Method for determining the concentration of intact microorganisms in a sample

Abstract

The present invention relates to a method of determining the concentration of intact microorganisms in a sample comprising optionally diluting an aliquot of the sample to provide a diluted aliquot at a dilution value; contacting at least a portion of an aliquot or of a diluted aliquot of the sample with first and second stains capable of binding to DNA, wherein the first stain is a fluorescent stain, is cell-permeable, and has a first emission wavelength, and the second stain is cell-impermeable, and is capable of acting as an acceptor molecule in a FRET pair with the first stain acting as a donor molecule; imaging the aliquot-stain mixture at the first emission wavelength and determining an image analysis value for the number of objects corresponding to intact microorganisms in the imaged mixture; and comparing the image analysis value for said aliquot to a pre-determined calibration curve, as well as an apparatus, a consumable and a kit therefor.

Claims

1. A method of determining the concentration of intact microorganisms in a sample derived from a sample containing microbial and non-microbial cells, said method comprising: (a) providing a sample containing microorganisms, wherein non-microbial cells in a sample containing microbial and non-microbial cells have been selectively lysed, or wherein non-microbial cells in a sample containing microbial and non-microbial cells have been selectively lysed and microbial cells have been recovered therefrom; (b) optionally diluting an aliquot of said sample to provide a diluted aliquot at a dilution value; (c) contacting at least a portion of an aliquot of the sample of step (a), or of a diluted aliquot of the sample either during or after dilution step (b), with first and second stains capable of binding to DNA to provide a sample-stain mixture, wherein said first stain is a fluorescent stain, is cell-permeable, and has a first emission wavelength, said second stain is cell-impermeable, and is capable of acting as an acceptor molecule in a FRET pair with the first stain acting as a donor molecule and has a higher DNA binding affinity than the first stain, such that the second stain is able to displace the first stain from DNA; (d) imaging the aliquot-stain mixture of step (c) at the first emission wavelength and determining an image analysis value for the number of objects corresponding to intact microorganisms in the imaged mixture, wherein said imaging is performed on a suspension of microorganisms; and (e) comparing the image analysis value for said aliquot to a pre-determined calibration curve, thereby to determine the concentration of intact microorganisms in said sample.

2. The method of claim 1, wherein: (i) an aliquot of the sample is diluted to provide a diluted aliquot at a dilution value, wherein contacting step (c) takes place either during or after dilution step (b), and wherein steps (c)-(e) are performed on the diluted aliquot; and/or (ii) wherein the method comprises diluting aliquots of said sample to provide two or more diluted aliquots at different dilution values, wherein said two or more aliquots are prepared simultaneously before or during step (c), or sequentially wherein a second or further diluted aliquot is prepared after steps (d) and/or (e); and/or (iii) wherein steps (c) and (d) are performed on two or more aliquots at different dilution values, and wherein step (e) comprises identifying an aliquot which comprises an image analysis value within the range of a pre-determined calibration curve, and comparing the image analysis value for said aliquot to said pre-determined calibration curve, thereby to determine the concentration of viable microorganisms in said sample.

3. The method of claim 2, wherein steps (c) and (d) are performed on each aliquot simultaneously, or wherein steps (c) and (d) are performed on each aliquot sequentially.

4. The method of claim 1, wherein: (i) when the non-microbial cells in the sample containing microbial and non-microbial cells have been selectively lysed in (a), said method does not comprise detecting the second stain; or (ii) when the non-microbial cells in the sample containing microbial and non-microbial cells have been selectively lysed and microbial cells have been recovered therefrom in (a), said method comprises detecting the second stain.

5. The method of claim 4, wherein when the non-microbial cells in the sample containing microbial and non-microbial cells have been selectively lysed and microbial cells have been recovered therefrom, step (d) comprises simultaneously imaging each aliquot-stain mixture to detect the first and second stains.

6. The method of claim 1, wherein the sample comprises microorganisms contained in a growth medium.

7. The method of claim 1, wherein the first and second stains are pre-mixed to form a stain solution, prior to contacting the aliquot of the sample or diluted aliquot of the sample, or portion thereof, in step (c).

8. The method of claim 1, wherein: (i) the fluorescence intensity of said first fluorescent stain at said first emission wavelength is enhanced when the stain is bound to nucleic acid; and/or (ii) said first fluorescent stain has excitation and emission wavelengths in the wavelength range 350-700 nm; and/or (iii) said first fluorescent stain is a green-fluorescent stain; and/or (iv) said first fluorescent stain is an unsymmetrical cyanine dye; and/or (v) the first fluorescent stain is a SYTO stain; and/or (vi) the first fluorescent stain is SYTO BC; and/or (vii) the second stain is a fluorescent stain; and/or (viii) the second stain is a red-fluorescent stain; and/or (ix) the red-fluorescent stain is propidium iodide.

9. The method of claim 1, wherein: (i) an image is obtained at one or more focal planes through the suspension; or (ii) said imaging comprises obtaining a series of 2-D images along an optical axis, wherein each image is obtained at a different position along the optical axis through a volume of the suspension.

10. The method of claim 9, wherein: (i) step (c) of contacting with the stains is performed at a temperature of greater than 4 C.; and/or (ii) in the contacting of step (c) the aliquot or diluted aliquot, or portion thereof, is incubated with the first and second stains for a time period of 1 to 20 minutes; and/or (iii) the imaging in step (d) is carried out at room temperature; and/or (iv) in the imaging step (d) it is identified whether the microorganisms are clustering or non-clustering and a calibration curve is used which is predetermined for clustering or non-clustering microorganisms; and/or (v) the images are analysed for fluorescence intensity and/or size of each enumerated object, and optionally morphology of each enumerated object; and/or (vi) the images are analysed for maximum fluorescence intensity, median fluorescence intensity and/or area of each enumerated object; and/or (vii) the images are analysed for maximum, median and/or mean fluorescence intensity and/or area of the population of objects; and/or (viii) the concentration of viable microorganisms is determined.

11. A method for determining the antimicrobial susceptibility of a microorganism in a sample, said method comprising: (i) providing a sample containing a viable microorganism wherein (i) non-microbial cells in a sample containing microbial and non-microbial cells have been selectively lysed, or wherein (ii) non-microbial cells in a sample containing microbial and non-microbial cells have been selectively lysed and microbial cells have been recovered therefrom; (ii) performing steps (b)-(e) as defined in claim 1 on said sample to determine the concentration of intact microbial cells in said sample; (iii) inoculating a series of test microbial cultures for an antibiotic susceptibility test (AST) using the sample in step (i), wherein the series of test microbial cultures comprises at least two different growth conditions, wherein the different growth conditions comprise one or more different antimicrobial agents, and each antimicrobial agent is tested at two or more different concentrations; and (iv) assessing the degree of microbial growth in each growth condition; wherein the concentration of microbial cells in said sample or said test microbial cultures is adjusted if necessary to a desired or pre-determined concentration; and wherein the degree of microbial growth in each growth condition is used to determine at least one MIC value for at least one antimicrobial agent, thereby to determine the antimicrobial susceptibility of said microorganism in said sample.

12. The method of claim 11, wherein: (i) based on the concentration determined in step (ii), the concentration of at least a portion of the sample of step (i) is adjusted to provide an inoculum for inoculating the test microbial cultures in step (iii); and/or (ii) the step of adjusting the concentration comprises a dilution based on the concentration determined in step (ii); and/or (iii) following step (ii), at least a portion of the sample of step (i) is diluted to provide an inoculum for step (iii); and/or (iv) wherein the concentration of microorganisms in the inoculated microbial test cultures is in the range 4.510.sup.580% or 510.sup.560%; and/or (v) at least one of the test microbial cultures comprises fastidious medium; and/or (vi) the concentration adjustment comprises culturing or further culturing the sample; and/or (vii) if the concentration of microorganisms in the sample is below 110.sup.6 microorganisms, the AST assay is not performed with the sample.

Description

(1) FIG. 1 shows the number of intact microorganisms measured in a H. influenzae (A) or P. aeruginosa (B) sample recovered from a blood culture, incubated with a stain solution at 4 C., room temperature, and 35 C.

(2) FIG. 2 shows the combined data for the relationship between the concentration of intact microorganisms in a sample, and the object number determined by imaging, for a range of different microorganisms. The data show good linearity, with a spread of approximately an order or magnitude between the different microorganisms. Additionally, S. aureus (a clustering microorganism) deviates from the main data.

(3) FIG. 3 shows the combined data from FIG. 2 (axes reversed) for all non-clustering microorganisms, with a regression line adapted to minimise the number of experimental points falling outside 60% boundaries from the best-fit line, per EUCAST recommendations.

(4) FIG. 4 shows the data from FIG. 3 for individual microorganisms. (AK. pneumonia; BH. influenza; CP. aeruginosa; DP. mirabilis; EE. faecalis; FS. pneumonia). The same calibration curve was found to be suitable for all non-clustering microorganisms tested.

(5) FIG. 5 shows the data and a calibration curve for S. aureus. A separate calibration curve was required for S. aureus, as this was the only clustering microorganism tested in these experiments.

(6) FIG. 6 shows the combined data from FIG. 2 (axes reversed) for all non-clustering microorganisms, with a regression line adapted to minimise the number of experimental points falling outside 80% boundaries from the best fit line. With the exception of H. influenzae, all data points above the LOD fell within these boundaries.

(7) FIG. 7 shows the data from FIG. 6 for individual microorganisms AK. pneumonia; BH. influenza; CP. aeruginosa; DP. mirabilis; EE. faecalis; FS. pneumonia) using a 80% boundary. The best fit curve was adjusted accordingly. The same calibration curve was found to be suitable for all non-clustering microorganisms tested.

(8) FIG. 8 shows the data and a calibration curve for S. aureus at the 80% boundary.

(9) FIG. 9 shows a schematic and simplified diagram of a first AST apparatus that is suitable for receiving a cell culture according to an embodiment of the present invention.

(10) FIG. 10 shows a schematic and simplified diagram of a second AST apparatus that is suitable for receiving a cell culture according to an embodiment of the present invention.

(11) FIG. 11 shows a schematic and simplified diagram of a third AST apparatus that is suitable for receiving a cell culture according to an embodiment of the present invention.

(12) FIG. 12 shows a schematic and simplified diagram of a fourth AST apparatus that is suitable for receiving a patient sample according to an embodiment of the present invention.

(13) FIG. 13 shows a schematic and simplified diagram of a fifth AST apparatus that is suitable for receiving a patient sample according to an embodiment of the present invention.

(14) FIG. 14 shows a schematic and simplified diagram of a sixth AST apparatus that is suitable for receiving a patient sample according to an embodiment of the present invention.

(15) FIG. 15 shows a schematic and simplified diagram of a concentration determination apparatus that is suitable for receiving a cell culture according to an embodiment of the present invention.

(16) FIG. 16 shows a consumable suitable for use in the AST apparatus of FIGS. 9 to 14 or the concentration determination apparatus of FIG. 15.

EXAMPLE 1

Concentration Determination and Setting Up an AST Assay

(17) A first aliquot of a sample (20 l) is diluted in 80 l PBS, and a 15 l aliquot of the diluted mixture is added to 15 l stain solution containing 10 M SYTO BC and 3 M propidium iodide to provide a first diluted aliquot/stain mixture. An aliquot of the sample/PBS mixture (20 l) is diluted further in 180 l PBS, and a 15 l aliquot is diluted in 15 l stain solution to provide a second diluted aliquot/stain mixture.

(18) The first and second diluted aliquot/stain mixtures are transferred to separate imaging wells to a depth of approximately 2 mm. 50 images are obtained of microorganisms in suspension for each well, spaced 30 m apart in the direction of the optical axis, using an emission filter at 502-561 nm to detect the SYTO BC emission peak at 509 nm. The images obtained are thresholded and subjected to analysis to determine the size, fluorescence intensity, and optionally morphology of each object corresponding to an intact microorganism to obtain an image analysis value for each diluted aliquot. Characteristics of the microorganisms in the sample are used to select a pre-determined calibration curve for use in the concentration determination step (e.g. to determine whether the sample is a clustering or a non-clustering microorganism). One of the diluted aliquots having an image analysis value within the range of a pre-determined calibration curve is identified. The concentration of intact microorganisms in the sample is determined by comparing the image analysis value for the selected diluted aliquot with the pre-determined calibration curve.

(19) An inoculum for inoculating a series of test microbial cultures for an antimicrobial susceptibility test (AST) assay is prepared by adjusting the concentration of at least a portion of the sample by dilution using a suitable growth medium (e.g. cation adjusted Mueller Hinton broth (CAMHB), if necessary. Optionally, a further inoculum in fastidious medium is also prepared. The inoculum (or inocula) is prepared to a concentration of 510.sup.5 CFU/ml and is added to a series of wells containing freeze-dried antimicrobial agents to prepare a series of test microbial cultures at at least two different growth conditions in an AST assay.

EXAMPLE 2

Effect of Stain Incubation Temperature on Concentration Determination

(20) 10 ml blood spiked with H. influenza or P. aerurinosa was added to a Bactec flask (BD) and incubated overnight until a positive culture result was obtained. Selective lysis of non-microbial cells present in the blood culture flask was performed using a lysis buffer and samples were filtered using a 0.2 m nylon mesh filter. Following filtration, samples were washed with cation adjusted Mueller Hinton broth (CAMHB) and resuspended by back-flushing CAMHB through the filter membrane.

(21) Diluted aliquots of the recovered microorganism samples were further diluted and contacted with stain solutions as outlined in Example 1. Diluted aliquot/stain mixtures were covered with foil, and incubated for 5 minutes at 4 C., room temperature, or 35 C. Each aliquot was imaged and the images analysed as outlined in Example 1 to determine an image analysis value for the number of objects corresponding to intact microorganisms. Repeats were performed for each diluted aliquot at each temperature. An image analysis value for a control sample was also determined. The image analysis values determined following preparation at each temperature was compared, and found to be similar for all temperatures for H. influenzae. Room temperature and 35 C. preparation were found to be similar for P. aeruginosa. Similar values were also determined for the control sample at all temperatures.

(22) Aliquots of the recovered microbial sample were diluted by a factor of 110.sup.4, 110.sup.5 and 110.sup.6 in PBS and plated to confirm microbial viability.

EXAMPLE 3

Preparing Pre-Determined Calibration Curves

(23) Data were collected for a number of different microorganisms at different concentrations, and the relationship between the number of objects counted and the concentration of intact microorganisms was plotted on a graph (FIG. 2). These data show that there is a linear relationship between the number of objects counted and the concentration of intact microorganisms for the majority of microbial species (albeit with a spread of approximately an order of magnitude for the number of objects counted for a given concentration between the different species measured).

(24) Data from the compiled runs for non-clustering microorganisms were combined to allow a best-fit line to be calculated (FIG. 3). The best-fit line was not generated to provide a mathematical best fit line, but rather was prepared to minimise the number of data points falling outside 60% limits, as per EUCAST guidelines. Data for individual microorganisms was compared with the generated best-fit line. Counts below the limit of detection are shown in a shaded region. Thus, the same best-fit line can be used to determine the concentration of both Gram-negative and Gram-positive bacteria in a sample.

(25) A separate best-fit line was calculated for S. aureus (FIG. 5), a clustering microorganism.

(26) A large proportion of the data points for H. influenzae fell outside the original 60% boundaries for the best fit curves. New best-fit curves based on 80% boundaries were generated for non-clustering (FIGS. 6 and 7) and clustering (FIG. 8) microorganisms. All data points above the limit of detection, with the exception of H. influenzae, fall within the new boundaries.

(27) FIG. 9 shows a schematic and simplified diagram of a first AST apparatus 1 that is suitable for analysing a cell culture sample. The first AST apparatus 1 comprises a processor 10 operable to control subsystems within the first AST apparatus 1, including a sample pipette 12 (i.e. a sample aliquoting device), a diluent pipette 14 (i.e. a diluent aliquoting device), a microscope 16 (i.e. a first imaging device 16), and a line camera 18 (i.e. a second imaging device).

(28) The first AST apparatus 1 receives a first consumable 30a in which is provided a sample container 31 (i.e. a container for receiving the sample), a diluent reservoir 35, a diluted aliquot container 36 (i.e. a container for receiving a diluted aliquot), a stain reservoir 37 comprising first and second stains, and an imaging well 38. The imaging well 38 has a viewable area of least 2 mm by 2 mm, and a depth (for example, 3 mm) which is sufficient to allow a liquid depth of at least 2 mm.

(29) The apparatus 1 also receives a second consumable 40a which comprises a plurality of wells 42 for test microbial cultures, wherein the wells 42 comprise a plurality of different antimicrobial agents, and each antimicrobial agent is provided at a plurality of concentrations.

(30) In use, a user loads a sample (not shown) into the sample container 31 in the first consumable 30a, and loads the first consumable 30a into the first AST apparatus 1.

(31) In use the processor 10 is configured to: dilute the cell culture sample in the diluted aliquot container 36 using diluent transferred by the diluent pipette from the diluent reservoir 35; transfer the diluted aliquot from the diluted aliquot container 36 to the imaging well 38; transfer the first and second stains from the stain reservoir 37 to the imaging well 38; image the imaging well 38 using the microscope 16, to determine the concentration of cells; transfer the diluted aliquot from the diluted aliquot container 36 to the wells 42; control the line camera 18 to image the wells 42 for assessing the degree of microbial growth in each well; and analyse the images in order to determine MIC values for the antimicrobial agents, in order to determine the antimicrobial susceptibility of the microorganism in the sample.

(32) The first and second stains are capable of binding to DNA to provide a sample-stain mixture. The first stain is a fluorescent stain, is cell-permeable, and has a first emission wavelength, and the second stain is cell-impermeable, and is capable of acting as an acceptor molecule in a FRET pair with the first stain acting as a donor molecule. In this example, the first stain is SYTO 9 and the second stain is propidium iodide.

(33) The processor 10 is operable to control the microscope 16 to image the imaging well 38 at the first emission wavelength. In use, the microscope 16 is focused on a plane inside the imaging well 38, for example parallel to the bottom, removed at a distance from the bottom (in this example, 0.2 mm from the bottom), and is configured to move the focal plane continuously through the liquid during the time of imaging, for example for a total of 1.5 mm during the image acquisition time (for example, 20-30 seconds).

(34) The processor 10 is operable to analyse the images obtained by the microscope 16 to determine an image analysis value for the number of objects corresponding to intact microorganisms in the imaged mixture. The processor 10 is configured to compare the image analysis value to a pre-determined calibration curve, thereby to determine the concentration of intact microorganisms in the sample.

(35) Two similar AST apparatuses are shown in FIGS. 10 and 11; both are also suitable for analysing a cell culture sample. The components of these apparatuses are broadly similar to those of the first AST apparatus 1, and discussion of the corresponding features is not repeated here. Instead, the discussion is focussed on the differences compared to the first AST apparatus 1 shown in FIG. 9. In particular, the differences relate to the location of the imaging well 38.

(36) FIG. 10 shows a schematic and simplified diagram of a second AST apparatus 2. Whereas in the first AST apparatus 1 the imaging well 38 is in the first consumable 30a, the imaging well is omitted from the first consumable 30b of the second AST apparatus 2, and is instead provided in the second consumable 40b.

(37) FIG. 11 shows a schematic and simplified diagram of a third AST apparatus 3. The third AST apparatus 3 receives the first consumable 30b (as described with reference to FIG. 10) and the second consumable 40a (as described with reference to FIG. 9), and also receives a third consumable 50. The imaging well 38 is provided in the third consumable 50.

(38) FIGS. 12 to 14 show schematic and simplified diagrams of three further AST apparatuses 4, 5, 6, which are suitable for receiving a patient sample (for example, a blood sample from a blood culture flask). FIGS. 12 to 14 are broadly similar to FIGS. 9 to 11, respectively. The difference in each case is that the first consumable 30c (which includes an imaging well) and 30d (which does not include an imaging well) include additional components in order to prepare the patient sample for analysis. The first consumables 30c and 30d include a lysis buffer reservoir 32, a filter 33 (i.e. a recovery means) and a resuspended cell container 34 (i.e. a container for receiving the sample).

(39) The processor 10 of the further AST apparatuses 4, 5, 6, shown in FIGS. 12 to 14 is further configured to: cause the sample pipette 12 to transfer an aliquot of the sample from the sample container 31 to a syringe (not shown) to be mixed with lysis buffer from the lysis reservoir 32; filter the lysed sample through the filter 33; recover microbial cells from the filter 33 by resuspending them from the filter 33 using culture medium (from a culture medium reservoir, not shown);

(40) FIG. 15 shows a concentration determination apparatus 7 which is similar to the third AST apparatus 3 shown in FIG. 11, except that the concentration determination apparatus 7 does not include the components which make the AST apparatus 3 of FIG. 11 suitable for performing AST analysis. The concentration determination apparatus 7 is adapted to perform a concentration determination analysis only. Compared to the third AST apparatus 3 shown in FIG. 11, the concentration determination apparatus 7 of FIG. 15 omits the second consumable 40a and the line camera 18. In this embodiment, the concentration determination apparatus 7 includes the first consumable 30b as described above with reference to FIG. 10, and the third consumable 50 as described above with reference to FIG. 11. That is, the imaging well 38 is provided in a separate consumable from the first consumable 30b. The concentration determination apparatus 7 is suitable for receiving a cell culture sample. In another embodiment (not shown), in which the concentration determination apparatus is suitable for receiving a patient sample (for example, a blood sample from a blood culture flask), the first consumable 30b is replaced by the first consumable 30d, such that the concentration determination apparatus includes a lysis buffer reservoir 32, a filter 33 (i.e. a recovery means) and a resuspended cell container 34 (i.e. a container for receiving the sample).

(41) In a similar embodiment (not shown), the concentration determination apparatus 7 could instead use the first consumable 30a (as described above with reference to FIG. 9) or the first consumable 30c (as described above with reference to FIG. 12). Both of these include the imaging well 38, in which case the third consumable 50 could be omitted. In the case that the first consumable 30a is used, the concentration determination apparatus is suitable for receiving a cell culture sample. In the case that the first consumable 30c is used, the concentration determination apparatus is suitable for receiving a patient sample (for example, a blood sample from a blood culture flask), because the apparatus then includes a lysis buffer reservoir 32, a filter 33,and a resuspended cell container 34.

(42) FIG. 16 shows a consumable 100 suitable for use as the second consumable 40a or 40b, or the third consumable 50. This consumable is described in detail in WO 2017/216314. As seen in FIG. 16, the consumable 100 has three layers. A first, optically flat, layer 110 forms a base layer. A second layer 114 is placed on top of the first layer 10 and is formed with volumes for holding fluids in wells 116 (corresponding to wells 42) that are connected via channels 118. The first layer 110 closes the bottoms of the wells 116. A third layer 120 covers the tops of the wells 116 and the channels 18. The third layer 120 includes openings 122 at one end of each of the channels 118 to allow for dispensing of fluid into each channel 118, and then along the channels 118 to fill all of the wells 116. The third layer 120 also includes vents 124 at the other ends of each of the channels 118 to allow for gas to leave the channels 118 as they are filled with the sample fluid(s). The vents 124 and optionally also the openings 122 may be covered by a gas permeable membrane.

(43) All of the layers 10, 14, 20 have a central hole 126 that is used during loading of the sample holder 100 into the apparatus 2. In this example the sample holder 100 has a circular geometry and it can be held in a similar fashion to a CD, thus being supported on a spindle platter and held for rotation with imaging elements above and/or below the sample holder 100. The central hole 126 forms the mounting to couple the sample holder 100 to a spindle platter in the apparatus 1, 2, 3, 4, 5, 6. The first layer 110 and the third layer 120 are transparent to light in the wavelengths used for imaging the samples and typically are transparent to visible light.