Effect of Chemotherapy on Fusobacterium nucleatum Abundance in Colorectal Cancer Patients: A Study on Relapsing Patients

Abstract

An intricate relationship exists, and interactions occur between the gut microbiota and colorectal cancer (CRC). Recent studies have indicated that inflammatory reactions stimulated by Fusobacterium nucleatum (Fn) lead to the development of CRC. Radical surgery combined with adjuvant chemotherapy is the primary treatment approach for most CRC patients. This study was designed to evaluate the abundance of Fn as part of the gut microbiota in patients with CRC compared to healthy individuals and to assess the effect of the gut microbiota Fn on patients undergoing adjuvant chemotherapy and those experiencing CRC relapse. There were 201 participants, comprising 50 healthy controls and 151 CRC patients. Stool samples were collected from three CRC groups (postoperatively, chemotherapy and relapse), and the fourth was the healthy control group. The amount of Fn in each sample was analyzed using quantitative loop-mediated isothermal amplification-phenol red (QLAMP-PhR), a novel biomolecular method that targets regions encoding the specific Fn FadA gene. Compared with healthy control stool samples, the Fn levels were significantly elevated in all CRC patient groups (P < 0.001), and it was significantly more frequent in the CRC relapse patients (group C) (P < 0.001). In addition, Fn abundance increased significantly in the distal colon compared to the proximal colon (P < 0.001). Both CRC relapse and chemotherapy exert significant reciprocal effects on the gut microbiota Fn of CRC patients. Microbiota-based intervention may be beneficial for patients during postoperative care, especially in CRC relapsing cases.

Registration: This study of the clinical trial has been registered in the ISRCTN registry with study registration number ISRCTN53358464. https://www.isrctn.com/ISRCTN53358464

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01279-6.

Introduction

Colorectal cancer (CRC) is the third most common type of cancer worldwide in both men and women [1]. For most CRC patients, the main treatment option is radical surgery plus adjuvant chemotherapy. Adjuvant chemotherapy is a standard treatment for patients with stage II and III CRC after surgery to remove the cancerous portion of the colon and surrounding lymph nodes [2]. However, the main obstacle to the treatment of CRC remains chemotherapeutic resistance, which leads to cancer recurrence and metastasis, ultimately leading to chemotherapy failure [3]. The relapse rate of patients with resectable CRC is more than 30% [4]. To overcome chemotherapeutic resistance, more effective strategies need to be developed to meet clinical needs [5, 6].

Fecal microbiota transplantation (FMT) involves restoring a healthy gut microbiota balance [7]. Dysbiosis, characterized by an imbalance in the gut microbial composition, has been associated with an increased risk of CRC [8]. Current studies show that the development and progression of CRC are strongly related to the intestinal microbiota, especially Fusobacterium nucleatum (Fn) [9, 10]. Radical surgeries, mechanical bowel preparation, and the prophylactic use of antibiotics lead to significant changes in the postoperative bowel environment [11]. In addition, the gut pathological state can lead to dysbiosis of the gut microbiota, leading to an increased population of harmful microorganisms and contributing to systemic adverse events [12, 13]. Numerous studies have confirmed that the gut microbiota changes before and after radical CRC resection, including an increase in aerobic bacteria and facultative anaerobic bacteria and a decrease in obligate anaerobes, butyric acid-producing bacteria, and tumor-related bacteria following CRC surgery [14, 15]. In addition, postoperative use of probiotics in CRC patients may reduce the incidence of infectious complications while improving intestinal microbiota and mucosal barrier function [16]. Probiotics have been investigated for their role in CRC prevention. Certain strains of probiotics, such as Lactobacillus and Bifidobacterium, have shown promising effects in modulating the gut microbiota and reducing inflammation, which may contribute to CRC risk reduction [17].

More than five main treatment types are currently used for advanced CRC: oxaliplatin, capecitabine, 5-fluorouracil (5FU), methotrexate, calcium folinate, irinotecan, gemcitabine, and immunotherapies (anti-PDL1 and anti-CLTA4) [18, 19]. Chemotherapy-induced gastrointestinal toxicity encompasses the full spectrum of adverse events associated with cancer treatment and is often referred to as mucositis. It affects up to 80% of patients, depending on the treatment type, and leads to more dramatic changes in the gut environment [20, 21]. The gut microbiota is closely linked to the pharmacological effects of chemical therapy and new targeted immunotherapy [22]. However, cancer chemical treatment can induce a variety of changes in the host microbiome and innate immune system, which may affect the outcomes and adverse events of chemotherapy [23, 24]. As an important member of the gut microbiota, Fn is a gram-negative anaerobic bacterium commonly found in the colonic tract of CRC patients. It can adhere to the colonic mucosa and attack epithelial cells, leading to inflammation and carcinogenesis [9, 25]. Clinical studies have shown that Fn also promotes the chemoresistance of CRC cells to chemotherapy drugs, which directly leads to the failure of chemotherapy in patients with advanced CRC. Therefore, it is important to inhibit the proliferation of tumor-induced Fn bacteria in the colon during CRC chemotherapy [26, 27]. In this study, a full set of stool samples was collected from CRC patients after radical surgery combined with chemotherapy and CRC relapse cases. These samples were then analyzed for the richness of Fn (copy/µl) using novel quantitative loop-mediated isothermal amplification (QLAMP) colorimetric biotechnology method tests. The aim of this study is not only to elucidate the effects of radical surgical interventions and anticancer drugs on the composition and function of Fn richness as a gut microbiota in CRC patients but also to demonstrate the ongoing short-term trend toward changes in gut microbial numbers after surgery and after repeated chemotherapy cycles. This provides invaluable theoretical evidence for gut microbiota improvement during postoperative care.

Materials and Methods

Bioethics Approved

This study was performed in accordance with the recommendations of the Declaration of Helsinki for biomedical research involving human subjects. This study was reviewed and approved by the University of Tishreen in Lattakia & Tishreen University Hospital Human Research Bioethics Board and Academic Science Research Committee. All volunteers provided written informed consent for their participation in this study and the use of their samples.

Participants and Samples

A total of 201 stool samples were obtained from 50 healthy control participants and 151 CRC patients (92 males (M), 59 females (F), with a mean age of 57.6 ± 7.8 years (Y) and a mean BMI of 23.94 kg/m²) referred to the Tishreen University chemotherapy center. The stool samples from CRC patients comprised 50 samples collected post-surgery and pre-first chemotherapy (Group A, 30 M, 20 F, 54.76 Y, 23.63 kg/m²), 51 samples from patients receiving chemotherapy after the first to fifth cycles (Group B, 31 M, 20 F, 59.29 Y, 23.82 kg/m²), and 50 samples from relapsed CRC patients (Group C, 31 M, 19 F, 58.86 Y, 24.38 kg/m²). Additionally, stool samples were gathered from 50 healthy controls (Group D, 31 M, 19 F, 57.4 Y, 24.0 kg/m²), matched to CRC patients by age, sex, and current living conditions.

Study Criteria

Strict inclusion and exclusion criteria were implemented to ensure the appropriate selection of CRC patients and healthy controls for a comparative analysis. CRC patients (Groups A, B, and C) had to be registered at Tishreen Hospital's chemotherapy center and diagnosed with CRC between November 2019 and November 2020, with confirmation via pathology reports. Patients could be at any stage or grade of CRC and might or might not have undergone chemotherapy or colon surgery. Healthy controls (Group D) were individuals without a history of CRC, colon polyps, chronic bowel diseases, or gastrointestinal issues for at least 3 weeks before the study. They also needed to share similar demographic characteristics with the patient group to minimize confounding variables. Exclusion criteria for CRC patients involved those with a history of other cancers, a known genetic predisposition to CRC, prior radiation therapy before sampling, or recent antibiotic usage. Healthy controls would be excluded if they had a personal cancer history, a family history of CRC among first-degree relatives, recent antibiotic intake, or recent use of antacid or PPIs medications. These rigorous criteria were established to ensure a uniform and dependable study population for the investigation of CRC and associated risk factors.

Sample Collection and Storage

This study was conducted on stool samples collected from the study participants to measure the quantity of Fn in the stool. Stool is considered to be a reflection of the microbial environment of the colon, representing the contents of the gastrointestinal tract [28]. It has been established that the microbiome associated with the colonic mucosa and stool are distinctly linked, indicating a true and accurate representation of the colonic microbiome through stool [29–31]. From each participant, the first bowel movement of the day was collected in the morning, and stool samples were collected on two occasions with a mean time interval of 48 h between the two time points. All stool samples were collected immediately after defecation in sterile cups and delivered to the laboratory of Tishreen University Hospital within 2 h. Stool samples were stored at -50 °C upon arrival at the microbiology laboratory. Due to the uneven distribution of microorganisms within stool [32, 33], to ensure an accurate representation of Fn content in stool and in accordance with the recommendations of a previous study (Gorzelak [34]), we homogenized and pooled the stool samples in the specimen before weighing the quantity taken for the study. Additionally, we calculated the mean value of two stool samples taken from the same participant with an interval of more than 48 h.

Tumor Site Classification

The CRC patients in the study were classified according to tumor site (ascending colon, transverse colon, descending colon, sigmoid colon, and rectum), and these proportions were graphically represented as shown in (Fig. 1A). A statistical analysis was conducted to correlate the quantitative results of Fn values in stool samples collected from CRC patients participating in the study based on tumor sites in all study groups.

Fig. 1.

A The percentage of participants in each CRC group at each tumor site is represented in the gut image. B QLAMP-PhR test using phenol red as an indicator; negative sample and control: phenol red color (violet—fuchsia), and positive sample and control: phenol red color change from violet-fuchsia to yellow-orange according to F. nucleatum DNA copies/sample

Sample Processing and Extraction

For the extraction of bacterial DNA, 10 g of each stool sample was measured (after thorough mixing and homogenization.) by a digital scale and prepared for the next step. Total microbial DNA was extracted from all stool specimens using a RIBO-prep nucleic acid extraction kit (AmpliSens® Federal Budget Institute of Science, Moscow, Russia) according to the manufacturer's protocol. DNA quality and concentrations were determined by a Nanodrop spectrophotometer (Thermo Scientific™ Multiskan™ GO Microplate spectrophotometer, Thermo Fisher Scientific Inc.) from the Atomic Energy Commission of Syria (AECS) in Damascus, Syria. Whole extracted DNAs were immediately stored at -50 °C.

The biomolecular method quantitative loop-mediated isothermal amplification-phenol red (QLAMP-PhR).

For comparison purposes, stool samples from CRC patients and healthy controls were examined for molecular evidence of Fn using QLAMP-PhR assays.

All QLAMP-PhR tests were performed in the microbiology and biomolecular laboratories of Tishreen University Hospital and in the laboratory departments of the Molecular Biology and Biotechnology Department of the Syrian Atomic Energy Commission (AECS).

LAMP Primer Design

The sequences of the four primers used in the current study were designed and handled specifically for this study. These primers target the Fn FadA gene based on GenBank accession number DQ012971.1. The primers were designed by Primer Explorer V5 software (https://primerexplorer.jp/e/). The first set (4 primers) was chosen and synthesized by the Atomic Energy Commission of Syria (AECS) in Damascus. The specific sequences of the primers are shown in Table 1.

Table 1.

LAMP Primers sets design by Primer Explorer V5 software, the first LAMP Primers set were chosen and synthesized by Atomic Energy Commission of Syria (AECS) in Damascus

ID:1	Dimer (minimum) dG = -1.68
Labela	5'pos	3'pos	Len	Tmb	5'dG	3'dG	GC ratec	Sequence (5′–3′)d
F3	36	53	18	57.48	-5.26	-5.58	0.50	T T C T G C T T C A G C A T T C G C
B3	213	233	21	56.32	-4.24	-4.15	0.38	A G T C T T T G A G C T C T T T G A G A T
FIP			44					T T G C T A A G T T T T G G T A T T C A G C A T C T T A G C A A A T G A T G C A G C A A G T
BIP			45					A A G C A A G A T T C A A T G A A G A A A G A G C T T T G T T C A T T T T G T G C T A G T G C

Quantitative colorimetric LAMP (QLAMP-PhR)

The bacterial numbers of the four groups were measured by QLAMP-PhR assays using Fn FadA gene-specific primers. All QLAMP-PhR tests were carried out in triplicate, and averaged numbers were used for calculation and analysis. Each reaction mixture had a total volume of 100 µl with final concentrations of 2 mM Tris/HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Tween 20, 0.8 M betaine, 8 mM MgSO₄, 1.4 mM each dNTP and 8 U Bst DNA polymerase (Jena Bioscience) and phenol red 15 µg/µl as a pH quantitative colorimetric indicator.

For each reaction, 40 p.mol FIP and BIP, 20 p.mol LB or LF (optional) and 5 p.mol F3 and B3 were used, and 1 µl DNA template (test tube), 1 µl DNA titrate dilutions (positive control) or 1 µl distilled water was used as a no-template control (negative control). The reactions were performed at 63 °C for 45 min and then cooled at room temperature for 5 min (Fig. 1B). The absorbance of each well was read at 560 nm with a microplate reader (Thermo Scientific™ Multiskan™ GO Microplate in the Atomic Energy Commission of Syria (AECS) in Damascus).

QLAMP-PhR specificity tests of DNA were performed, and all three QLAMP-PhR tests only amplified Fn, with no cross-reaction with any of the other organisms. All QLAMP-PhR sensitivity tests efficiently detected lower copies of Fn, and the LOD of the QLAMP-PhR test was 2 × 10^–3 ng/ul = 2 pg/ul = 823 copies/ul.

Bacterial Standard Strains

The bacterial standard strains used in this study were obtained from the American Type Culture Collection (ATCC) Fusobacterium nucleatum subsp. nucleatum [ATCC 25586].

Calibration Curve

Standard curves were constructed to determine the number of Fn in all groups present in each sample using tenfold serial dilutions of FadA genomic DNA from standard bacterial strains (Fusobacterium nucleatum subsp. nucleatum [ATCC 25586] was used as a reference strain) at known concentrations from pure cultures, corresponding to 10¹ to 10¹⁰ copies per gram of stool. Standard curves were created according to spectrophotometer absorption and normalized to the copy number of the FadA gene for each sample. The bacterial concentration from each stool sample was calculated and obtained from the standard curves and expressed as the quantity of bacteria per gram of stool.

Statistical Analysis

The statistical analysis was performed using SPSS V23 and is represented in the statistical Excel file. Independent sample t tests were used to compare the means of different variables between study groups. The linear correlation between the variables was estimated using the Pearson correlation method. A P < 0.05 value was considered statistically significant. All data are expressed as the mean ± standard deviation.

Fusobacterium nucleatum QLAMP-PhR Reaction

All stool samples were tested by the Quantitative Loop-Mediated Isothermal Amplification QLAMP colorimetric-phenol red method published at 10.17504/protocols.io.3byl4qjkovo5/v1. A numerical value of the Fn (DNA copy number) in each sample was then obtained and is listed in the statistical Excel file (sheets 1 and 2).

A digital value of the Fn amount in each sample was obtained by a linear standard QLAMP-PhR analysis curve. At 560 nm, we observed a linear relationship between Fn concentration (ng/µl) and absorption (A) by the following function:

$$\text{X}={\text{Log}}_{10}\left[\left(Fn\text{ng}/\text{ul}\right)/2\right]\to Fn\text{ng}/\text{ul}=2\times 10^{\text{X}},\text{1 ng}/\text{ul}=411,523Fn\text{copies}/\text{ul}$$

Results

The rate of CRC is increasing, and complications are associated with it, including the role of the microbiome, mainly Fusobacterium spp., which play a complex role in relation to chemoresistance [35].

In this study, QLAMP-PhR tests were conducted to compare the Fn abundance in stool samples from all CRC patients (groups A, B, and C). The results revealed that group C (recurrent CRC patients) exhibited the most enriched Fn among the three groups of CRC patients (Fig. 2A). Furthermore, when compared to healthy control participants (group D), CRC recurrence patients had the most enriched Fn. This finding suggests that Fn may have a role in recurrent CRC. It is important to note that recurrent CRC is attributed to chemoresistance. Additionally, Fn is the most overwhelming phylotype in CRC cases [36]. Therefore, Fn is intricately linked to the development of recurrent CRC. Moreover, the presence of abundant Fn may significantly exacerbate CRC chemoresistance.

Fig. 2.

Comparing study groups by Fn mean amounts (log₁₀ of DNA copy/µl) in several ways: A in each tumor site. B according to sex (Male vs. Female) C in transverse colon. D in ascending colon vs. descending colon. E in ascending colon vs. sigmoid colon. F in ascending colon vs. Rectum. G in proximal colon vs. distal colon body mass index (BMI) All the data in Fig. 2 is extracted from the statistical Excel in supplementary material

The results of the study, as shown in the statistical Excel file, demonstrated that the CRC groups (A, B, and C) exhibited higher concentrations of Fn in their stool samples compared to the healthy control (group D). Specifically, when comparing the CRC cohorts as a whole to the healthy controls, the CRC groups showed a significantly greater abundance of Fn (P = 4.2E-24) (Fig. 2A). Furthermore, when comparing each CRC group individually to the healthy controls, all three CRC groups (A, B, and C) displayed a significantly higher abundance of Fn (P = 1.7E-10 for groups A & D, P = 6.2E-09 for groups B & D, P = 1.5E-14 for groups C & D). Additionally, within the CRC groups, Group C was found to have a significantly greater abundance of Fn compared to groups A and B. However, no significant difference in Fn abundance was observed between groups A and B (Fig. 2A). Furthermore, the results indicated that there was a significant difference in Fn abundance between males and females in both the overall study participants and within each study group (P = 0.022, P < 0.05) (Fig. 2B).

In the course of treating patients with CRC, two groups were obtained. Group A underwent surgical resection without prior chemotherapy, whereas Group B underwent surgical resection and had received 4–8 cycles of chemotherapy at intervals of 7–21 days between each cycle. Results indicated that both groups had a similar presence of the Fn microbiome. This is because a portion of the Fn microbiome was removed during tumor surgery, and the remaining Fn microbiome present in the surroundings was reflected in the results obtained from stool samples, representing the entire gut environment. Nevertheless, the chemotherapy monitored in the second group did not impact the microbial availability. The minimal bactericidal effect of chemotherapy drugs affected only a small percentage, which did not influence its statistical significance. However, this should be considered if the progression of microbiome abundance (Fn) is monitored after each treatment cycle, as it is anticipated to decrease with each chemotherapy cycle. The impact of microbiome Fn bacteria seems notably pronounced in the recurrence group, unlike in the post-surgery and chemotherapy groups, due to the chronic impact of Fn bacteria on chemotherapy resistance and the activation of cellular pathways stimulating tumor and angiogenesis. While there is no specific statistical connection between the post-surgery group and the chemotherapy group, it suggests hints of a direct therapeutic effect on the microbiome. Further insights can be gained by tracking the Fn count in each round from the start of chemotherapy-CRC treatment and monitoring their recurrence in the future. The study's findings hold importance for researchers in the field of CRC treatment, particularly in examining the immediate impact of chemotherapy on the Fn bacterial microbiome in each chemotherapy cycle.

Results of Participants by Tumor Sites

In this case–control study, QLAMP-PhR analysis was conducted to evaluate the differences in the composition of the stool microbiota Fn in patients with CRC and healthy control individuals for all tumor sites (ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). After removing the outliers, intragroup analysis indicated meaningful differences in the abundance of Fn between participants in the CRC groups (statistical Excel file, sheet 3).

The study revealed several significant findings regarding the abundance of Fn in participants' stool samples and its relationship with the different tumor sites in CRC participants. Firstly, in the transverse colon patients, the levels of Fn were significantly higher in all CRC groups (A, B, and C) compared to the healthy control group (Group D) (P = 1.15E-5, P < 0.05) (Fig. 2C). Similarly, in the descending colon patients, Fn levels were significantly higher in all CRC groups compared to both the ascending colon patients (P = 6.5E-5, P < 0.05) and the healthy control groups (P = 2.9E-15, P < 0.05) (Fig. 2D). The same pattern was observed in the sigmoid colon patients, where Fn levels were significantly higher in all CRC groups compared to the ascending colon patients (P = 0.01, P < 0.05) and the healthy control group (P = 2.1E-10, P < 0.05) (Fig. 2E). Additionally, in the patients with CRC in the rectum, Fn levels were significantly higher in all CRC groups compared to both the ascending colon patients (P = 0.033, P < 0.05) and the healthy control groups (P = 1.2E-07, P < 0.05) (Fig. 2F). Furthermore, when comparing the proximal and distal colon sites, Fn levels were significantly higher in the patients with distal colon involvement in all CRC groups compared to the proximal colon patients (P = 0.013, P < 0.05) (Fig. 2G). However, when considering the BMI index values, there was no significant difference in Fn abundance among all study groups (P = 0.53, P > 0.05) according to the statistical analysis (Fig. 2H).

Despite previous knowledge indicating that microbes tend to co-localize on CRC tumors, our research revealed the presence of Fn bacteria throughout the entire colon, even after radical surgery. We conducted a quantitative analysis of Fn levels in 101 stool samples from CRC patients (Groups A and B), as well as 50 stool samples from CRC patients with recurrence (Group C). Consistent with previous reports [36, 37], we observed higher levels of Fn in recurrent patients with distal colon involvement compared to non-recurrent patients (Fig. 2A). Additionally, there was a notable enrichment of Fn in distal colon CRC patients compared to those with proximal colon involvement in both the recurrence and non-recurrence groups (Fig. 2G). This elevated Fn abundance potentially contributes to the promotion of chemoresistance in patients with distal colon involvement in CRC. Furthermore, pairwise comparisons of tumor sites (the transverse colon, descending colon, sigmoid colon, and rectum) revealed that recurrent CRC patients exhibited a higher abundance of Fn compared to non-recurrent patients in these tumor sites. In contrast to the proximal ascending colon, where Fn concentrations were higher in the non- recurrent patient groups compared to the recurrence group, it can be concluded that relapse predominantly affects the distal colon and, to a lesser extent, the proximal colon, which provides an explanation for the obtained results (Table 2).

Table 2.

Relationships between study groups based on Fn amount (Log₁₀ of copy/ul)

Relationships between groups (mean ± SD)		P value		Significantly different
Group A (2.65 ± 1.96)	Group B	0.199547809	(P > 0.05)	There is NOT a significant difference
	Group C	0.030336	(P < 0.05)	There is a significant difference
	Group D	1.7 × 10–10	(P < 0.05)	There is a significant difference
Group B (2.18 ± 1.74)	Group C	5.84 × 10–4	(P < 0.05)	There is a significant difference
	Group D	6.23 × 10–9	(P < 0.05)	There is a significant difference
Group C (3.54 ± 2.1)	Group D	1.45 × 10–14	(P < 0.05)	There is a significant difference
Group D (0.37 ± 0.88)	Group A,B and C (2.79 ± 2.01)	4.18 × 10–24	(P < 0.05)	There is a significant difference

The values are expressed as mean ± SD and were analyzed using the t-Test. Significance was considered at P < 0.05

Anaerosporobacter is rarely associated with human disease [38]. However, considering the anaerobic nature of Fn and its saccharolytic metabolic pathway, it is not surprising that our study found this bacterium predominantly in the distal colon, which provides a low oxygen (O₂) environment and a high protein fermentation environment.

The partial pressure of oxygen (pO₂) significantly decreases along the colon, starting from the proximal colon, moving to the middle colon, and reaching its lowest levels in the distal colon, where billions of anaerobic bacteria and pathogens reside. Moreover, the large intestine exhibits the highest concentrations of ammonia due to intense protein fermentation, resulting in increased levels of non-ionized ammonia that progressively rise along the colon, accompanied by a higher pH. This can be attributed to the interaction between protein fermentation and starch fermentation in the colon, which has implications for colonic oncogenesis and potentially promotes oncogenesis through the production of toxic byproducts. Additionally, the impact of elevated levels of short-chain fatty acids (SCFAs), such as butyrate, resulting from fermentation is more pronounced in the caecum and distal colon, where a high abundance of Fn is found [39]. Some studies have indicated that the high abundance of Fn can serve as a predictor of CRC aggressiveness, independent of other factors.

Types of Chemotherapy in Group B Patients (Table 3)

Table 3.

Fn mean amounts (Log₁₀ of copy/ul) by each type of chemotherapy intake in Group B patients

Chemotherapy drug	FOLFOXa	CAPOXb XELOXb	FOLFIRIc	XELIRId	FOLFOXIRIeFOLFIRINOXe
Number of CRC patients	12	11	14	8	6
Fn mean amounts by Log10 of copy/ul	4.5	4.3	4.3	4.4	4.4

^aCombination of the drugs folinic acid (Leucovorin), 5fluorouracil (5FU), and oxaliplatin (Eloxatin)

^bCombination of chemotherapy drugs: capecitabine (brand name Xeloda) and oxaliplatin (Eloxatin)

^cCombination of folinic acid, 5fluorouracil (5FU) and irinotecan

^dCombination of capecitabine (Xeloda) pills and irinotecan

^eCombination of folinic acid, 5fluouracil (5FU), irinotecan and oxaliplatin

Through comparing the results of Fn bacteria concentrations in stool samples from CRC patients in the group of patients undergoing chemotherapy, we observed a significant convergence in Fn bacteria concentrations among all patients, regardless of the type of chemotherapy treatment. This indicates that the variation in chemotherapy treatment type did not affect the presence of Fn bacteria. This can be explained by the slow effect of chemotherapy on Fn bacteria, as they reside within the biofilm environment, which reduces the accessibility of chemotherapy drugs to them. Additionally, this can be attributed to the variation among these patients in terms of timing of stool sampling for the study, according to the cycles of chemotherapy treatment, as well as the small number of patients in each type of treatment group (6–12 patients in each treatment type) who participated in the study.

Discussion

This study aimed to reduce potential confounding factors by matching CRC and control group participants based on factors such as age, sex, and lifestyle. The technical design of the study, which included the use of FadA-specific QLAMP primers, played a significant role in the study results. The study suggested that the richness of intestinal microbiota (Fn) in CRC patients differs from that in healthy individuals. This hypothesis was confirmed when comparing different groups of CRC patients (postoperative, chemotherapy, relapse) with their healthy counterparts (Fig. 2A). Previous studies have shown that Fn is more prevalent in gut tumor sites of CRC patients than in healthy tissue and that it varies before and after surgery, during chemotherapy, and post-chemotherapy [9, 25, 40–42].

In this study, the level of Fn in the three CRC groups was notably higher than in the healthy group, consistent with previous research by Tunsjø et al. [43], Wang et al. [44], and Flanagan et al. [45]. This higher abundance of Fn in CRC participant groups was also detected in tumor tissues of CRC patients. Interestingly, there was no significant difference in Fn concentration between the postoperative and chemotherapy groups in our study. This suggests that Fn levels may be linked to gut microbiota balance rather than the effects of an incomplete chemotherapy effect (during chemotherapy rounds) or changes in diversity. The relapse group showed relatively higher Fn concentrations compared to the other groups. Our findings align with Yu et al. [46], who found a correlation between high Fn levels and chemo-resistance in advanced CRC patients. Additionally, 5 out of 8 studies by Lauka et al. [47] indicated that higher Fn adherence to tumor tissues is associated with poor oncological outcomes.

The highest Fn levels were observed in the relapse group, followed by the postoperative group and then the chemotherapy group, with all CRC groups showing a significant increase in Fn abundance compared to the healthy group. These results support a strong association between Fn abundance and the occurrence, metastasis, and progression of CRC, consistent with Li et al. findings [48].

Furthermore, after removing outliers, it was noted that the highest Fn levels were in the latter parts of the colon (descending colon, sigmoid colon, and rectum) compared to the initial parts (ascending colon and transverse colon). This aligns with the findings of Alon‐Maimon et al. and Tunsjø et al. [26, 43]. The low Fn levels in the proximal colon support the notion that Fn may be activated in this region to promote CRC through various virulence mechanisms, similar to Cuellar-Gómez et al.'s findings [49].

While Andoh et al. [50] demonstrated a positive correlation between Fn and BMI, no such correlation was found in our study among the different participant groups.

Our study found that increased Fn abundance in CRC patients is linked to infection severity and higher rates of CRC recurrence. This is in line with prior studies showing a connection between gut microbiota and postoperative chemotherapy side effects. Dysbiosis of the gut microbiota correlates with chemotherapy response severity [51, 52]. For example, Oh et al. found that cancer chemotherapy negatively impacts the gut microbiome, leading to dysbiosis and affecting chemotherapy outcomes [53]. Rezasoltani et al. [54] discussed how gut microbiome changes induced by chemotherapy can reduce the effectiveness of immune checkpoint inhibitors in cancer immunotherapy.

We also observed that higher Fn abundance in CRC patients was associated with various chemotherapy types across treatment groups compared to healthy individuals, consistent with previous research. Sevcikova et al. [55] provided a review of the most recent data related showing that chemotherapy alters gut microbiome composition in cancer patients and identified specific bacterial taxa linked to chemotherapy-induced toxicity or resistance [56]. Our study supported the role of Fn abundance in the tumor area of CRC patients in the biofilm environment, enhancing microbiome involvement in chemotherapy resistance. Wei et al. explored how chemotherapy-induced dysbiosis impairs mucosal immunity and increases infection risk [57].

Conversely, some studies highlight positive effects of the gut microbiome on chemotherapy outcomes. A systematic review on the gut microbiome in chemotherapy outcomes suggested that modulating the gut microbiome could improve chemotherapy efficacy and reduce toxicity [23]. Beneficial gut bacteria have been shown to reduce inflammation and intestinal damage caused by chemotherapy, potentially mitigating side effects [58].

Restoring the natural microbial balance in the intestinal environment (dysbiosis modulation) contributes to improving chemotherapy efficiency in CRC patients. Our study showed that a decrease in Fn count correlated with reduced CRC recurrence rates and enhanced chemotherapy effectiveness. Bruneau et al. suggested that the gut microbiota might influence chemotherapy efficacy through various mechanisms like bacterial translocation, immune system regulation, and enzymatic degradation [59]. Deleemans et al. found that chemotherapy was linked to lower gut microbiota diversity, increased intestinal permeability, and elevated inflammatory markers [60].

In our study, we observed a decrease in Fn counts in CRC patients undergoing chemotherapy compared to post-surgery CRC patients, that emphasizing the role of chemotherapy in dysbiosis modulation. Andrew et al. (2004) noted that chemotherapy drugs like 5-FU and irinotecan can alter gut microbiota diversity and abundance, potentially leading to mucositis in CRC patients [61]. Furthermore, Vanlancker et al. [62] reported that 5-FU directly affects the composition of the human gut microbiota.

Studies have shown changes in gut microbial composition before and after chemotherapy in various conditions [63–65]. The impact of chemotherapy on the gut microbiota is significant, affecting gut permeability and mucosal barrier function [66]. However, limited research has explored global or specific microbial changes and their connections to chemotherapy adverse events.

The relationship between Fn and CRC is intricate, with evidence suggesting Fn's role in CRC development and progression. While our study indicated an association between CRC and Fn abundance, establishing a causal relationship between changes in Fn abundance and CRC incidence remains challenging. Targeting Fn or other gut bacteria could be a promising strategy for preventing or treating CRC, warranting further research in this area.

Conclusion

The simple incorporation of the LAMP technique, utilizing phenol red as a PH indicator, enables quick and precise quantitative identification of Fn in stool samples. This research contributes to our understanding of the intestinal microbiome Fn in patients with CRC. While there was no significant difference in Fn levels between the postoperative and chemotherapy groups, the higher presence of Fn in CRC patient cohorts, especially in those experiencing relapse, hints at a potential association between Fn abundance and resistance to chemotherapy. This suggests that modulating Fn may help improve the prognosis of CRC. Further exploration of strategies targeting Fn may offer new opportunities for preventing or managing CRC, underscoring the significance of comprehending the intricate interplay between gut microbiota and the progression of CRC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [A.A. Z, M.A. A., H. Y. and M. G.]. The first draft of the manuscript was written by [A.A. Z., Y. D. and M.A. K.], and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

Funding

The authors did not receive support from any organization for the submitted work. No funding was received to assist with the preparation of this manuscript. No funding was received for conducting this study.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors have no relevant financial or nonfinancial interests to disclose.

Ethical Approval

Ethical approval was granted as of October 16, 2019, according to the decision of research registration by the Tishreen University Council (No.299/15–10-2019), and recruitment started as of October 16, 2019. All participants gave their consent to anonymously publish their entered data at Tishreen University Hospital-Lattakia-Syria, which was compatible with the Declaration of Helsinki.

Consent for participation

Informed consent was obtained from all individual participants included in the study.

Consent for Publication

The authors affirm that human research participants provided informed consent for publication.

Patient and Public Involvement

Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.