Monika A. Krezalek

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

ABSTRACT 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.

Bacterial colonization and succession in a newly opened hospital

Patients share their microbiota with their rooms and with nursing staff, and this shapes the microbial ecology of the hospital environment. A new hospital teems with life Lax et al. conducted a yearlong survey of the bacterial diversity associated with the patients, staff, and built surfaces in a newly opened hospital. They found that the bacterial communities on patient skin strongly resembled those found in their rooms. The authors demonstrated that the patient skin microbial communities were shaped by a diversity of clinical and environmental factors during hospitalization. They found little effect of intravenous or oral antibiotic treatment on the skin microbiota of patients. The microorganisms that inhabit hospitals may influence patient recovery and outcome, although the complexity and diversity of these bacterial communities can confound our ability to focus on potential pathogens in isolation. To develop a community-level understanding of how microorganisms colonize and move through the hospital environment, we characterized the bacterial dynamics among hospital surfaces, patients, and staff over the course of 1 year as a new hospital became operational. The bacteria in patient rooms, particularly on bedrails, consistently resembled the skin microbiota of the patient occupying the room. Bacterial communities on patients and room surfaces became increasingly similar over the course of a patient’s stay. Temporal correlations in community structure demonstrated that patients initially acquired room-associated taxa that predated their stay but that their own microbial signatures began to influence the room community structure over time. The α- and β-diversity of patient skin samples were only weakly or nonsignificantly associated with clinical factors such as chemotherapy, antibiotic usage, and surgical recovery, and no factor except for ambulatory status affected microbial similarity between the microbiotas of a patient and their room. Metagenomic analyses revealed that genes conferring antimicrobial resistance were consistently more abundant on room surfaces than on the skin of the patients inhabiting those rooms. In addition, persistent unique genotypes of Staphylococcus and Propionibacterium were identified. Dynamic Bayesian network analysis suggested that hospital staff were more likely to be a source of bacteria on the skin of patients than the reverse but that there were no universal patterns of transmission across patient rooms.

Collapse of the Microbiome, Emergence of the Pathobiome, and the Immunopathology of Sepsis.

The definition of sepsis has been recently modified to accommodate emerging knowledge in the field, while at the same time being recognized as challenging, if not impossible, to define. Here, we seek to clarify the current understanding of sepsis as one that has been typically framed as a disorder of inflammation to one in which the competing interests of the microbiota, pathobiota, and host immune cells lead to loss of resilience and nonresolving organ dysfunction. Here, we challenge the existence of the idea of noninfectious sepsis given that critically ill humans never exist in a germ-free state. Finally, we propose a new vision of the pathophysiology of sepsis that includes the invariable loss of the host’s microbiome with the emergence of a pathobiome consisting of both “healthcare-acquired and healthcare-adapted pathobiota.” Under this framework, the critically ill patient is viewed as a host colonized by pathobiota dynamically expressing emergent properties which drive, and are driven by, a pathoadaptive immune response.

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

BackgroundDespite 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.MethodsHere, 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.ResultsResults 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.ConclusionThese 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 intestinal microbiome and surgical disease

As we have seen throughout this monograph, microbes play a key role in both human health, disease, response to surgery and pharmacologic intervention. Advancing the understanding of these complex relationships is in its infancy. Technological advances in analysis, large data management and interpretation of data are rapidly evolving to address disease states which have eluded investigator and clinicians. What has been learned so far, however is both exciting and humbling.

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

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.

The role of the microbiota in surgical recovery.

Purpose of reviewThe purpose of this review is to highlight new research findings in the complex bidirectional crosstalk that occurs between the intestinal microbiome and the host immune system in the context of surgical recovery and outcomes. Recent findingsSignificant evidence has been generated emphasizing the central role of the intestinal microbiome on surgical outcomes such as wound healing, surgical site infections and anastomotic leak. Current preventive strategies, including the use of some parenteral antibiotics, may actually exacerbate the problem by selecting for drug-resistant pathogens. SummaryA delicate balance exists between the human host and its microbial counterparts that is directly related to postsurgical healing. This balance can be easily altered in favor of the pathogen through perioperative and surgical interventions leading to intestinal dysbiosis and loss of colonization resistance. Current strategies to prevent infectious complications with the escalating use of broader and more powerful antibiotics are not an evolutionarily stable strategy. A more complete understanding of the ecological and molecular interactions of the host with its microbiome is necessary to uncover new therapeutic strategies that preserve the composition and function of the intestinal microbiome and constrain virulent pathogens through the course of surgical injury.

Influence of nutrition therapy on the intestinal microbiome.

Purpose of review This review describes the relationship between nutritional therapies and the intestinal microbiome of critically ill patients. Recent findings The intestinal microbiome of the critically ill displays a near complete loss of health-promoting microbiota with overgrowth of virulent healthcare-associated pathogens. Early enteral nutrition within 24 h of admission to the ICU has been advocated in medical and surgical patients to avoid derangements of the intestinal epithelium and the microbiome associated with starvation. Contrary to previous dogma, permissive enteral underfeeding has recently been shown to have similar outcomes to full feeding in the critically ill, whereas overfeeding has been shown to be deleterious in those patients who are not malnourished at baseline. Randomized clinical trials suggest that peripheral nutrition can be used safely either as the sole or supplemental source of nutrition even during the early phases of critical care. The use of probiotics has been associated with a significant reduction in infectious complications in the critically ill without a notable mortality benefit. Summary Focus of research is shifting toward strategies that augment the intestinal environment to facilitate growth of beneficial microorganisms, strengthen colonization resistance, and maintain immune homeostasis.

Media from macrophages co-incubated with Enterococcus faecalis induces epithelial cell monolayer reassembly and altered cell morphology

Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis.

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, HIF1&agr; 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 HIF1&agr; 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 HIF1&agr; (p = 0.039643). Similar results of decrease in wound infection and HIF1&agr; 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.