Abstract
It has been reported that microorganisms live social lives. They can use coordinated chemical and physical interactions to form complex communities. These communities are very intricate systems which coordinate microbial behavior. Little has been known about how these systems work in nature. Newer technologies, like microfluidics, are now available to better understand the small microbial communities that exist, also called microenvironments. Microenvironments are defined by Wessel, et al. (2013) as “small, defined regions of the environment.
In microbial communities, a microenvironment refers to the area immediately surrounding a single cell or small group of cells and is generally distinct from its environs on the basis of characteristics such as nutrient availability and mass transfer.” This has resulted in a greater knowledge of the microbial behavior and phenotypic heterogenicity that exists in microbial colonies. (Wessel, et al.,2013)
Biofilm is defined as a thin usually resistant layer of microorganisms (such as bacteria) that form on surfaces and can coat the surfaces. (Anonymous, 2018a) The microorganisms in a biofilm can be pathogenic or not and have formed complex multicellular structures on the surfaces. (Hall-Stoodley, et al., 2005) Previously, biofilm has been studied in the laboratory, using conventional methods including growth on or in nutrient media. Understanding the communities where microorganisms live may provide us with additional information in preventing and remediating issues with biofilm.
Industrial Concerns with Biofilm
It is frequently reported that biofilms are ubiquitous in industrial and drinking water systems. They can lead to biofouling in these systems. The effects of biofouling can be severe, e.g., damage to materials, production losses, and adversely affecting product quality. Biofouling is a biofilm development that exceeds a specified threshold of interference. Threshold limits need to be established for individual systems, with operators maintaining the system below these levels. It is not easy to apply control mechanisms from one system to another. It can be difficult to use biocides, as they are affected by things like the water quality and characteristics. Ideally, one should prevent the build-up of biofilm using regular disinfection or sterilization and when biofilms are formed to ensure that they do not achieve threshold values. (Murthy and Venkatesan, 2009)
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Developing a control program requires understanding of various biofilm components: thickness of slime layer, algal and bacterial species involved, amount of extracellular polymeric substances and inorganic components present. As such, it can be difficult to find a universal biocide. Monitoring should be performed to assess effectiveness. (Murthy and Venkateesan, 2009)
Typical concerns in the selection of a control mechanism include: cost, time constraints, and cleanliness (threshold values). In pharmaceutical applications, e.g., filling lines and processing areas there are also concerns with the potential for leaving residuals that can interfere with the product. (Murthy and Venkateesan, 2009)
Some of the newer technologies being used for biofi lm control include: ultrasound, electrical fi elds, hydrolysis of extracellular polymeric substances and methods altering biofi lm adhesion and cohesion.
These methods are in the very early laboratory stages. (Murthy and Venkateesan, 2009) On a commercial basis, ozonated water has been used in many operations. Depending upon the level of control desired, one can set the exposure level to provide sanitization, sterilization, and/or depyrogenation. Ozonated water has been shown to be eff ective in both the food and pharmaceutical industries.
Biofilm
There are two basic forms of microbial existence: free-floating or free-swimming (planktonic) and anchored-sessile forms (biofilms and spores). There is a much greater understanding of the free floating microbes, and standard treatments for remediation of these organisms. However, when the organisms are in a biofilm, they become more difficult to eradicate. It is reported that biofilms represent 90% of all microorganisms. Unfortunately, in a biofilm the organisms have sophisticated defense mechanisms and can be difficult to diagnose and eradicate. (Anonymous, 2018c)
When a biofilm is formed, the communities of microorganisms are enclosed in a protective extracellular polymeric substance (EPS) matrix. The matrix allows for adherence to living tissues, natural and artificial surfaces as well as to other microorganisms in the community.
The biofilm also provides a defense mechanism to prevent eradication. Another benefit is that the biofilm makes the microbes stronger and more resistant to attack, e.g., antimicrobials, disinfectants, and host immune defenses. (Anonymous, 2018c)
Biofilms are an accumulation of microbial cells. However, the biofilm has elaborate functional structures, which aid in the cell’s survival. The cells develop the extracellular matrixes that work to protect and aid the cells in the biofilm. The surface aids in the absorption of oxygen and aids in the collection of nutrients and the release of wastes. (Anonymous, 2018d)
Different microbes within the biomass have different functions (somewhat how human cells work together to form a tissue), some secrete the necessary extracellular matrix, and anchor the biomass in place. Others stay motile. Cells around the edge of the biofilm may be dividing to expand growth. Spore-forming organisms in the middle, may proliferate spores for survival and colonization of new locations. (Anonymous, 2018d)
There are a variety of ways in which the microbial communities can move: e.g., dendritic swarming and spiral migration. Dendritic swarming is a process where cells rapidly push outward in branching columns that are like “paving the surface.” Swarming occurs in nutrient rich environments, allowing the best use of the nutrients before other competitors can access them. Cells on the periphery of the biofilm develop extra flagellae (allowing more energy to swim). Other cells secrete surfactant to aid the motile cells slide rapidly over the surface. Swarming biofi lms grown on fl at laboratory plates remain distinct. They tend to coil in and around each other, but they do not cross. (Anonymous, 2018d)
Spiral migration occurs under laboratory conditions. The microbes grow in long chains or fi laments that curl either clockwise or counterclockwise. The advantages of this activity are not well known. Individual strains of bacteria spiral in different directions. (Anonymous, 2018d)
Biofilm Lifecycle
The typical lifecycle of biofi lm is described in Figure 1. There are three basic stages of the lifecycle including: attachment of the free microorganism to a moist (submerged) surface; the microorganism begins to grow including the production of slimy substances colonizing the microorganisms; and finally, detachment of small or large clumps of microorganisms that had been attached to the surface. Another term frequently used for the detachment phase is sloughing of microorganisms.
The detachment of microorganisms in a biofilm, is identified as a concern in the dissemination of infection and contamination of industrial systems. However, this is one of the least studied biofilm processes. The microbes in the biofilm show localized growth with discrete cell clusters. The clusters contain mature, mixed-species of microbes within the biofilms. Growth occurred in turbulent flow conditions in situ. When detachment occurred, the size of the biomass ranged from single cells to aggregates of about 500 μm, (Stoodley, et al. (2001)
Microorganisms have an amazing ability to adapt to changing environmental conditions. They want to survive. As such, they quickly modify their physiology according to the cell to cell interactions in their environment. There are a variety of signaling proteins available to aid in this adaptation and survival process. (Dubczak and McCullough, 2018)
Biofilm Formation and Organization
Cho, et al. (2007) indicated that biofilm can be formed by microorganisms to cope with local environmental challenges. Things like pitting in piping or nicks can provide the desirable environments. The microbial cells may seek-out small chambers or cavities, assemble there, and engage in quorum sensing behavior. Quorum sensing is defined as “a regulatory mechanism of microorganisms that involves the release of molecules which when present at threshold concentrations signal the expression of bacterial genes controlling specific group actions” (such as the formation of biofilms). (Anonymous, 2018b) There are many diff erent reasons that microbes may seek out these chambers or cavities. Some of the reasons include: environmental conditions, chemical stress, variable temperatures, fluid flow, and limited supplies of nutrients. (Cho, et al., 2007)
During biofi lm formation, the microbes must overcome the environmental conditions that caused them to isolate in these small areas. During this phase, they lay a foundation for a highly-organized mature structure. The microbes in these areas need to populate into very high densities, while having partial shelter in the small space. The confinement may be the reason for the quorum sensing initiation. The quorum sensing is believed to be a crucial step for the biofilm to progress. While there are many benefits to forming biofilms, there are also disadvantages.
They may include: the separation from the environment may lead to a reduced nutrient supply, concerns with water removal, the potential for disorganized expansion causing cell damage and even the hindrances to cell escape from the growth areas. (Cho, et al., 2007)
The organization of the cells in a biofilm are believed to be controlled by the cell to cell interactions. It is not intelligence as in a human. The advances in microfluidic devices with chemostatic microchambers have increased the ability to study these environments over many generations. This data showed that with E. coli cells, the spatial organization occurs by the cells themselves. This organization allowed the cells to increase the nutrient supply. It increased the efficiency of the cells ability to escape from the confined growth area. The study also indicated that the asymmetrical shape of the microbial species is significant in the biofilm’s organization. (Cho, et al., 2007)
The studies conducted also showed that the cells grew towards the exits (the cell’s escape path) connecting various chambers (areas of growth). This resulted in cellular relaxation – a reduction in cell stress.
The stress was postulated to be caused by the boundaries of the enclosed chambers or areas of growth. Cho, et al. (2007) indicated that the behavior of the biofilm cells mimicked the self-drive movement of a large crowd in a confined space. The growth appeared to show a bottleneck in the escape area, with a panic-like movement of the cells, blocking the exits. Think of it like a stampede. For microbes, however, only the relatively long cells have issues with their size versus the dimensions of the cavity exit. Understanding this concept allows one to identify an optimal cell length, so that the cells can undergo robust self-organization, but short enough to avoid the increased stress at confined exit spaces and potential block of the cell escape path. (Cho, et al., 2007)
The studies conducted by Cho’s team (Cho, et al., 2007) provided new potential mechanisms for the formation of complex biofilms. Interactions between the cells and the corresponding organization during the propagation and attachment phases are critical. Formation of biofilms are affected by the cell morphologies and the physical boundaries confining them. Cho, et al. (2007) indicated that this understanding may aid in the control, growth and treatment of biofilms.
Biofilm Attachment
The initial step for infection with some strains of Staphylococcus aureus in humans, has been shown to be the attachment of the organism to the surfaces of various materials, including both medical devices and human host tissues. When the organisms adhere, they advance to formation of biofilm. The microbes can express a variety of surface components – adhesive matrix molecules. They interact with the host proteins. The data shows that during the adherence phase, the organisms initially adhere to each other and then elaborate a biofilm. They have been able to identify some of the genes involved in these processes. (Atshan, et al., 2012)
Biofilm Growth
With the different species of microorganisms in the biofilm, the cells adapt and are able to increase their supply of nutrients allowing the cells to grow.
Detachment of Cell Clusters
Detachment of biofilm cells are a concern in pharmaceutical manufacturing as well as in clinical setting. However, it is one of the least studies areas of biofilm processes. New methods using digital time lapse microscopy along with fl ow cells designed for biofilm provided opportunities to study this mechanism of detachment. Detachment is comprised of two processes: erosion and sloughing (based upon the magnitude and frequency of the detachment event). Stoodley, et al. (2001) describes erosion as the continual detachment of single cells and small portion of the biofilm, without specifying size distribution and frequency of biofilm particulates. The authors describe sloughing as a rapid, massive loss of biofilm. (Stoodley, et al., 2001)
Using digital time-lapse microscopy, Stoodley, et al. (2001) was able to track single cells that were moving between biofilm clusters. They could also quantify the dynamic viscoelastic behavior and movement of the mature biofilms over solid surfaces in situ. The studies conducted showed a steady detachment of cell clusters over the monitoring periods. The quantity produced up to 3.6 x 109cfu/min.
The distributions appeared to resemble a Pareto distribution. In their data, they saw that the larger cell clusters detached less frequently, but they contained a disproportionally large proportion of the total detached biomass. (Stoodley, et al. (2001)
For pathogenic biofilm organisms (e.g., E. coli, Salmonella sp., and Listeria monocytogenes), the infectious dose could be as low as 10 cells of these organisms. Stoodley, et al. 2001), postulated that a single cluster that is detached may be enough to be infectious. If the same amount of microorganism is not dispersed, it may appear as a single colony-forming unit on a nutrient plate. They also postulated that the detached biofilm clusters can possess the protective properties, e.g., resistance to antibiotics and host resistance defense systems, as is shown with biofilms.
Stewart (1993) developed a specific mathematical framework for modeling biofilm detachment. This model showed that detachment is a growth-associated phenomenon. Detachment balances microbial growth. It determines how much biofilm is accumulated and the overall biofilm activity.
Bryers (1988) identified five types of detachment: erosion, sloughing, human intervention, predator grazing, and abrasion. The terms are defined as follows. Erosion is the continuous removal of individual cells, or small clusters of cells from the biofilm surface. Sloughing is a discrete process in which there is release of relatively large particles of biomass, typically greater in size that the thickness of the biofilm itself. The remaining three types of detachment are a result of external forces acting on the biofilm.
Prevalence of Biofilms
The United States National Institutes of Health, attribute biofilms responsible for 80% of the microbial infections in the human body. In fact, biofilms can affect every organ in the body, even skin. This has necessitated an increasing awareness of the proper diagnosis and management of biofilm-associated infections. (Anonymous, 2018c)
Scientists today realize that biofilms are a significant concern in both industrial and clinical settings. Malone, et al. (2017) published a systematic review of biofilms found in chronic patient wounds. Biofilms were found in 78.2% of the non-healing wounds studied. Cadalli-Tatar, et al. (2012) indicated that there is a prevalence of biofilms in patients that have chronic rhinosinusitis without polyps. Telang, et al. (2010) indicated an increase in antibiotic resistant microorganisms due to the presence of biofilms in some types of patients.
Conclusion
The emergence of many new technologies, not traditionally considered in microbiology, have allowed us to increase our understanding of biofilms and other microenvironments. As our knowledge increases, there is a potential to eliminate or better treat biofilms.
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