Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa

R E S E A R C H L E T T E R

Bio¢lm-detached cells, a transition froma sessile toa planktonicphenotype: a comparative studyofadhesionand physiologicalcharacteristics inPseudomonasaeruginosaCecile Rollet1,2, Laurent Gal1 & Jean Guzzo2

1UMR 1229 Microbiologie du Sol et de l’Environnement, Universite de Bourgogne, INRA, Dijon, France; and 2EA ReVV, Universite de Bourgogne, IUVV,

Dijon, France

Correspondence: Laurent Gal, UMR 1229

Microbiologie du Sol et de l’Environnement,

Universite de Bourgogne, INRA, 17 rue de

Sully, F-21000 Dijon, France. Tel.: 133 3 80

69 34 39; fax: 133 3 80 69 32 24; email:

[email protected]

Received 26 June 2008; accepted 9 October

2008.

First published online 27 November 2008.

DOI:10.1111/j.1574-6968.2008.01415.x

Editor: David Clarke

Keywords

Pseudomonas aeruginosa; detached cells; cell-

surface properties; biofilm-forming capacity;

antibiotic susceptibility.

Abstract

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high

incidence in clinical environments and its ability to form strong biofilms. During

biofilm development, sessile cells acquire physiological characteristics differentiat-

ing them from planktonic cells. But after treatment with disinfectants, or to ensure

survival of the species in hostile environments, biofilm cells can detach. This

complicates disinfection procedures. This study aimed to physiologically charac-

terize cells detached from a P. aeruginosa biofilm and to compare them with their

sessile and planktonic counterparts. We first tested planktonic growth kinetics and

capacities to form new biofilms. Then we investigated cell-surface properties. And

finally, we tested in vitro susceptibility to antibiotics. The results first indicated that

sessile and detached cells have similar planktonic growth kinetics and cell-surface

properties, distinguishable from those of planktonic cells. Interestingly, the three

populations exhibited different biofilm-forming capacities, suggesting that there is

a transitional phenotype between sessile and planktonic states, at least during the

first hours following cell detachment. It is important to consider this observation

when developing treatments to optimize disinfection processes. Surprisingly, the

three populations showed the same antibiotic susceptibility profile.

Introduction

In natural environments, most bacteria are able to develop

communities that adhere to various surfaces and are called

biofilms, while freely suspended cells appear to embody a

transitory growth mode (planktonic growth) (Jefferson,

2004). Pseudomonas aeruginosa is an excellent model to

study biofilms, as it is a good producer of exopolysacchar-

ides (Dunne, 2002) that favor bacterial adhesion and

colonization of surfaces. Naturally present in soil, on

vegetation, and in water from any source, this ubiquitous

Gram-negative bacterium is known as an opportunistic

human pathogen. It typically infects immunocompromised

individuals and is one of the main causes of chronic

respiratory infections in cystic fibrosis patients. It is espe-

cially well known for its resistance to antibiotics and

antiseptics, and its ability to adhere to surfaces and form

strong biofilms. This leads to serious therapeutic problems

in various infections. Pseudomonas aeruginosa is the main

colonizer of clinical environments, surgical materials

(catheters, equipment, implants, etc.), and pharmaceutical

products (antiseptic solutions, creams, powders, syrups, and

plasters), causing 10% of nosocomial infections.

Bacteria within biofilms are arranged in macrocolonies

surrounded by a matrix of extracellular polymeric sub-

stances (EPS) and separated by water channels ensuring the

diffusion of oxygen, nutrients, and waste products (Donlan,

2002). During the formation of these structures, sessile cells

acquire physiological characteristics that are different from

those of planktonic cells, altering in particular the produc-

tion of EPS, the growth rates, the pattern of genes expressed

(in cell adhesion and biofilm formation), and increasing the

resistance to sanitizing agents and antibiotics (Davies,

2003). Biofilms are consequently the source of persistent

contaminations. They are difficult to eradicate and are

involved in recurrent economic and health problems

FEMS Microbiol Lett 290 (2009) 135–142 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works.

(Costerton et al., 1999). Previous works identified distinct

stages during the process of biofilm development, including

(i) reversible and (ii) irreversible attachment, (iii) formation

of microcolonies, (iv) development of macrocolonies, form-

ing complex three-dimensional structures, and finally (v)

cell detachment (Donlan & Costerton, 2002; Sauer et al.,

2002). In several studies, it has been hypothesized that this

last step is essential to avoid nutrient and oxygen starvation

of bacteria within growing biofilms, and to allow coloniza-

tion of new surfaces (Allison et al., 1990; Sauer et al., 2002;

Kaplan et al., 2003; Hunt et al., 2004). Nevertheless, few

works have been carried out to elucidate the detachment

step: some studies were focused on mechanisms using a

molecular approach, but very few researches were carried

out to investigate the physiology of detached cells (Boles

et al., 2004; Bester et al., 2005; Ymele-Leki & Ross, 2007).

The majority of biofilm studies focused on the mechanisms

of bacterial adhesion and the changes occurring during the

switch from the planktonic state to the biofilm state

(O’Toole et al., 2000), and on the assessment of disinfection

procedures. However, it seems fundamental to carry out a

specific study of biofilm-detached cells to further elucidate

the potential risk of recontamination and to optimize

disinfection procedures. Therefore, we aimed to study the

phenotypic characteristics of biofilm-detached cells and

we completed our work with an original comparison of

detached cells with their well-described sessile and plank-

tonic counterparts, in order to investigate whether freshly

detached cells retain their biofilm characteristics.

Materials and methods

Bacterial strain and growth conditions

All experiments were conducted with the P. aeruginosa strain

A22, which was obtained from Institut Pasteur Collection

(CIP, Paris, France) and was isolated from a wound infec-

tion. This is a reference strain recommended by French

norms AFNOR (Chapalain & Puyhardy, 1997; Espigares

et al., 2003) to assess the efficacy of disinfectants. The strain

was stored at � 80 1C in tryptic soy broth (TSB; Biokar

Diagnostics, Pantin, France) containing 20% (v/v) glycerol.

A preculture was grown in 20 mL of TSB, and then the main

cultures were prepared by inoculating 200mL of the station-

ary-phase preculture into 20 mL of medium. Precultures

and cultures were incubated at 37 1C and at 150 r.p.m. in an

incubator shaker (Minitron, Infors AG, Bottmingen,

Switzerland) in 250-mL baffled round-bottomed flasks.

Biofilm preparation

Biofilms were developed under static conditions on AISI-

304 stainless-steel coupons of 2.5� 2.5 cm (Goodfellow,

Lille, France). This hydrophilic material is commonly used

in food-processing plants and in clinical environments. An

overnight TSB culture was used to inoculate (1/100, v/v)

MCDB 202 (Molecular Cellular Development of Biology

202, Cryo Bio System, l’Aigle, France) supplemented with

3.6 g L�1 glucose, and 30 mL of this bacterial suspension was

poured into a Petri dish containing three coupons placed flat

to develop biofilms on only one side. This mode of culture

ensures a large exchange surface with oxygen, allowing

better development of P. aeruginosa biofilms. Biofilms were

grown statically at 37 1C for 5 days.

Collection of cells, preparation of the differentcell suspensions, and enumeration

Three cell populations were investigated in this study:

originally planktonic cells, sessile cells, and biofilm-detached

cells. They received the same treatments during the

preparation of the suspension.

Planktonic cells were grown at 37 1C and 150 r.p.m. for

20 h in 20 mL of glucose-supplemented MCDB 202 med-

ium. Biofilm-resulting cells (sessile and biofilm-detached

cells) were sampled from colonized stainless-steel coupons

after they had been rinsed twice in a saline solution (0.85%

NaCl) under 240 r.p.m. agitation for 1 min on an orbital

shaker (IKAs KS 130 basic) to remove planktonic and

loosely attached cells. Sessile cells were detached by scraping

the colonized side of one coupon in the saline solution, with

a sterile disposable cell lifter (TPP; D. Dutscher, Brumath,

France). Biofilm-detached cells were collected using a device

designed in our laboratory to stimulate biofilm-cell

detachment (Fig. 1). Using a peristaltic pump (Watson

Fig. 1. Schematic representation of the device

developed to stimulate detachment of biofilm

cells and collect them.

FEMS Microbiol Lett 290 (2009) 135–142Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works.

136 C. Rollet et al.

Marlow 205S), a sterile saline solution was continuously

pumped into a flask containing one colonized coupon at a

constant flow rate (1 mL min�1) for 2 h. The flowthrough

containing detached cells was collected. The suspension

collected for the first hour was thrown away to remove

loosely attached cells.

Then the three cell suspensions were centrifuged twice

(3500 g for 5 min) and washed in a saline solution. Planktonic

and sessile cells were maintained at room temperature for 2 h

during the collection of detached cells. The three suspensions

were adjusted to about 105 CFU mL�1 and counted on tryptic

soy agar (TSA) after 24 h of incubation at 37 1C.

Planktonic growth revival experiment

The planktonic growth in liquid medium was monitored for

the three populations to compare their growth revival

abilities. Equal aliquots of each suspension (5 mL) were

incubated in 500-mL baffled round-bottomed flasks con-

taining 100 mL of TSB medium. Cultures were grown under

agitation (150 r.p.m.) at 37 1C. Growth kinetics were

followed by measuring the OD600 nm of culture aliquots at

different time intervals.

Biofilm formation assay

Biofilm experiments were conducted on 96-well polystyrene

microplates (Nunc; D. Dutscher, Brumath, France) to com-

pare the biofilm-forming capacities of the three P. aeruginosa

populations depending on their origin. Aliquots of each

suspension were used to inoculate TSB at different cell

concentrations (5� 102 and 104 CFU mL�1) and 100mL of

the suspension was dispensed per well. Control wells received

sterile TSB. Microplates were incubated at 37 1C for 24 h.

Biofilm formation was assessed by quantification of

attached cells after crystal violet staining as described

previously (Challan-Belval et al., 2006). For each experi-

ment, 15 replicates resulting from three independent inoculi

were analyzed.

Physicochemical characterization of the cellsurface

Hydrophobicity and the Lewis acid/base character of the

three P. aeruginosa populations were investigated according

to the microbial adhesion to solvents (MATS) method

(Bellon-Fontaine et al., 1996) with slight modifications.

The following pairs of solvents were used: chloroform

(acidic solvent)/hexadecane (apolar solvent) to determine

the Lewis basic character of bacterial populations, and ethyl

acetate (basic solvent)/decane (apolar solvent) to determine

the Lewis acid character.

Experimentally, 50 mL of TSB in 250-mL baffled round-

bottomed flasks was inoculated with equal aliquots (2.5 mL)

of each cell suspension, and incubated under agitation

(150 r.p.m.) for 24 h at 37 1C. It was technically mandatory

that this cell culture have the required cellular biomass

(c. 108 CFU mL�1) to perform MATS experiments. Then,

cells were washed three times with a saline solution, cen-

trifuged (7000 g for 10 min at 4 1C), and resuspended in

fresh saline solution to reach an OD of 1.0 at 600 nm (A0).

Then 2.4-mL aliquots of each bacterial suspension were

mixed with 0.4 mL of solvent by vortexing for 30 s. The mix

was allowed to stand at room temperature for 45 min.

One-milliliter aliquots were carefully taken from the

aqueous phase and the OD600 nm (A) was measured. The

percentage of cells associated with each solvent was

determined as follows: % affinity = (1�A/A0)� 100.

Experiments were conducted in nine replicates resulting

from three independent inoculi.

Antibiotic susceptibility testing

The three populations were analyzed in triplicate for their

in vitro susceptibilities to 14 antibiotics using the Kirby–Bauer

disc-diffusion assay (Bauer et al., 1966): antibiotic-impreg-

nated paper discs were placed on preinoculated Mueller–

Hinton agar plates to allow the antibiotic diffusion and the

formation of a concentration gradient into the agar. The

antibiotics (bioMerieux, Marcy l’Etoile, France) were

selected (shown in Table 1) and tested according to the

Antimicrobial Committee of the French Microbiology

Society guidelines (CASFM, 2008). After incubation (24 h,

37 1C), the inhibition zone diameters were measured for each

population and each antibiotic. The results were analyzed

using the zone diameter interpretative standards published by

the Antimicrobial Committee of the French Microbiology

Society (CASFM, 2008) for P. aeruginosa, in order to deter-

mine the equivalent minimal inhibitory concentration (MIC)

values corresponding to each inhibition diameter, and to

classify each population as sensitive, intermediate, or resistant

to each antibiotic.

Statistical analysis

All statistical analyses were performed using SIGMASTAT

V3.0.1 software (SPSS Inc.). To test the significance of the

differences in cell populations, a Kruskal–Wallis one-way

ANOVA was performed, followed by a Student–Newman–

Keuls pairwise multiple comparison for biofilm experi-

ments, and by Dunn’s pairwise multiple comparison for the

cell-surface investigation. For all statistical analyses,

Po 0.05 was considered statistically significant.

Results

Before comparing the physiological characteristics of the

three cell populations, their morphology was observed on a


137Physiology of biofilm-detached cells and recolonization

TSA plate to determine the presence or absence of variants

in each population. Five-day-old biofilm-resulting cells

(sessile and detached cells) and planktonic cells (stationary

phase) were grown for 24 and 48 h at 37 1C. The same

colonies were observed for the three populations: all

morphological criteria observed (size, texture, and

roughness) were similar, suggesting the absence of variants.

Planktonic growth kinetics

The three P. aeruginosa populations were compared for their

growth performance in batch cultures. A long lag phase was

observed because of the transfer of cells to a saline solution.

The growth curves had the same general profile (Fig. 2).

Nevertheless, when compared with originally planktonic cells,

both biofilm-resulting (detached and sessile) populations

exhibited longer lag phases and a 3-h delay was observed.

Under our experimental conditions, the specific growth

rates of biofilm-resulting populations were found to be 0.66

and 0.65 h�1 for detached cells and sessile cells, respectively,

which revealed no significant difference. These values

appeared to be different from that obtained with originally

planktonic cells, which exhibited a growth rate of 0.89 h�1.

Ability to form biofilms

The impact of the previous physiological state on the

biofilm-forming capacity was studied by quantifying new

biofilms on polystyrene microplates. The originally

planktonic population demonstrated significantly lower

production of biofilm compared with that of the sessile

population, irrespective of the inoculum concentration

(Fig. 3). Indeed, the amount of biofilm formed by the

planktonic population corresponded to an average of

63–73% of that formed by the originally sessile population

depending on the inoculum size. However, as expected, the

inoculum concentration directly influenced the quantity of

Fig. 2. Growth curves for batch cultures of the three populations of

Pseudomonas aeruginosa: originally planktonic cells (�), sessile cells (m),

and biofilm-detached cells (’). Growth is expressed as OD600 nm as a

function of time. Each point represents the mean value obtained from

four independent cultures. Vertical bars represent SDs.

Table 1. In vitro susceptibility of the three populations to the antibiotics tested by the disc-diffusion method�

Antibiotics (disc content)

Inhibition zone diameters (mm)w

MIC (mg mL�1)z SusceptibilityzPlanktonic cells Sessile cells Biofilm-detached cells

Amikacin (30 mg) 18 18 18 4–8 Sensitive

Aztreonam (30mg) 29 29 28 0.5 Sensitive

Cefepime (30 mg) 27 27 28 o 8 Sensitive

Ceftazidime (30 mg) 33 34 32 o 8 Sensitive

Ciprofloxacin (5 mg) 31 32 33 � 0.125 Sensitive

Colistin (50mg) 20 21 21 o 4 Sensitive

Fosfomycin (50 mg) 23 23 24 o 32 Sensitive

Gentamicin (10mg) 13 13 13 4 4 Resistant

Imipenem (10mg) 29 28 27 2 Sensitive

Netilmicin (30mg) 16 16 16 4 4 Resistant

Sulfamide (200mg) 0 0 0 4 256 Resistant

Ticarcillin (75 mg) 26 25 26 4 Sensitive

Ticarcillin/clavulanic acid (75/10 mg) 28 28 28 2 Sensitive

Tobramycin (10 mg) 20 20 20 o 4 Sensitive

�The MIC values and the corresponding susceptibility categories were determined from the inhibition zone diameters, according to the table of

correlation between the zone diameter interpretative standards and the equivalent MIC interpretative breakpoints published by the Antimicrobial

Committee of the French Microbiology Society.wValues represent average diameters obtained from three independent replicates.zThe MIC values and the assigned susceptibility categories are the same for the three populations.



biofilm. Concurrently, the quantity of biofilm formed by the

detached population appeared to be between the amount

produced by planktonic and that by sessile populations,

corresponding on average to 80–89% of the amount formed

by the sessile population.

Physicochemical properties of cell surfaces

If affinity values obtained with ethyl acetate (basic, polar)

and decane (apolar) did not appear to be significantly

different, those obtained with chloroform (acidic, polar)

were significantly higher than those obtained with hexade-

cane (apolar) for the three populations. Furthermore, the

affinity values were higher with chloroform than with ethyl

acetate (Fig. 4). These observations reflect the basic and

hydrophilic surface properties of P. aeruginosa CIP A22 cells.

With the chloroform/hexadecane pair, no significant

differences were found between the two biofilm-resulting

(detached and sessile) populations. However, the planktonic

population was different: its affinity toward these solvents

was much lower. Interestingly, the biofilm-resulting popula-

tions remained different from the planktonic population in

terms of physicochemical cell-surface properties even after a

24-h planktonic growth (see Materials and methods).

Susceptibility to antibiotics

The comparison of the measures obtained for each indepen-

dent antibiotic revealed no significant difference in the

inhibition diameters between the three populations.

According to the table of correlation between the zone

diameter interpretative standards and the equivalent MIC

interpretative breakpoints for P. aeruginosa (CASFM, 2008),

the inhibition diameters of the three populations therefore

correspond to the same MIC values and to the same

susceptibility categories for each independent antibiotic

(Table 1). The three populations exhibited the same profile

of susceptibility/resistance to the antibiotics.

Discussion

Sessile bacteria exhibit a phenotype that is distinct from that

of their planktonic counterparts; differences include differ-

ent growth rates and enhanced resistance to physicochem-

ical stresses (Costerton et al., 1987; Donlan & Costerton,

2002; Fux et al., 2005). Several studies have shown differ-

ential gene expression during the switch from planktonic to

biofilm growth (Christensen et al., 1998; O’Toole & Kolter,

1998; Davey & O’Toole, 2000). Thus, a sequence of five

developmental stages in biofilm growth was characterized in

P. aeruginosa (Sauer et al., 2002), demonstrating consider-

able alterations in cell physiology and phenotype through-

out the developmental cycle. The last described stage

involves the detachment of adherent bacteria. In this study,

we investigated, in particular, the physiological characteris-

tics of the biofilm-detached cells of a P. aeruginosa strain, in

comparison with their sessile and planktonically grown

counterparts, to observe whether the detached cells retain

their biofilm-like phenotype.

The device developed in this work to collect biofilm-

detached cells in a saline solution prevents the cells from

growing planktonically in the effluent. Such a system could

thus constitute a simple tool and provide a reproducible

device to accumulate and to study biofilm-detached cells.

Fig. 3. Biofilm formation of Pseudomonas aeruginosa originally plank-

tonic cells (&), sessile cells (’), and biofilm-detached cells ( ) measured

under batch conditions by a microtiter plate assay (crystal violet staining)

after 24 h of incubation at 37 1C. Histograms correspond to average

OD595 nm values measured on 15 replicate wells resulting from three

separate experiments. Error bars represent SDs. �Statistically significant

differences between the populations.

Fig. 4. Affinities of the three populations of Pseudomonas aeruginosa:

originally planktonic cells (&), sessile cells (’), and biofilm-detached

cells ( ), to various solvents used in the MATS test. Histograms

correspond to means of nine replicates resulting from three independent

populations. Error bars represent SDs. �Statistically significant differ-

ences between the populations.



As regards growth kinetics, both sessile and detached cells

seem to be in a different physiological state from that of the

originally planktonic cells. Indeed, the 3-h delay observed

during lag phases with biofilm-resulting populations

suggests that they need a period of adaptation to return to

the planktonic growth. Nevertheless, the originally plank-

tonic population still showed a higher growth rate than both

biofilm-resulting populations. These results first confirm

that sessile and planktonic bacteria exhibit different char-

acters as established previously (Costerton et al., 1987).

They are in agreement with published results (Xu et al.,

1998) showing that limiting diffusion of oxygen and

nutrients in biofilms alters bacterial growth rates. They also

indicate that detached cells are less able to return to the

planktonic mode compared with originally planktonic cells,

at least during the first hours following detachment.

Concurrently, we noted no significant difference between

detached and sessile cells in terms of growth rate and lag

phase, suggesting that these populations might have similar

physiological states at least during the first hours following

detachment, under our experimental conditions.

As the physiology of bacteria changes according to their

growth mode and as the physiological state affects bacterial

adherence, we were interested in the ability of the different

bacterial populations to form a biofilm. After 24 h of

growth, the sessile population had produced the most

biofilm, irrespective of the cell concentration of the

inoculum. Biofilm production by the originally planktonic

population was significantly lower, representing 63–73% of

the production shown by their sessile counterparts. Of

particular interest is the observation that the quantity of

biofilm produced by the detached population was between

that generated from sessile and planktonic populations. This

observation first suggests that detached cells exhibit a

distinct phenotype from those of both sessile and planktonic

cells. It also implies that detached cells have a greater

capacity to form biofilms than do planktonic cells, demon-

strating that this population should not be neglected, as

previously suggested (Ymele-Leki & Ross, 2007). The

differences observed may be due to differences in adhesion

capacities and growth rates.

As bacterial surface properties are known to play a crucial

role in bacterial adherence (Ly et al., 2006), the comparison

of the Lewis acid–base and the hydrophobic characters of

the three populations was of great interest to better

understand their biofilm-forming potential. When consid-

ering the affinity for solvents, we observed that the three

populations displayed the greatest affinity for chloroform,

suggesting that P. aeruginosa strain CIP A22 has a basic

character under our experimental conditions. As chloro-

form and hexadecane have the same van der Waals

properties, we first compared the affinities for these solvents:

the affinity for hexadecane (o 40%) revealed that the strain

adheres weakly to this apolar solvent, indicating that it is

rather hydrophilic. The affinity for ethyl acetate was lower

(no more than 25%) than that for chloroform, confirming

the nonacidic character of the strain, usually due to a

negatively charged cell surface. According to some authors

(Bellon-Fontaine et al., 1996; Pelletier et al., 1997), this

character can be linked to a large quantity of carboxylate and

sulfate groups on the microbial surface.

Comparing in particular the three populations, we ob-

served that the affinities of sessile and detached cells for both

chloroform and hexadecane were significantly increased in

contrast with originally planktonic cells. This indicates that

biofilm-resulting populations have a stronger basic charac-

ter. Our data are in agreement with other studies (Bellon-

Fontaine et al., 1996; Chavant et al., 2002), which associated

the electron-donor character with important Lewis acid–

base interactions during bacterial attachment to surfaces, as

this character is increased when cells were previously

attached. It is interesting to observe these differences be-

tween the populations after a 24-h cell culture (see Materials

and methods), which indicates that biofilm-resulting

populations can retain physiological biofilm characters after

they return to a planktonic growth mode (at least during

24 h). We can assume that some characters are hereditary.

But an in-depth study should be performed to better

understand the inheritance of the physiological characters.

Finally, the MATS results obtained with the chloroform/

hexadecane pair led to the assumption that whatever the

nature of the solvent is (polar or apolar), biofilm-resulting

populations have better adhesion capacities to solvents than

their planktonic counterpart, which could be correlated

with better capacities to adhere to surfaces and to form

biofilms. The absence of differences between the three

populations with the ethyl acetate/decane pair suggests that

surface interactions other than hydrophobicity could be

involved in affinity to solvents and adhesion (van der Waals

properties for example). If we consider particularly the results

obtained with hexadecane, we note that the biofilm-resulting

cells exhibited a better affinity for this apolar solvent than the

planktonic cells. This could suggest that the biofilm-resulting

cells adhere more easily on apolar supports such as

polystyrene, and justify that they form better biofilms on

polystyrene microplates than the planktonic cells.

In another study (Allison et al., 1990), it was reported

that detached cells of Escherichia coli biofilms were more

hydrophilic than sessile cells, while we observed no differ-

ence between detached and sessile cells. These differences in

observations could be linked to the nature and the proper-

ties of the strain studied, the procedure used to collect

cells, but also the method used to measure the surface

hydrophobicity.

Many studies reported that biofilms were more resistant

to antimicrobials than freely suspended cells; therefore, we



decided to compare the susceptibility of the three popula-

tions to antibiotics. As a result, the three populations

showed similar susceptibility profiles. First, this may be

surprising, because previous reviews reported that biofilms

were more resistant to antimicrobials than planktonic cells

(Gilbert et al., 1997; Donlan, 2002; Dunne, 2002; Davies,

2003). However, in our study, the three populations were

formed by cells collected and suspended in a saline solution,

which could explain why the mechanisms of resistance

associated with the protective matrix or implying cell-to-cell

signaling were lost. While we observed no significant

difference in sensitivity to antibiotics between planktonic

and biofilm-detached populations, another study (Bester

et al., 2005) reported that biofilm-detached cells were more

sensitive to a mix of disinfectants (glutaraldehyde and

isothiazolones) than planktonic cells. The differences in the

nature, the target, and the mode of action of the antimicro-

bial compounds (antibiotics or disinfectants) used in the

two studies could explain the difference of response

observed in both studies.

In summary, our findings demonstrate that biofilm-

detached cells are partially distinct from planktonically

grown cells and from sessile cells under our experimental

conditions. This led us to hypothesize that the physiological

changes that occurred during previous bacterial attachment

are temporarily and partially conserved after detachment, at

least for the first hours. Then, in addition to studies already

described (Gilbert et al., 1993; Sauer et al., 2002; Bester et al.,

2005), data on biofilm-forming ability support the theory

that the biofilm character progressively switches to the

planktonic phenotype after detachment.

These conclusions led us to believe that biofilm-detached

cells represent a high risk for recolonization of surfaces: they

appeared to be no more sensitive to antibiotics than plank-

tonic cells, and they adhere more easily to form biofilms. This

confirms the importance of considering detachment as a

distinct stage in the process of biofilm development. These

results should contribute to more effective management of

disinfection strategies, especially by making sure that cells

detached from contaminated surfaces will be rapidly killed or

removed to prevent the spread of contamination. An inter-

esting perspective would consist in studying more particularly

the impact of dispersive treatments (such as enzymatic

agents) on cell physiology just before the bacteria are

eliminated. Furthermore, it might be interesting to compare

the virulence of the three populations, because the ability to

develop biofilms is often associated with the expression of

virulence (De Kievit et al., 2001).

Acknowledgements

This work was supported and financed by the Conseil

Regional de Bourgogne, the Fond Social Europeen, and the

Laboratoires Protec society as part of the doctorate fellow-

ship of C.R. We thank Dr J.-P. Lemaitre for his helpful

discussions on antibiotic susceptibility profile determina-

tion, and Dr P. Lemanceau and Dr A. Hartmann for

welcoming C.R. in their research team UMR ‘Microbiologie

des Sols et de l’Environnement’ 1229 INRA/Universite de

Bourgogne.

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Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa