CPE Genomics in Hospital Reservoirs

Distinguish carbapenem-resistant Enterobacterales from carbapenemase-producing Enterobacterales using phenotypic and genomic evidence.

Explain how the major carbapenemase families differ in mechanism, genetic context, and epidemiological significance.

Identify the roles of insertion sequences, transposons, integrons, and plasmids in carbapenemase-gene mobilisation.

Explain why sinks, drains, toilets, and showers can select for and sustain CPE populations and resistance plasmids.

Distinguish clonal strain transmission from horizontal transfer of a resistance plasmid.

Formulate testable genomic hypotheses linking damp-environment reservoirs, colonisation, and patient infection.

Define the sample, isolate, host, event, location, and time units required for the 631-isolate investigation.

Construct a data dictionary for patient, colonisation, environmental, hospital, location, and sampling variables.

Standardise isolate identifiers, niche labels, taxonomic labels, and hospital-location categories.

Represent hospitals, wards, rooms, outlets, and patients as a consistent spatial hierarchy.

Clean sampling dates and derive epidemiologically meaningful time intervals.

Assess missingness, duplication, imbalance, and sampling intensity across sites, times, and niches.

Create an analysis-ready manifest that links metadata to short-read and long-read files without exposing patient identifiers.

Organise raw data, intermediate files, results, software environments, and provenance in a reproducible project structure.

Verify file integrity, isolate identity, and short-read pairing before analysis.

Interpret per-base quality, adapter content, duplication, read length, and GC profiles in Illumina reads.

Apply explicit rules for trimming, filtering, or retaining short reads based on quality-control evidence.

Interpret read-length, yield, and quality distributions for the 48 long-read datasets.

Detect likely contamination or mixed-isolate sequencing using read-level taxonomic and k-mer evidence.

Record auditable pass, caution, or fail decisions for every read set.

Select short-read or hybrid assembly strategies according to the data available for each isolate.

Generate draft assemblies for the full 631-isolate short-read collection.

Generate hybrid assemblies for the 48 cross-niche transmission candidates.

Polish hybrid assemblies using appropriate long-read and short-read evidence.

Assess circularisation and topology without treating assembler circularity claims as sufficient proof.

Detect misassemblies by examining read support and conflicts between assembly evidence.

Apply a genome-assembly acceptance rubric using completeness, contamination, fragmentation, size, and coverage metrics.

Assign Enterobacter and Citrobacter species using genome-wide similarity to suitable reference genomes.

Resolve disagreements among submitted species labels, genome-based taxonomy, and contamination evidence.

Generate consistent structural and functional annotations across the isolate collection.

Assign sequence types while recognising the limitations of MLST for species complexes and plasmid transmission.

Identify acquired antimicrobial-resistance genes using curated databases and explicit identity and coverage criteria.

Confirm carbapenemase gene alleles by inspecting read coverage and local assembly quality.

Classify carbapenemase genes as plasmid-borne, chromosomally integrated, or unresolved using convergent genomic evidence.

Choose among core-SNP, core-genome, and gene-content approaches for a defined relatedness question.

Construct a species-appropriate pangenome and core-genome alignment.

Identify and mask recombinant regions that could distort clonal relatedness.

Infer and quality-check phylogenies for the major Enterobacter and Citrobacter groups.

Calculate pairwise genomic distances within appropriate species and lineage strata.

Evaluate genomic-distance distributions before choosing operational cluster criteria.

Define candidate isolate clusters by combining genomic relatedness with epidemiological context rather than relying on a universal cutoff.

Encode the nested spatial relationships among hospitals, wards, rooms, damp outlets, and patient locations.

Visualise variation in sampling effort across hospitals, dates, niches, and species.

Overlay hospital, niche, date, and clinical source metadata on isolate phylogenies.

Construct timelines for genomic clusters containing environmental, colonisation, and infection isolates.

Evaluate whether observed time and location overlaps make proposed reservoir-to-patient links plausible.

Build isolate-level networks that combine genomic distance with spatial and temporal relationships.

Identify sampling bias, patient-movement gaps, and unsampled reservoirs that could confound transmission narratives.

Rank cross-niche candidates for hybrid-genome investigation using transparent genomic and epidemiological criteria.

Explain plasmid modular organisation and the limitations of replicon-based plasmid classification.

Identify candidate plasmid sequences in hybrid assemblies using multiple independent signals.

Validate complete plasmid circularity using long-read spanning and assembly-graph evidence.

Distinguish autonomous plasmids from chromosomal integrations and unresolved repeat-linked contigs.

Characterise plasmid replicons, relaxases, mating-pair systems, oriT signals, and predicted mobility.

Delineate the local insertion-sequence, transposon, and integron context of each carbapenemase gene.

Diagnose plasmid reconstruction ambiguity caused by repeats, cointegrates, or multiple related plasmids.

Build a curated collection that separates complete plasmids, high-confidence partial plasmids, and unresolved sequences.

Cluster plasmids using whole-sequence similarity while accounting for unequal completeness.

Compare plasmid gene content to distinguish conserved backbones from variable accessory regions.

Generate linear synteny diagrams for carbapenemase plasmids selected from different hosts and niches.

Generate interpretable circular comparisons of complete plasmids and their resistance regions.

Identify plasmid rearrangements, insertions, deletions, and cointegration events from comparative alignments.

Map short reads from the 631 isolates to hybrid-derived plasmid references and interpret breadth, depth, and ambiguity.

Distinguish evidence for the same plasmid, a related plasmid backbone, and a shared carbapenemase cassette.

Test whether host-genome and plasmid relationships support clonal spread, plasmid spread, or both.

Identify near-identical plasmids occurring across Enterobacter and Citrobacter lineages, species, or sequence types.

Compare carbapenemase cassette conservation when plasmid backbones differ.

Assess the temporal and spatial opportunity for each proposed cross-niche plasmid-transfer event.

Evaluate whether an event is more consistent with environment-to-patient transfer, patient-to-environment transfer, or an unsampled common source.

Explain why genomic similarity alone rarely proves transfer direction or direct transmission.

Assign transparent confidence grades to proposed horizontal-transfer events.

Define evidence-based gene categories related to adhesion, biofilm formation, metal tolerance, disinfectant tolerance, and stress response.

Screen Enterobacter and Citrobacter genomes for curated biofilm- and persistence-associated genes.

Distinguish core lineage-associated biofilm genes from accessory or mobile genes.

Determine whether selected persistence genes are chromosomal, plasmid-borne, or located on other mobile elements.

Test niche-associated gene enrichment while accounting for species and population structure.

Interpret genomic detection of a biofilm-associated gene as functional potential rather than proof of phenotype.

Integrate biofilm and stress-tolerance findings into cautious hypotheses of damp-environment persistence.

Create an isolate-level audit table connecting read QC, assembly QC, taxonomic assignment, typing, and plasmid confidence.

Perform sensitivity analyses for genomic-distance, plasmid-similarity, and mapping thresholds.

Triangulate host-genome, plasmid, spatial, temporal, and niche evidence in selected case studies.

Design a coherent figure set combining cohort summaries, phylogenies, timelines, networks, and plasmid comparisons.

Write reproducible methods for the short-read, hybrid-assembly, population-genomic, and plasmid analyses.

Report results using language calibrated to the strength and directionality of the evidence.

Explain the effects of incomplete sampling, assembly uncertainty, and metadata limitations on the study conclusions.

Package commands, environments, parameters, and outputs as a rerunnable analysis workflow.