| Background:Severe infection are common clinical symptom,if not treated promptly and effectively,it can cause serious complications.Antibiotics are the main treatment for infection for now,but the long-term large amount of antibiotic can lead to changes in the structure of the intestinal flora and can induce the emergence of drug-resistant bacteria and an increased risk of secondary infection,leading to a series of chain reactions that increase the difficulty of clinical treatment.Some studies have shown that ESBLs-producing gramnegative bacteria are the most predominant group of multi-drug resistant pathogenic bacteria in hospitals,while ESBLs-producing Escherichia coli and ESBLs-producing Klebsiella pneumoniae are representative groups of ESBLs-producing bacteria.After the long-term application of a large number of broad-spectrum antibiotics,the sensitive strains in the normal flora of the host are suppressed,while the few resistant bacteria originally at a disadvantage or from the external environment take advantage of the opportunity to colonize and multiply.This kind of colonization and mass reproduction that lead to disease caused by new infection due to imbalance of flora in the process of antibiotic treatment of original infectious diseases is called secondary infection.Escherichia coli is a conditional pathogenic bacteria settling in the intestinal tract,and ESBLs-producing Escherichia coli is one of the most common multi-drug resistant bacteria,and the secondary infections caused by them are more common in clinical practice,and new treatment methods,other than antibiotics,need to be found.Enteric probiotics are receiving increasing attention because of their important role in regulating intestinal flora and intestinal health.Intestinal probiotics refer to single microorganisms or well-defined mixed microorganisms that can colonize,inhibit the proliferation of harmful bacteria in the intestine,promote intestinal movement,improve intestinal function,and thus produce beneficial health effects.The main mechanisms by which intestinal probiotics exert their therapeutic effects include competitive rejection of pathogenic microorganisms,production of antimicrobial substances such as defensins,and enhancement of the intestinal epithelial barrier function and mucosal immune system.Bacillus,the most important group of probiotics,can inhibit the colonization and growth of pathogenic bacteria by releasing bacteriocins and reducing the ability of pathogens to adhere to epithelial cells in the intestine.Bacillus has also been found to produce cyclic lipopeptides,which have a variety of activities,including interactions with biofilms and antifungal,anti-inflammatory,antitumor,and antiviral properties.In addition,Bacillus can regulate the redox state of the intestinal environment through its metal ion chelating ability,antioxidant enzymes,regulatory signaling pathways and intestinal flora,thus inhibiting the growth and activity of intestinal pathogenic bacteria.Bacillus licheniformis,as a kind of Bacillus,has been widely used,and its live bacterial preparation,neurontin,can be used to relieve diarrhea and other intestinal inflammatory diseases.Studies have shown that Bacillus licheniformis can compete for oxygen consumption in the intestine,facilitating the growth of anaerobic bacteria in vivo and regulating the structural composition of the intestinal flora.Based on this,we hypothesized that B.licheniformis could alleviate the secondary infection caused by ESBLs-producing Escherichia coli and designed an experiment to test it.Objective: To explore whether Bacillus licheniformis,as a probiotic preparation,can alleviate secondary infections caused by ESBLs-producing Escherichia coli and to try to clarify its therapeutic mechanism.Methods: 1.Establish the model of secondary infection in mice induced by antibiotics(cefuroxime combined with levofloxacin)and drug-resistant bacteria(ESBLs producing Escherichia coli),and treatment of B.licheniformis was performed under this model and divided into three treatment groups according to the dose of B.licheniformis.2.Bacterial16 s r RNA high-throughput sequencing was used to analyze the changes of intestinal microbial community diversity and composition of mice in each treatment group,with emphasis on the abundance of Enterobacteriaceae.3.Mice were observed for mental,activity,hair and other physiological status.4.Histopathological sections were observed for intestinal pathological changes and inflammatory infiltration.real-time q PCR and Western blot(WB)methods were used to detect changes in intestinal tight junction protein expression and assess changes in intestinal permeability.5.Real-time q PCR,WB and ELISA methods were used to detect intestinal function related indicators,including changes in the expression of s Ig A,mucin,REG protein and transporter protein,and to assess the intestinal function in each treatment group.6.Real-time q PCR method was used to detect the changes of m RNA expression levels of inflammatory factors in the intestine of each treatment group,including IL-1β,IL-6,IL-12 and TNF-α.The levels of PCT,CRP and LPS in the intestine were detected by ELISA to assess the severity of inflammatory response.7.The changes of antioxidant enzymes SOD and GSH-PX activities in the intestinal tissues of mice in each treatment group were detected by commercial kits.Results: 1.In the mouse secondary infection model induced successfully by antibiotics and drug-resistant bacteria,the intestinal flora species diversity was significantly reduced to about 1/2 of the control group,and the relative abundance of Enterobacteriaceae was high,and intestinal flora disorders appeared.2.After 10 days of treatment with Bacillus licheniformis,there was no significant difference in intestinal flora species diversity between the treatment group and the model group,both of which were significantly reduced compared with the control group.However,the relative abundance of Enterobacteriaceae in the treatment group was reduced compared to the model group(p<0.05),implying that the growth and reproduction of drug-resistant bacteria were inhibited to some extent.The relative abundance of Akkermansiaceae,which has important probiotic effects on intestinal immunity and metabolism,increased compared to the model group(p<0.05).After 30 days of treatment with Bacillus licheniformis,the intestinal flora diversity of both model and treated mice was restored to the level of the control group.The abundance of Enterobacteriaceae decreased in all groups,but their percentage in the model group remained significantly higher than in the treatment group(p<0.05).And the abundance of Akkermansiaceae was higher in the treated group(p<0.05).3.At day 10,the body weight of mice in the model group decreased significantly(p<0.01)compared with the control group,while the body weight of mice in the high-dose Bacillus licheniformistreated group increased significantly(p<0.01)compared with the model group.At day 30,the body weight of mice in the model group was still significantly lower than that of the control group(p<0.05),while the body weight of mice in all three treatment groups was significantly higher than that of the model group(p<0.05).4.The results of histopathological sections showed that the mice in the model group showed significant inflammatory infiltration in the intestine,while the B.licheniformis treatment group showed less inflammatory infiltration than that of the model group.Real-time q PCR results showed that on day 10 and day 30,the mice in the model group showed a significant increase in body weight compared with the control group.The expression of intestinal tight junction proteins ZO-1,Occludin and Claudin-1 was significantly down-regulated at the gene level in the model group compared with the control group on day 10 and day 30(p<0.0001),and significantly increased in the treatment group compared with the model group(p<0.0001).WB results indicated that the trends of the above indicators at the protein level were consistent with the changes at the gene level.5.At both assay time points,the m RNA levels of mucin2 and mucin3 and the transport proteins SLC26A3,SLC5A8 and NHE3 were significantly reduced in the model group mice compared with the control group(p<0.0001);the expression of REG3 G protein was significantly upregulated(p<0.0001);and the s Ig A protein was significantly reduced(p< 0.0001).In contrast,mucin2 and mucin3 and the transporter proteins SLC26A3,SLC5A8,and NHE3 were upregulated at the gene level in the treatment group compared with the model group(p<0.0001);REG3G protein expression was significantly downregulated(p<0.0001);and s Ig A protein content was significantly increased(p<0.0001).6.At day 10,compared with the control group,the inflammatory factors IL-1β,IL-6,IL-12,IL-18,IL-33,TNF-α and inflammatory vesicles AIM2 and TLR4 were upregulated at the gene level in the model group,and all inflammation-related factors except TLR4 remained upregulated at the gene level at day 30;PCT protein content was significantly higher in the model group compared with the control group at day 10 and day 30.The Bacillus licheniformis treatment was able to reduce the upregulation of these indicators.7.The antioxidant enzymes SOD and GSHPX activities were significantly lower in the model group compared with the control group(p<0.0001),while SOD activity was significantly higher in the treatment group compared to the model group(p<0.01)and GSH-PX was more significantly higher(p<0.0001).Conclusions: The results of this study confirm that long-term high-dose antibiotic shock can disrupt the structure of the intestinal microbial population in mice,favoring the colonization of ESBLs-producing Escherichia coli,triggering an intestinal inflammatory response and affecting intestinal function.Bacillus licheniformis administration can inhibit the colonization and growth of ESBLs-producing Escherichia coli in the intestine,promote the recovery of intestinal flora to steady state,regulate intestinal permeability,reduce intestinal inflammatory response,and alleviate intestinal functional impairment,which has clinical therapeutic potential. |
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