Virulence Detection Of 41 S. Aureus Strains Isolated From Rabbits

Staphylococcus aureus is a worldwide pathogenic bacterium that can infect humans and animanls.It can cause necrotizing pneumonia,suppurative arthritis and septicemia in humans through infection,and it can also infect chickens,cows and rabbits.To investigate the carrying rate of Staphylococcus aureus virulence gene?genotype difference and relative rellationship in some areas of Sichuan.This study was based on the specific identification of femB gene and MRSA and MSSA's screening of 41 strains of Staphylococcus aureus in some areas of Sichuan,13 common virulence genes were detected by PCR,and the genotypic differences of Staphylococcus aureus in some areas of Sichuan were studied by the molecular typing methods of MLST and PFGE.The results showed that all 41 rabbit origin S.aureus can form surface smooth,thick,circular rise,moist,edge neat colony after incubated 24h in LB solid medium at 37?.Bacterial gram staining micriscipic examination is spherical,forming double or grape clusters of G~+coccus.The femB gene PCR identification results of 41 rabbit origin S.aureus strains obtained a length of about651bp of specific stripe,then 41 rabbit origin S.aureus strains were S.aureus combined with morphology and PCR detection.Meanwhile,the result of mecA gene amplification showed that 31 drug-resistant genes of 41 S.aureus strains were positive for mecA,which were confirmed as methoxicillin resistant staphylococcus aureus(MRSA),and the detection rate was 75.61%.The primers of 13 common virulence genes(sea?seb?sec?sed?see?hla?hlb?TSST-1?eta?etb?clfA?nuc?PVL)were synthesized.Taking the genomic DNA of 41 S.aureus from rabbits as the template,specific PCR amplification was used to identify the virulence gene of the strain according to the size of the amplified genome-specific band.The result of electrophoretic spectrum identification showed that 9virulence genes including sea,sec,see,hla,hlb,eta,clfA,nuc,PVL were detected in this test,but no seb,sed,TSST-1 and etb genes were detected.Even the results showed that the positive detection rates of nuc,eta,clfA and hla were the same in MSSA and MRSA strains,all of which were 100.0%.The positive detection rates of sea,sec,see and hlb and PVL in MSSA strains were 20.0%,90.0%,80.0%,100.0%and20.0%,respectively,which positive detection rates in MRSA strains were 6.5%,83.9%,80.6%,87.1%and3.2%,respectively,which positive detection rates in 41 strains were 9.7%,85.4%,80.5%,90.2%and 7.3%,respectively.It can be seen that MSSA strains maintain a relatively high virulence,while the MRSA strains are slightly weaker.Most strains(90.2%)carried two hemolysin genes(hla and hlb)simultaneously.Among them,Zigong and Leshan strains'virulence genes detected the most species,a total of 9 species;then 7virulence genes were detected by strains of Nanchong,Deyang,Chengdu and Meishan.Seven housekeeping genes of Staphylococcus aureus:arcC?aroE?glpF?gmk?pta?tpi?yqi were selected for MLST typing.The specific amplification was performed with PCR method based on genomic DNA of 41 S.aureus from rabbits.Sequence type determined by BLSAT in MLST database(http://pubmlst.org).Results showed that MLST typing received 2 STs sequence types(ST-398?ST-3320)and 1 clonal complexes CC-398,among which ST-3320 is a clone derivative of ST-398,and there are only single site differences between ST-3320 and ST-398.ST-398 was the dominant sequence type in this test,accounting for 97.6%.Only the N04 strain isolated from Leshan was ST-3320,and the sequence types of the other areas all was ST-398;Among them,10 MSSA strains all belonged to sequence type ST-398.31MRSA strains,30 of which belonged to sequence type ST-398,and only one was sequence type ST-3320.However,both MSSA strains and MRSA strains belonged to group CC-398.41 strains were buried in the plastic pieces,after protease K and cell lysis liquid relasing genomic DNA digested with 50 U Sma I restriction enzymes for 30 min,then observated the electrophoresis images after 16h under alternating current in the electrophoresis tank,took pictures and clusteried analysis with the Quantity One software.Results showed that PFGE clustering analysis was divided into 18 banding patterns(cluster A-R),among which 8 bands contain only a single type of strain(cluster B,E,G,I,J,K,N,Q),and the rest of the band types were all clustered with over 77%similarity.S.aureus in 8 bands isolated from different regions of Sichuan(cluster D,F,H,L,M,O,P,R).Only the strains in clusterA and clusterC were isolated from the same region(Zigong).In terms of similarity clustering,only clusterO and P contained both MSSA strains and MRSA strains.In this test,it was found that the carrying status of virulence factors of Staphylococcus aureus in some regions of Sichuan was relatively small,but the detection rate of virulence genes was relatively high,and the incidence rate of infection in rabbits was higher;the results of two genotypes of PFGE and MLST were relatively consistent,that is,the main strains of Staphylococcus aureus in some regions of Sichuan showed little genetic variation and relatively close relatives among the strains.