模拟胆汁体系中胆固醇结石内细菌对胆固醇晶体形成的作用

目的 探讨胆固醇结石中的细菌与结石形成的关系。方法 观察胆固醇结石中的细菌在模拟胆汁中的生长活性及其对胆固醇晶体成核时间(NT)的影响。结果 (1)模拟胆汁中大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、粪肠球菌、产孢梭菌和艰难梭菌生长旺盛;痤疮丙酸杆菌长势偏弱;脆弱类杆菌生长受抑制。(2)仅铜绿假单胞菌和粪肠球菌能缩短胆固醇晶体成核时间。(3)加入铜绿假单胞菌或粪肠球菌的胆汁,晶体形成呈现缓慢的阶段性演变过程。结论 铜绿假单胞菌和粪肠球菌在模拟胆汁中具有促成核活性,而痤疮丙酸杆菌无促成核作用。     

胆固醇结石患者胆囊细菌感染的研究

目的 比较胆固醇类结石患者和非胆石症患者胆道细菌感染情况.方法 用不依赖细菌培养的半定量PCR和16SrRNA序列对照法,检测76例胆固醇类结石患者胆囊黏膜、胆汁和胆石中细菌DNA,与34例非胆石患者对照.结果 胆石组和非胆石组胆汁细菌DNA阳性率分别为77%和67%,胆囊黏膜细菌DNA阳性率分别为64%和69%,两组间差异均无统计学意义(P＞0.05).胆石组细菌种类主要为大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、不动杆菌、链球菌、鞘氨醇单胞菌、脆弱类杆菌和痤疮丙酸杆菌,胆石组菌种比非胆石组丰富,非胆石组菌种基本上均能在胆石组中找到.结论 胆固醇结石与非胆石症患者胆道细菌感染率相似,胆固醇结石中存在细菌不足以证明细菌参与胆固醇结石的形成.     

细菌在含有游离胆红素的模拟胆汁体系中对胆固醇晶体形成的影响

目的探讨在含有游离胆红素(UCB)的模拟胆汁体系中细菌对胆固醇晶体形成的作用。方法大肠杆菌、铜绿假单胞菌和粪肠球菌分别加入含有不同浓度游离胆红素的模拟胆汁中,观察胆汁中胆固醇晶体形成时间(NT)的变化。结果游离胆红素缩短模拟胆汁中胆固醇晶体形成时间。铜绿假单胞菌和粪肠球菌缩短胆固醇晶体形成时间,但是同时加入游离胆红素后,NT未见进一步缩短。在CSI 0.6时单加铜绿假单胞菌的胆汁比单加UCB时的晶体形成时间更短。结论模拟胆汁体系中,细菌和游离胆红素对胆固醇晶体形成无协同作用。     

细菌对模拟胆汁热力学平衡的影响

目的:探讨细菌对模拟胆汁物理鄄化学平衡的影响。方法:观察加入大肠杆菌、粪肠球菌、铜绿假单胞菌和生理盐水的模拟胆汁中胆固醇晶体形成的时间和胆汁成分析出黏附的时间,测量胆汁黏度值和胆汁类脂成分溶解度的变化。结果:加入粪链球菌或铜绿假单胞菌的模拟胆汁,黏度值增加,试管壁上有黏附物质析出。加入大肠杆菌或生理盐水的模拟胆汁中,无类似变化。在不同的胆固醇饱和条件下,黏附物质析出的时间早于或等于胆固醇晶体形成的时间。胆固醇溶解度在加入粪链球菌或铜绿假单胞菌的模拟胆汁中,低于在加入大肠杆菌或生理盐水的模拟胆汁;胆汁酸盐或磷脂的溶解度无显著差异。胆汁析出的黏附物质中胆固醇是主要成分之一。结论:粪链球菌或铜绿假单胞菌增加模拟胆汁的黏度并有黏附物质形成,而降低胆固醇的溶解度。     

细菌促胆固醇晶体成核的机制研究

目的 定位铜绿假单胞茼、粪肠球菌促胆固醇晶体成棱活性来源,并探讨其作用途径.方法 分离细菌的外分泌物、细胞破坏后的上清和沉淀裂解组分.观察其在模拟胆汁体系中对胆固醇晶体形成的时间的影响,并对活性组分进行蛋白质提纯及促成核活性现察.结果 ①粪肠球菌和铜绿假单胞菌的外分泌组分和破壁上清组分能缩短胆固醇晶体成核时间(nucleation time,NT),破壁后沉淀物的裂解成分未见此活性.②大肠杆菌的外分泌组分不能缩短胆固醇晶体成核时间,但是其破壁上清组分能缩短胆固醇晶体成核时间.③从外分泌组分和破壁上清组分中提纯的蛋白质仍具有缩短胆固醇晶体成核时间的能力.结论 粪肠球菌和铜绿假单胞菌的外分泌组分和破壁上清组分,大肠杆菌的破壁上清组分具有促进胆固醇晶体形成的能力.蛋白质是重要的活性成分.     

外科感染患者细菌分布及耐药性的单中心研究

目的 分析外科感染患者细菌分布及其对常用抗菌药物的耐药性,为外科感染的规范化治疗提供依据.方法 回顾性调查分析2008年1月至201 1年12月外科感染患者送检标本的细菌鉴定及药物敏感性检测结果.结果 3257份临床标本共分离菌株3829株,革兰阴性杆菌占62.4％(以大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主);革兰阳性球菌占37.6％(以肠球菌、金黄色葡萄球菌及凝固酶阴性葡萄球菌为主),其中金黄色葡萄球菌、粪肠球菌检出率呈升高趋势.大肠埃希菌及肺炎克雷伯菌对亚胺培南、阿米卡星、哌拉西林/他唑巴坦等抗菌药物耐药率较低;铜绿假单胞菌和鲍曼不动杆菌对头孢类、碳青霉烯类及喹诺酮类抗菌药物耐药率较高,呈多药耐药性;所有葡萄球菌、粪肠球菌对万古霉素和替考拉宁敏感(100％),但耐万古霉素屎肠球菌检出率呈上升趋势(1.9％～7.5％).产超广谱β-内酰胺酶(ESBL)大肠埃希菌检出率为45.6％ ～61.5％;产ESBL肺炎克雷伯菌检出率呈波动表现;耐甲氧西林金黄色葡萄球菌检出率较高(21.1％ ～55.8％),耐甲氧西林表皮葡萄球菌检出率明显高于其他阳性球菌.结论 我院外科临床感染病原菌以革兰阴性杆菌为主,临床分离细菌耐药现象较为普遍,铜绿假单胞菌和鲍曼不动杆菌药物耐药率较高.     

由胆固醇结石提取DNA进行套式聚合酶链反应的研究

为探讨微生物在胆固醇结石形成中的作用，作者从３０例胆汁细菌培养阴性的胆囊胆固醇结石中提取ＤＮＡ，应用套式聚合酶链反应（ＮＰ－ＰＣＲ）技术从中特异性地扩增细菌ＤＮＡ片段。结果显示２６例（８６．６７％）胆固醇结石中有细菌ＤＮＡ存在。此外，用１６ＳｒＲＮＡ基因序列对照分析鉴定细菌种类，８例（２６．６７％）为与大肠杆菌相似的ＤＮＡ片段；７例（２３．３３％）为痤疮丙酸杆菌型ＤＮＡ序列；２例（６．６７％）ＤＮＡ片段与化脓性链球菌相关；７例（２３．３３％）ＤＮＡ片段具有多样性，可能有多种细菌混合感染；另外２例（６．６７％）ＤＮＡ分子量较低，归类于其它未鉴定细菌。作者认为多数胆固醇结石内有细菌ＤＮＡ存在，但这些微生物的确切作用，有待于进一步深入研究。     

我院临床检出细菌的耐药现状分析

目的对我院2000年各科室送检标本细菌的检出及对抗生素耐药的情况进行分析,为临床抗感染治疗提供参考依据.方法细菌鉴定及药酶试验采用全自动细菌生化分析仪(Vitek-Ams).结果全年分离病原菌1193株,其中革兰氏阳性菌440株,革兰氏阴性菌753株;革兰氏阳性菌中包括金黄色葡萄球菌227株、肠球菌61株,金黄色葡萄球菌中包括耐甲氧西林金黄色葡萄球菌(MRSA)122株,占金黄色葡萄球菌的54%.革兰氏阴性菌中构成比最大的前三种病原菌为铜绿假单胞菌(24%)、肺炎克雷伯菌(16%)和大肠杆菌(12%).大肠杆菌和肺炎克雷伯菌超广谱酶(ESBLs)的产酶率分别为43%和39%.结论我院主要感染病原菌为革兰氏阴性杆菌,而金黄色葡萄球菌为主要的革兰氏阳病原菌;我院主要致病菌的构成与国内大医院基本相同,多重耐药菌占很大的比例.     

T管胆汁细菌培养及对抗生素敏感性分析

目的：了解临床已无感染症状病人的T管引流胆汁的细菌感染情况及对抗生素的敏感性。方法：对2001年12月至2002年12月116例术后已无感染症状病人的T管引流胆汁的细菌培养和抗生素敏感性试验结果作统计分析。结果：T管胆汁细菌培养的阳性率为59．91％。158株微生物中包括23种细菌和3种真菌。肺炎克雷伯菌、铜绿假单胞菌、大肠埃希菌、表皮葡萄球菌、金黄色葡萄球菌、粪肠球菌和尿肠球菌占胆道细菌的72．75％，24例混合感染中13例感染铜绿假单胞菌，同时发现嗜麦芽窄食单胞菌和鲍曼不动杆菌等条件致病菌感染。细菌对头孢类抗生素具有较高的耐药率，对亚胺培南、万古霉素、阿米卡星有较高的敏感性。在不同病人相同菌种的药敏试验结果有较大差异，提示菌株不同。结论：临床无感染症状病人的T管引流胆汁具有较高的细菌感染率，二重感染和耐药菌株的增加是一旦发生胆汁性腹膜炎时严重感染的基础。     

细菌与胆固醇结石

20世纪60年代Maki等确立了细菌在棕色素结石发病机制中的重要作用。虽然19世纪Moynihan(1856)和Naun-yn(1892)朦胧地提出细菌学说,但自建立Small三角学说以来,医学界始终认为胆固醇结石的发病机制以非生理性胆固醇过饱和、胆汁促抗成核失衡以及胆囊局部因素为基础,不包括细菌。直至1995年,德国Swidsinski等的研究才又使人们关注细菌与胆固醇结石的关系。     

经ERCP途径获取胆汁培养病原菌分布及耐药性分析

背景与目的:胆道感染是临床常见急腹症,处理不及时可引发感染性休克,甚至死亡.因此,了解胆道感染患者胆汁中细菌的种类及药敏情况对指导临床治疗至关重要.本研究分析胆道疾病患者经内镜逆行胰胆管造影(ERCP)途径获取胆汁培养病原菌分布及耐药情况,以期为临床胆道感染患者的抗菌药物合理使用提供依据.方法:回顾性分析2016年1月...     

Human gallstones contain pronucleating nonmucin glycoproteins that are immunoglobulins.

OBJECTIVE: Pronucleating nonmucin glycoproteins in human cholesterol and black gallstones were isolated and identified. SUMMARY BACKGROUND DATA: Gallbladder bile contains nonmucin glycoproteins that are pronucleating of cholesterol monohydrate crystals. Little is known about the presence or activity of these proteins within gallstones. METHODS: Nonmucin glycoproteins were isolated from single cholesterol (n = 8), multiple cholesterol (n = 8), and black pigment (n = 8) gallstones by concanavalin A lectin-affinity chromatography. The proteins were separated by sodium dodecyl sulfate gradient electrophoresis. Western blot analysis was performed for Fab immunoglobulin fragments, and heavy chains from the immunoglobulin G, A, E, and M subclasses. A crystal observation time assay was performed on the combination of isolated nonmucin glycoproteins from gallstones and isolated Fab fragments. RESULTS: Nonmucin glycoproteins of molecular weights 10, 15, 17, 22, 28, and 208 kD were identified in gallstones. These six nonmucin glycoproteins shortened the crystal observation time by more than 50% (p < 0.01) compared with model bile. Western blot analysis confirmed the identity of the 22- and 28-kD proteins as immunoglobulin Fab fragments. These were seen in all gallstones, irrespective of the gallstone type. The isolated Fab 28-kD fragment from the gallstones of 23 patients shortened the extrapolated crystal observation time by 78% (p < 0.01). However, commercially available Fab fragments had no effect on either cholesterol crystal appearance or growth. CONCLUSIONS: Nonmucin glycoproteins that are pronucleating for cholesterol monohydrate crystals are also found in human cholesterol and black pigment gallstones. Fab immunoglobulin fragments were found in all gallstones irrespective of the gallstone type. Fab immunoglobulin fragments from gallstones shortened the crystal observation time but not crystal growth or total crystal content compared with model bile or commercially available Fab fragments. These data suggest that an antigen-immune (Fab) complex may contribute to cholesterol crystal function.     

Occlusive dressings. Does dressing type influence the growth of common bacterial pathogens?

We studied the effect of different occlusive dressings and of air exposure on the growth of four pathogenic bacteria in wounds. Partial-thickness wounds on domestic pigs were inoculated with Staphylococcus aureus, Clostridium perfringens, Bacteroides fragilis, or Pseudomonas aeruginosa. Each wound was covered with three dressings (DuoDERM, Opsite, or Vigilon), or left exposed to air. Groups of wounds were sampled at 24, 48, and 72 hours. Staphylococcus aureus reached high levels beneath all of the dressings and in the air-exposed wounds. The numbers of C perfringens and B fragilis were greatly reduced in the air-exposed wounds and slightly reduced in the Opsite-covered wounds. The numbers of P aeruginosa were greatest in the Opsite- and Vigilon-covered wounds. The results indicate that occlusive dressings are not indicated in wounds that clinically appear to be grossly contaminated or that may contain anaerobic organisms.     

Clinical and experimental studies on the formation of calcium bilirubinate gallstones

We identified the bacteria in the bile and measured the activity of bacterial beta-glucuronidase and analyzed the percentage of bilirubin glucuronide and unconjugated bilirubin by high performance liquid chromatography in the bile of human biliary stone disease. The percentage of positive bacterial infection in the bile are 72.0% with calcium bilirubinate gallstone, and 42.3% with cholesterol gallstone. The activity of beta-Glucuronidase (U/dl.hr.) was significantly higher in the bile of calcium bilirubinate gallstone than that of cholesterol gallstone (7013 +/- 5113 vs 3338 +/- 2615, mean +/- S.D.). Also, the percentage of unconjugated bilirubin (IX alpha) of the bile was significantly higher in calcium bilirubinate gallstone than in cholesterol gallstone (5.7 +/- 4.7% vs 2.6 +/- 2.0%, mean +/- S.D.). The beta-Glucuronidase activity of bacteria was as follows; E. coli 18752, K. pneumoniae 333, E.cloacae 124, S.faecalis 324, and B.fragilis 983. After 60 minutes' incubation at 37 degrees C of normal bile with E.coli, the percentage of unconjugated bilirubin (IX alpha) increased from 1.1% to 9.1%. Whereas, the other four bacteria did not increase the unconjugated bilirubin at all. We confirmed that the increase of unconjugated bilirubin caused by the high bacterial beta-Glucuronidase activity was the most important factor in the formation of calcium bilirubinate gallstone.     

Experimental polymicrobial osteomyelitis produced by both aerobic and anaerobic opportunistic pathogens

Experimental models of polymicrobial osteomyelitis were prepared using clinical isolates of Staphylococcus epidermidis and Enterococcus faecalis as aerobes and Bacteroides fragilis and Bacteroides bivius as anaerobes. These pathogens were used because of their opportunistic properties. Two 8 mm long silk threads with the microorganism were inserted into the bone marrow of a rat. The microorganism inoculated was about 10(5) c.f.u. for aerobes and 10(6) c.f.u. for anaerobes. Infected parts observed in the roentgenograms, histopathological changes, and bacterial counts all showed the evolution of osteomyelitis. It was found that our models caused osteomyelitis both when each of S. epidermidis, E. faecalis, B. fragilis, and B. bivius was implanted individually and in their combinations. The patterns of the radiological and the histological observations were almost similar in all cases examined, but their characteristics differed depending on the kinds and combinations of the pathogens.     

运甲状腺素蛋白在胆固醇结石胆囊组织中的表达及其促成核作用研究

目的 探讨运甲状腺素蛋白（transthyretin,TTR）基因及其蛋白在胆固醇结石患者胆囊组织中的表达情况,以及其在胆固醇结石形成中的促成核作用。方法 收集笔者所在的两所医院施行择期胆囊切除术的25例胆固醇结石患者和9例肝移植供体的正常胆囊组织,采用逆转录-聚合酶链反应（RT-PCR）技术及Western blot法检测2组患者胆囊组织中TTR基因及其蛋白的表达。构建Small和综合模拟胆汁体系,分别加入TTR和白蛋白（ALB）,再通过Holan成核时间法和Holzbach法分别检测成核时间和成核活性。结果 胆固醇结石组TTR mRNA及其蛋白的表达水平分别为1.51±0.78和3.95±0.09,均高于正常对照组的0.85±0.63和1.53±0.08 （P＜0.05）。在Small模拟胆汁体系中,观察至21 d时,TTR组的成核时间为（14.5±1.3） d,ALB组为（18.0±0.8） d,TTR的成核活性为0.81;在综合模拟胆汁体系中,TTR组的成核时间为（13.5±0.6） d,ALB组为（18.5±1.3） d,TTR的成核活性为0.73。TTR组的成核时间均较短（P＜0.01）。结论 TTR在胆固醇结石患者胆囊组织中的表达上调,并在体外模拟胆汁体系中表现出促成核活性,提示其参与了胆固醇结石的发生和发展过程。