Important Biofilms in Clinical Microbiology Essay
Important Biofilms in Clinical Microbiology Essay
The microorganisms growing in these biofilms have become resistant to the antimicrobial agents. These biofilms are now found on the medical devices and this creates a problem. Many diseases such as cystic fibrosis and the valve endocarditis are associated with biofilms. (Donlan and Costerton 2002, Watnick 2000).
The presence of these microorganisms on the surface is determined by the colony forming units. This is the traditional method of determination of the adherent cells on the surface. The disadvantage of this method is that the CFU varies for the different methods of identification such as fluorescent micro scoping and electron microscopy. This method is widely used for the identification of the low concentration of the bacteria on the sea water, sewage water and on the earth sludge. Important Biofilms in Clinical Microbiology Essay. The evaluation of the samples using the scanning electron microscope is difficult because of the requirement of the high vacuum. And the three-dimensional structure visualization of the structure is limited here. The determination of the biofilms thickness, density, porosity, roughness, bio-volume is required for the evaluation and the quantification of the biofilms. (Hannig et. al 2010).
Biofilms are clinically important. More than 80% of the infections cause4d in our body are based on the biofilms. The major infections in our body includes infections in the soft tissues, middle ear, dental implants, urogenital tract, eye, urinary tract prostheses, infections on the peritoneal membrane and the peritoneal dialysis catheters, hemodialysis catheters , cardiac implants in our body such as pacemakers, ventricular assist devices and prosthetic heart valves, internal fixation devices, tracheal and ventilator tubing and percutaneous sutures. Though the non-mucosal layer resists the entry of the pathogens into the body, the bacteria have been found to adopt numerous strategies for the action against the antimicrobial. The scientists say many reasons for this antimicrobial activity such as efflux, biofilms formation and the cell wall permeability and sometimes the expression of the genes that are mediating the inactivating enzymes. Important Biofilms in Clinical Microbiology Essay.
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Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Important Biofilms in Clinical Microbiology Essay. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.
INTRODUCTION
Biofilms have been described in many systems since Van Leeuwenhoek examined the “animalcules” in the plaque on his own teeth in the seventeenth century, but the general theory of biofilm predominance was not promulgated until 1978 (37). This theory states that the majority of bacteria grow in matrix-enclosed biofilms adherent to surfaces in all nutrient-sufficient aquatic ecosystems and that these sessile bacterial cells differ profoundly from their planktonic (floating) counterparts (37). The data on which this theory is predicated came mostly from natural aquatic ecosystems, in which direct microscopic observations and direct quantitative recovery techniques showed unequivocally that more than 99.9% of the bacteria grow in biofilms on a wide variety of surfaces. This predominance of biofilms was established in all natural ecosystems except deep groundwater and abyssal oceans, and we now realize that these sessile populations account for most physiological processes in these ecosystems (40).
Because bacterial biofilms cause very serious problems in industrial water systems, the people who manage these systems have been the first to develop methods to sample sessile bacteria and develop strategies to control their costly depredations.Important Biofilms in Clinical Microbiology Essay. Biofilm samplers, which are fitted into the walls of industrial pipes and vessels, are now widely used in industrial systems, and the biocides used to protect industrial installations are routinely tested for their efficacy in killing sessile bacteria.
This consensus that bacteria grow preferentially in matrix-enclosed biofilms in natural and industrial systems was not immediately accepted in the medical and dental areas in spite of the universal acceptance of dental plaque as a type of biofilm. However, new methods for the direct examination of biofilms soon showed that the organisms that cause many device-related and other chronic infections actually grow in biofilms in or on these devices (39). Gradually, important intellectual syntheses began to be made.
Once we concede that bacteria lack a complex nervous system that could enable them to determine their location vis-à-vis the animal body, we deduce that they have certain basic survival strategies that they employ wherever they are. In natural and industrial systems, they form biofilms, within which they are protected from antibacterial chemicals (including natural antibiotics), environmental bacteriophages, and phagocytic amoebae. For these reasons, it should come as no surprise that chronic biofilm infections resist antibiotic therapy and are phenomenally resistant to host clearance mechanisms such as antibodies and phagocytes.
For many centuries humans have suffered from acute bacterial infections (e.g., plague), in which planktonic cells of specialized pathogens mounted life-threatening attacks on our bodies. We have countered with vaccines and antibiotics, and these acute diseases are now largely under some measure of control. Important Biofilms in Clinical Microbiology Essay. However, organisms that have been successful for millions of years in the environment (e.g., Pseudomonas and Legionellaspp.) are now mounting successful attacks on our health care facilities. Obviously, they make full use of the biofilm strategy that has protected them so well in their native habitats. Compromised individuals, who might not have survived in earlier times, are especially susceptible to this new cohort of “environmental” pathogens that have invaded our homes and schools just as they have invaded our hospitals.
BIOFILMS DEFINED
Our definition of biofilm has evolved over the last 25 years. Marshall in 1976 (129) noted the involvement of “very fine extracellular polymer fibrils” that anchored bacteria to surfaces. Costerton et al. (37) observed that communities of attached bacteria in aquatic systems were found to be encased in a “glycocalyx” matrix that was found to be polysaccharide in nature, and this matrix material was shown to mediate adhesion. Costerton et al., in 1987 (41), stated that biofilm consists of single cells and microcolonies, all embedded in a highly hydrated, predominantly anionic exopolymer matrix. Characklis and Marshall in 1990 (28) went on to describe other defining aspects of biofilms, such as the characteristics of spatial and temporal heterogeneity and involvement of inorganic or abiotic substances held together in the biofilm matrix.
Costerton et al., in 1995 (40), emphasized that biofilms could adhere to surfaces and interfaces and to each other, including in the definition microbial aggregates and floccules and adherent populations within pore spaces of porous media. Costerton and Lappin-Scott (38) at the same time stated that adhesion triggered expression of genes controlling production of bacterial components necessary for adhesion and biofilm formation, emphasizing that the process of biofilm formation was regulated by specific genes transcribed during initial cell attachment. Important Biofilms in Clinical Microbiology Essay. For example, in studies of Pseudomonas aeruginosa, Davies and Geesey (47) have shown that the gene (algC) controlling phosphomannomutase, involved in alginate (exopolysaccharide) synthesis, is upregulated within minutes of adhesion to a solid surface. Recent studies have shown that algD, algU, rpoS, and the genes controlling polyphosphokinase synthesis are all upregulated in biofilm formation and that as many as 45 genes differ in expression between sessile cells and their planktonic counterparts (E. Pulcini, J. Costerton, and K. Sauer, personal communication).
A new definition for biofilm must therefore take into consideration not only readily observable characteristics, i.e., cells irreversibly attached to a surface or interface, embedded in a matrix of extracellular polymeric substances which these cells have produced, and including the noncellular or abiotic components, but also other physiological attributes of these organisms, including such characteristics as altered growth rate and the fact that biofilm organisms transcribe genes that planktonic organisms do not.
The new definition of a biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription. This definition will be useful, because some bacterial populations that fulfilled the earlier criteria of a biofilm, which involved matrix formation and growth at a surface, did not actually assume the biofilm phenotype. These “nonbiofilm” populations, which include colonies of bacteria growing on the surface of agar, behave like planktonic cells “stranded” on a surface and exhibit none of the inherent resistance characteristics of true biofilms. We can now speak of biofilm cells within matrix-enclosed fragments that have broken off from a biofilm on a colonized medical device and now circulate in body fluids with all the resistance characteristics of the parent community. Important Biofilms in Clinical Microbiology Essay.
HOW MICROORGANISMS FORM BIOFILMS
Now that we concede that bacteria form biofilms in essentially the same manner in whatever ecosystem they inhabit, it is important that we take full advantage of the elegant studies of this process that fill the environmental and industrial microbiology literature. The scientific and engineering community has already examined biofilm formation in some detail and has published a couple of books (30, 113) on this subject. Many aspects of biofilm formation are counterintuitive, and it may be useful to summarize these issues, so that the medical community does not repeat this work.
Perhaps the first surprise, for the medical community, is that bacteria form biofilms preferentially in very high shear environments (i.e., rapidly flowing milieus). Planktonic bacteria can adhere to surfaces and initiate biofilm formation in the presence of shear forces that dwarf those of heart valves and exceed Reynolds numbers of 5,000 (30).Important Biofilms in Clinical Microbiology Essay. The Reynolds number is a dimensionless number describing the turbulent flow of a liquid; if this number is high, turbulent flow exists; if it is low, laminar flow conditions prevail. Engineers speculate that turbulent flow enhances bacterial adhesion and biofilm formation by impinging the planktonic cells on the surface, but whatever the mechanism, biofilms form preferentially at high-shear locations in natural and industrial systems.
Studies of bacterial adhesion with laboratory strains of bacteria, many of which had been transferred thousands of times and lost their ability to adhere, first indicated that very smooth surfaces might escape bacterial colonization. Subsequent studies with “wild” and fully adherent bacterial strains showed that smooth surfaces are colonized as easily as rough surfaces and that the physical characteristics of a surface influence bacterial adhesion to only a minor extent (40). Once a biofilm has formed and the exopolysaccharide matrix has been secreted by the sessile cells, the resultant structure is highly viscoelastic and behaves in a rubbery manner (197). When biofilms are formed in low-shear environments, they have a low tensile strength and break easily, but biofilms formed at high shear are remarkably strong and resistant to mechanical breakage.
BIOFILM EXAMINATION AND MEASUREMENT
Our understanding of biofilms has developed as the methods for biofilm examination and characterization have evolved. Much of the early investigative work on biofilms relied heavily on the scanning electron microscope. This technique utilizes graded solvents (alcohol, acetone, and xylene) to gradually dehydrate the specimen prior to examination, since water of hydration is not compatible with the vacuum used with the electron beam. This dehydration process results in significant sample distortion and artifacts; the extracellular polymeric substances, which are approximately 95% water (28), will appear more as fibers than as a thick gelatinous matrix surrounding the cells. Important Biofilms in Clinical Microbiology Essay.
The use of transmission electron microscopy and specific polysaccharide stains like ruthenium red allowed researchers both to identify the nature of these extracellular fibers in biofilms and to better elucidate their association with the cells. Electron microscopy has been used for the examination and characterization of biofilms on medical devices (160, 187) and in human infections (66, 147). Because of its excellent resolution properties, the electron microscope will, in spite of its limitations, continue to be an important tool for the biofilm scientist. Figure 1 shows a typical scanning electron microscope image of a biofilm.
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Scanning electron micrograph of a biofilm on a metal surface from an industrial water system.
The development of the confocal laser scanning microscope (CLSM) in the 1980s provided researchers with the ability to examine biofilms in situ without the limitations encountered with the scanning electron microscope, albeit at lower magnifications. The trade-off in resolution was more than offset by the ability to examine the biofilm matrix unaltered and intact.
The use of both CLSM and epifluorescence microscopy requires that the organisms in the biofilms be stained with fluorescent stains. These stains are designed to emit light at specific wavelengths and can be used to probe specific cellular functions. For example, nucleic acid stains such as DAPI (4′,6′-diamidino-2-phenylindole), acridine orange, and Syto 9 will stain the DNA and RNA of all cells regardless of their viability. Other stains have been developed for probing cell viability.Important Biofilms in Clinical Microbiology Essay. Propidium iodide is taken up only by cells with damaged cytoplasmic membranes, and 5-cyano-2,3-ditolyl tetrazolium chloride is taken up and reduced to 5-cyano-2,3-ditolyl tetrazolium chloride-formazan only by cells that have a functioning cytochrome system. Using a suite of such stains allows the biofilm researcher to quantify all the cells and determine which ones are viable.
Fluorescent antisera and fluorescent in situ hybridization probes may enable us to identify specific organisms within a mixed biofilm community. Green fluorescent protein, a constitutively produced, plasmid-mediated molecule, can allow biofilms to be examined noninvasively, without fixation or staining (18). A confocal laser scanning microscopic image of a biofilm is shown in Fig. 2.
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Confocal laser scanning micrograph of a biofilm, showing cell clusters and water channels. Reproduced with the permission of Paul Stoodley.
In more common use are techniques that rely on removal of the biofilms or biofilm-associated organisms from the substratum by some type of mechanical force, such as vortexing or sonication, prior to examination and measurement. The most commonly used procedure for measurement of biofilms is the viable plate count procedure, in which the resuspended and dispersed biofilm cells are plated onto a solid microbiological medium, incubated, and counted.
Table 1 lists several of the methods that have been used by clinical microbiologists for the recovery and measurement of clinically relevant biofilms on indwelling medical devices. For most of these techniques, a determination of the recovery efficiency of the method (i.e., the percentage of cells that are actually recovered from the biofilm) is needed.Important Biofilms in Clinical Microbiology Essay. Methods that allow a determination of biofilm cell count in the implanted device without necessitating device removal, such as the endoluminal brush technique, could provide a distinct advantage for the clinical practitioner, potentially alleviating the need for device removal when the device is found not to contain intraluminal biofilms. These methods all rely on the quantification of biofilm cells as a measurement of total biofilm accumulation. Other methods have been used by biofilm researchers for measuring biofilms, including total protein (139), absorbance at either 550 nm (88) or 950 nm (201), tryptophan fluorescence (4), endotoxin (164), and total ATP (R. W. Walter and L. M. Cooke, paper no. 410, presented at the National Association of Corrosion Engineers Annual Conference, 1997). Any of these methods could be investigated for the measurement of clinically relevant biofilms. Important Biofilms in Clinical Microbiology Essay.
