Improvement and Recovery of Intestinal Flora Disorder Caused by Ciprofloxacin Using Lactic Acid Bacteria

Improvement and Recovery of Intestinal Flora Disorder Caused by Ciprofloxacin Using Lactic Acid Bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improvement and Recovery of Intestinal Flora Disorder Caused by Ciprofloxacin Using Lactic Acid Bacteria Xiumin Su, Li Su, Mengyuan Cao, Yulu Sun, Jinghan Dai, Yuanjie He, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4861156/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 9 You are reading this latest preprint version Abstract In this study, four lactic acid bacteria (LAB) strains demonstrating ciprofloxacin, bile salt, gastric fluid and intestinal fluid tolerance; as well as adhesion ability to Caco-2 and HT-29 cells were used to improve and recover the intestinal flora disorders caused by ciprofloxacin. Among which, Lactobacillus brevis 505 exhibited excellent adhesion ability to two kinds of cells and colonization ability to mouse intestinal. After ciprofloxacin treatment, certain recovery effect on cecum caused by ciprofloxacin in the mice was found during natural recovery (group 5C2), but it was challenging to fully restore the intestinal integrity to the initial level. After L. brevis 505 intervention (group 5C5), the intestinal damage to the colon and ileum caused by ciprofloxacin in mice was significantly alleviated, the recovery effect was better than that of natural recovery. Additionally, L. brevis 505 could effectively regulate INF-γ, sIgA and RegⅢγ increase induced by ciprofloxacin. Shannon and Simpson index of the intestinal flora of mice in 5C5 group were higher than those in other group, the relative abundance of Bifidobacterium and Lactobacillus in the mice in 5C5 group was increased, indicating that LAB can better restore the structure and abundance of intestinal microflora. Consequently, L. brevis 505 shows promise as a probiotic for gut microbiota restoration and rebuilding during antibiotic therapy. Lactic acid bacteria Intestinal flora disorder Mouse model Antibiotic Recovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Antibiotics are widely used to treat foodborne poisoning and disease caused by foodborne pathogens [ 1 , 2 ] . However, the use of antibiotics is a double-edged sword, their overuse has led to the breeding of multidrug-resistant pathogens, even superbacteria, and increased the risk of transferring antimicrobial resistance to other pathogens, posing a significant threat to human safety and health [ 3 ] . Overuse of antibiotics in humans could also lead to changes in the number, type and proportion of normal intestinal flora as well as rapid decline in diversity, evenness and taxonomic richness, which is known as "microbial imbalance" [ 4 , 5 ] . Previous research has demonstrated that antibiotic treatment not only leads to biological disorders in the host gut, but also causes inflammatory responses and a significant increase in intestinal antibiotic resistance genes (ARGs) [ 6 , 7 ] . Therefore, finding and discovering effective ways to mitigate the disruption of intestinal flora caused by antibiotics is crucial to mitigate the side effects of antibiotics, which have received widespread attention in recent years [ 8 ] . Indeed, several alternatives, including probiotics and their bacteriocins, phages, microecologics, supplemental prebiotics, or fecal transplants, have been suggested as promising options [ 9 , 10 ] . The application of LAB in food preparation has a long-standing history [ 11 ] . To be recognized as "general safe", after entering intestinal, LAB can reorganize the microbiome that produces single-chain fatty acids to restore host intestinal health, have physiological effects on regulating human intestinal flora, maintaining intestinal microbial balance, enhancing immunity, reducing cholesterol content, improving nutrient absorption and body's immune response [ 7 , 12 , 13 , 14 ] . However, antibiotics do not have the ability to recognize which bacterium should be killed and which should not be killed when they were used to inhibit and kill harmful bacteria in the intestinal. After entering the human body, they may kill or inhibit harmful and beneficial bacterial flora simultaneously, the symbiotic microbial subgroups will also be killed or inhibited indiscriminately, harming digestive flora in the intestinal tract and damaging the digestive system [ 15 , 16 , 17 ] . Meanwhile, many probiotic LAB are often intolerant to bile salts and gastrointestinal fluids. Therefore, it is of great significance to screen LAB that are resistant to common antibiotics, bile salt, gastric and intestinal fluid to recover the imbalance of intestinal flora caused by antibiotics. Fluoroquinolones, which have wide antibacterial spectrums and great inhibitory effect on gram-negative bacteria, are a class of antibiotics widely used in clinical practice. Although they are effective and safe drugs for various infectious diseases treatment, which can also destroy the dynamic balance of intestinal flora and cause intestinal flora disorders. Among them, ciprofloxacin is one of the most frequently used antibiotics for intestinal diseases treatment, having significant effect on the reduction of the diversity of intestinal flora other than to kill the invasive pathogens [ 18 ] . Therefore, in this study, foodborne LAB with strong bacteriostatic ability; bile salt, ciprofloxacin, gastric and intestinal fluid tolerance were screened and used to recover the intestinal flora disturbance and imbalance caused by ciprofloxacin to provide new microecological intervention programs when antibiotics were used for clinical treatment. 2. Materials and methods 2.1 Strain According to our previous tests, four LAB strains including GY-L082, GY-L096, 505 and 520 that exhibited relative strong bacteriostatic ability to Salmonella (ATCC 14028), E. coli O157:H7 (EK 274), Shigella dysenteriae , Yersinia enterocolitica (Y135) and Cronobacter sakazakii (ATCC BAA 894) were used in this study (Table S1 , Fig. S1 , Fig. S2 ). Yersinia enterocolitica 52203 and Y 1 , which have strong pathogenic ability, were used as pathogens for mice infection experiment. 2.2 Growth of LAB strain LAB was inoculated in MRS broth (Beijing Land Bridge Technology Co., Lt., Beijing, China) for growth study. Briefly, when LAB was cultured at 37℃ for 12 h under anaerobic conditions, optical density (OD) of the suspension was firstly measured by a UV Vis spectrophotometer (PerkinElmer, MA, USA) at 600 nm. Two hundred microliter of LAB suspension and MRS broth (used as blank control) were transferred into specific well of an automatic microbial growth curve plate (Nunc, Copenhagen, Denmark), respectively. The plate was incubated at 37℃, cell growth was monitored by enzyme micro-plate reader at 600 nm at 1 h interval, using a multimode plate reader (Tecan, InfiniteTM M200 PRO, Männedorf, Switzerland). For each LAB strain, 3 duplicates were performed. 2.3 Antibiotic susceptibility test The minimum inhibition concentrations (MICs) of ciprofloxacin were determined using broth microdilution method according to the guideline of the Clinical Laboratory Standard Institute (CLSI; the Clinical Laboratory Standard Institute, 2022). The ciprofloxacin was used in concentration ranges of 2–512 µg/mL. Wells in 96 well-plate were proportionally inoculated with the bacterial culture (1 × 10 8 colony-forming unit (CFU/mL)) and broth with ciprofloxacin at different concentrations, the plate was incubated at 37°C (for LAB) or 30℃ (for Yersina ) for 18–24 h. The MICs were determined as the lowest concentrations of ciprofloxacin at which the visible growth was inhibited. Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as quality control strains. 2.4 Acid tolerance assay of LAB The concentration of LAB suspension was adjusted to 10 8 CFU/mL by UV spectrophotometer, after 10% (v/v) of LAB cultures were inoculated into MRS broth with pH 2.5 and 4.0, respectively, the inoculated broths were incubated at 37℃ for 4 h. A similar set up was made for the control with pH was the normal pH of MRS broth. The cultures were sampled at 2 h and 4 h incubation, respectively. The living cells in the cultures were determined using the plate counting method on MRS agar plate. Agar plates were incubated at 37℃ for 48 h, then the CFU of LAB were counted. The following formula was used to calculate the survival rate of each strain in pH 2.5 and 4.0: Survival rate (%) = Δ CFU’s at different pH values /Δ CFU’s at control group × 100 2.5 Bile salt tolerance assay After 10% (v/v) of LAB cultures were inoculated into MRS broth that containing 0.3%, 0.5% and 1% (w/v) bile salt, the inoculated broths were incubated at 37℃ for 0–4 h. A similar set up of MRS broth without bile salts was made for the control. The cultures were sampled after 0 h, 2 h and 4 h incubation, respectively. The living cells in the cultures were determined using the plate counting method on MRS agar plates. Agar plates were incubated at 37℃ for 48 h, the CFU was counted. The following formula was used to calculate the survival rate of each strain in MRS broth containing 0.3%, 0.5% and 1% (w/v) bile salt: Survival rate (%) = Δ CFU’s at different bile salt concentrations /Δ CFU’s at control group × 100 2.6 Simulated gastric and intestinal fluid tolerance of LAB The living cells of LAB in simulated gastric and intestinal fluid was evaluated using previous protocol with some modifications [ 4 ] . To prepare simulated gastric fluid: 6.2 g /L NaCl, 2.2 g /L KCl, 0.22 g /L CaCl 2 , 1.2 g /L NaHCO 3 , 0.3% pepsin were fully dissolved, adjusted the pH of the fluid to 2.0 with 6 M HCl and then filtered using a 0.22 µm sterilized microporous filtration membrane to removed bacteria. For tolerance test, the suspension of overnightly cultured LAB was centrifuged and re-suspended in 0.85% saline, then added into the simulated gastric fluid, mixed thoroughly and placed in an oscillating water bath at 37℃ with a rotational speed of 75 r/min. After 2 h incubation, 100 µL cultures were taken out, gradiently diluted, and spread on MRS agar plate. After incubation at 37℃ for 48 h, the survival rate of LAB strain in simulated gastric fluid was calculated. To prepare simulated intestinal fluid: 4 g/L NaHCO 3 , 0.239 g/L KCl, 1.28 g/L NaCl and 0.1% pancreatin were fully dissolved, adjusted the pH of the fluid to 8.0 with 6 M HC1. After 2 h simulation, the gastric fluid with LAB cultures was centrifuged and re-suspended in the simulated intestinal fluid, and continued to be oscillated in an oscillating water bath. After 2 h incubation, 100 µL cultures were taken out, gradiently diluted, and spread on MRS agar plate. After incubating at 37℃ for 48 h, the survival rate of LAB strain in the simulated intestinal fluid was calculated. The calculation formula is as follows: Survival rate in simulated gastric fluid (%) = Δ CFU’s at 2 h / Δ CFU’s at 0 h× 100 Survival rate in simulated intestinal fluid (%) = Δ CFU’s at 4 h / Δ CFU’s at 2 h× 100 2.7 Adhesion ability to intestinal cells of LAB Caco-2 and HT-29 human colon cells were respectively used to determine the adhesion ability of LAB strain to colon cells. The cells were cultured in Dulbecco’s Modified Eagle’s (DMEM) broth containing 10% fetal bovine serum (F9665, Sigma) and 1% penicillin-streptomycin (P4458, Sigma) in an incubator filled with 5% CO 2 at 37℃ until the cell filled 90% of the bottom space of the culture dish. The cell suspension was firstly digested using 0.25% trypsin solution, and then inoculated into 24-well cell culture plates using complete DMEM culture medium with concentration at 1×10 5 cells /mL. Cells were cultured for 24 h to form monolayer cells. After monolayer cells formed, they were washed for 3 times with sterile PBS solution. Subsequently, 100 µL complete DMEM medium and LAB suspension (approximate 1×10 8 CFU/mL) were added, respectively. The plate was cultured in an incubator with 5% CO 2 at 37℃. Three parallels for each LAB. 2.7.1 Examination of LAB adhesion by Gram staining The adhesion of LAB to intestinal cells was examined by Gram staining. When the plate was washed 5 times with PBS, cells and LAB adhered to the cells were fixed for 30 min at room temperature with 3% paraformaldehyde. Gram staining was applied to the dried plate, the image was taken under a microscope. 2.7.2 Examination of LAB adhesion by transmission electron microscopy (TEM) The intestinal cells and adhered LAB were firstly centrifuged and washed with sterile PBS for 3 times, then cell pellets were fixed using glutaraldehyde (2.5%) overnight (4℃). They were further fixed (4℃, 2–4 h) using osmium tetroxide (OsO4; 1:1 dilution with PBS) and washed with sterile PBS for 3 times. After dehydration using ethanol with concentration gradients (30%, 50%, 70%, 80%, 90%, 100%), samples were transferred to propylene oxide and infiltrated, and embedded in spur’s resin, sections (70 nm) were obtained with an ultramicrotome and stained with Reynolds’ lead citrate prior to examination under a TEM (H-7650; Hitachi, Japan). 2.8 Pathogenic infection model construction and pathogenic ability evaluation 2.8.1 Animal treatment Specific pathogen free (SPF) BALB/c mice (4 weeks, male) were purchased from Huafukang Biotechnology Co., Ltd (Beijing, China). All animal procedures complict with all relevant ethical and were approved by the Northwest A&F University Animal Care Committee (XN2024-0321). After 7 days adaptation period, 27 mice were randomly divided into three groups as 1) normal group, only received PBS; 2) model group Y 1 , received Yersinia enterocolitis Y 1 suspension (1×10 9 CFU/mL) at a daily dose of 0.2 mL for 7 days; 3) model group 52203, received Yersinia enterocolitis 52203 suspension (1×10 9 CFU/mL) at a daily dose of 0.2 mL for 7 days. All mice were raised with standard mouse chow and water, the padding was changed daily. The room condition was maintained at 25 ± 2℃ with relative humidity of 50 ± 5% and 12/12-h light/dark cycle. 2.8.2 Sample collection After 7 days continuous gavage, the mice were sacrificed by intravenous injection of Fatal Plus (pentobarbital sodium), intestinal tissues were collected to detect histopathological changes and inflammatory factors. The mice feces were immediately collected and stored at -80℃ for further sequencing analysis. 2.8.3 Histomorphology of colon The length of colon tissue was measured after euthanasia. The dissected colon, cecum and ileal tissue were immediately placed in 4% paraformaldehyde fixative solution (PFS). After 48 h, the fixed tissues were embedded in paraffin, sectioned and stained using hematoxylin and eosin (H&E). The histomorphology of tissues was observed using a microscope. 2.8.4 Cytokine detection Pro-inflammatory cytokines (TNF-a, IL-6, and IL-1β) in mice serum were measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s instruction. 2.8.5 Colonization ability of Yersinia enterocolitica in mice intestine After fresh mice feces at 0, 1, 3, 7, 8, 11, and 14 d were collected aseptically and weighed, PBS was added at 1∶9 (w/v) into feces at 4℃ for vortex oscillations. When the suspension was serially gradient diluted, 100 µL dilution was uniformly spread on CIN plate (Beijing Land Bridge Technology Co., Lt.,), cultured at 30℃ for 24 h for Y. enterocolitica counting. 2.8.6 Microbial 16S rRNA gene sequencing Fresh mice feces in the control and the experimental group were collected aseptically after 7 days of intragastric administration, 6 parallel feces in each group were sent to Paisenol Biotechnology Co., Ltd. (Shanghai, China) at low temperature for microbial16s rRNA sequencing. 2.9 Improvement of intestinal flora disorder caused by ciprofloxacin using LAB 2.9.1 Animal treatments SPF BALB/c mice (4 weeks, male) were purchased from Huafukang Biotechnology Co., Ltd (Beijing, China). The animal experiment was reviewed and approved by the Animal Care and Use Committee of Northwest A&F University. After 7 days adaptation breeding, 54 mice were randomly divided into 6 groups, 9 mice in each group (Table S2 ). The mice in experimental group were treated with ciprofloxacin (100 µg/mL) at a daily dose of 0.2 mL for 7 days, then were treated with LAB 505 suspension (1×10 9 CFU/mL) at a daily dose of 0.2 mL for 14 days. The control group was given sterile PBS for 21 days. Mice all received standard mouse chow and water, the padding was changed daily. The room conditions were maintained at 25 ± 2℃ with relative humidity of 50 ± 5% and 12/12-h light/dark cycle. 2.9.2 Sample collection After experiment, the mice were sacrificed by intravenous injection of Fatal Plus. The cecum was weighed, and cecum index was calculated to be equal to cecum tissue weight (g) / mouse weight (g). Part of colon and cecum tissues were soaked in 4% PFS for pathological section detection, the remaining part was stored at -20℃ for inflammatory factors detection. Mice feces were immediately collected and stored at -80℃ for further microbial sequencing and gas chromatography (GC) analysis. 2.9.3 Short Chain Fatty Acids (SCFAs) analysis The content of SCFAs (i.e. acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid) in mice feces were determined using GC as described previously [ 4 ] . Briefly, The GC-2010 PLUS system and free fatty acid bonded elastic quartz capillary column (30 m×0.25 mm×0.25 um; Rt-Wax Shimadzu) were used. The flow rate of the mobile phase helium gas was 1.91 mL/min, the initial column temperature was 50℃. The temperature increasing was at 15℃/min to 120℃, then at 5℃/min to 170℃, at 50℃/min to 200℃, and finally maintaining at 200℃ for 1 min. The injection temperature was 250℃, the injection volume was 6 uL, the retention time of each sample was 17.67 min. 2.9.4 Histomorphology of colon The dissected colon and ileal tissues were immediately placed in 4% PFS after euthanasia. After 48 h, the fixed colon tissues were embedded in paraffin, sectioned and stained by hematoxylin and eosin (H&E). The histomorphology of tissues was observed using a microscope. 2.9.5 Cytokine detection The concentration of secretory immunoglobulin A (sIgA), interferon-γ (IFN-γ) and RegIIIy in mice serum was determined by ELISA kit. 2.9.6 16S rRNA gene sequencing of intestinal flora After 7, 14 and 28 days of intragastric administration, fresh fecal samples of mice were collected, stored at -80℃, and sent to Paisenol Biotechnology Co., Ltd. (Shanghai, China) for sequencing. DNA samples of mice feces were extracted and 16S rRNA was determined. 2.9.7 Determination of colonization ability of LAB Fresh feces of mice at 0, 3, 7, 9, 14, 17 and 24 d were collected aseptically and weighed, PBS was added at 1∶9 at 4℃ for vortex oscillations, 100 µL gradient dilutions was uniformly spread on MRS plate with ciprofloxacin and calcium carbonate supplemented, cultured at 37℃ for 48 h for counting. 2.10 Statistical analysis Microbial sequencing data of mouse fecal were bioinformatic analyzed using the QIIME2 platform. Statistical analyses were performed using SPSS 17.0 software. 3 Results 3.1 LAB strain screening used for intestinal flora disorder improvement and recovery 3.1.1 The growth of LAB Four LAB strains reached logarithmic stage after 5 h growth and to plateau stage quickly. There was no significant difference in the overall growth situation, showing good growth ability (Fig. S3 ). 3.1.2 Susceptibility of LAB and pathogenic bacteria to ciprofloxacin Four LAB strains were all resistant to ciprofloxacin. Among which, LAB 505 had strong tolerance ability to ciprofloxacin, the MIC of ciprofloxacin reached 128 µg/mL, while the MIC of ciprofloxacin to other 3 LAB strains was 16 µg/mL (Table 1 ). Two Yersinia enterocolitica strains (Y 1 and 52203) were all susceptible to ciprofloxacin. 3.1.3 Acid, bile salt, simulation of gastric and intestinal fluid tolerance of LAB in vitro When pH was 2.5, no significant difference was found among survival rate of GY-L082, GY-L096 and 520 for 2 h to 4 h treatment except for 505. When pH was 4, with the time increase, the survival rate of 4 strains decreased from about 60% to 20–30%. Strain 520 showed the best tolerance ability, its survival rate (64.71%) was significantly higher than that of GY-1082, GY-1096 and 505 ( P < 0.05) after 4 h treatment (Fig. 1A, B). GY-L082 and GY-L096 cannot grow when the concentration of bile salt was at 0.3%-1%, indicating they are susceptible to bile salt. After 4 h treatment, the survival rate of 520 was 26.69% when the concentration of bile salt was at 1%, indicating that 520 had a strong bile salt tolerance ability. Strain 505 showed certain bile salt tolerance ability (Table 2 ). Table 1 Ciprofloxacin susceptibility of four LAB. Strain Strain spp. MIC of ciprofloxacin(µg/mL) GY-L082 Lactiplantibacillus plantarum 16 GY-L096 Lactiplantibacillus plantarum 16 505 Lactobacillus brevis 128 520 Lactiplantibacillus plantarum 16 Table 2 Bile salt tolerance of four LAB. Strain Treatment time (h) Survival (%) 0.3% Bile salt 0.5% Bile salt 1% Bile salt GY-L082 0、2、4 - - - GY-L096 0、2、4 - - - 505 2 33.41 - - 4 33.51 - - 520 2 49.56 51.04 - 4 26.69 22.69 26.69 After 2 h treatment in gastric fluid, the minimum number of viable LAB decreased to 5.26 log CFU/mL with the survival rate was 58.14%. After 2 h treatment in intestinal fluid, almost more than 85% of the LAB cells were survival. Indicating 4 LAB strains can tolerate gastric fluid and intestinal fluid in certain extent (Fig. 1C). 3.1.4 Adhesion of LAB to intestinal cells Plate counting results showed the adhesion ability of 4 LAB strains to Caco-2 was significantly higher than those to HT-29 cells. No significant difference was found among adhesion rates of 4 LAB strains to HT-29 cells, however, the adhesion rate of 505 to Caco-2 cells was significantly higher than those of other LAB strains to this cell, indicating 505 had excellent adhesion properties (Fig. 1D; Fig. 2 ). 3.1.5 LAB strain selection used for intestinal flora disorder improvement and recovery According to above results on bacteriostatic ability, antibiotic susceptibility, acid and bile salt tolerance, survival ability in simulated gastrointestinal and intestinal fluid, cell adhesion ability, radar map directly and clearly shows that LAB strain 505 exhibits good characteristics in multiple indicators, which was selected and used for intestinal flora disorder improvement and recovery study (Fig. S4 ). 3.2 Pathogenic infection model construction and pathogenic ability evaluation 3.2.1 Effect of Yersinia enterocolica infection on mice tissue Compared with the colon length of the mice in control group (12 cm), that in Y 1 group (10 cm) and 52203 group (9.2 cm) was significantly shorter (Fig. S5 ). In 52203 group, mild swelling, goblet cells loss, local inflammatory cell infiltrated under mucous membrane of the colon; inflammatory cells abnormal infiltration, mucosa ulceration, villous structure disappearance in the cecum; inflammatory cells abnormal infiltration and goblet cells loss in the ileum; were detected. However, no significant pathological tissue change was detected in the colon, cecum and ileum in Y 1 group (Fig. S6). 3.2.2 Colonizing ability of Yersinia enterocolitica in the intestinal tract of mice During gavage period, the bacterial load of 52203 in intestinal tract of mice maintained more than at 10 5 CFU/g feces, while that of strain Y 1 maintained at 10 4 CFU/g feces (Table S3 ). 3.2.3 Cytokine change in mice tissue Secretion levels of cytokines TNF-α, IL-6 and IL-1β in the colon, cecum and spleen in mice serum in 52203 group (Fig. 3A) and Y 1 (Fig. 3B) were significantly increased after gavage compared with the control group. 3.2.4 Microbial diversity in mice fecal Compared with control group, the diversity of intestinal flora and the total number of species in mice fecal in Yersinia enterocolitis infected groups decreased. Shannon and Simpson indexes in 52203 group decreased more significantly ( P < 0.01) than those in Y 1 group, indicating that 52203 had more significant pathogenic effect on flora richness than Y 1 (Fig. 4A, B). On the contrary, Chao1 and ACE indices decreased more significantly in Y 1 group, indicating that Y 1 led to a more significant decrease in the total number of intestinal flora species ( P < 0.01) (Fig. 4C, D). PCoA map revealed that the clusters in Y 1 was far away from that in the control group, indicating that its microbial community structure was profoundly changed. In comparison, the microbial structure of the mice in 52203 group was similar to that of the control group (Fig. 4E). The top 25 OUT numbers of bacteria were selected for analysis (Fig. 5A and Fig. 5B). Pediococcus and Alistipes were predominant in the control group. Compared with those in the control group, the relative abundance of Lactobacillus and Bacteroides in Y 1 group increased significantly ( P 0.05). The relative abundance of Pediococcus and Alistipes in group Y 1 decreased, but there was no significant difference ( P > 0.05). The relative abundance of Pediococcus in group 52203 increased. Desulfovibrio and Alistipes decreased in relative abundance with no significant difference ( P > 0.05). 3.3 Improvement of intestinal flora disorder caused by ciprofloxacin using LAB 3.3.1 Colonization ability of Lactobacillus brevis 505 in vivo The concentration of Lactobacillus brevis 505 in the mice stool increased during the gavage, and could still be detected on the 9th and 14th day after gavage. No Lactobacillus brevis 505 was detected on the 17th and 21st days, indicating that it could colonize relatively stable in mice for at least 7 days (Table S4 ). 3.3.2 Alleviating effects of Lactobacillus brevis 505 on physiological damage caused by ciprofloxacin After 7 days of intragastric administration of ciprofloxacin, the mice showed soft stool, lazy activity and dull hair color, while the mice in control group did not show such phenomenon, indicating that ciprofloxacin caused intestinal disorders in mice. Compared with control group, cecum enlargement was found in the mice in 5C1 group ( P < 0.05). 5C5 group showed significant recovery effect, cecum index of the mice in this group was smaller than that in control group and 5C2 group. The treatment of 5C2 group had a recovery effect on cecum caused by ciprofloxacin, but it was not significant, and it was difficult to recover the cecum damage to the initial level (Table 3 ). Table 3 Effect of Lactobacillus brevis 505 on cecal index after ciprofloxacin treatment. Group Cecal index Control group 1.61 ± 0.22 a 52203 + CIP group (5C1) 1.88 ± 0.13 b 52203 + CIP + natural recovery group (5C2) 1.90 ± 0.18 b 52203 + CIP + 505 group (5C5) 1.62 ± 0.12 a 3.3.3 Recovery effect of Lactobacillus brevis 505 on intestinal injury caused by ciprofloxacin Ciprofloxacin treatment resulted in inflammatory infiltration of intestinal colon and ileal tissue of the mice, as well as epithelial detachment. Whether in the 5C2 group that depended on natural recovery or in the 5C5 group intervened with Lactobacillus brevis 505, both can alleviate the pathological symptoms caused by ciprofloxacin in the colon and ileum of the mice, the effect of Lactobacillus brevis 505 intervention in 5C5 group was better than natural recovery in the 5C2 group (Fig. S7). Secretion level of IgA (sIgA) and INF-γ significantly increased in the mice in 5C1 group ( P < 0.05). Natural recovery in 5C2 group and Lactobacillus brevis 505 intervention in 5C5 group had certain inhibitory effect on the increase of inflammatory factors. INF-γ level that significantly increased in mice in 5C5 group could be restored to the control level as well as other two inflammatory cytokines. The results showed Lactobacillus brevis 505 could effectively regulate INF-γ, sIgA and RegIIIγ increase induced by ciprofloxacin (Fig. S8). 3.3.4 Restorative effect of Lactobacillus brevis 505 on changes in SCFA caused by ciprofloxacin Ciprofloxacin treatment in 5C1 group resulted in a significant reduction of SCFA content in mice feces ( P < 0.05). For the decreased intestinal SCFA levels caused by ciprofloxacin, Lactobacillus brevis 505 intervention in 5C5 group significantly ( P < 0.05) increased SCFA level to that of the control and 5C2 group, which indicated that Lactobacillus brevis 505 intervention is more effective than natural recovery after the mice suffered ciprofloxacin (Fig. S9). 3.3.5 Restoring effect of Lactobacillus brevis 505 on ciprofloxacin-induced disorders of intestinal flora The microbial diversity and species abundance of intestinal flora of the mice in 5C1 group were the lowest. Except Simpson index, other indexes were significantly different from those of the control group ( P < 0.05). The microbial diversity and species abundance of intestinal flora of the mice in 5C2 group and 5C5 group increased, making their recovery was closer to that of the control group. Shannon and Simpson index of the intestinal flora of mice in 5C5 group were higher than those in the control group (Fig. S10). The use of ciprofloxacin led to changes of intestinal flora at the phylum level. Compared with the control group, the relative abundance of Firmicutes in the mice in 5C1 group was significantly increased while that of Bacteroidetes was significantly reduced. However, natural recovery in 5C2 group and Lactobacillus brevis 505 intervention in 5C5 group resulted in the decrease of Firmicutes phylum and increase of Bacteroides phylum. The recovery of intestinal flora of mice in 5C5 group was closer to that of the control group, indicating Lactobacillus brevis 505 can improve intestinal flora disorder caused by ciprofloxacin at a large extent (Fig. 6 , Fig. S11). The community abundance of the intestinal flora of the top 21 genera was further analyzed at genus level. As shown in Fig. 7 A, the average relative abundance of Pediococcus in the mice in 5C1 group increased, while the average relative abundance of Alistipes , Prevotella , and Lactobacillus decreased. The content of Bifidobacterium in the mice in 5C5 group significantly increased ( P < 0.05). Compared with that in control, 5C1 and 5C2 group, the average relative abundance of Rikenella and Candidatus Arthromitus in the mice in 5C5 group increased significantly as well. According to the cluster analysis in the heatmap, compared with that in 5C2 group, the recovered intestinal flora of mice in 5C5 group is closer to that of the control group, the relative abundance of Bifidobacterium and Lactobacillus in the mice in 5C5 group greatly increased (Fig. 7 B). 3.3.6 Improving effect of Lactobacillus brevis 505 on infections caused by Yersinia enterocolidis Lactobacillus brevis 505 can alleviate the inflammatory response in the colon, cecum and ileum; improve submucosal edema, abnormal inflammatory cell infiltration and goblet cell deficiency in the colon; recover abnormal infiltration of inflammatory cells in the cecum; the ulceration of the cecum mucosa; the disappearance of villi structure and reduce inflammatory cytokines TNF-α, IL-6 and IL-1β secretion in colon, cecum and spleen tissues of mice caused by Yersinia enterocolitis 52203 (Fig. S12, Fig. 8 , Fig. 9 ). Lactobacillus brevis 505 can also improve the decline of intestinal microbiota composition diversity and species abundance of the mice caused by Yersinia enterocolitidis 52203 (Fig. S13). The relative abundance of Lactobacillus , Pediococcus and Rikenella increased in the mice in 505 group significantly increased compared to that in 52203 group (Fig. 10A). The flora in the mice recovered by Lactobacillus brevis 505 is close to that of the control group, indicating that LAB can better restore the structure and abundance of intestinal microflora (Fig. 10B). 4. Discussion LAB is one kind of the most used and studied bacteria in human and animal probiotic preparations, widely present in humans and other animals and environment, can colonize in the intestine by secreting bacteriocins, synergistic with intestinal bacteria and other ways, and have physiological effects such as regulating intestinal flora and maintaining the balance of intestinal flora [ 19 ] . Over the years, potential probiotics have been screened for LAB from different sources, including animal gastrointestinal tracts as well as traditional fermented foods and dairy products [ 20 ] . One of the key properties of all potential probiotic strains is their antimicrobial activity, indicating they are able to inhibit other microorganisms by producing different metabolites or through competitive exclusion [ 10 ] . Probiotics are expected to pass through the gastrointestinal tract and survive in the presence of bile salts and acidic gastric juices, only strain with higher survival rate that will have more chance of exerting their beneficial effects [ 20 ] . The probiotic evaluation of new strains must include gastrointestinal tolerance, antimicrobial activity, antibiotic sensitivity, and mammalian cell adhesion [ 21 ] . In this study, a series of characteristics of 4 LAB strains obtained previously were screened and showed they have potential applications in fermented foods. Except for acid and bile salts tolerance, 3 Lactobacillus plantarum and 1 Lactobacillus brevis had high survival rate in simulated gastroenteric fluid, which was consistent with previous study that the survival rate of Lactobacillus plantarum in simulated gastrointestinal tract was high [ 4 ] . The adhesion ability of probiotics to intestinal epithelium is also a necessary prerequisite for its function to be fully utilized. Therefore, we used human intestinal cells including human HT-29 and Caco-2 cells to detect the adhesion characteristics of LAB to evaluate its probiotic potential. The adhesion ability of 4 LAB strains to Caco-2 cells was much greater than that of HT-29 cells, Lactobacillus brevis 505 showed stronger adhesion ability. Which is consistent with previous findings that adhesion of LAB is both host specific and strain specific [ 22 ] . Although these indicators are important for determining the performance of probiotics in vitro , the results of a single indicator do not necessarily represent the actual presence of probiotics in vivo [ 4 ] . Based on the results of in vitro , Lactobacillus brevis 505 was found to be a potential probiotic candidate. Yersiniasis is a common zoonotic foodborne disease with high infection and pathogenicity in young animals. 23 Enterocolitis is a common cause of foodborne bacterial diarrhea and gastroenteritis, in addition to infection with Campylobacter or Salmonella , and is of critical public health importance [ 23 , 24 , 25 ] . Severe infection with Yersinia enterocolitica can cause acute appendicitis, endocarditis, sepsis, serious complications, and even death [ 23 , 26 ] . Therefore, the use of antibiotics to treat enterocolitis infection is very necessary. The pro-inflammatory cytokine TNF-α is a key molecule in innate immunity to infection [ 27 ] . In this study, the colonization ability of Y. enterocolitica 52203 and Y 1 , pathological slices of mice after infection, and related inflammatory factors were determined. The colon, cecum and ileum slices of mice in group 52203 were damaged to varying degrees, inflammatory factors TNF-α, IL-6 and IL-1β increased significantly. Meanwhile, they all can reduce the diversity and abundance of intestinal flora of mice in different degrees. Science the pathogenic ability of strain 52203 was stronger than that of strain Y 1 , therefore, 52203 was selected as the strain for the pathogenic model construction before ciprofloxacin treatment. Probiotics are well known as an alternative strategy to antibiotics [ 28 ] . For the infection caused by Y. enterocolitica , many studies have reported that probiotics can be used to improve the infection. Shi et al. [ 23 ] found that L. brevis 23017 could inhibit the colonization of Y. enterocolitica in the intestinal tract of mice, alleviating the pathogenic effect of mice, confirmed that the function of L. brevis 23017 was to activate intestinal immune function, stimulate the secretion of SIgA in the intestinal tract of mice, and affect the expression level of inflammation-related cytokines. De Montijo-Prieto et al. [ 29 ] showed that the addition of a strain L. plantarum derived from Kefir could protect Y. enterocolitis from intestinal infection. This study explores the direct effect of L. brevis 505 on intestinal immunity and damage caused by Y. enterocolitica 52203. Through intestinal histopathological observation, treatment with L. brevis 505 showed significant improvement. In addition, the levels of proinflammatory cytokines (IL-6, TNF-α and IL-1β) were detected to be significantly decreased ( P < 0.05). These results indicate that L. brevis 505 is expected to control Y. enterocolitis infection in vivo , and is expected to be a substitute for antibiotics, thereby reducing the invasion and infection of pathogenic bacteria. Antibiotics have been used as medical treatments for nearly 100 years, improving survival rates for patients with previously refractory microbial infections [ 30 ] . A single dose of antibiotics is sufficient to induce intestinal flora dysregulation [ 31 ] . In susceptible hosts, antibiotic therapy may trigger intestinal ecological dysregulation and systemic bacterial translocation, its overuse is associated with an increased risk of ecological dysregulation and inflammatory disease [ 32 , 33 , 34 ] . In recent years, used as the most common probiotics, LAB have attracted much attention. Several animal studies and human clinical trials have demonstrated the protective effect of specific strains of probiotic Lactobacillus against intestinal diseases, including antibiotic-associated diarrhea. For example, L. acidophilus can regulate antibiotic-induced intestinal flora disturbance and diarrhea in mice and immune and microbiome dysregulation in bees [ 35 , 36 ] . LAB NS8 can improve the damage of antibiotics to intestinal flora and play a key role in maintaining the balance of intestinal flora [ 37 ] . Administration of L. rhamnosus and L. Swissoides prevented microbial dysregulation in mice models of IBD [ 38 ] . In this study, L. brevis 505 not only can colonize on the intestinal tract of mice, but also promote the recovery of antibiotic-induced intestinal ecological disorders and reduced inflammation. Although the levels of INF-γ, sIgA and RegIIIγ increased after ciprofloxacin treatment, L. brevis 505 could reduce the increase of cytokine levels. In addition, even ciprofloxacin significantly reduced the content of short-chain fatty acids in mice feces, L. brevis 505 could reversed this problem and increase the content of those short-chain fatty acids. Ding et al. [ 39 ] explored the alleviating and restoring effects of Lactobacillus on intestinal flora disorders by inadministration of Lactobacillus in young mice, and proposed that SCFAs are fermented products of intestinal beneficial bacteria, which can regulate intestinal pH value. Among them, the abundance changes of species such as butyric acid, one of the most significant SCFAs, and Bacteroidetes, which produce butyric acid, can also be used as indicators to determine intestinal health. [ 8 , 40 ] In healthy people treated with antibiotics, the total microbial population is significantly reduced, as are the richness and diversity of intestinal flora. In this study, the disturbance of intestinal flora caused by ciprofloxacin administration and the improvement effect of L. brevis 505 on it were investigated. The 16S rRNA sequencing results of mouse feces showed that ciprofloxacin significantly reduced the α-diversity of intestinal microbes in mice, the diversity and species abundance of the intestinal flora could be restored in L. brevis 505 intervention and natural recovery group. Sun et al. [ 2 ] found that at the phylum level, the Bacteroides and Firmicutes were the most predominant phylum in the mice without antibiotic treatment, accounting for 59.3% and 37.1% of total sequences, respectively. Shi et al. [ 30 ] found that several microbial taxa, including Enterobacteriaceae , Clostridium , Owenella and Klebsiella , were enhanced after ampicillin was given to NAR mice while studying the effects of LAB on antibiotics. In this study, after treatment with ciprofloxacin, at the phylum level, the relative abundance of Firmicutes in ciprofloxacin group increased significantly, the relative abundance of Bacteroidetes decreased significantly, and the Actinobacteria decreased. But there was an increase in Deferribacteres . Shi [ 4 ] showed that the relative abundance of Bacteroidetes decreased while that of firmicutes increased when ampicillin was used to establish an intestinal flora disturbance model. The results of this study are consistent with them. However, most studies have shown that Firmicutes decrease after antibiotic treatment [ 41 , 42 ] . The possible reason for this phenomenon was that Yersinia enterocolitica administration had a certain destructive effect on the intestinal flora of mice, or different mice, different antibiotic treatments and different days of administration had different effects on the intestinal flora of mice. Both L. brevis 505 intervention group and the natural control group recovered the changes of bacterial structure and abundance caused by ciprofloxacin, and the recovery results of L. brevis 505 intervention group were more similar to the control group. At the genus level, the average relative abundance of Pediococcus increased, while the average relative abundance of Alistipes , Prevotella and Lactobacillus decreased in ciprofloxacin group. Previous studies have shown that Alistipes has a protective effect against a number of diseases and ecological disorders, including colitis, cirrhosis, cardiovascular disease, and hepatocellular carcinoma, and that a decrease in Alistipes is associated with progression to a decompensated state of cirrhosis. [ 41 ] It was noteworthy that the mean relative abundance of Bifidobacterium , Rikenella and Candidatus Arthromitus in 5C5 group was significantly increased compared with control, 5C1 and 5C2 group ( P < 0.05). Studies have shown that Bifidobacterium can change SCFAs produced by microbial metabolism or activate signaling pathways by intervening in related microbial changes, so as to repair intestinal barrier function and effectively improve constipation symptoms. [ 43 ] These results suggest that ciprofloxacin administration can cause disruption of intestinal microflora in mice, with changes in resident microflora and increased risk of disease. The use of L. brevis 505 can improve the intestinal flora disorder caused by ciprofloxacin by increasing the proportion of beneficial bacteria, and its recovery effect is better than the 5C2 group. In conclusion, 4 LAB strains showed good probiotic properties. After the mice were treated via ciprofloxacin and resulted in intestinal damage and flora disorder, L. brevis 505 could effectively regulate INF-γ, sIgA and RegⅢγ increase induced by ciprofloxacin, improve the pathological symptoms in the colon and ileum, and recover the intestinal flora, which were even better than that in the natural recovery group. The relative abundance of Bifidobacterium and Lactobacillus in the mice in 5C5 group was increased to that of the control group, indicating that LAB can better restore the structure and abundance of intestinal microflora. Declarations Declaration of Competing Interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript. Author Contribution Xiumin Su and Li Su: Conceptualization, Writing - original draft, Writing - review & editing. Mengyuan Cao: Data curation, Visualization. Yulu Sun and Jinghan Dai: Validation. Yuanjie He, Wei Li, Wupeng Ge, Xin Lv, Qiang Zhang, Shenghui Cui, and Jia Chen: Investigation, Methodology. Baowei Yang: Data curation, Formal analysis, Methodology, Project administration, Supervision, Writing - review & editing. Acknowledgement This work was supported by the Ministry of Science and Technology of the People’s Republic of China (no. 2022YFC2303900), Department of Science and Technology of Shaanxi Province (no. 2024JC-YBQN-0178), Education Department of Shaanxi Provincial Government (no. 22JHQ068). Data Availability No datasets were generated or analysed during the current study. References Qiao M, Ying G, Singer A, Zhu Y (2018) Review of antibiotic resistance in China and its environment. Environ Int 110:160–172. https://doi.org/10.1016/j.envint.2017.10.016 Sun Y, Liu T, Si Y, Cao B, Zhang Y, Zheng X, Feng W (2019) Integrated metabolomics and 16S rRNA sequencing to investigate the regulation of Chinese yam on antibiotic-induced intestinal dysbiosis in rats. 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Supplementary Files Highlights.docx SupplementaryFigures.pptx SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted Editorial decision: Revision requested 25 Sep, 2024 Reviews received at journal 08 Sep, 2024 Reviews received at journal 20 Aug, 2024 Reviewers agreed at journal 19 Aug, 2024 Reviewers agreed at journal 18 Aug, 2024 Reviewers invited by journal 17 Aug, 2024 Editor assigned by journal 11 Aug, 2024 Submission checks completed at journal 11 Aug, 2024 First submitted to journal 05 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Baowei","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-08-05 10:15:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4861156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4861156/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-024-10401-5","type":"published","date":"2024-11-20T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64076195,"identity":"587f6203-3ba6-4fa1-ae3e-ca562324ec62","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eAcid tolerance of 4 LAB strains at 2 h. \u003cstrong\u003eB\u003c/strong\u003e Acid tolerance 4 LAB strains at 4 h. \u003cstrong\u003eC\u003c/strong\u003e Simulated gastric and intestinal fluid tolerance of 4 LAB strains. \u003cstrong\u003eD\u003c/strong\u003e Adhesion ability of 4 LAB strains to intestinal cells. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/209d8c9eec37fc9b74e39b0e.jpg"},{"id":64076206,"identity":"205ecc7f-05de-4c75-a599-9a5f57be18e4","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120543,"visible":true,"origin":"","legend":"\u003cp\u003eAdhesion of four LAB to intestinal cells. \u003cstrong\u003eA\u003c/strong\u003e Gram staining, 40× magnification. \u003cstrong\u003eB\u003c/strong\u003eTransmission electron microscope, scale bars, 1 μm.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/811ae9915c597f7a61c605a2.jpg"},{"id":64077137,"identity":"b806fffe-38eb-428a-bec8-cb3d17b5a751","added_by":"auto","created_at":"2024-09-06 09:09:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75883,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Yersinia enterocolitis on cytokine secretion level. \u003cstrong\u003eA\u003c/strong\u003e Effect of strain 52203 on cytokine secretion. \u003cstrong\u003eB\u003c/strong\u003e Effect of strain Y\u003csub\u003e1\u003c/sub\u003e on cytokine secretion. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/de56b151eb0c72c2cb9f6715.jpg"},{"id":64076667,"identity":"98ff32a0-2c59-477b-a072-35619d414001","added_by":"auto","created_at":"2024-09-06 09:01:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57153,"visible":true,"origin":"","legend":"\u003cp\u003eα and β diversity of intestinal flora in mice infected with \u003cem\u003eYersinia enterocolitica\u003c/em\u003e. \u003cstrong\u003eA, B \u003c/strong\u003eShannon and Simpson indices. \u003cstrong\u003eC, D\u003c/strong\u003e ACE and Chao1 indices, respectively, calculated after rarefying to an equal number of sequences reads for all samples. \u003cstrong\u003eE\u003c/strong\u003e Principle coordinate analysis (PCoA) based on bray Curtis distances. Each point represents a sample with colors representing different group. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/a000c2db6cdac801b65339b2.jpg"},{"id":64076200,"identity":"d5750808-0d3b-437b-bdad-820375624227","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86014,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of intestinal flora community in mice infected by \u003cem\u003eYersinia enterocolitis\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e Bar chart at genus level. \u003cstrong\u003eB\u003c/strong\u003e Heat map at genus level.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/972141771522284671d87057.jpg"},{"id":64076207,"identity":"9f48c398-5447-4916-9333-26a37618b304","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50864,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ciprofloxacin on intestinal microbial abundance at phylum level and improvement of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/d9ccc72e070add4c4ade86a9.jpg"},{"id":64076203,"identity":"1496770e-07b2-4d88-8db2-977c26d2fd92","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":95176,"visible":true,"origin":"","legend":"\u003cp\u003eImprovement of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on changes in flora abundance induced by ciprofloxacin. \u003cstrong\u003eA\u003c/strong\u003e Bar chart at genus level . \u003cstrong\u003eB\u003c/strong\u003eHeatmap at genus level.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/2778c9ed784d1bfee2bec78d.jpg"},{"id":64076201,"identity":"1ed55ad6-82be-4678-bf34-f1dcbab3e71f","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":126248,"visible":true,"origin":"","legend":"\u003cp\u003eRestorative effect of Lactobacillus brevis 505 on intestinal damage caused by \u003cem\u003eYersinia enterocolitica\u003c/em\u003e 52203.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/d453f50f4889a62617d3199b.jpg"},{"id":64076198,"identity":"81b7b766-0f94-4cb1-8c82-d29337118ef2","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":52096,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eYersinia enterocolitica\u003c/em\u003e 52203 exposure and \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on intestinal immune factors. \u003cstrong\u003eA\u003c/strong\u003eEffect of strain 505 on TNF-α content. \u003cstrong\u003eB\u003c/strong\u003eEffect of strain 505 on IL-6 content. \u003cstrong\u003eC\u003c/strong\u003eEffect of strain 505 on IL-1β content. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/17521418c2fcd3e6139f668f.jpg"},{"id":64076666,"identity":"092cbb40-ba35-4122-af13-a447a59010fb","added_by":"auto","created_at":"2024-09-06 09:01:29","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":85421,"visible":true,"origin":"","legend":"\u003cp\u003eImprovement of intestinal flora composition by Lactobacillus brevis 505. \u003cstrong\u003eA\u003c/strong\u003e Bar chart at genus level. \u003cstrong\u003eB\u003c/strong\u003e Heatmap at genus level.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/2056f39e130bea7df3e6ce68.jpg"},{"id":69834838,"identity":"0dafaaaa-c273-4eed-943b-758cde20cf2d","added_by":"auto","created_at":"2024-11-25 16:09:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2092717,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/ba2ae310-ff1c-4905-a816-3da163c162c4.pdf"},{"id":64076197,"identity":"ac56c195-d17d-4288-be66-17f2f4970b37","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12094,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/4f64ecfe6191fc362a2f0c6f.docx"},{"id":64076205,"identity":"d7ce9c77-206f-4229-b07b-0b5018151af3","added_by":"auto","created_at":"2024-09-06 08:53:29","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6018194,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/fe3958b810c865ae5e42f64d.pptx"},{"id":64076664,"identity":"f6415d4f-0b24-4e58-9b52-072b57246b3e","added_by":"auto","created_at":"2024-09-06 09:01:29","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26290,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4861156/v1/460715d319fef49c0aa442c6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improvement and Recovery of Intestinal Flora Disorder Caused by Ciprofloxacin Using Lactic Acid Bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntibiotics are widely used to treat foodborne poisoning and disease caused by foodborne pathogens\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, the use of antibiotics is a double-edged sword, their overuse has led to the breeding of multidrug-resistant pathogens, even superbacteria, and increased the risk of transferring antimicrobial resistance to other pathogens, posing a significant threat to human safety and health\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOveruse of antibiotics in humans could also lead to changes in the number, type and proportion of normal intestinal flora as well as rapid decline in diversity, evenness and taxonomic richness, which is known as \"microbial imbalance\"\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Previous research has demonstrated that antibiotic treatment not only leads to biological disorders in the host gut, but also causes inflammatory responses and a significant increase in intestinal antibiotic resistance genes (ARGs)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Therefore, finding and discovering effective ways to mitigate the disruption of intestinal flora caused by antibiotics is crucial to mitigate the side effects of antibiotics, which have received widespread attention in recent years\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Indeed, several alternatives, including probiotics and their bacteriocins, phages, microecologics, supplemental prebiotics, or fecal transplants, have been suggested as promising options\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe application of LAB in food preparation has a long-standing history\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. To be recognized as \"general safe\", after entering intestinal, LAB can reorganize the microbiome that produces single-chain fatty acids to restore host intestinal health, have physiological effects on regulating human intestinal flora, maintaining intestinal microbial balance, enhancing immunity, reducing cholesterol content, improving nutrient absorption and body's immune response\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, antibiotics do not have the ability to recognize which bacterium should be killed and which should not be killed when they were used to inhibit and kill harmful bacteria in the intestinal. After entering the human body, they may kill or inhibit harmful and beneficial bacterial flora simultaneously, the symbiotic microbial subgroups will also be killed or inhibited indiscriminately, harming digestive flora in the intestinal tract and damaging the digestive system\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, many probiotic LAB are often intolerant to bile salts and gastrointestinal fluids. Therefore, it is of great significance to screen LAB that are resistant to common antibiotics, bile salt, gastric and intestinal fluid to recover the imbalance of intestinal flora caused by antibiotics.\u003c/p\u003e \u003cp\u003eFluoroquinolones, which have wide antibacterial spectrums and great inhibitory effect on gram-negative bacteria, are a class of antibiotics widely used in clinical practice. Although they are effective and safe drugs for various infectious diseases treatment, which can also destroy the dynamic balance of intestinal flora and cause intestinal flora disorders. Among them, ciprofloxacin is one of the most frequently used antibiotics for intestinal diseases treatment, having significant effect on the reduction of the diversity of intestinal flora other than to kill the invasive pathogens\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Therefore, in this study, foodborne LAB with strong bacteriostatic ability; bile salt, ciprofloxacin, gastric and intestinal fluid tolerance were screened and used to recover the intestinal flora disturbance and imbalance caused by ciprofloxacin to provide new microecological intervention programs when antibiotics were used for clinical treatment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Strain\u003c/h2\u003e \u003cp\u003eAccording to our previous tests, four LAB strains including GY-L082, GY-L096, 505 and 520 that exhibited relative strong bacteriostatic ability to \u003cem\u003eSalmonella\u003c/em\u003e (ATCC 14028), \u003cem\u003eE. coli\u003c/em\u003e O157:H7 (EK 274), \u003cem\u003eShigella dysenteriae\u003c/em\u003e, \u003cem\u003eYersinia enterocolitica\u003c/em\u003e (Y135) and \u003cem\u003eCronobacter sakazakii\u003c/em\u003e (ATCC BAA 894) were used in this study (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). \u003cem\u003eYersinia enterocolitica\u003c/em\u003e 52203 and Y\u003csub\u003e1\u003c/sub\u003e, which have strong pathogenic ability, were used as pathogens for mice infection experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Growth of LAB strain\u003c/h2\u003e \u003cp\u003eLAB was inoculated in MRS broth (Beijing Land Bridge Technology Co., Lt., Beijing, China) for growth study. Briefly, when LAB was cultured at 37℃ for 12 h under anaerobic conditions, optical density (OD) of the suspension was firstly measured by a UV Vis spectrophotometer (PerkinElmer, MA, USA) at 600 nm. Two hundred microliter of LAB suspension and MRS broth (used as blank control) were transferred into specific well of an automatic microbial growth curve plate (Nunc, Copenhagen, Denmark), respectively. The plate was incubated at 37℃, cell growth was monitored by enzyme micro-plate reader at 600 nm at 1 h interval, using a multimode plate reader (Tecan, InfiniteTM M200 PRO, M\u0026auml;nnedorf, Switzerland). For each LAB strain, 3 duplicates were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Antibiotic susceptibility test\u003c/h2\u003e \u003cp\u003e The minimum inhibition concentrations (MICs) of ciprofloxacin were determined using broth microdilution method according to the guideline of the Clinical Laboratory Standard Institute (CLSI; the Clinical Laboratory Standard Institute, 2022). The ciprofloxacin was used in concentration ranges of 2\u0026ndash;512 \u0026micro;g/mL. Wells in 96 well-plate were proportionally inoculated with the bacterial culture (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e colony-forming unit (CFU/mL)) and broth with ciprofloxacin at different concentrations, the plate was incubated at 37\u0026deg;C (for LAB) or 30℃ (for \u003cem\u003eYersina\u003c/em\u003e) for 18\u0026ndash;24 h. The MICs were determined as the lowest concentrations of ciprofloxacin at which the visible growth was inhibited. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213 and \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 were used as quality control strains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Acid tolerance assay of LAB\u003c/h2\u003e \u003cp\u003eThe concentration of LAB suspension was adjusted to 10\u003csup\u003e8\u003c/sup\u003e CFU/mL by UV spectrophotometer, after 10% (v/v) of LAB cultures were inoculated into MRS broth with pH 2.5 and 4.0, respectively, the inoculated broths were incubated at 37℃ for 4 h. A similar set up was made for the control with pH was the normal pH of MRS broth. The cultures were sampled at 2 h and 4 h incubation, respectively. The living cells in the cultures were determined using the plate counting method on MRS agar plate. Agar plates were incubated at 37℃ for 48 h, then the CFU of LAB were counted. The following formula was used to calculate the survival rate of each strain in pH 2.5 and 4.0:\u003c/p\u003e \u003cp\u003eSurvival rate (%) = Δ CFU\u0026rsquo;s at different pH values /Δ CFU\u0026rsquo;s at control group \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Bile salt tolerance assay\u003c/h2\u003e \u003cp\u003eAfter 10% (v/v) of LAB cultures were inoculated into MRS broth that containing 0.3%, 0.5% and 1% (w/v) bile salt, the inoculated broths were incubated at 37℃ for 0\u0026ndash;4 h. A similar set up of MRS broth without bile salts was made for the control. The cultures were sampled after 0 h, 2 h and 4 h incubation, respectively. The living cells in the cultures were determined using the plate counting method on MRS agar plates. Agar plates were incubated at 37℃ for 48 h, the CFU was counted. The following formula was used to calculate the survival rate of each strain in MRS broth containing 0.3%, 0.5% and 1% (w/v) bile salt:\u003c/p\u003e \u003cp\u003eSurvival rate (%) = Δ CFU\u0026rsquo;s at different bile salt concentrations /Δ CFU\u0026rsquo;s at control group \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Simulated gastric and intestinal fluid tolerance of LAB\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe living cells of LAB in simulated gastric and intestinal fluid was evaluated using previous protocol with some modifications\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo prepare simulated gastric fluid: 6.2 g /L NaCl, 2.2 g /L KCl, 0.22 g /L CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 g /L NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.3% pepsin were fully dissolved, adjusted the pH of the fluid to 2.0 with 6 M HCl and then filtered using a 0.22 \u0026micro;m sterilized microporous filtration membrane to removed bacteria. For tolerance test, the suspension of overnightly cultured LAB was centrifuged and re-suspended in 0.85% saline, then added into the simulated gastric fluid, mixed thoroughly and placed in an oscillating water bath at 37℃ with a rotational speed of 75 r/min. After 2 h incubation, 100 \u0026micro;L cultures were taken out, gradiently diluted, and spread on MRS agar plate. After incubation at 37℃ for 48 h, the survival rate of LAB strain in simulated gastric fluid was calculated.\u003c/p\u003e \u003cp\u003eTo prepare simulated intestinal fluid: 4 g/L NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.239 g/L KCl, 1.28 g/L NaCl and 0.1% pancreatin were fully dissolved, adjusted the pH of the fluid to 8.0 with 6 M HC1. After 2 h simulation, the gastric fluid with LAB cultures was centrifuged and re-suspended in the simulated intestinal fluid, and continued to be oscillated in an oscillating water bath. After 2 h incubation, 100 \u0026micro;L cultures were taken out, gradiently diluted, and spread on MRS agar plate. After incubating at 37℃ for 48 h, the survival rate of LAB strain in the simulated intestinal fluid was calculated.\u003c/p\u003e \u003cp\u003eThe calculation formula is as follows:\u003c/p\u003e \u003cp\u003eSurvival rate in simulated gastric fluid (%) = Δ CFU\u0026rsquo;s at 2 h / Δ CFU\u0026rsquo;s at 0 h\u0026times; 100\u003c/p\u003e \u003cp\u003eSurvival rate in simulated intestinal fluid (%) = Δ CFU\u0026rsquo;s at 4 h / Δ CFU\u0026rsquo;s at 2 h\u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Adhesion ability to intestinal cells of LAB\u003c/h2\u003e \u003cp\u003eCaco-2 and HT-29 human colon cells were respectively used to determine the adhesion ability of LAB strain to colon cells. The cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s (DMEM) broth containing 10% fetal bovine serum (F9665, Sigma) and 1% penicillin-streptomycin (P4458, Sigma) in an incubator filled with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃ until the cell filled 90% of the bottom space of the culture dish. The cell suspension was firstly digested using 0.25% trypsin solution, and then inoculated into 24-well cell culture plates using complete DMEM culture medium with concentration at 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells /mL. Cells were cultured for 24 h to form monolayer cells. After monolayer cells formed, they were washed for 3 times with sterile PBS solution. Subsequently, 100 \u0026micro;L complete DMEM medium and LAB suspension (approximate 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL) were added, respectively. The plate was cultured in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. Three parallels for each LAB.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Examination of LAB adhesion by Gram staining\u003c/h2\u003e \u003cp\u003eThe adhesion of LAB to intestinal cells was examined by Gram staining. When the plate was washed 5 times with PBS, cells and LAB adhered to the cells were fixed for 30 min at room temperature with 3% paraformaldehyde. Gram staining was applied to the dried plate, the image was taken under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Examination of LAB adhesion by transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eThe intestinal cells and adhered LAB were firstly centrifuged and washed with sterile PBS for 3 times, then cell pellets were fixed using glutaraldehyde (2.5%) overnight (4℃). They were further fixed (4℃, 2\u0026ndash;4 h) using osmium tetroxide (OsO4; 1:1 dilution with PBS) and washed with sterile PBS for 3 times. After dehydration using ethanol with concentration gradients (30%, 50%, 70%, 80%, 90%, 100%), samples were transferred to propylene oxide and infiltrated, and embedded in spur\u0026rsquo;s resin, sections (70 nm) were obtained with an ultramicrotome and stained with Reynolds\u0026rsquo; lead citrate prior to examination under a TEM (H-7650; Hitachi, Japan).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Pathogenic infection model construction and pathogenic ability evaluation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Animal treatment\u003c/h2\u003e \u003cp\u003eSpecific pathogen free (SPF) BALB/c mice (4 weeks, male) were purchased from Huafukang Biotechnology Co., Ltd (Beijing, China). All animal procedures complict with all relevant ethical and were approved by the Northwest A\u0026amp;F University Animal Care Committee (XN2024-0321).\u003c/p\u003e \u003cp\u003eAfter 7 days adaptation period, 27 mice were randomly divided into three groups as 1) normal group, only received PBS; 2) model group Y\u003csub\u003e1\u003c/sub\u003e, received \u003cem\u003eYersinia enterocolitis\u003c/em\u003e Y\u003csub\u003e1\u003c/sub\u003e suspension (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL) at a daily dose of 0.2 mL for 7 days; 3) model group 52203, received \u003cem\u003eYersinia enterocolitis\u003c/em\u003e 52203 suspension (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL) at a daily dose of 0.2 mL for 7 days. All mice were raised with standard mouse chow and water, the padding was changed daily. The room condition was maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ with relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and 12/12-h light/dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Sample collection\u003c/h2\u003e \u003cp\u003eAfter 7 days continuous gavage, the mice were sacrificed by intravenous injection of Fatal Plus (pentobarbital sodium), intestinal tissues were collected to detect histopathological changes and inflammatory factors. The mice feces were immediately collected and stored at -80℃ for further sequencing analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3 Histomorphology of colon\u003c/h2\u003e \u003cp\u003eThe length of colon tissue was measured after euthanasia. The dissected colon, cecum and ileal tissue were immediately placed in 4% paraformaldehyde fixative solution (PFS). After 48 h, the fixed tissues were embedded in paraffin, sectioned and stained using hematoxylin and eosin (H\u0026amp;E). The histomorphology of tissues was observed using a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.4 Cytokine detection\u003c/h2\u003e \u003cp\u003ePro-inflammatory cytokines (TNF-a, IL-6, and IL-1β) in mice serum were measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.8.5 Colonization ability of \u003cem\u003eYersinia enterocolitica\u003c/em\u003e in mice intestine\u003c/h2\u003e \u003cp\u003eAfter fresh mice feces at 0, 1, 3, 7, 8, 11, and 14 d were collected aseptically and weighed, PBS was added at 1∶9 (w/v) into feces at 4℃ for vortex oscillations. When the suspension was serially gradient diluted, 100 \u0026micro;L dilution was uniformly spread on CIN plate (Beijing Land Bridge Technology Co., Lt.,), cultured at 30℃ for 24 h for \u003cem\u003eY. enterocolitica\u003c/em\u003e counting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.8.6 Microbial 16S rRNA gene sequencing\u003c/h2\u003e \u003cp\u003eFresh mice feces in the control and the experimental group were collected aseptically after 7 days of intragastric administration, 6 parallel feces in each group were sent to Paisenol Biotechnology Co., Ltd. (Shanghai, China) at low temperature for microbial16s rRNA sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Improvement of intestinal flora disorder caused by ciprofloxacin using LAB\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Animal treatments\u003c/h2\u003e \u003cp\u003eSPF BALB/c mice (4 weeks, male) were purchased from Huafukang Biotechnology Co., Ltd (Beijing, China). The animal experiment was reviewed and approved by the Animal Care and Use Committee of Northwest A\u0026amp;F University.\u003c/p\u003e \u003cp\u003eAfter 7 days adaptation breeding, 54 mice were randomly divided into 6 groups, 9 mice in each group (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The mice in experimental group were treated with ciprofloxacin (100 \u0026micro;g/mL) at a daily dose of 0.2 mL for 7 days, then were treated with LAB 505 suspension (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL) at a daily dose of 0.2 mL for 14 days. The control group was given sterile PBS for 21 days. Mice all received standard mouse chow and water, the padding was changed daily. The room conditions were maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ with relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and 12/12-h light/dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2 Sample collection\u003c/h2\u003e \u003cp\u003eAfter experiment, the mice were sacrificed by intravenous injection of Fatal Plus. The cecum was weighed, and cecum index was calculated to be equal to cecum tissue weight (g) / mouse weight (g). Part of colon and cecum tissues were soaked in 4% PFS for pathological section detection, the remaining part was stored at -20℃ for inflammatory factors detection. Mice feces were immediately collected and stored at -80℃ for further microbial sequencing and gas chromatography (GC) analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3 Short Chain Fatty Acids (SCFAs) analysis\u003c/h2\u003e \u003cp\u003eThe content of SCFAs (i.e. acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid) in mice feces were determined using GC as described previously\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Briefly, The GC-2010 PLUS system and free fatty acid bonded elastic quartz capillary column (30 m\u0026times;0.25 mm\u0026times;0.25 um; Rt-Wax Shimadzu) were used. The flow rate of the mobile phase helium gas was 1.91 mL/min, the initial column temperature was 50℃. The temperature increasing was at 15℃/min to 120℃, then at 5℃/min to 170℃, at 50℃/min to 200℃, and finally maintaining at 200℃ for 1 min. The injection temperature was 250℃, the injection volume was 6 uL, the retention time of each sample was 17.67 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.9.4 Histomorphology of colon\u003c/h2\u003e \u003cp\u003eThe dissected colon and ileal tissues were immediately placed in 4% PFS after euthanasia. After 48 h, the fixed colon tissues were embedded in paraffin, sectioned and stained by hematoxylin and eosin (H\u0026amp;E). The histomorphology of tissues was observed using a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.9.5 Cytokine detection\u003c/h2\u003e \u003cp\u003eThe concentration of secretory immunoglobulin A (sIgA), interferon-γ (IFN-γ) and RegIIIy in mice serum was determined by ELISA kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.9.6 16S rRNA gene sequencing of intestinal flora\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAfter 7, 14 and 28 days of intragastric administration, fresh fecal samples of mice were collected, stored at -80℃, and sent to Paisenol Biotechnology Co., Ltd. (Shanghai, China) for sequencing. DNA samples of mice feces were extracted and 16S rRNA was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e2.9.7 Determination of colonization ability of LAB\u003c/h2\u003e \u003cp\u003eFresh feces of mice at 0, 3, 7, 9, 14, 17 and 24 d were collected aseptically and weighed, PBS was added at 1∶9 at 4℃ for vortex oscillations, 100 \u0026micro;L gradient dilutions was uniformly spread on MRS plate with ciprofloxacin and calcium carbonate supplemented, cultured at 37℃ for 48 h for counting.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eMicrobial sequencing data of mouse fecal were bioinformatic analyzed using the QIIME2 platform. Statistical analyses were performed using SPSS 17.0 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.1 LAB strain screening used for intestinal flora disorder improvement and recovery\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 The growth of LAB\u003c/h2\u003e \u003cp\u003eFour LAB strains reached logarithmic stage after 5 h growth and to plateau stage quickly. There was no significant difference in the overall growth situation, showing good growth ability (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Susceptibility of LAB and pathogenic bacteria to ciprofloxacin\u003c/h2\u003e \u003cp\u003eFour LAB strains were all resistant to ciprofloxacin. Among which, LAB 505 had strong tolerance ability to ciprofloxacin, the MIC of ciprofloxacin reached 128 \u0026micro;g/mL, while the MIC of ciprofloxacin to other 3 LAB strains was 16 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two \u003cem\u003eYersinia enterocolitica\u003c/em\u003e strains (Y\u003csub\u003e1\u003c/sub\u003e and 52203) were all susceptible to ciprofloxacin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Acid, bile salt, simulation of gastric and intestinal fluid tolerance of LAB \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWhen pH was 2.5, no significant difference was found among survival rate of GY-L082, GY-L096 and 520 for 2 h to 4 h treatment except for 505. When pH was 4, with the time increase, the survival rate of 4 strains decreased from about 60% to 20\u0026ndash;30%. Strain 520 showed the best tolerance ability, its survival rate (64.71%) was significantly higher than that of GY-1082, GY-1096 and 505 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after 4 h treatment (Fig.\u0026nbsp;1A, B).\u003c/p\u003e \u003cp\u003eGY-L082 and GY-L096 cannot grow when the concentration of bile salt was at 0.3%-1%, indicating they are susceptible to bile salt. After 4 h treatment, the survival rate of 520 was 26.69% when the concentration of bile salt was at 1%, indicating that 520 had a strong bile salt tolerance ability. Strain 505 showed certain bile salt tolerance ability (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCiprofloxacin susceptibility of four LAB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrain spp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC of ciprofloxacin(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGY-L082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGY-L096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus brevis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBile salt tolerance of four LAB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eSurvival (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3% Bile salt\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5% Bile salt\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1% Bile salt\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGY-L082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0、2、4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGY-L096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0、2、4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter 2 h treatment in gastric fluid, the minimum number of viable LAB decreased to 5.26 log CFU/mL with the survival rate was 58.14%. After 2 h treatment in intestinal fluid, almost more than 85% of the LAB cells were survival. Indicating 4 LAB strains can tolerate gastric fluid and intestinal fluid in certain extent (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Adhesion of LAB to intestinal cells\u003c/h2\u003e \u003cp\u003ePlate counting results showed the adhesion ability of 4 LAB strains to Caco-2 was significantly higher than those to HT-29 cells. No significant difference was found among adhesion rates of 4 LAB strains to HT-29 cells, however, the adhesion rate of 505 to Caco-2 cells was significantly higher than those of other LAB strains to this cell, indicating 505 had excellent adhesion properties (Fig.\u0026nbsp;1D; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5 LAB strain selection used for intestinal flora disorder improvement and recovery\u003c/h2\u003e \u003cp\u003eAccording to above results on bacteriostatic ability, antibiotic susceptibility, acid and bile salt tolerance, survival ability in simulated gastrointestinal and intestinal fluid, cell adhesion ability, radar map directly and clearly shows that LAB strain 505 exhibits good characteristics in multiple indicators, which was selected and used for intestinal flora disorder improvement and recovery study (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Pathogenic infection model construction and pathogenic ability evaluation\u003c/h2\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Effect of \u003cem\u003eYersinia enterocolica\u003c/em\u003e infection on mice tissue\u003c/h2\u003e \u003cp\u003eCompared with the colon length of the mice in control group (12 cm), that in Y\u003csub\u003e1\u003c/sub\u003e group (10 cm) and 52203 group (9.2 cm) was significantly shorter (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). In 52203 group, mild swelling, goblet cells loss, local inflammatory cell infiltrated under mucous membrane of the colon; inflammatory cells abnormal infiltration, mucosa ulceration, villous structure disappearance in the cecum; inflammatory cells abnormal infiltration and goblet cells loss in the ileum; were detected. However, no significant pathological tissue change was detected in the colon, cecum and ileum in Y\u003csub\u003e1\u003c/sub\u003e group (Fig. S6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Colonizing ability of \u003cem\u003eYersinia enterocolitica\u003c/em\u003e in the intestinal tract of mice\u003c/h2\u003e \u003cp\u003eDuring gavage period, the bacterial load of 52203 in intestinal tract of mice maintained more than at 10\u003csup\u003e5\u003c/sup\u003e CFU/g feces, while that of strain Y\u003csub\u003e1\u003c/sub\u003e maintained at 10\u003csup\u003e4\u003c/sup\u003e CFU/g feces (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Cytokine change in mice tissue\u003c/h2\u003e \u003cp\u003eSecretion levels of cytokines TNF-α, IL-6 and IL-1β in the colon, cecum and spleen in mice serum in 52203 group (Fig.\u0026nbsp;3A) and Y\u003csub\u003e1\u003c/sub\u003e (Fig.\u0026nbsp;3B) were significantly increased after gavage compared with the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 Microbial diversity in mice fecal\u003c/h2\u003e \u003cp\u003eCompared with control group, the diversity of intestinal flora and the total number of species in mice fecal in \u003cem\u003eYersinia enterocolitis\u003c/em\u003e infected groups decreased. Shannon and Simpson indexes in 52203 group decreased more significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than those in Y\u003csub\u003e1\u003c/sub\u003e group, indicating that 52203 had more significant pathogenic effect on flora richness than Y\u003csub\u003e1\u003c/sub\u003e (Fig.\u0026nbsp;4A, B). On the contrary, Chao1 and ACE indices decreased more significantly in Y\u003csub\u003e1\u003c/sub\u003e group, indicating that Y\u003csub\u003e1\u003c/sub\u003e led to a more significant decrease in the total number of intestinal flora species (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;4C, D). PCoA map revealed that the clusters in Y\u003csub\u003e1\u003c/sub\u003e was far away from that in the control group, indicating that its microbial community structure was profoundly changed. In comparison, the microbial structure of the mice in 52203 group was similar to that of the control group (Fig.\u0026nbsp;4E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe top 25 OUT numbers of bacteria were selected for analysis (Fig.\u0026nbsp;5A and Fig.\u0026nbsp;5B). \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eAlistipes\u003c/em\u003e were predominant in the control group. Compared with those in the control group, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e in Y\u003csub\u003e1\u003c/sub\u003e group increased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e in 52203 group increased but had no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The relative abundance of \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eAlistipes\u003c/em\u003e in group Y\u003csub\u003e1\u003c/sub\u003e decreased, but there was no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The relative abundance of \u003cem\u003ePediococcus\u003c/em\u003e in group 52203 increased. \u003cem\u003eDesulfovibrio\u003c/em\u003e and \u003cem\u003eAlistipes\u003c/em\u003e decreased in relative abundance with no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Improvement of intestinal flora disorder caused by ciprofloxacin using LAB\u003c/h2\u003e \u003cdiv id=\"Sec41\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Colonization ability of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe concentration of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 in the mice stool increased during the gavage, and could still be detected on the 9th and 14th day after gavage. No \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 was detected on the 17th and 21st days, indicating that it could colonize relatively stable in mice for at least 7 days (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Alleviating effects of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on physiological damage caused by ciprofloxacin\u003c/h2\u003e \u003cp\u003eAfter 7 days of intragastric administration of ciprofloxacin, the mice showed soft stool, lazy activity and dull hair color, while the mice in control group did not show such phenomenon, indicating that ciprofloxacin caused intestinal disorders in mice.\u003c/p\u003e \u003cp\u003eCompared with control group, cecum enlargement was found in the mice in 5C1 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 5C5 group showed significant recovery effect, cecum index of the mice in this group was smaller than that in control group and 5C2 group. The treatment of 5C2 group had a recovery effect on cecum caused by ciprofloxacin, but it was not significant, and it was difficult to recover the cecum damage to the initial level (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Lactobacillus brevis 505 on cecal index after ciprofloxacin treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCecal index\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52203\u0026thinsp;+\u0026thinsp;CIP group (5C1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52203\u0026thinsp;+\u0026thinsp;CIP\u0026thinsp;+\u0026thinsp;natural recovery group (5C2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52203\u0026thinsp;+\u0026thinsp;CIP\u0026thinsp;+\u0026thinsp;505 group (5C5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Recovery effect of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on intestinal injury caused by ciprofloxacin\u003c/h2\u003e \u003cp\u003eCiprofloxacin treatment resulted in inflammatory infiltration of intestinal colon and ileal tissue of the mice, as well as epithelial detachment. Whether in the 5C2 group that depended on natural recovery or in the 5C5 group intervened with \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505, both can alleviate the pathological symptoms caused by ciprofloxacin in the colon and ileum of the mice, the effect of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 intervention in 5C5 group was better than natural recovery in the 5C2 group (Fig. S7).\u003c/p\u003e \u003cp\u003eSecretion level of IgA (sIgA) and INF-γ significantly increased in the mice in 5C1 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Natural recovery in 5C2 group and \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 intervention in 5C5 group had certain inhibitory effect on the increase of inflammatory factors. INF-γ level that significantly increased in mice in 5C5 group could be restored to the control level as well as other two inflammatory cytokines. The results showed \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 could effectively regulate INF-γ, sIgA and RegIIIγ increase induced by ciprofloxacin (Fig. S8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec44\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 Restorative effect of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on changes in SCFA caused by ciprofloxacin\u003c/h2\u003e \u003cp\u003eCiprofloxacin treatment in 5C1 group resulted in a significant reduction of SCFA content in mice feces (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For the decreased intestinal SCFA levels caused by ciprofloxacin, \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 intervention in 5C5 group significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased SCFA level to that of the control and 5C2 group, which indicated that \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 intervention is more effective than natural recovery after the mice suffered ciprofloxacin (Fig. S9).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec45\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5 Restoring effect of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on ciprofloxacin-induced disorders of intestinal flora\u003c/h2\u003e \u003cp\u003eThe microbial diversity and species abundance of intestinal flora of the mice in 5C1 group were the lowest. Except Simpson index, other indexes were significantly different from those of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The microbial diversity and species abundance of intestinal flora of the mice in 5C2 group and 5C5 group increased, making their recovery was closer to that of the control group. Shannon and Simpson index of the intestinal flora of mice in 5C5 group were higher than those in the control group (Fig. S10).\u003c/p\u003e \u003cp\u003eThe use of ciprofloxacin led to changes of intestinal flora at the phylum level. Compared with the control group, the relative abundance of Firmicutes in the mice in 5C1 group was significantly increased while that of Bacteroidetes was significantly reduced. However, natural recovery in 5C2 group and \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 intervention in 5C5 group resulted in the decrease of Firmicutes phylum and increase of Bacteroides phylum. The recovery of intestinal flora of mice in 5C5 group was closer to that of the control group, indicating \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 can improve intestinal flora disorder caused by ciprofloxacin at a large extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Fig. S11).\u003c/p\u003e \u003cp\u003eThe community abundance of the intestinal flora of the top 21 genera was further analyzed at genus level. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the average relative abundance of \u003cem\u003ePediococcus\u003c/em\u003e in the mice in 5C1 group increased, while the average relative abundance of \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e decreased. The content of \u003cem\u003eBifidobacterium\u003c/em\u003e in the mice in 5C5 group significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with that in control, 5C1 and 5C2 group, the average relative abundance of \u003cem\u003eRikenella\u003c/em\u003e and \u003cem\u003eCandidatus Arthromitus\u003c/em\u003e in the mice in 5C5 group increased significantly as well. According to the cluster analysis in the heatmap, compared with that in 5C2 group, the recovered intestinal flora of mice in 5C5 group is closer to that of the control group, the relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e in the mice in 5C5 group greatly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec46\" class=\"Section3\"\u003e \u003ch2\u003e3.3.6 Improving effect of \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 on infections caused by \u003cem\u003eYersinia enterocolidis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 can alleviate the inflammatory response in the colon, cecum and ileum; improve submucosal edema, abnormal inflammatory cell infiltration and goblet cell deficiency in the colon; recover abnormal infiltration of inflammatory cells in the cecum; the ulceration of the cecum mucosa; the disappearance of villi structure and reduce inflammatory cytokines TNF-α, IL-6 and IL-1β secretion in colon, cecum and spleen tissues of mice caused by \u003cem\u003eYersinia enterocolitis\u003c/em\u003e 52203 (Fig. S12, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e8\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 can also improve the decline of intestinal microbiota composition diversity and species abundance of the mice caused by \u003cem\u003eYersinia enterocolitidis\u003c/em\u003e 52203 (Fig. S13). The relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eRikenella\u003c/em\u003e increased in the mice in 505 group significantly increased compared to that in 52203 group (Fig.\u0026nbsp;10A). The flora in the mice recovered by \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 is close to that of the control group, indicating that LAB can better restore the structure and abundance of intestinal microflora (Fig.\u0026nbsp;10B).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLAB is one kind of the most used and studied bacteria in human and animal probiotic preparations, widely present in humans and other animals and environment, can colonize in the intestine by secreting bacteriocins, synergistic with intestinal bacteria and other ways, and have physiological effects such as regulating intestinal flora and maintaining the balance of intestinal flora\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Over the years, potential probiotics have been screened for LAB from different sources, including animal gastrointestinal tracts as well as traditional fermented foods and dairy products\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. One of the key properties of all potential probiotic strains is their antimicrobial activity, indicating they are able to inhibit other microorganisms by producing different metabolites or through competitive exclusion\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProbiotics are expected to pass through the gastrointestinal tract and survive in the presence of bile salts and acidic gastric juices, only strain with higher survival rate that will have more chance of exerting their beneficial effects\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The probiotic evaluation of new strains must include gastrointestinal tolerance, antimicrobial activity, antibiotic sensitivity, and mammalian cell adhesion\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this study, a series of characteristics of 4 LAB strains obtained previously were screened and showed they have potential applications in fermented foods. Except for acid and bile salts tolerance, 3 \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and 1 \u003cem\u003eLactobacillus brevis\u003c/em\u003e had high survival rate in simulated gastroenteric fluid, which was consistent with previous study that the survival rate of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e in simulated gastrointestinal tract was high\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe adhesion ability of probiotics to intestinal epithelium is also a necessary prerequisite for its function to be fully utilized. Therefore, we used human intestinal cells including human HT-29 and Caco-2 cells to detect the adhesion characteristics of LAB to evaluate its probiotic potential. The adhesion ability of 4 LAB strains to Caco-2 cells was much greater than that of HT-29 cells, \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 showed stronger adhesion ability. Which is consistent with previous findings that adhesion of LAB is both host specific and strain specific\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Although these indicators are important for determining the performance of probiotics \u003cem\u003ein vitro\u003c/em\u003e, the results of a single indicator do not necessarily represent the actual presence of probiotics \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Based on the results of \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 was found to be a potential probiotic candidate.\u003c/p\u003e \u003cp\u003eYersiniasis is a common zoonotic foodborne disease with high infection and pathogenicity in young animals.\u003csup\u003e23\u003c/sup\u003e Enterocolitis is a common cause of foodborne bacterial diarrhea and gastroenteritis, in addition to infection with \u003cem\u003eCampylobacter\u003c/em\u003e or \u003cem\u003eSalmonella\u003c/em\u003e, and is of critical public health importance\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Severe infection with \u003cem\u003eYersinia enterocolitica\u003c/em\u003e can cause acute appendicitis, endocarditis, sepsis, serious complications, and even death\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Therefore, the use of antibiotics to treat enterocolitis infection is very necessary.\u003c/p\u003e \u003cp\u003eThe pro-inflammatory cytokine TNF-α is a key molecule in innate immunity to infection\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In this study, the colonization ability of \u003cem\u003eY. enterocolitica\u003c/em\u003e 52203 and Y\u003csub\u003e1\u003c/sub\u003e, pathological slices of mice after infection, and related inflammatory factors were determined. The colon, cecum and ileum slices of mice in group 52203 were damaged to varying degrees, inflammatory factors TNF-α, IL-6 and IL-1β increased significantly. Meanwhile, they all can reduce the diversity and abundance of intestinal flora of mice in different degrees. Science the pathogenic ability of strain 52203 was stronger than that of strain Y\u003csub\u003e1\u003c/sub\u003e, therefore, 52203 was selected as the strain for the pathogenic model construction before ciprofloxacin treatment.\u003c/p\u003e \u003cp\u003eProbiotics are well known as an alternative strategy to antibiotics\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. For the infection caused by \u003cem\u003eY. enterocolitica\u003c/em\u003e, many studies have reported that probiotics can be used to improve the infection. Shi et al.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e found that \u003cem\u003eL. brevis\u003c/em\u003e 23017 could inhibit the colonization of \u003cem\u003eY. enterocolitica\u003c/em\u003e in the intestinal tract of mice, alleviating the pathogenic effect of mice, confirmed that the function of \u003cem\u003eL. brevis\u003c/em\u003e 23017 was to activate intestinal immune function, stimulate the secretion of SIgA in the intestinal tract of mice, and affect the expression level of inflammation-related cytokines. De Montijo-Prieto et al.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e showed that the addition of a strain \u003cem\u003eL. plantarum\u003c/em\u003e derived from Kefir could protect \u003cem\u003eY. enterocolitis\u003c/em\u003e from intestinal infection. This study explores the direct effect of \u003cem\u003eL. brevis\u003c/em\u003e 505 on intestinal immunity and damage caused by \u003cem\u003eY. enterocolitica\u003c/em\u003e 52203. Through intestinal histopathological observation, treatment with \u003cem\u003eL. brevis\u003c/em\u003e 505 showed significant improvement. In addition, the levels of proinflammatory cytokines (IL-6, TNF-α and IL-1β) were detected to be significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that \u003cem\u003eL. brevis\u003c/em\u003e 505 is expected to control \u003cem\u003eY. enterocolitis\u003c/em\u003e infection \u003cem\u003ein vivo\u003c/em\u003e, and is expected to be a substitute for antibiotics, thereby reducing the invasion and infection of pathogenic bacteria.\u003c/p\u003e \u003cp\u003eAntibiotics have been used as medical treatments for nearly 100 years, improving survival rates for patients with previously refractory microbial infections\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. A single dose of antibiotics is sufficient to induce intestinal flora dysregulation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In susceptible hosts, antibiotic therapy may trigger intestinal ecological dysregulation and systemic bacterial translocation, its overuse is associated with an increased risk of ecological dysregulation and inflammatory disease\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, used as the most common probiotics, LAB have attracted much attention. Several animal studies and human clinical trials have demonstrated the protective effect of specific strains of probiotic \u003cem\u003eLactobacillus\u003c/em\u003e against intestinal diseases, including antibiotic-associated diarrhea. For example, \u003cem\u003eL. acidophilus\u003c/em\u003e can regulate antibiotic-induced intestinal flora disturbance and diarrhea in mice and immune and microbiome dysregulation in bees\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. LAB NS8 can improve the damage of antibiotics to intestinal flora and play a key role in maintaining the balance of intestinal flora\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Administration of \u003cem\u003eL. rhamnosus\u003c/em\u003e and \u003cem\u003eL. Swissoides\u003c/em\u003e prevented microbial dysregulation in mice models of IBD\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eL. brevis\u003c/em\u003e 505 not only can colonize on the intestinal tract of mice, but also promote the recovery of antibiotic-induced intestinal ecological disorders and reduced inflammation. Although the levels of INF-γ, sIgA and RegIIIγ increased after ciprofloxacin treatment, \u003cem\u003eL. brevis\u003c/em\u003e 505 could reduce the increase of cytokine levels. In addition, even ciprofloxacin significantly reduced the content of short-chain fatty acids in mice feces, \u003cem\u003eL. brevis\u003c/em\u003e 505 could reversed this problem and increase the content of those short-chain fatty acids.\u003c/p\u003e \u003cp\u003eDing et al.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e explored the alleviating and restoring effects of \u003cem\u003eLactobacillus\u003c/em\u003e on intestinal flora disorders by inadministration of \u003cem\u003eLactobacillus\u003c/em\u003e in young mice, and proposed that SCFAs are fermented products of intestinal beneficial bacteria, which can regulate intestinal pH value. Among them, the abundance changes of species such as butyric acid, one of the most significant SCFAs, and Bacteroidetes, which produce butyric acid, can also be used as indicators to determine intestinal health.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn healthy people treated with antibiotics, the total microbial population is significantly reduced, as are the richness and diversity of intestinal flora. In this study, the disturbance of intestinal flora caused by ciprofloxacin administration and the improvement effect of \u003cem\u003eL. brevis\u003c/em\u003e 505 on it were investigated. The 16S rRNA sequencing results of mouse feces showed that ciprofloxacin significantly reduced the α-diversity of intestinal microbes in mice, the diversity and species abundance of the intestinal flora could be restored in \u003cem\u003eL. brevis\u003c/em\u003e 505 intervention and natural recovery group. Sun et al.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e found that at the phylum level, the Bacteroides and Firmicutes were the most predominant phylum in the mice without antibiotic treatment, accounting for 59.3% and 37.1% of total sequences, respectively. Shi et al.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e found that several microbial taxa, including \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eOwenella\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e, were enhanced after ampicillin was given to NAR mice while studying the effects of LAB on antibiotics.\u003c/p\u003e \u003cp\u003eIn this study, after treatment with ciprofloxacin, at the phylum level, the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e in ciprofloxacin group increased significantly, the relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e decreased significantly, and the \u003cem\u003eActinobacteria\u003c/em\u003e decreased. But there was an increase in \u003cem\u003eDeferribacteres\u003c/em\u003e. Shi\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e showed that the relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e decreased while that of firmicutes increased when ampicillin was used to establish an intestinal flora disturbance model. The results of this study are consistent with them. However, most studies have shown that \u003cem\u003eFirmicutes\u003c/em\u003e decrease after antibiotic treatment\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The possible reason for this phenomenon was that \u003cem\u003eYersinia enterocolitica\u003c/em\u003e administration had a certain destructive effect on the intestinal flora of mice, or different mice, different antibiotic treatments and different days of administration had different effects on the intestinal flora of mice. Both \u003cem\u003eL. brevis\u003c/em\u003e 505 intervention group and the natural control group recovered the changes of bacterial structure and abundance caused by ciprofloxacin, and the recovery results of \u003cem\u003eL. brevis\u003c/em\u003e 505 intervention group were more similar to the control group. At the genus level, the average relative abundance of \u003cem\u003ePediococcus\u003c/em\u003e increased, while the average relative abundance of \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e decreased in ciprofloxacin group. Previous studies have shown that \u003cem\u003eAlistipes\u003c/em\u003e has a protective effect against a number of diseases and ecological disorders, including colitis, cirrhosis, cardiovascular disease, and hepatocellular carcinoma, and that a decrease in \u003cem\u003eAlistipes\u003c/em\u003e is associated with progression to a decompensated state of cirrhosis.\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIt was noteworthy that the mean relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eRikenella\u003c/em\u003e and \u003cem\u003eCandidatus Arthromitus\u003c/em\u003e in 5C5 group was significantly increased compared with control, 5C1 and 5C2 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Studies have shown that \u003cem\u003eBifidobacterium\u003c/em\u003e can change SCFAs produced by microbial metabolism or activate signaling pathways by intervening in related microbial changes, so as to repair intestinal barrier function and effectively improve constipation symptoms.\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e These results suggest that ciprofloxacin administration can cause disruption of intestinal microflora in mice, with changes in resident microflora and increased risk of disease. The use of \u003cem\u003eL. brevis\u003c/em\u003e 505 can improve the intestinal flora disorder caused by ciprofloxacin by increasing the proportion of beneficial bacteria, and its recovery effect is better than the 5C2 group.\u003c/p\u003e \u003cp\u003eIn conclusion, 4 LAB strains showed good probiotic properties. After the mice were treated via ciprofloxacin and resulted in intestinal damage and flora disorder, \u003cem\u003eL. brevis\u003c/em\u003e 505 could effectively regulate INF-γ, sIgA and RegⅢγ increase induced by ciprofloxacin, improve the pathological symptoms in the colon and ileum, and recover the intestinal flora, which were even better than that in the natural recovery group. The relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e in the mice in 5C5 group was increased to that of the control group, indicating that LAB can better restore the structure and abundance of intestinal microflora.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiumin Su and Li Su: Conceptualization, Writing - original draft, Writing - review \u0026amp; editing. Mengyuan Cao: Data curation, Visualization. Yulu Sun and Jinghan Dai: Validation. Yuanjie He, Wei Li, Wupeng Ge, Xin Lv, Qiang Zhang, Shenghui Cui, and Jia Chen: Investigation, Methodology. Baowei Yang: Data curation, Formal analysis, Methodology, Project administration, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China (no. 2022YFC2303900), Department of Science and Technology of Shaanxi Province (no. 2024JC-YBQN-0178), Education Department of Shaanxi Provincial Government (no. 22JHQ068).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQiao M, Ying G, Singer A, Zhu Y (2018) Review of antibiotic resistance in China and its environment. 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Microbiome 9(1):39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-020-00991-x\u003c/span\u003e\u003cspan address=\"10.1186/s40168-020-00991-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactic acid bacteria, Intestinal flora disorder, Mouse model, Antibiotic, Recovery","lastPublishedDoi":"10.21203/rs.3.rs-4861156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4861156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, four lactic acid bacteria (LAB) strains demonstrating ciprofloxacin, bile salt, gastric fluid and intestinal fluid tolerance; as well as adhesion ability to Caco-2 and HT-29 cells were used to improve and recover the intestinal flora disorders caused by ciprofloxacin. Among which, \u003cem\u003eLactobacillus brevis\u003c/em\u003e 505 exhibited excellent adhesion ability to two kinds of cells and colonization ability to mouse intestinal. After ciprofloxacin treatment, certain recovery effect on cecum caused by ciprofloxacin in the mice was found during natural recovery (group 5C2), but it was challenging to fully restore the intestinal integrity to the initial level. After \u003cem\u003eL. brevis\u003c/em\u003e 505 intervention (group 5C5), the intestinal damage to the colon and ileum caused by ciprofloxacin in mice was significantly alleviated, the recovery effect was better than that of natural recovery. Additionally, \u003cem\u003eL. brevis\u003c/em\u003e 505 could effectively regulate INF-γ, sIgA and RegⅢγ increase induced by ciprofloxacin. Shannon and Simpson index of the intestinal flora of mice in 5C5 group were higher than those in other group, the relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e in the mice in 5C5 group was increased, indicating that LAB can better restore the structure and abundance of intestinal microflora. Consequently, \u003cem\u003eL. brevis\u003c/em\u003e 505 shows promise as a probiotic for gut microbiota restoration and rebuilding during antibiotic therapy.\u003c/p\u003e","manuscriptTitle":"Improvement and Recovery of Intestinal Flora Disorder Caused by Ciprofloxacin Using Lactic Acid Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-06 08:53:24","doi":"10.21203/rs.3.rs-4861156/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-25T23:34:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-08T20:51:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-20T21:51:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79048890704718996932300033781436507978","date":"2024-08-19T15:21:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63424792704818223018265294488833482472","date":"2024-08-18T22:00:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-17T10:51:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-12T01:40:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-12T01:39:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2024-08-05T10:08:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"63b223cd-3e3b-4c32-a827-876f05626bd1","owner":[],"postedDate":"September 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:00:57+00:00","versionOfRecord":{"articleIdentity":"rs-4861156","link":"https://doi.org/10.1007/s12602-024-10401-5","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2024-11-20 15:57:20","publishedOnDateReadable":"November 20th, 2024"},"versionCreatedAt":"2024-09-06 08:53:24","video":"","vorDoi":"10.1007/s12602-024-10401-5","vorDoiUrl":"https://doi.org/10.1007/s12602-024-10401-5","workflowStages":[]},"version":"v1","identity":"rs-4861156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4861156","identity":"rs-4861156","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}