MICROBIOLOGY
2009-2010
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DEVANGNA BHATIA
Microbiology Questions
General microbiology
1) Bacterial cell (morphology, staining reactions, classification of bacteria) Morphology: the form and structure of an organism. It includes shape and size-Shape: this can be of 3 main types:
- round (cocci) - regular (staphylococci) CCCM
flattened (meningococci) -d'^iotow* ' lancet shaped (pneumococci)
- elongated (rods) - straight (majority of them are like this; e.g. E.coli),
short (coccobacilli; e.g acinetobacters)
long (fibres) - these are mainly found in OLD cultures , __
slender - mycobacterium tuberculosis C^~r^;
robust - lactobacilli, bacillus .
with split ends -bifidobacteria Jy^C< LU
branching - nocardiae, actinomycetes
curved - Campylobacters
with flat ends - bacillus anthracis
spindle-shaped - fusobacteria
<sf^ club-shaped - corynebacteria
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^ Sr w v*"~~ pleomorPhic ~ haemophili ^>>^\ spiral - helicobacter, spirillum
Spiral bacteria (spirochetes): these are different to the spiral bacteria mentioned above! They are tightly coiled bacteria, o Uneven: borrelia o Delicate, regular: Treponema o Slender with bent ends: Leptospira
- Size:, pathogenic bacteria are generally between l-5fim Rods—the fibres can be upto 50 um long Some can be very large, e.g bacillus and Clostridium (1x10 urn) Others can be small, e.g. haemophilus (0.3 urn x 0.6 u.m)
Cocci can be found in:
- Clumps, e.g. Staphylococcus aureus
- Chains, e.g. Streptococcus pyogenes CCCCÖ
- Diplococci (lancet-like) e.g. Strep. Pneumoniae ^■ew.op^C^
- Diplococci (flattened) e.g. Neisseria gonorrhoeae
- Cocci in tetrads e.g. Micrococcus luteus
Rods can be found in:
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- Most of rods are found singly, e.g. E.coli
- Delicate Streptobacilli, e.g. haemophilus ducreyi
- Diplobacilli (in pairs) e.g. moraxella lacunata r^yľZŽ
- Robust rods with rounded ends e.g. Clostridium perfŕingens
- Robust rods which are fiat, with concave ends, found in bamboo cane-like chains, e.g. Bacillus anthracis
- club-like in palisades: Corynebacterium diphtheriae       fTTľ\Q \ •
- . slender, hinted palisades: Mycobacterium tuberculosis 'ß-
- branched and fragmented; Nocardia asteroides
- spindle-like: Fusobacterium fusiforme
- pleomorphic and small: Haemophilus influenza
Curved or Spiral rods:
- curved rods, crescent-shaped:^Vibrio cholerae
- thick spirals:' Spirillum minus
- uneven spirals: Borrelia recurrentis
- delicate, regular spirals •. Treponema pallidum
- Very fine spirals with bent ends : Leptospira icterohaemorrhagiae
Staining Reactions and Classification of bacteria: this depends on their morphology (above) and staining reactions (either G+ or G-).
QygrK| +^^^6^01: a^^^P|ayer in their cell wall, which is able to retain thscrvjlaj ^.ct, stain. This resutts^^^^^^^ÄM^ staining by Gram staining. Also these bacteria, lack the outer membrane which is found in G- bacteria. " E.g. Staphylococcus, Streptococcus, Bacillus, Lactobacillus, Corynebacterium...
Due t0 tnefe.s£fcjtofealfetejKSm the G- bacteria cell wall these bacteria can't retain the crystal violet stain, so it getst^ashedsout and the counter stain^^amn|colours all the G-bacteria »glli«l§Bffifc#^ Salmonella, Vibrio,Haemophilus,Pseudomonas, mycoplasma... .        ,„ ,±..,A<w \
2) Anatomy of bacterial cell -1 (contents of cytoplasm + cytoplasmic menrorane)'
Bacteria are prokaryotes meaning that they lack the membrane bound organelles found in eukaryotes, e.g. they don't have nucleus, mitochondria, Golgi apparatus or endoplasmic reticulum (ER). Some bacteria produce nutrient granules and store them in their cytoplasm, for use later on, e.g, glycogen or sulphur.
Other cytoplasmic contents are: vacuoles, plasmids, ribosomes and the nucleoid. The cytoplasmic (plasma/plasmatic) membrane is a phospholipid bilayer, containing fatty acids but lacking sterols. CJiannejs^caUe^prins^ can be found, which allow tbe- passive transport of several ions, sugars and amino acids across the outer membrane. The function of the phospholipid bilayer is to act as a permeability barrier for most molecules and it serves as the location for the transport of molecules into the cell.
3) Anatomy of the bacterial cell - II (cell wall, capsule, flagella, fimbriae, pili)
Virulence Factor: molecules produced by a pathogen that specifically cause disease Desiccation: drying out
Cell wall: The function of the bacterial cell wall is to provide structural integrity to the cell. However, its primary function is to protect the cell from internal turgor pressure caused by the higher concentration of proteins and other molecules inside the cell compared to the outside.
The bacterial cell wall contains ^flfilpplif which is located immediately outside of the cytoplasmic membrane. This peptidoglycan is responsible for the rigidity of the bacterial cell wall and determines the cell shape. It is relatively permeable, especially for small substrates. There are two main types of bacterial cell walls, Gram positive and Gram negative, which are differentiated by differences in their Gram staining characteristics.
The G+ cell wall has a thicker peptidoglycan layer, meaning that the cell wall is able to retain the crystal violet dye, hence staining it dark blue or violet.
On the other hand, the G- cell wall has a thin peptidoglycan layer and this cannot retain the dye, hence it gets decolourised (by ethanol) during staining. This results in the cell wail, staining a red or pink coloxir, due to the counter-staining with safranin. G- cell wall contains an outer membrane composed , ( of phospholipids and lipopoiysaccharides, which face the external environment.    t^-t^c—r^r^ p^P^0'
Capsule: They are mainly found in G- bacteria. Capsules are relatively impermeable layers, found outside the cell wall. They are wfter soluble, meaning that it is difficult to stain them, since many stains don't adhere to the capsule. A capsule is a well organised layer, which isn't easily washed off and it can be the causes of many diseases. ^
Their function is to help protect bacteria from phagocytosis and desiccation. Due to the fact that they help to protect bacteria against phagocytosis, they are considered a virulence factor. Capsules also contain water which protects bacteria against desiccation. Furthermore, bacterial capsules allow bacteria to adhere to surfaces and other cells.
Flagella: These are whip-like structures protruding from the bacterial cell wall and are responsible for bacterial motility (movement). The arrangement of flagella is unique to the species. Common forms include:
• Peritrichous - Many flagella found at numerous locations about the cell
• Polar - Single flagella found at one of the cell poles C^Z>~-—
• Lophotrichous - A bunch of flagella found at one cell pole £^Jf_
Flagella are complex structures, composed of many different proteins, including flagellin, which makes up the whip-like tube and a protein complex that extends through the cell wall and cell membrane to form a motor that causes the flagellum to rotate. This rotation is normally driven by proton motive force and is found in the body of the cell.
Fimbriae and Pili:
FJmbriae are protein niic^ofibrils that extend out from the outer membrane. They are usually short, half as thick as pin' and present in high numbers on the entire bacteriaTcell surface. The function of
fimbriae is to help the attachment of a bacterium to-a-surface (e.g. to form a biofilm) or to other cells (e.g. animal cells during pathogenesis). A few organisms (e.g. MyxocQ££M§) use fimbriae for motility to facilitate the assembly of multicellular structures. They are fouWm many G-j)acteria.
Pili are similar in structure to fimbriae but are much longer and present on the bacterial cell in low numbers. Pili are involved in the process of bacterial conjugation. Non-sex pili also aid bacteria in gripping surfaces. * ' h
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4) Anatomy of bacterial cell III - bacterial spores <eU-&U ccuWfc Noxae: agents capable of exerting a harmful effect on the body.
A bacterial spore or endospore is a dormant, tough, and non-reproductive structure, which is produced by G+ bacteria, Among human pathtfgenTc bacteria, only Clostridium and Bacillus produce spores.
The spores are spherical to oval in shape and are characterized by a thick spore wall and a high level of resistance to chemical and physical noxae. This ensures the survival of the bacterium in a dormant phase through long periods of starvation and other adverse environmental conditions. Also, they are highly heat resistant, meaning that during heat sterilisation procedures, very high temperatures are required to Kill them effectively, i.e. 100-120°C for lQjOmins.
They are also resistant to ultraviolet and gamma_j2diaiion, desiccation, lysozyme, starvation, and chemical disinfectants. Eridospores are commonly found in soil and water, where they may survive for long periods of time.
In unstained preparations, the spores are larger than lipid inclusion granules and often oval in shape.
The location of the endospore in the bacterium differs among different species and is useful in identification. The main types within the cell are
- Terminal: seen at the poles of cells - £... tete\l
- Subterminal: those between these two extremes, usually near the poles but close enough to the centre as well, so can't be considered either terminal or central - Qxdllvs su'ekilU
- Centrally placed: more or less in the middle - J&oiitw CttCM
- Lateral endospores: seen occasionally
5) Microbial growth, incl. conditions required
Bacteria reproduce by binary fission, which is a form of asexual reproduction and cell division.
- Period I (initiation): the cell grows and proteins that start the next step accumulate inside it.
- Period C (chromosome replication): begins in one spot and diverges out in opposite directions
- Period D (division): a supply of macromolecules is formed
The cytoplasmic membrane inserts between the replicated chromosomes and separates them The cell wall grows into the cell at a particular spot and forms a septum that ultimately divides the maternal cell into two daughter cells
Division of cocci can occur in one plane e.g. streptococci or in different planes e.g. staphylococci. Division of rods can occur in the transverse plane e.g. majority of chain rods or in the vertical plane, e.g. corynebacteria, mycobacteria.
The generation time is the time taken for the ^^^^^^^S OR duration of the growth cycle. On average it is usually about 30 minutes. Other examples: E.coli - 20mins, Mycobacterium tuberculosis (TBC) - about 12 hours.
Since the number of bacterium double generation time, we can say that bacteria multiply by geometric progression.
E.g. If the generation time is 30 min, after 24hrs theoretically one cell gives origin to 248 = 2.8xl014 cells. However the actual amount of cells produced, is approximately 5 orders less (i.e. around 109 cells). This amount of cells can be seen by the naked eye and in a liquid broth it appears cloudy or sedimentation occurs at the bottom or a pellicle is seen at the top. In a solid medium (agar), a bacterial colony is formed. {J
The result 109cells/24hrs applies for stationary cultures, in which nutrients are consumed and metabolites accumulate. The speed of multiplication changes depending on time and the growth of the bacterium can be illustrated by the use of a growth curve.
A growth curve depicts the number of viable cells in the logarithmic scale, depending on the age of culture.
Lao Exootenti
There are 4 growth phases, and they 'gradually change from one to the other:
- Lag ■- microbes are growing but not dividing. During this phase, bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
- Log (exponential) - cells are dividing at a constant speed. The relation between the no. of living cells and time is exponential. There are more than enough nutrients to allow the cells to grow..
- Stationary1- the no. of cells is staFle'- the no. of cells being produced = no. of cells dying. Here the growth rate slows down due to the lack of nutrients and the accumulation of metabolites.
j   -   Death - no. of cells dying > no. of cells being produced. Here the bacteria have run out of nutrients and die.
Growth conditions:
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Temperature; most bacteria grow optimally at human body temperature (37°0), e.g. E.coli, / which is part of the normal human intestinal micro flora/ Some can survive at higher/lower* temperatures than the human body temperature. .'
pH;)optimal conditions are between 6.7 - 7.5.? However some other bacteria, like vibrio cholera, can survive in pH conditions as high as 9.0 and gastric helicobacter can survive in acidic pH,
NaCl concentration: should be about 0.9% (physiological saline)/ Staphylococci/ can multiply on sweaty skin, where the concentrations are about io"%.     u/>d &\ feft *o sei^fcu* sn^M* Nutrients5: need to be in the correct balance of carbon, nitrogen, hydrogen, sulphur, iron etc. for synthesis of specific bacterial compounds. Some bacteria also require "growth factors,"1 i.e., organic compounds they are unable to synthesise themselves. /
^5)- * Osmotic Pressure: Bacteria are about 80-90% water; they require moisture to grow because
they obtain most of their nutrients from their aqueous environment.? 'S)-   Anaerobcs/acrobes:j oxygen may or may not be needed, depending on the species of bacteria
and the type of metabolism used to extract energy from food. In all cases, the initial
breakdown of glucose to pyruvic acid occurs during jfycojvsis, which produces a net gain of
two molecules of ATP. * -
6) Microbial metabolism
This is the way in which a microbe obtains energy and nutrients it requires, to survive and reproduce. The processes can be anabolic (synthesis of compounds and the consumption of energy) or catabolic (break down of substrates to gain energy). " '
Human pathogenic bacteria are always<^mosynthetB>, or^anojrophic_bacteria (or chemo-organotrophs).
Catabolic Reactions:
- Digestion - bacterial exoenzymes split the nutrient substrates into smaller molecules outside th~eceU '
- Uptake - nutrients are takenup by passiy£_d4fijsion, or more usually active transport through the membrane
- Preparation for Oxidation - phosphorylation etc
- Oxidation^ removal of electrons and H4 ions. The H2 atoms are then transferred to the hydrogen acceptor.
There are 3 types of catabolism:
1) Fermentation - breakdown of nutrients withouMhe need for oxygen. Only small amounts of energy are produced. Products are lactate, ethanol etc
2) Aerpbic Respiration - uses oxygen and a small amount of nutrient provides a large amount of energy. Products are CO2 andjj^O,
3) Anaerobic Respiration - another electron acceptor The type of catabolism is related to oxygen consumption:
1) Facultative anaerobes - they oxidise nutrients by respiration and fermentation. They can grow in all conditions. * '
2) Obligate (strict) aerobes - they can only reproduce in the presence of oxygen.
3) Obligate (strict) anaerobes - they die in the presence of oxygen. Their vital enzymes are inhibited by oxygen.
4) Micro-aerophile bacteria - grow in conditions with traces of oxygen.
5) Aerotolerant anaerobes - they don't utilise oxygen for growth but can survive in its' presence.
6) _Ca^nopiüle_bacteria^they^eed4ügher^mountsT)f eOä
7)  Media for microbial growth - examples
Microbes can be grown on a solid medium or in a liquid medium, usually called a liquid broth. There are 2 types of liquid media:
• Multiplying media: the most common and universal one, e.g. broth for aerobic culture and VL broth (Viande-Levure)- ,5+r;ci~(«j saaosju^
• Selectively multiplying media: only some bacteria can grow in this, e.g. selenite broth for salmonella
4) Micrc^aerophile bacteria - grow in conditions with traces of oxyget
5) Aerotoleralnt^anaerobes - they don't utilise oxyggfj-fet^growth biflsc^p^survive Inj its' presence.
6) Capnophile bacteria :rthey4ieedJ«grier amounts of CO2
7)  Media for microbial growth - examples
Microbes can be grown on a solid medium or in a liquid medium, usually called a liquid broth. There are 2 types of liquid media:
• Multiplying media: the most common and universal one, e.g. broth for aerobic culture and VL broth (Viande-Levure)
• Selectively multiplying media: only some bacteria can grow in this, e.g. seienite broth1 for -salmonella ------- ----------------- .....—
Liquid broths are used when there is only a small amount of microbes in the specimen and wjhen they multiply quickly.
Solid media: there are several types of this as well: --
• Selective solid media: similar to the liquid broth, only certatn bacteria can grow in this, from a mixture of several bacteria, e.g. blood agar with 10% NaCl Is used for staphylococci.
.....Sometimes antibiotics are also added for selectivity, e.g. blood agar with amikacin is selective
for streptococci and enterococci.
• Diagnostic media: they don't selectively grow any barterTa, since they don't suppress the growth of any microbe. Their contents, allow microbes to. grow and they can. be differentiated according to some properties, e.g. blood agar to observe haemolytic properties. * ".........................
Special kjnds of diagnostic media are chrpmogenic and fluorogenic media.
-  Chromogenic media: they contain a dye with bound specific substrate. The dye lfc>ses
- colour and is no longer a dye but a chromogen. - -------------
Bacteria are able to break down this specific substrate and change the specific substrate to the original dye. the media can contain more chromogens, depending on the number of species you want to differentiate. It is used for yeast cultures. .
- Fluorogenic media: works in the same way but uses a fluorescent dye instead. Blood Agar: oh this you can see haemolysis of RBCs, whether it is absent/partial ortotal It is
...... also possible to see vlridation (goes green). Total haemolysis shows up due to p-hemolytic
activity, whereas partial haemolysis is due to a-hemolysis activity and it appears green, y-
hemolysis refers to the lack of haemolytic activity. .........-.............
BA is an enriched medium, although it can also be classed as a diagnostic medium, therefore it can be classed as both (sometimes).
Endo Agar: it is a selective and diagnostic mediunrv used to grow some 6- bacteria (not jail!). The growing bacteria can be divided into or . Laqtose
positive bacteria are usually milder pathogens that lactose negative ones I        "    " | Other selective media: McConkey media, XLD and. MAL- these ones are selective and diagnostic media used to grow Salmonella.
Hajna medium is grown in test tubes, even though it is a solid medium. This is because it is used for biochemical testing and to see the difference between the lower part (no oxygen access) and the upper part (surface of the medium). j
Name	Class	Colour	Type	For
Broth	Liquid media	yellowish	multiplying	aerobes
VL-broth		darker		anaerobes
Selenite broth		pinkish	selective multiplying	Salmonella
Sabouraud Agar	solid media in test tubes!	white	selective (only with antibiotics)	Fungi
Lowentein-Jensen		green	enriched	TBC
Blood Agar	Solid media on Petri Dish	red	enriched + diagnostic	majority of bacteria
Endo Agar		pink	selective diagnostic	mostly enterobacteria
MUller-Hinton (MH) agar		white	special	antibiotic susceptibility
NaCl		mm	selective	staphylococci
VL-Agar		red	enriched + diagnostic	anaerobes
XLD (and MAL)			selective diagnostic	Salmonella
Chocolate Agar		HI	enriched	Haemophili, Niesseriae
Levinthal Agar		yellowish	enriched	Haemophili
Slanetz-Bartley		pink	selective diagnostic	Enterococci
8) Sterilisation
Sterilisation refers to any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) from a biological culture medium. It doesn't, remove prions. It can be done in various ways, such as use of heat, chemicals, irradiation, high pressure or filtration.
In microbiology, sterilisation is done in the following ways:
1) Hot steam under pressure: it should be saturated steam, which is only suitable for objects made of glass, metal, ceramics, china, textile, rubber and some plastics. Temperature ranges from 121-134°C
2) Hot air sterilisation: in apparatus with artificial air circulation: 180°C for 20mins, or 170°C for 30mins, or 160°C for lhour. Used for glass, china and metal
3) Hot water sterilisation under pressure: NOT USED ANY MORE!
4T~ GaWmaTay sterito^^ use-gloves---------------
5) Plasma sterilisation: in a high frequency electromagnetic field. This is a new method
6) Chemical sterilisation using formaldehyde vapours or ethylenoxide: in situations disabling the use of physical methods.
7) Fire sterilisation: for microbiological loops only. Incineration of waste
The method to use for sterilisation depends on the resistance of the materials to various temperatures, humidity, chemicals etc. Also, the temperature and time has to be sufficient enough to kill the microbes.
To check how effect the sterilisation was, indicators that change colour can be used at certain temperatures. This is the chemical method. There is a biological one as well, which consists of using resistant strains of Bacillus genus. Since they are resistant, their survival is assessed at the end of the sterilisation cycle.
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9) Disinfection
This means cleaning of surfaces by destroying all harmful microbes, i.e. all pathogenic organisms, which may cause an infection.
Disinfectants are anti-microbtal agents that can be used to destroy microorganisms. Some are suitable for surfaces or tools whereas others are suitable for the skin only. Disinfection can be done by various methods:
• Physical methods: by boiling
o At normal pressure for at least 30mins (in medicine) o in pressure vessels but for a shorter period of time
• Other physical methods such as filtration, sunrays, UV rays etc
• Chemical disinfectants:
o Oxidation reagents: peroxides, e.g. peracetic acid, it can be used for spores, fungi and TBC. Disadvantages - it is highly aggressive, causes decolourisation of textiles and instability of solutions
o  Hydrogen peroxide: similar to the above, but less effective and aggressive.
o Halogen preparations: hypochlorite, e.g. Sodium hypochlorite (bleach), calcium hypochlorite
o Chloramine
o  Iodine Solutions
The effectivity of various disinfectants to various organisms, can be found in booklets where they are divided into different groups, which are alphabetically arranged, e.g. A = effective to yeasts and bacteria, B = effective against viruses, C = bacterial endospores, T = TB mycobacterium, M= atypical mycobacterium and V - filamentous fungi.
10) Mechanism of antimicrobial drug action
"IDEALLY THE ANTIMICROBIAL AGENT SHOULD ACT AT A TARGET SITE PRESENT ON THE PATHOGEN, AND NOT C^N-JHE HOST CELL". /
Many microbial agents are today derived from natural products of fermentation (and they are usually also chemically^nodified for its improvementj^Some antimicrobial agents are synthetic as well, e.g. sulfonamides, quhnolones... /
An antimicrobial drug which is bactericidal^rneans that it kills the cells and the process is, hence, irreversible. An antimicrobial drug whjen is bacteriostatic stops bacterial cell growth and is reversible. Bacteriostatic drugs are gdoato use in patients with normal immune defense, which then can take care of the infectjjem by normal immune response. However, it's not suitable for immunocompromised patients^lowered immune response).
E.g. CLORAMPHENICOL inhibits growth of Escherichia\p!i (bacteriostatic) and kills Haemophilus influenza (bactericidal^ \.
* Antioarasitai agents against parasites ■   Antfmycotics against yeasts and molds
ntivirotics against viruses
■ Antitubercuiotics against mycobacteria
■ Antibiotics against bacteria
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General bacteriology
14. The strategy of antimicrobial chemotherapy
Antimicrobial chemotherapy is the use of chemical agents for antimicrobial action, which work by either killing it (bactericidal) or stopping its growth (bacteriostatic). The strategy of the antimicrobial agents is to have a target site exclusively present on the microbe and not on the host cell -> SELECTIVE TOXICITY.
Selective toxicity with desired properties of a new antimicrobial agent:
■ Selectivity for microbes rather than mammalian cells. £n,bdK» Cidal activity.
■ Slow emergence of resistance.
■ Narrow spectrum of activity.
■ Non-toxic side effects to the host.
■ Good body distribution. -To jmA H^2* i'^fi^f"* " Decrease in plasma protein-binding.
■ No interference with other drugs.
■ Easy administration.
The antimicrobial agents target cell wall, protein, and nucleic acid synthesis; metabolic pathways, and cell membrane functions.
Triangle relationship:
Antimicrobial agents
Human host
*■ Microorganisms
(Interfering with one side of the triangle will affect the other parts).
General bacteriology
15. Bacterial genetics
Bacteria can have primary resistance (i.e. innate resistance), and some bacteria can acquire it (secondary resistance).-^ccp^i
Bacteria can rapidly develop secondary resistance, as seen in many cases, e.g. Staphylococcus aureus.
Resistance can become present because of:
1) Single chromosomal mutation in one bacterial cell -> change in one protein. Although, this is rare. p
2) Series of mutations. ^ */• ™
When there is an antibiotic present, these mutations within the microbe become more   ^___—,
probable, and hence, the risk of acquiring secondary resistance increases. •„• ^ojXo^^^ Secondary resistance is acquired by genes on transmissible plasmids (the cell can even ^TaJ^vW acquire multiple resistance in this manner, so-called Infectious resistance). Resistance genes may also occur on transposons (jumping genes') -> these genes can integrate into chromosome (more stable location for the genes but slower spreading of genes than plasmids) or into plasmids.
An integron = site of multiple resistances (cassettes of resistance), It is found in plasmids, chromosomes and transposons.
Resistance gene cassette I
Integrons
\ I_
Transp osons j
| ▼ Chromosomeyplasmid
Mechanism of resistance:
■ The target site may be altered - lowered affinity.
■ Access to the target site may be altered (altered uptake, efflux mechanism, decreased cell wall permeability).
■ Enzymes that modify or destroy the antimicrobial agent may be produced e.g. beta-lactamases, amino-glycoside modifying enzymes, chloramphenicol acetyl transferases.
Epidemiologlcally important resistances:
■ MRSA (methicillin resistant staphylococci) - oxacillin or other beta-lactams cannot enter the cells. Many MRSAs are also resistant to other drugs, e.g. macrolides, lincosamides etc.-clu^/fio        fo^^feo u-v Pffil
■ VISA, VRSA (vancomycin-intermediate Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus) - staphylococci partially or fully resistant to glycopeptides (e.g. vancomycin).
■ VRE (vancomycin resistant enterococci) - they spread easily. Enterococci are found in many people's intestine.
General bacteriology
16. Pathogenicity and virulence
Pathogenicity = the ability of a microbe to be harmftďand cause disease1 (alter the state of health).
Infectiousness .= the ability to cause infection.
In a disease, symptoms are present i.e. disease x symptoms. The relation between infection and symptoms is following, infection x no symptoms (inapparent) or symptoms.
Microbes can also cause food poisoning apart from infections.
Three forms of symbiosis:
1, p^aŮsml- when both parts are benefitting from each other ^M) ■! f
2, Gamirn^^Msm -J when ofjjj^nf*^ the other part remains unaffected (©,-).
3, ParášitismV when one part benefits frdm the other part but at the same time negatively affecting it (©,©).
Microbe-4-> Host
Environment Illness is not a rule - peaceful coexistence is usually better for parasitism!
Pathogenicity depens on: \
1. Microbial species-pathogenic x nonpathogenic. \
2. Host specie^- susceptible x resistant. \
> Primaiy obligate pathogens: they cause infections in healthy individuals -^classical/ infe^ons| e.g. diphtheria, typhoid fever, plague, gonorrhoeae, tetanus, influenza, etc.
> Opportunistic (facultative) pathogens: members of normal flora reach other parts of f the body, where they shouldn't be present^ They can also spread to other locations when the person has decreased immunity] E.g. of facultative pathogens are E,coli (1% of normal colonic flora), Staphylococcus epidermidis (normal in skin and mucosal flora).
Virulence h the degree or measure of pathogenicity. It defines the property of a certain strain! which could be híglíiy^řuléríťor almost avirulent.flt is the relative ability of a pathogen to cause disease.
Indicator p£strain virulence: (ability to kill) j
- IJil^^^^W^) - it is the amount of microbe that is able to kill 50% of' experimental ahimal£' »
Attenuation:' it's a method causing artificial weakening of virulence '(e.g. to produce attenuated vaccines -> BCG vaccine against TBC).
Three elements of pathogenicity and virulence:
1) Communicability (transmissibiliry) - ability to be transmitted between hosts, ■/
General bacteriology
2) Invasiveness -3 ability to:
• a) Enter the host.!     *|      Ability to overcome the
• b) Multiply in iO      V     immune defense: of toe host.
• , c) Spread inside it.s J
3) Toxicity -fability to do harm to the host;
General bacteriology
17. Colonization and invasion ,   , . n . ^ .    .   -H   / -
Colonization means the colonization of a nonpathogenic microbe (or by a pathogen which does np_t cause pathological symptoms at that area) on a bodily surface. It is the establishment of a stable population of bacteria on the host.
Invasion is when the microbe enters the host, and it is usually through the mucosae. It is also commonly preceded by colonization (overcoming the possibility of commensalism (= symbiosis: ©,-)). For successful entry of the microbe it needs to adhere to the epithelium by means of adherence factors, and penetrate through the epith. by means of penetration factors.
Adherence factors: AfwW
1) Fimbriae (pili; - their end reacts with a receptor on the epithelial surface.
2) Nonfimbrial adhesions -hemagglutinins of yersinae, bordetellae, F protein of streptococcus pyogenes.
3) Viruses - they can envelope projections (hemagglutinin) of influenza virus. Glycoprotein gpl20_of HIV is also an adherence factor.
4) Parasites - suc3t themselves to the mucosae.
5) Micromyocetes - glucans and mannans of yeasts, & keratophilia of dermatophytes (skin moulds).
Penetration factors:
■ Direct penetration:
> Small cracks in skin.
> Small cracks in mucosa.
> Animal bite.
> Arthropod bite.
> Enzymes.
■ Forced penetration:
> Changing cellular framework (invasines).
> Ruffling (trouble) of epithelial surface (e.g. Salmonellae)
> Unknown mechanisms.
Multiplication:
■ Intracellular multiplication - better because of good nutrient supply, & defense against host immunity. E.g. of intracellular parasites are mycobacteria, rickettsiae, chlamydiae, usteriae, salmonp.liap- ~ '
■ Extracellular multiplication - stopped by antibacterial substances (e.g. complement, lysozym, antibodies) but mostly because of shortage of free iron (lactoferrin, transferin), and also high temperature.       "** ' "
Spreading of the microbe in the body:
■ Localized infections (common cold, salmonellosis, gonorrhea).
■ Systemic infections (influenza, meningitis).
■ Generalized infections (morbilli, typhoid fever, and even localized and systemic infections). l|>fWi$
> Way of spreading:
- Lymph -Blood
- Per continuitatem along nerves
Genera! bacteriology
Invasion by transmission:
■ The way of transmission - exit point of the body, and entry point of the host.
■ Microbe tenacity - degree of resistance to the external environment. E.g. Clostridium tetani, Giardi lamblia, and Helminth eggs (Taenia saginata) are all spore forming, so, that they can survive in the external environment.
"   Minimum infectious dose - amount of microbes required to start infection, An
immune person has a high infectious dose Coxiella burnetii, which causes Q fever, has an extremely low infectious dose of only 10° cells.
■ Behvaiour of the host - immune defense reflexes (cough, sneezing, diarrhea).
■ Way of elimination.- every biological substance is infectious.
■ Amount of eliminated substance.
■ Portal of entry.- better penetration through mucosa than skin. Some requires direct transmission (sexual contact, e.g. gonococci, treponemae), biological vector (tick, mosquitos -> arboviruses, borreliae), and transmission by water (leptospirae, shigellae).
General bacteriology
18. Aviodance of host defense mechanisms
Ability to overcome the innate immunity by:
0 -   Resisting complement.
0 -   Inhibiting complement activation.
(3) -   Protecting one's own surface.
g) -   Resisting phagocytosis by not getting engulfed, or by surving inside the phagocyte. -   Interfering with the cytokine functions.
\ Resisting complement system:
> Formation of capsule (shielding surface molecules), e.g. seen in meningococci, ' pneumococci
> Activation of inhibitors, e.g. gonococci add sialic acid to terminal saccharides.
> Production and regulation of factor H (= regulates the alternative pathway of the complement system), e.g. many viruses, E.coli, Strept. Pyogenes.
> Production of enzymes splitting C3b and C5a, e.g. Strept. Pyogenes and Pseudomonas aeruginosa.    |fo*cl<Yw<$$ töv^-p&v^t-<J~ Av$a^#w> .
> Protection of the surface, e.g. Salmonellae and E.coli in S phase, and flagellae of motile bacteria.
The ability to resist the complement system leads to seroresistance.
■ Inhibiting Chemotaxis - e.g. bordetellae, vaginal anaerobes, pseudomonads.
■ Leukocidins and lecithinase (causes myonecrosis and hemolysis) -produced by staphylococci, streptococci, pseudomonads, & Clostridia.
»   Formation of capsule - by agents of meningitis and pneumonia, e.g, Neisseria meningitidis, Haemophilus, Influenzae, E.coli, Streptococcus pneumoniae, Klebsiella pneumoniae.
■ Blocking phagolysosome formation, e.g. Chlamydia, Mycobacterium, Legionella, Toxoplasma.
■ Escaping phagosome - e.g. Rickettsia, Shigella, Listeria, Leishmania, Trypanosoma.
■ Production of antioxidants - e.g. staphylococci gonococci, meningococci.
■ Marked tenacity (i.e. persistency) - seen in Coxiella and Ehrlichia.
Overcoming acquired immunity:
The microbe attempts to avoid antibodies or lymphocytes by:
> Reproducing quickly, e.g. respiratory viruses, diarrheal agents, malarial plasmodia.
> Deceiving the immune system '
1. Hiding:*
■ Inside ganglions ~ HSV, VZV (Varicella zoster virus - one of the eight herpes viruses    chicken-pox in children).
■ On intracellular membranes - HSV, adenoviruses.
■ In infectious focuses - Mycobacterium tuberculosis, Echinococci.
Resisting phagocytosis: > Not allowing to become engulfed
> Surviving inside the phagocyte
General bacteriology
■ At privileged sites - agents of mucosal infections, Toxoplasma gondii in eye, retroviruses in cellular genome.
2. Changing one's own antigens:
■ Antigenic mimicry - Strept. pyogenes, Treponema pallidum, Mycoplasma pneumoniae.
■^Antigenic camouflage - Schistosomes (= blood proteins), Staphylococci ^    protein A; Streptococci -> protein G; cytomegalovirus -> ßtnG.
■"""Antigenic variability - trypanosomes, borreliae, gonococci, influenza , virus.
3. Inducing tolerance - cytomegalovirus (CMV - a herpes virus), rubella virus, leishmaniae, crypto cocci, maybe HIV.
> Suppressing immune response
■ Invading the immune system - HIV, measles virus.
■ Interference of cytokine formation - Mycobacterium leprae, protozoa.
■ Production of superantigens - staphylococci, streptococci.
■ Production of proteases - meningococci, gonococci, haemophili, pneuomococci.
■ Binding of Fc fragment (= fragment crystallizable region -> activates the immune system) - staphylocooci, streptococci, HSV.
19.Bacterial toxins and aggressins
Bacterial toxins is one of the stages of Bacterial pathogenesis.
• Entry into the host
• Adherence of the microorganism to the host cell
• propagation of organism
• Damage to the host cell by bacterial toxins
• Evasion of ho st secondary D efenses
Bacterial toxins are two type:
* Endotoxins
• Exotoxins
Exotoxins: are proteins secreted by both gram-negative and gram-positive.They are the most
poisonous substances known.
Exotoxins have 2 polypeptide components:
• one is responsible for binding to cell membrane •
• and one is responsible for the toxic effect
Such as Diphtheria toxhv.is an exotoxin secreted by Corynebacterium diphtheriae. It is an enzyme that block protein synthesis. Cholera toxins: result in ionic imbalance and loss of water,
Treatment: most antigens are inactivated by moderate heating(60 degree). In addition treatment with dilute formaldehyde destroys the toxic activity of most exotoxins —> toxoids( are usful in preparing vaccina^ * —-
y (yW& *-—-j,
Endotoxinsjareheat stable, lipopoiysaccharides (LPC), which areftot secretedjbut instead are
("integral componenf^nf the ce^Twallfcf gram-negative bacteria (not the gram-positive). (7 They are released into the hostscrrculation following bacterial cell lysis.
Main effect is fever, shock, hypotengion and thrombosis, result to septic shock. These effects are produced indirectly by activatioh^rcomplemenCandactivation of the coagulation cascade. Death can result from multiple organ failure" ~ "------r
Aggressins: invasive bacteria are those that can entexhfisJLcel^ or pentrate mucosal surfaces, spreading from the initial site of infections. Invasiveness is facilitated by several bacterial enzymes, most important are coUsgenase and hyaluronidase. There enzymes degrade components of the extracejluiarmafrjx, providing the bacteria with easier access to host cell surface. Invasion is followed by inflammation, which can be either pyogenic(involving pus formation) or granulomatous (having nodular inflammatory lesions), depending on the organism.
(, suk.-Wd l* -*u ^1 Hi ******10 .
20. Antigens, incl. antigen recognition and examples of bacterial antigens.
Antigen: is a substance capable of provoking the lymphoid tissues of an animal to respond by
generating an immune reaction directed specifically at the inducing substance.
It need to be foreign to the body, molecular weight that is higher then 5000 Da, chemical complex,
The reaction of an animal to contact with an antigen, called :
acquired immune response:
• humoral response
• cell mediated response
Antigen Determinants:
A response to antigen involves the specific interaction of components of immune system, antibodies
and lymphocytes, with epitopes on the antigen. The lymphocytes have receptors on their surface
that function as the recognition units, on B lymphocytes surface-bound immunoglobulin is the
receptor and on the T lymphocytes the recognition unit is known as T cell receptor.
The better the fit between the epitope and the paratope the stronger the non-covalant bonds formed
and consequently the higher the affinity of the interaction.      *        - fcyMM^iu det&aiwi vvgaa'I'
Antigenic determinants can be formed in two ways: ^P1   * ®
• Sequential epitopes U Rwrf" l^-C7tf/we&u4s?
• Conformational epitopes Ifi Wc^ltyl ^ \fa^M~<*»
Antigen Recognition: 4<^Wv, Two separate recognition systems:
• humoral immunity
• cell-mediated immunity
Humoral immunity: Antibody is the recognition molecule.-This glycoprotein is produced by plasma cells and circulates in the blood and other body fluid. Antibody is also present on the surface of the B lymphocytes. The interaction of this surface immunoglobulin with its specific antigen is responsible for the differentiation of these cells into plasma cells. Antibody molecules, whether free or on the surface of a B cell, will recognize free native antigen.
Cell-mediated immunity:-Sbs^Egsgfesejiss-aiitigen receptor will only bind to fragments of antigen thatare associated with products of the major histocompatibility complex (MHC). T cell recognition of antigen is said to be MHC-restricted, This MHC-restricted recognition mechanism has evolved because of the functions carried out by T lymphocytes.
The joint recognition of MHC molecules and antigen ensure that T cell makes contract with antigen on the surface of the appropriate target cell.
B cell receptor: antigen is found free in body fluids and as a transmembrane proteitj. on the surface of B lymphocytes, i.e. surface immunoglobulin, where it acts as the B cell antigen receptor.
T cell receptor: the T cell antigen receptors heterodimer composed of an alpha and beta or a gamma and a delta chain, mostly used is the alpha/beta heterodimer. the T cell receptors the molecule that is responsible for the recognition of specific MHC- antigen complexes, and will be different for every T cell.
Examples of bacterial antigens:
• Somatic or O-antigen: composed of repeating oligosaccharides. Such as gram negative-bacteria B. coli'
• flagellar protein or H-antigen: structure protein which make up flagella then enter in the organism with motility. Enteric bacilli ,
• Capsular or K-antigens: acidic polysaccharide, external to cell wall such as Salmonella.
21. Immunoglobulins. - bA MA6£ -4{&h'hi.
Antibodies:
• Glycoproteins.
• Presenting the serum and body fluids
• Induced when immunogenic molecules are introduced into the host's lymphoid system
• Reactive with, and bind specifically to, the antigen that induced their formation There are five distinct classes or isotypes of immunoglobulins: IgG, IgA, IgM, IgD and IgE. They differ from each other in size, charge, carbohydrate content and amino acid composition.
bAMA6£
Antibody structure: all antibody molecules have the same ba"sic"four cKain structure, composed of two light chains and two heavy chains.
IgG: .* /VlaiuAfi ji^ipev\AA
• Monomer y
• major immunoglobulin of serum „ -Wuvw
• exist as : IgGl, IgG2, IgG3, IgG4
• IgG is the major antibody of secondary response and is found in both serum and tissue fluid.
to fetus
IgA:   n. ^rov^^MOU^ Dimmer -^-^ruv ^ t)
Found in breast milk, mucosal areas, respiratory tract, urogenital tract. -> ^"ttfis^ Subclasses: IgAl, IgA2
IgM:     ZZ lw\/w.U^   /UCpti V&Ji   iA PxhlMAs,
•  Pentamer '
IgD:
Large size hence confined to intravascular pool
First antibody type to be produced during immune response.
Monomer I % $ e«cJ*W<^ ^ *
Many circulating B cells have IgD present on thejr sirrface.
IgE:
• Monomer
• Present in very low levels
• Found on the surface of rr^apejs,4ndbaspphil«s.
• Triggers Instamine release from mast cells and basophiles, involve inalkrgY^
22. Antibody function in Infection:
Primary function of an antibody is to bind the antigen that induced its formation. The binding of the antigen is mediated by the Fab portion and Fs region controls the biological defence mechanisms. For every antibody the paratope is different. J£
Neutralization:
Because antibodies are at least divalent they can form a complex with multivalent antigens. Depending on the physical nature of the antigen these innate complexes exist in various forms: Antibody excess, equivalence, antigen excess, monovalent antigen.
Complement activation:
Activation of the complement system is one of the most important antibody effecter mechanisms. Complement cascade is a complex group of serum proteins that mediate inflammatory reactions and cell lysis.
Cell binding and opsonisation:
Antibodies specific for particular antigens, such as bacteria, play a valuable role by binding to the surfaceand making the antigen more susceptible to phagpcytosi&and subsequent elimination:, process know asimgi^fei and is mediated by the!i&p^lSffi»'of the antibody The important residues are in the Ch2 domain near the hinge region, composed of disulphide bond.
23) Immune System
It is one of the homeostatic mechanisms of the body. It is a system of biological structures and processes within an organism. His main functions are the recognition of foreign or dangerous substances, the triggering complex reactions that eliminates, or attack to tumour or viral infected cells. In order to accomplish that he needs to detect and distinguish them from their onw healthy cells and tissues.
The immune system is divided in: gMraX&id r^ch^s
Innate, non specif immunity; is the first line of defence against infectious agents, and most potentials pathogens are checked before they establish an overt infection. Is quick|f (min-hours), recognizes foreign substance, eliminates them, has no memory.
Adaptative/ Specific immunity acquired immunity: produces a specific response to each infectious agent, and the effector mechanisms generated normally eradicate the offending material. It makes the distintion between self and non-self. Longer (requires        , ^ several days), but has immune memory. I fringe ^>
The immune system consists of a number of organs and several different cell types. All cells ^fc of the immune system - tissue cells and white blood cells or leukocytes- develop from pluripotent stem cells in the bone marrow. The production of leucocytes is through two    ^ g g main pathways of differentiation: Sr&^fc-)
Lymphoid lineage- T/lymphocytes, b/lymphocytes and (NK) natural killer cells.
Myeloid pathways- mononuclear phagocytes, monocytes and macrophages and granulocytes, basophils, eosinophils and neutrophils
24) Innate immunity Characteristics and humoral mechanisms
This kind of immunity doesn't depend on prior exposure to any particular organism. The innate defence mechanisms are ^opspecificlin the sense that they are iffectiye against aj wide range of potentially infectious agents.f
The components of the Innate immune system recognize structures that are unique to j microbes like complex of Jjpjds iand carbohydrates suchi as rp^t'i3%lycan of bacteria, / lipopdlysacharides of gram/negative bactirfa^ptej^iqic acid in gram/positive bacteria and -\ mahnose containing oligosaccharides found in manymicrobialmolecules./Therefore the WAw£e*v innate immune system is ^ble'to recognize non/self structuresfand react appropriately but sii| Awvii does not recognize self structure, so the potential autoimmunity is avoided. The fojcrpbiaj S WtW} products, recognized by the innate immune system (pXMPs- pathogen/associated molecular? WM. (      patterns) are essential for survival of the microorganisms and cannot be easily be discarded dmm. t     or mutated. J^fferen^^^^ ^
(£ r~     recognized by different pattern recognition receptors (PRRs)./On host cells and circulating ^ | ' molecules.
innate defences act as the initial response to microbial challenge and can eliminate the microorganisms from the host. When it is insufficient, it plays a critical role in the generation j of an effective acquired immune respohserCytokine produced by the innate immune system; signal^ that Tffi^ present and influence the type of acquired immune 1
response that develops. 1
' Humoral defence mechanisms
„<*,.••-«- ••• .....      - ■ - ,.■       , -
Lysozyme .}
Basic proteirj of low molecular wfreight found in relatively jii^h :cpnce)itrations in neutrophils7 as well in most tissue fluids {except CSF, sweat aryurine).^nctjons as#|juc^iytic enzyme^ ^%>^ splitting sugars of.the structural pepdidogiycan of the cell wall $f many gram-positive }J "bacfeha causing their lyJisJ In many pathogenic bacteria the peptidoglycan of the cell wall appears to be protected by other wall components (lipopoiysacharides), requiring the use of other enzymes to remove this protection. Basic polypeptides
A variety of basic proteins, derived from tissues and blood cells, have antibacterial properties, that depends on their ability to react non-specificaily with acid polysaccharides at the bacterial cell surface. In this group, spermine and spermidine, which can kill tubercle bacilli and some staphylococci. Protamine and histone (lysine and arginine proteins) are also included.
Acute/phase proteins
Their concentration rises dramatically in case of infection. Microbial products aas endotoxin stimulate macrophages to release IL-1. which stimulates liver to produce increased amounts of these proteins. ,
C/reactive protein binds phosphorylcholine residues^ the cell wall of microorganisms, activating the classical complement pathway.
al-antitrypsin, a2-macroglobulin, fibrinogen and serum amyloid A protein act* to limit the spread of the infectious agent or to stimulate the host response.
Interferon ** o< 0**^ £
Antiviral agents a- and (3 participate in innate immunity in the way that stop the processes that permit virus to spread.
Complement
is composed of a large number (about 30) of different serum proteins present in low concentrations in normal serum;some of them are enzymes, other control molecules and other structural proteins with no enzymatic activity.. Normally these proteins are inactivated but can be activated to form a an enzyme cascade. There are two pathways of complement activation, the alternative and the classical, that lead to the same consequences: Opsononization . ■
Cellular activation
The 2 pathways use different initiation processes. Component C3 forms the connection between both pathways, the binding of this molecule to a surface is the key process in complement activation,
Clissic Pathway,;--->  $0_<y?   /) 43
The classical pathway is triggered by activation of the Cl-complex (Clq, two molecules of Clr, and two molecules of Cls thus forming Clqr2s2), which occurs when Clq binds to IgM or IgG complexed with antigens (a single IgM can initiate the pathway, while multiple IgGs
<3
are needed), or when Clq binds directly to the surface of the pathogen. Such binding leads to conformational changes in the Clq molecule, which leads to the activation of two Clr {a serine protease) molecules. They then cleave Cls (another serine protease). The Clr2s2 component now splits C4 and then C2, producing C4a,C4b,C2a,and C2b. C4b and C2a bind to form the classical pathway C3-convertase (C4b2a complex), which promotes cleavage of C3 into C3a and C3b; C3b later joins with C4b2a (the C3 convertase) to make C5 convertase (C4b2a3b complex).
{^ernative pathway' §(X<^>   A 4 3
The alternative pathway is triggered by spontaneous C3 hydrolysis directly due to the breakdown of the thioester bond via condensation reaction (C3 is mildly unstable in aqueous environment) to form C3a and C3b. It does not rely on a pathogen-binding antibodies like the other pathways.^. C3b Is then capable of covalently binding to a pathogenic membrane surface if it is near enough, if there is no pathogen in the blood, the C3a and C3b protein fragments will be deactivated by rejoining with each other. Upon binding with a cellular membrane C3b is bound by factor B to form C3bB. This complex in presence of factor D will be cleaved into Ba and Bb. Bb will remain covalently bonded to C3b to form C3bBb which is the alternative pathway C3-convertase. The protein C3 is produced in the' liver.
The C3bBb complex, which is "hooked" onto the surface of the pathogen, will then act like a "chain saw," catalyzing the hydrolysis of C3 in the blood into C3a and C3b, which positively affects the number of C3bBb hooked onto a pathogen. After hydrolysis of C3, C3b complexes to become C3bBbC3b, which cleaves C5 into C5a and C5b. C5b with C6, C7, C8, and C9 (C5b6789) complex to form the membrane attack complex, also known as MAC, which is inserted into the cell membrane, "punches a hole," and initiates cells lysis. C5a and C3a are known to trigger mast cell degranulation.
IgA is associated with activating the alternative path.
25| Innate'Immunity- barriers & cell-mediated mechanisms / y Noh Specific barriers: Anatomical/Physiological f
fhejSkinfis a resistant barrier because of its outer horny layer consisting mainly of .keratin*
indigestible for most micro/organ ism si the drycondition of the skin and the high
concentration of salt of sweat |are inhibitory or lethal to many other microorganisms. The
^sebaceous's^CTetjInram^sweat contain also bactericidal and fungicidal fatty acids./
The'sticky mUcous covering the respiratory tract act as a trapping mechanism for inhaled
particles, the t:iJiCpus^"theWcreb"ohis''td oropharynx so that they are swalloed and the—r
acidic secretions of stomach destroy most of the microorganisms. \
Nasars:ecretions and saliva Contain mucopolysacharides capable of blocking virus/.
The washing action of teafsfand the flushing of urine are effective in stopihg invasion by
microorganisms.
The natural bacterial flora covering epithelial surface are protective in a number of ways: Their presence uses a niche that cannot be used by a pathogen Compete for nutrients
They produce by-products that inhibit the growth of other organisms.
Cell mediated mechanisms
Phagocytes- cells actively phagocytic (solid particles) and pinocytic (soluble material), contain enzymes to degrade ingested material intracelularly in specialised vacuoles, are an important link between the innate and the acquired immune mechanism. Neutrophils and mononuclear phagocytes engulf rapidly microorganisms Mononuclear phagocytes are named monocytes in the blood stream or macrophages in the tissues. (In connective tissue- histiocytes, kidney- mesangial cells, liver- kupfer cells, bone- osteoclasts, brain- microglia and in spleen, lymph node and thymus as the sinus-linning macrophages). The phagocytes are attracted by a process- chemotaxis. The chemotaxic factors include:
Products of the injured tissue
Factors from blood (C5a)
Substances produced by neutrophils and mast ceils (leukotrlenes and
histamine)
Bacterial products (formyl/methionine peptides) Phagocytosis involves 3 stages: recognition and binding; ingestion and digestion. Phagocytes have receptors on their surface that mediate the attachment of particles coated with the correct ligand - Fc portion of certain Immmunoglobulins isotypes and some of the components of the complement cascade- opsonins, this enhances the ingestion process where the particle is surrounded by cell membrane, which then invaginates and produces an endossome or phagosome within the cell. The microbicidal particles of the phagocyte are contained in lysosomes preventing the autodestruction of the cells by so toxic compounds. The phagosome and lysosome fuse forming a phagolysosome in which the ingested material is digested by a enzymatic complex. This is accompanied by increase in production of oxygen/dependent killing mechanisms (superoxides and peroxides) and the digestion.
Natural killer cells recognize changes in virus infected cell, cancer cells and destroy them by extracellular killing mechanism, damaging the membrane of the infected ceil. They are enhanced by interferons that appear to stimulate their production and rate of killing. Eosinophils polymorphonuclear leucocytes with bilobated nucleus and granules, in very low levels. They increase their number in parasitic infections and allergies, they are not efficient in phagocytosis but their granules contain susbstances toxic to parasites, that are to big to be internalized
26) Acquired immunity- general characteristics
This kind of immunity can be acquired in two ways:
Induced by overt clinical infection or unapparent
Deliberate artificial immunization Is dependent of exposure of the antigens of the invading microorganism to the cells of the immune system (macrophages and lymphocytes) to start a specific immune response to the material. This cells are pre-committed because of their surface receptors, to respond to a particular epitope on the antigen.
This response is divided in two that usually develop in parallel: Humoral and cell mediated. Humoral immunity depends on the appearance in the blood of antibodies produced by plasma cells. Cell mediated immunity refers to any response in which the antibody plays a
subordinate role. Depends mainly on the development of T cells that are specifically responsive to the inducing agent and is generally active against intracellular organisms. So we can say that acquired immunity is adaptive and has a high degree of specificity in distinction between seif and non-self. The reaction requires several days to be effectively triggered. There is immune memory.
27) Acquired immunity - humoral immunity
This kind of immunity is named like humoral because is found in the humours (body fluids), is mediated by secreted antibodies, which are produced in the cells of B lymphocytes. B ceils with co-stimulation (anthem antigen presented cell for example) transform into plasma cells which secrete antibodies. In this processes also co-operate CD4** T-helper2 cell. The antibodies will bind to the antigens presented in the surface of the invading microorganisms, acting like a marker for their destruction recognition of antigens associated with microorganisms or foreign substances. This recognition is coupled with the ability to initiate appropriate actions (e.g., antibody production) against these microorganisms or foreign substances.
Besides antibody production, also includes: functions of the antibody pathogen and toxin neutralization, classical complement activation, opsonin promotion of phagocytosis and pathogen elimination.
28) Acquired Immunity - cell mediated Immunity
Macrophages engulf antigens, process them internally, then display parts of them on their surface together with some of their own proteins. This sensitizes the T cells to recognize these antigens. All cells are coated with various substances. CD (cluster of differentiation) and there are more than one hundred and sixty clusters, each of which is a different chemical molecule that coats the surface," Every T and B cell has about 105 = 100,000 molecules on its surface. B cells are coated with CD21, CD35, CD40, and CD45 in addition to other non-CD molecules. T cells have CD2, CD3, CD4, CD28, CD45R, and other non-CD molecules on their surfaces.
The large number of molecules on the surfaces.of lymphocytes allows huge variability in the forms of the receptors. They are produced with random configurations on their surfaces. There are some 1018 different structurally different receptors. Essentially, an antigen may find a near-perfect fit with a very small number of lymphocytes, perhaps as few as one.
t cells are primed in the thymus, where they undergo two selection processes. The first positive selection process weeds out only those T cells with the correct set of receptors that can recognize the MHC molecules responsible for self-recognition. Then a negative selection process begins whereby T cells that can recognize MHC molecules compiexed with foreign peptides are allowed to pass out of the thymus.
Cytotoxic or killer T ceils (CD8+) do their work by releasing lymphotoxins, which cause cell lysis. Helper T cells (CD4+) serve as managers, directing the immune response. They secrete chemicals called lymphokiries that stimulate cytotoxic T cells and B cells to grow and divide,
attract neutrophils, and enhance the ability of macrophages to engulf and destroy microbes. Suppressor T cells inhibit the production of cytotoxic T cells once they are unneeded, lest they cause more damage than necessary. Memory T cells are programmed to recognize and respond to a pathogen once it has invaded and been repelled.
29) Immunodeficiency
Irftmunodeficreftcy/refers to a group of disorders in which the immune system 'does not ftfnctlon normally; Our bodies' immune cells attack and kill what they see as foreign invaders, usually bacteria, viruses, and fungi. Wheh: the immu^  system does not work ? properly,;a person is more likely to suffer from frequent andJonger lasting infections, often/ from organisms that don't normaiiy^   most people sick.^Congenital immunodeficiency is present at birth. Another type of immunodeficiency disorder is called acquired? ■nnmunodeficien'cyj which develops later in life.
The deficiency states seen are either due to defects in one of the components of the system itself or secondary to some other disease process affecting the normal functioning of some part of lymphoid tissues. This can be 'inherited, developmental or acquired! The individual affected is said to be immune compromised.
'Defective innate defence mechanisms can be presented in 2 forms:
-Where there is a quantitative deficiency in neutrophils that may be congenital (infantile ? agranulosis) or acquired as a result of replacement of bone marrow I5y tumour cells; or the toxic effect of drugs or cherfijcais. I
J. 4? Irt -("UAC .
-Where there is a qualitative deficiency in the functioning neutrophils which,-while intestinal bacteria normally, fail to digest them by an enzymatic defect. Susceptibility to bacterial and fungal diseases is common, but not to viral or protozoan.
Defective acquired immune defence mechanisms
-  Primary immunodeficiencies Caused by defined genetic defects, usually rare, but severe" I
Can arise from failure of any developmental pro/cesessfrbm stem cell to functional end; cell.sA complete lack of all leukocytes is seen in reticular dysgenesis' due to a defect in bone marrow stem cells in the foetus, normally leading to death of baby in one year due to recurrent intractable infections. DefecSintKe development of the common lymphoidf stem cell give rise to severe combined immunodeficiency (both T and B cells fail to/ develop/ but functional phagocytes are present). B cell defects give rise to several hypogammaglobulinaemias, low levels of gamma globulins (antibodies) in blood. Deficiencies of immunoglobulin synthesis is almost complete in x-linked Bruton's disease (cell mediated mechanisms function normally). Partial defects in immunoglobulin synthesis:
-wiskott-aldrich syndrome (low IgM, but high IgA and IgE) pyogenic infections, bleeding and eczema
-dysgammagiobuiinaemia- deficiency in one antybody class reduced IgA rest Is normal increased infections of upper and lower respiratory tracts.
?^ejjs|||(|fe<jts jead to more severe and persistent infections. Lack of t cells is normally 'associated to fts^jjh levels of :antibpd'(es^ Patients with this defect are "prone to viral,!
intrac^ rather than acute bacteria! /
infections (Digeorge syndrome)
Secondary immunodeficiencies: Consequence of some other disease (viral diseases are often immunosuppressive- measles, human immunodeficiency), treatment (irradiation ■ citotoxic drugs and steroids), environmental factors like exposure to drugs or chemicals. Usually frequent, but usually clinically mild (exceptions: HiV disease, secondary . aganulocytosis). Deficiency in immunoglobulins can be caused by deficiency in proteins due to renal disease or intestine in protein losing entheropaty. Malnutrition and iron deficiency can lead to depressed immuno-responsiveness, particularly in cell-mediated immunity. In contrast to the deficiency states raised immunoglobulins levels are found in certain disorders of the plasma cells due to malignant proliferation of a particular clone group of plasma cells (chronic lymphocytic leukaemia or multiple myeloma) malignant clones each produces a particular type of antibody. Usually with low synthesis of normal immunoglobulins and associated decrease of immune system to acute bacterial infections.
30) Immunity in bacterial infections
Inflammation ;
Al^r^feakl'ijg the mechanical barrlersj bacterial substances and phagocytosis, the ^bacteria starts to proliferate in the tissues!
'^he'!^ molecules are recognized by pattern recognition /
receptor along with tissue damaging; bacterial
products, trigger an inflammatory reaction. The resulting increase in permeability leads to exudation^,6?'serum proteins, including complement components^ ;antibodies/and clotting factors as well as phagocytic ceilst By chemotaxis the phagocytes are attracted to the site of inflammation. Anaphylatoxinsfgenerated by complement increase the vascular f permeability and encourage exudation/ These mediators also cause ;yasqdilatatiori, increasing blood flow in the area. The attack is primarily directed to the external components and secreted molecules. Bacteria are surrounded by a cytoplasmic rnembrane and Jpjeptidoglycan cell walljiysosomal enzymes and the outer lipid layer of Gram/negative bacteria by catldnic proteins and complement^. Associated to them can be a variety of other components such as proteins, capsules, liposacharides or teichoic acids. There are also structures associated with the motility (flagella or fimbriae' usually attacked by specific antibodies also responsible for inactivating bacterial enzymes and toxins) or adherence to the cells of the host. Ultimately in some situations cell mediated ; responses are required.
31) Passive Immunization f)
vf/o $J?PW
The objectives in immunization are, to produce, without arm the recipient, a degree of resistance sufficient to prevent clinical attack of the natural infection and to prevent the spread of the infection to susceptible in the community. In other words, the gains are both personal and also for the population. The degree of resistance conferred may not protect against an overwhelming challenge, but exposure may help to boost immunity.
Passive immunization is where pre-synthesized elements of the immune system are transferred to a person so that the body does not need to produce these elements itself. Antibodies are used in this kind of immunization begins to work very quickly, but it is short lasting, because the antibodies are naturally broken down, and if there are no B cells to produce more antibodies, they will disappear. Passive immunization occurs physiologically in pregnancy in transfer of antibodies from mother to fetus
Artificial passive immunization is used in clinical practice when it's necessary to protect a patient at a short notice and for a limited period. Antibodies, which may be antitoxic, antibacterial or antiviral, in preparations of human (homologous) or animal (heterologous) serum are injected to give temporary protection. Homologous antisera are much less likely to produce adverse reactions, and their protection last longer (3-6 months) against heterologous (few weeks).
Treatment of diphtheria is made with antiserum raised in horse against diphtheria toxin (equine diphtheria antitoxin), also a similar serum is used in botulism, and still some countries used equine tetanus antitoxin, that is being replaced by human tetanus immunoglobulin.
Pooled immunoglobulins
Protective levels of antibody to a range of diseases are present in pooled normal human serum. Human normal immunoglobulin (HNIG) is short term prophylaxis of hepatitis (A) and agammaglobulinaemia.
Specific immunoglobulins
Tetanus (human tetanus immunoglobulin HTIG);
Hepatitis B (HBIG)
Rabies (HRIG)
Varicella-zoster (ZIG)
32) Active immunization
Active immunization entails the introduction of a foreign molecule Into the body, which causes the body itself to generate immunity against the target. This immunity comes from the.T cells and the B cells with their antibodies.
agammaglobulinemia.
Specific immunoglobulins
Tetanus (human tetanus immunoglobulin HTI6);
Hepatitis B (HBIG)
Rabies (HRIG)
Varicella-zoster (ZIG)
32) Active immunization
Active immunization entails the introduction of a foreign molecule into the body, which causes the body itself to generate immunity against the target. This immunity comes from the T cells and the B cells with their antibodies.
Active immunization can occur naturally when a person comes in contact with, for example, a microbe. If the person has not yet come into contact with the microbe and has no pre-made antibodies for defense (like in passive immunization), the person becomes immunized. The immune system will eventually create antibodies and other defenses against the microbe. The next time, the immune response against this microbe can be very efficient; this is the case in many of the childhood infections that a person only contracts once, but then is immune.
Artificial active immunization is where the microbe, or parts of it, are injected into the person before they are able to take it in naturally. If whole microbes are used, they are pre-treated, attenuated vaccine.
Depending on the type of disease, this technique also works with dead microbes, parts of the microbe, or treated toxins from the microbe.
Types of vaccine
Toxoids, when the signs and symptoms of a disease can be attributed essentially to the effects of a single toxin, a modified form of toxin, that preserves his antigenicity but has lost his toxicity, like in case of tetanus and diphtheria.
Inactivated vaccines - If the disease is not mediated by a single toxin, it may be possible to stimulate the production of protective antibodies by using killed (inactivated organisms) this is donne in vaccines against Pertussis (whooping cough), Influenza and inactivated Polio (Salk) vaccine.
Attenuated live vaccines
When the inactivation procedure to make an inactivated vaccine destroys or modifies the protective antigenicity (immunogenicty) of the organisms, the solution is to use suspensions of living organisms that are reduced in their virulence (attenuated) but still immunogenic. Mumps, Measles and Rubella vaccines (combined), the live-virus Polio (Sabin) vaccine and the Yellow fever vaccine.
Other aerobic 5% Anaerobes 1%
Monomic«)biai etiology, agents originate in community
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