Microbial test standards for infrared spectroscopy and methods of use thereof

Abstract

This invention relates to a microbial test standard for infrared spectroscopy determination, the microbial test standard having at least two resealable containers that are impermeable to liquids when closed, each of the at least two resealable containers containing a predetermined amount of dried biomass of microorganisms. The dried biomass comprises vacuum-dried pellets. The microorganisms in the different containers differ in at least one characteristic, particularly selected from a group including species, subspecies, strains, serotypes, pathogenic types, toxin types, and variants, and the differences themselves are manifested by a predetermined intermicrobial spectral distance. This disclosure also includes a method for using the microbial test standard. The test standard and the method of use enable the determination and evaluation of the reproducibility and spectral specificity of infrared spectral measurements, as well as subsequent evaluation steps.

Description

[0001] This patent application is a divisional application of the patent application with application number 2018103543525, application date April 19, 2018, and invention title "Microbial testing standard for infrared spectroscopy determination and its usage method". Technical Field

[0002] This invention relates to test standards for distinguishing, identifying and / or characterizing microorganisms (especially bacteria) in infrared spectroscopy, particularly in Fourier transform infrared spectroscopy (FTIR), and preferably in transmission, as an aid to measurement and process control. Background Technology

[0003] The prior art is explained below with reference to specific aspects. However, this should not be construed as limiting. Other useful extensions and modifications known from the prior art beyond the relatively narrow scope of this description are also possible, and these extensions and modifications will readily become apparent to those skilled in the art upon reading the following disclosure.

[0004] Preferably, the IR spectrum acquired during transmission for microbial identification typically contains wavelengths within a predefined range, particularly in the mid-infrared region (approximately 4000 to 4000 cm⁻¹). -1 Several extinction signals or absorption bands in the spectrum are observed. As samples to be studied, microorganisms provide clear and identifiable spectral features in these spectra. These features reveal the superposition of contributions from all cellular components (e.g., cytoplasmic components, membrane and cell wall components, i.e., especially polysaccharides, DNA / RNA, and proteins)) and thus represent the phenotypic expression of the genome (DNA / RNA), or genetic fingerprint. The characteristics of this fingerprint form the basis for using infrared spectroscopy to distinguish, identify, and characterize microorganisms. For details on these measurement methods, refer to the overview in "Infrared Spectroscopy in Microbiology" in Dieter Naumann's Encyclopedia of Analytical Chemistry (RAMeyers (Ed.), pp. 102–131, John Wiley & Sons Ltd, Chichester, 2000).

[0005] Today, automated testing and recalibration routines can be used to detect and reliably compensate for unforeseen and undesirable fluctuations and changes in instrument settings and infrared spectrometer properties over a period of operation, which may affect the reproducibility and comparability of measurements.

[0006] However, external measurement conditions can vary during the operation period, sometimes even from one acquisition to the next. Many infrared spectrometers are not equipped with a regulated sample support chamber that ensures constant and stable environmental conditions, such as pressure, temperature, and humidity during and between acquisitions. It seems reasonable that changes in humidity, in particular, can affect the spectral properties of the prepared samples by, for example, the formation of hydration films or water of crystallization, and thus may adversely affect the comparability of similar samples or even identical replicas. These differences then become particularly apparent with respect to changes between winter (cold air, lower absolute humidity) and summer (warm air, higher absolute humidity). Therefore, a better process is needed to monitor infrared spectroscopy measurements to better account for these potential external influences and to evaluate IR measurements over short periods of time.

[0007] Simple forms of process control for IR measurements are already known. For example, in international application WO 99 / 16895 A1, four batches of microorganisms to be identified are each measured three times to monitor spectral reproducibility, and then the similarity of these replicas relative to each other is assessed.

[0008] A fundamental task of distinguishing, identifying, or characterizing microorganisms using IR spectroscopy is to differentiate strains and assess their phenotypic relationships with one another. Acquiring and comparing specific absorption spectra of microbial biomass allows for the determination and visualization of spectral distances using powerful clustering algorithms, making these relationships identifiable to the user. This presents the following challenges: On the one hand, high spectral specificity is required, especially to distinguish two different strains of the same microbial species; on the other hand, the same strains (even those from different sampling sites) must reliably belong to the same cluster in the analysis, meaning that in replicas applied to the sample carrier, they must have the minimum possible intramicrobial spectral distance relative to each other and between themselves to achieve reliable reproducibility of the measurements.

[0009] Therefore, it is necessary to provide microbial testing standards and appropriate methods for their use. When the identification and properties of the reference biomass being measured are known, the microbial testing standards and methods can be used to determine and evaluate the reproducibility and spectral specificity of IR spectral measurements, as well as subsequent evaluation steps. Summary of the Invention

[0010] According to a first aspect, the present invention relates to a microbial test standard for use in infrared spectroscopy determination, the microbial test standard having at least two resealable containers that are impermeable to liquid when closed, each of the at least two resealable containers containing a predetermined amount of dried biomass of microorganisms. The microorganisms in the different containers differ in at least one characteristic, which may be particularly selected from a group including species, subspecies, strains, serotypes, pathogenic types, toxin types, and variants, and the differences themselves are manifested by a predetermined intermicrobial spectral distance.

[0011] The test standard is preferably used as follows: At least two reference microorganisms, which have a predetermined intermicrobial spectral distance from each other, and an actual sample of an unknown microorganism are applied twice or more repeatedly to the same sample support plate to achieve spatial separation. The reference biomass is measured by means of IR spectroscopy before measuring the “actual” sample to be identified.

[0012] For the test to pass and for the overall measurement of the sample support plate to be valid, the intermicrobial spectral distance between the two reference microorganisms must be greater than a minimum spectral distance B to demonstrate sufficient spectral specificity. When applied twice repeatedly, four intermicrobial spectral distances can be calculated, each of which must meet the condition to pass the test. If each microorganism is applied with more than two replicates (e.g., five to ten), then 100% compliance is not required; specific quantiles of the intermicrobial spectral distances (e.g., two to ten percent, preferably five percent) may deviate from the limiting condition without causing test failure. A 5×5 sample spot (e.g.) generates 25 intermicrobial spectral distances between replicates of the two reference biomass; small deviations are permissible, such as one of these distances being less than B, without invalidating the overall measurement.

[0013] Assuming a large number of replicates are used, similar considerations for the maximum spectral distance A within microorganisms naturally apply to variations of this method. To indicate a minimum level of spectral reproducibility, the intramicrobial spectral distances of the replicates relative to each other and to themselves, or at least their larger quantiles (>90%), should be less than this specified maximum distance A.

[0014] Experienced practitioners can also carefully examine IR spectra and draw conclusions from them using (partially) non-compliance of test standards, rather than labeling IR measurements as failed measurements (warnings). This allows for repeatable measurements under modified and optimized conditions, where possible. For example, optimized humidity in the sample support chamber can yield better results. If the signal-to-noise ratio in the absorption band is too low, several acquisitions of the same sample spot can be combined to improve the database used for evaluation. Finally, the preparation of the microbial sample on the sample carrier can be examined to see if the microbial sample is applied with a substantially uniform layer thickness. If a substantially uniform layer thickness is not confirmed, preparation can be repeated while paying particular attention to this characteristic; and these optimization attempts can be monitored and checked using microbial test standards.

[0015] Beyond the obvious taxonomic hierarchy of species and subspecies, spectral distances between microorganisms can arise from the presence of microorganisms with varying detailed characteristics. The term "strain" (e.g.) describes the growth from a single organism and its location in a (usually state-run) collection for microbial strains, where the International Standardized Name of the strain is added to a nomenclature chain that includes species, subspecies, and variety type. The individual organisms within a strain share the same genome; the genetic makeup of different strains varies slightly, but this provides a wide variety of possible combinations of strains with different spectral characteristics and distances, which can be suitable as a standard for microbial testing in IR spectroscopy.

[0016] For example, other spectral specificities can be expressed by serotype or serogroup. The term serotype or serogroup (simply referred to as serological variant) is used to describe a variant within a bacterial subspecies that can be distinguished by serological testing. This is different from antigens on the cell surface and is identified in routine microbiology by means of specific antibodies. The classification hierarchy of serotypes is as follows: species > subspecies > serotype, for example, where the complete two items are: species name *Salmonella enterica*, subspecies *enterotype*, and serotype *Salmonella typhi*, which is simply *Salmonella typhi*.

[0017] A pathogenic type (derived from the Greek word for "disease") is a bacterial strain or group of strains that share the same pathogenicity, distinguishing it from other strains within a species or subspecies. Pathogenicity is indicated by a third or fourth addition to the two species names. For example, the bacterium *X. axonopodis*, which causes citrus canker, has various pathogenic types with different host specializations: *X. axonopodis* pv. citri is one of them. The abbreviation "pv." stands for "pathogenic type." Virulent strains of human pathogens also have pathogenic types, but in this case, the pathogenicity is indicated by a prefix before the name. Normally harmless enteric bacteria such as *Escherichia coli* have extremely dangerous pathogenic types, including enterohemorrhagic *E. coli* (EHEC), enteropathogenic *E. coli* (EPEC), enterotoxigenic *E. coli* (ETEC), enteroinvasive *E. coli* (EIEC), enteroaggregative *E. coli* (EAEC), and diffusely adherent *E. coli* (DAEC). Pathogenicity can also include different serotypes. Many known serotypes of EHEC exist, with approximately 60% of all identified EHEC serotypes being O157, O103, and O26. The serotype O157 / H7 is particularly dangerous. It should be understood that microorganisms harmless to humans are clearly preferred for routine laboratory microbiological testing standards.

[0018] In a broader sense, microorganisms can also be classified into variants that differ in several ways: other medically relevant characteristics, particularly their resistance to antibiotics (especially β-lactam and glycopeptide antibiotics), and their toxin formation (“toxin type”) or susceptibility to the same or similar phages (“phage type”). Generally, the term “biotype” is used if a group of microorganisms of a species or subspecies shares common biological characteristics. An example of an antibiotic-resistant variant is MRSA: methicillin-resistant Staphylococcus aureus.

[0019] The dried biomass can be sterilized to prevent microbial contamination that could distort measurements; in particular, the dried biomass can be prepared in the form of vacuum-dried pellets. This preparation method is especially suitable for rapidly processing test standard suspensions to produce replicas on sample carriers in the laboratory during rapid IR measurements.

[0020] In a preferred embodiment, the microorganism belongs to different strains of a bacterial species. In a particularly preferred embodiment, the species Escherichia coli should be used by way of example, wherein the dried biomass in different vessels may comprise the strains DH5α (DSM6897) and ML3 (DSM 1058). The two bacterial strains allow the determination of a defined inter-microbial spectral distance in order to thus monitor and evaluate the reproducibility between replicas of the strains and the spectral specificity between the respective measurements of different strains. This will be particularly advantageous when the strains regularly occur in the microbial sample and thus have sufficient practical relevance, which is the case for the Escherichia coli strains. The inter-microbial spectral distance may manifest itself as the distance between two principal components of a principal component analysis. In a further preferred embodiment, the spectral distance may also be Euclidean, which corresponds to the difference of n-dimensional spectral vectors. Depending on the resolution or the selected wavenumber range, the dimension may start in a two-dimensional range and extend to several hundreds or more, for example 20 < n < 2000, more precisely 400 < n < 600, for example n = 500.

[0021] The vessel may have a screw cap for fixing the opening and resealing. Even after a solvent has been added to the vessel to process the suspension of the microorganism, the suspension may be stored for a long period (e.g., up to ten weeks) for further use without adversely affecting the information value of the spectrum obtained therefrom.

[0022] According to a second aspect, the invention relates to a method of using such microorganisms for testing, comprising: - providing a sample carrier for infrared spectrometry, said sample carrier having an array of sample spots arranged in a matrix row-column arrangement, typically 48, 96 or 384; - resuspending the biomass in the vessel by adding a solvent such as distilled water and / or deionized water; - extracting a predetermined amount of the suspension from each vessel and then depositing the suspension on a predetermined number of sample spots, preferably repeating two or three times; - drying the suspension on the sample spots, if appropriate, by means of heat and convection; - preferably obtaining the infrared spectrum of the sample spots in transmission; - calculating the inter-microbial spectral distance between the spectral characteristics of the infrared spectra of different sample spots; and - determining whether the spectral distance exceeds a predetermined range. If this is the case, then the IR spectrum of the sample carrier may be marked with a corresponding (warning) label.

[0023] In different embodiments, the microbial spectral characteristics may be calculated preferably in the range of approximately 1300 and 800 cm -1The spectral distance is calculated within a predetermined wavenumber range to reduce the influence of interfering water absorption bands, and this spectral distance can correspond to a predetermined distance, such as the Euclidean distance directly corresponding to the spectral vector. Hierarchical clustering analysis (HCA) can also be used to analyze a large number of spectra. Alternatively, principal component analysis of microbial spectral features can be performed, and the distance then corresponds to a predetermined distance in the principal component space. Alternative procedures for distinguishing different microorganisms derived from supervised machine learning include Artificial Neural Network Analysis (ANN), Partial Least Squares Discriminant Analysis (PLS-DA), and Support Vector Machines (SVM). However, in these cases, what is crucial is not the spectral distance, but rather the correct assignment of the test spectrum to the expected category.

[0024] In addition to calculating the distance between microorganisms, it is also possible to calculate the intramicrobial spectral distance (i.e., the distance between replicas of the same microorganism) between the spectral characteristics of the infrared spectra of different sample spots for the purpose of checking spectral reproducibility.

[0025] (i) The predetermined range of intramicrobial spectral distances preferably extends to a maximum distance A, which is a measure of the reproducibility of the acquired distance, and (ii) the predetermined range of intermicrobial spectral distances preferably extends from a minimum distance B, which is a measure of the spectral specificity of the acquired distance. In various embodiments, a small amount (e.g., two percent to ten percent, precisely five percent) of spectral distance between different sample spot IR spectra may not satisfy the distance condition without generating a negative label or even a claim of invalidity for all measurements from this sample carrier, subject to the limitation that the vast majority of the distances satisfy the distance condition.

[0026] In terms of the uniform distribution of suspended microbial cells, resuspension can be promoted by agitating the vessel. Vessels with screw caps, in particular, ensure reliable protection of the surrounding environment from accidental release of microbial aerosols and are suitable for storing residual suspension for several weeks for later use. When the vessel is opened, mixing pipetting can be used additionally or alternatively as needed to promote resuspension.

[0027] In various embodiments, the shelf life of the suspension can be extended by adding ethanol. Following such measures, the prepared suspension can be stored for several weeks, specifically up to ten weeks, for subsequent use in measurement monitoring and process control.

[0028] It is particularly feasible to evaluate infrared spectra by using Fourier transform, because unknown recursive extinction signals with high sensitivity can be detected as a result. Attached Figure Description

[0029] The invention can be better understood by referring to the following description. The elements in the description are not necessarily drawn to scale, but are primarily intended to illustrate the principles of the invention (generally schematic). In the description, the same reference numerals denote corresponding elements in different views.

[0030] Figure 1A This is a schematic diagram of the first part of an example of a microbial testing standard.

[0031] Figure 1B A schematic illustration is provided of IR spectral measurements performed on a dry sample of a corresponding example having an extinction spectrum during transport.

[0032] Figure 1C A schematic illustration of the different processing steps performed on the extinction spectrum prior to evaluation is given.

[0033] Figure 2 This provides an illustrative illustration of possible results for determining the spectral distance between replicas of two bacterial strains in a two-dimensional principal component space. Detailed Implementation

[0034] Although the invention has been described and explained with reference to several different embodiments thereof, 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 as defined in the appended claims.

[0035] The dried biomass for microbial IR testing standards can be produced as follows: Different reference microorganisms are cultured overnight on a two-dimensional medium (e.g., Columbia sheep blood agar). The cultured cells are inoculated into several hundred milliliters of nutrient medium (e.g., LB lysogenic medium), and then allowed to grow again at 37°C with gentle agitation at several hundred rpm for several hours (e.g., 12 to 24 hours). The enriched culture can then be divided between several centrifuge dishes and centrifuged at several kJ for several minutes. The supernatant is treated, and the remaining microbial pellets are resuspended in water to remove residual nutrient medium. Renewal centrifugation and resuspension in water (supplemented as needed by adding proton solvents such as ethanol) produce a suspension in which the microbial content can be determined by measuring optical density (e.g., according to McFarland's standard). If the concentration of suspended microorganisms is sufficient, the suspension can be aliquoted into plastic dishes and dried therein (e.g., in a vacuum at a slightly elevated temperature) to form ready-to-use pellets with the liquid removed within the dish itself.

[0036] Figures 1A to 1C This outlines possible procedures for using microbial testing standards.

[0037] The dried biomass (2) for the two reference microorganisms can be supplied in a plastic container (4) with a screw cap (6). To prepare the reference sample, the screw cap (6) is removed and a certain amount of solvent, such as distilled water or deionized water, is added to resuspend the dried pellets (2). After the container (4) has been resealed, the procedure can be aided by photovibration or, alternatively, by repeatedly drawing the liquid into and then withdrawing it from the tip of a pipette (“mixed pipetting”) without forming any air bubbles (not shown).

[0038] After a few minutes, when the solid biomass has “dissolved” and is no longer visible, the lid (6) can be removed again and a certain amount of the microbial suspension (8) removed from the vessel (4), and the microbial suspension (8) applied to several sample spots (A to H; 1 to 12) on the IR spectroscopy sample carrier (10). This can be repeated two, three, or more times, as illustrated schematically by two sample carriers (10) with a 96-spot array (eight rows A to H, twelve columns 1 to 12). Generally, the statistical basis for spectral distance determination can be improved by increasing the number of replicates of the test standard, but this requires a smaller number of corresponding sample spots of the analytical sample actually to be identified on the sample carrier. For clarity, the latter is not shown here. The droplets of the test standard suspension are dried, which is assisted if necessary by thermal irradiation at a temperature slightly above room temperature and / or gas flow.

[0039] Next, using an IR spectrometer (12a, 12b), preferably during transport, the microorganisms of the reference standards (#1, #2) are measured in the same manner as the actual samples, as shown. An example of such a spectrometer is the TENSOR II FT-IR from Bruker Optik. The result of such measurements is an extinction spectrum, as illustrated by the example... Figure 1B The image is shown at the bottom. Optical density (OD) is plotted as a function of wavenumber, as is common in spectroscopic measurements. Under each condition, the acquired spectra are distinguished twice and smoothed within a specific number of data points. Then, the wavenumber range in which the absorption bands of microbial cells, particularly those producing carbohydrates and proteins (e.g., water absorption bands), are most prominent relative to background effects and thus provide the best signal-to-background ratio is selected. This is done at approximately 1,300 and 800 cm⁻¹. -1 The range between these two is particularly suitable for this situation; Figure 1C .

[0040] Vector normalization prepares the selected spectral range for subsequent evaluation of the spectral distance metric. One option for determining the spectral distance from a specific extinction signal of a microorganism is principal component analysis. The key issue here is selecting microorganisms for different vessels such that they can represent reliable, subtle, but clear differences in different components. While the spectral distance between microorganisms is essentially defined by a lower bound, it should not be set too high to assess spectral specificity within the performance limitations of the infrared spectrometer. Nor should it be set too low to offset the risk of random overlap caused by variance in the principal component data cloud, which represents a certain variance between measurements.

[0041] Figure 2 This is a schematic illustration of principal component analysis results based on the IR spectra of two bacterial strains, using an example. The intramicrobial spectral distance between replicas of the same microorganism must not exceed a certain peak A, as doing so would make identification impossible and cast doubt on the quality of the measurement. Furthermore, the intermicrobial spectral distance between different replicas of different microorganisms used must not fall below a certain lower limit B, as this would result in insufficient spectral specificity. The more replicas of each microbial suspension, the more statistically reliable the assessment obtained. However, the available space for actual microbial measurements on the sample carrier must be considered.

[0042] As in Figure 2 As can be seen, when the same microorganism is measured again, two roughly interpreted separate data clouds form in a two-dimensional principal component space. In each case, these clouds are close to each other relative to replicas of one organism, but at a considerable distance from replicas of another organism (>0.15 units in any of the examples shown). Since the microbial testing criteria are studied in the same measurement process as the actual microbial sample, this distance of the calculated principal components allows for the evaluation of the spectral specificity of the IR spectrometer. Simultaneously, the reproducibility of the IR measurements can be evaluated by means of the intramicrobial spectral distances of replicas of the same microorganism relative to each other and between themselves. If the data points from replicas of the same organism exceed the maximum permissible distance A, this is considered an indication of a problem with measurement reproducibility. Parallel measurements of the actual sample can then be labeled accordingly. If the data clouds of different microorganisms are even deeply embedded in each other, then the spectral specificity will be insufficient. This reduces the quality of identification, and it may even be necessary to label the identification / characterization results from the sample carrier in question as unreliable under the given measurement conditions.

[0043] The above use of principal component analysis should not be construed as limiting, but rather as illustrative examples. Alternative methods for determining spectral distances may also be used, such as using inherent Euclidean distances and / or employing hierarchical cluster analysis to perform the principles of this disclosure. Test spectra may also be classified using appropriately pre-trained ANNs (Artificial Neural Network Analysis), PLS-DA (Partial Least Squares Discriminant Analysis), or SVMs (Support Vector Machines).

[0044] The principles of the invention have been explained using two spectrally distinguishable microorganisms. However, those skilled in the art will recognize that more than two suitable microorganisms can be used to provide microbiological testing standards for infrared spectroscopy determination. In this respect, the basic concept can be expanded as needed.

[0045] The invention has been described above with reference to various specific examples and embodiments. However, it should be understood that various aspects or details of the described embodiments may be modified without departing from the scope of the invention. Specifically, features and measures disclosed with respect to different embodiments may be combined as needed, if it appears practicable to those skilled in the art. Furthermore, the above description serves only as an illustration of the invention and not as a limitation on the scope of protection; considering any possible equivalents, the scope is defined only by the appended claims.

Claims

1. A method for using microbial testing standards, comprising: A sample carrier for infrared spectroscopy is provided, the sample carrier having an array of sample spots. The biomass in the vessel is resuspended by adding a solvent. A predetermined amount of suspension is removed from each vessel, and then the suspension is deposited onto a predetermined number of sample spots. Dry the suspension on the sample spot. Infrared spectra were obtained from the sample spots; Calculate the intermicrobial spectral distance between the spectral characteristics of the infrared spectra of different sample spots; as well as Determine whether the spectral distance exceeds a predetermined range; The microbial test standard has at least two resealable containers that are impermeable to liquid when closed, each of the at least two resealable containers containing a predetermined amount of dried biomass of microorganisms, wherein the microorganisms in the different containers differ in at least one characteristic and the difference is manifested by a predetermined spectral distance in the infrared spectrum between the microorganisms, wherein the dried biomass comprises vacuum-dried pellets.

2. The method according to claim 1, wherein the spectral distance is calculated based on the spectral characteristics of microorganisms in a predetermined wavenumber range and corresponds to a predetermined distance.

3. The method of claim 2, wherein the predetermined wavenumber range is 1,300 cm⁻¹. -1 With 800 cm -1 between.

4. The method of claim 1, wherein the intermicrobial spectral distance between the spectral features of the infrared spectra of the different sample spots is calculated to check spectral reproducibility.

5. The method of claim 4, wherein the predetermined range of intermicrobial spectral distance extends to a maximum distance A, said maximum distance A being a measure of the reproducibility obtained, and the predetermined range of intermicrobial spectral distance extends from a minimum distance B, said minimum distance B being a measure of the spectral specificity obtained.

6. The method of claim 1, wherein the solvent used for resuspension contains distilled water and / or deionized water.

7. The method of claim 1, wherein resuspension is facilitated by agitating the vessel or by mixing the pipette.

8. The method of claim 1, wherein when the spectral distance exceeds the predetermined range, a warning label is used to mark the IR spectrum.

9. The method of claim 1, wherein multiple copies of the microbial suspension are applied to the sample carrier.

10. The method of claim 1, wherein the at least one characteristic is selected from the group consisting of: species, subspecies, strains, serotypes, pathogenic types, toxin types, and variants.

11. The method according to claim 1, wherein the microorganism belongs to a different strain of a bacterial species.

12. The method of claim 1, wherein the vessel has a screw cap.

Images