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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

Morphine Promotes Colonization of Anastomotic Tissues with Collagenase - Producing Enterococcus faecalis and Causes Leak.

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.

The Shift of an Intestinal "Microbiome" to a "Pathobiome" Governs the Course and Outcome of Sepsis Following Surgical Injury.

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.

C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

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.

Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak.

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.

Membership and behavior of ultra-low-diversity pathogen communities present in the gut of humans during prolonged critical illness.

UNLABELLED: 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.

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

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