| Staphylococcus aureus(Staphylococcus aureus,SA)is an important pathogen of clinical infection [1-3],in orthopedic infection pathogens in the first place [1].Staphylococcus aureus caused by bone and joint infection often recurrent,persistent healing,difficult to control osteomyelitis(OM)is a serious orthopedic infectious diseases,the recurrence rate is high,long duration,severe bone damage,these patients Resulting in a huge physical and economic burden.In this paper,139 strains of Staphylococcus aureus isolated from orthopedic patients were divided into OM group(60 isolates from 60 patients with OM)and 79 non-OM(77 isolates from 77 non-OM patients)with mec A-The MRB was identified by double B double PCR.The drug resistance profiles were compared between the two groups.At the same time,the molecular typing(MRSA identification,SCCmec classification,MLST typing,spa classification,agr typing)and virulence factor distribution between the two groups were evaluated to identify the association with the incidence of Staphylococcus aureus OM Potential indicators.Virulence factor is an important "weapon" of bacterial pathogenesis.Staphylococcus aureus can produce more than 50 virulence factors,causing a variety of diseases [10-12].γ-hemolysin is a pore-forming toxins(PFTs)produced by Staphylococcus,which can be divided into α-helix PFTs and β-barrel PFTs according to their secondary structure of transmembrane pores.Staphylococcus aureus can secrete a variety of β-PFTs,including α-solute,γ-lysin,kill leukotrienes,through the formation of β-barrel holes in the cell membrane,resulting in intracellular leakage and cell killing;The F and S components of γ-solubilizers and leukocidin are LukF and Hlg2,Luk F and Luk S,respectively,and several other β-PFTs have the same effect on F and S components.γ-lysin is similar to leukocidin,mainly in human and other mammalian blood cells,etc.,so that the pathogen in the Staphylococcus aureus,immune escape play an important role [21,22].RNA plays an important role in virulence regulation of S.aureus [14].RⅢ NAⅢ is the constituent of Agr’s Agr population induction control system and is the key effector of Agr system [12,13].Staphylococcus aureus Agr system consists of RNA Ⅱand RNA Ⅲtwo adjacent operatives,respectively,by the P2,P3 promoter control transcription.Among them,RNAⅢ is composed of 514 nucleotides of small RNA molecules,can form 14 hairpin stem ring structure,regulation of bacterial virulence gene expression.Almost all clinical strains of Staphylococcus aureus isolated from acute infection can produce RNA Ⅲ.The expression of multiple virulence factors can be regulated or controlled by direct base pairing.Currently known molecules of Streptococcus suis RNAⅢ can down-regulate molecules including SarH1,Lyt M,Lta S,oligopeptide permeability enzymes,etc.,can be upregulated molecules kill leukotriene,enterotoxin,extracellular protease,lipase,urease,mec A [15-18].Whether γ-lysin is regulated by RNA Ⅲ is not clear.Rot(repressor of toxins)was screened and identified as a regulatory factor by McNamara et al.[27] using a transposon mutant library,and further studies confirmed that rot’s inactivation would restore the QS system’s ability to attenuate endocarditis caused by S.aureus,Indicating that rot may be involved in the regulation of Staphylococcus aureus virulence factor spectrum,is a global regulatory factor,rot inhibition of toxin gene expression at the same time,the first class of virulence effects of Staphylococcus aureus may have a role in promoting.Boisset et al.[28] showed that rot expression was negatively regulated by RNAⅢ,and that the regulation function of S.aureus QS system on its population might be achieved by controlling rot [29].It is of great significance to study the changes of virulence factors and the regulation mechanism to understand the pathogenicity of Staphylococcus aureus and to find a new strategy for controlling S.aureus infection.The first part of this study is divided into OM group and non-OM group according to the medical records.The first part of this study is divided into OM group and non-OM group from September 2009 to September 2015,SNB was used to identify MRSA,and SCCmec,spa,MLST and agr were used to classify and detect various virulence factors.The differences of molecular types,drug sensitivity and virulence factors between the two groups were evaluated to identify Staphylococcus aureus OM incidence associated with the potential indicators.And on this basis,detection and comparison of drug resistance spectrum.The second part aims to explore the molecular mechanism of RNAⅢ-regulated γ-hemolysin: the homologous recombination principle is used to knock out the γ-hemolysin,RNAⅢ and Rot regulatory proteins of the Newman strain of Staphylococcus aureus respectively,and Newman-Δγ,Newman-ΔRNAⅢ and Newman-Δrot knockout strains.RT-qPCR and western-blot were used to analyze the relationship between RNAⅢ,Rot regulatory protein and γ-hemolysin.The main contents and results are as follows:1,Molecular classification of clinical strainsWe identified 139 strains of Staphylococcus aureus from the Southwest Hospital of Chongqing,which were divided into OM group and non-OM group.The spa,MLST and agr typing were identified and a series of related virulence factors were detected.(1)MRSA strain identification: MRSA was identified by mec A-femB double PCR method [7].The results showed that there were 34 MRSA in 139 strains.(2)Spa classification: 139 clinical Staphylococcus aureus spa classification,and the results of the summary.Results There were 54 spa types in 139 strains,with t189(17.27%,24/139)and t437(14.39%,20/139),accounting for 31.66% of all strains.In both groups,t189 and t437 were the most important types of spa(10/60,16.7% and 14/79,17.7% for t189,8/60,13.3% for t437 and 15.2% for 12/79).(3)MLST typing: MLST typing showed that 139 strains were divided into 33 types,including ST188(18.70%,26/139)and ST59(15.83%,22/139).OM group(ST3538)and non-OM group(ST3539)identified a new ST.(4)Agr type I(71.94%),agr II type 13(9.35%),agr Ⅲ type 14(10.07%),agr type IV 12 strains(8.63%).The agr I group was identified as the most common type(38/60,63.3% and 62/79,78.5%)in both groups.All of the above-mentioned predominant ST and spa type Staphylococcus aureus belonged to agrI.AgrⅢ / agr IV group was positively correlated with the incidence of OM(p = 0.0040)(5)Virulence genes and adhesion gene detection: 139 strains showed 24 strains were positive,positive detection rate was 18.2%.Analysis of pvl positive strains found 79.17%(19/24)from the OM patients,and longer course.The results showed that all isolates carried at least six adhesion-related and three exotoxin-associated virulence genes.Eno,clfA,clf B,icaD,hld and psmα genes were detected in all isolates.The following virulence genes were more than 50% in OM and OM groups,including ica A(98.3% and 100%),fibers(88.3% and 92.4%),hla(91.7% and 100%),hlg(96.7%),Hlgv(91.7% and 93.7%)and lukED(71.7% and 60.8%).FnbA,etb and luk M genes were not detected in any isolates.(6)There was no significant difference in antimicrobial resistance between OM patients.Eighteen and 22 multidrug-resistant strains(MDRs)were detected in OM and non-OM groups,but the difference was not statistically significant.Several antimicrobial agents showed relatively high rates of resistance in both groups,including penicillin(88.3% and 93.7%),erythromycin(45% and 53.2%),clindamycin(45% and 46.9%),Tetracycline(35% and 21.5%)and oxacillin(21.7% and 26.6%).But did not find linezolid,tigecycline and vancomycin-resistant strains.2,RNA regulation of Ⅲ γ-hemolysin molecular mechanism of the preliminary studyRNAⅢ is an intracellular effector of the Staphylococcus aureus population induction system,which is one of the largest regulatory RNAs known to control the expression of a large number of virulence genes.RNAⅢ regulates α-hemolysin,enterotoxin,esterase A and so on in Staphylococcus aureus.The overall regulation factor rot,which inhibits the expression of toxin gene,may promote the expression of the first class of virulence factors in Staphylococcus aureus.Therefore,we constructed RNAⅢ and rot knockout strains to study the regulation of γ-hemolysin.(1)Construction of Newman-Δγ,Newman-ΔRNAⅢ and Newman-Δrot knockout strainsThe desired gene was amplified by PCR from the genome of Staphylococcus aureus,and the target gene was cloned into Escherichia coli and Staphylococcus aureus shuttle plasmid PBT2 by restriction enzyme digestion.The knockout plasmid was constructed and transformed into gold The strain RN4220 was modified and transferred to Newman to induce and screen out knockout strains.(2)q-RTPCR detection of γ-hemolysin transcriptionWe used q-PCR to detect the expression levels of γ-hemolysin gene A,B and C,respectively.The expression of γ-hemolysin A subunit was down-regulated in ΔRNAⅢ,and the expression of γ-hemolysin A subunit was down-regulated in ΔRNAⅢ.The expression of γ-hemolysin C subunit was not significantly different.This result shows that RNAⅢ regulates gamma-hemolysin.(3)Western-blot detection of γ-hemolysin translationWestern-blot showed that γ-hemolysin A subunit was up-regulated in ΔRNAⅢ knockout strain and γ-hemolysin B subunit was up-regulated in ΔRNAⅢ knockout strain.The expression of γ-hemolysin C subunit was not significantly different.And the expression of these three subunits in Δrot knockout was significantly down-regulated.This result is not consistent with the qPCR results,suggesting that a factor in the wild strain inhibits the synthesis of the gamma-hemolysin A subunit,which is negatively regulated by RNAⅢ,so that the inhibition of RNAⅢ is knocked out Resulting in an increase in the expression of the gamma-hemolysin A subunit.In summary,we identified the differences between the two groups by dividing the clinical staphylococcus aureus into OM group and non-OM group by molecular typing,virulence factor test,agr typing and pvl detection.Identify potential indicators associated with the incidence of Staphylococcus aureus OM.To enrich the molecular epidemiological data of Staphylococcus aureus at the same time to provide a reliable basis for the monitoring of Staphylococcus aureus infection,combined with molecular typing and drug resistance characteristics,to further explore its epidemic causes and resistance mechanisms to strengthen the Staphylococcus aureus infection Prevention and control.And on this basis,to detect the drug resistance spectrum.After the detection of virulence factors,it was determined that γ-hemolysin was a toxin commonly found in Staphylococcus aureus,and there was no correlation between bacteria and the type of bacteria.Therefore,the actual operation of XQ wild strain could not be knocked out We can use Newman strains instead,for mechanism studies.Molecular resistance analysis of MRSA in molecular typing also provides a reference for clinical use,with practical clinical significance.On the other hand,we successfully knocked out RNAⅢ,rot,γ-hemolysin gene,and q PCR,western-blot at the level of transcription and translation were compared to determine the RNAⅢ,rot,γ-hemolysinbetween the existence of regulation Relationship,in order to further study the regulatory mechanism to do the bedding.The above findings extend our understanding and understanding of the relationship between RNAⅢ and toxins in Staphylococcus aureus. |
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