| Staphylococcus aureus is a widespread spread between humans and animals and is the main pathogenic bacterium that causes soft tissue,skin,wounds and other infections.Transmission of Staphylococcus aureus can cause local purulent infection,pneumonia and pericarditis,and severe animal symptoms of systemic infection such as sepsis and sepsis.Tigecycline is one of the effective drugs of choice for the clinical treatment of infections such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.Treatment of Staphylococcus aureus infection in sick livestock with antibiotics or antimicrobials is currently the most common and effective treatment.Tetracycline is a low toxicity,broad bactericidal spectrum of biotics,widely used in livestock farms,so in veterinary clinics,Staphylococcus aureus isolates have a high rate of resistance to tetracycline,but for tigecycline,which is also a tetracycline antibiotic,staphylococcal resistance rate is extremely low,and its resistance mechanism is rarely reported.In this study,the prevalence of tetracycline-resistant gene tet(L)was investigated for some pig slaughterhouses and farms isolated from Staphylococcus aureus in Henan Province,and the mechanism of resistance of Staphylococcus aureus to tigecycline was further explored,mainly including the isolation and identification of Staphylococcus aureus from pig sources,the detection of tet(L)gene carrier rate,drug susceptibility test,electrical conversion test,bacterial whole genome sequencing and bioinformatics analysis,drug resistance gene cloning and functional analysis.Establishment of mouse sepsis models and experimental treatment of tigecycline in vivo.Firstly,the tet(L)gene carrier rate of 330 staphylococcal isolates and 253 pig slaughterhouse isolates preserved in the laboratory were detected,and the results showed that the tet(L)gene carrier rate of 583 staphylococcus strains was 33.28%(194/583).Secondly,the tigecycline-resistant phenotype of 253 strains of staphylococcus from slaughterhouses was detected,from which the tigecycline-resistant strains XTB(methicillin-resistant Staphylococcus aureus),SZB(hemolytic staphylococcus),DMB(Staphylococcus aureus)were screened.Subsequently,after the resistance of tetracycline-resistant strain XTB was successfully transferred to Staphylococcus aureus RN 4220 by electroconversion test,the resistance of the recipient strain to tigecycline and elatronine increased by 16-fold(MIC from 0.125 μg/m L to 2 μg/m L).Staphylococcus aureus RN4220 was found through nanopore gene sequencing to obtain a plasmid carrying multi-drug resistance genes,including resistance genes tet(L)and ANT(4’)-Ia.The results of gene cloning and functional analysis showed that when tet(L)carried the upstream 300 bp regulatory region into Staphylococcus aureus RN4220,the MIC of tigecycline and epaecycline could reach 1 μg/m L,but the transfer of tet(L)gene carrying 200 bp upstream into Staphylococcus aureus RN4220 did not significantly improve its resistance to tigecycline.Through the sequencing results of XTB,SZB and DMB,as well as tigecycline-sensitive strains carrying tet(L)gene,it was found that there was a difference between the upstream regulatory region of 300 bp to 200 bp between tigecycline-sensitive strains and drug-resistant strains,but there was no difference in the upstream regulatory region of 300 bp between tigecycline-resistant strains.Finally,the plasmid of tigecycline-resistant strain XTB was transferred to Staphylococcus aureus ATCC 29213 for in vivo experimental treatment of tigecycline,and the results showed that the presence of antiplasmids reduced the therapeutic effect of endotigecycline.In summary,three tigecycline-resistant strains were found in Staphylococcus suis:XTB,DMB and SZB,and the mechanism of tigecycline resistance was explored on one of the strains of Staphylococcus aureus XTB,which was successfully metastained,and the situation that Staphylococci carried the resistance gene tet(L)was detected,which expanded the understanding of the mechanism of tigecycline resistance in Staphylococci and provided the prevalence of tet(L)in Staphylococci.The expression of Tet(L)was measured by real-time PCR,and it was found that the expression of tet(L)gene carrying the 300 bp upstream regulatory region in this study was significantly higher than that in the 200 bp upstream regulatory region,and it could confer Staphylococcal tigecycline resistance.Experimental treatment of tigecycline in mice has shown that the transfer of tigecycline-resistant plasmids will lead to a decrease in the therapeutic effect of tigecycline and may lead to the failure of clinical tigecycline,so there is an urgent need to take corresponding measures to prevent the spread of mobile genetic elements in human and animal clinically pathogenic strains. |
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