Automatic fergogram

Abstract

The present invention also relates to a corresponding method for use therein, the system comprising: a microwell plate comprising a plurality of wells, each of the plurality of wells configured to contain one of a plurality of phages, the microwell plate configured for a sample comprising the bacteria to be dispensed into the plurality of wells, at least one reagent and / or additive configured to be added to a mixture of the sample comprising the bacteria and one of the plurality of phages, and a control unit configured to interpret and present the results of the measurement in the form of a phagegram, the measurement being provided by at least one analytical device, the at least one analytical device configured to measure the interaction of the bacteria with each of the plurality of phages.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a system for determining the susceptibility of bacteria to multiple phages and for reporting said susceptibility and possible lytic effects in the form of an automated phagegram, and to corresponding methods for use therein. [Background technology]

[0002] background The problem of antimicrobial resistance (AMR) is growing worldwide and is a major threat to human and animal health in both modern veterinary and clinical settings. Today, pathogenic bacteria resistant to virtually all commercially available antimicrobials have emerged, and very few molecules are currently in development to develop new classes of antimicrobials. In a paper by Marston et al. ("Antimicrobial Resistance", JAMA 316(11)doi:10.1001 / jama.2016.1176), factors associated with AMR have been identified. The World Health Organization (WHO) has stated that antimicrobial resistance is one of the greatest threats to global health, food security, and development, estimating that at least 700,000 people die each year from drug-resistant diseases (WHO, Joint News Release, April 2019). Although initially limited to a few regions, AMR is now a challenge for health professionals worldwide, from clinical microbiologists and infectious disease specialists to veterinarians. The current situation of patients harboring multidrug-resistant bacteria highlights not only the clinical context but also the possible consequences of such a bacterial threat.

[0003] The advantages of using bacteriophages as antimicrobial agents over antibacterial drugs have been known for some time. A major advantage of phages is their species (or strain) and genus specificity, which allows phage therapy to eliminate target organisms without disturbing the local microbiome, reducing the chance of opportunistic infections. Phages require a relatively short administration period and may be used in combination with antibacterial drugs to delay resistance. A detailed overview of the advantages and some of the limitations and challenges of using phages as antibacterial therapeutics is presented in Nikolich et al. "Bacteriophage Therapy: Developments and Directions" Antibiotics, 2020, 9, 135, doi:10.3390 / antibiotics9030135, the preface of which is incorporated herein by reference.

[0004] Nevertheless, the largely species- or strain-specific behavior of phages and their particularly narrow host range may be considered as major drawbacks compared to antibacterial agents.

[0005] To overcome these shortcomings, the current common approach for phage therapy focuses on static mixtures or "cocktails" of multiple phage components selected to infect as many of the strains infecting patients and / or animals and / or the environment as possible. However, it is believed that any static phage cocktail can only treat a portion of patients, and that such cocktails may rapidly lose relevance as bacterial populations continue to change. Further information is presented in the article by Rohde et al. ("Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains", Viruses 2018,10(4),178;https: / / doi.org / 10.3390 / v10040178).

[0006] Also, personalized phage therapy plans would assemble a larger phage library than could be assembled in a cocktail and treat the patient only with phages that have been confirmed to be active against the patient's strain, creating a custom treatment for each patient's infection. Thus, a treatment would be developed directly tailored to the patient's infection, containing one or more phages that have lytic activity against the pathogen in that infection. Pirnay et al. ("The Magistral Phage", Viruses 2018,10(2),64;doi:10.3390 / v10020064) discusses the implementation of legal and regulatory frameworks for the preparation of custom-formulated phage medicines.

[0007] A further drawback is that current diagnostic methodologies for in vitro phage susceptibility testing are geared towards academic laboratory settings rather than clinical trial settings that are widely available to patients.

[0008] Konopacki et al., “PhageScore: A simple method for comparative evaluation of bacteriophage lytic activity”, Biochemical Engineering Journal, 161, 2020, doi.org / 10.1016 / j.bej.2020.107652, reported that OD 600nm Xie et al., in “Development and Validation of a Microtiter Plate-based Assay for Determination of Bacteriophage Host Range and Virulence”, Viruses 10,189, doi:10.3390 / v10040189, describe a method for evaluating the efficiency of bacteriophages by measuring bacterial growth curves using spot assays and microtiter plate liquid assays at two initial phage concentrations. 550nm discloses a study to determine the phage host range and virulence of 15 Salmonella phages against a panel of 20 Salmonella strains over a period of at least 12 hours.

[0009] In any study, testing the susceptibility of bacterial pathogen samples to the phage library would be time consuming, requiring a commercially unfeasible amount of manual labor by qualified staff.

[0010] Indeed, currently used techniques require exponentially growing cultures derived from pure culture isolates, which typically take a day to generate, at least another day to perform, and highly trained staff, the latter of which is not feasible in a professional clinical environment.

[0011] Because infections caused by bacterial pathogens can become dangerous or life-threatening within days or even a few hours before they can be identified, there is a need for a method to rapidly determine whether a particular bacterial strain is susceptible to a potentially therapeutic bacteriophage.

[0012] WO99 / 57304 relates to an assay for identifying bacteria present in a sample and a method for treating bacterial infections. The protocol disclosed therein involves growing a bacterial isolate overnight, dispensing the sample onto a microtiter plate and incubating the plate overnight, and determining the interaction of the phage with the bacteria by measuring the extent of bacterial growth after incubation with the phage.

[0013] WO2004 / 041156 relates to a method for identifying bacteria and selecting bacteriophages with therapeutic activity. Expression of a reporter molecule indicates whether the bacteriophage is able to infect bacterial cells in a sample. Samples taken from patients are incubated for 4-12 hours.

[0014] WO2017223101 relates to a method for formulating a phage cocktail directed against a bacterial pathogen, comprising constructing a bacterial diversity set comprising various strains of the same bacterial species, followed by constructing a storage phage library and a working phage library and screening the latter for delayed bacterial growth and / or lack of growth of phage-resistant bacteria.

[0015] Fischer et al., in “Microplate-Test for the Rapid Determinaton of Bacteriophage-Susceptibility of Campylobacter Isolates-Development and Validation”, PLoS ONE 8(1), doi:10.1371 / journal.pone.0053899, present a microplate susceptibility test for rapid determination of bacteriophage susceptibility of Campylobacter isolates and compare this simplified test with the traditional agar-coated plate assay. Plates are incubated under microaerobic conditions after solidification for 18 hours. Summary of the Invention [Problem to be solved by the invention]

[0016] Thus, there is a need to be able to quickly and easily determine which bacteriophages are suitable for treating a particular bacterial infection. [Means for solving the problem]

[0017] Summary of the Invention According to one aspect of the invention there is provided a system for determining susceptibility of a bacterium to a plurality of phages, the bacterium preferably being a bacterial pathogen, the system comprising: a microwell plate comprising a plurality of wells, each of the plurality of wells configured to contain one of a plurality of phages, the microwell plate further configured such that a sample comprising the bacteria is dispensed into each of the plurality of wells; at least one reagent and / or additive configured to be added to at least one mixture of the sample comprising the bacteria and a phage of the plurality of phages; and a control unit configured to interpret and present in the form of a phagegram the results of the measurements, said measurements being provided by at least one analytical device, said at least one analytical device being configured to measure the interaction of each of said plurality of phages with said bacterial pathogen.

[0018] More specifically, a system is provided for determining the susceptibility of a bacterium to a plurality of phages, the bacterium preferably being a bacterial pathogen, the system comprising: a microwell plate having a plurality of wells each capable of containing one of a plurality of phages and into which a sample comprising said bacteria can be dispensed; at least one reagent and / or additive for adding to at least one mixture of said sample comprising said bacteria and one phage of said plurality of phages; and a control unit configured to interpret and present in the form of a phagegram the results of the measurements, said measurements being provided by at least one analytical device, said at least one analytical device being configured to measure the interaction of each of said plurality of phages with said bacterial pathogen.

[0019] The advantage of the present invention is that the susceptibility of bacteria to multiple bacteriophages can be tested using one microwell plate or defined plate array. Thus, the phages interacting with the bacteria can be identified relatively quickly. The microwell plate is preferably disposable and is provided with a set or multiple bacteriophages, preferably selected for their ability to cause cell inactivation, preferably cell lysis, and preferably phage proliferation, against a specific species or genus of bacteria or bacterial pathogens and / or their strains, or against a collection of specific species or genus of bacteria or bacterial pathogens and their strains. Advantageously, the effect of the group of selected bacteriophages on the bacteria can be detected using one experimental technique, with one analytical device and / or software package.

[0020] A further advantage is that the measurements are presented in the form of an automated phagegram, which gives a direct overview of phage-bacteria interactions and allows the identification of bacterial species and / or phages causing cell inactivation, cell lysis and preferably phage proliferation in said bacterial species.

[0021] A further advantage of the present invention is that such a phargogram can be obtained in a relatively short time, especially compared to existing methods for obtaining such information. Indeed, the time from obtaining a usable sample from a patient to obtaining the phargogram can be reduced to a few hours, or even to about 1-2 to 4 hours if the raw sample can be used.

[0022] In an embodiment of the system according to the invention, the sample is an intact sample from a patient.

[0023] The advantage of this embodiment is that the system and method described herein can be directly applied in combination with intact samples, typically obtained directly from patients, animals, infected populations, or environments. Because the phage-bacteria interaction is specific, it may not be necessary to isolate and purify the bacteria or bacterial pathogen of interest. Thus, the phages selected for their ability to interact with the bacteria of interest in a sample will not react with unrelated bacteria that may be present in the sample. It may also not be necessary to obtain a pure culture isolate of the bacterial pathogen of interest from the patient sample and / or to incubate the sample taken from the patient to grow a number of colony forming units (CFU) that may be sufficient to contact the bacteriophages. Thus, invaluable time can be saved in research to identify the source of bacterial infection and identify the phages that interact with and / or cause cell lysis of the bacteria or bacterial pathogen.

[0024] By way of comparison, protocols for obtaining antibiograms have been developed previously that involve taking a patient sample to obtain a pure culture isolate of the infectious agent. This procedure typically requires waiting 18 hours for colonies of the pure culture isolate to grow. Only then, typically within an hour, can modern antibiograms produce a viable diagnosis of the antimicrobial susceptibility of the isolate.

[0025] In an embodiment of the system according to the present invention, the micro-well plate is disposable.

[0026] As used herein, the term "disposable" refers to something designed for a single use and then discarded.

[0027] The advantage of these embodiments is that microplates can be used that are pre-prepared and sealed and ready to use at the time of use. Such microplates may be accompanied by a protocol for use, which may include a computer program product or a script for a processor to deliver the appropriate (amount of) reagents and / or additives to each well of the microplate. Such microplates may be standard plates with a number of bacteriophages capable of inducing cell lysis in a particular bacterium and its strain, and may be configured to test for the presence of the particular bacterium in a sample.

[0028] In an embodiment of the system of the present invention, the plurality of wells are further configured to contain at least one group of selected phages, each of which is selected for its ability to interact with and / or inactivate one particular bacterial species and strain.

[0029] In an embodiment of the system of the present invention, the plurality of wells is associated with a plurality of wells for containing at least a group of selected phages, each of which is selected for its ability to interact with and / or inactivate one particular bacterial species and strain.

[0030] An advantage of these embodiments is that phages targeting different strains and / or different receptor types of a particular bacterial species, typically a particular bacterial pathogen, can be combined on one microplate to characterize that species or pathogen, allowing the development of a therapy or phage cocktail that can significantly reduce the risk of bacterial pathogens acquiring phage resistance. Preferably, the selected panel comprises a combination of phages such that interactions with the maximum number of strains of a bacterial species can be examined.

[0031] In an embodiment of the system according to the present invention, the at least one reagent and / or additive comprises at least one of a primer molecule, a DNA polymerase, a nucleoside triphosphate (dNTP), a molecular beacon probe capable of emitting fluorescence, a buffer, and a qPCR amplification compatible solvent and / or an enzyme. Furthermore, the at least one analytical device is configured to measure the interaction of the bacterium with each of the plurality of phages by measuring the abundance and / or differential concentration of phage DNA by using quantitative polymerase chain reaction after incubating the bacterium with each of the plurality of phages. Thus, the at least one analytical device is preferably configured to measure the susceptibility of the bacterium to each of the plurality of phages by measuring the abundance and / or differential concentration of phage DNA by using quantitative polymerase chain reaction after incubating the bacterium with each of the plurality of phages.

[0032] The advantage of these embodiments is that the interaction between bacteria and phages can be measured more quickly compared to currently available methods. Advantageously, several samples can be measured simultaneously. A qPCR measurement of a microplate containing a mixture of bacteria and phages can be performed in less than 2 hours. Furthermore, detailed information about phage proliferation can be calculated, whereas other detection methods are often limited to information about bacterial cell death. Such information allows the quantification of the pathogenicity of certain phages compared to others.

[0033] In an embodiment of the system of the invention, the at least one reagent and / or additive comprises at least one of a luciferin / luciferase complex, a luciferin / luciferase complex-compatible solvent and / or an enzyme configured to detect bacterial cell lysis by depletion of ATP released upon phage-mediated lysis and associated fluorescein light emission, and the at least one analytical device configured to measure interaction of each of the plurality of phages with the bacterium by measuring the light emission.

[0034] The advantage of these embodiments is that the interaction between bacteria and phages can be measured more quickly compared to currently available methods. Advantageously, several samples can be measured simultaneously. Bioluminescence measurements of microplates containing a mixture of bacteria and phages using luciferase / luciferin complexes can be performed within a few minutes, typically within about 1-2 minutes, or even less than 1 minute. Furthermore, measuring bioluminescence by adding luciferase / luciferin complexes gives accurate results, the technique is sensitive and fairly easy to perform.

[0035] In an embodiment of the system according to the invention, the control unit is further configured to provide a composition of phages based on the phagegram, wherein the composition of phages interacts with a maximum number of different binding receptor types on the bacterium or bacterial species of interest. Preferably, the composition contains no more than three different phage species. Preferably, each phage species interacts with a different type of binding receptor on the bacterial species of interest.

[0036] The advantage of these embodiments is that a composition of phages can be obtained that may be suitable for the treatment of bacterial infections caused by the bacteria or bacterial pathogens. Bacteria may have one or more specific receptor motifs arranged on their surface that mediate their binding and adsorption cascades. These receptor motifs may be found on cell surface molecules, including but not limited to sugars, glycoproteins, proteins, lipids, and lipoproteins. Phages can detect one or more specific receptor motifs, and therefore may be collectively referred to as "receptor groups" depending on the specific molecules on which they are found. A further advantage of these embodiments is that a system can provide a predictive companion diagnostic for bacterial infections caused by bacterial pathogens in a sample provided to the system.

[0037] In an embodiment of the system according to the invention, the control unit is further connected to a central database to report results thereto, including the obtained phragograms.

[0038] An advantage of these embodiments is that the results of multiple phage studies performed in a variety of clinical settings can identify phage host ranges for many relevant bacteria. Thus, host range trends can be differentiated by region, time, health system, and indication, allowing disease outbreaks to be identified. These outbreaks can be identified by "phage types," i.e., populations of specific phages capable of infecting specific strains that form unique identifiable signatures that can be tracked by phagegrams.

[0039] According to one aspect of the invention, there is provided a method for determining susceptibility of a bacterium to a plurality of phages, the method comprising: providing a first composition comprising the bacteria and a first microwell plate comprising a plurality of wells, each of the plurality of wells containing one of the plurality of phages; dispensing a first sample of the first composition into at least one well of the plurality of wells; for each of said at least one well, allowing a mixture of said first sample comprising said bacteria and bacteriophage contained in said well to incubate for a first period of time; adding at least one reagent and / or additive to each mixture; transferring and introducing at least one mixture into an analytical device and measuring the bacteria-phage interactions of the at least one mixture by the analytical device; and interpreting said measurements by said analysis device and presenting them in the form of a phragmogram by a control unit.

[0040] In an embodiment of the method according to the invention, the method further comprises, prior to the step of adding at least one reagent, transferring a second sample of the mixture to a second microwell plate, the second microwell plate being adapted to be compatible with the analytical device.

[0041] An advantage of these embodiments is that the use of a second microwell plate allows for the use of a microplate that is configured to be compatible with a particular analytical device, and further configured to facilitate the addition of additional reagents and / or additives prior to analysis by the analytical device.

[0042] In an embodiment of the method according to the invention, the first composition is an intact sample from a patient.

[0043] In an embodiment of the method according to the invention, the step of measuring the interaction of bacteria with phages by the analytical device implies measuring the abundance and / or concentration difference of phage DNA by use of quantitative polymerase chain reaction.

[0044] In an embodiment of the method according to the invention, the step of measuring the interaction of bacteria with phages by means of the analytical device implies detecting cell lysis by measuring bioluminescence upon addition of luciferase activity.

[0045] In an embodiment of the method of the present invention, the method further comprises the step of selecting a composition of phages based on the phagegram, the phages of the composition of phages interacting with the maximum number of different binding receptor types in the bacterium.

[0046] According to one aspect of the invention, there is provided a microwell plate comprising a plurality of wells, each for containing one of a plurality of phages, for use in the systems and methods described herein.

[0047] According to one aspect of the invention there is provided a computer program product comprising code means configured to cause a processor to carry out the functions of the above systems and methods.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS The above and other features and advantages of embodiments of the present invention will be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0049] [Figure 1] 1 generally illustrates a system and method for determining bacterial susceptibility to multiple phages according to an embodiment of the present invention. [Diagram 2] 2a-c generally illustrate a method for measuring bacterial susceptibility to multiple phages and selecting a composition of phages based on a phagegram. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description of the Embodiments definition The term "bacteriophage" or "phage" as used herein refers to any virus or virus-like entity that has a bacterial host. Phages form a highly diverse group of viruses.

[0051] The term "phageogram" as used herein refers to a collection of data, preferably in the form of a table, outlining the effect of multiple individual bacteriophages on either individual bacterial pathogens or mixed sample environments. In some respects, similar to an antibiogram, a phageogram provides a profile of an organism's resistance or susceptibility to a panel of phages.

[0052] The term "intact sample" refers to a sample containing bacteria (typically a pathogenic bacteria or infectious bacterial pathogen) taken from a human, animal, or environment during the formation or progression of a particular disease condition, in which the specific pathogen has not been isolated and monocultured.

[0053] As used herein, a bacterium is considered to be susceptible to a phage if interaction with the phage results in the death or inactivation of the bacterium, and preferably, the interaction also leads to the propagation of the phage within the bacterium, thereby leading to cell lysis and release of progeny phage.

[0054] The term "receptor" refers to a chemical group or molecule (site), such as a protein or sugar, present on the cell surface or inside the cell, that has affinity for a particular chemical group, molecule, or virus. Receptors are chemical sites on cells that are recognized by virus particles and are responsible for the specific induction of attachment.

[0055] The uses and / or objectives of the systems and methods described herein are: Identification of bacteria, typically bacterial pathogens, in samples from patients, either intact or in pure culture isolates thereof; identification of any phages that cause cell inactivation, more preferably cell lysis, and even more preferably phage multiplication in said bacteria in said sample, and / or The development of compositions or cocktails of phages for treating bacterial infections caused by said bacteria.

[0056] Although reference may be made herein to bacterial pathogens or pathogenic bacteria (these terms are used interchangeably herein), the systems and methods described herein may be applied to any bacterium or mixture of bacteria that is taxonomically defined at the variant, strain, species, genus, or family level.

[0057] In certain cases, a patient may already be diagnosed with a bacterial infection, and a bacterial pathogen of a particular species or genus may be suspected to have caused the infection. One may then attempt to study and measure the effectiveness of each of the group of selected bacteriophages against the bacterial pathogen in a sample, or a pure culture isolate thereof. Each of the group of selected bacteriophages is preferably selected based on its ability to affect at least one strain of the suspected pathogen species.

[0058] In an embodiment of the present invention, a bacteriophage may be selected because it may be effective against a particular bacterial pathogen species. Thus, a bacteriophage is considered effective if it causes cell lysis in that species, preferably if it can grow in the bacterial host and replicate its bacteriophage DNA within the host cell. In a specific embodiment, a bacteriophage may be selected because it may be effective against one of various strains of the bacterial pathogen.

[0059] Thus, the use of the system may be to provide a phageogram that predicts and validates what bacteria it is and / or results in the effectiveness of a group of selected bacteriophages against bacterial pathogens having one or more strains.The advantage of the system and method described herein is that it speeds up the handling and procedures, thereby saving valuable time in the search for effective treatment for patients.

[0060] Phages, like antibacterial drugs, can induce resistance in bacteria, and resistant mutants often lack the binding receptors that the phages use to identify bacterial targets, which are often also virulence factors involved in critical aspects of pathogenesis.

[0061] In embodiments of the invention, bacteriophages may be further selected for their ability to collectively utilize the greatest number of different receptors in bacterial pathogens, thus allowing for the targeting of multiple virulence factors simultaneously.

[0062] A typical application of the system for therapeutic design involves measuring the effectiveness of a panel of selected phages against a bacterial pathogen in a sample, the phages targeting the greatest number of host receptors and at least one of various strains of the bacterial pathogen, the sample being either a whole sample from a patient or a pure culture isolate thereof.

[0063] The present invention is based on the principle that highly specific interactions between phages and bacteria can be determined by measuring the different outcomes of phage expression upon contact with bacterial samples. The effect of the phage on the host bacteria can vary, ranging from complete lysis of the bacteria involved in phage propagation to no obvious effect. By identifying such phages that induce cell death with high efficiency in a particular host bacterial species or strain, preferably accompanied by phage propagation, treatments for patients with bacterial infections can be developed.

[0064] The present inventors have found that such phage expression can be measured when the phage is at different stages in its cycle.

[0065] Bacteriophages are known to multiply through the following steps: (1) Attachment to specific susceptible host cells; (2) Substitute the host's metabolism with that of the phage, causing cell growth to cease; (3) Replicate the bacteriophage DNA, (4) packaging the bacteriophage DNA into a phage particle to render it infectious; (5) Lysing the cells releases infectious particles, which can find new hosts.

[0066] It has now been found that techniques that measure phage growth in steps (3) and (5) of the cycle described above can be used to rapidly and reliably measure the interaction of phage with host bacteria.

[0067] 1 illustrates generally a system 100 and method for identifying bacteria in a sample according to an embodiment of the present invention, with the objective of identifying any phages that cause at least cell inactivation in said bacteria and / or developing a composition of phages for treating bacterial infection caused by said bacteria. Thus, the system 100 and method described herein measure the susceptibility of bacteria to multiple phages.

[0068] According to one aspect of the invention, a system 100 is provided. System 100 comprises a microwell plate 110 comprising a plurality of wells 120, each well of the plurality of wells configured to contain one of a plurality of phages, and microwell plate 110 further configured for a sample comprising bacteria, preferably pathogenic bacteria, to be dispensed into each well of the plurality of wells.

[0069] According to an embodiment of the present invention, the microwell plate 110 comprises at least 6 wells, preferably at least 12 wells, more preferably at least 24 wells, and even more preferably at least 48 wells. It is further understood that the microwell plate 110 can contain up to 9000 wells, preferably up to 5000 wells, more preferably up to 1000 wells, even more preferably up to 500 wells, and most preferably up to 200 wells. Typically, the microwell plate 110 can contain approximately 96 wells. The wells are usually arranged in a 2:3 rectangular matrix.

[0070] For purposes of the present invention, the term "microwell plate" is used interchangeably with the terms "microplate" or "microtiter plate."

[0071] According to an embodiment, the top of each well of the microplate 110 is sealed to retain the well contents and to separate the well contents from the environment. This seal (not shown) may be an individual seal for each well. Preferably, one common seal is used to seal all wells of the plurality of wells 120.

[0072] Preferably the wells are sealed by a transparent polymer sheet, which can be pierced, for example by the tip of a disposable pipette used by a liquid dispensing robot.

[0073] The microplate 110 may be made of any material that is substantially non-reactive with the contents of the wells, but is preferably made of or contains a polymeric material such as polycarbonate, polypropylene, or polystyrene. The microplate may be colored, such as black or white, to facilitate measuring luminescence or fluorescence. The microplate may also comprise different structural elements, at least one of which is made of or contains one of the materials described above. 96-well microplates typically have a working volume of 100 μl to 360 μl.

[0074] According to a preferred embodiment of the present invention, the wells of the microplate 110 are configured so that each well contains one type or species of phage.

[0075] Bacteriophage may be inserted by dispensing a liquid containing phage of one particular phage type into the well, which is subsequently removed by evaporation or freeze-drying, leaving the solid phage at the bottom of the well.

[0076] The phage may also be retained in a solid matrix, for example in a well, as part of a carrier such as a porous carrier, etc. Regardless of what method is used, the phage can be dissolved in a liquid medium, typically an aqueous medium containing the bacterial species of interest, within a few seconds.

[0077] Importantly, there is no significant cross-contamination between wells due to interactions between bacterial species and phage transferred from neighboring wells.

[0078] According to a preferred embodiment, each well of the microplate 110 contains at most one phage type, which has been selected for its ability to affect a species of the bacterial domain. Advantageously, this allows the identification of the presence of a particular species of bacteria by administering a sample containing said species to all used wells and observing the effect of the selected phage on said bacterial species, and observing the degree of susceptibility of said bacteria to any of the phages present on the microplate 110.

[0079] The bacterial species may be in the form of one or more of a number of different strains of that species. Phages that interact with a bacterial species are known to affect the strains of that species to different degrees. By measuring the interaction of a selected group of phages known to affect a certain bacterial species with the bacterial host, it can be advantageously determined which phage can better lyse the administered cells. Also, by testing various phages each known to have a potential effect on a particular bacterial species, at least one phage that can have the most significant effect on that strain can be selected for use in therapy.

[0080] According to an embodiment of the present invention, the microplate 110 contains at least one group of selected phages, which refers to a group of two or more phages that have the ability to affect at least one strain of a particular bacterial pathogen species.

[0081] Preferably, the selected panel is as complete as possible in order to allow as many strains and variants of a bacterial species as possible to be identified as susceptible through interaction with at least one of the panel of selected bacteriophages.

[0082] Preferably, the panel of selected bacteriophages is as complete as possible so as to target the maximum number of host receptors (meaning binding receptors) in that particular bacterial pathogen species.

[0083] According to an embodiment of the present invention, the number of phages may be optimized and adapted to the number of colony forming units (CFU) of bacteria in the sample. Indeed, if the number of phages in a well is too low relative to the number of target bacterial cells, the test will not work unless the lower limit of quantification is lower than necessary, since fewer cells will be infected and fewer copies will occur. However, if the concentration of phages in a well is too high relative to the concentration of target bacteria, the sensitivity must be higher than necessary, since the progeny phages detected may be overwhelmed by the number of phages added to the sample chamber. Preferably, regardless of the type or species of phages, the number of phages in each well of the microplate 110 is essentially the same, and the deviation of the average number of phages in all wells does not exceed 20%, preferably 15%, more preferably 10%, even more preferably 5%, and most preferably 2%. Preferably, the number of phages in each well of the microplate 110 is the same.

[0084] According to an embodiment of the present invention, the wells of the microplate 110 contain at least 10 plaque forming units (PFU). 6 Preferably, the PFU is at least 10 7 The maximum PFU is 10 11 Preferably, the PFU is at most 10 10 More preferably, the PFU is at most 10 9Typically, for phages occupying only one well of the microplate 110, one well may contain approximately 10 8 It may contain PFU of phage.

[0085] According to an embodiment of the present invention, the microplate 110 contains a group of selected phages that target multiple strains of one bacterial species. Alternatively, the microplate 110 contains two, three, four, or more groups of selected phages that each target two, three, four, or more different bacterial species. Advantageously, this allows the presence of at least two, three, four, or more different bacterial species to be identified.

[0086] By using two, three, four or more groups of selected phages in one microplate, several bacterial species can be putatively identified simultaneously and the susceptibility of these species to multiple phages can be tested.

[0087] Thus, neat (even mixed) samples can be tested, and the interactions measured between the phage and the neat sample can then be extrapolated to accurately indicate the presence of the bacteria of interest.

[0088] In embodiments of the present invention, the identification of any phage that cause cell inactivation in a bacterial pathogen of interest may exceed the capacity of a single microplate 110. In such cases, an array or multiple microplates 110 are used.

[0089] Preferably, a group of selected bacteriophages physically occupy a set of adjacent wells on the microplate 110 .

[0090] As a result, the phageogram can advantageously reveal at a glance what a bacterial species is presumed to be and its susceptibility to multiple phages.

[0091] According to an embodiment, the microwell plate 110 is a pre-prepared plate with a plurality of phages and a protection sealing the wells. The pre-prepared plate is adapted to be introduced into a typical laboratory robot, such as a liquid dispensing robot, and a sample containing a bacterial species can be dispensed into the wells to puncture the seal. The pre-prepared plate is typically a disposable microwell plate.

[0092] It was found that such pre-prepared microwell plates could be stored for at least 12 months when kept at a temperature of 20°C and humidity of 50%.

[0093] According to a preferred embodiment of the invention, at least one well of said plurality of wells may be filled with a "blank" composition that does not contain any concentration of a particular phage type above background concentration. Advantageously, the effect of the phage on the bacterial species can be determined by comparing measurements of the blank composition with measurements of other phage-containing wells.

[0094] According to one aspect of the invention, a method for determining the susceptibility of bacteria to multiple phages is provided.

[0095] The method comprises: providing a first composition 130 comprising the bacteria and a first microwell plate 110 comprising a plurality of wells 120, each of the plurality of wells 120 containing one of the plurality of phages; Dispensing a first sample 131 of the first composition 130 into at least one well of the plurality of wells 120; for each of said at least one well, allowing a mixture of said first sample 131 comprising said bacteria and the bacteriophage contained in said well to incubate for a first period of time; Optionally, transferring a second sample 141 of said mixture to a second microwell plate 150; adding at least one reagent and / or additive 160 to each mixture on the first microwell plate 110 or the second microwell plate 150; After the introduction of said at least one reagent and / or additive 160, the at least one mixture or a final sample 171 of said at least one mixture is transferred and introduced into an analytical device 180, and said final sample is measured by said analytical device for the interaction of bacteria and phages; and interpreting the measurements in the analysis device 180 by a control unit (not shown) and presenting them in the form of a phragmogram.

[0096] According to an embodiment of the present invention, the first composition 130 comprises at least one bacterial species of interest, which may be a bacterial pathogen or an infectious agent.

[0097] According to a preferred embodiment of the present invention, the first composition 130 may refer to or contain a patient sample (intact sample from a patient) containing the bacterial pathogen. The bacterial species is understood herein to refer to the species or genus of interest of the bacterial domain that can be identified by the method and for which treatment is desired. The patient sample may and most likely contains other species of bacteria, but due to the high specificity of the interaction between bacteriophage and host bacteria, it is believed that such other species will not interfere with the detection of possible phage expression.

[0098] According to another embodiment of the present invention, the first composition 130 refers to or contains a pure culture isolate of a patient's intact sample.

[0099] Independently of whether an intact sample or a pure culture isolate is used in the systems and methods described herein, the first composition 130 may further contain at least a buffer, such as McFarlan buffer or any other suitable buffer known in the prior art.

[0100] Typically, the first composition 130 may have a volume of at least 200 ml. The sample of bacterial species may be obtained from a patient (human or animal). The animal may be a cow or a livestock. Typically, the sample may be obtained from a patient suspected of having a bacterial infection.

[0101] The sample may include cells, tissues, biopsies, secretions or exudates, fluids, or stomach contents obtained from a subject. Examples of samples include, but are not limited to, blood, lymph, urine, skin scrapings or swabs, nasal secretions, earwax, serum, surface washings, plasma, cerebrospinal fluid, saliva, sputum, stool, vomit, milk, tears, sweat, biopsy tissue, bedding, litter, or egg wash.

[0102] However, the sample may be from within the environment, particularly if that portion of the environment is suspected of bacterial contamination and may further transmit the organism, hi another embodiment, the sample is from a location in the environment, such as a food source, a water source, a soil area, or a structure.

[0103] Given that the sample may have a variety of origins, the concentration of the target bacterial species in the sample may vary as a result. For a sample that is used directly in the first composition 130 without isolating the target bacterial species, the concentration should be at least 10 5 CFU / ml, preferably at least 10 6 CFU / ml, more preferably at least 10 7 It was found that the CFU / ml should be sufficient, however such low concentrations depend on the overall quality of the sample.

[0104] In a preferred embodiment of the present invention, the first composition 130 contains an intact patient sample, and the concentration of the bacterial species of interest in the composition 130 is at least 10 5 CFU / ml, preferably at least 10 6 CFU / ml, more preferably at least 10 7 CFU / ml.

[0105] In these embodiments, there is no need to isolate the bacterial species of interest and then grow it to a predetermined concentration as required due to the detection limit of the analytical technique. Thus, the analysis of infectious bacteria in a patient sample can be rapidly performed, ranging from identifying the specific bacterial strain in the patient sample to identifying at least one bacteriophage that affects the bacterial population. Identification of such bacteriophages can then lead to a treatment for the patient. By simultaneously detecting multiple reactive phages, the bacterial species that is the source of infection can be accurately identified.

[0106] Alternatively, a pure culture isolate of the bacterial species can first be obtained and then grown to a desired concentration, typically by amplifying the bacterial species of interest on a suitable medium.

[0107] In these other embodiments of the invention, the first composition 130 contains a pure isolate obtained from an intact patient sample, and the concentration of the target bacterial species in the composition 130 is at least 10 5 CFU / ml, preferably at least 10 6 CFU / ml, more preferably at least 10 7 CFU / ml.

[0108] In one step of the method of the present invention, a first sample 131 of the first composition 130 is dispensed into at least one well of the plurality of wells 120 of a microplate 110. Preferably, the microplate 110 is configured as described herein above.

[0109] The bacteria and phage are preferably mixed in a liquid medium in the well. The liquid for providing the liquid medium typically comes from a first sample 131 and comprises at least the bacterial species of interest and a buffer. By avoiding the use of a gel, the liquid medium advantageously facilitates the transfer of a sample of the mixture to a device for further analysis.

[0110] According to a preferred embodiment of the present invention, the liquid medium is selected such that it is capable of lysing essentially the entire population of bacteriophage in a few seconds.

[0111] According to the present invention, phage are placed in a well and a first sample 131 containing a bacterial species is added to the phage in the well.

[0112] In one step of the method of the present invention, the first sample 131 comprising the bacterial pathogen mixed with bacteriophage contained in the well is allowed to incubate for a first period of time.

[0113] In an embodiment of the present invention, the first period of time is at least 30 minutes, preferably at least 40 minutes, more preferably at least 50 minutes. The first period of time is preferably not longer than 2 hours, more preferably not longer than 90 minutes. Typically, the first period of time is about 1 hour. It has been found that, if an interaction can occur between the bacteriophage and the bacterial species, the bacteriophage can bind to the binding receptor of the bacterial species during this period of time.

[0114] During said first period of time, the mixing of the phage with the bacterial species of interest occurs at a temperature of at least 30° C., preferably at least 33° C., and most preferably at least 36° C. It is further understood that the mixing of both components occurs at a temperature of at most 40° C., preferably at most 39° C., and most preferably at most 38° C. Typically, the phage and the bacterial species of interest are mixed in the well at a temperature of about 37° C.

[0115] In a next step, a second sample 141 of the mixture is transferred into an analytical device 180 for measurement and further characterization.

[0116] Optionally, for each well, a second sample 141 containing a mixture of bacteriophage and bacterial species is transferred to a second microwell plate 150 configured to introduce the mixture into the analysis device 180 so that the samples in the wells can be measured by the device 180. The second microwell plate 150 is thus configured to be compatible with the analysis device 180 (which may in many cases require a relatively small sample volume). The transfer of the second sample 141 of the first microplate 110 to the second microplate 150 is typically performed by using a liquid pipetting robot. The analysis device 180 can then analyze and measure the samples on the second microplate 150 in a step of the method according to the invention. In a next step of the method according to the invention, a computational means or control unit may then process the measurements provided by the device 180 and provide them for interpretation and presentation in the form of a phragogram.

[0117] In embodiments of the systems and methods according to the invention, the calculation means or control unit is configured to direct the parts of the systems and steps of the methods provided herein so as to enable the phragmogram to be obtained automatically.

[0118] The control unit may be or contain a controller for directing, evaluating and processing the steps of the method, such as the transfer of samples between microplates, controlling the temperature of the mixtures and the microplates, controlling the addition of further reagents and / or additives 160, analysis of the samples or mixtures by the analysis device 180 and / or obtaining the results of the measurements in the form of a phagogram.

[0119] In embodiments of the present invention, the control unit may be further configured to communicate the phargogram results to a central server so that the phargogram can be advantageously shared with multiple system users.

[0120] In a preferred embodiment of the invention, further reagents and / or additives 160 are added to the phage / bacteria mixture prior to measurement on said analytical device 180. The administration time prior to measurement, the nature and number of such at least one reagent and / or additive 160 depends on the analytical technique selected. The at least one reagent and / or additive 160 may be added to the phage / bacteria mixture on the second microplate 150.

[0121] Alternatively, the mixtures in the wells of the first microplate 110 are analyzed by the analytical device 180. Any at least one reagent and / or additive 160 is added to the phage / bacteria mixtures in the wells of the first microplate 110 that is configured to be compatible with the device 180, such that the samples in the wells can be measured by the device 180.

[0122] In an embodiment of the invention, the further analysis comprises: Measuring the abundance of phage DNA in the second sample 141 using qPCR and fluorescence; and detecting cell lysis by measuring bioluminescence upon addition of luciferase.

[0123] The present invention is based inter alia on the inventors' insight that both of the above mentioned techniques allow to measure and identify the interaction of phages with host bacterial cells in patient samples within a reasonable time and ultimately to select appropriate phages or phage combinations suitable for the treatment of patients affected by said bacteria.

[0124] Both have been shown to reliably measure the interaction of bacteriophages with host bacteria. Typically, only one of the two techniques can be used at a time.

[0125] In an embodiment of the invention, the at least one analytical device 180 is configured to measure the interaction of each of the plurality of phages with the bacterial pathogen by measuring a concentration gradient of phage DNA by use of quantitative polymerase chain reaction (qPCR). Thus, the interaction of phages with the host bacteria is assessed by use of qPCR, a well-known laboratory technique based on the polymerase chain reaction.

[0126] The PCR process is generally as follows: (1) denaturation, which involves heating the sample to a temperature of about 94°C to 96°C to break the hydrogen bonds of the double-stranded DNA in the sample and obtain two single-stranded DNA molecules; (2) lowering the temperature of the sample to about 50°C to 65°C and annealing by binding a single-stranded primer to each of the single-stranded DNA molecules (typically using two different primers, each of which has a 3' end complementary to a target region of the single-stranded DNA molecule); (3) elongation by adding free dNTPs (nucleoside triphosphates containing deoxyribose) to allow DNA polymerase to synthesize new DNA double strands from the reaction mixture.

[0127] Under optimal conditions, the number of DNA sequences doubles with each extension step. With each successive cycle, the original template strand and all newly generated strands serve as templates for the next extension step, thereby exponentially amplifying a particular DNA target region. The denaturation, annealing, and extension processes constitute one cycle. Multiple cycles are required to amplify millions of copies of the target DNA.

[0128] While the PCR process is a fast and inexpensive method for amplifying DNA, qPCR or real-time PCR has the advantage that the amplification process can be measured and followed in real time, thus allowing the measurement of DNA concentration gradients through the use of fluorescence spectroscopy.Fluorescence spectroscopy or fluorimetry as used herein refers to analytical techniques for detecting and analyzing fluorescence in a sample.

[0129] In an embodiment of the invention, molecular beacon probes are used to measure the concentration of bacteriophage in a mixture of phage and host bacteria by measuring fluorescence in the mixture.

[0130] In a specific embodiment of the invention, such a molecular beacon probe is a TaqMan probe.

[0131] As used herein, a molecular beacon probe consists of a fluorophore covalently attached to the 5' end of the probe and a quencher at the 3' end. The quencher molecule quenches the fluorescence emitted by the fluorophore as long as the two are in close proximity.

[0132] As used herein, molecular beacon probes are typically designed to anneal within a specific region of a single-stranded DNA molecule that is complementary to the sequence of the probe and is amplified by a specific set of primers.

[0133] During the extension step, when the polymerase extends the primer to synthesize a new double strand, the annealed molecular beacon probe is degraded, releasing the fluorophore and removing the proximity between the fluorophore and quencher, resulting in detectable fluorescence that is directly proportional to the amount of fluorophore released. The resulting fluorescent signal allows a quantitative measurement of the accumulation of bacteriophage DNA material.

[0134] In the qPCR process, the primers, nucleic acid polymerase, and molecular probes are essentially selected in consideration of the bacteriophage that will be mixed with the bacterial host. Thus, prior to analysis by the analysis device 180, each specific mixture of bacteriophage and bacterial host in the wells of the first microplate 110 or the second microplate 150 can be provided by a selected specific composition containing at least one of the primers, nucleic acid polymerase, and molecular probe associated with the specific bacteriophage present in the well of the microplate 110 or in the mixture.

[0135] According to an embodiment of the invention, the at least one reagent and / or additive 160 added to the sample prior to analysis by the analysis device 180 comprises: at least one primer molecule; DNA polymerase, dNTPs, A molecular beacon probe capable of emitting fluorescence; A buffer; At least one of qPCR amplification compatible solvents and enzymes known in the prior art.

[0136] The at least one reagent and / or additive 160 may be specific to a combination of bacteriophage and host bacteria. Typically, the at least one reagent and / or additive 160 may be specific to the bacteriophage in the mixture.

[0137] The inventors have found that qPCR can be used to distinguish between phages that can productively infect the bacterial species of interest that may be present in a patient sample and those that cannot. This is done by directly measuring the abundance of phage DNA in infected cells. In other words, the replication (or lack thereof) of phage DNA in infected cells is used as an indicator and comparison point to evaluate the phage's ability to infect the bacteria of interest. This comparison is then made against the DNA still encapsulated in the surrounding non-infected cells in other phage / bacteria mixtures, and against negative controls.

[0138] The present invention is based on the insight that qPCR is so sensitive that it can measure even small changes in target abundance, and therefore can detect even relatively small changes in phage DNA abundance caused by replication. Advantageously, because of its target specificity, qPCR technology can be used directly on patient samples rather than on pure culture isolates, thereby saving significant time.

[0139] And in the embodiment described herein above, the analysis device 180 may refer to a qPCR analysis tool or instrument that can measure the concentration difference of phage DNA in real time. Advantageously, such an instrument is configured to perform parallel evaluation and measurement, which means that it is possible to obtain the concentration gradient of phage DNA in several wells simultaneously. Typically, the instrument is configured to measure the fluorescence in the wells of a 96-well microplate.

[0140] In an embodiment of the invention, the at least one analytical device 180 is configured to measure the interaction of each of the plurality of phages with the bacterium or bacterial pathogen by detecting cell lysis of the pathogen by measuring bioluminescence produced by the addition of a luciferase / luciferin complex.

[0141] Luciferase is used in a variety of applications. Assays to determine the presence or absence of microorganisms by using luciferase-mediated oxidation of bacterial ATP have been around for a long time. This assay is currently used in a variety of diagnostic and commercial kits to detect bacterial contamination.

[0142] The reaction catalyzed by the luciferase enzyme to produce light is as follows (Equation (1)):

[0143] [ka]

[0144] In the formula, PP i relates to pyrophosphate. In the luciferase reaction, light is emitted when luciferase acts on an appropriate luciferin substrate. The emission of photons can be detected by a light-responsive device such as a luminometer or modified optical microscope. The luciferase reaction allows the observation of biological processes.

[0145] The present invention is based on the previous insight that luciferase can act as an ATP sensor protein. Thus, luciferase is used to detect the efflux of ATP from lysed cells, thereby effectively displaying the release of ATP in real time through bioluminescence. Luciferase produces ATP-driven bioluminescence, allowing the detection of ATP in solution at very low concentrations with great sensitivity.

[0146] Most healthy bacterial cells produce approximately 2 × 10 ATP per cell. -18 mol of ATP, and commercially available kits and analytical tools typically have detection limits of 1 x 10 -17 mol ATP 1×10 -13mol. As a result, it is reasonable to expect that an assay based on this principle can detect healthy bacterial cells in the range of about 5 to about 50,000 cells. The inventors have found that even at the upper end of the detection limit range, a test based on the principle of measuring bioluminescence produced by a matched luciferin-luciferase combination reliably detects ATP released upon lysis of 2% of the viable cells present in very small amounts of pure culture isolates.

[0147] It has been found that the combination of luciferin and luciferase is not specific to the bacteriophage used, therefore, a suitable luciferin and luciferase combination may be used for any mixture of bacteriophage and host bacteria.

[0148] Further advantageously, it has been found that the combination of luciferin and luciferase, even at high concentrations, does not affect bioluminescence measurements.

[0149] According to an embodiment of the invention, the at least one reagent and / or additive 160 added to the sample prior to analysis by the analysis device 180 comprises: a luciferin-luciferase combination or complex, which allows detection of bacterial cell lysis by ATP depletion of lysed cells and concomitant light emission; a luciferin / luciferase complex compatible solvent and an enzyme known in the prior art.

[0150] And, in the embodiments described herein above, analytical device 180 may refer to a light-responsive instrument such as a luminometer or optical microscope capable of measuring the luminescence emitted by the reaction described in equation (1).

[0151] In a preferred embodiment of the invention, such an instrument is configured to allow parallel evaluation and measurement, meaning that the bioluminescence of a mixture in several wells can be measured simultaneously.

[0152] The instrument may be configured to measure bioluminescence in the wells of a 96-well microplate.

[0153] Alternatively, the instrument is configured to allow multiple mixtures to be measured sequentially. In an embodiment of the method according to the invention, the method further comprises a step of selecting a composition of phages based on the phagegram displayed by the control unit, the phages of the composition of phages being selected for their ability to interact with the greatest number of different binding receptor types on the bacteria.

[0154] Figures 2a-c illustrate these embodiments diagrammatically. According to these embodiments, the objective of the method is to suggest compositions or combinations of phage types based on the phagegram.

[0155] Preferably, such a composition may consist of 1-3 phages, each of which targets a receptor type or receptor family, preferably a different receptor type. A combination of phage types can be used to treat infections caused by the above bacteria.

[0156] According to an embodiment, the control unit is configured to carry out the steps of the method as described herein above based on the results of the measurements provided in the form of a phargogram by said at least one analytical device using the system as described herein above and according to the method as described herein above.

[0157] The control unit can interpret the results obtained by assigning phages to receptor groups (where phages in one receptor group interact with bacteria via a particular binding receptor type) and by ranking the phages in that group based on whether they were able to interact with the bacterial species.

[0158] Referring to Figures 2a-b, in a particular case, 14 phages that recognize five different receptor families or binding receptor types in a bacterial species of interest are found to have activity against that species. Five phages are found to target the α receptor family of that bacterial species, and three phages targeting the β receptor family, two phages targeting the δ receptor family, one phage targeting the γ receptor family, and three phages targeting the ε receptor family are identified as suitable to be considered as having the greatest interaction with the bacterium and capable of blocking infection caused by that bacterium.

[0159] Then, for each receptor family, the control unit is configured to select one phage from each receptor group that is most suitable for targeting the relevant receptor family, and to identify the best phage within that group.

[0160] To create a three-phage cocktail, three receptor families must be selected to target. This can be done by ranking each receptor family target from most to least likely to exhibit antimicrobial susceptibility to no antimicrobial susceptibility. Referring to Figure 2b, the α, β, and ε receptor families are retained.

[0161] To select phages within a receptor group, see FIG. 2c, the control unit can use the information provided by the phagegram and typically selects the phage that interacts best with the bacterial species, i.e. the phage to which the bacteria shows the highest sensitivity.

[0162] This process can automatically suggest a cocktail for a physician to prescribe, which may be tailored to the susceptibility of the patient's bacterial strain and the clinical context of that patient's infection.

[0163] Although the present invention has been described above with reference to specific embodiments, this is for the purpose of illustrating and not limiting the invention, the scope of which is defined by the claims, and the skilled person will readily appreciate that other combinations of features than those described herein may be made without departing from the scope of the invention as claimed.

Claims

1. A system (100) for determining the susceptibility of a bacterium to a plurality of phages, said system (100) comprising: A microwell plate (110) comprising a plurality of wells (120), each of the plurality of wells (120) adapted to contain one of a plurality of phages, the microwell plate (110) further adapted for a sample comprising the bacteria to be dispensed into each of the plurality of wells (120); at least one reagent and / or additive (160) suitable for being added to at least one mixture of said sample comprising said bacteria and one phage of said plurality of phages; a control unit configured to interpret and present in the form of a phagegram the results of the measurements, said measurements being provided by at least one analytical device (180), said at least one analytical device (180) configured to measure the interaction of each of said plurality of phages with said bacteria; The at least one reagent and / or additive (160) comprises at least one of a primer molecule, a DNA polymerase, a nucleoside triphosphate (dNTP), a molecular beacon probe capable of emitting fluorescence, a buffer, and a qPCR amplification compatible solvent and / or an enzyme; and the at least one analytical device (180) is further configured to measure the interaction of each of the plurality of phages with the bacteria by measuring the abundance and / or concentration differential of phage DNA by use of quantitative polymerase chain reaction; or The at least one reagent and / or additive (160) comprises at least one of a luciferin / luciferase complex, a luciferin / luciferase complex-compatible solvent, and / or an enzyme configured to detect bacterial cell lysis by depletion of ATP and associated fluorescein light emission; and The system (100), wherein the at least one analytical device (180) is further configured to measure an interaction of the bacteria with each of the plurality of phages by measuring the luminescence.

2. The system of claim 1 , wherein the sample is an intact sample from a patient.

3. The system of claim 1 or 2, wherein the microwell plate (110) is disposable.

4. The system of any one of claims 1 to 3, wherein the plurality of wells (120) is further configured to contain at least a group of selected phages, each of the group of selected phages being selected for its ability to interact with and / or inactivate one particular bacterial species and strain.

5. The system of any one of claims 1 to 4, wherein the control unit is further configured to provide a composition of phages based on the phagegram, the composition of phages interacting with a maximum number of different binding receptor types in the bacteria.

6. 1. A method for determining susceptibility of a bacterium to a plurality of phages, the method comprising: providing a first composition (130) comprising the bacteria and a first microwell plate (110) comprising a plurality of wells (120), each of the plurality of wells (120) containing one of the plurality of phages; dispensing a first sample (131) of said first composition (130) into at least one well of said plurality of wells (120); for each of said at least one well, allowing a mixture of said first sample (131) comprising said bacteria and the bacteriophage contained in said well to incubate for a first period of time; adding at least one reagent and / or additive (160) to each mixture; transferring and introducing at least one mixture into an analytical device (180) and measuring the bacteria-phage interactions of said at least one mixture by said analytical device (180); and interpreting said measurements by said analysis device (180) and presenting them in the form of a phragmogram by a control unit, The at least one reagent and / or additive (160) comprises at least one of a primer molecule, a DNA polymerase, a nucleoside triphosphate (dNTP), a molecular beacon probe capable of emitting fluorescence, a buffer, and a qPCR amplification compatible solvent and / or an enzyme; and the at least one analytical device (180) is further configured to measure the interaction of each of the plurality of phages with the bacteria by measuring the abundance and / or concentration differential of phage DNA by use of quantitative polymerase chain reaction; or The at least one reagent and / or additive (160) comprises at least one of a luciferin / luciferase complex, a luciferin / luciferase complex-compatible solvent, and / or an enzyme configured to detect bacterial cell lysis by depletion of ATP and associated fluorescein light emission; and The at least one analytical device (180) is further configured to measure an interaction of each of the plurality of phages with the bacteria by measuring the luminescence.

7. 7. The method of claim 6, further comprising the step of transferring a second sample (141) of the mixture to a second microwell plate (150) prior to the step of adding at least one reagent, the second microwell plate being configured to be compatible with the analytical device (180).

8. The method of claim 6 or 7, wherein the first composition (130) is an intact sample from a patient.

9. The method according to any one of claims 6 to 8, wherein the step of measuring the interaction of bacteria with phages by the analytical device (180) implies measuring the abundance and / or concentration difference of phage DNA by use of quantitative polymerase chain reaction.

10. The method according to any one of claims 6 to 8, wherein the step of measuring the interaction of bacteria with phages by the analytical device (180) implies detecting cell lysis by measuring bioluminescence upon addition of luciferase.

11. The method of any one of claims 6 to 10, further comprising selecting a composition of phages based on the phagegram, the phages of the composition of phages being selected for their ability to interact with the greatest number of different binding receptor types in the bacteria.

12. A computer program product comprising code means adapted to cause a processor of a control unit to carry out the functions of the system according to any one of claims 1 to 5 and the method according to any one of claims 6 to 11.

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