Materials and Reagents

1. 384-well microplate (Corning, catalog number: 3701)

2. 1.5 mL nuclease-free tube (Eppendorf, catalog number: 0030125150)

3. 0.2 mL PCR tube (Eppendorf, catalog number: 0030124332)

4. 10 μL tips (SANFICO, catalog number: TFR810)

5. 10 μL extended tips (SANFICO, catalog number: TFR810-L)

6. 200 μL tips (SANFICO, catalog number: TFR8200)

7. 1,000 μL tips (SANFICO, catalog number: TFR81000)

8. Quick-DNA/RNA Pathogen Miniprep kit (ZYMO RESEARCH, catalog number: R1042)

9. TIANSeq HiFi Amplification Mix (TIANGEN, catalog number: NG219)

10. NEBuffer^TM 3.1 (New England Biolabs, catalog number: B7203)

11. Nuclease-free water (Thermo Fisher, catalog number: 10977015)

12. Cas12a protein

    Note: In our experiments, the Cas12a protein expression and purification was performed as described (Li et al., 2018). We also recommend purchasing Cas12a from a biological company (e.g., New England Biolabs).

13. Primers for PCR amplification (primer sequences are shown in Table 1)

    Note: The sequence of primers was published in our previous article in Appendix 1, Table 1 (Wang et al., 2022). The primers can be ordered as PAGE-purified DNA oligonucleotide from a DNA synthesis company (e.g., GenScript Biotech or Sangon Biotech).

    
    

    Table 1. Primers sequences

    Species	Primers sequences (5′ > 3′)
    A. baumannii	forward primer	CACAGCGTTTAACCCATGCC
    	reverse primer	TATCGCCACCTGCACAGAAG
    E. coli	forward primer	GTTCCTGACTATCTGGCGGG
    	reverse primer	GCTTCCTGACTCCAGACACC
    K. pneumoniae	forward primer	CATGGGCATATCGACGGTCA
    	reverse primer	CCTGCAACATAGGCCAGTGA
    S. aureus	forward primer	AGGTGCAGTAGACGCATAGC
    	reverse primer	CATTCGCTGCGCCAATACAA
    P. aeruginosa	forward primer	TCTCTCTATCACGCCGGTCA
    	reverse primer	TCGCATCGAGGTATTCCAGC
    S. epidermidis	forward primer	CACGCATGGCACTAGGTACA
    	reverse primer	CGAAAAAGAGTTGTCCTTGTTGA
    S. capitis	forward primer	GGTTCAGTCATCCCCACGTT
    	reverse primer	CAGCTGCGACAACTGCTTAC
    E. faecalis	forward primer	CGGCAAGTTTGGAAGCAGAC
    	reverse primer	CAGCGCCTAGTCCTTGTGAT
    E. faecium	forward primer	ATCGGAAATCGGTGTGGCTT
    	reverse primer	TCAAATGCATCCCTGTGCCT
    S. maltophilia	forward primer	CGCCTCCCGTTTACAGATTA
    	reverse primer	TCGGCTCCACCACATACAC

    
    

14. crRNA used in Cas12a reaction (crRNA sequences are shown in Table 2)

    Note: The RNA can be ordered as a desalted RNA oligonucleotide or as PAGE-purified RNA oligonucleotide from an RNA synthesis company (e.g., GenScript Biotech or Sangon Biotech).

    
    

    Table 2. crRNA sequences

    Species	crRNA sequences (5′ > 3′)
    A. baumannii	UAAUUUCUACUAAGUGUAGAUGUAAAGAAGAAUUACUUGAA
    E. coli	UAAUUUCUACUAAGUGUAGAUACGGUCGCUAUGAUGGCAAG
    K. pneumoniae	UAAUUUCUACUAAGUGUAGAUACUGCGGUAAUCGCCAUCUU
    S. aureus	UAAUUUCUACUAAGUGUAGAUACAACGUGAACUUGCUGAGG
    P. aeruginosa	UAAUUUCUACUAAGUGUAGAUUUGUAAACACACAUGAGGAG
    S. epidermidis	UAAUUUCUACUAAGUGUAGAUUAAUAUUCAUCAAUUAGAGA
    S. capitis	UAAUUUCUACUAAGUGUAGAUAAUCACCUUAUUAAUCAAUA
    E. faecalis	UAAUUUCUACUAAGUGUAGAUGCACCAAAUAACACAGCUGA
    E. faecium	UAAUUUCUACUAAGUGUAGAUACUACCUGAACUUAUAUACA
    S. maltophilia	UAAUUUCUACUAAGUGUAGAUGCAUGACACCUGCGACCAGG

    
    

    Note: The backbone sequence of crRNA is 5′-UAAUUUCUACUAAGUGUAGAU-3′, and the following sequence is used to recognize the target DNA.

    
    

15. ssDNA-reporter (synthesized by Sangon Biotech)

    Note: Sequence of ssDNA-reporter: 5′-FAM-TTATT-BHQ1-3′. The feature of the ssDNA-reporter is that a fluorescent molecule that can generate a fluorescent signal (e.g., FAM) is coupled at one DNA end, and a quencher molecule (e.g., BHQ1) that can block the fluorescent molecule producing a fluorescent signal is coupled at the opposite DNA end.

16. Ethanol (Sangon Biotech, catalog number: A500737-0500)

Equipment

1. Centrifuge (Eppendorf, Centrifuge 5418 R)

2. Centrifuge for 0.2 mL PCR tube (Scilogex, catalog number: 914041419999)

3. PCR thermocycler (LongGene, T20D)

4. Fluorescence plate reader (Austria Tecan, Infinite M200 Pro)

5. NanoDrop One (Thermo Scientific, catalog number: ND-ONE-W)

6. Biological safety cabinet (Thermo Scientific, catalog number: 51028226)

7. Vortexer (Scilogex, catalog number: 821200049999)

8. 1–10 μL pipettes (Eppendorf, catalog number: 3123000020)

9. 2–20 μL pipettes (Eppendorf, catalog number: 3123000098)

10. 20–200 μL pipettes (Eppendorf, catalog number: 3123000055)

11. 100–1,000 μL pipettes (Eppendorf, catalog number: 3123000063)

Software

1. Primer design software, NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)

2. CrRNA design software, CHOPCHOP (https://chopchop.cbu.uib.no/)

3. Software to control the fluorescence plate reader, Tecan i-control (Austria Tecan)

4. Software for data analysis, Microsoft Excel

Procedure

1. Detection targets design

   Note: The primers and crRNAs used in this protocol for 10 pathogens (Table 1) have been experimentally and clinically validated with excellent effectiveness.

   1. Select detection targets. Manual screening of species-specific DNA data from bioinformatics; the screening principles are:

      1. To facilitate primer design, DNA length is preferably longer than 200 bp.

      2. DNA fragment without repetitive sequences.

      3. DNA fragment contains PAM sequences (5′-TTTN-3′) of Cas12a.

   2. Design crRNA.

      1. Find PAM sequences (5′-TTTN-3′) within the DNA target region.

      2. Select the sequence 20 bp downstream of the PAM sequence as the target of crRNA. Replace the T-base in the 20 bp target sequence with a U-base. Place the target sequence downstream of the crRNA backbone sequence (5′-UAAUUUCUACUAAGUGUAGAU-3′).

         Note: The base distribution of the target sequence should be balanced, with a GC content of 40%–80%. We also recommend using crRNA design software, e.g., CHOPCHOP (https://chopchop.cbu.uib.no/).

   3. Use NCBI Primer-BLAST primer design software to select primers.

      1. The input sequence is the upstream and downstream sequence of the crRNA target site.

      2. Set the Primer parameters as default parameters.

      3. Set Primer Pair Specificity Checking Parameters Database as Refseq representative genomes, and Organism as Homo sapiens (taxid: 9606).

      4. Pick a primer pair predicted without non-specific amplification.

   4. Experimental validation

      For example, for the 10 sets of primers and crRNAs used in this protocol, each set of primers and crRNAs needs to ensure that no non-specific signals are detected for the remaining pathogens and human genomic DNA.

      
      

2. DNA extraction from bronchoalveolar lavage fluid (BALF) sample

   Biosafety notes: DNA extraction procedure should be performed in a biological safety cabinet to ensure user safety. Wear biological protecting clothing, gloves, and masks during operation.

   DNA extraction was performed referring to the Quick-DNA/RNA Pathogen Miniprep kit instructions. The procedure is as follows:

   1. Add 800 μL of DNA/RNA shield to each 200 μL of BALF sample and mix well by using vortexer.

   2. Centrifuge at 16,000× g for 1 min. Transfer up to 200 μL of the supernatant into a 1.5 mL nuclease-free tube.

   3. Add 2 μL of proteinase K and mix well.

   4. Add 400 μL of pathogen DNA/RNA buffer. Mix well and incubate at room temperature for 5 min.

   5. Transfer the mixture into a Zymo-Spin IIC column in a collection tube and centrifuge at 16,000× g for 30 s. Discard the flowthrough.

   6. Add 500 μL of pathogen DNA/RNA wash buffer to the Zymo-Spin IIC column and centrifuge at 16,000× g for 30 s. Discard the flowthrough.

   7. Repeat the previous step.

   8. Add 500 μL of ethanol (95%–100%) to the Zymo-Spin^TM IIC column and centrifuge at 16,000× g for 1 min to ensure that the ethanol is removed. Discard the collection tube and transfer the Zymo-Spin^TM IIC column into a new nuclease-free 1.5 mL tube.

   9. Add 25–50 μL of nuclease-free water at 65 °C directly to the matrix of the column and centrifuge at 16,000× g for 1 min to elute the DNA.

   10. Measure DNA concentration using a NanoDrop and adjust DNA concentration to 100 ng/μL.

       Note: The DNA yield extracted from 200 μL of BALF is 8–43 μg. Depending on the patient's situation and operating procedure, typically 200 μL of BALF is sufficient for subsequent analysis. If the DNA yield is low, centrifuge the BALF sample at 12,000× g for 5 min, discard the supernatant, and retain 200 μL of bottom liquid.

       
       

3. PCR amplification enrichment of target DNA sequences to be tested

   1. For one BALF sample DNA, prepare 10 PCR reactions using amplification primers, one for each of the 10 pathogenic bacteria.

   2. Setting up the positive and negative controls: the templates for the positive and negative controls are the genomic DNA of the corresponding pathogen and nuclease-free water, respectively.

   3. Prepare the PCR reactions in a 0.2 mL PCR tube.

      Component	50 μL reaction
      2× HiFi amplification mix	25 μL
      Forward and reverse primers (10 μM)	2 μL of each
      Template DNA (100 ng/μL)	1 μL
      Nuclease-free water	20 μL

   4. Mix well and pulse-spin in a microcentrifuge.

   5. Place a 0.2 mL PCR tube in a PCR thermocycler.

   6. PCR procedure:

      98 °C	2 min
      98 °C	10 s	38 cycles
      52 °C	10 s
      72 °C	15 s
      72 °C	5 min
      4 °C	Forever

   7. After the PCR reaction, without the PCR purification step, directly use the PCR reaction solution as the detection substrate for the Cas12a reaction.

      
      

4. Detection of PCR products using Cas12a

   1. Prepare the reactions in a 384-well microplate on ice:

      Component	20 μL reaction
      ssDNA-reporter (10 μM)	1 μL
      PCR reaction solution	2 μL
      10× NEBuffer 3.1	2 μL
      Cas12a (10 μM)	1 μL
      crRNA (10 μM)	1 μL
      Nuclease-free water	13 μL

      Note: The PCR reaction solution for each pathogen requires the use of the corresponding crRNA. Preparation of premixes is recommended to improve experimental efficiency.

   2. With a pipette set at 15 μL and keeping the plate on ice, mix the reaction by pipetting the entire solution up and down 10 times.

      Note: Pipetting 10 times can ensure sufficient mixing of the reaction solution. Avoid excessive bubbles in the reaction solution; any trace bubbles will clear after 30 min of incubation at 37 °C in the next step.

   3. Place the plate in a fluorescence plate reader. Incubate at 37 °C for 30 min, then measure the fluorescence values of each reaction well (excitation wavelength: 485 nm; emission wavelength: 535 nm).

Data analysis

Note: The data analysis of this protocol is applied to the detection of pathogenic bacteria in patients with severe pneumonia in the ICU. For other studies, the analysis standards require adjustments accordingly.

1. For each sample, analyze the fluorescence values for each pathogen separately.

2. For each pathogen detected, the positive control (PC) is the genomic DNA of corresponding pathogen, and the negative control (NC) is nuclease-free water.

3. The fluorescence value of BALF sample (shown as F) is two-fold larger than the negative control, indicating that the SSBD method detected the corresponding pathogen.

4. In our previous article, the application of SSBD was for pathogen detection in ICU patients, so the degree of pathogenic infection is required to guide drug usage. The pathogen strength is determined to some extent by the fluorescence signal (Table 3). Set I (interval) as the point of distinction. I = (PC - 2NC)/3. When 2NC < F < 2NC+I, the pathogen strength level is defined as level I. When 2NC+I < F < 2NC+2I, the pathogen strength level is defined as level II. When F > 2NC+2I, the pathogen strength level is defined as level III.

   
   

   Table 3. Determining the presence of bacteria by fluorescence signal

   Fluorescence value	Test results	Bacterial DNA molarity
   F < 2NC	Undetected	N.A.
   2NC < F < 2NC+I	Detected level I	10-15 M–10-14 M
   2NC+I < F < 2NC+2I	Detected level II	10-14 M–10-13 M
   F > 2NC+2I	Detected level III	over 10-13 M

   NC: Value_{negative control}; F: Value_Sample; I = (Value_{positive control} - 2 Value_{negative control})/3

   
   

Example results

1. Using the data of two samples from our previously published article, we calculated the values of 2NC, 2NC+I, and 2NC+2I according to the fluorescence values of PC and NC, respectively. The fluorescence values of the samples are shown in Table 4.

   
   

   Table 4. Fluorescence values of two samples

   Species	Fluorescence value							Detection result
   	PC	NC	Sample 1	Sample 2	2NC	2NC+I	2NC+2I	Sample 1	Sample 2
   A. baumannii	9788	903	9300	9398	1806	4467	7127	Detected Level III	Detected Level III
   E. coli	8435	821	657	630	1642	3906	6171	Undetected	Undetected
   K. pneumoniae	8285	1791	1718	1730	3582	5150	6717	Undetected	Undetected
   S. aureus	8316	1008	1018	1003	2016	4116	6216	Undetected	Undetected
   P. aeruginosa	9323	1170	1164	1069	2340	4668	6995	Undetected	Undetected
   S. epidermidis	9309	1237	1210	1183	2474	4752	7031	Undetected	Undetected
   S. capitis	8832	1633	1949	1772	3266	5121	6977	Undetected	Undetected
   E. faecalis	8241	614	512	505	1228	3566	5903	Undetected	Undetected
   E. faecium	8432	1936	1692	1700	3872	5392	6912	Undetected	Undetected
   S. maltophilia	8194	1620	1549	1579	3240	4891	6543	Undetected	Undetected

   
   

2. The fluorescence value of A. baumannii in these two samples was higher than 2NC+2I. Therefore, the presence of A. baumannii in these two samples was detected, with a level III bacterial strength. For the rest of the pathogens, the fluorescence values of the samples were lower than 2NC, which indicates that the rest of the pathogens were not detected.

Notes

1. Optimizing PCR reactions can significantly avoid the generation of non-specific signals. When designing primers, BLAST the primers and select primers with excellent specificity. The suitable PCR reaction program is confirmed by experiment. Moreover, it is basically impossible for crRNA recognition sequences to exist in the non-specific amplification products generated during PCR. Therefore, combining the crRNA-Cas12a reaction with the PCR reaction greatly ensures the specificity of the detection. It is noteworthy that if crRNA is synthesized by in vitro transcription with RNA polymerase, the DNA used as in vitro transcription template needs to be removed to avoid contaminating crRNA (since the DNA templates can bind to crRNA to activate Cas12a).

2. Features considered important while selecting target:

   1. Target sequences are present in multiple strains of this species' genomes.

   2. Target sequences are not found in other species' genomes.

   3. Target without repetitive sequences.

   4. Target including PAM sequences.

      
      

      Table 5. Information of target DNA region used in the protocol

      Species	GenBank of genome	Target sequences (5′ > 3′)	Corresponding gene
      A. baumannii	CP115629.1	CACAGCGTTTAACCCATGCCATTGGTAAAGCTAAAGCAATGGAAATGTGCCTTACCGCTCGTCAAATGGGTGCAGTAGAGGCAGAACAAAGTGGTTTGGTTGCACGTGTGTTTAGTAAAGAAGAATTACTTGAACAAACCTTGCAAGCCGCTGAAAAAATCGCAGCACGGTCTTTAACTGCAAACATGATGCTCAAAGAAACCATTAATCGAGCTTTTGAGGTGAATCTCACCGAAGGTTTACGTTTTGAACGCCGCATGTTCCACTCCATTTTTGCAACTGCGGACCAAAAAGAAGGCATGCAGGCATTTGTTGAAAAACGGCAGGCAAATTTTAAAAATCAGTAAGAGATCAATAAAATGAATACAGAAAATCACTTAATGATTGAGCGACAAGGTAAGCTTGGCGTGATCACCTTAGATCGTGTGACTCATCTCAATGCATTGTCGCTAGATATGATTGAAGGAATTGGCGCCCAATTGGAGTTGTGGCGAAATGATGCTGCTGTTCAGGCGATCTTAATCAAATCAAATAGCCCAAAAGCCTTCTGTGCAGGTGGCGATA	enoyl-CoA hydratase/isomerase family protein
      E. coli	CP115173.1	GTTCCTGACTATCTGGCGGGGAATGGTGTGGTTTATCAAACCAGTGATGTGAAGTATGTGATTGCCAACAACAACTTGTGGGCCAGCCCGTTGGATCAACAGTTGCGCAACACCCTGGTTGCCAACCTGAGTACGCAACTGCCCGGCTGGGTGGTTGCCTCCCAGCCTCTGGGAAGCGCCCAGGACACGCTCAATGTTACCGTAACGGAGTTTAACGGTCGCTATGATGGCAAGGTCATTGTCAGTGGTGAGTGGCTGTTGAACCACCAGGGACAACTGATCAAACGTCCGTTCCGTCTGGAAGGAGTGCAAACTCAGGATGGTTACGATGAGATGGTTAAAGTGCTGGCCGGTGTCTGGAGTCAGGAAGC	membrane integrity-associated transporter subunit PqiC
      K. pneumoniae	CP115714.1	CATGGGCATATCGACGGTCACCGATTCGCCCGGACGCAGGCGAACATCTTTTTCCGCCACCCGGTCCGCCTGCAGCGCCTGGCCATCGCCGGCAAACAGCGACGGGTAGTCAGTATTGTCGAACGCTTGCCGGTCCTTCAGCTGATAAATACGCACCACCGTCGCCAGGGAGGCGCCTTTGGCGTTGTTATTCACGCCTTCCCGGGCGCGAAGATCCAGATGCAGGGTTTTCACCTGGGGGTAAAAAATGGACTGGGTCACGGAAACGGCGCCGTCTTTCACGGTCTGCGTCAGGCCGCAGCCGGTTAAGACGGTAACCATGAGGAATGCCAGCAACCTGGCGGAGGGTTTAACTGCGGTAATCGCCATCTTCATCGCTCTCCCTGCGGTGGATATTTTCCCGGACGCGCTGATAGCGCCCCAGATAAATCGTGACTCTGTCGTCAGCCTGTTTCTGCGCATCCAGAGGACGCAGCACAGCGGTACGGCCGAGCTGGACGGCATGCTCCTGCTGGCAGCAAAGTTGCGCATCCGGCAGTAAATGGCGGGCGACGCAAAGCTGCAGCCGCACGTCAAGATGAGATCCGAGCCAGACGTGCAGAAGCGCCATCAGGTCGCTGAACAGTTCGCCGCCGGGCAGCCAGCCGCGAACCTCATCAGGCTTTTCCGTGGCCAGCTGCAGCAATACCTGACCGTTCACATCCGTGGCGTGGGTTCCCATCACTGGCCTATGTTGCAGG	type VI secretion system baseplate subunit TssG; type VI secretion system lipoprotein TssJ
      S. aureus	CP063802.1	AGGTGCAGTAGACGCATAGCATCATCATATTGAATAGTAAAAACAAATAAAACATAGTAACGTGATTCAGTCGATGTAACAGTCGATAATGAGTCACGTTTTTTTATAGAAAAATATAAGACATAAAAATGTCATAATTTATAGTCGACAAATATCATACTGTATAAACATTTATCATTTTCTCAAGTGCCTTTTACACGATGGAATGAACTTACTTTTTACGAAATTATGCGTATTTTATAAACAAATATCATTGATATAACGGTAAATGTAAGCGTTTACAACAGAAATAACTGCATGCTACGATATTTTTGTAAATTCACTGATTCAAGTATTTTAAGTCAATATGAGGAGGGATGTTATGAGCGATTCTGAGAAAGAAATTTTAAAAAGAATTAAAGATAATCCGTTTATTTCACAACGTGAACTTGCTGAGGCAATTGGATTATCTAGACCCAGCGTAGCAAACATTATTTCAGGATTAATACAAAAGGAATATGTTATGGGAAAGGCATATGTTTTAAATGAAGATTATCCTATTGTTTGTATTGGCGCAGCGAATG	winged helix-turn-helix transcriptional regulator
      P. aeruginosa	CP080405.1	TCAGTGACATGAAGCCCGTCCCCGCGGCCAGGCTGCGCAGTTGCGCCTACGAGATGGGTTTTTCTCTCTATCACGCCGGTCAATGCTTGCGCGTGGTTCTCTAGGTTCGACCGAATCAAGAGTGTGTTTTCAGCAAAATAATGATAGTTTGTTGTAAACACACATGAGGAGGTCGTCATGAGCGCTCTCATCAAGGAACGTCCCAGCGCCGATGCCGTCCTGGCCAAGGCCGTCCTGGCCGCGCGCGAGCAATTGGGGCTGACGCAGCTCGAACTGGCCGGCATCGTCGGCGTCGATCGCAGCGCCATCAGCCGCTGGAAGACCCAGGGCCTGCGGGTGGACAGCAAGACCGGCGAGCTGGCTCTGCTGCTGGTGCGAGTCTATCGCGCACTGTATGCCCTGTTCGGCGGGCAGCAGGAGGACATGCGCCACTTCCTGCGCACTCCCAACCATCACCTGGCGGGCGAGCCGCTGGCACTGATGGGACAGGTGCAGGGCCTGGTTCATGTGCTGGAATACCTCGATGCGA	DUF2384 domain-containing protein; FAD-dependent oxidoreductase
      S. epidermidis	CP106834.1	CACGCATGGCACTAGGTACAAATCCAGTATAAACAGCGTAGATTAATAATAGTAAAATAGTTACACCTTTAATGAGAACTAGAGGTATATTTAAACGTACTAACAGTTGATAAACGAGGACAATAATTGTACCAACATGTGTACCACTTATAGCTAGTAAATGGTAAATACCTATATCTTTGATGTTAGATTTGTAATATTCATCAATTAGAGAGGTATCACCTGTTATCAACGCTAATATTCTTTCAGGATGCGTGACCCCAGATTTATGAATTATTTTGGTGATATAATTTTGATGATGATATATAGGAGTGAAAATATTGTTTTCTTGGCAAGACTTAAATTGAACTGTTGAAATATTCAACAAGGACAACTCTTTTTCG	DNA internalization-related competence protein ComEC/Rec2
      S. capitis	AP014956.1	GGTTCAGTCATCCCCACGTTTTGATCTTCAATATTCATGTTATAATTTTCTCCTTTTTAAAAGGTTTCATTTACCATTGTTTACTACAGTAAAGGTTGTTTTAAACATATTCGTTTTAATATTTTTAGAGTGAAGATTCTAAAACGTGTGATAAAGGTGGAATATACACATAAAAATTTCCGATATAGAAAAATAAAAAGCTTTCAATCACCTTATTAATCAATAAAAAGTGATTAAAAGCTTTAAATTAAAAGAGGATAAACATCAATTCATATCTCAAACGCTTCTAACATTTTTGAGTTAGATGACTGAATTTGAACTGTGTTTATATCAAGCTTTTGTCTATTCTAGCCATCTTAGAAAACTATATAGAATGAATTCTTATTTAAGAAATCAATGCAGTATTTCTTTAAACTTCTGTTACTTCGACTTCGATGTGTTCGATGCGATGATATGCACCATGATCAACAGGTCCTACAAATTCATTCATTTTCCAACCGTTTTTGATTGCAGATGCTACGAAAGCTTTAGCAGCGATGACTGCTTCTCTTGGAGATTTGCCGTTAGCTAGGTAAGCAGTTGTCGCAGCTG	putative pyridoxine kinase; FAD binding superfamily protein
      E. faecalis	CP046113.1	CGGCAAGTTTGGAAGCAGACAAAGGTATTTTTGCAGTATTGAATGTCATCATTGGGATTAATGGTTTTGTTTGGAATGGTACATTGGCTATCGCTGGTTTGATTTTCGCTTTTTCTTGGGGCTATAACTTAGCGAAAGCATACAATGTGAATGAGCTAGCTGGTGGGATTGTCAGTTTAGCTACCTTAATTTCAGGTGTTGCTTTTGCACCAAATAACACAGCTGAATTAGCTGTAAAAGTGCCAGAAAAGATTGCCAACGCAATTAATGGGGCAGAAATTGGTGCGTCAATTGCTAATAATCAATTGACTGTCAATCCATGGGGTTGGTTAAATCTCAACCATCTAAATGGTAACGCTTTCTTTACGGTGATGATTATGGGCGCTTTATCAACCATTATTTTTTGTAAATTAATGCAAGCGGATTTAACAATAAAAATGCCTGATTCTGTACCACCAGCAGTTTCAAAAGCGTTTGCGGCAATTTTACCAGCAACAATCGCATTGTATGTCGTAGCAATTATTAACTTTACGGTGTCAAAACTTTCAGGCGGTCAATTATTAATTGACTTAATTCAAAAATATATCGCGGAACCGTTCCTTGGGTTATCACAAGGACTAGGCGCT	PTS sugar transporter subunit IIC
      E. faecium	CP112862.1	ATCGGAAATCGGTGTGGCTTATGCGAAAGGCATCCCAGTGATCGGCTTATATACTGATACTCGACAGCAAGGCGGAACCCACCCGAAAAAAATCGCTGCACTACAAGAAACAGCTGAAAATCAATTTCACTACCTGAACTTATATACAATAGGTTTGATCAAATTGAACGGAAAAGTGGTTTCTTCAGAAATTGAGTTGCTTTCAGAAGTAAAAAGATTTTTAGATGGAGGGACTTTCAGTGATTAAAGAAATCAAAAAGGTGCAAATAGCTTTACTTGCTTTTGGTGTATTCGTTGTCTGCTATAATTTTTATGAATTTATCACACAAAAATATTCGACTTCTCAAGGAATCACATTTATCGTGGAATCTTTATTAGGGATTGCATTGATTTTCATGCCACAAGTCATTTTGAAAGTTTTCAAACTTAAAATACCAGCAGCAATCGTTTTGTTTTACTGGTTTTTCTTGTTCATCTCTGTCTTTTTAGGCACAGGGATGCATTTGA	nucleoside 2-deoxyribosyltransferase; hypothetical protein
      S. maltophilia	CP040440.1	CCCGCGAGAAGCTGGATGCGCTGGAAACCGCCGTGCGCGAGCTGGAAGGGCGCGGCGCCGCAGAATGAAAAAAGGAGGCGCACCGCGCCTCCCGTTTACAGATTATTTCCCGGATGGGGCCTTGCAGCAAGGCCCACCGGGCGCCCTAGACTCGCCGGCCTGTCGATCCGGCAGCACAGAGAGGGGCAGTGCCGATGGCGCAGGACACCGAATGGACACCCGTTTCGCATGACACCTGCGACCAGGTCGCCGGGCCGTTCTATCACGGCACCCGCGCTGACCTGGCGGTGGGCGAGCTGCTGAGCGCGGGCTTCCGCTCCAACTATCGCGACAGCGTGGTGATGAACCACATCTACTTCACCACGATTGCCAAGGGCGCCGGGTTGGCTGCGGAGATGGCCCGGGGTGAAGGGCGACCGCGCGTGTATGTGGTGGAGCCGA	accessory factor UbiK family protein; NAD(+)--rifampin ADP-ribosyltransferase

Acknowledgments

This protocol was adapted from our previously published work (Wang et al., 2022). This work was supported by National Natural Science Foundation of China (81927808).

Competing interests

The authors declare no competing interests.

Ethics

This study was registered in English at https://clinicaltrials.gov/ (NCT04178382) in November 2019.

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