6- Control of microorganisms

Question 1

What re the 6 antimicrobial control methods?

Answer 1

1) Sterilization: The killing or removal of all viable organisms (including endospores).
2) Inhibition: Effectively limiting microbial growth.
3) Decontamination: The treatment of an object to make it safe to handle.

4) Disinfection: Directly targets the removal of all pathogens, not necessarily all
microorganisms

5) Physical methods:
– Heat
– Radiation
– Filtration

6) Chemical methods (called antimicrobials):
– Used on external surfaces: Sterilants, disinfectants, sanitizers, antiseptics.
– Used internally: antibiotics, antivirals, antifungals.

Question 2

Heat- for antimicrobial control, what does it do?

Answer 2

Physical method 
• Heat is the most widely used
method for controlling microbial growth
– High temperatures denature
macromolecules
– Amount of time required to reduce viability tenfold is called the decimal reduction time (D).
– The decimal reduction time is
inversely correlated with the
temperature.
– The time necessary to kill a
defined fraction is independent of the initial cell concentration (90%).

• Different microorganisms have
different decimal reduction times:
A, mesophile; B, thermophile.

Question 3

Heat sterilization; thermal death time; autoclave

Answer 3

The thermal death time is the time needed to kill all cells at a given temperature.
It is dependent on the population size of the microorganism tested. Need to standardize the starting number of cells to be able to compare the sensitivity of different microorganisms.

• Endospores survive heat that would rapidly kill vegetative cells. A higher temperature is needed to kill endospores. Endospores can resist boiling
for a long time: 5 min for a decimal reduction (e.g. 90% dead).

• The autoclave is a sealed device that uses steam under pressure:
– Allows temperature of water to get above 100°C.
– At 15 psi (pound/square inch), steam reaches 121°C, sterilization is achieved
in 10-15 min.
– The object being sterilized will reach this temperature. Not suitable for heatsensitive
object/liquid.
– It is not the pressure that kills microorganisms, but the high temperature.

Question 4

Pasteurization; what is it? Flash and bulk pasteurisation

Answer 4

Physical method
Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids.
– Does not kill all organisms, it is not a method of sterilization.

• Reduces the microbial load, increases the shelf life of the product.

• Pasteurization reduces significantly the population of many pathogens:
• Listeria monocytogenes
• Salmonella enterica
• Campylobacter
• E. coli O157:H7
• Mycobacterium
• Flash pasteurization: 72°C for 15s.
• Bulk pasteurization: 65°C, 30min.

Question 5

Radiation; action?

Answer 5

Physical method
Microwaves, UV, X-rays, gamma
rays, and electrons can reduce
microbial growth

• UV has sufficient energy to cause modifications and breaks in DNA, which inhibit replication,
transcription and cause death.
– UV is useful for decontamination of surfaces
– Cannot penetrate solid, opaque, or light-absorbing surfaces

Question 6

What is ionizing radiation?

Answer 6

– Electromagnetic radiation that produces ions and other reactive molecules
– Generates electrons and hydroxyl radicals causing damage to DNA and proteins.
– Amount of energy required to reduce viability tenfold is analogous to D value

• Sources of radiation include cathode ray tubes (electrons), X-rays, and radioactive nuclides
• Radiation is used for sterilization in the
medical field and food industry

Question 7

Filter sterilization; dept filters; membrane filters

Answer 7

Physical method
• Filtration avoids the use of heat on sensitive liquids and gases
– Pores of filter are too small for organisms to pass through
– Pores allow liquid or gas to pass through

• Depth filters
– Fibrous sheet or mat made from an array of fiber (paper or glass).
– Used to sterilize liquid, air.
– HEPA filters

• Membrane filters
– Function more like a sieve
– A type of membrane filter is the nucleation track (nucleopore) filter.
– Filtration speed can be increase by syringe, pump, or vacuum

Question 8

Antimicrobial agents; bacteriostatic; bacteriolytic; bacteriocidal

Answer 8

Chemical method
Antimicrobial agents can be
classified as:
• Bacteriostatic: inhibit growth of microorganism.

• Bacteriocidal: kill microorganism.
• Bacteriolytic: kill microorganism by inducing lysis.

Question 9

How do we measure antimicrobial activity? MIC; MLC; MBC

Answer 9

• Minimum inhibitory concentration (MIC)
is the smallest amount of an agent needed to inhibit growth of a microorganism
• Varies with the organism used, inoculum size, temp, pH, etc.

• Minimum lethal concentration (MLC) is the lowest concentration of an agent that kills a test organism.
• Minimum bacteriocidal concentration (MBC) is the lowest concentration of an agent that kills a test bacterium.

• Decimal reduction time (at
concentration x), Decimal reduction concentration (after x minutes). “Kills 99.9% of germs in 30s”.

Viable counts: MLC, MBC

Question 10

Measuring antimicrobial activity; Disc diffusion assay; zone of inhibition

Answer 10

Disc diffusion assay
• Antimicrobial agent added to
filter paper disc.
• MIC is reached at some
distance from the disc.
• Zone of inhibition: Area of
no growth around the disc.

Question 11

Chemical antimicrobial agents for external use; why used?; 2 categories; sterilants; disinfectants; sanitizers; antiseptics; antimicrobial drugs

Answer 11

• Antimicrobial compounds are used to prevent spreading of a pathogen in the environment, prevent contamination of the host and cure superficial bacterial infections. These antimicrobial agents can be divided into two categories:

– Products used to control microorganisms in commercial and industrial applications. Examples: chemicals in paper, air-conditioning, cooling towers,
textile and paper products, fuel tanks.
– Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces: Sterilants, disinfectants, sanitizers, and antiseptics.

• Sterilants: destroy all forms of microorganisms, including endospores. Called COLD
STERILIZATION.

• Disinfectants/Sanitizers: applied to nonliving objects or surface (can be toxic for animals/humans). Do not kill endospores.

• Antiseptics: applied to the surface of living tissues or skin (must not be toxic for
animals/humans). Do not kill endospores.

• Antimicrobial drugs: Antibiotics, antifungals, antivirals: applied outside or inside the body of animals/humans (must not be toxic for animals/humans). Do not kill endospores.

Question 12

Antimicrobial drugs; classification; good antimicrobial drug has:

Answer 12

• Antimicrobial drugs are classified on the basis of
– Molecular structure
– Mechanism of action
– Spectrum of antimicrobial activity

• Can be used internally in humans or animals with minimal side effects (low toxicity).
• Can be either bacteriostatic or bactericidal. Usually have a specific target.

• A good antimicrobial drug has:
– NO severe side effects, must be far more toxic for bacteria than mammalian cells.
– Low risk/benefit ratio.
– Broad spectrum of activity to facilitate rapid medical intervention.
– Appropriate bioavailability and pharmacokinetics (must reach the site of infection).
– Low cost to develop and manufacture.

Question 13

Selective toxicity; who and what is it?; Salvarsan

Answer 13

Synthetic antimicrobial drugs
Paul Ehrlich studied selective
toxicity in the early 1900s

– Selective toxicity is the ability
to inhibit or kill a pathogen
without affecting the host

– Salvarsan – one of the first
antimicrobial drugs, used to
treat syphilis (Treponema
pallidum)

Question 14

Synthetic antimicrobial drugs;

growth factor analogs; sulfa drugs; isoniazid

Answer 14

Synthetic antimicrobial drugs

• Growth factor analogs are
structurally similar to growth
factors but do not function in the cell. Analogs similar to vitamins, amino acids, and other compounds.

• Sulfa drugs: discovered by
Gerhard Domagk in the 1930’s.
Sulfanilamide is an analogue of paminobenzoic acid. Bacteriostatic.

• Isoniazid is a growth analog
effective only against
Mycobacterium. Interferes with
synthesis of mycolic acid.

Question 15

Synthetic antimicrobial drugs; Nucleic acid base analog; quinolones

Answer 15

Synthetic antimicrobial drugs
• Nucleic acid base analogs have been formed by the addition of bromine or fluorine. Stop DNA replication, translation.

• Quinolones are antibacterial
compounds that interfere with DNA gyrase (control DNA supercoiling).

Question 16

What are antibiotics? What is β-Lactams?

Answer 16

• Antibiotics are antimicrobial agents naturally produced by a variety of bacteria and fungi to inhibit or kill other microorganisms.
– Less than 1% of known antibiotics are clinically useful
– Can be modified to enhance efficacy (semisynthetic antibiotics)

• The susceptibility of microbes to different antibiotics varies greatly
– Gram-positive and Gram-negative bacteria vary in their sensitivity to antibiotics. The cell wall is a major factor.

• β-Lactams are one of the most important groups of antibiotics of all time.
Include penicillins, cephalosporins, and cephamycins.

Question 17

β-Lactams Antibiotics; penicillins

Answer 17

• Penicillins
– Discovered by Alexander Fleming, isolated from Penicillium chrysogenum
(mold).
– Primarily effective against Gram-positive bacteria
– Some synthetic forms are effective against some Gram-negative bacteria
– Inhibit cell wall synthesis

Question 18

β-Lactams Antibiotics; Cephalosporins

Answer 18

• Cephalosporins
– Produced by the fungus Cephalosporium
– Same mode of action as the penicillins
– Commonly used to treat gonorrhea (Neisseria gonorrhea)

Question 19

Mode of action of β-Lactams

Answer 19

Transpep:dase (TPase) is a penicillin-binding protein.

• β-lactams are bactericidal or bacterioly:c (depending
on the species, growth phase, etc).

• Can be bacteriosta:c (in isotonic solu:ons).

Question 20

What antibiotics are for prokaryotes?; Aminoglycosides; Chloramphenicol

Answer 20

• Many antibiotics effective against Bacteria are also produced by Bacteria
(e.g. Streptomyces)

• Aminoglycosides: kanamycin,
neomycin, amikacin, streptomycin.
– Target 30S subunit of the
ribosome, cause misreading of
mRNA.
– Bactericidal.

• Chloramphenicol:
– bind to 23S rRNA and block
pep:de elonga:on.
– Bacteriostatic.

Question 21

What antibiotics are for prokaryotes?; Macrolides; Tetracyclines

Answer 21

Macrolides: erythromycin à
– Broad-spectrum antibiotics that target the 50S subunit of the ribosome, block protein
synthesis
– Bacteriostatic.

• Tetracyclines
– Broad-spectrum, bacteriostatic.
– Inhibit 30S ribosomal subunit, block protein synthesis.

Question 22

What antibiotics are for prokaryotes?; Daptomycin; Platensimycin

Answer 22

• Daptomycin:
– Also produced by
Streptomyces
– Used to treat Gram-positive
bacterial infections
– Forms pores in cytoplasmic
membrane

• Platensimycin
– New structural class of
antibiotics
– Broad-spectrum, effective
against MRSA and
vancomycin-resistant
enterococci

Question 23

What are antimicrobial drugs resistance?

Answer 23

The acquired ability of a microorganism to resist the effects of a
chemotherapeutic agent to which it is normally sensitive

Question 24

Antibiotic resistance- what do they do?

Answer 24

• Antibiotic producers are tolerant:
• Lack target sites (i.e. no
peptidoglycan)
• Modify target sites
• Lack of uptake mechanism

• Some microorganisms are
resistant to some antibiotics.

Antibiotic resistance mechanism:
• Destruction or modification of
the antibiotic (i.e. β-
lactamase)
• Modification of the target site
• Modification of uptake
mechanism
• Efflux pumps: reduce
intracellular concentration

Question 25

Acquisition of resistance?R-plasmid

Answer 25

Usually mediated by the acquisition of a
new gene (or groups of genes) that provide
the cells with a new function (i.e. antimicrobial resistance).

• Most drug-resistant bacteria isolated from patients contain drug-resistance genes
located on R plasmids.

• Evidence indicates that R plasmids predate
the antibiotic era (antibiotics come from nature).

• The use of antibiotics in medicine, veterinary medicine, and agriculture selects
for the spread of R plasmids.

• R plasmids can be transferred between
bacteria of the same species or related species.

Question 26

What does antiviral drugs do? tho the host?; protease inhibitor; fusion inhibitor

Answer 26

Most antiviral drugs also target host structures, resulting in toxicity (viruses use host cell machinery). Risk to the host may not justify the use of
antiviral.

• Antibiotics are ineffective.
• Most successful and commonly used antivirals are the nucleoside analogs
(e.g., AZT, Acyclovir): block reverse transcriptase and production of viral DNA (RNA viruses).

• Protease inhibitors inhibit the processing of large viral proteins into individual components.
• Fusion inhibitors prevent viruses from successfully fusing with the host cell.

Question 27

What are antifungal drugs? Why are they a problem? Targets?

Answer 27

• Fungi pose special problems for chemotherapy because they are eukaryotes: much of the cellular machinery is the same as that of animals and humans.

• A few drugs target unique metabolic processes not found in mammals:
– Ergosterol synthesis (Nystatin, Fluconazole).
– Cell wall synthesis (inhibitor of chitin synthesis).