Chapter Four · failure evidence
What Bacterial Resistance & Antibiotic Assays got wrong, from 29 dissertations
The evaluated records show that bacterial resistance evaluations and antibiotic assays frequently fail due to matrix interactions, physical vessel constraints, and readout limitations. Discrepancies between phenotypic tolerance and genetic resistance mechanisms further complicate accurate susceptibility classification and quantitative modeling. These records come from PhD theses at 13 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Agar and plate diffusion assays fail due to physicochemical binding, desiccation, or unsuitable growth parameters
Agar matrix components can bind antimicrobial compounds such as cationic peptides and weaken their activity, while desiccation and contamination disrupt long-term gradient plates. Furthermore, inappropriate inoculum densities or unsuitable strain growth conditions cause assays to produce no visible zones of inhibition or complete colony formation failures.
Tried and failed
large-scale agar plate gradient assay applied to bacterial lineage tracking and evolution. Outcome: unstable. Reason: Severe microbial and fungal contamination combined with rapid agar desiccation during incubation at 37°C.
Tried and failed
serial dilution colony forming unit plate count applied to bacteriocin sensitive bacterial isolates. Outcome: no signal. Reason: cultures failed to form colonies past early dilutions despite visible turbidity
Isolation, characterization, and potential clinical application of the M1-Pseudomonas aeruginosa pyocin · Texas Tech
Considered and rejected
Considered and rejected: Rejected agar-based susceptibility testing for streptococcins because interactions between cationic antimicrobial peptides and anionic agar components substantially reduce activity, adopting broth microdilution instead.
Genomic and experimental investigations into pneumococcal bacteriocins and their role in competition · Imperial
Tried and failed
standard disk diffusion antimicrobial susceptibility testing applied to weak food-grade antimicrobials against bacteria. Outcome: no signal. Reason: standard clinical inoculum density was too high to produce visible zones of inhibition for weak antimicrobials
Tried and failed
agar disk diffusion assay for antimicrobial susceptibility applied to acidic antimicrobial compounds against bacterial strains. Outcome: no signal. Reason: the specific antimicrobial formulations completely failed to produce zones of inhibition on agar plates
Lost to a baseline
One clinical P. aeruginosa strain (29130) failed to grow under tested conditions and was excluded from the antimicrobial sensitivity testing disc diffusion assay.
Developing bacteriophage and the predatory bacterium Bdellovibrio bacteriovorus as an alternative therapy for canine otitis externa · University of Nottingham Repository
Optical and fluorogenic viability assays fail due to low biomass sensitivity and dye interference
Optical density measurements and resazurin colorimetric assays lack the sensitivity to detect low bacterial densities, and extracellular polymeric substances can falsely inflate absorbance readings. Adding viability dyes or fluorescent reporters also reduces scatter resolution or fails to yield discernible signal changes under antibiotic exposure.
Considered and rejected
Considered and rejected: Rejected relying on optical density (OD600) for bacterial growth quantification because turbidity from EPS inflated absorbance without corresponding to viable cell replication
An Evaluation of Enriched Aerobic Bacterial Communities Capable of Biodegrading TCE Under Different pH and Temperature · TXST Digital Repository
Tried and failed
incorporating viability dyes in optical scatter assays applied to bacterial viability testing. Outcome: worse than baseline. Reason: Viability dye addition caused unexplained reduction in optical scatter distance between viability states.
Probabilistic Methods For Rapid Characterization Of Complex Biological Samples · Georgia Tech
Tried and failed
optical density and colorimetric viability assays applied to low-biomass liquid microbial cultures. Outcome: no signal. Reason: optical density and resazurin assays lacked sensitivity to detect bacterial concentrations below 10^3 CFU/ml
Pseudomonas aeruginosa in the era of modulator therapies for cystic fibrosis · Imperial
Tried and failed
reactive oxygen species fluorescent reporter assays applied to antibiotic susceptibility testing. Outcome: no signal. Reason: failed to produce discernible fluorescence changes in response to bacteriostatic or bactericidal agents
Probabilistic Methods For Rapid Characterization Of Complex Biological Samples · Georgia Tech
Tried and failed
low-inoculum fluorogenic assay for antimicrobial resistance detection applied to bacterial resistance phenotypic screening. Outcome: no signal. Reason: insufficient starting cell density prevented detectable enzyme activity and resistance expression under antibiotic pressure
Detection of Antibiotic Resistance in Enterococcus spp. by Growth Kinetics Measurements in the Presence of Vancomycin Using the Detection System from TECTA-PDS · Queens University Institutional Repository
Resistance detection assays fail when phenotypic tolerance is conflated with stable genetic mutations
Inoculating antibiotic plates or picking from intermediate inhibition zones frequently isolates transient persisters rather than true resistant mutants. Additionally, genotypic presence of resistance genes can fail to match in vitro susceptibility phenotypes, and standard phenotypic thresholds miss low-level non-standard resistance mutations.
Tried and failed
genotypic prediction of antimicrobial resistance phenotype applied to bacterial pathogen susceptibility testing. Outcome: no signal. Reason: isolate carried a full resistance gene on its chromosome but exhibited phenotypic susceptibility in vitro
Tried and failed
predicting evolutionary tolerance from baseline phenotypic classification applied to bacterial minimum inhibitory concentration evolution. Outcome: no signal. Reason: Baseline tolerance categories showed no significant association with parent, passaged, or adapted resistance levels
Tried and failed
re-isolation from intermediate inhibition zones for resistance applied to bacterial antimicrobial resistance selection. Outcome: did not generalise. Reason: re-isolated cells remained sensitive, displaying phenotypic tolerance rather than stable genetic resistance
Investigation of potassium tetraborate resistance in Dickeya spp. · Cornell
Tried and failed
automated liquid culture phenotypic susceptibility testing applied to detecting bacterial antibiotic resistance mutations. Outcome: did not generalise. Reason: critical concentration thresholds fail to detect low-level resistance conferred by specific non-standard target mutations
Considered and rejected
Considered and rejected: Standard practice of counting all colonies growing on antibiotic-supplemented agar plates as true resistant mutants, rejected because it conflates transient persisters with genetic mutants and skews resistance frequencies higher
UNDERSTANDING RESISTANCE AND PERSISTENCE OF MYCOBACTERIUM ABCESSUS TO COMMON CLINICAL DRUGS TO PROPOSE AN OPTIMIZED TREATMENT REGIMENT · JScholarship
Assay vessels and media conditions introduce physiological artifacts into susceptibility testing
Physical hardware such as Transwell inserts and standard microtiter plates can directly inhibit bacterial growth or fail to support adherence and media exchange during persistence screening. Similarly, testing in unmodified clinical matrices like pure urine or pre-incubating in minimal media triggers starvation-mediated tolerance and distorts testing accuracy.
Lost to a baseline
Pure urine inoculation at 10^8 CFU/mL resulted in lower AST accuracy across all antibiotics/dyes than 10^7 CFU/mL in 10% urine with 90% media.
DEVELOPMENT OF LOW-COST, MICRO-VOLUME ANTIMICROBIAL RESISTANCE ASSAYS FOR PATHOGENIC BACTERIA · Cornell
Considered and rejected
Considered and rejected: Rejected normalizing BCM treatments across bacterial species by CFU due to high dilution requirements (~10^9 PA vs ~10^8 EC) altering secreted factor concentrations.
Unveiling Macrophage-Biofilm Interactions: Implications for Cellular Metabolism and Wound Healing · Carleton University Institutional Repository
Considered and rejected
Considered and rejected: Rejected using PBS for competitive survival assays because 2-hour incubation in minimal media induced robust starvation-mediated antibiotic tolerance in both strains.
THE CONTRIBUTIONS OF THE OMPF AND OSMB GENES IN PROMOTING YERSINIA PSEUDOTUBERCULOSIS SURVIVAL DURING DOXYCYCLINE EXPOSURE · JScholarship
Considered and rejected
Considered and rejected: Rejected using Transwell membrane inserts during bacterial efficacy assays because inserts alone inhibited bacterial growth and interfered with efficacy readings
Considered and rejected
Considered and rejected: Rejected 96-well micro-well titer plates for persistence screening due to non-adherence of bacteria and the inability to dynamically exchange media during antibiotic treatment and washout
Predictive models fail to capture dynamic antibiotic responses and out of population resistance
Linear population loss terms underestimate inhibitory concentration thresholds and miss declines in bacterial growth yields. In genomic resistance modeling, unsupervised clustering methods fail to generalize across external populations and underperform compared to supervised baselines.
Tried and failed
linear loss terms in dynamic population models applied to antimicrobial treatment response modeling. Outcome: did not generalise. Reason: linear terms severely underestimated inhibitory concentration thresholds and failed to capture growth yield declines
Computational models for bacterial dynamics in community and treatment contexts · Georgia Tech
Tried and failed
density-based clustering with dimensionality reduction applied to bacterial genomic sequence resistance prediction. Outcome: did not generalise. Reason: failed out-of-population generalisation and achieved consistently lower accuracy than supervised baselines
Left open by the authors
Problems the authors named and did not get to.
Left open
Investigate the biological mechanisms causing pure-culture skim milk broth growth parameters to overestimate bacterial spoilage rates in HTST fluid milk. Blocker: Requires wet lab microbiological experiments and biological assays to determine mechanistic causes of growth differences
STRATEGIES TO IMPROVE THE SHELF LIFE OF FLUID MILK FROM BUSINESS IMPACTS TO SUSTAINABILITY · Cornell
Left open
Investigate mixed-species biofilm development and corrosion under regular media replenishment conditions. Blocker: Requires a wet microbiology and materials laboratory with specific bacterial strains, BioCells, and media replenishment apparatus
Designing and Optimizing a Biofilm Experiment for Space Flight · TXST Digital Repository
Left open
Integrate cell counts, cellular activity measurements, and antimicrobial resistance gene analysis in drinking water distribution system biofilms. Blocker: Requires physical wet-lab biofilm reactor experimentation and microbiological assays
Investigating the impacts of sub-inhibitory concentrations of ciprofloxacin on microbial abundance and diversity in a multi-species groundwater sourced drinking water biofilm · Queens University Institutional Repository
Left open
Monitor conjugative plasmid spread across multispecies biofilm communities under sub-inhibitory antibiotic exposure. Blocker: Requires a microbiological wet lab, bioreactor setup, and live multispecies biofilm communities.
Left open
Test the BAF solid media method using variable fluoranthene concentrations and non-volatile solvents like DMF. Blocker: Requires a wet microbiology laboratory, autoclaves, chemical reagents (fluoranthene, DMF), and bacterial/algal cultures.
Left open
Resolve the discrepancy with the Carballido-López group regarding MreB filament velocity changes across varying nutrient conditions. Blocker: Requires wet lab experiments with TIRF microscopy imaging of bacterial strains under different nutrient conditions.
Regulation of Cell Wall Synthesis and Role of Surface Sensing in Bacillus Subtilis · Harvard
Left open
Investigate why M1-sensitive Pseudomonas aeruginosa strains CF710 and CF753 fail to grow properly during serial dilution plate counts. Blocker: Requires a wet lab, bacterial cultures (CF710, CF753), and microbiological assay experiments
Isolation, characterization, and potential clinical application of the M1-Pseudomonas aeruginosa pyocin · Texas Tech
Left open
Measure dynamic contact angle and range expansion changes in bacterial colonies under varying nutrient levels and surface properties. Blocker: Requires a wet lab and white light interferometry equipment to culture and image live bacterial biofilms under dynamic conditions.
Biophysical basis of bacterial colony growth · Georgia Tech
Left open
Investigate the effects of DO, pH, ions, and TOC on nanoparticle combination disinfection efficacy using wet-lab batch testing. Blocker: Requires a microbiological wet lab, bacterial cultures (E. coli), water quality instrumentation, and nanoparticle handling facilities.
Impact of Low-Levels of Silver, Zinc and Copper Nanoparticles on Bacterial Removal and Potential Synergy for Point-of-Use Water Treatment Solutions · Carleton University Institutional Repository
Left open
Apply Kirby-Bauer disk diffusion assays using antimicrobial interventions to Salmonella, Listeria monocytogenes, and bacterial cocktail mixtures. Blocker: Requires wet-lab microbiology facilities, bacterial strains (Salmonella, Listeria), and antimicrobial testing reagents.
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