Transcript antibiotics

THE CONTROL OF
MICROORGANISMS BY
CHEMICAL AND PHYSICAL
FACTORS
EXERCISE 18.
THE ANTIBIOTIC SENSITIVITY TESTING
EXERCISE 19.
THE FILTER PAPER DISK METHOD:
A. EVALUATION OF DISINFECTANTS AND ANTISEPTICS
B. THE INHIBITORY ACTION OF HEAVY METALS AND OTHER
CHEMICALS
Microbial control by chemical and physical
“the use of antiseptics, disinfectants, antibiotics, ultraviolet
light (UV) and many other agents”
Basic terms used in discussing the control of microorganisms
1. Sterilization:
2. Disinfection:
The process of destroying all forms of life. A sterile
object is one free of all life forms, including endospores.
The reduction or elimination of pathogenic microorganisms in or
on materials, so they are no longer a health hazard.
3. Disinfectant:
Chemical agents used to disinfect inanimate objects but
generally to toxic to use on human tissues.
4. Antiseptic:
Chemical agents that disinfect, but are not harmful to
human tissues.
A metabolic product produced by one microorganism
that inhibits or kills other microorganisms.
5. Antibiotic:
6. Chemotherapeutic
Synthetic chemicals that can be used therapeutically.
antimicrobial chemical:
7. Cidal:
Kills microorganisms.
8. Static:
Inhibits the growth of microorganisms.
When evaluating or choosing a technique to
control microorgansims there are some
factors which may influence antimicrobial
activity:
1. the concentration and kind of a chemical agent used
2. the intensity and nature of a physical agent used
3. the length of exposure to the agent
4. the temperature at which the agent is used
5. the number of microorganisms present
6. the organism itself
7. the nature of the material bearing the microorganism.
EXERCISE 18: THE ANTIBIOTIC
SENSITIVITY TESTING
ANTIBIOTICs (anti: against; bios: life)
“metabolic by-products of one microorganism that is able to kill or
inhibit other microorganisms”
Alexander Fleming and the crude antimicrobial extract obtained
from Penicillium notatum was named as PENICILLIN
“FIRST DISCOVERED ANTIBIOTIC”
BACTERICIDAL
“physical and chemical agent that is able to kill some types of bacteria”
(e.g., penicillins, cephalosporins, streptomycin)neomycin)
BACTERIOSTATIC
“any agent that inhibits the growth and reproduction of some types of
bacteria but need not kill the bacteria”
(e.g., tetracyclines, erythromycin, sulfonamides)
Based on their origin, there are 2 general classes of
antimicrobial chemotherapeutic agents:
1. antibiotics: substances produced as metabolic products of one
microorganism which inhibit or kill other microorganisms.
2. antimicrobial chemotherapeutic chemicals: chemicals
synthesized in the laboratory which can be used therapeutically on
microorganisms.
Today the distinction between the two classes is not as
clear, since many antibiotics are extensively modified in the
laboratory (semisynthetic) or even synthesized without the help
of microorganisms.
Antimicrobial agents also vary in their spectrum..
BROAD SPECTRUM
NARROW SPECTRUM
tetracycline,
streptomycin,
cephalosporins,
ampicillin,
sulfonamides
penicillin G,
erythromycin,
clindamycin,
gentamicin
Drugs that are effective
against a variety of both
gram-positive and
gram-negative bacteria
a narrow spectrum is preferable
effective against just
gram-positive bacteria,
just gram negative bacteria,
or only a few species
cause less destruction of body’s normal flora
prevents superinfection by opportunistic microorganisms, drug toxicity, allergic rxns, selection of resistant
m/o
The antimicrobial activity of an
antibiotic
can be tested by several methods designed to determine the
smallest amount of the agent needed to inhibit the growth of a
microorganism.
Minimal Inhibitory Concentration (MIC)
The smallest amount of the agent needed to inhibit the growth of
a microorganism
Minimal Bactericidal Concentration (MBC)
the minimum amount of agent required to kill the microorganism
Commonly used methods to determine MIC and MBC are
“agar diffusion method” also known as Kirby-Bauer
method and “dilution method”.
Antimicrobials are usually regarded as bactericidal if
the MBC is no more than four times the MIC
Agar diffusion method
a petri plate containing a suitable medium is heavily inoculated by
spread plate technique with the microorganism whose antibiotic
sensitivity is to be determined
commercially available filter paper disks, each containing defined
concentrations of a specific antibiotic, are removed from individual
containers and are placed onto the agar surface
during the incubation antibiotics diffuse from the disk into the
agar
at some particular distance from each disk, the MIC for the
antibiotic is reached and microbial growth is inhibited
the MIC’s are recognized by the presence of “GROWTH
INHIBITION ZONES” (clear zones) surrounding the various
antibiotic disks used
Agar diffusion method
Escherichia coli exhibits varying
sensitivity to antibiotics.
Of the antibiotics used, this particular
strain of E. coli is most sensitive to
CTX 100 (Cefotaxime), showing no
growth in the zone surrounding the
disk
growth inhibition zones” are observed
and diameters of such zones are
measured milimetrically. The results
constitute an ANTIBIOGRAM.
Agar diffusion method (cont’d)
The relative diameters of the zones do not necessarily indicate
the relative activities of the chemotherapeutic agents used in
the test. The size of the growth inhibition zones can be
affected by several factors including:
1. the culture medium used
2. incubation conditions (temperature, oxygen availability,
time period and etc.)
3. the rate of diffusion of antibiotic
4. the concentrations of the antibiotics used
5. antibiotic sensitivity of the organism being tested.
The Kirby-Bauer test (agar diffusion
method) must be carefully standardized.
•Special agar, i.e. Mueller-Hinton agar, is used along with a
prescribed inoculum of broth.
•The antibiotic disks are also standardized to contain a specific
amount of antibiotic.
•A specified period of time of incubation at specified temperature,
“the clear zones” are measured.
These are compared with tables giving the interpretation of
measurement for each antibiotic.
Mueller Hinton Agar
This unsupplemented medium has been selected by the National Committee for Clinical Laboratory
Standards (NCCLS)1 for several reasons:5 this medium is low in sulfonamide, trimethoprim and
tetracycline inhibitors, provides satisfactory growth of most non-fastidious pathogens and
demonstrates batch-to-batch reproducibility.
Mueller Hinton Agar is often abbreviated as M-H Agar, and complies with requirements of the World
Health Organization.5 Mueller Hinton Agar is specified in FDA Bacteriological Analytical Manual6 for
food testing, and procedures commonly performed on aerobic and facultatively anaerobic bacteria.7
A variety of supplements can be added to Mueller Hinton Agar, including 5% defibrinated sheep or
horse blood, 1% growth supplement and 2% sodium chloride.
Principles of the Procedure
Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, and amino acids in
Mueller Hinton Agar. Starch is added to absorb any toxic metabolites produced. Agar is the
solidifying agent.
A suitable medium is essential for testing the susceptibility of microorganisms to sulfonamides and
trimethoprim. Antagonism to sulfonamide activity is demonstrated by para-aminobenzoic acid
(PABA) and its analogs. Reduced activity of trimethoprim, resulting in smaller growth inhibition
zones and inner zonal growth, is demonstrated on medium possessing high levels of thymide. The
PABA and thymine/thymidine content of Mueller Hinton Agar are reduced to a minimum, reducing
the inactivation of sulfonamides and trimethoprim.
Formula / Liter
Beef Extract...............................................................................2 g
Acid Hydrolysate of Casein..................................................17.5 g
Starch.....................................................................................1.5 g
Agar.........................................................................................17 g
Final pH 7.3 ± 0.1 at 25°C
Formula may be adjusted and/or supplemented as required to meet performance specifications.
Antimicrobial chemotherapy
SELECTIVE TOXICITY
WITHOUT seriously
harming the host
In order to be selectively toxic, a chemotherapeutic agent must interact with
some microbial function or microbial structure that is either not present or is
substantially different from that of the host.
In treating infections caused by prokaryotic bacteria,
the agent may inhibit peptidoglycan synthesis or alter bacterial
(prokaryotic) ribosomes. Human cells do not contain peptidoglycan and
possess eukaryotic ribosomes. Therefore, the drug shows little if any,
effect on the host
Eukaryotic microorganisms,
on the other hand, have structures and functions more
closely related to those of the host. As a result, the variety of agents selectively effective against
eukaryotic microorganisms such as fungi and protozoans is small when compared to the number
available against prokaryotes.
Viruses
are not cells and, therefore, lack the structures and functions altered by antibiotics so
antibiotics are not effective against viruses.
Commonly used antimicrobial chemotherapeutic agents
arranged according to their
Inhibit
peptidoglycan
synthesis
alter the
cytoplasmic
membrane
Inhibit
Protein
synthesis
mode of action:
Interfere
with DNA
synthesis
Principle of antibiotic spectrum

Different antibiotics target different kinds of
bacteria
◦ i.e., different spectrum of activity

Examples:
◦ Penicillin G (= original pen.) mainly streptococci (narrow
spectrum)
◦ Vancomycin only Gram-positive bacteria (intermediate
spectrum)
◦ Carbapenems many different bacteria (very broad
spectrum)
1. Antimicrobials acting on
the bacterial cell wall

Interfere with
synthesis of
peptidoglycan layer in
cell wall of actively
dividing bacteria
◦ eventually cause cell lysis
◦ bind to and inhibit
activity of enzymes
responsible for
peptidoglycan synthesis
 “penicillin-binding
proteins”
Antimicrobials acting on
the bacterial cell wall

Beta-lactams:
Penicillins
◦
◦
◦
◦
benzylpenicillin
flucloxacillin
ampicillin
piperacillin
B-lactam ring
Antimicrobials acting on
the bacterial cell wall

Beta-lactams:
Cephalosporins
◦ Orally active
 cephradine
 cephalexin
◦ Broad spectrum




cefuroxime
cefotaxme
ceftriaxone
ceftazidime
Cephalosporins
a house with a garage &
basement

synthetic side chains
change the spectrum
of action
Antimicrobials acting on
the bacterial cell wall

Unusual beta-lactams
◦ Carbapenems
 Imipenem, meropenem
◦ very wide spectrum

◦ Monobactams
 Aztreonam
◦ only Gram-negatives

Glycopeptides
◦ only Gram-positives, but
broad spectrum
◦ vancomycin
◦ teicoplanin

Antimicrobials acting on
nucleic acid synthesis

Inhibitors Of Precursor Synthesis
◦ sulphonamides & trimethoprim are synthetic,
bacteriostatic agents
 used in combination in co-trimoxazole
 Both of these drugs block enzymes in the bacteria pathway
required for the synthesis of tetrahydrofolic acid, a cofactor
needed for bacteria to make the nucleotide bases thymine,
guanine, uracil, and adenine.
◦ Sulphonamides inhibit early stages of folate synthesis
 dapsone, an anti-leprosy drug, acts this way too
◦ Trimethoprim inhibits final enzyme in pathway,
dihydrofolate synthetase.
 pyramethamine, an anti-toxoplasma and anti-PCP drug acts
this way too
2. Antimicrobials acting on
nucleic acid synthesis

Inhibitors of DNA replication
◦ Quinolones (e.g ciprofloacin) inhibit DNA-gyrase
◦ Orally active, broad spectrum

Damage to DNA
◦ Metronidazole (anti-anaerobes), nitrofurantoin (UTI)

Inhibitors of Transcription
◦ rifampicin (key anti-TB drug) inhibits bacterial RNA
polymerase
◦ flucytosine is incorporated into yeast mRNA
3. Antimicrobials acting on
protein synthesis

Binding to 30s Subunit
◦ aminoglycosides (bacteriocidal)
 streptomycin, gentamicin, amikacin.
30s subunit
mRNA
◦ tetracyclines

Binding to the 50s subunit
◦
◦
◦
◦
chloramphenicol
fusidic acid
macrolides (erythromycin,
clarithromycin, azithromycin)
◦ Agents that block TRANSCRIPTION
◦ i.e. rifampisin
◦
◦ Agents that block TRANSLATION
50s subunit
protein
These agents prevent bacteria from
synthesizing structural proteins and
enzymes
4. Antimicrobials acting on
the cell membrane

Alteration of the cytoplasmic membrane of microorganisms results in
leakage of cellular materials

amphotericin binds to the sterol-containing
membranes of fungi

polymyxins act like detergents and disrupt the
Gram negative outer membrane.
◦ Not used parenterally because of toxicity to mammalian cell
membrane

fluconazole and itraconazole interfere with the
biosynthesis of sterol in fungi
Mechanisms of resistance on
microorganisms against antibiotics
1. Producing enzymes which detoxify or inactivate the antibiotic,
e.g., penicillinase and other beta-lactamases.
2. Altering the target site in the bacterium to reduce or block
binding of the antibiotic, e.g., producing a slightly altered ribosomal
subunit that still functions but to which the drug can't bind.
3. Preventing transport of the antimicrobial agent into the
bacterium (reducing permeability of membranes), eg., producing an
altered cytoplasmic membrane or outer membrane.
4. Developing an alternate metabolic pathway that is not affected
by the drug; to by-pass the metabolic step being blocked by the
antimicrobial agent,
5. Increasing the production of the target,
6. Efflux of antimicrobial agent.
Mechanisms of resistance on
microorganisms against antibiotics

(cont’d)
Resistance can arise from chromosomal mutations,
or from acquisition of resistance genes on mobile
genetic elements (R plasmids mainly found in gram bacteria
◦ plasmids, transposons, integrons

Resistance determinants can spread from one
bacterial species to another, across large taxonomic
distances

Multiple resistance determinants can be carried by
the same mobile element
◦ Tend to stack up on plasmids
Spread of Antimicrobial Drug
Resistance





Inappropriate, extensive use of antimicrobial drugs is
leading to the rapid development of drug-resistance in
disease-causing microorganisms.
Drugs prescribed for treatment of a particular infection
have changed because of increased resistance of the
microorganism causing the disease.
Ex. Neisseria gonorrhoeae is now resistant to penicillin.
Antibiotic treatment is warranted in 20% of individuals who
are seen for clinical infectious disease, yet antibiotics are
prescribed up to 80% of the time. In up to 50% of cases
recommended doses or duration of treatments are not
correct.
This is compounded by patient noncompliance – how?
Spread of Antimicrobial Drug
Resistance (cont.)


Other indiscriminant, nonessential uses of
antibiotics contribute to the emergence of
resistant strains, ex. antibiotics are used in
agriculture both a growth-promoting substances
in animal feeds and as prophylactics – what does
that mean?
If the use of a particular antibiotic is stopped,
resistance to that antibiotic may be reversed
over time.
Impact of antibiotic resistance

Infections that used to be treatable with
standard antibiotics now need revised, complex
regimens:
◦ e.g., penicillin-resistant Strep. pneumoniae now requires
broad-spectrum cephalosporin

In some instances, hardly any antibiotics left:
◦ e.g., Multiresistant Pseudomonas aeruginosa
◦ e.g., Vancomycin-resistant Staph. aureus

Resistance rates worldwide increasing
The Search for New Antimicrobial
Drugs





The production of new analogs of existing antimicrobial
compounds is often productive since they mimic the
original drugs to some extent and have a predictable
mechanism of action, but may be different enough to act
on organisms resistant to the original form of the drug.
Application of automated robotic chemistry methods to
drug discovery = combinatorial chemistry.
Many different derivatives of an antimicrobial agent can be
generated in a short time, ex. 725 different tetracycline
derivatives from only 6 different reagents in only a few
hours.
According to the pharmaceutical industry, ~7 million
candidate compounds must be screened to yield a single
useful clinical drug. New drug discovery: 10-25 years and
$500 million for each new drug approved (FDA).
Computerized drug design can facilitate the design of
completely new drugs, ex. a protease inhibitor currently
used to treat HIV.
Antibiotic susceptibility testing
in the laboratory
EXERCISE 19.
THE FILTER PAPER DISK METHOD:
A. EVALUATION OF DISINFECTANTS AND ANTISEPTICS
B. THE INHIBITORY ACTION OF HEAVY METALS AND
OTHER CHEMICALS
DISINFECTION is the reduction or elimination of pathogenic
microorganisms in or on materials so that they are less of a health
hazard. The term DISINFECTANT is generally used for chemical agents
employed to disinfect inanimate objects, whereas the term
ANTISEPTIC is used to indicate a nontoxic disinfectant suitable for use
on animal tissue. Because disinfectants and antiseptics often work
slowly on some viruses (such as the hepatitis viruses), Mycobacterium
tuberculosis, and especially bacterial endospores, they are usually
unreliable for sterilization (the destruction of all life forms).
Originally the term antiseptic was applied to any agent that prevents sepsis, or
putrefaction. Since sepsis is caused by growing microorganisms, it follows that an
antiseptic inhibits microbial multiplication without necessarily killing them. By this
antiseptics are essentially
bacteriostatic agents.
definition, we can assume that
There are a number of factors which influence the antimicrobial
action of disinfectants and antiseptics, including:
1. The concentration of the chemical agent.
2. The temperature at which the agent is being used. Generally,
the lower the temperature, the lower the effectiveness.
3. The kinds of microorganisms present (endospore producers,
Mycobacterium tuberculosis, etc.).
4. The number of microorganisms present. The more organisms
present, the harder it is to disinfect.
5. The nature of the material bearing the microorganisms.
Organic material such as dirt and excreta interferes with some
agents.
The best results are generally obtained when the initial microbial
numbers are low and when the surface to be disinfected is clean and free
of possible interfering substances.
There are 2 common antimicrobial modes of action for disinfectants and
antiseptics:
1. They may damage the lipids and/or proteins of the semipermeable
cytoplasmic membrane of microorganisms resulting in leakage of cellular
materials needed to sustain life.
2. They may denature microbial enzymes and other proteins, usually by
disrupting the hydrogen and disulfide bonds that give the protein its threedimensional functional shape. This blocks metabolism.
Some of the more commonly used disinfectant groups are
listed below.
1. Phenol and phenol derivatives
These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective
against endospores. They alter membrane permeability and denature proteins.
2. Soaps and detergents
Detergents may be anionic or cationic. Anionic (negatively charged) detergents, such as
laundry powders, mechanically remove microorganisms and other materials but are not
very microbicidal. Cationic (positively charged) detergents alter membrane permeability
and denature proteins. They are effective against many vegetative bacteria, some fungi,
and some viruses. However, endospores, Mycobacterium tuberculosis, and
Pseudomonas species are usually resistant. They are also inactivated by soaps and
organic materials like excreta. Cationic detergents include the quaternary ammonium
compounds (zephiran, diaprene, roccal, ceepryn, and phemerol).
3. Alcohols
70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria,
enveloped viruses, and fungi. However, they are usually ineffective against endospores
and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols
denature membranes and are often combined with other disinfectants, such as iodine,
mercurials, and cationic detergents for increased effectiveness.
Some of the more commonly used disinfectant groups are
listed below (cont’d)
4. Acids and alkalies
Acids and alkalies alter membrane permeability and denature proteins and other molecules.
Salts of organic acids, such as calcium propionate, potassium sorbate, and methylparaben, are
commonly used as food preservatives. Undecylenic acid (Desenex®)
is used for dermatophyte infections of the skin. An example of an alkali is lye (sodium hydroxide).
5. Heavy metals
Mercury compounds (mercurochrome, metaphen, merthiolate) are only bacteriostatic and are not
effective against endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to
prevent gonococcal ophthalmia. Copper sulfate is used to combat fungal diseases of plants
and is also a common algicide. Selinium sulfide kills fungi and their spores.
6. Chlorine
Chlorine gas reacts with water to form hypochlorite ions, which in turn denature microbial
enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and
sewage. Sodium hypochlorite is the active agent in household bleach. Calcium hypochlorite,
sodium hypochlorite, and chloramines (chlorine plus ammonia) are used to sanitize
glassware, eating utensils, dairy and food processing equipment, and hemodialysis systems
Some of the more commonly used disinfectant groups are
listed below (cont’d)
7. Iodine and iodophores
Iodine also denatures microbial proteins and is usually dissolved in an alcohol solution to
produce a tincture. Iodophores are a combination of iodine and an anionic detergent
(such as polyvinylpyrrolidone) which reduces surface tension and slowly releases the
iodine. Iodophores are less irritating than iodine and do not stain. They are generally
effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses,
and some endospores. Examples include Wescodyne®, Ioprep®, Ioclide®, Betadine®,
and Isodine®
8. Aldehydes
Aldehydes, such as formaldehyde and glutaraldehyde, denature microbial proteins. Formalin
(37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of
microbial life. It is used in embalming, preserving biological specimens, and in preparing
vaccines. Alkaline glutaraldehyde (Cidex®), acid glutaraldehyde (Sonacide®), and
glutaraldehyde phenate solutions (Sporocidin®) kill vegetative bacteria in 10-30 minutes
and endospores in about 4 hours. A 10 hour exposure to a 2% glutaraldehyde solution
can be used for cold sterilization of materials.
9. Ethylene oxide gas
Ethylene oxide is one of the very few chemicals that can be relied upon for sterilization (after 4-12 hours
exposure). Since it is explosive, it is usually mixed with inert gases such as freon or carbon dioxide. Gaseous
chemosterilizers, using ethylene oxide, are commonly used to sterilize heat-sensitive items such as plastic
syringes, petri plates,
textiles, sutures, artificial heart valves, heart-lung machines, and mattresses. Ethylene oxide has very high
penetrating power and denatures microbial proteins. Vapors are toxic to the skin, eyes, and mucous
membranes and are also carcinogenic.
B. THE INHIBITORY ACTION OF HEAVY METALS AND OTHER
CHEMICALS
OLIGODYNAMIC ACTION
Heavy metals such as silver, copper and mercury, either alone or in certain
compounds have long been known to produce harmful effects on microorganisms.
.
The ability of extremely small quantities of certain metals to
exert toxic effects on microorganisms
Demonstration of the oligodynamic action and individual
reactions of selected microorganisms to the kinds and
concentrations of metals or heavy metal ions again using the
“FILTER PAPER DISK METHOD”
Oligodynamically active metals are used to control microorganisms in a
variety of circumstances. Such applications include the treatment of
various alcoholic beverages, milk and water, the preparation of antiseptic
agents and the impregnation of various fabric
If we are to compare antiseptics on the basis of their bacteriostatic
properties, the “FILTER PAPER DISK METHOD” is a simple satisfactory
method to use. In this method, a disk of filter paper is impregnated with
chemical agent to be tested and placed on a seeded nutrient agar plate. The
plate is incubated for a specified period and if the substance is inhibitory, a
clear zone of inhibition will surround the disk. The size of this zone is an
expression of the agent’s effectiveness and can be compared quantitatively
against other substances.
“FILTER PAPER DISK METHOD”