Bio Epidemiological Genomics Amr Surveillance

Version Compatibility

Reference examples tested with: ncbi-amrfinderplus 4.0+, resfinder 4.5+, rgi 6.0+ (CARD 3.3+), abritamr 1.0.14+, staramr 0.10+, hamronization 1.1+, tb-profiler 6.2+, mykrobe 0.13+, mob_suite 3.1+, plasmidfinder 2.1+, MobileElementFinder 1.0+, pandas 2.2+, BioPython 1.84+.

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags
• AMRFinderPlus: amrfinder --list_organisms for the current --organism catalogue
• WHO Mtb catalogue: tb-profiler list_db and verify the bundled WHO edition
• AMR database freshness: amrfinder -u (NCBI ReferenceGeneCatalog) and tb-profiler update_tbdb

If a flag or column name does not match (--species vs --organism, barcode_build vs barcode-build), introspect the installed package rather than retrying. AMRFinderPlus, RGI, and ResFinder all renamed columns between major releases.

AMR Surveillance

"What antibiotic-resistance determinants are in this assembly, and what susceptibility do they imply?" -> Combine acquired-gene detection, chromosomal point-mutation calling, mobile-element context, and curated phenotype mapping into a single per-isolate report fit for surveillance or clinical handover. Tool choice is determined by species (Mtb needs TB-Profiler, not AMRFinderPlus), reporting standard (WHO GLASS vs CARD vs in-house), and whether mobility matters (carbapenemase outbreak: yes; routine ESBL screen: less so).

• CLI: amrfinder -n assembly.fa --organism Klebsiella_pneumoniae --plus -- acquired + intrinsic-aware point mutations
• CLI: tb-profiler profile -1 r1.fq.gz -2 r2.fq.gz -p sample -- TB drug-resistance against WHO 2nd-edition catalogue
• CLI: hamronize amrfinderplus --analysis_software_version 4.0.3 --reference_database_version 2025-02-01.1 in.tsv > out.tsv -- normalise output across tools
• Python: pandas to join AMRFinderPlus + RGI + ResFinder + MOB-suite per-isolate for a single decision table

The Single Most Important Modern Insight -- Acquired AMR detection is not species-agnostic

A pan-species AMRFinderPlus run misses chromosomal point-mutation resistance because the point-mutation panels are species-specific and activated only when --organism is set. Running amrfinder -n mtb.fa without --organism returns "no AMR detected" on an XDR-TB genome because AMRFinderPlus has no Mtb organism mode -- the user must switch tools (TB-Profiler or Mykrobe + WHO 2nd-edition catalogue). Similarly, Salmonella gyrA T83I, Klebsiella mgrB inactivation, and E. coli QRDR mutations are silent without --organism Salmonella / Klebsiella_pneumoniae / Escherichia. For any cross-tool surveillance pipeline, AMRFinderPlus with --organism MUST be paired with a species-specific second tool (TB-Profiler for Mtb; ResFinder 4.0 with -s 'species' for PointFinder coverage; hAMRonization to merge). Andersson et al. Nat Rev Microbiol 17:479 (2019) further notes that heteroresistance at 0.1-1% allele frequency is widespread and invisible to default variant callers, compounding the failure mode for any single-tool workflow.

Algorithmic Taxonomy

Decision Tree by Scenario

Methodology evolves rapidly; before a high-stakes outbreak report, web-search "AMRFinderPlus organism modes 2026" and confirm the WHO Mtb catalogue edition currently bundled in TB-Profiler.

AMRFinderPlus With Species Mode

Goal: Produce a per-isolate AMR report including acquired genes, species-specific chromosomal point mutations, stress/virulence elements, and per-hit method and coverage metadata for downstream harmonisation.

Approach: Run amrfinder with -n for nucleotide assembly, --organism to activate the species-specific point-mutation panel, --plus to include stress / virulence / heat / metal, and --report_all when truncated-gene visibility matters. Pin the database via amrfinder -u -> verify --db matches a recorded date.

DB=$(amrfinder -V | grep -i database | awk '{print $NF}')

amrfinder \
    -n assembly.fa \
    --organism Klebsiella_pneumoniae \
    --plus \
    --threads 8 \
    -o sample.amrfinder.tsv

echo "DB version: ${DB}" >> sample.amrfinder.tsv

Column semantics worth inspecting: Element type (AMR / POINT / VIRULENCE / STRESS / etc.), Method (EXACTX / PARTIAL_CONTIG_END / HMM / etc. -- partial-contig hits flag assembly fragmentation), % Coverage of reference sequence, % Identity to reference sequence, Class / Subclass (drug-class summary). Method = PARTIAL_CONTIG_END is the smoking gun for plasmid-context fragmentation -- consider long-read confirmation.

TB-Specific Workflow

Goal: Generate a WHO-catalogue-aligned drug-resistance report for M. tuberculosis covering all 13 first-line + second-line + new/repurposed drugs, with lineage assignment via the Coll/Napier MTBC barcode (90-SNP version) and explicit handling of Group 3 (Uncertain) mutations.

Approach: TB-Profiler primary (it bundles the WHO catalogue and reports the Walker 2022 / 2023 association grouping); Mykrobe as an orthogonal cross-check on any R / XDR call that will drive treatment. NEVER collapse Group 3 mutations to "susceptible" -- the WHO catalogue's tiered scoring is load-bearing for clinical handover.

tb-profiler update_tbdb

tb-profiler profile \
    -1 reads_R1.fq.gz \
    -2 reads_R2.fq.gz \
    -p sample \
    --txt --csv --pdf \
    --dir tbprofiler_out

mykrobe predict \
    --sample sample \
    --species tb \
    --output sample.mykrobe.json \
    --format json \
    reads_R1.fq.gz reads_R2.fq.gz

Allix-Béguec et al. (CRyPTIC) NEJM 379:1403 (2018) established >99% negative predictive value for first-line drugs and is the basis for WGS-only DST policies; the same study showed sensitivity remains lower for bedaquiline, delamanid, linezolid, and clofazimine, so phenotypic DST is still required for second-line and new/repurposed agents in MDR/XDR-TB workups. The WHO 2023 catalogue (Walker et al. Lancet Microbe 3:e265, 2022 for the 2021 edition methodology; 2023 second edition data) grades each mutation Group 1 / Group 2 (Associated -- interim) / Group 3 (Uncertain) / Group 4 (Not Associated -- interim) / Group 5 (Not Associated). Reporting Group 3 as "S" actively misleads clinicians.

hAMRonization for Cross-Tool Reporting

Goal: Convert per-tool AMR output (AMRFinderPlus, ResFinder, RGI, abricate, staramr, ARIBA, TB-Profiler, Mykrobe) to the PHA4GE schema so a multi-laboratory or multi-tool surveillance dataset is comparable.

Approach: Invoke hamronize <tool> per input with mandatory provenance metadata (--analysis_software_version, --reference_database_version, --input_file_name), then hamronize summarize to merge.

hamronize amrfinderplus \
    --analysis_software_version 4.0.3 \
    --reference_database_version 2025-02-01.1 \
    --input_file_name sample.amrfinder.tsv \
    sample.amrfinder.tsv > sample.hamr.tsv

hamronize resfinder \
    --analysis_software_version 4.5.0 \
    --reference_database_version 2024-12-15 \
    --input_file_name sample.resfinder.json \
    sample.resfinder.json > sample.resfinder.hamr.tsv

hamronize summarize -t tsv -o cohort.hamr.tsv per_sample_hamr/*.tsv

Default reporting ontology for public-health output is NCBI ReferenceGeneCatalog. Two consequences: (1) CARD ARO terms need translation; (2) any custom gene additions need a corresponding NCBI accession before they appear in the harmonised stream.

Mobile-Genetic-Element Context

Goal: Determine whether a clinically actionable AMR gene (carbapenemase, mcr, ESBL) sits on a chromosome, a plasmid (and which incompatibility group / cluster), an integrative-conjugative element, or a transposon -- because "gene present" tells the infection-control team nothing about transmissibility.

Approach: Reconstruct plasmids from the assembly with MOB-suite mob_recon; type each plasmid with mob_typer; cross-reference AMR-gene coordinates against the plasmid contig list; annotate IS elements / integrons with MobileElementFinder. For surveillance-grade plasmid resolution, prefer long-read (R10.4.1 Q20+) or hybrid assemblies -- short-read draft assemblies routinely fragment plasmid contigs.

mob_recon \
    --infile assembly.fa \
    --outdir mob_out/ \
    --num_threads 4

mob_typer \
    --infile mob_out/plasmid_AA001.fasta \
    --out_file mob_out/plasmid_AA001.typed.tsv

mefinder find \
    --contig assembly.fa \
    --out mef_out/sample \
    --threads 4

Document MOB-suite version explicitly: v2 and v3 cluster codes are non-interoperable, and v3.1+ adds MGE reporting. Across longitudinal surveillance crossing the v2 -> v3 boundary, the same plasmid receives different cluster IDs and appears spuriously "novel".

Per-Method Failure Modes

AMRFinderPlus run pan-species on a Mycobacterium tuberculosis assembly

Trigger: amrfinder -n mtb.fa without --organism; AMRFinderPlus has no Mtb organism mode in any v4.x release.

Mechanism: The species-specific point-mutation panels are activated only when a recognised --organism is passed. Mtb resistance is overwhelmingly chromosomal point mutation (rpoB / katG / inhA / pncA / embB / gyrA / rrs). With no panel active, none of these are called.

Symptom: Empty AMR table on a phenotypically MDR/XDR isolate; lineage barcode not reported (AMRFinderPlus does not call MTBC lineage).

Fix: Switch tools. Use TB-Profiler (preferred -- bundles WHO catalogue + Coll/Napier barcode) or Mykrobe; the AMRFinderPlus catalogue does not cover MTBC.

OXA-48-like family collapsed to a single bucket

Trigger: Default per-isolate summarisers report the gene family (bla_OXA-48-like) rather than the allele (bla_OXA-244).

Mechanism: OXA-48 (potent carbapenemase), OXA-181 (Thr213Ala -- similar activity, different plasmid), OXA-232 (Arg214Ser -- reduced carbapenemase activity), and OXA-244 (Arg214Gly -- weakest, often phenotypically susceptible to ertapenem) share >95% identity. Pipelines that report family-level summary erase the clinically actionable variant identity.

Symptom: Surveillance report says "OXA-48-like detected", phenotype is borderline ertapenem-susceptible / meropenem-susceptible, clinical team is confused about whether ceftazidime-avibactam is needed.

Fix: Report the allele explicitly; never collapse to family. AMRFinderPlus reports the allele in Gene symbol -- preserve it through hAMRonization rather than summarising.

IS-element-mediated derepression invisible to gene-presence pipelines

Trigger: Phenotypic high-level AmpC hyperproduction in Enterobacter cloacae complex / Citrobacter freundii; or KPC over-expression on Tn4401b (100-bp promoter deletion).

Mechanism: Resistance is mediated by ISEcp1 / IS26 / IS10 insertion upstream of the ampC promoter producing a strong hybrid promoter, or by Tn4401 variant. Gene presence is unchanged. Short-read assemblies fragment IS elements and collapse repeats; promoter context is lost. AMRFinderPlus / RGI / ResFinder report the gene; the regulatory configuration is not annotated.

Symptom: AMR pipeline output identical for wild-type ampC carrier and the hyperproducer; clinical phenotype is divergent.

Fix: Long-read (Nanopore R10.4.1 Q20+ or PacBio HiFi) or hybrid assembly with explicit promoter-context inspection. For Tn4401 variant typing, manual blast of the transposon region. Tools that automate this are emerging but not yet a default in surveillance pipelines.

Heteroresistance below default variant-caller thresholds

Trigger: Routine Illumina WGS-AMR pipeline on a clinical isolate; resistance variant appears at 1-5% allele frequency.

Mechanism: Heteroresistance -- clonal subpopulations carrying resistance at 0.1-1% (Andersson, Nicoloff, Hjort Nat Rev Microbiol 17:479, 2019) -- is widespread and clinically meaningful (treatment failure under antibiotic pressure). Default variant callers (bcftools, lofreq default -q 20, GATK HaplotypeCaller) require minor-allele frequency of typically 10-20% to call. Assembly-based pipelines (AMRFinderPlus on assembly) collapse minor variants entirely.

Symptom: Surveillance pipeline calls "S" but clinical failure under therapy; subsequent sampling reveals high-level resistance.

Fix: For clinically critical drugs, supplement assembly-based AMR with deep-read variant calling at lower MAF thresholds (lofreq with -q 13 -a 0.01), or use targeted deep-amplicon sequencing. TB-Profiler reports allele frequency for resistance calls -- inspect for heteroresistance routinely.

WHO Mtb Group 3 mutations silently called "susceptible"

Trigger: TB-Profiler / Mykrobe run on an Mtb isolate carrying a mutation graded Group 3 (Uncertain) in the WHO catalogue.

Mechanism: Tools translate Group 3 calls to "no resistance prediction" in their summary; downstream summarisers and clinical handover forms collapse "no prediction" to "S". The Walker 2022 Lancet Microbe catalogue methodology explicitly separates Group 3 (Uncertain) from Group 4/5 (Not Associated) for exactly this reason.

Symptom: Patient treated as drug-susceptible; clinical failure; retrospective inspection finds Group 3 mutation that should have triggered phenotypic DST.

Fix: Read TB-Profiler JSON output, NOT the simplified TSV. Report Group 3 mutations explicitly with "uncertain significance -- phenotypic DST recommended". This is widely under-communicated in surveillance pipelines.

fosA / efflux-regulator hits overcalled as resistance

Trigger: Chromosomal fosA in Klebsiella pneumoniae / Enterobacter / Serratia reported as "fosfomycin resistant" by ResFinder / AMRFinderPlus.

Mechanism: Chromosomal fosA confers low-level fosfomycin resistance below the EUCAST clinical breakpoint (32 mg/L for urinary isolates). Treating chromosomal-fosA E. coli as fosfomycin-resistant withholds an oral option for uncomplicated UTI unnecessarily.

Symptom: All K. pneumoniae isolates flagged "fosfomycin-R" regardless of plasmid context or phenotype.

Fix: AMRFinderPlus with --organism Klebsiella_pneumoniae suppresses the intrinsic fosA report; verify the suppression worked. Cross-reference fosA hits with MOB-suite to determine plasmid context; only plasmid-borne or upregulated fosA should be reported as resistance.

Reconciliation: When AMR Tools Disagree

Quantitative Thresholds

Common Errors

Anticipated Reviewer Pushback

References

• Feldgarden M, Brover V, Haft DH et al (2021) AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci Rep 11:12728. doi:10.1038/s41598-021-91456-0
• Bortolaia V, Kaas RS, Ruppe E et al (2020) ResFinder 4.0 for predictions of phenotypes from genotypes. J Antimicrob Chemother 75(12):3491-3500. doi:10.1093/jac/dkaa345
• Alcock BP, Huynh W, Chalil R et al (2023) CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res 51(D1):D690-D699. doi:10.1093/nar/gkac920
• Phelan JE, O'Sullivan DM, Machado D et al (2019) Integrating informatics tools and portable sequencing technology for rapid detection of resistance to anti-tuberculous drugs. Genome Med 11:41. doi:10.1186/s13073-019-0650-x
• Hunt M, Bradley P, Lapierre SG et al (2019) Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe. Wellcome Open Res 4:191. doi:10.12688/wellcomeopenres.15603.1
• Walker TM et al (CRyPTIC / WHO) (2022) The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis. Lancet Microbe 3(4):e265-e273. doi:10.1016/S2666-5247(21)00301-3
• Allix-Béguec C et al (CRyPTIC) (2018) Prediction of susceptibility to first-line tuberculosis drugs by DNA sequencing. N Engl J Med 379(15):1403-1415. doi:10.1056/NEJMoa1800474
• Robertson J, Nash JHE (2018) MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genom 4(8):e000206. doi:10.1099/mgen.0.000206
• Carattoli A, Zankari E, García-Fernández A et al (2014) In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58(7):3895-3903. doi:10.1128/AAC.02412-14
• Johansson MHK, Bortolaia V, Tansirichaiya S et al (2021) Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder. J Antimicrob Chemother 76(1):101-109. doi:10.1093/jac/dkaa390
• Sherry NL, Horan KA, Ballard SA et al (2023) An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance. Nat Commun 14:60. doi:10.1038/s41467-022-35713-4
• Andersson DI, Nicoloff H, Hjort K (2019) Mechanisms and clinical relevance of bacterial heteroresistance. Nat Rev Microbiol 17(8):479-496. doi:10.1038/s41579-019-0218-1
• Liu YY, Wang Y, Walsh TR et al (2016) Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China. Lancet Infect Dis 16(2):161-168. doi:10.1016/S1473-3099(15)00424-7

Related Skills

• pathogen-typing - Strain context for AMR (Kleborate, MLST, cgMLST) feeds clonal interpretation of resistance dissemination
• transmission-inference - Outbreak transmission inference for resistant clones uses AMR + cgMLST jointly
• variant-surveillance - Lineage-level AMR-prevalence tracking for SARS-CoV-2-style surveillance (drug-resistance mutations in antiviral context)
• metagenomics/amr-detection - Community AMR / ARG quantification (NOT isolate-focused)
• variant-calling/variant-calling - Per-isolate SNP calling that feeds point-mutation panels
• variant-calling/filtering-best-practices - MAF threshold discipline for heteroresistance detection
• long-read-sequencing/long-read-alignment - Plasmid / promoter-context resolution for MGE-aware AMR
• comparative-genomics/whole-genome-alignment - Reference-based coordinate handling for point mutations
• clinical-databases/pharmacogenomics - Adjacent (host-side) pharmacogenetics, distinct from pathogen AMR
• workflows/somatic-variant-pipeline - End-to-end orchestration patterns (analogous to outbreak-pipeline workflows)