Intestinal tissues induce an SNP mutation in Pseudomonas aeruginosa that enhances its virulence: possible role in anastomotic leak

• Published in: PloS one Vol. 7; no. 8; p. e44326
• Main Authors: Olivas, Andrea D, Shogan, Benjamin D, Valuckaite, Vesta, Zaborin, Alexander, Belogortseva, Natalya, Musch, Mark, Meyer, Folker, Trimble, William L, An, Gary, Gilbert, Jack, Zaborina, Olga, Alverdy, John C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.08.2012; Public Library of Science (PLoS)
• Subjects: Anastomosis, Surgical - adverse effects; Anastomotic Leak - microbiology; Anastomotic Leak - pathology; Animal euthanasia; Animal tissues; Animals; Antibiotics; Apoptosis; Apoptosis - drug effects; Base Sequence; Bioengineering; Biology; Caenorhabditis elegans; Cancer; Chemoradiotherapy; Collagen; Collagenase; Colon; Colon - drug effects; Colon - metabolism; Colon - pathology; Colon cancer; Cytolysis; Endoscopy; Epithelial cells; Esophagus; Experiments; Gene polymorphism; Genetic aspects; Genetic transformation; Genotype & phenotype; Genotypes; In vivo methods and tests; Incidence; Intestine; Intestines - drug effects; Intestines - microbiology; Intestines - pathology; Intestines - ultrastructure; Laboratories; Leakage; Male; Medical personnel; Medicine; Microorganisms; Molecular Sequence Data; Mutation; Mutation - genetics; Pathogenesis; Pathogens; Phenotype; Phenotypes; Phosphates - pharmacology; Physiological aspects; Polyethylene glycol; Polyethylene Glycols - pharmacology; Polymorphism; Polymorphism, Single Nucleotide - genetics; Protective Agents - pharmacology; Pseudomonas aeruginosa; Pseudomonas aeruginosa - drug effects; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - isolation & purification; Pseudomonas aeruginosa - pathogenicity; Pyocyanin; Radiation; Rats; Rats, Wistar; Rectum; Reversion; Rodents; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Stop codon; Surgery; Swarming; Tight Junctions - drug effects; Tight Junctions - metabolism; Transformation; Virulence; Virulence (Microbiology); Wound Healing - drug effects; Zonula Occludens-1 Protein - metabolism; United States; United States > US; Illinois; Chicago Illinois
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0044326

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.