Method for determining antimicrobial susceptibility of a microorganism

Abstract

A method for determining the antimicrobial susceptibility of a microorganism in a clinical sample includes removing a test aliquot from a clinical sample culture before the culture reaches 0.5 McFarland units and isolating the microbial cells. The cells are transferred into a suitable culture medium for microbial growth and an AST assay is performed using the isolated microbes. The concentration of microbial cells in the microbial cells used to set up the AST assay is measured before measuring the degree of microbial growth in different conditions of the AST assay. Devices for determining the anti-microbial susceptibility of a microorganism in a clinical sample are also disclosed.

Claims

1. A method for determining the antimicrobial susceptibility of a microorganism in a clinical sample said method comprising: a) providing a clinical sample culture of a clinical sample in a culture vessel containing culture medium; b) removing a test aliquot from said clinical sample culture in said culture vessel, wherein said aliquot is removed when the culture in the culture vessel is less than 0.5 McFarland units; c) selectively isolating microbial cells from said test aliquot to separate microbial cells from non-microbial cells in said test aliquot; d) transferring said isolated microbial cells into a culture medium suitable for microbial cell growth thereby to prepare a microbial culture preparation; e) inoculating a series of test microbial cultures for an antibiotic susceptibility test (AST) using the microbial culture preparation of step (d), 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; f) assessing the degree of microbial growth in each growth condition; wherein the concentration of microbial cells in said microbial culture preparation is determined between steps (d) and (e) and/or the concentration of microbial cells is determined in the test microbial cultures during or after step (e) but prior to step (f), and optionally and if necessary the concentration of microbial cells in said microbial culture preparation and/or said test microbial cultures is adjusted 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 clinical sample.

2. The method of claim 1, wherein said clinical sample is blood or a blood fraction.

3. The method of claim 1, wherein said clinical sample culture is a primary culture of a clinical sample in a culture vessel containing culture medium.

4. The method of claim 1, wherein said clinical sample culture contains microbial cells derived from a separate clinical sample.

5. The method of claim 1, wherein the concentration of microbial cells in the clinical sample culture is less than 10.sup.8 CFU/ml.

6. The method of claim 5 wherein the concentration of microbial cells in the clinical sample culture is less than 10.sup.7 CFU/ml.

7. The method of claim 1, wherein non-microbial cells in the test aliquot are selectively lysed to obtain a microbial suspension and microbial cells are recovered from the microbial suspension.

8. The method of claim 7, wherein the microbial cells are recovered from said microbial suspension by filtration.

9. The method of claim 1, wherein the concentration of cells in the microbial culture preparation is adjusted to increase the number of cells by culturing the microbial culture preparation before step (e).

10. The method of claim 9, wherein the concentration of microbial cells in the microbial culture preparation is increased to a standard concentration of 0.5 McFarland units.

11. The method of claim 1, wherein the concentration of microbial cells in the microbial culture preparation is reduced by dilution before step (e).

12. The method of claim 11, wherein the concentration of microbial cells in the microbial culture preparation is reduced to a standard concentration.

13. The method of claim 7, wherein the concentration of microbial cells in the microbial culture preparation is determined again after or during the adjustment step.

14. The method of claim 1, wherein the concentration of microbial cells in the microbial culture preparation that is used to inoculate the series of test cultures in step (e) is not a standard concentration for AST, wherein the standard concentration is 0.5 McFarland units.

15. The method of claim 14, wherein the at least one MIC value obtained is adjusted based on the concentration of microbial cells in said culture to obtain a standard MIC value, wherein the standard MIC value is the MIC value of a microbial culture in which the concentration of microbial cells in the microbial culture is the standard concentration.

16. The method of claim 1, wherein the concentration of microbial cells in the test microbial culture is measured prior to step (f).

17. The method of claim 15, wherein the concentration of microbial cells in the test microbial culture is not the standard concentration for AST and wherein the at least one MIC value obtained is adjusted based on the concentration of microbial cells in said culture to obtain the standard MIC value.

18. The method of claim 1, wherein the assessing of the degree of microbial growth in step (f) is performed by determining the amount of microbial cell matter present in a test microbial culture.

19. The method of claim 18, wherein the determining of the amount of microbial cell matter includes determining at least one of: the amount of at least one of (a) microorganisms and (b) microbial colonies or aggregates of microorganisms; the number of at least one of (a) microorganisms and (b) microbial colonies or aggregates of microorganisms; the size of microbial colonies or aggregates of microorganisms.

20. The method of claim 18, wherein the determining of the amount of microbial cell matter present is performed by imaging.

21. The method of claim 20, wherein the determining of the amount of microbial cell matter present is performed by measuring the area of microbial biomass by the imaging.

22. The method of claim 20, wherein the imaging is bright field imaging.

23. The method of claim 20, further comprising fluorescently labeling the microbial cells prior to step (f) and wherein the assessing of the degree of microbial growth in step (f) includes determining the degree of microbial growth by fluorescence microscopy.

24. The method of claim 1 wherein said clinical sample is from a subject having, or suspected of having, sepsis or septic shock.

25. The method of claim 1 wherein the culture vessel is a blood culture flask.

26. The method of claim 23, further comprising filtering the test aliquot to remove resin particles prior to step (c), wherein said filter comprises a pore size of at least 100 m.

27. The method of claim 1, wherein the identity of the microorganism in the clinical sample is determined before step (b).

Description

(1) The invention will now be described in the detailed description and in the Examples below, with reference to the following drawings in which:

(2) FIG. 1 is a schematic diagram illustrating a device for determining the antimicrobial susceptibility of a microorganism in a clinical sample, according to an embodiment of the present invention.

(3) FIG. 2 shows a series of workflows indicating possible alternative embodiments of the present invention. FIG. 2A: Microbial cells are fluorescently labelled and are detected by fluorescence microscopy, Fluorescence-based AST requires a labelling step before microbial growth may be monitored (assessed). FIG. 2B: Workflow with a concentration adjustment step included after the concentration of microbial cells in the culture medium preparation. The adjustment step could be continued culturing or dilution of the sample to reach a pre-defined concentration before starting the AST. FIG. 2C: Non-fluorescent read-outmicrobial growth is assessed by imaging, or other technique which does not require fluorescent labelling.

(4) FIG. 3 shows examples of bright-field microphotographs of bacteria showing different morphology of bacteria after exposure to antibiotics for four hours. Images A-C show E. coli: A) no antibiotic, B) Ciprofloxacin and C) Meropenem. Image D shows S. aureus, which is known to form cellular aggregates in the presence of blood.

(5) FIG. 4 shows a comparison of measuring the concentration of microbial cells in a sample by imaging with conventional techniques. The concentration of microbial cells determined by imaging was found to correlate well with conventional techniques.

(6) FIG. 5 shows the effects of the concentration of microbial cells used to initiate the AST assay affect the MIC values obtained. FIG. 5A shows that different titers of gentamicin resistant E. coli may provide different MIC results. FIG. 5B shows the reproducibility of the present method where the same titer is used to initiate the AST assay: AST assays performed on separate days using the same microbial titer provided the same MIC results.

(7) FIG. 6 shows that MIC values obtained by measuring growth by imaging correlate well with the MIC values obtained by turbidity measurement (OD600). FIG. 6A: MIC determination by monitoring growth by imaging after incubation for 4 hours. FIG. 6B: MIC determination by monitoring growth by turbidity measurement after incubation for 24 hours.

(8) FIG. 7 shows that MIC values can reproducibly be measured by imaging; three separate AST assays were performed on separate days using separate samples adjusted to the same microbial titer level. FIG. 7A: MIC determination by monitoring growth by imaging after incubation for 4 hours. Data from each repeat was found to correlate well. FIG. 7B: Confirmation of MIC values by measuring turbidity after incubation for 24 hours.

DETAILED DESCRIPTION

(9) FIG. 1 is a schematic diagram illustrating a device for determining the antimicrobial susceptibility of a microorganism in a clinical sample, according to an embodiment of the present invention.

(10) The device 10 shown in FIG. 1 comprises: a test aliquot removal unit 12; an isolation unit 14; a transfer unit 16; a reservoir 17; an inoculation unit 18; a microtiter plate 20 comprising a series of test microbial cultures; a concentration determination unit 22; a concentration adjustment unit 24; an assessment unit 26; and a controller 28. In use, a culture vessel 1 (e.g. a blood culture flask) is placed inside the device 10.

(11) The test aliquot removal unit 12 comprises a needle 12a and syringe 12b, which together are provided as a consumable for a single use. The test aliquot removal unit 12 is operable to remove a test aliquot from the culture vessel 1.

(12) The isolation unit 14 comprises: a first filter 14a for removing resin beads present in the blood culture vessel from the test aliquot, the first filter having a pore size of approximately 100 m; a buffer reservoir 14b comprising a lysis buffer; a lysis reservoir 14c in which a portion of the test aliquot and lysis buffer are mixed and lysis takes place; a second filter 14d connected to the a lysis reservoir 14c for capturing microbial cells, the second filter 14d having a pore size of approximately 0.2 m; and a wash reservoir 14e for holding a wash buffer or culture medium for back-flushing the microbial cells off of the filter. The isolation unit 14 is provided as a single-use consumable.

(13) The transfer unit 16 comprises a fluidics or pipetting assembly for transferring the microbial cells back-flushed from the filter into a reservoir 17 to form a microbial culture preparation. Parts of the transfer unit 16 which come into contact with the sample (pipette-tips, syringes etc.) are provided as single-use consumables.

(14) The inoculation unit 18 comprises a pipetting assembly for inoculating the microtiter plate 20 using the microbial culture preparation. The wells of the microtiter plate (not shown) contain at least two different concentrations of at least one antimicrobial agent, a negative control (i.e. comprising only medium) and a positive control sample (i.e. comprising no antimicrobial agent). The microtiter plate 20 is provided as a single-use consumable. The concentration determination unit 22 comprises an imager for determining the concentration using an imaging method.

(15) The concentration adjustment unit 24 comprises a reservoir 24a comprising a diluent for diluting the microbial cells in the microbial culture preparation 17 and/or the concentration of microbial cells in the test microbial cultures. The concentration adjustment unit 24 also comprises a culturing unit 24b.

(16) The assessment unit 26 is an imaging unit comprising a bright field microscope and a camera. A specific area of the specimen is covered in a single xy-aligned image the size of which is dependent on the optical properties of the imaging apparatus. For each position in xy-space, one or more 2D images are collected at different intervals along the optical or z axis. Thus, a series, or stack of 2D images can be generated, providing 3D information of a sample volume. Once extracted, the 3D information inherent in the 2D image stacks is utilized to estimate/infer/deduce the total cell mass present in the analysed volume.

(17) The controller 28 is in communication with each stage of the device which is configured to carry out a controllable function (for example: the test aliquot removal unit 12; the isolation unit 14; the transfer unit 16; the inoculation unit 18; the concentration determination unit 22; the concentration adjustment unit 24; and the assessment unit 26). For clarity, the lines of communication between the controller and other parts of the device are not shown.

(18) The controller 28 is operable to operate in two modes. In the first, the controller 28 controls the device 10 to physically adjust the concentration of microbial cells in said microbial culture preparation 17 or in the test microbial cultures in the microtiter plate 20. Either the concentration (or number) of microbial cells in the microbial culture preparation 17 or in the wells of the microtiter plate 20 is physically increased if necessary (e.g. by culturing for a period of time to allow the microbial cells to grow) or physically decreased (e.g. by dilution using diluent in the concentration adjustment unit 24). The controller 28 may also control the concentration determination unit 22 to measure the concentration of microbial cells again after or during controlling the device 10 (or the concentration adjustment unit 24) to adjust the concentration of microbial cells.

(19) In the second mode, the controller 28 is configured such that the concentration of microbial cells is not physically adjusted, but instead a virtual adjustment (an algorithmic correction) is made, based on the measured concentration of the microbial cells. In either case, the controller 28 is configured to calculate a standard MIC value.

Example 1Method for Performing AST

(20) 5 ml of a cultured clinical sample is removed from a blood culture flask, and filtered using a 100 m filter to remove resin particles from the culture medium. The sample is mixed with a 10 ml of lysis buffer capable of selectively lysing any non-microbial cells present in the sample, and incubated for a sufficient amount of time for the non-microbial cells to be lysed. The resulting lysate is filtered through a 0.22 m filter. Liquid components of the lysate (including components of the lysed non-microbial cells) pass through the filter, whereas microbial cells present in the sample are retained on the filter.

(21) The retained microbial cells are resuspended in a suitable microbial culture medium to form a microbial culture preparation, by back-flushing the filter with culture medium (i.e. flowing the culture medium in the opposite direction to filtration). The concentration of microbial cells present in the microbial culture preparation is measured at this stage, and if necessary, diluted with additional culture medium in order to reduce the concentration, or allowed to grow further at this stage in order to increase the concentration.

(22) Once a suitable concentration of microbial cells has been obtained, 100 l aliquots of the microbial culture preparation are dispensed into wells of a microtiter plate. The wells of the microtiter plate contain at least two different concentrations of at least one antimicrobial agent. A negative control (i.e. comprising only medium) is also set up at this stage and a positive control sample (i.e. comprising no antimicrobial agent).

(23) The microtiter plate is placed in an oCelloScope reader in an InnuCell111 incubator. A bottom search for focus is performed on each inoculated well and each well is read at intervals of 1 hour. A total of 6 repeats are performed (a total of 7 images are taken at time points from 0 to 6 hours after the initiation of the AST assay. The degree of microbial growth in each growth condition is monitored by imaging, by measuring the amount of microbial biomass in each well.

Example 2Morphology of Bacteria in an AST Assay is Affected by the Growth Conditions Present

(24) Bacterial cells were spiked into blood culture flasks (BCF). Samples A-C were spiked with E. coli with blood at a concentration lower than 0.5 McFarland and subjected to sample clean-up and recovered in MH-media. Aliquots of the recovered bacteria were dispensed into a micro-titerplate with freeze dried antibiotics at varying concentrations and allowed to grow for four hours before imaging. Sample A did not contain an antibiotic. Sample B was added to Ciprofloxacin. Sample C was added to Meropenem. An image of the microbial cells after four hours' growth is shown in FIG. 3 A-C at a single concentration of each antibiotic, indicating that the morphology of microbial growth may be affected by the presence and nature of the antibiotic added to sample.

(25) Sample D was spiked with S. aureus, which is known to form aggregates in the presence of blood. The sample was cultured for a sufficient period of time to ensure that the aggregation of bacteria before sample clean-up. The same procedure was then performed as described for sample A-C.

Example 3Concentration Determination Using Imaging and Measuring Biomass in Microphotograph of Sample

(26) A sample of bacteria, (E. coli or S. aureus) was diluted in MH-media and aliquots of the dilution were dispensed into an optical microtiter plate. Viability of the bacteria in the original solution was determined by plating and counting CFU after overnight growth on TSA (Tryptic Soy Agar) plates. Images of the microtiter plate were acquired at T 0 h of the wells and the biomass of bacteria were recorded and to compared to expected viable count (see FIG. 4). Using the current method it is possible to measure the concentration of bacterial solutions well below 0.5 McFarland.

Example 4The MIC Value Obtained in an AST Assay May Vary Depending on the Initial Concentration of Microbial Cells

(27) E. coli grown in blood+BCF media were recovered via a clean-up procedure to remove blood components and only retain viable bacteria in MH-media. Adjustment of the recovered bacteria were made to ensure different titers are used at start of the AST. AST assays were set up to a starting concentration of 310.sup.5 CFU/ml (Lane A) and 210.sup.6 CFU/ml (Lane B) of a gentamicin resistant E. coli strain. Aliquots of the recovered bacteria were dispensed into a microtiter plate with freeze dried antibiotics at varying concentrations and allowed to grow for four hours before imaging. Images of the wells of the microtiter plate for each concentration of gentamicin are shown in FIG. 5A. Shown are microphotographs from a series of different concentration of antibiotics in two-fold dilutions ranging from 64 mg/l to 0.0625 mg/l of gentamicin. The higher bacterial titer (Lane B) show growth in presence of higher concentration of antibiotics, and thus has a higher MIC value. This demonstrates the requirement for concentration determination before AST. In contrast to this, the present method demonstrates good reproducibility when the same concentration of microbial cells is used to set up separate repeats of an AST assay. Lane A and Lane B were set up with bacterial cultures adjusted to have the same titer at the start of the AST on different days. Aliquots of a gentamicin sensitive strain of E. coli were dispensed into a microtiter plate as above, and allowed to grow for four hours before imaging. Images of the wells of the microtiter plate for each concentration of gentamicin are shown in FIG. 5B. The experiments performed on different days showed good reproducibility, highlighting the need to determine the concentration of microbial cells in the culture preparation to interpret the data obtained from the AST assay.

Example 5Determination of AST by Imaging

(28) Sample preparation as in Example 1 was performed on BCF-cultures spiked with E. coli and an aliquot was withdrawn with before the culture reached 0.5 McFarland units, i.e. before they were indicated positive in the blood culture cabinet. After recovery of bacteria from the filter, aliquots of the sample were directly added to an optical microtiter plate with freeze dried antibiotics (Sifin diagnostics GmbH.) containing selected antibiotics. The microtiter plate was imaged in an oCelloScope reader at time 0 (h) and 4 hours. The same microtiter plate was allowed to continue to grow for 24 hours and read with a turbimetric assay to control for the rapid AST generated by imaging.

(29) The data obtained using the rapid AST generated by imaging after 4 hours correlated well with the data obtained using a turbimetric assay after 24 hours (FIG. 6). Both the rapid AST and the 24 hour AST provided a MIC estimation of 1 g/ml, both starting from a culture which had not reached 0.5 McFarland units. The same strain was independently shown to yield an AST of 1 g/ml when tested using an EUCAST validated method at a certified lab, starting from a 0.5 McFarland culture.

(30) An aliquot of the sample was plated onto a TSA (Tryptic Soy Agar) plate to make an independent measurement of the concentration of bacteria present in the sample used to perform the AST in this experiment and this was found to be 210.sup.6 CFU/ml.

Example 6Further Determination of AST by Imaging

(31) E. coli were spiked into blood culture flasks (BCF) with blood at a concentration lower than 0.5 McFarland units. A sample was taken and subjected to sample clean-up and recovered in MH-media. Aliquots of the recovered bacteria were dispensed into a microtiter plate with freeze dried antibiotics at varying concentrations and allowed to grow for four hours before imaging. Algorithms for quantifying the biomass of bacteria were used to obtain quantitative data from the images. FIG. 7A shows triplicate measurements done on three different days of both the read-out after four hours using images and image algorithm analysis. Optical density measurement after 24 hours growth measured using OD600 are shown in FIG. 7B. AST determined by image analysis was found to correlate well with data obtained from measuring optical density. D02961, D02979 and D02992 denote the different experiments.

Example 7Determination of AST of Clinical Isolates

(32) Dilutions of two different bacterial clinical isolates were seeded into an aerobic blood culture flask (Bactec, Becton Dickinson) at an estimated concentration of either 1 or 10 CFU/ml blood. Before seeding, the BCF had been filled with human blood from healthy donors. Aliquots from the dilution series were plated on agar and grown over night for viable count. Target concentration was 10 or 100 CFU seeded in 0.5 ml per BCF, corresponding to approximately 1 or 10 CFU/ml blood. The seeded BCF were allowed to incubate for 8 hours at 35 C. and a 5 ml aliquot were taken for subsequent AST. Another aliquot from the same BCF was used for determination of viable count after 8 hours' growth in the BCF.

(33) A 5 ml aliquot of the cultured clinical isolate from the blood culture flask was filtered using a 100 m filter to remove resin particles from the culture medium. The sample was thereafter mixed with a 10 ml of lysis buffer capable of selectively lysing any non-microbial cells present in the sample, and incubated for a sufficient amount of time for the non-microbial cells to be lysed. The resulting lysate was filtered through a 0.2 m filter. Liquid components of the lysate (including components of the lysed non-microbial cells) passed through the filter, whereas microbial cells present in the sample were retained on the filter.

(34) The retained microbial cells were re-suspended in liquid Mueller Hinton broth medium to form a microbial culture preparation, by back-flushing the filter with culture medium (i.e. flowing the culture medium in the opposite direction to filtration). 100 l aliquots of the microbial culture preparation were then dispensed into wells of a microtiter plate.

(35) The wells of the microtiter plate contained between 5 and 13 different concentrations of an antibiotic and different antibiotics. A negative control (i.e. comprising a blood sample with no seeded bacteria) and a positive control sample (i.e. an aliquot of the sample from a seeded BCF but into a well with no antimicrobial agent) were also set up at this stage.

(36) The microtiter plate was placed in an oCelloScope reader in an InnuCell-111 incubator. Focus was performed on each inoculated well and each well was read at intervals of 1 hour. A total of 5 images were taken at time points from 0 to 4 hours after the initiation of the AST assay. The degree of microbial growth in each growth condition was monitored by imaging, by measuring the amount of microbial biomass in each well.

(37) Viable count of the sample before seeded into the BCF and after 8 hour of culture are as shown below in Table 1.

(38) TABLE-US-00001 TABLE 1 viable count of microbial cells before and after culture CFU Actual mL CFU/ml Exp. spiked/ blood/ Spike CFU/ after 8 h No.# Isolate BCF bottle** mL blood culture 1 QM006 96 17 6 7.0E+04 2 QM006 32 13 2 7.3E+04 3 QM006 103 14 7 1.4E+06 4 QM006 34 10 3 9.1E+05 5 QM006 69 11 6 9.8E+05 6 QM006 23 12 2 3.6E+05 7 QM006 103 10 10 2.8E+06 8 QM006 34 7 5 1.3E+06 9 QM006 95 16 6 1.1E+06 10 QM006 9 13 1 1.0E+05 1 QM171 95 12 8 4.7E+05 2 QM171 32 10 3 3.9E+05 3 QM171 88 11 8 1.2E+06 4 QM171 29 9 3 4.7E+05 5 QM171 91 13 7 6.3E+05 6 QM171 30 14 2 4.0E+05 7 QM171 99 12 8 7.1E+03 #Experiment number only to be used as a guide to Table 1. **Measured as the total volume recovered from the BCF minus the assumed 27.5 ml BCF media in the BCF and 0.5 ml seeded bacteria. All samples were taken well below 0.5 McFarland concentration in the cultured sample.
Microphotographs from the different wells were analysed to generate a numerical value of the biomass in the well and this was used as a marker of growth. From the growth a minimal inhibitory concentration was determined and compared to MIC obtained using a reference method, broth micro-dilution. The reference MIC value was conducted by taking one to three colonies of the bacterial strain from an agar plate with pure culture and adjust the concentration to 0.5 McFarland. The adjusted solution was then further processed in line with ISO 20776 guidelines and MIC were determined after 16-20 hours growth by estimating the turbidity in the sample. A sample was determined to Pass (P) if within +/1 two-fold dilution from the reference MIC.

(39) TABLE-US-00002 TABLE 2 MIC calculated from microphotographs. Experiment repeat no. Antibiotic Isolate 1 2 3 4 5 6 7 8 9 Reference P F % Passed % Passed Cefotaxime QM006 >8 >16 >16 >16 >16 >16 >16 >16 >16 >8 10 0 100 100 QM171 8 >16 >16 >16 >16 16 16 >8 7 0 100 Ceftazidime QM006 8 16 8 8 8 16 8 16 8 16 9 0 100 100 QM171 2 2 2 2 2 2 1 2 7 0 100 Ciprofloxacin QM006 >4 >4 >4 >4 >4 >4 >4 >4 >4 >4 10 0 100 100 QM171 0.5 0.5 0.5 0.5 0.5 0.5 0.125 0.25 7 0 100 Gentamicin QM006 16 16 16 16 16 16 16 16 16 >16 9 0 100 100 QM171 0.5 0.5 0.5 0.5 0.5 0.5 0.25 0.5 7 0 100 Meropenem QM006 0.5 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 9 0 100 100 QM171 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 7 0 100