Suppression of Francisella tularensis growth in the rat by co-infection with F. novicida

We have previously shown that when cultured in vitro, peritoneal rat macrophages infected with Francisella novicida spontaneously release nitric oxide in sufficient quantities to inhibit bacterial growth. However, it is not known whether F. novicida can have a similar antimicrobial effect in vivo. Here we show that a co-infection of F. novicida with Francisella tularensis can suppress the number of F. tularensis cells in rat spleens by as much as 100-fold.     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Chlorine disinfection of Francisella tularensis

Aims: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild‐type strains of Francisella tularensis. Methods and Results: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l^?1 FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild‐type F. tularensis strains at pH 8 and 5°C. Conclusions: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log₁₀. LVS was inactivated most quickly of the tested strains. Significance and Impact of the Study: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.     

Construction of a shuttle vector for use in Francisella tularensis

Abstract The characterisation of virulence factors of Francisella tularensis has been hampered by the lack of genetic system for the bacterium. In this study, a shuttle vector was constructed that can replicate autonomously in F. tularensis and Escherichia coli . To obtain this vector, the p15A replication origin of E. coli plasmid pACYC184 was introduced into a plasmid derivative of plasmid pFNL200, a plasmid which only can replicate in F. tularensis . The resulting shuttle vector, designated pKK202, harboured resistance genes for chloramphenicol and tetracycline. This vector might be used as a basis for the studies of virulence factors of F. tularensis .     

An optimized, multiplexed multi-locus variable-number tandem repeat analysis system for genotyping Francisella tularensis

We present a truncated, optimized, multiplexed multiple-locus variable-number tandem repeat analysis system for the molecular subtyping of Francisella tularensis that reduces time and cost requirements while retaining high discriminatory power.     

Cryptic plasmid pFNL10 from Francisella novicida-like F6168: The base of plasmid vectors for Francisella tularensis

Abstract The plasmid pFNL100 was created by ligation of Escherichia coli plasmid pBR328 and plasmid pFNL10 from Francisella novicida -like strain F6168. This plasmid was able to replicate and to express the genes for chloramphenicol and tetracycline resistance in both E. coli and F. tularensis . The origin of replication of pFNL10, needed for the replication of pFNL100 in F. tularensis , was mapped. A Sau 3A-deletion derivative of pFNL100, designated pFNL200, was constructed. This plasmid could replicate only in F. tularensis and was found to be stably inherited during cultivation both on solid medium and in liquid cultures.     

Genetic diversity of Francisella tularensis subspecies holarctica strains isolated in Japan

The recently developed MLVA has high discriminatory power for the typing of individual strains or isolates of Francisella tularensis. In the present study, MLVA was applied to 33 Japanese F. tularensis subspecies holarctica strains to examine the genetic diversity of F. tularensis isolated. Among the seven VNTR loci analyzed, Ft-M2, Ft-M10, and Ft-M20 loci showed high genetic polymorphism in Japanese strains, whereas Ft-M3 was most variable in non-Japanese strains. These results provide novel extended information about the genomic diversity among the strains of F. tularensis ssp. holarctica distributed in Japan and enable determination of whether a given isolate is indigenous to Japan by examining these loci using MLVA.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.     

应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

金标银染免疫渗滤法检测土拉弗朗西斯菌

目的:建立金标银染免疫渗滤法检测土拉弗朗西斯菌(土拉菌)的方法,评价其灵敏度、特异性、重复性及其应用。方法:以小鼠抗土拉菌脂多糖单克隆抗体作为捕获抗体包被硝酸纤维素膜、兔抗土拉菌多克隆抗体作为检测抗体标记胶体金,通过金标银染技术放大检测信号,建立金标银染免疫渗滤法检测土拉弗朗西斯菌体系;评价该方法的灵敏度、特异性和重复性;以经荧光定量PCR定量的土拉弗朗西斯菌为检测对象,比较金标银染免疫渗滤法和免疫层析法。结果:金标银染免疫渗滤法检测土拉弗朗西斯菌的最小检出量为1.0×103 CFU/mL,灵敏度高于免疫层析法;检测大肠杆菌、炭疽芽孢杆菌、布鲁菌和鼠疫耶尔森菌的结果均为阴性;密封保存的检测卡80 d内重复性良好,100 d后反应强度略有降低。结论:金标银染免疫渗滤法检测土拉弗朗西斯菌敏感性高、特异性强、重复性好,且方便快捷,不需要仪器设备,可作为快速检测土拉弗朗西斯菌的首选方法。     

Seroprevalence study of Francisella tularensis among hunters in Germany

In 2005 and 2006, Francisella tularensis unexpectedly reemerged in western Germany, when several semi-free-living marmosets (Callithrix jacchus) in a research facility died from tularemia and a group of hare hunters became infected. It is believed that hunters may have an elevated risk to be exposed to zoonotic pathogens, including F. tularensis. A previous cross-sectional study of the German population (n=6883) revealed a prevalence of 0.2%. Here, we investigated 286 sera from individuals mainly hunting in districts with emerging tularemia cases (group 1) and 84 sera from a region currently not conspicuous for tularemia (group 2). Methods included standard enzyme-linked immunosorbent assay (ELISA), Western blot analysis and indirect immunofluorescence assay. We found five out of the 286 hunters (1.7%; 95% CI 0.6-4.0%) in group 1 positive with standard ELISA and Western blot, but none in the Berlin area (group 2; 95% CI 0-0.04%). Group 1 showed an elevated risk for hunters to be seropositive for F. tularensis compared with the cross-sectional study (OR=7.7; P<0.001). This indicates a higher prevalence for tularemia in hunters of a suspected endemic region of Germany.     

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non‐Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H₂O₂ and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN‐γ, CD4 and CD8 double‐positive T cells and Th1 cells (CD3, CD4 and Tim3‐positive cells), and a decrease in the ratio of CD8‐positive CD4‐negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K‐sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.     

PCR和Southern Blot检测土拉弗氏菌气溶胶

为提高检测土拉弗氏菌的特异性和敏感性,建立了土拉菌ＰＣＲ及核酸杂交检测方法。运用平板计数、多聚酶链反应对土拉菌气溶胶稳定性进行了比较,结果表明ＰＣＲ具有较高灵敏度,并且在采样后３小时ＰＣＲ就可以得出定性结果,而平板计数则需要３～７天。采用ＰＣＲ法合成了土拉菌３７６－ｂｐ探针,分别对细菌菌液、５６８－ｂｐＰＣＲ产物和气溶胶样品进行杂交,结果表明菌悬液直接杂交可检出１０５ＣＦＵ左右的细菌,检测ＰＣＲ产物可达４０ｐｇ。ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹相结合有利于细菌的分离鉴定     

Phylogeography of Francisella tularensis ssp. holarctica in France

Aim: To investigate the phylogeography of French Francisella tularensis ssp. holarctica isolates. Methods and Results: Canonical SNPs and MLVA were used to genotype 103 French F. tularensis ssp. holarctica isolates. We confirmed the presence of one subclade, the central and western European group (B.Br.FTNF002‐00), and identified four major MLVA genotypes with no obvious geographical differentiation. Conclusions: The lack of geographical resolution among MLVA genotypes suggests rapid dispersal, convergent evolution or a combination of the two. Significance and Impact of the Study: This study expands knowledge of the phylogeography of one of the two dominant European F. tularensis ssp. holarctica subclades and illustrates the need for additional SNP discovery within this subclade.     

Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS

Abstract Lipopolysaccharide (LPS) from the live vaccine strain of Francisella tularensis ( F . tularensis LVS) was isolated and purified. The LPS did not stimulate lymphocytes from previously tularaemia-vaccinated individuals or lymphocytes from nonprimed individuals. However, serum antibodies from tularaemia vaccines reacted with the LPS whereas virtually no reactivity was found with antibodies from individuals not exposed to F. tularensis LVS. Antibodies of immunoglobulin class M displayed the antibody reactivity predominantly. The LPS failed to induce the mononuclear cell-derived cytokine interleukin-1 and only low levels of tumour necrosis factor were detected. Furthermore, no LPS endotoxin properties were found in galactosamine-treated mice or in the Limulus amoebocyte lysate assay. From these results it can be concluded that F. tularensis LVS possesses a lipopolysaccharide-like molecule, which does not exhibit properties of a classical endotoxin.     

Identification of Francisella tularensis in natural water samples by PCR

Abstract Francisella tularensis , the causative agent of the epizootic disease tularemia in mammals, can be isolated from mud and water. To study the spread and persistence of Francisella tularensis in water, different strategies for pre-treatment of natural water samples prior to identification of the bacterium by polymerase chain reaction (PCR) were evaluated. A method for handling of samples taken from natural waters was developed. Applied on natural water samples amended with F. tularensis , the method rendered identification by PCR reproducible and it resulted in an amplified Francisella -specific product in all samples from natural waters tested. In addition, by employing primers targeting conserved regions of the 16S rDNA the presence of bacteria was demonstrated in all samples investigated. The results presented will, in combination with other techniques that allow identification, improve studies on the epizootiology and epidemiology of the genus Francisella .     

Cytokine expression in the liver of mice infected with a highly virulent strain of Francisella tularensis

Abstract Cytokine mRNA expression was determined in the liver of mice subcutaneously inoculated with a lethal dose of the highly virulent strain FSC 041 of Francisella tularensis subvar. tularensis or a sublethal dose of the live vaccine strain of F. tularensis subvar. palaearctica . Expression of mRNA for TNF-α, IL-12, IFN-γ, and IL-10 was demonstrated within 48 h of inoculation, the kinetics being similar irrespective of bacterial strain used. Thus, the expression of a cytokine response believed to be important in the early host defence against live vaccine strain seemed insufficient to prevent the lethality of a more virulent strain.     

土拉弗朗西斯菌两种不同长度的外膜蛋白FopA抗原和抗体的制备

目的:根据外膜蛋白 FopA 的序列信息,建立土拉弗朗西斯菌 FopA蛋白全长(FopA-L)和部分(FopA-S)的特异性抗原的 BL21 表达系统,获得高活性的重组 FopA-L、FopA-S蛋白并制备相应的多克隆抗体,为土拉菌的监测、诊断和治疗提供依据。方法:通过 pET100 质粒构建FopA-L及FopA-S的表达载体,转化大肠杆菌BL21细胞并诱导表达FopA-L及FopA-S蛋白,螯合镍离子次氨基三乙酸(Ni-NTA)亲合层析纯化FopA蛋白,用重组蛋白免疫大耳白兔制备多克隆抗体,通过 ELISA、Western 印迹、胶体金免疫层析技术等方法进行检测。结果:构建了FopA-L及FopA-S的表达载体,获得相应的高表达目的蛋白 BL21 细胞株,用表达的蛋白为抗原成功制备了 FopA特异性的抗体,效价皆在1 ∶100000以上且特异性良好。结论:FopA-S与FopA-L两种抗原和相应抗体的制备为建立土拉菌快速检测方法奠定了基础。