Molecular Epidemiological Analysis Of Drug Resistance In Clinical Isolates Of Staphylococcus Aureus

Background and Objective:Among Gram-positive cocci,staphylococcus aureus is an important pathogenic Gram-positive bacteria,Which is one of the most common and pathogenic pathogens causing clinical infection and can cause multiple infections,such as respiratory tract infection,endocarditis,sepsis and urinary tract infection.Staphylococcus aureus can produce a variety of toxins and invasive enzymes,can cause serious infectious diaeases,and produce a variety of resistance genes,resulting in resistance to antibiotics.Drug resistance and virulence of Staphylococcus aureus have always been the two most concerned aspects in clinic.In this paper,the drug susceptibility of Staphylococcus aureus isolated from various clinical specimens in our hospital from 2012 to 2016 was retrospectively analyzed,and the relationship between drug resistance types and clinical drug use habits was analyzed in combination with the main isolation departments and drug resistance types of Staphylococcus aureus,so as to provide a strong basis for clinical rational use of antibiotic.Further,the drug resistance genes,virulence genes and biofilm genes of methicillin-resistant Staphylococcus aureus(MRSA)and methicillin-sensitive Staphylococcus aureus(MSSA)strains in our hospital were detected and multilocus sequence analysis(MLST)typing was performed to find the molecular epidemiological basis of MRSA and MSSA resistance.Finally,through Staphylococcus aureus biofilm experiment and Staphylococcus aureus infection mouse model,verify whether carrying drug resistance genes,virulence genes and biofilm genes affect biofilm and animale infectivity,and understand the inflammatory response caused by Staphylococcus aureus bacteremia in mice..Method:1.The isolation and culture of clinical specimens were conducted in accordance with the National clinical inspection operating procedures(3rd edition).The strains were identified and drug sensitivity test was conducted with automatic bacterial identification instrument VITEK-2 Compact,and the drug sensitivity test was measured with K-B method.2.mecA gene,fem B gene and pvlgenewere identified from clinical isolation of Staphylococcus aureus,and 21 PCR product of mecA gene positive were further sequenced According to the drug resistance phenotype,the 21 strains with mecA gene positive were divided into four groups as follows:(1)Fosfomycin sensitive group and moxifloxacin resistant group;(2)Fosfomycin resistance group,moxifloxacin resistance group;(3)Fosfomycin resistant group,moxifloxacin sensitive group;(4)Fosfomycin sensitive group,moxifloxacin sensitive group.mecA genes corresponding to four groups of different drug resistance phenotypes were analyzed by blasting with reference gene sequence(NCBI serial number: NC＿002952).3.Clinical isolates of Staphylococcus aureus were divided into four groups:mecA(+)/pvl(+);mecA(+)/pvl(-);mecA(-)/pvl(+);mecA(-)/pvl(-).PCR method was applied to detect drug resistance genes,virulence genes and biofilm genes.And AGR typing and MLST typing were also used to analyze the drug resistance genes,virulence genes and biofilm genes.Biofilm assay and mouse infection modle were conducted.Results:1.Comparision of drug resistance of MRSA and MSSAThe drug resistance of methicillin-resistant Staphylococcus aureus(MRSA)is higher than that of methicillin-sensitive Staphylococcus aureus(MSSA).Therefore,it is of great significance to strengthen the monitoring of drug resistance of MRSA to guide rational clinical drug use.Staphylococcus aureus mainly comes from orthopedics and neurosurgery.Check clinical medical records to understand the types of staphylococcus aureus drug resistance in these two major departments and analyze the relationship with clinical drug use habits,so as to provide a basis for better clinical selection of antibiotic treatment2.Analysis of molecular epidemiology1)mecA genes of four strains corresponding to different drug resistance phenotypes were sequenced.Using mecA sequence from NCBI(NC＿002952)as reference sequence,results from multiple sequence alignment showed that the absence of base A at site 1332 of MRSA mecA gene sequence may be related to fosfomycinresistance.and the absence of base G at site 1801 may be related to moxifloxacin resistance.2)Clinical isolates of Staphylococcus aureus were divided into four groups:mecA(+)/pvl(+);mecA(+)/pvl(-);mecA(-)/pvl(+);mecA(-)/pvl(-),PCR and ag R typing/MLST typing were performed to detect genes related to drug resistance,virulence and biofilm biosynthesis.The results showed that the higher drug resistance gene positive in mecA+/ pvl+ group,mecA+/ pvl-group and mecA-/ pvl-group than that in mecA-/ PVL + group,but the difference between groups was not statistically significant(P>0.05);The other drug resistance genes bla Z,erm A,erm B,dfr G and tet M were highly positivein each group.Virulence gene in mecA+/ pvl+ group was significantly higher than that in mecA+/ pvl-group,mecA-/ pvl+ group and mecA-/pvl-group.The positive rate of virulence gene in mecA-/ pvl+ group was the lowest(P<0.05),the positive expression rates of virulence genes sea+,hla+,hlb+ and agr1+were higher,while the expression rates of tst+,eta+,etb+,agr2+ and agr4+ were lower(P<0.05).The results showed that the positive expression rates of virulence genes hla+,hlb+,eta+ and agr3+ were higher in each group(P<0.05).3)Biofilm assay showed that the thickness of biofilm in mecA(+)/pvl(+)groups was significantly greater than that in mecA(+)/pvl(-),mecA(-)/pvl(-),andmecA(-)/pvl(+)groups(P<0.05).3.Infection ability tested in mouse modelThe animals were divided into four groups by being infected with different staphylococcus aureus strains: group A infected with mecA(+)/pvl(+);group B infected with mecA(+)/pvl(-);group C infected with mecA(-)/pvl(+);group D infected with mecA(-)/pvl(-).Serum was collected at time of 48 h and 7d from live mice and was tested for IL6,IL1,and CRP.1)IL6.The results showed that,at 48 hr serum IL6 concentration in group A was highest among four groups.IL6 concentration from group B was higher than that of group 3(p<0.05),and IL6 in group C was higher than that of group D(p<0.05).And the IL6 from serum taken at 7d showed the similar trend as serum taken at 48 hr.2)IL1.The IL1 concentration of serum taken at 48 hr and 7d showed no significantly diffierence among the four groups.3)CRP.Results showed that CRP concentration of serum at 48 hr in group A was higher than that in group B(p<0.05),but that in group B was higher than that in group C(p<0.05).CRP of Serum at 7d in group A was significantly higher than that in group C(p<0.05).Conclusion:1,the drug resistance of methicillin-resistant Staphylococcus aureus(MRSA)is higher than that of methicillin-sensitive Staphylococcus aureus(MSSA).Therefore,it is of great value to strengthen the monitoring of drug resistance of MRSA to guide rational clinical drug use.to understand the types of drug resistance of Staphylococcus aureus in these two major departments and analyze the relationship between them and clinical drug use habits,so as to provide a basis for better clinical selection of antibiotic treatment.2,methicillin-resistant Staphylococcus aureus(MRSA)resistance mecA.mecA(+)/pvl(+);mecA(+)/pvl(-);mecA(-)/pvl(+);mecA(-)/pvl(-),the carrying of drug resistance genes,virulence genes and biofilm genes in four groups of Staphylococcus aureus strains to guide clinical rational drug use.3,Staphylococcus aureus strains with mecA(+)/pvl(+)has significantly thickened biofilms and carriesstrong inflammatory responses in animal infection model.