Commensal Bacillus From Cow Milk Inhibits Staphylococcus Aureus Bio lm Formation and Mastitis in Mice


 Background: Mastitis, one of the most serious diseases in dairy industry, could cause tremendous economic losses worldwide and is commonly trigged by pathogen invasion. Staphylococcus aureus (S. aureus) -induced mastitis has been reported to play an important role in mastitis etiology characterized by high morbidity, recurrence, and increased antibiotic resistance, which may attribute to the formation of biofilm formation, a form of bacterial aggregation for better growth and resistance to adverse conditions. Probiotics Bacillus has been reported to disrupt bacteria quorum-sensing (QS) system, a central regulator for biofilm formation. However, whether commensal Bacillus affects S. aureus biofilm formation and consequent colonization during mastitis is still unknown. Results：Here, we identified that the Bacillus is associated with reduced colonization of S. aureus in the mammary gland of cows. Interestingly, Bacillus did not affect S. aureus growth but inhibited the biofilm formation of S. aureus by interfering with S. aureus QS signaling. The most obvious anti-biofilm effect was found in Bacillus subtilis H28, so it was selected for further study. We found that bacillus subtilis H28 treatment alleviated S. aureus-induced mastitis in mice, as showed by limiting pro-inflammatory cytokines production, enhancing barrier integrity, and reducing S. aureus burden. Consistently, Bacillus subtilis with the capacity to interfere S. aureus QS ameliorated S. aureus-induced NF-κB activation in mice mammary epithelial cells（MMECs）. Conclusions: Collectively, our results indicate that commensal Bacillus inhibits S. aureus colonization and alleviates S. aureus-induced mastitis by influencing biofilm formation, which suggests a potential strategy for the decolonization of S. aureus and acts as a basis for the prevention and treatment of S. aureus-related disease.


Introduction
Mastitis is one of the most serious diseases in dairy cows, especially in high-yielding cows (1). The high incidence of mastitis and the di culty of preventing and treating it has been one of the foremost diseases plaguing the health of the world's dairy industry (2). There is no effective vaccine for its prevention. Antibiotics are still the only means of clinical prevention and treatment of S. aureus (3). However, the non-standard use of antibiotics makes the drug increasingly serious resistant to S. aureus for a long time (4). In addition to improper production management and drug use, the complex biological functions of S. aureus also increase its bacterial resistance. It is reported that S. aureus has a strong bio lm-forming ability that was found to be signi cantly related to the severity of the disease (5). Much of the colonization and virulence production of S. aureus depends on its population sensing system (QS) and bio lm formation, and the microbial bio lm is responsible for the persistence of S. aureus-related disease (6). Therefore, di culty of mastitis control induced by S. aureus may be related to the long-term colonization of the mammary site by S. aureus. Bio lm is a kind of growth corresponding to planktonic bacteria in which bacteria adsorb to the surface of inert or active entities during the growth process to adapt to their living environment (7). S. aureus has a QS system, which is a central regulator in S. aureus pathogenicity (8). It can regulate adhesion and production of numerous virulence and pathogenic factors as well as the bio lm formation and heterogeneous resistance of S. aureus (9). The bacterial QS system is an intercellular communication mechanism used to synthesize, secrete and detect small signaling molecules to sense bacterial population densities and regulate the expression of speci c genes in response to environmental changes (10). The QS system allows bacteria to function as multicellular organisms because the concentration of extracellular self-inducers increases as the number of bacteria grows. After reaching a certain amount, this molecule diffuses back into the bacteria and regulates the transcription of different genes related to bio lm secretion and other (11). The QS system is important in order to bacterial bio lm formation.
Therefore, this QS system is a paramount target for the treatment of microbial bio lm associated infections (12). Once formed, bio lms protect bacteria from the action of traditional antibacterial drugs and exhibit multi-drug resistance, leading to the ineffectiveness of long-term antibiotic therapy (13). The bio lm also protects the bacteria from the immune response of the body and enables them to survive in a harsh environment (14). Therefore, the bio lm of S. aureus is considered an essential pathogenic feature.
Previous studies have found that live probiotic microorganisms are present in healthy milk, which considered to be the presence of self-or commensal microbiota. As one of the normal host microbiotas, Bacillus also is available in the mammary gland of cows (15). Different Bacillus strains showed antibacterial, antioxidant, and immunomodulatory activities in their hosts (16). Recently, probiotics such as bacillus have been utilized to prevent infection, because it is a nonpathogenic Gram-positive bacterium that can availably maintain a bene cial micro ora balance in the gastrointestinal tract of a mammalian host (17). B. subtilis-fermented fermentation products can promote the growth performance of immunestressed broilers and regulate the composition of intestinal micro ora (18). In addition, the exclusion of pathogens by inhibition of bacterial bio lms is another potential property of the proposed Bacillus strain (19). Evidence accumulated from animal and in vitro studies indicates that B. subtilis yields a variety of substances, such as surfaceins, iturins and fengycins, which may contribute to antibacterial, antiin ammatory, and immunomodulatory applications (17,18). Speci cally, a recent report showed that the secreted substance from B. subtilis abolished colonization with S. aureus by suppressing the production of the Arg-quorum-sensing signaling system (17). However, whether B. subtilis can reduce the colonization of mammary glands by S. aureus has not been reported.
Therefore, we hypothesized that there is interference between the mammary symbiotic bacterium Bacillus and the pathogenic bacterium S. aureus. Bacillus can reduce the colonization of S. aureus and thus relieve S. aureus-induced mastitis. Here, we found that Bacillus eliminated the colonization of the dangerous pathogen S. aureus in the mammary gland of cows. Further studies revealed that Bacillus disrupts the formation of bio lm and thus reduces the colonization of S. aureus probably by affecting the QS system of S. aureus. Using mice mastitis model, we also demonstrated that Bacillus ameliorated S. aureus-induced mastitis, as showed by improving mammary injury, limiting in ammatory markers, and promoting blood-milk barrier integrity. Collectively, our ndings that bacillus with the capacity to eliminate mammary gland pathogen colonization through disrupting bio lm formation protects against S. aureuseinduced mastitis in mice, suggests that probiotics that interfere with the formation of pathogenic bio lms may serve as a potential and effective st rategy to protect mastitis S. aureus-related disease.

Materials And Methods
Materials TNF-α and IL-1β ELISA kits were purchased from Biolegend (CA, USA). Tissue protein extract and BCA Protein Assay Kit were bought from Thermo (Thermo, MA, USA). Trypticase Soy Broth (TSB) and bacillus medium were purchased from Qingdao Haibo Biotechnology Co., Ltd (Qingdao, China). Crystal Violet Stain solution, 1% was acquired from Solarbio (Solarbio, Beijing, China). All the monoclonal antibodies, including β-actin, p65, IκB, the phosphorylation of p65, phosphorylation IκB, ZO-1, Occludin, and Claudin-3 were recruited from Cell Signaling Technology (Beverly, MA, USA). MPO kit was available from Nanjing Jiancheng Co., Ltd (Nanjing, China). Animals A total of 60 Balb/c mice (40 females and 20 males) aged 6-8 weeks were purchased from the Liaoning Changsheng Biotechnology Co. Ltd. The animal experiments were subject to approval by the Animal Ethics Committee of Jilin University (KT202103058). Females and males are mixed in miniature isolation cages in about a ratio of 2 to 1 after adapting to the environment with free food and water. The mastitis model was established complying with the experimental animal manual. This study is built on the Handbook on the Care and use of Experimental Animals published by the National Institutes of Health.
Bacteria and culture conditions Bacillus were isolated from healthy and mastitis milk samples from dairy cows in Baicheng, Jilin Province), ChiFeng, Inner Mongolia, and Weifang, Shandong Province, China. A total of 145 strains of Bacillus bacteria were separated and puri ed by the speci c culture of bacillus medium plate. S. aureus (ATCC 35556) was acquired from American Type Culture Collection. In the present study, S. aureus was inoculated for about 6 hours at the condition of 37 ℃ and 120 r/min, and the OD 600 was about 0.5 (concentration was approximately 10 8 CFU/mL). Meanwhile, B. subtilis bacteria were inoculated into the TSB broth medium by the same inoculation method, and then the OD 600 reached about 0.6 (concentration was approximately 10 8 CFU/mL) after 4 hours of growth.

Preparation of cell-free supernatant (CFS) from Bacillus culture and treatments
To prepare Bacillus CFS, Bacillus strains were cultured at 37 ℃ under shaking at 200 rpm overnight until the cultures reached an OD 600 of 0.6 ± 0.05. The CFS of bacterial culture was collected by centrifugation at 6000 g for 10 min, and then ltered through a 0.22 µm sterilizing-grade lter (Millipore, SLGV033RB, USA) to remove bacteria. To evaluate the effect of Bacillus CFS on S. aureus genes expression, overnight culture of S. aureus strains was collected by a centrifuge, washed with PBS, re-suspended at 10 8 CFU/mL in TSB/PBS (1:1 v/v, control) or TSB/B. subtilis CFS (1:1 v/v) and incubated in 6-well-plate at 37 ℃ for 3 h. Finally, bacteria were collected for RNA extraction and analysis of genes expression.
To determine the antibacterial effect of B. subtilis CFS on S. aureus, the Bacillus supernatant was obtained using the method described previously (18). Brie y, the supernatant of Bacillus bacteria was added into 96-well plates. 10 µL of S. aureus suspension (5 × 10 8 CFU/mL) from a fresh overnight culture was inoculated into 200 µL TBS (control), Bacillus CFS, TSB/ Bacillus CFS (1:1 v/v) or TSB/ Bacillus CFS (0.5:1.5 v/v) and incubated at 37 ℃ or 24 h. The growth of S. aureus was determined by monitoring OD 600 of the cell culture.

Bio lm formation and viability assay
To evaluate the effect of Bacillus CFS on S. aureus bio lm formation, 10 µL of S. aureus (5 × 10 8 CFU/mL) was added to 200 µL of TSB (control) or TSB/ Bacillus CFS (1:1 v/v) in each well on a 96-well plate and incubated at 37 ℃ for indicated time points without shaking. Next, the wells were washed three times with sterile PBS after the medium was removed. Finally, the plates were air-dried for 45 min and the adherent cells and matrix were stained with 1% crystal violet solution. To quantify the bio lm production, crystal violet was extracted by incubation in a solution (95% ethanol) at room temperature for 15 min, and absorbance was measured at 570 nm in a microplate reader.

RNA extraction and Quantitative real-time PCR (QRT-PCR)
Total RNA of S. aureus was extracted with a Bacterial RNA Extraction Kit (B518655-0050, Sangon Biotech, Shanghai, China) following the manufacturer's instructions. RNA purity was veri ed using a NanoDrop spectrophotometer (ND-1000, Nanodrop, USA). RNA was reversely transcribed using the 5× Prime Script RT Master Mix (RR036A, Takara, Shiga, Japan) according to the manufacturer's instructions. QRT-PCR was carried out using TB Green Premix Ex Taq II (RR820A, Takara, Shiga, Japan). Fold changes in level of chosing genes expression were determined using the 2 −ΔΔCt method.

Animal treatment and mastitis model
Totally forty female Balb/c mice 5-7 days after delivery were randomly divided into four groups: control group, S. aureus group (1 × 10 8 CFU per 100 µL PBS), B. subtilis H28 group (1 × 10 8 CFU per 100 µL PBS), B. subtilis H28 (1 × 10 8 CFU per 100 µL PBS) + S. aureus (1 × 10 8 CFU per 100 µL PBS) group. For induction of mastitis, S. aureus (1 × 10 8 CFU per 50 µL PBS) was an injected into each mammary gland of the mice using 100 µL syringes with a 30 gauge blunt needle. In the B. subtilis H28 group, the mammary gland of mice received an injection dose of intramammary with B. subtilis H28 (1 × 10 8 CFU per 100 µL PBS), later 2 hours S. aureus was an injected into each mammary gland. The control group mice received an injection dose of intramammary with an equal volume of sterile PBS. Twenty-four hours later, the mice were sacri ced and the mammary gland tissues were harvested and stored at −80°C for subsequent detection.
Histological analysis 24 hours after S. aureus infection, the mammary gland tissues of each group were collected and xed in 4% paraformaldehyde for 48 h. The sample was inserted into para n and cut into 4 µm sections. After depara nization, the sections were stained with hematoxylin and eosin (H&E), and histological analysis was conducted under an optical microscope. The main histopathological indicators are through hyperemia (grade 0-3, ranging from normal to severe, including normal, mild, moderate, and severe) and neutrophil in ltration (grade 0-5, grade 0, from nothing to transmural) to evaluate.
MPO activity assay MPO activity is a functional and activation marker of neutrophils. The mammary gland tissue was harvested and homogenized on ice with reaction buffer (weight/volume ratio 1:19). The detection method of MPO activity was carried out according to the manufacturer's instructions (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).

ELISA assay
The in ammatory cytokine was established using an ELISA kit, according to the manufacturer's instructions. 10% tissue homogenate was prepared and centrifuged at 4°C, 12000 r/min, for 10 min. The lipid layer was removed, and the middle supernatant was collected for detection. Use a microplate reader to test the read absorbance of the sample at 450 nm and 570 nm.

Mammary S. aureus load assay
To assess the S. aureus burden in the mammary gland, mammary tissues were aseptically obtained in mice and homogenized in 1 mL of PBS. A 10-fold dilution of the tissue homogenate was plated in mannitol high salt agar plates. Bacterial colonies were counted and calculated following plate incubation at 37 ℃ for 18 h. Results of bacterial burden were expressed on a log10 scale.

Immunohistochemistry (IHC)
The para n-embedded glass slides were dewaxed in xylene and different concentrations of alcohol. The antigen is restored with 0.01 M citrate buffer. Incubate in 3% H 2 O 2 and then in diluted goat serum. The sections were incubated with primary antibodies: Claudin 3 (1:500) at 4°C. After washing 3 times, the sections were incubated with HRP-labeled goats with anti-rabbit secondary antibody (1:500, ZS-Bio, Beijing, China) at 37°C for 15 min. This section was stained with DAB and observed under a microscope.

Statistical analysis
GraphPad Prism 5 (Manufacturer, La Jolla, CA, USA) was performed for statistical analysis. The data are presented as mean ± SEM. p < 0.05 indicated statistical signi cance by one-way ANOVA and Tukey's multiple comparisons test.

Results
Increased S. aureus colonization contributes to cow mastitis and is associated with reduced commensal Bacillus.
To investigate the correlation between S. aureus and Bacillus colonization in the mammary gland, we performed the culture-based analysis of milk samples with swabs taken from healthy non-mastitis cows and mastitis cows. The absolute abundance of total S. aureus and Bacillus on milk swabs was evaluated by manual colony counting for colony-forming units (CFUs) on a selective egg yolk mannitol salt agar and Bacillus culture medium respectively. As showed in Fig. 1A, we can clearly observe substantial differences in the quantity of S. aureus between health and mastitis cows that more colonization of S. aureus in mastitis cows. Meantime, we found that healthy cow had more Bacillus colonization than mastitis cows (Fig. 1B). Previous studies have found that laboratory isolates of Bacillus species can reduce the colonization of S. aureus (19). One explanation for the larger amount of S. aureus from mastitis milk compared to normal milk was that Bacillus from normal milk may affect colonization of S. aureus. Therefore, we next sought to further determine whether was a correlation between Bacillus and S. aureus. We started raising a striking correlation between the presence of Bacillus bacteria and the absence of S. aureus. We rst assessed the effect of the presence or absence of S. aureus colonization on the number of Bacillus. The results showed in that the number of Bacillus decreased in the presence of S. aureus colonization in both health and mastitis cows (Fig. 1C). We then analyzed the effect of Bacillus on S. aureus colonization and found that lower load of S. aureus was detected in healthy and mastitis cows when Bacillus was colonized (Fig. 1D). These results are mentioned that there is a negative correlation between S. aureus and Bacillus in the milk.
Commensal Bacillus inhibits S. aureus colonization through affecting the bio lm formation of S. aureus.
It has been declared in the literature that Bacillus can inhibit the population sensing system of S. aureus, thus reducing the colonization of the intestinal tract by S. aureus (18). We therefore hypothesize that Bacillus may exert a broad mechanism for comprehensively inhibit S. aureus colonization in the mammary gland. In the results of the ow disease survey, it was shown that the presence of Bacillus in the mammary gland could be a possibility determining factor for the absence of S. aureus in cows. We rst analyzed whether there is a growth-inhibitory effect of the Bacillus isolates on S. aureus. To detect antimicrobial growth effect, we randomly isolated about 145 individual Bacillus isolates from the milk samples and then conducted an unbiased analysis of antimicrobial activity by determining the capacity of the sterile conditioned supernatant of each individual isolate to inhibit S. aureus growth. However, we found no difference in the antibacterial effect between S. aureus-negative and -positive Bacillus ( Fig. 2A). Therefore, a growth-inhibitory effect not is able to explain the observed complete correlation between the presence of Bacillus and the absence of S. aureus, and rules out a bacteriocin-mediated phenomenon. The factors that are important in order to S. aureus mammary colonization are poorly understood. A previous study has implicated that quorum-sensing (QS) is a requirement for S. aureus to colonize the intestine, and discovering that secreted Bacillus function as QS blockers to achieve complete eradication of intestinal S. aureus (19). We then examined the anti-S. aureus bio lm capacity of S. aureus-negative and -positive Bacillus and found that S. aureus-negative Bacillus had higher anti-S. aureus bio lm capacity than S. aureus-positive Bacillus (Fig. 2B). We next determined the ability of bio lm formation between health and mastitis cow isolated S. aureus and showed that S. aureus from mastitis cow had higher bio lm formation capacity than S. aureus from non-mastitis. Collectively, these results suggest that Bacillus inhibits S. aureus colonization by affecting the bio lm formation of S. aureus.

Bacillus inhibits S. aureus bio lm formation by regulating QS signaling.
To detect anti-bio lm activity, we randomly isolated about 145 individual Bacillus isolates from each milk culture swab and then conducted an unbiased analysis of anti-bio lml activity by measuring the capacity of the sterile conditioned supernatant of each individual isolate to inhibit S. aureus bio lm ( Fig. 2A). The showed that the anti-bio lm effect of different Bacillus species varied signi cantly, and we performed the strain with the best anti-bio lm effect for an intensive investigation which was identi ed by 16S RNA sequencing and was named as Bacillus subtilis H28 (B. subtilis H28) (Fig. 3B). As shown in Fig. 3C, B. subtilis H28 strain did not produce any inhibition loop on agar. Moreover, bio lm formation in static S. aureus culture was evaluated by crystal violet staining. Results showed much faint staining in the culture of B. subtilis H28 treated S. aureus (Fig. 3D), which was signi cantly different from the group with the addition of S. aureus alone indicating that bacillus has an inhibitory effect on bio lm production of S. aureus. Moreover, we counted the S. aureus in the bio lm and showed that B. subtilis H28 signi cantly reduced the number of S. aureus in the bio lm (Fig. 3F).
To con rm whether the B. subtilis H28 supernatant had antibacterial activity, 5 × 10 8 CFUs of S. aureus were grown for 48 h in 25%, 50 , and 100 supernatant of B. subtilis H28 in polystyrene plates for 2 days at 37°C. As shown in Fig. 4A, different concentrations of B. subtilis H28 supernatant had no effect on bacterial growth. To test for the effects of B. subtilis H28 on bio lm formation in vitro, S. aureus was treated as similarly and bio lm was stained with crystal violet and determined at OD 570 nm. We found that B. subtilis H28 reduced cell attachment and biomass in a concentration-dependent manner (Fig. 4A).
To test whether B. subtilis H28 has a disruptive effect on bio lms already formed in vitro, 5 × 10 8 S. aureus cells were incubated in polystyrene plates at 37°C for 2 days. Afterwards, 25%, 50%, and 100% B. subtilis H28 supernatants were added and incubation was continued for 24 h. We demonstrated that B. subtilis H28 inhibited the bio lm already formed in a dose-dependent manner (Fig. 4B). However, a noninhibitory bacillus cannot destroy the bio lm formed by S. aureus (Fig. 4B).
Bio lm formation involves the expression and regulation of multiple genes (20). The QS system controls S. aureus bio lm formation and release of virulence factors (21). Researchers have indicated that the accessory gene regulator (Agr) system regulates the QS system of S. aureus (22). S. aureus secretes the polysaccharide intercellular adhesion (PIA) is a factor necessary for the bacterial aggregation phase of its bio lm formation, and PIA synthesis is mainly encoded by the ica manipulator (23,24). The RNAIII activating peptide is considered as an auto-inducible peptide that phosphocreeatine its target molecules, activates the Agr system, and regulates bio lm formation (25,26). We then analyzed the effect of B. subtilis H28 on the expression of genes involved in population sensing (ArgA and RNA III) and bio lm formation gene ica. We showed that B. subtilis H28 treatment signi cantly down-regulated the mRNA expression of all the above genes ( Fig. 4C-E). Taken together, these results indicate that Bacillus inhibited the bio lm formation of S. aureus by regulating the QS system.

B. subtilis H28 protects against S. aureus induced mastitis in mice
We then investigated whether B. subtilis H28 could ameliorate S. aureus induced mastitis. The results showed that S. aureus infection resulted in observable pathological damage of the mammary gland, including edema, in ammatory cell in ltration, and disarrangement of the mammary gland structure (Fig.  5A-B). However, pre-treatment with B. subtilis H28 signi cantly alleviated these pathological damages of the mammary gland induced by S. aureus (Fig. 5A-B). The mammary gland enumeration of bacterial burdens revealed that S. aureus mice harbored higher bacterial burdens, while B. subtilis H28 treatment did substantially reduce bacterial burdens in the mammary gland (Fig. 5C). S. aureus treated mice had higher MPO activity than control mice, while it was reduced by B. subtilis H28, and administration of B. subtilis H28 alone did not affect the MPO activity when compared with the control group (Fig. 5D). In addition, B. subtilis H28 inhibited the production of TNF-α and IL-1β, the major pro-in ammatory cytokines in mastitis, induced by S. aureus (Fig. 5E-F).

B. subtilis H28 improves the blood-milk integrity by increasing expressions of the tight junction proteins
To assess the effect of B. subtilis H28 on S. aureus-induced damage of the epithelial barrier integrity, we examined the expression of ZO-1, Occludin, and Claudin-3 by western blotting. Following S. aureus infection, the mammary gland markedly reduced the expression of ZO-1, Occludin, and Claudin-3 when compared to the mammary gland without S. aureus stimulation (Fig. 6A-C). However, B. subtilis H28 treatment increased the levels of ZO-1, Occludin, and Claudin-3 compared to S. aureus-infected mice ( Fig.  6A-C). Furthermore, we con rmed Claudin-3 level of immunochemistry and showed that B. subtilis H28 increased the Claudin-3 expression (Fig. 6D). To determine whether B. subtilis H28 inhibits the QS system and virulence, we measured the transcriptional level of AgrA, RNAIII, SarA through RTQ-PCR. We found that B. subtilis H28 could down-regulate the expression AgrA, RNAIII, and SarA as compared with that of the control group (Fig. 7A-C). Together, these results suggest that B. subtilis H28 inhibits S. aureus colonization and alleviates S. aureus-induced barrier damage by improving tight junctions.

B. subtilis H28 inhibited S. aureus-induced in ammatory response in mouse mammary epithelial cells
To verify the anti-in ammatory effect of B. subtilis H28, the mouse mammary epithelial cells were pretreated with B. subtilis H28 then stimulated with S. aureus. The activation of the NF-κB signaling pathway is responsible for the release of pro-in ammatory cytokines, such as TNF-α and IL-1β. Chemokines are a class of speci c small-molecule proteins that play critical roles in the recruitment and activation of leukocytes (27). CXCL3 controls the migration and adhesion of monocytes by interacting with a cell surface chemokine receptor called CXCR2, which affects the secretion of cytokines (28). We found that S. aureus increased TNF-α, IL-1 β, IL-6 and CXCL3 mRNA levels (Fig. 8A-D) but reversed by B. subtilis H28 treatment in a dose-dependent manner (Fig. 8A-D). Moreover, we found that B. subtilis H28 reduced the expression ica, RNAIII, and SarA compared to S. aureus treatment in a dose-dependent manner (Fig. 8E-F).

B. subtilis H28 limites the activation of NF-κB and increases tight junction protein expression in MMECs
In order to study the effect of B. subtilis H28 on the integrity of the mammary gland epithelial cell induced by S. aureus, we found that S. aureus reduced the expression of Claudin-3, ZO-1, and Occludin. However, treatment with B. subtilis H28 increased the levels of Claudin 3, ZO-1 and Occludin in a dose-dependent manner (Fig. 9A-C). This is compatible with the results of in vitro experiments. To test whether the antiin ammatory properties of B. subtilis H28 resulted from the regulation of the activation of the NF-κB, a predominant signaling pathway in S. aureus induced mastitis and associated with barrier injury, we assessed the levels of phosphorylation (p-) levels of p65 and IκB by western blotting and showed that the levels of p-p65 and p-IκB were signi cantly increased in the S. aureus group, while B. subtilis H28 treatment markedly inhibited the activation of the NF-κB signaling pathway in a dose-dependent manner ( Fig. 9A and E-F). Collectively, these results suggest that B. subtilis H28 alleviates S. aureus-induced mastitis by improving tight junction protein expression and limiting NF-κB pathway activation.

Discussion
Mastitis constitutes one of the aggressive diseases that affecting the development of dairy farming. S. aureus is the most frequent and important pathogen causing mastitis in dairy cows (29). Bacillus as one of the normal host-microbiota exists in the mammary gland of cows (30). But the functional Bacillus and their role in S. aureus-induced mastitis pathogenesis remain poorly de ned. The aim of our study was to elucidate the distinctive contribution of Bacillus in S. aureus mastitis. In studies, we found that there is interference between the mammary symbiotic bacterium Bacillus and the pathogenic bacterium S. aureus. Bacillus can down-regulate the gene expression of the QS system of S. aureus to inhibit the bio lm formation of S. aureus and thus reduce the amount of colonization of S. aureus. In treatment with Bacillus in mice mastitis, the result showed that preparations of the Bacillus were as effective as commonly used antibiotics relieving S. aureus-induced mastitis for the treatment of intramammary infections and did not show adverse effects on mammary tissue.
Probiotic nutrition is frequently claimed to be improve health. Probiotic bacteria obtained with food are thought to decrease colonization by pathogens, and thus reduce susceptibility to infection (31)(32)(33). Some probiotic strains produce bacteriocin proteins, which can kill phylogenetically related pathogenic bacteria (31). It is particularly noteworthy human data indicate that probiotic Bacillus can comprehensively eradicate intestinal as well as nasal S. aureus colonization (18). Epidemiological studies have established that there is interference between the mammary symbiotic bacterium Bacillus and the pathogenic bacterium S. aureus. Bacillus can reduce the colonization of mammary glands by S. aureus in dairy cows. it was shown that the presence of Bacillus in the gland mammary could be a potential determining factor for the absence of S. aureus in cows.
Several studies have reported that in addition to the potential probiotic properties of Bacillus strains, the Bacillus exhibited strong anti-cholesterol, anti-bio lm, and antioxidant properties, making the strain with additional functional abilities (34). B. subtilis exerts an antimicrobial effect against a broad spectrum of pathogens through direct bactericidal activity or indirect enhancement of immune response, such as interrupting quorum-sensing regulatory system by production of fengycins (18), inhibiting S. aureus adhesion and bio lm formation by production of surfactant (35), and the enhancing anti-microbial function of macrophage (36). We study has con rmed a potent inhibitory capacity of B. subtilis H28 against both planktonic and bio lm S. aureus in vitro, which may prominently suppress the expression of genes associated with S. aureus adhesion, bio lm formation.
QS plays an essential role in bio lm formation, in the production of virulence factors and antimicrobial resistance (37). There is evidence that quorum sensing in S. aureus is important for the construction and dissolution of bio lm communities (38). The QS system controls S. aureus bio lm formation and release of virulence factors (21,39). Researchers believe that the accessory gene regulator (agr) system regulates the QS in S. aureus (40). In fact, agr plays a pivotal role in regulating virulence factor expression (41), making it a potential therapeutic target (42). Bio lm formation is believed to require the adhesion of cells to a solid substrate, which creates multiple layers of cells. Intercellular adhesion requires PIA, which can be synthesized by-products of the intercellular adhesion (ica) locus (23,24). The RNAIII activating peptide is thought to be a type of auto-inducing peptide (25,26) that can phosphorylate its target molecule to activate the agr system, which increases the production of auto inducing peptides and AgrC to enhance the adhesion of the bacteria [36,37]. Recent ndings indicate that RNAIII is a regulatory mRNA molecule that not only regulates bio lm formation but also induces toxin production, such as enterotoxin, plasmacoagulase, hemolysin, and thermostable nuclease(26, 43). In addition, it is a fellow of the staphylococcal accessory regulator A (SarA) family of transcriptional regulators (44). Recent evidence indicates that SarA, a central regulatory element that controls the production of S. aureus virulence factors, is essential for the synthesis of polysaccharide intercellular adhesin (PIA) (45). We have shown that B. subtilis H28 could inhibit bio lm formation by S. aureus phenotypically. To determine whether B. subtilis H28 inhibits the QS system and virulence, we analyzed the effects of B. subtilis H28 on the expression of S. aureus genes involved in quorum sensing (AgrA and RNAIII) and bio lm formation (Ica and SarA). We found that B. subtilis H28 could down-regulate the expression of genes agrA, RNAIII, ica, and sarA associated with the QS system. B. subtilis H28 effectively inhibited the QS system of S. aureus in a dose-dependent manner, resulting in inhibition of bio lm formation. B. subtilis H28 might be considered a QS inhibitor because of its ability to block the cell-to-cell signal transduction that is regulated by the QS system, and thus inhibiting expression of QS-related genes. The speci c mechanism by which B. subtilis H28 inhibits the QS system requires further study and discussion, but the net outcome of QS inhibition is inhibition of bio lm formation. Therefore, we believe B. subtilis H28 is potential novel treatment against S. aureus bio lm-related infections.
Therefore, we further investigated in the next study whether B. subtilis H28 can have a protective effect against mastitis caused by S. aureus. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis (46). We took the same mammary gland infusion and an important nding in this study is that the intramammary infusion of B. subtilis H28 can signi cantly reduce S. aureus colonization to alleviate S. aureus-induced mastitis.
Our study suggests valuable translational applications regarding alternative strategies to combat antibiotic-resistant S. aureus. Bacillus-containing probiotics could be used in order to simple and safe S. aureus decolonization strategies. In that regard, it is particularly noteworthy that our data indicate that probiotic Bacillus can eradicate mammary S. aureus colonization. Such a probiotic approach would have numerous advantages over the present standard topical strategy involving antibiotics. Bacillus provides a reasonable reference for the treatment of in ammatory diseases. Therefore, our study provided support for the probiotic effect of Bacillus and Bacillus may have the potential as a promising candidate for the treatment of mastitis.
At the same time, the study has a few limitations. The speci c mechanisms responsible for the inhibition of the QS system by B. subtilis H28 need to be further investigated and discussed. In addition to this, it was observed Bacillus can reduce the colonization of mammary glands by S. aureus in cows and was con rmed in mice. However, all the experiments evaluating the effects of this defect were carried out in mice, which can be seen as a potential limitation. Further interventional studies in cows should be made to evaluate the proposed therapeutic strategy. Our future research will focus on the cows in vivo to identify the precise mechanism by which B. subtilis H28 inhibits S. aureus bio lm formation.

Conclusions
Collectively, our results indicate that commensal Bacillus inhibits S. aureus colonization and alleviates S. aureus-induced mastitis by in uencing bio lm formation, which suggests a potential strategy for the decolonization of S. aureus and acts as a basis for the prevention and treatment of S. aureus-related disease.

Declarations Author contributions
Min Qiu performed the article writing and result evaluation. Caijin Zhao performed histologic analysis and article revision. Xiaoyu Hu performed the nal revision of the article and provided expert opinion. Lianjun Feng, Siyuan Gao contributed to animal experiment. Naisheng Zhang and Yunhe Fu contributed to the study design.

Con icts of interest
The authors declare that they have no con icts of interest.

Funding
The present study was supported by the National Natural Science Foundation of China (31972749 and 31772812) and the China Postdoctoral Science Foundation (2020TQ0120 and 2020M681045). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. mastitis cows and mastitis cows. One-way analysis of variance was used for statistical analysis. *p < 0.05 and *p < 0.01 and ***p 0.001 indicate signi cant differences from each group. ns, no signi cance.  The values presented are the mean ± SEM (n=4). *p < 0.05 and *p < 0.01 and ***p 0.001 indicate signi cant differences from each group. ns, no signi cance.    (C) Relative expression of the Agr A was identi ed using qPCR. The values presented are the mean ± SEM(n=3). One-way analysis of variance was used for statistical analysis. *p < 0.05 and *p < 0.01 and ***p 0.001 indicate signi cant differences from each group. ns, no signi cance.  One-way analysis of variance was used for statistical analysis. *p < 0.05 and *p < 0.01 and ***p 0.001. ns, no signi cance.