrophages co-incubated with <i>Enterococcus faecalis</i> induces epithelial cell monolayer reassembly and altered cell morphology

Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis

Nanoparticles for intestinal sepsis prevention synthesized via inverse miniemulsion polymerization

Previous research has shown that phosphate becomes depleted in the intestinal mucosa following local surgical injury or disease, triggering bacterial virulence and sepsis. Consequently, replenishment of depleted phosphate levels has been shown to prevent bacterial virulence in vitro[3] and sepsis in vivo[1]. Inverse phase miniemulsion polymerization (IPMP) has been extensively used in recent years in the production of nanocapsules for drug delivery of water-soluble therapeutic compounds that can be rendered degradable with time while allowing for sustained release of the encapsulated agent. In previous work we have successfully encapsulated inorganic phosphate salts, such as potassium monophosphate[2], into nanoparticles formed using IPMP. Our in vitro studies, however, have shown that polyphosphate salts, specifically sodium hexametaphosphate (PPi), are more effective at suppressing bacterial virulence[3]. This study focuses on the production and encapsulation of sodium hexametaphosphate into nanoparticles for controlled and extended release. Previous studies demonstrated[3] that encapsulation of sodium hexametaphosphate presents a series of challenges affecting the reproducibility of the IPMP process. Sodium hexametaphosphate is a strong lipophobe whose presence induces a high degree of order for water molecules. This modification in water structure weakens the surfactant interaction with water molecules, actively affecting the stability of the emulsion. This process, known as “salting-out”, has been shown to shift the hydrophilic-lipophilic balance (HLB) of nonionic surfactants towards a more lipophilic value[4]. While this issue has been addressed in a variety of previous studies, no mathematical correlation currently exists describing the effect of salt concentration on the HLB of a specific surfactant. Since miniemulsions require combinations of different phase-soluble surfactants, this adds to the complexity in predicting the extent and strength of the electrolyte effect on the stability of the emulsion system. In this study, we adjusted the IPMP process to counter the unstabilizing force created by the presence of sodium hexametaphosphate in the aqueous phase of the system. A precursor solution containing PEG diacrylate (PEGDA) macromer and NVP comonomer were chosen to create the hydrogel matrix, due to its biocompatibility and the ability to control the crosslinking density. The emulsion was formed of water in cyclohexane with the help of two nonionic surfactants, Tween 20 and SPAN 80. The effect of variations in HLB ranging from 4.0 to 9.5 on emulsion droplet size was investigated, for which the optimum overall HLB occurred at 6.5, an increase of two HLB points over the theoretical required value without salt interference[2, 3]. The effects of total surfactant amounts, reaction time, temperature and initiator concentration on nanoparticle yield were also explored. A final emulsion with 3.2% w/v of surfactants, 2 hours of reaction time, 64ºC and an initiator concentration equal to 1% of the initial double concentration resulted in a maximum nanoparticle mass yield of ~39%. Finally, the particles were characterized in terms of crosslink density, showing an efficient encapsulation of the studied salt and a promising path for in-vivo testing. This study helped us develop a reproduceable formulation of an IPMP process that yields stable nanoparticles with suitable therapeutic levels of phosphates. [1] Hyoju, S.K. et al, “Oral Polyphosphate Suppresses Bacterial Collagenase Production and Prevents Anastomotic Leak Due to Serratia marcescens and Pseudomonas aeruginosa”, Annals of Surgery, Feb, 2017. [2]Vadlamudi, S. et al., “Inverse miniemulsion polymerization of phosphate-loaded hydrogel nanoparticles for sepsis prevention”, Unpublished master dissertation, Illinois Institute of Technology, Chicago, Illinois (2014) [3] Yin Y et al, “De Novo Synthesis and Functional Analysis of Polyphosphate-Loaded Poly(Ethylene) Glycol Hydrogel Nanoparticles Targeting Pyocyanin and Pyoverdin Production in Pseudomonas aeruginosa as a Model Intestinal Pathogen”. Annals of Biomedical Engineering. 45(4):1058-1068, 2017. [4] Shinoda, K., & Takeda, H. “The effect of added salts in water on the hydrophile-lipophile balance of nonionic surfactants: The effect of added salts on the phase inversion temperature of emulsions”. Journal of Colloid And Interface Science, 32(4), 642–646, 1970

Proceedings of the 2014 A.S.P.E.N. Research Workshop

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141886/1/jpen0167.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141886/2/jpen0167-sup-0001.pd

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH

<p>Abstract</p> <p>Background</p> <p>During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal <it>P. aeruginosa </it>to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation.</p> <p>Results</p> <p>In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with <it>P. aeruginosa </it>had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in <it>C. elegans</it>. Transcriptional analysis of <it>P. aeruginosa </it>demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses.</p> <p>Conclusion</p> <p>Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by <it>P. aeruginosa </it>can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients.</p

Infliximab Does Not Promote the Presence of Collagenolytic Bacteria in a Mouse Model of Colorectal Anastomosis

BACKGROUND: Previous work from our group has suggested a pivotal role for collagenolytic bacteria in the development of anastomotic complications. Tumor necrosis factor antagonists are a mainstay of treatment for patients with inflammatory bowel disease. The reported impact of these agents on key surgical outcomes such as anastomotic leak has been inconsistent. The objective of this study is to assess the impact of infliximab on the anastomotic microbiome in a mouse model of colon resection. DESIGN: BALB/c mice underwent colon resection with primary anastomosis. Mice were randomly assigned to receive either an intraperitoneal dose of saline (control) or 10 mg/kg of infliximab for 8 weeks prior to surgery. On postoperative day 7, the animals were sacrificed. Anastomotic tissues were analyzed by histology with TUNNEL staining as a marker of epithelial apoptosis. In order to assess compositional and functional changes of the local microbiome, anastomotic tissues were further analyzed by 16S rRNA V4 region sequencing and for the presence of collagenolytic strains that may impair anastomotic healing. The main outcome measures were microbiome community structure and the presence of collagenolytic bacteria. RESULTS: Infliximab-treated mice demonstrated an increase in epithelial apoptosis, consistent with the expected drug effect. Although infliximab modified the perianastomotic microbiome, no increase in the presence of collagenolytic bacteria was observed. CONCLUSIONS: Infliximab did not promote the emergence of collagenolytic bacteria or demonstrably impair anastomotic healing in a mouse model of colon resection and anastomosis