Introduction to Microbiology and Bacteriology PDF

Introduction to Microbiology & Bacteriology Presenter: C. Thoms-Rodriguez Department of Microbiology B Med Sci MBBS, DM OBJECTIVES ⚫ At the end of this lecture students should have had a general introduction to microbiology and become familiar with the following: ⚪ ⚪ ⚪ ⚪ classification of bacte...

Introduction to Microbiology & Bacteriology Presenter: C. Thoms-Rodriguez Department of Microbiology B Med Sci MBBS, DM OBJECTIVES ⚫ At the end of this lecture students should have had a general introduction to microbiology and become familiar with the following: ⚪ ⚪ ⚪ ⚪ classification of bacteria structure of bacteria laboratory diagnosis of bacterial infections pathogenesis and the host response for bacterial infections Introduction ⚫ Microbiology: the scientific study of microorganisms. The study of: ⚪ ⚪ ⚪ ⚪ bacteria-bacteriology viruses-virology fungi-mycology parasites-parasitology ⚫ Many disease-causing microorganisms have been identified and means of controlling their harmful effects have emerged from scientific study. Classification of living organisms KINGDOM Types of Organisms MONERA bacteria, blue-green algae, and spirochetes ?VIRUSES PROKAR YOTES (PRIMITIVE NUCLEUS) PROTISTA FUNGI protozoans and algae of various types molds, mushrooms, yeasts, mildews, and smuts PLANTAE mosses, ferns, woody and non-woody flowering plants ANIMALIAE sponges, worms, insects, fish, amphibians, reptiles, birds, and mammals EUKARY OTES Prokaryotes vs Eukaryotes CHARACTERISTIC EUKARYOTE PROKARYOTES SIZE >5UM 0-5 – 3.0 UM NUCLEUS Classic Membrane No nuclear membrane CHROMOSOMES Strands of DNA; Diploid Genome Single circular DNA; Haploid genome MITOCHONDRIA Present Absent GOLGI BODIES Present Absent ENDOPLASMIC RETICULUM Present Absent NUCLEAR STRUCTURES CYTOPLASMIC STRUCTURES Prokaryotes vs Eukaryotes CHARACTERISTIC EUKARYOTES PROKARYOTES Ribosomes (Sedimentation coefficient) 80S (60S + 40S) 70S (50S + 30S) Cytoplasmic membrane Contains Sterols Does not contain sterols Cell Wall Present for fungi otherwise absent Present and complex Reproduction Sexual and asexual Asexual (binary fission) Movement Complex flagellum if present Simple flagellum if present Respiration Via mitochondria Via cytoplasmic membrane CYTOPLASMIC STRUCTURES CONT. Basic Eukaryotic Cell Structure Basic Prokaryotic Cell Structure Structure of Viruses ⚫ Many diff structures ⚪ Helical, icosahedral, complex ⚫ Virus classification (Baltimore) ⚪ I: dsDNA viruses II: ssDNA viruses III: dsRNA viruses IV: (+)ssRNA viruses V: (−)ssRNA viruses VI: ssRNA-RT viruses VII: dsDNA-RT viruses BACTERIOLOGY History of Bacteriology ⚫ Development of microscopy: ⚫ Aristotle (384-322) –lens applications ⚫ 1590: Hans and Zacharias Janssen (Dutch lens grinders) mounted two lenses in a tube to produce the first compound microscope. ⚫ It was Antony Van Leeuwenhoek (1632-1723), a Dutch draper became the first man to make and use a real microscope. ⚪ (father of the microscope) History of bacteriology ⚫ 1660: Robert Hooke : improved upon the microcope; published drawings and detailed observations of biological materials made with the best compound microscope and illumination system of the time. ⚫ 1931: Ernst Ruska constructed the first electron microscope Antony Van Leeuwenhoek Joseph Lister ⚫ 1827 –1912: was an English surgeon and antiseptic surgery, who promoted the idea of sterile surgery while working at the Glasgow Royal Infirmary. ⚫ Lister successfully introduced carbolic acid to sterilise surgical instruments and to clean wounds, which led to reduced post-operative infections and made surgery safer for patients. Ferdinand Julius Cohn ⚫ (1828 –1898) German biologist classified algae and plants, and to define what distinguishes them from green plants. ⚫ His classification of bacteria into four groups based on shape (spherical, short rods, threads, and spirals Heinrich Herman Robert Koch ⚫ Robert Koch 1843 –1910 famous for isolating Bacillus anthracis (1877), the Tuberculosis bacillus (1882) and the Vibrio cholera (1883) and for his development of Koch's postulates. Koch’s Postulates ⚫ to establish that an organism is the cause of a disease it must be: ⚪ ⚪ ⚪ ⚪ found in all cases of the disease examined prepared and maintained in a pure culture capable of producing the original infection, even after several generations in culture retrievable from an inoculated animal and cultured again. ⚫ This eventually led to: ⚪ Development of pure culture techniques ⚪ Stains, agar, culture media, petri dishes ⚪ Bacillus for anthrax, and tuberculosis, vibrio for cholera Louis Pasteur ⚫ (1822-1895) discovered that most infectious diseases result from germs, known as the "germ theory of disease," (1857) ⚫ convinced surgeons germs existed carried diseases, and dirty instruments and hands spread germs and therefore disease. ⚫ Pasteur's pasteurization process killed germs and prevented the spread of disease. Ignaz Semmelweiss ⚫ (1818-1865), a Hungarian physician who decided that doctors in Vienna hospitals were spreading childbed fever (puerperal sepsis secondary usually to S. pyogenes) while delivering babies. He started forcing doctors under his supervision to wash their hands before touching patients 1847. History ⚫ 1882-3 Ziehl confirmed/modified staining technique today called Ziehl-Neelson stain ⚫ 1884-Gram described staining- aid in distinguishing bacterial groups ⚫ 1886-Escherich isolated the colon bacillus (E. coli) ⚫ 1898-Shiga isolated the variety of dysentery bacillus (Shigella) Classification Classification ⚫ Describes the diversity of bacterial species by naming and grouping organisms based on similarities. ⚫ Bacteria can be classified on the basis of: ⚪ ⚪ cell structure, cellular metabolism or on differences in cell components (DNA fatty acids, pigments, antigens and quinones) Classification ⚫ Identification of bacteria in the laboratory is particularly relevant in medicine, where the correct treatment is determined by the bacterial species causing an infection. ⚫ Consequently, the need to identify human pathogens was a major impetus for the development of techniques to identify bacteria. Phenotypic Classification ⚫ Bacteria are also then classified by several criteria: ⚫ Microscopic and Macroscopic morphologies: ⚪ Shape ⚪ Stain ⚪ Haemolytic properties on blood agar ⚪ Aerobic / anaerobic growth ⚪ Size, shape and odour of colonies ⚪ Growth characteristics/requirements ⚪ DNA content (G, C content) ⚪ homology Bacterial shapes Gram Positive Cocci (GPC): Staphylococci GPC:Streptococci Gram Negative Cocci: Neisseria Gram Negative Rods/Bacilli (GNB): E. coli Gram Positive Rods/ Bacilli (GPB): Bacillus spp. The Cell Wall All prokaryotes have a cell wall excepting Mycoplasmas. It helps to determine the shape of the bacteria and maintain its structural integrity. It has antigenic potential. The Cell Wall THE GRAM STAIN ```as it relates to cell structure! The difference in gram staining by prokaryotes, which puts them into two major groups ( GN and GP) is thought to be related to the difference in structure of their cell wall but the actual reason remains unclear. This will be evaluated further. There are organisms , acid fast bacilli who have an even different cell wall from these two major groups of bacteria and again their cell wall must be considered separately. Heamolysis on blood agar Phenotypic classification ⚫ Biochemical markers ⚪ Fermenting capacity, specific enzymes, ⚫ Biotyping/ serotyping ⚪ Epidemiological purposes ⚫ Antibiogram patterns and phage typing Environmental considerations pH – 6.8 Temperature psychrophilic 15-20oC mesophilic 30-37oC (most pathogens) Thermophilic 50-60oC Atmosphere O2 for Aerobes ANO2 for Anaerobes CO2 (5-10%) for Capnophiles Criteria Groups Examples SHAPE Cocci Staphylococcus, Neisseria Bacilli Bacillus, Listeria, Salmonella Spirilla Vibrio, Campylobacter Gram positive Gram Negative Acid-Fast Staphylococcus, Streptococcus, Bacillus Special Stains Clostridia spores STAIN Haemophilus, Escherichia, Salmonella Mycobacterium Criteria Groups Gas Strict Requirements Aerobes Examples Pseudomonas aeruginosa Facultative anaerobes Escherichia coli Strict anaerobes Clostridium spp. Capnophiles Neisseria spp. Specialised features Spores Clostridium spp. Enzymes Coagulase producing Staphylococcus Antibiotic Resistance MRSA Antigens Streptococcus (Lancefield groups), Chlamydia Gram Positive Cocci (GPC): Staphylococci GPC:Streptococci Gram Negative Cocci: Neisseria Gram Negative Rods/Bacilli (GNB): E. coli Gram Positive Rods/ Bacilli (GPB): Bacillus spp. Identification ⚫ Picture of bacterial cultures on Agar. ⚫ Can be differentiated based on morphology and colour of colonies. Basic Techniques used in Microbiology ⚫ Isolation of pathogenic bacteria from clinical specimen (patient) ⚫ Identification of isolate ⚫ Determination of antimicrobial susceptibility of isolate ⚫ Samples: blood, sputum, urine, swabs, aspirates etc. Isolation/Identification ⚫ Direct Microscopy: Darkfield e.g. spirochetes and Light microscopy e.g. for motility ⚫ Staining: Negative staining e.g. India ink for Cryptococccus ⚫ Gram stain: Differentiates bacteria into two groups: Gram positive bacteria - purple Gram negative bacteria - red Isolation/Identification ⚫ Other stains: ⚪ Ziehl Neelson (ZN) for TB ⚪ Auramine rhodamine for TB ⚪ Giemsa ⚪ Fontana-silver stain for spirochaetes. Isolation/Identification Culture: - to isolate and observe bacteria: Basal media e.g. Nutrient broth, Nutrient agar Enriched media e.g. Blood agar, “Chocolate” agar Differential Media e.g. MacConkey’s agar Isolation/Identification ⚫ Carbon source , enzyme activators ⚫ Nitrogen source (energy)/ minerals or ions Mg++, Ca++, Fe++, K+ Sulphur source ⚫ Growth factors e.g. Vitamin B, amino acids, blood, yeast extract ⚫ Phosphorus – trace elements Identification using Electron Microscopy ⚫ Electron micrograph of colonies on agar ⚫ Observe the single rods/ bacilli Serological Classification ⚫ Lancefield grouping: ⚪ A Serological classification dividing haemolytic streptococci into groups (A to O) which bear a definite relation to their sources, based upon precipitation tests depending upon group-specific substances that are carbohydrate in nature; e.g., ⚪ Group A ------- ----strains pathogenic for man; ⚪ Group B------------ strains from mastitis in cows , normal milk, human throat and vagina; ⚪ Group C------------ strains from various lower animals including a number from cattle; ⚪ Group D----------- strains from cheese; ⚪ Group E------------strains from milk; ⚪ Group F------------strains mainly from the human throat, associated with tonsillitis; ⚪ Group G----------- strains from man, monkeys and dogs; ⚪ Groups H, K, and O, non-pathogenic strains from normal human respiratory tract. Genotypic Identification ⚫ Identifies a degree of relatedness between organisms ⚫ The specific region of DNA that has proved to be the most informative is 16S rRNA ⚫ This gene is present in all bacteria, ⚫ e.g. The 16S rRNA gene of E. coli is 1,542 nucleotides long Genetic Identification ⚫ Initially classified by ratio of guanine to cytosine ⚫ Dna Hybridization ⚫ PCR ⚫ Nucleic Acid sequence analysis ⚫ Plasmid analysis ⚫ Ribotyping ⚫ Analysis of chromosomal DNA fragments Bacterial Pathogenesis and Host Response Bacterial Pathogenesis ⚫ Which is the pathogen: normal flora vs pathogen (koch’s postulates) ⚫ Bacteria use virulence factors to cause disease ⚫ Steps involved in the infectious process that often employs virulence factors ⚪ Entry into host-capsule allows organisms to avoid host defences ⚪ Adherence to host cells-pilli ⚪ Invasiveness-collagenase; hyaluronidase ⚪ Maintaining nutrition- Iron sequestering ⚪ Avoidance of killing (phagocytosis)- capsule; antigenic variation ⚪ Causing damage using further virulence factors-exo and endotoxins Host Response/Host-mediated Pathogenesis ⚫ The pathogenesis of many bacterial infections is caused by the host response rather than by bacterial factors. ⚫ Tissue damage is often caused by inflammatory mediators ⚫ Inflammation: ⚪ Rubor-redness ⚪ Calor-heat/fever ⚪ Dolor-pain ⚪ Tumor-swelling ⚪ Loss of funtion Host Response/Host-mediated Pathogenesis ⚫ When a person overcomes a bacterial infection it is a facilitated by the immune response ⚪ ⚪ Cell-mediated response-eg phagocytosis Humoral response-antibody dependent Nomenclature Nomenclature ⚫ Generally, a bacterial species can be defined as a group of different isolates or strains that: 1. share a large number of characteristics 2. can be distinguished from other species because of differences in certain characteristics. NOMENCLATURE ⚫ 1. 2. ⚫ ⚫ ⚫ All Living organisms have 2 names Generic/genus name: Staphylococcus Specific /species name (epithet): aureus Third name distinguishes varieties ⚪ Acinetobacter calcoaceticus var antitratus ⚪ Common names: Pneumococcus, Gonococcus Serological group names: Group A Streptococcus (a.k.a. S. pyogenes) Toxin product name : Clostridium perfringens type A Nomenclature ⚫ Proper family names can be recognized because they always end with "-aceae". The informal name for this important group of bacteria is the "enteric bacteria". ⚫ - In the same way: ⚪ ⚪ ⚪ ⚪ related families: order (Enterobacteriales) related orders: sections related sections: Kingdoms (phyla) eubacterial kingdoms: Domain (Eubacteria) Nomenclature ⚫ Domain:Bacteria Phylum:Proteobacteria Class:Gamma Proteobacteria Order:Enterobacteriales Family:Enterobacteriac eae Genus:Escherichia Species:coli Binomial name ⚫ Escherichia coli (Migula 1895) Nomenclature ⚫ Domain: Bacteria Kingdom: Eubacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: S. aureus Binomial name ⚫ Staphylococcus aureus (Rosenbach 1884) Rules of Nomenclature ⚫ The names are usually Latinized and italicized or underlined not both ⚫ The genus name begins with an upper case letter, the specific name does not. ⚫ The generic name may be abbreviated when designating a species. ⚫ Informal or trivial names are not underlined (nor italicized) and do not begin with upper case letters The End ACKNOWLEDGEMENTS: SPECIAL THANKS TO MRS L. RAINFORD AND DR. G. REYNOLDS FOR THEIR CONTRIBUTION OF SLIDES