Biofilm Formation Exercise 23 PDF

w V\ u er: w X w BIOFILM FORMATION LEARNING OBJECTIVES------------ 1. Define a biofilm and describe where and under what conditions biofilms form. 2. Outline the steps of biofilm formation, including critical structures and substances involve...

w V\ u er: w X w BIOFILM FORMATION LEARNING OBJECTIVES------------ 1. Define a biofilm and describe where and under what conditions biofilms form. 2. Outline the steps of biofilm formation, including critical structures and substances involved in this process. 3. Provide examples of both positive and negative consequences of biofilm formation. 4. Explain what happens during quorum sensing. 5. Compare and contrast the three main classes of quorum sensing systems. 6. Describe and explain what occurs during each step of the biofilm assay. 7. Perform a biofilm assay with the environmental isolate. 8. Identify possible sources of error in the biofilm assay and suggest appropriate solutions. 9. Correlate biofilm data with data from previous experiments and predict the type of quorum system present in the environmental isolate. INTRODUCTION--------------- Although the microbial cultures that have been provided to you so far in this course have,for the most part, been pure cultures of a single species of microorganism, microbes in nature rarely, if ever, exist as pure cultures. Most organisms live in communities that form on both living and nonliving surfaces where water and nutrients are plentiful, and these communities are known as biofilms. Microbial biofilms are ubiquitous; they are found within toilet bowls, water pipes, on rocks in creeks and streams, and on the teeth of humans and animals. They can be mechanically removed by scrubbing, but reappear quickly unless the water source is removed. Biofilms consist of all types of microbes, including bacteria,fungi, algae, and proto- zoa, and these species communicate with each other and can differentiate into separate sub- populations that fulfill different functions within the biofilm. Biofilms form when free-floating (planktonic) forms of microbes arrive at a surface and loosely attach. This step is known as the reversible attachment stage and it is influenced by temperature, pH, and other factors. After a period of time, some organisms enter the irrevers- ible attachment stage. Irreversible attachment depends on the presence of adhesins, various types of fimbriae, and culi (a protein produced by many members of the Enterobacteriaceae). Soon afterwards, organisms begin to reproduce and produce microcolonies. During biofilm maturation, the organisms produce an extracellular polymeric substance (EPS) which is composed of sug- ars, proteins, and nucleic acids. The EPS is secreted to the exterior of the cells and it allows the cells to begin sticking together. The numbers of organisms within the biofilm continue to grow as the organisms multiply and the biofilm begins to develop into a complex, three- dimensional structure which may be as thin as a few cell layers or as thick as several inches. The final step in biofilm development is transmission. In transmission, the biofilm propa- gates through the dispersion of clumps or individual cells. Biofilm communities exhibit behaviors and survival strategies that are markedly different from those exhibited by individual planktonic cells. Interestingly, planktonic and biofilm forms of a single organism may express different genes. In a process known as quorum sens- ing (OS), the cells within the biofilm respond in a density-dependent manner to environmen- tal signals. Signal molecules known as autoinducers (Als), which are produced by a few mem- bers of the biofilm, can be present in high concentrations within the biofilm. This occurs because the EPS prevents the Als from diffusing away from the biofilm. Once a threshold concentration of the Al has accumulated, transcription of certain genes may be activated. OS as a method of communication has been demonstrated to occur both between cells of the same species, but also between cells of different species. QS contributes to the develop- ment of many types of symbiotic relationships, the formation of endospores, competence (in bacterial transformation), apoptosis (programmed cell death), and the virulence of many different organisms The importance of biofilms has become much more apparent in recent years. Biofilms can tolerate antibiotic doses up to 1000 times greater than therapeutic doses which kill plank- tonic cells. A reevaluation of antibiotic doses may be required as all previous studies study- ing the effects of antibiotics on bacteria were performed using planktonic cells. Because of their resistance to antibiotics, their ability to disseminate, and their protection from the immune system, biofilms are thought to play a role in some chronic human infections. Examples include recurrent middle ear and sinus infections, diabetic foot ulcers, stomach ulcers, and recurrent pneumonia in patients with cystic fibrosis. Not only do biofilms affect human health, but they also affect natural environments. They can form the basis for food webs in aquatic systems, they can participate in bioremediation by breaking down contaminants bound to soil particles, and they are found in many plant-microbe symbiotic relation- ships. The formation of biofilms also affects most every water- based process and distribution system in the world, including pulp and paper manufacturing processes as well as air conditioning cooling towers. Biofilms in these environments can lead to the blocking of water disribution pipes, pipe corrosion, and general water contamination issues. In QS, transcription of a gene is induced by the interaction of a bacterially- produced signal molecule with a transcriptional activator. The three main classes of QS systems in bacteria are as follows: (1) Luxl/LuxR-type in gram-negative bacteria which use acyl-homoserine lactones (AHLs) as the signal molecule; (2) two- component-type systems found in gram-positive bacteria which use small oligopeptides as signal molecules; and (3) those found in both gram-negative and gram-positive bacteria that use the luxS-encoded auto inducer-2 (Al-2). One of ° the best-studied examples of Luxl/LuxR-type QS systems is the colonization of the Hawaiian bobtail squid, Euprymna scolopoes, by biofilms of the gram-negative bacterium, Aliivibrio fischeri. Research has now shown that hundreds of gram- negative bacteria use this class of QS to control a wide variety of cellular processes, but each species produces a unique AHL. Thus, signaling in this class of QS, as well as that which occurs in the two-component oligopeptide class, are intraspecies only. Only the Al-2 system allows for interspecies communication between gram-negative and gram-positive bacteria. The purpose of today's experiment is to investigate the ability of the environmental isolates to form biofilms on an abiotic surface. Each student's environmental isolate will be grown in triplicate for 48 hours in a 96-well microtiter plate. Planktonic cells will be removed through washing and cells that remain attached to the wells of the plate will be stained with a dye. Following the removal of the dye and washing of the wells, the plate will be dried. An indirect quantitative assessment of biofilm formation will be performed by solubilizing the surface- attached dye and reading absorbance of individual wells at 0D595 on a plate reader.