Medical Microbiology Prof. Dr. Jie YAN (严杰) Department of Medical Microbiology and Parasitology E-mail: med_bp@zju.edu.cn School of Medicine Zhejiang University Introduction to medical microbiology Microbes / Microorganisms •The word “microbe” comes from the Greek words mikros, meaning small life. So microbes / microorganisms are small living things that are too small to be seen by naked eye. •Microorganisms were probably the first organisms to appear on the earth. •However, these organisms were not seen until about 3 centuries ago when lenses powerful enough to make them visible were made. •Viruses, bacteria, fungi, protozoa and some algae are all in this category. Distribution •The distribution of microorganisms is universal in nature including air, soil, water, animals and human body. Relationship with human beings •There is a close relationship between microorganisms and human beings. • Beneficial activities: Most microbes are benefit to human beings, some are necessary (nitrogen and arbon cycles). • Harmful activities: Only a small portion of microbes cause human diseases, which called pathogenic microbes. Medical Microbiology • Medical microbiology is a branch of Microbilogy to study biological character, pathogenicity and immunoty, laboratory diagnosis, and prevention and control of pathogenic microbes. Microbes in nitrogen cycle Prokaryotes / Eukaryotes • The prokaryotic cell, in contrast to the eukaryotic cell, has no nuclear membranes, mitochondria, endoplasmic reticulum, Golgi body, phagosomes and lysosomes. • Prokaryotes generally possess only a single circular chromosome, which is bound to a specific site on the cell membrane - the mesosome. • Prokaryotic ribosomes are 70S (30S and 50S subunits) in size, whereas eukaryotic ribosomes are larger (80S, 40S and 60S subunits). Classification of microbes •According to organizational structure, microbes can be divided into three types: Prokaryotes (Eubacteria and Archaebacteria) Eukaryotes (fungi, Protozoa, algae) Acellular entities (viruses) •Eubacteria include Bacteria, Chlamydiae, Mycoplasmas, Richettiae, Spirochetes, and Actinomycetes. Some of them cause human diseases. Viruses •Viruses are very small particles and have no basic cell structure. A simplest virus consists of one core and one protein coat (capsid). The core composed with a nucleic acid molecule, either DNA or RNA. •Viruses are obligate parasites totally dependent on their host cells for replication. Fungi •Fungi is a kind of eukaryotic cells. So they have various organelles, for examples, nuclear membranes, mitochondria, endoplasmic reticulum, Golgi body, phagosomes and lysosomes. New challenge in medical microbiology •The numerous emerging and re-emerging infectious diseases such as AIDS, SARS, avian influenza, tuberculosis, viral hepatitis and so on. Bacteriology Morphology and Structure of Bacteria Size of bacteria • Unit for measurement of bacteria is micrometer (μm) • On the average, bacteria are 2-8 μm in length and 0.2-2.0 μm in diameter. Exceptions include some spiral shaped bacteria that can reach 4- 500 μm. 1000 Shape of bacteria • Spherical (Cocci, sing. Coccus ) • Rods (Bacilli, sing. Bacillus) • Spiral (Spiral bacteria) vibrio spirillum helicobacterium Spherical bacteria Different arrangements depending on the plane of division Diplococci: Pair of cells divide in one plane Streptococci: Chain of cells formed by dividing in one plane several times Tetrad: Divide in two planes Sarcinae: Divide in three planes Staphylococci: Divide in many planes and remain together as a cluster Rod-shaped bacteria • Considerable variation in length and diameter: 0.5-1 μm in width and 2-5 μm in length. • Most of rod-shaped bacteria are single arrangement. Diplobacilli: Bacilli that remain in pairs after they divide. Streptobacilli: Bacilli that remain in chains after they divide. Coccobacilli: A short Bacilli that nearly looks like a cocci. Spiral-shaped bacteria Divided into: Vibrio: comma shaped Spirillum: helical Structure of Bacteria • bacterial structures may be defined: Cell envelope Plasmids Flagella Pili Capsules Spores Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Sspores Cell envelope •Bacterial envelope is divided into cell membrane and cell wall (Gram positive) plus an outer membrane (Gram-negative). Gram-positive cocci Gram-negative bacilli (Gram-staining method) Cell wall: general component-peptidoglycan •Cell wall consists of peptidoglycan layer and attached structures. Gram-positive Gram-negative Peptidoglycan •glycan backbone: N-acetyl muramic acid and N-acetyl glucosamine are alternatively linked by -1,4 linkage. •4-peptide side chain: links to N-acetyl muramic acid. •peptide bridge: links side chains (gram-negative bacteria have no peptide bridges). •Penicillin can block the linkage between peptide side chain and bridges to kill gram-positive bacteria. Cell wall: characteristits of gram-positive bacteria •Peptidoglycan layer is thick (15-50 layers). •There are some special components such as teichoic acids, the major superficial antigen of gram-positive bacteria . Cell wall: characteristits of gram-negative bacteria •Peptidoglycan layer is thin (1-2 layers). •There is outer membrane located in outside of peptidoglycan layer but no any teichoic acids. Outer membrane •Outer membrane of a gram-negative bacterium is composed of phospholipids, membrane proteins and lipopolysaccharide (LPS) Lipopolysaccharide (LPS) •LPS is also called endotoxin (poisonous to mammal cells). •LPS has 3 regions: an external O antigen, a middle core, and an inner lipid A. • O antigen is a polysaccharide to act as the somatic antigen of gram-negative bacteria. •Core polysaccharide links O antigen with lipid A. •lipid A decides toxicity. Cell wall: function • Maintaining bacterial shape. • Resistance to osmotic pressure • Providing a platform for surface appendages such as flagella and pili. • Providing a pathogenic function to adhere host cells (For gram-positive bacteria, the major adhesin is teichoic acids. For Gram-negative bacteria, the major adhesin is pili and some of outer mambrane proteins). • Playing an essential role in bacterial division • Participating bacterial material exchange • Containing major antigens. Wall-less forms of bacteria • When bacteria are treated with 1) enzymes (e.g. lysozyme) with cell wall hydrolytic activity or 2) antibiotics inhibiting peptidoglycan synthesis, wall-less bacteria are generated which is called L-forms of bacteria. • L-forms of bacteria can cause chronic infections. • L-forms of bacteria are difficult to cultivate and usually require a medium with a right osmotic strength. • It is resistant to antibiotics (e,g. penicillin) and difficulty to detect (e.g. absence of O antigen). Electron micrograph of Staphylococcus A: L-form; B: wild type Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Spores Plasmids •Plasmids are small, circular / line, extra-chromosomal double-stranded DNA. •Usually present in multiple copies and are capable of self-replication. •Often code for pathogenic factors and antibiotic resistant factors. Are not essential for bacterial survival. Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Spores) Flagella: general description • Flagellum is composed of flagellin and provide motility. •It extends from cell envelope and projects as a long strand. •Flagellum is slender that can not be seen by light microscopy unless a special stain is applied. Flagella: structure • Basal body: a structure to insert into cell envelope. • Flagellin is an antigen (H antigens). Flagella: function •Motility of bacteria: move towards foodstuffs or away from toxic materials. •Identification of bacteria: According to the mobility and antigenicity of flagellin (H antigen). •Possible pathogencity: chemotaxis to the suitable sites in hosts for colonization. Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Spores Pili •Pili are hair-like strands of bacteria. •They are shorter and thinner than flagella, only visible under electron microscope. Pili •Pilus is composed of special protein called pilin. •Two types can be distinguished: Ordinary pili •Shorter, thinner, numerous for a bacterium •Relative to bacterial adhesion (adhering to host cells) •Contribute to virulence of some pathogenic bacteria Sex pili •Longer, coarser, only 1-4 for a bacterium •Relative to bacterial conjugation (a pattern of bacterial genetic material exchanges) •The recent data revealed the sex pili of some bacteria has the ability to adhere host cells. Ordinary pili Sex pili Recipient Donor bacterium Electron graph of pili Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Spores Capsules and slime layers •Capsule is a structure surrounding outside of cell envelope. •Usually, slime layer is thinner than capsule. •They are usually demonstrated by the negative staining or “capsule stain” which gives color to the background. Capsules and slime layers •They are usually composed of polysaccharide. However, in some certain bacilli, they are composed of polypeptide. • They are not essential to bacterial viability. • Some strains within a bacetrial species can produce a capsule, whereas the others can not. •Capsules are often lost during in vitro culture. •The capsules contribute to invasiveness (virulence) of bacteria by protecting them from phagocytosis by phagocytes. Important bacterial structures Cell envelope Plasmids Flagella Pili Capsules Spores Spores • Under adverse conditions, such as nutrient / water depletion, some bacteria form a thick wall inside the cytoplasmic membrane leading to a resting stage known as spores. •Spores contribute to bacterial resistance. Spores •One spore-forming bacterium can only produce one spore which has no propagation ability. •One spore germinates into one vegetative bacterial cell which can propagate / multiplication. •Spore can be seen after staining with dyes. Sometimes, it can also be seen as a colorless area by using conventional bacterial staining methods. •Spores are commonly found gram-positive bacilli. •Different sizes, shapes and positions of spores will help us to identify spore-forming bacteria. Structure of spores Core spore wall /core Cortex Coat Exosporium Classification of bacteria •Taxonomic terms: Family: a group of related genera. Genus: a group of related species. Species: a group of related strains. Type: sets of strains within a species (e.g. biotypes, serotypes). Strain: one line or a single isolate of a particular species. •The basic taxonomic group is species. strain type species genus family O157:H7 Coli Escherichia Enterobacteriaceae Classification of bacteria Staphylococcus aureus S. aureus Genus species 金黄色葡萄球菌 species genus Summary Structure of bacteria include essential structures of cell wall, cell membrane, cytoplasm, and nuclear material (nucleoid). Some bacteria also have one or more of the particular structures of capsule, flagella, pili, endospores. Structure of cell wall, cell wall structural differences between Gram-positive and Gram-negative bacteria, concept of plasmid, and functions of bacterial particular structures are the most important contents, because of their close association with bacterial pathogenesis. Growth, Propagation and Metabolismof Bacteria Nutrtion types of bacteria • Autotroph: can synthesize organic substances using CO2 as carbon source and N2 or NH3 as nitrogen source. The energy comes from oxidation of inorganic substances. • Heterotroph: use different organic substances, such as proteins, saccharides and lipids, as the nutrient substances or materials and energy source. ▲Saprophyte: dead bodies of animals and plants, or decomposed foods. ▲Parasite: living hosts (animals and/or human). Nearly all the pathogens are parasites. Nutrient substances of bacteria • Water: mediator for biological responses. • Carbon source • Nitrogen source • Inorganic salts: have many functions to act as a component of organic substance as well as to maintain enzymatic activity and osmotic pressure and pH, etc. • Growth factors: vitamins, some special amino acids, hemoglobin and coenzyme I or II (blood, serum) . Conditions of bacterial growth and propagation • • • • Nutrient substances pH: 7.2-7.4 for microbial pathogens. Temperature: 37ºC for microbial pathogens. Gas: O2 ▲Obligate aerobe: needs O2 during growth and propagation. ▲Microaerophilic bacteria: 5% O2. ▲Facultative anaerobe: grow and propagate in aerobic or anaerobic enviroment. ▲Obligate anaerobe: has no special enzymes (e.g. SOD and catalase) to deal with ROS such as O ¯2 and H2O2 produced in metabolism. Bacterial growth and propagation • Growth and propagation of a bacterial individual: binary fission (2n), a process in which a parent cell splits into two daughter cells with approximately equal size. a. Bacterial cell first can been seen to enlarge or elongate. a b. Followed by formation of membrane and new cell wall. b transverse c. The new membrane and cell wall grow inward from the outer layers. d. The cell divided into the two daughter cells. c d Bacterial growth and propagation • Generation time: under optimal conditions, the average time required for a population of bacteria to double in number. 20-30 min for most of bacteria (e.g. E. coli). • Colony: a bacterial cluster from propagation of a bacterium. ▲Obtain a pure bacterial species. ▲Often used for bacterial counting. Bacterial growth and propagation • Growth and propagation of a bacterial population: Growth curve Stationary Phase OD600 Log Phase Death Phase Lag Phase Time Bacterial growth and propagation • Phenomena of bacterial growth in liquid medium Broth (a common liquid medium) cultures can exhibit: (i) forming cloudiness in broth (growth with uniform turbid pattern), or (ii) forming a ring at the top of broth (growth with suspension pattern), or (iii) forming sediment at the bottom of broth (growth with sedimentary pattern). i ii iii Constructive metabolism of bacteria • Pyrogen: cause fever (LPS of G- bacteria and glycopeptide or glycolipid of G+ bacteria). • Toxins: exotoxins and endotoxin (LPS). • Invasive enzymes: e.g. collagenase (invasion and spreading) and coagulase (resist phagocytosis of macrophages). • Others: pigment, vitamine, antibiotic, bacteriocin (细菌素). Destructive metabolism of bacteria For identification of bacteria ! • Carbohydrate Fermentation Tests Positive: yellow color or yellow color with gas bubble Negative: red color and no gas bubble Destructive metabolism of bacteria Methyl Red (MR) Test hydrolyse pyruvate (丙酮酸) Destructive metabolism of bacteria Voges-Proskauer (VP) Test hydrolyse pyruvate (丙酮酸) → diacetyl (二乙酰) Destructive metabolism of bacteria Citrate Utilization Test The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Destructive metabolism of bacteria Indole Test hydrolyse tryptophan to produce indole Destructive metabolism of bacteria Hydrogen Sulfide (H2S ) Formation Test To determine the ability of a bacterium to produce hydrogen sulfide (H2S) by enzymatic reaction on amino acids such as cysteine, cystine and methionine. Positive result: The hydrogen sulfide combines with ferrous sulfide (Fe2S) in the triple sugar iron (TSI) agar to form a black to dark insoluble precipitate. Destructive metabolism of bacteria Urease Test Principle: The hydrolysis of urea by urease produces ammonia and carbon dioxide. The formation of ammonia alkalinizes the medium, and the pH is detected by the color change from light orange to pink-red. Positive result: pink-red color Negative result: light orange Death of Microorganisms Disinfection & Sterilization Concept and Definition Sterilization: A physical or chemical process to kill all microbial life including spores. Disinfection: A physical or chemical process to kill vegetative microbes, but not kill spores. Bacteriostasis: A physical or chemical process to inhibit bacterial growth / propagation in vitro and in vivo. Antisepsis: A physical or chemical process to inhibit bacterial growth / propagation in vitro, but not kill bacteria. Asepsis: a state of being free of living microbes. Antimicrobial agents ▲ Physical Agents: Heat, Radiation, Filtration, Low Temperature and Desiccation (Dry) ▲ Disinfectants and Antiseptics Physical antimicrobial agents: Heat ▲ A temperature of 100 ºC (boiling) usually for 2-5 min will kill all vegetative forms but not kill spores. ▲ A temperature of 121 ºC for 15-20 min will kill all microorganisms including spores (autoclave). ▲ Hot air sterilization by hot air ovens, heating at 160 ºC for 2 h, Physical antimicrobial agents: Ultraviolet Ray ▲ Microbial killing effect of sun light is due in large part to the action of ultraviolet light. ▲ Activity of ultraviolet (UV) ray depends on: i) Length of exposure: 30 min; ii) Wavelength of UV ray: 260 nm - 270 nm thymine-thymine dimmers within the one DNA strand will block base pairing and DNA replication. Summary The most important contents in this lecture are displayed as the followings: 1) Bacteria growth curve, especially the characteristics and application of log phase and maximum stationary phase. 2) Concepts of sterilization, disinfection and asepsis, and the temperature and time to kill bacteria including spores when using autoclaving and hot air sterilization. 3) The microbicidal mechanism and application limits of UV radiation. 4) The types (names) of bacteria based on the difference of O2 requirement in growth. 5) Concepts of bacterial colony, pyrogen and invasive enzymes.