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ABSTRACT: INTRODUCTION: Colorectal anastomotic leak, a feared complication, results in significantly increased patient morbidity, mortality, and hospital resource utilization. The overall incidence of colorectal anastomotic leak is approximately 11 % with increasing rate the closer the anastomosis is to the anal verge. Because surgeons are unable to reliably predict which anastomosis would fail, most will construct a diverting ileostomy for low colorectal anastomosis to circumvent the devastating complications of anastomotic failure. Despite extensive investigations on technical considerations of anastomosis construction, anastomotic leaks continue to occur at an unacceptably high rate. DISCUSSION: In this review, we examine the major known risk factors and technical considerations that have been implicated as factors in leakage. Although surgical technique has evolved over the past several decades with the advent of newer surgical staplers, laparoscopy, and robotics, we have not witnessed a decrease in the incidence of colorectal anastomotic leaks suggesting that the fundamental pathogenesis of anastomotic leak remains unknown. Among the factors contributing to anastomotic healing, intestinal bacteria remains largely overlooked even though compelling evidence exist that intraluminal microbes could play a major role in pathogenesis of anastomotic leak. Further investigation focusing on intestinal microbes could be one such avenue for uncovering the elusive cause of colorectal anastomotic leak.
Journal of Gastrointestinal Surgery 05/2013; · 2.83 Impact Factor
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ABSTRACT: BACKGROUND: During organ transplantation, it is inevitable that tissues undergo cold ischemia during harvest and transport before implantation. Polyethylene-based polymers have been proposed and tested as preservation agents, with promising results. We have previously reported that a high molecular weight polyethylene glycol (PEG) (15-20,000 MW; PEG 15-20) protects the intestinal epithelium against a variety of cellular stresses, including radiation injury and microbial invasion, by mechanisms that appear to involve lipid rafts. The aim of this study was to determine the preservation effect of PEG 15-20 on the integrity of intestine grafts harvested for subsequent transplantation. MATERIALS AND METHODS: We harvested intestinal grafts from mice using a complete surgical technique for intestinal transplantation and assessed them for the effect of PEG on graft tissue integrity. We preserved half of the grafts in histidine-tryptophan-ketoglutarate solution (HTK) alone and half in HTK-PEG 15-20 solution at 4°C for 24 h. We examined gross morphology, wet to dry ratios, histology, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick end labeling assay for apoptosis, goblet cell numbers, and bacterial localization studies to evaluate the effect of PEG on tissue integrity. RESULTS: Results demonstrated that PEG 15-20 had a superior preservation effect over HTK alone in all parameters tested. The effect of PEG was notable on attenuation of epithelial apoptosis, preservation of mucus-producing cells, and bacterial adherence to the epithelium. CONCLUSIONS: Taken together, these studies suggest that use of PEG 15-20 as a potential adjuvant during intestinal transplant may offer significant promise to prolong graft survival during organ harvest.
Journal of Surgical Research 03/2013; · 2.25 Impact Factor
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ABSTRACT: Abstract Background: Pseudomonas aeruginosa is an opportunistic, gram-negative pathogen associated with many hospital-acquired infections and disease states. In particular, P. aeruginosa has been identified as a crucial factor in the pathogenesis of neonatal necrotizing enterocolitis (NEC). This condition presents more frequently in infants fed a formula-based diet, which may be a result of the specific monosaccharide content of this diet. We hypothesized that P. aeruginosa would express virulence genes differentially when exposed to monosaccharides present in formula versus those in human milk. Methods: Using the results of a metabolomics study on infant diets and their resulting fecal samples, we identified several monosaccharides that distinguished milk from formula diets. Of these compounds, four were found to be metabolized by P. aeruginosa. We subsequently grew P. aeruginosa in tryptic soy broth (TSB) supplemented with these four monosaccharides and used quantitative reverse transcriptase-polymerase chain reaction to measure the expression of 59 major P. aeruginosa virulence genes. The results were standardized to an external control of P. aeruginosa grown in TSB alone. Results: P. aeruginosa did not respond differentially to the monosaccharides after 6 h of growth. However, after 24 h, the organism grown in arabinose (present in formula), xylose (present in human milk), and galactose (present in both formula and feces from milk-fed infants) displayed a significant increase in the expression of virulence genes in all categories. In contrast, P. aeruginosa grown in mannose (present in the feces of milk-fed infants) displayed a significant decrease in virulence gene expression. Conclusion: These results demonstrate the importance of nutrient content on the relative expression of virulence genes in pathogens that colonize commonly the gut of infants. Understanding the effect of current dietary formulas on virulence gene expression in various gut-colonizing pathogens may present a new approach to elucidating the differences between human milk and formula in the development of NEC.
Surgical Infections 03/2013; · 1.80 Impact Factor
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ABSTRACT: BACKGROUND: There is a growing recognition of the significance of host-pathogen interactions (HPIs) in gut biology leading to a reassessment of the role of bacteria in intestinal anastomotic leak. Understanding the complexities of the early postsurgical gut HPI requires integrating knowledge of both epithelial and bacterial behaviors to generate hypotheses of potential mechanisms of interaction. Agent-based modeling is a computational method well suited to achieve this goal, and we use an agent-based model (ABM) to examine alterations in the HPI affecting reestablishment of the epithelial barrier that may subsequently lead to anastomotic leak. METHODS: Computational agents representing Pseudomonas aeruginosa were added to a previously validated ABM of epithelial restitution. Simulated experiments were performed examining the effect of radiation on bacterial binding to epithelial cells, plausibility of putative binding targets, and potential mechanisms of epithelial cell killing by virulent bacteria. RESULTS: Simulation experiments incorporating radiation effects on epithelial monolayers produced binding patterns akin to those seen in vitro and suggested that promotility integrin-laminin associations represent potential sites for bacterial binding and disruption of restitution. Simulations of potential mechanisms of epithelial cell killing suggested that an injected cytotoxin was the means by which virulent bacteria produced the tissue destruction needed to generate an anastomotic leak, a mechanism subsequently confirmed with genotyping of the virulent P aeruginosa strain. CONCLUSIONS: This study emphasizes the utility of ABM as an adjunct to traditional research methods and provides insights into the potentially critical role of HPI in the pathogenesis of anastomotic leak.
Journal of Surgical Research 01/2013; · 2.25 Impact Factor
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ABSTRACT: Damage to an epithelial surface disrupts its mechanical and immunologic barrier function and exposes underlying tissues to a potentially hostile external environment. Epithelial restitution occurs quickly to reestablish the barrier and comprises a major part of the immediate host response to injured tissue. Pathways involving transforming growth factor beta and activation of epidermal growth factor receptor are both of critical importance, although cross-pathway interactions have been poorly characterized. Agent-based modeling has been showed to be useful in integrating disparate bodies of knowledge and showing the dynamic consequences of pathway structures and cellular population behavior and is used herein to create an in silico analog of an in vitro scratch assay. The In Vitro Scratch Agent-Based Model consists of agents representing individual epithelial cells in a simulated extracellular matrix. Agents sense signals from the damaged environment and produce effector molecules, leading to their healing behavior. The In Vitro Scratch Agent-Based Model qualitatively matched wound healing dynamics when compared against data from traditional experiments. Putative cross-talk mechanisms were then instantiated into the In Vitro Scratch Agent-Based Model and their relative plausibility examined, suggesting interaction at the receptor tyrosine kinase level. This highlights the utility of dynamic knowledge representation in the integration of pathways previously studied in separate contexts.
Wound Repair and Regeneration 10/2012; · 2.91 Impact Factor
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ABSTRACT: BACKGROUND: Secondary peritonitis continues to carry a high mortality rate despite the aggressive use of imaging, drainage, and antibiotics. Although host factors and microbial burden contribute to the outcome of peritonitis, we propose a role for bacterial virulence as a determinant of outcome from peritonitis. Bacterial virulence is an inducible trait that is activated in response to specific local "cues" that we have previously shown to be present in the mouse gut exposed to surgical stress and injury. METHODS: Pseudomonas aeruginosa was harvested after its intestinal inoculation into the cecum of mice subjected to surgical injury (30% hepatectomy) or sham surgery (controls). Harvested strains were then injected into the peritoneum of noninjured (naïve) mice and mortality determined. RESULTS: P. aeruginosa harvested from the intestines of surgically injured mice caused 100% mortality, whereas strains harvested from control mice caused no mortality. Among recovered strains, a distinct P. aeruginosa morphotype (wrinkled shape) was shown to cause lethal peritonitis compared to smooth-shaped strains, which were nonlethal. Wrinkled strains were associated with a tendency to elicit a more proinflammatory response in mice compared to smooth-shaped strains. CONCLUSION: Surgical injury transforms the morphotype of intestinal P. aeruginosa to express a hypervirulent response in the peritoneum of mice. Enhanced virulence of intestinal pathogens in response to surgical injury may play an important role in predicting the outcome of peritonitis.
Surgery 08/2012; · 3.10 Impact Factor
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ABSTRACT: Abstract Background: Although the cause of intestinal necrosis in diseases such as necrotizing enterocolitis (NEC) is multi-factorial, oxidants and bacteria likely are key factors. Many animal models of NEC generate histologic necrosis, but it is rare to see the gross necrosis that is observed in infants. Here, we present a novel model that produces full-thickness intestinal necrosis when chloramine-T, an oxidizing agent, is introduced directly into isolated intestinal loops. The aim of this study was to determine the role of bacteria in this model. Methods: Three-week-old mice underwent isolated ileal loop construction by suture ligation, and either chloramine-T or saline was injected into the loop. Intestines were then returned to the abdominal cavity, and the incision was closed for 24 h, after which the intestinal loops were analyzed histologically and microbiologically. To determine if bacteria home to the site of injury, some mice had intracecal injection of luminescent Pseudomonas aeruginosa (PA). These mice were sacrificed 2, 6, 8, and 24 h later, and luminescent bacteria were localized via photon camera imaging. Results: No gross necrosis was observed in the saline-treated group, but 59% of the animals in the chloramine-T-treated group had necrosis (p<0.001). Relative bacterial species numbers were calculated for untreated control animals and those with saline-treated loops and chloramine-T-treated loops without and with necrosis. Lactobacillus was detected in 60% of the control animals but was absent in all animals that underwent surgery. Methicillin-sensitive Staphylococcus aureus, PA, and Enterococcus faecium were present only in animals that underwent loop construction; however, bacterial communities did not differ according to loop treatment or the presence or absence of necrosis. After intracecal injection of PA, bacteria homed to the loop and proximal bowel even though the loop was discontinuous with the remaining bowel. Conclusions: Intraluminal chloramine-T causes full-thickness necrosis in three-week-old mice and is characterized by a loss of probiotic bacteria such as Lactobacillus. Necrotic loops of bowel become colonized rapidly with pathogenic bacteria by unconventional mechanisms. Oxidant stress and colonization by pathogenic bacteria may play important roles in intestinal necrosis across a wide spectrum of pathologic states, including NEC.
Surgical Infections 07/2012; 13(4):238-44. · 1.80 Impact Factor
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ABSTRACT: Bilioenteric anastomoses are commonly performed during liver transplantation, reconstruction following tumor resection, and repair of bile duct injury. Ischemia plays an important role in both short- and long-term complications related to biliary anastomoses. The authors describe a catheter-based, suture-free approach to bilioenteric anastomosis that aims to reduce or eliminate tissue trauma and foreign material that contribute to anastomotic ischemia. The device uses a novel "umbrella" anchoring mechanism to approximate bowel to the transected bile duct over a percutaneously placed transhepatic catheter. The authors performed an open choledochojejunostomy using the device in 7 pigs. The anastomosis was assessed with cholangiography, gross inspection, and histology at the time of necropsy. The procedure was well tolerated, with no evidence of anastomotic leak in any of the study animals. This alternative catheter-based approach to bilioenteric anastomoses is safe and effective, offers potential advantages over traditional approaches, and can be easily adapted to a minimal access surgery.
Surgical Innovation 06/2012; · 2.13 Impact Factor
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ABSTRACT: 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.
Annals of surgery 02/2012; 255(2):386-93. · 7.90 Impact Factor
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ABSTRACT: Necrotizing enterocolitis (NEC) is a complex disease involving prematurity, enteral feeding, and bacterial effects. We propose that the underlying initial condition in its pathogenesis is reduced ability of the neonatal gut epithelial cells (NGECs) to clear oxidative stress (OS), and that when such a NGEC population is exposed to enteral feeding, the increased metabolic OS tips the population toward apoptosis, inflammation, bacterial activation, and eventual necrosis. The multi-factorial complexity of NEC requires characterization with computational modeling, and herein, we used an agent-based model (ABM) to instantiate and examine our unifying hypothesis of the pathogenesis of NEC.
An ABM of the neonatal gut was created with NGEC computational agents incorporating rules for pathways for OS, p53, tight junctions, Toll-like receptor (TLR)-4, nitric oxide, and nuclear factor-kappa beta (NF-κB). The modeled bacteria activated TLR-4 on contact with NGECs. Simulations included parameter sweeps of OS response, response to feeding, addition of bacteria, and alterations in gut mucus production.
The ABM reproduced baseline cellular respiration and clearance of OS. Reduction in OS clearance consistent with clinical NEC led to senescence, apoptosis, or inflammation, with disruption of tight junctions, but rarely to NGEC necrosis. An additional "hit" of bacteria activating TLR-4 potentiated a shift to NGEC necrosis across the entire population. The mucus layer was modeled to limit bacterial-NGEC interactions and reduce this effect, but concomitant apoptosis in the goblet cell population reduced the efficacy of the mucus layer and limited its protective effect in simulated experiments. This finding suggests a means by which increased apoptosis at the cellular population level can lead to a transition to the necrosis outcome.
Our ABM incorporates known components of NEC and demonstrates that impaired OS management can lead to apoptosis and inflammation of NGECs, rendering the system susceptible to an additional insult involving regionalized mucus barrier failure and TLR-4 activation, which potentiates the necrosis outcome. This type of integrative dynamic knowledge representation can be a useful adjunct to help guide and contextualize research.
Surgical Infections 01/2012; 13(1):18-32. · 1.80 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(1):e30119. · 4.09 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(4):e34883. · 4.09 Impact Factor
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Andrea D Olivas,
Benjamin D Shogan,
Vesta Valuckaite,
Alexander Zaborin,
Natalya Belogortseva,
Mark Musch,
Folker Meyer,
William L Trimble,
Gary An,
Jack Gilbert,
Olga Zaborina, John C Alverdy
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ABSTRACT: 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<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.
PLoS ONE 01/2012; 7(8):e44326. · 4.09 Impact Factor
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ABSTRACT: 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.
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).
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.
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.
The Journal of trauma 12/2011; 71(6):1575-82. · 2.48 Impact Factor
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ABSTRACT: 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.
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.
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.
BMC Microbiology 09/2011; 11:212. · 3.04 Impact Factor
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ABSTRACT: There is a growing realization that alterations in host-pathogen interactions (HPI) can generate disease phenotypes without pathogen invasion. The gut represents a prime region where such HPI can arise and manifest. Under normal conditions intestinal microbial communities maintain a stable, mutually beneficial ecosystem. However, host stress can lead to changes in environmental conditions that shift the nature of the host-microbe dialogue, resulting in escalation of virulence expression, immune activation and ultimately systemic disease. Effective modulation of these dynamics requires the ability to characterize the complexity of the HPI, and dynamic computational modeling can aid in this task. Agent-based modeling is a computational method that is suited to representing spatially diverse, dynamical systems. We propose that dynamic knowledge representation of gut HPI with agent-based modeling will aid in the investigation of the pathogenesis of gut-derived sepsis.
An agent-based model (ABM) of virulence regulation in Pseudomonas aeruginosa was developed by translating bacterial and host cell sense-and-response mechanisms into behavioral rules for computational agents and integrated into a virtual environment representing the host-microbe interface in the gut. The resulting gut milieu ABM (GMABM) was used to: 1) investigate a potential clinically relevant laboratory experimental condition not yet developed--i.e. non-lethal transient segmental intestinal ischemia, 2) examine the sufficiency of existing hypotheses to explain experimental data--i.e. lethality in a model of major surgical insult and stress, and 3) produce behavior to potentially guide future experimental design--i.e. suggested sample points for a potential laboratory model of non-lethal transient intestinal ischemia. Furthermore, hypotheses were generated to explain certain discrepancies between the behaviors of the GMABM and biological experiments, and new investigatory avenues proposed to test those hypotheses.
Agent-based modeling can account for the spatio-temporal dynamics of an HPI, and, even when carried out with a relatively high degree of abstraction, can be useful in the investigation of system-level consequences of putative mechanisms operating at the individual agent level. We suggest that an integrated and iterative heuristic relationship between computational modeling and more traditional laboratory and clinical investigations, with a focus on identifying useful and sufficient degrees of abstraction, will enhance the efficiency and translational productivity of biomedical research.
Theoretical Biology and Medical Modelling 09/2011; 8:33. · 1.86 Impact Factor
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ABSTRACT: Translocation of bacteria and other luminal factors from the intestine following surgical injury can be a major driver of critical illness. Bile acids have been shown to play a key role in the loss of intestinal epithelial barrier function during states of host stress. Experiments to study the ability of nonionic block copolymers to abrogate barrier failure in response to bile acid exposure are described. In vitro experiments were performed with the bile salt sodium deoxycholate on Caco-2 enterocyte monolayers using transepithelial electrical resistance to assay barrier function. A bisphenol A coupled triblock polyethylene glycol (PEG), PEG 15-20, was shown to prevent sodium deoxycholate-induced barrier failure. Enzyme-linked immunosorbent assay, lactate dehydrogenase, and caspase 3-based cell death detection assays demonstrated that bile acid-induced apoptosis and necrosis were prevented with PEG 15-20. Immunofluorescence microscopic visualization of the tight junctional protein zonula occludens 1 (ZO-1) demonstrated that PEG 15-20 prevented significant changes in tight junction organization induced by bile acid exposure. Preliminary transepithelial electrical resistance-based studies examining structure-function correlates of polymer protection against bile acid damage were performed with a small library of PEG-based copolymers. Polymer properties associated with optimal protection against bile acid-induced barrier disruption were PEG-based compounds with a molecular weight greater than 10 kd and amphiphilicity. The data demonstrate that PEG-based copolymer architecture is an important determinant that confers protection against bile acid injury of intestinal epithelia.
Shock (Augusta, Ga.) 09/2011; 36(5):451-7. · 2.87 Impact Factor
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ABSTRACT: To determine whether a minimally invasive, colon-preserving approach could serve as an alternative to total colectomy in the treatment of severe, complicated Clostridium difficile-associated disease (CDAD).
C. difficile is a significant cause of morbidity and mortality worldwide. Most cases will respond to antibiotic therapy, but 3% to 10% of patients progress to a severe, complicated, or "fulminant" state of life-threatening systemic toxicity. Although the advocated surgical treatment of total abdominal colectomy with end ileostomy improves survival in severe, complicated CDAD, outcomes remain poor with associated mortality rates ranging from 35% to 80%.
All patients who were diagnosed with severe, complicated ("fulminant") CDAD and were treated at the University of Pittsburgh Medical Center or VA Pittsburgh Healthcare System between June 2009 and January 2011 were treated with this novel approach. The surgical approach involved creation of a loop ileostomy, intraoperative colonic lavage with warmed polyethylene glycol 3350/electrolyte solution via the ileostomy and postoperative antegrade instillation of vancomycin flushes via the ileostomy. The primary end point for the study was resolution of CDAD. The matching number of patients treated with colectomy for CDAD preceding the initiation of this current treatment strategy was analyzed for historical comparison.
Forty-two patients were treated during this time period. There was no significant difference in age, sex, pharmacologic immunosuppression, and Acute Physiology and Chronic Health Evaluation-II scores between our current cohort and historical controls. The operation was accomplished laparoscopically in 35 patients (83%). This treatment strategy resulted in reduced mortality compared to our historical population (19% vs 50%; odds ratio, 0.24; P = 0.006). Preservation of the colon was achieved in 39 of 42 patients (93%).
Loop ileostomy and colonic lavage are an alternative to colectomy in the treatment of severe, complicated CDAD resulting in reduced morbidity and preservation of the colon.
Annals of surgery 09/2011; 254(3):423-7; discussion 427-9. · 7.90 Impact Factor
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ABSTRACT: Important advances in the study of bacteria associated with the human gastrointestinal tract have significant implications for clinicians striving to meet the metabolic and nutritional needs of critically ill patients. This article offers a broad overview of the importance of the host-microbe relationship, discusses what is currently known about the role of gut microbes in nutrition and metabolism in the healthy human host, reviews how gut microbes are affected by critical illness, and discusses interventions that have already been used to manipulate the gut microbiome in patients in the intensive care unit.
Surgical Clinics of North America 08/2011; 91(4):771-85, viii. · 2.14 Impact Factor
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ABSTRACT: The surgical treatment of severe acute pancreatitis has significantly changed in the last several years with the advent of enhanced imaging techniques and minimally invasive surgery. Criteria for surgical intervention have been influenced by the use of step-up approaches that provide incremental multimodality interventions with repeat imaging. Here, we provide a brief introduction to the history, goals, indications, and technique of laparoscopic pancreatic necrosectomy. The technique requires a fundamental understanding of the natural history of pancreatitis and its complication. Laparoscopic approaches can be useful as primary and adjunctive therapy for the treatment of infected pancreatitic necrosis.
Journal of Gastrointestinal Surgery 07/2011; 15(7):1092-7. · 2.83 Impact Factor
