Biofilm Biofilm is a complex aggregation of microorganisms growing on solid substrate. A biofilm contains about 15% of microbial cells & 85% EPS (Extra polymeric substance).EPS composed of polysaccharides,proteins,and other polymers and water.
Biofilms are characterized by structural heterogeneity, genetic diversity and complex community interactions.
A biofilm formation often initiated by micro colonies from one type of organism. However biofilms quickly become heterogeneous as mixed cultures of bacteria, as well as fungi, algae and protozoa join the established structure and become intermixed. In fact with in a biofilm different types of microorganisms can coexist and form stable communities.
Biofilm formation is a survival strategy against environmental stress. Biofilms are resistant to phagocytic amoebae, and are considered to be 100% phage proof. Biofilms are much more resistant than planktonic cells to antimicrobial agents. For example chlorination of a biofilm is usually unsuccessful because the biocide only kills the bacteria in the outer layers of biofilm. The bacteria within the biofilm remains healthy and the biofilm can regrow. Repeated use of antimicrobial agents on biofilms can cause bacteria within the biofilm to develop an increased resistance to biocides.
In the industrial environments, biofilms can develop on the product contact surfaces of equipment’s and interiors of water purification and distribution system, which leads to clogs, corrosion and biological contamination of the medicinal products.
Stages of biofilm formation
There are three stages for biofilm formation. They are
1. Initial adhesion to a surface. The first colonists adhere to the surface initially through weak, reversible Van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion molecules such as Pilli. First colonies facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build matrix that holds the biofilm together.
2. Cell growth or reproduction and production of EPS. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment.
3. Detachment of sessile micro colonies. These cells travel to form new biofilms on other locations.
Quorum Sensing
Quorum sensing plays a major role in the initial stages of biofilm formation and biofilm dispersion. This phenomenon is a type of cell to cell signaling mechanism that enables a bacterium to regulate gene expression in response to population density. This type of intracellular communications occurs both within and between species.
Quorum sensing depends on production of diffusible signal molecules called auto inducers or pheromones. Once these molecules reach a high concentration, they start to interact with regulatory proteins that modulates gene expression. Besides bioluminescence, cell adhesion and cell detachment, a variety of other physiological process is regulated by quorum sensing and those includes swarming, motility sporulation, conjugation and production of virulent molecules.
Cell Adhesion
The initial cell adhesion surfaces is a process governed by long range forces, primarily van der Waals and electrostatic interactions. Initial stage of biofilm formation is dependent on several factors ,including bacterial cell properties (ex:hydrophobicity),the nature, type, shape and physiochemical properties of substratum as well as chemical composition, hydrodynamics and flow characteristics of liquid environment.
Biofilm Dispersion
Biofilm cells can be dispersed by three main process. Shedding, detachment and by physical process. Dispersion of biofilm cells can have detrimental effects on a process unit operation, a piece of equipment or water system. Dispersed film often maintain their biofilm phenotype, including antimicrobial resistance and the ability to attach new surfaces, thus posing a risk for systemic microbial colonization and potential product contamination.
Water Systems & Biofilm Formation
Important design features for prevention of biofilm formation in water systems are the material of construction, temperature of the system, and water flow. Usually stainless steel(SS316 or SS316L)piping is the preferred material due to ease of cleanability and suitability for heat sanitization.
WFI systems that are maintained circulating with a turbulent flow and at a high temperature (65–80°C) are deemed self-sanitizing. Purified water systems that are maintained circulating with a turbulent flow and at ambient temperature (25 ± 5°C) or cold WFI systems (0–5°C) are typically steam sanitized once a week, with user points
heat sanitized daily. These practices are often effective in preventing biofilm formation. Maintaining a circulating water system is critical because a one-way water system is basically a dead leg.
In pharmaceuticals, the risk of systemic microbial contamination in a water system is low due to proper purification steps and routine monitoring. Hence pharmaceutical water systems can be contaminated due to a failure in the maintenance or operating procedures designed to prevent introduction of microorganisms into the system. For example, nonsterile air that remains in a pipe, valve, or hose after drainage may be introduced into the system inadvertently. Carbon beds used for pretreatment of feed water can become a breeding ground for biofilms, and these units must be heat sanitized as backwashing does not work and can exacerbate the problem. Perforated heat exchangers can also lead to contamination of a water system. FDA recommends that heat exchangers not be drained of the cooling water when not in use to prevent pinholes from being formed in the tubing after they are drained as a result of corrosion of the stainless steel tubes in the presence of moisture and air.
Dead legs in piping’s can potentiates biofilm formation. Microbial contamination can also occur if pumps are not continuously in operation, resulting in a static reservoir area where water will become stagnant.
RO systems are used as pretreatment for highly purified waters. However, RO systems, if not of sanitary design, are prone to microbial contamination that often becomes established in the membrane filters and in the ball valves; the center of the valve can collect water when the valve is closed, and the stagnant water can harbor microorganisms and provide a starting point for the development of a biofilm. With the recognition of the dangers of potential biofilm formation in RO units, filter manufacturers recommend installing at least two units in series, and some manufacturers have installed heat exchangers immediately after the RO filters to heat the water to 75–80°C in an attempt to minimize microbial contamination. In addition, an ultraviolet (UV) light is often installed in the system
downstream from the RO units to aid in the control of microbial proliferation.
Although ozone and UV light have been used to control microbial contamination in water systems, both methods have pros and cons. The dissolved residual ozone remains in the system may pose safety concerns not only for employees but also for drugs formulated with the water. Another concern with using ozone is that ozonating
the incoming water breaks up many types of nutrients that otherwise would not be available for uptake by microorganisms. Therefore, for some water systems, ozone makes nutrients available to bacteria, and a biofilm bloom can develop immediately after the ozonater. The effectiveness of UV lights for control of microbial contamination is limited and dependent on where the unit is located, and whether the UV light is on continuously or just turned on when water is needed. UV light penetrates biofilms poorly. Much of the radiation gets trapped in the EPS matrix, so the sessile cells are protected from and resistant to the radiation.
Production Equipment’s & Biofilm Formation
The primary rule for biofilm prevention is to store equipment and materials as dry as possible. So cells cannot form biofilms in the absence of water or moisture. Care should be taken when connecting pipes, gauges, sensor probes, hoses, and other parts of equipment to ensure that connections do not create dead legs or dead zones where liquid can collect.
Product contact surfaces should be maintained smooth, with no imperfections or deterioration that would lead to microbial colonization. Corrosion of metals after exposure to water and chemicals is of great concern in the pharmaceutical industry. Water is the largest component used in pharmaceutical manufacturing and its use can lead to detrimental effects on metal surfaces such as rouging, corrosion, and biofilm formation.
Microbial contamination of pharmaceutical equipment occurs primarily with equipment and materials that do not meet sanitary design standards. The equipment used in pharmaceutical manufacturing should meet good engineering design and principles. Including aspects that relate to sterility and cleanability, dimensions and tolerances, surface finish, material joining, and seals.
Biofilm Control & Prevention
Biofilm control involves one of the following strategies: preventing the initial contamination of the material, attempting to minimize the initial microbial adhesion to the surface, killing of the biofilm cells via chemical or heat treatment, and removing the piece of equipment altogether and replacing it with a new and clean one.
Heat
Heat is very effective in removing biofilms, and it is the first choice for biofilm prevention and remediation. Companies should modify, whenever possible, equipment components so the system can be steamed in place. In order to do so, the equipment must be fitted with steam traps, and should have parts and components that can withstand heat. Another alternative is to disassemble the various equipment parts, autoclave them, and then reassemble the equipment using aseptic technique and under aseptic conditions.
Chemical Treatment
Equipment that cannot undergo steaming in place or autoclaving must be chemically sanitized prior to use. Chemical sanitization of equipment can be accomplished using caustic, acidic, and oxidizing agents such as hydrogen peroxide and sodium hypochlorite solutions. Oxidizing chemicals can actually dissolve the polysaccharide matrix and kill the bacteria. These solutions are very effective in biofilm removal but, unfortunately, not compatible with many materials.
