The strain was isolated from the feces of a 90-year-old woman in Du Jiang Yan City, Sichuan. 16 S-rDNA sequence analysis allows taxonomic identification of Lactobacillus strains to the species level (sequencing and taxonomic classification performed by Pasono Bio, Shanghai). The sequences were aligned in GenBank and the results showed that the strains were all Lactobacillus crispatus, which was named L.crispatus FSCDJY67L3. The strain was deposited on 17 October 2022 at the China General Microbial Strain Deposit and Management Center (CGMCCNo.25925). In this research paper, L. crispatus FSCDJY67L3 and Lactobacillus strains (strains from Jiangnan University, Wuxi, China) were grown in MRS medium at 37°C. Moreover, H. pylori SS1 (from the National Centre for Type Culture Collection) was grown in Brain-Heart Infusion broth with 5% fetal bovine serum at 37°C in a micro-oxygenated atmosphere (85% N₂, 10% CO₂, 5% O₂). The Lactobacillus strains and H. pylori bacteria were harvested by centrifugation at 8000 g for 10 min at 4 °C. They were subsequently washed twice with PBS and suspended in PBS and pH 4 artificial gastric juice (containing 0.9% NaCl and 0.3% pepsinogen), respectively, to achieve a concentration of 1×10⁹ CFU/mL for each sample.

The co-aggregation ability analysis was performed according to Collado et al. (16). Briefly, lactic acid bacteria suspension (2 mL) and H. pylori SS1 suspension (2 mL) were mixed as well as vortexed for 10 s to determine co-aggregation capacity. The mixture was incubated at 37°C for 2 h, and the OD600 value of the bacterial solution was measured. The co-aggregation ability was calculated by the equation as follows:

Where OD _{Lactobacillus} , OD _{H. pylori} , and OD _mixture represent, respectively, the absorbance at 600 nm of lactobacillus, H. pylori, and their mixture after incubating at 37°C for 2 h.

The sample preparation method was referred to by Holz et al. (15). L. crispatus FSCDJY67L3 and H. pylori SS1 suspensions were mixed in equal volumes to induce co-aggregation. After incubating for 2 h at room temperature, bacterial co-aggregates were obtained by centrifugation. Subsequently, the co-aggregates were suspended in a glutaraldehyde fixation solution (4%) and kept in a cold room (4°C) overnight. The fixed co-aggregates were then centrifuged, and a gradient dehydration process was carried out using ethanol solutions (70%, 80%, 90%, 95%, and 100%) for 10 min each. The dehydrated co-aggregates were allowed to air-dry at room temperature to ensure complete evaporation of organic reagents. Afterward, they were encapsulated in plastic wrap to prevent sample splatter and subjected to vacuum freeze-drying for 2 days. Following freeze-drying, the samples underwent palladium sputter coating and were observed using scanning electron microscopy (SEM) (HITACHI SU8100, Tokyo, Japan) at high magnification of 5000× and 10000×.

From November 2021 to December 2021, the study enrolled patients aged 18 to 65 years at the Sixth People’s Hospital of Yancheng City (Jiangsu Province, China). The subjects enrolled met the following inclusion criteria: (i) men and women aged from 18 to 65 years old, half from each group; (ii) confirmed diagnosis of H. pylori infection after study entry (≤3 months) by 14^C urea breath test, rapid urease test, or histology; (iii) without symptoms of discomfort, except for H. pylori infection; (iv) prior anti- H. pylori treatment was not received; and (v) understood the details of the study and provided written informed consent. Patients were excluded if they met any of the following criteria: a history of gastrointestinal surgery (except for appendectomy), pregnancy or lactation, a history of any severe disease or condition (e.g., severe cardiovascular, endocrine, hepatic, or renal dysfunction), a mental illness that could potentially hinder collaboration, lack of self-cognitive judgment, substance abuse (alcohol or drugs), or failure to meet the study requirements. Additionally, patients who had taken antibiotics or probiotics in the month prior to inclusion were also excluded. The following criteria were used to determine patient exclusion: (a) if the subject was unwell or had special physiological changes in their body not appropriate to continue participating in the study, (b) if the subject elected to opt out or terminate their participation in the study, and (c) if the subject failed to undergo laboratory procedures, take antibiotics or other probiotic products, undergo a medical examination, and take stool or blood-related samples during the study. If the subject failed to fill in the relevant scale on time, the subject was considered lost to follow-up.

The experimental scheme was approved and implemented by the Yan Cheng People’s Hospital Research Ethics Board (approval no. of the ethics committee: ET2021085) and registered in the Chinese Clinical Trial Registry (Registration Number: ChiCTR2100053710). The Declaration of Helsinki and pertinent local laws were followed during the planning and execution of this study. All study participants signed an informed consent form.

Gastrointestinal Symptom Rating Scale (GSRS) (17): All patients attended an interview for the recall of gastrointestinal symptoms. The 15-item GSRS to assess the severity and frequency of symptoms was reported. According to the degree of score statistics (0~3 points), the score is proportional to the severity of symptoms. The following symptoms were specifically investigated: abdominal pain, heartburn, acid regurgitation, sucking sensations in the epigastrium, nausea and vomiting, borborygmus, abdominal distension, eructation, increased flatus, decreased/increased passage of stools, loose/hard stools, sense of urgency of evacuation, and feeling of incomplete evacuation.

In this study, a double-blind, randomized, and placebo-controlled trial was used to evaluate the eradication rate and safety of L. crispatus FSCDJY67L3 in patients with confirmed H. pylori infection. Patients randomly received 2 g probiotics (5 × 10⁹ CFU/package L. crispatus FSCDJY67L3) or matching placebo twice a day for one month. In general, probiotic doses ranging from 1.0 × 10⁹ to 1.0 × 10¹⁰ colony-forming units (CFU) per day have been shown to be well tolerated in the general population (18). Blood samples were collected prior to the experiment and after the intervention for further analysis, including blood routine and blood biochemical indexes related to liver and kidney function. In addition, we compared the changes in the intestinal flora of subjects after the L. crispatus FSCDJY67L3 intervention using 16S rDNA amplicon sequencing. Chao1 and Shannon indexes were used to characterize the α-diversity of the intestinal flora in subjects after the L. crispatus FSCDJY67L3 intervention. PCoA analysis was used to characterize the β-diversity.

Fecal samples were collected before and after the experiment and were used to analyze changes in the composition of the gut microbiota. The resolved bacteria were extracted using the feces genome extraction kit from USA MP Company. After extraction, PCR was performed for the V3~V4 region of bacterial 16S rDNA. The PCR amplification system was as follows: 25 µL 2 × Premix Taq, 1 µL upstream primer 341F, 1 µL downstream primer 806R, 1 µL genomic DNA template, and 22 µL ddH2O. Amplification conditions were as follows: 95°C for 8 min; 95°C for 35 s, 52°C for 35 s, and 72°C for 40 s, 30 cycles; 72°C for 8 min. The gel was recovered using the ultra-thin agarose-gel purification and recovery kit from Beijing Tian Gen Biochemical Technology Co., LTD., and the purified fecal DNA was mixed according to the equal quality library samples and then sequenced by machine. QIIME1 software was used for data analysis after the microflora was disembarked.

SPSS22.0 software was used for data analysis, and GraphPad Prism 9 was used to make various statistical graphs. All data are expressed as mean ± standard deviation, and differences between groups were compared by one-way analysis of variance (ANOVA) with Duncan post-hoc analysis to correct for multiple comparisons. Differences were considered statistically significant when the p< 0.05.

In this study, Lactobacilli with excellent ability to co-aggregate H. pylori were selected from a large amount of Lactobacillus species ( Figure 1 ). Interestingly, L. crispatus FSCDJY67L3 exhibited the strongest co-aggregation with H. pylori among all Lactobacillus strains. The co-aggregation rate between L. crispatus FSCDJY67L3 and H. pylori was above 97.78%.

Cold field emission scanning electron microscopy was used to observe the morphology of strain co-aggregation. In previous studies, H. pylori morphology was generally S-shaped or spiraled. However, it may age into a cocoon or globular shape as the environment changes. L. crispatus FSCDJY67L3 was long and cylindrical and co-aggregated with H. pylori as shown in Figure 2 . It interacted with the surface of H. pylori in a strongly binding interaction. In addition, the binding site for H. pylori seemed to not be present on the flagellar structure of L. crispatus FSCDJY67L3, which is in agreement with the results reported by Holz et al. (15). Notably, L. crispatus FSCDJY67L3 also exhibited self-aggregation properties, suggesting that the formation of larger co-aggregates was facilitated.

In the present research, patients with H. pylori infection (total of 44) were recruited and randomized to complete the treatment protocol with a placebo (n=20) or with L. crispatus FSCDJY67L3 (n=24). Unfortunately, seven patients dropped out during the process of the study, including two in the L. crispatus FSCDJY67L3 group and five in the placebo group. There were no patients discontinued from treatment and no loss to follow-up. The basic information on the enrolled population is shown in Table 1 . There was no significant difference in age or sex among the groups of subjects.

A clinical trial in patients with a family history of gastric cancer demonstrated that eradication of H.pylori significantly reduced the risk of gastric cancer compared to persistent infection (19). Therefore, it is crucial to decrease the load of H. pylori in the human organism. The expiratory value of ¹⁴C is a widely used clinical indicator for the diagnosis of H. pylori infection, which reflects the H. pylori load in patients. Importantly, the sensitivity and specificity of ¹⁴C expiratory testing usually exceed 95% (20). As shown in Figure 3 , the reduction rate of ¹⁴C expiratory value in H. pylori-positive patients was significantly increased (67.19%) after the intervention of L. crispatus FSCDJY67L3. However, patients with increased ¹⁴C expiratory values showed a negative rate of reduction in expiratory values in the placebo group. This result indicated that L. crispatus FSCDJY67L3 could effectively reduce the H. pylori load in patients.

In this study, we evaluated the gastrointestinal symptoms presented by all patients, which were quantified with the GSRS score (21). There was a significant difference in gastrointestinal symptoms in H. pylori-positive patients before and after L. crispatus FSCDJY67L3 intervention. As seen in Figure 4 , the GSRS score of the placebo group changed from 3.47 ± 2.00 to 4.59 ± 2.01 (p>0.05) after the intervention. In addition, the GSRS score of the L. crispatus FSCDJY67L3 group decreased from 3.2 ± 1.01 to 2.5 ± 1.19 (p<0.05), indicating that the symptoms of gastrointestinal discomfort caused by H. pylori were significantly improved.

The changes in the number of white blood cells, red blood cells, hemoglobin concentration, platelet count, the content of basic phospholipase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin in patients with H. pylori infection in the L. crispatus FSCDJY67L3 and placebo groups are shown in Figures 5 , 6 . In the placebo and L. crispatus FSCDJY67L3 groups, there were no significant differences between the immune mediators in the serum of the patients before and after the intervention.

As shown in Figure 7 , the content of urea and uric acid refers to blood biochemical indicators of renal function in H. pylori-positive patients and showed no significant changes before and after the placebo and L. crispatus FSCDJY67L3 intervention. However, in the placebo group, the creatinine content of H. pylori-positive patients changed from 56.62 ± 17.94 before intervention to 74.05 ± 14.51 (p<0.01), and that of H. pylori-positive patients changed from 54.46 ± 7.82 before intervention to 70.30 ± 15.07 after intervention (p<0.005).

Previous research presented the theory that changes in intestinal flora are associated with a range of gastrointestinal diseases and systemic diseases (22). In addition, Luyi et al. demonstrated that stool samples from H. pylori-infected individuals showed reduced abundance of Clostridium perfringens and total anaerobic bacteria as compared to H. pylori-negative individuals (23). Therefore, the modification of the intestinal flora could be considered as a criterion to reflect the pathological status of the organism.

After intervention with the L. crispatus FSCDJY67L3, no significant changes in intestinal flora Chao1 and Shannon index were observed in H. pylori-positive infected patients ( Figure 8 ), indicating that L. crispatus FSCDJY67L3 intervention could not alter community richness and microbial diversity in H. pylori-positive infected individuals. The further the distance between the different groups in the PCoA analysis indicated the greater the difference in their intestinal flora. As shown in Figure 9 , there was no significant distance between the two groups before and after the intervention, suggesting L. crispatus FSCDJY67L3 could not contribute to the difference in intestinal flora in H. pylori-positive individuals.