Bacterial Spores & Sporulation: Case Study

• The module is Foundations of Infections & Infestations BMS171, for level 1, semester 2

Case Scenario

• A 33-year-old male driver involved in an accident sustained a severe wound to his left leg, which was contaminated.
• After a few days, the wound turned black with an offensive discharge.
• A Gram stain of the colonies grown on blood agar revealed Gram-positive spore-forming bacilli.
• Possible organisms and the steps of sporulation should be enumerated for this case

Bacterial Spores

• Sporulation occurs outside the body and is not a form of reproduction; one cell produces one spore that germinates into one cell.
• Examples of spore-forming organisms are:
  • Clostridium group
  • Bacillus group
• During sporulation, the nuclear material moves to one spot and is later surrounded by a thick spore membrane.
• Spores can be oval or rounded and may be the same thickness or bulge outside.
• Spores position may be:
  • Central
  • Subterminal
  • Terminal

Sporulation

• The sporulation process begins when nutritional conditions become unfavorable.
• The nuclear material moves to one spot in the cell and becomes surrounded by a thick spore membrane or coat consisting of peptidoglycan and a cytoplasmic membrane.
• The water content in the spore is decreased, which helps it resist heat.
• Diaminopimilic acid precipitates in the spore coat, forming a rigid resistant layer.
• The remaining bacterial cell undergoes autolysis.
• The original cell's metabolism stops and the bacterial DNA is replicated.
• The cytoplasmic membrane pinches, making a forespore.
• Vegetative bacterial DNA starts disintegrating as the cytoplasmic membrane engulfs the forespore in a second membrane.
• A cortex is deposited, containing calcium and dipicolinic acid.
• The spore coat forms around the endospore.
• The endospore matures, and the original cell lyses, releasing the endospore.

Germination

• Once formed, the spore has no metabolic activity and can remain dormant for many years.
• Upon exposure to water and appropriate nutrients:
  • Specific enzymes degrade the coat.
  • Water and nutrients enter the spore.
  • Germination occurs, leading to the formation of a metabolizing and reproducing bacterial cell.

Ribosomes

• Primarily composed of RNA and protein.
• In bacteria, ribosomes are formed from two subunits: a large 50S subunit and a small 30S subunit, making the entire ribosome a 70S subunit.
• Ribosomes are the site of translation, where messenger RNA (mRNA) is translated into a polypeptide chain.

Bacterial Nutrition

Basic Elements:

• Major elements include carbon, nitrogen, and water.
• Minor elements include phosphorus, sulfur, magnesium, calcium, and potassium.

Essential Metabolites and Growth Factors:

• Required in very small amounts for structure or as catalysts for growth, such as nucleotides and vitamins.

Gaseous Requirements for Bacteria

Oxygen:

• Obligate aerobes require the presence of oxygen to live.
• Facultative anaerobes can live in the presence or absence of oxygen and are the most pathogenic bacteria.
• Obligate anaerobes live only in the absence of oxygen due to a lack of peroxidase or catalase enzymes; oxygen combines with water to form toxic peroxides.
• Microaerophilic organisms thrive in minimal amounts of O2.

Carbon Dioxide

• CO2 is an essential element for the growth of most bacteria.
• Ordinary atmospheric CO2 levels are usually sufficient, but some organisms require higher concentrations (5-10%), which must be provided in the culture media.

Temperature

• Each organism has an ideal temperature for optimal growth.
• Most pathogenic bacteria grow best at 37°C.
• The temperature range (usually between 10-42°C for most pathogenic bacteria) falls between the minimal and maximal temperatures.
• Growth below the minimum temperature is called psychophilic, while growth above the maximum temperature is called thermophilic.

Hydrogen Ion Concentration

• Most pathogenic bacteria grow in a narrow pH range, with an optimum of 7.5.
• Some species, like Vibrio cholerae, tolerate alkaline media and are called alkalophilic.
• Acidophilic species, like Lactobacillus, tolerate acidic media.

Bacterial Products:

• Bacterial enzymes.
• Bacterial pigments.
• Bacterial toxins.
• Other important products include hemolysin and leucocidins.

Bacterial Enzymes

• Enzymes act on:
  • Proteins, called proteolytic enzymes
  • Carbohydrates (CHO), called saccharolytic enzymes
  • Lipids, called lipolytic enzymes.
• Bacteria also have respiratory enzymes such as dehydrogenases and oxidases.

Bacterial Pigments

Endopigment

• Remains bound to the bacteria.
• Staph. aureus produce golden yellow.

Exopigment

• Diffuses in the surrounding media.
• Pseudomonas aeruginosa produces:
  • Blue pigment called pyocyanin.
  • Yellow pigment called fluorescens.

Functions of Pigments

• Role in bacterial respiration and antibacterial action.

Bacterial Toxins

• Two types of bacterial toxins occur: Endotoxins and Exotoxins.

Endotoxins

• Remain bound to the body of the organism and are released only when the organism disintegrates.

Exotoxins

• These are diffusible, which diffuse into the surrounding medium and can be prepared
  • by growing the organism in fluid media
• then filtering the culture through a bacterial filter; the filtrate constitutes the toxin.

Exotoxin vs Endotoxin key differences

Bacterial Reproduction

• Bacteria multiply by simple binary fission, which involves:
  • Growth in size (elongation) of the bacterium, also known as the parent cell.
  • Division of the nuclear bodies, which begins with DNA replication.
  • Constriction originating from the cell wall inwards.
  • The bacterium then divides into two daughter cells.

Growth Curve

• Bacterial growth in a closed batch culture typically follows a growth curve consisting of four phases:
  • Lag phase
  • Log phase
  • Stationary phase
  • Phase of decline

The Lag Phase

• No multiplication occurs; hence, the growth rate is zero during the lag phase.
• Bacteria increase in size and prepare themselves for reproduction and division.
• This stage can vary from a few hours up to a few days.
• Correlates with the incubation period of disease in vivo.

Factors Affecting the Lag Phase Include:

• The nature of the organism
  • E. coli has a lag phase of fewer than approximately 60 minutes.
  • M. tuberculosis has a lag phase lasting a few days
• The size of the inoculum
  • A larger inoculum results in a shorter lag phase
• The stage from which the inoculum is taken.
  • If taken from the log phase, the lag phase will be shorter
• The more enriched the growth medium
  • The shorter the lag phase

Logarithmic Phase

• Follows the lag phase; cell division occurs at a maximum rate - cell numbers increase exponentially.
• The rate of division depends on the nature of the organism and environmental conditions
• This phase continues until nutrients in the medium become exhausted, or toxic metabolic products accumulate and inhibit growth.
• This phase transitions into the stationary phase.
• Correlates with the invasive period in vivo, and this is the best time antibiotics are most effective.

Stationary Phase

• The rate of division equals the rate of death so the quantity of live organisms remains constant.
• Factors for diminishing the rate of growth include
  • Exhaustion of nutrients and O2 starvation.
  • Accumulation of toxic materials.
• Correlates with symptoms and signs in vivo

Phase of Decline

• The number of organisms begins to decrease, and the rate of death exceeds the rate of growth.
• Correlates with the convalescent period in vivo, so antibiotics must be continued to completely eradicate the bacteria to avoid the development of antibiotic resistance.
• The increasing bacterial death depend on:
  • Nature of the organism.
  • Effect of temperature.
  • Effect of time.
  • Effect of pH.
  • Accumulation of toxic metabolites.