Rapid incubation and nucleic acid enrichment for identification and analysis of microbial samples

Abstract

The present disclosure relates to methods, compositions, and kits for the identification and analysis of microorganisms in a sample using nucleoside or nucleotide analogs.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Patent Application No. 62 / 840,322, filed Apr. 29, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Field of the Invention) The present disclosure relates to methods, compositions, and kits for the identification and analysis of microorganisms in a sample using nucleoside or nucleotide analogs.

Background Art

[0003] Determining whether a patient has a microbial infection is a common clinical challenge. Sepsis is the most common cause of death among hospitalized patients, with an estimated 200,000 deaths annually in the United States. However, sepsis is an ambiguous clinical syndrome with a wide range of clinical manifestations. Diagnosis is usually based on suspected infection combined with signs of organ failure. Early diagnosis and administration of antibiotics for sepsis is crucial because progression to severe sepsis or septic shock can have serious consequences. Unfortunately, differentiating sepsis from other inflammatory conditions is often difficult in critically ill patients. Detecting bacterial infection in the blood is a critical step in diagnosing sepsis and initiating antibiotic treatment. However, blood cultures are negative in 60-70% of patients with severe sepsis, and studies have shown >80% to be negative. In addition, conventional microbiological methods are too time-consuming to influence primary therapy against pathogenic bacteria. While the development of PCR and mass spectrometry has increased the possibility of identifying bacteria in blood samples, they often rely on time-consuming pre-analytical preparations such as blood cultures to increase the pathogen load. Proxy indicators of infection include elevated levels of circulating cytokines and acute-phase proteins such as C-reactive protein, although their concentrations also increase during physiological events such as childbirth or during pathological tissue injury such as burns. Typically, for sepsis, blood culture tests are performed to identify which type of bacteria or fungus caused the infection in the blood. Blood cultures are collected separately from other blood tests and are often taken multiple times from different veins. It can take several days to obtain the results of a blood culture. In vitro culture conditions result in only one-third to half of people with sepsis having blood cultures that are positive, meaning that bacteria do indeed grow and multiply under in vitro conditions. [Overview of the Initiative]

[0004] Currently, bacterial detection often requires culturing for two reasons: (1) to isolate bacteria for analysis, and (2) to reduce any contaminating background cells or other substances that could make analysis difficult or impossible. For example, in patients with sepsis, blood needs to be cultured to isolate the pathogen. Similarly, in food surveillance, samples need to be cultured to isolate contaminating microorganisms. Unfortunately, standard culture processes can take several days. In the case of sepsis, this lag time can lead to unnecessary antibiotic administration or misdiagnosis, potentially resulting in patient complications or death. In the food industry, this lag time delays information that could lead to recalls or other preventive measures. Therefore, rapid detection of active infections can enable measures that can mitigate problems and save lives.

[0005] While PCR detection methods may not require long-term culture, unlike unbiased sequencing techniques, PCR requires prior knowledge of the target organism's genome sequence. That is, researchers need to know what they are looking for, which may not be true for rare or undiscovered organisms. Furthermore, PCR-dependent methods only detect the presence of genetic material in a sample and cannot distinguish whether the material originates from living or dead organisms. In many cases, identifying active infections caused by living microorganisms is a critical consideration for treatment options and even for identifying contaminants in food or the environment.

[0006] This disclosure provides a method for identifying and analyzing viable and / or growing microorganisms in a sample, comprising: (a) obtaining a sample having or suspected having one or more types of microorganisms; (b) incubating the sample in the presence of one or more types of nucleosides or nucleotide analogs so that the one or more types of nucleosides or nucleotide analogs are incorporated into newly synthesized microbial nucleic acids; (c) labeling the newly synthesized microbial nucleic acids by contacting them with one or more types of nucleosides or nucleotide analogs or with labeling reagents that selectively bind thereto; (d) isolating or purifying the labeled newly synthesized microbial nucleic acids; and (e) determining the identity of viable and / or growing microorganisms in the sample based on sequencing or identity determination of the isolated or purified newly synthesized microbial nucleic acids. In another embodiment, the sample is obtained from a subject suspected of or having a microbial infection. In yet another embodiment, the subject is suspected of or has sepsis. In a further embodiment, the sample obtained in (a) is treated with a dehosting method before (b) to selectively remove non-microbial nucleic acids. In a further embodiment, this dehosting method selectively cleaves non-microbial DNA by (i) contacting the obtained sample with a recombinant protein comprising a binding domain that selectively binds to histone-bound non-microbial nucleic acids or non-microbial nucleic acids containing methylated CpG residues, and a nuclease domain that has nucleic acid cleavage activity, or (ii) an affinity agent bound to a solid substrate that selectively binds to histone-bound nucleic acids or selectively binds to methylated CpG residues of non-microbial nucleic acids.This includes removal by the use of an agent. In one particular embodiment, the sample is an environmental sample obtained from an environmental testing site. In another embodiment, the environmental site is tested for microbial contamination. In yet another embodiment, the sample is a sample obtained from food suspected of being microbially contaminated. In yet another embodiment, one or more types of microorganisms are bacteria, fungi, viruses, algae, archaea, and / or protozoa. In further embodiments, the bacteria may include Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, and Brucella suis. suis), Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriaediphtheriae), Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes monocytogenes), Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus aureus), Staphylococcus epidermidis, Staphylococcus saprofeticusSelected from saprophyticus), Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, and / or Yersinia pseudotuberculosis. In another embodiment, the fungi include Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp., and Blastomyces (spp.), Cadophora spp., Candida albicans, Cercospora apii, Chrysosporium spp., Cladosporium spp., Cladothrix asteroids, Coccidioides imithisimmitis), Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp., Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum floccosum), Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp., Langeronia sudanans soudanense), Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi, Loboa loboiLoboi), Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis *Lactis*, *Paracoccidioides brasiliensis*, *Petriellidium boydii*, *Phialophora* spp., *Piedraia hortae*, *Pityrosporum furfur*, *Pneumocystis jirovecii* (or *Pneumocystis carinii*), *Pullularia gougerotii*, *Pyrenochaeta romeroi*, *Rhinosporidium sebergii* Seeberi), Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum spp., Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., TorulaSelected from the genera Trichophyton (spp.), Trichophyton (spp.), Trichosporon (spp.), and / or Zopfia rosatii. In yet another embodiment, the viruses belong to the genera Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, Nupapillomavirus, and Alpha Polyomavirus. Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, Deltapolyomavirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus, Dependvirus (Dependovirus), Bocavirus, Orthohepadnavirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, Simiispumavirus, Coltivirus, Rotavirus, Seadonavirus, α-coronavirus (α-Coronavirus), β-Coronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus, Cosavirus Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebolavirus, Marburgvirus, Henipavirus, Morbillivirus Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Orthopneumovirus, Ledantevirus, Lyssavirus, Vesiculovirus, Mammarenavirus, Orthohantavirus,The following genera are selected: Orthonairovirus, Orthobunyavirus, Phlebovirus, α-Influenzavirus, β-Influenzavirus, γ-Influenzavirus, Quaranjavirus, Thogotovirus, and / or Deltavirus. In further embodiments, one or more types of nucleosides or nucleotide analogs are 2-ethynyl-adenosine, N6-propargyl-adenosine, 2'-(O-propargyl)-adenosine, 3'-(O-propargyl)-adenosine, 5-ethynyl-cytidine, 5-ethynyl-2'-deoxycytidine, 2'-(O-propargyl)-cytidine, 3'-(O-propargyl)-cytidine, 2'-(O-propargyl)-guanosine, 3'-(O-propargyl )-Guanosine, 5-Ethynyl-uridine, 5-Ethynyl-2'-Deoxyuridine, 2'-(O-Propargyl)-uridine, 3'-(O-Propargyl)-uridine, (2'S)-2'-Deoxy-2'-Fluor-5-Ethynyluridine, (2'S)-2'-Fluor-5-Ethynyluridine, 2'(S)-2'-Deoxy-2'-Fluor-5-Ethynyluridine, (2'S)-2'-Fluor-5-Ethynyluridine, 8-Azido-Adenosine, N, 6Selected from -(6-azido)hexyl-2'deoxyadenosine, 2'-azido-2'-deoxyadenosine, 5-azidomethyluridine, 5-(15-azido-4,7,10,13-tetraoxa-pentadecanoyl-aminoallyl)-2'-deoxyuridine, 5-(3-azidopropyl)-uridine, 5-azido-PEG4-uridine, 5-azido-PEG4-cytidine, 5-azido-PEG4-2'-deoxycytidine, 5-bromo-2'deoxyuridine, 5-bromouridine, 5-iodo-2'deoxyuridine, and 5-iodouridine. In further embodiments, one or more types of nucleosides or nucleotide analogs are 2-ethynyl-adenosine, N6-propargyl-adenosine, 2'-(O-propargyl)-adenosine, 3'-(O-propargyl)-adenosine, 5-ethynyl-cytidine, 5-ethynyl-2'-deoxycytidine, 2'-(O-propargyl)-cytidine, 3'-(O-propargyl)-cytidine, 2'-(O-propargyl)-guanosine, 3'-(O-propargyl) (gyl)-guanosine, 5-ethynyl-uridine, 5-ethynyl-2'-deoxyuridine, 2'-(O-propargyl)-uridine, 3'-(O-propargyl)-uridine, (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (2'S)-2'-fluoro-5-ethynyluridine, 2'(S)-2'-deoxy-2'-fluoro-5-ethynyluridine, and (2'S)-2'-fluoro-5-ethynyluridine. In yet another embodiment, one or more types of nucleosides or nucleotide analogs are selected from 8-azido-adenosine, N 6One or more types of nucleosides or nucleotide analogs are selected from -(6-azido)hexyl-2'deoxyadenosine, and one or more types of nucleosides or nucleotide analogs are selected from 2'-azido-2'-deoxyadenosine, and one or more types of nucleosides or nucleotide analogs are selected from 5'-azido-2'-deoxyadenosine, and one or more types of nucleosides or nucleotide analogs are selected from 5-bromo-2'deoxyuridine, 5-bromouridine, 5-iodo-2'deoxyuridine, and 5-iodouridine. In further embodiments, the sample is incubated for 5 to 180 minutes in the presence of one or more types of nucleosides or nucleotide analogs. In another embodiment, the sample is incubated for 30 to 120 minutes in the presence of one or more types of nucleosides or nucleotide analogs. In yet another embodiment, the labeling reagent is an antibody that binds with high specificity to one or more types of nucleosides or nucleotide analogs. In a particular embodiment, the antibody binds with high specificity to 5-bromo-2'deoxyuridine or iododeoxyuridine. In another embodiment, the labeling reagent binds to or to one or more types of nucleosides or nucleotide analogs via click chemistry, strained [3+2] cycloaddition, or Staudinger ligation. In yet another embodiment, the labeling reagent contains an azide group that binds to an alkynyl group-containing nucleoside or nucleotide analog via click chemistry. In further embodiments, the labeling reagent comprises an alkynyl group that binds to a nucleoside or nucleotide analog containing an azide group via click chemistry. In certain embodiments, the labeling reagent comprises a biotin group. In further embodiments, the labeling reagent comprising a biotin group is [ka] A selection is made from the following. In further embodiments, the labeling reagent further comprises a chemically cleavable linker or an enzymatically cleavable linker. In further embodiments, the cleavable linker is an acid-labile-based linker or a disulfide-based linker. In certain embodiments, the acid-labile linker comprises a hydrazone or a cis-aconityl group. In another embodiment, the enzymatically cleavable linker comprises a peptide linker or a β-glucuronide linker. In yet another embodiment, the pulldown agent is used to isolate or purify a newly synthesized labeled microbial nucleic acid. In further embodiments, the pulldown reagent is an antibody immobilized on a solid support, the antibody binds with high specificity to the labeling reagent or to one or more types of nucleosides or nucleotide analogs. In further embodiments, the pulldown reagent is streptavidin or avidin immobilized on a solid support, and the labeling reagent comprises a biotin group. In certain embodiments, the solid support is a nanomaterial or micromaterial, a bead, or a plate. In another embodiment, the labeling reagent or label is removed or cleaved from the isolated or purified newly synthesized microbial nucleic acid prior to (e) above. In yet another embodiment, the identity of the isolated or purified newly synthesized microbial nucleic acid is determined by using a microarray containing probes for nucleic acids from different microorganisms.In a further embodiment, the identity of isolated or purified newly synthesized microbial nucleic acids is determined by (i) amplifying the isolated or purified newly synthesized microbial nucleic acids using a first PCR-based method with a primer containing a fluorescent dye to form a labeled product, wherein the primer contains a sequence specific to a conserved microbial 16S rRNA gene region; (ii) applying the labeled product to a microarray containing probes containing unique 16S rRNA variable region sequences from 20 or more microorganisms; and (iii) determining the identity of viable and / or growing microorganisms based on imaging of the microarray of the fluorescent hybridization product and determining the identity of the microorganisms based on the sequences of the microarray probes. In another embodiment, the identity of isolated or purified newly synthesized microbial nucleic acids is determined or confirmed by sequencing the isolated or purified newly synthesized microbial nucleic acids. In yet another embodiment, isolated or purified newly synthesized microbial nucleic acids are sequenced using a transposome-based sequencing method. In further embodiments, sequencing of newly synthesized microbial nucleic acids is performed by (a) applying isolated or purified newly synthesized microbial nucleic acids to a bead-linked transposome, the bead-linked transposome mediating the simultaneous fragmentation of microbial nucleic acids and the addition of sequencing primers; (b) amplifying the microbial nucleic acid fragments with primers containing index and adapter sequences to form a library of amplified products; (c) washing and pooling the library of amplified products; (d) sequencing the library of amplified products; and (e) determining the identity of viable and / or growing microorganisms based on the correlation of sequences obtained from the library of amplified products with a database of known microbial sequences using bioinformatic analysis.In another embodiment, the newly synthesized microbial nucleic acid is RNA, and the microbial RNA is reverse transcribed into cDNA prior to (e) above, and the gene expression of viable and / or growing microorganisms can be determined based on analyzing the expression levels of gene products from the newly synthesized microbial RNA using a microarray and / or sequencing.

[0007] In certain embodiments, the Disclosure also relates to a method for determining the effectiveness of an antimicrobial agent that modulates the growth and proliferation of microorganisms(s) in a sample, comprising: (a) obtaining a sample having or suspected having one or more types of microorganisms; (b) dividing the sample into two samples, a control sample and a treatment sample; (c) incubating the control sample in the presence of one or more types of nucleosides or nucleotide analogs so that the one or more types of nucleosides or nucleotide analogs are incorporated into the newly synthesized microbial nucleic acid; (c') incubating the treatment sample in the presence of one or more types of nucleosides or nucleotide analogs and an antimicrobial agent so that the one or more types of nucleosides or nucleotide analogs are incorporated into the newly synthesized microbial nucleic acid; and (d) contacting the newly synthesized microbial nucleic acid with one or more types of nucleosides or nucleotide analogs or with a labeling reagent that selectively binds thereto, thereby converting the newly synthesized microbial nucleic acid into the new control sample and the treatment sample. The present invention provides a method for demonstrating that an antimicrobial agent is effective in regulating the growth and proliferation of microorganisms, comprising: (e) labeling newly synthesized microbial nucleic acids; (f) isolating or purifying the labeled newly synthesized microbial nucleic acids from a control sample and a treated sample; (g) determining the gene expression level and / or quantity or identity of the isolated or purified newly synthesized microbial nucleic acids in the control sample; (f') determining the gene expression level and / or quantity and identity of the isolated or purified newly synthesized microbial nucleic acids in the treated sample; and (g) comparing the gene expression level and / or quantity and / or identity of the isolated or purified newly synthesized microbial nucleic acids in the control sample with the gene expression level and / or quantity or identity of the isolated or purified newly synthesized microbial nucleic acids in the treated sample and determining any changes, wherein if the gene expression level of the newly synthesized microbial nucleic acids is reduced in the treated sample versus the control sample, or if the quantity and / or identity of the newly synthesized microbial nucleic acids is reduced in the treated sample versus the control sample, the antimicrobial agent is effective in regulating the growth and proliferation of the microorganism(s). In another embodiment, the antimicrobial agent is selected from antibiotics, antifungals, and antivirals. In further embodiments, the antibiotic isBacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezolocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin, pibmecillinam, ticalcillin, cefacetril, cefadroxil, cephalexin, cephaloglysin, cephalonium, cephaloridine, cephalothin, cefapillin, cefatolidine, cefazal, cefazedone, cefazolin, cefradiin, ceffloxazine, ceftezol, cefaclor, cefamandol, cefmetazole, cefonis Cefotetan, cefoxitin, cefprodil, cefuroxime, cefzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefozidime, cefotaxime, cefpimisole, cefpodoxime, cefteram, ceftibuten, ceftiofur, cefthiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, ceffluprenum, cefoselis, cefozopran, Cefpirome, cefquinome, ceftobiprole, cephthaloline, cefaclomezine, cephaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefibitril, cefmatylene, cefmepidium, cefobecin, cefoxazole, cefroti Cefrotil, cefsumide, cefuracetime, ceftioxide, aztreonam, imipenem, doripenem, ertapenem, meropenem, azithromycin, erythromycin, clarithromycin, dilithromycin, roxithromycin, telithromycin, clindamycin, lincomycin, amikacin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, streptomycin, tobramycin, flumequine, nalidixic acid,Oxolinic acid, pyromidic acid, pipemidic acid, losoxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, valofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, delafloxacin, clinafloxacin, gemifloxacin, prulifloxacin, sitafloxacin, trovafloxacin, sulf The following are selected: amethisole, sulfamethoxazole, sulfisoxazole, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, tigecycline, vancomycin, teicoplanin, teravancin, linezolid, cycloserine, rifampin, rifabutin, rifapentin, rifalazil, biomycin, capreomycin, bacitracin, polymyxin B, chloramphenicol, metronidazole, tinidazole, and nitrofurantoin. In further embodiments, the antifungal agent may include amorolfine, butenafine, naphthifine, terbinafine, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, ketoconazole, isoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, thioconazole, terconazole, albaconazole, efinaconazole, fluconazole, and Subconazole, itraconazole, posaconazole, ravconazole, terconazole, voriconazole, abafungin, amphotericin B, nystatin, natamycin, trichomycin, anidurafungin, caspofungin, micafungin, tolnaftate, flucytosine, butenafine, griseofulvin, cyclopirox, selenium sulfide, tavaborole. In another embodiment, the antiviral agent is acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir,Trifluridine, vidarabine, cytarabine, valacyclovir, tromatandine, pritelivir, amantadine, rimantadine, oseltamivir, peramivir, zanamivir, asunaprevir, boceprevir, silprevir, danoprevir, faldaprevir, glecaprevir, grazoprevir, narlaprevir, paritaprevir, simeprevir, sovaprevir, telaprevir, vaniprevir, vedroprevir, voxilaprevir, daclatasvir, Elbasvir, Ledipasvir, Odalasvir, Ombitasvir, Pibrentasvir, Rabidasvir, Ruzasvir, Samatasvir, Velpatasvir, Beklabuvir, Dasabuvir, Deleobuvir, Filibuvir, Setrobuvir, Sofosbuvir, Radalbuvir, Uprifosbuvir, Lamivudine, Terbivudine, Klevudine, Adefovir, Tenofvir / Disoproxil disoproxil), tenofovir alafenamide, enfuvirtide, maraviroc, bicriviroc, cenicriviroc, PRO 140, ibalizumab, fostemsavir, didanosine, emtricitabine, lamivudine, stabudine, zidovudine, amdoxovir, apricitabine, censavudine, elvucitabine, racivir, stampidine, 4'-ethinyl-2-fluoro-2'-deoxyadenosine, zalcitabine, efavirenz, nevirapine, delavirdin, etravirine, rilpivirine, doravirine, dolutegravir, elvitegravir, raltegravir, BI 224436,Cabotegravir, bictegravir, MK-2048, bevirimat, BMS-955176, amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, atazanavir, darunavir, tipranavir, dolutegravir, elvitegravir, raltegravir, BI The following are selected: 224436, cabotegravir, bictegravir, MK-2048, cobicistat, ritonavir, interferon-α, pegylated interferon-α, methisazone, rifampicin, imiquimod, reciquimod, podophyllotoxin, homivirsen, cidofovir, preconalil, favipiravir, galidesivir, remdesivir, mericitabine, MK-608, NITD008, moloxidine, tromantadine, and triazavirin. In further embodiments, the sample is obtained from a subject suspected of having or having a microbial infection. In certain embodiments, the subject is suspected of having or has sepsis. In other embodiments, one or more types of microorganisms are bacteria, fungi, and / or viruses. In yet another embodiment, the bacteria include Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia Helicobacter pylori, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leple (My, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus aureus), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, and / or Yersinia pseudotuberculosis (Yersinia In further embodiments, the fungus is selected from Absidia corymbifera, Absidia ramose, Achorion gallinae,Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp., Blastomyces spp., Cadophora spp., Candida albicans, Cercospora apii, Chrysosporium Cladosporium spp., Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp., Emmonsiella capsule capsulate), Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans,Species of the genus Fonsecaea, Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum, Keratinomyces, Langeronia soudanense, and Leptosphaeria senegalensis. senegalensis), Lichtheimia corymbifera, Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia (spp.), Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp,Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum species Selected from the genera Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp., Trichophyton spp., Trichosporon spp., and / or Zopfia rosatii. In further embodiments, the viruses may belong to the genera Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, and Nupapillomavirus.Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, Deltapolyomavirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus, Dependovirus, Bocavirus, Orthohepadnavirus, Gamma-retrovirus Virus genera (Gammaretrovirus), Deltaretrovirus, Lentivirus, Simispumavirus, Coltivirus, Rotavirus, Seardonavirus, α-Coronavirus, β-Coronavirus, Torovirus, Mamastrovirus Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus, Kobuvirus,Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebolavirus, Marburgvirus, Henipavirus, Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Orthopneumovirus, Ledantevirus, Lyssavirus Selected from the genera Vesiculovirus, Mammarenavirus, Orthohantavirus, Orthonairovirus, Orthobunyavirus, Phlebovirus, α-Influenzavirus, β-Influenzavirus, γ-Influenzavirus, Quaranjavirus, Thogotovirus, and / or Deltavirus. In certain embodiments, one or more types of nucleosides or nucleotide analogs are 2-ethynyl-adenosine, N6-propargyl-adenosine, 2'-(O-propargyl)-adenosine, 3'-(O-propargyl)-adenosine, 5-ethynyl-cytidine, 5-ethynyl-2'-deoxycytidine, 2'-(O-propargyl)-cytidine, 3'-(O-propargyl)-cytidine, 2'-(O-propargyl)-guanosine, 3'-(O-propargyl)-guanosine, 5-ethynyl-uridine, 5-ethynyl-2'-deoxyuridine, 2'-(O-propargyl)-uridine, 3'-(O-propargyl)-uridine,(2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (2'S)-2'-fluoro-5-ethynyluridine, 2'(S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (2'S)-2'-fluoro-5-ethynyluridine, 8-azido-adenosine, N, 6 Selected from -(6-azido)hexyl-2'deoxyadenosine, 2'-azido-2'-deoxyadenosine, 5-azidomethyluridine, 5-(15-azido-4,7,10,13-tetraoxa-pentadecanoyl-aminoallyl)-2'-deoxyuridine, 5-(3-azidopropyl)-uridine, 5-azido-PEG4-uridine, 5-azido-PEG4-cytidine, 5-azido-PEG4-2'-deoxycytidine, 5-bromo-2'deoxyuridine, 5-bromouridine, 5-iodo-2'deoxyuridine, and 5-iodouridine. In another embodiment, one or more types of nucleosides or nucleotide analogs are 2-ethynyl-adenosine, N6-propargyl-adenosine, 2'-(O-propargyl)-adenosine, 3'-(O-propargyl)-adenosine, 5-ethynyl-cytidine, 5-ethynyl-2'-deoxycytidine, 2'-(O-propargyl)-cytidine, 3'-(O-propargyl)-cytidine, 2'-(O-propargyl)-guanosine, 3'-(O-propargyl)-guanosine, 5-ethynyl-uridine, 5-ethynyl-2'-deoxyuridine, 2'-(O-propargyl)-uridine, 3'-(O-propargyl )-uridine, (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (2'S)-2'-fluoro-5-ethynyluridine, 2'(S)-2'-deoxy-2'-fluoro-5-ethynyluridine, and (2'S)-2'-fluoro-5-ethynyluridine are selected. In yet another embodiment, one or more types of nucleosides or nucleotide analogs are 8-azido-adenosine, N 6-(6-azido)hexyl-2'deoxy-adenosine is selected, and one or more types of nucleosides or nucleotide analogs are selected from 2'-azido-2'-deoxyadenosine, 5-azidomethyl-uridine, 5-(15-azido-4,7,10,13-tetraoxa-pentadecanoyl-aminoallyl)-2'-deoxyuridine, 5-(3-azidopropyl)-uridine, 5-azido-PEG4-uridine, 5-azido-PEG4-cytidine, and 5-azido-PEG4-2'-deoxycytidine. In certain embodiments, one or more types of nucleosides or nucleotide analogs are selected from 5-bromo-2'deoxyuridine, 5-bromouridine, 5-iodo-2'deoxyuridine, and 5-iodouridine. In another embodiment, both the control sample and the treated sample are incubated for the same duration of 5 to 180 minutes in the presence of one or more types of nucleosides or nucleotide analogs. In yet another embodiment, both the control sample and the treated sample are incubated for the same duration of 30 to 120 minutes in the presence of one or more types of nucleosides or nucleotide analogs. In a further embodiment, the labeling reagent is an antibody that binds with high specificity to one or more types of nucleosides or nucleotide analogs. In yet another embodiment, the antibody binds with high specificity to 5-bromo-2'deoxyuridine or iododeoxyuridine. In a particular embodiment, the labeling reagent binds to or to one or more types of nucleosides or nucleotide analogs via click chemistry, strain[3+2] cycloaddition, or Staudinger ligation. In yet another embodiment, the labeling reagent contains an azide group that binds to an alkynyl group-containing nucleoside or nucleotide analog via click chemistry. In yet another embodiment, the labeling reagent comprises an alkynyl group that binds to a nucleoside or nucleotide analog containing an azide group via click chemistry. In yet another embodiment, the labeling reagent comprises a biotin group. In a particular embodiment, the labeling reagent comprising a biotin group is [ka] Selected from. In another embodiment, the labeling reagent further comprises a chemically cleavable or enzymatically cleavable linker. In yet another embodiment, the cleavable linker is an acid-unstable linker or a disulfide linker. In yet another embodiment, the acid-unstable linker comprises a hydrazone or a cis-aconityl group. In yet another embodiment, the enzymatically cleavable linker comprises a peptide linker or a β-glucuronide linker. In a particular embodiment, the pull-down agent is used to isolate or purify newly synthesized labeled microbial nucleic acids. In another embodiment, the pull-down reagent is an antibody immobilized on a solid support, the antibody binding with high specificity to the labeling reagent or to one or more types of nucleosides or nucleotide analogs. In yet another embodiment, the pull-down reagent is streptavidin or avidin immobilized on a solid support, and the labeling reagent comprises a biotin group. In another embodiment, the solid support is a nanomaterial or micromaterial, a bead, or a plate. In yet another embodiment, the labeling reagent or label is removed or cleaved from the isolated or purified newly synthesized microbial nucleic acid prior to (f), (f') and (g) above. In yet another embodiment, the gene expression levels and / or quantities and / or identity of the isolated or purified newly synthesized microbial nucleic acids in the control sample and the treated sample are determined by using a microarray containing probes for nucleic acids from different microorganisms.In further embodiments, determining the gene expression level and / or quantity and / or identity of isolated or purified newly synthesized microbial nucleic acids in the control sample and the treated sample is done by (i) amplifying and labeling isolated or purified newly synthesized microbial nucleic acids from the control sample using a first PCR-based method with a primer containing a fluorescent dye, wherein the primer contains a sequence specific to the conserved microbial 16S rRNA gene region; (i') amplifying and labeling isolated or purified newly synthesized microbial nucleic acids from the treated sample using a first PCR-based method with a primer containing a fluorescent dye, wherein the primer contains a sequence specific to the conserved microbial 16S rRNA gene region; and (ii) labeling the product from the control sample from 20 or more microorganisms with unique 16S rRNA genes. (ii) applying a probe containing an rRNA variable region sequence to a first microarray, wherein the labeled product from the treated sample is applied to a second microarray, the second microarray being a replica of the first microarray, and (iii) imaging the first and second microarrays of the fluorescence hybridization product and determining whether there are any changes in the intensity, location, or absence of the fluorescence hybridization product between the microarrays, thereby determining that if there is a decrease in the intensity of the fluorescence hybridization product between the first and second microarrays, or if there is a change in the location or absence of the fluorescence hybridization product between the first and second microarrays, the antimicrobial agent is effective in regulating the growth and proliferation of the microorganism(s).In another embodiment, the effectiveness of an antimicrobial agent in regulating the growth and proliferation of microorganisms in a sample is determined or confirmed by sequencing newly synthesized microbial nucleic acids isolated or purified from the control and treated samples, and it is shown that the antimicrobial agent is effective in regulating the growth and proliferation of microorganisms if there is a decrease in the gene expression level of the newly synthesized microbial nucleic acids in the treated sample versus the control sample, or a decrease in the amount and / or identity of the newly synthesized microbial nucleic acids in the treated sample versus the control sample. In yet another embodiment, newly synthesized microbial nucleic acids isolated or purified from the control and treated samples are sequenced using a transposome-based sequencing method. In further embodiments, sequencing of newly synthesized microbial nucleic acids from control and treated samples is performed by (a) applying isolated or purified newly synthesized microbial nucleic acids from control and treated samples to bead-linked transpososomes, the bead-linked transpososomes mediating the simultaneous fragmentation of microbial nucleic acids and the addition of sequencing primers; (b) amplifying the microbial nucleic acid fragments with primers containing index and adapter sequences to form a library of amplified products; (c) washing and pooling the library of amplified products from control samples; (c') washing and pooling the library of amplified products from treated samples; (d) sequencing the library of amplified products from control samples; (d') sequencing the library of amplified products from treated samples; and (e) determining any changes in gene expression levels and / or amounts and / or identity of isolated or purified newly synthesized microbial nucleic acids from control and treated samples based on the use of bioinformatics analysis. In further embodiments, the newly synthesized microbial nucleic acid is RNA, and the microbial RNA is reverse transcribed into cDNA prior to (f), (f'), and (g) above, and the effectiveness of the antimicrobial agent that regulates the growth and proliferation of the microorganism(s) can be determined by determining the change in gene expression levels of the newly synthesized microbial nucleic acid from the control sample and the treated sample by using a microarray and / or sequencing. [Brief explanation of the drawing]

[0008] [Figure 1] This document illustrates an exemplary embodiment of a workflow for enriching newly synthesized DNA from rapid bacterial culture. Enrichment of newly synthesized DNA enables the genetic identification of viable bacteria in patient samples.

[0009] [Figure 2] This document illustrates an exemplary embodiment of a workflow for enriching newly synthesized RNA from rapid bacterial culture. Enrichment of newly synthesized RNA enables the evaluation of gene expression by viable bacteria in patient samples. [Modes for carrying out the invention]

[0010] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise. Thus, for example, a reference to "microorganisms" includes multiple such microorganisms, and a reference to "nucleoside analogues" includes one or more nucleoside analogues and their equivalents known to those skilled in the art.

[0011] Furthermore, unless otherwise stated, the use of "or" means "and / or". Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and not intended to be limiting.

[0012] Where the description of various embodiments uses the term "comprising," a person skilled in the art will further understand that in some specific examples, embodiments may be described using the language "essentially consisting of" or "consisting of."

[0013] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meanings as those commonly understood by those skilled in the art to which this disclosure pertains. Many methods and reagents disclosed herein are similar to or equivalent to those described herein, but exemplary methods and materials are disclosed herein.

[0014] All publications described herein are incorporated herein in whole by reference for the purpose of explaining and disclosing methodologies that may be used in connection with the descriptions herein. Furthermore, with respect to any terms presented in one or more publications that are similar to or identical to terms expressly defined herein, the definitions of terms expressly provided herein shall prevail in all respects.

[0015] This disclosure is not limited to the specific methodologies, protocols, and reagents described herein, and should therefore be understood to be subject to change. The terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the invention as defined solely by the claims.

[0016] Except in the operational examples or unless otherwise specified, all numerical values ​​representing amounts of components or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used to describe the present invention, “approximately” means ±1% in relation to percentages.

[0017] As used herein, the term "click chemistry" refers to a [3+2] cycloaddition reaction when carried out in the presence of a copper(I) catalyst. The copper(I) catalyst may include copper(I) ions or a copper(I) chelate moiety. The copper(I) chelate moiety may be "any entity characterized by the presence of two or more polar groups that can participate in the formation of a complex (containing two or more coordination bonds) with copper(I) ions" (see, e.g., Salic et al., U.S. Patent Application No. 20070207476, supra). Examples of copper(I) chelating agents include, but are not limited to, neocuproine and bathocuproinedisulfonate (see, e.g., Salic et al., U.S. Patent Application No. 20070207476 and Sharpless et al., U.S. Patent Application Publication No. 2003000516671). The [3+2] cycloaddition reaction, also known as a 1,3-dipolar cycloaddition, can occur between a 1,3-dipole and a dipolarophile. Examples of 1,3-dipoles include azides. Examples of dipolarophiles include alkynes.

[0018] As used herein, the term "dye" refers to a compound that emits light to produce a detectable signal that can be observed.

[0019] As used herein, the term "dual labeling" refers to a labeling process in which a nucleic acid is labeled with two detectable agents that produce distinguishable signals. A nucleic acid obtained from such a labeling process is said to be doubly labeled.

[0020] As used herein, the term "dye-labeled alkyne" refers to an alkyne that has been further modified to include a dye label.

[0021] As used herein, the terms "dye-labeled azide" and "azide dye molecule" refer to a compound or molecule having a reactive azide group that is also labeled with a dye. Examples, though not limited to them, include rhodamine-azide, Alexa Fluor(registered trademark) 350 azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 488 azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 555-azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 568-azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 568-azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 594 azide, Alexa Fluor(registered trademark) 633-azide (Molecular Examples include Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Alexa Fluor(registered trademark) 647-azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), Cascade Blue(registered trademark) azide (Molecular Probes(trademark) / Invitrogen(trademark), Carlsbad, CA), fluorescein-azide, coumarin-azide, bodipy-azide, cymarin-azide, or tetramethylrhodamine(TMR)-azide.

[0022] As used herein, the term “dye-labeled cycloalkyne” refers to a cycloalkyne that has been further modified to include a dye label. The term “cycloalkyne” refers to a compound or molecule that can be used in a strain[3+2] cycloaddition reaction to label DNA. In this context, examples of cycloalkynes include, but are not limited to, cyclooctin, difluorocyclooctin, heterocycloalkyne, dichlorocyclooctin, dibromocyclooctin, or diiodocyclooctin.

[0023] As used herein, the term “effective amount” refers to the amount of a substance, compound, molecule, drug, or composition that elicits a relevant response in a cell, tissue, or microorganism. For example, in the case of a microorganism in contact with a nucleoside analog, the effective amount is the amount of nucleoside incorporated into the microbial DNA.

[0024] As used herein, the terms “fluorescent phenotype” or “fluorescence” refer to a composition that exhibits a change in fluorescence upon binding to a biological compound or a sample of interest. Preferred fluorescent phenotypes of this disclosure include fluorescent dyes that have a high quantum yield in an aqueous medium. Exemplary fluorescent phenotypes include, among others, xanthenes, indoles, borapolyazindacenes, furans, benzofurans, and cyanines. The fluorescent phenotypes of the present invention may be substituted to alter the solubility, spectral properties, or physical properties of the fluorescent phenotype.

[0025] As used herein, the term “labeled” refers to a chemical moiety or protein that retains its original properties (e.g., spectral properties, conformation, and activity) when a portion of the labeling reagents of this disclosure is used in the methods of this disclosure. Exemplary “labeled” molecules may be directly detectable (fluorescent fluorophores), indirectly detectable (hapten or enzyme), or can be used for the detection and purification of nucleic acids incorporating nucleosides (e.g., biotin-streptavidin pull-down assay). Such “labeled” molecules include, but are not limited to, biotin labels, imino-biotin, or desthiobiotin-containing labels designed by click chemistry, e.g.

change

[0026] The terms “microorganism” and “microbe” are used interchangeably herein and refer to microscopic organisms that may exist as single cells or within colonies of cells. For the purposes of this disclosure, “microorganism” as used herein includes bacteria, fungi, viruses, algae, archaea, and protozoa.

[0027] As used herein, the term “microbial growth” refers to the proliferation and / or growth of microorganisms.

[0028] The terms “nucleoside analog” and “nucleotide analog” are used interchangeably and, as herein, refer to molecules or compounds structurally similar to natural nucleosides or nucleotides that are incorporated into newly synthesized microbial nucleic acids. In the case of nucleosides, upon entering the cell, they are phosphorylated to nucleotides and then incorporated into the newly formed nucleic acid polymer. Because nucleotides are less stable than nucleosides and have difficulty crossing the cell membrane due to their charge, their use typically requires additional steps and reagents for transfection to transport the nucleotides across the lipid bilayer. These nucleoside analogs are incorporated into nucleic acids (DNA or RNA) in a similar manner to natural nucleotides, and the correct polymerase enzyme recognizes the analogs as natural nucleotides, thus posing no problems in synthesis. These analogs include several different moieties used for final detection, including halogenated analogs (bromo, chloro, iodine, etc.) and those containing bioorthogonal moieties such as azides, alkynes, or phosphines.

[0029] As used herein, the term “pull-down reagent” refers to a reagent used to purify or isolate a nascent nucleic acid polymer, comprising one or more labeled nucleotide analogs disclosed herein. A “pull-down reagent” is typically conjugated to a solid support, such as a bead, and selectively binds to the labels disclosed herein. Typically, the label functions as a “tag” as described above. In exemplary embodiments, the pull-down reagent is streptavidin conjugated to a solid support, such as a bead, superparamagnetic microparticles or nanoparticles, or a plate. In other embodiments, the pull-down reagent is an antibody or other type of affinity ligand specific to the label or “tag” immobilized on a solid support, such as a bead, superparamagnetic microparticles or nanoparticles, or a plate.

[0030] As used herein, the term “Staudinger ligation” refers to a chemical reaction developed by Saxon and Bertozzi (E. Saxon and C. Bertozzi, Science, 2000, 287:2007-2010), which is a modification of the classical Staudinger reaction. The classical Staudinger reaction is a chemical reaction in which a combination of an azide and a phosphine or phosphine produces an aza-ylide intermediate, which hydrolyzes to yield a phosphine oxide and an amine. The Staudinger reaction is a mild method of reducing azides to amines, with triphenylphosphine commonly used as the reducing agent. In Staudinger ligation, an electrophilic trap (usually a methyl ester) is appropriately positioned at the triarylphosphinearyl group (usually in the ortho position relative to the phosphorus atom) and reacts with the azide to produce an aza-ylide intermediate, which can be reconstituted in an aqueous medium to produce a compound having an amide group and a phosphine oxide functional group. This Staudinger ligation is so named because it ligates (bonds / covalently bonds) two starting molecules together, whereas in the classical Staudinger reaction, the two products do not covalently bond after hydrolysis.

[0031] The terms “subject,” “patient,” and “individual” are used interchangeably herein and refer to animals from which samples can be obtained, in particular humans. This includes humans and non-human animals. Herein, the terms “non-human animal” and “non-human mammal” are used interchangeably herein and include all vertebrates, e.g., non-human primates (especially higher primates), mammals such as sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows, and non-mammals such as chickens, amphibians, and reptiles. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. “Mammal” refers to humans, non-human primates, domesticated animals and livestock, as well as any animal classified as a mammal, including zoo, sport, or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, and rabbits. The patient or subject may be any subset of those described above, e.g., all of the above, but excluding one or more groups or species such as humans, primates, or rodents. The subject may be male or female. The subject may be a fully developed subject (e.g., an adult) or a subject in development (e.g., a child, infant, or fetus).

[0032] Living, growing microorganisms (e.g., bacterial algae, archaea, protozoa, and fungi) continuously synthesize new DNA. In direct contrast, microorganisms that are no longer alive cease to synthesize DNA. Living, non-growing microorganisms can synthesize new DNA to repair and maintain their genomes, but the rate of new DNA synthesis is much lower than that of living, growing microorganisms. This disclosure provides methodologies and techniques that utilize the aforementioned differences in DNA synthesis to rapidly identify living, growing microorganisms in a sample, such as a blood sample from a patient, an environmental sample, or a sample from food suspected of contamination. Specifically, the methodologies and techniques presented herein enable the identification of living, growing microorganisms in a sample, regardless of whether the sample further contains non-living or non-growing microorganisms or is contaminated. More specifically, the methodologies and techniques presented herein provide selective enrichment and sequencing of newly synthesized microbial DNA obtained from one or more microorganisms in a sample, enabling the identification of living, growing microorganisms contained in the sample.

[0033] This disclosure also provides methods and compositions that can be used to selectively enrich DNA and RNA from specific organisms or similar groups of organisms in a mixed population. For example, in dehosting applications, it is desirable to enrich the DNA or RNA of an infectious organism from a background of DNA or RNA from an infected host. This method enables rapid enrichment of DNA and / or RNA of a targeted microorganism (e.g., bacteria) from a mixed population in order to identify the targeted organism. The enrichment requires conditions such that only the targeted organism can synthesize DNA and / or RNA. For example, culture medium conditions, temperature, and / or specific inhibitors can be used to selectively inhibit a targeted population or subpopulation in a sample.

[0034] As an example, blood can be obtained from a subject with or suspected of having sepsis and cultured under conditions in which mammalian cells in the blood sample have their DNA and / or RNA synthesis inhibited, while bacterial cells in the sample can continue to synthesize DNA and / or RNA. In this way, bacterial DNA and / or RNA can be selectively labeled. In one embodiment, the blood sample can be isolated and seeded or cultured in LB broth or other bacterial medium such that mammalian cells do not continue DNA and / or RNA synthesis (or DNA and / or RNA synthesis is substantially reduced), while the microbial population continues to undergo DNA and RNA synthesis, resulting in selective incorporation of, for example, EdU. In another embodiment, the temperature of the blood culture can be lowered so that the replication and synthesis of mammalian cells are inhibited, and only the replication and synthesis of microorganisms are maintained or regenerated when the temperature is returned to a higher temperature. The temperature can be lowered over a period of several minutes to several hours. In another embodiment, small molecule inhibitors of DNA and / or RNA synthesis that selectively target the mammalian DNA and / or RNA mechanism can be used. For example, one inhibitor, called alpha-amanitin, is derived from the Amanita mushroom and is responsible for approximately 100 deaths each year from undiscriminating mushroom hunting. RNAP inhibitors can be specific to organisms of a single taxonomy. Alpha-amanitin, for example, affects higher eukaryotes but not bacteria. Conversely, some drugs specifically affect bacterial RNAPs. The best-known of these is rifampin, which is produced by fungi and is now used as an anti-tuberculosis drug in the form of its derivative, rifampicin (Rif). Rif is specific to bacterial RNAPs. This specificity of the inhibitor arises for two reasons. Firstly, inhibitors are often produced by one organism that kills another, and the producing organism must evolve an inhibitor that does not commit suicide. Secondly, inhibitors usually bind to an unconserved portion of the enzyme, and in this case, sequence changes can prevent them from acting on all RNAPs.

[0035] This disclosure also provides embodiments aimed at dehosting a sample using the methods of this disclosure prior to the identification of nascent microbial nucleic acid synthesis. Such dehosting techniques and compositions relate, for example, to the selective cleavage of non-microbial nucleic acids in a sample containing both microbial and non-microbial nucleic acids, resulting in the sample being significantly enriched with microbial nucleic acids. Examples of dehosting methods are described in Feehery et al., PLoS ONE 8:e76096 (2013), Sachse et al., Journal of Clinical Microbiology 47:1050-1057 (2009), Barnes et al., PLoS ONE 9(10):e109061 (2014), Leichty et al., Genetics 198(2):473-81 (2014), Hasan et al., J Clin Microbiol 54(4):919-27 (2016), and Liu et al., PLoS ONE 11(1):e0146064 (2016), the disclosures of which are fully incorporated herein. Furthermore, commercially available kits for performing dehosting are also available, including the NEBNext Microbiome DNA Enrichment® kit, the Molzym MolYsis Basic® kit, and the MICROBEEnrich® kit.

[0036] In some embodiments, the dehosting methods and compositions disclosed herein utilize properties associated with non-microbial nucleic acids, including methylation at CpG residues, and their association with DNA-binding proteins such as histones. For example, in certain embodiments, the dehosting methods and compositions may utilize nucleic acid-binding proteins that selectively bind to non-microbial nucleic acids (e.g., histamine, restriction enzymes). In further embodiments, the dehosting methods and compositions may include recombinant proteins that selectively bind to and selectively degrade non-microbial nucleic acids, i.e., the recombinant proteins include both a non-microbial nucleic acid-binding domain and a nuclease domain. In certain embodiments, the nucleic acid-binding protein is a histone. Histones are found in the nuclei of eukaryotic cells and in certain archaea, namely the Thermoproteales and Euryarchaea, but not in bacteria or viruses. In further embodiments, histone-bound non-microbial nucleic acids can then be removed from a sample by using a substrate containing an affinity agent that selectively binds to a histone protein, i.e., a histone-binding domain. Examples of affinity agents that can bind to histone proteins include chromodomains, Tudor, Malignant Brain Tumor (MBT), plant homeodomains (PHD), bromodomains, SANT, YEATS, proline-tryptophan-tryptophan-proline (PWWP), bromo adjacent homology (BAH), Ankryin repeat, WD40 repeat, ATRX-DNMT3A-DNMT3L (ADD), or Zn-CW.In another embodiment, the histone-binding domains are HAT1, CBP / P300, PCAF / GCN5, TIP60, HB01 (ScESA1, SpMST1), ScSAS3, ScSAS2 (SpMST2), ScRTT109, SirT2 (ScSir2), SUV39H1, SUV39H2, G9a, ESET / SETDB1, EuHMTase / GLP, CLL8, SpClr4, MLL1, MLL2, MLL3, MLL4, MLL5, SET1A, SET1B, ASH1, Sc / Sp SET1, SET2 (Sc / Sp SET2), NSD1, SYMD2, DOT1, Sc / Sp DOT1, Pr-SET7 / 8, SUV4 20H1, SUV4 20H2, SpSet 9. It may contain a domain that specifically binds to histones or histone-binding fragments from proteins such as EZH2, RIZ1, LSD1 / BHC110, JHDM1a, JHDM1b, JHDM2a, JHDM2b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, CARM1, PRMT4, PRMT5, Haspin, MSK1, MSK2, CKII, Mst1, Bmi / Ring1A, RNF20 / RNF40, or ScFPR4.

[0037] In additional embodiments, the disclosure provides nucleic acid-binding proteins or nucleic acid-binding domains that selectively bind to DNA containing methylated CpGs. CG dinucleotide motifs ("CpG sites" or "CG sites") are found in regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in a linear sequence of bases along its 5'-3' direction. CpG islands (or CG islands) are high-frequency regions of CpG sites. CpG is an abbreviation for 5'-C-phosphate-G-3', i.e., cytosine and guanine separated by a single phosphate group. Cytosine in CpG dinucleotides can be methylated to form 5-methylcytosine. Cytosine methylation occurs throughout the human genome at many CpG sites. Cytosine methylation at CG sites also occurs throughout the genomes of other eukaryotes. In mammals, for example, 70%–80% of CpG cytosines may be methylated. In target pathogenic microorganisms such as bacteria and viruses, this CpG methylation is either absent or significantly lower than in the human genome. Therefore, dehosting can be achieved by selectively cleaving CpG-methylated DNA.

[0038] In some embodiments, the disclosure provides a dehosting method comprising a nucleic acid-binding protein or binding domain that binds to a CpG island or CpG site. In another embodiment, the binding domain comprises a protein or fragment thereof that binds to a methylated CpG island. In yet another embodiment, the nucleic acid-binding protein binding domain comprises a methyl-CpG-binding domain (MBD). An example of an MBD is a polypeptide of about 70 residues that folds into an alpha / beta sandwich structure comprising a layer of twisted beta sheets, backed by another layer formed by an alpha-1 helix and a C-terminal hairpin loop. Both of these layers are amphiphilic, with the alpha-1 helix and beta sheets arranged in parallel and the hydrophobic faces tightly packed together. The beta sheets consist of two long inner strands (beta-2 and beta-3) sandwiched between two short outer strands (beta-1 and beta-4). In a further embodiment, the nucleic acid-binding protein or binding domain comprises a protein selected from the group consisting of MECP2, MBD1, MBD2, and MBD4, or fragments thereof. In further embodiments, the nucleic acid-binding protein or binding domain includes MBD2. In certain embodiments, the nucleic acid-binding protein or binding domain includes a fragment of MBD2. In other embodiments, the nucleic acid-binding protein or binding domain includes MBD5, MBD6, SETDB1, SETDB2, TIP5 / BAZ2A, or BAZ2B, or fragments thereof. In yet another embodiment, the nucleic acid-binding protein or binding domain includes a CpG-methylated or demethylated protein, or fragments thereof. In further embodiments, CpG-bound nonmicrobial nucleic acids can then be removed from a sample by using a substrate containing an affinity agent that selectively binds to a nucleic acid-binding protein or binding domain bound to a CpG island or CpG site. Examples of affinity agents include antibodies or antibody fragments that selectively bind to nucleic acid-binding proteins or binding domains bound to a CpG island or CpG site. The affinity agent containing an antibody or antibody fragment may be bound to a substrate, or it may be bound by a secondary antibody that is itself bound to the substrate, thereby providing a means for separating and removing nonmicrobial nucleic acids from a sample.

[0039] In another embodiment, the disclosure provides a dehosting method using a nuclease or recombinant protein comprising a nuclease domain, thereby cleaving non-microbial nucleic acids into fragments. In the latter case, the recombinant protein may also comprise a nucleic acid protein-binding domain having activity toward nucleic acid-binding proteins (e.g., histamine, methyl-CpG-binding proteins). The nuclease or nuclease may include, but is not limited to, nonspecific nucleases, endonucleases, nonspecific endonucleases, nonspecific exonucleases, homing endonucleases, and restriction endonucleases. In another embodiment, the nuclease domain is derived from any nuclease in which the nuclease or nuclease domain does not have its own inherent target. In yet another embodiment, the nuclease domain is active when fused with other proteins. Examples of nonspecific nucleases include FokI and I-TevI. In some embodiments, the nuclease domain is FokI or a fragment thereof. In further embodiments, the nuclease domain is I-TevI ​​or a fragment thereof. In even further embodiments, FokI or I-TevI ​​or a fragment thereof is unmutated and / or wild-type. Further examples of nucleases include, but are not limited to, deoxyribonuclease I (DNase I), RecBCD endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, endonuclease I (endo I), micrococcal nuclease, endonuclease II (endo VI, exo III), neurospora endonuclease, S1-nuclease, P1-nuclease, mung bean nuclease I, ustilago nuclease (DNase I), AP endonuclease, and Endo R.

[0040] The microorganisms to be identified can be found in a variety of samples, including, but not limited to, patient samples (e.g., blood, urine, and cerebrospinal fluid), food samples (e.g., wheat flour, beef, and lettuce), or environmental samples (e.g., groundwater, and hospital construction swabs). The main advantage of the methods, compositions, and kits disclosed herein is that they allow for the identification of viable and / or growing microorganisms in a sample without the need for extensive culture of the microorganisms beforehand. Thus, microorganisms that cannot be cultured in vitro on conventional culture media or tissue cultures, such as Treponema pallidum (syphilis) and environmental bacteria, can be easily identified using the methods, compositions, and kits disclosed herein.

[0041] Furthermore, this specification discloses methods for labeling, purifying, and sequencing newly synthesized nucleic acids to identify and analyze viable microorganisms in patients, food, the environment, or other samples. The methods disclosed herein can be further used to screen test compounds (e.g., antibiotics) for their effects on viable microorganisms identified in a sample. The methods disclosed herein utilize nucleoside analogs that are “supplied” to microorganisms and incorporated into newly synthesized or nascent nucleic acids. With respect to microorganisms, any type of microorganism, including bacteria, fungi, viruses, algae, archaea, and protozoa, can be detected by the methods disclosed herein.

[0042] Bacteria are prokaryotes, lacking a nucleus and containing no organelles. Within the bacterial family, there are two classes: Gram-positive bacteria, which have thick cell walls, and Gram-negative bacteria, which have a thin layer sandwiched between an inner and outer membrane. Bacteria are incredibly diverse and, in terms of numbers, are the most successful organisms on Earth. They are the only microorganisms that can survive harmlessly in the human body and often assist in bodily functions such as digestion. Apart from viruses, bacteria are the most problematic in relation to diseases in humans, such as sepsis. Examples of bacteria that can be identified and analyzed using the methods, compositions and kits disclosed herein include, but are not limited to, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, and Brucella melitensis. **Leptospira perfringens*, *Clostridium tetani*, *Corynebacterium diphtheriae*, *Enterococcus faecalis*, *Enterococcus faecium*, *Escherichia coli*, *Francisella tularensis*, *Haemophilus influenzae*, *Helicobacter pylori*, *Legionella pneumophila*, *Leptospira interrogans*, *Leptospira santarosai*, *Leptospira weirii* Weilii), Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium typhimurium), Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidesExamples include *Epidermidis*, *Staphylococcus saprophyticus*, *Streptococcus agalactiae*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Treponema pallidum*, *Ureaplasma urealyticum*, *Vibrio cholerae*, *Yersinia pestis*, *Yersinia enterocolitica*, and / or *Yersinia pseudotuberculosis*.

[0043] Fungi are eukaryotes, meaning they possess a distinct nucleus and organelles. Their cells are larger than those of prokaryotes such as bacteria. Fungal colonies become visible to the human eye once they reach a certain level of growth, such as mold on bread. Fungi can be divided into three main groups: (1) molds, which exhibit filamentous growth and multicellular structures; (2) yeasts, which are typically non-filamentous and can be unicellular; and (3) mushrooms, which possess fruiting bodies for spore production. Fungi can be problematic due to immunodeficiency and can be significant pathogens to plants. Examples of fungi that can be identified and analyzed using the methods, compositions, and kits disclosed herein include Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp., and Blastomyces (spp.), Cadophora spp., Candida albicans, Cercospora apii, Chrysosporium spp., Cladosporium spp.), Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp., Emmonsiella capsulate, Endomyces geotrichum * geotrichum*, *Entomophthora coronate*, *Epidermophyton floccosum*, *Filobasidiella neoformans*, *Fonsecaea* spp., *Geotrichum candidum*, *Glenospora khartoumensis*, *Gymnoascus gypseus*, *Haplosporangium parvum*, *Histoplasma*, *Histoplasma capsulatum*, *Hormiscium dermatididis*, *Hormodendrum* genus spp.), Keratinomyces spp., Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp., Mucor Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp.), Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum species Examples include species of the genera Stachybotrys, Stachybotrys chartarum, Streptomyce, Tinea, Torula, Trichophyton, Trichosporon, and Zopfia rosatii.

[0044] Viruses are generally considered to be highly complex non-living molecules, typically containing a protein coat surrounding a genetic material RNA or DNA core, but lacking a semipermeable membrane. They can grow and multiply only in living cells and represent a large group of ultramicroscopic infectious agents that cause a variety of important diseases in humans, animals, and plants. Examples of viruses that can be identified and analyzed using the methods, compositions, and kits disclosed herein include the genera Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, and Nupapillomavirus. avirus), Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, Deltapolyomavirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus,Dependovirus, Bocavirus, Orthohepadnavirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, Simispumavirus, Coltivirus, Rotavirus, Seadonavirus, α-Coronavirus, β-Coronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus Cosavirus, Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebolavirus, Marburgvirus, Henipawy Henipavirus, Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Orthopneumovirus, Ledantevirus, Lyssavirus, Vesiculovirus, Mammarenavirus,Examples include the genera Orthohantavirus, Orthonairovirus, Orthobunyavirus, Phlebovirus, α-Influenzavirus, β-Influenzavirus, γ-Influenzavirus, Quaranjavirus, Thogotovirus, and / or Deltavirus.

[0045] Algae are a more difficult group of organisms to define, and by some definitions they include both prokaryotes and eukaryotes. Unlike other microorganisms, algae are typically photosynthetic organisms and are typically found in marine environments. Harmful algal blooms (HABs) are algal blooms that produce natural toxins, cause mechanical damage to other organisms, or otherwise adversely affect other organisms. HABs are often associated with large-scale marine fatal events and are linked to various types of shellfish poisoning. HABs include toxic or other harmful phytoplankton such as dinoflagellates of the genera Alexandrium and Karenia or diatoms of the genera Pseudo-nitzschia. Such algal blooms often exhibit a red or brown hue and are commonly known as red tides. The methods, compositions, and kits of this disclosure enable the identification of such algae from samples, such as environmental samples.

[0046] Archaea are prokaryotes that have a morphology similar to bacteria. Archaea differ from eukaryotes and bacteria in terms of genetic, biochemical, and structural characteristics. For example, archaea possess unique flagellin and ether-binding lipids, and their cell walls lack murein. Archaea share several characteristics with known pathogens that may reflect their disease-causing potential. Such characteristics include abundant access (i.e., opportunity) to the host, as well as the ability to colonize long-term and coexist with the endogenous flora within the host. The detection of anaerobic archaea in the human colon, vagina, and oral microbiome demonstrates their ability to colonize human hosts. The methods, compositions, and kits of this disclosure enable the identification of such archaea from samples, e.g., environmental samples, samples obtained from subjects, etc.

[0047] Protozoa are single-celled eukaryotes, either free-living or parasitic, that feed on other microorganisms or organic matter such as organic tissues and debris. Traditionally, protozoa range in size from about 1 micrometer to several millimeters or larger. All protozoa are heterotrophic, extracting nutrients from other organisms either by ingesting them whole or by consuming their organic residues and waste products. Some protozoa ingest food through phagocytosis, swallowing organic particles with pseudopods (like amoebas) or by taking in food through specialized mouth-like openings called cytostomes. Others ingest food through osmosis, absorbing dissolved nutrients through the cell membrane. Numerous protozoan pathogens are human parasites that cause diseases such as malaria (caused by Plasmodium parasite), amebiasis, giardiasis, toxoplasmosis, cryptoporidiasis, trichomoniasis, Chagas disease, leushmaniasis, African trypanosomiasis (sleeping sickness), amebic dysentery, Acanthamoeba keratitis, and primary amebic meningitis (naegleriasis). The methods, compositions, and kits of this disclosure enable the identification of such protozoa from samples, such as environmental samples or samples obtained from subjects.

[0048] The methods disclosed herein provide for the identification and analysis of the aforementioned microorganisms, particularly viable and / or proliferating microorganisms, from a sample. As described above, the sample may originate from a variety of sources, such as from a subject, from the environment, or from food. Any number of types of samples from a subject can be used with the compositions, methods, and kits disclosed herein, and these include, but are not limited to, blood, urine, saliva, middle ear aspirate, bile, vaginal secretions, pus, pleural fluid, synovial fluid, and abdominal abscesses. Thus, the methods, compositions, and kits disclosed herein are not particularly limited by the type and location of the sample obtained from the subject. Furthermore, the methods, kits, and compositions disclosed herein offer an improvement over standard methodologies for identifying microorganisms causing sepsis or urinary tract infections in patients, in that they can accurately identify the microorganism in question in a much faster manner than standard methodologies. Therefore, the appropriate antimicrobial agent(s) against the identified microorganism(s) can be administered much faster, thereby allowing for the combat and elimination of microbial infections in a more rapid manner, and preventing or mitigating side effects associated with microbial infections, such as septic shock, chills, fever, body aches, cognitive changes, fatigue, malaise, respiratory distress, abnormal cardiac infections, inflammation, nausea, vomiting, and anxiety. Furthermore, the methods, kits, and compositions disclosed herein can also determine whether an antimicrobial agent is effective in inhibiting or killing a microorganism. Thus, if a microorganism is resistant to a particular antimicrobial agent, the methods, kits, and compositions disclosed herein can make such a determination quickly, and as a result, another antimicrobial agent can be tried.

[0049] The methods, compositions, and kits of this disclosure can utilize both nucleosides and nucleotide analogs for identifying nascent microbial nucleic acid synthesis. As described in more detail below, the methods, compositions, and kits of this disclosure can utilize multiple types of nucleosides and nucleotide analogs, the use of which may be advantageous for establishing baseline nucleic acid synthesis and determining changes in the rate of nucleic acid synthesis, such as the addition of antimicrobial agents. Nucleosides are typically used in experiments where they are added to cell cultures or administered to animals, as they are readily taken up by living cells, where they are phosphorylated to nucleotides and then incorporated into the growing nucleic acid polymer. In contrast, nucleotides are more unstable, either before or after incorporation into cells, more susceptible to enzymatic cleavage, and generally less stable than nucleosides. In addition, due to the additional charge from the phosphate group, nucleotides are not readily transported into living cells and generally require a transfection step to obtain sufficient concentrations of nucleotides across the cell membrane. This is not ideal for either in vivo or ex vivo / in vivo experiments, where cellular perturbations should be kept to a minimum in order to accurately interpret the results. For these reasons, the following disclosures generally refer to nucleosides as analogs added to cells or animals, but this is not intended to be limited to nucleotides, which are equally important.

[0050] Nucleosides and nucleotide analogs can be analogs of any of the four DNA bases (adenine (A), cytosine (C), guanine (G), or thymine (T)) or any of the four RNA bases (adenine (A), cytosine (C), guanine (G), or uracil (U)), and include their triphosphate and phosphoramidite forms. Nucleosides and nucleotide analogs are incorporated into newly synthesized nucleic acids by polymerases present in microorganisms. Nucleosides and nucleotide analogs differ from naturally occurring nucleosides in that their phosphate backbone, pentose sugar, and / or ribose or deoxyribose are typically modified by synthetic chemical techniques. For example, a nucleotide or nucleoside can be modified to include a detectable label (e.g., a dye, a fluorophore), a bioorthogonal functional moiety (e.g., a moiety involved in specific chemical reactions such as click chemistry), or a biomolecule (e.g., an enzyme, an antibody, a biotin), any one of which can be used in the methods, compositions, and kits of this disclosure to identify newly synthesized and fabricated microbial nucleic acid polymers. In one embodiment, the nucleoside analog is a halogenated analog containing, but not limited to, bromo, chloro, and iodine moieties. Examples of halogenated analogs include, but are not limited to, 2'(S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (2'S)-2'-fluoro-5-ethynyluridine, 5-bromo-2'-deoxyuridine, 5-bromouridine, 5-iodo-2'deoxyuridine, and 5-iodouridine. With respect to halogenated analogs, antibodies have been specifically developed to bind to these analogs, such as bromo-2-deoxyuridine and iododeoxyuridine, with high affinity (see Dako, Carpinteria, CA; BD Bioscience, San Diego, CA; EMD Biosciences, Madison, WI). In another embodiment, the nucleoside or nucleotide analog comprises a bioorthogonal functional moiety, including, but not limited to, an azide, alkynyl, or phosphinyle moiety.Examples of nucleosides or nucleotide analogs containing bioorthogonal functional moieties include 2-ethynyl-adenosine, N6-propargyl-adenosine, 2'-(O-propargyl)-adenosine, 3'-(O-propargyl)-adenosine, 5-ethynyl-cytidine, 5-ethynyl-2'-deoxycytidine, 2'-(O-propargyl)-cytidine, 3'-(O-propargyl)-cytidine, 2'- (O-propargyl)-guanosine, 3'-(O-propargyl)-guanosine, 5-ethynyl-uridine, 5-ethynyl-2'-deoxyuridine, 2'-(O-propargyl)-uridine, 3'-(O-propargyl)-uridine, (2'S)-2'-deoxy-2-fluoro-5-ethynyluridine, (2'S)-2'fluoro-5-ethynyluridine, 8-azido-adenosine, N. 6 Examples include, but are not limited to, -(6-azido)hexyl-2'deoxyadenosine, 2'-azido-2'-deoxyadenosine, 5-azidomethyluridine, 5-(15-azido-4,7,10,13-tetraoxa-pentadecanoyl-aminoallyl)-2'-deoxyuridine, 5-(3-azidopropyl)-uridine, 5-azido-PEG4-uridine, 5-azido-PEG4-cytidine, and 5-azido-PEG4-2'-deoxycytidine.

[0051] In certain embodiments, the nucleoside analog comprises a bioorthogonal functional moiety that can undergo click chemistry, strain[3+2] cycloaddition, or Staudinger ligation with a functional group of a labeled reagent. In some embodiments, the reactive bioorthogonal moiety is supported by a base of the nucleoside. The base supporting the reactive bioorthogonal moiety may be a purine (e.g., adenine or guanine) or a pyrimidine (e.g., cytosine, uracil, or thymine). In certain embodiments, the base is uracil, and in some such embodiments, uracil supports the reactive bioorthogonal moiety at position 5. In certain embodiments, the base is adenine, and in some such embodiments, adenine supports the reactive bioorthogonal moiety. In certain embodiments, the bioorthogonal moiety is indirectly attached to the base, while in other embodiments, the bioorthogonal moiety is directly covalently bonded to the base. In certain embodiments, the reactive bioorthogonal moiety is supported by a sugar (ribose and deoxyribose) of the nucleoside. In certain embodiments, the bioorthogonal portion is indirectly attached to the sugar, while in other embodiments, the bioorthogonal portion is directly and covalently bonded to the sugar. In certain embodiments, the reactive bioorthogonal portion is attached to the phosphate portion of the nucleoside. The sugar supporting the reactive bioorthogonal portion can be covalently bonded to a purine (e.g., adenine or guanine) or a pyrimidine (e.g., cytosine, uracil, or thymine). In certain embodiments, the base is uracil, while in other embodiments, the base is adenine.

[0052] The reactive bioorthogonal moiety may be a 1,3-dipole such as a nitrile oxide, azide, diazomethane, nitrone, or nitrile imine. In certain embodiments, the 1,3-dipole is an azide. Alternatively, the reactive bioorthogonal functional moiety may be a parent dipole such as an alkene (e.g., vinyl, propylene) or an alkyne (e.g., ethynyl, propynyl). In certain embodiments, the parent dipole is an alkyne such as an ethynyl group.

[0053] These bioorthogonal functional moieties described above are unnatural, non-perturbative biochemical moieties with intrinsic chemofunctionality that can be modified by highly selective reactions. Specifically, these incorporated nucleosides are labeled using labeling reagents that include chemical means for selectively forming covalent bonds with the nucleosides in the presence of the cellular environment.

[0054] Analyzing complex cellular processes, including microbial growth, requires the ability to track biomolecules to function within their natural habitats. In recent years, bioorthogonal functional moieties have been used as a further method for tagging biomolecules. The use of bioorthogonal functional moieties has been described for the detection of metabolites and post-translational modifications that utilize azide moieties as biologically orthogonal functional moieties. Upon introduction into a target biomolecule, azides, either metabolically or chemically, can be probe-tagged using one of three highly selective reactions: Staudinger ligation, Cu(I)-catalyzed azide-alkyne cycloaddition, or strain-promoting [3+2] cycloaddition (see, e.g., Agard et al., J Am Chem Soc. 2004 Nov 24;1, 26(46):1 5046-7).

[0055] The bioorthogonal functional moieties can be used to label nucleic acids by incorporating nucleosides or nucleotide analogs. Thus, nucleic acids can be labeled using bioorthogonal labeling methods such as Staudinger ligation, Cu(I)-catalyzed [3+2] cycloaddition of azides and alkynes ("click chemistry"), or "copper-free" click chemistry, as independently described by Barry Sharpless and Carolyn Bertozzi. (See, for example, Sharpless et al., Angew Chem Int Ed Engl. 2002 Mar 15;41(6):1 053-7, Meldal et al., J.Org.Chem. 2002,67,3057; Agard et al., J Am Chem Soc. 2004 Nov 24;1 26(46):1 5046-7; U.S. Patent No. 7,122,703, U.S. Patent Application Publication No. 2003000516671). Click chemistry and Staudinger ligation are adapted to measure cell proliferation by directly detecting nucleotide incorporation. See Salic, et al., Methods and Compositions for Labeling Nucleic Acids, U.S. Patent Application Publications No. 20070207476 and 20070099222 (filed October 27, 2006).

[0056] A click chemistry technique for labeling nucleic acids involves treating cells with a first nucleoside or nucleotide analog containing a reactive unsaturated group so that a first nucleoside analog is incorporated into a newly synthesized microbial nucleic acid. The cells are then contacted with a labeling reagent containing a second reactive unsaturated group bound to the label, thereby causing a [3+2] cycloaddition between the first and second reactive unsaturated groups.

[0057] The following description of a [3+2] cycloaddition reaction for labeling microbial nucleic acids is provided for illustrative purposes only and is not intended to limit the scope of the invention.

[0058] As an example of labeling microbial DNA using click chemistry, a sample is treated with an effective amount of an alkyl-modified nucleoside analog, e.g., 5-ethynyl-2'-deoxyuridine (EdU), for a defined period of time so that EdU is incorporated into the newly synthesized DNA. After labeling with EdU, the labeled microbial DNA is reacted with an azide-disulfide-biotin linker in the presence of a copper(I) catalyst. A covalent bond is formed between the azide and the incorporated nucleoside analog via a [3+2] cycloaddition reaction, and the resulting complex can then be captured using a streptavidin conjugate substrate (e.g., beads). After washing the substrate, the microbial DNA is released from the substrate by adding a reducing agent such as dithiothreitol (DTT). The sequence of the microbial DNA can then be determined using standard methods (e.g., Illumina Nextera DNA Flex using PCR library amplification).

[0059] In a second example of labeling microbial DNA using click chemistry, the sample is treated with an effective amount of an azide-modified nucleoside analog, e.g., 5-azido-2'-deoxyuracil (AzdU), for a defined period of time so that AzdU is incorporated into the newly synthesized microbial DNA. After labeling with AzdU, the labeled microbial DNA is reacted with a dye-labeled alkyne in the presence of a copper(I) catalyst. A covalent bond is formed as a result of a [3+2] cycloaddition reaction between the azide and alkyne moieties. The dye labeling can then be measured using standard methods, including but not limited to flow cytometry, fluorescence microscopy, imaging, multi-well plate assays, or high-content screening.

[0060] In an example of RNA labeling using click chemistry, the sample is incubated for a defined period in the presence of an effective amount of an alkyl-modified nucleoside analog, e.g., 5-ethynyluridine (EU), so that EU is incorporated into the newly synthesized microbial RNA. After labeling with EU, the pathogenic microorganism is lysed and reacted with an azide-disulfide-biotin linker in the presence of a copper(I) catalyst. A covalent bond is formed between the azide and the incorporated nucleoside analog via a [3+2] cycloaddition reaction, and the resulting complex can then be captured using a streptavidin conjugate substrate (e.g., beads). After washing the substrate, the RNA is released from the substrate by adding a reducing agent such as dithiothreitol (DTT). The RNA is reverse transcribed into cDNA. A sequencing library can be prepared from the cDNA.

[0061] One alternative to click chemistry that utilizes strain[3+2] cycloaddition reactions without using a copper(I) catalyst is the “copper-free” click chemistry reaction described by Bertozzi et al., Compositions and methods for modification of biomolecules, U.S. Patent Application No. 20060110782.

[0062] For example, pathogenic microorganisms can be initially treated with an effective amount of an azide-modified nucleoside analog, such as AzdU, for a defined period of time, so that the azide-modified nucleoside analog is incorporated into the newly synthesized microbial DNA. After adding AzdU, cells are treated with an effective amount of a compound or molecule having a reactive cycloalkyne moiety so that a strain[3+2] cycloaddition reaction occurs between the azide and the cycloalkyne moiety. The cycloalkyne may be further modified to include dye labeling, which can be measured using standard methods such as flow cytometry, fluorescence microscopy, imaging, multi-well plate assays, or high-content screening, biotin labeling which may be used with pull-down reagents, HRP enzymes, etc. Examples of cycloalkynes that can be used in a strain[3+2] cycloaddition reaction to label DNA include, but are not limited to, cyclooctin, difluorocyclooctin, heterocycloalkyne, dichlorocyclooctin, dibromocyclooctin, or diiodocyclooctin. Other chemistry techniques known in the art can be applied to label microbial DNA. For example, the azide-phosphine chemistry described by Bertozzi et al., also known as Staudinger ligation, can be used to detect the incorporation of azide-modified nucleoside analogs, such as AzdU, into newly synthesized microbial DNA. See Bertozzi et al., Chemoselective ligation, U.S. Patent Application No. 20070037964. The pathogenic microorganism is first exposed to an effective amount of an azide-modified nucleoside analog, such as AzdU, for a defined period of time. The pathogenic microorganism is then reacted with the manipulated phosphine moiety. An example of a manipulated phosphine moiety is 2-diphenylphosphanyl methyl benzoate. When azide-phosphine chemistry is used to label microbial DNA, the manipulated phosphine moiety further includes dye molecules, biotin moieties, enzymes, etc. After the reaction between the azide and phosphine moieties has occurred, the biotin molecule can be used in a pull-down assay or the like.

[0063] To measure both baseline microbial growth and subsequent changes in microbial growth, this disclosure further provides the use of a second nucleoside or nucleotide analog labeled separately from the first nucleoside or nucleotide analog used. It is further conceivable that a third and / or fourth nucleoside or nucleotide analog may be used to measure the effectiveness of an antimicrobial agent (e.g., an antibiotic) against microbial growth or gene expression by a microbial agent. The baseline synthesis rate can be recorded by labeling the nucleic acid with the first nucleoside or nucleotide analog. It is not necessary to remove the first nucleoside or nucleotide analog before introducing the second nucleoside or nucleotide analog. Furthermore, removing the first nucleoside or nucleotide analog before introducing the second nucleoside or nucleotide analog may make accurate determination of the microbial growth rate difficult. In addition, the no-wash process makes the process suitable for high-throughput screening (HTS).

[0064] One of the key advantages of the compositions, methods, and kits disclosed herein is that, unlike standard protocols, the identification of microorganisms in a sample does not require a long culture step. Since DNA is always produced in living and growing organisms, the compositions, methods, and kits disclosed herein can identify microorganisms without the need to use a culture step to grow them. Instead, the compositions, methods, and kits disclosed herein utilize an incubation step in which the sample is incubated for a minimum amount of time in the presence of one or more types of nucleosides or nucleotide analogs so that the nucleosides or nucleotide analogs are incorporated into the newly synthesized microbial nucleic acids. Therefore, once a sample is obtained, it should be tested in the presence of one or more types of nucleosides or nucleotide analogs for approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, and 1 It may be incubated for 45 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, 190 minutes, 200 minutes, 220 minutes, 330 minutes, 240 minutes, 260 minutes, 280 minutes, 300 minutes, 350 minutes, 400 minutes, 500 minutes, 600 minutes, or any range including or between any two of the aforementioned time points, and including increments of that faction.

[0065] This disclosure further provides a method for labeling newly synthesized microbial nucleic acids containing nucleosides or nucleotide analogs with one or more labeling reagents. The labeling reagents disclosed herein specifically bind to nucleosides or nucleotide analogs. For example, the labeling reagent may be a primary antibody that can be conjugated to the label or conjugated by a secondary antibody covalently bound to the label, the primary antibody binding to the incorporated nucleoside or nucleotide analog. Examples of such primary antibodies include anti-BrdU antibodies, anti-ldU antibodies, and anti-CldU antibodies, all of which are commercially available from various vendors. However, other antibodies (as described above) that can selectively bind to the incorporated nucleoside or nucleotide analog are also conceivable. With respect to secondary antibodies, the secondary antibody can be bound to a substrate such as beads or plates. Thus, the secondary antibody can function as a pull-down reagent enabling the isolation or purification of newly synthesized microbial nucleic acids. Alternatively, the labeling reagent may be a compound containing a functional group (e.g., an azide) that is designed to react chemically with a nucleoside or nucleotide analog having a complementary bioorthogonal functional group (e.g., an alkynyl group), and may include labels such as a dye moiety, a fluorophore moiety, an affinity ligand (e.g., GST, biotin, histidine, etc.), or an enzyme (e.g., horseradish peroxidase). Examples of labeling reagents containing biotin labeling include: [ka]

[0066] As already mentioned above, the role of labeling is to enable the visualization or detection of nucleic acid polymers, such as newly synthesized microbial DNA, after labeling. Typically, the label (or detectable drug or portion) is selected so that it can be selectively bound by a pull-down reagent or so that it can be measured and its intensity can produce a signal that is, for example, related (e.g., proportional) to the amount of labeled nucleic acid polymer in the sample being analyzed. Thus, it is conceivable to use multiple labels to detect, identify, and quantify newly synthesized microbial nucleic acids, for example, a first label can be bound by a pull-down reagent to provide isolation of newly synthesized microbial nucleic acids, and second, third, or more labels can be used to produce a signal that is measured and whose intensity is related to the amount of labeled nucleic acid polymer in the sample being analyzed. The use of such multiple labels is particularly advantageous for determining the growth rate or new nucleic acid synthesis, or for testing the effect of administered drugs such as antibiotics. Furthermore, the labeling reagent may further include a chemically cleavable or enzymatically cleavable linker so that the label can be removed as needed. Any number of chemically cleavable linkers can be used, but generally, they must be linkers that can be cleaved under mild reaction conditions, such as acid-labile-based linkers, base-labile-based linkers, diazo linkers, or disulfide linkers. Examples of acid-labile linkers include hydrazones, enamines, enol ethers, imines, or linkers containing cis-aconityl groups. Examples of base-labile linkers include carbamate linkers and ester linkers. Alternatively, the cleavable linker may be an enzymatically cleavable linker. Examples of enzymatically cleavable linkers include peptide linkers or β-glucuronide linkers.

[0067] The methods for identifying and analyzing microorganisms in a sample described herein further provide for the isolation or purification of labeled microbial nucleic acids. In certain embodiments, labeled microbial nucleic acids may be purified or isolated using a pull-down reagent. In such cases, the newly synthesized microbial nucleic acid is labeled with a labeling reagent, which includes a label that binds to or to a nucleoside analog incorporated as described herein and can be selectively bound by an immobilized pull-down reagent. The labeling reagent may be an antibody or another type of affinity-based ligand (e.g., GST, biotin, histidine). The pull-down reagent may be a secondary antibody specific to the labeling reagent, or another type of drug or compound having high and selective affinity for the labeling reagent. For example, the labeling reagent may be a biotin-based molecule that binds to a nucleoside analog via click chemistry and can itself be selectively bound by a pull-down reagent containing avidin or streptavidin. Therefore, the interaction between biotin-based labeling reagents and streptavidin-based pull-down agents enables the isolation or purification of "labeled" microbial nucleic acids from "unlabeled" microbial nucleic acids and other microbial components. Typically, the pull-down reagents are immobilized on solid supports such as plates, beads, nanomaterials, or micromaterials (e.g., magnetic nanoparticles).

[0068] This disclosure also provides that the compositions, methods, and kits of this disclosure can detect the effectiveness of agents on microbial viability, growth, and proliferation. For example, an antimicrobial agent can be added directly to a sample, and the resulting effect on microbial viability, growth, and / or proliferation can be determined using the methods of this disclosure based on the detection or absence of newly synthesized nucleic acids. To obtain more controlled results, a sample can be divided into two samples, a "control" sample and a "treated" sample, thereby adding the antimicrobial agent to the "treated" sample and a vehicle control to the "control" sample, determining any difference in the production of newly synthesized microbial nucleic acids between the two samples, and demonstrating the effectiveness of the antimicrobial agent if the "treated" sample has less or no newly synthesized microbial nucleic acids compared to the "control" sample. Alternatively, the effect of an antimicrobial agent can be determined in a system in which a first sample is taken before administration of the antimicrobial agent, and second, third, or more samples are taken at one or more time points after administration of the antimicrobial agent. For example, blood samples can be obtained from septic human patients before and after antibiotic administration, thereby indicating the reduced percentage or absence of newly synthesized nucleic acids, which demonstrates the effectiveness of the antibiotic against the viability, growth, and / or proliferation of bacteria causing sepsis. Examples of antimicrobial agents that can be used with the compositions, methods, and kits of this disclosure include, but are not limited to, antibiotics, antifungals, antivirals, and antiparasitics. Antimicrobial agents are a type of antimicrobial substance that is active against bacteria and are the most important type of antimicrobial agent for combating bacterial infections. Antimicrobial agents are widely used in the treatment and prevention of such infections. They may either kill or inhibit the growth of bacteria. Examples of antibiotics that can be used with the compositions, methods, and kits disclosed herein include, but are not limited to, amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezolocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin, pibmecillinum, ticarcillin, and other penicillin derivatives, as well as cepacetril, cefadroxil, cephalexin, cephaloglysin,Cephalonium, cephaloridine, cephalothin, cefapillin, cefatoridine, cefazal, cefazedone, cefazolin, cefradiin, ceffloxazine, ceftezol, cefaclor, cefamandol, cefmetazole, cefonisid, cefotetan, cefoxitine, cefprodil, cefuroxime, cefzonam, cefcapene, cefdaroxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefozidime, cefotaxime, cefpimisole, cefpodoxime, cefteram, ceftibuten, cefthioflu, cef Cephalosporins such as thiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclizine, cefepime, ceffluprenum, cefoselis, cefozopran, cefpirome, cefquinome, ceftoviprole, ceftarolin, cefaclomedin, cephaloram, cefparol, cefcanel, cefedorol, cefempidone, cefetrizole, cefibitril, cefmatylene, cefmepidium, cefobesin, cefoxazole, cefofrotyl, cefsmid, cefracetim, ceftioxide, and monobacteria such as aztreonam. Carbapenem antibiotics such as imipenem, dalipem, ertapenem, and meropenem; macrolide antibiotics such as azithromycin, erythromycin, clarithromycin, dilithromycin, roxithromycin, and telithromycin; lincosamide antibiotics such as clindamycin and lincomycin; aminoglycoside antibiotics such as amikacin, gentamicin, kanamycin, neomycin, peromomycin, streptomycin, and tobramycin; flumequin, nalidixic acid, oxolinic acid, pyromidic acid, pipemid Quinolone antibiotics such as acid, soxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, varofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, delafloxacin, clinafloxacin, gemifloxacin, prulifloxacin, sitafloxacin, and trovafloxacin, sulfamethizol,Antifungal agents include sulfonamide antibiotics such as sulfamethoxazole, sulfisoxazole, and trimethoprim-sulfamethoxazole; tetracycline antibiotics such as demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, and tigecycline; glycopeptide antibiotics such as vancomycin and teicoplanin; lipoglycopeptide antibiotics such as teravancin; oxazolidinediones such as linezolid and cycloserine; rifamycin antibiotics such as rifampin, rifabutin, rifapentin, and rifaradyl; tuberactinomycin antibiotics such as biomycin and capreomycin; and other antibiotics such as bacitracin, polymyxin B, chloramphenicol, metronidazole, tinidazole, and nitrofurantoin. Antifungal agents are a type of antimicrobial substance that is active against fungi and are the most important type of antifungal agent for fighting fungal infections. Antifungal drugs are widely used in the treatment and prevention of such infections. These may either kill or inhibit the growth of fungi. Examples of antifungal agents that can be used with the compositions, methods, and kits disclosed herein include allylamine antifungal agents such as amorolfine, butenafine, naphthifine, and terbinafine; imidazole antifungal agents such as bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, ketoconazole, isoconazole, luliconazole, miconazole, omoconazole, oxyconazole, sertaconazole, sulconazole, thioconazole, and terconazole; and albaconazole, efinaconazole, fluconazole, and isabco. Triazole antifungal agents such as nazole, itraconazole, posaconazole, ravconazole, terconazole, and voriconazole; thiazole antifungal agents such as abafungin; polyene antifungal agents such as amphotericin B, nystatin, natamycin, and trichomycin; echinocandins such as anidurafungin, caspofungin, and micafungin; thiocarbamate antifungal agents such as tolnaftate; antimetabolites such as flucytosine; benzylamines such as butenafine; griseofulvin; cyclopirox; selenium sulfide.and other antifungal agents such as tavaborole. Antivirals are agents that prevent the entry, replication, spread, and / or maturation of viruses. Examples of antiviral agents that can be used with the compositions, methods, and kits disclosed herein include, but are not limited to, antiherpes agents such as acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, vidarabine, cytarabine, valacyclovir, tromatandine, and pretellivir; antiinfluenza agents such as amantadine, rimantadine, oseltamivir, peramivir, and zanamivir; and asunaprevir, boceprevir, silprevir, and danoprevir. NS3 / 4A protease inhibitors such as faldaprevir, glecaprevir, grazoprevir, nalulaprevir, paritaprevir, simeprevir, sovaprevir, telaprevir, vaniprevir, pedroprevir, and boxylaprevir; NS5A inhibitors such as daclatasvir, elbasvir, ledipasvir, odalasvir, ombitasvir, pibrentasvir, rabidasvir, ruzasvir, samatasvir, and velpatasvir; and NS5B inhibitors such as beclabuvir, dasabuvir, dereobvir, filibvir, cetrobuvir, sofosbuvir, radalbuvir, and uprifosvir. RNA polymerase inhibitors, anti-hepatitis B agents such as lamivudine, terbivudine, klevudine, adefovir, tenofvir disoproxil, and tenofovir alafenamide, entry / fusion inhibitors such as enfuvirtide, maraviroc, bicliviroc, senicliviroc, PRO 140, ivalizumab, and fostemsavir, reverse transcriptase inhibitors such as didanosine, emtricitabine, lamivudine, stabudine, zidovudine, amdoxovir, apricitabine, sensabudine, erbucitabine, rasivir, stampidine, 4'-ethinyl-2-fluoro-2'-deoxyadenosine, zalcitabine, efavirenz, nevirapine, delavirdin, etravirine, rilpivirine, and doravirine, dolutegravir, elvitegravir, raltegravir, BI 224436, cabotegravir, bictegravir,and other inegrase inhibitors such as MK-2048, maturation inhibitors such as bevirimat and BMS-955176, protease inhibitors such as amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, atazanavir, darunavir, and tipranavir, dolutegravir, elvitegravir, raltegravir, BI Other antiviral agents such as 224436, cabotegravir, bictegravir, and MK-2048 (integrase inhibitors), tenofovir disoproxil and tenofovir alafenamide (TAF) (nucleotide analogues / NtRTIs), cobicistat and ritonavir (pharmacokinetic enhancers), interferons such as interferon-α and pegylated interferon-α, methisazone, rifampicin, imiquimod, reximod, podophyllotoxin, homivirsen, cidofovir, preconalil, favipiravir, galidesivir, remdesivir, mericitabine, MK-608, NITD008, moloxidine, tromantadine, and triazavirin.

[0069] The compositions, methods, and kits disclosed herein also provide identification of microorganisms in a sample by identifying newly synthesized microbial nucleic acids using a microarray containing probes for nucleic acids from many different microorganisms. Typically, a microarray has probes for nucleic acids from different microorganisms in a range including 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 10000, 15000, 20000, 30000, 40000, 50000, 100000, or any two of the aforementioned values, or any range between them (including increments of those groups). The probes are typically designed to have sequences complementary to segments of one or more target organism genomes (e.g., 16S rRNA). Oligos may be spotted onto the array by mechanical deposition, sprayed with a modified inkjet printer head, or synthesized in situ by a series of photocatalytic reactions. Probes are placed on the array in a rectangular grid of "features," each containing many copies of the same oligo. The density of features on the array varies between platforms, from 20,000 spots per slide for a typical spotted array to millions for platforms such as NimbleGen and Affymetrix, which use in-situ synthesized oligos. The array may be subdivided into subarrays with gaskets, allowing multiple samples to be tested on a single slide. Randomly scattered duplicate feature regions across the array can be used to allow for correction of scratch and spatial effects. Some arrays include a negative control probe with a random sequence to provide a threshold level for background noise correction.Any number of pathogen detection microarrays have been described in the art, including ViroChip (Wang D. et al., PLoS Biol. 2003;1:E2), resequencing of pathogen microarrays (Leski T. et al. PLoS ONE. 2009;4:e6569), universal detection microarrays (Belosludtsev Y. et al., BioTechniques. 2004;37:654-8,66), GreeneChip (Quan et al., J Clin Microbiol. 2007;45:2359-64), and Lawrence Livermore microbial detection arrays (Gardner S. et al. BMC Genomics. 2010;11:668). For example, the Lawrence Livermore microbial detection array has probes for approximately 6,000 viruses and 15,000 bacteria, as well as fungi and protozoa. After applying newly synthesized microbial DNA to a microarray, a probe that detects a specific sequence will fluoresce and be read by a scanner. The raw data from the scanner is then analyzed using an algorithm. Bioinformatics is used to identify numerous nucleic acid sequences or probes that are signatures of pathogenic microorganisms.

[0070] The compositions, methods, and kits disclosed herein also provide identification of microorganisms in a sample by sequencing newly synthesized microbial nucleic acids incorporating the nucleosides or nucleotide analogs of this disclosure. Any number of sequencing methods can be used to sequence newly synthesized microbial DNA or microbial RNA reverse-transcribed to cDNA, including Sanger dideoxy chain termination sequencing (Sequencing technology based on the Sanger dideoxy chain termination sequencing method, in vitro transposition, next-generation sequencing platforms including 454 FLX® or 454 TITANIUM® (Roche), SOLEXA® Genome Analyzer (Illumina), HELISCOPE® Single Molecule Sequencer (Helicos Biosciences), and SOLID® DNA Sequencer (Life Technologies / Applied Biosystems) instruments), as well as other platforms developed by companies such as Intelligent Biosystems and Pacific Biosystems. The chemistry used to generate sequence information varies with respect to different next-generation sequencing platforms, but they all share the common characteristic of generating sequence data from a very large number of sequencing templates in which sequencing reactions are performed simultaneously. Generally, data from all of these sequencing reactions is collected using scanners and then assembled and analyzed using computers and powerful bioinformatics programs. Sequencing reactions are performed, read, assembled, and analyzed in a "massively parallel" or "multiplex" manner. The massive parallelism of these instruments has necessitated a change in thinking about the types of sequencing templates needed and how they are generated in order to obtain the maximum possible amount of sequencing data from these powerful instruments.Therefore, rather than requiring a genomic library of DNA clones in E. coli, it is important to consider in vitro systems for generating DNA fragment libraries containing a collection or population of DNA fragments generated from target DNA in a sample, where all combinations of DNA fragments in the collection or population represent sequences that qualitatively and / or quantitatively represent the sequence of the target DNA from which the DNA fragments were generated. In fact, in some cases, it is necessary to consider generating DNA fragment libraries consisting of multiple genomic DNA fragment libraries, each of which is labeled with a different address tag or barcode to enable identification of the source of each sequenced fragment.

[0071] Generally, these next-generation sequencing methods require fragmenting genomic DNA or double-stranded cDNA (prepared from RNA) into smaller ssDNA fragments and tagging at least one strand of the ssDNA fragment, or preferably both strands. In some methods, the tag provides a priming site for DNA sequencing using DNA polymerase. In some methods, the tag also provides a site for trapping the fragment on a surface such as a bead (for example, using the method described in U.S. Patent No. 7,323,305, for example, before emulsion PCR amplification for some of these methods). In most cases, the DNA fragment library used as a template for next-generation sequencing contains 5'- and 3'-tagged DNA fragments or "di-tagged DNA fragments". Generally, current methods for generating DNA fragment libraries for next-generation sequencing involve fragmenting the target DNA to be sequenced (e.g., genomic DNA after reverse transcription of RNA or target DNA including double-stranded cDNA) using an ultrasound device, nebulizer, or nuclease, and then attaching oligonucleotides consisting of adapters or tags to the 5' and 3' ends of the fragments (e.g., by ligation). Some next-generation sequencing methods use circular ssDNA substrates in the sequencing process. For example, U.S. Patent Applications Nos. 20090011943, 20090005252, 20080318796, 20080234136, 20080213771, 20070099208, and 20070072208 by Drmanac et al. disclose the generation of circular ssDNA templates for ultra-parallel DNA sequencing.Gunderson and Steemers' U.S. Patent Application No. 20080242560 discloses a method including: creating a digital DNA ball (see, for example, Figure 8 of U.S. Patent Application No. 20080242560), and / or locus-specific cleavage and amplification of DNA, such as genomic DNA, including amplification by multiple substitution amplification or whole-genome amplification (see, for example, Figure 17 therein), or by hyperbranched RCA (see, for example, Figure 18 therein) for generating amplified nucleic acid arrays (e.g., ILLUMINA BeadArrays®; ILLUMINA, San Diego Calif., USA).

[0072] In certain embodiments, this disclosure provides the use of transposome-based sequencing methods for identifying microorganisms in a sample. Such transposome-based sequencing methods are described in U.S. Patent Publications 2014 / 0162897, 2015 / 0368638, 2018 / 0245069, 2018 / 0023119, International Publications 20122103545, 20150160895, 2016130704, 2019028047, U.S. Patent No. 9,574,226, and European Patent No. 3,161,152, which are fully incorporated into this disclosure. The number of steps required to convert a target nucleic acid, such as DNA, into an adapter-modified template ready for next-generation sequencing can be minimized by using transposase-mediated fragmentation and tagging. This process, referred to herein as “tagmentation,” often involves modification of a target nucleic acid by a transposomal complex comprising a transposon enzyme that forms a complex with a transposon pair containing a single-stranded adapter sequence and a double-stranded transposon terminal sequence region, as well as any additional sequences designed for a particular purpose. Tagging results in simultaneous fragmentation of the target nucleic acid and ligation of the adapter to the 5' ends of both strands of the double-stranded nucleic acid fragment. If the transposomal complex is support-bound, the resulting fragment is bound to a solid support after the tagging reaction (directly in the case of a 5'-bound transposomal complex, or via hybridization in the case of a 3'-bound transposomal complex). Specifically, by using the transposases and transposon terminus compositions described herein, libraries of di-tagged linear ssDNA fragments or tagged circular ssDNA fragments (and their amplification products) can be generated from target microbial DNA (including double-stranded cDNA prepared from microbial RNA) for genome, subgenome, transcriptome, or metagenomic analysis or microbial RNA expression analysis (for example, for use in creating labeled targets for microarray analysis, for example, for analyzing copy number variations, for detecting and analyzing single nucleotide polymorphisms, and for searching for genes from environmental samples such as soil or water sources).

[0073] In certain embodiments, the transposome-based sequencing methods described herein use an in vitro transposition reaction to simultaneously cleave newly synthesized microbial DNA into fragments and affix a tag to the 5' end of each fragment. The in vitro transposition reaction can be carried out by assembling the reaction using either separate transposases and transposon end compositions, or a single transposome composition comprising a stable complex formed between the transposase and the transposon end composition. Thus, it will be understood that any transposome-based sequencing method describing the use of transposases and transposon end compositions may also use a transposome composition prepared from the transposases and transposon end compositions, and any transposome-based sequencing method describing the use of a transposome composition may also use separate transposases and transposon end compositions comprising the transposome composition.

[0074] A library of tagged DNA fragments can be generated from newly synthesized microbial DNA using the transposome-based sequencing method described herein, which involves incubating the newly synthesized microbial DNA in an in vitro transposition reaction with at least one transposase and a transposon end composition on which the transposase forms a transposition complex, wherein the transposon end composition comprises (i) a transposition chain showing a transposition transposon end sequence, and optionally, an additional 5'- sequence of the transposition transposon end sequence, and (ii) a non-transposition chain showing a sequence complementary to the transposition transposon end sequence, where newly The method comprises incubating the synthesized microbial DNA under conditions and for a sufficient time, such that multiple insertions may occur, each resulting in the attachment of a first tag containing or consisting of a transferred strand to the 5' end of the nucleotides of the target DNA, thereby fragmenting the target DNA and generating a population of annealed 5'-tagged DNA fragments, each fragment having a first tag at its 5' end, and then attaching the 3' end of the 5'-tagged DNA fragment to either the first or second tag, thereby generating a library of tagged DNA fragments (e.g., tagged circular ssDNA fragments or 5'- and 3'-tagged DNA fragments (or "di-tagged DNA fragments")). In further embodiments, the transposome-based sequencing method described above uses separate transposases and transposon end compositions, while in other embodiments, the transposome-based sequencing method is carried out using a transposome composition comprising a complex formed between a transposase and a transposon end composition.

[0075] This disclosure further provides microbial identification by using transposome-based sequencing methods in which transposome complexes are bound to a solid support. An example of a commercially available product using bead-linked transpososomes for sequencing is the Nextera® DNA Flex system provided by Illumina®. The Nextera® DNA Flex system can be used for microbial identification using the method described herein. A nucleic acid fragment library can be prepared using a transposome-based method in which two transposon terminal sequences (one linked to a tag sequence) and a transposase form a transposome complex. The transposome complex is used to fragment and tag target nucleic acids in solution to produce a sequencer-ready library. The transposome complex may be immobilized on a solid surface via biotin appended to the 5' end of one of the two terminal sequences. The use of immobilized transposomes offers significant advantages over liquid-phase approaches by reducing practical and overall library preparation time, cost, and reagent requirements, lowering sample input requirements, and enabling the use of unpurified or degraded samples as a starting point for library preparation. Exemplary dislocation procedures and systems for immobilizing transposomes on solid surfaces to result in uniform fragment size and library yield are described in detail in International Publications 2014 / 108810 and 2016 / 189331, each of which is incorporated herein by reference in whole. In certain bead-based tagging methods described in International Publication 2016 / 189331 and U.S. Patent Application Publication 2014 / 093916(A1), transposomes are bound to magnetic beads using biotin-streptavidin interactions.

[0076] Generally, transposomes are immobilized on substrates such as slides or beads using covalent or non-covalent partners, e.g., affinity elements and affinity-binding partners. For example, a transposome complex is immobilized on streptavidin-coated beads via a biotinylated linker bound to the transposome complex. Newly synthesized microbial nucleic acids are captured by the immobilized transposome complex, and the nucleic acids are fragmented and tagged ("tagged"). The tagged fragments are amplified, the desired amplicon is captured (e.g., via a hybridization probe), and the tagged fragments are sequenced.

[0077] The use of solid-supported transposome complexes for library preparation reduces the need for normalization of sample inputs to proceed to the library preparation process and normalization of library outputs before enrichment or sequencing steps. Using these complexes, libraries with more consistent insert sizes are produced compared to liquid-phase methods, even when various sample input concentrations are used. In some embodiments, the transposome complex is immobilized to a support via one or more polynucleotides (e.g., oligonucleotides), such as polynucleotides (oligonucleotides) containing the transposon terminal sequences. In some embodiments, the transposome complex may be immobilized via linkers attached to the ends of the transposon sequences, for example, to couple a transposase enzyme to a solid support. In some embodiments, both the transposase enzyme and the transposon polynucleotide (e.g., oligonucleotide) are immobilized to the solid support. When referring to the immobilization of molecules (e.g., nucleic acids, enzymes) to a solid support, the terms “immobilized,” “attached,” and “bonded” are used interchangeably herein, and both terms are intended to encompass direct or indirect, covalent or non-covalent bonding unless explicitly or contextually indicated. In certain embodiments of this disclosure, covalent bonding may be preferred, but generally, the requirement is that the molecule (e.g., nucleic acid, enzyme) remains immobilized or attached to the support under conditions where the support is intended to be used, for example, in applications requiring nucleic acid amplification and / or sequencing. In some examples, in bead-based tagging, the transposome may be bound to the bead surface via a ligand pair, e.g., an affinity element and an affinity-binding partner.

[0078] Transposon-based technologies can be used to fragment DNA, as exemplified in the workflows of NEXTERA® XT and Flex DNA Sample Preparation Kits (Illumina, Inc.), where newly synthesized microbial nucleic acids are processed with transposomal complexes that simultaneously fragment and tag ("tagmentation") targets, thereby creating a collection of fragmented nucleic acid molecules tagged with adapter sequences specific to the ends of the fragments.

[0079] A transposition reaction is a reaction in which one or more transposons are inserted into a target nucleic acid at a random or nearly random site. Components of a transposition reaction include a transposase (or other enzymes capable of fragmenting and tagging nucleic acids as described herein, e.g., integrase), a transposon element comprising a double-stranded transposon terminal sequence bound to the enzyme, and an adapter sequence bound to one of the two transposon terminal sequences. One strand of the double-stranded transposon terminal sequence is transposed to the single strand of the target nucleic acid, while the complementary transposon terminal sequence strand is not transposed (i.e., the untransposed transposon sequence). The adapter sequence may optionally or desiredly include one or more functional sequences (e.g., primer sequences).

[0080] Accordingly, in further embodiments, the identification and analysis of microorganisms in a sample comprises the steps of: providing a solid support on which the transposome complex described herein is immobilized; and contacting the isolated or purified microbial nucleic acid with the solid support under conditions sufficient to provide a plurality of 5'-tagged target fragments by fragmenting a target nucleic acid into a plurality of target fragments and ligating the 3' end of a first transposon to the 5' end of the target fragments. In further embodiments, the method further comprises amplifying the 5'-tagged target fragments. In even further embodiments, the method further comprises sequencing one or more of the 5'-tagged target fragments or their amplified products. In some aspects, the disclosure provides a library of newly synthesized 5'-tagged fragments of microbial nucleic acids produced by the method described herein.

[0081] In another embodiment, the present invention provides a kit comprising at least one nucleoside analog and a labeling reagent. The kit generally also includes instructions for using the nucleoside analog and the labeling reagent in one or more ways, typically to detect or measure changes in microbial nucleic acid synthesis.

[0082] In exemplary embodiments, the kit comprises a nucleoside or nucleotide analog containing a bioorthogonal functional moiety and a first labeling reagent capable of undergoing click chemistry with the bioorthogonal functional moiety. Additional kit components include pull-down reagents, buffers, other detection reagents, and reference materials.

[0083] The following embodiments are intended to illustrate, but not limit, the present disclosure. They are typical of what may be used, but other procedures known to those skilled in the art may also be used. [Examples]

[0084] An exemplary method for detecting and identifying bacteria in samples from septic patients. The methodology and techniques of this disclosure enable the detection of bacteria in septic patients. As shown in Figure 1, blood samples are cultured in a medium containing the nucleoside-labeling reagent 5-ethynyl-2'-deoxyuridine (EdU). After a short period of culture (from a few minutes to several hours), viable cells in DNA synthesis incorporate EdU into their genomes. If antibiotic resistance is to be examined, the antibiotic of the interest may be added to the culture medium. After rapid culture, the cells are lysed and the DNA can be optionally purified from the lysate. The newly synthesized EdU-containing DNA is then labeled with biotin via a click reaction with an azido-disulfide-biotin linker. After biotin labeling, the newly synthesized DNA is captured by streptavidin conjugate beads. After washing the beads, the DNA is released from the beads by adding dithiothreitol (DTT). To identify DNA-producing bacteria, sequencing libraries are prepared and sequenced using standard methods (e.g., Illumina Nextera DNA Flex using PCR library amplification). Bioinformatics analysis of the sequences reveals the identity of bacteria causing sepsis.

[0085] An exemplary method for rapidly analyzing microbial gene expression in a sample containing viable microorganisms. Using the methodology and techniques of this disclosure, microbial gene expression in a sample containing viable microorganisms can be rapidly analyzed (Figure 2). Instead of culturing with EdU, the sample is cultured with 5-ethynyluridine (EU) and then incorporated into freshly synthesized microbial RNA. After biotin labeling and streptavidin-based purification of the RNA, the RNA is reverse transcribed into cDNA. A sequencing library is prepared from the cDNA. Next, gene expression of viable pathogenic microorganisms in the sample is revealed by sequencing and bioinformatics analysis. This gene expression analysis can be used to identify genes causing disease phenotypes or to determine whether the microorganisms are responding to antibiotics. In addition to information on gene expression, strain identification can also be performed using RNA sequencing analysis. Since RNA is synthesized in viable, non-proliferating pathogenic microorganisms, RNA analysis may be able to identify contaminating or infectious pathogenic microorganisms that do not replicate in culture but are still viable.

[0086] It will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for identifying and analyzing viable and / or growing microorganisms in a sample, (a) Incubating a sample containing or suspected to contain one or more types of microorganisms in the presence of one or more types of nucleosides or nucleotide analogs, wherein the one or more types of nucleosides or nucleotide analogs are incorporated into newly synthesized microbial nucleic acids, selected from 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-uridine (EU), (b) Labeling the newly synthesized microbial nucleic acid by contacting it with an azide-linker-biotin labeling reagent that selectively binds to one or more types of nucleosides or nucleotide analogs, (c) Isolating or purifying the labeled newly synthesized microbial nucleic acid using streptavidin or an avidin solid support pull-down agent, (d) Cutting the linker to obtain microbial nucleic acids isolated or purified from the pull-down agent, (e) A method comprising determining the identity of the viable and / or growing microorganisms in the sample based on sequencing or identity determination of the isolated or purified newly synthesized microbial nucleic acid.

2. The method according to claim 1, wherein the sample is obtained from a subject suspected of having a microbial infection or who is actually suffering from a microbial infection.

3. The method according to claim 2, wherein the subject is suspected of having sepsis or is actually sepsis.

4. The method according to any one of claims 1 to 3, wherein the obtained sample is treated using a dehosting method before (b) to selectively remove non-microbial nucleic acids.

5. The aforementioned dehosting method, Nonmicrobial nucleic acids, (a) Selectively cleaving nonmicrobial DNA by contacting the obtained sample with a recombinant protein comprising a binding domain that selectively binds to nonmicrobial nucleic acids bound by histones (or multiple histones) or to nonmicrobial nucleic acids containing methylated CpG residues, and a nuclease domain that has the activity to cleave nucleic acids, or (b) The method according to claim 4, comprising removing by the use of an affinity agent bound to a solid substrate that selectively binds to nucleic acids bound by histones or selectively binds to methylated CpG residues of nonmicrobial nucleic acids.

6. The method according to claim 1, wherein the sample is an environmental sample obtained from an environmental testing site.

7. The method according to claim 6, wherein the environmental testing site is tested for microbial contamination.

8. The method according to claim 1, wherein the sample is a sample obtained from food suspected of being contaminated with microorganisms.

9. The method according to any one of claims 1 to 8, wherein the one or more types of microorganisms are bacteria, fungi, viruses, algae, archaea, and / or protozoa.

10. The aforementioned bacteria include Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Campylobacter jejuni. Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis Haemophilus influenzae, Helicobacter pyloripylori), Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis meningitidis), Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus piogenes Pyogenes), Treponema pallidum, Ureaplasma urealyticum, Vibrio choleraeThe method according to claim 9, selected from Yersinia cholerae, Yersinia pestis, Yersinia enterocolitica, and / or Yersinia pseudotuberculosis.

11. The aforementioned fungi include Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, and Arthroderma spp.), Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii gattii), Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp., Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp.), Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp., Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia kolimbifera corymbifera), Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis *Phialophora lactis*, *Paracoccidioides brasiliensis*, *Petriellidium boydii*, and other species of the genus *Phialophora*.), Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum species The method according to claim 9, comprising a species selected from the genera Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp., Trichophyton spp., Trichosporon spp., and / or Zopfia rosatii.

12. The aforementioned viruses include Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, Nupapillomavirus, Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, and Deltapolyomavirus. Deltapolyomavirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus, Dependovirus, Bocavirus, Orthohepadnavirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, SimiispumavirusColtivirus, Rotavirus, Seadonavirus, α-Coronavirus, β-Coronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebola Virus genera (Ebolavirus), Marburgvirus, Henipavirus, Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Orthopneumovirus, Ledantevirus, Lyssavirus, Vesiculovirus, Mammarenavirus, Orthohantavirus, Orthonairovirus, Orthobunyavirus, Phlebovirus, α-Influenzavirus, β-Influenzavirus, γ-InfluenzavirusThe method according to claim 9, selected from the genera Quaranjavirus, Thogotovirus, and / or Deltavirus.

13. The method according to any one of claims 1 to 12, wherein the sample is incubated for 5 to 180 minutes in the presence of one or more types of nucleosides or nucleotide analogs.

14. The method according to claim 13, wherein the sample is incubated for 30 to 120 minutes in the presence of one or more types of nucleosides or nucleotide analogs.

15. The method according to any one of claims 1 to 14, wherein the labeling reagent binds to or with one or more types of nucleosides or nucleotide analogs via click chemistry, strain [3+2] cycloaddition, or Staudinger ligation.

16. The labeling reagent is 【Chemistry 1】 The method according to claim 1, selected from the following.

17. The method according to any one of claims 1 to 16, wherein the linker of the labeled reagent includes a chemically cleavable linker or an enzymatically cleavable linker.

18. The method according to claim 17, wherein the severable linker is an acid-unstable linker or a disulfide linker.

19. The method according to claim 18, wherein the acid-unstable linker comprises a hydrazone or a cis-aconityl group.

20. The method according to claim 17, wherein the enzymatically cleavable linker includes a peptide-based linker or a β-glucuronide-based linker.

21. The method according to claim 1, wherein the solid support pull-down agent is a nanomaterial or micromaterial, beads, or a plate.

22. The method according to claim 1, wherein the isolated or purified newly synthesized microbial nucleic acid is sequenced using a transposome-based sequencing method.

23. The sequencing of the newly synthesized microbial nucleic acid is (a) Applying the isolated or purified newly synthesized microbial nucleic acid to a bead-bound transposome, wherein the bead-bound transposome mediates the simultaneous fragmentation of the microbial nucleic acid and the addition of sequencing primers; (b) Amplifying the microbial nucleic acid fragment with a primer containing an index and an adapter sequence to form a library of amplified products, (c) Washing and pooling the library of amplification products, (d) Sequence the library of amplification products, (e) The method of claim 22, wherein the identity of the viable and / or growing microorganisms is determined by using bioinformatics analysis to correlate sequences obtained from the library of amplified products with a database of known microbial sequences.

24. The method according to claim 1, wherein the newly synthesized microbial nucleic acid is RNA, and the microbial RNA is reverse transcribed into cDNA prior to (e) of claim 1, and the gene expression of the viable and / or growing microorganisms can be determined based on analyzing the expression level of the gene product from the newly synthesized microbial RNA using a microarray and / or sequencing.

25. A method for determining the effectiveness of an antimicrobial agent that regulates the growth and proliferation of microorganisms (multiple may be present) in a sample, (a) Dividing a sample containing or suspected to contain one or more types of microorganisms into two samples: a control sample and a treated sample, (b) Incubating the control sample in the presence of one or more types of nucleosides or nucleotide analogs, wherein the one or more types of nucleosides or nucleotide analogs are incorporated into newly synthesized microbial nucleic acids and selected from 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-uridine (EU), (b') Incubating the treated sample in the presence of one or more types of nucleosides or nucleotide analogs and an antimicrobial agent, wherein the one or more types of nucleosides or nucleotide analogs are incorporated into the newly synthesized microbial nucleic acid. (c) Labeling the newly synthesized microbial nucleic acids of the control sample and the treated sample by contacting the newly synthesized microbial nucleic acids with an azide-linker-biotin labeling reagent that selectively binds to one or more types of nucleosides or nucleotide analogs, (d) Isolating or purifying the labeled newly synthesized microbial nucleic acid from the control sample and the treated sample using streptavidin or an avidin solid support pull-down agent, (e) Determining the expression level and / or quantity or identity of the isolated or purified newly synthesized microbial nucleic acid in the control sample, (e') Determining the expression level and / or quantity and identity of the isolated or purified newly synthesized microbial nucleic acid in the treated sample, (f) Comparing the expression level and / or amount and / or identity of the isolated or purified newly synthesized microbial nucleic acid in the control sample with the expression level and / or amount or identity of the isolated or purified newly synthesized microbial nucleic acid in the treated sample and determining any changes, A method for demonstrating that the antimicrobial agent is effective in regulating the growth and proliferation of the microorganism(s) if, in the treated sample versus the control sample, the expression level of the newly synthesized microbial nucleic acid is reduced, or if, in the treated sample versus the control sample, the amount and / or identity of the newly synthesized microbial nucleic acid is reduced.

26. The method according to claim 25, wherein the antibacterial agent is selected from antibiotics, antifungal agents, and antiviral agents.

27. The aforementioned antibiotics include amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezolocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin, pibmecillinum, ticarcillin, cepacetril, cefadroxil, cephalexin, cephaloglysin, cephalonium, cephaloridine, cephalothin, cefapillin, cefatolidine, cefazal, cefazal, cefazol Lol, cephamandol, cefmetazole, cefonisid, cefotetan, cefoxitin, cefprodil, cefuroxime, cefzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefozidime, cefotaxime, cefpimisole, cefpodoxime, cefteram, ceftibuten, ceftiofur, cefthiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cef Epim, ceffluprenum, cefoselis, cefozopran, cefpirome, cefquinome, ceftoviprole, cephthaloline, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefibitril, cefmatylene, cefmepidium, cef Besin, cefoxazole, cefrotil, cefsumide, cefracetime, ceftioxide, aztreonam, imipenem, doripenem, ertapenem, meropenem, azithromycin, erythromycin, clarithromycin, dilithromycin, roxithromycin, telithromycin, clindamycin, lincomycin, amikacin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin,Streptomycin, Tobramycin, Flumequin, Nalidixic acid, Oxolinic acid, Pyromidic acid, Pipemidic acid, Rosoxacin, Ciprofloxacin, Enoxacin, Lomefloxacin, Nadifloxacin, Norfloxacin, Ofloxacin, Pefloxacin, Rufloxacin, Barofloxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Moxifloxacin, Pazufloxacin, Sparfloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Delafloxacin, Clinafloxacin, Gemifloxacin, Prulifloxacin, Sitafloxacin, Trobaf The method according to claim 26, selected from roxacin, sulfamethizol, sulfamethoxazole, sulfisoxazole, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, tigecycline, vancomycin, teicoplanin, teravancin, linezolid, cycloserine, rifampin, rifabutin, rifapentin, rifalazil, biomycin, capreomycin, bacitracin, polymyxin B, chloramphenicol, metronidazole, tinidazole, and nitrofurantoin.

28. The aforementioned antifungal agents include amorolfine, butenafine, naphthifine, terbinafine, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, ketoconazole, isoconazole, luliconazole, miconazole, omoconazole, oxyconazole, sertaconazole, sulconazole, thioconazole, terconazole, albaconazole, efinaconazole, fluconazole, isabconazole, and itora. The method according to claim 26, selected from conazole, posaconazole, ravconazole, terconazole, voriconazole, abafungin, amphotericin B, nystatin, natamycin, trichomycin, anidurafungin, caspofungin, micafungin, tolnaftate, flucytosine, butenafine, griseofulvin, cyclopirox, selenium sulfide, and tavaborole.

29. The aforementioned antiviral agents include acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, vidarabine, cytarabine, valacyclovir, tromatandine, pritelivir, amantadine, rimantadine, oseltamivir, peramivir, zanamivir, asunaprevir, boceprevir, silprevir, danoprevir, faldaprevir, glecaprevir, grazoprevir, narlaprevir, paritaprevir, simeprevir, sovaprevir, telaprevir, vaniprevir, and vedroprevir. ), voxilaprevir, daclatasvir, elbasvir, ledipasvir, odalasvir, ombitasvir, pibrentasvir, ravidasvir, ruzasvir, samatasvir, velpatasvir, beclabuvir, dasabuvir, deleobuvir, filibuvir, setrobuvir, sofosbuvir, radalbuvir, uprifosbuvir, lamivudine, terbivudine, klevudine, adefovir, tenofvir disoproxil, tenofvir alafenamide, enfuvirtide, maraviroc, vicriviroc, cenicriviroc, PRO140, Ibalizumab, Fostemsavir, Didanosine, Emtricitabine, Lamivudine, Stabudine, Zidovudine, Amdoxovir, Apricitabine, Censavudine, Elvucitabine, Racivir, Stampidine, 4'-Ethinyl-2-Fluoro-2'-Deoxyadenosine, Zalcitabine, Efavirenz, Nevirapine, Delavirdine, Etravirine, Rilpivirine, Doravirine, Dolutegravir, Elvitegravir, Raltegravir, BI 224436, cabotegravir, bictegravir, MK-2048, bevirimat, BMS-955176, amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, atazanavir, darunavir, tipranavir, dolutegravir, elvitegravir, raltegravir, BI The method according to claim 26, selected from 224436, cabotegravir, bictegravir, MK-2048, cobicistat, ritonavir, interferon-α, pegylated interferon-α, methisazone, rifampicin, imiquimod, reciquimod, podophyllotoxin, homivirsen, cidofovir, preconalil, favipiravir, galidesivir, remdesivir, mericitabine, MK-608, NITD008, moloxidine, tromantadine, and triazavirin.

30. The method according to any one of claims 25 to 29, wherein the sample is obtained from a subject suspected of having a microbial infection or who is actually suffering from a microbial infection.

31. The method according to claim 30, wherein the subject is suspected of having sepsis or has sepsis.

32. The method according to any one of claims 25 to 31, wherein the one or more types of microorganisms are bacteria, fungi, and / or viruses.

33. The aforementioned bacteria include Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Campylobacter jejuni. Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis Haemophilus influenzae, Helicobacter pyloripylori), Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis meningitidis), Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus piogenes Pyogenes), Treponema pallidum, Ureaplasma urealyticum, Vibrio choleraeThe method according to claim 32, selected from Yersinia cholerae, Yersinia pestis, Yersinia enterocolitica, and / or Yersinia pseudotuberculosis.

34. The aforementioned fungi include Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, and Arthroderma spp.), Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii gattii), Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp., Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp.), Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp., Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia kolimbifera corymbifera), Lobmyces loboi, Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp., Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis *Phialophora lactis*, *Paracoccidioides brasiliensis*, *Petriellidium boydii*, and other species of the genus *Phialophora*.), Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum species The method according to claim 32, selected from the genera Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp., Trichophyton spp., Trichosporon spp., and / or Zopfia rosatii.

35. The aforementioned viruses include Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lympho-cryptovirus, Rhadinovirus, Mastadenovirus, α-Papillomavirus, β-Papillomavirus, X-Papillomavirus, γ-Papillomavirus, Mupapillomavirus, Nupapillomavirus, Alphapolyomavirus, Betapolyomavirus, γ-Polyomavirus, and Deltapolyomavirus. Deltapolyomavirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, α-Torquevirus, β-Torquevirus, γ-Torquevirus, Cyclovirus, Gemycircular, Gemykibivirus, Gemyvongvirus, Erythrovirus, Dependovirus, Bocavirus, Orthohepadnavirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, SimiispumavirusColtivirus, Rotavirus, Seadonavirus, α-Coronavirus, β-Coronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus, Pegivirus, Orthohepevirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Ebola Virus genera (Ebolavirus), Marburgvirus, Henipavirus, Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Orthopneumovirus, Ledantevirus, Lyssavirus, Vesiculovirus, Mammarenavirus, Orthohantavirus, Orthonairovirus, Orthobunyavirus, Phlebovirus, α-Influenzavirus, β-Influenzavirus, γ-InfluenzavirusThe method according to claim 32, selected from the genus Quaranjavirus, the genus Thogotovirus, and / or the genus Deltavirus.

36. The method according to any one of claims 22 to 35, wherein both the control sample and the treated sample are incubated for the same amount of time from 5 minutes to 180 minutes in the presence of one or more types of nucleosides or nucleotide analogs.

37. The method according to claim 36, wherein both the control sample and the treated sample are incubated for the same amount of time (30 to 120 minutes) in the presence of one or more types of nucleosides or nucleotide analogs.

38. The method according to any one of claims 22 to 37, wherein the labeling reagent binds to or with one or more types of nucleosides or nucleotide analogs via click chemistry, strain [3+2] cycloaddition, or Staudinger ligation.

39. The labeling reagent is 【Chemistry 2】 The method according to claim 25, selected from the following.

40. The method according to any one of claims 22 to 39, wherein the labeling reagent further comprises a chemically cleavable linker or an enzymatically cleavable linker.

41. The method according to claim 40, wherein the severable linker is an acid-unstable linker or a disulfide linker.

42. The method according to claim 41, wherein the acid-unstable linker comprises a hydrazone or a cis-aconityl group.

43. The method according to claim 40, wherein the enzymatically cleavable linker includes a peptide-based linker or a β-glucuronide-based linker.

44. The method according to claim 25, wherein the solid support pull-down agent is a nanomaterial or micromaterial, beads, or a plate.

45. The method according to claim 28, wherein the labeling reagent or label is removed or cleaved from the isolated or purified newly synthesized microbial nucleic acid prior to (e), (e'), and (f) of claim 25.

46. The method according to any one of claims 25 to 45, wherein the expression level and / or amount and / or identity of the isolated or purified newly synthesized microbial nucleic acid in the control sample and the treated sample is determined by using a microarray comprising probes for nucleic acids from different microorganisms.

47. Determining the expression level and / or quantity and / or identity of the isolated or purified newly synthesized microbial nucleic acid in the control sample and the treated sample is: (i) Amplifying the isolated or purified newly synthesized microbial nucleic acid from the control sample using a primer containing a fluorescent dye and a first PCR-based method to form a labeled product, wherein the primer contains a sequence specific to the conserved microbial 16S rRNA gene region. (i') Amplifying the isolated or purified newly synthesized microbial nucleic acid from the treated sample using a primer containing the fluorescent dye and the first PCR-based method to form a labeled product, wherein the primer contains a sequence specific to the conserved microbial 16S rRNA gene region. (ii) Applying the labeled product from the control sample to a first microarray containing probes that include unique 16s rRNA variable region sequences from 20 or more microorganisms, (ii') Applying the labeled product from the processed sample to a second microarray, wherein the second microarray is a replica of the first microarray. (iii) Determining the effectiveness of an antimicrobial agent that modulates the growth and proliferation of microorganisms in a sample, based on imaging the first and second microarrays of the fluorescence hybridization products and determining whether there are any changes in the intensity, position, or absence of the fluorescence hybridization products between the microarrays, The method according to claim 46, wherein if there is a decrease in the intensity of the fluorescent hybridization product between the first microarray and the second microarray, or if there is a change in the position or absence of the fluorescent hybridization product between the first microarray and the second microarray, it is determined that the antimicrobial agent is effective in regulating the growth and proliferation of the microorganism(s).

48. The effectiveness of an antimicrobial agent that regulates the growth and proliferation of microorganisms (multiple) in a sample is determined or confirmed by sequencing the isolated or purified newly synthesized microbial nucleic acids from the control sample and the treated sample. The method according to any one of claims 25 to 47, which shows that the antimicrobial agent is effective in regulating the growth and proliferation of the microorganism(s) when there is a decrease in the expression level of the newly synthesized microbial nucleic acid in the treated sample versus the control sample, or a decrease in the amount and / or identity of the newly synthesized microbial nucleic acid in the treated sample versus the control sample.

49. The method according to claim 48, wherein the isolated or purified newly synthesized microbial nucleic acids from the control sample and the treated sample are sequenced using a transposome-based sequencing method.

50. The sequencing of the newly synthesized microbial nucleic acids from the control sample and the treated sample is as follows: (a) Applying the isolated or purified newly synthesized microbial nucleic acids from the control sample and the treated sample to a bead-bound transposome, wherein the bead-bound transposome mediates the simultaneous fragmentation of the microbial nucleic acids and the addition of sequencing primers. (b) Amplifying the microbial nucleic acid fragment with a primer containing an index and an adapter sequence to form a library of amplified products, (c) Washing and pooling the library of amplification products from the control sample, (c') Washing and pooling the library of amplification products from the processed sample, (d) Sequence the library of amplified products from the control sample, (d') Sequence the library of amplified products from the processed sample, (e) The method according to claim 49, wherein the method involves determining any changes in the expression level and / or amount and / or identity of the isolated or purified newly synthesized microbial nucleic acid from the control sample and the treated sample based on the use of bioinformatics analysis.

51. The method according to any one of claims 25 to 50, wherein the newly synthesized microbial nucleic acid is RNA, and the microbial RNA is reverse transcribed into cDNA prior to (e), (e'), and (f) of claim 25, and the effectiveness of the antimicrobial agent that regulates the growth and proliferation of the microorganism(s) can be determined by determining the change in the expression level of the newly synthesized microbial nucleic acid from the control sample and the treated sample by using a microarray and / or sequencing.

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