Combined phenotypic characterization of dominant Enterococcus strains isolated from aquaculture facilities and fish in Greece: growth kinetics, minimum inhibitory concentrations of antibiotics, and biofilm behavior.

Aquaculture is a key contributor to global food security, yet the emergence of antimicrobial-resistant bacteria within production systems poses a growing threat to food safety and environmental health. Enterococcus spp., commonly present in aquatic environments, are known for their resilience, antibiotic resistance, and biofilm-forming ability. Ιn this exploratory study, we conducted a combined phenotypic characterization of five dominant Enterococcus faecium and Enterococcus faecalis isolates from aquaculture facilities and farmed fish in Greece to evaluate their planktonic growth kinetics, minimum inhibitory concentrations (MICs) of antibiotics under modified experimental conditions, and biofilm-forming capacity under temperature conditions representative of aquaculture environments. Planktonic growth dynamics were modeled through the Baranyi and Roberts and Ratkowsky equations, while minimum inhibitory concentrations (MICs) of vancomycin, tetracycline, and streptomycin were determined using the broth microdilution method at 12 °C and 30 °C. Biofilm formation was tested on polystyrene microtiter plates at these two temperatures using the classical crystal violet staining method in two growth media: a well-established nutrient-rich laboratory medium, and a medium designed to mimic the natural seawater-associated environment. Growth modeling revealed that maximum specific growth rate (μmax) increased with temperature, whereas lag phase duration decreased. Estimated theoretical minimum growth temperatures (Tmin) ranged from 1.0 °C to 4.1 °C, indicating strain-specific cold adaptation. E. faecium strain 22 exhibited the broadest thermal adaptability and strongest biofilm capacity. Temperature-dependent differences in apparent antimicrobial inhibition were observed. All strains formed detectable biofilm biomass in the nutrient-rich brain heart infusion (BHI) broth, whereas fish feed infusion (FFI) broth did not yield detectable biofilm biomass for all strains except one under the conditions tested. In BHI, crystal violet measurements indicated higher biofilm biomass at the lower temperature. Overall, this work highlights temperature- and strain-dependent behavior in the five studied Enterococcus isolates under aquaculture-relevant conditions and illustrates how combining growth kinetics, antimicrobial inhibition and biofilm formation can inform exploratory hypotheses for risk assessment and hygiene strategies.