Biofilms are an adaptive mechanism that bacteria use in order to protect themselves from threats. This mechanism has become a grave problem as biofilms protect bacteria and are the underlying cause of resistance against antibiotics and biocides. It is estimated that every year, at least 10 million people could die if a control over antibiotic resistance is not found.
Biofilms in experiments are able to block the activity of antibiotics to 100 times, making them practically insensitive, creating such a phenomenon as bacterial resistance to antibiotics in clinical use. Recent studies have allowed scientists to trace antibiotic resistance and biofilm formation in bacteria to 350 to 500 million of years in the past. However, the misuse and overuse of antibiotics since the discovery of penicillin has allowed bacteria to adjust and supercharge the formation of biofilms to a point that in the coming years we now face the reality of more deaths per year than those caused by diabetes and cancer combined.
Bacteriophages, natural predators of bacteria, have their own mechanisms allowing them to reach their prey. As bacteria evolves and adjusts towards their threats, phages are also constantly evolving and adjusting their methods to match to those of bacteria.
Biofilm structure and formation
The biofilm structure is composed of an extracellular matrix which is composed of extracellular polymeric substances (EPS). The EPS consists of polysaccharides, proteins, lipids, DNA and other biological macromolecules. The size, shape and composition of a biofilm may vary in response to environmental conditions, preserving viability and metabolic activity of the bacteria within.
Today’s advanced imaging technology has revealed that biofilms have water channels and pores for the exchange of nutrients and waste products. The outer matrix plays a crucial role for the survival of bacteria in hostile environments, as it acts as a protective barrier against predators and the host’s immune system.
Contents enclosed within a biofilm are mainly water (90% – 97%), aggregations of cells and persistent cells. Persistent cells or slow growing cells are immune to the effects of antibiotics and help the growth or regrowth of the biofilm. The close proximity of species within the biofilm allow for horizontal gene transfer, allowing the continuation of genetic resistance. The biofilm complex as a whole acts as a community, where the residents within communicate by sending chemical signals that are known as quorum sensing molecules. Gene sequencing has uncovered a secondary messenger that has been identified to be responsible for biofilm formation is the cyclic di guanosine mono phosphate (cyclic di GMP).
Bacteriophages and Biofilms
The role of bacteriophages is to control the population of bacteria in the environment. Due to this, phages have the ability to evolve and adjust to bacterial protective mechanisms. The 2 main defenses that bacteria use to defend themselves against bacteriophages are; the alteration of the phage entry receptors and the development of immunity by the use of CRISPR and CRISPR-associated Cas 9 proteins. The constant adaption of bacteria and bacteriophages, keeps both evolving their methods against one another.
There are several ways by which bacteriophages can get through a biofilm and reach their target;
The amplification process involves a low number of bacteriophages that kill the host bacteria by the process of lysis replication. The high numbers of bacteria in comparison to the low numbers of phages, trigger the bacteriophages to balance out the ratio resulting in the attack and reduction of bacteria present. This method is referred to as “active penetration”, where the bacteriophages spread within a biofilm using lysis and diminish the bacteria population.
Bacteriophages can influence and destroy biofilms with the use of 3 enzymes; enzymes that hydrolyze extracellular polymeric substances in biofilms, enzymes that destroy the capsule of the bacterial cell and enzymes that destroy the cell wall in bacteria. The enzyme that has the capability to degrade the extracellular polymeric substances (EPS) of the biofilm causes degradation that leads to the breakdown of the biofilm and allows phages to reach the bacteria there were encapsulated within it.
Some bacteriophages have the ability to induce depolymerase enzyme expression. It is still unclear whether the induction of this enzyme is done by a bacteriophage from within or by the bacteria in a survival flight response. No gene has been found that would respond to this.
Attacking persistent cells
Unlike antibiotics, bacteriophages can attack and infect persistent cells the same way as they target normal bacteria. Persistent cells can be protected from prophages (lysogeny); however, they are not protected from lytic infection (via lysis). Analysis has shown that persistent bacteria become a target to phages once they switch from their slow growth to an activated normal speed, which subsequently is the state they use when forming or for the regrowth of the biofilm.
Phage therapy against Biofilms
Currently, it is not possible to patent natural bacteriophages based on their constantly changing and evolving nature. A medical patent is legal protection against market competition that a government grants to the inventor of a unique medical item or process. Phages are the most abundant forms of life, about 1031 are present in the global biosphere. It is estimated that approximately a few thousand have been sequenced to date.
In some countries, manufacturers look beyond the inability to patent phage products and produce natural virulent (lytic) bacteriophages that are used for treatments. Others are looking into the use of bacteriophage elements, that can be patented, in order to produce some sort of partial phage therapy effect to fight against biofilms.
Genetically modified phages
Biotechnological applications of bacteriophages include genetically modifying phages to increase the antibacterial phage activity by using filamentous non lytic phages, with a single strand DNA.
Enzybiotics are bacteriolytic enzymes which have been phage encoded, with the isolated holin and lysine gene to act as biocontrol agents.
Endolysin gives bacteriophages the opportunity to penetrate the membrane of a bacterium. Taking this into focus, certain companies were able to duplicate endolysin and patent this product.
Bacteriophages in combination treatment
Bacteriophages in combination with antibiotics have been identified to have potential in future therapy. Another therapeutic combination was the use of a polysaccharide lyase and DNase enzymes to break down the biofilm matrix, administrated together with bacteriophages. The use of bacteriophages with physical cleaning of wounds was seen to be effective in a rabbit ear model. In another research, it was noticed that bacteriophages were more effective on biofilm cells that were dislodged as opposed to those that were undisturbed, leading to a proposition of considering some disruption of the biofilm, prior to phage application for short-term treatment.
Bacteriophages as a biocontrol
Bacteriophages do not obliterate present bacteria. They have mechanisms to primarily control the population of bacteria. The use of bacteriophages for human therapy raises the concern of phage host ratio (PHR) in relation to the ratio of bacteria present. In the case of acute infections, a low dosage of bacteriophages should give a positive result against a prominent infection, as opposed to multiple dosing, which would be an effective approach towards chronic infections. It is important to note that bacteriophages do not only perform as antibacterial agents, but also have an immunoregulatory effect on the overall system.
Just recently, headlines of a deadly superbug outbreak of Salmonella enterica serotype Newport were reported. The multi-drug resistant bacteria was initially found in cows and was passed on to humans through meat and dairy products. Just prior to the superbug outbreak, the World Health Organization released a statement in regards to battling antibiotic resistance by strictly dividing the current and upcoming antibiotics under 3 categories; critical, high and medium priority. Biofilm formation has created a serious problem, however so has the misuse and over use of antibiotics. So far, WHO has not announced any alternative treatment plans except of that of antibiotics.
Louis-Charles Fortier* and Ognjen Sekulovic. Published in ResearchGate 2013 | DOI: 10.4161/viru.24498
David R. Harper, Helena M.R.T. Parracho, James Walker, Richard Sharp, Gavin Hughes, Maria Werthén, Susan Lehman, Sandra Morales. Published in ResearchGate 2014 | DOI: 10.3390/antibiotics3030270
Shilpa Deshpande Kaistha. Published in ECronicon 2017
V.V. Dryukker, A.S. Gorshkova. Published in Izvestia 2012
More questions on the topic:
- Is it possible that the mis-use or/and overuse of antibiotics, triggered the rapid formation of biofilms in bacteria we are facing to date?
- Will the possibility of the treatment or prevention of bacterial infections, that has been used for over 80 years in the ex-Soviet countries, be available in the US and EU in the near future?
- Will regulations change to accommodate the use of nature’s bacteria predator, bacteriophages, or will manufacturers need to continue modifying or/and using parts of phage mechanisms for future treatments?