INTRODUCTION:

Biofilms are architecturally complex communities formed of multicellular aggregates of sessile cells that are irreversibly attached to a substrate, surrounded by a self-produced extracellular matrix composed of polymeric slime with polysaccharides, proteins and nucleic acids and have a different phenotype from that of planktonic bacteria in relation to growth rate and gene expression^1-4.

One of the most crucial characteristics of biofilm infections is the significantly high resistance to antimicrobial agents. Consequently, biofilm-based infections are persistent and very difficult to eradicate ⁵. This review discusses the different mechanisms of biofilms to antimicrobial agents.

1. Slow growth and low oxygen concentration:

Biofilms contain different subpopulations of cells with differential physiological states that affect their susceptibility phenotypes. P. aeruginosa biofilms are composed of at least two distinct physiological subpopulations: that differ in metabolic activity, protein synthesis, oxygen concentration and growth; one is close to the substratum with low metabolic activity and reduced susceptibility to antibiotics and the other on the top with high metabolic activity and higher antimicrobial susceptibility ^6-10.

2. Increased mutation and genetic exchange rates:

The rate of mutation and horizontal gene transmission among biofilm cells is markedly higher than that among planktonic cells ^11,12. As a consequence, biofilm cells can exhibit multidrug resistance by classical resistance mechanisms against β-lactam antibiotics, aminoglycosides and fluoroquinolones such as production of drug inactivating enzymes, change of antibiotic targets and over expression of multidrug efflux pumps ^13,14.

3. Retarded penetration through the biofilm matrix barrier:

The biofilm matrix barrier increases the biofilm resistance to antimicrobial agents either by adsorption of the drug onto the matrix ¹⁵ or by chemical interaction with the drug molecules¹⁶.The negatively charged matrix limits the penetration of the positively charged molecules of antibiotics, such as amino glycosides. As a consequence, the penetration of these antimicrobials is significantly delayed ^17,18.

4. Overexpression of efflux pumps:

The role of efflux pumps in biofilm resistance to antimicrobial agents was investigated in many studies. The upregulation of the MexAB-OprM efflux pump makes the metabolically active biofilm cells highly resistant to colistin ¹⁰.The efflux-pump genes mexAB-oprM and mexCD-oprJ were induced in P. aeruginosa biofilm in the presence of azithromycin ¹⁹. The expression of a potential efflux system (encoded by PA1874–1877) is enhanced in biofilm cells and was involved in the high resistance toward tobramycin, gentamicin, and ciprofloxacin ²⁰.

5. Adaptive phenotypes:

A unique biofilm phenotype is induced by expression of different sets of genes and proteins than those of planktonic bacteria. This biofilm-specific phenotype could enhance antimicrobial resistance mechanisms against biofilms ²¹.

Biofilm development is linked to alterations in protein regulation. The most significant ones are proteins involved in resistance to oxidative damage, exopolysaccharide production and metabolism. Moreover, a significant up-regulation of proteins involved in anaerobic processes was observed during biofilm maturation, probably due to oxygen limitation of a large portion of the population ²².

In biofilms, genes encoding pili and flagella were repressed that indicate that they are no longer required in mature biofilms. Moreover, some genes that affect antibiotic sensitivity were repressed; e.g. the tolA gene that decreases aminoglycoside affinity for the cell outer membrane by affecting lipopolysaccharide structure is activated in P. aeruginosa biofilms ²³.

In another study, a set of 20 genes that were differentially expressed in biofilms exposed to high levels of the antibiotic tobramycin compared to untreated biofilms were found ²³. Among them two genes involved in stress responses and two efflux systems were activated ²³.

6. Persister cells and phenotypic variants:

Persisters are small fraction of the biofilm population which exhibit different phenotype²⁴. These cells are dormant or slowly dividing bacteria ^25-27. Persister cells are responsible for the high levels of resistance and survival of the population in biofilm communities ^24,26.

Antibiotic-resistant phenotypic variants may undergo transient phenotypic changes that are associated with the ability of the bacteria to form biofilms such as the high surface hydrophobicity that results in increased cell attachment and formation of biofilms with increased biomass, indicating that there is a link between phenotypic variation and biofilm resistance ²⁸.

7. General stress response:

Activation of the general stress response in bacteria enhances resistance to environmental stresses such as starvation, DNA damage, osmotic pressure, temperature and oxidative stress. Biofilm resistance to antibiotics may be linked to activation of the general stress response²¹.

The sigma factor rpoS is a general stress response regulator that activates expression of some genes that are needed for cell viability during stationary phase when cells undergo nutrient limitation. Similarly, nutrient limitation in biofilms could induce the expression of rpoS with subsequent physiological changes that protect against environmental stress and antimicrobial agents ²¹.

8. High cell density and quorum sensing (QS):

Quorum sensing is a cell-to-cell communication mechanism that allows bacteria to sense when a critical number (quorum) of bacteria are present in a limited space in the environment and respond by activating certain genes that regulate the production of virulence factors ^29-31,
3. Quorum sensing controls the development of the biofilm and determines the resistance of biofilms to antibiotic therapy ^32,33.

CONCLUSION:

Antimicrobial biofilm resistance is multifactorial. The emergence of a biofilm-specific phenotype increases biofilm resistance. The exopolysaccharide matrix delays antimicrobial penetration. Gradients of nutrient and oxygen are developed by the increase in cell density and result in decreased metabolic activity and growth rate. Moreover, activation of quorum-sensing systems is induced by increase in cell density. Furthermore, nutrient starvation and oxygen limitation induce the general stress response and up-regulation of efflux pumps. Environmental conditions induce the formation of phenotypic/persister variants that are resistant to high concentrations of antimicrobials. The understanding of resistance of biofilm bacteria to antimicrobial agents is very important for development of strategies for treatment of biofilm-based infections.

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Received on 20.12.2012 Modified on 25.12.2012

Research J. Pharm. and Tech. 6(1): Jan. 2013; Page 01-03