Bacteriology: Overview

Bacteriology is the branch of microbiology that deals with the morphology, structure, classification, and biochemistry of bacteria. The discipline of bacteriology arose during the 19th century from scientific attempts to prove the “germ theory of disease,” namely that diseases were caused by microscopic organisms invading host cells. Bacteria are prokaryotic single-celled microorganisms that are metabolically active and divide by binary fission. Some of these organisms play a significant role in the pathogenesis of diseases. Management of bacterial disease is generally with antibiotics; however, the choice of antibiotics may vary depending on the bacterial structure and metabolism.

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Introduction

  • Bacteria: 
    • Thought to be among 1st life forms on Earth
    • Inhabit almost every environment on Earth 
    • Live symbiotically with plants and animals 
    • Most bacteria have not been characterized.
  • Basic features:
    • Unicellular
    • Prokaryotes: lack envelope-enclosed nucleus
  • Identification:
    • Starts with Gram staining using microscopy
    • Cell culture is helpful in exact identification.
    • Sensitivities are considered to determine treatment options.
The process of laboratory diagnosis (2)

Process of laboratory identification:
Identification of bacterial pathogens follows a stepwise process that usually begins with Gram staining and is followed by growth in isolated culture.

Image by Lecturio.

Structure

Structure of bacteria

Structure of a prokaryote cell:
The cell envelope comprises a plasma membrane, seen here in green, and a thick peptidoglycan-containing cell wall (yellow layer). No outer lipid membrane is present, as seen in gram-negative bacteria. The red layer, known as the capsule, is distinct from the cell envelope.

Image: “Average prokaryote cell” by Mariana Ruiz Villarreal. License: Public Domain
  • Prokaryote cell organization:
    • Lack enveloped nucleus
    • DNA compressed into nucleoid
    • Lack membrane-bound organelles
    • Metabolism occurs in the cytoplasm.
  • Cell wall:
    • Provides mechanical stability 
    • Allows for the exchange of nutrients and waste
    • Peptidoglycan murein: 
      • Sugar and amino acid polymer that forms the basis of cell wall
      • N-acetyl glucosamine and N-acetylmuramic acid crosslinked with oligopeptides
      • Component of almost all bacterial walls (exception: mycoplasmas)
    • Thickness of cell wall determines Gram staining.
  • Plasma membrane:
    • Analogous to prokaryotic membranes
    • Composed of proteins and phospholipids 
    • Unlike eukaryotic organisms, lacks sterols
    • Envelops the cytoplasm of cells
  • Flagella:
    • Approximately 50% of prokaryotes move with help of flagella:
      • Monotrichic: 1 flagellum 
      • Polytrichic: > 1 flagellum
    • Consist of the protein, flagellin, and are not covered by cell membrane
    • Placement: monopolar, bipolar, or peritrich
  • Pili (fimbriae):
    • Structurally similar to flagella, but much smaller
    • Aid in adherence to host/other bacteria = virulence factor
    • Used to exchange genetic information during conjugation
  • Nucleoid:
    • Functional equivalent of prokaryotic nucleus
    • Lacks membrane
    • Contains genetic material:
      • Usually 1 chromosome
      • May have additional plasmids
  • Inclusions and vesicles:
    • Gas vesicles allow photosynthetic bacteria to float in water.
    • Organelles for photosynthesis and chemosynthesis
    • Carboxysomes contain key enzyme for fixing CO2.
    • Storage granules (e.g., sulfur, phosphate, calcium, glycogen)
Table: Structures of the bacterial cell with their chemical composition and functions
StructureChemical compositionFunction
Appendages
FlagellumProteinMotility
Pili/fimbriaeGlycoproteinAdherence to cell surface
Specialized structures
Spore
  • Keratin-like coat
  • Dipicolinic acid
  • Peptidoglycan
  • DNA
  • Gram+ only
  • Resists dehydration, heat, and chemicals
Cell envelope
CapsuleOrganized polysaccharide layerProtects against phagocytosis
Slime layerLoose network of polysaccharidesMediates adherence to surfaces
Outer membrane
  • Phospholipids
  • Proteins
  • Gram- only
  • Endotoxin: Lipid A induces TNF and IL-1.
PeriplasmPeptidoglycan in middleAccumulate components exiting gram- cells
Cell wallPeptidoglycan in sugar backboneNet-like structure gives rigid support.
Cytoplasmic membranePhospholipid bilayer sac
  • Site of oxidative and transport enzymes
  • Lipoteichoic acids induce TNF-α and IL-1.
TNF: tumor necrosis factor
IL-1: interleukin-1

Classification

Gram staining

Gram staining is a technique named after the bacteriologist Hans Christian Joachim Gram, and is used to differentiate between groups of bacteria based on the differences in the constituents of their cell walls. 

Differences between gram-positive and gram-negative bacteria cell

Differences between the cell membranes of gram-positive and gram-negative bacteria:
While both gram-positive and gram-negative bacterial cell walls contain peptidoglycan layers, the layer in gram-negative bacteria is much thinner. Gram-negative bacteria make up for this deficit by having another membrane layer outside the peptidoglycan layer.

Image by Lecturio.

Gram staining helps distinguish between gram-positive and gram-negative bacteria by staining cells red or violet.

Process of Gram staining:

  • Bacteria treated with crystal violet dye 
  • Stain is washed with alcohol.
  • Cells are counterstained with red safranin stain.

Staining of gram-positive bacteria: 

  • Cell wall contains a thick layer of peptidoglycan (murein).
  • Retain crystal violet stain and are not affected by safranin counterstain
  • Appear purple-blue

Staining of gram-negative bacteria: 

  • Cell wall has a thin layer of peptidoglycan (murein).
  • Do not retain crystal violet stain, but retain safranin counterstain
  • Appear pink-red

Staining plays important role in diagnostics/pathology: 

  •  Gram-positive bacteria:
    • More common commensal
    • Often found on the skin of healthy individuals
  • Gram-negative bacteria: 
    • Less sensitive to penicillin owing to additional lipid bilayer membrane
    • Contain 2nd membrane in the form of a lipid bilayer
    • Upon degradation, lipopolysaccharides of the 2nd membrane layer can be released as endotoxins.
    • Endotoxins are pyrogens that cause high fever and chills

Morphology

  • Bacteria display a wide diversity of shapes and sizes.
  • Most common morphologies are cocci and bacilli, which can occur in many arrangements
  • Arrangement:
    • Pairs
    • Tetrads
    • Clusters
    • Chains
    • Palisades
Table: Morphology and arrangement of bacteria
MorphologyArrangements
Cocci
  • Spherical
  • Immobile
  • Single or paired (Diplococci)
  • Twisted chains (Streptococci)
  • Clusters (Staphylococci)
Bacilli
  • Round-ended cylinders
  • Rods
Spirilla
  • Helical
  • Twisted
Spirochetes
  • Helical
  • Long, flagella-like filaments
  • Corkscrew-like movements
  • Subgroup: Treponema
SelenomasCylinders curved in a plane
Genus Haloquadratum
  • Unusual morphologies
  • Square, flat box-shaped
Vibrions
  • Round, comma-shaped bacteria
  • Flagellated
Bacterial cells different morphologies and arrangements

Bacterial cells with various morphologies and arrangements:
Bacteria exist in a wide variety of morphologies and arrangements. Cocci and bacilli in pairs and clusters are the most common.

Image: “Bacteria display a large diversity of cell morphologies and arrangements” by Mariana Ruiz. License: Public Domain

Classification overview

Microbiology flowchart gram-positive bacteria classification

Gram-positive bacteria:
Most bacteria can be classified according to a lab procedure called Gram staining.
Bacteria with cell walls that have a thick layer of peptidoglycan retain the crystal violet stain utilized in Gram staining but are not affected by the safranin counterstain. These bacteria appear as purple-blue on the stain, indicating that they are gram positive. The bacteria can be further classified according to morphology (branching filaments, bacilli, and cocci in clusters or chains) and their ability to grow in the presence of oxygen (aerobic versus anaerobic). The cocci can also be further identified. Staphylococci can be narrowed down on the basis of the presence of the enzyme coagulase and on their sensitivity to the antibiotic novobiocin. Streptococci are grown on blood agar and classified on the basis of which form of hemolysis they employ (α, β, or γ). Streptococci are further narrowed on the basis of their response to the pyrrolidonyl-β-naphthylamide (PYR) test, their sensitivity to specific antimicrobials (optochin and bacitracin), and their ability to grow on sodium chloride (NaCl) media.

Image by Lecturio. License: CC BY-NC-SA 4.0
Gram negative bacteria classification flowchart

Gram-negative bacteria:
Most bacteria can be classified based on a lab procedure called Gram staining.
Bacterial cell walls having a thin layer of peptidoglycan do not retain the crystal violet stain used for Gram staining. However, gram-negative bacteria retain the safranin counterstain and appear pinkish-red. These bacteria can be further classified according to morphology (diplococci, curved rods, bacilli, and coccobacilli) and their ability to grow in the presence of oxygen (aerobic versus anaerobic). Gram-negative bacteria can be accurately identified by culturing on specific media (triple sugar iron (TSI) agar), where their enzymes can be identified (urease, oxidase) and their ability to ferment lactose can be determined.
* Stains poorly on Gram stain
** Pleomorphic rod/coccobacillus
*** Require special transport media

Image by Lecturio.

Toxins

Endotoxins

  • Generated during the breakdown of bacterial cell wall when bacteria die
  • Activate host complement and coagulation cascades 
  • Cause septic shock
  • Non-disease-specific symptoms: 
    • Fever
    • Pain
    • Shock
    • Fatigue
    • Discomfort

Exotoxins

  • Produced and secreted
  • Can result in severe, disease-specific symptoms
  • 3 main categories:
    • Enterotoxins
    • Neurotoxins
    • Cytotoxins
  • Examples:
    • Cholera
    • Botulinum
    • Diphtheria
    • Tetanus toxins

Metabolism

Bacteria are heterotrophic organisms that need organic substances to survive.

Classified based on oxygen requirements:

  • Obligate aerobes (aerophilic): Oxygen is needed to maintain metabolism.
  • Obligate anaerobes: 
    • No respiratory enzymes present
    • Energy is generated via anaerobic glycolysis.
    • Oxygen is toxic.
  • Microaerophiles:
    • Oxygen needed for growth
    • If level is too high, growth stops
  • Facultative anaerobes: can grow in the presence or absence of oxygen

Reproduction

Bacteria can exchange genetic material:

  • Can integrate foreign DNA into their genome
  • Can recombine in the existing gene pool
  • Can transfer genetic traits between each other:
    • Conjugation: parasexual transfer through contact via pili
    • Transduction: through bacteriophages (viruses that infect bacteria)
    • Transformation: introduction of free, isolated, foreign DNA into genome
Bacterial types of genetic transmission

Schematic of types of genetic transmission in bacteria

Image by Lecturio.

Virulence Factors

Virulence is the ability of an organism to infect the host and cause disease. Virulence factors are molecules that assist the bacterium in colonizing the host and can be either secretory, membrane associated, or cytosolic in nature.

Table: Mechanism of virulence, virulence factors, and function
MechanismVirulence factorsFunction
Colonization
  • Teichoic acid (primarily in gram-positive organisms)
  • Adhesins
  • Biofilms
  • Flagella
  • Allows attachment to and invasion of host cell surfaces
  • Biofilms protect from antibiotic penetrance
  • Motility
Avoiding the immune system
  • Capsule
  • IgA protease
  • Protein A
Creates physical barrier blocking opsonization and phagocytosis
Bacterial nutritionSiderophores
  • Chelate iron
  • Iron absorption
Antigenic variation
  • Pili
  • Capsule and flagella expression
  • Antigenic drift
Camouflage of surface molecular markers that allow evasion of the immune system
Intracellular survival
  • Inhibition of phagosome-lysosome fusion
  • Exiting phagosomes before fusion occurs
  • Invasins
Prevents intracellular destruction of the bacteria
Type III secretion systemInjectisomeAllows bacteria to inject toxins into host cells
Inflammatory response
  • Bacteria-specific antibodies
  • Immune complexes
  • Peptidoglycan and teichoic acid
  • Mimic host cells (e.g., group A Streptococcus)
  • Delayed hypersensitivity reactions
  • Granuloma formation

References

  • Baron, S., Editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Introduction to Bacteriology.
  • Woese, C.R., Fox, G.E. November 1977. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America. 74 (11): 5088–90.
  • Cabeen, M.T., Jacobs-Wagner C. August 2005. Bacterial cell shape. Nature Reviews. Microbiology. 3 (8): 601–10.

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