Mucosal Cancer-Associated Microbes and Anastomotic Leakage after Resection of Colorectal Carcinoma

Abstract

Background:

Clinical and experimental evidence suggests that colorectal mucosal microbiota changes during colorectal carcinogenesis and may impair colorectal anastomotic wound healing. Thus, we hypothesized that amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with anastomotic leakage after resection for colorectal carcinoma.

Methods:

We analyzed 256 fresh frozen tissues of colorectal cancer from patients who underwent elective colorectal resection and anastomosis. Amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, in colorectal cancer tissues were measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable logistic regression analysis was conducted to assess associations of these microbes with anastomotic leakage, adjusting for patient and tumor characteristics, and surgery-related factors.

Results:

Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; P_trend = 0.004). The association of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with anastomotic leakage was not statistically significant.

Conclusions:

The amount of Bifidobacterium genus in colorectal tissue is associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection.

Introduction

More than 100 trillion microorganisms inhabit the human gastrointestinal tract and play important roles in health conditions, immunity, and diseases including colorectal cancer [1–3]. Human colorectal mucosal microbiota has been shown to change during the development and progression of colorectal neoplasia [4–6]. Experimental evidence from animal models demonstrates that intestinal microbes, such as Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, can influence the development and progression of colorectal neoplasms by damaging DNA, producing metabolites, and modulating host immune response and intestinal inflammation [3,7]. Consistent with these lines of experimental evidence, human studies have shown associations of these microbes with antitumor immune response, intestinal inflammation, and colorectal neoplasms [8–13]. In 1313 colorectal cancers from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus has been associated with the extent of signet ring cells in colorectal cancer tissue [14].

Although surgery with complete resection represents a potentially curative treatment for colorectal carcinoma, anastomotic leakage after colorectal resection remains the major cause for morbidity and mortality [15,16]. Emerging evidence from animal models indicates that colorectal mucosal microbes in colorectal tissue may impair colorectal anastomotic wound healing after colorectal resection [17–23]. However, the relationship between the complex intestinal microbiota and wound healing of colorectal anastomoses in humans cannot be completely recapitulated in animal models, analysis using human tissue is required for clinical application. We hypothesized that higher amounts of these colorectal cancer-associated microbes in human colorectal tissue might be associated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. A better understanding of roles of the intestinal microbes in the incidence of anastomotic leakage after resection of colorectal cancer may provide new opportunities to utilize the intestinal microbes for postoperative recovery in patients with colorectal cancers.

To test our hypothesis, we examined associations of the amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus, in human colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma.

Methods

Subjects and specimens

We analyzed 256 fresh frozen colorectal cancer tissue specimens from patients who underwent elective colorectal resection and anastomosis for colorectal cancer at Kumamoto University Hospital in Japan. All patients had histologically confirmed colorectal carcinoma. The pathologic diagnoses and the clinicopathological factors were established based on the American Joint Committee on Cancer/International Union Against Cancer staging system. Written informed consent was obtained from each patient. This study was approved by the Human Ethics Review Committee of the Graduate School of Medicine, Kumamoto University (Kumamoto, Japan).

All patients received mechanical bowel preparation with 2L of polyethylene glycol 1 day before surgery. None of the patients received preoperative oral antibiotics. All of the patients received 1 g of cefmetazole intravenously after the induction of anesthesia and within 60 minutes before skin incision. Subsequently, 1 g of cefmetazole was additionally given every 3 hours during surgery. The antibiotic was discontinued within 24 hours after surgery.

Colorectal carcinoma tissue specimens and normal colorectal mucosa tissue specimens that were at least 5 to 10 cm from the edge of the tumor tissues were cut using a sterile blade and placed in a cryotube and frozen immediately in liquid nitrogen within 30 minutes after colorectal resection. The frozen tissue was stored at −80°C until DNA extraction.

Quantitative polymerase chain reaction (PCR) for intestinal microbes

Genomic DNA was extracted from colorectal carcinoma tissue specimens and normal colorectal mucosa tissue specimens, using QIAamp DNA Mini Kit (Qiagen). We performed quantitative PCR assays to measure the amount of tissue DNA of Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus. Custom TaqMan primer/probe sets (Applied Biosystems) for the nusG gene of Fusobacterium nucleatum, the pks colibactin gene cluster of Escherichia coli, the 16S ribosomal RNA gene DNA sequences of Enterococcus faecalis and Bifidobacterium genus, and the reference human gene, SLCO2A1 were used as previously described [8,24–26]. Each reaction contained 12.5 ng of genomic DNA and was assayed in 10 μL reactions containing 1× final concentration LightCycler 480 Probe Master (Roche) and each TaqMan Gene Expression Assay (Applied Biosystems), in a 384-well optical PCR plate. Amplification and detection of DNA was performed with a LightCycler 480 Instrument II (Roche) using the following reaction conditions: 10 min at 95°C and 45 cycles of 15 sec at 95°C and 1 min at 60°C.

The primer and probe sequences for each TaqMan Gene Expression Assay were as follows: Fusobacterium nucleatum forward primer, 5’-TGGTGTCATTCTTCCAAAAATATCA-3’; Fusobacterium nucleatum reverse primer, 5’-AGATCAAGAAGGACAAGTTGCTGAA-3’; Fusobacterium nucleatum FAM probe, 5’-ACTTTAACTCTACCATGTTCA -3’; pks-positive Escherichia coli forward primer, 5’-TGCTATGTATTCACGCAAACG-3’; pks-positive Escherichia coli reverse primer, 5’-CGTTGCTCTCCATGACCTG-3’; pks-positive Escherichia coli probe, 5’-CTGGCTGG-3’; Enterococcus faecalis forward primer, 5’-AAACCTTTCCCTGGTGTTCA-3’; Enterococcus faecalis reverse primer, 5’-CGTTAATCCCTGTTGAAGCAA-3’; Enterococcus faecalis probe, 5’-TTCTGGCT-3’; Bifidobacterium genus forward primer, 5’-CGGGTGAGTAATGCGTGACC-3’; Bifidobacterium genus reverse primer, 5’-TGATAGGACGCGACCCCA-3’; Bifidobacterium genus FAM probe, 5’-CTCCTGGAAACGGGTG-3’; SLCO2A1 forward primer, 5’-ATCCCCAAAGCACCTGGTTT-3’; SLCO2A1 reverse primer, 5’-AGAGGCCAAGATAGTCCTGGTAA-3’; SLCO2A1 VIC probe, 5’-CCATCCATGTCCTCATCTC-3’.

Each specimen was analyzed in duplicate for each target in a single batch, and we used the average of the two cycle threshold (Ct) values for each target. The amount of Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2^−ΔCt method (where ΔCt = “the average Ct value of intestinal microbes” - “the average Ct value of SLCO2A1”) as previously described [8,27].

Definition of anastomotic leakage

The definition of anastomotic leakage was used as previously reported clinical trials [28,29]; peritonitis from any staple line, and pelvic abscess without radiologically proven leakage mechanism were included. Leakage was verified by clinical (inspection of drain contents), endoscopic (flexible sigmoidoscopy), or radiologic (rectal contrast study, computed tomography scan) interventions.

Statistical analysis

All statistical analyses were conducted using JMP (version 12.2, SAS Institute, Cary, NC) and all P values were two-sided. Neither the amounts of intestinal microbes, including Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus, nor the log-transformed value of the amounts of these intestinal microbes fit a normal distribution with the use of the Shapiro-Wilk test for normality (P < 0.0001). Thus, our primary hypothesis testing was the linear trend test in a logistic regression model to assess associations of the amounts of these intestinal microbes in colorectal carcinoma tissue (an ordinal predictor variable) with the incidence of anastomotic leakage after resection of colorectal carcinoma (a dichotomous outcome variable), as previously described [8,14,30]. Cases with detectable these microbes were categorized as low or high based on the median cutpoint amount of the microbes, while cases without detectable the microbes were categorized as “negative”. The linear trend test was performed using the ordinal predictor variable of the microbes (negative, low, and high) as a continuous variable in a logistic regression model. All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives [31].

We performed multivariable logistic regression analysis to adjust for potential confounders. The multivariable model initially included age (≤ 64 vs. 65–74 vs. ≥ 75), sex, body mass index (< 30 kg/m² vs. ≥ 30 kg/m²), diabetes (present vs. absent), serum albumin levels (< 3 g/dl vs. ≥ 3 g/dl), tumor location (colon vs. rectum), pT stage (pT1–3 vs. pT4), lymph node metastasis (negative vs. positive), distant metastasis (negative vs. positive), surgical approach (open or laparoscopy converted vs. laparoscopy), operating time (< 240 minutes vs. ≥ 240 minutes), intraoperative bleeding (< 300 mL vs. ≥ 300 mL), and diverting ileostomy (present vs. absent). A backward stepwise elimination with a threshold of P < 0.05 was used to select variables in the final models.

To assess associations between the dichotomous category (absence and present) of the incidence of anastomotic leak and categorical data, the chi-square test was performed.

Results

Patient and tumor characteristics, and surgery-related factors in relation to anastomotic leakage after resection of colorectal carcinoma

Anastomotic leakage was diagnosed in 29 (11%) of 256 cases. Patient and tumor characteristics, and surgery-related factors are summarized according to the incidence of anastomotic leakage in Table 1. Male sex and rectal tumor location were associated with anastomotic leakage (P < 0.001; with α level of 0.005).

Table 1.

Patient and tumor characteristics, and surgery-related factors according to the incidence of anastomotic leakage after resection of colorectal carcinoma

Characteristica	All patients (n = 256)	Patients without anastomotic leak (n = 227)	Patients with anastomotic leak (n = 29)	P valueb
Age (year)				0.37
≤ 64	90 (35%)	83 (36%)	7 (24%)
65–74	84 (33%)	72 (32%)	12 (41%)
≥ 75	82 (32%)	72 (32%)	10 (35%)
Sex				< 0.001
Men	152 (59%)	127 (56%)	25 (86%)
Women	104 (41%)	100 (44%)	4 (14%)
Body mass index (kg/m2)				0.14
< 30	247 (96%)	218 (96%)	29 (100%)
≥ 30	9 (3.5%)	9 (4%)	0
Diabetes				0.99
No	212 (83%)	188 (83%)	24 (83%)
Yes	44 (17%)	39 (17%)	5 (17%)
Serum albumin levels (g/dl)				0.67
< 3	21 (8.2%)	18 (7.9%)	3 (10%)
≥ 3	235 (92%)	209 (92%)	26 (90%)
Tumor location				< 0.001
Caecum, ascending colon, transverse colon	83 (32%)	78 (34%)	5 (17%)
Descending colon, sigmoid colon, rectosigmoid junction	128 (50%)	117 (52%)	11 (38%)
Rectum	45 (18%)	32 (14%)	13 (45%)
pT stage (depth of tumor invasion)				0.49
pT1–3	209 (82%)	184 (81%)	25 (86%)
pT4	47 (18%)	43 (19%)	4 (14%)
Lymph node metastasis				0.72
Negative	160 (62%)	141 (62%)	19 (66%)
Positive	96 (38%)	86 (38%)	10 (34%)
Distant metastasis				0.23
Negative	210 (82%)	184 (81%)	26 (90%)
Positive	46 (18%)	43 (19%)	3 (10%)
Surgical approach				0.06
Open or laparoscopy converted	67 (26%)	55 (24%)	12 (41%)
Laparoscopy	189 (74%)	172 (76%)	17 (59%)
Operating time (minutes)				0.010
< 240	62 (24%)	60 (26%)	2 (6.9%)
≥ 240	194 (76%)	167 (74%)	27 (93%)
Intraoperative bleeding (mL)				0.12
< 300	198 (77%)	179 (79%)	19 (66%)
≥ 300	58 (23%)	48 (21%)	10 (34%)
Diverting ileostomy				0.32
No	234 (91%)	209 (92%)	25 (86%)
Yes	22 (8.6%)	18 (7.9%)	4 (14%)

^aPercentage (%) indicates the proportion of cases with patient and tumor characteristics or surgery-related factors according to the incidence of anastomotic leakage.

^bTo assess associations between the dichotomous category of the incidence of anastomotic leakage and categorical variables, the chi-square test was performed. All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.

Associations of the amounts of colorectal cancer-associated microbes in tumor tissue with anastomotic leakage after resection of colorectal carcinoma

We measured the relative amounts of Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus in fresh frozen tumor tissue of 256 colorectal carcinoma cases, using the quantitative PCR assay. Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. We categorized colorectal carcinoma cases with detectable these four microbes as low or high based on the median cutpoint amount of the microbes.

Table 2 shows the distribution of colorectal carcinoma cases according to the amounts of the microbes and the incidence of anastomotic leakage. In our primary hypothesis testing, we conducted univariable and multivariable logistic regression analyses to assess associations of the amounts of the microbes in colorectal carcinoma tissue (as ordinal predictor variables) with the incidence of anastomotic leakage (as a dichotomous outcome variable) (Table 3). The amount of Bifidobacterium genus in colorectal carcinoma tissue was associated with an increased risk of anastomotic leakage in univariable (P_trend = 0.002) and multivariable logistic regression analysis (P_trend = 0.004). Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Table 3 and Supplementary Table S1). The association of the amount of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant (P_trend > 0.039; with α level of 0.005; Table 3).

Table 2.

Distribution of cases according to the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue and the incidence of anastomotic leakage after resection of colorectal carcinoma

The amount of microbes in colorectal carcinoma tissue	All patients (n = 256)	Patients without anastomotic leak (n = 227)	Patients with anastomotic leak (n = 29)	Ptrenda
Fusobacterium nucleatum				0.45
Negative	116 (46%)	104 (46%)	12 (41%)
Low	70 (27%)	63 (28%)	7 (24%)
High	70 (27%)	60 (26%)	10 (35%)
pks-positive Escherichia coli				0.13
Negative	162 (64%)	148 (65%)	14 (48%)
Low	47 (18%)	39 (17%)	8 (28%)
High	47 (18%)	40 (18%)	7 (24%)
Enterococcus faecalis				0.99
Negative	63 (25%)	55 (24%)	8 (28%)
Low	95 (37%)	86 (38%)	9 (31%)
High	98 (38%)	86 (38%)	12 (41%)
Bifidobacterium genus				0.002
Negative	167 (65%)	155 (68%)	12 (41%)
Low	44 (17%)	38 (17%)	6 (21%)
High	45 (18%)	34 (15%)	11 (38%)

^aP_trend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in univariable logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.

Table 3.

The association of the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma

The amount of microbes in colorectal carcinoma tissue		Univariable OR (95% CI)	Multivariable OR (95% CI)a
Model for anastomotic leakage after resection of colorectal carcinoma (n = 256, as an outcome variable)
Fusobacterium nucleatum	Negative	1 (reference)	1 (reference)
	Low	0.96 (0.34–2.52)	0.83 (0.27–2.34)
	High	1.44 (0.58–3.55)	1.23 (0.44–3.29)
	Ptrendb	0.45	0.73
pks-positive Escherichia coli	Negative	1 (reference)	1 (reference)
	Low	2.24 (0.77–6.15)	2.17 (0.66–6.76)
	High	1.92 (0.62–5.44)	2.71 (0.81–8.73)
	Ptrendb	0.13	0.039
Enterococcus faecalis	Negative	1 (reference)	1 (reference)
	Low	0.72 (0.26–2.02)	0.89 (0.30–2.74)
	High	0.96 (0.37–2.59)	0.88 (0.32–2.57)
	Ptrendb	0.99	0.83
Bifidobacterium genus	Negative	1 (reference)	1 (reference)
	Low	2.04 (0.67–5.62)	2.33 (0.72–6.95)
	High	4.18 (1.68–10.34)	3.96 (1.50–10.51)
	Ptrendb	0.002	0.004

Abbreviations: CI, confidence interval; OR, odds ratio.

^aThe multivariable logistic regression analysis model initially included age, sex, body mass index, diabetes, serum albumin levels, tumor location, pT stage, lymph node metastasis, distant metastasis, surgical approach, operating time, intraoperative bleeding, and diverting ileostomy. A backward stepwise elimination with a threshold of P < 0.05 was used to select variables in the final models.

^bP_trend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.

Associations of the amount of Bifidobacterium genus in colorectal carcinoma tissue with the patient and tumor characteristics, including tumor location, were not statistically significant (with α level of 0.005; Supplementary Table S2).

In our exploratory analysis, we measured the relative amount of Bifidobacterium genus in normal colorectal mucosa tissues that were at least 5 to 10 cm distant from the edge of the tumor in 209 colorectal carcinoma cases, using the quantitative PCR assay. The amount of Bifidobacterium genus in normal colorectal mucosa tissue appeared to be associated with an increased risk of anastomotic leakage in univariable (P_trend = 0.005) and multivariable logistic regression analysis (P_trend = 0.040; Supplementary Table S3).

Discussion

We conducted this study to test the hypothesis that higher amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. We found that higher amounts of colorectal cancer-associated microbes, such as Bifidobacterium genus, in human colorectal carcinoma tissue were associated with an increased risk of anastomotic leakage.

Although clinical studies have demonstrated associations of anastomotic leakage with patient and tumor characteristics (male sex, obesity, diabetes, and rectal tumor location), and surgery-related factors (operative time, intraoperative bleeding, and diverting ileostomy) [28,29], exact mechanisms of colorectal anastomotic leakage remain uncertain. Our human data suggest a possible link between colorectal mucosal microbes and anastomotic leakage, generating some mechanistic hypotheses for further investigation.

Experimental evidence from animal models demonstrates that Bifidobacterium genus in the gut appear to inhibit colorectal carcinogenesis through prevention of enteropathogenic infection or acidification, which can reduce secondary bile acid production [32]. However, in 1313 human colorectal cancer tissue specimens from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus is correlated with the extent of signet ring cells, which has been associated with worse patient survival [14,33]. Experimental evidence demonstrates that prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2) is essential for neovascularization of the colonic anastomosis and thereby promote colorectal anastomotic wound healing [34]. Bifidobacterium genus has been shown to decrease PTGS2 expression level in colonic cells [35]. Studies have shown the enrichment of Bifidobacterium genus in the hypoxic tumor microenvironment [36–38]. Although exact mechanisms underlying the association of Bifidobacterium genus with colorectal anastomotic leakage need to be elucidated by further investigations, Bifidobacterium genus might impair anastomotic wound healing through poor vascularization and tissue hypoxia in colorectal anastomotic tissues. It is also possible that Bifidobacterium genus may contribute to colorectal anastomotic leakage through microbe-microbe interactions as a member of human microbial ecosystems [39].

An experimental study of a rat model has shown that Enterococcus faecalis may impair colorectal anastomotic would healing by producing collagenases and activating host metalloproteinase MMP9 in anastomotic tissues [18]. In the current study, the association of the amount of Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant. Our findings need to be validated by further studies.

We recognize limitations of this study. First, we did not examine the microbiota and its metabolites in colorectal tissue, data on stool microbiota, or microbe-microbe interactions. Future comprehensive metagenomic analyses on colorectal tissue and stool may provide further insights on roles of intestinal microbiota in the development of colorectal anastomotic leakage. Nonetheless, given complex roles of interactions between microbial and host factors in human colorectal anastomotic wound healing, we believe that our human data on colorectal cancer-associated microbes in relation to the incidence of anastomotic leakage after colorectal resection represent valuable information. Second, we did not examine potential influences of mechanical bowel preparation with or without oral antibiotics before surgery on the amount of the colorectal cancer-associated microbes in colorectal tissue because all patients routinely received mechanical bowel preparation without oral antibiotics before elective resection of colorectal cancer in the current study. Mechanical bowel preparation has been shown to influence both the luminal and mucosal microbiota in the human colon [40,41]. Clinical studies have shown that preoperative mechanical bowel preparation may decrease the amount of Bifidobacterium genus in the human colon, and significantly reduce anastomotic leakage after colorectal resection [42–46]. Future investigations are needed to examine potential influences of mechanical bowel preparation with or without oral antibiotics before surgery on the intestinal and mucosal microflora in patients with colorectal cancer, and examine associations of the colorectal cancer-associated microbes in colorectal tissue with the incidence of anastomotic leakage after resection of colorectal cancer in patients who do not receive bowel preparation. Third, data on colorectal mucosal microbes from patients with benign colorectal disease were not available in the current study. Because colorectal mucosal microbiota in patients with colorectal cancer has been shown to differ significantly from that in patients with benign colorectal disease [4–6], future investigations are needed to examine associations of colorectal mucosal microbes with the incidence of anastomotic leakage after colorectal resection in patients with benign colorectal disease.

A major strength of this study includes the use of our database which integrates patient and tumor characteristics, surgery-related factors, and the amounts of colorectal carcinoma-associated microbes in colorectal cancer and normal colorectal tissues. The comprehensiveness of this colorectal cancer database enabled us to assess the association between the amounts of colorectal mucosal microbes and anastomotic leakage, controlling for potential confounders.

Conclusions

The amounts of colorectal cancer-associated microbes, such as Bifidobacterium genus, in colorectal tissue are associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated by further studies to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection.

Highlights.

• Higher amounts of colorectal cancer-associated microbes, such as Bifidobacterium genus, in colorectal mucosal tissue were associated with an increased risk of anastomotic leakage after resection for colorectal cancer.

• Upon validation, colorectal mucosal microbes may serve as potential targets for the prevention of anastomotic leakage after colorectal resection.

Abbreviations:

CI

confidence interval

Ct

cycle threshold

OR

odds ratio

PCR

polymerase chain reaction