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Introduction to Bacteria I & II

MICR570/ZMR/F12 4-5

Introduction to Bacteria

Lectures 4 & 5

OBJECTIVES: LECTURE 4 & 5

• Describe bacterial morphology – cell shape &arrangement

• Describe various staining t echniques

• Describe components of a bacterial cell

(Internal contents: Ribosomes, Mesosomes,Nucleoid, Plasmids, Cell Envelope: CytoplasmicMembrane, Cell Wall, Glycocalyx, ExternalStructures: Flagellum, Pili, Additional Feature:Endospores)

– Structure & Function of each – fundamental for

Growth, Control, Pathogenesis, etc

Lacking Cell Wall Rigid Cell Wall Flexible Cell WallMycoplasma Borrelia

Ureaplasma LeptospiraTreponemaSimple Filamentous

Actinomyces

MycobacteriumNocardia

Obl igate Int racel lu lar F ree- li vi ngChlamydia/Chlamydophila

RickettsiaEhrlichia

MEDICALLY IMPORTANT BACTERIA

Gram +ve Gram –ve

Cocci Rods Cocci Non-enteric Rods Enteric Rods

Staphy lococcus Bac il lus Moraxell a Bar tonell a Campylobacter S trep tococcus Clost ridi um Neisser ia Bordetell a Ente robac ter Enterococcus Corynebacterium Brucella Escherichia

Lactobacilli Burkholderia Helicobacter

Listeria Francisella KlebsiellaPropionibacterium Haemophilus Proteus

Leigonella Salmonella

Pseudomonas ShigellaVibrioYersinia

Adapt ed fr om Fig 1.4, p 4 Li ppin cot ’s Illu str ated Review s: Mic rob iolo gy. 2007

*ANGEL>Lessons>Dr. Ross’s Additio nal Material

BACTERIAL SIZE

Bacteria range: 0.2μm - 10μm (Typically 1 - 2μm)

Mycoplasma sp. 0.15-0.3μm dia (size of poxvirus)

(1μm=10-6m)

“Size ISEverything ”




MICR570/ZMR/F12 4-5

BACTERIAL SHAPES

Characteristi c Shapes

• Spherical - COCCOID (COCCUS, COCCI)

• C lindrical - ROD BACILLUS BACILLI

• Curved (Helical, Spiral) - VIBRIO

• Square - (Not infectious)

CELL ARRANGEMENT• TERMINOLOGY

COCCOIDAL BACTERIA

Single

Pairs DIPLOCOCCI Streptococcus pneumoniae

Chains STREPTOCOCCI

Clusters STAPHYLOCOCCI

Tetrads

Diplo, Strepto & Staphylo-bacillus DO NOT EXIST

“Cop out” terms: COCCOBACILLUS, PLEOMORPHIC

Streptococcus pyogenes

All Staphylococcus sp.

Sarcina sp.

STAINING TECHNIQUES

• Why required? Transparency & Size

• GRAMS STAIN - Hans Christian Gram (1884)

Differential stain - colour reaction with cells

www.wikimedia.org

Gram +ve

Cells on slide

Primary Stain (Crystal Violet)

Mordant (Gram’s Iodine) – increases

Gram -ve

affinity of primary stain for cell

Decolourizer (alcohol &/or acetone)

Counterstain (Safranin)




MICR570/ZMR/F12 4-5

Gram +ve Staphylococcus aureusGram -ve Escherichia coli

(1000x)

© Z. Ross 1992© Z. Ross 1992

WHY SHOULD PHYSICIANS EXAMINE

GRAM-STAINED SMEARS?

• Determine the adequacy of the specimen for culture

• Make a presumptive aetiologic diagnosis & early clinical

• Suggest a need for non-routine laboratory procedure

• Help make accurate interpretation of culture results

• Provide a better insight into the current infection

• ACID FAST STAIN - Paul Ehrlich

Differential stain (similar to Gram’s)

Used with Gram’s resistant bacteria

Mycobacterium: tuberculosis

Nocardia spp.i i i

3 Techniques:

Ziehl-Neelsen Staining (hot )

Kinyoun Stain (cold)

Fluorochrome stain (auramine-rhodamine)

. i i i .

1) Ziehl-Neelsen Staining (hot method) (oldest method)

Stain: hot basic carbolfuchsin; decolourize with acid-alkali

Solutions; counterstain: methylene blue or malachite green

Acid-fast Bacteria : RED/PINK

Non Acid-fast bacteria : BLUE/GREEN

2 Kin oun Stain col d m ethod

Same as ZN but does not require heating

3) Fluorochrome stain (auramine-rhodamine)

Same principle

1o

stain: fluorescent dyesCounterstain: potassium permanganate (oxidising agent)

Org’s: fluoresce yellowish/green against black background




MICR570/ZMR/F12 4-5

Mycobacterium tuberculosis (300x)

Ziehl-Neelsen Staining

Auramine Rhodamine Staining

www.cdc.gov

PROKARYOTIC CELL

STRUCTURES• INTERNAL: Cytoplasm/Protoplasm

Nucleoid

Ribosomes

Inclusions

Cellmembrane/Cytoplasmic membrane

Cell wall

Glycocalyx or S-layers

•EXTERNAL: Flagella

Pili

Fimbriae

(Endospores)

Cell envelope

INTERNAL CONTENTS

• PROTOPLASM / CYTOSOLMembrane bound

Granular appearance (Ribosomes)

Site of biochemical activity

Water 70-80% Acts as solvent (sugars, salts & Aa’s)




MICR570/ZMR/F12 4-5

RIBOSOMES

• RNA/PROTEIN bodies

(60% RNA, 40% Protein)

Composed of 2 sub units (70S)

Svedberg Units

(Sedimentation Coefficient)

• Sites of Protein Synthesis

MESOSOMES• Extensive invaginations of cytoplasmic membrane

Continuous with membrane

Function NOT KNOWN

Mainly seen in Gram +ve’s

E.g., Corynebacterium parvum

CHROMATIN AREA

• NO distinct membrane enclosed nucleus

• NO mitotic apparatus

• DNA aggregated in one area

(NUCLEOID)

BACTERIAL CHROMOSOME

• Single circular DNA (Chromatic Body): 3x109 daltons mw

(Exception Streptomyces & Borrelia sp (Linear);Rhodobacter sphaeroides (2 separate chromosomes)

• All genes Linked (No Histone Proteins)

BACTERIAL CHROMOSOME

E. coli 1100 - 1400μm length




MICR570/ZMR/F12 4-5

PAI’s (PATHOGENICITY ISLANDS)

• Distinct Genetic Elements on Chromosome

Characteristics:

– Carry 1 or more virulence genes

– Present only in pathogen genome

– - – -

– Differ from core genome (base composition & operon

useage)

– Frequently located next to tRNA genes

– Frequently associated with mobile genetic elements

(transposons)

– Often unstable

Examples of Pathogens with PAI’s

• Gram positive

– Listeria spp., S. aureus, Streptococcus spp.,

Enterococcus faecalis, Clostridium difficile

• Gram negative

– H. pylori, E. coli, Salmonella spp., Shigella spp., Yersinia

spp., L. pneumophilia, P. aeruginosa, V. cholerae,

Bacteroides fragilis

PLASMIDS

• Extrachromosom al DNA

Circular DNA smaller than chromosome

Self-replicating

• Supplemental to chromosomal DNA genetic info:

Ant ibi ot ic resistance (R plasmid)

Tolerance to toxic metals

Production of toxinsMating capabilities (F-plasmid)

CELL ENVELOPE

1. CYTOPLASMIC MEMBRANE• Plasma membrane - Inner membrane

General Membrane Structure

• Electron Microscopy: 2 densely staining layers separated

by non staining region

• 4-5nm thick: PHOSPHOLIPID 30-40% & PROTEIN 60-

70%

• Semi-permeable barrier

• No Sterols(except Mycoplasma sp.)

Used with permission: http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/




MICR570/ZMR/F12 4-5

• MEMBRANE FUNCTION

• Active transport (metabolites)

• Secretion extracellular enzymes & toxins

•

• Biosynthesis, export cell wall components

• Anchoring DNA (during cell division) (MESOSOME)

E. coli Electron Transport Chain

Chemotactic system

Picture used with permission from Brock: Biology of Microorganisms

2. CELL WALL

• Surrounds all Eubacteria (except Mycoplasma spp.)

• Important in bacterial characteristics

Structure & Function distinctive

Backbone: PEPTIDOGLYCAN Murein la er

Polysaccharide chains alternating NAM & NAG

NAG - (N-acetylglucosamine)

NAM - (N-acetylmuramic acid)

Provides rigidity & strength

Prevents osmotic lysis (dilute environments)

β,1-4 glycosidic bonds




MICR570/ZMR/F12 4-5

L-Lysine or

Used with Permission http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/

Peptidoglycan Layer StructureNAMNAG NAMNAG

Adapted with permission from

http://gsbs.utmb.edu/microbook/images/fig2_8.jpg

L-Lysine or

Used with Permission http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/

Comparison of Peptidoglycan Layer

See Figure 2.5, p14 & 15; Murray et al. 6th EditionUsed with Permission http://gsbs.utmb.edu/microbook/images/fig2_8.jpg




MICR570/ZMR/F12 4-5

GRAM +VE

BACTERIA

• THICK peptidoglycan

– (50-60% of dry weight)

TEICHOIC ACIDS (Lipoteichoic acid) (acidic anionic polysacc’s)

(CH2O (glucose), phosphate + alcohol (glycerol/ribitol)

Function: Bind protons (maintain low pH), cations (Ca2+ & Mg2+)

Act as ADHESINS, virus receptor sites

Additional CH2O’s & proteins (depends upon species)

E.g., M, T & R proteins:GROUP A streptococci; Protein A:Staph. aureus

Lipoteichoic acid (during disease)

causes:

• Dermal necrosis (Schwartzman reaction)

• Induction of cell mitosis at the site of infection

•

• Stimulation of non-specific immunity

• Adhesion to the human cell

• Complement activation

• Induction of hypersensitivity (anaphylaxis)

WHY DOGram +ve’s stain PURPLE?

GRAM -VE

BACTERIA

COMPOSITION:

COMPLEX MORPHOLOGY

• THIN Peptidoglycan (5-10% of dry weight)

• OUTER MEMBRANE (Braun Proteins)¾ PORINS (protein channels) Nutrient Transport

¾ LIPOPOLYSACC (ENDOTOXIN - fever, lysis RBC’s)




MICR570/ZMR/F12 4-5/

Lipopolysaccharide Structure

ENDOTOXIN

TOXIC

Endotoxin induction of:

• Fever

• Haemorrhagic necrosis (Shwartzman reaction)

• Disseminated Intravascular Coagulation

• Production of Tumor Necrosis Factor

• Activation of the Alternate Complement Pathway

• Stimulation of bone marrow cell proliferation

• Enhancement of the immune and the Limulus lysate

reaction (clotting of horseshoe crab amoebocyte lysates)

Core

Lipool igosaccharide (LOS)

• Bordetella pertussis, Neisseria meningitidis, C. jejuni

Lipid A

Taken from www.freepatents.com

WHY DO

Gram -ve’s stain RED/PINK?





COMPARISON OF GRM+VE /GRM-VE

CELL WALLS

Character + ve - ve

No’ of major layers 1 2

Chemical Makeup Peptidoglycan Lipopolysaccharide

e c o c ac

Lipoteichoic acid

popro e n

Peptidoglycan

Overall Thickness Thick (20-80nm) Thin (8-11nm)

Periplasmic Space In some In all

Porin Proteins No Yes

Permeability More permeable Less penetrable

ACID-FAST BACTERIA• Genera Mycobacterium & Nocardia

• Peptidoglycan + arabinose & galactose polymers

Arabinogalactan esterification → mycolic acids (waxy) 60%

Used with permission: http://www.cat.cc.md.us/courses/biol141/lecguide/unit1/prostruct/

EFFECT OF LYSOZYME

• LYSOZYME: breaks β1-4 bonds between NAM & NAG

• Produced: various organisms

– Present: body secretions; tear & saliva

• Destroys all or part of cell wall

SPHEROPLAST: portion of cell wall remains

PROTOPLAST: cell wall completely removed(Gram +ve more sensitive)

Wall degraded

With osmotic support (0.5M sucrose) cell NOT lyse





EFFECT OF PENICILLIN

• Penicillin (antibiotic) prevents cell wall formation

ONLY growing cells NO EFFECT on Mycoplasma sp.

• Inhibits formation normal cross-linkages in Peptidoglycan

• Binds irreversibly to PENICILLIN-BINDING PROTEINS (PBP)

Transpeptidases

• Result: Defective cell walls

NO protection from osmotic shock = Cell Death (Gram +ve)

Penicillin forms inactive complex with transpeptidase

Peptide cross-linkages NOT formed

PERIPLASMIC SPACE

NOTE: Gram -ve (2 membranes)

• Space between inner & outer membranes

• Some Gram +ve

• Gel-like area

• Loose network of peptidoglycan

Contains: nutrient transport proteins

nutrient acquisition enzymes (proteases)

detoxifying enzymes (β-lactamases)

membrane derived oligosaccharides (MDO)

osmoprotectants

3. GLYCOCALYX

(ALSO KNOWN AS CAPSULE, SLIME

LAYER & S-LAYER)

• External mucilaginous layer EPS (POLYSACCHARIDE)

Bacillus anthracis (POLYPEPTIDE)

• Surrounds cell, Non-vital

• Shows degree of organisation

– SLIME LAYER - poor organisation, weak attachment to

cell wall (Coagulase -ve: Staphylococcus epidermidis)

– CAPSULE - organised, adhere to cell wall

K antigen (M - Streptococcus pyogenes; Vi - Salmonella sp.)











Speed & Distance

20-90μm/sec

Approx equivalent:

6ft Human running 5 body lengths/sec

U. Bolt (Jamaica)

100m in 9.58secsSource: www.bbc.co.uk

16th August, 2009

http://www.youtube.com/watch?v=891M1TH99_8

AXIAL FILAMENTS

• Motile bacteria that LACK flagella

Example: Spirochetes - Leptospira

• Flagella-like filaments (Protein chemically & structurally)

• Long thin microfibril inserted into a hook

• Entire structure enclosed in periplasmic space

(Not exposed to external environment)

ENDOFLAGELLUM

AXIAL FILAMENTSUsed with permission www.microvet.arizona.edu

http://www.youtube.com/watch?v=ODYu--TNPDE

PILUS(s) PILI(pl)

FIMBRIA(s) FIMBRIAE(pl)

• Hollow, helical (9-10nm dia), thinner than flagella

• Filamentous, more numerous

• Protein composition (PILIN) (Classification & ID)

• F-PILUS (SEX PILUS) GRAM -VE BACTERIA ONLY

Entry of genetic material during conjugation

Used with permission fromThe Pasteur Institute, France





• FIMBRIAE: Attachment (common) Pili (type I pili)

Adhesion to surfaces

Predominantly Gram -ve, Some Gram +ve

(Corynebacterium renale, Actinomyces naeslundii)

Used with permission from

The Pasteur Institute, France

ADDITIONAL FEATURE

ENDOSPORES

• Few bacteria: Bacillus & Clostridium spp.

Important pathogenic bacteria

-

• Resistant: UV, irradiation, chemical disinfection, drying

• Require specialized stains (light microscopy)

Mature Bacillus subtilis spore (x3000)

N: DNA region PM: Protoplast membrane

Cx: Cortex D: Inner spore coat

SC: Outer spore coat

Picture used with permission from http://gsbs.utmb.edu/microbook.htm

• COAT: Keratin-like protein

Impermeable layer (resistance to antibacterials)

• CORTEX: Type of peptidoglycan (fewer cross-links)

• SPORE WALL: Peptidoglycan layer

Cell wall germinating vegetative cell

• CORE: Contains complete genetic material

Protein-synthesizing apparatusEnergy-generating system (Glycolysis)

Calcium-dipicolinic acid (10% dry wt, characteristic)





VEGETATIVE CELLS vs ENDOSPORES

Property Vegetative Cell Endospore

Surface Coat Typical

Gram +ve

Thick spore coat +

peptidoglycan spore wall

Microscopic appearance Non-refractile Refracti le

Calcium dipicolinic acid Absent Present in core

Heat resistance Low High

Radiation resistance Low High

Resistance to chemicals

(acids)

Low High

Sensitivity to Lysozyme Sensitive Resistant

Sensitivity to s tains/dyes Sensitive Resistant

Adapted from Todar, K. Structure & Function of Procaryotic Cells.http://texbookofbacteriology.net/structure.html

SPORULATION

TRUE DIFFERENTIATION

• NO MULTIPLICATION (NOT GROWTH)

• Sporulation: Vegetative cell → 1 spore

• Germination: 1 spore → vegetative cell

STEPS IN

SPORULATION:

Vegetative cell

DNA Condenses

Transverse wall begins to form

S ore material se arated fores ore,

formation

Vegetative cell grows around spore

Spore forms multilayered coating

Cell lysis frees spore



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