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#58 Tier 1: Exploratory 🧪🧪🧪 3/5 • Comparative Synthesis Comparative Genomics & Antimicrobial Resistome

Hierarchical Antimicrobial Resistance, Porin Disruption & Mobile HGT Island Calibration: Benchmarking Against NCBI AMRFinderPlus

Evaluating 2,253-node Reference Gene Hierarchy traversal, protein/rRNA/promoter point mutations, cefiderocol/carbapenem porin disruptions, and tetranucleotide Kullback-Leibler HGT island divergence across 10 ESKAPE benchmark cohorts

By Bryan P. White • 14 min read • Medium (Undergraduate Biology & Biochemistry)
✨ Executive Summary

Distinguishing horizontally transferred plasmid or transposon resistance cassettes from chromosomal point mutations and outer-membrane porin disruptions is critical for computational pathogen genomics and molecular epidemiology. We benchmarked a native five-stage hierarchical antimicrobial resistance (AMR) and horizontal gene transfer (HGT) island engine against NCBI AMRFinderPlus v4.2.7 (database release 2026-08-07.1) across ten multi-species ESKAPE cohorts and official NCBI validation panels. The native engine reproduces all 72 reference resistance, virulence, stress, point-mutation, and porin-disruption calls with 100.00% gene-symbol and hierarchy-node concordance (Jaccard = 1.0000) while providing a 9.67-fold tetranucleotide Kullback-Leibler divergence separation between mobile plasmid/integron cargo and intrinsic chromosomal mutations.

1. Algorithmic & Mathematical Architecture: Coupled Reference Gene Hierarchy Traversal and Compositional HGT Island Scoring

Accurately profiling the antimicrobial resistome, virulome, and biocide/metal stress repertoire of bacterial assemblies requires resolving three distinct evolutionary mechanisms that flat sequence-similarity searches frequently conflate:

  • Hierarchical Allele vs. Gene-Family Classification (fam.tsv Ancestor Climb): Within enzyme superfamilies such as class A, B, C, and D β-lactamases (blaTEM, blaCTX-M, blaKPC, blaNDM, blaPDC, blaOXA) or phosphoethanolamine transferases (mcr-1.1), a single amino acid substitution can shift substrate specificity from narrow-spectrum penicillins to extended-spectrum cephalosporins or carbapenems (Feldgarden et al., 2019; Feldgarden et al., 2021). In the NCBI Reference Gene Hierarchy (fam.tsv, comprising 2,253 curated nodes and 10,078 reference proteins in release 2026-08-07.1), each node v defines complete-length (covref ≥ 90%) and partial-length (50% ≤ covref < 90%) identity and reference-coverage thresholds (BlastRule). Given a query alignment with amino acid identity idobs = nident / Lalign and reference coverage covref = (qend − qstart + 1) / Lref, classification begins at the closest reference sequence's leaf node v0 and climbs ancestor pointers (v ← parent(v)) until a reportable node satisfies idobs ≥ idmin(v) and covref ≥ covmin(v). Exact 100.00% identity and 100.00% coverage on an allele node (reportable = 2 with a distinct symbol from its parent) receives method code ALLELEP (protein) or ALLELEX (translated nucleotide), whereas a 99.75% match to an allele leaf climbs to its parent gene family (for example, blaPDC-114 → blaPDC via BLASTP/BLASTX).
  • Curated Chromosomal Point Mutations & Susceptible Porin/Receptor Disruptions (POINTP/X, POINTN, POINT_DISRUPT): Clinical resistance to colistin, fluoroquinolones, macrolides, nitrofurantoin, cefiderocol, and carbapenems frequently arises not by acquiring a foreign enzyme, but via specific missense mutations in housekeeping sensors (such as Escherichia pmrB_C84R), non-coding rRNA/promoter substitutions (such as 23S_A2058T in the 50S peptidyl transferase center, blaTEMp_G162T, or ampC_T-14TGT), or loss-of-function frameshifts, nonsense stops, and insertion-sequence disruptions in outer-membrane entry channels (such as Klebsiella pneumoniae catecholate siderophore receptor cirA for cefiderocol and porin ompK35 for carbapenems; AMRProt-susceptible.tsv).
  • Information-Theoretic Horizontal Gene Transfer (HGT) Island Context: While reference gene cataloging identifies which resistance determinants are present, epidemiological risk assessment also depends on whether a locus is locked on the host chromosome or carried on a horizontally transmissible conjugative plasmid (IncFII, IncX3, IncL/M), composite transposon (Tn4401, Tn125), or Class 1 integron (intI1–attI1–qacEΔ1–sul1). Our native engine augments every called element with its 256-tetranucleotide Kullback-Leibler divergence DKL(Plocus ∥ Qhost) (in bits) and 64-codon Relative Synonymous Codon Usage (RSCU) cosine distance against the host chromosomal baseline:

DKL(Plocus ∥ Qhost) = Σk ∈ {A,C,G,T}4 Plocus(k) · log2( Plocus(k) / Qhost(k) )

To validate our five-stage native Rust implementation head-to-head against the external reference standard, we compiled the official NCBI AMRFinderPlus v4.2.7 C++ tool suite (amr_report, dna_mutation, disruption2genesymbol, and fasta_check) against the official 2026-08-07.1 database release and executed a ten-cohort calibration benchmark (Figure 1).

Figure 1. Five-Stage Hierarchical AMR, Point-Mutation, Porin-Disruption & HGT Island Architecture vs. NCBI AMRFinderPlus v4.2.7 
ALL (Root Hierarchy: 2,253 fam.tsv Nodes | 10,078 AMRProt.fa Reference Sequences | DB 2026-08-07.1)
 +-- AMR (Antimicrobial Resistance Core + Plus)
 |    +-- bla (Beta-lactamase Superfamily)
 |    |    +-- bla-A (Ambler Class A Serine Beta-lactamases)
 |    |    |    +-- blaKPC -> blaKPC-2  [ALLELEP: 100.00% id, 100.00% cov | Tn4401a / IncFII(K) D_KL = 0.425 bits]
 |    |    |    +-- blaCTX-M -> blaCTX-M-15 [ALLELEX: 100.00% id, 100.00% cov | ISEcp1 / IncF D_KL = 0.431 bits]
 |    |    |    +-- blaTEM -> blaTEM-156 [ALLELEP/X: 100.00% id] / blaTEM [PARTIAL/INTERNAL_STOP climb via brFam]
 |    |    +-- bla-B1 (Subclass B1 Metallo-Beta-lactamases)
 |    |    |    +-- blaNDM -> blaNDM-1  [ALLELEP: 100.00% id, 100.00% cov | Tn125 / ISAba125 D_KL = 0.481 bits]
 |    |    |    +-- blaVIM -> blaVIM-2  [ALLELEP: 100.00% id, 100.00% cov | Class 1 Integron D_KL = 0.438 bits]
 |    |    +-- bla-C (Ambler Class C AmpC Cephalosporinases)
 |    |    |    +-- blaPDC (99.75% id to PDC-114 -> climbs from allele leaf to parent blaPDC family via BLASTP/X)
 |    |    +-- bla-D (Ambler Class D OXA Carbapenemases/Oxacillinases)
 |    |         +-- blaOXA-48_fam -> blaOXA (87.92% cov to OXA-436 -> climbs to blaOXA-48_fam via PARTIALP/X)
 |    +-- PBP_resistant -> mecA [EXACTP: 100.00% id | S. aureus SCCmec Type IV Island D_KL = 0.395 bits]
 |    +-- mcr -> mcr-1 -> mcr-1.1 [ALLELEP: 100.00% id | IncX3 / ISApl1 Plasmid Replicon D_KL = 0.422 bits]
 |    +-- POINT / POINT_DISRUPT (Curated Organism-Specific Mutational & Porin-Disruption Resistome)
 |         +-- Protein Point Mutations (POINTP/X): pmrB_C84R (Colistin), nfsA_R15C / nfsA_K141Ter (Nitrofurantoin)
 |         +-- Nucleotide Point Mutations (POINTN): 23S_A2058T (Macrolide), blaTEMp_G162T, ampC_T-14TGT
 |         +-- Susceptible Gene Disruptions (POINT_DISRUPT): cirA_A169insTer10..Q562Ter (Cefiderocol), ompK35_R60LfsTer31 (Carbapenem)
 +-- VIRULENCE (Plus Scope) -> stxA2_acd / stxB2a [EXACTP/X: 100.00% id | Lambdoid Stx2a Prophage D_KL = 0.429 bits]
 +-- STRESS (Biocide & Metal Plus Scope) -> qacR / qacEdelta1 / merA [BLASTP/EXACTP] (arsR_K-12 suppressed in Escherichia)
🔧 Generated by: Native Horizontal Gene Transfer, Tetranucleotide KL-Divergence & Resistome Scanner

2. Ten-Cohort Calibration Sweep Across Official NCBI Validation Panels & Six ESKAPE Taxgroups

We evaluated 79 protein and nucleotide sequences across ten benchmark cohorts designed to test every classification branch of the AMRFinderPlus v4.2.7 state machine:

  1. Official NCBI Proteome Validation Panel (COHORT-01, Escherichia, 17 sequences): Tests exact allele calls (blaTEM-156 via ALLELEP), 1-mismatch ancestor family climbing (blaPDC-114 at 99.75% identity climbing to blaPDC via BLASTP; qacR at 99.47% via BLASTP), internal vs. contig-boundary partial truncations (blaOXA-436 at 87.92% coverage → blaOXA / blaOXA-48_fam via PARTIALP; aph(3'')-Ib at contig 5′ boundary → PARTIAL_CONTIG_ENDP; sul2 at 81.55% → PARTIALP), amino acid point mutations (pmrB_C84R and nfsA_R15C via POINTP), Shiga toxin subunits (stxA2, stxB2 via EXACTP), and organism-specific suppression of intrinsic housekeeping E. coli K-12 arsenical repressor arsR (BAE77793.1).
  2. Official NCBI Translated Nucleotide & Promoter/rRNA Panel (COHORT-02, Escherichia, 19 contigs): Evaluates six-frame translated ALLELEX, EXACTX, BLASTX, PARTIALX, PARTIAL_CONTIG_ENDX, premature stop detection (blaOXA-9 and blaTEM via INTERNAL_STOP), dual nonsense/missense calls (nfsA_R15C and nfsA_K141Ter via POINTX), and nucleotide-level promoter/rRNA mutations via POINTN (blaTEMp_G162T at NZ_CP095603.1, ampC_T-14TGT 2-bp promoter insertion at NZ_CP041538.1, and 23S_A2058T macrolide resistance at NC_004431.1).
  3. Official NCBI Susceptible Porin & Siderophore Disruption Panel (COHORT-03, Klebsiella pneumoniae, 7 contigs): Evaluates multi-HSP insertion-sequence disruptions (cirA_A169insTer10 with a 3,120-bp IS insertion and cirA_N184NRHSEWTer with a 4,023-bp insertion carrying blaCTX-M-15), frameshift indels (cirA_K633LfsTer8, cirA_Y253CfsTer5, ompK35_R60LfsTer31), and nonsense stop codons (cirA_Q562Ter, cirA_T98Ter) conferring clinical cefiderocol and carbapenem resistance via POINT_DISRUPT.
  4. Six Clinical ESKAPE MDR Plasmid/Integron/SCCmec Cassettes (COHORT-04 through COHORT-09) & One 0🧪 Housekeeping Negative Control (COHORT-10): Spans K. pneumoniae IncFII(K) Tn4401a (blaKPC-2, blaTEM-1, aac(6')-Ib4, sul1, qacEdelta1), Acinetobacter baumannii Tn125 (blaNDM-1, ble-MBL, aph(3')-VI, armA, blaOXA-23), E. coli ST131 IncF (blaCTX-M-15, blaOXA-1, aac(6')-Ib-cr, tet(A), dfrA17, mph(A)), Staphylococcus aureus USA300 MRSA SCCmec Type IV (mecA, blaZ, erm(C), qacA, merA), Pseudomonas aeruginosa ST235 Class 1 integron (blaVIM-2, aac(6')-Ib4, sul1, qacEdelta1, catB3), Salmonella enterica IncX3/SGI1 (mcr-1.1, blaSHV-12, floR, tet(G), aadA2), and a deliberate 0🧪 negative control of conserved E. coli K-12 housekeeping proteins (rpoB, dnaK, groEL, recA, plus suppressed arsR_K-12).
Figure 2. CLI Master Calibration Telemetry: Native Hierarchical AMR & HGT Engine vs. NCBI AMRFinderPlus v4.2.7 (DB 2026-08-07.1) 
========================================================================================================================================
TOOL COMPARISON #14: NATIVE RUST HIERARCHICAL AMR & HGT ENGINE vs. NCBI AMRFinderPlus v4.2.7 (DB 2026-08-07.1)
========================================================================================================================================
Cohort ID  | Organism Taxgroup       | Mode               | Seqs | Native | NCBI  | Jaccard | Node %  | Method % | Mean D_KL (bits)
-----------+-------------------------+--------------------+------+--------+-------+---------+---------+----------+-----------------
COHORT-01  | Escherichia             | PROTEIN+GFF        |   17 |     13 |    13 |  1.0000 | 100.00% |  100.00% |       0.356 bits
COHORT-02  | Escherichia             | NUCLEOTIDE         |   19 |     20 |    20 |  1.0000 | 100.00% |  100.00% |       0.325 bits
COHORT-03  | Klebsiella_pneumoniae   | NUCLEOTIDE_DISRUPT |    7 |      8 |     8 |  1.0000 | 100.00% |  100.00% |       0.089 bits
COHORT-04  | Klebsiella_pneumoniae   | PROTEIN            |    5 |      5 |     5 |  1.0000 | 100.00% |  100.00% |       0.440 bits
COHORT-05  | Acinetobacter_baumannii | PROTEIN            |    5 |      5 |     5 |  1.0000 | 100.00% |  100.00% |       0.485 bits
COHORT-06  | Escherichia             | PROTEIN            |    6 |      6 |     6 |  1.0000 | 100.00% |  100.00% |       0.422 bits
COHORT-07  | Staphylococcus_aureus   | PROTEIN            |    5 |      5 |     5 |  1.0000 | 100.00% |  100.00% |       0.418 bits
COHORT-08  | Pseudomonas_aeruginosa  | PROTEIN            |    5 |      5 |     5 |  1.0000 | 100.00% |  100.00% |       0.428 bits
COHORT-09  | Salmonella              | PROTEIN            |    5 |      5 |     5 |  1.0000 | 100.00% |  100.00% |       0.442 bits
COHORT-10  | Escherichia             | NEGATIVE_CONTROL   |    5 |      0 |     0 |  1.0000 | 100.00% |  100.00% |       0.036 bits
-----------+-------------------------+--------------------+------+--------+-------+---------+---------+----------+-----------------
TOTAL/MEAN | 6 ESKAPE Taxgroups      | 10 Benchmark Sets  |   79 |     72 |    72 |  1.0000 | 100.00% |  100.00% | 0.435 vs 0.045
========================================================================================================================================
🔧 Generated by: Native Horizontal Gene Transfer, Tetranucleotide KL-Divergence & Resistome Scanner

Across all ten cohorts, the native engine matches 72 out of 72 reference calls from AMRFinderPlus v4.2.7 with a gene-symbol Jaccard index of 1.0000, 100.00% fam.tsv hierarchy node concordance, 100.00% method classification concordance, and 0.00% identity or coverage deviation (Table 1). Simultaneously, the compositional HGT scanner separates mobile plasmid, transposon, prophage, and integron resistance cargo (mean DKL = 0.435 bits) from chromosomal point mutations and porin disruptions (mean DKL = 0.045 bits, a 9.67-fold compositional separation).

Table 1. Ten-Cohort Calibration Summary: Native Hierarchical AMR & HGT Engine vs. NCBI AMRFinderPlus v4.2.7 (NCBI RefSeq Verified)
Cohort ID Organism Taxgroup Assay Mode Input Sequences Total Length (bp/aa) Native Calls AMRFinderPlus Calls Symbol Jaccard Hierarchy Node Concordance (%) Method Concordance (%) Mean HGT D KL (bits) Dominant Mobilome / Genomic Context
COHORT-01 Escherichia Protein + GFF 17 4668 13 13 1.0000 100.00 100.00 0.356 Mixed plasmid AMR (blaTEM-156, blaPDC, vanG), Stx2a prophage, and chromosomal pmrB/nfsA
COHORT-02 Escherichia Nucleotide 19 31538 20 20 1.0000 100.00 100.00 0.325 6-frame ALLELEX/BLASTX/INTERNAL_STOP + POINTN (blaTEMp_G162T, ampC_T-14TGT, 23S_A2058T)
COHORT-03 Klebsiella pneumoniae Nucleotide Disrupt 7 24486 8 8 1.0000 100.00 100.00 0.089 7 chromosomal cirA/ompK35 POINT_DISRUPT loci + 1 mobile blaCTX-M-15 insertion
COHORT-04 Klebsiella pneumoniae Protein 5 1157 5 5 1.0000 100.00 100.00 0.440 IncFII(K) conjugative plasmid + Tn4401a transposon (blaKPC-2, blaTEM-1, sul1, qacEdelta1)
COHORT-05 Acinetobacter baumannii Protein 5 1182 5 5 1.0000 100.00 100.00 0.485 Tn125 / ISAba125 composite transposon island (blaNDM-1, ble-MBL, aph(3')-VI, armA, blaOXA-23)
COHORT-06 Escherichia Protein 6 1624 6 6 1.0000 100.00 100.00 0.422 Pandemic ST131 IncF MDR plasmid cassette (blaCTX-M-15, blaOXA-1, aac(6')-Ib-cr, tet(A))
COHORT-07 Staphylococcus aureus Protein 5 2262 5 5 1.0000 100.00 100.00 0.418 USA300 MRSA SCCmec Type IV + biocide/mercury island (mecA, blaZ, erm(C), qacA, merA)
COHORT-08 Pseudomonas aeruginosa Protein 5 1040 5 5 1.0000 100.00 100.00 0.428 ST235 Class 1 integron intI1–attI1 cassette (blaVIM-2, aac(6')-Ib4, sul1, qacEdelta1, catB3)
COHORT-09 Salmonella enterica Protein 5 1882 5 5 1.0000 100.00 100.00 0.442 IncX3 / ISApl1 & SGI1 island (mcr-1.1, blaSHV-12, floR, tet(G), aadA2)
COHORT-10 Escherichia Negative Control 5 2996 0 0 1.0000 100.00 100.00 0.036 0🧪 Negative control: conserved rpoB, dnaK, groEL, recA + suppressed intrinsic arsR_K-12
🔧 Generated by: NCBI Entrez E-Utilities • CRV Science Labs PoliteCacheManager • Native Horizontal Gene Transfer, Tetranucleotide KL-Divergence & Resistome Scanner

3. Focal-Region Sequence Alignments & Representative Locus Telemetry Across Method Classes

To illustrate how the native engine and AMRFinderPlus v4.2.7 evaluate distinct resistance mechanisms at single-residue and single-nucleotide resolution, Table 2 presents 18 representative calls spanning all 10 method classifications, followed by annotated focal-region alignments (Figure 3) across three diagnostic regimes: (A) the blaTEM-156 vs. blaKPC-2 Ambler Class A active-site Ser70-X-X-Lys73 and Omega-loop region, (B) the K. pneumoniae cirA catecholate siderophore receptor truncation and IS-insertion hotspot conferring cefiderocol resistance, and (C) the Escherichia 23S rRNA Domain V peptidyl transferase center (A2058T) and ampC promoter (T-14TGT) non-coding resistance mutations.

Table 2. Representative Locus-Level Call Concordance Across All Method Classes (ALLELEP/X, EXACTP/X, BLASTP/X, PARTIALP/X, INTERNAL_STOP, POINTP/X, POINTN & POINT_DISRUPT)
Cohort Contig / Protein ID Element Symbol Hierarchy Node Scope Type / Subtype Drug Class / Subclass Method Target Len Ref Len Ref Cov (%) Identity (%) Closest Ref Accession HGT D KL (bits)
COHORT-01 contig01 blaTEM-156 blaTEM-156 core AMR / AMR BETA-LACTAM / BETA-LACTAM ALLELEP 286 286 100.00 100.00 WP_061158039.1 0.427
COHORT-01 contig02 blaPDC blaPDC core AMR / AMR BETA-LACTAM / CEPHALOSPORIN BLASTP 397 397 100.00 99.75 WP_061189306.1 0.448
COHORT-01 contig03 blaOXA blaOXA-48_fam core AMR / AMR BETA-LACTAM / BETA-LACTAM PARTIALP 233 265 87.92 100.00 WP_058842180.1 0.440
COHORT-01 contig09 aph(3'')-Ib aph(3'')-Ib core AMR / AMR AMINOGLYCOSIDE / STREPTOMYCIN PARTIAL_CONTIG_ENDP 225 267 81.27 100.00 WP_001082319.1 0.441
COHORT-01 contig14 pmrB_C84R pmrB core AMR / POINT COLISTIN / COLISTIN POINTP 363 363 100.00 99.72 WP_001300761.1 0.059
COHORT-01 contig18 stxA2 stxA2_acd plus VIRULENCE / VIRULENCE STX2 / stxA2 EXACTP 319 319 100.00 100.00 TJA36680.1 0.444
COHORT-02 contig01 blaTEMp_G162T NA core AMR / POINT BETA-LACTAM / AMOXICILLIN-CLAVULANATE POINTN 984 1176 83.67 99.80 NZ_CP095603.1 0.415
COHORT-02 contig10 blaOXA blaOXA-9_fam core AMR / AMR BETA-LACTAM / BETA-LACTAM INTERNAL_STOP 274 274 100.00 99.64 WP_000722315.1 0.422
COHORT-02 contig15 23S_A2058T NA core AMR / POINT MACROLIDE / AZITHROMYCIN POINTN 2905 2905 100.00 99.97 NC_004431.1 0.041
COHORT-02 contig16 nfsA_K141Ter nfsA core AMR / POINT NITROFURAN / NITROFURANTOIN POINTX 240 240 100.00 99.17 WP_089631889.1 0.050
COHORT-02 contig17 ampC_T-14TGT NA core AMR / POINT BETA-LACTAM / CEPHALOSPORIN POINTN 247 245 100.00 99.19 NZ_CP041538.1 0.044
COHORT-03 cirA_A169insTer10 cirA_A169insTer10 cirA core AMR / POINT_DISRUPT BETA-LACTAM / CEFIDEROCOL POINTX 1696 657 100.00 99.85 WP_002912926.1 0.041
COHORT-03 ompK35_R60LfsTer31 ompK35_R60LfsTer31 ompK35 core AMR / POINT_DISRUPT BETA-LACTAM / CARBAPENEM POINTX 361 359 100.00 100.00 WP_004141771.1 0.042
COHORT-04 blaKPC-2 blaKPC-2 blaKPC-2 core AMR / AMR BETA-LACTAM / CARBAPENEM ALLELEP 293 293 100.00 100.00 WP_004199234.1 0.451
COHORT-05 blaNDM-1 blaNDM-1 blaNDM-1 core AMR / AMR BETA-LACTAM / CARBAPENEM ALLELEP 270 270 100.00 100.00 WP_004201164.1 0.495
COHORT-07 mecA mecA mecA core AMR / AMR BETA-LACTAM / METHICILLIN EXACTP 668 668 100.00 100.00 WP_000721306.1 0.432
COHORT-08 blaVIM-2 blaVIM-2 blaVIM-2 core AMR / AMR BETA-LACTAM / CARBAPENEM ALLELEP 266 266 100.00 100.00 WP_003158487.1 0.442
COHORT-09 mcr-1.1 mcr-1.1 mcr-1.1 core AMR / AMR COLISTIN / COLISTIN ALLELEP 541 541 100.00 100.00 WP_049589868.1 0.438
🔧 Generated by: Native Horizontal Gene Transfer, Tetranucleotide KL-Divergence & Resistome Scanner
Figure 3. Annotated Focal-Region Sequence Alignments Across Class A Beta-Lactamases, CirA Porin Disruptions & 23S rRNA Point Mutations 
========================================================================================================================
  FOCAL REGION A: AMBLER CLASS A BETA-LACTAMASE CATALYTIC MOTIF I (S70-X-X-K73) & SDN LOOP (RESIDUES 60..140)
========================================================================================================================
  Coord Ruler     : 60        70        80        90        100       110       120       130       140
                    |.........|.........|.........|.........|.........|.........|.........|.........|
  Domain Track    : [====== Catalytic S70-X-X-K73 Pocket ======]        [==== Omega / SDN Loop ====]
  blaTEM-1_Ref    : ERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGG
  blaTEM-156_Qry  : ERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGG  [ALLELEP 100.00%]
  blaTEM_IntStop  : ERFPMMSTFKVLLCGAVLSCVDAGQEQLGRSIHYSQNDLVEYSPVTEKHLTDGMTLRELCSAAITMSDNTAANLLLTTIGG  [INTERNAL_STOP *]
  blaKPC-2_Tn4401 : EPFAKLEQDFGGSIGVYAMDTGSGATVSYRAEERFPLCSSFKGFLAAAVLARSQQQAGLLDTPIRYGKNALVPWSPISEKYL  [ALLELEP KPC-2]
  blaCTX-M-15_Qry : ERFAMCSTSKVMAAAAVLKKSESEPNLLNQRVEIKKSDLVNYNPIAEKHVNGTMSLAELSAAALQYSDNVAMNKLIAHVGG  [ALLELEX CTX-M-15]
  Conservation    : * *   .. *   ..***  .     .*  . .   ..** *.*...*..  .*.. ** .*..  ***.* * *.....*
  Entropy (H_var) : ▁▁▁▅▃▅▃▃▃▁▅▃▃▃▃▁▁▁▅▅▅▅▃▅▅▅▃▁▅▅▃▃▅▅▅▃▅▃▁▁▅▁▃▁▃▃▁▁▃▃▅▃▁▁▃▅▃▁▁▅▃▁▁▃▅▅▁▁▁▃▁▃▁▅▁▃▅▅▃▁▁
------------------------------------------------------------------------------------------------------------------------
  FOCAL REGION B: KLEBSIELLA PNEUMONIAE CIRA SIDEROphore RECEPTOR CEFIDEROCOL-RESISTANT DISRUPTIONS (WP_002912926.1)
========================================================================================================================
  Coord Ruler (aa): 90       100       165       175       180       190       250       260       558       566
                    |.........|.........|.........|.........|.........|.........|.........|.........|.........|
  Domain Track    : [=== TonB Plug ===] [===== Beta-Barrel Extracellular Gate / IS Insertion Hotspot =====]
  CirA_WT_Ref     : SVTQRVAVTGPSSADAT...GSVSGGVVAAGDTRV...SLGVRGNSFDRDNYR...GKTLYGGLSYTY...VLGQWQYGLG
  cirA_T98Ter     : SVTQRVAV*GPSSADAT...GSVSGGVVAAGDTRV...SLGVRGNSFDRDNYR...GKTLYGGLSYTY...VLGQWQYGLG  [POINT_DISRUPT T98Ter]
  cirA_A169insTer : SVTQRVAVTGPSSADAT...GSVSGGVV[+3120bp IS_ins -> Ter10]...GKTLYGGLSYTY...VLGQWQYGLG  [POINT_DISRUPT A169insTer10]
  cirA_N184insTer : SVTQRVAVTGPSSADAT...GSVSGGVVAAGDTRV...SLGVRG[+4023bp IS26+blaCTX-M-15]->Ter        [POINT_DISRUPT + ALLELEX]
  cirA_Y253CfsTer : SVTQRVAVTGPSSADAT...GSVSGGVVAAGDTRV...SLGVRGNSFDRDNYR...GKTLCWWCRIL*...----------  [POINT_DISRUPT Y253CfsTer5]
  cirA_Q562Ter    : SVTQRVAVTGPSSADAT...GSVSGGVVAAGDTRV...SLGVRGNSFDRDNYR...GKTLYGGLSYTY...VLG*WQYGLG  [POINT_DISRUPT Q562Ter]
  Entropy (H_var) : ▁▁▁▁▁▁▁▁█▁▁▁▁▁▁▁▁...▁▁▁▁▁▁▁▁█████▁▁...▁▁▁▁▁▁█████████...▁▁▁▁█████▁▁▁...▁▁▁█▁▁▁▁▁▁
------------------------------------------------------------------------------------------------------------------------
  FOCAL REGION C: ESCHERICHIA 23S rRNA DOMAIN V PEPTIDYL TRANSFERASE CENTER (NC_004431.1:2045..2070)
========================================================================================================================
  Coord Ruler (nt): 2045      2055  2058  2065      2070
                    |.........|.....|.....|.........|
  Structure Track : ((((((....((((......))))...))))))
  23S_WT_Ref      : GGCGGCCGUAACUAUAACGGUCCUAAGGUAGCG
  23S_A2058T_Qry  : GGCGGCCGUAACUAUATCGGUCCUAAGGUAGCG  [POINTN: 23S_A2058T -> Azithromycin/Erythromycin/Telithromycin Resistance]
  Conservation    : ****************.****************
  Entropy (H_var) : ▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁█▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁▁
========================================================================================================================
🔧 Generated by: Native Secondary-Structure rRNA / ITS Compensatory Base Change (CBC) & Partitioned Systematics Engine

4. Phylogenetic Relationships Across Ambler Class A, B, C & D Beta-Lactamase Superfamilies

To contextualize the hierarchical fam.tsv node assignments across the β-lactamase superfamily, we constructed a Neighbor-Joining cladogram and Newick phylogeny across twelve representative reference enzymes from our benchmark cohorts spanning Ambler Class A serine β-lactamases (blaTEM-1, blaTEM-156, blaSHV-12, blaKPC-2, blaCTX-M-15, blaZ), Class B1 metallo-β-lactamases (blaNDM-1, blaVIM-2), Class C AmpC cephalosporinases (blaPDC-114, blaEC-28), and Class D OXA oxacillinases/carbapenemases (blaOXA-1, blaOXA-23, blaOXA-436) (Figures 4A and 4B).

Figure 4A. ASCII Neighbor-Joining Cladogram of Ambler Class A, B1, C & D Beta-Lactamase Families in the Benchmark Panel 
  +-- [Ambler Class A Serine Beta-Lactamases (S70-X-X-K73 / SDN / E166)]
  |    +-- [TEM/SHV Subclade (99% Bootstrap)]
  |    |    +--+-- blaTEM-1 (WP_000027057.1 | Broad-spectrum Class A | ALLELEP)
  |    |    |  +-- blaTEM-156 (WP_061158039.1 | TEM-156 allele | ALLELEP/ALLELEX)
  |    |    +-- blaSHV-12 (WP_004176269.1 | ESBL SHV-12 | ALLELEP)
  |    +-- [KPC/CTX-M/BlaZ Subclade (96% Bootstrap)]
  |         +--+-- blaKPC-2 (WP_004199234.1 | Carbapenemase Tn4401a | ALLELEP)
  |         |  +-- blaCTX-M-15 (WP_000239590.1 | Pandemic ESBL ISEcp1 | ALLELEX)
  |         +-- blaZ (WP_000733274.1 | S. aureus SCCmec/Plasmid Penicillinase | EXACTP)
  |
--+-- [Ambler Class C AmpC Cephalosporinases (Y150-X-N152 / K315-T316-G317)]
  |    +-- blaPDC-114 (WP_061189306.1 | Climbs to blaPDC family at 99.75% id | BLASTP/BLASTX)
  |    +-- blaEC-28 (WP_498365732.1 | Climbs to blaEC family at 97.89% id | BLASTX)
  |
  +-- [Ambler Class D OXA Carbapenemases & Oxacillinases (Carboxylated K73)]
  |    +--+-- blaOXA-436 (WP_058842180.1 | Climbs to blaOXA-48_fam at 87.92% cov | PARTIALP/X)
  |    |  +-- blaOXA-23 (WP_000018329.1 | A. baumannii Carbapenemase | ALLELEP)
  |    +-- blaOXA-1 (WP_000165853.1 | Class D Oxacillinase | ALLELEP)
  |
  +-- [Ambler Class B1 Dizinc Metallo-Beta-Lactamases (H116-X-H118-X-D120 / H196 / C221 / H263)]
       +-- blaNDM-1 (WP_004201164.1 | Tn125 / ISAba125 Metallo-Carbapenemase | ALLELEP)
       +-- blaVIM-2 (WP_003158487.1 | Class 1 Integron Metallo-Carbapenemase | ALLELEP)
🔧 Generated by: Native K2P / TN93 Distance, Neighbor-Joining & ABGD Systematics Engine • Native Horizontal Gene Transfer, Tetranucleotide KL-Divergence & Resistome Scanner
Figure 4B. Newick Tree Representation of the Calibrated Beta-Lactamase Superfamily Panel 
((((blaTEM_1_WP_000027057:0.0035,blaTEM_156_WP_061158039:0.0035)100:0.1420,blaSHV_12_WP_004176269:0.1680)99:0.2840,((blaKPC_2_WP_004199234:0.2910,blaCTX_M_15_WP_000239590:0.3120)94:0.1550,blaZ_WP_000733274:0.4180)96:0.2110)98:0.3450,(blaPDC_114_WP_061189306:0.2240,blaEC_28_WP_498365732:0.2410)99:0.3820,((blaOXA_436_WP_058842180:0.2650,blaOXA_23_WP_000018329:0.2890)95:0.1940,blaOXA_1_WP_000165853:0.3760)97:0.3510,(blaNDM_1_WP_004201164:0.3180,blaVIM_2_WP_003158487:0.3340)100:0.4620);
🔧 Generated by: Native K2P / TN93 Distance, Neighbor-Joining & ABGD Systematics Engine

Discussion & Key Takeaways

1. Signal Rating Assessment (3🧪 Comparative Synthesis): This ten-cohort benchmark spans 79 sequences and 72 verified resistance, virulence, stress, point-mutation, and porin-disruption calls across six ESKAPE taxgroups (Escherichia, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica), paired with a deliberate 0🧪 housekeeping negative control (COHORT-10). In accordance with our signal rating rubric, this multi-clade cross-engine synthesis earns a 3🧪 Comparative Synthesis rating.

2. Why Multi-HSP Split Chaining Is Essential for Susceptible Porin & Siderophore Receptor Disruptions: In clinical K. pneumoniae isolates resistant to cefiderocol or carbapenems, loss of function in cirA or ompK35 is frequently driven by insertion sequences (such as IS26 or ISKpn26) jumping directly into the coding sequence (as seen in cirA_A169insTer10 and cirA_N184NRHSEWTer). Standard single-HSP local alignment drops the downstream half of the split open reading frame, misclassifying an insertion disruption as a simple partial fragment. Retaining all secondary HSPs (--max-hsps 0) and evaluating split-ORF coordinates through disruption2genesymbol recovers all eight POINT_DISRUPT and embedded blaCTX-M-15 calls on test_disrupt.fa with 100.00% concordance.

3. Candidate Hypotheses for Downstream Genomic Surveillance: Pairing hierarchical fam.tsv gene typing with 256-tetranucleotide Kullback-Leibler divergence (DKL = 0.435 bits on mobile plasmid/integron cassettes vs. 0.045 bits on chromosomal point mutations) provides a fast computational signal that may help molecular epidemiologists prioritize horizontally transferable carbapenemase and colistin-resistance islands during outbreak surveillance.

References & Primary Sources

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  2. [2] Feldgarden M, Brover V, Haft DH, Prasad AB, Slotta DJ, Tolstoy I, Tyson GH, Zhao S, Hsu CH, McDermott PF, Tadesse DA, Morales C, Simmons M, Tillman G, Wasilenko J, Folster JP, Klimke W (2019). Validating the AMRFinder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates. Antimicrobial Agents and Chemotherapy 63(11):e00483-19. https://doi.org/10.1128/AAC.00483-19
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