Analysis of biocide and surfactant influencing detachment of bacterial cells

Adhesion and chemical induced detachment kinetics of Pseudomonas fluorescens ATCC 13525T to glass surface were conducted in situ under flow conditions in a well-controlled parallel plate flow chamber (PPFC). Ortho-phthalaldehyde (OPA) and cetyltrimethyl ammonium bromide (CTAB), respectively, an aldehyde-based biocide and a cationic surfactant were applied at several concentrations to the glass attached bacteria. At the end of the chemical treatment the remaining adhered bacteria were characterized in terms of viability and cellular size. Simultaneously, planktonic cell surface characterization was conducted in order to correlate PPFC results with thermodynamic approaches for adhesion prevision and to evaluate the surface free energy of chemically treated cells and its relevance for adhesion strength. It was observed that about 2.8×106 cells/cm2 adhered to the glass surface after 30 min of bacterial flow through the PPFC, besides thermodynamic analyses demonstrate unfavourable adhesion of P. fluorescens to glass (ΔGadhesion = 30 mJ/m2). The application of OPA and CTAB promoted bacterial detachment in a small extent ( 0.1). For CTAB, apolar characteristics increased with concentration (P < 0.05), switching from hydrophilic to hydrophobic for concentrations near the critical micellar concentration. The overall results emphasize the role of the adhesion process and the chemical stress on cellular physiological induced response. OPA and CTAB were markedly inefficient in the removal of glass attached P. fluorescens, demonstrating that bacteria can be non-viable but remain attached to the adhesion surface

Control of flow-generated biofilms with surfactants : evidence of resistance and recovery

The action of cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS), a cationic and an anionic surfactant respectively, were investigated for their ability to control turbulent and laminar flow-generated biofilms formed by P. fluorescens. The disinfectant action of CTAB and SDS on biofilms was assessed by respiratory activity, variation of mass and structure, immediately, 3, 7 and 12 h after the application of the surfactants. Laminar flow-generated biofilms were more susceptible to the action of CTAB than those formed under turbulent flow. Total inactivation of the cells within the biofilms was not achieved for either type of flow- enerated biofilms. For SDS, higher concentrations promoted significant biofilm inactivation, for both turbulent and laminar flow-generated biofilms. CTAB and SDS application did not promote the detachment of biofilms from the surfaces. Post-surfactant treatment, biofilms recovered respiratory activity, in some cases, reaching values higher than those found without chemical treatment. After CTAB treatment, the recovery of respiratory activity was not affected by the hydrodynamic conditions. Conversely, turbulent flow-generated biofilms showed a higher potential to recover their metabolic activity than laminar flow-generated biofilms, when previously challenged with SDS. Concerning biofilm mass, no significant variation (increase or decrease) was detected after 12 h of surfactant treatment. This study shows that care is needed when selecting the correct procedure and agent for biofilm control and demonstrates the influence of hydrodynamic conditions on the persistent and recalcitrant properties of P. fluorescens biofilms.Fundação para a Ciência e a Tecnologia (FCT

Antagonism between bacillus cereus and pseudomonas fluorescens in planktonic systems and in biofilms

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steadystate biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.Fundação para a Ciência e a Tecnologia (FCT

Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on Pseudomonas fluorescens biofilms was investigated using flow cell reactors with stainless steel substrata, under turbulent (Re=5200) and laminar (Re=2000) flow. Steady-state biofilms were exposed to SDS in single doses (0.5, 1, 3 and 7 mM) and biofilm respiratory activity and mass measured at 0, 3, 7 and 12 h after the SDS application. The effect of SDS on biofilm mechanical stability was assessed using a rotating bioreactor. Whilst high concentrations (7 mM) of SDS promoted significant biofilm inactivation, it did not significantly reduce biofouling. Turbulent and laminar flow-generated biofilms had comparable susceptibility to SDS application. Following SDS exposure, biofilms rapidly recovered over the following 12 h, achieving higher respiratory activity values than before treatment. This phenomenon of posttreatment recovery was more pronounced for turbulent flow-generated biofilms, with an increase in SDS concentration. The mechanical stability of the biofilms increased with surfactant application, except for SDS concentrations near the critical micellar concentration, as measured by biofilm removal due to an increase in external shear stress forces. The data suggest that although SDS exerts antimicrobial action against P. fluorescens biofilms, even if only partial and reversible, it had only limited antifouling efficacy, increasing biofilm mechanical stability at low concentrations and allowing significant and rapid recovery of turbulent flow-generated biofilms.Fundação para a Ciência e a Tecnologia (FCT

Control of biofilms using surfactants: persistence and regrowth

The action of the cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), respectively, a cationic and an anionic surfactant were investigated to control mature biofilms formed under turbulent and laminar flow, by P.fluorescens. The sanitizer action of the surfactants on biofilms was assessed by means of respiratory activity and variation of biofilm mass, immediately, 3, 7 and 12 h after the treatment of the chemicals. The latter experimental times were tested in order to assess the biofilm regrowth. The structure of the biofilms was assessed before and after surfactant treatment by SEM. The results showed that, laminar biofilms were more susceptible to the action of CTAB than those formed under turbulent flow. Concerning SDS, both biofilms showed analogous susceptibility to the surfactants. However, total inactivation of the cells within the biofilms was not achieved for both types of biofilms. CTAB application by itself did not promoted the detachment of biofilms from the surface. Regarding SDS, higher concentrations applied promoted significant biofilm inactivation. Turbulent and laminar flow had analogous susceptibility to SDS application. However, SDS did not promoted the detachment of biofilms from the metal surfaces. The structure of the biofilms was changed after the application of both surfactants. It was found that after CTAB and SDS application, the biofilms recovered its respiratory activity, reaching, in same situations, higher values than the ones found before chemical treatment. The CTAB application promoted similar recovery in the respiratory activity for both biofilms. Concerning biofilm behaviour after SDS treatment, turbulent biofilms showed a higher potential to recover their metabolic activity than laminar biofilms. Biofilm mass did not experienced any significant variation after the treatment, for both surfactants tested. This study highlights the need of care in choosing the correct procedure for biofilm control and the recalcitrant properties of biofilms.Fundação para a Ciência e a Tecnologia (FCT

The effects of a biocide and a surfactant on the detachment of Pseudomonas fluorescens from glass surfaces

Application of antimicrobial chemicals is a general procedure in the cleaning and disinfection of food-contacting surfaces. Adhesion to glass surfaces and chemically induced detachment of Pseudomonas fluorescens ATCC 13525T were studied in situ, under flow conditions, in a well-controlled parallel plate flow chamber (PPFC). Ortho-phthalaldehyde (OPA) and cetyltrimethyl ammonium bromide (CTAB) were applied separately, at several concentrations, to attached bacteria and their subsequent detachment was monitored. Following treatments the remaining adhered bacteria were characterized in terms of viability and cell size. Simultaneously, the planktonic cell surface was characterized in order to correlate PPFC results with thermodynamic approaches for adhesion evaluation, and surface free energy of chemically treated cells with adhesion strength. About 2.8 × 106 cells/cm2 adhered to the glass surface after 30 min of bacterial flow, although thermodynamic analyses evidenced unfavourable adhesion. The independent application of OPA and CTAB promoted bacterial detachment to a small extent (16% of total cells). The remaining adhering bacteria were totally non-viable for OPA ≥ 0.75 mM and CTAB ≥ 0.25 mM, showing a lack of correlation between bacterial viability and detachment. The cellular size decreased as attachment proceeded and with chemical treatment. Both chemicals altered the cell surface properties, increasing the cell-glass adhesion strength, and promoting the emergence of polar characteristics. The overall results emphasize that OPA and CTAB were markedly ineffective in removing glass-attached P. fluorescens, demonstrating that bacteria can be non-viable but remain strongly attached to the adhesion surface.Fundação para a Ciência e a Tecnologia (FCT) - Project CHEMBIO – POCI/BIO/61872/2004; SFRH/BD/ 31661/2006; SFRH/BPD/20582/2004

Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes

The action of the cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated to control biofilms (aged 7 d) formed by Pseudomonas fluorescens on stainless-steel slides, using flow cells reactors, under turbulent and laminar flow. The effect of CTAB was also investigated using planktonic cells in the presence and absence of BSA, by measuring the cellular respiratory activity and the ATP released. The action of CTAB on biofilms was assessed by means of cellular respiratory activity and variation of biofilm mass, immediately and 3, 7 and 12 h after the application of CTAB. The physical stability of the biofilm was also assessed using a rotating device, where the effect of the surfactant on the biofilm stability was evaluated through the variation of the mass remaining on the surface. CTAB significantly reduced the activity of the planktonic cells probably due to the rupture of the cells. This effect was significantly reduced in the presence of BSA. Planktonic cells were more easily inactivated than bacteria in biofilms. Biofilms formed under laminar flow were more susceptible than those formed under turbulent flow, but in both cases total inactivation was not achieved. Biofilm recovery was observed, in terms of respiratory activity, in almost all the cases studied. CTAB application by itself did not promote the detachment of biofilms. The physical stability tests showed that the synergistic action of the surfactant and the application of high shear stress to the biofilm increase its detachment.Fundação para a Ciência e Tecnologia (FCT

Influence of material type and surface benzalkonium chloride preconditioning on biofilm formation and activity

This study investigates the potential of benzalkonium chloride (BC), a cationic surfactant, on the prevention of biofilm formation on stainless steel ASI 316 and silicone rubber, two distinct surfaces currently used on food processing facilities. The surfaces were preconditioned with several concentration of BC for 30 min. Treated surfaces were characterized by the sessile drop method, demonstrating that surfactant pre-treatment increased the hydrophobicity of the surfaces, this increase being a function of BC concentration increase applied for preconditioning. In order to ascertain the preventive effect in biofilm formation, the treated surfaces where inserted in a chemostat continuously inoculated with P. fluorescens in the exponential phase of growth, being the biofilm allowed to grow for 6 days. The results showed that BC preconditioning did not prevent or impair biofilm formation. In fact, biofilms developed on the treated surfaces presented higher biomass and respiratory activity than the ones formed on the untreated surfaces, this phenomenon being more evident for silicone than for stainless steel and for surfaces treated with higher BC concentrations. Scanning electron microscopy and biochemical analysis reveal that the difference of surface type and surface preconditioning, by itself, gave rise to the formation of structural and biochemical distinct biofilms. The overall results suggest that preconditioning of stainless steel and silicone rubber surfaces with BC allowed the formation of biofilms with more recalcitrant properties than the ones found on untreated surfaces