[15] First stages of biofilm formation: Characterization and quantification of bacterial functions involved in colonization process

doi:10.1016/S0076-6879(01)36587-4

Methods in Enzymology

Volume 336, 2001, Pages 152-159

https://doi.org/10.1016/S0076-6879(01)36587-4 Get rights and content

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This chapter discusses the Bacterial colonization of abiotic materials and biofilm formation, having important detrimental consequences in medicine (contamination of catheters, prostheses, indwelling devices, and artificial organs), and in many economic fields. There is therefore, a strong need to design surface coating methods able to interfere with the colonization process in order to prevent, or at least to delay, biofilm development. To reach this objective, increasing attention is being paid to the physiology and genetics of the initial stages of adhesion. Bacterial appendages and adhesins responsible for the linkage of the first pioneering cells to the surface (or the conditioning film) are potential targets for antiadhesion molecules grafted or smeared on the surface. The determination of the mechanisms resulting in the movement of bacteria toward surfaces—the identification of the functions involved in the sensing of the particular microenvironments encountered at interfaces, and the description of the regulatory networks allowing the developmental processes necessary for the structural development of biofilms—would help find nontoxic surface treatments able to lead the microorganisms away from the locations they usually contaminate. This would be particularly important in the field of indwelling medical devices. Any delay in the colonization process could successfully increase the capacities of the antibiotic therapy and the immunological defenses to eradicate the infection. This chapter describes methods to obtain and analyze bacterial mutants with altered adhesion properties.

References (25)

GottenbosB. et al.
Methods Enzymol.
(1999)
CowanM.M. et al.
J. Microbiol. Methods
(1987)
DorelC. et al.
FEMS Microbiol. Lett.
(1999)
BosR. et al.
FEMS Microbiol. Rev.
(1999)
GenevauxP. et al.
FEMS Microbial. Lett.
(1999)
Prigent-CombaretC. et al.
Methods Enzymol.
(1999)
T. T. Le Thi, P. Lejeune, unpublished results...
JuckerB.A. et al.
J. Bacteriol.
(1996)
DykhuizenD.E. et al.
Microbiol. Rev.
(1983)
LarsenD.H. et al.
J. Bacteriol.
(1964)

MurgelG.A. et al.

Appl. Environ. Microbiol.

(1991)

R. Longin, unpublished observation...

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Moreover, as observed in the 3D biofilm complex, bacteria are metabolically present in different conditions, and removals of such biofilms require using high-dose antibiotics or surgery. Another possible risk of building a biofilm is its association with blood flow which may locate it in a new area (Le-Thi et al., 2001). In general, the process of biofilm formation is a highly regulated and evolutionary one.
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The microscopic examination was performed with an epifluorescence microscope (Olympus BX51, Olympus) equipped with a 100 W mercury lamp, XC digital camera and a set of filters: U-MNB2: 470–490 nm and U-MNG2: 530–550 nm for detection of green and red fluorescence, respectively. Thirty pictures were taken for each well to evaluate biofilm adhesion characteristics using a 9-stage adherence scale (Le Thi, Prigent-Combaret, Dorel, & Lejeune, 2001) with some modifications (Olszewska et al., 2016). Statistical analysis was conducted with Statistica software ver.
Biofilms pose a serious hygienic concern in the food processing environment due to the strong antimicrobial tolerance and thus higher risk of food contamination. The aim of this study was to investigate dual species biofilm formation of P. aeruginosa ATCC 27853 or S. aureus FI 2 with L. innocua FI 2 and biofilm inactivation using QAC-, tertiary alkyl amine-, and chlorine-based disinfectants; the effect of biofilm maturity and comparison with single species biofilms are reported. We used epifluorescence microscopy (EFM) coupled with fluorescence in situ hybridization (FISH) and LIVE/DEAD staining to evaluate species contribution and cell inactivation; cells were prepared using the LabTek Chamber Slide System. In dual species biofilms during 72-h incubation time, P. aeruginosa produced a higher and S. aureus slightly lower cell numbers than the companion species of L. innocua. In dual species biofilm inactivation experiments, P. aeruginosa was more resistant to disinfectant treatment of 24-h compared to 72-h; however, S. aureus was more resistant at 72-h compared to 24-h. In addition, P. aeruginosa was more resistant to tertiary alkyl amine- and chlorine-based disinfectants and S. aureus was more resistant to all three disinfectants in dual species biofilms compared to single species biofilms. In the LabTek™ experiment, when nutrients were repeatedly provided, FISH showed almost equal contribution of P. aeruginosa and L. innocua into the biofilm as well as a predominance of S. aureus when grown with L. innocua. Regardless of biofilm composition, the LIVE/DEAD staining revealed that the majority of cells had damaged membranes after the treatment with the QAC-based disinfectant, while approximately half of the cells remained intact following the treatment with the tertiary alkyl amine-based disinfectant. As for chlorine, cells in P. aeruginosa-L. innocua biofilm were mostly dead whereas cells in S. aureus-L. innocua biofilm were largely viable, and thus were protected under the applied conditions. This study shows that dual species biofilms with foodborne pathogens are more resistant to disinfectant treatment than their single species counterparts and that biofilm maturity as well as environmental conditions, i.e. access to nutrients, play an important role in their response to the treatments tested.
Biofilm formation by meat-borne Pseudomonas fluorescens on stainless steel and its resistance to disinfectants
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Differences in the experimental design, including incubation conditions, support structures, and evaluation methods, may explain these discrepancies. Nine stages of bacterial adherence have been defined by Thi, Prigent-Combaret, Dorel, and Lejeune (2001). The classification was based on viewing randomly selected fields during epifluorescence microscopy.
Microorganism biofilms can form on food-contact surfaces and lead to cross-contamination of bacteria during food processing. The dynamics of biofilm formation by Pseudomonas fluorescens associated with meat spoilage and the resistance of established biofilm to four disinfectants were investigated in this study. The results indicated that more than 4.5 log CFU/cm² of P. fluorescens were transferred to a stainless-steel surface under short-term (5 h) scenario, and bacteria transfer occurred even after only 10 min exposure. A mature biofilm was discovered after 5 days of incubation under long-term (7 days) scenario. The biofilm was characterized by more than 9.5 log CFU/cm² cells, 120 μm thick and a large amount of extracellular polymeric substances. Observation of mature biofilm were further confirmed by confocal laser scanning microscopy and attenuated total reflection fourier-transformed infrared spectroscopy. The established biofilm showed distinct resistance to four disinfectants, namely sodium hypochlorite (SH), chlorine dioxide (CD), slightly acidic electrolyzed water (sAEW) and acidic electrolyzed water (AEW). The cells reduction was significant dependent on the concentrations, almost 8.0 log CFU/cm² of biofilm cells were reduced by 40 mg/L AEW and 200 mg/L SH treatment for 30 min, below the detection limit (1.32 log CFU/cm²). However, less than 3 log CFU/cm² reduction of biofilm cells were obtained after treatment with 40 mg/L sAEW and 20 mg/L CD for 30 min, remaining large numbers of biofilm survival. Meanwhile, the cells reduction was time-dependent, and the numbers of surviving cells rapid declined along with increasing time, but there was no additional log reduction when treatment of 20 mg/L CD was increased from 15 to 30 min. The finding suggested that AEW has great potential to be used as an environmentally friendly intervention alternative for the elimination of P. fluorescens biofilm, substituting for the traditional sodium hypochlorite in the food industry.
Inhibition of quorum sensing-related biofilm of Pseudomonas fluorescens KM121 by Thymus vulgare essential oil and its major bioactive compounds
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Citation Excerpt :
In this work, the adhesion analysis was started when the Thymus vulgare oil and its selected phytochemicals switched-off AHLs production and down-regulated the flgA gene expression in the examined cells. To define the rate of the biofilm development process, the 9-degree adhesion scale described by Le Thi et al. (2001) was used. According to the method, when a particular degree of adhesion occurred with a minimum amount of 20%, the degree became the dominant one.
The purpose of these investigations was to evaluate the anti-quorum sensing and anti-biofilm potential of Thymus vulgare essential oil (EO) and its respective components. The behavior of the agents tested was validated via examination of their influence on the production of quorum sensing autoinductors (AHLs), motility, flagella (flgA) gene expression, and biofilm formation capacity by model Pseudomonas fluorescens KM121 strain. In this work, Thymus vulgare EO, carvacrol, and thymol at concentrations below the sub-MICs caused significant limitations (i.e., 90%, 80% and 78%, respectively) in the AHLs production in a 72-h-old P. fluorescens KM121 culture. All of the tested agents, which were appreciably effective against P. fluorescens KM121 AHLs production, significantly suppressed bacterial motility and reduced the mRNA level of flagella gene (−0.672, −0.776, and −0.576 for thyme EO, carvacrol and thymol, respectively). Changes in the expression of the AHL-related flgA gene significantly inhibited the biofilm formation on stainless-steel (type 316L) surfaces and only single bacterial cells were presented on the surfaces. The results of the present study propose Thymus vulgare EO as a candidate biocide against food-spoilage P. fluorescens biofilms.
Electrokinetic and bioactive properties of CuOSiO<inf>2</inf> oxide composites
2012, Bioelectrochemistry
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Bacterial attachment to the surface is crucial for the survival and development of bacterial biofilms, which contribute to enhanced biological, chemical and mechanical resistance. In order to assess the anti-adhesive activity of the CuO·SiO2 enriched polymers, the 9-degree scale of adhesion previously proposed by Le Thi et al. [19] was employed. A certain degree became dominant when the adhesion occurred with a minimum of 20%.
CuO∙SiO₂ hybrid oxide precipitated on a semi-technical scale was thoroughly characterised in terms of physicochemical properties. Its particle size distribution and SEM analysis were performed to establish dispersion and surface morphology. Chemical analysis provided information on the content of CuO and SiO₂ oxides in the hybrid systems. The oxide systems were also subjected to elemental analysis. Zeta potential determinations were evaluated to obtain information regarding the interactions between colloidal particles. The stability of copper silicates' water dispersions was estimated on the basis of zeta potential measurements. The obtained oxide systems were used as components of polymer composites with polyester resins, which were subjected to mechanical tests and bactericidal tests against Pseudomonas aeruginosa, a well known biofilm-forming microorganism. The anti-adhesive activity of the CuO·SiO₂ enriched polymers was assessed using a 9-degree scale of adhesion. A significant reduction in the P. aeruginosa biofilm development rate was achieved for Palatal A 400-01 resins enriched with both 2 and 8 phr of the filler. In the case of Aropol M 105 TB resins the introduction of CuO∙SiO₂ caused inhibition of bacterial colonisation but to a smaller extent. These results strongly indicate that the biological activity of Cu was maintained. The release of copper ions into the local environment was examined by atomic absorption spectrometry (AAS). Maximum values of 1.621 and 5.934 mg/dm³ of released copper were detected. The surface composition of both resins studied by energy dispersive X-ray spectroscopy (EDS) contributed to the data suggesting homogenous distribution of Si; however copper seemed to form local aggregates. The presented results may be of great significance for those dealing with materials tailored for specific needs.
Quantitative and morphological analysis of biofilm formation on self-assembled monolayers
2007, Colloids and Surfaces B: Biointerfaces
In spite of intensive studies over the past two decades, the influence of surface properties on bacterial adhesion and biofilm formation remains unclear, particularly on late steps. In order to contribute to the elucidation of this point, we compared the impact of two different substrates on the formation of bacterial biofilm, by analysing bacterial amount and biofilm structure on hydrophilic and hydrophobic surfaces. The surfaces were constituted by NH₂- and CH₃-terminated self-assembled monolayers (SAMs) on silicon wafers, allowing to consider only the surface chemistry influence because wafers low roughness. A strain of Escherichia coli K12, able to produce biofilm on abiotic surfaces, was grown with culture durations varying from 4 h to 336 h on both types of substrates. The amount of adhered bacteria was determined after detachment by both photometry at 630 nm and direct counting under light microscope, while the spatial distribution of adhered bacteria was observed by fluorescence microscopy. A general view of our results suggests a little influence of the surface chemistry on adherent bacteria amount, but a clear impact on dynamics of biofilm growth as well as on biofilm structure. This work points out how surface chemistry of substrates can influence the bacterial adhesion and the biofilm formation.

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[15] First stages of biofilm formation: Characterization and quantification of bacterial functions involved in colonization process

Publisher Summary

Methods Enzymol.

J. Microbiol. Methods

FEMS Microbiol. Lett.

FEMS Microbiol. Rev.

FEMS Microbial. Lett.

Methods Enzymol.

J. Bacteriol.

Microbiol. Rev.

J. Bacteriol.

Appl. Environ. Microbiol.