Microbiology: Prokaryotes, Eukaryotes & Parasites

Introduction to Microbes

• Microbiology is the study of microscopic living forms.
• Some microbes can be very large.

Learning Objectives

• Understand the classification and structure of microbes, especially bacteria.
• Know the differences between eukaryotes and prokaryotes.
• Learn how bacteria are classified.
• Understand the differences between Gram-negative and Gram-positive bacteria.

Medically Important Organisms

• Viruses range in size from 0.03um to 0.3um.
• Bacteria range in size from 0.2um to 2um.
• Fungi range in size from 4um to 40um, including mushrooms.
• Parasites, such as worms, range in size from 0.3cm to 12m.
• Organisms larger than 40um can typically be seen with the naked eye.

Taxonomic Hierarchy

• The taxonomic hierarchy includes the classifications of: Kingdom, Phylum, Class, Order, Family, Genus, and Species.
• An example of taxonomic hierarchy is: Kingdom-Prokaryotes; Phylum-Bacteria; Division-Firmicutes; Class-Bacilli; Order-Bacillales; Family-Staphylococcaceae; Genus-Staphylococcus; Species-aureus; Sub Species-anaerobius.
• Binomial nomenclature, as devised by Linnaeus has the following rules: Genus name is capitalized, species name is lowercase, both names are italicized or underlined.

Bacteria and Symbiosis

• The human body is covered in bacteria.
• Symbiosis refers to organisms living together.

Indigenous Microbiota

• Indigenous microbiota, also known as normal flora, is when there are more bacterial cells in/on organisms than their own cells.
• Normal Flora is found on the skin, in the mouth, upper airways, gastro-intestinal tract, and genitourinary tract.

Prokaryotes vs. Eukaryotes vs. Viruses

• Prokaryotes are represented by bacteria.
• Eukaryotes include fungi and parasites.
• Viruses do not fit into either category.

Differences Between Eukaryotes and Prokaryotes

• Eukaryotic cells contain a nucleus with a nuclear membrane enclosing multiple chromosomes.
• Prokaryotic cells have a single chromosome not enclosed in a nuclear membrane.
• Eukaryotic cells contains chitin or cellulose.
• Prokaryotes contain peptidoglycan, lipids, and proteins.
• Prokaryotes have no nuclear membrane, while Eukaryotes do.
• Prokaryotes feature a single, closed, circular, dsDNA chromosome, while Eukaryotes have multiple, linear chromosomes.
• Prokaryotes are haploid, while Eukaryotes are diploid or hapoid.
• Prokaryotes have 70S ribosomes while Eukaryotes have 80S ribosomes.
• Only Eukaryotes feature mitochondria, Golgi complex, and endoplasmic reticulum.
• The cytoplasmic membrane of a prokaryote lacks sterols, except in some cases, while eukaryotes have phospholipids and sterols.
• Prokaryotes have simple flagella; Eukaryotes have complex flagella.
• Prokaryotes generate energy via cytoplasmic membrane association; Eukaryotes use mitochondria.
• Prokaryotes do not have sexual reproduction; Eukaryotes may have sexual reproduction, which alternates with asexual reproduction.
• Prokaryotes have chromosomal or plasmid gene exchange via transformation, transduction, or conjugation.
• Eukaryotes have a diploid zygote formed from haploid germ cells; meiosis results in genetic recombination.

Bacterial Characteristics

• Bacteria are the smallest living cells, ranging from 0.1-10um.
• They have a cell wall and a cytoplasmic membrane.
• The cytoplasm contains ribosomes and a single double-stranded (DS) DNA molecule.
• Bacteria do not have organelles or a nucleus.
• They are free-living.
• Bacteria reproduce through binary fission in artificial media and can grow within a day.

Classification of Bacteria

• Phenotypic classification involves using Gram stain and morphology, growth requirements, biochemical reactions, and serological tests.
• Genotypic classification involves phylogenetic tree analysis, ribosomal RNA analysis, and molecular techniques like PFGE and sequencing.
• Gram stain and morphology involve determining if bacteria are Gram-positive or Gram-negative their shape, arrangement, and size..
• Bacteria shapes include cocci (round), bacilli (rod-like), cocco-bacilli (very short rods), spirochetes (spiral), and curved shapes (vibrio/spirillum).
• Bacterial arrangements includ clusters, chains, and Coryneform or chinese letters.
• Gram stain uses crystal violet (purple), iodine (black), decolouriser, and safranin/carbol fucshin (pink).
• Gram-positive bacteria retain crystal violet due to thicker cell walls and appear purple, while Gram-negative bacteria are decolorized and pick up safranin, appearing pink-red.
• Gram stain reaction is used in conjunction with the types (cocci/bacilli) and arrangement of the bacteria to make a presumptive identification.
• Gram-positive cocci include, staphylococcus aureus (in clusters), Streptococcus pneumoniae and Streptococcus pyogenes (in chains).
• Gram-positive bacilli can be spore-forming like bacillus and clostridium spp.
• Gram-positive bacilli can form chinese letters/club-shaped patterns; Corynebacterium (diphtheriae).
• Gram-positive bacilli can be short rods/coccobacilli-Listeria (monocytogenes)
• Gram-positive bacilli can be branching like; Nocardia spp and Actinomyces spp.
• Gram-negative cocci includes Neisseria (gonorrhoeae or meningitidis)
• Gram-negative bacilli includes Enterobacterales (E. coli, Klebsiella pneumonia, Proteus mirabilis, Salmonella spp, Shigella spp) and Pseudomonas aeruginosa.
• Gram-negative curved bacilli includes Vibrio (cholerae), Campylobacter spp.
• Growth requirements of bacteria include aerobic-oxygen is terminal electron acceptor and cannot grow in its absence (Eg. Pseudomonas aeruginosa).
• Growth requirements of bacteria include; anaerobic-depend on other substances (organic acids/alcohols etc) as electron acceptors (EG. Clostridium spp, Bacteroides spp).
• Growth requirements of bacteria include facultative anaerobes; preferentially utilize oxygen as a terminal electron acceptor, by can also metabolize in the absense of oxygen by reducing other compounds (EG. E.coli).

Structure of Bacteria

• Some bacteria have a slime layer, made of extracellular polysaccharide is loosely associated and facilitates colonization and intravenous catheters.
• Some bacteria have external capsule which is almost universally bacterial, hydrophilic gel, not readily stained and is smooth/muciod layer.
• The cell wall is internal to the capsule of bacteria and is present in almost all bacteria (except cell-wall-less Chlamydia and mycoplasma.
• The function of having a cell wall includes; rigidity/shape, protection from bursting/mechanical disruption, barrier from toxins and anti-phagocytosis.
• The main structural component of the bacterial cell wall is 'peptidoglycan', a mixed polymer of hexose sugars and amino acids
• Peptidoglycan (PG aka murein) is only found only in prokaryotes.
• Peptidoglycan is made of NAGA (N-acetyl-glucosamine) and NAMA (N-acetyl-muramic acid) alternating units 1:4 linkages.
• Lysozyme can break the Peptidoglycan bond.
• Peptidoglycans also features Tetrapeptide side chains with alternating L- & D-amino acids and features; cross bridges - sheets -peptide bonds (3rd aa to 4th(terminal).
• Penicillin binding proteins(PBP) are transpeptidases/co-peptidases are enzymes - export new polymers from cell & link them to inner aspect of the cell wall(ANTIBIOTICS!!).
• Autolysins remove old PG.
• Bacteria are classified according to their cell wall as Gram positive or Gram negative.
• Gram positive peptidoglycan forms a thick layer external to the cell membrane.
• Gram positive bacteria may have techoic acid – polymers of ribitol/glycerol from PG or cell membrane which stabilise cell wall, antigenic, adherence
• Gram positive bacteria have other cell wall structures EG M protein, S layer, C polysaccharide.
• On gram-negative organisms, the peptidoglycan layer is a thin, single layer.
• The peptidoglycan is overlaid by an outer membrane, anchored to lipoprotein molecules in the peptidoglycan layer.
• Omp (Outer membrane proteins) function as attachment points.
• Porins are pores which small hydrophilic solutes can pass.
• A periplasmic space between the CM and OM
• Lipopolysaccharide is also a feature of gram-negative organisms and includes a core polysaccharide Lipid A and O-sp polysaccharide side chains.
• O-sp polysaccharide side chains are major surface antigenic determinants in GNB.
• Another feature of Lipopolysaccharide is it possesses endotoxin activity, which are extremely toxic to man.
• Even in minute amounts, Lipopolysaccharide creates endotoxic shock(fever & shock)/Gram negative shock- high mortality & ICU admission

Cell Membrane

• Encloses cytoplasm.
• Composed of a phospholipid bilayer and is rich in proteins.
• Membrane has no sterols.
• The cell membrane contains enzymes for respiration, PG & lipid synthesis etc.
• Binds to the bacterial chromosome.

Cytoplasm

• Amorphous gel with enzymes, ions, granules, plasmids, ribosomes.
• Many ribosomes lead to faster growth rate
• Composed of 70S ribosomes (50 S + 30 S)

Nucleoid and Plasmids

• Nucleoid has a: Single chromosome
• Features about 4000 genes-ds DNA
• Coiled-1000 times length of cell! DNA gyrase & topoisomerase play a role in this in the nucleoid.
• Plasmids have; Extrachromosomal DNA
• Plasmids are small circular 1 type/>1 type and genes code of enzymes to protect cell from toxins EG abt

Flagella

• Motility is achieved via flagella.
• Flagella is composed of flagellin.
• Major parts of flagella are; Filament, hook, basal body.
• Flagella originate from cytoplasm.
• Position will dictate function; Peritrichous/lophotrichous/monotrichous

External Fimbria

• Fimbria (pili) are used for Non-flagellar hair-like projections on surface.
• Fimbria facilliate adhesion – virulence (eg neisseria).
• Sex pili found in Gram negatives -transfer of genetic material
• Compose of fimbrillin

Spores

• Allows bacteria to enter resting/dormant stage
• Allows for survival-can survive adverse conditions of Bacillus/Clostridia
• Spherical/oval in bacteria
• Possesses a definable location, size, shape
• Does not stain
• Needs sterilisation temp. to kill

Fungi

• Mycology is the study of fungi.
• Fungi are free-living and widely found in nature.
• Fungi is typically slow-growing.
• Fungi can be in; yeast/mould/both (dimorphic fungi).
• Many fungi that infect man are dimorphic and yeast at body temperature - 37°C
• Mould at room temperature - 25°C

Structure

• Fungal structure is Eukaryotic and has a nucleus, nuclear membrane, nucleolus, and linear chromosomes
• Also features organelles but has a Cytoplasmic membrane contains ergosterol & not cholesterol like human cells
• BUT have rigid cell wall – different structure from bacteria
• Rigid external cell wall comprising of; glucan- form fibrils to strengthen, Mannan – mannose-based polymers and Chitin- poly-NAGA which is Similar to crab shell
• Size varies (2-4um) yeast to visible with naked eye eg mushrooms
• Bud extends from yeast cell, constricts-new cell (blastoconidia)
• Hyphae – tube-like extensions - mould
• Aerial hyphae – "fluffy”. Bear reproductive structures
• Vegetative- portion grows into medium/soil
• Mycelium- intertwined mass of hyphae
• Septa-cross-walls that divide hyphae into subunits
• Conidia arise from from hyphae/ from conidiophore (stalk-like)

Reproduction

• Fungi is Haploid usually but Diploid during sexual reproduction
• Asexual - Multiply by budding- mitosis - conidia
• Sexual – produce spores - meiosis
• Three types of infection(mycoses) are recognized:
• Superficial mycoses where the fungus grows at the body surface on skin or hair.
• Cutaneous and subcutaneous mycoses where nails and deeper layers of the skin are involved.
• Systemic or deep mycoses with involvement of internal organs.

Yeasts and Molds

• Examples of Yeasts include; Candida spp (Candida albicans/Candida parapsilosis/Candida glabrata) and Cryptococcus neoformans
• Examples of Moulds include; Aspergillus spp and Rhizopus spp

Parasites

• Protazoa are Single celled and includes; Free-living/parasites and are mostly found in tropical & sup-tropical countries
• Helminths are Macroscopic - Multi-cellular worms <1mm - 1m!

Protozoa

• Generally 2 - >100um in size.
• Structure includes; Nucleus & cytoplasm
• Contains endoplasm (food vacuoles) and ectoplasm (locomotion (flagella/pseudopodia))
• Can be further divided into;
• Amoebae - Entamoeba histolytica.
• Ciliates eg Balantidium coli.
• Flagellates eg Trichomonas spp
• Sporozoa eg malaria (Plasmodium falciparam)
• Protozoa infect tissue and body organs as follows: Intracellular parasites in a wide variety of cells (red cells, macrophages, epithelial cells, brain, muscle).
• Extracellular parasites in the blood, intestine or urogenital system include;

Helminths

• Parasitic Worms
• 3 Classes, Tapeworms(Cestodes)- flat eg Taenia
• Flukes(Trematodes) – leaf-shape eg Schistosoma
• Roundworms(nematodes) eg Ascaris
• Tapeworms & flukes-flattened bodies with suckers/hooks
• Roundworms – long, cylindrical bodies
• Features Complex body structure with; Nervous/reproductive/alimentary systems
• Also features No circulatory system
• Suckers/hooks/teeth/plates for attachment at anterior features on parasitic helminths.
• A tough cuticle allows for body protection.
• Body forms are: Larval stages (100-200um) and Adults- cm - metres