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

Abstract

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.

Fig. 1. E. faecalis with high collagen-degrading activity is associated with anastomotic leak

A. Rat model of anastomotic leak. B. Incidence of leak between devascularized (Dvasc) versus non- devascularized (Anast) intestinal segments of the rat model (n=15, ^*p<0.01). C. Collagen-degrading activity of whole microbial communities between various groups of rats showing that microbial collagen-degrading activity discriminated between leaking versus non-leaking groups (n=10/group ^*p<0.01, Student t-test). D. Trichrome staining of representative rat intestinal segments demonstrates collagen depletion and leakage. Collagen shown by white arrow. E. Trichrome stain density between treatment groups discriminated between rats with and without anastomotic leak (^*p<0.05, Student t-test). F. Collagen-degrading activity among various cultured microorganisms shows non-uniform activity across E. faecalis strains. G. Collagen-degrading activity among various isolates of E. faecalis from sham-operated versus devascularized anastomotic tissues that either healed (heal) or leaked (leak). H. Collagen content of intestinal tissues of rats injected by E. faecalis E1 or E2 strains measured by Trichrome staining (3 slides/rat n=5/group, ^*p<0.01, Student t-test). I. Leak rates in rats with intestinal anastomoses that were transrectally inoculated with E1 and E2 strains of E. faecalis (n=5/group). J. Collagen-degrading capacity of E. faecalis demonstrating that the human strain V583 degraded collagen I in a GelE/SprE dependent manner (n=3/group). K. Leak rates in rats with intestinal anastomoses transrectally inoculated with the V583 derivative double mutant ΔgelEΔsprE versus the complemented double mutant ΔgelEΔsprE/gelE+sprE (n=20 in ΔgelEΔsprE group and n=17 in ΔgelEΔsprE/gelE+sprE). p<0.001, Student t-test.

Fig. 2. E. faecalis strains with high collagen-degrading activity also activate host intestinal MMP9

A. Zymography, a eletrophorectic technique to determine proteolytic activity that is indicated by Coomassie staining, of normal rat intestinal tissue exposed to supernatants obtained from high collagenase producing E2 strains of E. faecalis reveals host protease cleavage with the appearance of an 86 kDa band. The three bands on the left side (tissue extracts) represent proteins extracted from tissues; the three bands in the middle (tissue secreted products) represent proteins secreted by tissues; the two bands on the right (no tissue) show negative results for E. faecalis supernatants not exposed to tissues. Visualization of the proteolytic activity appears as clear bands over a deep blue background after Coomassie staining. Vehicle consisted of TH- (Todd-Hewitt broth) in which E. faecalis is usually grown. B. Western blots demonstrate that E2 supernatant cleaves MMP9 to its active form. C. Zymography demonstrates the ability of E2 to cleave human recombinant pro-MMP9 (r-MMP9). APMA, an agent known to induce MMP9 cleavage, was used as a positive control. D. The effect of the human laboratory strain of E. faecalis (V583) and its gelE and sprE mutants on human r-MMP9 cleavage is shown. E,E′. Zymography (E) and Western blot (E′) analyses of MMP9 activation in a murine macrophage cell line J774.1 incubated with E. faecalis V583 and its derivative mutants ΔGelE, ΔSprE, and ΔGelEΔSprE. F. Zymography analysis of MMP9 activation in the murine macrophage cell line J774.1 incubated with E. faecalis strains E1 and E2, and Proteus mirabilis is shown. G–L. Shown are effects of pharmacologic inhibition of MMP9 in rat tissues following colon resection, anastomosis, and devascularization: intestinal tissue MMP9 by Western blot (G), zymography (H), band densities of Western blot (I), anastomotic leak rate (J), collagen-degrading activity (K), and collagen content (L) (n=3, p<0.01, Student t-test).

Fig. 3. Antibiotic treatment of rats with anastomosis plus devascularization

Antibiotic treatment (Abx) of rats with anastomosis plus devascularization (Anast + Dvasc) attenuated MMP9 activation, and expression of HIF1-α (hypoxia inducible factor), iNOS (inducible nitric oxide) and MPO (myeloperoxidase). (A) Western blot analyses of rat tissues subjected to devascularization (Dvasc), anastomosis (Anast) or Anast + Dvasc, in the presence or absence of direct topical application of ciprofloxacin, metronidazole, and neomycin (topical Abx) via enema. Experiments were performed on 3 rats per group; displayed immunoblot is representative of all results. (B) The evaluation of band intensities using Image J software demonstrated the abundance of the active form of MMP9 in the rat group Anast + Dvasc, and loss of active MMP9 after topical application of antibiotics. n=3, p<0.01, Student t-test. (C) Zymography analysis confirmed the abundance of the active form of MMP9 in the Anast + Dvasc rat group, and loss of the active form of MMP9 by topical application of antibiotics.

Fig. 4. Topical antibiotic treatment applied to rat anastomotic tissues prevents leak

Effect of intramuscular (IM) cefoxitin (systemic antibiotic treatment) versus direct topical antibiotic treatment with ciprofloxacin, flagyl and neomycin via enema on post-operative day (POD) 0 and 6. (A–C) Shown are the effects of systemic and topical antibiotic treatment on leak rate, collagen-degrading activity of whole microbial communities, and intestinal collagen content in rats that underwent surgical resection, anastomosis, and devascularization (n=10, ^*P<0.01, Student t-test). (D, D′). Effect of IM cefoxitin versus topical antibiotic treatment on intestinal tissue MMP9 activity (n=3, ^*P<0.05, Student t-test).

Fig. 5. Isolation of bacterial species from resected intestinal samples from surgical patients

Shown are species identification, collagen-degrading activity, and MMP9 cleavage capacity among 68 bacterial isolates cultured from the proximal (p) and distal (d) ends of resected intestinal specimens from 11 patients undergoing colon surgery. A. Culture results of bacterial species and their collagen-degrading activity from patients #3 to #11; patients #1 and #2 were culture negative. B. Zymography demonstrating the ability of isolated strains of E. faecalis to activate cleavage of human recombinant pro-MMP9 (r-pro-MMP9) to its active form. C. Zymography (left panel) and Western blot (right panel) demonstrating the ability of P. aeruginosa human isolates to activate cleavage of human r-pro-MMP9 to its active form. P. aeruginosa positive control is a human stool isolate maintained in our laboratory that is known to have high collagen-degrading activity as well as the ability to cleave MMP9.

Fig. 6. Microbial composition of resected intestinal specimens from 11 patients undergoing elective colon surgery

A, B. 16S rRNA analysis at the phylum (A) and class (B) levels of the microbial community composition of the proximal (p) and distal (d) ends of resected colon samples from 11 patients. C. Ratio of Proteobacteria (disease-related) to Bacteroidetes (health promoting) in the 11 patients (Pt) who underwent elective colon surgery. D. Gammaproteobacteria was the most abundant class in patient #10.