Week 9: Bacterial Intolerance Notes

Bacterial Intolerance

Microorganisms exhibit varying capabilities in utilizing oxygen for cellular respiration.
Respiration involves the oxidation of substrates to generate energy essential for life.

Oxidation and Reduction (Redox) Reactions

Oxidation: The loss of a hydrogen ion and an electron by a substance.
Reduction: The gain of a hydrogen ion and an electron by a substance.
Redox Reactions: Paired oxidation and reduction reactions.

Oxidation Reduction Potential (ORP)

Some microorganisms possess enzyme systems that use oxygen as an electron acceptor, reducing it to water.
These cells have high redox potentials, known as Oxidation Reduction Potential (ORP), measured in millivolts (mV) in aquatic systems.
A more negative ORP value indicates a more reduced system.
High ORP values indicate abundant oxygen in the water, enhancing the efficiency of bacteria that decompose dead tissue and contaminants.

Oxygen Requirements of Bacteria

To determine a bacterium's oxygen requirement (aerobic, obligate anaerobe, aerotolerant, microaerophile, or facultative anaerobe), an oxygen gradient is established in thioglycollate tubes.
Culturing microaerophiles or obligate anaerobes requires specialized growth conditions and apparatus, such as a Candle Jar or a GasPak system commonly used in research and clinical labs.

Terminology

Aerotolerance: The ability of microorganisms to grow in the presence or absence of oxygen.
Cellular Respiration: A process involving oxidation (loss of electrons/hydrogen ions) and reduction (gain of electrons/hydrogen ions), known as redox reactions.
Oxidation Reduction Potential (ORP): Indicates oxygen availability in an environment.
- High ORP (positive): Oxygen-rich, supports aerobic bacteria.
- Low ORP (negative): Oxygen-poor, supports anaerobic bacteria.
Obligate Aerobes: Organisms requiring oxygen to grow, utilizing cellular respiration to metabolize substances (e.g., sugars or fats) for energy.
Obligate Anaerobes: Microorganisms killed by normal atmospheric concentrations of oxygen, metabolizing energy by anaerobic respiration or fermentation.
Facultative Anaerobes: Organisms that make ATP (energy) by aerobic respiration if oxygen is present, but switch to fermentation or anaerobic respiration if oxygen is absent.
Aerotolerant Anaerobes: Organisms that use fermentation to produce ATP (energy), do not utilize oxygen, but can protect themselves from reactive oxygen molecules.
Microaerophiles: Microorganisms requiring oxygen to survive, but at lower levels than in the atmosphere.
Capnophiles: Microorganisms that thrive in the presence of high concentrations of carbon dioxide.

Biofilms

Biofilm: A community of microorganisms where cells adhere to each other and often to a surface, embedded within a slimy extracellular matrix on moist or wet surfaces. Biofilms exhibit increased resistance to antibiotics and disinfectants.

Dental Caries

Dental Caries (Cavities): Breakdown of teeth due to acids produced by bacteria.

Anaerobic Habitats

Anaerobic habitats are widespread, including San Francisco Bay mud flats, cow guts, deep-sea hydrothermal vents, and the human gut and mouth.
The human mouth's plaque hosts a diverse "bacterial zoo."
Facultative anaerobes consume $O_2$ , creating anaerobic microenvironments suitable for obligate anaerobes.
Wherever organic matter accumulates, microbes consume $O2$ faster than it can be replaced, particularly underwater where $O2$ is poorly soluble.
Lakes/ponds stratify into aerobic (upper) and anaerobic (lower) zones in summer due to microbial growth on sediments.
Some bacteria such as streptococcus, Haemophilus require extra $CO_2$ .

Biofilms (Detailed)

Biofilms are complex aggregates of interacting microbial cells adhering to each other and surfaces via a polysaccharide matrix.
They facilitate communication among participants (quorum sensing), promoting survival and adaptation.
Bacterial biofilms in the mouth lead to dental caries.

Pellicle

Pellicle: A protein film in the mouth.

Microbial Flora of the Mouth

Niches for bacteria: teeth, tongue, pockets under gums.
Protection: Saliva washes bacteria away, lysozyme kills bacteria.
Over 1000 microbial species have been found in the human mouth, with millions of bacteria per mL of saliva.
Some are transient (from food), others are resident.
Niches for residents: attached to teeth, attached to tongue, under the gums.
Humans produce 1L of saliva a day, requiring microbes to adhere tightly.

Dental Caries (Specific Bacteria)

Key bacteria involved:
- Lactobacillus acidophilus
- Streptococcus mutans
These bacteria produce organic acids, especially lactic acid, by fermenting carbohydrates on tooth surfaces.
Continued presence of lactic acid causes decalcification and softening of dental enamel, resulting in tiny perforations (dental caries).

Sucrose and Cavities

Sucrose increases the risk of cavities:
- Sucrose + Strep mutans → Glucan (glycocalyx) → Biofilm Formation
- Sucrose + Strep mutans → Fructose + (S. Mutans, L. acidophilus, A.odontolyticus) → Lactic Acid (erodes enamel)
Saliva forms a coating for Strep attachment.
Strep produces fructose and glucan from sucrose (refined sugar).
Other bacteria stick to glucan, forming a community = plaque = biofilm.
Fructose production leads to extensive fermentation leading to acid production eroding enamel!
Streptococcus mutans: A facultatively anaerobic, gram-positive coccus, commonly found in the oral cavity and a significant contributor to tooth decay.

Snyder’s Test

Snyder's test is used to detect susceptibility to cavity formation.

Thioglycollate Tubes

Fluid Thioglycollate Medium is used to culture anaerobic organisms and determine their oxygen requirements.
The medium, a thick broth with a small amount of agar, has dissolved oxygen expelled during autoclaving.
During cooling, oxygen diffuses from top to bottom, creating an oxygen gradient.
Resazurin or methylene blue indicates oxygen location, turning pink where oxygen is present.
The medium is inoculated with a vertical stab, ensuring initial presence throughout.
Sodium thioglycollate binds oxygen (oxygen scavenger), acting as a reducing compound.
After incubation, growth position indicates the bacterium's oxygen requirements.
The media allows differentiation of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms.
Obligate anaerobic Clostridium species grow only at the bottom where no oxygen exists.

Anaerobic Habitats in the Lab

Thioglycollate broth: Identifies oxygen requirement of bacteria, medium consumes $O2$ so anaerobes can grow, and $O2$ diffuses in from the top, creating a range of $O_2$ availability
Examples:
- E. coli = facultative anaerobe
- Clostridium sporogenes = obligate anaerobe
- Staphylococcus aureus = facultative anaerobe
- Streptococcus pyogenes = microaerophile
- Pseudomonas aeruginosa = obligate aerobe

Candle Jar and Gas Pak

Atmospheric composition: 78% nitrogen, 21% oxygen, 1% trace gases.
Early attempts to isolate anaerobic organisms involved placing inoculated Petri dishes in a jar with a lit candle.
Sealing the jar limited oxygen, and the candle consumed oxygen until it extinguished, leaving an atmosphere of nitrogen, carbon dioxide, water vapor, and oxygen, creating a microaerophilic environment.
When bacteria are grown in the laboratory, their oxygen requirements need to be considered to accommodate the requirements of the specific bacteria.
An excellent way to determine the oxygen needs of your bacterium is to grow it in different oxygen environments and compare the quality and quantity of growth. We will be using three different environments to determine the oxygen needs of our organisms.

Oxygen Requirement of Microbes

Higher organisms require $O_2$ ; microbes vary in their oxygen needs.
Obligate aerobes depend on atmospheric $O2$ for growth, use $O2$ as TEA (e.g., Neisseria, Pseudomonas).
Facultative anaerobes do not require $O2$ for growth, but grow better with it, have two enzyme systems- one for with $O2$ , one for without, most pathogens and most “lab” bacteria are facultative (e.g., E. coli).
Aerotolerant anaerobes ignore $O_2$ and grow equally well whether it is present or not (e.g., Enterococcus faecalis).
Obligate (strict) anaerobes do not tolerate $O2$ and die in its presence, use anaerobic respiration (use sulfate-sulfate reducers $(SO4)$ , nitrate-denitrification $(NO_3)$ or sulfur as electron acceptors) or fermentation (e.g., Clostridium, and Bacteroides).
Microaerophiles are damaged by normal atmospheric levels of $O_2$ (20%) but require lower levels (2 to 10%) for growth (e.g., Micrococcus, Campylobacter).

Capnophiles (Detailed)

Prefer higher $CO_2$ (from 0.04% in air to 4% in jar).
Some human pathogens in intestines.

Oxygen Toxicity for Anaerobes

Anaerobic bacteria:
- $O_2$ → Metabolism → Toxic products for enzymes → No detoxifying pathway → Bacterial death
Toxic reaction products of oxygen: superoxide $(O2^-)$ , peroxide $(H2O_2)$ , and hydroxyl radical $(OH^-)$
Aerobic or facultative bacteria:
- $O_2$ → Metabolism → Toxic products → Detoxifying pathway → Nontoxic products
Aerobes have enzymes to detoxify: superoxide dismutase and catalase

Oxygen's Role

Oxygen is reactive (an oxidizing reagent).
Capable of degrading organic molecules.
Can generate toxic by-products.
Strong oxidizing reagents that react indiscriminately with any organic molecule (including DNA, proteins, etc.).
By-products include: superoxide, peroxide, and hydroxyl radical
Aerobes must have enzymes to eliminate these radicals: superoxide dismutase, and catalase

Candle Jar (Detailed)

Contains 8-10% $O2$ and 3-5% $CO2$ (normally present in the atmosphere at around 0.3%).
This environment is created by lighting a small candle in the sealed jar. The flame consumes most of the oxygen in the jar and elevates the $CO_2$ levels.
What classification(s) of organisms will grow in the candle jar?

Anaerobic Jar (Detailed)

Contains 0% $O2$ , 10-15% $CO2$ .
We create this oxygen-free environment by sealing the jar with a gas generator packet.
The chemicals in the packet react with all of the $O_2$ in the jar to create water and carbon dioxide within about 2.5 hours of sealing the jar.
An indicator strip in the jar will turn white (instead of blue) when the oxygen in the jar is consumed by the chemical reactions.
What classification(s) of organisms will grow in the anaerobic jar?

Snyder's Test

Purpose: to detect bacteria involved in dental caries (tooth decay).
Mechanism of dental caries formation: bacteria ferment carbohydrates (sugars) present on tooth surfaces, produce lactic acid as a metabolic product, lactic acid causes decalcification (removal of calcium) and softening of dental enamel , leads to tiny holes or cavities.
A variety of microorganisms are known to be involved in the formation of dental caries including Lactobacillus acidophilus, Streptococcus mutans and Actinomyces odontolyticus. These organisms in the oral flora produce organic acids, particularly lactic acid, by fermenting carbohydrates that adhere to the surface of teeth.
In the continued presence of lactic acid, dental enamel undergoes decalcification and softening, which results in the formation of tiny perforations called dental caries.
In order to test for the presence of these bacteria (which produce lactic acid, a media has been developed that measures the amount of acid produced by the action of these bacteria on sugar substrates.
Snyder's media is a differential media that contains glucose and a pH indicator Bromcresol green. Uninoculated media appears green in color at neutral pH, as acids are produced by the bacteria, the pH drops, turning the media yellow in color indicating a positive result.

Procedure

Melt solid Snyders agar that is in a tube in a microwave until it melts
Put molten snyder agar into a $42^oC$ water bath
Into a sterile tube containing 500 ul of sterile water, add 250 ul of saliva (750 ul total)
Remove the molten Snyders agar from the 42oC bath and add the 200 ul of the sample to the molten Snyders agar, swirl solution to mix.
Let the inoculated Snyders agar stand at room temperature until it solidifies
Place Solid inoculated media into a 35oC incubator for 24-72 hrs, and observe culture at 24, 48 and 72 hrs to look for a color change from green to yellow (positive result)

Additional notes

Your Saliva sample
Add 200 μl
Cool it down and Incubate in $37^o$ for 72 h
A melted and cooled to $45^o C$ Snyder agar