Interactions between biocide cationic agents and bacterial biofilms

Article abstract of DOI:10.1128/AAC.46.5.1469-1474.2002

The resistance of bacterial biofilms to physical and chemical agents is attributed in the literature to various interconnected processes. The limitation of mass transfer alters the growth rate, and physiological changes in the bacteria in the film also appear. The present work describes an approach to determination of the mechanisms involved in the resistance of bacteria to quaternary ammonium compounds (benzalkonium chloride) according to the C-chain lengths of those compounds. For Pseudomonas aeruginosa CIP A 22, the level of resistance of the bacteria in the biofilm relative to that of planktonic bacteria increased with the C-chain length. For cells within the biofilm, the exopolysaccharide induced a characteristic increase in surface hydrophilicity. However, this hydrophilicity was eliminated by simple resuspension and washing. The sensitivity to quaternary ammonium compounds was restored to over 90%. Staphylococcus aureus CIP 53 154 had a very high level of resistance when it was in the biofilm form. A characteristic of bacteria from the biofilm was a reduction in the percent hydrophobicity, but the essential point is that this hydrophobicity was retained after the biofilm bacteria were resuspended and washed. The recovery of sensitivity was thus only partial. These results indicate that the factors involved in biofilm resistance to quaternary ammonium compounds vary according to the bacterial modifications induced by the formation of a biofilm. In the case of P. aeruginosa, we have underlined the involvement of the exopolysaccharide and particularly the three-dimensional structure (water channels). In the case of S. aureus, the role of the three-dimensional structure is limited and drastic physiological changes in the biofilm cells are more highly implicated in resistance.

Full text of DOI:10.1128/AAC.46.5.1469-1474.2002

Interactions between Biocide Cationic
Agents and Bacterial Biofilms
C. Campanac, L. Pineau, A. Payard, G. Baziard-Mouysset
and C. Roques
Antimicrob. Agents Chemother. 2002, 46(5):1469. DOI:
10.1128/AAC.46.5.1469-1474.2002.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 1469–1474
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.5.1469–1474.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 5
Interactions between Biocide Cationic Agents and Bacterial Biofilms
C. Campanac,1 L. Pineau,1 A. Payard,2 G. Baziard-Mouysset,2 and C. Roques1*
Laboratoire de Bactériologie, Virologie et Microbiologie Industrielle,1 and Laboratoire de Chimie Pharmaceutique,2
Faculté des Sciences Pharmaceutiques, 31062 Toulouse cedex 04, France
Received 20 April 2001/Returned for modification 22 September 2001/Accepted 26 January 2002
The resistance of bacterial biofilms to physical and chemical agents is attributed in the literature to various
interconnected processes. The limitation of mass transfer alters the growth rate, and physiological changes in
the bacteria in the film also appear. The present work describes an approach to determination of the
mechanisms involved in the resistance of bacteria to quaternary ammonium compounds (benzalkonium
chloride) according to the C-chain lengths of those compounds. For Pseudomonas aeruginosa CIP A 22, the level
of resistance of the bacteria in the biofilm relative to that of planktonic bacteria increased with the C-chain
length. For cells within the biofilm, the exopolysaccharide induced a characteristic increase in surface hydro-
philicity. However, this hydrophilicity was eliminated by simple resuspension and washing. The sensitivity to
quaternary ammonium compounds was restored to over 90%. Staphylococcus aureus CIP 53 154 had a very high
level of resistance when it was in the biofilm form. A characteristic of bacteria from the biofilm was a reduction
in the percent hydrophobicity, but the essential point is that this hydrophobicity was retained after the biofilm
bacteria were resuspended and washed. The recovery of sensitivity was thus only partial. These results indicate
that the factors involved in biofilm resistance to quaternary ammonium compounds vary according to the
bacterial modifications induced by the formation of a biofilm. In the case of P. aeruginosa, we have underlined
the involvement of the exopolysaccharide and particularly the three-dimensional structure (water channels). In
the case of S. aureus, the role of the three-dimensional structure is limited and drastic physiological changes
in the biofilm cells are more highly implicated in resistance.
Bacterial biofilms associated with surfaces are complex
three-dimensional structures in which bacteria are embedded
in a matrix principally made of extracellular polymers. Biofilm
formation may be separated into the primary attachment of
bacteria to surfaces, followed by proliferation of the attached
bacterial cells, which leads to the accumulation of multilayered
clusters of cells and glycocalyx formation. This mode of growth
has often been implicated in surface contamination that leads
to resistance to particular physical and/or chemical treatments
(7, 23). This mode of growth is also considered essential for
infections that occur and persist at various sites in the human
body, especially in association with medical devices (6). Such
infections are characteristically resistant to antibiotics as well
as to host defenses (7, 16). Many in vitro (2, 36, 39) and in vivo
(35) studies have attributed the resistance to the biofilm’s
mode of growth (slow growth rate, adhesion steps), but it is
now thought that there are a number of contributory factors.
The exopolysaccharide (EPS) matrix may exclude and/or in-
fluence the penetration of antimicrobial agents (11, 30). It has
also been demonstrated that the abiotic part of the biofilm
plays a part in resistance because of its potent neutralization
effect on many compounds. Chlorine is known for its suscep-
tibility to organic and inorganic matter (41). Therefore, the
production of EPS can dramatically affect the activity of chlo-
rine (34). On the other hand, recent work has shown that a
large number of genes and regulatory systems are activated
during biofilm formation. Some of these are associated with
the adhesion step (17, 31, 38), whereas others are associated
with colonization and maturation of the biofilm (10, 13). This
leads to different phenotypes between planktonic and biofilm
cells. The mechanism(s) involved in biofilm maturation and
particularly in biofilm resistance to biocides may involve a wide
variety of profound changes. The present study was performed
to characterize the variations in the susceptibilities of two
different mature biofilms (biofilms of Pseudomonas aeruginosa
and Staphylococcus aureus) versus that of planktonic cells of P.
aeruginosa and S. aureus to cationic surfactants (quaternary
ammonium compounds [QACs]) according to the C-chain
lengths of the surfactants. The two species selected, P. aerugi-
nosa and S. aureus, are opportunistic human pathogens that
are increasingly the cause of hospital-acquired infections,
which is strongly correlated with the increasing rate of use of
implantable medical devices like catheters (21, 27). Modifica-
tion of the surface hydrophobicities of planktonic and biofilm
cells and the persistence of resistance after disruption of the
three-dimensional structure were also determined to focus on
the possibility that phenotypic changes limit the efficacies of
the chemicals tested against the two different biofilms.
MATERIALS AND METHODS
QACs. N-Alkylbenzyldimethyl ammonium chlorides were prepared by the
reaction between the N,N-alkyldimethylamine and benzyl chloride in refluxing
butanone (unpublished data). They were recrystallized from the butanone and
were obtained in a highly purified form (purity, Ͼ98%). The formula for the
QACs tested is (C9H13N-CnH2nϩ1)Cl, where n is equal to 8, 10, 12, 14, 16, or 18.
The compounds tested, named C8, C10, C12, C14, C16, and C18, were dissolved at
the appropriate concentrations in sterile distilled water.
Bacterial strains. The bacterial strains were provided by the Institut Pasteur
Collection (Paris, France). They included two bacteria (P. aeruginosa CIP A 22
and S. aureus CIP 53 154) recommended by French standards for the evaluation
of water-miscible antiseptics and the efficiencies of disinfectants. P. aeruginosa
* Corresponding author. Mailing address: Laboratoire de Bactéri-
ologie, Virologie et Microbiologie Industrielle, Faculté des Sciences
Pharmaceutiques, 35 Chemin des Maraˆıchers, 31062 Toulouse cedex
04, France. Phone: (33) 0562256860. Fax: (33) 0561259572. E-mail:
CH.ROQUES@wanadoo.fr.
1469

1470
CAMPANAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
CIP A 22 and S. aureus CIP 53 154 were maintained on Trypticase soy agar
(Biomérieux, Marcy l’Etoile, France) and were incubated under aerobic condi-
tions at 37°C for 24 to 48 h. The bacterial suspensions were freshly prepared in
sterile distilled water before each experiment.
Activities of the tested compounds against planktonic bacteria. The assays for
determination of the activities of the compounds were performed according to
French and European standards, and the end of the contact between the micro-
organisms and the compounds was obtained by dilution-neutralization (3). The
neutralizing medium was selected as indicated by the standards. Briefly, 1 ml of
compound solution (or 1 ml of sterile distilled water, which was used to establish
the harmlessness of the neutralizing medium) was added to 9 ml of neutralizing
medium. After 10 min of contact, 1 ml of a bacterial suspension adjusted to 103
CFU/ml was added. After homogenization, 1 ml of each mixture was placed in
duplicate on Trypticase soy agar medium, and the plates were incubated under
the conditions described above. The neutralizing medium selected permitted at
least 80% of the microorganisms to be recovered, indicating that it protected
them from the antimicrobial effects of the products and was totally harmless. The
method was validated for the neutralizing medium made of Trypticase soy broth
containing 5% Tween 80 (Sigma, Saint Quentin Fallavier, France), 2% lecithin
(Sigma), and 0.5% sodium thiosulfate (Sigma). For evaluation of the bactericidal
efficiencies of the compounds against planktonic cells, 1 ml of a bacterial sus-
pension adjusted to 108 CFU/ml was added to 9 ml of each dilution of the QACs
(or 9 ml of distilled sterile water as a control). After 5 min of contact at 20°C, 1
ml of each mixture was transferred to 9 ml of neutralizing medium and allowed
to remain in contact for 10 min after homogenization. Viable bacterial counts
were determined by duplicate inclusion of serial dilutions of homogenized sam-
ples in Trypticase soy agar. The agar plates were incubated aerobically, as
described above. The results were expressed as the log reduction (log reduction
ϭ log value for the control Ϫ log value for the assayed sample).
Biofilm formation. Bacterial biofilms were obtained by a previously described
procedure (32, 34). The nutrient broth used to promote bacterial adhesion and
biofilm formation consisted of Vitamin Assay Casamino Acids (0.1 g/liter;
Difco), yeast extract (0.1 g/liter), MgSO4 · 2H2O (0.2 g/liter), FeSO4 · 7H2O
(0.0005 g/liter), anhydrous Na2HPO4 (1.25 g/liter), KH2PO4 (0.5 g/liter), and
lactose (0.025 g/liter). This medium was circulated through a sterile loop of
Tygon (Bioblock, Illikirch, France) tube (inner diameter, 6.4 mm) at 100 ml/min.
The loop was maintained at 30°C and was connected to a discharge line and to
a feeding tank (supply pump; feeding rate, 3 ml/min). After the bioreactor was
filled with the adhesion broth, the loop was inoculated with 5 ml of a bacterial
suspension containing about 108 CFU/ml. The circulation pump was run for 30
min to allow the cells to spread around the loop before the supply pump was
turned on. After 65 h, stabilized and reproducible populations of adherent and
evacuated bacteria were obtained for the two strains tested (32, 34).
Bactericidal activities of the tested compounds against the biofilm. For eval-
uation of bactericidal activities of the tested compounds against the biofilm,
samples of the Tygon tube (2-cm pieces cut in half lengthwise) were removed
from the loop carrying the 65-h-old biofilm and were distributed among test
tubes containing 10 ml of dilutions of the compounds to be tested. The control
was dipped into sterile distilled water. After 5 min of contact at 20°C, the samples
were removed and placed in 10 ml of neutralizing medium for 10 min. The
adherent cells were immediately recovered by scraping them off with a sterile
cutter. The portions of the Tygon tube and the corresponding neutralizing
medium were then dispersed for 1 min with a vortex mixer (Bioblock) and by
sonication (600 W; Vibra cell; Bioblock) at 45 W for 5 s. Viable bacterial counts
were determined by placement of duplicate serial dilutions of homogenized
samples on Trypticase soy agar. The agar plates were incubated aerobically as
described above. The results were expressed as the log reduction (log reduction
ϭ log value for the control Ϫ log value for the assayed sample).
Protein and polysaccharide levels. Protein and polysaccharide levels were
determined for planktonic cells and attached cells. For planktonic cells, a bac-
terial suspension at an absorbance of 70 to 75% (measured at 640 nm) was
prepared. For biofilm cells, the samples of the Tygon tube (2-cm pieces cut in
half lengthwise) were removed, the adherent cells were recovered by scraping the
tubes with a sterile cutter, and the material was placed in 3 ml of distilled sterile
water. The protein level was determined by the method of Lowry et al. (24) by
using bovine serum albumin as a standard. The polysaccharide level was evalu-
ated by the phenol-sulfuric assay procedure by using glucose as a standard (14).
Recovery of sensitivity to QACs after disruption of biofilms. Samples of the
Tygon tube (2-cm pieces cut in half lengthwise) were removed from the loop
carrying the biofilm. The attached cells were immediately recovered in a phos-
phate-buffered (PBS) solution at 0.15 mol/liter by scraping the tube with a sterile
cutter. The test was performed as described above for determination of the
activities of the compounds against planktonic cells directly after disruption of
FIG. 1. Variations in bactericidal efficiencies (log reductions) of
QACs against planktonic cells of P. aeruginosa (n ϭ 2).
the biofilm or after three washes in a PBS solution at 0.15 mol/liter. The percent
recovery of sensitivity to QAC after disruption of the biofilm was expressed as
(log reduction of cells after disruption of the biofilm/log reduction of planktonic
cells) ϫ 100.
Evaluation of hydrophobicity of planktonic cells. Evaluation of hydrophobicity
was done by the microorganism adherence to hydrocarbons (MATH) method
first described by Rosenberg et al. (33) and modified by Mozes and Rouxhet (28).
Cell suspensions were prepared at 108 CFU/ml in PBS at 0.15 mol/liter. They
were directly used for the MATH assay or were first washed three times in a PBS
solution at 0.15 mol/liter. A total of 1.2 ml of each cell suspension was placed into
a glass test tube, and 0.6 ml of hexadecane was then added. After the tubes were
allowed to rest for 10 min, each glass tube was vortexed for 2 min and was left
for 15 min, during which time the two phases separated completely. The absor-
bance of the aqueous phase was then measured (␭ ϭ 640 nm). The blank
consisted of PBS without cells. The results are expressed as the proportion of the
cells which were excluded from the aqueous phase, determined by the equation
[(A0 Ϫ A)/A0] ϫ 100, where A0 and A are the initial and final optical densities of
the aqueous phase, respectively.
Evaluation of hydrophobicity of biofilm cells. Samples of the Tygon tube (15-
to 20-cm pieces cut in half lengthwise) were removed from the loop carrying the
biofilm. The biofilm cells were immediately recovered in a PBS solution at 0.15
mol/liter by scraping the tube with a sterile cutter. The test was then performed
as described above directly after disruption of the biofilm or after three washes.
Scanning electron microscopy observations. Biofilm samples were prepared
for scanning electron microscopy by fixation in 2.5% glutaraldehyde, dehydration
in ethanol, and air drying. The dried samples were mounted onto aluminum
stubs, sputter coated with gold and palladium (60:40), and imaged with a Hitachi
S-450 electron microscope (Centre de Microscopie Électronique Appliquée à la
Biologie, Université de Médecine, Toulouse, France).
RESULTS
P. aeruginosa. The variations in logarithmic reductions ac-
cording to QAC concentration are presented in Fig. 1 and 2 for
planktonic and biofilm P. aeruginosa cells, respectively. High
levels of bactericidal activity (Ͼ5 logs) were observed against
planktonic cells even at low concentrations (10Ϫ4 mol/liter) for
C12 to C18 compounds. A gradual loss of activity was noted
with a reduction in the C-chain length, and for the short-chain
compounds (C8 and C10), a dilution effect appeared. Biofilm
assays were characterized by resistance to the six QACs tested,
with the logarithmic reductions being lower than 2 even at
10Ϫ4 mol/liter. The optimal efficiency was observed for C12.
The loss of activity was thus dramatic for C14 to C18 com-
pounds, and the loss of activity increased according to the
C-chain length. In contrast, the bactericidal activities of short-
chain QACs (C8 and C10 compounds) were maintained, even if
the level of activity was low. The 65-h-old P. aeruginosa biofilm

VOL. 46, 2002
BIOCIDE CATIONIC AGENTS AND BIOFILMS
1471
FIG. 3. Variations in bactericidal efficiencies (log reductions) of
QACs against planktonic cells of S. aureus (n ϭ 2).
FIG. 2. Variations in bactericidal efficiencies (log reductions) of
QACs against a P. aeruginosa biofilm (n ϭ 2).
obtained for washed bacteria, but important but incomplete
(92%) recovery was obtained for unwashed bacteria.
was characterized by very much higher protein (50 times) and
polysaccharide (400 times) contents than planktonic cells. The
protein levels were 61.0 Ϯ 0.5 and 3,080 Ϯ 760 ng/106 CFU for
planktonic cells and biofilms, respectively; and polysaccharide
levels were 8.9 Ϯ 7.4 and 3,800 Ϯ 1,000 ng/106 CFU for plank-
tonic cells and biofilms, respectively (the values are means Ϯ
standard deviations; n ϭ 3 for planktonic cells and 25 for the
biofilm). The results are expressed according to the number of
cultivable cells and so indicate the levels of accumulation of
proteins and polysaccharides in the three-dimensional matrix.
The results for P. aeruginosa adherence to hexadecane are
presented in Table 1 according to whether the cells were plank-
tonic or were from the biofilm and whether they were washed.
After three washes, the hydrophobicities of planktonic cells
and those obtained from the biofilm were similar. However,
assays performed with unwashed bacteria indicated a higher
degree of hydrophilicity of biofilm bacteria (20.4%) than of
planktonic cells (41.4%) (P ϭ 0.002) and also a higher degree
of hydrophilicity of unwashed bacteria than of washed biofilm
cells (42.3%; P ϭ 0.05). The hydrophobicity of planktonic cells
was not modified by washing. Therefore, the variations in sur-
face properties observed for P. aeruginosa biofilm cells seem to
be linked to the extracellular matrix.
S. aureus. Planktonic S. aureus CIP 53 154 was characterized
by susceptibility to the C12 and C14 QACs, with the logarithmic
reductions being greater than 6 at 10Ϫ4 mol/liter (Fig. 3). A
paradoxical effect was observed for the C18 compound, and
further experiments are required to explain the effect, despite
the possible problem with the dissolution or distribution of this
hydrophobic compound in an aqueous solution. Figure 4 pre-
sents the variations in logarithmic reductions for the S. aureus
biofilm according to the QAC concentration. These results
show a more dramatic loss of sensitivity to QAC in comparison
with that of the P. aeruginosa biofilm, with logarithmic reduc-
tions being lower than 2 even at 10Ϫ3 mol/liter. The best
efficiency was always observed for the C12 and C14 compounds.
The mature biofilm had significantly higher levels of protein (9
times) and polysaccharide (70 times) than the planktonic cells.
The protein levels were 98.2 Ϯ 11.2 and 896 Ϯ 280 ng/106 CFU
for planktonic cells and the biofilm, respectively; and polysac-
charide levels were 12.0 Ϯ 9.2 and 901 Ϯ 270 ng/106 CFU for
planktonic cells and the biofilm, respectively (the values are
means Ϯ standard deviations; n ϭ 3 for planktonic cells and 7
for the biofilm). Note the similarity in protein and polysaccha-
ride levels for planktonic S. aureus and P. aeruginosa cells (see
above). In contrast, the values of both parameters were very
much higher for the P. aeruginosa biofilm than for the S. aureus
The rates of recovery of sensitivity to the C12 and C14 com-
pounds after disruption of the biofilm were as follows: 93 and
92%, respectively, for washed bacteria and 100 and 100%,
respectively, for unwashed bacteria (the values are means; n ϭ
2). Therefore, total recovery of sensitivity to C12 and C14 was
TABLE 1. Adherence of P. aeruginosa planktonic cells
or biofilm to hexadecane
% Adherencea
Cell
P
Planktonic cells
Biofilm
Unwashed
Washed
41.4 Ϯ 4.6
38.2 Ϯ 4.8
20.4 Ϯ 2.8
42.3 Ϯ 4.7
0.002b
Ͼ0.05b
P
Ͼ0.05c
0.05c
a Values are means Ϯ standard deviations (n ϭ 14 for planktonic cells and 10
for the biofilm).
b Unpaired t test.
c Paired t test.
FIG. 4. Variations in bactericidal efficiencies (log reductions) of
QACs against an S. aureus biofilm (n ϭ 2).

1472
CAMPANAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 6. A 65-h-old biofilm of P. aeruginosa observed by scanning
electron microscopy. Bar, 5 ␮m.
FIG. 5. A 65-h-old biofilm of. S. aureus observed by scanning elec-
tron microscopy. Bar, 5 ␮m.
28%, respectively [the values are means; n ϭ 2]). The addition
of three washes led to better recoveries of efficiency (84 and
61%, respectively), although it was still partial. These results
indicate a partial effect of the extracellular part of the biofilm,
but it is not the only phenomenon involved. These results
indicate that the extracellular matrix is only partially impli-
cated in S. aureus biofilm resistance to QACs and that modi-
fications of the parietal structure are involved.
biofilm. These results corroborate the scanning electron mi-
croscopy observations for the two 65-h biofilms, which show a
lower level of colonization of the Tygon tube surface for S.
aureus (Fig. 5), which was characterized by cluster formation,
than for P. aeruginosa, which was characterized by a total
overlap of the cells (Fig. 6). These observations correspond to
the cultivable counts determined for the two mature biofilms,
i.e., 7.5 to 8 log CFU/cm2 for the P. aeruginosa biofilm and 6 to
6.5 log CFU/cm2 for the S. aureus biofilm. The levels of adhe-
sion to hexadecane (Table 2) indicate that planktonic S. aureus
cells are more hydrophobic than planktonic P. aeruginosa cells,
and there was no difference between washed (88.3%) and
unwashed cells (79.5%) (P Ͼ 0.05). Washed biofilm cells are
significantly (P ϭ 0.002) more hydrophilic (69.8%) than plank-
tonic ones (88.3%). For unwashed biofilm bacteria (hydropho-
bicity, 69.2%), the decrease in hydrophobicity (biofilm versus
planktonic) is not significant (P Ͼ 0.05) but is sufficient to
suppress the difference between washed and unwashed bacte-
ria.
TABLE 2. Adherence of S. aureus planktonic cells or biofilm to
hexadecane
% Adherencea
Cell
P
Planktonic cells
Biofilm
Unwashed
Washed
79.5 Ϯ 6.6
88.3 Ϯ 2.5
69.2 Ϯ 2.6
69.8 Ϯ 5.3
Ͼ0.05b
0.002b
P
Ͼ0.05c
Ͼ0.05c
a Values are means Ϯ standard deviations (n ϭ 18 for planktonic cells and 13
for the biofilm).
b Unpaired t test.
c Paired t test.
After biofilm cells had been resuspended, determination of
the bactericidal efficiencies of C12 and C14 indicated that the
recovery of sensitivity was poor for unwashed bacteria (39 and

VOL. 46, 2002
BIOCIDE CATIONIC AGENTS AND BIOFILMS
1473
DISCUSSION
that expressed no structure (13) and with the increase in the
level of resistance to chlorine observed with mucoid P. aerugi-
nosa strains (34).
P. aeruginosa is able to rapidly produce structured biofilms
under the conditions described above. In the course of contin-
uous urine flow, Goto et al. (20) also noted that P. aeruginosa
cells adherent to a Teflon catheter formed microcolonies at 6 h
and a biofilm at 24 h. This maturation is characterized by EPS
accumulation and is characterized overall by a three-dimen-
sional structure that has been well defined by Costerton et al.
(9). The negatively charged EPS produced by P. aeruginosa has
been shown to bind to positively charged antibiotics such as
aminoglycosides, but the resulting reduction in the diffusion
coefficient within the biofilm has been shown to be insufficient
to explain the observed changes in the susceptibility of the
biofilm (29). The mucoid glycocalyx associated with the P.
aeruginosa biofilm also protects the stuck cells against neutral
antibiotics, indicating that factors other than charge are re-
sponsible for the reduced efficacies of antibiotics (22). Coste-
rton (8) observed that ␤-lactam antibiotics can penetrate the
entire thickness of the biofilm and so concluded that protection
of biofilm cells is not really linked to slime. This observation is
certainly true for hydrophilic antibiotics able to pass through
the water channels of P. aeruginosa biofilms. Because of this
specific architecture of the P. aeruginosa biofilm, molecules like
the C8 and C10 QACs have ready access to the water channel
systems, and this is aided by convective flow, which leads to
preservation of their bactericidal activities. In contrast, an in-
crease in the hydrophobicity of a molecule corresponds to a
progressive loss of bactericidal efficiency against a P. aerugi-
nosa biofilm according to the C-chain length of the QAC.
Concerning N-alkylbenzyldimethyl ammonium chlorhydrate
(40% C12, 50% C14, 10% C16), Gelinas and Goulet (19) also
indicated that it was more readily neutralized by organic mat-
ter than other disinfectants, a phenomenon which could be
attributed to the patterns of adsorption of the QAC. A second
hypothesis in biofilm resistance concerns the microenviron-
ment of the cells. When bacteria undergo nutrient deprivation
they respond in a number of ways, leading to a population of
cells with physiological functions and cell envelopes peculiar to
each nutrient limitation (15) and, therefore, the adoption of
atypical phenotypes. In the same way, reports of cell density-
regulated phenotypes in biofilms (4) and the presence of N-
acylhomoserine lactone-dependent regulation in biofilms (26)
have led to demonstration of the role of quorum sensing in the
final maturation of biofilms (13); this is especially the case for
P. aeruginosa cells (12, 18, 27). The bacteria in a biofilm adopt
a phenotype profoundly different from that of their planktonic
counterparts, and 30 to 40% of the proteins in the cell enve-
lope are different. Therefore, current work focuses on the
specific role of this genetic switch in biofilm resistance, espe-
cially resistance to antibacterial compounds with specific tar-
gets. Concerning nonspecific bactericidal products like QACs,
modification of the phenotype of attached P. aeruginosa cells
does not seem to play a key role in the reduction of the
efficiencies of the compounds. The disruption of the three-
dimensional structure alone and the elimination of EPS by
washing induce a loss of the cell surface-specific hydrophilicity
and restoration of the major part of the efficiency of QACs.
These results are in agreement with the loss of resistance to
sodium dodecyl sulfate of a lasI mutant P. aeruginosa biofilm
Attached S. aureus cells growing in nutrient-rich batch cul-
ture were found to go through the same growth phases as
equivalent planktonic cultures and to possess high levels of
viability (39). Biofilm formation in S. aureus is not as well
defined as that in gram-negative bacteria and coagulase-nega-
tive staphylococci (Staphylococcus epidermidis) (25). Neverthe-
less, some attachment factors have been identified (37), like
polysaccharide intercellular adhesin (10). In gram-positive bac-
teria, the signaling molecules are not homoserine lactones but
hydrophobic peptides (8), which also lead to a wide range of
modifications in gene expression. However, one of the most
important observations that we made is that the two mature
biofilms have very different structures, with the persistence of
individual microcolonies for the S. aureus biofilm and the low-
est protein and polysaccharide levels for the S. aureus biofilm.
Despite this “poor” S. aureus biofilm structure, its formation
led to more drastic resistance to QACs than the resistance of
the P. aeruginosa biofilm. The adhesion of S. aureus to a solid
substrate followed by cell-cell adhesion corresponds to a sig-
nificant reduction of cell surface hydrophobicity which is not
modified either by disruption of the biofilm or by washing. The
percentages of recovery of sensitivity indicate a partial role of
EPS (after washing) but a very important role of phenotypic
modifications, the numbers of which increase with the mole-
cule’s hydrophobicity. Akiyama et al. (1) reported on the re-
sistance to antimicrobial agents of an immature biofilm of S.
aureus formed on coverslips after incubation for 24 h at 37°C.
This observation for such a young biofilm structure could be
linked to an earlier physiological modification of the attached
cells and could corroborate the roles of parameters other than
the final structure in the resistance of S. aureus biofilms.
Thus, the presence of a glycocalyx will sometimes alter sus-
ceptibility or will sometimes be negligible, depending on the
circumstances (5), including the bacterial species concerned.
The structure and organization of microbial biofilms are de-
termined largely by surface properties, the nature and avail-
ability of nutrients, and fluid dynamic forces (40). With this in
mind, the mode of biofilm formation could modify responses to
antimicrobial agents. The results presented here describe pos-
sible mechanisms of resistance of biofilms and demonstrate the
variabilities in the ways that microorganism adapt and decrease
their susceptibilities according to environmental factors.
In a recent review, Costerton (8) underlines the fact that
planktonic cells appear to “explore” the surface that they en-
counter in a species-specific series of behaviors, leading to the
formation of isolated microcolonies or cell layers. This kind of
species-specific behavior may certainly be extended to the
mechanisms implicated in biofilm resistance if we consider that
the EPS polymers produced by different species of bacteria are
different, thus leading to different immediate surroundings and
to the upregulation of different genes. Therefore, we must
consider species-specific factors as well as molecule-specific
factors in biofilm resistance patterns, especially for the devel-
opment of new therapeutic concepts.
ACKNOWLEDGMENTS
This work was supported by the Medlore Company.

1474
CAMPANAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
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