Living Cells of Staphylococcus aureus Immobilized onto the Capillary Surface in Electrochromatography: A Tool for Screening of Biofilms

Abstract

Microorganisms attach to nonliving surfaces in many natural, industrial, and medical environments, enveloped within extracellular polymeric substances. The result is a biofilm. Biofilms are reported to exist in 65-80% of bacterial infections refractory to host defenses and antibiotics therapy and are regarded as a central problem in present-day medical microbiology. Understanding of the parameters governing the interaction of antimicrobials with biofilms is thus of great interest in any attempt to increase biocide efficacy. In this work, study was made of the feasibility of using open tubular capillary electrochromatography (CEC) in bacterial biofilm studies with living cells. Staphylococcus aureus was selected as model bacterium. First, S. aureus was shown, under various conditions, to form a biofilm on the inner wall of a fused-silica capillary coated with poly(L-lysine). Optimal conditions for biofilm formation, such as bacterial concentration, growing time, and the stability of the ensemble, were preliminarily defined with conventional 96-microtiter well plates. Continuous flushing of the capillary with fresh cells meant that no growth medium was needed. The presence of biofilm in the capillary was confirmed by atomic force microscopy. Interactions between S. aureus biofilms and different antibiomicrobial agents were studied by capillary electrochromatography. The effect of five antibiotics (penicillin G, oxacillin, fusidic acid, rifampicin, vancomycin) on biofilms was examined in terms of retention factors and reduced mobilities of the antibiotics. The antibiotic susceptibility profile for S. aureus is similar as the result of minimal inhibitory concentrations registered on the 96-microtiter well plates for both planktonic and biofilm cells. The results show, for the first time, that bacterial biofilms can be studied by CEC. The technique allows highly efficient and easy characterization of interactions between S. aureus biofilms and potentially active antimicrobial compounds under different conditions. Reagent and cell consumption are minimal.

Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions

“…Biofilm has been found in 65-80% of the bacterial infections, and is considered refractory to host defenses in antibiotic therapy [1]. Among the biofilm forming bacteria, Staphylococcus aureus is responsible for severe skin infections to such major diseases as bacteremia, endocarditis and osteomyelitis.…”

Anti-Biofilm Drug Susceptibility Testing Methods: Looking for New Strategies against Resistance Mechanism

“…The retention of analytes is due to a combination of electrophoretic migration and chromatographic retention. Some authors have shown that CEC technique is successful applied to the separation, identification, and characterization of mixtures of both living and dead bacteria contained in biofilm structure, very useful in biofilm metabolomics research [46].…”

Proteome Analyses of Staphylococcus aureus Biofilm at Elevated Levels of NaCl

“…Biofilm, an extracellular polymeric matrix secreted by certain microorganisms, has been found in 65%–80% of bacterial infections, and is considered refractory to host defenses in antibiotic therapy [ 1 ]. Among the biofilm-forming bacteria, Staphylococcus aureus is responsible for health problems ranging from minor skin infections to such major diseases as bacteremia, endocarditis and osteomyelitis.…”