Method for determining the susceptibility of bacteria to bacteriophages

Abstract

The present invention relates to a method based on the WST-8 reduction to formazan by NAD(P)H for determining the susceptibility of relevant bacteria isolates to certain bacteriophage infection allowing direct visualization of the results measured by the cell death of the target bacteria. The reduction of tetrazolium salts by NAD(P)H to formazan is used to determine the metabolic activity of cells, and as an indicator of cell viability, whilst WST-8 is used for the qualitative measurement of the phage killing activity of the targeted bacteria. Thus, the present invention falls within the field of medical biology, particularly in vitro methods of laboratory tests.

Description

[0001]DESCRIPTION METHOD FOR DETERMINING THE SUSCEPTIBILITY OF BACTERIA TO BACTERIOPHAGES Technical Field The present invention relates to a method based on the WST-8 reduction to formazan by NAD(P)H for determining the susceptibility of relevant bacteria isolates to certain bacteriophage infection allowing direct visualization of the results measured by the cell death of the target bacteria. The reduction of tetrazolium salts by NAD(P)H to formazan is used to determine the metabolic activity of cells, and as an indicator of cell viability, whilst WST-8 is used for the qualitative measurement of the phage killing activity of the targeted bacteria. Thus, the present invention falls within the field of medical biology, particularly in vitro methods of laboratory tests. Prior Art Antimicrobial resistance (AMR) is currently a major health threat. A recent study estimated that in 2019, 1.3 million people died from infections attributed directly to bacterial AMR (Murray et al, 2022). Therefore, it is urgent to develop alternative or complementary therapies to antibiotics use. Bacteriophages or phages have been used in the treatment of bacterial infections. In fact, this has been a reality for a long time, since even before the discovery of antibiotics phage therapy was already practiced. Several studies have described compassionate use cases of different antibiotic-refractory infections treated with phages, with good clinical outcomes. The growing interest in phage therapy and its increasing use in the treatment of infections both in isolated cases of patients with no therapeutic alternatives and in clinical trials requires the immediate response of clinical microbiology laboratories. These laboratories need to adapt their routine to respond in a timely manner, particularly regarding phage susceptibility testing. Double agar overlay plaque assay, or plaque assay, is the standard method to determine if a specific bacterial isolate is susceptible to infection by a specific phage allowing the determination of the concentration of phages present in biological samples. This quantitative ancient technique is based on the plating of serial dilutions of phage suspensions with a mixture of a bacterial host and a soft agar previously melted, that are poured into solid medium. After incubation of the plates, the visible phage plaques (lysis halos) are counted, and the titter determined as plaque forming units (pfu / mL). This method enables the visualization of the phage plaques, the study of the phage morphology and the phage enumeration. In the application of phage therapy in the treatment of compassionate use cases it is essential to evaluate characteristics of the phages that might allow predicting a good clinical outcome. With this objective, clinical microbiology laboratories use the plaque assay to select from the biobanks phages with the clearest and largest plaque areas, also evaluating pre-existing resistance by the presence of bacteria colonies within the plaques or turbid plaques that can suggest lysogeny or efficient defence mechanisms of the patient bacterial isolate. Phage cocktails prepared in a personalized and designed manner or fixed compositions with a wider commercial application aim, among other things, to broaden host ranges and avoid the emergence of phage resistance. However, the plaque assay method carries in these cases challenges such as difficulties in titrating the phages individually. One strategy is to use specific strains for each phage, which adds complications associated with plating efficiency, as well as variability and increased workload. Throughout clinical trials and at the time of the application of commercial phage cocktails, the phage susceptibility test of the bacterial isolates causing infection should be performed before the beginning of any treatment regimen to confirm the adequacy of the therapy. In this context, the use of plaque assay can be also rather limiting. Typically, phage plaques are visualized after necessary phages replication in the host cells, generate progeny, and release it from the cell by lysing it. Several parameters influence the phages activity against a bacterial strain, thus impacting the outcome of the infection. Furthermore, phages can kill bacterial cells without being able to replicate in it and without releasing progeny, thus presenting false results. Being dependent on the metabolic state of the host, this methodology imposes the use of grown bacterial cultures with overnight incubations resulting in a time-consuming assay, of about 36h to 48h, after the isolation of the bacterial pathogens from the patients. Document WO2004 / 041156, wherein a method to identifying bacteriophage infection of a host bacterium is disclosed by contacting two different bacteriophages with a sample comprising the target bacterium in the presence of an agent able of generating a detectable signal in response to a lytic activity of the bacteriophage, and wherein the reading of the results is performed fluorescence and luminescence and thus, the result readings cannot be directly visual, requiring specialized equipment for this purpose. The drawbacks of this method are already mentioned above. Document WO2017 / 223101 discloses a method of producing phage cocktails directed against bacterial pathogens, the method comprising the steps of constructing a bacterial diversity set including diverse strains of the same bacterial species, and subsequently constructing of an archival phage library and a working phage library, wherein the latter is screened for a delay in bacterial growth and / or a lack of appearance of phage- resistant bacterial growth. Also, this method uses fluorescence, absorption, and transmission assays to read the results, with the same drawbacks as mentioned for the previous document. Document WO2022 / 167597 discloses a system for measuring the sensitivity of a bacterium, the bacterium being preferably a bacterial pathogen, to a plurality of phages comprising the steps of contacting plurality of phages, the target bacterium and an agent, wherein the results are measured based on the interaction of the phages with the bacterium by the abundance and / or differential concentration of phage DNA using quantitative polymerase chain reaction or by measuring said light emission resulting from cell lysis through depletion of ATP and concomitant fluorescein. This system requires an expensive DNA-based amplification equipment (qPCR) that uses sophisticated and expensive reagents requiring specialized operators. Additionally, is also based on fluorescence and thus, result readings cannot be visual. This document is silent in what regards to the assessment of susceptibility analysis. Document Skusa R, Kohlen J, Podbielski A, Warnke P. Introducing "Rapid phage susceptibility testing" (RPST): an accelerated lytic phage activity test for routine diagnostic laboratories within eight hours. Diagn Microbiol Infect Dis. 2023 Dec;107(4):116054. (doi: 10.1016 / j.diagmicrobio.2023.116054. Epub 2023 Sep 3. PMID: 37748232) discloses a method of detection of visible lysis zones in tested phage and host strain combinations after eight hours. However, this method is greatly limited by the special growth behaviour of the host strain, for example anaerobic, fastidious, or slow-growing bacteria. As shown above, several alternative methods have been described to overcome some of the drawbacks referred before of the plaque assay methodology. Phage susceptibility tests ideally, should suit existing clinical laboratory protocols, have the possibility of standardization and incorporate as many as possible of the following specifications: present high sensitivity, specificity, and throughput, use inexpensive disposables or equipment requirement and be user friendly. The present invention answers all these questions by providing a method based on the WST-8 reduction to formazan by NAD(P)H for determining the susceptibility of relevant bacteria isolates to certain bacteriophage allowing direct visualization of the results measured by the cell death of the target bacteria. Description of Figures These and further aspects of the invention will be explained in greater detail by way of examples and with reference to the accompanying figures in which: Fig. 1: Represents the steps of the present invention for determining the susceptibility of bacteria to bacteriophages. The steps presented are performed for a bacterial isolate to be tested. Fig. 2 – Is a schematic representation of the WST-8 reduction to formazan by NAD(P)H which the method is based on. Fig. 3 – Is an example of a result obtained from the method for determining the susceptibility of the present invention for a bacterial isolate susceptible to phage infection. The wells with bacteria and medium are orange (alive bacteria), the wells with bacteria and SDS / EDTA are yellow (dead bacteria) and the wells with phage and bacteria are yellow (dead bacteria) which represents a phage susceptibility profile. Fig. 4 –A) Represents a plate design for the determination of S. aureus strains susceptibility with the method of the present invention for two independent experiments. B) Shows the results obtained in the determination of S. aureus strains susceptibility with the method of the present invention by using the pentaphage cocktail disclosed in the document WO2013 / 141730 after 30 min. at 37°C of incubation with colouring reagent. Lines B to H represents the 7 strains tested. Columns 1, 2, 7 and 8 are the bacteria control in TSB (duplicate for each strain), columns 3, 4, 9 and 10 are negative controls in SDS / EDTA (duplicate for each strain) and columns 5, 6, 11 and 12 are phage infection (duplicate for each strain). Result interpretation: orange means cell growth, yellowish indicates cell death. TSB control is in line A (wells 1, 2, 7 and 8). Fig. 5 – Shows the absorbance readings at 440 nm for 7 distinct S. aureus strains tested in the microplate represented in figure 4. Lines B to H are the 7 strains tested. Columns 1, 2, 7 and 8 are the bacteria control in TSB (duplicate for each strain), columns 3, 4, 9 and 10 are negative controls in SDS / EDTA (duplicate for each strain) and columns 5, 6, 11 and 12 are phage infection (duplicate for each strain). Fig. 6 –A) Represents a plate design for the determination of A. baumannii strains susceptibility with the method of the invention for two independent experiments. B) Shows the results obtained in the determination of A. baumannii strains susceptibility with the method of the present invention by using the pentaphage cocktail disclosed in the document WO2013 / 141730 after 30 min. at 37°C of incubation with colouring reagent. Lines B to H are the 7 strains tested. Columns 2, 3 and 4 are the bacteria control (triplicate for each strain), columns 6, 7 and 8 are negative controls (triplicate for each strain) and columns 10, 11 and 12 are phage infection (triplicate for each strain). Results interpretation: orange means cell growth, yellowish indicates cell death. TSB blank control is in line A (wells 2 to 4). Fig. 7 – Represents a plate design for the determination of E. coli strains susceptibility with the method of the invention for one experiment. Lines A to G are the 14 strains tested. Columns 1, 2 and 7, 8 are the bacteria control (duplicate for each strain), columns 3, 4 and 9, 10 are negative controls (duplicate for each strain) and columns 5, 6 and 11, 12 are phage cocktail wells (duplicate for each strain). TSB blank control wells H11 and H12. Fig. 8 – Shows the results obtained in the determination of E. coli strains susceptibility with the method of the present invention to E. coli cocktail after 1h at 37°C of incubation with colouring reagent. Results interpretation: orange means cell growth (not susceptible to phage cocktail), yellowish indicates cell death (susceptible to phage cocktail). TSB blank control wells H11 and H12. Fig. 9 - Shows the results obtained in the determination of K. pneumoniae strains susceptibility with the method of the present invention with the triple phage cocktail disclosed in the document WO 2021 / 125988 after 2h at 37°C of incubation with the colouring reagent. The plate layout is represented in the plate lid. Green wells are the bacteria controls with medium, red wells are SDS / EDTA controls, blue wells are bacteria with the triple phage cocktail (triplicates for all strains). Results interpretation: orange means cell growth (not susceptible to phage cocktail), yellowish indicates cell death (susceptible to phage cocktail). TSB blank control wells A2-4. Fig. 10 – Shows the results obtained in the determination of P. aeruginosa strains susceptibility with the method of the invention and with the triple phage cocktail disclosed in the document WO2018 / 106135 after 2h at 37°C of incubation with colouring reagent. Plate layout represented in the plate lid. Green wells are the bacteria controls with medium, red wells are bacteria and SDS / EDTA controls and blue wells with phage cocktail and bacteria (triplicates for all strains). Interpretation: Orange means cell growth (not susceptible to the triple phage cocktail), yellowish indicates cell death (susceptible to the triple phage cocktail). TSB blank control wells A2-4. Summary of the invention The present invention relates to a method for determining the susceptibility of relevant bacteria isolates to certain bacteriophages.The method of the invention comprises the steps of preparing a bacterial suspension with a target bacterial isolate, diluting to ½ of said suspension in a liquid enrichment culture medium and further treating the resulting liquid culture with individual phage suspensions or with a mixture of several phages, also known as page cocktails and then adding WST-8 and a colouring reagent to the final culture according to claim 1. The method of the present invention provides a way of assessing qualitative phage susceptibility that is rapid in turnaround time, of approximately 5 hours from patient isolate, a reduction of one to two working days compared to the gold standard method known as “plaque assay”. Further, this method is easy to perform, with the option of high-throughput testing by using 96-well microplates, thus allowing testing of several bacterial isolates simultaneously, and the possibility of automation, which is extremely difficult to establish for plate-based assay, in a reproductible and stable manner. Another advantage is the ability to use cocktails and / or individual page and the possibility to be used with therapeutic and non-therapeutic bacteriophages. The method of the present invention qualifies the clinical microbiology laboratories with a rapid, straightforward methodology which provides, from a bacterial isolate, a result in approximately 5 hours a reduction of one to two days work compared to the gold standard method. Abbreviations and Definitions In the scope of the present invention, the following words and expressions mean: ESKAPE group: an acronym comprising the scientific names of six highly virulent and antibiotic-resistant bacterial pathogens including: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., either Gram- positive or Gram-negative bacteria. Phage or bacteriophage cocktail: a composition comprising a mixture of more than one strain of phages or bacteriophages. Phage or bacteriophage cocktail of document WO2013 / 141730 and WO 2018 / 106135: phage cocktail comprising a mixture of at least two of the phages as disclosed as inventive in the documents WO2013 / 141730 in examples 5 and 6, and in WO 2018 / 106135. WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt. Description of the Invention The present invention relates to a method for determining the susceptibility of bacteria to bacteriophages, where the susceptibility is measured by the cell death of the target bacteria. The method of the invention comprises the preparation of a bacterial suspension with a target bacterial isolate, diluting said suspension in a liquid enrichment culture medium and further treating the resulting liquid culture with individual phage suspensions or with a mixture of several phages, also known as page cocktails and then adding WST-8 and a colouring reagent to the final culture. The method for determining the susceptibility of bacteria to bacteriophages comprises the following steps: a) providing a bacterial suspension of the target bacteria in a concentration of 1.0 x 108up to 1.0 x 1010cfu / mL, b) providing an individual phage suspension or a phage cocktail suspension comprising isolated strains of phages, each of said strains having a genome that comprises a nucleic acid sequence selected from the group of consisting of SEQ ID NO: l (F44 / 10), SEQ ID NO:2 (F125 / 10), SEQ ID NO:3 (F770 / 05), SEQ ID NO:4 (F510 / 08), and SEQ ID NO:5 (F1245 / 05), SEQ ID NO: 6 (F99 / 10), SEQ ID NO: 7 (F27 / 12), SEQ ID NO: 8 (F95 / 13), SEQ ID NO: 9 (F391 / 08), SEQ ID NO: 10 (F17 / 19), SEQ ID NO: 11 (F 58 / 19), SEQ ID NO: 12 (F 19 / 15), SEQ ID NO: 13 (F 72 / 21), SEQ ID NO: 14 (F142 / 21) or a variant thereof, said variant having at least 95% sequence identity to said nucleic acid sequence, wherein said suspension has a concentration of 1.0 x 109to 1.0 x 1010pfu / mL,c) contacting 0.5 to 1.5 mL of the bacterial suspension of step a) with 50 to 200 µL of a phage suspension of the step b), d) adding 10 to 200 µL of the colouring reagent that is previously prepared by adding 900 µL of WST-8 solution to 100 µL of electron donor (as indicated in Dojindo kit) to the suspension of step c). The preparation of a bacterial suspension with a target bacterial isolate can be made with any bacteria species or strains, hereinafter referred only as “target bacteria”. In an embodiment, the target bacterial isolates can be obtained from clinical samples of a patient, from laboratory cultures or from bacterial bank cultures. In another embodiment target bacterial isolates comprise pathogenic bacterium for mammals, more preferably is a pathogenic bacterium for humans. In a more preferred embodiment, the target bacteria belong to the ESKAPE group including Gram-positive and Gram-negative bacteria, namely Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter spp and E. coli. Suitable aqueous salt solutions in the scope of the present invention include NaCl or PBS in a concentration of 0,8% a 0,98%. In the scope of the invention bacterial suspensions comprise a bacterial concentration of 1.0 x 108to 1.0 x 1010cfu / mL, preferably 1.0 x 108to 1.0 x 109cfu / mL, more preferably of 1.0 x 108to 2.0 x 108cfu / mL, even more preferably of 1.5 x 108to 1.75 x 108cfu / mL. Suitable bacterial suspensions can be obtained by placing an isolate of the target bacterium in a concentration as described previously in an aqueous salt solution of NaCl or PBS in a concentration of 0,8% a 0,98%. In a preferred aspect of the invention a primary bacterial suspension having a concentration as described previously is prepared with an aqueous solution of NaCl 0,9% and the turbidity is adjusted to McFarland 0.5 to 2. This bacterial suspension is advantageously diluted from ½ to ¼ in a liquid medium for culture enrichment and further incubated for a period of 30 minutes to 3 hours, at a temperature of 30 to 37ºC, under agitation conditions. The enrichment of the bacterial suspension aims to bring the target bacteria into a metabolic state that is favourable to infection by the phage. Phage infection requires metabolically active bacteria, and this enrichment step brings the bacteria to this state and increases the number of bacteria present in the suspension. This enrichment of the medium is carried out in a common manner, i.e. well known to the experts in the field. Nutrient broth is used as liquid culture medium for enrichment of non-fastidious microorganism cultures. More rich media commonly used in bacteria cultures are Luria-Bertani (LB) broth and Tryptic soy broth (TSB). Specially enriched media can be prepared for more demanding microorganisms by adding extra substances in the basal medium, ex. for the growth of fastidious microorganisms with additional nutritional requirements that can be provided e.g. by brain heart infusion broth, Super Optimal Broth. In the scope of the present invention, phage cocktails of document WO2013141730 comprise a mixture of at least two of the phages as disclosed as inventive in the document WO2013 / 141730, namely isolated strains of phages, each of said strains having a genome that comprises a nucleic acid sequence selected from the group of consisting of SEQ ID NO: l (F44 / 10) of WO2013 / 141730 which corresponds to SEQ ID NO: 1 in the present invention, SEQ ID NO:2 (F125 / 10) of WO2013 / 141730 which corresponds to SEQ ID NO: 2 in the present invention, SEQ ID NO:3 (F770 / 05) of WO2013 / 141730 in the present invention, SEQ ID NO:4 (F510 / 08) of WO2013 / 141730 in the present invention, and SEQ ID NO:5 (F1245 / 05) of WO2013 / 141730 in the present invention, or a variant thereof, said variant having at least 95% sequence identity to said nucleic acid sequence and showing antibacterial activity against at least one of Staphylococcus aureus, Pseudomonas aeruginosa, and / or Acinetobacter baumannii. In the scope of the present invention, phage cocktails of document comprise a mixture of at least two of the bacteriophages as disclosed as inventive in the documents WO2018 / 106135 and WO2021 / 125988, namely isolated strains of phages, each of said strains having a genome that comprises a nucleic acid sequence selected from the group of consisting SEQ ID NO: l (F99 / 10) of WO2018 / 106135 which corresponds to SEQ ID NO: 6 in the present invention SEQ ID NO:3 (F27 / 12) of WO2018 / 106135 which corresponds to SEQ ID NO: 7 in the present invention, SEQ ID NO:5 (F95 / 13) of WO2018 / 106135 which corresponds to SEQ ID NO: 8 in the present invention, SEQ ID NO:6 (F391 / 08) of WO2018 / 106135 which corresponds to SEQ ID NO: 9 in the present invention , SEQ ID NO: 2 (F 17 / 19) of WO2021 / 125988 which corresponds to SEQ ID NO: 10 in the present invention, SEQ ID NO: 3 (F58 / 19) of WO2021 / 125988 which corresponds to SEQ ID NO: 11 in the present invention or variants thereof, said variants having at least 90% sequence identity to said nucleic acid sequence and showing antibacterial activity against Pseudomonas aeruginosa and / or Klebsiella pneumoniae. Preferred phage cocktails comprise: 1. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:l and SEQ ID NO:2, or said variant thereof. 2. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:l and SEQ ID NO:2 and a further third bacteriophage strain having a genome that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or said variant thereof. 3. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:l and SEQ ID NO:2, further comprising at least a third and a fourth bacteriophage strain, each having a genome that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:4, and SEQ ID NO:5, or said variant thereof. 4. At least two bacteriophage strains are the strains having genomes that comprise the nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO:4 or said variant thereof. 5. At least two bacteriophage strains are the strains having genomes that comprise the nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO:4 and further comprising further comprising at least a third bacteriophage strain, said third strain having a genome that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:5, or said variant thereof. 6. One of the two different isolated strains of bacteriophages comprises a nucleic acid sequence SEQ ID NO:5 and further comprises at least one isolated strain of a phage having genomes that comprise the nucleic acid sequences SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a variant thereof. 7. At least two different purified bacteriophages, having a genome that comprises a nucleic acid sequence of SEQ ID NO:6 (F99 / 10), SEQ ID NO:7 (F27 / 12), SEQ ID NO:8 (F95 / 13) andSEQ ID NO:9 (F391 / 08), or variants thereof. 8. A purified bacteriophage comprising a nucleic acid having a nucleotide sequence of SEQ ID NO: 6 (F99 / 10), SEQ ID NO:7 (F27 / 12), SEQ ID NO: 8 (F95 / 13), or variant thereof and having antibacterial activity against Pseudomonas aeruginosa or Klebsiella pneumoniae. 9. A purified bacteriophage having a genome which comprises at least 85% sequence identity to at least one nucleotide sequence selected from the group of SEQ ID NOs: 10 (F17 / 19) and 11 (F58 / 19) and having antibacterial activity against one or more strains of Klebsiella pneumoniae. The principles of reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. The application of WST-8 can be advantageously performed for enhancing the qualitative measurement of the phage killing activity. Therefore, in the scope of the present invention tetrazolium salt WST-8 is used for the qualitative determination of phage susceptibility. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] is one of numerous water-soluble tetrazolium salts (WST) used in studies of cell metabolic activity, cell viability, and cytotoxicity. In the presence of an electron mediator, such as 1-methoxy-5- methylphenazium methyl sulphate (a photochemically stable electron mediator between NAD(P)H and tetrazolium salts), WST is rapidly reduced to produce a formazan dye, as shown in the Scheme 1 below: This artificial chromogenic molecule produces a colour range from dark blue to dark red to orange, when in presence of a photochemically stable electron mediator between NAD(P)H and tetrazolium salts depending on the original tetrazolium salt used as the substrate for the reaction. This colour property allows the determination by monitoring the absorbance in the 430 to 500 nm range. Since the absorbance of formazan is proportional to the concentration of NAD(P)H, the WST-based assays are useful for various qualitative and quantitative assays. The results readout is colorimetric enabling a rapid visualization of the results. The method of the present invention can be carried out in microtubes or 96-well plates, preferably is performed in 96- well plates, so that it is possible to have more assays read at the same time in a very known and reliable way. For better assessment and comparative purposes of the efficiency of bacterial phage infection, a negative and a positive control were established. The positive control can be made using the culture medium, per se. The negative control is SDS / EDTA with concentrations between 3,46 mM to 346 mM (v / v) for SDS and 0,10 to 0,50 M (v / v) for EDTA. Therefore, the incubation of the bacterial cells with the SDS / EDTA solution provides a negative control, wherein the cells are dead in this condition. The result of the incubation of the bacterial cells with the phages establishes the level of phage infection and thus if they can kill or not the bacteria present in the suspension. Consequently, the bacteria susceptibility to the phages can be indirectly assessed in an easy and fast manner. When phages kill the bacteria the result of the incubation will be presented with the same colour as of the negative control, whereas when bacteria are resistant to phage action the result of the incubation is displayed with the same colour as of the positive control. In practice, and according to the present invention, cultures with bacteria and culture medium are orange indicating that the bacteria are alive; in opposition, bacteria with SDS / EDTA are yellow indicating that the bacteria are dead; when suspensions with phage and bacteria are yellow, thus dead bacteria, it is clearly shown that the target bacteria present a phage susceptibility profile. The use of in-method controls makes it easier to read the results by comparison, thus making it more robust and reliable. The incubation of the bacterial cells with medium is a positive control of cell viability, i.e. cells are alive in this condition. Aiming to obtain homogeneous and reliable readings, whenever the method is carried out in well-plates, the visible absorbance is measured at 440 nm (Abs440). The susceptibility rate (%) for a given target bacteria is calculated with the following equation: ^^^^^^^^^^^^^^ ^^^^ ^%^ = 100 100& wherein Abs440 Phage cocktail corresponds to the wells with bacterial suspension incubated with the phage cocktail disclosed in document WO2013 / 141730, Abs440 Control corresponds to the wells with bacterial suspension incubated with TSB and Abs440Blank is relating to wells TSB. The outcome of susceptibility rate was split in three categories depending on the percentage: bellow 20% is not susceptible; between 20 and 70% is partially susceptible; above 70% is susceptible. Further, the interpretation of the results is orange means cell growth means that bacteria are not susceptible to phage cocktail or individual phage, yellowish indicates that cell death occurs, and the bacteria is susceptible to phage cocktail. The method of the invention is suitable for use with pathogenic bacteria belonging to high-risk clones spread world-wide, that cause severe infections, difficult to treat or even with no alternative treatment to antibiotics and for which phage therapy is a potential option. One example is bacteria belonging to the ESKAPE group, which comprises Gram-positive and Gram-negative bacteria that evade or escape the action of commonly used antibiotics. This group includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp, and Escherichia coli. The World Health Organization (WHO) and the Centres for Disease Control and Prevention (CDC) listed the highest priority multidrug resistant pathogens (CDC 2019, WHO 2019), in which the urgent threats include carbapenem-resistant Acinetobacter species and carbapenem-resistant Enterobacteriaceae, comprising K. pneumoniae and E. coli. The method of the present invention can be used in laboratory settings to evaluate bacteriophage susceptibility of bacteriophages to be used as diagnostic tools (Grant 2021; Zelcbuch et al 2021) or also as nanocarriers targeting cancer cells (Foglizzo and Marchiò, 2021) and is presented in Figure 2. The steps presented are performed for a bacterial isolate to be tested. EXAMPLES Example 1 - Method for determining the susceptibility of Staphylococcus aureus The titter of each phage comprised in phage cocktail disclosed in document WO2013 / 141730, namely the bacteriophage strains F44 / 10 and F125 / 10, having a genome that comprises the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO:2 respectively, infecting Staphylococcus aureus), expressed as plaque forming units (pfu) per mL, was determined using the double-layer agar technique, using the protocol described in the article Kropinski AM, Mazzocco A, Waddell TE, et al. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol. 2009; 501:69-76. https: / / doi.org / 10.1007 / 978-1- 60327-164-6_7. The conditions for bacterial suspension enrichment, adsorption and phage infection were optimized, as described below. Based on these conditions we optimized the colouring reagent concentration and time of incubation to automatize the process in the microplate reader. After a sequence of optimizations, the final conditions were obtained to assess the effectiveness of the cocktail disclosed in document WO2013 / 141730 for S. aureus. A bacterial suspension was prepared by transferring 1 mL of NaCl 0,9 % to a 10 mL tube and with a disposable loop collect some material from the S. aureus bacterial isolate culture plate to test, was inoculated in the tube and vortex. The turbidity was adjusted to McFarland 0.5. Five mL of tryptic soy broth (TSB) was added to a new 10 mL tube, and 500 µL of the bacterial cell suspension were transferred. The tube was incubated at 37ºC and 200 rpm during 1 h. The enriched culture (900 µL) was transferred to three new 10 mL tubes marked with TSB, SDS / EDTA (Solution SDS 173 mM; EDTA 0.25M) or phage cocktail. Additionally, 100µL of TSB, SDS / EDTA or phage cocktail were added to the respective tubes. The tubes were incubated at 37ºC with no agitation for 10 minutes, following incubation with agitation at 37ºC with 200 rpm for 1 h. A volume of 190 µL of each culture was transferred to each well of a 96-well plate and 10 µL of the colouring reagent from Dojindo Microbial Viability Assay Kit – WST # 277M439-10 was prepared according with manufacturing instructions and added to the plate. The manufacturer’s instructions were modified to optimize the conditions to evaluate bacterial cell viability after the infection of phage cocktail. The colouring reagent was previously prepared with a dilution of 1:30 in sterile water and a new dilution of 1:10 in WST solution. The plate was incubated at 37ºC with no agitation for 30 minutes. For visual reading, the appearance of orange to yellow colour (Y) in the wells was observed. Atribuir valores às cores para se conseguir ler as tabelas? To validate the assay the wells with TSB must have the orange colour (O) that corresponds to alive bacteria, and the wells with SDS / EDTA must have the yellow colour (Y) that corresponds to dead bacteria. The phage susceptibility result of the bacterial isolate is given by a yellow colour (Y), as shown in Figure 3. For a standardized reading, the visible absorbance was measured at 440 nm (Abs440) using a Spark™ multimode microplate reader (TECAN, Spain). The susceptibility rate (%) for a given bacteria was calculated with the following equation: ^^^^ ^^^^^^^^^ℎ^ ^ !"^#^^^^ − ^^^^ ^^^^^^^^$^^^#^^^^^^^^^^^^^^ ^^^^ ^%^ = 100 − ^^^^^ ^^^^^^^^!"^^^"^ − ^^^^ ^^^^^^^^$^^^#× 100&Abs440Phage cocktail corresponds to the wells with bacterial suspension incubated with the phage cocktail disclosed in document WO2013 / 141730, Abs440 Control corresponds to the wells with bacterial suspension incubated with TSB and Abs440Blank corresponds to wells with TSB. The outcome of susceptibility rate was split in three categories depending on the percentage: bellow 20%: not susceptible; between 20 and 70%: partially susceptible; above 70%: susceptible. Results and discussion: Phages F44 / 10 and F125 / 10, corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 respectively of the present invention, comprised in the phage cocktail disclosed in the document WO2013 / 141730 had their titter determined by plaque assay (as shown in Table 1) to be considered as baseline for the method of the invention optimization. Table 1 – Shows the S. aureus phages concentration in the pentaphage cocktail disclosed in document WO2013 / 141730 used in the optimization of the method of the invention. Phage Component Concentration S. aureus bacteriophage F44 / 10 2.82 x108pfu / mL S. aureus bacteriophage F125 / 10 1.1x107pfu / mL Seven different S. aureus strains with distinct infection profiles tested by plaque assay (as shown in Table 2), were also tested for phage susceptibility by the method of the invention. Table 2 – Shows the individual profiles of phage infection, by plaque assay, for seven S. aureus tested strains. Sequence Strain Phage infection type STA 64 / 18 No infection ST5 F44 / 10 STA 67 / 18 USA100 (PFGE) F125 / 10 F44 / 10 STA 71 / 18 ST36 F125 / 10 STA 72 / 18 No infection ST59 STA 75 / 18 F125 / 10 ST5 STA 185 / 11 F44 / 10 - STA 108 / 15F125 / 10- Figure 4 shows the layout of the microplate and the visual result for the seven S. aureus strains used for the optimization of the method of the invention. The assessment of colour change by a measurement of absorbance at 440 nm overcomes any subjectivity and allows the automatic reading of the colourimetric reaction. To improve the readings of the absorbance in the present method, it was necessary to dilute more the electron donor, than what is suggested by the manufacturer of the WST-8 kit, because the bacteria control wells tended to be saturated (Abs440 > 4) without displaying a numerical result.The absorbance at 440 nm was measured (raw data are shown in Figure 5) and the susceptibility rate (%) was calculated as previously described. Furthermore, the timings established for the culture enrichment and phage treatment were optimized according to kinetic parameters associated with bacterial growth and phage infection, as well known by the person skilled in the art. Three independent experiments were performed based on the optimized protocol. The overall results obtained for S. aureus strains are presented in Table 3. Table 3 – Shows the susceptibility rate (%) of the pentaphage cocktail disclosed in document WO2013 / 141730 for S. aureus strains. Susceptibility rate (%) STA 108 / 15 93.57 96.28 97.98 95.94 1.82 Susceptible Legend: Susceptibility rate: < 20%, not susceptible; between 20 and 70%, partially susceptible; > 70%, susceptible. The results agreed with the infection profile shown in Table 2. It is important to highlight that the strain infection profile was defined by double-layer plaque assay, where each phage was tested individually, and the assay evaluated the productive phage infection that results in both cell lysis and release of phage progeny as described in Dennehy, J.J., Abedon, S.T. (2020). Phage Infection and Lysis. In: Harper, D.R., Abedon, S.T., Burrowes, B.H., McConville, M.L. (eds) Bacteriophages. Springer, Cham. https: / / doi.org / 10.1007 / 978- 3-319-40598-8_53-1. In fact, it was observed a partial susceptibility of the S. aureus strain (72 / 18) by phage cocktail disclosed in WO2013 / 141730 although this strain was not infected by any of the individual phages, F44 / 10 or F125 / 10, corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 respectively of the present invention. This might be explained by a greater action of the lytic enzymes involved in the initial stages of phage infection (Fernandes and São-José, 2018), and / or by the existence of abortive infection systems that results in non-productive phage infection. These bacterial anti-phage systems are not detectable by the plaque assay, interfering with phage replication at different stages after viral genome entry, working as suicide ”altruistic” systems aimed at preventing viral dissemination, thus protecting the cell population instead of the infected cell Also, according to plaque assay, it was expected infection of S. aureus strain by phage F44 / 10, however no susceptibility was observed (18.3%). F44 / 10 was present in the suspension with a titter of 2x108pfu / mL, nonetheless this amount of phage may not be sufficient to destroy all the cells, for example due to a low infection efficiency because of, for instance, differential receptor-phage binding affinity Molina F, Menor- Flores M, Fernández L, et al. Systematic analysis of putative phage-phage interactions on minimum-sized phage cocktails. Sci Rep. 2022; 12:2458. https: / / doi.org / 10.1038 / s41598-022-06422- 1. The results were consistent and in agreement with the infection profile determined by plaque assay, still these studies aimed at the optimization of the method of the invention conditions intended to demonstrate cell death, not phage enumeration, since both methodologies rely on different assumptions: plaque assay on phage replication and the invention method on cell viability. Example 2 Method for determining the susceptibility of Acinetobacter baumannii The method for determining the susceptibility of A. baumannii, followed the previously for S. aureus with a special focus on the optimization of the bacterial growth conditions considering this bacterium (Acinetobacter baumannii). Seven different strains as presented in Table 2, already tested individually with F1245 / 05, corresponding to SEQ ID NO: 5 of the present invention disclosed in document WO2013 / 141730 were used. Following the same progress workflow previously used for S. aureus, the final conditions to assess the effectiveness of the phage cocktail disclosed in document WO2013 / 141730 for A. baumannii were obtained. A bacterial suspension was prepared by transferring 1 mL of NaCl 0,9 % to a 10 mL tube and with a disposable loop collect some material from the A. baumannii bacterial isolate culture plate to test, was inoculated in the tube and vortex. The turbidity was adjusted to McFarland 0.5. Five mL of tryptic soy broth (TSB) was added to a new 10 mL tube, and 500 µL of the bacterial cell suspension were transferred. The tube was incubated at 37ºC and 200 rpm for 40 minutes. The enriched culture (900 µL) was transferred to three new 10 mL tubes marked with TSB, SDS / EDTA (Solution SDS 5%; EDTA 0.25M) or phage cocktail. Additionally, 100µL of TSB, SDS / EDTA or phage cocktail were added to the respective tubes. The tubes were incubated at 37ºC with no agitation for 10 minutes, following incubation with agitation at 37ºC with 200 rpm for 1 h. A volume of 190 µL of each culture was transferred to each well of a 96-well plate and 10 µL of the colouring reagent from Dojindo Microbial Viability Assay Kit – WST # 277M439-10 was prepared according with manufacturing instructions and added to the plate. The plate was incubated at 37 ºC with no agitation for 30 minutes. For visual read out, the appearance of orange to yellow colour in the wells was observed. To validate the assay the wells with TSB must have the orange colour that corresponds to alive bacteria, and the wells with SDS / EDTA must have the yellow colour corresponding to dead bacteria. A phage susceptibility result of the bacterial isolate is given by a yellow colour as shown in Figure 3. For a standardized reading, the visible absorbance was measured at 440 nm (Abs440) using a Spark™ multimode microplate reader (TECAN, Spain). The susceptibility rate (%) for a given bacteria was calculated with the following equation: ^^^^^^^^^^^^^^ ^^^^^^^^ ^^^ ^ℎ^ ^ !"^#^^^^^%^= 100 − ^^^^^^ − ^^^^ ^^^^^^^^$^^^#^^^^ ^^^ !"^^^"^ − ^^^^ ^^^ $^^^× 100& ^^^^^ ^^^^^ #In which the Abs440 phage cocktail corresponds to the wells with bacterial suspension incubated with the phage cocktail of WO2013 / 141730, Abs440 Control corresponds to the wells with bacterial suspension incubated with TSB and Abs440 Blank corresponds to wells with TSB. The outcome of susceptibility rate was split in three categories depending on the percentage: bellow 20%, not susceptible; between 20 and 70%, partially susceptible; above 70%, susceptible. Results and discussion: Seven different A. baumannii strains with distinct infection profiles, were evaluated for phage susceptibility with the method of the invention. Table 4 shows the previous infection profiles of each strain as well as their sequence types (ST) profiles. Table 4 – Shows the individual profiles of phage infection, by plaque assay, for seven A. baumannii tested strains. Sequence Strain Phage infection type ACB 1305 / 05 F1245 / 05 - ACB 2023 / 05 F1245 / 05 - ACB 1016 / 06 F1245 / 05 - ACB 142 / 13 No infection ST218 ACB 66 / 15 F1245 / 05 ST350 ACB 51 / 17 No infectionST106ACB 54 / 17 No infectionST945Following the example of S. aureus, a similar microplate layout was replicated, as shown in Figure 6, for the seven A. baumannii strains. The overall results obtained for three independent experiments with A. baumannii strains are presented in Table 5 and example of visual result are in Figure 6. The results were in accordance with the infection profile shown in Table 4. Increased susceptibility rates, with values above 90%, were observed for the strains susceptible to phage action, demonstrating the killing capability of phage F1245 / 05. The conditions to evaluate the efficacy of TP-102 for A. baumannii were achieved. It should be noted that in these trials was tested the effect of only one phage from the set of five comprised in phage cocktail of WO2013 / 141730 (F44 / 10 and F125 / 10 against S. aureus; F770 / 05 and F510 / 08 against Pseudomonas aeruginosa and F1245 / 05 against A. baumannii). This means that possible interactions between phages (Molina et al, 2022) that could affect their action are not likely to happen with A. baumannii in this situation. Curiously, when F1245 / 05 could not infect a strain, the values of Abs440nm were slightly higher in wells with phage cocktail when compared to bacterial control wells. In this circumstance, the susceptibility rate was negative, as it happened for ACB 142 / 13, ACB 51 / 17 and ACB 54 / 17 (Figure 6), being susceptibility rate considered equally zero (no susceptibility). Table 5 – Shows the susceptibility rate (%) of the pentaphage cocktail disclosed in patent application WO2013 / 141730 for A. baumannii strains. Susceptibility rate (%) Legend: Susceptibility rate: < 20%, not susceptible; between 20 and 70%, partially susceptible; > 70%, susceptible. Example 3 Method for determining the susceptibility of Pseudomonas aeruginosa The method for determining the susceptibility of P. aeruginosa, was based in the previously improved procedures for S. aureus and A. baumannii with a special focus on the optimization of the bacterial growth conditions considering this bacterium (Pseudomonas aeruginosa). Similar former approaches were used, resulting in the final conditions to assess the effectiveness of phage cocktail of WO2013 / 141730 (phage cocktail composed of two phages against S. aureus, namely F44 / 10 and F125 / 10, corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention, respectively; two phages against P. aeruginosa, namely F510 / 08 and F770 / 05, corresponding to SEQ ID NO: 4 and SEQ ID NO: 3 of the present invention, respectively and one phage against A. baumannii, namely F1245 / 05 corresponding to SEQ ID NO: 5 of the present invention) for P. aeruginosa. Five different strains, already tested individually with phage cocktail of WO2013 / 141730 by plaque assay were employed. A bacterial suspension was prepared by transferring 1 mL of NaCl 0,9 % to a 10 mL tube and with a disposable loop collect some material from the P. aeruginosa bacterial isolate culture plate to test, was inoculated in the tube and vortex. The turbidity was adjusted to McFarland 0.5. Five mL of tryptic soy broth (TSB) was added to a new 10 mL tube, and 500 µL of the bacterial cell suspension were transferred. The tube was incubated at 37ºC and 200 rpm for 2 h. The enriched culture (900 µL) was transferred to three new 10 mL tubes marked with TSB, SDS / EDTA (Solution SDS 173 mM; EDTA 0.25M) or phage cocktail. Additionally, 100 µL of TSB, SDS / EDTA or phage cocktail were added to the respective tubes. The tubes were incubated at 37ºC with no agitation for 10 minutes, following incubation with agitation at 37ºC with 200 rpm for 1 h. A volume of 190 µL of each culture was transferred to each well of a 96-well plate and 10 µL of the colouring reagent from Dojindo Microbial Viability Assay Kit – WST # 277M439-10 was prepared according with manufacturing instructions and added to the plate. The plate was incubated at 37ºC with no agitation for 2 h. For visual assessment the appearance of orange to yellow colour in the wells was observed. To validate the assay the wells with TSB must have the orange colour that corresponds to alive bacteria, and the wells with SDS / EDTA must have the yellow colour corresponding to dead bacteria. A phage susceptibility result of the bacterial isolate is given by a yellow colour (Figure 3). For a standardized reading, the visible absorbance was measured at 450 nm (Abs450) using a Spark™ multimode microplate reader (TECAN, Spain). The susceptibility rate (%) for a given bacteria was calculated from the following equation: ^^^^ ^^^ ^ℎ^ ^ !"^#^^^^ % = 100 − ^ ^^^^^ ^^^ − ^^^^ ^^^ $^^^#^^^^^^^^^^^^^^ ^ ^ ^ ^^^^^^^^^ ^^^^^^^^!"^^^"^ − ^^^^ ^^^^^^^^$^^^#× 100&In which the Abs450 Phage cocktail corresponds to the wells with bacterial suspension incubated with the phage cocktail disclosed in the document WO2013 / 141730, Abs450 Control corresponds to the wells with bacterial suspension incubated with TSB and Abs450 Blank corresponds to wells with TSB. The outcome of susceptibility rate was split in three categories depending on the percentage: bellow 20%, not susceptible; between 20 and 70%, partially susceptible; above 70%, susceptible. Results and discussion: Table 6 shows the previous infection profiles of each of the five P. aeruginosa strains evaluated by plaque assay. The overall results of the method of the invention for these strains, obtained with two independent experiments, are shown in Table 7. Table 6 – Shows the individual profiles of phage infection, by plaque assay, for five P. aeruginosa tested strains. Sequence Strain Phage infection type PSA 484 / 06 F510 / 08 - PSA 963 / 05 No infection - F770 / 05 PSA 433 / 07 ST676 F510 / 08 PSA 67 / 09 F770 / 05 - F770 / 05 PSA 637 / 06 - F510 / 08 Table 7 – Shows the susceptibility rate (%) of the pentaphage cocktail disclosed in patent application WO2013 / 141730 for P. aeruginosa strains.Susceptibility rate (%)Average 35.87 6.46 susceptible 9.23 5.25 Not susceptible 88.56 1.61 Susceptible -0.01 1.26 Not susceptible 93.49 0.00 Susceptible Legend: Susceptibility rate: < 20%, not susceptible; between 20 and 70%, partially susceptible; > 70%, susceptible. Comparing the results of plaque assay infection with susceptibility in the method of the invention, it was found that for strains that are infected by only one of the phages present in phage cocktail (F770 / 05 or F510 / 08 against Pseudomonas aeruginosa) of WO2013 / 141730, no susceptibility was obtained by the method of the invention. The P. aeruginosa 67 / 09 strain infected by F770 / 05 according plaque assay is not susceptible by the invention method and the P.aeruginosa 484 / 06 strain infected only by F510 / 08 showed partial susceptibility on the method of the invention. It should also be pointed out that the effectiveness of the phage cocktail of WO2013 / 141730 was completely observed in the strains that allowed themselves to be infected simultaneously by the two comprising phages (F770 / 05 + F510 / 08). In this case, the number of infecting phage particles available to infect these bacteria are twice as much as in the test of strains infected by only one of the phages present in the cocktail. Additionally, it was previously observed in vitro synergistic effect of these two phages against strain PSA 433 / 07 (Mendes et al, 2014), supporting the observed result (Table 7). The partial susceptibility observed for PSA 484 / 06 (35.87%) could be explained also by a low infection efficiency because of, for instance, differential receptor-phage binding affinity (Molina et al, 2022). Curiously, as happened for A. baumannii strains, negative susceptibility rates were determined, (Table 7), in which case susceptibility rate considered equally zero (no susceptibility). Overall, the results confirmed that the optimized conditions provided RSPT the capability to detect phage susceptibility in distinct P. aeruginosa strains. Example 4 - Method for determining the susceptibility of Klebsiella pneumoniae The method of the invention was also used for proof of concept for a quick analytical test for the determination of K. pneumoniae phage susceptibility. The method was carried out only with a final visual assessment of the results. A bacterial suspension was prepared by transferring 1 mL of NaCl 0,9 % to a 10 mL tube and with a disposable loop collect some material from K. pneumoniae bacterial isolate culture plate to test, was inoculated in the tube and vortex. The turbidity was adjusted to McFarland 0.5. Five mL of tryptic soy broth (TSB) was added to a new 10 mL tube, and 500 µL of the bacterial cell suspension were transferred. The tube was incubated at 37ºC and 200 rpm for 1 h. The enriched culture (900 µL) was transferred to three new 10 mL tubes marked with TSB, SDS / EDTA (Solution SDS 173 mM; EDTA 0.25M) or phage cocktail. Additionally, 100 µL of TSB, SDS / EDTA or phage cocktail were added to the respective tubes. The tubes were incubated at 37ºC with no agitation for 10 minutes, following incubation with agitation at 37ºC with 200 rpm for 1h. A volume of 190 µL of each culture was transferred to each well of a 96-well plate and 10 µL of the colouring reagent from Dojindo Microbial Viability Assay Kit – WST # 277M439-10 was prepared according with manufacturing instructions and added to the plate and added to the plate. The plate was incubated at 37ºC with no agitation for 2 h. For visual assessment, observe the appearance of orange to yellow colour in the wells. To validate the assay the wells of bacterial control with TSB must have the orange colour that corresponds to alive bacteria, and the wells with SDS / EDTA must have the yellow colour that corresponds to dead bacteria. A phage susceptibility result of the bacterial strain is reported by a yellow colour (Figure 3). Results and discussion: Seven distinct strains of K. pneumoniae were used in one assay carried out according to the layout presented at Figure 9. From the image of the plate in Figure 9 it is possible to observe that strains KLE 237 / 14, KLE 30 / 17, KLE 43 / 17 and KLE 57 / 17 wells presented yellow colour (analogous to SDS / EDTA control wells) demonstrating the death of the cells in the presence of the K. pneumoniae phages composing cocktail disclosed in WO2021125988 (F391 / 08, F17 / 19 and F58 / 19), corresponding to SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 of the present invention, respectively therefore these strains were considered susceptible to the cocktail action. Strain KLE 127 / 18 triplicate wells presented an orange colour less concentrated than the colour presented by the bacterial control wells and could be considered as having a partial susceptibility to the cocktail. The conditions for the final establishment of the methodology for this bacterium still need improvements, nevertheless it was here shown the potential for the application of the invention method. Example 5 - Method for determining the susceptibility of Pseudomonas aeruginosa P. aeruginosa strains were tested with the method of the invention with the phage cocktail disclosed in the document WO2018 / 106135 (F99 / 10, F27 / 12, and F95 / 13) corresponding to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 of the present invention, respectively. RPST was performed only with a final visual assessment of the results. A bacterial suspension was prepared by transferring 1 mL of NaCl 0,9 % to a 10 mL tube and with a disposable loop collect some material from P. aeruginosa bacterial isolate culture plate to test, was inoculated in the tube and vortex. The turbidity was adjusted to McFarland 0.5. Five mL of tryptic soy broth (TSB) was added to a new 10 mL tube, and 500 µL of the bacterial cell suspension were transferred. The tube was incubated at 37ºC and 200 rpm for 2h. The enriched culture (900 µL) was transferred to three new 10 mL tubes marked with TSB, SDS / EDTA (Solution SDS 173 mM; EDTA 0.25M) or phage cocktail. Additionally, 100 µL of TSB, SDS / EDTA or phage cocktail were added to the respective tubes. The tubes were incubated at 37ºC with no agitation for 10 minutes, following incubation with agitation at 37 ºC with 200 rpm for 1 h. A volume of 190 µL of each culture was transferred to each well of a 96-well plate and 10 µL of the colouring reagent from Dojindo Microbial Viability Assay Kit – WST # 277M439-10 was prepared according with manufacturing instructions and added to the plate and added to the plate. The plate was incubated at 37 ºC with no agitation for 2 h. For visual result, observe the appearance of orange to yellow colour in the wells. To validate the assay the wells with TSB must have the orange colour that corresponds to alive bacteria, and the wells with SDS / EDTA must have the yellow colour that corresponds to dead bacteria. A phage susceptibility result of the bacterial isolate is given by a yellow colour (as shown in Figure 3). Results and discussion: Seven distinct strains of P. aeruginosa were used in one assay carried out according to the layout presented in Figure 10. An image of the plate is shown in Figure 10 and it was possible to observe that strains 863 / 05 and 67 / 19 wells presented orange colour (analogous to bacterial control wells) demonstrating the viability of the cells in the presence of the P. aeruginosa phage cocktail, therefore these strains were considered not susceptible to the cocktail action. All the other strains were susceptible, supported by the fact that the respective wells present a yellow colour (analogous to SDS / EDTA control wells). The method for determining the susceptibility of bacteria to phages of the invention demonstrated the action of the phage cocktail of document WO2018 / 106135 against P. aeruginosa strains that are responsible for respiratory infections. Conclusions The conditions for assessing the susceptibility of various bacteriophage cocktails against Gram-negative and Gram- positive bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, by means of the method of the present invention, has proven to be suitable, rapid, easy to perform and accurate for a quick choice of the most appropriate phage therapy as a potential option when dealing with a serious infection that is difficult to treat or even as an alternative treatment with antibiotics. Plaque assay is the standard technique to determine the susceptibility of a bacterial isolate to a phage suspension. However, as previously referred, is a very time-consuming technique, it is not the most suitable for a rapid response, for example during a period of screening patients in a clinical trial. Thus, to fulfil the need for an expeditious technique, the procedure presented above was established. With this method, in four to five hours it is possible to determine whether a bacterial isolate is killed by phages. With this innovative assay is not possible to enumerate phage plaques, and that is not its purpose. Phage enumeration should continue to be done by plaque assay or any other appropriate technique, such as cytometry or even real time PCR. Although these methodologies nowadays are available in clinical microbiology laboratories, their utilization depends on specialized operators and is quite expensive. The described technique aims to rapidly evaluate if a certain phage suspension is active or not in a specific bacterial isolate, and that may help in the decision for example to recruit a patient into a clinical study, or in the suitability of that suspension to treat a given patient in compassionate use. Furthermore, this method can be very helpful, during an early stage of the assays, throughout the selection process of new potential therapeutic phages. Isolation rounds of new phages usually generate hundreds of phage plaques that need to be selected. This procedure enables the evaluation of multiple phages in diverse bacterial isolate panels as a high throughput system and has the possibility of being fully automatized. It should be noted that the results obtained with this technique are not comparable with the results of the plaque assay in terms of phage concentration. In the proposed methodology the outcome is cell viability, that is, if the phages can kill the cells, while the plaque assay determines the ability of the phages to perform a productive infection, that is, if they are capable of replicating in the bacteria and lyse it. According to what is known and described in the literature, bacteria have several mechanisms of resistance to phage infection that may impact the beginning of the phage life cycle but may also impact late stages. In these cases, the phages can infect bacteria, kill them and yet, due to these defence mechanisms of the bacteria, do not produce progeny and consequently do not generate visible phage plaques. These infections are not detected by plaque assay. Therefore, care must be taken not to confuse the results, as well as the applicability of the plaque assay technique with the suitability of this innovative methodology. Each has a different purpose. Currently, with the increasing number of treatments with phage therapy, as well as the increase in clinical trials to evaluate new treatments with phages, it is necessary to ensure a quick response from the clinical laboratory regarding the susceptibility to phage action. The method presented herein brings the great advantage of celerity, extremely important in treatments of severe clinical conditions. The decision of rapidly verify whether the phage therapy is appropriate or not can be the difference between life and death for a critically ill patient. In conclusion, the procedure described above is innovative and responds to a need in the current reality of the phage therapy as a valid alternative in the treatment of bacterial infections, namely those caused by multi-resistant bacteria. As used in the description, the definite and indefinite articles, in their singular form, are intended to be interpreted to include the plural forms as well, unless the context of the description explicitly indicates otherwise. THE SEQUENCE LISTING IS HEREIN PROVIDED AS ANNEX IN PAPER SUPPORT AND IN THE WIPO ST.26 FORMAT

Claims

CLAIMS 1. A method for determining the susceptibility of bacteria to bacteriophages comprising the following steps: bacterial suspension of the targetan aqueous salt solution, having said bacteriaa concentration of 1.0 x 108up to 1.0 x 1010cfu / mL, b) providing a bacterial culture of the target bacteria from step a)a liquid culture medium of Luria- Bertani (LB) broth, Tryptic soy broth (TSB)derivates thereof including the addition of compounds for improving the growth of the target bacteria, providing an individual phage suspension or a phage cocktail suspension comprising isolated strains of phages in an aqueous salt solution, wherein each of said strains has a genome that comprises a nucleic acid sequence selected from the group of SEQ ID NO: 1, corresponding to phage F44 / 10, SEQ ID NO:2, corresponding to phage F125 / 10, SEQ ID NO:3, corresponding to phage F770 / 05, SEQ ID NO:4, corresponding to phage F510 / 08, and SEQ ID NO:5, corresponding to phage F1245 / 05, SEQ ID NO: 6 corresponding to phage F99 / 10, SEQ ID NO: 7 corresponding to phage F27 / 12, SEQ ID NO: 8 F95 / 13, SEQ ID NO: 9 corresponding to phage F391 / 08, SEQ ID NO: 10 corresponding to phage F17 / 19, SEQ ID NO: 11 corresponding to phage F 58 / 19, SEQ ID NO: 12 corresponding to phage F 19 / 15, SEQ ID NO: 13 corresponding to F 72 / 21, SEQ ID NO: 14 corresponding to F142 / 21 or a variant thereof, said variant having at least 95% sequence identity to said nucleicsequence, and wherein said phage suspension has aconcentration of 1.0 x 109to 1.0 x 1010pfu / mL of each bacteriophage in that said aqueous salt solution, d) providing a positive control, wherein the target bacteria of the bacterial suspension of step a) is cultured in a liquid culture medium, e) providing a negative control, wherein the target bacteria culture of b) is incubated with solution of SDS / EDTA having the following concentrations of 3,46 mM to 346 mM (v / v) for SDS and 0,10 to 0,50 M (v / v) for EDTA, f) contacting 0.5 to 1.5 mL of the bacterial culture of the target bacteria of b) with 50 to 200 µL of a phage suspension of c), g) adding 10 to 200 µL of colouring reagent that is previously prepared by adding 900 µL of WST-8 solution to 100 µL of electron donor to the suspension or culture of f).

2. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, wherein of the target bacteria isolate in the bacterial suspension of a) is collected from a clinical sample of a patient, from laboratory cultures or from bacterial bank cultures.

3. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, wherein the target bacteria isolate of the bacterial suspension of a) is a pathogenic bacterium for mammals, more preferably is a pathogenic bacterium for humans.

4. A method for determining the susceptibility of bacteria to bacteriophages according to claim 3, wherein the targetbacteria isolate of the bacterial suspension is a bacteria belonging to the ESKAPE group including Gram-positive and Gram-negative bacteria, namely Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter spp. and E. coli.

5. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, wherein the aqueous salt solutions of the bacterial suspensions of a) are NaCl or PBS in a concentration of 0,8% a 0,98%.

6. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, wherein the bacterial suspension of a) comprises a bacterial concentration of 1.0 x 108to 1.0 x 1010cfu / mL, preferably 1.0 x 108to 1.0 x 109cfu / mL, more preferably of 1.0 x 108to 2.0 x 108cfu / mL, even more preferably of 1.5 x 108to 1.75 x 108cfu / mL in an aqueous salt solution of NaCl or PBS in a concentration of 0,8% a 0,98%.

7. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, further comprising incubating the bacterial suspension of a) wherein said suspension is first diluted from ½ to ¼ in a liquid culture medium of Luria-Bertani (LB) broth, Tryptic soy broth (TSB) or derivates thereof including the addition of compounds for improving the growth of the target bacteria followed by incubation at a temperature of 30 to 37ºC, under agitation conditions, during 30 minutes to 3 hours.

8. A method for determining the susceptibility of bacteria to bacteriophages according to claim 7, wherein the liquidculture medium is Luria-Bertani (LB) broth or Tryptic soy broth (TSB) or derivates thereof including the addition of compounds for improving the growth of the target bacteria.

9. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, wherein the phage suspension of c) comprises the following isolated strains of phages: a. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:2, or said variant thereof, b. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:2 and a further third bacteriophage strain having a genome that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or said variant thereof, c. At least two bacteriophage strains having a genome that comprises the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:2, further comprising at least a third and a fourth bacteriophage strain, each having a genome that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:4, and SEQ ID NO:5, or said variant thereof, d. At least two bacteriophage strains having genomes that comprise the nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO:4 or said variant thereof, e. At least two bacteriophage strains having genomes that comprise the nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO:4 and further comprising further comprising at least a third bacteriophage strain, said third strain having a genome that comprises a nucleicacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:5, or said variant thereof, f. One of the two different isolated strains of phages comprises a nucleic acid sequence SEQ ID NO:5 and further comprises at least one isolated strain of a phage having genomes that comprise the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a variant thereof. g. A purified bacteriophage comprising a nucleic acid having a nucleotide sequence of SEQ ID NO: 6 (F99 / 10), SEQ ID NO:7 (F27 / 12), SEQ ID NO: 8 (F95 / 13), or variant thereof and having antibacterial activity against Pseudomonas aeruginosa or Klebsiella pneumoniae. h. A purified bacteriophage having a genome which comprises at least 85% sequence identity to at least one nucleotide sequence selected from the group of SEQ ID NOs: 10 (F17 / 19) and 11 (F58 / 19) and having antibacterial activity against one or more strains of Klebsiella pneumoniae.

10. A method for determining the susceptibility of bacteria to bacteriophages according to claim 1, further comprising placing the bacterial culture of b), the positive control of d), the negative control of e) and the bacterial-phage culture of g), in individually microtubes or preferably in 96-well plates and reading the absorbance of each of the solutions at an absorbance range of 430 to 500 nm.

11. A method for determining the susceptibility of bacteria to bacteriophages according to claim 10, wherein the susceptibility of bacteria to bacteriophages is performedby reading at an absorbance range of 440nm and calculated according to the following equation: ^^^^^^^^^^^^^^ ^^^^ %^^^^ ^^^^^^^^^ℎ^ ^ !"^#^^^^ − ^^^^ ^^^^^^^^$^^^#^ ^= 100 − ^ ^^^^ ^^^ !"^^× 100& ^^^^^ ^"^ − ^^^^ ^^^^^^^^$^^^#wherein Abs440 Phage cocktail are the wells with bacterial suspension incubated with individual phage suspensions or phage cocktails, Abs440Control are the wells with bacterial suspension incubated with TSB, and Abs440 Blank are the wells only with culture liquid medium, and wherein the susceptibility rate is classified according to the resulting percentage, being: - bellow 20% = not susceptible, - between 20 and 70% = partially susceptible, and - above 70%, susceptible.

12. A method for determining the susceptibility of bacteria to bacteriophages according to claim 10, wherein the susceptibility of bacteria to bacteriophages is performed by visual reading.

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