![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, April 2000, p. 1093-1096, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Control of Staphylococcal Adhesion to Polymethylmethacrylate and
Enhancement of Susceptibility to Antibiotics by Poloxamer 407
Marie-Laure
Veyries,1,*
Francois
Faurisson,1
Marie-Laure
Joly-Guillou,2 and
Bernard
Rouveix3
Department of Microbiology and Hygiene, Louis
Mourier Hospital, Colombes,2 and INSERM
EPI 9933, Bichat-Claude Bernard Hospital,1
and Department of Pharmacology, Cochin
Hospital,3 Paris, France
Received 28 May 1999/Returned for modification 20 September
1999/Accepted 19 January 2000
 |
ABSTRACT |
We studied the antiadhesive effect of Poloxamer 407 (P407),
together with modifications in the antimicrobial susceptibility of
residual adherent staphylococci. Bacterial adherence was markedly inhibited (77% to more than 99.9%) whether polymethylmethacrylate was
exposed to P407 before or during the adherence assay.
Furthermore, residual adherent staphylococci appeared to be more
susceptible to antibiotic activity, suggesting that combination of P407
with antibiotics could be a promising approach to the prevention of infection of foreign material.
| |
TEXT |
Medical implants frequently have to
be removed because of bacterial infection. Bacterial adherence, a
prerequisite in these infections (7, 10, 29), is initially
reversible but later becomes irreversible (18). The
biomaterial surface is colonized, and bacteria develop an environment
that protects them from host defenses and antibiotics (14,
27). The best prophylactic approach would be to prevent bacterial
adhesion or to kill bacteria shortly after adhesion, during the
reversible phase. Poloxamers, nontoxic, inert surfactants, are a broad
group of compounds that were introduced commercially in the early 1950s
as food additives and in pharmaceutical preparations. Some of these
compounds have been reported to have unusual rheological
characteristics (26) which permit them to be administered in
liquid form and to gel in situ upon warming to body temperature.
Investigations have mainly focused on Poloxamer 407 (P407), which forms a gel at the lowest concentration, is the least
toxic, and is the most stable in solution (9). This material
has already been proposed as a matrix for drug delivery (11,
21). Some poloxamers have been shown to have abhesive (antiadhesive) activity, which makes them useful as detergents in
contact lens solutions (23). The prevention of
fibrinogen adsorption and platelet adhesion to surfaces (1,
17) could be useful in the prevention of medical-implant
infection. These poloxamers also prevent postsurgical tissue adhesion
(4, 24).
Polymethylmethacrylate (PMMA) is an acrylic cement widely used in
orthopedic surgery. It has the major disadvantage of promoting bacterial adherence (15). Antibiotics have been incorporated into this material for the prophylaxis of bone infections, but adherence studies have shown that bacteria can adhere to such pretreated PMMA and survive despite high antibiotic concentrations that
would be lethal to bacteria in suspension (15, 22).
The aim of this study was to assess (i) the in vitro abhesive effect of
P407 using PMMA coverslips as a support for bacterial adherence and
(ii) changes in the susceptibility of residual adherent bacteria to
antimicrobial agents.
Three staphylococcal strains known to adhere to PMMA were used:
Staphylococcus aureus MRGR3 was a generous gift from D. Lew (Geneva, Switzerland), and S. aureus A970862 and S. epidermidis RP62A (ATCC 35984) were kindly provided by J. Etienne
(Lyon, France). MRGR3 is a methicillin-resistant bloodstream isolate
from a patient with an infected indwelling device. A970862 was isolated
from a patient with septicemia. RP62A, a prolific slime producer, was first isolated by Christensen from an infected peritoneal dialysis catheter (2). Aliquots of each bacterial suspension were
maintained at 
80°C in Trypticase soy broth (TSB) (Difco, Detroit,
Mich.) with 10% glycerol (Difco). For each experiment, aliquots of 1 ml were cultured twice until the early stationary phase in TSB medium
at 37°C for 18 to 24 h. Cells were harvested by centrifugation, washed twice, and resuspended in pH 7.3 phosphate-buffered saline (PBS)
with Ca, Mg, and 0.25% glucose (Bio-Mérieux,
Campronne, France). The density of bacterial suspensions
was adjusted by optical density measurement (Unicam SP 1800 Ultraviolet
Spectrophotometer; Pye Unicam Ltd., Cambridge, England) and checked by
inoculation on Trypticase soy agar (TSA) (Difco).
MICs were determined by the broth dilution method using geometric
twofold dilutions in Mueller-Hinton broth (Difco) in the presence or
absence of P407 at two different concentrations (4 and 8%) for the
three strains and for each antibiotic tested. MICs were determined
after incubation at 37°C for 18 h. The antimicrobial agents were
vancomycin hydrochloride (Lilly, Saint-Cloud, France) and gentamicin
sulfate (Schering-Plough, Levallois-Perret, France). MICs in the
presence of 15% P407 were determined by the E-test method because of
marked viscosity at 37°C. A final inoculum of 107
CFU · ml
1 was prepared in Mueller-Hinton broth
containing 15% P407 and was inoculated by swabbing the surfaces of
Mueller-Hinton agar plates in three directions at 20°C. Strips of
vancomycin and gentamicin were placed in the plates and incubated at
37°C for 18 h.
P407 (Pluronic F-127) was a gift from BASF (Levallois-Perret, France).
P407 solutions were prepared in sterile PBS by the cold method
described by Schmolka (26). Freshly prepared solutions were
sterilized by filtration through a 0.22-µm-pore-size Millipore filter.
Adherence assay.
PMMA coverslips (total surface area, 1.6 cm2) were used as adherence supports. They were placed in
glass tubes containing PBS with bacteria at 107 CFU
· ml1 and 0, 4, or 15% (wt/wt) P407. The tubes were
then statically incubated at 37°C for 4 h. To remove nonadherent
bacteria after incubation, each specimen was washed eight times by
gentle shaking in distilled water (eight washes eliminated the majority
of nonadherent bacteria).
Quantification of adherence.
After the adherence assay and
rinses, each specimen was placed in a glass tube containing 1 ml of
filtered EDTA-trypsin solution (0.05%; GIBCO, Cergy Pontoise, France)
and incubated for 15 min at 37°C. A 4-ml volume of sterile saline was
then added to the trypsin solution, and the coverslips were sonicated
for 30 min in a Transsonic TP 690 (ELMA) ultrasonic cleaner. The
absence of any effect of trypsin and sonication on bacterial viability was checked first.
Quantitative cultures were then performed by plating 100 µl of the
solution, either undiluted or serially diluted 10-fold on TSA. The
results were expressed as numbers of CFU per square centimeter for an
initial suspension of 107 CFU · ml1.
When the number of adherent bacteria was 0 or below 10 CFU · cm2 (our detection limit), we recorded it as
<1 log10 CFU · cm2. The rate of
residual bacterial adhesion, expressed as a percentage, is given as the
number of adherent bacteria in treated samples divided by the number in
control samples.
To count bacteria not detached by trypsin and sonication, after three
rinses in distilled water, coverslips were pressed on TSA (10 times per
side) and TSA was incubated for 24 h at 37°C.
Interactions between P407 and antimicrobial agents.
No
difference was noted in the MICs of gentamicin and vancomycin
against the three strains when tested in the presence and absence
of P407 at various concentrations. The MICs of gentamicin and
vancomycin were 12 and 1.5 mg · liter1 for MRGR3,
0.25 and 1 mg · liter1 for A970862, and 6 and 1.5 mg · liter1 for RP62A.
A possible P407 effect on the antibiotic susceptibility of adherent
staphylococci was evaluated. Following the adherence assay in the
presence of 4% P407, PMMA coverslips (with residual adherent bacteria)
were incubated with an antimicrobial agent in brain heart infusion
broth (Difco). The vancomycin concentration studied was the MIC, and
the incubation time was 24 or 48 h. Gentamicin concentrations were
the MIC and twice the MIC, and the incubation time was 24 h.
Coverslips were rinsed three times before quantification of adherence
was done as described above.
Preincubation assay.
To determine whether P407 preferentially
anchors to PMMA or to bacterial surfaces, they were pre-exposed to P407
for 24 h before the adherence assay. PMMA coverslips were
incubated at room temperature with PBS and 0 (controls), 4, or 25%
P407 and/or vancomycin at 20 mg · ml1 and then
rinsed three times in sterile water. For bacterial pre-exposure, P407
was added to TSB. The adherence assay was then performed as usual with
PBS alone.
To identify a possible antibacterial effect of P407, bacterial
densities after 4 and 24 h of exposure to 4 and 15% P407 were compared to those of control suspensions.
The groups treated with P407 were compared with the controls by using
Student's t test.
In every case, the number of bacteria not detached by trypsin and
sonication was negligible in comparison with the number of detached
bacteria. The mean inoculum size was 107 CFU · ml1.
S. aureus and S. epidermidis adherence was
strongly inhibited by P407, whatever the strain's capacity for
adherence (Table 1). P407 comprises a central hydrophobic nucleus of
polyoxypropylene surrounded by hydrophilic sequences of
polyoxyethylene (POE) (8). A proposed mechanism of action is
that the hydrophobic central block of the poloxamer anchors to
the material and/or the bacterial surface, its protruding POE
chains forming a sterically stabilized barrier to adhesion. The POE
chains must be of sufficient length and mobility in aqueous solution to
ensure an effective barrier to adhesion. The polyoxypropylene block
size may also be a key factor in firm anchorage of the molecule to the
material and/or the bacterial surface (3). This powerful
nonspecific abhesive effect could result from an increase in surface
hydrophilicity (3, 12), as hydrophobicity and adherence
generally correlate (16, 25). In keeping with the hypothesis
that poloxamers create a hydrated layer around bacteria and /or
biomaterials, it is interesting that, as for the adherence of
Pseudomonas aeruginosa (23), we found that
increasing concentrations of P407 enhanced the inhibition of
staphylococcal adherence (Table 1).
The nonspecific nature of the abhesive effect is a great advantage, as
more than one type of bacterium is commonly isolated from infected
biomaterials (20). Moreover, prevention of the adherence of
some microorganisms, e.g., some strains of slime-producing S. epidermidis that are able to promote the adherence of organisms that would otherwise be nonadherent (5, 6), is of particular interest.
PMMA and bacterial surfaces were exposed to P407 before the adherence
assay in order to determine if P407 preferentially anchors to one
or the other. At similar P407 concentrations, pretreatment of PMMA was
less effective against S. aureus adhesion than was the
presence of P407 during the adherence assay (Table
2). The addition of vancomycin seemed to
increase the abhesive effect, but the difference was not significant.
Adherence of S. epidermidis was inhibited equally by
pretreatment of PMMA and by the presence of P407 in the adherence
assay, but it was less inhibited by pre-exposure of S. epidermidis to P407 (residual adherence, 52.78 and 28.49% at 4 and 15% P407, respectively). This suggested a stronger interaction with the PMMA surface than with the surface of S. epidermidis. Nevertheless, Portolés et al. (23)
observed that the adherence of P. aeruginosa to contact
lenses was inhibited (99%) when bacteria were pre-exposed to P407 but
not when the contact lenses were pretreated. Conversely,
poloxamer-treated and POE-grafted surfaces were shown to be
protein or S. epidermidis resistant (3, 17). The
differences in the above results may be explained by the
characteristics of the strains used, the different supporting
materials, and different methods of treating these materials.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Effect of 24-h PMMA pretreatment with P407 and/or
vancomycin on S. aureus and S. epidermidis
adherence to PMMA
|
|
Poloxamers have been shown to be active against mycobacteria (12,
13). In our work, P407 did not have significant intrinsic activity against staphylococci when it was present in the broth, even
if this was not a favorable medium for microbial growth (Table 3).
We have previously shown that combination of vancomycin with P407 does
not impair antimicrobial activity (28a). This observation is
interesting, as the two components might act synergistically against
implant colonization and infection. This was confirmed by our results,
as vancomycin and gentamicin were far more effective on residual
adherent staphylococci when P407 was present during the adherence assay
(Tables 4 and
5). While
the surface bacterial density on PMMA continued to increase in control
specimens when they were exposed to the antibiotic, it decreased
when bacteria had adhered in the presence of P407. Synergism
between P407 and antibiotics appeared to be a two-step phenomenon:
first, a decrease in the number of adherent bacteria, second, for
residual adherent bacteria, a qualitative modification of binding,
resulting in restoration of susceptibility to antibiotic activity. The
P407 barrier to adhesion may thus prevent the colonization of PMMA and
the resulting modifications of infecting bacteria, leaving them more
susceptible to antibiotic action.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Enhancement of vancomycin activity at 24 and 48 h on
residual adherent S. aureus MRGR3 after 4 h of PMMA
incubation with bacteria and 4% P407
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Enhancement of gentamicin activity at 24 h on
residual adherent S. aureus A970862 after 4 h of
PMMA incubation with bacteria and 4% P407
|
|
This study illustrates the potential of P407 for inhibiting the
attachment of S. aureus and S. epidermidis and
for increasing their susceptibility to antibiotics once they are
adherent. Moreover, poloxamer-coated surfaces have been shown to be
resistant to fibrinogen and platelet adhesion (1, 17). As
these two components are present in vivo and cover foreign-body
surfaces, thereby promoting bacterial adhesion (19, 28),
this last property should reinforce the inhibitory effect of P407 on
bacterial adherence. To validate this hypothesis, studies with a guinea
pig model of foreign-body infection which unites all of the in vivo
conditions are now under way in our laboratory. An effective way of
decreasing infection of biomaterials is to reduce surface adhesion and
rapidly kill bacteria that still adhere before biofilm formation. The
nonspecificity of the abhesive effect of P407, together with its
inhibition of antibiotic resistance mechanisms, suggests that the
combination of P407 with an antimicrobial agent could be a promising
approach to prevent infection of foreign materials in vivo.
Antibiotic-loaded poloxamer could be used to coat prosthetic material
at the time of surgery in order to protect it against primary infection
and early hematogenous infection.
| |
ACKNOWLEDGMENTS |
Part of this work was supported by Schering-Plough France.
We gratefully acknowledge the technical assistance of Guylène Bertrand.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM EPI 9933, Hôpital Bichat-Claude Bernard, 170, blvd. Ney, 75877 Paris Cedex 18, France. Phone: (33) 1 40 25 86 10. Fax: (33) 1 40 25 86 02. E-mail:
veyries{at}bichat.inserm.fr.
| |
REFERENCES |
| 1.
|
Amiji, M., and K. Park.
1992.
Prevention of protein adsorption and platelet adhesion on surfaces by PEO/PPO/PEO triblock copolymers.
Biomaterials
13:682-692[CrossRef][Medline].
|
| 2.
|
Baddour, L. M.,
D. L. Smalley,
A. P. Kraus,
W. J. Lamoreaux, and G. D. Christensen.
1986.
Comparison of microbiologic characteristics of pathogenic and saprophytic coagulase-negative staphylococci from patients on continuous ambulatory peritoneal dialysis.
Diagn. Microbiol. Infect. Dis.
5:197-205[CrossRef][Medline].
|
| 3.
|
Bridgett, M. J.,
M. C. Davies, and S. P. Denyer.
1992.
Control of staphylococcal adhesion to polystyrene surfaces by polymer surface modification with surfactants.
Biomaterials
13:411-416[CrossRef][Medline].
|
| 4.
|
Carr, M. E.,
S. L. Carr, and A. A. High.
1996.
Effects of Poloxamer 407 on the assembly, structure and dissolution of fibrin clots.
Blood Coagul. Fibrinolysis
7:109-113[Medline].
|
| 5.
|
Chang, C. C., and K. Merritt.
1991.
Effect of Staphylococcus epidermidis on adherence of Pseudomonas aeruginosa and Proteus mirabilis to polymethylmethacrylate (PMMA) and gentamicin-containing PMMA.
J. Orthop. Res.
9:284-288[CrossRef][Medline].
|
| 6.
|
Chang, C. C., and K. Merritt.
1992.
Microbial adherence on poly(methyl methacrylate) (PMMA) surfaces.
J. Biomed. Mater. Res.
26:197-207[CrossRef][Medline].
|
| 7.
|
Dougherty, S. H., and R. L. Simmons.
1989.
Endogenous factors contributing to prosthetic device infections.
Infect. Dis. Clin. N. Am.
3:199-209[Medline].
|
| 8.
|
Garcia Sagrado, F.,
M. Guzman,
J. Molpereces, and M. R. Aberturas.
1994.
Pluronic copolymers characteristics, properties, and pharmaceutical applications. Part I. Pharm. Technol. Eur., p. 46-56.
.
|
| 9.
|
Garcia Sagrado, F.,
M. Guzman,
J. Molpereces, and M. R. Aberturas.
1994.
Pluronic copolymerscharacteristics, properties, and pharmaceutical applications. Part II. Pharm. Technol. Eur., p. 38-44.
.
|
| 10.
|
Gristina, A. G.
1987.
Biomaterial-centered infection: microbial adhesion versus tissue integration.
Science
237:1588-1595[Abstract/Free Full Text].
|
| 11.
|
Henry, R. L., and I. R. Schmolka.
1989.
Burn wound coverings and the use of poloxamer preparations.
Crit. Rev. Biocompat.
5:207-220.
|
| 12.
|
Hunter, R. L.,
C. Jagannath,
A. Tinkley,
C. A. Behling, and F. Nolte.
1995.
Enhancement of antibiotic susceptibility and suppression of Mycobacterium avium complex growth by Poloxamer 331.
Antimicrob. Agents Chemother.
39:435-439[Abstract/Free Full Text].
|
| 13.
|
Jagannath, C.,
H. S. Allaudeen, and R. L. Hunter.
1995.
Activities of Poloxamer CRL8131 against Mycobacterium tuberculosis in vitro and in vivo.
Antimicrob. Agents Chemother.
39:1349-1354[Abstract].
|
| 14.
|
Jansen, B.,
G. Peters, and G. Pulverer.
1988.
Mechanisms and clinical relevance of bacterial adhesion to polymers.
J. Biomater. Appl.
2:520-543.
|
| 15.
|
Kendall, R. W.,
C. P. Duncan,
J. A. Smith, and J. H. Ngui-Yen.
1996.
Persistence of bacteria on antibiotic loaded acrylic depots.
Clin. Orthop. Relat. Res.
329:273-280.
|
| 16.
|
Kristinsson, K. G.
1989.
Adherence of staphylococci to intravascular catheters.
J. Med. Microbiol.
28:249-257[Abstract].
|
| 17.
|
Lee, J.,
A. Martic, and J. S. Tan.
1989.
Protein adsorption on Pluronic copolymer-coated polystyrene particles.
J. Colloid Interface Sci.
131:252-266[CrossRef].
|
| 18.
|
Mack, D.,
N. Siemssen, and R. Laufs.
1992.
Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis: evidence for functional relation to intercellular adhesion.
Infect. Immun.
60:2048-2057[Abstract/Free Full Text].
|
| 19.
|
McDevitt, D.,
P. François,
P. Vaudaux, and T. J. Foster.
1994.
Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.
Mol. Microbiol.
11:237-248[Medline].
|
| 20.
|
Merritt, K.
1988.
Factors increasing the risk of infections in patients with open fractures.
J. Trauma
28:823-827[Medline].
|
| 21.
|
Miyasaki, S.,
T. Tobiyama,
M. Takada, and D. Attwood.
1995.
Percutaneous absorption of indométhacin from Pluronic F127 gels in rats.
J. Pharm. Pharmacol.
47:455-457[Medline].
|
| 22.
|
Naylor, P. T.,
Q. N. Myrvik, and A. Gristina.
1990.
Antibiotic resistance of biomaterial-adherent coagulase negative and coagulase-positive staphylococci.
Clin. Orthop.
261:126-133.
|
| 23.
|
Portolés, M.,
M. F. Refojo, and F. L. Leong.
1994.
Poloxamer 407 as a bacterial abhesive for hydrogel contact lenses.
J. Biomed. Mater. Res.
28:303-309[CrossRef][Medline].
|
| 24.
|
Reigel, D. H.,
B. Bazmi,
S. R. Shih, and M. D. Marquardt.
1993.
A pilot investigation of Poloxamer 407 for the prevention of leptomeningeal adhesions in the rabbit.
Pediatr. Neurosurg.
19:250-255[Medline].
|
| 25.
|
Schadow, K. H.,
W. A. Simpson, and G. D. Christensen.
1988.
Characteristics of adherence to plastic tissue culture plates of coagulase-negative Staphylococci exposed to subinhibitory concentrations of antimicrobial agents.
J. Infec. Dis.
157:71-77[Medline].
|
| 26.
|
Schmolka, I. R.
1972.
Artificial skin. I. Preparation and properties of Pluronic F-127 gels for treatment of burns.
J. Biomed. Mater. Res.
6:571-582[CrossRef][Medline].
|
| 27.
|
Schwank, S.,
Z. Rajacic,
W. Zimmerli, and J. Blaser.
1998.
Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics.
Antimicrob. Agents Chemother.
42:895-898[Abstract/Free Full Text].
|
| 28.
|
Vaudaux, P. E.,
F. A. Waldvogel,
J. J. Morgenthaler, and U. E. Nydegger.
1984.
Adsorption of fibronectin onto polymethacrylate and promotion of Staphylococcus aureus adherence.
Infect. Immun.
45:768-774[Abstract/Free Full Text].
|
| 28a.
|
Veyries, M. L.,
G. Couarraze,
S. Geiger,
F. Agnely,
L. Massias,
B. Kunzli,
F. Faurisson, and B. Rouveix.
1999.
Controlled release of vancomycin form Poloxamer 407 gels.
Int. J. Pharm.
192:183-193[CrossRef][Medline].
|
| 29.
|
Zimmerli, W.,
D. Lew, and F. A. Waldvogel.
1984.
Pathogenesis of foreign body infection. Evidence for a local granulocyte defect.
J. Clin. Investig.
73:1191-1200.
|
Antimicrobial Agents and Chemotherapy, April 2000, p. 1093-1096, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
