Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies
© Crouzet et al.; licensee BioMed Central Ltd. 2014
Received: 22 November 2013
Accepted: 24 September 2014
Published: 30 September 2014
Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth.
Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated.
Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.
KeywordsPseudomonas aeruginosa Glass wool Adsorption mode Adhesion Biofilm
Bacteria form complex multicellular structures called biofilms . Biofilm formation is commonly considered to occur in four main stages: (1) bacterial attachment to a surface, (2) microcolony formation, (3) biofilm maturation and (4) detachment (also termed dispersal) of bacteria which may then colonize new areas . Bacteria within the biofilm, termed sessile bacteria, exist in a stationary or dormant growth phase  and exhibit phenotypes that are distinct from planktonic bacteria . In biofilms, bacteria display an exceptional resistance to environmental stresses, especially antibiotics . This makes biofilms a major public health problem as 60-80% of human microbial infections are caused by bacteria growing as a biofilm ,. The identification of biochemical pathways and biological factors critical to biofilm formation is important to prevent biofilm formation. Even with our general understanding of the basic structure and development of bacterial biofilms, knowledge of the underlying processes responsible for inducing the transition from planktonic to sessile cells is still unclear. This transition is thought to be a complex and highly regulated process resulting in a phenotypic change .
To identify the biological elements of sessile bacteria involved in biofilm physiology and especially in antibiotic resistance, studies were developed in multiple ways using several in vitro systems and surfaces . The simplest biofilm system is to set a liquid in a recipient and let the bacteria colonize the solid surface, as described by Zobell . Nowadays, multi-well plates are commonly used in this way to grow and quantify biofilms . Another technique is to add a substratum to a planktonic batch culture (named in this study “the immersion mode”). These systems are cost-effective and simple to implement, but the biofilms formed become increasingly heterogeneous over time. For instance, after 24 h of incubation, the biofilm is composed not only of elderly sessile cells but also of cells recently attached to the surface. In addition, sessile cells are potentially under the influence of surrounding planktonic cells , which may impact the results of the study. The latter issue could be solved by using a biofilm system in “flow-through” mode, meaning that the substratum to which the bacteria adhere is immersed in a continuous flow of culture medium . Flow-through systems need specialized equipment and often do not produce the large biomass essential for biochemical studies, except by multiplying the assays or increasing the adhesion surface, which may increase the heterogeneity of the population.
In addition to the variety of approaches, several surfaces with different physicochemical properties such as silicone, clay, metal, hydroxyapatite, polystyrene, polycarbonate and glass have been used to grow biofilms. Borosilicate glass has been validated by the American Society for Testing and Materials (ASTM) Committee (Surface Method E2871 -12) to study the effectiveness of disinfectants on biofilms (http://www.biofilm.montana.edu/content/astm-approves-method). Glass beads or glass wool fibers have been used in flow-through systems  or in immersion mode . Glass wool (GW) affords a large surface-to-volume ratio, so a small piece of GW allows the colonization of a large surface area , thereby obtaining a large biomass essential for performing biochemical and proteomic analyses. So far, our laboratory has used GW in the immersion mode in a large volume of culture medium . To facilitate biofilm formation and increase the sessile biomass, we investigated the use of GW in adsorption mode rather than in immersion mode. This approach utilizes the high retention capacity of GW, just like a sponge adsorbs a liquid. The rationale was to grow biofilms on the largest surface area with a minimal volume of culture medium adsorbed on GW. As bacteria were in close proximity to GW fibers, the probability for bacteria to encounter the surface was increased, so adhesion to the substratum should be promoted over time. In addition, no cells adhered to the surface of the vessel as biofilms were obtained in a system without contact to container walls. Thus, by using the adsorption mode, we expected to obtain a larger and more homogeneous population of sessile cells.
This paper presents the attachment and growth of Pseudomonas aeruginosa PAO1 on GW in the adsorption mode. The liquid adsorbed on GW formed a regular thin sheath around the fibers in which PAO1 grew like a planktonic culture. We showed that the colonization of the GW surface was very fast and depended in part on protein synthesis. The colonization profile was similar in complex and synthetic media. However, it was influenced by the bacterial concentration of the inoculum. Our system provides sufficient sessile biomass to perform proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.
Bacterial strain, growth conditions and biofilm formation
Pseudomonas aeruginosa PAO1 (CIP 104116) was provided by the Institut Pasteur (CRBIP, Paris, France). Strain PAO1 was grown either in lysogeny broth (LB: tryptone 10 g/L; yeast extract 5 g/L; NaCl 5 g/L; pH 7.2) or in a synthetic medium (SM: 60 mM K2HPO4; 30 mM KH2PO4; 7.5 mM (NH4)2SO4; 1 mM MgSO4,7H2O; 17 mM glucose; 10 μM FeSO4,7H2O; pH 7.2) previously described by Aspedon et al..
Planktonic and biofilm cultures were performed at 37°C under agitation (150 rpm). Overnight pre-cultures were obtained by inoculating 20 mL of medium with one bacterial colony. Afterwards, bacterial suspensions were prepared by diluting the pre-culture in fresh identical medium at 1/10, 1/100 or 1/1000 corresponding to≤109, ≈108 and≤107°CFU/mL, respectively. Planktonic cultures consisted of incubating 20 mL of bacterial suspension in a 100 mL Erlenmeyer flask. Biofilms were grown on GW fibers used either in immersion or adsorption modes. In immersion mode, a 1 g piece of GW was placed in 100 or 500 mL of medium with bacteria. In adsorption mode, 5 mL of bacterial suspension were adsorbed on 1 g GW.
Glass wool characteristics
Glass wool material was provided by the Sodipro Company (Echirolles, France, ref. number SCI03950). Calibrated pieces of GW (1 g) in distilled water (50 mL) were sterilized by autoclaving (121°C, 20 min). Water was removed by vacuum aspiration and the pieces were dried for 48 h at 80°C before use. The density of the GW fibers was determined experimentally. The mass of several randomly cut GW pieces was determined and their volume was estimated by measuring the volume increase after immersion in a known volume of water. The diameter of the fibers was directly determined by microscopy. In adsorption mode, the maximum volume that could be immobilized on a 1 g piece of GW was determined experimentally by adding mL per mL of liquid (water, LB and SM media) until liquid leaked. Then the value was refined by adding 0.1 mL per 0.1 mL until a drop appeared. Finally, the surface covered by the adsorbed volume was defined by using an aqueous solution of methylene blue 0.02% (w/v). The ratios tested were 10, 7.5, 5, 2.5 and 1 mL / g GW. After adsorption, GW pieces were placed in 100 mL Erlenmeyer flasks and were incubated at 37°C for 6 h under agitation (150 rpm). Then the stained parts were cut off and the colored and uncolored parts were dried for 48 h at 80°C. The percentage of covered surface was calculated by measuring the mass of colored and non-colored parts. The percentage of covered surface was also determined just after liquid adsorption onto GW by performing the same experiment without the 6 h incubation period.
Construction of PAO1 expressing eGFP
A PAO1 strain expressing eGFP was constructed for this study. The eGFP coding sequence was amplified from the plasmid pEGFP (Clontech, CA, USA) using the primers 5′-ATGGTGAGCAAGGGCGAGGAGCTGTTCACC-3′ and 5′-TTCTGCAG AGTCGCGGCCGCTTTACTTGTAC-3′ (containing a Pst I restriction site at 3′ end). The eGFP coding sequence was set under the control of the promoter region of the PA4249 gene. This gene has been shown to be constitutively expressed in planktonic and sessile PAO1 cells . The PA4249 promoter was amplified from PAO1 genomic DNA using the primers 5′- AAGGATCC CAAGTTCGGCCTGAGCCGTAACAA-3′ (containing a Bam HI restriction site at 5′ end) and 5′- TTGCTCACCATGGGCTTAACGCTCCTGATAC-3′. All primers were provided by Eurogentec. PCR cycles were done as follows: denaturation 95°C, 30 s; annealing 63°C, 45’s; elongation 72°C, 1 min performed with a Phusion Taq polymerase (New England Biolabs, MA, USA). The two amplicons were fused and the resulting DNA fragment carrying the construction - PA4249 promoter - eGFP CDS (named pPA4249-eGFP below) - was amplified and purified from agarose gel. The fragment was cloned into pUCP20 (kindly provided by Dr. Schweizer) by Pst I - Bam HI double digestion. pUCP20 is a high-copy plasmid replicating in E. coli and P. aeruginosa. PAO1 was transformed by the plasmid pUCP20-[pPA4249-eGFP] according to a protocol previously described  and transformants were selected on LB agar with carbenicillin 200 μg/mL. GFP fluorescence arising from transformants (515 nm) was checked with a Versafluor fluorometer (Biorad). The recombinant DNA was verified by DNA sequencing. The plasmid pUCP20-[pPA4249-eGFP] allowed the constitutive eGFP expression in strain PAO1 enabling bacteria to be self-labeling.
Flow-through washing process and bacterial quantification
One g pieces of GW used in immersion or adsorption modes were placed in a 50 mL syringe such that the fibers were parallel to the vertical axis of the syringe. Washing was performed with 100 mL PBS (NaCl 8 g/L, KCl 0.2 g/L, Na2HPO4 2H2O 1.44 g/L, KH2PO4 0.24 g/L) running down through GW by gravity. This step was completed in less than 40 seconds (volumetric flow rate = 2.6 × 0.1 mL.s-1). The planktonic and loosely attached bacteria were recovered in the flow-through. The GW piece was removed from the syringe and placed in 100 mL PBS. Sessile cells were harvested from GW by vortexing vigorously for 30’seconds. GW was then squeezed against the wall of the flask. To achieve maximum recovery of bacteria, this latter step was repeated three times in the same PBS bath . Ultimately the squeezed GW was discarded. The planktonic and sessile bacterial biomasses contained in PBS solutions were quantified by colony forming unit (CFU) counting. The number of CFU was determined by plating 0.1 mL aliquots of serial dilutions twice onto LB agar and incubating for 24 h at 37°C. All time points were performed in biological triplicate.
Microscopy experiments were performed on 1.5 mg pieces of GW loaded with 3.75 or 7.50 μl of LB (i.e. 2.5 or 5 mL/g GW) with or without bacteria (107°CFU/mL). Cell attachment and biofilm development, as well as the diameter of GW fibers, were determined by spinning-disk microscopy. The spinning-disk experiments were done on an inverted Leica DMI 6000 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a confocal head Yokogawa CSU-X1 (Yokogawa Electric Corporation, Tokyo, Japan) and a resolutive HQ2 camera (Photometrics, Tucson, USA). The diode laser used was at 491 nm. The objective used was a HCX PL Fluotar 40X oil 1.25 NA or HCX PL APO CS 63X oil 1.32 NA. The z stacks were performed with a piezo P721.LLQ (Physik Instrumente (PI), Karlsruhe, Germany). The mosaics were done with a motorized stage Scan IM (Märzhäuser, Wetzlar, Germany). The 37°C atmosphere was created with an incubator box and an air heating system (Life Imaging Services, Basel, Switzerland). This system was controlled by MetaMorph software (Molecular Devices, Sunnyvale, USA). The microscopic experiments were performed three times and more than twenty five frames were observed for each experiment.
Tetracycline effect on initial colonization of glass wool by P. aeruginosa
The study of tetracycline effect on P. aeruginosa adhesion was inspired from data reported by O Toole and Kolter . Stationary and exponential calibrated bacterial suspensions (108°CFU/mL) were prepared from an overnight culture. Briefly, stationary cells were obtained by diluting pre-culture overnight in LB (1:100). Exponential cells were obtained by inoculating fresh LB with pre-culture and incubated up to OD546nm = 0.3. These bacterial suspensions were treated or not for 1 h at the bacteriostatic concentration of tetracycline (i.e. 10 μg/mL and 150 μg/mL for exponential and stationary cultures, respectively). After antibiotic treatment, 5 mL were adsorbed on 1 g of GW incubated at 37°C for 20 min and sessile bacteria were quantified as mentioned above. The same experiment was performed using the microplate biofilm formation assay as previously described . Briefly, the diluted cultures were incubated in wells of microplates and biofilms formed after 20 min were quantified by staining with crystal violet.
Protein extraction and electrophoresis
The objective of this experiment was as follows: (1) to extract the protein content of relatively few planktonic or sessile bacteria (108 and 109°CFU); (2) to maintain the integrity of the proteome by directly lysing bacteria in situ; (3) to demonstrate the feasibility of the approach using GW. To obtain 108 sessile cells, 5 mL of LB at 107°CFU/mL were adsorbed onto 1 g of GW. After 3 h of incubation at 37°C, GW was washed as mentioned above (see “Flow-through washing process and bacterial quantification”). PBS was immediately removed by pipetting, leaving≤3 mL adsorbed on GW. The same number of planktonic cells was obtained from 170 μl of a 3 h-old LB planktonic culture (see “Bacterial strain, growth conditions and biofilm formation”). Similarly, 109 sessile cells were obtained by adsorbing 5 mL of LB at 109°CFU/mL onto 1 g of GW. After 1 h of incubation at 37°C, GW was washed and PBS was removed, leaving≤3 mL on GW. The same number of planktonic cells was obtained from 500 μl of a 1 h LB planktonic culture.
Cell lysis was performed by adding one volume of lysis buffer 2X (7 M urea, 2 M thiourea, 65 mM CHAPS, 20 mM DTT, 1 M NaCl) to one volume of sample. The mix was frozen (-80°C, 30 min) and thawed (35°C, 20 min). The proteins were concentrated by 15% TCA precipitation followed by two successive acetone washings. Proteins were suspended in 50 μl of the following solution: 7 M urea, 70 mM SDS, 20 mM DTT. Nine μl of protein extract were mixed with 3 μl of Laemmli buffer 4X and then 10 μl were loaded on an SDS-PAGE (12%). After electrophoresis, proteins were visualized by colloidal Coomassie blue staining. The gels were scanned with a GS-800 densitometer (BioRad).
Culture medium formed a sheath surrounding the glass wool fiber in adsorption mode
Discrimination of sessile bacteria from planktonic bacteria
In immersion systems, colonized surfaces like GW are removed from the batch and washed to separate the biofilm biomass from the planktonic bacteria. For our adsorption system, we developed a new approach to separate strongly attached bacteria from planktonic/ weakly attached bacteria without excessive handling of the material. The removal of planktonic and weakly attached bacteria was performed through washing the GW. This was achieved by flowing 20 volumes of PBS solution through the GW for one adsorbed volume, i.e. 100 mL of PBS for 5 mL adsorbed on GW [see Additional file 4]. For that purpose, GW was placed in a 50 mL syringe and flushed with PBS by gravity at a volumetric flow rate of 2.6 × 0.1 mL.s-1 (n = 9). Finally, the number of bacteria in the flow through and retained on the washed GW fibers was determined as described in Methods section. The efficiency of the process was tested with a 1 g piece of GW inoculated with 5 mL of complex (LB) or synthetic (SM) medium at three different bacterial concentrations (107, 108 or 109°CFU/mL). When the washing step immediately follows the adsorption step, the PBS “flow-through” contained more than 98% of CFU of the inocula (data not shown), thereby proving the effectiveness of this method to remove all unattached bacteria. This result was validated by microscopic observations since no adhered bacteria were observed on GW (data not shown). When the inoculated GW pieces were incubated for 24 h, 5.3 × 0.7 106°CFU/cm2 were still present on the GW after the PBS washing step. Microscopic observations of these GW pieces showed that the retained bacteria were not motile and, either in contact with the surface or with bacteria which were immobilized on the GW fiber. In our study, these bacteria that were not removed after PBS washing were then considered as adhered/sessile cells. So, the flow-through process was appropriate to estimate the sessile bacterial population and thus to study the GW colonization by P. aeruginosa.
P. aeruginosa grew in adsorption mode as in a standard planktonic culture…but survived longer in the former
Surface colonization and biofilm development respond to various signals such as the nutritional conditions of the environment . As the adsorption mode may influence bacterial growth and therefore biofilm formation, we examined P. aeruginosa growth in complex (LB) or synthetic (SM) medium adsorbed on glass wool by determining the number of CFU, without distinguishing unattached cells from sessile cells. These results were compared with planktonic cultures (PC) at identical initial bacterial concentrations in the same media. As this study was performed with 5 mL of medium initially at 107 or 109°CFU/mL adsorbed on a 1 g piece of GW, we compared total CFU from cultures on GW with CFU number obtained in 5 mL of PC.
After 24 h, the viability decreased in both PC and GW cultures but differently according to the medium. In LB-PC, the biomass was reduced by a factor≤3 log at 96 h compared to the biomass at 24 h, irrespective of the initial bacterial concentration (Figure 2A,B). In GW culture, the viability depended on the initial bacterial concentration. At 107°CFU/mL, the reduction was limited to the 24 h-48 h period, and then the number of viable bacteria remained stable (≈1010°CFU) (Figure 2A). At 109°CFU/mL, the biomass continually decreased up to 96 h (Figure 2B). In any case, the 72 h- and 96 h-old biomasses in GW cultures were greater by a factor > 1 log than the biomasses in PC, indicating that GW culture enhanced the long-term survival of P. aeruginosa. In SM-PC from 48 h to 96 h, whatever the initial bacterial concentration, we systematically observed the clumping of bacteria resulting in a huge free-floating aggregate in the medium. This prevented us from comparing the PC and GW biomasses. Even if a strict comparison was not possible, a long-term survival was observed when P. aeruginosa was grown in SM.
Thus growing bacteria in a limited volume of LB or SM medium adsorbed on GW had no negative effects on P. aeruginosa growth compared to a planktonic culture grown in the same conditions. The biomasses obtained after 24 h of incubation were similar in all conditions (PC vs GW, LB vs SM). Moreover, consistently with the properties of sessile cells in biofilms classically described in the literature , the survival of bacteria on GW was higher after several days of culture in comparison to planktonic cells.
Colonization of GW fibers by P. aeruginosa was very quick and dependent on the bacterial concentration
We also examined fiber colonization by microscopy using an LB inoculum at 107°CFU/mL (Figure 3C). After washing the GW, single and dispersed attached bacteria were visible after 1 h of incubation. At 3 h the density of the adherent cells had increased and at 6 h bacterial microcolonies were present on the fibers. These observations confirmed the previous result based on CFU follow-up (Figure 3A, LB medium).
Adhesion was enhanced in adsorption mode compared to immersion mode
Glass wool colonization by Pseudomonas aeruginosa involved protein synthesis
Adsorption mode allowed performing proteomic analysis of the first steps of colonization
The development and validation of practical, reproducible and representative laboratory growth systems for the study of biofilms is a challenge. A panoply of in vitro systems have been developed, which range in complexity from a bacterial colony growing directly on agar plates to sophisticated continuous culture fermentation systems . Each system has its own advantages, but the results of all these study systems are mixed. Biofilm physiology is dynamic  so analysis may be complicated by the action of several variables: nature of the substratum ,, the strain used and its degree of domestication , the carbon source , etc. For instance, a study has shown that surface hydrophobicity and charge have an effect on the initial colonization and attachment of cells onto the surface . Biofilm systems that allow the growth rate to be controlled more accurately and heterogeneity to be minimized are arguably more suited to studies aimed at characterizing cell attachment and biofilm development. Therefore, biofilm formation systems can be considered in terms of the degree of control they provide over various aspects of physiology and the ease with which they can be established, maintained and replicated.
So far in our laboratory, P. aeruginosa biofilms have been grown on GW as substratum. GW was chosen as it affords a large surface-to-volume ratio for bacterial colonization . Moreover, GW pieces are small and easy to handle. Conventionally, the surface is immersed in the culture medium and the sessile cells are collected, after washing and sonication ,,. Here we describe an improvement of our biofilm system. Instead of using the immersion mode, we tested the adsorption mode on GW. This makes it possible to work with a very small inoculum volume adsorbed and distributed over a large area and using a small piece of material. The fact that the culture volume was small did not impair bacterial growth. Furthermore, this configuration brought the bacteria near the support and enhanced their attachment to the surface. Comparison of the number of attached cells in immersion and adsorption modes showed the advantage of the latter system in obtaining more sessile biomass of P. aeruginosa. In adsorption mode, cell attachment was very efficient at short incubation times with more than 107 sessile CFU within 20 min of incubation. The sessile bacteria population can be considered as corresponding to cells adhered to the surface, as our washing protocol (flow-through process) eliminated planktonic cells and cells loosely adhered to the substratum. In addition, analysis of the adherent cell population is not skewed by sessile cells attached to the recipient walls, as in the immersion mode. Thus, in a very short time, we obtained a large and presumably homogeneous population of sessile bacteria that had just adhered onto the GW. This new approach should reduce heterogeneity with respect to sessile biomass especially during the attachment period, and could pave the way for molecular analysis of the early events of biofilm formation.
During this study, we noticed that the initial colonization pattern changed reproducibly with the characteristics of the bacterial inoculum. Clearly the physiological state of P. aeruginosa cells had an impact on the degree of adhesion. Stationary cells were attached two-fold more than exponentially growing P. aeruginosa. Likewise the inoculum concentration affected the profile of colonization, irrespective of the culture medium. Whereas colonization regularly increased from the beginning with the concentrated inoculum (109°CFU/mL), a kind of plateau occurred at the start of incubation (1 h - 3 h according to the medium used) with the more diluted inoculum (107°CFU/mL). Nevertheless, these two distinct responses did not at first prevent the same maximal amount of biomass occurring in all experimental conditions. It was obvious that this plateau was not due to a limited surface adhesion capacity in that a larger number of bacteria were able to attach to GW when a higher bacterial concentration was used. In 2000, Rice and coauthors described a surface-associated lag time for P. aeruginosa PAO1 . They analyzed the change from reversibly to irreversibly adsorbed cells on glass coverslips and the subsequent bacterial growth using confocal scanning laser microscopy. They found that P. aeruginosa cells that initially colonized the surface, also referred to as primary biofilm cells, experienced a lag in their growth; then the progeny of the first adhered population grew at the same rate as planktonic cells. They proposed that the lag phase occurred upon initial attachment of truly planktonic cells and was indicative of a physiological change from a planktonic to a sessile environment. Thus, attached cells at the plateau phase could correspond to a highly homogeneous population useful for the investigation of initial colonization. However, there are other reports where microorganisms do not exhibit a lag in replication after initial attachment ,. These data underline the critical need for understanding the nature of the inoculum and for controlling the physiological state of cells used to generate the initial biofilm. Indeed in our experiments using a higher concentration from the same PAO1 pre-culture was sufficient to eliminate the lag phase.
Our results further demonstrate that protein synthesis is required for the early phase of GW colonization, whatever the inoculum produced from exponential or stationary P. aeruginosa cells. In both conditions, tetracycline treatment prevented about half of the bacteria from adhering to GW after 20 min incubation. This result was reproduced with microplates, suggesting that protein synthesis is necessary for starting colonization, irrespective of the surface properties. The involvement of protein synthesis has already been described in P. fluorescens. The authors showed that the initial interaction with the abiotic surface required new protein synthesis but not for the subsequent step, i.e. the short-term maintenance of the attached cells. Our results and those of O Toole and Kolter  suggest that the initial colonization phase in Pseudomonas is a regulated process involving the synthesis of new proteins. This observation seems consistent with the processes that may involve hydrodynamic and physical-chemical interactions in the deposition of cells onto the surface .
Understanding the transition from the deposition stage, during which there is potential for bacterial removal, to the development of irreversible interactions with the surface is a way to develop strategies for preventing biofilm formation. Our biofilm system may make it possible to explore the early stages and to identify the molecular components by which bacteria deposit and, shortly after, attach irreversibly to surfaces. To demonstrate the potential of our biofilm system for analyzing bacterial adhesion, we tried to visualize the protein content of a small amount of sessile cells after 1 h or 3 h incubation. A very critical issue in sample preparation is the need to rapidly and efficiently quench all biological and enzymatic activities in order to capture an accurate “snap-shot” of the proteome. Rapid cell lysis should avoid changes in gene expression that result from the process of harvesting the cells and might perturb their state. Our system makes it possible to directly lyse sessile cells on their support and immediately freeze their protein content. The initial results showed that proteins extracted from a small amount of sessile bacteria (108°CFU) were clearly reachable on 1-D polyacrylamide gel. A comparison with the protein profile obtained from the same amount of planktonic bacteria reproducibly revealed some differences. These differences were consistent with the involvement of protein synthesis in the initial colonization of GW. These preliminary results show that the P. aeruginosa biofilm proteome can be studied soon after inoculation of GW either by SDS-PAGE analysis or another tool such as mass spectrometry. Indeed, mass spectrometry is more sensitive and requires a smaller amount of proteins compared with SDS-PAGE. It also covers a wider diversity of the proteome. Beyond the above-mentioned characteristics of our biofilm system, the adsorption mode on GW is easy to perform, reliable and adjustable. The physiological response of the organism tested by manipulation of a single variable can thus be easily monitored. The reduction of incubation volume can also be seen as an advantage when working with expensive media cultures. Furthermore, this tool can be miniaturized to monitor biofilm formation in a wide variety of experimental conditions. Finally, this new approach can be extended to other bacterial organisms.
So far, the literature did not report any molecular studies describing the very first steps of biofilm formation, in particular bacterial attachment. Our experimental approach based on adsorption of the medium onto GW fibers offers the opportunity to perform molecular and proteomic analysis of the early stages of colonization, in particular within the first hour of incubation. We showed that the colonization of the GW surface is very fast, irrespective of the medium, and depends on protein synthesis. Then our growth system should permit identification of proteins involved in cell attachment and decipher the very early steps in biofilm formation.
CLS, PC, CB and SV carried out the experimental studies. MC, VSB and SV designed the study. MC, BG, MB and SV were in charge of interpretation of the data. MC and SV wrote the manuscript and CLS, PC, CB, VSB, BG and MB were involved in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
The microscopy was done in the Bordeaux Imaging Center of the University Bordeaux Segalen (http://www.bic.u-bordeaux2.fr). We greatly acknowledge Christel Poujol for helping us in the microscopy experiments. We also thank Pr. HP Schweizer for providing us with the pUCP20 plasmid. This work was supported by the Institut Polytechnique de Bordeaux and the University Bordeaux Segalen.
- Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM: Microbial biofilms. Annu Rev Microbiol. 1995, 49: 711-745. 10.1146/annurev.mi.49.100195.003431.View ArticlePubMedGoogle Scholar
- Landini P, Antoniani D, Burgess JG, Nijland R: Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal. Appl Microbiol Biotechnol. 2010, 86 (3): 813-823. 10.1007/s00253-010-2468-8.View ArticlePubMedGoogle Scholar
- Stoodley P, Sauer K, Davies DG, Costerton JW: Biofilms as complex differentiated communities. Annu Rev Microbiol. 2002, 56: 187-209. 10.1146/annurev.micro.56.012302.160705.View ArticlePubMedGoogle Scholar
- Vilain S, Cosette P, Zimmerlin I, Dupont JP, Junter GA, Jouenne T: Biofilm proteome: homogeneity or versatility?. J Proteome Res. 2004, 3 (1): 132-136. 10.1021/pr034044t.View ArticlePubMedGoogle Scholar
- Høiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O: Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010, 35 (4): 322-332. 10.1016/j.ijantimicag.2009.12.011.View ArticlePubMedGoogle Scholar
- Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science. 1999, 284 (5418): 1318-1322. 10.1126/science.284.5418.1318.View ArticlePubMedGoogle Scholar
- Lewis K: Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001, 45 (4): 999-1007. 10.1128/AAC.45.4.999-1007.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- O Toole GA, Gibbs KA, Hager PW, Phibbs PV, Kolter R: The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa. J Bacteriol. 2000, 182 (2): 425-431. 10.1128/JB.182.2.425-431.2000.View ArticleGoogle Scholar
- Bjarnsholt T, Alhede M, Alhede M, Eickhardt-s’rensen SR, Moser C, K hl M, Jensen PO, Høiby N: The in vivo biofilm. Trends Microbiol. 2013, 21 (9): 466-474. 10.1016/j.tim.2013.06.002.View ArticlePubMedGoogle Scholar
- Zobell CE: The effect of solid surfaces upon bacterial activity. J Bacteriol. 1943, 46 (1): 39-56.PubMed CentralPubMedGoogle Scholar
- Merritt JH, Kadouri DE, O Toole GA: Growing and analyzing static biofilms.Curr Protoc Microbiol 2011, 1B.1.1-1B.1.18. doi:10.1002/9780471729259.mc01b01s22.,Google Scholar
- Vilain S, Brözel VS: Multivariate approach to comparing whole-cell proteomes of Bacillus cereus indicates a biofilm-specific proteome. J Proteome Res. 2006, 5 (8): 1924-1930. 10.1021/pr050402b.View ArticlePubMedGoogle Scholar
- Weiss Nielsen M, Sternberg C, Molin S, Regenberg B: Pseudomonas aeruginosa and Saccharomyces cerevisiae biofilm in flow cells. J Vis Exp. 2011, 47: pii: 2383-Google Scholar
- Revetta RP, Gomez-Alvarez V, Gerke TL, Curioso C, Santo Domingo JW, Ashbolt NJ: Establishment and early succession of bacterial communities in monochloramine-treated drinking water biofilms.FEMS Microbiol Ecol 2013, doi:10.1111/1574-6941.12170.,Google Scholar
- Jouenne T, Vilain S, Cosette P, Junter GA: Proteomics of biofilm bacteria. Curr Proteomics. 2004, 1: 113-129. 10.2174/1570164043379307.View ArticleGoogle Scholar
- Steyn B, Oosthuizen MC, MacDonald R, Theron J, Brözel VS: The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis. Proteomics. 2001, 1 (7): 871-879. 10.1002/1615-9861(200107)1:7<871::AID-PROT871>3.0.CO;2-2.View ArticlePubMedGoogle Scholar
- Costaglioli P, Barthe C, Claverol S, Brözel VS, Perrot M, Crouzet M, Bonneu M, Garbay B, Vilain S: Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation. Microbiol Open. 2012, 1 (3): 326-339. 10.1002/mbo3.33.View ArticleGoogle Scholar
- Aspedon A, Palmer K, Whiteley M: Microarray analysis of the osmotic stress response in Pseudomonas aeruginosa. J Bacteriol. 2006, 188 (7): 2721-2725. 10.1128/JB.188.7.2721-2725.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ: Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene. 1994, 148 (1): 81-86. 10.1016/0378-1119(94)90237-2.View ArticlePubMedGoogle Scholar
- Liu SC, Webster DA, Stark BC: An improved method of transformation in Pseudomonads. Biotechnol Tech. 1996, 10 (9): 683-686. 10.1007/BF00168480.View ArticleGoogle Scholar
- O Toole GA, Kolter R: Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol. 1998, 28 (3): 449-461. 10.1046/j.1365-2958.1998.00797.x.View ArticleGoogle Scholar
- Seneviratne CJ, Wang Y, Jin L, Wong SS, Herath TD, Samaranayake LP: Unraveling the resistance of microbial biofilms: has proteomics been helpful?. Proteomics. 2012, 12 (4-5): 651-665. 10.1002/pmic.201100356.View ArticlePubMedGoogle Scholar
- McBain AJ:In vitro biofilm models: an overview. Adv Appl Microbiol. 2009, 69: 99-132. 10.1016/S0065-2164(09)69004-3.View ArticlePubMedGoogle Scholar
- Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG:Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol. 2002, 184 (4): 1140-1154. 10.1128/jb.184.4.1140-1154.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Dalton HM, Poulsen LK, Halasz P, Angles ML, Goodman AE, Marshall KC: Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure. J Bacteriol. 1994, 176 (22): 6900-6906.PubMed CentralPubMedGoogle Scholar
- Vilain S, Cosette P, Hubert M, Lange C, Junter GA, Jouenne T: Comparative proteomic analysis of planktonic and immobilized Pseudomonas aeruginosa cells: a multivariate statistical approach. Anal Biochem. 2004, 329 (1): 120-130. 10.1016/j.ab.2004.02.014.View ArticlePubMedGoogle Scholar
- McLoon AL, Guttenplan SB, Kearns DB, Kolter R, Losick R: Tracing the domestication of a biofilm-forming bacterium. J Bacteriol. 2011, 193 (8): 2027-2034. 10.1128/JB.01542-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T: Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003, 48 (6): 1511-1524. 10.1046/j.1365-2958.2003.03525.x.View ArticlePubMedGoogle Scholar
- Boks NP, Kaper HJ, Norde W, van der Mei HC, Busscher HJ: Mobile and immobile adhesion of staphylococcal strains to hydrophilic and hydrophobic surfaces. J Colloid Interface Sci. 2009, 331 (1): 60-64. 10.1016/j.jcis.2008.11.025.View ArticlePubMedGoogle Scholar
- Rice AR, Hamilton MA, Camper AK: Apparent surface associated lag time in growth of primary biofilm cells. Microbial Ecol. 2000, 40 (1): 8-15. 10.1007/s002480000011.View ArticleGoogle Scholar
- Korber DR, Lawrence JR, Caldwell DE: Effect of motility on surface colonization and reproductive success of Pseudomonas fluorescens in dual-dilution continuous culture and batch culture systems. Appl Environ Microbiol. 1994, 60 (5): 1421-1429.PubMed CentralPubMedGoogle Scholar
- Lawrence JR, Caldwell DE: Behavior of bacterial stream populations within the hydrodynamic boundary layers of surface microenvironments. Microbial Ecol. 1987, 14 (1): 15-27. 10.1007/BF02011567.View ArticleGoogle Scholar
- Walker SL, Redman JA, Elimelech M: Role of cell surface lipopolysaccharides in Escherichia coli K12 adhesion and transport. Langmuir. 2004, 20 (18): 7736-7746. 10.1021/la049511f.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.