Microbial Biofilms Issues - Essay Example

Assessment of the microbial biofilms begins with first and foremost sourcing and or culturing them. These can be observed in situ or can be cultured for further studies and experimentation. Caves serve as a very lucrative and accessible natural source for biofilm observation. Another natural source is dental plaque. The microbial biofilms can be grown in PVC microtiter plates and stained with crystal violet for invitro assessment in simplest measures as desThese could be cultured in laminar flows or alternatively turbulent flow systems. The continuous culture systems chemostat and turbidostat could also be used to culture the biofilms. The type of environment they are grown in, effects their ultimate morphological, clustering and adhesion character. For Visualization, traditionally electron microscopy was the method of choice to examine microbial biofilms under high resolution. Unfortunately, sample preparation for electron microscopy results in dehydrated samples. Consequently, this approach provided a deceivingly simplistic view of biofilms, since the biofilm collapsed when water was removed. (Davey and Ootle, 2000) Phase microscopy was also an alternative to visualization of living biofilms. Biofilms are removed and either directly visualised or fixed and stained prior to examination. Several techniques for microscopy examination of biofilms in situ on the substrata supporting their growth have been used in this study. These have included transmission electron microscopy (TEM), scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), episcopic differential interference contrast microscopy (DIC) with and without fluorescence, Hoffman modulation contrast microscopy (HMC), atomic force microscopy (AFM). Cross-sections prepared for TEM analysis gives useful information about the spatial relationships of microorganisms within the biofilm matrix, whilst SEM enables the surface topology of the biofilms to be examined at a high magnification. The preparation required for TEM and SEM, may however, result in the inclusion of artefacts. ESEM and AFM allow direct visualisation of intact hydrated specimens at high magnification. AFM images may be rotated and manipulated to provide accurate measurement of individual microorganisms with relative ease. Light microscopy techniques, although unable to reproduce the high magnification of the methods described above, are still of importance in the examination of intact biofilms. HMC allows the in situ examination of biofilms, a clear image is produced without artefacts. DIC may be used to examine biofilms on opaque surfaces and if used in conjunction with fluorescent vital stains can be used to assess the viability of the microbial population. (Surman et al, 1996) Viability of the microbial biofilm populations can also be determined by LIVE/DEAD BacLight bacterial viability staining kit (Hentzer 2001) The application of confocal scanning laser microscopes (CSLM) to biofilm research radically altered our perception of biofilm structure and function (Laurence 1991). Before the use of CSLM, electron microscopy was the method of choice to examine microbial biofilms under high resolution. Unfortunately, sample preparation for electron microscopy results in dehydrated samples. Consequently, this approach provided a deceivingly simplistic view of biofilms, since the biofilm collapsed when water was removed. On the other hand, CSLM, which allows the visualization of fully hydrated samples, has revealed the elaborate three-dimensional structure of biofilms (Costerton 1995). CSLM is used very effectively to monitor Microbial Biofilms. It is used to investigate not only the presence and the viability of the biofilm consortium but also biofilm/substrata interactions. CSLM has been used very effectively to monitor biofilm development in flow cells. Flow cells are small continuous-flow systems with a viewing port that allows direct observation of the biofilm without disrupting the community. These systems are often once-flow, meaning that fresh medium enters the system, passes through the cell, and is collected as waste-the medium is not recycled through the flow cell. A number of descriptions of flow cell and related techniques have been reported. (Davey and Otoole 2000) For better visualization, these biofilms can also be tagged with autofluorescent proteins. When autofluorescent proteins (AFPs), such as green fluorescent protein (GFP) and Discosoma striata red fluorescent protein (DsRed), are excited with light of a specific wavelength, they emit light of a longer wavelength, without the further addition of substrates. Furthermore, the integration of AFP reporters with other markers and technologies is facilitating a systems approach to research in microbial ecology. (Larrainzar et al 2005) Major pathogens have developed a variety of strategies with which they adapt their genetic expression to meet the challenges of their ever-changing surrounding environment, e.g. within the host cell. These include specific sigma factors, two-component systems, repressors, positive regulators, as well as small regulatory RNAs. Biofilms is hence nothing but a logical mechanism adopted by the microbial flora to adapt to the culture conditions. Studies of these metabolic interactions and adaptations in both the bacteria and the host cell will lead to a better understanding of the mutual reactions in the course of infection. The aim is to carry out an extensive analysis of the gene activity of infected hosts and microbes during the course of infection. To identify genes which are typically expressed in biofilms, a micro-representational-difference analysis (micro-RDA) can be adopted. It is better from the c DNA RDA protocol in that minimal quantities of RNA are required and RNA need not be be fractionated Total RNA including Rrna fraction can be used for this study.(Becker et al 2001). Initial phylogenetic screening with 16S rRNA gene sequences can identified the diverse community with lineages across the breadth of the Bacteria. In growth limiting environments, the adaptive study of these biofilm interactions can lead to interesting observations like selfish competition for resources is replaced by cooperative and mutualistic associations as was observed by Barton and Jurado. DNA Microarray studies can be used to analyze large samples of biofilm microbial data for example from oral samples. The exopolymeric matrix was identified by histochemistry using the wheat germ agglutinin lectin method (Romero et al 2009). Studies on regulatory networks involved in the production of virulence factors and survival of pathogens in vitro and within the host will be accomplished by whole genome expression profiling using DNA arrays, expression studies and reporter gene fusions. Random mutagenesis or overexpression of genes will also be used in combination with proteome analysis. Furthermore, protein-protein interaction mapping can be undertaken as well to define the structure of complex regulators and regulons. Determination Biofilm of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay required for antibiotic susceptibility testing.antibiotic Adherent bacterial populations(biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as that planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of assay biofilm susceptibilities to antibiotics. The CBD produces 96 equivalen biofilms for the assay of antibiotic susceptibilities by the standard 96-well grown technology. The CBD offers a new technologyfor the rational selection of antibiotics effective antibiotic agains tmicrobial biofilms and for the screening of new effective antibiotic compounds. (Ceri et al 2005) Northern Blotting analysis to study differential patterns of gene expression in sessile cells as compared with their planktonic counterparts may represent an alternative mechanism responsible for the resistance properties associated with biofilm formation. (Ramage et al 2002) Horizontal gene transfer is defined to be the movement of genetic material between bacteria other than by descent in which information travels through the generations as the cell divides. It is most often thought of as a sexual process that requires a mechanism for the mobilization of chromosomal DNA among bacterial cells. Horizontal Gene Transfer can be monitored by confocal electronic microscopy. Plasmid horizontal transfer is assessed by including plasmids with RFP and other fluorescent markers. The overlap of these markers gives a distinct colour in the confocal microscopy as determined by Sorenson et al (2005) Metagenomics, or the culture-independent genomic analysis of an assemblage of microorganisms, has potential to answer fundamental questions in microbial ecology. Metagenomics include integrating statistical analysis of different studies. Its biggest advantage is its culture free analysis of microorganisms. Metagenomics includes on site cell lysis and nucleic acid extraction. Genome enrichment is done by Stable Isotope Probing (SIP). It enhances the screening of metagenomic libraries for a particular gene of interest, the proportion of which is generally smaller than the total nucleic acid content. RT-PCR is used to monitor environmental sequences as RNA markers are more sensitive than DNA markers. This is followed by metagenomic sequencing. The objectives have been to sequence and identify the thousands of viral and prokaryotic genomes as well as lower eukaryotic species present in small environmental samples such as a gram of soil or liter of seawater. References Djordjevic, Wiedmann, Landsborough (2002) Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation . American Society for Microbiology. 68(6): 2950-2958 Studying plasmid horizontal transfer in situ: a critical review Sren J. Srensen, Mark Bailey, Lars H. Hansen, Niels Kroer and Stefan Wuertz Nature Reviews Microbiology 3, 700-710 (September 2005) Hentzer Mortan et al (2001) Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function. Vol. 183. p. 5395-5401 Davey Mary and O'Toole George Microbial Biofilms: from Ecology to Molecular. Genetics. Microbiology and Molecular Biology Reviews. Vol 64. P.847-867 Surman S.B., Walker J.T., Goddard D.T., Morton L.H.G., Keevil C.W., Weaver W., Skinner A., (...), Kurtz J. (1996) Comparison of microscope techniques for the examination of biofilms Journal of Microbiological Methods,25(1),pp.57-70. Barton and Jurado Whats up down there Microbial Diversity in Caves http://quest.nasa.gov/projects/spacewardbound/docs/lifeincaves.pdf Romero Roberto Lawrence, J. R., D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell. 1991. Optical sectioning of microbial biofilms. J. Bacteriol. 173: 6558-6567. Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber, and H. M. Lappin-Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711-745. Danese, P. N., L. A. Pratt, and R. Kolter. 2000. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J. Bacteriol. 182:3593-3596. Read More