BACTERIAL BIOFILMS

1.- BACTERIAL CAPSULAR EXOPOLYSACCHARIDES

2.- BACTERIAL BIOFILMS: THE ROLE OF EXOPOLYSACCHARIDES IN CHRONIC PROCESSES

3.- LIPOSOMES: ANTIGEN VEHICLES

4.- LATEST GENERATION AUTOVACCINES

2.- BACTERIAL BIOFILMS: THE ROLE OF EXOPOLYSACCHARIDES IN CHRONIC PROCESSES

This second part deals with the role of bacterial exopolysaccharides in the formation of biofilms and in causing infectious processes to become chronic.

Biofilms in nature

Biofilms are bacterial communities which are enclosed within a matrix of polysaccharides produced by the bacteria and adhere to a living or inert surface (Fig. 1.1). In nature, biofilms constitute a protected growth modality that allows the bacteria to survive in hostile environments. The structures which make up these microcolonies contain canals through which nutrients circulate, and in different zones of the biofilm the cells express different genes, as if they were part of an organized structure. Bacterial biofilms colonize any humid surface, such as dental enamel, the stones on a riverbed, or an infected tissue. Occasionally, these bacterial aggregates release individual cells that disperse and rapidly multiply, thereby colonizing other places. The general idea is that the individualized bacteria are exposed to the agents found in the environment (including, for example, the possibility of becoming phagocytosed by an ameba, or of being carried downstream in a river), while inside the biofilm these cells are protected (Costerton, 1999).

Image 1.1. Bacterial fixation to the epithelium and the emission of chemical signals.

Infections mediated by bacterial biofilms

Many of the infections that affect animals are caused by bacteria that constitute part of the commensal flora of the host. Some examples of commensalism are provided by Staphylococcus aureus, S. epidermidis or S. hyicus, which colonize the skin; Streptococcus suis and S. agalactiae, which colonize the mucosal membranes; Pasteurella multocida, P. haemolytica, Actinobacillus pleuropneumoniae, Mycoplasma spp. or Haemophilus parasuis, which are found in the upper airways, etc. It is difficult to eradicate such infections from a farm, and in many cases they may even cause chronic infections in livestock.

When the bacteria colonize live tissues, they make use of adhesion mechanisms to avoid elimination from the body with the natural fluids such as nasal or vaginal mucus and the passage of food, or by mechanical forces such as sneezing, mastication or intestinal peristaltism. Adhesins normally participate in initial adhesion to the epithelial surfaces (e.g., Staphylococcus fibronectin receptor), together with fimbria (e.g., f41, 987P, etc., of Escherichia coli). In fact, similar adhesion mechanisms have been described for almost all bacterial species. However, these adhesins and fimbria are "hazardous" for the bacteria because their "adhesiveness" makes them easily recognizable by circulating phagocytes — with the resulting risk of rapid elimination.

Image 1.2. Activation of the mechanisms responsible for biofilm exopolysaccharide production and bacterial multiplication.

For defense purposes, bacteria have developed an interesting system. After adhering to the epithelial surface, they begin to multiply while emitting chemical signals that "intercommunicate" the bacterial cells. Once the signal intensity exceeds a certain threshold level, the genetic mechanisms underlying exopolysaccharide production are activated (Costerton, 1999; Fig. 1.2). In this way, the bacteria multiply embedded within an exopolysaccharide matrix, thus giving rise to the formation of a microcolony (Fig. 1.3). However, it is important to point out that these exopolysaccharides are both chemically and physically distinct from those forming the bacterial capsule (McKenney, 1998).

The advantages of family life: chronification

The microcolony induces an immune response, though the colony is too large to be phagocytosed. Indeed, enzymes released by the phagocytes surrounding the colony may damage the host tissues in proximity to the biofilm — a phenomenon that in turn favors growth of the colony.

On the other hand, the bacteria enclosed within the biofilm are extremely resistant to antibiotic treatments. Such resistance can be explained by three not necessarily exclusive hypotheses (Costerton, 1999; Gracia 1999)(Fig. 3):

Although antibiotics are able to penetrate the biofilm, the drug concentrations obtained may be insufficient in certain areas of the film.

The bacteria located at the base of the biofilm are metabolically inactive and are therefore resistant to certain antibiotics.

On the other hand, the bacteria possess active antibiotic degradation mechanisms that contribute to avoid the accumulation of effective drug concentrations in certain areas of the biofilm.

In any case, practice shows that in chronic processes mediated by biofilms, antibiotherapy is unable to eliminate the infection. As an example, and regardless of the amount of amoxicillin administered, it is not possible to eradicate Streptococcus suis or Staphylococcus hyicus from a farm, and both pathogens often become a recurrent problem. In chronic mastitis caused by Staphylococcus aureus in ruminants or rabbits, it is universally accepted that antibiotic treatment is entirely useless.

Image 1.3. Constituted microcolony from which bacteria without exopolysaccharides (i.e., in phase variation) are released to colonize other tissues.

In order to prevent these processes from becoming chronic, vaccines can be used targeted to different bacterial adhesion factors (for example, vaccines against K88, K99, 987P of E. coli), or alternatively early humoral responses can be induced against the exopolysaccharides responsible for biofilm formation — thereby avoiding the appearance of these microcolonies.

Phase variation

The microcolony continues to grow in volume, and the bacteria in proximity to the epithelium have difficulties in gaining access to nutrients from the external environment. Only those located in the upper layers of the colony are able to continue multiplying — a situation that creates bacterial populations with metabolic differences. Occasionally, for purely mechanical reasons, some bacteria are shed from the colony, or (more frequently) some bacteria stop producing exopolysaccharide and are thus "released" into the surrounding environment (Fig. 1.3). This phenomenon by which certain bacteria stop expressing certain genes is known as phase variation. These individualized cells in turn attempt to colonize other surfaces or individuals to form new microcolonies (Baselga, 1994).

The bacteria individualized in the external environment are easily phagocytosed by the inflammatory cells surrounding the microcolony, and are susceptible to antibiotic action.

The risks of chronic problems

However, during the lifetime of an animal, stressful situations are frequent and can lead to generalized immune depression.

In such situations the individualized bacteria may overcome the inflammatory barrier and spread to other tissues. In fact, the continuous appearance of such acute outbreaks is what characterizes chronic processes. For example, in chronic mastitis due to Staphylococcus aureus, a characteristic observation is the appearance of acute outbreaks in the animal every few weeks or months, without eventual healing in any case — while in situations of streptococcal meningitis or in dermatitis, it is normal for the process to manifest on a recurrent basis in a given farm for years.

These acute pathologies can be resolved on a point basis by antibiotic therapy, and sometimes even disappear spontaneously — though this is not the case in chronic infectious disorders. A strategy for controlling and preventing such processes would be to use the exopolysaccharides of the biofilm as vaccinal antigens. In this way the appearance of chronic problems in the farm can be avoided.

Bibliografía

1. Baselga, R.; Albizu, I.; Amorena, B. Staphylococcus aureus capsule and slime as virulence factors in ruminant mastitis. A review. 1: Vet. Microbiol. 1994 Apr ;39 (3-4): 195-204
2. Baselga, R.; Albizu, I,.; De La Cruz, M.; Del Cacho, E.; Barberán, M.; Amorena, B. Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence. 4: Infect. Immun. 1993 Nov.; 61 (11): 4857-62
3. Gracia, E.; Fernández, A.; Conchello, P.; Alabart, J.L.; Pérez, M.; Amorena, B. In vitro development of Staphylococcus aureus biofilms using slime-producing variants and ATP-bioluminescence for automated bacterial quantification. Luminescence 1999 Jan; 14 (1): 23-31
4. Costerton, J.W.; Stewart, P.S.; Greenberg, E.P. Bacterial biofilms: a common cause of persistent infections. Science 1999 May 21; 284 (5418): 1318-22
5. McKenney, D.; Hubner, J.; Muller, E.; Wang, Y.; Goldmann, D.A.; Pier, G.B. The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/adhesin. Infect Immun 1998 Oct; 66 (10): 4711-20
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