Pathogens are acquiring resistance faster than the introduction of new antibiotics. A growing number of infections such as pneumonia, tuberculosis, gonorrhoea, salmonellosis, etc are becoming harder to treat due to antibiotic resistance. Therefore, it is the need of the time to conduct research in order to reduce antibiotic resistance by microorganisms. The article will discuss biofilm, which is responsible for around 65% of all bacterial infections around the globe. It emphasizes the process of formation of biofilm, the contrast between bacterial biofilm communities and planktonic cells, mechanisms adopted by bacterial communities in developing resistance against antimicrobial drugs and approaches to control biofilm-related infections.
Biofilms are the microbial communities containing fungi and bacteria. Microorganisms synthesize and secrete a protective matrix that attaches them to either biotic or abiotic surfaces. . Figure 1 : Bacteria in a Biofilm[a] .
They form firmly attached microcolonies within two to four minutes. They then become increasingly resistant to antimicrobials within 6 to 12 hours and evolve into fully mature biofilm colonies that are extensively resistant to antibiotics. In a biofilm, bacteria of different species can form a colony, enabling them to develop resistance against antibiotics sooner. Biofilm provides protection to the microorganisms not only from altered pH, nutrient scarcity, mechanical forces, etc, but also blocks the access of bacterial biofilm communities from antibiotics and host immune cells.
Bacterial biofilm communities and Planktonic cells
Planktonic bacteria are free-living bacteria, which means that they are not sessile as that of bacteria in a biofilm. Bacterial biofilm communities differ from the planktonic bacteria in areas such as growth rate, gene expression, transcription and translation because these biofilm communities live in different microenvironments, which have higher osmolarity, greater nutrient scarcity and higher cell density than those of heterogeneous bacterial communities. Bacteria living within the biofilms are protected from a variety of environmental stressors such as desiccation, antimicrobial attacks by the immune system and ingestion by protozoans. This architecture makes them comparatively more advanced than planktonic bacteria. Microbial cells within biofilm have shown 10 to 1000 times more antibiotic resistance than planktonic cells.
Formation of biofilm
Thestudy of the process of formation of biofilm eases the understanding of the mechanisms by which bacterial communities develop resistance against antimicrobials.Formation of biofilm involves multiple steps as shown in figure2.
Cells secrete a protective matrix that helps them attach to a surface.
Figure2: Formation of biofilm[b]
Colonization and Maturation:
Many bacterial cells, even of heterogeneous species, get attached to each other and form a colony.
Consequent cell division increases the bacterial population. In a biofilm, bacteria form colonies by an extracellular matrix which possesses bacterial secreted polymers such as exopolysaccharides, extracellular DNA (e-DNA), proteins, and amyloidogenic proteins.
Bacterial cells detach from each other and take on planktonic stage, allowing them to become free and hence continue the cycle for the formation of biofilm.
The formation of biofilm in mycobacteria is similar to that of other bacterial communities. However, they do not produce the usual exopolysaccharide as they lack pilli. So, proteins help in the attachment and aggregation of mycobacterial cells in a biofilm.
Antimicrobial Resistance in Biofilm Structures
A unique property of polymicrobial biofilms is the mutual protective effects that different species of bacteria can provide to each other. For instance, antimicrobial-resistant bacteria which produce protective enzymes or antimicrobial binding proteins share these with non-antimicrobial resistant bacteria in a biofilm in the way that social animals share mutual coordination with each other to help the indigent one and improve lives, maintaining co-existence of all. Hence, non-resistant bacteria develop resistance against antibiotics with the help of the protective enzymes shared by others in the biofilm.
Another survival strategy adopted by bacteria in a biofilm is for a subpopulation to remain metabolically quiescent (to hibernate). Antimicrobials can only affect bacteria that remain metabolically active rather quiescent. Hence, the non-affected bacteria again share protective enzymes with the affected bacteria to develop resistance and fight collaboratively against the antibiotics.
Gram-positive bacteria have a cell wall made up of peptidoglycan, but Gram-negative bacteria have a more complex cell wall. Their outer membrane is made up of lipopolysaccharides (outer layer) and phospholipids (inner layer). This reduces the permeability of some antibiotics through the cell wall. Hence, poor diffusion of antibiotics through the biofilm polysaccharide is responsible for the antimicrobial resistance in biofilm structures.
Figure 3: Cell structure of Gram-positive and Gram-negative bacteria [c]
One of the antibiotic resistance mechanisms of biofilm communities is the uptake of resistance genes by horizontal gene transfer. Biofilm produces compatible conditions for horizontal gene transfer to occur, such as high cell density and increased genetic competence. Conjugation is the only mechanism of horizontal gene transfer in biofilms and may confirm the resistance to several antibiotics. It occurs through direct cell-to-cell contact as shown in figure 4.
Figure 4: Process involved in bacterial conjugation [d]
Lederberg and Tatum first discovered bacterial conjugation in E.coli. In this process, one bacterium acts as a donor, whereas the other acts as a recipient of the genetic material from the donor cell. The donor cell contains Fertility factor (F-factor) and hence is denoted as F+ ,whereas the recipient cell lacks it and is denoted as F– . Conjugation takes place only between a bacterium that possesses F-factor and one that lacks it. The F-factor in the donor cell produces a thin tube-like structure called pilus which helps it adhere to the recipient cell. Generally, the genetic material is in the form of a plasmid (extrachromosomal DNA) or transposons. Relaxosome helps in the transfer of genetic material from a donor bacterial cell to the recipient one. Plasmids carry resistance genes, which get passed to the recipient cell during the process. These new genes are incorporated into the recipient gene by recombination, thereby forming a recombinant gene. Hence, the recipient bacterial cell, which is non-antimicrobial resistant, develops resistance against antimicrobial drugs. A transposon is a DNA sequence that changes its position within its genomes, hence called jumping genes. Integrons also carry resistance genes which are transferred by the help of transposons from one cell to another. This wonderful mechanism helps bacteria to develop resistance in the biofilm communities.
Approaches to control Biofilm Related Infections
Biofilm-related infections are difficult to treat due to high antibiotic resistance in the community. Classical antibiotic chemotherapy is unable to completely eradicate bacterial cells, which are situated in the central region of the biofilm. The multicellular nature of biofilm communities is a key factor for developing antibiotic resistance. Disrupting any process involved in biofilm formation and maturation allows an increase in antibiotic efficacy, leading to quicker treatment of this persistent infection.
A panel of studies reported that at an early stage of biofilm development, antibiotics treatment was more effective, possibly because of the cells which are not completely adapted into biofilm communities. Enzymes like DNase-I Dispersin B (Dsp B) and a-amylase degrade e-DNA, biofilm matrix and exopolysaccharide respectively. Degradation of e-DNA prevents the horizontal transfer of resistance genes and that of biofilm matrix and exopolysaccharide disrupt the biofilm colony, affecting the formation and maturation of bacterial communities. The effectiveness of these enzymes in inhibiting maturation has already been reported in several microbes such as Streptococcus aureus, Vibrio cholera and Pseudomonas aeruginosa.
Quorum sensing (QS) is the coordination within the bacterial communities via cell-to-cell communication. Biofilm development is well organized and intracellular as well as intercellular signalling occurs during its development and maturation. Mutation in QS signalling genes inhibits the coordination among the bacterial communities. Natural products like halogenated furanone isolated from Delisea pulchra (marine algae) interrupt QS signalling. Garlic extract, usnic acid and azithromycin possess inhibitory activity against bacterial and fungal biofilms. Nitric oxide disperses the biofilms in P. aeruginosa and induces a switch to planktonic growth.
Photodynamic Therapy (PDT) is used to deliver light to the affected tissue for maximal damage of microbes. This therapy can be used to treat various infections caused by drug-resistant bacteria. Certain natural products show antibiofilm properties. For instance, 4-Phenylbutanoic acid shows high antibiofilm activity against Gram-positive and Gram-negative bacteria. Azadirachta indica (Neem) and Acacia extracts have an antimicrobial effect against certain bacteria like Streptococcus mutans and S. faecalis.
Figure 5: CRISPR Gene Editing [e]
Bacterial antibiotic resistance is one of the consequences of the bacterial biofilm communities which contribute to the chronic infections. These biofilm communities have few additional resistance mechanisms as compared to the planktonic ones which hamper the treatments option and leads to emergence as well as spreading of the chronic bad bugs. Therefore, what can be done to prevent this is the proper use of antibiotics at the early stage of showing symptoms of any bacterial infections. It is suggested to use CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) technology to suppress bacterial activity for antibiotic resistance led by their genes. For instance, luxS gene control QS signalling and fimbriae associated gene (fimH) mediates the formation of sex pilus. We can use this technology either to destroy or suppress these genes. This prevents the transfer of resistance genes to other bacteria even of heterogeneous species and also to upcoming generations. Besides, the suppression of genes that control DNA replication reduces antimicrobial resistance. RNA polymerase transcribes RNA from DNA. This enzyme can be degraded to prevent further multiplication and transfer of resistance to other bacteria in the biofilm.
Biofilm formation and maturation are one of the major causes of antibiotic resistance. The way that bacteria adapt to develop antibiotic resistance and help others in the bacterial communities in developing and transferring resistance genes is a major obstacle in the treatment of infections caused by antimicrobial-resistant bacteria. Inhibition of biofilm formation and maturation is a key phenomenon of controlling such infections.
- Turhan Karaguler, Hasan Kahraman, and Melek tuter. “Analyzing effects of ELF electromagnetic fields on removing bacterial biofilm” Elsevier (2017): 336-340: https://en.wikipedia.org/wiki/Bacterial_conjugation#/media/File:Conjugation.svg
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Hemshankar is a high school biology student from Nepal. He is passionate about learning immunology, molecular biology and human genetics. He is interested in using the ideas of science in improving human race by reducing the problems caused by pathogens. Besides, he enjoys learning philosophy of life.