WO2023275327A1

WO2023275327A1 - Amoebae for treating bacterial infections especially due to antibiotic-resistant bacteria

Info

Publication number: WO2023275327A1
Application number: PCT/EP2022/068201
Authority: WO
Inventors: Xavier Charpentier; Bénedicte COUPAT-GOUTALAND; Félix BORNIER
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique (Cnrs); Université Claude Bernard - Lyon 1; Ecole Normale Supérieure de Lyon
Priority date: 2021-07-01
Filing date: 2022-06-30
Publication date: 2023-01-05

Abstract

The present invention relates to amoebae isolates, for use in the treatment of bacterial infections especially due to antibiotic-resistant bacteria, e.g. A. baumannii, K. pneumoniae P aeruginosa and S. aureus drug resistance strain. The present invention also provides a combination of amoebae isolates of the invention with an antibiotic for use in the treatment of bacterial infections, especially from antibiotic-resistant bacterial infection. Indeed, the inventors isolated amoebae able to predate on human pathogens and belonging to five genera: Tetramitus, Acanthamoeba, Vermamoeba vermiformis, Vahlkampfia and Stemonitis. Interestingly, pathogens previously reported as resisting predation by domesticated amoebae were found to be consumed by natural isolates of the same amoebal genera/ species. Specifically, one isolate, Vermamoeba vermiformis M-2 B4 presented a high predatory activity against all tested pathogens, decimating outnumbering antibiotic-resistant bacterial populations by up to 6 orders of magnitude in 24 hours. These voracious amoebae species with high predatory activity may represent an untapped resource to control and fight populations of antibiotic-resistant pathogens.

Description

AMOEBAE FOR TREATING BACTERIAL INFECTIONS ESPECIALLY DUE TO ANTIBIOTIC-RESISTANT BACTERIA

FIELD OF THE INVENTION:

The present invention relates to amoebae species, for use in the treatment of bacterial infections especially due to antibiotic-resistant bacteria, e.g. Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus multidrug- resistant strains. The present invention also provides a combination of amoebae species with an antibiotic for use in the treatment of bacterial infections, especially from antibiotic-resistant bacterial infection.

BACKGROUND OF THE INVENTION:

Amoebae are unicellular eukaryotic microorganisms found in natural aquatic and terrestrial environments under temperate climate, but also under more extreme environments such as polar melt water, arid land or tropical forest (1, 2). These protists move and feed by emitting cytoplasmic extensions called pseudopods. While some are parasitic, such as Entamoeba histolytica (3), others who do not depend on a host are qualified as “Free-living” (1). Amoebae have at least a two-step life cycle: the trophozoite form and the cyst form (4). The trophozoite form is a vegetative state during which the cells are metabolically active, move, feed and reproduce (4). Under adverse environmental conditions (osmotic stress, temperature, pH, predators, antagonistic compounds...), they can adopt a quiescent state form, the cyst, in order to persist in their environment. Once favorable conditions return, they can excyst back to the trophozoite form. In the environment, amoebae graze naturally on bacteria, fungi or other protists that they engulf by phagocytosis into digestive vacuoles (1, 5). Thus, amoebae are predators that naturally regulate populations of multiple microorganisms in the environment and play an important ecological role (5, 6). Yet, the relationship of amoebae and bacteria are complex and can extend to mutualistic and parasitic interactions (2, 7). While predatory interactions can have ecological implications (8), parasitic interactions have attracted considerable interest in biomedical research. Indeed, some of these parasitic amoeba-resistant bacteria are also human pathogens (9), highlighting a role of amoebae as reservoir and/or means of dissemination (10) but also as training ground for pathogens (11). The latter hypothesis primarily resulted from the observation that amoebae could support the growth of the human pathogen Legionella pneumophila (12) and that this pathogen uses the same mechanisms to survive and replicate in human macrophages (13).

Since their discovery, antibiotics have revolutionized the medical treatment of patients with bacterial infections by saving numerous lives. They represent a major therapeutic medical tool, which can be used in several clinical settings, including infections, chemotherapies, transplantation, and surgery for examples.

However, antimicrobial resistance (AMR) has been observed at dangerously high levels worldwide (Spellberg et al. (2013) Engl. J. Med. 368:299-302) and alternative therapeutic strategies are urgently needed. Among the different resistance phenomena, the AMR involving the broad-spectrum cephalosporins, one of the major class of antibiotic used worldwide, has become a major public health issue (Rossolini et al. (2008) Clin. Microbiol. Infect. 14(suppl 1): 33-41). But the mechanisms underlying the AMR (to inactivate antibiotics or to prevent them from reaching their target) are multiple and also affect chronic bacterial infection. In 2017, the World Health Organization (WHO) issued a major warning and published a list of pathogens for which new therapeutic options are urgently needed (www. who. int/news/item/27-02 -2017- who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently -needed). This list includes all the bacterial pathogens considered in the present invention.

Hence, there is an important need in identifying new solutions for efficiently targeting and fighting AMR. The present invention meets this need.

SUMMARY OF THE INVENTION:

Therefore, the present invention relates to an amoebae, for use in the treatment of bacterial infection wherein the amoeba is selected from the list of amoeba species consisting of: Tetramitus sp., Acanthamoeba sp., Vermamoeba vermiformis, Vahlkampfia sp., and Stemonitis sp.

In particular, the present invention relates to an amoebae, for use in the treatment of bacterial infection in a patient in need thereof, wherein the amoeba is selected from the list of amoeba species consisting of: Tetramitus sp., Acanthamoeba sp., Vermamoeba vermiformis, Vahlkampfia sp., and Stemonitis sp.

The present invention refers also to use of an amoeba for the treatment of bacterial infection in a plant, wherein the amoeba is selected from the list of amoeba species consisting of : Tetramitus sp., Acanthamoeba sp., Vermamoeba vermiformis sp., Vahlkampfia sp. and Stemonitis sp. In a particular embodiment the bacterial infection is due to Gram-negative bacteria or Gram-positive bacteria.

In a particular embodiment, the bacterial infection is due to an antibiotic-resistant bacterial strain.

In a particular embodiment, the amoeba of Vermamoeba vermiformis sp. is selected from the list of amoeba strain consisting of: Vermamoeba vermiformis (GenBank: KY476315), Vermamoeba vermiformis CCAP 1534/16 (GenBank: KC161965); Vermamoeba vermiformis CCAP 1534/17 (GenBank: KC188996), Vermamoeba vermiformis M.ut.l (GenBank: KX856373), Vermamoeba vermiformis Pugl93F (GenBank: KP792389), Vermamoeba vermiformis Pugl97TW (GenBank: KP792392), Vermamoeba vermiformis Pugll02TW (GenBank: KP792394), Vermamoeba vermiformis Pugll04F (GenBank: KP792396), Vermamoeba vermiformis Pugll05F (GenBank: KP792397), Vermamoeba vermiformis TW EDP 1 (GenBank: KT266863), Vermamoeba vermiformis TW EDP 3 (GenBank: KT266865)

In a more particular embodiment, the amoeba Vermamoeba vermiformis is V vermiformis M-2 B4 strain (NCBI : MZ338393).

DETAILED DESCRIPTION OF THE INVENTION:

In the present invention, inventors aimed at testing the hypothesis that environmental amoebae can predate on a wide range of human pathogens bacteria, including multidrug resistant strains. Inventors have setup a culture-based approach to isolate undomesticated amoebae based on their capacity to feed on specific bacterial species, and characterize their predatory activity against other bacterial species and strains harboring distinct virulence traits or resistance determinants. Amoebae predating on human pathogens (A. baumannii or K. pneumoniae ) were found in five genera: Tetramitus sp, Acanthamoeba sp, Vermamoeba vermiformis, Vahlkampfia sp and Stemonitis sp. Interestingly, pathogens previously reported as resisting predation by domesticated amoebae were found to be consumed by natural isolates of the same amoebal genera/ species. Specifically, one isolate ( Vermamoeba vermiformis M-2 B4) presented a high predatory activity against all tested pathogens, decimating outnumbering antibiotic-resistant bacterial populations by up to 6 orders of magnitude in 24 hours. This invention documents the characteristics of trophic interactions between diverse undomesticated amoebae and bacteria. Voracious amoebae with high predatory activity may represent an untapped resource to control and fight populations of antibiotic-resistant pathogens.

More precisely inventors have found that amoebae species of the present invention have the following properties: i/ 104 isolated amoebae belonging to 5 genera, have a bactericidal activity on either clinical isolate strains of A. baumannii (strain AB5075 resistant to carbapenems) or K. pneumoniae (strain zt246 and 26425, the latter encoding an extended-spectrum- -lactamase) : (see figure 1) ii/ All tested amoebae proved effective at controlling and killing bacterial populations of A. baumannii with different kinetics and the two tested V. vermiformis showed both the stronger and faster bactericidal activity with a moderate (M-2 E5 strain) to strong (M-2 B4 strain) reduction in the A. baumannii population at 24 hours. The same amoeba were also efficient against the constitutively capsulated AB5075F-M strain (see figures 2B). iii / microscopy analyses allowed to conclude that pathogenic bacteria are phagocytosed in amoeba digestive vacuoles and that the observed bactericidal activity of the isolated amoebae results from trophic interactions (figures 3). iv/ 8 of the isolated amoebae were then tested against 4 antibiotic-resistant bacterial strains: A. baumannii 40288 (resistant to carbapenems), K. pneumoniae 26425 (resistant to cephalosporins), P. aeruginosa PP34 (resistant to carbapenems) and S. aureus SF8300 (resistant to methicillin). The amoeba V vermiformis M-2 B4 showed the greatest bactericidal activities of all tested amoebae (see figure 4) v/ V. vermiformis M-2 B4 has a bactericidal effect against highly virulent clinical isolates : clinical isolate K. pneumoniae 32536, a hypermucoviscous K1 K. pneumoniae capsular serotype considered hypervirulent (50) and resistant to phagocytosis by neutrophils (51). V vermiformis also had a bactericidal activity against two P. aeruginosa clinical isolates, including IHMA87, an exolysin-secreting clinical isolate cytotoxic to different eukaryotic cell lines (52, 53) (see figure 5)

Altogether these results provide new insights for treating bacterial infections, especially due to resistant and multi-resistant bacterial strains (Gram-negative or Gram- positive), using amoebae of the invention as main active principle ingredient or in combination with antibiotic agent.

Amoebae and uses of the invention

Accordingly a first aspect of the invention relates an amoeba, for use in the treatment of bacterial infections wherein the amoeba is selected from the list of amoeba species consisting of : Tetramitus sp, Acanthamoeba sp, Vermamoeba vermiformis, Vahlkampfia sp and Stemonitis sp. In particular embodiment, the bacterial infection occurs in a patient or in a plant in need thereof.

Thus, the invention refers to an amoeba, for use in the treatment of bacterial infections in a patient in need thereof, wherein the amoeba is selected from the list of amoeba species consisting of : Tetramitus sp,Acanthamoeba sp, Vermamoeba vermiformis, Vahlkampfia sp and Stemonitis sp.

The invention also refers to use of an amoeba for the treatment of bacterial infection in a plant in need thereof, wherein the amoeba is selected from the list of amoeba species consisting of : Tetramitus sp., Acanthamoeba sp., Vermamoeba vermiformis sp., Vahlkampfia sp. and Stemonitis sp.

In a particular embodiment the bacterial infection is due to a Gram-negative bacteria or a Gram-positive bacteria.

The term " amoeba " refers to unicellular eukaryotic microorganisms found in natural aquatic and terrestrial environments under temperate climate, but also under more extreme environments such as polar melt water, arid land or tropical forest (1, 2). These protists move and feed by emitting cytoplasmic extensions called pseudopods. While some are parasitic, such as Entamoeba histolytica (3), others who do not depend on a host are qualified as “Free-living” (1). Amoebae have at least a two-step life cycle: the trophozoite form and the cyst form (4). The trophozoite form is a vegetative state during which the cells are metabolically active, move, feed and reproduce (4). Under adverse environmental conditions (osmotic stress, temperature, pH, predators, antagonistic compounds...), they can adopt a quiescent state form, the cyst, in order to persist in their environment. Once favorable conditions return, they can excyst back to the trophozoite form. In the environment, amoebae graze naturally on bacteria, fungi or other protists that they engulf by phagocytosis into digestive vacuoles (1, 5). Thus, amoebae are predators that naturally regulate populations of multiple microorganisms in the environment and play an important ecological role (5, 6).

In older classification systems, most amoebae were placed in the class or subphylum Sarcodina, a grouping of single-celled organisms that possess pseudopods or move by protoplasmic flow. However, molecular phylogenetic studies have shown that Sarcodina is not a monophyletic group whose members share common descent. Consequently, amoeboid organisms are no longer classified together in one group (Adi, S.M., Bass, D., Lane, C.E., Lukes, 1, Schoch, C.L., Smirnov, A., et al. (2019) Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes. Journal of Eukaryotic Microbiology 66: 4-119). Recent classification places the various amoeboid genera in the following super groups:

1. Amoebozoa with major groups and genera :

• Lobosa:

• Acanthamoeba, Amoeba, Balamuthia, Chaos, Clydonella, Discamoeba, Echinamoeba, Filamoeba, Flabellula, Gephyramoeba, Glaeseria, Vermamoeba vermiformis (Hartmannella), Hollandella, Hvdramoeba Korotnevella (Dactylamoeba) , Leptomyxa, Lingulamoeba, Mastigina, Mayorella, Metachaos, Neoparamoeba, Paramoeba, Polychaos, Phreatamoeba, Platyamoeba, Protoacanthamoeba, Rhizamoeba, Saccamoeba, Sappinia, Stereomyxa, Thecamoeba, Trichamoeba, Trichosphaerium, Undo, Vannella, Vexillifera

• Conosa:

• Endamoeba, Entamoeba, Iodamoeba, Mastigamoeba, Mastigella, Pelomyxa, Dictyostelium, Physarum, Stemonitis

2. Rhizaria with major groups and genera :

• Cercozoa:

• Filosa:

• Monadofilosa: Gyromitus, Paulinella

• Granofilosea

• Chlorarachniophyceae

• Endomyxa:

• Proteomyxidea: orders Aconchulinida, Pseudosporida, Reticulosida

• Gromiidea

• Foraminifera

• Radiolaria

3. Excavata with major groups and genera :

• Heterolobosea:

• V ahlkampfiidae: Monopylocystis, Naegleria, Neovahlkampfia, Paratetramitus, Paravahlkampfia, Protonaegleria, Psalteriomonas, Sawyeria, Tetramitus, Vahlkampfia, Willaertia

• Gruberellidae: Gruberella, Stachyamoeba • Parabasalidea: Dientamoeba, Histomonas

• Other: Rosculus, Acrasis, Heteramoeba, Learamoeba, Stvsamoeba, Plaesiobystra, Tulamoeba

4. Heterokonta with genera :

• Chrvsonhvceae: Chrvsamoeba Rhizochrysis

• Xanthophvceae: Rhizochloris

• Labyrinthulomvcetes

5. Alveolata with genera :

• Dinoflagellata: Oodinium, Pfiesteria

6. Opisthokonta with genera :

• Nucleariida: Micronuclearia, Nuclearia

7. Ungrouped with genera :

• Adelphamoeba, Astramoeba, Dinamoeba, Flagellipodium, Flamella, Gibbodiscus, Gocevia, Malamoeba, Nollandia, Oscillosignum, Paragocevia, Parvamoeba, Pernina, Pontifex, Pseudomastigamoeba, Rugipes, Striamoeba, Striolatus, Subulamoeba, Theratromyxa, Trienamoeba, Trimastigamoeba, and over 40 other genera (Patterson, D. J.; Simpson, A. G. B.; Rogerson, A. (2000). "Amoebae of uncertain affinities". In: Lee, J. J.; Leedale, G. F.; Bradbury, P. An Illustrated Guide to the Protozoa, 2nd ed., Vol. 2, p. 804-827)

Amoebae useful in the present invention can be identified and isolated using technique well known in the Art, including SSU-rDNA sequencing (Small subunit ribosomal DNA) by PCR analyses. Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. For instance, as described in Experimental section, isolated amoebae were identified by amplifying a fragment of approximately 650-800 bp of the 18S small subunit ribosomal RNA (18S SSU-rRNA) region by PCR using specific primers F-566 (5’-CAGCAGCCGCGGTAATTCC-3’) (SEQ ID N°l) and R-1200 (5 ’ -CCCGTGTTGAGTCAAATTAAGC-3 ’) (SEQ ID N°2) (88). Reference sequences and representative sequences of different amoebae genera and Acanthamoeba genotypes were retrieved from the NCBI database. Accession number of each reference sequence is available in Table 2. According to the present invention, the amoeba is selected from the list of amoeba species consisting of: Tetramitus sp, Acanthamoeba sp, Vermamoeba vermiformis sp., Vahlkampfia sp and Stemonitis sp.

In a particular embodiment, the amoeba Tetramitus sp. is selected from the list of amoeba strain consisting of: Tetramitus entericus (GenBank: AJ224889) , Tetramitus rostratus (GenBank: M98051), Tetramitus dokdoensis (GenBank: KY463322) and Tetramitus waccamawensis (GenBank: AF011455)

In a more particular embodiment, the Tetramitus amoeba sp is Tetramitus entericus NM- 3 D4 strain (NCBI MZ338461) or Tetramitus entericus NM-2 E12 strain (NCBI MZ338467).

In a particular embodiment, the amoeba Acanthamoeba sp. is selected from the list consisting of: Acanthamoeba genotype T2 ( Acanthamoeba sp. E_5C/ GenBank: AB425955), Acanthamoeba genotype T3 ( Acanthamoeba griffini S-7 ATCC 30731/ GenBank: U07412), Acanthamoeba polyphaga Panola Mountain (GenBank: AF019052), Acanthamoeba genotype T4 ( Acanthamoeba castellanii Castellanii ATCC 50374/ GenBank: U07413 ), Acanthamoeba hatchetti 2HH (GenBank: AF260722 ), Acanthamoeba mauritaniensis (GenBank: AY351647) and Acanthamoeba genotype Til ( Acanthamoeba hatchetti BH-2 /GenBank: AF019068).

In a more particular embodiment, the amoeba Acanthamoeba mauritaniensis is Acanthamoeba M-2 D6 strain (NCBI MZ338413).

In a more particular embodiment, the amoeba Acanthamoeba hatchetti BH2 is Acanthamoeba M-2 B 6 strain (NCBI MZ338413)

In a particular embodiment, the amoeba Vermamoeba vermiformis species is selected from the list consisting of:, Vermamoeba vermiformis (GenBank: KY476315), Vermamoeba vermiformis CCAP 1534/16 (GenBank: KC 161965/ Vermamoeba vermiformis CCAP 1534/17 (GenBank: KC 188996), Vermamoeba vermiformis M.ut.l (GenBank: KX856373), Vermamoeba vermiformis Pugl93F (GenBank: KP792389), Vermamoeba vermiformis Pugl97TW (GenBank: KP792392), Vermamoeba vermiformis Pugll02TW (GenBank: KP792394), Vermamoeba vermiformis Pugll04F (GenBank: KP792396), Vermamoeba vermiformis Pugll05F (GenBank: KP792397), Vermamoeba vermiformis TW EDP 1 (GenBank: KT266863), Vermamoeba vermiformis TW EDP 3 (GenBank: KT266865).

In a more particular embodiment, the amoeba Vermamoeba vermiformis specie is V vermiformis M-2 E5 strain ((NCBI : MZ338394)or V vermiformis M-2B4 strain ((NCBI : MZ338393).

In a particular embodiment, the amoeba Vahlkampfia specie is Vahlkampfia inornata CCAP 1588/2 (Genbank : AJ224887). In a more particular embodiment, the amoeba Vahlkampfla is Vahlkampfla inornata 4ES El strain (NCBI : MZ338491).

In a particular embodiment, the amoeba Stemonitis species is Stemonitis aff. flavogenita Bl/2 (GenbankAY 321109 ) In a more particular embodiment, the amoeba Stemonitis species is Stemonitis aff. flavogenita 2ED3 strain (NCBI : MZ338495)

In preferred embodiment, the amoeba is V vermiformis M-2 B4 strain (NCBI : MZ338393). The 8 isolated specific amoebae strains ( Tetramitus NM-3 D4 strain , Tetramitus NM-

2 E12 strain, Acanthamoeba M-2 D6 strain , Acanthamoeba M-2 B6, V vermiformis M-2 E5 strain, V vermiformis M-2 B4 strain, Vahlkampfla -4ES El strain and Stemonitis -2ES D3 strain) tested against antibiotic-resistant bacterial strains (figure 2 and 4) can be also identified and isolated with fragment of the 18S small subunit ribosomal RNA (SSU-rRNA) region described in the following table 1.

TABLE 1

According to the present invention the bacterial infections is Gram-negative bacterial infection and Gram-positive bacterial infection. The term "Gram-negative" bacterial infection refers to a local or systemic infection with

Gram-negative bacteria. The proteobacteria are a major group of Gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enter obacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria. Medically relevant Gram-negative cocci include the four types that cause a sexually transmitted disease {Neisseria gonorrhoeae), a meningitis {Neisseria meningitidis), and respiratory symptoms {Moraxella catarrhalis, Haemophilus influenzae). Medically relevant Gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems {Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems {Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems {Helicobacter pylori, Salmonella Enteritidis, Salmonella Typhimurium). Gram-negative bacteria associated with hospital-acquired infection include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator- associated pneumonia in hospital intensive-care units.

In another particular embodiment of the invention, the Gram-negative bacteria according to the invention are selected from the group consisting of Escherichia coli, Pseudomonas spp, Salmonella spp, Klebsiella spp, Acinetobacter spp, E. corrodens, and Haemophilus influenza. In a more particular embodiment of the invention, the Gram-negative bacteria according to the invention is Pseudomonas spp, Acinetobacter spp and Klebsiella spp.

The term ‘ Pseudomonas bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term “ Pseudomonas bacteria ” refers to but it is not limited to Gram-negative bacteria Pseudomonas, e.g ; a bacterium of the Pseudomonas aeruginosa group such as P. aeruginosa group, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea.

In particular, the Pseudomonas according to the invention is Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common Gram-negative bacteria that can cause disease in animals, including humans. It is citrate, catalase, and oxidase positive. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, its versatility enables the organism to infect damaged tissues or those with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal (Balcht, et al., Informa Health Care, 1994). Because it thrives on moist surfaces, this bacterium is also found on and in medical equipment, including catheters, causing cross infections in hospitals and clinics.

The term “ Klebsiella bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term “ Klebsiella bacteria ” refers to but it is not limited to Gram-negative bacteria Klebsiella e.g, a bacterium of the Klebsiella pneumoniae group such as K. pneumoniae group, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella pneumoniae (species- type), Klebsiella pneumoniae subsp. Ozaenae, Klebsiella pneumoniae subsp. Pneumoniae, Klebsiella pneumoniae subsp. Rhinoscleromatis , Klebsiella quasipneumoniae, Klebsiella quasipneumoniae subsp. Quasipneumoniae, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella variicola.

In particular, the Klebsiella according to the invention is Klebsiella pneumoniae.

The term “ Acinetobacter bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term “ Acinetobacter bacteria ” refers to but it is not limited to gram-negative bacteria Acinetobacter, e.g; a bacterium of the Acinetobacter baumannii group such as Acinetobacter baumannii, Acinetobacter calcoaceticus , Acinetobacter genomospecies 3 and Acinetobacter genomospecies 13 (Ingela Tjemberg et Jan Ursing) grouped together in a group called the ' cinetobacter calcoaceticus -baumannii complex ". In particular, the Acinetobacter according to the invention is Acinetobacter baumannii. In a particular embodiment the Gram-negative antibiotic-resistant bacteria is selected from the list consting of A. baumannii, K. pneumoniae andP. aeruginosa drug resistance strain The term "gram-positive" bacterial infection refers to a local or systemic infection with gram-positive bacteria. Gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell wall. Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through a microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test.

In the classical sense, six Gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus and Staphylococcus are cocci (sphere-shaped). The remaining organisms are bacilli (rod-shaped) and can be subdivided based on their ability to form spores. The non-spore formers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus and Clostridium produce spores (Gladwin, et al (2007). Miami, Florida: MedMaster. pp. 4-5. ISBN 978-0-940780-81-1).

In particular embodiment of the invention, the Gram-positive bacteria according to the invention are selected from the group consisting of Staphylococcus, Streptococcus, Clostridium, Listeria, Bacillus and Corynebacterium.

In another particular embodiment of the invention, the Gram-positive bacteria according to the invention is Staphylococcus selected from the group consisting of S. aureus group ( S . argenteus, S. aureus, S. schweitzeri, S. simiae ) , S. auricularis group ( S . auricularis), S. carnosus group (S. carnosus, S. condimenti, S. debuckii, S. massiliensis, S. piscifermentans, S. simulans) , S. epidermidis group ( S . capitis, S. caprae, S. epidermidis, S. saccharolyticus ), S. haemolyticus group ( S. borealis, S. devriesei, S. haemolyticus, S. hominis) ; S. hyicus- intermedius group (S. agnetis, S. chromogenes, S. cornubiensis, S. felis, S. delphini, S. hyicus, S. intermedius, S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, S. schleiferi ), S. lugdunensis group (S. lugdunensis ); S. saprophyticus group ( S. arlettae, S. caeli, S. cohnii, S. equorum, S. gallinarum, S. kloosii, S. leei, S. nepalensis, S. saprophyticus, S. succinus, S. xylosus ), S. sciuri group (S. fleurettii, S. lentus, S. sciuri, S. Stepanovich, S. vitulinus ), S. simulans group (S. simulans) and S. warneri group ( S . pasteuri, S. warneri )

In more particular embodiment, the Gram-positive bacteria according to the invention is S. aureus group In more particular embodiment, the Gram-positive bacteria according to the invention is Staphylococcus aureus and more specifically a Staphylococcus aureus drug resistant strain.

In a more particular embodiment the Gram-positive antibiotic-resistant bacteria is Staphylococcus aureus drug resistant strain

The present invention aims in particular at fighting antimicrobial resistance, in particular antibiotic resistance.

Accordingly, in a particular embodiment bacterial infections is due to antibiotic-multi resistant bacteria.

By “antimicrobial resistance” or “AMR” is meant herein the phenomenon that a microorganism does not exhibit decreased viability or inhibited growth or reproduction when exposed to concentrations of the antimicrobial agent that can be attained with normal therapeutic dosage regimes in patients. It implies that an infection caused by this microorganism cannot be successfully treated with this antimicrobial agent.

As used herein, the terms "antibiotic" and "antimicrobial compound" are used interchangeably and refer to a compound which decreases the viability of a microorganism, or which inhibits the growth or reproduction of a microorganism. The term “antibiotic agent” has its general meaning in the art and refers to antibacterial agent, such as described in US2013/0029981.

Suitable main class of antibiotic agents include, without limitation:

1. b-lactam antibiotic (beta-lactam antibiotic) are the antibiotic agents that contain a beta-lactam ring in their molecular structure and containing a beta-lactam functionality. This b- lactam antibiotics includes penicillin and derivatives (penams), cephalosporins (cephems), monobactams, carbapenems and carbacephems. Most b-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics (in 2003 more than half of all commercially available antibiotics in use were b- lactam compounds)

By “cephalosporins” (cephems) is meant herein a subgroup of b-lactam antibiotics originally derived from the fungus Acremonium. Together with cephamycins, they constitute a subgroup of b-lactam antibiotics called cephems. Cephalosporins include ceftazidime.

By “monobactam” is meant herein a subgroup of b-lactam antibiotics, which are monocyclic and wherein the b-lactam ring is not fused to another ring. Monobactam include aztreonam.

By “carbapenems” is meant herein a subgroup of b-lactam antibiotics, which have a bactericide effect by binding to penicillin-binding proteins (CBPs) thus inhibiting bacterial cell wall synthesis This class of antibiotics is usually reserved for known or suspected multidrug- resistant (MDR) bacterial infections. Carbapenem include imipenem.

By “penicillin” and “penicillin derivatives” (penams) is meant herein a subgroup of b- lactam antibiotics, derived originally from common moulds known as Penicillium moulds; which includes penicillin G (intravenous use), penicillin V (use by mouth), procaine penicillin, and benzathine penicillin (intramuscular use). Penicillin antibiotics were among the first medications to be effective against many bacterial infections caused by staphylococci and streptococci. They are still widely used today, though many types of bacteria have developed resistance following extensive use. There are several enhanced penicillin families which are effective against additional bacteria; these include the antistaphylococcal penicillins, aminopenicillins and the antipseudomonal penicillins. They are derived from Penicillium fungi.

Example of Natural penicillin :Penicillin G, Penicillin K, Penicillin N, Penicillin O, Penicillin V.

Example of b-lactamase-resistant penicylin derivatives: Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin, Flucloxacillin.

Example of Aminopenicillins: Ampicillin, Amoxicillin, Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin, Epicillin.

Example of Carboxypenicillins: Carbenicillin, Ticarcillin, Temocillin.

Example of Ureidopenicillins: Mezlocillin, Piperacillin, Azlocillin.

Example of b-lactamase inhibitors penicylin derivatives: Clavulanic acid, Sulbactam, Tazobactam.

2. Aminoglycoside are the antibiotic agents directed to Gram negative bacteria that inhibit protein synthesis (targeting the small ribosome sub-unit of (30 Svedberg)) and contain as a portion of the molecule an amino-modified glycoside (Mingeot-Leclercq MP, et al (1999). Antimicrob. Agents Chemother. 43 (4): 727-37). The term “Aminoglycoside” can also refer more generally to any organic molecule that contains amino sugar substructures. Aminoglycoside antibiotics display bactericidal activity against Gram-negative aerobes and some anaerobic bacilli where resistance has not yet arisen but generally not against Gram positive and anaerobic Gram-negative bacteria.

Streptomycin is the first-in-class aminoglycoside antibiotic. It is derived from Streptomyces griseus and is the earliest modem agent used against tuberculosis. Streptomycin lacks the common 2-deoxystreptamine moiety present in most other members of this class. Other examples of aminoglycosides include the deoxystreptamine-containing agents, kanamycin, tobramycin, gentamicin, and neomycin. 3. Antibiotic agents which inhibit acid nucleic synthesis

Antibiotic agent which block DNA gyrase (topoisomerase specific to bacteria) : aminocoumarines, and quinolones (such as oxolinic acid).

Antibiotic agents which block the bacterial RNA polymerase: rifampicine.

4. Antibiotics which inhibit protein synthesis (other than Aminoglycoside)

Antibiotic agents which block the formation of the peptide bond: amphenicols (examples: chloramphenicol, thiamphenicol azidamfenicol and florfenicol)

Antibiotic agents which block elongation of the polypeptide chain: Tetracyclins (examples: tetracycline, oxytetracycline, doxycycline, aureomycine, eravacycline, sarecycline ,omadacy cline) macrolides (examples : erythromycin, azithromycin) and ketolides (examples : telithromycin, cethromycin and solithromycin).

4. Antibiotics which inhibit folate metabolism

Sulfonamides also called sulphonamides, sulfa drugs or sulpha drugs (examples: Sulfamethoxazole) andsulfanilamides.

5. New classes of antibiotics compounds

Four new classes of antibiotics have been brought into clinical use in the late 2000s and early 2010s: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), and lipiarmycins (such as fidaxomicin)

In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting bacterial resistance against cephalosporin, carbapenem and b-lactamase-resistant penicylin derivatives (i.e- methicillin).

In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting multi-resistance bacterial infection.

Bacteria are said to be multidrug-resistant (MDR) when, due to the accumulation of acquired resistance to several families of antibiotics, they are only sensitive to a small number of antibiotics usable in therapy (resistance to more than 3 different families).

Method of treatment

As described in the experimental data (figure 2 and 5), the treatment with isolated amoebae of the invention on gram-positive and gram-negative bacteria, such as A. haumannii, K. pneumoniae, P. aeruginosa and S. aureus drug resistant strains allows to prevent (or to treat) harmful consequence of bacterial infection. The invention also provides a method of treating bacterial infection in a patient or a plant in need thereof with amoeba selected from the list of genera consisting of : Tetramitus sp, Acanthamoeba sp, Vermamoeba vermiformis, Vahlkampfia sp and Stemonitis sp.

As used herein, the term “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human.

In a particular embodiment of the invention, said patient is suffering from an antibiotic- resistant bacterial infection or said patient is suffering from an injury in which an infection can develop. In a more particular embodiment of the invention, said patient is suffering from a skin bacterial infection.

As used herein, the term “plant” denotes any plant species and refers to mainly multicellular organisms, predominantly photosynthetic eukaryotes of the kingdom Plantae. By one definition, plants form the clade Viridiplantae (Latin name for "green plants"), a group that includes the flowering plants, conifers and other gymnosperms, ferns and their allies, homworts, liverworts, mosses, and the green algae, but excludes the red and brown algae..

In a particular embodiment the bacterial infection is due to Gram-negative bacteria or Gram-positive bacteria.

In a particular embodiment, the bacterial infection is an antibiotic-resistant bacterial infection.

The present invention also relates to a method for treating bacterial infection in a patient or in a plant at risk to develop such infection, such method involving the step of administering to a patient or in a plant in need thereof a therapeutically effective amount of isolated amoeba of the present invention.

In particular embodiment of the invention, the Gram-positive bacterial infection is a skin Gram-positive bacterial infection.

By a "therapeutically effective amount" is meant a sufficient amount to be effective, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient in need thereof will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The amoebae of the present invention can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracistemal and intraperitoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient. In a preferred embodiment amoeba of the present invention is administered by topical way. In a preferred embodiment, amoeba of the present invention, when used for treating plants is administered by pulverization way.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.])

Typically medicaments according to the invention comprise a pharmaceutically- acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.

Combination and Pharmaceutical compositions according to the invention

Another aspect of the invention relates to a combination of (i) an amoeba according to the invention, and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infection.

In particular embodiment, the bacterial infection occurs in a patient in need thereof or in a plant in need thereof.

In particular, the present invention relates to a combination of (i) an amoeba according to the invention, and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infection in a patient in need thereof.

The present invention refers also to use of an amoeba according to the invention for the treatment of bacterial infection in a plant, wherein the amoeba is simultaneous or sequential administered with an antibiotic.

As used herein, the terms "combination" refers to a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can vary. The combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 7, days before the second partner.

The present invention also provides a pharmaceutical composition comprising: i. an amoeba (as defined here above), ii. optionnaly an antiobiotic agent (as defined here above); and iii. a pharmaceutically acceptable carrier. for use in the prevention or the treatment of bacterial infection in a patient in need thereof.

Pharmaceutical compositions formulated in a manner suitable for administration to humans are known to the skilled in the art. The pharmaceutical composition of the invention may further comprise stabilizers, buffers, etc.

The compositions of the present invention may, for example, be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for administration by injection or for local administration.

The choice of the formulation ultimately depends on the intended way of administration, such as e.g. an intravenous, intraperitoneal, subcutaneous or oral way of administration, or a local or topical administration.

The pharmaceutical composition according to the invention may be a solution or suspension, e.g. an injectable solution or suspension. It may for example be packaged in dosage unit form.

In a preferred embodiment, the amoeba and an antibiotic agent of the invention is preferably administered by the oral route or intravenous route for the antibiotic agent and by local or topical for the amoeba of the invention.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1:. Phylogeny of 104 amoebae isolated from a composting site using A. baumannii strains or K. pneumoniae strains as food source. Partial SSU-rDNA tree inferred from the Maximum Likelihood approach (437 comparison sites) of amoebae isolated from environmental samples. Amoebae isolated are indicated in bold. Amoebae isolated on A. baumannii are labelled as NM or M. Amoebae isolated on K. pneumoniae are indicated as WT or ES. Accession numbers of reference sequences of different amoebae genera are available in Table 2. Nuclearia simplex (Opisthokonta) was used as outgroup.

Figure 2: Predation activity of selected isolated amoebae. Amoebae isolates selected on A. baumannii AB5075F (NM), the capsulated AB5075F-M (M) or on K. pneumoniae 26425 (ES) were tested for they ability to prey on A. baumannii AB5075F and the capsulated AB5075F-M. The size of an initial bacterial population (~10⁶ bacteria) of AB5075F (panel A) or the capsulated AB5075F-M (panel B) was determined by CFU count in the absence (open circles) or the presence of selected amoebae (black downward-facing triangles). CFU counts at time zero are based on enumeration of the bacteria deposited in the well. CFU counts at 24, 48 and 72h correspond to the number of bacteria recovered from the wells. If no CFU are detected, each sample is given the value of the detection limit (5 CFUs), corresponding to the number of CFU possibly present in the non-plated fraction of the collected sample.

Figure 3: Bactericidal activity of isolated amoebae results from trophic interaction. A. A. baumannii AB5075F was inoculated in minimal medium conditioned by V. vermiformis M- 2 B4 or Tetramitus sp. NM-2 E12 for 24 hours. B. Same experiment as in A., but with AB5075F inoculated in spent medium of bacteria (A. baumannii) alone or in co-culture with V. vermiformis M-2 B4 or Tetramitus sp. NM-2 E12. For both A and B, CFU counts were determined at t=0h, 24h and 48h and growth is expressed as the ratio of the logio of CFU counts at 24h and 48h (t_x) relative to the inoculated CFU count (to)..

Figure 4: Isolated amoebae display broad-spectrum bactericidal activity against clinical isolates. Encysted amoebae (10⁵ cysts) were presented with clinical isolates (10⁶ CFU) on solid medium. CFU counts were determined at each time point and expressed as the logio ratio of the bacterial population with and without amoebae. If no CFU are detected, each sample is given the value of the detection limit (5 CFUs).

Figure 5: V vermiformis M-2 B4 displays strong broad-spectrum bactericidal activity against clinical isolates. A. V. vermiformis M-2 B4 kills clinical isolates of K. pneumoniae and P. aeruginosa. Isolates of K. pneumoniae and P. aeruginosa (~10⁶ bacteria) were deposited alone (open circles) or exposed to V vermiformis M-2 B4 (black downward-facing triangles). CFU counts at time zero are based on enumeration of the bacteria deposited in the well. CFU counts at 24, 48 and 72h correspond to the number of bacteria recovered from the wells. If no CFU are detected, each sample is given the value of the detection limit (5 CFUs, grey area), corresponding to the number of CFU possibly present in the non-plated fraction of the collected sample. B. V. vermiformis M-2 B4 but not V vermiformis CDC-19 (ATCC 50237) is bactericidal to A. baumannii. Amoeba cysts or trophozoites (10⁵ cells) were presented with A. baumannii AB5075F or AB5075F-M (10⁶ CFU) on solid medium. CFU counts were determined at each time point and expressed as the logio ratio of the bacterial population with and without amoebae. If no CFU are detected, each sample is given the value of the detection limit (5 CFUs).

Figure 6: Mean of ear swelling through time. Three groups of 5 mice were established: one receiving several applications of Vermamoeba M-2 B4, one with Stemonitis - 2ES D3 and the other one S. aureus AD04E17. The tubes of amoebae at 107 amoebae/ml were diluted by half in NEM medium (500 pi of amoebae culture + 500 mΐ of NEM medium) and the tubes of S. aureus at 109 CFU/ml were diluted in the same way in NEM medium (500 mΐ of bacteria suspension + 500 mΐ of NEM medium). Mice were anesthetized by an intraperitoneal injection of 100 mΐ ketamine (30 mg/ml)/xylazin (6 mg/ml) mix diluted in IX PBS. Amoebae and bacteria were applied 4 times (day 0, 2, 6 and 8) on the intact ear skin of mice, and on both sides, using a cotton swab soaked in the suspensions. Ear swelling was measured at different times using a thickness gauge JZ 15 (Machine Impex Canada Inc, Canada). Error bars represent mean ± SD.

Figure 7: Mean of wound size through time. Two groups of 5 mice were established: one receiving several applications of Vermamoeba M-2 B4 and one receiving the vehicle NEM medium. Vermamoeba M-2 B4 was cultured and harvested as described before but diluted at 2.107 amoebae/ml in NEM medium. Mice were anesthetized with isoflurane by placing their snout in a facemask. Two full-thickness excisional wounds were generated on the back skin of each mouse using a 4-mm sterile biopsy punch (Stiefel Laboratories, USA) after depilation and disinfection. Amoebae or NEM medium were applied three times (day 0, 1 and 2) by depositing a 20 mΐ drop on the surface of each wound). Wounds were photographed at different times. Images were analyzed using ImageJ 1.53v software and wound size was determined. Error bars represent mean ± SD.

Figure 8: Mean percentage of wound closure through time. In the same experience describe in figure7, Images of wounds were analyzed using ImageJ 1.53v software and wound healing was expressed as a percentage of closure relative to the initial surface of each wound. . Error bars represent mean ± SD.

EXAMPLE:

Material & Methods

Bacterial growth conditions and strains

All bacterial isolates were cultivated in liquid or solid medium of Lysogeny Broth (LB) or Tryptic Soy Broth (TSB). Acinetobacter baumannii AB5075 is a human clinical isolate highly virulent in a mouse model (79) and resistant to carbapenems (80). A. baumannii 40288 is an animal clinical isolate from the ST25 lineage and resistant to carbapenems (81). AB5075F is a derivative of AB5075 that was naturally transformed with a syn-thetic PCR product to insert the genes encoding the super-folder GFP (sfgfp) and resistance to apramycin (82) at the attTn7 site downstream of the glmS gene (83). The 40288 strain used in this study was also naturally transformed to become resistant to apramycin. AB5075F-M is a naturally-occurring mutant of AB5075F harbor-ing a mutation S551L in the wzc gene. K. pneumoniae zt246 is sensitive to most classes of antibiotics (except first generation beta-lactams) while K. pneumoniae 26425 strain is resistant to cephalosporins (cefotaxim, ceftazidime, cefuroxime), fluoroquinolones (ofloxacine, enrofloxacine), the aminoglycoside tobramycin and sulfamides. K. pneumoniae 32536 is a K1 capsular serotype sensitive to most antibiotics except the beta-lactam amoxicillin. The hypermucoviscous phenotype of the K1 capsular serotype was confirmed by the for-mation of string of >5 mm, when an inoculation loop is stretched upward from colonies on an agar plate (84). P. aeruginosa PP34 is a clinical isolate producing the cytotoxic exoenzyme ExoU and is resistant to the fluoro-quinolones ciprofloxacin and moxifloxacin, to the cephalosporin cefepime and the carbapenem imipenem (85). P. aeruginosa CHA is a highly virulent clinical isolate with a mucoid phenotype (86). Staphylococcus aureus SF8300 is a USA300 clone and resistant to methicillin, erythromycin and cefotaxime (87).

Isolation of environmental free-living amoebae feeding on A. baumannii or K. pneumoniae

The samples used to isolate amoebae capable of feeding on A. baumannii or K. pneumoniae has been collect-ed from an open-air compost site in FArbresle, France. The first sample used to isolate amoebae that could grow on A. baumannii has been collected in January 2019. Five grams of sample were mixed with 10 mL of Page’s Amoeba Saline (PAS; 2 mM NaCl, 0.016 mM MgSCri, 0,.027 mM CaCk, 0.79 mM Na2HPC>4, and 0.99 mM KTkPCri) for 5 min using a vortex mixer. Serial dilutions of the suspension were spotted on Non-Nutrient Agar (NNA; 4.6 mM Na2HP04, 2.9 mM KH2PO4, 15 g/L bacteriological agar) medium coated with A. baumannii AB5075F lawns and incubated 7 days, 30°C. An equal number of petri dishes were coated with the strain A. baumannii AB5075F and with the constitutively capsulated A. baumannii AB5075F-M. Daily microscopical observations allowed to isolate emerging amoebae. Each isolated amoeba was then sub-cultured two more times on fresh NNA with the same bacterial lawn. Isolated amoebae were stored at -80°C in a Peptone Y east Glucose (ATCC Medium 712 PYG, 0.05 M CaCl2.2H₂0, 0.4 M MgS04.7H₂0, 0.25 MNa2HP04.7H₂0, 0.25 M KH2PO4, 0.1% sodium citrate dehydrate, 5 mM Fe(NH4)2(S04)2.6H20, 0.1 M glucose, pH 6.5) and Dimethylsufoxide 10% (DMSO) mix. Isolation of amoebae capable of phagocyting on K. pneumoniae has been done with another sample collected at the end of February 2019 from the same composting site. The new sample were processed as described above and dilutions were spotted on NNA medium coated with K. pneumoniae zt246 or K. pneumoniae 26425 strains. Amoebae able to grow on those bacteria were isolated and stored as described before.

Identification of isolated amoebae

To identify amoebae, one-week culture of each isolated amoeba were collected in 2 mL of Phosphate Buffered Saline IX (PBS; 0.13 M NaCl, 8 mM Na₂HPC>4. 2H₂0, 0.18 mM KH2PO4, 2.7 mM KC1) and heated at 80°C during 10 min. Amplification of approximately a 650 bp fragment of the 18S small subunit ribosomal RNA (SSU-rRNA) region was carried out by PCR using specific primers F-566 (5’-CAGCAGCCGCGGTAATTCC-3’) (SEQ ID N°l) and R-1200 (5 ’-CCCGTGTTGAGTCAAATTAAGC-3 ’) (SEQ ID N°2) (88). PCR products were then purified using AMPure XP magnet-ic beads (Beckman Coulter, USA) and then sequenced (Eurofms Genomics, Germany). Sequences were aligned in SeaView (version 5.0.4, Pole Rhone- Alpes de Bioinformatique Site Doua, Lyon, France) using the MUSCLE algorithm before being manually inspected. Reference sequences and representative sequences of different amoebae genera and Acanthamoeba genotypes were retrieved from the NCBI database. Accession number of each reference sequence is available in Table 2. Maximum- Likelihood reconstruction tree was carried out on 437 selected sites of SSU-rDNA using the PhyML 3.0 algorithm (89) of the ATGC Montpellier Bioinformatic Platform with a GTR model, optimized equilibrium frequencies, NNI type of tree improvement and 1000 bootstrap replicates.

Quantification of amoebae predation activities against A. baumannii AB 5075

A. baumannii AB5075F and AB5075F-M were cultured in LB for 3h and then washed two times in PBS. The suspensions were next adjusted to 10⁸ CFU/mL on the basis of absorbance, and the CFU count was verified subsequently by plating. Ten microliters of each bacterial suspension (~10⁶ bacteria) were spotted at the center of a well of a 24-well plates containing 2 mL of NNA/Gelrite (NNA, 10 g/L Gelrite (Carl Roth, Germany)) and then dried. Amoebae isolates were cultured on NNA medium on a lawn of E. coli K12 bacteria for 7 days at 30°C. Each amoeba was then collected and washed two times in PBS and incubated overnight in a PBS solution containing Penicillin-Streptomycin (1000 units/mL Penicillin, lmg/mL Streptomycin) mix (Thermo Fisher Scientific, USA). Amoebae suspensions were then washed two times with PBS and starved one week in a Neff s Encystment Medium (NEM, containing in 1 L of distilled water, 0.1 M KC1, 0.39 mM MgSCri, 0.3 mM CaCh, 0.9 mM NaHCCb, 0.2 mM 2-Amino-2-methyl-l, 3-propanediol, pH 8,8-9). On the day of the experiment, suspensions of amoebae were washed two times in PBS and then diluted to obtain a concentration of 10⁷ cysts/mL. Ten microliters of each suspension were spotted on top of the previously spotted bacteria in the 24-well plate. Plates were then incubated 72h at 30°C and the content of well was recovered by adding 150 pL of PBS, 2-3 glass beads followed by gentle shaking. The liquid (-100 pL) was recovered, and serial dilutions were plated on LB medium containing Apramycin at 30 pg/mL to determine CFU counts. Each experiment consists of three bacteria- cysts mixing (from three independent bacterial cultures) for which two replicates were used to determine CFU counts. Presented experiments have been conducted at least twice.

Quantification of amoebae predation against multiple antibiotic-resistant clinical isolates

Clinical isolates of human pathogens were grown in LB (A. baumannii, K. pneumoniae and P. aeruginosa) or TSB (S. aureus). Cultures were washed in PBS and diluted at 10⁸ CFU/mL as described before. Ten microliters of each bacterial suspension were placed in 24- well plates containing 2 mL of NNA/Gelrite and then dried. Selected isolated of amoebae were cultured, starved and adjusted to 10⁷ amoebae/mL as described above. Ten microliters of each suspension were spotted on top of the bacteria. Plates were then incubated 72h at 30°C and the content of well collected as described above to determine CFU counts. Viability of A. baumannii 40288 was evaluated by plating ton LB medium containing apramycin at 30 pg/mL. Isolates of K. pneumoniae and P. aeruginosa were plated and counted on LB medium containing ampicillin at 50 pg/mL while the S. aureus isolate was plated and counted on Tryptic Soy Agar medium (TSA) containing ampicillin at 50 pg/mL.

Effect of amoebae supernatant on bacterial viability

A. baumannii AB5075F were incubated (10⁶ CFU/mL) at 30°C for 48h in minimum acetate media (MAM; 0.07 M KH2PO4, 0.03 M Na₂HP0₄, 0.02 M (NH^SCri, 0.8 mM MgS04, 0.007 mM CaCh, 0,004 mM FeSC and 1 g/L of sodium acetate) alone as a control or co-inoculated with encysted Vermamoeba M-2 B4 or Tetramitus NM-2 E12 (10⁵ amoeba/mL). The amoebae tested were also inoculated (10⁵ amoeba/mL) alone in the same conditions. The different cultures were then centrifugated gently (600 g, 10 min) to prevent cell lysis. Bacteria were then inoculated (10⁶ CFU/mL) at 30°C in the resultant filtered supernatants (0.2 mM Acrodisc, Pall Corporation, USA) of different culture conditions or in fresh MAM medium. After 24h and 48h of incubation, the suspensions were plated on LB medium containing apramycin at 30 pg/mL at 37°C to determine CFU counts.

Microscopy observations of amoeba-bacteria interactions

Confocal microscopy. Tetramitus sp. and AB5075F were processed as for the quantification of amoebae predation. Contents of the wells were recovered in 100 pL of PBS and deposited between slide and slipcover to be observed under confocal microscopy using a Leica DMI4000 inverted microscope (Leica Microsystems, Germany) equipped with a Yokogawa W1 spinning-disk confocal head (Yokogawa, Japan).

Transmission electron microscopy. Tetramitus sp. amoebae were observed after co cultivation with A. baumannii AB5075F and suspended in 100 pL of PBS, centrifuged and then fixed for 15 minutes in 0.2 M sodium cacodylate (Caco) and 4% glutaraldehyde solution. The fixed cell suspensions were then washed in 0.2 M Caco, embedded in 2% agar and placed in 1% osmium tetroxide solution for lh. The samples were placed in contact with a 1% uranyl acetate solution for lh and then progressively dehydrated by placing them in ethanol baths of increasing concentration for 10 minutes. The samples were then embedded in an EPON resin. Ultrathin sections were made with a Leica Ultracut UCS Ultramicrotome (Leica Microsystems, USA), stained with a uranyl acetate and citrate solution and then observed with a Philips CM120kV transmission electron microscope (Philips, The Netherlands).

Holo-tomographic imaging (HTM). HTM was used in combination with epifluorescence, and performed as previously described (90). V. vermiformis M-2 B4 was observed when fed with AB5075F, on the 3D Cell-Explorer Fluo (Nanolive, Ecublens, Switzerland) using a 60* air objective (NA = 0.8) at a wavelength of l = 520 nm (Class 1 low power laser, sample exposure 0.2 mW/mm2) and USB 3.0 CMOS Sony IMX174 sensor, with quantum efficiency (typical) 70% (at 545 nm), dark noise (typical) 6.6 e-, dynamic range (typical) 73.7 dB, field of view 90 ^c 90 ^c 30 pm, axial resolution 400 nm, and maximum temporal resolution 0.5 3D RI volume per second. The theoretical sensitivity is 2.71 ^c 10-4.

Results Isolation and identification of amoebae feeding on A. baumannii or K. pneumoniae We set out to isolate amoebae based on their ability to feed on two multi drug resistant bacterial human pathogens: A. baumannii and K. pneumoniae. Two K. pneumoniae clinical isolates were selected, strain zt246 and 26425, the latter encoding an extended-spectrum-b- lactamase. Strain AB5075 of A. baumannii is a clinical isolate resistant to carbapenems. In order to ease microscopy observations, we genetically modified it to encode the super-folder GFP and is hereafter referred to as AB5075F. In order to test a possible role of capsule production in resistance to amoebal predation (18), we obtained a spontaneous mutant in the wzc gene, hereafter designated AB5075F-M, and which forms highly mucoid colonies and constitutively express a thick capsule (Data not shown). The two K. pneumoniae isolates and the two A. baumannii strains were then used as food source to isolate amoebae from a compost sample. In a first isolation campaign, suspensions of the diluted compost samples were deposited on non-nutritive agar plate coated with either the parental of mucoid strain. This resulted in the isolation of 57 amoebae with the ability to grow using A. baumannii as their sole nutrient source (Fig. 1). Of these, 28 were isolated in the presence of the A. baumannii AB5075F strain (solid blue dots, Fig. 1), while the remaining 29 were obtained on the constitutively capsulated mutant AB5075F-M (, Fig. 1). Following the same procedure, a second isolation campaign using another sample from the same composting site led to the isolation of 47 amoebae capable of growing using K. pneumoniae as the only nutrient source. Of these, 20 were isolated in the presence of the K. pneumoniae strain zt246 (Fig. 1) while the other 27 were isolated on the ESBL-producing K. pneumoniae strain 26425 (Fig. 1). All 104 amoebae isolates were purified and their phylogeny and taxonomic attribution was performed using the sequence of the 18S small subunit ribosomal RNA (SSU-rRNA) region (Fig. 1). In all, the amoebae belong to five genera: Tetramitus,Acanthamoeba, Vermamoeba vermiformis, Vahlkampfla and Stemonitis (Fig. 1). Amoebae identified as Tetramitus sp. distribute around Tetramitus entericus, Tetramitus rostratus, Tetramitus dokdoensis and Tetramitus waccamawensis (formerly Learamoeba waccamawensis ) (35) and may belong to different and possibly new species. Amoebae belonging to the Tetramitus genus have been isolated in aquatic or soil environments samples but yet remain poorly studied (36-38). Being one of the most frequently isolated amoeba genus in the environment, we were not surprised to isolate a large number Acanthamoeba isolates (39). Isolates cluster around different genotypes defined by the hypervariable regions found in the 18S SSU-rRNA gene (40, 41) and appear diverse, grouping with genotypes T2, T3, T4 and Til (Fig. 1). Amoebae isolated and identified as Vermamoeba vermiformis appear less diverse and this genus includes only one species (42, 43). This species had been frequently isolated from water environments, soil samples and also in a compost facility (38, 42, 44). The amoebae identified as Vahlkampfia group closer to the species Vahlkampfia inornata than to Vahlkampfia avara. Like Tetramitus, this amoeba genus has already been isolated from environment samples but remains poorly described and studied (35, 45, 46). Two amoebae were identified as Stemonitis (formerly Hyperamoeba ), amoebae close to slime moulds and whose phylogeny has long been a source of debate (47, 48). Also, and not displayed in the tree of Figure 1, we isolated one ciliate ( Telotrochidium matiense ) feeding on A. baumannii, two cibates belonging to Kreyellidae and Colpodidae families and one kinetoplastid microorganism ( Dimastigella trypaniformis ) feeding on K. pneumoniae strains. Tetramitus is the amoebic genus that has been most frequently isolated in the presence of the A. baumannii AB5075F strain (96%), followed by the Acanthamoeba genus (4%). The Vermamoeba vermiformis amoeba species has only been isolated in the presence of the mucoid mutant AB5075F-M. Tetramitus and Acanthamoeba amoebae were isolated in similar proportions on the wild-type and the constitutively capsulated mutant of AB5075 (Fig. 1). Acanthamoeba is the amoebic genus that has most frequently been isolated on both strains of K. pneumoniae, followed by V vermiformis. While the Tetramitus genus was dominant in the isolates feeding on A. baumannii, only one amoeba belonging to this genus was isolated on K. pneumoniae. In contrast, the isolation campaign on K. pneumoniae uncovered genera not found on A. baumannii. Six amoebae from the Vahlkampfia genus were isolated on both K. pneumoniae strains and two amoebae identified as Stemonitis sp., were isolated on K. pneumoniae zt246.

In all, we isolated 104 amoebae belonging to 5 genera, 3 ciliates and 1 kinetoplastid flagellate capable of feeding on either A. baumannii or K. pneumoniae. This indicates that diverse amoebae and protists can use these pathogens as food source. Indeed, even if the isolation on A. baumannii and K. pneumoniae were conducted independently, many amoebae isolated on one pathogen are phylogenetically undistinguishable from amoebae isolated on the other pathogen (Fig. 1). This suggests that some of the isolated amoebae would be able to feed on both species. Also, amoebae isolated on the wild-type and capsulated mutant are often found within the same clade, suggesting that the constitutive production of a thick capsule by the prey did not select specific amoebae.

Kinetics of amoebal predation on A. baumannii is not altered by constitutive expression of a thick capsule.

We then sought to characterize the predation activity of the isolated amoebae. In order to compare the activity of different amoebae and bacteria, we established a standardized protocol to monitor the effect of predation on populations of bacteria in excess relative to the number of amoebae. Amoebae were cultivated on lawns of the non-pathogenic E. coli K 12, harvested and starved to induce their development into cysts. About lxl 0⁵ cysts were then exposed to lxl 0⁶ bacteria (here A. baumannii wild-type) on a non-nutritive solid medium. In the absence of amoeba, this medium allowed the bacterial population size to slightly increase and remained steady for 72 hours (Fig. 2A, open circles). In contrast, in the presence of all 8 tested amoebae, this initial AB5075F population was either contained or even fell below the detection limit, with kinetics that differed between the tested amoebae (Fig. 2A, solid downward-facing triangles). The two tested Acanthamoeba isolates and Vahlkampfia -4ES El could not alter the A. baumannii population during the first 24 hours. Yet, they then were able to consume it during the next 48 hours, so that the A. baumannii population were about 10 to 100-fold lower than if it had not been exposed to amoebae. Two tested Tetramitus amoebae were effective more rapidly, being able to alter the population at 24 hours and to steadily consume it, reducing the original population by 3 to 4 orders of magnitude. The Stemonitis - 2ES D3 was also capable of containing the A. baumannii population at 24 hours and then consumed it rapidly, resulting in a reduction of the population by over 4 orders of magnitude at 72 hours. The two isolated V. vermiformis showed both the stronger and faster bactericidal activity with a moderate (M-2 E5) to strong (M-2 B4) reduction in the A. baumannii population at 24 hours leading to a dramatic reduction of the population that fell below the detection limit within 48 hours. The same amoebae, which were isolated on K. pneumoniae, A. baumannii wild-type or constitutively capsulated, were then tested against the constitutively capsulated AB5075F-M strain (Fig. 2B, solid triangles). V. vermiformis M-2 E5 seemed to reduce the bacterial population less efficiently, yet still managed to reduce it by over 2 orders of magnitude. For all other amoebae, the kinetic of bacterial population reductions were largely similar to those observed with the wild-type A. baumannii AB5075F (Fig. 2A, solid triangles). We conclude that under the tested conditions, constitutive expression of a thick capsule offered little to no protection to predation by undomesticated amoebae. All tested amoebae proved effective at controlling and killing bacterial populations of A. baumannii but with large differences in the extend and kinetic of control. These may be due to distinct morphological properties, trophic activity, excystation rate and/or production of bactericidal compounds Bactericial activity of amoebae results from trophic interaction Amoebae have been isolated based on their ability to feed on A. baumannii or K. pneumoniae and are thus expected to internalize bacteria in a digestive vacuole by phagocytosis, constituting the basis of their bactericidal activity. However, bactericidal activity of axenic isolates of V. vermiformis,Acanthamoebapolyphaga,A. castellanii,Acanthamoeba lenticulata and D. discoideum toward the rice pathogens Xanthomonas oryzae were reported to essentially stem from the production of antibacterial compounds (49). X. oryzae was rarely observed within the digestive vacuoles of the amoebae (49). To determine if the amoebae isolated in this study produce bactericidal or bacteriostatic compounds, we analysed bacterial growth in culture supernatants of V vermiformis M-2 B4 and Tetramitus NM-2 E12. To test this, bacteria were inoculated either in fresh medium or in the same medium previously incubated with V vermiformis M-2 B4 and Tetramitus sp. NM-2 E12. Both bacterial strains showed a similar growth in the fresh medium and in amoebal culture supernatants, indicating that the amoeba had not released bactericidal or bacteriostatic compounds (Fig. 3A). We then tested the possibility that amoebae only produce these compounds when presented with their bacterial preys. We thus tested growth of A. baumannii in co-culture supernatants of the bacterial strain and amoebae and in supernatants of the bacteria alone. In this situation bacterial growth is limited because the carbon source had been previously exhausted. But again, no bactericidal or bacteriostatic effect of amoebae co-cultivation could be detected after 24h of exposure (Fig. 3B). At 48h, a limited reduction is observed but is not statistically significant. We thus examined the fate of bacteria incubated with amoebae. Transmission electron microscopy of a Tetramitus sp. isolate incubated with A. baumannii revealed the presence of particles resembling A. baumannii within vacuolar compartments (Data not shown). Confocal fluorescence microscopy of Tetramitus incubated with GFP-expressing A. baumannii indeed confirmed numerous GFP-positive vacuoles (Data not shown). We also used holo-tomography imaging combined with epifluorescence in which GFP-positive spherical particles larger than bacteria were also observed associated with the highly motile V vermiformis M-2 B4 (Data not shown). In contrast to extracellular bacteria (yellow arrowheads), these fluorescent compartments moved along with the amoeba indicative of an intracellular localization (Data not shown). Thus, the results of microscopy analyses are consistent with A. baumannii being phagocytosed in digestive vacuole to support amoebal growth. We conclude that the observed bactericidal activity of the isolated amoebae is most likely due to their active feeding on bacteria.

Most isolated amoebae display broad-spectrum bactericidal activity

The fact that the bactericidal activity result from trophic interaction suggests that the isolated amoebae may kill diverse species that they may ingest, including antibiotic-resistant clinical isolates. We thus challenge 8 of the isolated amoebae with 4 antibiotic-resistant strains: A. baumannii 40288 (resistant to carbapenems), K. pneumoniae 26425 (resistant to cephalosporins), P. aeruginosa PP34 (resistant to carbapenems) and S. aureus (resistant to methicillin). Bacterial isolates (lxl 0⁶ cfu) were inoculated on solid medium with or without encysted amoebae (lxlO⁵ cysts). The ratio of bacterial cfu counts between these two conditions were determined daily and a fold change is reported as logio ratio (logioFC) (Fig. 4). Bactericidal activities were globally the highest toward A. baumannii 40288. For this clinical isolate, the presence of any of the tested amoebae reduced by two orders of magnitude the viable cfu count at 72h (logioFC>2). Most amoebae, with the exception of Vahlkampfia sp. -4ES El could also reduce the viable count of P. aeruginosa PP34 by over a 100-fold (logioFC>2) in 72h. However, bactericidal activity was globally less important against K. pneumoniae 26425 as the logioFC change was inferior to 2 for 5 out of 8 amoebae at 72h. For instance, Vahlkampfia sp. -4ES El, which could reduce the viable count of A. baumannii by a 1000-fold (logioFC>3) could reduce the viable count of K. pneumoniae by only 10-fold (logioFC~l). The gram positive S. aureus proved more resistant than the 3 tested gram-negative. Yet, Acanthamoeba sp. M-2 B6, V vermiformis M-2 B4, V vermiformis M-2 E5 and Vahlkampfia sp. -4ES El display a logioFC>2. The two Tetramitus sp. isolates did not display any bactericidal activity against S. aureus (logioFC~0), although they efficiently killed A. baumannii. Interestingly, V vermiformis M-2 B4 showed the greatest bactericidal activities of all tested amoebae. It could reduce the viable count of all tested species by over 4 orders of magnitude at 72h. Moreover, the bactericidal effect of this amoeba was also high at 24h, reducing the viable count of K. pneumoniae and P. aeruginosa PP34 by over 5 orders of magnitude (Fig. 4). Overall, all tested amoebae were effective at killing bacteria other than those on which they were selected as source of food. With the exception of Tetramitus, they show some killing activity against the gram-positive S. aureus. Altogether, the results indicate that environmental amoebae have broad spectrum bactericidal activity, yet the extent of which varies between amoebal species and isolates.

V. vermiformis M-2 B4 is bactericidal to highly virulent clinical isolates

We next tested V vermiformis M-2 B4, the most bactericidal isolate, against additional clinical isolates of K. pneumoniae and P. aeruginosa (Fig. 5A). Confirming its potent bactericidal activity, M-2 B4 reduced the viability of two K. pneumoniae clinical isolates by 5 logs at 24h. The clinical isolate 32536, a hypermucoviscous K1 capsular serotype considered hypervirulent (50) and resistant to phagocytosis by neutrophils (51), proved more resistant with no reduction of viable count at 24h. Yet, at 48h and 72h, the viable count in the presence of V. vermiformis M-2 B4 was at least lower by two orders of magnitude (Fig. 5A). V vermiformis M-2 B4 was then tested against another two P. aeruginosa clinical isolates, including IHMA87, an exolysin-secreting clinical isolate with cytotoxic to different eukaryotic cell lines (52, 53). V vermiformis M-2 B4 brought the viable cfu counts of the two isolates under the detection limit at 48h. The high bactericidal activities of V. vermiformis M-2 B4 prompted us to compare it to the widespread axenically-growing V. vermiformis CDC-19 (ATCC 50237) (54). While V vermiformis M-2 B4 could consistently lower the viable A. baumannii AB5075 population by over 5 orders of magnitude, V vermiformis CDC-19 did not impact on the viable count, whether the predation assay was initiated with cysts (like M-2 B4) or already active trophozoites (Fig. 5B). This suggests that environmental amoebae have a stronger bacterial activity than domesticated and axenically-growing isolates.

Discussion

We here report the isolation of free-living amoebae from compost, a medium in which many microorganisms coexist and promote the breakdown of organic matter (55). With the objective of testing that environmental free-living amoebae could predate on human pathogens, we isolated amoebae based on their ability to feed on A. baumannii and K. pneumonias. The isolation of amoebae able to grow on K. pneumoniae led to the identification of the same three amoebic genera observed during the isolation campaign on A. baumannii. However, proportion of each amoeba genus isolated on K. pneumoniae and A. baumannii differed. Prey size, presence of surface molecular patterns or hydrophobicity may influence the phagocytosis capacity and the isolation of specific amoebae (56, 57). Yet, the difference of amoebae genera proportions isolated between the two screening campaigns, and the specific isolation of Vahlkampfia sp. and Stemonitis sp. on K. pneumoniae, likely do not reflect a differential ability for amoebae to better phagocyte one of the two bacterial species. Indeed, we subsequently found that amoebae isolated on A. baumannii could feed on K. pneumoniae and vice-versa (Fig. 4). Rather, it is more likely that seasonal variations between the two screening campaigns could be the cause of the differences. The two campaigns for isolations on A. baumannii and A. pneumoniae were conducted using samples from the same open-air compost site but one month apart, in January and February respectively. While climate during January 2019 in the Auvergne-Rhone Alpes region was cold and rainy, the end of February 2019 was warmer and dry, and variations of climatic conditions been observed to influence on the abundance of different amoebae genera (5, 58, 59).

One of the features of pathogens proposed to limit predation by phagocytes is the production of an extracellular capsule. This was primarily supported in the fungus Cryptococcus neoformans, whose capsule protected it against A. castellanii (60). A protective role of the capsule against amoebic predation was also demonstrated in the case of K. pneumoniae against the social amoeba/) discoideum (18). Capsule production was found to shield S. aureus, K. pneumoniae and Streptococcus pneumoniae from phagocytosis by mammalian phagocytes (61-64). The two K. pneumoniae clinical isolates selected as food source display a mucoid phenotype on plates, as it is expected for this species known for expressing a thick capsule (65). A. baumannii also naturally displays a polysaccharidic capsule but whose production is stimulated by sub-inhibitory concentrations of antibiotics and increases bacterial virulence during infection (66). We thus included in our study, a constitutively mucoid strain with a mutation (S551L) in the autokinase domain of wzc and causing a regulation defect in the capsule production (66). Isolation of amoebae on this constitutively capsulated mutant of A. baumannii did not prove more challenging than on the parental strain. Overall, the constitutive production of a thick polysaccharide capsule by the A. baumannii did not seem to impact the predation capacity and the kinetic of killing by the different amoebae (Fig. 2). This was rather unexpected, given the above-mentioned reports that capsule production provided resistance to phagocytes. We cannot exclude that the difference in capsule production by the parental mutant and the wzc mutant observed on the agar plates is not retained under amoebal predation. It is also possible that capsule production offers some relative protection, but amoebae can overcome it when the capsulated bacteria are their only food source. Consistent with this, V vermiformis M-2 B4 could even predate on the hypermucoviscous capsular serotype K1 of K. pneumoniae, yet less efficiently than on other K. pneumoniae isolates (Fig. 5). Thus, although capsule production can alter predation kinetics, it does not constitute a bullet proof vest against predatory amoebae.

Differences in the kinetics of bactericidal activity were noticeable between isolates of different genera (Fig. 2 and 4). For instance, one isolate of V vermiformis is able to kill the bacterial population as early as 24h, while no isolates of Acanthamoeba could show bactericidal activity before the 48h time point. It should be noted that all amoebae were encysted at the time they were exposed to bacteria and that excystment speed could vary between amoebae and was shorter for V. vermiformis than Acanthamoeba (67, 68). While excystment into the first trophozoites was observed after approximately 9 hours for V vermiformis ATCC 50237 (68), we observed that a majority of cysts of V vermiformis excysted as trophozoites in as little as 3 hours and not cysts were observed at 6 hours (Fig. S3). Thus, amoebae that excysted faster could predate bacteria sooner and reduce bacterial viability more rapidly. Some amoebae, such as the Tetramitus and Vahlkampfia isolates displayed an initial ability to reduce the bacterial population by 10 to 100-fold but no further decline occurred over time, suggesting that predation stopped, establishing a form of equilibrium. Predation activity was found to be dependent of prey density for strains of Tetramitus, Hartmanella (renamed Vermamoeba vermiformis), Naegleria, and Vahlkampfia toward Rhizobium meliloti bacteria, possibly because feeding is limited by the ability to capture rare preys (69). The cessation of predation activity by these amoebae could also be linked to the production of molecules by P. aeruginosa and A. baumannii that respectively force the amoebae to encyst (70) or lead to their death (71). P. aeruginosa may also kill amoebae using their type III secretion system (72). However, the two members of the V vermiformis displayed strong and sustained bactericidal activity, bringing A. baumannii populations down to the detection limit (Fig. 2). Indeed, although the axenic V vermiformis laboratory strain CDC-19 could not predate on A. baumannii, the natural isolate M-2 B4 of the same species could efficiently kill this pathogen. Similarly, we observed that A. castellanii wild isolates could kill and feed on A. baumannii, while it was previously reported resistant to laboratory strains of A. castellanii (71, 73). It may be that laboratory strains have a diminished bactericidal activity. Laboratory-domesticated amoebae have been selected to grow axenically by feeding on liquid medium by micropinocytosis rather than by predating on bacteria through phagocytosis (74). Although reversible, axenic D. discoideum grow less efficiently on bacteria (75), indicating that axenic amoebae may partly be defective in predating pathogens. However, high bactericidal activity could also be unique to specific isolates, such as V. vermiformis M-2 B4 which could eliminate K. pneumoniae and P. aeruginosa but also consume the gram-positive pathogen S. aureus. V. vermiformis M-2 B4 could clear populations of P. aeruginosa, including the PP34 isolate which encodes the Type III secretion system and the ExoU toxin (52) involved in killing A. castellanii (72, 76), but also the highly virulent CHA isolate which can induce ExoU-independent oncosis in phagocytic cells (77) (Fig. 5). It is possible that the exceptionally high bactericidal activity of M-2 B4 is due to the fact that it is immune to mechanisms used by bacteria to mitigate predation by amoebae. Importantly, M-2 B4 was found by characterizing only a subset of the 104 amoebae isolated in this study and other isolates may display similar characteristics.

In conclusion, we report here that free-living amoebae capable of predating on human pathogens can be easily recovered from natural environments. Pathogens such as A. baumannii and K. pneumoniae, which were previously reported as resistant to killing by domesticated Acanthamoeba strains (71, 73, 78), were easily consumed by natural isolates of the same species. Our work supports the idea that laboratory axenically-growing amoebae poorly reflects the relationship of human pathogens and amoebae in natural environments, overestimating their chance of survival in the war against predatory amoebae. Rather, we propose that the natural environment is a rich source of diverse amoebae with broad-spectrum predatory activities against human pathogens, including against antibiotic resistant isolates.

Table 2. Amoebae SSU-rDNA sequences used as reference in the phylogenetic tree and retrieved from the NCBI database.

Table 3. Amoebae SSU-rDNA sequences of the amoeba identified and tested in the present invention with NCBI Accession Number.

EXAMPLE 2:

Material & methods

Cultivation of amoebae and bacteria

Environmental free-living amoebae were previously isolated from an open-air compost site in l’Arbresle (France) in early 2019 and described as capable of feeding on A. baumannii or K. pneumoniae strains as only nutrient source (1). It has been possible to axenize some of the isolated amoebae, like Stemonitis -2ES D3, by inoculating them from encystment tubes into a liquid culture medium (ATCC 1034 or PYG medium) supplemented with glucose and fetal bovine serum, antibiotics and a decreasing dose of dead E. coli K12.

Before each in vivo experiment, the Vermamoeba vermiformis M-2 B4 (Vermamoeba M-2 B4) amoeba was grown for several days at 30°C on Petri dishes containing NNA medium supplemented with a layer of live E. coli K12. The amoeba was harvested one day before the beginning of experiments, washed several times with IX PBS (0.13 M NaCl, 8mM Na2HP04.2H20, 0.18mM KH2P04, 2.7mM KC1) before being incubated overnight in a IX PBS and 1/10 penicillin-streptomycin [1,000 units/ml penicillin and 1 mg/ml streptomycin; Thermo Fisher Scientific, USA] mix. Then, amoebae were once again washed several times in IX PBS and diluted at 107 amoebae/ml in Neffs encystment medium (NEM) [containing in 1 liter of distilled water, 0.1 M KC1, 0.39mM MgS04, 0.3mM CaC12, 0.9mM NaHC03, and 0.2mM 2-amino-2-methyl-l, 3-propanediol [pH 8.8 to 9]].

Axenized amoeba Stemonitis -2ES D3 was cultured for a few days before experiments in 75cm2 tissue culture flask (Sarstedt, Germany) containing modified PYNFH medium (ATCC medium 1034) [10.0 g peptone, 10.0 g yeast extract, 1.0 g yeast nucleic acid, 15.0 mg folic acid, 1.0 mg hemin in 880 ml of distilled water; 100 ml heat-inactivated fetal bovine serum and 20 ml of buffer solution containing in 1 liter of distilled water 18.1 g KH2P04 and 25.0 g Na2HP04], 1/10 fetal bovine serum (FBS) (Cytiva, USA) and 1/100 penicillin-streptomycin mix. Amoebae were then harvested, washed several times in IX PBS and diluted at 107 amoebae/ml in NEM medium.

Staphylococcus aureus AD04.E17 (S. aureus AD04E17) was cultured at 37°C in Brain Heart Infusion Broth (BHI) (Thermo Fisher Scientific, USA), diluted in tubes containing 1 ml of culture at 109 CFU/ml and then stored at -80°C before experiments.

Study of the safety of amoebae application on healthy mouse ear skin

All in vivo experiments have been conducted under specific pathogen-free conditions with mice between 6 and 8 weeks of age. Animal experimental procedures were conducted with the approval of and in accordance with the guidelines for animal experiments of a local ethics committee (CECCAPP Lyon, France) and the Ministere de l’Enseignement Superieur, de la Recherche et de Elnnovation (Paris, France). The project is referenced as APAFIS#31427- 202102221603847 v2. Female wild-type 57BL/6J were purchased from Charles Rivers Laboratories (L’Arbresle, France).

To study the safety of amoebae application on healthy mouse skin, three groups of 5 mice were established: one receiving several applications of Vermamoeba M-2 B4, one with Stemonitis -2ES D3 and the other one S. aureus AD04E17. The tubes of amoebae at 107 amoebae/ml were diluted by half in NEM medium (500 pi of amoebae culture + 500 mΐ of NEM medium) and the tubes of S. aureus at 109 CFU/ml were diluted in the same way in NEM medium (500 mΐ of bacteria suspension + 500 mΐ of NEM medium). Mice were anesthetized by an intraperitoneal injection of 100 mΐ ketamine (30 mg/ml)/xylazin (6 mg/ml) mix diluted in IX PBS. Amoebae and bacteria were applied 4 times (day 0, 2, 6 and 8) on the intact ear skin of mice, and on both sides, using a cotton swab soaked in the suspensions. Ear swelling was measured at different times using a thickness gauge JZ 15 (Machine Impex Canada Inc, Canada). Mice were killed at day 13 and ears were cut and kept in IX PBS for further investigations.

Study of the safety of amoebae application on injured mouse skin

Female mice wild-type C57BL/6J between 6 and 8 weeks of age were purchased from Charles Rivers Laboratories as described before. To study the safety of amoebae application on injured mouse skin, two groups of 5 mice were established: one receiving several applications of Vermamoeba M-2 B4 and one receiving the vehicle NEM medium. Vermamoeba M-2 B4 was cultured and harvested as described before but diluted at 2.107 amoebae/ml in NEM medium. Mice were anesthetized with isoflurane by placing their snout in a facemask. Two full-thickness excisional wounds were generated on the back skin of each mouse using a 4-mm sterile biopsy punch (Stiefel Laboratories, USA) after depilation and disinfection. Amoebae or NEM medium were applied three times (day 0, 1 and 2) by depositing a 20 mΐ drop on the surface of each wound. After drying, a bandage was formed using a square of Jelonet (Smith & Nephew, UK) and a strip of Tegaderm (3M Company, USA). Wounds were photographed at different times and bandage were redone after each application of amoebae or NEM medium. Images were analyzed using ImageJ 1.53v software and wound healing was expressed as a percentage of closure relative to the initial surface of each wound. Mice were killed at day 13 and skin biopsies were kept in IX PBS for further investigations. Results:

Study of the safety of amoebae application on healthy mouse ear skin

Ear swelling was observed through time in the group of mice receiving receiving S. aureus AD04E17 (Fig 6). In contrast, no swelling was observed in the group of mice receiving amoebae (Fig. 6) . The health of the mice in the different groups was not affected by the application of amoebae or bacteria. The ears collected at the end of the experiments were placed on NNA medium coated with E. coli K12. Growth of Vermamoeba M-2 B4 or Stemonitis -2ES D3 was observed, thus indicating that they persisted on the surface of the ears. As expected, no amoebae could be grown from ears of mice that received bacteria only.

Study of the safety of amoebae application on injured mouse skin

Wounds receiving amoebae or NEM vehicle medium healed in as little as 10 days and the health status of mice was intact in both group (Fig 7 and 8). Thus, application of amoebae on injured skin had not negative effect on wound closure.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. An amoeba for use in the treatment of skin bacterial infection in a patient in need thereof wherein the amoeba is selected from the list of amoeba species consisting of : Tetramitus sp., Acanthamoeba sp., Vermamoeba vermiformis sp., Vahlkampfia sp. and Stemonitis sp.

2. The amoeba for use according to claim 1, wherein the skin bacterial infection is due to Gram-negative bacteria or Gram-positive bacteria.

3. The amoeba for use according to claim 1 or 2, wherein the skin bacterial infection is due to an antibiotic-resistant bacterial strain.

4. The amoeba for use according to claim 1 to 3, wherein, the amoeba of Tetramitus sp. is selected from the list of amoeba strain consisting of : Tetramitus entericus (GenBank: AJ224889), Tetramitus rostratus (GenBank: M98051), Tetramitus dokdoensis (GenBank: KY463322) and Tetramitus waccamawensis (GenBank: AF011455)

5. The amoeba for use according to claim 4, wherein the Tetrami tus amoeba is Tetramitus entericus NM3D4 strain (NCBI MZ338461) or Tetramitus entericus NM2E12 strain (NCBI MZ338467).

6. The amoeba for use according to claim 1 to 3, wherein the amoeba of Acanthamoeba sp. is selected from the list of amoeba strain consisting of:, Acanthamoeba genotype T2 ( Acanthamoeba sp. E_5C/ GenBank: AB425955), Acanthamoeba genotype T3 ( Acanthamoeba grifflni S-7 ATCC 30731/ GenBank: U07412), Acanthamoeba polyphaga Panola Mountain (GenBank: AF019052), Acanthamoeba genotype T4 ( Acanthamoeba castellanii Castellanii ATCC 50374/ GenBank: U07413), Acanthamoeba hatchetti 2HH (GenBank: AF260722), Acanthamoeba mauritaniensis (GenBank: AY351647), and Acanthamoeba genotype Til ( Acanthamoeba hatchetti BH-2 /GenBank: AF019068).

7. The amoeba for use according to claim 6, wherein the amoeba Acanthamoeba mauritaniensis is Acanthamoeba M-2 D6 strain (NCBI MZ338413).

8. The amoeba for use according to claim 6, wherein amoeba Acanthamoeba hatchetti BH-2 is Acanthamoeba M-2 B6 strain (NCBI : MZ338411) .

9. The amoeba for use according to claim 1 to 3, wherein the amoeba of Vermamoeba vermiformis sp. is selected from the list of amoeba strain consisting of:, Vermamoeba vermiformis (GenBank: KY476315), Vermamoeba vermiformis CCAP 1534/16 (GenBank: KC161965), Vermamoeba vermiformis CCAP 1534/17 (GenBank: KC 188996), Vermamoeba vermiformis M.ut.l (GenBank: KX856373), Vermamoeba vermiformis Pugl93F (GenBank: KP792389), Vermamoeba vermiformis Pugl97TW (GenBank: KP792392), Vermamoeba vermiformis Pugll02TW (GenBank: KP792394), Vermamoeba vermiformis Pugll04F (GenBank: KP792396), Vermamoeba vermiformis Pugll05F (GenBank: KP792397), Vermamoeba vermiformis TW EDP 1 (GenBank: KT266863), and Vermamoeba vermiformis TW EDP 3 (GenBank: KT266865).

10. The amoeba for use according to claim 9, wherein the amoeba of Vermamoeba vermiformis sp. is V vermiformis M-2 E5 strain (NCBI : MZ338394) or V vermiformis M-2 B4 strain (NCBI : MZ338393).

11. The amoeba for use according to claim 1 to 3, wherein the amoeba of Vahlkampfia sp. is Vahlkampfia inornata CCAP 1588/2 (Genbank : AJ224887).

12. The amoeba for use according to claim 11, wherein the amoeba is Vahlkampfia inornata -4ES El strain (NCBI : MZ338491).

13. The amoeba for use according to claim 1 to 3, wherein the amoeba of Stemonitis sp. is Stemonitis aff. flavogenita Bl/2 (GenbankAY321109 )

14. An amoeba for use according to claim 13, wherein the amoeba of Stemonitis sp. is Stemonitis -2ES D3 strain (NCBI : MZ338495).

15. The amoeba for use according to claim 2 to 14 wherein the Gram negative bacteria is selected from the list consisting of Pseudomonas spp, Acinetobacter spp and Klebsiella spp.

16. The amoeba for use according to claim 2 to 14 wherein the Gram-positive bacteria is selected from the list consisting of Staphylococcus, Streptococcus, Clostridium, Listeria, Bacillus and Corynebacterium.

17. Use of an amoeba as defined in claim 1 or 4 to 14 for the treatment of bacterial infection in a plant in need thereof.

18. Use of an amoeba according to claim 17, wherein the amoeba is simultaneous or sequential administered with an antibiotic.

19. A combination of (i) an amoeba according to claim 1 to 16, and (ii) an antibiotic for the simultaneous or sequential use in the treatment of skin bacterial infections due to antibiotic-resistant bacteria in a patient in need thereof.

20. A method of treating skin bacterial infection in need thereof in a patient or bacterial infection in a plant with amoeba as defined in claim 1 or 4 to 14.