SUMMARY RESULTS

Run name: 20231220

Time point: 3h

Table 1: Summary results
Sample Organism* Polymicrobial QC† Predicted resistance phenotype‡
SRR22957083 Streptococcus pyogenes no pass None detected
SRR26162843 Escherichia coli no pass Amikacin, Amoxicillin+Clavulanate, Ampicillin, Cefepime, Cefotaxime, Cefoxitin, Ceftriaxone, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Meropenem, Piperacillin, Piperacillin+Tazobactam, Sulfamethoxazole, Tetracycline, Tobramycin, Trimethoprim
* Organisms flagged with asterisks (*) indicate samples with Level 1 species identification due to a low number of reads.
† Samples failed QC if fewer than 2 % of reads and fewer than 10 reads total mapped to one species or if the median read length was < 500 bases or if the median read quality Q-score was < 10 .
‡ Predicted phenotype is derived from the Centre for Genomic Epidemiology ResFinder database does not represent all resistant determinants or point mutations. These phenotypes have not been completely validated in all organisms and may not represent the true phenotype for each antimicrobial. Predicted phenotypes only show those antimicrobials included in the Clinical and Laboratory Standards Institute document Performance Standards for Antimicrobial Susceptibility Testing: Informational Supplement M100 ED35:2025 Table 1 (Tier 1 and Tier 2). Please see Table 5 for the full list of reportable antimicrobials in Tier 1 and Tier 2 for each organism. “Could not predict” indicates AMR prediction is not supported for this organism or that there was not enough sequencing data to generate an assembly.

 

DETAILED RESULTS

 

SEQUENCING METRICS

Sequencing read quality and length metrics are found in Table 2.

Table 2: Sequencing quality and read length metrics
Real-time genome assembly stats
Estimated genome assembly stats
Read and Q-scores
Sample Completeness (%)* Assembled depth of coverage (fold)† Assembled size (Mb) Estimated depth of coverage (fold)‡ Estimated size (Mb)§ % reads > 1 kb and Q-score > 10 % bases > 1 kb and Q-score > 10 Number of reads > 1 kb Median read length (kb) Median read quality (Q-score)
SRR22957083 87.6 15.7 1.8 15.3 2.1 44.3 74.7 10846 1.82 14.2
SRR26162843 92.3 3.9 5.0 6.8 5.1 99.8 99.8 4236 5.05 17.6
* Assembly completeness values are derived from CheckM2. NA indicates no assembly for this isolate or that this isolate is a polymicrobial culture or fungus. Note AMR phenotypes may be missing if completeness < 90 %.
† Assembled depth of coverage was determined by mapping reads to the assembled genome.
‡ Estimated genome depth of coverage was determined by dividing total bases sequenced by estimated genome size (varies per organism). Values below 5X are flagged in red and values below 10X are flagged in yellow. NA indicates not enough reads to identify an organism in that sample.
§ Estimated size was obtained from the average size of all assembled RefSeq genomes for each genus in the Bacteria (taxid:2) dataset from the National Center for Biotechnology Information (NCBI). The estimated size is a sum of genome sizes for all organisms found in the sample.

 

ORGANISM IDENTIFICATION

The following species were identified for each sample in Figure 1.

Figure 1: Species ID and read count for each sample. These results are from Level 2 species identification which cannot identify species with low read counts (flagged with asterisks). Level 1 species identification for low read count samples can be found in Table 1. For more information of Level 1 and Level 2 species identification, please see Methods.

STAPHYLOCOCCUS AUREUS TOXINS

No Staphylococcus aureus isolates detected.

STREPTOCOCCUS PYOGENES emm-TYPING

Table 3: emm types predicted in Streptococcus pyogenes isolates
Sample Predicted emm type* emm cluster*
SRR22957083 EMM12.0 A-C4
* emm types and clusters generated via the emmtyper tool which uses the U.S. Centers for Disease Control and Prevention trimmed emm subtype database. NA indicates no emm type was detected, possibly due to an incomplete assembly.

 

ANTIMICROBIAL RESISTANCE

All samples had enough reads for assembly-based antmicrobial resistance gene detection with StarAMR and AMRFinder

StarAMR (assembly-based)

Table 4: Antimicrobial resistance genes detected by StarAMR
Sample Gene Percent identity (%)* Percent coverage (%)* Predicted resistance phenotype†
SRR22957083 None NA NA NA
SRR26162843 aadA2 99.87 100 Spectinomycin, Streptomycin
SRR26162843 aph(3’’)-Ib 100 100 Streptomycin
SRR26162843 aph(6)-Id 99.88 100.12 Streptomycin
SRR26162843 blaCMY-42 100 86.3 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefotaxime, Cefoxitin, Ceftazidime, Piperacillin, Piperacillin+Tazobactam, Ticarcillin, Ticarcillin+Clavulanic acid
SRR26162843 blaCTX-M-15 100 100 Amoxicillin, Ampicillin, Aztreonam, Cefepime, Cefotaxime, Ceftazidime, Ceftriaxone, Piperacillin, Ticarcillin
SRR26162843 blaNDM-5 100 100 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefepime, Cefixime, Cefotaxime, Cefoxitin, Ceftazidime, Ertapenem, Imipenem, Meropenem, Piperacillin, Piperacillin+Tazobactam, Temocillin
SRR26162843 blaOXA-181 100 100 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefepime, Ertapenem, Imipenem, Meropenem, Piperacillin, Piperacillin+Tazobactam
SRR26162843 blaTEM-1B 100 100 Amoxicillin, Ampicillin, Cephalothin, Piperacillin, Ticarcillin
SRR26162843 dfrA12 100 100 Trimethoprim
SRR26162843 erm(B) 98.95 100.26 Erythromycin, Lincomycin, Clindamycin, Quinupristin, Pristinamycin IA, Virginiamycin S
SRR26162843 gyrA (D87N) 99.5 100 Nalidixic acid;Nalidixic acid,Ciprofloxacin
SRR26162843 gyrA (S83L) 99.5 100 Nalidixic acid,Ciprofloxacin
SRR26162843 mph(A) 100 99.67 Erythromycin, Azithromycin, Spiramycin, Telithromycin
SRR26162843 parC (S80I) 99.87 100 Nalidixic acid,Ciprofloxacin
SRR26162843 parE (S458A) 97.41 100 Nalidixic acid,Ciprofloxacin
SRR26162843 qnrS1 99.85 100.15 Ciprofloxacin
SRR26162843 rmtB 100 100 Amikacin, Gentamicin, Tobramycin, Kanamycin, Arbekacin, Sisomicin, Isepamicin
SRR26162843 sul1 100 100 Sulfamethoxazole
SRR26162843 sul2 100 100 Sulfamethoxazole
SRR26162843 tet(A) 100 100 Doxycycline, Tetracycline
* Percent identity and percent coverage are relative to the StarAMR reference gene. Percent identity below 98 % and percent coverage below 90% are flagged in red and should be interpreted with caution.
† Predicted phenotype is derived from the Centre for Genomic Epidemiology ResFinder database and does not represent all resistant determinants or point mutations. These phenotypes have not been completely validated in all organisms and may not represent the true phenotype for each antimicrobial.

KmerResistance (read-based)

Table 5: Antimicrobial resistance genes detected by KmerResistance
Sample Gene Percent identity (%)* Percent coverage (%)* Predicted resistance phenotype†
SRR22957083 None NA NA NA
SRR26162843 aadA2_2_JQ364967 100 100 Spectinomycin, Streptomycin
SRR26162843 aph(3’’)-Ib_5_AF321551 100 100 Streptomycin
SRR26162843 aph(6)-Id_4_CP000971 99.88 100 Streptomycin
SRR26162843 blaCMY-42_1_HM146927 100 100 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefotaxime, Cefoxitin, Ceftazidime, Piperacillin, Piperacillin+Tazobactam, Ticarcillin, Ticarcillin+Clavulanic acid
SRR26162843 blaCTX-M-15_1_AY044436 100 100 Amoxicillin, Ampicillin, Aztreonam, Cefepime, Cefotaxime, Ceftazidime, Ceftriaxone, Piperacillin, Ticarcillin
SRR26162843 blaNDM-5_1_JN104597 100 100 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefepime, Cefixime, Cefotaxime, Cefoxitin, Ceftazidime, Ertapenem, Imipenem, Meropenem, Piperacillin, Piperacillin+Tazobactam, Temocillin
SRR26162843 blaOXA-181_1_CM004561 100 100 Amoxicillin, Amoxicillin+Clavulanic acid, Ampicillin, Ampicillin+Clavulanic acid, Cefepime, Ertapenem, Imipenem, Meropenem, Piperacillin, Piperacillin+Tazobactam
SRR26162843 blaTEM-1B_1_AY458016 100 100 Amoxicillin, Ampicillin, Cephalothin, Piperacillin, Ticarcillin
SRR26162843 dfrA12_8_AM040708 100 100 Trimethoprim
SRR26162843 erm(B)_1_JN899585 99.86 100 Erythromycin, Lincomycin, Clindamycin, Quinupristin, Pristinamycin IA, Virginiamycin S
SRR26162843 mph(A)_1_D16251 99.56 100 Erythromycin, Azithromycin, Spiramycin, Telithromycin
SRR26162843 mph(A)_2_U36578 99.78 101.09 Erythromycin, Azithromycin, Spiramycin, Telithromycin
SRR26162843 qnrS1_1_AB187515 100 100 Ciprofloxacin
SRR26162843 rmtB_1_AB103506 100 100 Amikacin, Gentamicin, Tobramycin, Kanamycin, Arbekacin, Sisomicin, Isepamicin
SRR26162843 sul1_2_U12338 100 100 Sulfamethoxazole
SRR26162843 sul2_2_AY034138 100 100 Sulfamethoxazole
SRR26162843 tet(A)_4_AJ517790 100 100 Doxycycline, Tetracycline
* Percent identity and percent coverage are relative to the Centre for Genomic Epidemiology ResFinder database. Percent identity below 98 % and percent coverage below 90 % are flagged in red and should be interpreted with caution.
† Predicted phenotype is derived from the Centre for Genomic Epidemiology ResFinder database and does not represent all resistant determinants or point mutations. These phenotypes have not been completely validated in all organisms and may not represent the true phenotype for each antimicrobial.

 

METHODS

  • Blood cultures flagged positive by the BACT/ALERT 3D automated microbial detection system (Biomerieux, Saint-Laurent, Canada) at HSC were aliquoted in vials sent to NML by courier
  • DNA was immediately extracted upon receipt using the QIAamp BiOstic Bacteremia DNA Kit (Qiagen) (approximate time: 2 h)
  • DNA was quantified on a Qubit using the Broad Range kit (approximate time: 15 mins)
  • The Rapid Barcoding Kit v14 (SQK-RBK114.24) was used for library preparation according to the manufacturer’s instructions (approximate time: 2 h)
  • The final library was loaded onto a R10.4.1 flow cell (approximate time: 15 mins)
  • Data was basecalled using Dorado v0.9.0 and the SUP (super accurate model) basecalling model v5.0.0
  • The venae pipeline v0.1.0 managed by Snakemake was run to analyze the sequencing results and output this report
  • Human reads were removed and discarded with NoHuman v0.3.0 using the Kraken2 Human Pangenome Reference Consortium database: v1 (2023-09)
  • Samples passed quality control (QC) measures if all of the following criteria were met:
    • More than 2 % of reads and more than 10 reads total mapped to one species
    • Median read length was > 500 bases
    • Median read quality Q-score was < 10
  • Assemblies were generated with Flye v2.9.4 using reads that were >1 kb in length
  • Species identification was performed with the following tools using reads that were >2 kb in length:
    • Level 1 identification for evaluting samples with low read counts: Kraken2 v2.1.4 using the plusPF-2024-09-04-16Gb database
    • Level 2 identification for evaluating samples with high read counts for more accurate polymicrobial identification: Sylph v0.8.0 using the bacteria dbv1 GTDB r220 (113,104 genomes 2024-04-24) database and the fungal RefSeq v0.3 -c200 2024-07-25 (595 genomes) database
  • Antimicrobial resistance gene detection was performed using the following tools and required a minimum > 90% identity to the reference gene:
    • StarAMR v0.11.0 using the following databases: ResFinder Tue, 24 May 2022 06:51 (db commit fa32d9a3cf0c12ec70ca4e90c45c0d590ee810bd), PlasmidFinder Wed, 18 Jan 2023 09:45 (c18e08c17a5988d4f075fc1171636e47546a323d), MLST v2.23.0
    • kmerResistance v2.2.0 using the ResFinder v2.4.0 (2024-08-06) database and the KmerFinder bacteria 2024-01-30 database
  • Staphylococcus aureus isolates were screened for the following toxin genes: eta, etb, tsst-1, sea, seb, sec, sed, see, seh, selk, sell, selq, lukK and lukS using the following tool with a minimum > 80% identity to the reference gene:
  • Streptococcus pyogenes isolates were screened for the M protein gene (emm) using the following tool with a minimum > 95% identity to the reference U.S. Centers for Disease Control and Prevention trimmed emm subtype database.
  • HTML results report generated with R v4.4.3

 

DISCLAIMER

  • Testing was performed, in whole or in part using a research-based test
  • Whole genome sequencing is not an ISO accredited test and results are validated and measured for quality based on in-house workflows
  • Interpretation of the results are based on in-house workflows
  • Predicted phenotype is derived from the Centre for Genomic Epidemiology ResFinder database does not represent all resistant determinants or point mutations.
    • These phenotypes have not been completely validated in all organisms and may not represent the true phenotype for each antimicrobial.
  • Predicted phenotypes in the Summary Table 1 only show those antimicrobials included in the Clinical and Laboratory Standards Institute document Performance Standards for Antimicrobial Susceptibility Testing: Informational Supplement M100 ED35:2025 Table 1 (Tier 1 and Tier 2).
    • Please see Table 6 for the full list of reportable antimicrobials in Tier 1 and Tier 2 for each organism
    • Note that this workflow cannot confirm the following Tier 1 and Tier 2 antimicrobials:
      • Enterobacterales: Ampicillin+Sulbactam
      • Enterobacterales, Salmonella, Shigella, Pseudomonas aeruginosa, Stenotrophomonas, Burkholderia, non-Enterobacterales Gram negatives, and Streptococcus pneumoniae: Levofloxacin
      • Staphylococcus species and Enterococcus species: Daptomycin
      • Streptococcus pneumoniae: Moxifloxacin
      • Neisseria meningitidis: Cefotaxime, Ceftriaxone, Penicillin
Table 6: Tier 1 and Tier 2 reportable antibiotics from CLSI Performance Standard M100 ED34:2024 Table 1
CLSI Table Organism Antibiotics
Table1A-1 Enterobacterales Amikacin, Amoxicillin+Clavulanate, Ampicillin, Ampicillin+Sulbactam, Cefazolin, Cefepime, Cefotaxime, Cefotetan, Cefoxitin, Ceftriaxone, Cefuroxime, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Levofloxacin, Meropenem, Piperacillin, Piperacillin+Tazobactam, Sulfamethoxazole, Tetracycline, Tobramycin, Trimethoprim, Trimethoprim+Sulfamethoxazole
Table1A-2 Salmonella, Shigella Ampicillin, Azithromycin, Cefotaxime, Ceftriaxone, Ciprofloxacin, Levofloxacin, Sulfamethoxazole, Trimethoprim, Trimethoprim+Sulfamethoxazole
Table1B-1 Pseudomonas Cefepime, Ceftazidime, Ciprofloxacin, Imipenem, Levofloxacin, Meropenem, Piperacillin, Piperacillin+Tazobactam, Tobramycin
Table1B-2 Acinetobacter Amikacin, Cefepime, Ceftazidime, Ciprofloxacin, Gentamicin, Imipenem, Levofloxacin, Meropenem, Minocycline, Piperacillin, Piperacillin+Tazobactam, Sulfamethoxazole, Tazobactam, Tobramycin, Trimethoprim, Trimethoprim+Sulfamethoxazole
Table1B-4 Stenotrophomonas Levofloxacin, Minocycline, Trimethoprim+Sulfamethoxazole
Table1B-5 Non-Enterobacterales Amikacin, Aztreonam, Cefepime, Ceftazidime, Ciprofloxacin, Gentamycin, Imipenem, Levofloxacin, Meropenem, Minocycline, Piperacillin, Piperacillin+Tazobactam, Sulfamethoxazole, Tobramycin, Trimethoprim, Trimethoprim+Sulfamethoxazole
Table1C Staphylococcus Azithromycin, Cefoxitin, Clarithromycin, Clindamycin, Daptomycin, Doxycycline, Erythromycin, Linezolid, Minocycline, Oxacillin, Penicillin, Sulfamethoxazole, Tetracycline, Trimethoprim, Trimethoprim+Sulfamethoxazole, Vancomycin
Table1D Enterococcus Ampicillin, Daptomycin, Gentamicin, Linezolid, Penicillin, Vancomycin
Table1E Haemophilus influenzae Amoxicillin+Clavulanate, Ampicillin, Ampicillin+Sulbactam, Cefotaxime, Ceftazidime, Ceftriaxone, Ciprofloxacin, Levofloxacin, Moxifloxacin, Sulfamethoxazole, Trimethoprim, Trimethoprim+Sulfamethoxazole
Table1G Streptococcus pneumoniae Cefotaxime, Ceftriaxone, Clindamycin, Doxycycline, Erythromycin, Levofloxacin, Meropenem, Moxifloxacin, Penicillin, Sulfamethoxazole, Tetracycline, Trimethoprim, Trimethoprim+Sulfamethoxazole, Vancomycin
Table1H-1 Streptococcus (Group A and B) Ampicillin, Clindamycin, Erythromycin, Penicillin, Tetracycline
Table1H-2 Streptococcus vgs Ampicillin, Clindamycin, Erythromycin, Penicillin, Tetracycline
Table1I Neisseria meningitidis Cefotaxime, Ceftriaxone, Penicillin

 

DATA DICTIONARY

  • ONT: Oxford Nanopore Technologies
  • Q-score: a quality score indicating the probability of correct identification of the nucleotides generated by DNA sequencing. A Q-score of 20 (Q20) represents an error rate of 1 in 100 (meaning every 100 bp sequencing read may contain an error).
  • QC: quality control measure. Samples are assigned pass/fail in the QC columns of the Summary table if total bases sequenced divided by genome size (average depth of coverage) is greater than 10-fold.
  • CLSI: Clinical and Laboratory Standards Institute
  • AMR: antimicrobial resistance