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E

WA

D

WORNICZEK1

, U

RSZULA

N

AWROT1

, A

LICJA

S

ENIUK1

, K

ATARZYNA

W

ŁODARCZYK1

,

R

AFAŁ

B

IAŁYNICKI

−B

IRULA2

The

in Vitro

Effect of a Silver−Containing Dressing

on Biofilm Development

Działanie opatrunku zawierającego srebro na rozwój biofilmu

in vitro

1 Department of Microbiology, Wrocław Medical University, Poland

2 Department of Dermatology, Venereology, and Allergology, Wroclaw Medical University, Poland Adv Clin Exp Med 2009, 18, 3, 277–281

ISSN 1230−025X

ORIGINAL PAPERS

© Copyright by Wroclaw Medical University

Abstract

Background.Biofilms are surface−associated aggregates of microorganisms responsible for a number of diseases in man. They play an important role in chronic infections, including those in wounds. One rapid therapeutic inter− vention is silver−containing dressings used to control wound bioburden.

Objectives.The aim of this study was to evaluate the antimicrobial effect of a silver−containing hydro−fiber dress− ing (Textus bioactiv) on bacterial and fungal biofilms.

Material and Methods.In in vitroexperiments, clinical strains of EnterococcusandStaphylococcusbacteria and

Candida fungi were used in a microtiter plate model.

Results.The results showed that the dressing prevented biofilm formation by EnterococciandStaphylococci, but not by Candida. The incubation of various species of yeast with silver−containing fibers did not inhibit the devel− opment of their biofilms.

Conclusions.Silver−containing Textus bioactiv dressing constitutes an efficacious way of rapid therapeutic inter− vention that may prevent an infected wound from developing a Gram−positive bacterial biofilm. Such activity may be not sufficient in the case of heavily Candida−infected wounds (Adv Clin Exp Med 2009, 18, 3, 277–281). Key words:silver, dressing, biofilm, wounds, Candida albicans, Enterococcus, lack of inhibitory action.

Streszczenie

Wprowadzenie. Biofilmy, związane z powierzchnią zbiorowiska drobnoustrojów, są odpowiedzialne za liczne choroby człowieka. Odgrywają ważną rolę w rozwoju zakażeń przewlekłych, w tym również infekcji ran. Jedną z szybkich metod terapeutycznych stosowanych w leczeniu ran zakażonych są opatrunki zawierające srebro. Cel pracy. W badaniu dokonano oceny działania opatrunku hydroabsorbcyjnego zawierającego srebro (Textus bio− activ) na rozwój biofilmów bakteryjnych i grzybiczych.

Materiał i metody. W doświadczeniach prowadzonych in vitrowykorzystano szczepy kliniczne bakterii z rodza− ju Enterococcus, Staphylococcus, grzyby z rodzaju Candida oraz model płytki titracyjnej.

Wyniki. Wyniki badań wykazały hamujące działanie opatrunku na rozwój biofilmów bakterii Enterococcus i Sta− phylococcusi brak oddziaływania na grzyby z rodzaju Candida. Włókna zawierające srebro, inkubowane z różny− mi gatunkami grzybów drożdżopodobnych, nie hamowały rozwoju ich biofilmów.

Wnioski. Zawierający srebro opatrunek Textus bioactiv jest skutecznym sposobem ochrony rany przed rozwojem biofilmów bakterii Gram−dodatnich. Aktywność „przeciwbiofilmowa” opatrunku w przypadku ciężkich zakażeń ran spowodowanych przez grzyby Candidamoże być niewystarczająca (Adv Clin Exp Med 2009, 18, 3, 277–281). Słowa kluczowe: srebro, opatrunek, biofilm, rany, Candida albicans, Enterococcus,brak działania hamującego.

In most environments, microorganisms are attached to a surface and are in close contact with each other. This gated community is known as a biofilm [1, 2]. Pathogens that adhere to damaged tissue form a biofilm which is difficult to eradicate

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delay or even prevent the healing process [7–9]. The profile of pathogens frequently isolated from various wounds includes coagulase−positive and coagulase−negative staphylococci, enterococci, Gram−negative rods, and yeasts [9, 10].

Since antibiotics fail to eradicate biofilms, new therapeutic strategies have been introduced. Silver− containing dressings are a part of the management of wounds [11]. The bactericidal properties of ionic silver result from its effective disruption of the organization of bacterial DNA [12]. Thus there are good indications for the use of silver dressings to remove or reduce the bioburden in burns and open wounds [13, 14]. One such dressing is the Textus bioactiv, designed for the local, active treatment of wounds. The dressing absorbs and neutralizes exu− dations with pathogens and necrotic tissue. The double−layer membrane is made of fibers with sil− ver zeolite, super−absorbing fibers, and fibers pre− venting the dressing from sticking to the wound. In clinical practice, the one−directional migration of silver ions from the zeolite to the absorbing fibers means a lack of bio−accessibility of silver to the tis− sue. The high absorption (42 g/100 cm2) and con−

trolled release of silver during Textus bioactiv usage should ensure an effective elimination of microorganisms [15].

The aim of this preliminary study was to assess the ability of the Textus bioactiv silver−con− taining dressing to prevent biofilm formation by bacteria and yeasts in vitro.

Material and Methods

The silver−containing Textus bioactive dress− ing and the nonsilver−containing Textus multi dressing (BioCELL® Biotechnologie GmbH,

Germany) were used in this study. The experi− ments were performed on clinical strains of Enterococcus faecalis (3 strains), E. faecium (3 strains), Staphylococcus aureusMSSA (2 strains), and S. epidermidis MRCNS (3 strains) bacteria and Candida albicans(3 strains), C. parapsilosis (2 strains), C. glabrata (2 strains), C. kefyr (2 strains), C. krusei (2 strains), and C. guiller− mondii(2 strains) yeasts. The strains were isolated from skin, blood, and urinary and respiratory tract infections of patients hospitalized in Wrocław (Poland). They were defined in authors’ previous studies (data not published) as producers of biofilms. The bacteria were cultured in Tryptic Soy Broth (TSB, Graso, Poland) and the fungi in 0.5% Yeast Nitrogen Basic medium with 0.5% glucose (Difco, USA). To allow the detection of a biofilm, a microtiter−plate model was used. Two types of control experiments, one of microbial cul−

ture with the non−silver−containing non−antimicrobial Textus−multi dressing and one with microbial culture alone, were performed for each experimental strain.

The dressings (with and without silver ions) were aseptically cut to a size of 0.4 cm2and placed

into microtiter plate wells containing the microbial cultures. The plates were incubated at 37°C for 1–2 days (bacteria) or for 2–6 days (fungi). Bio− films which formed in the wells were assessed by staining with 1% crystal violet and measuring the optical density (OD) at 570 nm. The results were graded in categories of strong (OD ≥2.0), moder− ate (1.0 ≤OD < 2.0), weak (0.1 < OD < 1.0), and no biofilm (OD ≤0.1). The culture and detection of biofilms was conducted according to Toledo− Arana et al. [16] (for enterococci), Ando et al. [17] (for staphylococci), and Li et al. [18] (for yeasts).

Results and Discussion

Textus bioactiv silver−containing dressing pre− vented Staphylococcus and Enterococcus biofilm formation. The spectrophotometric studies showed that the dressing was highly effective in killing planktonic bacteria and inhibiting the development of biofilm in vitrowithin 48 hours. Following the addition of the control (bacteria in TSB), all the bacterial biofilms were significant within 24–48 hours (Fig. 1a). The optical densities (thicknesses) of the observed biofilms were in the range of 0.8–1.8 for S. aureus, 0.4–1.0 for coagulase−nega− tive staphylococci, 1.4–2.2 for E. faecalis, and 0.8 to > 2.5 for E. faecium.However, contact times of 24–48 hours with the silver−containing dressing produced weak or no biofilms in all the tested bacterial strains. Studies performed in vivo by H. Hofman−Stefanek and A. Karolczyk [14] con− cerning the treatment of infected wounds in chil− dren also demonstrated a high efficacy of this dressing in the eradication of Gram−positive and Gram−negative bacteria. Hence Textus bioactiv enables a rapid elimination of bacterial pathogens and prevents biofilm formation, which favors the natural healing process.

Surprisingly, weak or no biofilms were obtained with the control dressing Textus multi, a result similar to that of Textus bioactive. The inhibitory properties of the non−silver−containing Textus multi indicate that in this experimental model, the activity of silver ions is not the only mechanism acting on biofilm development. Similar observations were made by Persival et al., who noted inhibitory properties of some fibers of the Hydrofiber®dressing. In the biofilm model, the

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strains

szczep

A1–2 Candida parapsilosis(2 strains) D1–2 Candida krusei(2 strains) B1–2 Candida kefyr(2 strains) E1–3 Candida albicans(3 strains) C1–2 Candida glabrata(2 strains) F1–2Candida guilliermondii (2 strains)

strains

szczep

Fig. 1 a, b. Biofilm production by selected strains of bacteria (a) and yeasts (b) in the presence of Textus bioactiv, Textus multi dressings, and culture broth without dressing. The figures show mean values of absorbance (optical den− sity OD) obtained from three experiments (each strain tested in triplicate). The arrows show absorbance over the detection limit

Fig. 1a, b.Wytwarzanie biofilmu przez wybrane szczepy bakterii (a) i grzybów drożdżopodobnych (b) w obecności opatrunków Textus bioactiv, Textus multi i w hodowli bulionowej (bez opatrunku). Wykresy przedstawiają średnie wartości absorbancji (gęstości optycznej OD) uzyskane w 3 eksperymentach (każdy szczep testowano

w 3 powtórzeniach). Strzałki pokazują wartości absorbancji przekraczające poziom detekcji czytnika

Textus bioactiv Textus multi (control 1) culture broth (control 2)

A1–3 Enterococcus faecalis(3 strains) B1–3 Enterococcus faecium(3 strains) C1–3 Staphylococcus aureus(2 strains) D1–3 Staphylococcus epidermidis(3 strains) OF optical density

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An interesting observation was the lack of inhibitory properties of Textus bioactiv on fungal biofilm formation. Strong and moderate biofilms (OD 1.2 to > 2.5) were clearly apparent for all strains after 2–6 days of exposure to ionic silver Textus bioactive and, in some cases, the non−silver− containing Textus multi (OD 0.2–2.5), whereas weak or no biofilms were noted in the control wells containing yeast cultures without dressing. All Candida species ultimately developed intensive biofilms, thicker than in the controls (Fig. 1b). The present authors’ previous study [10] indicated that Textus bioactiv soaked with suspensions of plank− tonic Candida cells showed strong candicidal activity. The dressing easily eradicated 102–105

CFU/ml Candida blastospores and killed up to 90% of suspensions with a density of 106CFU/ml.

In the present study it was observed that the dress− ing incubated with a heavy culture of Candida (106CFU/ml) in YNB medium strongly promoted

biofilm formation. This finding may suggest the possibility of a regulatory effect of silver ions and/or the fibers making up the dressings on processes inducing Candida morphogenesis and biofilm formation. It seems clear that in this exper−

iment the silver concentration was too low to inhibit the development of Candidabiofilm. The conditions for fungal growth (e.g. nutrient con− centration, pH) and the level of silver ion release (the concentration of calcium ions) in microplates are different from those in wounds. Therefore the results obtained cannot predict the dressing’s effectiveness in vivo. To the present authors’ knowledge there are no reports about treating Candida−infected wounds with Textus bioactive. In view of the present results one may only sup− pose that Textus bioactiv will probably effective− ly prevent single Candida cells from reaching uninfected wounds, but its fungicidal activity in the case of heavily Candida−infected wounds may be not sufficient.

In summary, the silver−containing Textus bioactiv dressing constitutes an efficacious way of rapid therapeutic intervention that will prevent an infected wound from developing a bacterial biofilm. It succeeded in eradicating Gram−positive pathogens in vitro within 1–2 days. The develop− ment of Candida biofilms in the presence of Textus bioactiv requires explanation on the basis of a further detailed study.

Acknowledgements. The authors thank BioCELL Biotechnologie GmbH, Germany, for providing them with the Textus active and Textus multi dressings.

References

[1] Hall−Stoodley L, Costerton JW, Stoodley P:Bacterial biofilms: from the natural environment to infectious dis− eases. Nature Rev 2004, 2, 95–108.

[2] Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002, 15, 167–193.

[3] Smith A:Biofilms and antibiotic therapy: is there a role for combating bacterial resistance by the use of novel drug delivery systems? Adv Drug Deliv Rev 2005, 57, 1539–1550.

[4] Costerton JW, Steward PS, Geenberg EP: Bacterial biofilms: a common cause of persistent infections. Science 1999, 284, 1318–1322.

[5] Wilson M:Bacterial biofilms and human disease. Sci Prog 2001, 84, 235–254.

[6] Costerton JW, Stewart PS: Biofilm and device−related infections. In: Persistent bacterial infections. Eds.: Nataro JP, Blaser MJ, Cunningham−Rundles S, ASM Press, Washington, 2000, 423–439.

[7] Gjødsbøl K, Christensen JJ, Karlsmark T, Jørgensen B, Klein BM, Krogfelt KA:Multiple bacterial species reside in chronic wounds: a longitudinal study. Int Wound J 2006, 1, 1–2.

[8] James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, Costerton JW, Stewart PS:Biofilms in chronic wounds. Wound Rep Reg 2008, 16, 37–44.

[9] Bjarnsholt T, Kirketerp−Møller K, Jensen PØ, Madsen KG, Phipps R, Krogfelt K, Høiby N, Givskov M: Why chronic wounds will not heal: a novel hypothesis. Wound Rep Reg 2008, 16, 2–10.

[10] Białynicki−Birula R, Nawrot U, Baran E, Włodarczyk K, Kołodziej T: Efficacy of antifungal action of Textus® dressing. Mikol Lek 2006, 13, 143–147.

[11] Leaper DJ:Silver dressings: their role in wound management. Int Wound J 2006, 3, 282–294.

[12] Hobot J, Walker M, Newman G, Bowler P: Effect of Hydrofiber®wound dressings on bacterial ultrastructure. J Electron Microsc 2008, 57, 67–75.

[13] Kaźmierski M, Puchała J, Chrapusta−Klimeczek A, Mańkowski P, Jankowski A: Silver containing dressings as applied in the treatment of burn in wounds. Zakażenia 2006, 4, 123–130.

[14] Hofman−Stefanek H, Karolczak A: Treatment of pediatric infected skin wounds with active dressing containing ionized silver. Zakażenia 2006, 1, 86–88.

[15] Ślęzak A, Kucharzewski M, Grzegorczyn S, Ślęzak I: Study of transport properties of the membranous dress− ing with silver ions. Polim Med 2005, 35, 3–11.

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[17] Ando E, Monden K, Mitsuhata R, Kariyama R, Kumon H: Biofilm formation among methicillin−resistant

Staphylococcus aureusisolates from patients with urinary tract infection. Acta Med Okayama 2004, 58, 207–214. [18] Li X, Yan Z, Xu J:Quantitative variation of biofilms among strains in natural populations of Candida albicans.

Microbiology 2003, 149, 353–362.

[19] Percival SL, Bowler P, Woods EJ: Assessing the effect of an antimicrobial wound dressing on biofilms. Wound Rep Reg 2008, 16, 52–57.

Address for correspondence:

Ewa Dworniczek

Department of Microbiology Wroclaw Medical University Chałubińskiego 4

50−368 Wrocław Poland

E−mail: ekoks@mbio.am.wroc.pl

Conflict of interest: None declared

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Figure

Fig. 1 a, b. Biofilm production by selected strains of bacteria (a) and yeasts (b) in the presence of Textus bioactiv,Textus multi dressings, and culture broth without dressing

References

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