Polymerase chain reaction detection of Streptococcus anginosus in digestive diseases and its immunomodulation in rat gastric cancer model

Abstract

BACKGROUND

Streptococcus anginosus (S. anginosus), a commensal bacterium of the oral cavity, has been associated with digestive infections; however, its role in tumorigenesis remains unclear.

AIM

To characterize S. anginosus colonization in digestive diseases and evaluate its immunomodulatory effects in a rat model of gastric cancer.

METHODS

We prospectively enrolled 93 patients (January-December 2025) undergoing gastroscopy/colonoscopy with pathologically confirmed diagnoses of erosive gastritis (n = 45), digestive polyps (gastric or intestinal; n = 14), peptic ulcers (n = 24), and digestive tract tumors (gastric or colorectal cancer; n = 10). Gastric/colonic mucosal biopsies and gastric fluid or fecal samples were collected. S. anginosus was detected by polymerase chain reaction using species-specific primers, and the positivity rates and relative loads were compared across diseases and sampling sites. Multivariate logistic regression was used to assess the association between S. anginosus positivity and digestive tract tumors. A chemically induced gastric cancer rat model was established, and rats were randomized into control, cancer model, and cancer + S. anginosus intervention groups.

RESULTS

Baseline sex, age, body mass index, and Helicobacter pylori status were comparable among the groups (P > 0.05), although patients with tumors were older (P < 0.05). S. anginosus positivity and relative load were the highest in tumors, intermediate in peptic ulcers and polyps, and the lowest in erosive gastritis (P < 0.05). Patients with tumors had higher mucosal and luminal positivity than those without tumors with higher loads in the tumor-adjacent vs distal mucosa (P < 0.05). Among the 10 patients with tumors, S. anginosus positivity was higher in those with severe mucosal inflammation, TNM III-IV, and nodal metastasis, and the bacterial load was positively correlated with inflammation scores and tumor invasion depth (P < 0.05). In rats, tumor nodule count, maximal diameter, and total tumor volume/weight were greater in the cancer model than in the controls and further increased with S. anginosus intervention (all P < 0.05). Serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels increased in cancer rats vs controls and further increased with S. anginosus (P < 0.05). CD4⁺ cell, CD8⁺ T-cell, and CD68⁺ macrophage infiltration scores were higher in the cancer group than in the control group and further increased in the S. anginosus-infected group (P < 0.05). Bacterial load correlated positively with IL-6, TNF-α, and immune infiltration scores (P < 0.05).

CONCLUSION

S. anginosus colonization is markedly evident in digestive tract tumors and is associated with inflammatory and clinicopathological features. In the rat model of gastric cancer, the increased levels of IL-6 and TNF-α and infiltration of immune cells (including CD8⁺ T cells) are consistent with the aggravation of tumor burden; however, the increased CD8⁺ T-cell infiltration does not necessarily indicate effective antitumor immunity, and their functional status and potential mechanisms related to arginine-ornithine metabolism still need further verification. S. anginosus may be an important microecological and immunomodulatory target in the development and progression of digestive tract tumors and warrants further in-depth research.

Key Words: Streptococcus anginosus; Polymerase chain reaction; Digestive tract neoplasms; Gastric cancer; Interleukin-6; Tumor necrosis factor-α; Immune cell infiltration

Core Tip: This study characterizes Streptococcus anginosus (S. anginosus) colonization across digestive diseases and its immunomodulatory role in gastric cancer. In a prospective cohort of 93 patients, S. anginosus positivity and relative load were highest in malignant tumors. Using a chemically induced gastric cancer rat model, S. anginosus colonization was shown to promote tumor progression, elevate serum interleukin-6 and tumor necrosis factor-α levels, and enhance infiltration of CD4⁺, CD8⁺ T cells, and CD68⁺ macrophages. The findings indicate that S. anginosus remodels the tumor immune microenvironment to favor carcinogenesis, suggesting its potential as a microbial and immunotherapeutic target.

INTRODUCTION

In recent years, the relationship between gut microecology and gastrointestinal tumors has attracted increasing attention. In addition to Helicobacter pylori (H. pylori), various oral and intestinal commensal bacteria are closely associated with the initiation and progression of esophageal, gastric, and colorectal cancer[1,2]. Streptococcus anginosus (S. anginosus), a member of the S. anginosus group (SAG) was originally regarded as an opportunistic pathogen of the oropharynx that causes angina and invasive infections[3]. In recent years, studies have found that SAG is not only associated with oral and oropharyngeal infections, but may also be involved in digestive system diseases. The presence of SAG in gastrointestinal cancers, such as esophageal and colon cancers, has gradually gained attention. Through the production of virulence factors (e.g., hemolysins, hyaluronidases, and proteases), SAG may play a procarcinogenic role in cancer occurrence and development. These virulence factors can damage host tissue structure and promote tumor cell invasion and metastasis. Additionally, SAG may indirectly facilitate carcinogenesis by inducing chronic inflammation. Moreover, multiple studies have detected a relatively high proportion of bacterial DNA or viable bacteria in esophageal, gastric, and colorectal cancer tissues, suggesting that they may be involved in the initiation and progression of gastrointestinal tumors[4,5].

Tumor-associated chronic inflammation and remodeling of the immune microenvironment are key factors in the occurrence of gastrointestinal tumors. Proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) promote tumor cell proliferation, invasion, and angiogenesis, as well as inhibit antitumor immunity, by activating signaling pathways including JAK/STAT and nuclear factor-kappa B[6,7]. The quantity and functional status of tumor-infiltrating CD4⁺ T cells, CD8⁺ T cells, and macrophages are closely correlated with prognosis and treatment response[8,9]. Microbial colonization can affect tumor behavior by altering the local inflammatory status and immune cell infiltration patterns[10]. At present, evidence remains limited regarding whether S. anginosus exhibits a characteristic distribution in the spectrum of digestive system diseases, as well as whether it participates in the remodeling of the gastric cancer immune microenvironment by regulating IL-6, TNF-α, and immune cell infiltration. Therefore, we designed an integrated research protocol comprising clinical and animal experiments. Polymerase chain reaction (PCR) was used to detect S. anginosus and analyze its distribution characteristics in different digestive system diseases and its relationship with clinicopathological factors, and a logistic regression model was used to explore its independent association with the occurrence of gastrointestinal tumors. For the animal experiment part, a rat model of gastric cancer combined with bacterial intervention is adopted to observe the effects of S. anginosus on IL-6, TNF-α, and immune cell infiltration. This study provides a new perspective on the pathogenesis of digestive system diseases and lays the foundation for the future development of therapeutic strategies for gastric cancer treatment based on microecological regulation.

MATERIALS AND METHODS

Case data

In total, 93 patients with pathologically confirmed gastrointestinal diseases who underwent gastroscopy or colonoscopy in our hospital between January and December 2025 were enrolled.

Inclusion criteria: (1) Age 18-80 years; (2) Confirmed diagnosis by endoscopic examination and pathology; and (3) Informed consent obtained from all patients.

Exclusion criteria: (1) Patients who had received treatment with antibiotics, proton pump inhibitors, or bismuth agents within the last 4 weeks; (2) Patients with a history of subtotal gastrectomy or extensive colorectal resection; (3) Patients who experienced severe cardiac, hepatic, or renal insufficiency or autoimmune diseases; and (4) Patients with other malignant tumors or hematological diseases.

Based on the pathological results, the 93 patients were divided into four groups: (1) The erosive gastritis group (n = 45); (2) Gastrointestinal polyp group (including gastric polyps and intestinal polyps, n = 14); (3) Peptic ulcer group (n = 24); and (4) Gastrointestinal tumor group (including gastric cancer and colorectal cancer, n = 10). General data [age, sex, body mass index (BMI)], H. pylori infection status, smoking and drinking history, and laboratory examination results were collected from all patients.

Specimen collection

During endoscopy, 2-4 biopsy specimens were obtained from the lesional and relatively normal mucosa, at least 5 cm away from the lesion in the stomach or colon, respectively. The specimens were placed in sterile Eppendorf tubes and immediately stored at -80 °C. Gastric juice and fresh feces were collected.

PCR detection

S. anginosus infection was detected using high-throughput sequencing and quantitative real-time PCR (qPCR). Briefly, the total bacterial genomic DNA was extracted using a commercial DNA extraction kit. PCR was performed using primers targeting species-specific S. anginosus sequences. For conventional PCR, the amplification products were detected by agarose gel electrophoresis, and the appearance of a specific band was defined as a positive result. For qPCR, the SYBR Green method was used, and the relative bacterial load was expressed as the ΔCt or 2^-ΔΔCt value. For high-throughput sequencing, the V3-V4 hypervariable region of the bacterial 16S rRNA gene was used as the amplification target. PCR amplification was performed using universal primers and the extracted total DNA. After purification with magnetic beads, the amplification products were used to construct a sequencing library, and paired-end sequencing was performed on a high-throughput sequencing platform (e.g., Illumina MiSeq). Raw sequencing data were subjected to quality control to remove adapter sequences and low-quality reads, followed by assembly to obtain valid high-quality sequences. Sequence clustering and taxonomic annotation were performed using bioinformatics software, QIIME, with reference to 16S rRNA databases, including SILVA, to obtain the bacterial classification information for each sample. Sequences annotated as S. anginosus were extracted. The relative abundance of S. anginosus was calculated as the ratio of its sequence number to the total number of valid sequences, and the results were compared with those obtained by qPCR. High-throughput sequencing revealed that sequences related to S. anginosus were detected and showed an elevated relative abundance in all qPCR-positive samples, whereas the relative abundance of this bacterium was extremely low or undetectable in qPCR-negative samples. These two methods showed good consistency and provided molecular evidence for the identification of S. anginosus infections.

Selection of experimental animals

Fifteen specific pathogen-free male Sprague-Dawley rats weighing 180-220 g were used in this study. The rats were purchased from a qualified experimental animal center (Guangzhou Center for Disease Control and Prevention). The housing and all experimental procedures were performed in strict accordance with relevant regulations on the administration and protection of laboratory animals.

Establishment of animal models and grouping

After 1 week of adaptive feeding, 15 rats were randomly divided into three groups (n = 5 per group): (1) Normal control; (2) Gastric cancer model; and (3) Gastric cancer + S. anginosus intervention. Animal housing and experimental procedures were performed in accordance with the laboratory animal ethics’ guidelines.

The gastric cancer model was established using a classic chemical carcinogenesis method (e.g., MNNG administration in drinking water combined with a high-salt diet). The detailed protocol was performed as described previously[11] with appropriate modifications. During carcinogenesis, the mental state and body weight of the rats were regularly monitored.

The identified S. anginosus strain was cultured and propagated in appropriate medium. Bacteria in the logarithmic growth phase were collected and resuspended in sterile normal saline at a final concentration of approximately 1 × 10 colony-forming units/mL.

According to the carcinogenic schedule described previously[11], weeks 12-24 of carcinogen treatment were defined as the middle-to-late stage. In the present study, from week 12 onward, the gastric cancer + bacterial intervention group received an intragastric administration of the bacterial suspension three times per week for 8 consecutive weeks. Equal volumes of sterile normal saline were administered to control and model groups.

Collection and detection of experimental specimens

Specimen collection: The rats were sacrificed at the end of the experiment. Laparotomy was performed to observe the condition of the gastric mucosa and tumor nodules. Tumor burden indicators including tumor number, maximum diameter, and total tumor volume/weight were recorded.

Detection methods: (1) Serum cytokine detection. Abdominal aortic blood was collected and centrifuged at 3000 rpm (r = 8 cm) for 10 minutes to separate the serum. Serum contents of IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay, with the results expressed as pg/mL; (2) Histopathological and immunohistochemical examinations. Gastric tumors and adjacent mucosal tissues were harvested, fixed, paraffin-embedded, and sectioned. Hematoxylin and eosin staining was performed to observe the gastric mucosal structure, degree of tumor differentiation, and inflammatory infiltration. Immunohistochemistry was performed to detect the expression of CD4, CD8, and CD68 using appropriate antibodies. Positive cells were counted in multiple randomly selected high-power fields, or semi-quantitative scoring was performed by image analysis software; and (3) Detection of S. anginosus presence/Load in gastric tissue: Gastric tumors and adjacent mucosal tissues (sampled by region, if necessary) were homogenized, and total DNA was extracted. The qPCR was performed using S. anginosus species-specific primers, and the relative DNA load of this bacterium in the gastric tissue was expressed as 2^-ΔΔCt. No-template controls and control tissue DNA controls were established to exclude contamination. This indicator was used for correlation analysis with serum IL-6, TNF-α levels, and immune cell infiltration scores.

Observation indicators

The pathological types and baseline characteristics of 93 patients with gastrointestinal diseases, including sex, age, BMI, H. pylori infection status, and smoking and drinking history, were collected and statistically analyzed. Compare the positive rate and bacterial load of S. anginosus were compared among patients with different disease types and sampling sites (lesional mucosa, distal mucosa, and gastric juice/feces). The positive rate of S. anginosus was compared among patients with different clinicopathological characteristics in the digestive tract tumor group, and the correlation between S. anginosus positivity and the degree of mucosal inflammation, tumor stage (TNM), and lymph node metastasis, etc., were analyzed. The degree of gastric mucosal inflammation was referenced to the updated Sydney scoring system, and semiquantitative grading was performed by two pathologists in a blinded manner according to the degree of lymphoplasmacyte and neutrophil infiltration, with scores of 1-3 representing mild, moderate, and severe inflammation, respectively.

With the presence or absence of digestive tract tumors as the dependent variable and sex, age, smoking, drinking, S. anginosus positivity, H. pylori infection, and other data as independent variables, a logistic regression model was established, and the odds ratio and 95%CI of each variable were intuitively displayed using a forest plot. The tumor burdens among the different model groups, including the number of tumors, maximum diameter, and total tumor volume/weight, were compared. The serum levels of IL-6 and TNF-α were compared among different model groups. The immune cell infiltration status (CD4⁺, CD8⁺ T cells, and CD68⁺ macrophages) was also compared in different model groups.

Statistical analysis

All data were analyzed using SPSS Statistics for Windows version 24.0 (IBM Corp., Armonk, NY, United States). Measurement data conforming to a normal distribution were expressed as mean ± SD. Intergroup comparisons were conducted using t-test or one-way analysis of variance. Measurement data with a non-normal distribution are presented as the median (interquartile range). For nonparametric tests, the Mann-Whitney U test/Wilcoxon rank-sum test was used for comparisons between two groups, and the Kruskal-Wallis H test was used for comparisons among multiple groups. For paired/repeated measures data, the Wilcoxon signed-rank test or Friedman test was performed. Enumeration data were described as n (%), and comparisons were carried out using the χ² test or Fisher’s exact test when appropriate. Correlation analysis was performed using Spearman’s rank correlation coefficient or Pearson’s correlation coefficient, according to the data distribution characteristics. Multivariate logistic regression analysis was conducted using a stepwise regression. Significance was defined as a two-tailed P value < 0.05.

RESULTS

Comparison of general data among 93 patients with gastrointestinal diseases

Based on the collation and analysis of the pathological results, the 93 patients with gastrointestinal diseases were classified into four groups based on the pathological type: (1) Erosive gastritis (n = 45); (2) Gastrointestinal polyp (including gastric polyps and intestinal polyps, n = 14); (3) Peptic ulcer (n = 24); and (4) Gastrointestinal tumor (including gastric cancer and colorectal cancer, n = 10). No significant differences were observed in sex, BMI, or H. pylori positivity among the four groups (P > 0.05). However, patients with gastrointestinal tumors were significantly older than those in the other groups (P < 0.05; Table 1).

Table 1 Comparison of general characteristics among patients with different diseases, mean ± SD/n (%).

Group	n	Gender (male/female)	Age (years)	BMI (kg/m2)	Helicobacter pylori positivity
Erosive gastritis group	45	23 (51.11)/22 (48.89)	50.66 ± 5.31	22.87 ± 3.02	13 (28.89)
Gastrointestinal polyp group	14	7 (50.00)/7 (50.00)	55.49 ± 5.78	23.04 ± 2.95	4 (28.57)
Peptic ulcer group	24	10 (41.67)/14 (58.33)	52.41 ± 5.02	23.22 ± 3.08	6 (25.00)
Gastrointestinal tumor group	10	4 (40.00)/6 (60.00)	66.20 ± 6.97a	22.41 ± 2.90	3 (30.00)
F/χ2	-	0.831	9.32	0.176	0.154
P value	-	0.843	< 0.001	0.913	0.991

^aP < 0.05: Compared with the erosive gastritis group, gastrointestinal polyp group, and peptic ulcer group.

BMI: Body mass index.

Comparison of S. anginosus positivity rate and relative bacterial load among patients with different gastrointestinal diseases

The gastrointestinal tumor group presented the highest positivity rate for S. anginosus, followed by the peptic ulcer and gastrointestinal polyp groups, whereas the erosive gastritis group had the lowest positivity rate. The differences in positivity rates among the four groups were significant (P < 0.05; Table 2). In addition, the relative bacterial load of S. anginosus in the gastrointestinal tumor group was significantly higher than that in the other groups (P < 0.05; Figure 1).

Figure 1 Comparison of relative bacterial load of Streptococcus pharyngitidis in patients with different gastrointestinal diseases. Compared with the erosive gastritis group, gastrointestinal polyp group, and peptic ulcer group, ^aP < 0.05.

Table 2 Comparison of Streptococcus pharyngitidis positivity rate and relative bacterial load among patients with different gastrointestinal diseases, n (%).

Group	n	Mucosal positivity	Luminal content positivity
Erosive gastritis group	45	6 (13.33)	5 (11.11)
Gastrointestinal polyp group	14	4 (28.57)	3 (21.43)
Peptic ulcer group	24	7 (29.17)	5 (20.83)
Gastrointestinal tumor group	10	8 (80.00)a	6 (60.00)a
χ2/H	-	10.213	8.014
P value	-	0.017	0.046

^aP < 0.05: Compared with the erosive gastritis group, gastrointestinal polyp group, and peptic ulcer group.

Comparison of S. anginosus positivity rate and bacterial load in different sample sites among patients with different gastrointestinal diseases

The positivity rates for S. anginosus in the mucosal and gastric juice/fecal specimens of patients with gastrointestinal tumors were significantly higher than those in those without tumors (P < 0.05; Table 3). In the gastrointestinal tumor group, the bacterial load of S. anginosus in the adjacent mucosa was higher than that in the distal mucosa (P < 0.05; Figures 2 and 3).

Figure 2 Comparison of relative bacterial load in pathological mucosa, distal mucosa, gastric juice/fecal matter among patients with different diseases. A: Pathological mucosa; B: Distal mucosa; C: Gastric juice/fecal matter. Compared with erosive gastritis group, gastrointestinal polyp group, and peptic ulcer group, ^aP < 0.05.

Figure 3 Comparison of relative bacterial load in different locations of gastrointestinal tumors. Compared with pathological mucosa, ^aP < 0.05.

Table 3 Comparison of Streptococcus pharyngitidis positivity rates and bacterial load in different anatomical sites among patients with various gastrointestinal diseases, n (%)/median (interquartile range).

Group	n	Lesional mucosa positivity	Distal mucosa positivity	Gastric juice/feces positivity	Relative bacterial load (lesional mucosa, 2-ΔΔCt)	Relative bacterial load (distal mucosa, 2-ΔΔCt)	Relative bacterial load (gastric juice/feces, 2-ΔΔCt)
Erosive gastritis group	45	6 (13.33)	3 (6.67)	4 (8.87)	0.18 (0.10, 0.32)	0.12 (0.06, 0.25)	0.16 (0.09, 0.28)
Gastrointestinal polyp group	14	4 (28.57)	1 (7.14)	2 (14.29)	0.34 (0.20, 0.55)	0.21 (0.12, 0.39)	0.30 (0.18, 0.48)
Peptic ulcer group	24	7 (29.17)	5 (20.83)	6 (25.00)	0.36 (0.22, 0.60)	0.24 (0.18, 0.41)	0.32 (0.20, 0.50)
Gastrointestinal tumor group	10	8 (80.00)a	6 (60.00)	5 (50.00)	0.92 (0.65, 1.35)a,b	0.61 (0.42, 1.06)a	0.82 (0.55, 1.20)a
χ2/H	-	10.213	7.86	8.40	44.0	38.6	42.7
P value	-	0.017	0.044	0.038	< 0.001	< 0.001	< 0.001

^aP < 0.05: Compared with the erosive gastritis group, gastrointestinal polyp group, and peptic ulcer group.

^bP < 0.05: Compared with the relative bacterial load of the distal mucosa.

Comparison of S. anginosus positivity rate among patients with different clinicopathological characteristics

Among the 10 patients with gastrointestinal tumors, the positivity rates for S. anginosus in those with severe mucosal inflammation, TNM stage III-IV tumors, and lymph node metastasis were significantly higher than those in patients with mild-to-moderate mucosal inflammation, TNM stage I-II tumors, or no lymph node metastasis (P < 0.05; Table 4).

Table 4 Comparison of Streptococcus pharyngitidis positivity rates among patients with different clinical-pathological characteristics, n (%).

Clinicopathological characteristics	n = 10	S. anginosus		P value
		Positive	Negative
Degree of mucosal inflammation				< 0.0011
Mild-moderate	6	3 (50.00)	3 (50.00)
Severe	4	4 (100.00)	0 (0.00)
TNM staging				< 0.0011
Stage I-II	6	2 (50.00)	4 (50.00)
Stage III-IV	4	4 (100.00)	0 (0.00)
Lymph node metastasis				< 0.0011
Present	3	3 (100.00)	0 (0.001)
Absent	7	3 (42.86)	4 (57.14)

¹: It indicates Fisher’s test.

S. anginosus: Streptococcus anginosus.

Correlation between the relative bacterial load of S. anginosus and pathological characteristics of patients in the gastrointestinal tumor group

Correlation analysis showed that the relative bacterial load of S. anginosus was positively correlated with the mucosal inflammation score and tumor invasion depth (P < 0.05; Table 5).

Table 5 Correlation between relative bacterial load of Streptococcus pharyngitidis and pathological characteristics in patients with gastrointestinal tumors.

Pathological characteristics	Relative bacterial load of S. anginosus
	r value	P value
Mucosal inflammation score	0.436	< 0.001
Tumor invasion depth	0.382	< 0.001

S. anginosus: Streptococcus anginosus.

Multivariate analysis of factors influencing gastrointestinal tumor onset

Multivariate logistic regression analysis was performed with the occurrence of gastrointestinal tumors (yes/no) as the dependent variable, and sex, age, smoking status, drinking status, S. anginosus positivity, and H. pylori infection status as independent variables. The results are presented visually using a forest plot (Figure 2C). After adjusting for confounding factors, age and S. anginosus positivity became independently correlated with the occurrence of gastrointestinal tumors, both serving as independent risk factors for gastrointestinal tumor onset (P < 0.05; Table 6 and Figure 4).

Figure 4 Forest plot of multivariate analysis influencing gastrointestinal tumors. OR: Odds ratio.

Table 6 Multivariate analysis of factors affecting gastrointestinal tumors.

Factors	β	SE	χ²	OR	95%CI	P value
Gender	-2.008	0.500	6.746	0.135	0.050-0.358	0.744
Age	1.890	0.609	11.029	1.389	1.642-2.598	0.004
Smoking	-2.000	0.509	6.841	0.139	0.113-0.161	0.722
Alcohol consumption	-2.011	0.513	6.802	0.135	0.049-0.366	0.613
S. anginosus positivity	1.486	0.736	14.539	2.385	1.846-5.102	< 0.001
Helicobacter pylori infection	-2.015	0.510	6.940	0.133	0.049-0.362	0.849

OR: Odds ratio; S. anginosus: Streptococcus anginosus.

Comparison of tumor burden among different rat model groups

Compared with the normal control group, obvious irregular hyperplasia and tumor nodule formation were observed in the gastric mucosa of rats in the gastric cancer and bacterial intervention groups. The number of tumor nodules in both the model and intervention groups was significantly higher than that in the control group (P < 0.05). Furthermore, the number of tumor nodules, maximum tumor diameter, total tumor volume, and total tumor weight were significantly higher in the intervention group than in the model group (P < 0.05; Table 7).

Table 7 Comparison of tumor burden between different model groups, mean ± SD.

Group	Tumor nodules (n)	Maximum diameter (mm)	Tumor volume (mm3)	Tumor weight (g)
Normal control group (n = 5)	0.11 ± 0.02	-	-	-
Gastric cancer model group (n = 5)	4.62 ± 0.21a	6.85 ± 0.66	185.46 ± 16.22	0.86 ± 0.02
Gastric cancer + S. anginosus intervention group (n = 5)	6.28 ± 0.36a,b	8.41 ± 0.52	248.63 ± 20.17	1.17 ± 0.09
F/t value	878.8	4.152	5.463	7.518
P value	< 0.001	0.003	0.001	< 0.001

^aP < 0.05: Compared with the control group.

^bP < 0.05: Compared with the model group.

S. anginosus: Streptococcus anginosus.

Comparison of serum IL-6 and TNF-α levels among different rat model groups

Compared with the normal control group, the gastric cancer model and bacterial intervention groups exhibited significantly higher serum levels of IL-6 and TNF-α. Moreover, the serum levels of IL-6 and TNF-α were significantly higher in the intervention group than in the model group (P < 0.05; Table 8).

Table 8 Comparison of serum interleukin-6 and tumor necrosis factor-α levels between different model groups, mean ± SD.

Group	IL-6 (pg/mL)	TNF-α (pg/mL)
Normal control group (n = 5)	20.02 ± 3.22	16.41 ± 2.33
Gastric cancer model group (n = 5)	76.48 ± 5.96a	60.58 ± 8.26
Gastric cancer + S. anginosus intervention group (n = 5)	102.71 ± 8.52a,b	84.23 ± 10.12
F value	225.6	101.0
P value	< 0.001	< 0.001

^aP < 0.05: Compared with the control group.

^bP < 0.05: Compared with the model group.

IL-6: Interleukin-6; S. anginosus: Streptococcus anginosus; TNF-α: Tumor necrosis factor-α.

Comparison of immune cell (CD4⁺ T cells, CD8⁺ T cells, and CD68⁺ macrophages) infiltration among different rat model groups

Hematoxylin and eosin staining showed evident inflammatory cell infiltration in the tumor tissues and surrounding mucosa of rats in the model and intervention groups, whereas the gastric mucosal structure in the control group was normal. The infiltration scores of CD4⁺ T cells, CD8⁺ T cells, and CD68⁺ macrophages in the model and intervention groups were higher than those in the control group (P < 0.05), and the infiltration of these immune cells in the intervention group significantly increased (P < 0.05; Table 9).

Table 9 Comparison of immune cell infiltration between different model groups, mean ± SD.

Group	CD4+ T	CD8+ T	CD68+ macrophages
Normal control group (n = 5)	8.42 ± 3.14	6.58 ± 1.16	5.87 ± 0.74
Gastric cancer model group (n = 5)	24.68 ± 6.22a	18.74 ± 2.52a	22.18 ± 3.06a
Gastric cancer + S. anginosus intervention group (n = 5)	36.55 ± 7.51a,b	26.47 ± 3.44a,b	29.85 ± 3.71a,b
F value	28.50	77.22	95.02
P value	< 0.001	< 0.001	< 0.001

^aP < 0.05: Compared with the control group.

^bP < 0.05: Compared with the model group.

S. anginosus: Streptococcus anginosus.

Correlation between the relative bacterial load of S. anginosus and serum IL-6, TNF-α levels, as well as immune cell infiltration scores

Correlation analysis results indicated that the relative bacterial load of S. anginosus was positively correlated with serum IL-6 and TNF-α levels and immune cell infiltration scores (P < 0.05; Table 10).

Table 10 Relationship between relative bacterial load of pharyngitis streptococcus and serum interleukin-6, tumor necrosis factor-α levels, and immune cell infiltration score.

Indicators	Relative bacterial load of S. anginosus
	r value	P value
IL-6	0.458	< 0.001
TNF-α	0.469	< 0.001
CD4+ T cell infiltration score	0.587	< 0.001
CD8+ T cell infiltration score	0.506	< 0.001
CD68+ macrophage infiltration score	0.545	< 0.001

Relative bacterial load of Streptococcus anginosus in this section refers to the relative DNA abundance of Streptococcus anginosus detected by speciesspecific quantitative real-time polymerase chain reaction in gastric tumor/adjacent mucosal tissues at the experimental endpoint. IL-6: Interleukin-6; S. anginosus: Streptococcus anginosus; TNF-α: Tumor necrosis factor-α.

DISCUSSION

Previous studies have reported that S. anginosus is frequently detected in esophageal cancer and head and neck tumors[12,13], and its DNA or viable bacteria have also been identified in the tissues of some gastric and colorectal cancer cases[14]. Currently, research on the role of SAG in gastrointestinal diseases remains relatively limited. However, existing evidence indicates that SAG may colonize the stomach and participate in the occurrence and progression of gastrointestinal disorders[15]. SAG can colonize the gastric mucosa and directly damage gastric epithelial cells via virulence factors, thereby inducing local inflammatory responses. Moreover, SAG may exacerbate dysbiosis of the gastric microecology through interactions with H. pylori or other gastric bacteria, thus promoting the development of gastritis, gastric ulcers, and gastric cancer[16-18].

The results of this study demonstrated that the positivity rate and relative bacterial load of S. anginosus in lesional mucosal tissues and luminal contents of patients with gastric and colorectal cancers were significantly higher than those in patients with non-tumor gastrointestinal diseases. Furthermore, in patients with gastric cancer, the bacterial load in the mucosa adjacent to the tumor is higher than that in the distal mucosa, suggesting that this bacterium preferentially colonizes the tumor microenvironment. Conversely, the analysis of clinicopathological characteristics revealed that among patients with gastrointestinal tumors, those with higher mucosal inflammation scores, advanced tumor T stage, and lymph node metastasis had a higher PCR positivity rate for S. anginosus. Correlation analysis indicated that the bacterial load was positively correlated with the degree of mucosal inflammation, tumor invasion depth, and lymph node metastasis. Further analysis using a logistic regression model and forest plots confirmed that advanced age and S. anginosus positivity were independent risk factors for the occurrence of gastrointestinal tumors, which is consistent with the findings of several domestic and international microecological studies[19,20].

However, systematic studies on the specific pathogenic mechanisms of SAG in gastrointestinal diseases, particularly its role in the immune response of the gastric mucosa, are lacking[21]. Therefore, in this study, a rat model of gastric cancer was established to systematically analyze the potential effect of S. anginosus on the immune microenvironment of gastric cancer, which provides a better theoretical basis for elucidating the association between S. anginosus and the occurrence and progression of gastrointestinal tumors.

In the rat models of gastric cancer in this study, obvious irregular hyperplasia and tumor nodules were observed in the gastric mucosa of rats in the gastric cancer model and bacterial intervention groups compared with the normal control group. The number, maximum diameter, and total tumor volume/weight of tumor nodules in the model and intervention groups were significantly higher than those in the control group; these indicators were higher in the intervention group than in the model group alone, suggesting that S. anginosus may promote the progression of gastric cancer.

Gastric cancer is inseparable from chronic inflammation and immune imbalance. IL-6 and TNF-α are classic proinflammatory cytokines that play important roles in the occurrence and development of gastric cancer[22]. In this study, serum levels of IL-6 and TNF-α in rats with gastric cancer were significantly higher in the model group than in the control group and were further elevated after intervention with S. anginosus, indicating that this bacterium can aggravate gastric cancer-related inflammation.

The underlying mechanism may involve the activation of innate immune cells by structural components or metabolites of the bacterium via pattern recognition receptors such as Toll-like receptors, which induce an inflammatory cascade. Bacterial colonization disrupts the mucosal barrier, facilitating the invasion of pathogens and toxins into the mucosa, and triggering persistent inflammation.

With regard to immune cell infiltration, tumor infiltrating lymphocytes and tumor associated macrophages have been confirmed to exert important effects on the prognosis and therapeutic response of gastric cancer[23,24]. Further immunohistochemical analysis in this study demonstrated increased infiltration of CD4⁺ and CD8⁺ T cells and CD68⁺ macrophages in the model group, which increased further after S. anginosus intervention.

Notably, an increased CD8⁺ T-cell infiltration score only reflects changes in the number or distribution of CD8⁺ cells and cannot be directly correlated with effective antitumor cytotoxicity. Notably, a 2024 study[25] suggested that streptococci could increase ornithine abundance through the arginine metabolic pathway, thereby inhibiting CD8⁺ T-cell differentiation and infiltration. This conclusion appears to contradict the increased CD8⁺ infiltration observed in the present study; however, the two findings are not necessarily contradictory. The possible reasons are as follows: (1) The present study used semiquantitative immunohistochemistry scoring, without distinguishing infiltration in the tumor parenchyma from accumulation in the stroma or inflammatory margin or evaluating the differentiation lineage and functional status of CD8⁺ T cells; and (2) Under the background of chemical carcinogenesis and persistent inflammation, bacterial intervention may cause “passive recruitment” of CD8⁺ cells through inflammatory chemotactic signals. However, under the combined effects of sustained elevation of IL-6 and TNF-α levels and metabolic inhibition (e.g., arginine-ornithine axis imbalance), their effector differentiation and cytotoxic function may be suppressed, ultimately coexisting with increased tumor burden.

Based on these findings, a more cautious interpretation is that S. anginosus intervention is associated with an increase in the number of CD8⁺ cells; however, whether they exert effective antitumor functions still needs to be verified by combined detection of effector molecules and exhaustion markers (such as granzyme B, interferon-gamma, programmed cell death-1, T cell immunoglobulin and mucin-domain containing 3, and lymphocyte activation gene 3), as well as arginine/ornithine metabolism indicators, and further compared with the aforementioned study at the mechanistic level[26].

Thus, S. anginosus acts as an important driver that sustains the “inflammation carcinogenesis” cascade. All these results indicate that the colonization of S. anginosus is closely related to inflammation and the immune microenvironment in the host.

In addition, the positivity rate for S. anginosus was higher in patients with gastrointestinal polyps than in patients with erosive gastritis, suggesting that it participates in the alteration of the mucosal microenvironment at the precancerous lesion stage. Combined with the relatively high positivity rate in the polyp group and considering the microbial migration route of the oral-gut axis, factors such as oral hygiene status, gastric acid suppression therapy, and disruption of the gastric mucosal barrier may affect colonization in the upper gastrointestinal tract.

Therefore, the pathogenic potential of different strains and their precise interactions with host immunity must be further clarified by combining techniques such as metagenomic sequencing, strain virulence typing, and single-cell immune profiling.

This study has certain limitations: (1) The clinical component was a single-center cross-sectional study with a limited sample size, thus, causal inferences should be interpreted with caution; (2) Animal experiments employ a single bacterial strain and a specific gastric cancer model, which cannot fully recapitulate the complexity of human diseases; (3) In-depth analyses of the functional status of tumor-infiltrating immune cells (e.g., exhaustion phenotypes and cytokine profiles) were not performed; and (4) Although terminal gastric tissue was assessed for S. anginosus DNA load using species-specific qPCR, this study did not verify persistent viable bacterial colonization or spatial localization via viable bacterial isolation and culture, in situ hybridization, or fluorescence in situ hybridization. Therefore, definitively distinguishing between persistent colonization and transient passage/residual DNA is challenging.

Future perspectives

Future studies should expand the clinical samples and adopt multicenter longitudinal designs. Integrating immunomics and metabolomics can help systematically elucidate the key pathways by which S. anginosus remodels the immune microenvironment in gastric cancer, explore its feasibility as an early screening biomarker and therapeutic target, and incorporate multi-timepoint dynamic detection, viable bacterial culture and identification, and in situ localization techniques. Controls, such as inactivated bacteria and bacterial supernatants, should be included to further exclude the nonspecific effects of intragastric administration and strengthen the causal inference.

CONCLUSION

In summary, S. anginosus exhibits an uneven distribution across a spectrum of gastrointestinal diseases and serves as an independent risk factor for the development of gastrointestinal tumors, suggesting that this bacterium is involved in the initiation and progression of gastrointestinal malignancies. In a rat model of gastric cancer, bacterial gavage intervention was associated with increased tumor burden, high serum levels of IL-6 and TNF-α, as well as enhanced infiltration of CD4⁺ and CD8⁺ T lymphocytes and CD68⁺ macrophages. These findings indicate that S. anginosus may contribute to gastric cancer progression by exacerbating inflammatory responses and remodeling the tumor immune microenvironment. Notably, increased CD8⁺ T-cell infiltration does not necessarily equate to effective antitumor immunity. In conjunction with previous studies reporting that streptococci can suppress CD8⁺ T-cell differentiation and infiltration via the arginine metabolism-ornithine pathway, the elevated CD8⁺ T-cell infiltration observed in this study was more likely to reflect a concomitant state of inflammation-driven cell recruitment and potential functional suppression. Its actual biological role requires the combined validation of CD8⁺ T-cell effector/exhaustion phenotypes and related metabolic indicators. However, that study lacked direct evidence of persistent bacterial colonization of the gastric mucosa at the experimental endpoint, such as bacterial culture, species-specific PCR, or in situ localization. Gavage-related stress and other confounders should be further excluded. Future studies should include multi-timepoint colonization verification, viable bacterial isolation, and spatial localization and use controls such as inactivated bacteria or bacterial supernatants to strengthen causal inference. Collectively, S. anginosus holds research value as a potential microbiota-associated biomarker and therapeutic target for gastrointestinal tumors. However, the immune metabolic mechanisms underlying its pro-tumorigenic effects require further clarification using large-scale samples and through in-depth mechanistic investigations.