SIGNIFICANCE OF MICROBIOLOGY IN PRACTICAL ACTIVITY OF DOCTORS. THE HISTORY OF MICROBIOLOGY. CLASSIFICATION AND STRUCTURE OF MICROORGANISMS. Chair of Microbiology, Virology, and Immunology

Lecture schedule 1.History of Microbiology. 2.Classification of bacteria. 3.Structure of bacterial cell

Microbiology is a science, which study most shallow living creatures - microorganisms. Before inventing of microscope humanity was in dark about their existence. But during the centuries people could make use of processes vital activity of microbes for its needs. They could prepare a koumiss, alcohol, wine, vinegar, bread, and other products. During many centuries the nature of fermentations remained incomprehensible.

Brueghel: The Triumph of Death (1560)

Microbiology learns morphology, physiology, genetics and microorganisms systematization, their ecology and the other life forms. Specific Classes of Microorganisms  Algae  Protozoa  Fungi (yeasts and molds)  Bacteria  Rickettsiae  Viruses  Prions

The Microorganisms are extraordinarily widely spread in nature. They literally ubiquitous forward us from birth to our death. Daily, hourly we eat up thousands and thousands of microbes together with air, water, food. On our skin, in mouth and nasal cavities, on mucous membranes and in bowels enormous amount of microorganisms live and act. Many of them are found in earth cortex and in the air, and in the ocean’s, sea’s, river’s water, on all of latitudes, mainlands and continents.

For the first time term “microbe" was offered by French scientist Sh. Sedillot in It derives from Greek “microbe", that means briefly living, or most shallow living creature. Science, which learns the microorganisms, was named by E. Duclaux microbiology. For short development period this science accumulated great factual material. The separate microbiological branches such as bacteriology, mycology, protistology, virology quickly appeared.

Comparative sizes of Bacteria

Periods of microbiology development Morphologic Physiologic Prophylactic

Development of microbiological science was interlinked with art of glass and diamonds grinding. This brought to creation of the first microscope by Hans and Zacharian Jansen in Holland in The discovery of microorganisms is associated with the name of Antony van Leeuwenhoek ( ).

In 1683 Leeuwenhoek described the basic bacterium forms. His scientific supervisions Leeuwenhoek described in special letters and sent off them to the London Royal Scientific Society. He sent away about 300 letters. The Leeuwenhoek’s letters brought on enormous surprise among English scientists. They opened a fantastic world of invisible creatures. He named them “living animals" (animalcula viva) and in one of letter wrote: “In my mouth there are more animacula viva, than peoples in all United Kingdom".

These wonderful discovery of Dutch naturalist were the embryo, with which science of bacteria developed. Namely from these times starts the so-called morphological period in microbiology history (XVII middle of age). It is also called micrographycal period, as the study of microorganism came only to description of their dimensions and forms. Biological properties and their significances for man still a long time remained incomprehensible.

However, using the primitive microscopes of that time it was difficult to determine the difference between separate bacteria species. Even celebrated founder of scientific systematization of all of living organisms Karl Linney renounced to classify the bacteria. He gave them general name “chaos".

In the second half of XIX century microbiology strongly affirms as independent science. Namely these sciences were fruitful soil, on which Pasteur's talent evinced. He studied wine "illness“, fermentation, made Pasteurization method, offered to grow microbes on artificial nutrient media, he proved, that on definite cultivation conditions the pathogenic bacteria lose its virulence, made vaccine against anthrax, rabies. Physiological period has began

Not less important are scientific works of celebrated German scientist R. Koch. He performed classic researches on etiology of anthrax, opened tuberculosis bacilli, cholera vibrio, proposed to isolate pure bacterial cultures on solid nutrient media (gelatin, potatoes), developed the preparations staining methods by aniline dye-stuffs, method of hanging drop for examination of bacteria motility, offered apparatus for sterilization

The Patriarch of world and Ukrainian microbiology - I. Metchnikov He studied inflammation pathology, phagocytosis, bases about antagonism of bacteria. From all microbes-antagonists I.Metchnikov preferred the lactic bacteria. On their base he offered three medical preparations - sour clotted milk, yogurt and lactobacillin. Now they are called by eubiotics. Classic Metchnikov's researches defined a prophylactic period in microbiology history.

In 1892 D. Ivanovskiy described an virus of mosaic tobacco illness – new class of infectious agents

Microorganisms constitute a very antique group of living organisms which appeared on the Earth's surface almost 3000 million years ago.

There are natural and artificial classifications system. Bergey's Manual of Determinative Bacteriology - the "bible" of bacterial taxonomy.

Classifying Bacteria Bergey’s Manual of Systematic Bacteriology –Classifies bacteria via evolutionary or genetic relationships. Bergey’s Manual of Determinative Bacteriology –Classifies bacteria by cell wall composition, morphology, biochemical tests, differential staining, etc.

Idea: Whitaker – five kingdoms (1969)

The Three-Domain System Figure 10.1 Idea: Woese- domains (1978) Prokaryotes

Comparison of the three domains Characteristic EubacteriaArchaeaEucarya Cell typeProkaryote Eukaryote Cell wall Peptidoglycan Varies Membrane lipids UnbranchedBranchedUnbranched Sensitive to antibiotics? YesNo Circular chromosome? Yes No (except in mitochondria and chloroplasts) Histones?NoYes

Prokaryotes

Classification Systems in the Procaryotae 1.Microscopic morphology 2.Macroscopic morphology – colony appearance 3.Physiological / biochemical characteristics 4.Chemical analysis 5.Serological analysis 6.Genetic and molecular analysis G + C base composition DNA analysis using genetic probes Nucleic acid sequencing and rRNA analysis

Bacterial Taxonomy Based on Bergey’s Manual Bergey’s Manual of Determinative Bacteriology – five volume resource covering all known procaryotes –classification based on genetic information – phylogenetic –two domains: Archaea and Bacteria –five major subgroups with 25 different phyla

Taxonomy Domain Kingdom Phylum Class Order Family Genus species

Major Taxonomic Groups of Bacteria Vol 1A: Domain Archaea –primitive, adapted to extreme habitats and modes of nutrition Vol 1B: Domain Bacteria Vol 2-5: –Phylum Proteobacteria – Gram-negative cell walls –Phylum Firmicutes – mainly Gram-positive with low G + C content –Phylum Actinobacteria – Gram-positive with high G + C content

Microbial Phylogeny Phylogeny of domain Bacteria –The 2nd edition of Bergey’s Manual of Systematic Bacteriology divides domain Bacteria into 23 phyla.

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Proteobacteria –The largest group of gram-negative bacteria –Extremely complex group, with over 400 genera and 1300 named species –All major nutritional types are represented: phototrophy, heterotrophy, and several types of chemolithotrophy –Sometimes called the “purple bacteria,” although very few are purple; the term refers to a hypothetical purple photosynthetic bacterium from which the group is believed to have evolved

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Proteobacteria (cont.) –Divided into 5 classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Proteobacteria (cont.) –Significant groups and genera include: »The family Enterobacteriaceae, the “gram-negative enteric bacteria,” which includes genera Escherichia, Proteus, Enterobacter, Klebsiella, Salmonella, Shigella, Serratia, and others »The family Pseudomonadaceae, which includes genus Pseudomonas and related genera »Other medically important Proteobacteria include genera Haemophilus, Vibrio, Camphylobacter, Helicobacter, Rickessia, Brucella

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Firmicutes –“Low G + C gram-positive” bacteria –Divided into 3 classes »Class I – Clostridia; includes genera Clostridium and Desulfotomaculatum, and others »Class II – Mollicutes; bacteria in this class cannot make peptidoglycan and lack cell walls; includes genera Mycoplasma, Ureaplasma, and others »Class III – Bacilli; includes genera Bacillus, Lactobacillus, Streptococcus, Lactococcus, Geobacillus, Enterococcus, Listeria, Staphylococcus, and others

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Actinobacteria –“High G + C gram-positive” bacteria –Includes genera Actinomyces, Streptomyces, Corynebacterium, Micrococcus, Mycobacterium, Propionibacterium Phylum Chlamidiae –Small phylum containing the genus Chlamydia

Microbial Phylogeny Phylogeny of domain Bacteria (cont.) Phylum Spirochaetes –The spirochaetes –Characterized by flexible, helical cells with a modified outer membrane (the outer sheath) and modified flagella (axial filaments) located within the outer sheath –Important pathogenic genera include Treponema, Borrelia, and Leptospira Phylum Bacteroidetes –Includes genera Bacteroides, Flavobacterium, Flexibacter, and Cytophyga; Flexibacter and Cytophyga are motile by means of “gliding motility”

Procaryotae Kingdom has 4 Divisions according to the structure of cell wall and Gram staining: Gracilicutes (gracilis - thin, cutis - skin) – Gram-negative bacteria, Firmicutes (firmus - firm) – Gram-positive bacteria, Tenericutes (tener – soft, tender) – microbes without cell wall, Mendosicutes (mendosus - mistaket) – microbes with atipical peptidoglican

Bacterial Nomenclature Binomial naming system –Two word naming system First word is genus name –Always capitalized Escherichia Second word is species name –Not capitalized coli When writing full name genus usually abbreviated –E. coli Full name always italicized –Or underlined

Species is population of microbes, which have the only source of origin, common genotype, and during the present stage of evolution are characterized by similar morphological, biochemical, physiological and other signs

If deviations from the typical species properties are found on examination of the isolated bacteria, then culture is considered a subspecies. Infrasubspecies subdivisions serovar (antigenic properties) morphovar (morphological properties) chemovar (chemical properties) biovar (biochemical or physiological properties) pathovar (pathogenic properties) phagovar (relation to phages)

The term clone was applied to population of cells derived from a single cell Population is an elementary evolutional unit (structure) of a definite species The term strain designates a microbial culture obtained from the different sources or from one source but in different time Or: A subgroup within a species with one or more haracteristics that distinguish it from other subgroups in the species

Morphological Classification of Bacteria Bacteria (Gk. bakterion - small staff) are unicellular organisms lacking chlorophyll. Morphologically, bacteria possess four main forms: spherical (cocci) rod-shaped (bacteria, bacilli, and clostridia) spiral-shaped (vibrios, spirilla and spirochaetes) thread-shaped (non-pathogenic)

Cocci groupings Coccus Diplococcus Streptococcus Tetrad Sarcinae Staphylococcus

Cocci (Gk. kokkos berry). These forms of bacteria are spherical, ellipsoidal, bean-shaped, and lanceolate. Cocci are subdivided into six groups according to cell arrangement, cell division and biological properties Micrococci (Micrococcus). The cells are arranged singly or irregularly. They are saprophytes, and live in water and in air ( M. roseus, M. luteus, etc.).

Diplococci (Gk. diplos double) divide in one plane and remain attached in pairs. These include: Meningococcus (causative agent of epidemic cerebrospinal meningitis, and gonococcus, causative agent of gonorrhoea and blennorrhoea) Pneumococcus (causative agents of pneumonia)

Streptococci (Gk. streptos curved, kokkos berry) divide in one plane and are arranged in chains of different length. Some streptococci are pathogenic for humans and are responsible for various diseases.

Tetracocci (Gk. tetra four) divide in two planes at right angles to one another and form groups of fours. They very rarely produce diseases in humans.

Staphylococci (Gk. staphyle cluster of grapes) divide in several planes resulting in irregular bunches of cells, sometimes resembling clusters of grapes. Some species of Staphylococci cause diseases in man and animals

Sarcinae (L. sarcio to tie) divide in three planes at right angles to one another and resemble packets of 8, 16 or more cells. They are frequently found in the air. Virulent species have not been encountered

Rods. Rod-shaped forms are subdivided into:  bacteria,  bacilli,  clostridia Bacteria include those microorganisms which, as a rule, do not produce spores (colibacillus, and organisms responsible for enteric fever, paratyphoids, dysentery, diphtheria, tuberculosis, etc.). Bacilli and clostridia include organisms the majority of which produce spores (hay bacillus, bacilli responsible for anthrax, tetanus, anaerobic infections, etc.)

According to their arrangement, cylindrical forms can be subdivided into three groups: monobacteriamonobacilli E. coliY. pestis C. tetani C. botulinum

diplobacteriadiplobacilli K. pneumoniae

streptobacteriastreptobacilli Haemophilus ducreyi (chancroid) Bacillus anthracis (anthrax)

Spiral-shaped bacteria Vibriones (L. vibrio to vibrate) are cells which resemble a comma in appearance. Typical representatives of this group are Vibrio cholerae, the causative agent of cholera, and aquatic vibriones which are widely distributed in fresh water reservoirs.

Spirilla (L. spira coil) are coiled forms of bacteria exhibiting twists with one or more turns. Only one pathogenic species is known {Spirillum minus} which is responsible for a disease in humans transmitted through the bite of rats and other rodents (rat-bite fever, sodoku)

Spirochaetes (L. spira curve, Gk. chaite cock, mane) differ from bacteria in structure with a corkscrew spiral shape Borrelia. Their cells have large, obtuse-angled, irregular spirals, the number of which varies from 3 to 10. Pathogenic for man are the causative agents of relapsing fever transmitted by lice (Borrelia hispanica), and by ticks (Borrelia persica, etc.). These stain blue-violet with the Romanowsky-Giemsa stain

Leptospira (Gk. leplos thin, speira coil) are characterized by very thin cell structure. The leptospirae form 12 to 18 coils wound close to each other, shaping small primary spirals. The organisms have two paired axial filaments attached at opposite ends (basal bodies) of the cell and directed toward each other. Leptospira interrogans which is pathogenic for animals and man cause leptospirosis

Treponema (Gk. trepein turn, nema thread) exhibits thin, flexible cells with 6-14 twists. The micro-organisms do not appear to have a visible axial filament or an axial crest when viewed under the microscope A typical representative is the causative agent of syphilis Treponema pallidum

Properties of prokaryotes and eukaryotes ProkaryotesEukaryotes The nucleoid has no membrane separating it from the cytoplasm Karyoplasm is separated from the cytoplasm by membrane Chromosome is a one ball of double twisted DNA threads. Mitosis is absent Chromosome is more than one, There is a mitosis DNA of cytoplasm are represented in plasmids DNA of cytoplasm are represented in organelles There aren’t cytoplasmic organelle which is surrounded by membrane There are cytoplasmic organelle which is surrounded by membrane The respiratory system is localized in cytoplasmic membrane The respiratory system is localized mitochondrion There are ribosome 70S in cytoplasm There are ribosome 80S in cytoplasm Peptidoglycan are included in cell’s wall (Murein) Peptidoglycan aren’t included in cell’s wall

The structure of procaryotes

Nucleus. The prokaryotic nucleus can be seen with the light microscope in stained material. It is Feulgen- positive, indicating the presence of DNA. Histonelike proteins have recently been discovered in bacteria and presumably play a role similar to that of histones in eukaryotic chromatin The DNA is seen to be a single, continuous, "giant" circular molecule with a molecular weight of approximately 3 X The unfolded nuclear DNA would be about 1-3 mm long (compared with an average length of 1 to 2 µm for bacterial cells)

Plasmids: R, Col, Hly, Ent, Sal Plasmids  small circular, double-stranded DNA  free or integrated into the chromosome  duplicated and passed on to offspring  not essential to bacterial growth & metabolism  may encode antibiotic resistance, tolerance to toxic metals, enzymes & toxins  used in genetic engineering- readily manipulated & transferred from cell to cell  There may be several different plasmids in one cell and the numbers of each may vary from only one to 100s in a cell

Prokaryotic Ribosome  A ribosome (70 S) is a combination of RNA and protein, and is the site for protein synthesis  Composed of large (50S) and small (30S) subunits  S = Svedverg unit, measures molecular size The 80S ribosomes of eukaryotes are made up of 40S and 60S subunits.

Storage granules –Metachromatic granules –Polysaccharide granules –Lipid inclusions –Sulfur granules –Carboxyzomes –Magnetosomes Gas vesicles Inclusions, granules

Volutin granules Corynebacterium diphtheriae Loeffler's techniqueNeisser's staining

Composted of A. The cytoplasmic membrane To act as a physical barrier btw cytoplasm and environments and selectively controls the movement of substaces into and out of the cell “Semipermeable” B. Cell wall The rigid layer that protect the fragile cytoplasmic membrane from rupturing To maintains cell’s shape C. Capsule or slime layer (glycocalyx) Cell Envelope

Cell membrane Peripheral Membrane Protein Integral Membrane Protein Peripheral Membrane Protein Phospholipid Bacterial plasma membrane are composed of 40 percent phospholipid and 60 percent protein. The phospholipids are amphoteric molecules with a polar hydrophilic glycerol "head" attached via an ester bond to two nonpolar hydrophobic fatty acid tails, which naturally form a bilayer in aqueous environments. Dispersed within the bilayer are various structural and enzymatic proteins which carry out most membrane functions.

Mesosome

The predominant functions of bacterial membranes are: 1. Osmotic or permeability barrier; 2. Location of transport systems for specific solutes (nutrients and ions); 3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase; 4. Synthesis of membrane lipids (including lipopolysaccharide in Gram- negative cells); 5. Synthesis of murein (cell wall peptidoglycan); 6. Assembly and secretion of extracytoplasmic proteins; 7. Coordination of DNA replication and segregation with septum formation and cell division; 8. Chemotaxis (both motility per se and sensing functions); 9. Location of specialized enzyme system.

Unique chemical structure –Distinguishes Gram positive from Gram-negative –bacteria and archaea bacterial species Rigidity of cell wall is due to peptidoglycan (PTG) –Compound found only in bacteria –Archaea –psudomurein or other sugars, proteins, glycoproteins Many antimicrobial interfere with synthesis of PTG Penicillin; Lysozyme Cell wall

Basic structure of peptidoglycan –Alternating series of two subunits N-acetylglucosamin (NAG) N-acetylmuramic acid (NAM) –Joined subunits form glycan chain Glycan chains held together by string of four amino acids –Tetrapeptide chain: L-ala-D-glu-DAP-D-ala L-ala-D-glu-Lys-D-ala Interpeptide bridge Structure of peptidoglycan

Structures associated with gram-positive and gram-negative cell walls. Differences of cell wall structure in Gram- positive and Gram negative cells

L Forms

Glycocalyx  Capsule Protects bacteria from phagocytic cells  Slime layer Enable attachment and aggregation of bacterial cells

Capsules Most prokaryotes contain some sort of a polysaccharide layer outside of the cell wall polymer Only capsule of B. anthracis consist of polypeptide (polyglutamic acid)

Capsule  The capsule is covalently bound to the cell wall.  Associated with virulence in bacteria.  Example: Streptococcus pneumoniae

Slime Layer  The slime layer is loosely bound to the cell.  Carbohydrate rich material enhances adherence of cells on surfaces  Example: Streptococcus mutans and “plaque formation”

Biofilms  The slime layer is associated with cell aggregation and the formation of biofilms  Example: Staphylococcus epidermidis biofilms on catheter tips

Adhesion Avoidance of immune response Protection from dehydration Protection of bacterial cells from engulfment by protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. They provide virulent properties of bacteria (S. pneumoniae, B. anthracis) General capsule function

Flagella 3 parts –filament – long, thin, helical structure composed of proteins –hook- curved sheath –basal body – stack of rings firmly anchored in cell wall rotates 360 o 1-2 or many distributed over entire cell functions in motility

Flagellar arrangements 1. Monotrichous – single flagellum at one end (cholera vibrio, blue pus bacillus), 2. Lophotrichous – small bunches arising from one end of cell (blue- green milk bacillus, Alcaligenes faecalis) 3. Amphitrichous – flagella at both ends of cell (Spirillum volutans), 4. Peritrichous – flagella dispersed over surface of cell, slowest E. coli, salmonellae of enteric fever and paratyphoids A and B

Bacterial Motility The rotation of the flagella enables bacteria to be motile. Flagella are important for:  Motility (dispersal)  Antigenic determinant  Number and location species specific

Pili and Fimbriae Short, hair-like structures on the surfaces of procaryotic cells Proteinaceuse filaments ( ~ 20 nm in diameter) Very common in Gram-negative bacteria Functions: –Adherence to surface/ substrates: teeth, tissues –Involved in transfer of genetic information btw cells –Have nothing to do with bacterial movement (Except the twitching movement of Pseudomonas) Fimbriae are smaller than flagella and are important for attachment

Bacterial endospores Bacterial spores are often called “endospore” (since they are formed within the vegetative cell) Produced in response to nutrient limitation or extreme environments Highly resistant Highly dehydrated (15% water) Metabolically inactive Stable for years Not reproductive Functions: to survive under an extreme growth conditions such as high temperature, drought, etc. Bacillus, Clostridium, Sporolactobacillus, Thermoactinomyces, Sporosarcina, Desulfotomaculum species sporulate

Spore

Spores Key compositions: –Dipicolinic acid (DPA) –Calcium (Ca 2+ ) Structure –Core / Cytoplasm –Plasma membrane –Core wall/ spore wall –Cortex –Spore coat –Exosporium

Endospores

The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor. Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components.

Spores are located: 1)Centrally (B. anthracis); 2) Terminally (С. tetani); 3) Subterminally (C. botulinum, C. perfringens)

The spores of certain bacilli are capable of withstanding boiling and high concentrations of disinfectants. They are killed in an autoclave exposed to saturated steam, at a temperature of  C, and also at a temperature of  C in a Pasteur hot-air oven.

Important Point: