双歧杆菌、保加利亚乳杆菌、嗜热链球菌三联活菌去除肠道产超广谱β-内酰胺酶大肠埃希菌的研究

目的研究双歧杆菌、保加利亚乳杆菌、嗜热链球菌三联活菌对肠道产超广谱双歧杆菌、保加利亚乳-内酰胺酶（ESBL）大肠埃希菌的除菌作用,为临床辅助治疗大肠埃希菌感染提供依据。方法对2013年1月-9月5个病区1358例人院患者采集肛拭子标本进行产ESBL大肠埃希菌定植筛查,选取产ESBL大肠埃希菌阳性患者306例,随机分为试验组与对照组。试验组140例在常规消毒隔离措施基础上口服双歧杆菌、保加利亚乳杆菌、嗜热链球菌三联活菌去除肠道产ESBL大肠埃希菌,对照组166例仅采取常规消毒隔离措施。3～7d后,两组再次取肛拭子进行筛查。结果试验组87例未再检出产ESBL大肠埃希菌,双歧杆菌、保加利亚乳杆菌、嗜热链球菌三联活菌去除肠道产ESBL大肠埃希菌有效率为62．14％;对照组125例再次检出产ESBL大肠埃希菌,肠道自行清除率为24．70％,两组差异有统计学意义（Х^2=43．761,P〈0．001）。结论双歧杆菌、保加利亚乳杆菌、嗜热链球菌三联活菌具有清除肠道产ESBL大肠埃希菌的作用,清除率与使用时间长短、使用抗菌物有关。     

大黄对脓毒症大鼠胃肠道菌群的影响

目的探讨大黄对脓毒症大鼠胃肠道菌群的影响。方法 36只健康SD大鼠分为4组,正常对照组、烫伤组各8只,脓毒症组、大黄组各10只。采用大鼠背部烫伤模型,烫伤后24h分2次给予内毒素(20mg/kg)进行"二次打击",大黄组内毒素打击后即予大黄(100mg/kg,2次/d)灌胃。24h后分别取胃、小肠、大肠内容物行定量培养、菌种鉴定。结果烫伤、脓毒症大鼠肠道细菌总数增加,且肠球菌数量增加大于肠杆菌;肠杆菌也不再是单一的大肠杆菌,出现了铜绿假单胞菌、肺炎克雷伯菌、阴沟杆菌、变形杆菌及其他菌;厌氧菌数量显著减少,真菌数量、菌种增多。应用大黄后肠道细菌总数有所下降,肠杆菌中的大肠杆菌比例增加;厌氧菌数量增多,与正常对照组无统计学差异;对念珠菌数量影响不显著,但可以使致病菌株数量减少。结论大黄能减少胃肠道细菌和真菌数量及种类,保护肠道正常菌群,减少肠源性感染的发生。     

多重PCR和基因芯片检验致病性大肠埃希菌

致病性大肠埃希菌包括肠道致病性和肠道外致病性两大部分。两部分中又各包括多种致病性大肠埃希菌。长期以来,此类细菌的检测缺乏可常规应用的鉴别手段,成为临床细菌学检验的薄弱环节。此类感染的普遍性和严重性迫切需要可靠的致病性大肠埃希菌的检测技术。该文重点介绍多重PCR技术同时检测多种致腹泻大肠埃希菌以及基因芯片技术同时检测多种肠道内和肠道外感染的大肠埃希菌。     

佳木斯地区尿路感染患者产ESBLs和AmpC酶大肠埃希菌的耐药性分析

大肠埃希菌是存在于人体肠道中的正常细菌之一,同时也是引起尿路感染最主要的致病菌。近年来随着广谱抗生素的不断更新及其在临床上的广泛应用,耐药菌株尤其是多重耐药菌大量出现并迅速传播。大肠埃希菌产超广谱β-内酰胺酶(ESBLs)和C类头孢菌素酶(AmpC酶)是耐-内酰胺类抗生素     

广谱抗生素对肠道菌群的选择作用

目的探讨广谱抗生素对烫伤、脓毒症大鼠肠道菌群的选择作用。方法56只健康SD大鼠被随机均分为正常对照组，烫伤罗氏芬治疗前组（单纯烫伤组）、烫伤罗氏芬治疗3d和9d组，脓毒症罗氏芬治疗前组（单纯脓毒症组）、脓毒症罗氏芬治疗3d和9d组。采用大鼠背部30％总体表面积Ⅲ度烫伤模型，于烫伤后24h内腹腔给予内毒素（20mg／kg）进行“二次打击”。“二次打击”24h后给予罗氏芬治疗，分别取胃、小肠、大肠内容物行细菌定量培养、菌种鉴定。结果应用罗氏芬治疗后大鼠胃肠内容物球菌数量明显增加（P〈0．05或P〈0.01），球／杆菌比严重倒置；正常对照组胃肠内容物中肠杆菌均为大肠杆菌，烫伤、脓毒症后出现肺炎克雷伯杆菌、变形杆菌，应用罗氏芬治疗后大肠杆菌减少或消失，代之以铜绿假单胞菌为主，肺炎克雷伯杆菌、鲍曼不动杆菌、阴沟杆菌、变形杆菌和其他杆菌等并存的肠道菌群。结论广谱抗生素破坏肠道微生态平衡，降低了肠道定植抗力，使条件致病菌、病原菌成为优势菌群，造成肠道菌群紊乱和抗生素相关性肠源性疾病的发生。     

肠致病性大肠埃希菌致病机理的研究进展

肠致病性大肠埃希菌（EPEC）致病性大肠埃希菌的一种，是引起儿童腹泻的主要致病菌之一。虽然目前对肠致病性大肠埃希菌的致病机制还不十分清楚，但肠致病性大肠埃希菌引起腹泻的四阶段模型为大多数学者所接受，现对肠致病性大肠埃希菌的致病机制作一综述。     

肺炎克雷伯菌多药耐药机制研究进展

肺炎克雷伯菌(KPN)属于革兰阴性杆菌,通常存在于人类肠道、呼吸道,是除大肠埃希菌外导致医源性感染的最重要条件致病菌[1].由于抗菌药物的大量使用,在选择性压力下多药耐药KPN菌株不断出现,耐药率日益上升.     

下呼吸道大肠埃希菌感染诊断与痰液半定量培养细菌数量的相关研究

目的 探索自然咳出痰标本半定量培养12级法筛检大肠埃希茵的茵量指标.方法 每份痰标本均同时涂片和接种.涂片观察标本质量,以白细胞吞噬菌或白细胞浓集区域优势茵为拟分离菌;培养采用半定量12级法,选相对数量大于该菌口咽部平均携带数量的涂片对应菌做鉴定及药敏试验(当地健康人群口咽部G-杆菌的携带概率为33％,平均携带茵量小于0.1,故临床标本G-杆菌量以≥0.1为筛检指标).查阅2005年6月1日～2011年5月31日住院病人痰内筛检出大肠埃希菌者病历,统计分析首次培养的细菌数量、标本质量与出院时临床诊断的关系.结果 大肠埃希菌163株,其首次培养的相对数量0.1的临床诊断率为43.33％,≥0.2的临床诊断率为50.38％;首次培养的标本质量为A+B级的临床诊断率为55.56％,C级的临床诊断率为38.18％;前者高于后者17.38％,但x2=4.89,P＜0.05.结论 按规定要求采集自然咳出痰标本,若其半定量12级法培养出涂片拟分离菌对应性大肠埃希菌的相对数量≥0.1时,应做药敏试验并报告医师.     

开菲尔来源乳杆菌益生作用的实验研究

目的 了解开菲尔来源乳杆菌抑制肠道病原菌黏附的效果以及对肠道正常菌群大肠埃希菌的影响.方法 分离纯化乳杆菌,待人结肠腺癌细胞株(Caco-2)培养至单层细胞后,将乳杆菌及其代谢产物分别与伤寒沙门菌、大肠埃希菌混合培养进行黏附和黏附抑制试验,记数50个Caco-2细胞黏附的细菌数目,取平均值,计算黏附抑制率,组内、组间相比较,对结果进行统计学分析.结果 与普通乳杆菌混合培养的伤寒沙门菌数相比,经开菲尔来源乳杆菌RG1,RG2混合培养的伤寒沙门菌数下降明显,差异具有统计学意义(P<0.05);经普通乳杆菌混合培养的大肠埃希菌数与RG1,RG2混合培养的大肠埃希菌数相比差异无统计学意义(P＞0.05),另外它们各自代谢产物在酸、中性环境下的抑制效果也有相同趋势.结论 RG1,RG2能抑制伤寒沙门菌的黏附,且抑制能力明显较普通乳杆菌强;另外其代谢产物也能抑制伤寒沙门菌的黏附;而RG1,RG2及其代谢产物对大肠埃希菌均无明显抑制效果.     

住院患者肠道大肠埃希菌质粒介导喹诺酮类耐药基因研究

大肠埃希菌是肠道内主要定植菌,是耐药基因的重要储存库~([1]),其耐药水平直接关系到临床感染株的耐药水平,另外,肠道正常定植的大肠埃希菌还可将所携带的耐药基因传递给致病性大肠埃希菌、沙门菌及志贺菌等不同菌属~[2].     

Manipulation of the size and clone of an intra-abdominal abscess in rats

A rat grading model of chronic sepsis was developed by inoculation of a small (0.8 ml) or a large (1.5 ml) fecal pellet consisting of sterile rat feces, agar and a known number and strain of bacteria. A uniform spherical abscess containingEscherichia coli andBacteriodes fragilis was formed in 100% of the animals that survived the initial peritonitis stage. The effects of a large biclonal abscess were compared with those of a small abscess and of a sham operation. The peritonitis stage with high mortality was followed by an abscess stage. In rats with a large abscess, net body weight did not increase and there was 16% mortality during the abscess stage. On the 7th day, severe hepatic energy deficiency and lactic acidosis occurred in the septic liver withB. fragilis bacteremia. Rats with small abscesses showed mild metabolic disturbances with no mortality. Standardization of rat models with chronic graded septic abscess is possible by controlling the size of the fecal pellet and the species and number of inoculated bacteria.     

大黄对脓毒症大鼠肠道菌群紊乱影响的实验研究

目的探讨中药大黄对脓毒症大鼠肠道菌群紊乱的影响。方法将42只SD雄性大鼠随机抽出12只为正常对照组,余下按照15 mg/kg腹腔注射内毒素方式建立内毒素脓毒症模型后,再随机分为模型组、大黄组,大黄组于制模后每日用大黄汤剂灌胃3次,模型组每日用相同剂量生理盐水灌胃3次,10天后用强迫排便法取各组大鼠新鲜大便进行乳酸杆菌、双歧杆菌、肠球菌、大肠杆菌4种菌的培养与计数,菌落计数以LgCFU/g计算。结果模型组与正常组相比较存在明显菌群紊乱,厌氧菌减少(P<0.05),需氧菌增多(P<0.05),厌氧/需氧比值降低(P<0.05);大黄组与模型组比较,有着明显的纠正菌群紊乱的作用,表现在厌氧菌增多(P<0.05),厌氧/需氧比值升高(P<0.05)。结论①脓毒症大鼠存在肠道菌群紊乱;②中药大黄有明显的纠正菌群紊乱的作用。     

PCR-DGGE蝴性结肠炎大鼠胃肠道中细菌多样性研究

目的探讨溃疡性结肠炎对大鼠胃肠道微生物多样性影响,为治疗溃疡性结肠炎提供可靠依据。方法采用2,4-二硝基氯苯乙酸复合法对健康SD大鼠进行溃疡性结肠炎造模;运用CTAB—SDS法提取肠道微生物总DNA,通过PCR．DGGE分析溃疡性结肠炎大鼠之间胃肠道微生物多样性差异。结果溃疡组、用药组和正常组大鼠的消化道中,不同部位细菌Shannon~数变化较大（1．64-3．05）,结肠中Shannon指数最高,其次是胃,小肠最低。在结肠,不同组的细菌多样性Shannon指数变化幅度不大,以用药组最高（3．05）,其次溃疡组（2．98）,正常组2．95,说明大鼠结肠在健康状态下细菌多样性最低,结肠炎后会发生一定的变化。此外,发现病变后的大鼠胃中拟杆菌属、梭菌属、普氏菌属、紫单胞菌属等厌氧细菌增加,病变后的结肠中乳杆菌属细菌明显减少,紫单胞菌属和梭菌属细菌明显增加;病变后小肠中部分乳杆菌种类消失,同时梭菌属和紫单胞菌属等不常见菌群的出现。结论溃疡性结肠炎大鼠胃、小肠、结肠中有益菌乳杆菌明显减少,普氏菌属、紫单胞菌属、梭菌属等明显增多,这些菌属可作为条件致病菌引起内源性感染,可能是造成溃疡性结肠炎的重要原因。     

脓毒症大鼠心脏组织基因表达变化的研究

目的 应用基因芯片技术初步分析脓毒症大鼠心脏组织细胞基因表达谱的变化.方法 雄性Wistar大鼠30只被随机分为脓毒症组和对照组,每组15只.采用盲肠结扎穿孔术(CLP)制备大鼠脓毒症模型,以透射电镜下心脏组织检查鉴定模型.应用含有22 523个大鼠基因cDNA克隆的表达谱基因芯片进行检测,以Cy3和Cy5两种荧光信号强度结果 比值均>2.0或<0.5的基因为脓毒症表达差异基因,用计算机软件筛选并分析脓毒症大鼠心脏组织术后24 h的基因表达变化,并初步分析表达差异基因与脓毒症之间的关系.结果 与对照组比较,脓毒症组大鼠心脏组织共筛选出418个出现差异表达的基因,占基因芯片总点数的1.86%,其中表达上调者200个,表达下调者218个.在已知功能基因中表达上调者84个,下调者74个,与应激反应、细胞信号转导、糖皮质激素受体、免疫反应、胰岛素样生长因子、细胞物质能量代谢等方面的相关基因功能有关.结论 脓毒症大鼠心脏组织出现一系列基因表达异常,用基因芯片检测技术可快速分析.     

依达拉奉对脓毒症大鼠心肌细胞肿瘤坏死因子α和一氧化氮的影响

目的:探讨依达拉奉对脓毒症大鼠心肌组织的肿瘤坏死因子α(TNF-α)和一氧化氮(NO)的影响.方法:54只SD大鼠随机分成对照组、脓毒症组、治疗组,建立腹腔注射脂多糖大鼠脓毒症模型(LPS 10 mg/kg),治疗组予以腹腔注射LPS 10 mg/kg和静脉注射依达拉奉3 g/kg,每组按造模后6h、12h、24h随机分成三个亚组,检测不同时点各组大鼠血清肌钙蛋白T(CTnT),心肌组织肿瘤坏死因子α(TNF-α)、一氧化氮(NO)含量,以及24 h后心肌细胞显微镜下改变.结果:在6h时、12h时和24 h时点各组血清中CTnT浓度、心肌匀浆TNF-α和NO含量,脓毒症组＞治疗组＞对照组,差异有统计学意义(P＜0.05).脓毒症组和治疗组心肌匀浆中的TNF-α.在6h时达到高峰,随着建模时间的延长,心肌匀浆中的TNF-α逐渐回落;同一组内各时点之间比较,差异有统计学意义(P＜0.05).显微镜显示治疗组与脓毒症组相比,心肌细胞断裂、溶解减轻,间质水肿明显改善.结论:依达拉奉可通过减少心肌TNF-α、NO生成对脓毒症大鼠心肌组织发挥一定的保护作用.     

The septic abscess wall: a cytokine-generating organ associated with portal venous cytokinemia,hepatic outflow fibrosis,sinusoidal congestion,inflammatory cell sequestration,hepatocellular lipid deposition,and focal cell death

An acute septic inflammatory response with access to the portal circulation was created in a rat model using an intra-abdominal abscess composed of a sterile agar pellet, or one contaminated with 102 Escherichia coli (E. coli) and 109 Bacteriodes fragilis (B. fragilis). After 3 days postimplantation, a well-formed intra-abdominal abscess occurred whose wall showed IL-6 DNA by PCR and IL-6 mRNA by in situ hybridization. Portal venous blood draining into the liver from the intra-abdominal abscess had increased levels of TNF-alpha, IL-1beta, and IL-6 in both sterile and septic groups compared with a control normal animal group. Increased levels of these cytokines were also found in suprahepatic inferior vena caval blood, but were correlated with the higher portal vein levels, suggesting a gradient from abscess wall to portal vein into the systemic circulation via the liver. Liver histology demonstrated sinusoidal congestion centering on the central vein, growing worse with progression from normal in control, to sterile, to septic. Similarly, the degree of intrahepatic myeloperoxidase-positive inflammatory cell infiltration and hepatocellular lipid deposition and apoptosis also increased from control, to sterile, to septic. Gene expression by in situ hybridization demonstrated a significant increase in IL-6 and fibrinogen mRNAs in cells surrounding the central vein in sterile and septic animals, being greatest in animals with sepsis, associated with an increased deposition of collagen in the central vein area, most prominent in the septic liver. The pericentral vein cells with IL-6 and fibrinogen mRNA increases paralleled the increases in cells containing IL-6 and fibrinogen mRNAs in the abscess walls of sterile and septic animals, respectively. The data suggest that an intra-abdominal abscess, especially when contaminated with gram-negative bacteria, induces mRNA-generated cytokine responses in the abscess wall that are related to increased portal venous levels of the inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 perfusing the liver. These, in turn, induce localized production of IL-6 and fibrinogen mRNAs in cells at the central vein area with resultant outflow fibrosis and increased inflammatory cell sequestration within the liver lobular sinuses. This is associated with a generalized inflammatory response and intrahepatic portal sinusoid congestion. There is also increased hepatocellular lipid deposition and apoptosis. Thus, the cytokine production of the abscess wall itself appears to be a major mediator of the septic hepatic response.     

Protection against an Escherichia coli-induced sepsis by alkaline phosphatase in mice

Alkaline phosphatase (AP) is a phosphate transferase present in bacteria and eukaryotes. In previous studies, we have shown that AP is able to dephosphorylate lipopolysaccharide (LPS) at physiological pH levels. Because LPS is the causative agent of gram-negative sepsis, we hypothesize that AP might be used as a medication during early stages of LPS-induced septic shock. We have demonstrated protective effects of AP when this enzyme was administered simultaneously with LPS. However, a major question of anti-LPS therapies is whether they are also effective after systemic infiltration of whole bacteria and if they also act in later stages of the disease. To test this, we explored the protective effects of AP from human placenta (plAP) in a bacterial challenge model in Balb/c mice. AP was intravenously administered 20 min after a bacterial intraperitoneal inoculation of 2 to 5 x 10 CFU of Escherichia coli suspended in a 100-microL volume of saline. It was shown that AP attenuated the systemic host response upon E. coli. Body temperature was normalized as compared with untreated septic mice. Also, serum nitric oxide levels 24 h after the injection of bacteria were reduced almost to control levels in mice that received AP. Moreover, survival after 24 h was significantly higher in the AP-treated group compared with the nontreated control group.     

Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock

Survivors of both human and animal bacterial shock develop a characteristic pattern of progressive changes in cardiovascular function over a period of 7-10 d. In this present study, we examined whether endotoxin (a product of Gram-negative bacteria) or TNF (a cytokine released from macrophages) could reproduce the same complex cardiovascular changes observed in septic shock over a period of 7-10 d. To test this hypothesis, we implanted a thrombin-fibrin clot containing purified endotoxin from E. coli into the peritoneal cavity of eight dogs, and infused TNF into eight different dogs. Over the next 10 d, serial simultaneous heart scans and thermodilution cardiac outputs were performed in these awake nonsedated animals. By day 2 after challenge with either endotoxin or TNF, animals developed a decrease (p less than 0.05) in both mean arterial pressure and left ventricular ejection fraction. With fluid resuscitation, animals manifested left ventricular dilatation (increased [p less than 0.05] end diastolic volume index), increased or normal cardiac index, and decreased or normal systemic vascular resistance index. In surviving animals, these changes returned to normal with 7-10 d. The time course of these changes was concordant (p less than 0.05) with that previously described in a canine model of septic shock using viable bacteria. During the 10-d study, control animals receiving sterile clots or heat-inactivated TNF had not significant changes in hemodynamics. The results from this canine model demonstrate that either endotoxin or TNF alone can produce many of the same hemodynamic abnormalities seen in human septic shock and in a canine septic shock model induced by live bacteria. These findings support the hypothesis that the action of endogenous mediators (TNF) responding to bacterial products (endotoxin) is the common pathway that produces the serial cardiovascular changes found in septic shock.     

Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide

Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay. In the in vivo study, adult male Wistar rats were given an intraperitoneal injection of 1 x 10(9) colony-forming units of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally 1 mg/kg Bac7(1-35), or isotonic sodium chloride solution (control group C1), 60 mg/kg of piperacillin and 1 mg/kg polymyxin B, 1 mg/kg of polymyxin B plus 60 mg/kg of piperacillin, and 1 mg/kg of Bac7(1-35) plus 60 mg/kg of piperacillin. Each group included 15 animals. Bac7(1-35) was found to completely inhibit the LPS procoagulant activity at approximately 10 microM peptide concentration, as determined by in vitro LAL chromogenic assay. Treatment with Bac7(1-35) resulted in significant decrease in plasma endotoxin levels and lethality rates compared with saline injected control animals. No statistically significant differences were noted between Bac7(1-35) and polymyxin B in reducing all variables measured. These results provide evidence for the ability of Bac7(1-35) to effectively bind LPS and protect animals from lethal effects of this molecule, and point to its potential use for the treatment of endotoxin-induced septic shock.     

Reduced clearance of leukocytes from lungs in septic rats

Leukocytes have previously been shown to sequestrate in the lungs and liver in association with traumatic and septic shock. In a rat model of gram-negative sepsis of intra-abdominal origin, a previously described in vivo technique was used for dynamic studies of leukocyte sequestration in different organs using white blood cells labeled with 111-indium-oxine. One group of rats was either studied immediately after induction of sepsis or for 6 h under a scintillation camera for continuous registration of the activity distribution (i.e., presence of leukocytes). Another group was studied 12 h after induction of sepsis for 60 min. The activity increased immediately over the lungs, indicating sequestration of the leukocytes during the first 6 h, but there was no significant difference in this respect between septic and control animals. It does not seem possible to study leukocyte sequestration dynamically in this way. When the labeled leukocytes were administered 12 h after induction of sepsis, however, the activity of septic animals' lungs was seen to remain elevated over the time period studied compared with control rats, in which the activity slowly decreased. In the liver and spleen, the activity increased in both groups, but significantly more so in control animals, which may be explained by disturbed leukocyte margination and cell turnover in the septic animals. This study has indicated that leukocyte distribution in different organs is affected by sepsis and this reaction can be studied using radiolabeled leukocytes.