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This document details the action of physical, chemical, and biological factors on bacteria, including definitions of key terms. It describes various agents and methods used to control bacterial growth, such as heat, chemicals, and bacteriophages. The document also briefly explores bacterial genetics and the interactions between bacteriophages and bacteria.
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THE ACTION OF PHYSICAL, CHEMICAL AND BIOLOGICAL FACTORS ON BACTERIA DEFINITIONS Bacteriostatic agent: stop the multiplication of bacteria. Multiplying recurs again after removal the agent. Bactericidal agent: to kill (definitively inactive) bacterial...
THE ACTION OF PHYSICAL, CHEMICAL AND BIOLOGICAL FACTORS ON BACTERIA DEFINITIONS Bacteriostatic agent: stop the multiplication of bacteria. Multiplying recurs again after removal the agent. Bactericidal agent: to kill (definitively inactive) bacterial cell, the process is irreversible: even after removing agent, bacterial multiplication does not occur. Sterilization = the action through which the suppression of any microbial flora is obtained. Disinfectant = a substance (agent) used for inactivation of microorganisms in some areas (tables, floors), but through its marked toxicity can not be applied on human tissues. Disinfection = the inactivation of microorganisms from surfaces (inanimate “objects"). Antisepsis = the application of bactericidal or bacteriostatic substances in order to kill or inhibit the growth of pathogenic flora in wounds. Asepsis includes all measures which prevent contamination with infectious agents. Conservation = preventing spoilage by microbial agents of degradable products such as food or drugs. I. Physical agents – action on bacteria Heat – dry heat sterilization: flaming, dry heat oven and moist heat sterilization: boiling, pasteurization, tyndallisation, autoclaving; dryness (desiccation); lyophilisation = drying gradual in vacuum at very low temperatures (- 40°C to - 70°C). Mechanical pressure Osmotic pressure Radiations Ultrasounds Electricity Filtering – thermo sensitive fluids are passed through sterilizing filters which capture microorganisms. II. Chemical agents – with action on bacteria Phenol and derivatives – with bactericidal action by bacterial cell lyses. Alcohols – with weaker power than phenol and act bactericidal only on vegetative forms. Halogens and halogenated compounds – most frequently used as antiseptics and disinfectants are chlorine and iodine, both with powerful bactericidal and sporicidal action. Acids and bases - The bactericidal action of acids and bases is due to brutal distortion of bacterial proteins. Salts – between salt, the heavy metals have the strongest bactericidal effect. Formaldehyde and Glutaraldehyde Gaseous disinfectants - Ethylene oxide is the best disinfectant gas for dry surfaces. Soaps and detergents (surfactants) Metabolic antagonists Dyes Others disinfectants – hydrogen peroxide, potassium permanganate. III. Biological agents - action on bacteria. Bacteriophages. 1. Bacteriophages They are viruses capable of bacteria cell lysis (lytic bacteriophages) or unable of lysis (symbiotic bacteriophages). A. Morphology and chemical composition of bacteriophages After the type of nucleic acid containing, bacteriophages can be: - DNA phages - RNA phages DNA PHAGES A typical bacteriophage particle morphologically includes: head and tail. The head contains a nucleic acid core and an protein shell. - nucleic acid core consists of DNA, comprising about 50% of dry weight of bacteriophages. - protein shell (capsid) gives polygonal shape of the head and consists of identical protein subunits. In fact, shell protein forms hexagonal prism in space. Tail of the bacteriophage is the part of bacteriophage used for attached to bacterial cell surface. Generally, the tail has three parts: lacunar middle, contractile sheath and basal terminal plate. The basal terminal plate has a hexagonal shape with attached „tail fibres". Bacteriophages tail can be found in two functional stages: relaxed stage and contracted stage. a) in relaxed stage, contractile sheath cover almost entirely the lacunar core and tail fibres are not visible; b) in contracted stage, contractile sheath is shortened, the lacunar core is visible on a larger stretch, and the terminal plate have obvious tail fibres. In addition to the typical morphology of bacteriophages, there may be other forms, atypical like: bacteriophages without tail (some DNA bacteriophages) filamentous bacteriophages. RNA PHAGES They present a special morphology: A core RNA single stranded with molecular weight of 4x106 Daltons and with a “self complementary complex" type tertiary structure. Capsid is composed of 180 subunits (capsomeres). RNA phages are involved in chromosome conjugation phenomena (fixing fimbria on donor bacterium F+), as in lysogenisation of intestinal bacteria. II. TYPES OF INTERRELATIONS BETWEEN BACTERIOPHAGES AND BACTERIA The bacteriophage is an bacterial virus of which host is an bacterium. Attaching phage of bacterial cell surface: by some receptors. In terms of genetic attachment, there are : Lyso-sensitive bacteria – which allow the attachment and the entry of bacteriophages. Lyso-resistant bacteria – which does not allow the attachment and the entry of bacteriophages. There are two categories of relationships bacteriophage – bacterium: Lytic type Symbiotic type 1. Lytic relation between bacteriophage and bacteria In this type of relationship, phages penetrate the bacterial lyso-sensitive cell, resulting in cell death (with some exceptions), and release of newly formed bacteriophage cell particles. Steps: A. Adsorption B. Penetration C. Intracellular replication D. Maturation of newly formed phage particles E. Release of newly formed phage particles A. Adsorption Due to the presence of the receptors on the surface of lyso- sensitive bacterial cell, phage particles will get adsorbed on the surface, by attaching the tail (tail fibres) to those receptors. On the surface of bacterial cells, can be attached up to 100 phages. The attachment on receptors is based on a specific property: the bacterial cell receptors are highly specific for certain bacteriophages. The specificity of phage receptors is due to the chemical nature of bacterial cell wall (available mucopeptides network). B. Penetration Once attached, the bacteriophage will “inject" its nucleic acid from the head into the bacterial cell, passing into the contracted stage from the relaxed stage of the tail. C. Intracellular replication C1. Replication of DNA phages A few minutes after entering the nucleic acid phage, DNA phage begins to "order" through its genes, in the bacterial host cell, the synthesis of "early proteins", which includes the enzymes (DNA polymerase, nucleoside-triphosphate-kinase, thymidylate-synthetases) necessary for increased synthesis of DNA phage. Synthesis for the affected cell host stops. The phage “copies" of DNA (resulting from "early“ enzyme action) determine, using the synthesis apparatus of the cell (ribosomes), the formation of "late proteins", which will assemble around phage DNA molecules, giving rise to head capsid and other protein parts of the newly formed phage particles. C2. Replication of RNA phages When RNA phage enter in the bacterial cell, it acts as a messenger to the ribosomes, where the first one summarizes RNA synthetize, which then provide formation of “copies" of RNA phage, which, in turn, "encode“ in ribosomes the synthesis of protein for capsid formation in the case of newly formed particles. Intermediate in the replication of RNA phages is the formation of a double-stranded RNA, resulting from RNA chain of phage particles entering the cell (chain "plus") relating complementarily to a chain resulting from action of RNA synthetize phage (chain "minus"). D. Maturation of newly formed phage particles Assembly between newly formed phage nucleic acid molecules in host cells and “late“ phage proteins, also newly synthesized, after the model “encoded" by the nucleic acid phage, results in new bacteriophage particles. E. Release of newly formed phage particles At one point, phage particles "fill" the space of bacterial cell, cause “explosion" of cell wall and cytoplasm membrane, with cell lysis, at the same time with outdoor release of newly formed phage particles able, in the presence of lyso-sensitive bacterial cells, to resume the lytic cycle. 2. Symbiotic relation between bacteriophage and bacteria Symbiotic relation between bacteriophage and bacteria means: entry of bacteriophage into the bacterial cell, without causing its lysis, with integration of phage genetic material into the bacterial chromosome; this phenomenon is called ‘lysogeny’. This type of bacteriophage-bacterium relationship following attachment and penetration of phages in the host cell, independent replication of nucleic acid phage does not occur, but it integrates into cellular DNA (bacterial chromosome), replicating at the same time with the DNA cellular replication (cell division). Bacteriophages able to establish a symbiotic relationship with carrier bacteria are called "prophages" or “temperate bacteriophages". Stages of “symbiotic phage infection" are: - adsorption - penetration of phage nucleic acid - "circularisation“ of DNA phage - coupling circular DNA phage with bacterial DNA. Bacteria containing inside the temperate phages are called "lysogene bacteria". They are resistant ('immune') to "infection" with another homologous bacteriophage. This phenomenon is due to the release in the lysogenic cell cytoplasm of a "repressor“ substance which inhibit the multiplication of the lytic phage inside the cell. Inducing agents act by determining synthesis of ‘inducer’ substances in the cell, inactivating ‘repressor’ substance, secondarily. The result: some new properties of the cell appear. This phenomenon of change of some properties of the bacterial cell, by prophage presence, is called ‘conversion’. For example – strains of Corynebacterium diphteriae by lysogenisation (wearing a temperate phage) from non-toxigenic can become toxigenic (diphtheria toxin-producing). BACTERIAL GENETICS Bacterial genetics It studies heredity and variability of bacteria. Heredity – is the overview property of all living things to transmit specific species characters in the offspring. Variability – is the hereditary change of bacteria. I. Bacterial heredity Characters of bacteria are genetically determined. Expression of these characters is manifested by phenotype. 1. Organization of genetic material in bacteria Bacterial genome consist of replicons, which are genetic configurations that can replicate independently: - Bacterial chromosome - Extrachromosomal genetic elements - Genetic transposable elements 1.1.Bacterial chromosome Most bacterial genes are in the bacterial chromosome, which encodes the information necessary for survival of the species under normal conditions. A. DNA structure DNA is composed of two complementary spiral strands with reverse polarity (Watson and Crick model). Each chain results from nucleotides polymerization. These nucleotides consist of: a deoxyribose molecule, a molecule of ortho- phosphoric acid, one of nitrogenous purine bases (adenine and guanine-A- G) and pyrimidine bases (cytosine and thymine-C-T). B. DNA replication The double helix structure of DNA allows identical semi-conservative replication at the initial site. The spiral opens like a zipper at the initial site for replication using DNA gyrase action. On each spiral a new, complementary spiral will be synthesize, with the participation of DNA polymerase (I, II, III) from the end of the 5 terminal to end of the 3 terminal. 1.2. Extrachromosomal genetic elements These genetic elements are represented by: bacteriophages, plasmids and transposons. 1.2.1. Plasmids They are autonomous genetic formations, extra-chromosomal, free inside the cytoplasm, represented by circular DNA molecules which can replicate independently of chromosome. There are more than 1000 different kinds of plasmids. Plasmids can be: conjugative: plasmids that can transfer alone to other bacteria - ex: antibiotic resistance plasmid – ‘R’ plasmid; non-conjugative: plasmids that cannot leave alone home bacteria, but only through another conjugative plasmid or bacteriophage – ex: plasmid encoding the secretion of β-lactamase from Staphylococcus aureus. Episomes: - extra-chromosomal genetic material that may replicate autonomously or become integrated into the chromosome. Genetic determinants from the plasmids are: - essential (encode information about the autonomous replication) - accessories (encoding nonessential phenotypic characters for bacterial cell survival under natural conditions) – ex: transfer genes (tra), toxins secretions genes, antibiotic resistance genes (R factor) Some plasmids have no phenotypic manifest effect. They are cryptic plasmids. Resistance plasmids to chemotherapy – ‘R’ - are circular DNA molecules, containing one or more genes which encode resistance to one or more antibiotic types. Genetic determinants carried by a plasmid are divided into two main classes: - genes coding for antibiotic resistance - "R", single or multiple - genes conferring plasmid the ability of transfer -"FTR“, even in the absence of F factor. Virulence plasmids : they have genetic determinants which produce the synthesis of virulence factors of bacteria. Examples: - enterotoxin (labile and thermo stable) of Escherichia coli; - colonization factors of Escherichia coli; - hemolysin (Staphylococcus aureus, Escherichia coli). F plasmid (plasmid for sex, fertility factor) contains genes for transfer. F plasmid can be transmitted by conjugation to other bacterial cells. It can integrate into the bacterial chromosome and can mediate chromosomal gene transfer from donor to recipient bacterial cell. Col Plasmids – contain genes that encode for the antibacterial polypeptides called bacteriocins, proteins which can kill other bacterial strains. 1.3. Genetic transposable elements - Fragments of insertions (IS) - Transposons (Tn) - Inverted sequence. II. Bacterial variability Variability of bacteria can be explained by: - phenotype variation: changing of bacterial properties by adapting to environmental conditions, without any intervening change in the bacterial genome; - genotype variation: result from genome modification. Variation in bacteria is achieved by: - mutations, - transfer of genetic material. 1.Mutations A mutation is a spontaneous change in bacterial genome, which changes the nucleotide sequence of a gene. It can be: point, inversions, deletions, insertions, secondary mutations. A. Depending on the number of affected bases mutations can be: point and extended. A.1. Point mutations – can affect one nucleoid within one gene and are reversible. A.2. Extended mutations – are alterations which exceed the limits of a codon, and may affect the larger sequences of one or more genes (polygenic mutations). B. After the manner of appearance, mutations can be: - spontaneous - induced B.1. Natural (spontaneous) mutations – are copying mistakes during self-replication due to self-replication speed. Ex – mutations that cause resistance of bacteria to antibiotics. B.2. Induced mutations – are caused by mutagens: X-rays, UV, biological agents, which determine the modifications of sequences of nitrogenous bases. Consequences of mutations By mutation individuals with new characters can arise – ex. Resistance to chemotherapy. 2. Transfer of genetic material It takes place between two bacterial cells: one know as donor, the other one called receiver. Those processes are: - transformation - transduction - conjugation - transposition 2.1. Transformation It is the transfer of genetic material from a donor cell to a receiver cell as pure DNA released by the donor cell lysis or by chemical extraction. Gene transformation is possible only if the receiver bacterium is in the state of competence, which allow incorporation of the foreign DNA. According to some authors the competent cells have on the surface an particular antigen, called ‘power factor’ and cell wall becomes more porous and electropositive, as they are charged by facilitating foreign DNA. 2.2. Transduction It is a chromosomal gene transfer from one bacterial cell to another, mediated by bacteriophages. Some bacteriophages are able to transfer any bacterial gene (generalized transduction) and some only specific genes (specialized transduction). A. Generalized transduction Is mediated by lytic phage, which after the penetration into the bacterial cell multiplies and causes lysis of the host cell. During lysis of the bacteria, its chromosome fragments. It is possible that one of these fragments with the size close to the phage genome, to integrate into the bacteriophage capsule, instead of phage genome. These bacteriophages will not be able to replicate, but can enter in other bacterial cell, injecting their DNA derived from the donor cell. DNA will integrate into the receiving cell genome by recombination, resulting in new characters such as: pathogenic bacteria changes, resistance to chemotherapeutic agents. B. Specialized transduction Is mediated by temperate phage. After entering in the bacterial cell DNA of temperate phage, DNA undergoes circularisation, then inserts itself into the chromosome by recombination as prophage (based on homology on 10 base pairs of DNA and bacterial phages). Prophage becomes an integrated part of the bacterial chromosome; it will replicate together with it, because it is subject of repression from the part of host genome. 2.3. Conjugation It is the transfer of genetic material (chromosomal or extra- chromosomal) from a donor to a recipient bacterium through a process of mating, which is achieved by direct contact of the two cells. This can transmit plasmids and chromosomal genes (through the F+ factor). The F+ factor can be located in a plasmid, favoring its transfer, or in a bacterial chromosome, favoring the transfer of chromosomal genes from a bacterium to another. 4. Transposition It means the integration into the genome of a transposable genetic element from the same DNA molecule or another but present in the same cell. Transposable genetic elements, by structure and mechanism of translocation, fall into three classes: I, II and III. - Class I consists of insertion sequences (IS) and transposons compounds. - Class II contain the transposons (TnA). - Class III: transposable bacteriophage (phage M11 and D108). ANTIBACTERIAL CHEMOTHERAPY 1. DEFINITION Chemotherapeutics = substances able to exercise in small doses, an inhibitory effect on bacteria. They can be: lethal (bactericidal) or only prevent the multiplication of the microorganisms (bacteriostatic). 2. CHEMOTHERAPY ACTION MECHANISMS Today there are several known mechanisms of action of chemotherapics: - metabolic competitive antagonism, - inhibition of bacterial cell wall synthesis, - alteration of cytoplasmic membrane function, - inhibition of bacterial protein synthesis, - inhibition of bacterial nucleic acid synthesis. 2.1. CELL WALL SYNTHESIS INHIBITORS 2.1.1. a. Betalactams – penicillins and cephalosporins Mechanism of action: Beta-lactams inhibit cell wall synthesis by their binding with enzymes involved in the final stage of this process (PBPs, penicillin binding proteins). Result: prevent cross-linking of polysaccharide chains with secondary accumulation of murein subunits that activate autolytic enzyme system which will lead to bacterial cell lysis. B. Glycopeptides – are polypeptides with big molecule like: Vancomycin and Teicoplanin – they interfere with peptidoglycan elongation, having bactericidal action during bacterial multiplication. C. Bacitracin – due his high toxicity is used only in local application for skin infections. D. Isoniazid, Cycloserine and Ethionamide – are used in tuberculosis. 2.1.2.SURFACTANTS THAT INJURES CYTOPLASMIC BACTERIAL MEMBRANE Mechanism of action: free amino groups acts like cationic detergents on the cell membrane, destroying its phospholipid structures. Of this class are polymyxins. Also in the same way acts Amphotericin B, Colistin, Imidazole, Gramicidin. 2.1.3. INHIBITORS OF PROTEIN SYNTHESIS A. Aminoglycosides Are bactericidal by: - blocking of formyl-methionyl-t-RNA linking to ribosomes, practically preventing the initiation of polypeptide chain synthesis. - an incorrect decoding information on m-RNA by their irreversible binding to ribosomes. B. Tetracyclines (Tetracycline, Doxycycline) – are bacteriostatic antibiotics that inhibit protein synthesis by blocking the attachment of aminoacyl-t-RNA molecules to ribosomes. C. Chloramphenicol – acts by blocking the production of peptides bonds between amino acids. D. Macrolides ( Clarithromycin, Azithromycin) – are bound by ribosomes blocking translocation of peptide chain during its synthesis. E. Lincosamides (Clindamycin) – they bind to ribosomes of prokaryotic cells, preventing peptide bond formation. F. Fusidic acid – blocks protein synthesis by binding elongation factor to polypeptide chain at the guanine-phosphate and the ribosome. 2.1.4. INHIBITORS OF BACTERIAL NUCLEIC ACIDS A. Rifampicins They bind to DNA-dependent RNA polymerase by blocking the synthesis of RNA. B. Quinolones (Nalidixic acid, Fluoroquinolones like Ciprofloxacin, Norfloxacin, Ofloxacin etc.) - are synthetic chemotherapeutics blocking DNA gyrase responsible for supra-twisting DNA. C. Sulphonamides – acts by blocking synthesis of folic acid and DNA. A particularly valuable chemotherapic is Co-trimoxazole (Bactrim or Septra), resulting from association of Trimethoprim with Sulfamethoxazole. It is used in urinary, respiratory and systemic infections. Sulfones are related to sulphonamides. Among them is Dapsone, used to treat leprosy. 3. RESISTANCE TO CHEMOTHERAPICS There are TWO TYPES OF BACTERIAL RESISTANCE TO ANTIBIOTICS: - natural resistance, which is a character of the species, genetically determined; - acquired resistance, which occurs in strains of susceptible species to a natural antibiotic; it is relative and it means the resistance of a strain to the antibiotic concentrations used in therapy. 3.1. ACQUIRED RESISTANCE MECHANISMS 3.1.1. The biochemical paths involved in resistance to antibiotics A. Production of enzyme by bacteria that inactivates the antibiotic, for example penicillinase secreted by Staphylococcus aureus that inactivates the penicillin beta lactam core or different types of beta lactamases produced by Gram negative bacteria. B. Decreased of wall or cytoplasmic membrane permeability of microorganism to antibiotics. C. Development in excess of complementary enzymes by bacteria which limit or cancel antibacterial action of the antibiotic. D. Alteration of intracellular target (modification of ribosomal protein). E. The increase of para-amino benzoic acid synthesis, which cancel the inhibitory action of sulphonamides. Acquired resistance can be: - chromosomal (by developing spontaneous mutations at a place which control the susceptibility to a particular antimicrobial product), - extrachromosomal (produced by plasmids). TYPES OF RESISTANCE A. Resistance to an antimicrobial agent can be: - Monovalent (monoresistance - when the germ is resistant to one antibiotic) - Multivalent (the germs resist to multiple antibiotics - multiple resistance). B. Depending on the rhythm of installation - fast rhythm of resistance (Streptomycin type) - intermediate rhythm (Erythromycin type) - slow rhythm (Penicillin type) - very slow rhythm (Vancomycin type) Bacterial taxonomy. The structure of the bacterial cell: characteristics; shape and dimensions of the bacteria; the structural components of the bacterial cell. The spore. Bacterial cell division. - Lecture 1 - DEFINITIONS MICROBIOLOGY – is the science that studies microorganisms. MICROORGANISMS (BACTERIA, MICROBES) – are prokaryote cells that are not visible with the naked eye, but with a microscope we can observe them, and their dimensions is between 1 – 10 µm. MICROORGANISMS are – bacteria, viruses, parasites and fungus. Basic Classification of Microorganisms Eukaryotes Prokaryotes Small in Size Large in size DNA not separated from Mitochondria present cytoplasm Mitochondria absent Membrane bound Nucleus Contains all enzymes for Contains all enzymes like production of metabolic energy Eukaryotes THE MORPHOLOGY AND STRUCTURE OF THE BACTERIAL CELL I. THE MORPHOLOGY OF BACTERIA Bacteria are unicellular asexual prokaryotes haploid (with unique set of genes), which are dividable into identical cells. 1. Dimensions Dimensions are expressed in micrometers and are between: 1-10. Exceptions: mycoplasma (0.3 to 0.8), anthrax 10, spirochetes 15-20, actinomyces can reach a length up to 500. 2. Form Cocci are: spherical (Staphylococcus), oval (Streptococcus), lanceolate – elongated as a candle flame (Streptococcus pneumoniae), kidney shaped (Neisseria). Bacilli are bacteria to form elongated rods with sizes between 1.5 to 10. Cocobacillus are slightly elongated bacteria, the intermediate forms between cocci and bacilli (Yersinia pestis, Bordetella pertussis). Vibrios are comma-shaped curved bacteria: Vibrio cholera. Spiral and spirochaetes are spiral bacteria, long, very thin (Treponema pallidum, Leptospira). 3. Settlement A. Cocci can be placed: in irregular heaps: grape-like clusters - Staphylococcus; in chains : Streptococcus; in tetrad (four individuals lying symmetrically): Micrococcus; in bales of 8 individuals lying symmetrically – Sarcina; In diplo, as two candle flames which come together by their bases - species Streptococcus pneumoniae; In diplo, as two beans that look face to face by their concavities - Neisseria. B. Bacilli can be found: isolated and in random positions one to the other - Enterobacteriaceae; short chains: Klebsiella; disposed in chains: genus Bacillus; chinese letters:Corynebacterium diphtheriae; in palisades: genus Mycobacterium. II. BACTERIAL CELL STRUCTURE The morphofunctional unit of the bacteria is the cell. Bacteria as cell type, are prokaryotic cells, which are different from the eukaryotic cells by structure and organization. General characteristics of prokaryotic cell: Dimensions – small – 1 to 10 µm Organization – unicellular (populations) Method of reproduction - Direct division (binary fission scissiparity) Nucleus Chromosome unique ring Double helix DNA molecule Nucleolus is absent Nuclear membrane is absent Cytoplasmic membrane – sterols are not present (except Mycoplasma) Cytoplasm - Is unpartitioned - Tubular organelles are absent - Free ribosomes in the cytoplasm - Specific organelles are present: mesosomes, oxysomes, plasmids Peripheral cell body layer - cell wall Annex Elements - cilia, fimbria, capsule Form of resistance - spore Bacteria have a very complex structure: - required/compulsory components - optional components. I. Mandatory components or intrinsic elements: nucleoid cytoplasm cytoplasmic membrane cell wall II. Optional components (cladding or extrinsic) components: cilia or flagella capsule fimbriae or pili I. Intrinsic elements A. Bacterial nucleus Bacterial nuclear material has no nucleolus and nuclear membrane. The nuclear material is dispersed in the cytoplasm, with areas of maximum concentration in the centre of cytoplasmic space. In electronic microscopy we can observe: no nuclear membrane and nucleolus is present, DNA is diffusely spread in the cytoplasm, the chromosome is unique, like ring, consisting of a double-stranded DNA molecule, long, twisted like a "hank’’. Function – the storage of the genetic information, it represent the place of chromosomal heredity and ensures all species specific characters. B. Cytoplasm Cytoplasm is between nucleus and cell membrane. Cytoplasm is a complex colloidal system consisting of about 80% water, organic small molecules, inorganic ions, enzymes and ribonucleic acids (ribosomal RNA, transport RNA, messenger RNA). In bacterial cytoplasm there can also be found: plasmids, vacuoles and inclusions. There are no: endoplasmic reticulum, Golgi apparatus, mitochondria. B.1. Ribosomes Are the main elements of the cytoplasm, they are spherical structures, smaller than eukaryotic cells ribosomes, and it is the base of protein synthesis of the cell.. Complete ribosomes have 70 S (Svedberg sedimentation constant), but in the absence of Mg ions, dissociation of ribosomes occurs in subunits 50S and 30S. B.2. Mesosomes Are membrane structures formed by cytoplasmic membrane invagination. They are present at Gram positive bacteria and occasionally in Gram negative bacteria. Function: - participate in bacterial chromosome replication and cell division. B.3. Plasmids Are extra-chromosome genetic units. They are circular DNA molecules, small, able to replicate independently of the bacterial chromosome and are responsible for extra-chromosomal heredity (they have a role in the transmission of some characters such as antibiotic resistance). B.4. Granulation inclusions They are inert, temporary structural formation, with different sizes, varying according to bacterial species and environmental conditions. They have as chemical composition: Glycogen (Enterobacteriaceea) Starch (sporulated anaerobe germs – Clostridium) Lipids (Bacillus) Polymetaphosphate or volutine inclusions (Babes and Ernst inclusions described at the diphtheria bacilli). B.5. Vacuoles Are spherical formations containing liquids or gases, surrounded by a coating layer lipoprotein. B.6. Oxysomes They are specific organelles of the prokaryotic cell. They are the headquarters of the redox enzymes. In them we can find cytochromes, cytochrome oxidase, flavin- enzymes, and also the enzymes of the Krebs cycle. C. Cytoplasmic membrane (also known as cell membrane or the plasma membrane) It appears as a very thin layer, surrounding the cytoplasm and adheres to the cell wall. It is a fine elastic membrane (6.5-7nm), with no mechanical resistance, it represents the area where the processes of osmotic permeability occur and it shows selective phenomena ("semi-permeable" membrane). Particularity: in the prokaryotic cell membrane sterols are absent, except Mycoplasma. Cytoplasmic membrane has the following functions: It is a semi-permeable membrane, which regulates the exchanges between bacterial cell and the external environment; Can produces hydrolytic enzymes; Participate in the chemotactic systems; It is the target cellular structure for detergents, that, used as disinfectant substances, alter his structure; It is also a target for some antibiotics interfering with the biosynthetic function of the membrane (polymyxins). D. Cell wall It ensures shape and stiffness of the cell. Structures: The basal layer The surface layer. The basal layer is similar in all bacteria; surface layer is differentiated for Gram positive bacteria, Gram negative bacteria and acid-alcohol resistant bacteria. D.1. The structure of the cell wall D.1.1. The basal layer This layer is composed of peptidoglycan (murein) network. Peptidoglycan network (murein) is the chemical structure responsible for the rigidity of the cell wall and provide shape and mechanical strength of the bacteria. It is present in all bacteria and have three parts: D.1.2. Surface layer It includes three types of special structures: Gram positive bacteria, Gram negative bacteria, acid-alcohol resistant bacteria. D.1.2.a. Gram-positive bacteria (special features) The cell wall of the Gram-positive bacteria is relatively thick, but with a simpler composition. Peptidoglycan is about 50 - 90% of the dry weight of the wall, has a thickness of 15-30 nm and contains up to 200 parallel mureine chains, linked into a three-dimensional thick network. D.1.2.b. Gram-negative bacteria (special features) The wall is thinner, but more complex structured, multi-layered. Special structural layer is much more complex than the Gram positive bacteria, consisting of: - periplasmic space - outer membrane - the lipopolysaccharide (LPS) wall. D.1.2.c. Alcohol acid-resistant bacteria These bacteria have cell walls similar to Gram-positive bacteria. Special structures contain lipids 30%, almost half of which are represented by mycolic acid and wax, which gives these bacteria tinctorial special characters and resistance to environmental factors. D.1.2.d. Bacteria with altered wall or L shaped They are bacteria with bad basal layer under the action of environmental factors (eg: lysozyme that lyses peptidoglycan or penicillin which inhibits his synthesis). Bacteria with the lack of the cell wall are called protoplasts and are coming from the Gram-positive bacteria. Bacteria with wall partially damaged are called spheroplasts and are coming from the Gram-negative bacteria. If the harmful factors disappear from the environment, L forms can turn into normal bacteria by resynthesis of the cel wall. D.1.2.e. Bacteria without cell wall These bacteria have a naturally free cell wall, do not have constant shape, their form is variable depending on the environment in which they live. Example: Mycoplasma (pear, elongated or filamentous). D.2. Functions of cell wall provides shape, mechanical and osmotic resistance of bacteria; involved in cell division and its growth; stores some enzymes in the periplasmic space of the Gram negative bacteria; has receptors for bacteriophages; has based surface antigens; is the seat of pathogenicity factors; has role in the sporulation; it’s the target for some antibiotics. II. Annex (extrinsic) elements A. Bacteria cilia (flagella) They are hair-like structures mobile, very long, thin, fragile. They are involved in locomotion. The number of cilia is characteristic to each species. They have been categorized in : atrichous (flagella is absent), monotrichous – a single flagellum at one pole (Vibrio cholera), lophotrichous – flagella are present at one pole (Pseudomonas fluorescens), amphitrichous – with flagella at both poles (Spirillum) and peritrichous – with flagella on the entire surface of the bacteria (Salmonella, E. coli, Proteus). The bacterial flagellum is made up of a protein = flagellin. The flagella structure (highlighted in electronic microscopy) is: - basal body - hook - basal filament A.1. Basal body (basal granulate) is a component by which attaches to the bacterial cell body. It is mounted entirely in the cell wall and cytoplasmic membrane. It consists of four parallel discs, arranged in two pairs on a rod passing through their center. Cilia are organs of locomotion for bacteria (mobility) and their mobility is associated with chemotaxis property. Chemotaxis property is directed movement to or from a chemical substance. Cilia are the site of flogging antigens (H), which are important in identification of some bacteria (Salmonella). In addition, it can act as receptor for viruses and in some cases it can cause adhesion to epithelia. B. Fimbriae (pili) Fimbriae or pili are extra short, rigid and thick extensions, especially highlighted at Gram-negative bacteria (Enterobacteriaceae, Neisseria gonorrhoea) and less at Gram positive bacteria (Streptococcus, Corynebacterium). These elements are stronger than flagella with avaible peritrichal disposal and with highlighter only in electronic microscopy. From the chemical point of view - the fimbriae are composed of pilin proteins. Functional pili are divided in two categories: B.1. Common pili or pili of adherence - They are in a great number (100 – 200) on the cell surface. - Role: in adherence to various surfaces (named adhesines), especially to epithelia; - They are virulence factors (ex. gonococcus) with anti phagocytosis role. - Their synthesis is determined by chromosomal genes. B.2. ‘’Sex’’ pili – are longer formations, in number of 1-4/cell, always coded by extra chromosomal genetic formations (plasmids). These pili are particularly important in transfer of genetic material between bacteria, forming bridges between cell donor and recipient during conjugation process. These are present mainly in Gram-negative bacteria (Enterobacteriaceae). B.3. Capsule Is an extracellular coating cell wall with a compact structure and a limited aqueous solubility. From the chemical point of view: has a polysaccharide structure, forming a tight network across the cell wall (Streptococcus pneumonia) or having a lamellar structure (Klebsiella). Rarely the capsule can have proteic nature (ex. anthrax bacillus). Role in pathogenicity of bacteria (virulence factor): capsule prevents phagocytosis of bacteria and by that they can escape from the defence mechanisms of the body. In encapsulated species the loss of capsule results in loss of virulence. The capsule is a structure with specific antigenic properties that allow differentiation of serotypes within the species. Example: over 88 antigenic types are described based on pneumococcal capsule. Capsule functions: - it protects bacteria from various antibacterial agents from environment such as: bacteriophage, complement, lysosyme and other bacteriolytic enzymes - it protects bacteria from phagocytes action (virulence factor) - the seat of capsular antigens which are important in the identification of these bacteria B.4. Microcapsule It has a discrete structure with a thickness below 0.2m. It is a virulence factor (Neisseria gonorrhoea). Glycocalyx It is an amorphous layer with a polysaccharide structure, usually seen as a network around one cell or several bacterial cells, even if not belonging to the same species. Glycocalyx is present in bacteria only in their natural competitive environment. It is a structure that can mediate adhesion : - nonspecific attachment on different types of surfaces - or a specific attachment to the surface of certain cell types. Ex: Pseudomonas aeruginosa can produce a thick mucus layer which increase antibiotic resistance. III. The spore The spore is the resistant form of bacteria in unfavorable terms of development. The bacterial spore has a high resistance to environmental factors, to physical agents (heat, radiation), chemical substances (acids, disinfectants), dyes (for highlighting them we use special stains). From a vegetative bacteria a single spore forms, which in favorable environmental conditions, will give birth to a single bacterial cell with all the original properties. Form: spherical or oval. Diameter is lower in sporulated aerobic bacilli, the diameter not exceeding bacteria (Genus Bacillus), while in anaerobic (Clostridium) the diameter of the spore is higher than the bacteria, producing its misshape. Position of the bacterial spore – may be central (Clostridial gas gangrene), subterminal (anthrax bacillus) or terminal (Clostridium tetani). In common stains spore appears as a colorless zone bacterial body. It is highlighted in particular coloration like Moller coloration. A. Spore structure From the inner to the outer spore consists of: 1. „Core” or protoplast of the spore – consists of nuclear material surrounded by cytoplasmic membrane. Here DNA is stored, the complete genome, some respiratory enzymes (cytochromes, flavoproteins) and a substance which play a role in heat resistance of the spore - calcium dipicolinate; 2. Spore wall (internal cortex) - is the primary celuler wall. 3. Spore cortex (outer membrane), the thickest layer of spore, contains a peptidoglycan, with particular structure, very sensitive to lysozyme. Autolysis of it is the key moment in the transformation of spore in vegetative form. 4. Spore coat - composed of chitin proteins with disulfide bonds. It is waterproof and cause resistance to some disinfectants. 5. Exosporium - contain lipoproteins and sugars and it is present only in some spores. C. Bacterial cell division Bacterial cell divide according to binary fusion - direct division - scissiparity, resulting in the appearance of two daughter cells with equal size and identical genetic material. Bacterial cell division comprises of two phases: septation and separation, nuclear division. 1). Septation and separation It starts with invagination of the cytoplasmic membrane in association with mesosome (often at their own level). This produces a completely transverse sept, thicker than the cell wall. 2). Nuclear division Bacterial chromosome segregation involves several sequences: - chromosome ring attachment to mesosome - separation of the two chains of a DNA chromosomal molecule eith the twisting of the two DNA chains in double direction, with their removal into space with the mesosome as leaning point - mesosome’s division along its axis; - appearance of the two daughter cells, each having one parental DNA chain, anchored to its own mesosome; - each daughter cell formation from of a new DNA complementary chain Result: each of the two daughter cells resulting from division will usually carry genetic material identical to that contained in the parental cell chromosome - Half of the parental cell DNA - ("semiconservative“ division model). BACTERIAL METABOLISM Definition: Bacterial metabolism = all biochemical reactions that occur in bacterial cell and the ones caused by the microorganism in the environment, reactions aimed at bacterial growth and multiplication. Includes: A. Catabolic reactions – the process through which the nutrient substrate descomposes, with energy release. B. Intermediate metabolic reactions – the energy resulting from the first reaction is stored in macroergic compounds. C. Anabolic reactions – the process through which bacterial cell syntetize their substances with energy consumption.
