Influence of strain, biomaterial, proteins, and oncostatic chemotherapy on staphylococcus epidermidis adhesion to intravascular catheters in vitro

Influence of strain, biomaterial, proteins, and oncostatic chemotherapy on Sfophylococcus epidermidis adhesion to intravascular catheters in vitro S. GALLIANI, A. CREMIEUX, P. VAN DER AUWERA, and M. VIOT BRUSSELS, BELGIUM, and NICE and MARSEILLE,FRANCE

Initial adhesion of four phenotypically different strains of Staphylococcus epidermidis to 16 silicone, polyurethane, or hydrophilic polyurethane catheters was assessed in vitro by a bacterial radiolabeling method. The effect of catheter exposure to plasma proteins, to an anticancer polychemotherapy (5-fluorouracil, doxorubicin, cyclophosphamide), or to both of them was determined. Bacterial adhesion on native catheters was dependent on the hydrophobicity of both bacteria and catheters. The four strains ~ested adhered preferentially to silicone catheters (p < 0.05); adhesion was moderate to polyurethane surfaces, whereas the least adhesion was obtained for hydrophilic polyurethane catheters. Adsorption of plasma proteins on the surface produced a marked decrease in adhesion on silicone (-66.2%; p < 0.001) and polyurethane (-32.8%; p < 0.01) catheters and a marked increase in adhesion on hydropNlic surfaces (+91.7%; p < 0.05). Chemotherapeutic treatment of the catheter produced a slight but not significant decrease in adhesion on silicone (-17.4%) and polyurethane (-19.8%) catheters and a marked increase in adhesion on hydrophUic polyurethanes (+ 148.2%; p < 0.001). The in vitro simulation of catheter use suggested that oncostatic drugs and plasma proteins play an important role in S. epidermidis adhesion to intravascular catheters. Overall, bacterial adhesion is lowest on hydrophilic polyurethane catheters before and after simulation of catheter use. (J LA~ CklN MED 1996;127:71-80)

Abbreviations: BSA = bovine serum albumin; CFU = colony- forming units; C-NS = coagulasenegative staphylococci; CPM = counts per minute; FAC = polychemofherapy regimen; i,e., administration of 5-fluorouracil, doxorubicin, and cyclophosphamide; PBS = phosphate-buffered saline solution

From the Clinique des Maladies Infectieuses et Laboratoire de Microbiologie, Service de M6decine et Laboratoire d'Investigation Clinique H. J. Tagnon, Institut Jules Bordet, Centre des Tumeurs de l'Universit6 Libre de Bruxelles; MICRAAM, Facult6 de Pharmacie, Marseille; and CAL, Nice. Supported by Socidt6 des Laboratoires Vygon, Ecouen, France; B. Braun Celsa, Chasseneuil, France; Soci~t6 Cordis, Valbonne, France; and Laboratoires Wuhrlin, Soplamed, Etablissements Vermed, Neuilly-en-Thelle, France. Presented in part at the Sixth European Congress of Microbial and Infectious Diseases, Seville, Spain, April 1992. Reprint requests: Michele Viot, Laboratoire de Microbiologie,Centre Antoine Lacassagne 36, voie Romaine, 06054 Nice, France. Copyright © 1996 by Mosby-Year Book, Inc. 0022-2143/96 $5.00 + 0 5/1/68839

~ ntravascular catheters, c o m m o n l y used in supportive care of patients with cancer, may be used for several weeks or even m o n t h s because they contribute to the patient's comfort, Prolonged catheterization constitutes a major risk for nosocomial infections, 1 which are responsible for the death of some patients. 2 C-NS are the most frequent organisms involved in catheter-related infections, 3'4 with Staphylococcus epiderrnidis accounting for approximately 60%. 4 Electron microscopic studies have shown that the surface of the catheter lumen is f r e q u e n t l y c o l o n i z e d by b i o f i l m - f o r m i n g b a c t e r i a (S. epiderrnidis being the m o s t c o m m o n o r g a n i s m 71

72

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Galliani et al.

isolated), despite no sign of infection. 5'6 The adhesion of the microorganism to the medical device constitutes a necessary and preliminary step for catheter-related infection. The colonization of the catheter proceeds either from the skin-catheter interface toward the external surface of the catheter 7 or from the hub toward the internal surface of the catheter, s,9 High quality and radio-opaque devices are being manufactured with new technologies that have improved the state of the catheter surface. Indeed, as suggested by Franson, 1° surface irregularities could facilitate bacterial colonization of the device. Recent studies with scanning electron microscopy 11'12 have shown cracks and bumps on the surfaces of catheters. According to the nature of the biomaterial and the manufacturer, the luminal surfaces of silicone and polyurethane catheters present either more or fewer hollows, bumps, and cracks. The surface of polyurethane catheters is generally smoother than the surface of silicone catheters. But exposure to drugs used for chemotherapeutic treatment of patients provoked more surface alterations: the particles of barium sulfate, included in the material to make it radio-opaque, were d e t a c h e d - resulting in the formation of additional hollows. Nevertheless, the opacified zone can be covered by an external and internal varnish by using a co-extrusion process. This treatment makes a catheter smoother and more resistant to the chemotherapeutic aggression. The first steps of bacterial attachment to polymer surfaces involve hydrophobic interactions, a3-15 and the adhesion of microorganisms may be different on a hydrophobic or hydrophilic substratum. 16'a7 Because of the role of the chemical nature of the device in bacterial adhesion, new biomaterials have been used in the manufacture of catheters (e.g., hydrophilic polyurethane or hydrophilic coatings). Polymers treated with antimicrobial agents are also of interest. Maki is has shown that a silver-impregnated cuff reduces the rate of catheter-related infections. The in vitro study of microbial adhesion on biomaterial must take into consideration the environmental factors regarding the patient catheter. The implanted device is rapidly covered by plasma proteins; fibrillar proteins such as fibronectin, fibrinogen, or laminin, along with other proteins, can modulate the interactions between the microorganism and the polymer substratum. Although fibrinogen and laminin 19 have been found to enhance C-NS adhesion, serum, 15'2° albumin, 15'19'21 fresh blood, plasma, and fibrin 2° were shown to inhibit adhesion,

and conflicting results have been reported with fibronectin. 1s'19'2°-22 The presence or the absence of bacterial receptors (adhesins) for fibrillar proteins can explain the stimulating or inhibiting effect of the latter on microbial adhesion. In a previous study concerning 20 clinical strains of S. epidermidis, we showed that the initial adherence to polystyrene surfaces depends mainly on strain hydrophobicity. Adhesion was usually impaired by plasma proteins but was enhanced for two strains, providing evidence for strain-to-strain heterogeneity. 23 Slime production was not detectable until after 7 hours of incubation (and did not occur in PBS), suggesting its later intervention in the proc e s s . 23

In the present study the initial adhesion of S.

epidermidis to various catheters made of hydrophilic and hydrophobic materials was investigated, as was the effect of plasma proteins adsorbed on the catheters and/or the effect of drugs applied under conditions simulating those of an oncostatic treatment. METHODS Bacterial strains. The four strains of S. epidermidis involved in this study were obtained at the Institut Jules Bordet, from blood cultures of patients with catheterrelated infection. They were identified by the API-StaphIdent system (Bio-M6rieux, Marcy l'Etoile, France) and the Coagulase-test (Wellcome, Dartford, England), with confirmation by a laboratory of reference (Statens Seruminstitut, Copenhagen, Denmark). These four strains were previously tested for hydrophobicity, slime production, and early adhesion to polystyrene microtiter plates with or without plasma proteins 23 (Table I). Hydrophobicity was measured according to the method of Rosenberg e t al., 24 which consists of measuring a partition coefficient in a xylene/water system by an optical density measurement. The production of slime was measured by the technique of Pfaller et al.25 that we have modified, as follows: bacteria adhered for 24 hours on microtiter plates, and the slime stained by safranin was detected by an optical density measurement. Bacterial adhesion was measured by an original radiolabeling method. 7 Radiolabeled bacteria adhered on plasma-pretreated and -untreated polystyrene microtiter plates during 15, 60, and 150 minutes, and adherent bacteria were lysed by lysostaphin. The strains, stored at -80 ° C as previously described,23 were grown on blood agar plates (Columbia; BioM6rieux) without prior thawing. Catheters. Sixteen catheters provided by five manufacturers were tested. Some of them were prototypes. Biomaterials were silicone (n = 6), polyurethane (n = 7), and hydrophilic polyurethane (n = 3). The manufacturers were as follows: Cordis Corp., Miami Lakes, Florida (n = 3); B. Braun Celsa, Chasseneuil, France (n = 3); Labora-

J L a b Clin M e d V o l u m e 127, N u m b e r 1

Table

I. C h a r a c t e r i s t i c s

Strain number

3 5 6 14

G a l l i a n i e t al.

of the four clinical

Hydrophobicily* (n = 9)

90 81 49 37

_+ 3 _+ 4 _+ 5 _+ 6

strains of

73

S. epidermidis

Slime productiont" (n = 10)

Adhesion on polystyrene:[; (n = 12)

25 1639 1868 53

1290 605 328 107

_+ 5 _+ 94 _+ 71 -+ 16

_+ 262 _+ 92 + 61 +_ 23

Influence of plasma proteins on adhesion~ (n = 12)

Inhibition ( - 9 7 % ) Inhibition ( - 9 6 % ) Inhibition ( - 9 5 % ) Stimulation (+516%)

Results are expressed as mean _+ SEM for (n) experiments. *Partition coefficient in a xylene biphasic system based on the method of Rosenberg et al.24 Results are expressed in percentage of adherence to xylene. tQuantitative staining method based on the method of Pfaller et al.25 and modified by stain extraction and spectrophotometric measurement. Results are expressed by the optical density value x 1000. :i:Radiolabeling method. 7 Results are expressed by the area-under-the-curve value calculated according to the kinetics from 15 to 150 minutes x 10,000. Values in parenthesesare percentages of inhibition of adhesion after a plasma precoating of polystyrene.

toires Wuhrlin, Soplamed, Etablissements Vermed, Neuilly-en-Thelle, France (n = 3); Socidt6 des Laboratoires Vygon, Ecouen, France (n = 6); and Ohmeda (Viggo), Windlesham, England (n = 1). Table II reports some characteristics of these catheters. Adhesion of bacteria was determined by using 1 cm long, longitudinally split segments of catheter to expose both external and internal surfaces to the bacteria. To reproduce in vitro the environmental conditions in which catheters are placed during patients' treatment, an experimental protocol was designed at the Centre Antoine Lacassagne, Nice, France. Because it produces the greatest alteration of the catheter surface, 12'26'27 the currently used FAC treatment was chosen as the chemotherapy regimen. The FAC treatment protocol used at the Centre Antoine Lacassagne for patients with cancer is performed according to the patient body surface administration of 5-fluorouracil, doxorubicin, and cyclophosphamide. 28 Between each drug administration, glucose solution is administered to the patient. This daily treatment is reproduced for 6 days, every 3 weeks for 6 months (total of 36 treatments). This treatment was simulated in vitro, under aseptic conditions at ambient temperature, by successive immersions of segments of catheters in the different drug solutions, according to standard doses calculated for an adult patient with a body surface of 1.60 m2: (1) immersion in 5-fluorouracil (total dose per treatment, 800 mg for 3 minutes), (2) immersion in doxorubicin (80 mg for 15 minutes), (3) immersion in cyclophosphamide (800 mg for 15 minutes). Segments of catheters were washed in glucose solution between exposure to each drug. The different catheter treatments applied for the bacterial adhesion study were as follows: (1) no treatment, (2) human plasma alone for 1 hour at 37° C, (3) BSA (reference: A-4503, 96% to 99% purity; Sigma, St. Louis, Mo.) alone at a concentration of 2 mg/ml for 1 hour at 37° C, (4) FAC alone, (5) FAC then plasma, (6) plasma then FAC. After each step, segments of catheter were washed 2 times in PBS to remove any free material. Before the treatment, each segment of catheter was

weighed, and the surface (mm 2) was calculated by using an appropriate formula that took into account the volume and density of each biomaterial. The state of the surface of catheters exposed to staphylococci was observed by scanning electron microscopy at the Centre Commun de Microscopie Electronique Appliqu6e, Facult6 des Sciences, Nice, France. Segments were fixed with 2% glutaraldehyde (in cacodylate buffer), dried by evaporation, and submitted, after silver lake sticking, to gold-palladium metallization. Radiolabeling of the bacteria. Bacterial strains were grown on blood agar plates (Columbia agar; BioM6rieux) overnight at 37 ° C. Several colonies were transferred into Mueller-Hinton Broth-II (BectonDickinson, Sparks, Md.), and the turbidity was adjusted to Mac Farland 0.5. After 2 hours of incubation at 37 ° C, they were radiolabeled with Me-3H-thymidine (85 Ci/mmol; Amersham International, Buckinghamshire, England) for 90 minutes at 37 ° C under constant agitation. Surplus tritiated thymidine was removed by centrifugation (3000 rpm, 10 minutes) and washing (three times) with PBS (Gibco, Paisley, Scotland), pH 7.2. The pellet resuspended with 1 ml of PBS was dispersed by sonic treatment for 1 minute at 26 watts (cell disrupter model W-225R; Heat Systems Ultrasonics; Farmingdale, N.Y.). The final suspension was adjusted spectrophotometrically (600 nm; UV/VIS; Perkin-Elmer Corp., Norwalk, Conn.) to between 5 × 107 and 5 × 108 CFU/ml. The control count was obtained from serial dilutions spread on Columbia agar plates. Radioactivity was measured by using 10 ml of scintillation fluid (U1tima Gold; Camberra Packard, Meriden, Conn.) and a [3-counter (LKB, Pharmacia, Uppsala, Sweden) and reported as CPM/ml of suspension. Efficiency of radiolabeling (CFU/CPM) was calculated with the number of CFU and the number of CPM in 1 ml of bacterial suspension. Adhesion a s s a y . Each segment of catheter was incubated with 2 ml of radiolabeled suspension for 3 hours at 37 ° C under gentle agitation. After removal of the suspension, the catheter was washed three times with PBS (pH 7.2) and transferred into a flask containing 1 ml of lyso-

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74

Table II. Catheters a n d descriptions Catheter code

Material

Silicone A B C E F G Polyurethane D H I d K L M Hydrophilic polyurethane N

P

Internal diameter (ram)

External diameter (ram)

10 to 20 11 to 15 11 to 15 13 13 20

1.4 to 1.8 1.24 to 1.28 1.2 to 1.39 1.26 1.32 1.28

0.5 0.9 1.0 1.0 1.0 1.2

1.0 2.0 2.3 2.0 2.8 2.0

Single Single Single Single Single Co-extruded/double varnish

Aliphatio polyurethane Aliphatic polyurethane Aromatic polyurethane

Single Single Co-extruded/ double varnish Co-extruded Co-extruded Co-extruded Single with silver coating

20 20 40

1.35 1.26 1.34

1.55 1.5 1.0

2.1 1.9 1.5

20 20 20 20

1.14 1.14 1.14 1.25

1.5 1.2 0.8 1.25

2.1 1.6 1.25 1.8

Co-extruded/double varnish with hydrophilic coating Single, structurally hydrophilic With hydrophilic coating

20

1.17

1.25

1.8

None

1.06 1.14

1.25 2.0

1.8 2.3

Aromatic polyurethane Aromatic polyurethane Aromatic polyurethane Aliphatic polyurethane

Aliphatic polyurethane

staphin 10 U/ml (ref. L7386; Sigma). Bacteria lysis, p e r f o r m e d for 1 hour at 37 ° C, 23'29 was used to release bacteria adhering to the catheter. Radioactivity was counted, after transfer of the lysate and the segment of catheter into 10 m l of scintillation fluid, with a [3-counter. Each measure was performed in triplicate. The results were expressed by the following formula: CPM/catheter × efficiency of labeling Adhesion -

Density (mg/mm 3)

Silicone platinum Silicone platinum Silicone platinum Silicone Silicone Silicone peroxide

Aliphatic polyurethane Polyurethane

O

Extrusion

Barium sulfate (% weight)

surface of the catheter

= bacteria/mm 2 of catheter Statistics. Fisher's exact test was computed with StatView 512 + software from Brain Power Inc. (1986 version).

RESULTS Because we consider that the four strains selected for this study are representative of the population of C-NS responsible for catheter-related infections, most of the results were expressed as a general mean obtained from the data of all tested strains. Bacterial adhesion on native catheters: Influence of

material and strain. Fig. 1 shows the results of bacte-

rial adhesion measured for each untreated catheter, expressed by the mean calculated with the 4 strains. Bacterial adhesion to untreated catheters was he-

mogeneous among the different catheters of each group, as follows: 7.6 × 103 to 34.5 × 103 bacteria/ mm 2 for silicone, 3.3 × 103 to 11.8 × 103 bacteria/ mm 2 for polyurethane, and 1.2 × 103 to 3.5 x 103 bacteria/mm 2 for hydrophilic polyurethane. Adhesion of S. epidermidis on native catheters differed according to the biomaterial and was significantly higher on silicone than on polyurethane and hydrophilic polyurethane catheters (p < 0.05). Statistical analysis concerning untreated catheters showed no significant difference between bacterial adhesion to aromatic (mean 5.9 _+ 0.8 × 103 bacteria/mm 2) and aliphatic (mean 7.1 + 0.8 × 103 bacteria/mm 2) polyurethane catheters. Likewise, no significant difference was observed between bacterial adhesion to structurally hydrophilic catheters (mean 1.2 _+ 0.1 × 103 bacteria/ram 2) and catheters with hydrophilic coating (mean 2.6 _+ 0.4 × 103 bacteria/ mm2). Bacterial adhesion was rather important on the silver-coated polyurethane catheter (code M; mean 8.7 × 103 bacteria/mm 2) as compared with the general polyurethane catheters. Bacterial adhesion on native silicone catheters was strain-dependent (Fig. 2). Strain 3, the most hydrophobic strain (90% adhesion to xylene), was the most adherent. Strains 5 and 6 (81% and 49%

J Lab Clin M e d V o l u m e 127, N u m b e r 1

Galliani e t al,

75

60 E 50-

40-

30-

20o 0

70 .<

10t {{ 0

I A

I B

I C

I E

I F

I G

I D

Silicone catheters

I H

I I

i J

I K

I L

I M

I N

Polyurethane catheters

I O

I P

Hydrophilic polyurethane catheters

C a t h e t e r code

Fig. 1. Adhesion of S. epidelT~nidis organisms to 16 native catheters: silicone (.4, B, C, E, F, G), polyurethane (D, H, I, J, K, L, M), or hydrophilic polyurethane (N, O, P). Results are expressed as a general mean, obtained from the data of all tested strains, in number of bacteria × 1000/mm 2 (mean _+ SEM, for a total of 48 measurements for each catheter).

5°iT l

E

40 4

/ I ~

[~

Sili . . . . . .

theters

[]

Polyurethane catheters

1

Hydr°philic polyurethane catheters

30-

la

T

20-

,0-I iiiiiiiii

iiiiiiiii!iii

iiiiiiiiiiill

r

ee_

-,e 3

5

6

14

Strain n u m b e r

Fig. 2. Adhesion of S. epidermidis organisms to different native catheters (silicone [n = 6], polyurethane In = 7], or hydrophilic polyurethane [n 3]). Results were obtained from each of the four strains and are expressed in number of bacteria × 1000/mm 2 (mean _+ SEM, for a total of 72 measurements for silicone, 84 for polyurethane, and 36 for hydrophilic polyurethane catheters).

a d h e s i o n to xylene, r e s p e c t i v e l y ) a d h e r e d to a l e s s e r e x t e n t o n n a t i v e s i l i c o n e c a t h e t e r s . S t r a i n 14 ( 3 7 % a d h e s i o n to x y l e n e ) a d h e r e d p o o r l y o n n a t i v e silicone catheters.

Effect of drugs on the state of the catheter surface.

E x a m p l e s o f g o o d q u a l i t y s i l i c o n e c a t h e t e r (/A) a n d p o l y u r e t h a n e c a t h e t e r w i t h a scaling s t r u c t u r e (IIA) a r e p r e s e n t e d in Fig. 3. T h e e x p o s u r e to F A C g r e a t l y

76

Galliani et al.

J Lab Clin Med January 1996

Fig. 3. Photomicrographs of native (A) or FAC treated (B) catheters after S. epidermidis adhesion (scanning electron microscopy). I, Silicone catheter; II, polyurethane catheter. Strain 3 adhering to the native silicone catheter (IA) and after FAC treatment, cracks on the surface, and lodging bacteria (IB) are shown. Strain 14 adhering to the native polyurethane catheter whose surface presented a scaling structure (IIA) and after FAC treatment, big scales (IIB) were observed.

altered the surface structure of the catheters. After the treatment, cracks appeared on the surface of the silicone catheter (IB) and big scales appeared on the surface of the polyurethane catheter (IIB). Nevertheless, bacteria adhered to both the smooth and altered parts (cracks and scales) of the surfaces of catheters. Bacterial adhesion on treated catheters: Influence of treatment and material. Mean bacterial adhesion to

catheters according to the kind of biomaterial and the type of treatment is presented in Fig. 4 (data obtained for the four bacterial strains). The presence of proteins on the surface of silicone and polyurethane catheters significantly reduced bacterial adhesion as compared with that measured on native devices (p < 0.001 for silicone; p < 0.01 for polyurethane). In contrast, bacteria adhered to a greater extent on hydrophilic polyurethane catheters when they were coated with proteins (p < 0.05), and adherent bacteria reached a level

similar to that observed with hydrophobic polyurethane catheters. After pretreatment of the devices with BSA, bacterial adhesion was greatly reduced on all kinds of biomaterials as compared with the adhesion measured on untreated devices and plasma-exposed catheters. Bacterial adhesion was lower on BSAcoated hydrophobic and hydrophilic polyurethane catheters as compared with plasma- exposed biomaterials (p < 0.001 for polyurethane; p < 0.01 for hydrophilic polyurethane). When catheters were treated by FAC, bacterial adhesion was decreased on silicone and polyurethane material (not significant), whereas it was increased on hydrophilic polyurethane catheters (p < 0.001) in comparison with bacterial adhesion to native catheters. F A C treatment increased bacterial adhesion to hydrophilic polyurethane catheters, and adherent bacteria reached a level similar to that observed with hydrophobic polyurethane catheters.

J Lab Clin M e d Galliani et al.

V o l u m e 127, N u m b e r 1

77

50

40-

,m

[]

Silicone catheters

[]

Polyurethane catheters

•

Hydrophilic polyurethane catheters

30

20

T m

10.< I

I

1

I

Untreated

Plasma

BSA

FAC

I

I

FAC+plasma Plasma+FAC

Catheter pretreatment

Fig. 4. Effect of catheter pretreatment on S. epidermidis adhesion. Silicone (n = 6), polyurethane (n = 7), or hydrophilic polyurethane (n = 3) catheters were pre-exposed to plasma, BSA, oncostatic chemotherapy (FAC), FAC plus plasma, or plasma plus FAC, before exposure to bacteria. The mean of bacterial adhesion for the four strains tested is expressed for each treatment and each kind of biomaterial by the number of adhering bacteria × 1000/mm 2 (mean _+ SEM, for a total of 288 experiments for silicone, 336 for polyurethane, and 144 for hydrophilic polyurethane catheters).

When the devices were exposed to FAC before being exposed to plasma proteins, bacterial adhesion was reduced on silicone catheters (p < 0.001), unchanged on polyurethane catheters, and increased on hydrophilic polyurethane catheters (p < 0.05) when compared with the adhesion measured on the same untreated biomaterials. The effect of proteins on bacterial adhesion previously observed was suppressed by a prior FAC treatment for hydrophobic catheters. When catheters were exposed to plasma before FAC, bacterial adhesion was reduced on all biomaterials (p < 0.001 for silicone and polyurethane;p < 0.05 for hydrophilic polyurethane) as compared with bacterial adhesion measured on native catheters. But when compared with adhesion to devices treated with plasma or BSA, only hydrophilic polyurethane catheters showed a reduced level of adherent bacteria. The sequential treatment (plasma plus FAC) on silicone catheters did not have the same favorable effect. The sheltering effect of proteins, which alone reduced bacterial adhesion, was suppressed when these catheters were exposed to a subsequent treatment with FAC. Bacterial adhesion on silicone and polyurethane devices treated by plasma and then by FAC was more reduced than bacterial adhesion on the same catheters treated with FAC alone. The data obtained from all treatments of cathe-

ters showed that bacterial adhesion was significantly higher (p < 0.001) on silicone than on polyurethane or hydrophilic polyurethane catheters, and it was significantly lower (p < 0.05) on hydrophilic polyurethane catheters than on ordinary polyurethane catheters. When all catheter treatments and all strains were considered together, the mean of bacterial adhesion showed no significant difference between aromatic (mean 4.3 + 0.3 × 103 bacteria/ mm 2) and aliphatic (mean 5.0 _+ 0.4 × 103 bacteria/ mm 2) polyurethane catheters. Likewise, statistical analysis concerning all treated catheters showed no significant difference between bacterial adhesion to structurally hydrophilic catheters (mean 2.9 +_ 0.3 × 103 bacteria/mm 2) and catheters with hydrophilic coating (mean 3.4 _+ 0.3 × 103 bacteria/mm2). DISCUSSION

The results presented in this study show that initial bacterial adhesion of S. epiderrnidis to native catheters was dependent on hydrophobicity of both the biomaterial and the strain. This is in agreement with the results of Pascual et al., 15 who showed in vitro that the most hydrophobic C-NS strains adhered to polytetrafluoroethylene catheters more than the slightly hydrophobic strains. As Kristinsson 3° demonstrated in a previous study, our results showed that S. epidermidis adhesion was lower on

78

Galliani et al.

hydrophilic catheters than on hydrophobic silicone or polyurethane catheters. In our experimental model, slime production was not involved in initial bacterial adhesion. According to Muller et al.,2° trypticase soy broth is the best test medium for distinguishing between adhesion of slime producers and that of nonproducers. In the present study, adhesion was measured in PBS, an unfavorable medium for slime production. We showed in a previous study that slime was produced only after 7 hours of incubation in trypticase soy broth. 23 The use of PBS and the short duration of incubation (3 hours) were the most appropriate conditions for studying the contribution of hydrophobicity to the first steps of catheter colonization. In vivo, other factors can interfere in the first steps of bacterial adhesion, including the presence of plasma proteins and physical or chemical interactions between the biomaterial and the drugs (chemotherapeutic treatment). Our previous study23 showed inhibition of bacterial adhesion of most C-NS strains by various plasma proteins, and chemotherapeutic treatment has been shown to impair the state of the internal surface of catheters. 11.12,27Is bacterial adhesion influenced by drug administration and/or by the presence of host proteins on the catheter? To answer this question, we considered together the results obtained for the four strains typical of the S. epiderrnidis species. Bacterial adhesion measured on silicone and polyurethane catheters was lower (-66.2%, p < 0.001, and -32.8%, p < 0.01, respectively) in the presence of plasma proteins than it was on untreated catheters, except for strain 14 (data not shown), which probably possesses receptors (adhesins) for fibrillar proteins. Concerning strains 3, 5, and 6, the affinity of the bacteria for the plasma proteins was lower than their affinity for the biomaterial. The hydrophobicity of the polymer was probably decreased in the presence of proteins such as fibrinogen, fibronectin, or albumin. Such a reduction in bacterial adhesion was not observed for surfaces with hydrophilic properties, on which bacterial adhesion was increased (+91.7%, p < 0.05). The negative effect of BSA on adhesion was obvious for all strains tested with silicone and polyurethane catheters. This is in agreement with previous studies, which showed the inhibitory effect of albumin on bacterial adhesion, 15'19'21 because of the hydrophilic properties of this protein. This is confirmed by the fact that bacterial adhesion is unchanged when this hydrophilic protein is adsorbed on already-hydrophilic polyurethane catheters. Holes, cracks, and scales were obviously produced

J Lab Clin Med January 1996

by FAC treatment (Fig. 3). Previous observations from Franson 1° when using scanning electron microscopy suggested that bacteria adhered preferentially on those surface irregularities. Our results show that this may be possible for hydrophilic polyurethanes on which bacterial adhesion was greater after a FAC treatment (+ 148.2%,p < 0.001). But in contrast, the alteration of the surface of hydrophobic silicone and polyurethane catheters by oncostatic treatment resulted in a decrease (respectively -17.4% and -19.8%, not significant) in bacterial adhesion (Fig. 4). The data obtained separately for each strain revealed an actual decrease in microbial adhesion on FAC-treated silicone catheters (the most hydrophobic material) for strains 3 and 5 (the most hydrophobic strains). Exposure to FAC alters the state of the catheter surface and also alters its properties--probably decreasing its hydrophobicity. The method described by Steinhauser, 3a which consists of measuring the contact angle formed between the material and one drop of an aqueous solution, would permit us to confirm our assumptions by a direct measurement of material hydrophobicity before and after the catheter treatment. In vivo, bacterial adhesion is probably influenced by both oncostatic chemotherapy and plasma proteins. In this study we tried two combinations, (1) exposure to FAC followed by plasma and (2) exposure to plasma followed by FAC. The effect of catheter exposure to FAC and plasma in combination was intermediate between the specific effects of the oncostatic treatment alone and the effect with proteins alone. The inhibitory effect of plasma proteins on bacterial adhesion to silicone catheters was lower when it was associated with FAC treatment. The increasing effect of FAC treatment on bacterial adhesion to hydrophilic surfaces did not occur when proteins were previously adsorbed on the catheters. The diverse treatments of the devices were aimed at reproducing in vitro the conditions encountered in the host. However, it remains difficult to know exactly what happens in the intravenous catheter through which various fluids are infused and blood is drawn. Protein coating and FAC treatment are probably closer to the real events happening in the implanted catheter. Protein coating in vivo principally occurs on the external surface of the device and intermittently on the intraluminal surface of the catheter when it is used to obtain blood samples. After protein coating on both external and internal surfaces of the catheter, the hydrophobicity of the polymer itself no longer seemed to have any importance regarding bacterial adhesion, but bacterial adhesion was greater on silicone than on polyurethane

J Lab Clin M e d V o l u m e 127, N u m b e r 1

and hydrophilic polyurethane catheters. In vivo, the intraluminal surface of the catheter covered by a protein coat is also exposed to oncostatic drugs. When oncostatic drugs are administered through protein-coated catheters, bacterial adhesion is lower on hydrophilic surfaces. Our study showed that under these two conditions (plasma alone and plasma followed by FAC), neither protein coating nor chemotherapeutic treatment increased S. epiderrnidis adhesion on hydrophobic catheters. Bacterial adhesion was increased on plasma-coated hydrophilic polyurethane catheters. It was reduced on hydrophilic polyurethane catheters that were exposed to plasma and then to FAC to the same extent as bacterial adhesion measured on the same untreated catheters. This result is of importance because it is probably close to the real events happening in vivo. Whatever the catheter treatment, hydrophilic catheters presented the best properties, with low adhesion of the microorganisms. The results of bacterial adhesion measured on untreated and treated catheters showed no significant difference between structurally hydrophitic catheters and catheters with hydrophilic coating. Likewise, no statistical difference was observed between aromatic and aliphatic polyurethane catheters. The silver-coated polyurethane catheter gave results similar to those obtained with ordinary polyurethane catheters, suggesting that initial bacterial adhesion was not prevented by silver. The negative effect of silver on bacterial growth and biofilm formation in situ may be detectable after a prolonged incubation time. In conclusion, we showed that initial bacterial adhesion to native catheters was strain- and polymer-dependent. When in vivo conditions were reproduced in vitro, neither the presence of proteins on the device nor the exposure of the protein-coated catheter to chemotherapeutic treatment increased S. epidermidis adhesion, except on hydrophilic surfaces coated with proteins alone. In both conditions, bacteria adhered more to silicone catheters than to polyurethane catheters, and hydrophilic polyurethane biomaterials presented the most favorable properties, with low initial bacterial adhesion. Other experiments confirmed that hydrophilic polyurethane remains the biomaterial of choice, even after a later bacterial colonization of the catheter in conditions favorable to slime production (unpublished data). The validity of this in vitro model for S. epiderrnidis adhesion needs to be confirmed by a prospective and comparative clinical trial.

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We thank C. Thibaudat, V. Duchateau, N. Palli, D. Daneau, and P. Grenier for technical assistance. REFERENCES

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