Microbiology Growth, diagnosis and an introduction to antimicrobials

Prokaryotes

• Little internal organization

• Much smaller than eukaryotes

Bacterial Structure

1. The shape of a bacteria can usually be determined with appropriate staining techniques using a light microscope.

• 2. Types

– a. Round (coccus)

– b. Rod-like (bacillus)

– c. Spiral

• 3. Cocci and bacilli often grow in doublets (called diplococci) or chains (streptococci). Cocci that grow in clusters are called staphylococci.

• 4. Some bacterial species are pleomorphic.

• 5. Antibiotics that affect cell wall biosynthesis (e.g., penicillin) may alter a bacteria's shape.

The Bacterial Cell: An Overview

• Most prokaryotes share fundamental traits.

• - Thick, complex outer envelope

• - Compact genome

• - Tightly coordinated cell functions

• Modern research shows that the cell’s parts fit together in a structure that is ordered, though flexible.

• Cytoplasm = Consists of a gel-like network

• Cell membrane = Encloses the cytoplasm

• Cell wall = Covers the cell membrane

• Nucleoid = Non-membrane-bound area of

the cytoplasm that contains the chromosome in the form of looped coils

• Flagellum = External helical filament whose rotary motor propels the cell

Biochemical Composition of Bacteria

•All cells share common chemical components

• Water

• Essenial ions

• Small organic molecules

• Macromolecules

Isolating Parts of Cells

• Cells must be broken up by techniques that allow subcellular parts to remain intact.

• Examples of such techniques include:

• - Mild detergent analysis

• - Sonication

• - Enzymes

• - Mechanical disruption

The Cell Wall

• The cell wall confers shape and rigidity to the cell, and helps it withstand turgor pressure.

• The bacterial cell wall, or the sacculus, consists of a single interlinked molecule.

Peptidoglycan Structure

• Most bacterial cell walls are made up of peptidoglycan (or murein).

• The molecule consists of:

• - Long polymers of two disaccharides

called N-acetylglucosamine [NAG] and N-acetylmuramic acid [NAM], bound to a peptide of 4-6 amino acids

• - The peptides can form cross-bridges connecting the parallel glycan strands.

• Peptidoglycan is the site of action of some antibiotics such as penicillin and the cephalosporins (active against Gram+ and

Gram- species and are often employed for the treatment of patients that are allergic to penicillins).

• In Gram-positive bacteria, it comprises up to 50% of the cell wall. In Gram-negative bacteria, it comprises only 2% to 10% of the cell wall.

Gram-positive and negative Bacteria

• Most bacteria have additional envelope layers that

provide structural support and protection.

• Envelope composition defines:

• Gram-positive bacteria (thick cell wall)

• - Example: Staphylococcus (G+ cocci)

• Capsule (not all species)

  - - Made of polysaccharides

• S-Layer (not all species)

  - - Made of protein

• Thick cell wall —

  - - Amino acid cross-links in peptidoglycan

  - - Teichoic acids for strength

• Plasma membrane-

• Grame-negative bacteria (thin cell wall)

• - Example: Pseudomonas (G- rod)

• The thin peptidoglycan layer consists of one or two sheets.

• Covered by an outer membrane, which confers defensive abilities and toxigenic properties on many pathogens

Teichoic acid (Gram +)

• Teichoic and teichuronic acids are water-soluble polymers, containing a ribitol or glycerol residue linked by phosphodiester bonds.

• They are found in Gram-positive cell walls or membranes.

• Teichoic acid is found in cell walls and is chemically bonded to peptidoglycan.

• Lipoteichoic acid is found in cell membranes and is chemically bonded to membrane glycolipid.

• Functions

• Contain important bacterial surface antigenic determinants, and lipoteichoic acid helps anchor the wall to the membrane.

• May account for half of the dry weight of a Gram-positive cell wall.

Periplasmic space

• The periplasmic space is found in Gram-negative cells.

• a. The periplasmic space refers to the

area between the outer membrane and

cell membrane.

• b. The periplasmic space contains Hydrated peptidoglycan, hydrolytic enzymes including β-lactamases, specific carrier molecules, and oligosaccharides.

The Cell Membrane

• The structure that defines the existence of a cell is the cell membrane.

• The cell membrane acts as a semipermeable barrier.

• Selective transport is essential for survival.

• - Small uncharged molecules, such as O2

and CO2, easily permeate the membrane by

diffusion.

• - Water tends to diffuse across the membrane in a process called osmosis.

Membranes also include planar molecules that fill gaps between hydrocarbon chains.

• In eukaryotic membranes, the reinforcing agents are sterols, such as cholesterol.

• In bacteria, the same function is filled by hopanoids, or hopanes.

Outer membrane (OM)

• An outer membrane is found in Gram-negative cells.

• The outer membrane is a phospholipid bilayer in which the phospholipids of the outer portion are replaced by lipopolysaccharides. The OM contains:

• 1. Embedded proteins, including matrix porins (nonspecific pores)

• 2. Some nonpore proteins (phospholipases and proteases)

• 3. Transport proteins for small molecules

The functions of the OM;

• 1. Protects cells from harmful enzymes and some antibiotics

• 2. Prevents leakage of periplasmic proteins.

LPS

• Lipopolysaccharide is found in the outer fold of the OM of Gram-negative cells.

• Structure

• 1. Lipopolysaccharide consists of lipid A, several long-chain fatty acids attached to phosphorylated glucosamine disaccharide units, and a polysaccharide composed of a core and terminal repeating units.

• 2. It is -ve charged and noncovalently cross-bridged by divalent cations.

• Functions

• 1. LPS is also termed endotoxin; the toxicity of LPS is associated with the lipid A.

• 2. Contains major surface antigenic determinants, including O antigen found in the polysaccharide component.

Nucleus

In bacteria, the nucleus is often termed a

nucleoid or nuclear body.

• 1. The bacterial nucleus is not surrounded by a nuclear membrane.

• 2. Composition: The nucleus consists of

polyamine and magnesium ions which are

bound to -ve charged, circular, supercoiled,

double-stranded DNA; small amounts of

RNA; RNA polymerase; and other proteins

Cytoplasm & Ribosomes

• Bacterial cytoplasm contains ribosomes and assorted nutritional storage granules.

• Bacterial cytoplasm contains NO organelles unlike the cytoplasm of eukaryotic cells.

• Bacterial ribosomes contain proteins and RNAs that are different from their eukaryotic counterparts.

• Ribosomes involved in protein biosynthesis are membrane bound.

• Many antibiotic drugs target ribosomes, inhibiting protein biosynthesis.

Pili

• Pili (fimbriae) are rigid surface appendages composed mainly of a protein called pilin.

• Ordinary pili (adhesins) are involved in bacterial adherence and Gram-positive cell conjugation.

• Sex pili are involved in attachment of donor and recipient bacteria in Gram-negative cell conjugation.

• Ordinary pili are the colonization antigens or virulence factors associated with some bacterial species.

• They also may confer antiphagocytic properties.

Cell Division

• Cell division, or cell fission, requires highly coordinated growth and expansion of all the cell’s parts.

• Unlike eukaryotes, prokaryotes synthesize RNA and proteins continually while the cell’s DNA undergoes replication.

• Bacterial DNA replication is coordinated with the cell wall expansion and ultimately the separation of the two daughter cells.

Bacterial growth - General

• a. Bacterial growth refers to an increase in bacterial cell numbers (multiplication), which results from a programmed increase in the biomass of the bacteria.

• b. It results from bacterial reproduction due to binary fission, which may be characterized by a parameter called generation time (the average time required for cell numbers to double).

• c. It may be determined by measuring cell concentration (turbidity measurements or cell counting) or biomass density (dry weight or protein determinations).

• d. It usually occurs asynchronously (i.e., all cells do not divide at precisely the same moment).

Bacterial cultivation

Bacterial cultivation refers to the propagation of bacteria based on their specific pH, gaseous, and temperature preferences.

• b. It is performed in either liquid (broth) or solid (agar) growth medium and requires an environment that

contains:

• 1. A carbon source

• 2. A nitrogen source

• 3. An energy source

• 4. Inorganic salts

• 5. Growth factors

• 6. Electron donors and acceptors

Nutrition

• 1. Heterotrophs require preformed organic

compounds (e.g., sugar, amino acids) for

growth.

• 2. Autotrophs do not require preformed

organic compounds for growth because

they can synthesize them from inorganic

compounds and carbon dioxide.

Growth medium

• Minimal essential growth medium

• This medium contains only the primary precursor compounds essential for growth.

• A bacterium grown in this medium must synthesize most of the organic compounds required for its own growth.

• Generation time is relatively slow.

• Complex growth medium

• This medium contains most of the organic compound building blocks (e.g., sugars, amino acids, nucleotides) necessary for growth.

• Generation time for a bacterium in complex medium is faster relative to its generation time in minimal essential medium.

• Fastidious bacteria are grown in this medium.

• Differential growth medium

• This medium contains a combination of nutrients and pH indicators to allow visual distinction of bacteria that grow on or in it.

• Colonies of particular bacterial species have a distinctive color.

• Selective growth medium

• This medium contains compounds that prevent the growth of some bacteria while allowing the growth of other bacteria.

• Dyes or sugars, high salt concentration, or pH are used to achieve selectivity.

• Many prokaryotes cannot be cultured in a laboratory.

• Bacterial and archaeal cells reproduce asexually.

Microbial Growth

A bacterial growth curve illustrates the dynamics of growth is seen in 4 phases:

1. During the lag phase, no cell division occurs while bacteria adapt to their new environment.

2. Exponential growth of the population occurs during the logarithmic (log) phase, Human disease symptoms usually develop during the log phase.

3. When reproductive and death rates equalize, the

population enters the stationary phase.

4. The accumulation of waste products and scarcity of resources causes the population to enter the decline (exponential death) phase.

• Endospores are a response to nutrient limitation.

• Endospores are a highly resistant structure formed by species of Bacillus and Clostridium when nutrient supplies are low.

• A stressed cell undergoes asymmetrical cell division, creating a small prespore and larger mother cell.

• The prespore contains:

Cytoplasm, DNA and dipicolinic acid, which stabilizes proteins and DNA.

• The mother cell matures the prespore into an endospore, then disintegrates, freeing the spore.

• Endospores: are resistant to desiccation and heat.

• When environmental conditions are again favorable, protective layers break down and the spore germinates into a vegetative cell.

Microscopy

• Most microbial agents are in the micrometer size range.

– Most bacterial and archaeal cells are 1–5 micrometers (µm) in length.

• Staining techniques provide contrast.

– The simple stain technique involves flooding a prepared specimen with basic dye.

– The negative stain technique uses acidic dye, which is repelled by cell walls, leaving clear cells on a dark background.

– In the Gram stain technique, cells are stained with crystal violet and Gram’s iodine solution and washed with a decolorizer.

– Safranin is applied as a counter stain.

– Gram-positive bacteria retain the crystal violet, whereas gram-negative bacteria do not.

• Optimal prokaryotic growth is dependent on several physical factors.

• Temperature

• Each prokaryotic species has an optimal temperature for growth and about a 30°C range of acceptable temperatures.

• Psychrophiles grow optimally below 15°C and make up the largest portion of all prokaryotes on Earth.

• Mesophiles thrive at the medium temperature range of 10° to 45°C, including pathogens that thrive in the human body.

• Thermophiles multiply best around 60°C, living in compost heaps and hot springs.

• Hyperthermophiles are Archaea that grow optimally above 80°C, found in seafloor hot-water vents.

• Oxygen

• Many prokaryotes are obligate aerobes, which require oxygen to grow.

• Anaerobes do not or cannot use oxygen; aerotolerant species are insensitive to oxygen, but obligate anaerobes are inhibited or killed by oxygen.

• Facultative prokaryotes grow either with oxygen or in reduced oxygen environments.

• Population measurements are made using pure cultures.

• A pure culture is a population consisting of only one species of prokaryote.

• The pour-plate isolation method allows separation of species through dilution of a sample

• The streak-plate isolation method spreads out individual cells to form discrete colonies of species.

• Population growth can be measured in several ways.

• Turbidity (cloudiness) of a sample can be measured with a spectrophotometer.

• One can perform a direct microscopic count.

• Number of cells can be estimated through the most probable number test.

• In the standard plate count procedure, colonies grow on a plate containing a sample of diluted broth.

• The number of colonies indicates the original number of viable (living) cells in the broth.

Infection control: Terminology

• 1. Sterility—total absence of viable microorganisms as assessed by no growth on any medium

• 2. Bactericidal—kills bacteria

• 3. Bacteriostatic—inhibits growth of bacteria

• 4. Sterilization—removal or killing of all microorganisms

• 5. Disinfection—removal or killing of disease-causing microorganisms

• 6. Sepsis—infection

• 7. Aseptic—without infection

• 8. Antisepsis—any procedure that inhibits the growth and multiplication of microorganisms