Method of Diagonosizing Pathogens and their Antimicrobial Susceptibility

Abstract

Disclosed are methods of identifying pathogens and determining their antimicrobial susceptibility. The methods comprise detecting biomarkers in a test sample, locating the sample in a phylogenetic tree based on biomarker information, obtaining drug susceptibility prediction rules based on the phylogenetic tree positioning of the sample, and determining the drug susceptibility of a pathogen according to the prediction rules. Further disclosed are an application of the phylogenetic tree in the preparation of pathogen identification and/or drug susceptibility diagnostic product, and a pathogen identification and drug susceptibility diagnostic kit.

Claims

1. A method of determining the drug susceptibility of a pathogen in a test sample, the method comprising: detecting biomarkers in a test sample; locating the sample in a phylogenetic tree based on biomarker information; obtaining drug susceptibility prediction rules based on the phylogenetic tree positioning of the sample; and determining the drug susceptibility of a pathogen according to the prediction rules.

2. The method of claim 1, wherein the biomarker information is metabolic fingerprints and/or nucleic acid sequences of a pathogen.

3. The method of claim 2, wherein the metabolic fingerprints are feature information of metabolites detected by mass spectrometry, preferably, the feature information is one or more of mass-to-charge ratio, retention time, and species abundance of the metabolites.

4. The method of claim 3, wherein the metabolites are water-soluble molecules with a mass-to-charge ratio between 50-1500 Da and a minimum abundance value of 2000.

5. The method of claim 2, wherein the nucleic acid sequences are antimicrobial resistance determinants in the genome of a pathogen, preferably, the antimicrobial resistance determinants are selected from the group consisting of antibiotic resistance genes, plasmids, chromosomal housekeeping genes, insertion sequences, transposons and integrons.

6. The method of claim 5, wherein the antibiotic resistance genes are selected from the group consisting of abarmA, abAPH(3′)-Ia, abOXA239, abNDM-10, abgyrA, abSUL-1, abSUL-2, abSUL-3, kpCTX-M-65, kpTEM-1b, kpIMP-4, kpKPC-2, kprmtB, IkpAAC(3′)-Iid, kpQNR-S, kpgyrA, kpparC, kptetA, kptetD, kpSUL-1, kpSUL-2, kpSUL-3, ecrmtB, ecAAC(3′)-Iid, ecgyrA, ectetA, ectetB, ecSUL-1, ecSUL-2, ecSUL-3, ecIMP-4, ecNDM-5, ecTEM-1b, ecCTX-M-14, ecCTX-M-55, ecCTX-M-65, ecCMY, paTEM-1b, paGES-1, paPER-1, paKPC-2, paOXA-246, parmtB, paAAC(3′)-Iid, paAAC(6′)-IIa, paVIM-2, pagyrA, efermB, eftetM, eftetL, efparC, efANT(6′)-Ia, stmecA, stmsrA, stermA, stermB, stermC, strpoB, stgyrA, stAAC(6′)-APH(2′), stdfrG, sttetK, sttetL, stcfrA, spbpb1a, sppbp2x, spbpb2b, spdfr sptetM, spermB, spgyrA, aat1a, acc1, adp1, mpib, sya1, vps13, zwf1b, fcy2, fur1, fca1, erg11, erg3, tac1, cdr1, cdr2, mdr1, pdr1, upc2a, fks1hs1, fks1hs2, fks2hs1, fks2hs2.

7. The method of claim 1, wherein the phylogenetic tree is obtained by liquid chromatography-tandem mass spectrometry technology and/or whole genome sequencing technology.

8. The method of claim 1, wherein the phylogenetic tree is selected from the group consisting of a metabolic spectrum phylogenetic tree constructed based on the species and amounts of metabolites, a whole genome phylogenetic tree constructed based on SNPs and InDels, and a core genome phylogenetic tree constructed based on antimicrobial resistance determinants and their upstream regulatory sequences.

9. The method of claim 1, wherein the drug susceptibility prediction rules comprise: different metabolite-based prediction rules are applied for different branches of the metabolic spectrum phylogenetic tree, and/or different sequence-based prediction rules are applied for different branches of the genomic phylogenetic tree.

10. The method of claim 9, wherein the metabolite-based prediction rules are selected from: 1) use independent prediction rules in the drug susceptibility determination of non-fermentative Gram-negative bacteria: when the test sample contains antimicrobial resistance determinants, follow the principle that the drug resistance profile predicted by antimicrobial resistance determinants is preferred over phylogenetic tree interpretation; when the test sample is located in the susceptibility branch of the metabolic spectrum phylogenetic tree, follow the principle that the drug resistance profile inferred by the phylogenetic tree is preferred over antimicrobial resistance determinants analysis and the sample is directly determined to be susceptible to all β-lactam antibiotics; 2) use independent prediction rules in the drug susceptibility determination of Enterobacteriaceae: when the test sample contains antimicrobial resistance determinants, follow the principle that the drug resistance profile predicted by antimicrobial resistance determinants is preferred over phylogenetic tree interpretation; when the test sample is located in the susceptibility branch of the metabolic spectrum phylogenetic tree, follow the principle that the drug resistance profile inferred by the phylogenetic tree is preferred over antimicrobial resistance determinants analysis and the sample is directly determined to be susceptible to β-lactams, β-lactamase inhibitors and cephamycins; 3) use independent prediction rules in the drug susceptibility determination of Gram-positive cocci: when the test sample contains antimicrobial resistance determinants, follow the principle that the drug resistance profile predicted by antimicrobial resistance determinants is preferred over phylogenetic tree interpretation; when the test sample is located in the susceptibility branch of the metabolic spectrum phylogenetic tree, follow the principle that the drug resistance profile inferred by the phylogenetic tree is preferred over antimicrobial resistance determinants analysis and the sample is directly determined to be susceptible to penicillins, macrolides, lincosamides, quinolones, aminoglycosides, glycopeptides and oxazolidinones; when the test sample is identified as Enterococcus faecalis and has the metabolic fingerprints of a sequence type 4 Enterococcus faecalis clone, the sample is directly determined to be resistant to penicillins; 4) use independent prediction rules in the drug susceptibility determination of Streptococcus pneumoniae: when the test sample contains antimicrobial resistance determinants, follow the principle that the drug resistance profile predicted by antimicrobial resistance determinants is preferred over phylogenetic tree interpretation; when the test sample is identified as Streptococcus pneumoniae and has the metabolic fingerprints of a Streptococcus pneumoniae clone with altered penicillin-binding protein patterns, the sample is directly determined to be resistant to penicillins; and/or 5) use independent prediction rules in the drug susceptibility determination of Fungi: strictly follow the principle that the drug resistance profile of a fungal strain is inferred on the basis of its closest relatives in the metabolic spectrum phylogenetic tree.

11. The method of claim 9, wherein the sequence-based prediction rules are selected from: 1) resistance of Enterobacteriaceae to carbapenems and quinolones is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of Enterobacteriaceae to aminoglycosides, tetracyclines, sulfonamides, β-lactams except carbapenems is determined by antimicrobial resistance determinants analysis; 2) resistance of non-fermentative Gram-negative bacteria to cephalosporins and carbapenems is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of non-fermentative Gram-negative bacteria to aminoglycosides, tetracyclines, sulfonamides, quinolones and β-lactamase inhibitors is determined by antimicrobial resistance determinants analysis; 3) resistance of Gram-positive cocci to penicillin, ampicillin, oxacillin and cefoxitin is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of Gram-positive cocci to macrolides, lincosamides, aminoglycosides, quinolones, glycopeptides and oxazolidinones is determined by antimicrobial resistance determinants analysis; 4) resistance of Streptococcus pneumoniae to penicillins and cephalosporins is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of Streptococcus pneumoniae to macrolides, lincosamides, aminoglycosides, quinolones, glycopeptides and oxazolidinones is determined by antimicrobial resistance determinants analysis; and/or 5) resistance of Fungi to triazoles and amphotericin B formulations is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of Fungi to echinocandins is determined by antimicrobial resistance determinants analysis.

12. Application of the phylogenetic tree of a pathogen in the preparation of an antimicrobial susceptibility diagnostic product, wherein the phylogenetic tree is obtained by liquid chromatography-tandem mass spectrometry technology and/or whole genome sequencing technology.

13. The application as claimed in claim 12, wherein the phylogenetic tree is selected from the group consisting of a metabolic spectrum phylogenetic tree constructed based on the species and amounts of metabolites, a whole genome phylogenetic tree constructed based on SNPs and InDels, and a core genome phylogenetic tree constructed based on antimicrobial resistance determinants and their upstream regulatory sequences.

14. The application as claimed in claim 12, wherein the antimicrobial susceptibility diagnostic product further comprises reagent and equipment for detecting the biomarker information in a test sample.

15. The application as claimed in claim 14, wherein the equipment for detecting the biomarker information is selected from the group consisting of liquid chromatography-tandem mass spectrometry and whole genome sequencing devices.

16. The application as claimed in claim 14, wherein the reagent for detecting the biomarker information is selected from the group consisting of a pathogen identification and drug susceptibility diagnostic kit based on liquid chromatography-tandem mass spectrometry, and a pathogen identification and drug susceptibility diagnostic kit based on whole genome sequencing technology.

17. A pathogen identification and drug susceptibility diagnostic kit, comprising: KIT1: a pathogen identification and drug susceptibility diagnostic kit based on liquid chromatography-tandem mass spectrometry; or, KIT2: a pathogen identification and drug susceptibility diagnostic kit based on whole genome sequencing technology; and, the phylogenetic tree of a pathogen in the test sample.

18. The kit as claimed in claim 17, wherein the pathogen identification and drug susceptibility diagnostic kit based on liquid chromatography-tandem mass spectrometry comprise bacterial standards, fungal standards, extraction buffer and resuspension buffer.

19. The kit as claimed in claim 17, wherein pathogen identification and drug susceptibility diagnostic kit based on whole genome sequencing technology comprise cell lysis reagents, primer mixture, target enrichment reagents, library preparation reagents, native barcoding reagents and sequencing reagents.

Description

DESCRIPTION OF DRAWINGS

[0056] FIGS. 1A to 1F as a whole show a metabolic spectrum phylogenetic tree constructed by 82 metabolic fingerprints of 24 bacterial species for bacterial identification. More particularly, FIG. 1A shows the top portion of the metabolic spectrum phylogenetic tree constructed by 82 metabolic fingerprints of 24 bacterial species for bacterial identification; FIGS. 1B-1E shows the middle portion of the metabolic spectrum phylogenetic tree constructed by 82 metabolic fingerprints of 24 bacterial species for bacterial identification; and FIG. 1F shows the bottom portion of the metabolic spectrum phylogenetic tree constructed by 82 metabolic fingerprints of 24 bacterial species for bacterial identification.

[0057] FIGS. 2A to 2G as a whole show the distribution of 16 blind samples in the metabolic spectrum phylogenetic tree for bacterial identification. The branches of the phylogenetic tree representing different species are indicated by different colors on the left, where black represents blind samples. More particularly, FIG. 2A shows the top portion of the distribution of 16 blind samples in the metabolic spectrum phylogenetic tree for bacterial identification; FIGS. 2B-2F show the middle portion of the distribution of 16 blind samples in the metabolic spectrum phylogenetic tree for bacterial identification; and FIG. 2G shows the bottom of the distribution of 16 blind samples in the metabolic spectrum phylogenetic tree for bacterial identification.

[0058] FIGS. 3A to 3E as a whole show a metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference. More particularly, FIG. 3A shows the top portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference; and FIGS. 3B-3D show the middle portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference; and FIG. 3E shows the bottom portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference.

[0059] FIGS. 4A to 4E as a whole show the distribution of 16 blind Acinetobacter baumannii samples in the metabolic spectrum phylogenetic tree for susceptibility inference. More particularly, FIG. 4A shows the top portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference; FIGS. 4B-4D shows the middle portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference; and FIG. 4E shows the bottom portion of the metabolic spectrum phylogenetic tree constructed by 87 representative Acinetobacter baumannii metabolic fingerprints for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0060] FIGS. 5A-5B as a whole show a metabolic spectrum phylogenetic tree constructed by 36 representative Enterococcus faecalis metabolic fingerprints for susceptibility inference.

[0061] FIGS. 6A-6C as a whole show the distribution of 6 blind Enterococcus faecalis samples in the metabolic spectrum phylogenetic tree for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0062] FIG. 7 is a metabolic spectrum phylogenetic tree constructed by 18 representative Streptococcus pneumoniae metabolic fingerprints for susceptibility inference.

[0063] FIG. 8 shows the distribution of 2 blind Streptococcus pneumoniae samples in the metabolic spectrum phylogenetic tree for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0064] FIGS. 9A to 9F as a whole show a metabolic spectrum phylogenetic tree constructed by 115 metabolic fingerprints representative of 22 fungal species for fungal identification. More particularly, FIG. 9A shows the top portion of the metabolic spectrum phylogenetic tree constructed by 115 metabolic fingerprints representative of 22 fungal species for fungal identification; FIGS. 9B-9E show the middle portion of the metabolic spectrum phylogenetic tree constructed by 115 metabolic fingerprints representative of 22 fungal species for fungal identification; and FIG. 9F shows the bottom portion of the metabolic spectrum phylogenetic tree constructed by 115 metabolic fingerprints representative of 22 fungal species for fungal identification.

[0065] FIGS. 10A to 10F as a whole show the distribution of 8 blind samples in the metabolic spectrum phylogenetic tree for fungal identification. More particularly, FIG. 10A shows the top portion of the distribution of 8 blind samples in the metabolic spectrum phylogenetic tree for fungal identification; FIGS. 10B-10E show the middle portion of the distribution of 8 blind samples in the metabolic spectrum phylogenetic tree for fungal identification and FIG. 10F shows the bottom portion of the distribution of 8 blind samples in the metabolic spectrum phylogenetic tree for fungal identification. The branches of the phylogenetic tree representing different species are indicated by different colors on the left, where black represents blind samples.

[0066] FIGS. 11A-11C as a whole show the distribution of 6 blind Candida tropicalis samples in the metabolic spectrum phylogenetic tree for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where blue indicates azole-susceptible, yellow indicates azole-resistant, and black represents blind samples.

[0067] FIGS. 12A to 12G as a whole show a genomic phylogenetic tree constructed by 165 representative Klebsiella pneumoniae genomes for susceptibility inference. More particularly, FIG. 12A shows the top portion of the genomic phylogenetic tree constructed by 165 representative Klebsiella pneumoniae genomes for susceptibility inference; FIGS. 12B-12F shows the middle portion of the genomic phylogenetic tree constructed by 165 representative Klebsiella pneumoniae genomes for susceptibility inference; and FIG. 12G shows the bottom portion of the genomic phylogenetic tree constructed by 165 representative Klebsiella pneumoniae genomes for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where yellow indicates ST11 clone, red indicates ST15 clone, and blue indicates susceptible to all antibiotics (including ST23 clone).

[0068] FIGS. 13A-13D as a whole show a genomic phylogenetic tree constructed by 93 representative Staphylococcus aureus genomes for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right.

[0069] FIG. 14 shows the genomic phylogenetic tree constructed by 25 representative Streptococcus pneumoniae genomes and the distribution of 2 blind samples in the genomic phylogenetic tree for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where yellow indicates susceptible to all antibiotics, blue indicates penicillin-resistant, and black represents blind samples.

[0070] FIGS. 15A-15C as a whole show a genomic phylogenetic tree constructed by 107 representative Candida albicans genomes for susceptibility inference. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where red indicates azole-resistant, yellow indicates 5-fluorocytosine-resistant, green indicates echinocandins-resistant, gray indicates Amphotericin-B-intermediate, and blue indicates susceptible to all antibiotics.

[0071] FIG. 16 shows the distribution of a respiratory sputum sample in the genomic phylogenetic tree of Acinetobacter baumannii.

[0072] FIG. 17 shows the distribution of a respiratory sputum sample in the genomic phylogenetic tree of Klebsiella pneumoniae.

DETAILED DESCRIPTION

[0073] The following embodiments further illustrate content of the present invention, but should not be construed as a limitation on the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention shall fall within the scope of the present invention. Unless otherwise specified, in each table (tables 1 to 24), ‘R’ indicates drug-resistant, ‘S’ indicates drug-susceptible, blank means no interpretation.

Example 1: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Bacterial Identification Based on LC-MS

[0074] 1. Sample Preparation and Detection

[0075] Step 1. Sample collection and identification: 430 clinical isolates were collected from 42 hospitals across china in a period between May 2017 and July 2019. All isolates were subjected to Sanger sequencing or the third-generation whole genome sequencing, as the gold standard for species identification.

[0076] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Columbia sheep blood agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous bacterial suspension.

[0077] Step 3. Cell breakage: An equal volume of bacterial standards was added to 180 μL of bacterial suspension, and sonicated at 80 Hz for 5 min.

[0078] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0079] Step 5. Mass spectrometry detection: The residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0080] 2. Bioinformatics Analysis and Database Construction

[0081] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. Bacteria of different species were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as: fold change>10, VIP>1, p value<0.05, CV<30%. The following 258 biomarkers were screened out:

TABLE-US-00001 No. Compound CP1 0.55_100.0746 m/z CP2 0.55_1284.0467 m/z CP3 0.55_1361.3539 m/z CP4 0.56_171.1479 m/z CP5 0.56_188.1747 m/z CP6 0.56_210.1554 m/z CP7 0.56_526.1755 m/z CP8 0.56_542.1480 m/z CP9 0.56_548.1544 m/z CP10 0.57_540.1899 m/z CP11 0.58_504.1929 m/z CP12 0.58_644.7665 m/z CP13 0.58_742.7425 m/z CP14 0.61_372.0036 m/z CP15 0.63_1038.331 m/z CP16 0.63_1054.3086 m/z CP17 0.63_1054.8086 m/z CP18 0.63_1101.2951 m/z CP19 0.63_1209.8945 m/z CP20 0.63_1217.3785 m/z CP21 0.63_1217.8830 m/z CP22 0.63_1225.8718 m/z CP23 0.63_1247.3924 m/z CP24 0.63_1247.8869 m/z CP25 0.63_1266.3725 m/z CP26 0.63_1266.8695 m/z CP27 0.63_1286.4402 m/z CP28 0.63_1369.4686 m/z CP29 0.63_1369.9676 m/z CP30 0.63_1377.9759 m/z CP31 0.63_1378.4733 m/z CP32 0.63_1380.4561 m/z CP33 0.63_1380.9560 m/z CP34 0.63_1388.4415 m/z CP35 0.63_1388.9458 m/z CP36 0.63_1418.4505 m/z CP37 0.63_1418.9547 m/z CP38 0.63_1429.4435 m/z CP39 0.63_1437.9370 m/z CP40 0.63_753.6939 m/z CP41 0.63_867.7757 m/z CP42 0.63_875.2647 m/z CP43 0.63_875.7657 m/z CP44 0.63_924.7535 m/z CP45 0.64_1038.8347 m/z CP46 0.64_1053.8045 m/z CP47 0.64_1147.8626 m/z CP48 0.64_1206.9203 m/z CP49 0.64_1207.4190 m/z CP50 0.64_1319.4207 m/z CP51 0.64_1471.4886 m/z CP52 0.64_1490.4942 m/z CP53 0.64_329.5271 m/z CP54 0.64_696.7194 m/z CP55 0.64_797.7422 m/z CP56 0.64_805.7445 m/z CP57 0.64_806.2437 m/z CP58 0.64_976.8049 m/z CP59 0.65_204.0868 m/z CP60 0.65_463.6322 m/z CP61 0.65_534.2027 m/z CP62 0.65_546.2036 m/z CP63 0.65_565.1722 m/z CP64 0.65_568.1855 m/z CP65 0.65_608.2409 m/z CP66 0.65_634.6881 m/z CP67 0.65_776.7318 m/z CP68 0.66_292.5782 m/z CP69 0.66_404.5512 m/z CP70 0.66_869.2594 m/z CP71 0.67_352.5631 m/z CP72 0.67_605.6739 m/z CP73 0.67_885.3152 m/z CP74 0.73_434.6168 m/z CP75 0.73_696.2561 m/z CP76 0.75_1008.3655 m/z CP77 0.75_495.2256 m/z CP78 0.75_515.6433 m/z CP79 0.75_523.6326 m/z CP80 0.77_649.6213 m/z CP81 0.77_649.9563 m/z CP82 0.80_247.3097 m/z CP83 0.80_348.2961 m/z CP84 0.82_937.3576 m/z CP85 0.82_937.8613 m/z CP86 0.85_572.7552 m/z CP87 0.86_892.2968 m/z CP88 0.87_275.1338 m/z CP89 0.87_574.2563 m/z CP90 0.87_645.1994 m/z CP91 0.87_912.2736 m/z CP92 0.92_196.9863 m/z CP93 0.97_233.1107 m/z CP94 1.15_187.1435 m/z CP95 1.20_330.0604 m/z CP96 1.28_384.0628 m/z CP97 1.35_535.1892 m/z CP98 1.39_154.1216 m/z CP99 1.39_171.1478 m/z CP100 1.46_583.3177 m/z CP101 1.48_147.1119 m/z CP102 1.69_239.0345 m/z CP103 1.78_736.8275 m/z CP104 1.80_737.0786 m/z CP105 1.80_737.3288 m/z CP106 1.84_268.1042 m/z CP107 1.95_119.0333 m/z CP108 1.95_136.0604 m/z CP109 1.95_268.1029 m/z CP110 1.97_1024.7863 m/z CP111 1.97_350.5713 m/z CP112 1.97_623.0645 m/z CP113 1.97_664.2311 m/z CP114 1.97_686.2108 m/z CP115 1.97_769.5926 m/z CP116 1.98_1025.4522 m/z CP117 1.99_768.8416 m/z CP118 1.99_769.0922 m/z CP119 1.99_769.3422 m/z CP120 2.06_243.6588 m/z CP121 2.18_330.0594 m/z CP122 2.18_393.1092 m/z CP123 2.24_277.5730 m/z CP124 2.24_798.3440 m/z CP125 2.24_798.5898 m/z CP126 2.24_798.8430 m/z CP127 2.25_1107.139 m/z CP128 2.25_1107.479 m/z CP129 2.25_667.9714 m/z CP130 2.25_668.6395 m/z CP131 2.25_830.6066 m/z CP132 2.25_830.8570 m/z CP133 2.25_831.1085 m/z CP134 2.26_862.3679 m/z CP135 2.26_862.6206 m/z CP136 2.26_862.8715 m/z CP137 2.26_863.1262 m/z CP138 2.26_894.3873 m/z CP139 2.26_894.8847 m/z CP140 2.27_710.6566 m/z CP141 2.27_710.9904 m/z CP142 2.27_894.6352 m/z CP143 2.29_1089.6800 m/z CP144 2.29_1089.8807 m/z CP145 2.29_1090.0818 m/z CP146 2.29_1204.3381 m/z CP147 2.29_1204.8394 m/z CP148 2.29_753.3411 m/z CP149 2.29_753.6760 m/z CP150 2.29_754.0095 m/z CP151 2.31_1111.2433 m/z CP152 2.31_1111.4422 m/z CP153 2.31_1264.0437 m/z CP154 2.31_1264.5437 m/z CP155 2.31_1264.7921 m/z CP156 2.32_1011.6423 m/z CP157 2.32_1011.8424 m/z CP158 2.32_1012.0425 m/z CP159 2.32_1012.2466 m/z CP160 2.32_1264.2959 m/z CP161 2.33JO63.6266 m/z CP162 2.33_605.6714 m/z CP163 2.33_624.6453 m/z CP164 2.34_1086.6690 m/z CP165 2.34_1086.8670 m/z CP166 2.34_1087.0677 m/z CP167 2.34_1087.2688 m/z CP168 2.34_1087.4686 m/z CP169 2.34_1087.6692 m/z CP170 2.34_1091.4661 m/z CP171 2.34_1112.4790 m/z CP172 2.34_1358.3389 m/z CP173 2.34_1358.5844 m/z CP174 2.34_1358.8369 m/z CP175 2.34_1359.0866 m/z CP176 2.34_590.6673 m/z CP177 2.34_912.3922 m/z CP178 2.35_1041.7903 m/z CP179 2.35_1112.6759 m/z CP180 2.35_1181.0990 m/z CP181 2.35_242.5698 m/z CP182 2.36_1249.7396 m/z CP183 2.36_170.9955 m/z CP184 2.39_98.0585 m/z CP185 2.41_507.5750 m/z CP186 2.41_511.5846 m/z CP187 2.41_720.5883 m/z CP188 2.41_793.2797 m/z CP189 2.41_815.2600 m/z CP190 2.42_492.0796 m/z CP191 2.43_1281.3409 m/z CP192 2.43_1281.8429 m/z CP193 2.43_768.6241 m/z CP194 2.44_455.5592 m/z CP195 2.44_455.8912 m/z CP196 2.45_1173.9496 m/z CP197 2.45_782.6249 m/z CP198 2.45_782.9596 m/z CP199 2.46_575.9609 m/z CP200 2.46_597.9710 m/z CP201 2.47_1101.9174 m/z CP202 2.47_1194.9504 m/z CP203 2.47_796.6309 m/z CP204 2.47_796.9648 m/z CP205 2.47_804.6253 m/z CP206 2.47_809.6124 m/z CP207 2.47_814.6012 m/z CP208 2.48_1013.8949 m/z CP209 2.48_1113.9199 m/z CP210 2.48_1121.9113 m/z CP211 2.48_1122.9206 m/z CP212 2.48_1205.1806 m/z CP213 2.48_1205.9352 m/z CP214 2.48_1215.6860 m/z CP215 2.48_1221.1778 m/z CP216 2.48_1221.6823 m/z CP217 2.48_1224.8961 m/z CP218 2.48_1229.8540 m/z CP219 2.48_1234.9331 m/z CP220 2.48_1241.9157 m/z CP221 2.48_1242.9173 m/z CP222 2.48_1245.9559 m/z CP223 2.48_608.4744 m/z CP224 2.48_608.9734 m/z CP225 2.48_810.6347 m/z CP226 2.48_810.8615 m/z CP227 2.48_810.9689 m/z CP228 2.48_811.3031 m/z CP229 2.48_817.9590 m/z CP230 2.48_818.6289 m/z CP231 2.48_828.6067 m/z CP232 2.48_836.5968 m/z CP233 2.48_999.0284 m/z CP234 2.48_999.7032 m/z CP235 2.49_1215.4607 m/z CP236 2.49_1215.9592 m/z CP237 2.49_1226.9496 m/z CP238 2.49_1488.0764 m/z CP239 2.49_1488.5825 m/z CP240 2.49_801.6338 m/z CP241 2.50_1226.1823 m/z CP242 2.50_1297.9967 m/z CP243 2.50_1308.9879 m/z CP244 2.50_1381.0357 m/z CP245 2.51_1013.0378 m/z CP246 2.51_1013.7082 m/z CP247 2.51_1143.9206 m/z CP248 2.51_1222.9671 m/z CP249 2.51_1236.9623 m/z CP250 2.51_1247.9518 m/z CP251 2.51_1255.9395 m/z CP252 2.51_1259.9411 m/z CP253 2.51_618.9763 m/z CP254 2.51_824.6365 m/z CP255 2.51_824.9703 m/z CP256 2.51_832.6299 m/z CP257 2.51_837.6194 m/z CP258 2.51_842.6097 m/z CP259 2.52_1034.6979 m/z CP260 2.52_651.1092 m/z CP261 2.52_655.1100 m/z CP262 2.52_770.5255 m/z CP263 2.52_771.0314 m/z CP264 2.52_815.6393 m/z CP265 2.52_815.9740 m/z CP266 2.53_1041.7178 m/z CP267 2.53_1048.7066 m/z CP268 2.53_1049.0365 m/z CP269 2.53_1053.6921 m/z CP270 2.53_1243.9642 m/z CP271 2.53_781.0341 m/z CP272 2.53_996.8791 m/z CP273 2.54_1257.9658 m/z CP274 2.54_838.6384 m/z CP275 2.54_838.9677 m/z CP276 2.54_846.6335 m/z CP277 2.56_1045.7110 m/z CP278 2.56_1046.0462 m/z CP279 2.56_1055.7225 m/z CP280 2.56_1062.7114 m/z CP281 2.56_1063.0460 m/z CP282 2.56_791.5357 m/z CP283 2.56_792.0383 m/z CP284 2.56_851.6234 m/z CP285 2.57_328.5501 m/z CP286 2.57_616.1518 m/z CP287 2.57_618.1475 m/z CP288 2.57_629.6244 m/z CP289 2.57_640.1282 m/z CP290 2.57_668.6512 m/z CP291 2.58_1159.0780 m/z CP292 2.58_528.0691 m/z CP293 2.58_580.0414 m/z CP294 2.58_580.5434 m/z CP295 2.58_691.6719 m/z CP296 2.58_773.7202 m/z CP297 2.60_1069.7234 m/z CP298 2.60_1076.7168 m/z CP299 2.60_1411.5904 m/z CP300 2.60_802.0401 m/z CP301 2.60_852.6441 m/z CP302 2.63_1133.9809 m/z CP303 2.65_344.4278 m/z CP304 2.65_908.9106 m/z CP305 2.66_1421.5652 m/z CP306 2.66_632.1659 m/z CP307 2.67_1250.6971 m/z CP308 2.67_1395.8226 m/z CP309 2.68_672.5593 m/z CP310 2.70_1343.0987 m/z CP311 2.71_102.1264 m/z CP312 2.71_119.0266 m/z CP313 2.71_472.6112 m/z CP314 2.73JO58.9230 m/z CP315 2.73_1279.0598 m/z CP316 2.73_1389.6516 m/z CP317 2.73_821.5490 m/z CP318 2.74_1070.9482 m/z CP319 2.74_1082.9834 m/z CP320 2.75_731.6471 m/z CP321 2.76_1092.0217 m/z CP322 2.76_1098.9938 m/z CP323 2.76_1132.0044 m/z CP324 2.76_1197.0118 m/z CP325 2.76_1459.7094 m/z CP326 2.76_626.0012 m/z CP327 2.77_687.6290 m/z CP328 2.77_822.6933 m/z CP329 2.78_1126.0419 m/z CP330 2.78_1299.6447 m/z CP331 2.78_1316.6255 m/z CP332 2.78_1338.6192 m/z CP333 2.78_1491.7467 m/z CP334 2.83_457.9436 m/z CP335 2.85_1182.0766 m/z CP336 2.85_1329.6451 m/z CP337 2.85_1444.7037 m/z CP338 2.86_1374.1612 m/z CP339 2.86_1411.6575 m/z CP340 2.86_1471.7506 m/z CP341 2.89_1108.1168 m/z

[0082] (2) Construction of a metabolic spectrum phylogenetic tree database: To optimize the representativeness and reproducibility of the database, a variety of enterobacteriaceae bacteria, non-fermenting bacteria and Gram-positive cocci were evenly covered and at least 10 isolates from each hospital were employed for metabolic fingerprints profiling test. To optimize the resolution of the database, closely-related species such as Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii, Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae which could not be accurately distinguished by the automated identification system such as VITEK2, were selected and added to the database.

[0083] Through biomarker analysis, identical clones were merged and 82 representative clones were selected for the database. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated, as shown in FIGS. 1A-1F.

[0084] 3. Database Validation and Result Interpretation

[0085] (1) Blind test: The metabolic fingerprints of 16 blinds were imported into the software 11BM SPSS Statistics 23 for cluster analysis. The species of each blind was determined based on its positioning in the phylogenetic tree. As is shown in FIGS. 2A-2G, the branches of the phylogenetic tree representing different species are indicated by different colors on the left, where black represents blind samples.

[0086] (2) Result interpretation: The species of 16 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. As is shown in Table 1, identification results predicted by method of his invention were in a 100 agreement with the gold standard (sequencing method) result. Notably, closely relates species such as Acinetobacter baumannii and Acinetobacter pittii, which can not be distinguished by automated identification systems such as VITEK2, were identified in complete agreement with the gold standard method.

TABLE-US-00002 TABLE 1 blind result by method of this result by gold standard agree- sample invention (sequencing) ment blind-1 Pseudomonas aeruginosa Pseudomonas aeruginosa yes blind-2 Escherichia coli Escherichia coli yes blind-3 Escherichia coli Escherichia coli yes blind-4 Stenotrophomonas maltophilia Stenotrophomonas maltophilia yes blind-5 Acinetobacter pittii Acinetobacter pittii yes blind-6 Enterococcus faecium Enterococcus faecium yes blind-7 Klebsiella variicola Klebsiella variicola yes blind-8 Acinetobacter baumannii Acinetobacter baumannii yes blind-9 Acinetobacter baumannii Acinetobacter baumannii yes blind-10 Enterobacter cloacae Enterobacter cloacae yes blind-11 Staphylococcus aureus Staphylococcus aureus yes blind-12 Klebsiella aerogenes Klebsiella aerogenes yes blind-13 Klebsiella pneumoniae Klebsiella pneumoniae yes blind-14 Staphylococcus haemolyticus Staphylococcus haemolyticus yes blind-15 Klebsiella pneumoniae Klebsiella pneumoniae yes blind-16 Staphylococcus epidermidis Staphylococcus epidermidis yes

Example 2: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Acinetobacter baumannii Based on LC-MS

[0087] Acinetobacter baumannii was selected as the representative of Gram-negative bacteria to illustrate the way a metabolic spectrum phylogenetic tree for drug susceptibility determination is constructed. Those skilled in the art can understand that this method is applicable to other Gram-negative bacteria including enterobacteriaceae and non-fermentative bacteria.

[0088] 1. Sample Preparation and Detection

[0089] Step 1. Drug susceptibility verification: The Acinetobacter baumannii isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-N335 cards, and the results were used as the gold standard (culture-based AST).

[0090] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Columbia sheep blood agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous bacterial suspension.

[0091] Step 3. Cell breakage: an equal volume of bacterial standards was added to 180 μL of bacterial suspension, and sonicated at 80 Hz for 5 min.

[0092] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0093] Step 5. Mass spectrometry detection: the residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0094] 2. Bioinformatics Analysis and Database Construction

[0095] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. The Acinetobacter baumannii clones of different resistance profiles were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as: fold change>10, VIP>1, p value<0.05, CV<30%. The following 102 biomarkers were screened out:

TABLE-US-00003 No. Compound CP1 0.67_885.3152 m/z CP2 0.80_247.3097 m/z CP3 0.82_937.3576 m/z CP4 0.82_937.8613 m/z CP5 0.86_892.2968 m/z CP6 0.87_275.1338 m/z CP7 0.87_574.2563 m/z CP8 0.87_645.1994 m/z CP9 1.46_583.3177 m/z CP10 1.95_119.0333 m/z CP11 1.95_136.0604 m/z CP12 1.95_268.1029 m/z CP13 2.18_330.0594 m/z CP14 2.18_393.1092 m/z CP15 2.24_277.5730 m/z CP15 2.35_242.5698 m/z CP16 2.39_98.0585 m/z CP17 2.41_507.5750 m/z CP18 2.41_511.5846 m/z CP19 2.42_492.0796 m/z CP20 2.60_1411.5904 m/z CP21 2.63_1133.9809 m/z CP22 2.65_908.9106 m/z CP23 2.66_1421.5652 m/z CP24 2.67_1250.6971 m/z CP25 2.67_1395.8226 m/z CP26 2.67_1395.8226 m/z CP27 2.68_672.5593 m/z CP28 2.70_1343.0987 m/z CP29 2.71_102.1264 m/z CP30 2.73_1058.9230 m/z CP31 2.73_1279.0598 m/z CP32 2.73_1389.6516 m/z CP33 2.74_1070.9482 m/z CP34 2.74_1082.9834 m/z CP35 2.75_731.6471 m/z CP36 2.76_1092.0217 m/z CP37 2.76_1098.9938 m/z CP38 2.76_1132.0044 m/z CP39 2.76119.0118 m/z CP40 2.76_1459.7094 m/z CP42 2.78_1126.0419 m/z CP43 2.78_1299.6447 m/z CP44 2.78_1316.6255 m/z CP45 2.78_1338.6192 m/z CP46 2.78_1491.7467 m/z CP47 2.85_1182.0766 m/z CP48 2.85_1329.6451 m/z CP49 2.85_1444.7037 m/z CP50 2.86_1374.1612 m/z CP51 2.86_1411.6575 m/z CP52 2.86_1471.7506 m/z CP53 2.88_1358.5844 m/z CP54 2.89_1108.1168 m/z CP55 2.911011.8289 m/z CP56 2.91_1012.8270 m/z CP57 2.91_1371.6639 m/z CP58 2.91_1372.2017 m/z CP59 2.91_915.8016 m/z CP60 2.92_1187.1433 m/z CP61 2.92_1206.1139 m/z CP62 2.92_1293.6244 m/z CP63 2.92_1329.7157 m/z CP64 2.92_1365.6693 m/z CP65 2.93_1269.6239 m/z CP66 2.94_948.1554 m/z CP67 2.95_1342.7146 m/z CP68 2.95_947.8219 m/z CP69 2.95_948.8201 m/z CP70 2.96_1257.2006 m/z CP71 2.97_1052.0905 m/z CP72 2.97_1421.7925 m/z CP73 2.97_1443.7972 m/z CP74 2.97_708.7048 m/z CP75 2.97_837.7936 m/z CP76 2.97_838.1319 m/z CP77 2.98_1009.0445 m/z CP78 2.98_1044.0853 m/z CP79 2.98_1055.0739 m/z CP80 2.98_1055.5707 m/z CP81 2.98_1063.0585 m/z CP82 2.98_1066.0654 m/z CP83 2.98_1066.5628 m/z CP84 2.98_1074.0492 m/z CP85 2.98_1077.0566 m/z CP86 2.98_1260.7389 m/z CP87 2.98_1356.6824 m/z CP88 2.98_666.0361 m/z CP89 2.98_703.7160 m/z CP90 2.98_704.0466 m/z CP91 2.98_709.0372 m/z CP92 2.98_921.9998 m/z CP93 2.98_998.0538 m/z CP94 3.00_1036.5737 m/z CP95 3.00_1093.1141 m/z CP96 3.00_1093.6160 m/z CP97 3.00_1094.1189 m/z CP98 3.00_1104.1054 m/z CP99 3.00_1104.6071 m/z CP100 3.00_1115.0977 m/z CP101 3.00_729.7483 m/z CP102 3.00_730.0794 m/z

[0096] (2) Resistance profile classification: According to the susceptibility properties of Acinetobacter baumannii to 12 antibacterial drugs including piperacillin, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, amikacin, levofloxacin, ciprofloxacin, TMP-SMZ and minocycline, its resistance profiles were classified into different types, named as A to S. The drug resistance profile classification and corresponding drug susceptibility are shown in Table 2.

TABLE-US-00004 TABLE 2 Resistance profile Suscepibility of Acinetobacter baumannii to 12 antibacterial drugs classification pipercillin A R R R R R R R R R R R R R B R R R R R R S S R R R S R C R R R R R R R R R R R S R D R R R R R R S S R R R R R E R R R R R R S S R R R R J F R R R R R R R S R R R R S G R R R R R R S S R R R R S H R R R R R R S S R R R S S I R R R R R R S S R R R S S J R R R R R R R S S R R R S K R R S R R R R R S R R R S L R R S R S S R R R R R R S M R R S R S S R R R R R S S N R R S R S S S R R R R S S O S S S R S S R R S S S S S P S S S R S S S R S S S R S Q S S S S S S S S S R R S S R S S S S S S S S S R R R S S S S S S S S S S S S S S S indicates data missing or illegible when filed

[0097] (3) Construction of a metabolic spectrum phylogenetic tree database for susceptibility determination: Through biomarker analysis, identical clones were merged and 87 representative Acinetobacter baumannii clones were selected for the database. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated, as shown in FIGS. 3A-3E.

[0098] 3. Database Validation and Result Interpretation

[0099] (1) Blind test: The metabolic fingerprints of 16 blinds were imported into the software IBM SPSS Statistics 23 for cluster analysis. The resistance profile of each sample was determined based on its positioning in the phylogenetic tree and the corresponding prediction rules. As is shown in FIGS. 4A-4E, the branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0100] (2) Result interpretation: The resistance profiles of 16 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. Use independent prediction rules: when the test sample contains metabolic fingerprints of a specific clone, follow the principle that the drug resistance profile predicted by metabolic fingerprints is preferred over phylogenetic tree interpretation.

[0101] The resistance profiles of the 16 blinds to 12 antibacterial drugs including piperacillin, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, amikacin, levofloxacin, ciprofloxacin, TMP-SMZ and minocycline predicted by method of the present invention are shown in Table 3.

TABLE-US-00005 TABLE 3 Resistance profiles Resistance profiles Prediction Agreement Agreement by VITEK2 (the inferred by rules (preliminary (final Sample gold standard) phylogenetic tree involved Corrections result) result) blind-1 G A208 contains G208 corrected to 1 False positive yes biomarkers, G208 (gentamicin, preferred over tobramycin. phylogenetic tree amikacin) blind-2 A A208 / / yes yes blind-3 A A195 / / yes yes blind-4 C C195 / / yes yes blind-5 F D540 contains F540 corrected to 1 False negative yes biomarkers, F540 (tobramycin) preferred over phylogenetic tree blind-6 A A208 / / yes yes blind-7 A A540 / / yes yes blind-8 A A381 / / yes yes blind-9 C C136 / / yes yes blind-10 A A191 / / yes yes blind-11 D D191 / / yes yes blind-12 A A93S / / yes yes blind-13 D D368 / / yes yes blind-14 A C195 / / 11 False negative 1 False (TMP-SMZ) negative (TMP-SMZ) blind-15 A A547 / / yes yes blind-16 D D784 / / yes yes

[0102] When analyzed solely using the phylogenetic tree, out of 16 blinds, 3 samples and 6 antibiotics displayed inconsistent results compared with the gold standard VITEK2 AST. However, once the phylogenetic tree was combined with metabolic fingerprints and specific prediction rule was applied, the results were corrected. Since the blind 1 contained specific G208 biomarkers (2.41_507.5750 m/z and 2.41_511.5846 m/z) and the blind 5 contained specific F540 biomarkers (3.00_1093.6160 m/z and 3.00_1094.1189 m/z), the prediction rule that when the test sample contains metabolic fingerprints of a specific clone, the drug resistance profile predicted by metabolic fingerprints is preferred over phylogenetic tree interpretation was applied. Therefore, the results of blind sample 1 and blind sample 5 were revised to G208 and F540, which were in agreement with the gold standard VITEK2 AST.

[0103] Finally, in one case of the 16 blinds, the resistance profile predicted by method of the present invention was inconsistent with that of the gold standard VITEK2 AST, that is, the false negative result of TMP-SMZ. As for 12 drugs involved in this study, a total of 192 drug results were analyzed, displaying 1 false negative result and no false positive result. The positive predictive value was 100% (168/168), the negative predictive value was 95.83% (23/24), the sensitivity was 99.41% (168/169), and the specificity was 100% (23/23). The performances meet the design requirements of the present method and the needs of clinical application, that is, the sensitivity and specificity being above 95%.

Example 3: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Enterococcus faecalis Based on LC-MS

[0104] Enterococcus faecalis was selected as the representative of Gram-positive cocci to illustrate the way a metabolic spectrum phylogenetic tree for drug susceptibility determination is constructed. Those skilled in the art can understand that this method is applicable to other Gram-negative bacteria including Enterococcus faecium. and the Staphylococcus spp.

[0105] 1. Sample Preparation and Detection

[0106] Step 1. Drug susceptibility verification: The Enterococcus faecalis isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-P639 cards, and the results were used as the gold standard (culture-based AST).

[0107] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Columbia sheep blood agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous bacterial suspension.

[0108] Step 3. Cell breakage: an equal volume of bacterial standards was added to 180 μL of bacterial suspension, and sonicated at 80 Hz for 5 min.

[0109] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0110] Step 5. Mass spectrometry detection: the residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0111] 2. Bioinformatics Analysis and Database Construction

[0112] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. The Enterococcus faecalis clones of different resistance profiles were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as:

TABLE-US-00006 No. Compound CP1 0.53_491.2411 m/z CP2 0.55_1409.6162 m/z CP3 0.55_1450.5881 m/z CP4 0.60_104.1128 m/z CP5 0.60_258.1144 m/z CP6 0.60_258.3054 m/z CP7 0.60_258.5580 m/z CP8 0.61_162.1183 m/z CP9 0.64_538.0832 m/z CP10 0.75_829.2745 m/z CP11 0.79_991.3231 m/z CP12 0.97_664.1133 m/z CP13 1.02_308.0948 m/z CP14 1.04_347.1912 m/z CP15 1.06_192.5755 m/z CP16 1.07_215.5787 m/z CP17 1.14_639.0793 m/z CP18 2.04_366.1352 m/z CP19 2.06_268.1078 m/z CP20 2.47_194.1219 m/z CP21 2.47_373.2003 m/z CP22 2.48_254.1650 m/z CP23 2.48_496.2471 m/z CP24 2.48_764.3693 m/z CP25 2.49_516.0883 m/z CP26 2.50_343.1991 m/z CP27 2.50_697.3339 m/z CP28 2.51_867.4025 m/z CP29 2.53_120.0864 m/z CP30 2.53_515.2600 m/z CP31 2.58_414.2292 m/z CP32 2.58_615.2853 m/z CP33 2.59_442.2250 m/z CP34 2.59_795.3734 m/z CP35 2.59_941.4294 m/z CP36 2.61610.3115 m/z CP37 2.61_757.3598 m/z CP38 2.63_711.3399 m/z CP39 2.66_188.0762 m/z CP40 2.66_718.3410 m/z CP41 2.66_724.3318 m/z CP42 2.66_824.3855 m/z CP43 2.68_585.3086 m/z CP44 2.71_642.3346 m/z CP45 2.71_856.4256 m/z CP46 2.72_813.4196 m/z CP47 2.74_576.3378 m/z CP49 2.76_646.9948 m/z CP50 2.76_684.3738 m/z CP51 2.84_659.3369 m/z

[0113] (2) Resistance profile classification: According to the susceptibility properties of Enterococcus faecalis to 11 antibacterial drugs including penicillin, ampicillin, vancomycin, linezolid, daptomycin, high-level gentamicin, erythromycin, levofloxacin, ciprofloxacin, tigecycline and tetracycline, its resistance profiles were classified into different types, named as A to S. The drug resistance profile classification and corresponding drug susceptibility are shown in Table 4.

TABLE-US-00007 TABLE 4 Resis- tance profile class- Susceptibility of Emterococcus faecalis to 11 antibacterial drugs ification penicillin ampicillin vancomycin linezolid daptomycin HLAR erythromycin levofloxacin ciprofloxacin tigecycline tetracycline A R R S S S R R R R S R B R R S S S R R R R S S C R R S S S S R R R S S D R R S S S S S R R S S F R R S S S S R S S S S F R R S S S S R S S S R G S S S S S R R R R S R H S S S S S R R S S S R I S S S S S R R R R S S J S S S S S S R S S S R K S S S S S S S R R S R L S S S S S S S R R S S M S S S S S S S S R g R S S S S S S S S 8 S S S

[0114] (3) Construction of a metabolic spectrum phylogenetic tree database for susceptibility determination: Through biomarker analysis, identical clones were merged and 36 representative Enterococcus faecalis clones were selected for the database. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated, as shown in FIGS. 5A-5B.

[0115] 3. Database Validation and Result Interpretation

[0116] (1) Blind test: The metabolic fingerprints of 6 blinds were imported into the software IBM SPSS Statistics 23 for cluster analysis. The resistance profile of each sample was determined based on its positioning in the phylogenetic tree and the corresponding prediction rules. As is shown in FIGS. 6A-6C, the branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0117] (2) Result interpretation: The resistance profiles of 6 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. Use independent prediction rules: a) when the test sample contains metabolic fingerprints of a specific clone, follow the principle that the drug resistance profile predicted by metabolic fingerprints is preferred over phylogenetic tree interpretation; b) when the test sample has the metabolic fingerprints of a sequence type 4 Enterococcus faecalis clone, the sample is directly determined to be resistant to penicillins.

[0118] The resistance profiles of the 6 blinds to 11 antibacterial drugs including penicillin, ampicillin, vancomycin, linezolid, daptomycin, high-level gentamicin, erythromycin, levofloxacin, ciprofloxacin, tigecycline and tetracycline predicted by method of the present invention are shown in Table 5.

TABLE-US-00008 TABLE 5 Resistance Resistance profiles by profiles VITEK2 inferred by Prediction Agreement Agreement (the gold phylogenetic rules (preliminary (final Sample standard) tree involved Corrections result) result) blind-1 D D4 / / yes yes blind-2 K K6 / / yes yes blind-3 J J179 / / yes yes blind-4 B B4 / / yes yes blind-5 S S537 / / yes yes blind-6 A M16 contains corrected 1 False negative yes ST4 to_A4 (penicillin, biomarkers, ampicillin, preferred HLAR, over erythromycin, phylogenetic levofloxacin, tree ciprofloxacin)

[0119] When analyzed solely using the phylogenetic tree, out of 6 blinds, 1 samples and 6 antibiotics displayed inconsistent results compared with the gold standard VITEK2 AST. However, once the phylogenetic tree was combined with metabolic fingerprints and specific prediction rule was applied, the results were corrected. Since the blind 6 contained specific ST4 biomarkers (1.06_192.5755 m/z, 1.07_215.5787 m/z, 1.14_639.0793 m/z 2.04_366.1352 m/z), the prediction rule that when the test sample has the metabolic fingerprints of a sequence type 4 Enterococcus faecalis clone, the sample is directly determined to be resistant to penicillins was applied. Therefore, the result was revised to A4, which were in 100%/agreement with the gold standard VITEK2 AST.

Example 4: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Streptococcus pneumoniae Based on LC-MS

[0120] Streptococcus pneumoniae was selected as the representative of the fastidious bacteria to illustrate the way a metabolic spectrum phylogenetic tree for drug susceptibility determination is constructed.

[0121] 1. Sample Preparation and Detection

[0122] Step 1. Drug susceptibility verification: The Streptococcus pneumoniae isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-GP68 cards, and specifically, the susceptibility of penicillin was double checked by disc diffusion method (OXOID, CT0043B), and the combined results were used as the gold standard (culture-based AST).

[0123] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Columbia sheep blood agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous bacterial suspension.

[0124] Step 3. Cell breakage: an equal volume of bacterial standards was added to 180 μL of bacterial suspension, and sonicated at 80 Hz for 5 min.

[0125] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0126] Step 5. Mass spectrometry detection: the residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0127] 2. Bioinformatics Analysis and Database Construction

[0128] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. The Streptococcus pneumoniae clones of different resistance profiles were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as: fold change>10, VIP>1, p value<0.05, CV<30%. The following 21 biomarkers were screened out:

TABLE-US-00009 No. Compound CP1 0.66_382.0899 m/z CP2 0.71_1355.4229 m/z CP3 0.72_166.0547 m/z CP4 0.72_295.0939 m/z CP5 0.85_1487.5082 m/z CP6 1.16_202.5253 m/z CP7 1.16_366.0977 m/z CP8 1.16_237.0537 m/z CP9 1.16_404.0425 m/z CP10 1.16_219.0439 m/z CP11 1.78_443.7582 m/z CP12 2.99_884.1424 m/z CP13 2.99_1317.2267 m/z CP14 2.98_1305.7357 m/z CP15 2.98_1306.2387 m/z CP16 2.99_871.1594 m/z CP17 2.99_870.8261 m/z CP18 2.99_878.8190 m/z CP19 2.97_1134.1406 m/z CP20 2.96_1275.6738 m/z CP21 2.26_1297.4318 m/z

[0129] (2) Resistance profile classification: According to the susceptibility properties of Streptococcus pneumoniae to 14 antibacterial drugs including penicillin, amoxicillin, cefepime, cefotaxime, ceftriaxone, ertapenem, meropenem, erythromycin, TMP-SMZ, levofloxacin, moxifloxacin, vancomycin, linezolid and tetracycline, its resistance profiles were classified into different types, named as A to S. The drug resistance profile classification and corresponding drug susceptibility are shown in Table 6.

TABLE-US-00010 TABLE 6 Resistance profile Susceptibility of Streptococcus pneumoniae to 14 antibacterial drugs classification penicillin ampicillin tetracycline A R R R R R R S S R S S S S R B R R R R R R S S R S S S S R C R R R R R R S S R S S S S S D R R R R R S S S R S S S S S E R R R R R R S S R S S S S S F R R R R R R S S S S S S S S G S S S S S S S S R S S S S S H S S S S S S S R S S S S S S I S S S S S S S S S S S S S S J S S S S S S S S S S S S S R S S S S S S S S S S S S S S S indicates data missing or illegible when filed

[0130] (3) Construction of a metabolic spectrum phylogenetic tree database for susceptibility determination: Through biomarker analysis, identical clones were merged and 18 representative Streptococcus pneumoniae clones were selected for the database. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated, as shown in FIG. 7.

[0131] 3. Database Validation and Result Interpretation

[0132] (1) Blind test: The metabolic fingerprints of 2 blinds were imported into the software IBM SPSS Statistics 23 for cluster analysis. The resistance profile of each sample was determined based on its positioning in the phylogenetic tree and the corresponding prediction rules. As is shown in FIG. 8, the branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where black represents blind samples.

[0133] (2) Result interpretation: The resistance profiles of 2 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. Use independent prediction rules: a) when the test sample contains metabolic fingerprints of a specific clone, follow the principle that the drug resistance profile predicted by metabolic fingerprints is preferred over phylogenetic tree interpretation; b) when the test sample has the metabolic fingerprints of a Streptococcus pneumoniae clone with altered penicillin-binding protein patterns, the sample is directly determined to be resistant to penicillins and cephalosporinase.

[0134] The resistance profiles of the 2 blinds to 14 antibacterial drugs including penicillin, amoxicillin, cefepime, cefotaxime, ceftriaxone, ertapenem, meropenem, erythromycin, TMP-SMZ, levofloxacin, moxifloxacin, vancomycin, linezolid and tetracycline predicted by method of the present invention are shown in Table 7.

TABLE-US-00011 TABLE 7 Resistance Resistance profiles by profiles Agree- VITEK2 inferred by Agreement ment (the gold phylogenetic Prediction Correc- (preliminary (final Sample standard) tree rules involved tions result) result) blind- G A contains corrected 5 False yes 1 penicillin to G negative biomarkers, (penicillins, preferred over cephalo- phylogenetic sporinase) tree blind- A A / / yes yes 2

[0135] When analyzed solely using the phylogenetic tree, out of 2 blinds, 1 samples and 5 antibiotics displayed inconsistent results compared with the gold standard VITEK2 AST. However, once the phylogenetic tree was combined with metabolic fingerprints and specific prediction rule was applied, the results were corrected. Since the blind 1 has metabolic fingerprints of a Streptococcus pneumoniae clone with altered penicillin-binding protein patterns, the prediction rule that when the test sample has the metabolic fingerprints of a Streptococcus pneumoniae clone with altered penicillin-binding protein patterns, the sample is directly determined to be resistant to penicillins and cephalosporinase was applied. Therefore, the result was revised to G, which were in 100% agreement with the gold standard VITEK2 AST.

Example 5: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Fungal Identification Based on LC-MS

[0136] 1. Sample Preparation and Detection

[0137] Step 1. Sample collection and identification: 420 clinical isolates were collected from 31 hospitals across china in a period between September 2015 and January 2019. All isolates were subjected to Sanger sequencing, as the gold standard for species identification.

[0138] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Chromogenic agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous fungal suspension.

[0139] Step 3. Cell breakage: An equal volume of fungal standards was added to 180 μL of fungal suspension, and sonicated at 80 Hz for 5 min.

[0140] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0141] Step 5. Mass spectrometry detection: The residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0142] 2. Bioinformatics Analysis and Database Construction

[0143] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. Fungi of different species were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as: fold change>10, VIP>1, p value<0.05, CV<30%. The following 72 biomarkers were screened out:

TABLE-US-00012 No. Compound CP1 0.53_312.6547 m/z CP2 0.53_455.2273 m/z CP3 0.53_462.7313 m/z CP4 0.53_601.3308 m/z CP5 0.53_639.3055 m/z CP6 0.53_651.3450 m/z CP7 0.53_675.3021 m/z CP8 0.53_783.4167 m/z CP9 0.53_797.4358 m/z CP10 0.55_1266.5582 m/z CP11 0.55_1284.5517 m/z CP12 0.55_1331.5422 m/z CP13 0.62_162.2207 m/z CP14 0.62_162.2590 m/z CP15 0.62_162.4624 m/z CP16 0.62_187.5477 m/z CP17 0.65_148.0955 m/z CP18 0.68_161.9788 m/z CP19 0.70_713.3483 m/z CP20 0.70_754.3701 m/z CP21 0.72_158.5687 m/z CP22 0.72_190.1422 m/z CP23 0.75_258.3427 m/z CP24 0.80_1315.4491 m/z CP25 0.88_1135.3840 m/z CP26 0.95_195.5753 m/z CP27 1.00_1354.3790 m/z CP28 1.02_371.0143 m/z CP29 1.02_599.0594 m/z CP30 2.25_472.9171 m/z CP31 2.44_611.9771 m/z CP32 2.46_254.2982 m/z CP33 2.46_531.9382 m/z CP34 2.46_588.6445 m/z CP35 2.46_598.6573 m/z CP36 2.46_882.9598 m/z CP37 2.48_102.0899 m/z CP38 2.48_407.5446 m/z CP39 2.49_318.5222 m/z CP40 2.49_318.8541 m/z CP41 2.49_391.9494 m/z CP42 2.49_734.0538 m/z CP43 2.50_417.9738 m/z CP44 2.54_643.9956 m/z CP45 2.54_647.5503 m/z CP46 2.56_1035.1450 m/z CP47 2.56_487.0010 m/z CP48 2.56_643.5541 m/z CP49 2.56_776.6147 m/z CP50 2.58_530.0289 m/z CP51 2.58_599.0957 m/z CP52 2.58_599.4973 m/z CP53 2.58_698.5924 m/z CP54 2.59_1032.1849 m/z CP55 2.59_758.0302 m/z CP56 2.60_453.5753 m/z CP57 2.63_1173.2365 m/z CP58 2.63_704.1436 m/z CP59 2.64_687.0079 m/z CP60 2.65_656.9775 m/z CP61 2.66_847.6572 m/z CP62 2.75_912.0944 m/z CP63 2.78_774.1840 m/z CP64 2.79_541.6204 m/z CP65 2.81_780.7309 m/z CP66 2.82_261.0359 m/z CP67 2.82_305.0075 m/z CP68 2.82_307.0062 m/z CP69 2.83_1014.1838 m/z CP70 2.44_569.9670 m/z CP71 2.44_853.9486 m/z CP72 0.62_162.1116 m/z

[0144] (2) Construction of a metabolic spectrum phyogenetic tree database: To optimize the resolution of the database, closely-related species such as Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis which could not be accurately distinguished by the automated identification system such as VITEK2, were selected and added to the database.

[0145] Through biomarker analysis, identical clones were merged and 115 representative clones were selected for the database. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated, as shown in FIGS. 9A-9F.

[0146] 3. Database Validation and Result Interpretation

[0147] (1) Blind test: The metabolic fingerprints of 8 blinds were imported into the software 11BM SPSS Statistics 23 for cluster analysis. The species of each blind was determined based on its positioning in the phylogenetic tree. As is shown in FIGS. 10A-10F, the branches of the phylogenetic tree representing different species are indicated by different colors on the left, where black represents blind samples.

[0148] (2) Result interpretation: The species of 8 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. As is shown in Table 8, identification results predicted by method of this invention were in a 100% agreement with the gold standard (sequencing method) result. Notably, closely relates species such as Candida parapsilosis and Candida metapsilosis, which can not be distinguished by automated identification systems such as VITEK2, were identified in complete agreement with the gold standard method.

TABLE-US-00013 TABLE 8 result by method result by gold standard sample of this invention (sequencing) agreement blind-1 Candida parapsilosis Candida parapsilosis yes blind-2 Candida metapsilosis Candida metapsilosis yes blind-3 Candida tropicalis Candida tropicalis yes blind-4 Candida tropicalis Candida tropicalis yes blind-5 Candida albicans Candida albicans yes blind-6 Candida albicans Candida albicans yes blind-7 Candida albicans Candida albicans yes blind-8 Candida albicans Candida albicans yes

[0149] The prediction results of the 8 blind samples based on the phylogenetic tree positioning analysis of the metabolic profile database were in 100% agreement with those of the gold standard (sequencing method). Particularly, the results of blind 1 and blind 2 revealed that the resolution of the present method for fungal identification was at the subspecies level.

Example 6: Construction and Validation of a Metabolic Spectrum Phylogenetic Tree Database for Candida tropicalis Based on LC-MS

[0150] Candida tropicalis is the second most common pathogen of the invasive fungal infection after Candida albicans, and its triazole resistance rate is much higher than that of Candida albicans (30% vs 5%), and thus the prediction of resistance is more valuable in Candida tropicalis. Therefore, Candida tropicalis was selected as the representative of fungi to illustrate the way a metabolic spectrum phylogenetic tree for drug susceptibility determination is constructed. Those skilled in the art can understand that this method is applicable to other Candida spp. or other yeasts.

[0151] 1. Sample Preparation and Detection

[0152] Step 1. Drug susceptibility verification: The Candida tropicalis isolates were tested for drug susceptibility using broth microdilution method following the M27-A3 and M27-S4 guidelines of NCCLS, and the results were used as the gold standard (culture-based AST).

[0153] Step 2. Preparation of bacterial suspension: Each test isolate was inoculated on Chromogenic agar plate, and after culturing overnight, an appropriate amount of colonies was scraped into a sterile saline tube with a disposable inoculation loop to prepare a homogeneous fungal suspension.

[0154] Step 3. Cell breakage: an equal volume of fungal standards was added to 180 μL of bacterial suspension, and sonicated at 80 Hz for 5 min.

[0155] Step 4. Extraction and concentration of metabolites: 340 μL of the sonication product obtained in step 3 was transferred to a 1.5 ml centrifuge tube, mixed with an equal volume of extraction buffer, shaked on ice for 3 min, and centrifuged quickly for 3-5 seconds. The liquid in the tube was spin down to the bottom of the tube and dried using a nitrogen blower.

[0156] Step 5. Mass spectrometry detection: the residue obtained in step 4 was resuspended with 140 μL of resuspension buffer, vortexed, centrifuged at high speed for 5 min. The supernatant was transferred to a new centrifuge tube, and centrifuged at high speed for 5 min. 100 μL of the supernatant was transferred to the sample introduction system of LC-MS, and 4 μL of the sample was injected each run for detection and analysis; preferably, the high-resolution LC-MS is Waters Q-TOF Synapt G2-Si quadrupole-time-of-flight mass spectrometer. The retention was achieved in gradient elution reversed-phase chromatography under the conditions as follows: water-acetonitrile-formic acid was used as the mobile phase system, the flow rate of the mobile phase was 0.4 ml/min, and the column temperature was 40° C.; the chromatographic column was a Waters HSS T3 column with a particle size of 1.8 μm, an inner diameter of 2.1 mm, and a column length of 100 mm; Mass spectrometry detection adopts electrospray ionization source (ESI), positive ion mode, multiple reaction monitoring scan mode (MRM) and MSeContinnum data independent acquisition mode.

[0157] 2. Bioinformatics Analysis and Database Construction

[0158] (1) Biomarker screening: Progenesis QI software was used to generate peak alignment, peak extraction, compound identification, and normalization on the raw data collected by mass spectrometry, and output feature information such as mass-to-charge ratio (m/z), retention time, and abundance. The Enterococcus faecalis clones of different resistance profiles were categorized into different groups for principal component analysis, with parameters for screening biomarkers set as: fold change>10, VIP>1, p value<0.05, CV<30%. The following 22 biomarkers were screened out:

TABLE-US-00014 No. Compound CP1 0.88_1135.3840 m/z CP2 0.95_195.5753 m/z CP3 1.00_1354.3790 m/z CP4 1.02_371.0143 m/z CP5 1.02_599.0594 m/z CP6 2.25_472.9171 m/z CP7 2.44_611.9771 m/z CP8 2.46_254.2982 m/z CP9 2.46_531.9382 m/z CP10 2.46_588.6445 m/z CP11 2.46_598.6573 m/z CP12 2.46_882.9598 m/z CP13 2.48_102.0899 m/z CP14 2.48_407.5446 m/z CP15 2.49_318.5222 m/z CP16 2.49_318.8541 m/z CP17 2.49_391.9494 m/z CP18 2.49_734.0538 m/z CP19 2.50_417.9738 m/z CP20 2.54_643.9956 m/z CP21 2.54_647.5503 m/z CP22 2.56_1035.1450 m/z

[0159] (2) Resistance profile classification: Since 5-flucytosine, amphotericin B and echinocandin-resistance is rarely observed in clinical isolates of Candida tropicalis, its resistant profiles are categorized into two types: azole-resistant (pan-resistant to triazoles, e.g., fluconazole, itraconazole, voriconazole) and azole-susceptible.

[0160] (3) Construction of a metabolic spectrum phylogenetic tree database for susceptibility determination: Through biomarker analysis, identical clones were merged and 60 representative Candida tropicalis clones were selected for the database, among which 27 were azole-resistant and 33 were azole-susceptible. The feature information of the biomarkers (retention time, mass-to-charge ratio, abundance) was imported into the software IBM SPSS Statistics 23, the retention time and mass-to-charge ratio information used as variable names, the compound abundance used as variable value. Using the analysis mode of ‘systematic cluster’, a dendrogram was generated.

[0161] 3. Database Validation and Result Interpretation

[0162] (1) Blind test: The metabolic fingerprints of 6 blinds were imported into the software IBM SPSS Statistics 23 for cluster analysis. The species of each blind was determined based on its positioning in the phylogenetic tree. As is shown in FIGS. 11A-11C, the branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the left, where blue indicates azole-susceptible, yellow indicates azole-resistant, and black represents blind samples.

[0163] (2) Result interpretation: The resistance profiles of 6 blinds were predicted based on their positioning in the phylogenetic tree and the corresponding prediction rules. Use independent prediction rules: strictly follow the principle that the drug resistance profile of a fungal strain is inferred on the basis of its closest relatives in the metabolic spectrum phylogenetic tree.

[0164] The resistance profiles of the 6 blinds to 6 antifungal drugs including 5-flucytosine, amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin predicted by method of the present invention are shown in Table 9.

TABLE-US-00015 TABLE 9 Resistance profiles inferred by Resistance profiles by broth microdilution branch phylogenetic (the gold standard) Agree- location tree 5-FC AMB FLU ITR VOR CAS ment blind- yellow pan-azole S S R R R S yes 1 resistant blind- blue susceptible to S S S S S S yes 2 all drugs blind- yellow pan-azole S S R R R S yes 3 resistant blind- yellow pan-azole S S R R R S yes 4 resistant blind- blue susceptible to S S S S S S yes 5 all drugs blind- blue susceptible to S S S S S S yes 6 all drugs

[0165] The prediction results of the 6 blind samples based on the phylogenetic tree positioning analysis of the metabolic profile database were in 100% agreement with those of broth microdilution (the gold standard).

Example 7: Construction and Validation of a Genomic Phylogenetic Tree Database for Klebsiella pneumoniae Based on WGS

[0166] Klebsiella pneumoniae was selected as the representative of Enterobacteriaceae to illustrate the way a genomic phylogenetic tree for drug susceptibility determination is constructed. Other Enterobacteriaceae can refer to this method for library construction and analysis.

[0167] 1. Sample Preparation and Detection

[0168] Step 1. Sample collection and drug susceptibility verification: 240 clinical Klebsiella pneumoniae isolates were collected from 23 hospitals across china in a period between January 2018 and March 2019. All isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-GN13/GN334 cards, and specifically, and the results were used as the gold standard (culture-based AST).

[0169] Step 2. Genomic DNA preparation: Strains were inoculated on Columbia sheep blood agar plates by streaking and placed in an incubator (37° C.) for 24 hours. The bacterial precipitate was then collected by centrifugation at 10,000 rpm for 2 mins, and the genomic DNA was purified from the pellets using DNeasy Blood and Tissue kit following the protocol by the manufacturer. DNA concentration was measured using Qubit fluorometer and the QC was performed on a NanoDrop™ spectrophotometer, DNA with OD 260/280 1.6-2.0 and 260/230 2.0-2.2 were accepted for nanopore sequencing library preparation.

[0170] Step 3. Library preparation: ONT Native Barcoding Kit 1D (EXP-NBD104 & 114) and Ligation Sequencing Kit 1D (SQK LSK109) were used in library preparation following the 1D Native barcoding genomic DNA protocol developed by ONT with a few modifications. The procedure is briefly described below, DNA extracted from each isolate were quantified on a Qubit 3.0 fluorometer and diluted to 20ng/μl with nuclease-free water (NF water), 50 μl diluted DNA was used as starting material and incubated with 7 μl Ultra II End-Prep reaction buffer and 3 μl Ultra II End-Prep enzyme mix (New England Biolabs, USA) for 5 minutes at 20° C. follow by 5 minutes at 65′C. The end-prepped DNA was then purified from the reaction mix using 1×(v/v) AMPure XP magnetic beads and eluted with 25 μl NF water. After quantification, elution containing 500ng DNA was obtained from each sample and topped up to 22.5 μl with NF water and mixed with 2.5 μl unique Native Barcode and 25 μl Blunt/TA Ligation Master Mix. The mixture was incubated at room temperature for 10 min and purified with 1×(v/v) AMPure XP magnetic beads, and the barcoded DNA was eluted with 26 μl NF water. In the final step, equal amounts of 12 individually barcoded DNA samples were pooled to 700ng in total, and extra NF water were added to 50 μl final volume. To the pooled DNA, 20 μl ONT Barcode Adapter Mix, 20 μl NEBNext Quick Ligation Reaction Buffer (5×) and 10 μl Quick T4 DNA Ligase was added in order and mixed thoroughly, and after 10 minutes incubation at room temperature, the BAM ligated DNA was purified from the reaction mix with 0.4×(v/v) AMPure XP magnetic beads, and eluted in 15 μl NF water.

[0171] Step 4. Nanopore sequencing: The sequencing consumable used in this study was ONT flowcell FLO-MIN106 R9.4. After flowcell priming, 75 μL prepared library (35 μL Running buffer, 25.5 μL loading beads, and 14.5 μL pooled library) was loaded. Sequencing was performed on an ONT MinION™ portable sequencing device, and set and monitored using ONT MinKNOW™ desk software. Samples were pooled and sequenced for about 6 hours or until 1 Gb of data for each sample were generated for database isolates and clinical specimens, respectively.

[0172] Step 5. Bioinformatics analysis: Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software. And the CANU 2.0 software was used to assemble the reads in fastq files into genomic assemblies or contigs with default parameters. The assembled genomes were analyzed using the local antibiotic resistance database for resistance determinants identification. Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for the identification of pathogens present in the sample and reads classified as Klebsiella pneumoniae were extracted for further resistance analysis. A local nBLAST tool was created for interrogating contigs of clinical samples for a panel of validated AMR determinants. Phylogenetic analysis was conducted with kSNP3 (version 3.1) based on pan-genome SNPs identified and a Kmer value of 31 was adopted.

[0173] 2. Database Construction and Analytical Logic Design

[0174] (1) Construction of a genomic phylogenetic tree: Through biomarker analysis, identical clones were merged and 165 representative Klebsiella pneumonia clones were selected for the database and a genomic phylogenetic tree database was built as shown in FIGS. 12A to 12G. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where yellow indicates ST11 clone, red indicates ST15 clone, and blue indicates susceptible to all antibiotics (including ST23 clone).

[0175] (2) Analytical Logic Design

[0176] (2a) Species identification: When the genome assembly size of the test sample is within the range of 5,200,000-5,600,000 bp, fragments of different lengths from multiple sites in the genome were selected to BLAST against the NCBI nucleotide database. The species of the pathogen is determined as Klebsiella pneumoniae only when the strain description shows Klebsiella pneumoniae and the per identity value exceeds 98%.

[0177] (2b) Drug susceptibility determination: When the test sample is located in the ST11 cluster (the yellow branch in FIGS. 12A to 12G) of the genomic phylogenetic tree, its resistance profiles to 23 antibacterial drugs including ampicillin-sulbactam, piperacillin-tazobactam, cefoperazone-sulbactam, amoxicillin-clavulanate, cefazolin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime, aztreonam, cefoxitin, cefotetan, meropenem, ertapenem, imipenem, gentamicin, tobramycin, amikacin, ciprofloxacin, levofloxacin, tetracycline and TMP-SMZ is inferred according to the following ST11-type interpretation rules, that is, the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the test sample is directly determined to be resistant to cephalosporins, cephamycins, carbapenems and quinolones. The drug species and antimicrobial resistance determinants used in this study are shown in Table 10.

TABLE-US-00016 TABLE 10 Resistance profiles Antimicrobial resistance inferred by determinants (partial) phylogenetic aac(6')- aph(3')- sul1/ Antibiotics tree rmtB Ib3 Ia 2/3 tet(A) Ampicillin- R sulbactam Piperacillin- R tazobactam Cefoperazone- R sulbactam Amoxicillin- R clavulanate Cefazolin R Cefuroxime R Cefotaxime R Ceftriaxone R Ceftazidime R Cefepime R Aztreonam R Cefoxitin R Cefotetan R Meropenem R Ertapenem R Imipenem R Gentamicin R R R Tobramycin R R S Amikacin R S S Ciprofloxacin R Levofloxacin R Tetracycline R TMP-SMZ R

[0178] When the test sample is located out of the ST11 cluster, its resistance profiles to 23 antibacterial drugs is inferred according to the following non-STI11-type interpretation rules, that is, the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, wherein the ST15-, ST23-type interpretation rules are detailed in Table 11.

TABLE-US-00017 TABLE 11 ST23 ST15 phylo- phylo- Antimicrobial resistance determinants genetic genetic CTX sul1 tree tree & aac(6′)- aph(3′)- sul2 Antibiotics inferrence inferrence TEM CTX DHA KPC rmtB IB3 Ia QnrS QRDR sul3 tetA Ampicillin- S R R R R sulbactam Piperacillin- S R R tazobactam Cefoperazone- S R R sulbactam Amoxicillin- S R R R R clavulanate Cefazolin S R R R R Cefuroxime S R R R R Cefotaxime S R R R R Ceftriaxone S R R R R Ceftazidime S R R R Cefepime S R R R Aztreonam S R R R R Cefoxitin S R R R Cefotetan S R R R Meropenem S R R Ertapenem S R R Imipenem S R R Gentamicin R R R Tobramycin R R S Amikacin R S S Ciprofloxacin R R R Levofloxacin R R R Tetracycline R TMP-SMZ R

[0179] 9. Database Validation and Result Interpretation

[0180] (1) Blind test: The genome-wide SNPs loci of library Klebsiella pneumoniae and 24 blinds were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. As is shown in FIGS. 12A to 12G, there were 10 blinds attributed to the ST11 cluster, and 14 blinds belonged to the non-ST 11 clusters, including 1 case of ST 15 type, 2 cases of ST23 type, and 11 cases of other ST types. The resistance profiles of each blind were determined according to the prediction rules of ST11, ST15, ST23, and non-ST 11-ST15-ST23 types, respectively. 7 blinds were identified as susceptible to all antibiotics involved in this study, and none of them contained any antimicrobial resistance determinants; the rest were pandrug-resistant or multidrug resistant.

[0181] (2) Result interpretation: Based on the positioning in the phylogenetic tree, the antimicrobial resistance determinants and the corresponding prediction rules, the resistance profiles of 24 blinds against 23 antibiotics were determined and compared with the gold standard VITEK2 AST result, as shown in Table 12.

TABLE-US-00018 TABLE 12 Resistance Resistance profiles profiles by the Positive Negative by present predictive predictive VITEK2 method value value Antibiotics R S R S (PPV) (NPV) Sentitivity Specificity Ampicillin- 15 9 15 9 100.00% 100.00% 100.00% 100.00% sulbactam Piperacillin- 13 11 12 12 100.00% 91.67% 92.31% 100.00% tazobactam Cefoperazone- 13 11 12 12 100.00% 91.67% 92.31% 100.00% sulbactam Amoxicillin- 16 8 16 8 100.00% 100.00% 100.00% 100.00% clavulanate Cefazolin 15 9 15 9 100.00% 100.00% 100.00% 100.00% Cefuroxime 15 9 15 9 100.00% 100.00% 100.00% 100.00% Cefotaxime 15 9 15 9 100.00% 100.00% 100.00% 100.00% Ceftriaxone 15 9 15 9 100.00% 100.00% 100.00% 100.00% Ceftazidime 13 11 12 12 100.00% 91.67% 92.31% 100.00% Cefepime 13 11 12 12 100.00% 91.67% 92.31% 100.00% Aztreonam 15 9 15 9 100.00% 100.00% 100.00% 100.00% Cefoxitin 12 12 12 12 100.00% 100.00% 100.00% 100.00% Cefotetan 12 12 12 12 100.00% 100.00% 100.00% 100.00% Meropenem 12 12 12 12 100.00% 100.00% 100.00% 100.00% Ertapenem 12 12 12 12 100.00% 100.00% 100.00% 100.00% Imipenem 12 12 12 12 100.00% 100.00% 100.00% 100.00% Gentamicin 12 12 12 12 100.00% 100.00% 100.00% 100.00% Tobramycin 9 15 9 15 100.00% 100.00% 100.00% 100.00% Amikacin 9 15 9 15 100.00% 100.00% 100.00% 100.00% Ciprofloxacin 14 10 15 9 93.33% 100.00% 100.00% 90.00% Levofloxacin 14 10 15 9 93.33% 100.00% 100.00% 90.00% Tetracycline 11 13 11 13 100.00% 100.00% 100.00% 100.00% TMP-SMZ 11 13 10 14 100.00% 92.86% 90.91% 100.00%

[0182] Among the 24 blinds, 1 case of false negative was found against piperacillin/tazobactam, cefoperazone/sulbactam, ceftazidime, cefpiramide, trimethoprim/sulfamethoxazole, respectively, and 1 case of false positive was found against ciprofloxacin and levofloxacin, respectively. In total, 5 false negative results and 2 false positive results were found, counting up 23 antibiotics (552 susceptibility results). When compared with the gold standard VI TEK2 AST, the positive predictive value, negative predictive value, sensitivity and specificity of the present method was demonstrated to be 99.32% (293/295), 98.05% (252/257), 98.32% (293/298), and 99.21% (252/254), respectively. The performances meet the design requirements of the present method and the needs of clinical application, that is, the sensitivity and specificity being above 95%.

Example 8: Construction and Validation of a Genomic Phylogenetic Tree Database for Staphylococcus aureus Based on WGS

[0183] Staphylococcus aureus was selected as the representative of Gram-positive cocci to illustrate the way a genomic phylogenetic tree for drug susceptibility determination is constructed. Other gram-positive cocci such as Coagulase-Negative Staphylococcus, Enterococcus faecalis can refer to this method for library construction and analysis.

[0184] 1. Sample Preparation and Detection

[0185] Step 1. Sample collection and drug susceptibility verification: 160 clinical Staphylococcus aureus isolates were collected from 20 hospitals across china in a period between June 2018 and June 2019. All isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-P639 card, and specifically, and the results were used as the gold standard (culture-based AST).

[0186] Step 2. Genomic DNA preparation: Strains were inoculated on Columbia sheep blood agar plates by streaking and placed in an incubator (37° C.) for 24 hours. The bacterial precipitate was then collected by centrifugation at 10,000 rpm for 2 mins, and incubated with lysozyme at a final concentration of 20 mg/mL at 37° C. for 30-60 min. The genomic DNA was purified from the pellets using DNeasy Blood and Tissue kit following the protocol by the manufacturer. DNA concentration was measured using Qubit fluorometer and the QC was performed on a NanoDrop™ spectrophotometer, DNA with OD 260/280 1.6-2.0 and 260/230 2.0-2.2 were accepted for nanopore sequencing library preparation.

[0187] Step 3. Library preparation: ONT Native Barcoding Kit 1D (EXP-NBD104 & 114) and Ligation Sequencing Kit 1D (SQK LSK109) were used in library preparation following the 1D Native barcoding genomic DNA protocol developed by ONT with a few modifications. The procedure is briefly described below, DNA extracted from each isolate were quantified on a Qubit 3.0 fluorometer and diluted to 20ng/μl with nuclease-free water (NF water), 50 μl diluted DNA was used as starting material and incubated with 7 μl Ultra II End-Prep reaction buffer and 3 μl Ultra II End-Prep enzyme mix (New England Biolabs, USA) for 5 minutes at 20° C. follow by 5 minutes at 65° C. The end-prepped DNA was then purified from the reaction mix using 1×(v/v) AMPure XP magnetic beads and eluted with 25 μl NF water. After quantification, elution containing 500ng DNA was obtained from each sample and topped up to 22.5 μl with NF water and mixed with 2.5 μl unique Native Barcode and 25 μl Blunt/TA Ligation Master Mix. The mixture was incubated at room temperature for 10 min and purified with 1×(v/v) AMPure XP magnetic beads, and the barcoded DNA was eluted with 26 μl NF water. In the final step, equal amounts of 12 individually barcoded DNA samples were pooled to 700ng in total, and extra NF water were added to 50 μl final volume. To the pooled DNA, 20 μl ONT Barcode Adapter Mix, 20 μl NEBNext Quick Ligation Reaction Buffer (5×) and 10 μl Quick T4 DNA Ligase was added in order and mixed thoroughly, and after 10 minutes incubation at room temperature, the BAM ligated DNA was purified from the reaction mix with 0.4×(v/v) AMPure XP magnetic beads, and eluted in 15 μl NF water.

[0188] Step 4. Nanopore sequencing: The sequencing consumable used in this study was ONT flowcell FLO-MIN106 R9.4. After flowcell priming, 75 μL prepared library (35 μL Running buffer, 25.5 μL loading beads, and 14.5 μL pooled library) was loaded. Sequencing was performed on an ONT MinION™ portable sequencing device, and set and monitored using ONT MinKNOW™ desk software. Samples were pooled and sequenced for about 6 hours or until 1 Gb of data for each sample were generated for database isolates and clinical specimens, respectively.

[0189] Step 5. Bioinformatics analysis: Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software. And the CANU 2.0 software was used to assemble the reads in fastq files into genomic assemblies or contigs with default parameters. The assembled genomes were analyzed using the local antibiotic resistance database for resistance determinants identification. Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for the identification of pathogens present in the sample and reads classified as Staphylococcus aureus were extracted for further resistance analysis. A local nBLAST tool was created for interrogating contigs of clinical samples for a panel of validated AMR determinants. Phylogenetic analysis was conducted with kSNP3 (version 3.1) based on pan-genome SNPs identified and a Kmer value of 31 was adopted.

[0190] 2. Database Construction and Analytical Logic Design

[0191] (1) Construction of a genomic phylogenetic tree: Through biomarker analysis, identical clones were merged and 93 representative Staphylococcus aureus clones were selected for the database and a genomic phylogenetic tree database was built as shown in FIGS. 13A-13D. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right.

[0192] (2) Analytical Logic Design

[0193] (2a) Species identification: When the genome assembly size of the test sample is within the range of 2,400,000-3,000,000 bp, fragments of different lengths from multiple sites in the genome were selected to BLAST against the NCBI nucleotide database. The species of the pathogen is determined as Staphylococcus aureus only when the strain description shows Staphylococcus aureus and the per identity value exceeds 98%.

[0194] (2b) Drug susceptibility determination: When the test sample is located in the CC5mecA+ or ST59mecA+ clusters, follow the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the sample is directly determined to be resistant to penicillin, oxacillin, cefoxitin and quinolones. When the test sample is located in the CC5mecA− or ST22mecA− clusters, follow the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the sample is directly determined to be susceptible to penicillin, oxacillin, and cefoxitin. When the test sample is located in the susceptibility branches of the genomic phylogenetic tree, follow the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the sample is directly determined to be susceptible to all antibiotics involved in this study. When the test sample is located in branches outside the above designated regions, the drug resistance profile is determined solely by antimicrobial resistance determinants.

[0195] The drug species and antimicrobial resistance determinants used in this study are shown in Table 13.

TABLE-US-00019 TABLE 13 Pen- Oxacillin, Quin- Macro- Clinda- TMP- Line- Van- Teico- Tetra- Rifam- Genta- icillin Cefoxitin olones lides mycin ICR SMZ zolid comycin planin cyclicnes picin micin Results CC5mecA+ R R R by CC5mecA− S S phylogenetic ST59mecA+ R R R tree ST22mecA− S S S S S S S S S S S S S S S S Results blaZ R by mecA R R anti- gyrA/ R microbial parC resistance determinants ermA R R R ermB ermC R R mphC/ R msrA dfrG R 23srRNA R vanA R R tetL/ R tetM/ tetK rpoB R AAC6-Ie- R APH2-Ia

[0196] 3. Database Validation and Result Interpretation

[0197] (1) Blind test: The genome-wide SNPs loci of library Staphylococcus aureus and 22 blinds were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. As is shown in FIGS. 13A-13D, there were 3 blinds attributed to the CC5mecA+ cluster, 2 blinds belonged to the CC5mecA− cluster, 4 blinds belonged to the ST59mecA+ cluster, and 5 blinds belonged to the S cluster. The resistance profiles of each blind were determined according to the prediction rules of CC5mecA+, CC5mecA−, ST59mecA+, and S types, respectively, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis.

[0198] (2) Result interpretation: Based on the positioning in the phylogenetic tree, the antimicrobial resistance determinants and the corresponding prediction rules, the resistance profiles of 22 blinds against 20 antibiotics were determined and compared with the gold standard VITEK2 AST result, as shown in Table 14 (For antibiotics without relevant drug-resistant samples, the positive predictive value and sensitivity are not counted).

TABLE-US-00020 TABLE 14 Resistance profiles by Resistance the Positive Negative profiles by present predictive predictive VITEK2 method value value Antibiotics R S R S (PPV) (NPV) Sentitivity Specificity Penicillin 20 2 20 2 100.00% 100.00% 100.00% 100.00% Oxacillin, 8 14 8 14 100.00% 100.00% 100.00% 100.00% Cefoxitin Quinolones 6 16 6 16 100.00% 100.00% 100.00% 100.00% Macrolides 15 7 14 8 100.00% 87.50% 93.33% 100.00% Clindamycin 10 12 9 13 100.00% 92.31% 90.00% 100.00% ICR 15 7 14 8 100.00% 87.50% 93.33% 100.00% Cefazolin 0 22 0 22 — 100.00% — 100.00% Vancomycin 0 22 0 22 — 100.00% — 100.00% Teicoplanin 0 22 0 22 — 100.00% — 100.00% Linezolid 0 22 0 22 — 100.00% — 100.00% TMP-SMZ 5 17 5 17 100.00% 100.00% 100.00% 100.00% Tetracycline 6 16 6 16 100.00% 100.00% 100.00% 100.00% Tigecycline 0 22 0 22 — 100.00% — 100.00% Rifampicin 1 21 1 21 100.00% 100.00% 100.00% 100.00% Gentamicin 7 15 7 15 100.00% 100.00% 100.00% 100.00%

[0199] Among the 22 blinds, 1 case of false negative was found against macrolides (erythromycin, clarithromycin, azithromycin), and lincosamides (clindamycin, ICR); no false positive case was found. In total, 5 false negative results and were found, counting up 20 antibiotics (440 susceptibility results). When compared with the gold standard VITEK2 AST, the positive predictive value, negative predictive value, sensitivity and specificity of the present method was demonstrated to be 100.00% (138/138), 98.34% (297/302), 96.50% (138/143), and 100.00% (297/297), respectively. The performances meet the design requirements of the present method and the needs of clinical application, that is, the sensitivity and specificity being above 95%.

Example 9: Construction and Validation of a Genomic Phylogenetic Tree Database for Streptococcus pneumoniae Based on WGS

[0200] Streptococcus pneumoniae was selected as the representative of the fastidious bacteria to illustrate the way a genomic phylogenetic tree for drug susceptibility determination is constructed.

[0201] 1. Sample Preparation and Detection

[0202] Step 1. Sample collection and drug susceptibility verification: 48 clinical Streptococcus pneumoniae isolates were collected from 18 hospitals across china in a period between May 2017 and October 2019. All isolates were tested for drug susceptibility using bioMérieux Vitek2 Compact System and AST-GP68 card, and specifically, the susceptibility of penicillin was double checked by disc diffusion method (OXOID, CT0043B), and the combined results were used as the gold standard (culture-based AST).

[0203] Step 2. Genomic DNA preparation: Strains were inoculated on Columbia sheep blood agar plates by streaking and placed in an incubator (37° C.) for 24 hours. The bacterial precipitate was then collected by centrifugation at 10,000 rpm for 2 mins, and incubated with lysozyme at a final concentration of 20 mg/mL at 37° C. for 30-60 min. The genomic DNA was purified from the pellets using DNeasy Blood and Tissue kit following the protocol by the manufacturer. DNA concentration was measured using Qubit fluorometer and the QC was performed on a NanoDrop™ spectrophotometer, DNA with OD 260/280 1.6-2.0 and 260/230 2.0-2.2 were accepted for nanopore sequencing library preparation.

[0204] Step 3. Library preparation: ONT Native Barcoding Kit 1D (EXP-NBD104 & 114) and Ligation Sequencing Kit 1D (SQK LSK109) were used in library preparation following the 1D Native barcoding genomic DNA protocol developed by ONT with a few modifications. The procedure is briefly described below, DNA extracted from each isolate were quantified on a Qubit 3.0 fluorometer and diluted to 20ng/μl with nuclease-free water (NF water), 50 μl diluted DNA was used as starting material and incubated with 7 μl Ultra II End-Prep reaction buffer and 3 μl Ultra II End-Prep enzyme mix (New England Biolabs, USA) for 5 minutes at 20° C. follow by 5 minutes at 65° C. The end-prepped DNA was then purified from the reaction mix using 1×(v/v) AMPure XP magnetic beads and eluted with 25 μl NF water. After quantification, elution containing 500ng DNA was obtained from each sample and topped up to 22.5 μl with NF water and mixed with 2.5 μl unique Native Barcode and 25 μl Blunt/TA Ligation Master Mix. The mixture was incubated at room temperature for 10 min and purified with 1×(v/v) AMPure XP magnetic beads, and the barcoded DNA was eluted with 26 μl NF water. In the final step, equal amounts of 12 individually barcoded DNA samples were pooled to 700ng in total, and extra NF water were added to 50 μl final volume. To the pooled DNA, 20 μl ONT Barcode Adapter Mix, 20 μl NEBNext Quick Ligation Reaction Buffer (5×) and 10 μl Quick T4 DNA Ligase was added in order and mixed thoroughly, and after 10 minutes incubation at room temperature, the BAM ligated DNA was purified from the reaction mix with 0.4×(v/v) AMPure XP magnetic beads, and eluted in 15 μl NF water.

[0205] Step 4. Nanopore sequencing: The sequencing consumable used in this study was ONT flowcell FLO-MIN106 R9.4. After flowcell priming, 75 μL prepared library (35 μL Running buffer, 25.5 μL loading beads, and 14.5 μL pooled library) was loaded. Sequencing was performed on an ONT MinION™ portable sequencing device, and set and monitored using ONT MinKNOW™ desk software. Samples were pooled and sequenced for about 6 hours or until 1 Gb of data for each sample were generated for database isolates and clinical specimens, respectively.

[0206] Step 5. Bioinformatics analysis: Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software. And the CANU 2.0 software was used to assemble the reads in fastq files into genomic assemblies or contigs with default parameters. The assembled genomes were analyzed using the local antibiotic resistance database for resistance determinants identification. Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for the identification of pathogens present in the sample and reads classified as Streptococcus pneumoniae were extracted for further resistance analysis. A local nBLAST tool was created for interrogating contigs of clinical samples for a panel of validated AMR determinants. Phylogenetic analysis was conducted with kSNP3 (version 3.1) based on pan-genome SNPs identified and a Kmer value of 31 was adopted.

[0207] 2. Database Construction and Analytical Logic Design

[0208] (1) Construction of a genomic phylogenetic tree: Through biomarker analysis, identical clones were merged and 25 representative Streptococcus pneumoniae clones were selected for the database and a genomic phylogenetic tree database was built as shown in FIG. 14. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where yellow indicates susceptible to all antibiotics, blue indicates penicillin-resistant, and black represents blind samples.

[0209] (2) Analytical Logic Design

[0210] (2a) Species identification: When the genome assembly size of the test sample is within the range of 2,000,000-2,300,000 bp, fragments of different lengths from multiple sites in the genome were selected to BLAST against the NCBI nucleotide database. The species of the pathogen is determined as Streptococcus pneumoniae only when the strain description shows Streptococcus pneumoniae and the per identity value exceeds 98%.

[0211] (2b) Drug susceptibility determination: When the test sample is located in the PEN-R cluster, follow the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the sample is directly determined to be resistant to penicillin and cephalosporins. When the test sample is located in the S branch of the genomic phylogenetic tree, follow the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis, and the sample is directly determined to be susceptible to all antibiotics involved in this study. When the test sample is located in branches outside the above designated regions, the drug resistance profile is determined solely by antimicrobial resistance determinants.

[0212] The drug species and antimicrobial resistance determinants used in this study are shown in Table 15.

TABLE-US-00021 TABLE 15 Peni- Amox- Cefe- Cefo- Ceftri- Erta- Mero- Erythro- TMP- Levo- Moxi- Vanco- Line- Tetra- cillin icillin pime taxime axone penem penem mycin SMZ floxacin floxacin mycin zolid cyclines Results PEN-R R R R R R by S S S S S S S S S S S S S S S phylo- genetic tree Results pbp2x R R R R R by pbp1a R R R R R anti- pbp2b R R R R R microbial erMA/B/C R resistance IsaA/E R determinants dfr R tetL/tetM R 23srRNA R gyrA/parC R R

[0213] 3. Database Validation and Result Interpretation

[0214] (1) Blind test: The genome-wide SNPs loci of library Streptococcus pneumoniae and 2 blinds were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. As is shown in FIG. 14, the blind 1 was belonged to neither PEN-R nor S cluster, and its drug resistance profile is determined solely by antimicrobial resistance determinants; the blind 2 was located in the PEN-R cluster, and its drug resistance profile is directly determined to be resistant to penicillin and cephalosporins, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis.

[0215] (2) Result interpretation: Based on the positioning in the phylogenetic tree, the antimicrobial resistance determinants and the corresponding prediction rules, the resistance profiles of 2 blinds against 14 antibiotics were determined and compared with the gold standard VITEK2 AST result, as shown in Table 16.

TABLE-US-00022 TABLE 16 cefepime, cefotaxime, mero- erythro- TMP- vanco- tetra- levofloxacin, Sample penicillin ceftriaxone ertapenem penem mycin SMZ mycin linezolid cycline moxifloxacin Blind 1 ermB dfr tetM by this S S S S R R S S R S method Blind 1 S S S S R R S S R S by AST blind 2 PEN-R PEN-R ermB dfr tetM by this R R S S R R S S R S method blind 2 R R S S R R S S R S by AST

[0216] By analyzing the sample positioning in the phylogenetic tree, combined with antimicrobial resistance determinants and specific prediction rules, the susceptibility of the two blinds inferred by this present method showed a 100% agreement with the gold standard VITEK2 AST result.

Example 10: Construction and Validation of a Genomic Phylogenetic Tree Database for Candida albicans Based on WGS

[0217] Candida albicans was selected as the representative of fungi to illustrate the way a genomic phylogenetic tree for drug susceptibility determination is constructed. Other Candida spp. or yeast-like fungi can refer to this method for library construction and analysis.

[0218] 1. Sample Preparation and Detection

[0219] Step 1. Sample collection and drug susceptibility verification: 120 clinical Candida albicans isolates were collected from 31 hospitals across china in a period between September 2015 and January 2019. All isolates were tested for drug susceptibility using broth microdilution method following the M27-A3 and M27-S4 guidelines of NCCLS, and the results were used as the gold standard (culture-based AST).

[0220] Step 2. Genomic DNA preparation: Strains were inoculated on Sabouraud plate or Chromogenic agar plate by streaking and placed in an incubator (37° C.) for 24 hours. The fungal precipitate was then collected by centrifugation at 10,000 rpm for 2 mins, and incubated with sorbitol sodium phosphate buffer and lysozyme at a final concentration of 1.2 mol/L and 20 mg/mL, respectively, at 37° C. for 30-60 min. The genomic DNA was purified from the pellets using DNeasy Blood and Tissue kit following the protocol by the manufacturer. DNA concentration was measured using Qubit fluorometer and the QC was performed on a NanoDrop™ spectrophotometer, DNA with OD 260/280 1.6-2.0 and 260/230 2.0-2.2 were accepted for nanopore sequencing library preparation.

[0221] Step 3. Library preparation: ONT Native Barcoding Kit 1D (EXP-NBD104 & 114) and Ligation Sequencing Kit 1D (SQK LSK109) were used in library preparation following the 1D Native barcoding genomic DNA protocol developed by ONT with a few modifications. The procedure is briefly described below, DNA extracted from each isolate were quantified on a Qubit 3.0 fluorometer and diluted to 20ng/μl with nuclease-free water (NF water), 50 μl diluted DNA was used as starting material and incubated with 7 μl Ultra II End-Prep reaction buffer and 3 μl Ultra II End-Prep enzyme mix (New England Biolabs, USA) for 5 minutes at 20° C. follow by 5 minutes at 65° C. The end-prepped DNA was then purified from the reaction mix using 1×(v/v) AMPure XP magnetic beads and eluted with 25 μl NF water. After quantification, elution containing 500ng DNA was obtained from each sample and topped up to 22.5 μl with NF water and mixed with 2.5 μl unique Native Barcode and 25 μl Blunt/TA Ligation Master Mix. The mixture was incubated at room temperature for 10 min and purified with 1×(v/v) AMPure XP magnetic beads, and the barcoded DNA was eluted with 26 μl NF water. In the final step, equal amounts of 12 individually barcoded DNA samples were pooled to 700ng in total, and extra NF water were added to 50 μl final volume. To the pooled DNA, 20 μl ONT Barcode Adapter Mix, 20 μl NEBNext Quick Ligation Reaction Buffer (5×) and 10 μl Quick T4 DNA Ligase was added in order and mixed thoroughly, and after 10 mins incubation at room temperature, the BAM ligated DNA was purified from the reaction mix with 0.4×(v/v) AMPure XP magnetic beads, and eluted in 15 μl NF water.

[0222] Step 4. Nanopore sequencing: The sequencing consumable used in this study was ONT flowcell FLO-MIN106 R9.4. After flowcell priming, 75 μL prepared library (35 μL Running buffer, 25.5 μL loading beads, and 14.5 μL pooled library) was loaded. Sequencing was performed on an ONT MinION™ portable sequencing device, and set and monitored using ONT MinKNOW™ desk software. Samples were pooled and sequenced for about 6 hours or until 1 Gb of data for each sample were generated for database isolates and clinical specimens, respectively.

[0223] Step 5. Bioinformatics analysis: Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software. And the CANU 2.0 software was used to assemble the reads in fastq files into genomic assemblies or contigs with default parameters. The assembled genomes were analyzed using the local antibiotic resistance database for resistance determinants identification. Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for the identification of pathogens present in the sample and reads classified as Candida albicans were extracted for further resistance analysis. A local nBLAST tool was created for interrogating contigs of clinical samples for a panel of validated AMR determinants. Phylogenetic analysis was conducted with kSNP3 (version 3.1) based on pan-genome SNPs identified and a Kmer value of 31 was adopted.

[0224] 2. Database Construction and Analytical Logic Design

[0225] (1) Construction of a genomic phylogenetic tree: Through biomarker analysis, identical clones were merged and 107 representative Candida albicans clones were selected for the database and a genomic phylogenetic tree database was built as shown in FIGS. 15A-15C. The branches of the phylogenetic tree representing different resistance profiles are indicated by different colors on the right, where red indicates azole-resistant, yellow indicates 5-fluorocytosine-resistant, green indicates echinocandins-resistant, gray indicates Amphotericin-B-intermediate, and blue indicates susceptible to all antibiotics.

[0226] (2) Analytical Logic Design

[0227] (2a) Species identification: When the genome assembly size of the test sample is within the range of 12,000,000-16,000,000 bp, fragments of different lengths from multiple sites in the genome were selected to BLAST against the NCBI nucleotide database. The species of the pathogen is determined as Candida albicans only when the strain description shows Candida albicans and the per identity value exceeds 98%.

[0228] (2b) Drug susceptibility determination: Resistance of Candida albicans to triazoles and amphotericin B formulations is determined by the positioning of a test sample in the genomic phylogenetic tree, following the principle that the drug resistance profile predicted by phylogenetic tree interpretation is preferred over antimicrobial resistance determinants analysis; resistance of Candida albicans to echinocandins is determined by antimicrobial resistance determinants analysis. The drug species and antimicrobial resistance determinants used in this study are shown in Table 17.

TABLE-US-00023 TABLE 17 Results by phylo- genetic Antimicrobial resistance determinants Antibiotics tree fcy2 fca1 fur1 erg11 fks1 fks2 Amphotericin S B 5- R R R Fluorocytosine Fluconazole R Itraconazole R Voriconazole R Caspofungin R R Micafungin R R

[0229] 3. Database Validation and Result Interpretation

[0230] (1) Blind test: The genome-wide SNPs loci of library Candida albicans and 12 blinds were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. As is shown in FIGS. 15A-15C, none of the 12 blinds was belonged to Amphotericin B-R or echinocandin-R cluster. The phylogenetic tree prediction and Antimicrobial resistance determinants analysis of the 12 blinds are detailed in Table 18:

TABLE-US-00024 TABLE 18 Results by phylogenetic tree Antimicrobial resistance determinants Ampho- Echino- (Amino acid substitution) tericin B candin fcy2 fca1 fur1 erg11 fks1 fks2 blind-1 S S — — — — — — blind-2 S S H80R — — — — — blind-3 S S — — — — — — blind-4 S S — G28D — — — — blind-5 S S — — — Y132H + — — R467K blind-6 S S — G28D — — — — hetero- geneous blind-7 S S — — — — — — blind-8 S S — — — — — — blind-9 S S — — — K259E + — — I471T blind-10 S S — — — — — — blind-11 S S — — R101C — — — blind-12 S S — — — — — —

[0231] (2) Result interpretation: Based on the positioning in the phylogenetic tree, the antimicrobial resistance determinants and the corresponding prediction rules, the resistance profiles of 12 blinds against 7 antibiotics were determined and compared with the gold standard broth microdilution method (culture-based AST), as shown in Table 19 (For antibiotics without relevant drug-resistant samples, the positive predictive value and sensitivity are not counted).

TABLE-US-00025 TABLE 19 Results by broth Results by Positive Negative microdilution this present predictive predictive method method value value Antibiotic R S R S (PPV) (NPV) Sentitivity Specificity 5- 4 8 4 8 100.00% 100.00% 100.00% 100.00% Fluorocytosine Amphotericin 0 12 0 12 — 100.00% — 100.00% B Fluconazole 2 10 2 10 100.00% 100.00% 100.00% 100.00% Itraconazole 2 10 2 10 100.00% 100.00% 100.00% 100.00% Voriconazole 2 10 2 10 100.00% 100.00% 100.00% 100.00% Caspofungin 0 12 0 12 — 100.00% — 100.00% Micafungin 0 12 0 12 — 100.00% — 100.00%

[0232] By analyzing the sample positioning in the phylogenetic tree, combined with antimicrobial resistance determinants and specific prediction rules, the resistance profiles of the 12 blinds inferred by this present method showed a 100% agreement with that of the gold standard broth microdilution method.

Example 11: Rapid Identification and Antibiotic Susceptibility Inference with Clinical Metagenomics Based on Nanopore Sequencing Technology and the Rapid Library Method

[0233] A case of a clinical respiratory sputum specimen containing Acinetobacter baumannii, collected in July 2019, was selected as the representative to illustrate the way a clinical sample is test and analyzed. Other non-fermenting Gram-negative bacteria can refer to this method for pathogen identification and drug susceptibility determination.

[0234] 1. Sample Preparation and Nucleic Acid Extraction

[0235] (1) Take 1 ml of sputum sample and add the same amount of the Digestion buffer, mix gently at room temperature for 15 mins. Recover 1 ml of liquefied sputum into a 1.5 ml tube, centrifuge at 12,000 rpm for 5 mins, discard the supernatant, and add 1 ml of PBS buffer to fully resuspend the pellet. Transfer the supernatant to a clean 1.5 ml tube, centrifuge at 12000×g for 5 min, and discard the supernatant;

[0236] (2) Add 500 μl 1×PBS to resuspend the pellet, centrifuge at 12000×g for 5 min, and discard the supernatant;

[0237] (3) Add 98 μl of ddH2O and 2 μl of 5% saponin to the precipitate, mix by pipetting with a pipette tip, and let it stand for 10 mins at room temperature;

[0238] (4) Add 500 μl of 1×PBS, mix by pipetting with a pipette tip, and centrifuge at 12000×g for 5 min. Discard the supernatant;

[0239] (5) Add 40 μl 1×PBS to resuspend the pellet, prepare the reaction solution according to the following reaction system, mix well and incubate at 37° C. for 15 min;

TABLE-US-00026 Components Volume Sample  40 μl 10 x reaction buffer 5.6 μl Thermo DNase  10 μl ddH2O 0.5 μl

[0240] (6) Use the Bacterial Genomic DNA Extraction kit (Tiangen, China) to extract and purify the genomic DNA from the sample, and elute with 50ul TE;

[0241] (7) Take 2ul of the extracted genomic DNA for quantification with Qubit reagent.

[0242] 2. Library Preparation

[0243] (1) Prepare the End-prep reaction system according to the following table in a 0.2 ml PCR reaction tube:

TABLE-US-00027 Components Volume DNA 45 μl Ultra II End-prep reaction  7 μl buffer   Ultra II End-prep enzyme mix  3 μl ddH20  5 μl

[0244] (2) Mix gently by finger tapping, centrifuge briefly, incubate at 20° C. for 5 mins, and then incubate at 65° C. for 5 mins. Add AMPure XP (Beckman) magnetic beads that have been mixed and equilibrated to room temperature, and transfer the mixture to a 1.5 ml tube. Invert and mix for 5 min;

[0245] (3) After instant centrifugation, place the centrifuge tube on a magnetic rack, remove the supernatant after the magnetic beads are enriched, and wash twice with 200ul freshly-prepared 70% ethanol;

[0246] (4) Dry the magnetic beads at room temperature for 2 mins, and add 31ul TE buffer. After mixing and incubating for 2 mins, place the tube on the magnetic frame again. After the magnetic beads are aggregated, take the supernatant for use;

[0247] (5) Prepare the barcoding reaction solution according to the following table in a 1.5 ml tube:

TABLE-US-00028 Components Volume End-preped DNA 30 μl Barcode Adapter 20 μl Blunt/TA Ligase Master Mix 50 μl

[0248] (6) Gently mix by finger tapping, centrifuge briefly and incubate at room temperature for 10 mins, add 100ul XP magnetic beads, mix by pipetting, and continue to invert and mix at room temperature for 5 minutes;

[0249] (7) After instant centrifugation, place the centrifuge tube on a magnetic rack, remove the supernatant after the magnetic beads are enriched, and wash twice with 200ul freshly prepared 70% ethanol;

[0250] (8) Dry the magnetic beads at room temperature for 2 min, and add 25ul TE buffer. Resuspend the beads and incubate at room temperature for 2 min, put the centrifuge tube back on the magnetic rack, and take the supernatant after the magnetic beads aggregate;

[0251] (9) Take 1 ul of the eluate for quantification with Qubit reagent;

[0252] (10) Prepare the PCR reaction solution in a 0.2 ml PCR tube as follows:

TABLE-US-00029 Components Volume PCR Barcode 1  2 μl 10 ng/μl adapter ligated template  2 μl LongAmpTaq 2x master mix 50 μl Nuclease-free water 46 μl

[0253] (11) PCR under the following conditions:

TABLE-US-00030 Temperature Time 95° C.  3 min 94° C. 15 s 15Cs 62° C. 15 s {open oversize brace} 65° C.  3 min 65° C.  3 min

[0254] (12) Add 100ul XP magnetic beads, mix by pipetting, and invert and mix at room temperature for 5 minutes;

[0255] (13) After instant centrifugation, place the centrifuge tube on a magnetic stand, remove the supernatant once the solution turns clear, and wash twice with 200ul freshly prepared 70% ethanol;

[0256] (14) Dry the magnetic beads at room temperature for 2 minutes, add 46ul TE buffer. After mixing and incubating for 2 minutes off the rack, place the centrifuge tube on the magnetic rack again. Once the solution turns clear, take the supernatant for use;

[0257] (15) Take 1 ul of the eluate for quantification with Qubit reagent;

[0258] (16) Prepare an End-prep reaction system in a 0.2 ml PCR reaction tube according to the table below:

TABLE-US-00031 Components Volume DNA 45 μl Ultra II End-prep reaction  7 μl buffer Ultra II End-prep enzyme mix  3 μl ddH20  5 μl

[0259] (17) Mix gently with finger tapping, centrifuge briefly, incubate at 20° C. for 5 minutes, then incubate at 65° C. for 5 minutes, add 60 μl of AMPure XP (Beckman) magnetic beads that have been mixed and equilibrated to room temperature, and transfer the mixture to a 1.5 ml centrifuge tube. Invert and mix at room temperature for 5 min;

[0260] (18) After instant centrifugation, place the centrifuge tube on a magnetic stand, remove the supernatant once the solution turns clear, and wash twice with 200ul freshly prepared 70% ethanol;

[0261] (19) Dry the magnetic beads at room temperature for 2 min, and add 61ul TE buffer, resuspend off the rack and incubate for 2 min, put the centrifuge tube back on the magnetic rack, and take the supernatant after the magnetic beads aggregate;

[0262] (20) Prepare a sequencing adapter ligation reaction system in a 1.5 ml centrifuge tube as follows:

TABLE-US-00032 Components Volume DNA 60 μl Ligation buffer (LNB) 25 μl NEBnext Quick T4 DNA Ligase 10 μl Sequencing adapter (AMX)  5 μl

[0263] (21) Close the tube, mix by finger tapping, centrifuge briefly, incubate at room temperature for 10 minutes, and add 40 μl of AMPure XP magnetic beads that have been mixed and equilibrated to room temperature. Continue to invert and mix at room temperature for 5 minutes;

[0264] (22) After a brief centrifugation, place the centrifuge tube on a magnetic stand. Once the solution turns clear, discard the supernatant, add 250 μl of short fragment washing buffer (SFB), cover the tube, and mix by finger tapping until the magnetic beads are suspended again. After centrifugation, put the centrifuge tube back on the magnetic rack, and once the solution turns clear, discard the supernatant;

[0265] (23) Repeat step 22;

[0266] (24) Dry the magnetic beads at room temperature for 30 seconds, add 15 μl of elution buffer (buffer EB), flick off the rack to resuspend, and incubate at room temperature for 10 minutes;

[0267] (25) After instant centrifugation, place the centrifuge tube on a magnetic stand, and once the solution turns clear, transfer the supernatant to a 1.5 ml tube for future use;

[0268] (26) Take 1 μl of DNA for quantification with Qubit reagent. The total amount of target DNA is 1-20 ng/μl. If the DNA concentration is too high, dilute it with elution buffer (Buffer EB).

[0269] 3. Sequencing

[0270] (1) Mix a tube of the flowcell priming buffer (FB) with 30 μl of flushing aid (FLT), vortex well, inject 800 μl through the injection hole into the chip, leave it at room temperature for 5 minutes, then open the injection hole (Spot on), and then Inject 200 μl of rinse mix from the initial well;

[0271] (2) Prepare the sequencing reaction in a 1.5 ml tube according to the table below:

TABLE-US-00033 Components Volume Sequencing buffer (SQB) 37.5 μl Library Loading beads (LB) 25.5 μl DNA library   12 μl

[0272] (3) After gently pipetting twice with a pipette, load 75 μl of the mixture into the sequencing flowcell through the sample loading hole. After the mixture has completely flowed into the flowcell, cover the sample hole first, and then close the initial hole;

[0273] (4) Sample was sequenced until 1 Gb of data for each sample were generated.

[0274] 4. Bioinformatics analysis

[0275] (1) Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software;

[0276] (2) Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for pathogen identification, and with Antimicrobial resistance for drug susceptibility prediction;

[0277] (3) WIMP analysis showed that Acinetobacter baumannii was positive with a total reads of 10096 as listed in Table 20

TABLE-US-00034 TABLE 20 Species Reads Corynebacterium striatum 43890 Homo sapiens 15657 Acinetobacter baumannii 10,096 Corynebacterium simulans 9,247 Streptococcus mitis 3,341 Streptococcus pneumoniae 1,511 Streptococcus sp. oral taxon 431 1,354 Corynebacterium diphtheriae 1,311 Corynebacterium aurimucosum 1,056 Corynebacterium resistens 1,010 Streptococcus pseudopneumoniae 728 Streptococcus oralis 621

[0278] (4) The antimicrobial resistance determinants including sul2, APH(3′)-Ia, OXA239, and gyrA(T) identified are listed in Table 21, and their corresponding resistance profiles are inferred as in Table 22;

TABLE-US-00035 TABLE 21 Antimicrobial resistance determinants abeM adeL abeS adeN ADC-22 adeR adeA ANT(3″)-IIb adeB APH(3″)-Ib adeC APH(3′)-Ia adeF APH(6)-Id adeG mphD adeH msrE adeI OXA-239 adeJ sul2 adeK TEM-122 tet(B) TEM-90 tetR

TABLE-US-00036 TABLE 22 Antibiotics Sulfa- Ceftaz- Cef- Cefox- Piper- Mero- Gen- Tobra- Levo- Cipro- Genes methoxazole idime Cefepime otetan icillin acillin penem Imipenem tamicin mycin Amikacin floxacin floxacin AMR Sul2 OXA239 APH(3′)-Ia gyrA(T) Pheno- R R R R R R R R R R R R R type

[0279] (5) The genome-wide SNPs loci of library Acinetobacter baumannii together with this test clinical specimen were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. The test sample was located in the Cluster 5 of phylogenetic tree, as shown in FIG. 16. Therefore, this Acinetobacter baumannii strain was determined resistant to all listed antibiotics, as shown in Table 23.

TABLE-US-00037 TABLE 23 Antibiotics Sulfa- Ceftaz- Cefox- Piper- Mero- Gen- Tobra- Levo- Cipro- Cluster methoxazole idime Cefepime Cefotetan icillin acillin penem Imipenem tamicin mycin Amikacin floxacin floxacin 1 S S S S S S S S S S S S S 2 R R R R R R R R R/I R/S R/S R R 3 R R R R R R R R R S/R S/R R R 4 S R R R R R R R R/S R/S S R R 5 R/S R R R R R R R R/S R/S R/S R R 6 R/S R R R R R R R R/S S S R R 7 S/R R R R R R R R R/S R/S R/S R R 8 R/S R R R R R R R R R R R R 9 S/R R R R R R R R R R R R R 10 R R R R R R R R R R R R R 11 R R R R R R R R R/I R/S R/S R R 12 R R R R R R R R R R R R R 13 R R R R R R R R R R R R R 14 S R R R R R R R R R R R R 15 R R R R R R R R R S S R R 16 R S R R R R R R R S S R R 17 S S S S S S S S S S S S S

Example 12: Rapid Identification and Antibiotic Susceptibility Inference with Clinical Metagenomics Based on Nanopore Sequencing Technology and the PCR Library Method

[0280] A case of a clinical respiratory sputum specimen containing Klebsiella pneumoniae, collected in August 2019, was selected as the representative to illustrate the way a clinical sample is test and analyzed. Other Enterobacteriaceae can refer to this method for pathogen identification and drug susceptibility determination.

[0281] 1. Sample Preparation and Nucleic Acid Extraction

[0282] (1) Take 1 ml of sputum sample and add the same amount of the Digestion buffer, mix gently at room temperature for 15 mins. Recover 1 ml of liquefied sputum into a 1.5 ml tube, centrifuge at 12,000 rpm for 5 mins, discard the supernatant, and add 1 ml of PBS buffer to fully resuspend the pellet. Transfer the supernatant to a clean 1.5 ml tube, centrifuge at 12000×g for 5 min, and discard the supernatant;

[0283] (2) Add 500 μl 1×PBS to resuspend the pellet, centrifuge at 12000×g for 5 min, and discard the supernatant;

[0284] (3′) Add 98 μl of ddH2O and 2 μl of 5% saponin to the precipitate, mix by pipetting with a pipette tip, and let it stand for 10 mins at room temperature;

[0285] (4) Add 500 μl of 1×PBS, mix by pipetting with a pipette tip, and centrifuge at 12000×g for 5 min. Discard the supernatant;

[0286] (5) Add 40 μl 1×PBS to resuspend the pellet, prepare the reaction solution according to the following reaction system, mix well and incubate at 37° C. for 15 min;

TABLE-US-00038 Components Volume Sample  40 μl 10 x reaction buffer 5.6 μl Thermo DNase  10 μl ddH2O 0.5 μl

[0287] (6) Use the Bacterial Genomic DNA Extraction kit (Tiangen, China) to extract and purify the genomic DNA from the sample, and elute with 50ul TE;

[0288] (7) Take 2ul of the extracted genomic DNA for quantification with Qubit reagent.

[0289] 2. Amplification of Antimicrobial Resistance Determinants

[0290] (1) Prepare the reaction solution according to the following table in a 0.2 ml PCR tube:

TABLE-US-00039 Components Volume Taq 2x master mix 20 μl DNA  5 μl Primer mix 10 μl ddH2O  5 μl Nuclease-free water 40 μl

[0291] (2) PCR under the following conditions:

TABLE-US-00040 Temperature Time 95° C. 3 min 94° C. 15 s 55° C. 1 mins 35Cs {open oversize brace} 68° C. 1 min 68° C. 3 min

[0292] 3. Library Preparation

[0293] (1) Prepare the End-prep reaction system according to the following table in a 0.2 ml PCR reaction tube:

TABLE-US-00041 Components Volume DNA 45 μl Ultra II End-prep reaction  7 μl buffer   Ultra II End-prep enzyme mix  3 μl ddH20  5 μl

[0294] (2) Mix gently by finger tapping, centrifuge briefly, incubate at 20° C. for 5 mins, and then incubate at 65° C. for 5 mins. Add AMPure XP (Beckman) magnetic beads that have been mixed and equilibrated to room temperature, and transfer the mixture to a 1.5 ml tube. Invert and mix for 5 min;

[0295] (3) After instant centrifugation, place the centrifuge tube on a magnetic rack, remove the supernatant after the magnetic beads are enriched, and wash twice with 200ul freshly-prepared 70% ethanol;

[0296] (4) Dry the magnetic beads at room temperature for 2 mins, and add 31ul TE buffer. After mixing and incubating for 2 mins, place the tube on the magnetic frame again. After the magnetic beads are aggregated, take the supernatant for use;

[0297] (5) Prepare the barcoding reaction solution according to the following table in a 1.5 ml tube:

TABLE-US-00042 Components Volume End-preped DNA 30 μl Barcode Adapter 20 μl Blunt/TA Ligase Master Mix 50 μl

[0298] (6) Gently mix by finger tapping, centrifuge briefly and incubate at room temperature or 10 mins, add 100ul XP magnetic beads, mix by pipetting, and continue to invert and mix at room temperature for 5 minutes;

[0299] (7) After instant centrifugation, place the centrifuge tube on a magnetic rack, remove the supernatant after the magnetic beads are enriched, and wash twice with 200ul freshly prepared 70% ethanol;

[0300] (8) Dry the magnetic beads at room temperature for 2 min, and add 25ul TE buffer. Resuspend the beads and incubate at room temperature for 2 min, put the centrifuge tube back on the magnetic rack, and take the supernatant after the magnetic beads aggregate;

[0301] (9) Take 1 ul of the eluate for quantification with Qubit reagent;

[0302] (10) Prepare the PCR reaction solution in a 0.2 ml PCR tube as follows:

TABLE-US-00043 Components Volume PCR Barcode 1-96  2 μl 10 ng/μl adapter ligated temμlate  2 μl Taq 2x master mix 50 μl Nuclease-free water 46 μl

[0303] (11) PCR under the following conditions:

TABLE-US-00044 Temperature Time 95° C.  3 min 94° C. 15 s 2Cs 62° C. 15 s {open oversize brace} 68° C.  3 min 68° C.  3 min

[0304] (12) Add 100ul XP magnetic beads, mix by pipetting, and invert and mix at room temperature for 5 minutes;

[0305] (13) After instant centrifugation, place the centrifuge tube on a magnetic stand, remove the supernatant once the solution turns clear, and wash twice with 200ul freshly prepared 70% ethanol;

[0306] (14) Dry the magnetic beads at room temperature for 2 minutes, add 46ul TE buffer. After mixing and incubating for 2 minutes off the rack, place the centrifuge tube on the magnetic rack again. Once the solution turns clear, take the supernatant for use;

[0307] (15) Take 1 ul of the eluate for quantification with Qubit reagent;

[0308] (16) Prepare an End-prep reaction system in a 0.2 ml PCR reaction tube according to the table below:

TABLE-US-00045 Components Volume DNA 45 μl Ultra II End-prep reaction  7 μl buffer   Ultra II End-prep enzyme mix  3 μl ddH20  5 μl

[0309] (17) Mix gently with finger tapping, centrifuge briefly, incubate at 20° C. for 5 minutes, then incubate at 65° C. for 5 minutes, add 60 μl of AMPure XP (Beckman) magnetic beads that have been mixed and equilibrated to room temperature, and transfer the mixture to a 1.5 ml centrifuge tube. Invert and mix at room temperature for 5 min;

[0310] (18) After instant centrifugation, place the centrifuge tube on a magnetic stand, remove the supernatant once the solution turns clear, and wash twice with 200ul freshly prepared 70% ethanol;

[0311] (19) Dry the magnetic beads at room temperature for 2 min, and add 61ul TE buffer, resuspend off the rack and incubate for 2 min, put the centrifuge tube back on the magnetic rack, and take the supernatant after the magnetic beads aggregate;

[0312] (20) The product obtained from identification library and the susceptibility library are mixed in equal amounts to form a library-pool;

[0313] (21) Prepare a sequencing adapter ligation reaction system in a 1.5 ml centrifuge tube as follows:

TABLE-US-00046 Components Volume DNA mix 60 μl Ligation buffer (LNB) 25 μl NEBnext Quick T4 DNA Ligase 10 μl Sequencing adapter (AMX)  5 μl

[0314] (22) Close the tube, mix by finger tapping, centrifuge briefly, incubate at room temperature for 10 minutes, and add 40 μl of AMPure XP magnetic beads that have been mixed and equilibrated to room temperature. Continue to invert and mix at room temperature for 5 minutes;

[0315] (23) After a brief centrifugation, place the centrifuge tube on a magnetic stand. Once the solution turns clear, discard the supernatant, add 250 μl of short fragment washing buffer (SFB), cover the tube, and mix by finger tapping until the magnetic beads are suspended again. After centrifugation, put the centrifuge tube back on the magnetic rack, and once the solution turns clear, discard the supernatant;

[0316] (24) Repeat step 23;

[0317] (25) Dry the magnetic beads at room temperature for 30 seconds, add 15 μl of elution buffer (buffer EB), flick off the rack to resuspend, and incubate at room temperature for 10 minutes;

[0318] (26) After instant centrifugation, place the centrifuge tube on a magnetic stand, and once the solution turns clear, transfer the supernatant to a 1.5 ml tube for future use;

[0319] (27) Take 1 μl of DNA for quantification with Qubit reagent. The total amount of target DNA is 1-20 ng/μl. If the DNA concentration is too high, dilute it with elution buffer (Buffer EB).

[0320] 4. Sequencing

[0321] (1) Mix a tube of the flowcell priming buffer (FB) with 30 μl of flushing aid (FLT), vortex well, inject 800 μl through the injection hole into the chip, leave it at room temperature for 5 minutes, then open the injection hole (Spot on), and then Inject 200 μl of rinse mix from the initial well;

[0322] (2) Prepare the sequencing reaction in a 1.5 ml tube according to the table below:

TABLE-US-00047 Components Volume Sequencing buffer (SQB) 37.5 μl Library Loading beads (LB) 25.5 μl DNA library   12 μl

[0323] (3) After gently pipetting twice with a pipette, load 75 μl of the mixture into the sequencing flowcell through the sample loading hole. After the mixture has completely flowed into the flowcell, cover the sample hole first, and then close the initial hole;

[0324] (4) Sample was sequenced until 1 Gb of data for each sample were generated.

[0325] 5. Bioinformatics Analysis

[0326] (1) Raw FAST5 reads files were base-called using the Guppy v3.2.4 basecalling software;

[0327] (2) Called fastq files of clinical specimens were analyzed with the EPI2ME WIMP rev.3.3.1 pipeline for pathogen identification, and with Antimicrobial resistance for drug susceptibility prediction;

[0328] (3) WIMP analysis showed that Klebsiella pneumoniae was positive with a total reads of 88638, as listed in Table 24;

TABLE-US-00048 TABLE 24 Species Reads Klebsiella pneumoniae 88,638 Homo sapiens 66,519 Escherichia coli 4,635 Rothiamucilaginosa 2,917 Streptococcus mitis 1,044 Acinetobacter baumannii 1,001 Corynebacterium striatum 861 Klebsiella variicola 527 Streptococcus sp. oral taxon 431 444 Streptococcus pneumoniae 374 Veillonellaparvula 353 Acinetobacter nosocomialis 310

[0329] (4) Results of Antimicrobial resistance determinants: none of the antimicrobial resistance determinants including CTX-M-65, TEM-1B, IMP-4, KPC-2, rmtB, AAC(3′)-Iid, QRDR, gyrA(T), tetA, tetD, sul1, sul2, sul3 were detected;

[0330] (5) The genome-wide SNPs loci of library Klebsiella pneumoniae together with this test clinical specimen were identified using kSNP3 (Version 3.1) (Standard mode, kmer=31), and a genomic phylogenetic tree based on SNP similarities were generated for cluster analysis. The test sample was located in the S branch of phylogenetic tree, as shown in FIG. 17. Therefore, this Klebsiella pneumoniae strain was determined to be susceptible to all antibiotics involved in this study, which is in agreement with the gold standard VITEK2 AST result.