Francisella Species Research Articles

Distribution of Haemaphysalis longicornis and associated pathogens: analysis of pooled data from a China field survey and global published data

Haemaphysalis longicornis, a vector of various pathogens with medical and veterinary importance, is native to eastern Asia, and recently reached the USA as an emerging disease threat. In this study, we aimed to identify the geographical distribution, hosts, and associated pathogens of H longicornis. Data were collected from multiple sources, including a field survey, reference book, literature review, and related websites. The thematic maps showing geographical distribution of H longicornis and associated pathogens were produced by ArcGIS. Hosts of H longicornis and positive rates for H longicornis-associated pathogens were estimated by meta-analysis. Ecological niche modelling was used to predict potential global distribution of H longicornis. H longicornis was found to be present in ten countries, predominantly in eastern Asia, the USA, Australia, and New Zealand. The tick was known to feed on a variety of domestic and wild animals, and humans. At least 30 human pathogens were associated with H longicornis, including seven species of spotted fever group rickettsiae, seven species in the family of Anaplasmataceae, four genospecies in the complex Borrelia burgdorferi sensu lato, two Babesia species, six species of virus, and Francisella, Bartonella, Coxiella, and Toxoplasma, which were mainly reported in eastern Asia. The predictive modelling revealed that H longicornis might affect more extensive regions, including Europe, South America, and Africa, where the tick has never been recorded before. H longicornis is relatively common in the world, and is associated with various human and animal pathogens. Authorities and health-care workers should be aware of the threat of the tick species to public health and veterinary medicine. Surveillance and further investigations should be enhanced globally. National Natural Science Foundation of China and National Key Research and Development Program of China.

Francisella novicida and F. philomiragia biofilm features conditionning fitness in spring water and in presence of antibiotics.

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.

Low genetic diversity among Francisella-like endosymbionts within different genotypes of Hyalomma dromedarii ticks infesting camels in Saudi Arabia.

Background and Aim:Hyalomma dromedarii ticks are vectors of disease agents and hosts of Francisella-like endosymbionts (FLEs). Knowledge about intraspecific genetic variation among H. dromedarii and its Francisella species is limited. The aims of this study were to investigate whether certain H. dromedarii genotypes are specialized in carrying specific Francisella species genotypes and scrutinize the population structure of H. dromedarii ticks in Saudi Arabia.Materials and Methods:We collected 151 H. dromedarii ticks from 33 camels from 13 locations in Saudi Arabia. The second internal transcribed spacer (ITS2), cytochrome c oxidase subunit-1(COI), and 16S rRNA genes were used for single- and multi-locus sequence typing and phylogenetic analyses. H. dromedarii-borne Francisella was screened using the tul4 gene and 16S rRNA Francisella-specific primers followed by amplicon Sanger sequencing.Results:Single-locus typing of ticks using ITS2, 16S rRNA, and COI genes yielded 1, 10, and 31 sequence types (ST), respectively, with pairwise sequence similarity of 100% for ITS2, 99.18-99.86% for COI, and 99.50-99.75% for 16S rRNA. COI sequence analysis indicated a lack of strict geographical structuration, as ST15 was found in both Saudi Arabia and Kenya. In contrast, multilocus sequence typing resolved 148 H. dromedarii ticks into 39 genotypes of ticks and three genotypes of FLEs. The ST2-FLE genotype was carried by the tick genotype ST35, while the ST1-FLE genotype and 41.89% of the ST3-FLE genotype were carried by the tick genotype ST32. Accordingly, there appeared to be no specialization of certain tick genotypes to harbor-specific FLE genotypes.Conclusion:For the 1st time, we have provided an overview of the population structure of H. dromedarii ticks and FLE strains. We found a low level of genetic diversity among FLEs and non-specialized circulation of FLEs among H. dromedarii ticks.

Outbreak of francisellosis (Francisella noatunensis subsp. orientalis) in cultured neon jewel cichlids Hemichromis bimaculatus from Morelos, Mexico

Francisellosis is a disease caused by different species of the bacterial genus Francisella and has been diagnosed in a wide variety of animals, including fish. Francisellosis in fish is characterized by the development of non-specific clinical signs as well as the presence of numerous granulomas in several organs (mainly spleen and kidney). Ten neon jewel cichlids Hemichromis bimaculatus were submitted for diagnosis from a farm located in Morelos, Mexico. Gross examination, wet preparations, cytology, histopathology and PCR were performed. Affected fish showed lethargy, erratic swimming, imbalance and gasping. At the post mortem examination, multiple granulomas were observed in the kidney and spleen. Microscopically, granulomatous inflammation was observed in several organs. Species-specific PCR assay using DNA from the affected tissues of H. bimaculatus as a template demonstrated the presence of F. noatunensis subsp. orientalis (Fno) by amplifying a hypothetical protein gene of the Fno species. The end diagnosis of francisellosis is important for Mexican ornamental aquaculture, since it is necessary to implement measures for treatment, prevention, control and diagnosis. This is the first report of francisellosis in the neon jewel cichlid.

First molecular detection of Francisella-like endosymbionts in Hyalomma and Rhipicephalus tick species collected from vertebrate hosts from Sardinia island, Italy.

Ticks are vectors of a wide variety of human and animal pathogens as well as non-pathogenic microorganisms acting as endosymbionts and whose role in ticks is still little known. Symbionts such as Francisella-like endosymbionts (FLEs) are members of Francisellaceae family with unknown pathogenicity, detected in both hard and soft ticks. A total of 236 ticks collected from several sites in Sardinia were screened for Francisella species by PCR using primers targeting a fragment of the 16S rRNA gene. DNA of Francisella was detected in 5.1% (12/236) of the ticks tested. Sequencing results revealed that seven Rhipicephalus sanguineus s.l., three Hyalomma marginatum, one Hy. lusitanicum, and one Rh. bursa ticks exhibited DNA with 99-100% similarity to Francisella-like endosymbionts isolated from different tick species all over the world. Further research is needed in order to better characterize FLE strains obtained in Sardinia and to better understand if their presence could be related to the infection with other zoonotic pathogens.

Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a “scatter screen”, used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.

Two-Component Systems in Francisella Species.

Bacteria alter gene expression in response to changes in their environment through various mechanisms that include signal transduction systems. These signal transduction systems use membrane histidine kinase with sensing domains to mediate phosphotransfer to DNA-binding proteins that alter the level of gene expression. Such regulators are called two-component systems (TCSs). TCSs integrate external signals and information from stress pathways, central metabolism and other global regulators, thus playing an important role as part of the overall regulatory network. This review will focus on the knowledge of TCSs in the Gram-negative bacterium, Francisella tularensis, a biothreat agent with a wide range of potential hosts and a significant ability to cause disease. While TCSs have been well-studied in several bacterial pathogens, they have not been well-studied in non-model organisms, such as F. tularensis and its subspecies, whose canonical TCS content surprisingly ranges from few to none. Additionally, of those TCS genes present, many are orphan components, including KdpDE, QseC, QseB/PmrA, and an unnamed two-component system (FTN_1452/FTN_1453). We discuss recent advances in this field related to the role of TCSs in Francisella physiology and pathogenesis and compare the TCS genes present in human virulent versus. environmental species and subspecies of Francisella.

The prevalence of Francisella spp. in different natural surface water samples collected from northwest of Iran

Francisella tularensis has a wide distribution in northern hemisphere of the world. Up to now, there was little information about the Francisella spp. situation in the environmental samples in Iran. In this study we aimed to determine the prevalence of Francisella spp. in the environmental samples in northwest of Iran. A total of 237 natural water samples from ponds, rivers, lakes, springs and other surface waters from north western provinces of Iran (Kurdistan and Western Azerbaijan) were collected from September to November 2015. All samples were cultured for Francisella and other bacterial species and Real Time TaqMan PCR was performed on the concentrated and DNA extracted samples. For detection of the presence of bacterial DNA in the samples, two different targets in the genome of Francisella, ISFtu2 and fopA were used. Among the tested surface water samples, 40 (17.09%; 95% CI: 12.67-22.33%) and 12 (5.13%; 95%CI: 2.81-8.56%) samples were positive for ISFtu2 and fopA respectively. None of them was positive in culture. The prevalence of Francisella spp. in the environmental samples in the west of Iran is high and it is comparable with Turkey, Iran's neighboring country. Use of higher copy number genes or IS like ISFtu2 could improve the detection of this organism in the environmental samples.

Application of multiplex quantitative polymerase chain reaction methods to detect common bacterial fish pathogens in Nile tilapia, Oreochromis niloticus, hatcheries in Costa Rica

Edwardsiella spp., Streptococcus spp., and Francisella noatunensis subsp. orientalis are some of the most important fish pathogens affecting global tilapia, Oreochromis spp., aquaculture. In Costa Rica, the aquaculture industry is dominated by freshwater‐cultured Nile tilapia, Oreochromis niloticus, which are raised in all seven national provinces. At present, little is known regarding the diversity of pathogens present in these facilities, and definitive identification of agents associated with disease outbreaks are rare. To evaluate the prevalence of common bacterial pathogens in these systems, this study used multiplex quantitative polymerase chain reaction (qPCR) assays targeting Edwardsiella, Streptococcus, and Francisella species as a diagnostic and surveillance tool. In 2017, seven different tilapia hatcheries were visited, and 350 fingerlings were subjected to necropsy and molecular diagnostic evaluation. Fish exhibiting gross signs of disease were subjected to histological and microbiological analysis. For the first time, Edwardsiella anguillarum was recovered and molecularly confirmed from diseased tilapia in Costa Rica. In addition, F. noatunensis subsp. orientalis was identified in a region of Costa Rica where it had not been previously reported.

A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look atF. adeliensis asa pioneer in the Francisella colonization of eukaryotic organisms.

Molecular Survey of Tularemia and Plague in Small Mammals From Iran.

Introduction: Plague and tularemia are zoonoses and their causative bacteria are circulating in certain regions of Iran. This study was conducted to investigate potential disease reservoirs amongst small wildlife species in different regions of Iran.Methods: Rodents, insectivores and hares from 17 different provinces of the country were collected in 2014 and 2015. Samples were taken from the spleens of the animals and Real-time PCR was applied to detect nucleic acid sequences that are specific to Francisella tularensis and Yersinia pestis, respectively.Results: Among 140 collected rodents, 25 distinct species were identified out of which five were the most common: Microtus paradoxus (21% out of 140 rodents), Apodemus witherbyi (12%), Microtus irani (11%), Mus musculus (11%) and Microtus socialis (10%). Seventeen insectivores were collected and identified as Crocidura suaveolens (82%) and C. leucodon (18%). Fifty-one hares were collected and identified as Lepus europaeus (57%), Lepus tolai (14%) and Lepus sp. (29%). Three out of 140 explored rodents (1.91%) were positive for F. tularensis, an A. witherbyi, a Mus musculus domesticus, and a Chionomys nivalis collected from Golestan, Khuzestan and Razavi Khorasan provinces, respectively. Two hares (3.92%) were F. tularensis-positive, a L. europaeus from Khuzestan and a Lepus sp. from the Sistan and Baluchistan province. None of the tested animals were positive for Y. pestis.Conclusion: This is the first report of direct detection of F. tularensis in mammals of Iran and the first-time observation of the agent in a snow vole, C. nivalis worldwide. The results indicate that tularemia is more widespread in Iran than previously reported including the Northeast and Southwestern parts of the country. Future studies should address genetic characterization of F. tularensis positive DNA samples from Iran to achieve molecular subtyping and rule out assay cross-reactivity with near neighbor Francisella species.

Galleria mellonella Reveals Niche Differences Between Highly Pathogenic and Closely Related Strains of Francisella spp.

Francisella tularensis, a highly virulent bacteria that causes the zoonotic disease tularemia, is considered a potential agent of biological warfare and bioterrorism. Although the host range for several species within the Francisella is known, little is known about the natural reservoirs of various Francisella species. The lack of knowledge regarding the environmental fates of these pathogens greatly reduces the possibilities for microbial risk assessments. The greater wax moth (Galleria mellonella) is an insect of the order Lepidoptera that has been used as an alternative model to study microbial infection during recent years. The aim of this study was to evaluate G. mellonella as a model system for studies of human pathogenic and closely related opportunistic and non-pathogenic strains within the Francisella genus. The employed G. mellonella larvae model demonstrated differences in lethality between human pathogenic and human non-pathogenic or opportunistic Francisella species. The F. novicida, F. hispaniensis and F. philomiragia strains were significantly more virulent in the G. mellonella model than the strains of human pathogens F. t. holarctica and F. t. tularensis. Our data show that G. mellonella is a possible in vivo model of insect immunity for studies of both opportunistic and virulent lineages of Francisella spp., that produces inverse results regarding lethality in G. mellonella and incapacitating disease in humans. The results provide insight into the potential host specificity of F. tularensis and closely related members of the same genus, thus increasing our present understanding of Francisella spp. ecology.

Golden jackals (Canis aureus) as hosts for ticks and tick-borne pathogens in Serbia

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens.In a study conducted during a three-year period (01/2010–02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses.The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected.From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Zinc Acquisition Mechanisms Differ between Environmental and Virulent Francisella Species.

ABSTRACTZinc is an essential nutrient for bacterial growth. Because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as Francisella must sense their environment and acquire zinc in response. In many bacteria, the conserved transcription factor Zur is a key regulator of zinc acquisition. To identify mechanisms of zinc uptake in Francisella novicida U112, transcriptome sequencing of wild-type and putative zur mutant bacteria was performed. Only three genes were confirmed as directly regulated by Zur and zinc limitation by quantitative reverse transcription-PCR. One of these genes, FTN_0879, is predicted to encode a protein with similarity to the zupT family of zinc transporters, which are not typically regulated by Zur. While a putative znuACB operon encoding a high-affinity zinc transporter was identified in U112, expression of this operon was not controlled by Zur or zinc concentration. Disruption of zupT but not znuA in U112 impaired growth under zinc limitation, suggesting that ZupT is the primary mechanism of zinc acquisition under these conditions. In the virulent Francisella tularensis subsp. tularensis Schu S4 strain, zupT is a pseudogene, and attempts to delete znuA were unsuccessful, suggesting that it is essential in this strain. A reverse TetR repression system was used to knock down the expression of znuA in Schu S4, revealing that znuA is required for growth under zinc limitation and contributes to intracellular growth within macrophages. Overall, this work identifies genes necessary for adaptation to zinc limitation and highlights nutritional differences between environmental and virulent Francisella strains.IMPORTANCE Francisella tularensis is a tier 1 select agent with a high potential for lethality and no approved vaccine. A better understanding of Francisella virulence factors is required for the development of therapeutics. While acquisition of zinc has been shown to be required for the virulence of numerous intracellular pathogens, zinc uptake has not been characterized in Francisella. This work characterizes the Zur regulon in F. novicida and identifies two transporters that contribute to bacterial growth under zinc limitation. In addition, these data identify differences in mechanisms of zinc uptake and tolerance to zinc limitation between F. tularensis and F. novicida, highlighting the role of znuA in the growth of Schu S4 under zinc limitation.

Development of nineteen Taqman real-time PCR assays for screening and detection of select highly pathogenic bacteria

ABSTRACTBackground: Here, we describe a set of six Taqman real-time PCR assays for screening of highly pathogenic bacteria, i.e. Bacillus, Brucella, Burkholderia, Coxiella, Francisella, and Yersinia species. Twelve specific assays are subsequently performed to identify the species that are classified as highly pathogenic and a general 16S Taqman real-time PCR assay is included to see if the sample contains bacteria.Methods: These assays were designed using all available genomes in the public database of bioterror agents. They were validated with a collection of reference strains, clinical isolates and one environmental sample.Results: These assays were tested against all the ring trials we participate among them the ones which were coordinated by Robert Koch Institute from a repository built up in the framework of the EU funded project ‘Efficient response to highly dangerous and emerging pathogens’ (EMERGE). All bacteria were accurately identified in food, clinical and environmental matrices.Conclusions: These assays are used routinely in our diagnostic laboratory to rapidly screen for and specifically detect select highly pathogenic bacteria of potential bioterrorism use. The platform can be used as an open array format in 96-well plates to screen for a single species or up to 6 agents in one run.Abbreviations: ATCC; American Type Culture Collection, B; Brucella; BLAST: Basic local alignment search tool; BSL; Biosafety level; Cq: Quantification cycle; DNA; Deoxyribonucleic acid, FAM; 6-carboxyfluorescein, FOHM; the Public Health Agency of Sweden, IAC; internal amplification control, LOD: Limit of detection, MGB; Minor groove binder, NCBI; National Center for Biotechnology Information, NFA; National Food Agency, PCR; polymerase chain reaction; PhHV-1; Phocine Herpesvirus 1, SVA; National Veterinary Institute, Tm: Melting Temperature.

Monitoring of Francisella noatunensis subsp. orientalis in farmed Nile tilapia (Oreochromis niloticus) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.

Shared features of cryptic plasmids from environmental and pathogenic Francisella species.

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We have identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. Genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.

Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan.

Francisella novicida is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Francisella tularensis Data published by us and others demonstrate that F. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, F. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that F. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

Contribution of methionine sulfoxide reductase B (MsrB) to Francisella tularensis infection in mice.

The essential mechanisms and virulence factors enabling Francisella species to replicate inside host macrophages are not fully understood. Methionine sulfoxide reductase (Msr) is an antioxidant enzyme that converts oxidized methionine into methionine. Francisella tularensis carries msrA and msrB in different parts of its chromosome. In this study, single and double mutants of msrA and msrB were constructed, and the characteristics of these mutants were investigated. The msrB mutant exhibited decreased in vitro growth, exogenous oxidative stress resistance and intracellular growth in macrophages, whereas the msrA mutant displayed little difference with wild-type strain. The double mutant exhibited the same characteristics as the msrB mutant. The bacterial count of the msrB mutant was significantly lower than that of the wild-type strain in the liver and spleen of mice. The bacterial count of the msrA mutant was lower than that of the wild-type strain in the liver, but not in the spleen, of mice. These results suggest that MsrB has an important role in the intracellular replication of F. tularensis in macrophages and infection in mice.