Biofilm Surveillance in Environment, Industry and Clinical

Biofilms are encountered in various systems and monitoring of biofilms is essential to ensure that microbial incidents are avoided as well as material degradation and infection.

4.1 Environmental Biofilms

Aquatic systems, drinking water distribution systems and wastewater treatment systems have biofilms that act as reservoirs of pathogens and cause biofouling. These biofilms have effects on the water quality, nutrient cycle, and microbe ecology (38).

4.2 Industrial Biofilms

A significant problem in food processing, cooling systems and pipelines is industrial biofilms. They decrease the productivity of the process, cause corrosion (biocorrosion), and represent a threat to food safety due to the contamination with disease-causing microorganisms.

4.3 Clinical Biofilms

The formation of biofilms is a common event in the medical device (catheters, implants), on chronic wounds, as well as on dental plaques, leading to their chronic infection. The microbes can escape the host immune system and antibiotic therapy due to the biofilm lifestyle and this makes the infections hard to eliminate (8).

4.4 Technologies of Real-Time Monitoring

The formation and activity of biofilm can be monitored continuously or in real-time, using modern methods:

•             Biosensors: Sense the metabolites, quorum sensing, or biofilm.

•             Electrochemical monitoring: Monitors growth of biofilm based on impedance or redox values.

•             Microfluidic systems: Visualization and manipulation of biofilms under controlled conditions under microfluidics.

•             opto method: Non-invasive tissue thickness and structure imaging of biofilm thickness and structure in real time.

5. Mechanisms of Biofilm Resistance

The biofilm of bacteria is exceptionally resistant to antimicrobial agents, environmental stress, and immune response of the host, and is therefore of great concern in clinical, industrial and environmental settings. Biofilm-associated bacteria, in contrast to planktonic bacteria, are entrenched in an extracellular polymeric substance (EPS) that is self-produced and serves as a protective barrier. The mechanisms of resistance are multifactorial and are a result of physical, chemical, genetic, and physiological adaptations which together contribute to the survival of the microbes. It is important to know these mechanisms in order to come up with appropriate measures to manage biofilms (15).

1. Extracellular Polymeric Substance (EPS) Barrier

The most evident factor that has contributed to biofilm resistance is the EPS matrix. The matrix is made of polysaccharides, proteins, lipids, and extracellular DNA (eDNA) that form a diffusion barrier that retards the entry of antimicrobial agents into the biofilm. The EPS may contain hydrophobic interaction and ion binding, which traps cationic or hydrophilic drugs and prevents them at the cell surface of the microbes (8). Also, EPS offers a wet environment to sustain the metabolic activity and stabilize the biofilm structure subjected to shear stress or mechanical perturbation. The physical resistance does not only slow down the action of antibiotics but also resists disinfectants, detergents and host immune factors, including antibodies and complement proteins.

2. Physiological Heterogeneity and Metabolic Dormancy

Due to chemical gradients of nutrients, oxygen and waste products within the EPS matrix, biofilms have a high level of physiological heterogeneity. The cells in the deeper layers tend to be under nutrient limitation as well as hypoxia resulting in low growth or metabolic quenching. Most of the antimicrobial agents especially those which act as cell wall synthesis or protein production inhibitors do not work so well on dormant or slow-growing cells, a factor that leads to tolerance, and not genetic resistance per se (32). Besides this, the outermost cells possess metabolic activity that has the ability to inactivate antimicrobials before penetrating to inner layers, forming a stratified system of defense.

3. Abnormal Gene Expression and Stress Response

The gene expression pattern of biofilm-associated bacteria is also different than that of planktonic cells. Biofilms upregulate genes related to the production of EPS, efflux pumps, stress response and repair of DNA, which help to increase the survival. The heat shock proteins, oxidative stress defenses systems and two-component regulatory systems are stress response systems that allow cell to endure extreme conditions like antibiotics, pH changes, or reactive oxygen species (23). Efflux pumps are very active in pushing antimicrobial molecules out of bacterial cells reducing the concentration of drugs within the cell and also leading to additional resistance.

4. The Horizontal Gene Transfer and Genetic Adaptation

Biofilm communities offer a perfect setting in which horizontal gene transfer (HGT) occurs and antibiotic resistance genes are disseminated among the resident bacteria. Conjugation, transformation and transduction in biofilms are more effective because of high cell proximity and cell stabilization by the eDNA within the EPS matrix. This increases the development of multi drug resistance traits, which may be maintained even after the dispersal of biofilms. The spontaneous mutation of the genes responsible of antimicrobial resistance is also selected by biofilms so that evolutionary adaptation can take place under the selection pressure (11).

5. Persister Cells

A portion of biofilm cells, referred to as transiently phenotypically tolerant persister cells, neither develop genetic resistance to antibiotics nor persistently express them. These cells go into a dormant or low-metabolic condition, and are able to withstand fatal levels of antimicrobials. After the treatment is removed, persister are able to recover and resocialize the biofilm, which lead to chronic and recurrent infections (13). The development of persisters is controlled by stress-induced toxin-antitoxin systems, quorum sensing and metabolic signals in the biofilm. They pose a specific challenge to the total elimination of biofilms with long-term or intensive antibiotics treatment.

6. Quorum Sensing and Co-ordinated Defense.

Quorum sensing (QS) is a cell-density-dependent system of communication that controls the process of biofilm development and defense. Bacteria produce autoinducers that induce the expression of EPS synthesis genes, virulence factor genes, and stress resistance genes. QS facilitates concerted biofilm behavior, like activating efflux pump or enzyme secretion that neutralizes antibiotics. QS enhances biofilm resistance by coordinating the defenses of the microbial community and plays a role in antimicrobial resistance in the population.

7. Exposure to Host Immune Defenses.

EPS and biofilm architecture of clinical biofilms inhibit phagocytosis and restrict immune cell and antimicrobial peptide penetration. The matrix protects the pathogens against neutrophil attacks, complement, and antibody recognition. Besides, a few bacteria in biofilms generate enzymes like catalases and proteases that inactivate reactive oxygen species or breakdown host defense molecules. Such defense mechanisms help the pathogens to survive in chronic wounds, indwelling medical devices and mucosal surfaces resulting in repeated infections and higher morbidity (9).

8. Maturity and Structural Complexity of biofilm.

The very mature biofilm structure is resistant. Biofilms consist of microcolonies between water channels, which facilitate the movement of nutrients but at the same time, it may limit diffusion of antimicrobials. The biofilm has micro environments with different PH, oxygen levels, and metabolic activity, zoning out areas of low activity of antimicrobials. The heterogeneity in the structure of the cells allows certain cells to survive even in the aggressive treatment, so that biofilm is preserved (14).