Microbiology
http://www.lab-initio.com/index.html
08.04.2015
Helmut Pospiech
Control of Microbial Growth
Some definitions:
• Sterilisation – the killing or removal of all viable organisms
(from a growth medium, instrument or surface)
• Decontamination – the treatment of an object or surface to
make it safe to handle
• Disinfection – the killing or removal of pathogenic
organisms
• Preservation – treatment to limit growth of (micro-)
organisms for a transient time (e.g. to extend the shelf life
of food stuff)
• Antibiosis – the killing or limitation of growth of microbial
pathogens within an other organism (human, cattle, plants
etc.)
Brock Biology of Microorganisms, 13th ed.
Physical Antimicrobial Control
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Heat sterilisation
Most vegetative cells of
mesophilic bacteria will die at
temperatures above 70°C
– Denaturation of enzymes and
nucleic acids
– Disruption of membranes
Endospores are most heat
resistant
– Geobacillus stearothermophilus
endospores are among the most
heat-resistant forms of life and
are used to control sterilisation
efficiency
The decimal reduction time D is
the time required to kill 90% of
the cells of a certain
microorganism
Brock Biology of Microorganisms, 13th ed.
Heat sterilisation
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Dry heat
– Glass ware and other heat resistant equipment
– Microorganisms are much more resistant to dry compared to moist heat
 2 hours at 160°C or
 1 hour at 180°C
Moist heat (autoclavation)
– Media, glass ware etc.
– Most commonly used method
– In automatic or semi-automatic instruments
 In water steam in the absence of air
at 2.1 atm (2.06 bar) pressure / 121°C
Pasteurisation
– Partial killing (“desinfection”) of foodstuff to remove pathogens and prolong
shelf life with minimal effect on nutritional value and appearance
• Kills pathogens such as Mycobacterium bovis/tuberculosis, Brucella spp., Salmonella,
Listeria monocytogenes, Campylobacter spp., Escherichia coli
• e.g. 65°C for 30 minutes (bulk pasteurisation) or 71°C for 15s (flash pasteurisation)
Autoclave
Brock Biology of Microorganisms, 13th ed.
Radiation Treatment
– Ultraviolet Radiation
• Wavelength 220-300 nm
(254 nm)
• Destroys mainly DNA
and thereby kills the
organism
• Used for surface and
water sterilisation
• Low penetration of most
materials (except clear
liquids)
Brock Biology of Microorganisms, 13th ed.
Radiation Treatment
– Ionising Radiation (IR)
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•
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High energy electromagnetic radiation
(e.g. Roentgen rays)
Destroys mainly nucleic acids and
proteins and thereby kills the organism
Variable resistance
Also destroys nutrient without changing
the outer appearance of food stuffs
Brock Biology of Microorganisms, 13th ed.
Radiation Practices
• IR sources:
– Cathode electron beams
– X-ray machines
– Nuclides such as 60Co or
137Cs
• Good penetration of
solids and liquids except
metals
• Use for food sterilisation
and decontamination
approved, but not
generally accepted
Brock Biology of Microorganisms, 13th ed.
Filter Sterilisation
•
Depth filter
– Fibrous sheet
• Borosilicate (glass) fibre
– HEPA (high-efficiency particulate air) filter
– Mainly air filters
•
Membrane filters
•
Micropore filters are produced of thin (10 µm)
Polycarbonate by radiation followed by
chemical etching
– Cellulose acetate, cellulose nitrate or
polysulfone
– High tensile strength
– Contain large numbers of small holes (pores)
– Pore size can be controlled during
manufacturing process)
– <0.2 µm pore size is required for sterilisation
– Viruses, mycoplasms and some spirlliae will
pass through 0.2 µm filters
Brock Biology of Microorganisms, 13th ed.
Filter Sterilisation
Brock Biology of Microorganisms, 13th ed.
Chemical Antimicrobial Control
• Antimicrobial component
– Natural or synthetic chemical that kills or inhibitd growth
of an microorganism
• -cidal indicates an agent that kills the microorganism
• -static indicates an agent that does not kill but inhibits growth of
an microorganism (e.g. bacteriostatic, fungizidal)
– Different effects
• Bacteriostatic
• Bacteriozidal
• Bacteriolytic
Brock Biology of Microorganisms, 13th ed.
Measuring the Antimicrobial Activity
The tube dilution technique is used to
determine the Minimum Inhibitory
Concentration of an antimicrobial agent in
respect of a certain microorganism
The disc diffusion technique is used to
determine the Susceptibility spectrum of a
certain microorganism against
several antimicrobial agent
Chemical Antimicrobial Agents for
External Use
Sterilants
• Destroy all microbial life
• Mainly used for materials that cannot
be autoclaved or irradiated
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Ethylene oxide
Formaldehyde
Peroxyacetic acid
Hydrogen peroxide
Hypochlorite (bleach)
Amylphenol
Disinfectants
• Kill microorganisms, but not
necessarily endospores
• Used on inanimate objects (e.g.
floors, tables etc.)
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Ethanol
Cationic detergents
Sanitizers
• Reduce microbial numbers, but do
not eliminate them completely
• E.g. for instruments used in food
chemistry
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Chloramine
Hypochlorite
Antiseptics and Germicides
• Kill or inhibit growth of
microorganisms
• Can be applied to living tissue
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Ethanol
Iodine
Hydrogen peroxide
Antimicrobial Agents Used In Vivo
• Compounds with selective
toxicity:
 Pathogen is more sensitive
than the host
 The greater the difference in
sensitivity, the more of the
agent can be applied
- off-target effects!
• Search for target (e.g.
essential metabolic feature
of the pathogen that is not
present in the host
• Synthetic Antimicrobial
Drugs
• Naturally Occuring
Antimicrobial Drugs:
Antibiotics
– Natural compound produced
by a microorganism with
antimicrobial activity against
other microorganisms
– Mainly secondary metabolites
– Producer always has natural
resistance against the
antibiotic produced
– Mainly by Steptomycetes and
related Gram-positive
bacteria
A “magic bullet” against
microorganisms?
Around 100 years ago, German chemists and physicians experimented with with dyes
(e.g. azodyes and metal-organics to find compounds that selectively kills bacteria and
could be used in treatment of infectious disease
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Paul Ehrlich
Salvasan to treat syphilis
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Scientific break-through
But severe side-effects
Difficult to administer because of low
solubility
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Gerhard Domagk
Sulfonamide (Sulfa drugs)
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The principle of using growth factor
analogues as synthetic drugs was born
Resistance require only one point mutation
Still used in combination with another folic
acid synthesis inhibitor, trimethoprim
Targets of Antimicrobial agents
Brock Biology of Microorganisms, 13th ed.
Other growth factor analogues
• P-fluorphenolalanine
– Amino acid analogue
• Nucleoside analogues
– Blocking nucleic acid synthesis
– Mainly used in the treatment
of viral and fungal infections
(and cancer)
• Isoniazid
– Nicotinamide analogue
– Interferes with mycolic acid
synthesis in Mycobacterium
– Used to treat tuberculosis
Brock Biology of Microorganisms, 13th ed.
Classes of Antibacterial Drugs
• Quinolones
– Inhibit DNA gyrase, a
topoisomerase only
present in bacteria
– Several quinolones are
now in heavy use against
several bacterial
diseases, e.g.
• Tuberculosis
• Anthrax
Brock Biology of Microorganisms, 13th ed.
Penicillins
• The first antibiotics discovered
by Alexander Fleming by
chance in 1929
• Produced by the mold
Penicillium chrysogenum
• Inhibitor of transpeptidase
(bacterial cell wall synthesis)
• Now only semi-synthetic
penicillins are used that can
be orally administered and
show improved spectrum
against different bacteria or
evasion of resistance
Brock Biology of Microorganisms, 13th ed.
The production of penicillin
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Original strains of Penicillium produced few
micrograms per liter culture
Optimised strains produce up to 60 g/l
penicillin now
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Feeding of different precursors that will be
incorporated by the producing
microorganism
 biosynthetic penicillin
Chemical or enzymatic cleavage of side chain
followed by chemical addition of synthetic
side chains
 semisynthetic penicillins
Brock Biology of Microorganisms, 13th ed.
Cephalosporins
•
By moulds of the genus
Cephalosporium
• Also β-lactam antibiotics
• Same mode of action as penicillin
• Mainly semi-synthetic cephalosporins
used
• Cephalosporin has to side chains that
can be modified, penicillin only one
• More possibilities to apply
combinatorial chemistry
 Testing systematically or structureguided combinations of new side
chains to obtain new derivatives with
improve properties, e.g.
-
Improved activity
Broader spectrum
Better administration
Brock Biology of Microorganisms, 13th ed.
Aminoglycosides
• E.g. Streptomycin
(Streptomyces griseus),
kanamycin, gentamycin,
neomycin, spectinomycin
etc.
• Target 30S subunit of the
ribosome
• Inhibit protein synthesis
• Useful against Gramnegative bacteria
• Serious side effects (e.g.
nephrotoxicity)
Brock Biology of Microorganisms, 13th ed.
Macrolides
• Lacton rings bonded to
sugars
• E.g. erythromycin
(Streptomyces erithreus)
• Targets the 50S subunit of
the ribosome
• Inhibits protein synthesis
• Broad spectrum antibiotics
• Particularly use against
legionellosis and for the
treatment of small children
Brock Biology of Microorganisms, 13th ed.
Tetracyclin
• By Streptomyces species
• Target 30S subunit of the
ribosome
• Inhibit protein synthesis
• Both natural and
semisynthetic tetracyclins in
use
• Second most commonly
used antibiotic
• Overuse in non-medical
applications (e.g. farming)
causes increasing occurence
of resistance
Brock Biology of Microorganisms, 13th ed.
Chloramphenicol
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By Streptomyces venezuelae
Nowadays exclusively produced by
chemical synthesis
– Probably the cheapest antibiotic
available
Inhibits protein synthesis by binding
the 50S subunit of the ribosome
Broad spectrum antibiotic
In industrial countries rarely used
because of developed resistance and
severe side effects
– Bone marrow toxicity
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Aplastic anemia
(loss of hematopoietic stem cells)
Bone marrow suppression
Leukemia
In low-income countries still used
because of the low price
Daptomycin
• Novel antibiotic with
new mode of action
• By Streptomyces species
• Cyclic peptide
• Forms pores in the
bacterial membrane
– Loss of membrane
potential
Brock Biology of Microorganisms, 13th ed.
Platensimycin
• Completely new class of
antibiotic
• by Streptomyces
platensis
• Disrupts bacterial lipid
synthesis
• Potential new ”last line
of defence” antibiotic
Brock Biology of Microorganisms, 13th ed.
Vancomycin
• Inhibits cell wall
synthesis
• After occurance of
resistance, a modified
Vancomycin has been
developed that evades
resistance
Brock Biology of Microorganisms, 13th ed.
The Antimicrobial Spectrum of Activity
Brock Biology of Microorganisms, 13th ed.
Brock Biology of Microorganisms, 13th ed.
Types of resistance
The appearance of antimicrobial drug
resistance
Types of resistance
Antifungal agents
Antifungal agents
How to find new antibiotics
• Systematic screening
– Mainly soil samples
around the world
•
It was said that the researcher
from pharma companies broad
along soil sample from all over
the world whenever they visited a
new place!
Brock Biology of Microorganisms, 13th ed.
Enrichment and Selection
•
Inoculum is the material
from which an
microorganism is isolated,
e.g.
– Soil
– Transformation of a cloning
experiment
– Sample from a diseased
patient
– Food sample from a
restaurant after a customer
got gastroenteritis
•
What if the bacteria of
interest represent only a
small fraction of all the
microorganisms present?
Brock Biology of Microorganisms, 13th ed.
Enrichment and Selection
• Creation of conditions that favour the microorganisms of
interest
– Omit nutrients of growth factors that other (contaminating)
microorganisms require, e.g.
• No nitrogen source for nitrogen-fixing bacteria
– Add chemicals that prevent the growth of other organisms, e.g.
• Ampicillin in culture medium to select for E. coli that have uptaken a
plasmid with an ampicillin resistance gene
– Choose sample preparation of growth conditions that favour the
microorganisms of interest or harm/kill the unwanted
microorganisms, e.g.
• Boiling of the inoculum to select for endospore formers (all vegetative
cells will die)
• Incubation at 37°C for human pathogenic bacteria
• pH 4-5 for lactic acid bacteria or yeasts
Inoculum
Enrichment
Selection
Pure Culture
Brock Biology of Microorganisms, 13th ed.