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OBJECTIVE: To investigate the role of bacterial- mediated plasminogen activation in the pathogenesis of anastomotic leak (AL) and its mitigation by tranexamic acid. SUMMARY BACKGROUND DATA: 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 plasminogen (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 tranexamic acid (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.

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.

To determine whether intestinal colonization with methicillin-resistant Staphylococcus aureus (MRSA) can be the source of surgical site infections (SSIs).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).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.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.Immune cells as Trojan horses carrying gut-derived MRSA may be a plausible mechanism of SSIs in the absence of direct contamination.

BACKGROUND Acinetobacter baumannii has emerged as an increasingly important and successful opportunistic human pathogen due to its ability to withstand harsh environmental conditions, its characteristic virulence factors, and quick adaptability to stress. METHODS We developed a clinically relevant murine model of A. baumannii traumatic wound infection to determine the effect of local wound environment on A. baumannii virulence. Mice underwent rectus muscle crush injury combined with ischemia created by epigastric vessel ligation, followed by A. baumannii inoculation. Reiterative experiments were performed using (1) a mutant deficient in the production of the siderophore acinetobactin, or (2) iron supplementation of the wound milieu. Mice were euthanized 7 days later, and rectus muscle analyzed for signs of clinical infection, HIF1α accumulation, bacterial abundance, and colony morphotype. To determine the effect of wound milieu on bacterial virulence, Galleria mellonella infection model was used. RESULTS The combination of rectus muscle injury with ischemia and A. baumannii inoculation resulted in 100% incidence of clinical wound infection that was significantly higher compared with other groups (n = 15/group, p < 0.0001). The highest level of wound infection was accompanied by the highest level of A. baumannii colonization (p < 0.0001) and the highest degree of HIF1α accumulation (p < 0.05). A. baumannii strains isolated from injured/ischemic muscle with clinical infection displayed a rough morphotype and a higher degree of virulence as judged by G. mellonella killing assay as compared with smooth morphotype colonies isolated from injured muscle without clinical infection (100% vs. 60%, n = 30 Log-Rank test, p = 0.0422). Iron supplementation prevented wound infection (n = 30, p < 0.0001) and decreased HIF1α (p = 0.039643). Similar results of decrease in wound infection and HIF1α were obtained when A. baumannii wild type was replaced with its derivative mutant [INCREMENT]BasD deficient in acinetobactin production. CONCLUSION The ability of A. baumannii to cause infections in traumatized wound relies on its ability to scavenge iron and can be prevented by iron supplementation to the wound milieu.

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.

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 β-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.

Current surgical dogma dictates that tissue ischemia and hypoxia are major contributing factors in anastomotic leak despite scant evidence. The aim of this study was to determine if tissue hypoxia is a feature of anastomotic leakage in rats following colon resection and segmental devascularization. Rats were randomly assigned to undergo sham operation, segmental colon devascularization alone, colectomy alone, or segmental devascularization plus colectomy. Tissue hypoxia present at the colon anastomosis site across the various treatment groups was determined at sacrifice on postoperative day 6. Pimonidazole HCl was injected 30 min prior to sacrifice. Anastomotic tissues were examined and scored for healing versus leakage using an anastomotic healing score (AHS). Collagen content, hypoxia, enteric smooth muscle and periendothelial stromal patterning, and apoptosis were evaluated histologically. No differences in tissue hypoxia were noted in the 16% of anastomotic tissues with poor healing compared to the remaining 84% of rats whose anastomoses healed well. No significant changes were found in cell death in the submucosa of any group. Consistent with previous findings, poor healing was associated with lower collagen content. Submucosal thickness correlated with increased arteriole diameter (R 2 = 0.25, p

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., 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.

BACKGROUND: Despite ever more powerful antibiotics, newer surgical techniques, and enhanced recovery programs, anastomotic leaks remain a clear and present danger to patients. Previous work from our laboratory suggests that anastomotic leakage may be caused by Enterococcus faecalis strains that express a high collagenase phenotype (i.e., collagenolytic). Yet the mechanisms by which the practice of surgery shifts or selects for collagenolytic phenotypes to colonize anastomotic tissues remain unknown. METHODS: Here, we hypothesized that morphine, an analgesic agent universally used in gastrointestinal surgery, promotes tissue colonization with collagenolytic E. faecalis and causes anastomotic leak. To test this, rats were administered morphine in a chronic release form as would occur during routine surgery or vehicle. Rats were observed for 6 days and then underwent exploratory laparotomy for anastomotic inspection and tissue harvest for microbial analysis. These results provide further rationale to enhanced recovery after surgery (i.e., ERAS) programs that suggest limiting or avoiding the use of opioids in gastrointestinal surgery. RESULTS: Results demonstrated that compared to placebo-treated rats, morphine-treated rats demonstrated markedly impaired anastomotic healing and gross leaks that correlated with the presence of high collagenase-producing E. faecalis adherent to anastomotic tissues. To determine the direct role of morphine on this response, various isolates of E. faecalis from the rats were exposed to morphine and their collagenase activity and adherence capacity determined in vitro. Morphine increased both the adhesiveness and collagenase production of four strains of E. faecalis harvested from anastomotic tissues, two that were low collagenase producers at baseline, and two that were high collagenase producers at baseline. CONCLUSION: These results provide further rationale to enhanced recovery after surgery (i.e., ERAS) programs that suggest limiting or avoiding the use of opioids in gastrointestinal 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.

We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

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.

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 (P i ) 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.

Phosphate signaling and acquisition are critical for the bacterial response to phosphate limitation, and bacteria express multiple factors to scavenge phosphate. We previously found that multidrug-resistant strains of Pseudomonas aeruginosa from critically ill patients can form unusual outer-surface appendages harboring PstS proteins. Here, we have expanded our investigation to DING proteins that like PstS belong to the family of high-affinity phosphate-binding proteins but have strong similarity with eukaryotic DING proteins. We demonstrate the localization of DING on PstS-containing outer-surface appendages in both multidrug-resistant strain MDR25 and the PA14 strain of P. aeruginosa. However, the number of cells producing appendages and the amount of appendages on each cell in PA14 were found to be negligible, unless overexpression of either PstS or DING was achieved by transformation with constructed plasmids. We further noticed that DING expression under low phosphate conditions was significantly higher in MDR25 compared to PA14 which may explain the greater abundance of appendages in MDR25. Our finding that DING proteins are localized on extracellular appendages provides an opportunity to study the interaction of bacterial DING with host proteins by mimicking the action of host DINGs.

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. SUMMARY BACKGROUND DATA: 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 as well as 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 following 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.

Sepsis following surgical injury remains a growing and worrisome problem following both emergent and elective surgery. Although early resuscitation efforts and prompt antibiotic therapy have improved outcomes in the first 24 to 48 h, late onset sepsis is now the most common cause of death in modern intensive care units. This time shift may be, in part, a result of prolonged exposure of the host to the stressors of critical illness which, over time, erode the health promoting intestinal microbiota and allow for virulent pathogens to predominate. Colonizing pathogens can then subvert the immune system and contribute to the deterioration of the host response. Here, we posit that novel approaches integrating the molecular, ecological, and evolutionary dynamics of the evolving gut microbiome/pathobiome during critical illness are needed to understand and prevent the late onset sepsis that develops following prolonged critical illness.

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.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.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.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.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.

The intestinal tract of a host exposed to extreme physiologic stress and modern medical intervention represents a relatively unexplored yet important area of infection research, given the frequency with which this site becomes colonized by highly pathogenic microorganisms that cause subsequent sepsis. Our laboratory has focused on the host tissue derived environmental cues that are released into the intestinal tract during extreme physiologic stress that induce the expression of virulence in colonizing pathogens with the goal of developing novel gut directed therapies that maintain host pathogen neutrality through the course of host stress. Here we demonstrate that maintenance of phosphate sufficiency/ abundance within the intestinal microenvironment may be considered as a universal strategy to prevent virulence activation across a broad range of pathogens that colonize the gut and cause sepsis, given that phosphate depletion occurs following stress and is a universal cue that activates the virulence of a wide variety of organisms. Using small animal models (Caenorhabditis elegans and mice) to create local phosphate depletion at sites of colonization of Pseudomonas aeruginosa, a common cause of lethal gut-derived sepsis, we demonstrate the importance of maintaining phosphate sufficiency to suppress the expression of a lethal phenotype during extreme physiologic stress. The molecular details and potential therapeutic implications are reviewed.

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.

Background We explored the possibility that the opportunistic pathogen, Pseudomonas aeruginosa senses low phosphate (Pi) as a signal of host injury and shifts to a lethal phenotype. Methods Virulence expression in P aeruginosa was examined in vitro under low phosphate conditions by assessing expression of the PA-I lectin, a barrier dysregulating protein, pyocyanin, and biofilm production, and PstS, a phosphate scavenging protein. Virulence expression in vivo was assessed using operatively injured mice (30% hepatectomy) intestinally inoculated with P aeruginosa . Results In vitro experiments demonstrated that acute phosphate depletion resulted in an increase ( P = .001) in the expression the PA-I lectin, biofilm, pyocyanin, and PstS. Operative injury caused a depletion (P = .006) of intestinal phosphate concentration and increased mortality (60%) owing to intestinal P aeruginosa , which was prevented completely with oral phosphate supplementation and restoration of intestinal phosphate, neither of which were observed with systemic (IV) administration. PstS gene expression was 32-fold higher in P aeruginosa recovered from the cecum after hepatectomy indicating inadequate intestinal Pi. Conclusions Operative injury-induced intestinal phosphate depletion shifts the phenotype of P aeruginosa to express enhanced virulence in vitro and lethality in vivo. Intestinal phosphate repletion may be a novel strategy to contain pathogens associated with lethal gut-derived sepsis.

Even under the most expert care, a properly constructed intestinal anastomosis can fail to heal, resulting in leakage of its contents, peritonitis, and sepsis. The cause of anastomotic leak remains unknown, and its incidence has not changed in decades. We demonstrate that the commensal bacterium Enterococcus faecalis contributes to the pathogenesis of anastomotic leak through its capacity to degrade collagen and to activate tissue matrix metalloproteinase 9 (MMP9) in host intestinal tissues. We demonstrate in rats that leaking anastomotic tissues were colonized by E. faecalis strains that showed an increased collagen-degrading activity and also an increased ability to activate host MMP9, both of which contributed to anastomotic leakage. We demonstrate that the E. faecalis genes gelE and sprE were required for E. faecalis-mediated MMP9 activation. Either elimination of E. faecalis strains through direct topical antibiotics applied to rat intestinal tissues or pharmacological suppression of intestinal MMP9 activation prevented anastomotic leak in rats. In contrast, the standard recommended intravenous antibiotics used in patients undergoing colorectal surgery did not eliminate E. faecalis at anastomotic tissues nor did they prevent leak in our rat model. Finally, we show in humans undergoing colon surgery and treated with the standard recommended intravenous antibiotics that their anastomotic tissues still contained E. faecalis and other bacterial strains with collagen-degrading/MMP9-activating activity. We suggest that intestinal microbes with the capacity to produce collagenases and to activate host metalloproteinase MMP9 may break down collagen in the intestinal tissue contributing to anastomotic leak.

We 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., IMPORTANCE During 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.

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.

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (≈ 10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies.

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient.

Pseudomonas aeruginosa is a model pathogen with which to advance the notion that microbial endocrinology plays a central role in the pathogenesis of bacteria and other microbes. P. aeruginosa is a gram-negative opportunistic pathogen that can infect a variety of host species, including Arabidopsis, Drosophila, Caenorhabditis elegans, rodents, and man. Like many opportunistic pathogens, virulence expression in P. aeruginosa is not an invariant phenotype. Some investigators consider P. aeruginosa to be an accidental pathogen to man given that it does not appear to have co-evolved with the human immune system; as such it has been assumed to be rarely part of the normal commensal flora. Yet more comprehensive genome-based analyses of the human intestinal microflora suggest that P. aeruginosa is present in up to 20% of normal healthy individuals (Marshall 1991). Although primarily considered to be a nosocomial pathogen that infects the injured and immunocompromised host, P. aeruginosa appears to be the most common cause of infection-related deaths among patients with cystic fibrosis, a genetic disorder of the respiratory epithelium. In this latter host, P. aeruginosa is a chronic colonizer that can persist for many years where it often exerts only moderate virulence.

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.