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Microbiology Exam 4 - Summary Brock Biology of Microorganisms
Microbiology for Health Professionals (BIOL2031C)
University of Cincinnati
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Microbiology Exam 4 Ch. 19 Taking the Measure of Microbial Systems Microbial Ecology is focused on how microbial populations assemble to form communities and how these communities interact with each other and their environments To study biodiversity, microbial ecologists must identify and quantify microorganisms in their habitats To study microbial activity, microbial ecologists must measure the metabolic processes that microorganisms carry out in their habitats More than of all microorganism species most estimates, have never been grown in lab cultures o Recognition of this fact, based on molecular diversity surveys of various microbial habitats, has stimulated the development of new methods for isolating microbes from nature in order to establish pure cultures One way to isolate microorganisms is the Enrichment Approach o A and useful method for isolating microorganisms from nature but one with limitations Enrichment is based on culturing in a selective growth medium, and thus the tools and methods used in this approach are considered culturedependent analyses 19 Enrichment Culture Microbiology For an enrichment culture, a medium and a set of incubation conditions are established that are SELECTIVE for the desired organism and COUNTERSELECTIVE for the undesired organisms o Enrichment cultures duplicate as closely as possible the resources and conditions of a particular ecological niche Inocula Enrichment cultures are established placing the inoculum (such as pond water) into selective media and incubating under specific conditions o In this way, many many common microbes can be isolated Example: Martinus Beijerinck (who conceptualized the enrichment culture technique, used enrichment cultures to isolate the bacterium Azotobacter Because Azotobacter is a rapidly growing bacterium capable of N2 fixation in air, enrichment using media devoid of fixed nitrogen, such as ammonia or nitrate, and incubation in air selects STRONGLY for this bacterium and its close relatives bacteria and anaerobic bacteria are counterselected in this technique Enrichment Culture Outcomes The isolation of the desired organism from an e. says nothing about the ecological importance or abundance of the organism in its habitat o A positive enrichment proves only that the organism was present in the sample The Winogradsky Column An artificial microbial ecosystem and a source of various bacteria for enrichment culture Prepared filling a glass cylinder about half full with ORGANICALLY rich, sulfuric mud into which carbon substrates are mixed o The substrates determine which organisms are enriched o Fermentative substrates, such as glucose, that can lead to acidic conditions and excessive gas formation (which can create gas pockets that disrupt the enrichment and let in air) are avoided o The mud is supplemented with small amounts of calcium carbonate (CaCO3) as a buffer and gypsum (CaSO4) as a source of sulfate o The mud is packed tightly in the cylinder, taking care to avoid trapping air, and then covered with pond water o The top of the cylinder is covered to prevent evaporation, and the container is placed near a window that receives diffuse sunlight for a period of months A diverse community of microbes develop o Algae and cyanobacteria develop quickly in the upper portions of the water producing O2 these organisms help to keep this zone of the column OXIC much as they do in the upper zones of a lake o Fermentative processes in the mud lead to the production of organic acids, alcohols, and H2, all suitable substrates for bacteria Hydrogen sulfide (H2S) from the sulfate reducers triggers the development of purple and green sulfur bacteria (anoxygenic phototrophs) that use sulfide as a PHOTOSYNTHETIC ELECTRON DONOR These organisms typically grow in patches in the mud on the sides of the column but may bloom in water itself if oxygenic phototrophs are scarce Besides supplying a ready source of inocula for enrichment cultures, columns can also be supplemented with a specific compound to enrich an organism in the inoculum that can degrade it o Once a crude enrichment has been established in the column, culture media can be inoculated for the isolation of pure cultures 19 Classical Procedures for Isolating Microbes Once a positive enrichment culture has been obtained, the next step is to attempt to get the enriched organism in pure culture Agar Dilution Tubes and the Technique In the agar dilution tube method, a mixed culture is diluted in tubes of molten agar medium, resulting in colonies embedded in the agar o This method is useful for purifying anaerobic organisms such as phototrophic sulfur bacteria and bacteria from samples taken from Winogradsky columns or other sources Another purification procedure is the serial dilution of an inoculum in a LIQUID MEDIUM until the final tube in the series shows no growth properties, offering the experimenter the capability to monitor several species simultaneously One drawback to the use of GFP is that to become fluorescent it requires O2, and thus it is NOT SUITABLE for tracking cells introduced into strictly anoxic habitats o However, fluorescent proteins that DO NOT require O2 are available to overcome this limitation These proteins are derived from bacterial and plant photosensory flavoproteins and are more thermally stable than the GFP, making them useful for tracking mildly thermophilic species Limitations of Microscopy None of them reveal the phylogenetic diversity of the microorganisms in the habitat under study o Powerful staining methods can reveal the phylogeny of organisms observed in a natural sample 19 Fluorescence In Situ Hybridization (FISH) Nucleic acid probe a DNA or RNA oligonucleotide complementary to a sequence in a target gene or when the probe and the target come together they hybridize o Nucleic acid probes can be made fluorescent attaching fluorescent dyes to them o The fluorescent probes can often be used to identify organisms that contain a nucleic acid sequence complementary to the probe (this is FISH) Phylogenetic Identification Using FISH Phylogenetic stains penetrate cells without lysing them and hybridize with rRNA directly in the ribosomes o The number of fluorescent probes bound to a cell reflects the number of its ribosomes As single microbial cells can contain tens of thousands of ribosomes, strong signals can be achieved 19 PCR Methods of Microbial Community Analysis rRNA genes are universal and contain several regions of high sequence conservation, it is possible to amplify them from all organisms using only a few different PCR primers, even though the organisms may be phylogenetically distantly related Genes such as those encoding rRNAs that have retained ancestral function while changing in sequence over time as species have diverged are called orthologs o Organisms that share the same or very closely related orthologous genes are called a phylotype Phylotype is widely used to describe the microbial diversity of a habitat based solely on nucleic acid sequences It is only when additional physiological and genetic information becomes available, typically after the organism is brought into laboratory culture, that proposing a genus and species name for a phylotype becomes possible 19 Environmental Genomics and Related Methods Metagenomics the molecular study of microbial communities o all genes in a given microbial community can be sampled, and if the experiment is properly designed, the information obtained can support a much deeper understanding of the structure and function of the community than can singlegene analysis Critical importance in evaluating a genome reconstructed from metagenomic DNA is to assess whether it contains all of the genes required a cell (for EX: all necessary tRNA and rRNA genes and genes encoding essential proteins such as DNA and RNA polymerases) and is therefore a legitimate genome candidate Currently no methods are available for translating metagenomic sequence data into fundamental physiological information about the microbial community, such as the maximum specific growth rate of different species, their saturation constants for nutrients, their optimum, minimum, and maximum pH or temperature for growth, or their speed of recovery from starvation o These realities once again underscore why it is important to culture new microbes from at present, there is no substitute for characterization to define many critical aspects of a functional biology Ch. 20 Microbial Ecosystems 20 General Ecological Concepts ecosystem a dynamic complex of plant, animal, and microbial communities and their abiotic surroundings, all of which interact as a functional unit o an ecosystem contains many different habitats, parts of the ecosystem best suited to one or a few populations o Some ecosystems are mostly or even exclusively microbial o Depending on several factors, microbial activities in an ecosystem can have minimal or profound impacts and can diminish or enhance the activities of both the microorganisms themselves and the macroorganisms that may coexist with them Species Diversity in Microbial Habitats Population a group of microorganisms of the SAME SPECIES that reside in the same place at the same time Community consists of populations of one species living in association with populations of one or more other species o The microbial species that reside in a certain habitat are those best able to grow with the nutrients and conditions that prevail there Diversity of microbial species in a community can be expressed in 2 ways: o Species richness the total NUMBER of different species can be expressed their diversity of phylotypes observed in a given community o Species abundance the PROPORTION of each species in the community The microbial species richness and abundance of a community are functions of the conditions that PREVAIL and the kinds and amounts of nutrients available in the habitat EX: in undisturbed soils, high species richness is common, with most species present at only moderate abundance Nutrient levels and Growth Rates Resources typically enter an ecosystem intermittently A large pulse of nutrients ex: carcass of a dead animal can be followed a period of nutrient deprivation o Microorganism thus face the existence It is common for them to produce storage polymers as reserve materials when resources are abundant and draw upon these reserves in period of starvation Extended periods of exponential microbial growth in nature are probably rare o Microorganisms typically grow in spurts, linked closely to the availability and types of resources o Because all relevant physicochemical conditions in nature are RARELY OPTIMAL for microbial growth at the same time, growth rates of microorganisms in nature are usually well below the maximum growth rates recorded in the lab Microbial Competition and Cooperation Competition among microorganisms for resources in a habitat may be intense, with the outcome dependent on several factors, including rate of nutrient uptake, inherent metabolic rates, and ultimately, growth rates Syntrophy process where microbes cooperate together to carry out transformations that neither can accomplish alone o These microbial partnerships are important for anoxic carbon cycling 20 Surfaces and Biofilms Surfaces are important microbial habitats, typically offering greater access to nutrients, protection for predation and physicochemical disturbances, and a means for cells to remain in a favorable habitat, modify the habitat from their own activities, and not be washed away o Moreover, flow across a colonized surface increases transport of nutrients to the surface, providing MORE RESOURCS that are available to planktonic cells (floating) in the same environment Microbial mats accumulations that often contain highly complex yet stable assemblages of phototrophic, chemolithotrophic, autotrophic and heterotrophic microbes Biofilms Assemblages of bacterial cells attached to a surface and enclosed in an adhesive matrix that is the product of excretion cells and cell death o The matrix is typically a mixture of polysaccharides, proteins and nucleic acids that bind the cells together o They trap the nutrients for microbial growth and help prevent the detachment of cells on dynamic surfaces, such as in flowing systems Biofilms are functional and growing microbial communities and not just cells trapped in a sticky matrix Biofilm microbial communities have inherent tolerance to ANTIBIOTICS and other antimicrobial chemicals o Reasons for this greater tolerance include slower growth rates in biofilms, reduced penetration of antimicrobial substances through the extracellular matrix, and the expression of genes that INCREASE tolerance to stress This explains why biofilms are responsible for many untreatable or chronic infections 20 Microbial Mats Macroscopically layers of different bacteria Supported phototrophic or chemolithotrophic autotroph bacteria The combination of microbial metabolism and nutrient transport controlled diffusion results in steep concentration gradients of different microbial nutrients and metabolites, creating unique niches at different depth intervals in the mats Cyanobacterial mats are primarily phototrophs that use light energy to synthesize new organic material from CO2 COMPLEMENTARY (as one uses waste other is used as nutrient) Ch.23 The Nodule Symbiosis mutualism of great importance to humans is that of leguminous plants and bacteria o The ability of legumes to grow without added nitrogen saves farmers millions of dollars in fertilizer costs yearly and reduces the polluting effects of fertilizer runoff Most bacterial symbionts of plants are called RHIZOBIA o Species of rhizobia are or Betaproteobacteria Infection of legume roots rhizobia leads to the formation of root nodules in which the bacteria fix gaseous Nitrogen (N2) o Nodulated legumes can grow well on unfertilized bare soils that are nitrogen deficient, while other plants grow only poorly on them Leghomoglobin and Groups In the absence of of its bacterial symbiont, a legume cannot fix N2 o RHIZOBIA can fix N2 when grown in pure culture under MICROAEROPHILIC conditions (due to nitrogenase getting inactivated from high levels of O2) o In the nodule, O2 levels are precisely controlled the protein leghemoglobin Leghemoglobin protein) production is INDUCED through the interaction of the plant and bacterial partners Functions as an cycling between the oxidized and reduced forms of iron to supply sufficient O2 for BACTERIAL RESPIRATION while keeping unbound O2 within the nodule low A group of related legumes that can be infected a particular rhizobial species is called a group o Each group consists of all the legume species that will develop nodules when inoculated with RHIZOBIA obtained from any other legume of the group o If legumes are inoculated with the correct rhizobial strain, nodules develop on their roots with the ingested material and thus a more complete degradation of the plant polymers Foregut versus Hindgut Fermenters Foregut fermentation (rumen) the microbial fermentation chamber PRECEDES the small intestine o Found in colobine monkeys, sloths and mcropod marsupials Hindgut Fermentation (caecum) they have only one stomach but use an organ called a cecum where microbial breakdown of fiber is done o Found in horses and rabbits o Foregut fermenters have an advantage over hindgut fermenters in that the cellulolytic microbial community of the foregut eventually passes through an acidic stomach As this occurs, most microbial cells are killed the acidity and become a protein source for animals 23 The Rumen and Ruminant Animals Ruminants have rumens which is an organ within which cellulose and other plant polysaccharides are digested microorganisms o Cows, sheep, goats Rumen function: constant temperature (39), narrow pH range and ANOXIC environment Microbial Fermentation in the Rumen Hay (has cellulose) is broken down to sugars which is bacterial fermented with the production of volatile fatty acids (VFAs), primarily acetic, propionic and butyric acids, and the gases CO2 and methane (CH4) o The VFAs pass through the rumen wall into the bloodstream and are OXIDIZED the animal as its main source of energy o The gaseous fermentation products (CO2 CH4) are released ERUCTATION (belching) Rumen bacteria are Fibrobacter succinogenes and Ruminococcus albus o F. succinogenes GN R. albus GP Ch.24 Overview of the Human Microbiome The sites of the human body inhabited microorganisms include the mouth, nasal cavities, throat, stomach. Intestines, urogenital tracts, and skin Future Benefits of Knowing the Human Microbiome This includes: o The development of biomarkers for predicting predisposition to specific diseases o The design of therapies targeting selected microbial species in particular body sites o personalized drug therapies o probiotics Microbial association with health or disease states are for the most part only CORRELATIONS, and a CAUSAL relationship between microbiome and the health status of the host has been well established in only a few instances QUESTIONS TO PONDER: o Do individuals share a core human microbiome? o Is there a correlation between the composition of microbiota colonizing a body site and host genotype? o Do differences in the human microbiome correlate with difference in human health? o Are differences in the relative abundance of specific bacterial populations important to either health or disease? 24 Gastrointestinal Microbiota Monogastric mammals (omnivorous humans) have only the stomach positioned before the intestines The tract has about 400 m2 of surface area and is home to a total of about 1013 microbial cells The stomach is home of a vibrant bacterial community with hundreds of phylotypes distributed between the gastric lumen (pH and the mucus layer of the wall (pH o Dominated Baceteroidetes (Prevotella) Firmicutes (Streptococcus, Veillonella, Lactobacillus) Actinobacteria Fusobacteria Proteobacteria H. pylori accounts for vast majority of stomach microbial biomass Pathogenic proteobacterium is typically transmitted orally between family members and can persist for decades in the gastric mucosa Chronic imflammation is a major risk factor for the development of ulcers and gastric malignancies Down to the small and large intestines, it becomes greatly anoxic environment and pH becomes LESS acidic o Duodenum acidic pH o Jejunum transition to neutral pH o Ileum transition to anoxia o Cecum transition to anoxia o Colon (strictly anoxic conditions) high of bacteria o Fusiform anaerobic bacteria is typically present Large Intestine In the colon, Bacteria are present in enormous numbers and large numbers of Archaea (methanogens) Facultative anaerobes consume any remaining oxygen, rendering the l. to be STRICTLY ANOXIC o As the air passed into the lower tract, the flow rate DECREASES, and organisms SETTLE onto the walls of the respiratory passages The walls of the entire respiratory tract are lined with ciliated epithelial cells (CILIARY ESCALATOR) and the cilia, beating upwards, push bacteria and other particulate matter toward the upper tract where they are then expelled in saliva and nasal secretions or are swallowed 24 Urogenital Tracts and Their Microbes Kidneys and bladder are sterile in healthy adults o However, epithelial cells lining the distal urethra are colonized facultatively aerobic GRAM NEGATIVE bacteria Proteus can be a urinary tract pathogen This is strong urease it generates ammonia from urea and uses the ammonia as a nitrogen source o However, ammonia also causes urine pH to become ALKALINE, and this can trigger other urinary tract conditions such as formation of KIDNEY STONES The vagina of adult females is weakly acidic (pH 5) and contains significant amounts of glycogen o Lactobacillus acidophilus ferments polysaccharide glycogen, producing lactic acid that maintains a local acidic environment Before puberty, L. acidophilus is ABSENT, the female vagina is NEUTRAL in pH and DOES NOT produce glycogen After menopause, glycogen production ceases, the pH rises, and the microbiota again resembles that found before puberty o The microbiota is not diverse in vagina since (in healthy female) it is dominated lactobacilli 24 The Skin and Its Microbes 1010 skin microorganisms covering the average adult o Although these numbers are much lower than the oral and gut communities, molecular analyses have shown that the skin harbors a diverse microbial community of bacteria and fungi (primarily YEAST) that vary significantly with location on the body as a function of the diversity of habitats These habitats consist of microenvironments of varying temperature, pH, moisture, sebum content and surface characteristics MOIST: Corynebacteria, Staphylococci DRY: Flavobacteria, Corynebacteria OILY: Propionibacteria, corynebacteria Sweat is high in salt and other antimicrobial substances such as free fatty acids and antimicrobial peptides and thus play a role in controlling diversity 24 Disorders Attributed to the Gut Microbiota Diseases include: obesity, type 2 diabetes, asthma, atopic dermatitis, liver disease, colorectal cancer, kidney stones, psoriasis, tooth decay and periodontitis The failure for immune cells to develop tolerance to the normal microbiota early in life is associated with different diseases, including allergies and chronic inflammation of the gut such as inflammatory bowel disease (IBD) o IBD is NOT caused a specific pathogenic microbe but rather an IMBALANCE between the immune system and the normal gut microbiota Antibiotics in early life increases the risk of IBD o IBD may follow the disruption of mucosal barrier integrity (leaky gut) a gut pathogen, permitting commensal bacteria to interact with and activate the adaptive immune this stimulates the proliferation and differentiation of T cells into effector cells that can persist in the intestine long after resolution of the infection The Role of the Gut Microbiota in Obesity: Mouse Models Normal mice had more total body fat than those raised under conditions, although both mouse populations were fed the same amount type of food o Studies of experimental colonization of mice with individual microbial species have demonstrated that colonization triggers the expression of host genes for glucose uptake and lipid absorption and transport in the ileum Mice that are GENETICALLY OBESE have microbial gut communities that differ from those of normal mice, with: o fewer Bacteroidetes o Proportional increase in Firmicutes o Greater number of Methanogens (methanogenic Archaea) Methanogens are thought to INCREASE the efficiency of microbial conversion of fermentable substrates consuming molecular hydrogen (H2) The working hypothesis is that H2 removal stimulates fermentation, making more fermentation products available for absorption the host and thus contributing to obesity Ch Microbial Adherence If a pathogen gains access to the specific tissue it infects, disease will occur only if it first ADHERES to those tissues, multiplies to yield many cells or viral particles, and then proceeds to damage tissues (or the entire organism) the release of toxic or invasive substances o Pathogen receptors (adhesins) have evolved to bind specifically to complementary molecules on the host cell cytoplasmic membrane, and the complementary nature of the pathogen and host cell receptors alert the pathogen that it has arrived on a suitable infection site Adherence Structures: Capsules Capsule (VIRULENCE FACTOR) surface contains specific receptors that facilitate adherence to host tissues, but the inherently sticky nature of the capsule itself also assists in the overall attachment process o They also protect pathogenic bacteria from host defenses o Pathogenicity and virulence ARE NOT uniform properties of a given pathogen and can differ dramatically between different strains of the same bacterial species or virus Virulence factors toxic or destructive substances produced the pathogen that directly or indirectly enhances invasiveness and host damage facilitating and promoting infection o Virulence is a QUANTIFIABLE ENTITY, especially if a pathogen is lethal and an experimental animal model is available LD50 the number of cells of a pathogen (or virions, for a viral pathogen) that kills of the animals in a test group Highly virulent pathogens frequently show little difference in the number of cells required to kill of a test of animals as compared with the LD50 Vibrio cholera is not especially virulent, as a large inoculum of this intestinal pathogen is necessary to initiate disease Virulence in Salmonella: Pathogenicity Islands and Plasmids Salmonella encodes a large amount of virulence factors o Siderophores organic molecules that bind iron (Fe) tightly and, in pathogenic bacteria, allow the bacteria to outcompete host sequestration systems for iron Several cytochromes on the ETC have Fe o With the exception of endotoxin, many of these virulence factors are encoded genes present on mobile DNA rather than on the chromosomes Pathogenicity island virulence factor genes clustered together on the chromosome These and R plasmids allow for the facile and rapid transfer (horizontal gene exchange) of virulence factors Can also contain genes encoding antibiotic resistance 25 Enzymes as Virulence Factors Following adherence, colonization, and infection a pathogen, invasiveness requires the breakdown of host tissues and access to nutrients released from host cells o In many classical bacterial pathogens, this is accomplished through the activity of enzymes that attack and destroy cells in one type of tissue or another EX: Staphylococcus aureus produces coagulase which PROMOTES fibrin (insoluble blood protein that triggers blood clots) clotting Coagulase allows bacterial cells to remain at site of infection (these clots protect the pathogen from host responses and its immune system) o Typically seen in boils and pimples o S. aureus strains are typically more virulent than strains, a likely reflection of the ability to evade INNATE IMMUNE RESPONSES such as phagocytosis and continue growth and tissue destruction for a longer period 25 Exotoxins Exotoxins toxic proteins released from the pathogens as it grows o These toxins travel from a site of infection and cause damage at distant sites o AB Toxins B subunit binds to a HOST CELL surface molecule, facilitating the transfer of the A subunit across the cytoplasmic membrane, where it damages the cell, contain toxins EX: Diptheria toxin produced the AEROBIC bacterium Corynebacterium diphtheria Diptheria inhibits protein synthesis in EUKARYOTIC CELLS After binding, subunit A cleaves from the AB complex, allowing subunit A to move across the host cytoplasmic membrane into the cytoplasm o Elongation factor 2 a protein that functions in growth of the polypeptide chain, catalyzes ADP to blocking the elongation step of translation and eventually cell host death Neurological Exotoxins: Botulinum and Tetanus Toxins o Clostridium botulinum and Clostridium tetani are bacteria (found in soil) which cause botulism and tetanus o Function as NEUROTOXINS o Both block the release of neurotransmitters that control muscle activities Cl. botulinum (botox) blocks acetylcholine release (no muscle contraction is muscle is in FLACCID PARALYSIS), preventing muscle contraction This can lead to death suffocation if the diaphragm muscles are severely affected Cl. Tetani grows in the body in deep wounds that become ANOXIC, such as punctures Glycine from the inhibitory interneurons then stops the release of acetylcholine the motor neurons and inhibits muscle contraction, allowing relaxation of the muscle fibers o Tetanus toxin BLOCK glycine release, so the motor neurons cannot be inhibited, resulting in the continual release of acetylcholine and UNCONTROLLED contraction of the muscle fibers, preventing muscle relaxation o Prolonged contraction may result in death due to asphyxiation Cholera Enterotoxin: Intestinal Distress o Massive fluid loss from the intestines, resulting in severe diarrhea, lifethreatening dehydration, and electrolyte depletion o The increased cAMP induced cholera enterotoxins (A subunits) blocks uptake small intestine epithelial cells and induces secretion of chloride and bicarbonate into the intestinal lumen
Microbiology Exam 4 - Summary Brock Biology of Microorganisms
Course: Microbiology for Health Professionals (BIOL2031C)
University: University of Cincinnati
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