Biofilms: Microbial Shelters Against Antibiotics

Biofilm as a physical barrier

The biofilm matrix consists of EPS, which include exopolysaccharides, minerals, proteins, and extracellular DNA.^18,22,23 The composition and physical properties of EPS vary greatly among different genera and within species. Hydrated networks formed by EPS facilitate cell-to-cell and surface adhesion and provide structural support for biofilm architecture.^4,22 Exopolysaccharides constitute the main EPS in many biofilms and a wide range of exopolysaccharides such as poly-N-acetyl-D-glucosamine, acetan, alginate, cellulose, gellan, pullulan, and xanthan serve as biofilm scaffolding for different microbial species.^24,25

Biofilm EPS form a dense barrier containing numerous anionic and cationic molecules (e.g., uronic acids, proteins, glycoproteins, glycolipids, eDNA, etc.) that can bind charged antimicrobial agents and provide shelter for microorganisms.^5,7,22 These chemical and structural features help impart antibiotic tolerance to cells embedded within the biofilm aggregates. ²⁶ The EPS can limit the diffusion of some antibiotics through several different mechanisms, thus mitigating their antibacterial activity against biofilm bacteria.^{12,13,23,27–30}

Reaction with, or sorption to, EPS may limit transport of antimicrobial agents into biofilm cells. ²⁹ For example, penetration of β-lactams (e.g., oxacillin, cefotaxime) and vancomycin was significantly reduced in Staphylococcus aureus and Staphylococcus epidermidis biofilms. ²⁹ Singh et al. assessed the penetration of these antibiotics through mature biofilms formed on polycarbonate membrane. Using a nitrocellulose membrane placed on the top of a biofilm, they assayed directly for antibiotic diffusion from the antibiotic disc positioned at the top of the biofilm to the agar plate, preinoculated with bacteria, thus enabling the production of a zone of inhibition. The experiment showed that oxacillin, cefotaxime, and vancomycin have limited penetration through S. epidermidis biofilm. ²⁹ Using infrared and Raman spectroscopies, Lu et al. also showed that the transport rate of ciprofloxacin and erythromycin into Campylobacter jejuni biofilms was reduced, compared to natural antibiotic diallyl sulfide from Allium, which freely penetrated the phospholipid bilayer. ³⁰

Charged EPS polymers may also inactivate or bind antibiotics.^26,31 In Pseudomonas aeruginosa biofilms, polyanionic exopolysaccharides, such as alginate, which are EPS overproduced by mucoid P. aeruginosa strains, decrease the activity of positively charged antibiotics such as aminoglycosides,^13,23,32,33 while alginate lyase enhances the activity of some aminoglycosides against P. aeruginosa biofilms. ³³ Interestingly, the host antimicrobial peptide LL-37 produced by polymorphonuclear cells promotes alginate overproduction in P. aeruginosa by inducing mucA mutations through interactions with the DNA polymerase DinB. ³⁴ Such interactions highlight the role of bacterial adaptation to host responses that leads to antibiotic tolerance. Pel exopolysaccharides, another EPS component of P. aeruginosa biofilms, are also able to sequester cationic antibiotics such as aminoglycosides and, thus, confer tolerance to these molecules. ³⁵ The mechanism responsible for this protection is not clear, but Pel is a cationic polysaccharide and is tightly associated with negatively charged eDNA. ³⁶ eDNAs are well known to bind cationic antimicrobials (e.g., aminoglycosides and antimicrobial peptides) and, thus, provide a protective shield effect.^13,36,37

Different mathematical models have shown that in addition to interaction and sorption, delayed antibiotic penetration can be affected by biofilm thickness and age.^12,38,39

Accumulation of β-lactamases within the P. aeruginosa biofilm matrix can lead to increased hydrolysis of antibiotics such as imipenem and ceftazidime.^40,41 P. aeruginosa cells growing in biofilms and exposed to imipenem showed increased expression of AmpC β-lactamases, followed by an increased number of AmpC within the matrix and higher tolerance to β-lactam antibiotics. ⁴¹ Ampicillin is also unable to reach the deeper layers of Klebsiella pneumoniae biofilms, which are associated with β-lactamase activity, while deletion of β-lactamase activity restores ampicillin diffusion. ⁴²

Slow growth, physiological heterogeneity, and persister cells

Biofilm cells are not completely eradicated even when antibiotics successfully penetrate biofilms.^43–46 Although diffusion limitation may occur with some compounds, other mechanisms are clearly involved in biofilm tolerance. During growth in biofilm structures, nutrient and oxygen concentration gradients develop, resulting in physiologically heterogeneous bacterial populations displaying different growth rates.^18,31,47 Since conventional antibiotics usually target cellular functions required in actively replicating cells (e.g., protein synthesis and peptidoglycan synthesis), metabolically inactive or slow growing cells are less affected. Studies on P. aeruginosa biofilms revealed that slow growing cells showed tolerance to ciprofloxacin and tobramycin, ⁴⁸ while antimicrobial peptides such as colistin were active against cells with low metabolic activity and persister cells. ⁴⁹

Persister cells are subpopulations of phenotypically dormant cells generated stochastically or under endogenous stress (e.g., oxidative stress and exposure to antibiotics) that are highly antibiotic tolerant.^{12,44,48,50,51} While persister cells are also found in planktonic populations, they are likely present in higher numbers in biofilms and likely contribute significantly to biofilm antibiotic tolerance. Toxin–antitoxin (TA) systems and the alarmone guanosine 5′-(tri)diphosphate 3′-diphosphate or (p)ppGpp are tightly associated with induced cell dormancy, persister cell formation, and antibiotic tolerance.^44,50–53 In Escherichia coli, the hipA (higher persister protein A) gene encodes for the toxin of type II hipAB TA system, and hyperactivation of HipA through mutation leads to 100 to 1,000-fold increases in persistence. ⁵⁰ Persistence in hyperactivated-HipA mutants was linked to an increased frequency of cells with high levels of (p)ppGpp. ⁵⁰ (p)ppGpp mediates cell dormancy by inhibiting PPX, an exopolyphosphatase, that degrades inorganic polyphosphate (polyP). ⁵¹ Together, polyP and Lon protease mediate degradation of the 11 antitoxins in E. coli K-12, leading to activation of toxins, inducing slow growth, cell dormancy, and antibiotic tolerance.^50,51

Nutrient starvation also induces (p)ppGpp production, which mediates a global stress response known as the stringent response. The stringent response and (p)ppGpp signaling contribute to multidrug tolerance in P. aeruginosa biofilms, as ofloxacin, gentamicin, meropenem, and colistin killing increased upon inactivation of the stringent response. ⁵⁴ Nutrient starvation also induced ofloxacin tolerance in E. coli K-12 biofilm through mechanisms dependent on the stringent and SOS response. ⁵⁵

SOS response, a DNA repair system, can also mediate antibiotic tolerance in biofilms. ⁵⁵ E. coli cells undergo DNA damage, leading to an increased drug tolerance through a mechanism dependent on TisB. This SOS-dependent small toxin binds to cell membranes and disrupts the proton motive force, leading to a drop in ATP levels and reduced susceptibility to multiple antibiotics, including ampicillin and streptomycin. Interestingly, a complete tolerance to ciprofloxacin, known to be effective against nongrowing cells, was also recorded. ⁵⁶

Genetic diversification and antibiotic resistance in biofilms

Biofilms are considered as a reservoir of genetic diversity that promotes bacterial adaptation, evolution, and survival in hostile environments. Biofilm growth favors genetic diversification^34,57–59 and may, thus, contribute to the development of drug resistance. Increased plasmid copy number may also occur during biofilm growth. Cook and Dunny demonstrated that in Enterococcus faecalis biofilm, plasmid copy number increases with increased transcription of plasmid-borne antibiotic resistance genes. However, this mechanism appears reversible upon transfer of biofilm cells to planktonic conditions. ⁶⁰

Several studies have revealed an interconnection between biofilm formation and the prevalence of antibiotic resistance.^61,62 Indeed, higher rates of multidrug-resistant patterns were observed in Acinetobacter baumannii, K. pneumoniae, S. aureus, and P. aeruginosa biofilm forming strains compared to nonforming ones. ⁶² Biofilms may contribute to the emergence and dissemination of antibiotic resistance genes through horizontal gene transfer (HGT) and integrative conjugative elements.^9,63,64 The polymicrobial nature of biofilms and close proximity between species may facilitate HGT. The highly hydrated matrix provides favorable conditions for the transfer of extracellular DNA and natural transformation.^9,33,65 HGT and conjugative plasmids play a key role in bacterial adaptation and evolution ⁶⁶ and occur at higher frequencies in biofilms than in their planktonic counterparts. ⁶⁴ For example, cocultures of two C. jejuni kanamycin or chloramphenicol-resistant strains lead to multiresistant C. jejuni through natural transformation, which occurs more frequently during biofilm compared to planktonic growth. ⁶¹ Merod and Wuertz demonstrated that the transformation frequency in Acinetobacter baylyi biofilms was correlated to the EPS surface area-to-biovolume, suggesting that architectural characteristics, but not relative amounts of EPS, affected natural transformation in A. baylyi. ⁶⁷

Efflux pumps in biofilm resistance

Efflux pumps are transport proteins involved in the extrusion of different metabolites, including antibiotics and secondary metabolites to prevent toxic accumulation.^58,68 They are thus implicated in antibiotic resistance and may promote biofilm antimicrobial resistance in several bacterial species, ⁶⁹ including Burkholderia cenocepacia, ⁷⁰ E. coli, ⁷¹ and P. aeruginosa.^49,72 The PA1874-1877 efflux pump in P. aeruginosa is more highly expressed in biofilms compared to planktonic cells and is involved in resistance to ciprofloxacin, gentamicin, and tobramycin. ⁷³ Similarly, De Kievit et al., observed that P. aeruginosa efflux pumps MexAB-OprM and MexCD-OprJ were overexpressed at the biofilm substratum, where bacterial cells are more susceptible to be exposed to stressful conditions. ⁷⁴ Overexpression of MexAB-OprM was linked to increased biofilm resistance to tetracycline. ⁷⁴

Conversely, uropathogenic E. coli biofilm exhibited a mutation in rapA (RNA polymerase-associated protein) that leads to reduced biofilm and increased susceptibility to penicillin G. ⁷⁵ Transcriptomic analysis demonstrated 22 genes downregulated in rapA mutants, one of which (yhcQ) encodes a putative multidrug resistance pump. ⁷⁵ Mutation in yhcQ gene increased penicillin G sensitivity, suggesting that the YhcQ multidrug resistance pump contributes to biofilm penicillin G resistance in uropathogenic E. coli. ⁷⁵

Multispecies impact on antibiotic tolerance

Although biofilms in nature are likely polymicrobial, most in vitro biofilm studies are carried out with single species. ⁷⁶ This overlooks the potential competitive advantage that multispecies biofilms confer, particularly in stressful and noxious environments.^76,77 For example, dental plaque, a model biofilm, may harbor up to 1,000 different Gram-positive and Gram-negative bacterial species that cooperatively form biofilms.^78,79 The distance between cells of different species affects biofilm architecture and density, ⁸⁰ while polymicrobial interactions influence biofilm antibiotic tolerance. For example, Inquilinus limosus and Dolosigranulum pigrum, unusual antibiotic-sensitive species isolated from the airways of cystic fibrosis patients, became more antibiotic tolerant upon coculture in biofilm conditions with P. aeruginosa. ⁸¹ Similarly, mixed-species biofilms composed of P. aeruginosa, Pseudomonas fluorescens Pf-5, and K. pneumoniae were more tolerant to tobramycin and SDS compared to mono-species biofilms, suggesting that increased tolerance stems from a cross protection beneficial to the entire community. ⁸² More recently, Manavathu et al. developed a polymicrobial biofilm of P. aeruginosa and Moraxella catarrhalis, both highly prevalent in the airways of cystic fibrosis patients, and found that the mixed biofilm had reduced susceptibility to cefepime. ⁸³

Biofilm matrices and EPS that sequester antibiotics may confer cross-species shelter. For example, Candida albicans exopolysaccharide, β-1,3-glucan, can bind ofloxacin. Mixed E. coli—C. albicans biofilms thus have increased ofloxacin tolerance compared to E. coli mono-species biofilms. ⁸⁴ Similarly, S. aureus cells embedded within Candida biofilm showed increased tolerance to vancomycin. ⁸⁵ Interestingly, Candida EPS itself were sufficient to confer vancomycin tolerance to S. aureus, suggesting that the Candida matrix mediates cross-species tolerance by sequestering vancomycin. ⁸⁵ Billings et al. demonstrated that the Psl polysaccharide provides an electrostatic barrier to colistin and contributes to colistin tolerance in P. aeruginosa biofilms, and this protective effect was transferable to non-Psl producers. ⁸⁶

Polymicrobial biofilms formed by M. catarrhalis and Streptococcus pneumoniae rendered both bacteria more resistant to β-lactam antibiotics and bacterial clearance. ⁸⁷ In the mixed biofilms, β-lactamase produced by M. catarrhalis provided passive protection to S. pneumoniae against amoxicillin killing. In turn, S. pneumoniae protected M. catarrhalis from azithromycin killing by an unknown mechanism. ⁸⁷ Antibiotic tolerance in multispecies biofilms remains poorly studied and will require a better understanding of the key elements of cell–cell interaction in mixed communities.

Antibiotics as biofilm signals

Subminimal inhibitory concentrations of antibiotics (sub-MICs) increase the risk of tolerance and resistance to antibiotics. ⁸⁸ Bacterial pathogens are potentially exposed to subinhibitory and ineffective doses of antibiotics: (1) during clinical therapy with inadequate dosing regimens; (2) following delayed drug penetration in biofilms;^22,89 and (3) in food-producing animals exposed to antibiotics as growth promoters to prevent infection in contaminated environments. ⁹⁰ Such sub-MIC antibiotics may have broad effects on bacterial gene expression, virulence, quorum sensing (QS), and biofilm formation.^91–95 The mechanisms associated with sub-MIC antibiotic-induced biofilm formation and antibiotic tolerance are dependent on the nature of the antibiotic, the dosage, and the bacterial species, among other things.

Multiple lines of evidence suggest that sub-MIC antibiotics increase EPS production. Using flow conditions, Berlutti et al., demonstrated that azithromycin influenced S. aureus biofilm development by increasing matrix production. ⁹⁶ Sub-MIC erythromycin, tetracycline, and quinpristin–dalfopristin induced the intercellular adhesion gene cluster (ica) expression in S. epidermidis,^97,98 leading to increased EPS expression and invasiveness. ⁹⁹ Sub-MIC tigecycline increased biofilm matrix poly-N-acetyl-glucosamine production ¹⁰⁰ and sub-MIC aminoglycoside stimulated EPS production in P. aeruginosa. ¹⁰¹ Similarly, sub-MIC imipenem activated genes involved in alginate biosynthesis in P. aeruginosa. ⁴¹ Colanic acid, an important biofilm component in E. coli, was induced in the presence of sub-MIC of certain β-lactam antibiotics. ¹⁰² Sub-MICs of β-lactam drugs (i.e., ampicillin and cefuroxime) upregulated genes involved in glycogen biosynthesis by nontypable Haemophilus influenzae, resulting in thicker biofilms. ⁹⁴ Similarly, exposure of Streptococcus mutans to sub-MICs of triclosan, a broad spectrum antimicrobial agent, upregulated expression of genes involved in biofilm formation and surface adhesion, such as gtfC (encoding a glucotransferase) and atlA (encoding a cell surface fibronectin-binding adhesin), leading to increased adherence to epithelial cells. ¹⁰³

More recently, Hathroubi et al., demonstrated that sub-MIC of penicillin G induced cell envelope stress, increased the expression of extracellular polysaccharide genes, and enhanced release of extracellular DNA in the biofilm matrix in Actinobacillus pleuropneumoniae, leading to autoaggregation and increased biofilm formation. ¹⁰⁴ Similarly, sub-MIC β-lactams also led to increased extracellular DNA and autoaggregation in S. aureus biofilms. ¹⁰⁵

Exposure to sub-MIC antibiotics also alters bacterial morphology. Sub-MIC penicillin induced bacterial filamentation and increased cell-surface hydrophobicity in Corynebacterium diphtheriae, leading to increased biofilm formation. ¹⁰⁶

Sub-MIC antibiotics may also modulate QS signaling, a form of cell–cell communication. Sub-MIC ampicillin induced Staphylococcus intermedius biofilm formation using the autoinducer-2/LuxS signaling pathway. ¹⁰⁷ In addition, the autoinducer type 2 synthase luxS was also upregulated when Streptococcus mutans was cultivated in the presence of sub-MIC triclosan. ¹⁰³

Taken together, these observations strongly suggest that sub-MIC of antibiotics may promote biofilm formation and modulate biofilm matrix composition, leading to greater persistence and protection against antibiotics.

Strategies to prevent biofilm formation

Various strategies have been examined to prevent biofilm formation on diverse surfaces (e.g., prosthetics, medical devices, industrial water pipe systems, food product packaging, etc.). One approach has focused on modifying abiotic surface properties by either coating or impregnating them with antibacterial agents, which are released in a controlled manner to inhibit surface bacterial adhesion and growth.^108,109 Unfortunately, such coated biomaterials usually have only short-term efficiency ¹⁰⁹ and may promote the emergence of multiresistant bacteria. ¹¹⁰

Alternatives to antibiotics that inhibit biofilm formation have been intensively investigated.^111–115 Bismuth thiols (BTs) have potent antimicrobial and antibiofilm activity against a wide range of bacterial species.^116,117 Sub-MICs of BTs reduce K. pneumoniae, methicillin-resistant S. aureus (MRSA), and P. aeruginosa biofilms substantially.^117–120 At sublethal doses, BTs reduce MRSA and P. aeruginosa biofilm formation in vitro under continuous flow biofilm growth conditions.^117,119,120 Biofilm formation was also reduced in vivo on subcutaneous BT-coated prosthetics contaminated with MRSA. ¹¹¹ Recently, BTs have been used to prevent biofilm-related wound infections. When administrated locally to infected open fracture wounds, BTs prevented S. aureus biofilm formation, disrupted preexisting biofilms, and sensitized bacteria to antibiotic treatment and immune defenses. ¹²¹ Indeed, several antibiotics can be potentiated or repurposed in combination with sub-MIC BTs, ¹²² even against highly resistant Burkholderia strains. ¹²³ By virtue of their activity at micromolar concentrations to enhance antibiotic activity, reduce virulence, and enable immune defenses, without inducing bacterial resistance, as well as low toxicity in mammals, BTs may address several unmet needs and present as ideal candidates in the fight against biofilm-related infections.

Metal ions have also been intensely investigated. Silver molecules are tolerated by eukaryotic cells and, thus, can be incorporated into medical devices and implants.^113,124 Using a coating technique called Sol-gel processing, Marini et al. fabricated a silver-doped organic–inorganic hybrid coating of tetraethoxysilane- and triethoxysilane-terminated poly(ethylene glycol)-block-polyethylene, which was effective in inhibiting E. coli and S. aureus biofilms. ¹²⁵ Nanotechnology further adds to silver's promise. Selenium and silver nanoparticles inhibit biofilm formation through mechanisms not fully understood, although likely implicating the antimicrobial properties of these metals.^114,126,127 Selenium nanoparticles synthetized from Se-reducing Bacillus sp. strain MSh-1 inhibited biofilms from several pathogens, including P. aeruginosa and S. aureus. ¹¹⁴ Similarly, silver nanoparticles inhibited biofilms formed by multidrug-resistant P. aeruginosa ¹²⁷ and S. epidermidis. ¹²⁸ The acylated homoserine AiiA lactonase purified from Bacillus licheniformis has QS inhibitory activity by inactivating acylated homoserine lactones, which are cell–cell communication signals implicated in biofilm formation. Coupling of AiiA with gold nanoparticles significantly enhanced the inhibition of Proteus biofilms compared to that of AiiA alone. ¹²⁹

Several thiazolidine-2,4-dione (TZD) derivatives, heterocyclic compounds belonging to the antidiabetic drug family, exhibit specific antibiofilm and antiadhesion activities against C. albicans. ^130,131 Incorporated into food packaging, TZD successfully interfered with several bacterial and fungal biofilms. ¹³²

Cationic antimicrobial peptides (CAMPs) are positively charged hydrophobic amino acid molecules that kill bacteria mainly through membrane perturbation ¹³³ and have a broad-spectrum antimicrobial activity, high potency, and low mammalian cell toxicity. They are novel antimicrobials that may also be attractive antibiofilm agents, either alone or as adjuncts to therapies against antibiotic-resistant biofilms. Several antibiofilm CAMPs were investigated, including AS10, a synthetic peptide, a variant of the mouse antimicrobial peptide LL-37. AS10 inhibited biofilm formation from various Gram-negative bacteria, including E. coli, P. aeruginosa, and Porphyromonas gingivalis. ¹³⁴ Similarly, the antimicrobial peptide 1018 also inhibited biofilm formation in a broad range of pathogens, putatively due to degradation of the alarmone signal ppGpp. ¹³⁵ More recently, a synthetic peptide innate defense regulator (IDR-1080), derived from the bovine neutrophil host defense peptide bactenecin, demonstrated antibiofilm activity against antibiotic-resistant P. aeruginosa and S. aureus biofilms. ¹¹² Urinary catheters coated with CWR11, a synthetic antimicrobial peptide, displayed potent antibiofilm properties against several urinary tract pathogens. ¹³⁶

Strategies to overcome pre-established biofilm

Once established, biofilms are very difficult to eradicate. Therapeutic approaches have focused on weakening and/or disrupting the biofilm architecture by targeting the EPS and biofilm matrix. Dornase alfa or Pulmozyme^®, a recombinant human DNase enzyme, can target extracellular DNA in biofilm matrix. It has been commercially developed for inhaled clinical use to treat the chronic lung disease in patients with cystic fibrosis. ¹³⁷ Tested against S. pneumoniae biofilms, Pulmozyme significantly reduced biofilm biomass and thickness. ¹³⁸ Other biofilm-disrupting enzymes have also been explored. The serine protease ESP produced by S. epidermidis ¹³⁹ and the elastase LasB by P. aeruginosa ¹⁴⁰ disrupt S. aureus biofilms. More recently, Yu et al. reported that PslG, a glycosyl hydrolase from P. aeruginosa, disrupted established biofilms from a wide range of Pseudomonas strains at nanomolar concentrations, while pretreatment with PslG sensitized biofilms to tobramycin and ciprofloxacin decreased the minimum biofilm eradication concentration by eight- and fourfold, respectively. ¹⁴¹ More recently, the combination of DispersinB^®, a polysaccharide-hydrolyzing enzyme that degrades poly-N-acetylglucosamine, with a silver wound dressing (Anticoat™) ¹⁴² or an antimicrobial peptide-based wound gel, ¹⁴³ demonstrated synergistic antibiofilm activity against chronic wound-associated biofilm infections, including MRSA. The use of biofilm disrupting enzymes such as polysaccharide-hydrolyzing enzymes, DNAse, and proteases could thus be a promising strategy to treat established biofilm-related infections, most likely in combination with conventional antibiotic therapy that can target newly released bacterial cells.^{43,46,122,144}

Bacteriophages may also hold promise against biofilms. These are highly specific self-replicating agents that can infect and rapidly lyse bacteria and may be used in a variety of applications, for example, food production,^145–147 and as potential alternative treatments against pathogenic bacteria, such as P. aeruginosa.^148–150 For example, combinations of bacteriophages and conventional antibiotics eradicated MRSA biofilm, while antibiotic treatment alone had no significant effect on the biofilm, indicating synergistic activity of the combined therapy. ¹⁵⁰ An anti-Enterococcus faecalis bacteriophage isolated from sewage effluent proved to be more effective than conventional antibiotics in disrupting a 2-week old biofilm of E. faecalis, demonstrating the superiority of phage therapy over antibiotherapy. ¹⁵¹ Lu and Collins engineered an enzymatic phage that expressed DispersinB ¹⁵² and reduced biofilm cell counts by two orders of magnitude greater than the nonengineered phage, leading to nearly complete biofilm removal. ¹⁵³

Interfering with biofilm structures may also be achieved by inhibiting QS systems, which are implicated in coordinating bacterial biofilm formation.^15,154,155 For example, TZDs act as QS inhibitors in Vibrio harveyi ¹⁵⁶ and P. aeruginosa ¹⁵⁷ and negatively affect biofilm formation without affecting bacterial growth. ¹⁵⁷ Trans-cinnamaldehyde also inhibited P. aeruginosa N-acyl-homoserine lactone synthase without affecting bacterial viability. ¹⁵⁸ 6-gingerol oil reduced P. aeruginosa biofilms by repressing QS pathways. ¹⁵⁹ Antifungal agents produced by streptomycetes, called maniwamycins, demonstrated high anti-QS potential in the soil bacterium Chromobacterium violaceum. ¹⁶⁰ However, recent studies have shown that loss of QS function emerged readily during chronic human biofilm-associated infections^57,161 and raises questions about the role of QS and the relevance of QS inhibitors as therapeutic agents in such infections.