An in vitro model of chronic wound biofilms to test wound dressingsand assess antimicrobial susceptibilities
Katja E. Hill1,2*, SladjanaMalic1, Ruth McKee1,2, Tracy Rennison3, Keith G. Harding2, David W.Williams1
and David W. Thomas1
1Wound Biology Group, Tissue Engineering and Reparative Dentistry, Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY,UK; 2Department of Wound Healing, Heath Park, Cardiff CF14 4XN, UK; 3Ethicon Wound Care, Gargrave, North Yorkshire BD23 3RX, UK
*Corresponding author. Tel: +44-29-2074-4252; Fax: +44-29-2074-2442; E-mail: email@example.com
Received 20 November 2009; returned 14 December 2009; revised 22 January 2010; accepted 6 March 2010
Objectives: The targeted disruption of biofilms in chronic wounds is an important treatment strategy and thesubject of intense research. In the present study, an in vitro model of chronic wound biofilms was developed toassess the efficacy of antimicrobial treatments for use in the wound environment.
Methods: Using chronic wound isolates, assays of bacterial coaggregation established that aerobic and anaero-bic wound bacteria were able to coaggregate and form biofilms. A constant depth film fermenter (CDFF) wasused to develop wound biofilms in vitro, which were analysed using light microscopy and scanning electronmicroscopy. The susceptibility of bacteria within these biofilms was examined in response to the mostfrequently prescribed chronic wound antibiotics and a series of iodine- and silver-containing commercialantimicrobial products and lactoferrin.
Results: Defined biofilms were rapidly established within 12 days. Antibiotic treatment demonstrated thatmixed Pseudomonas and Staphylococcus biofilms were not affected by ciprofloxacin (5 mg/L) or flucloxacillin(15 mg/L), even at concentrations equivalent to twice the observed peak serum levels. The results contrastedwith the ability of povidoneiodine (1%) to disrupt the wound biofilm; an effect that was particularly pro-nounced in the dressing testing where iodine-based dressings completely disrupted established 7 day biofilms.In contrast, only two of six silver-containing dressings exhibited any effect on 3 day biofilms, with no effect on7 day biofilms.
Conclusions: This wound model emphasizes the potential role of the biofilm phenotype in the observedresistance to antibiotic therapies that may occur in chronic wounds in vivo.
Keywords: chronic venous leg ulcer, constant depth film fermenter, antibiotic resistance, biocide resistance, coaggregation,lactoferrin
IntroductionChronic wounds harbour a diverse microflora and are a reposi-tory of complex polymicrobial communities (which include bothaerobic and anaerobic species).1 The precise role of these organ-isms in mediating the observed impairment of wound healing iscomplex and may include both direct and indirect mechan-isms.2,3 The importance of individual species, multiple speciesor microbial density in relation to healing, however, remainsunclear.46 Anaerobic species constitute 45% of the totalmicrobial population in non-infected venous leg ulcers,79
which increases to 49% in clinically infected chronic venous legulcers (CVLUs).7 Whilst all wounds are colonized by bacteria,not all wounds are clinically infected;4 the definition of inflam-mation and infection requiring clinical experience to avoid the
unnecessary prescription of antibiotics in this at-riskpopulation.10,11
Considerable attention has recently been focused on theability of bacteria within chronic wounds to form and exist in bio-films.1214 Bacterial biofilms consist of a complex microenviron-ment of single or mixed bacterial species encased within anextracellular polymeric substance (EPS) or glycocalyx which thebacteria themselves produce. The moist wound surface, withits adhesive, proteinaceous substrate and a ready supply of nutri-ents, represents (conceptually at least) the ideal environment forbiofilm development.15 Researchers have demonstrated thatbacteria within the wound environment possess the ability toform biofilms.12,13,16,17 Moreover, it has recently been suggestedthat acute partial thickness wounds13,18 may harbour bacterial
# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: firstname.lastname@example.org
J Antimicrob Chemotherdoi:10.1093/jac/dkq105
biofilms growing on the wound surface. In addition, EPS has beenvisualized by epifluorescence and light microscopy on chronicwound smears.16,19 Individual bacteria and bacterial microcolo-nies have also been observed using fluorescence in situ hybridiz-ation (FISH) on chronic wound biopsy sections.12,20 Such biofilmsmay play an important role in the ability of wounds to resist anti-microbial and antibiotic treatments.
In the formation of biofilms, coaggregation is a specific mech-anism of bacterial cell-to-cell adhesion that plays a key role inbiofilm formation. Coaggregation is mediated by specificgrowth-phase-dependent adhesinreceptor interactions21,22
with bacteria from biofilm communities showing an increasedtendency to coaggregate compared with planktonic bacteria.23
This coaggregation has also been shown to contribute a meta-bolic advantage by facilitating the survival of obligate anaerobicspecies in aerated environments.24 Apart from oral plaque bac-teria, coaggregation has also been shown to occur between bac-teria isolated from other ecosystems such as the gastrointestinaland urogenital tracts2528 as well as wastewater and food pro-cessing environments.2931 Despite the importance of coaggre-gation in biofilm establishment, the coaggregation phenotypeof chronic wound bacteria remains to be studied.
In attempts to model dental plaque biofilm formation in vitro,the constant depth film fermenter (CDFF) was developed.3234
The CDFF allows the generation of identical, multiple biofilms ofuniform depth for sequential analysis (including gene, proteinand cellular/structural analysis). Importantly, the flexibility of thesystem allows key parameters, including nutrient source,temperature, oxygen availability and substrata to be varied.Schematic representation of the CDFF has already been publishedelsewhere.35,36 The CDFF model has consequently been exten-sively used to study various aspects of biofilm physiology as wellas for testing antimicrobial therapies e.g. chlorhexidine, sodiumhypochlorite, tetracycline and silver.31,3739 In addition to thestudy of human disease causing biofilms, it has also been utilizedto model bacteria in other ecosystems, such as wastewater.33
In this study, we sought to develop a reliable in vitro model ofchronic wound biofilms, initially testing the coaggregating abilityof bacteria derived directly from chronic wounds, before estab-lishing biofilms in the CDFF system. The model was then usedto test and compare the efficacy of conventional antibacterialwound therapies on biofilms.
Materials and methods
Bacterial strains and mediaBacterial species were selected from a previous prospective study of 70patients with newly diagnosed CVLUs at the Wound Healing ResearchUnit in Cardiff, with informed consent.5,40,41 These included the mostfrequently encountered species, namely those of the Pseudomonas,Staphylococcus, Micrococcus and Streptococcus genera, as well as arange of strictly anaerobic bacteria (Table 1).
For the CDFF, seven bacterial species with good coaggregating abilitywere selected to represent the polymicrobial nature of chronic woundbeds. Davies et al.5 found that the mean number of organisms perwound (for both deep tissue or wound surface) was fewer than three,but had a range of one to six. Hence, up to six organisms were used atany one time for themodel wound biofilm, using both aerobic and anaero-bic species. In later experiments this number was reduced to four aerobicspecies. From our previous work,5,40 two of the most frequently isolated
wound bacteria were Staphylococcus aureus and Pseudomonasaeruginosa. Strains of S. aureus (D76, methicillin susceptible) andP. aeruginosa (D40), were therefore selected from wound isolates to becomponents of the biofilm consortium. In addition, Micrococcus luteus(B81) and Streptococcus oralis (B52) were selected, not only on the basisof their relative ability to coaggregate, but also on their relatively fastgrowth rates. This was done with the caveat that both pseudomonadsand staphylococci could potentially out-compete other slower-growingorganisms in mixed culture. The anaerobic bacteria selected included Pro-pionibacterium acnes (E67), Bacteroides fragilis (B11) and Peptostreptococ-cus anaerobius (B12), although only two of these species were used at anyone time in the mixed bacterial biofilm in the CDFF.
Aerobic isolates were routinely grown on blood agar No. 2 (BA; Lab M)and anaerobes on fastidious anaerobe agar (FAA; Lab M), both sup-plemented with 5% (v/v) defibrinated sheep blood. BA plates were incu-bated aerobically at 378C for 23 days. FAA plates were incubated in ananaerobic environment (10% CO2, 10% H2, 80% N2) also at 378C for up to7 days. Fastidious anaerobe broth (FAB; Oxoid) was used for liquid culture.
Coaggregation testingThe ability of bacteria from wounds to initiate biofilm formation wasinvestigated by testing chronic wound isolates for their ability to
Table 1. Bacterial isolates from CVLUs used in coaggregation assays
Wound isolate ID No. Bacterial isolate
G68 Staphylococcus aureusD58 Staphylococcus aureus (methicillin resistant)D76 Staphylococcus aureusC49 Staphylococcus aureusC72 Staphylococcus aureusD21 Staphylococcus aureusB60 Pseudomonas aeruginosaC21 Pseudomonas aeruginosaD40 Pseudomonas aeruginosaD49 Pseudomonas aeruginosaB43 Pseudomonas aeruginosaC31 P