WO2013014324A1 - Método para evaluar la integridad de la pared celular bacteriana - Google Patents
Método para evaluar la integridad de la pared celular bacteriana Download PDFInfo
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- WO2013014324A1 WO2013014324A1 PCT/ES2012/070575 ES2012070575W WO2013014324A1 WO 2013014324 A1 WO2013014324 A1 WO 2013014324A1 ES 2012070575 W ES2012070575 W ES 2012070575W WO 2013014324 A1 WO2013014324 A1 WO 2013014324A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the present invention falls within the field of the biotechnology industry, and mainly that related to microbiology, whose scope is within the health sector (human, veterinary, environmental and basic).
- the present invention relates to a method for assessing the integrity of the cell wall of the bacteria present in a culture in the presence of an antibiotic that acts at the level of the bacterial cell wall, which from a practical point of view, allows to determine quickly if a bacterium is sensitive or resistant to an antibiotic that acts at the level of the cell wall.
- the study of the sensitivity of microorganisms to antimicrobials is one of the most important functions of clinical microbiology laboratories. Its performance is developed through sensitivity tests or antibiogram, whose main objective is to evaluate in the laboratory the response of a microorganism to one or several antimicrobials.
- Some of the most commonly used methods in daily clinical practice include (i) diffusion methods, such as the disk-plate antibiogram based on the work of Bauer, Kirby et al. ⁇ Bauer A W, et.al. Am. J. Clin. Pathol 1966, 45: 493-496) or the epsilon test or E-test method (AB Biodisk, Sweden), or (ii) dilution methods, such as the agar dilution method or the broth microdilution method. Comparing diffusion methods with dilution methods, these are technically more complex and almost always more expensive, particularly when commercial micro-dilution panels are used. In the routine practice of the clinical microbiology laboratory, microdilution methods in liquid medium are the most used.
- the use of commercial panels is based on the use of semi-automatic incubation-reading-interpretation systems; this facilitates its use, but It has the disadvantage of increased spending.
- Some companies have introduced panels on the market in which the culture medium includes a fluorescent indicator that allows rapid results (less than 8 hours) to be obtained.
- a fluorogenic metabolizable compound is added to the culture medium (patent application WO / 1992/019763). If the body grows with the antibiotic, the metabolism of the bacteria results in the release of the fluorophore. If the organism does not grow, the fluorescence of the sample is increased.
- antibiotics that act at the level of the bacterial wall such as ⁇ -lactams
- the BACcelr8r TM is a platform under development by Accelr8, for the automatic identification of microorganisms and to study their resistance to antibiotics. It does not use culture or make it necessary to isolate bacteria. It works by cassettes, where each corresponds to a sample. Use an automated system, with a microscope controlled by a computer, a digital camera and analysis software. A pump maintains a medium flow with bacteria, in different conditions, through the cassette. The analysis of antibiotic resistance could be completed in 8 hours.
- Patent application US2004 / 0014066 describes a method to detect in a sample the activity of an antibiotic that affects cellular integrity comprising (a) providing a transformed microorganism comprising a nucleic acid encoding a promoter operably linked to a heterologous reporter gene capable of emitting a detectable signal, and (b) contacting the sample with the transformed microorganism, (c) observing said microorganism for said detectable signal, in which the promoter is regulated by a two component signal transduction system, where The components are (i) a receptor sensitive to changes in the envelope or cell membrane of the microorganism and (ii) a trans-acting factor that is activated in response to stimulation by the receptor and regulates the promoter.
- the skeleton of the bacterial cell wall is made up of a heteropolymer, peptidoglycan murein.
- This macromolecule is formed by an alternating sequence of N-acetyl-glucosamine (NAG) and N-acetylmuramic acid (NAM) linked by B-1,4 bonds.
- the chain is straight and unbranched, constituting the basic structure of the cell wall.
- N-acetylmuramic acid has a group of lactic acid that bonds with a small peptide chain (tetrapeptide).
- Typical amino acids in this chain include L-alanine, D-glutamic acid, m-diaminopimelic acid or L-Usine or D-alanine.
- Antibiotics that inhibit bacterial wall synthesis are different families of drugs that act on different steps of bacterial wall synthesis:
- Cycloserine is an analogue of D-alanine and competitively inhibits the binding of this amino acid to the enzymes D-alanine-D-alanine synthetase and alanine racemase, preventing its binding to the precursors of peptidoglycan.
- Bacitracin inhibits the recycling of undecaprenil, the lipid transporter of peptidoglycan to the outside of the cell.
- glycopeptides or glycopeptides are a class of peptides that contain bound sugars, as in the bacterial cell wall, having a great affinity to the precursors of this structure.
- the best known are vancomycin and teicoplanin. Vancomycin exerts its bactericidal action by inhibiting the synthesis of the bacterial cell wall, joining the fragment D alanine-D alanine (D-Ala-D-Ala) of the pentapeptide from the wall of Gram + bacteria, blocking the incorporation of peptides into the wall mobile.
- vancomycin would act by other mechanisms such as the affectation of the permeability of the cytoplasmic membrane and inhibition of RNA synthesis, which is exerted after the drug is bound to peptidoglycan.
- - ⁇ -Lactam antibiotics exert bactericidal function by interfering with the transverse junction or interpeptide bridge, necessary for crosslinking. They inhibit the activity of PBPs, serine proteases or transpeptidases, with which they bind irreversibly.
- antibiotics that interfere with wall synthesis are isoniazid, ethionamide and ethambutol. They are used in the treatment of mycobacterial infections, as well as cycloserine, cited above. Isoniazid has bactericidal activity in active replication phase. It affects synthesis of mycolic acid, interrupting the elongation of fatty acids. Ethionamide also inhibits mycolic acid synthesis. Ethambutol interferes with the synthesis of cell wall arabinogalactan. The resistance to these antibiotics is due to the lack of penetration into the bacteria and / or modification of their cell targets. Antibiotic resistance causes tens of thousands of deaths every year. Many of these deaths could be avoided with an antibiotic treatment well selected for their effectiveness. Given the resistance levels, it is necessary to perform the bacterial culture, followed by antibiogram. To complete all this, bacteria should grow 2-3 days, usually. The antibiogram itself usually requires one day of incubation, at least, in the usual fast-growing bacteria.
- the inventors also observed that, when the bacterial culture is mixed or contaminated, that is, there is a mixture of sensitive or resistant cells, said method would not be very suitable as a strict criterion of discrimination since despite releasing extracellular DNA fragments , the morphology of the bacteria is not altered by the action of the antibiotic, that is, both the sensitive and antibiotic resistant bacteria apparently show the intact cell wall.
- the inventors have designed a lysis solution that only affects bacteria whose cell wall has been previously damaged by the action of an antibiotic that acts at the level of the cell wall, and that when added to the bacterial culture, which has previously been exposed to the action of said antibiotic, the release of the bacterial nucleoid occurs then observing a bacterium with the damaged cell wall (Examples 1 to 5).
- the presence of the bacterial nucleoid after applying the lysis solution is indicative of the presence of antibiotic sensitive bacteria.
- the invention relates to a method for assessing the integrity of the bacterial cell wall in a pure culture in the presence of an antibiotic that acts at the level of the bacterial cell wall comprising:
- the invention in another aspect, relates to a method for assessing the integrity of the cell wall of the bacteria present in a culture in the presence of an antibiotic that acts at the level of the bacterial cell wall comprising: i) add to said culture an antibiotic that acts at the level of the bacterial cell wall,
- lysis solution is a specific lysis solution for those bacteria whose cell wall has been damaged by the antibiotic that acts at the level of the bacterial cell wall, and it comprises a buffer with a pH between 3 and 11.5, and
- the invention in another aspect, relates to a method for determining the sensitivity of a bacterium to an antibiotic that acts at the level of the bacterial cell wall, which comprises measuring the integrity of the cell wall of said bacterium by a method according to the present invention. , in which if the integrity of the bacterial cell wall has been damaged, then the bacterium is sensitive to the antibiotic.
- the invention relates to a method for designing a personalized antibiotic therapy to an individual suffering from a bacterial disease comprising
- the invention relates to a method for identifying a compound that acts at the level of the bacterial cell wall comprising:
- the candidate compound is a compound that acts at the level of the bacterial cell wall.
- the invention relates to a method for identifying a persistent or antibiotic tolerant bacterium that acts at the level of the bacterial cell wall in a culture of sensitive bacteria, which comprises assessing the integrity of the cell wall of the bacteria present. in said culture by a method according to the present invention, in which the bacterium whose cell wall integrity has not been damaged is identified as persister or tolerant.
- the invention relates to a lysis solution characterized in that it only affects the bacteria that have the bacterial wall damaged by the action of an antibiotic, which comprises a buffer and a pH between 3 and 11.5.
- the invention relates to the use of the lysis solution of the present invention to evaluate the integrity of the bacterial cell wall.
- the invention in another aspect, relates to a kit comprising the lysis solution of the invention.
- FIG. 1 Three different strains of Escherichia coli, Gram- bacteria, exposed to the antibiotic ⁇ -lactam amoxicillin, together with the inhibitor of beta-lactamases clavulanic acid, processed by the technique for evaluation of cell wall integrity. The incubation was in Mueller-Hinton liquid medium, during the exponential growth phase, at 37 ° C, with stirring, for 40 minutes. The antibiotic doses were chosen according to the cut-off points indicated by the Clinical and Laboratory Standards Institute (CLSI). Thus, the strain is considered sensitive when its minimum inhibitory concentration (MIC) is ⁇ 8/4 (amoxicillin 8 ⁇ / ⁇ .
- MIC minimum inhibitory concentration
- the first strain (top row: a, a ', a ") is sensitive, the second is intermediate (middle row: b, b', b") and the third is resistant (row from below: c, c ', c ").
- a, b, c control, without antibiotic, a', b ', c': 8/4; amoxicillin 8 ⁇ g / mL and clavulanic acid 4 ⁇ g / mL; a ", b", c ": 32/16; amoxicillin 32 ⁇ g / mL and clavulanic acid 16 ⁇ g / mL.
- Controls without antibiotics show bacteria without lysing. After the 8/4 dose, only the bacteria of the first strain, sensitive, appear Used, showing the nucleoids (a ').
- e 32 ⁇ g / mL.
- the MIC dose is already sufficient to observe the involvement of the wall by the antibiotic and the background of extracellular DNA fragments.
- Figure 3 Enterococcus faecium, Gram + bacteria, resistant to the antibiotic ⁇ -lactam ampicillin (MIC> 32), incubated with different doses of this antibiotic for 60 min.
- a control, without antibiotic
- b 32 ⁇ g / mL
- c 320 ⁇ g / mL. After 320 ⁇ g / mL, wall involvement is seen in some isolated cells, with a discrete background of extracellular DNA fragments.
- FIG. 4 E. coli sensitive to the antibiotic ⁇ -lactam ceftazidime, of the cephalosporin type. The strain was exposed to different doses for 60 minutes, to: control, without antibiotic; b: 1 ⁇ g / mL (MIC); c: 8 ⁇ g / mL. The MIC gives rise to a filamentous appearance of the cells, with wall involvement. After 8 ⁇ g / mL a large wall involvement, clearly showing the background of extracellular fragments of DNA.
- E. coli cells from a culture in liquid medium, growing in exponential phase, sensitive to amoxicillin / clavulanic acid, and exposed to the high dose of 32/16 for 90 minutes. After processing by the technique of the invention, in addition to the cells affected in the wall, which release the nucleoid, and from the background of extracellular DNA fragments, a cell that keeps its morphology intact (asterisk), which has not been clearly observed been affected by the antibiotic. This cell behaves like a "persister.”
- Figure 6 Graphs representing the percentages of cells in culture, cells without halo and cells with wall damage, of E. coli, from a culture in liquid medium, growing in exponential phase, sensitive to amoxicillin / acid clavulanic, and exposed to the high dose of 32/16 during different times. Above it is observed that the proportion of non-lysed cells is increasing in the culture over time, as the cells affected by the antibiotic disappear (HG: large halo + HP: small halo + F: Used with fragmented DNA) . Below, the same figure but whose data have been normalized according to the percentage of cells that remain in the culture.
- Figure 8 The same strains of E. coli, presented in Figure 1, were cultured for 24 hours in plaque and then for 40 min in liquid medium with doses 0 (a, b, c), 8/4 (low dose ; a ', b', c ') and 32/16 (high dose; a ", b", c ") of amoxicillin / clavulanic acid. The strains were processed without the lysis step of exposure to the lysis solution. The sensitive strain is presented in the top row (a, a ', a "); the intermediate in the middle row (b, b ⁇ b ”) and the resistant one in the bottom row (c, c ', c").
- the sensitive strain shows the homogeneous micro-granular-fibrillar homogeneous background of extracellular DNA fragments, detached by the cells. This background is evident after the low dose (8/4) and increases markedly after the high dose (32/16). In this way, the sensitive strain can be clearly differentiated from the others.
- Figure 9 A strain of ampicillin-sensitive E. coli was incubated with a dose of 8 ⁇ g / mL of said antibiotic, for 60 minutes, a and b: Fresh observation, a: control culture, without antibiotic; b: culture treated with ampicillin. The latter shows, among bacteria, a diffuse microgranular or fibrillar background, of extracellular fragments of bacterial DNA, c and d: methanol-fixed culture: acetic acid, c: control culture, without antibiotic; d: culture treated with ampicillin, in which the DNA background material between the bacteria is visualized, constituting aggregates of different shape and size.
- Figure 10 A strain of ampicillin-sensitive E. coli was incubated with a dose of 32 ⁇ g / mL of said antibiotic, for 60 minutes. Aliquots of a control culture without antibiotic and aliquots of the culture with the ampicillin treatment were included in microgels and stained with SYBR Gold. The control culture without antibiotic does not show micro granular-fibrillar background in the preparation (a), while the ampicillin treated does show such background (b). Incubation of the microgels with the DNAase I (c) or proteinase K (e) enzyme buffers does not affect said background.
- the Carnoy causes the micro-granular-fibrillar material of the fund to be added, encompassing bacteria.
- Consinction with DAPI evidences aggregates of the background material, with bacteria whose nucleoids stain intensely with DAPI.
- the added background material is also dyed with said dye, although in a more tenuous way.
- the hybrid total genomic DNA probe in both the cell nucleoids and the background material demonstrating that the latter corresponds to fragmented bacterial DNA.
- Figure 12 A strain of Acinetobacter baumannü, sensitive to imipenem, was incubated with this antibiotic 0.76 ⁇ g / mL, 1 hour. An aliquot of the culture was diluted and included in an agrose microgel, dehydrated in growing alcohols, dried and stained with SYBR Gold. A microgranular-fibrillar fund is seen that corresponds to DNA fragments in different degrees of stretching.
- the invention relates to a method for assessing the integrity of the bacterial cell wall in a pure culture in the presence of an antibiotic that acts at the level of the bacterial cell wall (hereinafter, "first method of the invention "), which includes: i) adding to said pure culture of said bacterium an antibiotic that acts at the level of the bacterial cell wall, and
- the term “cell wall” refers to the cell wall that surrounds a bacterial cell and is constituted by the peptidoglycan murein, which is formed by an alternating sequence of N-acetyl-glucosamine ( NAG) and N-acetylmuramic acid (NAM) linked by B-1,4 bonds.
- NAG N-acetyl-glucosamine
- NAM N-acetylmuramic acid
- cell wall and “bacterial cell wall” are equivalent and can be used interchangeably throughout the present description.
- the external agent is an antibiotic that acts at the level of the bacterial cell wall, that is, an antibiotic that blocks the synthesis of peptidoglycan so that the cell wall of the bacterium is damaged.
- pure culture is understood as that culture that contains only one type of microorganism.
- the different culture media, techniques and procedures for obtaining pure cultures are widely known in the state of the art and are routine practice for those skilled in the art (Rotger, R. (editor), 1997, Health and Clinical Microbiology, Editorial Synthesis, Madrid).
- the first stage of the first method of the invention comprises adding to said pure culture of said bacterium an antibiotic that acts at the level of the bacterial cell wall.
- antibiotic includes any chemical compound that eliminates or inhibits the growth of infectious organisms; said term, as used herein, includes any chemical compound produced by a living being, or synthetic derivative thereof, which at low concentrations eliminates or inhibits the growth of infectious organisms.
- a common property of all antibiotics is selective toxicity: the toxicity is higher for invading organisms than for animals or humans that host them.
- Antibiotics can be classified according to their structure, the microorganism they attack, their mechanism of action, their therapeutic target, etc.
- antibiotic that acts at the level of the bacterial cell wall is understood as the antibiotic that interferes with any of the stages of bacterial wall synthesis.
- An assay to determine if an antibiotic acts at the level of the cell wall is, for example, any of the assays described in the examples of the present patent application.
- the antibiotic that acts at the level of the bacterial cell wall is selected from the group consisting of a ⁇ -lactam antibiotic, an isoniazid, an ethionamide, an ethambutol, a cycloserine and a glycopeptide antibiotic.
- the ⁇ -lactam antibiotic is selected from the group consisting of penicillins, cephalosporins, cephamycins, carbacefem, carbapenems, monobactams and beta-lactamase inhibitors.
- beta-lactamase inhibitors are selected from the group consisting of clavulanic acid, sulbactam and tazobactam.
- the glycopeptide antibiotic is vancomycin or teicoplanin.
- the incubation time of the culture together with the antibiotic can vary over a wide range depending on whether the culture is in a stationary or exponential growth phase, whether the culture is carried out in plaque or in liquid medium, of the dose of antibiotic that is added to the culture, etc.
- the incubation time with the antibiotic can range from 5 minutes to 90 minutes, preferably, from 20 to 60 minutes.
- the incubation time is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 minutes.
- the amount of antibiotic to be added to the culture medium can also vary within a wide range; however, in a particular embodiment, the amount of antibiotic to be added to the culture medium is between 5 and 2,570 ⁇ / ⁇ ., although preferably, the amount of antibiotic to be added will be the minimum inhibitory concentration (MIC) for a given bacterium. As the person skilled in the art knows, in the state of the art there are widely accepted and standardized tables in which the MIC necessary to inhibit a specific microorganism is collected. In a particular embodiment, the amount of antibiotic that can be added to the culture medium is 0.06, 0.038, 0.38, 4, 8, 16, 20, 32, 160, 256, or 2,560 ⁇ g / mL.
- the incubation temperature of the culture with the antibiotic may range from 36 ° C to 38 ° C, preferably 37 ° C.
- the second stage of the first method of the invention [step ii)] comprises observing the presence of extracellular DNA fragments in the culture medium.
- the determination of the presence of DNA fragments in the medium can be carried out by microscopy or by any other alternative method to detect DNA released by the microorganisms to the culture medium, whether physical or chemical, between those that are included, without limitation to electrophoresis, antibodies, spectrophotometry, polymerase chain reaction, hybridization techniques, microarrays, microfluidics, nanoparticles, quantum dots, etc. These procedures are widely known in the state of the art and the implementation thereof is routine practice for the person skilled in the art.
- the first method of The invention comprises carrying out, between stages i) and ii), the step of immobilizing a sample of the culture of stage i) on a support.
- Said support can be a slide.
- said slide is a glass slide totally or partially coated with a standard agarose film.
- a standard agarose solution between 0.2% and 2% in distilled water is prepared in a Coplin jar or the like; it is covered with a perforated plastic sheet and placed in a microwave oven; the microwave oven is regulated to a power comprised between 300 W and 1,000 W, preferably at 500 W, occasionally stirring the container for a better dissolution of the agarose, leaving it until it boils.
- This procedure can also be performed using a thermostatic bath.
- the agarose solution becomes completely transparent, it will be ready to be deposited in vertical containers with a content between 10 and 250 mL.
- slides covered with an agarose film are deposited horizontally on a smooth, cold surface between 1 ° C and 15 ° C, preferably at 4 ° C, for example, glass or metal.
- This plate, with the slides is introduced into the refrigerator at 4 ° C for a minimum of 30 minutes, until it is verified that the agarose solution has gelled on the surface of the slide.
- the trays are removed from the refrigerator and the surface of the slides that was in contact with the plate with a blotting paper is cleaned.
- the slides are placed horizontally in an oven at a temperature between 37 ° C and 100 ° C, until the agarose dries completely and forms a thin film adhered to the glass. Slides so treated can be used immediately or store in a tightly closed box at room temperature for several months.
- concentration of microorganisms in a liquid sample is obtained and checked.
- the appropriate concentration for the analysis ranges between 0.1 and 20 million microorganisms per milliliter. If the sample is excessively concentrated, it is adjusted to the appropriate concentration by diluting it with culture medium or with buffered saline / phosphate solution (PBS) or similar, suitable according to the microorganism and according to the stability of the antibiotic to be tested.
- PBS buffered saline / phosphate solution
- a medium with characteristics similar to those of a suspension such as, for example, an agarose microgel.
- a low melting gelling solution of agarose is prepared at a concentration between 0.5 and 3% in distilled water or phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the fusion of this agarose is carried out using a microwave oven or a thermostatted bath, and is subsequently maintained between 30 ° C and 37 ° C in a tube introduced in a thermostated bath or stove.
- the culture sample and agarose solution are carefully mixed, so that the latter is at a concentration between 0.3% and 2%.
- the slides coated with agarose are placed on a smooth and cold glass or metal surface, with a temperature between 1 ° C and 15 ° C, avoiding air bubbles. It is recommended to deposit with a micropipette a drop (between 2 and 200 microliters ( ⁇ ⁇ )) of the mixture (prepared culture sample + agarose), placing a coverslip on top of the drop. Multiple drops can be pipetted, i.e. bacterial culture samples from stage i), on each slide. As a precaution, it is recommended to process each sample in duplicate and use a control sample each time the technique is applied.
- the plate with the slides is placed in a refrigerator at 4 ° C, for a period of time between 2 and 30 minutes until adequate gelation of the agarose occurs. Once the gelation has occurred, the coverslips are removed very gently, inside the same refrigerator and preventing damage to the microgel.
- the dried slides are incubated in a microwave oven at a power between 300 W and 1,000 W, preferably at 500 W, for a period of time between 1 and 10 minutes.
- An alternative, although less recommended for its duration, is to incubate the slides in an oven or stove at high temperature for a period of time between 1 and several hours.
- the slides already processed containing the sample can be stored in filing boxes at room temperature, in the dark, for months. This facilitates the separation of the sample treatment process and the subsequent stage of assessing the integrity of the cell wall of the microorganisms. Archiving allows repeated evaluation at different intervals of several samples of the same microorganism.
- the samples are treated, and after stabilizing and firmly adhering the DNA fragments to the slide, they can be stained and the integrity of the cell wall of the microorganisms can be evaluated.
- the observation of the presence of extracellular DNA fragments in the culture medium is carried out by staining.
- Staining or staining is an auxiliary technique in microscopy to improve the contrast in the image seen under a microscope. In biochemistry, this involves adding a dye specific for the molecule to be stained (DNA in the context of the present invention) to a substrate to qualify or quantify the presence of a certain compound. Stains can be used, among other things, to define and examine organelles within individual cells, or to label nucleic acids in gel electrophoresis.
- dyes or dyes are organic compounds that have some specific affinity for cellular materials. Many frequently used dyes are positively charged molecules (cations) and are intensively combined with negatively charged cell constituents, such as nucleic acids and acidic polysaccharides.
- Illustrative, non-limiting examples of cationic dyes include methylene blue, violet crystal and safranine. Methylene blue is a good simple dye that acts on all bacterial cells quickly and does not produce a color so intense that it obscures cellular details, which is especially useful. Sometimes, some dyes dye better only after the cell has been treated with another chemical, called mordant, which is not a dye by itself.
- a common mordant is tannic acid, which is combined with a cellular constituent and alters it so that it can now attack the dye.
- tannic acid which is combined with a cellular constituent and alters it so that it can now attack the dye.
- specific techniques for staining the DNA such as, for example, Feulgen staining, which consists in subjecting the material to hydrolysis with 1N hydrochloric acid at 60 ° C or with 5N hydrochloric acid at room temperature and then add the Schiff reagent By this technique it is possible to stain the nuclei of the bacterial cells.
- fluorescence microscopy is the technique of choice to visualize the DNA fragments given their greater sensitivity, for this it is necessary to perform the staining of bacteria with certain chemical compounds called fluorophores or fluorochromes. These compounds are capable of emitting fluorescence when excited with light at a suitable wavelength. At present there is a whole range of fluorochromes that provide not only information on cell viability, but also show certain physiological characteristics and in some structural cases of bacteria.
- fluorophores that detect respiratory activity eg, tetrazolium derivatives, etc.
- esterase activity eg, calcein-AM, carboxyfluorescein, etc.
- membrane potential eg, rhodamine 123, oxonol VI, carbocyanins, etc.
- membrane integrity eg, SYTO-9, SYTO-13, Sitox green, propidium iodide, etc.
- staining is carried out by using one or more fluorochromes.
- the samples can be stained with specific fluorochromes for DAPI type DNA (4 ', 6-diamidino-2-phenylindole), Hoechst 33258, Hoechst 33342, ethidium bromide, propidium iodide , etc.
- fluorochromes of greater sensitivity such as GelRed, EvaGreen, and other cyanine derivatives, such as the SYBR®, PicoGreen® (Invitrogen-Molecular Probes TM) families, the TOTO, YO YO variants, are preferred.
- fluorochromes include, but are not limited to, SYTOX Blue, chromomycin A3, mitramycin, acridine orange, SYTOX Green, thiazoyl orange, LDS 751, 7-AAD, SYTOX orange, DRAQ5.
- the fluorochromes are selected from the group consisting of Hoechst 33342, Hoechst 33258, DAPI, chromomycin A3, mitramycin, ethidium bromide, acridine orange, thiazoyl orange, 7-AAD, cyanine derivatives, and variants of fluorochromes TOTO, YOYO, BOBO, POPO, JOJO, LOLO, SYTOX, PO-PRO, BO-PRO, YO-PRO, TO-PRO, JO-PRO, PO-PRO and LO-PRO
- an "antifading" medium can be included (for example, Vectashi the d-Vector H-1000, DABCO; etc.).
- these media usually produce diffuse fluorescence and a clear background that makes contrasting the image difficult. Therefore, in general, it is preferable to use a high sensitivity and relative photostability fluorochrome, mounted in a buffered aqueous solution, and evaluate the sample with Relatively fast, before drying. If necessary, the slide can be washed and stained again.
- the images obtained can be studied by direct visual analysis or, preferably, by applying a digitalized image analysis software, obtained by analog or digital cameras, coupled to microscopy platforms.
- the integrity of the microorganism wall is evaluated by determining the presence of extracellular DNA fragments in the culture medium, where the presence of extracellular DNA fragments in the culture medium is indicative that the integrity of the bacterial cell wall has been damaged.
- the first The method of the invention would not be very suitable as a strict criterion of discrimination since both the antibiotic-sensitive bacteria apparently show the intact cell wall.
- the inventors have designed a lysis solution that only affects bacteria whose cell wall has been previously damaged by the action of the antibiotic that acts at the level of the cell wall.
- the invention relates to a method for assessing the integrity of the cell wall of bacteria present in a culture in the presence of a antibiotic that acts at the level of the bacterial cell wall (hereinafter, "second method of the invention"), comprising:
- lysis solution is a specific lysis solution for those bacteria whose cell wall has been damaged by the antibiotic that acts at the level of the bacterial cell wall, and it comprises a buffer with a pH between 3 and 11.5, and
- mixed culture is understood as a bacterial culture that contains two or more different species of bacteria.
- the presence of two or more different species of bacteria in a crop is due to contamination of the crop due to improper handling of the sample, so that in the context of the present invention, the terms “ mixed culture “and” contaminated culture “are equivalent and can be used interchangeably throughout the description.
- the bacteria present in the culture belong to the same species or to different species.
- steps i) and iii) of the second method of the invention are common to the first method of the invention [stages i) and ii), respectively]. Accordingly, all the particular explanations and embodiments mentioned above in relation to said steps are also applicable to the second method of the invention.
- the second method of the invention comprises, in addition to steps i) and iii) [common to steps i) and ii) of the first method of the invention], a step ii) not present in the first method of the invention comprising adding a lysis solution to the culture resulting from step i), wherein said lysis solution is a specific lysis solution for those bacteria whose cell wall has been damaged by the antibiotic that acts at the level of the bacterial cell wall, and comprises a buffer with a pH between 3 and 11.5.
- the lysis solution that specifically affects the bacteria that have the cell wall affected by the antibiotic that acts at the level of the bacterial cell wall basically comprises a buffer solution that has a pH between 3 and 11.5 but that Additionally, it may comprise other components, including, but not limited to, ionic detergents, non-ionic detergents, salts, etc. in different proportions.
- the buffer that is part of the lysis solution is tris (hydroxymethyl) aminomethane (Tris) of formula (HOCH 2 ) 3 CH 2 , which can also be used to prepare other buffer solutions, among which They include, but are not limited to, Tris-HCl, Tris-Gly, TAE (Tris-acetate-EDTA) and TBE (Tris-Borate-EDTA) buffers.
- Tris has a pKa of 8.06, which gives it effective buffering capacity in a pH range between 7.0 and 9.2.
- Tris base it is the basic, non-ionized form of the amine
- Tris-HCl is also used.
- buffer solutions include, but are not limited to, Hepes, Mops, Pipes, etc.
- sodium phosphate also called disodium
- hydrogen phosphate Na 2 HP0 4
- boric acid-borate triethylamine
- CABS 4- [cyclohexylamino] -l-butanesulfonic acid
- the lysis solution comprises, in addition to the buffer solution, up to 3% of an ionic detergent or a non-ionic detergent.
- ionic detergent refers to that compound that has a hydrophobic part and a hydrophilic part, which in solution forms positively charged ions (cationic detergent) or negatively (anionic detergent) and which allows to achieve emulsion.
- cationic detergent cationic detergent
- anionic detergent anionic detergent
- the terms "detergent”, “surfactant” and “surfactant” are synonymous, so they can be used interchangeably throughout the present description.
- cationic detergents include, but are not limited to, primary, secondary, tertiary and quaternary ammonium salts, whether linear or cyclic in structure, mixtures thereof, such as, for example, pyridine, piperizine salts, and derivatives thereof.
- Ammonium The term “derivatives of ammonium salts” includes those salts that incorporate at least two amino groups in the same structure, whether primary, secondary, tertiary and / or quaternary, such as, for example, guanine dyne, piperazine and imidazole salts. In this definition, amino acid salts would also be included, such as, for example, Usine, arginine, ornithine or tryptophan salts.
- ammonium salts in which the positive charge is on a phosphorus atom such as ditetradecyl iodide (trimethylethylphosphonium) methylphosphonate, ditetradecyl iodide (butyldimethylphosphonium) methylphosphonate, ditetradecyl iodide (iodide of dimethylisopropylphosphonium) methylphosphonate or arsenic (ditetradecyl iodide (trimethylarsonium) methylphosphonate, dioleyl iodide (trimethylphosphonium) methylphosphonate, instead of over the nitrogen atom.
- ditetradecyl iodide trimethylethylphosphonium
- ditetradecyl iodide butyldimethylphosphonium
- ditetradecyl iodide iodide of dimethylisopropylphosphonium
- arsenic ditetradecyl
- ammonium salts include, but are not limited to, tetraalkylammonium salts alkylbenzyl dimethyl ammonium or heterocyclic ammonium salts, such as cetyltrimethylammonium bromide (CTAB).
- CTAB cetyltrimethylammonium bromide
- ionic detergents include acyl amino acids, such as acyl glutamic, acyl peptides, sarcosinates, taurates, etc., carboxylic acids, such as saturated chain acids, carboxylic acid esters, carboxylic acid ethers.
- phosphoric acid esters such as acyl isothiates, alkyl aryl sulfonates, alkyl sulfonates, sulfosuccinates, etc.
- sulfuric acid esters such as alkyl ether sulfates and alkyl sulfates.
- the ionic detergent is a detergent selected from the group consisting of sodium dodecyl sulfate (SDS), alkylbenzene sulphonate, laurylsarcosine, hydrated salt of glycolic acid, and its salts.
- SDS sodium dodecyl sulfate
- alkylbenzene sulphonate alkylbenzene sulphonate
- laurylsarcosine hydrated salt of glycolic acid
- nonionic detergents include, but are not limited to, polysorbates, polyethylene glycol copolymers and polypropylene glycol copolymers, such as Tween, Span, Poloxamer.
- the nonionic detergent is selected from the group consisting of toctylphenoxypolyethoxyethanol, N, N-Bis (3-D-gluconamidopropyl) colamide, Brij (r) 35 P, N-decanoyl-N-methylglutamine, digitonin, dodecanoyl-N-methylglucamide, heptanoyl-N-methylglutamide, octylphenoxy poly (ethyleneoxy) branched ethanol, N-Nonanoyl- N-methylglucamine, Nonidet P 40, N-octanoyl-N-methylglutamine, Span 20 solution and polysorbate 20.
- the lysis solution further comprises up to a 3M concentration of a salt.
- salts that may be part of the lysis solution of the invention include, but are not limited to, carbonates, chlorides, phosphates, nitrates, nitrites, sulfates, citrates, carboxylates (acetates, formates, salicylates, etc.).
- the lysis solution comprises sodium chloride (NaCl).
- the lysis solution provided by this invention comprises Tris between 0.001 M and 2 M, SDS up to 3%, and NaCl up to a concentration of 3 M, at a pH between 3 and 11.5.
- the expert in the field understand that these compounds may be substituted for other equivalent compounds; for example, the SDS can be replaced by Triton X-100 up to 10%.
- the lysis solution provided by this invention comprises (approximately 0.2 M hydroxymethyl) -l, 3-propanediol (Tris), approximately 0.025% SDS, approximately 0.5 M or 0.05 M sodium chloride , and a pH of 10; In this case, the SDS can be replaced by Triton X-100 at approximately 5%.
- the lysis solution provided by this invention comprises (approximately 0.2M hydroxymethyl) -l, approximately 0.2M 3-propanediol, approximately 5% Triton X-100, approximately 1M sodium chloride, and a pH of 10.
- the lysis solution provided by this invention comprises approximately 0.3 M sodium phosphate, approximately 2% SDS, approximately 0.05 M ethylenediamine tetraacetic acid (EDTA), and a pH of 11.45.
- the preparations are incubated in the lysis solution for 0.5 to 120 minutes, preferably 1 to 35 minutes, more preferably for about 5 minutes; and at a temperature between 1 ° C and 45 ° C, preferably between 15 ° C and 40 ° C, more preferably between 22 ° C and 37 ° C.
- the incubation is carried out at a temperature of 22 ° C.
- the incubation is carried out at a temperature of 37 ° C.
- the evaluation of the presence of the bacterial nucleoid in the medium can be performed by microscopy or by any other alternative method to detect DNA released by the microorganisms at culture medium, whether physical or chemical, which includes, but is not limited to electrophoresis, antibodies, spectrophotometry, polymerase chain reaction, hybridization techniques, microarrays (microarrays), microfluidics, nanoparticles, quantum dots, etc. These procedures are widely known in the state of the art and the implementation thereof is routine practice for the person skilled in the art.
- the detection of the bacterial nucleoid is carried out by microscopy, for which it is necessary to immobilize a sample of the culture resulting from step i) [ie, after exposure of the bacterial culture to the action of the antibiotic] on a support, such as, for example, a slide.
- step ii) of the second method of the invention that is, the addition of the lysis solution that only affects bacteria whose cell wall has been affected by the antibiotic, can be carried out before or after immobilization. the bacteria on said support.
- the second method of the invention further comprises, before or after step (ii), immobilizing a sample of the culture on a support.
- the lysis solution is treated after immobilizing a sample of the culture resulting from stage i) on a support, such as a slide, it is immersed horizontally in a container containing the lysis solution .
- the preparations can be washed to remove the remains of this solution.
- the supports are introduced in a washing solution as smooth as possible, avoiding chelating agents or detergents; by way of illustration, the supports can be submerged horizontally in a container containing abundant distilled water or a buffer solution or physiological serum for a time between 1 and 60 minutes. Then, the sample is dehydrated. For this, solutions of increasing alcohol concentration can be used.
- the slides are lifted and immersed in containers with a series of increasing concentration of ethanol, between 5% and 100%, for 30 seconds to 60 minutes each and then the preparations are allowed to air dry.
- the temperature of the alcohols can range from -20 ° C to room temperature. It may be preferable to use alcohols at a temperature of -20 ° C to improve the precipitation of DNA, for 5 minutes each.
- the preparations can be dehydrated by incubating in solutions of different alcohols such as methanol, or by letting it air dry or in an oven.
- step iii) of the second method of the invention the presence of the bacterial nucleoid is determined either in the culture medium or in the support if the sample has been immobilized.
- bacterial nucleoid means the region that contains DNA in the cytoplasm of bacterial cells. Experimental evidence suggests that the nucleoid is primarily composed of DNA (60%), with small proportions of RNA and proteins. These last two components act as messenger RNA and as genome regulatory proteins. In the state of the art, the bacterial nucleoid is also known as "nuclear region” or “nuclear body.” The techniques previously explained to observe the presence of extracellular DNA fragments in the culture medium can also be used to observe the bacterial nucleoid as required by the second method of the invention.
- the technique of choice for determining the presence of the bacterial nucleoid is microscopy, then, as previously explained for the first method of the invention, it is convenient to stabilize and firmly adhere the DNA fragments to the slide since they can be detached. Once the samples are treated, and after stabilizing and firmly adhering the DNA fragments to the slide, they can be stained and the integrity of the cell wall of the microorganisms can be evaluated.
- the observation of the presence of the bacterial nucleoid in the culture medium is carried out by staining. By choosing the staining conditions conveniently, high image quality and high consistency of the evaluation results can be obtained.
- the different staining techniques as well as the dyes that can be used to visualize DNA by microscopy have been previously explained in the present description in relation to the first method of the invention and are applicable to the second method of the invention.
- the observation of the presence of the bacterial nucleoid in the culture medium is carried out by staining which, in a more particular embodiment, is carried out by the use of fiuorochromes which, in yet another embodiment
- fiuorochromes which, in yet another embodiment
- they are selected from the group consisting of fiuorochromes, they are selected from the group consisting of Hoechst 33342, Hoechst 33258, DAPI, chromomycin A3, mitramycin, ethidium bromide, acridine orange, thiazoyl orange, 7-AAD (7-aminoactinomycin D ), cyanine derivatives, variants of the TOTO, YOYO, BOBO, POPO, JOJO, LOLO, SYTOX, PO-PRO, BO-PRO, YO-PRO, TO-PRO, JO-PRO, PO-PRO and LO- PRO, etc.
- the implementation of the second method of the invention requires in an initial stage to add to an bacterial culture an antibiotic that acts at the level of the bacterial cell wall.
- an antibiotic that acts at the level of the bacterial cell wall is selected from the group consisting of a beta-lactam antibiotic (among which they include, but are not limited to penicillins, cephalosporins, cephamycins, carbacefem, carbapenemics, monobactamics and beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam, etc.), an isoniazid, an ethionamide, an ethambutol, a cycloserine and a glycopeptide antibiotic (including, but not limited to, vancomycin or teicoplanin).
- the practical application of the first and second method of the invention is to quickly and reliably find out if a bacterium is sensitive or resistant to an antibiotic that acts at the level of the bacterial cell wall. For this, it is sufficient to evaluate the integrity of the bacterial wall by any of the methods of the invention described above in the present description.
- the present invention relates to a method for determining the sensitivity of a bacterium to an antibiotic that acts at the level of the bacterial cell wall comprising assessing the integrity of the cell wall of said bacterium by any of the methods of the invention, in which if the integrity of the bacterial cell wall has been damaged, then the bacterium is sensitive to the antibiotic.
- the bacteria is not sensitive to the antibiotic, it can be a resistant bacterium or a bacterium called "persister" or tolerant.
- Both types of bacteria are not affected by the antibiotic that acts at the level of the bacterial cell wall, but they differ in that resistant bacteria have mutations in the DNA so that antibiotic resistance is permanent, while bacteria persist. they do not have resistance mutations in the DNA, being a reversible functional state. Therefore, other applications of the methods of the invention consist in the detection in a sample of resistant bacteria or "persister" bacteria.
- the invention relates to a method for identifying a persistent or antibiotic tolerant bacterium that acts at the level of the bacterial cell wall in a culture of sensitive bacteria, which comprises assessing the integrity of the cell wall of Bacteria present in said culture by the second method of the invention, in which the bacterium whose cell wall integrity has not been damaged is identified as persister or tolerant.
- Another of the practical applications presented by the methods of the invention relates to the design of a personalized antibiotic therapy for a person suffering from a bacterial disease, since by means of the methods of the invention it is possible to find out if the bacterium causing the bacterial disease It is sensitive or resistant to a certain antibiotic.
- the doctor may decide to administer to the individual suffering from said bacterial disease a therapy based on said antibiotic. If, on the contrary, the bacterium causing the bacterial disease is resistant to the antibiotic tested, then the doctor will choose a treatment that is not based on said antibiotic.
- the present invention relates to a method for designing a personalized antibiotic therapy to an individual suffering from a bacterial disease comprising
- "individual” means a member of any animal species, including, but not limited to, mammals, in particular, cattle (cows, bulls, oxen, yaks, etc.), cattle sheep (sheep, etc.), pigs (pigs, wild boars, etc.), goats (goats, etc.), horses or horses (horses, mares, zebras, etc.), camelids (camels, llamas, alpacas , etc.), rabbits, hares, bison, buffalo, deer, reindeer, deer, caribou, dogs, cats, mice, nonhuman primates (chimpanzees, gorillas, orangutans, macaques, gibbons, etc.).
- the mammal is preferably a human being of any sex, age or race.
- the "individual" or “subject” terms are synonyms and can be used interchangeably throughout this description.
- bacterial disease is understood as that disease that results from the infection of an individual by a bacterium.
- bacterial diseases include, but are not limited to, diseases caused by infection of bacteria of the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus , Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Heamophilus and Bordetella.
- the first stage of the method for designing a personalized antibiotic therapy for an individual suffering from a bacterial disease comprises isolating the bacterium causing said bacterial disease from a sample from said individual.
- the sample must be representative of the infectious process and must be taken from the correct anatomical site, must be collected in sufficient quantity to ensure an adequate examination, sterile devices, etc. must be used in the collection. More information on the procedures and materials used to collect samples can be found in Rotger, R. (editor), 1997 (cited ad supra).
- the invention relates to a method for identify a compound that acts at the level of the bacterial cell wall that comprises:
- the candidate compound is a compound that acts at the level of the bacterial cell wall.
- the first stage of the method for identifying compounds that act at the level of the cell wall comprises contacting a culture of a bacterium sensitive to antibiotics that act at the level of the cell wall in the presence of the candidate compound.
- antibiotic-sensitive bacteria include, but are not limited to, amoxicillin-sensitive E. coli strains, amplicillin-sensitive Enterococcus faecaelis strain, iminepen-sensitive Acinetobacter baumannii strain, strain of E. coli sensitive to ceftazidime, etc. More information on the sensitivity of bacteria to ⁇ -lactam antibiotics can be found in the state of the art (June 2010 CLSI Guidelines). Lysis solution of the invention
- the present invention is based on the fact that a consequence of the activity of antibiotics acting at the level of the cell wall on bacteria is the release of extracellular fragments of DNA to the culture medium. Additionally, when the bacterial culture is mixed or contaminated, that is, there is a mixture of sensitive or resistant cells, the observation of extracellular DNA fragments would not be very suitable as a strict criterion of discrimination since despite releasing Extracellular DNA fragments, the morphology of the bacteria is not altered by the action of the antibiotic, that is, both the sensitive and antibiotic resistant bacteria apparently show the intact cell wall. To solve this problem, the inventors designed a lysis solution that only affects bacteria whose cell wall has been previously damaged by the action of the antibiotic that acts at the level of the cell wall.
- lysis solution hereinafter lysis solution of the invention, characterized in that it affects only the bacteria that have the bacterial wall damaged by the action of an antibiotic, which comprises a buffer and a pH between 3 and 11.5.
- the lysis solution that specifically affects the bacteria that have the cell wall damaged by the antibiotic that acts at the level of the bacterial cell wall basically comprises a buffer solution that has a pH between 3 and 11.5 but additionally , as explained in previous inventive aspects, may comprise other components, including, but not limited to, ionic detergents, non-ionic detergents, salts, etc. in different proportions.
- the lysis solution of the invention further comprises up to 3% of an ionic detergent or a non-ionic detergent.
- said ionic detergent is a detergent selected from the group consisting of sodium dodecyl sulfate, alkylbenzene sulphonate, lauryl sarcosine, hydrated salt of glycolic acid, and mixtures thereof.
- said non-ionic detergent is selected from the group consisting of toctylphenoxypolyethoxyethanol, N, N-Bis (3-D-gluconamidopropyl) colamide, Brij (r) 35 P, N-decanoyl-N-methylglutamine, digitonin, dodecanoyl -N-methylglutamide, heptanoyl-N-methylglutamide, octylphenoxy poly (ethyleneoxy) branched ethanol, N-Nonanoyl-N-methylglutamine, Nonidet P 40, N-octanoyl-N-methylglutamine, Span 20 solution and polysorbate 20.
- the lysis solution further comprises up to a 3M concentration of a salt, for example, carbonates, chlorides, phosphates, nitrates, nitrites, sulfates, citrates, carboxylates (acetates, formates, salicylates, etc.);
- a salt for example, carbonates, chlorides, phosphates, nitrates, nitrites, sulfates, citrates, carboxylates (acetates, formates, salicylates, etc.);
- the lysis solution comprises sodium chloride (NaCl).
- the lysis solution provided by this invention comprises Tris between 0.001 M and 2 M, SDS up to 3%, and NaCl up to a concentration of 3 M, at a pH between 3 and 11.5.
- the person skilled in the art will understand that these compounds can be substituted by other equivalent compounds; for example, the SDS can be replaced by Triton X-100 up to 10%.
- the lysis solution provided by this invention comprises (approximately 0.2 M hydroxymethyl) -l, 3-propanediol (Tris), approximately 0.025% SDS, approximately 0.5 M or 0.05 M sodium chloride , and a pH of 10; In this case, the SDS can be replaced by Triton X-100 at approximately 5%.
- the lysis solution provided by this invention comprises (approximately 0.2M hydroxymethyl) -l, approximately 0.2M 3-propanediol, approximately 5% Triton X-100, approximately 1M sodium chloride, and a pH of 10.
- the lysis solution provided by this invention comprises approximately 0.3 M sodium phosphate, approximately 2% SDS, approximately 0.05 M ethylenediamine tetraacetic acid (EDTA), and a pH of 11.45. More details and explanations on the various particular embodiments of the lysis solution of the invention can be found in the description of the second method of the invention.
- the invention relates to the use of the lysis solution of the invention to evaluate the bacterial cell wall.
- Information on how the lysis solution of the invention can be used to evaluate the bacterial cell wall can be found in the description of the first and second methods of the invention.
- kit of the invention The implementation of the methods of the invention requires a series of components that can be arranged together in the form of a pack or kit, hereinafter, kit of the invention.
- Components useful for the implementation of the methods of the invention include, but are not limited to, buffer solution, lysis solution, dyes, sterile material for sample collection (swabs, swabs, tweezers, etc.), covers and slides, distilled water alcohols (ethanol), etc.
- the kit of the invention may contain instructions or indications that guide the person skilled in the art in implementing the methods of the invention.
- the invention relates to a kit comprising the lysis solution of the invention.
- the samples can be stained with specific fiuorochromes for EvaGreen or (green) or GelRed (red) type DNA.
- sample-agarose mixture was pipetted onto a previously coated slide (eg, with an agarose film) and the sample was covered with a 22x22 mm coverslip.
- the slide is placed on a cold plate of a refrigerator (4 ° C) for 5 minutes to allow the agarose to form a microgel with intact cells trapped inside.
- the coverslip was carefully removed and the slide was immediately immersed horizontally in a lysis solution for 5 minutes at 37 ° C for Gram + (gram positive) bacteria and at 22 ° C for Gram- (gram negative) bacteria.
- the slide was washed horizontally on a tray with plenty of distilled water for 3 minutes, dehydrated by incubating it horizontally in cold ethanol (-20 ° C) at increasing concentration (70%, 90% and 100%) for 3 minutes at each concentration and Air dried in an oven.
- the dried slide was incubated in a microwave oven at 750 W for 4 minutes and the DNA was stained with 25 ⁇ L of SYBR Gold (Molecular Probes, Eugene, OR, USA) diluted 1: 400 in TBE buffer (Tris-borate 0, 09 M, EDTA 0.002 M, pH 7.5) for 2 minutes in the dark, with a glass coverslip. After a brief wash in phosphate buffer pH 6.88 (Merck, Darmstadt, Germany) a 24x60 mm coverslip was added and the slides were visualized by fluorescence microscopy.
- the images were visualized in an epifluorescence microscope (Nikon E800), with a lOOx objective and fluorescence filters appropriate for FITC-SYBR Gold (465 nm excitation, 515-555 nm emission), PI-Cy3 (540/25 nm excitation , emission 605/55 nm) and DAPI (excitation 340-380 nm, emission 435-485 nm).
- FITC-SYBR Gold 465 nm excitation, 515-555 nm emission
- PI-Cy3 540/25 nm excitation , emission 605/55 nm
- DAPI excitation 340-380 nm, emission 435-485 nm.
- the images were captured with a high sensitivity CCD camera (KX32ME, Apogee Instruments, Roseville, CA, USA). Groups of 16-bit digital images were obtained and archived as .tiff files.
- Image analysis used a macro in the Visilog 5.1 program (Noesis, Gif sur Yvette, France). This allowed to determine the threshold, subtract the background and measure the size of the average width of the halo of the nucleoids in ⁇ , delimited between the peripheral end of the nucleoid and the outer limit of the cell body. In the case of unrecognized cell bodies, the nucleoid centroid was considered as an internal reference point to measure the width of the halo of the disseminated nucleoid.
- EXAMPLE 1 Confirmation of the operation of the technique: release of the bacterial nucleoid and diffuse remains of the wall and / or bacterial products, in bacteria sensitive to an antibiotic that acts at the level of the bacterial wall
- Three different strains of Escherichia coli were exposed to the ⁇ -lactam antibiotic amoxicillin, together with the clavulanic acid ⁇ -lactamase inhibitor, and processed by the technique for evaluating the integrity of the cell wall of the present invention.
- Bacteria that were growing in Mueller-Hinton liquid medium were incubated with the antibiotic in Mueller-Hinton liquid medium during the exponential growth phase, at 37 ° C, with shaking, for 40 minutes.
- the antibiotic doses were chosen according to the cut-off points indicated by the Clinical and Laboratory Standards Institute (CLSI).
- a strain is considered sensitive when its minimum inhibitory concentration (MIC) is ⁇ 8/4 (amoxicillin: 8 ⁇ g / mL and clavulanic acid: 4 ⁇ g / mL) and resistant when its MIC is> 32/16 (amoxicillin : 32 ⁇ g / mL and clavulanic acid: 16 ⁇ g / mL).
- MIC minimum inhibitory concentration
- E. coli isolated from hospital After the results of the previous experiment (Example 1), 11 different strains of E. coli were studied, isolated in a Microbiology Service. After growing in plaque with Mueller-Hinton medium, for 24 hours, they were exposed to amoxicillin, together with Clavulanic acid, in Mueller-Hinton liquid medium for 1 hour, after which they were processed by the technique for assessing the integrity of the cell wall according to the present invention. As in Example 1, the doses were 0, 8/4 and 32/16 (amoxicillin / clavulanic acid).
- beta-lactam antibiotics penicillins, cephalosporin and carbapenem
- Different bacterial strains grew on plaque with Mueller-Hinton medium, for 24 hours, then were exposed to the ⁇ -lactam antibiotic for 60 minutes in Mueller-Hinton liquid medium, a 37 ° C, under stirring, and finally processed by the technique for assessing the integrity of the cell wall according to the present invention.
- the strains and antibiotics used were:
- the antibiotic doses applied were 0, the MIC, determined in the Microbiology Laboratory using the microdilution and / or e-test technique, and those of the sensitivity and resistance cut-off points indicated by the Clinical and Laboratory Standards Institute (CLSI) for each strain. A dose 10 times higher than the MIC was also used.
- the antibiotic cut-off points vary for each strain:
- Escherichia coli Ampicillin-resistant strain (MIC> 256) and ceftazidime (MIC2).
- Figure 5 shows cells of a sensitive strain of E. coli, from a culture in liquid medium, growing in exponential phase, sensitive to amoxicillin / acid clavulanic, and exposed to a high dose (32/16) for 90 minutes.
- a high dose 32/16
- the "persister" cells when not growing, should progressively increase their relative proportion on the slide, as the incubation time with the antibiotic increases and the sensitive cells disappear from the culture. This phenomenon could also take place when the strain is incubated with progressively increasing doses of the antibiotic, for a fixed time. In fact, if the results were adjusted for the amount of cells present after incubation with the antibiotic, it would be obtained that the cells without halo (“persisters") would remain constant regardless of the dose of antibiotic or the incubation time with it , because they do not grow, while the rest of the sensitive cells, with halo, would decrease progressively, when disappearing from the culture. With these two types of experiments, among others, it can be determined if the non-lysed cells correspond to "persisters".
- the nucleoid frequency with fragmented DNA was determined according to the protocol described in Tamayo et al. ⁇ Tamayo M, Santiso R, Gosálvez J Bou Q Fernández JL.
- bacteria When bacteria are processed bypassing the incubation step in the lysis solution, they appear intact in their entirety, whether or not they are affected by the antibiotic. However, if after pertinent incubation with the dose of antibiotic for sensitive cutting, the homogeneous microdiffuse, granular-fibrillar background of DNA fragments detached by the cells can be clearly seen in the preparation, it can be clearly decided whether the strain is sensitive or not, respectively.
- This background assessment without using lysis to see the effect on the cell wall, can be done by staining with the fluorochrome the material included in microgel, or the material not including in microgel, either fixed or fresh.
- the liquid with the cells is included in an agarose microgel, which is dehydrated in alcohols and / or air dried or in an oven and stained with fluorochrome. It is the same process described in detail above, but without incubation with the lysis solution. The evaluation of the fund can be done quickly, after the 15-20 minutes it takes to perform the technique. The preparations are permanent. The result is shown in Figure 8, which shows the same strains presented in Figure 1, grown for 24 hours in plaque and then incubated for 40 minutes with doses 0, 8/4 (low) and 32/16 (high) amoxicillin / clavulanic acid. This more abbreviated system does not detect the "persisters" of the bacterial strain itself, nor discriminate sensitive cells from resistant, in case of mixed or contaminated culture.
- the background intensity of extracellular DNA fragments depends on the concentration of sensitive bacteria. If this is reduced, the fund may be very dim or almost unobservable.
- a strain of E. coli sensitive to amoxicillin / clavulanic acid incubated with a high concentration (32/16), from an OD600 of 0.07, shows very evident background at 15 minutes of incubation (OD600: 0.071 ; 42.5 million cells per mL, measured by Neubauer chamber; 5.62% viable cells) and remains intense until 2 hours of incubation (OD600: 0.033; 14.5 million cells per mL; 3.77 % of viable cells).
- the fund becomes scarce at 3 and 4 hours (OD600: 0.037 and 0.033 respectively; 14 million cells per mL at both times; 0.38% of viable cells at both times) and is no longer visible at 6 hours (OD600: 0.035; 13.5 million cells per mL; 0.13% viable cells). Although the total cells remain between 40-30% of the initial ones from 60 minutes to 6 hours, the intensity of the background of DNA fragments is reduced in that time until it is not detectable, possibly by degradation thereof.
- An aliquot of the culture without antibiotic and others with antibiotic doses can be mixed with a fixative.
- alcoholic, aldehyde or ketone fixatives such as methanol, ethanol, acetone, formaldehyde, glutaraldehyde, also acetic acid, picric acid, mercury chloride, dichromate ion, osmium tetroxide, etc, and mixtures such as methanol can be used: acetic, for example in a 3: 1 ratio, such as Carnoy liquid.
- formaldehyde the solution may be 0.1 to 50% aqueous, preferably 10%.
- the other fixatives they can be used in different proportions, from 0.1 to 100%.
- a proportion of 5-10% aqueous solution of microorganisms and 95-90%> of fixative is recommended.
- fixative over fresh observation is that the material can be stored for a long time and observed when convenient.
- a drop (a few microliters) is spread on the slide and allowed to dry.
- SYBR Gold fluorochrome (1: 400) is added, mounted with a coverslip and examined.
- Formaldehyde retains the background only briefly, so it is not recommended.
- the other fixers are more durable and give a clear background image.
- Methanol acetic acid is preferable. With methanol: acetic acid, the material spreads more easily on the slide and the background material and the bacteria are better adhered to the glass, while with the other fixatives they are more likely to detach when stained, unless it Incubate previously with dry heat. The background of extracellular DNA fragments is seen as a dispersed aggregate ( Figure 9). The extension of the material fixed on the slide takes about 8-10 minutes to dry, although when the slide is placed on a plate or stove at 37 ° C it dries in 5 minutes. If the methanol: acid: acetic acid (3: 1) is 95%, drying after the drop is rapid, in less than 1 minute.
- the fixings do not provide great advantage in terms of preparation and observation time, with respect to the preparation of the microgel, which is also permanent.
- the operability of the fixation or use of the microgel is practically similar.
- Fluorochrome is added to an aliquot of the culture, a coverslip is placed and examined directly with the fluorescence microscope. For example, about 10 ⁇ of the liquid culture with bacteria, 2 ⁇ of SYBR Gold fluorochrome (1: 400) are added. This is done in cultures with an antibiotic that acts on the cell wall, including a control culture, without an antibiotic. In the culture with the antibiotic, in addition to the floating bacteria, diffuse micro filamentous or granular intercellular material is seen, in continuous Brownian motion, which corresponds to the background emitted by the sensitive bacteria ( Figure 9). This determination of the sensitivity or resistance to the antibiotic that acts on the cell wall is the fastest and simplest, in 1 minute.
- the experiment was performed with an ampicillin sensitive E. coli strain and an imipenem sensitive A. baumanii strain. The first was incubated with 32 ⁇ g / mL ampicillin and the second with imipenem 0.76 ⁇ g / mL, for 60 minutes, in Mueller-Hinton liquid medium, at 37 ° C, while stirring. After incubation, each culture with the cells was included in microgels on the slide. A microgel of the control culture without antibiotic was placed on each slide, and 2 microgels of the antibiotic treated culture were placed. The size of each microgel is the one that corresponds to an 18xl8mm coverslip.
- microgels on slides were washed in proteinase K buffer (1% SDS, 2 mM EDTA) and on other slides, in DNAase I buffer (20 mM Tris-HCl, pH 8.3, 2 mM MgCl 2 ).
- proteinase K buffer 1% SDS, 2 mM EDTA
- DNAase I buffer 20 mM Tris-HCl, pH 8.3, 2 mM MgCl 2 .
- one of the microgels of the culture treated with ampicillin was incubated only with proteinase K buffer and the other microgel of the culture treated with ampicillin was incubated with 5 ⁇ of the proteinase K itself, 2 mg / mL, in its buffer.
- FISH Fluorescent in situ hybridization
- the DNA present on the slides was denatured, incubating them in 75% formamide / 2XSSC, pH7, at 67 ° C, 90 seconds. Subsequently they were passed through alcohols of 70%, 90% and 100%, at -20 ° C, 5 minutes each and allowed to dry. In each of them, at the level of the extension area, micro quantities of biotin-labeled total genomic DNA probe (4.3 ng / ⁇ in 50% formamide, 2XSSC, 10%) sulfate were pipetted dextran, 100 mM sodium phosphate, pH 7), putting an 18xl8mm coverslip. The probe was incubated overnight in a humid chamber.
- the non-hybridized probe was washed in 50% formamide / 2XSSC, pH 7, two 5-minute washes and then in 2XSSC, pH 7, two 3-minute washes each, at 37 ° C.
- the slides were incubated in antibody blocking solution (5% BSA, 4XSSC, 0.1% Triton X-100) for 5 minutes at 37 ° C and then in streptavidin-Cy3 (1: 200, in 1% BSA, 4XSSC, 0.1% Triton X-100), 30 minutes.
- the slides were counterstained with DAPI (1 ⁇ g / mL in Vectashield) and examined with a fluorescence microscope.
- microgranular-fibrillar background observed in the middle of the cultures of microorganisms where the antibiotic against the cell wall has been effective corresponds to extracellular DNA fragments, released by the microorganism.
- Examples 1 to 8 show the efficacy of the methods provided by the present invention for the rapid determination in situ of the bacterial sensitivity or resistance to antibiotics that act at the level of the cell wall, by example, inhibiting the biosynthesis of peptidoglycan.
- the technique for assessing the integrity of the cell wall is a quick and simple procedure that allows discriminating between resistant and antibiotic-sensitive strains that act at the level of the cell wall, for example, interfering with the biosynthesis of peptidoglycan. This methodology can be useful not only at the clinical level but also to perform basic studies on the mechanism of action of antibiotics that act at the level of the cell wall.
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CN201280046327.1A CN103946389A (zh) | 2011-07-26 | 2012-07-26 | 用于评价细菌细胞壁完整性的方法 |
MX2014000920A MX357457B (es) | 2011-07-26 | 2012-07-26 | Metodo para evaluar la integridad de la pared celular bacteriana. |
ES12817952T ES2908261T3 (es) | 2011-07-26 | 2012-07-26 | Procedimiento para evaluar la integridad de la pared celular bacteriana |
US14/234,875 US9976170B2 (en) | 2011-07-26 | 2012-07-26 | Method for evaluating bacterial cell wall integrity |
EP12817952.0A EP2738262B1 (en) | 2011-07-26 | 2012-07-26 | Method for evaluating bacterial cell wall integrity |
CA2842865A CA2842865C (en) | 2011-07-26 | 2012-07-26 | Method for evaluating bacterial cell wall integrity |
BR112014001871-5A BR112014001871B1 (pt) | 2011-07-26 | 2012-07-26 | Solução de lise, método para avaliar a integridade da parede celular das bactérias, método para prescrever uma terapia com antibiótico, método para identificar um composto que atua sobre a parede celular bacteriana, uso de uma solução de lise e kit que compreende uma solução de lise |
EP22152446.5A EP4050108A1 (en) | 2011-07-26 | 2012-07-26 | Lysis solution |
HK14110731A HK1197271A1 (en) | 2011-07-26 | 2014-10-27 | Method for evaluating bacterial cell wall integrity |
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CN105358982A (zh) * | 2013-07-04 | 2016-02-24 | Abm科技公司 | 用于快速确定细菌对抗生素的敏感性或耐药性的方法 |
US10472663B2 (en) | 2015-01-21 | 2019-11-12 | Abm Technologies, Llc | Procedure for the rapid determination of bacterial susceptibility to antibiotics that inhibit protein synthesis |
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EP3052642A2 (en) | 2013-09-30 | 2016-08-10 | AdvanDx, Inc. | Antimicrobial compound susceptibility test |
US9597319B2 (en) * | 2014-04-23 | 2017-03-21 | Case Western Reserve University | Compositions and methods of inhibiting metallo-β-lactamases |
CN107656056B (zh) * | 2017-08-29 | 2019-05-28 | 山东师范大学 | 一种基于细菌增长对细菌快速镜检的方法 |
GB201801022D0 (en) * | 2018-01-22 | 2018-03-07 | Q Linea Ab | Single dye concentration determination |
FR3103197A1 (fr) * | 2019-11-15 | 2021-05-21 | bioMérieux | Determination par spectrometrie de masse de la sensibilite ou de la resistance de bacteries a un antibiotique |
CN111458313A (zh) * | 2020-04-07 | 2020-07-28 | 上海交通大学医学院附属仁济医院 | 基于荧光d型氨基酸代谢标记的抗菌药敏试验检测方法 |
CN113188876B (zh) * | 2021-04-29 | 2022-10-25 | 西北农林科技大学 | 一种枣根尖染色体压片的制备方法 |
CN113106082B (zh) * | 2021-05-27 | 2022-11-04 | 云南师范大学 | 动物粪便宏基因组来源的丙氨酸消旋酶及其制备和应用 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105358982A (zh) * | 2013-07-04 | 2016-02-24 | Abm科技公司 | 用于快速确定细菌对抗生素的敏感性或耐药性的方法 |
US11319574B2 (en) | 2013-07-04 | 2022-05-03 | Abm Technologies, Llc | Method for the rapid determination of susceptibility or resistance of bacteria to antibiotics |
US10472663B2 (en) | 2015-01-21 | 2019-11-12 | Abm Technologies, Llc | Procedure for the rapid determination of bacterial susceptibility to antibiotics that inhibit protein synthesis |
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BR112014001871B1 (pt) | 2021-03-30 |
EP4050108A1 (en) | 2022-08-31 |
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US9976170B2 (en) | 2018-05-22 |
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