| Environmental quality is closely related to human health.In recent years,the environmental pollution problems still exist,threatening human health to varying degrees.The pathogenic microorganism pollution in the environment is the focus of public health attention.Due to the wide variety and vast sources of pathogenic bacteria existing in the medical microenvironment,as well as the drug resistance of some bacteria,these pathogenic microorganisms will increase the risk of acquired infections in the hospital environment.Bacterial infection acquired in the hospital microenvironment is a common adverse event,which is still challenging to be completely resolved.The incidence of nosocomial infection has been reported to be 5-10% and up to 30% of patients in intensive care units.In addition,the use of some invasive instruments can increase the risk of infection,resulting in a severe waste of social and economic resources,which is a significant public health problem threatening human health.Bacillus cereus has been widely distributed in various environments.It has been reported that Bacillus cereus was isolated from different parts of wards in various departments of hospitals,and it can cause severe infection in patients and spread in hospitals,becoming a nosocomial infection.In the early stage of this study,environmental samples were collected from different hospital departments and different disinfection environments(mainly ultraviolet disinfection).After laboratory culture,multiple bacterial samples were isolated.Among the environmental samples,a bacterial model with the highest abundance and strong virulence was found in pre-experimental animals.It was identified as Bacillus cereus ZJ4,whose morphological characteristics and virulence were similar to those of Bacillus cereus.In order to explore the toxic features and infective mechanism of its virulence factors,whole genome sequencing and protein profiling were used to identify the virulence genes and secreted virulence proteins of a Bacillus cereus bacterium from a medical microenvironment for the first time,and their virulence characteristics were analyzed.Two recombinant virulence proteins were expressed in vitro,and their cytotoxicity was studied.At the same time,the cytotoxicity and infectivity of bacteria to model animals were developed based on transcriptome and proteome.The main results are stated as follows:(1)The physiological and biochemical characteristics of bacteria ZJ4 are similar to those of Bacillus cereus.It was Gram-positive bacteria and could form endophytic spores with mobility and hemolysis.The optimal PH was about 7.0,and it could survive in an alkaline environment but not in an acidic one,and the sugar fermentation experiment shows that it could utilize different carbon source sugars.MIC antibacterial tests showed that ZJ4 was resistant to a variety of antibiotics and was only sensitive to Enrofloxacin and Ofloxacin.Phylogenetic analysis showed that ZJ4 belonged to the Bacillus cereus group and was closely related to Bacillus cereus ATCC 14579,and the genetic distance was 0.6522.(2)The genome size of Bacillus cereus ZJ4 was 5.6Mbp,containing one chromosome and six plasmids,the average content of GC was 35.92%,encoded 6058 genes,the average length of genes was 783.17 bp,and there were 107 t RNAs,45 r RNAs and 201 repeats in the non-coding region.Compared with other existing Bacillus cereus genomes,ZJ4 genome exhibits some significant characteristics.Its genome size is larger than other genomes,but its coding genes are slightly less than those of other bacteria,and there are relatively more symbiotic plasmids in ZJ4.The results of genome annotation showed that the coding genes were mainly related to the normal physiological metabolism of bacteria and exercised the functions of energy production and transformation as well as amino acid transport and metabolism.14 genomic islands were predicted in the genome,of which 10 genomic islands were located on chromosomes,and the other four genomic islands were located on plasmids.The secreted proteins contained toxic proteins such as hemolysin and enterotoxin.CARD resistance predicted 352 resistance genes,confirming resistance to multiple antibiotics.VFDB virulence factor predicted 528 virulence genes,among which 6 virulence genes had high sequence recognition threshold,namely Cyt K,Nhe A,Nhe B,Nhe C,Inh A,and ALO,and were indeed present in the ZJ4 genome after PCR detection.Among them,ALO gene was first identified in the type of Bacillus cereus bacteria.(3)203 proteins and 805 secreted proteins were identified by MALDI-TOF-MS mass spectrometry.GO and KEGG enrichment analysis showed that the proteins in the bacteria were mainly involved in carbohydrate metabolisms such as the tricarboxylic acid cycle and pentose phosphate pathway.In contrast,the function of secreted proteins was mainly involved in structural molecular activity,catalysis,REDOX,and other metabolic functions of microorganisms in different environments.In addition,a variety of toxic proteins have been identified in secreted proteins,such as cytotoxic proteins,immunosuppressive proteins,enterotoxin proteins,hemolysin proteins,insect insecticide proteins,and cholesterol-dependent toxins.These virulence proteins correspond to virulence genes in the genome of ZJ4.(4)Two virulence genes,Cyt K and ALO,were cloned and expressed by the prokaryotic expression technique.Subsequently,active recombinant Cyt K and ALO virulence proteins were obtained by purification and identification.In the cytological tests,the half inhibitory concentration(IC50)of Cyt K and ALO on glomerular mesangial cells(GMCs)was 3.672ng/μL and 3.927 ng/μL,respectively,and the proportion of normal cells decreased from 97.00%to 63.40% after 12 h of Cyt K treatment at IC50 concentration.The proportion of apoptotic cells increased from 0.20% to 36.67%.After 12 h treatment with ALO,the proportion of normal cells decreased from 96.40% to 67.70%,and the proportion of apoptotic cells increased from 0.24%to 31.60%.With the increase of treatment time with Cyt K and ALO,the percentage of apoptotic cells increased gradually.(5)Bacillus cereus ZJ4 could induce apoptosis of GMC cells after infection,and the phenomenon of apoptosis became more and more evident with the increase of infection time.Dual RNA-Seq analysis showed that the differentially expressed genes(DEGs)after bacterial infection were mainly involved in the TNF signaling pathway,MAPK,IL-17,and other inflammatory signaling pathways.The q PCR results showed that the expression of differential genes was consistent with the transcriptome trend.ZJ4 induces the expression of pro-inflammatory cytokines IL-2,IL-17,NF-κB,and TNF-α,forming an inflammatory response,and induces the expression of pro-apoptotic genes Dr5,Fas,Bax,and Bcx3,leading to cell apoptosis after infection.(6)After Bacillus cereus ZJ4 infected the silkworm in the blood cavity,the results of the histopathological section confirmed that ZJ4 affected the silkworm’s immune tissue fat body.The proteomics results identified 138 up-regulated proteins and 165 down-regulated proteins.The differential proteins were mainly involved in the defense response against bacteria,innate immune response,ribosome biogenesis,pyruvate metabolism,and other pathways.Moreover,the expression of βgrp,Spaetzli,Myd88,Tube,and Dorsal genes in the Toll pathway was significantly up-regulated.The levels of CAT,SOD,and ROS in the silkworm body also increased significantly.These results confirmed that after ZJ4 infection,a large number of ROS would be produced to reduce pathogenic bacteria.CAT and SOD enzyme genes were also overexpressed,producing corresponding enzymes to remove excess ROS.(7)After Bacillus cereus ZJ4 was intraperitoneally injected into mice,the bacterial load of ZJ4 detected in liver tissue was higher.The detection results of antioxidant enzyme activity and liver function indicators showed that ZJ4 infection could lead to liver function damage,with the increase of AST,ALT,ALP indicators,and the proliferation of antioxidant enzymes SOD and GSH.Liver pathological sections showed that ZJ4 infection caused liver cell damage,and the immunohistochemical(IHC)tests showed that the expression of inflammatory cytokines TNF-αand IL-6 in the liver was increased after ZJ4 infection.The expression levels of TLR4,TNF-α,TAP1,LY6 A,TGF-β,and Cyp51a1 genes and proteins were verified by q PCR and Western blot,revealing that TLR4 protein was the first to recognize bacteria and its secreted virulence factors,and the expression level increased.Subsequently,TGF-β and Cyp51a1 proteins were down-regulated,and TNF-α,TAP1,and LY6 A proteins were increased to activate the inflammatory signaling pathway.In this paper,through the experimental study of model organism silkworms,human cells and mice,the relevant research results suggest that there is still a risk of Bacillus cereus infection in the agro-ecological environment and hospital microenvironment,especially in the hospital environment.After the retention and accumulation in the hospital environment,bacterial drug resistance will be enhanced,virulence genes will be transferred horizontally,and the infectivity and pathogenicity of a variety of organisms will be enhanced,suggesting that ecological environment and the hospital microenvironmental health management should be strengthened,and improving the elimination measures against Bacillus cereus and adjusting the diagnosis and treatment plan of ecological environment pollution and related diseases that may be caused by Bacillus cereus,also provide a theoretical basis for the protection of ecological environment and the prevention of microorganisms in medical environment in the future. |
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