RESEARCH ARTICLE WOUND HEALING Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak Benjamin D. Shogan,1 Natalia Belogortseva,1 Preston M. Luong,1 Alexander Zaborin,1 Simon Lax,1 Cindy Bethel,1 Marc Ward,1 Joseph P. Muldoon,2 Mark Singer,2 Gary An,1 Konstantin Umanskiy,1 Vani Konda,1 Baddr Shakhsheer,1 James Luo,1 Robin Klabbers,1,3 Lynn E. Hancock,4 Jack Gilbert,1,5 Olga Zaborina,1* John C. Alverdy1*† 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 patho- genesis 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 ac- tivation 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 rec- on May 7, 2015 ommended 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. INTRODUCTION who demonstrated that repeated direct topical application of anti- The most devastating complication after removal of an intestinal seg- biotics onto anastomotic tissues accelerated healing and prevented stm.sciencemag.org ment (resection) and its reconnection (anastomosis) is an anastomotic leak in dogs undergoing colon resection and anastomosis when the leak. The clinical manifestations of an anastomotic leak range from ab- supplying blood vessels were divided in a manner that resulted in gross dominal pain with fever to septic shock. In its extreme form, anastomotic ischemia (2). Remarkably, despite the grossly visible presence of is- leak can cause peritonitis, sepsis, and even death. Leaks are particularly chemia, direct topical application of antibiotics not only prevented prevalent in patients undergoing surgery in high-risk regions of the anastomotic leak but also completely reversed the ischemia. Although intestine such as the rectum and esophagus (1). In the distal colon and from 1955 to 1984 oral antibiotics were introduced as a routine part of rectal area, the anastomotic leak rate can be excessive (30 to 40%), forcing the preparation of gastrointestinal surgery, they were soon replaced by surgeons to routinely perform a protective diverting stoma (ileostomy intravenous antibiotics owing primarily to the convenience of admin- Downloaded from and colostomy) to lessen the clinical effects of intestinal content spillage istration and the perception that they were equally efficacious in de- (1). This practice requires a second operation to close the diverting contaminating anastomotic tissues of potentially offending pathogens stoma, which itself carries significant morbidity and includes the risk (3, 4). Despite numerous studies demonstrating the benefit of adding of an anastomotic leak. Consequently, many patients and surgeons elect oral antibiotics before gastrointestinal surgery to prevent infection and to leave the stoma as a permanent solution to avoid a second high-risk anastomotic leak, most surgeons do not routinely administer oral an- surgery. Given this, there is little motivation among surgeons to elimi- tibiotics in preparation for gastrointestinal surgery (3, 4). We have nate the routine use of a diverting stoma in lower colorectal surgery be- recently published work that readdresses the role of bacteria in anas- cause they have accepted that the actual causes of anastomotic leaks tomotic leak in a more molecular context (5). We reported that expo- remain unknown and hence they are not preventable. sure of anastomotic tissues to pathogenic bacteria such as Pseudomonas That intestinal microbes play a key causative role in the patho- aeruginosa resulted in selection of a more virulent phenotype characterized genesis of anastomotic leak has been suggested for over 60 years. by high collagen-degrading activity, which was associated with anastomotic The most direct evidence was first reported in 1955 by Cohn and Rives, leak (5). We hypothesized that the capacity of intestinal bacteria to degrade collagen may be an important mechanism underlying anastomotic leak. To 1University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA. 2NorthShore identify additional and, perhaps, more common bacteria with collagen- University HealthSystem, Evanston, IL 60201, USA. 3Department of Surgery, Radboud degrading activity that might colonize anastomotic tissues after surgery, 4 University Nijmegen Medical Centre, Nijmegen, Netherlands. The University of Kansas, we next examined the microflora associated with anastomotic tissues using Lawrence, KS 66045, USA. 5Argonne National Laboratory, Argonne, IL 60439, USA. *Senior co-authors. 16S rRNA (ribosomal RNA) and PICRUSt (Phylogenetic Investigation †Corresponding author. E-mail: [email protected] of Communities by Reconstruction of Unobserved States) analyses in www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 1 RESEARCH ARTICLE rats after anastomotic surgery (6). Results A B C x104 C x104 demonstrated a 500-fold increase in the 1 cm 100 6 6 relative abundance of the genus Enterococcus * Dvasc 80 5 * 5 at the anastomotic site. The PICRUSt func- 60 4 4 tional analysis (7) predicted the predomi- Anast 40 3 3 nance of several bacterial virulence factors, 20 2 2 1 Dvasc 1 one of which, coccolysin [GelE (gelatin- 1 cm 0 + 0 Dvasc – activity, Collagen-degrading RFU/OD 600 nm 0 ase)], is responsible for collagen/gelatin % leak rate, Anastomotic Dvasc – ++– Anast + + Anast – – ++ Heal Leak degradation (8, 9). These findings, coupled with the observations that bacterial-derived D Sham Dvasc Anast Dvasc+Anast Dvasc+Anast heal leak collagenases are known to play an important C role in a variety of intestinal disorders such as those involving inflammation and necrosis, led us to explore the role of Enterococcus in anastomotic leak (10–15). Here, we demonstrate that among com- 4 mensal microbiota, Enterococcus faecalis E E F x10 strains with enhanced collagen-degrading 5 activity and the capacity to activate intestinal 60 60 4 50 50 40 3 tissue matrix metalloproteinase 9 (MMP9) 40 30 30 2 contribute to the pathogenesis of anasto- 20 motic leak. 10 20 10 1 Collagen content, % content, Collagen 0 * Dvasc – + – + 0 activity, Collagen-degrading RFU/OD 600 nm 0 Anast – – ++ Heal Leak E. coli E. hirae on May 7, 2015 E. faecalis M. morganii P. mirabilis E. gallinarum pneumoniae RESULTS K. G H I J Gelatin K 4 4 Collagen I Clinical outcome of rats after x10 x10 surgery demonstrates that while 50 100 Collagen IV 3 3 80 40 80 intestinal devascularization is 60 2 2 associated with leak, it does not 30 60 40 20 40 1 20 cause gross ischemia 1 % rate, Leak % rate, Leak 10 20 stm.sciencemag.org 0 0 We created an anastomotic leak model in % content, Collagen 0 0 Collagen-degrading activity, activity, Collagen-degrading RFU/OD 600 nm 0 E1 E2 ∆ 2 activity, Collagen-degrading RFU/OD 600 nm ∆ ∆ sprE rats by performing a 1-cm colon resection E1 E2 E1 E V583 gelE sprE ∆ ∆gelE Sham Heal Leak ∆gelE sprE (at the peritoneal reflection) and primary No bacteria No bacteria anastomosis, followed by devasculariza- ∆gelE∆sprE/gelE+sprE tion of a 2-cm segment of blood supply Fig. 1. E. faecalis with high collagen-degrading activity is associated with anastomotic leak. (A)Rat adjacent to the anastomosis (Fig. 1A). All model of anastomotic leak. (B) Incidence of leak between devascularized (Dvasc) versus nondevascularized rats that survived did well after surgery and (Anast) intestinal segments of the rat model (n =15;*P < 0.01). (C) Collagen-degrading activity of whole mi- were healthy-appearing at the time of sac- crobial communities between various groups of rats showing that microbial collagen-degrading activity dis- Downloaded from rifice [postoperative day 6 (POD6)] as criminated between leaking versus nonleaking groups (n =10pergroup;*P <0.01,Student’s t test). RFU, judged by their feeding pattern, movement relative fluorescence unit; OD, optical density. (D) Trichrome staining of representative rat intestinal segments in the cage, and stool passage. There were demonstrates collagen depletion and leakage. Collagen is shown by a white arrow. (E) Trichrome stain density occasional anesthesia-related deaths (<5) between treatment groups discriminated between rats with and without