Medical Microbiology: Introduction

Dr. Md. Abdullah Yusuf

Assistant Professor, Dept. of Microbiology

National Institute of Neurosciences & Hospital

Dhaka, Bangladesh

Email: ayusuf75@yahoo.com

Learning Objectives

Introduction and history of Microbiology• Important events of history• Koch’s postulates• Branches of Medical Microbiology• Role of Microbes in environment & medical

science

Important events of history

• Dutch Biologist “Anton Van Leeuwenhock” discovered “animicules” in a drop of water by his simple microscope in 1674

• Otto Muller (Danish biologist) organized bacteria into genus and species in1774

• In 1840, Friedrich Henle (German pathologist) proposed germ theory of disease

Conti…• Robert Koch

– Father of medical Microbiology• Luis Pasteur (Father of Modern Microbiology)

– identified causative agent of anthrax, rabies, plague, cholera and tuberculosis

• Discovery of penicillin– 1928 (Alexander Flemming)

• Discovery of sulphanilamide– 1935 (Gerhard Domagk’s)

• Discovery of streptomycin– 1928 (Selman Waksman)

Koch’s postulates1. The causative (etiological) agent must be

present in all affected organisms– but absent in healthy individuals

1. The agent must be capable of being isolated and cultured in pure form

2. When the cultured agent is introduced to a healthy organism,

– the same disease must occur1. The same causative agent must be isolated

again from the affected host

Limitation of Koch’s Postulate

• Genetic Diseases• Auto-immune Diseases• Non-culturable microbial diseases

Branches of Medical Microbiology

• Bacteriology– General– Systemic

• Virology– General

– Systemic

• Parasitology– Protogoology

– Helminthiology

• Mycology• Immunology

Difference between Virus And Bactria

• Structure• Nucleic Acid• Type of Nucleus• Ribosomes• Nature of Outer Surface• Motility• Method of Replication

Eukaryotes and Prokaryotes

• Properties• Differences• examples

What is bacterium?

• Prokaryotic cells– Pro means primitive– Karyote means nuceus

• Multiply by binary fission– 1divide to 2

• Devoid of chlorophil• ** single cell, living being, microscopic entity but

fantastic in structural organization, reproduction, metabolism and function

Eukaryotes• Eu means True (normal)• Karyote means nucleus• Much larger than most prokaryotes• Have subcellular, membrane-bound organelles• Includes all “higher" plants and animals• Microbiology includes

– Fungi– Protozoa– Algae– Multicellular Organisms (helminthes)

Fungus

• Eukaryotic organism • cell walls contains Chitin • Molds are multicellular

– masses of mycelia– composed of filaments called hyphae

• Yeasts are unicellular

Protozoa

• Eukaryotes• No Cell Wall• Surround and absorb food• May be motile via

– Pseudopods– cilia, or – flagella

Algae

• Eukaryotes• Cellulose cell walls• Use photosynthesis for energy• Produce molecular oxygen and organic

compounds

Virus

• Acellular• Consist of DNA or RNA• Core is surrounded by a protein coat• Coat may be enclosed in a lipid envelope• Viruses are replicated only when they are in a

living host cell• Living

Multicellular- helminthes

• Eukaryote• Multicellular animals• Parasitic flatworms and round worms are called

helminths• Microscopic stages in life cycles

Characteristics of Prokaryotes

Nuclear level

• Absence of – nuclear membrane– Nucleolie– Nucleoplasm– paired multiple chromosome– centriole

Conti..

Cytoplasmic level• Absence of membrane bound organelles

– Mitochondria– endoplasmic reticulum– golgi complex– Microtubules– Microfilaments

Conti..

• Presence of – Ribosome– Plasmid

• No protoplasmic streamming in the form of Brownian movement and Tyndall phenomena

• No change in definite shape and formation of pseudopodia

Characteristics of prokaryotes (Structural)

Cell coverings (cell envelope)• Innermost layer

– bilaminar phospholipid + Lipoprotein – cytoplasmic membrane (fluid stage) without

any sterol• Two membrane in Gram negative bacteria• Condensation at some points forming

mesosome

Conti…

2) Outer to Cell Membrane– cell wall containing specialized unique

chemicals called peptidoglycan

3) Outer to Cell Wall– investing layer either capsule/glycocalyx/slime

layer

4) S layer– Single type protein lattice outer to CW

Characteristics of Prokaryotes

• No genetic exchange during chromosome replication

• Show secretory and excretory activities• Show chemical selectivity, motility and alteration

of life stage (spore)• Wide range of temperature and pH tolerance• Heterogenous nutritional requirements and host

specificity• Can produce self protective molecule bacteriocin• Become infected by particular virus• Can acquire extrachromosomal DNA (plasmid)

Difference Between Eukaryotes and Prokaryotes

• Nature of Nucleus• Mitotic Division• Presence of histones in DNA• Membrane bound Organelles• Size of Ribosomes• Peptidoglycan in Cell Wall

Bacterial cells

Bacterial cells

• essential structures – chemical composition– Organization– Functions

• non-essential structures– chemical composition– Organization– Functions

Structures of bacteria

• Essential Structures (maintain viability)– Cell Wall– Cell Membrane– Mesosome– Ribosome– Nucleoid– Periplasm

Structures of bacteria

• Non-essential Structures (viability is not affected in their absence)– Flagella– fimbria (pili)– Capsule– Glycocalyx– slime layer– Plasmid– spore

Cell wall

• Component– peptidoglycan (PG)

– Teichoic acid– teichorunic acid– Polysaccharides– LPS– Lipoproteins

• PG backbone: NAG and NAM• Tetrapeptide side chains attached to NAM• Interpeptide bridge

Conti…

• Function– osmotic protection– integrity of cellular structures– virulence factors

– Fully permeable to ions, aminoacids and sugars

– makes it rigid

– determines shape– Acts as antigen– Used as serological diagnosis

Peptidoglycan

Gram positive cell envelope

• Covalently bound to the thick peptidoglycan are – teichoic acid (their backbones are usually phosphorus

containing polymers of ribitol or glycerol) or – teichuronic acid (glucuronic acid- containing

polysaccharides

• negatively charged molecules concentrate metal ions from the surroundings

Conti…

• Teichoic acids can also direct autolytic enzymes to sites of peptidoglycan digestion (autolysis).

• This is needed to insert sections of cell wall for growth and division.

• Lipoteichoic acid is primarily associated with the cell membrane.

The Gram negative cell envelope• Covalently linked to the thin peptidoglycan is the

Braun lipoprotein which binds the outer membrane to the cell wall.

• Like other membranes it contains proteins and phospholipids.

• Unlike other membranes it contains lipopolysaccharide

Conti…

• Lipopolysaccharide – helps to provide a permeability barrier

• LPS consists of three regions– an outer O antigen– a middle core– an inner lipid A region

• core contains several sugars– lipid A contains β hydroxyfatty acids

(uncommon in nature)– The molecule displays endotoxin activity

Conti…

• Porins in the outer membrane form channels to allow passage of small hydrophilic nutrients (such as sugars) through the outer membrane.

Cell membrane

• Phospholipid bilayer– Except triple layer in Mycoplasma

• Interspersed with lipoprotein• Selectively permeable

Cell Membrane

• Functions– Transport– Synthesis– Excretion– Secretion– Metabolism– division by mesosome

Nucleoid

• Double stranded coiled helical DNA molecule – remain in a single chromosome

• no introns• no non-coding sequences• no regulatory sequence• no long terminal repeats (LTRs)• Operon present

Nucleoid

• Function– bears genes and genetic characters– maintain metabolism

Ribosome

• 70 s in sedimentation co-efficient• Aggregated• Function

– Protein Synthesis

Non-essential structures

• Flagellum/-a (long whip like)– Filamentous Protein Appendages– Composed of protein as flagellin unit– Arise from cytoplasmic membrane– Account for most bacterial motility– “Run and tumble”– Chemotaxis, phototaxis, aerotaxis, and

magnetotaxis– Antigenic structure

Fig 3.42

Non-essential structures

Pilus/-i• Thin hair like appendages• Composed of protein as pillin subunit• Arise from cytoplasmic membrane• Function

– Attachment– Conjugation– Acts as virulence factors– Antigenic

Capsule/Glycocalyx

• External surface layer composed of polysaccharides Except– Polypeptide in Bacillus– Hyaluronic acid for Streptococci

• Gel like• Forming either capsule (compact, complete and

tight investing) or slime layer (loose meshwork)• Functions

– prevent phagocytosis– Attachment of bacteria– Acts as Antigen

Plasmid

• Extra-chromosomal Double stranded Circular DNA

• Independent of replication• Types

– Transmissible– non-transmissible

• Function– Carries gene for its own replication

– Carries gene antibiotic resistance factors

– Carries gene Proteins & toxin

Endospores

• Highly resistant structures formed at adverse environment

• Composition– Bacterial DNA– small cytoplasm– cell membrane– Peptidoglycan– very little water– thick keratin like coat

Endospores

• Highly resistant to– heat, moisture, chemicals, radiation and

antibiotics• Special component

– calcium salt of Dipicolinic acid• No metabolic activities, remain dormant for

many years• Two events: sporulation and germination

The endospore

Steps of Sporulation

Classification of bacteria

• Basis of Classification• differences between Gram positive and Gram

negative bacteria• atypical characters of bacteria in relation to

staining

Basis of Classification

• Morphological Classification• Staining Properties• Thickness of Wall• According to Motility

Morphological Classification

Morphology of Bacteria• Cocci

– rounded or oval (Staphylococcus)• Bacilli

– elongated rod like (Esch.coli)• Vibrio (from vibration)

– coma shaped (Vibrio cholerae)• Spiral bacteria

– long slender curved body with wave like spiral (Spirillum minus, Helicobacter pylori)

Others Shapes

• Cocco-bacillus– longer than coccus, shorter than bacillus

(Bordetella, Brucella)• Filamentous

– long curved body like ribbon with branching (Actinomycetes, Nocardia)

Spirochaetes

Thickness of Wall• Rigid thick wall Bacteria

– Free-Living (Extracellular)– Non-Free Living (Obligate Intracellular)

• Flexible thin walled Bacteria– Treponema– Borrela– Leptospira

• Wall-less Bacteria– Mycoplasma– Ureaplasma

Rigid thick wall Bacteria

• Free-Living (Extracellular)– Gram Positive– Gram Negative– Acid-Fast

• Non-Free Living (Obligate Intracellular)– Rickettsia– Chlamydia

Staining Properties

• Gram Positive

• Gram Negative

• Acid-Fast Bacteria

Gram Positive Bacteria• Cocci

– Streptococcus– Staphylococcus

• Bacilli– Spore Forming

• Bacillus– Non-spore forming

• Clostridium• Corynebacterium• Listeria

Gram Negative Bacteria

• Cocci– Neisseria gonorrhoae– Neisseria meningitidis

• Bacilli– Enterobacteriaceae– Non-Enterobacteriaceae

According to Motility

• Motile– Flagellated Bacteria– Non-Flagellated Bacteria

• Non-Motile– All Cocci

Bacillus with peritrichous flagella

Morphological study of bacteria

• Staining• Motility test

StainingTheoretical approach

• Types

• Laboratory procedures

• findings of – Gram’s– Zeihl-neelsen (Z-N)– Albert’s staining

Types of Staining

• Simple Staining

• Differential Staining

• Special Staining

Types of Staining

Simple Staining

• using a single dye– methylene blue– Leishman

Types of Staining

Differential Staining

• using primary and counter dye

• performing a step of decolourisation

• to see differential characters of bacteria– Gram’s stain– Ziehl-Neelsen stain

– Albert stain

Types of Staining

Special Staining

• to see unique property of particular bacteria– Fluorescense stain

Gram’s staining

• Named according to inventor Christian Gram• Based on the property of bacterial cell envelope• stained at first by a primary dye & mordanted• Decolorized by acetone or alcohol• Counter stain is added• Gram positive bacteria

– can resist decolourisation and retain the primary dye

• Gram negative bacteria – can not resist decolourisation and take the counter

dye

Gram Positive Bacteria

• Thick layer of Peptidoglycan• Negatively charged teichoic acid on surface• Polysaccharide

Gram Negative Bacteria

• Cell wall much more complex• Thin peptidoglycan layer, filled and surrounded

with periplasm (protein rich gel-like fluid)

• Unique outer membrane on top– Bilayer, yet outer layer is LPS layer (lipid A

and O specific polysaccharide) – LPS acts as endotoxin (lipid A)

• Gram neg. bacteria are less sensitive to medications because outer membrane acts as additional barrier.

Fig 3.34

Atypical cell envelope

• Acid-fast and related bacteria – Mycobacteria– Nocardia

• Presence of Mycolic acids – long, branch chained fatty acids

Gram un-stainable cell wall

• Atypical peptidoglycan• Spirochaetes, Mycobacterium,

– High lipid content

– Tight fluid mosaic

Thank You