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. Here, 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 metalloprotease-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 activity. We suggest that intestinal microbes with the capacity to produce collagenases and to activate host metalloproteinase MMP9 may break down collagen in the gut tissue contributing to anastomotic leak.

Bacterial growth as a biofilm on solid surfaces is strongly associated with the development of human infections. Biofilms on native heart valves (infective endocarditis) is a life-threatening disease as a consequence of bacterial resistance to antimicrobials in such a state. Enterococci have emerged as a cause of endocarditis and nosocomial infections despite being normal commensals of the gastrointestinal and female genital tracts. We examined the role of two-component signal transduction systems in biofilm formation by the Enterococcus faecalis V583 clinical isolate and identified the fsr regulatory locus as the sole two-component system affecting this unique mode of bacterial growth. Insertion mutations in the fsr operon affected biofilm formation on two distinct abiotic surfaces. Inactivation of the fsr-controlled gene gelE encoding the zinc-metalloprotease gelatinase was found to prevent biofilm formation, suggesting that this enzyme may present a unique target for therapeutic intervention in enterococcal endocarditis.Bacterial virulence is one of many adaptive responses generally believed to be controlled through signal transduction mechanisms (39). Signal transduction in bacteria is mainly the prerogative of the so-called two-component systems consisting of a sensory histidine kinase that senses the signal and relays the adaptive response through the transfer of a phosphoryl group to a response regulator, generally a transcriptional regulator, that modulates gene expression in response to the signal received. Two-component signal transduction pathways are responsible for controlling gene expression in a wide variety of cellular processes including sporulation, virulence, biofilm formation, and antibiotic production and resistance (for reviews, see references 15 and 28). A total of 17 two-component systems and one orphan response regulator have been identified on the genome of the Enterococcus faecalis strain V583 (14). In a study aimed at the systematic inactivation of all two-component systems present in E. faecalis and the analysis of their role in virulence, we identified the fsr system as the only one affecting biofilm formation when inactivated (our unpublished data).The fsr regulatory locus is comprised of three genes, designated fsrA, fsrB, and fsrC ( Fig. 1) (33). Recently, this system has been identified as a quorum-sensing locus which responds to the extracellular accumulation of a peptide lactone encoded at the C terminus of the FsrB protein (27). Accumulation of the peptide in the extracellular space is likely sensed by the FsrC histidine kinase, leading to the activation of the response regulator and transcription factor FsrA. The fsr system and the products of the genes it regulates have been shown to be important for virulence in several infection models, including mouse peritonitis, Caenorhabditis elegans infection, and rabbit endophthalmitis (26,32,37). The FsrABC proteins are necessary for the production of two secreted proteases, gelatinase (GelE) and serine protease (SprE) (33). Here, ...

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE ؊ mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE ؊ mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases-GelE and SprE-is responsible for regulating autolysis and the release of highmolecular-weight eDNA, a critical component for the development of E. faecalis biofilms.

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial UTIs and pose significant clinical challenges. Enterococcal species are among the predominant causative agents of CAUTIs. However, very little is known about the pathophysiology of Enterococcus-mediated UTIs. We optimized a murine model of foreign body-associated UTI in order to mimic conditions of indwelling catheters in patients. In this model, the presence of a foreign body elicits major histological changes and induces the expression of several proinflammatory cytokines in the bladder. In addition, in contrast to naïve mice, infection of catheterimplanted mice with Enterococcus faecalis induced the specific expression of interleukin 1␤ (IL-1␤) and macrophage inflammatory protein 1␣ (MIP-1␣) in the bladder. These responses resulted in a favorable niche for the development of persistent E. faecalis infections in the murine bladders and kidneys. Furthermore, biofilm formation on the catheter implant in vivo correlated with persistent infections. However, the enterococcal autolytic factors GelE and Atn (also known as AtlA), which are important in biofilm formation in vitro, are dispensable in vivo. In contrast, the housekeeping sortase A (SrtA) is critical for biofilm formation and virulence in CAUTIs. Overall, this murine model represents a significant advance in the understanding of CAUTIs and underscores the importance of urinary catheterization during E. faecalis uropathogenesis. This model is also a valuable tool for the identification of virulence determinants that can serve as potential antimicrobial targets for the treatment of enterococcal infections.

With the goal of identifying and characterizing traits of Enterococcus faecalis that play key roles in human disease, we identified an operon specifying synthesis of a capsular carbohydrate of the type most commonly expressed by clinical isolates. This surfaceexposed carbohydrate consists of glycerol phosphate, glucose, and galactose residues, and its biosynthesis is encoded by a determinant that includes 11 ORFs. Insertional inactivation of genes in this pathway yielded mutants with enhanced susceptibility to phagocytic killing in vitro and compromised in the ability to persist in regional lymph nodes in vivo.