Microorganisms in biofilms are resistant to antimicrobial agents, but not in the classic sense. Conventional antimicrobial resistance refers to acquired resistance in free-floating (ie, planktonic) bacteria (and other microorganisms), and occurs through antimicrobic inactivation (eg, by β-lactamases), modification of targets to which antimicrobial agents bind (eg, as occurs with vancomycin resistance in enterococci), or decreased access of antimicrobial agents to their target (eg, through efflux). Traditional antimicrobial resistance is conferred by mutation or acquisition of a new gene (or new genes) via genetic exchange, and, in general, is irreversible. Proximity of cells to one another within biofilms may provide an environment conducive to exchange of antimicrobial resistance genes (eg, on plasmids or transposons). “Conventional” antimicrobial resistance, however, generally is considered distinct from biofilm-associated antimicrobial resistance (however, in Pseudomonas aeruginosa, PvrR, a regulatory protein, modulates the phenotypic switch from antimicrobial-resistant to antimicrobial-susceptible and apparently also regulates biofilm formation).13
Orthopaedic surgeons are familiar with resistance associated with microorganisms in biofilms because orthopaedic-associated infections can be difficult to treat with antimicrobial agents (to which the organisms are susceptible using conventional tests) alone and often require surgical debridement and removal of foreign bodies (if present) for cure. Conversely, clinical experience suggests that biofilm-associated antimicrobial resistance is reversible because if the foreign body is removed, the organism can be eradicated with a conventional antimicrobial agent.
Although there is some understanding of the mechanism or mechanisms of biofilm-associated antimicrobial resistance, this is clearly an area replete with rapidly emerging data. The mechanism of biofilm-associated antimicrobial resistance seems to be multifactorial and may vary from organism to organism. Techniques that address biofilm susceptibility testing to antimicrobial agents may be necessary before antimicrobial regimens for orthopaedic prosthetic-device-associated infections can be appropriately defined in research and clinical settings. A variety of approaches are being defined to overcome biofilm-associated antimicrobial resistance. The purpose of this review is for me to examine the emerging understanding of mechanisms of biofilm-associated antimicrobial resistance.
The pathogenesis of many orthopaedic infections (eg, osteomyelitis and prosthetic joint infection) is related to the presence of microorganisms in biofilms. Biofilms are composed of microcolonies enclosed in a highly hydrated polymeric matrix surrounded by interstitial voids in which nutrients circulate between cells. Within biofilms, microorganisms develop into organized communities with structural and functional heterogeneity similar to that of a multicellular organism; interstitial voids between microcolonies can be considered, for example, to serve as a rudimentary circulatory system. Cell-to-cell signaling (ie, quorum-sensing) molecules induce biofilm microorganisms to change patterns of gene expression. At sufficient population density, such signals reach sufficient concentrations to activate genes involved in biofilm differentiation. Existence within a biofilm represents a basic survival mechanism of microorganisms in which microorganisms are protected through mechanisms that are in the process of being defined, from environmental influences including host immune responses (eg, opsonization, phagocytosis, and complement-mediated lysis) and normal levels of conventional antimicrobial agents. In conditions mimicking physiological shear, polymorphonuclear neutrophils attach to, penetrate, and produce cytokines in, maturing and fully matured Staphylococcus aureus biofilm29; nevertheless, they are unable to clear the bacteria.57 Actually, ineffective, “frustrated” attempts at phagocytosis on the part of polymorphonuclear neutrophils may result in release of cytotoxic and proteolytic substances contributing to tissue injury and ultimately to periprosthetic osteolysis.57
Phenotypic Studies of Biofilm-Associated Antimicrobia Resistance
In vitro data indicate that microorganisms in biofilms are substantially more resistant to killing by antimicrobial agents than are planktonic bacteria. In a study of Staphylococcus epidermidis clinical isolates, for example, all isolates were susceptible to vancomycin in the planktonic state; however, when grown as a biofilm, almost ¾ had minimum bactericidal concentrations (defined as 99.9% reduction in colony forming units) of greater than 2048 μg/mL of vancomycin.23
Biofilm antimicrobial resistance generalizes to a wide range of bacterial species and to fungi.11,27 In a study of Propionibacterium acnes prosthetic hip-associated isolates growing in in vitro biofilms on polymethylmethacrylate, for example, all isolates showed considerably greater resistance to cefamandole, ciprofloxacin and vancomycin in the biofilm than in the planktonic state.46 Likewise, Candida albicans isolates grown as biofilms are more resistant to fluconazole, voriconazole, nystatin, terbenafine, and amphotericin B, than are planktonic forms.27
Antimicrobial Resistance-Correlation with Biofilm Age
Biofilm resistance to antimicrobial agents begins at the attachment phase and increases as the biofilm ages.25 In a study of S. epidermidis biofilms, for example, vancomycin exhibited decreased killing as the biofilm aged from 6 hours to 2 days.32 This observation is paralleled in orthopaedic clinical practice where debridement with retention of an infected prosthesis is more successful for early in comparison to late postoperative infection (provided that it is done expeditiously).
The observation that a diverse array of microorganisms in biofilms exhibits increased antimicrobial resistance in the biofilm versus planktonic state led to an early impression that there might be a single, generalizable mechanism of biofilm-associated antimicrobial resistance. Recent data suggest, however, that this likely is a multifactorial process and that mechanisms may vary from one organism type to another. A substantial body of work has focused on Pseudomonas aeruginosa biofilms. Less is known about the mechanism of antimicrobial resistance in staphylococcal biofilms, which is more relevant to orthopaedic practice.
A variety of reasons for the increased antimicrobial resistance of microorganisms in biofilms have been postulated and investigated. Bacteria in biofilms are surrounded by an extracellular matrix that might physically restrict the diffusion of antimicrobial agents. Nutrient and/or oxygen depletion and/or waste product accumulation within the biofilm might cause some bacteria to enter a nongrowing (ie, stationary) state, in which they are less susceptible to growth-dependent antimicrobial killing. A subpopulation of bacteria might differentiate into a phenotypically resistant state. Finally, organisms in biofilms might express biofilm-specific antimicrobial resistance genes that are not required for biofilm formation.
Antimicrobic inactivation or slow diffusion does not seem to play an important role in Klebsiella pneumoniae biofilm quinolone or β-lactam (for β-lactamase-deficient mutants) resistance, P. aeruginosa carbapenem resistance, or C. albicans azole or polyene resistance.1,2,11 Anderl et al2 showed that ciprofloxacin penetrated Klebsiella pneumoniae colony biofilms despite these biofilms being relatively resistant to ciprofloxacin. Similarly, ampicillin-penetrated colony biofilms formed by a β-lactamase-deficient mutant Klebsiella pneumoniae strain (but not biofilms formed by a β-lactamase-producing Klebsiella pneumoniae strain) despite these biofilm cells being relatively resistant to ampicillin.2 Likewise, Coquet et al11 showed that when viable P. aeruginosa cells were entrapped in alginate gel layers to form artificial biofilmlike structures, impaired diffusion of imipenem into the alginate layer was not observed. Al-Fattani and Douglas1 used a filter disk assay to show penetration of flucytosine, fluconazole, amphotericin B, and voriconazole into single-species and mixed-species biofilms containing candida species.
Pseudomonas aeruginosa biofilms formed by an alginate-overproducing strain, however, show a highly structured architecture and are more resistant to tobramycin than biofilms formed by an isogenic nonmucoid strain, suggesting that an important consequence of the conversion to mucoidy in P. aeruginosa is an altered biofilm architecture associated with increased resistance to antimicrobics.18 Mah et al31 recently have identified a gene, ndvB, that when disrupted, results in P. aeruginosa cells that form biofilms with the characteristic P. aeruginosa architecture, but that do not develop high-level biofilm-specific resistance to antimicrobial agents. The ndvB locus is required for the synthesis of periplasmic glucose polymers that interact with tobramycin apparently preventing antimicrobial agents from reaching their sites of action as a result of sequestration in the periplasm.31 Whether such a process occurs in staphylococci is not known, but could hypothetically explain the poor activity of glycopeptides against S. epidermidis biofilms.26
Using deoxyribonucleic acid (DNA) microarrays, it has been shown that approximately 1% of genes show differential expression between biofilm and planktonic P. aeruginosa cells-about ½ are relatively activated and ½ are relatively repressed in biofilms.60 Likewise, the proteome of attached P. aeruginosa cells differs from that of planktonic P. aeruginosa and is related strongly to the nature of the substratum.56
Pseudomonas aeruginosa recovered from a biofilm and tested in nongrowing conditions with tobramycin shows higher resistance levels than planktonic cells but lower resistance levels than cells of an intact biofilm.7 Likewise, amphotericin B resistance of C. albicans biofilms is not simply caused by slow growth.4
The contribution of multidrug resistance pump-mediated efflux to antimicrobial resistance of P. aeruginosa biofilms was examined using strains overexpressing and lacking the MexAB-OprM pump. Resistance of P. aeruginosa biofilms to ofloxacin was dependent on the expression of MexAB-OprM, but only in the low concentration range. Biofilm resistance to ciprofloxacin, also a substrate of MexAB-OprM, did not depend on the presence of this pump. Instead, a small super-resistant cell fraction was considered primarily responsible for very high resistance of P. aeruginosa biofilms to quinolones.7 Although expression of genes encoding active efflux through ATP-binding cassette transporters and major facilitators of azoles from C. albicans are up-regulated during the course of biofilm formation and development, mutants carrying deletions of these genes, although hypersusceptible to fluconazole when planktonic, are resistant to fluconazole at late stages of biofilm growth.33,45
Anderl et al3 suggest that antimicrobial tolerance of biofilm bacteria is at least partially explained by nutrient limitation by experimentally characterizing the nutrient availability and growth status of Klebsiella pneumoniae biofilms. The average specific growth rates of biofilms and planktonic K. pneumoniae were 0.032 per hour and 0.59 per hour, respectively.3 Glucose did not penetrate the biofilm completely, and oxygen was shown to penetrate only the upper 100 μm.3 Transmission electron microscopy revealed that bacteria were affected by ampicillin near the biofilm periphery but not in the interior.3 Anderl et al3 concluded that K. pneumoniae have nutrient limitation locally within the biofilm, leading to zones in which they enter stationary phase and grow slowly or not at all and that in these inactive regions, bacteria are less susceptible to killing by antimicrobics.
Local oxygen limitation and the presence of nitrate may contribute to reduced susceptibility of P. aeruginosa biofilms causing infections in vitro.6 Whereas 2-day-old P. aeruginosa colony biofilms physiologically are heterogeneous and most of the cells occupy an oxygen-limited, stationary-phase state, 4-hour-old colony biofilms are still growing, active, and susceptible to antimicrobial agents when they are challenged in air. However, when challenged in anaerobic conditions, the level of killing by antimicrobics is reduced, compared with that for controls grown aerobically. Oxygen limitation seems, therefore, to explain a substantial amount of the protection afforded to 2-day-old P. aeruginosa colony biofilms. Nitrate amendment decreases the susceptibilities of the organisms to antimicrobial agents.
Roberts and Stewart49 used a mathematic model to investigate protection from antimicrobial killing afforded to microorganisms in biofilms based on localized nutrient limitation and slow growth. Their model assumed that the rate of killing was directly proportional to the local growth rate as calculated by using the local concentration of a single growth-limiting substrate with Monod kinetics.49 The concentration profile of this metabolic substrate was calculated by solving a reaction-diffusion problem.49 The model predicted stratified patterns of growth with zones of no growth in the biofilm interior, slow killing of biofilm microorganisms further retarded as the initial biofilm thickness increased, nonuniform spatial patterns of killing inside the biofilm, biofilm killing rates decreasing in a nonlinear way as the concentration of the growth-limiting substrate feeding the biofilm decreased, and heightened tolerance when external mass transfer resistance manifested.49 Overall these results suggest that nutrient limitation and slow growth contribute in an important way to the antibiotic tolerance of microorganisms in biofilms.49
Fux et al14 observed that the oxacillin resistance of detached S. aureus biofilm particles depends on embolus size and can be attributed to the nutrient-limited stationary-phase physiology of cells within emboli; they hypothesize that the detachment of such multicellular clumps may explain the high rate of symptomatic metastatic infections characteristic of S. aureus.
Antimicrobial Resistance-Laboratory Testing of Microorganisms in Biofilms
Techniques that address biofilm susceptibility testing may be necessary before antimicrobial regimens for orthopaedic prosthetic device-associated infections can be appropriately defined based on antimicrobial susceptibility testing results. Clearly, easy to use, standard, clinically validated methods for antimicrobial susceptibility testing of microbial biofilms are warranted. The Calgary Biofilm Device (commercially available as the MBEC Assay System, through MBEC Biofilms Technology Ltd., Calgary, Canada) for example, can be used to form bacterial biofilms and to determine minimum biofilm eradication concentration values of antimicrobial agents.36
Strategies for Control of Biofilms
Cell-to-cell signaling (ie, quorum-sensing) regulation of biofilm formation affords a novel potential target to control biofilms. Biofilms formed by P. aeruginosa quorum-sensing-deficient mutants are thinner with closer packed cells, have less visible extracellular matrix, and are more susceptible to kanamycin than are biofilms formed by wild-type strains.51 Synthetic derivates of natural furanone compounds found in the marine environment act as potent antagonists of P. aeruginosa quorum sensing and increase P. aeruginosa biofilm susceptibility to antimicrobial agents, such as tobramycin, in vitro; they also have been shown to promote clearance of P. aeruginosa in a murine pulmonary infection model.19 Ribonucleic acid III (RNAIII)-inhibiting peptide is a heptapeptide that disrupts biofilm formation in S. aureus and S. epidermidis apparently also by disrupting quorum-sensing mechanisms.5 Ribonucleic acid III-inhibiting peptide seems to be synergistic with conventional antimicrobial agents, and has been shown to eliminate graft-associated S. epidermidis infections in vivo suggesting that it could be used to coat medical devices to prevent staphylococcal infection5 (Fig 1).
Application of ultrasound, at levels without inhibitory or bactericidal activity against bacteria (67 kHz), enhances the inhibitory and bactericidal activity of gentamicin against planktonic cultures of P. aeruginosa and Escherichia coli.42 Even as the age of the culture increases, and the bacteria become more resistant to the effect of gentamicin alone, the application of ultrasound reverses this resistance.42 Pitt and Ross43 showed that low-frequency (70 kHz), low power density (< 2 W/cm2) ultrasound increased the growth rate of S. epidermidis, P. aeruginosa, and E. coli adhered to polyethylene (PE) surfaces compared with growth without ultrasound, and that ultrasound enhanced planktonic growth of S. epidermidis and other planktonic bacteria. Ultrasound also has been shown to increase transport of gentamicin across biofilms.8
Rediske et al47,48 subcutaneously implanted E. coli biofilms grown on PE disks in the backs of rabbits and applied low-frequency (28.48 kHz) and low-power density (300 mW/cm2) ultrasound treatment for 24 hours with and without systemic administration of gentamicin. Whereas exposure to ultrasound alone caused no considerable difference in bacterial viability, in the presence of gentamicin, there was a substantial reduction in bacterial viability (the bioacoustic effect). However, tissue damage to the skin was observed. A similar experiment was done with PE disks covered with S. epidermidis biofilms implanted subcutaneously in the backs of rabbits treated with or without systemic administration of vancomycin.9 Staphylococcus epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times were required than were required in the case of E. coli and gentamicin.9
Low electric current combined with an antimicrobial agent has also been shown to enhance the killing of biofilm-associated bacteria compared with the antimicrobial agent alone (the bioelectric effect). The bioelectric effect reduces the very high concentrations of aminoglycosides, tetracyclines or quinolones needed to kill biofilm bacteria to levels close to those needed to kill planktonic bacteria of the same species.10,12,20,21,24,41,55,58,59 The mechanism of the bioelectric effect is not completely defined and, to date, an in vivo bioelectric effect has not been shown.
Lysostaphin, a constituent of human secretions, is a glycylglycine endopeptidase that specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan and rapidly kills planktonic S. aureus at low concentrations (MIC90, 0.001 to 0.064 μg/mL) and S. epidermidis at higher concentrations (MIC90, 12.5 to 64 μg/mL).61 Wu et al61 showed that lysostaphin disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces and killed lysostaphin-susceptible, but not lysostaphin-resistant S. aureus in biofilms at concentrations as low as 1 μg/mL. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms.61 Lactoferrin, another constituent of human secretions, blocks biofilm development by P. aeruginosa.53
A variety of chemicals have been shown to be active against bacteria in biofilms. A combination of streptokinase and streptodornase has been shown by Nemoto et al34,35 to be active against S. aureus and P. aeruginosa biofilms. Other investigators tested the activity of enzymes against bacteria in biofilms; oxidoreductases were bactericidal against biofilms and a complex mixture of polysaccharide-hydrolyzing enzymes removed bacterial biofilm.22 Hatch and Schiller17 showed that alginate lyase permitted increased diffusion of aminoglycosides through alginate in P. aeruginosa. Yasuda et al62 studied interactions between clarithromycin and biofilms formed by S. epidermidis using a clarithromycin-resistant strain; treatment with a relatively low concentration of clarithromycin resulted in eradication of the “slime-like structure” and in a decrease in the amount of hexose. Allicin, which is derived from garlic, is a sulfur-containing compound formed in small quantities from the enzymatic action of allinase on alliin. Allicin has been shown to be active in vitro against S. epidermidis and C. albicans, and diminishes S. epidermidis and C. albicans biofilm formation.40,52
Staphylococcus epidermidis and S. aureus are often susceptible to rifampin, although emergence of rifampin resistance can be problematic. Use of combination therapy generally avoids this pitfall. Gagnon et al15 determined the effect of combinations of 13 different antimicrobics with rifampin against S. epidermidis biofilms in vitro. Synergy with rifampin was observed with cloxacillin, cephalothin, cefazolin, cefamandole, vancomycin, ciprofloxacin, tetracycline, and amikacin. Whereas tobramycin, erythromycin, clindamycin, fusidic acid did not influence the outcome; gentamicin unexpectedly showed antagonism with rifampin.15
In continuous-flow biofilm cultures using a medium mimicking cystic fibrosis bronchial secretions, P. aeruginosa was not eradicated from biofilms by 1 week of treatment with high concentrations of ceftazidime and gentamicin, to which the strains were susceptible by conventional testing.16 Addition of rifampin, however, which had little activity against the strains as measured by minimum inhibitory concentrations, led to nonstrain-specific elimination of bacteria from biofilms.16
The comparative activities of vancomycin, clindamycin, novobiocin, and minocycline, alone or in combination with rifampin, were tested in an in vitro model of colonization using the modified Robbins device with antibiotic-impregnated cement filling the lumen of catheter segments.44 The combination of minocycline and rifampin was the most active in preventing bacterial colonization of biofilm-producing strains of S. epidermidis and S. aureus to the catheter surfaces.44 A similar trend was observed when the inhibitory activities of polyurethane catheters coated with minocycline and rifampin were compared with the inhibitory activities of catheters coated with other antimicrobial agents.44 The inhibitory activities of catheters coated with minocycline and rifampin against S. epidermidis, S. aureus, and Enterococcus faecalis strains, were significantly better than those of catheters coated with vancomycin.44 The inhibitory activities of catheters coated with minocycline and rifampin against gram-negative bacilli and C. albicans were comparable to those of catheters coated with ceftazidime and amphotericin B, respectively.44
Rifampin penetrates biofilms formed by S. epidermidis but does not kill biofilm S. epidermidis.63 The combination of sparfloxacin or vancomycin with amikacin or rifampin show activity against S. epidermidis biofilms on catheters.37,38 Peck et al39 showed that the combination of erythromycin or rifampin and vancomycin was more active than vancomycin alone against S. epidermidis biofilms formed on polyurethane sheets.
The activity of tigecycline against S. epidermidis growing in an in vitro adherence biofilm model was determined. Tigecycline minimum bactericidal concentrations ranged from 1 to 8 μg/mL for S. epidermidis growing in biofilms, compared with 0.12 to > 32 μg/mL for planktonic cells.28 The killing activity of tigecycline against the adherent bacteria was at least fourfold better than that of vancomycin and daptomycin.28
Liposomal amphotericin B, amphotericin B lipid complex and the echinocandins, caspofungin and micafungin, show promising in vitro activity against candidal biofilms.27 In a rabbit model of silicone catheter-associated infection with C. albicans biofilms, antifungal lock therapy with liposomal amphotericin B was shown to be an effective treatment strategy compared with fluconazole or no treatment.50 Interestingly, a subinhibitory concentration of caspofungin induced decreased in vitro adherence of all fluconazole-susceptible C. albicans isolates but of only 60% of fluconazole-resistant isolates, suggesting a possible relationship between the activity of caspofungin in inhibiting a first step in the development of C. albicans biofilm and resistance to fluconazole.54
Although there is some understanding of the mechanism or mechanisms of biofilm-associated antimicrobial resistance, this clearly is an area replete with rapidly emerging data. The mechanism of biofilm-associated antimicrobial resistance seems to be multifactorial and may vary from organism to organism. Techniques that address biofilm susceptibility testing to antimicrobial agents are necessary before antimicrobial regimens for orthopaedic prosthetic device-associated infections can be appropriately defined in research and clinical settings. A variety of approaches are being defined to overcome biofilm-associated antimicrobial resistance.
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