Francisella tularensis, a tier 1 select agent, is the causative bacterium of tularemia, a zoonosis with a large animal reservoir. However, F. tularensis, like many other Francisella species (including F. novicida and F. philomiragia), is assumed to have an aquatic reservoir. The mechanisms of Francisella species persistence in surface water remain poorly characterized. In this study, we deeply investigated the long-term interactions of the tularemia agent F. tularensis subsp. holarctica, F. novicida or F. philomiragia with amoebae of the Acanthamoeba species. In amoeba plate screening tests, all the Francisella species tested resisted the attack by amoebae. In in vitro infection models, intra-amoebic growth of Francisella varied according to the involved bacterial species and strains, but also the amoeba culture medium used. In co-culture models, the amoebae favoured Francisella survival over 16 days. Such enhanced survival was likely dependent on direct contact between bacteria and amoebae for F. novicida and on amoeba-excreted compounds for F. novicida and for F. tularensis. In a spring water co-culture model, amoebae again enhanced F. novicida survival and preserved bacterial morphology. Overall, our results demonstrate that amoebae likely promote Francisella survival in aquatic environments, including the tularemia agent F. tularensis. However, bacteria-amoebae interactions are complex and depend on the Francisella species considered.

Legionellae are gram-negative bacteria most commonly found in freshwater ecosystems and purpose-built water systems. In humans, the bacterium causes Legionnaires’ disease (LD) or a Pontiac fever. In this study, the different waters (drinking water, pool water, cooling towers) in which Legionella pneumophila has been isolated were studied to assess the possible risk of bacterial spreading in the population. The influence of physical and chemical parameters, and interactions with Acanthamoeba castellanii on L. pneumophila, were analyzed by Heterotrophic Plate Count, the Colony-forming units (CFU) methods, transmission electron microscopy (TEM), and Sequence-Based Typing (SBT) analysis. During the study period (2013–2019), a total of 1932 water samples were analyzed, with the average annual rate of Legionella-positive water samples of 8.9%, showing an increasing trend. The largest proportion of Legionella-positive samples was found in cooling towers and rehabilitation centers (33.9% and 33.3%, respectively). Among the isolates, L. pneumophila SGs 2–14 was the most commonly identified strain (76%). The survival of Legionella was enhanced in the samples with higher pH values, while higher electrical conductivity, nitrate, and free residual chlorine concentration significantly reduced the survival of Legionella. Our results show that growth in amoeba does not affect the allelic profile, phenotype, and morphology of the bacterium but environmental L. pneumophila becomes more resistant to pasteurization treatment.

Tularemia is a zoonotic disease caused by Francisella tularensis. A large number of recent studies have provided an update on the disease characteristics and the distribution across Europe. In Croatia, most of the clinical cases, as well as the reports of the disease in animals, date from the 20th century. In that period, epidemic and epizootic research had given detailed information about endemic regions and their characteristics, including suspected animal hosts and vectors. The region along the middle course of the Sava River, called Middle Posavina, is described as an endemic region, i.e., a “natural focus” of tularemia, in Croatia. In the 21st century, cases of human tularemia are being reported sporadically, with ulceloglandular, oropharyngeal and typhoid forms of disease. A majority of the described cases are linked with the consumption of contaminated food or water. The disease outbreaks still occur in areas along the course of the river Sava and in northwest Croatia. In this review article, we have summarized epidemiologic and epizootic data of tularemia in the past and in recent Croatian history.

Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.