Heterogeneity of phenotypic and genotypic traits including organic-acid resistance in Escherichia coli O157 isolates

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing Escherichia coli (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.     

大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。     

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Aims: To develop a duplex real‐time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2‐encoding genes which are pivotal to EHEC‐mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non‐E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H^?, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H^?, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real‐time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real‐time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real‐time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.     

Molecular detection of bacterial indicators in cosmetic/pharmaceuticals and raw materials

PCR assays were compared with standard microbiological methods for rapid detection of the United States Pharmacopoeia (USP) bacterial indicators in artificially contaminated samples of raw materials and cosmetic/pharmaceutical products. DNA primers containing the specific sequences of the uidA gene of the β-glucuronidase enzyme for Escherichia coli, the membrane lipoprotein gene oprL for Pseudomonas aeruginosa, and the 16S ribosomal gene for Staphylococcus aureus were used for detection in the PCR reaction. Contaminated samples were incubated for 24 h at 35°C. After incubation in broth media with and without 4% Tween 20, samples were streaked on selective growth media. After 5–6 days, all microbial indicators were morphologically and biochemically identified using standard methods while detection and identification by the PCR-based assays was completed within 27–30 h. Rapid PCR detection of E. coli, S. aureus, and P. aeruginosa will allow a faster quality evaluation and release of raw materials and cosmetic/pharmaceutical products sensitive to microbial contamination. Received 21 June 1998/ Accepted in revised form 11 January 1999     

木薯ftsZ基因分离及在原核生物中功能初步鉴定

ftsZ基因是控制细胞分裂的关键基因,其蛋白能够在分裂位点形成一个环状结构而影响细胞分裂.为了研究木薯质体分裂与木薯淀粉品质形成的关系,根据木薯基因组数据库上的预测序列,设计引物,从木薯基因组中分离了与质体分裂相关的ftsZ家族3个新基因(ftsZ1,ftsZ2,ftsZ3).分别将它们与荧光蛋白基因(GFP)融合,构建了3个原核表达载体pET-fisZ1-GFP、pET-fisZ2-GFP、pET-fisZ3-GFP,并转化大肠杆菌BL21(DE3).通过荧光显微镜观察菌体的表型和分裂,初步鉴定了木薯质体分裂相关基因ftsZ家族对细胞分裂的作用.结果显示:尽管木薯与大肠杆菌的亲缘关系较远,ftsZ基因的同源性较低,但是两者表现出相似的功能,木薯ftsZ基因的表达能严重影响大肠杆菌细胞分裂.这一结果为进一步研究木薯ftsZ家族基因的功能奠定了基础.     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。