Intracellular Behaviour of Legionella Non-pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky

Legionellosis, an often-lethal pneumonia, is generally associated with contamination by Legionella pneumophila. This bacterium can persist in the environment and resist chemical treatment when it is internalized by amoebae. In addition, there is increasing medical proof that other Legionella species can be causative agents of Legionellosis. The objective of this study was to evaluate whether Legionella non-pneumophila (Lnp) strains were able to use the machinery of amoeba to multiply, or whether amoebae were able to limit their proliferation. Seven strains belonging to the species L. longbeachae, L. anisa, L. bozemanae, L. taurinensis, and L. dumoffii were cocultured with three amoebae, Acanthamoeba castellanii, Willaertia magna T5(S)44, and Willaertia magna C2c Maky, at two temperatures, 22 and 37 °C. We found that at 22 °C, all amoebae were able to phagocytose the seven Lnp strains and to avoid intracellular development, except for L. longbeachae, which was able to multiply inside W. magna T5(S)44. At 37 °C, four Lnp strains were able to hijack the machinery of one or two amoebae and to use it to proliferate, but none were able to multiply inside W. magna C2c Maky.


Introduction
The genus Legionella currently comprises more than 60 species (https://lpsn.dsmz.de/ genus/legionella) (accessed on 18 October 2021) [1], of which more than half have been shown to be pathogenic to humans [2][3][4]. These bacteria occur naturally in freshwater (lakes and rivers) and wet soils. From the natural environment, the bacteria colonise anthropogenic infrastructures, especially those containing stagnant water at a temperature between 25 and 45 • C. Free-living Legionella have difficulty surviving in the environment because of their nutritional requirements. They develop in biofilms or within protists such as ciliates and amoebae [5,6]. The presence of biofilm and certain materials such as iron, zinc, and aluminium favour their survival [7].
Legionella have developed mechanisms to invade FLA by inducing "coiled phagocytosis" to escape digestion by inhibiting the fusion of the phagosome with the lysosome [6,19]. After intracellular replication, Legionella progressively invade the cytoplasm, leading to lysis of the amoeba and the release of mobile Legionella. In some cases, the amoeba also The survival of seven L. non-pneumophila (Lnp) strains in the coculture medium was evaluated at 22 and 37 • C.
The seven bacterial strains had a better survival capacity at 22 • C than at 37 • C in the SCYEM medium (Figure 1a,b, respectively).

Amoeba Survival
The SCYEM medium allows the survival of the three amoebae during the four days of experimentation at 22 and 37 • C ( Figure S1). Survival of the three amoebae in the presence of bacteria was evaluated for four days at 22 and 37 • C in an SCYEM medium. At

Coculture Experiments
Considering the death of the Lnp strains in SCYEM medium at 37 • C, the phagocytosis effect by the amoebae could not be easily evaluated. However, the potential of the bacterial strains to hijack the amoeba machinery could be determined because if bacterial multiplication occurs, it can only be attributed to its ability to use amoebae as bioreactors, as no bacterial multiplication in the culture medium occurred.
In order to compare the behaviour of the three amoebae in the presence of the seven Lnp strains, an efficacy percentage was calculated according to the following formula (Equation (1)): where CFU (t0) is the number of intracellular bacteria at day zero and CFU (t) is the number of intracellular bacteria at day one, two, three, or four. To consider the bacterial decrease due to the culture medium effect, the same calculation was applied to the control conditions (bacteria in SCYEM without amoeba) (Equation (2)): where CFU c (t0) is the number of bacteria at day zero in the control flask and CFU c (t) is the number of bacteria in control flasks at day one, two, three, or four. The net efficacy percentage was calculated by subtracting the medium effect from the efficacy values of cocultures according to the following formula (Equation (3)): where E assay is the efficacy percentage of both the amoeba and the culture medium to decrease the number of intracellular bacteria; E control is the percentage of bacteria killed by the culture medium in control conditions. E f is the net efficacy of amoeba on Lnp strains.
If E f is positive, it means that the amoeba was efficient, and consequently, bacterial death was increased by the presence of the amoeba, whereas a negative E f means that the number of living Legionella cells increased, indicating that it was able to multiply within the amoeba (as Lnp strains are not able to multiply in the SCYEM medium).
At 22 • C, W. magna C2c Maky was efficient in phagocytosing and killing L. taurinensis, L. dumoffii, L. anisa 1025, and, in particular, L. bozemanae, with 98% efficacy after 72 h (Figure 3a). At 37 • C, the amoeba efficacy could not be proven, as the bacteria in the control condition died as fast as in the presence of W. magna C2c Maky (Figure 3d). However, at both temperatures, none of the seven Legionella were able to multiply in the presence of W. magna C2c Maky ( Figure S2).  Figure S2f) were able to multiply inside A. castellanii. A. castellanii was completely invaded by bacteria from day two (Figure 3e).
At 22 • C, W. magna T5(S)44 was efficient in phagocyting L. taurinensis, L. dumoffii, L. anisa 1025, and, in particular, L. bozemanae, with 95% efficacy after 48 h (Figure 3c), but it could not reduce the level of L. longbeachae, which was able to multiply inside the amoeba ( Figure S2a). At 37 • C, no efficacy could be established as the bacteria in the control condition died as fast as in the presence of W. magna T5(S)44 ( Figure 3f). Moreover, L. bozemanae was able to multiply within W. magna T5(S)44 cells that lost their efficacy from day two (Figure 3f) and allowed the bacterium to multiply ( Figure S2d).

Discussion
Even if L. pneumophila is responsible for most legionellosis outbreaks, L. non-pneumophila (Lnp) strains are also involved in legionellosis cases, and often their responsibility is under-recognized due to diagnostic bias [2,39,46]. Even though it is now well-known that among the approximately 60 species of Legionella, 50% are able to infect humans [47], the responsibility for 4% of legionellosis cases can still not be attributed to a known species [39]. An extensive detection of Lnp strains in water distribution systems demonstrated that 16% of the sampled water was contaminated with Legionella, and that Lnp strains were prevalent [48]. In the same way, an international survey demonstrated that 43 legionellosis cases among a total of 508 were due to Lnp strains, with the most prevalent being L. longbeachae [43]. Tools developed to study the behaviour of L. pneumoniae can be used to study Lnp. A clinical strain of L. bozemanae transformed with a GFP-expressing plasmid was able to infect and replicate within A. castellanii [49]. To increase knowledge of the replication of Lnp strains inside amoeba, we studied seven non pneumophila strains, three anisa isolates, and one isolate of strains bozemanae, dumoffii, longbeachae, and taurinensis (Table 1).
L. anisa ATCC 35291, isolated from a sink faucet in Illinois, USA, and strain DSM 17627 (ATCC 35292) from tap water in California, USA, were not associated with disease [50]. These two strains were not able to multiply within the three amoebae tested in this study. In contrast, L. anisa 1025 was isolated from a human lung and was responsible for the death of the patient [51]. This strain was able to hijack A. castellanii machinery at 37 • C and to multiply within the amoeba. Its closest relative is L. pneumophila strain Lens [52]. L. anisa is rarely encountered in legionellosis cases, being responsible for only 0.2% of cases [39,43]. However, it was responsible for an outbreak in California in 1988 [53] and was also isolated from a patient in Spain [42]. It is one of the most frequently isolated Lnp species from water systems [48]. L. bozemanae DSM 16523 (ATCC 33217) originated from a human lung tissue of a patient who died in 1959 (USA); its current name was proposed in 1980 [38,54]. L. dumoffii DSM 17625 (ATCC 33279) was isolated from a cooling tower in New York. For the last ten years, L. bozemanae and L. dumoffii were responsible for, respectively, 1 and 3% of legionellosis cases in New Zealand, and remained below 1% in other countries [39]. These two strains were able to multiply within A. castellanii at 37 • C, and L. bozemanae was also able to hijack the machinery of W. magna T5(S)44 at 37 • C.
L. longbeachae DSM 10572, originally designated as Long Beach 4, was isolated from transtracheal aspirates from a fatal case of pneumonia in 1980 [55]. In the case of L. longbeachae confirmed infections, its source was exclusively associated with soils, potting mixes, and composts [56]. It is responsible for 5% of legionellosis in the US and is endemic in New Zealand and Australia [2,57]. It was unable to multiply in A. castellanii and Mono Mac 6 cells [58]. This strain was not able to develop inside amoebae at 37 • C, but it could multiply within W. magna T5(S)44 at 22 • C.
L. taurinensis DSM 21897 (ATCC 700508) was isolated from a water sample of an oxygen bubble humidifier in a hospital in Turin (Italy), where nosocomial Legionella infection occurred [59,60]. It was able to multiply within A. castellanii at 37 • C, but not in the two W. magna strains.
These data show that some Lnp strains can use amoebae for their own benefit. There is no general rule for this; this property is strain-dependent. The optimal temperature for growing A. castellanii is 28 • C. At 37 • C, A. castelanii showed limited growth, which may determine the interaction with Legionella species. However, none of the seven Lnp strains were able to multiply within W. magna C2c Maky, which is considered to be a non-permissive amoeba. Other amoebae have this property such as Micriamoeba tesseris [61] or environmental FLA isolates [62].
These results on seven Lnp strains reinforced the previous results on three L. pneumophila strains [32] to demonstrate that W. magna C2c Maky must have a very efficient process to fight amoeba-resistant bacteria and is a good candidate to be used as a biological biocide to treat industrial waters instead of, or along with, chemical treatments.

Bacteria and Amoeba Survival in SCYEM Medium (Controls)
Survival of the 3 amoeba strains and 7 bacterial strains was monitored in SCYEM medium at both 22 and 37 • C for four days as described by Hasni et al. [32], except for the plating method. Samples were plated in duplicate with the easySpiral Pro apparatus (Interscience, Saint-Nom-la-Bretèche, France). Plates were incubated at 36 ± 2 • C, and CFUs were counted after 7 days with a Scan 500 reader (Interscience, Saint-Nom-la-Bretèche, France). Each condition was performed in triplicate and independently repeated (n = 6).

Coculture Assays
Amoeba and bacterial working solutions were mixed in equal quantity as described in Hasni et al. [32]. All flasks were left to stand for 2 h at 22 ± 2 • C or at 37 ± 2 • C to allow for amoeba-bacteria contact and the internalization of Legionella into amoebae. After the 2-hour contact step, each flask was gently shaken 10 times to detach amoeba cells, and the suspension was transferred into a 15 mL Flacon ® tube and centrifuged twice at 500× g for 10 min. The supernatant was removed and the cells were resuspended in 10 mL of fresh SCYEM medium. This step allowed for the removal of non-internalized (i.e., extracellular) Legionella. The suspensions were transferred into a new 25 cm 3 flask and incubated at 22 ± 2 • C or at 37 ± 2 • C for four days. This time point corresponded to the T0 time point of the assay. Each condition consisted of three independent flasks and was repeated independently (n = 6).

Bacteria and Amoeba Quantifications in Coculture Assays from T0 to T96h
At T0, T24h, T48h, T72h, and T96h, the supernatant was removed from each flask to detect only intracellular bacteria and replaced by 10 mL of sterile SCYEM according to Hasni et al. [32]. One millilitre was then sampled from each flask. Amoeba numbers were determined using a haemocytometer cell counting chamber method with Trypan blue. Intracellular Legionella CFUs were obtained after lysing amoeba with Triton™ X-100 at 0.02% v/v (final concentration) for 2 min according to Hasni et al. [32]. The samples were then serially 10-fold diluted in SCYEM and plated in duplicate with the easySpiral Pro apparatus. Plates were incubated at 36 ± 2 • C, and CFUs were counted after 7 days with a Scan 500 reader. Each condition was performed in triplicate and independently repeated (n = 6).

Microscopic Observations of Cocultures
Cocultures were stained by means of the Gimenez technique [63,64] at T0, T48h, and T96h. Cocultures (0.5 mL) were centrifuged at 1000× g for 5 min, 0.45 mL of the supernatant was removed, pellets were resuspended in the remaining 50 µL, and 25 µL was deposited onto glass slides. After 5 min at room temperature, the cells were thermally fixed on a flame and then stained using the Gimenez technique. Briefly, each of the glass slides was stained with fuchsin solution for 3 min and washed with water. Then, the glass slides were stained with malachite green for 10-15 s and washed, and this step was repeated twice. Finally, the glass slides were allowed to dry at room temperature. Bacteria were stained in purple, and amoebae in blue.
The observations were performed using a LEICA DM 2500 LED microscope (Leica Microsystemes SAS, Nanterre, France) under a 100× oil immersion objective.