Technical field

[0001] The present disclosure relates to the microorganism extraction and/or for the determination of the underlying mechanisms of resistance using and/or for the determination of minimum inhibitory concentration, a kit and uses thereof, preferably by flow cytometry. The present disclosure finds use in the diagnostic in medical or veterinary applications.

Background art

[0002] Antimicrobial resistance is a worldwide problem. The knowledge of the underlined mechanism of resistance has particular interest for patient treatment but also has extremely importance for hospital infection control and obvious epidemiological relevance.

[0003] The susceptibility profile can be determined by evaluating growth ability in the presence of different antimicrobials, which takes at least 18-24 hours, giving no information regarding most underlined mechanisms of resistance. Given the time required for the all procedure, empiric therapy is applied through broad-spectrum antimicrobials, encompassing severe individual and social consequences.

[0004] To clarify the underlined mechanism of resistance some phenotypic tests are available but are cumbersome and will need additional 24 hours incubation time (72 hours from the biological sample collection). This information is often too late for patient treatment and, almost useless regarding hospital control of infection. Such patient could meanwhile have been transferred to several departments and spread the organism to other patients, hospital staff or environmental. Diagnostic molecular methods have set the way for a new era in clinical microbiology however, regarding susceptibility, only a few genes have actually been correlated with antimicrobial resistance. Nevertheless, resistance is often due to multiple associated mechanisms probing the limitation and expensiveness of a genotypic approach. New methods such as detection of some Penicillin-binding proteins (PBP) that confer methicillin resistance, like PBP2a detection, seems an alternative for clinical laboratories; however, it will not detect the PBP2c recently described, also conferring methicillin resistance. Mass spectrometry is also promising regarding the detection of some mechanisms of resistance, but the information provided would be parallel to molecular biology and it would be difficult to detect all the possible resistant mechanisms.

[0005] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General Description

[0006] Flow cytometry has been proven to be a powerful tool in areas such as Hematology or Citopathology and its increasing use in Microbiology also envisages a great potential. Cell microbial populations can be discriminated in terms of susceptibility versus resistance phenotypes in a short period of time. Indeed, antimicrobial drug effects can be quickly detected and quantified using potent software programs which allow the collection of a large amount of data including cell size and shape and, using different fluorochromes to evaluate viability, membrane potential, membrane lesions, DNA quantification and many other morpho-functional parameters of the cells.

[0007] The present disclosure relates to an extraction and /or susceptibility tests for the main antimicrobials directly from blood positive cultures, urine or from pure cultures, towards bacteria and fungi. This novel approach will allow the determination of the antimicrobial susceptibility phenotype in just one hour, and/or the minimum inhibitory concentration (MIC) of a therapeutic agent. Thus, revolutionizing the clinical microbial diagnosis, and even more so if coupled to a rapid identification method such as mass spectrometry.

[0008] This disclosure shows improvements in terms of protocol methodology in comparison with the document WO2012/164547. I n table 1 is described a comparison between results of flow cytometry antimicrobial susceptibility tests performed directly from agar culture versus broth culture (incubation until exponential phase) and immediately performed from positive blood culture versus at least 6 hours after the blood culture turns positive (table 1). In the last case after a pre-incubation of the positive blood culture in broth medium, better results can be achieved regarding the flow cytometry antimicrobial susceptibility test (FAST) to a wide range of antibiotic classes (table 1).

In addition, the underlying resistance mechanisms such as efflux pumps, methicillin resistance by Staphylococcus aureus (MRSA), resistance or heterogeneous resistance to glycopeptides hGISA by S. aureus, vancomycin resistance and high level of resistance to aminoglycosides by Enterococcus, resistance to penicillin by Streptococcus pneumonia can be achieved. The presence of enzymes that can inactivate the antimicrobial agents namely cephalosporins and carbapenems could be present even in susceptible phenotype. Screening cut-off values should be tested in order to detect them. Those mechanisms could be inferred by flow cytometry in a time to result (TTR) of 1-2 hours in contrast to the current methodologies. Table 2 summarizes the market available methodologies for antimicrobial susceptibility testing and mechanisms of antimicrobial resistance. In order to optimize and validate the methodology of the present disclosure, an extensive collection of clinical isolates with a previously characterized antimicrobial susceptibility phenotype were used. Resistant strains well characterized molecularly were used as controls. The results were compared with the standard methods including CLSI (1 - Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Information Supplement M100-S23. Clinical and Laboratory Standards Institute 2013) and EUCAST documents (2 - Breakpoint tables for interpretation of MICs and zone diameters version 4.0. European Committee on Antimicrobial Susceptibility Testing 2014; 3 - EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. European Committee on Antimicrobial Susceptibility Testing 2013). Sensitivity and specificity of the method proved to be very high. This innovative product will bring quicker answers and will give relevant additional information, impossible to achieve with the actual methods on clinical laboratories.

[0009] The present disclosure relates to a method for detecting resistant microorganisms to a therapeutical agent and/or the minimum inhibitory concentration of a therapeutical agent, in a biological sample, comprising the steps of:

preparing a microbial suspension from the biological sample;

aliquoting the microbial suspension into microbial suspension portions; adding the therapeutical agent to each microbial suspension portion; incubating each microbial suspension portion with an initial concentration of the therapeutical agent and with a fluorescent marker, wherein the initial concentration of the therapeutical agent is different for each microbial suspension portion;

determining a final concentration of the therapeutical agent in each microbial suspension portion;

determining a minimal inhibitory concentration (MIC) of the therapeutical agent; performing a flow cytometric analysis and,

i.e when it is identified a microorganism exhibiting a phenotype resistant to said therapeutic agent, it is possible to characterize the underlined mechanism of resistance applying the previous methodology.

[0010] In an embodiment, the initial concentration of the therapeutical agent ranges from 0.002 to 256 μg/ml.

[0011] In an embodiment, the microbial suspension portions are at least 3 microbial suspension portions, preferably at least 5 microbial suspension portions, more preferably at least 10 microbial suspension portions.

[0012] In an embodiment, the incubating step is carried out for 5 hours, preferably 15 minutes - 5 hours, more preferably 1 hour - 3 hours, even more preferably 1.5 hours - 2 hours.

[0013] In an embodiment, the incubating step is carried out at 20-37 °C, preferably at 25-35 °C, more preferably at 28-32 °C.

[0014] In an embodiment, the step of preparing a microbial suspension from the biological sample comprises a centrifugation, a filtration or combinations thereof.

[0015] In an embodiment, the biological sample is selected from a peritoneal sample, pleural fluid sample, cerebrospiral fluid sample, urine sample, blood culture sample, broth culture sample or a colony sample.

[0016] In an embodiment, the method further comprises a step in vitro cell staining.

[0017] In an embodiment, the step of cell staining is made by incubating a fluorochrome with the microbial suspension, in particular at 20-37 °C, more in particular for at least 5 min. [0018] In an embodiment, the fluorochrome is selected from calcein AM, evans blue, fluorescein diacetate, propidium iodide staining, green fluorescent protein, lactate dehydrogenase, methyl violet, propidium iodide, DNA stain, Trypan Blue, cell labelling or staining (e.g., a cell-permeable dye (e.g., Carboxylic Acid Diacetate, Succinimidyl Ester (Carboxy-DFFDA, SE), a cell-impermeable dye, cyanine, phenantridines, acridines, indolesa nucleic acid stain, a cell permeant reactive tracer (e.g., intracellularly-activated fluorescent dyes CMRA, CMF2HC (4-Chloromethyl-6,8-Difluoro-7-Hydroxycoumarin), CMFDA (5- Chloromethylfluorescein Diacetate), CMTMR (5-(and-6)-(4-

Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine), CMAC (7-Amino-4-

Chloromethylcoumarin), CMHC (4-Chloromethyl-7-Hydroxycoumarin)) or any combinations thereof), fluorescent DNA dyes (e.g., DAPI, Hoechst family, SYBR family, SYTO family, SYTOX family, ethidium bromide, propidium iodide, acridines, or any combinations thereof); cytoplasma stain (e.g., calcofluor white, periodic acid-schiff stain, or any combinations thereof); metabolic stains (e.g., any diacetate dye (including, rhodamine based-dye, fluorescin, or any combinations thereof), resazurin/resorufin (alamar blue); ROS stains (e.g., DCFDA and related family, calcein-acetoxymethyl and related family); membrane stains (e.g., bodipy, FM 1-43, FM 4-64, and functionally equivalent thereof, CellMask™ stains, Dil, DiO, DiA); biologic stains (e.g., labeled antibodies, labeled chitin-binding protein), viable dyes such as FUN-1, Dioc 2(3), Dibac 3(4), or any combinations thereof.

[0019] In an embodiment, the microorganism is selected from the following list: a Staphylococcus spp, Streptococcus spp, Enterococcus spp; Enterobacteriacea, Escherichia coli, Klebsiella pneumonia, Proteus spp; Pseudomonas aeruginosa, Acinetobacter baummanii, Burkloderia cepaciae; Neisseria spp, Haemophilus spp; Mycobacteria spp, Nocardia spp; Legionella spp, anaerobic bacteria; Candida spp, Cryptococcus neoformans, Pneumocystis jirovecii, or moulds like Aspergillus spp; Giardia spp., or combinations thereof.

[0020] In an embodiment, the therapeutic agent is an antibacterial drug selected from the following list: penicillins, cephalosporins, carbepenems, monobactams, fluoroquinolones, aminoglycoside^ glycopeptides, streptogramins, tetracyclines, echinocandins, azoles, lincosamides, polyene pyrimidine analogues, aztreonam, macrolides, quinolones, aminoglycosides, glycopeptides, a lipopeptide, tetracyclines colistin, chloramphenicol, clindamycin, fosfomycin, linezolid, nitrofurantoin, sulphonamide, rifampin, trimethoprim, trimethoprim-sulfamethoxazole or tigecycline, polyenes, 5-fluorocytosine, or echinocandins, or combinations thereof.

[0021] In an embodiment, the penicillins are selected from benzylpenicillin, ampicillin, ampicillin+sulbactam, amoxicillin, amoxicillin plus clavulate, piperacillin, piperacillin plus tazobactam, ticarcillin, ticarcillin plus clavulanate, phenoxymethylpenicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, mecillinam, mezlocillin, or combinations thereof.

In an embodiment, cephalosporin is selected from a list consisting of cefaclor, cefadroxil, cefalexin, cefamandole, cefazolin, cefdinir, cefepime, cefetamet, cefixime, cefmetazole, cefonocid, cefoperazone, cefotaxime, cefotetan, cefoxitin, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephalothin, loracarbef, moxalactam, ceftalozane or combinations thereof. In particular, with or without enzymatic inhibitors such as clavulanic acid, avibactam, vaborbactam, and relebactam or tazobactam.

[0022] In an embodiment, the carbapenems may be selected from a list consisting of doripenem, ertapenem, imipenem, meropenem or combinations thereof. In particular, with or without enzymatic inhibitors such as clavulanic acid, avibactam, vaborbactam, relebactam or tazobactam.

[0023] In an embodiment, the monobactams may be selected from a list consisting of aztreonam, tigemonam, nocardicin A, tabtoxin or combinations thereof.

[0024] In an embodiment, fluoroquinolones may be selected from a list consisting of cinoxacin, ciprofloxacin, enoxacin, fleroxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, or combinations thereof.

[0025] In an embodiment, aminoglycosides may be selected from a list consisting of amikacin, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin or combinations thereof.

[0026] In an embodiment, glycopeptides may be selected from a list consisting of teicoplanin, telavancin, vancomycin, or combinations thereof. [0027] In an embodiment, streptogramins may be selected from a list consisting of azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, clindamycin, quinupristin-dalfopristin, or combinations thereof.

[0028] In an embodiment, tetracyclines may be selected from a list consisting of selected from a list consisting of: doxycycline and/or minocycline and/or tetracycline and/or tigecycline and/or chloramphenicol and/or colistin and/or daptomycin and/or fosfomycin and/or fusidic acid and/or linezolid and/or metronidazole and/or mupirocin and/or nitrofurantoin and/or polymyxin B and/or spectomycin and/or sulfonamides and/or trimethoprim and/or trimethoprim-sulfamethoxazole and/or combinations thereof.

[0029] In an embodiment, echinocandins may be selected from a list consisting of as anidulafungin and/or caspofungin and/or micafungin and/or combinations thereof.

[0030] In an embodiment, azoles may be selected from a list consisting of fluconazole and/or voriconazole and/or itraconazole and/or posaconazole and/or ketoconazole and/or combinations thereof.

[0031] In an embodiment, polyene may be amphotericin B.

[0032] In an embodiment, pyrimidine analogues may be flucytosine.

[0033] In an embodiment, the final concentration of the fluorochrome is from 0.125 μΜ - 150 μΜ.

[0034] This disclosure also relates to a microorganism extraction method from biological samples. In particular, when the biological sample is a blood culture.

[0035] The present disclosure for better results further discloses relates to a microorganism extraction method from biological sample comprising the steps of:

preparing a microbial suspension from the biological sample;

adding a lysing agent, in particular for better results can be use as lysing agent: triton, votex; preferably wait 5 minutes; more preferably use as lysing agent triton X 10 % (v/v);

adding hystopaque, centrifuging the sample, in particular 1300 rpm during about 1 min; reject the supernatant of the centrifuged sample; adding a saline solution to the remaining sample; adding an adequate culture medium.

[0036] The use of hystopaque, in the extraction method of the present disclosure, surprisingly shows the formation of a concentration gradient in the centrifugated sample. This solution it is especially relevant when the microorganism to extract is Staphylococcus. As show in table 3 and 4 this method (protocol B) improve the efficiency of the microorganism extraction.

[0037] In an embodiment, the biological sample is blood or a blood culture.

[0038] In an embodiment, the microorganism is a gram-positive bacterium, in particular Staphylococcus spp.

[0039] Another aspect of the present disclosure also relates to a kit for detecting the resistant microorganisms to a therapeutic agent and/or the minimum inhibitory concentration of a therapeutical agent, comprising the method now disclosed.

Brief Description of the Drawings

[0040] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

[0041] Figure 1 a-b: A typical example of a strain with MIC value of 1 μg/ml to meropenem after flow cytometry test. Note that at concentration 1 μg/ml the staining index (SI) is 2.80, being the lowest concentration that provokes significant cell depolarization.

[0042] Figure 2 : Screening test for detecting the presence of carbapenemase, then perform a flow cytometry analysis and determined the staining index SI, wherein

if SI of SN < to SI of carbapenem/cephalosporin indicates enzyme production; if SI of SN > to SI of carbapenem/cephalosporin indicates efflux and/or cell membrane impermeability. [0043] Figure 3: A typical example of a resistant strain that produce carbapenemase. The enzyme degraded meropenem so the effect of supernatant is significantly lower than the effect of the drug (mean SI 6.44 versus 89.50).

[0044] Figure 4: Resistance mechanism: characterization of the kind of enzymatic mechanism underlined to carbapenems resistance, then perform a flow cytometry analysis and determined the staining index SI, wherein

when SI with inhibitors APBA/PBA > cut-off indicates the presence of metallo-β - lactamase; when SI with inhibitors EDTA/DPA >cut-off indicates the presence of KPC; when SI with inhibitors PABA/PBA and EDTA/DPA >cut-off indicates the presence of metallo-β -lactamase and KPC; when SI with cloxacilin >cut— off indicates the presence of AMPc; when no synergic effect is seen indicates the presence of OXA-48 —like enzyme. Additionally, temocillin resistance (MIC>32 mg/L) could he performed.

[0045] Figure 5: A typical example of a resistance strain with a KPC. It was observed an increase of intensity of fluorescence after incubation with the inhibitor APBA (boronic acid).

[0046] Figure 6: A typical example of a resistance strain with a metallo-beta-lactamase. Note an increase of fluorescence after incubation with EDTA.

[0047] Figure 7: Resistance mechanism: confirmatory test - enzyme detection: AmpC detection, then perform a flow cytometry analysis and determined the staining index SI, wherein

when SI < cut-off AmpC detected.

[0048] Figure 8: A typical example of a resistance strain with an AmpC beta-lactamase. Note an increase of fluorescence after incubation with cloxacillin. [0049] Figure 9: Resistance mechanism: confirmatory test - efflux detection. Then perform a flow cytometry analysis and determined the staining index SI, wherein

SI with efflux inhibitors > cut-off indicates the presence of efflux, when SI with efflux inhibitors > cut-off indicates the presence of porin loss.

[0050] Figure 10 a-b: hVISA detection. Then perform a flow cytometry analysis and determined the staining index SI, wherein

MIC value will be the concentration that shows a SI > cut-off value determined using statistic analysis.

[0051] Figure 11 a-f: A typical example of a heterorresistant strain. The MIC value increase from 1 to 16 μg/ml after growth in broth with 4 μg/ml of vancomycin.

Detailed Description

[0052] The present disclosure relates to the determination of the underlying mechanisms of resistance using and/or for the determination of minimum inhibitory concentration, a kit and uses thereof, preferably by flow cytometry. The present disclosure finds use in the diagnostic in medical or veterinary applications.

[0053] In an embodiment, the characterization of the main mechanisms of resistance by flow cytometry was performed as described in the following embodiments.

[0054] In an embodiment, the peritoneal, articular, pleural fluid samples, cerebrospinal fluid or urine was analysed in the following way: perform a Gram stain or mass spectrometry analysis (MALDI-TOF MS) in order to guide the choice of antibiotics to be tested. This step provides information in terms of the presence of bacteria, in particular cocci or bacilli; Gram-positive or Gram-negative, and fungi in the biological sample. [0055] In an embodiment, if the sample is of cerebrospinal fluid, said sample may be concentrated by centrifugation, in particular at 13000 rpm for 1 minute, and the pellet may be resuspend in 1 ml of broth medium until 0.5 MacFarland turbidity; if it could not be obtained incubate in an Erlenmeyer with 10 ml of brain heart infusion or Mueller Hinton broth or Cation adjusted Mueller Hinton or Todd Hewitt broth or HTM broth or Columbia broth, Tioglycolate broth or buffer solution such as PBS, HEPES or MOPS at pH 6-8 until that turbidity is obtained (about 1-3 hours). These broths may be supplemented with 0%, 0.2%, 0.5%, 0.9%, 1%, 2%, 4%, 5%, 8% or 10% of one or more additives such as dextrose and/or casein and/or vitamin K and/or hemine and/or bovine calf serum and/or yeast extract and/or NaCI and/or calcium and/or magnesium and/or lysed horse blood whenever necessary, and incubated for 2 hours or 3 hours or 4 hours or 5 hours at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or capnophilic (2.5% to 5% C0 ₂ ) or anaerobic atmosphere.

[0056] In an embodiment, if the sample is urine, determine the microbial cell density of the urine sample using the flow cytometer to screen for positivity (more than 10 ⁵ cells/ml, only one microorganism).

[0057] In an embodiment transfer the sample, in particular 3 to 6 ml or 4 to 8 ml of the sample, to a separator serum tube (e.g. gel tube) and centrifuge in particular for 5 to 10 minutes at 1500 to 3000 g. Transfer 3 ml, or 4 ml or 5 ml or 6 ml of the supernatant to a new tube and add 3 ml of sterile saline and repeat the centrifugation step, in particular at 1500 to 3000g during 5 to 10 minutes. Discharge the supernatant. Make a microbial suspension by adding 1ml to the gel tube of a broth culture medium like brain heart infusion, or buffer solution such as PBS, HEPES or MOPS at pH 6-8. These broths may be supplemented with 0%, 0.2%, 0.5%, 0.9%, 1%, 2%, 4%, 5%, 8% or 10% of one or more additives such as dextrose and/or casein and/or vitamin K and/or hemine and/or bovine calf serum and/or yeast extract and/or sodium chloride (NaCI) and/or calcium and/or magnesium and/or lysed horse blood and/or choline chloride whenever necessary). Invert 3 times and vortex. Adjust the cell suspension to 0.5 McFarland and afterwards dilute 1:10 or 1:100 in broth culture medium or buffer solution such as PBS, HEPES or MOPS at pH 6-8 similar to those described above, and/or hemine and/or bovine calf serum and/or yeast extract and/or NaCI. [0058] In an embodiment, if the cell suspension does not reach 0.5 McFarland the process may need to be repeated, otherwise if the suspension reaches a 10 ⁵ to 10 ⁶ cells/ml the 1:10 and 1:100 are unnecessary.

[0059] In an embodiment, if cocci in grapes or yeasts are detected or as an alternative to the previous described method: transfer 1ml of the sample to an Eppendorf tube and add a lysing agent such as Triton X (10%v/v) for 5 min at room temperature (specially if red blood cells are present); transfer carefully that sample to another Eppendorf tube with Hystopaque or similar. Centrifuge 13000 g (1337 g) for 1 min and reject the supernatant. Add sterile and filtered saline solution and centrifuge again. Resuspend with the described broth media or buffer solutions and proceed according to IFU for AST by flow cytometry.

[0060] In an embodiment, pure cultures obtained from colonies were analysed as follows.

[0061] In an embodiment, perform a Gram stain or mass spectrometry analysis (MALDI-TOF MS) in order to guide the choice of antibiotics to be tested. This previous step will provide information in terms of the presence of bacteria (cocci or bacilli; Gram-positive or Gram- negative) and fungi in culture.

[0062] In an embodiment, a. In case of a culture of bacilli or cocci: From a 24 hour culture pick one or two colonies and prepare a culture in an Erlenmeyer with at least 10ml or 20 ml of brain heart infusion or Mueller Hinton broth or Cation adjusted Mueller Hinton or Todd Hewitt broth or HTM broth or Columbia broth, Tioglycolate broth or buffer solution such as PBS, HEPES or MOPS at pH 6-8 (these broths may be supplemented with 0%, 0.2%, 0.5%, 0.9%, 1%, 2%, 4%, 5%, 8% or 10% of one or more additives such as dextrose and/or caseine and/or vitamin K and/or hemine and/or bovine calf serum and/or yeast extract and/or NaCI and/or calcium and/or magnesium and/or lysed horse blood and/or choline chloride whenever necessary) and incubate for 2, 3 or 4 or 5 hours at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or capnophilic (2.5% to 5% C02) or anaerobic atmosphere, until exponential growth phase. Dilute the culture in a broth culture medium like brain heart infusion, or buffer solution such as PBS, HEPES or MOPS at pH 6-8 (these broths may be supplemented with 0%, 0.2%, 0.5%, 0.9%, 1%, 2%, 4%, 5%, 8% or 10% of one or more additives such as dextrose and/or caseine and/or vitamin K and/or hemine and/or bovine calf serum and/or yeast extract and/or NaCI and/or calcium and/or magnesium and/or lysed horse blood and/or choline chloride whenever necessary), in order to obtain an optical density (OD600nm) of 0.06 (corresponding approximately to lxlO ⁶ cells/ml). For some drugs such as colistin, a suspension could be made directly from the colonies, avoiding the incubation in broth, with similar results.

[0063] I n an embodiment, b. I n case of a culture of yeasts: From 1 or two colonies of a 24 hour culture make a microbial suspension on a sterile saline solution or broth culture medium such as Sabouraud broth or yeast peptone dextrose broth or a buffer saline such as PBS, HEPES or MOPS at pH 6-8 or water (supplemented with 1% or 2% or 4% of dextrose whenever necessary) and adjust the cell suspension to 0.5 McFarland (or at least 106 cells/ml) in a sterile saline solution or broth culture medium such as Sabouraud broth or yeast peptone dextrose broth or a buffer saline such as PBS, HEPES or MOPS at pH 6-8 or water (supplemented with 1% or 2% or 4% of dextrose whenever necessary). The final cell suspension concentration will yield 1 to 5xl0 ⁶ cells/ml.

[0064] I n an embodiment, the blood cultures and other broth cultures (e.g. peritoneal, articular or pleural fluid) samples were analysed as follows:

[0065] I n an embodiment, i. From positive blood cultures (with blood and other biological fluids), perform a Gram stain or mass spectrometry analysis (MALDI-TOF MS) in order to guide the choice of antibiotics to be tested. This previous step will provide information in terms of the presence of bacteria, in particular cocci or bacilli; Gram-positive or Gram- negative, and fungi in the biological sample.

[0066] I n an embodiment, 2 protocols could be used A and B. Protocol A only use centrifugation steps and could not be able to recover Staphylococcus in grapes or fungi. Protocol B uses a lysing agent and a gradient centrifugation and could be used in all the cases.

[0067] I n an embodiment, if the cell suspension does not reach 0.5 McFarland the process may be repeated, otherwise if the suspension reaches a 10 ⁴ to 10 ⁶ cells/ml the 1:10 and 1:100 are unnecessary.

[0068] I n an embodiment, the antimicrobial susceptibility test could be performed following a maximum of 24 hours after the blood culture turns positive; If the cell suspension does not reach 0.5 McFarland the process must be repeated, otherwise if the suspension reaches a 10 ⁴ to 10 ⁶ cells/ml the 1:10 and 1:100 are unnecessary. [0069] In an embodiment, if biological fluids (e.g. peritoneal or pleural fluid) are collected from patient in a blood culture bottle proceed with steps previously described above.

[0070] In an embodiment, the evaluation of antimicrobial phenotype by flow cytometry using screening cut-off values or breakpoint values was carried out as follows.

[0071] In an embodiment, transfer the same volume of the microbial suspension (prepared as described above) to a set of vials or wells or galleries that can take the form of, e.g., but not limited to, plates (e.g., multi-well plates) or tubes containers with penicillins such as benzylpenicillin and/or ampicillin and/or ampicillin+sulbactam and/or amoxicillin and/or amoxicillin plus clavulate and/or piperacillin and/or piperacillin plus tazobactam and/or ticarcillin and/or ticarcillin plus clavulanate and/or phenoxymethylpenicillin and/or oxacillin and/or cloxacillin and/or dicloxacillin and/or flucloxacillin and/or mecillinam and/or mezlocillin and/or cephalosporin such as cefaclor and/or cefadroxil and/or cefalexin and/or cefamandole and/or cefazolin and/or cefdinir and/or cefepime and/or cefetamet and/or cefixime and/or cefmetazole and/or cefonocid and/or cefoperazone and/or cefotaxime and/or cefotetan and/or cefoxitin and/or cefpodoxime and/or cefprozil and/or ceftaroline and/or ceftazidime and/or ceftibuten and/or ceftizoxime and/or ceftriaxone and/or cefuroxime and/or cephalothin and/or loracarbef and/or moxalactam and/or carbapenems such as doripenem and/or ertapenem and/or imipenem and/or meropenem and/or monobactams such as aztreonam and/or fluoroquinolones such as cinoxacin a nd/or ciprofloxacin and/or enoxacin and/or fleroxacin and/or gatifloxacin and/or gemifloxacin and/or grepafloxacin and/or levofloxacin and/or lomefloxacin and/or moxifloxacin and/or nalidixic acid and/or norfloxacin and/or ofloxacin and/or aminoglycosides such as amikacin and/or gentamicin and/or kanamycin and/or netilmicin and/or streptomycin and/or tobramycin and/or glycopeptides such as teicoplanin and/or telavancin and/or vancomycin and/or macrolides, lincosamides and streptogramins such as azithromycin and/or clarithromycin and/or erythromycin and/or roxithromycin and/or telithromycin and/or clindamycin and/or quinupristin-dalfopristin and/or tetracyclines such as doxycycline and/or minocycline and/or tetracycline and/or tigecycline and/or chloramphenicol and/or colistin and/or daptomycin and/or fosfomycin and/or fusidic acid and/or linezolid and/or metronidazole and/or mupirocin and/or nitrofurantoin and/or polymyxin B and/or spectomycin and/or sulfonamides and/or trimethoprim and/or trimethoprim- sulfamethoxazole and with echinocandins such as anidulafungin and/or caspofungin and/or micafungin and/or azoles such as fluconazole and/or voriconazole and/or itraconazole and/or posaconazole and/or ketoconazole and/or polyene such as amphotericin B and/or pyrimidine analogues such as flucytosine. These antimicrobials may be dehydrated or lyophilized or in solution. The concentrations range from 0 μg/ml (untreated or control) and 0.002 to 256 μg/ml using double dilutions allowing the determination of minimal inhibitory concentrations (MIC) values to all antimicrobials; in alternative breakpoints concentrations according CLSI or EUCAST could be tested or regarding carbapenems screening cut-off values according EUCAST protocol (December 2013) could be used; for ertapenem and meropenem from 0.125 to 8 μg/ml and for imipinem from 1 to 8 μg/ml (see example 1).

[0072] In an embodiment, incubate for at least 30 minutes, at least 1 hour, at least 2 hours, 2.5 hours, at least 3 hours at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or capnophilic (2.5% to 5% C0 ₂ ) or anaerobic atmosphere.

[0073] In an embodiment, the in vitro cells staining may also be carried out simultaneously with steps presented below.

[0074] In an embodiment, add the appropriate fluorochrome such as Calcein AM, Evans blue, Fluorescein diacetate, Propidium iodide staining, Green fluorescent protein, Lactate dehydrogenase, Methyl violet, Propidium iodide, DNA stain, Trypan Blue (a living-cell exclusion dye), cell labeling or staining (e.g., a cell-permeable dye (e.g., Carboxylic Acid Diacetate, Succinimidyl Ester (Carboxy-DFFDA, SE)), a cell-impermeable dye, cyanine, phenantridines, acridines, indolesa nucleic acid stain, a cell permeant reactive tracer (e.g., intracellularly-activated fluorescent dyes CMRA, CMF2HC (4-Chloromethyl-6,8-Difluoro-7- Hydroxycoumarin), CMFDA (5- Chloromethylfluorescein Diacetate), CMTMR (5-(and-6)-(((4- Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine), CMAC (7-Amino-4-

Chloromethylcoumarin), CMHC (4-Chloromethyl-7-Hydroxycoumarin) or any combinations thereof), fluorescent DNA dyes (e.g., DAPI, Hoechst family, SYBR family, SYTO family, SYTOX family, ethidium bromide, propidium iodide, acridines, or any combinations thereof); cytoplasma stain (e.g., calcofluor white, periodic acid-schiff stain, or any combinations thereof); metabolic stains (e.g., any diacetate dye (including, rhodamine based-dye, fluorescin, or any combinations thereof), resazurin/resorufin (alamar blue); ROS stains (e.g., DCFDA and related family, calcein-acetoxymethyl and related family); membrane stains (e.g., bodipy, FM 1-43, FM 4-64, and functionally equivalent thereof, CellMask™ stains, Dil, DiO, DiA); biologic stains (e.g., labeled antibodies, labeled chitin-binding protein), viable dyes such as propidium iodide, DHR, FUN-1, Dioc 2(3), Dibac 3(4) or any combinations thereof. The final concentration may range from 0.125 μΜ to 150 μΜ. One aliquot of the cells suspension will not be stained (negative or autofluorescence).

[0075] In an embodiment, incubate for at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or capnophilic (2.5% to 5% C0 ₂ ) or anaerobic atmosphere, in the dark. If the staining is simultaneous with the antimicrobial treatment, the incubation time is according the incubation time of the treatment.

[0076] In an embodiment, flow cytometry cell acquisition was carried out as follows. Sonicate the cells for about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes, if necessary.

[0077] In an embodiment, acquire cells in suspension in a standard flow cytometer with at least three PMTs equipped with standard filters, FL1: BP 530/30 nm; FL2: BP 585/42 nm; FL3: LP 670, and at least a 15 mW 488 nm Argon laser. The number of acquired events may round from 10000 to 50000 events. The flow cytometer use may be equipped with up to 3 lasers and/or up to 8 colours. Acquisition settings are defined using carboxylated highly green fluorescent polystyrene microsphere samples by adjusting voltage to the third logarithmic (log) decade of all fluorescence channels. Forward scatter (FSC) or side scatters (SSC) are used to trigger the signal. Globally, threshold parameters, compensations, detectors voltages, logarithmic or linear scale acquisition are defined according the dye used and/ or the type of microorganism, in order to optimize the sensitivity and reduce the background noise of the equipment.

[0078] In an embodiment, results of the acquisition of a gated or no gated population are obtained in a dot plot SSC versus FSC and/or SSC or FSC versus fluorescence channel (for e.g., FL1, FL2, FL3, FL4...) and/or in a histogram representing the number of events versus fluorescence.

[0079] In an embodiment, flow cytometry analysis and result interpretation was carried out as follows. Results may be expressed in percentage of stained cells and/or the distribution pattern and/or mean of the intensity of fluorescence and/or median of the intensity of fluorescence.

[0080] In an embodiment, for each tested antimicrobial concentration, a staining index (SI) may be calculated as the ratio between the percentage of stained cells or mean or median of intensity of fluorescence of antimicrobial treated cells and the percentage of stained cells or mean or median of intensity of fluorescence of untreated (control).

[0081] In an embodiment, cut-off values are determined according the staining index at each antimicrobial breakpoint concentration.

[0082] In an embodiment, in case of resistance phenotype or susceptibility only above screening cut-off values: facing a resistant phenotype or a susceptible phenotype only above screening cut-off values, the underlined mechanism of resistance should be investigated. In order to clarify the mechanism, several procedures should take place according to the type of microorganism.

[0083] One of the most important mechanisms of antimicrobial resistance is the production of enzymes with limited or broad-spectrum activity that inactivate the different antimicrobials mainly β-lactamics or azoles.

[0084] The present disclosure further relates to a screening test for β-lactamics, a test designed based on flow cytometry to evaluate the amount of active drug after incubating the test strain with a certain antimicrobial. If the strain is producing hydrolytic enzymes, the amount of the drug will decrease.

[0085] On the other hand, if the concentration of the drug after incubation with the test strain does not decrease and the antimicrobial effect is similar or greater when compared with what is expected, efflux pumps or cell membrane impermeability should be present.

[0086] The amount of drug will be evaluated incubating it with a type strain and measuring by flow cytometry its effect (see example 2).

[0087] In an embodiment, the procedure is the following: i. Incubate the different antimicrobials at different concentrations as previously described in 3.1 with the strain to be tested during 1 hour at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or anaerobic atmosphere; ii. Centrifugate at 12000 to 14000 rpm for at least 5 minutes or filtrate the vial and incubate the supernatant with a type strain, the most adequate, for example an ATCC strain (e.g. E. coli ATCC 25922, K. pneumonia ATCC 25955, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus 29213) with a known behavior when incubated with the tested drug; iii. Stain the cells with the adequate probe as described above to study the drug effect and analyze by flow cytometry as described in above. iv. Compare the effect of the supernatant with the expected effect. If the effect is inferior, this means that the amount of drug decreased, most probably was hydrolyzed, so we are facing an enzymatic mechanism of resistance; to clarify the enzymatic mechanism continue with above. If the effect corresponds to a higher concentration, we are facing an efflux or cell mem brane impermeability mechanism of resistance; to clarify if it is efflux or impermeability proceeds to above.

[0088] Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to β-lactam antibiotics like penicillins, cephamycins, and carbapenems.

[0089] Detection for Enterobacterales: Enterobacteriaceae group I was described on document WO2010/164547; regarding Enterobacteriacea group II (Enterobacter spp, Citrobacter freundii, Providencia stuartii, Serratia spp, and Hafnia alvei), Morganella morganii and Yersinia spp use cefepime with and without clavulanic acid.

[0090] I n an embodiment, the vast majority of carbapenemases are acquired enzymes, encoded by genes on transposable elements located on plasmids. Carbapenemases confer decreased susceptibility to carbapenems; majority is also resistant to cephalosporins however with some of these enzymes (OXA-48 like enzymes) the organism could be fully susceptible to cephalosporins.

[0091] I n an embodiment, minimal inhibitory concentration (MIC) values of meropenem or ertapenem superior to 0.125 mg/L or imipenem superior to lmg/L or >25 mm increase of ertapenem or meropenem and >23 mm in case of imipenem if disk diffusion method is used, in all Enterobacteriaceae should be submitted to carbapenemase detection. [0092] In an embodiment, the procedure for carbapenemases characterization (example 3) may be the following:

Associate different inhibitors to meropenem during 1 hour at ambient temperature or 35 °C ± 2 °C at static and/or 150 rpm and/or 180 rpm, in an aerobic or anaerobic atmosphere and repeat flow cytometry protocol previously described; compare the effect with and without inhibitors. The concentration of the inhibitors such as aminophenyl boronic acid (APBA) or phenyl boronic acid (PBA) inhibits class A carbapenemases or Klebsiella pneumonia carbapenemase (KPC), and dipicolinic acid (DPA) or ethylenediaminetetraacetic acid (EDTA) inhibits class B carbapenemases or metallo-beta-lactamases may range between 1.25 mM to 10 mM. There is no available inhibitor for class D carbapenemases. Also combinations such as APBA/PBA and/or EDTA/DPA and/or APBA/PBA+EDTA/DPA must be tested. Cloxacilin synergy, at concentrations between 125 to 1000 mg/l, should also be tested to distinguish from AmpC hyperproduction plus porin loss. If no synergic effect was found an ESBL plus porin loss or OXA-48 should be present. Susceptibility for temocillin should be also tested by flow cytometry and MIC values superior to 32 mg/L means OXA-48-like.

[0093] In an embodiment, AmpC-type cephalosporinases are Amber class C b-lactamases. They hydrolyse penicilins, cephalosporins (including the third-generation but not the fourth- generation compounds) and monobactams. In general, AmpC-type enzymes are poorly inhibited by the classic ESBL inhibitors, especially clavulanic acid. A cefoxitin MIC superior to 8 mg/L combined with a ceftazidime and/or cefotaxime MIC superior to 1 mg/L may be used as phenotypic criteria for investigation of AmpC production in group 1 Enterobacteriaceae.

[0094] In an embodiment, the procedure Acquire AmpC β-lactamase (example 4) may be the following: Associate cefotaxime and/or ceftazidime and/or cefoxitin with cloxacilin (125 to 1000 mg/l), or clavulanic acid or both, and perform the flow cytometric test for susceptibility. If a synergic effect between cloxacilin and cephalosporin is observed or between (cloxacilin + clavulanic) + cephalosporin, an AmpC should be present; if cloxacillin synergy is not detected, other mechanism e.g. porin loss should be present.

[0095] In an embodiment for the efflux or cell membrane impermeability detection may follow the next procedure (example 5). Note: efflux blockers (also valid for other drugs e.g. quinolones). [0096] I n an embodiment, the procedure is the following: determine the MIC to several antimicrobial (as previously described in before) by flow cytometry with and without different efflux blockers e.g. CCCP (1.25, 2.5 and 5mg/L), chlopromazide (20, 40 and 80 mg/L) or phenyl-arginine-beta-naphthylamide (50 to 200 mg/L) or l-(l-naphthylmethyl)- piperazine (15 to 150 mg/L). The presence of synergism (meaning lower MIC) indicates the presence of efflux; if no synergic effect is found, resistance is probably due to impermeabilization/efflux (not inhibited by those efflux blocker).

[0097] An embodiment of MRSA detection, Methicillin resistant S. aureus (MRSA) is a major cause of morbility and mortality. MRSA infections are endemic in both hospitals and the community in all parts of the world. It is due to the presence of a penicillin-binding protein (PBP2a encoded by gene mecA or the recently discovered PBP2 encoded by mecC). β-lactam agents, except the new class of cephalosporin's having anti-MRSA activity, have low activity.

[0098] I n an embodiment, susceptibility test to oxacillin and cefoxitin by flow cytometry should be performed; briefly breakpoint concentrations of both drugs should be incubated with Staphylococcus aureus suspension for 1 hour with the adequate fluorochrome for oxacillin and cefoxitin respectively. Resistance to one of the drugs should be considered as a MRSA.

[0099] An embodiment of glycopeptides resistance determination, regarding S. aureus high level of resistance to glycopeptides is VanA-mediated and non-VanA mediated low-level resistance isolates. These non-VanA mediated low-level resistance isolates could be glycopeptides intermediate S. aureus (GISA) and heteroresistant glycopeptides S. aureus (hGISA). The MIC values should always be determined when using vancomycin to treat a patient with severe S. aureus infection. In selected cases, e.g. when therapeutic failure is suspected, testing for hGISA may also be warranted.

[00100] I n an embodiment, the procedure is the following: Perform MIC determination of vancomycin and teicoplanin using flow cytometry protocol; briefly incubate 1 hour with different concentrations of the drug and the adequate fluorescent probe and if MI C value >8 mg/L consider GRSA, if MIC 4-8 mg/L consider GISA. If MIC <4 mg/L, incubate the cells with vancomycin, 4 mg/L for 4 hours, and then re-determine the MIC value by flow cytometry. If MIC value turns>=8 mg/L consider hGISA. [00101] In an embodiment, regarding Enterococcus spp are considered resistant to vancomycin (VRE) when MIC>4 mg/L. Clinically-relevant resistance is often mediated by plasmid-encoded VanA (resistance to both vancomycin and teicoplanin) and VanB ligases (usually remain susceptible to teicoplanin that replace the terminal D-Ala in the peptidoglycan with D-Lac. This substitution reduces the binding of glycopeptides to the target. Other Van enzymes of lower prevalence are VanD, VanE, VanG, VanL, VanM and VanN.

[00102] In an embodiment the method for the determination of glycopeptides resistance is the following: i. MIC determination incubating the strain with vancomycin and the adequate fluorescent probe for 1 hour and make flow cytometry analysis according. ii. To distinguish from strains VanA and VanB we should determine the MIC not only to vancomycin but also to teicoplanin. MIC values of 64-1024 mg/L of vancomycin and 8- 512 mg/L of teicoplanin should be VanA; MIC values of 1-1024 mg/L of vancomycin and 0.06-1 mg/L of teicoplanin (susceptible) should be VanB.

[00103] In an embodiment, for high level of resistance to aminoglycosides by Enterococcus perform a flow cytometric analysis incubating the testing strain with 128 and 512 mg/L of gentamycin or streptomycin for 1 hour with DIBAC. If susceptible consider synergic to b- lactamics.

[00104] An embodiment for the determination of Penicillin resistance on non-susceptible Streptococcus pneumonia, S. pneumoniae isolates with reduced susceptibility to penicillin (MIC >0.06) have PBPs with lower affinity for b-lactams. Strains with MIC of benzylpenicillin in the range 0.12 to 2 mg/L are considered susceptible in non-meningitis infections when a higher dose of penicillin is used, whereas for meningitis such strains must always be reported as resistant. Recommended test. Determination of MIC of benzylpenicillin is essential by microdilution or gradient test taking at least 24 h.

[00105] In an embodiment, oxacillin susceptibility by flow cytometry could be used as a screening method for the detection of penicillin non-susceptible pneumococci as well as to predict susceptibility to other b-lactams. The method is very sensitive but not highly specific. When susceptible to oxacillin it means that it is susceptible to all b-lactams (except for cefaclor which if reported should be reported intermediate. When it is resistant to oxacillin perform MIC determination to penicillin, cetotaxime and cetriaxone by microdilution or gradient test.

[00106] In an embodiment, the susceptibility to oxacillin by flow cytometry could also be used as a screening method for b-lactamics, incubating the cells for 2 h with breakpoint values of oxacillin and adequate probe; if susceptible infers susceptibility to b-lactams and whether is resistant evaluate the MIC values to penicillin, cefotaxime and ceftriaxone.

[00107] Another embodiment of the method of the present disclosure is the determination of MIC to penicillin, cefotaxime and ceftriaxone incubating the microbial cells in early exponential growth phase in Todd broth and adequate fluorescent probe for 2 hours and perform flow cytometric analysis.

[00108] In an embodiment, the determination from a positive blood culture perform a Gram staining and/or MALDI-TOF may have the following steps, please see table 3/4 Protocol B:

• Shake vigorously the blood culture (BC) 5 times and fill a vacuum blood collection gel tube (tube A) with about 5.5 ml. NOTE 1: If the filling was not complete repeat all the process.

• Extract 1 ml of the BC to an Eppendorf tube (A)

• Add 50 ul of Triton X 10% (or other non-toxic hemolytic agent)- vortex and wait 5 min at room temperature

• Add this 1 ml to the top of a 1 ml Hystopaque (B)

• Centrifuge 13000 rpm (11337 rpm)/lmin

• Reject the supernadant

• Add 1 ml sterile and filtered saline solution

• Reject the supernadant

• Add 1 ml sterile and filtered Muller-Hinton cation adjusted medium

• Adjust the suspension to 0.5 macFarland (around 5 ml) • NOTE 2: Save the remaining suspension of tube B in the fridge.

[00109] In alternative a simplest protocol could be used on Anaerobic lytic Blood cultures (BD) or in all the BC bottles except for Staphylococcus spp and yeasts (see table3/4 protocol A)

• Shake vigorously the blood culture 5 times and fill a vacuum blood collection gel tube (tube A) with about 5.5 ml. NOTE 1: If the filling was not complete repeat all the process.

• Invert the tube A 5 times (one complete turn of the wrist, 180 degrees, and back). Centrifuge the tube A at 1,500 x g during 5 min.

• Reject the supernadant and add 1 ml of sterile and filtered (0.22 micrometers) saline solution; transfer 1 ml to an Eppendorf tube (B).

• Centrifuge the tube B at 13,000 x rpm (11337 g) during 1 min and reject the supernatant.

• Add 1 ml of sterile and filtered (0.22 micrometers) Brain Heart broth to tube B and gently invert the tube to homogenize the solution.

• NOTE 2: Save the remaining suspension of tube B in the fridge.

• If polimicrobial population is observed, make subculture and take the FAST protocol for pure colonies

In an embodiment, the determination from selective or non-selective culture media perform a Gram staining and/or MALDI-TOF may have the following steps:

• With a swab take 2 isolated colonies from the culture media and inoculate in 20 mL of filtered Brain-Heart broth (tube A). Vortex for 3 seconds.

• Incubate at 37°C, in an orbital shaker (aprox. 550 rpm), during 1 to 2 hours (until evident growth).

• Centrifuge 8,000 x g during 10 min. Reject the supernatant. Add sterile saline solution until 0.5 McFarland. • Take 0.5 mL of that suspension and add 4.5 mL of filtered Brain Heart broth (tube B).

[00110] In an embodiment, the panel rehydration/ inoculation is the following:

• Transfer the bacteria suspension to the microplate kit according IFU;

• Incubate at 37°C, with agitation, for 15 to 60 min in the dark;

• Acquire on the flow cytometer using instrument set-up and analysis protocol below.

[00111] In an embodiment, the flow cytometry analysis should be performed within 3 hours with the AST (antimicrobial susceptibility test) panel always protected from light.

• Take the 0.5 mL of that suspension and add to 4.5 mL of filtered Brain Heart broth (tube C).

• If polymicrobial population is observed, make subculture

• Panel rehydration/ inoculation;

• Transfer 100 μί from the bacteria suspension to wells Al to C9 of the microplate;

• Incubate at 37°C, with agitation, for lh in the dark;

• Acquire on the flow cytometer using instrument set-up and analysis in the cytometer.

[00112] In an embodiment, the flow cytometry analysis should be performed within 3 hours with the AST panel always protected from light.

[00117] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[00118] It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

[00119] Flow diagrams of particular embodiments of the presently disclosed methods are depicted in figures. The flow diagrams do not depict any particular means, rather the flow diagrams illustrate the functional information one of ordinary skill in the art requires to perform said methods required in accordance with the present disclosure.

[00120] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[00121] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[00122] The above described embodiments are combinable.

[00123] The following claims further set out particular embodiments of the disclosure.