THE AQUATIC MICROBIAL FOOD WEB

THE AQUATIC MICROBIAL FOOD WEB

AND OCCURRENCE OF PREDATION-

RESISTANT AND POTENTIALLY PATHOGENIC

BACTERIA, SUCH AS FRANCISELLA

TULARENSIS

JOHANNA THELAUS

DEPARTMENT OF ECOLOGY AND ENVIRONMENTAL SCIENCE, UMEÅ UNIVERSITY, SWEDEN

FOI CBRN DEFENCE AND SECURITY, UMEÅ, SWEDEN

THE AQUATIC MICROBIAL FOOD WEB AND

OCCURRENCE OF PREDATION-RESISTANT

AND POTENTIALLY PATHOGENIC BACTERIA,

SUCH AS FRANCISELLA TULARENSIS

Av

JOHANNA THELAUS

Institutionen för Ekologi Miljö och Geovetenskap Umeå Universitet

Umeå 2008 ___________________________________________________ Akademisk avhandling, som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av doktorsexamen i ekologi kommer att offentligen försvaras i Lilla hörsalen, KBC -huset, Umeå universitet. Torsdagen den 22 maj 2008, kl. 10.00. Avhandlingen kommer att försvaras på engelska. Fakultetsopponent: Professor Farooq Azam Scripps Institution of Oceanography, UCSD La Jolla, USA.

Organisation Document type Umeå University Doctorial thesis Department of Ecology and Environmental Science SE- 901 87 Umeå Author Date of publication Johanna Thelaus 29 April 2008 Title The aquatic microbial food web and occurrence of predation-resistant and potentially pathogenic bacteria, such as Francisella tularensis Abstract

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. Key Words: bacteria, protozoa, predation, nutrient-availability, microbial food web, model, predation-resistance, Francisella, reservoir, biofilm formation, ecology Language: English ISBN: 978-91-7264-529-5 Number of pages: 49 + 4 papers

THE AQUATIC MICROBIAL FOOD WEB AND

OCCURRENCE OF PREDATION-RESISTANT

AND POTENTIALLY PATHOGENIC BACTERIA,

SUCH AS FRANCISELLA TULARENSIS

JOHANNA THELAUS

____________________________________________________________ DEPARTMENT OF ECOLOGY AND ENVIRONMENTAL SCIENCE, UMEÅ UNIVERSITY, SWEDEN

FOI CBRN DEFENCE AND SECURITY, UMEÅ, SWEDEN

UMEÅ 2008

PhD Thesis

Department of Ecology and Environmental Science

Umeå University

SE-901 87 Umeå

FOI, CBRN Defence and Security

Swedish Defence Research Agency

SE-901 82 Umeå

ISBN: 978-91-7264-529-5

© Johanna Thelaus 2008

Detta verk skyddas enligt lagen om upphovsrätt (URL 1960:729)

Printed in Sweden by Solfjädern Offset AB, Umeå, 2008

Cover: Bacterial isolates growing on an agar plate; from the field study

experiment described in paper III. Photo by Johanna Thelaus.

TABLE OF CONTENTS LIST OF PAPERS............................................................................................iv INTRODUCTION.............................................................................................1

The aquatic microbial food web Components and structure....................................................................... 1 The effect of increasing nutrient availability............................................... 4 Predation-resistant bacteria survive protozoan predation............................... 6

Francisella, the causative agent of tularemia The pathogenic bacterium Francisella....................................................... 7

OBJECTIVES................................................................................................10 METHODOLOGICAL CONSIDERATIONS...................................................... 11

Model simulations - An ecosystem model of the microbial food web.11 Bacterial edibility assay........................................................................13 Molecular methods............................................................................... 14

RESULTS AND DISCUSSION......................................................................... 16 Impact of increasing productivity on aquatic bacterial communities

Increasing predation pressure on bacteria................................................. 16 Occurrence of predation-resistant bacteria................................................ 20

Francisella tularensis in the aquatic microbial food web Occurrence of F. tularensis in a lake water system.....................................23 Francisella - a pathogenic predation-resistant bacterium............................. 25 A potential reservoir for F. tularensis holarctica....................................... 29 F. tularensis is able to form biofilms....................................................... 31

CONCLUSIONS AND FUTURE PERSPECTIVES.............................................. 34 A tentative model for the dispersal of F. tularensis holarctica from water to mammals................................................................................ 36

ACKNOWLEDGEMENTS.............................................................................. 36 REFERENCES.............................................................................................. 37 SVENSK SAMMANFATTNING....................................................................... 46 TACK.......................................................................................................... 48

iv

LIST OF PAPERS This thesis is based on the following four articles, which are reffered to in the text by their roman numerals. I. Thelaus J., Heacky P., Forsman M. and Andersson A. (2008)

Predation pressure on bacteria increases along aquatic productivity gradients. Aquatic Microbial Ecology. forthcoming. doi 10.3354/ame01200.

II. Thelaus J., Forsman M. and Andersson A. (2007) Role of

productivity and protozoan abundance for the occurrence of predation-resistant bacteria in aquatic systems. Microbial Ecology. early online. doi 10.1007/s00248-007-9320-4.

III. Thelaus J., Andersson A., Mathisen P., Forslund A-L. and

Forsman M. Influence of nutrient status and microbial food web structure on the fate of Francisella tularensis in lake water. Submitted manuscript.

IV. Ireland P., Thelaus J., Diaper H., Lever M.S., Forsman M.

and Titball R.W. Characterization of biofilm formation by Francisella tularensis. Submitted manuscript.

Paper I and II are reproduced with kind permission of Springer Science and Business Media and Inter-Research Science Publisher, respectively.

INTRODUCTION

1

INTRODUCTION The aquatic microbial food web

Ecological processes that are visible to the human eye normally take place at the top of the food web, for example predatory birds capturing fish in lakes. However, macro-scale processes are based upon organisms acting on a micro-scale. One of these micro-scale systems involves the microbes that occupy open waters in lakes and oceans. Microbial activity in aquatic environments became an established ecological paradigm, known as the “microbial loop”, in the early 1980s (Pomeroy 1974, Azam et al. 1983) (Fig. 1). At this time, it was acknowledged that bacteria play an important role in planktonic communities, functioning as decomposers of organic material and remineralizers of inorganic nutrients. Despite intensive research and advances in methodologies, the function of the microbial food web has still not been fully resolved. The microbial food web is the foundation of many ecological processes. In order to predict the effects of environmental change and pollution on lakes and oceans, it is important to understand the function and regulation of the system. Knowledge about aquatic microbial processes is also important for understanding how and when specific species are favoured in a particular environment. For example, what environmental factors determine the occurrence of bacterial pathogens in aquatic systems? Such knowledge may inform decision makers when producing management plans and help to avoid epidemics caused by bacterial pathogens, such as Legionella pneumophila and Vibrio cholerae, which are transmitted via water. Components and structure

Half of all global primary production occurs in the oceans (Falkowski et al. 1998, Field et al. 1998). The phytoplankton and photosynthetic bacteria use sunlight to fix carbon from carbon dioxide (CO2), inorganic phosphorus (P) and nitrogen (N), and incorporate these elements into cell components (autotrophy). Autotrophs, together with zooplankton and fish, make up the classical aquatic food chain. A large proportion of the material and energy produced by phytoplankton does not directly reach the top of this classical food chain (Fig. 1). Material, or dissolved organic matter (DOM) (Williams 1981), is lost from the food chain via several mechanisms, including phytoplankton exudation (Bjornsen 1988), sloppy feeding by predators (Strom et al. 1997) and the production of faecal pellets by metazooplankton (Jumars et al. 1989). Only

INTRODUCTION

2

3–5% of the phytoplankton primary production reaches the higher trophic levels, i.e. fish (Ducklow et al. 1986, Fenchel 1988).

Figure 1. The aquatic microbial food web. Phytoplankton (pico-, nano-, and microphytoplankton) use carbon dioxide (CO2) in photosynthesis (open symbols, autotrophs). Phytoplankton are grazed (dotted line) by heterotrophic protists (filled symbols); flagellates, ciliates and metazooplankton. Dissolved organic matter (DOM) is continuously lost from the food web (solid line) and utilized by heterotrophic bacteria, linking the DOM back into the food web via predation by the phagotrophic protists. Redrawn from (Fenchel 1988) Heterotrophic bacteria are the primary decomposers of DOM, with almost exclusive access to this source of nutrients, thus transforming DOM to cell components (heterotrophy) (Azam et al. 1983) (Fig. 1). All nutrients, oxygen and waste products pass through the bacterial cell surface (osmotrophy). Thus, the maximum rate of metabolism possible per bacterial biomass is limited by the surface area to volume ratio. The large value of this ratio in bacteria enables a rapid response to favourable conditions (Pomeroy et al. 2007). Bacteria are thought to consume 40–50 % of the DOM produced by phytoplankton as a result of primary production within aquatic systems (Larsson & Hagström 1982). Thus, bacteria constitute an important link between DOM and larger organisms, like zooplankton and fish, within the classical aquatic food chain (Pauly & Christensen 1995).

INTRODUCTION

3

In natural aquatic systems, nutrient availability is often low and bacteria have to compete for inorganic nutrients with phytoplankton. Since bacteria have a higher affinity for mineral nutrients due to their larger surface area to volume ratio, they out-compete the phytoplankton. However, the main source of bacterial carbon originates from phytoplankton; thus, bacterial growth may be limited by carbon when phytoplankton productivity is low. These mechanisms enable coexistance between bacteria and phytoplankton in aquatic systems (Thingstad & Lignell 1997). Further, protozoan predation on bacteria (bacterivory) may also facilitate the coexistence of bacteria and phytoplankton. Bacterial biomass is kept low by protozoan predators and phytoplankton may compete for the remaining nutrients, resulting in bacteria being nutrient limited. The main predators in the microbial food web are protozoa (flagellates, ciliates and amoeba) and metazoa (Valiela 1995) (Fig. 1). Phagotrophic amoebae are primarily associated with surfaces, grazing/feeding on bacterial biofilms and attached cells (Fenchel 1987, Pickup et al. 2007). The dominant groups of pelagic protozoa, the flagellates and ciliates, differ in size, feeding mechanisms and motion (Fenchel 1987). Flagellates graze on heterotrophic and small sized autotrophic bacteria, regulating bacterial numbers in the sea. Flagellate growth is limited by the availability of resource (Samuelsson et al. 2006). There is a large discrepancy between the observed maximum growth of flagellates on 107 bacteria mL-1 and the actual number of bacteria recorded in the sea, which amounts to 105 to 106 bacteria mL-1 (Boenigk & Arndt 2002). This leads to the limited fluctuations in bacterial and flagellate numbers often observed in field studies and experiments (Azam et al. 1983) (III). In turn, flagellate numbers are controlled by ciliate predation; however ciliates have also been shown to graze on bacteria (Zingel et al. 2007) and phytoplankton (Simek et al. 1995). Metazooplankton feed on ciliates and phytoplankton (Kivi et al. 1996). Metazooplankton predation regulates the protozoan community and, thus, also has an indirect effect on the bacterial community (Jürgens 1994). The importance of viruses and their role in the microbial food web has recently been acknowledged. The abundance of viruses has been shown to exceed the abundance of prokaryotes in both marine and freshwater systems (viral abundance up to 108 cells mL-1) (Wommack & Colwell 2000). The abundance and activity of viruses in aquatic environments are positively correlated with those of the host population (e.g. bacteria). Lytic

INTRODUCTION

4

phages (viruses specific to bacteria) can be viewed as bacterial predators and, thus, contribute to processes within the microbial food web via lysis of bacteria and the release of cell lysis products to the surrounding medium (Weinbauer 2004). The DOM produced and excreted by phytoplankton is utilized by bacteria and brought into the microbial food web via protozoan grazing, then further channelled into the classical food chain (5 % of primary productivity is transported back to the classical food chain via the microbial food web) (Fenchel 1988). Moreover, one important characteristic of the pelagic habitat is that it is very nutrient-poor, 99.99999% of the sea is water (Pomeroy et al. 2007). Thus, microhabitats such as marine snow or an exudate trace left behind a phytoplankton are important growth zones for the organisms in the microbial food web (Azam & Malfatti 2007). The effect of increasing nutrient availability

An immediate effect of increasing nutrient availability in fresh and saltwater ecosystems is increased growth of the basal resources in the food web, i.e. phytoplankton and heterotrophic bacteria (Cole et al. 1988). Increases in basal productivity cascade upwards through the food web via the predator–prey chain, and may result in increased predation on bacteria and phytoplankton (I). The ecosystem exploitation hypothesis describes a size-structured linear food web, where each predator grazes on the prey organisms below its own size. According to this hypothesis, the top predator would show an increase in biomass with increasing productivity; while lower trophic levels may show alternating responses with respect to biomass concentrations (Oksanen et al. 1981, Thingstad & Sakshaug 1990). Hence, in such a theoretical food chain the top trophic level (n) and level n-2, n-4 etc. will increase in biomass while level n-1, n-3 etc. will remain at a constant biomass. Thus, mechanisms controlling the structure of the microbial food web would shift between predation and resource control at different trophic levels. Accordingly, a hypothesis relating nutrient status to the relative importance of resource and predation control of heterotrophic bacteria has been proposed (Gasol et al. 2002, Pernthaler 2005). According to the authors, predation control of bacteria is stronger in low-productivity than in high-productivity aquatic systems. Thus, resource control of bacteria dominates in highly productive environments. The mechanism behind this process would be the cascade effect in the microbial food web. Efficient control of heterotrophic nanoflagellates, as a

INTRODUCTION

5

result of predation by ciliates in nutrient-rich systems, might release bacteria from predation pressure, leading to resource control of the bacterial community. However, several mechanisms are known to modify this linear pattern: omnivory is common in aquatic food webs, thus increasing the complexity of trophic interactions (Posch & Arndt 1996, Diehl 2003). Introducing heterogeneity of trophic levels alters the response to increasing productivity compared to that predicted by the exploitation ecosystem hypothesis (Leibold 1989, Abrams 1993). Furthermore, predation defence mechanisms occur commonly in phytoplankton and bacterial communities in nutrient-rich waters, possibly as a response to increased predation pressure (Jürgens & Güde 1994, Rousseau et al. 2000) (II). In a microcosm study, Hall et al. showed that an inedible producer influenced the degree of top-down versus bottom-up regulation of a two level community (Hall et al. 2006). In contrast to the hypothesis presented by Gasol et. al 2002 and Pernthaler 2005 (Gasol et al. 2002, Pernthaler 2005), it has been suggested that the availability of resource, or “bottom-up” control of bacteria, is more dominant in low productivity than in high productivity waters (Sommaruga 1995). Hence, the impact of predation (and viral lysis), or “top-down” control of bacteria, should be more important in highly productive systems (Sanders et al. 1992) (I, II). In support of these suggestions, the ratio of protozoan to bacterial biomass has been shown to increase from nutrient poor to nutrient-rich waters (Auer et al. 2004) (I, II). However, these observations provide only indirect evidence since protozoa may also predate other food items, such as small phytoplankton (Fig. 1). Rather than altering bacterial numbers or bacterial biomass, predation has been shown to influence the composition and morphology of the bacterial community (Sanders et al. 1992, Matz & Jürgens 2003) (II, III). Similar effects of predation have been reported for phytoplankton communities. Kivi et al. (1996) showed, in a microcosm study, that ciliate and crustacean predation resulted in similar phytoplankton biomasses, while the composition of phytoplankton communities varied due to predator preference for different prey (Kivi et al. 1996). These observations suggest that if increased nutrient availability leads to increased predation and predation defences in the lowest trophic levels, then resource control is likely to occur in nutrient-rich systems. This would lead to reduced energy transfer efficiency within the food web. Taken together, top-down and

INTRODUCTION

6

bottom-up regulation should be considered as balancing processes that regulate communities according to the environment (Bell 2002). Predation-resistant bacteria survive protozoan predation

Bacteria and phytoplankton may respond to high predation pressure by evolving predation defence mechanisms (predation-resistance). Bacteria show a wide range of predation-resistance mechanisms, which either prevent their capture and ingestion by protozoa, or hinder their degradation within the protozoa (Matz & Kjelleberg 2005). It is known from laboratory experiments that high predation pressure induces changes in bacterial morphology and community composition, resulting in higher proportions of bacteria that are resistant to protozoan predation (Jürgens et al. 1999b, Horner-Devine et al. 2003, Corno & Jürgens 2006) (II, III). This is reflected, for example, in increased bacterial swimming speed, adjustment of cell size, and formation of filaments and biofilms (Jefferson 2004, Matz & Kjelleberg 2005). Intracellular survival in protozoa is a mechanism used by several bacterial pathogens, allowing them to persist in natural ecosystems (Harb et al. 2000, Winiecka-Krusnell & Linder 2001). A number of predation-resistant bacteria, such as the pathogens Legionella pneumophila, Vibrio cholerae, Mycobacterium avium, Escherichia coli, Salmonella enterica and Francisella tularensis, are able to avoid degradation within protozoan predators (Gao et al. 1997, Abd et al. 2003, Rasmussen et al. 2005, Abd et al. 2007, Steinberg & Levin 2007, Thomas & McDonnell 2007) (III). The phagocytosis mechanisms involved in protozoan predation and macrophage defences against invading organisms are similar with respect to prey recognition, engulfment and degradation (Gao et al. 1997, Bandyopadhyay et al. 2004, Tenant & Bermudez 2006). These mechanistic similarities suggest that the development of bacterial predation-resistance traits may explain the intracellular life-style of bacterial pathogens (Cirillo et al. 1997, Harb et al. 2000, Steinberg & Levin 2007). Furthermore, a number of bacterial species use protozoa as environmental hosts or reservoirs (Greub & Raoult 2004). For example, Caedibacter sp. and Holospora sp. are both intracellular symbionts in the ciliate Paramecium (Fokin 2004). Several studies of bacteria and phytoplankton report a cost associated with the expression of predation-resistance mechanisms. The trade off between fast growth and effective competition for nutrients compared to predation-

INTRODUCTION

7

resistance and the ability to escape predation, change with increasing productivity (Bohannan & Lenski 2000a, Bohannan & Lenski 2000b, Chase et al. 2002, Jürgens & Matz 2002, Corno & Jürgens 2006, Steinberg & Levin 2007) (II). Meyer et al 2006 investigated the fitness trade-off between two genotypes (predation-resistance and superior growth rate under N-limiting conditions) in a rotifer–algae microcosm experiment (Meyer et al. 2006). An initial increase in the competitive, fast growing algal genotype was observed in low-productivity conditions; however, when rotifer densities increased in response to eutrophication, the predation-resistant algal genotype dominated. Taken together, current data suggest that in eutrophic and highly productive environments, where bacteria may be subject to intense predation pressure, strong selective forces might favour predation-resistant and potentially pathogenic bacteria (I, II and III). Francisella, the causative agent of tularemia

Francisella is a small (~ 1 μm) facultative intracellular bacterial pathogen that causes the zoonotic disease tularemia in a wide range of mammals (Francis 1921). The symptoms of tularemia in humans generally vary according to the route of infection, but include high fever, headache, nausea and swollen lymph nodes. Bites by ticks, biting flies and mosquitoes are common transmission routes (Keim et al. 2007). Further, there is a risk of inhaling the bacterium when handling contaminated animals. Tularemia is treated with antibiotics since there is currently no commercial vaccine; however a less virulent, live vaccine strain (LVS), is used for vaccination of laboratory personal, since the bacterium is highly virulent and as few as 10 bacteria can induce the disease in humans (Saslaw et al. 1961, Burke 1977, Rohmer et al. 2006). The pathogenic bacterium Francisella

The genus Francisella includes two commonly accepted species: F. tularensis and F. philomiragia (Hollis et al. 1989). Four subspecies of F. tularensis have been identified; these are distinguished by their distribution and virulence in humans and animals. They are: subspecies tularensis (type A), holarctica (type B), novicida and mediasiatica (Oyston et al. 2004, Svensson et al. 2005) (Table 1). Recently, it has been demonstrated, however, that type A can be divided into two populations: type AI and type AII, with distinct geographical distributions and differences in pathogenicity (Farlow et al. 2001, Johansson et al. 2004,

INTRODUCTION

8

Staples et al. 2006). It is extremely difficult to culture Francisella directly from environmental samples (i.e. soil and water) without passage through a host. Under laboratory conditions, the bacterium exhibits optimal growth at 37°C with 5 % ambient CO2 and requires a defined media supplemented by several amino acids (Sandström et al. 1984). The capacity for intracellular multiplication within its host plays an important role in F. tularensis virulence (Sjöstedt 2003, Lai et al. 2004). The bacterium has been shown to replicate in macrophages after escaping the phagosome (Sjöstedt 2006). The fate of F. tularensis between outbreaks remains unknown. Subspecies holarctica has been identified in waters close to known outbreaks of tularemia (Hopla 1974) (Table 1). Based on laboratory experiments, amoebae have been considered to be a potential environmental reservoir for the bacterium (Abd et al. 2003). The ability to survive within microorganisms, e.g. protozoa, might be a viable strategy for F. tularensis to survive in aquatic environments (III). F. tularensis subspecies holarctica live vaccine strain (LVS) has been shown to persist for several months when incubated in cold tap water. Under these conditions, F. tularensis enters a state where the bacteria are viable but non-culturable (VBNC) (Forsman et al. 2000). It has been suggested that the VBNC state is a survival mechanism for several bacteria during limiting conditions (Effendi & Austin 1995). Other bacteria that also possess the capacity to enter this state (i.e. Camphylobacter jejuni, Legionella pneumophila, Vibrio cholerae and vulnificus) have been shown to resuscitate upon passage through animals (Jones et al. 1991), amoeba (Steinert et al. 1997) and when incubated in a specific medium (Colwell et al. 1996, Whitesides & Oliver 1997). However, no study has yet been able to show resuscitation of F. tularensis from the VBNC state (Forsman et al. 2000). F. tularensis subsp novicida is also closely linked to water-borne transmission, although isolates exhibit lower virulence and only rarely cause disease (Sjöstedt 2005) (Table 1). The bacterium has been reported to be able to form a thin structured biofilm (Hassett et al. 2003) (IV). F. philomiragia is associated with water and soil; recently several fish pathogens have been identified as subspecies of F. philomiragia (Barns et al. 2005, Nylund et al. 2006, Mikalsen et al. 2007). Not all Francisella subspecies have been shown to have a strong association with water (Keim et al. 2007). F. tularensis subsp tularensis is often associated with dry environments in the presence of lagomorphs (rabbits and hares) (Table 1).

9

Tabel 1. Francisella species and subspecies and their geographical distribution, major vectors, known associations, virulence in mice and resistance to predation by the ciliate Tetrahymena pyriformis. Species/ subspecies

Geographical distribution

Major vectors Association *Virulence * Predation-resistance 8

F. tularensis tularensis AI North America (east) 1 ticks 1 lagomorphs 1 + + + 1 + + tularensis AII North America (west) 1 biting flies 1 lagomorphs 1 + 1 + holarctica B Northern hemisphere 2 tick 1, mosquito 2 water 3, rodents 1 + + 2 + + mediasiatica Central Asia 2 - rodents, hares 7 + + 2 + + novicida Global 2 - water borne 6 + 2 + + F. philomiragia Northern hemisphere 2 - seawater 5 + 4 + + + Lagomorphs = rabbits and hares, 1 (Staples et al. 2006), 2 (Keim et al. 2007), 3 (Berdal et al. 1996), 4 (Penn 2005), 5 (Wenger et al. 1989), 6 (Clarridge et al. 1996), 7 (Friend 2006), 8 (Paper III), * Virulence and predation-resistance scale (+ = low, + + = high, + + + = very high), - Not known

OBJECTIVES

10

OBJECTIVES

The aims of this thesis were to clarify if the predation pressure on bacteria increases as a result of increasing numbers of protozoa associated with increasing nutrient status in aquatic systems (Paper I), and to determined whether this leads to the development of predation-resistance in the bacterial communities (Paper II). Furthermore, I aimed to determine whether the tularemia bacterium, Francisella tularensis, is resistant to protozoan predation (Paper III), and to establish whether natural aquatic systems with high nutrient availability can act as a reservoir for the bacterium between epidemic outbreaks in mammals (Papers III and IV). I examined whether the tularaemia bacterium survives in the aquatic microbial food web (Paper III), and whether the bacterium has the ability to form a biofilm (Paper IV). The following questions were addressed: - Does the nutrient status of an aquatic ecosystem affect the protozoan predation pressure on bacteria? - Does the occurrence of predation-resistant bacteria increase with increasing nutrient availability in natural waters? - Does the microbial food web constitute a potential reservoir for the predation-resistant pathogenic bacterium F. tularensis? - Does F. tularensis form biofilms as a potential strategy for survival in aquatic environments?

METHODOLOGICAL CONSIDERATIONS

11


This section details the methodological developments and techniques that were important in order to complete the research outlined in this thesis. Model simulations – An ecosystem model of the microbial food web

To study how the dynamics of an aquatic food web were affected by nutrient addition, we constructed a size-structured ecosystem food web model (I). In particular, we wanted to investigate how the predation pressure on bacteria (protozoan predation per bacterial biomass) changed with changing nutrient availability in the water. The intention was to construct a general and simplified food web model that could mimic predation pressure on bacteria from marine and freshwater systems, and from nutrient poor (oligotrophic) to nutrient-rich (eutrophic) conditions. The organisms in the model were grouped by size (size structured) and included bacteria, phytoplankton, three size classes of protozoa and metazooplankton (Fig 2, available at http://www.bioras.com/index.html). The organisms were defined by their cell radius, carbon density and carbon to nitrogen (C:N) and carbon to phosphorous (C:P) ratios. The processes driving the model were physical constraints set by primary production, osmotrophy, respiration, resting metabolism and predation. We chose to build our model based on empirical parameters i.e. constants derived from experiments or observational data from field studies. Further, we included as few empirical parameters as possible in order to ensure that the simulation outputs were generally applicable. The ecosystem model was designed as a population dynamics model in a single volume of water, without any spatial resolution. Therefore, no random movement of organisms was incorporated. Each predator grazed on one trophic level, specifically the one below its own size (Fig. 2).


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DOC

Bacteria

Protozoa I

Protozoa II

Protozoa III

Metazooplankton

DINDIP

Picophytoplankton

Light Size (µm):

350

30

20

15

8

4

10,8

Nanophytoplankton

Microphytoplankton

Figure 2. Schematic view of the food web used in the model simulations. Primary producers used light and inorganic nutrients (DIN, DIP) for growth. Bacteria consumed dissolved organic carbon (DOC), excreted by primary producers or predators and assimilated inorganic nutrients. Bacteria and primary producers were grazed by heterotrophic flagellates, ciliates or metazooplankton. Predators also consumed other predator’s in the size class below their own. Organisms are arranged according to cell size, as indicated by the arrow to the right. Uptake, grazing or consumption (solid lines); excretion (dotted lines); DOC: dissolved organic carbon; DIN: dissolved inorganic nitrogen; DIP: dissolved inorganic phosphate The biomass of each species was integrated over time, and depended on growth rate, respiration, grazing and, for the primary producers, photosynthesis. Biomass was conserved in the model reactions, except in processes such as primary production, where mass was added. The simulator integrated values in steps of 0.005 h (18 seconds). Nineteen simulations were run sequentially from ultraoligotrophic to hypertrophic conditions; each simulation was initiated using the end values from the previous simulation. The increase in nutrient availability was caused by dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) loadings at a constant ratio (DIN : DIP = 36). All simulations were run for 365 days, at which point population dynamics appeared to be stable. The model was designed as a flow-through system, similar to a chemostat experiment, with a daily water exchange of 10 %.


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Bacterial edibility assay

In paper II, the effects of nutrient availability and protozoan abundance on the occurrence of predation-resistant bacteria were investigated in a field study based on a natural productivity gradient. Bacterial edibility (bacterial predation-resistance) was tested using the ciliate Tetrahymena pyriformis and a standardized optical clearance-rate test (II, III). Bacterial isolates were co-cultured with axenic protozoa in 0.5 mM NaCl (initial cell concentration of 108 bacteria mL-1 and 2–4 × 103 protozoa mL-1). The reduction in bacterial abundance due to protozoan grazing was monitored by measuring optical density once a day. For each day of the experiment, optical density of the samples (Nday, OD750) was corrected for autolysis of bacteria, by subtracting decreases in OD750 recorded for bacterial controls. The slope of the reduction in OD750 (k) over time was then calculated for each sampling day. The average k-value (kavg

OD) for the whole period of decrease was then calculated. Bacterial half-time (inedibility) was calculated according to (Eq. 1):

T1/2 = ( )ODavgk5.0ln (1)

By calculating the inverse of this parameter the bacterial degradation rate (edibility) was obtained. To my knowledge, no method has been described previously for direct screening of bacterial predation-resistance. Previous studies on predation-resistance in bacteria have been performed using microscopic observations of increasing swimming speed, filament formation and predator survival (Jürgens & Matz 2002). Even though the bacterial edibility assay used was not designed to distinguish between different predation defence mechanisms, it captures both mechanistically- (colony formation, increases in cell size) and chemically-mediated bacterial defence strategies (toxin production, survival in acidified phagosomes) (Matz & Kjelleberg 2005). The edibility of a bacterium may vary depending on the type of predator and its feeding strategies (Jürgens & Matz 2002). I observed a significant difference in bacterial edibility when using a flagellate (Ochromonas danica) compared to a ciliate (T. pyriformis) predator (II, III). However, for both predators, a bacterium that is known to be edible (E. coli) was found to be five times more edible than an inedible bacterium (F. tularensis subspecies holarctica). Thus, my standardized edibility test was able to assess the relative edibility of the tested bacterial strains. In my


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research, I investigated the role of the pelagic microbial food web in the occurrence of predation-resistant bacteria (II, III). Since experiments were performed on planktonic bacteria, a planktonic predator (ciliate) and not a surface feeder (amoeba) (Pickup et al. 2007) was used in the edibility experiments. Molecular methods

Polymerase chain reaction (PCR) detection of bacteria in eutrophic waters and soils can be problematic because of the inhibition of the PCR by humic substances (Tsai & Olson 1992). This may lead to a high incidence of false-negative results, due to difficulties in isolating pure DNA. Therefore, it is important to use DNA extraction protocols that efficiently eliminate the inhibiting substances (Whitehouse & Hottel 2007). The field study (II) was performed in an emerging tularemia area in southern Sweden (Örebro). I sampled 20 lakes in the area and screened for F. tularensis using 16S rRNA Francisella-specific primers Fr153F0.1 (5’-GCCCATTTGAGGGGGATACC-3´) and Fr1281R0.1 (5’-GGACTAAGA GTACCTTTTTGAGT-3´), as previously described (Barns et al. 2005). DNA was prepared from 2 mL of sample water that was centrifuged 16 000 x g for 1 hour; 1.9 mL of the supernatant was discarded. The remaining volume was treated according to instructions in the SoilMaster DNA Extraction Kit (Epicentre Biotechnologies) manual and modified for environmental water samples. The 16S rRNA PCR products were purified on SeaKem agarose gels, and excised bands were eluted using GenEluteTM Gel Spin Columns (Sigma-Aldrich). Products were cloned into the pCRII vector using a TOPO-TA cloning® kit according to the manufacturer’s (Invitrogen) protocol. Fifty clones were selected from each PCR product and stored in glycerol at -70 ˚C prior to sequencing. Plasmid DNA was isolated from overnight cultures using an E.Z.N.A.TM Plasmid Miniprep Kit (Omega bio-tek) and sequenced by MWG-Biotech (Ebersberg, Germany) using universal M13F and M13R primers. Good quality sequences (size: ~1150 bp) were deposited with GenBank; their accession nos. are DQ994171 to DQ994200. During the experiment with F. tularensis in lake water microcosms (III), I identified the need for a culture-independent quantitative method, to determine the occurrence of F. tularensis, as a complement to the microscopic counts. I employed a quantitative real-time PCR, using


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specific Francisella primers for the lpnA gene encoding a 17 kDa lipoprotein, a major membrane protein (Sjöstedt et al. 1990). The encoding gene has been used successfully as a target for Francisella specific real-time PCR (Sjöstedt et al. 1997). I included an internal control (rat DNA) in every PCR reaction, thus, results from different runs could be compared and quantified against a calibration curve. The real-time PCR assay generated reproducible results, with a detection limit (103 cells mL-1), similar to that described in a previous study of Francisella real-time PCR on soil samples using the same DNA extraction kit (SoilMaster DNA Extraction Kit from Epicentre Biotechnologies) (Whitehouse & Hottel 2007).

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION Impact of increasing productivity on aquatic bacterial communities Increasing predation pressure on bacteria

In Paper I, we described the effect of increasing nutrient availability on the predation pressure on bacteria in aquatic systems. The predation pressure on bacteria was defined as protozoan predation per bacterial biomass. Results from a computer model simulation and a meta-analysis of field data showed an increase in predation pressure on bacteria with increasing nutrient availability (10-fold and 100-fold respectively) (I). In the model simulation, bacterial production increased with increasing phytoplankton productivity within the aquatic system (estimated chl a 0.2 – 112 μg L-1) (I). This is in agreement with the meta-data and a study by Cole et. al. 1988, where a positive correlation between bacterial and primary productivity was observed in freshwater and marine ecosystems (Cole et al. 1988) (I). In an aquatic system with low nutrient availability, flagellates have been shown to be mainly resource limited/controlled (Berglund et al. 2005), indicating that biomass resulting from an increase in bacterial productivity would be consumed by the flagellates, thus bacterial biomass might remain unchanged. This hypothesis has been tested in enclosure experiments, where bacterial production increased markedly in response to the addition of carbon and nitrogen while the bacterial biomass was unaltered (Kuuppo et al. 2003). In the same study, increased bacterial productivity resulted in an increased biomass in the highest trophic level within the enclosures, i.e. in the ciliate community. The predators in our model simulation (predator I) and the meta-analysis (flagellates and ciliates) efficiently consumed the increased bacterial production that followed the increase in nutrient availability. Thus, an increasing growth rate of a relatively stable bacterial biomass resulted in an increase in the bacterial specific growth rate (bacterial production per bacterial biomass) (Fig. 3). Bacterial biomass remained relatively constant over the productivity range and the increasing bacterial specific growth rate sustained the growth of an increasing number of protozoan cells, as illustrated in Figure 3. This resulted in an increasing ratio of protozoan to bacterial biomass (by a factor of 10), observed in our model simulation and meta-analysis (chl a


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0.1–11μg L-1) (I). The ratio of protozoan to bacterial biomass was also observed to increase (a hundredfold) over a natural productivity gradient (chl a 1.4 - 31μg L-1) in southern Sweden (II). These results are in agreement with a field study of 55 lakes in northern Germany, where both flagellate and ciliate predators were included (Auer et al. 2004).

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Figure 3: A schematic model describing the dynamics of bacterial biomass, bacterial production and predator biomass over a productivity range. The model was built on the basis of results from our ecosystem model simulation and meta-analysis (I). According to Wright and Coffin 1984, a negative relationship between bacterial growth rate and bacterial abundance indicates that the bacteria are resource limited. Thus, if bacterial growth rate and abundance are not related, the bacteria may be limited by predation (Wright & Coffin 1984). We did not observe a decrease in the relative figures for bacterial specific growth rate and bacterial biomass over the nutrient range, suggesting that the bacterial communities were predation limited (I). In the model simulation and the meta-analysis of field data, we observed that the increase in protozoan predation over the productivity range exceeded the increase in bacterial biomass, indicating that protozoan predation was controlling the bacterial community in the more nutrient-rich environments (I). The increase in predation pressure (protozoan predation per bacterial biomass) on bacteria was best explained by an increase in bacterial production, but also by the increase in predator biomass (Fig. 3). Thus, our food web model predicted an increase in predation pressure, i.e. predation control of bacteria along aquatic productivity gradients with


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maximum predation control under hypertrophic conditions; this was verified by the meta-analysis of field data (I). The ecosystem model simulations resulted in elevated biomass of the top predators (metazooplankton) and varying increases in the four lower trophic levels in response to increased productivity (I), in part, concurring with the predictions of the exploitation ecosystem hypothesis (Oksanen et al. 1981, Thingstad & Sakshaug 1990). However, the results of our food web model were not in agreement with the results presented by Gasol et al. 2002, who suggested that flagellate predation controls bacteria mainly in low productivity conditions (Gasol et al. 2002). However, only flagellate predators were included in that study. We included three size classes of protozoa in our food web model, although only small protozoa (e.g. HNF) were bacteriovores (I). Despite this simplification, we found that the predation pressure on bacteria was similar in the model simulations to that found in the meta-analysis. Thus, using our generalized ecosystem food web model, we were able to simulate the predation pressure on bacteria in marine to freshwater systems and under conditions ranging from oligotrophic to eutrophic. Many protozoa utilize food sources other than bacteria, and omnivory, i.e. grazing on different trophic levels, is quite common in the protozoan community, especially among ciliates (Fenchel 1987, Posch & Arndt 1996). Increasing ciliate biomass would influence both predation on flagellates and competition for the bacterial resource. In a field study, I observed an increasing proportion of ciliates in the protozoan community along a natural productivity gradient (II). The importance of ciliates as bacterial predators would thus increase in nutrient-rich environments. Omnivorous ciliates and metazooplankton have been shown to be the main predators of bacteria under specific conditions (i.e. in turbid ecosystems and during population peaks) and the impact of these groups of predators may increase in more productive aquatic systems (Jürgens 1994, Zingel et al. 2007). It has been suggested that metazooplankton regulate the ciliate community in aquatic systems (Jürgens et al. 1999a). In productive and turbid environments, however, large metazooplankton, in particular, are inefficient predators and ciliates are released from predation control (Porter & McDonough 1984). Because the more nutrient-rich aquatic systems in my field study were generally shallow and turbid, the high biomass of ciliates in the eutrophic waters may be related to both high food availability and turbidity (II).


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Previous theoretical approaches to aquatic food web dynamics and trophic interactions have made use of linear lumped-parameter models and a maximum of three trophic levels (Leibold 1989, Sanders et al. 1992, Abrams 1993, Thingstad & Havskum 1999, Gasol et al. 2002). Unlike previous studies on bacterial predation pressure, we included omnivorous predators and covered a wider productivity range (oligotrophic – hypertrophic conditions) in our meta-analysis (chl a range from 0.1 to 280 μg L-1) as well as in our simulations (estimated chl a range from 0.2 to 112 μg L-1) (I) (Sanders et al. 1992, Gasol et al. 2002). Our ecosystem food web model is based on physical constraints and published empirical parameters, while other models e.g. lumped-parameter models, are based on differential equations and parameter selection (Leibold 1989, Sanders et al. 1992, Bohannan & Lenski 2000a). Nevertheless, both the ecosystem model and the lumped-parameter models show the increasing importance of predation control of the basal trophic levels with increasing productivity within the system. Thus, the food web structure seems to be more important than the choice of approach when modelling population dynamics. Our model simulation did not include predation defence mechanisms, although the meta-analysis could, in principle, have included some predation-resistant bacteria (I). Most of the studies included in the meta-analysis used the method described by Sherr and Sherr (1993), where in situ bacteria are stained with a fluorescing dye (Sherr et al. 1993). However, the recommended 1 μm filtration probably excludes many morphology-induced predation-resistant forms (e.g. clumped cells or filaments). The approaches used in my thesis, both the model simulation and the meta-analysis, showed an increasing predation pressure with increasing productivity. In reality, this could trigger selection of predation-resistant forms of bacteria in nutrient-rich environments. Viruses use structures on the bacterial cell surface to gain entry to the bacterial cell. These cell structures can, for example, be porins, transporters lipopolysacharides or pili (Weinbauer 2004). Bacterial resistance to phage infection could be achieved by several mechanisms; for example, the absence of receptor expression on the bacterial surface. Further, the bacterial surface receptors could be altered, masked or shielded, to prevent the virus gaining access to the cell. However, the development of phage-resistance also results in physical constraints on the bacterial cell. The modified bacteria, resistant to phage infection, are often much less efficient at acquiring the specific nutrient that is normally transported through the


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modified receptor. Thus, for a bacterium to benefit from phage-resistance, low competition for nutrients would be required, potentially in a nutrient-rich environment (Bohannan & Lenski 2000b). Increasing predation on bacteria by nanoflagellates has been shown to stimulate viral infection of bacteria (Simek et al. 2001). It has been speculated that this is the result of a trade-off effect, since phage-resistant bacteria may be more vulnerable to protozoan predation than phage-sensitive cells. Thus, phage-sensitive cells are likely to be favoured in waters with high predation pressure on bacteria (Weinbauer 2004). It is interesting to speculate whether bacteria resistant to protozoan predation also are more susceptible to phage infection. If this is the case, phage infection could play an important role in regulating the abundance of predation-resistant bacteria in highly productive environments. Thus, viral lyses could contribute to maintaining the flow of energy through the microbial food web in environments where nutrient availability and protozoan predation favour inedible bacteria. However, the ratio between bacterial production and protozoan predation was, on average, ~1 in the meta-analysis data, indicating that protozoa constituted the main predators on bacteria (I). This is in accordance with a study by Sanders et al. 1992, who reported that the balance between the bacterial production and bacterial loss rate was the result of flagellate predation (Sanders et al. 1992). Variations in the meta-data, however, do suggest the possibility of virus effects in the aquatic systems. Occurrence of predation-resistant bacteria

The increase in predation pressure on bacteria with increasing productivity (I, II) might also apply to other basal resources in the food web, e.g. primary producers. One indication that this is the case is that blooms of toxic and inedible phytoplankton are more common in eutrophic than in nutrient poor waters (Paerl et al. 2001). The correlation between increasing predation pressure and the initiation of defence mechanisms by phytoplankton and bacteria, indicates that there is a trade-off when expressing these mechanisms (Jürgens & Güde 1994, Chase et al. 2002, Selander et al. 2006). Predation pressure has to be strong enough to make it of evolutionary benefit for the organism to express energetically costly mechanisms to resist predation. Our studies suggest that high productivity aquatic environments constitute such situations (I, II, III). To investigate whether increasing nutrient availability in aquatic systems favours the occurrence of predation-resistant bacteria, I performed a field


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study of a natural productivity gradient (II). Bacteria from 20 lakes in southern Sweden (Örebro) were isolated on agar plates and tested for predation-resistance using a standardized edibility method with a ciliate protozoon (Tetrahymena pyriformis). Using quantile regression analysis, I found a decreasing prevalence of bacteria that were edible to the ciliate over the nutrient range. Thus, inedible, or predation-resistant bacteria were selected for as the nutrient availability within the system increased (II). In addition, the ratio of protozoan to bacterial biomass increased over the same range. This is in line with previous experimental studies that have shown a positive relation between protozoa predation, nutrient availability and predation-resistant forms of bacteria (Jürgens et al. 1999b, Matz & Jürgens 2003, Corno & Jürgens 2006). The force behind this selection is probably protozoan predation, whereas high nutrient availability is an indirect factor leading to higher protozoan biomass, but it also enables bacteria to express energetically costly resistance mechanisms in response to the increasing predation pressure (Chase et al. 2002). In addition to the selection for inedible bacteria, I found that microcolony formation or clumping of bacteria occurred commonly in the edibility experiment as a predation defence strategy. When the ciliate was added, 18% of the tested bacteria responded by forming microcolonies. Microcolony formation is one of the most reported bacterial predation defence mechanisms in nature (Matz & Jürgens 2003). The isolated bacteria used in the edibility test belonged to five dominant taxa recorded frequently in aquatic environments: α-, β-, and γ-Proteobacterium, Actinobacteria and Firmicutes (Zwart et al. 2002). The representativeness of the isolated bacteria in the aquatic bacterial communities could be questioned because, as a result of their low plating efficiency, it has been argued that colony-forming bacteria are not representative of natural aquatic communities (Jannasch & Jones 1959, Kogure et al. 1979). Nevertheless, I see no reason to assume that those bacteria that are capable of growing on agar plates would vary in their edibility over the productivity gradient. The composition and diversity of the bacterial communities along the natural aquatic productivity gradient was analyzed using denaturating gel gradient electrophoresis (DGGE), fragment analysis and sequencing (II). The DGGE method ideally results in the amplification of DNA sequences from all of the prokaryotic organisms present in a sample. However DGGE patterns derived from natural samples are complex. The resolution of the DGGE gel could be a limiting factor when analyzing neighbouring bands.


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However, DGGE provides a method for detecting the dominant bacterial populations that are PCR amplifiable (Torsvik et al. 1998). Bacterial diversity was found to decrease with increasing nutrient availability in the field study, indicating that predation and nutrient concentration were strong selective forces acting at the level of the bacterial species (II). This is in agreement with a previous study showing that bacterial diversity varies with primary productivity (Horner-Devine et al. 2003). Many of the DGGE sequences identified were representatives of the same taxon as the bacterial isolates used in the edibility test. Common groups were the Cytophaga–Flavobacterium–Bacteroides (CFB) group, although α-, β-, and γ-Proteobacteirum and Actinobacteria were also represented. CFB did not grow on plates, however, members of the group are known to form filaments in nutrient-rich waters regulated by growth and are, thus, less vulnerable to protozoan predation (Hahn et al. 1999, Jürgens et al. 1999b). The species composition of the bacterial communities was not significantly different in the oligotrophic compared to the eutrophic lakes (II). Some bacteria occurred generally across the whole gradient e.g., Pseudomonas and Sphingomonas. This suggests that the bacterial strains that are favoured in the nutrient-rich environments were also present in the oligotrophic lakes; in the latter case they were less dominant. The presence of inedible bacteria was also recorded across the whole gradient. Moreover, the edibility of Pseudomonas sp. isolates decreased significantly over the nutrient range. However, Serratia, Shigella and Streptococcus were among the most inedible bacteria and were only isolated from nutrient-rich waters. Taken together, the results from the field study show that there is selection for predation-resistant bacteria in nutrient-rich waters with intense protozoan predation. These findings provided a framework for further investigations into possible reservoirs of intracellular bacteria, such as F. tularensis, in eutrophic environments with high predation pressure on the bacteria.


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Francisella tularensis in the aquatic microbial food web Occurrence of F. tularensis in a lake water system

The region where I performed my field study (II) is an emerging tularemia area, with the highest reported tularemia incidences in Sweden during the past decade. Bacterial isolates from patients that were infected with tularemia in the study area have been identified as F. tularensis holarctica (Larsson et al. 2007). Since transmission of F. tularensis holarctica in Sweden is associated with water, I screened the aquatic systems in this area for the presence of Francisella, using Francisella-specific 16S rRNA primers. Twenty percent of the sampled aquatic systems were found to be positive for Francisella. In a phylogenetic analysis, the sequences obtained were found to group together with Francisella tularensis isolates (Fig. 4). In previous environmental studies, where the same basic techniques and primers were used, the targeted bacteria were found to consist of a mixture of more distantly related Francisella-like bacteria (Barns et al. 2005, Nylund et al. 2006). This implies that the aquatic environments from which I obtained 16S rRNA are more selective for F. tularensis than the soil and sediment samples analyzed by Barns et al. (Barns et al. 2005). Even though the occurrence of F. tularensis exhibited no clear relationships with the measured environmental parameters (chl a, bacterial diversity and bacterial-, protozoa- and phytoplankton-biomass), all the aquatic systems in which I detected F. tularensis could be characterized as eutrophic systems with low bacterial diversity (II). PCR detection is difficult in eutrophic waters due to the high level of background PCR-inhibiting substances (Tsai & Olson 1992). Thus, one possible error-source within my Francisella analysis could have been an underestimate of the number of samples in which Francisella was present.


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Figure 4. Phylogenetic analysis of environmental Francisella sequences obtained from water samples (bold, this study, Accession nos DQ994171 to DQ994200), environmental soil samples (Barns et al. 2005) and reference sequences from GenBank. To evaluate the phylogenetic position of the Francisella sequences obtained, reference sequences from GenBank were included in the ClustalW alignment performed using MEGA version 3.1 (Cole et al. 2003). A phylogenetic tree was generated using Maximum Parsimony analysis and bootstrapping.


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Francisella – a pathogenic predation-resistant bacterium The idea of water as a potential environmental reservoir for

F. tularensis between infection outbreaks originates from the isolation of holarctica (type B) strains in close association with water (Hopla 1974, Berdal et al. 1996). Moreover it has been speculated that the holarctica strains are adapted to aquatic environments based on reports that F. tularensis subspecies holarctica is able to survive in co-culture with amoeba and ciliates (Konstantinova et al. 2000, Abd et al. 2003). In support of this hypothesis, I identified F. tularensis in water samples from an emerging tularemia area (this study). In order to investigate differences between Francisella species and subspecies with respect to their adaptation to aquatic environments, I screened representatives of F. tularensis (subspecies holarctica, novicida, tularensis and mediasiatica) and F. philomiragia for differences in their ability to survive protozoan grazing (i.e. predation-resistance) (III). This study was performed using my standardized edibility test (II). I observed low edibility for all the Francisella species and subspecies tested when predated by the ciliate Tetrahymena pyriformis (III). However, there were significant differences in the edibility of different Francisella strains (Fig. 5). The bacterium Escherichia coli, used as a reference in the edibility test, was successfully degraded by the ciliate within 35 h; the resultant bacterial edibility was 0.028 h-1. All of the tested Francisella strains were less susceptible to ciliate predation, resulting in edibilities ranging from 0.0085 to 0.0017 h-1, corresponding to degradation times from 118 to 588 h (where a F. tularensis subspecies tularensis AII strain was the most edible and a F. philomiragia strain was the least edible) (III). The low edibility of F. tularensis subspecies holarctica (Fig. 5), supports the hypothesis that there is an aquatic environmental reservoir for the holarctica strains (Berdal et al. 1996). The virulent holarctica strain FSC200 was more resistant to degradation by protozoa than the attenuated vaccine strain LVS (Burke 1977, Rohmer et al. 2006). This indicates that the virulence mechanism used by F. tularensis holarctica strains also plays a role in surviving predation by protozoa.


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Figure 5. Edibility of Francisella philomiragia and Francisella tularensis subspecies holarctica, tularensis AI and AII, mediasiatica and novicida, grazed by the ciliate Tetrahymena pyriformis. Results based on n = 3 strains except for holarctica n = 8 The F. tularensis subspecies tularensis strains (type A) are historically associated with dry environments (Keim et al. 2007). My results show that tularensis type AII strains are clearly the most edible of the Francisella subspecies. The observed difference in edibility between the tularensis AI and AII strains is in line with the recent division of tularensis into two subgroups (Farlow et al. 2005). Type AI is highly virulent and is confined to the east of North America, while type AII is less virulent and confined to the west of North America (Farlow et al. 2005, Staples et al. 2006). Further, types AI and AII also differ at the genomic level: the two types have been shown to form two separate phylogenetic groups (Farlow et al. 2001, Johansson et al. 2004, Beckstrom-Sternberg et al. 2007). Moreover, their biogeography reflects the distribution of different potential vectors, namely Dermacentor variabilis and Amblyomma americanum ticks for type AI and Dermacentor andersoii ticks and deer fly (Chrysops discalis) for type AII (Farlow et al. 2005). My results support the theory that there are two distinct ecotypes of tularensis (types AI and AII), possibly resulting from a bottleneck effect after isolation of the subspecies tularensis in two distinct ecological islands (Keim et al. 2007). Such biogeographic isolation may explain the adaptative divergence into the highly virulent type AI and the less virulent type AII strains.


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During the edibility experiments, I observed that F. tularensis was ingested by protozoa but degraded very slowly. In co-culture with F. tularensis holarctica, the ciliate T. pyriformis excreted vesicles filled with bacteria (Fig. 6) (III). This indicates that the bacteria survive passage through the protozoa without being degraded. The normal cycling of the food vacuole through the ciliate cell starts with the formation of the vacuole at the oral cavity. After passage through the cell (including acidification and nutrient uptake steps), the matured vacuole is emptied of its contents at the cytoproct. The vacuole membrane is then transported back to the oral cavity and reused to form new food vacuoles (Allen & Wolf 1979). When T. pyriformis excretes vesicles, the entire intact food vacuole, including the membrane and its contents, is excreted. The excretion of vesicles seems to be dose-dependent and occurs when the multiplicity of infection (MOI) exceeds 1:1000 (protozoa to bacteria ratio) (unpublished data). This is probably a result of overfeeding: the ciliate ingests more bacteria than it can degrade and the efficiency of assimilation is reduced. Even so, the protozoan predator was able to multiply in co-culture with F. tularensis (from 103 cells mL-1 to approximately 105 cells mL-1), although the edibility experiments were performed in a low nutrient medium where the protozoa were dependent on predation as the only energy source. Therefore, there seems to be a commensal relationship between the F. tularensis subspecies and ciliated protozoa in the edibility experiment. This is in accordance with previous results, where co-cultures of subspecies holarctica and protozoa (ciliate T. pyriformis and amoeba Acanthamoeba castellanii) in liquid broth resulted in slowly decreasing bacterial numbers and a constant number of protozoa (Konstantinova et al. 2000, Abd et al. 2003).


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Figure 6. Co-culture of the ciliate T. pyriformis and F. tularensis subspecies holarctica carrying a plasmid with the green fluorescent protein (gfp) (upper pictures). Dose-dependent excretion of vesicles filled with bacteria by T. pyriformis (upper right and lower picture). Photos by Johanna Thelaus In contrast to the experiments with the F. tularensis strains, I observed low numbers or the absence of protozoa in the co-cultures of F. philomiragia and T. pyriformis, indicating that the ciliate was not able to utilize the bacterium as an energy source to sustain growth. The F. philomiragia strains were significantly less edible than all of the F. tularensis strains tested (Fig. 5) (III). Thus, the complete predation-resistance of F. philomiragia suggests that the bacterium either exerts an adverse effect on protozoa or possess a mechanism for escaping protozoan phagocytosis. Recently, F. philomiragia subspecies noatunensis (Mikalsen et al. 2007) and the closely related F. piscicida (Nylund et al. 2006, Ottem et al. 2007) were found to be fish pathogens. This further supports the hypothesis that F. philomiragia is adapted to survive in aquatic environments but is not dependent on protozoa. However, F. philomiragia may still be able to


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survive in protozoa provided that there is an alternative energy source available to maintain protozoan growth (i.e. other bacteria and soluble nutrients). The observed difference between F. philomiragia strains and F. tularensis strains could be expected, since the strains form two distinct species and F. philomiragia is known to be a marine bacterium. A potential reservoir for F. tularensis holarctica

I hypothesised that eutrophication, i.e. high availability of nutrients, followed by an increase in protozoan predation pressure on bacteria, exerts some control over the occurrence of predation-resistant F. tularensis in aquatic systems (I, II, III). This was tested by monitoring the survival of a fully virulent F. tularensis subspecies holarctica strain labelled with green fluorescent protein (gfp) in a microcosm experiment using natural lake water, and varying the food web complexity and nutrient availability. I observed a 150-fold increase in the abundance of F. tularensis subspecies holarctica during the first 16 days of the treatment in the presence of protozoan predators and with high nutrient availability (TOC 7.85 ± 0.88 mg L-1, total phosphorous 0.433 ± 0.118 mg L-1 and total nitrogen 3.006 ± 0.430 mg L-1) (III). Here, flagellates were the only predators on bacteria, and protozoan predation pressure (the ratio of protozoan biomass to bacterial biomass) was high, increasing from approximately 1:10 to 10:1, compared to a ratio of 1:100 often reported from surface waters (Azam et al. 1983, Fenchel 1988). Throughout the experiment, F. tularensis cells were observed in the planktonic form as well as inside protozoa. On days 5 and 16, large numbers of gfp-labelled holarctica were clearly observed within small colourless flagellates (~ 2 × 5 μm) in the treatment involving predation and high nutrients. A stronger gfp signal was recorded in these intracellular holarctica compared to the free-living ones, indicating a higher metabolic activity associated with the intracellular cells. This suggests that the bacterium is able to survive and multiply in nutrient-rich lake water with high protozoan predation pressure. Further, a specific interaction with small flagellates may provide the conditions needed for F. tularensis holarctica replication to occur. The distribution of tularemia outbreaks is often patchy, both spatially and temporally. The reasons for this remain largely unexplained. However, similar spatio-temporal patterns have been described for the bacteria Holospora and Caedibacter, which are symbionts of the ciliate Paramecium (Fokin 2004). It has also been demonstrated that F. tularensis


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shares characteristics at the phylogenetic level with bacterial symbionts such as Caedibacter taeniospiralis (~ 87 % 16S rRNA sequence similarity) (Beier et al. 2002). The inability to grow on a culture plate is characteristic of bacterial symbionts (Fokin 2004). Symbiotic bacteria down-regulate protein synthesis pathways and obtain specific proteins from the host cell. Thus, the bacterium may lose the ability to replicate outside its host. This may explain the failure to isolate F. tularensis directly from aquatic environments onto plates. The data from my microcosm study show that F. tularensis subspecies holarctica entered a viable but non culturable (VBNC) state after five days in co-culture with protozoa in nutrient-rich lake water (III). Low nutrient concentrations (TOC 6.41 ± 0.55 mg L-1, total phosphorous 0.008 ± 0.002 mg L-1 and total nitrogen 0.291 ± 0.028 mg L-1) and the absence of predators prolonged the ability of F. tularensis subspecies holarctica to grow on plates. It has been shown previously that F. tularensis subspecies holarctica vaccine strain LVS remains culturable for periods up to 70 days in sterile tap water at a temperature of 8°C (Forsman et al. 2000). Under poor environmental conditions, therefore, the bacterium may survive in sediments, on surfaces or, possibly, in biofilms. Thus, the bacterial cells maintain their ability to grow on surfaces. In support of this, preliminary data show that F. tularensis subspecies novicida has the ability to form a thin, structured biofilm (Hassett et al. 2003). Further, F. tularensis subspecies holarctica has been recorded, in a microcosm study, as persisting longer in sediment than in water (Mironchuk & Mazepa 2002). In my microcosm study, F. tularensis subspecies holarctica was avirulent after long incubation in lake water (120 days), irrespective of treatment. Thus, neither the availability of nutrients nor the presence of predators affected the preservation of virulence over long periods in water (i.e. 120 days) (III). However, I speculate that the intracellular F. tularensis observed in the non-pigmented small flagellates, under conditions with high-nutrient availability and intense predation pressure, may allow the bacterium to be transported further up through the aquatic and possibly the terrestrial food webs. Organisms of about 5 μm can be predated by zooplankton, mosquito larvae and larger ciliates (Merritt et al. 1992). This process is rapid and could, in principle, occur during the time that F. tularensis remains virulent in water.


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F. tularensis is able to form biofilms The ability of F. tularensis subspecies holarctica to grow on

plates was shown to be prolonged in nutrient depleted waters in the absence of predators (III). We hypothesised that the mechanism behind this was that the bacterium survives such poor environmental conditions due to associations with surfaces, or the ability to form biofilms. Biofilm formation has previously been shown to be a strategy used by several bacteria to survive harsh conditions, such as nutrient limitation, or as a defence against protozoan predation (Jefferson 2004). Previous results indicate that F. tularensis subspecies novicida has the ability to form a thin, structured biofilm (Hassett et al. 2003). We investigated the ability of F. tularensis to form biofilms, as a potential mechanism for the bacterium to survive in aquatic environments (IV). Using a microtitre assay, we established that all of the 85 F. tularensis strains that we tested (representatives from subspecies; holarctica, tularensis, mediasiatica and novicida) formed biofilms, but at varying rates. The characteristics of Francisella biofilms were investigated using F. tularensis holarctica live vaccine strain (LVS) as a model. Biofilm formation occurred between 30 and 37°C, but not at lower temperatures, suggesting that Francisella biofilm formation is temperature regulated (IV). This absence of LVS biofilm formation below 30° C was not due to growth limitation, since planktonic bacteria grew at a similar rate at 30 and 25° C. We cannot however, exclude the fact that a strong selection pressure, like protozoan predation or resource limitation, could potentially induce the formation of biofilms, as previously described for Vibrio cholerae (Matz et al. 2005). On the other hand, preliminary data showed that F. tularensis does not form biofilms at 25° C in co-culture with ciliated protozoa T. pyriformis (unpublished). We did not detect any extracellular polysaccharide (EPS) matrix joining the attached LVS cells, although a thin capsule-like structure surrounded the biofilm cells (IV). Capsule-like structures have been observed previously in F. tularensis (Hood 1977). The LVS biofilm cells were significantly more resistant to antimicrobial agents, compared to the growing planktonic cells. Increased tolerance to antibiotics is a characteristic of many bacterial biofilms. This resistance can be mediated by the physical barrier provided by the three-dimensional structure of the biofilm, preventing antibiotic substances from reaching the bacterial cells (Mah & O'Toole 2001). Moreover, the reduced bacterial growth rate in biofilms renders cells resistant to antimicrobials that target dividing cells.


32

The strong temperature regulation of Francisella biofilm formation suggests that it plays a role during the colonization of mammals rather than it being a strategy for survival in aquatic environments. This was further supported by the fact that biofilm formation by F. tularensis strains was only detected in nutrient-rich Chamberlains defined media (CDM) (Chamberlain 1965). In our microarray analysis, genes encoding the enzyme chitinase, chitin binding proteins and components of the type IV pilus (T4P) system, were up-regulated in biofilm cells compared to exponentially growing planktonic cells (IV). Type IV pili have previously been shown to be important for microcolony formation and biofilm maturation in Pseudomonas aeruginosa (O’toole 1998, Klausen 2003). Forslund et al. 2006 showed that a F. tularensis subspecies holarctica strain with a non-functional type IV pilus is attenuated in virulence in mice infected by the subcutaneous route (Forslund et al. 2006). This was interpreted as being an important role of the type IV pilus, allowing the bacterium to spread from the initial infection site to the spleen. At the site of infection in humans, for example a mosquito bite, a characteristic papule develops, often becoming a slow-healing ulcer with a crust (Eliasson et al. 2006). Further, cultures of F. tularensis can be readily obtained from these ulcers. This supports the hypothesis that F. tularensis colonizes sites of infection by the formation of biofilms. Intracellular biofilm-like phenotypes of E. coli and P. aeruginosa have been identified during infections, supporting the suggestion that biofilm formation by common pathogens could be clinically relevant (Anderson et al. 2003, Garcia-Medina et al. 2005). Considering all these data, it appears that F. tularensis biofilm formation may play a role in the colonization and infection of the mammalian host. Recently, temperature dependent growth and virulence of F. tularensis holarctica LVS has been demonstrated in a Drosophila melanogaster model (Vonkavaara et al. 2008). The temperature optima for LVS infection in Drosophila was determined to be 29° C, the highest temperature possible for the long-term culture of D. melanogaster. A similar temperature-dependent infection of fleas has been shown to include biofilm formation by Yersinia pestis (Vadyvaloo et al. 2007). The observed up-regulation of a chitinase gene and chitin binding protein in F. tularensis LVS biofilm cells indicates a possible interaction between F. tularensis and an insect vector (IV). It has previously been reported that F. tularensis subspecies novicida excretes chitinase and chitin binding proteins via a secretion system structurally homologous to a type IV pilus, although the


33

secretion seems to be functionally homologous to type II secretion (Hager et al. 2006). Thus, our results suggest that F. tularensis colonization of arthropod vectors could include biofilm formation and secretion of chitinase on chitin surfaces at relatively high temperatures for natural conditions. This implies a role for biofilm formation in the survival and transmission of F. tularensis in natural aquatic systems: a route that complements rather than contradicts the original hypothesis presented at the beginning of this thesis.

CONCLUSIONS AND FUTURE PERSPECTIVES

34


We constructed a size-structured ecosystem food web model showing that the protozoan predation pressure on bacteria increases with increasing nutrient availability in aquatic systems (I). The results were supported by meta-analysis of published data. This relationship was the result of an increasing growth rate for a relatively constant number of bacterial cells, maintaining the growth of an increasing number of protozoan cells. An extension of the ecosystem model, including modules such as bacterial predation-resistance, viruses and temperature, might broaden the applicability of the output from our original. I found that intense selection pressure from protozoan predators, dominated by ciliates, favoured predation-resistant forms of bacteria in nutrient-rich lakes (II). Bacterial predation-resistance was determined using a standardized edibility test, in co-culture with the ciliated protozoon Tetrahymena pyriformis. To my knowledge, no previous study has investigated the actual protozoan edibility of isolated bacteria along a natural productivity gradient. It has been suggested that the development of bacterial predation-resistance traits can explain the intracellular lifestyle of bacterial pathogens (Harb et al. 2000). It would be interesting to test the hypothesis that protozoa act as a “biological gym” for bacteria and to examine the role of protozoan “training camps” in the emergence of predation-resistant and pathogenic bacteria. This could be performed using an experimental setup where bacterial isolates are continuously co-cultured with axenic protozoa. Such an experiment has previously shown that bacterial isolates become less edible to a protozoan predator (Mathisen 2006). A similar approach could be used to test the biological gym hypothesis. After repeated co-culture with protozoa, the less edible bacteria could be tested for their ability to infect macrophages. In an emerging tularemia area, I found that F. tularensis was present in eutrophic aquatic systems with low bacterial diversity. Identification of the bacterium was performed using Francisella-specific 16S rRNA primers (this study). Development of a Francisella-specific fluorescence in situ hybridization (FISH) probe would provide a culture-independent technique for the


35

detection of Francisella in environmental samples. The Francisella-specific 16S rRNA primers used here and developed by Barns et. al. 2005 (Barns et al. 2005) seem to be appropriate for such an application. All F. philomiragia and F. tularensis strains were found to be relatively resistant to ciliate predation, however, the degree of resistance varied between the species and subspecies. This supports the hypothesis that the adaptation by Francisella to an aquatic environmental reservoir varies within the genus (III). I observed that F. tularensis subspecies holarctica survived and multiplied in nutrient-rich lake water with strong protozoan predation pressure on the bacteria (III). The bacterium occurred intracellularly and in close association with a small colourless flagellate (3 μm). Thus, the complexity of the microbial food web and the nutrient status of the water have an important impact on F. tularensis transmission. However, my results indicate that, over long periods, F. tularensis holarctica can survive in lake water but may lose its virulence. F. tularensis holarctica enters a VBNC state in lake water. This highlights the need to resolve the question of whether it is possible to resuscitate VBNC Francisella cells (III). This information is crucial to determine the role of aquatic environments as a reservoir for the bacterium between outbreaks. The dominant route for the transmission of F. tularensis holarctica in Sweden is mosquito bites. Future experiments should investigate further the interaction between holarctica and mosquitoes. Such experiments could help us to understand how the bacterium is transported from a potential aquatic reservoir to mammalian hosts and how this is linked to microbial food web processes. F. tularensis strains were found to have the ability to form biofilms (IV). F. tularensis biofilm formation is temperature regulated. Thus, biofilm formation may play a role in the colonization of arthropod vectors or the colonization of primary infection sites in hosts, rather than being a strategy for survival in aquatic environments.


36

A tentative model for the dispersal of F. tularensis holarctica from water to mammals

Aquatic environments with a high nutrient status and protozoan predation are favourable for the development of predation-resistant bacteria (I, II). Under such conditions, it has been shown that predation-resistant F. tularensis holarctica are able to replicate (III). It is possible that hypertrophic conditions and protozoan predation in general are not sufficient for this to occur, but rather that a specific interaction with small protozoa or other organisms is required for F. tularensis to replicate in lake water (III). Such a species-specific interaction might be necessary for resuscitation of the viable but not culturable (VBNC) state that F. tularensis enters after prolonged incubation in lake water. The dominant route for transmission of tularemia in Sweden, caused by F. tularensis holarctica, is mosquito bites. A microorganism that ingests but does not degrade F. tularensis is a potential reservoir, but is also a potential mode of transport for the bacterium to “travel” up through the size-structured microbial food web (III). Small protozoa (~3μm) are potential vectors, since they are in the feeding range of mosquito larvae (Merritt et al. 1992, Manasherob et al. 1994). Another possible route for holarctica transfer from water to mosquitoes is via the surface of the insect. The bacteria may come into contact with the fully developed mosquito at the water–air interface. F. tularensis colonization of the mosquito’s surface may include biofilm formation and degradation of chitin (IV). Since F. tularensis holarctica is highly virulent, approximately 10 bacterial cells are enough to cause disease in mammals. Thus, transmission of tularemia to a mammalian host by mosquitoes does not require high doses of the bacteria. At the infection site, bacteria may colonize the mosquito bite by biofilm formation, induced by the temperature shift to 37°C (IV). ACKNOWLEDGEMENTS

I would like to thank Agneta Andersson, Mats Forsman and Pia Haecky for valuable comments on earlier versions of this thesis. The research presented in this thesis was supported by grants from the Center for Environmental Research in Umeå (CMF, No.0322248), Umeå Marine Sciences Center, the Swedish Ministry of Defence (No.B400024) and the Swedish Research Council (No.60276201).

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I alla naturliga vatten finns en stor mängd mikroorganismer. Mikroorganismerna ingår i en födoväv där mindre mikroorganismer som bakterier och alger fungerar som föda till de något större, protozoer. Det är känt att en ökad tillgång på näring i ett sötvattensystem skapar många förändringar i vattnets födoväv. Detta kan bland annat leda till en förändrad artsammansättning av mikroorganismer och orsaka blomning av giftiga alger. Jag har i min avhandling visat att om det tillkommer näring i ett sådant system kommer de mindre mikroorganismerna (bakterier) att öka sin tillväxthastighet, detta gör att också rovdjuren (protozoer) får tillgång till mer byten och kan öka i antal. På så vis balanseras antalet bakterier av protozoer medan omsättningen av bakterier ökar. Detta medför att betningstrycket från protozoer på varje bakterie ökar dvs. att bakterier blir mer intensivt jagade av protozoer. Jag har i min avhandling visat att under stort betningstryck gynnas de bakterier som använder försvarsmekanismer för att inte bli uppätna av protozoer (predations resistens), detta gör att när näringstillgången ökar i ett vatten så ökar också förekomsten av predationsresistenta bakterier. I min avhandling har jag också visat att bakterien Francisella tularensis, som orsakar sjukdomen harpest, är predationsresistent och förekommer i näringsrika vatten i ett område med återkommande harpestutbrott. Harpest är en sjukdom som drabbar många djurarter bland andra människor och gnagare. Den främsta smittvägen av sjukdomen harpest till människor i Sverige, är via myggbett och närhet till vatten. Hur harpest överlever i naturen mellan sina utbrott är okänt. Jag har i min avhandling visat att både tillgång till mycket näring samt vattnets sammansättning av mikroorganismer (egentligen förekomst av protozoer) är viktiga för tillväxten och överlevnaden av F. tularensis. Mina resultat stöder teorin att vattnets mikroorganismer kan utgöra en naturlig tillflyktsort eller reservoar för den sjukdomsframkallande bakterien. Om så är fallet skulle också spridningen av sjukdomen från vatten till människor och djur kunna förklaras med att myggans larver under sin tid i vattnet äter bakterien och att bakterien sedan överlever inne i den vuxna myggan och sprids vidare via myggbett.


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Flera bakteriearter har visat sig kunna växa på ytor och då bilda en matta av bakterier med en tredimensionell struktur, så kallad biofilm. Bakterier som växer i biofilm är bättre skyddade mot stressfaktorer (till exempel låg näringstillgång) och mot rovdjur eller mot immunförsvarets attacker. Jag har i min avhandling visat att harpest bakterien kan bilda biofilm som är starkt temperatur reglerad (biofilm av F. tularenis bildas bara vid temperaturer över 30°C). Slutsatsen av min avhandling är att betningstrycket på bakterier från protozoer ökar då näringstillgången i vatten ökar och att detta gynnar predationsresistenta bakterier som till exempel F. tularensis, bakterien som orsakar harpest. Sammansättning av mikroorganismer och näringstillgången i vattnet är viktiga faktorer som kan påverka spridningen av den sjukdomsframkallande bakterien. Förmågan att bilda biofilm kan vara viktig för F. tularensis när bakterien orsakar sjukdom i däggdjur eller för att bakterien skall kunna spridas med insekter t.ex. myggor.

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TACK! Jag tycker att det känns bra att få skriva dessa rader i slutet av mitt alster och berätta att jag tycker om er alla en massa. Ni har på olika sätt bidragit till att jag nu till slut faktiskt är där jag är idag. För det mesta genom att förgylla min vardag och göra det riktigt behagligt att vara Johanna. Dessutom är det ju så att jag vet, att denna del av ”boken” kommer ganska många av er att läsa. Den vetskapen känns extra bra nu när jag har spenderat fyra år på att tillverka denna textmassa! Först vill jag tacka dig Agneta, för att du är en så bra handledare. Jag tycker att vårt samarbete har fungerat riktigt bra och jag kommer att sakna vårt lilla team! Mats vill jag ju så klart också tacka, du har under hela resan funnits till hands för snabb respons och bidragit med många mycket goda synpunkter. Stort tack också till alla på EMG och UMF, mina vistelser hos er har ju varit ganska sporadiska men jag känner mig alltid välkommen; Peder, Kristin, Ignacio, Rickard, Anna och Otilia jag önskar er lycka till i framtiden med alla era projekt och planer! Tack till Peter, utan ditt slit på lab hade jag inte hunnit; Erik L, för hjälp med analyser på alla möjliga och omöjliga typer av vattenprov; Pia H, för ett roligt och lärorikt samarbete kring vår ekosystem modell; Anna J, för ditt sällskap och stöd under våra första vingliga steg som doktorander. Jag vill också tacka hela högen på FOI i Umeå, ni har förgyllt mina arbetsdagar! Tjafs, flams och diskussioner i fikarummet, på lunchen, pubar, fester, på innebandyplan och på Hippologum har gjort att jag verkligen trivts under de här 4 åren. Särskilt tack till Linda och Malin som gjort kjolfredag till ett begrepp och kaffepaus till en religion. En och annan afterwork och utelunch behöver man ju faktiskt för att orka med vardagen!? Eva L, Stina, Anki, Ulla, Mona och Tina B; i min begreppsvärld är ni definitionen på expertstöd och mina närmsta systrar som känner till alla bekymmer som drabbar den som av någon anledning vill leta efter en viss liten bakterie ute i naturen. Kerstin S, Per W, Pär L, Daniel E och Johan – vi som på något vis alltid sitter lite över alla andra. Gamla medicinskt skydd’are, där allting började, Charlotta (min mentor då och för all framtid), Big-Mo, Jeanette, Anna-Lena, Solveig, Laila, Anna M, Kerstin K, Camilla, Linda, Elisabeth och Åke F. Forskningsstöd substrat; Sonja, Lotta, Lina. Maria L och Kerstin G i biblioteket som har varit

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otroliga på att snoka reda på ryska artiklar och förlänga lånetider av tegelstenar som behandlar ekologiska teorier. Marie L och Ondina som alltid har tagit sig tid när jag behövt er hjälp. Jag kallar er Intersport gänget, ni vet vilka ni är. Jag räknar också in era män och alla era coola kids, som bara blir fler och fler. Ni hjälper mig med perspektiv och endorfiner. Ni har dessutom lärt mig allt jag vet om hur man gör när man är vuxen: köper bil, köper hus, renovera hus, skaffar hund och föder barn. Jag ligger lite efter huvudklungan här, har bara kommit till etapp ett, men jag trivs bra i bak-suget efter er andra. Lisa, bara säg till innan du lägger in rycket för att jobba dig uppåt i klungan, vem vet, jag kanske hänger på! Volleyboll är en rolig sport som gör ont. Tack till Team Bechterews, Jalla och alla beachbrudar; Ylva, Pia, Charlotta och Louise, som ställer upp så att vi tillsammans kan göra något av det roligaste som finns. Pajala borna, emigrerade eller bofasta, Lundholms och Isaksson klanen. Britt-Mari och Ingemar, det känns alltid som att komma hem när jag kommer till er. Men jag förstår fortfarande inte hur det gick till när jag blev klubbmästare för Liviöjärvi IF? Kristina och Magnus, ni är som en härlig oas, lugn och avslappning infinner sig lätt när jag är hos er. Kan behövas för mig som lätt springer iväg och skrapar mina knän. Familjen Thelaus (Per, min allra bästa lillebror, Åsa, Mamma, Pappa och Farmor) ni kan berätta att jag är bäst när jag som mest behöver det utan att jag behöver be om det! Ett extra tack skall ni ha för att ni har byggt ”stugan” i Lövåsen, platsen dit man kan åka och få en känsla av vad som verkligen är värt att prioritera i livet. Åsa, you will always be my soldier! Jag behöver inte säga något mer, du vet. Lundensarna Samuel, Rebecka och Manne. Sist och allt annat än minst, min Erik, du ger mig en massa kärlek och du gör mig så nyfiken på framtiden. Jag, ”Johanna-no-tips”, är glad för att jag får del av ganska mycket Erkki, och minst 16 kramar per dag. Puss!

Umeå, april 2008

THE AQUATIC MICROBIAL FOOD WEB

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