Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Genetic variability among archival cultures of Salmonella typhimurium

The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies. In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in these populations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.     

焦磷酸测序检测食品中鼠伤寒沙门氏菌

根据鼠伤寒沙门氏菌的特异序列,分别设计扩增引物和测序引物,建立焦磷酸测序检测鼠伤寒沙门氏菌的方法。针对鼠伤寒沙门氏菌设计特异性扩增引物,对目标片段进行PCR扩增,然后制备单链模板,并利用测序引物进行焦磷酸测序。测序结果表明,6株不同来源的鼠伤寒沙门氏菌均可以扩增出碱基序列为TACAACCGGA GTGCACATTA ATCCCGCAGC的基因片段,而30株阴性对照菌株均未得到扩增。进行BLAST比对表明,该序列与GenBank中鼠伤寒沙门氏菌的碱基序列100%匹配。焦磷酸测序法是一种快速、准确的检测方法,可用于食品中鼠伤寒沙门氏菌的快速检测。     

减毒鼠伤寒沙门菌体内定位分析…

分析减毒鼠伤寒沙门菌口服感染后在小鼠体内定位的情况.将构建的红色荧光蛋白(RFP)原核质粒pYA33-DsRed,以电穿孔法转化减毒鼠伤寒沙门菌X4550,重组菌命名为X4550(33-DsRed).重组菌分别感染巨噬细胞RAW264.7和骨髓源树突状细胞(BMDC),并用流式细胞术检测红色荧光细胞荧光强度.此外,以不同剂量重组菌口服免疫BALB/c小鼠,并于免疫后1d、2d、3d、5d、7d取小鼠脾、肝、肠系膜淋巴结(MLN)、派伊尔氏结(PP)、腹股沟淋巴结(ILN)细胞,检测各组织器官中的红色荧光阳性细胞百分率.重组菌对RAW264.7细胞和BMDC均具有良好的侵袭力.口服小鼠后,第1d,仅在MLN及PP中检测到RFP阳性细胞,其中PP中阳性细胞达到1.4%;第2 d,在ILN中达到0.4%;第3 d,各个组织器官中RFP阳性细胞均有上升趋势,此时在脾、肝中也检测到RFP阳性细胞.第5 d,RFP阳性细胞均减少,第7 d则未检测到任何RFP阳性细胞.减毒鼠伤寒沙门菌具有良好的侵袭力,其黏膜移行方式以及对免疫组织器官靶向定位性,在优化黏膜疫苗以及提高疫苗免疫效力等方面都具有重要作用.     

鼠伤寒沙门菌动物模型活体成像技术的研究进展

鼠伤寒沙门菌的体内实验有利于开展食物中毒、胃肠炎、伤寒热等肠道传染病的防治。由于在活体内检测鼠伤寒沙门菌的动态变化存在瓶颈,使细菌致病机制的研究、疫苗及药物研发滞后。近年来应用小动物成像技术在活体中追踪转化了荧光素酶基因的鼠伤寒沙门菌越来越受到人们关注,综述该技术的应用现状及缺憾之处。     

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice

Abstract To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 μ and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100°C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100°C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.     

鼠伤寒沙门氏菌参与细菌竞争相关基因

[背景]细菌在环境中以复杂的微生物群落形式存在,细菌间的竞争是细菌生存的一种重要方式.鼠伤寒沙门氏菌是一种可引起胃肠道疾病的重要人畜共患病病原体,其在水源、食物或是宿主肠道等环境中均需与其他细菌进行相互作用以获得生存优势.[目的]通过转座子技术构建鼠伤寒沙门氏菌转座子插入突变体库,从中筛选鼠伤寒沙门氏菌与细菌竞争能力相...     

短双歧杆菌对鼠伤寒沙门氏菌的抑制

【背景】鼠伤寒沙门氏菌是主要的肠道病原菌之一,利用益生菌治疗肠道病原菌感染已成为一种新型、绿色的微生态疗法。【目的】研究筛选出的短双歧杆菌无细胞发酵上清液(Cell-free supernatant,CFS)对鼠伤寒沙门氏菌的体外抑制作用及机制。【方法】采用微量稀释法测定短双歧杆菌YH68 CFS对鼠伤寒沙门氏菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)和亚抑制浓度(Sub-inhibitory concentrations,SIC),并从鼠伤寒沙门氏菌的细胞形态、细胞膜通透性、膜完整性以及毒力基因表达的变化探讨YH68 CFS对鼠伤寒沙门氏菌的抑菌机理,同时检测YH68 CFS对鼠伤寒沙门氏菌粘附和侵袭肠上皮细胞HT29的影响。【结果】YH68 CFS (3×109 CFU/mL)对鼠伤寒沙门氏菌具有较好的抑制效果,抑菌圈直径为22.27±0.44 mm,最小抑菌浓度为250μL/mL,对鼠伤寒沙门氏菌的抑制机制是通过增加其细胞膜通透性破坏其完整性,形成难以修复的孔洞,最终达到抑菌的目的;亚抑制浓度为62.5μL/mL时YH68 CFS并不能影响鼠伤寒沙门氏菌的生长,但仍然能通过下调毒力基因表达的方式抑制其对肠上皮细胞的粘附和入侵。【结论】短双歧杆菌YH68对鼠伤寒沙门氏菌具有良好的抑菌作用,可作为治疗沙门氏菌感染的潜在益生菌。     

Purification and characterization of distinct type of mannose-sensitive fimbriae from Salmonella typhimurium

Abstract The fimbriae (E₄) of a virulent strain of Salmonella typhimurium were purified by ion exchange chromatography in an FPLC system. They had a channelled appearance under transmission electron microscope and showed a major structural subunit of 17-kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fimbriae were found to agglutinate guinea pig erythrocytes, but this effect was inhibited in presence of D-mannose. Immune sera raised against the Mono-Q purified fimbriae (E₄) showed cross-reactivity with the type-1 fimbriae (F₁) composed of 21-kDa fimbrin subunit, purified by a different method from the same strain.     

鼠伤寒沙门氏菌asd基因的克隆及序列分析

以鼠伤寒沙门氏茵标准株基因组DNA作为模板,用PCR的方法扩增鼠伤寒沙门氏菌的asd基因并克隆入质粒pUCl9,并对其进行测序,序列与献报道一致。同时将质粒pYA248上的链球菌asd基因进行了置换,观察了分别含有链球菌asd基因与鼠伤寒沙门氏菌asd基因的质粒在减毒鼠伤寒沙门氏菌X4072中的生长情况,结果表明含有鼠伤寒沙门氏菌的asd基因的高拷贝质粒pUCl9的菌株生长情况更好。为完善染色体／质粒平衡致死系统,构建减毒鼠伤寒沙门氏活菌疫苗奠定了基础。     

Kinetics of cobalamin repression of the cob operon in Salmonella typhimurium

Abstract The cob operon in Salmonella typhimurium encodes 25 proteins involved in the biosynthesis of cobalamin. Expression of the cob operon is negatively feedback regulated by cobalamin via a translational control mechanism. The concentration of cobalamin required to repress cob expression to half-maximal was determined in vivo and in vitro to 0.4 μM and 0.6 μM, respectively. These results suggest that cob expression in wild-type cells is partially repressed by de novo synthesized cobalamin.     

Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines

Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of chi(3985), a genetically modified organism, were variants and showed different macrorestriction profiles.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Abstract Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Calcium enhances Salmonella typhimurium invasion of HeLa cells

Salmonella typhimurium cultured with physiological levels of calcium are significantly enhanced in their ability to penetrate HeLa cells in vitro. Increased infectivity was not observed in magnesium-supplemented media, but was demonstrated in calcium-supplemented minimal defined or calcium-supplemented cation-deficient media. Invasion enhancement was observed for a number of S. typhimurium strains and ranged from 28–390% over calcium-deficient controls. Enhanced HeLa cell infectivity was not dependent on the presence of an autonomous 60-MDa plasmid.     

Salmonella typhimurium InvA expression probed with a monoclonal antibody to the C-terminal peptide of InvA

Abstract The Salmonella typhimurium InvA protein is a component of a sec -independent secretion apparatus necessary for full virulence of the bacteria. We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S. typhimurium and weakly in Y. enterocolitica , but not in several other species of bacteria, including S. flexneri. S. typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase. Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.     

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Abstract Diaminopimelic acid is incorporated into the peptidoglycan of Salmonella typhimurium in an efficient and quantitative manner. The amount of DAP incorporated is similar to the number of molecules estimated to exist in the Salmonella cell wall. In contrast, strains of E. coli , including those most used for studies of cell wall synthesis, are much less efficient in the incorporation of diaminopimelic acid. The lysine-requiring strains of E. coli appear to excrete diaminopimelic acid related material during growth and this accounts, in part, for the inefficient incorporation of radioactive diaminopimelic acid into Escherichia strains. In addition, the Escherichia strains are much less permeable to DAP than Salmonella strains. Cysteine and cystine inhibit the incorporation of DAP into the cell and this result suggests that Salmonella uses the cystine uptake system to allow DAP into the cell.     

Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice

Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C₅₇BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis.