Method of processing test samples for a metagenomic analysis

Abstract

The present invention provides improved methods and kits for isolating microbial nucleic acids, preferably microbial DNA, from a tissue sample comprising a eukaryotic tissue, said method comprising the steps of: a) treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, b) treating the sample of step a) with at least one nuclease, and c) processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA. The method significantly improves depletion of host cell DNA.

Description

[0001] 19 August 2025

[0002] 065812 K

[0003] Method of processing test samples for a metagenomic analysis:

[0004] FIELD OF THE INVENTION

[0005] The present invention pertains to the field of metagenomic analysis and to a method of processing tissue samples for metagenomic analysis. The method includes the processing of swabs as well as of solid tissue samples, such as cancer biopsies.

[0006] BACKGROUND OF THE INVENTION

[0007] Metagenomic analysis of the microbes associated with animal tissues is complicated by the relatively low concentration of bacteria and the comparatively smaller size of the bacterial genome, leading to a large excess of host DNA in seguencing. This reduces the amount of information available that can be assigned to the metagenomic analysis and increases costs for obtaining satisfying bacterial seguence information.

[0008] Methods exist, such as QIAamp DNA Microbiome Kit from QIAGEN, which can reduce host DNA in loosely dissociated host samples with higher bacteria content, such as buccal or genital swabs. These methods are based on a differential lysis of eukaryotic and bacterial cells, e.g. by the use of saponin, which attacks the cholesterol in the cell wall of eukaryotes. But these methods are not able to effectively deplete host DNA from tissue sample that have a higher content in fibrous materials and / or are characterized by a low ratio of bacterial cells to host cells.

[0009] Hence there is a need for methods that are especially designed to process such tissue samples for assigning them to metagenomic analysis. Furthermore, there is a need for providing improved methods for isolating microbial nucleic acids, such as microbial DNA, from diverse tissue samples.

[0010] SUMMARY OF THE INVENTION

[0011] The method according to the invention is for isolating microbial nucleic acids, preferably microbial DNA, from a tissue sample comprising a eukaryotic tissue, said method comprising the steps of: a) treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, b) treating the sample of step a) with at least one nuclease, and c) processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA. The method according to the invention enables metagenomic sequencing of tissue sample of eukaryotic cells, in particular of animal tissues, by first dissociating the tissue by means of enzymatic homogenization (also “tissue dissociation”; step a)), and then depleting host DNA enzymatically by nuclease digest (also “host DNA depletion”; step b)). The sample that had been the subject of tissue dissociation and host DNA depletion then is subject to further downstream processing (step c)) to isolate the microbial nucleic acids, in particular the microbial DNA, more preferred bacterial DNA. The isolated microbial DNA can subsequently be the subject of genetic analysis (step d)).

[0012] The isolation of the microbial nucleic acids also includes the isolation of the nucleic acids, in particular the DNA, of archaea. Archaea are defined as of single-celled microorganisms that, although similar in shape and size to bacteria, have a unique evolutionary history and biochemistry.

[0013] The present invention enables in particular the analysis of tissue samples for microbiome DNA, in particular for bacterial DNA, by effectively freeing bacteria from the associated eukaryotic cells and thoroughly depleting the DNA derived from the host cells. In the most preferred embodiment of the invention an animal tissue sample is investigated for its bacteriome. A bacteriome is the collective community of bacteria in a certain environment (here the tissue sample).

[0014] According to the invention a combination of collagenase and neutral protease is used to dissociate the eukaryotic tissue (e.g. the animal tissue) into smaller clusters of cells. By this step of tissue dissociation, the eukaryotic cells seem to become vulnerable to the action of the nuclease (e.g. benzonase), which will effectively deplete the host DNA, while leaving microbial (in particular the bacterial) cells and their DNA intact. This improves the investigation of solid tissue samples for its respective bacteriome, for example solid tissue samples from cancer (e.g. tissue samples derived from cancer biopsies) or other solid tissue samples (especially gut samples).

[0015] The microbial cells, in particular bacterial cells, can be collected as fraction, e.g. in form of a pellet, and the comprised microbial, in particular bacterial DNA can be isolated therefrom by disrupting and lysing the microbial cells and purifying the comprised DNA. For this processing step, standard methods can be used. Such standard methods may comprise for example a mechanical lysis (e.g. by using the PowerBead Pro tubes, QIAGEN), followed by binding (e.g. to silica), washing and eluting. The isolated microbial (in particular the bacterial) DNA can then be assigned to an analytical step, e.g. a genetic analysis such as new generation sequencing.

[0016] Surprisingly, it was found that the method of the invention renders a differential lysis after tissue dissociation, such as the known saponin digest, unnecessary. The method of the invention thus, preferably, does not include a step of saponin digest in step a) or b). This new method improves the efficiency of host depletion methods, and furthermore is applicable to tissue types that were previously not able to be processed.

[0017] The avoidance of using saponin for differential cell lysis has multiple advantages: saponin is an undefined mixture of detergents derived from plant ash - as such it is difficult to control its performance in a product and requires time-consuming functional testing each time a new lot is made. Additionally, some bacteria, like those which coopt host cell machinery to build their cell walls and thus can incorporate cholesterol, may be lysed during exposure to saponin buffer. Thus, excluding saponin from the protocol for tissue dissociation improves the microbial diversity seen due to the increased relative number of microbial reads sequenced. Therefore, it is advantageous that the method of the invention can be performed without a saponin digestion step.

[0018] Importantly, the invention is not only a significant improvement in methods for investigating tissue-associated bacteria, in particular in solid tissue samples, but also continues to be applicable to any types of tissues samples, including tissues from swabs, in particular oral microbiome samples, such as saliva or buccal swabs or other swabs, including in particular also genital swabs.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] The essential findings and advantages of the invention are briefly summarized as follows based on the discussions of the figures.

[0021] Figure 1 illustrates an exemplary general workflow according to the present invention for host DNA depletion for tissue samples. Breaking down the tissue matrix is the first essential step of the tissue microbiome protocol and is achieved via an enzymatic dissociation solution. Preferably mechanical homogenization (e.g., via TissueRuptor, QIAGEN) of each sample is performed prior to processing, to account for inhomogeneous distribution of bacterial cells within the tissue. For host DNA removal, eukaryotic cell lysis (step a)) is followed by enzymatic depletion of host nucleic acids (step b)), while bacterial cells remain pristine. Subsequent measures of step c) of the invention comprise in particular the mechanical lysis of bacterial cells using e.g. PowerBeadPro Tubes (QIAGEN) for maximum lysis across taxa without discrimination or loss of sensitive bacterial groups.

[0022] The sample with the microbial DNA, in particular with the bacterial DNA, can then be bound to silica to facilitate purification and washing, wherein the binding chemistry is optimized to remove any remaining RNA and host DNA fragments. Thereby the microbial DNA, in particular the bacterial DNA, is isolated. In the method of the invention as performed in the example section, binding of the microbial DNA occurs in the presence of a chaotropic salt. This ensures good binding of the microbial DNA to the solid phase. Although in embodiments an alcohol such as ethanol may be added for the microbial nucleic acid binding step, it is preferred that no C1-C5 alkanol, such as ethanol or isopropanol, is added during binding when aiming at isolating exclusively microbial DNA from the lysed bacterial sample. As is demonstrated by the working examples of the invention, where in contrast to the prior art methods (such as the QIAamp DNA Microbiome kit) no ethanol is added during the microbial DNA binding step, this may reduce RNA binding and may further improve removal of remaining host DNA fragments.

[0023] Figure 2 demonstrates the enhanced removal of host DNA in various types of tissues (including fibrous tissues and soft tissue) by use of the present invention in comparison to known commercially available kits.

[0024] The performance of the method according to the present invention (denominated here: “TissueP”) is compared to the QIAamp DNA Microbiome Kit (QIAamp) from QIAGEN, which features a host DNA removal solution optimized for sputum, saliva and swab samples. DNA yields obtained from different sample types are quantified by Qubit (QIAGEN) measurement of total DNA that serves as a proxy for host DNA, and 16S rRNA gene qPCR that represents bacterial DNA. Bacterial DNA yields obtained from buccal swab samples by use of the preset invention is in the same order of magnitude as the ones obtained from QIAamp workflow, while host DNA is almost completely removed from the original sample (FIG. 2 A). Total DNA yields is significantly reduced in tissue samples processed according to the present invention instead of QIAamp, without compromising bacterial DNA recovery (FIG. 2 A). A reduction in host-to- bacterial DNA ratio of 2-3 orders of magnitude is achieved by the method according to the present invention in comparison the commercially available DNeasy PowerSoilPro kit (PSP) from QIAGEN (FIG. 2 B). The improved performance is attributed to the tissue dissociation step a) that is comprised in the method of the present invention. Homogenization is achieved in both soft and fibrous tissues by processing the tissue samples according to the present invention while residual tissue pieces are still visible in the samples processed according to QIAamp workflow (FIG. 2 C; importantly: the asterisk in Fig. 2B and C indicate that the unit is ng / mg for both total and bacterial DNA in buccal swabs)

[0025] Figure 3 shows the microbial diversity detected in bovine omasum swabs using the method according to the present invention (“Tissue P”) in comparison to workflow of the QIAamp DNA Microbiome kit (QIAamp), QIAGEN, and DNeasy PowerSoilPro kit (PSP), QIAGEN. An overall improvement in the microbial diversity is observed by use of the method according to the present invention that allows for detection of all taxa detected by use of the commercially available kits and various additional taxa that could not be detected when processing the sample according to the workflow of the commercially available kits. Taxonomic profiles (species-level) are assigned by matches with the Unified Human Gastrointestinal Genome (UHGG) database carried out with CLC Microbial Genomics Module, QIAGEN, Aarhus, version 24.1.1).

[0026] Figure 4 shows the analysis of whole genome sequencing data from different tissue samples that had been processed in accordance with the present invention. It demonstrates that the method of the present invention enables the recovery of highly diverse low-abundance grampositive and -negative bacteria, thereby facilitating discrimination between largely similar samples. (A) Taxonomic profiles (species-level) assigned by matches with the Unified Human Gastrointestinal Genome (UHGG) database carried out with CLC Microbial Genomics Module, QIAGEN, Aarhus, version 24.1.1). (B) Average representation of Gram-positive and -negative bacterial taxa in the sequenced samples. (C) Host-bacterial read ratio in samples processed according to the present invention (Tissue P) in comparison to the workflow of the DNeasy PowerSoilPro kit (PSP), QIAGEN. Depicted values are means of quadruplicates. (D) Taxonomic composition of microbial communities in rat intestinal tissue. Tissue specimens have been processed in accordance with the present invention and following the workflow of the DNeasy PowerSoilPro kit (PSP), QIAGEN for host DNA depletion. Relative abundances were recovered from shallow (QNS390) and deep (QNS391) sequencing of the same library pool and subsequent taxonomic profiling against the UHGG database. The analysis is carried out using the CLC Microbial Genomics Module (QIAGEN, Aarhus, version 24.1.1). (E) Average number of taxa found per sample in rat intestinal tissue microbiomes. Relative abundances of taxa are categorized into low (<1 %), mid (>1 %) and high (>10 %).

[0027] In more detail: A high resolution is achieved by increasing the relative number of bacterial reads via host DNA removal (illustrated in FIG. 4 C). Host DNA depletion can further help keeping sequencing costs at bay by increasing the relative number of bacterial reads per sample. The method according to the present invention enables recovery of the main taxa from microbial communities, already at shallow sequencing depth (0 26k reads / sample) (FIG. 4 E). In comparison, much deeper sequencing (0 2,720k reads / sample) is required to adequately represent the tissue microbiome when the same sample pool is processed without host DNA depletion, for instance using the DNeasy PowerSoilPro protocol (Figure 4 D). Deeper sequencing of samples processed in accordance with the present invention captured on average 49 additional low-abundance taxa per sample, demonstrating that host DNA depletion according to the present invention preserves low abundance microdiversity. Accordingly, the present invention provides a reliable recovery of tissue microbial communities at different sequencing depths.

[0028] Figure 5 shows the bacterial DNA and total DNA detected following host DNA removal by use of the enzymes collagenase and dispase from different suppliers in comparison to enzyme mixtures with saporin comprising buffer is added. The QIAamp DNA Microbiome kit (QIAamp) was used as a reference. Supplier 1 is QIAGEN, Supplier 2 is Worthington and Supplier 3 is Merck. Total DNA was quantified via Qubit (Qub), QIAGEN, and bacterial DNA via 16S rRNA gene qPCR (16S).

[0029] The bars indicate the amount of bacterial DNA detected by 16S PCR - higher values indicate more bacteria was found. The dotted line indicates the amount of total DNA detected - lower values indicate a higher efficiency of host DNA removal. It is demonstrated that according to the invention, host DNA depletion is just as effective using the enzyme mixture without a detergent (e.g. saponin) as when using the saponin comprising buffer. Thus, the use of saponin is not necessary. A dramatic improvement in host DNA depletion is observed compared to QIAamp: a reduction of total DNA from ~1200ng to ~35ng, or 34-fold. That “contamination test” indicates if there is an extraneous microbial signature that comes from the enzymes used; hence this serves as a control to make sure that the enzymes are not contaminating the results with microbial DNA that does not derive from the sample.

[0030] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention provides a method for isolating microbial nucleic acids, preferably microbial DNA, from a tissue sample comprising a eukaryotic tissue, said method comprising the steps of: a) treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, b) treating the sample of step a) with at least one nuclease, and c) processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA.

[0033] The isolated microbial DNA can be the subject of genetic analysis. Thereby the method of the invention enables to investigate examine tissue sample comprising eukaryotic tissue, in particular animal tissue, for the microbiome DNA therein, in particular for its bacteriome. The method relates preferably to the processing of animal tissue samples, most preferred tissue samples of human origin. Most relevant, and preferred, are tissue samples that are to be investigated for their microbiome, in particular for the bacteria that could be comprised therein. Hence, in one specifically preferred embodiment the invention is a method of processing animal tissue possibly including bacteria for providing analytical samples for a metagenomic analysis. The genetic analysis, most preferably, is based on sequencing, such as new generation sequencing. Next generation sequencing methods are well-known and established in the art and thus readily available to the skilled person.

[0034] Microbes can be hosted within the cells of a eucaryotic tissue or can be found in the interstitial spaces between the cells of the tissue. Hereinafter no difference will be made between these situations. The eukaryotic cells of the tissue will be denominated as “host cells”; the DNA of the eukaryotic cells will be denominated as “host DNA”. In the present context, the term “metagenomic analysis” is understood as an analysis of genetic material recovered from a sample, aiming at analyzing the collective genomes of the microbial community present in that sample. As mentioned above, preferably the microbes are bacteria. The focus of the invention lies on bacterial DNA and bacterial cells and the possible collective bacterial genome of the tissue under investigation.

[0035] The method of the invention comprises at least three steps, namely step a) which is treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, step b) which is treating the sample that had been the subject of step a) with a nuclease, and step c) which is the processing of the sample resulting from step b) in order to provide an analytical sample that is suitable for the genetic analysis.

[0036] In step a) the tissue is dissociated. Hence this step is also called “tissue dissociation” (or “breaking down the tissue matrix”). The result of this step is “dissociated tissue”. In the present invention the tissue dissociation is based on enzymatic homogenization. Tissue dissociation is a process where a tissue is broken down in individual cells or cell clusters.

[0037] In step b) the host cell DNA is digested with the nuclease. This step is also called “host DNA depletion” or “DNA removal”. It encompasses any reduction or removal of the host DNA from a sample. The effective reduction of the host cell DNA is in particular relevant when aiming at analyzing the microbial DNA in a sample in which the host cell DNA (as e.g. the human DNA in a clinical sample) can vastly outnumber the microbial DNA, leading to a significant background signal. Hence, the invention also relates to a method of enriching the ratio of the microbial DNA versus the host cell DNA, in particular the ratio of bacterial DNA to host cell DNA in an analytical sample, making it easier to sequence and analyze the sample for the bacterial DNA.

[0038] In a preferred embodiment, after the nuclease digestion in step b), the nuclease is inactivated, preferably by enzymatic degradation.

[0039] A well-known method of enzymatic degradation is the treatment with proteinase K. The reaction is often performed by adding proteinase K to the sample containing the enzyme (here the nuclease) and heating the sample e.g. up to 56°C for 30 minutes to several hours. This temperature range is optimal for Proteinase K activity and helps to ensure complete degradation of the nuclease. Hence, in one preferred embodiment of the invention, the sample having been the subject of tissue dissociation and host cell depletion is treated with proteinase K for the inactivation of the nuclease, before the microbes / bacteria are disrupted.

[0040] Preferably, the microbial cells, in particular the bacterial cells, remain intact during the processing of step a) and step b).

[0041] The term "neutral protease" is used herein to define an enzyme with proteolytic activity at a pH between 7 and 8. Most preferred is a neutral protease with an activity at about pH 7. According to the invention, the neutral protease preferably is selected from the group consisting of trypsin, elastase and neutral proteases form Bacillus spec., such as Bacillus subtilis, Bacillus licheniformis, Bacillus thermoproteolyticus or Bacillus polymyxa. The neutral protease derived from Bacillus thermoproteolyticus is known as thermolysin (CAS No 9032- 08-0). The most preferred embodiment of the invention is the neutral protease derived from Bacillus polymyxa. This enzyme is also known under the name “dispase” (CAS No 9034-01- 3), which will also be used hereinafter. Dispase has a mild enzymatic activity that minimizes damage to cell surface proteins. It targets specific proteins in the extracellular matrix, allowing for selective tissue dissociation.

[0042] The most preferred embodiment of step a) is treating the sample with the neutral protease dispase and a collagenase.

[0043] A collagenase breaks down collagen. Collagenases are endopeptidases that digest native collagen. Collagens are the major fibrous component of animal extracellular connective tissue (see also below). Bacterial collagenases differ from vertebrate collagenases in that they exhibit broader substrate specificity. Unlike animal collagenases that split collagen in its native triplehelical conformation, bacterial collagenase is unique because it can degrade both waterinsoluble native collagens and water-soluble denatured ones. It can attack almost all collagen types and is able to make multiple cleavages within triple helical regions. According to the invention bacterial collagenase is preferred. In a specifically preferred embodiment of the invention, the bacterial collagenase is derived from Clostridium histolyticum.

[0044] Various types of collagenases derived from Clostridium histolyticum are known, namely collagenase of type I, II, III and IV and type B. They all slightly differ in terms of their activities. According to the present invention the collagenase of type IV (CAS No 9025-66-7) and type B (CAS No 9001-12-1) are preferred. The type IV collagenase is less active than the other types and is enriched in clostripain activity, which is a cysteine protease that degrades non-collagen proteins.

[0045] The most preferred embodiment of step a) is treating the tissue sample with a collagenase IV and dispase.

[0046] In step a) the sample can be treated with both enzymes, the collagenase and the neutral protease, together, i.e. simultaneously, or sequentially. The simultaneous treatment of the tissue sample with both enzymes is preferred. In general, it is preferred to accomplish the treatment in such a way that the enzymes can exhibit their proteolytic function on the tissue dissociation synergistically.

[0047] The treatment according to step a) is performed at a temperature that is suitable for the biological activity of the enzymes. A preferred temperature is around 37°C. The time period of the treatment can vary; a suitable and preferred incubation time is around 30 min; if adequate the sample can be mixed or shaken (e.g. by rotating at 600 rpm).

[0048] The two proteases can be added to the tissue sample in step a) separately or as a fixed combination. In order to simplify the procedure, adding the enzymes as a fixed combination, i.e. as a composition comprising both enzymes, is preferred. Fixed combinations of a neutral protease and a collagenase are commercially available, e.g. of collagenase and dispase (e.g. STEMxyme® from Worthington or SCR139 from Merck). Hence, in a preferred embodiment of the invention the treatment of step a) is accomplished by adding to the tissue sample a composition comprising a collagenase and a neutral protease, in particular collagenase IV and dispase.

[0049] The ratio of the two proteases that are added to the tissue sample can widely vary. In a preferred embodiment of the invention, the ratio of the collagenase to the neutral protease is 1 :5 to 10:1 , preferably from 1 :2 to 8:1 , more preferred from 1 :1 to 5:1 , most about 5:1 , based on enzyme activity units (II). The same ratios apply when the enzymes are added to the tissue sample in a composition comprising both enzymes.

[0050] In step b) the sample that had been the subject of step a) is treated with a nuclease. Notably, this does not mean that the sample resulting from step a) is directly treated with a nuclease without further manipulation. Preferred, step a) comprises further steps such as e.g. a separation or purification step. This includes preferably separating the treated sample into a solid fraction and a liquid fraction, optionally wherein the separation is assisted by centrifugation or sedimentation, preferably centrifugation.

[0051] When a separation step is applied, that the liquid fraction is preferably discarded, optionally as supernatant, and the solid fraction can be kept as sample of step a) for the further treatment of step b). Hence, in a preferred embodiment the solid fraction obtained thereby is subjected as sample of step a) to the nuclease treatment in step b).

[0052] Furthermore, it may also be advantageous to perform prior to step a) an upstream step, e.g. a step in which the tissue sample is homogenized, e.g mechanically homogenized. Mechanical processing of the sample, such as grinding and mincing, has proven to be particularly advantageous. Hence, in one preferred embodiment of the invention, the method includes a step before step a), wherein the tissue sample is homogenized, preferably it is mechanically homogenized.

[0053] The treatment according to step b) is performed at a temperature that is suitable for the biological activity of the nuclease. A preferred temperature is around 37°C. The time period of the treatment can vary; a suitable and preferred incubation time is around 30 min; if adequate the sample can be mixed or shaken (e.g. by rotating at 600 rpm). Preferably, in step b) a digestion buffer is added.

[0054] As explained above, treating the sample with a nuclease results in host DNA depletion. Preferred nuclease according to the invention are selected form the group consisting of DNase, RNase, exonuclease or endonuclease. The selection of the nuclease is not specifically limited. In a specific embodiment a nuclease is used that has both DNAse and RNAse activity. Examples for such preferred nucleases is benzonase (CAS No 9033-24-3), saltonase (supplied by QIAGEN; Cat. No: EN32-050) or DNAsel. Especially preferred is benzonase.

[0055] Benzonase is a type of nuclease that nonspecifically degrades all forms of DNA and RNA into smaller oligonucleotides and nucleotides. Benzonase works efficiently over a wide range of conditions and is known to be used in applications where the removal of contaminating nucleic acids is critical, such as in protein purification or virus production. Benzonase functions by hydrolyzing the phosphodiester bonds in nucleic acids, specifically targeting both DNA and RNA. It acts as an endonuclease, cleaving these molecules into smaller oligonucleotides and eventually into nucleotides. The enzyme exhibits broad specificity, meaning it degrades singlestranded, double-stranded, linear, and circular forms of DNA and RNA. This non-specificity is crucial for applications where complete nucleic acid removal is necessary, such as in protein purification.

[0056] A further functionally similar enzyme to benzonase is DNase I (Deoxyribonuclease I; CAS No 9003-98-9). Like benzonase, DNase I degrades DNA, but it specifically cleaves DNA into smaller fragments by hydrolyzing the phosphodiester bonds within the DNA strand. DNase I is known to be used to remove DNA contamination from RNA samples or to reduce DNA viscosity in cell lysates, much like benzonase. However, DNase I primarily targets DNA, while benzonase degrades both DNA and RNA. As outlined above, in the context of the present invention, benzonase is the most preferred embodiment of the present invention.

[0057] As pointed out above, the method according to the invention allows the avoidance of using a saponin, or any other detergent in the step of tissue dissociation. Hence, in one preferred embodiment of the invention, the sample is not treated with saponin in step a). It is also possible to perform the method without the use of any detergent in step a). Notably, this does not exclude the optional use of a detergent in the further downstream processing of the sample after step a), in particular after step a) and or b). In particular the lysis of the bacterial cells after host DNA depletion can comprise the use of a detergent.

[0058] According to one embodiment, the method of the invention comprises

[0059] - mechanically homogenizing the tissue sample prior to step a); performing step a), wherein step a) comprises ■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably, the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,

[0060] ■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),

[0061] ■ optionally wherein during step a) the tissue sample is not treated with saponin;

[0062] - performing step b), wherein step b) comprises

[0063] ■ treating the solid fraction obtained in step a) with at least one nuclease,

[0064] ■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;

[0065] - performing step c), wherein step c) comprises isolating microbial nucleic acids, preferably microbial DNA, from the nuclease inactivated sample of step b).

[0066] The method of the invention is particularly useful for processing tissues that comprise components of extracellular matrix (ECM). The extracellular matrix (ECM) is a complex network of macromolecules that provides structural and biochemical support to surrounding cells. Components of the ECM are particularly, fibrous proteins (such as collagen, elastin, laminin), proteoglycans (such as glycosaminoglycans (GAGs)) and matrix metalloproteinases (MMPs). Accordingly, in one preferred embodiment of the invention, the sample tissue comprises tissue that is associated with ECM components selected from the list consisting of fibrous proteins, proteoglycans and MMPs. Most preferred is tissue which is associated with collagen.

[0067] It follows that a particularly preferred embodiment of the invention is the processing of a tissue sample that comprises connective tissue and / or fibrous cells. It also follows, that in a preferred embodiment of the invention, the samples comprise non-liquid tissue, such as blood or lymph. Preferably the tissue sample consists of non-liquid tissues. In a preferred embodiments the sample tissue comprises one or more of the following tissues: muscles, tendons, ligaments, fascia, cartilage, adipose tissue, blood vessels and nerves or mixtures thereof.

[0068] The sample that is particularly suitable for the method according to the invention is a sample including human tissue. In a specifically preferred embodiment it is a clinical sample, preferably from a biopsy or a swab, a biopsy is preferred. The biopsy for example is a colon biopsy, breast biopsy or any other solid tissue samples (particularly a gut sample). Notably, according to the invention also an environmental probe can be processed as a sample. A clinical sample from a human is especially preferred. The method of the invention further comprises step c). This third step of the invention follows the tissue dissociation and the host DNA depletion. The sample is further processed in order to provide the isolated microbial or bacterial DNA for the genetic analysis. Step c) can optionally encompass various steps, in particular the bacterial cell lysis (i) and DNA purification (ii). Preferably the bacterial cell lysis is assisted by mechanical and chemical disruption.

[0069] As noted, step c), i.e. processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA, may be divided in two core sub-steps, namely step c) (i), bacterial cell lysis, and step c) (ii), microbial nucleic acid isolation, preferably microbial DNA isolation.

[0070] As disclosed herein, bacterial cells remain intact under the conditions of steps a) and b).

[0071] In a specific embodiment the mechanical disruption of the bacterial cells in step c) are assisted by disrupting particles. The disrupting particles can have one or more of the following features:

[0072] (i) the particles are crystalline particles;

[0073] (ii) the particles comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), yttrium-stabilized zirconium, quartz, aluminium oxide, silicon carbide, ceramic, glasses (e.g. silicon dioxide glass or silica) or a combination of the foregoing;

[0074] (iii) the particles are substantially spherical;

[0075] (iv) the particles have a size that lies in the range selected from 0.05mm to 0.9mm, 0.07mm to 0.8mm, 0.08mm to 0.75mm and 0.09mm to 0.7mm;

[0076] (v) the particles are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide) or yttrium-stabilized zirconium having on average a size that lies in the range of 0.08mm to 0.7mm, preferably 0.09mm to 0.6mm, wherein preferably, yttria-stabilized zirconium beads are used as disrupting particles.

[0077] Such particles may advantageously be used in step c) (i), i.e. to support bacterial cell lysis. For the lysis of the bacterial cells, preferably, at least one lysis buffer is added to assist the chemical lysis of the bacterial cells. It is most preferred that the step of bacterial cell lysis comprises the addition of disrupting particles and at least one lysis buffer and chemical and mechanical disruption of comprised bacterial cells.

[0078] It is yet even more preferred, that the cell lysis comprises the following steps:

[0079] - adding a first lysis buffer and disrupting particles to the sample of step b), in which the nuclease is preferably inactivated by proteolytic digestion, and mechanically disrupting the sample in the presence of the first lysis buffer; - separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction, optionally wherein separation is assisted by centrifugation or sedimentation, preferably centrifugation and

[0080] - adding a second lysis buffer to the liquid fraction and incubating the mixture, optionally incubation is performed at elevated temperature, such as at a temperature within the range of 30°C - 85°C, 40°C - 80°C and 50°C to 75°C.

[0081] The bacterial cell lysis can be further specified as follows, as a particularly preferred embodiment with one or more, preferably all, of the following characteristics:

[0082] (aa) the first lysis buffer comprises a detergent, preferably an anionic detergent SDS;

[0083] (bb) the first lysis buffer does not comprise a chaotropic salt;

[0084] (cc) the second lysis buffer comprises a chaotropic salt, preferably a guanidinium salt such as GTC, and a detergent;

[0085] (dd) the second lysis buffer comprises the chaotropic salt in a concentration of 1 M-6M, preferably 3M-5M, and a non-ionic detergent, optionally wherein the non-ionic detergent is present in a concentration of 5%-30% (v / v); and / or

[0086] (ee) the second lysis buffer assists in establishing the DNA binding conditions in step c) (ii).

[0087] Further even more specific embodiments of the measure of step c) are disclosed as part of the subsequently discussed items and claims.

[0088] Due to the effective tissue dissociation and the subsequent effective host DNA depletion, the invention is particularly suitable for the processing of tissue samples which are typically characterized by a low ratio of bacteria cells to host cells. In preferred embodiments of the invention tissue samples are processed with a bacterial to host cell ratio of 1 :10 000 or less, more preferred 1 :25 000 or less, even more preferred 1 :50 000 or less, most preferred 1 :100 000 or less.

[0089] Advantageously, the ratio of bacterial cells to host cells in the sample having been the subject of step a) and b) is enriched, and preferably is at least 1 :1000, more preferred at least 1 :100, most preferred at least 1 :10. Accordingly, the method of the invention can result in a substantial enrichment of the ratio of bacterial cells versus host cells compared to the original tissue sample. The enrichment can be at least one order of magnitude, preferably at least two orders of magnitude, most preferred at least three orders of magnitude.

[0090] As a consequence of the advantageous enrichment of the bacterial cells, the ratio of host DNA to bacterial DNA in the analytical sample is reduced. The reduction amount to 2 or 3 orders of magnitude. Preferably, in the analytical sample, this ratio is 200 or less, preferably 100 or less, more preferred 50 or less, even more preferred 10 or less.

[0091] The method according to the invention is suitable for preparing a sample for the analysis of DNA of Gram-negative bacteria as well as of Gram-positive bacteria. Gram-negative bacteria are generally more difficult to be extracted than Gram-positive cells due to differences in their cell wall structures and the composition of their outer layers. Hence, the suitability of the method according to the invention particularly also for Gram-negative bacteria is of great benefit.

[0092] The improved efficiency of the method according to the invention, leading to a reduced host DNA load in the analytical sample, substantially improves the read out reflecting the microbial diversity that can be sequenced and identified in the metagenomic analysis. This effect is even further improved when the treatment of the tissue sample with saponin is avoided (see also the comments in the description of Figure 3 above). Accordingly, the method of the invention is specifically useful to increase the specificity of the taxonomic profiling in a metagenomic analysis of sample originating from a eukaryotic sample with a low load of microbial cells.

[0093] These advantages allow to subject the sample from steps a), b) and c) to genetic analysis even without a prior target amplification of bacterial DNA or the target sequencing of the bacterial DNA, e.g. by 16 S PGR. The analytical sample, preferably, can be the subject of next generation sequencing.

[0094] As outlined above the method of the invention includes step c) which relates to the downstream processing of the samples that had been the subject matter of step a) and step b). Possible means for processing to sample to optimize is applicability for a genetic analysis can comprise one or more of the following steps: disrupting the bacterial cells, isolating the bacterial DNA, washing the bacterial DNA, purifying the bacterial DNA and or eluting the bacterial DNA.

[0095] It has been experienced that it is advantageously to disrupt the bacterial cells by using yttria- stabilized zirconia beads. Furthermore, in a preferred embodiment the bacterial DNA is bound to a column comprising a silica-containing nucleic acid binding support, such as a silica membrane, for washing and purification.

[0096] The method according to the invention, most preferably, can be accomplished with the following measures: homogenizing a tissue sample to be investigated for its bacteriome step a) dissociating the tissue by adding to the sample a collagenase and a neutral protease (such as dispase); (step a)) centrifugating and discarding the supernatant step b) depleting the host DNA by adding to the pellet a DNA digest buffer comprising a nuclease with DNAse activity and RNAse activity (such benzonase); deactivating the nuclease (such as by incubation with proteinase K) step c) adding tissue lysis buffer and mechanically disrupting the bacterial cells (such as with yttria-stabilized zirconia beads as disrupting particles) separating the beads from the sample adding a pathogen lysis buffer purifying the bacterial DNA (such as with a spin column with a silica membrane)

[0097] - washing the column with a washing buffer eluting the column with an elution buffer.

[0098] The eluate can then be subjected to a genetic analysis (such as next generation sequencing).

[0099] According to one embodiment, the method of the invention comprises

[0100] - mechanically homogenizing the tissue sample prior to step a);

[0101] - performing step a), wherein step a) comprises

[0102] ■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably, the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,

[0103] ■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),

[0104] ■ wherein during step a) the tissue sample is not treated with saponin;

[0105] - performing step b), wherein step b) comprises

[0106] ■ treating the solid fraction obtained in step a) with at least one nuclease,

[0107] ■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;

[0108] - performing step c), wherein step c) comprises

[0109] (i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises

[0110] ■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer; separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction; ■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature; and

[0111] (ii) purifying microbial DNA from the lysed sample.

[0112] According to one embodiment, the method of the invention comprises

[0113] - mechanically homogenizing the tissue sample prior to step a);

[0114] - performing step a), wherein step a) comprises

[0115] ■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,

[0116] ■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),

[0117] ■ wherein during step a) the tissue sample is not treated with saponin;

[0118] - performing step b), wherein step b) comprises

[0119] ■ treating the solid fraction obtained in step a) with at least one nuclease selected from benzonase and DNAsel, preferably benzonase,

[0120] ■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;

[0121] - performing step c), wherein step c) comprises

[0122] (i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises

[0123] ■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer, wherein the first lysis buffer comprises an anionic detergent;

[0124] ■ separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction;

[0125] ■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature, wherein the second lysis buffer comprises a chaotropic salt; and

[0126] (ii) purifying microbial DNA from the lysed sample. As disclosed herein, the isolated microbial nucleic acids comprise pure microbial DNA, preferably bacterial DNA. Host cell DNA is effectively depleted by steps a) and b) by freeing bacteria from the associated eukaryotic host cells and thoroughly depleting the DNA derived from the host cells in step b). As bacterial cells remain intact during processing of steps a) and b), after bacterial lysis in step c) (i), bacterial DNA can be efficiently isolated from the lysed sample in step c) (ii) with high purity and low host cell contamination.

[0127] According to one embodiment, step c) (ii) comprises isolating bacterial DNA from the lysed sample. Step c) (ii) may comprise

[0128] (aa) binding DNA to a nucleic acid binding solid phase;

[0129] (bb) washing the bound DNA, optionally wherein at least two washing steps are performed; (cc) eluting the bound DNA.

[0130] Step c) (ii) may comprise binding the bacterial DNA to a silica containing nucleic acid binding solid phase, optionally wherein the nucleic acid containing solid phase is comprised in a column or is provided by magnetic silica particles, wherein nucleic acid binding is supported by the presence of a chaotropic salt.

[0131] In one embodiment, the method of the invention comprises

[0132] - mechanically homogenizing the tissue sample prior to step a);

[0133] - performing step a), wherein step a) comprises

[0134] ■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,

[0135] ■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),

[0136] ■ wherein during step a) the tissue sample is not treated with saponin;

[0137] - performing step b), wherein step b) comprises

[0138] ■ treating the solid fraction obtained in step a) with at least one nuclease, preferably selected from benzonase and DNAsel, after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion; performing step c), wherein step c) comprises

[0139] (i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises

[0140] ■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer, wherein the first lysis buffer comprises an anionic detergent, preferably SDS;

[0141] ■ separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction;

[0142] ■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature, wherein the second lysis buffer comprises a chaotropic salt and assists in establishing the DNA binding conditions in step c) (ii) (aa); and

[0143] (ii) purifying bacterial DNA from the lysed sample, wherein step c) (ii) comprises

[0144] (aa) binding the bacterial DNA to a silica containing nucleic acid binding solid phase, wherein bacterial DNA binding is supported by the presence of the chaotropic salt that is comprised in the second lysis buffer of step c) (i);

[0145] (bb) washing the bound DNA, optionally wherein at least two washing steps are performed;

[0146] (cc) eluting the bound DNA.

[0147] When aiming at isolating microbial DNA devoid of RNA, it is preferred that no ethanol is added in step c) (ii) (aa). Ethanol may support, as do other C1-C5 alkanols, the binding of RNA to a silica containing nucleic acid solid phase. To reduce RNA binding to the solid phase, it is thus preferred that no ethanol is present in the DNA binding mixture of step c) (ii) (aa). The absence of ethanol in binding step c)(i)(aa) may furthermore reduce the binding of short host DNA fragments to the solid phase in case some host DNA fragments may have remained in the nuclease treated sample that is obtained after performing step (b). Hence, while ethanol may be added in embodiments in step c) (ii) (aa), it is preferred that no ethanol is added during the microbial DNA binding step to enhance the purity of the isolated microbial DNA. This embodiment, wherein no ethanol is added in step c)(i)(aa), was also used in the working example. According to one embodiment, no C1-C5 alkanol is added in step c) (ii) (aa). Hence, in embodiments, no C1-C5 alkanol is present in the DNA binding mixture of step c) (ii) (aa).

[0148] Furthermore, the method of the invention may comprise a step of analyzing the recovered microbial nucleic acids, which preferably is microbial DNA. In one embodiment of said analysis step the isolated microbial DNA is used as analytical sample for genetic analysis. The genetic analysis may comprise a metagenomic analysis. The genetic analysis may be based on next generation sequencing.

[0149] Analyzing may comprise performing a PCR, qPCR, and / or DNA sequencing. Analyzing may comprise whole metagenome or 16S rRNA gene sequencing.

[0150] In an especially preferred embodiment of the invention, the method as described here above is used for the analysis of the oral microbiome samples (e.g. saliva, buccal swabs), genital swabs, solid tissue samples in cancer (e.g. colon biopsy, breast biopsy) or other solid tissue samples (esp. gut samples).

[0151] According to a further aspect, a kit is provided for processing a tissue sample for isolating microbial nucleic acids suitable for genetic analysis, in particular for a metagenomic analysis, comprising a collagenase, a neutral protease and a nuclease, preferably comprising a composition comprising the collagenase and the neutral protease.

[0152] Also provided is a kit for processing a tissue sample for genetic analysis is provided, in particular for a metagenomic analysis, comprising a collagenase, a neutral protease and a nuclease, preferably a composition comprising the collagenase and the neutral protease. The neutral protease preferably is dispase, the nuclease preferably has both, a DNAse and an RNAase activity, such as benzonase, and the collagenase preferably is collagenase IV.

[0153] Advantageously, the kits of the invention may further comprises one or more of the following means: means for disrupting bacterial cells and means for isolating, washing, purifying and / or eluting bacterial DNA. The means for disrupting the bacterial cells preferably are yttria- stabilized zirconia beads. Particularly advantageously, the kit comprises means for washing and purifying bacterial DNA.

[0154] The kits of the invention may be used for performing the method of the invention. Details were described above and it is referred to the above disclosure.

[0155] The following items are also disclosed as aspects and embodiments of the invention.

[0156] 1. A method for isolating microbial nucleic acids, preferably microbial DNA, from a tissue sample comprising a eukaryotic tissue, said method comprising the steps of: a) treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, b) treating the sample of step a) with at least one nuclease, and c) processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA. 2. The method according to item 1 , wherein the neutral protease and the collagenase can act synergistically in dissociating the eukaryotic tissue.

[0157] 3. The method according to any one of item 1 or 2, wherein the tissue sample in step a) is treated with the collagenase and the neutral protease simultaneously or sequentially, preferably simultaneously.

[0158] 4. The method according to any one of the above items, wherein the neutral protease is selected from the group consisting of trypsin, elastase and neutral proteases from Bacillus spec., preferably from Bacillus subtilis, Bacillus licheniformis, Bacillus thermoproteolyticus or Bacillus polymyxa.

[0159] 5. The method according to any one of the above items, wherein the neutral protease is thermolysin or dispase, preferably dispase.

[0160] 6. The method according to any one of the above items, wherein the collagenase is bacterial collagenase, preferably derived from Clostridium histolyticum.

[0161] 7. The method according to any one of the above items, wherein the collagenase is selected from the group consisting of collagenase I, collagenase II, collagenase III, collagenase IV, and collagenase type B.

[0162] 8. The method according to any one of the above items, wherein in step a) a composition comprising the collagenase and the neutral protease is added to the tissue sample, preferably a composition comprising collagenase IV and dispase.

[0163] 9. The method according to item 8, wherein ratio of the collagenase to neutral protease in the composition is from 1 :5 to 10:1 , preferably from 1 :2 to 8:1 , more preferred from 1 :1 to 5:1 , most preferred it is about 5:1 , based on enzyme activity units (II).

[0164] 10. The method according to any one of the above items, wherein the at least one nuclease is selected from the group consisting of DNase, RNase, exonuclease or an endonuclease.

[0165] 11 . The method according to any one of the above items, wherein the at least one nuclease is benzonase or DNAsel, preferably benzonase.

[0166] 12. The method according to any one of the above items, wherein in step a) the tissue sample is not treated with saponin, preferably is not treated with any detergent.

[0167] 13. The method according to any one of the above items, wherein the tissue sample comprises connective tissue.

[0168] 14. The method according to any one of the above items, wherein the tissue sample comprises fibrous cells.

[0169] 15. The method according to any one of the above items, wherein the tissue sample comprises one or more of a component of extracellular matrix.

[0170] 16. The method according to item 15, wherein the component of the extracellular matrix is selected from the group consisting of fibrous proteins (such as collagen, elastin, laminin), proteoglycans (such as glycosaminoglycans (GAGs)) and matrix metalloproteinases (MMPs).

[0171] 17. The method according to any one of the above items, wherein the tissue sample comprises collagen. 18. The method according to any one of the above items, wherein the tissue sample originates from a clinical specimen, in particular a biopsy, or a swab, or from an environmental specimen.

[0172] 19. The method according to any one of the above items, wherein the tissue sample originates from a human.

[0173] 20. The method according to any one of the above items, wherein the tissue sample comprises animal cells, such as human cells, and bacteria.

[0174] 21. The method according to any one of the above items, wherein the tissue sample comprises gram negative bacteria.

[0175] 22. The method according to any of the above items, wherein the tissue sample is homogenized before step a), preferably is mechanically homogenized.

[0176] 23. The method according to any of the above items, wherein during treatment in step a) the sample is incubated at elevated temperature, e.g. at 30°C to 40°C, such as 37°C.

[0177] 24. The method according to item 23, wherein during step a) the sample is incubated at elevated temperature for at least 15 min, at least 20 min, at least 25min, such as 30min.

[0178] 25. The method according to any of the above items, wherein step a) comprises separating the treated sample into a solid fraction and a liquid fraction, optionally wherein separation is assisted by centrifugation or sedimentation, preferably centrifugation.

[0179] 26. The method according to item 25, wherein step a) comprises discarding the liquid fraction, optionally as supernatant, and keeping the solid fraction as sample of step a) for step b).

[0180] 27. The method according to item 25 or 26, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b).

[0181] 28. The method according to any of the above items, wherein step a) comprises adding a DNA digestion buffer but no nuclease in step a).

[0182] 29. The method according to any of the above items, wherein a DNA digestion buffer is added in step b).

[0183] 30. The method according to any of the above items, wherein after performing the nuclease digestion step during step b), the nuclease is inactivated.

[0184] 31. The method according to item 30, wherein inactivating the nuclease comprises enzymatic degradation, optionally wherein inactivating the nuclease comprises performing a proteinase K digestion step.

[0185] 32. The method according to any of the above items, wherein under the conditions of steps a) and b) bacterial cells remain intact.

[0186] 33. The method according to any of the above items, wherein processing step c) comprises

[0187] (i) bacterial cell lysis and

[0188] (ii) DNA purification.

[0189] 34. The method according to item 33, wherein in step c) (i) bacterial cell lysis is assisted by mechanical and chemical disruption.

[0190] 35. The method according to item 33 or 34, wherein mechanical disruption is assisted by disrupting particles.

[0191] 36. The method according to item 35, wherein the disrupting particles have one or more of the following features: (i) the particles are crystalline particles;

[0192] (ii) the particles comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), yttrium-stabilized zirconium, quartz, aluminium oxide, silicon carbide, ceramic, glasses (e.g. silicon dioxide glass or silica) or a combination of the foregoing;

[0193] (iii) the particles are substantially spherical;

[0194] (iv) the particles have a size that lies in the range selected from 0.05mm to 0.9mm, 0.07mm to 0.8mm, 0.08mm to 0.75mm and 0.09mm to 0.7mm;

[0195] (v) the particles are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide) or yttrium-stabilized zirconium having on average a size that lies in the range of 0.08mm to 0.7mm, preferably 0.09mm to 0.6mm, wherein preferably, yttria-stabilized zirconium beads are used as disrupting particles. The method according to any one of items 33 to 36, wherein step c) (i) comprises adding at least one lysis buffer to assist chemical lysis. The method according to any one of items 33 to 36, wherein step c) (i) comprises the addition of disrupting particles and at least one lysis buffer and chemical and mechanical disruption of comprised bacterial cells. The method according to any one of items 33 to 38, wherein step c) (i) comprises

[0196] - adding a first lysis buffer and disrupting particles to the sample of step b), in which the nuclease is preferably inactivated by proteolytic digestion, and mechanically disrupting the sample in the presence of the first lysis buffer;

[0197] - separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction, optionally wherein separation is assisted by centrifugation or sedimentation, preferably centrifugation;

[0198] - adding a second lysis buffer to the liquid fraction and incubating the mixture, optionally incubation is performed at elevated temperature, such as at a temperature within the range of 30°C - 85°C, 40°C - 80°C and 50°C to 75°C. The method according to item 39, wherein step c) (i) has one or more, preferably all, of the following characteristics:

[0199] (aa) the first lysis buffer comprises a detergent, preferably an anionic detergent SDS;

[0200] (bb) the first lysis buffer does not comprise a chaotropic salt;

[0201] (cc) the second lysis buffer comprises a chaotropic salt, preferably a guanidinium salt such as GTC, and a detergent;

[0202] (dd) the second lysis buffer comprises the chaotropic salt in a concentration of 1 M-6M, preferably 3M-5M, and a non-ionic detergent, optionally wherein the non-ionic detergent is present in a concentration of 5%-30% (v / v); and / or

[0203] (ee) the second lysis buffer assists in establishing the DNA binding conditions in step c) (ii). The method according to any one of items 33 to 40, wherein step c) (ii) comprises isolating bacterial DNA from the lysed sample, optionally wherein step c) (ii) comprises

[0204] (aa) binding DNA to a nucleic acid binding solid phase;

[0205] (bb) washing the bound DNA, optionally wherein at least two washing steps are performed; (cc) eluting the bound DNA. 42. The method according to any one of items 33 to 41 , wherein step c) (ii) comprises binding the bacterial DNA to a silica containing nucleic acid binding solid phase, optionally wherein the nucleic acid containing solid phase is comprised in a column or is provided by magnetic silica particles, further optionally wherein nucleic acid binding is supported by the presence of a chaotropic salt and / or a C1-C5 alkanol.

[0206] 43. The method according to any one of the above items, wherein the isolated microbial nucleic acids comprise microbial DNA, preferably bacterial DNA.

[0207] 44. The method according to any one of the above items, wherein the ratio of bacteria cells to host cells in the tissue sample is 1 :10 000 or less, more preferred 1 :25 000 or less, even more preferred 1 :50 000 or less, most preferred 1 :100 000 or less.

[0208] 45. The method according to any one of the above items, wherein in the isolated microbial DNA, the ratio of bacterial DNA to host DNA is enriched compared to the ratio in the tissue sample, preferably the ratio is enriched by at least two or three orders of magnitude.

[0209] 46. The method according to any one of the above items, wherein the ratio of host DNA to bacterial DNA in the isolated DNA is 200 or less, preferably 100 or less, 50 or less or 10 or less.

[0210] 47. The method according to any one of the above items, comprising

[0211] (e) analyzing the recovered microbial DNA.

[0212] 48. The method according to item 47, wherein in step (e) the isolated microbial DNA is used as analytical sample for genetic analysis.

[0213] 49. The method according to item 48, wherein the genetic analysis is a metagenomic analysis and / or wherein the genetic analysis is based on next generation sequencing.

[0214] 50. The method according to any one of items 47 to 49, wherein analyzing comprises performing a PCR, qPCR, and / or DNA sequencing.

[0215] 51. The method according to any one of items 47 to 50, wherein analyzing comprises whole metagenome or 16S rRNA gene sequencing.

[0216] 52. A kit for processing a tissue sample for isolating microbial nucleic acids suitable for genetic analysis, in particular for a metagenomic analysis, comprising a collagenase, a neutral protease and a nuclease, preferably comprising a composition comprising the collagenase and the neutral protease.

[0217] 53. The kit according to item 52, wherein the neutral protease is dispase, the nuclease is benzonase and the collagenase preferably is collagenase IV.

[0218] 54. The kit according to item 52 or 53, wherein the kit further comprises one or more of the following means: means for disrupting bacterial cells and means for isolating, washing, purifying and / or eluting bacterial DNA.

[0219] 55. The kit according any one of the items 52 to 54, wherein the means for disrupting the bacterial cells are disrupting particles, preferably disrupting particles as defined in any one of the preceding items, more preferably yttria-stabilized zirconia beads.

[0220] 56. The kit according to any one of items 52 to 55, further comprising one or more, two or more, or all of the following:

[0221] (aa) a first and / or a second lysis buffer, optionally having the characteristics as defined in item 40; (bb) at least one washing buffer;

[0222] (cc) at least one elution solution;

[0223] (dd) a protease, preferably proteinase K;

[0224] (ee) a nucleic acid binding solid phase, which preferably is a silica containing nucleic acid binding solid phase, optionally wherein the silica nucleic acid containing solid phase is comprised in a column.

[0225] 57. Use of a kit as defined in any one of items 52 to 56 in a method as defined in any one of items 1 to 51.

[0226] This invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole.

[0227] EXAMPLE SECTION

[0228] The following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner.

[0229] 1. Exemplary workflow for the method according to the present invention

[0230] An exemplary workflow for the method according to the present invention is described in the following. This workflow is particularly suitable to isolate pure microbial DNA, in particular bacterial DNA, while efficiently depleting human and animal host nucleic acids. Various different tissue samples can be used as staring material. The workflow of the invention dramatically improves host depletion and bacterial DNA isolation in tissue samples. The workflow allows isolating bacterial DNA from intact bacteria. The isolated microbial DNA is of high quality and can used for a variety of applications, including qPCR and whole metagenome or 16S rRNA gene sequencing. Advantageously, the method allows to avoid the use of saponin for differential sample lysis.

[0231] Exemplary equipment & Reagents used

[0232] • Microcentrifuge (with rotor for 2 ml tubes)

[0233] • Shaker-incubator

[0234] • Equipment for sample disruption and homogenization, one of: o Vortex Genie 2 and Vortex Adapter for 24 (1 .5-2 ml) tubes (cat. no. 13000-V1-24) o TissueLyser II (cat. no. 85300), TissueLyser III (cat. no. 9003240) with adapter sets for use with the PowerBead Pro Tubes (TissueLyser Adapter Set 2 x 24, cat. no. 69982), or 2ml Tube Holder, cat. no. 11993, in conjunction with Plate Adapter Set, cat.no. 11990)

[0235] • PowerBead Pro Tubes (QIAGEN, comprising zirconia particles for bead beating)

[0236] • Ethanol (96-100 %)

[0237] • Phosphate-buffered saline (PBS, w / o Mg2+or Ca2+)

[0238] • EM: The enzyme mixture (EM) comprises a combination of collagenase and dispase. In the below example, STEMxyme® 1 & 2 Collagenase / Neutral Protease (Dispase®) from Worthington was used as EM. This is a combination of animal origin-free Clostridium histolyticum collagenase (Code: CLSAFB) and animal origin free Bacillus polymyxa neutral protease (Dispase®) with a minimum of 250 CLS (Collagenase Liberated Substrate) units and 1,000 or 2,000 caseinase units per mg dry weight, respectively.

[0239] Notes before starting

[0240] - Dissolve lyophilized enzyme mix (EM) in 6 ml PBS. Mix gently by inverting. For long-term storage store aliquots at -15 to -25°C.

[0241] - Tissue samples: mince or grind sample before starting the protocol, e.g. using TissueRuptor II (cat.no. 990890).

[0242] - Swab samples: swirl in 220 pl DNA digestion buffer (e.g. Buffer RDD, QIAGEN) for at least 20s and dry off by pressing against the wall of the tube multiple times. Do not add any additional buffer at step 2.

[0243] - Sputum samples: highly viscous samples should be pre-treated, e.g. with Sputasol (Oxoid) solution

[0244] - If a precipitate has formed in a lysis buffer, dissolve by incubation at 56°C. In the below example, lysis buffers ATL (QIAGEN) and APL2 (QIAGEN) were used at the indicated lysis stages. In the below example, an antifoaming agent was added to the lysis buffer ATL (100 pl Reagent DX to 15 ml Buffer ATL). Mix well. After preparation, the mixture is stable for 6 months at room temperature (15-25°C).

[0245] - Washing buffers are preferably used to further purify the microbial DNA. In the below examples, Buffers AW1 and AW2 from QIAGEN were used as washing buffers. Ensure that the washing buffers have been prepared according to the instructions.

[0246] Procedure

[0247] 1. Add up to 100 mg of (homogenized) tissue, 220 pl of a DNA digestion buffer (e.g. Buffer RDD, QIAGEN) and 30 pl EM stock to a 2 ml collection tube (in that order). Mix well and incubate at 37 °C for 30 min at 600 rpm in a heating block or water bath.

[0248] 2. Centrifuge at 10,000 x g for 1 min. Discard the supernatant. Do not disturb the pellet as this will result in loss of bacterial material. Add 190 l DNA digestion buffer (e.g. Buffer RDD, QIAGEN) and 2.5 pl Benzonase, mix by vortexing briefly and incubate at 37 °C for 30 min at 600 rpm in a heating block or water bath. Add 20 pl Proteinase K, mix well and incubate at 56 °C for 30 min at 600 rpm in a heating block or water bath. Add 200 pl lysis buffer. The lysis buffer used at this step preferably comprises a detergent, such as the anionic detergent SDS. In this working example, Buffer ATL (containing Reagent DX) was used. Mix well to avoid loss of sample material. The mixture is then transferred into a vessel comprising disrupting particles for mechanical disruption of comprised bacteria (e.g. PowerBead Pro Tube, QIAGEN). To ensure that the disrupting particles have settled at the bottom, the vessel (e.g. PowerBead Pro Tube, QIAGEN) can be briefly centrifuged prior to transferring the mixture. Mechanically disruption of the sample, which is assisted by the disrupting particles. Mechanical disruption may be advantageously achieved by using one of the following methods: a) Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 24 (1.5-2 ml) tubes (cat. no. 13000-V1-24). Orient the tube caps to point toward the center of the vortex adapter. Vortex at maximum speed for 10 min. b) Use a TissueLyser II / III. Place the PowerBead Pro Tube into the TissueLyser Adapter Set 2 x 24 (cat. no. 69982) or 2ml T ube Holder (cat. No. 11993) and Plate Adapter Set (cat. no.1190). Fasten the adapter into the instrument and shake for 5 min at speed 30 Hz. Re-orient the adapter so that the side that was closest to the machine body is now furthest from it. Shake again for 5 min at speed 30 Hz. After mechanical disruption, centrifuge the vessel (e.g. PowerBead Pro Tube) comprising the disrupted sample and disrupting particles. Centrifugation can be performed e.g. at 10,000 x g for 1 min. Transfer the supernatant to a fresh microcentrifuge tube. Add 200 pl of a lysis buffer. At this stage, a lysis buffer comprising a chaotropic salt, preferably a guanidine salt, and a non-ionic detergent may be used. In this working example, lysis buffer APL2 (QIAGEN) was used. Mix by pulse vortexing for 30 s. Incubate at 70 °C for 10 min and briefly spin the tube to remove condensation. Carefully apply up to 700 pl of the mixture from step 9 to a nucleic acid binding spin column. Preferably, the spin column comprises a silica material such as a silica membrane for efficient nucleic acid binding. In this working examples, the QIAamp UCP Mini Column was used. Close the cap and centrifuge at 6,000 x g for 1 min. Discard the flow-through. Put the column back into the collection tube to repeat step 10 with any remaining mixture from step 9. Transfer the QIAamp UCP Mini Column to a fresh collection tube. Carefully open the cap and add 500 pl washing buffer without wetting the rim. A washing buffer comprising a guanidinium salt and ethanol may be used at this step (e.g. Buffer AW1, QIAGEN). Close the cap and centrifuge at 6,000 x g for 1 min. Place the spin column (e.g. QIAamp UCP Mini Column) into a fresh 2 ml collection tube and discard the filtrate.

[0249] 13. Add 500 pl wash buffer (e.g. an ethanol containing wash buffer such as Buffer AW2, QIAGEN) to the QIAamp UCP Mini Column without wetting the rim. Centrifuge at full speed (20,000 x g) for 3 min.

[0250] 14. Place the QIAamp UCP Mini Column into a fresh 2 ml collection tube. Discard the filtrate. Centrifuge at full speed (20,000 x g) for 1 min.

[0251] 15. Place the QIAamp UCP Mini Column into a fresh 1 .5 ml tube and apply 50 pl elution solution (e.g. Buffer AVE, QIAGEN) directly onto the center of the membrane. Close the lid and incubate at room temperature for 5 min.

[0252] 16. Centrifuge at 6,000 x g for 1 min to elute the DNA.

[0253] 2. Prior art workflows The workflow according to the present invention was compared with prior art protocols, such as the QIAamp DNA Microbiome Kit and the DNeasy PowerSoilPro kit. The prior art protocols were performed according to the manufacturer’s instructions. Details of the performed experiments are provided in the description of the figures and the figures show the results. The results clearly support the important advantages of the method of the present invention.

Claims

CLAIMS1. A method for isolating microbial nucleic acids, preferably microbial DNA, from a tissue sample comprising a eukaryotic tissue, said method comprising the steps of: a) treating the tissue sample with a collagenase and a neutral protease that is different from the collagenase, b) treating the sample of step a) with at least one nuclease, and c) processing the sample of step b) to isolate microbial nucleic acids, preferably microbial DNA.

2. The method according to claim 1 , wherein the tissue sample in step a) is simultaneously treated with the collagenase and the neutral protease.

3. The method according to claim 1 or 2, wherein the neutral protease is thermolysin or dispase, preferably dispase, and wherein the collagenase is bacterial collagenase, preferably derived from Clostridium histolyticum.

4. The method according to any one of the above claims, wherein in step a) a composition comprising the collagenase and the neutral protease is added to the tissue sample, preferably a composition comprising collagenase IV and dispase.

5. The method according to claim 4, wherein ratio of the collagenase to neutral protease in the composition is from 1 :5 to 10:1 , preferably from 1:2 to 8:1 , more preferred from 1 :1 to 5:1 , most preferred it is about 5:1 , based on enzyme activity units (U).

6. The method according to any one of the above claims, wherein the at least one nuclease is selected from the group consisting of DNase, RNase, exonuclease or an endonuclease.

7. The method according to any one of the above claims, wherein the at least one nuclease is benzonase or DNAsel, preferably benzonase.

8. The method according to any one of the above claims, wherein in step a) the tissue sample is not treated with saponin, preferably is not treated with any detergent.

9. The method according to any one of the above claims, wherein the tissue sample has one or more of the following characteristics:(i) the tissue sample comprises connective tissue;(ii) the tissue sample comprises fibrous cells;(iii) the tissue sample comprises one or more of a component of extracellular matrix, optionally wherein the component of the extracellular matrix is selected from the group consisting of fibrous proteins (such as collagen, elastin, laminin), proteoglycans (such as glycosaminoglycans (GAGs)) and matrix metalloproteinases (MMPs);(iv) the tissue sample comprises collagen; and / or(v) the tissue sample originates from a human.

10. The method according to any one of the above claims, wherein the tissue sample originates from a clinical specimen, in particular a biopsy, or a swab, or from an environmental specimen.

12. The method according to any one of the above claims, wherein the tissue sample comprises animal cells, such as human cells, and bacteria.

13. The method according to any one of the above claims, wherein the tissue sample comprises gram negative bacteria.

14. The method according to any of the above claims, wherein the tissue sample is homogenized before step a), preferably is mechanically homogenized.

15. The method according to any of the above claims, wherein during treatment in step a) the sample is incubated at elevated temperature, e.g. at 30°C to 40°C, such as 37°C, optionally wherein during step a) the sample is incubated at elevated temperature for at least 15 min, at least 20 min, at least 25min, such as 30min.

16. The method according to any of the above claims, wherein step a) comprises separating the treated sample into a solid fraction and a liquid fraction, optionally wherein separation is assisted by centrifugation or sedimentation, preferably centrifugation.

17. The method according to claim 16, wherein step a) comprises discarding the liquid fraction, optionally as supernatant, and keeping the solid fraction as sample of step a) for step b).

18. The method according to claim 16 or 17, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b).

19. The method according to any of the above claims, wherein after performing the nuclease digestion step during step b), the nuclease is inactivated.

20. The method according to claim 19, wherein inactivating the nuclease comprises enzymatic degradation, optionally wherein inactivating the nuclease comprises performing a proteinase K digestion step.

21. The method according to any of the above claims, wherein under the conditions of steps a) and b) bacterial cells remain intact.

22. The method according to any one of the above claims, wherein the method comprises- mechanically homogenizing the tissue sample prior to step a);- performing step a), wherein step a) comprises■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably, the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),■ optionally wherein during step a) the tissue sample is not treated with saponin;- performing step b), wherein step b) comprises■ treating the solid fraction obtained in step a) with at least one nuclease,■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;- performing step c), wherein step c) comprises isolating microbial nucleic acids, preferably microbial DNA, from the nuclease inactivated sample of step b).

23. The method according to any of the above claims, wherein processing step c) comprises(i) bacterial cell lysis and(ii) DNA purification.

24. The method according to claim 23, wherein in step c) (i) bacterial cell lysis is assisted by mechanical and chemical disruption.

25. The method according to claim 23 or 24, wherein step c) (i) comprises the addition of disrupting particles and at least one lysis buffer and chemical and mechanical disruption of comprised bacterial cells.

26. The method according to any one of claims 23 to 25, wherein step c) (i) comprises- adding a first lysis buffer and disrupting particles to the sample of step b), in which the nuclease is preferably inactivated by proteolytic digestion, and mechanically disrupting the sample in the presence of the first lysis buffer;- separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction, optionally wherein separation is assisted by centrifugation or sedimentation, preferably centrifugation;- adding a second lysis buffer to the liquid fraction and incubating the mixture, optionally incubation is performed at elevated temperature, such as at a temperature within the range of 30°C - 85°C, 40°C - 80°C and 50°C to 75°C.

27. The method according to claim 26, wherein step c) (i) has one or more, preferably all, of the following characteristics:(aa) the first lysis buffer comprises a detergent, preferably an anionic detergent such as SDS;(bb) the first lysis buffer does not comprise a chaotropic salt;(cc) the second lysis buffer comprises a chaotropic salt, preferably a guanidinium salt such as GTC, and a detergent;(dd) the second lysis buffer comprises the chaotropic salt in a concentration of 1 M-6M, preferably 3M-5M, and a non-ionic detergent, optionally wherein the non-ionic detergent is present in a concentration of 5%-30% (v / v); and / or(ee) the second lysis buffer assists in establishing the DNA binding conditions in step c) (ii).

28. The method according to any one of the above claims, wherein the method comprises- mechanically homogenizing the tissue sample prior to step a);- performing step a), wherein step a) comprises■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably, the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),■ wherein during step a) the tissue sample is not treated with saponin;- performing step b), wherein step b) comprises■ treating the solid fraction obtained in step a) with at least one nuclease,■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;- performing step c), wherein step c) comprises(i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer;■ separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction;■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature; and(ii) purifying microbial DNA from the lysed sample.

29. The method according to any one of the above claims, wherein the method comprises- mechanically homogenizing the tissue sample prior to step a);- performing step a), wherein step a) comprises■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),■ wherein during step a) the tissue sample is not treated with saponin;- performing step b), wherein step b) comprises■ treating the solid fraction obtained in step a) with at least one nuclease selected from benzonase and DNAsel, preferably benzonase,■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion;- performing step c), wherein step c) comprises(i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer, wherein the first lysis buffer comprises an anionic detergent;■ separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction;■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature, wherein the second lysis buffer comprises a chaotropic salt; and(ii) purifying microbial DNA from the lysed sample.

30. The method according to any one of the above claims, wherein the isolated microbial nucleic acids comprise microbial DNA, preferably bacterial DNA.

31. The method according to any one of claims 23 to 30, wherein step c) (ii) comprises isolating bacterial DNA from the lysed sample, optionally wherein step c) (ii) comprises(aa) binding DNA to a nucleic acid binding solid phase;(bb) washing the bound DNA, optionally wherein at least two washing steps are performed;(cc) eluting the bound DNA.

32. The method according to any one of claims 23 to 31 , wherein step c) (ii) comprises binding the bacterial DNA to a silica containing nucleic acid binding solid phase, optionally wherein the nucleic acid containing solid phase is comprised in a column or is provided by magnetic silica particles, wherein nucleic acid binding is supported by the presence of a chaotropic salt.

33. The method according to any one of the above claims, wherein the method comprisesmechanically homogenizing the tissue sample prior to step a); performing step a), wherein step a) comprises■ adding a composition comprising the collagenase and the neutral protease to the homogenized tissue sample, wherein preferably the neutral protease is dispase and the collagenase is a bacterial collagenase, and incubating the sample at elevated temperature for at least 15min,■ separating the treated sample into a solid fraction and a liquid fraction, wherein the obtained solid fraction is subjected as sample of step a) to the nuclease treatment in step b),■ wherein during step a) the tissue sample is not treated with saponin; performing step b), wherein step b) comprises■ treating the solid fraction obtained in step a) with at least one nuclease, preferably selected from benzonase and DNAsel,■ after performing the nuclease digestion step during step b), inactivating the nuclease in the sample of step b), preferably by proteolytic digestion; performing step c), wherein step c) comprises(i) bacterial cell lysis, wherein bacterial cell lysis in step (c) (i) comprises■ adding a first lysis buffer and disrupting particles to the nuclease inactivated sample of step b), and mechanically disrupting the sample in the presence of the first lysis buffer, wherein the first lysis buffer comprises an anionic detergent, preferably SDS;■ separating the mechanically disrupted bacterial sample into a solid fraction and a liquid fraction;■ adding a second lysis buffer to the liquid fraction and incubating the mixture at elevated temperature, wherein the second lysis buffer comprises a chaotropic salt and assists in establishing the DNA binding conditions in step c) (ii) (aa); and(ii) purifying bacterial DNA from the lysed sample, wherein step c) (ii) comprises(aa)binding the bacterial DNA to a silica containing nucleic acid binding solid phase, wherein bacterial DNA binding is supported by thepresence of the chaotropic salt that is comprised in the second lysis buffer of step c) (i);(bb)washing the bound DNA, optionally wherein at least two washing steps are performed;(cc) eluting the bound DNA.

34. The method according to any one of the above claims, wherein no ethanol is added in step c) (ii) (aa).

35. The method according to any one of the above claims, wherein no C1-C5 alkanol is added in step c) (ii) (aa).

36. The method according to any one of the above claims, wherein in the isolated microbial DNA, the ratio of bacterial DNA to host DNA is enriched compared to the ratio in the tissue sample, preferably the ratio is enriched by at least two or three orders of magnitude.

37. The method according to any one of the above claims, wherein the ratio of host DNA to bacterial DNA in the isolated DNA is 200 or less, preferably 100 or less, 50 or less or 10 or less.

38. The method according to any one of the above claims, comprising a step of analyzing the recovered microbial DNA.

39. The method according to claim 38, wherein the step of analyzing is characterized by one or more of the following features:(i) in said analysis step the isolated microbial DNA is used as analytical sample for genetic analysis;(ii) in said analysis step the isolated microbial DNA is used as analytical sample for genetic analysis, wherein the genetic analysis is a metagenomic analysis and / or wherein the genetic analysis is based on next generation sequencing; and / or(iii) analyzing comprises whole metagenome or 16S rRNA gene sequencing.

40. A kit for processing a tissue sample for isolating microbial nucleic acids suitable for genetic analysis, in particular for a metagenomic analysis, comprising a collagenase, a neutral protease and a nuclease, preferably comprising a composition comprising the collagenase and the neutral protease.

41. The kit according to claim 40, wherein the neutral protease is dispase, the nuclease is benzonase and the collagenase preferably is collagenase IV.

42. The kit according to claim 40 or 41 , wherein the kit further comprises one or more of the following means: means for disrupting bacterial cells and means for isolating, washing, purifying and / or eluting bacterial DNA.

43. The kit according any one of the claims 40 to 42, wherein the means for disrupting the bacterial cells are disrupting particles, preferably disrupting particles as defined in any one of the preceding claims, more preferably yttria-stabilized zirconia beads.

44. The kit according to any one of claims 40 to 43, further comprising one or more, two or more, or all of the following:(aa) a first and / or a second lysis buffer, optionally having the characteristics as defined in claim 27;(bb) at least one washing buffer;(cc) at least one elution solution;(dd) a protease, preferably proteinase K;(ee) a nucleic acid binding solid phase, which preferably is a silica containing nucleic acid binding solid phase, optionally wherein the silica nucleic acid containing solid phase is comprised in a column.

45. Use of a kit as defined in any one of claims 40 to 44 in a method as defined in any one of claims 1 to 39.

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