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    • 专利摘要:
    kit and method of detection microorganisms resistant to a therapeutic agent the present invention relates to a method for the detection of microorganisms resistant to a therapeutic agent in a biological sample, comprising the following steps: a. inoculate said sample, uncultivated, in a first tube without at least a first tube with, and in a second tube without at least one therapeutic agent; preferably further including at least one lysis agent and / or buffer, and / or suitable culture medium, or placing the sample in a serum separation tube; and incubate it; B. add a fluorescent marker to both tubes; ç. perform a fluorescence or growth analysis to obtain one or more fluorescence or growth parameters for each of the two tubes; wherein the phenotype of resistant microorganisms from the biological sample to said therapeutic agent is obtained by comparing the one or more fluorescence parameters between the two tubes.
    公开号:BR112013031110B1
    申请号:R112013031110-0
    申请日:2012-06-04
    公开日:2020-09-29
    发明作者:Cidália Irene Azevedo Pina Vaz；Acácio Agostinho Gonçalves Rodrigues；Isabel Cristina Santos Silva De Faria Ramos Antunes；Rita Mafalda Dos Santos Rocha Do Rosário；Ana Sofia Quinta E Costa De Oliveira Morais；Ana Teresa Pinto E Silva
    申请人:Universidade Do Porto；
    IPC主号:
    专利说明:

    Technical Domain of the Invention
    [001] The present invention relates to a kit and method of detecting microorganisms resistant to a therapeutic agent and to the determination of the underlying resistance mechanisms. Background of the invention
    [002] Nowadays, there is a great need to shorten and improve the current laboratory procedures for the detection and, above all, the susceptibility assessment of microorganisms. An ideal diagnostic technology would provide a susceptibility profile in time to allow the institution of appropriate therapy based on this.
    [003] In the case of the most serious infections associated especially with septic shock, speed is essential. It has been> shown that the risk of mortality would increase substantially per hour if appropriate antimicrobial therapy was postponed.
    [004] Current gold standard diagnostic methods are based on culture that is slow and labor intensive, providing phenotypic identification and antimicrobial susceptibility tests, requiring 48-72 h after sample collection. Empirical therapy should generally be started often with a broad spectrum of antibiotics, leading to increased resistance to antimicrobials.
    [005] Using classic tests, biological products such as urine should be grown in solid media and only then could susceptibility identification and assessment occur. Bloods are usually inoculated in broth (blood cultures) and analyzed automatically; when the result is positive, they are plated on a solid medium and only. After at least 24 hours, colonies are formed to assess susceptibility. On the other hand, another 24 hours are necessary to determine the susceptibility profile, since they are based on the ability to grow in the presence of a drug picture.
    [006] Clinical resistance could be related to the lack of antimicrobial susceptibility of the strain, but also to low levels of active antimicrobial drug in local infection. Biochemical protocols are available for only a few drugs, for example, vancomycin, gentamicin or amikacin, and they could be inactive. Summary of the Invention
    [007] This invention relates to an antimicrobial susceptibility test performed on pure microbial cultures - a laboratory culture containing a single species of the organism - or directly from biological samples - not cultivated - by flow cytometry. Some preferred modalities include not requiring the use of a pure culture obtained from a biological product - which takes at least 24 hours for microorganisms to grow - then an antimicrobial susceptibility test is performed directly from biological samples - not grown such as urine, blood or water cultures by fluorescence analysis as a flow cytometry.
    [008] The subject disclosed refers to a method for detecting microorganisms resistant to a therapeutic agent in a biological sample, comprising the following steps: a. inoculate said sample in a first tube with, and in a second tube without at least one therapeutic agent and incubate it; B. add a fluorescent marker to both tubes; ç. perform a fluorescence analysis to obtain one or more fluorescence or growth parameters for each of the two tubes; wherein the phenotype of resistant microorganisms from the biological sample to said therapeutic agent is obtained by comparing the one or more fluorescence parameters between the two tubes.
    [009] The subject disclosed also refers to a method to detect resistant microorganisms (a bacterium, a fungus or a protist) to a therapeutic agent in a biological sample. That is, directly from biological samples - not cultivated - as a urine sample, blood culture, water or a suspension of microorganisms obtained from pure cultures, comprising the following steps: a. inoculate said sample, uncultivated, in a first tube with, and in a second tube without at least one therapeutic agent; preferably also including at least one lysis agent and / or a buffer, and / or suitable culture medium, or placing the sample in a serum separation tube; and incubate it; B. add a fluorescent marker to both tubes; ç. perform a flow cytometry analysis to obtain one or more flow cytometry parameters for each of the two tubes; wherein the phenotype of resistant microorganisms from the biological sample to said therapeutic agent is obtained by comparing the one or more parameters of flow cytometry between the two tubes.
    [010] The material disclosed also refers to a method for the detection of resistant microorganisms. (a bacterium, a fungus or a protist) for a therapeutic agent in a biological sample. That is, directly from biological samples - without culture - such as a urine sample, culture of blood, water or a suspension of microorganisms, obtained from pure cultures, comprising the following steps: a. inoculate said sample, without culture, in a first tube with, and a second tube without, at least, a therapeutic agent; preferably further including at least one lysis agent and / or a buffer, and / or a suitable culture medium, or placing the sample in a serum separation tube, and incubating it; B. add to both. tubes of a fluorescent marker; ç. perform a flow cytometry analysis to obtain one or more flow cytometry parameters for each of the two tubes; wherein the resistant phenotype of microorganisms from the biological sample to which said therapeutic agent is obtained by comparing one or more flow cytometry parameters between the two tubes.
    [011] In another embodiment, one or more flow cytometry parameters comprise (m) forward scatter and / or lateral scatter and / or fluorescence parameters. The parameters of the fluorescence dispersion signal can be intensity, spectral profile and / or cell count.
    [012] In another embodiment of the present invention, the disclosed method further comprises one or more of the preparatory steps: • identifying the type of microorganism present in the biological sample and, therefore, selecting the therapeutic agent (s), namely, if the type is gram-positive or gram-negative; • concentrate the biological sample and remove, if present, any debris, for example, debris from blood, urine or other biological products; preferably by centrifugation, filtration or using a separating serum tube and centrifuge.
    [013] In another embodiment of the present invention, the sample incubation time can vary between 30 min and 2 h, preferably between 30 and 60 min.
    [014] In another embodiment of the present invention, the sample incubation temperature is between 30 and 40 ° C, preferably between 25 and 35 ° C.
    [015] In another embodiment of the present invention, the reaction time of step b) with the fluorescent marker can be between 5 to 60 minutes.
    [016] In another embodiment of the present invention, for determining the resistance mechanisms of microorganisms, the biological sample in step c) may also comprise an enzyme inhibitor, for example, in the case of detection of the presence of broad-spectrum beta-lactamases (ESBLs). The preferred enzyme inhibitor may be clavulanic acid or tazobactam, or mixtures thereof, among others.
    [017] In another preferred aspect of the present invention, the method further comprises the next step in order to detect the resistance mechanisms of microorganisms such as, for example, due to the presence of carbapenemases - enzymes that degrade carbapenemas, an important resistance mechanism ( the Hodge test is recommended, taking another 24 hours, being complicated and difficult to interpret): a. Inoculate said sample with a carbapenema and incubate it; B. removing the microorganism from the biological sample, preferably by filtration; ç. add a strain with known behavior - that is, a control strain with a dose-effect curve known as, for example, incubating a serial concentration of a carbapenema with a microorganism such as Escherichia coli, strain ATCC 25922, by flow cytometry; d. add a fluorescent marker to both samples; and. perform a flow cytometry analysis to obtain one or more flow cytometry parameters for each of the two tubes; f. compare the generated profile with a control strain.
    [018] In the presence of carbapenemases, the strain tested will degrade carbapenema, so that the amount of active drug is reduced; the effect of carbapenemas on the type strain is reduced. If the effect of a given concentration of carbapenema is less than expected, the microorganism is a producer of carbapenemase.
    [019] In a more preferred embodiment of the present invention, the method further comprises the next step in order to detect the resistance mechanisms of microorganisms such as, for example, due to the presence of carbapenemases: i. obtain a flow cytometry dose-effect curve, incubate a serial concentration of a carbapenema with a microorganism such as Escherichia coli, strain ATCC 25922, by flow cytometry ii. target a strain resistant to carbapenemas, remove the microorganism by filtering the test tube containing the strain after incubation with a certain concentration of the carbapenema; iii. this filtrate is then incubated with the type strain (ATCC) and its effect is analyzed by flow cytometry; its effect is compared with the expected result in relation to the dose-effect curve.
    [020] In another preferred modality, the therapeutic amount of any drug in a biological fluid could still be assessed based on the same methodology.
    [021] In a more preferred method of detecting microorganisms resistant to a therapeutic agent in a biological sample, said microorganism is Staphylococcus spp, Streptococcus spp, Enterococcus spp; Enterobacteriacea such as Escherichia coli, Klebsiella pneumonia, Proteusspp; non-fermenting bacteria such as Pseudomonas aeruginosa, Acinetobacter baummanii or Burkloderia cepaciae; Neisseriaspp, Haemophilus spp; Mycobacteriumspp and Nocardiaspp; Legionellaspp and anaerobic bacteria; fungi like Candidaspp, Cryptococcus neoformans, Pneumocystis jirovecii and molds like Aspergillusspp; parasites such as Giardia spp or another similar microorganism.
    [022] In another preferred embodiment of the method for detecting microorganisms resistant to a therapeutic agent in a biological sample, the therapeutic agent can be an antibacterial drug such as penicillins, cephalosporins, other beta-lactams (carbepenemas, aztreonam) macrolides, quinolones, aminoglycosides , glycopeptides, a lipopeptide, tetracyclines and others (for example, colistin, chloramphenicol, clindamycin, fosfomycin, linezolid, nitrofurantoin, sulfon.amide, rifampicin, trimethoprim, trimethoprim-sulfamethoxazole or tigecycline); an antifungal such as polyenes, 5-fluorocytosine, azoles or echinocandins, or another similar therapeutic agent.
    [023] In another preferred embodiment of the method for detecting microorganisms resistant to a therapeutic agent in a biological sample, the fluorescent marker may be a nucleic acid dye, a metabolic dye, a membrane potential dye, a probe for organelles, a fluorescent marker of cell morphology and fluid flow, probe for cell viability, proliferation and function or probe for reactive oxygen species. Most preferably, the fluorescent marker is the acridine dye, cyanine dye, fluorone dye, oxazine dye, phenanthridine dye or a rhodamine dye.
    [024] In a different modality, antifungal susceptibility to molds can additionally be achieved by inoculating spores in Bactec Mycosis IC / F flasks or in MGIT tubes with and without antifungals such as: amphotericin B, voriconazole and fluconazole; the time for the fluorescence emission to become positive will be recorded by the Bactec equipment. Vials with strains susceptible to antifungals will take at least twice as long to become positive compared to the control vial - without antifungal.
    [025] It is also the subject of the present invention a kit for the detection of microorganisms resistant to a therapeutic agent comprising the method described above.
    [026] In a further embodiment of the present invention, the kit can additionally contain the following solutions: a therapeutic agent and / or a fluorescent marker.
    [027] In yet another embodiment of the present invention, the kit may additionally comprise the following solutions: a buffer, a lysis agent and / or a culture medium and / or an enzyme inhibitor or a control strain.
    [028] A fluorescence device for carrying out the methods of any of the preceding claims is still the subject of the present invention. The fluorescence could be a flow cytometer or a BACTEC MGIT - mycobacterial growth indicator tube.
    [029] The disclosed subject allows the assessment of the susceptibility of a microorganism - namely, infectious agents - to the main antimicrobial agents of colonies or biological samples; molds have a different protocol. The disclosed subject also allows the detection of resistance mechanisms and even the quantification of the drug in microbiological assays in different biological samples. General Description of the Invention:
    [030] The laboratory methods for the susceptibility profile used in the routine clinical laboratory require a minimum time of 48 hours. This delay is due to the fact that the known methods are based on the growth of microorganisms. Instead, the assessment of antimicrobial susceptibility by fluorescence analysis, namely, flow cytometry, based on the assessment of the cell's physiological state after exposure to the drug for a short period of time. The determination of morphological lesions or physiological activity is not possible by the classical methodology.
    [031] Many developments have emerged in the field of microbiology, particularly in relation to the identification of microorganisms, using molecular biology techniques. However, this technology has a limitation that can only be applied to organisms whose genome is known. Another disadvantage concerns its high cost. In addition, molecular biology only occasionally provides information on the susceptibility of microorganisms, since, only occasionally, the presence of a specific gene is known to confer resistance to a particular drug. This direct relationship is established in the specific case of rifampicin in relation to Mycobacterium tuberculosis resistance to methicillin resistance in Staphylococcus aureus.
    [032] The characteristics that allow the test to be performed directly from the product are: • the fact that these products have a sufficient cell density (preferably greater than 105) for flow cytometry scanning. Blood culture is subjected to incubation prior providing, after that when it is positive, the microorganism in the cell density necessary to carry out susceptibility tests; Urinary samples, the criteria for infection corresponding to a cell density greater than 105 cells per milliliter. • in both products, microorganisms are found in an exponential phase - this is necessary because the evaluation of the susceptibility of some types of antimicrobial drugs needs to have microorganisms in the exponential phase due to their mode of action.
    [033] The innovative application of flow cytometry is the possibility to perform a multiparametric assessment of a population of microorganism cells dynamically, over time, and correlate this with the study of their replication capacity. To demonstrate a lethal activity of a drug quickly by the conventional method, we must prove that the cell is unable to replicate. By flow cytometry, we might be able to demonstrate, after a few minutes of incubation time with a specific drug, a serious damage to the cell membrane, 25 representing death. In addition, mechanisms of action and, even more importantly, resistance mechanisms could be investigated by flow cytometry. In addition, this new approach allows the study of drug combinations in common use in critically ill patients, evidence of previous 30 in vitrode synergistic effect. Description of the Figures
    [034] The following figures provide preferred modalities to illustrate the description and should not be observed as limiting the scope of the invention.
    [035] Figure 1: Outline of the methodology for a rapid assessment of antimicrobial susceptibility directly from positive blood cultures or urine samples prior to the acquisition of flow cytometry.
    [036] Figure 2: Scheme of two distinct modalities of the present invention - the method of detecting the susceptibility of a microorganism to a therapeutic agent using blood cultures or positive urine samples implemented and the results obtained with typical sensitive and resistant strains.
    [037] Figure 3: Flow cytometry histograms of a typical example of a susceptible Staphylococcus aureusum (MSSA) and oxacillin resistant (MRSA) after 2 hours of incubation and staining with EDA.
    [038] Figure 4: Flow cytometry histograms of a typical example of an susceptible and resistant vancomycin Enterococcus faecalis after the 1 hour incubation time and FDA staining.
    [039] Figure 5: Flow cytometry histograms of a typical example of a quinolone-resistant Escherichia coli, namely ciprofloxacin after one hour of incubation and staining with SYBR Green.
    [040] Figure 6: Flow cytometry histograms of a typical example of Escherichia coli susceptible and resistant to cefotaxime (CTX) after incubation for one hour with the drug and stained with DiBAC4 (3); in the case of resistance to CTX, incubate with CTX with and without clavulanic acid (CIA) and stained with DiBAC4 (3). Positive ESBL strains behave as susceptible when incubated with CLA.
    [041] Figure 7: Flow cytometry histograms of the typical example of the Candida albicans strain after incubation for one hour with anidulafungin, fluconazole or combinations of both and stained with DiBAC4 (3). The. control cells (without antifungals) and C after incubation with the drugs (Cl - anidulafungin, C2 - fluconazole and C3 - anidulafungin and fluconazole) Detailed Description of the Invention
    [042] Classical susceptibility methods are based on pure cultures in solid media. The suspensions of microorganisms are prepared and, accordingly, appropriate susceptibility plates / panels (example: Gram-negative bacilli, Gram-positive cocci; fungi, etc.) in relation to the automatic methods (VITEK 1 or VITEK 2 - BioMerieux; BO Phoenix System - Becton-Dickinson Biosciences; MicroScan WalkAway system - Dade Behring).
    [043] Other manual tests are available such as broth macrodilution and microdilution tests, E test (AB BIODISK), disk-diffusion test and dry form (TREK diagnosis).
    [044] All of these are based on the ability of the study microorganism to replicate in the presence of different antimicrobial agents.
    [045] First, the biggest disadvantage is that they are made from colonies in a solid medium, which need at least 24 h to grow. Although semi-automatic methods are available, they are based on detecting growth that takes time to deliver results (minimum 18 to 24 hours), they require highly experienced professionals and are expensive.
    [046] Biomolecular methods (not implemented in every laboratory routine).
    [047] Molecular methods represented a revolution for the microbiology laboratory diagnostic test, mainly related to the identification of microorganisms and, only rarely, can they help in the susceptibility assessment (Multiplex PCR, Micromatrices, etc.).
    [048] In fact, there are several genes involved in resistance mechanisms, often leading to sequencing steps. There is usually a lack of correlation between the genotype and the phenotype. Although they are highly sensitive and specific - they do not provide any information on viability and are expensive. 1. Preparation of a microbial suspension 1.1 Blood cultures or urine sample
    [049] From a positive blood culture or urine sample: - Perform a Gram stain (maximum: 10 minutes) or (Maldi-TOF) mass spectrometry analysis (Maldi-TOF) to guide the subsequent analysis. This previous step will provide us with essential information about the microorganism, namely, in terms of: - Bacteria: coconuts or bacilli; gram-positive or gram-negative - leading to the choice of antibiotics to be tested; - Fungi • Filter (in order to remove hematological cells and large debris) and transfer the blood culture or urine to a vial containing a blood lysis solution (aqueous solution of NH4C1, KHCO3 and EDTA), or • Transfer to a separator tube serum (for example, a vacoutainer gel tube) and centrifuge. The blood cells will be at the bottom of the gel, and the microorganisms at the top of the gel. • Make a microbial suspension in filtered sterile culture medium. 1.2 From pure cultures
    [050] Make a microbial suspension from a colony in sterile and filtered broth culture medium (for example: Mueller-Hinton) and incubate until the exponential growth phase. 1. Incubate the bacterial suspension at 37 ° C and 150 beats per minute, until the bacteria reach the start of the exponential growth phase (preferably at an optical density of 0.2 at a wavelength of 600 nm) (0.22 pm) in filtered and sterile Mueller-Hinton broth (Difco ™). 2. Dilute the culture broth to an O.D. = 0.06 (about 106 cells / mL, which is the ideal cell density to read on a flow cytometer) or alternatively adjusted to 0.5 MacFarland and then preferably diluted 1: 100 in broth culture . 2. In vitro antimicrobial treatment Obtain cell density for the sensitivity test by flow cytometry, which can be, for example, 106 cells / mL. - Transfer the microbial suspension to a set of vials with different antimicrobial drugs (at breakdown concentrations) including a control vial (without antimicrobial drug); Incubate the flasks according to the antimicrobial drug (0.5 to 2 hours); - Add the appropriate fluorochrome (depending on the antimicrobial drug) according to the previous optimized protocol. 3. Flow cytometry analysis
    [051] The acquisition settings are defined using microsphere samples, adjusting the voltage to the third logarithmic ten (log) of all fluorescence channels. The FSC threshold is used.
    [052] The samples are analyzed in the fluorescence channel FL1, FL2 and FL3, depending on the fluorescent probe, using two point lots: SSC versus FSC and SSC versus fluorescence.
    [053] Numerical results are expressed using the fluorescence intensity and / or the percentage of cells stained.
    [054] The equipment is a flow cytometer (for example, a FACSCalibur BD Biosciences, Sydney, Australia) with three PMTs equipped with standard filters (FL1: BP 530/30 nm; FL2: BP 585/42 nm; FL3: LP 670 nm), a 488 nm 15 mW argon laser and operating with Quest Pro cell software (version 4.0.2, BDBiosciences, Sydney). The flow cytometry data file format used was FCS 2.0a. Examples
    [055] The following examples provide preferential modalities and should not be considered as limiting the scope of the invention. Example No. 1 Evaluation of Escherichia coli susceptibility to quinolones • Microbial agent: Escherichia coli • Drug tested: quinolone - ciprofloxacin [2 breakpoint concentrations: 1 and 4 pg / mL] • Fluorochrome used: SYBR Green, a dye that permeates the membrane that attaches to a double-stranded DNA structure. 1. Distribute the bacterial suspension at a concentration of 106 cells / mL to the series of flasks containing: • Ciprofloxacino at 1 pg / mL; • Ciprofloxacino at 4 pg / mL; • Viable control (without ciprofloxacin). 2. After thirty minutes of incubation at 35 ° C and 150 beats per minute, centrifuge the set of flasks at 10,000 rpm for five minutes. 3. Add SYBR Green (prepared in TE, pH = 7.5) at a final dilution of 1: 100,000 for 30 minutes in the dark. 4. Perform acquisition by flow cytometry at 530 / 30nm - FL1; compare the fluorescence intensity of treated cells with untreated cells. Results interpretation criteria:
    [056] Ciprofloxacin works by inhibiting DNA gyrase and blocks DNA replication leading to DNA with less than two strands that is the target for the fluorochrome SYBR Green. Therefore, strains susceptible (MIC values <1 pg / mL) to ciprofloxacin are expected to show a reduction in double-stranded DNA - corresponding to a lower fluorescence intensity.
    [057] Calculate a staining index (SI) based on the ratio between the intensity of green fluorescence (530/30 nm; FL-1) of treated cells and untreated cells (viable control). • Bacteria are susceptible if SI <1 pg / mL ciprofloxacin; • Bacteria are resistant if SI 2 1 with 4 pg / mL ciprofloxacin. Example No. 2 Assessment of the susceptibility of gram-negative bacteria to carbapenemas • Microbial agent: Pseudomonas aeruginosa • Drug tested: carbapenemas - meropenem [3 breakpoint concentrations: preferably 1, 2 or 4 pg / mL] • Fluorochrome used: bis- (1,3-dibutylbarbituric acid) trimethine oxonol (DÍBAC4 (3) - a voltage-sensitive lipophilic anion for measuring membrane potentials; depolarized cells show the highest fluorescence intensity, while polarized cells show a lower value 1. Distribute the bacterial suspension at a concentration of 106 cells / mL to the series of vials containing: • Meropenem at 1 pg / mL; • Meropenem at 4 pg / mL; • Viable control (without meropenem). thirty minutes of incubation at 35 ° C and 150 beats per minute, add DiBAC4 (3) in a final concentration of 1 pg / mL for 30 minutes in the dark 3. Perform the acquisition by flow cytometry at 530 / 30nm - FL1; compare fluore intensity cells treated with untreated cells. Results interpretation criteria:
    [058] Meropenem is a bactericide that acts by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. The depolarization of the membrane can be quickly detected by DiBAC4 (3) in susceptible strains. Depolarized cells show greater fluorescence intensity, while polarized cells show a lower value.
    [059] Calculate a staining index (SI) based on the ratio between the intensity of green fluorescence (530/30 nm; FL-1) of depolarized cells after treatment with the antimicrobial and control cells (untreated). • Bacteria are susceptible if SI <1 pg / mL of I meropenem; • Bacteria are resistant if SI> 1 with 4 pg / mL of meropenem; Example No. 3 Assessment of Staphylococcus aureus susceptibility to oxacillin • Microbial agent: Staphylococcus aureus • Drug tested: oxacillin [2 concentrations: 2 and 4 pg / mL] • Fluorochrome used: FDA (fluorescein diacetate), a fluorescent indicator of viability cell phone; viable cells after hydrolysis by esterases, FDA forms fluorescein. 1. Distribute the bacteria suspension with the concentration of. 106 cells / ml for the vial series containing: • oxacillin at 2 pg / ml • oxacillin at 4 pg / ml; • Viable control (without oxacillin). 2. After 120 minutes of incubation at 35 ° C, add FDA 1 pg / mL and incubate for 60 minutes in the same conditions. 3. Perform the acquisition by flow cytometry at 530 / 30nm - FL1; compare the fluorescence intensity of treated cells with untreated cells. Results interpretation criteria:
    [060] Oxacillin is a bactericide that acts by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. Therefore, it is expected that strains susceptible (MIC values 2 pg / mL) to oxacillin show a reduction in metabolic activity - corresponding to the lowest fluorescein intensity of fluorescein.
    [061] Calculate a staining index (SI) based on the ratio between the intensity of green fluorescence (530/30 nm; FL-1) of treated cells and untreated cells (viable control). • Bacteria are susceptible if SI <1 with 2 pg / mL of oxacillin; • Bacteria are resistant if SI 1 with 4 pg / mL oxacillin. Example No. 4 Assessment of susceptibility of gram positive bacteria to vancomycin Microbial agent: Enterococus faecali • Tested drug: glycopeptide - vancomycin [2 breakpoint concentrations: 4 to 16 pg / mL] • Fluorochrome used: FDA (fluorescein diacetate) , a fluorescent indicator of cell viability; viable cells after hydrolysis by esterases, FDA forms fluorescein, increasing the emission of fluorescence. 1. Distribute the bacterial suspension at a concentration of 106 cells / mL to the series of vials containing: • 4 pg / mL vancomycin; • vancomycin at 16 pg / mL; • Viable control (without vancomycin). 2. After 60 minutes of incubation at 35 ° C and 150 beats per minute, add FDA 1 pg / mL and incubate for 30 minutes under the same conditions. 3. Perform the acquisition by flow cytometry at 530 / 30nm - FL1; compare the fluorescence intensity of treated cells with untreated cells. Results interpretation criteria:
    [062] Vancomycin is a bactericide that acts by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. Therefore, it is expected that strains susceptible (MIC values 2 pg / mL) to vancomycin show a reduction in metabolic activity corresponding to the lower fluorescence intensity of fluoresceins.
    [063] Calculate a staining index (SI) based on the ratio between the intensity of green fluorescence (530/30 nm; FL-1) of treated cells and untreated cells 5 (viable control). • Bacteria are sensitive if SI <1 with 4 pg / mL vancomycin; • Bacteria are resistant if SI 1 with 16 pg / mL vancomycin. Example No. 5 - Study of resistance mechanisms Resistance by broad-spectrum β-lactamases (ESBLs) • Microbial agent: Klebsiella pneumonia • Drug tested: cefotaxime (CTX) and ceftazidime (CAZ) 2 breakdown concentrations: 15 - 1 and 4 pg / mL for CTX - 4 and 16 pg / mL for CAZ • Drug inhibitor tested: clavulanic acid (CLA) - 4 pg / mL • Fluorochrome used: bis- (1,3-dibutylbarbituric acid) trimetine-oxonol (DÍBAC4 ( 3) - a voltage sensitive lipophilic anion for the measurement of membrane potentials 1. Distribute the bacterial suspension at a concentration of 106 cells / mL to the series of flasks 25 containing: • CTX at 1 pg / mL; at 4 pg / mL; • CTX at 1 pg / mL + 4 pg / mL CLA; • CTX at 4 pg / mL + 4 pg / mL CLA ;. • CAZ at 4 pg / mL; • CAZ at 16 pg. / mL; • CAZ at 4 pg / mL + 4 pg / mL CLA; • CAZ at 16 pg / mL + 4 pg / mL CLA; • Viable control (without cephalosporins). 2. After thirty minutes of incubation at 35 ° C and 150 beats per minute, add DiBAC4 (3) in a final concentration of 1 p g / mL for 30 minutes in the dark. 3. Perform flow cytometry analysis with appropriate protocol and reading at 530 / 30nm - FL1. Results interpretation criteria:
    [064] ESBLs are detected based on their ability to hydrolyze cephalosporins (CTX and CAZ) but are inhibited by clavulanic acid (CIA), that is, it is expected to detect bacteria that are resistant to cephalosporins through the production of ESBLs when they become susceptible to exposure to cephalosporins associated with ASD.
    [065] Calculate a staining index based on the ratio of the green fluorescence intensity (530/30 nm; FL-1) of depolarized cells (dead cells, cells with high fluorescence intensity) after treatment with the highest concentrations of both cephalosporins (4 pg / mL of cefotaxime (CTX)) and 16 pg / mL of ceftazidime (CAZ) with and without the presence of CLA - in this specific case, called the CLA index. CLA index> 1.5 (for at least one cephalosporin) of ESBL-producing bacteria CLA index 1.5 of ESBL-producing bacteria Example No. 6 Susceptibility assessment of a positive blood culture drug combination for yeasts
    [066] Filter and use the lysis agent or use a separating tube and centrifuge • Perform a Gram - yeast or analysis of MaldiTof - Candida albicans • Drugs tested: anidulafungin and fluconazole isolated and in combination • Fluorochrome used: FUN-1, one fluorescent marker used to study the viability of yeasts NOTE: non-viable cells show greater fluorescence intensity than viable cells (untreated cells) • After sixty minutes of incubation with the antifungal isolated and in combination at 37 ° C, add FUN -1 at a final concentration of 0.5 pg / mL for 30 minutes in the dark. Perform flow cytometry acquisition at 575 nm - FL2; a fluorescence index is calculated as the sum of the ratios between the fluorescence of the cells treated with the combination of antifungals and the fluorescence of the cells treated with each individual antifungal. The association is classified as synergistic, indifferent or antagonistic. Results interpretation criteria:
    [067] In vitro drug interaction assessments are determined according to the heart index (SI). The SI is calculated as the sum of the mean fluorescence intensity displayed by the cells treated with the drug combination (0.5 x MIC of each drug) divided by the fluorescence of the drug alone. Therefore, SI = (MIF AND + FLU / MIF AND) + (MIF AND + FLU / MIF FLU) (MIF ratio between the average fluorescence intensity of treated cells and viable cells). An association is defined as "antagonism" for SI <1, "indifferent" for SI between 1 to 4 and "synergistic" for SI> 4. Example No. 7 Antifungal susceptibility of Aspergillus fumigatus • Prepare a dense suspension of Aspergillus fumigatus; • Inoculate 107 spores / mL in a series of Bactec Mycosis IC / F flasks or in MGIT tubes of Bactec automated equipment 10 (Becton Dickinson); • Each series includes a bottle or tube without antifungal (control), one with amphotericin B, one with posaconazole and one with voriconazole; • Vials or tubes are inserted into the respective 15 Bactec equipment and the time to produce a positive result is recorded Results interpretation criteria:
    [068] Susceptible strains incubated with antifungals could take more than twice as long to become 20 positive compared to the control. MGIT tubes provide faster results than Bactec vials (for example, control samples take 7 ± 1 hour in Bactec vials versus 11 + 1 hour in MGIT tubes) Example No. 8 Microbiological antimicrobial quantification of a biological fluid • Use a strain control with a known flow cytometry dose-effect curve when incubated with the antimicrobial to be quantified; • Incubate the biological sample with the control strain; • Label the two exemplary samples with an appropriate fluorescent marker to assess the antimicrobial effect; • obtain the fluorescence signals from the flow and dispersion cytometer to generate a profile of said 5 microorganism. Results interpretation criteria:
    [069] Compare the flow cytometry profile generated with the curve of the control sample and infer the concentration present in the sample.
    [070] The invention, obviously, is not limited in any way to the described modalities and a person normally skilled in the art will foresee many possibilities for modifying them without departing from the basic idea of the invention, as defined in the attached claim table.
    [071] The following claims establish particular embodiments of the invention.
    权利要求:
    Claims (23)
    [0001]
    1. Method for the detection of microorganisms resistant to a therapeutic agent in a biological sample characterized by the said biological sample being an uncultivated sample, comprising the following steps: a. inoculate said sample, in a first tube with, and in a second tube without, at least one therapeutic agent and incubate it; B. add a fluorescent marker to both tubes; ç. perform a fluorescence analysis to obtain one or more fluorescence parameters for each of the two tubes; wherein the phenotype of resistant microorganisms from the biological sample to said therapeutic agent is obtained by comparing one or more fluorescence parameters between the two tubes.
    [0002]
    2. Method for the detection of microorganisms resistant to a therapeutic agent in a biological sample characterized by the said biological sample being an uncultivated sample, comprising the following steps: a. inoculate the referred sample, in a first tube with, and in a second tube without, at least one therapeutic agent and incubate it; B. add a fluorescent marker to both tubes; ç. perform a flow cytometry analysis to obtain one or more flow cytometry parameters for each of the two tubes; wherein the phenotype of microorganisms resistant from the biological sample to said therapeutic agent is obtained by comparing one or more flow cytometry parameters between the two tubes.
    [0003]
    Method according to claim 1 or 2, characterized in that the one or more flow cytometry parameters comprises forward dispersion and / or lateral dispersion and / or fluorescence parameters.
    [0004]
    Method according to any one of claims 1, 2 or 3, characterized in that the parameters of the scattering fluorescence signal comprise intensity, spectral profile and / or cell count.
    [0005]
    Method according to any one of claims 1, 2, 3 or 4, characterized in that it further comprises one of the preparation steps: i. identify the type of microorganism present in the biological sample and choose the therapeutic agent or agents accordingly, that is, whether the type is Gram-positive or Gram-negative bacteria or fungi; ii. concentrate the biological sample and remove, if present, any debris.
    [0006]
    6. Method according to any one of claims 1, 2, 3, 4 or 5, characterized by the fact that the biological sample is a sample of uncultivated urine, culture of blood, water or a suspension of microorganisms, obtained from from pure cultures.
    [0007]
    7. Method according to claim 3, characterized by the fact that step ii) comprises a centrifugation or filtration.
    [0008]
    Method according to any one of claims 1, 2, 3, 4, 5, 6 or 7, characterized in that step a) further includes at least one lysis agent.
    [0009]
    Method according to any one of claims 1, 2, 3, 4, 5, 6, 7 and 8, characterized in that step a) further includes a plug.
    [0010]
    Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that step a) the sample is inoculated in a suitable culture medium.
    [0011]
    11. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized by the fact that the sample incubation time in step a) is between 30 min - 2 hours.
    [0012]
    12. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, characterized in that the sample incubation temperature is between 25-35 ° Ç.
    [0013]
    13. Method according to claim 12, characterized in that the reaction time of step b) with the fluorescent marker is 5-60 minutes.
    [0014]
    14. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, or 13, characterized by the fact that the biological sample in step c) comprises also an enzymatic inhibitor, in case of detection of the presence of extended spectrum beta-lactamases (BLEEs).
    [0015]
    15. Method according to claim 14, characterized by the fact that the enzyme inhibitor is a clavulanic acid or tazobactam.
    [0016]
    16. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, characterized by further comprising the following steps: i . inoculate said sample with a carbapenem and incubate it; ii. removing the microorganism from the biological sample, preferably by means of filtration; iii. add a strain with known behavior; iv. add a fluorescent marker to the two samples; v. perform a flow cytometry analysis to obtain one or more flow cytometry parameters for each of the two tubes; saw. compare the generated profile with the control strain.
    [0017]
    17. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, characterized by the fact that said microorganism is a bacterium, fungus or protist.
    [0018]
    18. Method, according to claim 16, characterized by the fact that the bacterium is a Gram-positive or negative; variable Gram or bacteria unable to stain Gram, such as Mollicutes or acid-resistant bacteria.
    [0019]
    19. Method, according to claim 18, characterized by the fact that said microorganism is a Staphylococcus spp, Streptococcus spp, Enterococcus spp, Enterobacterales, Escherichia coli, Klebsiella pneumonia, Proteus spp, Pseudomonas aeruginosa, Acinetobacter baummanii, Burkeria cepkacter Neisseria spp, Haemophilus spp, Mycobacterium spp, Nocardia spp, Legionella spp, anaerobic bacteria, Candida spp, Cryptococcus neoformans, Pneumocystis jirovecii, or molds such as Aspergillus spp-, Giardia spp.
    [0020]
    20. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, characterized by the fact that the therapeutic agent is an antibacterial drug, such as penicillins, cephalosporins, carbepenems, aztreonam, macrolides, quinolones, aminoglycosides, glycopeptides, a lipopeptide, colistin tetracyclines, chloramphenicol, clindamycin, phosphomycin, rheifin, amine, ammonia, line , trimethoprim, sulfamethoxazole-trimethoprim or tigecycline, polyenes, 5 - fluorocytosine, azoles or echinocandins.
    [0021]
    21. The method of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 , characterized by the fact that the fluorescent marker is a nucleic acid dye, a metabolic dye, a membrane potential dye, an indicator for organelles, a fluorescent marker of cell morphology and fluid flow, an indicator for cell viability, proliferation and function or indicator for reactive oxygen species.
    [0022]
    22. Method according to claim 21, characterized in that the fluorescent marker is an acridine dye, cyanine dye, fluorone dyes, oxazin dye, phenanthridine dye, or a rhodamine dye.
    [0023]
    23. Kit for the detection of microorganisms resistant to a therapeutic agent, to be used in the method described in any one of claims 1 or 2, characterized by comprising: Therapeutic agent; Fluorescent marker; J Buffer; Lysis agent; Culture medium.
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    同族专利:
    公开号 | 公开日
    BR112013031110A2|2016-12-06|
    WO2012164547A1|2012-12-06|
    EP2714922A1|2014-04-09|
    DK2714922T3|2017-11-27|
    JP2017113018A|2017-06-29|
    CN103717747A|2014-04-09|
    JP2014515276A|2014-06-30|
    US9290790B2|2016-03-22|
    PT2714922T|2017-12-29|
    ES2652367T3|2018-02-01|
    PL2714922T3|2018-02-28|
    NO2714922T3|2018-02-17|
    US20140199703A1|2014-07-17|
    JP6587643B2|2019-10-09|
    EP2714922B1|2017-09-20|
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    法律状态:
    2017-04-04| B11A| Dismissal acc. art.33 of ipl - examination not requested within 36 months of filing|
    2017-06-06| B04C| Request for examination: reinstatement - article 33, solely paragraph, of industrial property law|
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-07-02| B06T| Formal requirements before examination|
    2020-01-28| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|
    2020-07-14| B09A| Decision: intention to grant|
    2020-09-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    PT105744|2011-06-03|
    PT10574411|2011-06-03|
    PCT/IB2012/052807|WO2012164547A1|2011-06-03|2012-06-04|Kit and method of detecting the resistant microorganisms to a therapeutic agent|
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