SYMBIOSIS IN THE COLD: IDENTIFICATION AND CHARACTERIZATION OF A NEW FRANCISELLA ENDOSYMBIONT FROM THE POLAR CILIATE, EUPLOTES PETZI

Ciliates of the genus Euplotes are commonly found in polar environments, and different species isolated from Arctic and Antarctic coastal seawaters are currently studied for their genome evolution and adaptation. In analyzing whole genome sequences of a wild-type E. petzi strain collected from Terra Nova Bay (Antarctica), it appeared that more than 3% of the assembled contigs had a bacterial origin and overlapped (one contig containing rDNA operon included) with DNA sequences of the gammaproteobacterium Francisella (represented by extremely infectious species to a wide array of different organisms man included). Given that an Euplotes species of temperate seawaters, E. raikovi, has already been found to host a Francisella species (namely F. endociliophora), we searched for and succeeded in isolating Francisella-like endosymbionts from E. petzi cells. Colonies of these endosymbionts (grown optimally at a temperature range from 4 to maximum 30 °C) have been analyzed for their genome and found to represent a new clade with a basal position in the Francisella phylogenetic tree. This clade is unequivocally distinct from F. endociliophora (living in E. raikovi) as well as from all the other well recognised Francisella clades. The finding that Francisella is adapted to live in the extreme environmental conditions of the polar regions implies that this bacterium is much more common and geographically widespread than previously known, and that free-living Euplotes species may represent a natural reservoir of Francisella in every aquatic environments

Association between Legionella species and humic substances during early summer in the northern Baltic Sea

Climate change is projected to cause alterations in northern coastal systems, including humification and intensified nutrient loads, which can lead to ecosystem imbalances and establishment of new bacterial species. Several potential pathogens, such as different species of Legionella, hide in the environment between infections, some by living inside protozoan host cells. Knowledge about the occurrence of Legionella in natural waters is missing, which disable risk assessments of exposure. We performed a study of the species diversity of Legionella in the northern Baltic Sea (Gulf of Bothnia) during early summer to map their occurrence and to identify possible environmental drivers. We detected Legionella and potential protozoan hosts along gradients of the Gulf of Bothnia. We also for the first time present third generation full-length 16S rRNA amplicon sequencing (Nanopore) to resolve environmental species classification of Legionella, with a method suitable to study all bacteria. Our data show that full length 16S rRNA sequences is sufficient to resolve Legionella while the standard short Illumina sequences did not capture the entire diversity. For accurate species classification of Legionella, harmonization between the Nanopore classification methods is still needed and the bias toward the well-studied Legionella pneumophila need to be resolved. Different Legionella species occurred both in the Bothnian Sea and in the Bothnian Bay and their abundance were linked to humic substances and low salinity. The relative abundance of Legionella was higher in the humic-rich northern waters of the Bothnian Bay. The link between Legionella species and humic substances may be indirect via promotion of the heterotrophic microbial food web, allowing Legionella species and similar bacteria to establish. Humic substances are rich in iron, which has been shown crucial for growth of Legionella species and other pathogens. Considering climate change projections in this regional area, with increased humification and freshwater inflow, this bacterial niche containing potential pathogens might become more widespread in the future Baltic Sea. This study demonstrates the significance of DNA sequencing to monitor public health relevant bacteria like Legionella species in the environment. Including sequencing of bacteria and protozoa in the environmental monitoring programs could be used to identify ecosystem imbalances, which enable appropriate responses to emerging diseases

The aquatic microbial food web and occurence of predation-resistant and potentially pathogenic bacteria, such as Francisella tularensis

All natural aquatic systems harbour a vast variety of microorganisms. In the aquatic microbial food web, the larger microorganisms (i.e. protozoa) feed on the smaller microorganisms (i.e. bacteria and phytoplankton). An increase in nutrient availability results in changes of the microbial food web structure, like altered community composition and blooms of toxic phytoplankton. In this thesis work I hypothesised that nutrient-rich aquatic environments, with strong protozoan predation, favour the occurrence of predation-resistant bacteria like F. tularensis, and that the microbial food web may provide a reservoir for the bacterium between outbreaks. By using a size-structured ecosystem food web model it was shown that the protozoan predation pressure on bacteria, defined as protozoan predation per bacterial biomass, increases with increasing nutrient availability in aquatic systems (estimated chlorophyll a 0.2 to 112 μg L-1). This dynamics was caused by increasing growth-rate of a relatively constant number of bacterial cells, maintaining the growth of an increasing number of protozoan cells. The results were supported by meta-analysis of field studies. Thus my results suggest that protozoa control the bacterial community by predation in nutrient-rich environments. In a field study in a natural productivity gradient (chlorophyll a 1.4 to 31 μg L-1) it was shown that intense selection pressure from protozoan predators, favours predation-resistant forms of bacteria. Thus, the abundance of predation-resistant bacteria increases with increasing nutrient availability in lakes. Furthermore, I could demonstrate that the bacterium Francisella tularensis, the causative agent of tularemia, was present in eutrophic aquatic systems in an emerging tularemia area. Isolated strains of the bacterium were found to be resistant to protozoan predation. In a microcosm study, using natural lake water, high nutrient availability in combination with high abundance of a small colourless flagellate predator favoured the occurrence of F. tularensis holarctica. In laboratory experiments F. tularensis strains were able to form biofilm at temperatures between 30-37°C, but not below 30°C. In conclusion, I have shown that the protozoan predation pressure on bacteria increases with increasing nutrient availability in aquatic systems. Predation-resistant forms of bacteria, such as F. tularensis are favoured in nutrient-rich environments. The complexity of the microbial food web and nutrient-richness of the water, influence the transmission of the pathogenic F. tularensis holarctica. However, over long periods of time, the bacterium survives in lake water but may lose its virulence. The temperature-regulated biofilm formation by F. tularensis may play a role in colonization of vectors or for colonization of hosts, rather than for survival in aquatic environments

Microbial Interactions - Underexplored Links Between Public Health Relevant Bacteria and Protozoa in Coastal Environments

The co-existence of bacteria and protozoa in aquatic environments has led to the evolution of predation defense mechanisms by the bacteria. Some of the predation-resistant bacteria (PRB) are also pathogenic to humans and other mammals. The links between PRB and protozoa in natural aquatic systems are poorly known, but they are important in predicting outbreaks and determining the long-term consequences of a contamination event. To elucidate co-occurrence patterns between PRB (16S rRNA) and bacterivorous protozoa (18S rRNA), we performed a field study in a coastal area in the northern Baltic Sea. Interactions between bacteria and protozoa were explored by using two complementary statistical tools. We found co-occurrence patterns between specific PRB and protozoa, such as Legionella and Ciliophora, and we also found that the interactions are genotype-specific as, for example, Rickettsia. The PRB sequence diversity was larger in bays and freshwater inlets compared to offshore sites, indicating local adaptions. Considering the PRB diversity in the freshwater in combination with the large spring floods in the area, freshwater influxes should be considered a potential source of PRB in the coastal northern Baltic Sea. These findings are relevant for the knowledge of survival and dispersal of potential pathogens in the environment

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

The Tick-Borne Diseases STING study : Real-time PCR analysis of three emerging tick-borne pathogens in ticks that have bitten humans in different regions of Sweden and the Aland islands, Finland

A milder climate has during the last decade contributed to an increased density and spreading of ixodid ticks, thus enhancing their role as emerging vectors for pathogenic microorganisms in northern Europe. It remains unclear if they contribute to the occurrence of infections caused by the bacteria Bartonella spp., Francisella tularensis subspecies holarctica and the parasite Toxoplasma gondii in Sweden and on the Åland islands, Finland. In this study, we want to improve understanding of the tick-borne transmission of these pathogens. Volunteers were recruited at primary healthcare centers. Ticks and blood samples were acquired from participants recruited in 2008 and 2009. Health questionnaires were completed, and medical records were acquired where applicable. Feeding time was estimated and screening of pathogens in the ticks was performed through real-time PCR. Ticks (n = 1849) were of mixed developmental stages: 76 larvae, 1295 nymphs, 426 adults and 52 undetermined. All analyzed ticks were considered negative for these pathogens since the CT-values were all below the detection limit for Bartonella spp. (1663 ticks), Francisella spp. (1849 ticks) and Toxoplasma gondii (1813 ticks). We assume that infections with these pathogens are caused by other transmission pathways within these regions of Sweden and the Åland islands, Finland

Table_1_Galleria mellonella Reveals Niche Differences Between Highly Pathogenic and Closely Related Strains of Francisella spp..DOCX

<p>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.</p

Image_1_Galleria mellonella Reveals Niche Differences Between Highly Pathogenic and Closely Related Strains of Francisella spp..TIFF

<p>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.</p

Association between Legionella species and humic substances during early summer in the northern Baltic Sea

Climate change is projected to cause alterations in northern coastal systems, including humification and intensified nutrient loads, which can lead to ecosystem imbalances and establishment of new bacterial species. Several potential pathogens, such as different species of Legionella, hide in the environment between infections, some by living inside protozoan host cells. Knowledge about the occurrence of Legionella in natural waters is missing, which disable risk assessments of exposure. We performed a study of the species diversity of Legionella in the northern Baltic Sea (Gulf of Bothnia) during early summer to map their occurrence and to identify possible environmental drivers. We detected Legionella and potential protozoan hosts along gradients of the Gulf of Bothnia. We also for the first time present third generation full-length 16S rRNA amplicon sequencing (Nanopore) to resolve environmental species classification of Legionella, with a method suitable to study all bacteria. Our data show that full length 16S rRNA sequences is sufficient to resolve Legionella while the standard short Illumina sequences did not capture the entire diversity. For accurate species classification of Legionella, harmonization between the Nanopore classification methods is still needed and the bias toward the well-studied Legionella pneumophila need to be resolved. Different Legionella species occurred both in the Bothnian Sea and in the Bothnian Bay and their abundance were linked to humic substances and low salinity. The relative abundance of Legionella was higher in the humic-rich northern waters of the Bothnian Bay. The link between Legionella species and humic substances may be indirect via promotion of the heterotrophic microbial food web, allowing Legionella species and similar bacteria to establish. Humic substances are rich in iron, which has been shown crucial for growth of Legionella species and other pathogens. Considering climate change projections in this regional area, with increased humification and freshwater inflow, this bacterial niche containing potential pathogens might become more widespread in the future Baltic Sea. This study demonstrates the significance of DNA sequencing to monitor public health relevant bacteria like Legionella species in the environment. Including sequencing of bacteria and protozoa in the environmental monitoring programs could be used to identify ecosystem imbalances, which enable appropriate responses to emerging diseases

The ciliate Euplotes petzi is the natural reservoir of the bacterium Francisella in the Antarctic region.

Euplotes petzi is a strict psychrophilic ciliate isolated from coastal waters of Terra Nova Bay (Antarctica). In analyzing the results from a draft genome sequencing of total E. petzi DNA preparations, we found that more than 3% of the assembled contigs had a bacterial origin. All the bacterial DNA sequences, including one containing rRNA genes, overlapped with DNA sequences of the genus Francisella, which comprises a large number of species classified as facultative intracellular γ-proteobacteria potential noxious to their hosts. Based on these findings, we undertook the isolation, characterization and genome determination of the Francisella-like bacteria – tentatively named F. adeliensis sp. nov. – hosted in the cytoplasm of E. petzi. Genome sequence comparisons provide evidence that F. adeliensis forms a new clade in the Francisella phylogenomic tree. In addition to being well separated from all the recognised pathogenic Francisella species, this clade is distinct also from F. endociliophora, symbiont in the congeneric species E. raikovi, as well as from the Allofrancisella species that are collectively known as ‘environmental francisellas’. The inclusion of F. adeliensis in the Francisella phylogenomic tree does not support the separation of this genus between ‘intracellular’ and ‘environmental’ forms