Journal of Antimicrobial Chemotherapy (1998) 42, 755760

JAC

Resistance of articial biolms of Pseudomonas aeruginosa to imipenem and tobramycin

L. Coquet, G.-A. Junter and T. Jouenne*
Laboratoire des Polymres, Biopolymres, Membranes, UMR 6522 du CNRS, 543 Chemin de la Bretque, BP 73, 76233 Bois Guillaume Cedex, France
Viable cells of Pseudomonas aeruginosa were entrapped in alginate gel layers and incubated in a minimal glucose (15 g/L)yeast extract (2 g/L)salt medium to form articial biolm-like structures. After cultivation for 2 days, the biomass distribution inside the polymer was highly heterogeneous. The cell number reached approximately 1011 cells/g gel in the outer regions of the gel structures whereas the inner areas were less colonized (c. 108 cells g/gel). Killing of immobilized organisms by imipenem and tobramycin were compared with free-cell experiments (inoculum c. 109 cells/mL). Sessile-like bacteria displayed a higher resistance to the two antibiotics used alone or in combination than did suspended cells. Exposure for 10 h to 20 MIC imipenem and 15 MIC tobramycin reduced the number of viable immobilized bacteria to 0.3% and 3%, respectively, of the initial cell population, whereas these antibiotic concentrations were much more efcient (bactericidal) against free-cell cultures (5 log kill in 6 h). A synergic effect of tobramycin and imipenem was detected on bacterial suspensions but not on biolm-like structures. Effective diffusivity measurements showed that the diffusion of imipenem in the alginate layer was not hindered. A slight but signicant enhancement of -lactamase induction in immobilized cells as compared with their suspended counterparts was insufcient to explain the high resistance of sessile-like bacteria.

Introduction
Pseudomonas aeruginosa is particularly pathogenic when associated with the nidus of an implanted medical device, and is often resistant to antimicrobials, especially those used to treat nosocomial infections.1 Chronic infection of the lower respiratory tract by alginate-overproducing P. aeruginosa is a distinctive feature of patients with inherited cystic brosis. 2 The most efcient antimicrobials against individual or planktonic (oating) bacteria, e.g. chlorine, do not eradicate the same species growing deep within a glycocalyx-encased biolm.3 The physical and biological mechanisms that lower the susceptibility of biolm micro-organisms to antibiotics have not been fully elucidated. Conicting results do not allow determination of the actual effect of exopolysaccharide produced by attached bacteria on drug penetration.4 Although extensively investigated, the inuence of natural5 or articial6,7 immobilization on microbial physiology is still controversial. Articial biolms are useful laboratory tools, serving as simplied models for more complex biolm phenomena,
*Corresponding author. Tel: 33-2-35-61-42-80; Fax:

to conduct fundamental biolm research under controlled and reproducible conditions.8,9 In a previous paper,10 we described a simple articial model structure of Escherichia coli biolms which consists of a plane agar gel layer entrapping viable cells, the polysaccharide matrix simulating microbial glycocalyx. We have shown that immobilized cells are more resistant to aminoglycoside and -lactam antibiotics than are free organisms.10,11 In the present study, we adapted this model to P. aeruginosa biolms, substituting alginate for agar to approach clinical reality more closely. The efcacy of imipenem and tobramycin, alone or in combination, against the biolm-like structures was evaluated. The induction of -lactamase by imipenem in free and immobilized bacteria was also investigated.

Materials and methods

Bacterial strain and inoculum preparation
Mucoid P. aeruginosa PAO was kindly provided by Dr Nicole Orange (Laboratoire de Microbiologie du Froid,
33-2-35-61-34-96; E-mail: thierry.jouenne@univ-rouen.fr

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1998 The British Society for Antimicrobial Chemotherapy

L. Coquet et al. Evreux, France). The organisms were collected from stock cultures on nutrient agar slants (Difco Laboratories, Detroit, MI, USA) and grown overnight at 37C in a minimal salt medium (MGM medium) which had the following composition per litre of distilled water: TrisNH2, 0.6 g; TrisHCl, 15 g; NH4Cl, 0.5 g; CaCl2, 0.05 g; MgS04, 7H2O, 0.05 g; FeSO4, 7H2O, 0.005 g; MnSO4, H2O, 0.005 g; glucose, 15 g; yeast extract (Difco), 2 g. After centrifugation of the bacterial suspension (1500g for 15 min), the pellet was washed and suspended in 1 mL of MGM. Populations of the inocula were estimated by optical density (OD) measurements at 546 nm referred to a calibration curve. water. They were placed in Petri dishes containing 10 mL of MGM medium supplied with a bactericidal concentration of antibiotic. The inhibitors were used alone (15 MIC, i.e. 75 mg/L tobramycin, and 20 MIC, i.e. 80 mg/L imipenem) or in combination (10 MIC, i.e. 40 mg/L imipenem, and 5 MIC, i.e. 25 mg/L tobramycin). Dishes were incubated for 10 h at 37C under soft agitation (20 oscillations/min). Every 2 h, one of the immobilizedcell structures was removed from its dish, and the gel disc was thoroughly rinsed with sterile distilled water and crushed in 10 mL of sterile phosphate buffer (0.1 M phosphate buffered saline from Sigma; pH 7) using a blender. Appropriate dilutions of the resulting mixture were plated out on MuellerHinton agar plates. Colonies were counted after incubation of the plates for 24 h at 37C. All counts were performed in triplicate. Killing assays were also performed on free-cell cultures (initial cell content c. 109 cfu/mL) in 50 mL Erlenmeyer asks containing 15 mL of MGM medium supplemented with the tested antibiotic or the antibiotic mixture. Suspended cultures were exposed to the same inhibitor concentrations as immobilized cells. Control growth curves in the absence of drugs were also obtained for immobilized and suspended cultures.

Antibiotics and MIC determinations

Imipenem was kindly provided by Merck Sharp & Dohme-Chibret (Paris, France). Tobramycin was purchased from Sigma-Aldrich (St Louis, MO, USA). MICs were determined by a standard broth microdilution technique using MuellerHinton medium (Difco) and inocula of 106 cfu/mL. The MIC was dened as the lowest antibiotic concentration inhibiting visible growth after incubation at 37C for 24 h. Values of 4.0 and 5.0 mg/L were obtained for imipenem and tobramycin, respectively.

Preparation of biolm-like structures

A 2% (w/v) alginate (Prolabo, Fontenay-sous-Bois, France) solution was inoculated with a known amount of P. aeruginosa cells. The polymerbacteria mixture was then poured into the cylindrical cavity of a mould obtained by superimposing two rigid plastic plates, the upper one having a cylindrical opening. The opening was then covered with a hydrophilic microporous membrane (RA mixed ester cellulose lter from Millipore, Saint-Quentin en Yvelynes, France; mean pore diameter, 1.2 m) that, in contact with the alginate solution, was held in place by another perforated plastic plate. This structure was immersed for 2 h in a 5.5 g/L CaCl2 solution. After polymerization, the gel was washed by immersion for 1 h in a 0.55 g/L CaCl2 solution. The alginate disc (thickness 3 mm; surface 10 cm2; cell content 106 cfu/g gel) was then removed from the mould, placed into a Petri dish, and covered with a perforated plastic plate equipped with a plastic grid. The structure was maintained on the bottom of the dish by a silicone grease that also ensured its watertightness. The plate was lled with 10 mL of MGM medium and incubated for 48 h at 37C under slight agitation (20 oscillations/min) in an air convection incubator. A series of six identical immobilized-cell discs was necessary for each killing assay.

Diffusion measurement
Diffusion measurement was performed as previously described.10

Protein extraction and -lactamase assays

A culture of P. aeruginosa (initial cell contents, 109 cfu/ mL) was performed at 37C in an Erlenmeyer ask containing 15 mL of MGM medium supplemented with imipenem (4 mg/L) as inducer. After incubation for 4 h, the organisms were harvested (10,000g for 15 min) and the pellet resuspended in 50 mL of a lysis buffer of the following composition: Tris, 50 mM; NaCl, 29 g/L; EDTA, 2.9 g/L; PMSF, 0.17 g/L; DTT, 0.15 g/L; glycerol, 50 g/L; Triton X-100, 10 g/L. Bacterial cells were disrupted by sonic treatment (20 W) at 4C using an ultrasonicator (at 5 s intervals for 8 min). The sonicated cells were sedimented by centrifugation (10,000g for 15 min) and the supernatant was used as the source of -lactamase. Protein contents were determined according to the method of Lowry et al.12 The same procedure was followed to extract proteins from immobilized cells. Once the immobilized-cell gel discs had been crushed as described above, gel particles were eliminated by ltration through a glass-bre membrane (GF/C from Whatman, Maidstone, UK) under vacuum, and bacterial cells present in the ltrate were collected by centrifugation (1500g for 15 min). -Lactamase activity was determined spectrophotometrically. The reaction mixture, whose temperature was

Susceptibility tests
At the end of the incubation period, the immobilized-cell structures were collected and rinsed with sterile distilled

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Resistance of sessile-like P. aeruginosa cells to antibiotics maintained at 30C, consisted of 3 mL of phosphate buffer (pH 7.0) containing 0.36 g/L penicillin-G (Sigma). The decrease in OD at 240 nm was recorded for 15 min after the addition of 60 L of crude -lactamase solution (protein extract). The drop in OD value was related to the number of enzymatic units using a calibration curve obtained with a commercial penicillinase (Sigma). One unit of penicillinase activity (UE) was dened as the amount of enzyme that hydrolyses 1 M of penicillin-G in 1 min at 25C and pH 7. mycin induced a decrease limited to 3 log units in the exposed cell population. The exposure for 10 h to 25 mg/L tobramycin caused no killing of immobilized cells whereas the killing effect of 40 mg/L imipenem on sessile-like organisms was similar (P 0.02) to that observed at a concentration of 75 mg/L (data not shown). No signicant synergic effect of the antibiotic association was observed against articial biolm structures (Figure 2b): the time kill curve obtained with the combination was similar (P 0.02) to that observed with 75 mg/L imipenem (Figure 1b). Studies of the biomass distribution inside the gel discs after exposure to the antibiotics highlighted the formation of cell-killing gradients in the articial biolms (not shown). A decrease of 97% in the number of viable P. aeruginosa cells located in the outer region (250 m thick) of the gel disc could be observed after exposure to 80 mg/L imipenem or 75 mg/L tobramycin. The number of viable organisms in this outer gel region was reduced to 0.01% of the initial cell number after exposure to the antibiotic combination. No killing effect was detected in the inner areas of the polymer matrix, whatever the treatment.

Data analysis
All experiments were performed at least in triplicate. Results were expressed as mean standard deviation (SD). Calculations were performed using a scientic graphic software package (Sigmaplot from Jandel Scientic, Corte Madera, CA, USA). Results were analysed using Students t test. For signicant differences, P 0.05.

Results
Formation of biolm-like structures
We rst investigated the spatiotemporal evolution of the biomass inside the alginate layer during the 48 h preincubation period in MGM medium (not shown). The slight heterogeneity of the biomass distribution in the gel observed after incubation for 24 h was highly amplied by extending the incubation period to 48 h. At this time, the cell concentration reached 1011 cfu/g gel in the outer region of the gel disc whereas the inner areas were noticeably less colonized (c. 108 cfu/g gel). Microscopic examinations highlighted the presence of underlying bacterial microcolonies.

Susceptibility tests
Timekill curves (Figure 1) highlighted the resistance to imipenem and tobramycin of entrapped cells as compared with planktonic bacteria. The exposure of alginate discs for 10 h to tobramycin 75 mg/L reduced the number of viable immobilized bacteria to 3% of the initial cell population. This percentage was lowered to 0.3% after the exposure of immobilized cells for 10 h to imipenem 80 mg/L. The two antibiotics at the tested concentrations were much more efcient (bactericidal) against free-cell cultures (5 log kill in 6 h). Tobramycin and imipenem exerted a synergic effect against free-cell cultures. Used together at the concentrations of 40 mg/L imipenem (i.e. 10 MIC) and 25 mg/L tobramycin (i.e. 5 MIC), they displayed a bactericidal effect (7 log kill after exposure for 10 h) (Figure 2a), whereas imipenem alone was bacteriostatic and tobra-

Figure 1. Susceptibility of (a) planktonic and (b) immobilized P. aeruginosa cells to ( ) 80 mg/L imipenem; ( ) 75 mg/L tobramycin; ( ) control without antibiotic. Bars: SD.

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L. Coquet et al. Table. Comparison of -lactamase induction by imipenem in free-cell cultures and in articial biolmsa Controlb Planktonic cells Immobilized cells 3.0 2.7 0.8 0.5 Imipenem at MIC 3.7 6.1 0.2 1.1

a Results are expressed in UE/mg proteins, where UE is the amount of enzyme that hydrolysed 1 mol of penicillin-G in 1 min at 25C and pH 7. b Without imipenem.

Figure 2. Effect of the association of imipenem and tobramycin against (a) planktonic and (b) sessile-like P. aeruginosa cells. ( ) 40 mg/L imipenem; ( ) 25 mg/L tobramycin; ( ) 40 mg/L imipenem and 25 mg/L tobramycin; ( ) control without antibiotic. Bars: SD.

Diffusivity measurements
The effective diffusivity (De) of imipenem in the cell-free gel layer, i.e. (2.9 1.0) 105 cm2/s, did not signicantly differ from the De value in water ((1.05 0.02) 105 2 cm /s).

-Lactamase assays
No signicant (P 0.2) -lactamase induction by imipenem was observed in free-cell conditions (Table). However, articial immobilization in alginate induced a signicant increase in the -lactamase activity recovered from P. aeruginosa cells.

Discussion
The distribution of P. aeruginosa cells in alginate gel discs after pre-incubation for 48 h, similar to that previously reported for agar-entrapped E. coli cells,10 conrms that our model structure of biolms applies to the P. aeruginosaalginate association. In fact, this cell

distribution is consistent with the physical characteristics of natural biolms. The microcolony is the basic growth unit of biolms and some authors consider bacterial growth to be sessile in nature if microcolonies are produced.13 Moreover, biolms usually display the highest cell concentration close to the solidliquid interface.14,15 Imipenem presents a broad activity spectrum against Gram-positive and Gram-negative bacteria, including P. aeruginosa.16 This carbapenem has been described as an antibiotic that shows substantial bactericidal activity against slow-growing bacteria17 and is effective against some E. coli biolms.18 Tobramycin as an aerosol has proved efcient in the treatment of lung infections.19 Nevertheless, these two antibiotics, at high concentration, were inefcient against biolm-like organisms, which corroborates published data on the enhanced resistance of natural20,21 or articial8,9 P. aeruginosa biolms to antibacterial agents. The synergic effect (i.e. an enhanced killing effect higher than 2 log units22) of tobramycin and imipenem against free-cell cultures is in agreement with results obtained by Giamarellos-Bourboulis et al.22 on the association of carbapenems with amikacin. Contradictory observations have been reported by Saiman et al.,19 however, who showed that antibiotic combinations including tobramycin were usually very active against P. aeruginosa, except those regimens that contained imipenem. Although inefcient against alginate-entrapped organisms in terms of overall cell counts (i.e. including the whole gel structure), the two antibiotics, alone or mixed, displayed a killing effect on bacteria close to the gelliquid interface. Such spatially non-uniform efcacy of antimicrobials against sessile organisms has been observed by others. Using an epiuorescence micrography technique, Huang et al.,23 for instance, demonstrated the formation of gradients of respiratory activity in biolms exposed to chloramine. In the same way, Korber et al.,24 using uorescent molecular probes and scanning confocal laser microscopy, described gradients in nucleic acid staining and cell elongation in biolms of Pseudomonas uorescens treated with eroxacin.

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Resistance of sessile-like P. aeruginosa cells to antibiotics Among the different hypotheses suggested to explain the low and heterogeneous efcacy of antimicrobial agents against sessile bacteria, a direct role for the glycocalyx as a diffusion barrier has been advanced.9 Here, no restricted diffusion of imipenem in cell-free alginate was observed, the obtained De value being consistent with published data on other -lactam antibiotics.10,25 The high brittleness of cell-loaded alginate discs after incubation for 2 days in MGM medium did not permit diffusivity measurements in the biolm-like structures. However, we have shown previously that the diffusion of small molecules (e.g. antibiotics) through agar gels is not affected by the presence of gel-entrapped organisms.10 In contrast with imipenem, published data on tobramycin diffusivity in 2% (w/v) alginate give a De value nine times lower than in water.4,26 It seems improbable that the possibly restricted diffusion of tobramycin in alginate discs might explain the aminoglycoside inefcacy against immobilized cells, as the duration of exposure (10 h) was sufcient to balance the slightly reduced penetration rate of the antibiotic in the gel layer. Nichols et al.27 have shown that the restricted diffusion of tobramycin through binding to alginate is not a major mechanism of biolm resistance to this antibiotic. Resistance of biolms to aminoglycosides more probably arises from a low uptake of the antibiotics by oxygendeprived organisms.11 Another suggestion attributes the enhanced resistance of biolms to antimicrobial agents to physiological differences between xed and suspended organisms28bearing in mind that the physiology of sessile bacteria probably depends on cell location within the biolm. Therefore, the production yields of many biological compounds from bacteria grown on surfaces are higher than those obtained using free cultures.28 Here, alginate-entrapped P. aeruginosa cells displayed enhanced -lactamase synthesis in the presence of imipenem as compared with their suspended counterparts. Yang & Livermore29 reported that exposure to imipenemwhich they showed to be a strong inducer of class I -lactamasestriggered the production of -lactamase in biolm cells of P. aeruginosa. In contrast to our results, however, they found that the highest median level of -lactamase activity achieved was lower in biolms than in induced planktonic bacteria. Nevertheless, the increase in -lactamase activity of sessile-like organisms in the present studyvery limited though signicantseems insufcient to explain their high resistance to imipenem. Most antibiotics have to enter the target organism by crossing the cell membrane(s) before exerting their lethal effect. The penetration of antibiotics into Gram-negative bacteria is a particularly complex phenomenon, since these bacteria possess double membrane layers. A number of experimental data indicate that small hydrophilic molecules, including -lactam antibiotics, may enter E. coli cells through non-specic diffusion channels formed by outer membrane proteins, i.e. porins.30,31 The outer membrane of P. aeruginosa is less permeable to hydrophilic solutes than that of E. coli,32 which may explain the broad antibiotic resistance of P. aeruginosa. Experimental results strongly indicate that most P. aeruginosa porins play a trivial role, if any, in antibiotic diffusion.33 The only exception is the OprD2 porin channel, which has been shown to be the pathway of imipenem diffusion.33 In a previous paper,7 we showed that the porin protein OmpF was underexpressed in immobilized E. coli cells that displayed enhanced resistance to latamoxef. This underexpression of OmpF may be due to change in the environmental conditions (osmolarity, for instance) inside the polymer matrix.28 Do the environmental conditions prevailing in the alginate matrix modify OprD2 functioning (e.g. expression level, conformation) in immobilized bacteria? Yoshihara & Nakae have shown, for instance, that Ca 2+ ions, implied in alginate gelation, activate the OprD2 channel by causing conformational change of the protein.34 Current investigations are devoted to answering these questions of fundamental interest.

References
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J. & Livermore, D. M. (1989). Interactions of meropenem with class I chromosomal -lactamases. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 20717. 30. Jaff, A., Chabbert, Y. A. & Semonin, O. (1982). Role of porin proteins OmpF and OmpC in the permeation of -lactams. Antimicrobial Agents and Chemotherapy 22, 9428. 31. Yoshimura, F. & Nikaido, H. (1985). Diffusion of -lactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrobial Agents and Chemotherapy 27, 8492. 32. Yoshimura, F. & Nikaido, H. (1982). Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. Journal of Bacteriology 152, 63642. 33. Nakae, T. (1995). Role of membrane permeability in determining antibiotic resistance in Pseudomonas aeruginosa. Microbiology and Immunology 39, 2219. 34. Yoshihara, E. & Nakae, T. (1993). Calcium ion-mediated opening of the channel gate in the Pseudomonas aeruginosa porin. Biochemical and Biophysical Research Communications 194, 14605. Received 23 March 1998; returned 7 May 1998; revised 29 May 1998; accepted 1 July 1998

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