Resistance to Antimicrobics

Biofilms possess a remarkable resistance to antibiotics sometimes more than 1000 times that of planktonic cells. Although the cause of this resistance is still under intensive investigation, several mechanisms have been advanced.

Failure of the Antimicrobic to Penetrate the Biofilm Matrix

In the early history of biofilm research this was a favored hypothesis. However, research by a number of investigators showed that in most cases, antibiotics readily penetrate to the substratum surface. Except in specific circumstances, lack of penetration is not considered a significant contributor to antimicrobic resistance.

The movie shown below was created from time-lapse photography of a dye penetrating a biofilm over a period of eight minutes. The main point of this clip is to illustrate just how long it takes a substance to reach microbes in a biofilm as compared to microbes in planktonic form.

 

Degradation of the Agent

Microbial enzymes may degrade the antibiotic or disinfectant, as they penetrate the biofilm. The degradation of penicillin by the enzyme penicillinase (β-lactamase), or the decomposition of hydrogen peroxide by catalase are examples.

Metabolic Heterogeneity (Quiescence)

A biofilm contains a vast array of different metabolic niches which vary in oxygen concentration, nutrient and ionic concentrations as well as concentrations of waste materials. Cells within the biofilm matrix vary in growth rate from actively growing to essentially dormant. Presumably, in every biofilm, there are niches in which certain cells are metabolically quiescent due to nutrient deprivation.

Nutrient limitation, whether it be in stationary phase culture or in the depths of a biofilm, may increase resistance by reducing metabolic activity. These cells, having fewer metabolic points of attack, are less susceptible to the action of antimetabolites like antibiotics and disinfectants.

Phenotypic Versatility

Within moments of the time a bacterial cell adheres to a surface it undergoes a remarkable change. Many genes are repressed and others are induces the sum amounting to as much as 40 percent of the total bacterial genome. Many of the newly synthesized proteins play a role in antimicrobic resistance. Some constitute efflux pumps capable of excreting antibiotics at a rate that keeps the concentration below lethal levels.

Persister Cells

Investigator Kim Lewis has found evidence of genes that in a small proportion of the biofilm population produce "toxins" that inhibit certain critical metabolic functions such as translation, transcription and DNA replication.

With these vital functions inhibited, cells expose fewer critical targets to antibiotics and disinfectants and are thus much more resistant. Lewis finds these "persister cells" in late log phase planktonic culture as well as in biofilms, but in the planktonic state these persisters are destroyed by phagocytic neutrophiles or protozoa. In biofilms the persisters are protected from predation as stated earlier.

Originally thought to be involved in scheduled cell death (Apoptosis-like), these genes are now viewed as producing altruistic persister cells whose primary function is survival. Following exposure to an antimicrobic which kills much of the biofilm, these persisters reactivate by producing antitoxin proteins that inactivate the toxins and permit resumption of metabolic activity and growth. These persister cells are not mutants since cells surviving antibiotic treatment produce once again a population of normally antibiotic cells and a small fraction of recalcitrant persisters.