CUG-China focus on the biofilm-associated problems this year. Microbes can exist in either planktonic or biofilm mode. Unlike the free-swimming planktonic counterparts, bacteria in biofilms colonize surfaces and interfaces by anchoring themselves with self-produced extracellular polymeric substances (EPS) such as polysaccharide, proteins and nucleic acids, eventually forming a complex three-dimensional matrix that encases the cells.
Compared with the planktonic mode of life, the biofilm mode confers several key potential advantages for the bacteria which include protection against predation, increased resistance to antimicrobial agents and facilitated genetic exchange. As such, the biofilm mode of life is the predominant form of existence for bacteria in natural and engineered environments where they enable their communities to strive even in extreme and hostile conditions. Currently, extensive studies on a wide range of pathogenic microorganisms have shown the formation of biofilms on human tissues and medical implants, often results in intractable chronic infections. The three-dimensional biofilm structure formed by EPS can effectively protect bacteria and become a natural barrier to prevent antibiotics from penetrating the biofilm. In a biofilm mode, antibiotics can only kill the plankton on the top layer of the biofilm, but cannot penetrate deep enough to form effective concentrations. Approximately 10-1000 times more antibiotic doses are typically required to kill pathogen biofilms compared to planktonic cells. Meanwhile, The natural barrier mentioned above can also separate bacteria from the immune system of hosts, so that the phagocytes and enzymes secreted by the hosts cannot effectively attack bacteria. Therefore, biofilms are also resistant to human immune cells. All these indicate that bacteria in the biofilm mode show higher tolerance to host immune response and antibiotics, making it difficult to eradicate infections based on biofilms.
Fig. 1 Some proposed-biofilm associated resistance mechanisms: (1) Antimicrobial agents may fail to penetrate beyond the surface layers of the biofilm. Outer layers of biofilm cells absorb damage. Antimicrobial agents action may be impaired in areas of waste accumulation or altered environment . (2) Antimicrobial agents may be trapped and destroyed by enzymes in the biofilm matrix. (3) Altered growth rate inside the biofilm. Antimicrobial agents may not be active against nongrowing microorganisms. (4) Expression of biofilm-specific resistance genes. (5) Stress response to hostile environmental conditions.[2]
To learn more about biofilm and its formation mechanisms, we interviewed Prof. Xiaoli Zeng, from the Institute of Hydrobiology, Chinese Academy of Sciences. She has been doing the research about the function of intracellular second messenger cyclic diguanylate (c-di-GMP) in Cyanobacteria for many years. She told us that some chemicals can affect biofilm formation by interfering with chemical signaling systems essential in biofilm formation. One of the important signal networks mediated by c-di-GMP controls biofilm formation and dispersal in a variety of bacteria. C-di-GMP can promote biofilm growth through binding with different effector proteins to positively regulate the expression of exopolysaccharide and adhesin, as well as negatively mediate cell motility which is driven by flagella or pili. There have been numerous studies showing that maintaining a low level of c-di-GMP efficiently attenuates biofilm formation, and that provoking a decrease in intracellular c-di-GMP level can disperse preformed biofilms.
Fig. 2 c-di-GMP effects biofilm formation[1]
After discussing with Prof. Zeng, we shifted our focus from biofilm to c-di-GMP. Detecting the dynamic change of c-di-GMP concentration is an essential entry point for the identification of biofilm-inhibiting/dispersing agents that target c-di-GMP metabolism. Currently, there are two commonly used methods for c-di-GMP measurement. One is phenotype-based screens for motility or EPS production. Although these assays can achieve high-throughput screens, they show indirect detection of c-di-GMP with low sensitivity and cannot perform real-time monitoring. Another highly sensitive method is Liquid Chromatograph-Mass Spectrometry (LC-MS) analysis of cell extracts. One challenge for the MS-based method, however, is to achieve high-throughput screen and real-time monitoring of dynamic changes because of the complex sample preparation and long analysis time.
In order to overcome all the disadvantages above and effectively screen c-di-GMP-targeted drugs, by using synthetic biology approaches, we will build a biosensor that can gauge the concentration of c-di-GMP in different bacterial cells, and then conduct screening of drugs that can disperse biofilms.
Fig. 3 Compare of different c-di-GMP detecting methods
[1] Watnick P, Kolter R. Biofilm, city of microbes[J]. J Bacteriol, 2000,182(10):2675-2679.
[2] Jenal U, Reinders A, Lori C. Cyclic di-GMP: second messenger extraordinaire[J]. Nat Rev Microbiol, 2017,15(5):271-284.