A Novel Nonantibiotic Gut-directed Strategy to Prevent Surgical Site Infections.

OBJECTIVE: To determine the efficacy of an orally delivered phosphate-rich polymer, Pi-PEG, to prevent surgical site infection (SSI) in a mouse model of spontaneous wound infection involving gut-derived pathogens. BACKGROUND: Evidence suggests that pathogens originating from the gut microbiota can cause postoperative infection via a process by which they silently travel inside an immune cell and contaminate a remote operative site (Trojan Horse Hypothesis). Here, we hypothesize that Pi-PEG can prevent SSIs in a novel model of postoperative SSIs in mice. METHODS: Mice were fed either a standard chow diet (high fiber/low fat, SD) or a western-type diet (low fiber/high fat, WD), and exposed to antibiotics (oral clindamycin/intraperitoneal cefoxitin). Groups of mice had Pi-PEG added to their drinking water and SSI incidence was determined. Gross clinical infections wound cultures and amplicon sequence variant analysis of the intestinal contents and wound were assessed to determine the incidence and source of the developing SSI. RESULTS: In this model, consumption of a WD and exposure to antibiotics promoted the growth of SSI pathogens in the gut and their subsequent presence in the wound. Mice subjected to this model drinking water spiked with Pi-PEG were protected against SSIs via mechanisms involving modulation of the gut-wound microbiome. CONCLUSIONS: A nonantibiotic phosphate-rich polymer, Pi-PEG, added to the drinking water of mice prevents SSIs and may represent a more sustainable approach in lieu of the current trend of greater sterility and the use of more powerful and broader antibiotic coverage.

Defining Microbiome Readiness for Surgery: Dietary Prehabilitation and Stool Biomarkers as Predictive Tools to Improve Outcome.

OBJECTIVES: Determine whether preoperative dietary prehabilitation with a low-fat, high-fiber diet reverses the impact of Western diet (WD) on the intestinal microbiota and improves postoperative survival. BACKGROUND: We have previously demonstrated that WD fed mice subjected to an otherwise recoverable surgical injury (30% hepatectomy), antibiotics, and a short period of starvation demonstrate reduced survival (29%) compared to mice fed a low-fat, high-fiber standard chow (SD) (100%). METHODS: Mice were subjected to 6 weeks of a WD and underwent dietary pre-habilitation (3 days vs 7 days) with a SD prior to exposure to antibiotics, starvation, and surgery. 16S rRNA gene sequencing was utilized to determine microbiota composition. Mass spectrometry measured short chain fatty acids and functional prediction from 16S gene amplicons were utilized to determine microbiota function. RESULTS: As early as 24 hours, dietary prehabilitation of WD mice resulted in restoration of bacterial composition of the stool microbiota, transitioning from Firmicutes dominant to Bacteroidetes dominant. However, during this early pre-habilitation (ie, 3 days), stool butyrate per microbial biomass remained low and postoperative mortality remained unchanged from WD. Microbiota function demonstrated reduced butyrate contributing taxa as potentially responsible for failed recovery. In contrast, after 7 days of prehabilitation (7DP), there was greater restoration of butyrate producing taxa and survival after surgery improved (29% vs 79% vs 100%: WD vs 7DP vs SD, P < 0.001). CONCLUSIONS: The deleterious effects of WD on the gut microbiota can be restored after 7 days of dietary prehabilitation. Moreover, stool markers may define the readiness of the microbiome to withstand the process of surgery including exposure to antibiotics and short periods of starvation.

Prevention of Anastomotic Leak Via Local Application of Tranexamic Acid to Target Bacterial-mediated Plasminogen Activation: A Practical Solution to a Complex Problem.

OBJECTIVE: To investigate the role of bacterial- mediated plasminogen (PLG) activation in the pathogenesis of anastomotic leak (AL) and its mitigation by tranexamic acid (TXA). BACKGROUND: AL is the most feared complication of colorectal resections. The pathobiology of AL in the setting of a technically optimal procedure involves excessive submucosal collagen degradation by resident microbes. We hypothesized that activation of the host PLG system by pathogens is a central and targetable pathway in AL. METHODS: We employed kinetic analysis of binding and activation of human PLG by microbes known to cause AL, and collagen degradation assays to test the impact of PLG on bacterial collagenolysis. Further, we measured the ability of the antifibrinolytic drug TXA to inhibit this process. Finally, using mouse models of pathogen-induced AL, we locally applied TXA via enema and measured its ability to prevent a clinically relevant AL. RESULTS: PLG is deposited rapidly and specifically at the site of colorectal anastomoses. TXA inhibited PLG activation and downstream collagenolysis by pathogens known to have a causal role in AL. TXA enema reduced collagenolytic bacteria counts and PLG deposition at anastomotic sites. Postoperative PLG inhibition with TXA enema prevented clinically and pathologically apparent pathogen-mediated AL in mice. CONCLUSIONS: Bacterial activation of host PLG is central to collagenolysis and pathogen-mediated AL. TXA inhibits this process both in vitro and in vivo. TXA enema represents a promising method to prevent AL in high-risk sites such as the colorectal anastomoses.

Western Diet Promotes Intestinal Colonization by Collagenolytic Microbes and Promotes Tumor Formation After Colorectal Surgery.

BACKGROUND & AIMS: The Western diet, which is high in fat, is a modifiable risk factor for colorectal recurrence after curative resection. We investigated the mechanisms by which the Western diet promotes tumor recurrence, including changes in the microbiome, in mice that underwent colorectal resection. METHODS: BALB/c male mice were fed either standard chow diet or Western-type diet (characterized by high fat, no fiber, and decreased minerals and vitamins) for 4 weeks; some mice were given antibiotics or ABA-PEG20k-Pi20 (Pi-PEG), which inhibits collagenase production by bacteria, but not bacterial growth, in drinking water. Colorectal resections and anastomoses were then performed. The first day after surgery, mice were given enemas containing a collagenolytic rodent-derived strain of Enterococcus faecalis (strain E2), and on the second day they were given mouse colon carcinoma cells (CT26). Twenty-one days later, distal colons were removed, and colon contents (feces, distal colon, and tumor) were collected. Colon tissues were analyzed by histology for the presence of collagenolytic colonies and by 16S ribosomal RNA sequencing, which determined the anatomic distribution of E faecalis at the site of the anastomosis and within tumors using in situ hybridization. Mouse imaging analyses were used to identify metastases. RESULTS: Colorectal tumors were found in 88% of mice fed the Western diet and given antibiotics, surgery, and E faecalis compared with only 30% of mice fed the standard diet followed by the same procedures. Colon tumor formation correlated with the presence of collagenolytic E faecalis and Proteus mirabilis. Antibiotics eliminated collagenolytic E faecalis and P mirabilis but did not reduce tumor formation. However, antibiotics promoted emergence of Candida parapsilosis, a collagenase-producing microorganism. Administration of a Pi-PEG reduced tumor formation and maintained diversity of the colon microbiome. CONCLUSIONS: We identified a mechanisms by which diet and antibiotic use can promote tumorigenesis by colon cancer cells at the anastomosis after colorectal surgery. Strategies to prevent emergence of these microbe communities or their enzymatic activities might be used to reduce the risk of tumor recurrence in patients undergoing colorectal cancer surgery.

Enterococcus faecalis exploits the human fibrinolytic system to drive excess collagenolysis: implications in gut healing and identification of druggable targets.

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing.NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

Oral Polyphosphate Suppresses Bacterial Collagenase Production and Prevents Anastomotic Leak Due to Serratia marcescens and Pseudomonas aeruginosa.

OBJECTIVE: The objective of this study was to determine the effect of polyphosphate on intestinal bacterial collagenase production and anastomotic leak in mice undergoing colon surgery. BACKGROUND: We have previously shown that anastomotic leak can be caused by intestinal pathogens that produce collagenase. Because bacteria harbor sensory systems to detect the extracellular concentration of phosphate which controls their virulence, we tested whether local phosphate administration in the form of polyphosphate could attenuate pathogen virulence and prevent leak without affecting bacterial growth. METHODS: Groups of mice underwent a colorectal anastomosis which was then exposed to collagenolytic strains of either Serratia marcescens or Pseudomonas aeruginosa via enema. Mice were then randomly assigned to drink water or water supplemented with a 6-mer of polyphosphate (PPi-6). All mice were sacrificed on postoperative day 10 and anastomoses assessed for leakage, the presence of collagenolytic bacteria, and anastomotic PPi-6 concentration. RESULTS: PPi-6 markedly attenuated collagenase and biofilm production, and also swimming and swarming motility in both S. marcescens and P. aeruginosa while supporting their normal growth. Mice drinking PPi-6 demonstrated increased levels of PPi-6 and decreased colonization of S. marcescens and P. aeruginosa, and collagenase activity at anastomotic tissues. PPi-6 prevented anastomotic abscess formation and leak in mice after anastomotic exposure to S. marcescens and P. aeruginosa. CONCLUSIONS: Polyphosphate administration may be an alternative approach to prevent anastomotic leak induced by collagenolytic bacteria with the advantage of preserving the intestinal microbiome and its colonization resistance.

Can Methicillin-resistant Staphylococcus aureus Silently Travel From the Gut to the Wound and Cause Postoperative Infection? Modeling the "Trojan Horse Hypothesis".

OBJECTIVE: To determine whether intestinal colonization with methicillin-resistant Staphylococcus aureus (MRSA) can be the source of surgical site infections (SSIs). BACKGROUND: We hypothesized that gut-derived MRSA may cause SSIs via mechanisms in which circulating immune cells scavenge MRSA from the gut, home to surgical wounds, and cause infection (Trojan Horse Hypothesis). METHODS: MRSA gut colonization was achieved by disrupting the microbiota with antibiotics, imposing a period of starvation and introducing MRSA via gavage. Next, mice were subjected to a surgical injury (30% hepatectomy) and rectus muscle injury and ischemia before skin closure. All wounds were cultured before skin closure. To control for postoperative wound contamination, reiterative experiments were performed in mice in which the closed wound was painted with live MRSA for 2 consecutive postoperative days. To rule out extracellular bacteremia as a cause of wound infection, MRSA was injected intravenously in mice subjected to rectus muscle ischemia and injury. RESULTS: All wound cultures were negative before skin closure, ruling out intraoperative contamination. Out of 40 mice, 4 (10%) developed visible abscesses. Nine mice (22.5%) had MRSA positive cultures of the rectus muscle without visible abscesses. No SSIs were observed in mice injected intravenously with MRSA. Wounds painted with MRSA after closure did not develop infections. Circulating neutrophils from mice captured by flow cytometry demonstrated MRSA in their cytoplasm. CONCLUSIONS: Immune cells as Trojan horses carrying gut-derived MRSA may be a plausible mechanism of SSIs in the absence of direct contamination.

Critical role of microbiota within cecal crypts on the regenerative capacity of the intestinal epithelium following surgical stress.

Cecal crypts represent a unique niche that are normally occupied by the commensal microbiota. Due to their density and close proximity to stem cells, microbiota within cecal crypts may modulate epithelial regeneration. Here we demonstrate that surgical stress, a process that invariably involves a short period of starvation, antibiotic exposure, and tissue injury, results in cecal crypt evacuation of their microbiota. Crypts devoid of their microbiota display pathophysiological features characterized by abnormal stem cell activation as judged by leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) staining, expansion of the proliferative zone toward the tips of the crypts, and an increase in apoptosis. In addition, crypts devoid of their microbiota display loss of their regenerative capacity as assessed by their ability to form organoids ex vivo. When a four-member human pathogen community isolated from the stool of a critically ill patient is introduced into the cecum of mice with empty crypts, crypts become occupied by the pathogens and further disruption of crypt homeostasis is observed. Fecal microbiota transplantation restores the cecal crypts' microbiota, normalizes homeostasis within crypts, and reestablishes crypt regenerative capacity. Taken together, these findings define an emerging role for the microbiota within cecal crypts to maintain epithelial cell homeostasis in a manner that may enhance recovery in response to the physiological stress imposed by the process of surgery. NEW & NOTEWORTHY: This study provides novel insight into the process by which surgical injury places the intestinal epithelium at risk for colonization by pathogenic microbes and impairment of its regenerative capacity via loss of its microbiota. We show that fecal transplant restores crypt homeostasis in association with repopulation of the microbiota within cecal crypts.

Emergence of the P2 phenotype in Pseudomonas aeruginosa PAO1 strains involves various mutations in mexT or mexF.

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF.

Phosphate-containing polyethylene glycol polymers prevent lethal sepsis by multidrug-resistant pathogens.

Antibiotic resistance among highly pathogenic strains of bacteria and fungi is a growing concern in the face of the ability to sustain life during critical illness with advancing medical interventions. The longer patients remain critically ill, the more likely they are to become colonized by multidrug-resistant (MDR) pathogens. The human gastrointestinal tract is the primary site of colonization of many MDR pathogens and is a major source of life-threatening infections due to these microorganisms. Eradication measures to sterilize the gut are difficult if not impossible and carry the risk of further antibiotic resistance. Here, we present a strategy to contain rather than eliminate MDR pathogens by using an agent that interferes with the ability of colonizing pathogens to express virulence in response to host-derived and local environmental factors. The antivirulence agent is a phosphorylated triblock high-molecular-weight polymer (here termed Pi-PEG 15-20) that exploits the known properties of phosphate (Pi) and polyethylene glycol 15-20 (PEG 15-20) to suppress microbial virulence and protect the integrity of the intestinal epithelium. The compound is nonmicrobiocidal and appears to be highly effective when tested both in vitro and in vivo. Structure functional analyses suggest that the hydrophobic bis-aromatic moiety at the polymer center is of particular importance to the biological function of Pi-PEG 15-20, beyond its phosphate content. Animal studies demonstrate that Pi-PEG prevents mortality in mice inoculated with multiple highly virulent pathogenic organisms from hospitalized patients in association with preservation of the core microbiome.

Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration.

OBJECTIVE: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. BACKGROUND DATA: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. METHODS: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a µ opioid receptor antagonist. RESULTS: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. CONCLUSIONS: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1.

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir-a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the "red death" response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR-PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS+Fe(3+) complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS+Fe(3+) complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR-PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.

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.

BACKGROUND: 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 P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation. RESULTS: 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 P. aeruginosa had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in C. elegans. Transcriptional analysis of P. aeruginosa 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. CONCLUSION: Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by P. aeruginosa 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.

Structure-function aspects of PstS in multi-drug-resistant Pseudomonas aeruginosa.

The increasing prevalence of multi-drug-resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface-expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic DeltaPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%-100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant DeltaHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14-55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium.

Spatial Compartmentalization of the Microbiome between the Lumen and Crypts Is Lost in the Murine Cecum following the Process of Surgery, Including Overnight Fasting and Exposure to Antibiotics.

The cecum is a unique region in the mammalian intestinal tract in which the microbiome is localized to two compartments, the lumen and the crypts. The microbiome within crypts is particularly important as it is in direct contact with lining epithelial cells including stem cells. Here, we analyzed the microbiome in cecum of mice using multiple techniques including metagenomics. The lumen microbiome comprised Firmicutes and Bacteroidetes whereas the crypts were dominated by Proteobacteria and Deferribacteres, and the mucus comprised a mixture of these 4 phyla. The lumen microbial functional potential comprised mainly carbon metabolism, while the crypt microbiome was enriched for genes encoding stress resistance. In order to determine how this structure, assembly, and function are altered under provocative conditions, we exposed mice to overnight starvation (S), antibiotics (A), and a major surgical injury (partial hepatectomy [H]), as occurs with major surgery in humans. We have previously demonstrated that the combined effect of this "SAH" treatment leads to a major disturbance of the cecal microbiota at the bottom of crypts in a manner that disrupts crypt cell homeostasis. Here, we applied the SAH conditions and observed a loss of compartmentalization in both composition and function of the cecal microbiome associated with major shifts in local physicochemical cues including decrease of hypoxia, increase of pH, and loss of butyrate production. Taken together, these studies demonstrated a defined order, structure, and function of the cecal microbiome that can be disrupted under provocative conditions such as major surgery and its attendant exposures.IMPORTANCE The proximal colon and cecum are two intestinal regions in which the microbiome localizes to two spatially distinct compartments, the lumen and crypts. The differences in composition and function of luminal and crypt microbiome in the cecum and the effect of physiological stress on their compartmentalization remain poorly characterized. Here, we characterized the composition and function of the lumen-, mucus-, and crypt-associated microbiome in the cecum of mice. We observed a highly ordered microbial architecture within the cecum whose assembly and function become markedly disrupted when provoked by physiological stress such as surgery and its attendant preoperative treatments (i.e., overnight fasting and antibiotics). Major shifts in local physicochemical cues including a decrease in hypoxia levels, an increase in pH, and a loss of butyrate production were associated with the loss of compositional and functional compartmentalization of the cecal microbiome.

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

Oral PEG 15-20 protects the intestine against radiation: role of lipid rafts.

Intestinal injury following abdominal radiation therapy or accidental exposure remains a significant clinical problem that can result in varying degrees of mucosal destruction such as ulceration, vascular sclerosis, intestinal wall fibrosis, loss of barrier function, and even lethal gut-derived sepsis. We determined the ability of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect the intestine against the early and late effects of radiation in mice and rats and to determine its mechanism of action by examining cultured rat intestinal epithelia. Rats were exposed to fractionated radiation in an established model of intestinal injury, whereby an intestinal segment is surgically placed into the scrotum and radiated daily. Radiation injury score was decreased in a dose-dependent manner in rats gavaged with 0.5 or 2.0 g/kg per day of PEG 15-20 (n = 9-13/group, P < 0.005). Complementary studies were performed in a novel mouse model of abdominal radiation followed by intestinal inoculation with Pseudomonas aeruginosa (P. aeruginosa), a common pathogen that causes lethal gut-derived sepsis following radiation. Mice mortality was decreased by 40% in mice drinking 1% PEG 15-20 (n = 10/group, P < 0.001). Parallel studies were performed in cultured rat intestinal epithelial cells treated with PEG 15-20 before radiation. Results demonstrated that PEG 15-20 prevented radiation-induced intestinal injury in rats, prevented apoptosis and lethal sepsis attributable to P. aeruginosa in mice, and protected cultured intestinal epithelial cells from apoptosis and microbial adherence and possible invasion. PEG 15-20 appeared to exert its protective effect via its binding to lipid rafts by preventing their coalescence, a hallmark feature in intestinal epithelial cells exposed to radiation.

Genomic Characterization and Virulence Potential of Two Fusarium oxysporum Isolates Cultured from the International Space Station.

Two isolates of Fusarium oxysporum, ISS-F3 and ISS-F4, were cultured from the dining table on the International Space Station (ISS). Genomic analyses using EF-1α sequences, presence/absence of effector proteins, k-mer comparisons, and single nucleotide polymorphisms indicate that these two strains are genomically different from 65 known sequenced strains. Functional analysis revealed that ISS-F3/F4 had higher relative abundances of polyketide synthase domains than a non-plant-pathogenic soil isolate, used for biocontrol properties (Fo47), and a clinical isolate (FOSC-3a). Putative secondary metabolite analysis indicates that ISS-F3/F4 may produce yet-unreported polyketides and nonribosomal peptides. While genomic analysis showed that these ISS strains are unlikely to be plant pathogens, a virulence assay using an immunocompromised Caenorhabditis elegans model of fusariosis revealed that they were virulent and may represent opportunistic pathogens in animals, including humans. However, its effects on the health of immunocompromised humans warrant further study. IMPORTANCE This is the first study to isolate and characterize F. oxysporum isolates from a built environment, as well as one that has been exposed to space. The characterization and analysis of these two genomes may have important implications for the medical, agricultural, and food industries as well as for the health of the crew who coinhabit the ISS with these strains.

Mice Fed an Obesogenic Western Diet, Administered Antibiotics, and Subjected to a Sterile Surgical Procedure Develop Lethal Septicemia with Multidrug-Resistant Pathobionts.

Despite antibiotics and sterile technique, postoperative infections remain a real and present danger to patients. Recent estimates suggest that 50% of the pathogens associated with postoperative infections have become resistant to the standard antibiotics used for prophylaxis. Risk factors identified in such cases include obesity and antibiotic exposure. To study the combined effect of obesity and antibiotic exposure on postoperative infection, mice were allowed to gain weight on an obesogenic Western-type diet (WD), administered antibiotics and then subjected to an otherwise recoverable sterile surgical injury (30% hepatectomy). The feeding of a WD alone resulted in a major imbalance of the cecal microbiota characterized by a decrease in diversity, loss of Bacteroidetes, a bloom in Proteobacteria, and the emergence of antibiotic-resistant organisms among the cecal microbiota. When WD-fed mice were administered antibiotics and subjected to 30% liver resection, lethal sepsis, characterized by multiple-organ damage, developed. Notable was the emergence and systemic dissemination of multidrug-resistant (MDR) pathobionts, including carbapenem-resistant, extended-spectrum ß-lactamase-producing Serratia marcescens, which expressed a virulent and immunosuppressive phenotype. Analysis of the distribution of exact sequence variants belonging to the genus Serratia suggested that these strains originated from the cecal mucosa. No mortality or MDR pathogens were observed in identically treated mice fed a standard chow diet. Taken together, these results suggest that consumption of a Western diet and exposure to certain antibiotics may predispose to life-threating postoperative infection associated with MDR organisms present among the gut microbiota.IMPORTANCE Obesity remains a prevalent and independent risk factor for life-threatening infection following major surgery. Here, we demonstrate that when mice are fed an obesogenic Western diet (WD), they become susceptible to lethal sepsis with multiple organ damage after exposure to antibiotics and an otherwise-recoverable surgical injury. Analysis of the gut microbiota in this model demonstrates that WD alone leads to loss of Bacteroidetes, a bloom of Proteobacteria, and evidence of antibiotic resistance development even before antibiotics are administered. After antibiotics and surgery, lethal sepsis with organ damage developed in in mice fed a WD with the appearance of multidrug-resistant pathogens in the liver, spleen, and blood. The importance of these findings lies in exposing how the selective pressures of diet, antibiotic exposure, and surgical injury can converge on the microbiome, resulting in lethal sepsis and organ damage without the introduction of an exogenous pathogen.

De Novo Synthesis of Phosphorylated Triblock Copolymers with Pathogen Virulence-Suppressing Properties That Prevent Infection-Related Mortality.

Phosphate is a key and universal "cue" in response to which bacteria either enhance their virulence when local phosphate is scarce or downregulate it when phosphate is adundant. Phosphate becomes depleted in the mammalian gut following physiologic stress and serves as a major trigger for colonizing bacteria to express virulence. This process cannot be reversed with oral supplementation of inorganic phosphate because it is nearly completely absorbed in the proximal small intestine. In the present study, we describe the de novo synthesis of phosphorylated polyethylene glycol compounds with three defined ABA (hydrophilic/-phobic/-philic) structures, ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20, and linear polymer PEG20k-Pi20 absent of the hydrophobic block. The 10k, 16k, and 20k demonstrate the molecular weights of the poly(ethylene glycol) block, and Pi10, Pi14, and Pi20 represent the repeating units of phosphate. Polymers were tested for their efficacy against Pseudomonas aeruginosa virulence in vitro and in vivo by assessing the expression of the phosphate sensing protein PstS, the production of key virulence factor pyocyanin, and Caenorhabditis elegans killing assays. Results indicate that all phosphorylated polymers suppressed phosphate sensing, virulence expression, and lethality in P. aeruginosa. Among all of the phosphorylated polymers, ABA-PEG20k-Pi20 displayed the greatest degree of protection against P. aeruginosa. To define the role of the hydrophobic core in ABA-PEG20k-Pi20 in the above response, we synthesized PEG20k-Pi20 in which the hydrophobic core is absent. Results indicate that the hypdrophobic core of ABA-PEG20k-Pi20 is a key structure in its protective effect against P. aeruginosa, in part due to its ability to coat the surface of bacteria. Taken together, the synthesis of novel polymers with defined structures and levels of phosphorylation may elucidate their antivirulence action against clinically important and lethal pathogens such as P. aeruginosa.