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7/27/2019 Lectures 04 _ 05 Slides
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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 techniques
• 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
Obligate Intracellular Free-livingChlamydia/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
Adapted from Fig 1.4, p 4 Lippincot’s Illustrat ed Reviews: Microbiology. 2007
*ANGEL>Lessons>Dr. Ross’s Additional Material
BACTERIAL SIZE
Bacteria range: 0.2Pm - 10Pm (Typically 1 - 2Pm)
Mycoplasma sp. 0.15-0.3Pm dia (size of poxvirus)
(1Pm=10-6m)
“Size ISEverything ”
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5
BACTERIAL SHAPES
Characteristic 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)
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Introduction to Bacteria I & II
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.
3 Techniques:
Ziehl-Neelsen Staining (hot)
Kinyoun Stain (cold)
Fluorochrome stain (auramine-rhodamine)
. .
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 coldmethod
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
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Introduction to Bacteria I & II
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)
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Introduction to Bacteria I & II
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 - 1400Pm length
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Introduction to Bacteria I & II
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
• Extrachromosomal DNA
Circular DNA smaller than chromosome
Self-replicating
• Supplemental to chromosomal DNA genetic info:
Antibiotic 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/
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Introduction to Bacteria I & II
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)
E,1-4 glycosidic bonds
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Introduction to Bacteria I & II
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
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Introduction to Bacteria I & II
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)
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Introduction to Bacteria I & II
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
Lipooligosaccharide (LOS)
• Bordetella pertussis, Neisseria meningitidis, C. jejuni
Lipid A
Taken from www.freepatents.com
WHY DO
Gram -ve’s stain RED/PINK ?
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
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 esterificationo mycolic acids (waxy) 60%
Used with permission: http://www.cat.cc.md.us/courses/biol141/lecguide/unit1/prostruct/
EFFECT OF LYSOZYME
• LYSOZYME: breaks E1-4 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
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Introduction to Bacteria I & II
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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 (E-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.)
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
FUNCTION:
• ADHERENCE: to other species members (colonies)
to surfaces
E.g., Streptococcus mutans (dental caries)
EPS glucose & fructose polymer (GLUCANS & FRUCTANS)
• ANTIGENIC ACTIVITY: EPS antigenic
E.g., S. pneumoniae, Haemophilus influenzae: Identification
• ANTIPHAGOCYTIC: (Defense Mechanism: ingestion of foreign objects by blood or tissue cells)
E.g., Streptococcus pneumoniae - resists phagocytosis
• PREVENTION OF NEUTROPHIL KILLING OF ENGULFED
BACTERIA: lysosome contents do not have direct access to
the interior of the bacterial cell
• PREVENTION OF PMN LEUKOCYTE MIGRATION TO SITE
OF INFECTION:
Bacteroides fragilis: capsule of succinic acid.
• TOXICITY TO HOST CELL:
E.g., B. fragilis capsule induces abscess formation
• PROTECTION:
anaerobes from oxygen toxicity, desiccation & nutrient loss
QUELLUNG REACTION
• Swelling Reaction: Capsule presence by microscopy
• Antiserum + Bacteria o Swelling
• Specific antisera: capsular (K/M) antigens for TYPING
EXTERNAL APPENDAGES
FLAGELLUM (s), FLAGELLA (pl)
• Not essential for survival
• Flexible, non-vital (Never on cocci)
• Organ of Motility
• Possible role in colonization?
www.wikimedia.org
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
FLAGELLUM ARRANGEMENT
• Polar: Monotrichous
E.g. Vibrio sp.
Amphitrichous
E.g. Spirillum sp.
Lophotrichous
E.g. Pseudomonas sp.
• Lateral: Peritrichous
E.g. Proteus sp.
Composed of 3 parts
• HELICAL FILAMENT
Inserted into hook
Composed of protein (FLAGELLIN: hauch H antigen)
|20nm dia, 1-7Pm length
• HOOK
Short curved structure; anchors filament into basal body
• BASAL BODY
Contains rod & 1 or 2 sets of double plates (rings)
Located in cytoplasmic membrane & cell wall
2 rings Gram +ve; 4 rings Gram -ve bacteria
Used with permission http://www.arn.org/docs/mm/flag_labels.jpg
TAXIS
• involuntary movement of organism in response to a stimulus
• CHEMOTAXIS: Response to a chemical substance
• AEROTAXIS: to air
• PHOTOTAXIS: to light
• MAGNETOTAXIS: to magnetic field
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
• CHEMOTAXIS
Chemical receptors PROTEIN (20) Attractant/(10) Repellant
Located in cytoplasmic membrane
+ve response: org swims fromlow [ ] tohigh[ ]
(i.e., up concentration gradient)
-ve response: down concentration gradient
Non response: random walk (uniform [ ])
• Rotation of basal body plates is reversible
Clockwise (CW) or Counterclockwise (CCW)
In General:
• Rotation in CCW direction
Flagella sweep around cell in common axis
• Reversal to CW direction
Flagella disperse
Result: tumbling motion
Movement
• Random movement - absence of [ ] gradienttumble
run
• Movement in attractant gradient. Tumbling frequency
reduced
Increasing concentration
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
Speed & Distance
20-90Pm/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
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
• 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: D NA 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)
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Introduction to Bacteria I & II
MICR570/ZMR/F12 4-5/
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 stains/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 o 1 spore
• Germination: 1 spore o vegetative cell
STEPS IN
SPORULATION:
Vegetative cell
DNA Condenses
Transverse wall begins to form
S ore mater ial se arated fores ore,
formation
Vegetative cell grows around spore
Spore forms multilayered coating
Cell lysis frees spore
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Introduction to Bacteria I & II
In contrast to a human cell, the
bacterial cell:
• May have a capsule (K antigen)
• May have an outer membrane (Only Gram-ve)
• May have a periplasmic space (between outer & inner
membranes - Only Gram-ve)
• Has a rigid cell wall (except Mycoplasma)
– Thick: Gram+ve; Thin: Gram-ve
In contrast to a human cell, the
bacterial cell:
• Has a cytoplasmic membrane, lacking sterols (except
Mycoplasma)
– Contains: energy-producing cytochrome & oxidative phosphorylation
system, membrane permeability (transport) systems, various
ol mer-s nthesizin s stems,ATPase
• Has cytoplasmic membrane invagination (mesosome)
– Attachment site for the chromosome
• May have flagellum (arise from plasma membrane, protrude
through cell wall (H antigen)
– Considered a virulence factor
In contrast to a human cell, the
bacterial cell:
• Has fimbriae/pili (originate in plasma membrane and
protrude through cell wall (Pilin protein/adhesins)
– Considered major virulence factors
• Has ribosomes attached to plasma membrane and free in
cytoplasm (70S)
• May have endospore within cytoplasm (resists adverse
conditions)
• Has a nucleus lacking a nuclear membrane
In contrast to a human cell, the
bacterial cell:
• Has a haploid (single) chromosome
• May have a circular plasmid (small relative to chromosome)
codes for virulence factors
Recommended