Biofilm-Specific Antibiotic Resistance
One of the most intriguing and clinically relevant features of microbial biofilms is their significantly higher antibiotic resistance relative to their free-floating counterparts, which generates serious consequences for therapy of biofilm-associated infections. The MIC of antibiotics to biofilm-growing bacteria may be up to 1000-fold higher than that of planktonic bacteria. Multiple biofilm-specific mechanisms are operated simultaneously in a reversible and transient manner contributing to the high levels of antibiotic resistance of biofilms, and these are distinct from the well-characterized intrinsic resistance mechanisms (e.g., expression of antibiotic-degrading enzymes, inducible decrease in antibiotic influx, inducible increase in antibiotic efflux and alteration in antibiotic target sites) employed by planktonic cells.
Delayed Antibiotic Diffusion into Biofilms
The biofilm matrix can act as a barrier to delay the diffusion of antibiotics into biofilms because antibiotics may either react chemically with biofilm matrix components or attach to anionic polysaccharides. If the time required for an antibiotic to penetrate biofilms is longer than the duration of antibiotic treatment, the slower penetration will explain the antibiotic resistance. Antibiotics have been shown to readily penetrate biofilms in some cases, but poorly in others depending on particular antibiotics and biofilms. The binding of the positively charged aminoglycosides to the negatively charged biofilm matrix polymers of P. aeruginosa will delay the penetration of aminoglycosides, while the penetration of fluoroquinolones occurs immediately and without delay. The penetration of oxacillin and cefotaxime (β-lactams), and vancomycin and teicoplanin (glycopeptides) is significantly reduced through Staphylococcus aureus biofilms, whereas that of amikacin (aminoglycoside), and rifampicin and ciprofloxacin (fluoroquinolones) was unaffected. The antimicrobial activity of antibiotics will resume in any of the following cases: the biofilm matrix becomes saturated due to the full adsorption/reaction of antibiotic molecules; the time required for an antibiotic to penetrate biofilms is shorter than the duration of antibiotic treatment; and the replenishment of biofilm matrix proceeds at a rate slower than the adsorption/reaction/diffusion of antibiotic molecules. It is thought that the reduced diffusion may only provide an initial short-term protective effect.
Subpopulations of Persister Cells
Biofilms contain a small reversible subpopulation of so-called persister cells that adopt a slow- or nongrowing lifestyle through the emergence of small colony variants and are highly tolerant to extracellular stresses, such as antibiotic treatment. Many antibiotics are less effective against slow- or non-growing cells compared with fast-growing ones because these antibiotics target growth-specific factors; thereby, the slow growth rates of biofilm-growing cells will render them less susceptible to antibiotics. For instance, β-lactams are only active against dividing bacterial cells, while fluoroquinolones are able to kill nongrowing cells, but are more effective in killing cells that are rapidly growing and dividing; the effectiveness of fluoroquinolones on biofilm-growing P. aeruginosa is greater when compared with β-lactams, while both fluoroquinolones and β-lactams are less effective against biofilm-growing P. aeruginosa compared to planktonic cells. After antibiotic treatment, only persister cells may survive, creating the reservoirs of surviving cells that may regrow to cause a relapsing chronic infection, which has been clearly described for cystic fibrosis-associated lung infections caused by P. aeruginosa and for candidiasis by C. albicans. The strategy of triggering cells to enter into persister cell fate is to overproduce the toxins that inhibit cellular processes and growth, which is mediated by toxin–antitoxin modules.
Starvation-Induced Stress Responses
In the laboratory, bacteria become highly resistant to antibiotics when nutrients are limited in the media. Starvation is also found in biofilms owing to nutrient consumption by peripheral cells and reduced diffusion of oxygen and nutrients through biofilms. Starvation induces the stringent response that is characterized as the repression of growth and division, in addition to the stimulation of amino acid synthesis in order to promote bacterial survival. The starvation-induced stringent response is a determinant of biofilm-specific antimicrobial resistance in P. aeruginosa, because inactivation of this protective mechanism greatly sensitizes P. aeruginosa biofilms to various classes of antibiotics and markedly enhances the efficacy of antibiotic treatment in experimental infections of P. aeruginosa.
The bacterial SOS response is a global response to DNA damage in which the cell cycle is arrested while DNA repair and mutagenesis are induced. Multiple SOS response genes are significantly induced in mature E. coli biofilms compared with planktonic cells, and the starvation-induced SOS response is a molecular mechanism leading to biofilm-specific antibiotic resistance of E. coli. Although the nutrient limitation-induced stringent response and SOS response have been linked to the formation of highly antibiotic-resistant populations within biofilms, the manner in which these two distinct stress responses become integrated to promote biofilm-specific antibiotic resistance remains unclear.
Glucan, a cyclic polymer of 12–15 β-(1,3)-linked glucose molecules, is synthesized by a plasma membrane-bound glucan synthase complex, extruded through the periplasmic space and linked covalently to cell wall components; notably, glucan can be also deposited as a component of the biofilm matrix.[25,26] The deletion of ndvB, a gene encoding a glucosyltransferase required for glucan synthesis, attenuates the high-level antibiotic resistance of P. aeruginosa biofilms, but has no effect on the kinetics of biofilm formation and the architecture of P. aeruginosa biofilms. Increased ndvB mRNA levels, as well as elevated glucan synthesis, are observed in biofilm-growing P. aeruginosa cells compared with planktonic organisms. The glucan can be found in both the cell wall and the biofilm matrix, and it binds to and sequesters antibiotic molecules away from their cellular targets.[25,27]
The elevated glucan synthesis in C. albicans biofilm cells also contributes to biofilm-specific resistance to antifungal drugs.[26,28] Glucan is produced by the primary glucan synthase Fks1p and deposited in the biofilm matrix, and extracellular glucan sequesters antifungals to render the biofilm resistant to antifungal agents. Two glucan transferases, Bgl2p and Phr1p, and an exoglucanase Xog1p – acting in a complementary fashion downstream of Fks1p – are responsible for the delivery of glucan to the biofilm matrix, resulting in accumulation of glucan in the mature biofilm matrix. Deletion of any of the above four genes enhances biofilm susceptibility to antifungals, but does not affect biofilm formation.
In addition to sequestration of antibiotic molecules, glucan is involved in activating the expression of multiple ethanol oxidation genes in biofilm-growing P. aeruginosa cells and, moreover, these genes are not expressed in planktonic cultures in the absence of known inducers. Inactivation of ethanol oxidation genes increases the sensitivity of P. aeruginosa biofilms to antibiotic treatment, indicating the contribution of ethanol oxidation to biofilm-specific antibiotic resistance. However, the mechanism by which ethanol oxidation promotes antibiotic resistance is still unclear.
Extracellular DNA can contribute to <1–2% of the biofilm matrix composition in P. aeruginosa. It is required for attachment and aggregation of microcolonies during biofilm development and also functions as a mere structural support to maintain biofilm architecture. The extracellular DNA within the biofilm matrix binds to and sequesters the cations, and the resulting cation-limited environment within biofilms activates the two-component regulatory systems PhoP/Q and PmrA/B that are required for the expression of multiple antibiotic resistance genes in P. aeruginosa. Extracellular DNA also plays an important role in antifungal resistance in mature A. fumigatus biofilms; however, the underlying mechanisms are as yet unclear.
Multidrug efflux pumps are well-known to mediate antibiotic efflux to contribute to antibiotic resistance in planktonic cells. At least nine efflux pumps of P. aeruginosa have been shown to account for this organism's high intrinsic resistance to antibiotics. A novel P. aeruginosa efflux pump encoded by PA1874–1877, which is more highly expressed in biofilm-growing cells than in planktonic counterparts, has been shown to contribute to the biofilm-specific antibiotic resistance of P. aeruginosa. Complete deletion of the PA1874–1877 genes in P. aeruginosa results in an increase in sensitivity to tobramycin, gentamicin and ciprofloxacin, but no impairment in planktonic resistance; an induced expression of cloned PA1874–1877 genes in planktonic P. aeruginosa cells increases their resistance to antibiotics. Notably, a double mutation of ndvB and PA1874–1877 makes the resulting mutant more sensitive to antibiotics than either mutant strain alone, indicating that ndvB and PA1874–1877 mediate two different mechanisms of biofilm-specific antibiotic resistance in P. aeruginosa. In addition, a putative E. coli efflux pump, YhcQ, two Burkholderia cenocepacia pumps, RND-3 and RND-9, and an Aspergillus fumigatus pump, AfuMDR4, are all overexpressed in a biofilm model and have also been implicated in biofilm-specific antibiotic resistance.
Type VI Secretion System
The type VI secretion system is bacterial protein injection machinery with roles in biofilm formation, virulence and interbacterial interaction. In total there are three type VI secretion system loci on the genome of P. aeruginosa, namely HSI-I, HSI-II and HSI-III.HSI-I has been shown to account for the delivery of toxins to arrest the growth of bacteria lacking immunity to these toxins, and thus provides P. aeruginosa cells with a major advantage for in vitro and in vivo growth. The tssC1 gene of HSI-I is much more highly expressed in biofilm cells than in planktonic cells. Mutation of tssC1 has no effect on biofilm formation, but leads to a huge increase in antibiotic susceptibility. Deletion of tssC1 only has a minor effect on planktonic resistance, while overexpression of tssC1 in planktonic cells increases antibiotic resistance. These observations indicate that HSI-1 is involved in the biofilm-specific antibiotic resistance of P. aeruginosa.
Biofilm-specific pathways responsible for antibiotic resistance may be tightly regulated by one or more regulators. The expression of P. aeruginosa glucan synthase gene ndvB is under the positive control of BrlR, a MerR-family transcriptional activator. BrlR is produced only under biofilm-specific growth conditions, and not under planktonic growth conditions. It is essential for biofilm-specific antibiotic resistance of P. aeruginosa; however, the inactivation of brlR has no effect on biofilm formation. BrlR is the first characterized DNA-binding regulator that modulates biofilm-specific genetic determinants of antibiotic resistance. Further studies on how BrlR itself is regulated and what its downstream targets are may reveal additional novel mechanisms of biofilm-specific antibiotic resistance.
Rlm1p is one of the two master transcription factors of the PKC1–SLT2 pathway that is important for controlling cell wall glucan production in response to stress in Saccharomyces cerevisiae, and Smi1p is an intrinsically disordered protein that participates in this pathway via a protein–protein interaction with the Sit2p kinase. Both Rlm1p and Smi1p are required for the production/deposition of cell wall and matrix glucan during biofilm growth of C. albicans, and contribute to biofilm-specific antibiotic resistance. Smi1p positively regulates expression of the glucan synthase gene FKS1 via the transcription factor Rlm1p; however, the detailed role of the PKC1–SLT2 pathway in the control of glucan synthesis and biofilm-specific antibiotic resistance needs to be elucidated in C. albicans.
Future Microbiol. 2013;8(7):877-886. © 2013 Future Medicine Ltd.