Basics of Medical Microbiology PDF

Faculty of Applied Medical Science October 6 University Basics of Medical Microbiology By Ass Prof Dr/ Amal Sabry Othman Dr/ Nashwa Abbas Ahmed Contents...

Faculty of Applied Medical Science October 6 University Basics of Medical Microbiology By Ass Prof Dr/ Amal Sabry Othman Dr/ Nashwa Abbas Ahmed Contents 2  Title page Introduction…………………………………………………………1 Taxonomic position of microorganism………………………….….2 Cell structure and function………………………….………………6 Bacterial growth and physiology……………………………….…17 Media for bacterial growth……………………………….………. 24 Bacterial viruses……………………………………………………27 Antimicrobial chemotherapy………………………………………31 The control of microorganisms……………………………….…...43 Bacterial genetics………………………………………………….52 Pathogenesis and microbial infection……………………………..63 References……………………………………………………….73 CHAPTER (1) Introduction To Microbiology 3  (Taxonomic position of Microorganisms) OBJECTIVES __________________________________ Upon completion of this chapter, the reader should be able to: 1. Explain the nomenclature system used for all living things. 2. Explain how relatedness of organisms is determined. Microbiology: is the study of microorganisms, which are minute in size (unicellular or cell-cluster) microscopic organisms. Classification of bacteria Taxonomy is the science of classification, identification, and nomenclature. Classification is the orderly arrangement of bacteria into groups. There is a hierarchy to the organization. The broadest groups are kingdoms and the smallest subsets are species. Kingdom Division Class Order Family Genus Species Subspecies (biotype, serotype, phagetype) All organisms are given two names, genus and species. This nomenclature system derives the names from Latin and Greek. The genus name is always capitalized while the species name never is The old system of classification was based on phenotypic characteristics. Bacteria were classified into: 4 1- Higher bacteria: i.e. actinomyces (filamentous branching organism)2- Lower bacteria: Simple unicellular organisms classified on the basis of: a- Shape ( fig. 2). Cocci: mono, diplo, chain, tetrads or clusters. Bacilli: rod-shaped. Vibrios: Comma-sheped. Spirilla: Spiral-shaped. Fig. (2) Typical shapes and arrangements of bacterial cells. b- ability to form spores. c- Method for energy production: glycolysis for anaerobes and cellular respiration for aerobes. d- Nutritional requirements e- Reaction to Gram stain: Gram positive (violet color) and Gram negative (pink color) bacteria according to the different kind of bacterial cell walls. 5 f- Pathogenicity: Saprophytes: live on dead material, soil, water, dust…etc. They never cause disease. Parasitic: live in the body of living organism. It may be Pathogenic: cause disease. e.g. Mycobacterium tuberculosis. or Non pathogenic: commensal bacterial flora e.g. Staphylococcus epidermidis (on the skin), E. coli (in the intestine). They are found in healthy individuals. They live on skin, in mouth, throat, GIT…etc. They are non pathogenic and may be benificial to body under the normal conditions. If the body defense or immunity are lowered, they can be introduced into different body sites causing dieases. They are considered as opportunistic pathogens The new system of classification is based on genotypic characteristics which introduce new criteria for bacterial rlatedness. e.g.: 1- Nucleotide base composition: The parameter most often used is the mol percent of guanine plus cytosine (G+C) in the total DNA. For any one species, the G+C content is relatively fixed. 2- Nucleic base homology: organisms can be classified into groups on the basis of the homology of their DNA base sequences. 6 3- Genome squencing: It is based on sequence homologies in ribosomal RNA. It has provided new insights into the evolutionary relationships among the bacteria. CHAPTER (2) Cell Structure And Function OBJECTIVES ______________________________ Upon completion of this chapter, the reader should be able to: 1-Describe the differences between prokaryotes and eukaryotes. 7  2-Describe the structure of the cell wall and its differences in gram- negative and gram-positive bacteria. There are two main types of cell in the world – the simpler cells such as bacteria are prokaryotic cells, and cells like that of fungi, protozoa, algae and slime molds are eukaryotic cells. The difference is that a eukaryotic cell has a nucleus, a membrane-bound section which holds the cell's main DNA. N.B: Viruses, are not strictly classed as living organisms. They depend on their host for living and replication. They are the smallest infective agent. Fig.(1) and table (1) show the differences between prokaryotic and eukaryotic cells. Prokaryotes Fig. (1) Structure of prokaryotic and eukaryotic cells. Table (1): The differences between prokaryotic and eukaryotic cells Prokaryotes Eukaryotes 1- cell wall Present Absent (except in mycoplasma) 8 2-cytoplasmic No sterol Contain membrane contain sterol- mesosome lipoprotein and No lipoprotein mesosome 3- Cytoplasmic structure: Mitochondria Absent Present 4-Rough Absent Present endoplasmic reticulum 5-Nucleus Absent Has Nucleoli Single Has chromosome Multiple chromosome No histones Histones associated with DNA 6- Division Simple Mitosis binary division 7- Size Relatively Larger small Example Bacteria Fungi, human cell Bacteria are unicellular organisms. Their cell consists of three essential components: 1- Bacterial genome (chromosome and genetic materials) 2- Cytoplasm 3- Cell wall Non essential components: capsules, flagella, pili and endospores. (Fig. 2) 9  Fig. (2): Bacterial cell structure A-Cell wall: It is the outermost layer surrounding the cell membrane. Bacteria can be divided into two groups on the basis of staining with the Gram stain: Gram positive bacteria remain stained by crystal violet on washing. Gram negative do not. All bacteria have a cell membrane where oxidative phosphorylation occurs (since there are no mitochondria). Cell wall function: 1-Maintain the shape of the cell 2- Support the weak cytoplasmic membrane 3-play a role in cell division 4- Responsible for staining properties of the organisms. 5- It is rigid and protects the cell from osmotic lysis. In Gram positive bacterial cell wall, the cell wall peptidoglycan layer is a much thicker layer than in Gram negative bacteria. It is formed of alterating N-acetyl muramic 10 acid and N-acetyl glucosamine.They are connected by chain of 4 amino acids (tetrapeptides) cross linked by identical penta peptides. (Fig 2) It is the site of action of penicillins, cephalosporins and lysozyme enzyme. Teichoic acids: Polymers of glycerol phosphate or ribitol phosphate located in the outer layer of gram positive cell wall. Fig (3): Bacterial cell wall cross linking Gram negative bacterial cell wall: 1- Have an additional outer membrane (OM). The outer membrane is the major permeability barrier in Gram negative bacteria. It is composed of lipopolysaccharide (LPS) called endotoxin with special channels consist of protein molecules called porins. 11 2- The space between the inner and outer membranes is known as the periplasmic space in which degradative enzymes are stored.( Fig 3) 3- A thin layer of peptidoglycan Gram positive bacteria lack a periplasmic space; instead they secrete exoenzymes and perform extracellular digestion. Digestion is needed since large molecules can not readily pass across the outer membrane (if present) or cell membrane. Fig (3): Gram negative cell wall Cell wall of acid-fast bacteria: Mycobacteria have unusual cell wall can not be gram stained due to high lipid content (fatty mycolic acids) of the cell wall. Wall-less forms of Bacteria: I- Mycoplasma: The only group of bacteria that exist naturally without cell wall. It has no defined shape due to lacking the rigid cell wall. It is resistant to antibiotics which destroy bacterial cell wall. II-When bacteria are treated with 12 1) enzymes that are lytic for the cell wall e.g. lysozyme or 2) antibiotics that interfere with biosynthesis of peptidoglycan, wall-less bacteria are often produced. Usually these treatments generate non-viable organisms. Wall-less bacteria that can not replicate are referred to as spheroplasts in gram negative bacteria or protoplasts in gram positive bacteria. Wall-less bacteria that can replicate are called (L forms). B- Cytoplasmic membrane: It lies just inside the peptidoglycan layer. It is a phospholipid bilayer containing protein. Function: 1- selective permeability: Molecules move across the membrane by simple diffusion or active transport that require energy to transport molecules against concentration gradient. 1- Respiration and energy production 2- Excretion of toxic and hydrolytic enzymes outside the cell. 3- Biosynthesis of cell wall and other proteins 4- It playes an important role during cell division (it is the site of DNA attachment during replication and by forming the transverse septum). c-Cytoplasm: It is a viscous watery solution or soft gel that contains a variety of organic and inorganic solutes. a- Mesosomes: 13 They are invaginations of cytoplasmic membrane Function: They increase the surface area of the membrane that increase the efficiency of permeability and active transport. Site of attachment of DNA during cell division. Site of respiratory activity of the cell. b- Ribosomes: These are the site of protein synthesis. Composed of 60% RNA and 40% protein. Have sedimentation constant of 70S being composed of 2 subunits, small 30S and large 50S subunits. The 2 subunits separate except during protein synthesis where they aggregate to form polyribosomes. c- Inclusion granules: Round granules present in the cytoplasm Store energy or reserve nutrients concerned with cell metabolism. D-Bacterial cell Genome: It is the total gene content of a cell. It consists of: I-Nucleoid: It forms the bacterial chromosome on which 2000 gene responsiple for growth and multiplication are carried. 14  Consists of DNA and small amount of RNA and RNA polymerase. It is a single circular double stranded DNA. II- Plasmid: Extrachromosomal double stranded circular DNA Capable of replication independent of the bacterial chromosome. Can transefer to other bacteria by conjugation, transformation or transduction. III- Transposons Also called jumping genes They are pieces of DNA that move from one site to another within or between the DNAs of bacteria, plasmids or bacteriophages.. These elements move within the bacterial cell and can be transferred to other cells. There is documen- tation that the gene for vancomycin resistance has been transferred from Enterococcus to Staphylococcus aureus. Non essential components (Structures outside the cell wall): a-Capsule: These are structures surrounding the outside of the cell envelope. They usually consist of polysaccharide; however, in certain bacilli they are composed of a polypeptide (polyglutamic acid). They are not essential to cell viability and some strains within a species will produce a capsule, whilst others do not. 15  Capsules of pathogenic bacteria inhibit ingestion and killing by phagocytes. Capsules are often lost during in vitro culture. b-Flagella Some bacterial species are mobile and possess locomotory organelles - flagella (Fig. 4). Those that do are able to taste their environment and respond to specific chemical foodstuffs or toxic materials and move towards or away from them (chemotaxis). Flagella are embedded in the cell membrane, extend through the cell envelope and project as a long strand. Flagella consist of a number of proteins including flagellin. They move the cell by rotating with a propeller like action. Axial filaments in spirochetes have a similar function to flagella. Flagella are complex structures that give the bacterium motility. Flagella can be located at one end (monotrichous) of the cell, at both ends (lophotrichous), or around the cell (peritrichous). Fig (5). Fig (5): Arrangement of flagella c-Pili (fimbriae): Pili are hair-like projections of the cell. 16  Some are involved in sexual conjugation and others allow adhesion to host epithelial surfaces in infection. Fig (6) Fig (6): Pili d-Endospores (spores) These are a dormant form of a bacterial cell produced by certain bacteria when starved. The spore is resistant to adverse conditions (including high temperatures and organic solvents). The spore cytoplasm is dehydrated and contains calcium dipicolinate which is involved in the heat resistance of the spore. Spores are commonly found in the genera Bacillus and Clostridium. 17  CHAPTER (3) Bacterial Growth And Physiology OBJECTIVES ______________________________ Upon completion of this chapter, the reader should be able to: 1-Describe the differences between autotrophic and heterotrophic bacteria. 2-recognize the effect of temperature, pH, light……on bacterial growth. 3-describe the bacterial growth curve with correspondence to human infection Bacterial growth is measured as an increase in numbers rather than size. The conditions necessary for reproduction of bacteria are highly variable. Growth requirements: Bacteria have certain requirements (source of energy, mineral salts, suitable temperature, suitablr pH, and suitable concentration of certain gases O 2 /CO 2 ) A-Nutrients: Bacteria are classified into: 1-Autotrophic bacteria: Can utilize simple organic materials e.g. CO 2 as a source of carbon and ammonia as source of nitrogen and form complex organic metabolites from these simple materials. 18  They can obtain energy from light (photosynthetic bacteria) or from chemical reactions by oxidation of inorganic materials (chemolithotrophic bacteria). 2-Heterotrophic bacteria: They can not synthesize complex organic substance from simple inorganic materials. They drive their energy by oxidation or fermentation of organic compounds Most of them are of medical importance. Physical conditions for growth Growth factors are organic compounds essential for growth of bacteria which is unable to synthesize from available nutrients. Growth factors are organized into three categories. 1. purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA) 2. amino acids: required for the synthesis of proteins 3. vitamins: needed as coenzymes and functional groups of certain enzymes B-The Effect of Oxygen Bacteria are classified into 4 groups according to oxygen requirements (fig. 1) 1- Obligate aerobes require O 2 for growth; they use O 2 as a final electron acceptor in aerobic respiration. e.g. Mycobacterium tuberculosis 19 2- Obligate anaerobes (occasionally called aerophobes) do not need or use O 2 as a nutrient. In fact, O 2 is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation or anaerobic respiration.e.g. Closteridium spp. 3- Facultative anaerobes (or facultative aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O 2 ) they grow by fermentation or anaerobic respiration, but in the presence of O 2 they switch to aerobic respiration. e.g. Staphylococcus spp. 4-Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O 2. They live by fermentation alone whether or not O 2 is present in their environment. e.g. Streptococcus spp. 5- Microareophilic: are bacteria that require oxygen levels below 0.2atmosphere (less O 2 and high CO 2 ) e.g. Neisseria spp. Figure 1. The relative growth requirements for oxygen. The medium without bacteria is yellow, while bacterial growth is indicated by the green color. For example, obligate aerobes and anaerobes, grow respectively only at the top (A) and bottom (D) of a tube of liquid medium. Obligate aerobes often form a film (scum) or pellicle, that floats on the top of the liquid. Facultative aerobes have the best of both worlds, as they are able to grow under both aerobic and anaerobic 20 conditions (C). Microaerophilic bacteria require a little bit of oxygen, but too much is toxic (B). C-The Effect of pH on Growth Acidophiles:.Microorganisms which grow at an optimum pH well below neutrality (7.0) are called Neutrophiles Those which grow best at neutral pH. Alkaliphiles those that grow best under alkaline conditions. D-The Effect of Temperature on Growth Microorganism will exhibit a range of temperature over which it can grow, defined by three cardinal points in the same manner as pH. Mesophiles: Organisms with an optimum temperature near 37 degrees (the body temperature of warm-blooded animals). Thermophiles: Organisms with an optimum temperature between about 45 degrees and 70 degrees. Extreme thermophiles or hyperthermophiles: Some Archaea with an optimum temperature of 80 degrees or higher and a maximum temperature as high as 115 degrees. Psychrophiles: The cold-loving organisms defined by their ability to grow at 0 degrees. Psychrotroph: Are psychrophile which usually has an optimum temperature of 10-15 degrees. E-Water Availability 21 water activity : The availability of water for a cell in the atmosphere (relative humidity) or its presence in solution or a substance. The only common solute in nature that occurs over a wide concentration range is salt [NaCl]. Halophiles: Microorganisms that require some NaCl for growth. Mild halophiles require 1-6% salt moderate halophiles require 6-15% salt. extreme halophiles that require 15-30% NaCl for growth are found among the archaea. Halotolerant : Bacteria that are able to grow at moderate salt concentrations, even though they grow best in the absence of NaCl. osmophiles is usually reserved for organisms that are able to live in environments high in sugar. Xerophiles: Organisms which live in dry environments (made dry by lack of water). F-Light: Darkness provide a favourable condition for bacterial growth and viability. 22 Bacterial reproduction Reproduction Occurs by binary fission - one cell splits into two cells, offspring are genetically identical to parent.(Fig. 2). Fig (2): reproduction by binary fission Generation time (doubling time): It is the time required by bacteria to double its number. Measurement of bacterial growth: Bacterial count: measures the number of bacteria. 1- Total cell count: number of living and dead bacterial cells. 2- Viable count: number of living bacterial cells only. 23 Growth Curves A growth curve of an organism in a closed environment, like a test tube of broth, can be made by graphing the time in hours (or days) versus the number of viable cells. It consists of four phases: (fig. 3). Lag phase, the organisms are shocked, and trying to adjust to the new environment. There are many changes in bacterial gene expression as enzymes and other proteins that are needed for growth are synthesized. This phase is clinically correspond to incubation period in human Log phase, conditions are very good. There is adequate nutrition, toxic waste products have not built up in the surroundings, and the cells grow as fast as their metabolism and the environment permits. This phase is clinically correspond to active disease or invasion period. Stationary phase, the appearance of new organisms is equal to the death rate. The bacteria are getting crowded, food is more limited, and waste products are building up. This phase is clinically correspond to active disease. Death phase. The death rate increase and exceed the multiplication ratedue to nutrient exhaustion. This phase is clinically correspond to convalescence period. Fig. (3): Bacterial growth curve 24  CHAPTER (4) Media for Bacterial Growth OBJECTIVES Upon completion of this chapter, the reader should be able to: 1.List the four types of media and give examples of each type. 2.Explain how the different types of media can be used to detect and isolate pathogens. 3. Explain how to inoculate culture media. 4. List the incubation options with respect to temperature and atmosphere. 5. Explain how enzyme tests can be used to identify organisms and list the use of the following tests: cata-lase, oxidase, indole, PYR, hippurate hydrolysis. 6. Explain turbidity and how it is measured. Cultivation is the process of growing organisms in vitro (in an artificial environment) and not in their actual in vivo site. When growing organisms in the laboratory, nutritional requirements must be considered. Some organisms are fastidious and cannot grow without a particular nutrient. Media can be in liquid (broth) or solid (agar) form. The indication of growth in broth is the development of turbidity or cloudiness. For bacteria to be visible to the naked eye, there need to be 106 bacteria per milliliter. One cell gives rise to a single colony. All cells in that colony have the same genetic and phenotypic characteristics. Cultures from this single colony are considered "pure." There are four categories of media: enrichment, supportive (nutritive), selective, and differential. Enrichment media: contain specific nutrients required for growth of a particular organism. Regan Lowe charcoal agar is enriched to support the growth of Bordetella pertussis. Supportive media: also called nutritive media, support most nonfastidious organisms. 25  Selective media: inhibit the growth of some organisms while allowing other groups to grow. CNA agar selects for gram-positive organisms by inhibiting the growth of gram-negative ones. Differential media: contain a factor that can be used to distinguish pertain characteristics. XLD agar differentiates Salmonella spp. and Shigella spp. from other enterics. Below are some artificial media routinely used in microbiology. This list is not complete. Chocolate agar—similar to blood agar except that cells have been lysed to release hemin and NAD that will support the growth of Haemophilus and Neisseria Columbia CNA with blood—addition of colistin and nalidixic acid suppresses the growth of most gram-negative organisms Hektoen enteric (HE) agar—bile salts and dyes selectively slow the growth of nonpathogenic enterics and allow Salmonella spp. and Shigella spp. to grow MacConkey agar—most frequently used selective and differential medium for gram-negative bacilli Sheep blood agar—supports all but most fastidious organisms Thayer-Martin agar—enriched and selective medium for N. meningitidis and N. gonorrhoeae These media will not support the growth of obligate intracellular parasites. Viable host cells are required for culture of chlamydia, rickettsiae, and rickettsiae-like organisms. There are several methods of providing optimum incubation conditions that include incubators, candle jars, and various atmosphere-generating systems. Most bacteria will grow within 24 to 48 hours, but some require longer incubation times. Putting a known amount of specimen on the plate allows quantitation of colony-forming units. This method is used for urine cultures. 26 Colonies are evaluated for morphology to provide preliminary information and to determine the need for subculture and organism identification. Evaluation of colonies includes: Type of media (selective, differential, nutrient) that support growth Relative quantities of each colony type to determine the predominant organism Colony characteristics that include size, pigmentation, odor, surface appearance, and changes in the agar (e.g., pitting or hemolysis) Gram stain of suspect colonies with subculture for additional pure growth, if necessary Once an organism has been isolated in culture, it must be identified. Two basic identification schemes are used. Identification is accomplished by genotypic characterization using one of the molecular methods available. Or identification is based on phenotypic observations of morphology and metabolic activities The more parameters tested for each organism, the better the probability of correct identification. This information is stored in computer databases for fast and easy access. Commercially available identification systems are widely used and take various formats. Conventional biochemical reactions have been miniaturized from test tubes into microtitre tray format Other identification systems are fully automated and have miniaturized this microtitre plate format into smaller "cards. CHAPTER (5) Bacterial Viruses 27  Objectives: Upon the completion of this chapter the student would be able to: 1-Explain the difference between viruses and bacteria 2- Explain how can virus infect specific bacteria Viruses are smaller and less complex than bacteria. Viruses Contain a protein coat Some are enclosed by an envelope Most viruses infect only specific types of cells in one host Host range is determined by specific host attachment sites and cellular factors Obligate intracellular parasites generally species specific hosts = invertebrates, vertebrates, plants, protists, fungi, bacteria, archaea Comparison: Viruses and Bacteria Viruses Bacteria size(nm) 24-1000nm avg. 1000-3000nm (0.5mm max) intracellular parasite yes no (exceptions) binary fission no yes DNA + RNA no (not both) yes ATP production no yes ribosomes no yes antibiotic sensitivity no yes can pass through filters yes no (exceptions) Helical Viruses Polyhedral VirusesEnveloped Viruses Bacterial viruses or bacteriophages are viruses that infect bacteria. Fig. (1) 28  Fig (1): Bacteriophage structure Growing Viruses Viruses must be grown in living cells. Bacteriophages form plaques on a lawn of bacteria. Animal viruses may be grown in living animals or in embryonated eggs. Animal and plants viruses may be grown in cell culture. Continuous cell lines may be maintained indefinitely. Virus Identification Cytopathic effects - Serological tests Detect antibodies against viruses in a patient. Use antibodies to identify viruses in neutralization tests, viral hemagglutination, and Western blot. Nucleic acids Multiplication of Bacteriophages (Lytic Cycle) Attachment: Phage attaches by tail fibers to host cell. Penetration: Phage lysozyme opens cell wall, tail sheath contracts to force tail core and DNA into cell. Biosynthesis: new particles of virus is formed Maturation: Assembly of phage particles. Release: by lytic or lysogenic Lytic cycle: Phage causes lysis and death of host cell. Lysogenic cycle: Prophage DNA incorporated in host DNA (replicate together). 29 A- Lytic cycle When viruses reproduce by the lytic cycle, they break open, or lyse, their host cells, resulting in the destruction of the host. Before viral infection the cell is busy replicating its own DNA and transcribing its own genetic information to carry out biosynthesis, growth and cell division. After infection, the viral DNA takes over the the biosynthetic machinery of the host cell and uses it to produce the parts needed for production of new virus particles. Viral DNA replaces the host cell DNA as a template for both replication (to produce more viral DNA) and transcription (to produce viral mRNA). Viral mRNAs are then translated, using host cell ribosomes, tRNAs and amino acids, into viral proteins such as the coat proteins. The process of DNA replication, synthesis of proteins, and viral assembly is a carefully coordinated and timed event. The overall process of lytic infection is diagramed in the figure below. Discussion of the specif steps follows. B-Lysogenic cycle Lysogenic or temperate infection rarely results in lysis of the bacterial host cell. Lysogenic viruses, such as lambda phage which infects E. coli, have a different strategy for their replication. After penetration, the virus DNA integrates into the bacterial chromosome and it becomes replicated every time the cell duplicates its chromosomal DNA during normal cell division Temperate viruses usually do not kill the host bacterial cells they infect. Their chromosome becomes integrated into a specific scection of the host chromosome. These bacteria are called lysogen 30  F b Fig. (3). Adsorption, penetration and injection of bacteriophage T4 DNA into an E. coli cell. 31  Chapter 6 Antimicrobial Chemotherapy Antimicrobial chemotherapy To kill or inhibit microbial infection using chemical (eg antibiotics) An antibiotic Large group of chemical substances, produced by microorganisms or made synthetically having the capacity to inhibit or destroy bacteria and other microorganisms in inhibit solutions, it is used in the treatment of infectious diseases. Bactericidal A substance capable of killing bacteria, upon its removal the bacteria can’t grow again. Bacteriostatic A substance capable of inhibiting the growth or multiplication of bacteria, upon the removal of bacteriostat the bacteria usually start to grow again. Sources of Antibacterial agents: Antibacterial agents may be Natural - mainly fungal sources Semi-synthetic - chemically-altered natural compound Synthetic - chemically designed in the lab The original antibiotics were derived from fungal sources. These can be referred to as “natural” antibiotics. Organisms develop resistance faster to the natural antimicrobials because they have been pre-exposed to these compounds in nature. Natural antibiotics are often more toxic than synthetic antibiotics. Benzylpenicillin and Gentamicin are natural antibiotics. Semi-synthetic drugs were developed to decrease toxicity and increase effectiveness. Ampicillin and Amikacin are semi-synthetic antibiotics. Synthetic drugs have an advantage that the bacteria are not exposed tothe compounds until they are released. They are also designed to have greater effectiveness and less toxicity. Moxifloxacin and Norfloxacin are synthetic antibiotics -2-  There is an inverse relationship between toxicity and effectiveness on moving from natural to synthetic antibiotics Spectrum activity of antibiotics - Broad spectrum antibiotics: They are active against wide range of bacteria eg: Tetracycline. - Narrow spectrum antibiotics: They are active against one or few types of bacteria eg: Vancomycin. - Antibiotics are usually classified based on their structure and/or their mechanism 1. Structure - molecular structure. - ß-Lactams - Beta-lactam ring - Aminoglycosides - vary only by side chains attached to basic structure 2. Mechanism- how the drug works or its mode of action. In these discussions, we will primarily use the functional classification. Mechanism of action of antibiotics: An ideal antimicrobial agent should have selective toxicity, it must kill or inhibit the growth of a microorganism in concentrations that are not harmful to the cells of the host. The mechanism of action of antibiotic must depend on the inhibition of the metabolic channel that is present in the microbe. Basic mechanisms of antibiotic action against bacteria -3-  Mechanism of action of antibiotics 1) Inhibition of cell wall synthesis: - The bacterial cell wall in an ideal point of attack by selective toxic agents. B-Lactams (eg Penicillin) inhibit the final steps in peptidoglycan synthesis while glycopeptides (eg: Vanconmycin) inhibits the early steps in peptidoglycan synthesis. 2) Alteration of cell membrane functions: - Some antibiotics cause disruption of the cytoplasmic membrane nucleotides leading to cell death eg :polymyxins. -4- 3) Inhibition of protein synthesis : - Antibiotics inhibit protein synthesis in bacteria without interfering with protein synthesis in human cells.this selectivity is due to the differences between bacterial and human ribosomal proteins bacteria have 70s ribosomes (with 50s and 30s subunits) while human cells have 80s ribosomes (with 60s and 40s subunits) chloramphenicol and erythromycin act on 50s subunits , while tetracycline and aminoglycosides act on 30s subunits.. 4) Inhibition of Nucleic acid synthesis : - They act on any of the steps of DNAor RNA replication.Quinolones and Novobiocin inhibit DNA by blocking DNA gyrase , while Rifampicin inhibits RNA synthesis by blocking RNA polymerase. Sulfonamides inhibit nucleotide synthesis. 5) Antimetabolite activity : - Antibiotics compete with an essential metabolite for the same enzyme.Para-amino benzoic acid(PABA) in an essential metabolite for many organisms. They use it as a precursor in folic acid synthesis which is essential for nucleic acid synthesis.Sulphonamides are structural analogues to PABA so they emits into the reaction in its place and compete of the active center of the enzyme thus inhibiting folic acid synthesis. -5- Resistance to Antimicrobial agents 1- Non genetic resistance : a- Metabolic inactivity Most antimicrobials act on replicating cells. Non replicating organisms are resistant to antibiotics, tubercle bacilli can survive in tissue for long time their resistance to antibiotic is due to their metabolic inactivity. b- Loss of target structure L- Forms of bacteria may lose their cell wall which is the target site of the drug. c- Intrinsic resistance Natural resistance of bacteria as the lack of the receptor sites of the drug. 2- Genetic acquired resistance: - a- Plasmid mediated resistance Resistance factors (R) are classes of plasmids that mediate resistance to antimicrobial agents. Plasmids carry genes that code for the production of enzymes that inactivate antimicrobial agents. b-Transposon mediated resistance Many transposons carry genes.Since they move between plasmids and chromosoms they can transfere this property to bacteria (transposition). d- Chromosomal drug resistance -6-  This develops as a result of spontaneous mutation in a gene that controls susceptibility to an antimicrobial agent. What is the Ideal Antibacterial? Selective target. Narrow spectrum – does not kill normal flora. Few adverse reactions – toxicity, allergy…. Various routes of administration. Good absorption. Good distribution to site of infection. Emergence of resistance is slow. Complications of antimicrobial chemotherapy 1-Development of drug resistance 2-drug toxicity 3-Super-infection 4-Hypersensitivity Chemoprophylaxis Chemoprophylaxis refers to the administration of a medication for the purpose of preventing disease or infection. ANTIBIOTIC SUSCEPTIBILITY TESTING Various methods of antibiotic susceptibility testing are: 1. Quantitative Methods 2. Qualitative Methods 3. Automated Susceptibility Tests -7- 4. Newer Non-Automated Susceptibility Tests 5. Molecular Techniques Quantitative Methods: In these tests, the minimum amount of antibiotic that inhibits the visible growth of an isolate or minimum inhibitory concentration (MIC) is determined. Bacterial isolate is subjected to various dilutions of antibiotics. The highest dilution of antibiotic that has inhibited the growth of bacteria is considered as MIC. These tests can be performed on broth or agar. 1. Broth dilution methods a. Macrobroth dilution MIC tests b. Microbroth dilution MIC tests 2. Agar dilution methods Macro-broth dilution tests: A serial two-fold dilution of antibiotic are made in test tubes from zero to maximum concentration that is achieved in vivo without toxic effect on patient. The inoculum density of bacterial isolate to be tested is standardized with 0.5 McFarland turbidity standard. The suspension should have a final inoculum of 5 x 105cfu/ml. 1ml of bacterial suspension is added to rows of antibiotic solution and incubated at 37oC overnight. The lowest concentration of antibiotic that completely inhibits visual growth of bacteria (no turbidity) is recorded as MIC. Micro-broth dilution tests: -8- A polystyrene tray containing 80 wells is filled with small volumes of serial two-fold dilutions of different antibiotics. The inoculum suspension and standardization is done according to McFarland standard. The bacterial inoculum is then inoculated into the wells and incubated at 37oC overnight. MIC is determined as in macro-broth dilution test. Agar dilution method: A serial two-fold dilution of the antibiotic is prepared in Mueller-Hinton agar. The bacterial inoculum is standardized according to McFarland standard. Using calibrated loops a volume of 0.001-0.002 ml is inoculated on the surface of agar and incubated at 37oC overnight. The lowest concentration of antibiotic that inhibits visible growth on surface of nutrient agar is taken as MIC. Qualitative Methods: These tests categorize a bacterial isolate as sensitive, intermediate or resistant to a particular antibiotic. Disk diffusion test: In this method the standardized bacterial isolate is spread on an agar plate and then paper disc containing specific concentration of antibiotics are placed and incubated at 37oC overnight. If the isolate is susceptible to the antibiotic, it does not grow around the disk thus forming a zone of inhibition. Strains resistant to an antibiotic grow up to themargin of disk. The diameter of zone of -9- inhibition must be measured and result read from the Kirby Bauer chart as sensitive, intermediate or resistant. Automated Susceptibility Methods: Determination of bacterial growth in wells containing antimicrobial agent are performed in short period of time using computer-assisted instruments. Various techniques include: Turbidimetric detection: VITEK (report ready in 4-15 hours) Flourimetric detection: AutoSCAN Walkaway (report ready in 3.5-7 hours) Newer Non Automated Susceptibility Tests: Alamar system, E-test and Spiral gradient endpoint system Molecular Techniques: Detection of gene coding for resistance to one or several drugs by techniques such as PCR and DNA hybridization. - 10 -  Chapter 7 The Control of Microorganisms Control of microorganisms can be achieved by a variety of chemical and physical methods.sterilization is generally achieved by using physical means such as heat, radiation and filtration, or by chemical agents. - Sterilization: It is the killing of all living forms of microbes including spores. - Disinfection: It is the destruction of most but not all pathogenic microbes or their spores. Disinfectants are categorized according to their spectrum of activity into : a) High level disinfectants : These kill all microbial pathogens but not all bacterial spores eg: gluteraldehyde b) Intermediate level disinfectants :- These kill all microbial pathogens but not bacterial spores. eg : isopropyl alcohol. c) Low level disinfectants : These kill most vegetative bacteria (except tubercle bacilli) , e.g. : quaternary ammonium compounds. - 11 - - Antiseptics : They are chemical agents that are non-toxic to living tissues. They can be safely applied to the skin and mucous membrane, but are not suitable for systemic administration eg: ethyl alcohol and chlorohexidine. - Decontamination: It is the procedure that reduces pathogenic microorganisms to a level where items are safe for handling, use or disposal. It can be done by cleaning, disinfection and sterilization. Methods of Sterilization 1- Sterilization by heat : - 12 -  Heat in the most different and inexpensive method of sterilization it can be used in two forms:- 1- Dry heat: kills by destructive oxidation of essential cell constituents. It is less efficient than moist heat. However, it is less expensive and is not corrosive. Dry heat sterilizer is the main method of sterilization by dry heat. It is an isolated double walled metal chamber that is heated by electricity and has a thermostat that maintains the chamber air constantly at the chosen temperature. It uses a temperature of 160 oC for 2 hr or 170 oC for 1 hr. This method is used for the sterilization of glass-ware, ointments, powders, oil and metallic instruments. 2- Moist heat: This method of sterilization kills microorganisms by protein denaturation. It is used as : o a) Moist heat at a temperature below 100 C : Pasteurization Pasteurization of milk is the best example, by heating either at 63 oC for 30 min. or at 72 oC for 15-20 sec. and immediately cooling to below 10 oC. This process will destroy all the non-spore forming pathogens that may be found in milk i.e. M.tuberculosis ,Salmonella and Coxiella brunette. - 13 -  b) Moist heat at a temperature of 100 oC : Boiling at 100 oC for 20 min. is sufficient to kill all vegetative bacteria, and hepatitis B virus, but not all bacterial spores. This method may be used for the disinfection of surgical and medical equipment when sterility is not essential, and in emergency if no sterilizer is available. c) Steam sterilization at a temperature above 100 oC: The autoclave When water is heated in a closed chamber at a pressure above atmospheric, the boiling point of water rises above 100 oC. The autoclave can be used at 121 oC for 20 min. ( at double atmospheric pressure = 2 bars ) or at 134 oC for 3-6 min ( at 3 bars ).The autoclave is used for sterilization of surgical instruments, bed linen, surgical dressings, gowns, cotton and for any culture media not destroyed by heat. It is the most efficient method of sterilization due to: 1- The high temperature reached. 2- The high penetrating power of steam under pressure. 3- When steam condenses on an article, it liberates a large amount of latent heat to its surface. 3- It is not toxic and is not time consuming. Monitoring sterilization procedures: - 14 - The efficiency of any sterilization procedure should be checked routinely by: 1- Mechanical methods: using a printout or graph that monitors the time, temperature and pressure of the sterilization cycle daily. 2- Chemical indicators or: These are chemically impregnated paper strips that are placed inside and outside the packs during sterilization. The strips change color at the proper temperatures and time. 4- Biological indicators: Paper strips impregnated with bacterial spores are placed within the load and at the end of sterilization they are tested for viability of the spores. Geobacillusstearothermophilus spores which are killed at 120 oC in about 20 min. are used for monitoring steam sterilization. Bacillus atrophaeus spores are used to monitor dry heat and ethylene oxide sterilization. II- Sterilization by irradiation Certain types of irradiation are used to control the growth of microorganisms. These include both ionizing and non-ionizing radiation. a- Ionizing radiation: have short wave length and high energy, giving them great penetrating power. The effect of ionizing radiation is due to the production of highly reactive free radicals, which disrupt the structure of macromolecules such as DNA and proteins. - 15 -  eg. Gamma rays from the isotope cobalt 60 which is used for sterilization of disposable plastic items that cann’t with stand heat as syringes, catheters, gloves. b- Non-ionizing radiation: The most widely used is the ultra-violet radiation ( UV), its wave length is around 260 nm so it has low penetration power. UV radiation is absorbed by nucleic acids and proteins, the absorbed energy causes the rupture of chemical bonds so the normal cellular function is impaired. UV lamps are usually found in operation rooms, food preparation area and laboratory safety cabinets. III- Sterilization by filtration : It is a mechanical method for the exclusion of microorganisms from fluids which are destroyed by heat as serum, plasma hormones and broth culture media, or for the sterilization of air in operation rooms. a- Membrane filters : Commonly made from nitrocellulose or poly-carbonate, they are manufactured as discs of varying diameters and varying pore size. The liquid pass through by means of pressure or suction.It is important that an appropriate pore size is chosen for any given task (for bacteria a pore size of 0.22 um is commonly used). b- Air filters : - 16 - Large volumes of air may be rapidly freed from microorganisms by passage through high efficiency particle arresters (HEPA). They are mainly used to decontaminate the air input into the operation rooms. IV- Gaseous Sterilization: Ethylene oxide (EO) gas is effective against all microorganisms. It is used for sterilization of large items of medical equipments and materials such as plastics that would be damaged by heat treatment The materials to be treated are placed in a special chamber which is sealed and filled with the gas in a humid atmosphere at 40-50 oC for several hours. It is highly toxic so all items must be thoroughly flushed with sterile air after treatment to remove any traces of it. Methods of Disinfection: Chemical substances are mainly used as disinfectants for inanimate objects (endoscopes, floors, top of benches, etc ….). They are also used as antiseptics for topical application on living tissues. The most commonly used are:- 1- Alcohols : Ethanol and isopropanol are most commonly used at a concentration of 70 %. Alcohols cause denaturation of proteins, also dissolve lipids and thus have a disruptive effect on microbial membranes and the envelope of certain viruses. Although spores are often resistant. The use of alcohols is limited to those materials that can withstand their solvent action. - 17 -  2- Halogens : Chlorine gas in compressed form is used in the disinfection of municipal water supplies and swimming pools. Sodium hypochlorite is used as a disinfectant in homes and hospitals. Iodine is used as skin antiseptic, its effect is enhanced by being dissolved in ethanol ( 1% I 2 in 70% ethanol ) Phenolics(Dettol, Lysol) are very effective against gram - Vebacteria, they are used in the laboratory to clean spilled cultures on working areas. Surfactants (surface active agents) as quaternary ammonium compounds are used as antiseptics in detergents and soaps. - 18 -  Chapter 8 Bacterial Genetics Genetics is the process of trait inheritance from parents to off- springs. The same happens with bacteria where the parent cells passes its genetic information to its off-springs this information takes the form of genes A Gene is a sequence of DNA that usually encode a polypeptide Bacterial chromosome:- It is a double standard DNA Molecule Which is circular and wounded on itself to form the nuclear mass. Chemical structure - 19 - It is formed of phosphate group and sugar (dexyribose) alternating with one another, to which one attached the Purine (adenine and guanine) and pyrimidine (thymine and cytosine) bases. The double helix is stabilized by hydrogen bonds between the opposite strands. The bonds are formed between adenine and pyrimidine bases on the opposite strands. The bonds are formed only between the adenine and thymine or guanine and cytosine. The chromosome is functionally subdivided into segments or genes. The sequence of the nucleotides in each gene determine the sequence of amino acids, hence the structure of the protein formed. Genes essential for bacterial growth are carried on the chromosome, few genes associated with specialized function are carried on plasmids. DNA replication:- 1- The two strands of each chromosome separate by the action of helicase enzyme. - 20 - 2- Enzyme primase initiates the synthesis of RNA primer each DNA strand acts as a template for the production of new complementary strand, new nucleotides are being added according to the rules of base –pairing. 3- On formation of RNA primase on each of the DNA strands of the replication Fork. DNA polymerase I changes the Primer RNA to DNA. 4- Hydrogen bonds are formed between the old poly- nucleotide strand and the newly synthesized one. Steps of DNA replication This mechanism is known as the "semi conservative replication" of DNA because each daughter molecule comprises one parental strand and one newly synthesized strand Gene expression ( protein synthesis) - 21 - It is the process by which the sequence of nucleotides in a gene determine the sequence of the amino acids in the protein, it takes place through two steps - Transcription - Translation 1- Transcription :- 1- The two strands of the chromosome are separated. 2- Messenger RNA (mRNA) is formed by the action of the enzyme RNA polymerase using DNA as a template. 3- The mRNA attached to the ribosome where the protein will be constructed. 4- Each of the amino acids found in the cytoplasm is transferred to certain RNA molecule Called transfer RNA (tRNA) , for each amino acid, these is a specific tRNA, which attached to the amino acid at one end, its other end has a triplet of bases (anti-codon) complementary to the triplet of bases (codon) on mRNA. 2- Translation:- 1- m RNA and t RNA come together and the surface of the ribosome. 2- Each tRNA finds its complementary nucleotide triplet on m RNA. 3- Each amino acid is linked with the adjacent one to from a polypeptide chain. - 22 - Thus entire mRNA is translated into the corresponding sequence of amino acids which are linked to form polypeptide chains so proteins are formed. Steps of protein synthesis Types of RNA involved in protein synthesis - 23 - Plasmid Plasmid is a small, circular, double-stranded DNA molecule that is distinct from the cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. Plasmids have a wide range of lengths, from roughly one thousand DNA base pairs to hundreds of thousands of base pairs. When a bacterium divides, all of the plasmids contained within the cell are copied such that each daughter cell receives a copy of each plasmid. Bacteria can also transfer plasmids to one another through a process called conjugation. Scientists have taken advantage of plasmids to use them as tools to clone, transfer, and manipulate genes. Plasmids that are used experimentally for these purposes are called vectors. Researchers can insert DNA fragments or genes into a plasmid vector, creating a so-called recombinant plasmid. This plasmid can be introduced into a bacterium by a process called transformation. Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities. - 24 - Functions of plasmids: Antibiotic resistance, heavy metal resistance, virulence, environmental adaptability & persistence, andmetabolic functions that allow utilization of different nutrients are some of the known functions coded byplasmids. Another important feature includes plasmid-mediated biodegradation of a variety of toxicsubstances such as toluene and other organic hydrocarbons and herbicides. Transposable genetic elements: - These are non – replicating DNA segments that are capable of insetting themselves into other DNA molecules. Transposition relies on the ability of the transposable elements to synthesize its own specific recombination enzymes. They are valuable tools for gene manipulation. 1- Transposons Pieces of DNA move from one site to another between DNAs of bacteria, they replicate as a part of recipient DNA. They carry the genes for drug resistance, toxin production or other metabolic processes. - 25 - 2- Insertion sequences They are simple types of transposons that have fewer bases they carry the genetic information that codes for their integration, they are found in multiple copies at the end of larger transposons. 3- Pathogenicity islands (PAIs) DNA segments containing mobile genetic elements, the genes that encode the virulence factors in bacteria (e.g adhesion, invasion, exotoxins, ……) are clustered in PAIs. - 26 -  Bacterial variation Bacterial variation may be phenotypic or genotypic Types of Bacterial variations Phenotypic variation: It is a change in bacterial characters due to environmental influence it is reversible and not heritable. Example:- 1- Change in the colony morphology. 2- Increase in pigment production by Staphylococci at room temperature. Genotypic variation - 27 - It a change in bacterial characters due to genetic changes it is irreversible and heritable Example:- 1- Mutation. 2- Gene transfer. Mutation A Mutation is a permanent change in the DNA sequence of a gene. Mutation in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene.It may be due to substitutions of one base by another, deletion of bases or addition of new bases. Mutation by be:- Spontaneous as a result of an error in replication or induced as a result of stimulation by a physical or chemical agent. Gene Transfer There are three types of gene transfers leading to bacterial variation. 1- Transformation Dying bacterial cells release DNA which can be taken by other cells, this process is enhanced by Nacl or heat shock. 2- Conjugation Direct contact of two bacterial cells and formation of cytoplasmic bridge, the genome is transferred from the donor to the recipient. 3- Transduction - 28 - During the lytic phage cycle a piece of DNA enter another phage, when this phage inject another bacterial cell gene transfer occurs. Genetic Engineering Incorporation of new different species, allowing the manufacture of genetic material in the laboratory. Applications:- 1- Production of hormones. 2- Gene therapy. Chapter 9 PATHOGENESIS OF BACTERIAL INFECTIONS - 29 - A pathogen is a microorganism that is able to cause disease in a plant, animal or insect. Pathogenicity is the ability to produce disease in a host organism. Microbes express their pathogenicity by means of their virulence, a term which refers to the degree of pathogenicity of the microbe. Hence, the determinants of virulence of a pathogen are any of its genetic or biochemical or structural features that enable it to produce disease in a host. The relationship between a host and a pathogen is dynamic, since each modifies the activities and functions of the other. The outcome of such a relationship depends on the virulence of the pathogen and the relative degree of resistance or susceptibility of the host, due mainly to the effectiveness of the host defense mechanisms. Saprophytic bacteria are those which live freely in nature on.decaying organic matter, in soil or water Parasitic bacteria are those which live in/on a living host. they can multiply in the host tissues causing disturbance of physiologic functions, thus leading to disease and are called.pathogenic bacteria Many of the parasitic bacteria live on the external or internal surfaces of the body without causing diseases. These are called commensals and they constitute the largest group of normal body flora. These organisms may even be beneficial to the host, e.g. commensals in the gut digest polysaccharides and are a - 30 - source of certain vitamins. They also compete with pathogenic.organisms for nutrition thus inhibits their growth Under certain conditions, commensal bacteria may cause disease and should be considered as potential pathogens or : opportunities. Some of these conditions are 1- Lowered host defense mechanisms e.g. immunosuppressed,.diabetic or leukemic patients 2- Alteration of the host tissues, e.g. Streptococcus viridans, a normal inhabitant of the mouth and throat may reach the blood stream and cause endocarditis if the host has a predisposing.heart lesion 3- Change in the natural habitat of the organisms, e.g. E. coli is a normal inhabitant of the intestine. If it reaches the renal system through blood or lymphatics, it causes urinary tract infection. INFECTION and DISEASE Infection is the process by which the parasite enters into a relationship with the host. Disease is the destruction of host tissues by the organisms due to invasion of tissues, toxin :production or other virulence factors. It requires the following.1- A source of infection which may be man , animal or soil 2- Mode of transmission e.g. insect bite, faecal contamination of food, inhalation of droplets, sexual contact, blood transfusion - 31 - 3- A portal of entry into the host, e.g. gastrointestinal, genitourinary or respiratory tracts, skin and mucous membranes,. through abrasions, insect bites or injections 4- Multiplication of the parasite within the host : The parasite may multiply locally at the portal of entry or it may spread.through the tissues, blood or lymphatics to reach a target organ 5- Portal of exit from the host, e.g. in urine, stools, respiratory or genital discharges or from the blood by insects or injections.from which the organism will be transmitted to another host Carriers are apparently healthy individuals carrying a pathogenic organism without showing clinical manifestations..They can transmit this organism to other susceptible individuals Carriers may be transient carriers (during the incubation period) or permanent (chronic) carriers as in hepatitis B. :Carriers are considered as serious sources of infection because 1. They do not show manifestations of the disease. 2. They communicate with the public without being noticed. 3. They carry the organism in the inter-epidemic period. Factors that govern disease production: The outcome of any infection depends on: 1. Microbial factors (Pathogenicity and virulence ) 2. Host resistance factors (natural and acquired immunity) Microbial factors.Pathogenicity: the ability of an organism to cause disease - 32 - Virulence: the degree of pathogenicity. In pathogenic species of bacteria, e.g. C. diphtheriae, some strains are highly virulent producing severe disease. Others are moderately virulent producing mild disease, while others are avirulent i.e. unable to produce disease. This depends on the ability of different strains.to produce a toxin and on its potency Virulence is genetically determined by genes carried on plasmids, phages, chromosomes and pathogenicity islands (PAIs), which are mobile genetic DNA segments inserted into the chromosome. The genes that encode many virulence factors e.g. adhesions, invasion and exotoxins are clustered adjacent to.each other on PAIs Virulence factors of bacteria: These are certain structures or products that help microorganisms to overcome body defense mechanisms and :cause disease. These are :Adherence Factors (adhesions) I- Certain bacteria have specialized structures or substances that help their adhesion to host cell mucous membranes thus allow them to start the disease process. Mutants that lack these structures are often avirulent. For example, the pili of N. gonorrhoeae and E. coli mediate the attachment of the organisms to the urinary tract epithelium. Lipoteichoic acid found on the fimbriae causes adherence of Streptococci to - 33 - buccal mucosa.Glycocalyx of Staph.epidermidis and S.viridians.allows their adherence to the endothelium of heart valves Bacterial biofilms: these are aggregates of interactive bacteria attached to a solid surface or to each other and encased in an exopolysaccharide matrix, forming a slimy coat on solid surfaces. The initial step in biofilm formation is colonization of the surface through multiplication of bacteria and adhering to the surface and to each other by their flagella or pili and by production of extracellular polysaccharides. Bacteria in biofilms are protected from the immune mechanism of the host and are resistant to antibiotics. They are important in human infections that are persistent and difficult to treat e.g. staph. epidermidis and staph.aureus infections of central venous catheters,.intraocular lenses and artificial prostheses :Invasive Factors II- This is the ability to invade tissues, multiple and spread rapidly causing the inflammatory process. This may be partly due to the:- 1- Antiphagocytic action of certain surface components that protect the bacteria from phagocytosis and destruction, e.g. the capsule of many organisms e.g. Pneumococci. The “M” proteins found on the fimbriae of S. pyogenes. Protein A of.staph.aureus 2- Extracellular Enzymes: These are substances produced by - 34 - some bacteria that help the spread, invasion and establishment :of microorganisms into the tissues, these include a- Collagenase and hyaluronidase, which degrade collagen and hyaluronic acid respectively, thereby allowing the bacteria to spread through subcutaneous tissues. They are especially.important in cellulitis caused by S. pyogenes Lecithinase produced by C. perfringensbreaks down b-.lecithin c-Coagulase is produced by Staph. aureus in the presence of certain serum factors, it coagulates plasma with the deposition of a fibrin wall around staphylococcal lesions which helps them to persist. It also causesdeposition of fibrin on the surface of the individual organisms protecting them from phagocytosis. d-Streptokinase (fibrinolysin) and streptodornase (deoxyribonuclease) are produced by S. pyogenes. The former causeslysis of fibrin clots and the latter breaks down DNA; a viscous constituent of pus and inflammatory exudates, both facilitate spread of organisms in tissues. e-Immunoglobulin A (IgA)protease which is produced by N. gonorrhoeae, N.meningitidis, H.influenzae and S. pneumoniae. It degrades the secretory IgA on mucous surfaces and thus.eliminates protection of the host by antibody Ability to survive intracellularly: This is a property of Ill- some organisms which use several different mechanisms to allow them to survive and grow intracellularly escaping - 35 - intracellular killing by phagocytic cells. Examples are M. tuberculosis which survive by inhibiting phagosome- lysosome.fusion variation: Many pathogens change their Antigenic IV-.surface antigens and evade the host's immune system :Toxin Production V- Toxins are bacterial products which have a direct harmful action on tissue cells. They fall into two groups: a- Exotoxins: They are protein toxins, secreted by living bacteria and diffuse freely into the surrounding medium i.e. extracellular toxins. The production of most exotoxins is controlled by genes in plasmids or bacteriophages rather than by chromosomal genes. Toxins are specific in action and can be sub-classified as neurotoxins, enterotoxins, or cytotoxins according to their mode of action and the organs affected. Cl. tetani exotoxin is coded on plasmid, C. diphtheriae and Cl. botulinum toxins are coded on phage. b- Endotoxins: These are integral part of the cell wall of gram negative bacteria from which they are liberated when the cell dies and disintegrates. The toxicity of the endotoxin is associated with the lipid. Organisms producing endotoxins.include E. coli, and Meningococci Endotoxins are the most important cause of endotoxic or septic shock. All endotoxins produce generalized non-specific toxic effects in the form of fever, hypotension, disseminated - 36 -  intravascular coagulation (DIC), shock and sometimes death due to massive organ failure. Differences between Exotoxins and Endotoxins Property Exotoxins Endotoxins Location of Plasmid or bacteriophage Bacterial chromosome genes Composition Proteins Lipopolysaccharides Action Specific (binds to specific Non-specific (fever and receptors on specific host shock). No specific cells). No fever receptors Heat stability Labile, destroyed at 60°C.Stable at 100°C for 1 hr Diffusibility.Diffusible Not diffusible. Integral Excreted by living cells part of the cell wall Immunogenicity Strong, induce high titer of Weak immunogenicity antitoxin Toxicity Strong Weak Convertibility to Yes No toxoid Produced by Gram positive bacteria mainly Gram negative bacteria * Toxoidis toxin treated usually with formalin so that it loses toxicity but.retains immunogenicity * Peptidoglycan-teichoic acid, released when gram positive cells die, may also cause effects similar to endotoxins. - 37 - References: Diagnostic Microbiology, Twelfth ed. Bailey and Scotts.2010 McGill Laboratory Biosafety Manual - Second edition, 1997. Sterilization and disinfection in the laboratory. - 38 -