U.S. patent application number 17/521410 was filed with the patent office on 2022-06-02 for identification of microorganisms using maldi-tof-ms on-plate extraction.
This patent application is currently assigned to BECTON DICKINSON AND COMPANY. The applicant listed for this patent is BECTON DICKINSON AND COMPANY. Invention is credited to William B. Brasso, Liping Feng, Susan M. Kircher, Adrien P. Malick, John D. Mantlo, Xiao Mo, Tuan-Linh Ngoc Nguyen, Jon E. Salomon, Song Shi, Vanda White.
Application Number | 20220170065 17/521410 |
Document ID | / |
Family ID | 1000006137679 |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220170065 |
Kind Code |
A1 |
Feng; Liping ; et
al. |
June 2, 2022 |
IDENTIFICATION OF MICROORGANISMS USING MALDI-TOF-MS ON-PLATE
EXTRACTION
Abstract
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.
Inventors: |
Feng; Liping; (Baltimore,
MD) ; Brasso; William B.; (Columbia, MD) ;
Kircher; Susan M.; (Hanover, PA) ; White; Vanda;
(Baltimore, MD) ; Shi; Song; (Reisterstown,
MD) ; Mo; Xiao; (Ridgewood, NJ) ; Nguyen;
Tuan-Linh Ngoc; (Carlisle, PA) ; Malick; Adrien
P.; (Granite, MD) ; Salomon; Jon E.; (Mount
Dora, FL) ; Mantlo; John D.; (Sykesville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BECTON DICKINSON AND COMPANY |
Franklin Lakes |
NJ |
US |
|
|
Assignee: |
BECTON DICKINSON AND
COMPANY
Franklin Lakes
NJ
|
Family ID: |
1000006137679 |
Appl. No.: |
17/521410 |
Filed: |
November 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
16734826 |
Jan 6, 2020 |
11193158 |
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17521410 |
|
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|
15472759 |
Mar 29, 2017 |
10557162 |
|
|
16734826 |
|
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|
13600702 |
Aug 31, 2012 |
9631221 |
|
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15472759 |
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61649483 |
May 21, 2012 |
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61530620 |
Sep 2, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
H01J 49/40 20130101;
C12Q 1/04 20130101; G01N 33/4833 20130101; H01J 49/164 20130101;
G01N 33/6851 20130101 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; G01N 33/68 20060101 G01N033/68; G01N 33/483 20060101
G01N033/483 |
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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] 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
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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 .mu.L 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.
[0008] 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
[0009] 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.
[0010] 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).
[0011] 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.
[0012] 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
[0013] FIG. 1 is a flowchart that illustrates the prior art method
of extracting proteins from pure microbial isolates for
identification by mass spectrometry.
[0014] 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.
[0015] 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
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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, .alpha.-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).
[0022] 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-.beta.-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%.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.degree. 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
[0038] In all examples below, the MALDI matrix solution was
prepared by dissolving 2.5 mg of HCCA in 250 .mu.L 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:
[0039] 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.
[0040] 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").
[0041] 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 .mu.L) 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 .mu.L) is added to the tube and vortexed thoroughly.
The mixture is centrifuged at maximum speed for 2 minutes at
10,000.times.g in a microcentrifuge. The ethanol is decanted and
the sample is centrifuged again for 2 minutes at 10,000.times.g
speed in a microcentrifuge. Excess ethanol is removed with a
pipette. Formic acid (70% solution; 50 .mu.L) is added to the
pellet and vortexed thoroughly. Acetonitrile (100% solution; 50
.mu.L) is added to sample. The sample is centrifuged at
10,000.times.g for 2 minutes in a microcentrifuge. The supernatant
(1 .mu.L) is pipetted onto a MALDI plate and allowed to air dry.
The dried sample is overlaid with 1 .mu.L 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
[0042] From a PBC sample, the pellet of isolated microorganisms was
resuspended in 600 .mu.L 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 .mu.L) was spotted
onto the MALDI plate. The sample was allowed to air dry. Formic
acid (1-2 .mu.L of 70% formic acid solution) was overlaid onto the
dried, spotted sample and allowed to air dry. The dried sample was
overlaid with 1 .mu.l of matrix solution (HCCA) and allowed to air
dry before identification by MALDI-TOF MS.
[0043] 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 .mu.L of a 70% aqueous solution) was overlaid
onto the sample and allowed to air dry. The dried sample was
overlaid with 1 .mu.l of MALDI matrix solution (HCCA) and allowed
to air dry before identification by MALDI-MS.
[0044] 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-US-00001 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:
[0045] A liquid sample (0.5 .mu.L) was applied to a target plate
and covered with 0.5 .mu.L 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 .mu.L 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.TM. 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
[0046] The pellet of isolated microorganism from a PBC sample was
resuspended in 600 .mu.L 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 .mu.L) was spotted onto
the MALDI plate. The sample was allowed to air dry. Formic acid
(1-2 .mu.L of a 70% solution) was overlaid onto the sample and
allowed to air dry. The dried sample was overlaid with 1 .mu.L 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:
[0047] Yeast colonies were resuspended in 600 .mu.l of water in a
microcentrifuge tube. The turbidity of the sample was visually
adjusted to greater than 2.0 McFarland. Sample (0.5 .mu.L) was
pipetted onto a MALDI plate. Formic acid (0.5 .mu.L 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 .mu.L 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:
[0048] Yeast colonies were resuspended in 600.mu.1 of water in a
microcentrifuge tube. The turbidity of the sample was visually
adjusted to greater than 2.0 McFarland. Sample (1 .mu.L) was
pipetted onto a MALDI plate and allowed to air dry. Formic acid (1
.mu.L of a 50% solution in water) was pipetted onto the dried
sample and allowed to air dry. MALDI matrix solution (1 .mu.L of
HCCA) was pipetted onto the dried sample and allowed to air dry
before identification by MALDI-MS.
Exemplary Control Method 2:
[0049] 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 .mu.L) 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 .mu.L of HCCA) and allowed to air
dry before identification by MALDI-TOF MS.
Exemplary Embodiment:
[0050] Yeast colonies were resuspended in 600 .mu.L of water in a
microcentrifuge tube. The turbidity of the sample was visually
adjusted to greater than 4.0 McFarland. Sample (1-1.5 .mu.L) was
pipetted onto a MALDI plate and allowed to air dry. Formic acid
(1-1.5 .mu.L 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.
[0051] 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-US-00002 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
[0052] Yeast colonies were resuspended in 600 .mu.L 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
.mu.L each) were pipetted onto a MALDI-TOF MS plate and allowed to
air thy. Formic acid (1-1.5 .mu.L) 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 .mu.L
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
[0053] 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-US-00003 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
[0054] 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
.mu.L) 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 .mu.L 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.
[0055] 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-US-00004 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
[0056] Bacterial colonies from a sub-culture were resuspended in
600 .mu.L water. The turbidity of the sample was visually adjusted
to greater than 4.0 McFarland. Three aliquots of the bacterial
suspension (1.5 .mu.L) for each microorganism were spotted onto a
MALDI-TOF MS plate, and then immediately followed by sequential
additions of 1.5 .mu.L of 70% formic acid in ethanol and 1.5 .mu.L
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.
[0057] 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-US-00005 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):
[0058] Yeast colonies were resuspended in 600 .mu.L of water in a
microcentrifuge tube. The turbidity of the sample was visually
adjusted to greater than 2.0 McFarland. Three samples (1 .mu.L) for
each microorganism were pipetted onto a MALDI-TOF MS plate and
allowed to air dry. Neat formic acid (1 .mu.L) was pipetted onto
the dried samples and allowed to air dry. Matrix solution (1 .mu.L
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)
[0059] Yeast colonies from a sub-culture were resuspended in 600
.mu.L of 50% ethanol in a microcentrifuge tube. The turbidity of
the sample was visually adjusted to greater than 4.0 McFarland.
Three samples (1 .mu.L) for each microorganism were pipetted onto a
MALDI-TOF MS plate and allowed to air dry. Formic acid (1 .mu.L of
70% solution in ethanol) was pipetted onto the dried samples and
allowed to air dry. Matrix solution (1 .mu.L 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)
[0060] Yeast colonies from a sub-culture were resuspended in 600
.mu.L of water in a microcentrifuge tube. The turbidity of the
sample was visually adjusted to greater than 4.0 McFarland. Three
samples (1 .mu.L) for each microorganism were pipetted onto a MALDI
plate and allowed to air dry. Ethanol (1 .mu.L 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
.mu.L) onto the dried samples and allowed to air dry. Matrix
solution (1 .mu.L 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)
[0061] Yeast colonies from a sub-culture were resuspended in 600
.mu.L of 50% acetone in a microcentrifuge tube. The turbidity of
the sample was visually adjusted to greater than 4.0 McFarland.
Three samples (1 .mu.L) for each microorganism were pipetted onto a
MALDI-TOF MS plate and allowed to air dry. Formic acid (1 .mu.L of
70% concentration in acetone) was pipetted onto the dried samples
and allowed to air dry. Matrix solution (1 .mu.L 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)
[0062] Yeast colonies from a sub-culture were resuspended in 600
.mu.L of water in a microcentrifuge tube. The turbidity of the
sample was visually adjusted to greater than 4.0 McFarland. Three
samples (1 .mu.L) for each microorganism were pipetted onto a
MALDI-TOF MS plate and allowed to air dry. Acetone (1 .mu.L, 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 .mu.L HCCA) was overlaid onto the dried samples and
allowed to air dry before identification by MALDI-MS.
[0063] 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-US-00006 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:
[0064] Yeast colonies from a pure culture plate were directly
smeared onto a MALDI-TOF MS plate. Without drying, the matrix
solution (1 .mu.l 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
[0065] Yeast colonies from a pure culture plate were directly
smeared onto a MALDI-TOF MS plate and allowed to air dry. Formic
acid (1 .mu.L of 70% formic acid solution in water) was pipetted
onto the smear and allowed to air dry. Matrix solution (1 .mu.L 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
[0066] Yeast colonies from a pure culture plate were directly
smeared onto a MALDI-TOF MS plate and allowed to air dry. Organic
solvent (1 .mu.L 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 .mu.L of 70%
solution in water) was pipetted onto the dried sample and allowed
to air dry. Matrix solution (1 .mu.L of HCCA) was overlaid onto the
dried sample and allowed to air dry before identification by
MALDI-TOF MS.
[0067] 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-US-00007 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
[0068] 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 .mu.L) for each microorganism were
pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic
acid (1 .mu.L of 70% solution in ethanol) was pipetted onto the
dried samples and allowed to air dry. Matrix solution (1 .mu.L of
HCCA) was overlaid onto the dried samples and allowed to air dry
before identification by MALDI-TOF MS.
[0069] 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-US-00008 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-US-00009 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
[0070] 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.
[0071] 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.
* * * * *