Biofilms and Antimicrobial Resistance 

What is a biofilm?

A biofilm is a population of microorganisms suspended and organized within a self-produced matrix that is primarily made of polysaccharides, proteins, lipids, DNA and RNA. Several pathogenic fungi and bacteria including Candida albicans, Clostridium difficile, Escherichia coli, Staphylococcus aureus ("Staph") and Pseudomonas aeruginosa are known to form biofilms

The development of a biofilm generally progresses in stages starting from separated planktonic ("free-floating") cells. The free cells then adhere to a surface and communicate with each other through a process called quorum sensing and begin producing the biofilm. As the cells multiply they begin to mature into a complex, three-dimensional structure. The end phase of biofilm production involves cells dispersing from the biofilm to establish another biofilm elsewhere or return to a planktonic form. [2]

Biofilms offer several advantages for biofilm-forming microbes. Once cells are established into a biofilm, they become much more resilient to environmental stresses including low O2, temperature fluctuations, antimicrobials, and the host's immune system. [3] Additionally, passing of genetic information is much more efficient in the protective environments biofilms offer. [4]

Significance of Biofilms

The formation of a biofilm inside of a patient's body can cause serious recurring or chronic infections. Completely removing or destroying a biofilm in a patient's body can be especially difficult as only one surviving organism in a biofilm is required to repopulate. Several antimicrobial medications are not able to sufficiently treat biofilms. [1] Common biofilm-related medical conditions include gingivitis, acute ear infection, and persisting urinary tract infection. More severe concerns for biofilm formation include biofilm-forming P. aeruginosa infections, the leading cause of death in cystic fibrosis patients.

Biofilm-forming infections are particularly concerning for patients with implanted medical devices. Found on a variety of medical devices including catheters, prosthetic joints, pacemakers, and even contact lenses, a biofilm gives microorganisms a stable dwelling safe from antimicrobials and the hosts' immune system. A serious biofilm infection may warrant the removal of an implanted medical device which can cause undue risk and expense to the patient.

It should be noted that the vast majority of bacteria exist in biofilms and only a small percentage of biofilm-forming microbes are pathogenic. Many times, biofilms are a natural state of cellular organization, are clinically insignificant, and may even be beneficial to human health. Here, we will focus on where biofilms and disease overlap and where serious public health concerns arise.

How do biofilms promote antimicrobial resistance?

The initial explanation for the antimicrobial-resistant properties of biofilms is that the extracellular matrix of biofilms physically restricts the diffusion of antibiotics. While this does occur to a certain extent, it doesn't accurately account for the whole picture. Organisms within a biofilm secrete degradative enzymes within and outside of a biofilm that destroy or inactivate incoming antibiotics rendering them useless as they slowly filter in. The slowed diffusion and degradation work synergistically; the degradation doesn’t always work fast enough to render the antimicrobial ineffective, and the diffusion is slowed but inevitable without the degradation step [5]. In addition to preventing the diffusion of antibiotics, nutrients and oxygen are also prevented from reaching too deeply into a biofilm. The lack of nutrients and oxygen creates a starvation-induced stressed state in some cells called "persister cells" which have been shown (in vitro) to make microbes more resistant to antibiotics [2].

Most antibiotics specifically target enzymes that are active in normal cellular growth and division processes. Persister cells generally are not sensitive to these antibiotics because the targeted enzymes are essentially inactive. Persister cells are of great concern as they may be able to revert back to a normal state and restore the biofilm population. [2,6]

In addition to structural barriers, degradative enzymes, and a physically resistant state, bacteria within a biofilm up-regulate genes responsible for the production of efflux pumps. Efflux pumps are transport proteins that "pump" antimicrobial drugs out of a cell before they can work on intracellular enzymatic machinery. Organisms within a biofilm have been found to express more efflux pump genes than planktonic cells making them extremely difficult to fight with traditional antibiotics. [7]

How can we fight resistant biofilms?

Researchers from around the world have formulated a variety of strategies to explore anti-biofilm treatment. Here are just a few:

* Stop the formation of a biofilm before it begins. Sun, Liao and Wang have found that magnolol and honokiol (both organic compounds) restricted biofilm formation in vitro, with a dramatic decrease in filamentation from C. albicans as it fails the first step of biofilm formation: adhering to a surface. [8]

* Encourage the dispersal phase sooner. Pushing biofilms into their final stage will cause cells to leave the biofilm and revert back to a more vulnerable, planktonic state. Warner, Cheng, Yildiz and Linington designed a series of benzo[1,4] oxazines and found that one of them could induce dispersal in Vibrio cholerae and showed excellent numbers as a biofilm inhibitor, leading the way for macrolide antibiotics azithromycin and erythromycin to clear the infection. [9]

* Target the extracellular matrix in addition to the cells within the biofilm. Rajendran et al. Removed the extracellular DNA of an Aspergillus fumigatus strain with DNAase and found that it became susceptible to the antifungal compounds amphotericin B and caspofungin. [10]

* Prevent biofilm-related cellular communication Without quorum sensing, the biofilm becomes weaker and less organized. Farnesol is able to re-sensitize MRSA to antimicrobials in this paper by Rizk, Meiller, James and Shirtliff. [11]

* Utilize competition between microbes. Many antimicrobials, including penicillin, were discovered because one microorganism had developed the ability to compete with the other. Hogan, Vik and Kolter found that Candida albicans's biofilm development was affected by the presence of Pseudomonas aeruginosa which inhibited the establishment of C. albicans filamentation. [12]

Biofilms are just one of the ways that microorganisms protect themselves from antimicrobials. Read more on other microbial resistance strategies and how the scientific community plans to overcome them here: http://www.toku-e.com/Overcoming-Multi-Drug-Resistant-Microbes.aspx

 

Sources

[1] Bjarnsholt, T. (2013). The role of bacterial biofilms in chronic infections. Apmis, 121, 1-58. http://doi.org/10.1111/apm.12099

[2] Sun, F., Qu, F., Ling, Y., Mao, P., Xia, P., Chen, H., & Zhou, D. (2013). Biofilm-associated infections: Antibiotic resistance and novel therapeutic strategies. Future Microbiology, 8(7), 877-886. http://doi.org/10.2217/fmb.13.58

[3] Davies, D. (2003). Understanding biofilm resistance to antibacterial agents. Nature Reviews Drug Discovery, 2(2), 114-122. http://doi.org/10.1038/nrd1008

[4] Molin, S., & Tolker-Nielsen, T. (2003). Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure. Current Opinion in Biotechnology, 14(3), 255-261. http://doi.org/10.1016/S0958-1669(03)00036-3

[5] Lewis, K. Riddle of Biofilm Resistance. Antimicrobial Agents and Chemotherapy 45.4 (2001): 999-1007. Web. http://doi.org/10.1128/AAC.45.4.999-1007.2001

[6] Huang, C. T., Yu, F. P., McFeters, G. A., & Stewart, P. S. (1995). Nonuniform spatial patterns of respiratory activity within biofilms during disinfection. Applied and Environmental Microbiology, 61(6), 2252–2256. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167496/

[7] Gibbons, J. G., Beauvais, A., Beau, R., Mcgary, K. L., Latge, J., & Rokas, A. (2011). Global Transcriptome Changes Underlying Colony Growth in the Opportunistic Human Pathogen Aspergillus fumigatus. Eukaryotic Cell, 11(1), 68-78. http://doi.org/10.1128/ec.05102-11

[8] Sun, L., Liao, K., & Wang, D. (2015). Effects of Magnolol and Honokiol on Adhesion, Yeast-Hyphal Transition, and Formation of Biofilm by Candida albicans. PLoS ONE, 10(2). http://doi.org/10.1371/journal.pone.0117695

[9] Warner, C. J., Cheng, A. T., Yildiz, F. H., & Linington, R. G. (2015). Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae. Chem. Commun., 51(7), 1305-1308. http://doi.org/10.1039/c4cc07003h

[10] Rajendran, R., Williams, C., Lappin, D. F., Millington, O., Martins, M., & Ramage, G. (2013). Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms. Eukaryotic Cell, 12(3), 420-429. http://doi.org/10.1128/ec.00287-12

[11] Jabra-Rizk, M. A., Meiller, T. F., James, C. E., & Shirtliff, M. E. (2006). Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility. Antimicrobial Agents and Chemotherapy, 50(4), 1463-1469. http://doi.org/10.1128/aac.50.4.1463-1469.2006

[12] Hogan, D. A., Vik, Å, & Kolter, R. (2004). A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. Molecular Microbiology, 54(5), 1212-1223. http://doi.org/10.1111/j.1365-2958.2004.04349