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AMBO UNIVERSITY WOLISO CAMPUS DEPARTMENT OF NURSING MEDICAL MICROBIOLOGY FOR NURSING STUDENTS BY፡ AMSALU MEKONNEN (BSC, MSC, DPHM)
CHAPTER ONE HISTORY AND SCOPE OF MICROBIOLOGY AND MEDICAL MICROBIOLOGY
INTRODUCTION TO MICROBIOLOGY What is Microbiology? • Micro - too small to be seen with the naked eye • Bio - life • Logus-logy - study of o Microbiology: is a subject which deals with living organisms that are individually too small to be seen with the naked eye. Amsalu M 3 02/23/2022
Microbiology • Is the science dealing with the study of very small or microscopic organisms and infectious agents which cannot be seen by the naked eye: –Include bacteria, protozoa, viruses, and fungi –Note :Viruses = are infectious agents‖ or ―infectious particles,‖ rather than microorganisms. 4 Amsalu M 02/23/2022
Organisms included in the study of Microbiology 1. Bacteria Bacteriology 2. Protozoans Protozoology 3. Algae Phycology 4. Parasites Parasitology 5. Yeasts and Molds Mycology – Fungi 6. Viruses Virology Amsalu M 5 02/23/2022
5 Kingdoms of Living Organisms • 1. Animalia • 2. Plantae • 3. Fungi • 4. Protista • 5. Monera - Bacteria and Cyanobacteria • Eukaryotic vs. Prokaryotic Amsalu M 6 02/23/2022
Classification System • 3 Domains 1978 Carl Woese – 1. Bacteria • Unicellular prokaryotes with cell wall containing peptidoglycan – 2. Archaea • Unicellular prokaryotes with no peptodoglycan in cell wall – 3. Eukarya • Protista • Fungi • Plantae • Animalia Amsalu M 7 02/23/2022
Uses of Microbes Benefit Humans • 1.Bacteria are primary decomposers - recycle nutrients back into the environment (sewage treatment plants) • 2. Microbes produce various food products – cheese, pickles, sauerkraut, green olives – yogurt, soy sauce, vinegar, bread – Beer, Wine, Alcohol Amsalu M 8 02/23/2022
3. Microbes are used to produce Antibiotics • Mold – Penicillium notatum (Penicillin) • 1928 Alexander Fleming 4. Bacteria synthesize chemicals that our body needs, but cannot synthesize • Example: E. coli – B vitamins - for metabolism – Vitamin K - blood clotting • Escherichia coli – Dr. Escherich – Colon (intestine) Amsalu M 9 02/23/2022
5. Biochemistry and Metabolism • Very simple structure • rapid rate of reproduction • provides “instant” data 6. Microbial antagonism • Our normal microbial flora prevents potential pathogens from gaining access to our body Amsalu M 10 02/23/2022
7. Insect Pest Control • Using bacteria to control the growth of insects • Bacillus thuringiensis – caterpillars – bollworms – corn borers 8. Bioremediation • Using microbes to clean up pollutants and toxic wastes • Exxon Valdez - 1989 • 2 Genera – Pseudomonas sp. – Bacillus sp. Amsalu M 11 02/23/2022
9. Recombinant DNA Technology, Gene Therapy, Genetic Engineering • Bacteria can be manipulated to produce enzymes and proteins they normally would not produce – Insulin – Human Growth Hormone – Interferon 10. Food Chains • Marine and fresh water microorganisms Amsalu M 12 02/23/2022
 Beneficial aspects: – Inhibit growth of pathogens by • occupying space, • Competing for food and • secreting waste products, toxins, antibiotics, etc. – Many microorganisms are involved in the decomposition of dead organisms and the waste products of living organisms. Beneficial and harmful aspects of microorganisms 13 Amsalu M 02/23/2022
– Some microorganisms aid in the digestion of food and, in some cases, produce substances that are of value to the host (vitamins K and B1) – Many microorganisms are essential in various food and beverage industries – Certain bacteria and fungi produce antibiotics that are used to treat patients with infectious diseases. Beneficial and harmful aspects Cont… 14 Amsalu M 02/23/2022
– Microbes are essential in the field of genetic engineering. – e. g. production of insulin, various types of growth hormone, interferons, and materials for use as vaccines.  Harmful aspects – Microorganisms cause : infectious diseases and food spoilage. Beneficial and harmful aspects Cont… 15 Amsalu M 02/23/2022
Bacteria - what comes to mind? • Diseases • Infections • Epidemics • Food Spoilage • Only 1% of all known bacteria cause human diseases • About 4% of all known bacteria cause plant diseases • 95% of known bacteria are non-pathogens Amsalu M 16 02/23/2022
Microbes do benefit us, but they are also capable of causing many diseases • Pneumonia Whooping Cough • Botulism Typhoid Fever Measles • Cholera Scarlet Fever Mumps • Syphilis Gonorrhea Herpes 1 • Chlamydia Tuberculosis Herpes 2 • Meningitis Tetanus RMSV • Strep Throat Lyme Disease AIDS • Black Plague Diarrhea Gangrene Amsalu M 17 02/23/2022
History of the Study of Microorganisms • 1665 Robert Hooke – “little boxes” - “cells” – Cell Theory - all living things are made up of cells Amsalu M 18 02/23/2022
Anton van Leeuwenhoek 1674 - 1st person to actually see living microorganisms “wee animalcules” Amsalu M 19 02/23/2022
Spontaneous Generation • Theory that life just “spontaneously” developed from non-living matter • Example: – toads, snakes and mice - moist soil – flies and maggots - manure and decaying flesh Amsalu M 20 02/23/2022
Experiments to disprove Spontaneous Generation • Francesco Redi 1668 • Rudolph Virchow 1858 – Theory of Biogenesis • Cells can only arise from preexisting cells • Louis Pasteur 1861 Amsalu M 21 02/23/2022
History (cont.) • 1861 Pasteur – Proved Microorganisms are present in nonliving matter – Microbes can be destroyed by heat •Aseptic Technique • Fermentation mediated by yeast, not air – Pasteurization to prevent wine and beer spoilage (by bacteria) Amsalu M 22 02/23/2022
FIELDS IN MICROBIOLOGY • Includes: – Agricultural Microbiology – Industrial Microbiology – Environmental Microbiology – Medical Microbiology – Microbial Genetics and Genetic Engineering 23 Amsalu M 02/23/2022
Medical Microbiology – Involves the study of pathogens, the diseases they cause, and the body‘s defenses against disease. – Concerned with : • Epidemiology and transmission of pathogens • Disease prevention measures and treatment of infectious diseases • Aseptic techniques • Immunology and the production of vaccines to protect people and animals against infectious diseases. 24 Amsalu M 02/23/2022
Subdivision of Medical Microbiology • Bacteriology: the study of structure, functions and activities of bacteria • Mycology: is the scientific study of fungi. • Virology: is the scientific study of viruses. • Immunology: is the scientific study of immunity.. • Parasitology: is study parasite & their interaction of the host. 25 Amsalu M 02/23/2022
Brief History of Microbiology  The Microscope  The theory of abiogenesis and biogenesis  Germ Theory of disease 26 Amsalu M 02/23/2022
The First Microscope 27 Microbes were first observed by Antonie van Leeuwenhoek using a simple microscope (1673) Reported his ―animalcules‖ to the Royal Society of London Amsalu M 02/23/2022
Cont… • Antony Van Leeuwenhoek (1632-1723)- –father of Microbiology – He was the first to describe ―animalcules‖ (single celled organism) using simple microscope with one lens. – is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria. – He was the first who properly described the different shapes of bacteria. 28 Amsalu M 02/23/2022
The theory of Abiogenesis vs. Biogenesis • Although Leeuwenhoek was not concerned about the origin of micro-organism; many other scientists were searching for the origin of microorganisms • Two major theories were formulated: 1. Theory of Abiogenesis 2. Theory of Biogenesis  Theory of Abiogenesis: deals with the theory of spontaneous generation; stating that living things originated from non-living things. • Example: maggots from decaying meat or mushrooms from rotting wood 29 Amsalu M 02/23/2022
An experiment to disprove spontaneous generation • Francesco Redi (1626-1697) :  used two flasks and placed meat in each of the two flasks – One of the flasks left open – Another one tightly sealed • He observed that the flies laid eggs on the open flask and then the maggots developed. • Proved that no maggots appeared in meat when flies were prevented from laying eggs. 30 Amsalu M 02/23/2022
Francesco Redi‘s experiments with meat 31 uncovered covered Maggots No maggots Disproved that maggots arise from decaying meat!! Amsalu M 02/23/2022
Theory of Biogenesis  Theory of Biogenesis states that life comes from pre- existing life.  Louis Pasteur (1822-1895 GC)- was the scientist who disproved the theory of abiogenesis Experiment of Louis Pasteur: • He designed a large curved flask (Pasteur goose neck flask) and placed a sterile growth broth medium. • Air freely moved through the tube; but dust particles and microbes were trapped in the curved portion of flask. • Microbial growth in the broth was not seen. • Therefore he proved that micro-organisms entered to substrates through the air and micro-organisms did not evolve spontaneously. 32 Amsalu M 02/23/2022
Cont… 33 Amsalu M 02/23/2022
Pasteur’s Swan-Necked Flasks 34 Amsalu M 02/23/2022
Major contribution of Louis Pasteur 1. Microbial theory of fermentation 2. Principles and practice of sterilization and pasteurization 3. Control of diseases of silk worms 4. Development of vaccines against anthrax and rabies. 5. Discovery of streptococci 35 Amsalu M 02/23/2022
History (cont.) • 1857-Louis Pasteur saves France’s wine • 1861 Pasteur – Proved Microorganisms are present in nonliving matter – Microbes can be destroyed by heat technique • Fermentation mediated by yeast, not air – Pasteurization to prevent wine and beer spoilage (by bacteria) Amsalu M 36 02/23/2022
1) Good wine contained yeast 2) Sour wine contained bacteria 1) (Bacteria that use alcohol and produce acetic acid spoil wine by turning it to vinegar (acetic acid). 3) He reasoned that if wine is heated to destroy the harmful bacteria it wouldn’t spoil (process known as Pasteurization) Amsalu M 37 02/23/2022
Ignaz Semmelweis (1818-1865) • Taught medicine in Vienna • No one connected germs with disease yet • Puerperal fever “childbirth fever” caused 25-30% mortality • Nearby obstetric hospital had only a 2% death rate Amsalu M 38 02/23/2022
Ignaz Semmelweis (cont.) • He made some observations • Medical Students working on cadavers moved from the dissecting room to the maternity ward • Midwives –Stayed only in maternity ward Amsalu M 39 02/23/2022
Ignaz Semmelweis (cont.) • Ordered students to wash hands and medical instruments in chlorinated lime • Mortality dropped to 1.3% • By 1848, 0% mortality Amsalu M 40 02/23/2022
The Golden Age of Microbiology • 1857-1914 • Beginning with Pasteur‘s work, discoveries included • the relationship between microbes and disease, • immunity, and • antimicrobial drugs 41 Amsalu M 02/23/2022
Golden Age of Microbiology 1857 - 1914 • Pasteur – Pasteurization – Fermentation • Joseph Lister – Phenol to treat surgical wounds – 1st attempt to control infections caused by microoganisms • Robert Koch – Koch’s Postulates • Edward Jenner – vaccination • Paul Erlich – 1st synthetic drug used to treat infections – Salvarsan - arsenic based chemical to treat Syphilis • “salvation” from Syphilis Amsalu M 42 02/23/2022
Germ Theory of Disease Germ Theory of Disease: the theory stating microorganisms are the causative agent of disease. • Before the time of Pasteur, effective treatments for many diseases were discovered by trial and error, • but the causes of the diseases were unknown. • The discovery of yeast that play a crucial role in fermentation lead scientists to belief that microbes might be involved in causing disease. 43 Amsalu M 02/23/2022
Germ Theory of Disease • Pasteur proposed that wine spoiling in an analogy for disease (bacterial growth made the wine “sick”) • germ theory – some diseases are caused by microorganisms Amsalu M 44 02/23/2022
Germ Theory of Disease • Hard for people to believe that diseases were caused by tiny invisible “wee animalcules” • Diseases, they thought, were caused by: – demons – witchcraft – bad luck – the wrath of God – curses – evil spirits Amsalu M 45 02/23/2022
Edward Jenner (country doctor) –Milkmaid didn’t get smallpox b/c they contracted the milder form of cowpox –Immune system cannot distinguish btw cowpox/smallpox –Scratched a farmboy w/ a needle bearing fluid from cowpox –Small pox Vaccine - Vacca-cow - Vaccination w/ cowpox provided immunity for smallpox Amsalu M 46 02/23/2022
Robert Koch • -1st to prove that bacteria actually caused diseases • 1876 • Microbial Etiology of Infectious Disease – etiology - the cause of a disease • Established “scientific rules” to show a cause and effect relationship between a microbe and a disease – Koch’s Postulates Amsalu M 47 02/23/2022
Pure Culture Key to Studying Microbes Definition: Pure culture is a population of organism, all of which are the progeny of a single organism -In nature, microbes almost never occur as pure cultures Amsalu M 48 02/23/2022
Koch’s Postulates • 1. The same organisms must be found in all cases of a given disease. • 2. Organism can be isolated and grown in pure culture. • 3. The isolated organism must reproduce the same disease when inoculated into another animal • 4. The original organism must again be isolated from the experimentally infected animal. Amsalu M 49 02/23/2022
Exceptions to Koch’s Postulates 1. Some organisms have never been grown in pure culture on artificial media Treponema pallidum - Syphilis Amsalu M 50 02/23/2022
Mycobacterium leprae Leprosy Never been grown in pure culture on artificial media Abdominal cavity of the Seven Banded Armadillo Exceptions to Koch’s Postulates Amsalu M 51 02/23/2022
Exceptions to Koch’s Postulates • In exclusively human diseases, it is not morally acceptable to inoculate a deadly pathogen into a “human guinea pig” • HIV Amsalu M 52 02/23/2022
Koch established the Microbial Etiology of 3 important diseases of his day • 1. Cholera (fecal-oral disease) – Vibrio cholerae • 2. Tuberculosis (pulmonary infection) – Mycobacterium tuberculosis • 3. Anthrax (sheep and cattle) – Bacillus anthracis Amsalu M 53 02/23/2022
• In general, germ theory is Proposed by Louis Pasteur in 1857. • But given experimental support and proofed by Robert Koch (1843-1910) – The first proof that bacteria actually cause disease came from Robert Koch in 1876. – Koch discovered rod -shaped bacteria now known as Bacillus anthracis in the blood of cattle that had died of anthrax. – Koch played a major role in establishing that microorganisms cause specific diseases. 54 Amsalu M 02/23/2022
Germ Theory of Disease  1876 - Robert Koch provided proof that a bacterium causes anthrax using experimental steps now called the Koch’s Postulates  He was the first to use agar as solid culture medium in bacteriology. 55 Amsalu M 02/23/2022
• Koch‘s Postulates – a method of determining the etiologic (causative) agent of infectious diseases 1. The suspected etiologic agent must be found in every case of the disease and be absent in healthy hosts 2. The suspected etiologic agent must be isolated in pure culture and identified 3. The suspected etiologic agent is inoculated into a healthy, susceptible host and that host must come down with the same disease 4. The same etiologic agent as in step 2 must be isolated and identified in the second diseased animal Koch’s Postulates 56 Amsalu M 02/23/2022
Limitations to Koch’s postulate • Many healthy people carry pathogens but do not exhibit symptoms of the disease. • Some microbes are very difficult or impossible to grow in vitro (in the laboratory) in artificial media. E.g. Treponema pallidum, viruses, reckettsia and chylamidia • Certain diseases develop only when an opportunistic pathogen invades imuno-compromised host. 57 Amsalu M 02/23/2022
Major achievements of Robert Koch 1. Discovery and use of solid medium in bacteriology 2. Discovery of causative agents of tuberculosis, anthrax and cholera. 3. Koch’s postulates 58 Amsalu M 02/23/2022
Paul Ehrlich-hospital dermatologist • Chemotherapy- Treatment using chemical substances • 1910 Paul Ehrlich - ”Magic bullet” –Salvarsan (arsenic derivative) •Preparation 606 –Syphilis Amsalu M 59 02/23/2022
• Alexander Fleming 1928 – Scottish researcher • Discovered Penicillin (fungus) by accident • Was convinced that nasal mucus had antibacterial effects • Left his Staphylococcus culture on an agar plate for 2 weeks-went on vacation-came back &found mold on his plate which prevented bacterial growth – (a Mycology lab underneath him had this rare spore drift) Amsalu M 60 02/23/2022
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QUESTIONS
CHAPTER TWO TAXONOMIC CLASSIFICATION, NOMENCLATURE AND IDENTIFICATION OF MICROORGANISMS
– Taxonomy: The science of classification, especially the classification of living forms. – It helps : • To establish relationships between one group of organisms and another and to differentiate them. • Provides a common reference for identifying organisms already classified. • A basic and necessary tool for scientists. TAXONOMIC CLASSIFICATION, NOMENCLATURE AND IDENTIFICATION OF ORGANISMS 64 Amsalu M 02/23/2022
• There are three interrelated areas in Taxonomy: 1) Nomenclature: is naming an organism by international rules according to its characteristics 2) Classification: the arrangement of organisms into taxonomic groups (taxa) on the basis of similarities or relationships. 3) Identification: refers to the practical use of a classification scheme. 65 Amsalu M 02/23/2022
Phenotypic Characteristics to Classify Prokaryotes Includes size and shape, staining characteristics, and metabolic capabilities Genotypic Characteristics to Classify Prokaryotes  Based on the comparison of the nucleotide sequences of the rDNA.  DNA hybridization  Comparing the Sequences of 16S Ribosomal Nucleic Acid  DNA Base Ratio (G+C content) How organisms are classified? 66 Amsalu M 02/23/2022
• Provides a crude comparison of genomes • Prokaryotic G+C content is the most variable – If two organisms differ in their G+C content by more than about 10%, their genomes have quite different base sequences. – The G+C content of strains within a particular species is constant. 67 Amsalu M 02/23/2022
• Taxonomic classification categorizes organisms in a hierarchical order, with the species being the basic unit. • The species designation gives a formal taxonomic status to a group of related isolates or strains – permits their identification 68 Amsalu M 02/23/2022
• Species—a group of related isolates or strains. – Note that members of a species are not all identical; individual strains may vary in minor properties. • Genus—a collection of related species. • Family—a collection of similar genera. – In prokaryotic nomenclature, the name of the family ends in the suffix -aceae. • Order—a collection of similar families. – In prokaryotic nomenclature, the name of the order ends in the suffix –ales • Class—a collection of similar orders. – In prokaryotic nomenclature, the name of the class ends in the suffix -ia. 69 Amsalu M 02/23/2022
• Phylum or Division—a collection of similar classes. • Kingdom—a collection of similar phyla or divisions. • Domain—a collection of similar kingdoms. – The domain is a relatively new taxonomic category that reflects the characteristics of the cells that make up the organism. 70 Amsalu M 02/23/2022
• The domain system contains three members 1) Bacteria (formerly eubacteria) 2) Archaea (archaebacteria)=share the prokaryotic type of cellular configuration. 3) Eukarya 71 Amsalu M 02/23/2022
Nomenclature (binomial nomenclature) • Organisms are named using binomial nomenclature • Binomial nomenclature employs the names of the two level taxa, genus and species to name an organism. • According to Binomial nomenclature : a) Genus comes before species (e.g., Escherichia coli) b) The first letter of the genus name is always capitalized (e.g., Escherichia) c) Species name is never capitalized (e.g., coli) d) Both names are always either italicized or underlined e.g., Escherichia coli or Escherichia coli e) The genus name may be used alone, but not the species name (i.e., saying or writing ―Escherichia “ alone is legitimate while saying or writing ― coli” is not) 72 Amsalu M 02/23/2022
EUKARYOTIC AND PROKARYOTIC CELL  EUKARYOTIC CELL • Eu- true • Karyote- nucleus • The eukaryotic cell has a true membrane bound nucleus, usually containing multiple chromosomes, a mitotic apparatus, a well defined endoplasmic reticulum and mitochondria Eukaryotes . Algae . Protozoa . Fungi 73 Amsalu M 02/23/2022
Cont… PROKARYOTIC CELL • Pro- primitive • Karyote- nucleus The prokaryotic cell possesses naked DNA without associated basic proteins divides by binary fission Prokaryotes . Bacteria 74 Amsalu M 02/23/2022
Cont… • The distinguishing features between Eukaryotic cell and Prokaryotic cell • Features Prokaryotic cell Eukaryotic cell • Size 1m 10m • Nuclear membrane absent Present • Chromosome Single Multiple • Nucleolus absent Present • Histones absent Present • Sexual reproduction absent Present • Mitochondria absent Present • Endoplasmic reticulum absent Present • Lysosomes absent Present • Peptidoglycan present absent • Cell membrane composition Phospholipids &Protein Sterols 75 Amsalu M 02/23/2022
BACTERIOLOGY Definition: Bacteriology: the study of structure, functions and activities of bacteria. Medical Bacteriology: - the study of medically important bacteria 02/23/2022 Amsalu M 76
Bacterial Cell General property: • Typical prokaryotic cell • Contain both DNA and RNA • Most grow in artificial media • Replicate by binary fission • Almost all contain rigid cell wall • Sensitive to antimicrobial agent 77 Amsalu M 02/23/2022
STRUCTURE OF BACTERIA Bacterial structure is considered at three levels: 1. Cell envelope: Cell wall and cell membrane. 2. Cellular element internal the cell envelope: Mesosomes, ribosomes, chromosomes (nucleoid) and cytoplasmic granules, spores… 3. Cellular element external to the cell envelope: Flagellum, Pilus and Glycocalyx,….. 78 Amsalu M 02/23/2022
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1. Cell envelope • External covering outside the cytoplasm • Composed of two basic layers: –cell wall and cell membrane • Maintains cell integrity A. Cell wall Multi layered structure and constitutes about 20% of the bacterial dry weight. Average thickness is 0.15-0.5 µm 81 Amsalu M 02/23/2022
• Main structural component–Peptidoglycan:-is repeating disaccharides units and Polypeptides Functions of cell wall • Determines cell shape • prevents lysis (bursting) or collapsing due to changing osmotic pressures • Provides staining characteristics to the bacterium • Site of action of antibody • Contains toxic components to host 82 Amsalu M 02/23/2022
• Based on their cell wall structure two different groups of bacteria are demonstrated by Gram stain: –Gram-positive bacteria: thick cell wall composed primarily of thick peptidoglycan and teichoic acid –Gram-negative bacteria: composed of outer membrane, thin peptidoglycan layer, and periplasmic space 83 Amsalu M 02/23/2022
Cell wall 84 Amsalu M 02/23/2022
Gram-positive Cell Wall • Thick, homogeneous sheath of peptidoglycan: –function in cell wall maintenance and enlargement during cell division –stimulate a specific immune response • Have Teichoic acid 85 Amsalu M 02/23/2022
Gram-negative Cell Wall • Composed of an outer membrane and a thin peptidoglycan layer and also has periplasmic space • Outer membrane is: – outermost layer contains lipopolysaccharides (LPS), lipoproteins and phospholipids – is an important factor in evading phagocytosis and actions of complements. – Provides a barrier to certain antibiotics (e.g.penicillin), digestive enzymes (e.g. lysozyme), detergents, heavy metals, bile salts, and certain dyes. – contains porin proteins in upper layer – regulate molecules entering and leaving cell 86 Amsalu M 02/23/2022
Cont… Lipopolysaccharide (LPS) - is one component of outer membrane: Has three components a) lipid A b) a core polysaccharide and c) an O polysaccharide. A) Lipid A – Lipid portion of the LPS – Embedded in the top layer of the outer membrane – Functions as an endotoxin. B) Core polysaccharide – Its role is structural i.e. to provide stability. 87 Amsalu M 02/23/2022
Cont… c) O polysaccharide – Extends outward from the core polysaccharide – Functions as an antigen and is useful for distinguishing species of gram-negative bacteria. • For example, the foodborne pathogen E. coli O157:H7 • Note : gram negative bacteria do not contain teichoic acids and Gram positive don‘t contain outer membrane. 88 Amsalu M 02/23/2022
Bacteria Classification 89 Amsalu M 02/23/2022
Components of cell wall of Gram negative bacteria • Peptidoglycan • Periplasmic space • Lipoprotein • Phospholipid Outer membrane • Lipopolysaccharide Components of cell wall of Gram positive bacteria • Peptidoglycan • Teichoic acid 90 Amsalu M 02/23/2022
B. Cell membrane (cytoplasmic membrane) • It accounts for 30% of the dry weight of bacterial cell. • Phospholipid bilayer with embedded proteins • It is composed of • 60% protein, • 20-30% lipids and • 10-20% carbohydrate • Functions in: – Regulates the transport of nutrients and waste products into and out of the cell. 91 Amsalu M 02/23/2022
2. Cellular element internal to the cell envelope A. Mesosomes • Convoluted invagination of cytoplasmic membrane often at sites of septum formation. • It is involved in DNA segregation during cell division. B. Ribosomes • Cytoplasmic particles which are the sites of protein synthesis. • It is composed of RNA (70%) and proteins (30%) 92 Amsalu M 02/23/2022
Cont… C. Inclusion bodies • Represent accumulated food reserve D. Chromosome – single, circular, double-stranded DNA molecule that contains all the genetic information required by a cell – DNA is tightly coiled around a protein, aggregated in a dense area called the nucleoid. (nuclear region of bacteria ) 93 Amsalu M 02/23/2022
Cont… • Plasmids: bacteria may have extra chromosomal genetic material named as Plasmids small circular, double- stranded DNA • which may facilitate survival and propagation of the micro- organism • provide virulence and drug resistance. 94 Amsalu M 02/23/2022
Cont… E. Spores – Resistant and metabolically inactive – produced by some Gram positive genera: Clostridium, Bacillus • Such bacteria have a 2-phase life cycle: ovegetative cell : metabolically active and growing oendospore : –Dormant cell –Produced when starved –when exposed to adverse environmental conditions; capable of high resistance and very long-term survival – sporulation -formation of endospores – germination- return to vegetative growth 95 Amsalu M 02/23/2022
 Resistance of spore is linked to:  high levels of calcium and Presence of dipicolinic acid thick coat  Longevity verges on immortality – can live million years.  Resistant to ordinary cleaning methods and boiling  Pressurized steam at 120oC for 20-30 minutes will destroy the spore 96 Amsalu M 02/23/2022
3. Cellular element external to the cell envelope A. Glycocalyx (capsule and slime layer)  Coating of molecules external to the cell wall, made of sugars and/or proteins  Two types: 1. slime layer - loosely organized and attached 2. capsule - highly organized, tightly attached  Functions: ◦ protect cells from dehydration and nutrient loss ◦ inhibit killing by white blood cells by phagocytosis contributing to pathogenicity ◦ attachment - formation of biofilms 97 Amsalu M 02/23/2022
Cont… B. Flagellum It is the organ of locomotion in bacterial cell. It is composed of protein named as flagellin. The flagellar antigen in motile bacterium is named as H antigen  consists of three parts. These are: • Filament • Hook and • Basal body The basal body and hook are embedded in the cell surface while the filament is free on the surface of bacterial cell. 98 Amsalu M 02/23/2022
Cont… Based on flagella arrangements bacteria can be: • Atrichous: Bacteria with no flagellum. • Monotrichous: Bacteria with single polar flagellum. • Lophotrichous: Bacteria with bunch of flagella at one pole. • Amphitrichous: Bacteria with flagella at both poles. • Peritrichous: Bacteria with flagella all over their surface. 99 Amsalu M 02/23/2022
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Cont… C. Fimbriae and Pili – Are shorter, and thinner than flagella – Found on many gram-negative bacteria a) Fimbriae (singular: fimbria ) – Can occur at the poles or can be evenly distributed over the entire surface of a bacterium. – Important for adherance. b) Pili (singular: pilus) – Are usually longer than fimbriae – Number only one or two per cell. – Involved in DNA transfer from one to the other 101 Amsalu M 02/23/2022
Classification of bacteria • Bacterial classification depends on the following characteristics. 1. Morphology and arrangement 2. Staining 3. Cultural characteristics 4. Biochemical reactions 5. Antigenic structure 6. Base composition of bacterial DNA • Morphology and staining of bacteria are the commonly used characteristics to classify bacteria. 102 Amsalu M 02/23/2022
1. Morphology of bacteria • When bacteria are visualized under light microscope, the following morphology are seen. 1. Cocci (singular coccus): spherical or oval bacteria measuring about 0.5-1.0μm in diameter. • They are found in single, pairs, chains or clusters. • Micrococci:- Cocci occurring single. • Diplococci:- arranged in pair • Streptococci :- chain forming cocci • Staphylococci:- bunches of cocci or groups or grapes of cocci 103 Amsalu M 02/23/2022
Shape(cocci) and their arrangement 104 Amsalu M 02/23/2022
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2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tapered, square or swollen ends; with a size measuring 1-10μm in length by 0.3-1.0μm in width. 106 Amsalu M 02/23/2022
• Bacilli: stick- like bacteria Bacilli (rod Shape) 107 Amsalu M 02/23/2022
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3. Spiral: Spiral shaped bacteria with regular or irregular distance between twisting. • Coiled or cork-skrew shaped • Eg. Spirilla and spirochaetes 109 Amsalu M 02/23/2022
Spiral bacteria 110 Amsalu M 02/23/2022
Cont… 2. Staining of bacteria • staining is the process of coloring of colorless object using stains (dyes) • Uses of staining • To observe the morphology, size and arrangement of bacteria • To differentiate one group of bacteria from the other group. 111 Amsalu M 02/23/2022
Cont… Type of staining methods –Simple staining method –Differential staining method –Special staining method 112 Amsalu M 02/23/2022
Cont… 1. Simple staining method It is a type of staining method in which only a single dye is used. Two kinds of simple stains A. positive staining:- The bacteria or its parts are stained by the dye E.g. Methylene blue stain, Crystal violent stain B. Negative staining: - the dye stains the background and the bacteria remain unstained E.g. Indian ink stain 113 Amsalu M 02/23/2022
Cont… 2. Differential staining method  Multiple stains (dyes) are used to distinguish different group of bacteria  React differently with different types of bacteria  2 Most common: –Gram Stain –Acid-Fast Stain (Ziehl- Neelson stain) 114 Amsalu M 02/23/2022
A. Gram stain • Gram Stain: Named after Christian Gram who invented it • Most bacteria are differentiated by their gram reaction due to difference on their cell wall structure. Principle: • Bacteria are stained with Crystal violet and Iodine, then subjected to decolorization with alcohol/acetone. Bacteria that retain the primary stain (Crystal violet) are Gram-positive. Bacteria that loose the primary stain (Crystal violet) are Gram-negative 115 Amsalu M 02/23/2022
Required reagents: – crystal violet: primary stain – Gram‘s Iodine: mordant – Acetone-Alcohol: decolorizer – Safranin: counterstain Procedure: 1. Prepare the smear from the culture or from the specimen. 2. Allow the smear to air-dry completely. 3. Rapidly pass the slide (smear upper most) three times through the flame. 4. Cover the fixed smear with crystal violet for 1 minute and wash with distilled water. 116 Amsalu M 02/23/2022
5. Tip off the water and cover the smear with gram‘s iodine for 1 minute. 6. Wash off the iodine with clean water. 7. Decolorize rapidly with acetone-alcohol for 30 seconds. 8. Wash off the acetone-alcohol with clean water. 9. Cover the smear with safranin for 1 minute. 10. Wash off the stain wipe the back of the slide. Let the smear to air-dry. 11. Examine the smear with oil immersion objective to look for bacteria. • Interpretation: – Gram-positive bacterium ……………Purple – Gram-negative bacterium …………..Pink(red) 117 Amsalu M 02/23/2022
Principle of staining technique: 118 1- Crystal violet: all cells are stained violet. 2- Iodine acts as a mordant (fixes the dye to the cell) all cells remain violet. 3-Alcohol acetone decolorizes gram negative cells only: Gram-positive remains violet , gram-negative becomes colorless. 4-Counter stain with Safranin: Gram- positive remains violet while gram negative becomes red. Amsalu M 02/23/2022
Cont… • Result  Gram (+) Purple  Gram (-) Red • Difference - due to structure of cell wall –Gram (+) Thick cell wall –Gram (-) Thin cell wall 119 Amsalu M 02/23/2022
Gram-Positive 120 Amsalu M 02/23/2022
Gram-Positive 121 Amsalu M 02/23/2022
Gram-Negative 122 Amsalu M 02/23/2022
Gram-Negative 123 Amsalu M 02/23/2022
B. Ziehl- Neelson staining Method • Ziehl- Neelson stain (Acid fast stain) is used to stain Mycobacterium and other acid fast organisms which cannot be stained with gram stain.  once the mycobacterium is stained with the primary stain it can not be decolorized with acid. So named as Acid fast bacteria 124 Amsalu M 02/23/2022
• Reagents required: – Carbol-fuchsin: primary stain – Acid-Alcohol: decolorizer – Methylene blue/Malachite green: counterstain Procedure for Ziehl-Neelson staining method 1. Prepare the smear from the primary specimen and fix it by passing through the flame and label clearly 2. Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin solution. 3. Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait for 5 minutes. 125 Amsalu M 02/23/2022
4. Wash off the stain with clean water. 5. Cover the smear with 3% acid-alcohol solution until all color is removed and wait for 3 minutes. 6. Wash off the stain and cover the slide with 1% methylene blue and wait for one minute. 7. Wash off the stain with clean water and let it air-dry. 8. Examine the smear by adding a drop of oil under the oil immersion objective to look for acid fast bailli. • Interpretation: – Acid fast bacilli…………..Red – Back ground………………Blue 126 Amsalu M 02/23/2022
Cont… Result  Acid fast bacilli . . .Red  Back ground . . . . Blue 127 Amsalu M 02/23/2022
3. Special Stains A. Capsule Staining method B. Spore staining method 128 Amsalu M 02/23/2022
GROWTH, NUTRITION & GENETICS OF BACTERIA
BACTERIAL NUTRITION • Bacteria, like all cells, require nutrients for growth. • Most important elements required by bacteria can be divided into two main categories: Physical requirements include: –Temperature, pH, and osmotic pressure. Chemical requirements include –Sources of carbon, nitrogen, sulfur, phosphorus, trace elements, and organic growth factors. 130 Amsalu M 02/23/2022
 Chemical requirements • The four most important elements of bacteria are carbon, hydrogen, oxygen and nitrogen. Carbon • Organisms require a carbon for the synthesis of numerous organic compounds that comprise bacterial cell. • Depending on their requirements, bacteria can be classified as 1. Autotrophs: Free-living, non-parasitic bacteria which use carbon dioxide as carbon source. • The energy needed for their metabolism can be obtained from: Sun light-Photoautotrophs Inorganic compounds by oxidation-Chemoautotrophs 131 Amsalu M 02/23/2022
2. Heterotrophs: Parasitic bacteria require more complex organic compounds as their source of carbon and energy. • Human pathogenic bacteria are heterotrophs. • The principal source of carbon is carbohydrate which are degraded either by oxidation, in the presence of oxygen, or by fermentation, in the absence of oxygen, to provide energy in the form of ATP. 132 Amsalu M 02/23/2022
Hydrogen and oxygen • Obtained from water. • Essential for the growth and maintenance of cell.  Based on oxygen requirements and tolerance, bacteria are classified as:  Obligate aerobes - require oxygen to live  Obligate anaerobes - grow in the absence of oxygen ; exposure to oxygen kills anaerobes  Facultative anaerobes - grow in the presence or absence of oxygen.  Microaerophiles- grow best at reduced oxygen tension 133 Amsalu M 02/23/2022
134 Amsalu M 02/23/2022
Nitrogen • For protein synthesis & also for DNA and RNA synthesis • Obtained from organic molecules like proteins and inorganic molecules like ammonium salts and nitrates. NB: Main source of nitrogen is ammonia, in the form of ammonium salt. 135 Amsalu M 02/23/2022
 Physical Requirements Temperature  Temperature tolerance range: The minimum and maximum temperature at which a micro-organism can grow  which is different in different species of bacteria.  Optimal growth range of temperature: The temperature at which the bacteria grow best. 136 Amsalu M 02/23/2022
 Based on different optimal growth temperature requirement, bacteria are divided into: Psychrophilic bacteria: 00C-20 0C Mesophilic bacteria: 20-450C Thermophilic bacteria: 50-600C • NB: Most human pathogens and many of the normal flora of human bodies have an optimal temperature of 370c; Therefore they are mesophilic bacteria. 137 Amsalu M 02/23/2022
PH • It is a measure of acidity and alkalinity.  Neutrophilic bacteria grow best at near neutral PH value. • Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5.  Acidophilic bacteria prefer to grow at low PH value (acidic medium).  Alkalinophilic bacteria prefer to grow at high PH value (alkaline medium). 138 Amsalu M 02/23/2022
Salinity Salt content of the medium affects bacterial growth.  Halophilic bacteria grow best at high salt concentration.  Moderate halophiles require 3% salt concentration.  Extreme halophiles require 15% salt concentration. 139 Amsalu M 02/23/2022
BACTERIAL GROWTH • It is an orderly increase in all the components of an organism. • It is an increment in cell number. Generation time • It is the time taken for a bacterial population to double.  time required for a cell to divide • bacteria divide into two equal daughter cells and double the number. i.e binary fission 140 Amsalu M 02/23/2022
• The generation time varies with: the species the amount of nutrients, the temperature, the pH, and other environmental factors. Bacterial growth phases  The normal bacterial growth curve has four phases: 1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase 141 Amsalu M 02/23/2022
1. Lag phase • The period of adaptation with active macro molecule synthesis like DNA, RNA, various enzymes and other structural components. • It is the preparation time for reproduction; no increase in cell number 2. Exponential (log) phase • The period of active multiplication of cells. • Cell division proceeds at a logarithmic rate, and determined by the medium and condition of the culture. 142 Amsalu M 02/23/2022
3. stationary phase (Equilibrium phase) • The period when the bacteria have achieved their maximal cell density or yield. • There is no further increase in viable bacterial cell number. • The growth rate is exactly equal to the death rate. • A bacterial population may reach stationary growth when one of the following conditions occurs: – The required nutrients are exhausted – Inhibitory end products are accumulated – Physical conditions do not permit a further increase in population size. 143 Amsalu M 02/23/2022
4. Decline phase • The period at which the rate of death of bacterial cells exceeds the rate of new cell formation. • There is drastic decline in viable cells. • Few organisms may persist for so long time at this period at the expense of nutrients released from dying micro-organisms. 144 Amsalu M 02/23/2022
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CULTIVATION OF BACTERIA IN CULTURE MEDIA • Culture- is the growth of particular microorganism on or within a medium as a result of inoculation and incubation under controlled conditions. • Culture media- is a liquid or solid nutrient material containing the required nutrients for cultivation of microorganisms. Uses: Isolation& identification of microorganisms Transportation of sample Performing anti-microbial sensitivity tests 146 Amsalu M 02/23/2022
Types of culture media Basic /Simple / All purpose media • It is a media that supports the growth of micro- organisms that do not require special nutrients. • Eg. Nutrient Broth, Nutrient Agar Enriched media • Media that are enriched with whole blood, lyzed blood, serum, special extracts or vitamins to support the growth of fastidious bacteria. • Eg. Blood Agar, Chocolate Agar 147 Amsalu M 02/23/2022
Selective media • Media which contain substances ( Eg. Antibiotics) that prevent or slow down the growth of bacteria other than pathogens for which the media are intended. • Eg. Modified Thayer –Martin Agar • Salmonella-Shigella( SS) agar Differential media • Media to which indicator substances are added to differentiate bacteria. • Eg. TCBS Agar differentiates sucrose fermenting yellow colonies of Vibrio cholerae from non-sucrose fermenting blue colonies of other Vibrio species. 148 Amsalu M 02/23/2022
 Forms of culture media 1. Solid culture media 2. Semisolid culture media 3. Fluid culture media 149 Amsalu M 02/23/2022
Culture media 150 Amsalu M 02/23/2022
Cont… 151 Amsalu M 02/23/2022
BACTERIAL GENETICS 152  Most of the genetic information in a bacterial cell is contained with in chromosome . A single molecule of DNA arranged as a double in closed loop.  Bacterial inherited characteristics are encoded in DNA. These are:  Chromosome  Extra chromosome (Plasmid) Amsalu M 02/23/2022
153  Chromosome- Bacterial chromosome is circular double stranded DNA attached to bacterial cell membrane  Plasmids: are self replication extra chromosomal DNA molecules.  Plasmids are not essential to the life of the cell but they may have selective advantage for these organism  R-factor (Resistance)  Extra cellular toxin Amsalu M 02/23/2022
Genetic variation in Bacteria 154 GeneticVariation in Bacteria can occur by Mutation Gene-Transfer Amsalu M 02/23/2022
Mutation 155 Bacterial mutations occur when the information in a bacterial chromosome may alter by different means There are two types of mutation: Spontaneous mutation Induced mutation Amsalu M 02/23/2022
Gene-Transfer 156 The transfer of genetic information can occur by either of the three methods: Conjugation Transudation Transformation Amsalu M 02/23/2022
Conjugation 157 A process where by DNA is transferred from one bacterium to another by cell to cell physical contact (through sex pilli) Plasmids are the genetic elements most frequently transferred by conjugation Amsalu M 02/23/2022
158 Fig. Transfer/replication process of a conjugative plasmid Amsalu M 02/23/2022
Transduction 159  Is a method of gene transfer in which a virus (phage) acts as a vehicle for carrying DNA from a donor bacterium to recipient bacterium. Amsalu M 02/23/2022
Transformation 160  A process by which a bacterium acquire DNA fragments or genes from surroundings.  Usually this occurs in microbial culture. Amsalu M 02/23/2022
161 Fig. Transformation process in bacteria Amsalu M 02/23/2022
QUESTIONS ?????????
GRAM NEGATIVE RODS Enterobacteriaceae: A. Lactose-fermenters  Escherichia spp, Klebsiella spp.  Enterobacter spp & Citrobacter spp. B. Non-lactose fermenters- Salmonella spp, Shigella spp and Proteus spp Non- Enterobacteriaceae: Pseudomonas, Vibrio, Helicobacter & Campylobacter 163 02/23/2022 Amsalu M.
Family enterobacteriaceae Characteristics • Named as coli forms or enterobacilli. • Most of them found as normal flora in intestinal tract of humans and animals. • Are Gram-negative, non-spore forming, aerobic and facultative-anaerobic bacteria. • Most are motile, grow over a wide range of temperature in ordinary media. • All ferment glucose, reduce nitrate to nitrite and are oxidase negative • Release endotoxin from their cell wall and some release exotoxin. • Most of them have possessed three types of antigens: H antigen, K-antigin and O(somatic) antigen. 164 02/23/2022 Amsalu M.
Antigenic Structures and Virulence Factors Complex surface antigens contribute to pathogenicity and trigger immune response: • H – flagellar Ag • K – capsule and/or fimbrial Ag • O – somatic or cell wall Ag – all have 165 02/23/2022 Amsalu M.
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Enterobacteriaceae: Types of Disease • Intestinal (diarrheal) infection • Extra-intestinal infection • Urinary tract (primarily cystitis) • Respiratory (nosocomial pneumonia) • Wound (surgical wound infection) • Bloodstream (gram-negative bacteremia) • Central nervous system (neonatal meningitis) 167 02/23/2022 Amsalu M.
GENUS: ESCHERICHIA Main species of medical importance is Escherichia coli. Escherichia coli (E.coli) Characteristics of • Normal flora in human and animal gastrointestinal tract. • Found in soil, water & vegetation. • Most are motile; some are capsulated. Clinical features • Urinary tract infection- cystitis, pyelonephritis • Wound infection- appendicitis, peritonitis. • Neonatal septicemia & meningitis • Escherichia coli-associated diarrheal diseases • The strains of E. coli that cause gastroenteritis are subdivided into five major groups: 168 02/23/2022 Amsalu M.
1. Enteropathogenic E.coli (EPEC)  causes outbreaks of self-limiting infantile diarrhea  they also cause severe diarrhea in adults antibiotic treatment shorten the duration of illness and cure diarrhea. 2. Enteroinvasive E.coli (EIEC) Non-motile, non-lactose fermenting E. coli invade the mucosa of the ileum and colon, and causes shigellosis-like dysentery in children in developing countries and travelers to these countries. 169 02/23/2022 Amsalu M.
3. Enterotoxigenic E.coli (ETEC)  Colonization factor of the organism promote adherence to epithelial cells of small intestine followed by release of enterotoxin which causes toxin-mediated watery diarrhea in infants & young adults. It is also an important cause of traveler‘s diarrhea 4. Entero haemorrhagic E.coli ( EHEC) causes hemorrhagic colitis (severe form of diarrhea), and hemolytic uremic syndrome characterized by acute renal failure, hemolytic anemia and low platelet count. 5. Enteroaggressive E.coli (EAEC) Adhere to human intestinal mucosal cells and produce shiga-like toxin and hemolysin, and causes acute and chronic diarrhea in persons in developing countries Produce food-borne illness in developed countries 170 02/23/2022 Amsalu M.
PREVENTION AND CONTROL Restricting use of antibiotics and avoiding unnecessary use of urinary catheters. Maintenance of high hygienic standards to reduce risk of gastroenteritis. 171 02/23/2022 Amsalu M.
 GENUS: SALMONELLA • Salmonella can colonize virtually all animals, including poultry, reptiles, livestock, rodents, domestic animals, birds and humans. • Species of medical importance are: S. typhi, S. paratyphi & S. enteritidis. 172 02/23/2022 Amsalu M.
Clinical features 1. Typhoid fever and paratyphoid fever: -Both are caused by S. Typhi and S. paratyphi respectively, and transmitted by fecal-oral route via contaminated food and drinks. - Incubation period: 10-14 days, but depend on bacterial load and immune status of hosts. Predisposing factors: - Reduced gastric acidity - Disrupted intestinal microbial flora - Compromised local intestinal immunity • Both manifest with persistent fever, headache, malaise, chills, enlargement of liver and spleen, and skin rashes. • Paratyphoid fever is milder than typhoid fever 173 02/23/2022 Amsalu M.
Complications: Intestinal perforation, Lower gastrointestinal bleeding, Dissemination to different body organs including meninges and brain. 2. Bacteremia with focal lesions • Causative agent: S. typhimurium • Manifests with blood stream invasion with focal lesions in lungs, bones and meninges. • Intestinal manifestation is often absent. 3. Gastroenteritis - It is caused by S. enteritidis & S. typhimurium - It manifests with initial watery diarrhea, and later bloody mucoid diarrhea associated with crampy abdominal pain and tenesmus. 174 02/23/2022 Amsalu M.
LABORATORY DIAGNOSIS Specimen: • Blood, stool, urine and serum for typhoid fever. • Stool for gastroenteritis. Gram reaction: Gram-negative rods Culture: • Selective medium-favour growth of Salmonella and Shigella over other enterobacteriaceae. • Example: Salmonella-Shigella (SS) agar, XLD agar, Deoxycholate-Citrate agar. • Serology • Widal Test 175 02/23/2022 Amsalu M.
TREATMENT Ciprofloxacin, chloramphenicol, trimethoprim, SXT or a broad spectrum cephalosporin. PREVENTION AND CONTROL Proper sewage treatment Chlorination of water supply, Pasteurization of milk. hand washing prior to food handling Cultures of stool samples from food handlers to detect and treat carriers.  Proper cooking of poultry, eggs and meat are important preventive measures. 176 02/23/2022 Amsalu M.
GENUS: SHIGELLA Characteristics: • They are Non-lactose fermenting gram-negative rods. • They are non-motile. • All Shigella have O antigens (polysaccharides) in their cell walls. - These antigens are used to divide in to four groups. 1. Shigella dysenteriae 2. Shigella flexneri 3. Shigella sonnei 4. Shigella boydii 177 02/23/2022 Amsalu M.
Epidemiology Shigellosis is only a human disease (except primates like monkeys) that can be transmitted from person to person. The five F‘s (fingers, flies, food, fomites, and faeces) are the principal factors in transmission. =Route of infection is fecal-oral route Epidemic out breaks of the disease occurs in:- Day care centers - nurseries custodial institutions. 178 02/23/2022 Amsalu M.
Shigellosis • It is characterized by abdominal cramp, sudden onset of bloody mucoid diarrheas, fever, generalized muscle ache and weakness. • Dehydration & electrolyte and acid-base disturbance. • High prevalence in: poor sanitation, poor personal hygiene & polluted water supply. • Young children are frequently affected. Treatment – Ciprofloxacin, ampicillin, tetracycline, trimethoprim - sulfamethoxazole and chloramphenicol Prevention and Control Proper sewage disposal chlorination of water, and  personal hygiene. 179 02/23/2022 Amsalu M.
Genus Klebsiella 180 Characteristics • Non-motile, lactose-fermenting, capsulated, gram- negative rods. • Main species of medical importance: K. pneumoniae, K. rhinoscleromatis & K. ozenae Klebsiella pneumoniae • It is found as a commensal in the intestinal tract, and also found in moist environment in hospitals. • It is an important Nosocomial (hospital acquired) pathogen. • It causes: Pneumonia, Urinary tract infection, Septicemia and meningitis (especially in neonates), Wound infection and peritonitis 02/23/2022 Amsalu M.
181 Klebsiella rhinoscleromatis  It causes rhinoscleroma of nose and pharynx to extensive destruction of nasopharynx. Klebsiella ozenae  It causes ozaena manifesting with foul smelling nasal discharge leading to chronic atrophic rhinitis. Laboratory diagnosis of klebsiella species  Specimen: Sputum, urine, pus, CSF, body fluid  Smear: Gram-negative rods  Culture: Large, mucoid, lactose-fermenting colonies on MacConkey agar 02/23/2022 Amsalu M.
Genus: Proteus 182 General characteristics • are found in the intestinal tract of humans and animals, soil, sewage and water. • They are gram-negative, motile, non-capsulated, pleomorphic rods. • produce the enzyme urease and phenylalanine diaminase. • Certain species are very motile and produce a striking swarming colonies on blood agar plate • Cell wall O antigens of certain strains of Proteus (such as OX -2, OX-19 and OX-K) cross react with antigens of several species of rickettsia  Species of medical importance are: » P. mirabilis » P. vulgaris 02/23/2022 Amsalu M.
Pathogenesis and clinical manifestations 183 P. mirabilis causes:  Urinary tract infection, Septicemia, Abdominal and wound infection P. vulgaris  Isolated in wound infection and urinary tract infection.  Important Nosocomial pathogen. 02/23/2022 Amsalu M.
184 Laboratory diagnosis  Specimen: Urine, pus, blood, ear discharge  Smear: Gram-negative rods  Culture: Produce characteristic swarming colonies over the surface of blood agar.  Are non-lactose fermenting colonies on MacConkey agar. 02/23/2022 Amsalu M.
GRAM NEGATIVE SHORT RODS: COCCOBACILLI GENUS: HAEMOPHILUS • Characteristics: • This is a group of small gram-negative, non-spore forming, non-motile, pleomorphic bacteria that require enriched media for growth. • Present in upper respiratory tract as a normal microbial flora in healthy people. The main species of medical importance are: Haemophilus influenzae, H. ducreyii & H. aegypticus. 185 02/23/2022 Amsalu M.
A. Haemophilus influenzae Characteristics: - Gram-negative Coccobacilli. - may produce a capsule (six capsular types have been distinguished) or may be unencapsulated - is a normal flora of the URT in healthy humans, and may also colonize the conjunctiva and genital tract. - Humans are the only natural hosts, and colonization begins shortly after birth. - transmitted by respiratory droplets 186 02/23/2022 Amsalu M.
Diseases caused by H. influenzae, therefore, fall into two categories: First, disorders such as otitis media, sinusitis, epiglotitis, and bronchopneumonia result from contiguous spread of the organism from its site of colonization in the respiratory tract. Second, disorders such as meningitis, septic arthritis, and cellulitis result from invasion of the bloodstream, followed by localization of H. influenzae in these and other areas of the body. 187 02/23/2022 Amsalu M.
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Prevention and control Active immunization with conjugated PRP (polyrabitol phosphate) vaccines prevents most H.influenzae type b infections. Generally given to children younger than two and is also effective in preventing invasive disease, and reduces respiratory carriage of Hib. Rifampin can be used as prophylaxis. 189 02/23/2022 Amsalu M.
B. Heamophilus ducreyii – Slender, gram-negative, ovoid bacilli, slightly larger than H. influenzae. - It causes genital ulcer is known as chancroid (soft chancre), a sexually transmitted ulcerative disease. - In addition to the painful genital lesions, inguinal lymphadenopathy may occur. - If untreated, this progresses to formation of a bubo (a swollen lymph node), which then suppurates (forms pus). 190 02/23/2022 Amsalu M.
Note: Open genital sores facilitate the transmission of HIV, and H. ducreyi infection is frequently associated with AIDS. Treatment  It is treated by erythromycin, cotrimoxazole and third generation cephalosporins if they are sensitive  Azithromycin is also effective against H. ducreyi infections C. Heamophilus aegypticus – It causes contagious purulent conjunctivitis 191 02/23/2022 Amsalu M.
GENUS: BORDETELLA Characteristics: • They are small, encapsulated, aerobic, non-motile, gram- negative coccobacilli that grow singly or in pairs • Bordetella species of medical importance: B. pertussis and B. parapertussis • The former causes the disease pertussis (also known as whooping cough), and the latter causes a mild pertussis- like illness. • Whooping cough is a highly contagious disease and a significant cause of morbidity and mortality worldwide . 192 02/23/2022 Amsalu M.
• The major mode of transmission of Bordetella is via droplets spread by coughing from early cases and possibly carriers. • But the organism survives only briefly outside the human respiratory tract. • The incubation period for pertussis generally ranges from one to three weeks. Clinical features: • The disease can be divided into three phases: Catarrhal stage, Paroxysmal stage & Convalescence stage 193 02/23/2022 Amsalu M.
A. During catarrhal stage, the patient is highly infectious but not very ill manifesting with mild coughing and sneezing (nonspecific symptoms, and then progresses to include a dry, nonproductive cough). B. During paroxysmal stage, the patient presents with explosive repetitive cough with characteristic ‘whoop’ upon inhalation leading to exhaustion, vomiting, cyanosis and convulsion.  Large amounts of mucus are typically produced. C. During convalescence stage, the patient presents with prolonged cough – During this period, secondary complications such as infections (for example, otitis media and pneumonia) and CNS dysfunction (for example, encephalopathy or seizures) may occur. 194 02/23/2022 Amsalu M.
• Treatment – Erythromycin is the drug of choice for infections with B. pertussis, both as chemotherapy (where it reduces both the duration and severity of disease), and as chemoprophylaxis for household contacts. – For erythromycin treatment failures, trimethoprim- sulfamethoxazole is an alternative choice. Prevention: – Two forms of vaccine are currently available (one of killed whole cells, and one that is acelullar, containing purified B. pertussis proteins). – Both are formulated in combination with diphtheria and tetanus toxoids. • To protect infants who are at greatest risk, immunization is generally initiated when the infant is two months old. 195 02/23/2022 Amsalu M.
Gram negative rods not grouped under family Enterobacteriaceae • Genus Pseudomonas • Genus Vibiro • Genus Helicobacter • Genus Campylobacter 196 02/23/2022 Amsalu M.
Genus፡ Pseudomonas • Gram-negative motile aerobic rods having very simple growth requirement. • Can be found in water, soil, sewage, vegetation, human and animal intestine. • Species of medical importance: - P. aeruginosa - P. pseudomallei 197 02/23/2022 Amsalu M.
Pseudomonas aeruginosa • Can be found in the intestinal tract, water, soil and sewage and is frequently found in moist environments in hospitals, (sinks, cleaning buckets, drains, etc). • It is able to grow in some eye drops, saline and other aqueous solutions. • Many infections with P. aeruginosa are opportunistic hospital-acquired, affecting those already in poor health and immuno-suppressed. • Infections are often difficult to eradicate due to P. aeruginosa being resistant to many antimicrobials. 198 02/23/2022 Amsalu M.
• Infections caused by P. aeruginosa include: • Skin infections, especially burn sites, wounds, pressure sores, and ulcers (often as secondary invader). Septicaemia may develop. • Urinary infections, usually following catheterization or associated with chronic urinary disease. • Respiratory infections especially in patients with cystic fibrosis or conditions that cause immuno-suppression. • External ear and eye infections often secondary to trauma or surgery. 199 02/23/2022 Amsalu M.
Treatment • P. aeruginosa is resistant to most of the commonly used antibiotics. • Antimicrobials that usually show activity against Pseudomonas include aminoglycosides, polymyxin, and some penicillins and cephalosporins. 200 02/23/2022 Amsalu M.
GENUS: VIBRIOS Important properties: • Curved ―Comma-shaped‖ actively motile gram- negative rods. • They are single or occasionally united in S shapes or spirals.. • Found in fresh water, shellfish and other sea food. • They are motile by a single polar flagellum. • Readily killed by heat and drying • Species of medical important include: – Vibrio cholerae and – Vibrio parahaemolyticus 201 02/23/2022 Amsalu M.
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Vibrio cholerae • V. cholerae is halotolerant and can grow in alkaline conditions Antigenic structure: • has a complex cell wall which includes lipopolysaccharides, the carbohydrate components of which are designated as O antigens that confer serologic specificity. • Many vibrios share a single heat-labile flagellar H antigen. • There are now 206 somatic antigens (O antigens) in the V. cholerae V. cholerae O1 and O139 – are responsible for causing Cholera which can occur in epidemic or worldwide pandemics. • occasionally, non-01/non-0139 V. cholerae causes cholera- like disease. 203 02/23/2022 Amsalu M.
• Virulence factors – Cholera toxin (CT) • CT consists of a number of subunits: one A or enzymatic subunit and five identical B or binding subunits. 204 02/23/2022 Amsalu M.
Pathogenesis & Pathology • V. cholerae is pathogenic only for humans. • A person with normal gastric acidity may have to ingest as many as 1010 or more V. cholerae to become infected when the vehicle is water, because the organisms are susceptible to acid. • When the vehicle is food, as few as 102-104 organisms are necessary because of the buffering capacity of food. • Any medication or condition that decreases stomach acidity makes a person more susceptible to infection with V. cholerae. 205 02/23/2022 Amsalu M.
• Virulent V. cholerae organisms attach to the microvilli of the brush border of epithelial cells. • There they multiply and liberate cholera toxin (CT) and perhaps mucinases and endotoxin. • CT disrupts ion fluxes without causing apparent damage to the mucosa. • CT decreases the net flow of sodium into the tissue. • This produces a net flow of chloride and water out of the tissue into the lumen, causing the massive diarrhea and electrolyte imbalance that is associated with this infection. 206 02/23/2022 Amsalu M.
Epidemiology Organisms are responsible for major pandemics with significant mortality in developing countries. The main animal reservoirs are marine shellfish The disease can spread by consumption of contaminated food and water. Direct person-to person spread is rare. 207 02/23/2022 Amsalu M.
Some infections are asymptomatic or cause mild diarrhea Incubation period is 1-4 days Can cause severe disease resulting in abrupt watery diarrhea (20-30L/day) and vomiting ―Rice-water stool‖ is characteristic There are no RBC or WBC in the stool. Results in severe fluid and electrolyte loss, dehydration, shock, acidosis Can progress to coma and death Mortality rate in untreated cases is 60% Patients with suspected cholera need to be treated prior to laboratory confirmation, because death by dehydration can occur within hours. Clinical Significance 208 02/23/2022 Amsalu M.
Treatment  Fluid and electrolyte replacements are crucial. Antibiotic therapy reduces the bacterial burden, exotoxin production, and duration of diarrhea. • Antibiotics (doxycycline is the drug of choice) can shorten the duration of diarrhea and excretion of the organism. Prevention and control  Ensuring clean water and food supply. The use of tetracycline for prevention is effective in close contacts but cannot prevent the spread of a major epidemic. 209 02/23/2022 Amsalu M.
210 Genus፡ Campylobacter General characteristics  Campylobacters are motile, curved, oxidase-positive, thermophillic, Gram- negative rods similar in morphology to vibrios.  Campylobacters cause both diarrhoeal and systemic diseases and are among the most widespread causes of infection in the world.  The cells have one polar flagella and attached at their ends giving pairs ―S‖ shapes or a ―seagull‖ appearance.  C. jejuni, and C. coli are by far the most common  Domestic animals such as dogs may also carry the organisms and probably play a significant role in transmission to humans
211  The most common source of human infection is undercooked poultry, but outbreaks have been caused by contaminated rural water supplies and unpasteurized milk Species of medical importance - Campylobacter jejuni - Campylobacter coli Virulence factors  Lipopolysaccharides with endotoxic activity.  Cytopathic extra cellular toxins.  Enterotoxins.
212 Pathogenesis and clinical manifestations  Campylobacter jejuni accounts for 90 to 95% of human campylobacter infections in most parts of the world.  Campylobacter jejuni and Campylobacter coli cause enteritis which may take the form of toxigenic watery diarrhoea or dysentery.  The organisms are able to produce enterotoxins and cytotoxins.  In developing countries, C. jejuni and C. coli cause disease mainly in children under 2 years.
213  The jejunum and ileum are the first sites to be come colonized followed by the colon and rectum.  In well developed infections, mesenteric lymph nodes are enlarged.  Diarrhoea is due to disruption of intestinal mucosa due to cell invasion by campylobacters and the production of toxins.  The toxin blocks the cell cycle of host cells but its precise role is not yet clear.
214 Laboratory diagnosis • Specimen: Fresh diarrhoeal or dysenteric specimens containing blood, pus and mucus • Microscopy: Typical ‗S‘ shaped gram-negative rods. • Typical darting motility (swift and sudden) of the bacteria under dark field microscopy or phase contrast microscopy
215 Culture  Campylobacter species are strictly microaerophilic, requiring incubation in an atmosphere of reduced oxygen (5–10%) with added carbon dioxide (about 10%).  C. jejuni and C. coli are thermophiles, i.e. they will grow at 42-43 ºC and 36–37 ºC but not at 25 ºC.  Incubation at 42-43 ºC helps to identify C. jejuni and C. coli. Blood agar: C. jejuni and C. coli produce non-haemolytic spreading, droplet-like colonies on blood agar.  Campylobacter species are oxidase and catalase positive.
216 Treatment Erythromycin or ciprofloxacin are drugs of choice for C. jejuni enterocolitis. Prevention and control 1.Proper cooking of foods. 2.Avoiding contact with infected human or animals and their excreta. 3.Pasteurizing of milk and milk products. 4.No vaccine available.
Genus: Helicobacter Characteristics; • spiral shaped gram- negative bacilli, with polar flagella Species of medical importance Helicobacter pylori • Helicobacters are urease positive. • Urease production is a consistent finding in Helicobacter species of humans that colonize the stomach (acidic environment). – This is uncommon in species found in the intestine. • H. pylori have now been associated with gastritis, peptic ulcers, gastric adenocarcinoma and gastric mucosa- associated lymphoid type B – cell lymphomas. 217 02/23/2022 Amsalu M.
Helicobacter pylori • The organism survives in the mucous layer that coats the epithelium, and causes chronic inflammation of the mucosa. • Transmission is by person to person contact, and probably also by contaminated water and food. Diseases H. pylori causes gastritis and peptic ulcers. Infection with H. pylori is a risk factor for gastric carcinoma. 218 02/23/2022 Amsalu M.
Virulence factors  Vaculating toxin (Vac A): has been associated with pore formation in host cell membranes, the loosening of the tight junctions between epithelial cells, thus affecting mucosal barrier permeability.  Cytotoxin A (Cag A) : gene is a marker of increased risk of both peptic ulceration and gastric malignancy. 219 02/23/2022 Amsalu M.
Pathogenesis and clinical manifestations • Multiple factors can contribute to the gastric inflammation, alteration of gastric acid production and others. • Initial colonization is facilitated by blockage of acid production by a bacterial acid inhibitory protein and neutralization of gastric acids by the ammonia produced by bacterial urease activity. – Urease : urea → CO₂ + NH₃ • The actively motile Helicobacter can then pass through gastric mucus and adhere to the epithelial cells. • Localized tissue damage is mediated by urease by products, mucinase, phospholipase and the activity of vaculating cytotoxin that induce epithelial cell damage. 220 02/23/2022 Amsalu M.
Treatment Combination of a proton pump inhibitor (e.g. omeprazole) and one or more antibiotics (e.g. tetracycline, clarithromycin, amoxicillin, and metronidazole). • usually selected from clarithromycin, metronidazole, amoxicillin and tetracycline), combined with acid suppressants and/or bismuth compounds. Prevention and control • Improving sanitary hygiene 221 02/23/2022 Amsalu M.
QUESTIONS ?????????
223 HYPERSENSITIVITY REACTIONS & AUTOIMMUNITY • An Immune response eliminates antigen without extensively damaging the host’s tissue. Under certain circumstances, however, this response can have deleterious effects, resulting in significant tissue damage or even death. This inappropriate/heightened immune response is termed hypersensitivity or allergy.
224 Hypersensitive Reactions cont… Although the word hypersensitivity implies an increased response, the response is not always heightened but may, instead, be an inappropriate immune response to an antigen. Hypersensitive reactions may develop in the course of either humoral or cell-mediated responses. Hypersensitivity reactions require pre-sensitized (immune) state of the host .
225 Classification of Hypersensitivity Based on mechanics involved and time taken for the reactions hypersensitivity reactions are four types : – Type I (Anaphylactic) Reactions – Type II (Cytotoxic) Reactions – Type III (Immune Complex) Reactions – Type IV (Cell-Mediated) Reactions • The first three occur within the humoral branch and are mediated by antibody or antigen-antibody complexes. • A fourth type depends on reactions within the T cell- mediated branch, and is termed delayed-type hypersensitivity, or DTH(type IV).
226 A. Type I (Anaphylactic) Reactions • It is also known as immediate hypersensitivity. • The reaction takes 15-30 minutes from the time of exposure to the antigen. • Type I hypersensitivity is mediated by IgE. • The primary cellular component in this hypersensitivity is mast cell or basophil. • The mechanism of reaction involves preferential production of IgE.
227 Type I reactions can be systemic or localized Systemic anaphylaxis • Is a shock-like and often fatal state whose onset occurs within minutes of a type I hypersensitive reaction. • A wide range of antigens have been shown to trigger this reaction in susceptible humans, including the venom from bee, ant stings, drugs such as penicillin, insulin, and antitoxins • If not treated quickly, these reactions can be fatal. • Epinephrine is the drug of choice for systemic anaphylactic reactions.
228 Localized anaphylaxis (atopy) In localized anaphylaxis, the reaction is  limited to a specific target tissue or organ,  Often involving epithelial surfaces at the site of allergen entry. The tendency to manifest localized anaphylactic reactions is inherited and is called atopy. Atopic allergies include:  Allergic rhinitis (hay fever)  Asthma  Atopic dermatitis (eczema)  Food allergies
229 Type II (Cytotoxic) Reactions • Mediated, primarily, by antibodies of IgM or IgG class and complement • The reaction time is minutes to hours. • Affect a variety of organs and tissues. • The antigens are normally endogenous, although exogenous chemicals (haptens) that can attach to cell membranes can also lead to type II hypersensitivity.
230 • It includes: – blood transfusion reactions – drug-induced hemolytic anemia – Hemolytic Disease of the Newborn • Hemolytic disease of the newborn develops when maternal IgG antibodies specific for fetal blood-group antigens cross the placenta and destroy fetal red blood cells. • Caused by Rh incompatibility
231 Type III (Immune Complex) Reactions  When large amounts of antigen bind to antibody, immune complexes can form.  Mediated by neutrophils  If antigen is in excess, large complexes form; because these are not easily cleared by the phagocytic cells, they can cause tissue-damaging  The antibodies here are mostly of IgG class, although IgM may also be involved.  The antigen may be exogenous (chronic bacterial, viral or parasitic infections), or endogenous  e.g., systemic lupus erythematous-SLE).
232 Type IV (T Cell-Mediated) Reactions  Delayed type hypersensitivity (DTH) response does cause extensive tissue damage mediated by T cells  In many cases tissue damage is limited  The response plays an important role in defense against intracellular pathogens and contact antigens.  The hallmarks of a type IV reaction are the delay in time required for the reaction to develop and the recruitment of macrophages  DTH response is important in host defense against parasites and bacteria that live within cells, where circulating antibodies cannot reach them.
233 • Generally, the pathogen is cleared rapidly with little tissue damage. • However, in some cases, especially if the antigen is not easily cleared, a prolonged DTH response can itself become destructive to the host as the intense inflammatory response develops into a visible granulomatous reaction. • Macrophages present intracellular antigens to T cells and T-helper 1 cells (Cytotoxic T lymphocytes) release toxins that harm tissues • Example: contact dermatitis to nickels, gold jewelries etc.
Introduction to Vaccines 234 • Vaccines: biological substances that stimulate the person’s immune system to produce an immune response identical to that produced by the natural infection. • Vaccines can – prevent the debilitating and, in some cases, fatal infectious diseases. – help to eliminate the illness and disability of polio, measles, and rubella
235 • Vaccines protect the – vaccinated individual, – protect society. • A community with many vaccinated people – protects the few who cannot be vaccinated—such as young children. – indirectly protects unvaccinated from exposure to disease = HERD IMMUNITY • Aim of an ideal vaccine: – To produce the same immune protection with the natural infection but without causing disease – To generate long-lasting immunity – To interrupt spread of infection
Criteria for Effective Vaccines 236 • The World Health Organization (WHO) has stated that the ideal vaccine would have the following properties: – Affordable worldwide – Heat stable – Effective after a single dose – Applicable to a number of diseases – Administered by a mucosal route – Suitable for administration early in life
Active and Passive Immunization 237 • Active immunity- immunity produced in response to natural infection • Many diseases stimulate an immune response in host, those who survive the disease are protected from second infection – naturally acquired active immunity • Artificially acquired active immunity - is stimulated by initial exposure to specific foreign macromolecules through the use of vaccines, to artificially establish state of immunity. • Passive immunity- immunotherapy (administration of immunity components from external source)
238 • Artificially acquired passive immunity- The individual receives protective molecules (anti bodies) or cell (lymphocytes) produced in another individual. • Naturally acquired passive immunity- refers to antibodies transferred from mother to fetus across the placenta and to the newborn in colostrum and breast milk during the first few months of life. • Passive immunization does not activate the immune system, it generates no memory response and the protection provided is transient/temporary
3/15/2015 239
Types of vaccines 240 • Many common vaccines use – Inactivated (killed) organisms, but still antigenic – Attenuated-live/altered/weakened microorganisms. Lost their pathogenicity due to cultured in abnormal conditions – Specific substances (e.g., protein, polysaccharide) from pathogen, capable of producing an immune response
1. Whole-Organism Vaccines 241 • Attenuated organisms: they lost ability to cause significant disease (pathogenicity) but to attenuate, grow a pathogenic bacterium or virus for prolonged periods under abnormal culture conditions – retains capacity for short term growth within inoculated host. – capacity for transient growth, permits prolonged immune-system exposure to attenuated epitopes, increased immunogenicity and production of memory cells. • As a consequence, these vaccines often require only a single immunization. • A major disadvantage is the possibility that they will revert to a virulent form. • Whole killed organisms- they are killed but still immunogenic
2. Purified Macromolecules 242 • Derived from pathogens. • Are specific, purified macromolecules . • Avoid some risks associated with attenuated or killed whole organism vaccines. • Three general forms of such vaccines are in current use: – inactivated exotoxins, – capsular polysaccharides, and – recombinant microbial antigens/DNA molecules
243 • Isolate gene encoding immunogenic protein, clone it, and express/insert in bacterial, yeast, or mammalian cells using recombinant DNA technology. • Example for human use is hepatitis B vaccine developed by cloning the gene for surface antigen of hepatitis B virus (HBsAg) and expressing it in yeast cells. • The recombinant yeast cells are grown in large fermenters, and HBsAg accumulates in the cells. • The yeast cells is disrupted, releases the recombinant HBsAg, which is purified by biochemical techniques.
QUESTIONS ?????????
GRAM POSITIVE COCCI • There are two most important genera of gram-positive cocci. –Genus Staphylococci –Genus Streptococci 245 02/23/2022 Amsalu M.
GENUS: STAPHYLOCOCCI Characteristics: Gram positive non spore-forming, spherical cells, usually arranged in grape-like clusters (in group) Can readily grow in ordinary media under aerobic and micro- aerophilic conditions , grow most rapidly at 370c relatively resistant to drying , heat, and 9% Nacl, thus can persist for long periods on fomites Produce catalase, which differentiate them from the streptococci. Some of them are normal flora of the skin and mucus membrane of human 246 02/23/2022 Amsalu M.
Cont… The pathogenic staphylococci often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins The genus Staphylococcus has more than 35 species. The three main species of clinical importance are: Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. Less common staphylococcal species: Staphylococcus lugdenensis, Staphylococcus hominis & Staphylococcus warneri 247 02/23/2022 Amsalu M.
Staphylococcus aureus Epidemiology: • Produce coagulase, which differentiate them from the the other staphylococci species. • Present in most environments frequented by humans • Carriage rate for healthy adults is 20-60%. • Carriage is mostly in anterior nares, skin, nasopharynx, • Predisposition to infection include: – poor hygiene and nutrition, tissue injury, preexisting primary infection, diabetes, immunodeficiency. – Increase in community acquired methicillin resistance - MRSA 248 02/23/2022 Amsalu M.
249 Virulence factors of S. aureus Enzymes: • Coagulase – coagulates plasma and blood; produced by 97% of human isolates; diagnostic • Hyaluronidase – digests hyaluronic acid of connective tissue • Staphylokinase – digests blood clots • DNase – digests DNA • Lipases – digest oils; enhances colonization on skin • Penicillinase – inactivates penicillin 02/23/2022 Amsalu M.
250 Virulence factors of S. aureus Toxins: • Hemolysins (α, β, γ, δ): 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-1 (TSST-1) - induces fever, vomiting, shock, systemic organ damage 02/23/2022 Amsalu M.
S. aureus disease Range from localized to systemic Localized cutaneous infections – invade skin through wounds, follicles, or glands – folliculitis – superficial inflammation of hair follicle; usually resolved with no complications but can progress – furuncle –inflammation of hair follicle or sebaceous gland progresses into abscess or 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 251 02/23/2022 Amsalu M.
252 (a) Superficial folliculitis in which raised, domed pustules form around hair follicles. (b) In deep folliculitis the microorganism invades the deep portion of the follicle and dermis. (c) A furuncle arises when a large abscess forms around a hair follicle. Folliculitis: Infection of hair follicle. 02/23/2022 Amsalu M.
253 (d) A carbuncle consists of a multiocular abscess around several hair follicles. 02/23/2022 Amsalu M.
Impetigo: Crusted superficial skin lesion 254 lesions of impetigo. This disease is characterized by isolated pustules that become crusted. (e) Impetigo on the neck of 2-year-old male. 02/23/2022 Amsalu M.
255 Toxigenic disease – food intoxication – ingestion of heat stable enterotoxins; results in gastrointestinal distress – staphylococcal scalded skin syndrome – toxin induces bright red blisters, then desquamation of the epidermis – toxic shock syndrome – toxemia leading to shock and organ failure Systemic infections – Osteomyelitis – infection of bone – Endocarditis : Infection of heart tissue – Pneumonia: Infection of lung parenchyma. 02/23/2022 Amsalu M.
S. epidermidis and S .saprophyticus  They are opportunistic pathogens.  They are coagulase negative  S. epidermidis is frequently associated with endocarditis, bacteremia, wound infection and urinary tract infection (UTI).  S. saprophyticus is now recognized as an important cause of UTI. staphylococcal species are important agents of hospital- acquired infections associated with the use of implanted prosthetic devices and catheters. 256 02/23/2022 Amsalu M.
Treatment and prevention Penicillin sensitive staphylococci: penicillin, ampicillin Penicillin resistant staphylococci: cloxacillin, oxacillin, floxacillin Methicillin resistant staphylococci: Vancomycin  Prevention and control Source of infection is shedding human lesions, the human respiratory tract and skin contact spread of infection occur in hospitals. Treatment of nasal carriers with topical antiseptics or Rifampicin and anti-staphylococcal drug. There is no effective vaccine against S. aureus. Infection control procedures, such as barrier precautions and disinfection of hands and fomites, are important in the control of nosocomial S. aureus epidemics. 257 02/23/2022 Amsalu M.
GENUS: STREPTOCOCCI Characteristics: They are non-motile, non-sporeformer, gram-positive facultative anaerobes.  Spherical or oval cells characteristically forming chains or pairs during growth Grow well on media enriched with blood, serum or glucose. Some species and strains are capsulated. Sensitive to drying, heat and disinfectants They are widely distributed in nature and are found in upper respiratory tract, gastrointestinal tract and genitourinary tract as normal microbial flora.
Cont… Classification of streptococci • Can be classified based on: 1. Hemolytic pattern as beta, alpha, and gamma hemolytic on blood agar. 2. Serological property (antigenic composition of group specific cell wall substance): Lancefield grouping - A to H and K to V 3. Biochemical (physiological) property. 259 02/23/2022 Amsalu M.
Species Lance field group Hemolysis Biochemical property S. pyogenes A beta Bacitracin sensitive S. agalactiae B beta Bacitracin resistant E. faecalis D Alpha or beta, Growth in 6.5%NaCl or gamma S. bovis D Alpha or beta No growth in 6.5%NaCl S. pneumoniae no alpha Bile soluble, sensitive to optochin Viridian group no alpha Not bile soluble, resistant to optochin 260 02/23/2022 Amsalu M.
Lancefield grouping of streptococci: Streptococci produce group specific carbohydrates identified using group specific antiserum. It is designated A-H and K-V. The clinically important streptococci are grouped under A, B, C, D, F and G. NB: Viridans streptococci and Streptococci pneumoniae are not grouped under Lancefield Classification. 261 02/23/2022 Amsalu M.
Human Streptococcal Pathogens • S. pyogenes • S. agalactiae • Viridans streptococci • S. pneumoniae • Enterococcus faecalis 262 02/23/2022 Amsalu M.
Group A streptococci (S. pyogenes) The most pathogenic member of the genus. It is present as a commensal in the upper respiratory tract (nasopharynx) in a variable proportion of healthy individuals and on the skin. The pathogen is spread from person to person through respiratory droplets. It produces different types of enzymes and exotoxins. 263 02/23/2022 Amsalu M.
264 • Produces surface antigens: – C-carbohydrates – protect against lysozyme – Fimbriae - adherence – M-protein – contributes to resistance to phagocytosis • Extracellular enzymes streptokinase – digests fibrin clots hyaluronidase – breaks down connective tissue DNase – hydrolyzes DNA Virulence Factors 02/23/2022 Amsalu M.
265 Virulence Factors • Extracellular toxins: streptolysins – hemolysins streptolysin O (SLO) and streptolysin S (SLS) – both cause cell and tissue injury pyrogenic toxin (erythrogenic) – induces fever and typical red rash superantigens – strong monocyte and lymphocyte stimulants; cause the release of tissue necrotic factor 02/23/2022 Amsalu M.
Diseases cause S. pyogenes Skin infections Impetigo (pyoderma) – superficial lesions • is a small vesicle surrounded by an area of erythema. • The vesicle enlarges over a period of days, becomes pustular, and eventually breaks to form a yellow crust. • is associated with insect bites, poor hygiene, and crowded living conditions 266 02/23/2022 Amsalu M.
Streptococcal pharyngitis – strep throat • is most frequent between the ages of 5 and 15 years • is characterized by acute sore throat, malaise, fever, and headache. • Infection typically involves the tonsillar pillars, and soft palate, which become red, swollen, and covered with a yellow-white exudate. 267 02/23/2022 Amsalu M.
268 02/23/2022 Amsalu M.
269 Mbio2053 02/23/2022 Amsalu M.
270  Necrotizing fasciitis (Streptococcal gangrene): Extensive and rapidly spreading necrosis of skin and subcutaneous tissue. Necrotizing fasciitis The disease,as shown here on a thigh, is commonly produced by so-called flesh-eating Streptococcus pyogenes. 02/23/2022 Amsalu M.
Systemic infections • Scarlet fever • Septicemia • Pneumonia • Streptococcal toxic shock syndrome 271 02/23/2022 Amsalu M.
Long-Term Complications of Group A Infections Acute glomerulonephritis: occurs usually following a streptococcal infection of the skin. There is inflammation of the kidney due to deposition of immune complex in the glomeruli. It clinically manifests with generalized body edema, elevated blood pressure, protein and blood in the urine  Acute rheumatic fever – Due to immunological damage to the heart valves and muscle following Streptococcal upper respiratory tract infection. – It clinically presents with fever, malaise, migratory non-suppurative polyarthritis, carditis 272 02/23/2022 Amsalu M.
Group B: Streptococcus agalactiae • Regularly resides in human vagina, pharynx and large intestine • Can be transferred to infant during delivery and cause severe infection – most prevalent cause of neonatal pneumonia, sepsis, and meningitis – Pregnant women should be screened and treated. • Wound and skin infections and endocarditis in debilitated people 273 02/23/2022 Amsalu M.
Group D Enterococci • Group D: – they are commensals of the GIT – they can be enterococci (E. faecalis) or non-enterococci (S. bovis) – cause opportunistic urinary, wound, and skin infections, particularly in debilitated persons 274 02/23/2022 Amsalu M.
Viridans Streptococci • Large complex group – Streptococcus mutans, S. oralis, S. salivarus, S. sanguis, S. milleri, S. mitis • Most numerous and widespread residents of the gums and teeth, oral cavity and also found in nasopharynx, genital tract, skin • Not very invasive; but dental or surgical procedures facilitate their entrance 275 02/23/2022 Amsalu M.
Viridans Group • Bacteremia, meningitis, abdominal infection, tooth abscesses • Most serious infection – subacute endocarditis – blood- borne bacteria settle and grow on heart lining or valves • S. mutans produce slime layers that adhere to teeth, basis form plaque and cause dental caries. • Persons with preexisting heart conditions should receive prophylactic antibiotics before surgery or dental procedures. 276 02/23/2022 Amsalu M.
Pneumococci (S. pneumoniae) – Are fastidious lancet shaped gram positive cocci arranged in pairs (diplococci) – they are facultative anaerobes – Possess a capsule of polysaccharide that permits typing with specific antisera (more than 80 serotypes) Epidemiology – S. pneumoniae can be found as a commensal in the upper respiratory tract. – Most infections are caused by endogenous spread from the colonized nasopharynx. 277 02/23/2022 Amsalu M.
Virulence factors • Capsule interferes with phagocytosis • Pneumolysin disrupts cells and cilia • Other Virulence – Pneumococcal surface protein A (PspA: interferes with complement action 278 02/23/2022 Amsalu M.
Clinical syndrome • Pneumococci cause pneumonia, meningitis, endocarditis, Otitis media, bacteremia and infection of the upper respiratory tract. • The following factors lower host resistance and predispose to pneumococcal infection: Alcohol or drug intoxication or other cerebral impairment that can depress the cough reflex and aspiration or secretion. Abnormality of the respiratory tract Trauma to the head that causes leakage of spinal fluid through the nose predisposes to pneumococcal meningitis. 279 02/23/2022 Amsalu M.
Treatment, prevention and control – drugs of choice are penicillin, erythromycin and cotrimoxazole. – immunization with polyvalent polysaccharide vaccine provides long lasting immunity (at least 5 years). 280 02/23/2022 Amsalu M.
281 02/23/2022 Amsalu M.
Gram negative cocci Family Neisseriaceae
GRAM NEGATIVE COCCI GENUS: NEISSERIA • Characteristics: – They are non-motile, gram negative intracellular diplococci – rapidly killed by drying, sunlight, heat, and disinfectants. – Each cocci is kidney-shaped – grow best on complex media under aerobic conditions containing 5% CO2 – Oxidase positive. The main species of medical importance are: N. meningitidis N. gonorrhoeae. 283 02/23/2022 Amsalu M.
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micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
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micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
micro biology I (2022 UPDTD).pdf
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micro biology I (2022 UPDTD).pdf

  • 1. AMBO UNIVERSITY WOLISO CAMPUS DEPARTMENT OF NURSING MEDICAL MICROBIOLOGY FOR NURSING STUDENTS BY፡ AMSALU MEKONNEN (BSC, MSC, DPHM)
  • 2. CHAPTER ONE HISTORY AND SCOPE OF MICROBIOLOGY AND MEDICAL MICROBIOLOGY
  • 3. INTRODUCTION TO MICROBIOLOGY What is Microbiology? • Micro - too small to be seen with the naked eye • Bio - life • Logus-logy - study of o Microbiology: is a subject which deals with living organisms that are individually too small to be seen with the naked eye. Amsalu M 3 02/23/2022
  • 4. Microbiology • Is the science dealing with the study of very small or microscopic organisms and infectious agents which cannot be seen by the naked eye: –Include bacteria, protozoa, viruses, and fungi –Note :Viruses = are infectious agents‖ or ―infectious particles,‖ rather than microorganisms. 4 Amsalu M 02/23/2022
  • 5. Organisms included in the study of Microbiology 1. Bacteria Bacteriology 2. Protozoans Protozoology 3. Algae Phycology 4. Parasites Parasitology 5. Yeasts and Molds Mycology – Fungi 6. Viruses Virology Amsalu M 5 02/23/2022
  • 6. 5 Kingdoms of Living Organisms • 1. Animalia • 2. Plantae • 3. Fungi • 4. Protista • 5. Monera - Bacteria and Cyanobacteria • Eukaryotic vs. Prokaryotic Amsalu M 6 02/23/2022
  • 7. Classification System • 3 Domains 1978 Carl Woese – 1. Bacteria • Unicellular prokaryotes with cell wall containing peptidoglycan – 2. Archaea • Unicellular prokaryotes with no peptodoglycan in cell wall – 3. Eukarya • Protista • Fungi • Plantae • Animalia Amsalu M 7 02/23/2022
  • 8. Uses of Microbes Benefit Humans • 1.Bacteria are primary decomposers - recycle nutrients back into the environment (sewage treatment plants) • 2. Microbes produce various food products – cheese, pickles, sauerkraut, green olives – yogurt, soy sauce, vinegar, bread – Beer, Wine, Alcohol Amsalu M 8 02/23/2022
  • 9. 3. Microbes are used to produce Antibiotics • Mold – Penicillium notatum (Penicillin) • 1928 Alexander Fleming 4. Bacteria synthesize chemicals that our body needs, but cannot synthesize • Example: E. coli – B vitamins - for metabolism – Vitamin K - blood clotting • Escherichia coli – Dr. Escherich – Colon (intestine) Amsalu M 9 02/23/2022
  • 10. 5. Biochemistry and Metabolism • Very simple structure • rapid rate of reproduction • provides “instant” data 6. Microbial antagonism • Our normal microbial flora prevents potential pathogens from gaining access to our body Amsalu M 10 02/23/2022
  • 11. 7. Insect Pest Control • Using bacteria to control the growth of insects • Bacillus thuringiensis – caterpillars – bollworms – corn borers 8. Bioremediation • Using microbes to clean up pollutants and toxic wastes • Exxon Valdez - 1989 • 2 Genera – Pseudomonas sp. – Bacillus sp. Amsalu M 11 02/23/2022
  • 12. 9. Recombinant DNA Technology, Gene Therapy, Genetic Engineering • Bacteria can be manipulated to produce enzymes and proteins they normally would not produce – Insulin – Human Growth Hormone – Interferon 10. Food Chains • Marine and fresh water microorganisms Amsalu M 12 02/23/2022
  • 13.  Beneficial aspects: – Inhibit growth of pathogens by • occupying space, • Competing for food and • secreting waste products, toxins, antibiotics, etc. – Many microorganisms are involved in the decomposition of dead organisms and the waste products of living organisms. Beneficial and harmful aspects of microorganisms 13 Amsalu M 02/23/2022
  • 14. – Some microorganisms aid in the digestion of food and, in some cases, produce substances that are of value to the host (vitamins K and B1) – Many microorganisms are essential in various food and beverage industries – Certain bacteria and fungi produce antibiotics that are used to treat patients with infectious diseases. Beneficial and harmful aspects Cont… 14 Amsalu M 02/23/2022
  • 15. – Microbes are essential in the field of genetic engineering. – e. g. production of insulin, various types of growth hormone, interferons, and materials for use as vaccines.  Harmful aspects – Microorganisms cause : infectious diseases and food spoilage. Beneficial and harmful aspects Cont… 15 Amsalu M 02/23/2022
  • 16. Bacteria - what comes to mind? • Diseases • Infections • Epidemics • Food Spoilage • Only 1% of all known bacteria cause human diseases • About 4% of all known bacteria cause plant diseases • 95% of known bacteria are non-pathogens Amsalu M 16 02/23/2022
  • 17. Microbes do benefit us, but they are also capable of causing many diseases • Pneumonia Whooping Cough • Botulism Typhoid Fever Measles • Cholera Scarlet Fever Mumps • Syphilis Gonorrhea Herpes 1 • Chlamydia Tuberculosis Herpes 2 • Meningitis Tetanus RMSV • Strep Throat Lyme Disease AIDS • Black Plague Diarrhea Gangrene Amsalu M 17 02/23/2022
  • 18. History of the Study of Microorganisms • 1665 Robert Hooke – “little boxes” - “cells” – Cell Theory - all living things are made up of cells Amsalu M 18 02/23/2022
  • 19. Anton van Leeuwenhoek 1674 - 1st person to actually see living microorganisms “wee animalcules” Amsalu M 19 02/23/2022
  • 20. Spontaneous Generation • Theory that life just “spontaneously” developed from non-living matter • Example: – toads, snakes and mice - moist soil – flies and maggots - manure and decaying flesh Amsalu M 20 02/23/2022
  • 21. Experiments to disprove Spontaneous Generation • Francesco Redi 1668 • Rudolph Virchow 1858 – Theory of Biogenesis • Cells can only arise from preexisting cells • Louis Pasteur 1861 Amsalu M 21 02/23/2022
  • 22. History (cont.) • 1861 Pasteur – Proved Microorganisms are present in nonliving matter – Microbes can be destroyed by heat •Aseptic Technique • Fermentation mediated by yeast, not air – Pasteurization to prevent wine and beer spoilage (by bacteria) Amsalu M 22 02/23/2022
  • 23. FIELDS IN MICROBIOLOGY • Includes: – Agricultural Microbiology – Industrial Microbiology – Environmental Microbiology – Medical Microbiology – Microbial Genetics and Genetic Engineering 23 Amsalu M 02/23/2022
  • 24. Medical Microbiology – Involves the study of pathogens, the diseases they cause, and the body‘s defenses against disease. – Concerned with : • Epidemiology and transmission of pathogens • Disease prevention measures and treatment of infectious diseases • Aseptic techniques • Immunology and the production of vaccines to protect people and animals against infectious diseases. 24 Amsalu M 02/23/2022
  • 25. Subdivision of Medical Microbiology • Bacteriology: the study of structure, functions and activities of bacteria • Mycology: is the scientific study of fungi. • Virology: is the scientific study of viruses. • Immunology: is the scientific study of immunity.. • Parasitology: is study parasite & their interaction of the host. 25 Amsalu M 02/23/2022
  • 26. Brief History of Microbiology  The Microscope  The theory of abiogenesis and biogenesis  Germ Theory of disease 26 Amsalu M 02/23/2022
  • 27. The First Microscope 27 Microbes were first observed by Antonie van Leeuwenhoek using a simple microscope (1673) Reported his ―animalcules‖ to the Royal Society of London Amsalu M 02/23/2022
  • 28. Cont… • Antony Van Leeuwenhoek (1632-1723)- –father of Microbiology – He was the first to describe ―animalcules‖ (single celled organism) using simple microscope with one lens. – is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria. – He was the first who properly described the different shapes of bacteria. 28 Amsalu M 02/23/2022
  • 29. The theory of Abiogenesis vs. Biogenesis • Although Leeuwenhoek was not concerned about the origin of micro-organism; many other scientists were searching for the origin of microorganisms • Two major theories were formulated: 1. Theory of Abiogenesis 2. Theory of Biogenesis  Theory of Abiogenesis: deals with the theory of spontaneous generation; stating that living things originated from non-living things. • Example: maggots from decaying meat or mushrooms from rotting wood 29 Amsalu M 02/23/2022
  • 30. An experiment to disprove spontaneous generation • Francesco Redi (1626-1697) :  used two flasks and placed meat in each of the two flasks – One of the flasks left open – Another one tightly sealed • He observed that the flies laid eggs on the open flask and then the maggots developed. • Proved that no maggots appeared in meat when flies were prevented from laying eggs. 30 Amsalu M 02/23/2022
  • 31. Francesco Redi‘s experiments with meat 31 uncovered covered Maggots No maggots Disproved that maggots arise from decaying meat!! Amsalu M 02/23/2022
  • 32. Theory of Biogenesis  Theory of Biogenesis states that life comes from pre- existing life.  Louis Pasteur (1822-1895 GC)- was the scientist who disproved the theory of abiogenesis Experiment of Louis Pasteur: • He designed a large curved flask (Pasteur goose neck flask) and placed a sterile growth broth medium. • Air freely moved through the tube; but dust particles and microbes were trapped in the curved portion of flask. • Microbial growth in the broth was not seen. • Therefore he proved that micro-organisms entered to substrates through the air and micro-organisms did not evolve spontaneously. 32 Amsalu M 02/23/2022
  • 35. Major contribution of Louis Pasteur 1. Microbial theory of fermentation 2. Principles and practice of sterilization and pasteurization 3. Control of diseases of silk worms 4. Development of vaccines against anthrax and rabies. 5. Discovery of streptococci 35 Amsalu M 02/23/2022
  • 36. History (cont.) • 1857-Louis Pasteur saves France’s wine • 1861 Pasteur – Proved Microorganisms are present in nonliving matter – Microbes can be destroyed by heat technique • Fermentation mediated by yeast, not air – Pasteurization to prevent wine and beer spoilage (by bacteria) Amsalu M 36 02/23/2022
  • 37. 1) Good wine contained yeast 2) Sour wine contained bacteria 1) (Bacteria that use alcohol and produce acetic acid spoil wine by turning it to vinegar (acetic acid). 3) He reasoned that if wine is heated to destroy the harmful bacteria it wouldn’t spoil (process known as Pasteurization) Amsalu M 37 02/23/2022
  • 38. Ignaz Semmelweis (1818-1865) • Taught medicine in Vienna • No one connected germs with disease yet • Puerperal fever “childbirth fever” caused 25-30% mortality • Nearby obstetric hospital had only a 2% death rate Amsalu M 38 02/23/2022
  • 39. Ignaz Semmelweis (cont.) • He made some observations • Medical Students working on cadavers moved from the dissecting room to the maternity ward • Midwives –Stayed only in maternity ward Amsalu M 39 02/23/2022
  • 40. Ignaz Semmelweis (cont.) • Ordered students to wash hands and medical instruments in chlorinated lime • Mortality dropped to 1.3% • By 1848, 0% mortality Amsalu M 40 02/23/2022
  • 41. The Golden Age of Microbiology • 1857-1914 • Beginning with Pasteur‘s work, discoveries included • the relationship between microbes and disease, • immunity, and • antimicrobial drugs 41 Amsalu M 02/23/2022
  • 42. Golden Age of Microbiology 1857 - 1914 • Pasteur – Pasteurization – Fermentation • Joseph Lister – Phenol to treat surgical wounds – 1st attempt to control infections caused by microoganisms • Robert Koch – Koch’s Postulates • Edward Jenner – vaccination • Paul Erlich – 1st synthetic drug used to treat infections – Salvarsan - arsenic based chemical to treat Syphilis • “salvation” from Syphilis Amsalu M 42 02/23/2022
  • 43. Germ Theory of Disease Germ Theory of Disease: the theory stating microorganisms are the causative agent of disease. • Before the time of Pasteur, effective treatments for many diseases were discovered by trial and error, • but the causes of the diseases were unknown. • The discovery of yeast that play a crucial role in fermentation lead scientists to belief that microbes might be involved in causing disease. 43 Amsalu M 02/23/2022
  • 44. Germ Theory of Disease • Pasteur proposed that wine spoiling in an analogy for disease (bacterial growth made the wine “sick”) • germ theory – some diseases are caused by microorganisms Amsalu M 44 02/23/2022
  • 45. Germ Theory of Disease • Hard for people to believe that diseases were caused by tiny invisible “wee animalcules” • Diseases, they thought, were caused by: – demons – witchcraft – bad luck – the wrath of God – curses – evil spirits Amsalu M 45 02/23/2022
  • 46. Edward Jenner (country doctor) –Milkmaid didn’t get smallpox b/c they contracted the milder form of cowpox –Immune system cannot distinguish btw cowpox/smallpox –Scratched a farmboy w/ a needle bearing fluid from cowpox –Small pox Vaccine - Vacca-cow - Vaccination w/ cowpox provided immunity for smallpox Amsalu M 46 02/23/2022
  • 47. Robert Koch • -1st to prove that bacteria actually caused diseases • 1876 • Microbial Etiology of Infectious Disease – etiology - the cause of a disease • Established “scientific rules” to show a cause and effect relationship between a microbe and a disease – Koch’s Postulates Amsalu M 47 02/23/2022
  • 48. Pure Culture Key to Studying Microbes Definition: Pure culture is a population of organism, all of which are the progeny of a single organism -In nature, microbes almost never occur as pure cultures Amsalu M 48 02/23/2022
  • 49. Koch’s Postulates • 1. The same organisms must be found in all cases of a given disease. • 2. Organism can be isolated and grown in pure culture. • 3. The isolated organism must reproduce the same disease when inoculated into another animal • 4. The original organism must again be isolated from the experimentally infected animal. Amsalu M 49 02/23/2022
  • 50. Exceptions to Koch’s Postulates 1. Some organisms have never been grown in pure culture on artificial media Treponema pallidum - Syphilis Amsalu M 50 02/23/2022
  • 51. Mycobacterium leprae Leprosy Never been grown in pure culture on artificial media Abdominal cavity of the Seven Banded Armadillo Exceptions to Koch’s Postulates Amsalu M 51 02/23/2022
  • 52. Exceptions to Koch’s Postulates • In exclusively human diseases, it is not morally acceptable to inoculate a deadly pathogen into a “human guinea pig” • HIV Amsalu M 52 02/23/2022
  • 53. Koch established the Microbial Etiology of 3 important diseases of his day • 1. Cholera (fecal-oral disease) – Vibrio cholerae • 2. Tuberculosis (pulmonary infection) – Mycobacterium tuberculosis • 3. Anthrax (sheep and cattle) – Bacillus anthracis Amsalu M 53 02/23/2022
  • 54. • In general, germ theory is Proposed by Louis Pasteur in 1857. • But given experimental support and proofed by Robert Koch (1843-1910) – The first proof that bacteria actually cause disease came from Robert Koch in 1876. – Koch discovered rod -shaped bacteria now known as Bacillus anthracis in the blood of cattle that had died of anthrax. – Koch played a major role in establishing that microorganisms cause specific diseases. 54 Amsalu M 02/23/2022
  • 55. Germ Theory of Disease  1876 - Robert Koch provided proof that a bacterium causes anthrax using experimental steps now called the Koch’s Postulates  He was the first to use agar as solid culture medium in bacteriology. 55 Amsalu M 02/23/2022
  • 56. • Koch‘s Postulates – a method of determining the etiologic (causative) agent of infectious diseases 1. The suspected etiologic agent must be found in every case of the disease and be absent in healthy hosts 2. The suspected etiologic agent must be isolated in pure culture and identified 3. The suspected etiologic agent is inoculated into a healthy, susceptible host and that host must come down with the same disease 4. The same etiologic agent as in step 2 must be isolated and identified in the second diseased animal Koch’s Postulates 56 Amsalu M 02/23/2022
  • 57. Limitations to Koch’s postulate • Many healthy people carry pathogens but do not exhibit symptoms of the disease. • Some microbes are very difficult or impossible to grow in vitro (in the laboratory) in artificial media. E.g. Treponema pallidum, viruses, reckettsia and chylamidia • Certain diseases develop only when an opportunistic pathogen invades imuno-compromised host. 57 Amsalu M 02/23/2022
  • 58. Major achievements of Robert Koch 1. Discovery and use of solid medium in bacteriology 2. Discovery of causative agents of tuberculosis, anthrax and cholera. 3. Koch’s postulates 58 Amsalu M 02/23/2022
  • 59. Paul Ehrlich-hospital dermatologist • Chemotherapy- Treatment using chemical substances • 1910 Paul Ehrlich - ”Magic bullet” –Salvarsan (arsenic derivative) •Preparation 606 –Syphilis Amsalu M 59 02/23/2022
  • 60. • Alexander Fleming 1928 – Scottish researcher • Discovered Penicillin (fungus) by accident • Was convinced that nasal mucus had antibacterial effects • Left his Staphylococcus culture on an agar plate for 2 weeks-went on vacation-came back &found mold on his plate which prevented bacterial growth – (a Mycology lab underneath him had this rare spore drift) Amsalu M 60 02/23/2022
  • 63. CHAPTER TWO TAXONOMIC CLASSIFICATION, NOMENCLATURE AND IDENTIFICATION OF MICROORGANISMS
  • 64. – Taxonomy: The science of classification, especially the classification of living forms. – It helps : • To establish relationships between one group of organisms and another and to differentiate them. • Provides a common reference for identifying organisms already classified. • A basic and necessary tool for scientists. TAXONOMIC CLASSIFICATION, NOMENCLATURE AND IDENTIFICATION OF ORGANISMS 64 Amsalu M 02/23/2022
  • 65. • There are three interrelated areas in Taxonomy: 1) Nomenclature: is naming an organism by international rules according to its characteristics 2) Classification: the arrangement of organisms into taxonomic groups (taxa) on the basis of similarities or relationships. 3) Identification: refers to the practical use of a classification scheme. 65 Amsalu M 02/23/2022
  • 66. Phenotypic Characteristics to Classify Prokaryotes Includes size and shape, staining characteristics, and metabolic capabilities Genotypic Characteristics to Classify Prokaryotes  Based on the comparison of the nucleotide sequences of the rDNA.  DNA hybridization  Comparing the Sequences of 16S Ribosomal Nucleic Acid  DNA Base Ratio (G+C content) How organisms are classified? 66 Amsalu M 02/23/2022
  • 67. • Provides a crude comparison of genomes • Prokaryotic G+C content is the most variable – If two organisms differ in their G+C content by more than about 10%, their genomes have quite different base sequences. – The G+C content of strains within a particular species is constant. 67 Amsalu M 02/23/2022
  • 68. • Taxonomic classification categorizes organisms in a hierarchical order, with the species being the basic unit. • The species designation gives a formal taxonomic status to a group of related isolates or strains – permits their identification 68 Amsalu M 02/23/2022
  • 69. • Species—a group of related isolates or strains. – Note that members of a species are not all identical; individual strains may vary in minor properties. • Genus—a collection of related species. • Family—a collection of similar genera. – In prokaryotic nomenclature, the name of the family ends in the suffix -aceae. • Order—a collection of similar families. – In prokaryotic nomenclature, the name of the order ends in the suffix –ales • Class—a collection of similar orders. – In prokaryotic nomenclature, the name of the class ends in the suffix -ia. 69 Amsalu M 02/23/2022
  • 70. • Phylum or Division—a collection of similar classes. • Kingdom—a collection of similar phyla or divisions. • Domain—a collection of similar kingdoms. – The domain is a relatively new taxonomic category that reflects the characteristics of the cells that make up the organism. 70 Amsalu M 02/23/2022
  • 71. • The domain system contains three members 1) Bacteria (formerly eubacteria) 2) Archaea (archaebacteria)=share the prokaryotic type of cellular configuration. 3) Eukarya 71 Amsalu M 02/23/2022
  • 72. Nomenclature (binomial nomenclature) • Organisms are named using binomial nomenclature • Binomial nomenclature employs the names of the two level taxa, genus and species to name an organism. • According to Binomial nomenclature : a) Genus comes before species (e.g., Escherichia coli) b) The first letter of the genus name is always capitalized (e.g., Escherichia) c) Species name is never capitalized (e.g., coli) d) Both names are always either italicized or underlined e.g., Escherichia coli or Escherichia coli e) The genus name may be used alone, but not the species name (i.e., saying or writing ―Escherichia “ alone is legitimate while saying or writing ― coli” is not) 72 Amsalu M 02/23/2022
  • 73. EUKARYOTIC AND PROKARYOTIC CELL  EUKARYOTIC CELL • Eu- true • Karyote- nucleus • The eukaryotic cell has a true membrane bound nucleus, usually containing multiple chromosomes, a mitotic apparatus, a well defined endoplasmic reticulum and mitochondria Eukaryotes . Algae . Protozoa . Fungi 73 Amsalu M 02/23/2022
  • 74. Cont… PROKARYOTIC CELL • Pro- primitive • Karyote- nucleus The prokaryotic cell possesses naked DNA without associated basic proteins divides by binary fission Prokaryotes . Bacteria 74 Amsalu M 02/23/2022
  • 75. Cont… • The distinguishing features between Eukaryotic cell and Prokaryotic cell • Features Prokaryotic cell Eukaryotic cell • Size 1m 10m • Nuclear membrane absent Present • Chromosome Single Multiple • Nucleolus absent Present • Histones absent Present • Sexual reproduction absent Present • Mitochondria absent Present • Endoplasmic reticulum absent Present • Lysosomes absent Present • Peptidoglycan present absent • Cell membrane composition Phospholipids &Protein Sterols 75 Amsalu M 02/23/2022
  • 76. BACTERIOLOGY Definition: Bacteriology: the study of structure, functions and activities of bacteria. Medical Bacteriology: - the study of medically important bacteria 02/23/2022 Amsalu M 76
  • 77. Bacterial Cell General property: • Typical prokaryotic cell • Contain both DNA and RNA • Most grow in artificial media • Replicate by binary fission • Almost all contain rigid cell wall • Sensitive to antimicrobial agent 77 Amsalu M 02/23/2022
  • 78. STRUCTURE OF BACTERIA Bacterial structure is considered at three levels: 1. Cell envelope: Cell wall and cell membrane. 2. Cellular element internal the cell envelope: Mesosomes, ribosomes, chromosomes (nucleoid) and cytoplasmic granules, spores… 3. Cellular element external to the cell envelope: Flagellum, Pilus and Glycocalyx,….. 78 Amsalu M 02/23/2022
  • 81. 1. Cell envelope • External covering outside the cytoplasm • Composed of two basic layers: –cell wall and cell membrane • Maintains cell integrity A. Cell wall Multi layered structure and constitutes about 20% of the bacterial dry weight. Average thickness is 0.15-0.5 µm 81 Amsalu M 02/23/2022
  • 82. • Main structural component–Peptidoglycan:-is repeating disaccharides units and Polypeptides Functions of cell wall • Determines cell shape • prevents lysis (bursting) or collapsing due to changing osmotic pressures • Provides staining characteristics to the bacterium • Site of action of antibody • Contains toxic components to host 82 Amsalu M 02/23/2022
  • 83. • Based on their cell wall structure two different groups of bacteria are demonstrated by Gram stain: –Gram-positive bacteria: thick cell wall composed primarily of thick peptidoglycan and teichoic acid –Gram-negative bacteria: composed of outer membrane, thin peptidoglycan layer, and periplasmic space 83 Amsalu M 02/23/2022
  • 85. Gram-positive Cell Wall • Thick, homogeneous sheath of peptidoglycan: –function in cell wall maintenance and enlargement during cell division –stimulate a specific immune response • Have Teichoic acid 85 Amsalu M 02/23/2022
  • 86. Gram-negative Cell Wall • Composed of an outer membrane and a thin peptidoglycan layer and also has periplasmic space • Outer membrane is: – outermost layer contains lipopolysaccharides (LPS), lipoproteins and phospholipids – is an important factor in evading phagocytosis and actions of complements. – Provides a barrier to certain antibiotics (e.g.penicillin), digestive enzymes (e.g. lysozyme), detergents, heavy metals, bile salts, and certain dyes. – contains porin proteins in upper layer – regulate molecules entering and leaving cell 86 Amsalu M 02/23/2022
  • 87. Cont… Lipopolysaccharide (LPS) - is one component of outer membrane: Has three components a) lipid A b) a core polysaccharide and c) an O polysaccharide. A) Lipid A – Lipid portion of the LPS – Embedded in the top layer of the outer membrane – Functions as an endotoxin. B) Core polysaccharide – Its role is structural i.e. to provide stability. 87 Amsalu M 02/23/2022
  • 88. Cont… c) O polysaccharide – Extends outward from the core polysaccharide – Functions as an antigen and is useful for distinguishing species of gram-negative bacteria. • For example, the foodborne pathogen E. coli O157:H7 • Note : gram negative bacteria do not contain teichoic acids and Gram positive don‘t contain outer membrane. 88 Amsalu M 02/23/2022
  • 90. Components of cell wall of Gram negative bacteria • Peptidoglycan • Periplasmic space • Lipoprotein • Phospholipid Outer membrane • Lipopolysaccharide Components of cell wall of Gram positive bacteria • Peptidoglycan • Teichoic acid 90 Amsalu M 02/23/2022
  • 91. B. Cell membrane (cytoplasmic membrane) • It accounts for 30% of the dry weight of bacterial cell. • Phospholipid bilayer with embedded proteins • It is composed of • 60% protein, • 20-30% lipids and • 10-20% carbohydrate • Functions in: – Regulates the transport of nutrients and waste products into and out of the cell. 91 Amsalu M 02/23/2022
  • 92. 2. Cellular element internal to the cell envelope A. Mesosomes • Convoluted invagination of cytoplasmic membrane often at sites of septum formation. • It is involved in DNA segregation during cell division. B. Ribosomes • Cytoplasmic particles which are the sites of protein synthesis. • It is composed of RNA (70%) and proteins (30%) 92 Amsalu M 02/23/2022
  • 93. Cont… C. Inclusion bodies • Represent accumulated food reserve D. Chromosome – single, circular, double-stranded DNA molecule that contains all the genetic information required by a cell – DNA is tightly coiled around a protein, aggregated in a dense area called the nucleoid. (nuclear region of bacteria ) 93 Amsalu M 02/23/2022
  • 94. Cont… • Plasmids: bacteria may have extra chromosomal genetic material named as Plasmids small circular, double- stranded DNA • which may facilitate survival and propagation of the micro- organism • provide virulence and drug resistance. 94 Amsalu M 02/23/2022
  • 95. Cont… E. Spores – Resistant and metabolically inactive – produced by some Gram positive genera: Clostridium, Bacillus • Such bacteria have a 2-phase life cycle: ovegetative cell : metabolically active and growing oendospore : –Dormant cell –Produced when starved –when exposed to adverse environmental conditions; capable of high resistance and very long-term survival – sporulation -formation of endospores – germination- return to vegetative growth 95 Amsalu M 02/23/2022
  • 96.  Resistance of spore is linked to:  high levels of calcium and Presence of dipicolinic acid thick coat  Longevity verges on immortality – can live million years.  Resistant to ordinary cleaning methods and boiling  Pressurized steam at 120oC for 20-30 minutes will destroy the spore 96 Amsalu M 02/23/2022
  • 97. 3. Cellular element external to the cell envelope A. Glycocalyx (capsule and slime layer)  Coating of molecules external to the cell wall, made of sugars and/or proteins  Two types: 1. slime layer - loosely organized and attached 2. capsule - highly organized, tightly attached  Functions: ◦ protect cells from dehydration and nutrient loss ◦ inhibit killing by white blood cells by phagocytosis contributing to pathogenicity ◦ attachment - formation of biofilms 97 Amsalu M 02/23/2022
  • 98. Cont… B. Flagellum It is the organ of locomotion in bacterial cell. It is composed of protein named as flagellin. The flagellar antigen in motile bacterium is named as H antigen  consists of three parts. These are: • Filament • Hook and • Basal body The basal body and hook are embedded in the cell surface while the filament is free on the surface of bacterial cell. 98 Amsalu M 02/23/2022
  • 99. Cont… Based on flagella arrangements bacteria can be: • Atrichous: Bacteria with no flagellum. • Monotrichous: Bacteria with single polar flagellum. • Lophotrichous: Bacteria with bunch of flagella at one pole. • Amphitrichous: Bacteria with flagella at both poles. • Peritrichous: Bacteria with flagella all over their surface. 99 Amsalu M 02/23/2022
  • 101. Cont… C. Fimbriae and Pili – Are shorter, and thinner than flagella – Found on many gram-negative bacteria a) Fimbriae (singular: fimbria ) – Can occur at the poles or can be evenly distributed over the entire surface of a bacterium. – Important for adherance. b) Pili (singular: pilus) – Are usually longer than fimbriae – Number only one or two per cell. – Involved in DNA transfer from one to the other 101 Amsalu M 02/23/2022
  • 102. Classification of bacteria • Bacterial classification depends on the following characteristics. 1. Morphology and arrangement 2. Staining 3. Cultural characteristics 4. Biochemical reactions 5. Antigenic structure 6. Base composition of bacterial DNA • Morphology and staining of bacteria are the commonly used characteristics to classify bacteria. 102 Amsalu M 02/23/2022
  • 103. 1. Morphology of bacteria • When bacteria are visualized under light microscope, the following morphology are seen. 1. Cocci (singular coccus): spherical or oval bacteria measuring about 0.5-1.0μm in diameter. • They are found in single, pairs, chains or clusters. • Micrococci:- Cocci occurring single. • Diplococci:- arranged in pair • Streptococci :- chain forming cocci • Staphylococci:- bunches of cocci or groups or grapes of cocci 103 Amsalu M 02/23/2022
  • 104. Shape(cocci) and their arrangement 104 Amsalu M 02/23/2022
  • 106. 2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tapered, square or swollen ends; with a size measuring 1-10μm in length by 0.3-1.0μm in width. 106 Amsalu M 02/23/2022
  • 107. • Bacilli: stick- like bacteria Bacilli (rod Shape) 107 Amsalu M 02/23/2022
  • 109. 3. Spiral: Spiral shaped bacteria with regular or irregular distance between twisting. • Coiled or cork-skrew shaped • Eg. Spirilla and spirochaetes 109 Amsalu M 02/23/2022
  • 111. Cont… 2. Staining of bacteria • staining is the process of coloring of colorless object using stains (dyes) • Uses of staining • To observe the morphology, size and arrangement of bacteria • To differentiate one group of bacteria from the other group. 111 Amsalu M 02/23/2022
  • 112. Cont… Type of staining methods –Simple staining method –Differential staining method –Special staining method 112 Amsalu M 02/23/2022
  • 113. Cont… 1. Simple staining method It is a type of staining method in which only a single dye is used. Two kinds of simple stains A. positive staining:- The bacteria or its parts are stained by the dye E.g. Methylene blue stain, Crystal violent stain B. Negative staining: - the dye stains the background and the bacteria remain unstained E.g. Indian ink stain 113 Amsalu M 02/23/2022
  • 114. Cont… 2. Differential staining method  Multiple stains (dyes) are used to distinguish different group of bacteria  React differently with different types of bacteria  2 Most common: –Gram Stain –Acid-Fast Stain (Ziehl- Neelson stain) 114 Amsalu M 02/23/2022
  • 115. A. Gram stain • Gram Stain: Named after Christian Gram who invented it • Most bacteria are differentiated by their gram reaction due to difference on their cell wall structure. Principle: • Bacteria are stained with Crystal violet and Iodine, then subjected to decolorization with alcohol/acetone. Bacteria that retain the primary stain (Crystal violet) are Gram-positive. Bacteria that loose the primary stain (Crystal violet) are Gram-negative 115 Amsalu M 02/23/2022
  • 116. Required reagents: – crystal violet: primary stain – Gram‘s Iodine: mordant – Acetone-Alcohol: decolorizer – Safranin: counterstain Procedure: 1. Prepare the smear from the culture or from the specimen. 2. Allow the smear to air-dry completely. 3. Rapidly pass the slide (smear upper most) three times through the flame. 4. Cover the fixed smear with crystal violet for 1 minute and wash with distilled water. 116 Amsalu M 02/23/2022
  • 117. 5. Tip off the water and cover the smear with gram‘s iodine for 1 minute. 6. Wash off the iodine with clean water. 7. Decolorize rapidly with acetone-alcohol for 30 seconds. 8. Wash off the acetone-alcohol with clean water. 9. Cover the smear with safranin for 1 minute. 10. Wash off the stain wipe the back of the slide. Let the smear to air-dry. 11. Examine the smear with oil immersion objective to look for bacteria. • Interpretation: – Gram-positive bacterium ……………Purple – Gram-negative bacterium …………..Pink(red) 117 Amsalu M 02/23/2022
  • 118. Principle of staining technique: 118 1- Crystal violet: all cells are stained violet. 2- Iodine acts as a mordant (fixes the dye to the cell) all cells remain violet. 3-Alcohol acetone decolorizes gram negative cells only: Gram-positive remains violet , gram-negative becomes colorless. 4-Counter stain with Safranin: Gram- positive remains violet while gram negative becomes red. Amsalu M 02/23/2022
  • 119. Cont… • Result  Gram (+) Purple  Gram (-) Red • Difference - due to structure of cell wall –Gram (+) Thick cell wall –Gram (-) Thin cell wall 119 Amsalu M 02/23/2022
  • 124. B. Ziehl- Neelson staining Method • Ziehl- Neelson stain (Acid fast stain) is used to stain Mycobacterium and other acid fast organisms which cannot be stained with gram stain.  once the mycobacterium is stained with the primary stain it can not be decolorized with acid. So named as Acid fast bacteria 124 Amsalu M 02/23/2022
  • 125. • Reagents required: – Carbol-fuchsin: primary stain – Acid-Alcohol: decolorizer – Methylene blue/Malachite green: counterstain Procedure for Ziehl-Neelson staining method 1. Prepare the smear from the primary specimen and fix it by passing through the flame and label clearly 2. Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin solution. 3. Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait for 5 minutes. 125 Amsalu M 02/23/2022
  • 126. 4. Wash off the stain with clean water. 5. Cover the smear with 3% acid-alcohol solution until all color is removed and wait for 3 minutes. 6. Wash off the stain and cover the slide with 1% methylene blue and wait for one minute. 7. Wash off the stain with clean water and let it air-dry. 8. Examine the smear by adding a drop of oil under the oil immersion objective to look for acid fast bailli. • Interpretation: – Acid fast bacilli…………..Red – Back ground………………Blue 126 Amsalu M 02/23/2022
  • 127. Cont… Result  Acid fast bacilli . . .Red  Back ground . . . . Blue 127 Amsalu M 02/23/2022
  • 128. 3. Special Stains A. Capsule Staining method B. Spore staining method 128 Amsalu M 02/23/2022
  • 130. BACTERIAL NUTRITION • Bacteria, like all cells, require nutrients for growth. • Most important elements required by bacteria can be divided into two main categories: Physical requirements include: –Temperature, pH, and osmotic pressure. Chemical requirements include –Sources of carbon, nitrogen, sulfur, phosphorus, trace elements, and organic growth factors. 130 Amsalu M 02/23/2022
  • 131.  Chemical requirements • The four most important elements of bacteria are carbon, hydrogen, oxygen and nitrogen. Carbon • Organisms require a carbon for the synthesis of numerous organic compounds that comprise bacterial cell. • Depending on their requirements, bacteria can be classified as 1. Autotrophs: Free-living, non-parasitic bacteria which use carbon dioxide as carbon source. • The energy needed for their metabolism can be obtained from: Sun light-Photoautotrophs Inorganic compounds by oxidation-Chemoautotrophs 131 Amsalu M 02/23/2022
  • 132. 2. Heterotrophs: Parasitic bacteria require more complex organic compounds as their source of carbon and energy. • Human pathogenic bacteria are heterotrophs. • The principal source of carbon is carbohydrate which are degraded either by oxidation, in the presence of oxygen, or by fermentation, in the absence of oxygen, to provide energy in the form of ATP. 132 Amsalu M 02/23/2022
  • 133. Hydrogen and oxygen • Obtained from water. • Essential for the growth and maintenance of cell.  Based on oxygen requirements and tolerance, bacteria are classified as:  Obligate aerobes - require oxygen to live  Obligate anaerobes - grow in the absence of oxygen ; exposure to oxygen kills anaerobes  Facultative anaerobes - grow in the presence or absence of oxygen.  Microaerophiles- grow best at reduced oxygen tension 133 Amsalu M 02/23/2022
  • 135. Nitrogen • For protein synthesis & also for DNA and RNA synthesis • Obtained from organic molecules like proteins and inorganic molecules like ammonium salts and nitrates. NB: Main source of nitrogen is ammonia, in the form of ammonium salt. 135 Amsalu M 02/23/2022
  • 136.  Physical Requirements Temperature  Temperature tolerance range: The minimum and maximum temperature at which a micro-organism can grow  which is different in different species of bacteria.  Optimal growth range of temperature: The temperature at which the bacteria grow best. 136 Amsalu M 02/23/2022
  • 137.  Based on different optimal growth temperature requirement, bacteria are divided into: Psychrophilic bacteria: 00C-20 0C Mesophilic bacteria: 20-450C Thermophilic bacteria: 50-600C • NB: Most human pathogens and many of the normal flora of human bodies have an optimal temperature of 370c; Therefore they are mesophilic bacteria. 137 Amsalu M 02/23/2022
  • 138. PH • It is a measure of acidity and alkalinity.  Neutrophilic bacteria grow best at near neutral PH value. • Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5.  Acidophilic bacteria prefer to grow at low PH value (acidic medium).  Alkalinophilic bacteria prefer to grow at high PH value (alkaline medium). 138 Amsalu M 02/23/2022
  • 139. Salinity Salt content of the medium affects bacterial growth.  Halophilic bacteria grow best at high salt concentration.  Moderate halophiles require 3% salt concentration.  Extreme halophiles require 15% salt concentration. 139 Amsalu M 02/23/2022
  • 140. BACTERIAL GROWTH • It is an orderly increase in all the components of an organism. • It is an increment in cell number. Generation time • It is the time taken for a bacterial population to double.  time required for a cell to divide • bacteria divide into two equal daughter cells and double the number. i.e binary fission 140 Amsalu M 02/23/2022
  • 141. • The generation time varies with: the species the amount of nutrients, the temperature, the pH, and other environmental factors. Bacterial growth phases  The normal bacterial growth curve has four phases: 1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase 141 Amsalu M 02/23/2022
  • 142. 1. Lag phase • The period of adaptation with active macro molecule synthesis like DNA, RNA, various enzymes and other structural components. • It is the preparation time for reproduction; no increase in cell number 2. Exponential (log) phase • The period of active multiplication of cells. • Cell division proceeds at a logarithmic rate, and determined by the medium and condition of the culture. 142 Amsalu M 02/23/2022
  • 143. 3. stationary phase (Equilibrium phase) • The period when the bacteria have achieved their maximal cell density or yield. • There is no further increase in viable bacterial cell number. • The growth rate is exactly equal to the death rate. • A bacterial population may reach stationary growth when one of the following conditions occurs: – The required nutrients are exhausted – Inhibitory end products are accumulated – Physical conditions do not permit a further increase in population size. 143 Amsalu M 02/23/2022
  • 144. 4. Decline phase • The period at which the rate of death of bacterial cells exceeds the rate of new cell formation. • There is drastic decline in viable cells. • Few organisms may persist for so long time at this period at the expense of nutrients released from dying micro-organisms. 144 Amsalu M 02/23/2022
  • 146. CULTIVATION OF BACTERIA IN CULTURE MEDIA • Culture- is the growth of particular microorganism on or within a medium as a result of inoculation and incubation under controlled conditions. • Culture media- is a liquid or solid nutrient material containing the required nutrients for cultivation of microorganisms. Uses: Isolation& identification of microorganisms Transportation of sample Performing anti-microbial sensitivity tests 146 Amsalu M 02/23/2022
  • 147. Types of culture media Basic /Simple / All purpose media • It is a media that supports the growth of micro- organisms that do not require special nutrients. • Eg. Nutrient Broth, Nutrient Agar Enriched media • Media that are enriched with whole blood, lyzed blood, serum, special extracts or vitamins to support the growth of fastidious bacteria. • Eg. Blood Agar, Chocolate Agar 147 Amsalu M 02/23/2022
  • 148. Selective media • Media which contain substances ( Eg. Antibiotics) that prevent or slow down the growth of bacteria other than pathogens for which the media are intended. • Eg. Modified Thayer –Martin Agar • Salmonella-Shigella( SS) agar Differential media • Media to which indicator substances are added to differentiate bacteria. • Eg. TCBS Agar differentiates sucrose fermenting yellow colonies of Vibrio cholerae from non-sucrose fermenting blue colonies of other Vibrio species. 148 Amsalu M 02/23/2022
  • 149.  Forms of culture media 1. Solid culture media 2. Semisolid culture media 3. Fluid culture media 149 Amsalu M 02/23/2022
  • 152. BACTERIAL GENETICS 152  Most of the genetic information in a bacterial cell is contained with in chromosome . A single molecule of DNA arranged as a double in closed loop.  Bacterial inherited characteristics are encoded in DNA. These are:  Chromosome  Extra chromosome (Plasmid) Amsalu M 02/23/2022
  • 153. 153  Chromosome- Bacterial chromosome is circular double stranded DNA attached to bacterial cell membrane  Plasmids: are self replication extra chromosomal DNA molecules.  Plasmids are not essential to the life of the cell but they may have selective advantage for these organism  R-factor (Resistance)  Extra cellular toxin Amsalu M 02/23/2022
  • 154. Genetic variation in Bacteria 154 GeneticVariation in Bacteria can occur by Mutation Gene-Transfer Amsalu M 02/23/2022
  • 155. Mutation 155 Bacterial mutations occur when the information in a bacterial chromosome may alter by different means There are two types of mutation: Spontaneous mutation Induced mutation Amsalu M 02/23/2022
  • 156. Gene-Transfer 156 The transfer of genetic information can occur by either of the three methods: Conjugation Transudation Transformation Amsalu M 02/23/2022
  • 157. Conjugation 157 A process where by DNA is transferred from one bacterium to another by cell to cell physical contact (through sex pilli) Plasmids are the genetic elements most frequently transferred by conjugation Amsalu M 02/23/2022
  • 158. 158 Fig. Transfer/replication process of a conjugative plasmid Amsalu M 02/23/2022
  • 159. Transduction 159  Is a method of gene transfer in which a virus (phage) acts as a vehicle for carrying DNA from a donor bacterium to recipient bacterium. Amsalu M 02/23/2022
  • 160. Transformation 160  A process by which a bacterium acquire DNA fragments or genes from surroundings.  Usually this occurs in microbial culture. Amsalu M 02/23/2022
  • 161. 161 Fig. Transformation process in bacteria Amsalu M 02/23/2022
  • 163. GRAM NEGATIVE RODS Enterobacteriaceae: A. Lactose-fermenters  Escherichia spp, Klebsiella spp.  Enterobacter spp & Citrobacter spp. B. Non-lactose fermenters- Salmonella spp, Shigella spp and Proteus spp Non- Enterobacteriaceae: Pseudomonas, Vibrio, Helicobacter & Campylobacter 163 02/23/2022 Amsalu M.
  • 164. Family enterobacteriaceae Characteristics • Named as coli forms or enterobacilli. • Most of them found as normal flora in intestinal tract of humans and animals. • Are Gram-negative, non-spore forming, aerobic and facultative-anaerobic bacteria. • Most are motile, grow over a wide range of temperature in ordinary media. • All ferment glucose, reduce nitrate to nitrite and are oxidase negative • Release endotoxin from their cell wall and some release exotoxin. • Most of them have possessed three types of antigens: H antigen, K-antigin and O(somatic) antigen. 164 02/23/2022 Amsalu M.
  • 165. Antigenic Structures and Virulence Factors Complex surface antigens contribute to pathogenicity and trigger immune response: • H – flagellar Ag • K – capsule and/or fimbrial Ag • O – somatic or cell wall Ag – all have 165 02/23/2022 Amsalu M.
  • 167. Enterobacteriaceae: Types of Disease • Intestinal (diarrheal) infection • Extra-intestinal infection • Urinary tract (primarily cystitis) • Respiratory (nosocomial pneumonia) • Wound (surgical wound infection) • Bloodstream (gram-negative bacteremia) • Central nervous system (neonatal meningitis) 167 02/23/2022 Amsalu M.
  • 168. GENUS: ESCHERICHIA Main species of medical importance is Escherichia coli. Escherichia coli (E.coli) Characteristics of • Normal flora in human and animal gastrointestinal tract. • Found in soil, water & vegetation. • Most are motile; some are capsulated. Clinical features • Urinary tract infection- cystitis, pyelonephritis • Wound infection- appendicitis, peritonitis. • Neonatal septicemia & meningitis • Escherichia coli-associated diarrheal diseases • The strains of E. coli that cause gastroenteritis are subdivided into five major groups: 168 02/23/2022 Amsalu M.
  • 169. 1. Enteropathogenic E.coli (EPEC)  causes outbreaks of self-limiting infantile diarrhea  they also cause severe diarrhea in adults antibiotic treatment shorten the duration of illness and cure diarrhea. 2. Enteroinvasive E.coli (EIEC) Non-motile, non-lactose fermenting E. coli invade the mucosa of the ileum and colon, and causes shigellosis-like dysentery in children in developing countries and travelers to these countries. 169 02/23/2022 Amsalu M.
  • 170. 3. Enterotoxigenic E.coli (ETEC)  Colonization factor of the organism promote adherence to epithelial cells of small intestine followed by release of enterotoxin which causes toxin-mediated watery diarrhea in infants & young adults. It is also an important cause of traveler‘s diarrhea 4. Entero haemorrhagic E.coli ( EHEC) causes hemorrhagic colitis (severe form of diarrhea), and hemolytic uremic syndrome characterized by acute renal failure, hemolytic anemia and low platelet count. 5. Enteroaggressive E.coli (EAEC) Adhere to human intestinal mucosal cells and produce shiga-like toxin and hemolysin, and causes acute and chronic diarrhea in persons in developing countries Produce food-borne illness in developed countries 170 02/23/2022 Amsalu M.
  • 171. PREVENTION AND CONTROL Restricting use of antibiotics and avoiding unnecessary use of urinary catheters. Maintenance of high hygienic standards to reduce risk of gastroenteritis. 171 02/23/2022 Amsalu M.
  • 172.  GENUS: SALMONELLA • Salmonella can colonize virtually all animals, including poultry, reptiles, livestock, rodents, domestic animals, birds and humans. • Species of medical importance are: S. typhi, S. paratyphi & S. enteritidis. 172 02/23/2022 Amsalu M.
  • 173. Clinical features 1. Typhoid fever and paratyphoid fever: -Both are caused by S. Typhi and S. paratyphi respectively, and transmitted by fecal-oral route via contaminated food and drinks. - Incubation period: 10-14 days, but depend on bacterial load and immune status of hosts. Predisposing factors: - Reduced gastric acidity - Disrupted intestinal microbial flora - Compromised local intestinal immunity • Both manifest with persistent fever, headache, malaise, chills, enlargement of liver and spleen, and skin rashes. • Paratyphoid fever is milder than typhoid fever 173 02/23/2022 Amsalu M.
  • 174. Complications: Intestinal perforation, Lower gastrointestinal bleeding, Dissemination to different body organs including meninges and brain. 2. Bacteremia with focal lesions • Causative agent: S. typhimurium • Manifests with blood stream invasion with focal lesions in lungs, bones and meninges. • Intestinal manifestation is often absent. 3. Gastroenteritis - It is caused by S. enteritidis & S. typhimurium - It manifests with initial watery diarrhea, and later bloody mucoid diarrhea associated with crampy abdominal pain and tenesmus. 174 02/23/2022 Amsalu M.
  • 175. LABORATORY DIAGNOSIS Specimen: • Blood, stool, urine and serum for typhoid fever. • Stool for gastroenteritis. Gram reaction: Gram-negative rods Culture: • Selective medium-favour growth of Salmonella and Shigella over other enterobacteriaceae. • Example: Salmonella-Shigella (SS) agar, XLD agar, Deoxycholate-Citrate agar. • Serology • Widal Test 175 02/23/2022 Amsalu M.
  • 176. TREATMENT Ciprofloxacin, chloramphenicol, trimethoprim, SXT or a broad spectrum cephalosporin. PREVENTION AND CONTROL Proper sewage treatment Chlorination of water supply, Pasteurization of milk. hand washing prior to food handling Cultures of stool samples from food handlers to detect and treat carriers.  Proper cooking of poultry, eggs and meat are important preventive measures. 176 02/23/2022 Amsalu M.
  • 177. GENUS: SHIGELLA Characteristics: • They are Non-lactose fermenting gram-negative rods. • They are non-motile. • All Shigella have O antigens (polysaccharides) in their cell walls. - These antigens are used to divide in to four groups. 1. Shigella dysenteriae 2. Shigella flexneri 3. Shigella sonnei 4. Shigella boydii 177 02/23/2022 Amsalu M.
  • 178. Epidemiology Shigellosis is only a human disease (except primates like monkeys) that can be transmitted from person to person. The five F‘s (fingers, flies, food, fomites, and faeces) are the principal factors in transmission. =Route of infection is fecal-oral route Epidemic out breaks of the disease occurs in:- Day care centers - nurseries custodial institutions. 178 02/23/2022 Amsalu M.
  • 179. Shigellosis • It is characterized by abdominal cramp, sudden onset of bloody mucoid diarrheas, fever, generalized muscle ache and weakness. • Dehydration & electrolyte and acid-base disturbance. • High prevalence in: poor sanitation, poor personal hygiene & polluted water supply. • Young children are frequently affected. Treatment – Ciprofloxacin, ampicillin, tetracycline, trimethoprim - sulfamethoxazole and chloramphenicol Prevention and Control Proper sewage disposal chlorination of water, and  personal hygiene. 179 02/23/2022 Amsalu M.
  • 180. Genus Klebsiella 180 Characteristics • Non-motile, lactose-fermenting, capsulated, gram- negative rods. • Main species of medical importance: K. pneumoniae, K. rhinoscleromatis & K. ozenae Klebsiella pneumoniae • It is found as a commensal in the intestinal tract, and also found in moist environment in hospitals. • It is an important Nosocomial (hospital acquired) pathogen. • It causes: Pneumonia, Urinary tract infection, Septicemia and meningitis (especially in neonates), Wound infection and peritonitis 02/23/2022 Amsalu M.
  • 181. 181 Klebsiella rhinoscleromatis  It causes rhinoscleroma of nose and pharynx to extensive destruction of nasopharynx. Klebsiella ozenae  It causes ozaena manifesting with foul smelling nasal discharge leading to chronic atrophic rhinitis. Laboratory diagnosis of klebsiella species  Specimen: Sputum, urine, pus, CSF, body fluid  Smear: Gram-negative rods  Culture: Large, mucoid, lactose-fermenting colonies on MacConkey agar 02/23/2022 Amsalu M.
  • 182. Genus: Proteus 182 General characteristics • are found in the intestinal tract of humans and animals, soil, sewage and water. • They are gram-negative, motile, non-capsulated, pleomorphic rods. • produce the enzyme urease and phenylalanine diaminase. • Certain species are very motile and produce a striking swarming colonies on blood agar plate • Cell wall O antigens of certain strains of Proteus (such as OX -2, OX-19 and OX-K) cross react with antigens of several species of rickettsia  Species of medical importance are: » P. mirabilis » P. vulgaris 02/23/2022 Amsalu M.
  • 183. Pathogenesis and clinical manifestations 183 P. mirabilis causes:  Urinary tract infection, Septicemia, Abdominal and wound infection P. vulgaris  Isolated in wound infection and urinary tract infection.  Important Nosocomial pathogen. 02/23/2022 Amsalu M.
  • 184. 184 Laboratory diagnosis  Specimen: Urine, pus, blood, ear discharge  Smear: Gram-negative rods  Culture: Produce characteristic swarming colonies over the surface of blood agar.  Are non-lactose fermenting colonies on MacConkey agar. 02/23/2022 Amsalu M.
  • 185. GRAM NEGATIVE SHORT RODS: COCCOBACILLI GENUS: HAEMOPHILUS • Characteristics: • This is a group of small gram-negative, non-spore forming, non-motile, pleomorphic bacteria that require enriched media for growth. • Present in upper respiratory tract as a normal microbial flora in healthy people. The main species of medical importance are: Haemophilus influenzae, H. ducreyii & H. aegypticus. 185 02/23/2022 Amsalu M.
  • 186. A. Haemophilus influenzae Characteristics: - Gram-negative Coccobacilli. - may produce a capsule (six capsular types have been distinguished) or may be unencapsulated - is a normal flora of the URT in healthy humans, and may also colonize the conjunctiva and genital tract. - Humans are the only natural hosts, and colonization begins shortly after birth. - transmitted by respiratory droplets 186 02/23/2022 Amsalu M.
  • 187. Diseases caused by H. influenzae, therefore, fall into two categories: First, disorders such as otitis media, sinusitis, epiglotitis, and bronchopneumonia result from contiguous spread of the organism from its site of colonization in the respiratory tract. Second, disorders such as meningitis, septic arthritis, and cellulitis result from invasion of the bloodstream, followed by localization of H. influenzae in these and other areas of the body. 187 02/23/2022 Amsalu M.
  • 189. Prevention and control Active immunization with conjugated PRP (polyrabitol phosphate) vaccines prevents most H.influenzae type b infections. Generally given to children younger than two and is also effective in preventing invasive disease, and reduces respiratory carriage of Hib. Rifampin can be used as prophylaxis. 189 02/23/2022 Amsalu M.
  • 190. B. Heamophilus ducreyii – Slender, gram-negative, ovoid bacilli, slightly larger than H. influenzae. - It causes genital ulcer is known as chancroid (soft chancre), a sexually transmitted ulcerative disease. - In addition to the painful genital lesions, inguinal lymphadenopathy may occur. - If untreated, this progresses to formation of a bubo (a swollen lymph node), which then suppurates (forms pus). 190 02/23/2022 Amsalu M.
  • 191. Note: Open genital sores facilitate the transmission of HIV, and H. ducreyi infection is frequently associated with AIDS. Treatment  It is treated by erythromycin, cotrimoxazole and third generation cephalosporins if they are sensitive  Azithromycin is also effective against H. ducreyi infections C. Heamophilus aegypticus – It causes contagious purulent conjunctivitis 191 02/23/2022 Amsalu M.
  • 192. GENUS: BORDETELLA Characteristics: • They are small, encapsulated, aerobic, non-motile, gram- negative coccobacilli that grow singly or in pairs • Bordetella species of medical importance: B. pertussis and B. parapertussis • The former causes the disease pertussis (also known as whooping cough), and the latter causes a mild pertussis- like illness. • Whooping cough is a highly contagious disease and a significant cause of morbidity and mortality worldwide . 192 02/23/2022 Amsalu M.
  • 193. • The major mode of transmission of Bordetella is via droplets spread by coughing from early cases and possibly carriers. • But the organism survives only briefly outside the human respiratory tract. • The incubation period for pertussis generally ranges from one to three weeks. Clinical features: • The disease can be divided into three phases: Catarrhal stage, Paroxysmal stage & Convalescence stage 193 02/23/2022 Amsalu M.
  • 194. A. During catarrhal stage, the patient is highly infectious but not very ill manifesting with mild coughing and sneezing (nonspecific symptoms, and then progresses to include a dry, nonproductive cough). B. During paroxysmal stage, the patient presents with explosive repetitive cough with characteristic ‘whoop’ upon inhalation leading to exhaustion, vomiting, cyanosis and convulsion.  Large amounts of mucus are typically produced. C. During convalescence stage, the patient presents with prolonged cough – During this period, secondary complications such as infections (for example, otitis media and pneumonia) and CNS dysfunction (for example, encephalopathy or seizures) may occur. 194 02/23/2022 Amsalu M.
  • 195. • Treatment – Erythromycin is the drug of choice for infections with B. pertussis, both as chemotherapy (where it reduces both the duration and severity of disease), and as chemoprophylaxis for household contacts. – For erythromycin treatment failures, trimethoprim- sulfamethoxazole is an alternative choice. Prevention: – Two forms of vaccine are currently available (one of killed whole cells, and one that is acelullar, containing purified B. pertussis proteins). – Both are formulated in combination with diphtheria and tetanus toxoids. • To protect infants who are at greatest risk, immunization is generally initiated when the infant is two months old. 195 02/23/2022 Amsalu M.
  • 196. Gram negative rods not grouped under family Enterobacteriaceae • Genus Pseudomonas • Genus Vibiro • Genus Helicobacter • Genus Campylobacter 196 02/23/2022 Amsalu M.
  • 197. Genus፡ Pseudomonas • Gram-negative motile aerobic rods having very simple growth requirement. • Can be found in water, soil, sewage, vegetation, human and animal intestine. • Species of medical importance: - P. aeruginosa - P. pseudomallei 197 02/23/2022 Amsalu M.
  • 198. Pseudomonas aeruginosa • Can be found in the intestinal tract, water, soil and sewage and is frequently found in moist environments in hospitals, (sinks, cleaning buckets, drains, etc). • It is able to grow in some eye drops, saline and other aqueous solutions. • Many infections with P. aeruginosa are opportunistic hospital-acquired, affecting those already in poor health and immuno-suppressed. • Infections are often difficult to eradicate due to P. aeruginosa being resistant to many antimicrobials. 198 02/23/2022 Amsalu M.
  • 199. • Infections caused by P. aeruginosa include: • Skin infections, especially burn sites, wounds, pressure sores, and ulcers (often as secondary invader). Septicaemia may develop. • Urinary infections, usually following catheterization or associated with chronic urinary disease. • Respiratory infections especially in patients with cystic fibrosis or conditions that cause immuno-suppression. • External ear and eye infections often secondary to trauma or surgery. 199 02/23/2022 Amsalu M.
  • 200. Treatment • P. aeruginosa is resistant to most of the commonly used antibiotics. • Antimicrobials that usually show activity against Pseudomonas include aminoglycosides, polymyxin, and some penicillins and cephalosporins. 200 02/23/2022 Amsalu M.
  • 201. GENUS: VIBRIOS Important properties: • Curved ―Comma-shaped‖ actively motile gram- negative rods. • They are single or occasionally united in S shapes or spirals.. • Found in fresh water, shellfish and other sea food. • They are motile by a single polar flagellum. • Readily killed by heat and drying • Species of medical important include: – Vibrio cholerae and – Vibrio parahaemolyticus 201 02/23/2022 Amsalu M.
  • 203. Vibrio cholerae • V. cholerae is halotolerant and can grow in alkaline conditions Antigenic structure: • has a complex cell wall which includes lipopolysaccharides, the carbohydrate components of which are designated as O antigens that confer serologic specificity. • Many vibrios share a single heat-labile flagellar H antigen. • There are now 206 somatic antigens (O antigens) in the V. cholerae V. cholerae O1 and O139 – are responsible for causing Cholera which can occur in epidemic or worldwide pandemics. • occasionally, non-01/non-0139 V. cholerae causes cholera- like disease. 203 02/23/2022 Amsalu M.
  • 204. • Virulence factors – Cholera toxin (CT) • CT consists of a number of subunits: one A or enzymatic subunit and five identical B or binding subunits. 204 02/23/2022 Amsalu M.
  • 205. Pathogenesis & Pathology • V. cholerae is pathogenic only for humans. • A person with normal gastric acidity may have to ingest as many as 1010 or more V. cholerae to become infected when the vehicle is water, because the organisms are susceptible to acid. • When the vehicle is food, as few as 102-104 organisms are necessary because of the buffering capacity of food. • Any medication or condition that decreases stomach acidity makes a person more susceptible to infection with V. cholerae. 205 02/23/2022 Amsalu M.
  • 206. • Virulent V. cholerae organisms attach to the microvilli of the brush border of epithelial cells. • There they multiply and liberate cholera toxin (CT) and perhaps mucinases and endotoxin. • CT disrupts ion fluxes without causing apparent damage to the mucosa. • CT decreases the net flow of sodium into the tissue. • This produces a net flow of chloride and water out of the tissue into the lumen, causing the massive diarrhea and electrolyte imbalance that is associated with this infection. 206 02/23/2022 Amsalu M.
  • 207. Epidemiology Organisms are responsible for major pandemics with significant mortality in developing countries. The main animal reservoirs are marine shellfish The disease can spread by consumption of contaminated food and water. Direct person-to person spread is rare. 207 02/23/2022 Amsalu M.
  • 208. Some infections are asymptomatic or cause mild diarrhea Incubation period is 1-4 days Can cause severe disease resulting in abrupt watery diarrhea (20-30L/day) and vomiting ―Rice-water stool‖ is characteristic There are no RBC or WBC in the stool. Results in severe fluid and electrolyte loss, dehydration, shock, acidosis Can progress to coma and death Mortality rate in untreated cases is 60% Patients with suspected cholera need to be treated prior to laboratory confirmation, because death by dehydration can occur within hours. Clinical Significance 208 02/23/2022 Amsalu M.
  • 209. Treatment  Fluid and electrolyte replacements are crucial. Antibiotic therapy reduces the bacterial burden, exotoxin production, and duration of diarrhea. • Antibiotics (doxycycline is the drug of choice) can shorten the duration of diarrhea and excretion of the organism. Prevention and control  Ensuring clean water and food supply. The use of tetracycline for prevention is effective in close contacts but cannot prevent the spread of a major epidemic. 209 02/23/2022 Amsalu M.
  • 210. 210 Genus፡ Campylobacter General characteristics  Campylobacters are motile, curved, oxidase-positive, thermophillic, Gram- negative rods similar in morphology to vibrios.  Campylobacters cause both diarrhoeal and systemic diseases and are among the most widespread causes of infection in the world.  The cells have one polar flagella and attached at their ends giving pairs ―S‖ shapes or a ―seagull‖ appearance.  C. jejuni, and C. coli are by far the most common  Domestic animals such as dogs may also carry the organisms and probably play a significant role in transmission to humans
  • 211. 211  The most common source of human infection is undercooked poultry, but outbreaks have been caused by contaminated rural water supplies and unpasteurized milk Species of medical importance - Campylobacter jejuni - Campylobacter coli Virulence factors  Lipopolysaccharides with endotoxic activity.  Cytopathic extra cellular toxins.  Enterotoxins.
  • 212. 212 Pathogenesis and clinical manifestations  Campylobacter jejuni accounts for 90 to 95% of human campylobacter infections in most parts of the world.  Campylobacter jejuni and Campylobacter coli cause enteritis which may take the form of toxigenic watery diarrhoea or dysentery.  The organisms are able to produce enterotoxins and cytotoxins.  In developing countries, C. jejuni and C. coli cause disease mainly in children under 2 years.
  • 213. 213  The jejunum and ileum are the first sites to be come colonized followed by the colon and rectum.  In well developed infections, mesenteric lymph nodes are enlarged.  Diarrhoea is due to disruption of intestinal mucosa due to cell invasion by campylobacters and the production of toxins.  The toxin blocks the cell cycle of host cells but its precise role is not yet clear.
  • 214. 214 Laboratory diagnosis • Specimen: Fresh diarrhoeal or dysenteric specimens containing blood, pus and mucus • Microscopy: Typical ‗S‘ shaped gram-negative rods. • Typical darting motility (swift and sudden) of the bacteria under dark field microscopy or phase contrast microscopy
  • 215. 215 Culture  Campylobacter species are strictly microaerophilic, requiring incubation in an atmosphere of reduced oxygen (5–10%) with added carbon dioxide (about 10%).  C. jejuni and C. coli are thermophiles, i.e. they will grow at 42-43 ºC and 36–37 ºC but not at 25 ºC.  Incubation at 42-43 ºC helps to identify C. jejuni and C. coli. Blood agar: C. jejuni and C. coli produce non-haemolytic spreading, droplet-like colonies on blood agar.  Campylobacter species are oxidase and catalase positive.
  • 216. 216 Treatment Erythromycin or ciprofloxacin are drugs of choice for C. jejuni enterocolitis. Prevention and control 1.Proper cooking of foods. 2.Avoiding contact with infected human or animals and their excreta. 3.Pasteurizing of milk and milk products. 4.No vaccine available.
  • 217. Genus: Helicobacter Characteristics; • spiral shaped gram- negative bacilli, with polar flagella Species of medical importance Helicobacter pylori • Helicobacters are urease positive. • Urease production is a consistent finding in Helicobacter species of humans that colonize the stomach (acidic environment). – This is uncommon in species found in the intestine. • H. pylori have now been associated with gastritis, peptic ulcers, gastric adenocarcinoma and gastric mucosa- associated lymphoid type B – cell lymphomas. 217 02/23/2022 Amsalu M.
  • 218. Helicobacter pylori • The organism survives in the mucous layer that coats the epithelium, and causes chronic inflammation of the mucosa. • Transmission is by person to person contact, and probably also by contaminated water and food. Diseases H. pylori causes gastritis and peptic ulcers. Infection with H. pylori is a risk factor for gastric carcinoma. 218 02/23/2022 Amsalu M.
  • 219. Virulence factors  Vaculating toxin (Vac A): has been associated with pore formation in host cell membranes, the loosening of the tight junctions between epithelial cells, thus affecting mucosal barrier permeability.  Cytotoxin A (Cag A) : gene is a marker of increased risk of both peptic ulceration and gastric malignancy. 219 02/23/2022 Amsalu M.
  • 220. Pathogenesis and clinical manifestations • Multiple factors can contribute to the gastric inflammation, alteration of gastric acid production and others. • Initial colonization is facilitated by blockage of acid production by a bacterial acid inhibitory protein and neutralization of gastric acids by the ammonia produced by bacterial urease activity. – Urease : urea → CO₂ + NH₃ • The actively motile Helicobacter can then pass through gastric mucus and adhere to the epithelial cells. • Localized tissue damage is mediated by urease by products, mucinase, phospholipase and the activity of vaculating cytotoxin that induce epithelial cell damage. 220 02/23/2022 Amsalu M.
  • 221. Treatment Combination of a proton pump inhibitor (e.g. omeprazole) and one or more antibiotics (e.g. tetracycline, clarithromycin, amoxicillin, and metronidazole). • usually selected from clarithromycin, metronidazole, amoxicillin and tetracycline), combined with acid suppressants and/or bismuth compounds. Prevention and control • Improving sanitary hygiene 221 02/23/2022 Amsalu M.
  • 223. 223 HYPERSENSITIVITY REACTIONS & AUTOIMMUNITY • An Immune response eliminates antigen without extensively damaging the host’s tissue. Under certain circumstances, however, this response can have deleterious effects, resulting in significant tissue damage or even death. This inappropriate/heightened immune response is termed hypersensitivity or allergy.
  • 224. 224 Hypersensitive Reactions cont… Although the word hypersensitivity implies an increased response, the response is not always heightened but may, instead, be an inappropriate immune response to an antigen. Hypersensitive reactions may develop in the course of either humoral or cell-mediated responses. Hypersensitivity reactions require pre-sensitized (immune) state of the host .
  • 225. 225 Classification of Hypersensitivity Based on mechanics involved and time taken for the reactions hypersensitivity reactions are four types : – Type I (Anaphylactic) Reactions – Type II (Cytotoxic) Reactions – Type III (Immune Complex) Reactions – Type IV (Cell-Mediated) Reactions • The first three occur within the humoral branch and are mediated by antibody or antigen-antibody complexes. • A fourth type depends on reactions within the T cell- mediated branch, and is termed delayed-type hypersensitivity, or DTH(type IV).
  • 226. 226 A. Type I (Anaphylactic) Reactions • It is also known as immediate hypersensitivity. • The reaction takes 15-30 minutes from the time of exposure to the antigen. • Type I hypersensitivity is mediated by IgE. • The primary cellular component in this hypersensitivity is mast cell or basophil. • The mechanism of reaction involves preferential production of IgE.
  • 227. 227 Type I reactions can be systemic or localized Systemic anaphylaxis • Is a shock-like and often fatal state whose onset occurs within minutes of a type I hypersensitive reaction. • A wide range of antigens have been shown to trigger this reaction in susceptible humans, including the venom from bee, ant stings, drugs such as penicillin, insulin, and antitoxins • If not treated quickly, these reactions can be fatal. • Epinephrine is the drug of choice for systemic anaphylactic reactions.
  • 228. 228 Localized anaphylaxis (atopy) In localized anaphylaxis, the reaction is  limited to a specific target tissue or organ,  Often involving epithelial surfaces at the site of allergen entry. The tendency to manifest localized anaphylactic reactions is inherited and is called atopy. Atopic allergies include:  Allergic rhinitis (hay fever)  Asthma  Atopic dermatitis (eczema)  Food allergies
  • 229. 229 Type II (Cytotoxic) Reactions • Mediated, primarily, by antibodies of IgM or IgG class and complement • The reaction time is minutes to hours. • Affect a variety of organs and tissues. • The antigens are normally endogenous, although exogenous chemicals (haptens) that can attach to cell membranes can also lead to type II hypersensitivity.
  • 230. 230 • It includes: – blood transfusion reactions – drug-induced hemolytic anemia – Hemolytic Disease of the Newborn • Hemolytic disease of the newborn develops when maternal IgG antibodies specific for fetal blood-group antigens cross the placenta and destroy fetal red blood cells. • Caused by Rh incompatibility
  • 231. 231 Type III (Immune Complex) Reactions  When large amounts of antigen bind to antibody, immune complexes can form.  Mediated by neutrophils  If antigen is in excess, large complexes form; because these are not easily cleared by the phagocytic cells, they can cause tissue-damaging  The antibodies here are mostly of IgG class, although IgM may also be involved.  The antigen may be exogenous (chronic bacterial, viral or parasitic infections), or endogenous  e.g., systemic lupus erythematous-SLE).
  • 232. 232 Type IV (T Cell-Mediated) Reactions  Delayed type hypersensitivity (DTH) response does cause extensive tissue damage mediated by T cells  In many cases tissue damage is limited  The response plays an important role in defense against intracellular pathogens and contact antigens.  The hallmarks of a type IV reaction are the delay in time required for the reaction to develop and the recruitment of macrophages  DTH response is important in host defense against parasites and bacteria that live within cells, where circulating antibodies cannot reach them.
  • 233. 233 • Generally, the pathogen is cleared rapidly with little tissue damage. • However, in some cases, especially if the antigen is not easily cleared, a prolonged DTH response can itself become destructive to the host as the intense inflammatory response develops into a visible granulomatous reaction. • Macrophages present intracellular antigens to T cells and T-helper 1 cells (Cytotoxic T lymphocytes) release toxins that harm tissues • Example: contact dermatitis to nickels, gold jewelries etc.
  • 234. Introduction to Vaccines 234 • Vaccines: biological substances that stimulate the person’s immune system to produce an immune response identical to that produced by the natural infection. • Vaccines can – prevent the debilitating and, in some cases, fatal infectious diseases. – help to eliminate the illness and disability of polio, measles, and rubella
  • 235. 235 • Vaccines protect the – vaccinated individual, – protect society. • A community with many vaccinated people – protects the few who cannot be vaccinated—such as young children. – indirectly protects unvaccinated from exposure to disease = HERD IMMUNITY • Aim of an ideal vaccine: – To produce the same immune protection with the natural infection but without causing disease – To generate long-lasting immunity – To interrupt spread of infection
  • 236. Criteria for Effective Vaccines 236 • The World Health Organization (WHO) has stated that the ideal vaccine would have the following properties: – Affordable worldwide – Heat stable – Effective after a single dose – Applicable to a number of diseases – Administered by a mucosal route – Suitable for administration early in life
  • 237. Active and Passive Immunization 237 • Active immunity- immunity produced in response to natural infection • Many diseases stimulate an immune response in host, those who survive the disease are protected from second infection – naturally acquired active immunity • Artificially acquired active immunity - is stimulated by initial exposure to specific foreign macromolecules through the use of vaccines, to artificially establish state of immunity. • Passive immunity- immunotherapy (administration of immunity components from external source)
  • 238. 238 • Artificially acquired passive immunity- The individual receives protective molecules (anti bodies) or cell (lymphocytes) produced in another individual. • Naturally acquired passive immunity- refers to antibodies transferred from mother to fetus across the placenta and to the newborn in colostrum and breast milk during the first few months of life. • Passive immunization does not activate the immune system, it generates no memory response and the protection provided is transient/temporary
  • 240. Types of vaccines 240 • Many common vaccines use – Inactivated (killed) organisms, but still antigenic – Attenuated-live/altered/weakened microorganisms. Lost their pathogenicity due to cultured in abnormal conditions – Specific substances (e.g., protein, polysaccharide) from pathogen, capable of producing an immune response
  • 241. 1. Whole-Organism Vaccines 241 • Attenuated organisms: they lost ability to cause significant disease (pathogenicity) but to attenuate, grow a pathogenic bacterium or virus for prolonged periods under abnormal culture conditions – retains capacity for short term growth within inoculated host. – capacity for transient growth, permits prolonged immune-system exposure to attenuated epitopes, increased immunogenicity and production of memory cells. • As a consequence, these vaccines often require only a single immunization. • A major disadvantage is the possibility that they will revert to a virulent form. • Whole killed organisms- they are killed but still immunogenic
  • 242. 2. Purified Macromolecules 242 • Derived from pathogens. • Are specific, purified macromolecules . • Avoid some risks associated with attenuated or killed whole organism vaccines. • Three general forms of such vaccines are in current use: – inactivated exotoxins, – capsular polysaccharides, and – recombinant microbial antigens/DNA molecules
  • 243. 243 • Isolate gene encoding immunogenic protein, clone it, and express/insert in bacterial, yeast, or mammalian cells using recombinant DNA technology. • Example for human use is hepatitis B vaccine developed by cloning the gene for surface antigen of hepatitis B virus (HBsAg) and expressing it in yeast cells. • The recombinant yeast cells are grown in large fermenters, and HBsAg accumulates in the cells. • The yeast cells is disrupted, releases the recombinant HBsAg, which is purified by biochemical techniques.
  • 245. GRAM POSITIVE COCCI • There are two most important genera of gram-positive cocci. –Genus Staphylococci –Genus Streptococci 245 02/23/2022 Amsalu M.
  • 246. GENUS: STAPHYLOCOCCI Characteristics: Gram positive non spore-forming, spherical cells, usually arranged in grape-like clusters (in group) Can readily grow in ordinary media under aerobic and micro- aerophilic conditions , grow most rapidly at 370c relatively resistant to drying , heat, and 9% Nacl, thus can persist for long periods on fomites Produce catalase, which differentiate them from the streptococci. Some of them are normal flora of the skin and mucus membrane of human 246 02/23/2022 Amsalu M.
  • 247. Cont… The pathogenic staphylococci often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins The genus Staphylococcus has more than 35 species. The three main species of clinical importance are: Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. Less common staphylococcal species: Staphylococcus lugdenensis, Staphylococcus hominis & Staphylococcus warneri 247 02/23/2022 Amsalu M.
  • 248. Staphylococcus aureus Epidemiology: • Produce coagulase, which differentiate them from the the other staphylococci species. • Present in most environments frequented by humans • Carriage rate for healthy adults is 20-60%. • Carriage is mostly in anterior nares, skin, nasopharynx, • Predisposition to infection include: – poor hygiene and nutrition, tissue injury, preexisting primary infection, diabetes, immunodeficiency. – Increase in community acquired methicillin resistance - MRSA 248 02/23/2022 Amsalu M.
  • 249. 249 Virulence factors of S. aureus Enzymes: • Coagulase – coagulates plasma and blood; produced by 97% of human isolates; diagnostic • Hyaluronidase – digests hyaluronic acid of connective tissue • Staphylokinase – digests blood clots • DNase – digests DNA • Lipases – digest oils; enhances colonization on skin • Penicillinase – inactivates penicillin 02/23/2022 Amsalu M.
  • 250. 250 Virulence factors of S. aureus Toxins: • Hemolysins (α, β, γ, δ): 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-1 (TSST-1) - induces fever, vomiting, shock, systemic organ damage 02/23/2022 Amsalu M.
  • 251. S. aureus disease Range from localized to systemic Localized cutaneous infections – invade skin through wounds, follicles, or glands – folliculitis – superficial inflammation of hair follicle; usually resolved with no complications but can progress – furuncle –inflammation of hair follicle or sebaceous gland progresses into abscess or 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 251 02/23/2022 Amsalu M.
  • 252. 252 (a) Superficial folliculitis in which raised, domed pustules form around hair follicles. (b) In deep folliculitis the microorganism invades the deep portion of the follicle and dermis. (c) A furuncle arises when a large abscess forms around a hair follicle. Folliculitis: Infection of hair follicle. 02/23/2022 Amsalu M.
  • 253. 253 (d) A carbuncle consists of a multiocular abscess around several hair follicles. 02/23/2022 Amsalu M.
  • 254. Impetigo: Crusted superficial skin lesion 254 lesions of impetigo. This disease is characterized by isolated pustules that become crusted. (e) Impetigo on the neck of 2-year-old male. 02/23/2022 Amsalu M.
  • 255. 255 Toxigenic disease – food intoxication – ingestion of heat stable enterotoxins; results in gastrointestinal distress – staphylococcal scalded skin syndrome – toxin induces bright red blisters, then desquamation of the epidermis – toxic shock syndrome – toxemia leading to shock and organ failure Systemic infections – Osteomyelitis – infection of bone – Endocarditis : Infection of heart tissue – Pneumonia: Infection of lung parenchyma. 02/23/2022 Amsalu M.
  • 256. S. epidermidis and S .saprophyticus  They are opportunistic pathogens.  They are coagulase negative  S. epidermidis is frequently associated with endocarditis, bacteremia, wound infection and urinary tract infection (UTI).  S. saprophyticus is now recognized as an important cause of UTI. staphylococcal species are important agents of hospital- acquired infections associated with the use of implanted prosthetic devices and catheters. 256 02/23/2022 Amsalu M.
  • 257. Treatment and prevention Penicillin sensitive staphylococci: penicillin, ampicillin Penicillin resistant staphylococci: cloxacillin, oxacillin, floxacillin Methicillin resistant staphylococci: Vancomycin  Prevention and control Source of infection is shedding human lesions, the human respiratory tract and skin contact spread of infection occur in hospitals. Treatment of nasal carriers with topical antiseptics or Rifampicin and anti-staphylococcal drug. There is no effective vaccine against S. aureus. Infection control procedures, such as barrier precautions and disinfection of hands and fomites, are important in the control of nosocomial S. aureus epidemics. 257 02/23/2022 Amsalu M.
  • 258. GENUS: STREPTOCOCCI Characteristics: They are non-motile, non-sporeformer, gram-positive facultative anaerobes.  Spherical or oval cells characteristically forming chains or pairs during growth Grow well on media enriched with blood, serum or glucose. Some species and strains are capsulated. Sensitive to drying, heat and disinfectants They are widely distributed in nature and are found in upper respiratory tract, gastrointestinal tract and genitourinary tract as normal microbial flora.
  • 259. Cont… Classification of streptococci • Can be classified based on: 1. Hemolytic pattern as beta, alpha, and gamma hemolytic on blood agar. 2. Serological property (antigenic composition of group specific cell wall substance): Lancefield grouping - A to H and K to V 3. Biochemical (physiological) property. 259 02/23/2022 Amsalu M.
  • 260. Species Lance field group Hemolysis Biochemical property S. pyogenes A beta Bacitracin sensitive S. agalactiae B beta Bacitracin resistant E. faecalis D Alpha or beta, Growth in 6.5%NaCl or gamma S. bovis D Alpha or beta No growth in 6.5%NaCl S. pneumoniae no alpha Bile soluble, sensitive to optochin Viridian group no alpha Not bile soluble, resistant to optochin 260 02/23/2022 Amsalu M.
  • 261. Lancefield grouping of streptococci: Streptococci produce group specific carbohydrates identified using group specific antiserum. It is designated A-H and K-V. The clinically important streptococci are grouped under A, B, C, D, F and G. NB: Viridans streptococci and Streptococci pneumoniae are not grouped under Lancefield Classification. 261 02/23/2022 Amsalu M.
  • 262. Human Streptococcal Pathogens • S. pyogenes • S. agalactiae • Viridans streptococci • S. pneumoniae • Enterococcus faecalis 262 02/23/2022 Amsalu M.
  • 263. Group A streptococci (S. pyogenes) The most pathogenic member of the genus. It is present as a commensal in the upper respiratory tract (nasopharynx) in a variable proportion of healthy individuals and on the skin. The pathogen is spread from person to person through respiratory droplets. It produces different types of enzymes and exotoxins. 263 02/23/2022 Amsalu M.
  • 264. 264 • Produces surface antigens: – C-carbohydrates – protect against lysozyme – Fimbriae - adherence – M-protein – contributes to resistance to phagocytosis • Extracellular enzymes streptokinase – digests fibrin clots hyaluronidase – breaks down connective tissue DNase – hydrolyzes DNA Virulence Factors 02/23/2022 Amsalu M.
  • 265. 265 Virulence Factors • Extracellular toxins: streptolysins – hemolysins streptolysin O (SLO) and streptolysin S (SLS) – both cause cell and tissue injury pyrogenic toxin (erythrogenic) – induces fever and typical red rash superantigens – strong monocyte and lymphocyte stimulants; cause the release of tissue necrotic factor 02/23/2022 Amsalu M.
  • 266. Diseases cause S. pyogenes Skin infections Impetigo (pyoderma) – superficial lesions • is a small vesicle surrounded by an area of erythema. • The vesicle enlarges over a period of days, becomes pustular, and eventually breaks to form a yellow crust. • is associated with insect bites, poor hygiene, and crowded living conditions 266 02/23/2022 Amsalu M.
  • 267. Streptococcal pharyngitis – strep throat • is most frequent between the ages of 5 and 15 years • is characterized by acute sore throat, malaise, fever, and headache. • Infection typically involves the tonsillar pillars, and soft palate, which become red, swollen, and covered with a yellow-white exudate. 267 02/23/2022 Amsalu M.
  • 270. 270  Necrotizing fasciitis (Streptococcal gangrene): Extensive and rapidly spreading necrosis of skin and subcutaneous tissue. Necrotizing fasciitis The disease,as shown here on a thigh, is commonly produced by so-called flesh-eating Streptococcus pyogenes. 02/23/2022 Amsalu M.
  • 271. Systemic infections • Scarlet fever • Septicemia • Pneumonia • Streptococcal toxic shock syndrome 271 02/23/2022 Amsalu M.
  • 272. Long-Term Complications of Group A Infections Acute glomerulonephritis: occurs usually following a streptococcal infection of the skin. There is inflammation of the kidney due to deposition of immune complex in the glomeruli. It clinically manifests with generalized body edema, elevated blood pressure, protein and blood in the urine  Acute rheumatic fever – Due to immunological damage to the heart valves and muscle following Streptococcal upper respiratory tract infection. – It clinically presents with fever, malaise, migratory non-suppurative polyarthritis, carditis 272 02/23/2022 Amsalu M.
  • 273. Group B: Streptococcus agalactiae • Regularly resides in human vagina, pharynx and large intestine • Can be transferred to infant during delivery and cause severe infection – most prevalent cause of neonatal pneumonia, sepsis, and meningitis – Pregnant women should be screened and treated. • Wound and skin infections and endocarditis in debilitated people 273 02/23/2022 Amsalu M.
  • 274. Group D Enterococci • Group D: – they are commensals of the GIT – they can be enterococci (E. faecalis) or non-enterococci (S. bovis) – cause opportunistic urinary, wound, and skin infections, particularly in debilitated persons 274 02/23/2022 Amsalu M.
  • 275. Viridans Streptococci • Large complex group – Streptococcus mutans, S. oralis, S. salivarus, S. sanguis, S. milleri, S. mitis • Most numerous and widespread residents of the gums and teeth, oral cavity and also found in nasopharynx, genital tract, skin • Not very invasive; but dental or surgical procedures facilitate their entrance 275 02/23/2022 Amsalu M.
  • 276. Viridans Group • Bacteremia, meningitis, abdominal infection, tooth abscesses • Most serious infection – subacute endocarditis – blood- borne bacteria settle and grow on heart lining or valves • S. mutans produce slime layers that adhere to teeth, basis form plaque and cause dental caries. • Persons with preexisting heart conditions should receive prophylactic antibiotics before surgery or dental procedures. 276 02/23/2022 Amsalu M.
  • 277. Pneumococci (S. pneumoniae) – Are fastidious lancet shaped gram positive cocci arranged in pairs (diplococci) – they are facultative anaerobes – Possess a capsule of polysaccharide that permits typing with specific antisera (more than 80 serotypes) Epidemiology – S. pneumoniae can be found as a commensal in the upper respiratory tract. – Most infections are caused by endogenous spread from the colonized nasopharynx. 277 02/23/2022 Amsalu M.
  • 278. Virulence factors • Capsule interferes with phagocytosis • Pneumolysin disrupts cells and cilia • Other Virulence – Pneumococcal surface protein A (PspA: interferes with complement action 278 02/23/2022 Amsalu M.
  • 279. Clinical syndrome • Pneumococci cause pneumonia, meningitis, endocarditis, Otitis media, bacteremia and infection of the upper respiratory tract. • The following factors lower host resistance and predispose to pneumococcal infection: Alcohol or drug intoxication or other cerebral impairment that can depress the cough reflex and aspiration or secretion. Abnormality of the respiratory tract Trauma to the head that causes leakage of spinal fluid through the nose predisposes to pneumococcal meningitis. 279 02/23/2022 Amsalu M.
  • 280. Treatment, prevention and control – drugs of choice are penicillin, erythromycin and cotrimoxazole. – immunization with polyvalent polysaccharide vaccine provides long lasting immunity (at least 5 years). 280 02/23/2022 Amsalu M.
  • 282. Gram negative cocci Family Neisseriaceae
  • 283. GRAM NEGATIVE COCCI GENUS: NEISSERIA • Characteristics: – They are non-motile, gram negative intracellular diplococci – rapidly killed by drying, sunlight, heat, and disinfectants. – Each cocci is kidney-shaped – grow best on complex media under aerobic conditions containing 5% CO2 – Oxidase positive. The main species of medical importance are: N. meningitidis N. gonorrhoeae. 283 02/23/2022 Amsalu M.