NCBI AMRFinderPlus抗性基因检测工具结构化详解 (Guide to NCBI AMRFinderPlus for Antimicrobial Resistance Gene Detection)

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一、工具概述

• NCBI AMRFinderPlus 是由美国国家生物技术信息中心（NCBI）开发的微生物耐药性和相关基因检测工具。
• Identifies not just acquired AMR genes but also point mutations, virulence, metal, biocide, and stress genes by leveraging curated reference data and Hidden Markov Models (HMMs) from NCBI.

二、数据库与依赖资源

• 核心数据库为“细菌抗菌药物耐药基因参考数据库”（Bacterial AMR Reference Gene Database），BioProject号：PRJNA313047，包含人工整理的耐药基因/HMM模型与命名体系(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047/)。
• NCBI BioProject: PRJNA313047 — the official umbrella record for resistance genes, supporting scholarly citation and comparative studies.

三、功能支持与检测范围

• 支持蛋白序列和装配后的基因组（核酸序列）输入，可检测：
  • 获得性耐药基因
  • 关键点突变
  • 毒力因子/金属/应激/生物杀灭剂耐受基因（通过 --plus 选项）
• Detects both acquired resistance genes and resistance-associated point mutations; the “Plus” option includes stress, metal, and virulence determinants.

四、安装与更新

• 推荐用 Bioconda 或 GitHub 安装，定期更新数据库：
  • Bioconda: mamba install ncbi-amrfinderplus / mamba update ncbi-amrfinderplus
  • Check version: amrfinder -V

五、使用流程（命令结构）

• 典型命令与输入选项：
  • 装配基因组核酸序列： amrfinder -n assembly.fna -o amr.tsv --plus
  • 预测蛋白序列： amrfinder -p proteins.faa -o amr.tsv --plus
  • 需要突变规则的物种（如大肠杆菌、沙门氏菌等），可添加 --organism Escherichia
• If your input is protein (for specificity): amrfinder -p proteins.faa -o amr.tsv --plus
• Add --organism for species-specific detection rules (e.g., Salmonella, E. coli).

六、输出结果解读

• 输出结果包括：
  • 基因/等位基因名称
  • 类别
  • 检测方法（Exact / BLAST / HMM）
  • Percent identity \& coverage
• Reference Gene Hierarchy explains why HMM hits may be mapped to gene families.

七、与ResFinder工具对比

• AMRFinderPlus 强调数据库人工整理、层级命名与HMM深度，支持点突变/毒力因子类别；
• ResFinder primarily uses BLAST for acquired resistance genes; both are widely used.
• 两者结果可互补，建议在关键案例均报告并比较结论。

八、β-内酰胺酶检测相关

• AMRFinderPlus 能精准识别各类 β-内酰胺酶以及特定等位基因（KPC、NDM、OXA-48等），但药物抑制剂敏感性需结合表型MIC和人工比对。

九、实践建议与数据管理

• 分析前应QC（覆盖度、污染、N50等），优先用蛋白输入以降低假阳性；务必记录运行软件与数据库版本。
• Reference all results and analyses to your BioProject for project transparency and reproducibility.

十、数据提交与BioProject关联

• 强烈推荐提交数据到NCBI，关联BioProject，实验报告/论文需标明引用。合规数据将进入 Pathogen Detection/Isolate Browser，便于下游标准化分析与溯源。
• Follow NCBI pathogen/AMR submission guidelines for maximum interoperability and future-proofing.

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主要优点总结

• 全面覆盖AMR基因、点突变至毒力因子，有权威数据库与持续更新支持，多模式比对结果提升准确率。适用于宏/微生物组耐药基因监测、临床感染溯源、研究数据管理等多场景。

Citation: Reference Database: BioProject PRJNA313047 Official tool page: https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/

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This structured guide is suitable for scientific communication, teaching, or lab documentation. Please cite the BioProject and include the official tool URL when republishing.

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What AMRFinderPlus is good for

• Detects acquired AMR genes and key resistance-associated point mutations from assembled genomes (protein or nucleotide).
• It also optionally reports virulence, stress/biocide/metal genes (“Plus”).
  国家生物技术信息中心+1

Powered by NCBI’s curated Bacterial AMR Reference Gene Database (with HMMs and a naming hierarchy), used in NCBI’s Pathogen Detection pipeline.
journals.asm.org+2国家生物技术信息中心+2

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Database you’ll cite/use

Bacterial Antimicrobial Resistance Reference Gene Database
BioProject accession: PRJNA313047. Keep this in methods/notes.
国家生物技术信息中心

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Install & update (typical options)

• Bioconda or GitHub.
• After install, update DB regularly.
• Check versions with amrfinder -V.
  GitHub+2bioconda.github.io+2

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Minimal commands you’ll actually run

On an assembly (nucleotide FASTA): amrfinder -n assembly.fna -o amr.tsv --plus

If you have predicted proteins (often best for specificity): amrfinder -p proteins.faa -o amr.tsv --plus

Add organism when supported for point-mutation rules (e.g., Salmonella, E. coli, Campylobacter, S. aureus): amrfinder -n assembly.fna --organism Escherichia -o amr.tsv 国家生物技术信息中心+1

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Reading the output (what to look for)

• You’ll get gene/allele names, class, method (Exact/Blast/HMM), and % identity/coverage.
• Use the Reference Gene Hierarchy for how borderline hits are named and why an HMM hit maps to a family.
  国家生物技术信息中心

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How it complements (and differs from) ResFinder

• AMRFinderPlus: includes a curated hierarchy, extensive HMMs, and point-mutation rules for several taxa; “Plus” genes beyond classic AMR.
  国家生物技术信息中心+1

• ResFinder: strong for acquired genes via BLAST rules.
  Both are widely used; results can differ slightly by database scope and curation. Consider running both for critical cases and reconciling (multiple reviews/benchmarks discuss trade-offs).
  PubMed Central+1

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β-lactamase & BLI angle (important in the context)

• AMRFinderPlus will identify β-lactamase families and many specific alleles (e.g., KPC, OXA-48-like, NDM, CTX-M).
• Neither AMRFinderPlus nor ResFinder guarantees inhibitor efficacy (e.g., avibactam, vaborbactam) because allele-specific substitutions, expression, porins/efflux affect phenotype.
• Use the call to narrow likely classes (KPC vs OXA-48 vs MBL) and confirm with MICs.
  国家生物技术信息中心+1

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Tie-in with BioProject

• BioProject is the umbrella record linking all your project’s sequence data (assemblies, SRA reads, BioSamples).

• Create one for your study; reference it in your reports.
  国家生物技术信息中心

• If you submit to NCBI (recommended), follow the Pathogen/AMR submission guidance so your data appear in Pathogen Detection/Isolate Browser and benefit from standardized pipelines.
  国家生物技术信息中心+1

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Practical checkpoints / best practices

• QC first (coverage, contamination, N50); subpar assemblies yield spurious or missed hits.

• Run with proteins when available (often fewer false positives); still keep nucleotide run if you lack annotation.
  国家生物技术信息中心

• Record software & DB versions in your methods (e.g., “AMRFinderPlus vX.Y; database YYYY-MM-DD; PRJNA313047”).
  biocontainer-doc.readthedocs.io

• Corroborate critical clinical calls (e.g., suspected KPC escape mutants) with phenotypic MICs and, if needed, manual allele review.
  PubMed Central

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Run through an interpretation plan (what AMR genes/mutations to report, and how to summarize likely β-lactam/BLI implications).

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Metagenomic Analysis – Patient Sample

Key Findings

• Primary suspected pathogen: Klebsiella pneumoniae
  • Clinically meaningful if abundant with good genome coverage; confirm with orthogonal methods.
• Background organisms/contaminants:
  • Skin flora (e.g., Staphylococcus haemolyticus, Cutibacterium acnes, Corynebacterium spp.)
  • Environmental bacteria (e.g., Pseudomonas fluorescens, Dietzia, Massilia, Sphingomonas)
  • Non-human DNA and synthetic controls (e.g., Tapinoma sessile, synthetic constructs)
  • Taxonomically non-informative bins.

Next Steps

1. Verification:
   • Confirm K. pneumoniae via culture or PCR.
   • Cross-check read abundance and genome breadth for clinically relevant taxa.
2. AMR Profiling:
   • Run AMRFinderPlus or ResFinder on Klebsiella-assigned contigs to identify β-lactamases (KPC, OXA-48-like, NDM, CTX-M) and other resistance genes.
   • Document software and database versions for reproducibility.
3. Reporting:
   • Only report organisms that are both high-abundance and match the clinical scenario/specimen type.
   • Consider all others likely contaminants or non-informative.

Table: Species Interpretation

Species/Taxon	Likelihood	Interpretation/Notes
Klebsiella pneumoniae	High (pathogen)	Common pathogen; clinically relevant if abundant
Moraxella (genus)	Uncertain	Specify species/abundance for clinical interpretation
Staphylococcus haemolyticus	Background/Contam	CoNS; likely skin flora contamination
Cutibacterium acnes, Corynebacterium spp.	Background	Typical skin flora/commensals
Micrococcus, Rothia, Anaerococcus	Background	Skin/oral commensals, rarely pathogenic
Pseudomonas fluorescens, Dietzia, Massilia, etc.	Environmental	Soil, water, or industrial source; not pathogenic here
Tapinoma sessile, synthetic constructs	Contaminant	Non-patient origin DNA, likely process or control contamin
Malassezia (fungus)	Background	Skin yeast; relevant only in dermatologic context
[Unassigned], Bacteria, bacterium	Non-informative	Unresolved taxonomic assignment

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Summary: Klebsiella pneumoniae is the likely pathogen based on abundance and breadth; confirmation and AMR genotyping are recommended. Most other taxa are consistent with background skin, environment, or contamination and should generally not be included in clinical reporting unless concordant with the sample type and clinical scenario.