Lecture 6

Medically Significant Bacteria

• Mycobacterium tuberculosis affects ~10 million people worldwide and causes ~1.6 million deaths
• Clostridium perfringens causes ~1 million infections per year in the USA.
• Escherichia coli causes yearly outbreaks food supply.
• Cutibacterium acnes has an acne treatment market of $7 billion.
• Helicobacter pylori is the strongest risk factor for gastric cancer and the second leading cause of cancer-related deaths worldwide.

Introduction to Microbial Growth

• Microbes "growing" means an increasing number of them.
• Microbes accumulate into colonies, which are groups of cells large enough to be seen without a microscope.
• Microbial populations can become incredibly large rapidly.
• Understanding microbial growth helps control and encourage it.

Requirements for Microbial Growth

• Physical requirements include temperature, pH, and osmotic pressure.
• Chemical requirements include carbon, nitrogen, sulfur, phosphorus, trace elements, and oxygen.

Physical Requirements: Temperature

• Most bacteria grow within a limited temperature range.
• Psychrophiles are cold-loving and grow best between 0-20°C.
• Psychrotrophs are also cold-loving and grow best between 0-30°C, which can lead to low-temperature food spoilage.
• Mesophiles are moderate-temperature-loving and grow best between 12-45°C; this is the range of normal microbiota.
  • The optimum temperature for many pathogenic bacteria is 37°C.
• Thermophiles are heat-loving and grow best between 40-80°C.
• Hyperthermophiles are heat-loving and grow best between 65-113°C.

Range of Temperature

• Minimum growth temperature is the lowest temperature at which a species can grow.
• Optimum growth temperature is the temperature at which a species reproduces fastest.
• Maximum growth temperature is the highest temperature at which a species can grow.
• Optimum temperature is affected by enzymatic systems of the cell.

Temperatures for Food Safety

• High temperatures destroy microbes, even though lower temperatures take more time.
• Very slow bacterial growth occurs at certain temperatures.
• Rapid bacterial growth may occur at some temperatures and produce toxins.
• Many bacteria can survive, and some may grow, but refrigerator temperatures allow slow growth of spoilage bacteria and very few pathogens.
• No significant growth occurs below freezing.

Physical Requirements: pH

• Bacteria grow best between pH 6.5 and 7.5.
• Molds/yeasts have a wider pH range than bacteria, typically between pH 5 and 6.
• Very few bacteria can grow at an acidic pH of <4, making this useful for food preservation, such as pickling.
• Growth media can include buffers to minimize pH changes.
• Acidophiles can grow in highly acidic environments, such as bacteria growing at pH 1 in coal mine drainage water.

Physical Requirements: Osmotic Pressure

• In hypotonic environments, solute concentration is lower in the environment than in the cytoplasm.
  • Cells leak solutes to release osmotic stress, with the cell wall helps maintain a cell shape.
• In hypertonic environments, the solute concentration is higher in the environment than in the cytoplasm.
  • Water flows out of the cell, which is known as plasmolysis.
• Extreme/obligate halophiles require high salt concentrations, such as up to 30% NaCl.

Chemical Requirements: Nitrogen

• Nitrogen is a component of proteins, DNA, and ATP.
• Nitrogen is primarily used to form amino groups in amino acids.
• Bacteria decompose protein-containing material for a nitrogen source.
• Some bacteria use nitrogen fixation to directly use gaseous nitrogen (N2) from the atmosphere.

Chemical Requirements: Carbon, Sulfur, Phosphorus & Trace Elements

• Carbon is the structural backbone of living matter
• Carbon is needed for the organic compounds that make up a living cell.
• Sulfur is used in amino acids and vitamins.
• Most bacteria decompose protein for the sulfur source.
• Phosphorus is essential for synthesizing DNA, RNA, and ATP.
• Phosphorus is also found in membranes (phospholipids).
• Chemical elements required in small amounts are trace elements; these present in tap water.

Bacterial Oxygen Preferences

• Obligate aerobes require high O2 for growth, such as M. tuberculosis.
• Obligate anaerobes cannot tolerate oxygen, for example, C. perfringens.
• Facultative anaerobes can grow with or without oxygen, like E. coli.
• Aerotolerant anaerobes tolerate oxygen but do not use it, such as C. acnes.
• Microaerophiles require low concentrations of oxygen, exemplified by H. pylori.

Oxygen: A Double-Edged Sword

• Oxygen is beneficial as a terminal electron acceptor during respiration.
• Aerobic respiration can generate reactive oxygen species (ROS) that are toxic to cells without detoxifying enzymes.
• Organisms that can tolerate oxygen can detoxify harmful forms of oxygen.
• Superoxide dismutase (SOD) catalyzes the reaction: O2- + O2- + 2H+ → H2O2 + O2
• Catalase converts hydrogen peroxide to water and oxygen: 2H₂O₂ → 2H₂O + O₂
• Peroxidase converts hydrogen peroxide and hydrogen to water: H₂O₂ + 2H+ → 2H₂O

Clostridium perfringens

• Clostridium perfringens is a gram-positive, endospore-forming anaerobe found in soil, raw meat, and animal intestines.
• Clostridium perfringens is responsible for ~1 million food poisoning cases in the USA.
• This causes gas gangrene (clostridial myonecrosis), which is a rare but fatal infection leading to renal failure/sepsis if not treated right away.
• 20-30% of treated cases are fatal.
• Diabetic patients w/ decreased blood flow to the feet and poor circulation have weakened skin, leading to foot ulcers/impaired wound healing.
• Removal of damaged tissue and debris (debridement) from the wound is necessary.
• Antibiotic treatment is also needed.
• Amputation is needed in 1 of every 5 people with gas gangrene to prevent further damage.

Biofilms

• Bacteria live in communities in a thin, slimy layer encasing the bacteria.
• Coordinated functional microbial communities adhere to surfaces.
  • Bacteria communicate cell-to-cell by quorum sensing.
• Bacteria secrete an inducer (signaling chemical) to attract other bacteria.
• Biofilms can be single or multiple species.
• Biofilms shelter bacteria from harmful environmental factors and share nutrients.
• Process of forming pillars:
  • It begins with free-swimming (planktonic) bacteria that secrete an inducer via quorum sensing and form a protective extracellular matrix.

Biofilms in Human Health

• Biofilms are more resistant to microbicides.
• Biofilms prevent antibiotics from penetrating.
• Biofilms are involved in 70% of human bacterial infections.
• These can occur on catheters, heart valves, contact lenses, and teeth.

Culture Media

• Culture medium is a nutrient material prepared for microorganism growth in a laboratory.
• Inoculum is microorganisms introduced into culture to initiate growth.
• Culture is the microorganisms growing in/on a culture medium.
• Criteria for culture medium: contain correct nutrients, proper pH, suitable oxygen level, and proper temperature.
  • Must also be sterile, meaning that the medium only contains the desired microorganism and its offspring

Culture Media: Solid

• Agar can be used as a solidifying agent and has the properties of a complex polysaccharide-based thickener.
• Agar is useful for culture media in Petri plates, slants, and deeps.
• Agar is generally not metabolized nor degraded by microorganisms.
  • Agar is liquified at around 100°C but solidifies at around 40°C.

Types of Culture Media

• Chemically defined media has an exact chemical composition.
  • Fastidious organisms require many growth factors provided in their growth media.
• Complex media consists of extracts and digests of yeasts, meat, or plants; chemical composition varies from batch to batch.
  • Nutrient broth and nutrient agar are examples of complex media.

Anaerobic Growth Media and Methods

• Reducing media is used for cultivating anaerobic bacteria killed by exposure to oxygen.
  • They contain chemicals that combine with oxygen to deplete it.
• Methods for growing anaerobes:

1. Specialized anaerobic jars
   • These use a packet that enables hydrogen and atmospheric oxygen to combine and form water, removing oxygen
   • Anaerobic indicator turns blue when oxidized.
2. Anaerobic chambers
   • Oxygen-free chambers

Special Culture Techniques

• Some bacteria cannot be grown on artificial laboratory media and are obligate intracellular organisms.
  • For example, Mycobacterium leprae is grown in armadillos, and Chlamydia trachomatis is grown in tissue culture.

Selective and Differential Media

• Selective media suppresses unwanted microbes and encourages desired microbes.
  • Bismuth sulfite agar inhibits gram-positive and most gram-negative bacteria but encourages Salmonella Typhi.
• Differential media allows distinguishing of colonies of different microbes on the same plate.
  • Blood agar identifies Streptococcus pyogenes, which lyses red blood cells.

Selective and Differential Media: Mannitol Salt Agar

• Staphylococcus aureus has high salt tolerance, and uses mannitol for energy generation, resulting in acid and byproduct formation.
• Some media have both selective and differential characteristics.
• For example a medium high in salt, and with a pH indicator

Obtaining Pure Cultures

• A pure culture contains only one species or strain.
• A colony is a population of cells all arising from a single cell or spore or from a group of attached cells.
  • A colony often called a colony-forming unit (CFU)
• The streak plate method is used isolate pure cultures

Bacterial Division

• Bacterial growth is represented as an increase in the number of cells.
• Binary fission is a symmetrical process and the major means of growth in prokaryotes.
  • One (mother) cell divides to form two genetically equivalent progeny cells of equal size.
• Budding: (Asymmetrical): parent cell retains its identity while the bud increases in size until it separates as a complete new cell like Caulobacter does

Bacterial Growth Curve

• Characteristics:
  • A few bacteria are inoculated into a liquid growth medium
  • The population of bacteria is counted at regular intervals
  • The Log10 of the number of bacteria (y-axis) is plotted against time (x-axis)
• Growth phases:
  • Lag phase: little to no increase in number but intense metabolic activity
  • Log phase: cells increase rapidly in number
  • Stationary phase: cell growth slows, diminished nutrients, accumulation of waste occurs
• In stationary phase bacteria approach the carrying capacity
  • Death phase: cell deaths exceed the amount of new cells

Generation Time

• Cell number doubles with each binary fission.
• Generation (aka doubling) time (td) is the time it takes for a number of cells to double
• Most bacteria doubling times are 1-3 hours but can take >24 hours Ta can vary according to nutrients, growth conditions etc.
• Initial number of cells x 2 # of generations = number of cells

Direct Measurement: Viable Plate Count

• Viable cell counts measure only living bacteria (cells that can reproduce).
• Serial dilutions are made from a liquid culture sample; after, each dilution spread on a solid medium.
• Count the number of colonies after incubation.
• The # of reproducing cells in the original sample is reflected in the # of colonies.

Direct Measurement: Microscope Count

A volume of a cell suspension placed on a slide is used in conjunction with:

• Calculation of average number of bacteria per viewing field
• A Petroff-Hausser cell counter

Indirect Methods: Turbidity

• Turbidity: measurment of cloudiness w/ a spectrophotometer
• The amount of metabolic product = proportional to number of bacteria
• As bacteria #’s increases: Less light reaches detector
• As bacteria #’s increases: Absorbance is higher (optical density- OD)
• This method does not differentiate between live and dead cells.