Molecular diversity and high virulence of Legionella pneumophilastrains isolated from biofilms developed within a warm spring of a thermal spa
https://doi.org/10.1186/1471-2180-13-17
© Chaabna et al.; licensee BioMed Central Ltd. 2013
Received: 24 July 2012
Accepted: 14 January 2013
Published: 28 January 2013
Abstract
Background
Several cases of legionellosis have been diagnosed in the same French thermal spa in 1986, 1994 and 1997. L. pneumophila serogroup 1 (Lp1) strains have been isolated from several patients, but the source of contamination was not identified despite the presence of different Lp1 in water samples of the three natural springs feeding the spa at this period.
Results
Our strategy was to investigate L. pneumophila (Lp) strains from natural biofilms developed in a sulphur-rich warm spring of this contaminated site. Biofilm analysis revealed the presence of three Lp serogroups (Lp1, Lp10 and Lp12). Surprisingly, Lp10 and Lp12 were not reported in the previous described studies from water samples. Besides, the new seven Lp1 we isolated exhibit a high molecular diversity and have been differentiated in five classes according to their DNA genome patterns obtained by PFGE and mip sequences. It must be noted that these DNA patterns are original and unknown in databases. Interestingly, the 27 Lp environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acanthamoeba castellanii than those of known Lp1 epidemic strains.
Conclusion
The characteristics of Legionella pneumophila Lp1 strains isolated from the warm spring are in agreement with their presence in biofilms and their probable long-term persistence in this ecosystem.
Keywords
Background
The human pathogen Legionella pneumophila causes a severe pneumonia so-called legionellosis or Legionnaires’ disease (LD); this Gram negative bacterium was identified after the 1976 Philadelphia outbreak during the American Legion convention where 29 people succumbed [1]. Further outbreaks were associated with aerosol-producing devices like showers, cooling towers, whirlpools and fountains, but Rowbotham was the first to show a link between Legionella ecology and LD [2, 3]. Actually, L. pneumophila is ubiquitous in aquatic environment and is able to multiply intracellularly in fresh water protozoa.
L. pneumophila displays 15 serogroups but the majority of human cases are due to the serogroup1 (Lp1) (84% worldwide, 95% in Europe) [4, 5]. Lp1 is frequently found in the environment and accounts for 28% of environmental isolates in France. Other Legionella species, as L. anisa, L. dumoffii and L. feeleii that frequently colonize the water distribution systems, are rarely involved in human disease [4]. These data suggest that the high frequence of LD involving Lp1 is not due to its predominance in the environment but rather linked to a higher virulence than other species or serogroups of Legionella. The only exception is Legionella longbeachae accounting for 30% of human cases in Australia and New-Zealand, and even 50% of cases in South Australia [6]. In contrast to L. pneumophila, L. longbeachae is found predominantly in potting soil and transmitted by inhalation of dust of contaminated soils.
A lot of attention has been paid to the identification of Lp1virulence factors. It is now recognized that the co-evolution between eukaryotic hosts and L. pneumophila had led to the selection of a set of virulence factors which allow this bacterium to exploit host cellular processes; among these factors, eukaryotic-like proteins, encoded by genes identified on the basis of genome sequence analysis, are involved in different steps of the Legionella intracellular cycle [5, 7–10]. Recently, comparison of Legionella genome sequences has shown that some genes encoding the lipopolysaccharide biosynthesis were specific of Lp1 and constitute specific markers for the molecular typing [11].
We focused our attention on the identification and virulence capacities of different serogroups of L. pneumophila strains present in the French thermal spa where five cases of legionellosis were diagnosed in 1986, following by two cases in 1994 and 1997 [12, 13]. In order to determine the source of infection, water samples had been collected throughout the water distribution system as well as the three natural springs (S, sulphur; A, alum and P, cold) and two bore holes feeding the system. Eighty one L. pneumophila strains belonging to five serogroups (27 Lp1, 1Lp2, 62 Lp3, 3 Lp6 and 9 Lp13) had been identified from water samples collected over a two-year period (1997–1998); thus this water system appeared mainly contaminated by Lp1 and Lp3, also present in two natural spring (S and A). Nevertheless, comparative analysis of genomic DNA, by PFGE (“Pulse Field Gel Electrophoresis”), of both clinical Lp1 isolated from patients and environmental Lp1 isolates did not allow identifying the source of infection.
In this study, our goal was to identify legionellae directly virulent towards protozoa and as a consequence with the ability to survive in a specific environment, like the spring S characterized by a temperature of 37°C and a high level of sulphates and thiosulphates as the calcium and sodium salts [12]. Thus, we isolated legionellae from natural biofilms developed on glass slides immersed in this contaminated spring. After typing by different approaches, the DNA genome diversity of these environmental Lp strains was analyzed, and their virulence and cytotoxicity towards the amoeba Acanthamoeba castellanii were compared to those of well-known French clinical isolates (Lp1 strains Lens, Paris and Lorraine).
Results
Phenotypic analyses and serotyping of environmental L. pneumophilaisolates
Typing of 30 environmental Legionella isolates from biofilms developed in the contaminated S spring
Serotyping | Molecular typing | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Campaign | Environm. isolates | Catalase activity | Oxoid kit | bioMérieux reagents | 16S rRNA | mip | lpg 1905 | lpg 0774 | wzm | Typing |
LAXB | 1 | - | Lp2-14 | Lp10 | + | + | + | - | - | Lp |
2 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
3 | - | Lp2-14 | Lp10 | + | + | + | - | - | Lp | |
4 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
5 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
6 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
7 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
8 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
9 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
10 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
11 | - | NSR* | NSR* | + | + | + | - | - | Lp | |
12 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
13 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
14 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
15 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
16 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
17 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
18 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
19 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
20 | - | Lp2-14 | Lp10 | + | + | + | - | - | Lp | |
21 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
22 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
23 | - | Lp2-14 | Lp12 | + | + | + | - | - | Lp | |
24 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
25 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
LAXA | 21 | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 |
22 | - | Lp2-14 | Lp10 | + | + | + | - | - | Lp | |
23 | - | Lp2-14 | Lp10 | + | + | + | - | - | Lp | |
53 | - | NSR* | NSR* | - | + | - | - | - | non L | |
54 | - | NSR* | NSR* | - | + | - | - | - | non L | |
Clinical strains | Lp1 Lens | - | Lp1 | Lp1 | + | + | + | - | + | Lp1 |
Lens dotA | - | Nd§ | Nd§ | + | + | + | - | + | Lp1 | |
Lp1 Paris | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
Lp1 Lorraine | - | Lp1 | Lp1 | + | + | + | + | + | Lp1 | |
Lp3 | - | L | Lp3 | Nd§ | + | + | - | - | Lp | |
Lp8 | - | L | Lp6 | Nd§ | + | + | - | - | Lp | |
L. anisa | ++ | L | L. anisa | + | + | - | - | - | L | |
L. taurinensis | + | Nd§ | Nd§ | Nd§ | + | Nd§ | Nd§ | Nd§ | L | |
L. micdadei | ++ | L | Nd§ | Nd§ | Nd§ | Nd§ | Nd§ | Nd§ | L | |
L.longbeachae | + | L | L. longbeachae | + | + | - | - | - | L |
DNA analysis and molecular diversity of environmental L. pneumophilastrains
Examples of PCR Amplification of several Legionella pneumophila genes: lpg0774 , lpg1905 , wzm and mip . The ladder was the GeneRuler 1kb DNA ladder (Fermentas SM0311).
Thus, the LAXB environmental strains we isolated from the spring S mainly belong to Lp12 (15 isolates) and to a lesser extend to Lp1 (6 isolates) and Lp10 (3 isolates); it is interesting to underline that the isolate LAXB11 was classified as L. pneumophila only at the molecular level, and not by serotyping which could suggests a new serogroup. With regard to the LAXA strains, Lp10 (2 isolates) and Lp1 (1 isolate) were also identified, but Lp12 was not detected. Two isolates, LAXA53 and LAXA54, were classified as non Legionella species and were indeed further identified as Mycobacterium isolates on the basis of their 16S rRNA sequences using a different set of 16S rRNA primers (data not shown). The small number of Lp isolated in the LAXA campaign does not allow to draw any conclusion about the persistence of Lp between August and December 2010.
Legionella pneumophila typing. The dendogramm represents the relationships of environmental and clinical strains of Legionella pneumophila. Patterns were generated by pulse field gel electrophoresis (PFGE) of total bacterial DNA and then clustered by unweighted pair group method with arithmetic averages algorithm.
Classification of the 27 environmental L. pneumophila strains according to serogroup (sg), pulsotype (PST) and mip sequence
Class | Sg | PST | mip | Environmental isolates | Isolate number | |
---|---|---|---|---|---|---|
1 | sg1 | PST1 | mip1 | LAXB8, LAXB12 | 2 | 7 Lp1 |
2 | sg1 | PST1 | mip2 | LAXB6 | 1 | |
3 | sg1 | PST2 | mip2 | LAXA21 | 1 | |
4 | sg1 | PST2 | mip3 | LAXB24, LAXB25 | 2 | |
5 | sg1 | PST5 | mip3 | LAXB22 | 1 | |
6 | sg10 | PST4 | mip2 | LAXA22, LAXA23 | 2 | 5 Lp10 |
7 | sg10 | PST4 | mip3 | LAXB1, LAXB3, LAXB20 | 3 | |
8 | sg12 | PST3 | mip2 | LAXB2, LAXB4, LAXB5, LAXB7, LAXB9, LAXB13, LAXB14, LAXB15, LAXB16, LAXB17, LAXB18, LAXB19, LAXB21, LAXB23, LAXB10* | 15 | 15 Lp12 |
27 | 27 |
Phylogenetic tree (Neighbor-joining) of mip sequences from L. pneumophila sg 1 clinical and environmental ( mip1, mip2 and mip3) strains and L. non-pneumophila strains.
Cytotoxicity to Acanthamoeba castellani
Quantification of cytotoxicity and virulence of environmental L. pneumophila strains towards the amoeba Acanthamoeba castellanii . Lp1 dotA : dotA mutant of Lp1 Lens; Lp1 clin: means of cytotoxicities (a) and virulences (c) of three clinical Lp1 strains (Lens, Paris and Lorraine). These means of cytotoxicities (b) and virulences (d) of three clinical Lp1 strains were compared to those of five independent pulsotypes (PST1 to PST5) of environmental Lp1 strains.
Virulence towards Acanthamoeba castellani
Lp1 clinical strains involved in LD outbreaks (Lens, Lorraine) and the worldwide epidemic and endemic strain Paris were used as virulent references. 1 × 105 and 4, 5 × 105 extracellular clinical Lp1 cells were present in 3 μl samples taken after a 24 h and 48 h period of A. castellanii infection, respectively (Figure 4c). In the same periods, legionella cells released from amoeba cells infected with the dotA mutant were 100-fold less numerous. Interestingly, the number of extracellular Legionellae cells resulting from amoeba infections with environmental strains was very close to that of clinical Lp1 with the exception of extracellular Lp12 strains associated with a 10-fold increase after a 48 h-period of infection. No significant difference of virulence was observed between the different classes of environmental Lp1 at 48 h post-infection, even if some strains appeared to present a weak delay of virulence at 24 h post-infection (Figure 4d).
A co-infection experiment was also conducted in A. castellanii with two representative strains of Lp1 (LAXB24) and Lp12 (LAXB2) environmental isolates. Duplex PCR analysis (using wzm and lpg1905 primers) of extracellular bacteria revealed that 95% of 40 clones analyzed belonged to Lp12 strain (LAXB2), indicating the rapid and advantageous development of this Lp12 strain in competition to the Lp1 strain.
Discussion
Our original approach of isolation of L. pneumophila cells from natural biofilms allowed to extend the knowledge of Legionellae populations contaminating a French Alpine thermal spa where several successive cases of LD occurred from 1986 to 1997. Other previous studies had reported the presence of five sg (1, 2, 3, 6 and 13) of free-living L. pneumophila isolated from water samples collected in different sites of the spa [12, 13].
In contrast to these previous results, our work revealed that the sg 12 appears as the major population of L. pneumophila in biofilms developed within the spring S, a very original environment; besides, our results suggest that the 15 environmental Lp12 we isolated correspond probably to a unique strain; actually, all these Lp12 isolates could not differentiated at the DNA level (the same pulsotype PST3 the same mip2 sequence) or at the level of cytotoxicity towards Acanthamoeba castellanii. All these data raise the hypothesis of a probable recently-emerged Lp12 strain with a capacity of rapid development in this specific environment, and more particularly within protozoa present in the spring S. This hypothesis is also supported by the co-infection experiment that pointed out the potential advantage of Lp12 strain in competition with Lp1 strain during amoeba infection. This probable emergence of Lp12 gives also an explanation to the absence of detection of Lp12 free-cells in water samples analyzed in other reports [12, 13]. The absence of Lp12 from the LAXA strains we isolated in August 2010 could suggest an emergence of this strain in the spring S between the month of August and the month of December.
A similar hypothesis could be drawn for the sg 10, also absent from previous reports related to this thermal spa; the five Lp10 environmental isolates also characterized by a unique pulsotype (PST4); however, differences in two mip sequences (mip2 and mip) strongly suggests two Lp10 strains also recently appeared well-adapted in this site.
In contrast to Lp12 and Lp10, environmental Lp1 strains were already described in water samples collected from the three springs that fed the thermal spa. Unfortunately, Lp1 previously isolated from this thermal spa in 1988 and 1999 were no longer available; as a consequence, it is not possible to determine if the five classes of Lp1 we isolated result from a genetic evolution from a unique or several parental strain(s). Interestingly, the three distinct DNA patterns of environmental Lp1 were original and quite different of other known Lp1 clinical isolates involved in outbreaks. Besides, these environmental Lp1 were characterized by a higher toxicity and virulence towards amoebae than the Lp1 clinical isolates implied in outbreaks. At this stage, the possibility of a virulence decrease of Lp1 clinical isolates resulting from numerous times transfers in the laboratory cannot be ruled out. However, in our hands, no attenuation of virulence has been pointed out during the past 7 years. We can suppose that this high virulence of environmental isolates to amoebae is in relation with a long-term persistence of Lp1 probably in biofilms within the spring S. It is now recognized that the intracellular multiplication of Lp1 in amoebae enhanced their capacity of virulence towards alveolar human macrophages [20, 21]. Taking into consideration their very high virulence capacity, these environmental Lp1 strains constitute good candidates for the recurrent LD observed in this French thermal spa. Moreover, the high virulence trait of Lp12 strains isolated in the spring S must also be taken into consideration. Indeed, a Lp12 strain has already been involved in a legionnaires disease in the past [22]. The whole-genome sequence of this clinical isolate Lp12 strain 570-CO-H has been recently characterized [23]. However, high virulence in amoebae does not completely correlate to high virulence in humans. Thus, higher virulence of environmental strains (Lp1, Lp10 and Lp12) compared to references Lp1 outbreaks strains does not absolutely mean higher risk of legionellosis. This hypothesis needs to be validated by further studies to assess the virulence of these environmental isolates towards human macrophages.
Conclusion
This study highlights the role of mixed biofilms (protozoan and bacteria) of a site in the multiplication of virulent legionellae. Indeed, it has demonstrated the high virulence of environmental Legionella pneumophila serotype 1 isolates towards amoebae, a natural host in water spring; this is known to enhance Legionella virulence trait towards human macrophages. Moreover, it has shown the persistence capacity of Legionella pneumophila species in such an ecosystem. Finally, it also pointed out the biodiversity of Legionella pneumophila in their natural environment.
Methods
Environmental isolates
Glass slides were dipped into the contaminated spring S of a French Alpine thermal spa. After 15 days of incubation, the glass slides were covered with natural biofilms. These biofilms were harvested by scraping the glass slides and resuspended in 5 mL sterile water. Then, these suspensions were submitted to ultrasounds during 1 min in order to break up the aggregates formed by biofilms and to release bacterial cells. Bacterial suspensions were treated at 50°C during 30 min, and then submitted to an acidic treatment during 5 min by addition of 200 mM KCL/HCl pH 2.0. Aliquots (100 μL) were spread on agar GVPC medium (Oxoid, France) containing L-cysteine, iron pyrophosphate, ACES, charcoal and antibiotics (polymixin B, vancomycin, cicloheximide). After a 5 day-period incubation at 37°C, bacterial colonies with a fritted glass appearance were picked up and isolated again on GVPC. New independent colonies were picked up and suspended in cryotubes containing beads and bacterial preservers for storing at −20°C.
The Acanthamoeba castellani strain is an environmental isolate provided by F. Pernin (Institut des Sciences Pharmaceutiques et Biologiques - Faculté de pharmacie – Université Lyon 1, Lyon, France).
Reference bacterial strains
Reference strains obtained from the National Centre of Legionella (Bron, France) were used as controls in different assays: L. pneumophila serogroup 1 (Lens, Paris, Lorraine), L. pneumophila ATCC 35096 (sg 8) and ATCC 33155 (sg 3), L. anisa G12108, L. longbeachae ATCC 35096, L. micdadei ATCC 33218 and L. taurinensis ATCC 700508. The dotA mutant is derived from the strain Lens and shows a severe defect of virulence and cytotoxicity [19]. Routinely, Legionellae were grown on buffered charcoal yeast extract (BCYE) agar (Oxoid, France) or in BYE liquid medium. E. coli DH5α was cultivated on Lysogeny Broth (LB) agar medium at 37°C and Lactococcus lactis subsp. lactis IL1403 was grown at 30°C on M17 agar medium [24].
Serotyping of Legionellae
Legionella isolates were identified by polyclonal antisera coupled to latex-beads. Firstly, the Legionella latex test from Oxoid (DR0800M) allowed a separate identification of Legionella pneumophila serogroup 1 and serogroups 2–14, and the identification of seven non-pneumophila species: L. longbeachae 1 and 2, L. bozemanii 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei and L. anisa. Secondly, the 15 monovalent latex reagents prepared by bioMérieux allow the separate identification of 15 serogroups of L. pneumophila (bioMérieux, Craponne, France) [25].
In situassay of catalase activity
The presence of bacterial catalase activity was detected using H2O2 as the substrate. A bacterial colony was picked up with a sterile loop and diluted into a 15 μL drop of 10% (vol:vol) H2O2, loaded on an empty Petri dish. The rapid formation (in a few seconds) of oxygen bubbles indicates a positive result. E. coli DH5α was used as the positive control (Cat+) and Lactococcus lactis IL1403 as the negative one (Cat-).
Molecular identification and DNA amplification by PCR
Couples of primers used in this study
Gene | Primer name | Primer sequence | Amplicon size (pb) | Reference |
---|---|---|---|---|
16S RRNA | Leg225 | 5′ AAGATTAGCCTGCGTCCGAT | 654 | [18] |
Leg858 | 5′ GTCAACTTATCGCGTTTGCT | |||
mip | mipLesnsens | 5′ ATGAAGATGAAATTGGTGACTGCAG | 607 | [11] |
mipLensrev | 5′ CAACGCTACGTGGGCCATA | |||
lpg1905 | lpg1905sens | 5′ TTGCCTAAAACTCACCACAGAA | 528 | [18] |
lpg1905rev | 5′ ATGCCGCCCAAAATATACC | |||
lpg0774 | lpg0774sens | 5′ TGCTAACAACCACTATCCCAAA | 155 | [18] |
lpg0774rev | 5′ GTTTCAATAAAAGCGTGCTCCT | |||
wzm | wzmsens | 5′ ATGACCTCAATATCCTCAAAAACTCAG | 833 | [11] |
wzmrev | 5′ TTATGCTCCATGTGATGAAATGC |
DNA amplification was performed with the 2 × PCR Master Mix DNAzyme II (Finnzymes) containing 0.04 U/μL DNAzyme™ II DNA polymerase, 400 μM of each dNTP, 3 mM MgCl2, 100 mM KCl and 20 mM Tris–HCl pH 8.8 (and stabilizers). The PCR mixture (25 μL) contained the 2 × PCR Master Mix DNAzyme II (12.5 μL), 10 mM forward and reverse appropriate primers (1.0 μL each) (Table 1), and the bacterial lysate (8.0 μL). Amplification of DNA was performed in a Ep-gradient Mastercycler (Eppendorf) at initial denaturation of 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min with a final extension at 72°C for 8 min. Reactions mixtures were then held at 10°C. 8 μL of the PCR amplification mixture was analyzed by gel electrophoresis in a 0.8% agarose gel stained with ethidium bromide (1.0 μg/mL) and photographed under U.V. transillumination.
Purification and sequencing of PCR mipproducts
PCR mip products were analyzed by gel electrophoresis in a 0.8% agarose gel (50 mL) stained with 3 μL SYBR Safe DNA gel strain (Invitrogen). DNA products were visualized under blue U.V. transillumination and picked up with a band of agarose gel. Then PCR products were purified using GeneCleanR Turbo Kit (MP Biomedicals) according to the manufacturer’s instructions. Finally, the purified PCR products were suspended in 10 μL sterile water and then stored at −20°C. Sequencing was performed by GATC Biotech SARL (Mulhouse, France).
PFGE subtyping
Legionella isolates were subtyped by pulsed field gel electrophoresis (PFGE) method as described previously [26]. Briefly, legionellae were treated with proteinase K (50 mg/mL) in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8) for 24 h at 55°C, and DNA was digested with 20 IU of SfiI restriction enzyme (Boehringer Mannheim, Meylan, France) for 16 h at 50°C. Fragments of DNA were separated in a 0.8% agarose gel prepared and run in 0.5× Tris-borate-EDTA buffer (pH 8.3) in a contour-clamped homogeneous field apparatus (CHEF DRII system; Bio-Rad, Ivry sur Seine, France) with a constant voltage of 150 V. Runs were carried out with increasing pulse times (2 to 25 s) at 10°C for 11 h and increasing pulse times (35 to 60 s) at 10°C for 9 h.
Then, the gels were stained for 30 min with a ethidium bomide solution and PFGE patterns were analyzed with GelComparII software (Applied Maths, Saint-Martens-Latem, Belgium).
Quantification of Legionella virulence towards the amoeba Acanthamoeba castellanii
Legionellae were grown on BCYE agar and A. castellanii cells in PYG medium (Moffat and Tompkins, 1992) for five days at 30°C prior to infection. A. castellanii cells were first seeded in plates of 24 multiwell to a final concentration of 5 × 106 cells per ml in PY medium (PYG without glucose. Plates were incubated during two hours at 30°C to allow amoeba adhesion. Then, Legionellae were added to an MOI (“multiplicity of infection”) of 5 (in duplicate). In order to induce the adhesion of bacterial cells to the monolayer of amoeba cells, plates were spun at 2000 × g for 10 min and incubated for 1 h at 30°C. Non-adherent bacteria were removed by four successive washings of PY medium. This point was considered as the initial point of infection (T0) and the plates were incubated at 30°C. Extracellular cultivable bacteria released from amoebae were quantified at 1 day and 2 days post-infection as follows. Aliquots (100 μL) of the supernatants were taken and diluted in sterile water to the final 10-6 dilution. Aliquots (3 μL) of the serial dilutions (10-1 to 10-6) were immediately spotted to the surface of agar BCYE plates. Independent bacterial colonies of serial dilutions were numbered after 5 days at 30°C.
In the co-infection experiment, the same cells amount of each strain was added to achieve a final MOI of 5. Extracellular bacteria (10-5 and 10-6 dilutions) were plated on BCYE agar, 48 h post-infection. Independent bacterial colonies were picked-up after 3 days at 30°C to perform a PCR analysis.
Cytotoxicity to Acanthamoeba castellani
To quantify the viable A. castellanii cells remaining after infection with Legionellae (MOI 5), a monolayer of amoebae cells at the final concentration of 1 × 106 cells per ml in a 96 multiwell plate was washed (fourfold) with PY and then treated with 10% Alamar blue (Invitrogen). Cytotoxicity of each Legionella strain was tested in triplicate. After an overnight incubation at 30°C, measurements were performed at the optical density (OD) of 570 nm and corrected for background at OD600 nm with a μQuant microplate reader (Biotek Instruments Inc., Winooski, USA) The relative degree of amoeba mortality corresponds to the cytotoxicity and was expressed as the ratio of the OD value of infected monolayer to that of the uninfected one as following: [1-(mean OD value of infected/mean OD value of uninfected)] × 100%.
Declarations
Acknowlegdments
This study is supported by grants from the Centre National de la Recherche Scientifique and the Université Lyon 1. Z. Chaabna was the recipient of a fellowship from the Axelera Chemical Environmental competitiveness Cluster (LEGIOSECURE program). The authors are grateful to Claire Andréa for skilful technical assistance.
Authors’ Affiliations
References
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