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JP-7857280-B2 - Blood cell lysis agents for isolating bacteria from blood cultures

JP7857280B2JP 7857280 B2JP7857280 B2JP 7857280B2JP-7857280-B2

Inventors

  • マッセイ クリストファー
  • ツァン ジンドン
  • ヤップ アクセル エイ

Assignees

  • ベクトン・ディキンソン・アンド・カンパニー

Dates

Publication Date
20260512
Application Date
20210819
Priority Date
20200820

Claims (15)

  1. A step of producing a processed sample by contacting a sample containing blood cells and at least one microorganism with a lysis buffer, wherein the lysis buffer contains a somatic cell digestive agent (SDA) capable of lysing the blood cells in the sample, and the SDA is given by the following formula 1 (In the formula, x is an integer from 2 to 20, and y is an integer from 6 to 11) This is a compound of the form, A method for processing a sample, comprising the step of lysing blood cells in the sample thereby.
  2. The method according to claim 1, (a) y is an integer between 8 and 10. (b) x is an integer between 5 and 15. (c) The concentration of SDA in the lysis buffer is 0.01 g/L to 10 g/L. and/or (d) The concentration of the SDA in the lysis buffer is 0.01% (w/w) to 10% (w/w). The above method.
  3. The method according to claim 1 or 2, wherein the SDA is nonoxynol-9.
  4. A method according to any one of claims 1 to 3, (a) The sample is derived from a blood culture of a subject suspected of being infected. (b) The sample includes a positive blood culture sample which has been determined to contain at least one microorganism. (c) The at least one microorganism is selected from the group including Gram-positive bacteria, Gram-negative bacteria, and yeasts. (d) The at least one microorganism includes one or more of the following: Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pneumoniae . (e) The contact step includes ultrasonic treatment, osmotic shock, chemical treatment, or any combination thereof. and/or (f) The lysis buffer comprises one or more proteinases and/or one or more nucleases. The above method.
  5. A method according to any one of claims 1 to 4, (a) comprising the step of isolating at least one microorganism from the treated sample to produce at least one isolated microorganism, (b) The steps include isolating at least one microorganism from the treated sample to produce at least one isolated microorganism, preparing an inoculum from the at least one isolated microorganism, and analyzing the at least one microorganism obtained from the inoculum, (c) Isolating at least one microorganism from the processed sample to produce at least one isolated microorganism, and depositing at least a portion of the pellet containing the at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of the at least one microorganism by mass spectrometry , or The process involves isolating at least one microorganism from the processed sample to produce at least one isolated microorganism, depositing at least a portion of the pellet containing the at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of the at least one microorganism by mass spectrometry, and drying the deposited sample. Steps and The step of treating the deposited sample with a volatile acid solution, wherein the volume percentage of the volatile acid is at least 70% of the volatile acid solution combined with the deposited sample, and/or (d) A step of contacting the sample with the choline-containing solution before, simultaneously with, and/or after contact with the lysis buffer, The choline-containing solution is given by the following formula 2 (In the formula, R1 , R2 , and R3 independently represent groups selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and X represents the load group.) The step comprises a quaternary ammonium salt containing an N,N,N-trimethylethanolammonium cation, selected from the group consisting of the following: The above method.
  6. The method according to claim 5, In step (a) of claim 5, (i) The step of isolating the at least one microorganism from the treated sample includes the step of separating the at least one microorganism from lysed blood cells, (ii) The step of isolating the at least one microorganism from the treated sample includes the step of separating the at least one microorganism from the lysed blood cells, The steps include: centrifuging the processed sample to produce a pellet and a supernatant; The step of discarding the supernatant while retaining the pellet containing at least one isolated microorganism, and/or (iii) The method further comprises the steps of preparing a plate pure culture from at least one isolated microorganism and analyzing the microorganism obtained from the plate pure culture, The above method.
  7. The method according to claim 5, In step (c) of claim 5, (i) The volatile acid solution is an aqueous solution of a volatile acid or a volatile solution in an organic solvent. (ii) The volatile acid solution, when combined with the deposited sample, is a formic acid aqueous solution with a volume percentage of 70% to 90%. (iii) The step of treating the deposited sample with a volatile acid solution includes the step of drying the deposited sample treated with the volatile acid solution, (iv) The step of treating the deposited sample with a volatile acid solution includes the step of placing a matrix on top of the treated deposited sample, (v) The step of treating the deposited sample with a volatile acid solution includes the step of drying the treated deposited sample on which the matrix is arranged, and/or (vi) The process includes treating the deposited sample with an organic solvent and drying the deposited sample treated with the organic solvent before treating the deposited sample with a volatile acid solution. The above method.
  8. A method according to claim 5, wherein in step (d) of claim 5, (a) The choline-containing solution comprises choline chloride, phosphorylcholine, or both. (b) When the sample is in contact with the choline, the final concentration of choline is 0.25% by volume or 1% by volume or more, or the concentration of choline in the sample during the contact step is in the range of 0.25% by volume to 10% by volume. and/or (c) The contact step comprises incubating the sample with the choline-containing solution for up to 20 minutes, wherein the incubation temperature is room temperature. The above method.
  9. A method according to any one of claims 1 to 8, (a) The dissolution buffer further contains an antifoaming agent, or the dissolution buffer does not contain an antifoaming agent. (b) The lysis buffer further comprises at least one thiol, (c) The solubilizing buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilizing buffer is 0.01 g/L to 80 g/L. (d) The lysis buffer further comprises a nutritional base solution containing (d1) one or more of the following: (i) casein peptone in the lysis buffer at a concentration of 8 g/L to 35 g/L, (ii) sodium chloride in the lysis buffer at a concentration of 2 g/L to 10 g/L, (iii) soy peptone in the lysis buffer at a concentration of 1.5 g/L to 15 g/L, and (iv) potassium phosphate in the lysis buffer at a concentration of 0.5 g/L to 5 g/L, and (d2) at least one other nutrient. (e) The lysis buffer further comprises one or more of the following: nutrient medium, isotonic buffer, peptone, and salt. (f) The lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof. and/or (g) The lysis buffer further comprises at least one additional nonionic surfactant, the lysis buffer further comprises a saponin, or the lysis buffer does not contain the additional nonionic surfactant . The above method.
  10. A method according to any one of claims 1 to 9, (a) The method further comprises the step of identifying the at least one microorganism, (b) The SDA does not harm the at least one microorganism. (c) The at least one of the microorganisms remains intact in the presence of the SDA. (d) The method yields a MALDI score at least 5% higher than the comparative method using a lysis buffer that does not contain the SDA. (e) The lysis buffer selectively dissolves at least 1% , at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the blood cells in the sample. (f) The ratio of lysed blood cells to lysed cells of at least one microorganism after the contact step is at least 2:1. (g) After the contact step, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells of the at least one microorganism remain intact and/or viable. (h) The lysis buffer does not contain a buffering agent. (i) The solubilizing buffer is acidic. (j) The above method does not include density gradient centrifugation. and/or (k) The lysis buffer is (i) Saponins; (ii) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96/97, CHAPS, Octyl β-D-Glucopyranoside, Saponin, Nonaethylene glycol monododecyl ether (C12E9, polidocenol), Sodium dodecyl sulfate, N-Lauryl sarcosine, Sodium deoxycholate, Bile salt, Hexadecyltrimethylammonium bromide, SB3-10, SB3-12, Amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, Non-surfactant sulfobetaine (NDSB) One or more surfactants selected from the group consisting of 201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin; (iii) One or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96/97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers; (iv) One or more surfactants selected from the group consisting of sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO ; (v) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and/or (vi) a polyoxyethylene surfactant that does not contain the structure C12-18 / E9-10 (wherein C12-18 represents the carbon chain length of 12 to 18 carbon atoms, and E9-10 represents 9 to 10 oxyethylene hydrophilic head groups), The above method.
  11. The method according to any one of claims 5 to 10, wherein the mass spectrometry of step (c) of claim 5 comprises one or more of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS) n , matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), silicon desorption/ionization (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCJ-MS/MS, APCI-(MS) n , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero .
  12. A lysis buffer containing a somatic cell digestive agent (SDA) capable of lysing blood cells, wherein the SDA is defined by the following formula 1 A lysis buffer is a compound of the form (where x is an integer from 2 to 20, and y is an integer from 6 to 11), Blood cells and/or their debris, A composition comprising at least one microorganism.
  13. The composition according to claim 12, (a) y is an integer between 8 and 10. (b) x is an integer between 5 and 15. (c) The concentration of SDA in the lysis buffer is 0.01 g/L to 10 g/L. and/or (d) The concentration of the SDA in the lysis buffer is 0.01% (w/w) to 10% (w/w). The above composition.
  14. The composition according to claim 12 or 13, wherein the SDA is nonoxynol-9.
  15. A composition according to any one of claims 12 to 14, (a) The lysis buffer comprises one or more proteinases and/or one or more nucleases. (b) The composition further comprises a choline-containing solution comprising at least one quaternary ammonium salt comprising an N,N,N-trimethylethanolammonium cation selected from the group consisting of the following formula 2, In the formula, R1 , R2 , and R3 independently represent groups selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and (i) X represents the load base, (ii) X is selected from the group consisting of chlorides, fluorides, nitric acid, and bicarbonate. and/or (iii) The choline-containing solution comprises choline chloride, phosphorylcholine, or both. (c) The solubilating buffer further contains an antifoaming agent, or the solubilating buffer does not contain an antifoaming agent. (d) The lysis buffer further comprises at least one thiol, (e) The solubilizing buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilizing buffer is 0.01 g/L to 80 g/L. (f) The lysis buffer further comprises a nutritional base solution containing (f1) one or more of the following: (i) casein peptone in the lysis buffer at a concentration of 8 g/L to 35 g/L, (ii) sodium chloride in the lysis buffer at a concentration of 2 g/L to 10 g/L, (iii) soy peptone in the lysis buffer at a concentration of 1.5 g/L to 15 g/L, and (iv) potassium phosphate in the lysis buffer at a concentration of 0.5 g/L to 5 g/L, and (f2) at least one other nutrient. (g) The lysis buffer further comprises one or more of the following: nutrient medium, isotonic buffer, peptone, and salt. (h) The lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof. (i) The lysis buffer further comprises at least one additional nonionic surfactant, the lysis buffer comprises a saponin, or the lysis buffer does not contain the additional nonionic surfactant. (j) The lysis buffer does not contain a buffering agent. (k) The solubilizing buffer is acidic. (l) The solubilizing buffer is (i) Saponins; (ii) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96/97, CHAPS, Octyl β-D-Glucopyranoside, Saponin, Nonaethylene glycol monododecyl ether (C12E9, polidocenol), Sodium dodecyl sulfate, N-Lauryl sarcosine, Sodium deoxycholate, Bile salt, Hexadecyltrimethylammonium bromide, SB3-10, SB3-12, Amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, Non-surfactant sulfobetaine (NDSB) One or more surfactants selected from the group consisting of 201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin; (iii) One or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96/97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers; (iv) One or more surfactants selected from the group consisting of sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO ; (v) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and/or (vi) A polyoxyethylene surfactant that does not contain the structure C12-18 / E9-10 (wherein C12-18 represents the carbon chain length of 12 to 18 carbon atoms, and E9-10 represents 9 to 10 oxyethylene hydrophilic head groups), (m) The at least one of the microorganisms remains intact in the presence of the SDA. and/or (n) The SDA does not damage the at least one type of microorganism. The above composition.

Description

Related Applications This application claims priority to U.S. Provisional Application No. 63/068,278, filed on 20 August 2020, pursuant to Section 119(e) of the U.S. Patent Act. The entire contents of these applications are expressly incorporated herein by reference throughout them. Background: This disclosure generally relates to the field of microbial isolation and identification. Description of Related Techniques Sepsis is a serious medical condition resulting from a severe reaction of the host's immune system to infection. It can cause widespread inflammation and impaired blood flow. As sepsis progresses, the body's organs can become deprived of oxygen and nutrients, leading to permanent damage and eventual failure. If left undiagnosed or even untreated, the heart can fail, leading to septic shock, multiple organ failure, and death. Blood cultures are necessary to detect the presence of bacteria or yeast in the blood of patients with sepsis. If microorganisms are present (positive blood culture ("PBC")), the microorganisms must be identified and their antibiotic susceptibility determined in order to provide appropriate treatment. PBC samples are used for isolation, identification, and antimicrobial susceptibility testing ("AST"). Microorganisms are often identified by mass spectrometry or phenotypic growth-based methods, including MALDI-TOF/MS, such as Phoenix® ID. To identify microorganisms, perform phenotypic analysis, and conduct AST testing, it is necessary to isolate intact and/or viable microorganisms from blood cells and other materials in the collected sample. For microbial identification by mass spectrometry, substances known to interfere with MALDI-TOF/MS identification, such as blood cell components, other cellular debris, and salts, must be thoroughly removed from the microbial sample. Furthermore, the microbial sample must be of sufficient quantity to achieve reliable identification. Phenotypic identification methods, such as Phoenix™ ID, require intact, viable microorganisms free from substances that may interfere with the assay's enzyme substrates. For AST testing, such as Phoenix™ AST, the microbial sample must contain viable, unchanging microorganisms capable of growing in the presence of antibiotics during the assay, if resistance mechanisms exist. For all methods, it is crucial that residual blood or culture medium components are present in sufficient quantity and purity, as carryover can interfere directly or unduly by increasing microbial concentration (turbidity). Current techniques for isolating viable microorganisms from PBC samples involve microbial subculturing, which can take up to 72 hours. This can lead to delayed treatment or treatment with inappropriate antibiotics. For example, isolating a specific strain of a microorganism from a PBC sample while maintaining the viability of organisms such as Streptococcus pneumoniae (S. pneumoniae) is particularly difficult. Part of this difficulty stems from the activation of autolytic enzymes by Streptococcus pneumoniae, which causes the microbial cells to "self-destruct." See “Streptococcus pneumoniae Antigen Test Using Positive Blood Culture Bottles as an Alternative Method To Diagnose Pneumococcal Bacteremia”, Journal of Clinical Microbiology, Vol. 43, No. 5, May 2005, pp. 2510–2512. The current method for isolating microorganisms, including Streptococcus pneumoniae, from sepsis patients involves inoculation of blood culture bottles. Once a positive signal is achieved, a portion of the PBC sample is taken and Gram-stained, and another portion is used to subculture the microorganism. Downstream tests, such as identification by MALDI-TOF/MS, phenotypic identification, and AST testing, are performed using the microbial colonies from the subculture. Further techniques for isolating viable microorganisms from PBC samples often involve liquid separation methods that include a lysis buffer containing a surfactant to lyse blood cells in the PBC sample. After lysis, the lysed blood cells can be removed while retaining the microorganisms. However, the use of these lysis buffers often results in impaired, damaged, or infertile microorganisms, making them insufficient for specific growth-based identification methods such as AST testing. Currently available sample preparation methods and compositions suffer from various shortcomings. For example, (i) insufficient viability due to the interaction of coarse surfactants on the microbial cell wall after sample preparation to support growth-based identification and AST methods; (ii) resulting in inconsistent identification of microorganisms at the seed level across a panel of microorganisms; and/or (iii) inability to isolate viable microorganisms from a PBC sample that are free from interfering substances and enable multiple downstream tests from a single PBC sample, such as MALDI-TOF/MS identification and AST testing. Therefore, efficient hematopoietic agents are needed to isolate microorganisms fr