Introduction to Bacteriology PDF

Assist. Prof. Dr. Mujahid Kh. Ali  General Bacteriology Introduction We may define microbiology as the study of organisms too small to be seen clearly as individuals by the unaided human eye. It deals with structure, morphology, and the relationships between microorganisms...

Assist. Prof. Dr. Mujahid Kh. Ali  General Bacteriology Introduction We may define microbiology as the study of organisms too small to be seen clearly as individuals by the unaided human eye. It deals with structure, morphology, and the relationships between microorganisms and other organisms, importance of microorganisms, as well as control methods. Such organisms include: Bacteria, Fungi, Viruses, Protozoa and Algae. In general, any organism has a diameter of less than 1 mm will be considered as microorganism. Although microbiology dawn in the end of 19th century, many civilizations like Mesopotamia and Egypt dealt with such organism without seeing them. They used some of them to produce wine without knowing their role in the fermentation process, also they tried to cure disease caused by those microorganisms by medicinal plants. Furthermore, they used to protect their food from spoilage by salting or drying without knowing the causes of this spoilage. Bacteriology: is the branch and specialty of biology that studies the morphology, ecology, genetics and biochemistry of bacteria as well as many other aspects related to them. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species. Because of the similarity of thinking and working with microorganisms other than bacteria, such as protozoa, fungi, and viruses, there has been a tendency for the field of bacteriology to extend as microbiology. Beginning of microscopy The very best unaided human eyes fail to see object less than 100 μm in diameter. Nor can the eye clearly perceive as separate objects (i.e. resolve) particles separated by distances less than this. Microorganisms range downward in diameter from the 50 μm in diameter of animal tissue cells to the diameters of bacteria (1-5 μm) and those of viruses ( 0.25 μm). They are beyond the unaided eye ability. Louis Pasteur (1822–1895) was a French biologist, microbiologist and chemist renowned for his discoveries of the principles of vaccination, microbial fermentation and pasteurization. He is remembered for his remarkable breakthroughs in the causes and prevention of diseases, and his discoveries have saved many lives ever since. He reduced mortality from puerperal fever, and created the first vaccines for rabies and anthrax. His medical discoveries provided direct support for the germ theory of disease and its application in clinical medicine. He is best known to the general public for his invention of the technique of treating milk and wine to stop bacterial contamination, a process now called pasteurization. He is popularly known as the "father of microbiology" In 1884, Robert Koch proposed a series of postulates that have been applied broadly to link many specific bacterial species with particular diseases known as Koch's postulates: 1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms. 2. The microorganism must be isolated from a diseased organism and grown in pure culture. 3. The cultured microorganism should cause disease when introduced into a healthy organism. 4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. Koch's postulates played a role into identifying the relationships between bacteria and specific diseases. Since then, bacteriology has had many successful advances like effective vaccines, for example, diphtheria toxoid and tetanus toxoid. Bacteriology has also provided discovery of antibiotics. Structure of bacteria Morphology Bacteria are the smallest organisms that all machinery required for growth and self-replication, their diameter is usually about 1 μm. The high microscope reveals two principles forms of Eubacteria, spherical organisms called cocci and cylindrical ones called bacilli. Cocci appear in number of different patterns depending upon the planes in which they divide. When cocci appear in pairs they are known as diplococci, while if in chain they are called streptococci, and they are called staphylococci if they were in cluster. Cocci that remain adherent often splitting successively in two or three perpendicular direction yielding tetrads or cubical packets are known as sarcina. Bacillus when unusually short are referred as coccobacilli, when tapered at both ends as fusiform, when growing in long threads as filaments form, when curved as vibrio and when spiral as spirillum or spirochete. Arrangement of bacilli In 1981, square bacteria had been discovered; they 2-4 μm in diameter, halophilic (Archaebacteria), produce stains similar to bacterial rhodopsin. Pleomorphism Bacteria appear in number of different forms. Environmental conditions are affecting the size and shape of bacteria, which is seen obviously in bacilli forms other than cocci forms. Structure of bacterial cell The cell envelope The layers that surround the prokaryotic cell are called cell envelope. The structure and organization of the cell envelope differ in Gram positive and Gram negative bacteria. The Gram positive cell envelope It is relatively simple, consisting of two or three layers: the cytoplasmic membrane, a thick peptidoglycan layer (PG) and in some bacteria an outer layer called capsule. The Gram negative cell envelope It is a highly complex, multilayered structure. The cytoplasmic membrane (called inner membrane) is surrounded by a single layer of peptidoglycan to which is anchored a complex layer known as outer membrane, and the capsule may also be present. The space between inner membrane and outer membrane referred to as periplasmic space. Extracellular polysaccharides Many bacteria synthesize large amounts of extracellular polymer when growing in their natural environment. With one exception (the poly D- glutamic acid capsule of Bacillus anthracis) the extracellular material is polysaccharides which is also called glycocalyx. When the glycocalyx forms a condensed well defined layer closely surrounding the cell, it is called capsule; when it forms masses of polymers are formed that appear to be totally detached from the cell in which cells may be entrapped, in these instances the extracellular polymers may be referred to simply as a slime layer. The glycocalyx layer contributes to the invasiveness of pathogenic bacteria in protecting them from phagocytosis. Furthermore, it plays a role in the adherence of bacteria to surfaces in their environment, including the cells of plant and animal hosts. A biofilm is an aggregate of interactive bacteria attached to a solid surface or to each other. Biofilms are important in human infections that are persistent and difficult to treat. The cell wall The layers of the cell envelope lying between the cytoplasmic membrane and the capsule are called cell wall. In Gram positive bacteria, the cell wall consists mainly of peptidoglycan, teichoic acids, and polysaccharides. While in Gram negative bacteria, the cell wall includes the peptidoglycan, outer membrane, lipopolysaccharide (LPS), and lipoprotein. Gram positive and Gram negative cell wall The functions of cell wall 1 Protects the cell from osmotic pressure. 2 Plays an essential role in cell division. 3 Various layers of the wall are the sites of major antigenic determination of the cell surface. 4 Lipopolysaccharide is responsible for the endotoxin activity.  Chemical composition of the cell wall A- The peptidoglycan layer It is a complex polymer consisting or three parts: 1. A backbone composed of alternating subunit of N-acetyl glucosamine and N-acetylmuramic acid linked together by β 1-4 glycosidic bond. 2. A set of identical tetrapeptide side chains attached to N- acetylmuramic acid. 3. A set of identical peptide cross-bridge (the terminal COOH to NH2 of neighboring tetrapeptide). Peptidoglycan structure All peptidoglycan layers are cross linked, which means that each peptidoglycan layer represents a single giant molecule. In Gram positive bacteria there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall materials. In Gram negative bacteria, it appears to be only one or two sheets, comprising 5-10% of the wall materials. B- Special components of Gram positive cell wall 1 Teichoic acid Most Gram positive cell walls contain amount of teichoic acid, which may account for up to 50% of the dry weight of the wall and 10% of the dry weight of total cell. Teichoic acids are water soluble polymers containing ribitol or glycerol residues joined through phosphodiester linkage. There are two types of teichoic acids; wall teichoic acid covalently linked to peptidoglycan; and lipoteichoic acid (membrane teichoic acid), covalently linked to membrane glycolipid and concentrated in mesosome. Some Gram positive species lack wall teichoic acid but all appears to contain lipoteichoic acid. The function of teichoic acids is still a matter of speculation: a. The main function of teichoic acids is to provide rigidity to the cell wall by attracting cations such as magnesium and sodium. b. Teichoic acids provide an external permeability barrier to Gram positive bacteria. c. Limiting the ability of autolysins to break the β (1-4) bond between the N-acetyl glucosamine and the N-acetylmuramic acid. d. They have role in cell elongation and division. e. Functions in biofilm formation and adhesion. 2 Teichuronic acid The teichuronic acids are similar polymers, but the repeat units include sugar acids instead of phosphoric acids. They are synthesized in place of teichoic acids when phosphate is limiting. 3 Polysaccharides The hydrolysis of Gram positive cell wall has yielded neutral sugars such as mannose, arabinose, galactose, rhamnose, glucosamine and acidic sugars.  C- Special components of Gram negative cell wall 1 Lipoprotein Molecules of an unusual lipoprotein cross-link the outer membrane and peptidoglycan layers. The lipoprotein contains 57 amino acids. Their function is to stabilize the outer membrane and anchor it to the peptidoglycan layer. 2 Outer membrane The outer membrane is a bilayered structure; its inner leaflet resembles in composition that of the cytoplasmic membrane while the phospholipids of the outer leaflet are replaced by lipopolysaccharide (LPS) molecules. The functions of outer membrane are: a. Prevents leakage of periplasmic space proteins. b. Protects the enteric bacteria from bile salts and hydrolytic enzymes. c. Contains the minor proteins, which are involved in the transport of specific molecules such as vitamin B12 and iron- siderophore complexes. d. Has a special channels, consisting of proteins called porins that permit the passive diffusion of low molecular weight hydrophilic compounds like sugars, amino acids, and certain ions. e. Contains numbers of enzymes like proteases and phospholipases. 3- Lipopolysaccharide The lipopolysaccharide of Gram negative cell wall consists of a complex lipid called lipid A, to which is attached a polysaccharide made up of a core and a terminal series of repeat units (O antigen). Lipopolysaccharide is attached to the outer membrane by hydrophobic bound.  Lipopolysaccharides structure The function of lipopolysaccharide: a) Stabilizes the membrane and provides a barrier to hydrophobic molecules. b) Lipopolysaccharide, which is toxic to animals, has been called the endotoxin of Gram negative bacteria because it is firmly bound to the cell surface and is released only when the cells are lysed. All of the toxicity is associated with the lipid A. c) Polysaccharide represents a major surface antigen of the bacterial cell so called O-antigen, and is responsible for the antigenic specificity. The periplasmic space The space between the cytoplasmic membrane and outer membrane, called the periplasmic space, contains the peptidoglycan layer and a gel-like solution of proteins. The periplasmic space is approximately 20-40% of the cell volume. Its proteins include binding proteins for specific substrates (e.g. amino acids, sugars, vitamins, and ions) and the hydrolytic enzymes. Cytoplasmic membrane It is also called cell membrane, composed of proteins and phospholipids. The membrane of prokaryotic cell is differing from those of eukaryotic cells by the absence of sterols except Mycoplasma. Function of cytoplasmic membrane are: 1 Selective permeability and transport of solutes. 2 Electron transport and oxidative phosphorylation, in aerobic species. 3 Excretion of hydrolytic exoenzymes. 4 Bearing the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids. 5 Bearing the receptors and other proteins of the chemotactic and other sensory transduction systems. At least 50% of the cytoplasmic membrane must be in the semifluid state in order for cell growth to occur. Differences between Gram positive and Gram negative bacteria Characteristics Gram Positive Gram Nagative Gram Reaction Retain crystal Can be decolorized violet dye and stain to accept counterstain blue-purple (Safranin) and stain pink-red Cell wall thickness 20-30 nm 8-12 Cell wall Smooth Wavy Peptidoglycan Thick (Multi- Thin (single layer= Layer layered) unilayer) Teichoic acid Present in many Absent Periplasmic space Absent Present Outer membrane Absent Present Porins Absent Occurs in outer membrane Lipopolysaccharid None High e (LPS) content Lipid and Low (Acid fast High (Presence of lipoprotein bacteria have lipids outer membrane) content linked to peptidoglycan Toxins Exotoxins Exotoxins & Endotoxin Resistance to High Low drying  Microbial genetics Nucleic acids types The genetic information of prokaryotic and eukaryotic microorganisms encoded within the DNA (deoxyribonucleic acid) molecule and sometimes (as in viruses) in the RNA (ribonucleic acid) molecule. These molecules are known as macromolecules and they are responsible for the transition of hereditary information from one generation to the other. Another macromolecule found in the cell is the protein, which is the result of the genetic code into its structural or functional form. The structure of nucleic acids and their replication The genetic information of a cell forms a GENOME. The genome of a microorganism is divided into segments consisting of DNA nucleotides sequences known as a GENE. These genes may have structural or functional, metabolic functions. DNA structure The DNA is a double helix where each strand is composed of a sequence of nucleotides; phosphodiester bonds link these nucleotides to each other. Each nucleotide is formed of a deoxyribose sugar, a nitrogen base and a phosphate group. Four nitrogen bases are found in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T). A and G are purines, while C, T and U are pyrimidines. The primary structure of DNA It is resembled by the sequence of nucleotides in a single strand. In this structure when the nitrogen base is bound to the sugar it is known as a nucleoside, when a phosphate group is linked to the nucleoside it is known as nucleotide. A DNA molecule always carries a negative charge due to the PO4 groups. These charges are neutralized by alkaline proteins known as histones in eukaryotes, histone-like proteins in prokaryotes. Figure: Secondary structure of DNA Models for DNA replication There were three basic models for DNA replication that had been proposed by the scientific community after the discovery of DNA's structure. These models are illustrated in the diagram below: 1. Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand. 2. Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule). 3. Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA. Structure of RNA molecule An RNA molecule is usually single stranded; it has a sequence of ribonucleotides each is formed of a ribose sugar, a nitrogen base (A, G, C, and Uracil (U) instead of thymine), and a phosphate group. A ribonicleoside is formed of a ribose sugar and a nitrogen base. Types of RNA and steps in proteins synthesis There are three types of RNA (mRNA, tRNA, and rRNA). Their roles will be described within the process of protein synthesis. 1-Messenger RNA (mRNA): It is formed in the nucleus of eukaryotes and nuclear region of prokaryotes. It carries the information transcribed from the DNA to the ribosomes (in the cytoplasm) where protein is synthesized. It is transcribed from a single strand of DNA and is complementary to that strand. mRNA is a single strand with a sequence of ribonucleotides to be translated by the ribosomes to the required protein. Transcription is a non-symmetric process since only one strand of DNA is transcribed (except in some viruses where DNA is single stranded by nature). mRNA is synthesized by a DNA- dependent RNA polymerase known also as transcriptase. Transcription is the first step in protein synthesis. The second step in protein synthesis is translation; it requires the presence of the other two types of RNA; transfer RNA tRNA and ribosomal RNA (rRNA). 2-Transfer RNA (tRNA) It is also known as soluble RNA has a distinguished clover leaf structure and two recognition sites; one binds to an activated amino acid, the second is known as the anticodon that recognizes the codon on the mRNA. 3- Ribosomal RNA (rRNA) rRNA is a type of non-coding RNA that is a primary and permanent component of ribosomes. As non-coding RNA, rRNA itself is not translated into a protein, but it does provide a mechanism for decoding mRNA into amino acids and interacting with the tRNAs during translation by providing peptidyl transferase activity. The genetic code Every codon is made of three nucleotides coding for one amino acid, and since there are four nitrogen bases the probabilities of the number of genetic codes are 43 = 64. There are 20 amino acids therefore there could be more than one code for most amino acids. For each amino acid there is one or more tRNA that carries the specific activated amino acid to the ribosome. Protein synthesis There are three stages in proteins synthesis: 1 Activation: when each amino acid is activated by a specific amino acyl synthetase. This is also known as the initiation stage. The starting signal is f-met (formyl-methionine). This stage requires If1, If2, and If3 (If= initiation factor, which is a protein). 2 Elongation stage: it is achieved by the following steps: i) Attachment of the activated amino acid to a tRNA molecule. The specificity of the tRNA is determined by the anticodon sequence on the anticodon arm. ii) The mRNA is attached to the ribosome at a specific site. iii) The amino acid is transferred and carried by the tRNA to the ribosome at the site bound to the mRNA codon complementary to the anticodon of the tRNA where each C is bound to G and each A to U. iv) Translocation on the ribosome takes place and other amino acids are placed in position, these amino acids are then connected together by a peptide bond formed by the peptidyl transferase enzyme. 3- Termination stage: termination of translation occurs when a stop codon enters the ribosome and three release factors (Rf1, Rf2, and Rf3) help in releasing the tRNA, mRNA, and proteins from the ribosomes. Mutation in bacteria The term mutation applies to all heritable changes in nucleotide sequence arising within an organism, they may be: 1- Spontaneous (naturally occurring) 2- Induced by some mutagenic agents, this could be chemical or physical. When the mutant is not altered phenotypically but only genotypically it is known as Silent mutation. A mutation could occur by deletion, insertion of a nucleotide, transition, (purine into pyrimidine or pyrimidine into purine) or transversion (purine into purine or pyrimidine into pyrimidine). A point mutation occurs when one nucleotide is inserted or deleted. Bacterial mutants Several types of mutants are known: 1 Antibiotic or drug resistant mutants. 2 Mutants that differ in their fermentation products. 3 Auxotrophs: are mutants that lack the ability to synthesize organic compounds necessary for their growth therefore they are supplemented with vitamins or amino acids and need to grow on rich media. 4 Phenotypic mutants: they are altered phenotypically by a change in morphology or color of the colonies. 5 Mutation in cell surface and antigenic structure. 6 Phage resistant mutants. 7 Mutations with altered structures (i.e. loss of flagella, capsule, or spore formation). Growth and multiplication Growth means an increase in size, number, weight, and mass. It is a group of reactions and events led to an increase the macromolecules number and then cell division and reproduction. Cell cycle A group of steadily successive events are interrupted with periods which depending on environmental conditions. The required time from the beginning to the end of division known as generation time and the resulting growth called growth rate. Growth rate and generation time Generation time (doubling time): The time for a single cell to undergo fission. It takes short time in prokaryote (ex: 20-25 min in E coli). While in eukaryotes it takes two hours to several days. Generation time varies with: 1 Species of M.O. 2 Nutrients. 3 Environmental conditions: PH, and temperature. 4 Growth phase.  Prokaryotic cell cycle: Most of studies on prokaryotic cell cycle were done on E.coli because of it is easy to handle. Prokaryotic cell cycle includes: 1. First stage : This period is still under speculating. Mostly, under the optimal condition it disappears due to the shortage of generation time, also the environmental conditions greatly affect the cell. 2. Second stage: A- A stage of DNA synthesis abbreviated as C instead of S, it means chromosome replication. B-It required most of cycle time. C- It controls the continuity of the cycle, since when the DNA synthesis is interrupted the cell will not divide. D- It is affected, a little, by the environmental conditions. 3- Third and fourth stages: A- After the DNA synthesis stage, there is a gap before the cell is dividing into tow daughter cells. B- It represents both third and fourth stages, G2 and M. C- It referred as to D. D-It is affected, a little bit, by the environmental conditions.  Growth curve of bacteria: When bacteria are inoculated into a new culture media, it shows a characteristic growth curve which has four phases: 1- Lag phase: During this phase, bacteria exhibit growth in size but no increase in cell number and the bacteria are preparing for synthesis of DNA, various enzymes, and other components, which are for cell division.The lag phase varies in length with the conditions of the M.O and the nature of the media, this mean that the phase may be long if the inoculum is from an old culture or if the culture is refrigerated. 2- Logarithmic(exponential) phase: During this period the cells divide steadily at a constant rate. The log of the number of cells is plotted against time results in a straight line. Under appropriate conditions, the growth rate is maximal during this phase, and the population is most nearly uniform in terms of chemical compositions of cells, metabolic activity and other physiological characteristics. 3- Stationary phase: During this phase the growth rate is equal to the death rate. Food begins to run out, poisonous waste products accumulate, PH changes, hydrogen acceptors are used up, and energy transfers are diminished. The rate of fission begins to decline, and the organisms die in increasing numbers.  4- Death (decline) phase: In this phase , the number of viable bacterial cells begins to decline, signaling the onset of the death phase.The kinetics of bacterial death, like those of growth, are exponential.