金黄色葡萄球菌流行病学及致病机制研究

发布时间：2018-01-14 03:32

本文关键词：金黄色葡萄球菌流行病学及致病机制研究　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：[目的] 金黄色葡萄球菌是引起人类感染性疾病的主要病原菌,可分泌多种毒力因子,引起人体各个组织部位的感染,是血流感染最常见病原菌之一,近年来由于多重耐药菌的不断出现,尤其是hVISA和VISA的出现,给临床诊治带来极大的困难。本课题收集湖北省11家医院和中南地区(湖北省,湖南省和河南省)6家医院共768株金黄色葡萄球菌,分析其耐药性及分子流行病学特征,以便为临床抗感染治疗提供依据；采用PAP/AUC、MET和MHA5T三种方法检测1ⅥSA,旨在寻找出一种可用于临床检测hVISA经济、准确且简便易行的方法；建立小鼠败血症模型,检测小鼠体重、生存率、炎症反应、血液及组织细菌载量、组织病理学等变化,观察mgrA基因对小鼠败血症进程的影响,探讨金黄色葡萄球菌败血症的致病机制,为金黄色葡萄球菌败血症临床诊治提供新思路。 [方法] 一.金黄色葡萄球菌毒力基因检测及流行病学研究 1.收集湖北省11家医院2010年5月1日至2010年12月31日分离的416株金黄色葡萄球菌及中南地区6家医院2011年6月1日至2012年5月31日分离的352株金黄色葡萄球菌并通过革兰染色、触酶试验、血浆凝固酶试验和PCR的方法重新鉴定,琼脂稀释法检测其对常用抗菌药物的最小抑菌浓度(MIC)。 2.采用多重PCR方法对MRSA菌株进行SCCmec、MLST、spa和agr分型,阳性标本进行测序并扩增分离自中南地区6家医院无菌体液标本中的金黄色葡萄球菌31个毒力基因。 3.使用WHONET5.6软件分析药敏结果,其他数据统计采用SPSS18.0软件,以卡方检验或Fisher's精确检验比较组间差异(P0.05,表明差异具有统计学意义)。二、异质性万古霉素中介金黄色葡萄球菌检测方法及流行情况研究 1.使用PAP/AUC、MET和MHA5T三种方法检测hVISA,并以PAP/AUC法作为金标准,比较MHA5T和MET法检测hVISA的敏感性及特异性。 2.数据使用SPSS18.0软件进行统计,组间比较采用卡方检验或Fisher's精确检验(P0.05,表明差异具有统计学意义)。三、mgrA基因表达对金黄色葡萄球菌败血症进程的影响 1.重叠延伸PCR+同源重组的方法敲除金黄色葡萄球菌Newman株mgrA基因。 2.建立BALB/c小鼠败血症模型,实验分为两组,尾静脉分别注射野生型Newman株和mgrA基因敲除株,即Newman组和mgrA knockout组,分析mgrA基因对小鼠败血症进程的影响,实验分为四个部分： (1)注射6×106CFU菌落,每组10只BABL/c小鼠,监测至14天结束,比较两组小鼠生存率。 (2)注射3×106CFU菌落,每组10只BABL/c小鼠,观察至14天,观察两组小鼠的体重变化。 (3)注射6×106CFU菌落,每组每个时间点10只BABL/c小鼠监测至120h后结束,摘除眼球取血,流式细胞仪检测CD4+和CD8+T淋巴细胞,ELISA方法检测小鼠血清IL-6和TNF-a水平。 (4)注射剂量：3×106CFU菌落,观察至14天结束。分析两组小鼠血液和组织(肝脏、脾脏和肾脏)细菌载量及组织病理学差异(HE染色)。细胞因子水平、血液和组织细菌载量用mean±SD表示。 3.所有数据均采用SPSS18.0软件进行分析,生存曲线分析使用Kaplan-Meier法,小鼠体重改变采用Manne-Whitney U-test法,细胞因子水平、血液和组织细菌载量结果用Student's t-test进行分析,P0.05具有统计学差异。 [结果] 一.金黄色葡萄球菌毒力基因检测及流行病学研究 1.湖北省11家医院416株金黄色葡萄球菌共检出209株MRSA, MRSA检出率为50.2%。SCCmec分型分别为Ⅲ型,占82.8%(173/209)；Ⅳ型占13.4%(28/209)、Ⅱ型2.4%(5/209)和V型,占1.4%(3/209)。 2.中南地区6家医院352株金黄色葡萄球菌共检出118株MRSA,检出率为33.5%。SCCmec分型主要为Ⅲ型,占80.5%(95/118),SCCmecⅣ型和Ⅱ型分别占8.5%(10/118)和5.1%(6/118)。agr分型分别为Ⅰ型,占81.4%(96/118),Ⅱ型,占15.3%(18/118),Ⅲ型,占1.7%(2/118),Ⅳ型占1.7%(2/118)。 3.中南地区6家医院分离自无菌体液标本的184株金黄色葡萄球菌中MRSA流行率为41.8%(77/184)。MRSA和MSSA主要的流行克隆分别为ST239-SCCmecⅢ-t030-agr-I(43/77,占55.8%)和ST188-MSSA-tl89-agr-I(22/107,占20.6%)。 4. MSSA菌株中pvl, tst, hlb, hlg-v, seb, sed和sei的流行率明显高于MRSA菌株(P0.05),98株MSSA(53.3%)同时携带≥10个毒力基因,明显高于MRSA菌株(33.8%,26/77)(p=0.004),24株non-hVISA(40.0%)同时携带≥10个毒力基因,明显高于VISA菌株(11.8%,2/17)(P=0.041)。与其它CCs克隆相比,CC1和CC398克隆表皮剥脱素基因检出率更高(P0.05),CC1和CC59克隆中pvl基因检出率更高(P0.05)。二、异质性万古霉素中介金黄色葡萄球菌检测方法及流行情况研究 1.与PAP/AUC法相比,MET法的敏感性和特异性分别为81.6%和92.4%,MHA5T法的敏感性和特异性分别为92.1%和62.6%。 2.327株MRSA中共检出hVISA58株,hVISA流行率为17.7%。其中湖北省11家医院209株MRSA中有共检出hVISA38株,流行率为18.2%；中南地区6家医院118株MRSA中共检出hVISA20株,流行率为16.9%。 3.58株hVISA的主要流行克隆为ST239-SCCmecⅢ-t030-agrI型,占84.5%(49/58)。耐药性监测发现hVISA对妥布霉素和左氧氟沙星的耐药率明显高于VSSA(P0.05)。三、mgrA基因表达对金黄色葡萄球菌败血症进程的影响 1.两组小鼠生存率和体重：Newman组有7只小鼠死亡(病死率70%),而mgrA knockout组仅有1只小鼠死亡(病死率10%),两组间病死率具有明显差异(logrank=4.739, P=0.029)。在两组小鼠分别注射3×106CFU剂量的细菌时监测发现,Newman组小鼠比mgrA knockout组体重下降更明显((P0.05)。 2.两组小鼠CD4+/CD8+T淋巴细胞比值和血清TNF-a, IL-6水平：CD4+/CD8+T淋巴细胞比值在感染细菌24h达到高峰,在24h(P=0.0011)、8h(P=0.0025)和72h(P=0.0243)时,mgrAknockout组CD4+/CD8+T细胞比值明显低于Newman组。败血症诱导后12h血清IL-6水平达到峰值,之后迅速下降,4h(P0.0001)、12h(P0.0001)、24h(P0.0001)、48h(P=0.0003)和72h(P=0.0012)时,Newman组的血清IL-6水平显著高于mgrA knockout组。血清TNF-a水平在败血症诱导后4h升至最高,Newman组明显高于mgrA knockout组(P=0.0032)。 3.两组小鼠血液和组织细菌载量：Newman组小鼠肾脏和肝脏组织的细菌载量明显高于mgrA knockout组(Log10CFU/g肾脏7.53±0.23vs6.02±0.33, P0.001; Log10CFU/g肝脏：5.82±0.26vs4.84±0.29,P=0.024)。两组小鼠脾脏细菌载量没有明显差异(Log1oCFU/g脾脏：4.67±0.19vs4.51±0.54,P=0.404)。 4.组织病理学改变：在注射细菌后1d Newman组小鼠出现轻微的组织病理学改变,而mgrA knockout组没有发现明显改变。2d和4d时,两组小鼠肝脏和肾脏出现轻至中度病理学改变,脾脏出现轻微的病理学改变,脾脏体积明显增大。Newman组病理学改变明显比mgrA knockout组严重,7d时,两组小鼠均出现严重的病理学变化。 [结论] 1.中南地区分离自无菌体液标本中的MRSA菌株,主要流行克隆为ST239-MRSA-SCCmecⅢ-t030-agrⅠ型,占55.8%,表明本地区存在耐药株的传播,医疗部门应采取有效的措施控制耐药克隆的传播,如加强手部卫生、隔离患者、严格注意无菌操作、加强医院环境卫生等。 2.湖北省11家医院及中南地区6家医院768株金黄色葡萄球菌中hVISA流行率达17.7%。因hVISA主要由糖肽类药物选择性压力产生,故临床应重视合理使用抗生素。 3. PAP/AUC法是目前检测hVISA最准确的方法,但由于操作较为繁琐,难以应用于临床,MHA5T法检测hVISA的敏感性高达92.1%,推荐其可用于临床hVISA的初筛,初筛阳性的标本可再使用PAP/AUC法进行确证,以减少工作量和E-test实验条的使用。 4. mgrA基因在金黄色葡萄球菌败血症进程中发挥重要作用,在小鼠败血症模型中,mgrA可增加小鼠的死亡率,加剧败血症的发生和发展,与mgrA knockout组相比,Newman组小鼠具有更激烈的炎症反应,更多的血液和组织细菌载量和更严重的组织病理学变化, mgrA基因表达有助于金黄色葡萄球菌逃避宿主巨噬细胞的吞噬及杀伤,粘附至组织引起更严重的疾病,加剧败血症的发生和发展。
[Abstract]:[Objective]
Staphylococcus aureus is a major pathogen of human infectious disease bacteria can secrete a variety of virulence factors, cause all human tissue infection, is one of the most common pathogen of bloodstream infection, in recent years due to the emergence of multi drug resistant bacteria, especially hVISA and VISA, and brings great difficulties to the clinical diagnosis and treatment. This paper collected 11 hospitals in Hubei province and the South Region (Hubei Province, Hunan province and Henan province) in 6 hospitals of a total of 768 strains of Staphylococcus aureus, analyze its drug resistance and molecular epidemiology characteristics, so as to provide evidences for clinical anti infection treatment; using PAP/AUC, MET and MHA5T three kinds of detection methods of 1 SA, to find a can be used for clinical detection of hVISA economic, simple and convenient tool; establish a mouse model of sepsis, detection of mice weight, survival rate, inflammation, blood and tissue bacterial load, organization We observed the effect of mgrA gene on septicemia in mice, and discussed the pathogenic mechanism of Staphylococcus aureus septicemia, so as to provide new ideas for the clinical diagnosis and treatment of Staphylococcus aureus septicemia.
[method]
1. Detection and epidemiological study of virulence gene of Staphylococcus aureus
1. collected from 11 hospitals in Hubei province from May 1, 2010 to December 31, 2010 of 416 strains of Staphylococcus aureus and the central area of 6 hospitals from June 1, 2011 to May 31, 2012 of 352 strains of Staphylococcus aureus and the Gram staining, catalase test method, plasma coagulase test and PCR re examination, to detect the minimal inhibitory concentration of antibiotics the agar dilution method (MIC).
2. multiplex PCR method was used to classify MRSA strains by SCCmec, MLST, spa and agr. The positive samples were sequenced and amplified, and 31 virulence genes of Staphylococcus aureus isolated from 6 hospitals in South China were collected.
3., using WHONET5.6 software to analyze drug sensitivity results, the other data were statistically analyzed by chi square test or Fisher's exact test using SPSS18.0 software. The difference between groups was statistically significant (P0.05).
Two, study on the detection methods and epidemic situation of Staphylococcus aureus mediated by heterogeneous vancomycin
1. PAP/AUC, MET and MHA5T were used to detect hVISA, and PAP/AUC was used as the gold standard. The sensitivity and specificity of hVISA were compared by MHA5T and MET methods.
2. the data were calculated using SPSS18.0 software. The group was compared with the chi square test or the Fisher's accurate test (P0.05, indicating that the difference was statistically significant).
Three, the effect of mgrA gene expression on the process of Staphylococcus aureus septicemia
1. overlapping extended PCR+ homologous recombination method knocks out the mgrA gene of Staphylococcus aureus Newman strain.
2., BALB/c mice septicemia model was established. The experiment was divided into two groups. The wild type Newman and mgrA gene knockout strains were injected into the caudal vein, namely Newman group and mgrA knockout group. The influence of mgrA gene on sepsis process of mice was analyzed. The experiment was divided into four parts.
(1) injection of 6 x 106CFU colonies, each group of 10 BABL/c mice, monitored to the end of the 14 day, compared the survival rate of the two groups.
(2) injection of 3 x 106CFU colonies, each group of 10 BABL/c mice, observed for 14 days, to observe the body weight changes of the two groups.
(3) 6 x 106CFU colonies were injected, and 10 BABL/c mice at each time point were monitored until 120h. The eyeballs were removed. Blood and CD4+ and CD8+T lymphocytes were detected by flow cytometry. The levels of IL-6 and TNF-a in serum of mice were detected by ELISA.
(4) injection dose: 3 * 106CFU colonies, observed to the end of 14 days. Analysis of two groups of mice blood and tissue (liver, spleen and kidney) bacterial load and histopathological difference (HE staining). Cytokine levels, blood and tissue bacterial load was expressed by mean + SD.
3. all data were analyzed by SPSS18.0 software. Survival curve was analyzed by Kaplan-Meier method. The weight change of mice was determined by Manne-Whitney U-test method, cytokine level, blood and tissue bacterial load results were analyzed by Student's t-test, P0.05 had statistical difference.
[results]
1. Detection and epidemiological study of virulence gene of Staphylococcus aureus
1., 209 strains of MRSA were detected in 416 strains of Staphylococcus aureus in 11 hospitals in Hubei province. The detection rate of MRSA was 50.2%.SCCmec type, which accounted for 82.8% (173/209), type IV accounted for 13.4% (28/209), type II 2.4% (5/209) and V type, accounting for 1.4% (3/209).
6 2. hospitals in central and southern regions of 352 strains of Staphylococcus aureus were detected in 118 strains of MRSA, the detection rate of 33.5%.SCCmec type is mainly type III, accounting for 80.5% (95/118), SCCmec type IV and type II accounted for 8.5% (10/118) and 5.1% (6/118).Agr type were type I, accounting for 81.4% (96/118), type II, accounting for 15.3% (18/118), type III, accounting for 1.7% (2/118), type IV accounted for 1.7% (2/118).
3. the prevalence of MRSA in 6 strains of Staphylococcus aureus isolated from 6 hospitals in central and southern China is 41.8% (77/184).MRSA and MSSA. The main epidemic clones are ST239-SCCmec III -t030-agr-I (43/77, 55.8%) and ST188-MSSA-tl89-agr-I (22/107, 20.6%).
4. MSSA strains PVL, TST, HLB, hlg-v, SEB, SED and SEI prevalence rate was significantly higher than that of MRSA strain (P0.05), 98 strains of MSSA (53.3%) carrying more than 10 virulence genes at the same time, significantly higher than that of MRSA strains (33.8%, 26/77) (p=0.004), 24 strains (40%) carrying more than 10 non-hVISA virulence genes at the same time, significantly higher than that of VISA strains (11.8%, 2/17) (P=0.041). Compared with other CCs clones, CC1 and CC398 cloned exfoliatin gene high detection rate (P0.05), CC1 and CC59 cloned PVL gene in high detection rate (P0.05).
Two, study on the detection methods and epidemic situation of Staphylococcus aureus mediated by heterogeneous vancomycin
1. compared with the PAP/AUC method, the sensitivity and specificity of the MET method were 81.6% and 92.4% respectively. The sensitivity and specificity of the MHA5T method were 92.1% and 62.6%., respectively.
A total of 2.327 strains of MRSA were detected hVISA58 strains. The prevalence of hVISA was 17.7%.. Among the 209 hospitals in 11 hospitals in Hubei Province, there were 18.2% hVISA38 strains detected. The prevalence rate was 18.2%. In the 6 hospitals of central and southern China 118 strains of MRSA were detected hVISA20 strains, the prevalence rate was 16.9%..
The main epidemic clones of 3.58 strains of hVISA ST239-SCCmec type III -t030-agrI, accounting for 84.5% (49/58). Drug resistance monitoring found that hVISA resistant to tobramycin and levofloxacin was significantly higher than that of VSSA (P0.05).
Three, the effect of mgrA gene expression on the process of Staphylococcus aureus septicemia
1. two groups of mice survival rate and body weight: Newman group of 7 mice died (mortality rate 70%), while the mgrA knockout group only 1 mice died (mortality rate 10%), the mortality rate between the two groups have significant differences (logrank=4.739, P=0.029). In the two group were injected with the dose of 3 x 106CFU bacteria respectively monitoring found that the mice in Newman group than mgrA group significantly decreased (knockout weight (P0.05).
2. two groups of mice CD4+/CD8+T lymphocytes and the ratio of serum TNF-a, IL-6 levels: CD4+/CD8+T lymphocyte ratio in bacterial infection peaked at 24h in 24h (P=0.0011), 8h (P=0.0025) and 72h (P=0.0243), mgrAknockout group, CD4+/CD8+T cell ratio was significantly lower than that of Newman group. The serum level of IL-6 induced by sepsis after 12h reached the peak, then decreased rapidly 4h, (P0.0001), 12h (P0.0001), 24h (P0.0001), 48h (P=0.0003) and 72h (P=0.0012), serum IL-6 level of Newman group was significantly higher than that in mgrA knockout group. The serum level of TNF-a in sepsis induced 4H rose to the highest in Newman group was significantly higher than that in mgrA knockout group (P=0.0032).
3. two groups of blood and tissues of mice with bacterial load: Newman mice kidney and liver tissue of the bacterial load was significantly higher than that of mgrA group knockout (Log10CFU/g kidney 7.53 + 0.23vs6.02 + 0.33, P0.001; Log10CFU/g of the liver: 5.82 + 0.26vs4.84 + 0.29, P=0.024). The two groups of mice spleen bacterial load did not differ significantly (Log1oCFU/g spleen: 4.67 + 0.19vs4.51 + 0.54, P=0.404).
4. changes of histopathology: histopathologic slight in group Newman after injection of bacteria 1D mice and mgrA change, knockout group found no significant changes in.2d and 4D, two groups of mice liver and kidney appeared mild to moderate pathological changes of spleen, slight pathological change, the spleen volume increased significantly in.Newman group the pathological changes were significantly higher than mgrA group knockout seriously, 7d, two groups of mice showed severe pathological changes.
[Conclusion]
The 1. south region of MRSA isolates from sterile specimens, the main epidemic ST239-MRSA-SCCmec clone for type III -t030-agr, accounting for 55.8%, showed the presence of dissemination of resistant strains in the region, health departments should take effective measures to control the spread of resistant clones, such as strengthening the hand hygiene, isolation of patients, strict aseptic operation, strengthen the hospital the environment and health.
2. the prevalence of hVISA in 768 strains of Staphylococcus aureus in 11 hospitals and 6 hospitals in central and southern Hubei reached 17.7%., because hVISA was mainly caused by selective pressure of glycopeptide drugs. Therefore, the rational use of antibiotics should be emphasized in clinical practice.
3. PAP/AUC method is currently the most accurate detection of hVISA, but because the operation is complex, difficult to clinical application, the sensitivity of detection of hVISA MHA5T by up to 92.1%, which can be used for clinical recommended hVISA screening, screening positive samples can be confirmed by the method of PAP/AUC, to reduce the workload and the use of E-test.
4. mgrA genes play an important role in Staphylococcus aureus septicemia in the process, in a mouse model of sepsis, mgrA can increase the mortality of mice, increased the occurrence and development of sepsis, compared with mgrA knockout group, Newman group of mice with inflammatory reaction more intense, more changes of blood and tissue bacterial load and more serious pathology, the expression of mgrA gene contributes to the phagocytosis and killing of Staphylococcus aureus evade the host macrophage adhesion to tissue, cause more serious diseases, aggravate the occurrence and development of sepsis.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R515.3

【参考文献】