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.     

Genotyping of the Francisella tularensis strains isolated from natural foci of tularemia in the Rostov region by multilocus VNTR analysis

On the basis of an analysis of the VNTR alleles' distribution in 109 strains of F. tularensis it was established that 19 genotypes of the disease causative agent circulated in the Rostov Region from 1945 to 2002. The microbe-provoked infection episodes can be divided into polyclonal, monoclonal and cluster ones. A retrospective analysis of the genotypes' distribution is indicative of that strains of similar or of closely-related genotypes circulate simultaneously in the studied territory. All investigated F. tularensis strains could be differentiated into two groups; strains, whose genotypes are encountered almost evenly within the entire Region's territory, belong to group 1; and strains of group 2 displayed a trend towards being geographically bound. Isolations of cultures with similar (close) genotypic features made in prolonged time periods suggest that a part of F. tularensis clones can persist for a long time in environmental foci. A set of strains described by genotype can provide a foundation for a database of the tularemic microbe culture within the geo-information system of the South Federative Okrug of Russia.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

Genotypic diversity of oscillatoriacean strains belonging to the genera Geitlerinema and Spirulina determined by 16S rDNA restriction analysis

Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated.     

Features of the interaction of Escherichia coli and Francisella tularensis RNA polymerases with hybrid plasmids bearing fragments of Francisella tularensis chromosomal DNA]

Hybrid plasmids containing the fragments of Francisella tularensis chromosomal DNA and capable of tet-gene expression both in Escherichia coli and Francisella tularensis cells were constructed. The regions of francisella chromosomal DNA binding the RNA-polymerases of Escherichia coli and Francisella tularensis were found by the electron microscopy technique. Interconnection of those regions with the expression of tet-gene of the hybrid plasmids was demonstrated.     

Comparative characteristics of biological properties of Francisella tularensis strains isolated in the USSR]

With a large collection of the strains of F. tularensis isolated it has been recently shown that cultures belonging to holarctica and mediaasiatica circulate in the endemic foci of the USSR. By their biological and genetic properties the natural strains of F. tularensis were homogeneous and represented type cultures of F. tularensis. Various ecological conditions in the natural environment did not change within the last 20 years the sensitivity of the tularemia microbe to the antibacterial drugs.     

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.     

Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.     

Taxonomic heterogeneity of the collection strains of fluorescent pseudomonads

Typing of 13 strains of fluorescent pseudomonads from the Belarusian collection of nonpathogenic microorganisms (BIM) by ERIC-PCR and BOX-PCR revealed high level of genetic heterogeneity in bacteria, most of which have been previously identified as Pseudomonas fluorescens according to the classical scheme. Evaluation of the similarities of the 16S rRNA gene sequences and their phylogenetic analysis excluded affiliation of the bacteria under study within the same species and allowed them to be distributed within three relatively distant clusters of the genus Pseudomonas phylogenetic tree. While eight strains fell into the phylogenetic group of P. fluorescens, only one of them could be identified as P. fluorescens. Four strains clustered within the P. vancouverensis phylogenetic group, formed by new species, which have been described mainly according to the evaluation of genome relationships. One bacterium was related to a stable branch that did not contain any type strains of the known Pseudomonas spp. These results indicate taxonomic heterogeneity of collection strains of the fluorescent pseudomonads and demonstrate the necessity of identification of them considering the requirements of phylogenetic bacterial taxonomy.     

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.     

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.     

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

Background

Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.

Results

Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.

Conclusion

The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.     

Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Background

Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods

This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results

Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion

We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.     

Discrimination of Francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis

Francisella tularensis causes the zoonotic disease tularemia, and is considered a potential bioterrorist agent due to its extremely low infection dose and potential for airborne transmission. Presently, F. tularensis is divided into four subspecies; tularensis, holarctica, mediasiatica and novicida. Phenotypic discrimination of the closely related subspecies with traditional methods is difficult and tedious. Furthermore, the results may be vague and they often need to be complemented with virulence tests in animals. Here, we have used surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to discriminate between the four subspecies of F. tularensis. The method is based on the differential binding of protein subsets to chemically modified surfaces. Bacterial thermolysates were added to anionic, cationic, and copper ion-loaded immobilized metal affinity SELDI chip surfaces. After binding, washing, and SELDI-TOF-MS different protein profiles were obtained. The spectra generated from the different surfaces were then used to characterize each bacterial strain. The results showed that the method was reproducible, with an average intensity variation of 21%, and that the mass precision was good (300-450 ppm). Moreover, in subsequent cluster analysis and principal component analysis (PCA) data for the analyzed Francisella strains grouped according to the recognized subspecies. Partial least squares-discriminant analysis (PLS-DA) of the protein profiles also identified proteins that differed between the strains. Thus, the protein profiling approach based on SELDI-TOF-MS holds great promise for rapid high-resolution phenotypic identification of bacteria.     

Genotypic diversity and environmental variability affect the invasibility of experimental plant populations

The world is experiencing increasing climatic variability, an ongoing loss of biodiversity and a growing spread of invasive species. Previous experimental studies demonstrated that the invasibility of plant populations is reduced with increasing resident genetic diversity and is promoted by environmental fluctuations, but their combined effect has so far not been considered. In a growth chamber experiment, we tested whether the genotypic diversity of experimental populations of Arabidopsis thaliana (1, 3 or 6 genotypes) and temperature fluctuations affect population invasion by Senecio vulgaris, and how these factors interact. We found that genotypic diversity tended to increase the invasion resistance of experimental plant populations in terms of the biomass ratio between the species, and that temperature fluctuations strongly favoured Arabidopsis (biomass: +49%) over Senecio (–28%). However, there were no interactions between environmental fluctuations and genotypic diversity. Nevertheless, the magnitude of net diversity effects and transgressive overyielding depended on temperature conditions, indicating that increased environmental variability can influence diversity mechanisms. Our study shows that, although genotypic diversity and environmental variability did not interact, these two factors independently affected the invasibility of plant populations.     

Genotypic and phenotypic heterogeneity of Streptococcus thermophilus strains isolated from dairy products

AIMS: Forty strains of Streptococcus thermophilus isolated from dairy products were identified and typed by a polyphasic approach which included phenotypic and genotypic criteria. METHODS AND RESULTS: Strains were identified by sugar fermentation pattern and species-specific PCR. Phenotypic diversity was evaluated by a chemometric model taking into account some biochemical characteristics (e.g. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by PCR fingerprinting. The overall phenotypic and genotypic information was elaborated on a multivariate statistical basis by principal components analysis and cluster analysis, respectively. When acidifying and peptidase activities were considered, PCA indicated that most of the strains isolated from Pecorino Toscano cheese were separable from the others. Similarly, most of the starter culture strains tended to separate from the cheese isolates. CONCLUSIONS: A wide strain heterogeneity among Strep. thermophilus strains isolated from dairy products was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: A computerized analysis of genotypic and phenotypic information could be applied successfully to differentiate and characterize reliably and rapidly isolates occurring in different dairy products and to comprehend the technological role of specific Strep. thermophilus strains in dairy technology.     

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

AIM: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. METHODS AND RESULTS: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD-PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. CONCLUSIONS: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.     

Host-pathogen o-methyltransferase similarity and its specific presence in highly virulent strains of Francisella tularensis suggests molecular mimicry

Whole genome comparative studies of many bacterial pathogens have shown an overall high similarity of gene content (>95%) between phylogenetically distinct subspecies. In highly clonal species that share the bulk of their genomes subtle changes in gene content and small-scale polymorphisms, especially those that may alter gene expression and protein-protein interactions, are more likely to have a significant effect on the pathogen's biology. In order to better understand molecular attributes that may mediate the adaptation of virulence in infectious bacteria, a comparative study was done to further analyze the evolution of a gene encoding an o-methyltransferase that was previously identified as a candidate virulence factor due to its conservation specifically in highly pathogenic Francisella tularensis subsp. tularensis strains. The o-methyltransferase gene is located in the genomic neighborhood of a known pathogenicity island and predicted site of rearrangement. Distinct o-methyltransferase subtypes are present in different Francisella tularensis subspecies. Related protein families were identified in several host species as well as species of pathogenic bacteria that are otherwise very distant phylogenetically from Francisella, including species of Mycobacterium. A conserved sequence motif profile is present in the mammalian host and pathogen protein sequences, and sites of non-synonymous variation conserved in Francisella subspecies specific o-methyltransferases map proximally to the predicted active site of the orthologous human protein structure. Altogether, evidence suggests a role of the F. t. subsp. tularensis protein in a mechanism of molecular mimicry, similar perhaps to Legionella and Coxiella. These findings therefore provide insights into the evolution of niche-restriction and virulence in Francisella, and have broader implications regarding the molecular mechanisms that mediate host-pathogen relationships.