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.
Highlights
When patients undergo removal and re-connection of a segment or whole portion of the gastrointestinal tract, a significant number will develop anastomotic leaks despite being operated on by highly qualified surgeons in high volume centers [1]
Four groups of rats were studied: rats subjected to resection and anastomosis only (Group I), rats subjected to resection and anastomosis + cecal P. aeruginosa (Group II), rats subjected to preoperative radiation + resection and anastomosis (Group III), and rats subjected to preoperative radiation + resection and anastomosis + cecal P. aeruginosa (Group IV) (Fig. 1A)
In order to define the role of P. aeruginosa on anastomosis healing, we first developed a novel anastomosis model in which rats were subjected to preoperative fractioned radiation similar to clinical practice
Summary
When patients undergo removal (resection) and re-connection (anastomosis) of a segment or whole portion of the gastrointestinal tract, a significant number will develop anastomotic leaks despite being operated on by highly qualified surgeons in high volume centers [1]. Cohn first proposed in 1955 that the microbial content of the gut plays a central role in the pathogenesis of anastomotic leak [4] In his experiments, dogs were subjected to colon anastomosis and division of the mesenteric blood vessels to cause ischemia and delayed healing. Shardley was the first to suggest that P. aeruginosa might play a causative role in anastomotic leak [5], and performed the first randomized prospective placebo blinded trial with antibiotics confirming a role for microbes in human anastomotic leak [6] Despite this and other similar compelling observations, a microbial mechanism for anastomotic leak is generally not accepted, and around the world, anastomotic leak is posited to be primarily a problem of poor technique and/or poor wound healing [7,8,9]
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