GENERAL BACTERIOLOGY FULL.pdf

GENERAL BACTERIOLOGY ISU PREPARED BY | DR | H M H DR : H M H 2024 Introduction to Microbiology Microbe Microbes are minute living things that are usually unable to be viewed with the naked eye. For example: Bacteria, fungi, protozoa,...

GENERAL BACTERIOLOGY ISU PREPARED BY | DR | H M H DR : H M H 2024 Introduction to Microbiology Microbe Microbes are minute living things that are usually unable to be viewed with the naked eye. For example: Bacteria, fungi, protozoa, algae, viruses Some are pathogenic Many are beneficial What is Microbiology Defined as : the study of microorganisms too small to be seen with the naked eye. Micro = Very small, Bio = living and Ology= Study or Science of These organisms include viruses, bacteria, algae, fungi, and protozoa. Microbiologists are concerned with characteristics and functions such as morphology, cytology, physiology, ecology, taxonomy, genetics, and molecular biology. Organisms included in the study of Microbiology 1. Bacteria 2. Protozoans 3. Algae 4. Parasites 5. Yeasts and Molds Fungi 6. Viruses Bacteriology Protozoology Phycology Parasitology Mycology Virology Microorganisms cells Microorganisms have two different types of cells 1- Prokaryotic cells Genetic material is not enclosed by the nuclear membrane. Absence of nuclear membrane -No true nucleus (Viruses, Bacteria, and Prion) 2- Eukaryotic cells Genetic material is enclosed by the nuclear membrane. Presence of nuclear membrane. True nucleus (Fungi, protozoa, Algae, animals, and plants) Basic Classification of Microorganisms Scope and Relevance of Microbiology Microorganisms are exceptionally diverse, are found almost everywhere, and affect human society in countless ways. Microbiology has further diversified into: Food Microbiology Environmental Microbiology Medical Microbiology Agricultural Microbiology- water-soil Industrial Microbiology Public Health Microbiology Immunology Pharmaceutical Microbiology Microbiology divisions Microbiology branches can be divided based on the : Type of organisms studied and their role. The main branches include virology, bacteriology, mycology, protozoology, phycology, parasitology, and nematology. Other branches based on Medical divisions include microbial ecology, environmental microbiology, medical microbiology, veterinary microbiology, soil microbiology, industrial microbiology, and food microbiology. Taxonomy Kingdom Phylum (pl: Phyla) Class Order Family , Genus (pl: Genera) , Species (pl: Species) Naming and Classifying Microorganisms Carolus Linnaeus (1735) established the system of scientific nomenclature. Each organism has two names: the genus and Species. The genus is capitalized and the species is lowercase. Are italicized or underlined. Eg. Staphylococcus aureus Bacteria : Prokaryotes , Bacteria are microscopic, single-celled organisms that have no nucleus and a cell wall made of peptidoglycan , Most bacteria are much smaller than our own cells, though a few are much larger and some are as small as viruses. They usually do not have any membrane-wrapped organelles (e.g., nucleus, mitochondria, endoplasmic reticulum), but they do have an outer membrane. Most bacteria are also surrounded by at least one layer of cell wall. Bacteria are a huge and diverse group. Its members have many shapes, sizes, and functions, and they live in just about every environment on the planet. Fungi : Eukaryote , Fungi are single-celled or multicellular organisms with nuclei and cell walls made of chitin. They also have membrane-wrapped organelles, including mitochondria. Unlike plants, fungi cannot make their food. , Familiar fungi include yeasts, molds, and mushrooms. Yeasts live as small, individual cells, between the size of bacteria and our cells. Molds and mushrooms are the fruiting bodies of fungi that live as long, microscopic fibers. Fungi are important decomposers in most ecosystems. Their long, fibrous cells can penetrate plants and animals, breaking them down and extracting nutrients. Several species of fungi, mostly yeasts, live harmlessly on the human body. Viruses : Viruses are microscopic particles made of nucleic acids, proteins, and sometimes lipids(DNA or RNA)Viruses can’t reproduce on their own. Obligate intracellular parasites. Instead, they reproduce by infecting other cells and hijacking their host’s cellular machinery. Viruses are specialized to infect a certain host, and often a specific cell type within that host. HIV, for example, infects a certain type of immune cell in primates. Other viruses infect plants, animals, bacteria, or archaea. In our bodies, viruses infect not only our cells but also other microbes that live in our bodies. Viruses that infect bacteria are called bacteriophage. Viruses that infect archaea come in unusual shapes: some have two tails, and others are shaped like bottles or flowers. Protozoa : Eukaryote Protozoa (pro-toe-ZO-uh) are one-celled organisms, like bacteria. But they are bigger than bacteria and contain a nucleus and other cell structures, making them more like plant and animal cells. Protozoa love moisture. So intestinal infections and other diseases they cause, like amebiasis and giardiasis, often spread through contaminated water. Some protozoa are parasites. This means they need to live on or in another organism (like an animal or plant) to survive. For example, the protozoa that causes malaria grows inside red blood cells, eventually destroying them. Some protozoa are encapsulated in cysts, which help them live outside the human body and in harsh environments for long periods of time.  Algae :Eukaryotes. Members of a group of predominantly aquatic photosynthetic organisms , of the kingdom Protista. Algae have many types of life cycles, and they range in size from microscopic Micromonas species to giant kelps that reach 60 meters (200 feet) in length. Their photosynthetic pigments are more varied than those of plants, and their cells have features not found among plants and animals. Relation of microorganisms Microorganisms can be found as 1- Transient passengers(normal flora or commensals) 2- parasitic(using the host nutrients) 3- Pathogenic( Disease causing) 4- Having a beneficial role. Why study microbes? Major impact on health Microbe is talented and can live under extreme condition Responsible for disease in humans, animals, plant Protect against disease Used in bioremediation Major impact on the environment: major decomposers, nutrient cycling, making plastic, spoiling food, making food, producing light Scope and Importance of microbiology 1. Production of antibiotic Eg :penicillin from penicillium. 2. Production of enzymes ,vaccines ,alcoholic and other pharmaceutical product. 3. Diagnosis of disease and treatment Eg :ELISA ,Widal test. 4. Treatment of industrial waste and material 5. Plant growth promotion 6. Sterile product preparation 7. Sterilization( the process of killing microorganisms.)Eg :moist heat sterilization ,dry heat sterilization , and membrane filtration. 8. Steroid biotransformation.Eg :progesterone ,testosterone. 9. Identification of microorganisms.Eg :a morphological ,cultural ,or microscopic study. 10. Testing of pharmaceutical products and raw materials. Beneficial roles of bacteria Protection( colonization of surfaces) Immunization Secretion of enzymes- share in the digestion of foods Synthesis of vitamins and antibiotics Economic concern- food industry(cheese and wine) Disintegration of decayed maters and wastes Harmful roles of bacteria Still major cause of death and misery worldwide (meningitis, food poisoning) Hospital infection Antibiotic resistant infection Food spoilage History of microbiology Anton van Leeuwenhoek (1632–1723): was the first microbiologist and the first person to observe bacteria using a single-lens microscope of his own design. Louis Pasteur (1822–1895): Pasteur developed a process (today known as pasteurization) to kill microbes. pasteurization is accomplished by heating liquids to 63° to 65°C for 30 minutes or to 73° to 75°C for 15 seconds. Robert Koch (1843–1910): was a pioneer in medical microbiology and worked in cholera, anthrax and tuberculosis. He was awarded a Nobel prize in 1905 (Koch's postulates) he set out criteria to test. Alexander Fleming (1929): Discovered penicillin. History of microbiology The Germ Theory of Disease 1835: Agostino Bassi showed a silkworm disease was caused by a fungus. 1865: Pasteur believed that another silkworm disease was caused by a protozoan. 1840s: Ignaz Semmelweis advocated handwashing to prevent transmission of fever from patient to another. 1860s: Joseph Lister used a chemical disinfectant to prevent surgical wound infections after looking at Pasteur’s work showing microbes are in the air, can spoil food, and cause animal diseases. Development Of Microbiology In This Century Microorganisms become useful experimental subjects Discovery of the relationship between the genes and enzymes by Beadle and Tatum in 1941 The discovery of point mutations by Luria and Delbruck in 1943 Discovery of DNA as the genetic material by Avery, MacCLeod and McCarty in 1944. Microbiology becomes major contributor to the rise of molecular biology. New discoveries in microbiology led to the development of recombinant technology and genetic engineering. Bacterial Cell Structure & Function Bacteria Unicellular, prokaryotes Have both DNA and RNA Divided by Binary fission No mitochondria Rigid cell wall containing peptidoglycan Size and Shape of Bacteria Size: Average bacteria 0.5 - 2.0 um in diameter. RBC is 7.5 um in diameter. Shape: o Spherical (coccus) o Rod (bacillus) o Spiral o Pleomorphic (variable shapes) Arrangement: Can be: single, double, chains, in group, or irregular Bacterial cell structure Surface layers: capsule, cell wall, cell membrane Cytoplasm: nuclear material, ribosome, mesosome, inclusions etc. Appendages: flagella, pili or fimbriae Special structure: Spore Cell Wall Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a protein-sugar (polysaccharide) molecule. The wall gives the cell its shape and surrounds the cytoplasmic membrane, protecting it from the environment. It also helps to anchor appendages like the pili and flagella, which originate in the cytoplasm membrane and protrude through the wall to the outside. The strength of the wall is responsible for keeping the cell from bursting when there are large differences in osmotic pressure between the cytoplasm and the environment. Cell wall composition varies widely amongst bacteria and is one of the most important factors in bacterial species analysis and differentiation. A technique devised by Christian Gram in 1884, uses a staining and washing technique to differentiate between the two forms. When exposed to a gram stain, gram-positive bacteria retain the purple color of the stain because the structure of their cell walls traps the dye. In gram-negative bacteria, the cell wall is thin and releases the dye readily when washed with an alcohol or acetone solution. Cell wall of bacteria Cytoplasmic Membrane A layer of phospholipids and proteins, called the cytoplasmic membrane, encloses the interior of the bacterium, regulating the flow of materials in and out of the cell. This is a structural trait bacteria share with all other living cells; a barrier that allows them to selectively interact with their environment. Membranes are highly organized and asymmetric having two sides, each side with a different surface and different functions. Membranes are also dynamic, constantly adapting to different conditions. Cell membrane function Control permeability Transport protons for cellular metabolism Contain enzymes to synthesize and transport cell wall substance and for metabolism Secret hydrolytic enzymes Regulate cell division Cytoplasm 80% water, nucleic acids, proteins, carbohydrates, lipids, and inorganic ions etc……. The cytoplasm, or protoplasm, of bacterial cells, is where the functions for cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed of water, enzymes, nutrients, wastes, and gases and contains cell structures such as ribosomes, a chromosome, and plasmids. The cell envelope encases the cytoplasm and all its components. Unlike the eukaryotic (true) cells, bacteria do not have a membrane enclosed nucleus. The chromosome, a single, continuous strand of DNA, is localized, but not contained, in a region of the cell called the nucleoid. All the other cellular components are scattered throughout the cytoplasm. Cytoplasm Bacterial chromosomes: a single large circular double stranded DNA no histone proteins. The only proteins associated with the bacterial chromosomes are the ones for DNA replication, transcription etc. Ribosome: protein synthesis Mesosomes: A large invagination of the plasma membrane, irregular in shape. o Increase in membrane surface, which may be useful as a site for enzyme activity in respiration and transport. o May participate in cell replication by serving as a place of attachment for the bacterial chromosome Inclusions: Not separate by a membrane but distinct. Granules of various kinds: o glycogen, o polyhydroxybutyric acid droplets (PHB) i.e. fat droplets o inorganic metaphosphate (metachromatic granules) - in general, starvation of cell for almost any nutrients leads to the formation of this to serve as an intracellular phosphate reservoir. Plasmid One of those components, plasmids, are small, extrachromosomal genetic structures carried by many strains of bacteria. Like the chromosome, plasmids are made of a circular piece of DNA. Unlike the chromosome, they are not involved in reproduction. Only the chromosome has the genetic instructions for initiating and carrying out cell division, or binary fission, the primary means of reproduction in bacteria. Plasmids replicate independently of the chromosome and, while not essential for survival, appear to give bacteria a selective advantage. Ribosomes Ribosomes are microscopic "factories" found in all cells, including bacteria. They translate the genetic code from the molecular language of nucleic acid to that of amino acids— the building blocks of proteins. Proteins are the molecules that perform all the functions of cells and living organisms. Bacterial ribosomes are similar to those of eukaryotes, but are smaller and have a slightly different composition and molecular structure. There are sufficient differences between bacterial ribosomes and eukaryotic ribosomes that some antibiotics will inhibit the functioning of bacterial ribosomes, but not a eukaryote's, thus killing bacteria but not the eukaryotic organisms they are infecting. II. CELL SURFACE LAYER 1. Capsule or slime layer o Many bacteria are able to secrete material that adheres to the bacterial cell but is actually external to the cell. o It consists of polypeptide and polysaccharide on bacilli. Most of them have only polysaccharide. o It is a protective layer that resists host phagocytosis. o Medically important. Appendages Flagella (singular, flagellum): are hairlike structures that provide a means of locomotion for those bacteria that have them. They can be found at either or both ends of a bacterium or all over its surface. The flagella help the bacterium move toward nutrients; away from toxic chemicals; or, in the case of the photosynthetic cyanobacteria; toward the light. o Flagella: Long, thin extensions that allow some bacteria to move about freely in aqueous environments. Endoflagella: Wind around bacteria, causing movement in waves. Motility – movement o Swarming occurs with some bacteria. Spread across Petri Dish. o Proteus species most evident Arrangement basis for classification: o Monotrichous: 1 flagella o Lophotrichous: tuft at one end o Amphitrichous: both ends o Peritrichous: all around bacteria 2. Pili or Fimbriae o Pili: Many species of bacteria have pili (singular, pilus), small hair like projections emerging from the outside cell surface. Without pili, many disease-causing bacteria lose their ability to infect because they're unable to attach to host tissue. Specialized pili are used for conjugation, during which two bacteria exchange fragments of plasmid DNA. o Shorter than flagella and straighter, smaller. Only on some gram( – ve) bacteria. o Function: adhere. One of the invasive mechanisms of bacteria. Some pathogens cause diseases due to this. If mutant (fimbriae) not virulent. Prevent phagocytosis. Pili (Fimbrae) Two classes: o ordinary pili ‘colonization antigens’. Protein, attachment to host cells. Can be involved in host cell invasion e.g. Neisseria meningitidis o sex pili, role in conjugation (transfer of plasmid DNA) Origin: Cell membrane Position: common pili , numerous over the cell, usually called fimbriae sex pili Structure: composed of proteins which can be dissociated into smaller unit Pilin. It belongs to a class of protein Lectin which bond to cell surface polysaccharide. Special Structure Endospores o Spore former: sporobactobacilli and sporosarcinae - no medical importance. Bacillus and Clostridium have medical importance. o Position: median, sub-terminal and terminal have small water, high calcium content and dipicolinic acid (calcium dipicolinate) o Extremely resistant to heat, UV, chemicals etc. May be due to many S containing A.A for disulfide groups. o Vegetative/spore-containing cells (1) Endospores Resistant structure: o Heat, irradiation, cold o Boiling >1 hr still viable o Takes time and energy to destroy spores Location important in classification: o Central, Subterminal, Terminal Bacillus stearothermophilus –spores: o Used for quality control of heat sterilization equipment Bacillus anthracis - spores: o Used in biological warfare o Bacterial Taxonomy (Naming, Classification and Identification) Taxonomy: Is the science of the classification of organisms, to show evolutionary relationships among organisms. Is a way to provide a universal method of identification of an organism. Includes: o Identification: is the process of studying and recording the identical and distinguishing features. o Nomenclature: the process of assigning names to the various taxonomic rankings of each living organism. o Classification: is the orderly arrangement of organisms into groups, preferably in a format, that shows evolutionary relationships. Basis of Taxonomy: Phenetic system: groups organisms based on observable characteristics. e.g. motile v/s non-motile bacteria. Phylogenetic system: groups organisms based on genetic similarity and evolutionary relatedness. Nomenclature of Bacteria: There is a, quite a complicated, set of rules for the naming of Bacteria and Archaea. They must have two names: the first refers to the genus and the second refers to the species. The names can be derived from any language but they must be Latinized. Take for example Staphylococcus aureus. The genus name is capitalized and the species name is lowercase. The name is utilized to indicate that is Latinized. Nomenclature: Scientific name (Systematic Name): o Binomial System of Nomenclature: ▪ Genus name + species name ▪ Italicized or underlined ▪ Genus name is capitalized and may be abbreviated ▪ Species name is written in small letters and never abbreviated ▪ A genus name may be used alone to indicate a genus group eg Staphylococci ▪ A species name is never used alone ▪ eg: Bacillus subtilis ▪ B. subtilis Common or descriptive names (trivial names): o eg: tubercle bacillus (Mycobacterium tuberculosis) o meningococcus (Neiserria meningitidis) o Group A streptococcus (Streptococcus pyogenes) What is the importance of identifying bacteria? Accurate and definitive microorganism identification, including bacterial identification and pathogen detection, is essential for correct disease diagnosis, treatment of infection, and trace-back of disease outbreaks associated with microbial infections. Methods of identification of bacteria: Morphological classification: (shape, size etc..). Staining reaction: (Gram +ve or –ve). Growth requirement and Growth medium. Oxygen requirement: Aerobic or anaerobic). Biochemical classification: (Glucose fermentation). Serological classification: (Ags or Abs +ve). Antimicrobial sensitivity methods. Molecular classification: (Genetic relatedness). Morphological classification: Different shapes of bacteria are used to categorize bacteria. Different shapes of a bacterial cell are: o Spherical- Cocci: ▪ Monococcus: bacteria exist as a single spherical cell. ▪ Diplococcus: Cells are arranged in pairs after cell division. ▪ Streptococcus: the cocci are joined in a plane and arranged in a chain pattern. ▪ Tetrads: Tetrads are arranged in a group of 4 cells.. ▪ Staphylococcus: Cells are arranged in an irregular cluster, which looks like grapes.. ▪ Sarcinae: Sarcinae bacteria are anaerobic gram-positive bacteria. They occur as a group of 8 cells. It is found in the family Clostridiaceae. o Rod-shaped- Bacilli: ▪ The term bacillus has been applied in a general sense to all cylindrical or rodlike bacteria. o Spiral bacteria: ▪ These bacteria are spiral or helical in shape. o Comma shaped- Vibrio: ▪ These are curved and appear like a comma. ▪ These are mostly gram-negative bacteria. ▪ They are known to cause various foodborne diseases. Staining reaction: Gram’s stain: o Gram’s positive (Violet colour) o Gram’s negative (Red in colour) Ziehl Neelsen stain: for TB o Acid-fast bacilli. o Non acid fast bacilli. Growth requirement: Bacteria could be classified into different groups according to their needs. o Halophilic: are organisms that live in extremely salty environments. o Capnophilic: in the presence of high concentrations of carbon dioxide (CO2) o Autotrophes: an organism that can produce its own food using light, water, carbon dioxide, or other chemicals. o Heterotrophs: an organism that eats other plants or animals for energy and nutrients. o Thermophilic: an organism—a type of extremophile—that thrives at relatively high temperatures, between 41 and 122 °C (106 and 252 °F). o Fastidious: any organism that has very complicated nutritional requirements, meaning it will not grow without specific factors present or in specific conditions. o Chemotrophs: Derives energy from chemicals. o Phototrophs: Derives energy from sunlight. Oxygen requirement: Obligatory (strict) aerobic: requires oxygen. eg. Pseudomonas. Obligatory (strict) anaerobic: killed by oxygen. eg. Clostridium bacteria that cause tetanus and botulism. Facultative anaerobes: grow in the presence or absence of oxygen with different metabolic strategies. eg. E. coli, Staphylococcus, yeasts, and many intestinal bacteria. Microaerophilic bacteria: require reduced levels of oxygen. eg. Campylobacter. Aerotollerant: does not require oxygen but is not killed. eg. Lactobacillus. Biochemical classification: Biochemical/Physiological Properties Used for the identification of bacteria include: o nutrient utilization (carbohydrate utilization, amino acid degradation, lipid degradation), o resistance to inhibitory substances (high salt, antibiotics, etc.), o enzyme production (catalase, coagulase, hemolysins, etc.) and motility. Most bacteria are identified and classified largely on the basis of their reactions in a series of biochemical tests. Some tests are used routinely for many groups of bacteria (oxidase, nitrate reduction, amino acid degrading enzymes, fermentation or utilization of carbohydrates); others are restricted to a single family, genus, or species (coagulase test for staphylococci, pyrrolidonyl arylamidase test for Gram-positive cocci). Serological classification: Serological tests /immunological techniques identify aerobic/anaerobic bacterial infections in a laboratory by detecting antigens or antibodies in urine, blood, saliva, or cerebrospinal fluid (CSF). can be used to detect viral & bacterial antigens and antibodies (IgG and IgM), to help diagnose diseases and check immune status. A range of techniques are utilized including ELISA, agglutination, direct and indirect immunofluorescence, and Western blotting. Bacterial genetic Identification: DNA-based approaches used in the identification and classification of species of bacteria include DNA-DNA hybridization, DNA fingerprinting, and DNA sequencing. DNA fingerprinting methods for bacterial identification center primarily on the use of the polymerase chain reaction (PCR). Antimicrobial sensitivity: Testing for bacterial growth, the laboratory will perform antibiotic susceptibility testing (AST), if appropriate. The term susceptibility, also referred to as sensitivity, implies that the cultured bacterial pathogen growth may be inhibited, or not, by exposure to specific antibiotics. Resistant - indicates that clinical efficacy has not been reliable in treatment studies. Intermediate - implies clinical applicability in body sites where the drug is physiologically concentrated or when a high dosage of the drug can be used. Susceptible - implies that an infection due to the organism may be treated with the concentration of antimicrobial agent used unless otherwise contraindicated. Antimicrobials Selective toxicity of a drug: Inhibits or kills pathogens but has little or no toxic effect on the human cell. Broad spectrum antibiotics: Active against several microorganisms. Narrow spectrum antibiotics: Active against one or very few types. A bactericidal drug = kills the bacteria. A bacteriostatic drug = inhibits the growth of the bacteria, here 1- bacteria can grow again when the drug is withdrawn. 2- host defense mechanisms are required to kill the bacteria. Bactericidal drugs are useful in: 1- life-threatening infections, 2- when the leucocyte count is below 500/ microliter. Modes of actions of antimicrobials: 1- inhibitors of cell wall synthesis. 2- inhibitors of protein synthesis. 3- inhibitors of nucleic acid synthesis. 4- alteration of cell membrane function. 5- additional drug mechanisms. 1- Inhibitors of cell wall synthesis a) Penicillins & cephalosporins b) Carbapenems & monobactams c) Vancomycin 1- Inhibitors of cell wall synthesis a) Penicillins & cephalosporins: Inhibit the bacterial enzyme transpeptidase, the enzyme that cross-link peptidoglycan. Penicillins & cephalosporins are called betalactam drugs, i.e. an intact beta-lactam ring is required for activity. Beta-lactamases are enzymes that cleave the beta-lactam ring & so inactivate the drug. MRSA = Methicillin-Resistant Staphylococcus Aureus, treated by vancomycin. Hypersensitivity to penicillins is a problem. Cephalosporins: Structurally similar to penicillin. 1st, 2nd, 3rd & 4th cephalosporin generations. b) Carbapenems & monobactams Beta-lactam drugs. Structurally different from penicillins & cephalosporins.  c) Vancomycin: A glycopeptide i.e. not a beta-lactam drug, Bactericidal, Inhibits transpeptidase. 2- Inhibitors of protein synthesis. Drugs acting on 30 S subunit: 1- Aminoglycosides 2- Tetracyclines Drugs acting on 30 S subunit: 1- Aminoglycosides: Bactericidal drugs against gram negative rods bacteria. Inhibition of the initiation complex. Toxic to the kidneys, cannot be given orally. Drugs acting on 30 S subunit: 2- Tetracyclines: Block the binding of aminoacyl t-RNA to the ribosome. Drugs acting on 50 S Subunit: 1- Chloramphenicol. 2- Erythromycin. 1- Chloramphenicol: Blocks the enzyme peptidyl transferase. Can cause bone marrow suppression. 2- Erythromycin: Blocks release of t-RNA after it delivered its amino acid. 3- Inhibitors of nucleic acid synthesis 1- Sulfonamides & trimethoprim: Inhibit nucleotide synthesis. Trimethoprim inhibits the enzyme dihydrofolate reductase. A combination of sulfamethoxazole & trimethoprim is often used. 2- Quinolones: (e.g. ciprofloxacin) block DNA gyrase - the enzyme that unwinds DNA strands so that they can be replicated. 3- Rifampicin: Inhibits RNA polymerase that synthesizes mRNA. 4- Alteration of cell membrane function Antifungal drugs: Against ergosterol in fungi. 5- Additional drug mechanisms Isoniazid: Inhibits the synthesis of mycolic acid found in the cell wall of Mycobacteria. Metronidazole: Against anaerobic bacteria & certain protozoa. Acts by taking away the electrons that the organisms need to survive, it also forms toxic intermediates that damage DNA. Chemoprophylaxis To prevent infectious diseases, such as: 1- surgical wound infections. 2- opportunistic infections in immunocompromised patients. 3- serious pathogens.  Antimicrobial drugs resistance Mechanisms: 1- Enzymatic degradation of the drug. 2- Modification of the drug’s target. 3- Reduced permeability. 4- Active export of the drug. Nongenetic basis of resistance: Abscess. Foreign bodies, Penicillin will not affect the bacteria that are not growing. Combination of drugs: Indifference. Synergistic, increased effect when both drugs are given together Antagonistic effect: decreased effect when both drugs are given together. Minimal inhibitory concentration (MIC): Is the lowest concentration of the drug that inhibits the growth of the bacteria. Minimal bactericidal concentration(MBC): Is the lowest concentration of the drug that kills the bacteria. Bacterial vaccines By inducing active & passive immunity Active immunity: By vaccines consisting of: 1- capsule: Streptococcus pneumoniae. 2- toxoids (exotoxins that lost the ability to cause a disease but retain ability to induce antibody formation): diphtheria & tetanus. 3- purified bacterial proteins: acellular pertussis vaccine & anthrax vaccine. 4- live attenuated bacteria: BCG for tuberculosis, consists of live attenuated Mycobacterium bovis. 5- whole killed bacteria: Cholera, plague. Passive immunity: By antitoxins (antibodies against the toxins). e.g. tetanus & diphtheria. Passive-Active immunity: Such as in an unimmunized person who has sustained a contaminated wound. Both the toxoid & antitoxin should be given but at different sites. Laboratory Diagnosis By 3 approaches: 1. Bacteriologic methods: o Bacteria are colorless, i.e. cannot be seen by the microscope without staining. o a- Staining: ▪ Gram stain to see the: ▪ Shape (cocci or bacilli), ▪ Color (gram positive or gram negative), ▪ Arrangement (diplococci, streptococci or staphylococci). ▪ Ziehl-Neelsen stain (ZN) for Mycobacterium tuberculosis. ▪ Other stains, according to the suspected bacteria. o b- Isolate the bacteria to study its characteristics. 2. Immunologic methods (antibody detection). 3. Nucleic acid-based methods (genome detection i.e. DNA detection). 1- Bacteriologic methods: 1st day: Obtain a proper specimen, according to good knowledge of the pathogenesis of the suspected bacteria then: 1-Staining: ▪ Gram stain to know the: ▪ Shape (cocci or bacilli), ▪ Color (gram positive or gram negative), ▪ Arrangement (diplococci, streptococci or staphylococci). ▪ Ziehl-Neelsen stain (ZN) for Mycobacterium tuberculosis. ▪ Other stains, according to the suspected bacteria. Gram negative cocci gram positive rods Gram negative rods Some bacteria can not be stained by gram stain, so: o b- Ziehl-Neelsen stain for Mycobacterium tuberculosis. o c- other stains, according to the suspected bacteria. 2- Culture: Inoculate specimen in the suitable culture medium & incubate overnight in suitable temperature, pH, humidity & presence or absence of oxygen. Tools use for bacterial isolation Types of media 2nd day: Complete identification of some organism by: o Looking for colonies formation & their color, appearance, smell, hemolysis, pigments etc. o Gram stain of the culture. o Special tests e.g. coagulase test for Staphylococcus aureus. 2nd day: Complete identification of some organism by: o Looking for colonies formation & their color, appearance, smell, hemolysis, pigments etc. o Gram stain of the culture. o Special tests e.g. coagulase test for Staphylococcus aureus. o Sugar fermentation tests for gram negative rods. Oxidase test Indole test 3rd day: Complete identification of gram negative rods. Antibiotic sensitivity tests. 4th day: Read antibiotic sensitivity tests result. Final report. Some causes of negative results in bacteriologic approach: Organism cannot be stained by gram stain e.g. Mycobacterium tuberculosis or Cannot be cultured in bacterial cultures e.g. Chlamydia. Recent antibiotic therapy. Hazards of culture e.g. anthrax. So we do either: 2- Immunologic methods 1- Serology: i.e. detection of a specific antibody in the patient’s serum (IgM or IgG). 2- Detect antigen in the patient’s serum or 3- Nucleic acid-based methods 3- Detect nucleic acids in the specimen e.g. polymerase chain reaction (PCR). Normal Flora Definition: Normal flora: Bacteria and fungi that are permanent residents of certain body sites. Normal flora: Microorganisms that are frequently found on or within the body of healthy persons. Types of normal flora: Resident flora: Flora that are constantly present in-on the body. They prevent the permanent colonization of the body by other organisms. E.g., E. coli in the intestine. Transient flora: Flora that can be found normally from time to time. E.g., Meningococcus may be found in the nasopharynx. Normal flora prevent colonization of body pathogens Member of the intestinal flora synthesize vit. K and some B vitamins. Increase over all immune status of the host against pathogen The endotoxin help in the defense mechanism of the body Significance of normal flora: Can cause disease: o In other parts of the body o In immunocompromised individuals Protective: If suppressed, pathogens may grow. Nutritional: B vitamins and vitamin K synthesis. Examples of normal flora: Skin: Staphylococcus epidermidis (predominant) Throat: Viridans streptococci Colon: Major location in the body, mainly Bacteroides. Nose: Staphylococcus aureus. Host Defenses Against Bacterial Infections 1. Innate Immunity: Non-specific (against many microorganisms) Includes: o Physical barriers: Intact skin and mucous membranes (burns predispose to infections) o Cells: Neutrophils o Proteins: Complement o Low pH: Of skin, stomach, and vagina In response to most bacterial infections, there is an increased number of neutrophils. Killing of bacteria within neutrophils. 2. Adaptive (Acquired) Immunity: Specific Includes: o Antibodies o Cells (CD4 & CD8) Can be: o Active o Passive Adaptive (Acquired) Immunity: 1. Active Immunity: Mediators: Antibody and T cells Advantages: Long duration (years) Disadvantages: Slow onset. 2. Passive Immunity: Mediators: Antibody only (IgG across placenta) Advantage: Immediate availability Disadvantage: Short duration (months) Bacterial Vaccines: Inducing active and passive immunity Active Immunity: By vaccines consisting of: o Capsule: Streptococcus pneumoniae o Toxoids (exotoxins that lost the ability to cause a disease but retain ability to induce antibody formation): Diphtheria and tetanus o Purified bacterial proteins: Acellular pertussis vaccine and anthrax vaccine. o Live attenuated bacteria: BCG for tuberculosis, consists of live attenuated Mycobacterium bovis. o Whole killed bacteria: Cholera, plague. Passive Immunity: By antitoxins (antibodies against the toxins): E.g., tetanus and diphtheria. Passive-Active Immunity: Such as in an unimmunized person who has sustained a contaminated wound. Both the toxoid and antitoxin should be given but at different sites. Sterilization And Disinfection Objectives 1. Define the terms sterilization, disinfectant and antiseptic. 2. Classify the different methods of sterilization (physical and chemical methods). 3. Know and realize that heat is the most important method of sterilization and its application in medical practice. 4. Know the importance of non-heat sterilization methods and their use for sterilization of heat sensitive objects. Introduction 1. Cleaning is defined as removing any visible soil. Cleaning is typically achieved by manually combining water with enzymes or detergents and using a brush to physically remove visible debris. It is important to note that clean does not mean sterile or disinfected. 2. Decontamination is defined as removing pathogenic organisms to make objects safe enough to handle, use, or dispose of. 3. Disinfection is the process of eliminating most pathogenic organisms except spores. This is usually accomplished with liquid chemicals or wet pasteurization removing of pathogenic microorganisms or reducing their number (not all microorganisms). Unable to destroy spores and some non-enveloped viruses. 4. Sterilization is the process of eliminating all microorganisms, and is commonly achieved through chemical and/or physical means. 5. Germicide is an agent that can eliminate microorganisms. Agents with ‘cide/cidal’ suffixes have some type of killing action that is referenced in the name (i.e. fungi-cide). Note that germicides include both antiseptics and disinfectants (remember disinfectant differs from disinfection). a. Antiseptic germicides are agents that can be applied to living tissue (i.e. skin). b. Disinfectant is any germicide agent that can be applied to non-living tissue (i.e. not skin). It is only used to disinfect surfaces of inanimate objects because of the risk of injury to tissue. Factors influencing ability to kill microbes Strength of the killing agent Time that the agent has to act Temperature of environment Type of microbe Environment around the area to be decontaminated Number of microbes to be killed Sterilization All living cells, viable spores, viruses, and viroids are either destroyed or removed from an object or habitat Methods of sterilization: o Physical methods: ▪ Heat: ▪ Hot-air sterilizer ▪ Autoclaving ▪ Radiation ▪ Filtration ▪ Ultrasound ▪ Dryness, Low temperature o Chemical agents Methods of sterilization Physical or chemical methods. 1. Physical methods. a. Heat: Exposure of the objects to heat will kill microbes by coagulation of protein, denaturation of enzymes, and oxidation. b. Filtration: Sterilization through removing microbes from fluids by exposing them to small-size filters. Used for heat-sensitive fluids like serum, CHO, and urea. c. Radiation: Exposure to irradiation causes the denaturation of proteins and enzymes. I. Physical methods Heat: Dry heat Moist heat Heat: Heat sterilization is the most effective method of sterilization, where the elimination of microbes is achieved by the destruction of cell constituents and enzymes. It kills microbes by coagulation of protein. It is done by two methods: a. Dry heat This method is used on objects that are sensitive to moisture. Moisture-free heat or dry heat is applied on the surface or objects such that there is denaturation and lysis of proteins which leads to oxidative damage, and ultimately the microbial cell dies out or may even burn. Some methods of dry heat sterilization include incinerators, hot air ovens, and flaming techniques **b. Moist Heat Sterilization: **It is one of the best methods of sterilization. Moist heat sterilization is done with the help of an instrument called an autoclave. An autoclave works on the principle of producing steam under pressure. Thus moist heat sterilization is also known as steam sterilization. The water is boiled in an autoclave at 121-134℃ at a pressure of 15psi. This leads to coagulation of proteins in the microorganism, and they are effectively killed. a. Dry heat sterilization Dry heat sterilization is not as effective and efficient as wet heat (steam) sterilization. Dry heat sterilization should be used only for materials that might be damaged by moist heat or that are impenetrable to moist heat. The advantages for dry heat sterilization are that is nontoxic, it does not harm the environment, it penetrates materials, and it is noncorrosive for metal devices and sharp instruments. The disadvantages for dry heat are the slow rate of heat penetration, and microbial killing makes this a time-consuming method. Types of dry heat Red heat Direct flames Incineration Hot-air sterilizer o 160-170°C for 2 hours----spores , o Glass Petri dishes and pipettes Infrared Microwave 1. Red Heat Red heat sterilization is the process of instant sterilization by holding the instruments in a Bunsen flame till they become red hot. This method is based on dry heat sterilization is commonly used for sterilization of instruments like incubation loops, wires, an d points of forceps. This usually takes about 15-30 seconds. Learning this technique is essential to everything else you do in microbiology. 2. Flame Slowly passing of an objects to the Bunsen flame will reduce the number of microorganisms. In the case of flaming, the instrument is dipped in alcohol or spirit before burning it in a gas flame. This process doesn’t ensure sterility and is no t as effective as red hot sterilization. The Bunsen flame gives partially sterile area around it in which used for work (preparation of smears, cultivation, subculture, etc…). Used for sterilization of the mouth of bottle, flasks, containers and test tubes. 3. Incineration Incineration is the process of sterilization along with a significant reduction in the volume of the waste. It is usually conducted during the final disposal of the hospital or other residues. The scraps are heated till they become ash which is then disposed of later. This process is conducted in a device called an incinerator. It treats objects to heating over 250C until they become black. Done for used equipment. 4. Hot air oven A hot air oven is a method of dry heat sterilization which allows the sterilization of objects that cannot be sterilized by moist heat. It uses the principle of conduction in which the heat is first absorbed by the outer surface and is then passed into the inner layer. A hot air oven consists of an insulated chamber that contains a fan, thermocouples, temperature sensor, shelves and door locking controls. The commonly-used temperatures and time that hot air ovens need to sterilize materials are 170°C for 30 minutes, 160°C for 60 minutes, and 150°C for 150 minutes. These ovens have applications in the sterilization of glassware, Petri plates, and even powder samples.  Check the efficiency BoweiDick test (Adhesive tape). Browne’s tube No 3. (Red ----- green). b. Moist heat Less than 100°C Pasteurization of milk: Used heat at temperatures sufficient to inact ivate harmful organisms in milk. o Holding method (65 °C for 30 min) o Flash method (72 ° C for 20 sec) o Ultrahigh-temperature(UHT) sterilization-140 to 150°C for 1 to 3s To prevent diseases like : o Typhoid fever, Brucellosis, Tuberculosis and, Q fever Inspeciation: o Heating at 80 ° C for 30 - 1hrs until coagulation of protein. o Used for preparation of dorset egg medium and L.J medium. Preparation of vaccine: o By heating at 56 ° C for 30-60 min. At 100°C Steaming (Koch steamer): o Single exposure of the microbe to steam at 100°C for 90 min. o Tyndalization: o Steaming at 100°C for 30 min for 3 successive days. o 1st day kill vegetative bacteria and germinate sporulated one ---- Put on the bench. o 2nd day kills all vegetative bacteria ---- Put on the bench. o 3rd day insure complete sterilization. Boilling: o At 100C for 30 min. Above 100°C (Autoclaving) Depends on steam and pressure. Steam is a hot sticky air able to penetrate through things. Pressure will rise the temperature from 100°C to 121°C Kills microbes and their spores by coagulation of protein and denaturation of enzymes. Make complete killing of bacteria, their spores, fungi and their spores, parasites, and viruses including Envelop and non Envelop viruses. Thermal death point and thermal death time: o 121°C (15 bound or 1.1 bar) for 15 min. o Flash autoclaving at 134°C for 4-5min Autoclaving Need to maintain autoclaves and monitor effectiveness Temperature & pressure charts Chemical indicators (Browne’s tubes, Bowie-Dick test) Spore tests Advantages of Autoclave o Temp. > 100 C therefore spores killed. o Condensation of steam generates extra heat. o The condensation also allows the steam to penetrate rapidly into porous materials. Monitoring of Autoclaves 1. Physical method: use of thermocouple to measure accurately the temperature. 2. Chemical method: it consists of heat sensitive chemical that changes color at the right temperature and exposure time. e.g. a)- Autoclave tape b)- Browne’s tube. 3. Biological method: whe re a sp ore-bearing organism is added during the sterilization process and then cultured later to ensure that it has been killed. Irradiation Irradiation is the process of exposing surfaces or objects to different kinds of radiation for sterilization. Exposure to irradiation causes denaturation of proteins and enzymes. Used for plastic syringes, disposable plastic and dental equipments It is of two types: o **Non-ionising Radiation: **Ultraviolet radiation is exposed to the object, which is absorbed by nucleic acids of the microorganisms. This leads to the formation of pyrimidine dimers in the DNA strand, which causes the replicative error, and eventually, the microbe dies. Generally used in irradiation of air in certain areas such as operating rooms and tuberculosis labs. o **Ionising Radiation: **Upon exposure to ionising radiations such as gamma rays and X- rays, reactive oxygen species such as hydrogen peroxide and superoxide ions are formed that oxidise the cellular components of the microbe, and they die. Infrared radiation Infrared radiation (IR) is a method of thermal sterilization in which the radiation is absorbed and then converted into heat energy. For this purpose, a tunnel containing an IR source is used. The instruments and glassware to be sterilized are kept in a tray are then passed through the tunnel on a conveyer belt, moving at a controlled speed. During this movement, the instruments will be exposed to the radiation, which will result in a temperature of about 180°C for about 17 minutes. IR is applicable for mass sterilization of packaged items like syringes and catheters. UV radiation Ultraviolet is light with very high energy levels and a wavelength of 200-400 nm. One of the most effective wavelengths for disinfection is that of 254 nm. Filtration Filtration Sterilization through removing of microbes from fluids by exposing to small size filter. Used for heat sensitive fluids like serum, CHO and urea. This is a mechanical method of sterilization in microbiology. This method uses membranous filters with small pores to filter out the liquid so that all the bigger particles and microbes cannot pass through. The three steps of filtration are sieving, adsorption and trapping. Further, it is capable of preventing the passage of both viable and nonviable particles and can thus be used for both the clarification and sterilization of liquids and gases. Generally removes most bacteria but viruses and some small bacteria e.g. Chlamydia & Mycoplasma may pass through Main use: for heat labile substances e.g. sera, antibiotics Different filtration units is used according to the amount of filtrate and pores of the filter: Seitz filter ---- use asbestos Chamberland filter ------ use ceramic Sintered filter ----- use glass filter. Milipore or membrane filter ---- use filter paper Factors affecting filtration Filters o Type o Thickness. o Pore size Material filtered o Quantity o Type o viscosity Sterilization by Chemical Methods Useful for heat sensitive materials e.g. plastics and lensed endoscopes). 1. Ethylene Oxide Chamber * Ethylene oxide alkylates DNA molecules and thereby inactivates microorganisms. * Ethylenes oxide may cause explosion if used pure so it is mixed with an inert gas.Requires high humidity (50- 60% ).Temperature : 55-60°C and exposure period 4-6 hours. 2. Activated alkaline Gluteraldehyde 2% * Immerse item in solution for about 20 mins. If Mycobacterium tuberculosis or spores present then immersion period 2-3 hours. Disinfection * Disinfection is a process that reduces the number of microorganisms present to a level at which they do not present a risk. Disinfection differs to sterilization whereby the object is free of all viable microorganisms. Chemical disinfectants do not generally kill all the microorganisms that they come in contact with and do not disinfect all microorganisms equally well, for example most do not kill bacterial spores. The type of disinfectant selected will depend on the microorganism, the method of application and the nature of the material to be disinfected. Physical disinfection methods includes drying or application of heat (or thermal inactivation), or the use of irradiation methods (most commonly ultraviolet light). PHYSICAL (1) Boiling - Household use, temporary, expensive, emergency measure. - Kills bacterial, viruses + other microorganisms. (2) U-V light - effective for bacteria + viruses if Turbidity is low (a) Simple storage in glass containers - effective but not very practical CHEMICAL 1. Phenolic Group Examples: Phenol crystal, Dittol, Lysol, Cresol Mechanism: Injures lipid-containing plasma membranes, leading to leakage of cellular contents. Advantages: Remains active in the presence of organic materials, stable and persistent for long period of time. Applications: Suitable for Disinfecting pus, blood, sputum. Activity: Effective against Gram-positive, Gram-negative bacteria, Mycobacterium, and viruses. 2. Bisphenols Structure: Contains two phenol groups. Examples: o Hexaclorophenol: Used for surgical and microbial control (excessive use in infants can lead to neurological damage). o Triclosan: Found in antimicrobial soaps, inhibits fatty acid synthesis, affecting plasma membrane integrity. 3. Biguanides Example: Chlorohexidine Mechanism: Broad spectrum activity as disinfectant of skin and mucus membrane due to it is ability to bind to mucus membrane. Cause injury to plasma membrane. 4. Halogens Iodine: o Broad-spectrum activity against to many bacteria, spores, fungi, and some viruses. o Mechanism: Binds to amino acids in enzymes and proteins. Iodophores (Betadine, Isodine) is non pigmented iodine comprise from iodine + organic materials to release the iodine slowly. Used for skin disinfection and wounds. Chlorine: o in shape of gas or solution combined with water. o Applications: Used for Swimming pools, drinking water, sewage treatment. 5. Alcohols Mechanism: Denatures proteins, disrupts membranes, dissolves lipids. They able to act and Evaporate (Volatile). Applications: Skin disinfectant, vein puncture (70% concentration). 6. Heavy Metals Examples: Silver, Mercury, Copper Mechanism: denaturation of proteins when bind to it Applications: o Silver nitrate: Used as eye drops to prevent ophthalmia neonatorum by Neisseria gonorrhoeae. o Silver sulfadiazine (Flamazine): Used for treating burn infections. o Copper sulfate: Used to destroy green algae (effective in 1 part/million of water). 7. Dyes Examples: Gentian violet, Crystal violet, Eosine Activity: are very Effective antiseptics. 8. Surface Active Agents Examples: Soap, detergents Mechanism: Mechanical removal of microbes by scrubbing dead tissue so reduce their number. Quaternary Ammonium Compounds: o Positively charged molecules that kill Gram-positive bacteria. o Affect plasma membranes and alter cell permeability. o Example: Citerimide 9. Aldehydes Examples: Formaldehyde, Glutaraldehyde Mechanism: Inactivate proteins. Applications: Disinfecting hospital instruments, benches, and rooms. 10. Ethylene Oxide Gas Mechanism: Denatures proteins. Activity: Bactericidal, fungicidal, virucidal (kills viruses). Applications: Sterilizing plastics in a closed chamber (similar to autoclave). Considerations: Used at high temperatures (60°C) for 4-24 hours. Carcinogenic. Factors Influencing Disinfectant Activity: 1. Temperature: Activity directly proportional to temperature. 2. Concentration: Directly proportional to concentration up to a point – optimum concentration. After this level no advantage in further increases in concentration. 3. Inactivation: Disinfectants can be inactivated by: o Dirt o Organic matter (proteins, pus, blood, mucus, feces) o Non-organic materials (cork, some plastics) 4. Time: Disinfectants need time to work. 5. Range of Action: : Disinfectants not equally effective against the whole spectrum of microbes. e.g. Chlorhexidine less active against Gram negative bacteria than Gram positive cocci. Hypochlorites and Gluteraldehyde are more active against hepatitis viruses than most other disinfectants. Hospital Disinfection Methods: Article Disinfectant Floors, walls Phenolics fluids 1-2% Surfaces tables Hypochlorite, Alcohol Skin Surgeons’ hands Chlorhexidine, Iodine alcohol Patient skin 70% Alcohol, Iodine Endoscopes Gluteraldehyde 2% (Cidex), subatmospheric steam Thermometers 70% Alcohol Important Points: Sterility: Any instrument or item used for sterile body sites should be sterile. Disinfection: Any instrument or item used for non-sterile body sites can be disinfected. Hand Washing: The most important method to prevent hospital-acquired infection. Disinfectant Levels: Tests of Disinfectant Activity: In-Use Test: o Used to determine the proper disinfectant concentration to be used and to check if the used disinfectant is working properly or not: o Take 1ml from used disinfectant to 9 ml of nutrient broth. Immediately transfer 0.02ml into 10 different area of well dried nutrient agar. o Incubate one plate at 37C for 3 days and the other at R.T for 7 days. o Growth in more than 5 drops- Failure of Disinfectant o No growth ------- good disinfectant o Growth in less than 5 drop --- success but needs increasing the concentration.