Strains Of Francisella Tularensis Research Articles

Protective potential of outer membrane vesicles derived from a virulent strain of Francisella tularensis.

Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.

The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains

ABSTRACT Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1–3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

The Effectiveness of MALDI ToF Mass Spectrometry in Identification of <i>Francisella tularensis</i> Strains

The aim of the study was to evaluate the effectiveness of MALDI‑ToF mass spectrometry in the identification of collection and newly isolated strains of tularemia pathogen using the database “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”.Materials and methods. We investigated 142 strains of Francisella tularensis, including 59 collection strains and 83 newly isolated ones. Bacteriological, molecular-genetic and proteomic research methods were used to identify them. The acquisition of mass spectra, analysis, generation and expansion of reference libraries were performed on a mass analyzer “Microflex LT” using FlexControl v. 3.3, FlexAnalysis v. 3.3, and MALDI Biotyper 3.0 software packages. The cluster analysis was performed using the BioNumerics 7.6 software.Results and discussion. The possibility of identifying tularemia pathogen has been assessed using the extended database for MALDI Biotyper 3.0 “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”. During identification to the species level, the significance of mass spectrometry results for collection strains and newly isolated ones was 91.5 % and 97.6 %, respectively. In determining the genus appurtenance, the reliability of identification was 100 %. Thus, the MALDI‑ToF mass spectrometry method allows for accurate species and genus identification of F. tularensis strains. Based on the cluster analysis of 66 F. tularensis strains in BioNumerics 7.6 software using «Pearson correlation» and the UPGMA algorithm, the possibility of subspecies differentiation has been evaluated. Due to the similarity of protein profiles of F. tularensis strains, a clear differentiation into subspecies could not be achieved. It is necessary to use other options for sample preparation, new generation devices with higher resolution, as well as apply additional approaches and analysis tools for successful subspecific differentiation.

Improvement of Approaches to the Verification of the Vaccine Strain <i>Francisella tularensis</i> 15 NIIEG during Long-Term Storage

The aim of the study was to improve the methods for verifying the vaccine strain Francisella tularensis 15 NIIEG during long-term storage under current conditions.Materials and methods. The paper summarizes the results of studying the phenotypic and genetic properties of lyophilized cultures of the vaccine strain F. tularensis 15 NIIEG (1953, 1966, 1969, 1987, 1990, 2003, 2012 and 2013) stored at SCEMAP for a period of one to 60 years.Results and discussion. Previous studies have revealed that freeze-dried cultures of F. tularensis 15 NIIEG generally had the characteristics of the vaccine strain, with the exception of deviations from the regulatory requirements for residual virulence and specific safety. The stability of preservation of deletions in the pilA and pilE genes (the region of differentiation RD19) and the genes encoding lpp lipoprotein (RD18) in the vaccine strain, which was stored for various periods of time in a lyophilized state, has been confirmed. The vaccine-strain-specific mutation C178404T (by the genome of F. tularensis LVS strain, GenBank NCBI no. CP009694) has been identified, and an approach to determine it has been developed. The data obtained are promising as regards using the above deletions in the RD18/RD19 regions in combination with the C178404T mutation to assess the authenticity of the vaccine strain using molecular genetic methods. Thus, the conducted retrospective analysis of the data on the cultures of tularemia microbe vaccine strain from the 1940s to 2013 and the gathered experimental data, made it possible to supplement the uniform requirements for the manufacture, study, maintenance, storage and movement of F. tularensis 15 NIIEG vaccine strain with new evidence. Based on the results obtained, the authors have drawn a draft methodological recommendations of the federal level “Vaccinal strain Francisella tularensis 15 NIIEG: order of handling”.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

ABSTRACTThe life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays.IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.

In silico functional and structural characterization revealed virulent proteins of Francisella tularensis strain SCHU4.

Francisella tularensis is a pathogenic, aerobic gram-negative coccobacillus bacterium. It is the causative agent of tularemia, a rare infectious disease that can attack skin, lungs, eyes, and lymph nodes. The genome of F. tularensis has been sequenced, and ~16% of the proteome is still uncharacterized. Characterizations of these proteins are essential to find new drug targets for better therapeutics. In silico characterization of proteins has become an extremely important approach to determine the functionality of proteins as experimental functional elucidation is unable to keep pace with the current growth of the sequence database. Initially, we have annotated 577 Hypothetical Proteins (HPs) of F. tularensis strain SCHU4 with seven bioinformatics tools which characterized them based on the family, domain and motif. Out of 577 HPs, 119 HPs were annotated by five or more tools and are further screened to predict their virulence properties, subcellular localization, transmembrane helices as well as physicochemical parameters. VirulentPred predicted 66 HPs out of 119 as virulent. These virulent proteins were annotated to find the interacting partner using STRING, and proteins with high confidence interaction scores were used to predict their 3D structures using Phyre2. The three virulent proteins Q5NH99 (phosphoserine phosphatase), Q5NG42 (Cystathionine beta-synthase) and Q5NG83 (Rrf2-type helix turn helix domain) were predicted to involve in modulation of cytoskeletal and innate immunity of host, H2S (hydrogen sulfide) based antibiotic tolerance and nitrite and iron metabolism of bacteria. The above predicted virulent proteins can serve as novel drug targets in the era of antibiotic resistance.

Biological Properties and Genetic Characteristics of Francisella tularensis Strains Isolated in the Territory of the Rostov Region in 2020

Objective of the study was to investigate biological properties and genetic characteristics of tularemia agent strains isolated from natural foci of the Rostov Region in 2020.Materials and methods. Field material from natural foci of the Rostov Region was examined by serological, bacteriological, biological, and molecular-genetic methods. Cultural-morphological, biochemical, antigenic and pathogenic properties of isolated cultures were studied. Protein profles were obtained through MALDI-TOF MS using mass spectrometer Autoflex speed III Bruker Daltonics and Flex Control of Biotyper software. The genetic characteristics of the strains were determined by VNTR and INDEL typing and SNP analysis.Results and discussion. Six strains of tularemia pathogen were isolated from mouse-like rodents using biological method. The investigation of their biological features and data of PCR analysis and INDEL typing with canonical markers showed that all strains are typical representatives of the Francisella tularensis subsp. holarctica biovar EryR. VNTR typing by six genetic loci revealed that all strains belong to four individual genotypes. The strain isolated in 2020 in the Salsky district was identical to the strain which was isolated in the same area in 1989. Based on the whole genome sequencing of two strains, we established that they are closest to the cultures isolated in Turkey (2009, 2012) and Khanty-Mansiysk (2013) by the studied set of SNP markers. Thus, we found that both identical (or closely related) clones of the tularemia agent and new strains with unique genotypes which previously were not described for the Rostov Region can circulate in natural foci of this region for a long period of time.

Investigation of the ability of Francisella tularensis strains to form biofilms

The study results of the Francisella tularensis stains’ ability to form biofilm on the substrate surface are presented. The qualitative analysis of biofilm forming in the different culture conditions at the surface of glass vials has been performed. It was shown that all studied strains could form a biofilm, but most optimal conditions for biofilm formation are temperature at 22 ± 1 °C and culture exposition during 192 hours.

Itaconate deficiency results in prolonged bacterial burdens and enhanced T cell responses during Francisella tularensis infection

Abstract Shifts in cellular metabolism and associated metabolites regulate immune cell function. Recently, it has been shown that the metabolite itaconate plays a critical role in modulating inflammatory responses. Using animals that cannot synthesize itaconate due to an absence of aconitate decarboxylase 1 (AOCD KO), we previously established the lack of itaconate increased susceptibility to high dose challenge with the live vaccine strain (LVS) of Francisella tularensis in terms of both bacterial clearance and survival. However, ACOD KO mice that survived sublethal LVS infection had improved survival against a lethal challenge with virulent F. tularensis (Ftt). Since appropriate coordination of both macrophage and T cell function is essential for surviving Ftt, we next determined if the improved survival of ACOD KO mice was due to alterations in macrophage and/or T cell function. LVS immune ACOD KO mice had increased numbers of lung resident and splenic effector CD4+ and CD8+ T cells compared to WT mice following resolution of infection. Utilizing an in vitro macrophage-T cell co-culture system we did not observe any difference in the ability of WT and ACOD KO T cells to control LVS replication in macrophages when T cells were co-cultured with genetically matched macrophages. However, when macrophage sources were mixed, we observed that regardless of the source of LVS immune T cells, WT macrophages controlled bacterial replication significantly better than ACOD derived macrophages. Together, these findings suggest that the lack of itaconate results in increased and prolonged bacterial burdens in vivo, which leads to greater expansion of effector T cells that confer enhanced protection during Ftt challenge.

Comparison of tul4, fopA, 16S rRNA and RD1 gene regions of Francisella tularensis strains isolated from Sivas, Turkey

Tularemia is a zoonotic disease that can infect animals and people. It is known that tularemia outbreaks in Turkey is water-borne and causative agent F. tularensis subsp. holarctica. Eight F. tularensis were isolated from the water samples in tularemia outbreaks observed in Sivas during 2011-2013. In this study; tul4, fopA, RD1 and 16S rRNA gene regions of eight F. tularensis isolates were amplified by PCR method and investigated by sequencing method. Sequence analysis of the gene regions were compared with each other and the samples found in GenBank. All samples were found to be similar in terms of the partial sequence of the tul4 and fopA gene region. In terms of 16S rRNA gene region, Belkent2012 and Belkent2013 isolates and Çiçekoğlu and Döllük isolates were detected similar among themselves. Bahçeiçi and Karaören isolates were found similar at RD1 gene regions. In the sequence comparison of GenBank in terms of examined gene regions, 94-100% similarity was determined with F. tularensis subsp. holarctica LVS and F. tularensis subsp. holarctica PHIT-FT049. As a result of our study; the tul4 and fopA gene regions of eight F. tularensis subsp. holarctica isolates were found to have identical and different base sequence origins in 16S rRNA and RD1 gene regions in tularemia outbreaks in Sivas. In order to reveal the phylogeography of F. tularensis’ in Turkey, it is necessary to produce new isolates from epidemic regions and to investigate them with advanced molecular techniques.

Deficiency in CCR2 increases susceptibility of mice to infection with an intracellular pathogen, Francisella tularensis LVS, but does not impair development of protective immunity.

CCR2 is the major chemokine receptor that regulates appropriate trafficking of inflammatory monocytes, but the role of this chemokine receptor and its ligands during primary and secondary infection with intracellular infections remains incompletely understood. Here we used murine infection with the Live Vaccine Strain (LVS) of Francisella tularensis to evaluate the role of CCR2 during primary and secondary parenteral responses to this prototype intracellular bacterium. We find that mice deficient in CCR2 are highly compromised in their ability to survive intradermal infection with LVS, indicating the importance of this receptor during primary parenteral responses. Interestingly, this defect could not be readily attributed to the activities of the known murine CCR2 ligands MCP-1/CCL2, MCP-3/CCL7, or MCP-5/CCL12. Nonetheless, CCR2 knockout mice vaccinated by infection with low doses of LVS generated optimal T cell responses that controlled the intramacrophage replication of Francisella, and LVS-immune CCR2 knockout mice survived maximal lethal Francisella challenge. Thus, fully protective adaptive immune memory responses to this intracellular bacterium can be readily generated in the absence of CCR2.

Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity.

Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBP chr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs.

Genomic trajectories to fluoroquinolone resistance in Francisella tularensis subsp. holarctica live vaccine strain

Objectives: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness.Methods: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis.Results: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC, and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss.Conclusion: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures.

A role for the Sts phosphatases in negatively regulating IFNγ‐mediated production of nitric oxide in monocytes

IntroductionThe atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap‐70. Mice lacking Sts expression (Sts −/−) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts −/− mice have not been definitively established, Sts −/− bone marrow‐derived monocytes (BMMs) demonstrate greater clearance of intracellular LVS following ex vivo infection, relative to wild type cells. To determine how the Sts proteins regulate monocyte bactericidal properties, we analyzed responses of infected cells.MethodsMonocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony‐forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse‐transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme‐linked immunosorbent assay.ResultsWe demonstrate that Sts −/− monocyte cultures produce elevated levels of interferon‐γ (IFNγ) after infection, relative to wild type cultures. Sts −/− monocytes also demonstrate heightened responsiveness to IFNγ. Specifically, Sts −/− monocytes produce elevated levels of antimicrobial NO following IFNγ stimulation, and this NO plays an important role in LVS restriction. Additional IFNγ‐stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts −/− cells. Both Sts‐1 and Sts‐2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFNγ‐induced NO in Sts −/− monocytes is abrogated following chemical inhibition of Syk kinase.ConclusionOur results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFNγ.

Whole-Genome Sequencing and Phylogenetic Analysis of <i>Francisella tularensis</i> Vaccine Strain 15 NIIEG

Objective of the study was to conduct whole-genome sequencing of the vaccine strain Francisella tularensis 15 NIIEG and determine, based on the results, its phylogenetic relationships and the genetic organization features.Materials and methods. Whole-genome sequencing of F. tularensis 15 NIIEG strain was performed on Ion PGM (Ion Torrent, USA) and MinIon (Oxford Nanopore Technologies, UK) platforms. Alignment of readings obtained to the whole-genome of F. tularensis subsp. holarctica LVS (CP009694, USA, 2015) was performed using the software package DNASTAR Lasergene 15.3. Hybrid assembly of reads into contigs was performed by means of Unicycler v. 0.4.4, using data obtained by semiconductor sequencing technology (Ion PGM) and nanopore sequencing (MinIon). Phylogenetic analysis was performed on the basis of single nucleotide polymorphism (SNPs) data located in the core part of F. tularensis genome. Maximum parsimony algorithm was used to construct a dendrogram using the obtained data of common SNP-matrix.Results and discussion. The close relations of F. tularensis 15 NIIEG strain with F. tularensis LVS vaccine strain used in the countries of Western Europe and North America was confirmed. Searching for common single mutations characteristic of F. tularensis 15 vaccine strains of NIIEG and LVS, permitted to find 5 unique SNPs that distinguish them from all other 228 F. tularensis strains used in the comparison. Comparative genomic analysis ofF. tularensis 15 NIIEG vaccine strain and virulent strains revealed in its structure two extensive 526 bp deletions (genes pilA and pilE) and 1480 bp (genes encoding lipoprotein). Similar deletions are also present in the genome of the F. tularensis LVS vaccine strain.

Stochastic dynamics of Francisella tularensis infection and replication.

We study the pathogenesis of Francisella tularensis infection with an experimental mouse model, agent-based computation and mathematical analysis. Following inhalational exposure to Francisella tularensis SCHU S4, a small initial number of bacteria enter lung host cells and proliferate inside them, eventually destroying the host cell and releasing numerous copies that infect other cells. Our analysis of disease progression is based on a stochastic model of a population of infectious agents inside one host cell, extending the birth-and-death process by the occurrence of catastrophes: cell rupture events that affect all bacteria in a cell simultaneously. Closed expressions are obtained for the survival function of an infected cell, the number of bacteria released as a function of time after infection, and the total bacterial load. We compare our mathematical analysis with the results of agent-based computation and, making use of approximate Bayesian statistical inference, with experimental measurements carried out after murine aerosol infection with the virulent SCHU S4 strain of the bacterium Francisella tularensis, that infects alveolar macrophages. The posterior distribution of the rate of replication of intracellular bacteria is consistent with the estimate that the time between rounds of bacterial division is less than 6 hours in vivo.

RM-CSF efficiently replaces L929 in generating mouse and rat bone marrow-derived macrophages for in vitro functional studies of immunity to intracellular bacteria

Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization.

Genetic diversity of Francisella tularensis in Poland with comments on MLVA genotyping and a proposition of a novel rapid v4-genotyping

We investigated the genotypes of Francisella tularensis (F. tularensis) strains isolated in Poland during the period 1953–2013 and studied their genetic relationship to F. tularensis strains isolated in other countries using MLVA. We examined the mosquito and tick samples collected in Poland for the presence of F. tularensis DNA using PCR. Our results revealed a high genetic diversity among the strains of F. tularensis collected from Poland, suggesting that the bacterium is commonly found in the environment. However, we did not detect F. tularensis DNA in ticks and mosquitoes, showing that the arthropod bites might not be the main source of infection.We also propose the application of a practical assay called v4-genotyping that can be directly performed on the clinical and environmental samples.In addition, we discovered genetic variations among Schu S4 reference strains used in various laboratories and showed that MLVA analysis should not be based on amplicon sizes only because point mutations occurring within the MLVA loci might not always be manifested by a change in the amplicon size.