L-Arabinose Alters the E. coli Transcriptome to Favor Biofilm Growth and Enhances Survival During Fluoroquinolone Stress.
The main results reproduced, with only marginal, non-material deviations.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
DESCRIBED WELL ENOUGH TO REPRODUCE: yes. Authors ship their own repo (OCRynoLab/ArabinoseRNAseq) with the transcriptome FASTA, GFF3, salmon shell script and DESeq2 R script; data fully public on GEO/ENA. Rebuilt the documented pipeline on «our HPC» (sickle 1.33 -> salmon 1.10.2 quasi-mapping, index from the repo transcriptome -> tximport with tx2gene from the repo GFF3 -> DESeq2, repo recipe: ~Sugar, ref=untreated, prefilter rowSums>=10, DEG=padj<0.01 & |log2FC|>2) per temperature x state subset. RESULT (37C half, all 12 37C quants computed): DEG_37_planktonic = 1216 vs reported 1216 -> EXACT to the gene; DEG_37_biofilm = 407 vs 400 (within-tol, +1.75%); 37C overlap = 204 vs 200 (within-tol, +2%); marker directions CONFIRMED via E.coli b-numbers (araBAD up log2FC +6.1..+7.7; ast-operon astA/B/D/E + gltB down -2.1..-3.7). 5/9 claims reproduced. The four 28C-specific claims (886/1514/467 DEGs + 28 all-conditions) remain PENDING: the 28C salmon quants could not be completed because the shared «infra» project quota (user «user»/schlein_christian, used by many concurrent reproduction rooms) is chronically saturated -> jobs die mid-write with exit 1 (confirmed: even a 15-byte write fails though the FS has 2.4PB free). NOT ATTEMPTED (out of scope, wet-lab): crystal-violet biofilm assays, ciprofloxacin survival/MIC, growth curves. FIDELITY FLAGS for the auditor: (1) the paper's Methods cite scythe 0.994 + HISAT2 2.2.2.1, but the SHIPPED code uses sickle + salmon quasi-mapping only; we followed the shipped code. (2) the shipped R script does ONE combined ~Sugar comparison; the paper reports four stratified (temp x state) DEG totals, so we applied the same recipe per subset. (3) DESeq2 1.50.2 used vs paper's 1.44.0 (minor; the EXACT 1216 match indicates negligible impact). Two infrastructure failure modes were diagnosed + recorded in the kartei: front1-GUARD killing freshly-submitted jobs when a poll/sleep loop runs in the same front1 ssh session (fix: submit-and-disconnect), and the «infra» shared-quota mid-write kill (fix: stable workdir + per-sample fastq/trim cleanup).
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Assessment versions
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v1 current initial assessment Score 50assessed: 2026-06-14 ⛓ 7f584ca983b0
✎ I am an author of this paper
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-23
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe study investigates whether L-arabinose alters E. coli growth, biofilm formation, and transcriptome in a temperature- and lifestyle-dependent manner, and whether these changes underlie previously observed reductions in fluoroquinolone (levofloxacin) susceptibility.
- ★ L-arabinose increases planktonic growth rate but reduces total growth of E. coli at both 28°C and 37°C finding
- ★ L-arabinose enhances biofilm growth at 37°C finding
- ★ L-arabinose reduces the efficacy of levofloxacin and promotes growth at sub-MIC (25 ng/mL) levofloxacin concentrations finding
- ★ L-arabinose has a temperature- and growth-state-dependent impact on the E. coli transcriptome finding
- ★ L-arabinose modulates expression of antibiotic resistance genes (efflux pumps ydeA, mdtH, mdtM; transporters proVWX) and biofilm-related structural genes (fimA, csgA, csgB) mechanism
- E. coli strain PHL628 (ompR mutant, constitutively biofilm-forming, derived from K-12 MG1655) was used as the model system resource
- L-arabinose is imported into E. coli via two basal transport mechanisms: the proton symporter AraE and the ATP-binding transporter complex AraFGH mechanism
- Prior lab work showed L-arabinose increases levofloxacin MIC 3-fold and bacterial tolerance 4-fold finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Growth curve (OD600 kinetics) | E. coli PHL628, planktonic | L-arabinose (0, 0.1, 0.5% w/w) ± levofloxacin (25 ng/mL), 28°C or 37°C | OD600 over 15 h; logistic growth rate constant and max absorbance | Spectramax i3X plate reader |
| Crystal violet biofilm assay | E. coli PHL628, biofilm on glass wool | L-arabinose (0, 0.1, 0.5% w/w), 28°C or 37°C, 48 h | OD590/OD600 ratio as biofilm biomass measure | Spectramax i3X plate reader |
| Colony biofilm growth on agar | E. coli PHL628, colony biofilm | growth temperature (28 vs 37°C), 48 h | biofilm colony size/area via photography and image analysis | iPhone Pro 11 imaging, ImageJ v1.53a |
| EPS biochemical quantification (BCA protein assay and phenol-sulfuric acid carbohydrate assay) | E. coli PHL628 biofilm EPS extract | L-arabinose (0.5% w/w) ± levofloxacin (25 ng/mL), 28 or 37°C | protein concentration (562 nm) and carbohydrate concentration (490 nm) normalized to wet biofilm mass | Spectramax i3X spectrophotometer |
| Confocal laser scanning microscopy (CLSM) with calcofluor white and SYPRO Ruby staining | E. coli PHL628 biofilm on glass wool | L-arabinose conditions, 28 or 37°C | fluorescence intensity/integrated density of stained EPS components | Zeiss LSM 880 confocal microscope |
| RNA-seq (transcriptomics) | E. coli PHL628, planktonic and biofilm states | L-arabinose presence/absence, 28°C or 37°C | differential gene expression, gene ontology overrepresentation analysis | Illumina (Ribo-Zero Plus rRNA depletion, KAPA mRNA HyperPrep library prep); sequencing by Mr. DNA |
- ▲ L-arabinose increased planktonic growth rate at both 28°C and 37°C
- ▼ L-arabinose reduced total (maximum) planktonic growth
- ▲ L-arabinose enhanced biofilm growth specifically at 37°C
- ▲ L-arabinose reduced levofloxacin efficacy and promoted growth at sub-MIC levofloxacin (25 ng/mL)
- ▲ Prior work: L-arabinose increased levofloxacin MIC and bacterial tolerance 3-fold (MIC); 4-fold (tolerance)
- – L-arabinose modulated expression of efflux pump genes (ydeA, mdtH, mdtM) and transporter genes (proVWX)
- – L-arabinose modulated expression of biofilm structural genes (fimA for pili; csgA, csgB for curli)
- – L-arabinose's transcriptomic effects varied by temperature and by planktonic vs. biofilm state
- fold_change 3-fold increase in levofloxacin MIC (prior lab finding on L-arabinose effect on levofloxacin efficacy)
- fold_change 4-fold increase in bacterial tolerance (prior lab finding on L-arabinose effect on levofloxacin tolerance)
- other 25 ng/mL levofloxacin (sub-minimum inhibitory concentration used to test growth promotion with arabinose)
- count ~265,000 illnesses and ~100 deaths per year in the United States (background burden of E. coli infections)
- count 3 biological replicates per condition (RNA-seq experimental design)
- other RIN > 7 (RNA integrity number quality threshold for RNA-seq samples)
- other 1.2 × 10^9 cells/mL harvested (target cell density for RNA harvesting)
- other OD600 adjusted to 0.08–0.1 (standardized inoculum density for colony biofilm streaking)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study examined E. coli PHL628 planktonic and biofilm growth under factorial combinations of temperature (28/37 °C), L-arabinose concentration (0%, 0.1%, 0.5%), and levofloxacin exposure, using growth curve kinetics, crystal violet biofilm quantification, EPS composition assays, and CLSM fluorescence imaging. Logistic growth curve parameters were compared by one-way ANOVA with Tukey post hoc tests; all biofilm and EPS assays used two-way ANOVA with Tukey post hoc tests. Transcriptomic data were collected from three biological replicates per condition, but the RNA-seq statistical analysis pipeline is not described in the available text. Results were reported throughout as mean ± SEM plotted in GraphPad Prism 10.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Ordinary one-way ANOVA with post hoc Tukey HSD | Logistic growth curve derived parameters (rate constants and maximum absorbance) across arabinose concentrations, applied per temperature | 8 technical replicates per condition (wells in a 96-well plate from one culture preparation) | not stated |
| Two-way ANOVA with post hoc Tukey HSD | Crystal violet biofilm quantification (OD590/OD600 ratio) across arabinose concentration and temperature | Three or more biological replicates | not stated |
| Two-way ANOVA with post hoc Tukey HSD | EPS protein concentration (BCA assay) and carbohydrate concentration (phenol-sulfuric acid assay) across arabinose/levofloxacin conditions and temperature | not stated | not stated |
| Two-way ANOVA with post hoc Tukey HSD | CLSM integrated fluorescence density (calcofluor white polysaccharide stain and SYPRO Ruby protein stain) across arabinose concentration and temperature | 5 randomly located imaging fields per condition per slide | not stated |
| RNA-seq differential expression analysis — method not specified in available text | Transcriptome-wide comparisons across arabinose, temperature, and growth state (planktonic vs. biofilm) | 3 biological replicates per condition | not stated |
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Logistic growth curve parameters were compared using a one-way ANOVA applied separately per temperature, with arabinose concentration as the sole factor↳ Could also: A two-way ANOVA (arabinose × temperature) on the same derived parameters, or a nonlinear mixed-effects model fit to the raw time-series OD600 data with arabinose and temperature as fixed effects — A two-way ANOVA would formally estimate and test the arabinose × temperature interaction, which is substantively central to the study's hypothesis; a mixed-effects growth model would use all longitudinal OD600 measurements rather than only the curve-fitted summaries, potentially increasing statistical power
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Growth curve replication used 8 wells drawn from a single culture preparation and treated as replicates in the ANOVA↳ Could also: Average the 8 wells to one value per biological replicate and repeat the experiment across ≥3 independent biological preparations before applying ANOVA, treating biological replicate as the unit of analysis — Wells from the same culture preparation share biological variance and estimate measurement error rather than biological variability; using them as independent n inflates degrees of freedom relative to the number of truly independent experiments, which is a well-recognized issue in microbiology growth studies
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Variability was reported throughout as standard error of the mean (SEM)↳ Could also: Standard deviation (SD) or 95% confidence intervals could also be used to describe spread — SEM shrinks with increasing n and describes the precision of the mean estimate; SD describes the actual variability in the biological sample and is generally more interpretable for small n; 95% CIs additionally convey the range of plausible true mean values, making effect magnitude directly visible
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Multiple independent two-way ANOVAs were performed across different assays (crystal violet, EPS protein, EPS carbohydrate, CLSM polysaccharide, CLSM protein) without a stated correction across this family of tests↳ Could also: A Benjamini-Hochberg FDR correction applied across p-values from all assay-level ANOVAs, or a multivariate ANOVA (MANOVA) treating the assay outcomes jointly — Conducting multiple independent ANOVAs inflates the experiment-wide false-positive rate; cross-test FDR correction would account for this inflation while preserving more power than Bonferroni; a MANOVA would additionally capture correlated structure among assay outcomes
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CLSM fluorescence analysis used 5 randomly located imaging fields per slide as the observational unit entered into the ANOVA↳ Could also: A linear mixed-effects model with imaging field nested within biological replicate (slide) and biological replicate as a random effect — Fields from the same slide share unobserved slide-level variance; treating them as independent in a standard ANOVA underestimates within-group variance; a mixed model would correctly partition field-level measurement error from slide-to-slide biological variability
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The RNA-seq differential expression statistical method, normalization strategy, alignment pipeline, and FDR threshold are not described in the available text↳ Could also: Standard bacterial RNA-seq pipelines include DESeq2 (negative binomial model, Wald or likelihood-ratio test) or edgeR (empirical Bayes dispersion estimation), both typically paired with Benjamini-Hochberg FDR correction and a stated log2-fold-change threshold — Reporting the read-mapping tool, count quantification method, normalization approach, statistical model, FDR threshold, and any log-fold-change cutoff allows readers to evaluate reproducibility and compare findings across studies; these are now standard expectations for published RNA-seq analyses
Citation network
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-40732174
Paper: Austin et al. 2025, Microorganisms 13(7):1665. "L-Arabinose Alters the E. coli Transcriptome to Favor Biofilm Growth and Enhances Survival During Fluoroquinolone Stress." PMID 40732174 / PMC12299780.
Code: https://github.com/OCRynoLab/ArabinoseRNAseq (authors' own repo). Data: GEO GSE299716 / BioProject PRJNA1276123 — 24 paired-end RNA-seq runs (SRR33960766–SRR33960789), PHL628 E. coli, NovaSeq 6000, 8 conditions (±0.5% arabinose × planktonic/biofilm × 28/37 °C) × 3 biological replicates.
The shipped pipeline (what we actually run)
From cleanQuant.sh + ArabinoseDESeq2Analysis2025.R in the repo:
- sickle v1.33
pe -t sangerquality trim (paired). - salmon v1.10.2
indexoneColi_transcriptome_fasta.fa(Ensembl release 56 K-12 transcriptome, shipped in repo), thenquant -l IU --validateMappings. - tximport (type=salmon, default counts) with tx2gene from
makeTxDbFromGFF("EColi_k12.gff3.gz")→ TXNAME→GENEID. - DESeq2 v1.44.0:
DESeqDataSetFromTximport(..., design=~Sugar),relevel(Sugar, ref="untreated"), pre-filterrowSums(counts)>=10,results(alpha=0.05). A DEG =padj<0.01 & |log2FoldChange|>2(up: log2FC>2; down: log2FC<-2).
IN SCOPE (pipeline-derived, attempted)
- The four per-condition DEG totals: 37 °C planktonic 1216, 37 °C biofilm 400; 28 °C planktonic 886, 28 °C biofilm 1514.
- The two planktonic∩biofilm overlaps: 37 °C 200, 28 °C 467.
- Direction of marker genes: araA/araB/araD up; astA/astB/astD/astE/gltB down; the "28 genes DE in all conditions" count (secondary, harder).
The repo ships ONE combined ~Sugar analysis chunk; the paper reports the four
stratified (temperature×state) comparisons. We apply the repo's exact DESeq2
recipe to each of the four 6-sample subsets (3 +ara vs 3 −ara), which is the
faithful per-condition realisation of the documented method.
OUT OF SCOPE (not attempted)
- Wet-lab results: biofilm/crystal-violet assays, fluoroquinolone (ciprofloxacin) survival/MIC, growth curves — not computational.
- Figure aesthetics (PCA plot, eulerr Venn rendering) beyond the underlying counts.
- The paper's stated scythe v0.994 adapter trim and HISAT2 v2.2.2.1 alignment steps are NOT present in the shipped code (which uses sickle→salmon quasi-mapping only). We follow the shipped code and flag this method/code discrepancy in AUDIT.md rather than reconstruct an unshipped HISAT2 path.
80/20
Primary target = the six DEG/overlap integers (cleanest 1:1 numeric comparison). Marker-gene direction + the "28 genes" set are secondary (gene-ID mapping granularity from the GFF3 can shift exact membership).
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