BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present disclosure relates to methods for determining a host range and potency of a phage.

Discussion of the Related Art

[0002] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[0003] Multiple drug resistant (MDR) bacteria are emerging at an alarming rate. Currently, it is estimated that at least 2 million infections are caused by MDR organisms every year in the United States leading to approximately 23,000 deaths. Further, the overuse of antibiotics as well as bacteria’s natural evolution will likely lead to the generation of more virulent microorganisms. Genetic engineering and synthetic biology may also lead to the generation of additional highly virulent microorganisms.

[0004] For example, Staphylococcus aureus are gram positive bacteria that can cause skin and soft tissue infections (SSTI), pneumonia, necrotizing fasciitis, and blood stream infections. Methicillin resistant S. aureus (“MRSA”) is an MDR organism of great concern in the clinical setting as MRSA is responsible for over 80,000 invasive infections, close to 12,000 related deaths, and is the primary cause of hospital acquired infections. Additionally, the World Health Organization (WHO) has identified MRSA as organisms of international concern.

[0005] In view of the potential threat of rapidly occurring and spreading virulent microorganisms and antimicrobial resistance, alternative clinical treatments against bacterial infection are being developed. One such potential treatment for MDR infections involves the use of phage. Bacteriophages ("phages") are a diverse set of viruses that replicate within and can kill specific bacterial hosts. The possibility of harnessing phages as an antibacterial was investigated following their initial isolation early in the 20th century, and they have been used clinically as antibacterial agents in some countries with some success. Notwithstanding, phage therapy was largely abandoned in the U.S. after the discovery of penicillin, and only recently has interest in phage therapeutics been renewed.

[0006] The successful therapeutic use of phage depends on the ability to administer a phage strain that can kill or inhibit the growth of a bacterial isolate associated with an infection. Empirical laboratory techniques have been developed to screen for phage susceptibility on bacterial strains (i.e., efficacy at inhibiting bacterial growth). However, these techniques are time consuming and subjective, and involve attempting to grow a bacterial strain in the presence of a test phage. After many hours, an assessment of the capability of the phage to lyse (kill) or inhibit bacterial growth is estimated (the hostphage response) by manual, visual inspection.

[0007] One such test is the plaque assay which is a semi-solid medium assay which measures the formation of a clear zone in a bacterial "lawn" resulting from placement of a test phage and infection of the bacteria. Although the plaque assay is simple, plaque morphologies and sizes can vary with the experimenter, media and other conditions. More recently, an automated high throughput, indirect liquid lysis assay system has been developed to evaluate phage growth using the OmniLog™ system (Biolog, Inc; Henry M, Biswas B, Vincent L, Mokashi V, Schuch R, Bishop-Lilly KA, Sozhamannan S. Development of a high throughput assay for indirectly measuring phage growth using the OmniLog™ system. Bacteriophage 2012 Jul 1;2(3):159-167. doi: io.4i6i/bact.2i44O. PMID: 23275867; PMCID: PMC3530525.). The OmniLog™ system is an automated plate-based incubator system coupled to a camera and computer which, using redox chemistry, employs cell respiration as a universal reporter. The wells in the plate each contain growth medium, a tetrazolium dye, a (host) bacterial strain and a phage (along with co ntrol/calib ration wells). During active growth of bacteria, cellular respiration reduces the tetrazolium dye and produces a color change. Successful phage infection and subsequent growth of the phage in the host bacteria results in reduced bacterial growth and respiration and a concomitant reduction in color. The camera collects images at a plurality of time points, and each well in an image is analyzed to generate a color measure. This can be referenced to the initial color, or a reference color, so that a time series dataset of color change over time is collected (i.e., a colorimetric assay). The time series dataset for each well (i.e., host-phage combination) is graphed, and a user then (subjectively) reviews each of the growth curves of the graphs (e.g., 96 graphs for a 96-well plate). Interpretation of the growth curves is quite a challenging task as not only is there biological variability, but there may also be variability due to experimental sources such as medium and dye related effects which may affect interpretation of the growth curves. Thus, the user must use his/her experience, intuitive and implicit knowledge to interpret the graphs and estimate the host -phage response. This leads to increased variability or quality as the interpretation is subjective and dependent on the skill level and/or the attentiveness of the user reviewing the graphs on a particular day. Further, there is limited precision as most OmniLog™ systems only generate output data every 15 minutes.

[0008] Thus, there is a need to develop improved methods for determining a host range for a phage (i.e., the host-phage response), and/or the potency by which a phage infects, kills and/or inhibits the growth of one or more selected bacteria strain (e.g., a bacteria strain causative of an infection in a subject), to assist in the selection of phage (or at least candidate phage) that may be put to successful therapeutic use ("phage therapy").

SUMMARY OF THE INVENTION

[0009] According to a first aspect, there is provided a method of determining a host range of a phage, comprising the steps of: a) Providing a multiwell plate for the generation of one or more host-phage response result, wherein the wells of said plate are arranged in rows, and wherein one or more of said rows may be used in conducting the method, and wherein each row of said one or more rows used in conducting the method comprises: at least a first test well comprising suitable liquid growth media, a test phage and a test bacteria strain to provide a host -phage response result; a growth media control well comprising said growth media (i.e., no test phage or test bacteria present); a bacterial cell growth control well comprising said growth media and the test bacteria strain (i.e., no test phage present); at least one well comprising a diluted source preparation of the test bacteria strain in said growth media; and optionally, a positive control well comprising said growth media, the test phage and the test bacteria strain; b) Incubating said plate for a sufficient duration and under conditions suitable for growth of the test bacteria strain(s) in said growth media; and c) Measuring a characteristic indicative of growth of the test bacteria strain(s) in the wells; wherein, where the measured characteristic in the wells of a row used in conducting the method indicates that the host-phage response of the test phage in the at least first test well of that row includes inhibition of growth and/or killing of the test bacteria strain present in said well(s), then that test bacteria strain forms at least part of the host range of the test phage of that row.

[0010] In one embodiment, the multiwell plate of step a) of the method of the first aspect enables the test phage to be assayed with a plurality of different bacterial strains, such that the test bacteria strain provided in the wells of at least two rows, and more preferably, all rows, are different. In this way, the method of the first aspect may be conducted to identify whether or not a plurality of different selected bacterial strains are included in the host range of the test phage. The phage may be selected from phage "stocks" or a library of phage stocks, while the different bacterial strains may be bacterial strains causative of, for example, medically-significant infections including those caused by multiple drug resistant (MDR) bacteria such as MRSA. Otherwise, the phage may be selected from phage "stocks" or a library of phage stocks, and the different bacterial strains may be selected on the basis that they represent a group, or subset of a group, known to be causative of, or most commonly causative of, a certain infection type or types (e.g., urinary tract infections, ulcers and/or infections, such as for example, diabetic foot osteomyelitis, prosthetic joint infections, and/or eye infections, such as for example, blepharitis). Thus, by performing the method of the first aspect across the phage contained in a library of phage stocks, the method enables the elucidation of the host range for each phage within the library, which can then be used to assist in the selection of phage(s) from that library for use in a phage therapy of an infection caused by a particular, identified bacteria strain(s). Looking at it in a different way, by performing the method of the first aspect across the phage contained in a library of phage stocks, the method enables the evaluation of a library of phage stocks for "phage coverage" of the different bacterial strains causative of, or most commonly causative of, a certain infection type or types. For example, the method can be conducted to determine whether a library of phage stocks provides "phage coverage" of the different bacterial strains causative of, or most commonly causative of, urinary tract infections, ulcers and/or infections, such as for example, diabetic foot osteomyelitis, prosthetic joint infections, and/or eye infections, such as for example, blepharitis.

[0011] In another embodiment, the multiwell plate of step a) of the method of the first aspect enables a plurality of test phage to be assayed with the test bacteria strain (i.e., a single test bacteria strain is assayed against a number of different test phage), such that the test phage provided in the wells of at least two rows, and more preferably, all rows, are different.

[0012] In a second aspect, there is provided a method of determining potency of a phage to infect, kill and/or inhibit the growth of a test bacteria strain(s), comprising the steps of: a) Providing a multiwell plate for the generation of one or more host-phage response result, wherein the wells of said plate are arranged in rows, and wherein one or more of said rows may be used in conducting the method, and wherein each row of said one or more rows used in conducting the method comprises: at least a first test well comprising suitable liquid growth media, a test phage and a test bacteria strain to provide a host -phage response result; a growth media control well comprising said growth media (i.e., no test phage or test bacteria present); a bacterial cell growth control well comprising said growth media and test bacteria strain (i.e., no test phage present); at least one well comprising a diluted source preparation of the test bacteria strain in said growth media; and optionally, a positive control well comprising said growth media, the test phage and the test bacteria strain; b) Incubating said plate for a sufficient duration and under conditions suitable for growth of the test bacteria strain(s) in said growth media; and c) Measuring a characteristic indicative of a rate of killing or growth inhibition of the test bacteria strain(s) in the wells; wherein, where the measured characteristic in the wells of a row used in conducting the method indicates that the host-phage response of the test phage in the at least first test well of that row includes inhibition of growth and/or killing of the test bacteria strain present in said well(s) at, at least, a level(s) of a phage which potently infects, kills and/or inhibits growth of a host bacteria, then the test phage of that row is considered to potently infect, kill and/or inhibit growth of the test bacteria strain.

[0013] In one embodiment, the multiwell plate of step a) of the method of the second aspect enables the test phage to be assayed with a plurality of different bacterial strains, such that the test bacterial strain provided in the wells of at least two rows, and more preferably, all rows, are different. In this way, the method of the second aspect may be conducted to identify phage that potently infect, kill and/or inhibit growth one or more of a plurality of different selected bacterial strains. The phage may be selected from phage "stocks" or a library of phage stocks, while the different bacterial strains may be bacteria strains causative of, for example, medically-significant infections including those caused by multiple drug resistant (MDR) bacteria such as MRSA. Otherwise, the phage may be selected from phage "stocks" or a library of phage stocks, and the different bacterial strains may be selected on the basis that they represent a group, or subset of a group, known to be causative of, or most commonly causative of, a certain infection type or types (e.g., urinary tract infections, infections and/or ulcers, such as for example, diabetic foot osteomyelitis, prosthetic joint infections, and/or eye infections, such as for example, blepharitis). Thus, by performing the method of the second aspect across the phage contained in a library of phage stocks, the method enables the identification of phage within the library that may potently infect, kill and/or inhibit growth the test bacterial strain(s), which can then be used to assist in the selection of phage(s) from the library for use in a phage therapy of an infection caused by a particular, identified bacteria strain(s). [0014] In another embodiment, the multiwell plate of step a) of the method of the second aspect enables a plurality of test phage to be assayed with the test bacteria strain (i.e., a single test bacteria strain is assayed against a number of different test phage), such that the test phage provided in the wells of at least two rows, and more preferably, all rows, are different.

[0015] In some embodiments, the multiwell plate of step a) of the above methods comprise, in each row, 2 to 8 test wells comprising the liquid growth media, test phage and the test bacteria strain to provide a plurality of host -phage response results (i.e., generating a dataset). Those skilled in the art will understand that obtaining results from multiple "repeat" or replicate tests provides greater statistical confidence in results as parameters such as a lag time or hold time from multiple tests may be averaged and/or allow identification of outliers.

[0016] In some embodiments, the multiwell plate of step a) of the above methods comprise, in each row at least two wells (i.e., first and second bacterial source wells) comprising a diluted source preparation of the test bacteria strain in said growth media. In such embodiments, a first well comprises a diluted source preparation of the test bacteria strain (preferably, a preparation of the bacteria in the liquid growth media with an optical density (OD) in the range of 0.05-0.5, and more preferably, between 0.075 to 0.125, and even more preferably 0.090 to 0.110), and a second well is provided with a pre- loaded amount of the liquid growth media so as to readily enable a 1/10 dilution of the bacteria preparation of the first well. For instance, the second well is pre-loaded with 90 pL of the growth media, and 10 pL of the bacteria preparation of the first well is added to the second well to produce a 100 pL volume of a bacteria preparation that is a 1/10 dilution of the bacteria preparation of the first well. This diluted bacteria preparation (i.e., of the second well) can then be used to enable a further 1/10 dilution in the other wells of the row where the bacteria strain is required (i.e., the first and any other test well(s), the bacterial cell growth control well and the positive control well) in an analogous manner. For instance, each test well(s) may be pre-loaded with 80 pL of the growth media, and by adding to each test well a 10 pL volume of the bacteria preparation from the second bacterial source well along with a 10 pL volume of a phage preparation (i.e., a preparation comprising the test phage in the liquid growth media), each test well will be provided with a 100 pL volume comprising growth media, test phage and the test bacteria strain, such that the bacteria has effectively been diluted 1/10 as compared to the bacteria preparation of the second bacterial source well (nb. the phage is concomitantly diluted 1/10). The positive control well, if provided on the multiwell plate, may be likewise prepared. Similarly, the bacteria preparation of the second bacterial source well may be used in the preparation of the bacterial cell growth control well; that is, the bacterial growth control well may be pre-loaded with 90 pL of the growth media, to which 10 pL of the bacteria preparation of the second bacterial source well is added, resulting in a 100 pL final volume and achieving a 1/10 dilution of the bacteria. As such, the multiwell plate of step a) is provided with a substantially equivalent amount of the test bacteria in the test well(s), the bacterial growth control well, and the positive control well (if provided). In addition, each of the test well(s) and the positive control well (if provided) will be provided with a substantially equivalent amount of the test phage. The growth media control well will preferably be provided with a substantially equivalent volume of the liquid growth media to that provided in the test well(s), the bacterial growth control well, and the positive control well (if provided) (e.g., 100 pL).

[0017] In some embodiments, the multiwell plate of step a) of the above methods may further comprise a phage sterility control well(s) comprising said growth media and test phage strain (i.e., no test bacteria present). Such a well may, for example, comprise 90 pL of the growth media, into which is added a 10 pL aliquot of a test phage preparation (e.g., a master phage preparation of, preferably, 1 x io~ PFU/mL) to produce a total volume in that well of 100 pL and achieve a 1/10 dilution of the phage (e.g., to 1 x 10 ⁶ PFU/mL). However, in other embodiments, sterility of the phage preparation may be tested on a separate (i.e., "second") multiwell plate.

[0018] In some embodiments, the amount of test phage provided in the test well(s) will be in the range of o.5-1.5 x 10 ⁶ PFU/mL, and more preferably, in the range of 0.8-1.2 x 10 ⁶ PFU/mL. In some particular embodiments, the amount of test phage provided in the test well(s) is about 1.0 x 10 ⁶ PFU/mL.

[0019] In some embodiments, the amount of the test bacteria strain in the test well(s) will be in the range of o.5-1.5 x 105 CFU/mL, and more preferably, in the range of 0.8-1.2 x 105 CFU/mL. In some particular embodiments, the amount of test phage provided in the test well(s) is about 1.0 x 105 CFU/mL.

[0020] In further embodiments, the amount of the test bacteria strain in the test well(s) will be in the range of o.5-1.5 x io CFU/mL, and more preferably, in the range of 0.8-1.2 x 107 CFU/mL. In some particular embodiments, the amount of test phage provided in the test well(s) is about 1.0 x 107 CFU/mL.

[0021] In further embodiments, the amount of the test phage and test bacteria in the test well(s) are selected to provide a multiplicity of infection (MOI) in the range of 1 x 10’4 to 1 x 103. Where the multiwell plate is provided with two or more test wells, then the MOI for each of the test wells may be the same or different. For example, where in each row of the multiwell plate, there are 8 test wells then the methods of the present invention allow for the testing of different MOI values (e.g., to test an MOI range for variation in host -phage response).

[0022] In further embodiments, the method described herein can further comprise multiple test phage, which could then allow one to evaluate phage/phage synergy. Similarly, the method could also further comprise antibiotics, which could then allow one to evaluate phage/antibiotic synergy. Finally, both multiple phage and antibiotics can be included in the method as described herein to evaluate both phage/phage and phage/antibiotic synergy simultaneously.

[0023] In some embodiments, step b) of the above methods comprises incubating the multiwell plate at an appropriate pH (e.g., a substantially neutral pH such as about pH 7.4) and typical temperature for bacterial growth (e.g., a temperature in the range of about 35°C to about 39°C, but preferably about 37°C), and for a duration which, in the absence of infective phage, is sufficient for the test bacteria strain(s) to achieve logarithmic growth. In any case, the duration of the incubation may be selected from, for example, a time in the range of 5 to 72 hours, and more preferably in the range of 5 to 60 hours. In some particular embodiments, the duration of the incubation is about 48 hours. [0024] In some preferred embodiments, the multiwell plate of step a) of the above methods will comprise 8 rows of wells, wherein each row preferably comprises 12 wells (i.e., as found in a conventional multiwell plate with 96 wells provided in an 8x12 array). It will, however, be appreciated that multiwell plates with other numbers of wells and arrays may be suitable such as, for example, multiwell plates with 8 wells (e.g., in a 1x8 array), 12 wells (e.g., in a 1x12 array), 16 wells (e.g., in a 2x8 array), 24 wells (e.g., in a 3x8 array), 48 wells (e.g., in a 6x8 or 4x12 array) and 64 wells (e.g., in a 8x8 array). Figure 1 provided hereinafter is a schematic plot of one particular embodiment of the layout of an 8x12 multiwell plate suitable for use in the methods of the first and second aspects, wherein each row is provided with 7 test wells, a positive control well, a growth media control well, a bacterial cell growth control well, and first and second bacterial source wells.

[0025] In the process of providing the multiwell plate of step a) of the above methods, the multiwell plate of the particular embodiment shown in Figure 1 maybe pre- loaded as follows:

Test wells: each pre-loaded with 80 pL of growth media

Positive Control wells: each pre-loaded with 80 pL of growth media

Growth Media Control wells: each pre-loaded with 100 pL of growth media

Bacterial Cell Growth Control wells: each pre-loaded with 90 pL of growth media

First and Second Bacterial Source wells: each pre-loaded with 90 pL of growth media; and thereafter: using a micropipette, a 10 pL aliquot of a test phage preparation (e.g., a master phage preparation of, preferably, 1 x io" PFU /mL) is added to each of the test wells and the positive control well; and about 10 pL of a bacterial preparation with an OD of, preferably, 0.09 to 0.11, or about 1.0 x 10 ⁸ CFU/mL, is added to the first bacterial source well (to produce a total volume in that well of 100 pL), from which 10 pL is then drawn and added to the second bacterial source well (to produce a total volume in that well of 100 pL and achieve a 1/10 dilution). A 10 pL aliquot of the resultant diluted bacteria preparation of the second bacterial source well is then added to each of the test wells and the positive control well (thereby producing a total volume in that well of 100 pL and achieving a further 1/10 dilution of the bacteria, such that the amount of bacteria present maybe about 1 x 1<U CFU/mL and the amount of phage present is about 1 x io ⁶ PFU/mL).

[0026] In one particular embodiment, the multiwell plate of step a) of the above methods are OmniLog™ microarray assay plates (Biolog, Hayward, CA, United States of America) or other multiwell plates suitable for use in the OmniLog™ system or similar automated plate-based incubator systems and readers, such as for example Biotek’s LogPhase™ 600 or Cytation™ 7 can be used instead of the OmniLog™ system. In fact, any microplate reader that is capable of reading tetrazolium dye (or any other dye that is capable of measuring bacteria growth) or optical density and/or incubate and/or shake the plate can be used.

[0027] For example, and as described above, the OmniLog™ system is an automated plate-based incubator system coupled to a camera and computer, which uses cell respiration as a reporter of bacterial growth by measuring color change caused by the reduction of tetrazolium dye (i.e., reduction of the tetrazolium dye to its insoluble formazan, which has a purple color). Thus, by employing the use of the OmniLog™ system a time series of datapoints for each well can be obtained and analyzed to measure (or estimate) a characteristic indication of growth such that and the methods of the first and second aspects can reveal whether a test phage can infect and grow in the test bacteria strain to, for example, enable a determination of whether or not that bacteria strain forms at least part of the host range of the test phage. However, any dye or method that is capable of detecting directly or indirectly cell growth or measuring optical density can also be used instead.

[0028] Accordingly, in some embodiments, the said growth media may include a suitable amount of a reporter of bacterial growth (cell respiration) such as, for example, tetrazolium dye and related compounds and water soluble salts such as XTT (2,3-bis-(2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid e), MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium) in combination with phenazine methosulfate (PMS), and WST-8 (2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetr azolium). In such embodiments, the characteristic of growth of the test bacteria measured in step c) of the above methods is cellular respiration (i.e., through color change caused by reduction of a reporter such as tetrazolium dye or via a change in optical density). The reporter may be included in the growth media at any suitable concentration such as, for example, 1% v/v. [0029] The growth media may be selected from, for example, any of the liquid media types typically used for bacterial culture and which are well known to those skilled in the art. For example, the growth media may be, for example, Tryptic Soy Broth (TSB) media, Luria Broth (LB Broth), Basal Media (e.g., Nutrient Broth, Peptone Water etc.) and the like.

[0030] The amounts of the test phage and test bacteria strain present in the test well(s) are preferably selected so as to provide a useful host-phage response result(s) by the end of the incubation of step b) of the above methods (e.g., 5 to 72 hours, and preferably, about 48 hours). The amounts of test phage and the test bacteria strain described above in connection to a multiwell plate of the particular embodiment shown in Figure 1, are examples of selected respective amounts expected to provide a useful hostphage response result(s) (i.e., a prolonged suppression of bacterial logarithmic growth) by the end of a 48 hour incubation.

[0031] The amounts of the test phage and test bacteria strain present in the test well(s) may also be preferably selected to provide a multiplicity of infection (MOI) in the range of 1 x io ^_ 4 to 1 x 103 (i.e., an MOI in the range of 1000 down to 0.0001). An MOI of 1 corresponds to a ratio where there is an equal number of phage and bacterial cells present (i.e., a 1:1 ratio) such as, for example, where one million phage is added to one million bacterial cells. In some embodiments, the MOI used in the test well(s) may be in the range of 0.0001 to 0.001, 0.001 to 0.01, 0.01 to 0.1, 0.1 to 1, 1 to 10, 10 to 100, or 100 to 1000. Where the multiwell plate is provided with two or more test wells, then the MOI for each of the test wells may be the same or different. Indeed, in some embodiments, the methods may be conducted to test for the effect of a range of different MOIs on the phage-host response. For example, where the multiwell plate is provided with, for example, 8 test wells, then the MOI of each test well may vary, incrementally, across the row to test for the effect of different MOIs within the range of 1000 down to 0.0001; e.g., a first test well may have a MOI of 1000, a second test well may have a MOI of 100, a third test well may have a MOI of 10 etc. such that an eighth test well may have a MOI of 0.0001. However, such embodiments of the methods may also be performed to test for the effect of different MOIs (i.e., on the phage-host response) within narrower log 10 ranges such as 0.0001 to 0.001, 0.001 to 0.01, 0.01 to 0.1, 0.1 to 1, 1 to 10, 10 to 100, and 100 to 1000. The MOI range may extend over several decades, such as 0.01 to 100 or 0.1 to 100. In addition, in some embodiments, as has been described above, the methods may be performed such that a single test phage is tested against a plurality of different bacterial strains (e.g. tests with different bacteria may be conducted in each row of the multiwell plate) or such that multiple test phage are tested against a single test bacteria strain (e.g. tests with different phage may be conducted in each row of the multiwell plate), and it will be readily understood by those skilled in the art that the MOI may vary depending upon the particular combination of phage and bacterial cells. Moreover, the choice of a MOI value or MOI range for a specific bacteria strain may be made on the basis of calibration data or historical data. For instance, the selection of a MOI value or MOI range may be obtained by performing calibration experiments by adjusting the density of the cell concentration and/or the phage to test a range of MOI values. Replication and an analysis of the consistency of results (e.g., by using a measure such the coefficient of variation, or a validity/quality test may be performed by comparing the measure of variability with a threshold) may then be used, for example, to determine a MOI value or MOI range for the particular bacteria strain(s) in future tests.

[0032] A time series of measurements of growth maybe obtained for each well over the incubation period. Each time point is a measurement of a parameter indicative of growth of the bacteria in the well (in the presence of phage) and which may be plotted as a host -phage response curve. The parameter indicative of growth may be a characteristic of cell respiration, such as color change caused by reduction of tetrazolium dye which may be obtained from an optical measurement of a well using the OmniLog™ system or by any method that measures a change in optical density. The time series datasets may be plotted and/or analyzed to measure or estimate a characteristic indicative of growth or a rate of killing, such as hold time. The hold time for a phage host pair is a measure of the inhibition of logarithmic bacterial growth by a phage and maybe calculated as the length of time (or simply time) by which lag phase has been extended, or log phase has been delayed, relative to a control.

[0033] A computer implemented method may be used to analyze the time series dataset for each test well to estimate a hold time for the test well. As mentioned above, the multi-well plate of step a) of the above methods may comprise, in each row, 2 to 8 test wells to provide a plurality of host -phage response datasets or results. For each test well, the computer implemented method may be used to measure or estimate a characteristic indicative of growth or a rate of killing and may comprise estimating one or more parameters which are used to estimate the characteristic indicative of growth or rate of killing. In one form the computer implemented method comprises fitting one or more functions to each time series dataset for each test well and for each bacterial cell growth control well, wherein the one or more functions are used to estimate a characteristic indicative of growth or rate of killing such as a lag time for the respective time series dataset. A hold time for a test well may be obtained by subtracting the lag time of a bacterial cell growth control well (or an average of several control wells) corresponding to the bacteria in the test well, from the lag time of the test well. The one or more functions may be a sigmoidal function such as a Gompertz function, a Logistic function and/or a Richards function, or some other function including polynomial functions. In one form, the fitted function may be used to estimate a plurality of growth curve summary parameters including one or more of a max height, a slope, a lag time, and an area under curve, and the goodness of fit comprises one or more of a co-efficient of determination (R2), an error term, a residual term, or a summary statistic of residuals. Samples of test phage test bacteria combinations maybe replicated in multiple wells of a multiwell plate. A measure of variability of the replicates may be calculated, such as the coefficient of variation, and a validity (or quality) test may be performed by comparing the measure of variability with a threshold. The threshold may be selected based on expected variation, calibration data or historical data. For example, bacteria growing in biofilms are more heterogeneous than planktonic bacteria in liquid cell cultures, which may lead to high variability between replicates in the assay, and thus a threshold of CV < 50% may be used for planktonic bacteria and a threshold of CV < 75% may be used for biofilm bacteria. Other thresholds, including larger thresholds (e.g. 90%CV or 8s%CV) may be used based on the desired level of confidence or strictness of the validity test. If the test phage test bacteria combination passes the validity test, then a summary measure of the hold times of the replicates may be calculated, such as the mean or median of the replicates. This summary measure may then be reported as the hold time of the test phage test bacteria combination.

[0034] A test phage that achieves an inhibition of logarithmic bacterial growth (e.g., hold time) in the test well(s) of > 4 hours as compared to the bacteria in the bacterial cell growth control well, can be regarded as a "matching phage", meaning that the test bacteria strain forms at least part of the host range of the test phage. Further, where a test phage achieves an inhibition of logarithmic bacterial growth in the test well(s) for a period > 8 hours > 9 hours > 10 hours, > 11 hours, > 12 hours, > 13 hours, > 14 hours, > 15 hours, > 16 hours, > 17 hours, > 18 hours, > 19 hours, > 20 hours, > 21 hours, > 22 hours, > 23 hours, > 24 hours, > 25 hours, > 26 hours, > 27 hours, > 28 hours, > 29 hours, > 30 hours, > 31 hours, > 32 hours, > 33 hours, > 34 hours, > 35 hours, > 36 hours, > 37 hours, > 38 hours, > 40 hours, > 41 hours, > 42 hours, > 43 hours, > 44 hours, > 45 hours, > 46 hours, > 47 hours, > and/or 48 hours (i.e., as compared to the bacteria in the bacterial cell growth control well), that matching phage is considered to inhibit the growth and/or kill the test bacteria strain at a level corresponding to that of a phage which potently infects, kills and/or inhibits growth of a host bacteria, and accordingly then, the test phage of that row is considered to potently infect, kill and/or inhibit growth of the test bacteria strain. Such a phage, or for example, one that shows a somewhat lesser level of potency (e.g., achieves inhibition of logarithmic bacterial growth in the test well(s) for a period > 8 hours as compared to the bacteria in the bacterial cell growth control well), may be selected as a candidate for phage therapy to treat an infection caused by the test bacteria strain.

[0035] To assist with interpreting the results from the computational analysis of the wells in a multi-well plate, the data or results may be displayed as a heat map comprised of a plurality of cells where each cell is visual representation of the estimated hold time of a test well. The heat map may be a quantized heat map in which the heat map displays a predetermined set of colors where each color is associated with a predefined range of hold times, and the method comprises determining the predetermined range of hold times that include the estimated hold time of the test well, and displaying the associated color as the visual representation of the test well in the cell of the heat map. For example, red may be used for hold time of less than 1 hour, orange for hold times of 1-3 hours, yellow for hold times of 4-7 hours, green for hold times of 8-20 hours and blue for hold times of more than 20 hours. The heat map may be displayed on a screen or printed out. When displayed on a screen hovering over a cell may display information regarding the cell such as the phage and bacteria in the well corresponding to the cell.

[0036] Accordingly, the invention further relates to a method of phage therapy comprising administering a therapeutic phage formulation to an affected individual (i.e., a subject suffering from an infection), wherein the phage has been selected by determining that the phage potently infects, kills and/or inhibit growth of the bacteria causative of the infection. Further, the invention also relates to a therapeutic phage formulation comprising a phage that has been selected by determining that the phage potently infects, kills and/or inhibit growth of the bacteria causative of an infection to be treated.

[0037] In further preferred embodiments, the described methods can also be used to measure phage/phage synergy, phage/antibiotic synergy, and even phage/phage/antibiotic synergy by modifying the starting materials to include multiple phages and/or antibiotics in the test wells.

[0038] Further, the test bacteria may be provided to the wells "free" in a liquid preparation (e.g., as a planktonic preparation) or provided as a biofilm. If a biofilm is provided, then the methods can be tested on a single phage, or can also be used to test phage/phage synergy, phage/antibiotic synergy, and even phage/phage/antibiotic synergy by just modifying the starting materials. The methods provided herein can easily be modified to provide rapid testing of each of these parameters.

[0039] Where the methods use a test bacteria in the form of a biofilm, the multiwell plate used for the generation of one or more host -phage response result need not include at least one well comprising a diluted source preparation of the test bacteria strain in the growth media.

[0040] Thus, in a third aspect, there is provided a method of determining potency of a test sample comprising one or more test phage, optionally in combination with an antibacterial agent, to infect, kill and/or inhibit the growth of a test bacteria strain(s) present in a biofilm, comprising the steps of: a) Providing a multiwell plate for the generation of one or more host-phage response result, wherein the plate comprises: at least a first test well comprising suitable liquid growth media, a test sample comprising a test phage(s) optionally in combination with an antibacterial agent, and a test bacteria strain provided in the form of a biofilm, to provide a hostphage response result; a growth media control well comprising said growth media (i.e., no test phage(s), antibacterial agent or test bacteria present); at least a first bacterial cell growth control well comprising said growth media and test bacteria strain (i.e., no test phage present); and optionally, at least a first phage control well comprising said growth media and the test phage(s); b) Incubating said plate for a sufficient duration and under conditions suitable for growth of the test bacteria strain(s) in said growth media; and c) Measuring a characteristic indicative of a rate of killing or growth inhibition of the test bacteria strain(s) in the at least first test well; wherein, where the measured characteristic of the test bacteria strain(s) in the at least first test well indicates that the host-phage response of the test phage(s) in the test sample includes inhibition of growth and/or killing of the test bacteria strain present in said well at, at least, a level(s) of a phage which potently infects, kills and/or inhibits growth of a host bacteria, then the test sample of that well, optionally in combination with an antibacterial agent, is considered to potently infect, kill and/or inhibit growth of the test bacteria strain.

[0041] As such, the multiwell plate may be provided so that there are multiple test wells potentially enabling the determination of potency of numerous different test samples, each of which might comprise, for example, different single phage or different combinations of phage (e.g. for potential use in a composition comprising a phage "cocktail"), and optionally, combinations of one or more phage with one or more antibacterial agent (e.g. one or more antibiotics, one or more bactericides, and/or one or more other therapeutic molecules such as small molecules or biologies that have bactericidal activity). In this way, it can be readily appreciated that the methods can be used to measure phage/phage synergy, phage/antibiotic synergy, and phage/phage/antibiotic synergy.

[0042] The test bacteria strain is provided to the test wells either free in a liquid preparation such as a liquid growth media (a planktonic preparation) or in the form of a biofilm.

[0043] Those skilled in the art will appreciate that a biofilm of a bacteria may be prepared in vitro by culturing a suitable bacteria (which may be a bacterial strain obtained from a patient sample) in the presence of a solid surface, such as provided by, for example, a polymeric film or planar surface, or a particle or bead comprised of a polymeric material or other inert material such as glass. In some embodiments, the test bacteria is preferably provided to the test wells in the form of a biofilm attached to the surface of a bead such as a glass bead with a diameter of about 1 to 5 mm, and preferably, a glass bead of about 4 mm (which are suitable for, for example, conventional 96-well multiwell plates, wherein one biofilm-coated bead per test may be sufficient). Otherwise, biofilm produced through in vitro culture may, for example, be removed from a surface (if necessary) and processed into consistently-sized pieces (e.g., 2 mm x 2 mm substantially planar pieces) by any method that would be apparent to those skilled in the art (e.g. vigorous agitation and/or microdissection).

[0044] The measured characteristic indicative of a rate of killing or growth inhibition of the test bacteria strain(s) in the test wells, may be the hold time achieved by the test sample (e.g. a first test phage, or the first test phage in combination with an antibacterial agent, or a first test phage in combination with one or more other test phage (i.e., a second test phage and/or third test phage etc.) with or without an antibacterial agent), which is a measure of the inhibition of logarithmic bacterial growth by the test sample and may be calculated as the length of time (or simply time) by which lag phase has been extended, or log phase has been delayed, relative to a control. In some embodiments, the hold time is calculated from time series datasets obtained by measuring cell respiration (a parameter indicative of growth) by color change caused by reduction of tetrazolium dye (e.g., by optical measurement of a well using the OmniLog™ system), or by measuring a change in optical density in the wells.

[0045] Further, where the test sample achieves an inhibition of logarithmic bacterial growth in the test well(s) for a period > 8 hours (i.e., a hold time of > 8 hours),

> 9 hours > 10 hours, > 11 hours, > 12 hours, > 13 hours, > 14 hours, > 15 hours, > 16 hours, > 17 hours, > 18 hours, > 19 hours, > 20 hours, > 21 hours, > 22 hours, > 23 hours,

> 24 hours, > 25 hours, > 26 hours, > 27 hours, > 28 hours, > 29 hours, > 30 hours, > 31 hours, > 32 hours, > 33 hours, > 34 hours, > 35 hours, > 36 hours, > 37 hours, > 38 hours,

> 40 hours, > 41 hours, > 42 hours, > 43 hours, > 44 hours, > 45 hours, > 46 hours, > 47 hours, > and/or 48 hours (i.e., as compared to the test bacteria in the bacterial cell growth control well), that test sample is considered to inhibit the growth and/or kill the test bacteria strain at a level corresponding to that of a phage which potently infects, kills and/or inhibits growth of a host bacteria, and accordingly, maybe selected as a candidate phage therapy to treat an infection caused by the test bacteria strain.

[0046] Where the at least one test well is provided with an antibacterial agent (i.e., to create a test sample comprising an antibacterial agent in combination with one or more phage), then the antibacterial agent may, in some preferred embodiments, comprise an antibiotic compound. Suitable antibiotics may be selected from, for example, compounds of the following antibiotic classes: aminoglycosides (e.g. gentamycin and neomycin), carbapenems (e.g. imipenem), cephalosporins (e.g. cephalexin), fluoroquinolones (e.g. levofloxacin and ciprofloxacin), glycopeptides and lipoglycopeptides (e.g. vancomycin), macrolides (e.g. erythromycin and azithromycin), monobactams (e.g. aztreonam), oxazolidinones (e.g. linezolid andtedizolid), penicillins (e.g. penicillin G, amoxicillin and ampicillin), polypeptides (e.g. actinomycin and polymyxin B), rifamycins (e.g. rifampicin), sulfonamides (e.g. sulfamethizole), streptogramins (e.g. quinupristin and dalfopristin), and tetracyclines (e.g. tetracycline and doxycycline); as well as other miscellaneous antibiotic compounds such as chloramphenicol, clindamycin, daptomycin, fosfomycin, lefamulin, metronidazole and mupirocin.

BRIEF DESCRIPTION OF THE FIGURE(S)

[0047] Embodiments of the present disclosure will be discussed with reference to the accompanying figure(s) wherein:

[0048] Figure 1 is a schematic plot of the layout of a multiwell plate used to generate a plurality of host -phage response results (generating a dataset) according to an embodiment. Concentrations of phage and bacteria are indicated in "lEx" format corresponding to 1 x io ^x (e.g., 1E6 PFU/mL of phage is 1 x 10 ⁶ PFU/mL). In the embodiment illustrated in this figure, the amount of bacteria present in the wells of column 12, namely 1 x io" CFU/mL, corresponds to a bacteria preparation with an optical density (OD) in the range of 0.090 to 0.110. Each row of the test plate comprises: Wells 1-7 - test wells ("Assay Wells"), Well 8 - "Positive Control" well, Well 9 - growth media control ("Media Control") well, Well 10 - bacterial cell growth control ("Cell Control") well, and Wells 11-12 - wells for providing a source(s) of diluted bacteria preparation (Cell Dilution) wells;

[0049] Figure 2A is a schematic plot of the host-phage response results corresponding to each well in the multiwell plate of Figure 1 according to an embodiment; [0050] Figure 2B is a schematic plot of the normalized host-phage response dataset of results shown in Figure 2A according to an embodiment;

[0051] Figure 2C is a plot illustrating several growth curve summary parameters according to an embodiment;

[0052] Figure 2D illustrates the function forms (equations) for three sigmoidal functions;

[0053] Figure 2E is a plot of several sigmoidal functions according to an embodiment;

[0054] Figure 3 is a schematic diagram of a computing apparatus according to an embodiment;

[0055] Figure 4A is a series of plots of host-phage response datasets showing short lag time, medium lag time and large lag times according to an embodiment;

[0056] Figure 4B is a color coded heat map (matrix plot) of hold times calculated from estimated lag times of host -phage response dataset for multiple phages and the same bacteria host according to an embodiment; [0057] Figure 5 provides results obtained using a method according to the invention as described in Example 2. The lines of the figure represent the growth (expressed as relative respiration units) of a bacteria (strain A) in the absence (control) or presence of one of five different phage strains (phage 1-5), and particularly illustrates (by suppression of bacterial growth) which of the phage potently inhibit growth of the bacteria; and

[0058] Figure 6 provides results obtained using a method according to the invention as described in Example 3. The results are presented as a heat map, effectively summarizing the relative duration of bacterial growth inhibition achieved by "matching phage" (i.e., a phage that achieved inhibition of bacterial growth > 4 hours as compared to a "no phage" bacterial control).

[0059] Figure 7 is a schematic plot of the layout of a 96 well plate used for a Host Range Quick Test (HRQT) protocol using four replicates per treatment sample spread across 4 columns of a row;

[0060] Figure 8 is a schematic plot of the layout of a 96 well plate used for a Host Range Quick Test (HRQT) protocol using 8 replicates for each treatment sample spread across 8 rows of a column;

[0061] In the following description, like reference characters designate like or corresponding parts throughout the figures.

DETAILED DESCRIPTION

[0062] As used in the specification and claims, the singular form "a", "an" and "the", include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. "A phage formulation" can mean at least one phage formulation, as well as a plurality of phage formulations, i.e., more than one phage formulation. Also, as understood by one of skill in the art, the term "phage" can be used to refer to a single phage or more than one phage. [0063] The present invention can "comprise" (open ended) or "consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, "comprising" means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise. As used herein, "consisting essentially of’ means that the invention may include ingredients in addition to those recited in the claim, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed invention.

[0064] The term "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term “about” means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ±1-5%. In even further embodiments, "about" should be understood to mean+/-5%.

[0065] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0066] All ranges recited herein include the endpoints, including those that recite a range "between" two values. Terms such as "about," "generally," "substantially," "approximately" and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

[0067] Where used herein, the term "and/or" when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition is described as containing characteristics A, B, and/or C, the composition can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination. [0068] As used herein, the term "host range" refers to the spectrum of host bacterial strains that can be infected by a particular phage type. In the context of the method of the first aspect, the method determines whether the test bacteria strain forms the host range, or more likely, forms part of the host range of the test phage.

[0069] The term "host-phage response" as used herein, refers to an outcome of contact or interaction between a phage and a bacterium including any or all of infection, inhibition of bacterial growth, and killing of the bacteria.

[0070] The term “multiplicity of infection” or “MOI” as used herein refers to the ratio of the number of phage particles to the number of bacterial cells in a given system such as a test well of a multiwell plate as described herein. In preferred embodiments, when Gram positive bacteria having an OD6oo of 0.1. and consistently having a cell density of around 1 x 10 ⁸ CFU/mL, the MOI is 10 while Gram negative bacteria consistently having a cell density of around 1 x io CFU/mL, the MOI was around 100. However, MOI values for use in the methods of the present invention may be selected to be in the range of 1000 down to 0.0001, and may, in accordance with some embodiments, be a specific value or values within a sub-range as may be appropriate for a particular bacterial strain based on calibration data or historical data. The MOI may, for example, be varied within the test well(s) of a particular row of a multiwell plate cells so as to test the effect of varying the MOI on the host -phage response by varying the amount of the phage and/or bacterial cells in the respective wells. Moreover, different bacterial strains may well have different optimal MOI values or MOI ranges for use in the methods (which as mentioned, maybe based on calibration data or historical data). The selection of a MOI value or MOI range may be obtained by performing calibration experiments by adjusting the density of the bacterial cells and/or the phage to test a range of MOI values. Replication and an analysis of the consistency of results, may then be used to determine a MOI value or MOI range for the particular bacteria strain in future test. Consistency of results may be assessed using a measure such as the coefficient of variation, or a validity/quality test may be performed by comparing the measure of variability with a threshold). Once a target MOI value or MOI range is determined for a particular bacterial strain then this may be used for future tests. In the case that a target MOI value or MOI range is not known, then a default MOI may be used based on, for example, similar strains. A default MOI value may be 100 for Gram negative bacteria and 10 for Gram positive bacteria. [0071] The term "potency" as used herein in relation to phage refers to the efficacy and/or vigor by which a phage infects, inhibits and/or kills a host bacterium. One measure of phage potency, as contemplated herein, is the duration that a phage may suppress logarithmic growth in a suitable assay. In some embodiments, a measure of phage potency may be made in the context of, for example, a combination of two or more different phage (e.g. a phage "cocktail"), or in combination with one or more antibacterial agent (e.g. one or more antibiotics, one or more bactericides, and/or one or more other therapeutic molecules such as small molecules or biologies that have bactericidal activity). [0072] The term "biofilm" is well understood by those skilled in the art and refers to a colony formation of one or more layers of bacteria, that is typically encased within an adhesive polysaccharide material (excreted by the cells) and which is attached to a surface. The biofilm formation may, for example, protect the bacterial cells from the immune system of an infected subject, and/or provide resistance to antibacterial agents, which may fail to penetrate into the biofilm.

[0073] As used herein, a "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. In other preferred embodiments, the “subject” is a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), or an ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In other embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or rabbit animals) may be employed. Preferably, a "subject" encompasses any organisms, e.g., any animal or human, that may be suffering from a bacterial infection, particularly an infection caused by a multiple drug resistant bacterium.

[0074] As understood herein, a "subject in need thereof” includes any human or animal suffering from a bacterial infection, including but not limited to a multiple drug resistant bacterial infection, a microbial infection or a polymicrobial infection.

[0075] A "therapeutic phage formulation", "therapeutically effective phage formulation", “phage formulation” or like terms as used herein are understood to refer to a composition comprising one or more phage which can provide a clinically beneficial treatment for a bacterial infection when administered to a subject in need thereof. [0076] As used herein, the term "composition" encompasses "phage formulations" as disclosed herein which include, but are not limited to, pharmaceutical compositions comprising one or more purified phage. "Pharmaceutical compositions" are familiar to one of skill in the art and typically comprise active pharmaceutical ingredients formulated in combination with inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and/or diluents. The term "pharmaceutically acceptable" is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism (or at least nontoxic in amounts typically used). Examples of pharmaceutically acceptable excipients, carriers, buffers, and/or diluents are familiar to one of skill in the art and can be found, e.g., in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, Pa. For example, pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffering substances, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling or viscosity enhancing additives, flavoring agents, and colors. Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Pharmaceutically acceptable diluents include, but are not limited to, water, saline, and glycerol.

[0077] As used herein, the term “estimating” encompasses a wide variety of actions. For example, “estimating” may include calculating, computing, processing, determining, deriving, investigating, looking up (e.g., looking up in a table, a database or another data structure), ascertaining and the like. Also, “estimating” may include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory) and the like. Also, “estimating” may include resolving, selecting, choosing, establishing and the like. Measuring may encompass measuring one or more parameters and estimating a value based on the one or more measured parameters.

[0078] Embodiments of the method may be used to identify phage for including in phage formations for treating patients with bacterial infections, and in particular Multiple Drug Resistant Infections. The methods can also be used to identify phage that can be used to clean up bacterial contaminated areas, such as for cleaning up an industrial site. These phage formulations may include two or more phage with different mechanisms of action as described above. [0079] The present invention is hereinafter further described with reference to the following non-limiting example(s) and accompanying figures.

EXAMPLE(S)

Example 1 Host Range Quick Test (HRQT) protocol

[0080] This example describes one particular embodiment of a phage susceptibility test according to the present invention, denoted as a Host Range Quick Test (HRQT).

Phage Preparation - pre-titer and Normalization

[0081] An appropriate bacterial host strain is selected and grown by inoculating 3mL of sterile Tryptic Soy Broth (TSB) media contained within sterile i4mL round bottom culture tubes with a loopful of bacteria and incubation at 37°C with shaking (e.g., in a shaking incubator set to 175 RPM) for a minimum of 1 hour or until turbid. The tubes are then stored at 4°C until required.

[0082] Phage strains are selected from phage "stocks" or a library of phage stocks, and 10-fold serial dilutions performed on each phage strain from 10 ¹ to io ^-8 in SM buffer or PBS.

[0083] From the stored tubes of bacteria, and appropriate volume of the bacterial culture are transferred into smL round bottom culture tubes, on top of which is added 3 mL of molten top agar (per each tube). The contents of the tubes are then poured into respective standard Tryptic Soy Agar (TSA) plates and left, undisturbed for 3-5 minutes. Then, utilizing the diluted phage, the plates are given a spot titer with 10 pL of each phage dilution from 10-5 through 10-8. After allowing the spots to dry, the plates are then incubated overnight in a stationary 37°C incubator overnight. Finally, the plates are removed from the incubator and the phage titer observed and recorded; plates with a titer of < 1.0 x 10 ⁸ PFU/mL are discarded while those with a titer of > 1.0 x 10 ⁸ PFU/mL can be used for the further steps of the phage preparation.

[0084] That is, the phage are then diluted to a standardized volume of imL and a standardized titer of 1.0 x 10 ⁸ PFU/mL with 1X PBS in microcentrifuge tubes.

[0085] Sample Calculations:

If the titer = 1.23 x 109 PFU/mL

(titer) (volume phage to add) = (standardized titer) (standardized final volume)

(1.23 x 109 PFU/mL) (volume phage to add) = (1.00 x 10 ⁸ PFU/mL) (lOOOmL) (volume phage to add) = [(1.00 x io ⁸ PFU/mL) (1000 mL)]/(i.23 x 109 PFU/mL)];

Such that the volume phage to add = 81 pL phage.

(total volume)- (Volume phage to add) = volume of PBS

1000 pL - phage = volume of PBS 1000 pL - 81 pL = 919 pL PBS

[0086] The diluted phage preparations may then be stored for up to 1 month at 4°C until ready to use.

Assay Preparation

[0087] Tetrazolium dye can be prepared in standard TSB as set below:

1. An appropriate number of OmniLog™ microarray assay plates and lids are obtained for the phage to be assayed, and each plate labeled with the name or an identifier (ID) of the specific bacterial cell strain.

2. An appropriate volume of 1% v/v 1X Tetrazolium dye in TSB (Tz/TsB) is prepared and passed through a 0.22 pm Steriflip® filter (or equivalent). Each plate requires approximately 9mL of Tz/TSB. The Tz/TSB may be stored at 4°C, covered with aluminium foil to prevent light exposure until ready to use.

[0088] Master phage plates can be prepared as follows:

1. A single step 1:10 dilution of the desired phage can be performed using the above mentioned Tz/TSB preparation as the diluent in a 96-well microtiter dish. Well identifiers (IDs) should be recorded for each phage in each Master Phage Plate.

2. The Master Phage Plates can be stored at 4°C until ready for use.

[0089] Cell preparations for assaying can be prepared by, for example:

1. Inoculating an appropriate volume of bacterial cells into 10 mL TSB and incubate in a shaking incubator set to 175 RPM. The bacterial cells may be selected from stored bacteria "stocks" or a library of bacteria stocks. Additionally or alternatively, the bacterial cells may include cells from, or cultured from, samples obtained from a subject in need thereof treatment for a bacterial infection. The initial ODeoo must be between 0.005 - 0.015.

2. Embodiment 1

Dispensing a 1 mL aliquot of the cells into a new, labeled cuvette, and measuring the ODeoo values approximately every 60 minutes, or sooner based on the previous OD, until a range of 0.20 - 0.50 is achieved. And once the cells have reached an ODeoo range of 0.20 - 0.50, diluting the cells with cold TSB to a range of 0.090 - 0.110. The cells can then be placed on ice until ready for use (but preferably within 2 hours). Embodiment 2

In an alternative, the cells may be prepared directly prior to use by inoculating TSB with colonies pulled from a streaked plate and thereafter adjusting the cells and/or medium to achieve a culture preparation with an OD6oo in the range of 0.090 - 0.110.

[0090] Test plates are prepared as follows:

1. Test media, test phage strains and bacterial cells are aliquoted into the microarray assay plates as follows:

(i) 80 pL Tz/TSB into each well of lanes 1-8.

(ii) 90 pL Tz/TSB into each well of lanes 10-12.

(iii) 100 pL Tz/TSB into each well in lane 9.

(iv) 10 pL of the desired phage preparation is transferred from the Master phage plate into each well of lanes 1-8.

(v) 10 pL of the desired diluted bacterial cell preparation is aliquoted into each well in lane 12.

(vi) 10 pL of the desired bacterial cells is transferred from each well of lane 12 to the corresponding well of lane 11.

(vii) 10 pL of the desired bacterial cells is transferred from each well of lane 11 to the corresponding well in lane 10.

(viii) 10 pL from each well of lane 11 to the corresponding wells of each of lanes 1-8.

(ix) A lid is placed on each plate once completed.

[0091] Following the steps (i) to (ix) generates test plates with a multiwell layout as shown in Figure 1. This protocol and layout readily enables the generation of a plurality of host-phage response results/datasets (e.g., for inhibition of bacterial logarithmic growth).

[0092] Phage sterility control plates can be prepared as follows:

1. Test media and test phage strains are aliquoted into the microarray assay plate as follows:

(i) 90 pL Tz/TSB into each well of lanes 1-8.

(ii) 100 pL Tz/TSB into each well of lane 9.

(iii) 10 pL of the desired phage preparation is transferred from the Master Phage plate into each well of lanes 1-7.

(iv) A lid is placed on each plate once completed. HROT Batch Input

[0093] A computing system may be configured to execute computer software to collect and subsequently analyze the results of an assay using an OmniLog system.

[0094] In one embodiment the user opens the software which provides a user interface. The user may be presented with a batch plate loading screen, and may be asked to assign a 3-6 letter project code and a project name for a specific run. The incubation time may be preset to 48 hours (2 Days) and reads taken at 15 min intervals. The user may override the preset values if required. The user may navigate to a Plate Information Screen where they can enter the Project Code, Assay Plate ID (The cell strain on that specific plate), Phage Plate No. (Master Phage Plate No.), technician initials, and any comments. The user may repeat for all plates to be included in the current batch/run. The data is then saved.

[0095] The batch is then loaded into the Omnilog incubator by placing all assay plates into their appropriate slots in the incubator. The user then confirms that that all plates listed are properly loaded in the incubator reader and click “Load Batches”. The incubation and read cycle will then be started.

[0096] Following the 48-hour incubation period, the user logs back into the software and selects the desired plate for analysis. A host phage response dataset is received or accessed by the computing system. This may be sent by the apparatus (e.g., OmniLog™) which generated the dataset, or a computing apparatus associated with the apparatus (e.g., a control computer), or the data may be accessed from a storage device. The dataset comprises a time series dataset for a host-phage combination in which a host bacterium is grown in the presence of a phage. Each data point in the time series dataset associated comprises a measurement of a parameter indicative of the growth of the host bacteria in the presence of the phage at a specific time.

[0097] The data may be provided an electronic format, such as a CSV input file exported by an OmniLog™ (or similar host response) apparatus. Figure 1 is a schematic plot of the layout 100 of a multiwell plate (e.g., 96 wells) used to generate a plurality of host -phage response datasets according to an embodiment. Figure 2A is a schematic plot of the host-phage response datasets corresponding to each well in the multiwall plate of Figure 2A according to an embodiment. This shows two good phage 212 and poorly performing phage 214. These are arranged in a 7x8 grid of assay wells 210 (rows A to H and columns 1-12) and a set of control wells 220 comprising positive control wells 221, media control wells 222, cell control wells 223, and two cell dilutions 224, 225. The media control wells 222 in column 9 contain just growth media to allow detection of possible contamination. The cell control wells in column 10 comprise bacteria grown in media in the absence of phage and can be used to detect contamination or other growth issues. In some cases, the same host bacteria are grown in each well of a row with different phage in each column. In some cases, the same host bacteria may be grown in each well of the plate (apart from media controls). In some cases there maybe a mix and match of bacteria and phage in wells (e.g. 4 different bacteria and 9 different phage). A master phage plate file (in csv format) may also be provided which contains well-wise phage information. This file is used for the annotation of the final results. Other metadata relating to the experimental run may also be provided such as operator’s name, project ID, master phage plate ID and date of experiment.

[0098] The protocol can, however, be readily modified beyond the selection of a single phage to selection of a combination of phage (e.g. for use in a composition comprising a phage "cocktail"), optionally in combination with one or more antibacterial agent (e.g. one or more antibiotics, one or more bactericides, and/or one or more other therapeutic molecules such as small molecules or biologies that have bactericidal activity), for phage therapy to treat.

[0099] Further, the protocol can be readily modified by the selection of the amount of the test phage and the amount of the test bacteria strain to provide, a target MOI value or such that the MOI is within a desired MOI range. In one set of experiments, bacteria were grown to an early- to mid-log phase followed by a dilution to an OD6oo value of 0.1 and tested with phage at an amount of 1 x io~ PFU/mL Since it has been found that Gram positive bacteria with an OD6oo of 0.1. consistently had a cell density of around 1 x 10 ⁸ CFU/mL and that Gram negative bacteria consistently had a cell density of around 1 x io~ CFU/mL, in these experiments, the MOI was around 100 for the Gram negative bacteria and 10 for the Gram positive bacteria. However, as has been described above, MOI values for use in the methods of the present invention may be selected to be in the range of 1000 down to 0.0001, and may, in accordance with some embodiments, be a specific value or values within a sub-range as may be appropriate for a particular bacterial strain based on calibration data or historical data. The MOI may, for example, be varied within the test well(s) of a particular row of a multiwell plate cells so as to test the effect of varying the MOI on the host-phage response by varying the amount of the phage and/or bacterial cells in the respective wells. Moreover, different bacterial strains may well have different optimal MOI values or MOI ranges for use in the methods (which as mentioned, may be based on calibration data or historical data). The selection of a MOI value or MOI range may be obtained by performing calibration experiments by adjusting the density of the bacterial cells and/or the phage to test a range of MOI values. Replication and an analysis of the consistency of results, may then be used to determine a MOI value or MOI range for the particular bacteria strain in future test. Consistency of results may be assessed using a measure such as the coefficient of variation, or a validity/quality test may be performed by comparing the measure of variability with a threshold). Once a target MOI value or MOI range is determined for a particular bacterial strain then this may be used for future tests. In the case that a target MOI value or MOI range is not known, then a default MOI may be used based on, for example, similar strains. A default MOI value may be too for Gram negative bacteria and 10 for Gram positive bacteria.

[00100] Where the HRQT protocol is to be used with a combination of phage(s) and an antibiotic, the antibiotic solution is prepared to 100X the final test concentration in Tz/TSB medium. Typically, the concentration of the antibiotic used in the test sample is at least 1X the reported minimum inhibitory concentration (MIC) for the particular test bacteria strain. The antibiotic may be tested to ensure that it shows no effect on the Tetrazolium dye development (inhibitory or enhancement).

[00101] For combinations of phage, preferably the combinations comprise no more than three different test phage strains (i.e., preferably, phage combinations consist of 2 or 3 different phage strains). However, in some embodiments more than three different phage may be used as described herein.

[00102] Test samples comprising phage(s) (with or without antibiotic) of, for example, 100 pL may be prepared as follows in Table 2, which provides a representative example for an HRQT protocol investigating "test phage 1" either alone or in combination with other phage (i.e., test phage 2 and/or test phage 3) or with an antibiotic.

[00103] Table 1: Sample Preparation

[00104] Figure 7 shows an alternative layout of a 96 well microassay plate for the Host Range Quick Test (HRQT) protocol with four replicates per treatment sample allowing 20 different phage-host combinations to be trialed. The four replicates for each treatment sample are each spread across 4 columns of a row, in rows A to G. Row H is used for controls, with 4 replicate treatment free cell controls in columns 1-4, and 4 replicate media treatment controls in columns 5-8. Columns 9-12 may be used for an additional treatment sample or as shown in Figure 7, and 4 phage treatment controls may be placed in columns 9 to 12 comprising lOOpL of the phage in goopL of Tz/TSB media with no bacterial culture. In another embodiment these wells of row H may be used to provide antibiotic control wells (i.e. , wherein a bacteria is treated with an antibiotic only, or particularly one that may be present in a test sample in a test well elsewhere on the microassay plate). This allows 20 or 21 different phage treatments to be assessed in a single HRQT assay. [00105] Once the data is received normalization and quality control/quality assurance may be performed. These may be performed manually by the user, or preferably the computer software is configured to automatically perform a quality assessment of each plate. The quality assurance checks may include checking the phage control plate(s) for any indication of log phase growth that may invalidate that phage(s) throughout the entire test plate and checking the media control wells (column 9 Figure 1) to determine if any log phase growth is observed. If so, then that horizontal lane is voided (i.e. not analyzed) on that plate.

[00106] For example, as shown in Figure 2B, the media control wells 222 in column

9 have a general linear increase over time. Thus, in one embodiment a normalization step is performed in which each time series dataset is normalized based on an associated control curve. For example, for each dataset in columns 1-7 of a row, the media control curve 221 in column 9 of the same row is subtracted. Figure 2B is a schematic plot of the normalized host-phage response datasets shown in Figure 2A.

[00107] Other quality assurance checks may also be made to identify anomalous media control wells 222 or anomalous host cell only wells 223. Datasets associated with any identified anomalous media control wells or anomalous bacterial host cell only wells may be excluded. For example, an entire row may be discarded if the host growth curve in cell control well (column 10) is flat or has an unusual shape such as non-sigmoidal or with an unusual lag times which may indicate contamination or other growth issue with the bacteria host. For example, E. coli typically has a lag time in the range of 3-5 hours, and thus a lag time outside of this range may indicate an issue with the bacteria. Various rule based or threshold-based quality assurance procedures may be used. Further a machine learning method may also be used to perform quality assurance. This may be trained on labelled control wells 220 and used to identify anomalous control wells.

[00108] The user may manually record, or computing system may determine the duration of the bacteria (host) cell control Lag phase. With reference to Figure 1, column

10 contains bacteria cell controls, which are analyzed to determine the lag time - that is the duration (e.g. number of hours) of the Lag phase. This may be an estimate of an inflection point (the inflection time) between the end of the lag phase/start of the log phase, or some other parameter that identifies the end of the lag phase. This is recorded for each bacteria control cell. This is then repeated for each of the assay test wells containing phage and bacteria (columns 1-8). The hold time can then be calculated for each phage bacteria host pair in each test well. This is the time in which lag phase has been extended (or log phase has been delayed) relative to the control and is calculated as:

Phage/host hold time = (Lag time of test well) - (Lag time of control) (1)

[00109] For example the lag time of the control cell in column 10 may be 4 hours and the lag time of a phage-host test well in column 2 is 6 hours giving a phage/host hold time of 6-4 = 2 hours. That is the phage has inhibited growth of the bacteria by two hours. This can be performed manually for each test well or using a computer implemented method to automatically analyze each test well as discussed below. Lag times typically range between 1-4 hours, although the most fastidious (e.g., slow growing) bacteria could have longer lag times (e.g., greater than 4 hours.) In some embodiments the Lag time of control may be a single value (if there is no replication), or a representative single value such as the median or average of several replicate controls (e.g. average of each replicate cell in column 10).

[00110] A validity test (or quality test)_ may be applied to the HRQT assay for each test sample for such as requiring that a measure of variability of the replicates of the test samples are less than a predefined threshold. In one embodiment the measure of variability is the coefficient of variation (CV) which is the ratio of the standard deviation to the mean (CV = a/ii). In one embodiment the threshold is 50% (CV < 0.5), although larger thresholds such as 67%, 75%, 80%, 90% or 95% may be used. Choice of the threshold maybe made based on the expected variation based on historical or calibration results, expected heterogeneity (e.g bacterial populations in biofilms exhibit high heterogeneity than well mixed liquid cultures) or a desired level of confidence (or strictness) such as using a low threshold value (0.5) for high confidence or a high value (0.9) for lower confidence to ensure only clear outliers (ie high variability) are excluded. Robust alternatives to the co-efficient of variation may also be used such as versions based on medians and interquartile ranges, IQR, (eg RCV _Q = 0.75 x IQR/median) or median absolute deviation, MAD, (eg RCV _M = 1.4826 x MAD /median). If the HRQT assay passes the validity test it will be considered a valid assay, and if the validity test is failed then the HRQT assay ought to be regarded as invalid for that test sample and repeated.

[00111] In some embodiments the "Reportable Result" for the HRQT assay may be a summary measure of the hold time, such as the mean or median hold time for a sample (middle value for an odd number of replicates and average of the 2 middle value for an even number of replicates). This may be rounded to the nearest whole hour. Thus, for example, if the repeat hold times for a particular test sample is 5, 4, 5 and 6 hours, then the mean value if 5 hours. Repeat test results are valid for the sample (CV=o.i6), and the Reportable Result = 5 hours.

[00112] In embodiments where antibiotic combinations are tested, antibiotic synergy/antagonism to a phage may be determined. This may be defined as a difference greater than 10 hours in the mean hold times between phage along and the antibioticphage combination test wells. However other suitable values (ie different from 10 hours) could be used based on the particular case and any associated, prior knowledge, e.g. known phage hold times, or based on measured differences using the antibiotic in previous experiments.

[00113] Once any normalization and quality assurance has been performed, automated model fitting and parameter estimation may then be performed, for example to obtain an estimate of the lag time for each dataset, along with other model parameters that characterize the growth curve. A typical growth curve 242 is shown in Figure 2C has an S or sigmoidal shaped profile with a lag phase 243, growth phase 244 and stationary phase 245 up to end time point 241. The lag phase 243 can be characterized by a lag time 246 during which there is minimal observable growth of the bacteria - that is the curve is substantially flat. During the growth phase 244 the bacteria grows typically at a reasonably constant growth rate, until the growth begins to stabilize and the curve flattens into the stationary (or stabilization) phase 245 where it asymptotically approaches a maximum height 249. The growth phase 248 can be characterized as following an approximately linear path with rolled off ends at the transition between the adjacent phases, with the linear growth characterized by a constant slope 247. The first derivative of the curve 248 can be used to delineate the phase transitions, for example to estimate the inflection time.

[00114] The lag time can be defined as the time interval from the starting time to the time point where the growth curve starts to show evidence of an increase (and thus the end of the lag phase). It may be defined in a statistical model or could be set as time point when some threshold value or condition is satisfied or met. For example, it could be the first time point where the observed value is equal to some percentage of the maximum value, such as 1%, 2%, 5% or 10%. Alternatively, it could be defined based on a large deviation, such as the first value exceeding a measure variability (e.g. 2, 3 or 5 standard deviations calculated over a time window since the start of the experiment. In another embodiment, the lag time could be the intercept between the horizontal baseline starting level and the tangent line to maximum growth rate during the exponential phase of the growth curve (on a log scale). Statistical models, such as ANOVA and a may also be used to estimate the lag time, for example for example by using a model to estimate the best fit baseline values and growth rates in order to estimate the intercept point. In one embodiment the inflection time may be estimated and used as the lag time. The lag time may be used to calculate a hold time which is the lag time from which the lag time for a control is subtracted, thus representing a measure of deviation from a standard lag time. [00115] In the case that a phage inhibits the growth of the host bacteria, the growth curve will have a substantially flat profile (i.e., is not sigmoidal) which can be characterized as having a lag time equal to the end time. Thus, a preliminary step may comprise determining the maximum height of the time series dataset, and if the maximum height is less than a threshold maximum height, then classifying the time series dataset as flat and setting the lag time to the end time point and terminating (or bypassing) the fitting process. The threshold maximum height may be determined from the analysis of historical growth curves obtained using the OmniLog™ or similar platform. The threshold may be varied based on the specific equipment used to generate the host phage growth curves.

[00116] One or more functions (or models) may then be fitted for each phage host dataset (i.e. for each test well). A function maybe fit over the entire datasets, or for a portion of the dataset for example from an initial time point to an end time point. For example, this maybe from the start time (oh) to an end time (24, 36, 48 hours etc.). The end time point may be arbitrarily set to a time point sufficient for growth of the host bacteria to have stabilized and may be set prior to the end of the dataset. For example, an experiment may run for 48 hours but the end time point may be set to an earlier time such as 24 or 36 hours. The plurality of time points may be every time point in the dataset, at periodic intervals (e.g., every 15 minutes) or uniformly across the dataset. The interval between time points at which fitting is performed need to not be constant though (for example, some points could be lost due to data corruption, or the apparatus may not sample the wells at precisely regular intervals. For example, a fully loaded OmniLog™ machine takes around 15 minutes to sample every well in every plate, and thus data for each well may be collected at approximately 15 minutes intervals.

[00117] At each time point we fit one or more candidate functions over the fitting time window. The fitting time window may be from the initial time point (i.e., start time of the assay) to the current time point. The fitting fits a model of the candidate function to estimate a set of growth curve summary parameters (the fitted model parameters) comprising at least a lag time. A goodness of fit parameter may also be obtained. As shown in Figure 2C, the model may fit parameters such as a max height 249, a slope 247, a lag time 246, and an area under curve. The goodness of fit parameter measures how well the candidate function (or the model of the candidate function) matches the observed dataset, and is typically is a parameter that summarizes the difference between the observed value and the expected value from the model. In some embodiments the goodness of fit is the co-efficient of determination (R ² ), although others maybe used such as a parameter based upon an error term or a residual term, or a summary statistic of residuals.

[00118] To cope with the inherent variability of host-phage growth curves, embodiments of the model fitting method may fit one or more candidate functions at each time point and we select a best fit function for the current time point from the one or more fitted candidate functions based on the goodness of fit parameters. Each of candidate functions comprise a different functional form, at least one of which is a sigmoidal function (i.e., has an S shaped form). The sigmoidal functions may be a Gompertz function, a Logistic function or a Richards function, the functional forms 260 of which are shown in Figure 2D. A comparative plot 260 of several sigmoidal functions with different functional forms is shown in Figure 2E. Other candidate functions such as a third order or fifth order polynomial fit, linear models (including generalized linear models, non-linear regression models, may also be used. The candidate functions have different functional forms to capture different curve shapes, and deviations from a pure growth curve to capture the observed variability due to various experimental effects (e.g., dye and media effects). Various functions may be fitted to obtain a best fit (and best fit function), for example based on best goodness of fit and/or other parameters. If none of the goodness of fit estimates pass a threshold goodness of fit (e.g., R2< 0.6) then we classify the time series dataset as flat and set the lag time to the end time point and may cease model fitting for this dataset. Once the best fit is obtained, the fitted growth curve parameters including lag time, R2 and other data are stored, such as by saving to a CSV (comma-separated values) file. If hold time is not estimated by the model then the hold time may be calculated using the estimated lag time and lag time of the associated control. Individual model results for each replicate may be combined as discussed above, and quality criteria applied (eg CV < 0.5) and a reportable value obtained (e.g. mean replicate rounded to nearest hour). [00119] A “matching phage” is defined by inhibition of bacterial growth. In one embodiment a matching phage is defined as a hold time of more than 3 hours, and more preferably, 4 hours or more, and even more preferably, longer than 4 hours. This is further illustrated in Figure 4A which is a series of plots of host-phage response dataset showing short lag time (5I1), medium lag time (14I1) and large lag times (48I1) according to an embodiment. The hold time can be calculated from a control bacteria lag time (i.e., hold time = lag time with phage - control lag time), or using a reportable value (e.g. mean) estimated from several replicate values.

[00120] To assist in interpretation of data a heat map (matrix plot) may be prepared. A set of colors are used in the heat map where each color is associated with a predefined range of hold times. Each each phage-host combination is then represented in the heat map as a colored cell using the color associates with the range that the hold time of the cell falls within. Each colored cell in the heat map correspond to a separate cell in the HRQT assay, or each colored cell may correspond to the reportable result (i.e. valid average) of a phage-host combination determined from replicate samples in the HRQT. This is illustrated in Figure 4B which is a color coded matrix plot of hold times calculated from estimated lag times or hold time of host-phage response dataset for multiple phages and the same host according to an embodiment. The legend 412 defines colors for each of a plurality of time windows. The original colours have been converted to gray scale and correspond to: <1 hour (white - originally red), 1-3 hours (light gray - originally orange), 3-8 hours (very light gray - originally yellow), 8-20 hours (gray - originally green) and > 20 hours (dark gray - originally blue). Phage which inhibits bacterial growth (i.e., good phage) have large hold times (> 20 hours) and ineffective phage (i.e., poor phage) have short hold times (<1 hour). Other colors may be selected and similarly the number of classes (ie number of cells & colors) and the associated thresholds may be modified whilst retaining similar information. E.g. the 8-20 and >20 classes could be combined, and/or the 1-3 and 3-8 could be combined. Similarly, the exact thresholds could be varied provided they are capturing similar information. E.g, 8 hours could be changed to 6 hours or 10 hours, or the 20 could be reduced to 12 or increased to 24 hours depending upon how conservative a threshold for lag time is desired (these times may be more convenient for monitoring purposes).

[00121] The heat map may be displayed on a screen or printed out to assist in identifying or selecting phage for treatment of patients. For example phage in blue cells may be selected and a report generated and saved. When displayed on a screen hovering over a cell may display information regarding the cell such as the phage and bacteria in the well corresponding to the cell.

[00122] Once the plates have been read the user may select unload all done plates and the physical plates may then be removed from the incubator and disposed of in biohazard bins.

Example 2 HRQT assay of phage for infectivity of an E. coli strain

[00123] In this example, five different phage strains (phage 1-5) were selected from phage stocks and assayed, using the HRQT protocol of the Example 1, against an E. coli strain (bacterial strain A) to assess whether or not this bacteria forms part of their respective host range.

[00124] The results of the HRQT assay are shown graphically in Figure 5, where each line represents the bacterial growth (expressed as relative respiration units) of strain A in the absence (control) or presence of one of the different phage strains (i. e. , in the presence of one of the phage 1-5). Lines (curves) in Figure 5 have been converted to grayscale. Original red curve 510 represents the bacterial control, original brown long dash line 511 represents phage 1, original green dash-dot curved 512 represents phage 2, original light blue dotted line 513 represents phage 3, original dark blue solid line 514 represents phage 4 and original pink solid line 515 represents phage 5. The curves obtained with phage being present can be compared to the control curve 510 to enable the selection of phage resulting in prolonged suppression of bacterial growth. For instance, as shown in Figure 5, the uninfected bacteria (control) began logarithmic growth (log phase growth) after about 4 hours of incubation, whereas in the presence of an effective phage, the log phase growth of the bacteria was prevented over the duration of the assay (i.e., 48 hours) with phage 1 (e.g. a hold time of 48-4 = 40 hours), and delayed for phage 2, 4 and 5. Similar bacterial growth was observed when incubated with phage 3 (dotted lines) indicating that this phage does not infect the E.coli isolate (strain A) used in the assay (hold time of o hours).

[00125] Thus, in this example, since logarithmic growth was not prevented or delayed in the presence of phage 3, it has been determined that the strain A bacteria is resistant to the phage. In contrast, phage 1 was considered the most effective as it prevented breakthrough of bacterial growth (log phase growth) over the duration the assay. Phage 2, phage 4 and phage 5 delayed breakthrough of log phase growth for (i.e. had hold times of) 5 hours, 9 hours and 26 hours respectively. [00126] The durability (efficacy) of the phages can be interpreted by the length of time that any one phage holds bacterial growth from entering log phase. While each of phage 1, phage 2, phage 4 and phage 5 can be considered as "matching phage" since the inhibition of bacterial growth achieved by each was > 4 hours compared to the "no phage" bacterial control (i.e., as per the HRQT protocol of Example 1), in this example, phage 1 would be selected out of the four as a candidate for phage therapy to treat this bacteria strain.

Example 3 HRQT assay for selection of candidate phage for treating urinary tract infections

[00127] In this example, 70 different phage strains (phage 1-70) were selected from phage stocks and assayed, using the HRQT protocol of the Example 1, against 37 E. coli strains isolated from a UTI patient population. The results of the HRQT assay are presented in Figure 6 as a heat map 600 of cells 602 using the color legend shown in Figure 4B. The rows 601 represent phage, and columns 603 represent bacterial isolates This heat map shows results obtained after 48 hours, effectively summarized by the use of a color indicating the relative duration of bacterial growth inhibition within a predetermined range (e.g. each color represents a specific range). A "matching phage" 603 is one that achieved inhibition of bacterial growth (i.e. a hold time) > 4 hours as compared to a "no phage" bacterial control. The colors have been converted to grayscale and represent a range of inhibition times as compared with the bacterial control (alone) and are as follows: white (originally red) = < 1 hour, light gray (originally orange) = 1-3 hours, very light gray (originally yellow) = 4-7 hours, gray (originally) green =8-20 hours and dark gray (originally blue) = >20 hours. One E. coli bacterial strain that does not have a matching phage is highlighted in the header of the figure in purple.

[00128] The HRQT assay enabled an evaluation of the "phage coverage" of the UTI patient population with the 70 selected different phage strains.

Example 4 Host Range Quick Test (HRQT) protocol modified for use with bacterial biofilms

[00129] The HRQT protocol described in Example 1 is particularly suited for use wherein the test bacteria is grown by the planktonic mode of bacterial growth (e.g. where the test bacteria is provided free in a liquid preparation such as a liquid growth media; that is, a planktonic preparation). The protocol can, however, be readily modified for test bacteria provided as a biofilm. In this way, the HRQT protocol can be used, for example, for the selection of a single phage or a combination of phage (e.g. for use in a composition comprising a phage "cocktail"), optionally in combination with one or more antibacterial agent (e.g. one or more antibiotics, one or more bactericides, and/or one or more other therapeutic molecules such as small molecules or biologies that have bactericidal activity), for phage therapy to treat, for example, an infection involving bacteria in the form of a biofilm.

Biofilm Preparation

[00130] In this example, the test bacteria are bacterial cells sourced from a patient sample, but in some embodiments, bacteria of interest may be obtained from laboratory stocks or culture collection. The bacterial cells are streaked onto a sterile TSA plate and incubated at 37°C±2°C in a stationary incubator overnight or until colonies are sufficiently formed for use for inoculation. In some embodiments bacterial cells are inoculated into a liquid culture from the stock or culture collection and incubated at 37C in a shaking incubator at 175±SRPM.

[00131] Biofilm preparations may be prepared, for example, by adding up to 15 beads suitable for biofilm growth (e.g. sterile Glasperlen 4 mm beads; #1.04016, Millipore-Sigma, St Louis, MO, United States of America) to each well of a standard 6- well plate, with 8 ml of TSB media per well. Each well is then inoculated with one colony of cells from the incubated TSA plate and incubated at 37°C±2°C for a period of 18-24 hours to allow the bacteria to grow as a biofilm around each bead.

[00132] Using a sterile tweezer, the incubated beads are transferred to a suitable dish (e.g. a sterile large (100 x 15 mm) petri dish), 25 mL of PBS can be added, and then turbulence generated (sufficient to move the beads around the dish) by, for example, pipetting the PBS up and down with a serological pipette vigorously five times. The PBS is then removed from the dish using a serological pipette (nb. a micropipette can be used to remove much of the residual PBS). This washing process can be repeated for an additional three times, before adding a further 25 mL of fresh, sterile PBS, and storing the covered dish at, for example, ambient temperature until use.

Preparation of test samples (comprising phage(s) or phage(s) /Antibacterial Agent [00133] Phage strains are selected from phage "stocks" or a library of phage stocks, and each phage preparation is diluted, if necessary, to 4 x io ^-8 PFU/mL (also denoted herein as 4e8 PFU/mL) in TSB media including 1% v/v 1X Tetrazolium (Tz) dye as described in Example 1. The particular dye may be selected depending upon the species of the test bacteria such as set out, for example, in Table 1 below, wherein Tetrazolium Dye type "A" is Biolog product Cat#7422i (Biolog Inc., Hayward, CA, United States of America) and Tetrazolium Dye type "D" is Biolog product Cat#74224.

[00134] Table 2: Tetrazolium Dye Type per Bacterial Species

[00135] Where the HRQT protocol is to be used with a combination of phage(s) and an antibiotic, the antibiotic solution is prepared to 100X the final test concentration in Tz/TSB medium. Typically, the concentration of the antibiotic used in the test sample is at least 1X the reported minimum inhibitory concentration (MIC) for the particular test bacteria strain. The antibiotic should be tested to ensure that it shows no inhibitory effect on the Tetrazolium dye development.

[00136] For combinations of phage, preferably the combinations comprise no more than three different test phage strains (i.e., preferably, phage combinations consist of 2 or 3 different phage strains).

[00137] Test samples comprising phage(s) (with or without antibiotic) of, for example, 100 pL may be prepared as follows in Table 3, which provides a representative example for an HRQT protocol investigating "test phage 1" either alone or in combination with other phage (i.e., test phage 2 and/or test phage 3) or with an antibiotic.

[00138] Table 3: Sample Preparation

Test Plate Preparation and Assaying

[00139] Using a 96-well OmniLog™ microassay assay plate, up to 21 different treatment sample types may be readily assayed using a format as shown in Figure 7. Each treatment type may preferably be tested in four "repeat" (i.e., replicate) "horizontal" wells. For example, "Sample 1" as shown in Figure 7 may be, for example, a treatment of a test bacteria (i.e., a test bacteria provided as a biofilm on bead) with a single phage type; wherein the repeat test wells are wells 1-4 on row A. Thus, as shown in Figure 7, the microassay plate is formatted with test wells occupying the complete row of each of rows A-G. While in row H, the microassay plate is provided with four repeat bacterial cell growth control well tests (shown in Figure 7 as "Treatment-Free Biofilm Control" wells 1- 4 of row H) and four repeat growth media control well (shown in Figure 7 as "Media Treatment Control" wells 5-8 of row H). The remaining four wells of row H (i.e., wells 9- 12) may be used for assaying a further treatment test sample type ("Sample 21") if required. However, in one alternative embodiment, these wells of row H may be used to provide antibiotic control wells (i.e. wherein a biofilm-coated bacteria is treated with an antibiotic only, particularly one that may be present in a test sample in a test well elsewhere on the microassay plate). In another alternative embodiment, these "sample 21" wells of row H may be used to provide "phage treatment control" wells (i.e., phage sterility control wells), wherein test phage used in a test sample elsewhere on the microassay plate, is provided in a 100 pL amount of TZ/TSB with one sterile bead (i.e. a bead without any biofilm).

[00140] Bacterial populations within biofilms have limited interactions with their neighbors, such a low diffusion of solutes and particulates, and as a result they possess high heterogeneity in their nutrient access and physiological state, and as a result phage- bacteria interactions and bacterial susceptibility are expected to be different than in well mixed liquid cultures. This may result in more variability in a biofilm HRQT assay compared to liquid culture HRQT assay as discussed in Example 1. Thus in one embodiment a larger number of replicates may be performed for a biofilm HRQT assay compared to a liquid culture assay. For example in Example 1, 4 replicates were used and thus for a biofilm assays may use six replicates or eight replicates to improve robustness. Figure 8 shows an alternative microassay plate layout for a biofilm HRQT assay using eight replicates for each treatment sample. That is each of the 8 rows (A to H) in each of columns 1 to 9 are replicate sample (or treatment) with treatment controls in column 10 and cell A of column 11, 7 media controls in cells B-H of column 11 and 8 replicate biofilm treatment free controls in column 12.

[00141] Each of the test wells in the microassay plate (i.e., the "Sample" wells) are provided with a total volume of 100 pL of the Tz/TSB liquid growth media; an initial volume of 75 pL of Tz/TSB is provided to each of the test wells, to which a 25 pL aliquot of a sample treatment (e.g. a test sample comprising a test phage, combination of test phages, or combination of test phage(s) and an antibiotic) is added. One bead coated with a biofilm of a test bacteria is added to each of the test wells.

[00142] The growth media control wells in the microassay plate are each provided with 100 pL of the Tz/TSB growth media and one sterile bead (i.e., a bead without any biofilm). Accordingly, the growth media control wells are provided with no test phage(s), antibiotic or test bacteria.

[00143] The bacterial cell growth control wells in the microassay plate are each provided with 100 pL of the Tz/TSB growth media and one bead coated with a biofilm of a test bacteria (i.e., the bacterial cell growth control wells are provided with no test phage(s) or antibiotic).

[00144] This protocol and layout readily enables the generation of a plurality of hostphage response results/datasets (e.g., for inhibition of bacterial logarithmic growth) in a manner substantially as described in Example 1. Typically, the incubation period used for the HRQT protocol of this example will similarly be of 48 hours (2 days) duration with reads taken at 15 min intervals. As described above in relation to Example 1, the MOI may be selected to have a target value or MOI range, and will typically be within the range of 1000 down to 0.0001, but for a specific bacterial strain, a specific MOI value or subrange of MOI values may be used. [00145] Following the incubation period, the user may log into the HRQT software and review the data for the microassay assay plate. This review will involve a confirmation that the various control wells have provided the anticipated results. That is, that:

1. The bacterial cell growth control wells show log phase growth of the test bacteria;

2. The growth media control wells show no indication of log phase growth of any bacteria; and that

3. Any phage treatment control wells show no indication of log phase growth of any bacteria.

[00146] If the results of bacterial cell growth control wells and/or the growth media control wells are not as anticipated, then the HRQT assay as a whole ought to be regarded as invalid. On the other hand, if a phage treatment control well produces a result indicating that there has been log phase growth of any bacteria, then the user may opt to discard only the results from test wells that included the phage type of that particular phage treatment control well.

[00147] The user may manually record, or the computing system may determine the duration of the test bacteria (host) cell control lag phase in a manner substantially as described in Example 1. More particularly, the lag phase duration for each test well and the bacterial cell growth control wells is determined. As discussed above the lag phase duration is the time, in hours, between the test start and a point indicating where log phase growth begins, such as an inflection point. As discussed above in relation to example 1, the lag time or hold time may be obtained using automated model fitting and parameter estimation on the cell results. . As in example (1) the hold time for each test well may be calculated according to:

Hold Time test well — Lag Duration test well ^— Lag Duration biofilm control (2)

The lag phase duration for the biofilm control maybe a single value (if there is no replication), or a representative single value such as the median or average of several replicate controls (e.g Lag Duration biofilm control — Lag Duration biofilm control average).

A validity test (or quality test) may be applied to the HRQT assay for a test sample such as requiring that a measure of variability of the replicates of the test samples are less than a predefined threshold. In one embodiment the measure of variability is the coefficient of variation (CV) which is the ratio of the standard deviation to the mean (CV = (r/fi). As discussed above biofilm HRQT assay maybe more variable than liquid cell culture HRQT Assays (Example 1) and thus a higher threshold such as 75% (CV < 0.75) may be used. However smaller or larger thresholds such as 50% 67%, 75%, 80%, 90% or 95%may be used with the choice of the threshold made based on the expected variation based on historical or calibration results, expected heterogeneity (e.g bacterial populations in biofilms exhibit high heterogeneity than well mixed liquid cultures) or a desired level of confidence such as using a low threshold value (0.5) for high confidence or a high value (0.9) for lower confidence to ensure only clear outliers (ie high variability) are excluded. Robust alternatives to the co-efficient of variation may also be used such as versions based on medians and interquartile ranges, IQR, (eg RCV _Q = 0.75 x IQR/median) or median absolute deviation, MAD, (eg RCV _M = 1.4826 x MAD/median). If the HRQT assay passes the quality criteria it will be considered a valid assay, and if the quality criteria is failed then the HRQT assay ought to be regarded as invalid for that test sample and repeated. [00148] The "Reportable Result" for the HRQT assay may be a summary measure, such as the mean or median hold time (middle value for an odd number of replicates and average of the 2 middle value for an even number of replicates). This may be rounded to the nearest whole hour. Thus, for example, if the repeat hold times for a particular test sample is 5, 4, 5 and 6 hours, then the mean value if 5 hours. Repeat test results are valid for the sample (CV=o.i6), and the Reportable Result = 5 hours. This indicates that the particular phage(s) of the test sample can be considered as "matching" and the particular test sample might then be selected as a candidate for phage therapy of an infection involving a biofilm of the particular test bacteria. The results may be reported using a heat map representation as discussed in relation to Example 1.

[00149] Embodiments described herein thus advantageously provide automated methods for analyzing /interpreting host phage response data that are designed to be robust to the intrinsic variability observed in host phage response datasets. Embodiments of the method fits a range of functions over a range of time points from the start time to an end time and at each time point including fitting one or more sigmoidal functions such as Gompertz, Logistic and Richards functions (although others could be used). The best fitting function can thus be obtained and saved to allow robust automated estimation of lag time from which a hold time can be obtained. The automated analysis saves considerable manual labor and time, with a pair of human experts taking around 45 minutes to analyze 48 plates. In contrast embodiments of the method can analyze 48 plates in around 10 minutes. Further the method is robust and results can be automatically exported to a data storage, web server, or laboratory information management system, along with associated experimental meta data.

[00150] Figure 3 depicts an exemplary computing system (300) with a number of components that may be used to perform the analysis methods described herein. For example, an input/output ("I/O") interface 330, one or more central processing units ("CPU") (340), and a memory section (350). The I/O interface (330) is connected to input and output devices such as a display (320), a keyboard (310), a disk storage unit (390), and a media drive unit (360). The media drive unit (360) can read/write a computer- readable medium (370), which can contain programs (380) and/or data. The I/O interface may comprise a network interface and/or communications module for communicating with an equivalent communications module in another device using a predefined communications protocol (e.g., Bluetooth, Zigbee, IEEE 802.15, IEEE 802.11, TCP/IP, UDP, etc.).

[00151] A computing system may comprises one or more processors including multi-core CPUs and Graphical Processing Units (GPUs) operatively connected to one or more memories which store instructions to configure the processor to perform embodiments of the method. Figure 3 depicts an exemplary computing system configured to perform any one of the computers implemented methods described herein. In this context, the computing system may include, for example, one or more processors (CPUs, GPUs), memories, storage, and input/output devices (e.g., monitor, keyboard, disk drive, network interface, Internet connection, etc.). However, the computing system may include circuitry or other specialized hardware for cariying out some or all aspects of the processes. The computing system may be a computing apparatus such as an all-in-one computer, desktop computer, laptop, tablet or mobile computing apparatus, server, and any associated peripheral devices. The computer system may be a distributed system including server based systems and cloud-based computing systems. In some operational settings, the computing system may be configured as a system that includes one or more units, each of which is configured to carry out some aspects of the processes either in software, hardware, or some combination thereof. For example, the user interface maybe provided on a desktop computer or tablet computer, whilst the fitting may be performed on a server based system including cloud based server systems, and the user interface is configured to communicate with such servers. The user interface may be provided as a web portal, allowing a user on one computer to upload datasets which may be processed on a remote computing apparatus or system (e.g., server or cloud system) and which provides the results (i. e. , the report) back to the user, or to other users on other computing apparatus.

[00152] The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. For a hardware implementation, processing maybe implemented within one or more application specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), processors including CPU and GPUs, controllers, micro-controllers, microprocessors, other electronic units designed to perform the functions described herein, or a combination thereof. Software modules, also known as computer programs, computer codes, or instructions, may contain a number a number of source code or object code segments or instructions, and may reside in any computer readable medium such as a RAM memory, flash memory, ROM memory, EPROM memory, registers, hard disk, a removable disk, a CD-ROM, a DVD-ROM, a Blu-ray disc, or any other form of computer readable medium. In some aspects the computer-readable media may comprise non- transitory computer-readable media (e.g., tangible media). In another aspect, the computer readable medium may be integral to the processor. The processor and the computer readable medium may reside in an ASIC or related device. The software codes may be stored in a memory unit and the processor may be configured to execute them. The memory unit may be implemented within the processor or external to the processor, in which case it can be communicatively coupled to the processor via various means as is known in the art. A computer program maybe written, for example, in a general -purpose programming language (e.g., Pascal, C, C++, Java, Python, JSON, etc.) or some specialized application-specific language to provide a user interface, perform the function or model fitting, and export results.

[00153] A non-transitory computer-program product or storage medium comprising computer-executable instructions for carrying out any of the methods described herein can also be generated. Anon-transitory computer-readable medium can be used to store (e.g., tangibly embody) one or more computer programs for performing any one of the above-described processes by means of a computer. Further provided is a computer system comprising one or more processors, memory, and one or more programs, wherein the one or more programs are stored in the memory and configured to be executed by the one or more processors, the one or more programs including instructions for carrying out any of the methods described herein.

[00154] Those of skill in the art would understand that information and signals may be represented using any of a variety of technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and chips maybe referenced throughout the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

[00155] Those of skill in the art would further appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software or instructions, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

[00156] Throughout the specification and the claims that follow, unless the context requires otherwise, the words “comprise” and “include” and variations such as “comprising” and “including” will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00157] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00158] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.