While perioperative protocols have resulted in major improvements in patient recovery, current approaches designed to modify gut microbial composition for optimized wound healing remain poorly understood and suboptimized. Here, we tested the impact of perioperative bowel preparation on colonic anastomotic and surgical site wound healing across two different diets and assessed microbiota-associated physiologic impacts. We predicted that SBP would have the most significant impact in reducing diet and surgically induced dysbiosis resulting in a reduction in AL and enhanced wound healing. Conversely, we observed no differences in AL and wound healing rates resulting from exposure to bowel preparation in mice with and without diet-induced dysbiosis.
SBP is a widely implemented intervention, as high as 79% among colorectal surgeons in the United States, to prepare patients for gastrointestinal surgery.19 AL complicates up to 14% of all colorectal surgical operations,20 while SSI complicate up to 27% of all operations.20,21 Thus, identifying and refining tools to reduce these complications remain critical needs. While many clinical outcomes studies have been performed evaluating the efficacy of each component, most trials have not included OA which has added confusion regarding the optimal protocol. A Cochrane review of 18 randomized trials with 5,805 patients did not detect significant differences in rates of AL with the use of MBP following colorectal operations.22 Indeed, these trials and data served as the foundation upon which the enhanced recovery after surgery (ERAS) protocols recommended against the routine use of MBP.10 However, evaluating each individual component may not accurately reflect an increased efficacy when MBP and OA are used in combination. Accordingly, multiple studies have shown a reduction in AL with combined MBP and OA prior to undergoing colonic surgery.23–26 Even with our robust number of operations, neither AL nor superficial wound healing was impacted by any of the bowel preparation interventions, despite impacts on the microbial composition. As expected, mice receiving oral antibiotics had the greatest changes in microbial composition. Given the deleterious impact of WD on metabolism and healing,27,28 it was also expected that WD fed mice would exhibit poorer healing capacity. While this was observed by the three-fold higher rate of wound breakdown in WD fed mice relative to SD fed mice, we did not observe a difference in overall AL rates between the two diet cohorts. Differences among diet with respect to AL may have been mitigated due to the uniform implementation of DietGel post-operatively. This was done purposefully to evaluate the clinical impact of the preoperative diets and interventions. However, even with the same postoperative diet, there was still major separation in the microbiota composition driven by preoperative diet.
A tool identifying the presence of AL would be highly valuable in the clinical setting, thus our application of our previously developed index.16 The intestinal microbiota has been proposed as a primary vehicle through which genetics and environment interact to directly affect gut homeostasis and healing. Distinct subpopulations of the microbiota preferentially colonize sites of damaged mucosa in response to local environmental cues and drive epithelial wound healing.11 It is then logical to expect that a predictive tool may be created that reflects dysbiosis, which may predict anastomotic breakdown thus allowing for interventions early in the postoperative period. It is noteworthy that no differences were observed in the composition of mice that had AL due to diet, suggesting that AL may be driven by stochastic microbiota processes unrelated to overall community composition. Utilizing this tool in applying it to each intervention and diet, we observed no differences in the scores produced from each intervention further supporting the conclusion that any effect on clinical outcomes is unlikely to occur directly through changes produced in the microbiota.16 This is further supported by our human studies in which we have observed greater abundances of Enterococcus, Lactobacillus, and Streptococcus in AL.15 Enterococcus has been mechanistically linked to the development of AL via the production of matrix metalloproteinase 9, which degrades collagen and results in tissue breakdown.12 Enterococcus and Streptococcus are also potent bacteriocin and lactic acid producing bacteria.29,30
We measured fecal levels of SCFA to explore changes in microbial metabolites, critical in the regulation of colonic inflammation and health. Dysbiotic microbiota with depleted SCFA have been shown to dysregulate immune function, propagate intestinal and systemic inflammation, and impede wound healing.11 SCFA levels were not significantly different among the cohorts, thus suggesting that they may not mediate clinical benefits related to bowel preparation. In our human investigations, we have observed significant reductions in butyrate shortly following surgery, in line with our observations that SCFA are unlikely to mediate any benefit of bowel preparation in surgery.15 Interestingly, we did observe a difference in fecal levels of IgA. As expected, mice fed WD had significantly increased levels of fecal IgA. This is consistent with others who have demonstrated increased IgA driven by the presence of increased pro-inflammatory microbiota.31–33 We and others have previously shown that fecal IgA levels inversely correlate with alpha diversity.15,34 Given the inflammation driven by a robust process such as AL, we expected fecal IgA to discriminate between the presence or absence of AL regardless of baseline diet. While this was true among WD fed mice, this was not so for SD fed mice. While diet had a strong role in influencing baseline levels of IgA, it is likely that the immune response elicited by surgery and the development of AL are also markedly different within the background of the different diets. This difference may suggest that each additional inciting event promotes a more robust inflammatory response, as demonstrated by markedly increased levels in WD fed mice with AL. Future studies are required to delineate whether IgA may serve as a clinical marker for the detection of AL.
To evaluate direct host effects of bowel preparation, RNAseq was performed on the colonic tissue adjacent to the anastomosis due to the potential confounding effects of significant inflammation should there be the presence of AL. Interestingly, we observed significant differences in leak versus no leak cohorts in both the SD, SBP and WD water cohorts, and no differences among the SD water and WD SBP cohorts, regardless of leak status. These observations may suggest that mice fed SD have a more resilient microbial community that was only disturbed enough to impact host immunity in the SBP group. In contrast, WD-fed mice may have much less resilient communities such that any intervention perturbed or improved the community to reduce immunogenicity. Altogether, no clear mechanistic pattern recognition was seen by SBP that may account for enhanced surgical recovery and healing.
Murine models have shown that the injured mucosa is markedly different from intact mucosa and is characterized by a microenvironment consisting of cellular migration, proliferation, and tissue remodeling.13 It has also been shown that mucous-adherent microbiota at the anastomosis may have a different composition when compared to expelled stool.35 To evaluate this in our model, we compared the microbiome composition of expelled stool to that at the anastomosis, and furthermore in the serum to evaluate for bacterial translocation. We identified that expelled stool was highly similar in composition to that at the anastomosis, and an accurate reflection of its composition. Interestingly, no serum samples from mice on regular chow showed detectable levels of microbiota, whereas four samples from WD fed mice achieved sufficient sequence reads. Although compositionally different from stool, this is consistent with bacterial translocation that is known to occur in the setting of WD-induced gut inflammation.36,37
There are important limitations to this study. This is a murine preclinical model which attempts to mimic the clinical situation. While we attempted to maintain translational accuracy, further studies are required in patients to verify our observations. Intravenous antibiotics are routinely given at the time of surgery to reduce wound infections. We purposefully did not administer perioperative antibiotics as this would have significantly impacted the microbiota and confounded the outcomes of this study.38 While our study involved a robust number of murine operations (n = 144), we may have been underpowered to identify significant differences in AL resulting from bowel preparation. Power calculations based on our observed leak rates suggest that ~ 200 mice/arm would be necessary to allow for detection of significant differences in leak rates. We attempted in-depth investigations to overcome this potential, without clear underlying mechanisms to potentially explain a clinical benefit induced by bowel preparation. All mice were placed on DietGel following surgery, which we expect to potentially have some influence on the microbiome.