Preparation of live microorganism samples and microorganisms for subsequent mass spectrometric measurement and evaluation

Abstract

The present invention relates to a method for preparing a sample of living microorganisms and microorganisms for subsequent mass spectrometric measurement and evaluation. Results that can be derived from such a measurement can be used, in particular, for faster identification of microorganisms in a sample of microorganisms according to species / subspecies and / or for rapid determination of the resistance / susceptibility of microorganisms to antimicrobial substances and / or further characteristics of the microorganisms; for example, in terms of pathogenicity, virulence and metabolism. According to a preferred embodiment form of the invention, the preparation is carried out particularly directly on a mass spectrometric sample carrier.

Description

Technical Field

[0001] This invention relates to a method for preparing live microbial samples and for the subsequent mass spectrometry measurements and evaluation of the microorganisms. The results obtained from such measurements can be used, in particular, to more quickly identify microorganisms in a microbial sample based on species / subspecies and / or to rapidly determine the resistance / susceptibility of microorganisms to antimicrobial substances and / or to further characterize the microorganisms; for example, in terms of pathogenicity, virulence, and metabolic properties. According to a preferred embodiment of the invention, preparation is performed particularly directly on a mass spectrometry sample carrier. Background Technology

[0002] Infectious diseases remain one of the major problems in medicine. Infections can occur independently, but are particularly common as a complication of other diseases or as a result of immunosuppressive therapy and / or the use of foreign bodies in patients. In recent years, advancements in modern medicine, along with the associated increase in complex surgical procedures and immunosuppressive therapies, and the use of foreign bodies, have become factors contributing to increased infection rates. Examples of such factors include solid organ and bone marrow transplants.

[0003] The increasing prevalence of multidrug-resistant pathogens is particularly concerning; these include bacteria (including MRSA-resistant Staphylococcus aureus; VRE-resistant enterococci; and ciprofloxacin, meropenem, or tobramycin-resistant Pseudomonas aeruginosa) or fungi (including fluconazole or voriconazole-resistant Candida albicans). Infections caused by these pathogens are especially difficult to treat with antimicrobial agents. Since the antimicrobials initially administered within the framework of so-called “empirical” or “computational” therapy often do not include multidrug-resistant pathogens in their activity spectrum, early detection of resistance is crucial for treatment success. Rapid identification of resistant microorganisms allows for timely switching to antimicrobials effective against these pathogens. For simplicity, these will be referred to as antibiotics below; the term refers not only to substances effective against bacteria but also to drugs against fungi and other microorganisms. This early switch to the appropriate antimicrobial therapy is essential for treatment success.

[0004] Currently, there is a particular lack of phenotypic (i.e., culture-based) testing systems or individual tests that can provide sensitivity test results in just a few hours. Phenotypic resistance means that a microorganism will grow despite the presence of an antibiotic. Phenotypic antibiotic sensitivity means that this growth is inhibited in the presence of the tested antibiotic if applied at a sufficient concentration. Phenotypic sensitivity tests represent the gold standard; one reason being that the test results are independent of the resistance mechanism on which they are based. While certain resistance genes can be detected in a short time using molecular biology, for example by polymerase chain reaction (PCR), such detection can only be used for certain resistance mechanisms; other resistance mechanisms cannot be detected. Furthermore, such molecular tests only detect known genetically encoded resistance mechanisms. Therefore, unless a specific resistance gene is detected, it is impossible to make a reliable account of a pathogen's sensitivity to antibiotics. In addition, even if a resistance gene is detected, these methods do not always allow for reliable predictions of phenotypic resistance. This is because gene expression can vary; despite the presence of the gene, a microorganism may still be phenotypically sensitive to antibiotics.

[0005] Furthermore, genetic testing is not feasible for many resistance mechanisms. Methods that enable rapid detection of specific resistance mechanisms through phenotype include, for example, the detection of β-lactamases produced by some bacteria. β-lactamases are bacterial enzymes that can cleave β-lactam antibiotics, thus rendering them ineffective. Detection can be accomplished by detecting β-lactam cleavage, for example, by color changes of pH indicators or by means of MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization-MALDI; TOF-Time-of-Flight; Mass Spectrometry-MS). In MALDI-TOF MS, uncleaved β-lactams and / or their cleavage products are determined by mass spectrometry. While these methods may be advantageous in certain situations, their general drawback is that they only detect one specific resistance mechanism and cannot provide a general, definitive indication of pathogen susceptibility or resistance.

[0006] Therefore, there is an urgent need for a method that, on the one hand, achieves growth-based phenotypic sensitivity testing and the resulting generalizability, as with conventional testing methods, but on the other hand, is significantly faster than conventional methods. The general aim of such rapid testing is to provide results within a few hours, i.e., within 1–4 hours. The feasibility of this time target depends first on the experimental method, and secondly on the characteristics of the microorganisms being tested; for example, their growth rate.

[0007] The recently developed MALDI-TOF MS-based method “MBT ASTRA” for quantifying microbial growth using internal standards demonstrates the feasibility of general growth-based sensitivity testing via MALDI-TOF MS (Lange et al., Journal of Clinical Microbiology, December 2014, Vol. 52, No. 12, pp. 4155-4162; and K. Sparbier et al. / Methods 104(2016) 48-54). However, methods described to date require multiple processing steps, implying significant labor costs in the laboratory. This cost reduces the acceptability of the method and thus hinders or even prevents the introduction of this inherently patient-beneficial method into routine diagnostics.

[0008] In view of the foregoing explanation, there is a need to provide a method that simplifies and accelerates the preparation of live microbial samples for subsequent mass spectrometry measurements. Other objectives of the present invention will become immediately apparent to those skilled in the art upon reading the following disclosure. Summary of the Invention

[0009] The methods described herein represent alternative approaches for very rapid and simple MS-based microbial measurements; for example, for species / subspecies identification and / or resistance / susceptibility testing and / or further pathogen characterization. This disclosure relates particularly to methods for sample handling / preparation and data analysis algorithms.

[0010] According to a first preferred aspect, the present invention relates to a method for preparing a live microbial sample for subsequent mass spectrometry measurements, comprising the steps of: (a) providing a flat sample carrier containing a plurality of sample spots (so-called “spots”); (b) applying at least one live microbial sample present in a droplet of nutrient medium to at least one sample spot; (c) placing the sample carrier in an incubation chamber having a defined atmosphere for a predetermined period of time to stimulate microbial growth; (d) removing residual liquid from the droplet of nutrient medium after the predetermined period of time to expose microbial deposits on the sample spot; (e) preparing the sample spot for desorption ionization; (f) transferring the sample carrier to a desorption ionization source of a mass spectrometer to generate ions from the prepared sample spot and obtain at least one corresponding mass spectrum; and (g) comparing the obtained mass spectrum with a set of reference data to determine at least one characteristic of the microbial sample.

[0011] A first preferred aspect of the invention is based in particular on the novel and surprising discovery that flat mass spectrometry sample carriers, serving as substrates for ionizing treated samples in a suitable ion source, have already been used in preliminary steps as substrates for promoting microbial growth through incubation. This dual functionality simplifies laboratory workflows and reduces the time required for diagnostic procedures by avoiding complex and error-prone manual sample transfer steps and eliminating the need for separate preparation containers, such as microtiter plates. This procedural simplification facilitates the establishment of rapid, reliable, and comprehensive mass spectrometry measurements of microorganisms in clinical diagnostics.

[0012] In various embodiments, the reference dataset may include a reference spectrum derived from a previously obtained mass spectrometry library, wherein, during the identification process, at least one feature of step (g) may include the species or subspecies of microorganisms in the microbial sample. In this simple variant, droplets of nutrient medium act as a pure growth reactor on the mass spectrometry sample carrier. Experts will recognize that microorganisms multiply faster in liquids due to factors such as the overall flushing of the nutrient medium than on flat nutrient media, such as an agar layer in a petri dish; thus, the proposed method offers a time advantage, which is crucial for patient survival in the clinical field.

[0013] In certain cases, the reference dataset can be derived from mass signals not originating from microorganisms contained in the mass spectrometer obtained in step (f). For example, the mass signal of one (or more) reference substances (internal standards) added during the preparation of the microbial sample, which may be used for quantification.

[0014] In a preferred embodiment, in step (b), the same microbial sample is applied in parallel to several sample points, where some droplets of the nutrient medium contain antimicrobial substances and others do not. Mass spectrometry sample carriers are particularly suitable for a wide range of resistance / sensitivity tests because they provide ample space to monitor microbial growth in the presence of different antimicrobial substances (or different concentrations of the same antimicrobial substance). For example, the question of whether a microorganism exhibits a sensitive response to a specific antimicrobial substance (or the concentration at which it begins to respond in this way) can be elucidated very quickly and reliably, indicating its effectiveness as a drug.

[0015] In a particular embodiment of this method, some of the nutrient medium droplets containing antimicrobial substances may contain enzyme inhibitors, while others may not. The use of β-lactamase inhibitors as enzyme inhibitors has significant clinical and therapeutic implications. If the presence of β-lactam antibiotics does not inhibit the growth of the studied microorganism, but is inhibited in the presence of a combination of β-lactam antibiotics and β-lactamase inhibitors, then this extension of the resistance / susceptibility test can indicate resistance induced by β-lactamase.

[0016] In various embodiments, the reference dataset may be a recently obtained sample spot mass spectrometer on which nutrient medium droplets without any antimicrobial substance or enzyme inhibitor have been applied in step (b); and, during characterization, at least one characteristic in step (g) may include the resistance / susceptibility of the microorganisms in the microbial sample to the antimicrobial substance or a combination of antimicrobial substance and enzyme inhibitor. In this way, the minimum inhibitory concentration of the antibiotic relative to the microorganism can be determined by applying several drops of nutrient medium with different concentrations of antimicrobial substance (and / or inhibitor, if applicable) in step (b), and the effectiveness as the concentration increases or decreases can be assessed.

[0017] In another embodiment, the reference dataset can be a mass spectrometry of sample spots on a second sample carrier, wherein the microorganisms are incubated for a short time, or possibly not at all, for use as the reference dataset. Here, the microbial sample is preferably applied to the sample spot in a droplet of nutrient medium without any antimicrobial substances or without a combination of antimicrobial substances and enzyme inhibitors. The amount of microbial cells initially applied to the sample spot is preferably the same for both the microbial sample and the reference dataset mass spectrometry.

[0018] Preferably, at least one characteristic of step (g) is derived from the differences in microbial growth, whereby microbial growth is reflected in the properties or intensity of the microbial-specific mass signal characteristics in the obtained mass spectrometer; depending on whether growth inhibition caused by antimicrobial substances alone or in combination with enzyme inhibitors is detected. In a simple variant, if the amount of microorganisms in the microbial sample measured prior to incubation is insufficient for reliable identification, microbial growth can be assessed based on successful species identification. The algorithm serves as a well-known example of an identification method. A similarity index (the so-called "log(Score)") greater than or equal to 1.7 is sufficient to confirm an acceptable and reliable identification of the microbial species. A similarity index greater than or equal to 2.0 indicates high reliability.

[0019] In various embodiments, the microbial sample in step (b) can be quantified in a droplet of nutrient medium such that the amount is slightly below the detection limit of the mass spectrometry measurement. In this way, the length of time the sample carrier must remain in the incubation chamber to stimulate microbial growth can be minimized. This is because growth reaching or slightly exceeding the detection limit can itself serve as an indication of the presence or characteristics of microorganisms, compared to measurements that do not contain information beyond background signals.

[0020] In various implementations, the temperature and humidity in the incubation chamber in step (c) can be set to approximately 36°C (which may be necessary or even mandated for incubation or sensitivity testing) and near saturation, respectively, to create optimal or desired growth conditions for the microorganisms under study. The goal is generally to create conditions in the incubation chamber that most clearly reveal differences in growth. A temperature of 36°C roughly corresponds to human body temperature and is suitable for microorganisms that specifically use humans as hosts. For example, in veterinary, food, or environmental diagnostics, other temperatures may likely be identified as optimal, depending on the preferred host or surrounding environment of the microorganism. High air humidity, close to 100%, is specifically used to prevent premature evaporation of droplets in the nutrient medium, ensuring that the volume of liquid available for microbial growth remains approximately constant over a predetermined period, typically up to several hours, during which time the sample carrier remains in the incubation chamber (usually 1–18 hours).

[0021] In various embodiments, removal of residual liquid (after the incubation step) may include gently wiping away the droplet supernatant with an absorbent material or pipetting it. These variants have the advantage that residues of substances present in the nutrient medium droplets are largely removed from the sample spot along with the liquid, which reduces chemical background in subsequent mass spectrometry measurements. Alternatively, the liquid can be evaporated for a short time, for example, using a hot air blower. In this case, the substances in the liquid nutrient medium precipitate onto the microbial sediment, which is then at least partially prepared together for subsequent mass spectrometry measurements.

[0022] In various embodiments, the preparation in step (e) may include preparative extraction of microbial proteins / peptides from microbial sediments and / or washing of the microbial sediments and / or embedding the microbial sediments in a laser absorption matrix for subsequent ionization by matrix-assisted laser desorption / sorption spectroscopy (MALDI). In the case of extraction, the amount of microbial-specific mass signal in the obtained mass spectrum can be increased, thereby improving the validity of the measurement; particularly when the aim is to identify the species / subspecies of the microorganism under study. One or more washing steps are particularly suitable for removing almost ubiquitous salts from microbial sediments that would otherwise reduce ionization efficiency. Therefore, the preparation method can be further optimized. Examples of matrix materials are 2,5-dihydroxybenzoic acid, sinapic acid, or α-cyano-4-hydroxycinnamic acid. The MALDI method has proven to be a very important and reliable tool in ion-based microbial research. Furthermore, it allows for pulsed ion generation, which is particularly suitable for obtaining mass spectra in the form of time-of-flight dispersions.

[0023] However, it is also conceivable that other types of desorption ionization, which do not require the application of a matrix material, can be used in conjunction with the method; for example, desorption electrospray ionization (DESI) or ionization by secondary ion mass spectrometry (SIMS). In very specific cases, the preparation in step (e) may consist of only a short waiting time, for example, a few minutes, without any further treatment of the microbial deposits.

[0024] Advantageously, the mass spectrometry in step (f) is obtained by time-of-flight dispersion. Time-of-flight mass spectrometers are currently considered the "gold standard" for both clinical and non-clinical microbiology analysis due to their high resolution, rapid measurement time, and broad mass acceptance. An example of a mass spectrometer operating based on the time-of-flight principle is a commercially available model from Bruker Daltonik GmbH. series.

[0025] In various embodiments, the microbial sample may be dispensed in step (b) (i) as a suspension in a droplet of nutrient medium at at least one sample point, or (ii) first deposited in cellular form on at least one sample point and then immersed in the dispensed droplet of nutrient medium. In other variants, antimicrobial substances (and, where possible, enzyme inhibitors) may be applied to the sample point together with or separately from the microbial sample and / or nutrient medium. In principle, it is also conceivable to reverse the order of deposition and dispensing, thereby first dispensing the droplet and then introducing the microbial sample into it.

[0026] According to a further preferred aspect, this disclosure relates to a method for preparing microorganisms for subsequent mass spectrometry measurements, comprising the steps of: (a) providing a flat sample carrier containing a plurality of sample spots, such as MSP 48 / 96 target polished steel from Bruker Daltonik GmbH. (b) Applying intact microorganisms, cultured and / or isolated at the point of departure from the flat sample support, in a droplet of nutrient medium to at least one sample spot on the flat sample support, preferably using a nanopipette or micropipette; the amount of droplet transferred may be between 1 and 10 μL; (c) Holding the flat sample support in place for a predetermined set time to allow microbial deposits to form on the sample spot, preferably about 10 to 60 minutes; (d) After the predetermined set time, removing any residual liquid from the nutrient medium droplet to expose the microbial deposits; (e) Preparing the sample spot for desorption ionization; preferably using a MALDI matrix material; (f) Transferring the sample support to the desorption ion source of a mass spectrometer to generate ions from the prepared sample spot and obtain at least one corresponding mass spectrum; and (g) Comparing the obtained mass spectrum with a reference data set to determine at least one characteristic of the microorganism.

[0027] The inventors have determined that microbial deposits on flat surfaces can form after a relatively short settling period of up to one hour, allowing the microorganisms deposited therein in the form of a "biofilm" to be gently removed from the residual liquid and reliably detected using subsequent mass spectrometry measurements. Utilizing this surprising discovery, according to a second aspect, the culture (or incubation) of microorganisms for promoting growth and preparation for mass spectrometry measurements, carried out on the same mass spectrometry sample carrier according to the first aspect of the invention, is performed on different substrates. This spatial separation opens up possibilities for application, particularly in the automation of workflows, because in clinical settings, automated culture protocols can be more easily performed in containers, such as the wells of standardized microtiter plates, compared to on flat surfaces, for example using MALDI-TOFMS carriers. In principle, embodiments of the method according to the first aspect are also applicable to the method according to the second aspect, provided they can be coordinated with each other.

[0028] In different implementations, the reference dataset may have a reference spectrum taken from a previously obtained mass spectrometry library, and in the identification process, at least one feature in step (g) includes a species or subspecies of microorganism.

[0029] In various embodiments, prior to the application in step (b), the microorganisms can be cultured in a liquid nutrient medium in at least one container remote from the flat sample carrier and transferred from there to the sample point; culture in an environmentally conditioned / temperature-controlled incubator preferably requires approximately 4 to 24 hours. As previously mentioned, culture is particularly useful for susceptibility testing of microorganisms—preferably in the presence and absence of antibiotics. Culture is not absolutely necessary for identifying or determining specific proteins in mass spectrometry that can indicate specific virulence factors or other microbial characteristics, and therefore does not need to be part of the protected method. Existing microbial suspensions can be used; for example, microbial suspensions generated due to other processes in the laboratory. For example, a bacterial suspension can be generated, for instance, by dissolving bacterial colonies present on an agar plate in a liquid; prior to this, bacteria can be cultured on a solid medium (such as agar) that allows colonies to grow (which may have already existed in this form for several days). Another example: MALDI The kit uses liquid from positive blood culture flasks containing blood and liquid medium from sepsis patients, and in positive cases, also containing cultured pathogens. The kit further enriches the pathogens from this positive liquid using a lysis / centrifugation method, followed by protein extraction and MALDI measurement of the proteins. Alternatively, a few microliters of positive blood culture can be applied to the sample spot. Even without additional heat during culturing, microbial cells (i) precipitate and (ii) adhere to the surface of the carrier simply at room temperature; (iii) moreover, most species even proliferate slightly at room temperature.

[0030] In various further embodiments, the same microorganisms can be cultured in several (external) containers, and sometimes antimicrobial substances can be added to the liquid nutrient medium, but sometimes they are not. Additionally, enzyme inhibitors can sometimes be added to the liquid nutrient medium in different containers containing antimicrobial substances, and sometimes they are not. One example is a β-lactamase inhibitor.

[0031] In various embodiments, the reference dataset may be a mass spectrometer of recently acquired sample points with microbial deposits derived from a liquid nutrient medium free of antimicrobial substances or enzyme inhibitors, and during characterization, at least one feature in step (g) may include the microorganism's sensitivity to antimicrobial substances or a combination of antimicrobial substances and enzyme inhibitors. In a particular variant, the same microorganism may be cultured in different (external) containers with different concentrations of liquid nutrient medium, and during characterization, at least one feature in step (g) may include the minimum inhibitory concentration of the antimicrobial substance on the microorganism.

[0032] Preferably, at least one characteristic in step (g) is derived from differences in microbial growth, which can manifest, for example, using MALDI. The difference between the reliability and failure identification performed by the algorithm.

[0033] At the start of cultivation, the microorganisms can be quantified so that their quantity is slightly below the detection limit of mass spectrometry; for example, approximately 10 micrograms per milliliter of nutrient medium. 5 CFU, in particular, results in a concentration of at least approximately 100 microorganisms per sample spot.

[0034] In different embodiments, removing residual liquid from the flat sample carrier in step (d) may include wiping off the supernatant of the droplet with an absorbent material or pipetting it.

[0035] The preparation in step (e) may include preparative extraction of microbial proteins / peptides from microbial deposits on a flat sample carrier and / or washing of the microbial deposits and / or embedding the microbial deposits into a laser-absorbing matrix material for subsequent ionization by matrix-assisted laser desorption (MALDI) in step (f).

[0036] The mass spectrometry in step (f) is preferably obtained by time-of-flight dispersion (in a time-of-flight mass spectrometer).

[0037] In different implementations, microorganisms may be (i) dispensed into an (external) container as a suspension in a liquid nutrient medium, or (ii) first introduced into an (external) container in cellular form, followed by infusion of a liquid nutrient medium; for example, the amount of nutrient medium may be between 10 and 100 microliters.

[0038] In various embodiments, the wells (or wells) in the microtiter plate can be used as (external) containers for culturing. Alternatively, the sample carrier plate from step (a) can be divided into a first flat section with flat sample spots and a second section with depressions on its surface, wherein these depressions serve as (external) containers (away from the first flat section), and in step (b) the intact microorganisms growing therein are transferred from there to the flat sample spots in the first flat section. Attached Figure Description

[0039] The invention can be better understood by referring to the following description. The elements in the illustrations are not necessarily drawn to scale, but are primarily used to illustrate the principles of the invention (mainly schematically). In the illustrations, the same reference numerals denote corresponding elements in different views.

[0040] Figure 1A schematic diagram of an embodiment of a mass spectrometer 10 with a linear axial flight path 2 terminating at a detector 4 and having an upstream ion source 6 for matrix-assisted laser desorption / resorption (MALDI), as is commonly used for mass spectrometry analysis of microbial cells.

[0041] Figures 2A to 2G schematically and exemplary illustrate methods for preparing microorganisms in microbial samples for identification.

[0042] Figures 3A to 3H illustrate schematic diagrams of possible methods for testing the resistance / susceptibility of microbial samples.

[0043] Figure 4A and Figure 4B The results of the resistance / susceptibility test based on the principles of this method are shown.

[0044] Figures 5A to 5I schematically and exemplary illustrate a method for preparing microorganisms according to a second preferred aspect of the present invention.

[0045] Figure 6 An embodiment of a combined sample carrier having multiple segments is shown, which on the one hand are flat and thus can be used as a mass spectrometry sample carrier (and possibly for preparation thereon), and on the other hand have an embedded container for culturing / incubating microorganisms in a liquid nutrient medium. Detailed Implementation

[0046] Although the invention has been described and explained with reference to several embodiments, those skilled in the art will recognize that various changes in form and detail may be made without departing from the scope of the technical teachings defined in the appended claims.

[0047] According to the first preferred aspect of this disclosure, it has been surprisingly found that preparing live microbial samples directly on a mass spectrometry sample carrier can be used alone to culture microorganisms to generate a sufficient number of microorganisms at the sample spot for mass spectrometry detection. In this simple yet surprisingly effective embodiment, nutrient medium droplets suspended or immersed without live microbial cells also serve as a proliferation reactor.

[0048] Figures 2A to 2G illustrate schematic diagrams of this culture method, which involves culturing directly in droplets on a sample carrier, followed by mass spectrometry measurements.

[0049] A flat sample carrier 12 is coated with droplets 14 of a microbial suspension at different locations (“dots”), wherein the liquid contains a nutrient medium, such as a cation-regulated Mueller-Hinton medium or an Iso-Sensitest medium. The droplets 14 may have a volume of 1–10 μL, for example, 2, 4, 6, or 8 μL, depending on the requirements. The sample dots may be marked on the carrier surface or applied at sufficient distances to other unmarked surfaces of the carrier 12. For example, an AnchorChip plate with 96 marked dots can be used as the carrier 12 (Bruker Daltonik GmbH, Bremen, Germany). Alternatively, the sample dots may also be defined by droplets of the applied nutrient medium 14, which will not diffuse if the surface of the sample support 12 is sufficiently hydrophobic. Figure 2A.

[0050] In this embodiment, a carrier 12 coated with five droplets 14, each containing a different microorganism, is placed in an incubation chamber 16, where it can be maintained, for example, for up to approximately 18 hours in a defined controlled atmosphere at 36°C and nearly 100% relative humidity. It should be noted that the application of the microbial sample can, of course, also be performed in the incubation chamber 16; therefore, the relevant method steps of depositing the sample onto the sample support 12 and placing the sample support 12 in the incubation chamber 16 can be reversed in all conceivable embodiments of the process described in this disclosure.

[0051] For example, the humidity in incubation chamber 16 can be adjusted using a sodium chloride solution. During the time spent in incubation chamber 16, microorganisms can proliferate by dissimilating the nutrient medium in droplet 14. As the initially clear nutrient medium in droplet 14 becomes turbid (not shown) after several hours, the proliferation becomes visible. However, this turbidity is not necessary for microbial growth that can be detected by mass spectrometry. But if the incubation time is long enough, it can serve as a visual process control marker. Figure 2B. The inventors were surprised to find that, simultaneously, the proliferating microorganisms settle in sufficient quantity at the boundary between droplet 14 and the supporting surface, which greatly facilitates subsequent liquid consumption (dehumidification) or drying. Figure 2C.

[0052] In one variant, residual liquid in the droplet supernatant can be carefully aspirated using a micropipette or nanopipette 18 until the microbial cell clusters 20 deposited on the carrier surface are almost completely exposed (Figures 2D-2E). Surprisingly, due to the sedimentation behavior described above, sufficient microorganisms remain on the sample spot after the residual liquid of the nutrient medium is removed in this manner, thus providing a basis for generating a detectable mass signal in the mass spectrometer detector.

[0053] Subsequently, the microbial deposits 20 exposed on the sample spots can be coated with a matrix material to allow direct ionization on the sample carrier 12 via matrix-assisted laser desorption (pipette tip 22 with tile-patterned block shading). Figure 2F. Furthermore, additional preparation steps can be inserted prior to this, such as the addition of substances for protein / peptide extraction and / or washing steps (not shown); these are well known to those skilled in the art. The thus prepared sample spots are bombarded with laser 24 in the ion source of the mass spectrometer, thereby generating ions from the precipitated and prepared microbial membrane 20 on the sample spots, which are then supplied to the connected mass spectrometer for measurement. Figure 2G.

[0054] Using known evaluation algorithms, such as MALDI (Bruker Daltonik GmbH, Bremen, Germany) The species or subspecies of cultured microorganisms can be reliably determined from a specific mass signal in a mass spectrometer by comparison with a reference spectrum in a spectral database. The procedure for this assessment is well-known to those skilled in the art and requires no further explanation here.

[0055] In addition to the pure identification measurements performed directly on the mass spectrometry sample carrier after microbial culture as described above, this disclosure also provides methods and test kits for processing microbial samples for resistance / sensitivity testing, as described below.

[0056] Routine devices used for sensitivity testing typically test a large number of antibiotics simultaneously (e.g., 12 to 18), contained in standardized so-called “panels.” Results are usually not available on the same business day due to the longer incubation and reaction times required; the typical incubation period is 10–24 hours (so-called “overnight incubation”). In actual clinical situations, testing for only one or a few antibiotics is sufficient, provided they are of interest in a specific patient and their disease, or have already been used in a specific patient. However, if, for example, no clinical improvement is observed with antibiotic administration, then it is urgent to verify efficacy. Therefore, especially in cases of life-threatening infections such as sepsis, it is crucial that these test results be provided to the responsible physician within a significantly shorter timeframe, such as a few hours, to aid in treatment decision-making, rather than the following day.

[0057] For this reason, this specification focuses specifically on methods and apparatus (test kits, consumables, and other tools) for conducting rapid individual tests, namely, tests against a specific microorganism or a specific antibiotic against a microbial community. This does not preclude the use of the same, similar, or derived principles, where possible, for matching simultaneous testing against multiple antibiotics to test large numbers of antibiotics simultaneously or rapidly.

[0058] One characteristic of the method described according to the first preferred aspect is that the resistance / sensitivity test, or at least most of the required procedural steps, is performed directly on a mass spectrometry sample support (e.g., a MALDI-TOF MS support), i.e., a flat conductive plate suitable for this purpose, such as one made of polished steel or ceramic, which serves as the ionization substrate in the ion source of the mass spectrometer during measurement. In this method, the sensitivity test is growth-based (culture-based), i.e., the method is a phenotypic sensitivity test and therefore independent of the resistance mechanism on which it is based (if present).

[0059] The growth of microorganisms, both with and without antibiotics (the latter representing growth control), can also be performed directly on the MALDI-TOF MS vector, and measurements are subsequently taken thereon. This fundamentally distinguishes the method described according to the first preferred aspect from existing MALDI-TOF MS-based methods, in which microorganisms are cultured externally on the MALDI-TOF MS vector. In these methods, cultures with and without antibiotics (for growth control) are first performed in the wells of culture vessels or microtiter plates, where microorganisms or microbial proteins are then isolated and subsequently transferred to the MALDI-TOF MS vector for measurement. However, this requires a series of cumbersome manual steps, making it difficult to integrate these existing methods into routine diagnostic work. In contrast, the method proposed according to the first aspect allows for the very simple and rapid preparation of processed samples.

[0060] Similar to Figures 2A to 2G, Figures 3A to 3H exemplarily illustrate the work steps described herein and serve to schematically demonstrate their possible implications. However, those skilled in the art will recognize that certain processing steps can be performed in different forms. Professionals will use the workflows presented herein as a directional aid and, where possible, adapt them to their general skills and knowledge if this appears advantageous or useful to them.

[0061] Antibiotics (e.g., antibiotic solutions in liquid nutrient media) can be mixed with the microbial suspension in a culture vessel or directly on the sample spot of the MALDI-TOF MS support 12 (shaded droplets 14*). This is typically accompanied by growth control on other areas of the MALDI-TOF MS support, i.e., culturing the microbial suspension in a nutrient medium without added antibiotics (unshaded droplets 14). A very small amount of suspension is preferably applied to the sample spot; for example, 1-10 μL. Therefore, sensitivity testing is performed in microdroplets 14, 14*, preferably with a volume of about 4-8 μL. Even smaller volumes can be used in principle, for example, in the form of nanodroplets. The initial microbial concentration in the droplets 14, 14* can be slightly below the detection limit of a conventional MALDI time-of-flight mass spectrometer with a linear flight path, and thus, for example, about 5 × 10⁵ cfu / mL (cfu-colony forming unit). Figure 3A.

[0062] In the illustrated embodiment, the MALDI-TOF MS carrier 12 containing the test solution is then incubated in an incubator at high humidity in a so-called "humidity chamber" 16. The purpose of the humidity chamber 16 is to prevent premature evaporation of the droplets 14,14* during incubation, and it can take the form of, for example, a box with a lid made of plastic, into which the MALDI-TOF MS carrier can be easily placed. The humidity chamber 16 can have a similar form to conventional transport or storage containers used for commercial MALDI-TOF MS carriers (Bruker Daltonik GmbH, Bremen, Germany), and is preferably designed such that the MALDI-TOF MS carrier 12 can be placed deep enough inside that the lid does not contact the droplets 14,14* on the surface of the MALDI-TOF MS carrier 12. A small amount of liquid, such as 0.1 to 5 ml of water or NaCl solution, can be added to the humidity chamber 16 to humidify the atmosphere within the chamber 16, thereby setting a high ambient humidity (close to 100%) and preventing the nutrient medium droplets 14,14* from evaporating themselves. Figure 3B.

[0063] In droplets 14, 14*, a high concentration of microorganisms in a small volume of liquid is rapidly achieved during incubation (provided growth is not inhibited by antimicrobial substances). After a sufficient period of time, the humidity chamber 16 with the MALDI-TOF MS carrier 12 can be removed from the incubator (not shown). The MALDI-TOF MS carrier 12 is then removed from the humidity chamber 16, and the droplets 14, 14* on the carrier are dried. Drying can be passive, such as in air, or accelerated, for example, by actively generated airflow, thermal effects, a combination of both, or other methods. Due to the very small volume of liquid in droplets 14, 14* (nanoli to microliters), drying is very rapid. However, this simple droplet drying (e.g., with hot air) may have the disadvantage that not only microbial cells, but also proteins and other components of the liquid nutrient medium are enriched at the sample sites on the MALDI-TOF MS support 12 and interfere with MALDI-TOF MS measurements. This potential problem can be addressed by directly isolating microbial cells from the nutrient medium on the MALDI-TOF MS carrier 12.

[0064] In both drying and separation cases, the aim is to remove as much residual liquid as possible from the nutrient medium droplets 14,14* to prepare sample spots for subsequent preparation. As previously stated, the inventors have determined in their studies that microbial cells tend to precipitate during incubation lasting several hours, primarily accumulating directly on the surface of the MALDI-TOF MS support 12. To some extent, the cells even adhere (“stick”) to the surface of the support 12 and form a kind of “microbial biofilm,” while the liquid component forms a “supernatant” above it. Without providing a well-developed scientific explanation for this behavior of microorganisms in droplets on a plate, it is speculated that the physical interaction between the plate surface and the microbial cells, and the adhesion process caused by the biochemical and biophysical properties of the cell surface, are related to this preferred deposition. Figure 3C.

[0065] Using this new discovery, the nutrient medium (residual liquid) in the supernatant can be removed from droplets 14, 14* on the MALDI-TOF MS carrier 12, as illustrated elsewhere with reference to Figure 2D. Alternatively, the liquid can be simply wiped away to expose the microbial deposits from the liquid. Absorbent, low-lint wipes 26 can be used for this purpose, as commonly used in biological / chemical laboratories; for example, KimWipes. TMHere, separation occurs immediately and can be explained, in particular, by capillary action. Separation can be achieved, for example, by manual wiping with a folded cloth (e.g., absorbent paper, blotting paper, a soft cloth for cleaning sensitive surfaces) or by using a special device. Such a device, for example, could have a sheet or “pad” made of absorbent material, positioned specifically at a distance above the MALDI-TOF MS carrier 12 for uniform, rapid, and standardized wiping, a distance that allows fluid contact with the droplets 14,14* to be established and removed again after a relatively short absorption time of a few seconds. (Figures 3D-3F)

[0066] In an alternative implementation of wiping, the contact between the droplet and the absorbent fabric is not established vertically (perpendicular to the sample carrier surface, as shown in Figures 3D-3F), but rather near the surface of the sample carrier at the side edge of the droplet. This ensures that (i) residual liquid in the nutrient medium is absorbed more quickly and completely, and (ii) cells preferentially accumulating at the center of the sample spot at the surface will not come into contact with the absorbent fabric in any case, reducing the risk of accidental cell removal. This different scheme of liquid absorption can further reduce background in mass spectrometry, thereby further improving the quality of the measurement.

[0067] These forms of removing residual liquid also result in substantially dehumidified (or reduced, exposed residual liquid) microbial deposits 20 at the corresponding sample sites with very little potential interference from nutrient media residue, which are used as the basis for subsequent mass spectrometry measurements.

[0068] The separation of cells from the liquid nutrient medium, achieved through wiping or pipetting, provides high-quality and efficient measurements of MS spectra. Another advantage of this separation compared to (passive) drying of droplets is that it occurs very rapidly (immediately or instantly), resulting in significant time savings and allowing for immediate further sample processing. However, in some embodiments, wiping may be accompanied by a heated drying gas stream to more thoroughly consume any residual liquid.

[0069] This method is equally effective for separating cells from liquid media, such as centrifugation followed by removal of the supernatant, but can be performed directly on a MALDI-TOF MS carrier without the time cost. Separation can be further enhanced—for example, by using specialized MALDI-TOF MS carriers, such as anchor carriers (AnchorChip, Bruker Daltonik GmbH, Bremen), or MALDI-TOF MS carriers with individual flattened conical sample spots. Shallow but slightly conical pores can enhance cell sedimentation. Other methods, such as washing liquid samples directly on the carrier, can also be used.

[0070] To enhance the formation of microbial deposits (adhesion) on the sample carrier surface during the growth phase, the sample spot surface can be coated with various adhesion-promoting substances; for example, proteins or sugars. Preferably, substances are chosen that do not interfere with the measurement and / or comparison of microbial mass spectrometry with a reference dataset. This can be achieved, for example, by placing the mass signal of these substances outside the mass range to be evaluated, which is typically between m / z 2000 and m / z 20000, e.g., between m / z 3000 and m / z 15000. Alternatively, substances (e.g., proteins) with adhesion-promoting properties can be selected that can also serve as standards; i.e., as markers of good mass for measurement and / or as intensity markers. In this case, the mass signals of these standards are within the mass range to be evaluated. Furthermore, materials with enhanced adhesion properties and / or enhanced surface properties can be used from the outset as materials for fabricating MALDI-TOF MS carriers.

[0071] As described above, after drying or separating the droplets of liquid nutrient medium from the corresponding sample points, such as before introducing the carrier into the MALDI-TOF MS instrument and measuring microbial biomolecules, such as proteins or peptides, the sample points are coated with a matrix (with a pipette tip 22 in a tile-shaped shade) for MALDI-TOF MS analysis. Figures 3G-3H.

[0072] Before or simultaneously with the application of the matrix material, various substances that aid in measurement (not shown), such as formic acid or acetonitrile, can be added to improve the extraction of microbial proteins. Deionized water droplets can also be applied as washing droplets to the microbial sediment and removed again to remove salts. After measurement, the results can be evaluated according to algorithms, which are explained in more detail below. Here, the growth of microorganisms in the presence of antibiotics is evaluated and assessed. The basic principle is to inhibit the growth of susceptible microorganisms in the presence of antibiotics, while resistant microorganisms can still grow despite the presence of antibiotics. Simultaneous growth control, i.e., testing antibiotic-free microbial suspensions on the same MS sample carrier, facilitates the evaluation and corresponding evaluation algorithms. Figures 3B and 3C schematically illustrate the comparison of growth and sedimentation behavior for susceptible (solid lines) and resistant (dashed lines).

[0073] In a variant of this method, microorganisms can be cultured on a composite microtiter plate with and without antibiotics, wherein a flat, smooth mass spectrometry sample carrier (e.g., a MALDI-TOF MS carrier) forms the bottom and, together with a removable top portion containing through-holes, forms a mesh of holes as described in patent application CA 2 467 131 A1 (Figure 10 therein). The provided reaction vessel or well (e.g., as a test kit) already contains antibiotics, for example, in solution, powder, or lyophilized form, before the addition of the microbial suspension. After a sufficient incubation period with or without microbial growth, residual liquid droplets are removed, for example by drying, and the top is removed from the MALDI-TOF MS plate. MALDI matrix preparation and MALDI-TOF MS measurements as described above can then be performed.

[0074] In another embodiment, the MALDI-TOF MS vector is not incubated in a separate incubator; instead, the incubation function can be directly integrated into the MALDI-TOF mass spectrometer or into the entire system, for example, as an incubation unit or incubation module. This allows for automation and further reduces the necessary manual preparation steps. Another embodiment sets the heating device directly into the humidity chamber, whereby the humidity chamber can function as an incubator, thus eliminating the need for a separate incubator.

[0075] The use of mass spectrometry sample carriers that have been pretreated with antibiotics, such as in dry powder or other forms, at the sample sites can further make it easier for users to perform sensitivity tests.

[0076] Figure 4A Results of a resistance / susceptibility test for an example of the facultative anaerobic, Gram-negative bacillus *Klebsiella pneumoniae* to meropenem, a carbapenem-derived β-lactam antibiotic, are presented. Sample preparation was performed directly on sample carriers, as shown in Figures 3A-3H. Droplet volumes were dispensed in 6 μL volumes; antibiotic concentrations were 2 μg / mL; and residence time in appropriately regulated incubation chambers was 4 h. The commercial product MALDI was used. The software module was used to evaluate MALDI time-of-flight mass spectrometry. A meropenem-resistant strain (top mass spectrometer) and a meropenem-sensitive strain (bottom mass spectrometer) were tested separately. Growth controls without any antibiotics, prepared on the same MALDI sample support plate, are shown in the spectra on the right.

[0077] It is clear that in the case of the resistant strain, the characteristics of the specific mass signal in the two upper spectra are almost indistinguishable. Therefore, it can be concluded that it is resistant, as bacterial growth is not significantly inhibited in the presence of meropenem, and the species can be reliably identified. On the other hand, in the case of the sensitive strain, the specific mass signal characteristics are only visible in the growth control spectrum (lower right). However, in the presence of meropenem (lower left spectrum), Klebsiella cells clearly cannot reproduce (or hardly reproduce). The prominent mass signal separated in the lower left spectrum belongs to a reference substance added to the droplets of the nutrient medium for microbial quantification, but it was not considered in the study specifically described here. Under these growth-deficient conditions, the evaluation software was unable to determine the species of microorganism due to a lack of data; particularly when the initial amount of microbial biomass was below the mass spectrometry detection limit. The conclusion drawn is that this Klebsiella strain responds sensitively to this specific antimicrobial substance.

[0078] Figure 4B Use a bar chart to illustrate the situation from Figure 4A The experiment statistically analyzed the growth behavior of Klebsiella pneumoniae in the presence of meropenem at five different droplet sizes of 2, 4, 6, 8, and 10 μL. It can be seen that the relative growth of susceptible strains reliably remained below the significant growth threshold of 0.4, while for resistant strains it was well above the threshold; the 4 μL droplet was an exception, where measurements below this threshold were occasionally observed, although the median was significantly greater than 0.4.

[0079] Resistance / susceptibility testing can be performed on microbial samples obtained from cultures or directly from biological materials. In the prior art, mature cultures, which have been incubated on solid media such as agar for 16–24 hours and exist as colonies after such incubation time, are commonly used for sensitivity testing. Testing with mature cultures cultured in liquid nutrient media is also possible.

[0080] However, in many cases, it is advantageous to perform sensitivity testing directly from the material to be analyzed in order to significantly reduce the time until results are obtained. Positive blood cultures serve as an example of such material, which is crucial for rapid pathogen diagnosis. Currently, the procedure in blood culture diagnostics typically involves first placing a blood sample collected from a patient into a special blood culture bottle containing a liquid nutrient medium. These bottles are then placed in an automated incubator, where microbial growth is continuously monitored, for example, by measuring carbon dioxide levels. When a blood culture bottle reports a positive result, the liquid from it is smeared onto a solid medium, which is then incubated for 16–24 hours. The resulting colonies are used for identification and antibiotic susceptibility testing. These colonies are also applicable to the susceptibility testing methods described herein, etc.

[0081] However, identification and sensitivity testing directly from reported positive blood cultures saves the time required for culturing on solid media, thus allowing results approximately one day earlier. To achieve this, samples must be pretreated to enrich the microorganisms. This can be done, for example, by lysis / centrifugation or lysis / filtration. In the lysis / centrifugation method, blood cells are first lysed by adding a lysis agent, such as a surfactant, and then the microorganisms are concentrated by centrifugation. In a selective washing step, a washing buffer is added and the microorganisms are concentrated again by centrifugation. Identification is then performed immediately or after protein extraction. This identification method has been used as... The identification kit (BrukerDaltonik GmbH, Bremen, Germany) has been developed and is commercially available (NGMorgenthaler et al., International Journal of Microbiology Volume 2015, Article ID 827416, 10 pages).

[0082] This method, or a similar method, can also be used as a sample pretreatment (microbial enrichment) for the resistance / susceptibility tests described herein via MALDI-TOFMS. This significantly reduces the time required to obtain results.

[0083] As an alternative, subcultures derived from positive blood cultures or other materials that have been incubated very briefly on a solid medium can be used for the MALDI-TOF MS-based sensitivity test described herein. Identification (Idelevich et al., Clin Microbiol Infect, 2014; 20: 1001-1006) and sensitivity testing (Idelevich et al., J Clin Microbiol. 2014; 52: 4058-4062) using subcultures derived from positive blood cultures that have been incubated very briefly on a solid medium have recently been demonstrated. Here, the solid medium is incubated only briefly after subculture (smear), typically 1.5 to 6 hours, and the resulting “young” microbial biomass is used for identification and sensitivity testing. Although this method does not allow for direct testing immediately after a blood culture reports a positive result, it is still very rapid compared to routine testing from mature colonies incubated for 16–24 hours. The advantage of this method is that it requires no additional consumables or extra work; the solid medium is observed only at an earlier stage, and testing is performed from “young” biomass.

[0084] A particular advantage is that sensitivity testing can be performed directly from the blood without pre-incubating the blood sample in a blood culture machine. Sample preparation for enriching microorganisms can be performed as described above for testing from reported positive blood cultures.

[0085] Direct MALDI-TOF MS-based identification of microorganisms directly from blood is currently difficult due to the low concentration of microbial cells in the blood after microbial concentration has been concentrated and no pre-culture has been performed. However, direct MALDI-TOF MS-based sensitivity testing can be performed directly from blood using the method described herein. After isolating microorganisms from the blood, a microbial suspension is prepared in a liquid nutrient medium and mixed with antibiotics, as is typically done in sensitivity testing. This suspension, along with, where possible, a growth control, is then applied as droplets to a MALDI-TOF MS support and incubated directly therein. Even if the initial microbial cell count in the blood is very low, the microorganisms will multiply after a certain incubation period, at least in the growth control, or, if phenotypic resistance is present, in the mixture of sample and antibiotics, which can be detected by MALDI-TOF mass spectrometry. Thus, the sensitivity test is performed based on the same principles described in relation to the first preferred aspect of this disclosure.

[0086] Microorganisms can be identified directly from mature colonies cultured on solid media using MALDI-TOF. However, samples must undergo pretreatment to be identified directly from research materials (e.g., from positive blood cultures). This can be achieved, for example, by the lysis / centrifugation method described above. However, this method requires additional processing steps, which is time-consuming and makes it more difficult to integrate into routine laboratory diagnostics.

[0087] The methods described in this application for detecting and identifying droplets directly on a MALDI-TOF MS carrier, or for droplet sensitivity testing, according to the first preferred aspect, can be implemented not only individually but also in combination, with this combination being particularly meaningful when testing directly from research materials, such as positive blood cultures. When sensitivity testing is combined with identification in this manner, in MALDI-TOF MS measurements, not only is the growth of the control measurement compared with the growth of the sample with added antibiotics, corresponding to the algorithm described for the sensitivity test, but uninhibited microbial growth in the control measurement can also be used for conventional MALDI-TOF MS identification. However, when the incubation period is sufficiently long—still very short compared to the usual 16-24 hours—the microbial biomass is sufficient for identification. The advantages of this combined method are that (i) additional processing steps, such as those used for lysis / centrifugation methods, can be omitted, (ii) the results of sensitivity testing and identification can be obtained promptly and simultaneously, and (iii) the time until sensitivity testing and identification are completed is shorter compared to conventional testing from mature colonies.

[0088] This combined approach can be applied to testing from mature or young colonies as well as directly from materials, such as from positive blood cultures or blood.

[0089] Another implementation of the method described herein enables rapid and simple detection of microbial resistance mechanisms. This can be achieved, for example, through a combination test. That is, in addition to a suspension consisting of microorganisms and antibiotics, and a suspension containing only microorganisms (a growth control without antibiotics), a suspension consisting of microorganisms, antibiotics, and a substance that specifically counteracts the microorganism's potential resistance to the antibiotic (i.e., based on a specific resistance mechanism) is also tested.

[0090] One example of this is through bacterial detection of β-lactamase formation. β-lactamases are bacterial enzymes that can cleave β-lactam antibiotics and thus render them ineffective. Examples of β-lactamases include ampC-β-lactamase, extended-spectrum β-lactamase (ESBL), carbapenemase, etc. Each type of β-lactamase cleaves a specific spectrum of antibiotics and, moreover, possesses different characteristics (e.g., gene location on plasmids or chromosomes), which to varying degrees limits the supply of antibiotics available for treatment and allows for different rates of spread of the corresponding bacterial strains. Therefore, rapid identification of potential resistance mechanisms is crucial, particularly in investigations that may require the introduction of hospital hygiene and sanitation measures.

[0091] The action of β-lactamases can be specifically inhibited by adding specific β-lactamase inhibitors (such as clavulanic acid for ESBL, or valasubamide for meropenem). This principle is used not only in treatment but also in diagnosis to detect β-lactamases as a basis of resistance. For example, test plates impregnated with antibiotics and test plates impregnated with antibiotics plus β-lactamase inhibitors are commercially available. Bacterial cultures are spread onto a solid medium such as an agar plate, and these test plates are placed in the plate; after 16–24 hours, the inhibition zone is measured (agar diffusion test). If a specific difference in the diameter of the inhibition zone is achieved between the test plate with antibiotics and the test plate with antibiotics plus β-lactamase inhibitors, this indicates the presence of a specific β-lactamase.

[0092] Besides its simpler implementation, the advantages of the combined testing method described herein are particularly evident in the fact that results are available within just a few hours compared to the agar diffusion method, which, for example, requires over 12 hours and is therefore often only available the following day. This speed advantage stems from the fact that, firstly, microbial growth in liquid nutrient media is significantly faster than in solid media; secondly, due to the smaller liquid volume, high concentrations of microorganisms are quickly achieved in the droplets; and thirdly, mass spectrometry measurements, such as by MALDI-TOF MS, ensure more sensitive and rapid growth detection than visual observation of growth on solid media, as in the case of the agar diffusion method.

[0093] Compared to the identification of β-lactamases by detecting β-lactam cleavage using MALDI-TOF MS (mass signal of undivided β-lactam or cleavage products) mentioned at the beginning, the MALDI-TOF MS-based method using a combination assay described here has an important advantage: the detection of β-lactam cleavage is an indirect method; a positive result indicates that the β-lactam antibiotic has been cleaved, thus concluding that the antibiotic is ineffective against this bacterial strain. However, effectiveness can also depend on other factors, such as the antibiotic dosage. In the combination assay described herein, the effect of the β-lactamase inhibitor on microbial growth is also directly determined, i.e., whether resistance is inhibited. These results have considerable clinical relevance.

[0094] According to the previous implementation, the combined test described herein can be used for testing from mature or young colonies, and can also be used for testing directly from materials, such as from positive blood cultures or blood.

[0095] In addition to applying the described method as a single rapid test—that is, testing a specific antibiotic against a specific microorganism—several antibiotics can be tested simultaneously (multiplex testing). This has the advantage of simultaneously generating a complete antimicrobial spectrum against the microorganisms in a microbial sample. Furthermore, multiple concentrations of each antibiotic can be tested simultaneously, allowing the determination of the minimum inhibitory concentration (MHK). The MHK is the lowest concentration of antibiotic that inhibits the growth of a microorganism. The MHK is a measure of a microorganism's sensitivity to an antibiotic. First, the MHK allows classification of microorganisms into "sensitive," "intermediate," or "resistant" categories; second, the MHK provides information about the "susceptibility" of a microorganism to a specific antibiotic. For multiplex testing, many sample pads of the MALDI-TOFMS carrier can be coated in parallel. For example, carriers with 96, 384, or 1536 sample spots can be used.

[0096] Microbial growth can be determined using different evaluation algorithms. Here, the growth of microorganisms treated with antibiotics can be compared with the growth of microorganisms without antibiotics (growth control).

[0097] A possible algorithm for detecting microbial biomass is proposed. A specific lower detection limit is a characteristic of the MALDI-TOF MS method, namely, the minimum amount of microbial biomass (approximately 10^6 ppm per point). 4 Or 10 5The detection of microorganisms (amount of microbial cells) is achieved in the sense that this quantity produces a recognizable mass signal in the mass spectrometer. The limit of detection depends on many factors, including instrument characteristics and settings. According to the algorithm described here, microorganisms in a liquid nutrient medium can be applied to the sample spot at a concentration (amount) below the limit of detection of the MALDI-TOF MS measurement method. That is, if a MALDI-TOF MS measurement is to be performed without further processing of the microbial sample, it is impossible to detect any microbial signature in the mass spectrum against an omnipresent background. It can be directly concluded that if the microorganism is sensitive to the antibiotic being tested, its growth is inhibited, and the microbial biomass is almost undetectable after the incubation period because it does not exceed the limit of detection. If the microorganism is resistant to the antibiotic being tested, the microorganism can grow during incubation as it does in the growth control (without antibiotics), and the microbial mass can be detected, i.e., the mass signal of the corresponding specific microorganism in the measurement spectrum.

[0098] To improve the accuracy of the method and avoid the possibility of misinterpreting "accidental" features in the mass spectrum that resemble specific microbial mass signals, even in cases involving small amounts of microbial biomass (typically below the detection limit), quantitative or relative quantification of the microbial biomass can be used (where possible, in combination). This can be achieved, for example, by comparing the so-called "area under the curve" (AUC) and / or peak intensity using internal standards ("MBT-ASTRA", Bruker Daltonik GmbH, Bremen, Germany) or other statistical methods, which are well-known to those skilled in the art and need not be explained in detail here. In particular, the reference dataset used for comparison with the microbial mass characteristics in the obtained mass spectrum can be derived or determined from the mass signals of internal standards or reference materials in the same mass spectrum.

[0099] An algorithm for spatial resolution is proposed as another possible variant. Here, laser irradiation is performed within a precisely defined spatial grid using the MALDI-TOF MS method—for example, 1000 irradiations distributed over a defined region of prepared sample points on a MALDI-TOF MS carrier. For example, the number of “successful” irradiations is compared between antibiotic-containing microbial samples and antibiotic-free microbial samples (growth controls), i.e., the irradiations that produce mass spectra with detectable microbial characteristics.

[0100] This algorithm can also be used, for example, to supplement the aforementioned algorithms for detecting microbial biomass, in order to improve the accuracy of the detection method and reduce the probability that "accidental" features, even in the case of small amounts of microbial biomass (usually below the detection limit), are misinterpreted as significant growth. That is, for example, a few successful irradiations cannot be interpreted as growth, but are considered accidental and unimportant.

[0101] Figures 5A to 5I illustrate embodiments of the method according to the second preferred aspect of this disclosure. Since many steps are similar to those of the methods described above, related embodiments can also be used in this embodiment; the following description is limited to the essential differences from the method according to the first preferred aspect of this disclosure.

[0102] The essential difference lies in the fact that the cultivation / incubation of microorganisms and the preparation for mass spectrometry measurements are not performed on the same planar substrate, such as a mass spectrometry sample carrier, but on separate substrates (or substrate segments). The microbial suspension in a liquid nutrient medium is added to container 28; for example, the wells in a microtiter plate 30. The inoculum can be approximately 10... 6 CFU / ml; the volume of the nutrient medium is approximately 50 to 250 μL; preferably 100 μL. For resistance / sensitivity testing, well 28 may (e.g., as part of a test kit) already contain antimicrobial substances, for example, in solution, powder, or lyophilized form, prior to the addition of the microbial suspension. Alternatively, these antibiotics may be added to the nutrient medium at a later stage. Figure 5A.

[0103] The well plate 30 is placed in the incubator 16 and incubated therefor a specific time, for example, 4 to 18 hours, to promote microbial growth. As already illustrated, the microorganisms have a tendency to form deposits (“microbial biofilms”) on the bottom and lower part of the sidewalls of the wells 28. Figures 5B-5C.

[0104] Remove the well plate 30 from the incubator 16. To remove a sufficient volume of nutrient medium containing a large quantity of intact, roughly uniformly distributed microorganisms from the wells 28, the sediment can be stirred, for example by a few up-and-down movements of the pipette tip 18 or by gently agitating the well plate 30 shortly before removal, so that the microorganisms can be sampled at a higher concentration and evenly distributed with the liquid nutrient medium. The volume of liquid removed can be between 1 and 10 μL. Figures 5D-5E.

[0105] As an alternative to this treatment method, where possible, the formation of microbial deposits in the wells 28 during incubation can be prevented or avoided from the outset by carefully agitating the well plate 30 (not shown) during incubation 16. This eliminates the need for dispersion and / or subsequent agitation with the aid of the pipette tip 18.

[0106] The extracted liquid containing intact microorganisms was placed as droplet 14 on the sample spot of the flat mass spectrometer sample carrier 12. Figure 5F.

[0107] Then it is left to stand or remain still for approximately 10 to 60 minutes, during which time microorganisms are given the opportunity to accumulate or deposit at the interface between the droplet liquid and the carrier surface. In principle, the settling period can be shortened as the concentration of microorganisms in the droplet 14 on the sample carrier 12 increases; in other words, at high concentrations, the settling period can be at the lower limit of the preferred range; at low concentrations, waiting for a longer period may be advantageous. Figure 5G.

[0108] As explained previously in different aspects, residual liquid in the nutrient medium can be removed from the sample site after a settling period, for example, by an absorbent fabric (cloth 26) that makes fluid contact with the droplets 14 on the sample site on the side of the carrier surface and simply absorbs most of the liquid. Of course, as mentioned above, other types of liquid removal, such as pipetting, can also be used. Figure 5H.

[0109] The microbial sediment 20 exposed in this manner can now be further prepared as described above and measured in a mass spectrometer. For example, peptides / proteins of the microorganisms can be extracted and / or the sediment 20 can be washed and / or the sediment 20 can be embedded in a MALDI matrix material. Figure 5I.

[0110] Figure 6 The combined well / sample carrier 32 is illustrated schematically and exemplary, having recesses 34 (which may represent multiple recesses) in which microorganisms can be cultured, and having flat sections 36 spaced therebetween, with sample spots, which can be used as a substrate for mass spectrometry sample preparation. In the ion source of the mass spectrometer, interference of the recesses 34 with the electric field can be reduced, for example, by pre-flush covering the recesses 34 (not shown).

[0111] The principles described here are not necessarily limited to the MALDI-TOF MS measurement method, but can in principle be implemented using other detection or differentiation methods, such as other mass spectrometry detection methods or methods for determining intrinsic fluorescence.

[0112] Besides the illustrative embodiments, other embodiments of the invention are also possible. With the knowledge gained from this disclosure, those skilled in the art can readily design further advantageous preparation methods and mass spectrometry measurement methods for live microbial samples and microorganisms, which are also covered by the scope of the claims.

Claims

1. A method for preparing microorganisms for subsequent mass spectrometry measurements, the method comprising the following steps: (a) Provide a flat sample carrier containing several sample points; (b) The intact microorganisms cultured and / or isolated at the exit of the sample carrier are applied in a droplet of nutrient medium containing culture medium to at least one sample point on the flat sample carrier, wherein prior to application in step (b), the microorganisms are cultured in a liquid nutrient medium in at least one container remote from the flat sample carrier and transferred from there to the sample point. (c) Keep the flat sample carrier in place for a predetermined time to allow for the formation of adherent microbial deposits at the sample sites; (d) After a predetermined settling time, remove the residual liquid from the nutrient medium droplets to expose the microbial deposits, wherein removing the residual liquid includes wiping off the supernatant of the droplets with an absorbent material or removing the droplets. (e) Prepare sample spots for desorption ionization; (f) Transfer the sample carrier to the desorption ion source of the mass spectrometer to generate ions from the prepared sample spot and obtain at least one corresponding mass spectrum; as well as (g) The obtained mass spectra are compared with a reference dataset to determine at least one characteristic of the microorganism.

2. The method according to claim 1, wherein the reference dataset has a reference spectrum taken from a previously obtained mass spectrometry library, and in the identification process, at least one feature in step (g) includes a species or subspecies of microorganism.

3. The method according to claim 1, wherein the same microorganisms are cultured in several containers, and some liquid nutrient media contain antimicrobial substances or a combination of antimicrobial substances and enzyme inhibitors, while others do not contain antimicrobial substances.

4. The method of claim 3, wherein the reference dataset is a recently obtained sample spot mass spectrometer containing microbial deposits derived from a liquid nutrient medium free of antimicrobial substances or a combination of antimicrobial substances and enzyme inhibitors, and in characterization, at least one feature in step (g) includes the sensitivity of the microorganisms to antimicrobial substances or to a combination of antimicrobial substances and enzyme inhibitors.

5. The method according to claim 4, wherein the same microorganism is cultured in different containers with different concentrations of liquid nutrient medium, and when characterizing, at least one feature in step (g) includes the minimum inhibitory concentration of the antimicrobial substance on the microorganism.

6. The method according to claim 5, wherein at least one feature in step (g) is derived from differences in microbial growth.

7. The method of claim 6, wherein at the start of cultivation, the microorganisms are quantified such that their quantity is slightly below the detection limit of mass spectrometry.

8. The method according to claim 1, wherein the predetermined settling time in step (c) is between 10 and 60 minutes.

9. The method according to claim 1, wherein the microorganisms: (i) are dispensed into the container as a suspension in a liquid nutrient medium, or (ii) are first introduced into the container in cellular form and then infused with the liquid nutrient medium.

10. The method according to claim 1, wherein the orifice in the microtiter plate serves as a container.

Images