| [Objective]Staphylococcus aureus is the major pathogen of human infectious diseases, which can cause various diseases by secreting a variety of virulence factors, is common pathogen in Bloodstream infection. In recent years, because of the emergence of multiple drug-resistant bacteria, especially hVISA and VISA, which has brought great difficulties and challenges to clinical medicine. We collected768Staphylococcus aureus isolated from11hospitals in Hubei Province and6hospitals in Central-southern China (Hubei province, Hunan province and Henan province), Antimicrobial susceptibility and molecular epidemiological characteristics were analyzed, in order to provide the basis for clinical anti-infection treatment. hVISA was detected using PAP/AUC, MET and MHA5T method, aimed at finding a simple method that can be used in laboratory screening of hVISA. Establishing mouse model of sepsis, the changes of mouse body weight, survival, inflammation, bacterial load in blood and tissues, and histopathology were detected, investigated the impact of mgrA gene on progression of Staphylococcus aureus sepsis and explored the pathogenesis of Staphylococcus aureus sepsis, in order to provide new ideas for diagnosis and treatment of Staphylococcus aureus sepsis.[Methods]一. Study on detection of virulence genes and epidemiology of Staphylococcus aureus1.416strains of Staphylococcus aureus from11hospitals in Hubei Province from Mayl,2010to December31,2010and352strains of Staphylococcus aureus from6hospitals in in Central-southern China from June1,2011to May31,2012were collected. Re-identification were done using Gram stain, catalase, coagulase and PCR methods, minimum inhibitory concentration (MIC) of16antibiotics were detected by agar dilution method.2. SCCmec, MLST, spa and agr typing of MRS A strains were detected by Multiplex PCR, positive samples were sequenced.31virulence genes of Staphylococcus aureus were were isolated Sterile body fluid from Central-southern China were detected by Multiplex PCR.3. Antimicrobial susceptibility results were analyzed using WHONET5.6software, other data were analyzed using SPSS18.0software(Chi-square test or Fisher’s exact test), P<0.05was considered statistically significant.二. Study on detection method and prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus1. PAP/AUC, MET and MHA5T methods were used for hVISA detection, and investigated the prevalence of hVISA. PAP/AUC method as the gold standard to compare the sensitivity and specificity of MHA5T and MET methods.2. Antimicrobial susceptibility results were analyzed using WHONET5.6software, other data were analyzed using SPSS18.0software(chi-square test or Fisher’s exact test), P<0.05was considered statistically significant.H. The impact of mgrA on progression of Staphylococcus aureus sepsis1. mgrA gene of Staphylococcus aureus wild-type Newman strain were knockouted using splicing by overlap extension PCR+homologous recombination method.2. Establishing BALB/c mice sepsis model, all mice were divided into two groups and injected intravenously wild-type strain Newman strain and mgrA knockout strain, respectively(the Newman group and mgrA knockout group). The impact of mgrA on progression of Staphylococcus aureus sepsis was observed, experiment was divided into four sections(1).6×106CFU Staphylococcus aureus were injected and observed for14days. Survival differences between the two groups were observed.(2).3×106CFU Staphylococcus aureus were injected and observed for14days. Weight changes in two groups were observed. (3).6×106CFU Staphylococcus aureus were injected and observed forl20h. Blood was obtained by enucleation. CD4+and CD8+T lymphocytes were detected by flow cytometry. Serum IL-6and TNF-a levels were detect with ELISA.(4).3*106CFU Staphylococcus aureus were injected and observed for14days, blood and tissues (liver, spleen and kidneys) bacterial loads and histopathological differences (H&E staining) were observed. Cytokine levels,blood and tissues bacterial counts were expressed as the mean+SD3. All data were analyzed using SPSS18.0software. Statistical analysis of survival curves was estimated by Kaplan-Meier analysis, log-rank test was used. Weight change was performed by Manne-Whitney U-test. Data sets of Cytokine levels,blood and tissues bacterial countswere analyzed using Student’s t-test. P value<0.05was considered significant.[Results]一. Study on virulence genes and epidemiology of Staphylococcus aureus1.209MRS A were detected in416Staphylococcus aureus were collected from Hubei province a, the prevalence of MRS A was50.2%. SCCmec typing were:Ⅲ type, accounting for82.8%(173/209), IV type, accounted for13.4%(28/209), Ⅱ type, accounted for13.4%2.4%(5/209) and V type, accounted for1.4%(3/209).2.118MRSA were detected in352Staphylococcus aureus were collected from6hospitals in Central-southern China, the prevalence of MRSA was33.5%. The mainly SCCmec type was Ⅲ type, accounted for80.5%(95/118), Ⅳ and Ⅱ type accounted for8.5%(10/118) and5.1%(10/118), respectively, agr type were agr-I, accounting for81.4%(96/118), agr-Ⅱ,15.3%(18/118), agr-Ⅲ,1.7%(2/118), agr-ⅣV,1.7%(2/118).3. The prevalence of MRSA from184sterile body fluid specimens was41.8%(77/184), the prevalence of hVISA was high, up to22.1%(17/77). The major MRSA and MSSA clone were ST239-S’CCmecⅢ-t030-agr-I (43/77,55.8%) and ST188-MSSA-t189-agr-I (22/107,20.6%), respectively. 4. The prevalence of pvl, tst, l1b, hlg-v, seb, sed, and sei in MSSA strains were significantly higher than MRSA strains (P<0.05).98MSSA isolates (53.3%) carried≥10virulence genes simultaneously, which was significantly higher than that of MRSA isolates (33.8%,26/77)(P=0.004).24VSSA isolates(40.0%) carried≥10virulence genes simultaneously, which was significantly higher than that of hVISA isolates (11.8%,2/17)(P=0.041). Compared with otherCCs isolates, CClandCC398isolates harbored higher frequency of exfoliatin genes (P<0.05), CClandCC59harbored higher frequency of pvl gene (P<0.05), only CC1isolates harbored lukED gene.二. Study on detection method and prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus1. Compared with PAP/AUC method, the sensitivity and specificity of MET method were81.6%and92.4%, respectively, the sensitivity and specificity of MHA5T method were92.1%and62.6%, respectively.2.58hVISA isolates were detected among327MRSA strains, prevalence of hVISA was17.7%. A total of38isolates were identified as hVISA from209MRSA strains in Hubei province, the prevalence of hVISA was18.2%. A total of20isolates were identified as hVISA among118MRSA strains from6hospitals in Central-southern China. The prevalence of hVISA was16.9%.3. The major epidemic clone of hVISA was ST239-SCCmec-Ⅲ-t030-agrl, accounting for84.5%(49/58). The resistance rates of hVISA strains to Tobramycin and Levofloxacin were significantly higher than VSSA strains (P<0.05).三. The impact of mgrA on progression of Staphylococcus aureus sepsis1. The impact of mgrA gene on survival and weight of sepsis mice:Seven mice died in Newman group(mortality70%), while only one mice died in mgrA knockout group (mortality10%), the mortality rate between two groups had significant difference(log rank=4.739, p=0.029). Weight of Newman group lost significantly more than that mgrA knockout group when were injected with a dose of3×106CFU bacteria. 2. Comparison of CD4+/CD8+T lymphocyte ratio and serum TNF-a, IL-6levels between two groups:The experimental results showed that CD4+/CD8+T lymphocyte ratio peaked at24h after injection, however, at24h (P=0.0011),48h (P=0.0025) and72h (P=0.0243), CD4+/CD8+T cells of mgrA knockout group were significantly lower than that Newman group. Serum IL-6level reached a peak at12h after sepsis induced, then declined rapidly. However, serum IL-6level of Newman group were significantly higher than mgrA knockout group at4h (P<0.0001),12h (P<0.0001),24h (P<0.0001),48h (P=0.0003) and72h (P=0.0012). Serum TNF-a level peaked at4h after induction of sepsis, and the TNF-a level of Newman group was significantly higher than mgrA knockout group (P=0.0032).3. Comparison of blood and tissues bacterial load between two groups:Bacterial load of kidney and liver tissue in Newman group was significantly higher than mgrA knockout group (Log10CFU/g kidneys:7.53±0.23vs6.02±0.33, P<0.001; Log10CFU/g liver:5.82±0.26vs4.84±0.29, P=0.024). However, two bacterial load of spleen had no significant difference.(Log10CFU/g spleen:4.67±0.19vs4.51±0.54, P=0.404).4. Histopathological analysis:Newman Group showed mild histopathological changes at1d after injection, and mgrA knockout group found no significant changes. Observed to2d and4d, liver and kidneys of two groups showed mild to moderate histopathological changes, and spleen showed slight pathological changes, however, the volume of spleen increased significantly. However, Newman group had significantly more severe histopathological changes than mgrA knockout group, two groups showed severe pathological changes at7d.[Conclusions]1. The major epidemic clone of isolated from sterile body fluid specimens among Central-southern China wes ST239-MRSA-S’CCmecⅢ-t030-agrI type (5.8%), indicating the presence of drug-resistant clone spreading in the region, health care system should take effective measures to control spread of drug-resistant clones, such as strengthening the hand hygiene, isolation of patients, strict attention to aseptic technique to enhance the hospital environment and public health. 2. The drug resistance is serious in11hospitals from Hubei Province and China and6hospitals from Central-southern China, the prevalence of hVISA was high, up to17.7%should be paid attention. The the emergence of hVISA phenotype is due to selective pressure of glycopeptide drugs, it was suggested that glycopeptide drug use should be strict controlled, antibiotics should be reasonable used based on susceptibility results.3. PAP/AUC method is the most accurate method of detecting hVISA, but the operation is more complicated, difficult to apply in clinical. MHA5T method had high sensitivity(92.1%)and can be clinical used as the screening method of hVISA, screening positive samples should be confirmed using PAP/AUC method, in order to reduce the workload and usage of E-test test strips,4. In a mouse model of sepsis, mgrA could increase mortality of mice, increase occurrence and development of sepsis, compared with mgrA knockout group, Newman group of mice with more intense inflammatory response, more blood and tissue bacterial load and more severe histopathological changes. mgrA play an important role in the process of Staphylococcus aureus sepsis, in a mouse model of sepsis, we found mgrA expression help Staphylococcus aureus to evade the host macrophage phagocytosis and killing, adhesion to tissue and cause more severe diseases, accelerating the occurrence and development of sepsis. |
