IDENTIFICATION OF MICROORGANISMS USING MALDI-TOF-MS ON-PLATE EXTRACTION

Rapid methods that identify sepsis-causing bacteria or yeast aid the physician in critical therapeutic decision-making, thus decreasing patient mortality rates. The methods described herein employ plating microorganisms directly on to a MALDI-MS plate, adding concentrated formic acid, and identifying the microorganism by mass spectrometry. Optionally, an organic solvent may be combined with the formic acid, or added to the sample before or after the concentrated formic acid is added thereto. The methods enable direct extraction of proteins from microorganisms without the need for liquid protein extraction methods and yields positive identification results for gram-positive bacteria, gram-negative bacteria and yeast in minutes.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Patent Application No. U.S. patent application Ser. No. 16/734,826, filed on Jan. 6, 2020, allowed, which is a divisional application of Ser. No. 15/472,759, filed on Mar. 29, 2017, and issued as U.S. Pat. No. 10,557,162, on Feb. 11, 2020, which application is a continuation of U.S. patent application Ser. No. 13/600,702, filed on Aug. 31, 2012, and issued as U.S. Pat. No. 9,631,221 on Apr. 25, 2017, which claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/649,483 filed May 21, 2012 and U.S. Provisional Patent Application No. 61/530,620 filed Sep. 2, 2011, the disclosures of which are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Sepsis is a serious medical condition caused by an overwhelming response of the host immune system to infection. It can trigger widespread inflammation, which can give rise to impaired blood flow. As sepsis progresses, the body's organs can be starved for oxygen and nutrients, causing permanent damage and eventual failure. Left improperly diagnosed or otherwise untreated, the heart weakens and septic shock can occur, leading to multiple organ failure and death. Blood cultures are required to detect the presence of bacteria or yeast in the blood of sepsis patients, to identify the microorganism(s) present and guide treatment. The conventional separation and identification of microorganism(s) from blood cultures takes at least 24-48 hours, which results in many of the septicemia patients being initially treated with inappropriate antibiotics. It is therefore desirable to separate and identify microorganisms from a positive culture (blood, cerebrospinal fluid etc.) rapidly.

Recently, certain proteomic technologies/tools, such as Matrix-Assisted Laser Desorption Ionization Time of Flight mass spectrometry, (“MALDI-TOF MS”), have been shown to provide a rapid and accurate identification of bacteria and/or fungi from a positive blood culture (“PBC”). The microorganism in the PBC sample can be sub-cultured prior to MALDI identification. In the alternative, microorganisms can be isolated from the PBC sample using various sample preparation methods without the need for subculturing. The microorganism isolates are generally directly smeared onto a MALDI plate to yield about 70-80% identification accuracy. For isolates failing to yield any identification, a follow-up liquid extraction method is typically used to extract proteins from the microorganism for improved identification by MALDI-TOF MS. Although these liquid protein extraction methods generally yield better identification accuracy, such methods not only require several centrifugation steps, but also are time-consuming.

Schmidt et al (Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Eur. J. Clin. Microbiol. Infect Dis, 23 Jun. 2011) discloses a method of identifying bacteria from positive blood cultures by spotting a liquid sample of the isolated bacteria onto a MALDI plate and overlaying 25% formic acid directly to the spotted liquid sample. Therefore, the final concentration of formic acid in the bacterial sample is less than 25%. The Schmidt method results in 86.6% identification accuracy for gram-negative bacteria and 60% identification accuracy for gram-positive bacteria. Schmidt does not test this method in Yeast.

Hyman et al (U.S. Patent Publication No. 2010/0120085, Published May 13, 2010), discloses a similar method as Schmidt, in which intact isolated microorganisms in solution are directly smeared onto a MALDI plate. The liquid sample is then overlaid with roughly an equal volume of 50% formic acid. Therefore, the final concentration of formic acid added to the sample is approximately 25%. This method was tested on 14 different species of bacteria and yeast. Although this method resulted in 91.1% identification, the data does not indicate how effective this method is with regard to gram-positive bacteria, gram-negative bacteria, or yeast.

Haigh et al. “Improved Performance of Bacterium and Yeast Identification by a Commercial Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry System in the Clinical Microbiology Laboratory,” J. Clin. Microbiol. (Jul. 6, 2011) describes a method in which neat formic acid is used to extract microbial proteins smeared directly onto a MALDI plate. This method, however, was unable to successfully identify all strains of yeast and gram-positive bacteria.

Herendael et al. “Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)”, Eur. J. Clin. Microbiol. Infect Dis (23 Aug. 2011) describes two methods for the identification of yeast. The standard extraction method described in Herendael et al., is a conventional liquid extraction method (this method is the “prior art procedure” of Example 1 hereinbelow). In the short extraction method described in Herendael et at, one colony was picked from an agar plate and applied directly to the target MALDI plate. Formic acid (1 μL at 70% concentration) was added to the sample and the sample was allowed to dry. The dried sample was overlaid with MALDI matrix, allowed to dry further, and analyzed by MALDI-MS. The short extraction method provided identical results as the standard extraction method although the MALDI scores were lower with the short extraction method. Nearly all of the isolates (97.6%) could be identified with the short extraction method; however 17.1% of these identifications fell below the reliable threshold level of 1.7.

Accordingly, there is a need to develop a rapid and accurate method of identifying all classes of bacteria and yeast without the need for liquid protein extraction prior to identification with MALDI-TOF MS.

SUMMARY OF THE INVENTION

Various embodiments of the disclosed method enable direct identification of microorganisms from positive blood cultures (“PBC”) or pure isolates by mass spectrometry without the need for a liquid protein extraction. In one embodiment of this method, identification of a microorganism from a pure isolate is achieved by obtaining a sample containing at least one microorganism, depositing at least a portion of the pure isolate sample (direct smear or microbial suspension) on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry, treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid, drying the sample, placing a MALDI matrix solution over the treated sample, and identifying the microorganism by MALDI mass spectrometry.

Optionally, a PBC sample is first processed to isolate the microorganism, followed by identification of the microorganism without performing a liquid protein extraction. This embodiment is achieved by: i) obtaining a PBC sample determined to contain at least one microorganism; ii) adding a lysis buffer to the sample to lyse the blood cells; iii) while the microorganism remains intact and viable, isolating the intact microorganism; iv) depositing the isolated microorganism on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; v) treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; vi) placing a MALDI matrix solution over the treated sample; and vii) identifying the microorganism by mass spectrometry. In another embodiment, the PBC sample is sub-cultured to produce a pure culture of the microorganism prior to identifying the microorganism. In one embodiment, the sample can be dried before treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; placing a MALDI matrix solution over the treated sample; and/or identifying the microorganism by mass spectrometry (i.e., before the treating step v) above).

The described methods can be used to isolate and/or identify a spectrum of microorganisms including but not limited to, gram-positive bacteria, gram-negative bacteria, fungi, mycobacterium, or yeast.

In another embodiment, a kit is provided for the detection of microorganisms in a sample. The kit includes, for example, one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart that illustrates the prior art method of extracting proteins from pure microbial isolates for identification by mass spectrometry.

FIG. 2 illustrates one embodiment of the disclosed method in which proteins are extracted from isolated microorganisms directly on a MALDI plate using concentrated formic acid.

FIG. 3 illustrates one embodiment of the disclosed method in which proteins are extracted from isolated microorganisms directly on a MALDI plate using concentrated formic acid in an organic solvent.

 DETAILED DESCRIPTION

FIG. 1 illustrates the prior art methods of identifying microorganisms from PBC. These methods are known to one having ordinary skill in the art. For example, these methods are described in Nassiff et al WO 2010/100612 A1, the disclosure of which is incorporated herein by reference. A positive blood culture 100 is obtained. The microorganism is isolated from the remaining blood cells and debris either by liquid separation 110 or by sub-culturing the microorganism 120 to produce a pure culture of microorganism 130. To identify the microorganism, microorganism separated by liquid separation 110 or the pure culture 130 can be directly smeared onto a MALDI plate 140 for MALDI-MS identification 170 or undergo liquid protein extraction 150 prior to transferring to the MALDI plate 160 and identification by MALDI-MS 170.

In one embodiment of the invention, direct identification of microorganisms is achieved from pure isolates or a PBC sample by mass spectrometry without requiring a liquid protein extraction. In one embodiment, the microorganism from pure isolates is identified by: i) obtaining a sample suspected to contain at least one microorganism; ii) depositing at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry in a manner that controls any dilution of a volatile acid and/or an organic solvent to be combined with the deposited sample; iii) treating the sample with at least one reagent; such reagents including a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; iv) placing a MALDI matrix solution over the treated sample; and v) identifying the microorganism by mass spectrometry. In one embodiment the volatile acid is at least 70% formic acid. In another embodiment the volatile acid is at least 80% formic acid. In another embodiment the volatile acid is at least 90% formic acid. Unless otherwise specified herein, the formic acid solutions are aqueous solutions. In another embodiment the volatile acid is at least 100% formic acid (e.g. neat). In another embodiment, the sample is treated with at least 70% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In another embodiment, the sample is treated with at least 80% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In another embodiment, the sample is treated with at least 90% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In one embodiment, the sample deposited on the solid surface is allowed to dry prior to adding the volatile acid, to prevent the sample from diluting the volatile acid. In another embodiment, the volatile acid is dried prior to placing the MALDI matrix solution over the sample. Examples of the volatile acids that may be used in the various embodiments of the invention include, but are not limited to, formic acid, acetic acid, trifluoracetic acid and hydrochloric acid. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the disclosed methods are used to isolate and/or identify gram-positive bacteria, gram-negative bacteria, or yeast. In another embodiment, the disclosed methods are used to isolate and/or identify Streptococcus pneumoniae. In another embodiment, the disclosed methods are used to isolate and/or identify yeast, including Cryptococcus neoformans.

In one embodiment, identification of microorganisms having thickened or hydrophobic cell wall complexes, for example, yeast and mycobacteria, is performed by using any of the methods described herein, which deploy an organic solvent, among other reagents, for on-plate extraction. In a preferred embodiment, Cryptococcus neoformans is the microorganism targeted for identification. Without being bound by a particular theory, it is believed that the organic solvent dissolves or disperses interfering substances in the cell wall and/or bound extracellular material, such as polysaccharides or lipids, and makes the cell wall and intracellular proteins more susceptible and more accessible, respectively, to the extraction methods.

After the sample is combined with the reagent(s), the combination of sample and reagents is dried. Drying is defined as allowing the liquid to evaporate sufficiently so as not to dilute any liquid subsequently added. While the sample can be dried in ambient air, a heating source, such as a heating block, hot plate, heating oven or infrared heating lamp can be used to accelerate the evaporation of the liquid portion of the combined sample and reagents. These drying methods do not change the spectrum of the sample upon identification by MALDI.

After the extracted sample is dried, it is combined with additional reagents for downstream sample assay using MALDI. Any MALDI matrix solution known to those skilled in the art can be used in the disclosed methods. These matrix solutions include, but are not limited to, α-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (SPA), 3-hydroxypicolinic acid (HPA), 3.4-dihydroxycinnamic acid, 2-(4-hydroxyphenylazo)-benzoic acid, 2-amino-4-methyl-5-nitropyridine, and 2,4,6-trihydroxy acetophonone (THAP).

In one embodiment, the methods described herein can also include using a non-ionic detergent capable of solubilizing proteins as an extraction reagent in addition to those described above. The detergent does not interfere with the MALDI ionization process. These detergents can be incorporated into the initial microorganism suspension or subsequent volatile acid and/or organic solvent treatment. Typical detergents suitable for MALDI include, for example, n-octyl-β-D-glucopyranoside (OG), saponin, triton, and those described in International Publication No. WO 2010/100612, incorporated herein by reference. In one embodiment, the concentration of detergent is in the range of 1% to 5%. In another embodiment, the concentration of detergent is 2%.

Referring to FIG. 2, which illustrates the protein extraction method of one embodiment of the disclosed methods, an isolated microorganism 200 is obtained (i.e. pure culture from sub-culturing or liquid separation as described in FIG. 1). The isolated microorganism 200 is added directly to the MALDI plate 210. The isolated microorganism 200 on MALDI plate 210 is allowed to air dry 220 to produce dried, isolated microorganism 230. Ninety percent formic acid 240 is applied to the dried, isolated microorganism sample 230 to obtain extracted microorganism sample 250 which contains solubilized proteins of the microorganism. Extracted microorganism sample 250 is allowed to air dry 260 to produce dried, extracted microorganism sample 270. MALDI matrix 280 is added to dried, extracted microorganism 270, allowed to air dry 285, and identified by MALDI-MS 290.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) drying the sample; iv) treating the sample with at least 70% formic acid in an aqueous solution or at least 70% formic acid in an organic solvent; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In yet another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) treating the sample with an organic solvent; iv) drying the sample; vii) treating the sample with formic acid and an organic solvent; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. In one embodiment, the organic solvent is ethanol, methanol, isopropanol, or acetone. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In yet another embodiment, the sample suspected of containing at least one microorganism is resuspended in a solution prior to deposition onto the solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry. One skilled in the art will appreciate that the concentration of the microbial suspension will be optimized for the various disclosed embodiments. For example, in one embodiment, a higher concentration of microbial suspension may be required, such as a concentration greater than 0.5 McFarland (for example, about 0.75 McFarland and higher), to better ensure proper identification of the microorganism (or a reliable indication of the absence of the microorganism) by MALDI-MS. In another embodiment, the concentration of the microbial suspension can be adjusted for use in the various disclosed embodiments as well as in additional assays, such as those described in U.S. patent application Ser. No. 13/177,031 (which is incorporated by reference herein), which include, for example, antimicrobial susceptibility testing (AST). In one embodiment, the microbial suspension is at least about 2.0 McFarland or higher.

One skilled in the art is aware that MALDI identification results are affected by the amount of cells deposited onto the MALDI plate. A standardized microbial suspension provides a uniform dispersion of cells, leading to more precise and reproducible results. In one embodiment, the microbial suspension is standardized prior to deposition onto the solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry. The microbial suspension can be optionally adjusted to a certain McFarland standard. Creation of the standardized microbial suspension can be accomplished by various methods well known to those skilled in the art, for example, using an inoculation loop, microdropper, or other physical methods. In a preferred embodiment, a microbial suspension is adjusted to a McFarland standard of at least 0.5 prior to deposition onto a MALDI plate. In another embodiment, a microbial suspension is adjusted to a McFarland standard in the range of 0.5-10 McFarland prior to deposition onto a MALDI plate.

Referring to FIG. 3, which illustrates the protein extraction method of one embodiment of the disclosed methods, an isolated microorganism 300 is obtained (i.e. pure culture from sub-culturing or liquid separation as described in FIG. 1). The isolated microorganism 300 is resuspended in organic solvent 305 to produce resuspended microorganism 310. The isolated microorganism 300 can be resuspended in any suitable organic solvent 305 such as, for example, ethanol, methanol, acetone, or ethyl acetate. Any concentration of organic solvent 305 may be used, and the appropriate concentration for a particular application is readily determined by one skilled in the art. In alternative embodiments, the concentration of organic solvent 305 is in the range of about 30% to about 70%. In another alternative embodiment, the organic solvent is ethanol. The resuspended microorganism 310 is deposited onto a MALDI plate 315. The resuspended microorganism 310 on MALDI plate 315 is allowed to air dry 320 to produce dried, isolated microorganism 325. Seventy percent formic acid in 30% organic solvent 330 is used to extract the dried, isolated microorganism 325. The dried, isolated microorganism 325 on MALDI plate 315 with 70% formic acid in 30% organic solvent 330 is allowed to air dry 335 to produce dried, extracted microorganism 340. MALDI matrix 345 is added to dried, extracted microorganism 340 and allowed to air dry 350 before identification by MALDI-MS 355.

The isolated microorganism 300 is obtained according to any of the exemplary methods described herein or by other methods known to one having ordinary skill in the art.

In one embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in an organic solvent; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an organic solution at a concentration greater than 0.5 McFarland. On one embodiment, the concentration of formic acid is at least 70%. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid and an organic solvent; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an aqueous solution at a concentration greater than 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in an organic solvent solution; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid in the organic solvent; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an organic solvent solution at a concentration greater than 0.5 McFarland. In one embodiment, the sample is resuspended in organic solvent solution with an organic solvent concentration of about 30% to about 70%. In another embodiment, the organic solvent is ethanol. In yet another embodiment, the microorganism to be detected is yeast. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) combining the microorganism resuspended in water with an organic solvent so that the final concentration of organic solvent in the combined solution is in the range of 30%-70%; iv) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; v) drying the sample; vi) treating the sample with an aqueous formic acid solution or formic acid in the organic solvent; vii) drying the sample; viii) placing a MALDI matrix solution over the treated sample; ix) drying the sample; and x) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in water at a concentration greater than about 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with an organic solvent; vi) drying the sample; vii) treating the sample with formic acid in the organic solvent; viii) drying the sample; ix) placing a MALDI matrix solution over the treated sample; x) drying the sample; and xi) identifying the microorganism by mass spectrometry. In this embodiment, the organic solvent is ethanol and the sample suspected of containing at least one microorganism is resuspended in water at a concentration greater than 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In yet another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear or suspension at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) treating the sample with a mixture of formic acid, an organic solvent such as ethanol or acetonitrile, and a MALDI matrix solution; iv) drying the sample; and v) identifying the microorganism by mass spectrometry. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) drying the sample; iv) treating the sample with an organic solvent solution that is at least about 70% organic solvent; v) drying the sample; vi) treating the sample with a formic acid solution that is at least about 70% formic acid; vii) drying the sample; viii) treating the sample with concentrated (100%) organic solvent; ix) drying the sample, x) placing a MALDI matrix solution over the treated sample; xi) drying the sample; and xii) identifying the microorganism by mass spectrometry. In one embodiment, the organic solvent is ethanol. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

In an alternative embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing, via direct smear or from a microbial suspension, at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) fixing the microorganism with an organic solvent, e.g. ethanol, a fixative, e.g. formaldehyde, or by applying heat, generally up to about 37° C. (i.e. approximately body temperature); iv) treating the fixed sample with at least 70% formic acid; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. Fixatives such as formaldehyde are well known to one skilled in the art and are not described in detail herein. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification.

EXAMPLES

In all examples below, the MALDI matrix solution was prepared by dissolving 2.5 mg of HCCA in 250 μL of a solution that is 2.5% trifluoroacetic acid, 47.5% deionized water, and 50% acetonitrile. All the mass spectrometry data was recorded on Bruker Microflex LT with Biotyper software with the following MALDI score key definition. A MALDI ID score of greater than 2.0 indicates a satisfactory identification to the Genus and Species level. A MALDI ID score of 1.7-1.999 indicates a satisfactory identification to the Genus level. A MALDI ID score of less than 1.699 indicates an unacceptable or not reliable identification.

Example 1: Comparison of Extraction Methods Prior Art Procedure:

The method and results from this prior art procedure are reported in and describe the findings of Haigh et al. “Improved Performance of Bacterium and Yeast Identification by a Commercial Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry System in the Clinical Microbiology Laboratory”, J. Clin. Microbiol. (Jul. 6, 2011) and Bizzini et al. “Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory”, J. Clin. Microbiol. (Mar. 10, 2010). A portion of isolated microorganism(s), including gram-positive and gram-negative bacteria, is directly smeared onto a MALDI-TOF MS plate and air dried. The dried sample is overlaid with 1 of matrix solution (HCCA) and air dried before identification by MALDI-TOF MS.

The MALDI analysis results in an overall correct identification of 70.3-75.6%. Therefore, at least 25% of the samples are not identified using this method (referred to as the “direct smear method”).

For those samples not identified by the direct smear method above, a liquid extraction method is performed on the microorganism sample prior to identification. Water (300 μL) is added to an Eppendorf microcentrifuge tube. A large single colony from a sub-culture of the microorganism is transferred to the centrifuge tube containing water and the sample is vortexed thoroughly. Ethanol (900 μL) is added to the tube and vortexed thoroughly. The mixture is centrifuged at maximum speed for 2 minutes at 10,000×g in a microcentrifuge. The ethanol is decanted and the sample is centrifuged again for 2 minutes at 10,000×g speed in a microcentrifuge. Excess ethanol is removed with a pipette. Formic acid (70% solution; 50 μL) is added to the pellet and vortexed thoroughly. Acetonitrile (100% solution; 50 μL) is added to sample. The sample is centrifuged at 10,000×g for 2 minutes in a microcentrifuge. The supernatant (1 μL) is pipetted onto a MALDI plate and allowed to air dry. The dried sample is overlaid with 1 μL of MALDI matrix solution (HCCA) and air dried before identification by MALDI-MS. The liquid extraction procedure results in up to 100% identification of those microorganisms not identified by the direct smear method.

Exemplary Embodiment

From a PBC sample, the pellet of isolated microorganisms was resuspended in 600 μL water or 2 mM OG solution in a microcentrifuge tube. The turbidity was visually adjusted to greater than 0.5 McFarland. Each sample (1-1.5 μL) was spotted onto the MALDI plate. The sample was allowed to air dry. Formic acid (1-2 μL of 70% formic acid solution) was overlaid onto the dried, spotted sample and allowed to air dry. The dried sample was overlaid with 1 μl of matrix solution (HCCA) and allowed to air dry before identification by MALDI-TOF MS.

From a sub-culture, a single colony was touched with a toothpick and a very thin layer of microorganism was smeared directly onto MALDI target plate. The sample was allowed to air dry. Formic acid (1-2 μL of a 70% aqueous solution) was overlaid onto the sample and allowed to air dry. The dried sample was overlaid with 1 μl of MALDI matrix solution (HCCA) and allowed to air dry before identification by MALDI-MS.

The list of organisms tested can be found in Table 1 below. The MALDI-TOF MS analysis from both the microorganism isolated from a PBC sample and from a sub-culture resulted in 95% correct identification, including 95% identification of gram-positive bacteria (organism and type), 100% identification of gram-negative (organism and type), and 100% identification of yeast samples. These results illustrate that the methods described in this example can accurately identify microorganism(s) from a PBC sample or from a sub-culture without the need for the complex and time-consuming prior art liquid extraction methods previously described. Specifically, no separate extraction step was required to identify microorganisms not identified by the methods in this example. In addition, the results demonstrate that these methods result in significantly higher percent identification of a variety of organisms including gram-positive bacteria and gram-negative bacteria, compared to the direct smear method.

TABLE 1 Organism Organism Type Strain Number Acinetobacter baumanii gram-negative ENF 11091 Enterobacter aerogenes gram-negative 13048 Enterobacter cloacae gram-negative 35030 Escherichia coli gram-negative 25922 Escherichia coli gram-negative 35218 Klebsiella pneumoniae gram-negative 33495 Klebsiella pneumoniae gram-negative 700603  (ESBL-producer) Proteus mirabilis gram-negative 29906 Pseudomonas aeruginosa gram-negative 27853 Pseudomonas aeruginosa (Carbap-R) gram-negative ENF 14620 Serratia marcescens gram-negative SCENF FR197 Stenotrophomonas maltophilia gram-negative 13637 Enterococcus faecalis gram-positive 29212 Enterococcus faecalis VRE gram-positive 51299 Enterococcus faecium gram-positive 19434 Enterococcus faecium VRE gram-positive 700221  Staphylococcus aureus gram-positive 25923 Staphylococcus aureus gram-positive 29213 Staphylococcus aureus MRSA gram-positive POS 3421 Staphylococcus aureus MRSA gram-positive 43300 Staphylococcus epidermidis gram-positive SCPOS 3568 Staphylococcus epidermidis gram-positive 14990 Staphylococcus haemolyticus gram-positive POS 3569 Staphylococcus sciuri gram-positive 29062 Streptococcus agalactiae gram-positive 12386 Streptococcus agalactiae gram-positive 13813 Streptococcus pneumoniae gram-positive 49619 Streptococcus pneumoniae gram-positive  6303 Streptococcus pneumoniae gram-positive 700670  (P-resistant) Streptococcus pyogenes gram-positive 19615 Viridans Streptococci gram-positive POS 3177 (Strep salivarius) Viridans Streptococci gram-positive 49456 (Strep mitis) Candida albicans yeast 18804 Candida albicans yeast 24433 Candida glabrata yeast  2001 Candida parapsilosis yeast 22019

Example 2: Comparison of Extraction Protocols Prior Art Procedure:

A liquid sample (0.5 μL) was applied to a target plate and covered with 0.5 μL of formic acid (FA: 25%; AnagnosTec) and allowed to air dry. Therefore, the total formic acid concentration was 12.5%, The dried sample was overlaid with 0.5 μL of 20 mg 2, 5-dihydroxybenzoic acid (DHB; AnagnosTec). The matrix sample was crystallized by air drying at room temperature for 5 minutes. Measurements were performed with a Shimadzu Biotech AXIMA Assurance™ mass spectrometer equipped with a 337-nm nitrogen laser. This method correctly identified 60% of gram-positive bacteria and 86.6% gram-negative bacteria. The method and results of this example summarize the findings of Schmidt et al., “Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry,” Eur. J. Clin. Microbiol Infect Dis, 23 Jun. 2011.

Exemplary Embodiment

The pellet of isolated microorganism from a PBC sample was resuspended in 600 μL water or 2 mM OG solution in a microcentrifuge tube. The turbidity was visually adjusted to greater than 0.5 McFarland. Sample (1-1.5 μL) was spotted onto the MALDI plate. The sample was allowed to air dry. Formic acid (1-2 μL of a 70% solution) was overlaid onto the sample and allowed to air dry. The dried sample was overlaid with 1 μL of matrix solution (HCCA) and allowed to air dry before identification by MALDI-TOF MS. This method correctly identified 95% of gram-positive bacteria and 100% gram-negative bacteria, These results illustrate that the use of formic acid at a concentration well above 12.5% significantly increases the rate of identification of both gram-positive and grain-negative bacteria.

Example 3: Comparison of Extraction Methods with Various Yeast Strains Prior Art Method:

Yeast colonies were resuspended in 600 μl of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Sample (0.5 μL) was pipetted onto a MALDI plate. Formic acid (0.5 μL of a 25% solution in water) was pipetted onto the sample and allowed to air dry. Therefore, the final concentration of formic acid was 12.5%. Matrix solution (1 μL HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS. This prior art method is the same method as disclosed in Schmidt et al., Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Eur. J. Clin. Microbiol. Infect Dis, 23 Jun. 2011.

Exemplary Control Method 1:

Yeast colonies were resuspended in 600μ1 of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Sample (1 μL) was pipetted onto a MALDI plate and allowed to air dry. Formic acid (1 μL of a 50% solution in water) was pipetted onto the dried sample and allowed to air dry. MALDI matrix solution (1 μL of HCCA) was pipetted onto the dried sample and allowed to air dry before identification by MALDI-MS.

Exemplary Control Method 2:

Yeast colonies were resuspended in 6004 of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Sample (1-1.5 μL) was pipetted onto a MALDI plate and allowed to air dry. No formic acid extraction was performed; rather the dried sample was directly overlaid with matrix solution (1 μL of HCCA) and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment:

Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Sample (1-1.5 μL) was pipetted onto a MALDI plate and allowed to air dry. Formic acid (1-1.5 μL of a 60%, 70%, or 90% solution of formic acid in water) was pipetted onto the dried sample and allowed to air dry. Matrix solution (14 of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS.

The results are summarized in Table 2 below and indicate a positive identification, “Yes”, or a failed identification, “No”, by MALDI-TOF MS. The results illustrate that the on-plate extraction methods described herein, when used with at least 70% formic acid, are sufficient to identify various yeast strains. In contrast, the prior art and control methods, which utilize 50% or less formic acid, failed to identify any of the yeast strains.

TABLE 2 12.5% Formic 50% 60% Acid Formic Formic No Formic (Prior Acid Acid 70% 90% Strain Acid Art (Control (Control Formic Formic Organism Number Extraction Method) Method 1) Method 2) Acid Acid Candida ATCC No No No No Yes Yes albicans 145 Candida ATCC No No No No Yes Yes parapsilosis 147 Candida ATCC No No No No Yes Yes glabrata 394

Example 4: Comparison of Exemplary Extraction Protocols with 90% Formic Acid+/−Ethyl Acetate for the Identification of Various Yeast Strains Exemplary Embodiment

Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Duplicate samples (1-1.5 μL each) were pipetted onto a MALDI-TOF MS plate and allowed to air thy. Formic acid (1-1.5 μL) of either a 90% solution in water or a 90% solution in ethyl acetate was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. In all, four samples (two samples for each formic acid solution) for each organism were prepared and subjected to MALDI-TOF MS analysis

The MALDI score results are summarized in Table 3 below. These results illustrate an improved identification performance when formic acid extraction buffer is prepared in an organic solvent, for example, ethyl acetate, compared to formic acid in an aqueous solution.

TABLE 3 Strain 90% Formic Acid 90% Formic Acid Organism Number in Water in Ethyl Acetate Candida YST 2.098 2.123 glabrata  26 2.211 2.038 Cryptococcus YST 1.579 1.494 neoformans  54 1.425 1.573 Candida YST 1.626 2.006 parapsilosis  194 1.826 2.014 Candida YST 1.766 1.856 parapsilosis  792 1.532 1.805 Cryptococcus YST 1.437 1.561 neoformans 1162 1.481 1.741 Candida YST 1.754 1.837 albicans 1235 1.656 1.813 Cryptococcus YST 1.344 1.386 neoformans 1479 1.436 1.442 Cryptococcus YST 1.552 1.617 neoformans 1481 1.693 1.772

Example 5: Comparison of Exemplary Extraction Protocols with Formic Acid+/−Acetonitrile or Formic Acid+/−OG Buffer for the Identification of Various Yeast Strains Exemplary Embodiment

Yeast colonies were resuspended in 600 of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Duplicate samples (1-1.5 μL) were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Various formic acid extraction buffers were prepared including: 70% formic acid in water; 90% formic acid in water; 70% formic acid in 20 mM OG buffer in water; and, 90% formic acid in 20 mM OG buffer in water. A volume of the extraction buffer (1-1.5 was pipetted onto the dried samples and allowed to air dry. In one set of samples treated with 90% formic acid in water, a subsequent extraction step was performed by overlaying the dried sample with 50% acetonitrile in water and allowing the sample to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. A total of eight samples were prepared for each organism (two for each extraction buffer) and evaluated by MALDI-TOF MS.

The MALDI-TOF MS score results are summarized in Table 4 below. The results illustrate that even microorganisms that are often difficult to identify, such as yeast, can be reliably identified by using an extraction buffer containing 90% formic acid, by preparing the formic acid extraction buffer in OG buffer or by performing a subsequent solvent only extraction step using an organic solvent such as acetonitrile.

TABLE 4 90% formic acid in water 70% formic followed by acid in 90% formic 90% formic 50% 20 mM OG acid in 20 mM Strain 70% formic acid in acetonitrile in buffer in OG buffer in Organism Number acid in water water water water water Candida glabrata ATCC 1.89 1.845 1.956 1.856 1.884  2001 1.932 1.96 1.968 1.931 1.979 Candida ATCC 1.673 2.093 1.932 2.016 2.019 albicans 18804 1.643 1.927 2.009 1.998 2.093 Candida ATCC <0 1.718 1.642 1.787 2.036 parapsilosis 22019 <0 2.131 1.861 1.871 1.976 Candida ATCC 1.58 1.687 1.874 1.684 2.06 albicans 24433 1.356 1.835 1.9 1.788 1.893 Candida glabrata BQ141 2.076 1.997 2.088 2.205 2.229 1.912 2.064 2.051 2.172 2.171 Candida glabrata BP154 1.941 1.845 1.996 2.154 2.165 1.867 2.034 2.181 1.977 2.199

Example 6: Identification of Various Bacteria According to One Embodiment Exemplary Embodiment

Bacterial colonies from a sub-culture were resuspended in 600 μL water. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three aliquots of the bacterial suspension (1.5 μL) for each microorganism were spotted onto a MALDI-TOF MS plate, and then immediately followed by sequential additions of 1.5 μL of 70% formic acid in ethanol and 1.5 μL of matrix solution (HCCA). The samples were allowed to air dry before identification by MALDI-TOF MS. The same experiment was repeated on a subsequent day by preparing a second bacterial suspension for each microorganism and performing the extraction procedure again preparing three samples for each microorganism.

The MALDI-TOF MS score results are summarized in Table 5 below. These results illustrate that drying the sample after applying the extraction buffer and before applying the matrix solution is not required to obtain positive identification of the microorganism. In addition, these results demonstrate the reproducibility of the extraction procedure. Specifically, overall, two different sample suspensions produce substantially similar identification results.

TABLE 5 Strain Organism Number Suspension 1 Suspension 2 Escherichia coli 25922 2.292 2.140 2.260 2.254 2.207 2.246 Enterococcus 29212 2.445 2.567 faecalis 2.385 2.565 2.263 2.524 Staphylococcus 3569 2.275 2.194 haemolyticus 2.098 2.181 2.005 2.227 Enterococcus 700221 2.316 2.482 faecium 2.377 2.444 0.000* 2.320 Staphylococcus 2885 1.253 1.931 haemolyticus 1.638 2.010 1.532 1.939 Streptococcus 13813 2.276 2.527 agalactiae 2.320 2.419 2.196 2.442 Streptococcus 49456 2.154 2.196 pneumoniae 2.286 2.191 2.157 2.138 Klebsiella 700603 2.189 0.000* pneumoniae 2.054 2.006 2.126 2.057 *These results are considered anomalies in the overall context of the data, and not viewed as a failure in the extraction protocol.

Example 7: Exemplary Embodiments of the Disclosed Methods Compared to Control Methods for the Identification of Yeast Strains Exemplary Control Method (Process 22):

Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Neat formic acid (1 μL) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—on Plate Extraction from Ethanol Suspension (Process 18)

Yeast colonies from a sub-culture were resuspended in 600 μL of 50% ethanol in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% solution in ethanol) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—on Plate Extraction from Water Suspension Followed by Ethanol (Process 19)

Yeast colonies from a sub-culture were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI plate and allowed to air dry. Ethanol (1 μL of 70% concentration) was pipetted onto the dried samples and allowed to air dry. A solution of 70% formic acid/30% ethanol was pipetted (1 μL) onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—On Plate Extraction From Acetone Suspension (Process 23)

Yeast colonies from a sub-culture were resuspended in 600 μL of 50% acetone in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% concentration in acetone) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—on Plate Extraction from Water Suspension Followed by Acetone (Process 24)

Yeast colonies from a sub-culture were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Acetone (1 μL, of 70% concentration) was pipetted onto the dried samples and allowed to air dry. Formic acid (1 ul of 70% formic acid in acetone) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-MS.

The MALDI-TOF MS score results for each of the methods in this example are summarized in Table 6 below. The results illustrate that 70% formic acid with an organic solvent produces superior results than with neat formic acid alone for the identification of yeast strains independent of the suspension used. The type of organic solvent used with formic acid did not affect the overall results. In addition, the organic solvent can be added during suspension of the sample, prior to treatment with formic acid, and/or during formic acid treatment without decreasing the rate of identification of various yeast strains.

TABLE 6 Strain Process Process Process Process Process Organism Number 22 18 19 23 24 Candida YST 1.693 2.060 2.067 2.145 2.022 glabrata  26 1.752 2.095 2.137 2.167 1.913 1.766 2.097 2.129 2.172 1.898 Cryptococcus YST 1.823 1.870 1.826 1.862 1.525 neoformans  54 1.794 0.000 1.990 1.917 1.282 1.918 1.816 1.872 1.902 0.000 Candida YST 1.927 1.842 1.949 1.942 1.814 parapsilosis 194 1.968 1.881 2.093 2.007 1.864 2.053 1.974 2.105 1.926 1.860 Candida YST 1.399 1.784 1.743 1.763 1.650 parapsilosis 792 1.255 1.871 1.771 1.932 1.706 1.645 1.733 1.835 1.836 1.769 Cryptococcus YST 1162 1.325 1.550 1.681 1.815 1.591 neoformans 1.489 1.776 1.657 1.671 1.431 1.504 1.623 1.741 1.566 1.562 Candida YST 1235 1.148 2.072 2.024 1.744 1.968 albicans 1.460 2.065 2.004 1.700 1.960 1.365 2.037 1.987 1.837 1.981 Cryptococcus YST 1479 1.390 0.000 1.899 1.913 1.705 neoformans 1.445 1.931 1.821 1.948 1.689 1.413 1.941 1.918 2.069 1.712 Cryptococcus YST 1481 1.299 0.000 2.071 2.020 1.860 neoformans 1.315 2.139 2.076 1.905 1.824 1.485 2.022 2.093 1.981 1.799 Candida YST 1.478 2.176 2.213 2.140 2.147 albicans 145 1.783 2.226 2.045 1.926 2.005 1.598 2.103 2.228 1.860 2.068 Candida YST 2.073 2.113 2.074 2.202 2.084 parapsilosis 147 1.882 2.021 2.052 2.169 2.107 2.021 2.010 2.064 2.137 2.070 Candida YST 2.022 2.010 2.076 2.171 2.188 parapsilosis 200 1.961 2.003 1.946 2.155 1.992 1.914 2.013 2.105 2.202 1.938 Candida YST 2.039 2.284 2.036 2.073 1.997 parapsilosis 214 2.012 2.082 2.082 2.163 2.050 1.991 2.034 2.182 2.156 2.030 Candida YST 1.408 1.741 1.520 1.708 1.341 dubliniensis 305 1.492 1.791 1.467 1.676 1.220 1.565 1.639 1.575 1.262 1.249 Candida YST 1.928 2.213 1.970 2.024 2.141 glabrata 394 1.745 2.255 2.089 2.022 2.108 2.011 2.279 2.128 2.094 2.136 Candida YST 1033 1.499 2.096 2.130 1.535 1.911 albicans 1.366 2.107 2.015 1.747 1.863 1.503 2.070 2.135 1.496 2.012 Candida YST 1035 1.538 2.154 2.148 2.047 1.973 albicans 1.796 2.120 2.187 1.887 2.033 1.760 2.130 2.236 1.933 1.921 Candida YST 1045 2.029 2.276 2.278 2.251 2.219 glabrata 1.904 2.286 2.179 2.232 2.175 1.986 2.247 2.297 2.253 2.145 Cyptococcus YST 1074 1.405 2.084 1.786 1.699 1.918 neoformans 1.298 2.057 1.954 1.850 1.832 1.427 2.068 1.940 1.812 1.890 Candida YST 1383 1.454 2.109 2.122 2.020 2.040 albicans 1.560 2.198 2.096 1.987 2.054 1.752 2.159 2.096 1.758 2.027 Candida YST 1454 1.808 2.283 2.265 2.147 2.008 glab*rata 1.969 2.257 2.274 2.143 2.097 1.862 2.220 2.301 2.128 1.949

Example 8: Comparison of Exemplary Embodiments of the Disclosed Methods With Control Methods for the Identification of Yeast Strains Exemplary Control:

Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate. Without drying, the matrix solution (1 μl of HCCA) was overlaid onto the sample and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—70% Formic Acid in Water

Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% formic acid solution in water) was pipetted onto the smear and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS.

Exemplary Embodiment—70% Organic Solvent followed by 70% Formic Acid in Water

Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate and allowed to air dry. Organic solvent (1 μL of 70% solution in water) was pipetted onto the smear and allowed to air dry. The organic solvent was methanol, ethanol, isopropanol, or acetone. Formic acid (1 μL of 70% solution in water) was pipetted onto the dried sample and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS.

The MALDI-TOF MS score results for each of the methods in this example are summarized in Table 7 below. The results indicate that for various yeast strains, using formic acid as an on-plate extraction solution provides a consistently higher rate of identification than the direct smear method without the use of formic acid. In addition, an additional extraction step with an organic solvent, prior to the formic acid on-plate extraction, results in a higher MALDI-TOF MS score than using the formic acid extraction alone.

TABLE 7 Direct Smear Methanol Ethanol with Followed Followed Acetone no Formic by by Isopropanol Followed Strain Formic Acid Formic Formic Followed by by Formic Organism Number Acid Only Acid Acid Formic Acid Acid Candida ATCC 0 1.709 2.044 2.083 2.098 2.01 albicans 18804 Candida ATCC 1.308 1.619 2.277 2.289 2.108 2.13 albicans 24433 Candida ATCC 1.329 1.889 2.128 2.209 2.18 2.172 glabrata  2001 Candida ATCC 0 1.736 1.887 1.775 1.883 1.951 parapsilosis 22019 Candida ATCC 0 1.421 1.779 1.886 1.83 1.949 neoformans 60234

Example 9: Identification of Various Yeast and Bacteria Strains Using Exemplary Extraction Methods with Formic Acid/Ethanol Solution Exemplary Embodiment

Yeast or bacterial colonies were resuspended in water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. An aliquot of the suspension was diluted 1:1 with 95.5% ethanol. Three aliquots of the diluted suspension (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% solution in ethanol) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS.

The MALDI-TOF MS score results are summarized in Table 8 (yeast strains) and Table 9 (bacterial strains) below. Table 8 and 9 illustrate that on-plate extraction methods in which a microbial suspension is prepared by resuspending a microbial pellet in an organic solvent, for example, ethanol, followed by extraction with formic acid in ethanol provide 100% identification by MALDI-TOF MS for various yeast and bacteria strains.

TABLE 8 Strain MALDI-TOF Organism Number MS score Candida glabrata YST 2.013  26 2.184 2.189 Cryptococcus YST 1.856 neoformans  54 2.068 1.912 Candida parapsilosis YST 1.929  194 1.957 2.113 Candida parapsilosis YST 1.962  792 1.831 1.844 Cryptococcus YST 1.733 neoformans 1162 1.688 1.728 Candida albicans YST 2.031 1235 1.860 1.949 Cryptococcus YST 1.966 neoformans 1479 1.974 1.966 Cryptococcus YST 2.115 neoformans 1481 2.186 2.136 Candida albicans YST 2.176  145 2.226 2.103 Candida parapsilosis YST 2.113  147 2.021 2.010 Candida parapsilosis YST 2.010  200 2.003 2.013 Candida parapsilosis YST 2.284  214 2.082 2.034 Candida dubliniensis YST 1.741  305 1.791 1.639 Candida glabrata YST 2.213  394 2.255 2.279 Candida albicans YST 2.096 1033 2.107 2.070 Candida albicans YST 2.154 1035 2.120 2.130 Candida glabrata YST 2.276 1045 2.286 2.247 Cryptococcus YST 2.084 neoformans 1074 2.057 2.068 Candida albicans YST 2.109 1383 2.198 2.159 Candida glabrata YST 2.283 1454 2.257 2.220

TABLE 9 Strain MALDI-TOF Organism Number MS score Acinetobacter ENF 2.359 baumanii 11091 2.403 2.356 Enterobacter 13048 2.410 aerogenes 2.409 2.427 Enterobacter cloacae 35030 2.178 2.097 2.042 Escherichia coli 25922 2.374 2.231 2.334 Escherichia coli 35213 2.387 2.164 2.219 Klebsiella 33495 2.282 pneumoniae 2.335 2.351 Klebsiella 700603 2.200 pneumoniae 2.046 2.190 Proteus mirabilis 29906 2.261 2.298 2.324 Pseudomonas 27853 2.276 aeruginosa 2.367 2.325 Pseudomonas ENF 2.258 aeruginosa 14620 2.275 1.999 Serratia marcescens FR197 2.139 2.081 2.054 Stenotrophomonas 13637 2.128 maltophilia 1.784 2.139 Enterococcus faecalis 29212 2.318 2.216 2.269 Enterococcus faecalis 51299 2.272 (VRE) 2.291 2.353 Enterococcus faecium 19434 2.373 2.389 2.271 Enterococcus faecium 700221 1.862 VRE 1.977 1.894 Staphylococcus aureus 25923 2.379 2.285 2.391 Staphylococcus aureus 29213 2.331 (MSSA) 2.305 2.387 Staphylococcus aureus 3421 2.324 (MRSA) 2.323 2.370 Staphylococcus aureus 4330 2.245 (MRSA) 2.187 2.200 Staphylococcus 3568 1.953 epidermidis 1.777 1.861 Staphylococcus 77 2.051 epidermidis 2.039 2.001 Staphylococcus 356 0.000 haemolyticus 2.071 1.883 Staphylococcus sciuri 29062 1.910 1.751 1.865 Streptococcus 12386 2.227 agalactiae 2.134 2.269 Streptococcus 13813 2.229 agalactiae 2.189 2.304 Streptococcus 49619 2.143 pneumoniae 1.923 2.067 Streptococcus 6303 2.124 pneumoniae 2.001 2.019 Streptococcus 700670 2.058 pneumoniae 2.321 2.185 Streptococcus 19615 2.380 pyogenes 1.721 2.474 Streptococcus 3177 1.864 salivarius 1.712 1.943 Streptococcus mitis 49456 2.119 2.021 1.995

The various embodiments described and illustrated herein, demonstrate various methods for identifying microorganism(s) in a sample resulting in a consistently superior rate of identification with fewer steps compared to the prior art methods. The on-plate extraction methods are easily amenable to automated procedures as there is no need for centrifugation. In addition, the extraction plate can be heated in order to increase the speed of the various drying steps resulting in a fast and accurate process for identifying microorganism(s) in a sample.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A kit for preparing a positive blood culture sample for identification of microorganisms therein, the kit comprising:

a lysis buffer, wherein the lysis buffer is selected to lyse blood cells in the sample while microorganisms, if any, remain intact;
a volatile acid solution, wherein a volume percent of the volatile acid is at least 70% of the volatile acid solution; and
a matrix.

2. The kit of claim 1, wherein the volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoracetic acid and hydrochloric acid.

3. The kit of claim 1, further comprising an organic solvent, wherein the organic solvent is selected from the group consisting of ethanol, methanol, isopropanol, or acetone.

4. The kit of claim 1, wherein the kit comprises a volatile acid solution that is 70% volatile acid.

5. The kit of claim 3, wherein the organic solvent is a solution is 100% organic solvent.

6. The kit of claim 3, wherein the organic solvent is a solution is 30% organic solvent to about 70% organic solvent.

7. The kit of claim 1, where the kit further comprises a fixative, wherein the fixative is selected from the group consisting of an organic solvent and a fixative.

8. The kit of claim 7, wherein the organic solvent is ethanol.

9. The kit of claim 7, wherein the fixative is formaldehyde.

10. A method for characterizing at least one microorganism in a sample for identification of microorganisms therein, the method comprising:

(a) obtaining a sample containing at least one microorganism;
(b) suspending the sample in a solution consisting of water to form a microbial suspension;
(c) combining the microbial suspension with an organic solvent;
(d) depositing at least a portion of the microbial suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by mass spectrometry;
(e) drying the sample;
(f) treating the sample with a solution selected from the group consisting of formic acid in water and formic acid in an organic solvent, wherein the formic acid is at a volume percent of at least 70% when combined with the sample;
(g) drying the sample;
(h) placing a matrix over the treated sample;
(i) drying the sample; and
(j) identifying the at least one microorganism by a mass spectrometry.

11. The method of claim 10, wherein a final concentration of organic solvent in the microbial suspension is in a range of about 30% by volume to about 70% by volume.

12. The method of claim 10, wherein a concentration of the sample in the water is greater than about 0.5 McFarland.

13. The method of claim 10, wherein the organic solvent is selected from the group consisting of ethanol, methanol, isopropanol, acetonitrile, acetone, and ethyl acetate.

14. The method of claim 10, wherein the method further comprises adding a non-ionic detergent to the sample either when the sample is obtained or when suspended in water or both.

15. The method of claim 10, wherein, after the sample is obtained in step a):

i) adding a lysis buffer to the sample;
ii) concentrating the at least one microorganism in the sample; and
iii) separating a concentrated portion of the sample from a remainder; and then suspending the separated sample in water.
Patent History
Publication number: 20220170065
Type: Application
Filed: Nov 8, 2021
Publication Date: Jun 2, 2022
Applicant: BECTON DICKINSON AND COMPANY (Franklin Lakes, NJ)
Inventors: Liping Feng (Baltimore, MD), William B. Brasso (Columbia, MD), Susan M. Kircher (Hanover, PA), Vanda White (Baltimore, MD), Song Shi (Reisterstown, MD), Xiao Mo (Ridgewood, NJ), Tuan-Linh Ngoc Nguyen (Carlisle, PA), Adrien P. Malick (Granite, MD), Jon E. Salomon (Mount Dora, FL), John D. Mantlo (Sykesville, MD)
Application Number: 17/521,410
Classifications
International Classification: C12Q 1/04 (20060101); G01N 33/68 (20060101); G01N 33/483 (20060101);