Method, device and kit for the phenotypical measurement of the effectiveness of antibiotics against microorganisms in a biological sample material

Abstract

The present invention relates to a method, a device for carrying out the method and a kit for quickly determining antibiotic sensitivity and antibiotic resistance by directly measuring bacterial growth in bodily fluids. The present invention relates to the field of health, microbiology, diagnostics and prognosis, and of therapy guidance by evaluating the effectiveness of antibiotics and antibiotic combinations in human and veterinary medicine. The described embodiments relate to determining bacterial growth in samples of bodily fluids ex vivo in the presence of antibiotics added to the samples. The bacterial growth is determined in a device which enables the effectiveness of antibiotics to be immediately monitored in a kit after addition of an aliquot of a bodily fluid in that electric resistance, current flow, impedance or optical measurements are recorded using (fluorescent) dyes, the data are compared with reference values and the data are evaluated by way of programmed calculations and adapted mathematical methods.

Description

[0001] Method, device and kit for the phenotypic measurement of the efficacy of antibiotics against microorganisms in a biological sample material

[0002] Description

[0003] The present invention relates to a method for the phenotypic measurement of the effectiveness of antibiotics against microorganisms in a biological sample material according to claim 1, a device for carrying out the method according to the invention according to claim 18 and a kit according to claim 28.

[0004] State of the art:

[0005] Currently, phenotypic antibiotic efficacy tests are performed in the clinical setting in the presence of resistance following a blood culture to increase the number of pathogens present [1 ,2],

[0006] The automated systems used for this purpose, which are often based on turbidity measurements, require 48 hours or more, including blood culture, to obtain the results.

[0007] This also applies, for example, to the commercially available automated systems Vitek2® from bioMerieux, Marcy-l'Etoile, France and BD Phoenix™ from Becton Dickinson, Winnersh, UK, which appear to be clinically equivalent [(1 ), (2)].

[0008] The Vitek2® system is a broth microdilution method that, unlike traditional agar diffusion, determines the minimum inhibitory concentration (MIC) of each antibiotic and identifies potential resistance mechanisms for each antibiotic family for each pathogen. Furthermore, the system offers the possibility of identifying unknown pathogens.

[0009] The Vitek2® system is an automated, computer-assisted procedure in which (as with agar diffusion) the pathogen cultured during routine bacteriological testing serves as the starting material. The isolated pathogen is homogenized in a NaCl solution to produce a standardized bacterial solution. Sample data is recorded electronically, and the corresponding antibiotic test card is assigned.

[0010] The BD Phoenix™ System for Automated Identification and Susceptibility Testing provides detection of known and emerging antimicrobial resistances. Automated nephelometry is used as the detection system. Furthermore, data management monitors and analyzes the results and transmits them directly to laboratories and clinicians.

[0011] It is also known from the scientific literature that bacterial growth or the effect of antibiotics can be determined more quickly by the absence of bacterial growth, for example by means of fluorescence [(3), (4)], microscopic evaluation [(5)], or electrochemical measurement methods such as differential pulse voltametry (DPV) [(6), (7)] or electrochemical impedance spectroscopy (EIS) [(8), (9)].

[0012] For example, Azizi et al. (2018) reveal in (3) that: Increasing resistance to antimicrobials presents physicians in clinical practice with the challenge of prescribing antibiotics that are effective against bacterial infections.

[0013] Conventional antibiotic susceptibility tests (AST) are labor-intensive and time-consuming. Emerging technologies such as microfluidics could enable faster AST testing times, according to (3). The authors use a specially designed nanoliter-sized microchamber / microarray-based microfluidic platform to shorten AST testing times, allowing the relatively rapid determination of minimum inhibitory concentrations of various antibiotics. Bacterial suspensions, with or without antibiotics, are filled into small, nanoliter-sized chambers, and the change in fluorescence intensity resulting from resazurin reduction, which correlates with bacterial growth, is measured. Document (3) demonstrates the reproducibility, functionality, and efficiency of the chosen platform for numerous clinical wild-type bacterial isolates, including Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis.The time to the final test result varies between ~1- 3 h, depending on the growth rates of the different bacterial species, but following cultivation of the pure culture in medium overnight (at least 12 h).

[0014] Kim et al. (2019) describe on-chip methods in their review in (4), with most microfluidic studies using specialized devices, assays, or original techniques to determine bacterial growth rates on the chip. They provide an overview of chip-based measurements and classify them into four main categories: fluorescence imaging, metabolic activity indicators, label-free optical imaging, and rotational measurement with magnetic beads. According to (4), the aforementioned categories are described as advantageous over conventional methods, along with their general properties and examples of on-chip studies.

[0015] Baltekin et al. (2017) describe in (5) an AST test system using direct single-cell imaging, which provides the test result in less than 30 minutes. In the method according to (5), bacterial cells are extracted directly from samples with a low bacterial count (10 4 CFU / mL) were captured using a specially designed microfluidic chip and their individual growth rates were monitored using microscopy.

[0016] Nemr et al. (2019) describe in (6) a method for detecting methicillin-resistant Staphylococcus aureus (MRSA) directly in patient nasal swabs without prior culture and with minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. The bacterium is captured based on antibody recognition of a membrane-bound protein marker that confers β-lactam antibiotic resistance. MRSA is then identified using a strain-specific antibody functionalized with alkaline phosphatase, through electrochemical detection via potential change of a p-aminophenol / quinone imine redox system after p-aminophenyl phosphate has been converted by alkaline phosphatase into the electrochemically active p-aminophenol.

[0017] Crane et al. (2021) describe in (7) the detection of bacterial growth in an AST test system using graphene-based screen-printed electrodes modified with resazurin and measured by differential pulse voltammetry (DPV). The screen-printed electrodes were used as part of a surface-modified resazurin-based detection method, detecting a change in the potentials of a resazurin / resorufin - dehydroresorufin redox system.

[0018] Resazurin can be reduced intracellularly by metabolically active bacteria or electrochemically at the surface of the working electrode, allowing the determination of antibiotic-sensitive and antibiotic-resistant bacteria.

[0019] Hannah et al. (2020) disclose in (8) a diagnostic sensor test to quickly determine the correct antibiotic for treating an infection. The sensor consists of a screen-printed gold electrode coated with an antibiotic-infused hydrogel to monitor bacterial growth. Electrochemical growth profiles of Escherichia coli (E. coli) (ATCC 25922) were measured in the presence and absence of the antibiotic streptomycin. The results show a clear difference between the E. coli growth profiles depending on the presence of streptomycin, with a time frame of 2.5 hours significantly faster than the current gold standard of culture-based ASTs. However, Hannah et al. (2020) also used an overnight culture before performing the measurement. The tests were evaluated using electrochemical impedance spectroscopy (EIS).

[0020] A similar test system is described by Hannah et al. (2019) in (12).

[0021] Spencer et al. (9) describe an AST that can also provide results within one hour, using an actively dividing bacterial culture as the starting material. The bacteria are incubated for 30 minutes in the presence of an antibiotic, and then approximately 10 5 Cells were analyzed individually using microfluidic impedance cytometry for 2–3 minutes. The measured electrical properties reflect the phenotypic response of the bacteria to the action of a specific antibiotic within a 30-minute incubation window. The results are consistent with those obtained with classical broth microdilution assays [(10)] for a range of antibiotics and bacterial species.

[0022] Furthermore, WO2022178142A1 describes methods and systems using genetic identification of polymicrobial samples, e.g., polymicrobial infections, as well as methods for determining the suitability of one or more compositions for inhibiting the growth of a polymicrobial sample, e.g., providing information regarding the probability of success in inhibiting the growth of the polymicrobial sample with one or more compositions. Document WO2022178142A1 also describes methods for providing information to a user regarding the polymicrobial sample, such as, but not limited to, information regarding the suitability of the compositions for inhibiting the growth of a polymicrobial sample.

[0023] Non-limiting examples of organisms that can be tested for according to WO2022178142A1 and / or that are present in the polymicrobial infection include: one or a combination of: Acinetobacter baumannii, Actinotignum schaalii, Aerococcus urinae, Aerococcus urinae, Alloscardovia omnicolens, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Chlamydia, Citrobacter freundii, Citrobacter koseri, Clostridium difficile, Corynebacterium riegelii, Klebsiella aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Pantoea agglomerans, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, Streptococcus pyogenes, Viridans group streptococci, Trichomonas vaginalis,Ureaplasma urealyticum, HHV-6, HHV-7, BK virus, JC virus, HSV 1&2, adenovirus, or CMV. Given the state of the art described above, the object of the present invention is to provide an improved test system for an antibiotic susceptibility test, particularly for the diagnosis of polymicrobial infections.

[0024] In order to significantly accelerate diagnosis time and administer a proven effective antibiotic to the patient as quickly as possible, it is necessary to avoid separate blood cultures and combine both steps (cultivation and testing) in a single procedure. This results in the need for a suitable test that can be performed directly on the patient sample (particularly in the case of blood plasma, which is a correspondingly viscous liquid) or a partial dilution of the same. This allows a statement on the effectiveness of the antibiotic to be obtained within the first 8 to 24 hours, at a bacterial concentration in the sample material of 1000 CFU / ml [(10), (11)] or less, which is ideally confirmed using redundant measurement methods.

[0025] Therefore, it is necessary within the scope of the present invention to have an interference-resistant and error-minimizing test setup in which a statement about all potentially present pathogens - aerobic as well as anaerobic - is possible without prior knowledge about the identity of the pathogen (untargeted).

[0026] The process-technical solution to the above problem is achieved by the characterizing features of claim 1. The device-technical solution is achieved according to the characterizing features of claim 18.

[0027] A kit according to claim 28 also solves the problem.

[0028] • The methods described here include: (1) detection of organisms (e.g., bacteria or other infectious agents) of the infection or polymicrobial infection, (2) antibiotic susceptibility testing to determine the sensitivity or resistance of the pathogens in the sample to an antibiotic, another therapeutic agent, or a combination thereof. (3) Together, the data from (1) and (2) can be applied to provide one or more therapeutic solutions for the present infection.

[0029] • Direct incubation in a microfluidic system, preferably direct introduction of the taken biological sample without additional buffer or medium - sample is passed into chambers with pre-filled antibiotics - with parallel measurement of DPV and EIS for redundant results (DPV-metabolic / EIS-cell integrity) and meaningfulness.

[0030] • Polymer selection of the chip according to aerobic or anaerobic cultivation conditions, or gas exchange via membrane to chamber

[0031] • Combinatorial analysis approach using change point detection, for example, using PELT (Pruned Exact Linear Time; or a comparable algorithm) in combination with significance testing compared to control samples. This specifically checks whether detected changes in the curves differ from the controls.

[0032] Further possible evaluation approaches:

[0033] Random forest

[0034] Dynamic Time Warping

[0035] Machine learning model

[0036] Principal Component Analysis (PCA)

[0037] Benefits achieved:

[0038] • Direct cultivation and measurement in one process by implementing measurement chambers with controllable incubation properties.

[0039] The method may further comprise subjecting a portion of the sample to a pooled phenotypic antibiotic resistance test, wherein the phenotypic antibiotic resistance test identifies either one or more therapeutic agents to which the polymicrobial infection is resistant and / or one or more therapeutic agents to which the polymicrobial infection is susceptible. The one or more organisms of the polymicrobial infection in the sample are not isolated prior to the phenotypic antibiotic resistance testing. The method may further comprise applying the results of genetic identification tests, genetic resistance marker tests, and the pooled antibiotic susceptibility tests to a database, e.g., to predetermined thresholds of a database. The analysis identifies one or more therapeutic agents effective for treating the polymicrobial infection (e.g.,a "therapeutic solution"). The method may further comprise administering to the patient at least one identified therapeutic agent, wherein at least one therapeutic agent is effective for treating the polymicrobial infection.

[0040] • Implementation of microfluidic distribution structure to reduce the error rate and the labor required to load the test cassette while stabilizing the test conditions.

[0041] • Scalability of the number of chambers for parallel testing of several antibiotics or antibiotic-inhibitor combinations or concentration series thereof.

[0042] • Largely pathogen-independent

[0043] • In some embodiments, the organisms are bacteria.

[0044] However, the present invention is not limited to bacterial infectious agents and may also include viruses, fungi, protozoa, bacteria, or a combination thereof.

[0045] • To combine the advantages of phenotypic characterization with the time savings of eliminating the need for blood culture, an approach based on electrochemical detection was used, for example, but not limited to, impedance spectroscopy (EIS) and differential pulsed voltametry (DPV).

[0046] • Easy filling of the test cassette following simple preparation of the patient sample (for serum, e.g. centrifugation and subsequent dilution)

[0047] • The measured values ​​obtained as a time series are collected and can be evaluated directly in the device using automated algorithms (change point detection) or transmitted via suitable interfaces to other devices for evaluation and data backup. The evaluation using the algorithm can be performed online (parallel to the test run with continuously updated data) or offline (after the test run).

[0048] • The evaluation is performed by using an algorithm to determine significant changes in the curves of the individual measurement methods and parameters. These can be output individually or combined into a score that provides an overview for the expert. It is also assumed that typical curves of specific pathogens can be explicitly assigned to a pathogen or at least a grouping using an established database, which contributes to pathogen identification.

[0049] • In certain embodiments, the method further comprises creating a report that communicates the data set. In certain embodiments, the report includes one or more charts and / or one or more tables. In certain embodiments, the method further comprises providing the report to a healthcare professional, wherein the report includes recommendations for treating the patient.

[0050] Further development of the invention:

[0051] • Polymicrobial infections (WO2022178142A1 ; 0022)

[0052] • Combination of different antibiotics (WO2022178142A1 ; 0023, 0053 or 0054)

[0053] • Any feature or combination of features described herein falls within the scope of the present invention, provided that the features included in such a combination are not inconsistent with each other, as is apparent from the context, this description, and the knowledge of one skilled in the art. Additional advantages and aspects of the present invention are apparent from the following detailed description and claims. (WO2022178142A1; 0029)

[0054] • In certain embodiments, the test for phenotypic antibiotic resistance testing comprises, for example, but not exclusively, the determination of the concentration of a redox-active substance which correlates with pathogen growth and can be determined via differential pulse voltammetry, cyclic voltammetry or suitable conventional electrochemical measuring methods, fluorescence, chemiluminescence, absorbance measurement or a combination thereof, or the measurement of the change in electrical properties of the suspension including the interface of the electrode and the pathogens and antibiotics, in particular, but not exclusively, the capacitance, impedance, conductivity or the phase angle by means of electrochemical impedance spectroscopy.

[0055] • In certain embodiments, phenotypic antibiotic resistance testing comprises placing fractions of the sample, prior to performing the test, in one or more media optimized for growth conditions of specific pathogens or growth of a broad spectrum of pathogens.

[0056] • The steps described herein may be performed in any order or simultaneously.

[0057] Description of one or more embodiments:

[0058] • Those skilled in the art will understand that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The features of the illustrated embodiments may be combined in any combination or arrangement. The drawings are not necessarily to scale; in some cases, various aspects of the inventive subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate understanding of various features. In the drawings, like reference numerals generally refer to like features (e.g., functionally similar and / or structurally similar elements).

[0059] • Figure 1 shows a schematic process of the phenotypic antibiotic resistance testing with the test cassettes shown in the following figures.

[0060] • Figure 2 shows a possible embodiment of a test cassette with a single anaerobic measuring chamber.

[0061] • Figure 3 shows an exemplary embodiment of a test cassette with a single aerobic measuring chamber with headspace.

[0062] • Figure 4 shows a possible schematic test sequence.

[0063] • Figure 5 shows a possible embodiment of a test cassette with several measuring chambers without an outlet.

[0064] • Figure 6 shows a possible embodiment of a test cassette with several measuring chambers, which has outputs with devices for corresponding sterile filters or gas-permeable membranes.

[0065] • Figure 7 shows a possible embodiment of a test cassette with several measuring chambers in which gas exchange with the environment takes place through a gas-permeable membrane.

[0066] • Figure 8 shows a possible embodiment of a test cassette with multiple measuring chambers, in which gas exchange with a gas takes place in the present head region, analogous to Figure 3. • Figure 9 shows a possible embodiment of a test cassette with multiple measuring chambers arranged one above the other.

[0067] • Figure 10 shows a possible embodiment of a test cassette with several measuring chambers, corresponding to Figure 9, in the side view.

[0068] • Figure 11 shows exemplary results of fluorescence detection of the metabolic activity of various bacterial strains in serum samples in the absence or presence of antibiotics. Row A lists bacterial strains with existing antibiotic resistance, and row B lists strains without antibiotic resistance.

[0069] • Figure 12 shows representative results of the DPV measurement method for determining the metabolic activity of various bacterial strains in serum samples in the absence or presence of antibiotics. The times of detection of significant growth are highlighted by vertical lines indicating "growth detected."

[0070] • Figure 13 shows representative results of the EIS measurement method for determining the growth of various bacterial strains in serum samples in the absence or presence of antibiotics. The times of detection of significant growth are highlighted by vertical lines.

[0071] Figure 14 shows the representative results of the EIS and DPV measurement methods for determining the growth of various bacterial strains in serum samples in the absence or presence of antibiotics. Bacterial growth was determined using a combined approach of all measurement parameters. The times of detection of significant growth are highlighted by vertical lines.

[0072] Detailed figure description and examples

[0073] Figure 2 shows a test cassette with a measuring chamber 2, equipped with a measuring electrode 1, for example, a screen-printed electrode, which can be inserted or applied directly to the material. The active substances to be tested are present in the measuring chamber at the appropriate concentration, so that when the sample is added via the inlet 4, which enables sterile addition and subsequent sterility, and distributed via the channel structures 3, the concentration to be tested is predefined depending on the chamber. The inlet 4 can ensure sterility, as shown, through a thread with a corresponding cap, or through a septum or other conventional methods.

[0074] Anaerobic cultivation conditions should be established by selecting an oxygen-impermeable test cassette material, for example, but not exclusively, polymethyl methacrylate, or by appropriate anaerobic ambient conditions in the measuring device chamber.

[0075] Figure 3 shows a test cassette with a measuring cassette similar to Figure 2, characterized in that the measuring chamber has a headspace 5, which, at the appropriate fill level, ensures gas exchange with the sample during the test duration. If the appropriate gas composition is present in the headspace above the sample, aerobic cultivation conditions prevail.

[0076] Figure 4 shows a workflow for loading a multi-chamber test cassette, as described in Figure 5, which can be replaced by other test cassettes in an analogous manner, which, after being sterilely loaded with an appropriately prepared sample, is inserted into the measuring device 6. The cassettes can be inserted only in the specified orientation due to geometric features, for example, but not exclusively, notches or shape, so that the measuring electrodes located thereon are directly connected to the designated contacts of the measuring device 7 and consequently the measuring chambers predefined with active ingredient are assigned to the correct contacts and are identified by the software, for different cassettes, for example, but not exclusively, via barcode, QR code, manual input or electronically and are loaded and assigned according to the existing database.In addition to the necessary measurement electronics and evaluation software 8, which is either installed internally and operated via corresponding output and input components, or externally and connected via suitable interfaces, the measuring device 7 has one or more measuring device chambers that have adjustable parameters, such as, but not limited to, temperature, humidity, and gas composition. These parameters can be stored in the database and, when a labeled cassette is inserted, can be applied automatically according to the listed parameters, or they can be defined manually.

[0077] Figure 5 shows a possible embodiment of a multi-chamber test cassette in a round arrangement without outlets, wherein the arrangement of the measuring chambers 2, distribution channels 3 and measuring electrodes 1 or their contacts or connections is not limited to the arrangement shown, but can be present in any arrangement on the test cassette, characterized in that several measuring chambers are loaded simultaneously and a sample with different active ingredients or combinations of active ingredients and different concentrations of these is tested for effectiveness and compared with corresponding control samples, for example, but not exclusively, without active ingredients, pathogens or preparations, as well as any combinations thereof. For suitable loading of the measuring chambers without air inclusions and to avoid non-wetted measuring electrodes due to these, the illustrated embodiment is prepared without air or with a vacuum.

[0078] Figure 6 shows a possible embodiment of a multi-chamber test cassette with outputs 9 of each measuring chamber to corresponding outlets 10, which are prepared for the attachment of corresponding filters, as shown, or are provided with gas-permeable membranes or filters integrated into the test cassette, which enable pressure and material equalization of the measuring chambers under sterile conditions with the environment of the measuring device chamber, so that gases present in the measuring chambers and distribution channels can escape when the measuring chambers are loaded with sample and the formation of air bubbles is avoided.

[0079] Figure 7 shows a possible embodiment of a multi-chamber test cassette, characterized in that the cassette is sealed 12 with a gas-permeable membrane 11, for example, but not exclusively, by gluing, welding, or with the aid of solvents, so that gas exchange can occur between the measuring chamber and the measuring device chamber. Depending on the set parameters of the measuring device chamber, aerobic or anaerobic cultivation conditions are thereby achieved. It is also possible to seal only part of the test cassette with a membrane, while the remaining part can be present according to other embodiments, so that different conditions can be tested on one test cassette.

[0080] Figure 8 shows a possible embodiment of a multi-chamber test cassette with headspace above each measuring chamber, analogous to that described in Figure 3 for a single-chamber test cassette.

[0081] Figure 9 shows a possible embodiment of a multi-chamber test cassette, with several measuring chambers, measuring electrodes and distribution channels in a "stacked" arrangement, which allows a 3-dimensional arrangement 14 in addition to the 2-dimensional arrangement 13 while maintaining the previously described possibilities and all measuring chambers are still connected to the inlet via the distribution channels for practical loading of the measuring chambers.

[0082] Figure 10 shows the possible embodiment of a multi-chamber test cassette from Figure 9 in the lateral view of a sectional plane with corresponding 3-dimensional arrangement 14 of the measuring chambers 2, measuring electrodes and distribution channel structures 3, which connect them to the inlet 4 for loading with sample.

[0083] Figure 11: Example of a fluorescence measurement corresponding to the results in Figure 11. A bacterial culture was diluted to 250 colony forming units (CFU) / ml in 25% / 75% serum / LB medium with 0.2 mM resazurin, and 200 μl were transferred into a transparent test cassette without an electrode or, for example, a microtiter plate containing 100 pg / ml of the corresponding antibiotic. Samples without bacteria or antibiotic were used as references. The fluorescence measurements were performed in a commercially available fluorescence measuring device, such as a microplate reader, at an extinction wavelength of 535 nm and an emission wavelength of 590 nm.

[0084] Example of an electrochemical measurement according to the results in Figures 12-14. A bacterial culture was diluted to 250 CFU / ml in 25% / 75% serum / LB medium with 0.2 mM resazurin, and 200 μl were transferred into a test cassette containing 100 pg / ml of the corresponding antibiotic per chamber. DPV and EIS measurements were performed using a connected potentiostat under controlled ambient conditions with appropriate measurement protocols and evaluated using a Python script.

[0085] Non-patent literature

[0086] (1) Jin, WY; Jang, S.J.; Lee, M.J.; Park, G.; Kim, M.J.; Kook, JK; Kim, DM; Moon, DS; Park, YJ Evaluation of VITEK 2, MicroScan, and Phoenix for Identification of Clinical Isolates and Reference Strains. Diagnosis Microbiol. Infect. Dis. 2011, 70 (4), 442-447. https: / / doi.Org / 10.1016 / j.diagmicrobio.2011.04.013.

[0087] (2) Mittman, S. A.; Huard, R. C.; Della-Latta, P.; Whittier, S. Comparison of BD Phoenix to Vitek 2, MicroScan MICroSTREP, and Etest for Antimicrobial Susceptibility Testing of Streptococcus Pneumoniae. J. Clin. Microbiol. 2009, 47 (11 ), 3557-3561 . https: / / doi.Org / 10.1128 / JCM.01137-09.

[0088] (3) Azizi, M.; Zaferani, M.; Dogan, B.; Zhang, S.; Simpson, K. W.; Abbaspourrad, A. Nanoliter-Sized Microchamber / Microarray Microfluidic Platform for Antibiotic Susceptibility Testing. Anal. Chem. 2018, 90 (24), 14137-14144. https: / / d0i.0rg / l 0.1021 / acs.analchem.8b03817.

[0089] (4) Kim, S.; Masum, F.; Jeon, J. S. Recent Developments of Chip-Based Phenotypic Antibiotic Susceptibility Testing. BioChip J. 2019, 13 (1 ), 43-52. https: / / doi.Org / 10.1007 / sl 3206-019-3109-7.

[0090] (5) Baltekin, Ö.; Boucharin, A.; Tano, E.; Andersson, D. I.; Elf, J. Antibiotic Susceptibility Testing in Less than 30 Min Using Direct Single-Cell Imaging. Proc. Natl. Acad. Sei. U. S. A. 2017, 114 (34), 9170-9175. https: / / d0i.0rg / l 0.1073 / pnas.1708558114.

[0091] (6) Nemr, C. R.; Smith, S. J.; Liu, W.; Mepham, A. H.; Mohamadi, R. M.; Labib, M.; Kelley, S. O. Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus Aureus. Anal. Chem. 2019, 91 , 2847-2853. https: / / doi.org / 10.1021 / acs.analchem.8b04792.

[0092] (7) Crane, B.; Hughes, J. P.; Rowley Neale, S. J.; Rashid, M.; Linton, P. E.; Banks, C. E.; Shaw, K. J. Rapid Antibiotic Susceptibility Testing Using Resazurin Bulk Modified Screen-Printed Electrochemical Sensing Platforms. Analyst 2021 , 146 (18), 5574-5583. https: / / d0i.0rg / l 0.1039 / d1 an00850a.

[0093] (8) Hannah, S.; Dobrea, A.; Lasserre, P.; Blair, E. O.; Alcorn, D.; Hoskisson, P. A.; Corrigan, D. K. Development of a Rapid, Antimicrobial Susceptibility Test for E. Coli Based on Low-Cost, Screen-Printed Electrodes. Biosensors 2020, 10 (11 ). https: / / doi.Org / 10.3390 / bios10110153.

[0094] (9) Spencer, D. C.; Paton, T. F.; Mulroney, K. T.; Inglis, T. J. J.; Sutton, J. M.; Morgan, H. A Fast Impedance-Based Antimicrobial Susceptibility Test. Nat. Commun. 2020, 11 (1 ). https: / / doi.org / 10.1038 / s41467-020-18902-x.

[0095] (10) Yagupsky, P.; Nolte, F. S. Quantitative Aspects of Septicemia. Clin. Microbiol. Rev. 1990, 3 (3), 269-279.

[0096] (11 ) Boardman, A. K., Campbell, J., Wirz, H., Sharon, A., Sauer-Budge, A. F. Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device. PLoS One 2015,, 10 (2), 1-13. . https: / / doi.org / 10.1371 / journal.pone.0116837.

[0097] (12) Hannah, S.; Addington, E.; Alcorn, D.; Shu, W.; Hoskisson, PA; Corrigan, DK Rapid Antibiotic Susceptibility Testing Using Low-Cost, Commercially Available Screen-Printed Electrodes. Biosense. Bioelectron. 2019, 145 (September), 111696.

[0098] Table 1: Overview of antibiotic susceptibility results by measurement method for treatment without antibiotic (None), with kanamycin (K), or with oxytetracycline (O). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each replicate compared to the agar disk diffusion test result.

[0099]

Claims

Patent claims 1 . In vitro method for the phenotypic measurement of the effectiveness of antibiotics against microorganisms contained in at least one liquid or fluidized biological sample material from a patient, wherein the sample material is optionally prepared beforehand and is transferred into at least three measuring chambers (2) in which the sample material is incubated under desired, defined ambient conditions for a preselected time in the presence or absence of different antibiotics and after the preselected incubation time, the growth or inhibition of the microorganisms to be tested in the presence of the antibiotics is recorded and wherein no prior cultivation of the microorganisms takes place, characterized in thatTo detect the growth of the microorganisms, at least one signal for the change in concentration of a redox-active substance and at least one signal via an electrochemical impedance measurement is generated, and the signals are displayed in time-dependent curves or patterns; and at least partial areas of the curves or patterns are analyzed by means of a computer in comparison with at least one control, and curve or pattern changes are recorded, and the changes are attributed to the growth or inhibition of the microorganisms.

2. Method according to claim 1, characterized in that the sample material is selected from tissue samples and body fluids, the body fluids being selected from: lung fluid, mucus of nasal, oral, tracheal or vaginal origin, puncture, serum, plasma, cerebrospinal fluid (CSF), sputum, saliva, semen, urine, wound secretion.

3. Method according to claim 1 or 2, characterized in that the biological sample material is subjected to a preparation by at least one of the following methods directly before the measurement: centrifugation, dilution with suitable media, filtration, solid phase extraction and shaking.

4. Method according to at least one of the preceding claims, characterized in that the sample material is supplied to the measuring chambers (2) via a distribution microfluidic system (3).

5. Method according to at least one of the preceding claims, characterized in that at least one of the following parameters is selected as the desired ambient conditions: ambient gas, in particular air or inert gas, humidity of the ambient gas, oxygen content of the ambient gas, temperature, pressure of the ambient gas, exclusion of light or exposure.

6. The method according to at least one of the preceding claims, characterized in that the redox-active substance is selected from the group consisting of: resazurin, triphenyltetrazolium chloride, and derivatives thereof; and those which have an oxidative peak in the range 0 to -1 V or a reductive peak in the range 0 to 1 V.

7. Method according to at least one of the preceding claims, characterized in that the signal for the change in concentration of the redox-active substance is obtained from at least one of the following methods: differential pulse voltammetry, cyclic voltammetry, fluorescence measurement, chemiluminescence measurement 8. Method according to at least one of the preceding claims, characterized in that the signal is measured via an electrochemical impedance measurement using different measuring frequencies, in particular 1, 10, 10 3 , 10 5 or 10 6 Hz occurs.

9. Method according to at least one of the preceding claims, characterized in that the microorganism is selected from bacteria, viruses, fungi, protozoa.

10. The method according to claim 9, characterized in that the microorganisms include bacteria which are selected in particular from: Acinetobacter baumannii, Actinotignum schaalii, Aerococcus urinae, Aerococcus urinae, Alloscardovia omnicolens, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Chlamydia, Citrobacter freundii, Citrobacter koseri, Clostridium difficile, Corynebacterium riegelii, Klebsiella aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Pantoea agglomerans, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, Streptococcus pyogenes, viridans group streptococci, Trichomonas vaginalis, and Ureaplasma urealyticum. 11 . Method according to at least one of the preceding claims, characterized in that it is carried out in parallel for several antibiotics and these are presented in concentration series in order to determine the minimum inhibitory concentration (MIC) of the antibiotic, also ex vivo.

12. Method according to at least one of the preceding claims, characterized in that the measured values ​​obtained are collected as a time series and displayed as a curve or pattern and are evaluated directly in a device for carrying out the method according to one of the preceding claims by machine algorithms which are selected from the group consisting of: change point detection, in particular PELT (Pruned Exact Linear Time), Random forrest, Dynamic-Time-Warping, Machine learning model and Principal Component Analysis (PCA).

13. Method according to claim 12, characterized in that the evaluation is carried out by means of an algorithm online, parallel to the measurement with continuously updated data or offline following the measurement.

14. Method according to at least one of the preceding claims, characterized in that AI algorithms, in particular deep learning, are used for automated evaluation.

15. Method according to at least one of the preceding claims, characterized in that a score is obtained from the measurement data obtained, which score provides an overview of the chances of success of using a tested antibiotic.

16. Method according to at least one of the preceding claims, characterized in that the determination of an MIC is carried out for organisms of a polymicrobial infection.

17. Method according to at least one of the preceding claims, characterized in that it provides a result for common clinically relevant microorganisms within the first 5 to a maximum of 24 hours.

18. Device for carrying out the method according to one of claims 1 to 17, wherein a measuring arrangement is provided on a carrier, which has at least one measuring electrode (1) and at least one closed measuring chamber (2) or at least one measuring chamber connected to the environment, as well as at least one sample inlet (4), characterized in that at least one head space (5) connected to the measuring chamber (2) is provided for setting a gas environment.

19. Device according to claim 18, characterized in that the measuring electrode (1) is a screen-printed electrode which is inserted into suitable recesses in the device or is arranged directly on the carrier.

20. Device according to claim 18 or 19, characterized in that channel-shaped distributor structures (3) are provided between the sample inlet (4) and the measuring chamber (2).

21. Device according to at least one of claims 18 to 20, characterized in that the sample inlet (4) has an internal or external thread.

22. Device according to at least one of claims 18 to 21, characterized in that the carrier is rectangular.

23. Device according to at least one of claims 18 to 21, characterized in that the carrier has the shape of a circular disc; and that a plurality of measuring arrangements are provided on the carrier.

24. Device according to claim 23, characterized in that the circular support has a plurality of sample inlets (4) arranged centrally and / or eccentrically, which are in fluid communication with the individual measuring chambers (2) of the measuring arrangements via the distributor structures (3).

25. Device according to at least one of claims 18 to 24, characterized in that the measuring chambers (2) are sealed from the environment by a gas-permeable or a gas-impermeable membrane in order to maintain aerobic or anaerobic conditions within the measuring chambers (2) and to ensure sterility during a test run.

26. Device according to at least one of claims 18 to 25, characterized in that the measuring chambers (2) are coated with a material which enables the growth of microorganisms and which Contains antibiotics, whereby each measuring arrangement contains at least one antibiotic to be tested.

27. Device according to at least one of claims 18 to 26, characterized in that the sample inlets (4) can be sterilely closed off from the environment.

28. Device according to at least one of claims 18 to 27, characterized in that the measuring arrangement is stacked one above the other.

29. Kit for carrying out the method according to one of claims 1 to 17, comprising: at least one device according to one of claims 18 to 28 and a measuring device for the automated detection of a growth of microorganisms; Control organisms and a panel of different antibiotics for checking and / or calibrating the measurement systems; as well as different carriers containing measurement systems loaded with different antibiotics or capable of being loaded from the antibiotic panel, which are tested under aerobic and anaerobic environmental conditions for the phenotypic measurement of the efficacy of antibiotics against microorganisms.

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