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THE RAPID EXPOSURE OF OXYGEN IN THE COLON DURING COLORECTAL SURGERY PROMOTES BACTERIAL COLLAGENASE PRODUCTION LEADING TO ANASTOMOTIC LEAK IN MICE
Bidisha Barat
*, Mohsen Rouhani Ravari, Nicholas R. Suss, Benjamin D. Shogan
The University of Chicago Medicine, Chicago, IL
Introduction: Anastomotic leakage is a devastating complication. Previous work from our lab has shown that the expression of bacterial collagenase by microbiota that colonize anastomotic tissue impairs anastomotic healing. Which intracolonic factors promote collagenase expression during the perioperative period are unknown. During colon resection, there is a rapid transition from an anaerobic to an aerobic environment. If this dramatic environmental shift drives bacterial collagenase production is unknown. We hypothesize that aerobic conditions drive increased bacterial collagenase production resulting in anastomotic leak.
Methods: In these studies,
Proteus mirabilis was chosen as the test bacterium because it has been highly associated with anastomotic leak in previous models. Aerobic conditions were created in an incubator (21% FIO
2) whereas an anaerobic chamber was used for anaerobic conditions (0% FIO
2). Functional collagenase was measured with fluorescent collagen and RT-PCR was used to measure collagenase gene expression (ZapA). In the murine model, mice underwent a colon resection and anastomosis and were sacrificed on postoperative day (POD) 6 to assess for anastomotic healing. 100uL of
P. mirabilis (OD=0.5) was given via enema on the day of surgery to colonize the anastomosis.
Results: Despite similar growth, switching
P. mirabilis from an anaerobic to aerobic environment significantly increased the functional collagenase (Fig1A). Similarly, RT-PCR analysis demonstrated that in just 2 hours of exposure to an aerobic environment, there was increased expression of the collagenase gene (
zapA)(Fig1B). Experiments with the collagenase-deficient mutant (
zapA MUT
) produced no collagenase under both aerobic and anaerobic conditions, demonstrating that production of collagenase by oxygen was via ZapA (Fig1C). To determine the role of collagenase in anastomotic leak, we colonized a mouse anastomosis with
P. mirabilis. We found that colonization with wild-type
P. mirabilis promoted anastomotic leak whereas colonization with the collagenase-deficient mutant had no effect (Fig1D). Since a colon resection cannot be performed without creating aerobic conditions, we investigated other ways to prevent collagenase production, such as probiotic strains that could compete with pathogenic bacteria. Supernatants from one such strain,
Lactobacillus paracasei prevented functional
P. mirabilis collagenase production and ZapA expression
in vitro (Fig2A/B). Precolonization of mice with
L. paracasei gavage on preop day 1-4 and POD 1 prevented anastomotic leak in mice colonized with
P. mirabilis (Fig2C).
Conclusion: Upregulation of
P. mirabilis zapA by oxygen significantly increases collagenase production resulting in anastomotic leak in mice. Preventing this interaction by perioperative exposure to
L. paracasei could be a novel strategy to reduce anastomotic leaks.
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