Laboratory Diagnosis of Viral Disease, Viral Replication, Genetics & Pathogenesis, Virology Structure and Classification, Antibacterial Drugs, Diagnosis of Bacterial Infection, Bacterial Growth and Death, Bacterial Pathogenesis, Bacterial Genetics, B...

Prophage

A phage genome that has been inserted into a specific site on the bacterial chromosome.

Axial Filaments

(endoflagella) For motility Flagellin covered by outer sheath (membrane) Only found in Spirochetes

Uptake of mobile genetic elements

(phages, virulence plasmids and pathogenicity islands), as well as the loss of chromosomal-DNA regions in different E. coli lineages, has enabled the evolution of separate clones, which belong to different E. coli pathotypes and are associated with specific disease symptoms. LEE, locus of enterocyte effacement; PAI, pathogenicity island; pEAF, enteropathogenic E. coli adhesion-factor plasmid; pENT, enterotoxin-encoding plasmids; Stx, Shiga-toxin-encoding bacteriophage.

Lysogenic Cycle

A phage replication cycle in which the viral genome becomes incorporated into the bacterial host chromosome as a prophage and does not kill the host.

Structural Components of Bacteria

1. Cell Wall 2. Plasma Membrane 3. Cytoplasm 4. Ribosomes 5. Nucleoid 6. +/- Plasmids 7. +/- Capsule 8. +/- Biofilms 9. +/- Pili/Fimbriae 10. +/- Flagella 11. +/-Axial Filaments 12. +/- Spores

Viral Isolation and Growth

1. Chicken eggs (Embryonated) - still used for some viruses 2. Animals - mainly for research purposes 3. Cell culture: Three types of cell lines: monolayer growth Primary cells - e.g. Monkey Kidney cells. These are essentially normal cells obtained from freshly killed adult animals. These cells have limited life span, and can only be passed (cultured) once or twice. Semi-continuous cells (diploid cell line)- e.g. Human embryonic kidney cells and skin fibroblasts. These are cells taken from embryonic tissue, and may be sub-cultured up to 50 times. Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM. These are immortalized cells i.e. tumor cell lines and may be sub-cultured indefinitely.

Growth requirements for bacteria

1. Essential elements: Hydrogen, Oxygen, Carbon, Nitrogen, Phosphorus and Sulphur 2. Mineral sources: K, Mg, Fe, Cu, Co, Mn, Mb, Zn 3. Organic growth factors: Growth factors or bacterial vitamins May be essential or accessory Vitamins: thiamine, riboflavine, nicotinic acid, pyridoxine, folic acid and vitamin B-12

Immunopathogenesis

1. Excessive immune response: innate, specific and inflammatory responses - Examples NK and CTLs - host cell death Activated neutrophils and macrophages: bystander damage Complement activation: anaphylatoxins, inflammation Granuloma formation: interfere with normal function 2. Molecular mimicry: Induction of autoimmune diseases: eg, M protein of Streptococcus pyogenes and heart protein (Rheumatic fever) 3. Immune complex deposition: In Bacterial endocarditis nephritis, arthritis, vasculitis

Mechanism of antibiotic resistance

1. Inactivation (cleavage) of antibiotics by enzymes 2. Active Efflux of drugs by membrane-bound multiple drug resistant (MDR) efflux pumps 3. Chemical Modification of the antibiotic: eg. transfer of acetyl group, methyl group 4. Decrease cell permeability of cell 5. Hyper-production of target 6. Mutation or post-translational modification of drug target 7. Formation of biofilm

Gene Transfer

1. Transformation: - the transfer of "naked" DNA. (DNA fragment and or plasmid) 2. Conjugation: the passage of plasmids/genetic elements through direct physical contact between two bacteria 3. Transduction: the transfer of genetic information through virus (phage). 4. Transposition: the movement of small pieces of DNA called transposons to different locations in the genome and between plasmids. These are sometimes called "jumping genes."

Environmental factors affecting growth

1. Water 2. Oxygen (obligate aerobes, microaerophiles, facultative anaerobes, aerotolerant anerobes and obligate anerobes) 3. Carbon dioxide 4. Temperature (Psychrophilic: below 20, meso-philic: 25-40, Thermophilic: 55-80 0 C) 5. Hydrogen ion concentration (optimal pH 7.2-7.4) 6. Light: Most prefer to grow in darkness except phototrophic 7. Osmotic pressure: Osmotically stabilized with 0.5% NaCl in growth media

Phenol

1st disinfectant used rarely used, replaced by phenol derivatives Act by dissolving lipid solvents - disrupts cell membranes Effective disinfectants but too toxic for antisepsis Not sporicidal, not effective against non-enveloped viruses

Protein Synthesis inhibitors

30s Aminoglycosides Tetracyclines 50s Macrolides Chloramphenicol Lincosamides Oxazolidinones Streptogramin

Bacterial Plasma membrane

4 important functions: Active transport of molecules into the cell Energy generation by oxidative phosphorylation Contains polymerizing enzymes for synthesis of cell wall Secretion of enzymes and toxins Components Phospholipid bilayer: similar to eukaryotic cells: Except that sterols are absent Some integral and/or peripheral glyco/lipoproteins participate in transport and/or metabolism

Viral Composition

A Complete virus is called a virion or a viral particle Genome + capsid = Nucleocapsid

Gas Pak system

A commercial product, available in the form of a disposable packet of aluminium foil containing pellets of sodium borohydride and cobalt chloride and of citric acid and sodium bi-carbonate, is now widely used for preparing anaerobic jars. The chemicals generate hydrogen and carbon di oxide inside the jar when water (10ml) is added. Hydrogen combines with Oxygen in the presence of a catalyst (eg. Alumina pellets coated with palladium) present in the undersurface of the lid of the jar. After the inoculated plates are placed inside a large air-tight jar, it is incubated at 370 C. The gas pak technique is simple and effective.

Lac Operon

A gene system whose operator gene and three structural genes control lactose metabolism in E. coli Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter, and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. LacY encodes lactose permease, a protein which becomes embedded in the cytoplasmic membrane to enable transport of lactose into the cell. Finally, lacA encodes galactoside O-acetyltransferase.

Pathogen

A microbe that has the ability to cause disease

Infrared combustion technology

A mobile thermal processing system that uses electrically-powered silicon carbide rods to heat organic wastes to combustion temperatures. Waste is fed into the primary chamber and exposed to infrared radiant heat (up to 1,850 °F) provided by silicon carbide rods above the conveyor belt. A blower delivers air to selected locations along the belt to control the oxidation rate of the waste feed. Any remaining combustibles are incinerated in an afterburner.

Suppression mutation

A mutation (second) that restores the function of a gene inactivated by previous mutation (first): Example ATA to AAA causes Tyr to Phe AAA to ATA causes Phe back to Tyr

Stages of Disease Progression

A primary infection refers to an infection you acquire for the first time A secondary infection is an infection that occurs during or after treatment of another pre-existing infection. It may result from the treatment itself or from changes in the immune system. For example, a vaginal yeast infection that occurs after antibiotic treatment of a bacterial infection is a secondary infection Types of infection: acute, chronic primary, secondary

Syncytia formation

A process wherein cells are caused by viruses to fuse together into giant cells. Multi-nucleated syncytial cells are often the result of infections by enveloped viruses that induce cell fusion.

Sigma Factor

A protein that associates with RNA polymerase that facilitates its binding to specific promoters a protein for initiation of RNA synthesis May associate with the RNA polymerase before binding to the promoter region or it may bind to promotor and act as a docking station for the polymerase. The specific sigma factor used to initiate transcription of a given gene will vary, depending on the gene and on the environmental signals needed to initiate transcription of that gene. Some sigma factors are inserted into the plasma membrane to "sense" the environment - may be targets for the adaptive immune system Turns on or off genes DNA- dep - RNA polymerase requires specific Sigma factors for accuracy and affinity of binding to the promoter New sigma factors allow new phenotypic expressions

One step viral growth curve

A single cycle of virus growth can be studied by determining the titer of infectious virus (e.g. by plaque assay) over time. Viral titer is stable during the brief period of virus attachment. Following attachment, viral titer declines precipitously as the virus undergoes disassembly, transcription, translation, and genome replication. That interval is termed the eclipse. Viral titer begins to increase as progeny virions, which are fully infectious, are assembled. Eclipse phase: corresponds to the period during which the input virus becomes uncoated. As a result, no infectious virus can detected during this time (any infectious virus detected is simply virus that is still stuck on the cell membrane) (Time period between entry and assembly/maturation) Latent period. During this period, no extracellular virus can be detected. (Time period between entry and release) Burst size : amount of infectious virus produced, per infected cell

Autoclave

A sophisticated pressure cooker. In its simplest form, it consists of a chamber in which the air can be replaced with pure saturated steam under pressure. Air is removed either by evacuation of the chamber before filling it with steam or by displacement through a valve at the bottom of the autoclave, which remains open until all air has drained out. The latter, which is termed a downward displacement autoclave, capitalizes on the heaviness of air compared with saturated steam. When the air has been removed, the temperature in the chamber is proportional to the pressure of the steam; autoclaves are usually operated at 121°C, which is achieved with a pressure of 15 pounds per square inch. Under these conditions, spores directly exposed are killed in less than 5 minutes, although the normal sterilization time is 10 to 15 minutes to account for variation in the ability of steam to penetrate different materials and to allow a wide margin of safety. The use of saturated steam in the autoclave has other advantages. Latent heat equivalent to 539 cal/g of condensed steam is immediately liberated on condensation on the cooler surfaces of the load to be sterilized. The temperature of the load is thus raised very rapidly to that of the steam. Condensation also permits rapid steam penetration of porous materials such as surgical drapes by producing a relative negative pressure at the surface, which allows more steam to enter immediately. Autoclaves can thus be used for sterilizing any materials that are not damaged by heat and moisture, such as heat-stable liquids, swabs, most instruments, culture media, and rubber gloves

Antimetabolites

A substance that interferes with the normal metabolic processes within cells, typically by combining with enzymes or mimic an important or a required metabolite Inhibition of Folic acid Synthesis Sulfonamides (several derivatives) Trimethoprim Para-aminosalicylic acid (PAS) Dapsone

Antiseptic

A substance/chemical that can be applied to tissue and inhibits the growth of microorganisms.

Viral neuraminidase

A type of neuraminidase found on the surface of virus (eg. Paramyxo virus, orthomyxovirus ) that enables the virus to be released from the host cell. Neuraminidases are enzymes that cleave sialic acid groups from glycoproteins and are required for influenza virus replication.

Lytic cycle

A type of viral (phage) replication cycle resulting in the release of new phages by lysis (and death) of the host cell. Eclipse phase: Entry of nucleic acid into the cell and first appearance of infectious intracellular phage particle Latent phase: Time interval between infection of a cell to the first release of infectious phage particles.

Complex virus

A virus with a complicated structure, such as a bacteriophage. Unusual morphology e.g. Poxvirus (enveloped), bacteriophage

Prokaryotes vs Eukaryotes

Protein synthesis in bacteria =n-formyl methionine as the first amino acid; in eukaryotes it is methionine

Activation of G protein

A-B toxin causes ADP ribosylation Increase in adenylate cyclase activity - Increase in cAMP In the respiratory epithelium, the high levels of cAMP results in increased respiratory secretions and mucous production and contributes to coughing. 1. Cholera toxin (Vibrio cholerae)/Heat labile toxin (Escherichia coli) - Act on small intestine Cause watery diarrhea/secretory diarrhea 2. Pertussis toxin (Bordetella pertussis): Respiratory epithelium pathogen The high levels of cAMP results in increased respiratory secretions and mucous production and contributes to coughing.

Inactivation of EF-2

A-B toxin causes ADP ribosylation Leads to Inhibition of protein synthesis 1. Diphtheria toxin: Corynebacterium diphtheriae Upper Respiratory tract pathogen; results in cell death. Toxin can enter circulation and affect heart, kidney, nerves Toxin gene carried by temperate phage 2. Exotoxin A: Pseudomonas aeruginosa Pneumonia in cystic fibrosis patients

Increase of antibiotic resistance

A. Increased use of antibiotics Selects for antibiotic resistance phenotypes. Administration of antibiotics without need 1/3 of outpatient prescriptions for antibiotics are unneeded Unregulated prescriptions Use of broad-spectrum antibiotics Use of antibiotics in agriculture Bystander effect on normal flora Serve as a link to pass on resistance genes When the antibiotics attack pathogenic (disease causing) bacteria, they also affect benign bacteria (innocent bystanders) in their path. They eliminate the drug susceptible bystanders that could otherwise limit the expansion of the pathogens and they simultaneously encourage the growth of resistant bystanders. Propagation of these resistant non pathogenic bacteria increases the reservoir of resistant traits in the bacterial population as a whole and raises the odds that such traits will spread to pathogens. In addition, sometimes the growing populations of bystanders themselves become agents of disease. B. Increased mobility of world's population C. Hospital setting: Selection and maintenance of antibiotic resistance genes

Pathogenicity

Ability to cause disease

Types of viral infection

Abortive: in non-permissive cells; replication does not occurNon permissive cell: may lack a receptor, important enzyme pathway, transcriptional activator or express an antiviral mechanism that will not allow replication of the virus Viral mutants, which cause abortive infections, do not multiply and disappear. In-apparent: little to no damage or rapid resolution eg. Cytomegalovirus in healthy Acute/Lytic : in permissive cells: host cell death - eg. Adenovirus Persistent: starts with acute phase, remain long A. Chronic (productive): Hepatitis B virus B. Latent (limited viral macromolecular but no virus synthesis): eg, Herpes virus C. Recurrent (periods of latency then virus production): eg, Herpes virus D. Transforming- Immortalization - oncogenesis - oncogenic virus eg. Papillomavirus

Non-Gram-staining bacterium

Acid Fast: Mycobacterium Cell wall less: Mycoplasma Atypical cell wall: Chlamydia, Chlamydophila, Orientia, Ehrlichia and Anaplasma, Rickettsia Spirochetes: Treponema Borrelia Leptospira

Sulfonamide and Trimethoprim

Act in the same pathway toward the synthesis of folic acid and eventually to purines and DNA. Sulfonamides are structural analogue to PABA. Sulfas mimic para-aminobenzoic acid needed by bacteria for folic acid synthesis. Sulfas inhibit bacterial folic acid synthesis at the level of the enzyme dihydropteroate synthetase Trimethoprim impairs folic acid synthesis at the level of dihydrofolate reductase. Human cells are not affected by these drugs, because unlike bacterial cell human cells require preformed folic acid Pharmacology: Usually used together Broad spectrum Bacteriostatic Urinary tract infections Gastrointestinal tract infections Respiratory infections Mechanisms of Resistance Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. Alteration (mutation) of enzymes dihydropteroate synthetase and dihydrofolate reductase Overproduction of PABA and or dihydrofolic acid Many bacteria, including those found in the human intestinal tract such as E. coli, generate PABA from chorismate, a biochemical intermediate. Folic acid dependency Decreased permeability

Innate Response Against Viruses

Activation of NK cells Infected cells produce interferon 1 Dendritic cells/macrophages produce IL-12, TNF alpha, etc. Toll like receptors TLR3 and TLR7, TLR8 found in NK cells: recognize viral PAMP These lead to activation of NK: which in turn results in -Killing of infected cells -Production of interferon gamma and IL-12 -favouring the development of Th1 (promote cell mediated immunity) -activation of CD8 and macrophages. Once NK are activated by either by IFN alpha/beta, IL-12, TNF alpha or by binding ssDNA/dsRNA through TLR3/TLR7/TLR8, they produce Interferon gamma which favour the development of Th1 and also activates CD8 and macrophages.

Multi-site mutations

Also known as Macrolesion. Alteration of DNA involving large no. of base pairs, More comprehensive changes in the DNA, involve loss (deletion), gain (addition), duplication or inversions

Phase variation (conversion)

Also known as programmed gene rearrangement Switch genes on/off: results in phenotype switching that is faster than mutation Transcriptional regulation of alternative genes. This is not mutation: no gene is changed, Only the direction of the promoter or position of a gene is changed. There are several ways this is achieved The changes may affect host cell binding and virulence Eg. Pili of Neisseria gonorrheae Several genes encoding proteins with the same or similar functions and the ability to express only one of the these genes at a time. In effect this is achieved by maintaining only one active promoter at a time and/or moving genes to a position downstream of an active promoter.

Fluorescence Microscope

An optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to look at organisms on a slide. Up to 2000x magnification.

Molecular Version of Koch's Postulates

An organism under investigation should be a member of pathogenic strains; and gene in question should be found in all pathogenic strains but absent from nonpathogenic strains The gene(s) coding for the responsible virulence factor(s) must be expressed during infection Inactivation of the gene (s) associated with virulence should lead to measurable decrease in pathogenicity Reversion of the inactivated gene/s should lead to restoration of pathogenicity. originally described by Dr.Stanley Falkow

Significance of conjugation

Antibiotic resistance, R Plasmids Plasmid carrying multiple antibiotic resistance genes Rapid spread The whole process of conjugation takes about 100 min Virulence genes Pili genes that mediate adherence to epithelial cells Exotoxins genes can be transferred.

Serological analysis

Antibody detection/titer ELISA Heamagglutination inhibition assay Neutralization tests: block cytopathic effects Western blot Rapid tests eg, for HIV Neutralization (prevention of cytopathic effect) Hemagglutination inhibition assay

Lipopolysaccharide (LPS)

Antigen - surface antigens on Gram negative bacteria Target for specific immune response Serotyping of bacteria Core polysaccharide - contributes to the integrity of membrane Conserved among related Gram negatives Lipid A - long fatty acid: Endotoxin: Inflammation, hypotension, shock some variation across species

Culture medium

Any material that contains the required nutrition and supports the growth of the microbes and is prepared in laboratory. It could be in liquid form - broth Solid/Semisolid - broth + a solidifying agent (agar)

Peplomers (spikes)

Are proteinaceous projections that extend from the capsid; or viral glycoproteins inserted on envelope Importance of Peplomers Attachment of the virus to host cells, act as viral attachment proteins (VAPs), Determine tissue and host specificity Some may have enzymatic activity to aid entry or exit of the virus Antigens - target for the immune system Have other useful properties—as antigens useful in classification, hemagglutination

Antimicrobial Susceptibility Testing

Are used to make the best drug of choice to treat infections Diffusion Tests Dilution Tests Automated Tests Phenotype testing/Enzyme testing Molecular tests: Genotype testing

Assembly and Release

Assembly The site of assembly depends on the site of replication of genome Assembly can be in the nucleus or cytoplasm Nucleocapsid assembly Procapsid formation - empty capsid proteins assemble first then gets filled with the genome OR, Capsid proteins assemble around the genome. Release Naked viruses: released by cell lysis or exocytosis Enveloped viruses: Viral proteins are delivered to cellular membranes. Nucleocapsid moves underneath the membranes (nuclear, golgi, or cell membrane) with viral proteins; and leave by budding.

Bacterial Cell Morphology

Basically there are three shapes Rods/bacilli (Coccobacilli) Cocci Spiral (spirochete, spirilla) Some show variation of these three basic structures Most bacteria are monomorphic Few are pleomorphic (many-shaped)

Categories of Infectious Agents

Bacteria - Prokaryotes, unicellular Virus - acellular, has either DNA or RNA Fungi - eukaryotes, unicellular/multicellular Parasites - eukaryotes, unicellular/multicellular Prions - abnormal proteins/ proteinaceous infectious agents, no nucleic acid

Bacterial Growth

Bacteria grow in size and number. Growth in number - population growth Population size increases by cell division They divide by binary fission A single cell divides to give two identical daughter cells The number of cells (population size) = 2n (n = generation number) This is exponential growth The doubling time - generation time varies Escherichia coli - 20 min Mycobacterium tuberculosis - 24 hrs

Intrinsic Resistance

Bacteria lacking the target - e.g, lack of peptidoglycan. Cell wall less: Mycoplasma, Ureaplasma Cell wall without typical peptidoglycan: Chlamydia, Chlamydophila,Orientia, Ehrlichia and Anaplasma, Rickettsia The antimicrobial cannot enter the bacterium eg. Gram negative bacteria due to the outer membrane; Acid fast bacteria due to mycolic acid

Naturally Cell Wall-Less

Bacteria that do not produce peptidoglycan, but have unusual form of cell wall. These are Chlamydia, Chlamydophila, Ehrlichia, Orientia and Anaplasma. Are resistant to antimicrobials which target peptidoglycan synthesis Components: (Picture) No peptidoglycan present Assume a variety of shapes Sterols within a phospholipid Bi-layer membrane; Maintain fluidity Cannot be stained using the Gram stain procedure Resistant to antibiotics that target cell wall Examples of Cell Wall-Less Bacteria: Mycoplasma spp. Ureaplasma spp.

Types of Culture Media

Basal (Simple) Media: contains peptone, meat extract, NaCl & H20 eg., Nutrient agar Enriched Media: contains additional substances like in blood, serum, eggs or extracts from animal tissue that encourage the growth of organism eg, Blood agar, Chocolate agar Selective media: allows growth of only the desired microbes. The purpose is to inhibit the growth of unwanted organisms eg, LJ medium for M. tuberculosis Enrichment media: purpose same as selective media but liquid eg, Selenite F broth for Shigella Differential Media: are used to distinguish among different organisms eg. MacConkey agar, EMB agar Transport media: eg. Stuart's medium for Gonococci

Nutritional classification of bacteria

Based on their energy requirements and on their ability to synthesize essential elements Autotrophs: can synthesize all their organic compounds Photo-autotrophs: derive their energy from sunlight Chemo-autotrophs: obtain their energy from chemical reactions Heterotrophs: Unable to synthesize their own metabolites and depend on preformed organic compounds Majority of the pathogenic bacteria are heterotrophs

Cell Wall Synthesis Inhibitors

Beta Lactams -Penicillins -Cephalosporins -Carbapenems -Beta Lactamase Inhibitors: are structural analogues of penicillins/cephalosporins and have little antibacterial activity but bind strongly to beta lactamases thus protect penicillins/cephalosporins. Vancomycin Bacitracin For t/t of mycobacterial infection Cycloserine Isoniazid (INH) Ethionamide Ethambutol Pyrazinamide

Penicillin resistance

Beta-lactamase production: Genes carried on plasmids : spread easily Betalactamase activity: have various degree of spectrum Penicillinase : inactivates penicillins Cephalosporinase: Inactivates cephalosporins, Extended-spectrum batalactamase (ESBL); inactivates both drugs (P+C) Carbapenemase: against Penicillins + Cephalosporins + Carbapenems + Monobactems Alteration of Penicillin Binding Protein (form PBP 2A) Eg. By Methicillin resistant Staphylococcus aureus (MRSA) (mec A gene mediated: alter target)

Penicillin

Betalactam Antibiotic Structure: Nucleus: 6-Aminopenicillanic Acid Beta Lactam Ring Thiazolidine Ring (5 membered) R group R group altered in Semisynthetic Penicillins Access of beta lactamase enzyme to the beta lactam ring of semisynthetic penicillin is blocked by modification of the side chain with the addition of large aromatic rings, containing bulky methyl or ethyl groups.

Tegument

Between capsid and envelope-containing viral proteins and enzymes for replication

Adhesion Factors

Binding specific molecules on host cells Determines tissue/ host specificity Pili, capsule, biofilm Cell wall structure: lipoteichoic acid Proteins on the cell wall Eg. Escherichia coli - urinary tract infection - has P fimbriae binds to Gal-Gal dimer on P antigen found on uroepithelial cells F protein found on cell wall of Streptococcus pyogenes - binds to fibronectin

Bacterial Classification

Binomial nomenclature: Genus and species: Staphylococcus aureus Classification/taxonomy: based on- Phenotype: based on morphology, staining properties, biochemical properties (biotyping), Genetics: DNA studies Antigenic variation: serotyping Phage typing : Sensitivity to bacteriophage

Variations of Microscopes

Bright-field (eg, Compound microscope)- most widely used; specimen is darker than surrounding field; best for preserved and stained specimens Dark-field - brightly illuminated specimens surrounded by dark field; used to detect live and unstained specimens - mostly for spirochetes Phase contrast : Uses parallel beam of lights which passes through objects of different densities. The wavelength of one beam moves out of 'phase' relative to other beam of light. Used for detailed analysis of internal structure in living cells UV/ Fluorescent microscope: uses ultraviolet light and fluorescent compounds - mostly for immunofluorescent (eg. DFA) stained smear - for fast diagnosis Such as CSF, Tissue biopsy, culture isolate Electron microscope: uses beams of electrons - mostly used for viruses, Types- Scanning and Transmission EM

Live attenuated influenza vaccine

Called FluMist (in the form of nasal spray), uses a temperature-sensitive mutant that can be reassorted with any new virulent influenza strain that appears. The reassorted virus will have the genes for the internal proteins from the attenuated virus (and hence will be attenuated) but will display the surface proteins of the new virulent antigenic variant.

Toll like receptors

Can directly activate NK cells in case of viral infection

Dilution test

Can measure MIC: Minimal inhibitory concentration MBC: Minimal bactericidal concentration Can be automated - results in few hrs Series of dilutions of the drug in broth media - inoculate same number of bacteria in every tube and determine the lowest concentration of drug that inhibits the growth of a particular bacteria, Minimum inhibitory concentration (MIC) = eg (below) 3.1 g/mL Does not mean this kills the bacterial isolate, only inhibits Minimal bactericidal Concentration (MBC) = 6.2 µg/ml that kills at least 99% of the bacteria For some infections -e.g, endocarditis- it may be necessary to determine amount of antibiotic that kills the bacteria, and also immunocompromised patients should be given a dose that would kill -the bacteria Bactericidal drugs usually have an MBC equal or very similar to the MIC, whereas bacteriostatic drugs usually have an MBC significantly higher than the MIC (Ref: Levinson)

Interference

Capsule: antiphagocytic Biofilm: prevent complement and antibodies Avoid killing: Inhibition of phagosome function or escape into the cytoplasm Killing of phagocytic cell by producing toxins Avoiding Ig mediated opsonization; bind Fc of IgG -Protein A of Staphylococcus aureus and -M protein of Streptococcus pyogenes Cleavage of antibodies/complement IgA proteases, C5a proteases

Opportunistic pathogens

Cause disease in immun-ocompromised individuals or when normal flora enter body site other than normal site or overgrowth Microbes with small IDs have greater virulence. True pathogens (primary pathogens): cause disease in immuno-competent individuals

Mechanisms of viral transformation

Cell growth is controlled by the maintenance of a balance in the external and internal growth activators (accelerators) and by growth suppressors, such as p53 and the retinoblastoma (RB) gene product (brakes). Oncogenic viruses alter the balance by removing the brakes or by enhancing the effects of the accelerators.

Adaptive Response Against Viruses

Cell-mediated: CD4 Th1 and CD8 Tc: killing infected cells, targets the intracellular phase of the virus Humoral immunity: primarily targets extracellular phase/cell surface expressed antigen Antibodies against viral attachment proteins (VAP): prevent infection Opsonization complement activation by antibody against virus Elimination via ADCC by NK cells (Ab provides specificity) Neutralization: Prevents spread by blocking extracellular virus particles

Point mutations

Change in single nucleotide Base pair substitution (Transition-most frequent, Transversion) Silent mutation: does not produce obvious effect Also called microlesion, changes in single nucleotides, may not necessarily result in detectable change in protein synthesis. Generally reversible. Two classes: I. Base pair substitution: Single base pair is substituted for another pair Transition-Substitution of one base pair but the purine, pyrimidine orientation is preserved. Happen by replacement of one pyrimidine by another pyrimidine, and one purine by another purine. Most frequent type of mutation Transversion: Pyrimidine is replaced by purine and vice versa, less frequently observed II. Silent mutation: It is a change at the DNA level only that does not result in any change of amino acid in the encoded protein. This is because more than one codon may encode an amino acid eg. GGT to GGA still gives proline.

Botulinum toxin

Clostridium botulinum toxin blocks/inactivates SNARE proteins needed for acetylcholine release causing flaccid paralysis

A-B toxins neurotoxins

Clostridium botulinum: toxin inactivates proteins [SNARE proteins (synaptobrevin and syntaxin required for the release of acetylcholine] Synaptobrevins are small integral membrane proteins of secretory vesicles Tetanus toxin affects anterior horn cells of the spinal cord and brain stem. Cleaves synaptobrevin and blocks release of inhibitory transmitter from inhibitory terminals produces hyperflexia and spasms of skeletal muscles.

Tetanospasmin

Clostridium tetani: Blocks release of neuro inhibitor (glycine and gamma amino butyric acid) causing spastic paralysis--tetanus

Operons

Clusters of co-regulated genes: with a promoter, operator and structural genes These genes are regulated such that they are all turned on or off together. Grouping related genes under a common control mechanism allows bacteria to rapidly adapt to changes in the environment. Prokaryotic genes to be regulated in the same manner are grouped together in operons, under the control of a single promoter and operator. Transcription of an operon is dependent upon whether or not the repressor (encoded by a nearby gene) binds to the operator region. Inducible or Repressible

Inclusion bodies

Collections of replicating virus particles either in the nucleus or cytoplasm: Have diagnostic importance Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections respectively.

Virus

Comes from the Latin word for "poison" Submicroscopic - measured in nm - electron microcopy Acellular - inert, not considered to be "living" Incapable of self-replication They are obligate intracellular infectious agent Must encode any required processes not provided by the host cell Replicate by assembly of individual components within host cell 1890s - First identified as filterable infectious agents (passed through filters that removed bacteria) - It was suggested that the infection might be caused by a toxin produced by bacteria (The agent is now known as tobacco mosaic virus) 1901 - first human virus discovered - Yellow Fever Virus 1909 - poliovirus, 1933 - influenza virus, 1963-hepatitis B virus

Pili (Fimbrae)

Commonly on Gram Negatives For adhesion and/or transport of bacterial material (sex pili) Fimbrins, adhesins, pilins - capable of inducing a specific immune response Adhesins in pili may be species specific and some are capable of undergoing antigenic variation; avoid immune response and/or bind to different host cells Bacteria are constantly losing and reforming pili as they grow in the body and the same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses

Protoplast

Complete Removal of the cell wall of a Gram positive bacteria results in the formation of protoplasts, which lyses unless it is osmotically stabilized

Smallpox (Variola) virus

Complex Structure Ds DNA, enveloped Complex appearance: brick like external structure and complex internal structure

Gram Negative Cell Wall

Components: Outer membrane: Phospholipid bilayer Lipopolysaccharide ( LPS) Porins proteins Lipoprotein Thin layer of peptidoglycan Periplasmic space: contain enzymes, such as nutrient transport enzymes

Gram Positive Cell wall

Components: Thick multilayered peptidoglycan Teichoic acids (TA) Lipoteichoic acids (LTA) Pentaglycine aa bridge cross -linking of peptidoglycan layers Teichoic acid and lipoteichoic acid. (water soluble polymers that contain ribitol or glyceral phosphate) These are species specific; some may be conserved across a genus. They are immunogenic and trigger a specific immune response but they may also undergo antigenic variation.

Satellite/defective viruses

Composed of protein and nucleic acid Mutation or deletion of part of their genome makes dependent on other viruses for replication Adeno-associated virus - replicates only in cells infected with adenovirus Delta agent - naked strand of RNA expressed only in the presence of hepatitis B virus

Peptidoglycan Monomer

Composed of: NAM: N-acetylmuramic acid NAG: N-acetylglucosamine * L-lysine could be replaced by another amino acid; D-aminopimelic acid (unique to bacteria) Unique to bacteria are NAM D forms of aa and D-aminopimelic acid Lysozyme: breaks the glycosyl bond - the bond that joins the two sugars Synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol

Transfer of Fertility factor

Conjugation occurs turning an F- cell into an F+ cell The F factor must be completely transferred to ensure a copy of the tra operon (transfer operon) present. The tra operon drives the conjugation process Maintaining the plasmid in the cytoplasm is energy requiring. If there is no external pressure to maintain the plasmid, it may be lost over time = Curing

Significance of Transposition

Consequence of integration: Disruption of normal gene sequence Insertion of new genetic information Can carry several genes (Pathogenicity Islands-several genes under the control of a single promoter)-Gene products include toxins, adhesins, antibiotic resistance, etc

Bacterial growth curve

Consists of the following phases: I. Lag phase: No appreciable multiplication of cells although they may increase considerably in size and show marked metabolic activity (total count remains same) This initial period is the time required for adaptation to the new environment during which necessary enzymes and metabolic intermediates are built up in adequate quantities for multiplication to proceed II. Log or exponential phase: Cells start dividing by binary fission and their number increase exponentially or by geometric progression with time Linear relationship between time and the logarithm of no. of cells Cells at this phase are smaller and stain uniformly Many antibiotics are effective at this stage III. Stationary phase: Cell division comes to halt due to depletion of nutrients and accumulation of toxic products The no. of progeny cells formed is just enough to replace the no. of cells that die Viable count remains stationary (However, total count is more) Cells are Gram variable and show irregular staining Sporulation occurs at this stage Also, many bacteria may produce exotoxins. IV. Phase of decline: Decrease in population due to death of cells Cause may be: nutritional exhaustion, toxic accumulation, action of autolytic enzymes etc. Involution forms (irregular, atypical form) are common

Mannitol Salt Agar

Contains a high concentration (~7.5%-10%) of salt (NaCl). It is also a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the pH indicator phenol red for detecting acid produced by mannitol-fermenting staphylococci. Selective - high osmotic pressure & % NaCl Differential - mannitol fermentation

Regulation of Gene Expression

Coordinated gene expression may be obtained by I. Operons: Clusters of co-regulated genes: with a promoter, operator and structural genes Operons could be Inducible or Repressible II. Sigma factor: a protein for initiation of RNA synthesis III. Quorum sensing: gene expression depends on population size Most metabolite regulatory mechanisms involve control of transcription of the gene into messenger RNA, rather than control of translation of the messenger RNA In order to adapt quickly and effectively to changes, bacteria have developed mechanisms to coordinate and regulate the expression of multicomponent genes. Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density.

Pure Culture

Culture with only one type of microbe. Pure culture techniques : several Streak plate: most commonly used

Drugs that inhibit cell wall synthesis

Cycloserine: an analogue to D-alanine: inhibits peptidoglycan synthesis Isoniazid (INH); prodrug; inhibits mycolic acid synthesis Ethionamide: derivative of INH, inhibits mycolic acid synthesis Pyrazinamide: prodrug; inhibits mycolic acid synthesis Ethambutol: Interferes with synthesis of arabinogalactan (part of the cell wall of acid fast bacteria), bacteriostatic All above drugs are used in the treatment of mycobacterial infections: (acid fast bacteria) First line essential: Rifampin, Isoniazid, Pyrazinamide, Ethambutol First line supplemental: Streptomycin, Rifabutin, Rifapentine Second line: Quinolones, Amikacin, Kanamycin, Capreomycin, Ethionamide, Para-aminosalicylic acid, cycloserine

Membrane Disrupting Toxins

Cytolysins /cytolytic toxins Promote leakage of water and ions, disrupting cell function or cell lysis Two actions: 1. Pore (channel) former Eg, Streptolysin O produced by Streptococcus pyogenes 2. Destruction of phospholipid bilayer Eg. lecithinase (alpha toxin) produced by Clostridium perfringens (gas gangrene) Some are known Hemolysins, leukocidins Some are known as RTX or CDC pore formers RTX - because they have repeats of amino acid sequence CDC - cholesterol dependent cytolysins

Immunofluorecent staining of cell culture

DEAFF: detection of early antigen fluorescent foci Detection o viral antigens in cell culture Detection of early proteins (expressed at the early stage of viral replication) by monoclonal antibodies tagged with fluorescent dye This is a method used for the early diagnosis of CMV infection. In immunocompromised patients, The specimen is inoculated into cell culture which is examined 24 hours later by immunofluorescence for expressed CMV encoded early proteins. The monoclonal antibodies must be able to cover most, if not all strains of CMV.

Virulence

Degree of pathogenicity; measured by (ID50) or (LD50) Infectious dose (ID50) number of a pathogen enough to cause disease Lethal dose (LD50): number of a pathogen enough to kill a host

Heavy metals

Denature proteins Silver nitrate: used to prevent eye infection in new born babies from gonococcal infections

Aldehydes

Denature proteins and modify nucleic acids Glutaraldehyde: in 2% solution (Cidex) used as sterilant for heat sensitive instruments Formaldehyde - disinfectant, preservative; toxicity limits use formalin - 37% aqueous solution

Bacterial Cell Group Arrangement

Depends on the line of consecutive cell divisions and the tendency of daughter cells to remain attached to each other after cell division

Rifamycin

Derivatives: Rifamycin, Rifampin/Rifampicin, Rifabutin Mechanism of Action Inhibits DNA-dependent RNA polymerase - prevents RNA synthesis Pharmacology Bactericidal, Broad spectrum Mycobacterium tuberculosis, Gram positives, some Gram negatives (but not all) Mechanism of Resistance Alteration of RNA polymerase

Lincosamides

Derivatives: Clindamycin (mainly), Lincomycin Mechanism: bind to the 23S rRNA of the 50S ribosomes and cause premature dissociation of the peptidyl-tRNA Bacteriostatic: effective against Gram positive cocci and against Gram -ve anaerobes. Inactive against Gram negative aerobes Resistance: methylation of the 23S rRNA Side effects: suppression of normal flora of GIT increases the risk of Clostridium difficile colitis

Carbapenems

Derivatives: Imipenem, meropenem Mechanism of action: Inh. cell wall synthesis Pharmacology: Bactericidal, broadest spectrum compared to penicillins and cephalosporins, Effective - Gram negative and positives; and also strict anaerobes Active against most beta-lactamases producing organisms including Extended spectrum beta-lactamase producers 'Drugs of last resort' against bacteria resistant to multiple antibiotics Resistance: Enzymatic inactivation by Carbapenemase (a form of beta-lactamases); resistant Gram negative rods on the increase. Klebsiella pneumoniae and Escherichia coli Strains are Multidrug resistant Outbreak with CRE in US in 2015

Streptogramins

Derivatives: Quinupristin, Dalfopristin Mechanism of Action: Both drugs given at the same time: synergistic action Inhibit protein synthesis: Dalfopristin - binds to 50s and prevent peptide chain elongation Quinupristin: causes premature release of peptide Pharmacology: bactericidal, Narrow spectrum: Gram positive Recommended for vancomycin resistant Staphylococcus and Enterococcus

Quinolones and fluoroquinolones

Derivatives: several Nalidixic acid - original drug not in use any more, resistance high Ciprofloxacin, Ofloxacin, Norfloxacin, Mechanism of Action Inhibits bacterial DNA topoisomerases II (gyrase) and topoisomerases IV. Topoisomerases are required for DNA replication, recombination and repair. Fluoroquinolones are derivatives of the Quinolones because they have fluorine . Not recommended for children and pregnant women, affects bone and cartilage. Pharmacology: Bactericidal - broad spectrum Oral administration Mechanisms of Resistance Alteration of target site: bacterial DNA gyrase/topoisomerases Decreased permeability: Alteration of outer membrane proteins Export through efflux pumps

Genotype test

Detect resistant gene: ex. Van A and Van B genes for Vancomycin DNA hybridization Sequencing PCR (Amplification) Rapid, detect presence of the resistant gene but it may not mean expression of the gene

Haemadsorption

Detection of viral peplomers (hemagglutinins on infected cells) Occurs as a consequence of the insertion of viral peplomers (that have the ability to bind to RBC) into the surface of infected cells, chicken RBCs can now bind to these peplomers on the surface of the infected cells phenomenon is particularly useful in detecting infection by viruses that cause little cytopathic effect. useful for enveloped viruses that express hemagglutinins (surface gp - binds to RBC)

Electron Microscopy

Detection of virus in specimens Study of Size, Morphology Detection and identification of virus can be enhanced by using virus specific ab (Ab agglutinate virus) or by immunofluorescence

Acid Fast Staining

Differential Staining Common method is Ziehl-Neelsen (Z-N) stain - used to stain Acid fast bacteria (AFB) Requires heating of a primary stain, decolorizing with an acid/acid-alcohol solution, and then pouring a secondary dye.

Gram Staining

Differential Staining A process by which components of bacterial cell walls are bound to Gram's stain. Depending on the amount of peptidoglycan in their cell walls, bacteria stain differently and are classified as Gram-negative or Gram-positive.

Stoke's Method

Diffusion test Müeller-Hinton agar plate is inoculated by streaking the control strains evenly across the upper and lower thirds of the plate,and the test strains between the control ,leaving a distance of not more than 5mm on each side of the control strain. Measure the radius of the inhibition zone from the edge of the disc to the edge of the zone. Sensitive (S): Zone radius is wider than or equal to,or not more than 3mm smaller than the control. Intermediate (I): Zone Radius is > 2 mm but smaller than the control by > 3mm. Resistant (R): No zone of inhibition or zone radius measures 2mm or less

Kirby-Bauer method

Diffusion test Place paper disks impregnated with known conc. of antibiotics on a smear of culture on semisolid/solid media (eg, MHA: Mueller-Hinton agar), measure zone of inhibition in mm The size of the zone is compared with standard zone of inhibition to determine the sensitivity of the organism to that drug Report as: Resistant (R) -equal to or less than 14 mm Intermediate (R) -15-16 mm Sensitive (S)- equal to or more than 17mm Zone of inhibition measured in the test is compared with the standard given by CLSI guidelines Cheap, easy, but not quantitative

UV microscopy

Direct Fluorescent Antibody Staining (DFA) Detection and identification of viruses in patient's tissue/specimen or viral culture

Bacterial chromosome

Double stranded DNA Single chromosome, circular Haploid No introns No nuclear membrane Highly coiled into supercoils Two enzymes DNA gyrase (Topoisomerase II) Topoisomerase IV In haploid organisms a mutation on a gene has profound effect than in Eukaryotes. Role of DNA gyrase and Topoisomerase and action of Flouroquinolones: In Gram negative organism: DNA gyrase becomes the primary target for Flouroquinolones In Gram positive organism: Topoisomerase IV is the primary target, DNA gyrase remains as a secondary target

Bacterial DNA

Double-stranded molecule twisted into a helix (similar to a spiral staircase). The two strands are comprised of a sugar-phosphate backbone and attached bases. 4 nucleobases, sugar, phosphate Thymine - Adenine, 2 Hydrogen bonds Guanine - Cytosine 3 Hydrogen bonds Genetic code; same like eukaryotic cells The smallest bacteria - Mycoplasma - 0.58 x106 base pairs and carry 475 potential genes A typical bacteria, Escherichia coli, 4.639 x 106 base pairs and carry 4288 potential genes

ADCC (antibody-dependent cell-mediated cytotoxicity)

During replication of a virus some of the viral proteins are expressed on the cell surface membrane of the infected cell. Antibodies can then bind to these viral proteins. Next, the NK cells which have Fc Receptors will bind to that antibody, inducing the NK cell to release proteins such as perforin and proteases known as granzymes, which causes the lysis of the infected cell to hinder the spread of the virus

Maturation

During this time, viral coded envelope glycoproteins are inserted in the host cell's membranes by the Golgi apparatus. Viruses that will be released by exocytosis obtain their envelopes by budding from the nuclear membrane, the endoplasmic reticulum, and/or from the Golgi complex. These enveloped viruses are then packaged in exocytic vesicles for release from the host cell by exocytosis. Matrix proteins for negative stranded RNA viruses line and promote the adhesion of nucleocapsid with glycoprotein-modified membrane. As more interaction occur, the membrane surrounds the nucleocapsid, and the virus buds from the membrane.

Antibiotic sensitivity test

E.g Optochin sensitivity for alpha hemolytic Streptococcus spp.

Disinfection

Elimination of disease producing (pathogenic) Micro-organism. Frequently toxic to apply to tissue but are commonly used on inanimate surfaces.

LPS (lipopolysaccharide)

Endotoxin Lipid A component of LPS of the cell wall of Gram-negatives Released after the bacteria die Activates several aspects of the immune system including the coagulation system. Acts as PAMP : activates macrophages, PMN, mast cells, endoth. cells, platelets Activates complement: alternative pathway B cell polyclonal activation Effects Low concentrations signal inflammation necessary for clearing infection High concentrations: Endotoxic shock, " SIRS"- systemic inflam. response syndrome Symptoms: Fever, Hypotension and DIC (disseminated intravascular coagulation) , inflammation, multi-organ failure, death

Bacterial Toxins

Endotoxins and Exotoxins Note: Teichoic and lipoteichoic acid, peptidoglycan of Gram positive can cause fever, acute phase response similar to that caused by Endotoxin

Viral Structure

Envelope: lipid bilayer from host cell and viral glycoproteins Peplomers/Spikes: viral glycoprotein, binding to host cell Matrix protein- mediate attachment of capsid with the envelope Packaged enzymes: facilitate initial viral replication (polymerases, proteases). May be located inside the nucleocapsid or in the space between the capsid and envelope (tegument) Other proteins: structural proteins: part of virion non-structural proteins: not in virion, involved in early events of replication

Enveloped vs Naked Viruses

Enveloped Environmentally labile: Spread - must stay wet, large droplets, secretions, blood and organ transplant Enter cells by fusion with cytoplasmic membrane lipids Usually released by budding through the cell membrane with no cell death Cell to cell spread by syncytia may occur Naked Environmentally stable, Easily spread by water, dust, food, hand to hand, small droplets, fomites, etc. Naked viruses usually enter cells through receptor mediated avenue Naked viruses most often exit host cells by lysis and death of the cell

Herpes virus

Enveloped with Icosahedral capsid ds DNA, tegument between capsid and envelope-containing viral proteins and enzymes for replication

HIV

Enveloped with helical capsid Genome: Segmented RNA, Two identical positive ss RNA strands Surface glycoproteins - for adhesion and entry Packed enzymes: Reverse transcriptase, integrase , and proteases

Paramyxovirus

Enveloped with helical capsid Non Segmented, negative sense RNA genome, carry RNA dep. RNA polymerase F: mediates cell entry by inducing fusion between the viral envelope and the cell membrane HN/ H, G: attachment proteins, with Hemagglutinin and/or neuraminidase activity

Rabies virus

Enveloped with helical capsid bullet shaped, negative sense ss RNA Virus

Influenza virus

Enveloped with helical capsid Segmented, negative sense ssRNA genome Peplomers: 1. Hemagglutinins - binds and agglutinate RBC 2. Neuraminidase: an enzymes ; splits polymers of sialic (acetylneuraminic) acid to get released RNA dep. RNA polymerase

Mechanisms of Aminoglycosides Resistance

Enzymatic Modification of the drug: -Acetylation by acetyltransferases -Adenylation by adenyltransferases -Phosphorylation by phosphotransferases Transport Interference: decreased uptake Mutation of ribosomal binding Site Accelerated Export of Drug

DNA Replication in Prokaryotic Cells

Enzymes & proteins required for bacterial replication are similar to those found in eukaryotic cells but different enough to be targets for specific antibiotics Replication requires uncoiling of supercoiled DNA. Antibiotics of class flouroquinolones bind to DNA gyrase (topoisomerase II) and inhibit uncoiling of supercoiling.

Transglycosylase

Enzymes facilitate formation of glycosidic bond to link peptidoglycan monomers

E-test

Epsilometer test MIC determined from the test is compared with the standard given by CLSI guidelines A strip of paper with decreasing concentration of antibiotics Advantage : quantitative: measures Minimum Inhibitory Concentration (MIC)

Spontaneous mutation

Error during replication Mutation rate in viruses is high due to the poor fidelity of viral polymerases Mutation rate > in RNA than DNA virus because RNA viruses do not have error-checking mechanism Viruses are subject to the same type of mutations as other organisms Different types of mutations - resulting in variety of mutants Lethal mutant - due to inactivation of essential genes, can't replicate Defective mutant- deletion/mutation of gene/s: able to replicate with the help of other viruses Host range mutant - change of host/tissue specificity Attenuated mutant - causes less serious/no disease (vaccines) Conditional mutant: (eg temperature sensitivity of influenza live vaccine - can multiply at lower temp of URT but not LRT

Toxicity of oxygen

Especially harmful to obligate anaerobes Obligate anaerobes lack superoxide dismutase and catalase and/or peroxidase, and therefore undergo lethal oxidations by various oxygen radicals when they are exposed to O2 Superoxide dismutases are a class of enzymes that catalyze the conversion of superoxide into oxygen and hydrogen peroxide. Catalase convert hydrogen peroxide to oxygen and water. Tolerant anaerobes with low catalase and peroxidase levels are known

Alcohols

Ethanol and isopropanol Dissolve lipids (cell membranes) and denature proteins Bactericidal against many, but not against spores and non-enveloped virus More effective at 70% than 100%, because more effective in presence of water Commonly used to disinfect skin, and items such as thermometers

Antigenic Variation

Existing in multiple types Antigenic/phase variation: programmed gene rearrangement Switch genes on/off : NO MUTATION but results in phenotype switching Transcriptional regulation of alternative genes. Help the bacterium evade the host immune system.

Invasion or Spreading Factors

Exoenzymes : act on the extracellular matrix Tissue degrading enzymes Examples : Proteases, phospholipases hyaluronidase, collagenase, fibrinolysin (streptokinase) Induction of phagocytosis and spread without leaving the cell: Eg. Shigella and Listeria

Secretion systems

Exotoxins are released through special apparatus Some transport the toxins directly into the extracellular spaces Others inject the toxin directly to the target host cell via structures that serve as "molecular syringe " Eg of bacteria with Type III secretion system: Pseudomonas, Shigella, Salmonella, Yersina pestis Bacterial secretion system are named as Type I, II, III, IV, V and VI. Type I, II, V deliver the toxin to the extracellular space. Type III, IV, and VI are syringe like apparatus

Plasmids

Extra-chromosomal DNA, circular One or more in a cell, Can be lost or gained Replicate independent of the main chromosome Can integrate into the main chromosome (episome) Carries genes - for specialized function, but not essential for survival such as R-plasmid - antibiotic resistance gene

Plasmids

Extrachromosomal genetic material (circular, DsDNA) Non essential genetic information May have more than one copy of a single plasmid

Transpeptidase

Facilitate formation of peptide cross-links between the amino acid layers of peptidoglycan to make the wall strong Inhibited by penicillins

Electron Microscope

Focuses a beam of electrons through the specimen or onto its surface TEM or SEM

Western blot

For detection of antiviral antibodies: The viral antigens are separated by electrophoresis, then transferred onto nitrocellulose membrane. The membrane is now reacted with the patient's serum containing the specific antiviral antibodies. The antibodies are now detected by secondary enzyme labeled antibody and substrate.

Flagella

For motility Gen. in rod shaped bacteria Flagellin proteins Variation in aa sequence - antigenic variation provides serologic classification Capable of being regenerated Number and position vary Generally found in many Gram negative bacteria. However, some of the Gram positive organisms like Vagococcus, two species of Enterococci (Enterococcus casseliflavus and E. gallinarum) have flagella

Ethylene oxide

Gas Bactericidal even against spores Great penetrating power because it is a gas Alkylates protein and nucleic acid Used to sterilize bulky stuff such as bedding materials heat sensitive medical materials, like plastic gloves, syringes Highly toxic, so need of special chamber

Focus Assay

For viruses with oncogenic potential

Superantigens

Form bridges between the MHC II (beta chain) of macrophages or other APCs and TCR of T cells Rate of T cell activation: 1/5 versus 1/10,000 to 100,000 (Superantigen Vs Other antigens) High levels of cytokines in circulation (cytokine storm) - Toxic shock syndrome - life threatening multi organ involvement. Examples: Toxic Shock Syndrome Toxins, TSST (Staphylococcus aureus) Streptococcal pyrogenic exotoxin (Spe) A and C (Streptococcus pyogenes) Toxic shock syndrome can affect most organ systems in the body, including the skin, lungs, liver, kidneys, blood, GI, and pancreas. Fever, rash, low pb etc

Viral Nomenclature

Formal names: 1. Family names end in - viridae, e.g Orthomyxoviridae, Reoviridae 2. Genera names end in virus eg. Myxovirus, Reovirus Common names based on; 1. Target tissue—Rhinovirus, hepatitis virus 2. Geography—St. Louis encephalitis virus, Coxsackie virus 3. Shape—Coronavirus 4. Transmission--Arboviruses (arthropod borne) viruses

Koch's Postulates

Four criteria designed to establish a causative relationship between a microbe and a disease

Serotyping

Further Identification of isolates: detection of expressed antigens Slide agglutination: identify antigens present on the surface of specific microorganisms Use of Fluorescent dye tagged antibodies - DFA

Transformation

Gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome. Successful gene transfer is dependent on the absence of restriction endonucleases Most important mechanism of genetic transfer in Gram positive microorganisms such as Streptococcus pneumoniae Cells must be competent Under the control of extracellular Competence Factor (CF) Uptaken DNA-Sensitive to endonucleases Occurs in nature and it can lead to increased virulence. In addition transformation is widely used in recombinant DNA technology.

Types of Transduction

Generalized - transfer of any bacterial gene from the disrupted cell Specialized - transfer of specific genes after integration of phage into bacterial chromosome

Bacterial Genome

Genes on Chromosome and extra chromosomal element (if any) Chromosome Plasmid Prophage

Nucleoid

Genome not enclosed within a nuclear envelope Most bacterial chromosomes are single, circular, highly coiled and haploid Double stranded DNA No introns Ultrastructure: fine structure, especially within a cell, that can be seen only with the high magnification obtainable with an electron microscope. Has no histones (protein) in the chromosome

Gram Positive vs. Gram Negative Bacteria

Gram positive to take a positive stain and associated with respiratory and soft tissue infections Gram negative to obtain a negative stain and associated with genitourinary or gastrointestinal tract infections

Bacterial ribosomes

Has two units Differ from eukaryotic cell ribosome in size and chemical composition Bacterial ribosome size -70s with 50s and 30s subunits Eukaryotic cell ribosome size - 80s with 60s and 40s subunits Site for antibiotic action The Svedberg unit (S) offers a measure of particle size based on the rate of the sedimentation in the centrifugation

Medical Significance of Spores

Highly resistant to heat and chemicals Not killed by boiling, but are killed at 121 0C The spore coat is impermeable to many chemicals - Only few disinfectants can kill spores (sporicidal disinfectants) Can survive for many years in the environment - eg. soil Any thing that is contaminated in soil may contain spores tetanus and gas gangrene by spore forming bacteria Their use as sterilization controls: B. stearothermophilus: in autoclave B. subtilis and C. tetani: in hot air oven

Mode of Transmission

Human to human: Direct contact, No direct contact, Transplacental, Blood borne Non human to human: from soil source, water source, animal source, fomite source Major Routes of entry: 3I's + sexual + Mother to child Ingestion, Inhalation, Injection -deep wounds, arthropod , Sexual contact. In addition vertical transmission from mother to unborn fetus (transplacental) during delivery and after delivery via breast feeding.

Laboratory Diagnosis of Viral Disease

I. Direct microscopic examination of clinical specimen Light/compound microscopy: Cytopathic effect (CPE). UV microscopy: Detection of viral antigens by fluorescent Ab staining Electron microscopy, immunoelectron microscopy II. Culture: cell culture (sometimes, inoculation of eggs or animals) Identification: Assessment of Cytopathic effect (CPE), Hemadsorption and other characters Detection of viral antigens by UV microscopy Quantification: viral titer-plaque assay Assessment of oncogenic potential (focus assay) III. Serological analysis: antibody detection/titer ELISA, Hemagglutination inhibition assay Neutralization tests: block cytopathic effects Western blot IV. Detection of viral antigens in sample: ELISA, rapid tests etc. V. Molecular analysis of genetic material PCR and In-situ hybridization: in clinical specimen or in culture specimen

DNA Viruses

I: ds DNA: replication is the same as for Eukaryotic DNA and can make mRNA from dsDNA, eg. Herpes virus use host DNAdep-DNApoly.(but varies) and DNA dependent RNA polymerase. _dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses) (Polyoma, papillomaviruses use host coded DNA polymerase whereas Adeno and Herpesviruses use viral coded DNA polymerases) II: ss DNA: replication passes through ds DNA then to mRNA, eg. parvovirus use a host DNAdep---DNApoly and DNA dependent RNA polymerase -ve strand or antisense ) DNA (e.g. Parvoviruses) VII: Partially ds DNA: replicate through RNA intermediate - Uses host DNA dep-RNA poly and code their own RNAdep-DNApoly. Eg. Hepatitis B (hepadnaviridae) VII: dsDNA-RT viruses (e.g. Hepadnaviruses i.e Hepatitis B virus)

Baltimore Classification

I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses) (Polyoma, papillomaviruses use host coded DNA polymerase whereas Adeno and Herpesviruses use viral coded DNA polymerases) II: ssDNA viruses (+ strand or "sense"/-ve strand or antisense ) DNA (e.g. Parvoviruses) III: dsRNA viruses (e.g. Reoviruses) IV: (+)ssRNA viruses (+ strand or sense) RNA (e.g. Picornaviruses, Togaviruses) V: (−)ssRNA viruses (− strand or antisense) RNA (e.g. Orthomyxoviruses (influenza virus), paramyxoviruses (measles, mumps, respiratory syncytial virus etc), Rhabdoviruses (rabies virus etc)] VI: ssRNA-RT viruses (+ strand or sense) RNA with DNA intermediate in life-cycle (e.g. Retroviruses) VII: dsDNA-RT viruses (e.g. Hepadnaviruses i.e Hepatitis B virus)

RNA Viruses

III: dsRNA: have positive and negative sense RNAs; negative sense RNA is used as a template for mRNA synthesis, Carries RNAdep-RNApoly (viral) to make the mRNA and also the genome; eg. Rota virus IV: ss +ve RNA: can directly act as mRNA, but during replication of their genome, forms a negative strand RNA (template) and then mRNA; code (viral) RNAdep-RNApoly [picornavirus (eg.rhinovirus), toga virus (chickengungya virus) etc. V: ss -ve RNA: must bring (carry) RNAdep-RNApoly (viral) to make mRNA (which also acts as a template for replication of their genome); orthomyxovirus (influenza virus), paramyxo virus (measles), rabies virus etc. VI: ss +ve RNA passing through a DNA intermediate: use their reverse Transcriptase to make ds DNA and host DNA dep-RNApoly to make mRNA (retrovirus- HIV)

Phenotypic mixing coat component

If two different viruses infect a cell, progeny viruses may contain coat components derived from both parents and so they will have coat properties of both parents. This is called phenotypic mixing. IT INVOLVES NO ALTERATION IN GENETIC MATERIAL, the progeny of such virions will be determined by which parental genome is packaged and not by the nature of the envelope.

Dark Field Microscope

Illumination comes from the sides, thus making the background appear dark, and the organism on the slide appear light(bright).

Peptidoglycan Cross-linking

In Gram negatives : cross linking between D-alanine and L-lysine is a direct covalent bond In Gram positives: 5 molecules of glycine make a bridge between D-alanine and L-lysine

Immunological diagnosis

In Vitro: Serology; Antibody or Antigen. In Vivo: skin tests : eg. PPD test

Categorization of Gram Positive Cocci

In general, the following characteristics Cytoplasmic lipid membrane Thick peptidoglycan layer Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence. Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase. A much smaller volume of periplasm than that in gram-negative bacteria.

Genotypic diagnosis

Includes the cause, and use of laboratory tests (endophenotypic). Based in genetic make up

Phenotypic Diagnosis

Includes the signs, symptoms, course, outcome, and response to treatment. Observable characteristics Isolation and characterization of pathogens Microscopic features - shape, size, staining reaction, cell structures Cultural Characteristics: Growth on artificial media; colony appearance; texture, size, shape, pigment, growth requirements Biochemical Characteristics - detection of presence or absence of particular enzymes or metabolic pathways Biotyping Serotyping: characterize based on antigen type Sensitivity to Antibiotics: for identification purpose

R group of penicillin

Increases resistance to stomach acids Increases absorption in the GI Resistance to penicillinases (beta lactamases) Increases spectrum of activity But no improvement in allergic reactions

Gray baby syndrome

Infants skin appears gray with vomiting and shock- due to toxic concentration of Chloramphenicol. This occurs due to reduced glucuronyl transferase (responsible for detoxification of chloramphenicol) activity in infants.

Nucleic Acid antibiotics

Inhibit Nucleic Acid Synthesis and Function DNA Metronidazole Quinolones /Fluoroquinolones RNA Rifampin

Penicillins Mechanism of Action

Inhibit Peptidoglycan Synthesis-act on penicillin binding proteins (primarily transpeptidases) Transpeptidases Transglycosylase enzymes Carboxypeptidases Accumulation of peptidoglycan monomers (since they are unable to cross link in +nce of penicillin) leads to activation of autolytic Enzymes (Murine hydrolase): Lysis of cells Bactericidal in actively growing bacteria

Cleavage of rRNA

Inhibition of protein synthesis Removes adenine from specific sites on the 28s rRNA of the 60s ribosome Shiga/Vero toxin : Enterohemorrhagic E. coli and Shigella spp. In the intestine: Bloody diarrhea If it enters the blood: Hemolytic uremic syndrome (HUS) - death of RBC HUS, is a disease characterized by hemolytic anemia (anemia caused by destruction of red blood cells), acute kidney failure (uremia), and a low platelet count (thrombocytopenia).

Recombination

Integration of donor DNA to the recipient chromosome/genome Stabilizing genetic information after gene transfer This results in new combinations of genes on the chromosomes involved Types A. Homologous DNA Recombination B. Site-Specific Recombination

Recombination

Interaction between two genetically different viruses Exchange of genetic material between different but related viral strains, occurring when infecting a cell simultaneously. By 2 mechanisms: Either by crossing over within the regions of significant base sequence homology (common with ds DNA viruses eg, Herpes 1 and 2) or, By exchange of segmented genome Reassortment-when two viruses with segmented genomes are simultaneously present and segments are mixed in progeny capsid Role in emergence of Epidemic Influenza strains

Reassortment of genes

Interaction between two genetically different viruses The mixing of the genetic material of a species into new combinations in different individuals. In particular, occurs among influenza viruses, whose genomes consist of eight distinct segments of RNA. (Genetic Shift) : Major changes

Negri Bodies

Intracytoplasmic inclusion body in rabies Is an accumulation of components of rabies virions in cytoplasm of neural cells.

Cowdry type A

Intranuclear inclusion body in Herpes infection

Inversion

Is a chromosome rearrangement in which a segment of a chromosome is reversed end to end Through the utilization of specific recombinases, a particular DNA sequence is inverted, resulting in an ON to OFF switch and vice versa of the gene located within or next to this switch. Many bacterial species can utilize inversion to change the expression of certain genes for the benefit of the bacterium during infection.

Capsid Assembly

Is assembled around the viral genome. In addition, viral-coded envelope glycoproteins are inserted in the host cell's membranes by the Golgi apparatus. The viral envelope will be added later as the virus buds from the membrane (eg. Cytoplasmic) of the host cell during the release stage.

Generalized Transduction

Is transduction in which potentially any bacterial gene from the donor can be transferred to the recipient. Phages that mediate this process generally breakdown host DNA into smaller pieces and package their DNA into the phage particle by a "head-full" mechanism. Occasionally one of the pieces of host DNA is randomly packaged into a phage coat. Thus, any donor gene can be potentially transferred but only enough DNA which can fit into a phage head can be transferred. If a recipient cell is infected by a phage that contains donor DNA, donor DNA enters the recipient. In the recipient a generalized recombination event can occur which substitutes the donor DNA and recipient DNA .

Transposons

Jumping Genes Mobile genetic elements The smallest transposon carries Insertion sequences -minimal genetic information required for transport and replication Transposases- transport; Resolvase or recombinase (Rec) is a nuclease which is involved in DNA recombination Can integrate and excise and take their gene to another place while leaving itself behind Can move to and from any DNA--phage, plasmid, bacterial genomes May carry additional genes such as genes coding antimicrobial resistance

Cell Wall Less Forms

L forms Produced by the action of Lysozyme Antibiotics: cell wall synthesis inhibitors (beta-lactam (penicillin) Protoplast: from Gram positive (Stable L forms) Spheroplasts: from Gram negative (retain the outer membrane). Unstable L forms : they can divide and revert back to make peptidoglycan if moved to antibiotic free environment Formation of L forms may lead to infection after antibiotic treatment. Stable L-forms: L-forms that are unable to revert to the original bacteria.

Oxazolidinones

Linezolid Mechanism of Action Binds to 50S and interfere with the formation of the initiation complex. Pharmacology: bacteriostatic, Gram positive, reserved for Vancomycin resistant Enterococcus faecium (VRE/VREF) Methicillin resistant Staphylococcus aureus (MRSA)/VRSA

Bactoprenol

Lipid carrier molecule that transports peptidoglycan precursors across the cytoplasmic membrane

Attenuation

Loss of/decreased virulence

Bacteriophage Life cycles

Lytic Cycle Virulent phage Leads to generalized transduction Lysogenic Cycle Temperate phage Leads to specialized transduction

Biofilms (Glycocalyx)

Made of mucopolysaccharide surround populations of cells to provide: Adhesion (devices & even in tissue) Protection against: Phagocytosis Dehydration Antibiotic/antibodies penetration

Bacterial Spores

Means of survival Formed in response to adverse conditions Occurs in unfavorable conditions such as starvation, desiccation, presence of disinfectants and in extreme temperature DNA replicates and separates, spore septum begins to form Double membrane encloses the DNA and cytoplasm Double membrane matures and becomes core wall Mother cell swells Spore coat develops Exosporium basal layer develops It contains- DNA, cell membrane, cytoplasm, cell wall, surrounded by a Spore Coat : contains high concentration of calcium bound to dipicolinic acid, plus a keratin-like protein Dehydrated : It contains about 70% less water than the mother cell. No detectable metabolic activity - makes them resistant to unfavorable conditions. Produced by some Gram positive & one Gram neg. Bacteria I. Gram positive bacilli: Obligatory aerobic: Genus Bacillus eg, B. anthracis, B. subtilis Obligatory anaerobic: Genus Clostridia eg. C. tetanus, C. welchii, C. botulinum Cocci: Gram positive cocci: Sporosarcina II. Gram Negative: Bacilli: Coxiella burnetii

Viral size and shape

Measured in nm Ranges from 18 nm to 300nm Assume different shapes

Immunological Methods

Measuring specific antibody levels against a particular pathogen - IgM - current infection IgG: paired sera- 4x increase OR : A single serum sample - determine titer and compare it with an upper limit of normal if it is established for that particular pathogen Detection of an antigen specific to a particular pathogen in a clinical (patient) sample in Vitro *Immunological techniques discussed in Immunological tools:

Filtration

Mechanical removal using filters Any particle bigger than the diameter of the filter pores gets trapped. The most common bacterial filters are nitrocellulose membranes with pore size of 0.22 micrometers Used for heat sensitive liquids, such as enzymes and solutions for intravenous administration Also used to sterilize air in safety cabinet

Replication Sites

RNA viruses replicate in the cytoplasm DNA viruses replicate in the Nucleus With a few exceptions Orthomyxo in the Nucleus and Poxviruses in the Cytoplasm In both compartments: Retroviruses and Hepadnaviruses

Metronidazole

Mechanism of Action: Nitro group is reduced in vivo to form the active drug that disrupts DNA under anaerobic condition Pharmacology Initially as anti protozoan (Trichomonas) Bactericidal, narrow spectrum Effective against anaerobes, not aerobes Sensitivity testing for anaerobes is not performed routinely. Therefore, resistance is under-reported. The proposed mechanisms of resistance 1. Decreased drug uptake or increased efflux 2. Decreased drug activation/change in the biological target 3. Increased oxygen scavenging capabilities (SOD/catalase/ peroxidase) 4. Enhanced activity of DNA repair enzymes

Resistance in General

Mechanism of Resistance Intrinsic resistance Bacteria lacking peptidoglycan Seven major pathogens Chlamydia, Chlamydophila, Mycoplasma, Ureaplasma, Orientia, Ehrlichia and, Anaplasma Mutation of porin proteins - decrease permeability among some Gram negatives Inactivating enzymes: on plasmids Beta lactamase, some have extended spectrum (ESBL) activity gene carried on plasmid - could carry genes for other drug resistance - Multidrug R plasmids Alteration of Penicillin Binding Protein (form PBP 2A) Eg. Methicillin resistant Staphylococcus aureus (MRSA) (mec A gene mediated: alter target)

Adsorption/ Attachment to Target Cell

Mediated by molecular interaction between virus and target cell Viral attachment structures: Surface structures on the capsid or envelope -Viral attachment proteins (VAP)/peplomers/spikes -Determine host/tissue specificity -High mutation rate in VAP can result in change in host specificity or avoid immune response eg. Influenza virus - hemagglutinin (HA) HIV - glycoprotein (gp)120 Receptors on target cell: are proteins or carbohydrates on glycoproteins and glycolipids Examples -CD4 and CxCR4/CCR5 (a chemokine receptors) on T helper cells, and Macrophages - for HIV -ICAM-1 on epithelial cells for rhinovirus -Sialic acid on epithelial cells for influenza virus Permissiveness = allowing Entry + Replication

Conjugation

Mediated by the F factor, a plasmid that contain tra operons: (transfer region) where the genes coding for sex pili and other "fertility" factors are found Origin of transfer (OriT): where one of strands breaks and is transferred to the recipient Insertion sequence (For Hfr): a region that facilitate integration to the main chromosome Requires direct cell contact between 2 cells

Categorization of Gram Negative Rod

Medically relevant gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems (Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi).

Categorization of Gram Negative Cocci

Medically relevant gram-negative cocci include the four types that cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus influenzae).

Semisynthetic Penicillins

Methicillin, nafcillin, oxacillin, ampicillin, amoxycillin Resistant to beta-lactamase Increased stability to stomach acid Increased spectrum of activity Activity - similar to natural penicillins with increased activity against Gram negative rods and beta-lactamase producing organism

Culture

Method of multiplying microbial organisms by letting them reproduce in predetermined culture media Pure culture: culture with one type of bacteria, theoretically derived from a single cell

Determinants of Bacterial Pathogenesis

Mode of Transmission Adherence factors Ability to invade, spread and cause inflammation Toxin production: Products that kill or alter physiology of host cells Surviving host defenses Immunopathogenesis: Collateral damage due to host immune response or immune complication

Chemical Agents

Mode of action: disrupt cell membrane, denature proteins, break DNA. The rate of killing by a chemical depends on: Concentration of the chemical: Effectiveness usually is directly related to concentration. But not always E.g Alcohol - 70% is more effective than 95% Time of exposure Physiological condition of the microbe (eg. Vegetative Vs. spore) Presence of organic matter - affects exposure Population size - the number of bacterial cells Disruption of cell membranes: Alcohol, Detergents, Phenols (Acronym-ADP) Modification of proteins: 3H-Halogens (chlorine, Iodine), Heavy metals, Hydrogen peroxide; Alcohol, Aldehydes (Formaldehyde and Gluteraldehyde), Ethylene oxide Modification of nucleic acids: Aldehyde (formaldehyde and Gluteraldehyde), Ethylene oxide and Dyes (Crystal Violet, Malachite green)

Camouflage

Molecular mimicry: antigenic similarity with host Hiding within cells - intracellular growth

CPE (Cytopathic Effect) on cell culture

Morphological changes of human corneal epithelial cells infected with a virus. A: Normal human corneal epithelial cells exhibited a cobblestone appearance. B: After cells were infected with HSV-1, the cytopathic effect could be seen at 8 h p.i. The space between infected cells increased for 12 h (C) and 24 h (D), the cobblestone appearance disappeared and many giant multinucleated cells could be seen. Magnification: 200X.

A-B Toxins

Most bacterial toxins fall into this class Two units: A and B B unit binds to host cell receptor Determines the host cell specificity of the toxin After entering the cell the A unit cleaves or modifies a target molecule within the cell. The A unit is translocated through host plasma membrane after B binds (directly or through endocytosis).

Syncytia Formation

Multi-nucleated syncytial cells are often the result of infections by enveloped viruses that induce cell fusion.

Adenovirus

Naked with Icosahedral capsid Capsomeres- hexons and pentons 12 vertices (with pentons) and 20 triangular faces (with hexons). Each face has no.of capsomers. Peplomers (fibers) - projections

Antibiotic terminology

Narrow spectrum: active against one or few types of pathogens Broad spectrum: active against a wide variety of pathogens Bactericidal: Kills the pathogen, irreversible Bacteriostatic: Inhibit the growth of the pathogen, reversible Empirical therapy: initial selection of antibiotics based on clinical findings Bacteriostatic drug: on withdrawal of drug - growth of agent. Assists the host defense by limiting the populations size

Types of Staining Methods

Negative staining: Stain the background, not the object of interest: which will be transparent against colored back ground Eg. Capsule staining (India ink) Simple staining - one dye is used; reveals shape, size, and arrangement Differential staining - use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain and endospore stain)

Side effects of aminoglycosides

Nephrotoxic 8th cranial nerve damage Hearing loss and/or loss of balance drug level monitoring required These drugs are ineffective in abscess. Do not penetrate cell wall of Gram positives. They are generally given with cell wall inhibitors to treat an infection with them.

Viral Genetics

New viral strains with properties different from the parent/wild type can arise by Spontaneous mutation Mutants can be point mutants (one base replaced by another) or insertion/deletion mutants. Recombination enables a virus to pick up genetic information from viruses of the same type and occasionally from unrelated viruses or even the host genome and/or Interaction between two genetically different viruses that infect the same cell Recombination Phenotypic mixing Complementation

Molecular Methods

Nucleic acid based tests Highly specific, quite sensitive, safe, and fast Historically used for pathogens difficult to culture, highly contagious, nowadays for rapid diagnosis Most commonly used are Amplification: Polymerase chain reaction (PCR) - require primers Hybridization; Detection and localization in biopsy:- requires labelled probes DNA Probes labelled with enzymes or fluorescent compounds Sequence analysis: 16s RNA subunit used to identify bacteria

Oxygen and Bacterial Growth

Obligate aerobes: require oxygen. Grow best at atmospheric concentration of oxygen. Microaerophiles: require oxygen at a lower concentration than the atmospheric conc. Facultative anaerobes: can grow in the presence or absence of oxygen. Aerotolerant anaerobes: They do not use oxygen but can grow in the presence of oxygen. Obligate anaerobes: Get inhibited by oxygen Examples: Obligate aerobes: Pseudomonas, Bacillus Micro-aerophiles: Helicobacter, Campylobacter Facultative anaerobe: Escherichia coli, Vibrio cholerae (They are generally aerobes but can grow in the anaerobically environment) Aerotolerant Anaerobes: Lactobacilli, Clostridium carnis, C. histolyticum, C. tertium etc. Obligate anaerobes: Clostridium tetani, Bacteroides.

Replicating virus

Often produce histological changes in infected cells. These changes may be characteristic or non-specific. Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or cytoplasm. Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections respectively. Although not sensitive or specific, histology nevertheless serves as a useful adjunct in the diagnosis of certain viral infection.

Biotyping

Presence or absence of biochemical markers The formation of distinct biochemical end-products eg. carbohydrates fermentation Presence of certain bacterial enzymes and or toxins eg. Catalase, oxidase, hemolysins Use of specific nutrients - eg. E.coli using acetate as sole source of carbon Information from selective, differential media and enrichment media E.g Lactose fermentation and other colony characteristics

Hydrogen peroxide

Oxidizing agent Attacks sulfydryl groups in proteins and modifies them kills most bacteria, kills spores Antiseptic - to clean wounds and contact lenses

Halogens

Oxidizing agents, Denature proteins kill the micro-organism by cross-linking essential sulfydryl groups in enzymes to form the inactive disulfide Chlorine: used in purification of water and as disinfectants on inanimate objects (Sod. hypochlorite) Iodine: Two forms, Tincture of iodine (2% solution of iodine and potassium iodide in ethanol) is used to prepare the skin prior to blood culture. Iodophores are complexes of iodine with detergents that are frequently used to prepare the skin prior to surgery because they are less irritating than tincture of iodine As antiseptics - to clean skin or disinfect wounds

Moist heat

Pasteurization: aim is to kill pathogenic bacteria in milk and some canned foods. 62 oC for 30 min followed by rapid cooling, or 72 oC for 15 seconds followed by rapid cooling 1350 C (275 0F) for 1 to 2 seconds- kills bacteria and most spores Boiling: at atmospheric pressure, 100 oC at sea level, can kill vegetative cells in 10 min. , but not spores. Not a means of sterilization. Tyndallization : intermittent boiling (three days in succession), can attain sterility, including spores. May be used for solutions that contain heat sensitive substance Exposure of 1000 C for 20 min on 3 successive days. Autoclaving: steam under pressure, 121 oC at 15 lb/sq inch for 15-20 min kills all microorganism including spores, but not prions 121 oC for 1 hr to destroy prions Sterilization control: Spores of Bacillus stearothermophilus

Coordinated expression of several genes

Pathogenicity Islands : Assemblage of several virulence genes in one or more contiguous DNA sequences Usually a transposon inserted into the chromosome or a plasmid Can be transferred as a unit and could be turned on by single stimulus Eg. Salmonella - 25 genes can be turned on by a change in pH in a phagocytic vesicle within macrophages

Naturally occurring Penicillins

Penicillin G Acid labile, Narrow spec Penicillin V Acid Stable, Narrow spec Active against Gram positive bacilli and cocci, Gram negative cocci, and spirochetes Poor activity against G -ve rods Side effects: allergic reaction Most clinical isolates have developed resistance to these drugs.

Heat

Physical Agent Denature proteins Dry heat: Incineration - sterilization by combustion Direct flaming : used to sterilize heat resistant materials for immediate use. Eg. Inoculating loop Dry hot air (Hot air oven): 160 oC for 1hr, used to sterilize heat resistant materials like glass ware Infrared combustion technology

Colistin

Polymyxin B, and Polymyxin E Structure: Basic polypeptide Mechanism of Action Cationic detergent Disrupts cell membrane Pharmacology: Bactericidal, narrow spectrum Gram negative bacilli (not for Gram positives) Side effects include neurotoxicity and acute renal tubular necrosis

Application of Cell Culture

Presumptive identification of virus: by study of Cytopathic effect, Hemadsorption (Infected cell with hemagglutinin expressed on its surface + RBCs= Adsorption of RBCs on the surface of infected cell) Hemagglutination (Virus with hemagglutinin +RBCs= Agglutination) Interference (with the formation of CPE) and Decrease in acid production by infected cells Definitive identification of virus (grown in culture): by Neutralization*, Hemagglutination inhibition*, (*will be discussed under serology-slide no. 23) Immunofluorescence, Complement fixation, ELISA Others: Plaque assay: quantification of viral particles, titration Focus assay: determine oncogenic potential

Phenotypic mixing

Progeny virus acquiring coat components of both the viruses; or genome of one virus surrounded by capsids or both capsids and envelope of other virus May occur between related viruses, e.g. different members of the Picornavirus family, or between genetically unrelated viruses, e.g. Rhabdo- and Paramyxo- viruses. In the latter case the two viruses involved are usually enveloped since it seems there are fewer restraints on packaging nucleocapsids in other viruses' envelopes than on packaging nucleic acids in other viruses' icosahedral capsids.

Virulence factor

Properties of a pathogen that help it cause disease

Prion

Proteinaceous infectious agent Composition: only protein - no DNA or RNA Encoded by host cell genes Forms from post translational modification of gene products Normal protein (PrPc ) - has alpha-helical form Abnormal protein (PrPsc )- has beta-pleated sheet Extremely resistant to usual sterilization techniques Cause slow fatal neurodegenerative diseases in human and animals transmissible spongiform encephalopathies - Eg Creutzfeldt-Jacob disease (CJD)

Capsid

Proteins made up of repeated subunits known as capsomeres covers and protects the nucleic acid may have special host cell attachment properties arrangement of capsomeres determine the shape of the virus Icosahedral or helical

Exotoxins

Proteins secreted by many bacteria Three types: A-B Toxin Membrane damaging Toxins Superantigens

Bacterial Cell Walls

Provides shape and helps cell to maintain its integrity under changing environmental conditions Importance from the medical point of view carries virulence factors It is a site of action for many antibiotics It is a site of action for body defense chemicals, such as lysozyme It is important in bacterial classification Gram positive, Gram negative, acid fast Made up of a polymer - Peptidoglycan (Murein) composed of multiple units of -N-acetylmuramic acid (NAM), with tetra-peptide chain (4 amino acids) coming off of the NAM. -N-acetylglucosamine (NAG) along with few other constituents

Detergents

Quaternary ammonium salts "Surfactants" disrupt cell membrane Composed of a long-chain lipid soluble- hydrophobic end and polar hydrophilic end. Effective against Gram negative cell and enveloped viruses Used as skin antiseptics

Nitrocef test

Rapid test for beta lactamase production are intended for the rapid testing of isolated colonies of Neisseria gonorrhoeae, Moraxella (Branhamella) catarrhalis, Staphylococcus spp., Haemophilus influenzae, Enterococcus spp., and anaerobic bacteria of the genera Bacteroides, Clostridium, Porphyromonas, Fusobacterium, and Prevotella. Disks are impregnated with nitrocefin (a chromogenic cephalosporin). As the amide bond in a beta-lactam ring is hydrolyzed by a beta-lactamase, nitrocefin changes color from yellow to red. Bacteria which produce beta-lactamase in significant amounts produce this yellow to red color change

Griffith's experiment

Reported in 1928 by Fredrick Griffith, was the first experiment suggesting that bacteria are capable of transferring genetic information through a process known as transformation Rough strain: Non capsulated (Non virulent) Smooth Strain: Capsulated (Virulent)

Culture Characteristics

Requirements: oxygen, Temp, pH, osmotic pressure, growth factors, etc Colony morphology: Appearance on culture media - Selective, differential, enriched Size, Texture (rough, smooth) hemolysis, pigment etc...

Site-Specific Recombination

Requires no homology It is an integration not exchange Requires restriction endonucleases and sites for the endonucleases on both DNA Foreign DNA may be circular or linear End result is the sum of the existing genome plus the integrated DNA Three major roles: Conjugation: Integration of a fertility factor to make an Hfr cell Transduction: Integration of temperate phage DNA into bacterial chromosome to create a prophage Transposition: Movement and insertion of transposons

Carboxypeptidase

Restriction enzyme that cuts to the left of carboxy terminal Remove terminal D alanine from the pentapeptides in peptidoglycan Inhibited by penicillins

Different types of mutations

Result in variety of mutants: Lethal mutant - due to inactivation of essential genes, can't replicate Defective mutant- deletion/mutation of gene/s: able to replicate with the help of other viruses Host range mutant - change of host/tissue specificity Attenuated mutant - causes less serious/no disease (vaccines) Conditional mutant: (eg temperature sensitivity of influenza live vaccine - can multiply at lower temp of URT but not LRT

Chlorhexidine

Salts dissociate and release the positively charged chlorhexidine cation. The bactericidal effect is a result of the binding of this cationic molecule to negatively charged bacterial cell walls. At low concentrations of chlorhexidine, this results in a bacteriostatic effect; at high concentrations, membrane disruption results in cell death.

Interferon Response

Secreted by infected cells bind to common interferon receptor on infected as well as non infected cells. The interferon receptor is linked to cytoplasm signalling molecules [Janus family of tyrosine kinases/Janus Kinase (JAK)] and which in turn is linked to STAT (Signal Transducer and Activator of Transcription). Activated STAT enters the nucleus and turns on several genes, including those proteins that help to inhibit viral replication. These include: -Oligoadenylate synthetase, activates an endoribonuclease (Rnase L) that degrades viral RNA. -The second gene activated by interferon is a serine-threonine kinase called PRK kinase - inactivates eIF-2 (an initiation factor) by phosphorylation, thus prevents protein synthesis, inhibits viral replication. -Another interferon induced protein called Mx protein, blocks viral transcription.

Eosin Methylene blue (EMB) agar and MacConkey agar

Selective and differential media Only Gram negative bacteria grow (Coliforms) Differentiate between lactose fermenter and non fermenter LF: Lactose fermenting colonies NLF: Non lactose fermenting colonies EMB agar: contains dyes that are toxic for Gram positive bacteria and bile salt which is toxic for Gram negative bacteria other than coliforms. EMB is the selective and differential medium for coliforms Lactose fermenters in EMB agar give a distinctive metallic green sheen (due to the metachromatic properties of the dyes) MacConkey agar: LF colonies: pink, NLF: gray/colourless

Amendments to Koch's postulates

Serological: antibodies or antigens specific to the pathogen in question should be detected Molecular: nucleic acid specific to the pathogen in question should be detected

Penetration and Uncoating

Several mechanisms Enveloped viruses Fusion: Enveloped virus fuses with host cell membranes, pH dependent; At neutral pH- fusion on the surface and release of the genome takes place. Endocytosis: At acidic pH the whole viral particle is engulfed (Endocytosis) and release takes place in the cytoplasm Naked viruses Endocytosis : Naked viruses are taken up by receptor mediated phagocytosis or, Viropexis: direct penetration of the genome Naked virus binds to receptor sites on cellular membrane and inject the nucleic acid. Uncoating Capsid digested or opened up AND nucleic acid released

Blood agar

Sheep Blood agar- enriched and differential media Enriched: with blood Differential: hemolytic character (alpha, beta and gamma/non)

Viroids

Single circular RNA, no protein coat; identified only in plants

Bacterial cell size

Size ranges between 0.2-5 µm The smallest bacteria, Mycoplasma = largest virus (Pox virus), measures about 0.1µ to 0.2µ m in diameter

Viral Plaque Assay

Titration of Infectious Virus/ quantification of viral particles Cells lyse upon virus release Virus infects neighboring cells Clear zone is created as infection spreads outwards Clear zones are called plaques

High Frequency Plasmids

Some F plasmids can be integrated into the host chromosome because they contain insertion sequences. These are called Hfr plasmids Can transfer chromosomal genes from the donor to the recipient

Competence

Some bacteria are able to take up DNA naturally. However, these bacteria only take up DNA in a particular time during their growth cycle when they produce a specific protein called a competence factor. At this stage the bacteria are said to be competent. Other bacteria are not able to take up DNA naturally. However, in these bacteria competence can be induced in vitro by treatment with chemicals (e.g. CaCl2). The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane

Limitations of Koch's postulates

Some pathogens are found as normal flora in certain % of the population Not all microorganisms are cultivable, eg, Treponema pallidum, Mycobacterium leprae Finding an experimental animal model for all microorganism is difficult Some diseases have a synergistic etiology of two or more agents.

Frame shift mutation

Sometimes, during replication, one or a few adjacent base pairs (nucleotides) have been inserted to or deleted from the DNA. Shifts reading frame. Missense mutation: base changes result in production of different amino acids but the resulting protein may be functional or non functional (which depends on the importance of area affected by the mutation) Non sense mutation: One or more of 3 non sense codons (UAG, UAA, UGA), normally cause termination of the polypeptide chain elongation. If a nonsense codon is formed within a gene by mutation of a sense codon, the protein synthesis is terminated prematurely and only partial polypeptide is produced

Categorization of Gram Positive Rods

Special techniques for endospore staining include the Schaeffer-Fulton stain (uses malachite green and safranin; spores appear green) and the Moeller stain (uses carbol fuchsin and methylene blue, spores appear red)

Enzyme test

Spot test: rapid colorimetric assays Detect Beta lactamases; eg. ESBL (Nitrocef test) Chloramphenicol modifying enzyme-Chloramphenicol acetyltransferase Faster

Homologous DNA Recombination

Stabilizes linear genes after genetic transfer It is an exchange of piece of DNA: loss of old information, replaced by new Requires long region of homology and a series of recombination enzymes (recA, recB, etc)

Stages of Viral Replication

Stages: Adsorption/Attachment - Adsorption: binding of virus to specific molecule on host cell Entry/penetration - the virus enters host cell Uncoating- the viral nucleic acid is released from the capsid Macromolecular Synthesis- viral components are produced Assembly- new viral particles are constructed Release - assembled viruses are released by budding (exocytosis) or cell lysis

Beta Lactamase Inhibitors

Structural analogues of beta-lactams: have little antibacterial activity but bind strongly to beta lactamase thus protect penicillins/cephalosporins. Given together with beta lactams Clavulanic Acid Augmentin=Clavulanic Acid+ Amoxycillin Sulbactam Unasyn=Sulbactam + Ampicillin Tazobactam Zosyn=Tazobactam + Piperacillin Avibactam Avycaz=Avibactam +Ceftazidime (Cephalosporin) Activity: improved against beta-lactamase producing bacteria

Dapsone and Para-Aminosalicylic Acid

Structure Similar to sulfonamides Mechanism of action Similar to sulfonamides Pharmacology Narrow Spectrum: Mycobacterium Mechanisms of Resistance Same as sulfonamides

Cephalosporins

Structure: 7-Aminocephalosporanic Acid Betalactam ring + Dihydrothiazine Ring (6 membered) R group: Change in R group - 1st, 2nd, 3rd, 4th, 5th generations Mode of action and uses: same as penicillins Ist generation: Cephalexin, Cefazolin, narrow spectrum: Gram +ve 2nd generation: Cefuroxime, cefoxitin: expanded spectrum to a few Gram negatives 3rd generation: Ceftriaxone, ceftazidime : broader spectrum, more Gram negative bacteria 4th generation: Cefepime, Cefpirome: extended spectrum, against Gram negative including Pseudomonas aeruginosa, also against Gram +ve 5th generation: ceftobibrole (powerful antipseudomonal activity), ceftaroline

Aminoglycosides

Structure: Amino-sugars linked by glycosidic bonds to other sugar derivatives Various derivatives Streptomycin, Amikacin, Gentamicin, Tobramycin Neomycin, Kanamycin, Netilmicin Mechanism of Action Inhibit protein synthesis by interfering with the function of the 30S ribosome Inhibit initiation complex and cause misreading of mRNA Oxygen dependent uptake Pharmacology Bactericidal (binds irreversibly)- narrow spectrum Effective against aerobes- Gram negatives Streptomycin - Mycobacterium Poorly absorbed in GI Penetrate poorly in abscess (Penetration requires O2)

Macrolides

Structure: Large (13-16 Carbon) lactone ring and two sugars one or more deoxy sugars, usually cladinose and desosamine Derivatives: Erythromycin, Azithromycin, Clarithromycin Mechanism of Action Exert their effect by their reversible binding to the 23S rRNA of the 50S ribosomal unit. Binding to 50s ribosome Prevents the release of tRNA after formation of peptide bond Pharmacology: Bacteriostatic: Broad spectrum, Gram positive and negative, Chlamydia, and Mycoplasma Drug of choice for penicillin allergic patients Mechanisms of Resistance Alteration of 50S ribosomal subunit Enzymatic modification Side effects: GIT disturbances due to suppression of the normal flora

Vancomycin

Structure: Large glycopeptide Mechanism of Action: Inhibits cross linking of peptidoglycan by binding to terminal D alanines Pharmacology: Bactericidal, Narrow spectrum; effective against Gram positives ADR: Red man syndrome: Histamine released from mast cell/basophils Resistance: Van A and Van B genes replace last D alanine with a lactic acid - Gene carried on plasmids and transposon (multi-drug R) Resistant strains of Enterococcus spp (known as VRE) and Staphylococcus aureus problematic (known as VRSA) world wide VRE-Vancomycin resistant enterococci VRSA-Vancomycin resistant Staphylococcus aureus

Surviving host defenses

To avoid immune attack by Interference: Camouflage: Antigenic Variation Coordinated expression of several genes (Pathogenicity islands) Camouflage: Disguise, concealment

Chloramphenicol

Structure: Nitrobenzene structure, no derivatives Mode of action: Binds to 50S: Inhibits protein synthesis by inhibiting peptidyl transferase or elongation stage Pharmacology: bacteriostatic Broad spectrum: including anaerobes Side effects: bone marrow toxicity- aplastic anemia; Gray-Baby syndrome Resistance: enzymatic modification and decreased permeability. Inhibits mitochondrial protein synthesis (mitochondria have 50s ribosome) in host cell - a reason for its toxicity in the bone marrow. Another explanation for toxicity is the drug acting as hapten.

Bacitracin

Structure: a large, complex cyclic peptide Mechanism of Action Binds to Bactoprenol and inhibits transportation of peptidoglycan precursors Bactericidal Pharmacology: narrow spectrum, Gram positive, nephrotoxic, Topical application Resistance: decreased penetration

Tetracyclines and derivatives

Structure: consists of four joined cyclic rings. Mechanism: binds to 30s - prevents binding of tRNA to mRNA Pharmacology: Bacteriostatic, broad spectrum- Many G+ve, G-ve bacteria; Mycoplasma, Rickettsia, Chlamydia -transported actively into prokaryotes but not into eukaryotes because of energy-dependent transport system present in prokaryotes, but not in eukaryotic cells. Side effects: Discoloration of teeth Calcium and Iron chelator - not for children under 8 and pregnant women Suppress Normal flora: Gastrointestinal, Vaginal tract Photosensitivity Mechanisms of Resistance Active efflux: most common Decrease permeability Alteration of ribosomal binding site Enzymatic modification (acetylation)

Capsules (Slime layer)

Surround single cells Composition: Mucopolysaccharides Multivalent - poor immunogen in children <2 Polypeptides Multivalent - still a poor immunogen Function Adhesion Protection against: Phagocytosis Dehydration Antibiotic penetration Gycocalyx = capsule (means sugar coat) Multivalent antigens: having multiple binding sites for antibodies Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose (six-carbon sugar) or a hexuronic acid, linked to a hexosamine (six-carbon sugar containing nitrogen)

Inclusion Bodies

The Negri body is an accumulation of rabies virions (belongs to Rhabdoviridae) in cytoplasm of neural cells Intranuclear inclusion body: From lesion of CMV (belongs to herpesviridae) infection-Cowdry type A (Owl' s eye)

HBsAg Rapid Test Principle

The Rapid Test is based on the principle of chromatographic immunoassay for qualitative detection of the surface antigen of hepatitis B virus (HBsAg) in human whole blood, serum, and plasma samples. Monoclonal and polyclonal antibodies are employed to identify HBsAg specifically. This HBsAg Rapid Test employs chromatographic lateral flow device. Colloidal gold conjugated monoclonal antibodies reactive to HBsAg are dryimmobilized onto a nitrocellulose membrane strip. When the sample is added, it migrates by capillary diffusion trough the strip rehydrating the gold conjugate. If present, HBsAg will bind with the gold conjugated antibodies forming particles. These particles will continue to migrate along the strip until the Test Zone (T) where they are captured by anti-HBs antibodies immobilized there and a visible red line appears.

Quorum sensing

The ability of bacteria to sense the presence of other bacteria via secreted chemical signals. Regulation of gene expression in response to fluctuations in cell-population density. Some genes are only turned on when bacteria are present in high concentrations. Many bacteria can use "quorum sensing" to detect presence of specific autoinducer chemicals used for measuring cell density. These autoinducers are specific peptides (Gram positive bacteria) or homoserine lactones (Gram-negative bacteria). Autoinducer is produced in low amounts by all cells, diffuses freely across membrane, so concentration inside and outside cell is same. As cell number increases, autoinducer conc. increases until it is sufficient to bind to activator, turn on transcription of specific genes. Example: some virulence genes of pathogens are controlled by quorum sensing. Until a sufficient density of cells is present, cells don't turn on these factors. Signal molecules produced by individual cells do not have an effect until the bacterial population density is sufficient to provide a concentration of molecules, which then cross cell membranes and activate the manufacture of such cellular products as toxins, enzymes or surfactants

Aseptic

The absence of microorganisms in tissue

Acid Fast Cell Walls

The cell envelopes of these organisms are considerably more complex than other bacteria. Mycolic acid (long, branch chained fatty acids) covalently bound via a polysaccharide to peptidoglycan. Other mycolic acid-containing compounds and other complex lipids form a thick waxy membranous layer outside the peptidoglycan layer. Components: Peptidoglycan Lipid based polymers woven through out, e.g, Mycolic acid Trehalose dimycolate Lipoarabinomannan Arabinogalactan Cell wall resists intracellular killing Examples Mycobacteria, Nocardia

Spheroplast

The complexity of the Gram negative cell wall results in innate resistance to enzymatic destruction of cell wall. Thus, Gm -ve bacteria with damaged cell walls become Spheroplasts i.e, they assume spherical shape even in a non-isotonic medium because of their resistance to differences in osmotic pressure between the extracellular and intracellular compartments.

Enveloped viruses

The envelope is derived from the host cell membrane or nuclear membrane depending on the site of replication (cytoplasm or nucleus). The proteins on the envelope are viral proteins (rarely are host cell proteins found on the envelope). Enveloped with icosahedral capsid e.g. herpes virus, yellow fever virus, rubella virus Enveloped with helical capsid e.g. rabies virus, influenza virus, parainfluenza virus, mumps virus, measles virus

Gene flow in bacteria

The genetic information is stored as a continuous segment of DNA, and the messenger RNA can immediately direct the synthesis of the corresponding protein. In higher organisms, the gene is usually split, and the messenger RNA has to be processed by splicing before it can be translated into a protein. Unlike prokaryotes, most genes in higher eukaryotic cells contain large amounts - as much as 98% in the human genome regions called introns that are not part of the code for the final protein. These are interspersed among the coding regions or exons that actually code for the final protein.

Nutritional factors

The most important elements necessary for synthesis of bacterial structural components (Carbohydrate, lipid, protein, nucleic acid) are hydrogen, oxygen, carbon, nitrogen, phosphorus and Sulphur. Hydrogen and Oxygen are made available from water added to the culture medium. Carbon and Nitrogen: Carbohydrate (sugars) is the principal source of carbon which is degraded by the bacteria either by oxidation or by fermentation. The oxidation or fermentation process provides energy in the form of ATP. Nitrogen is a major component of protein and nucleic acids and its main source is ammonia, usually in the form of an ammonium salt Sulphur and Phosphorus: Sulphur forms part of the structure of several co-enzymes and cysteinyl and methionyl side chains of proteins. Most bacteria use sulphate as a sulphur source and reduce it to hydrogen sulphide. Phosphorus is required as a component of nucleic acids, ATP, co-enzymes NAD and flavins. Mineral sources: required in trace amounts. Can be provided in tap water or as contaminants of other medium ingredients.

Human T-cell lymphotropic virus 1

The only human oncogenic retrovirus identified, uses more subtle mechanisms of leukemogenesis. It encodes a protein (TAX) that transactivates gene expression, including genes for growth-stimulating cytokines (e.g., interleukin-2 [IL-2]).

Septic

The presence of microorganisms in tissue.

Viropexis

The process by which different classes of viruses, particularly picornaviruses and papovaviruses, enter the host cell in which they will be able to replicate. The hydrophobic structures of the capsid proteins may be exposed after viral binding to the cell (see VAP, viral attachment protein). These structures help the virion or the viral genome slip through the membrane.

Hemagglutination

The red blood cells that are attached to virus particles form a lattice that coats the well

Quorum sensing

The regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities

Transcapsidation

The situation where a coat is entirely that of another virus, e.g. a retrovirus nucleocapsid in a rhabdovirus envelope. This kind of phenotypic mixing is sometimes referred to as pseudotype (pseudovirion) formation/Transcapsidation. The pseudotype described above will show the adsorption-penetration-surface antigenicity characteristics of the rhabdovirus and will then, upon infection, behave as a retrovirus and produce progeny retroviruses. This results in pseudotypes having an altered host range/tissue tropism on a temporary basis.

RNA Viruses

There are 13 families of RNA viruses (i.e more than DNA viruses) All RNA viruses replicate in the host cell cytoplasm except orthomyxo (e.g influenza) viruses (retroviruses in both the cytoplasm and nucleus) RNA of RNA viruses varies in its structure— ss or ds ssRNA is either positive (like mRNA) sense, negative sense Some may be segmented All negative sense RNA viruses are enveloped All negative sense RNA viruses carry an RNA dependent-RNA polymerase Only RNA viruses may be arthropod borne

DNA Viruses

There are 7 families Genome/DNA structure varies Double stranded linear (eg, herpes virus) Double stranded circular (complete) [eg, human papilloma virus] Partially double stranded circular - only in hepadnaviruses Single stranded linear - only in parvoviruses All replicate in the host cell nucleus except for poxviruses All have a neoplastic potential since they have an intra- nuclear residence and might damage host DNA They may also cause latent infection

Radiation

There are two types of radiation used in killing microbes- Ionizing and Non ionizing Ionizing radiation: X-rays, Gamma rays Have higher energy and greater penetrating power than ultraviolet light Create oxygen and hydrogen radicals from water Denature proteins and DNA Used to sterilize heat sensitive materials, such as gloves, syringes Non-ionizing radiation: UV light Destroy DNA by forming bonds between neighboring thymine molecule. Most lethal waves 240 nm-280 nm Poorly penetrate objects Used to sterilize air, operating theater (OT), surfaces Treatment of public water supply

Mutations in Bacteria

They reproduce fast, some divide within 20 min. Thus the probability of mutation is high. Because of their haploid state, bacterial mutations are expressed quickly, no recessive or dominant pattern Can occur as Spontaneous mutation: polymerase enzyme errors or Induced mutations: caused by mutagens - chemicals, radiation

Macromolecular Synthesis and Replication

Transcription of most viruses results in synthesis of early and late mRNA transcripts. The early transcripts encode regulatory proteins and enzymes required for DNA replication. This proteins are catalytic and only few are needed. The late transcripts encode mainly structural proteins of the virion. Replication of the genome usually initiates the transition to transcription of the late genes. Initiation of nucleic acid synthesis precedes transcription of the late genes.

Specialized Transduction

Transduction is transduction in which only certain donor genes can be transferred to the recipient. Different phages may transfer different genes but an individual phage can only transfer certain genes. Specialized transduction is mediated by lysogenic or temperate phage and the genes that get transferred will depend on where the prophage has inserted in the chromosome. During excision of the prophage, occasionally an error occurs where some of the host DNA is excised with the phage DNA. Only host DNA on either side of where the prophage has inserted can be transferred (i.e. specialized transduction). After replication and release of phage and infection of a recipient, lysogenization of recipient can occur resulting in the stable transfer of donor genes. The recipient will now have two copies of the gene(s) that were transferred. Legitimate recombination between the donor and recipient genes is also possible. Phage genome may pop out of chromosome with the exact genomic information it entered with - it is then non-transducing phage Phage genome may pop out of chromosome with a piece of bacterial chromosome which was adjacent to the insertion site - then it is a transducing phage. Since there is a size limitation on the amount of genomic material which can be packaged into a capsid, it must leave some of the original genome behind

Transduction

Transfer of bacterial genetic information using bacteriophage as the vehicle Bacteriophages Ds DNA viruses Complex structure, tadpole shaped-hexagonal head and cylindrical tail Head-icosahedral capsid with DNA 2 types: Virulent or lytic phage Temperate or lysogenic phage The phage coat protects the DNA in the environment so that transduction, unlike transformation, is not affected by nucleases in the environment. Not all phages can mediate transduction. In most cases gene transfer is between members of the same bacterial species. However, if a particular phage has a wide host range then transfer between species can occur. The ability of a phage to mediate transduction is related to the life cycle of the phage.

Generalized sequence of the stages of infection

Transmission from an external source into the portal of entry Evasion of primary host defenses such as skin or stomach acid Adherence to mucous membranes, usually by bacterial pili Colonization by growth of the bacteria at the site of adherence Disease symptoms caused by toxin production or invasion accompanied by inflammation Host response, both nonspecific and specific immunity, during steps 3,4 and 5 Progression or resolution of the disease

Hemagglutination Assay

Two-fold serial dilutions of a virus are prepared, mixed with a specific amount of red blood cells, and added to the wells of a plastic tray. The red blood cells that are not bound by the virus sink to the bottom. The red blood cells that are attached to virus particles form a lattice that coats the well Used to quantitate viruses that express hemagglutinins on their surface Reading (Example) Sample A: titer of 256 B: no virus detected D: titer of 512

Translation in prokaryotes

Unique microbial initiation, elongation, and termination factors 1st aa - N-formyl methionine ( fMet) (soluble PAMP = recognized by innate immunity as foreign) Targeted by antimicrobial agents Eg, tetracyclines, Macrolides etc. Most metabolic regulatory mechanisms involve control of transcription of the gene into messenger RNA, rather than control of translation of the messenger.

Sterilization

Use of physical procedures or chemical agents to destroy all microbial forms, including the bacterial spores. Physical means: heat and radiation Mechanical means : filtration Chemical means: Alcohol, H2O2

Salmonella

Use phase variation to switch between different types of the protein flagellin. As a result, flagella with different structures are assembled. Once an adaptive response has been mounted against one type of flagellin, or if a previous encounter has left the adaptive immune system ready to deal with one type of flagellin, switching types renders previously high affinity antibodies, TCRs and BCRs ineffective against the flagella.

Light Microscopy of viruses

Used to monitor the cytopathic effect. Morphological change: Change in shape, such as the rounding of cells, aggregation, degeneration Loss of attachment to culture dish, loss of contact inhibition Syncytia formation (multinucleated cells) Inclusion bodies: aggregation of viral proteins or particles in the cytoplasm or nucleus, margination of chromatin Haemadsorption - cells acquire the ability to stick to mammalian red blood cells by expressing viral hemagglutinin antigens on their surface

Bright field Microscope

Uses the visible light source under the stage to produce a clear image. The Diaphragm adjusts the amount of light entering the stage. It consists of three objectives- the scanning power(usually 4x-6x), the low power(10-12x), and high power(40x). Maximum useful magnification: 1000-2000X eg, Compound Microscope

Viral Evasion of the Immune Response

Various ways: few examples Existence in multiple Antigenic variants: eg. Influenza virus Latency- wait until the immune response weakens Infection of immuno-privileged sites within the body e.g. HSV in sensory ganglia in the CNS Interfere with antigen presentation Down regulation of MHC I expression, e.g. Adenoviruses Down regulation of LFA-3 and ICAM-1. Eg EBV LFA3: Lymphocyte function-associated antigen 3 (CD58): is a cell adhesion molecule expressed on Antigen Presenting Cells (APC), particularly macrophages. It binds to CD2 (LFA-2) on T cells and is important in strengthening the adhesion between the T cells and Professional APCs Inhibit TAP Transporter associated with Antigen Processing proteins Interfere with KAL and KAR i.e with NK recognition and killing, Eg. CMV Direct infection of the cells of the immune system, eg HIV

Detection of viral antigens

Viral antigens can be detected in the patient's blood or body fluids UV microscopy: by use of florescent dye tagged antibodies Serology: ELISA (Most common) Detection of p24 antigen of HIV, HBsAg of Hepatitis B Rapid tests Eg, for HBsAg of Hepatitis B

Pathogenicity Islands (PAIs)

Virulence cassettes: Unique area in the bacterial genomes where several genes are clustered, favoring their co-inheritance and co-expression May contain virulence genes Antibiotic resistance genes

Viral classification

Viruses are classified on the basis of their genome, capsid structure, envelope and replication strategy: Based on Genome: DNA: dsDNA; Gapped dsDNA; ssDNA; RNA: dsRNA; (+) ssRNA; (+) ssRNA with iDNA; (-) ssRNA. Based on Capsid Structure (capsid symmetry): Helical Icosahedral Complex Based on the presence/absence of Envelope Enveloped Non enveloped (naked)

Oncogenic human viruses

Viruses associated with human cancer

Naked viruses

Viruses that lack an envelope Naked with Icosahedral capsid: Capsomeres arranged in 20 triangles that form a symmetric figure - polyhedral e.g. poliovirus, adenovirus, hepatitis A virus Naked with Helical capsid: capsomeres arranged in a hollow coil that appear like a rod e.g. tobacco mosaic virus. So far no human viruses with this structure are known

Helix distortion mutations

When UV radiation induces dimerization of adjacent nucleotides, particularly thymines. Results cyclobutane ring in DNA, which prevents faithful replication

Complementation

When a defective virus is aided in its replication by a helper virus (complete virus/ or other defective virus) Can occur when either one or both of the two viruses that infect the cell have a mutation that results in a nonfunctional protein Non mutated virus (helper virus) complements the mutated one (defective virus) by making functional protein

Hfr Conjugation (Hfr × F-)

When the F factor integrates with the main chromosome 1.An F+ plasmid inserts into the donor bacterium's nucleoid to form an Hfr male. 2. The sex pilus adheres to an F- female (recipient). One donor DNA strand breaks in the middle of the inserted F+ plasmid. 3. The sex pilus retracts and a bridge forms between the two bacteria. One donor DNA strand begins to enter the recipient bacterium. The two cells break apart easily so the only a portion of the donor's DNA strand is usually transferred to the recipient bacterium. 4. The donor bacterium makes a complementary copy of the remaining DNA strand and remains an Hfr male. The recipient bacterium makes a complementary strand of the transferred donor DNA. 5. The donor DNA fragment undergoes genetic exchange (recombination) with the recipient bacterium's DNA. 6. The recipient bacterium gain new genes from the donor, but does not receive tra operon, thus remain F negative-.

Colony

a visible mass of microbial cells on solid/semisolid media that theoretically arose from one cell

A-B Toxins Mode of Action

a. ADP-ribosylation of Elongation factor-2 G-protein - increase in cAMP Intracellular signalling cAMP and G protein: G proteins (guanine nucleotide-binding proteins) are a family of proteins involved in transmitting chemical signals outside the cell, and causing changes inside the cell b. Cleavage of rRNA The ADP-ribosyl group is removed from the coenzyme NAD (see dashed line) and is covalently attached to a host cell target protein. This causes the inactivation/hyperactivation of that target protein. c. Interference with release of neurotransmitters (Neurotoxin)

Anaerobic Culture

a. Gas Pak system b. Anaerobic chamber c. Reducing medium - Thioglycollate broth

Red man syndrome

in the past attributed to impurities found in vancomycin preparations, earning the drug the nickname 'Mississippi mud'. But reports of the syndrome persisted even after improvements in the compound's purity. Studies have shown that an unknown percentage of the population may be prone to releasing a large amount of histamine in response to vancomycin. The hypersensitivity reactions that can arise due to vancomycin are due to its effect on the mast cells. In tissue culture, vancomycin causes degranulation of peritoneal mast cells in rats. An anaphylactoid reaction (Vs. The anaphylactic reaction-which is mediated by IgE) is caused by the degranulation of mast cells and basophils, resulting in the release of histamine independent of preformed IgE or complement. The extent of histamine release is related partly to the amount and rate of the vancomycin infusion. The name comes from the red rash that develops on the face, neck, and torso (trunk) of affected people

Viral Genome

nucleic acid RNA or DNA (not both) Single (ss) or double-stranded (ds) Linear, circular ssRNA, either positive strand or negative strand RNA may be segmented Usually haploid, some diploid