本文關(guān)鍵詞：金黃色葡萄球菌流行病學(xué)及致病機制研究　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 金黃色葡萄球菌 MRSA SCCmec hVISA PAP/AUC 毒力基因 基因敲除 mgrA 敗血癥

【摘要】：[目的] 金黃色葡萄球菌是引起人類感染性疾病的主要病原菌,可分泌多種毒力因子,引起人體各個組織部位的感染,是血流感染最常見病原菌之一,近年來由于多重耐藥菌的不斷出現(xiàn),尤其是hVISA和VISA的出現(xiàn),給臨床診治帶來極大的困難。本課題收集湖北省11家醫(yī)院和中南地區(qū)(湖北省,湖南省和河南省)6家醫(yī)院共768株金黃色葡萄球菌,分析其耐藥性及分子流行病學(xué)特征,以便為臨床抗感染治療提供依據(jù)；采用PAP/AUC、MET和MHA5T三種方法檢測1ⅥSA,旨在尋找出一種可用于臨床檢測hVISA經(jīng)濟(jì)、準(zhǔn)確且簡便易行的方法；建立小鼠敗血癥模型,檢測小鼠體重、生存率、炎癥反應(yīng)、血液及組織細(xì)菌載量、組織病理學(xué)等變化,觀察mgrA基因?qū)π∈髷⊙Y進(jìn)程的影響,探討金黃色葡萄球菌敗血癥的致病機制,為金黃色葡萄球菌敗血癥臨床診治提供新思路。 [方法] 一.金黃色葡萄球菌毒力基因檢測及流行病學(xué)研究 1.收集湖北省11家醫(yī)院2010年5月1日至2010年12月31日分離的416株金黃色葡萄球菌及中南地區(qū)6家醫(yī)院2011年6月1日至2012年5月31日分離的352株金黃色葡萄球菌并通過革蘭染色、觸酶試驗、血漿凝固酶試驗和PCR的方法重新鑒定,瓊脂稀釋法檢測其對常用抗菌藥物的最小抑菌濃度(MIC)。 2.采用多重PCR方法對MRSA菌株進(jìn)行SCCmec、MLST、spa和agr分型,陽性標(biāo)本進(jìn)行測序并擴增分離自中南地區(qū)6家醫(yī)院無菌體液標(biāo)本中的金黃色葡萄球菌31個毒力基因。 3.使用WHONET5.6軟件分析藥敏結(jié)果,其他數(shù)據(jù)統(tǒng)計采用SPSS18.0軟件,以卡方檢驗或Fisher's精確檢驗比較組間差異(P0.05,表明差異具有統(tǒng)計學(xué)意義)。 二、異質(zhì)性萬古霉素中介金黃色葡萄球菌檢測方法及流行情況研究 1.使用PAP/AUC、MET和MHA5T三種方法檢測hVISA,并以PAP/AUC法作為金標(biāo)準(zhǔn),比較MHA5T和MET法檢測hVISA的敏感性及特異性。 2.數(shù)據(jù)使用SPSS18.0軟件進(jìn)行統(tǒng)計,組間比較采用卡方檢驗或Fisher's精確檢驗(P0.05,表明差異具有統(tǒng)計學(xué)意義)。 三、mgrA基因表達(dá)對金黃色葡萄球菌敗血癥進(jìn)程的影響 1.重疊延伸PCR+同源重組的方法敲除金黃色葡萄球菌Newman株mgrA基因。 2.建立BALB/c小鼠敗血癥模型,實驗分為兩組,尾靜脈分別注射野生型Newman株和mgrA基因敲除株,即Newman組和mgrA knockout組,分析mgrA基因?qū)π∈髷⊙Y進(jìn)程的影響,實驗分為四個部分： (1)注射6×106CFU菌落,每組10只BABL/c小鼠,監(jiān)測至14天結(jié)束,比較兩組小鼠生存率。 (2)注射3×106CFU菌落,每組10只BABL/c小鼠,觀察至14天,觀察兩組小鼠的體重變化。 (3)注射6×106CFU菌落,每組每個時間點10只BABL/c小鼠監(jiān)測至120h后結(jié)束,摘除眼球取血,流式細(xì)胞儀檢測CD4+和CD8+T淋巴細(xì)胞,ELISA方法檢測小鼠血清IL-6和TNF-a水平。 (4)注射劑量：3×106CFU菌落,觀察至14天結(jié)束。分析兩組小鼠血液和組織(肝臟、脾臟和腎臟)細(xì)菌載量及組織病理學(xué)差異(HE染色)。細(xì)胞因子水平、血液和組織細(xì)菌載量用mean±SD表示。 3.所有數(shù)據(jù)均采用SPSS18.0軟件進(jìn)行分析,生存曲線分析使用Kaplan-Meier法,小鼠體重改變采用Manne-Whitney U-test法,細(xì)胞因子水平、血液和組織細(xì)菌載量結(jié)果用Student's t-test進(jìn)行分析,P0.05具有統(tǒng)計學(xué)差異。 [結(jié)果] 一.金黃色葡萄球菌毒力基因檢測及流行病學(xué)研究 1.湖北省11家醫(yī)院416株金黃色葡萄球菌共檢出209株MRSA, MRSA檢出率為50.2%。SCCmec分型分別為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.中南地區(qū)6家醫(yī)院352株金黃色葡萄球菌共檢出118株MRSA,檢出率為33.5%。SCCmec分型主要為Ⅲ型,占80.5%(95/118),SCCmecⅣ型和Ⅱ型分別占8.5%(10/118)和5.1%(6/118)。agr分型分別為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.中南地區(qū)6家醫(yī)院分離自無菌體液標(biāo)本的184株金黃色葡萄球菌中MRSA流行率為41.8%(77/184)。MRSA和MSSA主要的流行克隆分別為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的流行率明顯高于MRSA菌株(P0.05),98株MSSA(53.3%)同時攜帶≥10個毒力基因,明顯高于MRSA菌株(33.8%,26/77)(p=0.004),24株non-hVISA(40.0%)同時攜帶≥10個毒力基因,明顯高于VISA菌株(11.8%,2/17)(P=0.041)。與其它CCs克隆相比,CC1和CC398克隆表皮剝脫素基因檢出率更高(P0.05),CC1和CC59克隆中pvl基因檢出率更高(P0.05)。 二、異質(zhì)性萬古霉素中介金黃色葡萄球菌檢測方法及流行情況研究 1.與PAP/AUC法相比,MET法的敏感性和特異性分別為81.6%和92.4%,MHA5T法的敏感性和特異性分別為92.1%和62.6%。 2.327株MRSA中共檢出hVISA58株,hVISA流行率為17.7%。其中湖北省11家醫(yī)院209株MRSA中有共檢出hVISA38株,流行率為18.2%；中南地區(qū)6家醫(yī)院118株MRSA中共檢出hVISA20株,流行率為16.9%。 3.58株hVISA的主要流行克隆為ST239-SCCmecⅢ-t030-agrI型,占84.5%(49/58)。耐藥性監(jiān)測發(fā)現(xiàn)hVISA對妥布霉素和左氧氟沙星的耐藥率明顯高于VSSA(P0.05)。 三、mgrA基因表達(dá)對金黃色葡萄球菌敗血癥進(jìn)程的影響 1.兩組小鼠生存率和體重：Newman組有7只小鼠死亡(病死率70%),而mgrA knockout組僅有1只小鼠死亡(病死率10%),兩組間病死率具有明顯差異(logrank=4.739, P=0.029)。在兩組小鼠分別注射3×106CFU劑量的細(xì)菌時監(jiān)測發(fā)現(xiàn),Newman組小鼠比mgrA knockout組體重下降更明顯((P0.05)。 2.兩組小鼠CD4+/CD8+T淋巴細(xì)胞比值和血清TNF-a, IL-6水平：CD4+/CD8+T淋巴細(xì)胞比值在感染細(xì)菌24h達(dá)到高峰,在24h(P=0.0011)、8h(P=0.0025)和72h(P=0.0243)時,mgrAknockout組CD4+/CD8+T細(xì)胞比值明顯低于Newman組。敗血癥誘導(dǎo)后12h血清IL-6水平達(dá)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)時,Newman組的血清IL-6水平顯著高于mgrA knockout組。血清TNF-a水平在敗血癥誘導(dǎo)后4h升至最高,Newman組明顯高于mgrA knockout組(P=0.0032)。 3.兩組小鼠血液和組織細(xì)菌載量：Newman組小鼠腎臟和肝臟組織的細(xì)菌載量明顯高于mgrA knockout組(Log10CFU/g腎臟7.53±0.23vs6.02±0.33, P0.001; Log10CFU/g肝臟：5.82±0.26vs4.84±0.29,P=0.024)。兩組小鼠脾臟細(xì)菌載量沒有明顯差異(Log1oCFU/g脾臟：4.67±0.19vs4.51±0.54,P=0.404)。 4.組織病理學(xué)改變：在注射細(xì)菌后1d Newman組小鼠出現(xiàn)輕微的組織病理學(xué)改變,而mgrA knockout組沒有發(fā)現(xiàn)明顯改變。2d和4d時,兩組小鼠肝臟和腎臟出現(xiàn)輕至中度病理學(xué)改變,脾臟出現(xiàn)輕微的病理學(xué)改變,脾臟體積明顯增大。Newman組病理學(xué)改變明顯比mgrA knockout組嚴(yán)重,7d時,兩組小鼠均出現(xiàn)嚴(yán)重的病理學(xué)變化。 [結(jié)論] 1.中南地區(qū)分離自無菌體液標(biāo)本中的MRSA菌株,主要流行克隆為ST239-MRSA-SCCmecⅢ-t030-agrⅠ型,占55.8%,表明本地區(qū)存在耐藥株的傳播,醫(yī)療部門應(yīng)采取有效的措施控制耐藥克隆的傳播,如加強手部衛(wèi)生、隔離患者、嚴(yán)格注意無菌操作、加強醫(yī)院環(huán)境衛(wèi)生等。 2.湖北省11家醫(yī)院及中南地區(qū)6家醫(yī)院768株金黃色葡萄球菌中hVISA流行率達(dá)17.7%。因hVISA主要由糖肽類藥物選擇性壓力產(chǎn)生,故臨床應(yīng)重視合理使用抗生素。 3. PAP/AUC法是目前檢測hVISA最準(zhǔn)確的方法,但由于操作較為繁瑣,難以應(yīng)用于臨床,MHA5T法檢測hVISA的敏感性高達(dá)92.1%,推薦其可用于臨床hVISA的初篩,初篩陽性的標(biāo)本可再使用PAP/AUC法進(jìn)行確證,以減少工作量和E-test實驗條的使用。 4. mgrA基因在金黃色葡萄球菌敗血癥進(jìn)程中發(fā)揮重要作用,在小鼠敗血癥模型中,mgrA可增加小鼠的死亡率,加劇敗血癥的發(fā)生和發(fā)展,與mgrA knockout組相比,Newman組小鼠具有更激烈的炎癥反應(yīng),更多的血液和組織細(xì)菌載量和更嚴(yán)重的組織病理學(xué)變化, mgrA基因表達(dá)有助于金黃色葡萄球菌逃避宿主巨噬細(xì)胞的吞噬及殺傷,粘附至組織引起更嚴(yán)重的疾病,加劇敗血癥的發(fā)生和發(fā)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R515.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 李麗;田磊;張蓓;朱旭慧;陳中舉;王斌;朱琴;孫自鏞;;Mohnarin 2009年度報告:中南地區(qū)細(xì)菌耐藥監(jiān)測[J];中國臨床藥理學(xué)雜志;2011年07期

2 汪復(fù);朱德妹;胡付品;阮斐怡;倪語星;孫景勇;徐英春;張小江;胡云健;艾效曼;俞云松;楊青;孫自鏞;李麗;賈蓓;黃文祥;卓超;蘇丹虹;魏蓮花;吳玲;張朝霞;季萍;王傳清;薛建昌;張泓;李萬華;徐元宏;沈繼錄;單斌;杜艷;;2009年中國CHINET細(xì)菌耐藥性監(jiān)測[J];中國感染與化療雜志;2010年05期

3 朱德妹;汪復(fù);胡付品;蔣曉飛;倪語星;孫景勇;徐英春;張小江;胡云健;艾效曼;俞云松;楊青;孫自鏞;陳中舉;賈蓓;黃文祥;卓超;蘇丹虹;魏蓮花;吳玲;張朝霞;季萍;王傳清;王愛敏;張泓;孔菁;徐元宏;沈繼錄;單斌;杜艷;;2010年中國CHINET細(xì)菌耐藥性監(jiān)測[J];中國感染與化療雜志;2011年05期

4 楊青;俞云松;孫自鏞;陳中舉;徐英春;張小江;汪復(fù);朱德妹;胡付品;卓超;蘇丹虹;張泓;孔菁;張朝霞;季萍;王傳清;王愛敏;倪語星;孫景勇;單斌;杜艷;胡云建;艾效曼;徐元宏;沈繼錄;黃文祥;賈蓓;魏蓮花;吳玲;;2011年中國CHINET呼吸道病原菌分布和耐藥性監(jiān)測[J];中國感染與化療雜志;2013年05期

，

本文編號：1421863