Publicação
Use of biocides and surfactants to control Pseudomonas fluorescens biofilms: role of the hydrodynamic conditions
| Resumo: | Biofilms constitute a protected growth modality that allows the bacteria to survive in hostile environments. The most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, current disinfection practices show often inefficacy in the control of biofilms. The main goals of this work were the development of effective strategies, based on the application of chemical agents, in order to control biofilms formed by Pseudomonas fluorescens, and the evaluation of the impact of the hydrodynamic conditions under which biofilms were formed, in the phenotypic characteristics of the biofilms, as well as, in their susceptibility to antimicrobial agents. The antimicrobial agents tested were two non-oxidizing aldehyde-based biocides (ortho-phthalaldehyde - OPA and glutaraldehyde - GTA), two oxidizing biocides (sodium hydroxide - NaOH and sodium hypochlorite - SHC) and two surfactants (cetyltrimethyl ammonium bromide - CTAB and sodium dodecyl sulfate - SDS). The antimicrobial agents were selected due to their emergent and practical application in several industrial areas. The bacterial biofilms were developed on stainless steel surfaces in a flow cell reactor and in a bioreactor with a rotating device, being implemented different hydrodynamic conditions (laminar and turbulent flow) for biofilm formation. The action of the antimicrobial agents was assessed, mainly, by the determination of the biofilm respiratory activity, as well as, by the quantification of the biofilm mass removed, being tested several concentrations, contact times and strategies of application. The bacteria grown in biofilms under different flow regimes were phenotypically characterized in terms of outer membrane proteins expression (OMP), metabolic activity, biochemical composition and structure, being their phenotypes compared with bacteria in planktonic state. The respirometric method based on the assessment of the bacterial respiratory activity, the basic tool in the evaluation of the efficiency of the antimicrobial agents and in the characterization of the metabolic state of the bacteria, was validated by comparison with two reference methods (assessment of viability by “Live/Dead” stains and culturability in a solid medium). Within the scope of this thesis, and for further comparison, it was assessed the antimicrobial properties of the biocides OPA and GTA and of the surfactants CTAB and SDS in the control of planktonic cells. These experiments revealed that, in the range of concentrations tested, OPA was more efficient in the bacterial respiratory inactivation than GTA, causing total bacterial inactivation. Concerning the surfactants, both caused reduction of the bacterial activity, but only CTAB caused total bacterial inactivation. However, the chemical agents reacted differently with the bacteria, being CTAB the unique that promoted cellular disruption. The bacterial phenotype was affected with the application of the chemical agents, since the OMP expression and the cellular pellet colour changed after chemical treatment. The presence of bovine serum albumin (BSA) in the bacterial cultures (in order to simulate dirty conditions found in industrial systems) reduced significantly the antimicrobial action of the several chemical agents. Concerning biofilm formation in the flow cells, it was found that the flow regime and the sessile mode of life itself caused significant modifications in the metabolism, morphology and constitutive biochemical composition of the P. fluorescens cells. The application of biocides (OPA and GTA) and surfactants (CTAB and SDS) to the biofilms developed in the flow cells, under different hydrodynamic conditions, revealed that, independently of the concentration, exposure time and strategy of application, every chemical agent was more effective in the inactivation of laminar biofilms than turbulent biofilms, being OPA the only chemical that caused total inactivation. The entire chemicals tested exhibited poor ability in the removal of biofilms from the surfaces, independently of the flow regime under which biofilms were formed. It was verified a post-antimicrobial agents application effect for both biofilms, since after chemical treatment, biofilms recovered with the time their metabolic activity and viability, an evidence of the capability of biofilms to regrow. The comparison of these results with the ones obtained with the planktonic cells emphasises the higher resistance of biofilm microorganisms to disinfection when compared with their freely suspended counterparts. The results also underscore the inadequacy of planktonic testing methods for evaluating antimicrobial agents to be used as a means to control biofilms. This fact contradicts the presuppositions of the European Standard – EN 1276 (1997), where the bactericidal activity of disinfectants to be used in food, industrial, domestic and institutional areas are tested using cells in planktonic state. The biofilms grown in the bioreactor with the rotating device showed an accentuated inherent mechanical stability, i.e., a strong cohesion face to sudden changes in the surrounding hydrodynamic conditions. These biofilms were treated with OPA, GTA, NaOH, SHC, CTAB and SDS with the purpose to ascertain if after chemical treatment the mechanical stability of the biofilms (the biofilm behaviour face to external mechanical stress) was changed. It was found that biofilms previously treated with CTAB, NaOH, SHC, OPA and SDS (for concentrations near the critical micellar concentration) the biofilm mechanical stability decreased. Concerning GTA and SDS (for low concentrations), it was found an increase in the biofilm mechanical stability. These results highlight that even the synergistic chemical and mechanical treatments did not induce total biofilm eradication. Finally, with the purpose to ascertain if a strain can represent a specie, the biofilm formation ability, as single or mixed populations, of two P. fluorescens native strains from a dairy industry was assessed using the flow cell reactor and the bioreactor rotating system. Similar characteristics were found comparing the biofilms formed by the different strains, as single or mixed biofilms. The phenotypic characteristics of the native strains were comparable with the ones obtained with the type strain, even when native strains were used to form mixed biofilms. The results collected in this work allowed to conclude the role of the flow regime under which biofilms are formed in their susceptibility to antimicrobial agents, as well as, in the success/fallibility of procedures for biofilm control, fundamentally when extreme conditions are tested. The biofilms recovered their metabolic activity and viability, after chemical treatment, even when they presented merely residual activity. The submission of biofilms, previously exposed to antimicrobial agents, to hydrodynamic conditions different from the ones biofilms were developed did not cause total biofilm eradication from the surfaces contributing, therefore, for biofilm recalcitrance. |
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| Autores principais: | Simões, M. |
| Ano: | 2005 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Biofilms constitute a protected growth modality that allows the bacteria to survive in hostile environments. The most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, current disinfection practices show often inefficacy in the control of biofilms. The main goals of this work were the development of effective strategies, based on the application of chemical agents, in order to control biofilms formed by Pseudomonas fluorescens, and the evaluation of the impact of the hydrodynamic conditions under which biofilms were formed, in the phenotypic characteristics of the biofilms, as well as, in their susceptibility to antimicrobial agents. The antimicrobial agents tested were two non-oxidizing aldehyde-based biocides (ortho-phthalaldehyde - OPA and glutaraldehyde - GTA), two oxidizing biocides (sodium hydroxide - NaOH and sodium hypochlorite - SHC) and two surfactants (cetyltrimethyl ammonium bromide - CTAB and sodium dodecyl sulfate - SDS). The antimicrobial agents were selected due to their emergent and practical application in several industrial areas. The bacterial biofilms were developed on stainless steel surfaces in a flow cell reactor and in a bioreactor with a rotating device, being implemented different hydrodynamic conditions (laminar and turbulent flow) for biofilm formation. The action of the antimicrobial agents was assessed, mainly, by the determination of the biofilm respiratory activity, as well as, by the quantification of the biofilm mass removed, being tested several concentrations, contact times and strategies of application. The bacteria grown in biofilms under different flow regimes were phenotypically characterized in terms of outer membrane proteins expression (OMP), metabolic activity, biochemical composition and structure, being their phenotypes compared with bacteria in planktonic state. The respirometric method based on the assessment of the bacterial respiratory activity, the basic tool in the evaluation of the efficiency of the antimicrobial agents and in the characterization of the metabolic state of the bacteria, was validated by comparison with two reference methods (assessment of viability by “Live/Dead” stains and culturability in a solid medium). Within the scope of this thesis, and for further comparison, it was assessed the antimicrobial properties of the biocides OPA and GTA and of the surfactants CTAB and SDS in the control of planktonic cells. These experiments revealed that, in the range of concentrations tested, OPA was more efficient in the bacterial respiratory inactivation than GTA, causing total bacterial inactivation. Concerning the surfactants, both caused reduction of the bacterial activity, but only CTAB caused total bacterial inactivation. However, the chemical agents reacted differently with the bacteria, being CTAB the unique that promoted cellular disruption. The bacterial phenotype was affected with the application of the chemical agents, since the OMP expression and the cellular pellet colour changed after chemical treatment. The presence of bovine serum albumin (BSA) in the bacterial cultures (in order to simulate dirty conditions found in industrial systems) reduced significantly the antimicrobial action of the several chemical agents. Concerning biofilm formation in the flow cells, it was found that the flow regime and the sessile mode of life itself caused significant modifications in the metabolism, morphology and constitutive biochemical composition of the P. fluorescens cells. The application of biocides (OPA and GTA) and surfactants (CTAB and SDS) to the biofilms developed in the flow cells, under different hydrodynamic conditions, revealed that, independently of the concentration, exposure time and strategy of application, every chemical agent was more effective in the inactivation of laminar biofilms than turbulent biofilms, being OPA the only chemical that caused total inactivation. The entire chemicals tested exhibited poor ability in the removal of biofilms from the surfaces, independently of the flow regime under which biofilms were formed. It was verified a post-antimicrobial agents application effect for both biofilms, since after chemical treatment, biofilms recovered with the time their metabolic activity and viability, an evidence of the capability of biofilms to regrow. The comparison of these results with the ones obtained with the planktonic cells emphasises the higher resistance of biofilm microorganisms to disinfection when compared with their freely suspended counterparts. The results also underscore the inadequacy of planktonic testing methods for evaluating antimicrobial agents to be used as a means to control biofilms. This fact contradicts the presuppositions of the European Standard – EN 1276 (1997), where the bactericidal activity of disinfectants to be used in food, industrial, domestic and institutional areas are tested using cells in planktonic state. The biofilms grown in the bioreactor with the rotating device showed an accentuated inherent mechanical stability, i.e., a strong cohesion face to sudden changes in the surrounding hydrodynamic conditions. These biofilms were treated with OPA, GTA, NaOH, SHC, CTAB and SDS with the purpose to ascertain if after chemical treatment the mechanical stability of the biofilms (the biofilm behaviour face to external mechanical stress) was changed. It was found that biofilms previously treated with CTAB, NaOH, SHC, OPA and SDS (for concentrations near the critical micellar concentration) the biofilm mechanical stability decreased. Concerning GTA and SDS (for low concentrations), it was found an increase in the biofilm mechanical stability. These results highlight that even the synergistic chemical and mechanical treatments did not induce total biofilm eradication. Finally, with the purpose to ascertain if a strain can represent a specie, the biofilm formation ability, as single or mixed populations, of two P. fluorescens native strains from a dairy industry was assessed using the flow cell reactor and the bioreactor rotating system. Similar characteristics were found comparing the biofilms formed by the different strains, as single or mixed biofilms. The phenotypic characteristics of the native strains were comparable with the ones obtained with the type strain, even when native strains were used to form mixed biofilms. The results collected in this work allowed to conclude the role of the flow regime under which biofilms are formed in their susceptibility to antimicrobial agents, as well as, in the success/fallibility of procedures for biofilm control, fundamentally when extreme conditions are tested. The biofilms recovered their metabolic activity and viability, after chemical treatment, even when they presented merely residual activity. The submission of biofilms, previously exposed to antimicrobial agents, to hydrodynamic conditions different from the ones biofilms were developed did not cause total biofilm eradication from the surfaces contributing, therefore, for biofilm recalcitrance. |
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