Microbiology Lecture 1

Microorganisms and Their Importance

• Microorganisms make up the largest mass of living material on Earth
• The first cells appeared between 3.8 and 3.9 billion years ago, and life was exclusively microbial until ~1 billion years ago
• Microorganisms play a crucial role in cleaning up pollutants (bioremediation)

Macronutrients and Micronutrients

• Macronutrients:
  • Carbon: required by all cells, makes up ~50% of a typical bacterial cell, and is a major element in all classes of macromolecules
  • Nitrogen: makes up ~13% of a typical bacterial cell, and is a key element in proteins, nucleic acids, and other cell constituents
  • Phosphorus: necessary for the synthesis of nucleic acids and phospholipids
  • Sulfur: found in sulfur-containing amino acids, vitamins, and coenzyme A
  • Magnesium: stabilizes ribosomes, membranes, and nucleic acids, and is required for many enzymes
  • Calcium: helps stabilize cell walls in microbes, and plays a key role in heat stability of endospores
• Micronutrients:
  • Trace elements: boron, chromium, cobalt, iron, manganese, nickel, zinc, and molybdenum
  • Growth factors: organic compounds required in small amounts by certain organisms, such as vitamins, amino acids, purines, and pyrimidines

Culture Media

• Selective media: contains compounds that selectively inhibit the growth of some microbes, but not others
• Differential media: contains an indicator that detects particular chemical reactions occurring during growth
• Pure culture: a liquid culture or a plate containing only a single kind of microbe

Microscopy

• Light microscopy:
  • Bright-field: specimens are visualized due to differences in contrast between the specimen and surroundings
  • Positively charged dyes can be used to stain cells and improve their contrast
  • Differential stains: separate bacteria into groups, such as Gram-positive and Gram-negative
• 3D microscopy:
  • DIC (differential interference contrast) microscopy: gives structures a 3D appearance
  • AFM (atomic force microscopy): can image and measure the size of material at the nanoscale
  • CSLM (confocal scanning laser microscopy): applications include thick biofilms and microbial ecology

Microbial Metabolic Diversity

• Chemoorganotrophs: obtain energy from the oxidation of organic molecules
• Chemolithotrophs: obtain energy from the oxidation of inorganic molecules
• Phototrophs: contain pigments that allow them to use light as an energy source
• Autotrophs: use CO2 as their carbon source
• Heterotrophs: require one or more organic molecules for their carbon source
• Extremophiles: inhabit extreme environments, such as boiling hot springs and high-pH environments

Microbial Structure and Function

• Cytoplasmic membrane: embedded proteins, stabilized by hydrogen bonds and hydrophobic interactions
• Cell walls:
  • Gram-positive bacteria: contain up to 90% peptidoglycan
  • Gram-negative bacteria: outer membrane with porins and periplasm
• Cell surface structures:
  • Capsules and slime layers: polysaccharide or protein layers that assist in attachment to surfaces
  • Fimbriae: filamentous protein structures that enable organisms to stick to surfaces
  • Pili: filamentous protein structures that facilitate genetic exchange between cells

Microbial Inclusions

• Polyphosphates: accumulations of inorganic phosphate
• Sulfur globules: composed of elemental sulfur
• Magnetosomes: magnetic storage inclusions
• Gas vesicles: confer buoyancy in planktonic cells
• Endospores: highly differentiated cells resistant to heat, harsh chemicals, and radiation

Locomotion

• Flagellum: structure that assists in swimming and motility
• Gliding motility: flagella-independent motility

Microbial Ecology

• Microbes account for ~50% of all biomass on Earth
• Commensalism: one species benefits, and the other is neither harmed nor helped
• Ammensalism: one species doesn't benefit, but the other is harmed
• Most microbial growth takes place on the surfaces of soil particles
• Geosmin: formed by certain bacteria that gives a characteristic earthy aroma when rain falls after a dry spell of weather

Biofilms

• Assemblages of bacterial cells adhered to a surface and enclosed in an adhesive matrix
• Initiated by attachment of a cell to a surface followed by expression of biofilm-specific genes
• Formation of AHL (acylated homoserine lactones) and matrix formation
• Both intra- and interspecies signaling likely occur in biofilms (quorum sensing)
• Biofilms resist physical forces, phagocytosis, and penetration of toxins
• Allows cells to remain in a favorable niche and provides a consistent source of nutrients

Mutualistic Interactions

• Legume–root nodule symbiosis: mutualistic relationship between leguminous plants and nitrogen-fixing bacteria
• Microbe-insect symbiosis: horizontal and vertical transmission of symbionts
• Epibionts consortia: microbial mutualisms in freshwaters
• Lichens: mutualistic relationship between a fungus and an alga (or cyanobacterium)### Symbiotic Relationships
• Primary symbionts: required for host reproduction, restricted to specialized region bacteriome, and found in bacteriocytes
• Secondary symbionts: not required for host reproduction, not always present in every individual, can invade different cells and live extracellularly, and must provide a benefit such as nutrition, protection from environment stresses, or protection from pathogens

Microbial Interaction with Termites

• Termites decompose cellulose and hemicellulose
• Termite gut consists of foregut, midgut, and hindgut
• Posterior alimentary tract of higher termites (Termitidae) contains a diverse community of anaerobes, including cellulolytic anaerobes
• Lower termites contain anaerobic bacteria and cellulolytic protists

Marine Invertebrates at Hydrothermal Vents and Gas Seeps

• Association is mostly mutualistic
• Bacteria supply organic compounds, while animals supply electron donors for energy metabolism and a safe place to live

Symbiosis with Leeches

• Leeches are parasitic annelids (segmented worms) that feed on vertebrate blood and secrete anticoagulants and vasodilators
• In the digestive tract, the crop has Aeromonas and a Rikenella-like bacterium, while the bladder is packed with Ochrobactrum

Reef Building Corals

• Coral skeleton is a very efficient light-gathering structure
• Large surface area of animal body is well-suited to capture light
• Found in clear tropical waters (low nutrient condition for animals)
• Phototrophic symbionts include cyanobacterium, rhodophytes, chlorophytes, dinoflagellates, and diatoms
• Most ecologically significant symbiosis is between stony coral and the dinoflagellate Symbiodinium
• Bleaching is the loss of color caused by lysis of symbiont, which can be triggered by high temperature and high light, impairing the photosynthetic apparatus of the dinoflagellate

Microbes in Mammalian Gut

• Microbial associations with certain animals led to their ability to catabolize plant fibers
• Plant fibers are composed of insoluble polysaccharides, with cellulose being the most abundant component
• Two digestive plans: foregut fermentation and hindgut fermentation
• Ruminants, such as cows, sheep, and goats, possess a special digestive organ (the rumen) that contains a diverse community of microbes, including cellulolytic microbes

Human Microbiome

• We are more microbes than human, with 10-100 times more bacterial cells than human cells
• Important for proper maturation of the immune system, preventing growth of pathogenic bacteria in the mucosa, reducing predisposition to allergies, lactose, and gluten insensitivities
• Atherosclerosis is associated with the metabolism of L-carnitine by the intestinal microbiota

Bacterial Growth

• Increase in number of cells, rather than size
• Accumulate in colonies on solid medium
• Cell division: each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell
• Binary fission: cell division following enlargement of a cell to twice its minimum size
• Growth: increase in the number of cells
• Generation time: time required for microbial cells to double in number
• Budding division results in a totally new daughter cell

Batch Culture

• A closed-system microbial culture of fixed volume
• Typical growth curve for population of cells grown in a closed system is characterized by four phases: lag, exponential, stationary, and death
• Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval
• Generation time (G) is defined as the time (t) per generation (n = number of generations): G=t/n

Continuous Culture

• Constant working volume, open-system microbial culture
• In bioreactor, affluent and effluent flows have the same volume/hr rate
• Maintain proper pH conditions, and can connect acid and base inputs to regulate pH with a sensor
• Find growth rate first to maintain steady-state, and low dilution rate corresponds to higher growth

Chemostat

• Most common type of continuous culture device
• Both growth rate and population density of culture can be controlled by adjusting the flow of fresh medium/limiting nutrient
• Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out
• Chemostat cultures are sensitive to the dilution rate and limiting nutrient concentration

Chemostat Variations

• Turbitostat: feedback is between the turbidity of the culture vessel and the dilution rate
• Auxostat: measurement taken from growth vessel to control the media flow rate (pH, oxygen tension, metabolite concentration, etc.)
• Retentostat: use biomass, mainly for anaerobic conditions
• Fed-batch: measuring major C or N sources and using that information to feed culture, more manual method

Microbial Growth Measurements

• Microscopic counts: Petroff-Hausser counting chamber with grids
• FACs: flow cytometer using laser-based technology to count and sort cells
• Viable counts: measurement of living, reproducing population
• Spread-plate method: measurement of living, reproducing population
• Turbidimetric method: measurement of microbial growth based on the optical density of the culture

Environmental Factors Affecting Microbial Growth

• Temperature: can grow in many different temperatures, with optimal temperature for growth
• Psychrophile: low temperature (cold lovers), permanently cold environments
• Psychrotolerant: can grow at 0 degrees but optimum is a little higher, adapted due to evolution
• Mesophile: midrange temperature optima, warm-blooded animals, human intestinal and gut microflora
• Thermophile: high temperature (heat lovers), 45-80°C
• Hyperthermophile: very high temperature (above 80°C)

pH

• Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)
• Acidophile: organisms that grow best at low pH
• Pectinase enzymes can digest pectin, a major component of fruit, and are activated at low pH

Osmotic Effects

• Range between 0-1, water activity
• Halotolerant: organisms that can tolerate some reduction in water activity of the environment but generally grow best in the absence of the added solute
• Halophile: organisms that grow best at reduced water potential, with a specific requirement for some NaCl to grow
• Osmophile: organisms that live in environments high in sugar as solute (high osmotic pressure)
• Xerophile: organisms able to grow in very dry environments (less than 0.75 aw)

Oxygen

• Aerobe: requires oxygen to live
• Anaerobe: does not require oxygen and may even be killed by exposure
• Facultative organism: can live with or without oxygen
• Aerotolerant anaerobe: can tolerate oxygen and grow in its presence even though they cannot use it
• Microaerophile: can use oxygen only when it is present at levels reduced from that in air

Microbial Growth Control

• Sterilization: the killing or removal of all viable organisms within a growth medium
• Inhibition: effectively limiting microbial growth
• Decontamination: the treatment of an object to make it safe to handle
• Disinfection: directly targets the removal of all pathogens, not necessarily all microorganisms

Heat Sterilization

• The most widely used method of controlling microbial growth
• Denature macromolecules like enzymes and proteins
• Amount of time required to reduce viability tenfold is called the decimal reduction time

Radiation Sterilization

• Microwaves, UV, and X-rays can reduce microbial growth
• UV is useful for decontamination of surfaces
• Ionizing radiation: electromagnetic radiation that produces ions and other reactive molecules

Filter Sterilization

• Filtration to avoid the use of heat on sensitive liquids and gases
• Pores of filter are too small for organisms to pass through
• HEPA (High efficiency particulate air) filters for biosafety applications

Chemical Antimicrobials

• Bacteriostatic: inhibit growth, but cells can survive if concentration is low
• Bacteriocidal: can kill bacteria, dead cells are not destroyed and can interfere with Optical Density
• Bacteriolytic: kill by lysing bacteria, cytoplasmic contents are released

Chemical Growth Control

• Minimum inhibitory concentration (MIC) is the smallest amount of any chemical agent needed to inhibit growth of a microorganism
• Disc diffusion assay (Kirby-Bauer method): tiny small paper disks, put antibiotics on it, look for areas of inhibition

Antimicrobials in vivo

• Antimicrobial drugs are classified on the basis of mechanism of action, spectrum of antimicrobial activity, molecular structure### Quinolones
• Interfere with DNA gyrase, preventing supercoiling of DNA
• Active against both Gram-positive (G+) and Gram-negative (G-) forms of bacteria
• Examples: Ciprofloxacin, Moxifloxacin used to treat UTI, Anthrax, TB, and Respiratory diseases in animals

Fungal Cell Walls

• Major component of fungal cell walls is chitin, which can be targeted by drugs

Antibiotics

• Naturally produced antimicrobial agents, with less than 1% being clinically useful
• Can be modified to enhance efficacy (semisynthetic antibiotics)
• Susceptibility of microbes to different antibiotics varies greatly
• Broad-spectrum antibiotics are effective against both G+ and G- bacteria (e.g., Tetracycline)

Antibiotic Targets

• Ribosomes (inhibiting protein synthesis)
• Cell wall
• Cytoplasmic membrane
• Lipid biosynthesis
• DNA replication and transcription

β-Lactam Antibiotics

• One of the most important groups of antibiotics
• Examples: Penicillins, Cephalosporins, Cephamycins
• Over half of all antibiotics used worldwide
• Target cell wall synthesis

Penicillin

• Discovered by Alexander Fleming in 1929
• Primarily effective against Gram-positive bacteria
• Some synthetic forms are effective against some Gram-negative bacteria
• Penicillin G is naturally occurring, while others are synthetic

Cephalosporins

• Produced by fungus Cephalosporium
• Same mode of action as Penicillins
• Commonly used to treat Gonorrhea
• Example: Ceftriaxone has a 6-member dihydrothiazine ring, making it resistant to most β-lactamases

Antibiotics from Prokaryotes

• Aminoglycosides: contain amino sugars bonded by glycosidic linkage
• Inhibit protein synthesis
• Target 30S subunit of ribosome
• Clinically effective against Gram-negative bacteria
• Not commonly used today due to serious side effects