Method for determining the identity and antimicrobial susceptibility of a microorganism

Abstract

A method for detecting and characterizing a microorganism in a clinical sample includes introducing a clinical sample to a first culture vessel containing the culture medium; removing a test aliquot; separating DNA from the test aliquot; and performing nucleic acid tests on the DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in the microorganism. If a microorganism is identified, an antimicrobial susceptibility test is performed wherein microbial growth in the antimicrobial susceptibility test is monitored by accessing growth or markers for growth and wherein the type and concentration of antimicrobial agents used in the antimicrobial susceptibility test is determined by the identity of the microorganism and the antimicrobial resistance markers detected. A device for performing the method is also provided.

Claims

1. A method for detecting and characterizing a microorganism in a clinical sample, said method comprising: a) introducing a clinical sample taken from a patient to a first culture vessel containing culture medium; b (i)) optionally preculturing said clinical sample in said first culture vessel; b (ii)) optionally removing a portion of the clinical sample/medium mixture or, if precultured, the clinical sample culture from said first culture vessel, and introducing said portion to a second culture vessel containing culture medium, and optionally preculturing said portion in said second culture vessel; c) removing a test aliquot from said first and/or second culture vessel; d) after step (c) culturing or continuing to culture said clinical sample and/or portion in said first and/or second culture vessel; e) separating or enriching microbial DNA or microbial cells containing said DNA from said test aliquot; f) performing nucleic acid tests on said DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in said microorganism, wherein said nucleic acid tests are performed using: i) one or more nucleic acid probes and/or primers for microbial identification, a said probe or primer being capable of hybridizing specifically to, or a said primer being capable of selectively amplifying, a nucleotide sequence which is identificatory of a given microorganism; and ii) one or more nucleic acid probes and/or primers for antimicrobial resistance marker detection, a said probe or primer being capable of hybridizing specifically to, or a said primer being capable of selectively amplifying, a nucleotide sequence representing a genetic antimicrobial resistance marker; and detecting whether or not said probes and/or primers have hybridized to said DNA and/or said primers have been extended; and g) performing an antimicrobial susceptibility test on said cultured clinical sample and/or said culture clinical portion from step (d) without any further sub-culture, wherein microbial growth in said antimicrobial susceptibility test is monitored by assessing growth or markers for growth, wherein the assessment of growth or markers of growth includes obtaining an image of said cultured clinical sample and/or portion, and determining an area of microbial biomass in the image, and wherein the type and concentration of antimicrobial agents used in said antimicrobial susceptibility test is determined by the identity of the microorganism and antimicrobial resistance markers detected in step (f), and optionally continuing to culture said clinical sample and/or portion in said first and/or second culture vessel.

2. The method of claim 1, said method comprising: h) if no microorganism strain is identified in step (f), further culturing said clinical sample and/or portion to enable further microbial identification and antimicrobial susceptibility tests to be performed to identify the microorganism and determine its antimicrobial resistance profile.

3. The method of claim 1 wherein said clinical sample is from a subject having, suspected of having, or at risk from sepsis.

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

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

6. The method of claim 1 wherein the culture medium contains an agent which neutralizes the presence of any antimicrobial agents present in the clinical sample.

7. The method of claim 1, wherein steps (c) to (g) are repeated one or more times.

8. The method of claim 7, wherein steps (c) to (f) are performed without an initial preculture step, and if no microorganism is identified in step (f), steps (c) to (f) are repeated after a preculture step (b).

9. The method of claim 1, wherein the probes or primers of step (f)(i) are designed or selected for the identification of a microorganism in a panel of sepsis-causing microorganisms.

10. The method of claim 1 wherein in step (f)(i) and/or (ii) PCR primers are used.

11. The method of claim 1 wherein in step (f)(i) and/or (ii) hybridization probes are used.

12. The method of claim 11 wherein said hybridization probes are padlock probes comprising 5′ and 3′ ends that can hybridize to an identificatory or antimicrobial resistance marker nucleotide sequence.

13. The method of claim 12 wherein a circularized padlock probe is detected by rolling circle amplification (RCA).

14. The method of claim 13, wherein the RCA is or comprises circle-to-circle amplification (C2CA) in which a RCA product is cleaved into monomers, and the monomers are circularized and used as templates for a further RCA reaction; and/or wherein an RCA product is detected by cleaving the RCA product into monomers, hybridizing said monomers onto an array and detecting the monomers on the array; and/or wherein an RCA product is detected microscopically or by imaging or in a flow-cytometry-like method.

15. The method of claim 1, wherein for the nucleic acid tests of step (f), separated DNA is immobilized using capture probes capable of hybridizing to the DNA.

16. The method of claim 1 wherein step (g) is performed if a single microorganism is identified in step (f).

17. The method of claim 2 wherein if two or more microorganisms are identified in step (f), step (h) is performed.

18. A method for detecting and characterizing a microorganism in a clinical sample, said method comprising: a) introducing a clinical sample taken from a patient to a first culture vessel containing culture medium; b (i)) optionally preculturing said clinical sample in said first culture vessel; b (ii)) optionally removing a portion of the clinical sample/medium mixture or, if precultured, the clinical sample culture from said first culture vessel, and introducing said portion to a second culture vessel containing culture medium, and optionally preculturing said portion in said second culture vessel; c) removing a first test aliquot from said first and/or second culture vessel; d) after step c), culturing or continuing to culture said clinical sample and/or portion thereof in said first and/or second culture vessel; e) separating or enriching microbial DNA or microbial cells containing said DNA from said first test aliquot; f) performing nucleic acid tests on said DNA to identify the microorganism and to detect the presence or absence of one or more genetic antimicrobial resistance markers in said microorganism, wherein said nucleic acid tests are performed using: i) one or more nucleic acid probes and/or primers for microbial identification, a said probe or primer being capable of hybridizing specifically to, or a said primer being capable of selectively amplifying, a nucleotide sequence which is identificatory of a given microorganism; and ii) one or more nucleic acid probes and/or primers for antimicrobial resistance marker detection, a said probe or primer being capable of hybridizing specifically to, or a said primer being capable of selectively amplifying, a nucleotide sequence representing a genetic antimicrobial resistance marker; and detecting whether or not said probes and/or primers have hybridized to said DNA and/or said primers have been extended; and g) providing an antimicrobial susceptibility test device, which when a microorganism is identified in step (f), performs an antimicrobial susceptibility test using said cultured clinical sample and/or said cultured portion from step (d), without any further sub-culture, wherein microbial growth in said antimicrobial susceptibility test is monitored by assessing growth or markers for growth, wherein the assessment of growth or markers of growth includes obtaining an image of said cultured clinical sample and/or portion, and determining an area of microbial biomass in the image, and wherein the type and concentration of antimicrobial agents used in said antimicrobial susceptibility test is determined by the identity of the microorganism and antimicrobial resistance markers detected in step (f), and optionally continuing to culture said clinical sample and/or portion in said first and/or second culture vessel.

Description

(1) The invention will now be described in more detail in the Examples below with reference to the following drawings in which:

(2) FIG. 1 depicts in (A) a sample microarray panel for the detection of microorganisms in a sample, and in (B) the fluorescence intensity of the signal generated for a range of microorganisms. In FIG. 1A, the spots at the four corners and in the middle of the left hand side of the image are reference spots used for image alignment. The three brighter spots indicate that nucleotide sequences specific for E. coli are detected in the sample using the detection method of the present invention. FIG. 1B shows the signal generated for a range of different microorganisms when probes specific for E. coli are used, and indicates that the method of the present invention is capable of detecting the presence of a specific microorganism—in this case E. coli—in a sample.

(3) FIG. 2 shows the relative growth of bacteria grown in MH broth supplemented with ciprofloxacin at a range of different concentrations, relative to a positive control sample grown in the absence of ciprofloxacin. Cells were stained with Vybrant® DyeCycle™ Orange stain and counted by automated microscopy.

(4) FIG. 3 shows the relative growth of bacteria grown in MH broth supplemented with a number of different antibiotics, each at a range of different concentrations, relative to positive control samples grown in the absence of any antibiotics (A—Cefotaxime, B—Ciprofloxacin, C—Gentamicin, D—Meropenem, E—Ceftazidime, F—Piperacillin+Tazobactam). Cells were stained with Vybranti® DyeCycle™ Orange stain and counted by Aquila 400. These data indicate that differential growth can be detected within 4 hours, MIC values were calculated for each of the antibiotics at 4, 6 and 24 hours, and at a range of cut-off values.

(5) FIG. 4 depicts in (A) the workflow required for Amplified Single Molecule Detection (ASMD) of bacteria within a sample to determine antibiotic susceptibility of a microorganism, and in (B) shows the differential growth of bacteria grown in MH broth supplemented with Ciprofloxacin or Cefotaxime, relative to a positive control sample grown in the absence of either antibiotic. In FIG. 4B, the RCA products indicating the presence of specific microbial DNA sequences are detected by ASMD, and counted by Aquila 400.

(6) FIG. 5 depicts a scheme for detecting specific microbial DNA sequences for use in probe design. A biotinylated capture oligonucleotide complementary to a region of microbial DNA binds a target DNA fragment and immobilises it onto a solid phase. A padlock probe, comprising two target-complementary parts (5′/3′-arms) and a backbone with sites for detection (DO—generic), restriction digestion and priming (RO—generic), and array oligo hybridization (AO—unique) binds to its target via its complementary 5′ and 3′ end sequences, and can be ligated into a circle prior to RCA amplification and C2CA reaction.

(7) FIG. 6 presents the results of the experiment in Example 6 to detect microorganisms in 10 clinical samples and 1 spiked sample, and shows the fluorescent signals detected (RFU, relative fluorescent units) on a microarray when probes specific for different bacteria were used. 8 of the clinical samples were found to be negative, one clinical sample was found to contain E. coli, one clinical sample was found to contain S. pneumonia, and the sample spiked with E. coli was found to contain E. coli, showing that specific microorganisms can be detected in clinical samples; and

(8) FIG. 7 presents the results of the experiment in Example 7 and shows the time taken to obtain a positive blood culture result (TPP, time to positivity) for sets of blood culture flasks (BCFs) spiked with 50,000 CFU/ml and 500 CFU/ml blood in the BCF respectively at the time of sampling. TPP was found not to differ significantly between the set.

EXAMPLES

Example 1

Blood Culture and Microbial Identification and Characterisation by Molecular Tests

(9) Blood spiked with 500 μl of E. coli bacterial suspension to give a concentration of 10.sup.3 CFU/ml was added to a BacT/ALERT FA plus (Biomerieux) blood culture flask with a needle through the septa. For pre-culture we used an own designed Multirotators from Grant Instruments (Grant-bio PTR-35) re-designed to blood culture flask (BCF) agitators. Samples were incubated for 4 hours at 35° C.

(10) 5 ml of the sample was aspirated, equivalent to 1 ml of whole blood, using a syringe.

(11) Enrichment of bacterial DNA was carried out by the method from Molzym (Germany) without the need for centrifugation.

(12) Preparation of lysis buffer by addition of 800 μl ES to 160 μl MoIDNase. Mix by pipetting 6× and add all of the MoIDNase solution to 12800 μl lysis buffer followed by mix by pipetting 6×.

(13) Preparation of Proteinase K. Add 1000 μl ES to 400 μl Proteinase K and mix by pipetting 6×.

(14) Lysis

(15) Add 5 ml sample withdrawn from the BCF to 4300 μl Lysis-MoIDNase buffer and mix by pipetting 8×. Incubate at 45° C. for 10 min. Inactivating MoIDNase and add 350 μl Proteinase K to the lysate and mix by pipetting 8×. Incubate at 45° C. for 10 min.

(16) Sample capture to column and wash, 3 loops

(17) Add 605 μl lysate to a filter column (Molzym, Germany), apply 15 seconds vacuum. Add 500 μl RS to the filter column and apply 15 seconds vacuum. Add 605 μl lysate to a filter column (Molzym, Germany), apply 30 seconds vacuum. Add 500 μl RS to the filter column and apply 15 seconds vacuum. Add 605 μl lysate to a filter column (Molzym, Germany), apply 45 seconds vacuum. Add 500 μl RS to the filter column and apply 15 seconds vacuum. Continue with 45 seconds vacuum after sample addition until all sample have been added. More than one filter may be used in order to filter the lysate, if required, if the filter becomes clogged during filtration.

(18) Preparation of lysis buffer for microbes

(19) Add 5.6 μl 3-mercaptoethanol to 600 μl RL and mix by pipetting 6×. Add all pre-mixed BM-RL to 80 μl BugLysis and mix by pipetting 6×

(20) Lysis of microbes

(21) Add 170 μl of the prepared lysis mixture to the column and apply vacuum for 200 ms. Incubate at 45° C. for 10 min. Add 280 μl prepared Proteinase K-solution to 520 μl Buffer RP. Mix by pipetting 6×. Add 200 μl PK-RP to column. Apply vacuum for 200 ms. Incubate at 45° C. for 10 min

(22) Binding of microbial DNA to column

(23) Move column to room tempered block. Wait for 2 minutes. Add 500 μl CSAB to column. Apply vacuum for 2 s.

(24) Wash 1

(25) Add 500 μl WB to column. Apply vacuum for 2 s.

(26) Wash 2

(27) Add 500 μl WS to column. Apply vacuum for 2 s.

(28) Drying of membrane

(29) Apply vacuum for 10 min

(30) Elution

(31) Add 200 μl ET to column

(32) Apply vacuum for 200 ms.

(33) Incubate at RT for at least 5 min

(34) Apply vacuum for 1 min

(35) Repeat once to make two elutions.

(36) The molecular test was carried out on the enriched bacterial DNA sample by the method in Göransson et al, 2012 (supra). During the process of molecular identification of the bacteria the remaining blood sample was kept under agitators.

(37) Padlock probes and target capture probes were ordered from Integrated DNA Technologies (Munich, Germany). The probes were designed to detect unique motifs in each bacteria, selected via bioinformatics tools. The hybridization of capture probes and ligation of padlock probes to the target DNA were performed simultaneously, and was achieved by incubating fragmented and denatured genomic DNA in 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% Triton® X-100, 100 nM padlock probe, 50 nM capture probe, 0.2 μg/μl BSA (New England Biolabs, MA, USA), and 250 mU/μl Ampligase (Epicentre Biotechnologies, WI, USA) at 55° C. for 5 min. The target DNA along with reacted padlock probes were captured onto magnetic particles via the biotinylated capture probes. This was achieved by adding 50 μg Dynabeads MyOne™ Streptavidin T1 beads (Invitrogen) to the hybridization/ligation reaction and incubating the sample at room temperature for 3 min. Excess probes were eliminated by washing once with 100 μl washing buffer containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1 M NaCl, and 0.1% Tween-20. The elimination of excess line& padlock probes is performed, since these may otherwise interfere negatively with the subsequent RCA reaction.

(38) Reacted probes were amplified by C2CA, which includes serial enzymatic reactions starting with RCA. The RCA reaction was initiated by the addition of 20 μl ligation mixture containing 1×phi29 DNA polymerase buffer (Fermentas, Lithuania; 33 mM Tris-acetate (pH 7.9 at 37° C.), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween-20, 1 mM DTT), 100 μM dNTPs, 0.2 μg/μl BSA, 25 nM primer, and 100 mU/μl phi29 DNA polymerase. The reaction was incubated at 37° C. for 11 min, and inactivated at 65° C. for 1 min. The RCA products were digested at 37° C. for 1 min by the addition of 3 units of Alul (New England Biolabs), 600 nM replication oligonucleotide, 0.2 μg/μl BSA in 1×phi29 DNA polymerase buffer, and the reaction was terminated at 65° C. for 1 min. Ligation, amplification and labelling reactions were performed by the addition of a mixture containing 1.36 mM ATP, 100 μM dNTPs, 0.2 μg/μl BSA, 28 mU/μl T4 DNA ligase and 120 mU/μl phi29 DNA polymerase in 1×phi29 DNA polymerase buffer to a final volume of 50 μl. The reactions were incubated at 37° C. for 7 min, and terminated at 65° C. for 1 min. The above was repeated once. After the final RCA the products were digested once again into monomers. The RCPs were now ready for analysis.

(39) The digested sample was transferred to a microarray, incubated at 55° C. for 30 minutes followed by a wash with 1×SSC in RT. The hybridized RCA monomers is then labelled via hybridization of a detector oligo at 10 nM concentration in 2×SSC at 55° C. for 30 minutes, washed twice in 1×SSC at RT and spun dry.

(40) The array was then scanned in an array scanner and the result analysed using array image analysis software as follows.

(41) The array image is evaluated in order to detect spots corresponding to one or several pathogens. A lit spot corresponds to a detected pathogen, with a redundancy of three spots per pathogen. Further, the array has reference spots used for image alignment, which are always lit, and protocol reference spots which—if lit—verify that the individual steps of the molecular protocol succeeded. The analysis is divided into the following steps: 1. The reference spots are detected and the image is aligned accordingly. 2. The spot intensities and backgrounds are measured and the background corrected values are calculated. 3. The measured intensities are compensated for pathogen specific background, such as e.g. unspecific binding of DNA from probe sets corresponding to other pathogens. 4. The array intensity data is used to provide an answer on pathogen id(s) and a quality value a. The algorithm takes the intensity value of all replicate spots corresponding to a specific pathogen into account when calculating the ID answer. b. The ID answer may be qualitative (reporting presence of pathogen), and/or quantitative (reporting an indication of the amount of pathogen present in the sample) c. The protocol reference spots are evaluated in order to verify that the molecular protocol succeeded. 5. The result is collected and may be transferred to downstream AST analysis and/or reported out in a result report. The result includes: a. The ID of the detected pathogen(s) b. Semi quantitative values for each detected pathogen c. A quality value indicating the success rate of the molecular protocol.

(42) A sample array image is shown in FIG. 1A. The five spots along the edges of the plate are reference spots and are used for image alignment. Brighter spots indicate a detected pathogen (E. coli). FIG. 1B shows that a fluorescent signal was specifically generated for E. coli.

Example 2

AST Testing on the Blood Culture of Example 1

(43) 5 ml from the remaining sample in the blood culture bottle, that had continued to be under culture conditions during the molecular typing, was drawn from the flask.

(44) The drawn sample was enriched for bacteria and at the same time the culture media was changed, in this case to Mueller-Hinton media (MH-media). E. coli bacteria were recovered via filtration through a 0.2 μm filter and then the recovered bacteria were resuspended in MH-media, 63 μl aliquots of the bacterial suspension were transferred to selected wells in a microtiterplate.

(45) Each well had a different concentration of antibiotics as well as different antibiotics. As the microorganism identified in Example 1 was E. coli in this case, the following antibiotics were selected for use in antimicrobial susceptibility testing: Ciprofloxacin, Piperacillin+Tazobactam, Cefotaxime, Ceftazidim, Meropenem and Gentamicin. A series of six different concentrations was prepared for each antibiotic based on known clinical MIC values. A seventh well containing MH broth and no antibiotic was used as a positive control. The microtiterplate was incubated at 35° C. for 4 hours before the plate was read. 7 μl 10 μM Vybrant® DyeCycle™ Orange stain (Molecular Probes® Life Technologies) was added to each well and incubated for 30 min at 37° C. Each microtiterplate well was imaged and the number of bacteria were counted. The differential growth relative to the positive control was used to determine a MIC value for the bacteria. The result for Ciprofloxacin is shown in FIG. 2.

Example 3

Determination of AST Profiles Using Counting of Individual Bacteria in a Flow Cytometry Type Instrument (Aquila 400, Q-linea AB, Sweden)

(46) Bacteria were grown in blood culture as described in Example 1. 5 ml from the remaining sample in the blood culture bottle, that had continued to be under culture conditions during the molecular typing, was drawn from the flask.

(47) The sample was enriched for bacteria and at the same time the culture media was changed, in this case to Mueller-Hinton media (MH-media). E. coli bacteria were recovered via filtration through a 0.2 μm filter and the recovered bacteria were resuspended in MH-media to a concentration of approximately 10.sup.8 CFU/ml in Müller Hinton broth before being diluted to approximately 10.sup.6 CFU/ml in Müller Hinton Broth.

(48) Antibiotic solutions were prepared in a series of 2:1 serial dilutions in Müller Hinton broth at 10×test concentrations. The range of antibiotic concentrations was chosen based on identity of the bacteria and the antibiotic. As the microorganism identified in Example 1 was E. coli in this case the following antibiotics were selected for use in the antimicrobial susceptibility test: Ciprofloxacin, Piperacillin+Tazobactam, Cefotaxime, Ceftazidim, Meropenem and Gentamicin. A series of eight different concentrations was selected based on known clinical MIC values. Eight sample tubes containing 100 μl antibiotic solution at eight different concentrations and 800 μl Müller Hinton broth were prepared. One additional tube contains 900 μl Müller Hinton broth and no antibiotic as positive control. 100 μl bacterial suspension (10.sup.6 CFU/ml) was added to all nine tubes. A negative control sample comprising 1000 μl Müller Hinton broth (i.e. no bacteria) is prepared as negative control.

(49) All tubes were incubated at 35° C. and samples were taken after 0, 4, 6 and 24 hours. Bacterial samples were prepared for counting as in Example 2. Bacterial AST profiles were determined using a flow cytometry based counting of individual bacteria in an Aquila 400 instrument (Q-linea AB, Sweden). Aquila 400 analysis was performed using the alexa 488 laser.

(50) It was evident from the analysis performed at different time points that 4 hours was sufficient for detecting differential bacterial growth at the different concentration of antibiotics and thus to determine antibiotic susceptibility. Shorter times have been shown in the literature so this is not unexpected. Differential bacterial growth relative to the positive control sample for each antibiotic is shown in FIG. 3. MIC values for each antibiotic at different time points and cut-off values were calculated based on the differential bacterial growth. Values obtained by this method compared favourably with previously reported clinical MIC values and are shown below. The results show excellent precision and sensitivity obtained by counting individual bacteria instead of averaging measurements.

(51) Cefotaxime

(52) MIC with Macrobroth dilution is 0.125 μg/ml and another lab has determined MIC with E-test to ≤0.5 μg/ml.

(53) TABLE-US-00001 Cut-off 4 hours 6 hours 24 hours  5% — 0.06 0.125 10% — 0.06 0.125 15% 0.06 0.03 0.06 20% 0.06 0.03 0.06
Ciprofloxacin

(54) MIC with Macrobroth dilution is 0.016 μg/ml and another lab has determined MIC with E-test to ≤0.03 μg/ml.

(55) TABLE-US-00002 Cut-off 4 hours 6 hours 24 hours  5% 0.016 0.008 0.008 10% 0.008 0.008 0.008 15% 0.008 0.008 0.008 20% 0.008 0.004 0.008
Gentamicin

(56) MIC with Macrobroth dilution is 0.5-1 μg/ml and another lab has determined MIC with E-test to 1 μg/ml.

(57) TABLE-US-00003 Cut-off 4 hours 6 hours 24 hours  5% 1 0.5 1 10% 0.5 0.5 1 15% 0.5 0.5 1 20% 0.5 0.5 1
Meropenem

(58) MIC with Macrobroth dilution is 0.25-0.5 μg/ml and another lab has determined MIC with E-test to ≤0.25 μg/ml. MIC estimated with Macrobroth dilution and with Aquila 400 is too high because old stock of antibiotics was used.

(59) TABLE-US-00004 Cut-off 4 hours 6 hours 24 hours  5% — — 0.5 10% — — 0.5 15% — 0.5 0.25 20% 0.5 0.25 0.125
Ceftazidime

(60) MIC with Macrobroth dilution is 0.25 μg/ml and another lab has determined MIC with E-test to ≤0.5 μg/ml.

(61) TABLE-US-00005 Cut-off 4 hours 6 hours 24 hours  5% 0.25 0.125 0.25 10% 0.25 0.125 0.25 15% 0.125 0.125 0.25 20% 0.125 0.125 0.25
Piperacillin and Tazobactam

(62) MIC with Macrobroth dilution is 4-8 μg/ml and another lab has determined MIC with E-test to 2 μg/ml.

(63) TABLE-US-00006 Cut-off 4 hours 6 hours 24 hours  5% 4 4 4 10% 4 4 4 15% 4 4 4 20% 4 2 2

Example 4

Method with AST Determination by AST Using Padlock Probes and C2CA Amplification

(64) Blood cultures spiked with E. coli were set up and molecular typing was performed as described in Example 1. 5 ml from the remaining sample in the blood culture bottle, that had continued to be under culture conditions during the molecular typing, was drawn from the flask and the sample was enriched for bacteria and changed into MH-media as in Example 2.

(65) 63 μl aliquots were transferred to selected wells in a microtiterplate. Each well had a different concentration of antibiotics as well as different antibiotics. In one microtiterplate 12 different antibiotics had been placed, each at four different concentrations and one blank, i.e. no antibiotics. After 4 hours growth in the microtiterplate each well were subjected to bacterial lysis to release the bacterial DNA.

(66) Antibiotic susceptibility testing was performed using Amplified Single Molecule Detection (ASMD) where the DNA from pathogens is extracted followed by digital counting of amplification products, rather than by direct labelling of bacteria. The molecular detection test was carried out on the bacterial DNA sample by the method in Göransson et al, 2012.

(67) Padlock probes and target capture probes were ordered from Integrated DNA Technologies (Munich, Germany).

(68) The probes were designed to detect unique motifs in each bacteria, selected via bioinformatics tools. the hybridization of capture probes and ligation of padlock probes to the target DNA were performed simultaneously, and was achieved by incubating fragmented and denatured genomic DNA in 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% Triton® X-100, 100 nM padlock probe, 50 nM capture probe, 0.2 μg/μl BSA (New England Biolabs, MA, USA), and 250 mU/μl Ampligase (Epicentre Biotechnologies, WI, USA) at 55° C. for 5 min. The target DNA along with reacted padlock probes were captured onto magnetic particles via the biotinylated capture probes. This was achieved by adding 50 μg Dynabeads MyOne™ Streptavidin T1 beads (Invitrogen) to the hybridization/ligation reaction and incubating the sample at room temperature for 3 min. Excess probes were eliminated by washing once with 100 μl washing buffer containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1 M NaCl, and 0.1% Tween-20. The elimination of excess linear padlock probes is necessary, since these would otherwise interfere negatively with the subsequent RCA reaction.

(69) Reacted probes were amplified by C2CA, which includes serial enzymatic reactions starting with RCA. The RCA reaction was initiated by the addition of 20 μl ligation mixture containing 1×phi29 DNA polymerase buffer (Fermentas, Lithuania; 33 mM Tris-acetate (pH 7.9 at 37° C.), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween-20, 1 mM DTT), 100 μM dNTPs, 0.2 μg/μl BSA, 25 nM primer, and 100 mU/μl phi29 DNA polymerase. The reaction was incubated at 37° C. for 11 min, and inactivated at 65° C. for 1 min. The RCA products were digested at 37° C. for 1 min by the addition of 3 units of Alul (New England Biolabs), 600 nM replication oligonucleotide, 0.2 μg/μl BSA in 1×phi29 DNA polymerase buffer, and the reaction was terminated at 65° C. for 1 min. Ligation, amplification and labelling reactions were performed by the addition of a mixture containing 1.36 mM ATP, 100 μM dNTPs, 0.2 μg/μl BSA, 28 mU/μl T4 DNA ligase and 120 mU/μl phi29 DNA polymerase in 1×phi29 DNA polymerase buffer to a final volume of 50 μl. The reactions were incubated at 37° C. for 7 min, and terminated at 65° C. for 1 min. The above was repeated once.

(70) After the last RCA reaction fluorescent labelled oligonucleotides complementary to the RCP was added at a concentration of 5 nM each. The reaction was incubated at 65° C. for 2 minutes followed by 5 minutes at 37° C. and allowed to cool down.

(71) The samples were then injected into Aquila 400, (U.S. Patent Application No. 61/979,319) and number of RCP products were recorded as described in Jarvius et al, 2006 (Jarvius J., et al, Nature Methods 3, 725-727 (2006). The number of RCP products detected for the bacterial sample grown at each concentration of Ciprofloxacin and Cefotaxime is shown in FIG. 4. This data was used to estimate an MIC concentration for each antibiotic.

Example 5

Strategy for Probe Design

(72) Identification of the pathogen can performed in multiplex. For this each included pathogen has up to three specific capture oligonucleotides used to fish out the target DNA. Each pathogen also has up to three specific padlock probes hybridizing to the target near to the respective capture oligonucleotides. The process for designing ASMD probe sets can be divided into two main steps: finding optimal target regions and designing the probe sequences.

(73) Below is a further breakdown of the design process: Finding genomic targets—Acquire microbial genome sequences from a genome database (e.g. NCBI)—Partition genome sequences into target and background groups. —If more than one target genome, search for sequences common to all. —Apply a set of filters to remove low-complexity candidates (homopolymers, high/low %-GC, repeats, etc.)—Background filtering: candidates with high sequence similarity to genomes in background partition are discarded. —Report accepted candidates. Make probes—Load genomic targets to which probes will be designed (selected in the step above)—Choose settings (probe length, melting temperatures, hetero/homodimer filter, ligation filter, etc.)—Find optimal probe sequences. —Present passed candidates and a short design summary.

(74) The probes used for filtering and recognition of genomic DNA targets are made up as shown in FIG. 5. The capture probe brings target fragments onto solid phase, and the padlock then binds to its target, gets ligated into a circle and is amplified in the subsequent C2CA reaction. The padlock has two target-complementary parts (5′/3′-arms) and a backbone with sites for detection (generic), restriction digestion and priming (generic), and array oligo hybridization (unique). The backbone parts are referred to as DO, RO, and AO in FIG. 5. The capture oligo consists of a target complementary part, a CT-linker and a biotin for attachment to solid phase.

Example 6

Blood Culture and Microbial Identification of Clinical Samples by Molecular Tests

(75) Blood from patients, at an ICU, with suspected sepsis were drawn into Bactec (Becton Dickinson) blood culture flask and cultured in a blood culture cabinet. After four hours of incubation, well before the flasks have indicated positivity in the blood culture cabinet, a sample was drawn and used for molecular tests.

(76) 5 ml of the sample was aspirated, using a syringe and potential pathogen DNA was extracted as described in Example 1.

(77) The molecular test was carried out on the enriched bacterial DNA sample by the method in Goransson et al, 2012 (supra).

(78) Padlock probes and target capture probes were ordered from Integrated DNA Technologies (Munich, Germany). The probes were designed to detect unique motifs in each bacteria, selected via in-house developed bioinformatics tools. The hybridization of capture probes and ligation of padlock probes to the target DNA were performed simultaneously, and was achieved by incubating fragmented and denatured genomic DNA in 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% Triton® X-100, 100 nM padlock probe, 50 nM capture probe, 0.2 μg/μl BSA (New England Biolabs, MA, USA), and 250 mU/μl Ampligase (Epicentre Biotechnologies, WI, USA) at 55° C. for 5 min. The target DNA along with reacted padlock probes were captured onto magnetic particles via the biotinylated capture probes. This was achieved by adding 50 μg Dynabeads MyOne™ Streptavidin T1 beads (Invitrogen) to the hybridization/ligation reaction and incubating the sample at room temperature for 3 min. Excess probes were eliminated by washing (once) with (100 μl) washing buffer containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1 M NaCl, and 0.1% Tween-20. The elimination of excess linear padlock probes is performed, since these may otherwise interfere negatively with (the) subsequent RCA reaction.

(79) Reacted probes were amplified by C2CA, which includes serial enzymatic reactions starting with RCA. The RCA reaction was initiated by the addition of 20 μl ligation mixture containing 1×phi29 DNA polymerase buffer (Fermentas, Lithuania; 33 mM Tris-acetate (pH 7.9 at 37° C.), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween-20, 1 mM DTT), 100 μM dNTPs, 0.2 μg/μl BSA, 25 nM primer, and 100 mU/μl phi29 DNA polymerase. The reaction was incubated at 37° C. for 11 min, and inactivated at 65° C. for 1 min. The RCA products were digested at 37° C. for 1 min by the addition of 3 units of Alul (New England Biolabs), 600 nM replication oligonucleotide, 0.2 μg/μl BSA in 1×phi29 DNA polymerase buffer, and the reaction was terminated at 65° C. for 1 min. Ligation, amplification and labelling reactions were performed by the addition of a mixture containing 1.36 mM ATP, 100 μM dNTPs, 0.2 μg/μl BSA, 28 mU/μl T4 DNA ligase and 120 mU/μl phi29 DNA polymerase in 1×phi29 DNA polymerase buffer to a final volume of 50 μl. The reactions were incubated at 37° C. for 7 min, and terminated at 65° C. for 1 min. The above was repeated once. After the final RCA the products were digested once again into monomers. The RCPs were now ready for analysis.

(80) The digested sample was transferred to vessel containing a microarray, incubated at 55° C. for 30 minutes followed by a wash with 1×SSC in RT. The hybridized RCA monomers is then labelled via hybridization of a detector oligo at 10 nM concentration in 2×SSC at 55° C. for 30 minutes, washed twice in 1×SSC at RT and spun dry.

(81) The array was then scanned in an array scanner and the result analysed using image analysis software. The data shown have been normalized and only data are above background times 3 standard deviations from background are classified as true signals.

(82) Data from 10 clinical samples plus one spiked sample is shown in FIG. 6. The spiked sample was spiked with E. coli into a blood culture flask with blood from a negative sample. The amount of bacteria spiked in corresponds to the amount that would have been present if the original amount were 10 CFU E. coli ml blood into BCF and then grown for four hours. Eight of the clinical samples were found to be negative by blood culture, and two were found to be positive by blood culture. The positive samples were later identified as one E. coli and one S. pneumoniae using standard techniques.

(83) The only positive signals obtained were from the expected array features for each sample. One of the clinical samples was found to contain E. coli, and one of the clinical samples was found to contain S. pneumoniae in this assay. The spiked sample was also confirmed to contain E. coli. No signal above background, defined as 3 times standard deviation of the average signal in a set of negative samples, was seen in any of the samples later confirmed to be negative using traditional confirmatory assays. For E. coli two array features per sample (Q1101 and Q799) gave rise to a signal, this is because two different probe systems both detecting E. coli but reporting on different array features were used in this experiment.

Example 7

Time to Positivity After Withdrawal of an Aliquot from the Blood Culture Flask

(84) 10 sets of blood culture flasks were spiked with bacteria. 5 sets were spiked with 500 CFU/ml blood and 5 sets were spike with 50.000 CFU/ml blood. 10 ml of blood from healthy donors were drawn per blood culture flask (with 30 ml blood culture medium) to give a total volume of 40 ml/culture flask. Before the blood culture flasks were put into the blood culture cabinet an aliquot of 5 ml were withdrawn. Every 30 minutes we controlled if the blood culture flasks have indicated positivity in the blood culture cabinet.

(85) The time-to-positivity (TTP) is shown in FIG. 7, and was not found to differ significantly between the set. No significant difference was found between flasks that had an aliquot removed prior to culture, and flasks which did not have an aliquot removed.

Example 8

Aseptic Sampling of an Aliquot from a Blood Culture Flask

(86) 24 blood culture flasks (BCFs) divided into three groups of 8 were treated as indicated:

(87) Group 1: Piercing of septum with a needle

(88) Group 2: 10{circumflex over ( )}8 CFU/ml broth with E. coli swabbed on septum before piercing with a needle

(89) Group 3: No piercing of septum

(90) A sterile single use needle were used for each BCF.

(91) The BCFs were put into a blood culture cabinet and allowed to stand for five days. No growth were indicated in any of the group 1 or group 3 BCFs. One out of eight BCFs in group 2 became positive within five days, indicating a potential need for decontamination of septa before taking an aliquot if bottle is heavily contaminated (see Table 1).

(92) TABLE-US-00007 TABLE 1 E. coli 10{circumflex over ( )}8 CFU/ml No Piercing of contaminated cap piercing septum with before piercing of of Sample needle septum with needle septum 1 Neg Neg Neg 2 Neg Neg Neg 3 Neg Neg Neg 4 Neg Neg Neg 6 Neg Neg Neg 7 Neg Pos Neg 8 Neg Neg Neg DEHN 200057US02 4417101 1