wound biofilms: lessons learned from oral biofilms

PERSPECTIVE ARTICLE

Wound biofilms: Lessons learned from oral biofilmsKimberly A. Mancl, BS; Robert S. Kirsner, MD, PhD; Dragana Ajdic, PhD

Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida

Reprint requests:

Dr. D. Ajdic, Department of Dermatologyand Cutaneous Surgery, University ofMiami Miller School of Medicine,1600 NW 10th Ave, RMSB 2089, Miami,FL 33136, USA.Tel: +1 305 243 0475;Fax: +1 305 243 6191;Email: [email protected]

Manuscript received: September 27, 2012Accepted in final form: December 25,2012

DOI:10.1111/wrr.12034

ABSTRACT

Biofilms play an important role in the development and pathogenesis of many chronicinfections. Oral biofilms, more commonly known as dental plaque, are a primarycause of oral diseases including caries, gingivitis, and periodontitis. Oral biofilms arecommonly studied as model biofilm systems as they are easily accessible; thus,biofilm research in oral diseases is advanced with details of biofilm formation andbacterial interactions being well elucidated. In contrast, wound research has relativelyrecently directed attention to the role biofilms have in chronic wounds. This reviewdiscusses the biofilms in periodontal disease and chronic wounds with comparisonsfocusing on biofilm detection, biofilm formation, the immune response to biofilms,bacterial interaction, and quorum sensing. Current treatment modalities used by bothfields and future therapies are also discussed.

Microorganisms colonize the human skin, mouth, digestive,and reproductive tracts, and it is estimated that the number ofbacterial cells colonizing the human body exceeds the numberof our own cells.1 Bacteria have been studied for hundreds ofyears as scientist Anton van Leeuwenhock, credited as thefather of microbiology, first observed bacteria with a micro-scope in 1683.2 One of the first samples he studied was dentalplaque, and from that time plaque has served a crucial role infurthering human understanding of microorganisms.

While bacteria have been extensively studied, it was rela-tively recently that a fundamental shift occurred in researchregarding bacteria and various disease states. This shift wasthe knowledge that bacteria form biofilms. Biofilms are three-dimensional structures consisting of densely packed aggrega-tions of microbes that attach to each other and to a surface andare encased in a self-synthesized extracellular polymeric sub-stance (EPS).3 It is estimated that biofilms are composed of75–95% EPS and only 5–25% bacteria.2 In contrast to bio-films, planktonic bacteria, which have been the traditionalfocus of microbiology research, are free-floating microorgan-isms in an aqueous environment. The transition of planktonicmicroorganisms to a biofilm lifestyle can be damaging to ahost as biofilms notoriously resist elimination by hostdefenses and antibiotics.4

The details regarding biofilm formation are important asunderstanding these pathways is crucial to the development offuture antibiofilm therapy. The biofilm life cycle has distinctstages including attachment of planktonic cells to a surface,growth of the cells into a mature biofilm colony, and eventualdispersal of the cells from the colony into the surroundingenvironment.5 All stages are complex, involving various envi-ronmental signals, signal transduction pathways, and effectormolecules.

Attachment is initiated when planktonic bacteria form areversible bond to a surface, mediated by various physical andchemical forces as well as bacterial and host cell adhesins thatare proteins considered to be key factors in the attachmentstage.5 The bacteria then multiply on the surface and synthe-size EPS, which defines the second stage of biofilm develop-ment, also known as maturation. The extracellular polymericmatrix, which is composed of polysaccharides, proteins,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), andlipids, is important because it offers biofilm protection as wellas mediates cell-to-cell and cell-surface interactions thatallow for biofilm stabilization.6,7

The detachment of cells from the biofilm colony and theirrelease into the environment comprise the final stage ofbiofilm development known as dispersion. This stage contrib-utes to bacterial survival and disease transmission.5 Maturebiofilm is composed of microbial masses in species-specificconfigurations with intermixed channels that allow for theexchange of nutrients and waste products, transfer of geneticmaterial, transportation of signaling molecules used inquorum sensing (QS), and other bacterial interactions.5

Biofilms occur commonly in nature, and they are known toform in water, waste pipes, on ship’s hulls, and in food-processing areas.7 They are a topic of extensive research asthey are implicated in the pathogenesis of many infections,with the US Centers for Disease Control and Prevention esti-mating that more than 65% of microbial infections are asso-ciated with biofilms.8 Biofilms have a well-established role inoral diseases such as dental caries, gingivitis, and periodon-titis, and more recently they have been implicated in thepathogenesis of chronic wounds.8

Biofilms create a therapeutic problem as they are oftenresistant to conventional antimicrobial treatment. Thus, the

Wound Rep Reg (2013) 21 352–362 © 2013 by the Wound Healing Society352

understanding of biofilms and ways to control them is impera-tive to the successful treatment of a multitude of diseases.This review aims to provide a comparison and discussionregarding biofilms in oral and wound disease states andcurrent modalities of treatment, with a goal of focusing on theliterature from oral biofilm investigation and how this can beapplied to the understanding and future investigation of bio-films in chronic wounds.

BIOFILMS IN ORAL DISEASES ANDCHRONIC WOUNDSThe common inflammatory diseases, gingivitis and periodon-titis, are collectively referred to as periodontal disease. Gin-givitis, estimated to affect 50–90% of adults worldwide, is amild form of periodontal disease that is reversible with effec-tive oral hygiene.9,10 Periodontitis, a more severe form ofperiodontal disease, occurs when inflammation extends deepinto tissues and causes loss of gingival tissue, the periodontalligament, and alveolar bone.9 Mild periodontitis is estimatedto be present in 21.6% of dentate US adults, while moderateto severe disease affects approximately 13%.11 The economicimpact of periodontal disease is striking, with treatment costsestimated to exceed $14 billion per year in the United States.12

In addition to periodontal diseases, chronic skin woundsplay a major role in health care. Chronic wounds, defined aswounds that have failed to proceed through an orderly andtimely reparative process, typically affect patients withcomorbid medical conditions.13 The major chronic woundsfaced by clinicians are diabetic foot ulcers, venous leg ulcers,pressure ulcers, and ulcers resulting from peripheral vasculardisease. It is estimated that 1–2% of people will develop achronic wound during their lifetime in developed countries,and in the United States chronic wounds affect approximately6.5 million patients.13 The economic impact of wounds is alsostaggering as upwards of $25 billion is spent annually in theUnited States on the treatment of chronic wounds.13

The most widely accepted cause of periodontal diseases isthe formation of tooth biofilm with pathogenic microorgan-isms, commonly known as dental plaque. In fact, research hasshown that within 1 day of discontinuation of oral hygieneprocedures (e.g., brushing one’s teeth), biofilms begin todevelop, and within 10–21 days, gingivitis develops.9 Becauseof the widespread presence and easy accessibility of dentalbiofilms, they are commonly studied as model systems forbiofilm development and therapeutic techniques.14 Listgartenet al. described the complex nature and architecture of oralbiofilms decades ago through the use of light and electronmicroscopy on extracted teeth and epoxy resin crowns. Theirwork suggested that certain microorganisms are consistentwith states of periodontal health and disease.15,16 Though atthat time they were unable to determine the specific speciespresent, their work laid the foundation for future oral biofilmresearch.

While biofilms have been widely accepted and studied inoral diseases for many years, recent research indicating thatmany chronic diseases result from bacterial biofilms initiatedwound research’s focus on biofilms as a possible cause ofsuboptimal wound healing.17–19 One of the first associations ofbiofilms and wounds came from the electron microscopicexamination of sutures and staples removed from healed sur-gical wounds.20 Other studies then characterized in vitro and

in vivo models for biofilm development in wounds. A studyby Harrison-Balestra et al. showed that wound-isolatedPseudomonas aeruginosa displays characteristics of a maturebiofilm within 10 hours of in vitro growth, thus suggestingthat bacteria in wounds rapidly develop biofilms.21 Charleset al. utilized a tissue-engineered skin equivalent, Graftskin,and demonstrated time-dependent biofilm development onartificially created wounds inoculated with pathogenicP. aeruginosa and Staphylococcus aureus.22

Recently, a landmark study by James et al. investigated thepresence of biofilms, evident by aggregated bacterial coloniessurrounded by an extracellular matrix, in acute and chronicwounds using light and scanning electron microscopy(Figure 1).17 They found that only 6% of acute wounds hadbiofilms, whereas 60% of chronic wounds exhibited biofilmformation, though it was not clear whether those chronicwounds with or without biofilms were refractory or healing.While biofilms are thought to be present in chronic woundsmore so than acute wounds, both murine and porcine in vivomodels have shown acute wound biofilms.23–27 Thus, althoughbiofilms may be an important factor as to why chronicwounds do not follow the typical wound healing pattern, theymay not be the only factor.

Interestingly, another study by Akiyama et al. showed thatS. aureus cultures from patients with impetigo, furuncle, andatopic dermatitis grew biofilms on coverslips within 72 hoursof incubation at 37 °C in the presence of plasma.28 Theyinferred that biofilms also form in vivo. The implications ofbiofilms in not only wounds but also other dermatologic dis-eases including atopic dermatitis, acne, candidiasis, impetigo,and bullous diseases are now being recognized.29,30

Detection of microorganisms in oral and

wound biofilms

Research regarding oral biofilm composition has advancedconsiderably, mainly because of advanced technologies usedfor biofilm detection. While a study by Moore and Mooreshowed more than 500 distinct bacterial species in dentalplaque specimens via the use of culture methods,31 newer

Figure 1. Scanning electron micrograph of a chronic woundrevealing the presence of a mixed species biofilm. Copyright2008 Wiley. Used with permission from James et al.17

Mancl et al. Wound and oral biofilms

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research indicates that many more bacterial species arepresent that are uncultivable. Culture independentapproaches, in particular the use of 16S ribosomal RNA(rRNA) gene sequencing, have revolutionized the understand-ing of bacterial species diversity in oral niches. Multiplestudies utilizing this technique revealed previously unidenti-fied species in the oral environment.32–36

Zijnge et al. investigated the location of periodontitis-associated species in vivo using a panel of 16S and 18S rRNAtargeted fluorescence in situ hybridization (FISH) probes.14

Their research not only revealed the dominant bacterialspecies in plaque but also showed that there are progressivedifferences in biofilm cell physiological activity depending onbacterial depth in the biofilm.14 This finding was consistentwith previous in vitro studies.37 FISH overcomes the tradi-tional culture limits, allows for positional information regard-ing bacteria in intact biofilms, and can relatively simply beextended to newly identified species and phylotypes.

Similar to oral biofilm detection, culture has been the stan-dard method used to detect bacterial infection in wounds.38

Chronic wounds often have low bacterial burden measured bystandard laboratory assays, however, and lack overt clinicalsigns of infection, making lack of clinical suspicion a con-founding problem in wound biofilm identification.18,39 Culturetechniques likely fall short in wounds as they do in oralbiofilms at identifying all microorganisms present. This isbecause easily cultivable bacteria, such as S. aureus, areselected for and commonly used laboratory methods detectmostly planktonic bacteria.38,40 Multiple articles highlight thelimitations of culture methods in identifying skin and woundbacterial species and the increased microorganism diversityelucidated with molecular techniques such as 16S rRNA andFISH.41–46 Other novel approaches to detect biofilm organismsin chronic wounds are based on detection of QS moleculesfound in debridement specimens.47,48

Gjodsbol et al. utilized molecular and culture techniques todetermine the microbial community of venous ulcers. Theyfound that S. aureus was the most common wound bacteria,followed by Enterococcus faecalis, P. aeruginosa, coagulase-negative staphylococci, Proteus species, and anaerobic bacte-ria.49 Dowd et al. investigated bacterial species present inchronic wounds of diabetic foot ulcers, venous leg ulcers, andpressure ulcers using molecular amplifications followed bypyrosequencing, shotgun Sanger sequencing, and denaturinggradient gel electrophoresis.40 They found that across allwound types, major populations of bacteria included Staphy-lococcus, Pseudomonas, Peptoniphilus, Enterobacter,Stenotrophomonas, Finegoldia, and Serratia spp. Otherresearchers identified additional genera such as Streptococ-cus, Porphyromonas, Anaerococcus, and Prevotella inchronic wounds.45,46 Gardner et al. demonstrated an associa-tion between diabetic foot ulcer microorganisms and clinicalfactors including ulcer depth, ulcer duration, and glycemiccontrol using 16S rRNA sequencing.46 The previous studiesshow the identification of bacteria in wounds not traditionallythought of as wound pathogens and the diversity of microor-ganisms present in wounds.

As oral biofilm and now skin research have shown theidentification of multiple additional species with the advent ofmolecular microbial identification techniques, further use of16S rRNA probes along with FISH should be sought.However, while possible to detect biofilm organisms usingthese methods, molecular methods have historically been

expensive and time intensive and therefore generally not usedin the everyday diagnosis of biofilm organisms.38,50 With con-tinual advances in technology, hopefully 16S rRNA genesequencing will become widely available to clinicians androutine clinical microbiology laboratories.

Biofilm formation and composition

In dental plaque, bacteria have been shown to form biofilmsin an organized, concerted way with initial attachment ofpioneer species subsequently followed by other colonizingspecies.51 It is thought that each microorganism modifies theenvironment, which allows for successful community devel-opment. In 1998, a landmark paper published by Socranskyet al. described this sequential formation of dental plaquewith each successive colonization composed of increasinglyperiopathogenic bacteria. The end result was colonization byPorphyromonas gingivalis, Treponema denticola, and Tan-nerella forsythia, a group of bacteria known to be present inperiodontitis.52

As described earlier, attachment is the initial phase inbiofilm formation. Within minutes of professional cleaning, acollection of host-derived molecules termed the acquired pel-licle coats the enamel surface of teeth. The acquired pellicleserves as the source of receptors for the primary colonizers ofdental plaque. Examples of these receptors for various oralspecies include mucins, agglutinins, proline-rich proteins,phosphate-rich proteins (statherin), and enzymes such asalpha-amylase..53 Streptococci represent 60–90% of the earlycolonizers with other bacteria also involved.54 Crucial to thedevelopment of the initial biofilm are the interactions amongthese bacteria, many of which have been elucidated for dentalbiofilms.53

Secondary, or late, colonizers bind to the primary coloniz-ers, and the sequential bacterial binding results in the forma-tion of nascent surfaces that bridge with the nextcoaggregating bacterial cell. Late colonizers include manyperiodontal pathogens such as P. gingivalis, T. denticola,Eubacterium spp., Prevotella intermedia, Aggregatibacteractinomycetemcomitans, Tannerella forsythensis, and Seleno-monas flueggei.53 In dental biofilm formation, Fusobacteriumnucleatum, also considered a periodontal pathogen, is uniquein that it acts as a bridge between primary and secondarycolonizers.53 It is the most numerous gram-negative species athealthy sites, but it also markedly increases in sites of peri-odontal disease.31 An increase in plaque mass is observed atsites suffering from periodontal disease with increasedspecies diversity. A rise in gram-negative and obligate anaero-bic species is also observed.1 The proportion of certainbacterial species, such as Streptococcus mutans or P. gingiva-lis, is increased in patients with caries or periodontitis,respectively.2

Biofilms have only recently been shown to exist in chronicwounds; hence, research regarding wound biofilm formationand composition is significantly behind the known biofilmintricacies in dental plaque. Chronic wound microbiology isshown to be complex with a wide variety of microorganismsimplicated in delaying wound healing55; however, little isactually known regarding how these organisms form a woundbiofilm. S. aureus and P. aeruginosa are traditionally recog-nized as major wound pathogens, but they likely representonly a fraction of microbes involved in chronic wounds. Thepolymicrobial nature of wound infections and the role of

Wound and oral biofilms Mancl et al.


anaerobic bacteria have been emphasized by Bowler et al. andJames et al.17,56

It is reasonable to suggest that wound biofilms may form ina manner similar to oral biofilms with initial colonization ofskin flora and secondary colonization of wound pathogenssuch as S. aureus and P. aeruginosa. The advent of moleculartechniques with continual elucidation of species diversity inchronic wounds will serve as a foundation for future studiesdesigned to determine if there is also a sequential woundbiofilm formation.

Bacterial interaction in biofilms

While little is known regarding bacterial interactions inwounds, oral research offers guidance regarding processesthat may also occur in wound biofilm formation. Coaggrega-tion and coadherence are two mechanisms of interaction usedby bacteria to establish specific patterns of spatiotemporaldevelopment in the oral biofilm. Coaggregation describes theability of genetically distinct cells to recognize one another insuspension with resultant clumping.53,57 It is thought to occurvia physical interactions among bacteria that have beenshown to display specific recognition patterns with partnercells.58 Testing among 300 oral bacteria indicated that greaterthan 90% underwent coaggregation, illustrating the impor-tance of this interaction.2 Coadhesion, the recognition of aspecific bacterium in suspension to one already attached to asubstratum,53 and coaggregation bridges, a third bacteriumbridging two bacteria together, are also important bacteriainteractions.

While it is known that many species participate in thesequential colonization of the oral cavity to form biofilms, theexact mechanisms are still a subject of much research. Studieshave shown that many aggregations are inhibited by lactose,with a study by McIntire et al. reporting that Streptococcusand Actinomyces aggregation was inhibited 90% of the timeby lactose and d-galactose.59 Bacteria cooperate extensivelyvia coaggregation and other mechanisms to facilitate survivaland biofilm formation. For example, both F. nucleatum andP. intermedia help P. gingivalis, an acid-sensitive bacterium,survive in the oral environment by producing ammonia,which results in a more neutral environment. Bacteria alsocompete with one another in the process of oral biofilm for-mation mainly via bacteriocin synthesis, QS, and hydrogenperoxide excretion. Bacteriocins are bacterial peptide scap-able of lysing other bacteria, thus enhancing competitivenessof the producing species and consequently their ability toform biofilms.60 Streptococci possess the strongestbacteriocin-producing ability among oral bacteria. Furtherinformation regarding the detailed interactions of manypathogenic bacteria can be obtained in a comprehensivereview by Huang et al.2

With regard to wounds, studies have found a correlationbetween the presence of multiple bacterial species and non-healing wounds rather than just one bacterial species. Thissuggests that, similar to oral biofilms, cooperation andsynergy exist in wounds among the various speciespresent.41,55,61 Dowd et al. also have introduced the concept offunctional equivalent pathogroups as genetically distinct bac-teria that symbiotically co-occur in appropriate mixtures toproduce a pathogenic biofilm community.40 This concept isvery similar to oral biofilm knowledge that certain bacteria

are responsible for working together in various stages to formpathogenic biofilms.

Biofilm communication through QS

Microorganisms communicate via the secretion of one ormore agents in response to changes in bacterial density andthe surrounding environment. This communication process isintegral to bacterial cooperation and competition and istermed quorum sensing. QS plays a critical role in biofilms asit regulates biofilm maturation and development throughinducing differential protein expression. Numerous bacterialactivities are thought to be controlled by QS systemsincluding bacterial surface adhesion, extracellular matrix pro-duction, virulence factor expression, spore formation, biosur-factant synthesis, competency, and bioluminescence.2,62,63

Various QS systems have been described with N-acyl-homoserine lactone (AHL) and 4-quinolone systems used bygram-negative bacteria, oligopeptide (AgrD) systems by grampositives, and Autoinducer-2 (AI-2)/luxS systems by bothgram-positive and gram-negative organisms.2,64

AI-2 functions as an important signal molecule in multi-species biofilms as it promotes biofilm formation and matu-ration. In fact, the luxS gene encoding AI-2 is conservedamong many species of oral pathogenic bacteria.65,66 OralStreptococci that lack the luxS gene do not express AI-2,which decreases bacterial communication and results in loosebiofilm formation with significantly decreased bacterial den-sity.2 S. mutans’ QS system also involves competence-stimulating peptide, which interestingly has been shown tocause these organisms’ growth arrest and death when presentin large concentrations in biofilms. Thus, QS systems mayprovide future therapeutic options in dental caries and chronicwounds as more information is learned about them.67

Similar to oral biofilms, wound biofilms use QS as a meansof regulating the expression of biofilm-specific genes. Gram-positive bacteria communicate mainly via peptide signalssuch as autoinducing peptide 1 (AIP-1) produced by S. aureusand the delta toxin from Staphylococcus epidermidis andS. aureus. AIP-1 may play a role in biofilm dispersal throughup-regulating the matrix degrading proteases aureolysin andserum protease 1.5 Gram-negative bacteria communicatemainly via AHLs, with wound pathogen P. aeruginosabiofilm dispersal having been shown to be regulated by mul-tiple AHL signaling networks.3,5

Immune response to biofilms

The innate immune response plays a role in the pathogenesis ofboth periodontal disease and chronic wounds. Important in theactivation of the innate immune response is the presence ofvarious cell pattern recognition receptors (PRRs), such astoll-like receptors and nucleotide-binding oligomerizationdomain receptors (NOD)-like receptors, which survey theextracellular and intracellular environments and detectmicrobe-associated molecular patterns present on or releasedfrom microbial cells.1 Activation of PRRs stimulates the innateimmune response and triggers the production of cytokines andother inflammatory mediators. Abnormal expression of PRRsthat bind bacterial lipopolysaccharide has been linked to pre-disposition to periodontal disease, thus reflecting the impor-tance of oral tissue host detection systems.1



The clinical entity gingivitis is known to be caused by thehost’s response to the accumulation of dental plaque, as dem-onstrated by experimental gingivitis studies in the 1960s.68

Likewise, chronic inflammation of the periodontium is initi-ated by periodontal pathogens in subgingival biofilms. Thehost response contributes to the development of periodontaldisease through the recruitment of pro-inflammatory cells(neutrophils and macrophages), inflammatory mediatorrelease (interleukin-1 [IL-1] and prostaglandin E2), and tissuedestruction via matrix metalloproteinases (MMPs).7,51 Thefirst responders to inflammation, neutrophils, are shown to behyperresponsive in periodontal diseases, and they contributeto the disease process through releasing destructive reactiveoxygen species (ROS), proteinases, and other factors thatdamage the host gingiva and periodontal ligaments.1,69,70 Neu-trophils also increase pro-inflammatory cytokines such as leu-kotriene B4, IL-6, tumor necrosis factor-a (TNF-a), andIL-1b, and increased levels of these cytokines are consistentlyfound in studies of gingival crevicular fluid of patients withgingivitis and periodontitis.71,72

Adaptive immunity also plays a role in the pathogenesis ofperiodontal diseases. Page and Schroeder observed that acuteinflammation dominates early periodontal lesions, but laterperiodontal lesions have a predominance of T cells, B cells,and plasma cells.72,73 The gingivitis lesion is primarily a Tcell–mediated response, while the periodontitis lesion is pre-dominantly a B cell and plasma cell response, thus implyinga switch in immune response in the development of periodon-titis.74 Control of this shift is mediated by a balance betweenTh1 and Th2 subsets of T cells, with T regulatory and Th17cells also possibly involved in immunoregulation of periodon-tal disease.74

In wounds, normal healing in response to injury or traumato the skin consists of three phases: inflammatory, prolifera-tive, and remodeling. During the inflammatory phase, theexposed extracellular matrix triggers the coagulation andplasminogen cascades, and these along with wound bacteriatrigger the innate immune system.39 Neutrophils and mac-rophages are abundant during this phase, and eventually mac-rophages down-regulate inflammatory signals to move thewound into the proliferative phase after wound debris andbacteria are cleared.75 The proliferative phase is composedmainly of angiogenesis, as well as fibronectin and collagendeposition via fibroblasts with the formation of granulationtissue. The remodeling phase lasts up to 2 years and, duringthis time, collagen fibers reorganize and mature, returning thetissue to near original strength.39

Similar to periodontal disease, chronic wounds are thoughtto induce a sustained inflammatory response as the hostattempts to eliminate the biofilm. The role biofilms play is notyet clear, however. Some research implies that biofilmreleases planktonic bacteria and consequently maintains thisresponse.76 Other thoughts are that nonhealing wounds areassociated with compromised hosts that exhibit an inflamma-tory response incapable of managing the initial wound bacte-ria effectively. Bacteria, in particular P. aeruginosa,contribute to this as they combat the host’s immune responsevia the production of various substances.77–79 Either way,chronic wounds are thought to become fixed in the inflamma-tory wound healing stage, with the body unable to success-fully progress to the proliferative stage.39

Numerous studies have supported the notion that a chronicinflammatory response occurs in chronic wounds. Pro-

inflammatory cytokines such as interferon (IFN)-a, IFN-g,TNF-a, and IL-1, frequently produced in response to bacteria,are elevated in chronic wounds.23 Trengove et al. observedincreased cytokine and metalloprotease levels in chronicwounds compared with acute wounds.80 The inflammatoryresponse also results in numerous neutrophils and macroph-ages surrounding biofilms that secrete high levels of ROS,proteases (MMPs), and elastase. Neutrophil-derived MMP 8and elastase, and macrophage derived MMPs 2 and 9 arecommonly increased in chronic wounds.38 While proteases aidin dislodging biofilms from tissues, they also damage normaland healing tissues. As mentioned previously, the inflamma-tory response is not always successful in removing thebiofilm, and it has been hypothesized that the inflammatoryresponse actually increases biofilm survival. An ineffectiveinflammatory response can result in increased vascular per-meability and the production of wound exudates as fibrinslough builds up, which serves as biofilm nutrition.18

While innate immune mechanisms are mostly observed inchronic wounds, S. aureus biofilms have been shown to elicitan adaptive immune response in ocular biofilm.81 As researchhas shown the importance of adaptive immunity in the patho-genesis of periodontal diseases with differences between gin-givitis and periodontitis, adaptive immunity may also play arole in chronic wound pathogenesis and the conversion of anacute to chronic wound. The role of adaptive immunity inresponse to biofilms opens the possibility for the developmentof biofilm-specific diagnostics and even vaccines, an interest-ing prospect to both oral and wound biofilm research.

Impact of the host environment on biofilm species

and architecture

The host environment is selective and determines whichorganisms are able to colonize, grow, and become minor ormajor members of a microbial community, with the host-microflora interaction important in health and disease. Dentalresearch has shown that the distribution of oral microflora isnot random, but that most species prefer a specific oral sitewith microflora composition varying among locations such asthe tongue, buccal mucosa, and teeth.1,5 Aas et al. investigatedthe oral microflora composition at nine sites in healthy sub-jects and found that some species were common to all sites,while other species were site specific. They also determinedthat most sites had 20–30 predominant species and thatperiodontitis-associated species were not detected in thehealthy gingiva samples.82 For skin, bacteria colonize skinsites based on specific location and the underlying physiologyof the skin, with certain bacteria associated with most, dry,and sebaceous environments.83 In wounds, Dowd et al.showed that bacterial populations differed based on woundtypes, with pressure ulcers having 62% obligate anaerobes.40

Other studies have indicated that P. aeruginosa and S. aureusvary at different wound locations and appear nonrandomlydistributed in wounds.44,84

Composition and metabolic capability of an oral microbialcommunity are impacted by the environment. Differences ofover 2 °C have been measured between periodontally healthytissue and diseased states.1,85 Subgingival sites with increasedtemperatures likely resulting from the inflammatory responsehave been shown to have elevated proportions of periodontalpathogens.1,86 In addition to temperature, pH influences the



growth and survival of microorganisms. After sugar consump-tion, the oral environmental pH falls due to acidic fermenta-tion products, and this results in selection of acid-tolerant oralmicroorganisms, many of which are pathogenic.1,87 Organ-isms such as S. mutans and lactobacilli become prevalent andshift the biofilm composition to one that predisposes to caries.This shift emphasizes the role that the environment and hostcharacteristics, including diet, play in oral disease formation.

The environment also influences biofilm architecture.88

Robinson et al. investigated the effects of mechanicalmanipulation, pH, ionic strength, and surfactant (0.25%sodium lauryl sulfate) on in vivo biofilm architecture.88 Theyfound that biofilms were robust, and only surfactant (the con-centration used similar to that in toothpastes) removed or

significantly reduced biofilm as evidenced by decreasedbiomass. The knowledge of biofilm architecture and thepotential to manipulate it raise the possibility of future biofilmtherapeutics and periodontal disease prevention. A summaryof the literature presented in the previous chapters comparingoral and wound biofilms is shown in Table 1.

TREATMENT MODALITIES

Debridement

The mainstay of oral hygiene maintenance is the mechanicalremoval of bacterial biofilm from teeth via tooth brushing,

Table 1. Comparison of oral and wound biofilms

Oral biofilms Wound biofilms

Species identification Culture techniques reveal numerousorganisms but are thought tounderestimate species diversity

Culture techniques are widely used but likelydetect mostly planktonic bacteria andunderestimate the diversity of bacterialspecies present

Molecular analysis utilizing 16S rRNA andFISH reveal over 700 species

16S rRNA and FISH studies show increasedbacterial diversity in wounds

Formation and composition Primary colonizers (Streptococci and others)Crucial bridging bacteria (Fusobacterium

nucleatum)Late colonizers (periodontal pathogens)

Staphylococcus aureus, Pseudomonasaeruginosa, and anaerobes play a role

All bacterial species have not been identifiedyet

Bacterial interactions Coaggregation and coadherence bridges allmethods of bacterial biofilm interactions

Various species assist in the survival of otherspecies

Complex interactions elucidated for manyoral microorganisms

Functional equivalent pathogroups proposedas distinct bacterial groups thatsymbiotically exist in appropriate mixtures

Correlation between nonhealing wounds andthe presence of multiple bacterial speciesvs. one bacterial species

Bacterial communication Autoinducer-2 functions as an importantsignal molecule with the luxS geneconserved among oral pathogens

Gram-positive species: peptidesGram-negative species: homoserine lactones

Autoinducing peptide-1 and delta toxin serveas signal molecules for S. aureus

P. aeruginosa thought to communicate viaN-acyl-homoserine lactones

Immune response Innate: abundant neutrophils andmacrophages, tissue destruction by matrixmetalloproteinases and ROS, andinflammatory cytokines (IL-1, PGE2, TNF-a,IL-1b)

Adaptive: T cells—gingivitis; B cells andplasma cells—periodontitis

Innate: abundant neutrophils andmacrophages, tissue destruction by matrixmetalloproteinases and ROS, andinflammatory cytokines (IFN-a, IFN-g,TNF-a, and IL-1)

Environmental influence Supragingival and subgingival oral biofilmsare composed of different species

P. aeruginosa and S. aureus vary at differentwound locations and appear nonrandomlydistributed

Bacterial species prefer certain sites in theoral cavity

Variation in species has been observed withdiffering wound types

FISH, fluorescence in situ hybridization; IL-1, interleukin-1; IL-1b, interleukin-1b; IFN-a, interferon-a; IFN-g, interferon-g; PGE2,prostaglandin E2; ROS, reactive oxygen species; rRNA, ribosomal ribonucleic acid; TNF-a, tumor necrosis factor-a.



flossing, or using other devices to remove plaque. Denti-frices and mouthwashes are adjuncts containing various bio-cides, surfactants, polymers, or other components aimed atreducing biofilm, and these methods have been shown toreduce gingivitis.9 Physical removal of biofilm bacteria isthe standard means of managing biofilms in dentistry and inindustry because it is the most effective method currentlyavailable.39

In wounds, debridement is also thought to be essential foreffective biofilm control, with authors suggesting that sharpdebridement is the best way to effectively remove and sup-press biofilm.89,90 Interestingly, a study by Nusbaum et al.investigating a variety of debridement techniques found thatplasma-mediated bipolar radiofrequency ablation-treated bio-films had the lowest methicillin-resistant Staphylococcusaureus (MRSA) counts as compared with hydrosurgery, sharpdebridement, and no debridement.91 A decrease in MRSAcounts was observed in all treatment groups relative to nodebridement, though.91 Studies have also shown that biofilmsare able to rapidly recover from mechanical disruption toreform within 24 hours, thus implying that debridement maylead to only a brief window during which antimicrobial treat-ments are more effective in reducing both planktonic andbiofilm microorganisms.18

Antibiotics and antiseptics

Biofilms evade the host’s immune system and some studiesestimate biofilms to be 1,000–1,500 times more resistant toantibiotics than planktonic cells.92 Sedlacek and Walkerstudied subgingival oral species in biofilm and planktonicstates and found that almost all biofilm bacterial cells dis-played a higher minimum inhibitory concentration thanplanktonic cells.93 Multiple mechanisms for biofilm antibioticresistance were suggested including that biofilms contain asubpopulation of specialized survivor cells, the drug targetmay be modified or unexpressed, the biofilm may contain lesssusceptible slow-growing bacteria, and the agent may notadequately penetrate the biofilm.10 The latter is supported byRobinson et al. as they demonstrated increased biomassdensity from the saliva plaque interface inwards and adecreased frequency of channels and voids.88

In patients that lack signs of a local wound infection orsystemic bacteremia, systemic antibiotic therapy has beenshown to be minimally helpful with efficacy as low as25–30%.94 Topical antibiotics have also been widely used inchronic wounds with mixed results as wound biofilm bacteriaare more resistant to topical antibiotics than planktonic bac-teria.94 Davis et al. investigated biofilm resistance to antibiot-ics by applying Mupirocin cream or triple antibiotic ointmentafter 15 minutes (representing planktonic bacteria) or 48hours (representing biofilm bacteria) to a partial-thicknessporcine wound that was inoculated with S. aureus.27 Theyfound that both treatments reduced planktonic bacteria buthad decreased efficacy on S. aureus biofilms.

The use of antiseptics in the control of biofilm as examinedby Kunisada et al. revealed that in vitro P. aeruginosa bio-films were reduced when exposed to 0.2% povidone-iodine,but no inhibition was detected on exposure to 0.2% solutionsof chlorhexidine gluconate, benzalkonium chloride, or alkyl-diaminoethylglycine hydrochloride.95 Nakagawa et al. alsofound that povidone-iodine had rapid bactericidal activityagainst the causative bacteria of periodontal disease.96

Antibiofilm agents

With increased knowledge of biofilm formation, antibiofilmagents have been investigated in the role of biofilm control.Although disrupting community functions and defenses doesnot directly kill the bacteria, the perturbation allows otherconcomitant therapies and natural host mechanisms to workmore effectively to promote healing.39 Lactoferrin, a compo-nent of tears, mucus, and human milk, is thought to preventbiofilm formation via altering bacterial motility and decreas-ing bacterial surface attachment. Lactoferrin use in the dis-ruption of Pseudomonas biofilms was described by Singhet al.4 Lactoferrin has also been shown to act synergisticallywith xylitol, which impairs matrix development.94

A study regarding biofilm-based wound care (BBWC) forwound treatment in patients with critical limb ischemiashowed a statistically significant increase in wound healingwith BBWC as compared with a previous study by Fifeet al.97,98 The study used standard wound care therapy as wellas lactoferrin and xylitol compounded in a methylcellulosegel as the antibiofilm agents. The authors concluded thatmanaging wounds assuming that biofilms are present resultsin an improvement in healing outcomes.97

Further knowledge of biofilm molecular communicationhas led to the potential of a new therapeutic modality forbiofilms: QS inhibitors. Preventing or confusing bacterialcommunication may decrease the expression of biofilm andvirulence genes.18 In fact, a recent study by Novak et al.showed that knocking out QseBC, a two-component regulatorsystem involved in controlling AI-2 signaling for dentalpathogen A. actinomycetemcomitans, resulted in reducedalveolar bone loss than a wild-type murine model.99 Thus,modulation of microbial signaling may represent a suitabletherapeutic approach to oral and other biofilm diseases.8

Polysaccharides are an interesting potential therapeutic ofboth dental plaque and wound biofilms as studies have shownthat bacterial mutants deficient in polysaccharide productionexhibit increased biofilm formation. Valle et al. identified thefirst antibiofilm polysaccharide produced by extra-intestinalEscherichia coli.100 Other antibiofilm polysaccharides havesince been discovered including PsI and PeI produced byP. aeruginosa and crude polysaccharide (released polysaccha-ride or r-EPS) from Lactobacillus acidophilus. While thedetails surrounding antibiofilm polysaccharides need to beelucidated, they have potential to be applied in medical andindustrial settings as most exhibit broad-spectrum biofilm-inhibiting ability. Bacterial biofilms, therefore, constituteuntapped sources of natural bioactive molecules capableof antagonizing adhesion or biofilm formation of otherbacteria.100,101

Biofilm extracellular enzymes

Integral to the success of microorganisms is the final stage ofthe biofilm life cycle, dispersion, which allows bacteria torelease from the biofilm and colonize other areas. Dispersion,however, results in an increased number of easier-to-eradicateplanktonic bacteria and, thus, is a therapeutic topic of interest.One method of dispersion bacteria use is the production ofextracellular enzymes such as glycosidases, proteases, anddeoxyribonucleases, which degrade the adhesive componentsin the biofilm matrix. The glycoside hydrolase dispersin B,produced by the periodontal pathogen A. actinomycetemcomi-



tans, is known to degrade poly-N-acetyl glucosamine, a matrixpolysaccharide that functions in the attachment of A. actino-mycetemcomitans to abiotic surfaces and other microbes.5

Nonoral bacteria, including two common wound pathogensP. aeruginosa and S. aureus, also have been shown to produceenzymes that degrade the biofilm matrix. P. aeruginosa pro-duces alginate lyase, of which increased expression promotesbiofilm cell detachment and antibiotic effectiveness.102,103

Regarding S. aureus, mutant strains that were no longercapable of producing extracellular proteases aureolysin andSp1 resulted in increased biofilm formation and decreasedplanktonic cells.104 In addition, deoxyribonucleases, known asthermonuclease or micrococcal nucleases, are also implicatedin cell detachment in S. aureus biofilms.105

Vaccination and probiotics

As knowledge of the specific bacterial colonizers in periodon-tal diseases increased, this paved the way for the developmentof new treatment strategies, including the potential of vacci-nation against oral pathogens. Numerous studies have docu-mented effective vaccination against oral pathogens.8 Onestudy by Liu et al. observed decreased bacterial coaggrega-tion, biofilm formation, and gum inflammation when avaccine targeting F. nucleatum, a bacterium important inbridging primary and secondary colonizers during oralbiofilm formation, was used.106

Other studies investigating the use of probiotics to replaceperiodontal pathogens with normal oral flora have beenundertaken recently. A pilot human clinical trial assessing theprobiotic mouthwash, ProBiora, found that the mouthwashsubstantially decreased levels of periodontal pathogens, thuswarranting future studies as probiotics may represent a usefulaid in maintaining dental and periodontal health.107 Similartherapeutic approaches regarding chronic wounds may be dis-covered as more knowledge is gained regarding chronicwounds and biofilms.

Laser and ultrasound

Another therapeutic modality currently being investigated isthe use of laser treatment in the control of biofilms anddisease. In wounds, low-level laser therapy (LLLT) is thoughtto accelerate wound healing by increasing the proliferation ofcells involved in wound healing and the synthesis of collagenwhile also decreasing the inflammatory response.108 Lasersalso have been shown to cause disaggregation of microorgan-isms.109 In periodontal disease, results from LLLT studieshave shown significant bactericidal potential without damag-ing oral tissues.108 A study by Basso et al. that evaluated LLLTon biofilms formed by S. mutans and Candida albicans foundthat LLLT reduced both cell viability and biofilm growth.Interestingly, when S. mutans was associated with C. albicansin a dual species biofilm, it was resistant to therapy withLLLT. This highlights the role and importance of understand-ing microorganism biofilm interactions.

In addition to lasers, ultrasonic therapy has been used toinvestigate oral biofilms as well as wound healing. Nishikawaet al. studied the efficacy of noncontact ultrasonic waves onS. mutans biofilms and observed significant biofilm reductionin vitro as well as moderate biofilm removal in vivo.110 Like-wise, dental plaque biofilm was significantly reduced with the

use of an ultrasound toothbrush without bristle contact in anin vitro assay of S. mutans’ biofilm formed on hydroxyapatitedisks.111 Noncontact ultrasound therapy has also been inves-tigated in wounds as a study by Escandon et al. found asignificant reduction in wound area as well as decreased bac-terial counts, inflammatory cytokines, and pain over a 4-weektreatment period in 10 patients with chronic venous ulcers.112

While this study did not directly involve biofilms, the resultsmay have been related to decreased biofilm in chronic woundsas dental research has shown beneficial results of ultrasoundon biofilms.

FUTURE CONSIDERATIONSBoth oral and wound biofilms are topics of extensive research.While oral biofilms are accepted as the cause of both gingi-vitis and periodontitis, wound biofilms are a relatively newconcept and the role they play in chronic wounds is still beingelucidated. This article highlights many similarities betweenoral diseases and chronic wounds, implying that biofilmslikely play a significant role in chronic wound pathogenesis.Wound research can learn a considerable amount regardingbiofilms and the role they play in wounds through looking atwhat researchers have discovered regarding biofilms in oraldiseases.

Wound research has started to utilize knowledge from oralbiofilms regarding the use of advanced bacterial identificationtechniques, including 16S rRNA and FISH, as culturemethods are inadequate in detecting the diversity of microor-ganisms present in both wounds and oral disease. The tech-niques used to identify bacterial species present in biofilmsare crucial to furthering biofilm knowledge. While oralbiofilm research has elucidated the sequential process ofbiofilm formation on teeth and many of the bacterial interac-tions that occur in oral biofilms, these are largely unknown forwound biofilms. Knowledge of both mechanisms will aid indetermining future biofilm therapies.

The role of the innate and adaptive inflammatory immuneresponse in the pathogenesis of both chronic wounds andperiodontal disease serves as further evidence of the similari-ties between these fields of study. Wound research is begin-ning to investigate the role immunity may play in chronicwounds, and this research should be advanced as immunity iswell known to have a role in periodontal disease. Thus, thisarticle highlights the remarkable similarities between woundand oral biofilm-associated diseases and suggests that despitethe specific differences, future wound biofilm studies willbenefit from following the path of oral biofilm research anddiscoveries.

ACKNOWLEDGMENTOur research is supported by grant DE021424 (to DA) fromthe National Institute of Dental and Craniofacial Research.

Conflict of Interest: None of the authors has an actual orpotential conflict of interest.

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wound biofilms: lessons learned from oral biofilms

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