Biofilms

Kinkajou: Tells us how biofilms form.

Erasmus: There are four stages of biofilm development:

• Initial attachment:

• Irreversible attachment:

• Development or Maturation :

• Dispersion:

  Cell Biofilm Killing

Erasmus: Bacteria initially adhere to a surface using electrostatic forces (Van der Waals forces). This force acts between charged molecules on the bacterial surface and charged molecules on the surface to which they are adhering.

Strangely enough though, most researchers have shown that hydrophobic surfaces are more likely to be colonised. So this means that water inhibits the bonding process, (being in effect a highly charged and interactive molecule, probably even more chemically reactive in marine environments.)
Physical factors are also important in bacterial attachment. Factors such a surface roughness encourage colonisation. Surfaces coated by molecular conditioning films are more easily colonised. Other characteristics of the aqueous medium, such as pH, nutrient levels, ionic strength, presence of substrate cations and temperature, may play a role in the rate of microbial attachment to a substratum. BiofilmDevelopmentStages2.gif

Kinkajou: So, I take it the next stage is the irreversible attachment stage.

Erasmus:  Yes. Bacteria may then secrete other chemicals which enhance molecular adhesion, leading to the formation of irreversible attachments to the surface on which they are residing. Bacteria can also create fimbriae which are cell wall extrusions. Most fimbriae have a high proportion of hydrophobic amino acid residues which assist attachment to surfaces.

Other bacteria may not be able to attach to many surfaces, but may be able to attach to the extracellular polymeric substance (EPS), secreted by “early coloniser” bacteria. Biofilms seem to utilise specialist attributes of many different bacterial species.

BiofilmLifeCycle2.gif

Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The next stage of biofilm formation is known as
maturation or development.

Kinkajou: So bacteria in effect can work together?

Erasmus:  Biofilm cells can coordinate behaviour via intercellular "communication" using biochemical signalling molecules. This process is called quorum sensing. For example, a single bacterium through this chemical communication process can sense how many other bacteria are in close proximity, and can therefore adjust its metabolic behaviour in dense population environments to join a biofilm.
Quorum sensing can occur within a single bacterial species but also between different species. There is evidence that some of these chemical signals, produced by cells and passed into the external environment after passing through the outer bacterial cell membranes, may be interpreted not just by members of the same species, but by other microbial species. Bacteria can produce chemical signals ("talk") and other bacteria can respond to them ("listen". The process is described generally as cell-cell communication or cell-cell signalling. This communication can result in coordinated behaviour of microbial populations

In bacterial planktonic populations, the chemical signals released by bacterial cells are not concentrated enough to cause changes of genetic suppression in neighbouring bacteria. However in biofilms the presence of the extracellular polymeric substance (EPS) allows for a build-up in the concentrations of the signalling molecules leading to changes in cellular metabolic behaviour

Quorum sensing occurs through the up or down regulation of gene expression in response to changes in cell population type and cell population density.  In this process bacteria produce and release signalling molecules called auto-inducers which cause an alteration of gene expression in other bacteria within the biofilm. Gene expression can be altered at both transcriptional and translational levels.

Quorum sensing is believed to be important in processes such as symbiosis, sexual reproduction such as through conjugation, changes in the production of enzymes, motility, and spore formation amongst others. Gram-negative bacteria often use acylated homo-serine lactones is auto-inducers. Gram-positive bacteria often use oligo-peptides to communicate. These auto-inducers may be active within and between different species of bacteria.

Pseudomonas aeruginosa in some studies have found to secrete a long signalling molecules as well as a short signalling molecule. In cystic fibrosis patients the short signalling molecule predominates, presumably indicating that biofilms are present in the lung of these patients. In planktonic bacterial cultures the long form the signalling molecule predominates. When the lungs strains of pseudomonas aeruginosa were introduced into broth media they reverted to the production of long signalling molecules. When reintroduced into biofilm compatible situations, these bacteria then reverted to short chain signalling molecule production.

Quorum Sensing Microbial

This supports the contention that planktonic and biofilm behaviours are genetically coded within bacteria.

Complex molecular sugar structures known as polysaccharide matrices (or extracellular polymeric substance (EPS)) typically enclose bacterial biofilms. However many environmental chemicals, particles or components may also be present.

Quorum sensing is due to up and down-regulation of a number of genes present within the bacteria. Changes can appear as soon as minutes after attachment. Enzyme systems favouring bacterial function in limited oxygen environments such as the extra polymeric substance (EPS) need to be up regulated, allowing the bacteria to use fermentation as an energy source. Currently the regulatory pathways for quorum sensing are poorly understood. What is obvious is that “Gene expression” is different in biofilm bacteria as opposed to planktonic bacteria. Some studies have shown that quorum sensing can even trigger individual microbes to limit the growth, essentially favouring their fellow bacteria.

It is likely that an understanding of signalling and communication mechanisms between bacteria may well enhance infection control.

Kinkajou: You mentioned genetic DNA exchange as occurring in biofilms previously.

Erasmus: Gene Transfer can and does occur between bacteria in biofilms.  Biofilms also provide an environment suited to exchange of extra-chromosomal DNA such as that existing within plasmids within the bacterial cell wall. Conjugation through the formation of sex pili linking bacteria occurs at much greater rate between cells in biofilms than between planktonic cells. Researchers have shown that the incorporation exogenous DNA by bacterial conjugation is mediated by quorum sensing. In one studied organism Streptococcus mutans. DNA exchange occurred at frequencies between 10 to 600 times higher in biofilms than in planktonic bacterial cells.

  Altering Cell Biochemistry with Biofilm Stage

Kinkajou: Dispersion is the final step in biofilm growth, I believe.

Erasmus: Dispersion is a critical process in the replication of biofilms, enabling them to spread and colonise new surfaces. Enzymes within the extracellular matrix have a role to play in breaking down portions of the biofilm structure. Messenger chemicals such as cis-2-decenoic acid and nitrous oxide have been shown to be important agents in the dispersion of pseudomonas aeruginosa and even yeasts such as candida.

Main processes active in dispersion are detachment via erosion or shearing forces (encompassing the continuous removal of small fragments of the biofilm), sloughing (shedding of large fragments of the biofilm) and abrasion (microscopic shedding due to collision between particles within the environment in the biofilm.

Detachment mechanisms are also species specific. Some organisms recolonise their environment much faster than others, perhaps in response to depletion of nutrients within the biofilm environment.

However, biofilm bacteria can move in numerous ways that allow them to easily infect new tissues. Biofilms may move collectively, by rippling or rolling across the surface, or by detaching in clumps. Sometimes, in a dispersal strategy referred to as “swarming/seeding”, a biofilm colony differentiates to form an outer “wall” of stationary bacteria, while the inner region of the biofilm “liquefies”, allowing planktonic cells to “swim” out of the biofilm and leave behind a hollow mound.

Kinkajou: So tell us a bit about how biofilms enhance the ability of bacteria to colonise and grow in the causation of human disease.

Erasmus: Biofilms make bacteria less susceptible to antimicrobial agents. Several different mechanisms have been proposed.

• Reduced exposure of bacteria to toxic substrate such as antibiotics may well render biofilms are susceptible to anybody therapy. Colonised wounds in patients may well form an example of this.
  
  

• Altered micro environments due to metabolic processes from a number of bacteria could contribute to an ability to resist some antibiotics.
  
  

• Large-scale bacterial colonies may be able to neutralise antibiotics within their immediate environment. A lone bacterial cell would be able to affect this change. Larger groups of bacterial cells may well have a greater neutralising power.
  
  

• Within the biofilm some cells may “turn off”. These persister cells hide in the EPS and can reactivate at irregular intervals resulting in rapid recolonisation of the biofilm environment. Also the presence of diverse gene expression in the biofilm colonisers can result in variable sensitivity to specific antibiotics. Cells in bio- static growth phase will be less susceptible to cell wall targeted antibiotics, simply because they are not involved in constructing cell wall structures. The slower growth phase of biofilm bacteria may also contribute to some reduced antibiotic sensitivity.
  
  

• Within complex biofilms, plasmid DNA exchange is accelerated, facilitating anybody resistance under selective pressure from circulating anybody substrates.

• The presence of the extracellular polymeric substance (EPS), can also limit the ability of the immune system to access and destroy bacteria. Since many antibiotics facilitate cell killing reactions, this immune mechanism would be substantially inhibited.  (E.g. the presence of clindamycin increases the phagocytosis of bacteria by PMNs up to 4 times greater than that of penicillin exposed bacteria.)

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Kinkajou: Does the biofilm phenomenon suggest any
new strategies for disease control?

Erasmus:  Depending on the organism and type of antimicrobial and experimental system, biofilm bacteria can be up to a thousand times more resistant to antimicrobial attack than free-swimming bacteria of the same species. Some authors have suggested specific management protocols which may have some advantage over typical high dose antibiotic therapy.

Suggestions include use of an A2RA medication called Olmesartan, limiting vitamin D intake (due to the immune regulatory and inhibitory effects of this vitamin D on human cell expression”, and low-dose pulsed antibiotic protocols. Olmesartan is thought to act similar to an antimicrobial peptide (cathelicidin) which has antimicrobial, anti-attachment and anti-biofilm activity against Staphylococcus aureus.

After antibiotics are applied to a biofilm, a number of cells called “persisters” are left behind. Thus, a dose of antibiotics – particularly in immuno-compromised patients – eradicates most of the biofilm population but leaves a small fraction of surviving persisters behind.

Unfortunately, in the same sense that the beta-lactam antibiotics promote the formation of L-form bacteria, persister cells are actually preserved by the presence of an antibiotic that inhibits their growth. Thus, paradoxically, dosing an antibiotic in a constant, high-dose manner (in which the antibiotic is always present) helps persisters persevere. But persisters remain alive and resurrect the biofilm, causing the infection to relapse.

But in the case of low, pulsed dosing, where an antibiotic is administered, withdrawn, then administered again, the first application of antibiotic will eradicate the bulk of biofilm cells, leaving persister cells behind. Withdrawal of the antibiotic allows the persister population to start growing. Since administration of the antibiotic is temporarily stopped, the survival of persisters is not enhanced. This causes the persister cells to lose their phenotype (their shape and biochemical properties), meaning that they are unable to switch back into biofilm mode. A second application of the antibiotic should then completely eliminate the persister cells, which are still in planktonic mode.

Researchers have suggested the feasibility of a pulsed or cyclical biofilm eradication approach depends on the rate at which persisters lose resistance to killing and regenerate new persisters. Others have speculated that allowing the concentration of an antibiotic to drop could potentially lead to resistance towards the antibiotic. Management suggestions include using two or more antibiotics at one time. The Marshall Protocol has used a total of five bacteriostatic antibiotics, usually taken two or three at a time, to overwhelm biofilm bacteria and reduce resistance concerns.

It has been suggested that successful cases of antimicrobial therapy of biofilm infections result from a fortuitous optimal cycling [pulsed dosing] of an antibiotic concentration that eliminated first the bulk of the biofilm and then the progeny of the persisters that began to divide.
Mathematical modelling of low, pulsed dosing of antibiotics against biofilms bacteria has been suggested to be a superior way of targeting treatment-resistant biofilm bacteria.

The biofilm phenomenon also should be considered in strategies for food handling. Many food spoilage microorganisms are ubiquitous in the environment. Animals especially are a common source of human colonising bacteria but bacteria such as Pseumonads are very metabolically capable and can exist easily in the environment independent of people or other animals. Escherichia coli are common organisms used to indicate environment contamination by human /animal waste and can survive to some extent within the natural environment independently. If they contaminate throughout the food processing chain they will compromise the safety and shelf-life of the food product.

Many vegetable products when highly magnified as for example by an atomic force microscope reveal that the surface of many of these food items contains many deep clefts and pits. Many of the pathogens that cause food- borne illness, such as Shigella, E. coli, and Listeria, make sticky, sugary biofilms that can access these crevices, stick like glue, and hang on like crazy. Once the pathogenic organism gets on the product, no amount of washing will remove it. The microbes attach to the surface of produce in a sticky biofilm, and washing just isn’t very effective.

The danger arises when a pathogen becomes present, when you’re dealing with a food product that’s minimally processed, and the pathogen is capable of forming a biofilm in the particular foodstuff being harvested. Cells in these biofilms are very good at aligning themselves in the subsurface areas of produce, resulting in it being impossible to remove them by washing. In meat processing, it has been found that even germs such as Listeria monocytogenes are capable of forming biofilms. Biofilms in drinking water systems can serve as a significant environmental reservoir for pathogenic microorganisms, as well.

Biofilm Persistors
Kinkajou: The answer, Old Dog?

Erasmus: Research needs to be done. Perhaps food needs to be pre-processed to limit biofilm formation and the constitution of friendly bacterial biofilms may prevent the development of pathogenic ones.

Kinkajou: So what’s the conclusion?

Erasmus: I think as usual the problems are more about preconceptions and economics, rather than science. The research and the science is easy. It’s the people who are the problem.
Let’s use the example of medical infections.

Doctors will often swab a throat to test for which germs are causing the throat infection. At the pathology laboratory, the microbiologists will report if “one” germ predominates, otherwise an answer of “mixed normal flora’ is typically given. Generally, if a single germ is found, these germs could easily be regarded as “normal flora” as well.

So the only time a positive result will be given is when a single germ predominates in the plated culture specimens. (Bacteria are cultured or grown by inoculating a plate of growth media- usually an “agar” (polysaccharide) media with a sample of germs derived from the throat swab. If you think about it, the throat is a highly contaminated environment growing typically a large range of bacteria, most likely existing in complex mixed biofilms- as a survival mechanism to enable them to stick to the throat and not get washed away.

Water System Biofilms

The only time a single germ will predominate or be reported on a throat swab sample is when:

• Surface bacteria swarm over a biofilm (therefore not “necessarily” pathogenic), but easily sampled from the surface.
  

• Non-typical bacteria(obviously pathogenic) are found ( E.g. No one would argue that Neisseria gonorrhoea organisms in a throat swab are normal flora)
  

• A fairly pure culture biofilm is sampled from one area
  
  (I am sure that biofilms exist in a precarious coexistence of their bacterial members. If a patch of biofilm is “torn off”, the resulting raw patch is recolonised much like virgin growth forest).  Hence, sampling will reveal this monoculture regrowth.
  
  

• A single germ is present in high numbers for any reason, whether pathogenic or not.
  
  

• Perhaps the pathogenic organism is growing rampantly enough to appear as the predominant bacteria on a plate.
  
  
  Cell Membrane Biofilm Killing
  

Problems:

• The problem is that the pathogenic bacteria would typically be invading the human throat tissues and exist under the biofilm layers making them typically difficult to sample for testing. (The medical ethos is that if you find lots of it that is what the infection must be).
  
  

• There is no recognition that certain bacterial profiles may be normal. Conversely, there is no recognition that some cultured bacterial profiles can indicate poor health or can be abnormal.
  
  

• There is no method of culturing biofilms as an entity.
  
  

• There is no way of inhibiting gene expression alterations to reveal which bacteria are growing as biofilms and which are actively freely mobile or invasive.
  
  

• There are many bacteria that are never cultured because it cost as much as an extra 50 cents per plate for the more specialised culture media. Many germs have quite specific requirements for growth. Biofilms through the intermediary of other bacteria, may well provide the requirements for growth for many fastidious organisms.
  So many pathogens may well not be known or may be unable to be identified because routine testing techniques (usual agar plates) are not adequate for the task or identifying them.

 

Kinkajou: Don’t doctors ever wonder why the swab results they get focus so much on single germ identification in complex multi-bacterial environments? Why the germs they identify respond to treatment when they should not or conversely why they don’t respond to treatment when they should?

Goo: I expect that many never learn something that they have not been taught. It is a common failing.

Erasmus: Biofilms are obviously deserving of much more research and understanding. Yet it is entrenched knowledge and preconceptions as to the role of free planktonic type bacteria and the economics of changing what we already do, that are the biggest barriers to our growing understanding. Many doctors regard scientific research as a sideshow to their own understanding of the world which they learnt often up to decades ago in medical school, from people whose own knowledge is even decades older than their own. Change is a slow thing.










Bioremediation Biofilms