1. Fundamental Medical Bacteriology I: Morphology & Structure
Amy Hinkelman, Ph.D.
Assistant Professor of Microbiology and Immunology
Levine 182;
MSBS 570 Microbiology & Immunology Fall 2017

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3. Learning Objectives
Compare and contrast Gram positive vs. Gram negative bacteria.
Identify the major shapes and arrangements of bacteria.
Describe the major functions of bacterial structures and discuss any significance of these structures in human disease or as drug targets.
Explain how LPS of Gram negative and teichoic acids of Gram positive bacteria can induce septic shock.
Explain the significance of spores in human disease and identify the spore-forming bacteria.

4. Required Reading
Levinson, 2014, Review of Medical Microbiology and Immunology
Chapter 2 (skip genetics-related material)

5. Bacterial Nomenclature

6. Prokarya -aceae -oideae Genus
Domain
Phylum
Class
Order
-aceae
Family
-oideae
Subfamily
Genus
Genus

7. Taxonomic names: Genus species—Bacteria
Species – share an overall similar pattern of traits
Genus species
Bacillus anthracis, Haemophilus influenzae
Genus is capitalized; species is lower-cased
Italicized (or underlined if hand-written)
Abbreviation for species is sp. or spp. (plural)
FYI:
Historically by traits
Now that we have sequencing, there has been some shuffling based on genetics

8. Differences in structural or functional genes
Species
Strains
Serotypes
Differences in structural or functional genes
Differences in surface antigens

9. Strain and Serotype—Bacteria
Strain – a culture derived from a single parent that differs in structure or metabolism from other cultures of that species
Genetic variant of a species (antibiotic-resistant strains)
Serotype—distinguish strains based on antigenic differences against surface antigens

10. Common Names—Bacteria
Not taxonomic nomenclature!!!
Used in common language including the clinical setting
Examples (FYI):
Gonococcus—Neisseria gonorrhoeae
Pneumococcus—Streptococcus pneumoniae
GAS—group A Strep—Streptococcus pyogenes
GBS—group B strep—Streptococcus agalactiae

11. Morphology of bacteria: shape, size and arrangements
Bacterial structures and their functions

12. Bacterial Structure: Shapes
Shape has been used to help identify & classify bacteria!!!
Vary in size and arrangement but most fall into one of 3 basic shapes:
Coccus: spherical
“coccus” = berry

13. Bacterial Structure: Shapes
Shape has been used to help identify & classify bacteria!!!
Vary in size and arrangement but most fall into one of 3 basic shapes:
Bacillus:
Bacillus—rod
Coccobacillus—very short and plump
Vibrio—gently curved
Bacilli
Vibrio
“bacillus” = stick

14. Bacterial Structure: Shapes
Shape has been used to help identify & classify bacteria!!!
Vary in size and arrangement but most fall into one of 3 basic shapes:
Spiral-shaped: helical
Spirochete—spring-like
Spirillum—twisted rod
Spirillum
Spirochete

15. Common Bacterial Shapes
Cocci
Bacilli
Vibrio
Spirillum
Spirochete

16. What determines the shape of a bacterium???

17. Pleomorphism: Atypical bacteria
Variation in cell shape and size within a single species
Some species are noted for their pleomorphism
Mycoplasma pneumoniae

18. Size varies: 0.2 – 5 μm
Smallest Bacteria: Mycoplasma ≈ poxviruses (largest viruses)
Longest bacilli ≈ length of yeasts and RBCs

19. Bacterial Arrangements
Dependent on:
Shape
Pattern of division
How cells remain attached after division

20. Common Bacterial Arrangements
Division in Diplococci Streptococci
1 plane
Cocci:
Singles
Diplococci
Streptococci
Irregular clusters: Staphylococci
Division in
multiple planes Irregular clusters
“diplo-” = double
“strepto-” = twisted chain
“staphylo-” = bunch of grapes

21. Common Bacterial Arrangements
Bacilli:
Diplobacilli
Streptobacilli

22. Diplococci
Streptococci
Staphylococci
Diplobacilli
Streptobacilli

23. Diplococci
Streptococci
Staphylococci
Diplobacilli
Streptobacilli

24. Diplococci
Streptococci
Staphylococci
Diplobacilli
Streptobacilli

25. Diplococci
Streptococci
Staphylococci
Diplobacilli
Streptobacilli

26. Diplococci
Streptococci
Staphylococci
Diplobacilli
Streptobacilli

27. Morphology of bacteria: shape, size and arrangements
Bacterial structures and their functions

28. External to Cell Wall Cell Ribosomes wall Cell membrane Fimbriae
Capsule
External to
Cell Wall
Slime
layer
Cytoplasm
Flagellum
Actin filaments
Chromosome
(DNA)
Inclusion body
Sex Pilus

29. Appendages Function: Motility: Flagellum; Axial Filaments
Attachment: Fimbriae or pili
Transfer of DNA: Sex pilus

30. Bacterial flagella—rotational movement
Three Parts:
Filament – long, thin, helical structure composed of protein flagellin
Hook – curved sheath
Basal body – stack of rings firmly anchored in cell wall and cell membrane
Filament
Hook
Outer
membrane
L ring
Cell wall
Basal
body
Rod
Rings
Periplasmic
space
Rings
Cell
membrane

31. Bacterial flagella—rotational movement
Rotate 360o
Moves bacterial cell through environment
Antigenic
*Virulence factor:
E. coli travel to the bladder
Gram + and - 31

32. Many rod-shaped bacteria have flagella
FYI
Monotrichous
Lophotrichous
Amphitrichous
Peritrichous
Many rod-shaped bacteria have flagella

33. Periplasmic/Internal Flagella: Axial Filaments—twisting movementPF PC OM
Internal flagella
Motility—contraction; corkscrew-like
Spirochetes
Outer Membrane
Periplasmic
flagella (PF)
Peptidoglycan
Cell membrane

34. Appendages Function: Motility: Flagellum; Axial Filaments
Attachment: Fimbriae or pili
Transfer of DNA: Sex pilus

35. For attachment: Fimbriae (“fringe”); also called pili*
Fine, hairlike projections
Composed of glycoprotein
Mostly Gram – but also some Gram +

36. Sex Pilus Function to join bacterial cells for partial DNA transfer
called conjugation
Composed of pilin
Rigid, tube-like
Only Gram -

37. Outer Bacterial Coatings
Glycocalyx
Capsule
Slime layer
“Sugar coat”
Gram + and -

38. Glycocalyx Composition Polysaccharide
Exception is B. anthracis (polypeptide capsule)
Vary from species to species and even can distinguish within a species “serotypes”

39. Glycocalyx Composition 2 Types Slime layer Capsule Polysaccharide
Exception is B. anthracis (polypeptide capsule)
2 Types
Slime layer
Capsule

40. Functions of glycocalyx
Evasion of immune response
Protects against phagocytosis

41. Functions of glycocalyx
Attachment to surfaces
Colonization
Aid in formation of biofilms

42. Functions of glycocalyx
Retention of nutrients and water
Prevent dehydration and nutrient loss

43. Functions of glycocalyx
Evasion of immune response
Protects against phagocytosis
Attachment to surfaces
Colonization
Aid in formation of biofilms
Retention of nutrients and water
Prevent dehydration and nutrient loss

44. Cell Envelope Cell Ribosomes wall Cell membrane Fimbriae Capsule Slime
layer
Cytoplasm
Underlined structures are essential to all prokaryotes; most have a cell wall
Flagellum
Actin filaments
Chromosome
(DNA)
Inclusion body
Sex Pilus

45. The Cell Envelope External covering outside the cytoplasm
Composed of 2 basic layers:
Cell wall
2 major types (plus some atypical bacterial cell walls)
Cell membrane
Similar in structure to eukaryotic except no steroids/sterols
Different in function of eukaryotes
Takes on many of the organelle functions seen in eukaryotes
Electron transport, energy production, transport, ion pumps
Exception: Mycoplasma spp.

46. Cell Wall: Functions Maintains shape
Maintains strong structural support
Prevents lysis due to osmotic changes
Point of anchorage for flagella
Contributes to pathogenesis

47. Bacteria are classified by the Gram Stain
Gram-positive bacteria:
Peptidoglycan THICK cell wall
Cell membrane
Gram-negative bacteria:
Outer cell membrane
Thin peptidoglycan layer
Cell wall

48. Peptidoglycan: only in bacteria
Repeating, continuous framework of long glycan chains
N-acetylmuramic acid (NAM)
N-acetylglucosamine (NAG)
Linked by short peptide chains
Rigid support & protection against osmotic pressure changes

49. Peptidoglycan: sugar backbone with peptide crosslinks
Cross-linked by
transpeptidases
Peptide crosslinks can vary among species

50. Since only found in bacterial cells—major drug target!!!

51. (penicillins & cephalosporins)
Peptidoglycan: sugar backbone with peptide crosslinks
Cross-linked by
transpeptidases
β-lactam antibiotics
(penicillins & cephalosporins)
Peptide crosslinks can vary among species

52. Gram-Negative Bacteria
BOTH inner and outer membranes
Periplasmic space between the two membranes
Thin peptidoglycan layer
Outer membrane contains lipopolysaccharide (LPS) and lipoproteins
Porin proteins—allow entry of nutrients (& certain drugs)
Cell membrane
Peptidoglycan
Cell Envelope
Cell wall
Outer membrane
Gram (–)
Cell membrane
Outer
membrane
Peptidoglycan
Periplasmic space

53. LPS: Aka…ENDOTOXIN Causes tissue damage esp. to endothelium
Part of the outer leaflet of outer membrane of Gram negative
Causes fever and Septic SHOCK!
Strong stimulus for macrophages via TLR
IL-1 (fever & inflammation)
IL-6 (fever & inflammation)
Nitric oxide (vasodilation)
TNF (inflammation)
Antigenic—differences in sugar component
“endotoxin” b/cpart of the bacterium rather than synthesized & secreted (exotoxins)
Released when bacterium dies (w/a few exceptions)
Causes tissue damage esp. to endothelium

54. LPS: Aka…ENDOTOXIN Structure: O Antigen Outer polysaccharide
Up to 25 repeating units of 3-5 sugars
Used to identify certain Gram -
Core Polysaccharide
5 sugars linked to Lipid A
Lipid A
Phosphorylated disaccharide w/fatty acids
TOXIC effects
Extracellular space

55. Gram-Positive Bacteria
Peptidoglycan: several layers thick
Teichoic acid (attached to peptidoglycan) & Lipoteichoic acid (attached to cell membrane)
Virulence (G+ endotoxin)
Attachment
Antigenic
Cell wall close to plasma membranethin periplasmic space
Peptidoglycan
Cell membrane
Gram (+)
Cell wall (peptidoglycan)
Cell membrane
Cell Envelope

56. Teichoic acids: can also act as ENDOTOXIN for some Gram Positive
Not all and weaker
Part of the cell wall of Gram positive
Causes fever and Septic SHOCK!
Strong stimulus for macrophages via TLR
IL-1 (fever & inflammation)
IL-6 (fever & inflammation)
Nitric oxide (vasodilation)
TNF (inflammation)
“endotoxin” b/cpart of the bacterium rather than synthesized & secreted (exotoxins)
Released when bacterium dies
Causes tissue damage esp. to endothelium

57. Structures of Gram-Positive and Gram-Negative Bacterial Cell Walls
Lipoteichoic acid
Phospholipids
Lipopolysaccharides
Teichoic acid
Peptidoglycan
Envelope
Envelope
Cell membrane
Membrane
protein
Membrane
protein
Periplasmic
space
Phospholipid
Peptidoglycan
Porin
Outer
membrane layer
Membrane
proteins
Lipoprotein
Teichoic acid
Lipopolysaccharide

58. Biomedical Significance: Gram Positive
Lipoteichoic acids are antigenic
Actions on Gram +
Lysozyme hydrolyzes linkages between NAG and NAM
Penicillin inhibits synthesis of peptide cross bridges
Inhibits transpeptidases
Often more sensitive to penicillin than Gram negative
Disinfectants damage cell wall

59. Biomedical Significance: Gram Negative
LPS very toxic in disease
Actions on Gram -
More susceptible to mechanical breakage but less so to chemical breakdown (less permeable due to OM)
Lysozyme requires pretreatment to destabilize membrane (not as effective)
β-lactamases in periplasmic space: degrade penicillins & derivatives
Requires different, more aggressive drugs
Drugs must cross outer membrane

60. The Gram Stain Gram positive - retain crystal violet and stain purple
Differential stain: distinguishes cells with a Gram positive cell wall from those with a Gram negative cell wall
Gram positive - retain crystal violet and stain purple
Gram negative - lose crystal violet and stain red from safranin counterstain
Important basis of bacterial classification & identification
Practical aid in diagnosing infection and guiding drug treatment!!! (Typically sample taken & culturedget enough to identify)

61. The Gram Stain Procedure
Microscopic Appearance of Cell
Chemical Reaction in Cell
(very magnified view)
Gram (+)
Gram (–)
Gram (+)
Gram (–)
Step
1 Crystal Violet
(primary dye)
Both cell walls stain with the dye.
2 Gram’s iodine
(mordant)
Dye crystals
trapped in cell
No effect
of iodine
3 Alcohol
(decolorizer)
Crystals remain
in cell.
Outer wall is
weakened; cell
loses dye.
4 Safranin
(red dye
counterstain)
Red dye
has no effect.
Red dye stains
the colorless cell.

62. Gram Positive Bacteria

63. Gram Negative Bacteria

64. Gram-Positive vs. Gram-Negative Cell Walls
1 layer
MUCH thicker
Outer membrane
Teichoic & lipoteichoic acids
Stain purple
Sensitive to chemical disruption
2 layers
Thinner
Outer membrane w/porin proteins
Lipopolysaccharide (LPS)
Stain pink
Sensitive to mechanical disruption

65. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

66. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

67. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

68. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

69. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

70. Diplococci Streptococci Staphylococci Diplobacilli Streptobacilli
Gram Negative
Gram Positive

71. Cell Wall: Exception Examples
Mycobacteria: M. tuberculosis & M. leprae
Mycolic acid (wax-like lipid coat)Acid-fast stain
Mycoplasmas: Mycoplasma pneumoniae
Lack cell walls, very small (have sterols)
Chlamydiae & Rickettsiae
Intracellular, very small

72. Internal Structures Cell Ribosomes wall Cell membrane Fimbriae Capsule
Slime
layer
Cytoplasm
Flagellum
Actin filaments
Chromosome
(DNA)
Inclusion body
Sex Pilus

73. What is contained within the bacterial cytoplasm?
Chromosome (nucleoid region)
Plasmids (extra-chromosomal DNA)
Ribosome (protein & rRNA)
70S (50S + 30S)
Granules / Inclusion bodies
Reserve deposits
Cytoskeleton

74. Nucleoid Region: contains chromosomal DNA but NOT membrane-bound
Chromosome
Single, circular, double-stranded DNA (~2000 genes)
Essential
NO introns or histones

75. Plasmids: extrachromosomal DNA in the cytoplasm
Free small circular, double-stranded DNA
Not essential to bacterial growth & metabolism
Often carry virulence factor (e.g. toxins) or selective advantage (e.g. antibiotic resistance genes)
Used in genetic engineering

77. Bacterial Ribosome Drug target b/c different than eukaryotic cells!!!
Ribosome (70S)
Ribosomes
Site of protein synthesis
60% rRNA and 40% protein
2 subunits: large (50S) + small (30S) = 70S ribosome
Drug target b/c different than eukaryotic cells!!!
Large
subunit
(50S)
Small
subunit
(30S)

78. Bacterial Internal Structures
Inclusions and granules
Intracellular storage bodies
Vary in size, number, and content
Bacterial cell can use them when environmental sources are depleted

79. Some bacteria can form endospores to survive environmental changes
Bacillus spp. and Clostridium spp.

80. Endospores Wait…why is it pink??? Dormant Structure 2-phase Life Cycle
Nutrient deprivation, water unavailable
2-phase Life Cycle
vegetative cell (metabolically active)
spore (inert, resting condition)
Gram + rods (Clostridium; Bacillus)
Wait…why is it pink???

81. Endospores EXTREMELY HARDY!!!
Withstands extremes in heat, drying, freezing, radiation, and chemicals
Resistant to ordinary cleaning methods & boiling
Pressurized steam at 120oC for minutes will destroy
Not a means of reproduction (not a true “spore” like fungal spores)
Thick, keratinlike coat

82. Germination of spores
Germinating agent (water, nutrients) triggers germination
Stimulates hydrolytic enzymes
Digest coat & expose core to water
Core rehydrates

83. Biomedical Significance: Bacterial Spores
Survive boiling for several hours
Resistant to ordinary cleaning methods
Found in soil & dust (sporedisease)
Examples (FYI):
Clostridium perfringens: gangrene
Clostridium tetani: tetanus
Clostridium botulinum: botulism
Bacillus anthracis: anthrax
Bacillus cereus: food poisoning
Why medical supplies and instruments must be autoclaved!!!