Microbiology Lecture Notes PDF

MICROBIOLOGY CIMS 0121 Mr. A khalif ©2012 1 jimmy ongori 2013 03/11/2013 21:18  LECTURE ONE INTRODUCTION AND HISTORY OF MICROBIOLOGY 2 jimmy ongori 2013 03/11/2013 21:18...

MICROBIOLOGY CIMS 0121 Mr. A khalif ©2012 1 jimmy ongori 2013 03/11/2013 21:18  LECTURE ONE INTRODUCTION AND HISTORY OF MICROBIOLOGY 2 jimmy ongori 2013 03/11/2013 21:18 Lectures on;  Monday 10.00 – 12.00  Tuesday 4-6pm 3 jimmy ongori 2013 03/11/2013 21:18  INTRODUCTION Microbiology is the study of microorganisms - organisms which are of microscopic dimensions. These organisms are too small to be perceived by the unaided human eye. If an object has a diameter of less than 0.1mm, the eye can not perceive it at all, and very little detail can be perceived in an object with a diameter of I mm. 4 jimmy ongori 2013 03/11/2013 21:18  INTRODUCTION Cont’ Generally organisms with a diameter of 1 mm or less are microorganisms and fall into the broad domain of microbiology. Since most microorganisms are only a few thousandths of a millimetres in size they can only be seen with the aid of microscope These include protozoa, algae, fungi and bacteria. Viruses are ultramicroscopic and have an obligate parasitic relationship, but they still come under the domain of microbiology. 5 jimmy ongori 2013 03/11/2013 21:18  What is Microbiology?  Micro - too small to be seen with the naked eye  Bio - life  Ology - study of 6 jimmy ongori 2013 03/11/2013 21:18  Organisms included in the study of Microbiology 1. Bacteria  Bacteriology 2. Protozoans  Protozoology 3. Algae  Phycology 4. Parasites  Parasitology 5. Yeasts and Molds  Fungi  Mycology 6. Viruses  Virology 7 jimmy ongori 2013 03/11/2013 21:18 EARLY HISTORY OF MICROBIOLOGY Historians are unsure who made the first observations of microorganisms, but the microscope was available during the mid-1600s, and an English scientist named Robert Hooke made key observations. In 1665 he observed strands of fungi among the specimens of cells he viewed. He proposed the Cell Theory - all living things are made up of cells 8 jimmy ongori 2013 03/11/2013 21:18  History of Microbiology In the 1674, Anton van Leeuwenhoek was 1st person to view microorganisms Made careful observations of microscopic organisms, which he called animalcules. Until his death in 1723, van Leeuwenhoek revealed the microscopic world to scientists of the day and is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria. 9 jimmy ongori 2013 03/11/2013 21:18  Spontaneous generation Theory that life just ―spontaneously‖ developed from non-living matter After van Leeuwenhoek died, the study of microbiology did not develop rapidly because microscopes were rare and the interest in microorganisms was not high. In those years, scientists debated the theory of spontaneous generation, which stated that microorganisms arise from lifeless matter such as beef broth. 10 jimmy ongori 2013 03/11/2013 21:18 History  In 1668 the theory of spontaneous generation was disputed by Francesco Redi, who showed that fly maggots do not arise from decaying meat (as others believed)  To prove this he covered the meat with gauze to prevent entry of flies. Hence they could not deposit their eggs. No maggots appeared 11 jimmy ongori 2013 03/11/2013 21:18 Schleiden and Schwann Formulated Cell Theory: cells are the fundamental units of life and carry out all the basic functions of living things Pasteur, FR and Tyndall, UK (1861) Finally disproved Spontaneuous Generation. Joseph Lister, UK (1867) Used phenol (carbolic acid) to disinfect wounds First aseptic technique in surgery 12 jimmy ongori 2013 03/11/2013 21:18 Robert Koch, (1876) Postulates – Germ theory (1876) Identified microbes that caused anthrax (1876), tuberculosis (1882) and cholera (1883) Developed microbiological media & streak plates for pure culture (1881) Gave rise to Koch’s postulates 13 jimmy ongori 2013 03/11/2013 21:18  Koch’s Postulates 1. The specific causative agent must be found in every case of the disease. 2. The disease organism must be isolated from the lesions of the infected case and maintained in pure culture. 3. The pure culture, inoculated into a susceptible or experimental animal, should produce the symptoms of the disease. 4. The same bacterium should be re-isolated in pure culture from the intentionally infected 14 animal. jimmy ongori 2013 03/11/2013 21:18  Pasteur’s Contribution to Microbiology  Pasteur showed that microorganisms are not evenly distributed in the atmosphere and that their number varies from place to place.  For this, he took a large number of sealed flasks containing boiled and cooled infusions and opened a few at a time for a short period at various places and resealed. 15 jimmy ongori 2013 03/11/2013 21:18 Pasteur designed special “swan-necked flasks” with a boiled meat infusion Shape of flask allowed air in (vital force) but trapped dust particles which may contain microbes 16 jimmy ongori 2013 03/11/2013 21:18 Out of the 20 flasks which he opened and resealed on a dusty road, 8 showed spoilage Out of the 20 that he opened on the top of a mountain, only five showed spoilage Out of 20 that he opened near a glacier, only one showed spoilage. During the short time that the flasks were open, air had rushed into the flasks carrying along with it the microorganisms. After resealing and incubation, only those flasks which got the microbes from the air showed growth and spoilage. 17 jimmy ongori 2013 03/11/2013 21:18 From these experiments he concluded that the air contained microbes and the number of microorganisms in the atmosphere varied from place to place He also made an intensive study of the beer and wine manufacturing processes and causes of souring and spoilage of beer and wines. He found that wine spoilage was caused by the growth of undesirable contaminating microbes which produced the so called "disease". 18 jimmy ongori 2013 03/11/2013 21:18 The solution to this problem lay in preventing the growth of undesirable organisms. After considerable experimentation Pasteur showed that wine did not undergo spoilage if it was held for a few minutes at 50 to 60°C. In the same way, he found that beer could also escape the ―disease‖ by heating to 50-55° C. This gave rise to the new process of preserving wine, fruit juices, milk etc., and was called "Pasteurization". 19 jimmy ongori 2013 03/11/2013 21:18  The Germ theory of Disease 1835: Agostino Bassi showed a silkworm disease was caused by a fungus. 1865: Pasteur believed that another silkworm disease was caused by a protozoan. 1840s: Ignaz Semmelwise advocated hand washing to prevent transmission of puerperal fever 1860s: Joseph Lister used a chemical disinfectant to prevent 20 jimmy ongori 2013 surgical wound infections 03/11/2013 21:18 1796: Edward Jenner inoculated a person with cowpox virus. The person was then protected from smallpox. Called vaccination from vacca for cow The protection is called immunity 21 jimmy ongori 2013 03/11/2013 21:18  Chemotherapy – treatment with chemicals  1910: Paul Ehrlich developed a synthetic arsenic drug, salvarsan, to treat syphilis.  1930s: Sulfonamides were synthesized  1928: Alexander Fleming discovered the first antibiotic - penicillin, that killed Staphylococcus aureus. 22 jimmy ongori 2013 03/11/2013 21:18 DEFINITION OF SOME TERMS i. Pathogen – disease causing microorganism ii. Infection – the process of transmission of disease by a disease causing MO iii. Pathogenesis – process by which disease starts and develops within the body 23 jimmy ongori 2013 03/11/2013 21:18  Benefits of MOs  Maintain balance of environment (microbial ecology)  Basis of food chain  Nitrogen fixation  Photosynthesis  Digestion, synthesis of vitamins  Manufacture of food and drink, drugs  Genetic engineering  Synthesis of chemical products  Recycling sewage  Bioremediation: use microbes to remove toxins (oil spills)  Use of microbes to control crop pests  Normal microbiota 24 jimmy ongori 2013 03/11/2013 21:18  LECTURE TWO BACTERIAL GROWTH 25 jimmy ongori 2013 03/11/2013 21:18  GROWTH Growth is the orderly increase in the sum of all the components of an organism. Increase in size that results when a cell takes up water or deposits lipid or polysaccharide is not true growth. Cell multiplication is a consequence of growth; in unicellular organisms, growth leads to an increase in the number of individuals making up a population or culture. 26 jimmy ongori 2013 03/11/2013 21:18  GROWTH Cont’ Microbial concentrations can be measured in terms of;  cell concentration (the number of viable cells per unit volume of culture) or  biomass concentration (dry weight of cells per unit volume of culture). Culture - is a method of propagating/multiplying microbial (living tissue cells) in media conducive to their growth 27 jimmy ongori 2013 03/11/2013 21:18  GROWTH Cont’ Most Bacteria Reproduce by Binary Fission The cell doubles in size Replicates the chromosome (DNA) Forms a septum in the center Synthesizes a Cell Wall at the Septum Daughter cells separate. 28 jimmy ongori 2013 03/11/2013 21:18  Binary fission 29 jimmy ongori 2013 03/11/2013 21:18  GROWTH Cont’  All microorganisms undergo similar growth patterns.  Each growth Curve has 4 Phases  Lag phase  Logarithmic phase  Stationary phase  Death phase 30 jimmy ongori 2013 03/11/2013 21:18  Microbial Growth Curve Stationary phase # cells / ml Log phase Death phase Lag phase Time 31 jimmy ongori 2013 03/11/2013 21:18  1. LAG PHASE The lag phase represents a period during which the cells, depleted of metabolites and enzymes as the result of the unfavorable conditions that existed at the end of their previous culture history, adapt to their new environment. Enzymes and intermediates are formed and accumulate until they are present in concentrations that permit growth to resume. 32 jimmy ongori 2013 03/11/2013 21:18  LAG PHASE If the cells are taken from an entirely different medium, it often happens that they are genetically incapable of growth in the new medium. In such cases a long lag may occur, representing the period necessary for a few mutants in the inoculum to multiply sufficiently for a net increase in cell number to be apparent. 33 jimmy ongori 2013 03/11/2013 21:18  2. LOGARITHMIC/EXPONENTIAL PHASE During the exponential phase, the cells are in a steady state. New cell material is being synthesized at a constant rate, but the new material is itself catalytic, and the mass increases in an exponential manner. This continues until one of two things happens: either one or more nutrients in the medium become exhausted, or toxic metabolic products accumulate and inhibit growth. 34 jimmy ongori 2013 03/11/2013 21:18  2. LOG PHASE For aerobic organisms, the nutrient that becomes limiting is usually oxygen. Population doubles every generation Microbes are sensitive to adverse conditions e.g.  Antibiotics  Anti-microbial agents 35 jimmy ongori 2013 03/11/2013 21:18  3. STATIONARY PHASE Eventually, the exhaustion of nutrients or the accumulation of toxic products causes growth to cease completely. In most cases, however, cell turnover takes place in the stationary phase: There is a slow loss of cells through death, which is just balanced by the formation of new cells through growth and division. When this occurs, the total cell count slowly increases although the viable count stays constant. 36 jimmy ongori 2013 03/11/2013 21:18  3. STATIONARY PHASE Cells begin to encounter environmental stress due;  Lack of nutrients  Lack of water  Not enough space  Metabolic wastes  Oxygen  pH 37 jimmy ongori 2013 03/11/2013 21:18  4. DEATH PHASE (PHASE OF DECLINE) After a period of time in the stationary phase, which varies with the organism and with the culture conditions, the death rate increases until it reaches a steady level. Death due to limiting factors in the environment In most cases the rate of cell death is much slower than that of exponential growth. After the majority of cells have died, the death rate decreases drastically, so that a small number of survivors may persist for months or even years. This persistence may in some cases reflect cell turnover, a few cells growing at the expense of nutrients released from cells that. 38 jimmy ongori 2013 03/11/2013 21:18  REQUIREMENTS FOR GROWTH 39 jimmy ongori 2013 03/11/2013 21:18 Growth refers to increase in number of cells In terms of a single cell it is seen as an increase in size and mass over time Growth requirements can be divided into; 1. Physical – temperature, pH, osmotic pressure 2. Chemical – water, micro and macro-nutrients, organic growth factors 40 jimmy ongori 2013 03/11/2013 21:18  TEMPERATURE It is one of the most important environmental variables affecting microbial growth Every MO possesses a characteristic range of temperatures over which it can grow For a particular MO there is a minumum, maximum and optimum temperature  Minimum – lowest temp’ at which the spp. Can grow  Optimum – temp’ at which spp grows best  Maximum – highest temp’ at which growth is possible 41 jimmy ongori 2013 03/11/2013 21:18  TEMPERATURE Psychrophilic 0-200 C but grow best at low temperatures (15–20 °C) Mesophilic forms grow best at 30–37 °C Thermophilic forms grow best at 50–60 °C. Some organisms are hyperthermophilic and can grow above the temp’ of boiling water, which exists under high pressure in the depths of the ocean. Most organisms are mesophilic; 30 °C is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals. 42 jimmy ongori 2013 03/11/2013 21:18  Psychrophiles Some can grow at 00C, but will not grow beyond 200 C Some, optimum is 20-300 C – common in food spoilage because can grow at refrigerator temp’ 43 jimmy ongori 2013 03/11/2013 21:18  pH Every organism has a range of pH over which growth is possible and an optimal pH Most bacteria (neutralophiles) grow between pH 6.5 - pH 7.5 Acidophilic bacteria – grow in acidic pH eg Lactobacillus which produce acid. Most fungi grow at 1.5-2.0 Alkalophiles – can grow upto pH10.5 44 jimmy ongori 2013 03/11/2013 21:18  OSMOTIC PRESSURE Hypertonic environments, increase salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure 45 jimmy ongori 2013 03/11/2013 21:18  The Requirements for Growth: Physical Requirements 46 jimmy ongori 2013 03/11/2013 21:18 Figure 6.4  OXYGEN Many forms of life require oxygen for aerobic respiration Some forms of oxygen can actually be toxic Metabolism, UV light, chemical reactions can create toxic forms of oxygen Aerobes have enzymes to detoxify toxic forms of oxygen 47 jimmy ongori 2013 03/11/2013 21:18  Toxic Forms of Oxygen  Singlet oxygen: O boosted to a higher-energy state  Superoxide free radicals: O2  Peroxide anion: O22 48 jimmy ongori 2013 03/11/2013 21:18  OXYGEN Obligate aerobes – require O2 to survive Facultative anaerobes – can grow in the absence of O2 eg E. coli Obligate anaerobes – unable to use molecular O2 eg Clostridia Microaerophilic – aerobic but grow only in O2 concentrations lower than those in the air 49 jimmy ongori 2013 03/11/2013 21:18  Oxygen (O2) obligate Faultative Obligate Aerotolerant Microaerophiles aerobes anaerobes anaerobes anaerobes 50 jimmy ongori 2013 03/11/2013 21:18  ASSIGNMENT Write short notes on the following; Chemical requirements for microbial growth eg carbon, nitrogen, sulphur etc Classify MOs according to requirements eg autotrophs etc 51 jimmy ongori 2013 03/11/2013 21:18  LECTURE 3 CULTURE MEDIA 52 jimmy ongori 2013 03/11/2013 21:18 Culture is the term given to microorganisms that are cultivated in the lab for the purpose of studying them. Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth (that is, multiplication) in order to cultivate these microorganisms in large numbers to study them. 53 jimmy ongori 2013 03/11/2013 21:18 Organisms have varying needs hence, culture media have also been formulated with different ingredients. Culture media may be found as;  liquid (called broth)  semi-solid  solid. Media are solidified by the addition of solidifying agents such as agar (inert compound). Varying the concentration of agar will yield varying degrees of solidification. 54 jimmy ongori 2013 03/11/2013 21:18  Culture media may be classified as: 1. Synthetic media (Defined) 2. Complex (Non-synthetic) media Synthetic media contain only ingredients for which a complete chemical formula is known. Complex media contain at least one ingredient for which a chemical formula is not known (such as milk, egg, malt, animal tissues) 55 jimmy ongori 2013 03/11/2013 21:18 Culture media can also be classified based on the function they perform in determining various characteristics of organism that are able to grow on/in them  e.g. Differential, Selective media. 56 jimmy ongori 2013 03/11/2013 21:18 The primary function of culture media is to be able to grow particular organisms on/in them. Media should be devoid of any other living organisms. This is done through the process of sterilization (a process by which all living organisms and their spore forms are killed and the medium is made sterile) Sterilized through the process of autoclaving (using high temperatures that will kill all living organisms under increased pressure for specified periods of time – in an appliance called the autoclave) 57 jimmy ongori 2013 03/11/2013 21:18  Types of media For the cultivation of bacteria, a commonly used medium is nutrient broth, a liquid containing proteins, salts, and growth enhancers that will support many bacteria. To solidify the medium, an agent such as agar is added. Agar is a polysaccharide that adds no nutrients to a medium, but merely solidifies it. The medium that results is nutrient agar. Very few bacteria can decompose agar 58 jimmy ongori 2013 03/11/2013 21:18  COMPLEX MEDIA Many media for microorganisms are complex, reflecting the growth requirements of the microorganisms. Usually contain complex materials of biological origin eg milk, blood or yeast extract For instance, most fungi require extra carbohydrate and an acidic environment for optimal growth. The medium employed for these organisms is potato dextrose agar, also known as.  For protozoa, liquid media are generally required, and for rickettsiae and viruses, living tissue cells must be provided for best cultivation. 59 jimmy ongori 2013 03/11/2013 21:18  Selective Media Inhibits the growth of some bacteria while selecting for the growth of others eg 1. Brilliant Green Agar  dyes inhibit the growth of Gram (+) bacteria  selects for Gram (-) bacteria  Most G.I.T infections are caused by Gram (-) bact. 2. EMB (Eosin Methylene Blue)  dyes inhibit Gram (+) bacteria  selects for Gram (-) bacteria  G.I. Tract infections caused by Gram (-) bacteria 60 jimmy ongori 2013 03/11/2013 21:18  Differential Media Differentiates between different organisms growing on the same plate Example: Blood Agar medium (TSA with 5% sheep blood) used to differentiate different types of Streptococci o Alpha-Hemolysis- incomplete RBC lysis o Beta Hemolysis- complete RBC lysis o Gamma Hemolysis- no RBC lysis 61 jimmy ongori 2013 03/11/2013 21:18 62 jimmy ongori 2013 03/11/2013 21:18  Selective and Differential Media Mannitol Salt Agar  used to identify Staphylococcus aureus  High salt conc. (7.5%) inhibits most bacteria  Sugar - Mannitol  pH Indicator (Turns Yellow when acidic) MacConkey’s Agar  used to identify Salmonella  Bile salts and crystal violet (inhibits Gram (+) bact.)  Sugar - lactose  pH Indicator  Many Gram (-) enteric non-pathogenic bacteria can ferment lactose, Salmonella can not jimmy ongori 2013 63 03/11/2013 21:18  Enrichment Broths ―Encourage‖ the growth of a particular type of microbe; Addition of ―nutrients‖ enrich for microbial group of interest eg o Cellulose broth- enriches for microbes which degrade cellulose o Petroleum Broth- enriches for microbes which could eat an oil spill. 64 jimmy ongori 2013 03/11/2013 21:18 Bacterial Morphology Arrangement 1. Bacilli a.Streptobacilli b. Bacilli 2. Cocci a. Cocci b. Doplococci c. Streptococci d. Staphylococci 65 jimmy ongori 2013 03/11/2013 21:18  Bacterial Morphology Arrangement 3 Spiral a. Vibrio b. Spirillum c. Spirochete 66 jimmy ongori 2013 03/11/2013 21:18  Rod-Shaped Bacteria 67 jimmy ongori 2013 03/11/2013 21:18  Spherical Bacteria Diplococcus Streptococcus Staphylococcus 68 jimmy ongori 2013 03/11/2013 21:18  Spiral-Shaped Bacteria Borrelia burgdorferi Spirochete: 69 jimmy ongori 2013 03/11/2013 21:18  METHODS OF STUDY 70 jimmy ongori 2013 03/11/2013 21:18  OPTICAL METHODS 1. Light microscope a. Bright field microscope b. Phase contrast microscope c. Dark field microscope 2. Fluorescence Microscope 3. Differential Interference Contrast (DIC) Microscope 4. The Electron Microscope 5. Confocal Scanning Laser Microscope 71 jimmy ongori 2013 03/11/2013 21:18  1. The Light Microscope The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Several types of light microscopes are commonly 72 used in microbiology: jimmy ongori 2013 03/11/2013 21:18  a. Bright-Field Microscope  Is most commonly used in microbiology and consists of two series of lenses (objective and ocular lens)  Generally uses a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 μ in diameter are magnified to about 0.2 mm and so become clearly visible.  Specimens are rendered visible because of differences in contrast between them and the surrounding medium.  Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium.  Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope. 73 jimmy ongori 2013 03/11/2013 21:18  b. Phase Contrast Microscope Was developed to improve contrast differences between cells and surrounding medium, making it possible to see living cells without staining them; with bright-field microscopes, killed and stained preparations must be used. Takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase contrast microscope, leading to the formation of a dark image on a light background. 74 jimmy ongori 2013 03/11/2013 21:18  c. Dark-Field Microscope Light microscope in which the lighting system has been modified to reach the specimen from the sides only. This creates a "dark field" that contrasts against the highlighted edge of the specimens Resolution by dark-field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete which is less than 0.2 μm in diameter and therefore cannot be observed with a bright-field or phase contrast microscope 75 jimmy ongori 2013 03/11/2013 21:18  FLUORESCENCE MICROSCOPE Used to visualize specimens that fluoresce - the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent substances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. 76 jimmy ongori 2013 03/11/2013 21:18 Widely used in clinical diagnostic microbiology eg the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, When applied to a specimen containing M tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background. 77 jimmy ongori 2013 03/11/2013 21:18 The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent- antibody (FA) technique or immunofluorescence. In this technique, specific antibodies (eg, antibodies to Legionella pneumophila) are chemically labeled with a fluorochrome If the specimen contains L pneumophila, the fluorescent antibodies will bind to antigens on the surface of the bacterium, causing it to fluoresce when exposed to ultraviolet light. 78 jimmy ongori 2013 03/11/2013 21:18 Differential Interference Contrast (DIC) Microscope  DIC microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens where they are recombined into a single beam.  Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle differences in cell structure. Structures such as spores, vacuoles, and granules appear three dimensional.  DIC microscopy is particularly useful for observing unstained cells because of its ability to generate images that reveal internal cell structures that are less apparent by bright-field techniques. 79 jimmy ongori 2013 03/11/2013 21:18  The Electron Microscope The high resolving power of the electron microscope has enabled scientists to observe the detailed structures of prokaryotic and eukaryotic cells. Two types of electron microscopes in general use: the transmission electron microscope (TEM), and the scanning electron microscope (SEM). The TEM was the first to be developed and employs a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. TEM can resolve particles 0.001 m apart. Viruses, with diameters of 0.01–0.2 m, can be easily resolved. The SEM is useful for providing three-dimensional images of the surface 80 of microscopic objects. jimmy ongori 2013 03/11/2013 21:18  Confocal Scanning Laser Microscope  A laser beam is bounced off a mirror that directs the beam through a scanning device. Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a given vertical layer within the specimen.  By precisely illuminating only a single plane of the specimen, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the laser beam.  Cells are often stained with fluorescent dyes to make them more visible. Alternatively, false color images can be generated by adjusting the microscope in such a way as to make different layers take on different colors. The CSLM is equipped with computer software to assemble digital images for subsequent image processing. 81 jimmy ongori 2013 03/11/2013 21:18  STERILIZATION AND DISINFECTION 82 jimmy ongori 2013 03/11/2013 21:18  Sterilization and Disinfection Sterilization is defined as the process where all the living microorganisms, including bacterial spores are killed.  Sterilization can be achieved by physical, chemical and physiochemical means. Disinfection: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually involves the removal of vegetative pathogens  Chemicals used in disinfection are called disinfectants.  Not all disinfectants can kill all microorganisms. Some methods of disinfection such as filtration do not kill bacteria, they separate them out. 83 jimmy ongori 2013 03/11/2013 21:18  Sterilization and Disinfection Sterilization is an absolute condition while disinfection is not. The two are not synonymous. Decontamination is the process of removal of contaminating pathogenic microorganisms from articles by a process of sterilization or disinfection. It is the use of physical or chemical means to remove, inactivate, or destroy living organisms on a surface so that the organisms are no longer infectious. 84 jimmy ongori 2013 03/11/2013 21:18  Sterilization and Disinfection Asepsis: is the employment of techniques (eg gloves, air filters, uv rays etc) to achieve microbe-free environment. Antisepsis: is the use of chemicals (antiseptics) to make skin or mucus membranes free of pathogenic microorganisms. Bacteriostasis: is a condition where the multiplication of the bacteria is inhibited without killing them. Bactericidal: Chemicals that can kill or inactivate bacteria. Have various names such as bactericidal, virucidal, fungicidal, microbicidal, sporicidal, tuberculocidal or germicidal. 85 jimmy ongori 2013 03/11/2013 21:18  The Ideal Disinfectant  Resistant to inactviation  Broadly active (killing pathogens)  Not poisonous (or otherwise harmful)  Penetrating (to pathogens)  Not damaging to non-living materials  Stable  Easy to work with  Otherwise not unpleasant 86 jimmy ongori 2013 03/11/2013 21:18 Disinfectant Performance…  Is dependent on Disinfectant concentrations  Is dependent on length (time) of administration  Is dependent on temperature during administration (usual chemical reaction 2x increase in rate with each 10°C increase in temperature)  Microbe type (e.g., mycobacteria, spores, and certain viruses can be very resistant to disinfection—in general vegetative cells in log phase are easiest to kill)  Substrate effects (e.g., high organic content interferes with disinfection—stainless steel bench easier to disinfect than wood)  It is easier (and faster) to kill fewer microbes than many 87 microbes jimmy ongori 2013 03/11/2013 21:18 88 jimmy ongori 2013 03/11/2013 21:18  PHYSICAL METHODS 1. Sunlight 2. Heat a) Dry heat – red heat, flaming heat, incineration, hot air oven, infra red heat b) Moist heat – at 1000 C, >1000 C, < 1000 C 3. Vibration 4. Radiation – non-ionizing and ionizing 5. Filtration 89 jimmy ongori 2013 03/11/2013 21:18  PHYSICAL METHODS OF STERILIZATION 1. Sunlight o The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it o It is responsible for spontaneous sterilization in natural conditions - due to combination of ultraviolet rays and heat. 90 jimmy ongori 2013 03/11/2013 21:18  PHYSICAL METHODS OF STERILIZATION 2. Heat  Heat is considered to be most reliable method of sterilization of articles that can withstand heat.  Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those articles that cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration of exposure. 91 jimmy ongori 2013 03/11/2013 21:18  PHYSICAL METHODS OF STERILIZATION Factors affecting sterilization by heat are:  Nature of heat: Moist heat is more effective than dry heat  Temperature and time: are inversely proportional. As temperature increases the time taken decreases.  Number of microorganisms: More the number of microorganisms, higher the temperature or longer the duration required.  Nature of microorganism: Depends on species and strain  Spores are highly resistant to heat. 92 jimmy ongori 2013 03/11/2013 21:18  a) DRY HEAT: i. Red heat: bacteriological loops, tips of forceps etc sterilized by holding them in Bunsen flame till they become red hot. Limited to articles that can be heated to redness in flame. ii. Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to redness eg scalpels, mouth of test tubes, glass slides and cover slips are passed through the flame a few times. iii. Incineration: destroying contaminated material by burning in incinerator eg soiled dressings, pathological material and bedding etc. This technique results in the loss of the article, hence suitable only for those articles that have to be disposed. jimmy ongori 2013 93 03/11/2013 21:18 iv. Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high temp’ (160o C) for one hour in an electrically heated oven. Even distribution of heat throughout the chamber achieved by a fan. Articles sterilized: Metallic instruments (forceps, scalpels, scissors), glasswares (petri-dishes, pipettes, flasks, all-glass syringes), swabs, and some pharmaceutical products. Increasing temperature by 10 degrees shortens the sterilizing time by 50 percent. This is the only method of sterilizing oils and powders. jimmy ongori 2013 94 03/11/2013 21:18 v. Infra red rays  Infrared rays bring about sterilization by generation of heat.  Articles to be sterilized are placed in a moving conveyor belt and passed through a tunnel that is heated by infrared radiators to a temperature of 180oC for 7.5 minutes.  Articles sterilized include metallic instruments and glassware. jimmy ongori 2013 95 03/11/2013 21:18  b) MOIST HEAT  Moist heat acts by coagulation and denaturation of proteins. At temperature below 100oC: i. Pasteurization:  This process was originally used by Louis Pasteur.  Currently this procedure is used in food and dairy industry. There are two methods of pasteurization, the holder method (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds) followed by quickly cooling to 13oC. 96 jimmy ongori 2013 03/11/2013 21:18  PHYSICAL METHODS OF STERILIZATION  Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15 sec and 149oC for 0.5 sec. This method is suitable to destroy most milk borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may survive pasteurization. Others; ii.Vaccine bath iii. Serum bath iv.Inspissation 97 jimmy ongori 2013 03/11/2013 21:18  At temperature 100oC: i) Boiling  Boiling water (100oC) kills most vegetative bacteria and viruses immediately.  Certain bacterial toxins such as Staphylococcal enterotoxin are heat resistant.  Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for sterilization.  When absolute sterility is not required, certain metal articles and glasswares can be disinfected by placing 98 them jimmy ongori 2013 in boiling water for 10-20 minutes. 03/11/2013 21:18  ii) Steam at 100oC  Free steam at 100oC, An autoclave can be used. A steamer is a metal cabinet with perforated trays to hold articles.  The bottom of steamer is filled with water and heated, steam generated sterilizes the articles when exposed for a period of 90 minutes. Media eg DCA and selenite broth sterilized by steaming.  Sugar and gelatin in medium may get decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes for 3 successive days. This process is known as tyndallisation (after John Tyndall) or fractional sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure and the spores that germinate by next day are killed in subsequent days. The success of process depends on the germination of spores. 99 jimmy ongori 2013 03/11/2013 21:18 At temperature above 100oC: Autoclave:  Sterilization can be effectively achieved at a temperature above 100oC using an autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature at which the water boils also increases.  In an autoclave the water is boiled in a closed chamber. As the pressure rises, the boiling point of water also raises.  At a pressure of 15 lbs inside the autoclave, the temperature is said to be 121oC. Exposure of articles to this temperature for 15 minutes sterilizes them. 100 jimmy ongori 2013 03/11/2013 21:18  To destroy the infective agents associated with spongiform encephalopathies (prions), higher temperatures or longer times are used; 135oC or 121oC for at least one hour are recommended. Advantages of steam: It has more penetrative power than dry air, it moistens the spores (moisture is essential for coagulation of proteins Articles sterilized: Culture media, dressings, certain equipment, linen etc. Disadvantages: Drenching and wetting of articles may occur, trapped air may reduce the efficacy, takes long time to cool 101 jimmy ongori 2013 03/11/2013 21:18  AUTOCLAVE 102 jimmy ongori 2013 03/11/2013 21:18 RADIATION  Two types of radiation are used, ionizing and non-ionizing.  Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative power. Since radiation does not generate heat, it is termed "cold sterilization". i) Non-ionizing rays:  Rays of wavelength longer than the visible light are non-ionizing.  UV rays are generated using a high-pressure mercury vapor lamp.  UV rays induce formation of thymine-thymine dimers, which inhibits DNA replication. 103 jimmy ongori 2013 03/11/2013 21:18  RADIATION  UV readily induces mutations in cells irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation are inactivated within seconds.  UV rays don’t kill spores  UV rays are used to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. Disadvantages - harmful to skin and eyes. 104 jimmy ongori 2013 03/11/2013 21:18  Ionizing Rays  Used to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals, petri dishes, antibiotics, vitamins, hormones, glasswares and fabrics.  Sterilization is accomplished in few seconds. 105 jimmy ongori 2013 03/11/2013 21:18  FILTRATION  Filtration does not kill microbes, it separates them out.  Membrane filters with pore sizes between 0.2-0.45 μm are commonly used to remove particles from solutions that can't be autoclaved.  Used to remove microbes from heat labile liquids such as serum, antibiotic solutions, sugar solutions, urea solution.  Filtration is aided by using either positive or negative pressure using vacuum pumps. 106 jimmy ongori 2013 03/11/2013 21:18 Types of filters 1. Earthenware filters: These filters are made up of diatomaceous earth or porcelain. 2. Asbestos filters 3. Sintered glass filters: These are made from finely ground glass that are fused sufficiently to make small particles adhere to each other. 4. Membrane filters 5. Air Filters: They are usually used in biological safety cabinets. jimmy ongori 2013 107 03/11/2013 21:18  CHEMICAL METHODS  Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics. 108 jimmy ongori 2013 03/11/2013 21:18  Agent Mechanisms of Action Comments Surfactants Membrane Disruption; Soaps; detergents Chemical Antimicrobials increased penetration Quats (cationic Denature proteins; Antiseptic - benzalconium detergent) Disrupts lipids chloride, Cepacol; Disinfectant Organic acids High/low pH Mold and Fungi inhibitors; e.g., and bases benzoate of soda Heavy Metals Denature protein Antiseptic & Disinfectant; Silver Nitrate Halogens Oxidizing agent Antiseptic - Iodine (Betadine) Disrupts cell membrane Disinfectant - Chlorine (Chlorox) Alcohols Denatures proteins; Antiseptic & Disinfectant Disrupts lipids Ethanol and isopropyl Phenolics Disrupts cell membrane Disinfectant Irritating odor Aldehydes Denature proteins Gluteraldehyde - disinfectant (Cidex); Formaldehyde - disinfectant Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic; 109 jimmy ongori 2013 Hydrogen peroxide – disinfectan; 03/11/2013 21:18 Benzoyl peroxide – antiseptic Chemical Methods of Microbial Control Types of Disinfectants 1. Phenols and Phenolics: u Phenol (carbolic acid) was first used by Lister as a disinfectant. u Rarely used today because it is a skin irritant and has strong odor. u Phenolics are chemical derivatives of phenol, Destroy plasma membranes and denature proteins. u Cresols: eg Lysol u Biphenols 110 jimmy ongori 2013 03/11/2013 21:18 2. Halogens: Effective alone or in compounds. A. Iodine: u Tincture of iodine (alcohol solution) was one of first antiseptics used, denatures proteins. u Stains skin and clothes, somewhat irritating. u Iodophors: Compounds with iodine that take several minutes to act. Used as skin antiseptic in surgery. Not effective against bacterial endospores eg; Betadine B. Chlorine: u When mixed in water forms hypochlorous acid, Used to disinfect drinking water, pools, and sewage. u Sodium hypochlorite - active ingredient of bleach. u Chloramines: Consist of chlorine and ammonia. Less effective as germicides. 111 jimmy ongori 2013 03/11/2013 21:18 3. Alcohols: u Kill bacteria, fungi, but not endospores or naked viruses. u Act by denaturing proteins and disrupting cell membranes. Evaporate, leaving no residue. u Used to mechanically wipe microbes off skin before injections or blood drawing. u Not good for open wounds, because cause proteins to coagulate. u Ethanol: Optimum concentration is 70%. u Isopropanol: Better disinfectant than ethanol. Also cheaper and less volatile. 112 jimmy ongori 2013 03/11/2013 21:18 4. Heavy Metals: u Include copper, selenium, mercury, silver, and zinc. A. Silver: 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. B. Copper; Copper sulfate is used to kill algae in pools and fish tanks. C. Selenium: Kills fungi and their spores. Used for fungal infections. Also used in dandruff shampoos. E. Zinc: Zinc chloride is used in mouthwashes. u Zinc oxide is used as antifungal agent in paints. 113 jimmy ongori 2013 03/11/2013 21:18 5. Quaternary Ammonium Compounds: u Widely used surface active agents. u Effective against gram +VE bacteria, less effective against gram -VE bacteria. Also destroy fungi, amoebas, and enveloped viruses. u Zephiran, Cepacol u Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and nontoxic. u Diasadvantages: Form foam. Organic matter interferes with effectiveness. Neutralized by soaps and anionic detergents. 114 jimmy ongori 2013 03/11/2013 21:18 6. Aldehydes: Include some of the most effective antimicrobials. Inactivate proteins by forming covalent crosslinks with several functional groups. A. Formaldehyde gas: Excellent disinfectant. Commonly used as formalin Formalin is used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines. Irritates mucous membranes, strong odor. Also used in mortuaries for embalming. 115 jimmy ongori 2013 03/11/2013 21:18 B. Glutaraldehyde: Less irritating and more effective than formaldehyde. One of the few chemical disinfectants that is a sterilizing agent. A 2% solution of glutaraldehyde (Cidex) is: Bactericidal, tuberculocidal, and viricidal in 10 minutes. Sporicidal in 3 to 10 hours. Commonly used to disinfect hospital instruments. Also used in mortuaries for embalming. 116 jimmy ongori 2013 03/11/2013 21:18 7. Gaseous Sterilizers: Chemicals that sterilize in a chamber similar to an autoclave. A. Ethylene Oxide: Kills all microbes and endospores, but requires exposure of 4 to 18 hours. Toxic and explosive in pure form. Highly penetrating. Many hospitals have ethylene oxide chambers to sterilize mattresses and large equipment. 117 jimmy ongori 2013 03/11/2013 21:18 8. Peroxygens (Oxidizing Agents): Oxidize cellular components of treated microbes. Disrupt membranes and proteins. A. Ozone: Highly reactive form of oxygen. Used along with chlorine to disinfect water. Helps neutralize unpleasant tastes and odors. More effective killing agent than chlorine, but less stable and more expensive. Made by exposing oxygen to electricity or UV light. 118 jimmy ongori 2013 03/11/2013 21:18 B. Hydrogen Peroxide: Used as an antiseptic. Not good for open wounds because quickly broken down by catalase present in human cells. Effective in disinfection of inanimate objects. Sporicidal at higher temperatures. C. Benzoyl Peroxide: Used in acne medications. 119 jimmy ongori 2013 03/11/2013 21:18  D. Peracetic Acid: One of the most effective liquid sporicides available. Kills bacteria and fungi in less than 5 minutes. Kills endospores and viruses within 30 minutes. Used widely in disinfection of food and medical instruments because it does not leave toxic residues. 120 jimmy ongori 2013 03/11/2013 21:18  BACTERIA 121 jimmy ongori 2013 03/11/2013 21:18  The bacteria (singular: bacterium) are a large group of unicellular microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water Bacteria are vital in recycling nutrients, with many steps in nutrient cycles depending on these organisms, such as the fixation of nitrogen The study of bacteria is known as bacteriology, a branch of 122 jimmy ongori 2013 03/11/2013 21:18 microbiology.  There are about ten times as many bacterial cells in the human flora of bacteria as there are human cells in the body, with large numbers of bacteria on the skin and as gut flora. Majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, and a few are beneficial. A few species of bacteria are pathogenic and cause infectious diseases. Bacteria are important in sewage treatment, the production of cheese and yoghurt through fermentation, as well as in biotechnology, and the manufacture of antibiotics and other chemicals. 123 jimmy ongori 2013 03/11/2013 21:18  Once regarded as plants, bacteria are now classified as prokaryotes. Unlike cells of animals and other eukaryotes, bacterial cells do not contain a nucleus and rarely harbour membrane-bound organelles. Although the term bacteria traditionally included all prokaryotes, the scientific classification changed after the discovery in the 1990s that prokaryotes consist of two very different groups of organisms that evolved independently from an ancient common ancestor. These are called Bacteria and Archaea. Bacteria were first observed by Antonie van Leeuwenhoek in 1676, using a single-lens microscope of his own design. He called them "animalcules" 124 jimmy ongori 2013 03/11/2013 21:18  The name bacterium was introduced much later, by Christian Gottfried Ehrenberg in 1838. Louis Pasteur demonstrated in 1859 that the fermentation process is caused by the growth of microorganisms, and that this growth is not due to spontaneous generation. (Yeasts and molds, commonly associated with fermentation, are fungi.) Along with Robert Koch, Pasteur was an early advocate of the germ theory of disease. Robert Koch was a pioneer in medical microbiology and worked on cholera, anthrax and tuberculosis. In his research into tuberculosis, Koch finally proved the germ theory. In Koch's postulates, he set out criteria to test if an organism is the cause of a disease these postulates are still used today. 125 jimmy ongori 2013 03/11/2013 21:18 PROKARYOTES AND EUKARYOTES "True" bacteria (which include all bacteria that infect man) are members of one kingdom (the eubacteria). A group of organisms often found in extreme environments form a second kingdom (archaebacteria, Archaea). Morphologically, the two kingdoms of organisms appear similar, especially in the absence of a nucleus, and thus are classified together as prokaryotes. 126 jimmy ongori 2013 03/11/2013 21:18 PROKARYOTES AND EUKARYOTES However, they have major biochemical differences. Most archaea live in environments such as hot sulfur springs where they experience temperatures as high as 800 C and a pH 2. - called thermoacidophiles. Others live in methane-containing (methanogens) or high salt (extreme halophiles) environments. Members of the Archaea are not human pathogens 127 jimmy ongori 2013 03/11/2013 21:18  SUMMARY OF DIFFERENCES PROKARYOTIC CELLS EUKARYOTIC CELLS Small cells (10 μm) Always unicellular Often multicellular No nucleus or any membrane-bound Always have nucleus and other organelles membrane-bound organelles DNA is circular, without proteins DNA is linear and associated with proteins to form chromatin Ribosomes are small (70S) Ribosomes are large (80S) No cytoskeleton Always has a cytoskeleton Cell division is by binary fission Cell division is by mitosis or meiosis 128Reproduction jimmy ongoriis always asexual 2013 Reproduction is asexual or sexual 03/11/2013 21:18  EUKARYOTIC CELL 129 jimmy ongori 2013 03/11/2013 21:18  THE PROTOTYPE BACTERIAL CELL 130 jimmy ongori 2013 03/11/2013 21:18  ASSIGNMENT  Draw a diagram of a Gram negative and Gram positive bacterial cell wall and briefly describe the differences 131 jimmy ongori 2013 03/11/2013 21:18 BACTERIAL STRUCTURES Not all bacteria possess all of these components. 1. The cell envelope  Bacteria can be divided into two groups on the basis of staining with the Gram stain; Gram positive bacteria remain stained by crystal violet on washing, Gram negative do not.  All bacteria have a cell membrane where oxidative phosphorylation occurs (since there are no mitochondria). Outside the cell membrane is the cell wall which is rigid 132 jimmy ongori 2013 03/11/2013 21:18 and protects the cell from osmotic lysis.  In Gram +ve bacteria, the cell wall peptidoglycan layer is much thicker than in Gram -ve bacteria. Gram -ve bacteria have an additional outer membrane. The outer membrane is the major permeability barrier in Gram negative bacteria.  The space between the inner and outer membranes is known as the periplasmic space. Gram -ve bacteria store degradative enzymes in the periplasmic space.  Gram +ve bacteria lack a periplasmic space; instead they secrete exoenzymes and perform extracellular digestion.  Digestion is needed since large molecules can not readily pass across the outer membrane (if present) or cell membrane. 133 jimmy ongori 2013 03/11/2013 21:18 THE BACTERIAL CELL WALL As in other organisms, the bacterial cell wall provides structural integrity to the cell. In prokaryotes, the primary function of the cell wall is to protect the cell from internal turgor pressure caused by the much higher concentrations of proteins and other molecules inside the cell compared to its external environment. The bacterial cell wall differs from that of all other organisms by the presence of peptidoglycan, which is located immediately outside of the cytoplasmic membrane. 134 jimmy ongori 2013 03/11/2013 21:18 THE BACTERIAL CELL WALL Peptidoglycan is responsible for the rigidity of the bacterial cell wall and for the determination of cell shape All bacterial cell walls (with a few exceptions e.g. Mycoplasma) contain peptidoglycan, not all cell walls have the same overall structures. There are two main types of bacterial cell walls, Gram positive and Gram negative, which are differentiated by their Gram staining characteristics. 135 jimmy ongori 2013 03/11/2013 21:18 TYPES OF BACTERIAL CELL ENVELOPES Mycobacteria The Mycobacteria have a cell envelope which is not typical of Gram positives or Gram negatives. The mycobacterial cell envelope does not consist of the outer membrane characteristic of Gram negatives, but has a significant peptidoglycan- arabinogalactan-mycolic acid wall structure which provides an external permeability barrier. 136 jimmy ongori 2013 03/11/2013 21:18 The Gram positive cell wall The Gram positive cell wall has a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during the Gram staining procedure. The Gram negative cell wall It contains a thin peptidoglycan layer adjacent to the cytoplasmic membrane. This is responsible for the cell wall's inability to retain the crystal violet stain upon decolourisation with ethanol during Gram staining. In addition to the peptidoglycan layer, the Gram negative cell wall also contains an outer membrane composed by phospholipids and lipopolysaccharides whose chemical structure is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains. 137 jimmy ongori 2013 03/11/2013 21:18 Plasmids  These are extra-chromosomal DNA, usually present in multiple copies, that often code for pathogenesis factors and antibiotic resistance factors. Some forms are also involved in bacterial replication. Flagella  Some bacterial species are mobile and possess locomotory organelles – flagella. Those that do are able to taste their environment and respond to specific chemical foodstuffs or toxic materials and move towards or away from them (chemotaxis).  Flagella are embedded in the cell membrane, extend through the cell envelope and project as a long strand. They move the cell by rotating with a propeller like action. 138 jimmy ongori 2013 03/11/2013 21:18 Pili (synonym: fimbriae)  The types of pili varies both among and between species. Pili are hair- like projections of the cell.  Some are involved in sexual conjugation and others allow adhesion to host epithelial surfaces in infection. Capsules and slime layers  Surround the outside of the cell envelope. When more defined, they are referred to as a capsule when less defined as a slime layer or glycocalyx.  usually consist of polysaccharide; however, in certain bacilli they are composed of a polypeptide.  Capsules of pathogenic bacteria inhibit ingestion and killing by phagocytes. 139 jimmy ongori 2013 03/11/2013 21:18 Endospores (spores) (Read and make notes on spores)  These are a dormant form of a bacterial cell produced by certain bacteria when starved; the actively growing form of the cell is referred to as vegetative. The spore is resistant to adverse conditions (including high temperatures and organic solvents).  The spore cytoplasm is dehydrated and contains calcium dipicolinate which is involved in the heat resistance of the spore.  Spores are commonly found in the genera Bacillus and Clostridium. 140 jimmy ongori 2013 03/11/2013 21:18 CELL MORPHOLOGY Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are about one tenth the size of eukaryotic cells and are typically 0.5–5.0 μm in length. A few species eg Thiomargarita namibiensis – are up to half a millimetre long and are visible to the unaided eye. Among the smallest bacteria are members of the genus Mycoplasma, which measure only 0.3 micrometres. Most bacterial species are; spherical, called cocci ( Greek kókkos, grain/seed) or rod-shaped, called bacilli (Latin baculus, stick). Some rod-shaped bacteria, called vibrio, are slightly curved or comma- shaped; others, can be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. 141 jimmy ongori 2013 03/11/2013 21:18 CELL MORPHOLOGY Cont’ This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton Many bacterial species exist as single cells, others associate in characteristic patterns: Neisseria form diploids (pairs) Streptococcus form chains Staphylococcus group together in "bunch of grapes" clusters. Bacteria can also be elongated to form filaments, for example the Actinobacteria. Filamentous bacteria are often surrounded by a sheath that contains many individual cells. Bacteria are classified by direct examination with the light microscope through its morphology and aggregation. 142 jimmy ongori 2013 03/11/2013 21:18 COCCI (singular - coccus)  Are any bacteria whose overall shape is spherical or nearly spherical. Describing a bacterium as a coccus, or sphere, distinguishes it from bacillus, or rod. This is the first of many taxonomic traits for identifying and classifying a bacterium Basic forms; 1. Pairs – diplococci eg Neisseria gonorrhoeae 2. Groups of 4 or 8 known as tetrads or sarcina eg Micrococci 3. Bead-like chains, or streptococci eg Streptococcus pneumoniae 4. Grapelike clusters, or staphylococci eg Staphylococcus aureus 143 jimmy ongori 2013 03/11/2013 21:18 Bacillus  Although Bacillus refers to the genus, the word bacillus may also be used to describe any rod-shaped bacterium, and in this sense, bacilli are found in many different taxonomic groups of bacteria.  Bacilli are usually solitary, but can combine to form diplobacilli, streptobacilli, and palisades. Coccobacillus A type of rod-shaped bacteria. The word coccobacillus reflects an intermediate shape between coccus and bacillus. Coccobacilli rods are so short and wide that they resemble cocci. Haemophilus influenzae and Chlamydia trachomatis are coccobacilli. 144 jimmy ongori 2013 03/11/2013 21:18 145 jimmy ongori 2013 03/11/2013 21:18  PATHOGENESIS OF BACTERIAL INFECTION PATHOGENICITY TOXIGENICITY VIRULENCE 146 jimmy ongori 2013 03/11/2013 21:18  The pathogenesis of bacterial infection includes the initiation of the infectious process and the mechanisms leading to the development of signs and symptoms of bacterial disease.  The outcome of the interaction between bacteria and host is determined by characteristics that favour establishment of the bacteria within the host and their ability to damage the host as they are opposed by host defense mechanisms.  Among the characterics of bacteria are;  Adherence to host cells  Invasiveness  Toxigenity  Ability to evade the host´s immune system. 147 jimmy ongori 2013 03/11/2013 21:18 Pathogenesis of bacterial infection  Humans and animals have abundant normal microflora.  Most bacteria do not produce disease but achieve a balance with the host that ensures the survival, growth, and propagation of both the bacteria and the host.  Sometimes bacteria are pathogenic (e.g. Salmonella typhi) but infection remains latent or subclinical and the host is a "carrier" of the bacteria.  Analysis of infection and disease through the application of principles such as Koch´s postulates leads to classification jimmy ongori 2013 of bacteria as pathogenic or non-pathogenic. 148 03/11/2013 21:18  Some bacterial species are always considered to be pathogens, and their presence is abnormal. Eg Mycobacterium tuberculosis and Yersinia pestis (plague).  Some sp. are part of the normal flora of humans but can cause disease. Eg Escherichia coli is part of the GIT flora of normal humans, but it is also a comon cause of urinary tract infection and traveller´s diarrhea, 149 jimmy ongori 2013 03/11/2013 21:18 The infectious process  Infection indicates multiplication of M.O.s  Prior to multiplication, bacteria must enter and establish themselves within the host.  The most frequent portals of entry;  the respiratory (mouth and nose)  gastrointestinal  urogenital tracts  abnormal areas of mucous membranes and skin (e.g. cuts, burns) are also frequent sites of entry. 150 jimmy ongori 2013 03/11/2013 21:18  The infectious process  Once in the body, bacteria must attach or adhere to host cells - usually epithelial cells.  After the bacteria have established a primary site of infection, they multiply and spread.  Infection can spread directly through tissues or via the lymphatic system to bloodstream.  Bloodstream infection is known as bacteremia.  Bacteremia allows bacteria to spread widely in the body and permits them to reach tissues suitable for their multiplication. 151 jimmy ongori 2013 03/11/2013 21:18  TERMINOLOGIES  Infection:  Multiplication of an infectious agent within the body.  Multiplication of the bacteria that are part of normal flora is generally not considered an infection.  Pathogenicity:  The ability of an infectious agent to cause disease.  Virulence:  The degree or extent of pathogenicity  The quantitative ability of an agent to cause disease.  Virulence involves invasiveness and toxigenicity. 152 jimmy ongori 2013 03/11/2013 21:18  Virulence factors  These are traits or features that allow or enhance the microorganism’s ability to cause disease, include; i. adhesion organelles, ii. toxin production iii. evasion of the host’s immune response iv. resistance to antibiotics v. ability to invade host tissues vi. enhanced intracellular survival and growth 153 jimmy ongori 2013 03/11/2013 21:18  Toxigenicity:  ability of a microorganism to produce a toxin that contributes to the development of disease.  Invasion:  The process whereby bacteria, parasites, fungi and viruses enter the host cells or tissues and spread in the body.  Pathogen:  A microorganism capable of causing disease.  Opportunistic pathogen:  An agent capable of causing disease only when the host´s resistance is impaired (e.g. the patient is immunocompromised). jimmy ongori 2013 154 03/11/2013 21:18  Bacterial virulence factors 1. TOXINS  Toxins produced by bacteria are generally classified into two groups:  Exotoxins  Endotoxins - Only found in gram negative bacteria 155 jimmy ongori 2013 03/11/2013 21:18 Endotoxins of gram-negative bacteria  Endotoxins are toxic components of the bacterial cell envelope.  The classical and most potent endotoxin is lipopolysaccharide derived from bacterial cell walls and are liberated when the bacteria lyse.  The pathophysiologic effects of LPS are similar regardless of their bacterial origin  Relatively stable; withstand heating at temperatures above 60°C for hours without loss of toxicity  Usually produce fever in the host 156 jimmy ongori 2013 03/11/2013 21:18  Exotoxins  Many gram-positive and gram-negative bacteria produce exotoxins of considerable medical importance - that modify, by enzymatic action, or destroy certain cellular structures.  Examples - botulism, anthrax, cholera and diphtheria.  Vaccines have been developed for some of the exotoxin- mediated diseases and continue to be important in the prevention of disease.  These vaccines—called toxoids—are made from exotoxins, which are modified so that they are no longer toxicjimmy ongori 2013 157 03/11/2013 21:18  1. Diphtheria toxin (Corynebacterium diphtheriae)  It is a Gm +ve rod that can grow on the mucous membranes of the upper respiratory tract or in minor skin wounds. Some strains produce diphtheria toxin. 2. Tetanospasmin (toxin of Clostridium tetani)  C. tetani is an anaerobic gram-positive rod  It contaminates wounds, and the spores germinate in the anaerobic environment of the devitalized tissue. The vegetative forms of Clostridium tetani produce toxin tetanospasmin.  Toxin reaches the CNS and acts by blocking release of an inhibitory mediator in motor neuron synapses.  Extremely small amount of toxin can be lethal for 158 humans. jimmy ongori 2013 03/11/2013 21:18 3. Botulotoxin (toxin of Clostridium botulinum)  Clostridium botulinum is found in soil or water and may grow in foods if the environment is appropriately anaerobic.  It is the most potent toxin known – causes botulism  It is heat-labile and is destroyed by sufficient heating.  Toxin is absorbed from the GIT and carried to motor nerves, where it blocks the release of acetylcholine at synapses and neuromuscular junctions. Muscle contraction does not occur, and paralysis results. 159 jimmy ongori 2013 03/11/2013 21:18 4. Toxins of Clostridium perfringens  Spores of Clostridium perfringens are introduced into the wounds by contamination with soil or faeces. In the presence of necrotic tissue, spores germinate and vegetative cells produce several different toxins.  Many are necrotizing and hemolytic and favour the spread of gangrene 5. Streptococcal erythrogenic toxin  Some strains of hemolytic streptococci produce a toxin that results in a rash, as in scarlet fewer. 160 jimmy ongori 2013 03/11/2013 21:18 6. Toxic shock syndrom toxin - 1 (TSST-1)  Some Staphylococcus aureus strains growing on mucous membranes (e.g. on the vagina in association with menstruation), or in wounds, produce TSST-1.  Toxic shock syndrome is characterized by shock, high fewer, and a diffuse red rash 7. Exotoxins associated with diarrheal diseases  Vibrio cholerae toxin  Staphylococcus aureus enterotoxin  Other enterotoxins are produced; Yersinia enterocolitica, Vibrio parahaemolyticus, Aeromonas species 161 jimmy ongori 2013 03/11/2013 21:18  Enzymes  Many bacteria produce enzymes that play an important role in the infectious process; i. Collagenase: degrades collagen, the major protein of fibrous connective tissue, and promotes spread of infection in tissue. ii. Coagulase: Staphylococccus aureus produce coagulase, which coagulates plasma. Coagulase contributes to the formation of fibrin walls around staphylococcal lesions, which helps them persist in tissues. iii. Hyaluronidases: hydrolyze hyaluronic acid, a constituent of substance of connective tissue (e.g. staphylococci, streptococci and anaerobes) and aid in 162 their spread jimmy ongori 2013 through tissues. 03/11/2013 21:18  Enzymes iv. Streptokinase: many hemolytic streptococci produce streptokinase (fibrinolysin), a substance that activates a proteolytic enzyme of plasma. It dissolves coagulated plasma and aids in the spread of streptococci through tissues. Streptokinase is used in treatment of acute myocardial infarction to dissolve fibrin clots. v. Hemolysins and leukocidins: Many bacteria produce substances that are cytolysins - they dissolve red blood cells (hemolysins) or kill tissue cells or leukocytes (leukocidins). 163 jimmy ongori 2013 03/11/2013 21:18 Antiphagocytic factors  Many bacterial pathogens are rapidly killed once they are ingested by polymorphonuclear cells or macrophages.  Some pathogens evade phagocytosis or leukocyte microbidical mechanisms e.g. Streptococcus pneumoniae have polysaccharide capsules. Adherence factors  Once bacteria enter the body of the host, they must adhere to cells of a tissue surface. If they do not adhere, they would be swept away by mucus and other fluids  Adherence is followed by development of microcolonies and subsequent complex steps in the pathogenesis of infection. 164 jimmy ongori 2013 03/11/2013 21:18  STAINING 165 jimmy ongori 2013 03/11/2013 21:18  Staining Staining is a technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, muscle fibers or connective tissue), classifying different blood cells. 166 jimmy ongori 2013 03/11/2013 21:18  Staining In biochemistry it involves adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound Biological staining is also used to mark cells in flow cytometry, and in gel electrophoresis. In vivo staining is the process of dyeing living tissues By causing certain cells or structures to take on contrasting colours, their morphology or position within a cell can be distinguished 167 jimmy ongori 2013 03/11/2013 21:18  Staining In vitro staining involves colouring cells or structures that are no longer living. Certain stains are often combined to reveal more details and features than a single stain alone. A counter-stain is a stain that makes cells or structures more visible, when not completely visible with the principal stain. For example, crystal violet stains only Gram +ve bacteria during Gram staining. A safranin counterstain is applied which stains all cells, allowing the identification of Gram -ve bacteria. 168 jimmy ongori 2013 03/11/2013 21:18  GRAM STAINING The method is named after its inventor, Hans Christan Gram (1853–1938) Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (Gram +ve and Gram -ve) based on the physical properties of their cell walls In a modern molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are more specific than differential staining. 169 jimmy ongori 2013 03/11/2013 21:18  The Gram stain is the most widely used staining procedure in bacteriology.  It is called a differential stain since it differentiates between Gram+VE and Gram-VE negative bacteria.  Bacteria that stain purple with the Gram staining procedure are termed Gram +VE; those that stain pink are said to be Gram -VE. The terms have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. 170 jimmy ongori 2013 03/11/2013 21:18  Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls.  The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall which confers rigidity is peptidoglycan. 171 jimmy ongori 2013 03/11/2013 21:18  Principles Gram Staining 172 Gram stains are performed on body fluid or biopsy  Gram staining tests the bacterial cell wall's ability to retain crystal violet dye during solvent treatment.  Safranin is added as a mordant to form the crystal violet/safranin complex in order to render the dye impossible to remove.  Ethyl-alcohol solvent acts as a decolorizer and dissolves the lipid layer from gram-negative cells. This enhances leaching of the primary stain from the cells into the surrounding solvent.  Ethyl-alcohol will dehydrate the thicker gram-positive cell walls, closing the pores as the cell wall shrinks.  For this reason, the diffusion of the crystal violet-safranin staining is inhibited, jimmy ongori 2013 so the bacteria remain stained. 03/11/2013 21:18  STAINING MECHANISM Gram +ve bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which are stained purple by crystal violet Gram -ve bacteria have a thinner layer (10% of cell wall), which are stained pink by the counter-stain. There are four basic steps of the gram stain: 1. Applying a primary stain (crystal violet) to a heat- fixed (death by heat) smear 2. The addition of a trapping agent (gram's iodine) 3. Rapid decolorization with alcohol or acetone 4. Counterstaining with safranin. 173 jimmy ongori 2013 03/11/2013 21:18  PROCEDURE FOR G/STAIN (1) Fix smear by heat. (2) Cover with crystal violet for 30-60 seconds (3) Wash with water. Do not blot. (4) Cover with Gram's iodine for 30-60 seconds (5) Wash with water. Do not blot. (6) Decolorize for 10–30 seconds with gentle agitation in acetone-alcohol 174 jimmy ongori 2013 03/11/2013 21:18 (7) Wash with water. Do not blot. (8) Cover for 10–30 seconds with safranin (2.5% solution in 95% alcohol). (9) Wash with water and let dry. (10) Examine under oil immersion objective  Gram-positive bacteria stain dark blue or violet  Gram-negative organisms will appear red or pink because they are counterstaine 175 jimmy ongori 2013 03/11/2013 21:18  Step 1. Staining with crystal violet.  Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.  Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram negative) and not others (Gram positive).  Step 4. Counterstaining with safranin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink. 176 jimmy ongori 2013 03/11/2013 21:18 177 jimmy ongori 2013 03/11/2013 21:18  ZIEHL–NEELSEN STAIN The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl (1859 to 1926), and Friedrich Neelsen (1854 to 1898) It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is helpful in diagnosing M. tuberculosis since its lipid rich cell wall makes it resistant to Gram stain.  The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol and methylene blue. Acid-fast bacilli will be bright red after staining. jimmy ongori 2013 03/11/2013 21:18 178  Acid-Fast Principles  Primary stain penetrates cell wall  Intense decolourization does not release primary stain from the cell wall of AFB  Colour of AFB-based on primary stain  Counterstain provides contrasting background 179 jimmy ongori 2013 03/11/2013 21:18  PROCEDURE 1. Heat fix the smear 2. Cover with carbol fuchsin stain 3. Heat the until vapour just begins to rise, do not overheat. Allow the heated stain to remain on the slide for 5 minutes 4. Wash off the stain with clean water 5. Cover the stain with 3% acid alcohol for 5 minutes or until the stain is fully decolorized ie pale pink 180 03/11/2013 21:18 jimmy ongori 2013  Procedure cont’ 6. Wash with clean water 7. Cover the smear with methylene blue counter stain for 1 minute 8. Wash with clean water 9. Air dry on a rack 10. Examine using 100X oil immersion objective 181 jimmy ongori 2013 03/11/2013 21:18  THE COCCI 182 jimmy ongori 2013 03/11/2013 21:18  COCCI  The cocci are one of the most commonly encountered bacteria (the other being bacilli)  Cocci refers to the spherical shape  Can exist as;  Single cells  Diplococcus eg Neisseria  Long chains – Streptococcus, Enterococcus and Lactococcus  Irregular clumps – Staphyloccus  Tetrads – genus Micrococcus  Cubical packets of 8 cells – genus Sarcina 183 jimmy ongori 2013 03/11/2013 21:18  STAPHYLOCCUS  Are gram-positive spherical cells, usually arranged in grape-like irregular clusters.  Some are members of the normal flora of the skin and mucous membranes of humans; others cause suppuration, abscess formation, a variety of pyogenic infections, and even fatal septicaemia.  Pathogenic staphylococci often haemolyse blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins.  The most common type of food poisoning is caused by a heat-stable staphylococcal enterotoxin. 184 jimmy ongori 2013 03/11/2013 21:18  Staphylococci rapidly develop resistance to many antimicrobial agents  The genus Staphylococcus has at least 35 species.  The three main species of clinical importance are S. aureus, S. epidermidis, and S. saprophyticus.  S aureus is a major pathogen for humans. Almost every person will have some type of S aureus infection during a lifetime, ranging in severity from food poisoning or minor skin infections to severe life-threatening infections. jimmy ongori 2013 185 03/11/2013 21:18 General Characteristics  Gram-positive cocci, nonmotile,  Facultative anaerobes  Cells occur in grapelike clusters  Do not form spores but may have capsules  Salt-tolerant: allows them to tolerate the salt present on human skin  Tolerate desiccation: allows survival on environmental surfaces (fomites) jimmy ongori 2013 186 03/11/2013 21:18  STAPHYLOCOCCUS AUREUS  Many neonates, most children and adults become transiently colonized by S. aureus.  The organism is carried in the nasopharynx, occasionally on their skin and clothing and more rarely in the vagina, in the rectum and or perineal area.  One of the commoner causes of opportunistic infections in the hospital and community; including pneumonia, osteomyelitis, septic arthritis, bacteremia, endocarditis, abscesses/boils and other skin infections. 187 jimmy ongori 2013 03/11/2013 21:18  Virulence factors of S. aureus Enzymes:  Coagulase – coagulates plasma and blood; produced by 97% of human isolates; it is diagnostic  Hyaluronidase – digests connective tissue  Staphylokinase – digests blood clots  DNase – digests DNA  Lipases – digest oils; enhances colonization on skin  Penicillinase – inactivates penicillin (very important in resistance to penicillins) 188 jimmy ongori 2013 03/11/2013 21:18  Virulence factors of S. aureus Toxins:  Hemolysins – lyse red blood cells  Leukocidin – lyses neutrophils and macrophages  Enterotoxin – induce gastrointestinal distress  Exfoliative toxin – separates the epidermis from the dermis  Toxic shock syndrome toxin (TSST) – induces fever, vomiting, shock, systemic organ damage 189 jimmy ongori 2013 03/11/2013 21:18  Epidemiology  Present in most environments frequented by humans  Readily isolated from fomites  Carriage rate for healthy adults is 20-60%, mostly in anterior nares, skin, nasopharynx, intestine  Predisposition to infection include: poor hygiene and nutrition, tissue injury, pre-existing primary infection, diabetes, immunodeficiency  Increase in community acquired methicillin resistance - MRSA 190 jimmy ongori 2013 03/11/2013 21:18  Staphylococcal Disease 1. Localized cutaneous infections  Folliculitis – superficial inflammation of hair follicle  Furuncle – boil; inflammation of hair follicle or sebaceous gland progresses into abscess/pustule  Carbuncle – larger and deeper lesion created by aggregation and interconnection of a cluster of furuncles  Impetigo – bubble-like swellings that can break and peel away; most common in newborns  Abscess - Predominant pathogen in about 50% of skin abscesses 191 jimmy ongori 2013 03/11/2013 21:18  2. Systemic Disease i. Toxic shock syndrome - TSS toxin is absorbed into the blood and causes shock. Toxic shock syndrome is manifested by onset of high fever, vomiting, diarrhea, myalgias, rash, and hypotension with cardiac and renal failure in the most severe cases.  Occurs within 5 days after the onset of menses in young women who use tampons, but can occur in children or in men with staphylococcal wound infections. TSS -associated S aureus can be found in the vagina, on tampons, in wounds or other localized infections, or in the throat but virtually 192 never in2013the bloodstream. jimmy ongori 03/11/2013 21:18  TSS -associated S aureus can be found in the vagina, on tampons, in wounds or other localized infections, or in the throat but virtually never in the bloodstrea ii. Bacteremia - presence of bacteria in the blood iii.Endocarditis - occurs when bacteria attack the lining of the heart iv.Pneumonia - inflammation of the lungs v. Osteomyelitis - inflammation of the bone marrow and the surrounding bone jimmy ongori 2013 193 03/11/2013 21:18  STAPHYLOCOCCAL FOLLICULITIS 194 jimmy ongori 2013 03/11/2013 21:18  STYE 195 jimmy ongori 2013 03/11/2013 21:18  STAPHYLOCOCCAL FURUNCLE - “ BOIL” 196 jimmy ongori 2013 03/11/2013 21:18 197 jimmy ongori 2013 03/11/2013 21:18  Staphylococcal Scalded Skin Syndrome. 198 jimmy ongori 2013 03/11/2013 21:18  Staph skin infections Furuncle Deep folliculitis (infected hair follicle superficialfolliculitis Carbuncle Multiple abcesses Around many hair follicles 199 jimmy ongori 2013 03/11/2013 21:18 Scalded skin Staph impetigo syndrome Diagnostic Laboratory Tests  Specimens - pus, blood, tracheal aspirate, or spinal fluid for culture, depending upon the localization of the process.  Smears - appear as gram positive cocci in clusters in Gram-stained smears of pus or sputum.  Culture - Specimens planted on blood agar plates give rise to typical colonies in 18 hours at 37 °Cs. jimmy ongori 2013 200 03/11/2013 21:18  Clinical Concerns and Treatment  95% have penicillinase and are resistant to penicillin and ampicillin  MRSA – methicillin-resistant S. aureus – carry multiple resistance  Some strains have resistance to all major drug groups except vancomycin  Abscesses have to be surgically perforated  Systemic infections require intensive lengthy therapy  Amoxil/cloxacilin, vancomicin 201 jimmy ongori 2013 03/11/2013 21:18  Prevention  Hand antisepsis is the most important measure in preventing nosocomial infections  Also important is the proper cleansing of wounds and surgical openings, aseptic use of catheters or indwelling needles, an appropriate use of antiseptics 202 jimmy ongori 2013 03/11/2013 21:18 Staphylococcus epidermidis  Staphylococcus epidermidis is a less common cause of opportunistic infections than S. aureus, but is still significant. It is a mediator of nosocomial infections (e.g. catheters, shunts, surgery). It is a major component of the skin flora Staphylococcus saprophyticus  This organism is a significant cause of urinary tract infections. It is also coagulase-negative and is not usually differentiated from S. epidermidis clinically. 203 jimmy ongori 2013 03/11/2013 21:18  STREPTOCOCCI 204 jimmy ongori 2013 03/11/2013 21:18  General Characteristics of Streptococci  Gram-positive, spherical arranged in long chains; commonly in pairs  Non-spore-forming, nonmotile  Can form capsules and slime layers  Facultative anaerobes  Most parasitic forms are fastidious and require enriched media  Small, non-pigmented colonies on culture  Sensitive to drying, heat, and disinfectants 205 jimmy ongori 2013 03/11/2013 21:18  Are subdivided into groups by antibodies that recognize surface antigens.  The most important groupable streptococci are A, B and D.  Three types of hemolysis reaction (alpha, beta, gamma) are seen after growth of streptococci on sheep blood agar.  Alpha refers to partial hemolysis with a green coloration, Beta refers to complete clearing and gamma means there is no lysis.  Group A and group B streptococci are beta hemolytic, whilst D are usually alpha or gamma.  Streptococcus pneumoniae and viridans ("green") streptococci are alpha hemolytic.  Thus, the hemolysis reaction is important in grouping jimmy ongori 2013 206 streptococci. 03/11/2013 21:18  Human Streptococcal Pathogens  S. pyogenes  S. agalactiae  Viridans streptococci  S. pneumoniae  Enterococcus faecalis 207 jimmy ongori 2013 03/11/2013 21:18 β-Hemolytic S. pyogenes -  Pyogenes means pus producing  Individual cocci are spherical or ovoid and are arranged in chains.  Are non-motile  Most group A strains produce capsules - impede phagocytosis.  Are Lancefield Serological Group A  Have hair-like pili projecting through the capsule - important in the attachment of streptococci to epithelial cells  It is the most serious streptococcal pathogen  Strict parasite  Inhabits throat, nasopharynx, occasionally skin jimmy ongori 2013 208 03/11/2013 21:18  Virulence Factors Extracellular toxins:  Streptolysins – hemolysins; streptolysin O (SLO) and streptolysin S (SLS) – both cause cell and tissue injury  Erythrogenic toxin (pyrogenic) – induces fever and typical red rash  Superantigens – strong monocyte and lymphocyte stimulants; cause the release of tissue necrotic factor Extracellular enzymes:  Streptokinase (fibrinolysin) – digests fibrin clots  Hyaluronidase – breaks down connective tissue  DNase – hydrolyzes DNA 209 jimmy ongori 2013 03/11/2013 21:18  Epidemiology  Humans are the only reservoir  Inapparent carriers  Transmission – contact, droplets, food, fomites  Portal of entry - skin or pharynx  Children predominant group affected for cutaneous and throat infections  Systemic infections and progressive sequelae possible if untreated 210 jimmy ongori 2013 03/11/2013 21:18 PATHOGENICITY 1. Skin infections  Impetigo (pyoderma) – superficial lesions that break and form highly contagious crust; often occurs in epidemics in school children  Erysipelas – pathogen enters through a break in the skin and eventually spreads to the dermis and subcutaneous tissues; can remain superficial or become systemic jimmy ongori 2013 211 03/11/2013 21:18 2. Throat infections Streptococcal pharyngitis  The most common infection due to - hemolytic S pyogenes is streptococcal sore throat or pharyngitis. S pyogenes adhere to the pharyngeal epithelium by means of surface pili.  The illness may persist for weeks.  May be characterized by intense nasopharyngitis, tonsillitis, and intense redness and edema of the mucous membranes, with purulent exudate 212 jimmy ongori 2013 03/11/2013 21:18  ERYSIPELAS 213 jimmy ongori 2013 03/11/2013 21:18  Streptococcal skin infections 214 jimmy ongori 2013 03/11/2013 21:18  Pharyngitis and tonsillitis 215 jimmy ongori 2013 03/11/2013 21:18 3. Systemic infections  Scarlet fever – strain of S. pyogenes carrying a prophage that codes for pyrogenic toxin; can lead to sequelae  Septicemia  Pneumonia  Streptococcal toxic shock syndrome jimmy ongori 2013 216 03/11/2013 21:18  Long-Term Complications of Group A Infections Rheumatic fever - This is the most serious complication because it results in damage to heart muscle and valves.  Certain strains of group A streptococci contain cell membrane antigens that cross-react with human heart tissue antigens.  Rheumatic fever is often preceded by S pyogenes infection 1–4 weeks earlier Acute glomerulonephritis – This sometimes develops 3 weeks after S pyogenes skin infection  AGN may be initiated by antigen-antibody complexes on the 217 glomerular basement membrane. 03/11/2013 21:18 jimmy ongori 2013 Diagnostic Laboratory Tests  Specimens - depend upon the nature of the infection. A throat swab, pus, or blood for culture. Serum for antibody tests.  Smears - often show single cocci or pairs rather than definite chains.  Culture - on blood agar plates.  Antigen Detection Tests - for rapid detection of group A streptococcal antigen from throat swabs.  Serologic Tests - A rise in the titer of antibodies to many group A streptococcal antigens can be estimated. Such antibodies include antistreptolysin O (ASO) – it is the most widely used. jimmy ongori 2013 218 03/11/2013 21:18 Treatment  Penicillin G, erythromycin.  Antimicrobial drugs are also very useful in preventing reinfection with -hemolytic group A streptococci in rheumatic fever patients. Prevention (1) Detection and early antimicrobial therapy of respiratory and skin infections with group A streptococci. (2) Antistreptococcal chemoprophylaxis in persons who have suffered an attack of rheumatic fever. jimmy ongori 2013 219 03/11/2013 21:18  α-Hemolytic Streptococci: Viridans Group  Large complex group  Streptococcus mutans, S. oralis, S. salivarus, S. sanguis, S. milleri, S. mitis 