ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1995, p. 1038–1044 Vol. 39, No. 50066-4804/95/$04.0010Copyright q 1995, American Society for Microbiology

Combination Effect of Fosfomycin and Ofloxacin againstPseudomonas aeruginosa Growing in a BiofilmHIROMI KUMON,1* NORIAKI ONO,1 MAIKO IIDA,2 AND J. CURTIS NICKEL3

Department of Urology, Okayama University Medical School, Okayama 700,1 and Pharmaceutical Research Center,Meiji Seika, Yokohama 222,2 Japan, and Department of Urology, Queen’s University,

Kingston, Ontario K7L 2V7, Canada3

Received 6 December 1994/Returned for modification 6 February 1995/Accepted 23 February 1995

We examined the combined effect of fosfomycin and ofloxacin againstPseudomonas aeruginosa in biofilms by usingan in vitro experimental system with a modified Robbins device. Sessile cells in a mature or immature biofilm,developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterialeffects. A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drugindependently produced no detectable decrease in the bioactivity of sessile cells were used. Exposure of the cells ina mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500mg/ml)and ofloxacin at three or 10 times the MIC (18.75 or 62.5 mg/ml) resulted in reduction of the bioactivity to 1.5 to4.5% after 72 h. Young sessile cells in an immature biofilm were more susceptible to this combination therapy. Witha combination of fosfomycin at three times theMIC and ofloxacin at three times theMIC, complete eradication wasconfirmed by both ATP assay and scanning electron microscopy.

Pseudomonas aeruginosa is a problematic organism in acuteand chronic human infections, including urinary tract infec-tions (14). In chronic infections, the cells are protected by anextracellular polymeric substance (glycocalyx) and exhibit re-sistance to most antibiotics at high doses (4, 11, 17). In our invitro studies with a modified Robbins device, only fluoroquino-lones showed a considerable killing effect on P. aeruginosa inbiofilms (15). However, the effect of quinolones is limited andthere is no available single drug which is sufficiently activeagainst the cells in a biofilm to be of clinical significance.In the present study, we examined the combination of fos-

fomycin with ofloxacin against P. aeruginosa in biofilms. It isbelieved that the barrier to drug penetration formed by theexopolysaccharide and the low growth rate of bacteria in bio-films confer relative drug resistance (11, 15). Fluoroquinolonesare drugs which penetrate exopolysaccharide and attack slowlygrowing cells relatively well (11, 15, 17). Fosfomycin [(2R-cis)-(3-methyloxiranyl)-phosphonic acid] is a unique antibioticwhich is chemically unrelated to any other known antimicrobialagent. Fosfomycin is actively taken into bacterial cells throughtheir two nutrient transport systems (12) and inhibits the initialstep in cell wall synthesis by inhibiting phosphoenolpyruvatesynthesis (10, 12). This characteristic entry and action of fos-fomycin were not necessarily affected by the growth rate (9),and the penetration of fosfomycin was not inhibited by Pseudo-monas exopolysaccharides in the penetration assay using asandwich cup method (17). Therefore, we selected fosfomycinfor use in combination with a fluoroquinolone for eliminationof P. aeruginosa in biofilms.

MATERIALS AND METHODS

Bacteria and culture conditions. P. aeruginosaOP14-210 (15, 24) isolated froma patient with a catheter-associated urinary tract infection was used throughoutthis study. The culture conditions used to produce an adherent biofilm wereessentially identical to those reported previously by Nickel et al. (23). Artificialurine (19) supplemented with 0.4% nutrient broth, containing logarithmic-phase

cells, was pumped from a reservoir through a modified Robbins device (15, 23)by a peristaltic pump set to deliver 40 ml/h. Disks cut from a silicon plate (1 mmthick; Create Medic Co., Ltd., Yokohama, Japan) were attached to samplingplugs in the device, and these disks were aseptically removed from the system foranalysis.Antimicrobial agents and determination of MICs. Ofloxacin (Daiichi Phar-

maceutical Co., Ltd., Tokyo, Japan), fosfomycin (Meiji Seika Kaisha, Tokyo,Japan), gentamicin (Sigma Chemical Co.), and piperacillin (Toyama ChemicalCo., Ltd., Tokyo, Japan) were tested. In the present study, artificial urine sup-plemented with 0.4% nutrient broth was used in the determination of the MICsof these agents. The MIC was determined by the conventional tube dilutionmethod with a final inoculum of 5 3 105 CFU/ml. The MICs for P. aeruginosaOP14-210 measured in artificial urine were 6.25 mg/ml for ofloxacin, 50.0 mg/mlfor fosfomycin, 6.25 mg/ml for gentamicin, and 12.5 mg/ml for piperacillin. TheseMICs were about two to four times higher than those determined with Mueller-Hinton broth. The chemical stability of ofloxacin, fosfomycin, gentamicin, andpiperacillin in the artificial urine used was examined under several conditions,and the antipseudomonal activities of the antibiotic solutions employed in theexperiment showed no significant changes for at least 72 h.Exposure of sessile cells to antibacterial agents. (i) Experiments with sessile

cells in a mature biofilm. After 8 h of contact with artificial urine containinglogarithmic-phase P. aeruginosa, a thick biofilm consistently developed on eachsilicon disk in the device (15, 23, 24). At time zero, a further influx of cells wasstopped after the 8-h period of contact with cells and flow of antibiotic-contain-ing medium was initiated. Artificial urine containing ofloxacin and/or fosfomycinflowed through the modified Robbins device during the treatment period. Treat-ment was carried out with ofloxacin at concentrations of one, three, 10, 20, 50,and 80 times the MIC and with fosfomycin at concentrations of one-half of theMIC and one, three, five, and 10 times the MIC. Disks were removed from thedevice at 8, 24, and 48 h, and the sessile bacteria on the disks were examined byATP assay (15, 24) and scanning electron microscopy (SEM). Treatment was alsoconducted with fosfomycin at 1/32, one-eighth, and one-half of the MIC and one,three, and 10 times the MIC combined with ofloxacin at three and 10 times theMIC, and the cells were examined at 48 and 72 h.(ii) Experiments with sessile cells in an immature biofilm. After 3 h of contact

with the artificial urine containing P. aeruginosa, microcolonies developed atscattered sites on each silicon disk in the device (15, 23). At time zero, treatmentwas started with ofloxacin at a concentration of three times the MIC, fosfomycinat three times the MIC, ofloxacin at three times the MIC plus fosfomycin at threetimes the MIC, gentamicin at 400 mg/ml (64 times the MIC), or piperacillin at400 mg/ml (32 times the MIC). Disks were removed for ATP assay and SEM at5, 12, 48, and 72 h after the treatment.ATP assay. Instead of viable counts, an ATP bioluminescence assay was used

to evaluate the biological activity of sessile cells on each disk in the modifiedRobbins device as described previously (15, 24). After being washed with distilledwater, the sessile cells on each disk were boiled for 5 min with 500 ml of distilledwater in a glass tube. After low-output ultrasonication, the ATP-extracted solu-tion was stored at 2708C until measurement. For the assay, 10 ml of luciferase at100 mg/ml (Sigma Chemical Co.) and 100 ml of each ATP-extracted solution weremixed in a final 800 ml of 50 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-

* Corresponding author. Mailing address: Department of Urology,Okayama University Medical School, 2-5-1, Shikata-cho, Okayama700, Japan. Phone: 81-86-223-7151. Fax: 81-86-231-3986.

1038

ethanesulfonic acid) buffer (pH 7.75) with 5 mM MgSO4. After the addition of200 ml of 0.125 mM D-luciferin (Sigma Chemical Co.), the emitted light wasmeasured with an LB9505AT Automatic Luminescence Analyzer (Berthold).The area under the curve of the light emission between 12 and 60 s was employedas a reliable parameter in measuring the ATP concentration in each sample.All assays were performed in triplicate with three disks, and the mean values

were calculated. The bioactivity (ATP content) of all sessile cells on a single diskafter 8 h of contact with P. aeruginosa in this system was defined as 1.0, and theresults at each point were expressed as the mean value relative to this 1.0.SEM. The cells on each disk were fixed with 2.5% glutaraldehyde in phos-

phate-buffered saline (0.2 M, pH 7.4), postfixed with 2% tannic acid and 1%OsO4 (16), and dehydrated in an ethanol series. The specimen was then dried ina critical-point dryer, coated with platinum-palladium, and observed with aHitachi S-570 scanning electron microscope.

RESULTSExperiments with sessile cells in a mature biofilm. After 8 h

of contact with artificial urine containing logarithmic-phase P.aeruginosa, a thick biofilm developed on each silicon disk. Thesessile cells at time zero possessed 3.30 3 10210 mol of ATP(3.30 3 10210 6 0.28 3 10210; n 5 30), and this value isexpressed as 1.0 in the graphs shown here for convenience. Inthe control study, bioactivities assessed by the ATP assay re-mained almost constant throughout the experiments, althoughthey slightly increased after the medium was exchanged forfresh artificial urine at time zero (Fig. 1).Ofloxacin showed bactericidal effects on the sessile cells in a

dose-dependent fashion, and ofloxacin at concentrations above20 times the MIC produced an obvious decrease in bioactivity(Fig. 1). Although ofloxacin at concentrations ranging from 1to 10 times the MIC produced no detectable decreases in thetotal bioactivity of sessile cells on each disk, morphologicalchanges similar to those obtained with concentrations equal tomore than 20 times the MIC were observed even with a con-centration equal to the MIC or three times the MIC (see Fig.4B). Ofloxacin at a concentration of 80 times the MIC (500mg/ml) did not completely eradicate the viable biofilm; a total

of about 3.5% of the bioactivity remained 48 h after the treat-ment, and thick biofilms with numbers of elongated cells andnumerous amorphous material were still shown to be presentby SEM. On the other hand, fosfomycin produced no detect-

FIG. 1. Kinetics of killing of sessile cells of P. aeruginosa OP14-210 in amature biofilm by ofloxacin at various concentrations. Bioactivity is expressed asthe relative ATP content of cells as described in Materials and Methods. Sym-bols: v——v, MIC (6.25 mg/ml); E, three times the MIC; å, 10 times the MIC;Ç, 20 times the MIC; ■, 50 times the MIC; h, 80 times the MIC; v v,control.

FIG. 2. Kinetics of killing of sessile cells of P. aeruginosa OP14-210 in amature biofilm by fosfomycin at various concentrations. Bioactivity is expressedas the relative ATP content of cells as described in Materials and Methods.Symbols: F, one-half of the MIC (25.0 mg/ml); E, MIC; å, three times the MIC;Ç, five times the MIC; ■, 10 times the MIC.

FIG. 3. Kinetics of killing of sessile cells of P. aeruginosa OP14-210 in amature biofilm by a combination of fosfomycin at various concentrations com-bined with ofloxacin at three times the MIC. Bioactivity is expressed as therelative ATP content of cells as described in Materials and Methods. Symbols:v——v, fosfomycin at 1/32 of the MIC (1.56 mg/ml); E, one-eighth of the MIC;å, one-half of the MIC; Ç, MIC; ■, three times the MIC; h, 10 times the MIC;v----v, ofloxacin only at three times the MIC; v v, control.

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FIG. 4. Morphological changes in sessile cells in a mature biofilm 72 h after treatment with a combination of fosfomycin and ofloxacin. Panels: A, control withoutdrugs; B, control with ofloxacin only at three times the MIC; C, control with fosfomycin only at three times the MIC; D, fosfomycin at three times the MIC plus ofloxacinat three times the MIC.

1040 KUMON ET AL. ANTIMICROB. AGENTS CHEMOTHER.

able decreases in the bioactivity of sessile cells, even at 10 timesthe MIC (500 mg/ml), as illustrated in Fig. 2. However, ultra-structural changes produced by fosfomycin, which were mod-erate and not drastic, were detected at concentrations of morethan three times the MIC (see Fig. 4C).A synergistic effect of ofloxacin and fosfomycin was clearly

detected when concentrations at which each drug indepen-dently produced no detectable decrease in bioactivity wereused. As demonstrated in Fig. 3, when ofloxacin was used at aconcentration of three times the MIC and fosfomycin was usedat concentrations above one-eighth of the MIC (6.25 mg/ml),they showed a significant enhancing effect in decreasing thebioactivity. Morphologically, the synergistic effect was also ev-ident (Fig. 4). The number of elongated cells covered withamorphous materials, which were dominant in sessile cellstreated with ofloxacin only (Fig. 4B), was decreased and thenumber of bloated and/or rounded cells, which were charac-teristic for cells treated with fosfomycin at concentrations ofmore than three times the MIC (Fig. 4C), was increased in cellstreated with the combination (Fig. 4D). At the same time,however, a total of 1.5 to 4.5% of the bioactivity of the sessilecells and numbers of morphologically intact and active cellsstill remained 72 h after the combination treatment.The synergistic effect of fosfomycin at each concentration

combined with ofloxacin at a concentration equal to 10 timesthe MIC was almost identical to that observed when it wascombined with ofloxacin at three times the MIC (Fig. 3). Evenwhen fosfomycin and ofloxacin were combined and both werepresent at 10 times the MIC, the antibacterial effects did notnecessarily increase compared with those of the other effectivecombinations studied, and a total of about 3% of the bioactiv-ity of the cells remained 72 h after the treatment.Experiments with sessile cells in an immature biofilm. After

3 h of contact with artificial urine containing logarithmic-phaseP. aeruginosa cells, microcolonies developed at scattered siteson each disk (Fig. 5). Although the bioactivity of sessile cells attime zero showed a slight variation (Fig. 6), the ATP contentsafter an additional 5 h (8 h after the initial contact) in thecontrol were similar to those obtained in the above-describedexperiments (Fig. 1 to 3). Against sessile cells in an immaturebiofilm, ofloxacin at three times the MIC, fosfomycin at threetimes the MIC, and piperacillin at 32 times the MIC did notshow marked killing effects and changes in bioactivity with timewere identical to those in the control (Fig. 6). On the otherhand, gentamicin at 64 times the MIC decreased bioactivityand prevented formation of a mature biofilm until 48 h (Fig.7A). However, the subsequent growth was rapid enough toform a complete biofilm within the next 24 h. Interestingly,morphological changes detected in sessile cells 72 h after treat-ment with gentamicin were minimal (Fig. 8A). The morphol-ogy of a gentamicin-treated biofilm was almost identical to thatof a control biofilm, although that of an ofloxacin-, fosfomy-cin-, or piperacillin-treated biofilm showed drug-specificchanges (Fig. 8B, C, and D).On the other hand, a synergistic effect of ofloxacin and

fosfomycin against the sessile cells in this immature biofilm wasevident. Bioactivity decreased completely within 48 h (Fig. 6),and complete destruction of the sessile cells was also detectedby SEM (Fig. 7B). There was no evidence of regrowth in thefollowing 24 h (Fig. 6).

DISCUSSION

Recently, bacterial biofilms have been detected on a numberof living and inert surfaces within the human body (8, 13, 15,21). Establishment of a biofilm is the prelude to the develop-

ment of various chronic, intractable infections, such as bioma-terial associated infection and pulmonary infection in patientswith cystic fibrosis (8, 13). P. aeruginosa is a major pathogen inbiofilm-associated infections, and eradication of the organismin a biofilm with antibiotics is not achievable (1, 11, 15). Thebiofilm mode of growth protects the sessile bacteria in vivo orin vitro from concentrations of antibiotics which swiftly killtheir planktonic counterparts (11, 15, 23). In 1985, Nickel et al.(23) demonstrated that the P. aeruginosa alginate contributeddirectly to the tobramycin resistance of sessile cells in an invitro experimental model using a modified Robbins device.Although antibiotic resistance of the cells in a biofilm is alsodue to their reduced growth rate (5, 11), our subsequent resultsobtained with the same in vitro model demonstrated that onlyfluoroquinolones showed a considerable killing effect on thesessile P. aeruginosa in a mature biofilm (15). However, theeffects of quinolones against sessile cells were also limited asdemonstrated in the present study (Fig. 1 and 5).We used sessile cells at two different stages for assessment of

synergistic effects of fosfomycin and ofloxacin, since the degreeof antibiotic resistance of sessile cells in biofilms depends quitestrongly on their maturation or aging (2, 4, 11). Instead ofviable counts, an ATP bioluminescence assay was used to eval-uate the biological activity of sessile cells in biofilms (15, 24),since conventional viable cell counts were made unreliable andtroublesome by the intimate cell-to-cell connections which oc-cur with this mode of growth. The ATP assay could be easilyapplied to estimate the effects of antimicrobial agents on bio-film bacteria, although the ATP content expresses total bioac-tivity rather than the total number of sessile cells on each disk.In the present study, we set the total bioactivity of the cellsadherent to a silicon disk after contact for 8 h at 1.0 for

FIG. 5. SEM of sessile cells in an immature biofilm developed 3 h aftercontact with artificial urine containing bacteria.

VOL. 39, 1995 ERADICATION OF P. AERUGINOSA IN A BIOFILM 1041

convenience. This value corresponded to 3.30 3 10210 mol ofATP and also to 1.49 3 108 CFU of planktonic counterparts atthe stationary phase in artificial urine.A clear synergistic effect on sessile cells at two different

stages was obtained with the combination of fosfomycin andofloxacin. Against sessile cells in a mature biofilm, significantenhancement of bactericidal activity was detected when fosfo-mycin at concentrations of one-eighth of the MIC to 10 timesthe MIC was combined with ofloxacin at a concentration ofthree or 10 times MIC. However, a total of 1.5 to 4.5% ofbioactivity of the sessile cells persisted 72 h after the treatment,and SEM also demonstrated a considerable number of mor-phologically intact cells. On the other hand, the combination offosfomycin at three times the MIC and ofloxacin at three timesthe MIC showed complete eradication of young sessile cells inan immature biofilm, which was confirmed by both ATP assayand SEM (Fig. 6 and 7B). Subsequent regrowth after 72 h oftreatment was not detected (data not shown). These resultsindicate that eradication of a biofilm-associated infection isbest carried out as early as possible, even if an effective che-motherapy such as the present combination is employed. Adelay in implementing antibiotic therapy may result in failureof the therapy.It was very interesting that a high concentration of gentami-

cin (400 mg/ml, 64 times the MIC) could neither eliminateyoung sessile cells completely (Fig. 7A) nor prevent them fromforming a mature biofilm (Fig. 8A). In general, aminoglyco-sides are regarded as the most potent antibiotic againstpseudomonal infections and are used frequently in clinicalpractice. Anwar et al. (2) reported that young sessile bacteriawere more resistant than planktonic cells to tobramycin butthey could still be eradicated if higher concentrations of tobra-mycin were used. This was also true in the present study withgentamicin until 48 h after treatment. However, the subse-

FIG. 6. Kinetics of killing of sessile cells of P. aeruginosa OP14-210 in animmature biofilm. Symbols: v——v, ofloxacin at three times the MIC (18.75mg/ml); E, fosfomycin at three times the MIC (150 mg/ml); å, gentamicin at 64times the MIC (400 mg/ml); Ç, piperacillin at 32 times the MIC (400 mg/ml);■, fosfomycin at three times the MIC plus ofloxacin at three times the MIC;v v, control.

FIG. 7. Morphological changes in sessile cells in an immature biofilm 48 hafter treatment. Panels: A, gentamicin at 400 mg/ml; B, fosfomycin at three timesthe MIC plus ofloxacin at three times the MIC.

1042 KUMON ET AL. ANTIMICROB. AGENTS CHEMOTHER.

quent growth was rapid enough to form a complete maturebiofilm within the next 24 h. Since old sessile bacteria usuallyare more resistant than young sessile bacteria (2, 23), amino-glycosides are even less effective against these sessile cells. This

is probably due to their positively charged hydrophilic prop-erty, in which they interact with the negatively charged bacte-rial glycocalyx, retarding diffusion of the drug into the biofilmmatrix (7, 15, 20).

FIG. 8. Morphological changes in sessile cells in an immature biofilm 72 h after treatment. Panels: A, gentamicin at 400 mg/ml; B, ofloxacin at three times the MIC;C, fosfomycin at three times the MIC; D, piperacillin at 400 mg/ml.

VOL. 39, 1995 ERADICATION OF P. AERUGINOSA IN A BIOFILM 1043

Fosfomycin is an antibiotic with an extremely low molecularweight of 138, produced by strains of Streptomyces (10), and ischaracterized by structural features of an epoxide ring and acarbon-phosphorus bond. Fosfomycin is also characterized byits action, which inhibits the first step of peptidoglycan biosyn-thesis, and is synergistic in combination with many other anti-microbial agents (6). The entry and accumulation of fosfomy-cin, which are mediated by stereospecific nutrient transportsystems present in various bacteria, including P. aeruginosa (9,12), were still active in cells in the stationary phase, suggestinga potential effect against sessile cells with a low growth rate.Moreover, fosfomycin did not react with the negativelycharged bacterial glycocalyx in our preliminary experiments(17), indicating that the diffusion of fosfomycin into bacterialbiofilms might not be retarded by the penetration barrierformed by the glycocalyx (11). For these reasons, fosfomycinwas selected as a potential antibiotic for combination therapywith ofloxacin in the present study.Although fosfomycin did not independently show any de-

tectable ability to reduce the bioactivity of sessile cells, even atfive or 10 times the MIC, bloating and/or rounding changeswere evident morphologically at concentrations above threetimes the MIC. Enhanced bactericidal activity in the combina-tion treatment with ofloxacin was clearly detected even at fos-fomycin concentrations below the MIC. Nevertheless, syner-gistic effects were not clearly demonstrated when fosfomycinwas combined with ofloxacin at extremely high concentrationsequal to more than 100 times the MIC (data not shown).Although there are no directly supporting data, it may bepossible that fosfomycin altered the membrane permeability ofsessile cells by affecting their cell wall synthesis, which resultedin enhanced uptake of ofloxacin. To clarify the precise mech-anism of this synergistic action of fosfomycin and ofloxacin,ultrastructural and biochemical studies are being conducted.The effective concentrations of fosfomycin and ofloxacin

used in the experiments described here are easily achievable inthe urine of patients treated with clinical oral doses of thesedrugs (3, 18). Therefore, this combination may be helpful inthe treatment of urinary biofilm infections such as catheter-associated infections. The feasibility of using fosfomycin andofloxacin combination therapy to treat such patients needs tobe examined. The synergistic action of fosfomycin and ofloxa-cin may also be effective in eradicating chronic infections un-der the restricted in vivo pharmacodynamics within affectedtissues. We are now conducting in vivo studies using a ratexperimental model of chronic bacterial prostatitis (22) to de-termine the effectiveness of combination therapy in curing thisdifficult chronic infection.

ACKNOWLEDGMENT

Part of this work was supported by a Grant-in-Aid for ScientificResearch (05671318) from the Ministry of Education, Science andCulture, Japan.

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