Crystal violet (CV) staining is widely used to classify clinical isolates of Klebsiella pneumoniae as non-adherent, weak, moderate, or strong biofilm producers. However, a measurement of total adherent biomass does not necessarily indicate the number of viable bacteria present within a biofilm or the capacity of those bacteria to survive antimicrobial exposure. This study examined the relationships among CV biomass, three-dimensional biofilm architecture, viable sessile burden, and antibiotic survival in a panel of 60 non-duplicate clinical K. pneumoniae isolates. The isolates were characterised by CV staining (OD570), quantitative culture (log10 CFU/cm2), confocal laser scanning microscopy combined with COMSTAT analysis, and 24-hour antimicrobial challenges involving meropenem, ciprofloxacin, and colistin at 10× and 100× isolate-specific minimum inhibitory concentrations (MICs). Architectural measurements included biovolume, mean thickness, roughness, substratum coverage, and average diffusion distance. Minimum biofilm eradication concentrations (MBECs) were measured using a peg-lid method. CV biomass showed only a weak correlation with viable sessile burden (Spearman’s ρ = 0.31) and weak associations with log10 reductions achieved by the tested antibiotics. Average diffusion distance and initial viable burden were the most informative regression predictors of reduced killing; the meropenem model explained 61% of the reported variation in log10 reduction. Notably, 15 isolates classified as non-adherent or weakly adherent by CV nonetheless contained at least 107 CFU/cm2 and showed less than a 1-log10 reduction following meropenem exposure at 100× MIC. Conversely, six strongly staining isolates exhibited reductions greater than 3 log10. These observations indicate that total adherent biomass, viable bacterial burden, architectural organisation, and antibiotic survival are related but distinct biofilm characteristics. CV staining alone is therefore insufficient for evaluating biofilm-associated antimicrobial tolerance, and complementary assessments of viability and survival are warranted in clinical-isolate research.
