Second Unit MICRO xP

Normal growth conditions

• 20-40˚ C

• pH 6-8

• High nurtients

• 0.9% salt

• sea-level pressure (1 atm)

Extremophiles

Grow in extreme conditions

• anything other than the “normal”

• need unique adaptations to make them tolerent to extreme conditions

Cardinal conditions

the values of the minimum, maximum, and optimum growth conditions that support growth of an organism

Temperature

Psychrophiles

< 15˚ C

Mesophiles

20-45˚ C

Most microbes that we encounter

• don’t die below minimum, just don’t grow

Thermophiles

45-80˚ C

Hyperthermophiles

> 80˚ C

Use of heat to control microbial growth

Mechanisms of action: proteins (enzymes) can be denatured, and membranes can be disrupted

• amount of killing is proportional to both temperature and time of exposure to heat

• Higher temperature and longer = increase killing

Use of heat to control microbial growth: Boiling

100˚ C = 212˚ F

kills vegetative cells and viruses but NOT bacterial endospores

Use of heat to control microbial growth: Autoclaving

121˚ C = 250˚ F

Uses steam under pressure, moist heat penetrates cells well

kills vegetative cells, viruses, AND bacterial endospores (dependent on time) (>15 min)

Use of heat to control microbial growth: Dry heat sterilization

requires long time to kill endospores

used for objects

Use of heat to control microbial growth: Pasturization

Traditional: (50-75˚ C for 30 min) - Louis Pasteur’s orginial method

Flash: (50-75˚ C for 15 sec, then rapid cooling) - used to reduce microbial numbers without altering flavor (milk, wine, beer); lengthens shelf life

UHT (ultra high temp): (135˚ C for 1-2 sec) - used to sterilize milk to enable storage at room temp

Use of cold to control microbial growth

Refrigeration

• bacteria do not grow or grow slowly

Freezing

• bacteria die slowly, so are still present after the food is taken out of the refrigerator

Growing cells acidify the area they are in by:

• Transport

• PMF

• Metabolism

But too acidic or basic = bad

Internal pH – maintained at pH 5-8

pH <5 damages membrane and inactivates many enzymes → lethal

Use of pH to control microbial growth

Used in food preservation

• low pH, cells cant easily adapt, dead cells

Osmolarity

= Water availability 

measure of the degree of water availability - inversely related to osmolarity (measure of the solute molecules in a solution)

Limiting water availability to microbes prevents growth

• water limitation may result from high osmolarity

  • salted foods - beef jerky, ham, bacon

  • sugared foods - jellies and jams

• Water limitation may result from desiccation (absence of water)

  • bread, crackers, cheerios

Oxygen

Organisms differ in their need for oxygen

• oxygen-based metabolism use oxygen as terminal electron acceptor in a process called respiration

• Oxygen readily forms reactive oxygen species (ROS)

  • their high reactivity damages cellular components

Oxygen relationships to microorganisms: Aerobes

Cultures usually grown with vigorous shaking to give them O2

Oxygen relationships to microorganisms: Anaerobes

Do not use O2 as a terminal electron acceptor in respiration

• live in O2 free environments

Oxygen Summary

Use of radiation to control microbial growth

Kills by damaging DNA (causes breaks)

• is poor at penetrating many materials (glass or plastic)

• used for sterilizing surfaces

Ionizing radiation

X-rays & Gamma rays

• has higher energy than UV radiation so penetrates materials well produces ions when it hits biological molecules (very damaging

• used for sterilizing antibiotics, surgical supplies, food, plastic supplies, etc. 

Filtration (for liquids)

Physical removal of microbes - depends on pore size

For liquids:

• membranes usually have pore sizes of 0.2 or 045 um to remove bacteria

• do not remove viruses

Filtration (for air)

Physical removal of microbes - depends on pore size

For air:

• N95 filters remove particle. Use both:

  • mechanical filtration: remove viruses in water droplets

  • electrostatic filtration: special mask fabric is charged → attracts and binds viruses)

Decontamination

reducing microbes to a relatively safe level

Type of decontamination: Disinfection

Disinfection

killing, inhibiting, or removing microbes that may cause disease

Sterilization

Complete killing or removal of all organisms

• autoclaving

• gas sterilization (ethylene oxide, chlorine) for items destoryed by autoclaves

Chemical antimicrobial agents

chemicals that kill or inhibit the growth of microorganisms

• disinfectants

• sterilants

• chemotherapeutic agents (chemical agents that are used to treat disease)

Antibiotics

“anti-life”

chemicals that are produced by microbes that can kill or inhibit the growth of other microbes

include some synthetic chemicals modeled after antibiotics

Cidal agents

agents that kill microbes

(bacteriocidal)

Lytic agents

a subset of cidal agents that kill bacteria by lysing them

(bacteriolytic)

Static agents

agents that reversibly inhibit pathogen growth

Reversible: growth resumes when the antimicrobial agent is gone

(bacteriostatic)

Minimum inhibitory concentration (MIC)

the lowest concentration of a chemical that prevents growth of an organism

• add the same number of microbes to each tube, add increasing concentrations of a chemical, and evaluate growth

Zone of inhibition

a zone of clearing in a bacterial lawn around a filter disk soaked in an antimicrobial agent

• as agent diffuses out, the concentration decreases

  • the larger the zone, the lower the concentration required to kill the microbe

Selective toxicity

the ability to kill or inhibit the growth of a pathogen while damaging the host as little as possible

Spectrum of activity:

1. Broad spectrum - effective against many species ( if too broad can kill benificial organisms)

2. Narrow spectrum - effective against few or a single species (ideal if we only want to kill target)

Properties of chemotherapeutic agents

Most antibiotics were discovered as natural microbial products but have been modified to:

→ increase their toxicity to the microbes

→ decrease their toxicity to humans

→ narrow their spectrum of activity

Mode of actions of antibiotics

Knowing themode can tell us:

• if an antibiotic is likely to work on a pathogen

• the probability that the pathogen will become resistant to the antibitoic

Mode of action of antimicrobial drugs

Cell wall synthesis inhibitors

Inhibit enzymes that form cross links in peptidoglycan

• target of the majority of antibiotics

• high selective toxicity

• Penicillins & Cephalosporins

Protein synthesis inhibitors

Halt protein synthesis

• Generally bind to prokaryotic ribosomes

• High selective toxicity

• At high concentrations can inhibit mitochondria

• Often bacteriostatic (once removed, ribosomes resume function)

• Macrolides

Nucleic acid synthesis and transcription inhibitors

interfere with DNA or RNA production by targeting enzymes or blocking the building blocks, preventing cell growth and replication

• Cell components involved in these functions are relatively similar in prokaryotes and eukaryotes

• Quinolones

Cell membrane disruptors

agents that target and compromise the integrity of the cell membrane, leading to cell death or dysfunction

• Membrane structure is similar in prokaryotes and eukaryotes

  • low selective toxicity (so — not great for us!)

• Some specific targets have been found:

  • Dapotymcin - binds to specific lipids in the bacteria membrane, causing pore formation and depolarization

  • Platensimycin - inhibits fatty acid biosynthesis in bacteria

Growth factor analogs = Anti-metabolites

Chemicals that resemble growth factors but block metabolic pathways by competitively binding to metabolic enzymes

Sulfa Drugs = sulfonamides

Synthetic compounds that compete for the active site of an enzyme involved in folic acid synthesis 

• folic acid is required for purine and pyrimidine biosynthesis

• humans do not synthesize folic acid, so are not affected by sulfa drughs

  • High selective toxicity!

How do microorganisms become resistant to antibiotics?

1. Modification of the drug target through mutation

2. Introduction of a resistance gene

3. Formation of a biofilm

Modification of the drug target through mutation

Spontaneous mutations change the targets so that it no longer binds an antibiotic

Introduction of a resistance gene, which may:

• destroy the antibiotic before it gets in (enzymatic inactivation)

  • Beta - lactamase breaks Beta - lactam ring in penicillins)

• Chemically modify the antibitoic so that it is no longer active

  • add phosphategroup to kanamycin and inactivate it)

• Pump the antibiotic back out of the cell before it can do damage

  • pump out tetracycline

Formation of a biofilm

Enhances resistance by many mechanisms, such as:

• slowing diffusion so antibitoic doesnt reach many cells

• promoting cells generally are not damaged by antibiotics

Major cause of antibiotic resistance

Extensive use of antibiotics: 

1. Antibiotics are used as an additive in animal feed

2. Antibiotics are overperscribed

3. Antibiotics are improperly used

Effect: many bacterial infections are becoming untreatable

Strategies to reduce the emergence of antibiotic resistance

1. Use high doses to kill all bacteria

2. Complete full antibiotic treatment program

3. Use a narrow spectrum antibiotic whenever possible

4. Develop new antibiotics

10 million times more _____ on Earth than stars in the sky

Viruses!

Ivanowski (1892)

Took sap from diseased plant, filtered it for bacteria, applied to a healthy plant = diseased plant

• something smaller than bacteria that was infectious was there!

Beijerinck (1900)

This “toxin” had many properties of living organisms - could reproduce but needed a host

Called this entity a “virus” = poison or venom

More history

What is a virus?

a non-cellular particle that contains a genome

• lacks independant metabolism

• needs a host to reproduce

• obligate intracellular parasite

Virion

A complete viral particle; the form of a virus that occurs extracellularly (outside a host cell)

Impacts viruses have on host cell

• Host cell Lysis

• May insert into genome and replicate with host cell

  • may be there for long time, then when jump out lyse host

  • may help host (few cases)

Virus structures

1. Nucleic acid: DNA or RNA

2. Capsid: protein coat, individual proteins are capsomers

3. Envelope: Bilayer membrane from host that the virus takes as it escape the host cell

Virions do not contain __________ or __________

cytoplasm or ribosomes

Accessory proteins

contained in the envelope of virus, within the caspid, or between envelope and caspid

• Some help entry into, or release from, host cells

  • lysozyme-like enzymes (bacterial viruses)

  • Neuraminidases (spike proteins)

• Some help transcribe or replicate the viral genome

  • Polymerases - particularly important for RNA viruses

Viral sizes and shapes: Rod-Shaped

Filamentous (helical) virus

• capid like hallow tube

• Proteins arranged in helix

• resulting rod may be rigid or flexible

• length is determined by genome size

• filament can be inside an envelope

Viral sizes and shapes: Spherical viruses

Icosahedral virus

• polyhedron

• efficient approximation of a sphere

• Icosahedral can be inside an envelope

Viral sizes and shapes: Complex virus

• often have icosahedral head and helical tail

• may also have additional structures (tail fibers & collar)

Viral genomes

May be:

DNA

• single stranded (ss) DNA

• Double stranded (ds) DNA

RNA

• ssRNA

• dsRNA

Most DNA viruses are ______. Most RNA viruses are ______.

Most DNA viruses are dsDNA.

Most RNA viruses are ssRNA.

____ most common among bacterial virues.

____ most common among plant virues.

dsDNA most common among bacterial viruses.

ssRNA most common among plant viruses.

All types represented among animal viruses. 

Genome structure

DNA may be linear or circular

RNA is always linear, may be segmented

How do we grow viruses? (Cultivation of Bacteriophage)

Viruses depend on a host to replicate — so for bacteriophages, we need bacteria

Growth of bacteriophage on an agar plate: Plaques

Plaques: zone of clearing that results from cell lysis

Phage count = # plaque-forming units/ml

= #PFU/ml

= Phage titer

Use PFU because not every virion is infective

Cultivation of animal viruses

Animal organ → separate into individual cells → grow in tissue culture

Add virus → Add layer of agar → Plaques form in the monolayer of tissue

Steps to viral infection

Steps to viral infection: Attachment

Virion recognizes host cell → adsorbs to surface

Steps to viral infection: Penetration

viral genome penetrates the host cell

• bacterial viruses: inject the genome and caspid remains outside

• animal viruses: virion enters the host cell and genome is then released within the cell

Steps to viral infection: Replication

Viral genome and proteins are synthesized through redirection of the host cell machinery

Steps to viral infection: Assembly

Caspids are formed, and genomes are packaged in caspids to form virions

Steps to viral infection: release

Virions are released from the host; the host cell dies

Examples of bacteriophage

T4 phage → infects E. coli

• lyses host cells → death

Lambda (λ) phage → infects E. coli

• inserts into genome and replicates with host cell → can hide for generations then jumps out of the genome and lyses the host

Both are dsDNA viruses

Host recognition and attachment

Phage attach to Bacterial receptors: specific bacterial cell surface components

Receptors are required for infection: determine the “host specificity” of a phage

Penetration of the genome into the host cell

= injection of genome

• tail tube is injected down

• Lysozyme is released - degraded peptidoglycan

• DNA is injected

Synthesis of viral genome and proteins

Host DNA, RNA, and protein is halted; Host DNA degraded

Host cell machinery is redirected to synthesize viral constituents

• express “early genes” first (encode proteins that promote genome replication)

• express “middle & late genes” second (caspid proteins, tail fibers, collar proteins)

Assembly of caspids and packaging to form virions

• Caspids form with help of scaffolding proteins - hold capsid “prohead” together

• Nanomotor assembles on prohead

• Nanomotor uses ATP to pack DNA in (displaces scaffolding proteins)

• Tails and tail fibers are attached

• ~200 T4 virions are assembled inside host cell

Release from the host cell

Once the host cell is full of mature virions:

• a lysozyme-like enzyme helps degrade peptidoglycan

• another enzyme helps break cytoplasmic membrane\

• the cell lyses

One-step growth curve

Measure how long it takes to replicate and how many virions each cell releases

One-step growth curve PARTS:

Eclipse period: period during which no infective virions are released

Burst period = rise period = maturation phase

Burst size: number of virions released per bacterium

• information on a given phage: how long to replicate, hoe many virions released

Latent period

= the shortest time necessary for viral reproduction

Time course of events in T4 phage infection

Virulent phage

= lytic phage

a phage that experiences only a lytic life cycle (aka cell pops open and dies)

(ex. T4 phage)

Temperate phage

= lysogenic phage

a phage that experiences lytic and lysogenic cycles (cen lyse a cell or incorporate into the genome)

(ex. lambda phage)

Lysogenic life cycle

bacteriophage infect and the genome is replicated with the host chromosome

Prophage

a phage that is intergrated into the host’s genome

many bacteria have many prophage

Lysogeny

a state in which a phage remains within a bacterial cell and reproduces as the host cell reproduces

Induction

the process of initiating phage reproduction in a lysogenic cell

What triggers induction?

Conditions that threaten host cell survival

• low nutrient avaliability, DNA damage, shofts in pH/temp

• phage need healthy host to multiply → stressed host bad

Most bacteriophages are _____________ phages

lysogenic (temperate)

How do bacteria defend themselves against phage?

• lose the receptor required for phage adsorption

• produce enzymes that break down foreign DNA (restriction enzymes)

• Use an adapative immune system against current and future infections (CRISPR/Cas system)

CRISPRs

bacterial defense system against phage

DNA regions with repeated DNA sequences that are interspersed with short DNA sequences (“spacers”) acquired from infecting phages

Clustered Regularly Interspaced Short Palindromic Repeat