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 anastomotic leak (*P <0.05, within 24 hours. In these cases, additional Student’s t test). (F) Collagen-degrading activity among various cultured microorganisms shows non-uniform rats were operated on and added to the activity across E. faecalis strains. (G) Collagen-degrading activity among various isolates of E. faecalis from groups to achieve equal numbers in each sham-operated versus devascularized anastomotic tissues that either healed (heal) or leaked (leak). (H)Col- lagen content of intestinal tissues of rats injected with E. faecalis E1 or E2 strains measured by Trichrome stain- group. All rats were sacrificed on POD6 ing (three slides per rat, n =5pergroup;*P<0.01,Student’s ttest). (I) Leak rates in rats with intestinal anastomoses and underwent laparotomy for gross in- that were transrectally inoculated with E1 and E2 strains of E. faecalis (n =5pergroup).(J) Collagen-degrading spection of the anastomosis and detection capacity of E. faecalis demonstratingthatthehumanstrainV583degradedcollagenIinaGelE/SprE-dependent of leakage. No leaks were observed in rats manner (n = 3 per group). (K) Leak rates in rats with intestinal anastomoses transrectally inoculated with subjected to anastomosis alone (Anast). In the V583 derivative double mutant ∆gelE∆sprE versus the complemented double mutant ∆gelE∆sprE/gelE+sprE contrast, 50% of rats subjected to anastomo- (n =20in∆gelE∆sprE group, n =17in∆gelE∆sprE/gelE+sprE group; P < 0.001, Student’s t test). sis with devascularization (Anast + Dvasc) developed an anastomotic leak (Fig. 1B). The devascularization (fig. S1B). Finally, in a separate group of rats, we used confocal laser en- procedure did not cause grossly visible ischemia (fig. S1A). To confirm domicroscopy after fluorophore injection to examine the microscopic this, we performed endoscopy above and below the anastomotic suture blood supply of the colon just below the anastomosis and demonstrated line in selected rats that did not demonstrate any gross areas of ischemia that the segmental devascularization did not grossly interrupt the blood

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 2 RESEARCH ARTICLE supply (fig. S1C). Confocal images demonstrated normal filling of the the colon for surgery that involves elimination of the normal flora with blood vessels with the fluorophore (shown by arrows on fig. S1C) in antibiotics and depletion of mucosal mucus with a purgative bowel colon segments with or without devascularization. preparation (19). Results demonstrated that E2, but not E1, caused anastomotic leak associated with depletion of intestinal collagen (Fig. 1, Collagen-degrading activity of E. faecalis is associated with H and I). Further characterization of E1 and E2 demonstrated that anastomotic leak E2 had a greater capacity to degrade gelatin and collagen I (but not We measured collagen-degrading activity in whole bacterial com- collagen IV) (Fig. 1J). We also observed this capacity to degrade col- munities recovered by swabbing anastomotic tissues at the time of lagen I but not collagen IV with the well-characterized human isolate sacrifice and performing assays for collagen degradation capabilities E. faecalis V583 (Fig. 1J). Purified GelE from E. faecalis, a zinc metal- on the entire recovered microbiota. Results demonstrated that the loproteinase, has been shown to cleave numerous substrates including collagen-degrading activity of microbes enabled discrimination be- collagen fragments (20). GelE encoded by the gelE gene is cotranscribed tween leaking versus nonleaking anastomotic sites; microbes with in a quorum sensing–dependent manner with sprE (extracellular serine increased collagen-degrading activities predominated in leaking anas- protease) that encodes the serine protease SprE (21). We therefore used tomotic tissues (Fig. 1C′). Histologic examination demonstrated visible mutants derived from E. faecalis V583 including ∆gelE, ∆sprE, and collagen depletion in rats subjected to anastomosis and devasculariza- ∆gelE∆sprE and observed that both GelE and SprE are involved in tion that had anastomotic leakage compared to rats without anasto- the degradation of collagen I (Fig. 1J). Complementation of ∆gelE motic leakage (Fig. 1D). Similarly, collagen content was significantly with gelE and ∆gelE∆sprE with gelE+sprE led to higher collagenase different between rats with leaking versus nonleaking anastomoses activity compared to the wild-type strain. However, complementation (Fig. 1E′; P <0.05,Student’s t test), whereas there were no overall dif- of ∆sprE with sprE did not lead to increased collagen-degrading activ- ferences in collagen content between nondevascularized and devascu- ity as compared to the noncomplemented mutant (fig. S5). As others larized anastomoses (Fig. 1E). There was a clear association between have suggested, it is possible that a critical balance between GelE and enhanced collagen-degrading activity (Fig. 1C′) and attenuated collagen SprE is required for their proper function (22). The collagen-degrading content within the anastomotic leak group (Anast + Dvasc) (Fig. 1E′). activity of E2 and the E. faecalis V583 mutant was significantly sup-

In addition, measurements of collagen-degrading activity of whole- pressed by the zinc chelator 1,10-phenanthroline when tested using on May 7, 2015 tissue extracts demonstrated a significant difference between leaking either gelatin or collagen I as a substrate (fig. S6A), confirming the in- versus healed anastomotic tissues within the group of rats undergoing volvement of a zinc metalloproteinase. However, suppression of gelatin anastomosis and devascularization (fig. S2). and collagen I degradation by 1,10-phenanthroline was not complete. To identify the bacterial strains with high collagen-degrading We observed residual collagen-degrading activity in both strains (fig. activity, we cultured bacteria recovered from anastomotic tissues by S6B), which suggests involvement of nonmetalloproteinase activity, swabbing. E. faecalis and Proteus mirabilis, members of the commen- thereby supporting the role of SprEincollagendegradation. sal microbiota, had the highest collagen-degrading activity among all To define the role of GelE and SprE in anastomotic leak in our rat isolates. P. mirabilis strains uniformly had high activity, whereas isolates model, we introduced the V583 derivative double mutant ∆gelE∆sprE stm.sciencemag.org of E. faecalis showed both high and low collagen-degrading activities or ∆gelE∆sprE complemented with gelE plus sprE via enema similar to (Fig. 1F). Notably, the mean collagen-degrading activity of E. faecalis experiments with E1 and E2. Rats were sacrificed on POD6, and anas- strains was significantly increased (P < 0.01) in rats with anastomotic tomotic leak rates were determined by gross inspection. Results dem- leakage compared to those without (Fig. 1G). No such correlation was onstrated a significant difference in leak rates between the two groups observed with P. mirabilis because all isolates were high collagenase (P < 0.0001), with ∆gelE∆sprE causing a low rate of leakage of 5% (1 of producers. Therefore, we decided to focus on E. faecalis because its 20 rats) and ∆gelE∆sprE complemented with gelE+sprE causing a high collagen-degrading activity might be a potential discriminator be- rate of leakage of 70% (12 of 17 rats) (Fig. 1K). tween anastomotic leakage and nonleakage. We called low collagenase- Downloaded from producing E. faecalis strains E1 and high collagenase producers E2. Activation and cleavage of intestinal MMP9 are associated Time-dependent measurements of collagen-degrading activity revealed with anastomotic leak and can be induced by E. faecalis in a that E2 strains started to degrade collagen significantly earlier (P < 0.01) GelE/SprE-dependent manner and at a higher level compared to E1 strains (fig. S3). To determine other In addition to bacterial collagen–degrading proteases, host extra- phenotypic differences between E1 and E2 strains, we screened these cellular matrix–degrading enzymes can also contribute to anastomotic strains in Caenorhabditis elegans–killing assays and observed that the leakage. Among them, intestinal MMP9 has been shown to play an E2 strain was more virulent than E1 (fig. S4). This finding agreed with important role in the pathogenesis of anastomotic leak (23). Normally, previous work showing that collagenase plays a key role in the virulence as part of the response to tissue injury, zinc-dependent MMP9 degrades and lethality of E. faecalis (16–18). Next, to define the role of collagen- components of the extracellular matrix including collagen for re- degrading activity among E. faecalis strains in the pathogenesis of anas- modeling and wound healing. Excessive activation of MMP9 could tomotic leak, we selected a pair of strains from our isolates from rat tip the balance during anastomotic healing such that collagen degra- anastomoses that were low (E1) and high collagenase producers (E2). dation results in leakage instead of healing. Other inflammatory sig- We introduced live cultures of E1 and E2 (~106 colony-forming units nals could be also triggered in addition to, or as a result of, MMP9 in 0.1 ml of solution) via enema into rats after colon resection and anas- activation (24) and could participate in excessive inflammation at tomosis without devascularization and monitored the development of the site of the anastomosis. Zymography analysis of tissue extracts anastomotic leak after sacrifice on POD6 as in previous experiments. demonstrated higher MMP9 activity in leaking versus healed anas- Rats were administered systemic cefoxitin and mucolytics (systemic at- tomoses among groups of rats subjected to anastomosis plus devas- ropine, topical N-acetyl cysteine) to mimic the clinical preparation of cularization (fig. S7). On the basis of our observation that leaking

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 3 RESEARCH ARTICLE anastomotic tissues were colonized by high collagenase-producing firm that this band corresponded to MMP9, we performed Western E. faecalis, we next hypothesized that E. faecalis may activate MMP9 in blot analysis using anti-MMP9 antibodies. Results confirmed that the anastomotic tissues. It has been previously demonstrated that certain band was MMP9 (Fig. 2B). To examine direct cleavage of MMP9 by bacteria can activate tissue MMP9 (25, 26); however, this does not the E. faecalis isolates, we used recombinant pro-MMP9 (r-MMP9) as seem to have been demonstrated for E. faecalis to our knowledge. a substrate. Results demonstrated that only the E2 strain was able to To test this, we incubated rat colon explants isolated from normal activate pro-MMP9 (Fig. 2C). We hypothesized that either GelE or healthy rats with live bacterial cells and cell-free supernatants of E1 SprE is involved in the proteolytic cleavage of pro-MMP9. To test this, and E2 strains. Zymography, an electrophoretic method for we incubated r-MMP9 with the human E. faecalis isolate V583 and its measuring proteolytic activity, was performed on tissue extracts and derivative mutants DgelE and DsprE (8). Zymography demonstrated revealed that the E. faecalis E2 strain induced cleavage of host pro- that the GelE-deficient mutant failed to cleave MMP9, whereas the teases, resulting in the appearance of an 86-kD band (Fig. 2A). To con- SprE-deficient mutant cleaved MMP9, leading us to conclude that

A Tissue-secreted B Tissue extracts products No tissue

180 kD (dimer MMP9) 98 kD (profom MMP9) Cleaved form 86 kD (active form MMP9)

E2

Vehicle (TH) Vehicle (TH) Vehicle (TH) E1 supernatantE2 supernatant E1 supernatantE2 supernatantE1 supernatantE2 supernatant E2 supernatant C D E F Proform Proform Proform on May 7, 2015

Active Active Active form form E’ form E1 E2

V583 ∆gelE ∆sprE sprE P. mirabilisNo bacteria ∆ ∆ Active r-MMP9 gelE E. faecalis form V583 (no r-MMP9) r-MMP9 + APMA r-MMP9 (no bacteria) ∆ ∆ J771.4 V583 gelE sprE r-MMP9 + E2 supernatant ∆gelE∆sprE r-MMP9 + E1 supernatant I J KL 4 stm.sciencemag.org G x10 MMP9 1.6 * 50 50 4 1.2 40 40 3 30 30 0.8 2 * % rate, Leak 20 20 H Dvasc + Anast Dvasc + Anast Dvasc + Anast

+ vehicle (DMSO) + MMP9 inhibitor 0.4 1 * Downloaded from Collagen content, % content, Collagen 10 MMP9, arbitrary units MMP9,

10 activity, Collagen-degrading RFU/OD 600 nm Proform 0 0 0 0 Active form MMP9 inhibitor – – + –– + + + DMSO Leak – – –– No inhibitor Dvasc +Anast MMP9 inhibitor No MMP9 inhibitor MMP9 inhibitor MMP9 inhibitor control RAW 264.7 no leak leak Fig. 2. E. faecalis strains with high collagen-degrading activity also E2 to cleave human recombinant pro-MMP9 (r-MMP9). APMA (p-amino- activate host intestinal MMP9. (A) Zymography (an electrophoretic tech- phenylmercuric acetate), an agent known to induce MMP9 cleavage, was nique to determine proteolytic activity indicated by Coomassie staining) of used as a positive control. (D) The effect of the human laboratory strain of E. normal rat intestinal tissue exposed to supernatants obtained from high faecalis (V583) and its gelE and sprE mutants on human r-MMP9 cleavage is collagenase-producing E2 strains of E. faecalis reveals host protease cleav- shown. (E and E′) Zymography (E) and Western blot (E′) analyses of MMP9 age with the appearance of an 86-kD band. The three bands on the left side activation in a murine macrophage cell line J774.1 incubated with E. faeca- (tissue extracts) represent proteins extracted from tissues; the three bands lis V583 and its derivative mutants ∆GelE, ∆SprE, and ∆GelE∆SprE. (F)Zymo- in the middle (tissue-secreted products) represent proteins secreted by tis- graphy analysis of MMP9 activation in the murine macrophage cell line sues; the two bands on the right (no tissue) show negative results for E. J774.1 incubated with E. faecalis strains E1 and E2 and P. mirabilis.(G to L) faecalis supernatants not exposed to tissues. The proteolytic activity ap- Effects of pharmacologic inhibition of MMP9 in rat tissues after colon resec- pears as clear bands over a deep blue background after Coomassie stain- tion, anastomosis, and devascularization: intestinal tissue MMP9 by West- ing. Vehicle consisted of Todd-Hewitt (TH) broth in which E. faecalis is ern blot (G), zymography (H), band densities of Western blot (I), anastomotic usually grown. (B) Western blots demonstrate that E2 supernatant cleaves leak rate (J), collagen-degrading activity (K), and collagen content (L) (n =3; MMP9 to its active form. (C and C′) Zymography demonstrates the ability of P < 0.01, Student’s t test). DMSO, dimethyl sulfoxide.

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 4 RESEARCH ARTICLE

GelEwasresponsiblefortheactivationofMMP9(Fig.2D).Toveri- MMP9 inhibitor. Results demonstrated that pharmacologic inhibition fy the involvement of GelE in the activation of MMP9, we performed of MMP9 suppressed MMP9 production in anastomotic tissues (Fig. 2, ex vivo experiments using the mouse peritoneal macrophage cell line G to I) and prevented anastomotic leak (Fig. 2J) in association with sup- J774. J774 macrophages were incubated with V583 and its derivative pression of the collagen-degrading activity of whole colonizing micro- mutants DgelE, DsprE, and ∆gelE∆sprE, and MMP9 was measured by biota (Fig. 2K) and preservation of intestinal collagen content (Fig. 2L). both zymography (Fig. 2E) and Western blot (Fig. 2E′). Surprisingly, We next determined the influence of the intestinal microbiota on we observed the opposite effect with the mutants whereby SprE, not key inflammatory mediators present in intestinal tissues in rats 6 days GelE, appeared to be required for MMP9 cleavage (Fig. 2, E and E′). after resection, anastomosis, and segmental devascularization. These We speculated that the discrepancy between human recombinant mediators included HIF-1a (hypoxia-inducible factor–1a), iNOS MMP9 versus naturally occurring MMP9 from macrophages may (inducible nitric oxide synthase), and MPO (myeloperoxidase) in be due to protein folding differences influencing cleavage activity. addition to MMP9 activation (Fig. 3). We directly applied a triple anti- GelE is a metalloproteinase with broad substrate specificity and is biotic solution (ciprofloxacin, metronidazole, and neomycin) to anas- known to cleave unfolded proteins (27), whereas SprE is a serine pro- tomotic tissues via enema both immediately after surgical anastomotic tease with narrow substrate specificity (22) and thus may be incapable of construction and again on POD1. Rats were then treated with and cleaving proteins that are not properly folded. To verify the exclusive without antibiotic enemas and assigned to the following groups: role of SprE, we next examined the complemented mutants ∆gelE/ (i) sham operation; (ii) devascularization alone (Dvasc)—that is, no re- gelE, ∆sprE/sprE, and ∆gelE∆sprE/gelE+sprE for their ability to acti- section or anastomosis; (iii) resection + anastomosis without devas- vate macrophage MMP9. Results indicated that complementation of cularization (Anast); and (iv) resection + anastomosis + devascularization ∆sprE with sprE restored MMP9 activation and increased the forma- (Anast + Dvasc). All rats were sacrificed on POD6, and proteins were tion of the truncated form of MMP9 (fig. S8). Complementation of the extracted from colon or anastomotic segments and analyzed by West- double mutant ∆gelE∆sprE with gelE+sprE also restored MMP9 activa- ern blot for MMP9, HIF-1a,iNOS,andMPO.Datademonstratedthat tion (fig. S8). Surprisingly, complementation of ∆gelE with gelE the MMP9 expression in tissues exposed to both anastomosis and de- enhanced MMP9 activation, suggesting that GelE is important for vascularization was attenuated when rats were exposed to antibiotics

E. faecalis activation of MMP9 (fig. S8). However, we cannot invoke a spe- (Fig.3,AandB,andfig.S9).Zymographyconfirmedthatthehighest on May 7, 2015 cific and independent role for either GelE or SprE in MMP9 activation. level of MMP9 activation was in the Anast + Dvasc group and that this Finally, given that P. mirabilis expressed high collagen-degrading increase was attenuated by topical antibiotics (Fig. 3C). HIF-1a expres- activity, we measured its MMP9 cleavage activity using the macro- sion was similarly induced by devascularization alone or anastomosis phage MMP9 assay. As seen in Fig. 2F, P. mirabilis did not cleave alone compared to the untreated tissues. A clear synergistic effect on in- MMP9, consistent with its lack of association with anastomotic leak. flammatory mediators was seen when anastomotic construction was Thus, pathogens with the dual capacity to degrade collagen and cleave combined with devascularization, an effect that could be markedly at- MMP9, such as E. faecalis, appear to be associated with anastomotic tenuated when anastomotic tissues were exposed to the triple topical leak in this rat model. antibiotics. This synergism was also observed for iNOS and MPO, where stm.sciencemag.org To define the role of MMP9, we performed reiterative studies in topical antibiotic treatment attenuated expression of both inflammatory separate groups of rats using a specific MMP9 inhibitor (MMP9 inhib- markers. Together, these results indicate that the intestinal microbiota itor I, Calbiochem, catalog #444278-500UG). All rats underwent colon contributes to the amplification of MMP9, HIF-1a,andinflammation resection + anastomosis + devascularization with and without the during anastomotic surgery.

ACB

* Downloaded from RAW 264.7 Leak MMP9 30

α 20 HIF-1 Pro-MMP9 Active MMP9 iNOS 10 Truncated MMP9

MPO arbitrary units MMP9, Pro-MMP2 Active MMP2 Dvasc +++ –––– + 0 Dvasc –––– ++++ Anast –– ++ –– ++ ShamDvascAnast DvascAnast Topical Abx –––– + +++ Anast –– ++ –– ++ Dvasc +Anast Dvasc +Anast Topical Abx –––– + +++

No Abx Topical Abx Fig. 3. Antibiotic treatment of rats with anastomosis plus devascular- formed on three rats per group; displayed immunoblot is representative ization. Antibiotic treatment (Abx) of rats with anastomosis plus devascu- of all results. (B) The evaluation of band intensities using ImageJ software larization (Anast + Dvasc) attenuated MMP9 activation and expression of demonstrated the abundance of the active form of MMP9 in the rat group HIF-1a, iNOS, and MPO. (A) Western blot analyses of rat tissues subjected to Anast + Dvasc and loss of active MMP9 after topical application of antibio- devascularization (Dvasc), anastomosis (Anast), or Anast + Dvasc, in the tics. n =3;*P < 0.01, Student’s t test. (C) Zymography analysis confirmed the presence or absence of direct topical application of ciprofloxacin, metroni- abundance of the active form of MMP9 in the Anast + Dvasc rat group and dazole, and neomycin (topical Abx) via enema. Experiments were per- loss of the active form of MMP9 by topical application of antibiotics.

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 5 RESEARCH ARTICLE

We next compared the topical antibiotic regimen with parenteral to protect the underlying intestinal epithelium from invasion by path- cefoxitin, the most common antibiotic used as a single agent for pro- ogenic bacteria as well as to induce a health-promoting immune re- phylaxis in colon surgery as recommended by the Center for Medical sponse. Under normal conditions, Proteobacteria represent less than Services Surgical Quality Improvement Project (SCIP) (3). It has been 1% of the indigenous microbiota in the colon; however, when they well documented that cefoxitin and related cephalosporins do not predominate, they are often associated with intestinal pathology. eliminate E. faecalis. E. faecalis has been shown to “bloom” in the in- Several of our patients displayed a reversal in the ratio of health-related testine after a single parenteral dose of cefoxitin (28). As might be pre- Bacteroidetes to disease-related Proteobacteria (Fig. 6, A and B), the dicted, only the topical antibiotic regimen prevented anastomotic opposite of what would normally be expected. Of particular note was leak (Fig. 4A) in association with suppression of bacterial collagenase the ratio of Proteobacteria to Bacteroidetes in the swab from the distal (Fig. 4B), preservation of intestinal collagen (Fig. 4C), and lower ex- end of the colon of patient #10 that showed a ratio of Proteobacteria to pression of intestinal MMP9 (Fig. 4, D and D′, and fig. S10). High Bacteroidetes of 3:1, indicative of a highly imbalanced microbiota collagenase-producing strains of E. faecalis were recovered from (Fig. 6, A and C). In this patient, the phylum Proteobacteria (with anastomotic tissues in rats treated with cefoxitin (fig. S11). No E. faecalis an abundance of 50.12%) comprised mainly the class Gammaproteo- strains were recovered from rats treated with the topical antibiotic bacteria (44.47%) (Fig. 6, B and D) and the family Enterobacteriaceae regimen administered via enema (fig. S11). (43.1%), which encompasses the greatest number of human pathogens. Thus, among all 11 patients, patient #10 had the most pronounced re- Bacterial strains present on human colon tissues at the time versal of the ratio of Bacteroidetes to Proteobacteria (3:1) and was of anastomotic construction are capable of degrading culture-positive with a collagenolytic P. aeruginosa strain that cleaved collagen and activating MMP9 MMP9. This patient was discharged after surgery and then readmitted We next determined whether human colon anastomotic segments for an intestinal obstruction and a suspected abdominal abscess. Subse- harbored microbial organisms that expressed the “leak phenotype” quently, the patient developed a clinical course characteristic of an anas- defined by their ability to degrade collagen and activate MMP9. Eleven tomotic leak including small-bowel obstruction up to the point of the consecutive patients undergoing colon surgery were studied under In- ileocolic anastomosis, perianastomotic inflammation and free air, and

stitutional Review Board (IRB) protocol approved by the University of fluid collection. The patient was treated with percutaneous drainage, on May 7, 2015 Chicago. During surgery, once the colon segment was resected, the nasogastric tube decompression, and broad spectrum antibiotics. An distal and proximal ends were immediately swabbed for microbial anastomotic leak, however, was not confirmed with a contrast enema analysis. Swabs were used to isolate DNA for 16S rRNA analysis and, because this was a right hemicolectomy, and a full-contrast enema or in addition, were cultured to test individual growing organisms for colonoscopy would have been risky and difficult. The patient’scourse collagen-degrading activity and their ability to cleave human r-MMP9. gradually resolved with conservative management alone. Finally, al- All 11 patients received intravenous cefoxitin prophylaxis according to though 1 of the 11 patients harbored a collagenolytic E. faecalis strain, the SCIP recommendations, and all recovered from surgery and were this patient did not develop a clinical leak, perhaps owing to less dis- discharged home (3). One patient (patient #10) was readmitted because ruptioninthispatient’s indigenous microbiota. stm.sciencemag.org of complications. Of 64 cultured strains, only two species, P. aeruginosa and E. faecalis, showed significant collagenase activity (Fig. 5A) and cleaved MMP9 (Fig. 5, B and C). 16S rRNA analysis demonstrated dis- DISCUSSION ruption of the normal microbial community structure and member- ship distribution in anastomotic tissues among the 11 patients. This is Data from the present study offer the possibility that members of in- not surprising given that patients received purgative intestinal cleans- testinal commensal microflora, such as E. faecalis, may contribute to ing solutions and antibiotics before surgery. Normally, the dominant anastomotic leak. Here, we show in rats that the current method of phyla in the colon are Bacteroidetes and Firmicutes, which are known antibiotic prophylaxis used in humans (that is, systemic administration Downloaded from

ACBD D x104 * * 1.6 100 5 50 * MMP9 1.2 80 4 * 40 * 60 3 30 0.8 No Abx Topical Abx IM cefoxitin 40 2 20 0.4 Leak rate, % rate, Leak 20 1 10 Collagen content, % content, Collagen MMP9, arbitrary units MMP9,

0 activity, Collagen-degrading RFU/OD 600 nm 0 0 0 –– – + IM cefoxitin –– + –– – + + –– – + –– – + Topical Abx + –– ––+ + ––+ No Abx Leak Topical Abx IM cefoxitin Fig. 4. Topical antibiotic treatment applied to rat anastomotic tissues whole microbial communities, and intestinal collagen content in rats that prevents leak. Effect of intramuscular (IM) cefoxitin (systemic antibiotic underwent surgical resection, anastomosis, and devascularization (n =10; treatment) versus direct topical antibiotic treatment with ciprofloxacin, fla- *P < 0.01, Student’s t test). (D and D′) Effect of intramuscular cefoxitin versus gyl, and neomycin via enema on POD 0 and 6. (A to C) Effects of systemic topical antibiotic treatment on intestinal tissue MMP9 activity (n =3;*P < 0.05, and topical antibiotic treatment on leak rate, collagen-degrading activity of Student’s t test).

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 6 RESEARCH ARTICLE

A x104 10

8

6

4

2 Collagen-degrading activity, activity, Collagen-degrading RFU/OD 600 nm 0 -1-d -2-d -1-d -1-d -1-d -1-d -1-d -1-d -1-d -1-d -1-d -1-p -2-p -1-p -1-p -1-p -1-p -1-p -1-p -2-p -1-p -1-p -1-p E. coli E. E. coli E. coli E. E. coli E. coli E. E. coli E. E. coli E. coli E. coli E. E. coli E. coli E. E. coli E. E. coli E. E. coli E. S. mitis -1-d S. S. mitis -1-d S. S. mitis -1-p S. E. faecalis E. E. faecalis E. C. tertium C. E. faecium E. -1-d gordonii S. -1-d gordonii S. -1-p gordonii S. -1-d salivarius S. -1-d salivarius S. -1-d salivarius S. -1-d salivarius S. -1-p salivarius S. -1-p salivarius S. S. sanguinis -1-d S. S. anginosus -1-d S. anginosus -2-d S. S. anginosus -1-d S. S. anginosus -1-p S. anginosus -2-p S. anginosus -1-p S. S. anginosus -1-p S. P. aeruginosa -1-d P. aeruginosa -2-d P. S. gallolyticus S. S. gallolyticus S. -1-d marcescens S. S. intermedius -1-d S. K. pneumoniae -1-d K. K. pneumoniae -1-d K. K. pneumoniae -1-p K. pneumoniae -1-p K. pneumoniae -2-p K. Enterobacter sp-1-d Enterobacter sp-2-d -1-d parasanguinis S. -1-d parasanguinis S. -1-d parasanguinis S. -1-d parasanguinis S. -1-p parasanguinis S. -1-p parasanguinis S. -2-p parasanguinis S. -1-p parasanguinis S. -1-p parasanguinis S. E. avium/raffinosus E. E. avium/raffinosus E. α -Hemolytic strept-1-p Corynebacterium sp-1-d Corynebacterium sp-2-d Corynebacterium sp-1-p Enterobacter cloacae -1-p Enterobacter cloacae

Patient #3 #4 #5 #6 #8 #9 #10 #11 B

r-MMP9 Cleaved form

-1-p -1-p -1-p -1-p -d -1-d -1-d -1-d -1-d -1-p -1-d

E. coli No bacteria on May 7, 2015 salivarius E. faecium E. faecalis No bacteria Pt.10 - K. pneumoniae-1-p Pt.11 - E. faecalis Pt.11 - E. faecalis Pt.11 - E. faecalis Pt.11 - E. cloacae Pt.11 - E. faecalis Pt.11 - E. faecalis Pt.11 - Pt.11 - (live bacteria) (secreted products) Pt.10 - S. lostridium tertium (secreted products) Pt.11 - S. marcescens-1-d (live bacteria) (secreted products) Pt.11 - (secreted products) Pt.10 - S. parasanguinis-1-d C Pt.10 - C No r-MMP9 r-MMP9 No r-MMP9 r-MMP9

r-MMP9 r-MMP9

Cleaved form Cleaved form stm.sciencemag.org

d d -2- ical strain inosa-1-d uginosa-1-d No bacteria No bacteria - clin P. aer P. aeruginosa-2-d

Pt.10 - positive control positive control Pt.10 - P. aeruginosa Pt.10 - -1-dP. aeruginosapositive-2-d control Pt.10 - P. aeruginosapositive-2-d control Pt.10 - P. aeruginosa Pt.10 - P. aeru Pt.10g - P. aeruginosa P. aeruginosa - cli Pt.10nical strain- P. aeruginosa-1-P. aeruginosa - clinical strain P. aeruginosa - clinical strain Downloaded from Fig. 5. Isolation of bacterial species from resected intestinal samples ability of isolated strains of E. faecalis to activate cleavage of human recom- from surgical patients. Shown are species identification, collagen-degrading binant pro-MMP9 (r-MMP9) to its active form. (C) Zymography (left panel) activity, and MMP9 cleavage capacity among 68 bacterial isolates cultured and Western blot (right panel) demonstrating the ability of P. aeruginosa from the proximal (p) and distal (d) ends of resected intestinal specimens human isolates to activate cleavage of human r-MMP9 to its active form. from 11 patients undergoing colon surgery. (A) Culture results of bacterial P. aeruginosa positive control is a human stool isolate maintained in our species and their collagen-degrading activity from patients #3 to #11; pa- laboratory that is known to have high collagen-degrading activity as well tients #1 and #2 were culture-negative. (B) Zymography demonstrating the as the ability to cleave MMP9. of cefoxitin) fails to eliminate E. faecalis in association with anasto- biotic prophylaxis (30). Here, we found that E. faecalis contributes to motic leak. A recent study examining the fecal microbiota of patients anastomotic leak through its collagenolytic and MMP9-activating undergoing colorectal surgery for cancer before and after surgery functions. Our rat model suggests that there may exist a leak phenotype demonstrated that, despite the use of both intravenous (that is, a second- among intestinal microbes that colonize anastomotic tissues. This phe- generation cephalosporin) and oral antibiotics (kanamycin and metro- notype appears to be a function of a given bacterial strain’s collagen- nidazole), E. faecalis and P. aeruginosa were among the most dominant degrading activity and its ability to cleave host intestinal MMP9 to its pathogens remaining in feces after surgery with a several log-fold in- active, extracellular matrix–degrading form (31). MMPs, and in particu- crease in their concentration and prevalence over the course of recovery lar MMP9, are associated with inflammation (32, 33) in general and and hospitalization (29). Others have also demonstrated the high anastomotic leak (34–37) specifically. We show that E. faecalis can ac- prevalence of Enterococcus after colon surgery despite adequate anti- tivate MMP9 in a manner that is dependent on GelE and SprE, both of

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 7 RESEARCH ARTICLE

A B The observation that E. faecalis degrades collagen I and activates MMP9 to its active form suggests a dual mechanism by which E. faecalis might complicate anastomotic healing. Some evidence for this was provided by the observation that either bacterial elimination with top- ical antibiotic treatment or MMP9 in- hibition prevented anastomotic leak. It is important to recognize, however, that parenteral cefoxitin did not kill E. faecalis in our rat model nor did it prevent leak- age. It is noteworthy that the most com- monly used antibiotics in colon surgery (that is, second- and third-generation ce- Pt.6 - d Pt.7 - d Pt.8 - d Pt.9 - d Pt.6 - d Pt.7 - d Pt.8 - d Pt.9 - d Pt.6 - p Pt.7 - p Pt.8 - p Pt.9 - p Pt.6 - p Pt.7 - p Pt.8 - p Pt.9 - p Pt..1 - d Pt..2 - d Pt..3 - d Pt..4 - d Pt..5 - d Pt..1 - d Pt..2 - d Pt..3 - d Pt..4 - d Pt..5 - d Pt..1 - p Pt..2 - p Pt..3 - p Pt..4 - p Pt..5 - p Pt..1 - p Pt..2 - p Pt..3 - p Pt..4 - p Pt..5 - p Pt.10 - d Pt.11 - d Pt.10 - d Pt.11 - d Pt.10 - p Pt.11 - p Pt.10 - p Pt.11 - p phalosporins) do not eliminate E. faecalis in the gut but in fact allow it to proliferate phylum_Bacteroidetes phylum_Bacteroidetes; class_Bacteroidia and predominate (28, 30, 49). Although phylum_Firmicutes phylum_Firmicutes; class_Clostridia many have advocated the use of non- phylum_Proteobacteria; class_Gammaproteobacteria absorbable oral antibiotics in colon surgery phylum_Proteobacteria including the use of a combination of C D oral kanamycin and erythromycin (50), whether they actually eliminate collagen- Distal part of removed colon

degrading E. faecalis and other such or- on May 7, 2015 Proximal part of removed colon ganisms from anastomotic tissues remains 50 unknown. Our results from patients undergoing 3 colon surgery were not powered to deter- 40 minethepositivepredictivevalueofthe 30 collagenase/MMP9-cleaving phenotype 2 of bacterial strains on anastomotic leak. However, our rat data suggest that it 20 stm.sciencemag.org maybeworthtestingthepredictivevalue 1 10 of the collagenase/MMP9-cleaving bac- terial phenotype on anastomotic leak in 0 0 a clinical trial of patients undergoing co- Ratio Proteobacteria/Bacteroidetes lorectal surgery. How the disruption of Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Pt. 8 Pt. 9 Pt. Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Pt. 8 Pt. 9 Pt. Pt. 10 Pt. 11 Pt. Pt. 10 Pt. 11 Pt.

Abundance of Gammaproteobacteria, % of Gammaproteobacteria, Abundance normal microbiota community structure Fig. 6. Microbial composition of resected intestinal specimens from 11 patients undergoing elective due to prolonged illness and antibiotic colon surgery. (A and B)16S rRNA analysis at the phylum (A) and class (B) levels of the microbial community treatment affects proliferation of bacteria Downloaded from composition of the proximal (p) and distal (d) ends of resected colon samples from 11 patients. (C)Ratioof expressing the leak phenotype remains to Proteobacteria (disease-related) to Bacteroidetes (health-promoting) in the 11 patients (Pt) who underwent be defined. Our preliminary survey of 64 elective colon surgery. (D) Gammaproteobacteria was the most abundant class in patient #10. bacterial strains isolated from human anastomotic tissues demonstrated that only P. aeruginosa and E. faecalis expressed which appear to play a key role in anastomotic leak. In these ex- the collagen-degrading/MMP9-cleaving phenotype. However, the periments, we used V583, a vancomycin-resistant strain of E. faecalis background microbial composition in the patient harboring P. aeruginosa (38). It should be noted that vancomycin-resistant E. faecalis strains was highly disrupted, and this was the only patient with a suspected anas- represent only a minority of all vancomycin-resistant enterococci in- tomotic leak. Given these findings, we are currently planning a clinical fections (39–45). However, risk factors for vancomycin-resistant E. faecalis trial in which we will use serial endoscopic surveillance of anastomotic acquisition include previous antibiotic exposure and invasive procedures tissues after lower colon surgery over the entire course of healing (21 days). (43, 44), factors that are present in patients undergoing colorectal surgery. We plan to directly swab anastomotic tissues during the serial endoscopic Although the prevalence of vancomycin-resistant E. faecalis among pa- exam and identify tissue-associated microbial composition and phenotype; tients undergoing colorectal surgery is unknown, perhaps it should be the microbial community structure, phenotype, and species that develop examined in light of our results. over time will be investigated. Notably, E. faecalis degrades collagen I and activates MMP9, which There are some limitations to our study. First, we did not culture in turn degrades collagen IV (46). Both collagen I and IV play an impor- obligate anaerobes at anastomotic sites, which may play a contributory tant role in maintenance and repair of the extracellular matrix (47, 48). role in anastomotic leak. It is known that anaerobic bacteria such as

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 8 RESEARCH ARTICLE

Porphyromonas (Bacteroides) gingivalis (51), Bacteroides fragilis (52), Rat model of colorectal anastomosis and Clostridium histolyticum (53) produce collagenases. Application Adult, male Wistar rats (250 to 300 g) (Charles River Laboratories) of metagenomics/metatranscriptomics complemented with proteomics were used for all experiments. Rats underwent general anesthesia and metabolomics would be an ideal approach to more completely and laparotomy with segmental colon resection at the peritoneal define the roles that microbial community structure, membership, reflection and primary colorectal anastomosis. Devascularization and function play in the pathogenesis of anastomotic leak. Although of the anastomotic segment was carried out by dividing the feed- we attempted to do this in our human samples using 16S rRNA anal- ing blood vessel 1 cm above and below the anastomotic suture line ysis, our clinical study was not set up nor powered to fully use this ap- (55). Full details of the anastomotic surgery and other related procedures proach. We are planning a clinical trial in which the microbes present such as involving treatment with antibiotics and MMP9 inhibitors, anas- on anastomotic tissues can be comprehensively analyzed over the full tomotic leak evaluation, blood vessel visualization, and intestinal course of healing and their role in leak determined. Second, our rat collagen content are outlined in the Supplementary Materials. model has certain limitations in terms of recapitulating the anasto- motic leak that occurs in humans. Most anastomotic leaks are discovered Bacterial strains on computed tomography (CT) scans and show up as inflammation Bacterial strains isolated in this study were used in defined experi- or fluid collections adjacent to the anastomotic site. Leaks are not di- ments for collagen-degrading activity and MMP9 activation assays. rectly visualized during surgery because most patients do not undergo Among them, E. faecalis E1 and E. faecalis E2 were additionally used reoperation. The precise definition of anastomotic is thus debated, and in rat model. E. faecalis V583 and its derivative mutants ∆gelE, ∆sprE, both contrast enema and CT scanning are known to carry significant ∆gelE∆sprE, and complemented mutants ∆gelE/gelE, ∆sprE/sprE,and false-negative rates (54). Our approach was to directly examine all ∆gelE∆sprE/gelE+sprE were provided by L.E.H. (56). All strains were anastomoses in rats for evidence of dense adhesion, dehiscence, inflam- stored in 10% glycerol stock at −80°C. Only cells freshly plated from mation, and purulence that clearly contrasted with a healthy non-inflamed stock were used in experiments. Cells from stock were plated onto anastomosis without adhesions. We believe that this assessment is in tryptic soy broth (TSB) plates, grown overnight at 37°C, and were fur- line with what might be seen clinically in patients. ther used as designed. For complemented mutants, spectinomycin m

In summary, we present evidence that microbial pathogenesis may (500 l/ml) was added to TSB. on May 7, 2015 underlie anastomotic leak and may involve pathogens, such as E. faecalis, that break down collagen and cleave host tissue MMP9. Microbial culture and anastomotic tissue analysis In a separate experimental run, four groups of 10 rats each were ran- domly assigned to normal unoperated controls, devascularization MATERIALS AND METHODS alone (Dvasc), resection + anastomosis only (Anast), and resection, anastomosis, and devascularization (Dvasc + Anast). Colorectal Study design anastomotic tissues were then inspected for leak, opened, and The rationale for the study was based on performing a low colon anas- swabbed for microbial analysis. Swabs were cultured on medium spe- stm.sciencemag.org tomosis at the junction of the rectum and colon where the highest inci- cific for Gram-negative and Gram-positive bacteria, and bacterial spe- dence of anastomotic leak occurs clinically. On the basis of preliminary cies were identified as previously described (57, 58). Tissues were data, power analyses were carried out to determine the number of animals collected and analyzed for MMP9 and inflammatory markers. in each group required for statistical significance. Rules for stopping the experiment were to be enacted if ratsappearedmoribundorinanydis- Enterococcal inoculation of colorectal anastomosis tress whereby they were sacrificed immediately and excluded from To test the hypothesis that E. faecalis strains can cause anastomotic results. However, this was not the caseforanyanimalsinthestudy.There leak, we altered the animal model described above. As previously de- were five anesthetic deaths that occurred shortly after the initial surgery scribed, 1 hour before laparotomy, rats were given two 5-ml rectal Downloaded from (within 6 hours). These animals were excluded from the study analysis. enemas of atropine (0.2 mg/ml; Sigma-Aldrich) and N-acetyl-L-cysteine Devascularization was performed to divide the blood vessels supplying (20 mg/ml; Sigma-Aldrich), followed by two 5-ml rectal enemas of the removed segment of colon to mimic the clinical performance of in- 0.9% NaCl to remove the protective mucus layer (59). Additionally, testinal surgery. The overall objectives of the study were to develop a to rid the intestine of normal flora, cefoxitin (50 mg/kg) was given via model in which a significant (that is, 50%) leak rate was observed to an- intramuscular injection 30 min before incision. After completion of alyze tissues and determine the molecular mechanisms of anastomotic the anastomosis, E. faecalis strains were intestinally inoculated via a leak. Standard measurements (that is, histology and gross observation) 5-ml rectal enema of 1:100 diluted overnight culture in Todd-Hewitt were used to determine leak, and Western blotting, microbial analyses, broth (THB). As described above, the animals were sacrificed on and zymography were used to determine molecular markers of leak. POD6 to evaluate for anastomosis integrity. Randomization of rats to the various groups was performed by random- ly picking rats housed in groups of five to one group or another. All Human studies analyses, including the gross determination of leak, were performed Under approval from the IRB at the University of Chicago and North in a blinded fashion where the analysis was performed by a person Shore University Hospital IRB11-0481, 11 consecutive patients un- blinded to treatment. Sample size and replicates are included in dergoing colon surgery were consented to participate in the study. the figure legends. End point selection was based on using gross inspec- When the colon sample was removed by the operating surgeon, tion of the anastomotic sites on POD6 as the hard end point of the study the distal and proximal ends were immediately swabbed for 16S given that it is a common time at which an intestinal anastomosis leaks rRNA analysis and aerobic culture. In each case, whether the patient clinically. recovered and was discharged uneventfully or whether the patient

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 9 RESEARCH ARTICLE developed an anastomotic leak was determined by communicating staining was used as a loading control. The membranes were blocked with the operating surgery. in tris-buffered saline containing Tween 20 (pH 7.4) added with 4% nonfat dry milk (LabScientific) for 1 hour and incubated with Collagen-degrading activity primary anti-MMP9, iNOS (Abcam), HIF-1a,andMPO(NovusBio- Collagen-degrading activity was assessed as previously described (5) logicals) antibodies, followed by the corresponding horseradish peroxidase– using an EnzChek Gelatinase/Collagenase Assay Kits (Molecular coupled secondary antibody. The dilution of antibodies was performed Probes). Details are outlined in Supplementary Materials. as recommended by the vendors. The membranes were developed using ECL Western Blotting Detection Reagents (GE Healthcare). MMP9 activation assays Densitometry analyses were performed using ImageJ software. We have used human recombinant MMP9, murine macrophage MMP9, and intestinal tissue MMP9 to test for the MMP9 activation. Detailed Collagen-degrading activity in anastomotic tissues methods are displayed below. Tissue extracts from the above-described preparation were used to mea- Recombinant MMP9. Recombinant human proenzyme MMP9 sure total collagen-degrading activity in anastomotic tissues. Tissue (r-MMP9) (Calbiochem, catalog #PF038) was used as a substrate. extracts containing 60 mg of total protein were suspended in buffer m The r-MMP9 was diluted to a final concentration of 1 g/ml in the as- containing 50 mM tris-HCl, 0.15 M NaCl, 5 mM CaCl2,1mMZnCl2 say buffer [50 mM tris-HCl (pH 7.5), 10 mM CaCl2, and 0.05% Triton (pH 7.6), supplemented with 10 ml of fluorescent gelatin (1 mg/ml) in X-100]. Detection of MMP9-cleaving activity was performed as pre- total volume of 200 ml in 96-well plates. Incubation was performed at viously described (60). Briefly, 30 ml of bacterial culture grown in THB 37°C under static conditions, and fluorescence was measured at 480/ 8 for 6 hours [~OD 600 nm = 1.0, which corresponded about 5 × 10 520 nm. All experiments were performed in triplicate. Zymography cells/ml as measured by plating of 10-fold dilutions] was incubated assay is described in details in the Supplementary Materials. with 20 ml of r-MMP9 (1 mg/ml) for 2 hours. After centrifugation, 5 ml of the supernatant was mixed with an equal volume of 2× SDS loading C. elegans killing assay buffers and subjected to zymography. The positive control included E. faecalis E1 and E2 strains were tested for virulence/killing capacity

4-aminophenylmercuric acetate (Sigma) known to induce autocleavage by assessing mortality of C. elegans fed on E. faecalis lawns (62). Full on May 7, 2015 of MMP9 (61). Fresh 100 mM stock of APMA in DMSO was prepared, details are outlined in the Supplementary Materials. and MMP9 was activated by adding APMA to a final concentration of 1 mM, followed by incubation at 37°C for 3 hours. 16S rRNA analysis of bacterial community structure Murine macrophage MMP9. The murine macrophage cell line Composition of the bacterial microflora was analyzed by 16S rRNA (J774) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) V4 iTAG amplicon sequencing analysis (63, 64) using DNA recovered (Invitrogen) supplemented with 10% fetal bovine serum. The macro- from tissue swabs. Full details are outlined in the Supplementary Materials. phage cells were harvested and resuspended in DMEM to a concen- tration of 2 × 106 cells/ml. The cells were seeded on a 12-well plate, 2 ml Statistical analysis stm.sciencemag.org for each well, incubated for 1 hour, and allowed to adhere to plate sur- Statistical analysis was performed using SigmaPlot software. Student’s t face. E. faecalis strains were grown in THB to log phase, and density of tests were used when analyzing the differences between two means, cells was adjusted to OD 600 nm = 1.0. Bacterial cell suspensions (30 ml) whereas analysis of variance (ANOVA) with Bonferroni correction were added to each well. After 4-hour co-incubation, supernatants were was used when more than two means were compared. Kaplan-Meier collected, clarified by centrifugation for 10 min at 10,000g, and analyzed survival plot was analyzed using SPSS software. Significance was de- by zymography and Western blot. termined as a P value <0.05. Intestinal tissue MMP9. E. faecalis strains E1 and E2 were grown overnight in THB, followed by centrifugation (5000 rpm, 6 min) to Downloaded from separate bacterial cells and secreted fraction (supernatant). Bacterial SUPPLEMENTARY MATERIALS pellet was then diluted in fresh THB to OD 600 nm = 0.2, and 500 ml m www.sciencetranslationalmedicine.org/cgi/content/full/7/286/286ra68/DC1 of culture suspension was added to 100 g of colon tissues. Similarly, Materials and Methods 500 ml of filter-sterilized supernatant was added to 100 mg of colon tissues. Fig. S1. Images of selected rat colon segments after colon resection with and without segmen- After 4 hours of incubation at 37°C, colon tissues and their conditioned tal devascularization. media were collected and subjected to zymography and Western blot. Fig. S2. Collagenolytic activity of tissue extracts from leaking anastomosis compared to non- leaking anastomosis. Fig. S3. Time course–dependent degradation of gelatin by E. faecalis isolates E1 and E2. Western blot Fig. S4. Kaplan-Meier survival curves of C. elegans feeding on E1 and E2 strains of E. faecalis. The resected colon tissues were weighted, immediately placed in liquid Fig. S5. Gelatin degradation by complemented mutants. nitrogen, and kept at −80°C. The ice-cold lysis buffer [50 mM tris-HCl Fig. S6. Effect of zinc-chelating compound 1,10-phenanthroline on collagen-degrading activity (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, and phenylmethylsulfo- in E. faecalis E2 and V583 strains. Fig. S7. Zymography of tissue extracts in leaking tissues. nyl fluoride] was added to ~80 mg of grinded tissues (1:10; 80 mg of Fig. S8. Zymography analysis of macrophage activation of MMP9 by wild-type V583, its mutants tissues/800 ml of buffer). After vortexing and 15-min incubation on ice, lacking GelE and SprE, and its complemented mutants ∆gelE/gelE, ∆sprE/sprE,and∆gelE∆sprE/gelE+sprE. the lysates were centrifuged at 10,000 rpm for 15 min to remove the Fig. S9. Western blot analyses to identify MMP9 in tissues of rats subjected to devascularization debris. The supernatants (15 ml that contained ~40 mg of protein) were (Dvasc), anastomosis (Anast), or Anast + Dvasc, in the presence or absence of direct topical application of ciprofloxacin, metronidazole, and neomycin via enema. boiled for 5 min with Laemmli sample buffer, electrophoresed through Fig. S10. Effect of topical antibiotics on the activation of MMP9. 10% SDS–polyacrylamide gels, and then transferred onto poly- Fig. S11. Gelatin degradation by individual strains recovered from anastomotic tissues after intra- vinylidene difluoride membranes (Immobilon-P, Millipore). Ponceau muscular injection of cefoxitin or topical application of ciprofloxacin, metronidazole, and neomycin.

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 10 RESEARCH ARTICLE

REFERENCES AND NOTES 25. S. de Bentzmann, M. Polette, J. M. Zahm, J. Hinnrasky, C. Kileztky, O. Bajolet, J. M. Klossek, A. Filloux, A. Lazdunski, E. Puchelle, Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactiva- 1. P. Matthiessen, O. Hallböök, J. Rutegård, G. Simert, R. Sjödahl, Defunctioning stoma re- tion of epithelial matrix metalloproteinase–2. Lab. Invest. 80, 209–219 (2000). duces symptomatic anastomotic leakage after low anterior resection of the rectum for 26. T. Okamoto, T. Akaike, M. Suga, S. Tanase, H. Horie, S. Miyajima, M. Ando, Y. Ichinose, H. Maeda, cancer: A randomized multicenter trial. Ann. Surg. 246, 207–214 (2007). Activation of human matrix metalloproteinases by various bacterial proteinases. J. Biol. Chem. 2. I. Cohn Jr., J. D. Rives, Antibiotic protection of colon anastomoses. Ann. Surg. 141, 707–717 272, 6059–6066 (1997). (1955). 27. C. M. Waters, M. H. Antiporta, B. E. Murray, G. M. Dunny, Role of the Enterococcus faecalis 3. R. J. Deierhoi, L. G. Dawes, C. Vick, K. M. Itani, M. T. Hawn, Choice of intravenous antibiotic GelE protease in determination of cellular chain length, supernatant pheromone levels, prophylaxis for colorectal surgery does matter. J. Am. Coll. Surg. 217, 763–769 (2013). and degradation of fibrin and misfolded surface proteins. J. Bacteriol. 185, 3613–3623 (2003). 4. M. J. Englesbe, L. Brooks, J. Kubus, M. Luchtefeld, J. Lynch, A. Senagore, J. C. Eggenberger, 28. L. Kager, A. S. Malmborg, C. E. Nord, R. Pieper, The effect of short-term cefoxitin prophylaxis V. Velanovich, D. A. Campbell Jr., A statewide assessment of surgical site infection on the colonic microflora in patients undergoing colorectal surgery. Infection 10,338–340 following colectomy: The role of oral antibiotics. Ann. Surg. 252, 514–519 (2010). (1982). 5. A. D. Olivas, B. D. Shogan, V. Valuckaite, A. Zaborin, N. Belogortseva, M. Musch, F. Meyer, 29. S. Ohigashi, K. Sudo, D. Kobayashi, T. Takahashi, K. Nomoto, H. Onodera, Significant W. L. Trimble, G. An, J. Gilbert, O. Zaborina, J. C. Alverdy, Intestinal tissues induce an SNP changes in the intestinal environment after surgery in patients with colorectal cancer. J. mutation in Pseudomonas aeruginosa that enhances its virulence: Possible role in anas- Gastrointest. Surg. 17, 1657–1664 (2013). tomotic leak. PLOS One 7, e44326 (2012). 30. N. Komen, J. Slieker, P. Willemsen, G. Mannaerts, P. Pattyn, T. Karsten, H. de Wilt, E. van der Harst, 6. B. D. Shogan, D. P. Smith, S. Christley, J. A. Gilbert, O. Zaborina, J. C. Alverdy, Intestinal W. van Leeuwen, C. Decaestecker, H. Jeekel, J. F. Lange, Polymerase chain reaction for Enter- anastomotic injury alters spatially defined microbiome composition and function. Microbiome ococcus faecalis in drain fluid: The first screening test for symptomatic colorectal anastomotic 2,35(2014). leakage. The Appeal-study: Analysis of parameters predictive for evident anastomotic leakage. 7. M. G. Langille, J. Zaneveld, J. G. Caporaso, D. McDonald, D. Knights, J. A. Reyes, J. C. Clemente, Int. J. Colorectal Dis. 29,15–21 (2014). D. E. Burkepile, T. RL Vega, R. Knight, R. G. Beiko, C. Huttenhower, Predictive functional profiling 31. E. Hadler-Olsen, B. Fadnes, I. Sylte, L. Uhlin-Hansen, J. O. Winberg, Regulation of matrix of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, metalloproteinase activity in health and disease. FEBS J. 278,28–45 (2011). – 814 821 (2013). 32. I. Stamenkovic, Extracellular matrix remodelling: The role of matrix metalloproteinases. 8. L. R. Thurlow, V. C. Thomas, S. Narayanan, S. Olson, S. D. Fleming, L. E. Hancock, Gelatinase J. Pathol. 200, 448–464 (2003). contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis. Infect. 33. S. Chakrabarti, K. D. Patel, Regulation of matrix metalloproteinase-9 release from IL-8- Immun. 78, 4936–4943 (2010). stimulated human neutrophils. J. Leukoc. Biol. 78, 279–288 (2005). 9. J. Zeng, F. Teng, B. E. Murray, Gelatinase is important for translocation of Enterococcus faecalis 34. B. Pasternak, P. Matthiessen, K. Jansson, M. Andersson, P. Aspenberg, Elevated intra- across polarized human enterocyte-like T84 cells. Infect. Immun. 73, 1606–1612 (2005). peritoneal matrix metalloproteinases-8 and -9 in patients who develop anastomotic leak- 10. D. J. Harrington, Bacterial collagenases and collagen-degrading enzymes and their age after rectal cancer surgery: A pilot study. Colorectal Dis. 12, e93–e98 (2010). on May 7, 2015 potential role in human disease. Infect. Immun. 64, 1885–1891 (1996). 35. M. S. Agren, T. L. Andersen, U. Mirastschijski, I. Syk, C. B. Schiødt, V. Surve, J. Lindebjerg, 11. J. L. Gnerlich, J. H. Ritter, J. P. Kirby, J. E. Mazuski, Simultaneous necrotizing soft tissue J. M. Delaissé, Action of matrix metalloproteinases at restricted sites in colon anastomosis infection and colonic necrosis caused by Clostridium septicum. Surg. Infect. 12, 501–506 repair: An immunohistochemical and biochemical study. Surgery 140,72–82 (2006). (2011). 36. P. M. Krarup, M. Eld, K. Heinemeier, L. N. Jorgensen, M. B. Hansen, M. S. Ågren, Expression 12. L. J. Schlapbach, O. Ahrens, P. Klimek, S. Berger, U. Kessler, Clostridium perfringens and and inhibition of matrix metalloproteinase (MMP)-8, MMP-9 and MMP-12 in early colonic necrotizing enterocolitis. J. Pediatr. 157, 175 (2010). anastomotic repair. Int. J. Colorectal Dis. 28, 1151–1159 (2013). 13. J. Bos, L. Smithee, B. McClane, R. F. Distefano, F. Uzal, J. G. Songer, S. Mallonee, J. M. Crutcher, 37. M. S. Agren, L. N. Jorgensen, J. M. Delaissé, Matrix metalloproteinases and colon anasto- Fatal necrotizing colitis following a foodborne outbreak of enterotoxigenic Clostridium mosis repair: A new indication for pharmacological inhibition? Mini Rev. Med. Chem. 4, perfringens type A infection. Clin. Infect. Dis. 40, e78–e83 (2005). 769–778 (2004). 14. J. M. Rein, B. Cosman, Bacteroides necrotizing fasciitis of the upper extremity. Case report. 38.I.T.Paulsen,L.Banerjei,G.S.A.Myers,K.E.Nelson,R.Seshadri,T.D.Read,D.E.Fouts, stm.sciencemag.org Plast. Reconstr. Surg. 48, 592–594 (1971). J.A.Eisen,S.R.Gill,J.F.Heidelberg,H.Tettelin,R.J.Dodson,L.Umayam,L.Brinkac, 15. M. Engelbert, E. Mylonakis, F. M. Ausubel, S. B. Calderwood, M. S. Gilmore, Contribution of M.Beanan,S.Daugherty,R.T.DeBoy,S.Durkin,J.Kolonay,R.Madupu,W.Nelson,J.Vamathevan, gelatinase, serine protease, and fsr to the pathogenesis of Enterococcus faecalis endo- B.Tran,J.Upton,T.Hansen,J.Shetty,H.Khouri,T.Utterback,D.Radune,K.A.Ketchum, phthalmitis. Infect. Immun. 72, 3628–3633 (2004). B. A. Dougherty, C. M. Fraser, Role of mobile DNA in the evolution of vancomycin-resistant 16. G. Kayaoglu, D. Ørstavik, Virulence factors of Enterococcus faecalis: Relationship to endo- Enterococcus faecalis. Science 299, 2071–2074 (2003). dontic disease. Crit. Rev. Oral Biol. Med. 15, 308–320 (2004). 39. V. L. Merquior, A. R. Faria, F. S. Rocha, J. M. Morais, F. P. Neves, L. M. Teixeira, Emergence 17. A. K. Jha, H. P. Bais, J. M. Vivanco, Enterococcus faecalis mammalian virulence-related and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infect. Immun. Brazil. Mem. Inst. Oswaldo Cruz 107, 557–560 (2012). 73, 464–475 (2005). 40. B. Zheng, H. Tomita, T. Inoue, Y. Ike, Isolation of VanB-type Enterococcus faecalis strains Downloaded from 18. E. N. Vergis, N. Shankar, J. W. Chow, M. K. Hayden, D. R. Snydman, M. J. Zervos, P. K. Liden, from nosocomial infections: First report of the isolation and identification of the pheromone- M. M. Wagener, R. R. Muder, Association between the presence of enterococcal virulence responsive plasmids pMG2200, Encoding VanB-type vancomycin resistance and a Bac41- factors gelatinase, hemolysin, and enterococcal surface protein and mortality among pa- type bacteriocin, and pMG2201, encoding erythromycin resistance and cytolysin (Hly/Bac). tients with bacteremia due to Enterococcus faecalis. Clin. Infect. Dis. 35, 570–575 (2002). Antimicrob. Agents Chemother. 53, 735–747 (2009). 19. P. Bucher, P. Gervaz, J. F. Egger, C. Soravia, P. Morel, Morphologic alterations associated 41. M. A. Vilela, S. L. Souza, I. C. Palazzo, J. C. Ferreira, M. A. Morais Jr., A. L. Darini, M. M. Morais, with mechanical bowel preparation before elective colorectal surgery: A randomized trial. Identification and molecular characterization of Van A-type vancomycin-resistant Entero- Dis. Colon Rectum 49, 109–112 (2006). coccus faecalis in Northeast of Brazil. Mem. Inst. Oswaldo Cruz 101, 715–719 (2006). 20. P. L. Mäkinen, D. B. Clewell, F. An, K. K. Mäkinen, Purification and substrate specificity of a 42. M. López, E. Cercenado, C. Tenorio, F. Ruiz-Larrea, C. Torres, Diversity of clones and geno- strongly hydrophobic extracellular metalloendopeptidase (“gelatinase”)fromStreptococcus types among vancomycin-resistant clinical Enterococcus isolates recovered in a Spanish faecalis (strain 0G1-10). J. Biol. Chem. 264,3325–3334 (1989). hospital. Microb. Drug Resist. 18, 484–491 (2012). 21. X. Qin, K. V. Singh, G. M. Weinstock, B. E. Murray, Characterization of fsr, a regulator control- 43. K. Hayakawa, D. Marchaim, E. T. Martin, N. Tiwari, A. Yousuf, B. Sunkara, H. Pulluru, H. Kotra, ling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF. J. Bacteriol. A. Hasan, S. Bheemreddy, P. Sheth, D. Lee, S. Kamatam, P. Bathina, P. Nanjireddy, I. K. Chalana, 183,3372–3382 (2001). S.Patel,S.Kumar,A.Vahia,K.Ku,V.Yee,J.Swan,J.M.Pogue,P.R.Lephart,M.J.Rybak,K.S.Kaye, 22. M. Kawalec, J. Potempa, J. L. Moon, J. Travis, B. E. Murray, Molecular diversity of a putative Comparison of the clinical characteristics and outcomes associated with vancomycin-resistant virulence factor: Purification and characterization of isoforms of an extracellular serine Enterococcus faecalis and vancomycin-resistant E. faecium bacteremia. Antimicrob. Agents glutamyl endopeptidase of Enterococcus faecalis with different enzymatic activities. J. Bac- Chemother. 56, 2452–2458 (2012). teriol. 187, 266–275 (2005). 44. A. M. Omotola, Y. Li, E. T. Martin, K. Alshabani, D. Yadav, M. Sarkar, S. D. Thapa, V. Kumar, 23. M. Stumpf, U. Klinge, A. Wilms, R. Zabrocki, R. Rosch, K. Junge, C. Krones, V. Schumpelick, A. Mahabashya, S. Ahmad, A. Bhargava, D. Marchaim, J. M. Pogue, M. J. Rybak, K. S. Kaye, Changes of the extracellular matrix as a risk factor for anastomotic leakage after large K. Hayakawa, Risk factors for and epidemiology of community-onset vancomycin-resistant bowel surgery. Surgery 137, 229–234 (2005). Enterococcus faecalis in southeast Michigan. Am. J. Infect. Control 41,1244–1248 (2013). 24. C. Medina, M. W. Radomski, Role of matrix metalloproteinases in intestinal inflammation. J. 45. N. Gangurde, M. Mane, S. Fatale, Prevalence of multidrug resistant Enterococci in a tertiary Pharmacol. Exp. Ther. 318, 933–938 (2006). care hospital in India: A growing threat. Open J. Med. Microbiol. 4,11–15 (2014).

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 11 RESEARCH ARTICLE

46.M.Gioia,S.Monaco,P.E.VanDenSteen,D.Sbardella,G.Grasso,S.Marini,C.M.Overall, 61. T. Sorsa, T. Salo, E. Koivunen, J. Tyynelä, Y. T. Konttinen, U. Bergmann, A. Tuuttila, E. Niemi, G. Opdenakker, M. Coletta, The collagen binding domain of gelatinase A modulates O. Teronen, P. Heikkilä, H. Tschesche, J. Leinonen, S. Osman, U.-H. Stenman, Activation of degradation of collagen IV by gelatinase B. J. Mol. Biol. 386,419–434 (2009). type IV procollagenases by human tumor-associated trypsin-2. J. Biol. Chem. 272, 47. P. Brasken, M. Lehto, S. Renvall, Fibronectin, laminin, and collagen types I, III, IV and V in 21067–21074 (1997). the healing rat colon anastomosis. Ann. Chir. Gynaecol. 79,65–71 (1990). 62. A. Zaborina, K. Romanowskia, S. Gerdesb, C. Holbrooka, F. Lepinec, J. Longa, V. Poroykoa, 48. P. Braskén, S. Renvall, M. Sandberg, Fibronectin and collagen gene expression in healing S. P. Diggled, A. Wilkee, K. Righettid, I. Morozovaa, T. Babrowskia, D. C. Liua, O. Zaborinaa, experimental colonic anastomoses. Br. J. Surg. 78, 1048–1052 (1991). J. C. Alverdya, Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa 49. L. Kager, I. Ljungdahl, A. S. Malmborg, C. E. Nord, R. Pieper, P. Dahlgren, Antibiotic prophylaxis PAO1. Proc. Natl. Acad. Sci. U.S.A. 106, 6327–6332 (2009). with cefoxitin in colorectal surgery: Effect on the colon microflora and septic complications— 63. Q. Wang, G. M. Garrity, J. M. Tiedje, J. R. Cole, Naive Bayesian classifier for rapid assignment A clinical model for prediction of the benefit and risks in using a new antibiotic in prophylaxis. of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 Ann. Surg. 193, 277–282 (1981). (2007). 50. H. H. LeVeen, S. Wapnick, G. Falk, O. Olivas, D. Bhat, M. Gaurdre, M. Patel, Effects of pro- 64. J. G. Caporaso, C. L. Lauber, W. A. Walters, D. Berg-Lyons, J. Huntley, N. Fierer, S. M. Owens, phylactic antibiotics on colonic healing. Am. J. Surg. 131,47–53 (1976). J. Betley, L. Fraser, M. Bauer, N. Gormley, J. A. Gilbert, G. Smith, R. Knight, Ultra-high-throughput 51. A. Bodinka, H. Schmidt, B. Henkel, T. F. Flemmig, B. Klaiber, H. Karch, Polymerase chain microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6, reaction for the identification of Porphyromonas gingivalis collagenase genes. Oral Microbiol. 1621–1624 (2012). Immunol. 9, 161–165 (1994). 52. J. S. Moncrief, R. Obiso Jr., L. A. Barroso, J. J. Kling, R. L. Wright, R. L. Van Tassell, D. M. Lyerly, Acknowledgments: We thank the Core Facility Human Tissue Resource Center, University of T. D. Wilkins, The enterotoxin of Bacteroides fragilis is a metalloprotease. Infect. Immun. 63, Chicago for tissue sectioning, staining, and examination of slides. Funding: This study was 175–181 (1995). funded by a pilot and feasibility grant (J.C.A.) from the NIH Digestive Diseases Research Core 53. M. D. Bond, H. E. Van Wart, Purification and separation of individual collagenases of Centers (P30 DK42086). Author contributions: B.D.S. participated in study design, performed Clostridium histolyticum using red dye ligand chromatography. Biochemistry 23, 3077–3085 animal experiments, collagenase assays, and histochemistry analysis, collected samples, ana- (1984). lyzed data, performed statistics, and wrote portions of the manuscript. N.B. performed Western 54. V. N. Kornmann, B. van Ramshorst, A. B. Smits, T. L. Bollen, D. Boerma, Beware of false- blots, zymography, and macrophage assays, participated in study design, and wrote portions negative CT scan for anastomotic leakage after colonic surgery. Int. J. Colorectal Dis. 29, of the methods. P.M.L. performed collagenase assays and participated in study design. A.Z. 445–451 (2014). performed C. elegans experiments and participated in study design. B.S. performed animal 55. F. Daams, K. Monkhorst, J. van den Broek, J. C. Slieker, J. Jeekel, J. F. Lange, Local ischaemia experiments and mutational analysis and wrote portions of the methods. J.L. performed does not influence anastomotic healing: An experimental study. Eur. Surg. Res. 50,24–31 animal experiments and mutational analysis and wrote portions of the methods. R.K. per- (2013). formed zymography and Western blots with N.B., performed animal experiments and muta- 56. V. C. Thomas, L. R. Thurlow, D. Boyle, L. E. Hancock, Regulation of autolysis-dependent tional analysis, and wrote portions of the methods. M.W. performed animal experiments and extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm participated in study design. V.K. performed endoscopy and confocal laser endomicroscopy. C.B. on May 7, 2015 development. J. Bacteriol. 190, 5690–5698 (2008). performed bacterial identification. S.L., G.A., L.E.H., J.G., and K.U. analyzed the data; J.P.M. and 57.A.Zaborin,D.Smith,K.Garfield,J.Quensen,B.Shakhsheer,M.Kade,M.Tirrell,J.Tiedje, M.S. collected the human samples; and O.Z. and J.C.A. designed all the experiments, analyzed J. A. Gilbert, O. Zaborina, J. C. Alverdy, Membership and behavior of ultra-low-diversity the data, and wrote the paper. Competing interests: The authors declare that they have no pathogen communities present in the gut of humans during prolonged critical illness. competing interests. mBio 5, e01361–e013614 (2014). 58.K.Romanowski,A.Zaborin,V.Valuckaite,R.J.Rolfes,T.Babrowski,C.Bethel,A.Olivas, Submitted 15 September 2014 O. Zaborina, J. C. Alverdy, Candida albicans isolates from the gut of critically ill patients Accepted 17 April 2015 respond to phosphate limitation by expressing filaments and a lethal phenotype. PLOS Published 6 May 2015 One 7, e30119 (2012). 10.1126/scitranslmed.3010658 59. E. B. Kistler, T. Alsaigh, M. Chang, G. W. Schmid-Schönbein, Impaired small-bowel barrier stm.sciencemag.org integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory Citation: B. D. Shogan, N. Belogortseva, P. M. Luong, A. Zaborin, S. Lax, C. Bethel, M. Ward, shock. Shock 38, 262–267 (2012). J. P. Muldoon, M. Singer, G. An, K. Umanskiy, V. Konda, B. Shakhsheer, J. Luo, R. Klabbers, 60. M. R. Oggioni, G. Memmi, T. Maggi, D. Chiavolini, F. Iannelli, G. Pozzi, Pneumococcal zinc L. E. Hancock, J. Gilbert, O. Zaborina, J. C. Alverdy, Collagen degradation and MMP9 metalloproteinase ZmpC cleaves human matrix metalloproteinase 9 and is a virulence activation by Enterococcus faecalis contribute to intestinal anastomotic leak. Sci. Transl. Med. factor in experimental pneumonia. Mol. Microbiol. 49, 795–805 (2003). 7, 286ra68 (2015). Downloaded from

www.ScienceTranslationalMedicine.org 6 May 2015 Vol 7 Issue 286 286ra68 12 Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak Benjamin D. Shogan et al. Sci Transl Med 7, 286ra68 (2015); DOI: 10.1126/scitranslmed.3010658

Editor's Summary

Can our gut microbes prevent wound healing?

In a new study, Shogan et al. examined whether the bacterium Enterococcus faecalis, normally present in the intestine, contributes to anastomotic leak, the most feared complication after intestinal surgery. They demonstrated that intestinal E. faecalis can produce a tissue-destroying enzyme that affects the normal healing process by breaking down collagen, a protein that is critical to fully seal the intestine after its removal and reconnection. E. faecalis also activates a host gut enzyme, MMP9, further contributing to anastomotic leak. Finally, the authors demonstrated that the most common antibiotic used in intestinal surgery does not eliminate E. faecalis and thus does not prevent anastomotic leak.

A complete electronic version of this article and other services, including high-resolution figures,

can be found at: on May 7, 2015 http://stm.sciencemag.org/content/7/286/286ra68.full.html Supplementary Material can be found in the online version of this article at: http://stm.sciencemag.org/content/suppl/2015/05/04/7.286.286ra68.DC1.html Related Resources for this article can be found online at: http://stm.sciencemag.org/content/scitransmed/4/137/137rv5.full.html http://stm.sciencemag.org/content/scitransmed/3/106/106ra106.full.html http://stm.sciencemag.org/content/scitransmed/6/254/254ra127.full.html stm.sciencemag.org Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at: http://www.sciencemag.org/about/permissions.dtl Downloaded from

Science Translational Medicine (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved. The title Science Translational Medicine is a registered trademark of AAAS.