| Background:Influenza A virus(IAV)is a respiratory pathogen that is transmitted by droplet or contact.IAV is prone to antigenic drift and antigenic transformation,resulting in seasonal pandemics.Secondary bacterial infections post IAV infection leads to severe pneumonia and even death.With the increasing use of antibiotics worldwide,the proportion of community and clinically-acquired methicillin-resistant Staphylococcus aureus(MRSA)is increasing.MRSA exhibits varying degrees of resistance to all types of antibiotics except vancomycin.IAV-MRSA co-infection has become the main cause of severe pneumonia and death in influenza patients.The pathological mechanism of IAV-MRSA co-infection is a current research hotspot.Researchers have conducted extensive research on the interaction between IAV and host immunity,the virulence and invasiveness of IAV and MRSA,and the host tissue immune tolerance.In recent years,with the development of sequencing technology and bioinformatics,researchers have discovered that the host microbiome has an important impact on host physiology,metabolic regulation and immune function,as well as complex host behaviors.The disruption of the host microbiota caused by IAV infection is closely related to the severity of secondary bacterial co-infection pneumonia.However,current research mainly analyzes the overall microbiota of the respiratory or intestinal tract,and lacks research on the key species that play a role in the microbiota,especially the study of the interaction mechanism between the key species and the host.Mucosal immunity is the first line of defense against pathogen infection.Immunoglobulin A(Ig A)as an important antibody of mucosal immunity is closely related to the host microbiota.Previous studies have found that probiotic intervention can increase the content of host intestinal or respiratory Ig A,resist pathogen infection by binding with pathogens,enhancing the colonization ability of commensal bacteria,and regulating bacterial metabolism.Therefore,this study analyzed the microbiota characteristics of IAV-MRSA co-infected pneumonia mice and screened out key species,explored the effects of key species intervention on co-infected pneumonia and related mucosal immune mechanisms,and provided a reference for the development of probiotic therapy for infectious diseases.Objectives:(1)Analysis of the respiratory flora,immune response and plasma metabolic profile of IAV-MRSA co-infected mice,and explore the key host bacteria closely related to IAV-MRSA co-infection.(2)Validate the therapeutic effects of key bacteria on IAV-MRSA co-infected mice and explore the related mechanismsMethods:(1)A non-lethal IAV-MRSA co-infection mouse model was established by simultaneously infecting mice with IAV(strain A/Puerto Rico/8/34)and low-dose MRSA.Real-time quantitative PCR was used to detect the pathogen load of IAV and MRSA in nasal and bronchoalveolar lavage fluid(BALF)of mice at 4 and 13 days post infection(dpi).Hematoxylin and eosin(HE)staining was used to assess pathological lung damage at 4 dpi.Full-length 16 S r RNA gene sequencing was used to determine the distribution characteristics of nasal and BALF microbiota at 4 and 13 dpi.Flow cytometry and ELISA were used to detect changes in lung dendritic cell cells,macrophages,natural killer(NK)cells,B cells,regulatory T(Treg)cells,γδT cells,CD4+/CD8+ T cells,C-C motif chemokine ligand 2(CCL-2),interleukin-6,8,9(IL-6,IL-8,and IL-9),interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α)at 4 dpi.Splenic B cell and CD4+/CD8+ T cell levels were also measured.Liquid chromatography-mass spectrometry was used to detect changes in plasma metabolites at4 dpi.Finally,the correlations between lower respiratory tract microbiota,immune cells,plasma metabolites were used to identify key species negatively correlated with co-infection-induced host immune response and metabolic disorder.(2)The characterization of the microbiota in the nasal and oral pharynx,BALF,and feces of the mice infected with IAV on day 4 was analyzed and compared using full-length 16 Sr RNA gene sequencing.The correlation between microbiota of different sites was analyzed by Spearman analysis.The changes of key species in different sites of IAV-infected mice were analyzed by Kruskal-Wallis test.(3)A mouse model of lethal pneumonia with IAV-MRSA co-infection was constructed by secondary MRSA infection on the 4th day after IAV infection.Based on the screened key species,the IAV-MRSA co-infected mouse model of lethal pneumonia was intervened by dropping and drinking water with the key species for 4 days before MRSA infection after IAV infection.The mortality rate of mice was observed,and the BALF pathogen load of IAV and MRSA in the mice at 3 dpi of IAV-MRSA co-infection was detected using real-time quantitative PCR.The pathological damage of lung tissue was visualized using HE staining to evaluate the intervention effect of key species on IAV-MRSA co-infected mice.To further study the intervention mechanism of key species,the pathogen load of IAV in BALF before MRSA infection after key species intervention was detected using real-time quantitative PCR,and the characteristics of microbiota in BALF and feces were analyzed using full-length 16 S r RNA gene sequencing.The levels of BALF secretory Ig A,BALF H1N1-reactive Ig A,serum microbiota-reactive Ig A,lung Ig A+ cells,lung Ig A+ plasma cells,lung Treg cells,lung Th17 cells,lung interleukin-21(IL-21),lung tumor necrosis factor β(TNF-β),lung B-cell activating factor(BAFF),and lung proliferation-inducing ligand(APRIL)were detected using flow cytometry and ELISA methods to evaluate the effect of key species on T cell-dependent(TD)Ig A response and T cell-independent(TI)Ig A response.A model of gut microbiota depletion was established by adding mixed antibiotics to the drinking water of mice for two weeks.Flow cytometry and ELISA were used to measure the levels of BALF secretory Ig A,serum microbial-reactive Ig A,lung Ig A+cells,lung Ig A+ plasma cells,lung Treg cells,lung Th17 cells,lung IL-21,lung TNF-β,lung BAFF and lung APRIL to evaluate the impact of gut microbiota depletion on Ig A response.Results:(1)This study successfully established a non-lethal pneumonia model of IAV-MRSA co-infection in mice.The results showed that co-infection led to significant weight loss(P<0.01)and lung tissue damage(P<0.05)in mice at 4 dpi.IAV(P<0.001)and MRSA(P<0.001)loads were significantly increased in BALF from IAV-MRS co-infected mice compared with IAV or MRSA infected alone at 4 dpi.The full-length16 S r RNA gene sequencing results showed that IAV-MRSA co-infection caused long-term disruption of the nasopharyngeal and lung microbiota in mice,especially a significant decreased in the relative abundance of Lactobacillus murinus in BALF at both 4 dpi and 13 dpi.For innate immunity,co-infected mice showed immune characteristics of IAV and MRSA infections,including decreased levels of lung macrophages and NK cells and increased levels of γδT cells,but the changes were not significant.For adaptive immunity,co-infected mice showed immune characteristics similar to those of IAV infection,characterized by significant increases in CD4+/CD8+T cells and B cells in spleen(P<0.05)and elevated levels of cytokines IL-9,IFN-γ,TNF-α,IL-6,and IL-8(P<0.05)in lung tissue.Additionally,liquid chromatography mass spectrometry analysis of plasma metabolites in different infection groups revealed that the metabolic profiles of IAV infected mice and IAV-MRSA co-infected mice were similar.The representative metabolite of IAV-MRSA co-infected mice was Methylpentenolone(upregulated),while those of MRSA infected mice and IAV infected mice were Tyrosylglutamine(upregulated)and Lyso PC(downregulated).Correlation analysis of the lower respiratory tract microbiota,host immune cells,and plasma metabolites showed that Lactobacillus murinus is a key species and positively correlated with lung macrophages and NK cells,negatively correlated with spleen B cells and CD4+/CD8+ T cells and strongly correlated with multiple plasma metabolites,which may be a potential target for alleviating IAV-MRSA co-infection.(2)In this study,the results of full-length 16 S r RNA gene sequencing showed that Lactobacillus murinus dominated the oral-pharyngeal,nasal-pharyngeal,lung,and intestinal microbiota in normal mice,while IAV infection altered the composition of the microbial community in the upper and lower respiratory tract and the intestine,leading to a reduction in the relative abundance of Lactobacillus murinus in all sites.In addition,in normal mice,the similarity between the lung and nasal-pharyngeal microbiota was higher than that between the lung and oral-pharyngeal microbiota,while IAV infection increased the similarity between the lung and nasal-pharyngeal microbiota and decreased the similarity between the lung and oral-pharyngeal microbiota.These results indicate that IAV infection increases the influence of the nasal-pharyngeal microbiota on the lung microbiota,making it easier for pathogens to reach the lungs from the nasal-pharyngeal region.(3)This study successfully established a lethal pneumonia model of IAV-MRSA co-infection in mice,with a 100% mortality within seven days.The non-lethal co-infection models suggested that Lactobacillus murinus plays an important role in IAV-MRSA co-infection pneumonia.IAV infection also reduced the relative abundance of Lactobacillus murinus in the respiratory and intestinal tracts of mice.After infecting mice with IAV,we intervened with Lactobacillus murinus for 4 days through nasal drops and drinking water,and stopped the intervention when secondary MRSA infection occurred.The results showed that Lactobacillus murinus intervention significantly reduced the mortality(P<0.01),and lung IAV(P<0.001)and MRSA(P<0.01)load in IAV-MRSA co-infected mice.Meanwhile,full-length 16 Sr RNA gene sequencing showed that Lactobacillus murinus intervention increased its absolute abundance and restored the disruption of the intestinal microbiota caused by IAV infection,but had no significant effect on the lung microbiota.Flow cytometry and ELISA revealed that Lactobacillus murinus intervention significantly increased the level of BALF secretory Ig A,serum microbe-reactive Ig A,lung Ig A+ plasma cells,and lung APRIL(P<0.05),but had no significant effect on Treg cells,Th17 cells,IL-21,and TNF-β levels in lung.These results suggested that Lactobacillus murinus could increase the BALF secretory Ig A,serum microbe-reactive Ig A,and lung Ig A+ plasma cells though enhancing TI-Ig A response.To further verify the role of the gut microbiota in host Ig A response,we used long-term antibiotic water to successfully construct a gut microbiota-depleted mouse model,which suggested that gut microbiota depletion mice may reduce the BALF secretory Ig A and serum Ig A content by inhibiting the lung APRIL level(P<0.05).Conclusion:Based on the IAV-MRSA co-infection non-lethal models,combined with flow cytometry and full-length 16 Sr RNA gene sequencing technology,we identified a key bacterial strain,Lactobacillus murinus,which is closely related to the host innate immune cells and IAV-MRSA co-infection.Then,we found that Lactobacillus murinus predominates in the oropharynx,nasopharynx,lungs,and intestines of normal mice,while IAV infection reduces the relative abundance of Lactobacillus murinus in all locations.Therefore,we hypothesized that supplementation with Lactobacillus murinus can reduce the mortality rate of IAV-MRSA co-infected lethal pneumonia model.Finally,we used nasal drip and drinking water to intervene with Lactobacillus murinus,we found that Lactobacillus murinus can regulate the dysbiosis of the gut microbiota caused by IAV infection and reduce the mortality rate of IAV-MRSA co-infected mice by increasing the levels of lung secretory Ig A and serum microbiota-reactive Ig A though enhancing the TI-Ig A response.These results not only help to better understand the role of the host microbiota in respiratory infections,but also provide important references for the development of probiotic therapies of respiratory infectious diseases. |
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