Trissa Babrowski

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+Fe3+ 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+Fe3+ 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.

Candida albicans isolates from the gut of critically ill patients respond to phosphate limitation by expressing filaments and a lethal phenotype.

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 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 &mgr; 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.

The human microbiome and surgical disease.

Objective:The purpose of this review article is to summarize what is currently known about microbes associated with the human body and to provide examples of how this knowledge impacts the care of surgical patients. Background:Pioneering research over the past decade has demonstrated that human beings live in close, constant contact with dynamic communities of microbial organisms. This new reality has wide-ranging implications for the care of surgical patients. Methods and Results:Recent advances in the culture-independent study of the human microbiome are reviewed. To illustrate the translational relevance of these studies to surgical disease, we discuss in detail what is known about the role of microbes in the pathogenesis of obesity, gastrointestinal malignancies, Crohn disease, and perioperative complications including surgical site infections and sepsis. The topics of mechanical bowel preparation and perioperative antibiotics are also discussed. Conclusions:Heightened understanding of the microbiome in coming years will likely offer opportunities to refine the prevention and treatment of a wide variety of surgical conditions.

Host Stress and Virulence Expression in Intestinal Pathogens: Development of Therapeutic Strategies Using Mice and C. elegans

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.

Pseudomonas aeruginosa potentiates the lethal effect of intestinal ischemia-reperfusion injury: the role of in vivo virulence activation.

BACKGROUND Experimental models of intestinal ischemia-reperfusion (IIR) injury are invariably performed in mice harboring their normal commensal flora, even though multiple IIR events occur in humans during prolonged intensive care confinement when they are colonized by a highly pathogenic hospital flora. The aims of this study were to determine whether the presence of the human pathogen Pseudomonas aeruginosa in the distal intestine potentiates the lethality of mice exposed to IIR and to determine what role any in vivo virulence activation plays in the observed mortality. METHODS Seven- to 9-week-old C57/BL6 mice were exposed to 15 minutes of superior mesenteric artery occlusion (SMAO) followed by direct intestinal inoculation of 1.0 × 10(6) colony-forming unit of P. aeruginosa PAO1 into the ileum and observed for mortality. Reiterative studies were performed in separate groups of mice to evaluate both the migration/dissemination pattern and in vivo virulence activation of intestinally inoculated strains using live photon camera imaging of both a constitutive bioluminescent P. aeruginosa PAO1 derivative XEN41 and an inducible reporter derivative of PAO1, the PAO1/lecA:luxCDABE that conditionally expresses the quorum sensing-dependent epithelial disrupting virulence protein PA 1 Lectin (PA-IL). RESULTS Mice exposed to 15 minutes of SMAO and reperfusion with intestinal inoculation of P. aeruginosa had a significantly increased mortality rate (p < 0.001) of 100% compared with <10% for sham-operated mice intestinally inoculated with P. aeruginosa without SMAO and IIR alone (<50%). Migration/dissemination patterns of P. aeruginosa in mice subjected to IIR demonstrated proximal migration of distally injected strains and translocation to mesenteric lymph nodes, liver, spleen, lung, and kidney. A key role for in vivo virulence expression of the barrier disrupting adhesin PA-IL during IIR was established since its expression was enhanced during IR and mutant strains lacking PA-IL displayed attenuated mortality. CONCLUSIONS The presence of intestinal P. aeruginosa potentiates the lethal effect of IIR in mice in part due to in vivo virulence activation of its epithelial barrier disrupting protein PA-IL.

Resection of Intracaval Leiomyomatosis Using Abdominal Approach and Venovenous Bypass

BACKGROUND Intravenous leiomyomatosis is the venous involvement of a histologically benign uterine tumor. This uncommon tumor can present contemporaneously with the primary uterine tumor or in a delayed fashion. Tumor extends up the venous system, via the iliac or ovarian veins, and can involve portions or all of the inferior vena cava and can extend into the heart as well. Complete resection of this tumor is the therapeutic goal. Previous reports have described the use of combined thoracic and abdominal approaches, cardiopulmonary bypass, circulatory arrest, and a single report of an entirely abdominal approach to resection without bypass. METHODS AND RESULTS We present a review of the existing literature describing surgical intervention for intravenous leiomyomatosis and describe two cases of tumor extending up the intra-abdominal vena cava. Using venovenous bypass without need for thoracotomy, we were able to resect both tumors with minimal blood loss and no hemodynamic instability. CONCLUSIONS We suggest that venovenous bypass is an excellent tool in resection of these tumors and should be considered for many cases in lieu of full cardiopulmonary bypass or circulatory arrest.

IP089. Gut Bacteria Influence the Growth of Abdominal Aortic Aneurysms in Mice

TEVAR, Thoracic endovascular aortic repair. Models are adjusted for age, sex, race, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, coronary artery disease, hemodialysis, presentation, prior abdominal aortic aneurysm repair, proximal and distal zone, maximum preoperative aortic diameter, American Society of Anesthesiologists class, and statins. e112 Abstracts Journal of Vascular Surgery June 2018

Anatomic and clinical characterization of the narrow distal aorta and implications after endovascular aneurysm repair

Objective: The purpose of this analysis was to compare 1‐year clinical outcomes after endovascular repair of abdominal aortic aneurysms with the EXCLUDER device in patients with standard and narrow aortic bifurcations (AOBs). Methods: Data were prospectively collected from a 1055‐participant subset of the multicenter Global Registry for Endovascular Aortic Treatment (GREAT) treated for abdominal aortic aneurysm repair between August 2010 and September 2015. There were 117 patients with a narrow AOB (NB; defined as <16 mm) and 938 patients with a standard bifurcation (SB). The 30‐day and 1‐year morbidity, mortality, and reintervention outcomes were analyzed, with Kaplan‐Meier survival curve analysis conducted on freedom from mortality and freedom from reintervention. Results: The mean distal aortic neck diameter was 12.4 mm in the NB cohort and 25.3 mm in the SB cohort (P < .001), with NB patients also exhibiting significantly smaller diameter proximal aortic necks (P < .001). Patients in the NB cohort were more often female (25.6% vs 15.1%; P = .004) and with more severe comorbidity burden. There was a significantly higher rate of surgical cutdown access in the NB cohort (P < .001). Procedural survival was 100% in both groups. The 30‐day mortality and safety outcomes were similar; however, all‐cause mortality was significantly higher in the SB cohort through 1 year (P = .02). The 1‐year freedom from mortality was estimated as 92.1% in the SB cohort and 99.1% in the NB cohort. Freedom from reintervention was estimated as 95.1% in the SB cohort and 92.8% in the NB cohort at 1 year. Through 1‐year follow‐up, 24 SB patients (2.6%) and 4 NB patients (3.4%) exhibited an endoleak requiring reintervention (P > .99). Type II endoleaks represented 72% and 60% of treated endoleaks, respectively. Through 1 year, 10 SB patients (1.0%) and 2 NB patients (1.7%) exhibited occlusive/thrombotic events (P = .54). There were no reported instances of kinking, migration, fracture, compression, or dissection through 1 year in either cohort. One SB patient experienced thoracic aortic aneurysm rupture. Conclusions: The 1‐year outcomes after endovascular aneurysm repair with the EXCLUDER device were comparable in the NB and SB cohorts. A narrow AOB was not found to be associated with a higher incidence of later limb occlusions or endoleaks. Female patients were disproportionately more likely to have a narrow AOB, which correlated with narrowed proximal necks and access vessels, and a more severe comorbidity burden.