APPARATUS AND METHODS FOR MICROBIOLOGICAL ANALYSIS

• US 20140051113A1
• Filed: 04/30/2013
• Published: 02/20/2014
• Est. Priority Date: 05/01/2012
• Status: Active Grant

• Current Assignee: Oxoid Ltd. (Ireland)
• Original Assignee: Oxoid Ltd. (Ireland)
• Inventors: Stephenson, James L. Jr., Gvozdyak, Oksana, Grist, Roger, Campbell, Clay, Jardine, Ian D.
• Granted Patent: US 9,074,236 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: G01N 30/7233 interfaced to liquid or supercritical fluid chromatograph interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: H01J 49/0031 Step by step routines describing the use of the apparatus H01J49/0081 takes precedence
• CPC Class Codes: H01J 49/0036 Step by step routines describing the handling of the data generated during a measurement
• CPC Class Codes: H01J 49/165 Electrospray ionisation

Abstract

Methods and systems for identification of microorganisms either after isolation from a culture or directly from a sample. The methods and systems are configured to identify microorganisms based on the characterization of proteins of the microorganisms via high-resolution/mass accuracy single-stage (MS) or multi-stage (MSⁿ) mass spectrometry. Included herein are also discussion of targeted detection and evaluation of virulence factors, antibiotic resistance markers, antibiotic susceptibility markers, typing, or other characteristics using a method applicable to substantially all microorganisms and high-resolution/mass accuracy single-stage (MS) or multi-stage (MSⁿ) mass spectrometry.

Claims

1. A method for identifying one or more microbes, comprising:

a. providing a sample containing one or more microbes;

b. disrupting the one or more microbes present in the sample to provide a fluid extract;

c. separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract and preparing a solution of the soluble protein fraction;

d. ionizing a stream or flow of the solution of soluble protein fraction to form one or more ionized proteins;

e. analyzing the one or more ionized proteins with a mass spectrometer, wherein analyzing comprises;

i. in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the soluble proteins in the sample;

ii. determining molecular weights for the proteins from the one or more mass spectra;

iii. using the determined molecular weights to search a first database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the database;

iv. in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the one or more ionized proteins of the sample and fragmenting the precursor ions by fragmentation means to produce a plurality of product ions;

v. using mass-to-charge ratios of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of product ion m/z values, amino acid sequence, or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database; and

f. using information obtained in step (e) for one or more proteins from each of the one or more microbes in the sample to identify at least one of the microbes.

2. The method of claim 1, wherein step (b) is effective for Gram-positive bacteria, Gram-negative bacteria, yeast, viruses, mycobacteria and filamentous fungi.

3. The method of claim 1, wherein the ionization is electrospray ionization.

4. The method of claim 1, wherein in the first mass spectrometry step the mass spectra for the one or more of the proteins in the sample have a mass accuracy of at least 15 parts-per-million or better.

5. The method of claim 1, wherein the fragmentation means is collision-induced dissociation.

6. The method of claim 1, wherein in the second mass spectrometry step the mass-to-charge ratios for the product ions have a mass accuracy of at least 15 parts-per-million or better.

7. The method of claim 1 or claim 2, wherein the solution of soluble proteins is streamed directly into an ionization apparatus without chromatographic separation.

9. The method of claim 1, wherein steps (b)-(f) are performed using automated instrumentation.

17. The method of claim 1, wherein step (c) is performed using a solid-phase extraction device comprising bonded silica sorbents, polymeric sorbents, or chelating agents.

21. The method of claim 1, wherein prior to step (b), the sample is pre-treated with a solvent that facilitates disruption of the one or more microbes.

22. The method of claim 1, wherein prior to step (b) the sample is pre-treated with one or more organic solvents, provided that if only one organic solvent is used, the one organic solvent is other than ethanol.

8. A method for identifying one or more microbes, comprising:

a. providing a sample suspected of containing one or more microbes;

b. disrupting the one or more microbes present in the sample to provide a fluid extract;

c. separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract and preparing a solution of the soluble protein fraction;

d. ionizing a stream or flow of the solution of soluble protein fraction to form one or more ionized proteins;

e. analyzing the one or more ionized proteins with a mass spectrometer, wherein analyzing comprises;

i. in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the soluble proteins in the sample;

ii. determining molecular weights for the proteins from the one or more mass spectra;

iii. using the determined molecular weights to search a first database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the database;

iv. in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the one or more ionized proteins of the sample and fragmenting the precursor ions by fragmentation means to produce a plurality of product ions;

v. using mass-to-charge ratios of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of amino acid sequence or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database; and

f. automatically analyzing a sufficient number of proteins to identify at least one of the one or more microbes in said sample.

10. The method of claim 8, wherein at least one of the first or the second mass spectrometry step comprises selecting said one or more precursor ions from a mass range window, and repeating said second mass spectrometry step until a sufficient number of mass range windows have been covered to identify at least one of the one or more microbes in said sample.

18. The method of claim 8, wherein step (c) is performed using a solid-phase extraction device comprising bonded silica sorbents, polymeric sorbents, or chelating agents.

23. The method of claim 8, wherein prior to step (b), the sample is pre-treated with a solvent that facilitates disruption of the one or more microbes.

24. The method of claim 8, wherein prior to step (b) the sample is pre-treated with one or more organic solvents, provided that if only one organic solvent is used, the one organic solvent is other than ethanol.

11. A method for identifying and characterizing one or more microbes, comprising:

a. providing a first aliquot of a sample containing one or more microbes;

b. disrupting the one or more microbes present in the first aliquot to provide a fluid extract;

c. separating a first soluble protein fraction from an insoluble protein fraction present in the fluid extract and preparing a first solution of the soluble protein fraction;

d. ionizing a stream or flow of the first solution of soluble protein fraction to form one or more first ionized proteins;

e. analyzing the one or more first ionized proteins with a mass spectrometer, wherein analyzing comprises;

i. in a first mass spectrometry step, acquiring one or more first mass spectra representative of one or more of the soluble proteins in the sample;

ii. determining molecular weights for the soluble proteins based upon the one or more first mass spectra;

iii. using the determined molecular weights to perform a first search of a first database containing molecular weights of known microbial proteins, and selecting a first subset of candidate microbes from the first database on the basis of the determined molecular weights of soluble proteins from the sample;

iv. in a second mass spectrometry step, selecting one or more first precursor ions of the one or more first ionized proteins and fragmenting the first precursor ions by fragmentation means to produce a first plurality of first product ions;

v. using mass-to-charge ratios of the first plurality of first product ions to perform a first search of a second database containing molecular weights of known microbial proteins and at least one of product ion m/z values, amino acid sequence or post-translational modification information of said known microbial proteins, wherein the first subset of candidate from the first database is used to limit the first search of the second database;

f. using information obtained in step (e) for one or more soluble proteins from each of the one or more microbes in the sample to identify at least one of the microbes to the genus species level;

g. using information from step (f) to automatically select a second analytical method from a list of pre-defined analytical methods, the second method also using mass spectrometry;

h. providing a second aliquot of the fluid extract or of a second fluid extract of the same sample;

i. separating a second soluble protein fraction from an insoluble protein fraction present in the second aliquot and preparing a second solution of the second soluble protein fraction;

j. subjecting the second solution to chromatographic separation to provide a subset of soluble proteins;

k. ionizing a stream or flow of the subset of soluble proteins to form one or more second ionized proteins;

l. analyzing the one or more second ionized proteins with a mass spectrometer, wherein analyzing comprises;

i. in a third mass spectrometry step, acquiring one or more second mass spectra representative of one or more soluble proteins in the subset of soluble proteins;

ii. determining molecular weights for the one or more soluble proteins in the subset of soluble proteins from the one or more second mass spectra;

iii. performing a second search of the first database and selecting a second subset of the candidate microbes from the database on the basis of the determined molecular weights of the one or more soluble proteins in the subset of soluble proteins;

iv. in a fourth mass spectrometry step, selecting one or more second precursor ions of the one or more second ionized proteins and fragmenting the second precursor ions by fragmentation means to produce a second plurality of product ions;

v. using mass-to-charge ratios of the second plurality of product ions to perform a second search of the second database, wherein the second subset of tentatively identified microbes from the first database is used to limit the second search of the second database;

m. using information obtained in step (l) to identify and, optionally to quantitate, one or more soluble proteins indicative of any of;

strain and/or serovar identification, antibiotic resistance, antibiotic susceptibility, virulence or any combination thereof.

15. The method of claim 11, wherein the first and second analytical methods are performed using the same fluid extract.

16. The method of claim 11, wherein the second analytical method is performed using a second fluid extract of the same sample.

19. The method of claim 11, wherein step (c) is performed using a solid-phase extraction device comprising bonded silica sorbents, polymeric sorbents, or chelating agents.

20. The method of claim 11, wherein chromatographic separation of step (j) comprises rapid, time-compressed chromatography which provides partial separation of proteins in the second solution of the second soluble protein fraction.

25. The method of claim 11, wherein prior to step (b), the sample is pre-treated with a solvent that facilitates disruption of the one or more microbes.

26. The method of claim 11, wherein prior to step (b) the sample is pre-treated with one or more organic solvents, provided that if only one organic solvent is used, the one organic solvent is other than ethanol.

27. The method of claim 11, wherein steps (a)-(m) are performed using automated instrumentation.

12. A method for identifying and characterizing one or more microbes in a sample, the method comprising:

a. performing a first analytical method using mass spectrometry to detect and identify one or more proteins from each of the one or more microbes;

b. using the identity of the one or more proteins from each of the one or more microbes to further identify at least one of the microbes in the sample;

c. using information from step (b) to automatically select a second analytical method from a list of pre-defined analytical methods, the second method also using mass spectrometry;

d. performing the second analytical method on the sample to determine if proteins indicative of antibiotic resistance markers, antibiotic susceptibility markers and/or virulence factors are present in the sample and, optionally, quantifying the antibiotic resistance markers, antibiotic susceptibility markers and/or virulence factors present in the sample.

13. The method of claim 12, wherein the first analytical method incorporates direct infusion of a solution of one or more soluble proteins derived from a cellular extract of the one or more microbes into an electrospray apparatus.

14. The method of claim 12, wherein the second analytical method incorporates chromatographic separation of a solution of one or more soluble proteins derived from a cellular extract of the one or more microbes prior to introduction of the solution into an electrospray apparatus.

28. A system for mass spectrometry of a sample suspected of containing one or more microbes comprising:

a. a sample handling device configured to manage a plurality of samples;

b. reagents and means for disrupting the one or more microbes present in the sample to provide a fluid extract;

c. reagents and means for separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract;

d. a mass spectrometer capable of ionizing, fragmenting and detecting one or more soluble proteins in the soluble protein fraction; and

e. a control unit linked to one or more of;

the means for disrupting the one or more microbes, the means for separating the soluble protein fraction, and the mass spectrometer, wherein the control unit optionally serves as a data processing unit to process data from the mass spectrometer.

29. The system of claim 28, further comprising a chromatography system including at least one chromatography column.

30. The system of claim 28, wherein the means for separating the soluble protein fraction comprises at least one solid-phase extraction device comprising bonded silica sorbents, polymeric sorbents, or chelating agents.

31. The system of claim 28, further comprising a chromatography system including a chromatography column, wherein the means for separating the soluble protein fraction comprises a first and second solid-phase extraction devices comprising bonded silica sorbents, polymeric sorbents, or chelating agents;

wherein the first solid-phase extraction device is directly in-line with the mass spectrometer and the second solid-phase extraction device is directly in-line with the chromatography column.