General Bacteriology – Review Class

General Bacteriology –Review Class

Professor Md. Akram HossainMMC

December 13 Prof. Md. Akram Hossain1

Classify disease agents. State the differences among bacteria, viruses and fungi

� Bacteria – Prokaryote kingdom� Fungi, Protozoa – Protist kingdom (Eukaryotes)� Helminths – Animal Kingdom (Eukaryotes)� Viruses - Noncellular

Ponits of differences among diseases agentsPonits of differences among diseases agents1. Cells2. Nucleic acid content3. Nucleic acid Type4. Ribosomes5. Mitochondria6. Motility7. Replication 8. Outer coat

December 132 Prof. Md. Akram Hossain

Who is the founder of microbiology & Medical microbiology?

Robert Koch - Founder of Medical Microbiology

Louis pasteur, Founder of Microbiology


To know how to suggest is the art of teaching

AmielAmiel


� What is prokaryotes and Eukaryotes? What is the basic difference between them? What are the other differences?� Literal meaning of Prokaryotes (pro – primitive, Karyotes-

nucleus) – cells with primitive nucleus and Eukaryotes (Eu-advanced) - cells with true or advanced nucleus.

� Eukaryotes have nucleus with nuclear membrane whereasProkaryotes do not have true nuclei with nuclear membrane,rather they have nucleoid.rather they have nucleoid.

� Other differences include� No. of chromosome� Nucleoli� Mitochondria� Golgi complex� Endoplasmic reticulum


Classify bacteria. Examples of anaerobic bacteria

Wall less bacteria

e.g. Mycoplasma

Flexible thin Walled bacteria

Rigid thick walled bacteria e.g. Mycoplasma

e.g. Treponema, Borrelia, Leptospira

bacteria

Free living (Extracellular)

Non Free living(Obligate intracellular)

e.g. Chlamidya, rickettssia1. Gram positive – a. Cocci – Streptococci

b. Bacilli - (Sporing, Non sporing)2. Gram negative – a. Cocci

b. Bacilli ( Facultative, Aerobic, Anaerobic)3. Acid fast December 136 Prof. Md. Akram Hossain

Classification of bacteria (contd)

Extracellular bacteria ( Free living)

Gram positive

• Cocci – Staphylococci, Streptococci

• Bacilli –

1. Spore forming-

Gram negative• Cocci –Neisseria

• Bacilli –

1. Facultative

a. Straight1. Spore forming-

• Aerobic – Bacillus

• Anaerobic - Clostridum

2. Non sporeforming

• Non filamentous –Corynebacterium, listeria

• Filamentous –Actinomyces, Nocardia

a. Straight

i. Resp –

ii. Zoonotic

iii. Enteric

b. curved

2. Aerobic -

3. Anaerobic -


� What are the essential structures of bacterial cell?1. Cell wall2. Cell membrane3. Nucleoid4. Cytoplasm5. Periplasmic space (GNB)

� What are the nonessential structures of bacterial cell?

1. FlagellaFlagella2. Fimbria3. Capsule / slime layer4. Spores 5. Plasmid6. Transposon

� What are the functions of essential and nonessential structures?


Clinical significance of periplasmic space

1. Binding proteins for transport of nutrients ( aminoacids,sugars, vitamins & ions)

2. Hydrolytic enzymes – alk phsophatase & 5nucleotidase – breakdowns nontransportable totransportable ions

3. Detoxifying enzymes - Beta lactamases, aminoglycoside phosphorylase – drug resistance

4. Osmoregulation – due to presence of highly branchedof D glucose


What are functions or clinical significance of capsule?1. Acts as virulence factor by Protecting the bacteria from

phagocytosis2. Helps in diagnosis by capsular swelling test & other

immunological test3. Acts as agent for vaccine preparation.

What are functions or clinical significance of Flagella?1. Organ of motility – acts as virulence factor2. Helps in diagnosis due its antigenicity


To be proud of learning, is the greatest ignorance.

Jeremy Taylor


� What are spores? Mentions functions or clinical significance of spore? Why they are so tough? Name some spore bearing bateria.

1. Spores highly dormant stage bacteria formed in unfavourable environmental conditions.

2. Spores highly resistant due to following features

� Thick coats – Core, Cortex, Coat, Exosporium

� Dehydrated state-

� Metabolically inert –

� keratin like proteins - coat

� Calcium dipicolinic acid - cortex� Calcium dipicolinic acid - cortex

� Clostridium tetani, Cl.perfringenes, Cl. Botulinum, Bacilus anthracis

� Clinical significance –

� Difficulty in sterilization – ( autoclaving, sporicidal)

� Dreadful diaseases – Tetanus, Gas gangrene, anthrax

� Difficulty in diagnosis –

� Biological weapon- Anthrax


What are L –forms of bacteria? Why they are so called? Mentionsfunctions or clinical significance of them?

L – forms are cell wall deficient forms of bacteria. Theydevelop spontaneously or in the presence of penicillin,lysozyme or other agents that interfere with cell wallsynthesis. They require a solid mediumwith right osmoticstrength.strength.They were named after the name Lister Institute of London,where discovered.Clinical significance

Latent infection / chronic infectionResistance to cell wall active agents.


� What are different types of stains?� Simple stains:

� Most are basic. Crystal violet, and methylene blue are examples of simple stains.

� Differential stains:� bind to some types of bacteria but not others,therefore

providing a method of differentiating between different types of bacteria.

� Gram stain (gram positive vs. gram negative)� Acid-fast stain for bacteria that do not take up stain well (e.g.: � Acid-fast stain for bacteria that do not take up stain well (e.g.:

Mycobacteria)

� Special stains� Capsule stain with India ink (wet mount) – Negative stain� Endospore stain� flagella stain

� Fluorescent dyes� Auramine and rhodamine for Mycobacteria� Immunofluorescence uses


Education is the best provision for old age.provision for old age.

Aristotle (384 BC - 322 BC


Prokaryotic Cell Morphology

Coccus - cocciRod (bacillus)CoccobacillusCoccobacillusVibrioSpirillumSpirochete

Also possible: pleomorphic bacteria


Cell Groupings Binary division can lead

to diplococci, chains(Streptococcus), packets or clusters(Staphylococcus)


� What is the basic structure of cellwall?� Peptidoglycan composed of alternate strands of 2

major subunits: NAG and NAM

� cross linked by Tetrapeptide chain attached to NAM

� Interpeptide bridge in gram positive cell wall

� IN GNB, Lipoprotein layer, outer memberane andLPS


Gram Positive Bacteria

� Thick layer of peptidoglycan� Negatively charged teichoic acid on

surface


It is the mark of an educated mind to beable to entertain a thought withoutaccepting it.

Aristotle (384 BC - 322 BC)


1. Cell wall much more complex2. Thin peptidoglycan layer3. filled and surrounded with periplasm (protein

rich gel-like fluid)

How GNB cell wall differs from that of GPB?

rich gel-like fluid)

4. Unique outer membrane on top.

1. Bilayer, yet outer layer is LPS layer (lipid A and O specific polysaccharide)

2. LPS acts as endotoxin (lipid A)


What are requirement for bacterial growth?

� Physical1. Temperature2. pH3. Osmotic pressure

� Chemical1. Carbon1. Carbon2. Nitrogen, sulphur, Phosphorous3. Trace elements: potassium, magnesium, calcium, iron,

copper, molybdenum, and zinc. Are needed as cofactors for enzymes.

4. Oxygen� Both useful and harmful

5. Organic growth factors may be needed� Vitamins, Amino acids, Purine, Pyrimidines


What are the growth factors or bacterial vitamins?

� Some bacteria require organic compounds which can not be synthesized by themselves and be supplied from external sources.

Two types� Two types� Essential growth factors - purines or pyrimidines or amino

acids required after mutation of any bacteria� Accessory growth factors – X and V factors for the good

growth of H. influenzae.


� What are the different types of media?� Based on composition

� Synthetic� Complex media

� Basal media – Nutrient agar� Selective media – MacConkeys agar, BTA

Differential media – MA, � Differential media – MA, � Enriched media- BA, CA, LF etc

� Based on consistency� Solid or agar media� Liquid or broth media� Semi solid media


Classify microbes according to oxygen requirement?

» obligate aerobeRequires oxygen.

» facultative anaerobeGrows with or

without oxygen.

» obligate anaerobe Grows only in

absence of oxygen.

» aerotolerant anaerobe Grows in

presence of oxygen, but does not use it.

» microaerophile Grows in low oxygen

concentration.



� Why is oxygen is harmful?� It is a strong oxidizing agent. It pulls electrons off

other molecules.� What is formed in presence of oxygen?

� Singlet oxygen� Superoxide free radical: O2

-

� Hydrogen peroxide: H202

� Hydroxyl radical: OH-� Hydroxyl radical: OH-

� Toxic compounds in turn pull electrons off other molecules, e.g., lipids, proteins, and nucleic acids.

� Result: Cell is harmed if it cannot get rid of toxic forms of oxygen.� To get rid of toxic oxygen compounds, cells need:

� Superoxide dismutase (SOD)� 02

-. + 02-. + 2H+ H202 + 02

� Catalase� 2H202 2H20 + 02


What are phases of growth curve ?


What is the Principle of diagnosis Lab Dx ofMicrobial diseases ?

� Demonstration of causative agents by microscopycommonly practiced for parasitic diseases & fungaldiseases

� Isolation and identification of causative agent by� Isolation and identification of causative agent byartificial culture - (C/S) commonly practiced forbacterial diseases.

� Detection of antibody or antigen from blood or otherbody fluids practiced for viral diseases & somebacterial & parasitic diseases

� Detection of Nucleic acid segment (DNA or RNA)


Good teaching is one-fourth preparation and three-fourths theater.

Gail Godwin


� Specimen : according to site of infection� Microscopic Examination :

� Light microscopy / DGM / FM / EM� Unstained preparation� stained smear -Gram, AFB, Fluorescence

� Isolation & identification : Bacteriological, fungal, protozoal Culture, Cell culture for viruses

What are the Steps involved in Lab Dx of Microbial diseases?

protozoal Culture, Cell culture for viruses� Identification by standard biochemical & serological test

� Immunological test : detection of Ag or Ab by LAT, ELISA etc

� Detection of gene: NA based techniques by PCR etc

� Special tests : Toxigenicity tests


What is an antibiottic ? What is the basic Basic principles ofantimicrobial therapy

Ans:

Antibiotic is a substance produced by another microorganisms naturally or synthetically that inhibits the growth other microorganisms.

Basic principles-Basic principles-

1. Selective toxicity

- to exploit differences in structure & metabolism of pathogens and host cells (to kill organisms not man)

2. Reach the site of infection at inhibitory concentrations

3. Penetrate and bind to target, avoiding inactivation and extrusion


How can you classify antimicrobials?

Ans:

1. By origin-� Antibiotics- natural products of fungi, & bacteria which kill the

microbes e.g. Penicillins,

� Synthetic compound- e.g. trimethoprim

2. Whether bactericidal or bacteriostatic� Bactericidal- kills organisms � Bactericidal- kills organisms

� Bacteriostatic - prevents organisms from multiplying, host defences can cope with static population

3. By target site / mode of action

� Cell wall synthesis

� Cell membrane function

� Protein synthesis (Ribosome)

� Nucleic acid synthesis

4. By chemical structure


Q: What are the Mechanism of action of antibiotics

Ans: Following are mechanisms-� Cell wall synthesis inhibition

Cycloserine, Glycopeptides( vancomycin, teicoplanin) Bacitracin Beta-lactams (penicillins, cephalosporines, monobactams)

� Cell membrane permeability impairement-� Cell membrane permeability impairement-Polymyxine, polyenes, azole

� Inhibition of bacterial protein synthesisAminoglycosides, Macrolides,Tetracyclines,Chloramphenicol, Fusidic acid

� Inhibition of bacterial synthesis of nucleic acids� Trimethoprin,Sulphonamide, quinolone, Rifampicin,

Metronidazole,


No man can be a good teacher unless be has feelings ofwarm affection towards his pupils and a genuine desireto impart to them what he himself believes to be of value.

Bertrand Russel


Which organisms frequently develops resistance?

Ans:� Staphylococcus aureus, � Enterococcus faecalis, � Enterococcus faecium, � Pseudomonas aeruginosa � Mycobacterium tuberculosisis


How microorganisms develops resistance ?

1. Production of drug destroying enzymes � penicillinase, Beta-lactamase by staphylococci

2. Change in the permeability to the drug-� tetracyclines resistance by Ps. aeroginosa)Changes in receptors which decrease antibiotic 3. Changes in receptors which decrease antibiotic binding and action

� Change in PBP in MRSA

4. Altered metabolic pathway� Sulphonamide resistant bacteria utilize preformed folic acid

and do not require the presence of PABA


How can you delay emergence of drug resistance?

� Prescribed only when needed� Adequate dose & duration� Narrow-spectrum� Narrow-spectrum� Limit use of newer drugs� Combination of drugs in special cases� Minimize giving antibiotics to livestock


When combination of drugs used?

� Severe infection� Mixed infections� Prevention of resistance – tuberculosis� Prevention of resistance – tuberculosis� Decreased toxicity� Enhanced antibacterial action


� How a bacteria cause disease? What are the virulence factors of bacteria?� By different mechanisms known as virulence

factors which varies with bacteria� Adherence by pilli, CFA� Damage of mucosal defense by IgA protease� Interfering phagocytosis by capsule� Invasiveness� Intracellular multiplication� Toxins - exotoxin, endotoxin� enzymes - coagulase, hyaluronidase, etc.


What are the biological effects of endotoxin?

� Following are the biological effects1. Fever – due to release of interleukin -1 (endogenous pyrogen)

by macrophage which acts on hypothalamus.2. Hypotension, Shock and impaired perfusion of internal

organs– due to release of TNF and nitrous oxide from organs– due to release of TNF and nitrous oxide from activated macrophages which causes vasodilation.

3. DIC - due to activation of coagulation cascade through factor XII (Hageman factor)

4. Inflammation - due to activation of alternate pathway C3a, C5a.


What will result if antibiotic is administered late in shock due to GNB?

� If left too late, antibiotics worsen the situation by lysing bacteriasituation by lysing bacteria


What is Normal Flora ? What is the problem?

Organisms frequently found on or withinbody of healthy individuals� Most are bacteria, but some are fungi, and

protozoa� Some are found only on body; others also

found in environmentfound in environment

� Problem: some people have transientnormal flora (pathogens)

� Example: about 10% of population havemeningococcus or pneumococcus asnormal flora


What is the Clinical importance of normal flora?

� Opportunistic infections: normal flora in unusual sites; for example:� Bacteriodes from intestine into deeper tissues as a

result of trauma (or surgery)� Staphylococci from skin and nose� Streptococci and Gram— cocci from throat and � Streptococci and Gram— cocci from throat and

mouth

� Depends on pathogen and on defenses of host:� Candida (yeast) causes pneumonia in people

undergoing cancer chemotherapy� Pneumocystis carinii (common inhabitant of lung)

causes pneumonia and death in AIDS patients


Education is the ability to listen to almostanything without losing your temper or yourself confidence.

� Robert Frost (1874 - 1963)December 1352 Prof. Md. Akram Hossain

What is the beneficial effects of normal flora?� Antigenic stimulation by normal flora

� Serve as defense mechanism even in low concentration

� Bacterial stimulation leads to production of IgA that is secreted through mucus membranes

� Keeps out invadersMechanisms:� Mechanisms:

� Physical advantage of previous occupancy� Some produce bacteriocins or antibiotics

� Role in human nutrition and metabolism� E. coli and Bacteriodes synthesize vitamin K� Metabolism of key compounds involves excretion

from liver into intestine and their return to the liver


Which are the sterile sites of body? What is its clinical importance?

Sterile sites in the body includeBloodCerebrospinal fluidSynovial fluidSynovial fluidDeep tissues

pathogens in these definitely indicate disease


God heals and the doctortakesthefeetakesthefee

Franklin


� What is the difference between Selective toxicity &

Toxicity or side effects?

� Between antibacterial and antifungal agents which one

will be more selectively toxic and which one will be

toxic? Why?

� What are the target structures for antibiotics?� What are the target structures for antibiotics?

� Why some antibiotics some times can not act

efficiently?

� What is transferable and nontransferable drug

resistance?

� What is the role of plasmid in drug resistance ? How it

is transferred from one bacteria to another?December 1357 Prof. Md. Akram Hossain

•What is sterilization? Disinfection and antisepsis?

•Sterilization A process by which an object is freed from all viable microorganisms including spores.•Disinfection is a process of destruction of vegetative forms of pathogenic organisms capable of producing forms of pathogenic organisms capable of producing infection.•Antisepsis is process that is applied to prevent infection in a living surface.

•How will prevent milk borne diseases? Is it a process of sterilization?

•Pasteurization.•How will you sterilize bronchoscope, endoscope ?

•GluteraldehydeDecember 1359 Prof. Md. Akram Hossain

Write down the methods of sterlization. What is the principle of autoclaving?

� Methods of sterilization are –1. Heat

2. Ionising irradiation

3. Filtration

4. Sterilizing gases

5. Sterilizing liquids5. Sterilizing liquids

� Principle of autoclaving� Steamsaturated at a high pressure and temperature is a better

sterilizing agent then dry heat. Bacteria are intrinsically moresusceptible to moist heat as bacterial protein coagulates rapidly.

� Saturated steamheats the article rapidly by releasing latent heatwhich kills bacteria

� Steamcontracts in volume (1600 ml steamat 1000 C condenses intoI cc water) and enhances penetration.


What is the most efficient process of sterilization? Why?� Autoclaving is the most efficient process of

sterilization due to following reasons-� Moist heat kills organisms and spores at lower

temperature and in sorter duration because water helps indisruption of noncovalent bonds e.g. hydrogen bonds andthereby disrupts secondary and tertiary structure of proteins. ( 20 hrs at 1000C by dry heat vs 15 min at 1210C bu moistheat)heat)

� Penetrating power of moist heat is better than dry heat dueto

� Low density of steam� Condensation of steamand contraction of its volume creates

negative pressure on the surface (1600 ml of steamcondenses into1 ml of water.)

� Liberation of latent heat 518 calories for 1 ml of water.� Liquid or watery solution or articles retain their structure in moist

heat but not dry heat.


Write down some chemical sterlising / sporocidal agent agents. Uses of Ethylene oxide, Iodine, Gluteraldehyde

� Gaseous� Ethylene oxide� Gluteraldehyde� Formaldehyde

Hydrogen peroxide� Hydrogen peroxide

� Liquids� Iodine � Iodophores (Iodine + solublizing agent or carrier)� Chlorine releasing agents – Hypochlorous acid, sodium

hypochlorite at higher conc.� Formaldehyde -40% conc� Gluteraldehyde – 2% concentration


What is Koch’s Postulates ? What are its components? When it can not be proved?

The postulate of Robert Koch that establishes the etiologicalrelationship of microbe with disease.

1. Bacterium found in all patients having disease and it or its products found in all body parts affected

2 The bacterium should be isolated and grown in pure culture3 Pure culture inoculated into susceptible animal should produce3 Pure culture inoculated into susceptible animal should produce

disease

4 Same bacterium re-isolated in pure culture from experimentalanimal

# Some microbes can not cultured in artificial media – e.g. Treponemmapallidum, M.leprae

# Some microbes can not produce in experimental animals e.g. N. gonorrhoeae


Men learn while they teach.

Seneca


Best wishes

for

all studentsall students


Documents

General Bacteriology – Review Class