Intestinal Tissues Induce an SNP Mutation in Pseudomonas aeruginosa That Enhances Its Virulence: Possible Role in Anastomotic Leak

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.

Are gut bacteria associated with the development of anastomotic leaks?

“…They reported that exposure of anastomotic tissues to pathogenic bacteria, such as P. aeruginosa, resulted in selection of a more virulent phenotype caused by a single nucleotide point mutation (SNP). This phenotype is characterized by high collagendegrading activity against collagen types I and IV, which is associated with anastomotic leak [14]. The surgical procedure of cutting and reuniting intestinal tissue is apparently the trigger for this complex reaction.…”

“…The surgical procedure of cutting and reuniting intestinal tissue is apparently the trigger for this complex reaction. The latter authors hypothesized that the capacity of intestinal bacteria to degrade collagen may be an important mechanism underlying anastomotic leak [14]. To identify additional and perhaps more common bacteria with collagendegrading activity that might colonize anastomotic tissue after surgery, they examined the microflora associated with anastomotic tissues in rats after anastomotic surgery [28].…”

“…The use of non-microbicidal drugs to create microenvironmental conditions that suppress the virulence of pathogens is an attractive strategy to minimize the negative consequences of intestinal microbiome disruption caused by antibiotic use [14,28,29,32,33]. It has been shown previously that phosphate is depleted in the intestinal tract following surgical injury, that this depletion is a major "cue" that triggers bactericidal virulence, and that the maintenance of luminal phosphate abundance in the gut prevents virulence expression [12,14,32,33].…”

“…It has been shown previously that phosphate is depleted in the intestinal tract following surgical injury, that this depletion is a major "cue" that triggers bactericidal virulence, and that the maintenance of luminal phosphate abundance in the gut prevents virulence expression [12,14,32,33]. Since inorganic phosphate is not a suitable agent for phosphate delivery to the site of host-pathogen interaction, because it is readily absorbed in the small intestine, other ways must be found [32].…”

“…This allows for prolonged release of polyphosphates, which can be exploited as a target for virulence suppression of lethal pathogenic phenotypes in the gastrointestinal tract [32]. After all, phosphate bound to polyethylene glycol was able to reverse a P. aeroginosa destructive phenotype and prevent AL in the rat [14]. Maybe this or similar nonmicrobicidal drugs will prove to be clinically effective in the future to prevent AL.…”