Lecture 2 NEW Microbiology 24-03-23 PDF

Lecture 2 Content: Classification of bacteria, Growth, Survival, and Death of Microorganisms Physiology Cultivation of microorganisms 1 Identification of bacteria Understanding of differences existing in bacteria is significant because...

Lecture 2 Content: Classification of bacteria, Growth, Survival, and Death of Microorganisms Physiology Cultivation of microorganisms 1 Identification of bacteria Understanding of differences existing in bacteria is significant because each infectious agent has specifically adapted to a particular mode(s) of: transmission, the capacity to grow in a human host (colonization), and, a mechanism(s) to cause disease (pathology) 2 Bacterial taxonomy Bacterial taxonomy (Gk. Taxon) = arrangement; eg, the classification of organisms in an ordered system that indicates a natural relationship. Identification, classification, and nomenclature are three separate but interrelated areas of bacterial taxonomy. 3 Universal tree of life 4 Identification Identification is the practical use of a classification scheme: (1) to isolate and distinguish specific organisms among the mix of complex microbial flora, (2) to verify the authenticity or special properties of a culture in a clinical setting, and, (3) to isolate the causative agent of a disease. 5 Koch’s postulates Rules to prove an organism causes a disease - four criteria designed to establish a causative relationship between a microbe and a disease Organism consistently isolated from diseased individuals Organism cultivated in pure form Signs and symptoms induced after inoculation Same organism isolated from experimentally infected individual 6 Identification schemes Identification schemes are not classification schemes, although there may be some superficial similarity. For example, the popular literature has reported Escherichia coli as the causative agent of hemolytic uremic syndrome (HUS) in infants. There are hundreds of different strains that are classified as E. coli but only a few that are associated with HUS. These strains can be “identified” from the many other E. coli strains by antibody reactivity with their O-, H-, and K-antigens 7 Classification Classification is the categorization of organisms into taxonomic groups. Experimental and observational techniques are required for taxonomic classification for establishing a taxonomic rank: biochemical, physiologic, genetic, and morphologic properties. This area of microbiology is dynamic as the tools continue to evolve (eg, new methods of microscopy, biochemical analysis, and computational nucleic acid biology) 8 Nomenclature Nomenclature refers to the naming of an organism by an established group of scientific and medical professionals. Linnaean taxonomy is used for the formal ranks of: kingdom, phylum, class, order, family, genus, species, and subtype 9 Classification of E.coli 10 CRITERIA FOR IDENTIFICATION OF BACTERIA Growth on Media Complex media: carbon source, acid hydrolysate or enzymatically degraded source of biologic material (eg, casein) supplements e.g. vitamins, intact red blood cells Nonselective Media –supports growth of many different bacteria e.g. blood agar, chocolate agar) Selective media - is used to eliminate (or reduce) the large numbers of irrelevant bacteria in the specimens of interest. The basis for selective media is the incorporation of an inhibitory agent that specifically selects against the growth of irrelevant bacteria. 11  Examples of selective media McConkey agar (contains bile) selects for Gram negative rods Colombia CAN agar (containing colistin and nalidixic acid) that selects for Gram-positive cocci. Differential Media 12 Gram staining and microscopy The Gram stain was developed by Christian Gram in 1884 and can differentiate between the two types of cell walls Gram positive and Gram-negative. Christian Gram 1884 13  Gram stain mechanism The cells are first stained with the primary stain, crystal violet – 1 min. Wash with water Mordant, Gram's iodine – 1 min The iodine forms a complex with the crystal violet and the crystal violet-iodine complex becomes "trapped" inside the peptidoglycan. Wash with water Decolorize (using acetone-alcohol) – 5-10 sec The cells for a period of time long enough to dissolve the outer membrane of Gram-negative cells and pull the crystal violet-iodine complex through the thin layer of peptidoglycan. Counter-stain with safranin, to stain the Gram-negative cells pink. Gram-positive cells will also stain with safranin but it will not be seen on top of the purple crystal violet-iodine remaining in the cells. 14 Gram positive and Gram negative cell wall structure 15 Staining methods Simple staining - only one dye is used, differentiation between bacteria is impossible. Differential staining – more than one dye is used, differentiation between bacteria is possible (e.g. Gram stain, Fast-acid stain). Special staining – more than one dye is used, special structures are seen. 16 Acid-fast stain Used to stain organisms that resist conventional staining. Used to stain Mycobacterium tuberculosis High lipid concentration in cell wall prevents uptake of dye Once stained difficult to decolorize. 17  Differential Staining Methods - Acid-Fast Staining Acid-fast Stain: ACID-FAST Cell Color Cell color Mycobacterium and many Nocardia species are STAIN called acid-fast because during an acid-fast staining procedure they retain the primary dye Procedure Reagent Acid fast Non acidfast carbol fuchsin despite decolorization with the bacteria bacteria powerful solvent acid-alcohol. Nearly all other Primary dye Carbolfuchsin RED RED genera of bacteria are nonacid-fast. The Decolorizer Acid-alcohol RED COLORLESS acid-fast genera have lipoidal mycolic acid in their cell walls. It is assumed that mycolic acid Couterstain Methylene blue RED BLUE prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential stain. 18 19  Biochemical Tests (1) Catalase test Catalase activity can be used, e.g. to differentiate between the Gram-positive cocci; the species staphylococci are catalase positive, whereas the species streptococci are catalase negative. 20 Biochemical tests (2) Oxidase test Oxidase test can be used to distinguish organisms by detecting the presence or absence of a respiratory enzyme, cytochrome C, the lack of which differentiates the Enterobacteriaceae from other Gram-negative rods. 21 Immunologic Tests—Serotypes, Serogroups, and Serovars Cholera spp. subtyping The designation “sero” simply indicates the use of antibodies (polyclonal or monoclonal) that react with bacterial cell surface structures such as lipopolysaccharide (LPS), flagella, or capsular antigens. The terms “serotype,” “serogroups,” and “serovars” are, for all practical purposes, identical—they all use the specificity of these antibodies to subdivide strains of a specific bacterial species. In certain circumstances (eg, an epidemic), it is important to distinguish among strains of a given species or to identify a specific strain. This is called subtyping and is accomplished by examining bacterial isolates for characteristics that allow discrimination below the species level. 22 Clonality Clonality with respect to isolates of microorganisms from a common source outbreak (point source spread) is an important concept in the epidemiology of infectious diseases. Etiologic agents associated with these outbreaks are generally clonal; i.e. they are the progeny of a single cell and, for all practical purposes, are genetically identical 23 Genetic Diversity Developments in DNA sequencing now make it possible to investigate the relatedness of genes or genomes by comparing sequences among different bacteria. It should be noted that genetic instability can cause some traits to be highly variable within a biologic group or even within a specific taxonomic group. For example, antibiotic resistance genes or genes encoding toxins may be carried on plasmids or bacteriophages. Many organisms are difficult to cultivate, and in these instances, techniques that reveal relatedness by measurement of DNA sequence analysis may be of value. 24 25 CLASSIFICATION SYSTEMS Numerical taxonomy became widely used in the 1970s. For these assays, an individual bacterial colony must be isolated and used to inoculate the test format. One example of this is the Analytical Profile Index (API), which uses numerical taxonomy to identify a wide range of medically important microorganisms. APIs consist of several plastic strips, each of which has about 20 miniature compartments containing biochemical reagents. Almost all cultivatable bacterial groups and more than 550 different species can be identified using the results of these API tests. The limitation of this approach is that it is a static system. As such, it does not allow for the evolution of bacteria and routine discovery of new bacterial pathogens. 26 Nucleic Acid–Based Taxonomy plasmid profile analysis, restriction fragment endonuclease analysis, repetitive sequence analysis, ribotyping, 16S ribosomal sequencing, and genomic sequencing 27  Plasmid Analysis Plasmids are extrachromosomal genetic elements. These can be isolated from an isolated bacterium and separated by agarose gel electrophoresis to determine their number and size. Plasmid analysis has been shown to be most useful for examining outbreaks that are restricted in time and place (eg, an outbreak in a hospital), particularly when they are combined with other identification methods. 28 Restriction Endonuclease Analysis Restriction enzymes cleave DNA into discrete fragments = one of the most basic procedures in molecular biology. Restriction endonucleases recognize short DNA sequences (restriction sequences), and they cleave double stranded DNA within or adjacent to this sequence. Restriction sequences range from 4 to more than 12 bases in length and occur throughout the bacterial chromosome. Restriction enzymes that recognize short sequences (eg, 4-base pairs) occur more frequently than those that are specific for longer sequences (eg, 12-base pairs). Several subtyping methods use restriction endonuclease– digested DNA approaches. One method involved restriction of plasmid DNA, while another approach analysis of genomic DNA. Separation of these large DNA fragments is accomplished by a technique called pulsed field gel electrophoresis (PFGE), which requires specialized equipment. 29 Ribosomal RNA sequencing Ribosomes have an essential role in protein synthesis for all organisms and as such are indisposable. Genetic sequence encodings both ribosomal RNAs (rRNA) and ribosomal proteins (both of which are required to comprise a functional ribosome) are highly conserved throughout evolution and have diverged more slowly than other chromosomal genes. Comparison of the nucleotide sequence of 16S rRNA from a range of prokaryotic sources revealed evolutionary relationships among widely divergent organisms and has led to the elucidation of a new kingdom, the archaebacteria. 30 Classification of microorganisms according to nutrition type 31 THE MAJOR CATEGORIES AND GROUPS OF BACTERIA - The Eubacteria Gram-Negative Eubacteria B. Gram-Positive Eubacteria ❑ phototrophic ❑ chemosynthetic heterotrophs ❑ nonphototrophic aerobic, aerobic, anaerobic, anaerobic, facultatively anaerobic facultatively anaerobic, o simple asporogenous microaerophilic species o sporogenous bacteria o structurally complex actinomycetes 32 THE MAJOR CATEGORIES AND GROUPS OF BACTERIA - The Eubacteria C. Eubacteria Lacking Cell Walls mycoplasmas (class Mollicutes) They do not synthesize the precursors of peptidoglycan They are enclosed by a plasma membrane L-forms that can be generated by breaking down the cell wall Unlike L-forms mycoplasmas never revert to the walled state Mycoplasmas are highly pleomorphic organisms and range in size from vesicle-like forms to very small (0.2 µm), too small to be captured on filters that routinely trap most Mycoplasma genitalium bacteria 33 THE MAJOR CATEGORIES AND GROUPS OF BACTERIA - The Archaebacteria Archaebacteria ❑ lack a peptidoglycan cell wall, ❑ characteristic rRNA sequence chemolithotrophs, heterotrophs, facultative heterotrophs o extremophiles o mesophiles 34 Bacterial growth - The Measurement of Microbial Concentrations Microbial concentrations Viable cell count (titer) - the number of viable cells per unit volume of culture (e.g. cell/mL) Turbidity Density - biomass concentration i.e. dry weight of cells per unit volume of culture 35  Growth curve of bacteria A population growth curve for any particular species of bacterium may be determined by growing a pure culture of the organism in a liquid medium at a constant temperature. Samples of the culture are collected at fixed intervals (e.g., every 30 minutes), and the number of viable organisms in each sample is determined. The data are then plotted on logarithmic graph paper. The logarithm of the number of bacteria per milliliter of medium is plotted against time. 36 1.Lag phase: After a liquid culture broth is inoculated, the multiplication of bacteria does not start immediately. It takes some time to multiply. The time between inoculation and beginning of multiplication is known as lag phase. In this phase, the inoculated bacteria become acclimatized to the environment, switch on various enzymes, and adjust to the environmental temperature and atmospheric conditions. During this phase, there is an increase in size of bacteria but no appreciable increase in number of bacterial cells. The cells are active metabolically. The duration of the lag phase varies with the bacterial species, nature of culture medium, incubation temperature, etc. It may vary from 1 hour to several days. 37 2. LOG Phase This phase is characterized by rapid exponential cell growth (i.e., 1 to 2 to 4 to 8 and so on). The bacterial population doubles during every generation. They multiply at their maximum rate. The bacterial cells are small and uniformly stained. The microbes are sensitive to adverse conditions, such as antibiotics and other antimicrobial agents. Growth rate is the greatest during the log phase. The log phase is always brief, unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent removal of waste products. When plotted on logarithmic graph paper, the log phase appears as a steeply sloped straight line. 38 3.Stationary phase: After log phase, the bacterial growth almost stops completely due to lack of essential nutrients, lack of water oxygen, change in pH of the medium, etc. and accumulation of their own toxic metabolic wastes. It is during this phase that the culture is at its greatest population density. However, death rate of bacteria exceeds the rate of replication of bacteria. Endospores start forming during this stage. Bacteria become Gram variable and show irregular staining. Many bacteria start producing exotoxins. 39 4.Decline phase: During this phase, the bacterial population declines due to death of cells. The decline phase starts due to (a) accumulation of toxic products and autolytic enzymes and (b) exhaustion of nutrients. Involution forms are common in this stage. Some cells assume various shapes, becoming long, filamentous rods or branching or globular forms that are difficult to identify. Some develop without a cell wall and are referred to as protoplasts, spheroplasts, or L-phase variants (L-forms). When these involuted forms are inoculated into a fresh nutrient medium, they usually revert to the original shape of the healthy bacteria. 40  Food curve and The Chemostat Cells can be maintained in the exponential phase by transferring them repeatedly into fresh medium of identical composition while they are still growing exponentially. This is referred to as balanced growth. 41 GROWTH IN BIOFILMS Planktonic growth – cells grow individually A variety of stressors trigger biofilm formation, and each microorganism is unique in the signals it responds to. Biofilms begin with a single bacterium attaching on a surface followed by binary fission and ultimately to the formation of an intimate community of progeny bacteria Biofilms - often consist of several different microbial species The glycocalyx also serves to keep the biofilm community intact. Bacteria within a biofilm produce small molecules, such as homoserine lactones, which are taken up by adjacent bacteria and functionally serve as a colony “telecommunication” system, informing individual bacteria to turn on certain genes at a particular time (Quorum Sensing). These signals are known as quorum sensors 42 Control of bacterial growth Sterilization - killing all the organisms, including spores Disinfection – elimination of the vegetative cells but not the spores Pasteurization- the process of applying heat, usually to milk or cheese, for a specified period for the purpose of killing or retarding the growth of pathogenic bacteria. Aseptic - free of, or using methods to keep free of, microorganisms. 43 Physical and chemical agents affecting bacterial growth A. Heat Application of heat is the simplest means of sterilizing materials, provided the material is itself resistant to heat damage. A temperature of 100°C will kill all but the spores of eubacteria within 2–3 minutes in laboratory-scale cultures; A temperature of 121°C for 15 minutes is used to kill spores B. Radiation Ultraviolet (UV) radiation that has a wavelength of about 260 nm causes thymidine dimers resulting in the inability of bacterial DNA to be replicated. This is generally bactericidal, but not against spores. 44 Physical and chemical agents affecting bacterial growth C. Alcohols These agents effectively remove water from biologic systems. Thus, they functionally act as “liquid desiccants.” alcohol, isopropyl alcohol, and n-propanol exhibit rapid, broad-spectrum antimicrobial activity against vegetative bacteria, viruses, and fungi but are not against spores. B. Aldehydes Compounds like glutaraldehyde or formaldehyde are used for low-temperature disinfection and sterilization of instruments, endoscopes, and surgical tools. They are normally used as a 2% solution to achieve sporicidal activity. These compounds are generally bactericidal and sporicidal. 45 Physical and chemical agents affecting bacterial growth C. Peroxygens Hydrogen peroxide (H2O2 ) has broad-spectrum activity against viruses, bacteria, yeasts, and bacterial spores. Sporicidal activity requires higher concentrations (10–30%) of H2O2 and longer contact times. D. Phenols Phenol and many phenolic compounds have antiseptic, disinfectant, or preservative properties. In general, these are not sporicidal 46 Physical and chemical agents affecting bacterial growth Depending on the mechanism of action, different biocides are bacteriostatic, bactericidal, and/or sporicidal. Biocide activity is dependent on time and concentration. This activity can be reversed by agent removal, substrate competition, and agent inactivation 47 SOURCES OF METABOLIC ENERGY Fermentation – glycolise, substrate phosphorylation Respiration Photosynthesis 48  Movement of materials across the membrane Movement across the membrane may be by passive processes, in which materials move from areas of higher to lower concentration, and no energy is expended by the cell. In simple diffusion, molecules and ions move until equilibrium is reached Osmosis is the movement of water from areas of high to low concentration across a selectively semi permeable membrane until equilibrium is reached. In facilitated diffusion, substances are transported by transporter proteins across membranes from areas of low to high concentration. In active transport, materials move from areas of low to high concentration by transporter 49 proteins, and the cell must expend energy. Movement of materials across the membrane (2) 50 Movement of materials across the membrane (3) Channel proteins span the membrane and make Carrier proteins can change their shape to move a target hydrophilic tunnels across it, allowing their target molecule from one side of the membrane to the other. Like molecules to pass through by diffusion. Channels channel proteins, carrier proteins are typically selective for one are very selective and will accept only one type of or a few substances. Often, they will change shape in response to molecule (or a few closely related molecules) for binding of their target molecule, with the shape change moving transport. the molecule to the opposite side of the membrane. Channel and carrier proteins transport material at different rates. In general, channel proteins transport molecules much more quickly than do carrier proteins. 51 The Movement Of Materials Across Membranes (4) In group translocation, energy is expended to modify chemicals and transport them across the membrane. Once inside the cell the chemical modification prevents the transported substance from moving out of the cell. Example: glucose is converted to glucose-6- phosphate as it passes into the cell. This traps it and allows the movement of more glucose into the cell even when extracellular concentrations may be low. 52  Nutrition – carbon source Nutrients in growth media must contain all the elements necessary for the biologic synthesis of new organisms. Some bacteria can use photosynthetic energy to reduce carbon dioxide at the expense of water. T These organisms are referred to as autotrophs, bacteria that do not require organic nutrients for growth. Other autotrophic microorganisms are the chemolithotrophs, organisms that use an inorganic substrate such as hydrogen or thiosulfate as a reductant and carbon dioxide as a carbon source. Heterotrophs require organic carbon for growth, and the organic carbon must be in a form that can be assimilated. 53 Nitrogen Source Nitrogen is a major component of proteins, nucleic acids, and other compounds, accounting for approximately 5% of the dry weight of a typical bacterial cells The ability to assimilate N2 reductively via NH3 , which is called nitrogen fixation, is a property unique to prokaryotes, and relatively few bacteria can break the nitrogen–nitrogen triple bond Production of NH3 from the deamination of amino acids is called ammonification Some autotrophic bacteria (eg, Nitrosomonas, Nitrobacter spp.) can convert NH3 to gaseous N2 under anaerobic conditions; this process is known as denitrification. 54 Sulfur source Some autotrophic bacteria can oxidize sulphur to sulphate (SO4 2−). Most microorganisms can use sulphate as a sulphur source, reducing the sulphate to the level of hydrogen sulphide (H2S). Hydrogen sulfide test Some microorganisms can assimilate H2S directly from the growth medium, but this compound can be toxic to many organisms. 55 Phosphorus Source Phosphate (PO4 3−) is required as a component of ATP, nucleic acids, and such coenzymes as NAD, NADP, and flavins. In addition, many metabolites, lipids (phospholipids, lipid A), cell wall components (teichoic acid), some capsular polysaccharides, and some proteins are phosphorylated. Phosphate is always assimilated as free inorganic phosphate (Pi ) 56 ENVIRONMENTAL FACTORS AFFECTING GROWTH Nutrients Hydrogen-Ion Concentration (pH) Neutralophiles grow best at a pH of 6.0–8.0, Acidophiles have optima as low as pH 3.0, Aalkaliphiles have optima as high as pH 10.5 57 ENVIRONMENTAL FACTORS AFFECTING GROWTH Temperature Psychrophilic- grow best at low temperatures (–5 to 15°C) Psychrotrophs - temperature 20°C -30°C, but grow well at lower temperatures Mesophylic - 30–37°C Thermophylic - 50–60°C Hyperthermophylic – growth at the temperature of boiling water e.g. Yersinia enterocolitica ENVIRONMENTAL FACTORS AFFECTING GROWTH - Aeration Aerobic Facultatively aerobic Strictly (obligate) aerobic Anaerobic Facultatively anaerobic Strictly (obligate) anaerobic Microaerophylic – require small amount of oxigen along with CO2 59 CULTIVATION METHODS - Culture media Classification by consistency Classification based on functional use or Liquid - broth application Solid - 1,5%-2% agar Basal media Semi- solid – 0,3%- 0.7% agar Enriched media Selective media Enrichments media Differentiantial media Transport media Anaerobic media Classification by nutrition components Simple e.g. pepton water Complex e.g. blood media, components not always known Synthetic – used for research purposes, all components and their ratio is known 60 Cultivation of bacteria – isolation of microorganisms in pure culture Methods: Streak culture Lawn culture Stroke culture Pour plate culture Liquid culture 61 62 Aerobic and Anaerobic Cultivation ▪Anaerobic gas pack method ▪Evacuation – replacement method 63 Dilution method 64 Reading Chapters: 3. Classification of Bacteria 4. Growth, Survival, and Death of Microorganisms 5. Cultivation of Microorganisms 6. Microbial Metabolism