Microbiology and Infectious Diseases PDF

Bacteria; classification, structure, growth and metabolism-1,2,3 Prof. Dr. Necla TÜLEK Atilim University, Faculty of Medicine 04 November 2024 Objectives Describes the bacterial structures and the function of the different bacterial comp...

Bacteria; classification, structure, growth and metabolism-1,2,3 Prof. Dr. Necla TÜLEK Atilim University, Faculty of Medicine 04 November 2024 Objectives Describes the bacterial structures and the function of the different bacterial components Learns the different classification schemes for grouping bacteria, Describes the different types of bacteria Discuss the distinguishing characteristics of Gram positive and Gram negative bacteria. Learns the growth, biochemical, and genetic characteristics that are used in differentiating bacteria  Common noun bacteria Singular bacterium Bacteria range in size from about 0.2 to 5 μm The bacteria range in size from Mycoplasma, the smallest, to Bacillus anthracis, one of the largest BACTERIAL STRUCTURE  Two Basic Types of Cells Prokaryotic Cell Eukaryotic Cell The prokaryotic cell is simpler than the eukaryotic cell at every level, with one exception: The cell envelope is more complex. Images: Prokaryotic cell diagram & From the Virtual Microbiology Classroom on ScienceProfOnline.com Eukaryotic cell diagram, M. Ruiz  The Bacterial Chromosome or “Nucleoid” The nucleoid is the area of the cytoplasm in which DNA is located. The DNA of most prokaryotes is a single, circular molecule; however, there are important exceptions. For instance, the genome of Vibrio cholerae, the causative agent of cholera, is composed of two circular chromosomes. Borrelia burgdorferi, the spirochete that causes Lyme disease, is composed of a linear chromosome and multiple circular and linear plasmids. The size of bacterial genomes varies widely, with the smallest genome containing just over 130 genes and the largest containing approximately 11,600 genes. By contrast, human DNA has approximately 25,000 genes. Bacterial nucleoid contains no nuclear membrane, no nucleolus, no mitotic spindle, and no histones. Haploid Bacterial DNA released from E. coli cell Haploid refers to the presence of a single set of chromosomes in an organism's cells  Plasmid Small extra piece of chromosome/genetic material. 5 - 100 genes Not critical to everyday functions. Can provide genetic information to promote: - Antibiotic resistance - Virulence factors (exotoxins - Some structures (pili..) - Promote conjugation (transfer of genetic material between bacteria through cell- to-cell contact) Image: Prokaryotic Cell Diagram: M. Ruiz, Bacterial conjugation, Adenosine Transposons Transposons are pieces of DNA that move readily from one site to another either within or between the DNAs of bacteria, plasmids, and bacteriophages. Because of their unusual ability to move, they are nicknamed “jumping genes.” Transposons can code for drug resistant enzymes, toxins, or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alter the expression of nearby genes.  Bacterium Cytoplasm Also known as proto-plasm. Cytoplasm of bacterial cells is gel-like and contains the chromosome, ribosomes,various macromolecules and small molecules in water solution Location of growth, metabolism, and replication. The cytoplasm has two distinct areas when seen in the electron microscope: 1. An amorphous matrix that contains ribosomes, nutrient granules, metabolites, and plasmids. 2. An inner, nucleoid region composed of DNA. From the Virtual Microbiology Classroom on ScienceProfOnline.com  Some inclusions in bacterial cells Inclusion Composition Function Glycogen poly-glucose Reserve carbon and energy source Poly-betahydroxybutyric lipid Reserve carbon and energy acid (PHB) source Poly-phosphates polymers of PO4 Reserve phosphate, possibly high-energy PO4 Sulfur globules elemental S Reserve energy and or electrons Magnetosomes magnetite (iron oxide) Provide orientation in magnetic field Gas vesicles protein shells inflated with Provide buoyancy in aquatic gases environments Parasporal crystals protein Produced by endospore- forming Bacilli - toxic to insects The cytoplasm contains several different types of granules that serve as storage area for nutrients  Ribosome Function Ribosomes function in protein synthesis. Amino acids are assembled into proteins according to the genetic code on the surfaces of ribosomes during the process of translation.  Bacterium Cytoskeleton  Cellular "scaffolding" or "skeleton" within the cytoplasm.  Actin homologs (eg, MreB and Mbl) perform a variety of functions, helping to determine cell shape, segregate chromosomes, and localize proteins within the cell.  Nonactin homologs (eg, FtsZ) and unique bacterial cytoskeletal proteins (eg, SecY and MinD) are involved in determining cell shape and in regulation of cell division and chromosome segregation. Image: Prokaryotic Cell: M. Ruiz Endospores Endospore forming bacteria left to right: Clostridium botulinum, Bacillus brevis, Bacillus thuringiensis Endospores are produced as intracellular structures within the cytoplasm of certain bacteria, most notably Bacillus and Clostridium species. Provides resistance to dehydration, heat, and chemicals Endospore formation is NOT a mechanism of reproduction. Rather it is a mechanism for survival in deleterious environments. Spore formation (sporulation) occurs when nutrients, such as sources of carbon and nitrogen, are depleted. During the process of spore formation, one vegetative cell develops into one endospore. The sequential steps of endospore formation in a Bacillus species. The process of endospore formation takes about six hours. Eventually the mature endospore is released from its “mother cell” as a free spore Free endospore Endospore within mother cell Vegetative cell  Resting (dormant) cells show no signs of life primarily due to lack of water in the spore Highly resistant to heat (boiling), acids, bases, dyes ( don’t stain) irradiation, disinfectants, antibiotics, etc. The spore forms inside the cell and contains bacterial DNA, a small amount of cytoplasm, cell membrane, peptidoglycan, very little water, and most importantly, a thick, keratinlike coat that is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals. This resistance may be mediated by dipicolinic acid, a calcium ion chelator found only in spores.  Medically-important Endospore-forming Bacteria Bacillus anthracis causes anthrax Bacillus cereus causes food poisoning Clostridium tetani causes tetanus Clostridium botulinum causes botulism Clostridium perfringens causes food poisoning and gas gangrene Clostridium difficile causes antibiotic-induced diarrhea and pseudomembranous colitis  The Plasma Membrane (cytoplasmic membrane) Just inside the peptidoglycan layer of the cell wall lies the cytoplasmic membrane, which is composed of a phospholipid bilayer similar in microscopic appearance to that in eukaryotic cells. They are chemically similar, but eukaryotic membranes contain sterols, whereas prokaryotes generally do not. The only prokaryotes that have sterols in their membranes are members of the genus Mycoplasma. Composed of 60% protein and 40% phospholipid Arranged as a bilayer The membrane bilayer is formed by phospholipid Section of a cytoplasmic membrane molecules made up of glycerol and fatty acids Functions of the cytoplasmic membrane 1. Selective permeability and transport of molecules into the cells; 2. Electron transport and oxidative phosphorylation in aerobic species (energy generation); 3. Secretion of enzymes and toxins. 4. Contain the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids (Biosynthetic functions) 5. Bear the receptors and other proteins of the chemotactic and other sensory transduction systems. Functions of the cytoplasmic membrane Membrane is semi- permeable. Osmotic or permeability barrier: the membrane is impermeable to macromolecules Transport systems in bacteria Special transport processes—Iron (Fe) is an essential nutrient for the growth of almost all bacteria. Under anaerobic conditions, Fe is generally in the +2 oxidation state and soluble. However, under aerobic conditions, Fe is generally in the 3 oxidation state and insoluble. The internal compartments of animals contain virtually no free Fe; it is sequestered in complexes with such proteins as transferrin and lactoferrin. Some bacteria solve this problem by secreting siderophores— compounds that chelate Fe and promote its transport as a soluble complex Excretion of hydrolytic exoenzymes and pathogenicity proteins All organisms that rely on macromolecular organic polymers as a source of nutrients (eg, proteins, polysaccharides, and lipids) excrete hydrolytic enzymes that degrade these polymers to subunits small enough to penetrate the cell membrane. In Gram-positive bacteria, proteins are secreted directly across the cytoplasmic membrane, but in Gram-negative bacteria, secreted proteins must traverse the outer membrane as well.  Proteins secreted by the type II system are transported across the OM by a multiprotein complex. This is the primary pathway for the secretion of extracellular degradative enzymes by Gram-negative bacteria. Elastase, phospholipase C, and exotoxin A are secreted by this system in Pseudomonas aeruginosa. Some extracellular proteins—eg, the IgA protease of Neisseria gonorrhoeae and the vacuolating cytotoxin of Helicobacter pylori—are secreted by Type V. The type I and IV secretion systems have been described in both Gram-negative and Gram-positive bacteria, but the type II, III, V, and VI secretion systems have been found only in Gram-negative bacteria.  Cell Walls Cell wall is a structure that completely surrounds the cell protoplast. The cell wall is the outermost component common to all bacteria (except Mycoplasma species, which are bounded by a cell membrane, not a cell wall). Cell Walls Why study bacterial cell walls? They are essential structures in bacteria. They are made of chemical components found nowhere else in nature. They may cause symptoms of disease in animals. They are the site of action of some of our most important antibiotics.  Primary function of the bacterial cell wall To prevent rupture or osmotic lysis of the cell protoplast (osmotic protection) Lysis of a pair of dividing E. coli cells  The bacterial cell wall owes its strength to a layer composed of a substance variously referred to as murein, mucopeptide, or peptidoglycan In addition to providing osmotic protection, the cell Wall plays an essential role in cell division as well as serving as a primer for its own biosynthesis  Chemical nature of bacterial cell walls Bacterial cell walls always contain murein, which is a type of peptidoglycan Chemical nature of murein accounts for the function of the cell wall. Murein is only found in the cell walls of bacteria Peptidoglycan is made up of 2 amino sugars N-acetyl-glucosamine = G N- acetylmuramic acid = M 4 amino acids L-alanine = L-ala D-glutamic acid = D-glu diaminopimelic acid = DAP D-alanine = D-ala the tetrapeptide side chains and the peptide cross-bridges vary from species to species Diaminopimelic acid a unique element of bacterial cell walls  Assembly of the peptidoglycan This is a critical step for bacterial survival The cross-linking reaction is a transpeptidation. One peptide bond (produced inside the cell) is traded for another (outside the cell) with the release of D-alanine. The enzymes that catalyze the reaction are called D-alanine, D-alanine transpeptidase-carboxypeptidases. The transpeptidases and carboxypeptidases are called penicillin- binding proteins (PBPs) because they are targets for penicillin and other β-lactam antibiotics. Peptidoglycan extension and cross-linking is necessary for cell growth and division. Clinical Correlate Peptidoglycan synthesis is a major target for antimicrobial chemotherapy for the following reasons: The process is utilized only in prokaryotes. Its inhibition has few side effects on eukaryotic cells (human cells). As effective as antibiotics are, bacteria have become very clever at getting around them and becoming resistant to antibiotic  Other characteristics of bacterial cell walls Gram-positive cell walls contain teichoic acids The teichuronic acids are similar polymers, Teichoic acids are thought to stabilize the Gram positive cell wall and may be used in adherence. The teichoic acids constitute major surface antigens of those Gram-positive species Other characteristics of bacterial cell walls Gram-negative cell walls include an outer membrane Other characteristics of bacterial cell walls Outer membrane of Gram-negatives has two important properties 1. It protects the cells from permeability by many substances including penicillin and lysozyme. 2. It is the location of lipopolysaccharide (endotoxin) which is toxic for animals. The Bacterial Cell Envelope LPS is comprised of lipid A (the toxic portion), and polysaccharide. LPS is highly immunogenic and binds specifically to receptors (LPS receptor, or TLR-4) to activate macrophages. LPS can also non-specifically activate B cells without the help of T cells. LPS can be serotyped to classify bacteria.  Cell wall of Gram-positive or Gram negative bacteria Peptidoglycan is a complex polymer composed of alternating N-acetylglucosamine and N-acetylmuramicacid connected by β1→4 linkages; teichoic and teichuronic acids, which may account for up to 50% of the dry weight of the wall Gram negative bacteria have a small peptidoglycan layer but have an additional three components that lie outside of the peptidoglycan layer: lipoprotein, outer membrane, and lipopolysaccharide. Comparision of the Gram positive and Gram negative bacterial cell walls Property Gram-positive Gram-negative Thickness of wall thick (20-80 nm) thin (10 nm) Number of layers 1 2-3 Peptidoglycan (murein) >50% 10-20% content Teichoic acids in wall present absent Protein/lipoprotein 0-3% >50% content Lipopolysaccharide 0 13 content Sensitivity to penicillin sensitive resistant Sensitivity to lysozyme sensitive resistant Cell Walls of Acid-Fast Bacteria Mycobacteria (e.g., Mycobacterium tuberculosis) have an unusual cell wall, resulting in their inability to be Gram-stained. These bacteria are said to be acid-fast because they resist decolorization with acid–alcohol after being stained with carbolfuchsin. This property is related to the high concentration of lipids, called mycolic acids, in the cell wall of Mycobacteria. Nocardia asteroides, a bacterium that can cause lung and brain infections in immunocompromised individuals, is weakly acid-fast. The meaning of the term “weakly” is that if the acid-fast staining process uses a weaker solution of hydrochloric acid to decolorize than that used in the stain for Mycobacteria, then N. asteroides will not decolorize.  Glycocalyx Some bacteria have an additional layer outside of the cell wall called the glycocalyx. The glycocalyx is a polysaccharide coating that is secreted by many bacteria. It covers surfaces like a film and allows the bacteria to adhere firmly to various structures (e.g., skin, heart valves, prosthetic joints, and catheters). The glycocalyx is an important component of biofilm This additional layer can come in one of two forms: 1. Slime Layer - Glycoproteins loosely associated with the cell wall. - Slime layer causes bacteria to adhere to solid surfaces The slime layer of Gram+ Streptococcus mutans allows it to accumulate on tooth enamel Other bacteria in the mouth become trapped in the slime and form a biofilm & eventually a buildup of plaque. Etymology: Greek glykýs, meaning “sweet” + Greek kalux, meaning “husk” or “shell”. 2. Capsule Polysaccharides firmly attached to the cell wall. Adhesive power of capsules is a major factor in the initiation of some bacterial diseases. Capsule also protect bacteria from being phagocytized by cells of the hosts immune system. It is a determinant of virulence of many bacteria. Capsular polysaccharides are used as the antigens in certain vaccines because they are capable of eliciting protective antibodies. For example, the purified capsular polysaccharides of 23 types of S. pneumoniae are present in the current vaccine. Specific identification of an organism can be made by using antiserum against the capsular polysaccharide. In the presence of the homologous antibody, the capsule will swell greatly. This swelling phenomenon, which is used in the clinical laboratory to identify certain organisms, is called the Quellung reaction. cell recognition cell adhesion protection permeability barrier From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Prokaryotic Cell Diagram: M. Ruiz, Other Images Unknown Source  Surface Appendages Some bacteria have distinct appendages that allow them to move about or adhere to solid surfaces. Flagella: Long, thin extensions that allow some bacteria to move about freely in aqueous environments. (singular: flagellum) Axial filament (endoflagella): Wind around bacteria, causing movement in waves. Images: Helicobacter pylori ; Axial filament  Surface Appendages  Fimbriae: Most Gram-negative bacteria have these short, fine appendages surrounding the cell. Gram+ bacteria don’t have. No role in motility. Help bacteria adhere to solid surfaces. Major factor in virulence. (singular: fimbria)  Pili:Tubes that are longer than fimbriae, usually shorter than flagella. Use for movement, like grappling hooks, and also use conjugation pili to transfer plasmids. (singular = pilus) From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: E. coli fimbriae, Manu Forero; Bacterial conjugation, Adenosine Bacterial Structures Identification- Classification  Bacteria range in size from about 0.2 to 5 μm. The smallest bacteria (Mycoplasma) are about the same size as the largest viruses (poxviruses) and are the smallest organisms capable of existing outside a host. The longest bacteria rods are the size of some yeasts and human red blood cells (7 μm). Sizes of representative bacteria, viruses, yeasts, protozoa, and human red cells. General Concepts-Definitions Taxonomy divides organisms into categories and gives them scientific names. Identification, classification, and nomenclature are three separate but interrelated areas of bacterial taxonomy. Identification is the recognition of the essential character of an organism Nomenclature is the naming of organisms. Classification is the grouping of organisms into groups based on phylogeny and phenotype, Classification is how living things are organized into categories, and nomenclature is how organisms are named. In a microbiologic context, classification is the categorization of organisms into taxonomic groups. Identification Identification is the practical use of a classification scheme to distinguish certain organisms from others (1) to isolate and distinguish specific organisms among the mix of complex microbial flora, (2) to verify the authenticity or special properties of a strain in a clinical setting, (3) to isolate the causative agent of a disease. Assigning Specific Names The binomial system of nomenclature The generic (genus) name followed by the species name Generic part is capitalized, species is lowercase Both are italicized or underlined if italics aren’t available Staphylococcus aureus Criteria for identification- classification of bacteria Phenotypic Growth on media (selective/nonselective/differential media) Colony appearance Bacterial microscopy apperance Gram stain Characteristic growth and metabolic properties, Biochemical tests Immunologic tests—Antigenicity Genotypic Growth on a media Macroscopic distinction The initial distinction between bacteria can be made by growth characteristics on different nutrient and selective media. The bacteria grow in colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike. The cultural characteristics of a bacterium on an agar plate - called colony morphology Culture media is a gel or liquid that contains nutrients and minerals, and is used to grow bacteria or microorganisms. They are also termed growth media. Bacteria that require a medium with various growth factors or other components and are hard to grow are referred to as fastidious.  Colony morphology Size Shape :round, circular, irregular, punctiform (tiny, pinpoint) Color: White, Cream/Buff, Purple, Gray, Yellow, Red, Green/Blue-Green, Pink/Rose, Orange, Black centers Texture: Dull , shiny, mucoid (slimy) , dry , moist, rough, smooth, sticky, Molar Tooth, powdery Height (elevation) :Convex (rounded), concave (depressed/sunken in), flat, raised,umbonate (raised in the center). Edge : Smooth, no irregularities, undulate (wavy), lobate, filamentous, rhizoid (branched), swarming, spreading, Hemolysis zone Colony scant: Flowery , fruit-like, bleach, ammonia, malodorous, putrid Optical properties: Opaque, Transparent, Translucent, Shiny, Dull/Matte Different species of bacteria can produce very different colonies. Colony morphology is one of the first steps in characterizing and identifying a bacterial culture. https://www.tuyenlab.net/2016/08/microbiology-use-of-colonial-morphology_20.html The sum of their characteristics provides the colony with distinguishing characteristics, such as color, size, shape, to lyse erythrocytes (haemolytic- nonhaemolytic) The ability to resist certain antibiotics, To ferment specific sugars (e.g., lactose, to distinguish E. coli from Salmonella), To lyse erythrocytes (hemolytic properties), To hydrolyze lipids (e.g., clostridial lipase) can also be determined using the appropriate growth media  Bacterial morphology The microscopic appearance The microscopic appearance, including the size, shape, and configuration of the organisms (cocci, rods, curved, or spiral). A spherical bacterium, such as Staphylococcus, is a coccus; A rod-shaped bacterium, such as E. coli, is a bacillus A spiral bacterium such as Treponema is a spirillum. Some bacteria are variable in shape and are said to be pleomorphic (heterogeneous shape). The shape of a bacterium is determined by its rigid cell wall. The microscopic appearance of a bacterium is one of the most important criteria used in its identification.  Bacterial Shapes In addition to their characteristic shapes, the arrangement of bacteria is important. For example, certain cocci occur in pairs (diplococci), some in chains (streptococci), and others in grapelike clusters (staphylococci). These arrangements are determined by the orientation and degree of attachment of the bacteria at the time of cell division Some bacteria form aggregates, such as the grapelike clusters of Staphylococcus aureus or the diplococcus (two cells together) observed in Streptococcus or Neisseria species.  Binary fission 1. Prokaryote cells grow by increasing in cell number (as opposed to increasing in size). Cell division, of a bacterium 2. Replication is by into two daughter cells, in a binary fission, the process called binary fission splitting of one cell into two 3. Therefore, bacterial populations increase by a factor of two Incomplete cleavage of the septum (double) every can cause the bacteria to remain generation time. linked, forming chains (e.g., streptococci) or clusters (e.g., 4. Growth leads to an staphylococci). increase in the number of single bacteria making up a population, referred to as a culture. Cocci: Pairs Division in one plane Diplococci Neisseria Cocci: Chains Division in 2 Planes Streptococcus Cocci: Clusters Division in 3 planes Staphylococcus Cocci: Tetrads Division in three planes Micrococcus Cocci: 8-cell group Divides in 3 planes Sarcina Binary Fission Results Cocci Pairs Chains Tetrads Cubes Clusters Bacillus Separate Pairs Chains Rods: Straight E. coli Rods: Club-Shaped Corynebacterium Rods: Branching Actinomyces Rods: Comma form Vibrio Rods: Spore Formers Spiral Forms Pleomorphic Biochemical reactions Clinical microbiology laboratories typically will identify a pathogen in a clinical sample, purify the microorganism by plating a single colony of the microorganism on a separate plate, then perform a series of biochemical studies that will identify the bacterial species. Such as catalase positive- negative Citrate negative- positive Indol negative-positive Lactose negative-positive…. Phenotypic classification systems Gram stain Discovered by H.C. Gram in 1884 it remains an important and useful technique to this day. It allows a large proportion of clinically important bacteria to be classified as either Gram positive or negative based on their morphology and differential staining properties. Gram positive bacteria stain blue-purple and Gram negative bacteria stain red. The difference between the two groups is believed to be due to a much larger peptidoglycan (cell wall) in Gram positives, as a result the iodine and crystal violet precipitate in the bacteria Some bacteria such as mycobacteria (the cause of tuberculosis) are not reliably stained due to the large lipid content of the peptidoglycan. Alternative staining techniques (Kinyoun or acid fast stain) are therefore used. An intact cell wall is necessary for a positive reaction, and Gram-positive bacteria may fail to retain the stain if the organisms are old, dead, or damaged by antimicrobial agents. Rarely, a Gram-negative organism (eg, Acinetobacter) will appear Gram positive.  Serologic systems The designation “sero” simply indicates the use of antibodies (polyclonal or monoclonal) that react with specific bacterial cell surface structures such as lipopolysaccharide (LPS), flagella, or capsular antigens. Selected antisera can be used to classify different bacterial species. This may be based on either carbohydrate or protein antigens from the bacterial cell wall or the capsular polysaccharide. (Group A streptococcal M proteins or O and H polysaccharide antigens of salmonella). Environmental Reservoirs: When considering likely pathogens it is also important to know which of the different species are found in different locations. endogenous (i.e., on or within the human body) exogenous (somewhere in the environment). Nonculture methods for the identification of pathogenic microorganisms PCR-assisted approach using rRNA as a target to identify pathogenic microorganisms in situ. The first phase of this approach involves the extraction of DNA from a suitable specimen, The PCR amplification of the ribosomal DNA, The sequencing of the rRNA sequence information, and a comparative analysis of the retrieved sequences. This yields information on the identity or relatedness of the sequences in comparison with the available database. This approach has been used in the identification of pathogenic microorganisms. Description of the major categories and groups of bacteria Bergey’s Manual of Systematic Bacteriology serves as an aid in the identification of bacteria that have been described and cultured. I-Gram-negative eubacteria that have cell walls II-Gram-positive bacteria that have cell walls III-Cell wall-less eubacteria IV-Archaebacteria Major Categories and Groups of Bacteria That Cause Disease in Humans as Part of an Identification Scheme Described in Bergey’s Manual of Determinative Bacteriology, 9th Ed.-2  Bacterial metabolism, growth  Growth Requirements: on the basis of need for oxygen Bacteria can be grouped on the basis of their need for oxygen to grow. Facultatively anaerobic bacteria can grow in high oxygen or low oxygen content Strictly anaerobic bacteria grow only in conditions where there is minimal or no oxygen present in the environment. (such as bacteroides, clostridium) Strict aerobes only grow in the presence of significant quantities of oxygen. Pseudomonas aeruginosa, an opportunistic pathogen, is an example of a strict aerobe. Microaerophilic bacteria grow under conditions of reduced oxygen and sometimes also require increased levels of carbon dioxide. Neisseria species (e.g., the cause of gonorrhea) are examples of micraerophilic bacteria.  What do bacteria need? Bacteria have well defined requirements Proper nutrients, Oxygen, pH, Nitrogen, Salts, Temperature  Iron is so important that many bacteria secrete special proteins (siderophores) to concentrate iron from dilute solutions, Our bodies will sequester iron to reduce its availability as a means of protection. Siderophores, such as enterobactin produced by E. coli, are secreted by the bacteria, capture iron by chelating it, then attach to specific receptors on the bacterial surface, and are actively transported into the cell where the iron becomes available for use.  Where do get materials? Nutrition is a process by which chemical substances called nutrients are acquired from the surrounding environment and used in cellular activities such as metabolism and growth. Bacteria acquire energy from oxidation of organic or inorganic molecules, or from sunlight. Growth requires raw materials: some form of carbon. Autotrophs vs. heterotrophs Auto=self; hetero=other; troph=feeding. Autotrophs use carbon dioxide Heterotrophs use pre-formed organic compounds (molecules made by other living things). Humans and medically important bacteria are heterotrophs. 85  Essentials of bacterial nutrition Macronutrients: Needed in larger amounts, play principal role in cell structure and metabolism Needed in large quantities: Carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur. H and O are common. Sources of C, N, P, and S must also be provided. Macronutrients needed in smaller amounts: Mineral salts such as Ca+2, Fe+3, Mg+2, K+ Micronutrients = trace elements; needed in very small amounts; e.g. Zn+2, Mo+2, Mn+2 for enzyme and pigment structure and function.  How they get nutrients? Permeability and transport The cytoplasmic membrane forms a hydrophobic barrier impermeable to most hydrophilic molecules. Several mechanisms (transport systems) exist that enable the cell to transport nutrients into and waste products out of the cell. There are three general transport mechanisms involved in membrane transport: passive transport, active transport, and group translocation (Group translocation is a protein export or secretion pathway found in plants, bacteria, and archaea)..  Microbial metabolism Microbial metabolism consists of the biochemical reactions bacteria use to break down organic compounds and the reactions they use to synthesize new bacterial parts Energy for the new constructions is generated during the metabolic breakdown of the substrate. The occurrence of all biochemical reactions in the cell depends on the presence and activity of specific enzymes. Thus, metabolism can be regulated in the cell either by regulating the production of an enzyme itself or by regulating the activity of the enzyme (via feedback inhibition, in which the products of the enzymatic reaction or a succeeding enzymatic reaction inhibit the activity of the enzyme).  Bacterial metabolism Many of the principles of metabolism are universal. The broad differences between bacteria and human eukaryotic cells can be summarized as follows: Speed. Bacteria metabolize at a rate 10 to 100 times faster. Versatility. Bacteria use more varied compounds as energy sources and are much more diverse in their nutritional requirements. Simplicity. To synthesize macromolecules in a streamlined way. Uniqueness. Some biosynthetic processes, such as those producing peptidoglycan, lipopolysaccharide, and toxins, are unique to bacteria.  Bacterial metabolism Bacterial metabolism is highly complex. The bacterial cell synthesizes itself and generates energy by as many as 2000 chemical reactions. These reactions can be classified according to their function in the metabolic processes of fueling, biosynthesis, polymerization, assembly.  Biochemical Pathways from Glucose to Pyruvic Acid The starting carbohydrate for bacterial fermentations or oxidations is glucose. When bacteria use other sugars as a carbon source, they first convert the sugar to glucose, which is processed by one of three pathways. These pathways are designed to generate pyruvic acid, a key three-carbon intermediate. The three major biochemical pathways bacteria use to break down glucose to pyruvic acid are: (1) the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (2) the pentose phosphate pathway, (3) the Entner-Doudoroff pathway Pyruvate can be further processed either fermentatively or oxidatively. These reactions, which occur under both aerobic and anaerobic conditions  Pyruvic acid is a key metabolic intermediate. Bacteria process pyruvic acid further using various fermentation Fermentation pathways. Each pathway yields different end products, which can be analyzed and used as phenotypic markers Fermentation is the transfer of electrons and protons via NAD+ directly to an organic acceptor. Pyruvate occupies a pivotal role in fermentation. Fermentation is an inefficient way to generate ATP, and consequently huge amounts of sugar must be fermented to satisfy the growth requirements of bacteria anaerobically. Large amounts of organic acids and alcohols are produced in fermentation. Which compounds are produced depends on the particular pathway of fermentation used by a given species, and therefore the profile of fermentation products is a diagnostic aid in the clinical laboratory. End products of fermentation pathways. Nicotinamide Adenine Dinucleotide  Aerobic Utilization of Pyruvate (Oxidation) The most important pathway for the complete oxidation of a substrate under aerobic conditions is the Krebs or tricarboxylic acid (TCA) cycle. In this cycle, pyruvate is oxidized, carbon skeletons for biosynthetic reactions are created, and the electrons donated by pyruvate are passed through an electron transport chain and used to generate energy in the form of ATP. This cycle results in the production of Krebs tricarboxylic acid cycle allowing acid and the evolution of carbon complete oxidation of a substrate. dioxide  Respiration (not an act of breathing) Respiration is an efficient energy generating process Obligate aerobes and facultative anaerobes carry out aerobic respiration, in which oxygen is the final electron acceptor. Certain anaerobes can carry out anaerobic respiration, in which inorganic forms of oxygen, such as nitrate and sulfate, act as the final electron acceptors  Fermentation involves direct transfer of proton and electron via NAD+ to organic receptor acceptor ATP-generating efficiency is low Large amounts of organic acids and alcohols are produced in fermentation. Respiration uses electron chain for which oxygen is usually the terminal acceptor Respiration is efficient energy producer Connections to fermentation and respiration pathways allow the reoxidation of reduced coenzyme nicotinamide adenine dinucleotide (NADH) to NAD+ and the generation of ATP.  Carbohydrate Utilization and Lactose Fermentation The ability of microorganisms to use various sugars (carbohydrates) for growth is an integral part of most diagnostic identification schemes. The fermentation of the sugar is usually detectedby acid production and a concomitant change of color resulting from a pH indicator present in the culture medium. Bacteria generally ferment glucose preferentially over other sugars, so glucose must not be present if the ability to ferment another sugar is being tested. Bacterial growth  Bacterıal Growth Bacterial growth refers to an increase in bacterial cell numbers (multiplication) which results from a programmed increase in the biomass of the bacteria. Growth usually occurs asynchronously, i.e. all cells don’t divide at precisely the same moment. Whenever adequate nutrition and conducive environmental factors are available a bacterium enlarges and eventually divides by binary fission to form two daughter cells  Cell division Bacterial growth is the asexual reproduction. The replication of the bacterial chromosome also triggers the initiation of cell division. This requires metabolism, which produces cell material from the nutrient substances in the environment; regulation, which coordinates the progress of the hundreds of independent biochemical processes in an orderly way; finally, cell division, which produces two independent living units from one.  Binary Fission in Prokaryotes The replication of the bacterial chromosome triggers the initiation of cell division  Generation time The growth rate of a bacterial culture depends on three factors: the species of bacterium, the chemical composition of the medium, and the temperature. The average time required to for a population to double (doubling time- replication time ) in number. Compared to the growth rates of most other living things, bacteria are notoriously speedy. The average generation (doubling) time is 30-60 minutes under optimum conditions. Escherichia coli double every 20 minutes Mycobacterium tuberculosis double every 12 to 24 hours Longest generation time occurs in Mycobacterium leprae (10-30 days).  Culture Systems “growth curve” 1. Lag phase occurs when bacteria are adjusting to them medium. 2. In log or exponential phase, the cells are growing as fast as they can, limited only by growth conditions and genetic potential. During this phase, almost all cells are alive, they are most nearly identical, and they are most affected by outside influences like disinfectants. This phase will continue as long as cells have adequate nutrients and the environment is favourable. 3- Due to nutrient depletion and/or accumulation of toxic end products, replication stops and cells enter a stationary phase where there is no net change in cell number. 4- Death phase occurs when cells can no longer maintain viability and numbers decrease as a proportion.  Obligate intracellular growth Most bacterial pathogens of humans are capable of growing on artificial media in the clinical laboratory. The term artificial means that the medium is composed of purified chemicals such as sugars, amino acids, and salts, such as sodium chloride. Often blood is added in the form of sheep’s blood, but that is for nutritional purposes, not because the bacteria need to grow within the red blood cells. However, certain bacterial pathogens of humans, notably Chlamydia and Rickettsia and Ehrlichia and Anaplasma , can only grow within living cells and are referred to as obligate intracellular pathogens. The main reason for this is that they lack the ability to produce sufficient adenosine triphosphate (ATP) and must use ATP produced by the host cells.  Environmental conditions Acidity Water activity Temperature Oxygen Toxins such as ethanol can hinder or kill bacterial growth. This is used beneficially for disinfection and in food preservation. Antimicrobials  pH The pH of the medium of growth of bacteria has profound effect upon the multiplication of organisms. Most pathogenic bacteria require a pH of 7.2-7.6 for their optimal growth. Some bacteria can flourish in the presence of considerable degree of acidity and are termed acidophilic, e.g. Lactobacillus species. Some others are very sensitive to acid, but are tolerant of alkali, e.g. Vibrio cholerae.  Water activity: halophiles, osmophiles, and xerotolerant Water is critical for life; remove some, and things can’t grow. Halophiles/halotolerant: relationship to high salt. Marine bacteria; archaea and really high salt. Osmophiles: can stand hypertonic environments whether salt, sugar, or other dissolved solutes Fungi very good at this; grandma’s wax over jelly. The capability to survive under dry environment varies from organism to organism. The Gonococcus and Treponema pallidum die quickly in dry conditions but Staphylococcus aureus and tubercle bacilli can survive for weeks or months under similar conditions. Xerotolerant: dry. Subject to desiccation. Fungi best Bread, dry rot of wood Survival of bacterial endospores.  Effect of temperature Low temperature Enzymatic reactions too slow; enzymes too stiff Lipid membranes no longer fluid High temperature Enzymes denature, lose shape and stop functioning Lipid membranes get too fluid, leak DNA denatures As temperature increases, reactions and growth rate speed up. At high end of range, critical enzymes begin to denature, work slower. Growth rate drops off rapidly with small increase in temperature.  Temperature Psychrophile 0o to 18o C Psychrotroph 20°C to 30°C Important in food spoilage Mesophile 25°C to 45°C More common Disease causing pathogens Thermophiles 45°C to 70°C Common in hot springs and hot water heaters Hyperthermophiles 70°C to 110°C Live at very high temperatures, high enough where water threatens to become a gas Usually members of Archaea Found in hydrothermal vents Psychorophile is a microorganism that grows optimally below 15°C and is capable of growing at 0°C. It is obligate with respect to cold and generally cannot grow above 20°C. Room temperature is lethal to the organism. Storage in refrigerators incubates rather than inhibits. They are rarely, if ever, pathogenic to man. Mesophile are organisms that grow at moderate temperatures the optimal range being 20–40°C. Thermophile is a microbe that grows optimally at temperatures greater than 45°C. Such heat loving microbes live in soil and water associated with volcanic activity and in habitats directly exposed to sun. Most thermophiles are spore-forming sps. of Bacillus and Clostridium and a small number are pathogens.  Miscellaneous conditions Radiation (solar, UV, gamma, ionizing radiation) Can all damage cells; bacteria have pigments to absorb energy and protect themselves. Endospores are radiation resistant. Deinococcus radiodurans: extremely radiation resistant Extremely efficient DNA repair, protection against dessication damage to DNA. Barophiles/barotolerant: microbes from deep sea Baro- means pressure. Actually require high pressure as found in their environment. 110