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Antimicrobial Agents and Chemotherapy, January 2004, p. 151-160, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.151-160.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biofilm Formation by Stenotrophomonas maltophilia: Modulation by Quinolones, Trimethoprim-Sulfamethoxazole, and Ceftazidime

Giovanni Di Bonaventura,^1,2 Ilaria Spedicato,^1,2 Domenico D'Antonio,³ Iole Robuffo,⁴ and Raffaele Piccolomini^1,2*

Dipartimento di Scienze Biomediche, Laboratorio di Microbiologia Clinica,¹ Centro di Scienze dell’Invecchiamento,² Università "G. D'Annunzio"Istituto per i Trapianti d'organo e Immunocitologia, Consiglio Nazionale delle Ricerche, Sezione di Chieti, Chieti,⁴ Servizio di Microbiologia Clinica, Dipartimento di Ematologia ed Oncologia, Ospedale Spirito Santo, ASL Pescara, Italy³

Received 13 January 2003/ Returned for modification 8 July 2003/ Accepted 4 October 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
We investigated the in vitro effects of seven fluoroquinolones (ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and rufloxacin), compared to those of trimethoprim-sulfamethoxazole (SXT) and ceftazidime on total biomass and cell viability of Stenotrophomonas maltophilia biofilm. S. maltophilia attached rapidly to polystyrene, within 2 h of incubation, and then biofilm formation increased over time, reaching maximum growth at 24 h. In the presence of fluoroquinolones at one-half and one-fourth the MIC, biofilm biomass was significantly (P < 0.01) reduced to 55 to 70% and 66 to 76% of original mass, respectively. Ceftazidime and SXT did not exert any activity. Biofilm bacterial viability was significantly reduced by all antibiotics tested at one-half the MIC. At one-fourth the MIC all antibiotics, except levofloxacin, significantly reduced viability. Treatment of preformed biofilms with bactericidal concentrations (500, 100, and 50 µg/ml) of all fluoroquinolones caused, except for norfloxacin, significant reduction of biofilm biomass to 29.5 to 78.8, 64.1 to 83.6, and 70.5 to 82.8% of original mass, respectively. SXT exerted significant activity at 500 µg/ml only. Ceftazidime was completely inactive. Rufloxacin exhibited the highest activity on preformed biofilm viability, significantly decreasing viable counts by 0.6, 5.4, and 17.1% at 500, 100, and 50 µg/ml, respectively. Our results show that (i) subinhibitory (one-half and one-fourth the MIC) concentrations of fluoroquinolones inhibit adherence of S. maltophilia to polystyrene and (ii) clinically achievable concentrations (50 and 100 µg/ml) of rufloxacin are able to eradicate preformed S. maltophilia biofilm.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The use of synthetic materials for temporary or permanent implantation—i.e., central venous catheters, urinary catheters, intraocular lenses, and prosthetic heart valves—has been accompanied by the emergence of implant-associated infection. The bacterial infections following colonization and biofilm formation on these prosthetic materials represent the principal cause of morbidity in patients undergoing prosthetic implantation (6). The production of extracellular slime or glycocalyx is a crucial factor in the adherence of bacteria and their protection from host defense mechanisms and effects of antimicrobial agents. It has become clear that biofilm-grown cells express properties distinct from those of planktonic cells, one of which is an increased resistance to antimicrobial agents. Standard antimicrobial treatments typically fail to eradicate biofilms, which can result in chronic infection and the need for surgical removal of afflicted areas.

Stenotrophomonas maltophilia is being reported with increasing frequency as an important nosocomial pathogen. It is an opportunistic pathogen colonizing patients in intensive care settings, especially those with underlying debilitating conditions such as immunosuppression, malignancies, and implantation of foreign devices (catheters, respiratory therapy equipment, etc.). Bacterial adherence is the first step in the pathogenesis of infections of mucosal surfaces or prostheses. S. maltophilia strains of both clinical and environmental origin have been reported to adhere to abiotic (13, 21, 22) and living (10) surfaces. De Oliveira-Garcia et al. (12) found that S. maltophilia produces flagella as the bacteria spread on the abiotic surface. While previous studies of biofilm development and species interaction have focused largely on Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa, little is known about S. maltophilia. Further, nothing is currently known about antibiotic activity against S. maltophilia biofilms.

In this study, for the first time, we investigated the in vitro effects of seven quinolones (ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and rufloxacin), as well as two commonly used antibiotics for the therapy of S. maltophilia infections (SXT and ceftazidime), on biofilm formation (adherence) and biofilms preformed by S. maltophilia. The presence of microorganisms (dispersed or in microcolonies) was determined and biofilms, including glycocalyx formation, were analyzed semiquantitatively by scanning and transmission electron microscopy (SEM and TEM, respectively).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. Fifty S. maltophilia clinical strains were recently isolated from neutropenic patients admitted to the Hematology Department of the Pescara General Hospital for hematological malignancies. In order to avoid collecting repeat specimens, only one isolate per species was allowed to be collected per patient. All isolates were identified with the mini-API system (ID 32 GN; bioMérieux Italia S.p.A., Rome, Italy) and kept frozen at -70°C until use. Before use, the organisms were subcultured twice on Mueller-Hinton agar (Oxoid S.p.A., Garbagnate Milanese, Milan, Italy) for 24 h at 37°C.

Antimicrobial agents and determination of MICs. The antimicrobial agents tested against biofilm formation and biofilm preformed by S. maltophilia included ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, rufloxacin, ceftazidime, and SXT (1:20 ratio). Reagent-grade antibiotic powders of known potency were purchased from Sigma-Aldrich S.r.l. (Milan, Italy) except as follows: ceftazidime, grepafloxacin, and levofloxacin were from Glaxo Wellcome S.p.A. (Verona, Italy) and moxifloxacin was from Bayer S.p.A. (Milan, Italy). Stock solutions (1,000 µg/ml) of each antimicrobial agent were stored at -70°C until use. MICs of the antimicrobial agents tested were determined by the NCCLS broth microdilution technique (M100-S12) (30). The concentrations of the drugs ranged from 0.25 to 256 µg/ml, except for ceftazidime (1 to 512 µg/ml) and SXT (0.6 to 1,280 µg/ml). The MIC was defined as the lowest drug concentration which inhibited visible growth after incubation at 37°C for 18 h.

Biofilm formation (adherence) assay. Overnight cultures of S. maltophilia in 3 ml of Trypticase soy broth (TSB) (Oxoid) were washed, diluted with fresh TSB, and standardized to contain 5 x 10⁵ to 1 x 10⁶ CFU/ml. Aliquots (200 µl) of standardized inoculum were added to the wells of sterile flat-bottom polystyrene tissue culture plates (Iwaki; Bibby srl, Riozzo di Cerro al Lambro, Milan, Italy), and incubated at 37°C over a series of intervals (30 min and 1, 2, 4, 8, 16, and 24 h) in a closed, humidified plastic container. The medium was then discarded, and nonadherent cells were removed by being washed three times in sterile phosphate-buffered saline (PBS; pH 7.3) (Sigma-Aldrich).

Quantitation of S. maltophilia biofilms was performed by both viable cell enumeration and a spectrophotometric method previously described by Christensen et al. (7), with minor modifications. For plate counts, biofilms were removed from microtiter wells by scraping and then vortexed vigorously to disperse the cells. Cell counts were estimated by plating serial dilutions of these suspensions. The spectrophotometric method measures the total biofilm biomass, including bacterial cells and extracellular matrix. Briefly, slime and adherent organisms were fixed by incubating them for 1 h at 60°C (1) and then staining them with Hucker crystal violet for 5 min. After through washing with water to remove excess stain, the plates were dried for 30 min at 37°C. The extent of biofilm was determined by measuring the absorbance of the stained adherent film with a microplate reader (Sunrise; Tecan Italia srl, Cologno Monzese, Milan, Italy) at a wavelength of 492 nm. The low cutoff was chosen by using the criteria described by Christensen et al. (7), i.e., it represented approximately 3 standard deviations (SDs) above the mean optical density (OD) of control wells.

Effect of sub-MICs of antimicrobial agents on S. maltophilia biofilm formation. Each drug was tested at one-half, one-fourth, and one-eighth the MIC to study its effect on S. maltophilia biofilm formation. Various concentrations of antimicrobial agents prepared in 100 µl of TSB were added to microtiter wells containing 100 µl of the inoculum standardized as described above (biofilm formation assay). After 24 h of incubation, quantitation of biofilms was performed as described for the biofilm formation assay. Drug-free medium was used in control wells.

Effects of antimicrobial agents on S. maltophilia preformed biofilm. Biofilm formation by S. maltophilia was carried out in 96-well flat-bottom tissue culture plates as described for the biofilm formation assay. After 18 h of incubation at 37°C, the supernatant from each well was gently aspirated by a micropipette. Each well was then washed three times with PBS without disturbing the adherent film. Antimicrobial agents at different concentrations (50, 100, and 500 µg/ml) in 200 µl of TSB were then added to the wells. TSB without antimicrobial agent was added to the control wells. The plates were incubated at 37°C for 6 h. After incubation, quantitation of biofilms was performed as described for the biofilm formation assay. Drug-free medium was used in control wells. Isolates for which the MIC was higher than the antimicrobial concentration tested were not considered.

Electron microscopy analysis. SEM and TEM analyses were carried out with S. maltophilia strain SM33, selected because it was the highest producer of slime among 20 strains considered. Biofilms were allowed to grow on cell culture polystyrene dishes (Iwaki) with (ciprofloxacin for biofilm formation and moxifloxacin for preformed biofilm) or without (kinetic of biofilm formation) the antibiotic. The samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red for at least 4 h. After being washed with PBS, the samples were then dehydrated in a series of aqueous ethanol solutions (30 to 100%).

(i) SEM. The specimens were mounted on aluminum stubs with conductive carbon cement, allowed to dry for 3 h, and then coated with 15-nm Au film with an agar automatic sputter coater. After processing, samples were observed with a Philips XL30CP scanning electron microscope in the high-vacuum mode at 15 kV.

(ii) TEM. The samples were embedded in Spurr resin. Ultrathin sections (60 to 80 nm) were mounted on 200-mesh copper grids, stained with uranyl acetate and lead citrate, and observed with a Zeiss electron microscope.

SEM and TEM images were processed for display using Photoshop software (Adobe Systems Inc., Mountain View, Calif.).

Statistical analysis. Data were normalized to the control, which was taken as 100%. All assays were carried out in triplicate and repeated twice, and the results are presented as means ± SDs. A one-way analysis of variance was used to detect the existence of differences in activity against adherence and preformed biofilm among different groups. Where significant differences existed, comparison with the control (strain without antibiotic) was carried out by the Dunnett test for multiple comparisons. Pairwise comparisons among antibiotics were done by the Newman-Keuls multiple comparison test. Differences were considered statistically significant at a P value of <0.01.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reliability of biofilm formation assays. We performed a series of preliminary experiments in order to evaluate variability among S. maltophilia biofilms formed on independent wells of the same microtiter plate. No statistically significant differences were noted among 96 independent biofilms formed on the same plate: repeated quantitative assays on adherent control strain S. maltophilia SM33 invariably resulted in OD readings within the expected ranges (range, 0.411 to 0.505; mean ± SD, 0.423 ± 0.035; standard error of the mean, 0.026; 95% confidence interval, 0.371 to 0.475), validating the equivalency between each biofilm formed in multiple independent wells. A high correlation (Pearson r = 0.99) was observed between OD readings and viable cell enumerations.

Biofilm formation by S. maltophilia. The results of the biofilm production assay monitored by the spectrophotometric method revealed ODs ranging from 0.046 to 0.413: by a low cutoff OD of 0.056, 44 of 50 (88%) S. maltophilia isolates were positive for glycocalyx production. Striking differences in formation ability were observed among biofilm-positive strains (data not shown).

The 20 highest biofilm-producing S. maltophilia strains were selected for further studies. The mean kinetic of biofilm formation by 20 selected S. maltophilia isolates on the surface of polystyrene wells over 24 h is illustrated in Fig. 1. Bacteria were shown to attach rapidly, within 2 h of incubation, and then the biofilm formation increased over time up to a mean OD of 0.310 at 24 h. The ODs of the 24-h biofilm ranged from 0.413 to 0.091. As suggested by the SD values, there was a strain-to-strain variation in biofilm formation, probably in relation to their individual propensity to adhere to polystyrene. Because maximum attached growth observed had occurred in the test strains by 24 h, we have considered 24 h of incubation as the endpoint for all subsequent biofilm assays.

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FIG. 1. Biofilm formation by S. maltophilia determined by the spectrophotometric method. Results are the means and SDs for 20 selected S. maltophilia isolates.

Figure 2 shows scanning electron micrographs of the S. maltophilia biofilm mode of growth on polystyrene surface over 24 h of incubation. Adherent bacterial cells were observed at 2 h sticking to the polystyrene surface in a random manner (Fig. 2A), and after 4 h the biofilm covered just 10% of the surface, growing in specific clusters of cells (microcolonies) (Fig. 2B). These clusters extended over time and cohered to each other (Fig. 2C and D) to cover the entire surface of the well within 24 h: mature biofilm consisted of a dense network of cells deeply embedded in an extracellular matrix (Fig. 2E). As shown by TEM observation (Fig. 2F), the cotton-like material (glycocalyx) surrounded the bacteria and modulated their adherence to the polystyrene surface.

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FIG. 2. (A to E) Scanning electron micrographs of S. maltophilia SM33 biofilms on polystyrene surfaces at 2, 4, 8, 16, and 24 h, respectively. Magnification: x1,000 (A to D) and x2,000 (E). (F) Transmission electron micrographs of 24-h biofilm produced by S. maltophilia SM33. Arrows indicate glycocalyx surrounding bacteria. The asterisk indicates the biofilm limit line in contact with the polystyrene surface. Bar = 0.5 µm.

Effect of sub-MICs of antimicrobial agents on adherence of S. maltophilia. The in vitro activities of the antimicrobial agents tested against 20 adherent S. maltophilia isolates are shown in Table 1. Among the antibiotics tested, SXT was the most active compound, inhibiting all the isolates at a ratio of 0.5 to 9.5 µg/ml (100% susceptibility). Moxifloxacin was the most active fluoroquinolone against S. maltophilia, being about twice as active as levofloxacin and grepafloxacin, while norfloxacin was less active than the other agents.

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TABLE 1. Comparative in vitro activities of seven fluoroquinolones, SXT, and ceftazidime against 20 S. maltophilia biofilm-producing isolates

Relative efficacies of antibiotic treatments for biomass and viability of S. maltophilia adhering to polystyrene are presented in Fig. 3. The analysis of variance test showed that all fluoroquinolones tested at the subinhibitory concentrations significantly (P < 0.01) reduced the biomass of S. maltophilia biofilm and that this effect was generally dose dependent, resulting in 55 to 70%, 66 to 76%, and 74 to 87% reduction in the presence of one-half, one-fourth, and one-eighth the MIC of fluoroquinolones, respectively. Particularly, all fluoroquinolones tested at one-half the MIC exhibited comparable activities in significantly (P < 0.01) reducing biomass. At one-fourth the MIC, only levofloxacin did not exhibit significant activity. No significant activity was recorded when fluoroquinolones were tested at one-eighth the MIC. Ceftazidime and SXT were inactive on S. maltophilia biomass whatever the concentrations tested.

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FIG. 3. Relative effects of antibiotics tested at one-half (small-checkered bars), one-fourth (large-checkered bars), and one-eighth (striped bars) the MIC on biomass (A) and viability (B) of 20 S. maltophilia biofilms in formation. Results are expressed as means ± SDs. CIP, ciprofloxacin; LVX, levofloxacin; MXF, moxifloxacin; OFX, ofloxacin; GRX, grepafloxacin; NOR, norfloxacin; RFX, rufloxacin; CAZ, ceftazidime.

Spectrophotometric results were strongly supported by SEM observations (Fig. 4A and B). With respect to control, where bacterial cells were coated with an extracellular material, bacteria grown in the presence of ciprofloxacin were more distinctly outlined. Extracellular material decreased as the antibiotic concentration increased.

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FIG. 4. Scanning electron micrographs of antibiotic activity against S. maltophilia SM33 biofilm. (A and B) Ciprofloxacin at one-fourth (A) and one-half (B) the MIC for 24 h during biofilm formation. (C and D) Rufloxacin at 100 (C) and 500 (D) µg/ml against preformed S. maltophilia biofilm. Magnification: x2,500 (A to C) and x2,000 (D).

As for biomass, the overall effect on biofilm bacterial viability was generally dose dependent. Compared to control ([1.1 ± 0.5] x 10⁸ cells/cm²), all antibiotics tested at one-half the MIC affected cell viability significantly (P < 0.01), although SXT activity was significantly lower than that of ciprofloxacin. At one-fourth the MIC all antibiotics, except for rufloxacin and ofloxacin, induced a significant reduction. At one-eighth the MIC all antibiotics reduced viability, although not significantly.

Effects of antimicrobial agents on biofilm preformed by S. maltophilia. The activities of antimicrobial agents tested at concentrations of 50, 100, and 500 µg/ml against total biomass and bioactivity of S. maltophilia biofilm are shown in Fig. 5. All of the quinolones, except norfloxacin, significantly (P < 0.01) reduced preformed biofilm biomass. Compared to controls, OD was reduced to 29.5 to 78.8%, 64.1 to 83.6%, and 70.5 to 82.8% of the original value at concentrations of 500, 100, and 50 µg/ml, respectively. Ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, ofloxacin, and rufloxacin significantly (P < 0.01) reduced biofilm biomass at 500 µg/ml, although moxifloxacin was significantly more active than other antibiotics. In fact, moxifloxacin tested at 500 µg/ml caused eradication of biofilm biomass for 10 of 20 (50%) isolates tested, reducing it up to 95% for 12 (60%) isolates. At 100 µg/ml, only ofloxacin caused significant (P < 0.01) reduction. At 50 µg/ml, ciprofloxacin and ofloxacin had similar activities, causing significant (P < 0.01) reduction. SXT yielded significant (P < 0.01) reduction at 500 µg/ml only. In contrast, ceftazidime did not reduce the biomass of S. maltophilia preformed biofilm at any bactericidal concentration tested.

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FIG. 5. Relative effects of antibiotics tested at 500 (small-checkered bars), 100 (large-checkered bars), and 50 (striped bars) µg/ml on biomass (A) and viability (B) of 20 preformed S. maltophilia biofilms. Results are expressed as means ± SDs. CIP, ciprofloxacin; LVX, levofloxacin; MXF, moxifloxacin; OFX, ofloxacin; GRX, grepafloxacin; NOR, norfloxacin; RFX, rufloxacin; CAZ, ceftazidime.

Relative effects of antibiotic treatments on viability of preformed S. maltophilia biofilm were not dose dependent. Compared to control ([1.2 ± 0.6] x 10⁸ cells/cm²), rufloxacin exhibited the most activity, significantly (P < 0.01) decreasing viable counts in S. maltophilia biofilm to 0.6, 5.4, and 17.1% of original values at 500, 100, and 50 µg/ml, respectively. A >99.9% reduction was caused by rufloxacin in 15 (75%), 14 (70%), and 7 (35%) of 20 biofilms tested at 500, 100, and 50 µg/ml, respectively. Ofloxacin, SXT, norfloxacin, and grepafloxacin were significantly active at 500 µg/ml only, reducing the biofilm viability to 1, 4.4, 42.1, and 43.7%, respectively; moxifloxacin, ofloxacin, and SXT showed comparable activity, significantly higher than that of other antibiotics.

Figure 4C and D show scanning electron micrographs of S. maltophilia biofilms grown for 18 h on a polystyrene surface and then treated for 6 h with bactericidal concentrations of rufloxacin. A dose-dependent effect of the antibiotic on the preformed biofilm was evident with regard to the cell number and the quantity of extracellular material. At 500 µg/ml rufloxacin caused relevant ultrastructural changes in S. maltophilia cells.

As indicated by TEM analysis (Fig. 6), 18-h biofilms treated for 6 h with moxifloxacin at 100 µg/ml showed both a lytic phase of cells with necrosis (Fig. 6A to E) and DNA condensation forming apoptotic bodies (Fig. 6A to D). We also observed glycocalyx degradation with consequent separation of the bacteria from the polystyrene surface (Fig. 6D to F).

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FIG. 6. Transmission electron micrographs of S. maltophilia 18-h biofilm treated for 6 h with 100 µg of moxifloxacin/ml show both a lytic phase of cells with necrosis (A to E; arrows) and DNA condensation forming apoptotic bodies (A to D; arrowheads). Note glycocalyx degradation with consequent separation of the bacteria from the polystyrene surface (D to F; asterisks). Bars = 0.5 µm.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major risk factor for S. maltophilia infection in hospitalized patients is the implantation of medical devices such as central venous catheters (29), urinary tract catheters (25), prosthetic heart valves (24), and intraocular (19) and contact (9, 23) lenses. This evidence suggests that S. maltophilia is able to adhere to abiotic surfaces: both clinical and environmental isolates have been reported to adhere to glass and to several types of plastic materials including intravenous cannulae, polyvinyl chloride, and Teflon (13, 21). In contrast, there are few data for the role of adherence to living surfaces in the pathogenesis of S. maltophilia. de Abreu Vidipò et al. (10) showed adherence of clinical S. maltophilia strains to human epithelial respiratory cells in vitro, mainly along intercellular junctions. However, Grant et al. (17) were unable to demonstrate the adherence of the bacterium to cultured hamster tracheal cells. In the present study we found a strain-to-strain variation in adherence, as suggested by the large SDs, probably in relation to the propensities of individual strains to adhere to polystyrene, confirming the findings of other authors for coagulase-negative staphylococci (26, 35).

Microtiter plate systems for quantifying adherence and biofilm formation have been investigated with many different organisms and stains (7, 31). These techniques have been widely used because they are simple, reproducible, and quantitative methods. However, the staining measurements reflect the total amount of biofilm (sessile cells plus exopolysaccharide matrix) but do not give any information about its viability. Thus, we also considered measuring cell viability by scraping biofilms formed on polystyrene microtiter plates. Our results show that both the modified Christensen and viable count techniques that we used are highly reproducible in assessing S. maltophilia biofilm production. Although some variability in values was seen, the level of biofilm formation was highly consistent between experiments, discriminating strains which produced strong biofilms from strains which produced weak biofilms.

It is well known that antimicrobial agents of a variety of classes prevent bacterial adherence when used at very low concentrations (i.e., sub-MICs), probably due to the antibiotic effects on the bacterial cell wall or membrane (2, 4, 34). To the best of our knowledge, no data exist concerning antibiotic activity against S. maltophilia adherence. In the present study, for the first time, we tested the effects of several antibiotics on S. maltophilia adherence. The study antibiotics were chosen for several reasons. SXT and ceftazidime were tested because they are frequently used in the therapy of S. maltophilia infections (11). Fluoroquinolones were chosen because of their interesting activity against gram-negative (3, 16, 20) and gram-positive (35) bacterial biofilms; further, because of the growing frequency of S. maltophilia isolates resistant to traditional antimicrobial therapies including the "gold standard" SXT, fluoroquinolones have been used increasingly for treatment of S. maltophilia infections.

Our results showed that all fluoroquinolones tested at one-half the MIC significantly reduced the adherence of S. maltophilia to polystyrene in a dose-dependent manner. In particular, ciprofloxacin, grepafloxacin, moxifloxacin, and norfloxacin were the most active drugs since they significantly reduced both the biomass and viability of S. maltophilia biofilm at one-fourth the MIC also. Ofloxacin and rufloxacin tested at one-fourth the MIC caused reduction of biomass without an effect on cell viability, suggesting an active role for these fluoroquinolones in affecting slime production. Kadry et al. (22) reached a similar conclusion about the effect of low concentrations of ciprofloxacin and other antimicrobial agents on the adherence of mucoid S. epidermidis to intraocular lenses. The mechanism by which fluoroquinolones inhibit the adherence of S. maltophilia was not investigated in this study. However, as already reported for clarithromycin by Yasuda et al. (36) against S. epidermidis biofilm, it may be hypothesized that drugs suppress (or interfere with) the synthesis of extracellular matrix or destroy it, once produced, independently of their mechanism of action. This could be possible for S. maltophilia biofilm also, as suggested by the SEM analysis performed in the present study. Ceftazidime and SXT tested at one-half and one-fourth the MIC caused only a reduction in viability. None of the antibiotics showed significant activity if tested at one-eighth the MIC.

Biofilms, products of bacterial adherence, are structured communities of bacterial cells enclosed in a self-produced exopolysaccharide matrix and adherent to an inert or living surface (8). Establishment of a biofilm is the prelude to the development of various chronic, intractable infections, such as biomaterial-associated infections and pulmonary infection in patients with cystic fibrosis (27). Thus, understanding of biofilm dynamics is crucial to develop better control strategies.

Biofilm formation by S. maltophilia has been postulated but never before shown. In the present study, we characterized the kinetic of S. maltophilia biofilm formation: bacteria attach rapidly to polystyrene, after 2 h of incubation, and then the biofilm formation increases over time, reaching maximum intensity at 24 h of culture. The speed with which S. maltophilia colonizes the polystyrene surface suggests that merely replacing an old medical device with a new one could be ineffective, since it may result in planktonic bacteria adhering to the new device and the infection continuing.

Despite various efforts, treatment of an infection after biofilm is established is frequently futile because of the reduced susceptibility of biofilm to antibiotics. The nature of the resistance of biofilms, in spite of much research, remains an enigma. At least three mechanisms have been proposed to account for recalcitrance of biofilms to antimicrobial agents (28): (i) failure of the antimicrobial to penetrate the biofilm, (ii) slow growth and the stress response, and (iii) induction of a biofilm phenotype.

Before now, nothing was known about antibiotic activity against S. maltophilia biofilms. Our results showed that rufloxacin, ofloxacin, and grepafloxacin tested at 500 µg/ml caused significant (P < 0.01) reductions in both biomass and viability of S. maltophilia biofilm, decreasing viability to 0.6, 4.4, and 43.7%, respectively, with rufloxacin and ofloxacin being significantly more active than grepafloxacin. However, rufloxacin showed the most activity against preformed S. maltophilia biofilm, since it exhibited a significant (P < 0.01) activity at 100 and 50 µg/ml as well, reducing viability to 5.4 and 17.1% of original values, respectively. Further, rufloxacin induced a >99.9% reduction in viability in 15 (75%), 14 (70%), and 7 (35%) of 20 biofilms tested at 500, 100, and 50 µg/ml, respectively. These results suggest that rufloxacin, by an unknown mechanism, is able to penetrate S. maltophilia biofilm with a strain-specific efficiency, as suggested by the high strain-to-strain variation, probably due to chemical and physical heterogeneity of S. maltophilia biofilms.

Differences between antibiotics in the MICs obtained by the classical microdilution assay were not related to killing value differences obtained in biofilm tests, suggesting that other mechanisms and/or factors are required for antimicrobial activity. Further, this confirms that drug effects on stationary and adherent microorganisms, but not MICs (evaluated on planktonic organisms), are useful for predicting the outcome of device-related infections. Ceftazidime was totally ineffective in removing S. maltophilia biofilms in our assay. Similarly, Ishida et al. (20) found that ceftazidime, in contrast to levofloxacin, is inactive against P. aeruginosa biofilm, since it shows poor bactericidal activity against nongrowing cells and a low rate of diffusion through the biofilm layer.

The concentrations of fluoroquinolones tested in our study against preformed S. maltophilia biofilms are generally higher than those reached in serum when applied by the intravenous or oral route. Nevertheless, data from our study might have clinical significance because these concentrations are easily achievable when standard dosages of drugs are infused through the vascular catheters in the "antibiotic lock" technique (5, 14, 18). Further, topical application may be possible. However, continued surveillance for resistance is required since fluoroquinolone-resistant strains have been increasing (33).

One of the more radical hypotheses for biofilm resistance to antibiotics is that an "altruistic" majority of sublethally damaged cells in a population commit suicide (apoptosis), thereby providing some protection to the survivors (persisters) (15). Spoering and Lewis (32) suggested that persisters are responsible for the resistance of biofilms to killing since they have a disabled programmed cell death mechanism. The persisters thereby benefit from the self-sacrifice of the other cells and maintain the gene pool. The TEM analysis that we performed on antibiotic-treated S. maltophilia biofilm confirms this hypothesis, showing cell lysis characterized by necrosis and DNA condensation forming apoptotic bodies. This suggests that bactericidal antibiotics, i.e., fluoroquinolones, cause damage that activates programmed cell death.

In conclusion, this study shows that (i) subinhibitory concentrations of fluoroquinolones (ciprofloxacin, grepafloxacin, moxifloxacin, and norfloxacin in particular) greatly reduce adherence of S. maltophilia to plastic surfaces; (ii) bactericidal concentrations of fluoroquinolones (rufloxacin, in particular) reduce S. maltophilia preformed biofilms; (iii) high concentrations of ceftazidime and SXT, which are under many circumstances drugs of choice for S. maltophilia infection, are needed to affect adherence and preformed biofilms; and (iv) individual studies of the isolate responsible are needed to eradicate a particular infection. The clinical relevance of using fluoroquinolones for the prevention and treatment of prosthetic infections, i.e., intravascular catheter-related bloodstream infections, needs to be confirmed in vivo with an appropriate animal model.

 
This work was in part supported by a grant from the Ministero della Salute-Ricerca Sanitaria 2003 (Italy). I.S. is also the recipient of a grant from the Dipartimento di Scienze Biomediche, Università "G. D'Annunzio," Chieti, Italy.

 
* Corresponding author. Mailing address: Centro Scienze dell'Invecchiamento, Lab. 27, 5° Livello, Università "G. D'Annunzio," Via dei Vestini 31, 66100 Chieti, Italy. Phone: (39) 0871 355 52 83. Fax: (39) 0871 355 52 82. E-mail: r.piccolomini{at}dsb.unich.it.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
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Antimicrobial Agents and Chemotherapy, January 2004, p. 151-160, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.151-160.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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