Anti-microbial sensitivity testing of microbes in a urine sample

Abstract

The invention relates to a method of conducting an anti-microbial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents; and associated methods.

Description

[0001] Anti-microbial sensitivity testing of microbes in a urine sample

[0002] The present invention relates to methods to determine anti-microbial susceptibility of microbes in a urine sample.

[0003] Urinary tract infections (UTIs) are among the most common bacterial infections, affecting millions of individuals annually. Accurate and timely detection of bacterial concentration in urine samples is critical for diagnosing UTIs and guiding appropriate antimicrobial therapy. Traditional culture-based techniques remain the gold standard for bacterial detection and quantification. These involve inoculating urine samples on selective media, incubating them for 24-48 hours, and counting bacterial colonies to determine concentration. Although these methods are highly sensitive and specific, they are time-consuming and require significant pre-processing steps.

[0004] Antibiotic Susceptibility tests (AST) require a further overnight incubation step from pure cultures isolated in the first step. Leading to a minimum of 48 h before antibiotic susceptibility is known, this leads to empirical prescribing and potential delay for inappropriate use of antibiotics.

[0005] In addition to culture techniques, other methods of bacterial detection and quantification, such as flow cytometry, polymerase chain reaction (PCR), and microscopy, have been explored. Flow cytometry allows for rapid bacterial enumeration by analyzing suspended particles, but it often requires pre-treatment steps like sample staining or filtration to isolate bacteria from the urine matrix. Similarly, PCR provides high sensitivity and specificity by detecting bacterial DNA, yet it necessitates complex sample preparation steps such as DNA extraction and amplification. Microscopic methods, including direct counting with a hemocytometer or specialized imaging systems, demand careful sample preparation to minimize debris and enhance visualization of bacteria. These pre-processing requirements can introduce additional delays, costs, and variability into the diagnostic workflow.

[0006] The dependence on pre-processing steps, coupled with the intrinsic time demands of these methods, underscores the need for more efficient approaches. Delays in obtaining bacterial concentration results can hinder timely diagnosis and treatment, potentially leading to prolonged patient discomfort, disease progression, or inappropriate antimicrobial use.The present invention seeks to overcome the limitations of existing anti-microbial susceptibility test (AST) methods by developing a technique that minimizes or eliminates pre-processing steps while providing rapid, accurate, and reliable measurement of microbial growth and antibiotic susceptibility directly in samples. This innovation aims to significantly reduce the time to diagnosis, streamline laboratory workflows, and support the early and effective management of antibiotic use for UTIs.

[0007] According to a first aspect of the present invention, there is provided a method of conducting an antimicrobial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents, the method comprising:

[0008] providing a urine sample comprising the microbes to be tested;

[0009] diluting the urine sample by at least two different dilution factors, to provide at least two different dilutions of the urine sample;

[0010] i) placing one dilution of the urine sample into at least two microcapillaries of a multiplexed microcapillary device, wherein a first microcapillary is used for a control urine sample comprising no anti-microbial agents, a second microcapillary is used for a test urine sample comprising an anti-microbial agent;

[0011] ii) repeating step i) for each dilution factor of the urine sample;

[0012] iii) determining the doubling time of the microbes in the control urine sample using the difference in growth kinetics between the at least two dilution factors of the control urine sample; and

[0013] iv) determining the doubling time of the microbes in the test urine sample using the difference in growth kinetics between the at least two dilution factors of the test urine sample; determining whether the doubling time of the microbes is increased in the presence of the anti-microbial agent or a particular concentration of the anti-microbial agent relative to the control comprising no anti-microbial agents, wherein an increased doubling time indicates inhibition by the anti-microbial agent.

[0014] The method of the invention may be used to determine the minimum inhibitory concentration of an anti-microbial and / or the presence of anti-microbial resistance of microbes in the urine sample. Antibiotic susceptibility may be confirmed by an inhibition of growth of microbes in a microcapillary in the presence of an antimicrobial. The MIC of the antibiotic may be determined as the lowest concentration at which the microbes are inhibited from growing. In one embodiment, where growth of the microbes is detected in the control and / or in the presence of a concentration of the antimicrobial agent and growth of the microbes is inhibited at a higher concentration of the anti-microbialagent, the higher concentration may be an indication of the minimum inhibitory concentration (MIC) of the anti-microbial agent.

[0015] Advantageously, the accuracy of the measurement of anti-microbial susceptibility can be improved whilst not having to pre-process the sample prior to testing. Advantageously, the use of serial dilution removes the need for time consuming and costly pre-processing of the sample prior to its testing, such as sedimentation, filtering and resuspending at a known bacterial density according to existing tests. For example, the maximum wait time for a threshold infection of a Gram-negative bacterial infection could be in the order of about 6h, which is a significant improvement from the >48 h under the current standard clinical laboratory testing pathways. For testing antibiotic resistance, false resistance results can potentially be avoided by testing serial dilutions and ignoring dilutions where rapid detection indicates high starting cell density. However, delayed growth even at high cell densities can give us confidence in antibiotic susceptibility. The confidence in susceptibility and resistance results can be increased taking the results from 2 or more dilutions. Advantageously, determining the minimum inhibitory concentration of an antimicrobial and / or antimicrobial resistance of the microbes in the sample can allow the selection of an antimicrobial or an antimicrobial combination, that is capable of treating a subject's infection, such as a UTI.

[0016] Determining the doubling time using the difference in growth kinetics between dilutions

[0017] The doubling time may be determined according to the following equation:

[0018]

[0019] wherein At is the change in growth time between two dilutions. The growth time may be measured by observing how long before growth is detected. The growth time may be measured at the same growth stage of each dilution, for example at half the maximum growth of the growth curve of the microbe, for example as measured by optical density / turbidity, and / or in an embodiment that uses a growth indicator dye, the half maximum fluorescence or colour change of the growth indicator dye. Preferably the growth time is measured at the same point during the exponential growth stage. In one embodiment, the growth time is measured at the point that each dilution reaches the same density of microbes during their growth. Preferably, the same relative point on the curve is taken for each dilution, between the inflection point and the end of the exponential phase.

[0020] In one embodiment, the cell number (e.g. cfu / ml) may be estimated for each dilution and / or in the neat sample, for example using the growth kinetics of the microbes. False resistance may be avoidedby interpreting the susceptibility on higher dilution factors, such as dilutions comprising less than 107cfu / ml. Sample dilutions within a 105to 107cfu / ml range of microbial cell density, may provide a higher confidence (e.g. more accurate) result for AST determination. Therefore, false resistance may be avoided by interpreting dilutions comprising about 105to 107cfu / ml.

[0021] The multiplexed microcapillary device

[0022] The multiplexed microcapillary device may comprise a multiplexed microcapillary film. In a preferred embodiment, the multiplexed microcapillary device comprises a fluoropolymer microcapillary film (MCF) that contains a parallel array of multiple microcapillaries, such as about 5-10 microcapillaries. The multiplexed microcapillary device may comprise a hydrophilic polymer coating, for example to modify the internal walls of the capillaries allowing sample uptake by capillary action. Suitable microcapillary film is described, for example, by Edwards et al. (Lab Chip, 2011, 11 , 4267 —4273), which is herein incorporated by reference.

[0023] The multiplexed microcapillary device may comprise a multiplexed microcapillary film, for example with a range of different antibiotic concentrations in the capillaries or a range of different antibiotics. Each capillary may have one anti-microbial agent, or more than one type of anti-microbial agent, such as two or more antibiotic combinations.

[0024] The capillaries may be microcapillaries (i.e. having a diameter in the 1-1000 micrometer range). The multiplexed microcapillary device may comprise microcapillary film. The volume of the sample in each capillary may be about 0.5-3 microliters, preferably 1 microliter.

[0025] In one embodiment a further test sample may be provided by loading each dilution into a third microcapillary comprising:

[0026] a) the same anti-microbial agent provided in the second microcapillary, but at a different concentration, or

[0027] b) a different anti-microbial agent relative to the anti-microbial agent provided in the second microcapillary.

[0028] In a preferred embodiment, the multiplexed microcapillary device comprises a multiplexed microcapillary film comprising a first control capillary having no anti-microbial agent, and a second capillary comprising an anti-microbial agent at a first concentration. The multiplexed microcapillaryfilm may comprise a third capillary with an increased concentration of the same anti-microbial agent as the second capillary. A fourth, fifth, sixth, or more, capillary may be provided wherein each additional capillary comprises an increased concentration of the anti-microbial agent relative to the previous capillaries (or vice versa with decreasing concentrations of the anti-microbial agent). The increasing / decreasing concentrations may be used to determine the minimum inhibitory concentration (MIC) of the anti-microbial agent for microbes in the sample.

[0029] A fourth, fifth, sixth, or more, capillary may be provided wherein each capillary comprises a different anti-microbial agent.

[0030] The capillary contents and number may be replicated for each different dilution factor of the sample, thereby allowing the measurement of the different growth kinetics for each dilution for each of the test samples and control samples.

[0031] The anti-microbial agent may be pre-loaded in the microcapillaries prior to the sample addition.

[0032] At least two of the dilutions of unknown microbial concentration may be selected for determination of the microbial growth and / or antimicrobial sensitivity. A first lower-factor dilution of unknown microbial concentration may be used to verify presence of microbes and / or determine the growth inhibition of such microbes, but may not be relied upon to provide an accurate result for antimicrobial sensitivity, and a second higher-factor dilution of unknown microbial concentration may be used to determine the growth inhibition or lack thereof to provide a significantly higher confidence of the antimicrobial sensitivity result relative to the lower-factor dilution. In one embodiment, a first lower factor dilution of unknown microbial concentration may be used to verify the presence and growth of the microbes in the sample and a second higher dilution factor of unknown microbial concentration may be used to determine antimicrobial sensitivity or resistance. Additional higher factor dilutions in series may be provided to give ever more confidence in the antimicrobial sensitivity result relative to the lower-factor dilutions. Additional serial dilution factors may advantageously increase the likelihood of providing a dilution that can provide a high confidence result within 18 hours, or less, such as 6 hours or less.

[0033] The anti-microbial agentThe anti-microbial agent may comprise a test agent that may be tested for potential as an antimicrobial agent. Therefore, in one embodiment, the term "anti-microbial agent" may be substituted for "test agent" herein.

[0034] The anti-microbial agent may comprise any agent that is capable of, or potential for, inhibiting the growth of the microbes in the sample. The anti-microbial agent may comprise an antibiotic. In an embodiment wherein the sample comprises yeast, the antimicrobial agent may comprise an antifungal.

[0035] The antibiotic may comprise any selected from the group comprising cephalexin, ampicillin, amoxicillin, amoxicillin-clavulanic acid, nitrofurantoin cefoxitin, cefotaxime, ciprofloxacin, cefuroxime, ceftazidime, co-trimoxazole, meropenem, ofloxacin, nitrofurantoin, amikacin, gentamicin, Fosfomycin, trimethoprim, ertapenem, penicillins, cephalosporins, carbapenems, monobactams, fluoroquinolones, aminoglycosides, tetracyclines; or combinations thereof.

[0036] Advantageously, determining the minimum inhibitory concentration of an antimicrobial and / or antimicrobial resistance of the microbes in the sample can allow the selection of an antibiotic or antibiotic combination that is capable of treating a subject's UTI.

[0037] The Sample

[0038] In a preferred embodiment, the sample is of unknown microbial concentration (e.g. prior to conducting the method herein). Dilutions of the sample loaded into the microcapillaries may be of unknown microbial concentration. The microbial concentration may not be determined prior to dilution and / or prior to measuring growth of the microbes in the microcapillaries.

[0039] Preferably the sample is diluted before placing it into the microcapillaries. The sample may be diluted 1:10, or more. Alternatively, the sample may be diluted 1:100, or more. In one embodiment, the sample may be diluted 1:200, or more. In another embodiment, the sample may be diluted 1:1000, or more. In another embodiment, the sample may be diluted 1:2000, or more. In another embodiment, the sample may be diluted 1:4000, or more. In one embodiment, the sample may be diluted up to 1:5000. In another embodiment, the sample may be diluted between 1:2 and 1:10,000. In another embodiment, the sample may be diluted between 1:10 and 1:4000. In a preferred embodiment, the sample may be diluted between 1:10 and 1:1000. Multiple serial dilutions ofdifferent factors may be provided, for example multiple serial dilutions of the sample may be provided in different microcapillaries. The sample may be diluted by at least three different dilution factors, to provide at least three different dilutions of the sample. In another embodiment, the sample may be diluted by at four different dilution factors, to provide at least four different dilutions of the sample. In another embodiment, the sample may be diluted by 2-6 different dilution factors, to provide at least 2-6 different dilutions of the sample. In one embodiment, the sample maybe diluted about 1:5 to 1:20, about 1:100 to 1:500, and about 1:3000 to 1:10,000. In one embodiment, the sample may be diluted about 1:10, about 1:200 and about 1:4000.

[0040] The dilution may be with a microbial growth medium. The dilution may be with a bacterial growth medium, such as Mueller Hinton broth or a functionally equivalent medium thereof. The growth medium may be suitable for maintaining a culture of bacteria, such as Gram-negative bacteria. The growth medium may be suitable for maintaining a culture of Gram-negative and / or Gram-positive bacteria. The growth medium may be capable of maintaining a culture of Escherichia spp. (such as E. coli), Klebsiella spp., Pseudomonas spp. (such as Pseudomonas aeruginosa), Enterococcus spp., Proteus spp., Enterobacter spp., Staphylococcus spp. (such as coagulase-negative staphylococci), or Acinobacter spp.; or combinations thereof.

[0041] In another embodiment, the microbial growth medium may be a yeast growth medium.

[0042] The sample may be diluted with a growth indicator dye in addition to the medium.

[0043] Preferably, the sample is diluted with Mueller Hinton broth. More preferably, the sample is diluted with Mueller Hinton broth and growth indicator dye.

[0044] Preferably the sample is diluted, but otherwise not pre-processed prior to loading it into the microcapillaries. Preferably the sample is diluted, but otherwise not pre-processed prior to the addition of the metabolic dye. The microbes in the sample may not be subject to one or more of staining, filtration, inoculation, pelleting, resuspending or incubation prior to being added to the microcapillaries. The sample may be diluted and the microbes in the sample may not be subjected to one or more of staining, filtration, inoculation, or incubation prior to being added to the microcapillaries. The microbes in the sample may be diluted and the microbes in the sample may not be subjected to pelleting and resuspension, for example to adjust their concentration prior to dilution or to remove urine matrix from the sample.The sample may be from a subject who is a mammal, preferably a human. In another embodiment the sample is from a subject who is a non-human animal, such as a non-human mammal. The sample may be from a subject who is suffering from or suspected to be suffering from a disease or condition, such as an infection. The sample may be from a subject known or suspected to be suffering from a UTL The UTI may comprise cystitis.

[0045] The sample may be a fresh sample (i.e. not stored or frozen). Alternatively the sample may be a stored sample, for example stored in a universal or boric acid container. In one embodiment, the sample may have been stored in a container comprising boric acid. The sample may comprise or not comprise a bacteriostatic agent, such as boric acid.

[0046] Advantageously, the dilutions of the present method have been found to overcome the presence of a bacteriostatic agent, such as boric acid used in sample containers. In particular, a linear relationship can be established at higher dilutions where the boric acid has been diluted out sufficiently to not interfere with bacterial growth and / or the antimicrobial sensitivity testing.

[0047] Measuring Growth

[0048] Measuring the growth kinetics of the microbes in the microcapillaries may be by any means known to the skilled person. In one embodiment, a growth indicator dye is used, wherein the change in colour and / or fluorescence of the dye indicates active metabolism of microbes present. The growth indicator dye may comprise a metabolic dye. Additionally or alternatively, growth may be measured by detecting an increase in turbidity of the sample in the microcapillaries.

[0049] In one embodiment, the sample further comprise a growth indicator dye, and a change in colour and / or fluorescence of the growth indicator dye is measured over a period of time which indicates microbial growth in the microcapillaries. The method may comprise irradiating the sample in the microcapillaries with light and measuring colour and / or fluorescence intensity with a light detector to measure the change in colour and / or fluorescence intensity of the growth indicator dye over a period of time which indicates microbial growth.

[0050] In one embodiment, the method comprises detecting any increase in turbidity in the microcapillaries over a period of time which indicates microbial growth in the microcapillaries. The method maycomprise irradiating the sample in the microcapillaries with light to detect light scatter, which indicates the presence and growth of microbes within the microcapillaries. Detection of the light scatter may comprise the use of a light detector to measure an increase in light scatter (turbidity) over a period of time which indicates microbial growth.

[0051] In one embodiment, the sample further comprise a growth indicator dye, and a change in colour and / or fluorescence of the growth indicator dye is measured over a period of time which indicates microbial growth in the microcapillaries; and the method further comprises detecting any increase in turbidity in the microcapillaries over a period of time which indicates microbial growth in the microcapillaries.

[0052] In a preferred embodiment, the colour / fluorescence change measurement and the irradiation measurement steps are provided on the same sample in the same microcapillary. The colour / fluorescence change measurement and the turbidity measurement steps may be provided on the same sample in the same microcapillary sequentially (i.e. one before the other). The colour / fluorescence change measurement and the turbidity measurement steps may be immediately after each other, such as within less than 5 minutes, preferably within 1 minute. The colour change / fluorescence measurement and the turbidity measurement steps may be within 30 seconds of each other.

[0053] Each of the colour / fluorescence change and the turbidity measurement steps maybe repeated at least once (i.e. there are at least two measurements of the colour / fluorescence and the turbidity properties of the sample in the microcapillaries). Such repeated detections / measurements may be separated by the period of time. The colour / fluorescence change and the turbidity measurement steps may be repeated periodically, such as about every 1-5 minutes, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 30 seconds, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 1 minute, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 5 seconds, or more.

[0054] The period of time for measuring the microbial growth may be 0 minutes to 18 hours (i.e. the time between at least two measurements). The period of time for measuring the microbial growth may be 10 minutes to 18 hours. The period of time for measuring the microbial growth may be 1 to 18 hours.The period of time for measuring the microbial growth may be 18 hours, or less. The period of time for measuring the microbial growth may be 6 hours, or less.

[0055] The period of time for measuring the change in colour / fluorescence of the growth indicator dye may be 10 minutes to 18 hours (i.e. the time between at least two measurements). The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 1 to 18 hours. The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 18 hours, or less. The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 6 hours, or less.

[0056] The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 10 minutes to 18 hours (i.e. the time between at least two measurements). The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 1 to 18 hours. The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 18 hours, or less. The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 6 hours, or less.

[0057] The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the light scatter from the microbes in the microcapillaries may be 18 hours, or less. The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the light scatter from the microbes in the microcapillaries may be 6 hours, or less. The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the turbidity from the microbes in the microcapillaries may be from 10 minutes to 18 hours.

[0058] The growth indicator dye

[0059] The growth indicator dye may comprise any molecule that has a detectable change in colour and / or fluorescence intensity upon metabolization by microbes. The growth indicator dye may comprise metabolic dye. In a preferred embodiment, the metabolic dye comprises or consists of resazurin.

[0060] Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a phenoxazine dye that is weakly fluorescent, nontoxic, cell-permeable, and redox-sensitive. Resazurin is blue to purple in colour and weaklyfluorescent, and it is irreversibly reduced to resorufin, which is pink in colour and strongly fluorescent, by microbial metabolism.

[0061] In another embodiment, the growth indicator dye may comprise a pH indicator dye, such as phenol red, methylene blue or fluorescein. pH indicator dyes will change colour based on the pH of the media which is affected by microbial metabolism. In another embodiment, the growth indicator dye may comprise a sugar conjugate dye, such as MUG (4-Methylumbelliferyl-a-D-galactopyranoside).

[0062] Advantageously, growth indicator dye conversion, such as resazurin to resorufin, is proportional to the number of microbes present. The higher the microbial concentration the faster the reaction which can be calibrated to a calibration curve.

[0063] The growth indicator dye may, such as resazurin, may be provided in sufficient concentration to provide a detectable colour and / or fluorescence change in the presence of microbial metabolism. The growth indicator dye may, such as resazurin, may be provided in sufficient concentration to provide a detectable colour and / or fluorescence change in the presence of microbial metabolism, but at a concentration that is not toxic to microbes in the sample. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 5 pg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 50 pg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 0.1 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 0.2 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of about 0.005 mg mL-1to about 1 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of about 0.25 mg mL-1.

[0064] The light detector may be any means that can measure the intensity and wavelength of light emanating from the microcapillaries. The light detector may be any means that can detect and measure a change of colour and / or fluorescence in the sample in the microcapillaries, for example from blue to pink, and / or from lower fluorescence to higher fluorescence. The change in colour and / or fluorescence of the metabolic dye may be determined by measuring a change in fluorescence intensity. The detected wavelength may be at the appropriate, such as maximal, emission for the metabolic dye or its converted product, such as resorufin. In a preferred embodiment, the light detector is a camera. Preferably the camera is a digital camera. The camera may be a smartphone camera.The same light detector, such as the same camera, may be used for both the colour / fluorescence change measurement and the turbidity measurement steps described herein.

[0065] The light detector, such as a camera, may determine the colour / fluorescence intensity of the growth indicator dye and the determined colour / fluorescence intensity may be converted to a microbial concentration value, such as CFU / ml, for example by reference to a standard. The standard may be a predetermined standard, such as a chart or table, or the standard maybe one or more microcapillaries comprising microbes of a known concentration and measured under the same conditions as the sample (i.e. the test sample).

[0066] In one embodiment, the detection of an increase in the turbidity from the microbes in the microcapillaries is an indication of growth and concentration of the microbes wherein the concentration of the microbes is determined in CFU / ml relative to a standard. The level of turbidity may be converted to a microbial concentration value, such as CFU / ml, for example by reference to a standard. The standard may be a predetermined standard, such as a chart or table, or the standard may be one or more microcapillaries comprising microbes of a known concentration and measured under the same conditions as the sample (i.e. the test sample).

[0067] The turbidity detection may otherwise be known to the skilled person as an irradiation and light scatter measurement. The light may be irradiated at an optimum wavelength and intensity for detection of microbial cells. In one embodiment, the detection of light scatter is at 600 nm (ODgoo), optionally at an intensity capable of allowing the detection of light scatter.

[0068] The level of turbidity may be determined by a turbidimeter or a nephelometer. Nephelometric detection is based on a diode laser that directs a light beam through the sample. If there are no insoluble particles in solution, the light beam will simply pass through. The detector, set to the side of the path of the laser light beam (usually at a 90° angle), will measure no turbidity. In case a solid, insoluble matter is present in solution, the laser beam will be scattered in different directions by the particles and an optic system will collect and direct the scattered light to the detector. The higher the amount of particles scattering the light beam, the more light reaches the detector, the more Nephelometric Turbidity Units (NTU) will be measured. Nephelometry, particle density is a function of the scattered light directed to the detector.In a preferred embodiment, the same camera, such as a digital camera, is used to determine the colour / fluorescence intensity of the sample and the level of turbidity.

[0069] The microbes

[0070] The microbes in the sample may be single cell microbes, such as bacteria or yeast.

[0071] The microbes in the sample or potentially in the sample may be bacteria, such as Gram-negative and / or -positive bacteria. The bacteria in the sample or potentially in the sample may be any of the group consisting of Escherichia spp. (such as E. coli), Klebsiella spp., Pseudomonas spp. (such as Pseudomonas aeruginosa), Enterococcus spp., Proteus spp., Enterobacter spp., Staphylococcus spp. (such as coagulase-negative staphylococci), and Acinobacter spp.; or combinations thereof. In one embodiment, the bacteria in the sample or potentially in the sample is E. coli. The bacteria in the sample or potentially in the sample may be antibiotic-resistant bacteria. The antibiotic-resistant bacteria may be resistant to multiple antibiotics.

[0072] In one embodiment, the microbes comprise yeast. The yeast may be Candida spp., such as one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans.

[0073] Other Aspects

[0074] According to another aspect of the present invention, there is provided a method of selecting an antimicrobial for treatment of a subject with an infection, the method comprising determining the antimicrobial sensitivity or resistance of microbes in a sample of the subject in accordance with the invention herein, and selecting an antimicrobial or antimicrobial combination that is capable of treating the infection.

[0075] According to another aspect of the present invention, there is provided a method of determining the incidence of antimicrobial sensitivity of a microbial infection in a subject, the method comprising the determination of the antimicrobial sensitivity of microbes in a sample from the subject in accordance with the invention herein.The incidence of antimicrobial sensitivity may comprise or consist of one or more of the presence of antimicrobial sensitivity, the level of antimicrobial sensitivity, or type of antimicrobial sensitivity, of microbes in a sample from the subject.

[0076] The sample may be a urine sample. The patient may have a UTI.

[0077] Such determination may inform treatment options for the infection, such as a UTI.

[0078] The methods herein may further comprise the treatment of the subject for the infection, such as a UTI, such as by administration of a therapeutic agent for treatment of the infection (such as treatment for a UTI).

[0079] According to another aspect of the present invention, there is provided a method of treatment of a subject for an infection, the method comprising the determination of the antimicrobial sensitivity or resistance of microbes in a sample from the subject in accordance with the invention herein; and administration of a therapeutic agent for treatment of the infection.

[0080] In one embodiment, the subject has a UTI, and the sample is a urine sample.

[0081] The therapeutic agent may be an antimicrobial, such as an antibiotic. The choice (e.g. type or combination) of the antimicrobial may be determined by the method of the invention herein. The therapeutic agent may be an antimicrobial or antimicrobial combination to which the microbe is susceptible, e.g. as determined by the methods herein.

[0082] According to another aspect of the present invention, there is provided a method of conducting an anti-microbial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents, the method comprising:

[0083] i) providing a urine sample comprising the microbes to be tested;

[0084] ii) diluting the urine sample by at least two different dilution factors, to provide at least two different dilutions of the urine sample;

[0085] iii) culturing the at least two different dilutions of the urine sample and using the difference in growth kinetics between the at least two different dilutions to determine the doubling time of microbes in the urine sample, wherein each dilution factorincludes a control sample having no anti-microbial agent present, and one or more test samples comprising an anti-microbial agent;

[0086] iv) determining whether the doubling time of the microbes is increased in the test sample in the presence of the anti-microbial agent or a particular concentration of the antimicrobial agent relative to the control sample having no anti-microbial agents, wherein an increased doubling time indicates inhibition by the anti-microbial agent. The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0087] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0088] Figure 1 - Example workflow, (a-b) A urine sample or other sample containing bacteria is diluted at least 2 times in Mueller-Hinton Broth and resazurin solution, (c) Microcapillary devices containing 10-parallel capillaries are dipped into each dilution and the sample is loaded by capillary action, (d) The fluorescence change caused by bacteria metabolising resazurin is captured over time and (e) differential fluorescence measurements between the different conditions relates to bacteria present in the sample and growth or inhibition in the presence of different antimicrobials.

[0089] Figure 2 - Growth characteristics of an E. coli isolate measured by fluorescence intensity of resazurin to resorufin conversion. The fluorescence intensity is monitored at regular intervals. The growth curve shows a simple four parameter logistic curve fit. The lag phase, slope, half maximum and maximum fluorescence are highlighted.

[0090] Figure 3 - Growth characteristics differ between different organisms. A shows the growth curve for an E. coli isolate. B) shows the growth curve for a Staphylococcus saprophyticus isolate. C) shows the growth curve for serial dilution of an E. coli isolate. D) shows the growth curves for serial dilutions for a S. saprophyticus isolate. Black arrows indicate the time difference between each serial dilution.

[0091] Figure 4 - Calibration curve of several urine samples containing Gram-negative bacteria, serially diluted and the number of bacteria present (CFU / mL) plotted against the time for resazurin conversion. This allows an estimation of the number of bacteria present in a urinesample to calculated based on the time taken for the resazurin dye to be converted. A similar calibration curve can be produced for turbidity measurements.

[0092] Figure 5- Example of analysis of AST directly from a urinesamplesfor four first-line antibiotics. In this example the urine sample contains a Gram-negative bacteria which is resistant to cefoxitin (CFX), ampicillin (AMP) and ampicillin-clavulanic acid (AMC) and susceptible to nitrofurantoin (NIT) the order of antibiotics are shown in Fig3a. The samples are processed as shown in Figure 1 and the time-lapse images are presented as a single (b) image, in which each horizontal pixel indicates a still image taken every 5-minutes. (c) The rapid conversion of resazurin to resorufin in the lowest dilution factor (1:10) indicates a bacterial density of greater than 108CFU / mL and dilutions indicating a bacterial doubling time of 34 minutes, (d-e) Indicate the 4-paramret logistic curve fit of the fluorescence intensity over time, (d) Even at this high bacterial density, there is a delay in growth in the nitrofurantoin containing capillaries (e) which is confirmed in the subsequent dilutions. This shows that even at high bacterial density and rapid result, differential growth can accurately identify antibiotic susceptibility.

[0093] Figure 6 - Example of analysis of AST directly from a urine sample. In this example, a high cell density in the lowest dilution factor indicates a false resistant result until subsequent dilutions are analysed, (a) shows the layout of antibiotics in each test strip. The samples are processed as shown in Figure 2 and the time-lapse images are presented as a single (b) image, in which each horizontal pixel indicates a still image taken every 5-minutes. In the lowest dilution factor (1:10) this contains the highest density of bacteria and based on the generation of fluorescence in capillaries 1-8, would indicate the bacteria in the sample are resistant to cefoxitin (CFX), ampicillin (AMP) and ampicillin-clavulanic acid (AMC) and susceptible to nitrofurantoin (NIT). This is further shown in the fluorescence intensity against time graph (c) which shows no delay the capillary 5 containing 8 mg / L ampicillin compared to capillary 1 which contains no antibiotic. However, following a (d) further dilution of the sample, we see complete inhibition of growth in all antibiotic containing capillaries compared to the growth controls. This indicating the bacteria in the sample are susceptible to all antibiotics tested.

[0094] Figure 7 - Example of analysis of AST directly from a urine sample stored in boric acid containers, (a) shows the layout of antibiotics in each test strip. The samples are processed as shown in Figure 1 and the time-lapse images are presented as a single (b) image, in which each horizontal pixel indicates a still image taken every 5-minutes. (c) shows the 4-parameterlogistic curve fit for the fluorescence intensity against time. In low dilution factors, the boric acid inhibits the growth of bacteria. This effect is reduced in further dilutions, (d) the linear relationship between time for resazurin conversion and bacterial cell density is lost at low dilution factors. This effect is reduced in further dilutions.

[0095] Example 1 - Measurement for the rapid detection of antibiotic resistance / susceptibility testing of bacteria directly from urine

[0096] A method for detecting the susceptibility and resistance to antibiotics of bacteria in urine, in the presence or absence of a bacteriostatic agent boric acid, where a urine sample is serially diluted and split into multiple ~1 microlitre compartments, with or without antibiotics, and with growth monitored at time intervals in all dilutions, and the delayed growth observed in the presence of boric acid is still different in resistant vs susceptible organisms.

[0097] Determination of bacteria characteristics, growth rates and estimation of cell density

[0098] The determination of growth rates and the cell density in a urine sample is important for accurate antibiotic susceptibility testing. Reference AST methods require a fixed inoculum between 2-8 xlO5CFU / mL to produce a valid result. However, clinical samples such as urine will contain a range of cell densities unknown until after culture. Therefore, the estimation of the number of bacteria present in a test is important. For analysis of antibiotic susceptibility, the type of organism present is also important, as different organisms can have different breakpoints which indicate susceptibility / resistance. Using multiple dilutions of a urine sample, the growth characteristics of the bacteria in the sample can be analysed and the antibiotic susceptibility accurately reported.

[0099] The microcapillary AST device is dipped into each dilution which is loaded into the device by capillary action. The sample is then incubated in a reader that is capable of monitoring the fluorescence conversion of resazurin to resorufin and the light scatter of bacterial cells in the capillaries (Figure 1). The microcapillary AST device contains a growth control which does not contain any antibiotics. The growth of the bacteria can be monitored over time to produce a growth curve (Figure 2). These growth curves are different depending on organisms in the sample (Figure 3a). For example, the time taken to convert resazurin and the slope of the curve will change dependent on organism and cell density. The time taken between each serial dilution can be used to calculate the doubling time of the organism present (Figure 3c-d). The same relative point on the curve is taken for each dilution, between theinflection point and the end of the exponential phase. For example, If the time taken for resazurin conversion with 10sCFU / mL takes 3 h and 105takes 4 h. The difference between a ten-fold dilution is 1 h. This can be converted to doubling time by the following equation:

[0100]

[0101] wherein At is the change in growth time between two dilutions.

[0102] This example would indicate an organism with a doubling time of 18 minutes. The doubling time will vary depending on the organism or organisms in the sample.

[0103] Illustration of calculating doubling time from difference in time to growth detection between two 10-fold dilutions of bacterial cells

[0104] The change in time between two 10-fold dilutions, termed At, is measured by observing how long before growth is detected, with the dilutions A and B having the relationship: 10 x ndiiution B= ^dilution A

[0105] ndiiutionA 'sthe number of cells in dilution A, and ndiiution Bis the number of cells in dilution B.

[0106] Dilution B has lOx fewer cells, therefore growth is detected later by a length of time At.

[0107] The doubling time is defined as the time taken for bacterial growth to double the cell numbers. For any period of time, you can calculate a growth factor by taking the period of time, dividing it by the doubling time to see how many doubling times have occurred, and then calculating 2 to the power of the number of doubling times.

[0108] Therefore, after a difference in time of At the number of cells will increase by a factor of 2 to the power At

[0109]

[0110] of t divided by the doubling time, i.e. increase in cell number of 2 ^doubling

[0111] For 10-fold dilutions, this increase in cells will be lOx, thus we have equation 1:

[0112]

[0113] We can take logs in base 2 from this expression as follows:

[0114]

[0115] Rearranging this, we can calculate the doubling time from the change in growth detection using equation 2:

[0116]

[0117] If we take log base 2 of 10, we get 3.321928, and can simply divide the difference in time by this number.

[0118] For example, if we have observed growth in dilution A of 5h but 6hl5minutes for dilution B, we have At = Ih 15 minutes = 75 minutes and therefore calculate doubling time of 22.6 minutes (1 decimal place).

[0119] Alternatively, for a slower growing bacteria detection of dilution A might be 5h but dilution B has growth detected after 7h. In this example, we have At = 2h = 120 minutes and therefore calculate a doubling time of ~36 minutes.

[0120] Analysis of AST direct from urine samples

[0121] Figure 1 shows the urine sample process. Urine samples, either containing boric acid or without are serially diluted in a resazurin, Mueller-Hinton broth mix. This may be done in a 96 well plate format. Each microcapillary test strip (a single 10 capillary test strip) is placed into each dilution. Antibiotics are already loaded within the capillaries. The test is sealed to avoid evaporation during incubation. As the bacteria actively metabolise in the presence or absence of antimicrobials, the weakly fluorescent resazurin is converted into the highly fluorescent resorufin. The analysis of antibiotic susceptibility and resistance is based on a combination of the time taken for resazurin to be converted by the bacteria and differential growth compared to the growth control in the presence of antibiotics.

[0122] The time to resazurin conversion in proportional to the number of bacteria present in a sample (Figure 4). However, in cell densities greater than 108CFU / mL, rapid conversion occurs (within 1 hr) making it difficult to predict the number of bacteria in these samples.

[0123] Figure 5 shows the AST analysis of a urine samples following 3 serial dilutions. In this example, ( FigSb) a full 18 h timelapse series following bacterial conversion of resazurin to resorufin is displayed as a single image, in which each pixel wide horizontal line represents the fluorescence intensity of every 5 minute interval. In this case, we can follow the fluorescence generation of bacterial metabolism displayed as time 0 at the top of the image and the fluorescence intensity after 18 h at the bottom of the image.The serial dilution allows several bacterial cell densities to be analysed per sample. This can give us information on the number of cells present in the sample and the doubling time of the bacteria present. The time taken for each sample to convert resazurin (Fig 5c) can be compared to a calibration curve such as Figure 2. The rapid conversion of resazurin by urine following a 1:10 dilution in Figure 5 gives confirmation of bacteria present in the sample within one hour. Indicates a high density of bacteria in the sample around 5xl08colony forming units per ml (CFU / mL).

[0124] Samples which contain a high density of bacteria can show a false resistant result. This is a well characterised effect of a high inoculum. This results in a low confidence for a resistant result at higher cell densities. However, a delayed growth in the presence of antibiotics, at a high cell density indicates good confidence of a susceptible result. In Figure 5, the bacteria in the sample are susceptible to the antibiotic nitrofurantoin (NIT). Even at the 1:10 dilution, we see delayed growth in the presence of this antibiotic compared to the growth control (Fig 5d) which is confirmed and the effect increased in lower cell densities and high dilution factors (Fig 5e). The second dilution increases the confidence in the antibiotic susceptibility result within 3 hours and the third dilution increases confidence in resistant result within 4 hours. In this example, the bacteria in the sample are resistant to the three other antibiotics tested, cefoxitin (CFX), ampicillin (AMP) and amoxicillin-clavulanic acid (AMC). In these cases, there is no delayed growth in the presence of these antibiotics regardless of the cell density present in the test.

[0125] Analysis of AST direct from urine samples showing high inoculum results in false resistant result:

[0126] High cell densities are known to cause false resistant results. In Figure 6, we show that at low dilution factor, we see no differential growth in the presence of CFX, AMP and AMC at 1:10 dilution. This would normally indicate a resistant result. The time taken for resazurin conversion would indicate a cell density of lxlO8CFU / mL indicating a high cell density of which we cannot be confidant of the resistant results in the 1:10 dilution. Fig 6c shows there is no delay in growth in the presence of ampicillin compared to the growth control in a 1:10 dilution. However, a further dilution, 1:200 shows a complete inhibition of growth of bacteria in all antibiotics tested. This brings the cell density to what would approximately be used for a standard AST, 5xl05CFU / mL and a confidant susceptibility result within 6 hours.

[0127] Analysis of AST direct from urine samples stored in boric acid containers:Due to the high volume of urine samples tested in the community, often urine samples are placed in boric acid containers to control bacterial growth. Boric acid is a bacteriostatic agent that reduces the change in bacterial numbers while they are being transported to the laboratory for testing. Boric acid in the urine sample can affect the growth of bacteria within the microcapillary test.

[0128] In this example, we show how at low dilution factors, higher concentration of boric acid can reduce bacterial growth, which is then rescued by dilution and analysis of further dilutions. In this example (Fig 7), the bacteria in the urine are susceptible to all antibiotics tested and show complete inhibition of growth in the presence of antibiotics over the duration of the test. However, the highest cell density containing sample, 1:10, would normally show the fastest growth. However, the boric acid in the sample has delayed this and the subsequent dilution, 1:200 shows faster resazurin conversion (Fig 7c-d) and a further dilution rescues the linear relationship between resazurin conversion and bacterial density (Fig7c).

Claims

CLAIMS1. A method of conducting an anti-microbial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents, the method comprising:providing a urine sample comprising the microbes to be tested;diluting the urine sample by at least two different dilution factors, to provide at least two different dilutions of the urine sample;i) placing one dilution of the urine sample into at least two microcapillaries of a multiplexed microcapillary device, wherein a first microcapillary is used for a control urine sample comprising no anti-microbial agents, a second microcapillary is used for a test urine sample comprising an anti-microbial agent;ii) repeating step i) for each dilution factor of the urine sample;iii) determining the doubling time of the microbes in the control urine sample using the difference in growth kinetics between the at least two dilution factors of the control urine sample; andiv) determining the doubling time of the microbes in the test urine sample using the difference in growth kinetics between the at least two dilution factors of the test urine sample; determining whether the doubling time of the microbes is increased in the presence of the anti-microbial agent or a particular concentration of the anti-microbial agent relative to the control comprising no anti-microbial agents, wherein an increased doubling time indicates inhibition by the anti-microbial agent.

2. The method according to claim 1, wherein the minimum inhibitory concentration of the antimicrobial and / or the presence of anti-microbial resistance of microbes in the sample is determined by confirming inhibition of growth of microbes in a microcapillary in the presence of a particular antimicrobial or antimicrobial concentration3. The method according to claim 1 or 2, wherein the multiplexed microcapillary device comprises a fluoropolymer microcapillary film (MCF) that contains a parallel array of multiple microcapillaries.

4. The method according to any preceding claim, wherein the volume of the sample in each capillary is 0.5-3 microliters.

5. The method according to any preceding claim, wherein the multiplexed microcapillary device comprises a multiplexed microcapillary film comprising a first control microcapillary having no antimicrobial agent, and a second microcapillary comprising an anti-microbial agent at a first concentration; andoptionally the multiplexed microcapillary film comprises a third microcapillary with an increased concentration of the same anti-microbial agent as the second microcapillary; and further optionally a fourth, fifth, sixth, or more, microcapillary is provided wherein each additional capillary comprises an increased concentration of the anti-microbial agent relative to the previous microcapillaries.

6. The method according to any preceding claim, wherein at least two of the dilutions of unknown microbial concentration may be selected for determination of the microbial growth and / or antimicrobial sensitivity, optionally wherein the microbial cell number is estimated and false resistance is avoided by interpreting dilutions comprising less than 107cfu / ml.

7. The method according to any preceding claim, wherein a first lower factor dilution of unknown microbial concentration is used to verify the presence and growth of the microbes in the sample and a second higher dilution factor of unknown microbial concentration is used to determine antimicrobial sensitivity or resistance.

8. The method according to any preceding claim, wherein the urine sample comprises a bacteriostatic agent.

9. The method according to any preceding claim, wherein the anti-microbial agent comprises a test agent that is to be tested for potential as an anti-microbial agent.

10. The method according to any preceding claim, wherein the anti-microbial agent comprises an antibiotic or antibiotic combination; orWherein the antimicrobial agent comprises an anti-fungal.

11. The method according to any preceding claim, wherein the sample is a urine sample.

12. The method according to any preceding claim, wherein sample is of unknown microbial concentration.

13. The method according to any preceding claim, wherein the sample is diluted 1:10, or more.

14. The method according to any preceding claim, wherein sample is diluted between 1:10 and 1:4000.

15. The method according to any preceding claim, wherein the sample is diluted by at least three different dilution factors, to provide at least three different dilutions of the sample.

16. The method according to any preceding claim, wherein the sample is diluted about 1:5 to 1:20, about 1:100 to 1:500, and about 1:3000 to 1:10,000.

17. The method according to any preceding claim, wherein a growth indicator dye is used to measure microbial growth and / or turbidity increase is used to measure microbial growth.

18. The method according to any preceding claim, wherein period of time for measuring microbial growth is less than 18 hours.

19. A method of selecting an antimicrobial for treatment of a subject with an infection, the method comprising determining the antimicrobial sensitivity or resistance of microbes in a sample of the subject in accordance with any of claims 1-18, and selecting an antimicrobial or antimicrobial combination that is capable of treating the infection.

20. A method of determining the incidence of antimicrobial sensitivity of a microbial infection in a subject, the method comprising the determination of the antimicrobial sensitivity of microbes in a sample from the subject in accordance with any of claims 1-18.

21. A method of treatment of a subject for an infection, the method comprising the determination of the antimicrobial sensitivity or resistance of microbes in a sample from the subject in accordance with any of claims 1-18; andadministration of a therapeutic agent for treatment of the infection.

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