Biofilm-Associated Infections

Antibiotic Resistance and Novel Therapeutic Strategies

Fengjun Sun; Feng Qu; Yan Ling; Panyong Mao; Peiyuan Xia; Huipeng Chen; Dongsheng Zhou


Future Microbiol. 2013;8(7):877-886. 

In This Article

Promising Therapeutic Strategies

Successful treatment of biofilm-associated infections is hampered due to high levels of resistance of biofilm-growing pathogens to antibiotics. It is difficult to completely kill microbial cells in a biofilm (especially those in the center) by classical antibiotic treatment strategies, such as antibiotic prophylaxis, early aggressive antibiotic therapy and chronic suppressive antibiotic therapy, making biofilm-associated infections recurrent and chronic.[10,45] In recent years, various new antibiofilm approaches have been developed by application of novel natural, synthetic or bioengineered agents as alternatives to classical antibiotic treatment because the modes of action of these novel antibiofilm agents are much less susceptible to the emergence of resistance compared with classical antibiotic treatment.

Antimicrobial Peptides

Antimicrobial peptides (AMPs) are cationic, amphipathic peptides that are 15–30 amino acids in length, which are a part of the innate immune system of animals and plants in nature, but can also be produced by bacteria and fungi. AMPs can bind to negatively charged structural molecules on the microbial membrane with a broad spectrum of antimicrobial activity and reducin the likelihood of the bacteria developing resistance, and thus they potently act on slow- or non-growing bacteria, as observed in biofilms.[46]

AMPs can be optimized through modifying their primary amino acid sequences to enhance their effectiveness and stability, making them good templates for developing novel anti-infectious therapeutic agents.[47] The use of synthetic, modified AMPs, which have the advantage of a relatively low cost of production in larger quantities, has emerged as an attractive antibiofilm strategy.[46] However, the sensitivity of AMPs to physiological salt concentrations, ionic strength, pH and proteolytic activity in body fluids challenges the clinical application of AMPs.

Recently, researchers have designed so-called specifically targeted AMPs (STAMPs) that exhibit killing specificity against one[48] or two[49] bacterial species. A typical STAMP consists of two functionally independent moieties conjoined in a linear fusion peptide sequence: an optimized antimicrobial domain acting as a killing moiety, and a preselected species-specific binding domain providing specific binding to a selected pathogen and facilitating targeted delivery of the attached antimicrobial peptide.[48] Such a fusion peptide is relatively stable and retains its antimicrobial activity under a range of physiological conditions in body fluids.[47] Targeted antimicrobial therapy using STAMPs has been shown to be effective against the biofilm-forming cariogenic pathogen Streptococcus mutans, for which STAMPs selectively eliminate S. mutans from a mixed-species environment without affecting closely related noncariogenic oral streptococci.[48]

Biofilm Matrix-Degrading Enzymes

Another promising biofilm-controlling strategy makes use of biofilm matrix-degrading enzymes, especially DNase I, which degrades extracellular DNA, and α-amylase and DspB, which both degrade extracellular polysaccharides. Both natural and recombinant human DNase I can disrupt the biofilms of various medically important bacteria and fungi.[50,51] The cleavage of extracellular DNA by DNase I leads to an alteration of biofilm architecture, which permits increased penetration of antibiotics, and thus the addition of DNase I enhances the efficacy of antibiotics.[52] α-amylase has been proven to be an antibiofilm agent of S. aureus, Vibrio cholerae and P. aeruginosa, not only inhibiting biofilm formation, but also effective in degrading preformed mature biofilms of these pathogens.[53] DspB is a soluble β-N-acetylglucosaminidase that was originally purified from Actinobacillus actinomycetemcomitans[54] and it has a broad-spectrum activity to dissolve matrix exopolysaccharide of bacterial biofilms.[55–57] The combinational application of DspB and antibiotics/disinfectant/surfactant has a synergistic effect on bacterial pathogens such as A. actinomycetemcomitans, Staphylococcus epidermidis and S. aureus, characterized as reduced bacterial colonization on indwelling medical devices and a prolonged superior antimicrobial and antibiofilm activity.[55–57]

Therapeutic approaches using biofilm matrix-degrading enzymes aim to inhibit or reduce the cell-to-cell and cell-to-surface associations during the development of biofilms, detach the established biofilm colonies and render the biofilm or dispersed cells sensitive to killing by antibiotics or by the host immune defense. The pure enzymes are required for biomedical applications; however, the current high cost of industrial enzyme production makes the application of these enzymes relatively expensive. Nevertheless, a combination of biofilm matrix-degrading enzymes and antibiotics is a promising, highly effective tool for removing medically important biofilms.


Bacteriophages, the natural parasites of bacteria, have long been used as agents for treating bacterial infections.[58] Bacteriophages have the potential to negate the protection afforded by biofilms[59] owing to at least three reasons: replication of the phage in bacterial host cells during its infection and the lytic cycle results in the production and release of a large number of progeny phages, leading to a radial cell lysis around infection sites; phages produce polysaccharide depolymerases to degrade the biofilm matrix, causing a breakdown of biofilm matrix;[60] and phages have host specificity, leaving normal bacterial flora undisturbed.[61] Progeny phages can propagate radially through a biofilm[62] and thus, theoretically, a single dose of phage will be able to treat a biofilm-associated infection.[63] However, the safety concerns of the medical application of phage therapy arise from the largely unknown risks of introducing an organism that is capable of replication and perhaps transmission and spread into the patients, although humans are exposed to phages from birth and phages can be frequently isolated from the human GI tract and oral cavity.[59]

Phage-resistant bacteria can rapidly evolve through mutation and cause subsequent phage attack to fail. Coadministration of antibiotics and phages has a synergistic effect since modification of biofilm architecture by phages can increase susceptibility to antibiotics.[63,64] Phage and antibiotic resistance are unlikely to evolve simultaneously and, moreover, bacteria resistant to one agent can still be killed by the second agent; therefore, the combined phage–antibiotic therapy is less likely to fail due to the emergence of resistance.

By using synthetic biology technologies, bacteriophages can be engineered to express the biofilm matrix-degrading enzyme DspB, which greatly enhances the efficacy of biofilm removal by this enzymatic phage strategy compared with nonenzymatic phage treatment.[65] An engineered M13mp18 phage overexpressing the LexA3 protein (a repressor of the SOS response) to attack the SOS network of antibiotic-resistant bacteria could be used as an adjuvant for antibiotic therapy.[66] The above engineered phage strategies not only enhance bacterial killing, but are also hoped to reduce the incidence of antibiotic resistance.

Ultrasonic Treatment

Low-frequency ultrasound has been advocated for many years to treat chronic rhinosinusitis, an immunological disease affected by immunological disorders, environmental factors and the presence of microbial biofilms.[67] Low-frequency ultrasound alone is generally not effective in killing biofilm-growing bacteria, but the combination of low-frequency ultrasound and antibiotics is promising for biofilm removal.[68,69] Therapeutic ultrasound can not only enhance transport of antibiotics across biofilms due to physical fragmentation of compact biofilm barriers, but also the uptake of antibiotics due to an increase in membrane permeability of bacterial cells, thereby increasing sensitivity of biofilm-growing bacteria to antibiotics.[67–69] In addition, ultrasound can increase the rates of transport of oxygen and nutrients to bacterial cells within biofilms, making them reanimate from hibernation to resume active metabolism;[70] on the other hand, most antibiotics have beneficial effects on bacterial cells with active metabolism.

Ultrasound-targeted microbubbles (UTMs) have recently been developed as experimental tools for organ- and tissue-specific drug/gene delivery.[71] UTMs can also enhance antibiotic transport and improve antibiotic activity against biofilms, and thus antibiotics combined with UTMs may provide an efficient and noninvasive alternative to treat device-related biofilm infections.[72]

Quorum-Sensing Inhibitors

Quorum sensing (QS) is a signaling system that facilitates intra- and inter-species communication in bacteria and modulates the expression of a wide array of genes and phenotypes, especially those including virulence and biofilm formation.[73] QS inhibitors are compounds able to quench the action of QS. Most prokaryotes as well as some eukaryotes, such as certain traditional medicinal plants, can produce QS quenchers that are generally regarded as safe.[74] Specific screening systems have been developed to identify novel QS inhibitors among natural and synthetic compound libraries.[75]

As QS is not directly involved in the processes essential for bacterial growth, inhibition of QS by using QS inhibitors does not impose as harsh a selective pressure for development of resistance as classical antibiotic treatment.[76] The use of QS inhibitors provides alternative therapeutic methods for treating biofim-associated infections. For instance, a marine algae, Delisea pulchra, produces several halogenated furanone compounds capable of interfering with bacterial QS signaling, and both natural and synthetic furanones show potential in preventing bacterial biofilm-associated infections.[77,78] Usnic acid, a secondary lichen metabolite, possesses inhibitory activity against bacterial and fungal biofilms most likely through interfering with QS signaling.[79,80] QS inhibitors can also be developed as an adjuvant to antibiotic administration, as QS inhibitors can serve to increase the susceptibility of biofilm-growing bacteria to antibiotics.[81]

Other Natural Products as Antibiofilm Agents

Multiple bacterial and actinomycetic species have been shown to produce bioactive agents with antibiofilm properties, and are promising sources for developing novel drugs against antibiotic-resistant biofilms. Methanolic extract of a coral-associated actinomycete induces a reduction in biofilm formation of S. aureus.[82] Bioactive extracts from multiple coral-associated bacteria have been shown to inhibit biofilm formation of Serratia marcescens, an opportunistic pathogen causing severe urinary tract infections in hospitalized individuals.[83] A novel natural product, 4-phenylbutanoic acid, from the marine bacterium Bacillus pumilus shows a very high inhibitory activity against biofilm formation in multiple Gram-positive and -negative species.[84]