The aim of this work was to compare the glucose
uptake of biofilms formed by four different Staphylococcus
epidermidis strains as well as to compare between
sessile and planktonic cells of the same strain. Biofilm cells
showed a lower level of glucose uptake compared to
planktonic cells. Moreover, glucose uptake by cells in the
sessile form was strongly influenced by biofilm composition.
Therefore, this work helps to confirm the phenotypic
variability of S. epidermidis strains and the different
behaviour patterns between sessile and free cells.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/ESP/42688/2001; 
SFRH/BD/19265/2004

A Resch

C Eiff Von

CA Fux

Cláudia Sousa

D Mack

III MC Walters

J Azeredo

Joana Azeredo

M Dubois

Mariana Henriques

N Cerca

Pilar Teixeira

Rosário Oliveira

S Hennig

S Møller

S Stepanović

SE Cramton

World Journal of Microbiology and Biotechnology

Sousa, Cláudia

Henriques, Mariana

Azeredo, Joana

Teixeira, P.

Oliveira, Rosário

Universidade do Minho: RepositoriUM

ORIGINAL PAPERStaphylococcus epidermidis glucose uptake in biofilm versusplanktonic cellsCla´udia Sousa Æ Mariana Henriques ÆJoana Azeredo Æ Pilar Teixeira Æ Rosa´rio OliveiraReceived: 16 May 2006 / Accepted: 3 June 2007 / Published online: 15 July 2007 Springer Science+Business Media B.V. 2007Abstract The aim of this work was to compare the glu-cose uptake of biofilms formed by four different Staphy-lococcus epidermidis strains as well as to compare betweensessile and planktonic cells of the same strain. Biofilm cellsshowed a lower level of glucose uptake compared toplanktonic cells. Moreover, glucose uptake by cells in thesessile form was strongly influenced by biofilm composi-tion. Therefore, this work helps to confirm the phenotypicvariability of S. epidermidis strains and the differentbehaviour patterns between sessile and free cells.Keywords Biofilms  Glucose uptake  Planktonic cells Staphylococcus epidermidisIntroductionStaphylococcus epidermidis is a coagulase-negativestaphylococcus (CNS) that is one of the main nosocomialpathogens associated with infections of implanted medicaldevices (Von Eiff et al. 2002). CNS adheres to such de-vices and has the ability to form biofilms which constitutesone important virulence factor and is considered the mainresponsible for S. epidermidis pathogenesis (Henning et al.2007). Bacteria in biofilms are more resistant (1,000 upfold) to antibiotics and to the host immune defence systemthan their planktonic counterparts (Cerca et al. 2005),suggesting that cells in a biofilm may have altered meta-bolic activity (Resch et al. 2005). This strong resistanceregularly requires the removal of the infected biomaterialand leads to substantial morbidity and mortality (Macket al. 2004). Thus, it is extremely important to know theessential differences, especially concerning physiology andmetabolic activity, between a planktonic cell and its sessilecounterpart adhered to a surface living in a biofilm.The aim of this work was to compare the glucose uptakeof biofilms formed by four different S. epidermidis strains,and between sessile and planktonic cells of the same strain.Thus, the influence of biofilm characteristics such as totalbiomass formed, cell concentration and polysaccharidescontent on the glucose uptake was also evaluated.Materials and methodsAll strains used in this work (LE7, 9142, IE186 and IE214)are clinical isolates and were kindly provided by DR. G. B.Pier, Channing Laboratory, Department of Medicine,Brigham and Women’s Hospital, Harvard Medical School,Boston.For all the biofilm assays, cells were first grown for 24 hin 15 ml of TSB (Merck, Darmstadt, Germany) at 37 Cusing an orbital shaker (130 rev/min). After this period,50 ll of each suspension were transferred to 30 ml of freshTSB broth and incubated for 18 h (late exponential phase),at 37 C and 130 rev/min. Then, the cells were centrifuged(Sigma 4K10, B. Braun, Germany) for 5 min, at 10,500gand 4 C, washed twice with a saline solution (0.9% NaCl(Merck) in distilled water), and sonicated (UltrasonicProcessor, Cole-Parmer, Illinois, USA) during 10 s, withan amplitude of 22% (previously optimized to avoid celldisruption). The cellular suspension was adjusted to a finalconcentration of approximately 1 · 109 cells/ml, determinedC. Sousa  M. Henriques  J. Azeredo  P. Teixeira (&) R. OliveiraIBB-Institute for Biotechnology and Bioengineering, Centre forBiological Engineering, Universidade do Minho, Campus deGualtar, 4710-057 Braga, Portugale-mail: pilar@deb.uminho.pt123World J Microbiol Biotechnol (2008) 24:423–426DOI 10.1007/s11274-007-9478-4by optical density at 640 nm. Acrylic was used as sub-stratum and cut into 2 cm · 2 cm squares. Prior to use, thecoupons were washed with sterile distilled water and with asolution of 70% ethanol diluted in sterile distilled water.Each clean acrylic coupon was placed into an individualwell of a 6-well tissue culture plate (Sarstedt, Newton, NC,USA) containing 5 ml of TSB enriched with 0.25% ofglucose (Merck). For every strain, an inoculum of 50 llwith 1 · 109 cells/ml was added to each well. Assays wereperformed in triplicate and repeated 3 times. The plateswere incubated for 192 h (eight days) at 37 C in an orbitalshaker (120 rev/min). The medium was withdrawn andreplaced by fresh TSB + 0.25% glucose every 12 h (fed-batch mode). The total attached biomass to the acryliccoupons was measured by methanol fixation, crystal violetstaining and acid acetic elution as previously described(Stepanovic´ et al. 2000). The eluted dye of each well wasremoved and placed in a 96-well microtitre plate (Sarstedt,Newton, NC, USA) and its absorbance was read in anELISA reader (Bio-Tek Instruments Inc., Vermont, USA)at 570 nm. In order to determine the biofilm cell concen-tration, the acrylic coupons with biofilm were washed twicewith NaCl 0.9%. Then, the biofilm was removed from thecoupons by scrapping with a spatula and resuspended into25 ml of TSB + 0.05% Tween 20 (Merck). Next, the sus-pension was sonicated (20 s with 22% of amplitude),centrifuged (5 min, 10,500g, 4 C), resuspended in 30 mlof NaCl 0.9% and sonicated again (10 s, 22% amplitude) topromote biofilm disruption. The absorbance was readat 640 nm. The number of c.f.u in biofilm for eachS. epidermidis strain was determined by plating onto TSAin triplicate using decimal dilutions and incubating for24–48 h. The extraction of the exopolymeric matrix, forthe quantification of the polysaccharides, was assessedusing the Dowex resin extraction method as described byAzeredo et al. (2003). The biofilm matrix polysaccharideswere quantified by the method of Dubois et al. (1956). Inorder to determine the glucose uptake in the biofilm and toremove the cells loosely attached and residual medium,each coupon was consecutively transferred to three 100 mlglass beakers containing distilled water, and was allowed torest there for approximately 10 s. Then the coupons weretransferred to a new 6-well tissue plate containing 5 ml of0.10% glucose solution (w/v). The glucose concentration inthe medium was measured immediately after transferringthe coupons and also 60 min later, maintaining the platesunder slow agitation. Glucose was quantified with theenzymatic kit Glucose–TR (Spinreact, SA, Spain) using theELISA reader at 505 nm.For the assays to determine the glucose uptake by theplanktonic cells, all strains were incubated in 15 ml of TSBand grown for 24 (±2) h at 37 C in an orbital shaker(130 rev/min). Then, 100 ll of each cell suspension weretransferred to 60 ml of fresh TSB and incubated for 18 h(to reach late exponential phase) at 37 C and 130 rev/min.Cells were harvested by centrifugation (for 5 min at10,500g and 4 C) and resuspended in TSB to the sameconcentration as previously determined by c.f.u. plating ofbiofilm cells. For all strains, 1 ml of the suspension ob-tained was centrifuged at 9,500g and the pellet resuspendedin 1 ml of 0.10% glucose solution. These samples werethen transferred to the wells of a 6-well tissue culture platecontaining 4 ml of 0.10% glucose solution. Glucose pres-ent in the medium was measured at time zero and after60 min with the enzymatic kit Glucose–TR, as describedfor biofilm assays.The data obtained was analyzed using a statistical pro-gram, SPSS (Statistical Package for the Social Sciences).One-way ANOVA with Tukey test was used to comparethe results of the different assays. All tests were performedwith a confidence level of 95%.Results and discussionIn this study, strain variability in terms of biofilm forma-tion ability was assessed through the crystal violet assay(Fig. 1) and the data obtained revealed a significantly(p < 0.05) higher ability of biofilm formation by strains9142, IE186, IE214 in contrast to strain LE7. This showsthat crystal violet staining is a reliable method, having theadvantage of being easy and expedite, i.e., providing theresults almost immediately, with a great objectivity andaccuracy. The assays to determine the glucose uptake bycells in suspension were performed with the same cellularconcentration of the biofilm assays. This cellular concen-tration was determined by c.f.u. plating of the biofilm cells,in terms of c.f.u./ml, for the four S. epidermidis strains.Strain 9142 presented the highest cellular concentration(1.5 · 1018 c.f.u./ml) in comparison with strains IE2140.00.40.81.21.6LE7                       9142                      IE186                     IE214StrainA 570nmFig. 1 Biofilm biomass of four Staphylococcus epidermidis strains,accumulated over 192 h, expressed as crystal violet absorbance(A570nm). Results represent means plus standard deviations (errorsbars) from three independent experiments424 World J Microbiol Biotechnol (2008) 24:423–426123(2.7 · 1017 c.f.u./ml) and IE186 (3.9 · 1017 c.f.u./ml). Thenumber of cells on LE7 biofilms was very low(6.5 · 1016 c.f.u./ml) compared to the other three strainswhich are strong biomass producers (Fig. 1). As far as theamount of polysaccharides present in the polymeric matrixis concerned, strain IE214 produced the largest amount ofpolysaccharides (0.442 mg/g(dry weight/wet weight) ± 0.173)and this is the most likely explanation for the large value ofits total biomass production. In the same way, LE7 was thestrain that produced the smallest amount of polysaccha-rides (0.189 mg/g(dw/ww) ± 0.045) and is the one that pro-duced the least quantity of total biomass. Strain 9142 andstrain IE186 presented intermediary values, 0.378 mg/g(dw/ww) ± 0.172 and 0.228 mg/g(dw/ww) ± 0.092, respectively.The present study shows that biofilm glucose uptake candiffer between strains of the same species (Fig. 2). Inabsolute terms, strain 9142 biofilms removed from the li-quid phase the highest amount of glucose (23.98%), fol-lowed by the biofilms of strains IE186 (12.7%) and IE214(8.29%), while strain LE7 displayed a significantly lessglucose uptake (1.11%). These results correlate with thenumber of c.f.u./ml in the biofilms. This is also in agree-ment with previous studies that showed that the amount ofbiofilm produced by individual S. epidermidis strains ishighly variable phenotypically and regulated by severalfactors (Cramton et al. 2001; Mack et al. 2004). The per-centage of glucose uptake by planktonic cells (Fig. 2)shows the same tendency but is significantly higher thantheir biofilm counterparts. This would be expected becauseit has already been shown that cells in sessile form displaya lower metabolic activity (Walters et al. 2003). Thereduction of the percentage of glucose uptake fromplanktonic cells to biofilm cells is on average twofold forthe three highest biofilm producing strains. Low levels ofglucose uptake might be an indicative of a lower metabolicactivity and consequently slow growth has been consideredone of the main reasons for biofilm resistance and successagainst antimicrobial agents (Fux et al. 2005). Biofilmscells are immersed in a matrix which provides the bacterialpopulation protection from the host defence mechanismsand antimicrobial agents. In addition, the surface attachedpopulations are exposed to a different nutrient environmentthan the planktonic cells (Møller et al. 1995). Thus, manyfactors may affect the response of biofilm cells in terms ofmetabolic activity and antibiotic susceptibility.ConclusionsThis study shows that the determination of glucose uptakeby cells in suspension and in biofilm is an expedite methodto evaluate the different behaviour of S. epidermidis strainsin both forms. According to the results obtained, the four S.epidermidis strains revealed different abilities of biofilmformation, with sessile cells displaying lower glucose up-take in comparison to planktonic cells for all the strainsstudied. In the sessile form, glucose uptake seems to bedependent on the strain specific properties of the biofilmsuch as the cell concentration and the production ofextracellular matrix.Acknowledgements The authors acknowledge the financial supportof FCT, through the project POCTI/ESP/42688/2001 and the grantSFRH/BD/19265/2004. The authors also acknowledge to AnthonyDanko for his precious help in the improvement of the manuscript.ReferencesAzeredo J, Henriques M, Sillankorva S, Oliveira R (2003) Extractionof exopolymers from biofilms: the protective effect of glutaral-dehyde. Water Sci Technol 47:175–179Cerca N, Martins S, Cerca F, Jefferson KK, Pier GB, Oliveira R,Azeredo J (2005) Comparative assessment of antibiotic suscep-tibility of coagulase-negative staphylococci in biofilm versusplanktonic culture as assessed by bacterial enumeration or rapidXTT colorimetry. J Antimicrob Chemother 56:331–336Cramton SE, Gerke C, Go¨tz F (2001) In vitro methods to studystaphycococcal biofilm formation. Methods Enzymol 336:239–255Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)Colorimetric method for determination of sugars and relatedsubstances. Anal Chem 28:350–356Fux CA, Costerton JW, Stewart PS, Stoodley P (2005) Survivalstrategies of infectious biofilms. Trends Microbiol 13:34–40Hennig S, Wai SN, Ziebuhr W (2007) Spontaneous switch to PIA-independent biofilm formation in an ica-positive Staphylococcusepidermidis isolate. Int J Med Microbiol 297:117–122Mack D, Becker P, Chatterjee I, Dobinsky S, Knobloch JK-M, PetersG, Rohde H, Herrmann M (2004) Mechanisms of biofilmformation in Staphylococcus epidermidis and Staphylococcusaureus: functional molecules, regulatory circuits, and adaptiveresponses. Int J Med Microbiol 294:203–212Møller S, Kristensen CS, Poulsen LK, Carstensen JM, Molin S (1995)Bacterial growth on surfaces: automated image analysis for0102030405060LE7                       9142                     IE186                     IE214Strainkatpu esocu lG %eBiofilm Planktonic cellsFig. 2 Glucose uptake expressed in % for the four Staphylococcusepidermidis strains in biofilm and in the planktonic form. Resultsrepresent means plus standard deviations (errors bars) from threeindependent experimentsWorld J Microbiol Biotechnol (2008) 24:423–426 425123quantification of growth rate-related parameters. Appl EnvironMicrobiol 61:741–748Resch A, Rosenstein R, Nerz C, Gotz F (2005) Differential geneexpression profiling of Staphylococcus aureus cultivated underbiofilm and planktonic conditions. Appl Environ Microbiol71:2663–2676Stepanovic´ S, Vukovic´ D, Dakic´ I, Savic´ B, Sˇvabic´-Vlahovic´ M (2000)A modified microtiter-plate test for quantification of staphylo-coccal biofilm formation. J Microbiol Methods 40:175–179Von Eiff C, Peters G, Heilmann C (2002) Pathogenesis of infectionsdue to coagulase-negative staphylococci. Lancet Infec Dis2:677–685Walters III MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS(2003) Contributions of oxygen limitation, and low metabolicactivity to tolerance of Pseudomonas aeruginosa biofilms tociprofloxacin and tobramycin. Antimicrob Agents Chemother47:317–323426 World J Microbiol Biotechnol (2008) 24:423–426123

Staphylococcus epidermidis glucose uptake in biofilm versus planktonic cells

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Staphylococcus epidermidis glucose uptake in biofilm versus planktonic cells

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