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

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

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

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

Modeling Acinetobacter baumannii wound infections: The critical role of iron.

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.

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.

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

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

Depletion of intestinal phosphate after operative injury activates the virulence of P aeruginosa causing lethal gut-derived sepsis.

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.