Potency and penetration of telavancin in staphylococcal biofilms Authors: Kelly R. Kirker, Steve T. Fisher, & Garth James NOTICE: this is the author’s version of a work that was accepted for publication in International Journal of Antimicrobial Agents. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in International Journal of Antimicrobial Agents, 46, 4, October 2015. DOI: http://dx.doi.org/10.1007/s00792-015-0790-x. Kirker KR, Fisher ST, James GA, "Potency and penetration of telavancin in staphylococcal biofilms," Int J Antimicrob Agents Oct 2015 46(4):451–455. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu Potency and penetration of telavancin in staphylococcal biofilms Kelly R. Kirker ∗, Steve T. Fisher, Garth A. James Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA Abstract Due to the emergence of staphylococcal biofilm infections, the need fo antibiotics is c he p penetration of iffer models. Multip Stap aureus ATCC TCC heterogeneous and sensitive Staph trea flow reactors t Afte biofilm growth in r 1.65 to 2.17 wa qualitatively u in fluorescently l in Fluorescently no a the biofilm stru Introduction Multidrug-r emerged as a major source both of hospital- and community-acquired infec-tions. Healthcare-associated infections alone cost US hospitals an estimated $45 billion dollars annuall associated wit eradicate [2]. B several mechan to penetrate the within the biofi within the bio antibiotics to biofilms is criti Telavancin i lipoglycopeptid vancomycin bu chain [3]. Like synthesis; how that disrupts ba ability [4]. The telavancin has staphylococci t The effect of te biofilm models exhibited subst icro The s, a [7] h such as cell density and antibiotic tolerance, can vary depending on the biofilm model used [8]. For example, Staphylococcus epidermidis ATCC 12228, used in this study, ∗ Corresponding author at: Center for Biofilm y in 2007 [1]. Many of these infections are h bacterial biofilms, which are diffi-cult to iofilm resistance to antibiotics is a result of isms, which may include failure of the agent full depth of the biofilm, inhibited diffusion lm, and phenotypic heterogeneity of bacteria film [2]. As a result, the need for advanced treat multidrug-resistant staphylococci cal. s a semisynthetic, bactericidal e. It has a core structure similar to t is modified to include a lipophilic side vancomycin, telavancin inhibits cell wall ever, it has a second mechanism of action cterial cell membrane potential and perme- se two modes of action may explain why greater bactericidal activity against han van-comycin [4]. lavancin has been studied in a number of . Using a Sorbarod model, telavancin antial has been reported to be a non-biofilm forming strain in 96-well plate models using crystal violet assays, yet this strain readily formed biofilms in more robust biofilm culture systems such as flow cells [8]. Furthermore, despite the aforementioned tela- vancin studies, there are no direct experimental visualisations of the antibiotic penetrating into a biofilm, nor are there studies com-pleted on robust biofilms grown at the solid–liquid–air interface. The aim of this project was to evaluate the potency and penetration of telavancin against staphylococcal biofilms using two different biofilm models. Quantitative analysis of potency was evaluated by plate count to determine log reductions of the antibiotic-treated biofilms relative to control biofilms grown in drip flow reactors (DFRs). Penetration was evaluated qualitatively using confocal scanning laser microscopy to image the penetration of fluorescently labelled telavancin into a biofilm grown in a flow cell. 2. Materials and methods 2.1. Quantitative analysis of telavancin potency A quantitative analysis of antibiotic potency was performed using a Model DFR 110-4 drip flow reactor (BioSurface Technologiesru-cial. The aim of this investigation was to evaluate t telavancin against staphylococcal biofilms using two d le staphylococcal strains, including meticillin-sensitive 29213, vancomycin-intermediate S. aureus A ly vancomycin-intermediate S. aureus ATCC 700698 ylococ-cus epidermidis ATCC 12228, were grown and o determine log reductions due to telavancin treatment. and 24 h of treatment, mean log reductions for telavanc depending on the bacterial strain tested. Penetration sing confocal scanning laser microscopy to image the abelled antibiotic into a staphylococcal biofilm grown labelled telavancin rapidly penetrated the biofilms with cture. esistant staphylococcal infections have antim [5]. plate cell Keywords: Biofilm Staphylococcal Telavancin r advanced otency and ent biofilm hylococcus 700787, meticillin- ted in drip- r 3 days of anged from s evaluated filtration of a flow cell. lteration in bial activity against Staphylococcus aureus strains Calgary Biofilm Device [6,7], 96-well flat-bottom nd biofilms grown on polystyrene disks inside a flow ave also been used. 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Antibiotic efficacy was evaluated using single- species biofilms of meticillin-susceptible S. aureus ATCC 29213, vancomycin vancomycin susceptible Single-s tioning the Dickinson & channel wi test strain. strength TS period of 3 halted, the the treatme treatment s Inc., South (DMSO), di used as a ne were drain tional steril biofilms we (Becton Dic vortexing, ing. The re 10-fold wit tryptic soy bated at ro was counte the numbe cally (base was then ca flow experi sented as t performed PA). 2.2. Fluores Telavanc (Sigma, St L [11]. Telava 5g/mL, m and allowed turewas pu Healthcare in the ima otics were (antibiotic) extinction c belled antib by measur (GenesysTM Waltham, M 2.3. Flow ce Thepene lococcal bi Capillary fl were inocu aureus ATCC fusion of 10 at 37 ◦C. Fo on the stag (Leica Microsystems, Inc., Buffalo Grove, IL). Using a 63× water immersion objective, an image planewas selected near the bottom of the biofilm cluster where it was attached to the glass surface, and 108g/mL telavancin was introduced into the flow cell at a te of sly c cenc . Thi ge a ce in cenc anal sulti rsus er, t or ea that , the ed a the cenc r eac also oftw ults uanti r 3 cont 0.26, , S. a , res r tela 1.39 h tel rpar on an an lo log d the u vanc 65± 700 analy l wa ed as ed as cal a ffect ent ( ain-d uores lar ex eterm tivel d an ed by mg/ h ex-intermediate S. aureusATCC 700787, heterogeneously -intermediate S. aureus ATCC 700698 and meticillin- Staphylococcus epidermidis ATCC 12228. pecies staphylococcal biofilms were initiated by condi- slideswith 10%-strength tryptic soy broth (TSB) (Becton Co., Sparks, MD) for 10min and then inoculating each th 1mL of an overnight culture (108 CFU/mL) of the The biofilm was allowed to grow at 37 ◦C with 10%- B medium flowing at a rate of 10mL/h/channel for a days. After the growth period, flow to the reactor was reactor was set to a horizontal position and 20mL of nt solution was added to the appropriate channel. The olution consisted of 4mg/mL telavancin (Theravance San Francisco, CA) in 50% acidified dimethyl sulfoxide luted to 80g/mL in sterile water. Sterile saline was gative control. Following 24h at 37 ◦C, the treatments ed and the chambers were rinsed with 20mL of addi- e saline. The slides were removed from the reactor. The re scraped into 10mL of Dey–Engley neutralising broth kinson & Co.) and were then disaggregated by 30 s of 2min of sonication and an additional 30 s of vortex- sulting bacterial suspension was then serially diluted h phosphate-buffered saline (PBS) and was plated on agar (Becton Dickinson & Co.). The plates were incu- om temperature for 24–48h and the number of CFU d. Based on the dilution and surface area of the slide, r of CFU per unit area was calculated and logarithmi- 10) transformed. The log reduction of each treatment lculated relative to the saline-treated control. Each drip ment was repeated four times and the results are pre- he mean± standard deviation. Statistical analysis was usingMinitab v.16 software (Minitab Inc., State College, cent labelling of telavancin in was labelled with fluorescein isothiocyanate (FITC) ouis, MO) using standard protein labelling procedures ncin was dissolved in carbonate/bicarbonate buffer at ixed with 75L of FITC solution (10mg/mL in DMSO) to react at room temperature for 1h. The reactionmix- rifiedusingfiltration columns (PDMidiTrapTM G-10;GE Biosciences, Pittsburgh, PA) and was used immediately ging experiments. Concentrations of labelled antibi- determined using the relative absorbance at 280nm versus 490nm (fluorophore) and the respective molar oefficients. Molar extinction coefficients for the unla- iotic and the labelling reagent (FITC) were determined ing the absorbance versus concentration at 280nm 10S UV-Vis Spectrophotometer; Thermo Scientific, A). ll imaging tration of fluorescently labelled telavancin into staphy- ofilms was assessed using capillary flow cells [12]. ow cells (Model FC91; BioSurface Technologies Corp.) lated with 250L of a 108 CFU/mL suspension of S. 29213 in TSB. Biofilms were grown for 24h with per- %-strength TSB medium at a flow rate of 1.0mL/min llowing biofilm growth, the flow cells were mounted e of a Leica SP5 confocal scanning laser microscope flow ra taneou fluores 12min Ima rescen fluores image The re sity ve Howev rately f values values identifi age of fluores ters fo were 7.6.4 s data). 3. Res 3.1. Q Afte ment, 8.12± 29213 12228 ties fo 6.38± for eac counte tributi Me mean sity of for tela and 1. aureus linear interva includ includ statisti icant e treatm not str 3.2. Fl Mo were d respec labelle achiev of a 10 for eac1.0mL/min at room temperature. Images were simul- ollected at a wavelength of 500–550nm (e.g. green e, labelled antibiotic) and transmitted light every 3 s for s experiment was repeated three times. nalysis was conducted by comparing the mean fluo- tensity of individual biofilm clusters with the mean e intensity of the bulk fluid using MetaMorph® 7.7.0.0 ysis software (Molecular Devices, Downingtown, PA). ng data sets resulted in a mean fluorescence inten- time for multiple biofilm clusters for both treatments. he photomultiplier tube settings were optimised sepa- ch experimental run, resulting in fluorescence intensity could not be directly compared. To normalise these peak mean intensity value for each data set was nd the remaining data were calculated as a percent- peak value. Therefore, the percentage of peak mean e versus timewas determined formultiple biofilm clus- h labelled antibiotic treatment. The collected images used to create time-lapse videos using Imaris x64 are (Bitplane AG, South Windsor, CT) (Supplementary tative analysis of telavancin potency days of biofilm growth and 24h of saline treat- rol biofilms had mean log densities of 8.12±0.40, 8.08±0.31 and 7.93±0.16 log10 CFU/cm2 for S. aureus ureus 700698, S. aureus 700787 and S. epidermidis pectively (Fig. 1). The respective mean log densi- vancin-treated biofilms were 5.95±1.54, 6.05±1.37, and 6.28±1.38 log10 CFU/cm2 (Fig. 1). The log density avancin group was significantly lower than its control t (P<0.05) using Student’s t-test assuming one-tail dis- d unequal variances. g reductions were then calculated by subtracting the ensity of the treated biofilm from the mean log den- ntreated control. As a result, the mean log reductions in treatments were 2.17±1.37, 2.07±1.26, 1.70±1.57 1.43CFU/cm2 for S. aureus 29213, S. aureus 700698, S. 787 and S. epidermidis 12228, respectively. A general sis of variance (ANOVA) model with a 95% confidence s then fitted to the log reduction data. ‘Experiment’ was a random effect, whilst ‘strain’ and ‘treatment’ were fixed effects with a potential interaction. Results of this nalysis indicated that there was no statistically signif- of strain (P=0.943) or interaction between strain and P=0.871). Thus, the effect of telavancin treatment was ependent in these experiments. cent labelling of antibiotics tinction coefficientsdetermined for telavancinandFITC ined to be 2808.1mol−1 cm−1 and 72,833mol−1 cm−1, y, and were used to determine the concentration of tibiotic following purification. Optimum labelling was dissolving 5g in 1mL of PBS and then adding 75L mL FITC solution. These solutions were prepared fresh periment. 8.00 9.00 12 . epid * * * * Fig. 1. Mean b L). Re *Significantly 3.3. Flow ce Staphylo biofilms in biofilms for ters in the m middleof th Time-lapse tomof the b every3 s for antibiotic. T biofilm rath selected be antibiotic p sitated a sc biofilm. The techniques Using co within the intensity w was also id cently label (Fig. 2)with mean maxi alteration o exposure. 4. Discussi Telavanc ment of ad infections d is also app hospital-ac bacterial pn (MRSA) on able [14]. H ted ofte ent i e go phy t rob he D inuo she gh in d tis ts th0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 29213 700698 700787 Lo g C FU /c m 2 Test Strain S. aureus S iofilm log densities for biofilms treatedwith saline (control) and telavancin (80g/m different from control (P<0.05). ll imaging coccus aureus 29213 formed extensive, heterogeneous the glass capillary flow cell after 24h of growth. Thick med in the corners of the flowcell and also as large clus- iddle of the tube walls. These isolated biofilms in the e tubewallswereused for thepenetrationexperiments. images were taken of a single focal plane near the bot- iofilm clusterwhere itwas attached to the glass surface 12min, startingca. 40 sprior to introducing the labelled he technique of capturing one image plane within the er than an entire z-stack of the entire biofilm was associa [2]. An treatm [12]. Th into sta agains In t is cont ing low Althou infecte nutriencause preliminary experiments indicated that labelled enetration occurred rapidly. The rapid diffusion neces- an rate that was not achievable if imaging the entire refore, imagining one image plane was selected, using similar to those previously published [12]. mputer image analysis, a more densely packed region biofilm was selected to measure mean fluorescence ith time, and an identical region within the flow path entified to capture background fluorescence. Fluores- led telavancin rapidly penetrated the biofilm clusters a characteristic S-shaped curve (Fig. 3) [12,13],with the mum fluorescence intensity plateauing at ca. 600 s. No f biofilm structure was observed during the antibiotic on in is approved in the USA and Canada for the treat- ult patients with complicated skin and skin-structure ue to susceptible Gram-positive pathogens. Telavancin roved in the USA and Europe for the treatment of quired bacterial pneumonia and ventilator-associated eumonia due to S. aureus [meticillin-resistant S. aureus ly in Europe] when no other alternatives are suit- ospital-acquired staphylococcal infections are often more, the s a model ma ous biofilm robustbiofi DFR biofilm (12228) use ‘non-biofilm assays but and the stu In a ph for telavan (10mg/kg/d telavancin because it the recomm tration, the 2.16±1.37, aureus 2921 12228, resp for a treatm Whilst t of telavanc penetrating microscope FITC-labelle228 Control Telavancin ermidis sults are themean± standard deviation of four separate experiments. with bacterial biofilms, which are difficult to eradicate n-cited explanation for the failure of antibiotic biofilm s the failure of the antibiotic to penetrate the biofilm al of this projectwas to visualise telavancin penetration lococcal biofilms and to assess the efficacy of telavancin ust staphylococcal biofilms. FR system, biofilms are grown on a solid surface that usly wetted with medium at a slow flow rate, provid- ar stress conditions at a solid–liquid–air interface [9]. vitromodel systems cannot replicate conditionswithin sue, the DFR simulates slow flow of body fluids and at may flow through a biofilm-infected tissue. Further- ubstrate material used in this study (hydroxyapatite) is terial for bone and certain orthopaedic implants. Previ- studies have not examined the effects of telavancin on lmsgrown in thismanner [5–7]. The robustnatureof the was further demonstrated by the S. epidermidis strain d in this study. This strain had been reported to be a -former’ based on simple 96-well plate-based biofilm readily formed biofilms in more complex systems [8] dy described herein. armacokinetic study, the peak plasma concentration cin at the recommended daily therapeutic dosage ay) was 82.2±27.3g/mL [15]. Therefore, 80g/mL was selected as the DFR treatment concentration aligned with the peak plasma concentration using ended daily therapeutic dosage. Using this concen- mean log reductions for telavancin treatments were 2.07±1.26, 1.07±1.57 and 1.65±1.43CFU/cm2 for S. 3, S. aureus 700698, S. aureus 700787 and S. epidermidis ectively. The variability in these results are quite typical ent using the DFR [10]. he DFR provided quantitative data for the potency in, it did not provide for visualisation of the drug the biofilms. The flow-cell reactor was designed for imaging [12] and allowed for the visualisation of d telavancin penetration into staphylococcal biofilms. Fig. 2. Combin fluorescently l the first appea -40 -20 0 20 40 60 80 100 120 0 M ax im um F lu or es ce nc e In te ns ity (% ) Fig. 3. Image versus time fo cently labelled individual tela ance of the lab However, th compared w At 80g/m cent signal.ed transmitted light and fluorescence images illustrating a cross-section of Staphylococc abelled telavancin with time. Both the biofilm and the bulk fluid regions used to measure rance of the labelled antibiotic in the focal plane. 100 200 300 400 500 600 700 Time (Seconds) analysis results showing percentage of peak fluorescence intensity r Staphylococcus aureus ATCC 29213 biofilms treated with fluores- telavancin. Results represent themean± standard deviation of three vancin-treated biofilms. Time zero corresponds to the first appear- elled antibiotic in the focal plane. e telavancin concentration utilised had to be increased ith the concentration used in the DFR experiments. L, FITC-labelled telavancin produced a weak fluores- Increasing the concentration to 108g/mL produced a stronger flu tration was The flow-c penetrated curve (Fig. vancin prod other staph system. Biofilm nisms, whi full depth and phenot The study d otic efficacy into such b penetrated be involve techniques the efficacy resistant bi Funding vance, Inc. ( by Therava Competi Ethical aus aureus ATCC 29213 biofilm and the accumulation of 108g/mL of mean fluorescence intensity are outlined. Time zero corresponds to orescent signal sufficient for visualisation. This concen- still within the peak plasma concentration range [15]. ell experiments demonstrated that telavancin rapidly the S. aureus 29213 biofilms in a characteristic S-shaped 3). Since the DFR results (Fig. 1) revealed that tela- uced similar results regardless of the strain tested, the ylococcal strains were not examined in the flow-cell resistance to antibiotics is a result of several mecha- ch may include failure of the agent to penetrate the of the biofilm, inhibited diffusion within the biofilm, ypic heterogeneity of bacteria within the biofilm [2]. escribed herein presents methods to evaluate antibi- on robust biofilms and to visualise their penetration iofilms. The results indicated that telavancin rapidly staphylococcal biofilms. Thus, other mechanisms must d in the antibiotic tolerance of these biofilms. The described may prove useful in further examining of advanced antibiotics designed to treat multidrug- ofilms. : Financial support for thisworkwas provided by Thera- South San Francisco, CA). Telavancin was also supplied nce, Inc. ng interests: None declared. pproval: Not required. Appendix A. Supplementary data Supplementary data associatedwith this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015. 05.022 References [1] Scott II RD. The direct medical costs of healthcare-associated infections in U.S. hospitals and the benefits of prevention. Atlanta, GA: US Centers for Disease Control and Prevention; 2009. p. 1–13. [2] Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–22. [3] Leadbetter MR, Adams SM, Bazzini B, Fatheree PR, Karr DE, Krause KM, et al. Hydrophobic vancomycin derivatives with improved ADME properties: dis- covery of telavancin (TD-6424). J Antibiot (Tokyo) 2004;57:326–36. [4] Higgins DL, Chang R, Debabov DV, Leung J, Wu T, Krause KM, et al. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity inmethicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2005;49:1127–34. [5] Gander S, KinnairdA, FinchR. Telavancin: in vitro activity against staphylococci in a biofilm model. J Antimicrob Chemother 2005;56:337–43. [6] LaPlanteKL,Mermel LA. In vitro activities of telavancin andvancomycin against biofilm-producing Staphylococcus aureus, S. epidermidis, and Enterococcus fae- calis strains. Antimicrob Agents Chemother 2009;53:3166–9. [7] SmithK,Gemmell CG, LangS. Telavancin shows superior activity tovancomycin with multidrug-resistant Staphylococcus aureus in a range of in vitro biofilm models. Eur J Clin Microbiol Infect Dis 2013;32:1327–32. [8] Dice B, Stoodley P, Buchinsky F, Metha N, Ehrlich GD, Hu FZ. Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro. Biofouling 2009;25:367–75. [9] Goeres DM, Hamilton MA, Beck NA, Buckingham-Meyer K, Hilyard JD, Loet- terle LR, et al. A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor. Nat Protoc 2009;4: 783–8. [10] Buckingham-Meyer K, Goeres DM, Hamilton MA. Comparative evalua- tion of biofilm disinfectant efficacy tests. J Microbiol Methods 2007;70: 236–44. [11] Hlady V, Buijs J, Jennissen HP. Methods for studying protein adsorption. Meth- ods Enzymol 1999;309:402–29. [12] Stewart PS, Davison WM, Steenbergen JN. Daptomycin rapidly pene- trates a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother 2009;53:3505–7. [13] Rani SA, Pitts B, Stewart PS. Rapid diffusion of fluorescent tracers into Staphy- lococcus epidermidis biofilms visualized by time lapse microscopy. Antimicrob Agents Chemother 2005;49:728–32. [14] Theravance I. Package insert; 2013. http://www.vibativ.com/ [accessed 01.06.15]. [15] Saraf LJ, Wilson SE. Telavancin, a new lipoglycopeptide antimicrobial, in complicated skin and soft tissue infections. Infect Drug Resist 2011;4: 87–95. Corp., Bozeman, MT) [9,10] equipped with hydroxyapatite- coated glass microscope slides (Clarkston Chromatography, South Williamsport, PA). Antibiotic efficacy was evaluated using single- species biofilms of meticillin-susceptible S. aureus ATCC 29213, vancomycin-intermediate S. aureusATCC 700787, heterogeneously vancomycin-intermediate S. aureus ATCC 700698 and meticillin- susceptible Staphylococcus epidermidis ATCC 12228. Single-species staphylococcal biofilms were initiated by condi- tioning the slideswith 10%-strength tryptic soy broth (TSB) (Becton Dickinson & Co., Sparks, MD) for 10min and then inoculating each channel with 1mL of an overnight culture (108 CFU/mL) of the test strain. The biofilm was allowed to grow at 37 ◦C with 10%- strength TSB medium flowing at a rate of 10mL/h/channel for a period of 3 days. After the growth period, flow to the reactor was halted, the reactor was set to a horizontal position and 20mL of the treatment solution was added to the appropriate channel. The treatment solution consisted of 4mg/mL telavancin (Theravance Inc., South San Francisco, CA) in 50% acidified dimethyl sulfoxide (DMSO), diluted to 80g/mL in sterile water. Sterile saline was used as a negative control. Following 24h at 37 ◦C, the treatments were drained and the chambers were rinsed with 20mL of addi- tional sterile saline. The slides were removed from the reactor. The biofilms were scraped into 10mL of Dey–Engley neutralising broth (Becton Dickinson & Co.) and were then disaggregated by 30 s of vortexing, 2min of sonication and an additional 30 s of vortex- ing. The resulting bacterial suspension was then serially diluted 10-fold with phosphate-buffered saline (PBS) and was plated on tryptic soy agar (Becton Dickinson & Co.). The plates were incu- bated at room temperature for 24–48h and the number of CFU was counted. Based on the dilution and surface area of the slide, the number of CFU per unit area was calculated and logarithmi- cally (base 10) transformed. The log reduction of each treatment was then calculated relative to the saline-treated control. Each drip flow experiment was repeated four times and the results are pre- sented as the mean± standard deviation. Statistical analysis was performed usingMinitab v.16 software (Minitab Inc., State College, PA). 2.2. Fluorescent labelling of telavancin Telavancin was labelled with fluorescein isothiocyanate (FITC) (Sigma, St Louis, MO) using standard protein labelling procedures [11]. Telavancin was dissolved in carbonate/bicarbonate buffer at 5g/mL, mixed with 75L of FITC solution (10mg/mL in DMSO) and allowed to react at room temperature for 1h. The reactionmix- turewas purifiedusingfiltration columns (PDMidiTrapTM G-10;GE Healthcare Biosciences, Pittsburgh, PA) and was used immediately in the imaging experiments. Concentrations of labelled antibi- otics were determined using the relative absorbance at 280nm (antibiotic) versus 490nm (fluorophore) and the respective molar extinction coefficients. Molar extinction coefficients for the unla- belled antibiotic and the labelling reagent (FITC) were determined by measuring the absorbance versus concentration at 280nm (GenesysTM 10S UV-Vis Spectrophotometer; Thermo Scientific, Waltham, MA). 2.3. Flow cell imaging Thepenetrationof fluorescently labelled telavancin into staphy- lococcal biofilms was assessed using capillary flow cells [12]. Capillary flow cells (Model FC91; BioSurface Technologies Corp.) were inoculated with 250L of a 108 CFU/mL suspension of S. aureus ATCC 29213 in TSB. Biofilms were grown for 24h with per- fusion of 10%-strength TSB medium at a flow rate of 1.0mL/min at 37 ◦C. Following biofilm growth, the flow cells were mounted on the stage of a Leica SP5 confocal scanning laser microscope (Leica Microsystems, Inc., Buffalo Grove, IL). Using a 63× water immersion objective, an image planewas selected near the bottom of the biofilm cluster where it was attached to the glass surface, and 108g/mL telavancin was introduced into the flow cell at a flow rate of 1.0mL/min at room temperature. Images were simul- taneously collected at a wavelength of 500–550nm (e.g. green fluorescence, labelled antibiotic) and transmitted light every 3 s for 12min. This experiment was repeated three times. Image analysis was conducted by comparing the mean fluo- rescence intensity of individual biofilm clusters with the mean fluorescence intensity of the bulk fluid using MetaMorph® 7.7.0.0 image analysis software (Molecular Devices, Downingtown, PA). The resulting data sets resulted in a mean fluorescence inten- sity versus time for multiple biofilm clusters for both treatments. However, the photomultiplier tube settings were optimised sepa- rately for each experimental run, resulting in fluorescence intensity values that could not be directly compared. To normalise these values, the peak mean intensity value for each data set was identified and the remaining data were calculated as a percent- age of the peak value. Therefore, the percentage of peak mean fluorescence versus timewas determined formultiple biofilm clus- ters for each labelled antibiotic treatment. The collected images were also used to create time-lapse videos using Imaris x64 7.6.4 software (Bitplane AG, South Windsor, CT) (Supplementary data). 3. Results 3.1. Quantitative analysis of telavancin potency After 3 days of biofilm growth and 24h of saline treat- ment, control biofilms had mean log densities of 8.12±0.40, 8.12±0.26, 8.08±0.31 and 7.93±0.16 log10 CFU/cm2 for S. aureus 29213, S. aureus 700698, S. aureus 700787 and S. epidermidis 12228, respectively (Fig. 1). The respective mean log densi- ties for telavancin-treated biofilms were 5.95±1.54, 6.05±1.37, 6.38±1.39 and 6.28±1.38 log10 CFU/cm2 (Fig. 1). The log density for each telavancin group was significantly lower than its control counterpart (P<0.05) using Student’s t-test assuming one-tail dis- tribution and unequal variances. Mean log reductions were then calculated by subtracting the mean log density of the treated biofilm from the mean log den- sity of the untreated control. As a result, the mean log reductions for telavancin treatments were 2.17±1.37, 2.07±1.26, 1.70±1.57 and 1.65±1.43CFU/cm2 for S. aureus 29213, S. aureus 700698, S. aureus 700787 and S. epidermidis 12228, respectively. A general linear analysis of variance (ANOVA) model with a 95% confidence interval was then fitted to the log reduction data. ‘Experiment’ was included as a random effect, whilst ‘strain’ and ‘treatment’ were included as fixed effects with a potential interaction. Results of this statistical analysis indicated that there was no statistically signif- icant effect of strain (P=0.943) or interaction between strain and treatment (P=0.871). Thus, the effect of telavancin treatment was not strain-dependent in these experiments. 3.2. Fluorescent labelling of antibiotics Molar extinction coefficientsdetermined for telavancinandFITC were determined to be 2808.1mol−1 cm−1 and 72,833mol−1 cm−1, respectively, and were used to determine the concentration of labelled antibiotic following purification. Optimum labelling was achieved by dissolving 5g in 1mL of PBS and then adding 75L of a 10mg/mL FITC solution. These solutions were prepared fresh for each experiment. Lo g C FU/ cm 2 Test Strain S.aureus S.epidermidis * * * * Fig. 1.Mean biofilm log densities for biofilms treated with saline (control) and telavancin (80 g/mL). Results are the mean±standard deviation of four separate experiments. *Significantly different from control (P< 0.05). 3.3. Flow cel imaging Staphylococcus aureus29213 formed extensive, heterogeneous biofilms in the glass capilary flow cel after 24 h of growth. Thick biofilms formed in the corners of the flow cel and also as large clus- ters in the middle of the tube wals. These isolated biofilms in the middle of the tube wals were used for the penetration experiments. Time-lapse images were taken of a single focal plane near the bot- tom of the biofilm cluster where it was attached to the glass surface every 3 s for 12 min, starting ca. 40 s prior to introducing the labeled antibiotic. The technique of capturing one image plane within the biofilm rather than an entirez-stack of the entire biofilm was selected because preliminary experiments indicated that labeled antibiotic penetration occurred rapidly. The rapid diffusion neces- sitated a scan rate that was not achievable if imaging the entire biofilm. Therefore, imagining one image plane was selected, using techniques similar to those previously published[12]. Using computer image analysis, a more densely packed region within the biofilm was selected to measure mean fluorescence intensity with time, and an identical region within the flow path was also identified to capture background fluorescence. Fluores- cently labeled telavancin rapidly penetrated the biofilm clusters (Fig. 2) with a characteristic S-shaped curve (Fig. 3)[12,13], with the mean maximum fluorescence intensity plateauing at ca. 600 s. No alteration of biofilm structure was observed during the antibiotic exposure. 4. Discussion Telavancin is approved in the USA and Canada for the treat- ment of adult patients with complicated skin and skin-structure infections due to susceptible Gram-positive pathogens. Telavancin is also approved in the USA and Europe for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia due toS. aureus[meticilin-resistantS. aureus (MRSA) only in Europe] when no other alternatives are suit- able[14]. Hospital-acquired staphylococcal infections are often associated with bacterial biofilms, which are difficult to eradicate [2]. An often-cited explanation for the failure of antibiotic biofilm treatment is the failure of the antibiotic to penetrate the biofilm [12]. The goal of this projectwasto visualise telavancin penetration into staphylococcal biofilms and to assess the efficacy of telavancin against robust staphylococcal biofilms. In the DFR system, biofilms are grown on a solid surface that is continuously wetted with medium at a slow flow rate, provid- ing low shear stress conditions at a solid–liquid–air interface[9]. Although in vitro model systems cannot replicate conditions within infected tissue, the DFR simulates slow flow of body fluids and nutrients thatmayflow through a biofilm-infected tissue. Further- more, the substrate material used in this study (hydroxyapatite) is a model material for bone and certain orthopaedic implants. Previ- ous biofilm studies have not examined the effects of telavancin on robust biofilms grown in this manner[5–7]. The robust nature of the DFR biofilmwasfurther demonstrated by theS. epidermidisstrain (12228) used in this study. This strain had been reported to be a ‘non-biofilm-former’ based on simple 96-wel plate-based biofilm assays but readily formed biofilms in more complex systems[8] and the study described herein. In a pharmacokinetic study, the peak plasma concentration for telavancin at the recommended daily therapeutic dosage (10 mg/kg/day)was 82.2±27.3 g/mL[15]. Therefore, 80g/mL telavancin was selected as the DFR treatment concentration because it aligned with the peak plasma concentration using the recommended daily therapeutic dosage. Using this concen- tration, the mean log reductions for telavancin treatments were 2.16±1.37, 2.07±1.26, 1.07±1.57 and 1.65±1.43 CFU/cm2forS. aureus29213,S. aureus700698,S. aureus700787 andS. epidermidis 12228, respectively. The variability in these results are quite typical for a treatment using the DFR[10]. Whilst the DFR provided quantitative data for the potency of telavancin, it did not provide for visualisation of the drug penetrating the biofilms. The flow-cel reactor was designed for microscope imaging [12]and alowed for the visualisation of FITC-labeled telavancin penetration into staphylococcal biofilms. Fig. 2.Combined transmitted light and fluorescence images ilustrating a cross-section ofStaphylococcus aureusATCC 29213 biofilm and the accumulation of 108 g/mL of fluorescently labeled telavancin with time. Both the biofilm and the bulk fluid regions used to measure mean fluorescence intensity are outlined. Time zero corresponds to the first appearance of the labeled antibiotic in the focal plane. Ma xi mu m F luo res ce nc e I nte nsi ty (% ) Time (Seconds) Fig. 3.Image analysis results showing percentage of peak fluorescence intensity versus time forStaphylococcus aureusATCC 29213 biofilms treated with fluores- cently labeled telavancin. Results represent the mean±standard deviation of three individual telavancin-treated biofilms. Time zero corresponds to the first appear- ance of the labeled antibiotic in the focal plane. However, the telavancin concentration utilised had to be increased compared with the concentration used in the DFR experiments. At 80 g/mL, FITC-labeled telavancin produced a weak fluores- cent signal. Increasing the concentration to 108 g/mL produced a stronger fluorescent signal sufficient for visualisation. This concen- tration was stil within the peak plasma concentration range[15]. The flow-cel experiments demonstrated that telavancin rapidly penetrated theS. aureus29213 biofilms in a characteristic S-shaped curve (Fig. 3). Since the DFR results (Fig. 1) revealed that tela- vancin produced similar results regardless of the strain tested, the other staphylococcal strains were not examined in the flow-cel system. Biofilm resistance to antibiotics is a result of several mecha- nisms, whichmay include failure of the agent to penetrate the ful depth of the biofilm, inhibited diffusion within the biofilm, and phenotypic heterogeneity of bacteria within the biofilm[2]. The study described herein presents methods to evaluate antibi- otic efficacy on robust biofilms and to visualise their penetration into such biofilms. The results indicated that telavancin rapidly penetrated staphylococcal biofilms. Thus, other mechanisms must be involved in the antibiotic tolerance of these biofilms. The techniques describedmay prove useful in further examining the efficacy of advanced antibiotics designed to treat multidrug- resistant biofilms. Funding: Financial support for this work was provided by Thera- vance, Inc. (South San Francisco, CA). Telavancin was also supplied by Theravance, Inc. Competing interests: None declared. Ethical approval: Not required. Appendix A. Supplementary data Supplementary data associatedwith this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015. 05.022 References [1] Scott II RD. The direct medical costs of healthcare-associated infections in U.S. hospitals and the benefits of prevention. Atlanta, GA: US Centers for Disease Control and Prevention; 2009. p. 1–13. [2] Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–22. [3] Leadbetter MR, Adams SM, Bazzini B, Fatheree PR, Karr DE, Krause KM, et al. Hydrophobic vancomycin derivatives with improved ADME properties: dis- covery of telavancin (TD-6424). J Antibiot (Tokyo) 2004;57:326–36. [4] Higgins DL, Chang R, Debabov DV, Leung J, Wu T, Krause KM, et al. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity inmethicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2005;49:1127–34. [5] Gander S, KinnairdA, FinchR. Telavancin: in vitro activity against staphylococci in a biofilm model. J Antimicrob Chemother 2005;56:337–43. [6] LaPlanteKL,Mermel LA. In vitro activities of telavancin andvancomycin against biofilm-producing Staphylococcus aureus, S. epidermidis, and Enterococcus fae- calis strains. Antimicrob Agents Chemother 2009;53:3166–9. [7] SmithK,Gemmell CG, LangS. Telavancin shows superior activity tovancomycin with multidrug-resistant Staphylococcus aureus in a range of in vitro biofilm models. Eur J Clin Microbiol Infect Dis 2013;32:1327–32. [8] Dice B, Stoodley P, Buchinsky F, Metha N, Ehrlich GD, Hu FZ. Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro. Biofouling 2009;25:367–75. [9] Goeres DM, Hamilton MA, Beck NA, Buckingham-Meyer K, Hilyard JD, Loet- terle LR, et al. A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor. Nat Protoc 2009;4: 783–8. [10] Buckingham-Meyer K, Goeres DM, Hamilton MA. Comparative evalua- tion of biofilm disinfectant efficacy tests. J Microbiol Methods 2007;70: 236–44. [11] Hlady V, Buijs J, Jennissen HP. Methods for studying protein adsorption. Meth- ods Enzymol 1999;309:402–29. [12] Stewart PS, Davison WM, Steenbergen JN. Daptomycin rapidly pene- trates a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother 2009;53:3505–7. [13] Rani SA, Pitts B, Stewart PS. Rapid diffusion of fluorescent tracers into Staphy- lococcus epidermidis biofilms visualized by time lapse microscopy. Antimicrob Agents Chemother 2005;49:728–32. [14] Theravance I. Package insert; 2013. http://www.vibativ.com/ [accessed 01.06.15]. [15] Saraf LJ, Wilson SE. Telavancin, a new lipoglycopeptide antimicrobial, in complicated skin and soft tissue infections. Infect Drug Resist 2011;4: 87–95.