大肠埃希菌耐药性水平传播实验研究

目的研究重症监护病房（ICU）患者标本中分离的大肠埃希菌的耐药情况以及耐药性水平传播的实验研究。方法采取双纸片法（K-B）检测细菌的耐药性;产超广谱β-内酰胺酶（ESBLs）大肠埃希菌为供体菌,耐利福平大肠埃希菌（对其他抗生素敏感）作为受体菌进行接合实验;采用聚合酶链反应（PCR）技术扩增整合子和耐药基因。结果30株大肠埃希菌中产ESBLs菌株检出率为46．7％;接合培养后,接合菌携带23kb和25kb大质粒,而无供体菌中一系列小质粒;供体菌和接合菌均携带I型整合子。结论大肠埃希菌耐药性严重,且呈多重耐药性;产ESBLs菌株可通过质粒和整合子将耐药基因转移给敏感菌,导致耐药性传播。     

温度与pH值对长江水系产超广谱β-内酰胺酶大肠埃希菌耐药基因转移影响分析

研究温度和pH值对长江水系中产超广谱β-内酰胺酶(Extended-Spectrum β-Lactamases,ESBL)大肠埃希菌(Escherichia coli)耐药基因转移影响规律,为今后介水疾病的预防与控制提供理论依据。采用滤膜法分离、梅里埃微生物分析系统鉴定菌株;将由长江水系分离出的产ESBL大肠埃希菌与大肠埃希菌NK5449进行接合,观察不同温度和pH值条件下接合频率变化情况;用纸片扩散法测定耐药谱;用PCR方法分析产ESBL供体菌与转移接合子β-内酰胺酶编码基因(bla),并对供、受体菌及转移接合子进行随机扩增多态性分析,判别转移接合子与供、受体菌的同源性。温度和pH值对产ESBL大肠埃希菌耐药基因水平转移影响明显,发生接合最适宜的pH值为7.1。温度对接合频率的影响具有双重性,相同条件下,某些大肠埃希菌接合频率随环境温度的降低率急剧下降,但某些大肠埃希菌的接合频率随环境温度下降有所上升。温度和pH值对产ESBL大肠埃希菌接合频率有重要影响。     

转座子Tn916对固氮螺菌Azospirillum brasilense的接合诱变及氨分泌菌株的筛选

以携带质粒pAM120(Ap~r,Tc~r/Tn916)的大肠杆菌(E.coli CG120)为供体菌株,与受体菌巴西固氮螺菌(Azospirillum brasilense)采用滤膜接合法进行接合转移,在选择平板上得到具较高频率的接合子(10~(-5)/每个供体菌,选择四环素抗性)。从846株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株3株。在无氮培养基上,其氨分泌量可达7.5～14.0mmol/L。用乙炔还原法分析氨分泌突变株在不同浓度氮源上的固氮活力,发现20mmol/L NH Cl的存在不抑制其固氮活性,固氮活力与无氮条件下野生株的活力相差不多。无选择压力下细胞分裂50代后的稳定性实验证明,转座子Tn916在氨分泌突变株中的稳定性在50％～80％之间。以固氮螺菌氨分泌突变株为供体菌株,对E.coli HBX1进行反向接合转移实验,证实Tn916确实存在于氨分泌固氮螺菌接合子中。     

蚯蚓粪中乳酸菌的分离及其在大肠杆菌O157:H7中的抗菌活性

本研究利用SL培养基从蚯蚓粪中分离到54株具有产酸性能的菌株,并以E. coli O157:H7 (EDL933株)作为指示菌株,采用点种法检测分离菌株的抑菌活性.结果表明其中6个菌株对指示菌具有拮抗作用,通过形态特征,结合16S rDNA序列分析,初步鉴定该6个菌株分别为食物魏斯特菌(Listeria welshimeri)、乳酸片球菌(Pediococcus acidilactici)、短乳杆菌(Lactobacillus brevis)和格氏乳球菌(Lactococcus garvieae).分离到的乳酸菌对E. coli O157:H7 (EDL933株)具有显著的抑制作用,发酵温度和初始pH值影响发酵液的抑菌作用,优化环境因子可以促进拮抗菌对E. coli O157:H7的抑制作用.本研究为进一步分离抗菌产物用于人畜共患病的预防和治疗提供了理论依据.     

酶解法制备姜汁及其抑制大肠埃希菌O157：H7的研究

目的探究酶解对生姜出汁率及姜汁悬浮稳定性的影响,并测定经过酶解处理及未经酶解处理的生姜姜汁对大肠埃希菌O157：H7的抑菌活性。方法新鲜的生姜清水洗净后切块,放人组织捣碎机中打浆,所得浆液利用果胶酶和淀粉酶进行酶解。采用共培养法测定了制得的姜汁对大肠埃希菌O157：H7的抑菌活性。结果最适酶解条件为,加入3nag淀粉酶60℃酶解50min,灭酶后将pH调至4,再加入3mg果胶酶于45℃酶解50min。结果显示经酶解处理的姜汁对大肠埃希菌O157：H7的抑菌率远大于未经酶解处理的姜汁,酶解处理的姜汁其抑菌率最高达到86％。结论该方法能够有效提高生姜的出汁率及姜汁对大肠埃希菌O157：H7的抑菌效果。     

TaqMan探针荧光PCR检测肠出血性大肠埃希菌O157∶H7方法的建立

肠出血性大肠埃希菌(Enterohemorrhagie Escherichia coli,EHEC)O157∶H7是一种重要的传染病病原菌,以EHEC O157∶H7标准菌株rfbE保守区设计一对特异引物和一条探针,建立了检测EHEC O157∶H7核酸的荧光定量PCR检测方法.实验结果表明荧光定量PCR检测方法特异性好,最低检测限为20 cfu/mL,线性范围是102～108 cfu/mL.稳定性试验表明批内变异系数和批间变异系数分别为2.06％和2.45％.     

TaqMan探针荧光PCR检测肠出血性大肠埃希菌O157:H7方法的建立

肠出血性大肠埃希菌(Enterohemorrhagie Escherichia coli,EHEC)O157:H7是一种重要的传染病病原菌,以EHEC O157:H7标准菌株rfbE保守区设计一对特异引物和一条探针,建立了检测EHEC O157:H7核酸的荧光定量PCR检测方法。实验结果表明荧光定量PCR检测方法特异性好,最低检测限为20 cfu/mL,线性范围是102～108cfu/mL。稳定性试验表明批内变异系数和批间变异系数分别为2.06%和2.45%。     

玉溪市大肠埃希菌O157菌株的流行率、表型与毒力因子特征

目的:检测并分析大肠埃希菌O157(Escherichia coli O157)在玉溪市奶牛、猪、鸡和腹泻病人中的带菌率及其特征,为E.coli O157感染症的防治和该菌分离鉴定技术的建立提供科学依据.方法:将玉溪市最大的一个奶牛场、1个猪屠宰场、2个生禽市场和市医院检验科作为监测点,采集牛、猪、鸡、腹泻病人粪便标本数分别为70、250、350和400例,用免疫磁性分离法和免疫色层技术进行E.coli O157菌株的分离培养,纯化菌株经3种鉴别培养基培养以及法国生物梅里埃公司全自动微生物鉴定系统VITEK 32 GNI 或肠杆菌科鉴定系统API 20 E试条、血清学、噬菌体型、亚碲酸盐抗性、聚合酶链反应等检测与分析.结果:牛、猪、鸡、腹泻病人E.coli O157的带菌率分别为1.4%、2.0%、0和1.2%,分离到4株E.coli O157∶H7、6株E.coli O157∶Hund和1株E.coli O157∶HNM,认识了11株E.coli O157的形态学、生物化学、血清学、噬菌体型、亚碲酸盐抗性、毒力因子等特征.结论:单一动物群或人群可同时携带多株E.coli O157,甚至同一个体也会携带2株E.coli O157;E.coli O157菌株间的表型与毒力因子特征存在一定差异,基因指纹技术可确认并证实菌株间的差异性.     

餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发

为了应对餐饮等食品中病原菌快速检测的需求、研究建立病原菌筛查方法,选取痢疾志贺氏菌（Shigella dysenteriae）、金黄色葡萄球菌（Staphylococcus aureus）、副溶血性弧菌（Vibrio parahaemolyticus）、阴沟肠杆菌（Enterobacter cloacae）、产气肠杆菌（Enterobacter aerogenes）、沙门氏菌（Salmonella）、蜡样芽胞杆菌（Bacillus cereus）、大肠埃希氏菌O157∶H7（Escherichia coli O157∶H7）、单核细胞增生李斯特氏菌（Listeria monocytogenes）等9种病原菌开展多重实时荧光PCR方法研究工作。为了节约预增菌时间与提升检测效率,研发了适用于多种病原菌预增菌的通用型培养基,采取高温裂解法提取菌液核酸,利用PMA染料灭活死亡细菌DNA,筛选出活菌DNA,采用多重实时荧光PCR技术检测目标菌,该方法可在16 h内完成检测,对于目标病原菌的检测低限可达103 CFU·mL-1。     

伤寒沙门菌耐药质粒体内接合转移的研究

目的研究伤寒沙门菌耐药质粒pRST98在小鼠体内向大肠埃希菌的接合转移,比较质粒在体内、外接合转移的异同。方法由于伤寒沙门菌是一种只对人类致病的病原菌,因此将伤寒沙门菌的耐药质粒pRST98导入遗传背景明确的鼠伤寒沙门菌低毒株RIA中,用接合子pRST98/RIA口饲BALB/c小鼠进行体内接合转移。结果在体外伤寒沙门菌很容易将pRST98转移给大肠埃希菌E.coliK12W1485(F-)RifrLac+,该接合子又可将pRST98转移给鼠伤寒沙门菌RIA,但在不同宿主菌中耐药标志的表达有差异。未经人工感染小鼠肠道分离的大肠埃希菌耐药情况严重,口饲pRST98/RIA后出现了部分耐药标志与pRST98耐药谱相同的大肠埃希菌,但有些抗生素的耐药标记未能表达。质粒检测显示体内形成的接合子均含耐药质粒pRST98。结论伤寒沙门菌耐药质粒pRST98在动物体内、外均可转移给大肠埃希菌,但同一质粒在体内、外大肠埃希菌株中耐药标志表达有差异,即使在同一小鼠体内分离的不同接合子,pRST98/E.coli菌株耐药性亦有不同,显示耐药质粒表达的多样性和复杂性。     

A novel DNA microarray design for accurate and straightforward identification of Escherichia coli safety and laboratory strains

Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut.

Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains.

In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.     

Virulence properties of rough and smooth strains of Vibrio cholerae O1

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.     

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.     

果胶酶产生菌的分离及培养条件研究

从黑龙江省巴彦县亚麻厂不同发酵时期的沤麻液中 ,分离得到两株果胶酶产生菌MHZP 0 1和MHZP 0 2 ,经初步鉴定为芽孢杆菌属 (Bacillus)。在温度为 33～ 37℃ ,pH 5～ 6,工业级原料C N(质量比 )为 1∶1时 ,为这两株菌的最佳培养条件。     

Starch Gel Electrophoresis: An Effective Method for Separation of Pathogenic and Nonpathogenic Naegleria Strains*

SYNOPSIS. Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.     

浓香型白酒糟醅及窖泥产香功能菌的研究进展

浓香型白酒主要香气物为己酸乙酯、乳酸乙酯、乙酸乙酯和丁酸乙酯四大酯类,白酒微生物发酵过程中酯类合成主要是酸与醇在酯化酶的作用下产生,因此产香功能菌包括香气及其前体物产生菌和酯化酶产生菌.本文综述了浓香型白酒糟醅及窖泥主要产香功能菌的来源、组成、鉴定方法、功能特性、相互关系及其在浓香型白酒生产过程中的应用等方面的研究进展,旨为浓香型白酒重要微生物的研究提供思路,为其发酵过程控制提供理论依据.     

Field efficacy of a mixture of atoxigenic Aspergillus flavus Link:Fr vegetative compatibility groups in preventing aflatoxin contamination in maize (Zea mays L.)

Competitive exclusion of aflatoxin producers by endemic atoxigenic strains of Aspergillus flavus is a proven tool for aflatoxin management being adapted for use in Africa. Field efficacy of an experimental formulation consisting of four native atoxigenic strains (La3303, La3304, La3279 and Ka16127) was evaluated on maize in 2007 and 2008 in four agroecological zones in Nigeria. The four atoxigenic strains were individually formulated on sterile sorghum grain and subsequently mixed in equal proportions. The blended product was applied on soil (40 kg/ha), 2–3 weeks before flowering. Grains from treated and untreated fields were analyzed for aflatoxins at harvest and after storage. Proportions of the A. flavus population composed of each of the four applied strains in soil before treatment and in harvested grains were determined using vegetative compatibility analyses. Application of the strain mixture resulted in reduced aflatoxin content and significantly (P < 0.05) increased the combined frequencies of the vegetative compatibility groups (VCGs) of the applied strains recovered from the soil and grain. Aflatoxin reductions of 67–95% were associated with a 74–80% combined incidence of the VCGs of the four atoxigenic strains on the treated crops. The applied atoxigenic strains remained with the crop into storage and reduced postharvest increases in contamination. The results suggest that the evaluated multi-strain formulated product has potential to contribute to reduced aflatoxin contamination in Nigeria. This is the first report of a field evaluation of an endemic strain mixture effective at reducing aflatoxin contamination during crop development.     

Replication of small effect quantitative trait loci for behavioral traits facilitated by estimation of effect size from independent cohorts

Quantitative trait locus (QTL) mapping is often done in a single segregating population, such as a backcross or an intercross. Both QTL location and effect size are then estimated from the same dataset. This approach results in an over-estimate of effect size for two reasons: (1) LOD scores, which are maximized over numerous point-wise tests, are correlated with estimated effect size and (2) small effect QTLs are often undetected in underpowered experiments, yielding inflated effect sizes for detected QTLs (the Beavis effect). When it is impractical to maintain or generate large population sizes, an alternative is to use two populations, one for initial detection and localization and a second for a locus-matched estimate of effect size, not conditioned on significance. Recombinant inbred (RI) panels are eminently suitable for this approach, as each strain genotype can be sampled repeatedly. We present mapping results from the LXS RI panel for two behavioral phenotypes relating to ethanol response: low-dose ethanol activation and loss of righting following high-dose injection. Both the phenotypes were measured in two or three independent cohorts, which were then used to re-estimate effect size. Many small-effect QTLs replicated using this approach, but in all cases, effect size, in the replicate cohorts, was reduced from the initial estimate, often substantially. Such a reduction will have important consequences for power analyses in which sample sizes are determined for subsequent confirmation studies.