The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms

Aims:  The hypothesis that surrogate planktonic pathogens ( Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested.
Methods and Results:  Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0·15 and 1·5 N m⁻². The amount of surrogate pathogens introduced ranged from approximately 10⁵ CFU ml⁻¹ to 10¹⁰ spheres ml⁻¹. The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0·4–3·0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated.
Conclusions:  Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination.
Significance and Impact of the Study:  This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.     

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. Steady-state 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.     

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage φIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, φIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.     

Differential efficacy of a chlorine dioxide-containing sanitizer against single species and binary biofilms of a dairy-associated Bacillus cereus and a Pseudomonas fluorescens isolate

AIMS: Daily exposure to 100 p.p.m. chlorine dioxide of single species and binary biofilms of dairy-associated Bacillus cereus DL5 and Pseudomonas fluorescens M2, attached to stainless steel surfaces in a laboratory flow system, was studied. METHODS AND RESULTS: Surfaces were sampled daily before and after sanitizer treatment and cells and spores dislodged and enumerated by standard methods. Duplicate surfaces were prepared for confocal scanning laser microscopy (CSLM) and scanning electron microscopy. Higher counts of Ps. fluorescens M2 were obtained in single species biofilms, microcolonies stained green (viable) in CSLM images and were closely packed on attachment surfaces. By contrast, higher counts of B. cereus DL5 were obtained in binary biofilms, microcolonies stained green in CSLM images, but were more spread out. Lower spore counts were obtained for B. cereus DL5 in binary biofilms. The survival of Ps. fluorescens M2 cells after exposure to chlorine dioxide was apparently enhanced by the presence of B. cereus DL5 in binary biofilms. By contrast, B. cereus DL5 showed increased susceptibility to sanitizer treatment in the presence of Ps. fluorescens M2. CONCLUSIONS: Co-cultured bacteria in biofilms influence each other with respect to attachment capabilities and sanitizer resistance/susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Binary biofilms endemic in food-processing industries can survive sanitation regimes and may represent reservoirs of product contamination leading to subsequent spoilage and/or food safety risks.     

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.     

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

Abstract

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm², and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.     

Studies on the behaviour of Pseudomonas fluorescens biofilms after Ortho-phthalaldehyde treatment

A relatively novel biocide, ortho-phthalaldehyde (OPA), was tested to control biofilms formed by Pseudomonas fluorescens on stainless steel surfaces. The toxic action of OPA was assessed in terms of inactivation and removal of the biofilm by means of, respectively, the determination of the respiratory activity and the variation in the dry weight of the biofilms. For comparison, the activity of OPA against suspended bacteria was also evaluated. The results showed that higher concentrations of OPA and longer exposure times are needed to inactivate P. fluorescens biofilms than planktonic populations, thus denoting that sessile bacteria have a reduced susceptibility to OPA. This appears to be associated with the reaction with the proteins of the matrix, as demonstrated by the reduction of the antimicrobial action of OPA in the presence of a protein (bovine serum albumin). The application of OPA appeared to cause little effect in the removal of biofilms from the metal slides since the mass of biofilm that remained on the surfaces, after biocide treatment, was within the same range as those observed in the control tests. These results suggest that, with OPA application, biofilms can be inactivated but stay attached to the surfaces, decreasing thereby the success of the chemical treatment.     

The structure of Pseudomonas fluorescens biofilms in contact with flowing systems

Using a specially designed flow system Pseudomonas fluorescens has been grown on the inside of glass tubes under carefully controlled conditions. Results show that films developed from water flowing at 0–5 m.s^‐1 are less compact and thicker than with a water velocity of 2–5 m.s^‐1. In the latter all the cells are aligned in the direction of flow whereas in the former the individual cells directly attached to the surface, are randomly distributed with groups of cells lying parallel to the tube axis.     

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C₆) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C₈) fatty acyl chain.     

Effects of the interactions between glutaraldehyde and the polymeric matrix on the efficacy of the biocide against pseudomonas fluorescens biofilms

Glutaraldehyde (GTA) is a widely used biocide due to its high effectiveness. The experimental work reported here was carried out to assess the effectiveness of GTA in controlling biofilms formed by Pseudomonas fluorescens on stainless steel slides, and to compare efficacy against both planktonic and sessile micro‐rganisms. The tests were performed using two concentrations of GTA (50 and 100mg 1^‐1), biofilms of two ages (7 and 15 d), several pH values (5,7 and 9) and a range of exposure times (from 0 (control) to 1,3,7 and 24 h). The action of GTA on biofilm and planktonic populations was assessed by means of activity tests, zeta potential, and the wet weight of the biofilms. Biofilms were not completely removed after treatment with GTA in any of the conditions studied. The higher GTA concentration was more effective in reducing the bacterial activity of the biofilm. The biocide proved to be more effective for longer exposure times. GTA showed good antimicrobial activity against P. fluorescens in suspension, with higher activity at pH 9. The findings of this study suggest that when GTA is used to control biofilms, it reacts with one of the components of the matrix, the proteins, thereby reducing its antimicrobial action.     

The use of cyclic voltammetry to detect biofilms formed by Pseudomonas fluorescens on platinum electrodes

The development of an electrochemical detector to monitor the in situ formation of biofilms is described. The detector consisted of an electrochemical cell containing three electrodes, whose response to the application of a potential profile to the working electrode was sensitive to the amount of biofilm present on the surface. The electrochemical technique used was repetitive cyclic voltammetry. Differences between the response of the uncolonised electrode and after Pseudomonas fluorescens biofilms of different ages were grown on its surface were determined. The results show that cyclic voltammetry applied to platinum electrodes can be used to detect young biofilms. The development of the shape of the voltammogram as the potential is cycled may constitute a means of providing information on the coverage of the surface. Observation of the platinum electrodes before and after the electrochemical measurements showed that even after 30 min of recycling, most of the cells were still adhered to the surface, although some may have lost viability.     

Adhesion and removal kinetics of Bacillus cereus biofilms on Ni-PTFE modified stainless steel

Biofilm control remains a challenge to food safety. A well-studied non-fouling coating involves codeposition of polytetrafluoroethylene (PTFE) during electroless plating. This coating has been reported to reduce foulant build-up during pasteurization, but opportunities remain in demonstrating its efficacy in inhibiting biofilm formation. Herein, the initial adhesion, biofilm formation, and removal kinetics of Bacillus cereus on Ni-PTFE-modified stainless steel (SS) are characterized. Coatings lowered the surface energy of SS and reduced biofilm formation by > 2 log CFU cm^?2. Characterization of the kinetics of biofilm removal during cleaning demonstrated improved cleanability on the Ni-PTFE coated steel. There was no evidence of biofilm after cleaning by either solution on the Ni-PTFE coated steel, whereas more than 3 log and 1 log CFU cm^?2 of bacteria remained on the native steel after cleaning with water and an alkaline cleaner, respectively. This work demonstrates the potential application of Ni-PTFE non-fouling coatings on SS to improve food safety by reducing biofilm formation and improving the cleaning efficiency of food processing equipment.     

The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

Bacteriophage Phi S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms

This communication focuses on the efficacy of a specific lytic phage, phage Phi S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26 degrees C. At this temperature, bacteriophage Phi S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases.     

Properties of alkyl hydroxycinnamates and effects on Pseudomonas fluorescens.

Alkyl esters of six hydroxycinnamic acids, shown to be active antimicrobial agents when tested against Pseudomonas fluorescens, were further investigated for their effects against this organism. There was no statistically significant adaptation by this organism to either of the methyl esters of caffeic, rho-coumaric, cinnamic, or rho-hydroxybenzoic acids. Mixtures of these compounds taken two at a time gave at least additive effects, with some mixtures showing synergism. Preliminary work was also performed to determine the mode of inhibitory action for these compounds. The inhibition of oxygen utilization by the methyl esters correlated well with growth inhibition. Short-term lethality studies showed that none of the alkyl esters or methyl or propyl paraben produced any bacteriocidal effects. Oil-water partition coefficients were determined for these compounds and were shown to have no correlation with growth inhibitions. These all point to a specific mode of action, based in part on cellular energy depletion, rather than the nonspecific membrane-disrupting effects of other phenolic antimicrobial agents.     

Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Foam Separation of Pseudomonas fluorescens and Bacillus subtilis var. niger

An experimental investigation established the effect of the presence of inorganic salts on the foam separation of Pseudomonas fluorescens and of Bacillus subtilis var. niger (B. globigii) from aqueous suspension by use of a cationic surfactant. For P. fluorescens, 5.0 mueq/ml of NaCl, KCl, Na(2)SO(4), K(2)SO(4), CaCl(2), CaSO(4), MgCl(2), or MgSO(4) produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 x 10(5) up to 1.6 x 10(6) to 2.8 x 10(7) cells per milliliter (initial suspensions contain from 3.3 x 10(7) to 4.8 x 10(7) cells per milliliter). The exceptional influence of magnesium was overcome by bringing the cells into contact first with the surfactant and then the salt. For B. subtilis, the presence of 5.0 mueq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 x 10(4) to about 4.0 x 10(5) cells per milliliter. This occurred regardless of the sequence of contact as long as the surfactant contact period was sufficient. The presence of salts increased collapsed foam volumes with P. fluorescens and decreased collapsed foam volumes with B. subtilis.     

Changes in the electrochemical interface as a result of the growth of Pseudomonas fluorescens biofilms on gold

In this study, variations in corrosion potential and polarization resistance of thin-film gold electrodes as a result of the growth of Pseudomonas fluorescens biofilms on them are presented. The growth of the volumetric cell fraction of biofilms, as determined by optical sectioning and digital image analysis of phase-contrast images, was found to be exponential during at least 10 hours of incubation. As a consequence of biofilm growth, an exponential decay of the corrosion potential of gold was observed. Most importantly, an increase in polarization resistance of the interface was observed following a strong linear dependence on the mean thickness of biofilms (r = 0.997), as a consequence of oxygen consumption and diffusion limitations. The results presented indicate that the measurement of polarization resistance may be a suitable technique that could be applied easily in industrial or biotechnological systems for monitoring the formation of biofilms.