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ANTIBIOTICS
ANTIBIOTICS
Antimicrobial agents: Any chemical substance inhibiting the growth or causing the death of a microorganism
is known as an antimicrobial agent. Although a wide range of chemicals have these properties if a sufficiently
high concentration is used the term is usually restricted to those substances that are effective at conc.
Suitable for practical application. It includes antimicrobial, antifungal and anti viral agents.
History:
The development can be traced as 3 eras (periods). Pre – Ehrilch era, before 1891, wherein only 3
compounds were used in treatment i.e.; Cinchona for treatment of malaria, mercury for treatment of
syphilis and ipecacuanha for amoebic dysentery.
Ehrilich era:- The greatest achievement was done by an organic chemist Paul Ehrilch who was struck by the
fact that certain dyes like methylene blue specifically killed and stained certain bacterial cells and he
reasoned that chemical substances might be produced that could unite with and destroy the parasitic agents
of the disease without causing injury to the host cells . He called them “magic bullets “.
Ehrilch discovered an arsenic compound called Salvarsan 606 for the treatment of syphilis. Ehrilich
contributed a lot of ideas and he is called “father of modern chemotherapy”.
Post Ehrilch era: - Another milestone in the history of chemotherapy was by Domagk in 1935 who discovered
Prontosil for treatment of streptococcal infections. . Prontosil is converted into sulfanilamide in the
mammalian body and it inhibits the microbial activity.
Sir Alexander Fleming in 1929 discovered that strain of mould Pencillium notatum excreted a substance
cabpable of inhibiting Staphlococcus aureus. Pencillin was isolated in pure form by Florey, Chain and others.
Definitions:
Disinfectant:- This is a term applied to chemicals used to kill potentially infectious organisms and are
normally used in the treatment of inanimate objects , surfaces , water , etc and are not meant to come into
direct contact with man.
Antiseptics (Mild disinfectants):
This term refers to relatively non toxic and non irritant antimicrobial agents that may be appled topically to
the body surface either to kill or to inhibit the growth of pathogenic microorganisms.
Chemotherapeutic Agents:
This term describes the chemicals that are used to kill or inhibit the growth of microorganisms already
established in the tissues of the body. Another term which expresses clearly the functions of this group of
agents is Antimicrobial drugs. Chemotherapeutic agents must act at a concentration that can be tolerated by
the host tissues and therefore they must have a selective toxicity for the microorganisms compared with the
host. Chemotherapeutic agents must also have maximum curative capacity within a short time.
Cidal or Static Agents:
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If a substance merely causes cessations of growth of the microorganisms which is reversed when the
chemical is removed, it is called a Static agent. It may be bacteriostatic or fungistatic.
If the substance kils the microorganisms, it is called a cidal agent. It may be bactericidal or fungicidal.
Antibiotics:
These are naturally occurring antimicrobial agents produced by microorganisms that in small quantities
inhibit the growth or kill unrelated species of microorganisms.
Sources of antibiotics:
Main sources of antibiotics are bacteria and fungi and actinomycetes. Depending on the sources the
antibiotics are classified as those obtained from fungi, those obtained by bacteria, those synthesized in the
laboratory or those produced by the microorganisms and modified in the laboratory.
From Fungi – Cephalosporins from Cephalosporium, Pencillin from Pencillium.
From bacteria – Bacitracin from Bacillus subtilis , Chorophenicol from Streptomyces Venezuela .
Synthetic – Etambutol, Nitrafurans and
Semi-synthetic-Rifampin from Rifampicin ( Streptomyces mediterranei ), Amikacin (from Kanamycin ).
Classification of Antibiotics
Classification of Antibiotics is based on different criteria.
Based on their spectrum of activity:Group-1 Active against Gram-positive bacteria and gram-negative cocci
Ex: Pencillins, Erythromycin
Group- 2 Mainly against Gram-negative bacilli.
a) For systemic infection: - Aminoglycosides, Polymyxins.
b) For urinary tract infections:- Nitrofurantoin, Nalidixic acid
Group -3 Broad spectrum antibiotics
Ex: Sulphanamides Tetracycline
Group-4 Specific antibacterials
a) Active against anaerobic organisms.
Ex: metronidazole, lincomycin
b) for tuberculosis
Ex: Streptomycin, Isoniazid
b) for Chlamydia , Rickettsia , Mycoplasma
Ex: Erythromycin, Tetracyline
Group -5 Antifungal, antiviral agents
a) Antifungal agents: polyenes, Nystatin
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b) Antiviral : Idoxuridine , Amantadine
Antimalignancy antibiotics: Actinomycin ,Mitomycin
Based on the structure:-Lactam antibiotics – pencillin , Cephalosporins , loe mol.wt. -Lactam.
Aminglycosides, Macrolides, Tetracyclines, Polyenes, Nitrofurans.
Based on their source
Natural, Synthetic and Semi-synthetic
Based on whether they are cidal or static.
Ex: Bacteriocidal : Pencillin , Aminoglycosides
Bacteriostatic: Sulphanamides , Tetracyclines.
Based on their mechanism of actions
1) Inhibitors of cell wall synthesis.
2) Inhibitors of cell membrane.
3) Inhibitors of protein synthesis.
4) Inhibitors of nucleic acid synthesis.
ANTI MICROBIAL DRUGSGROWTH FACTOR ANALOGS
Sulfa Drugs
Sulfa drugs, discovered by Gerhard Domagk in the 1930s, were the first widely used growth factor analogs
that specifically inhibited the growth of bacteria. The Sulfanilamide, the simplest sulfa drug, is an analog of
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p-aminobenzoic acid, which is itself a part of the vitamin folic acid, a nucleic acid precursor. Sulfanilamide
blocks the synthesis of folic acid, thereby inhibiting nucleic acid synthesis. Sulfanilamide is selectively toxic in
bacteria because bacteria synthesize their own folic acid, whereas most animals obtain folic acid from their
diet.
Sulfa drugs. (a) The simplest sulfa drug, sulfanilamide.
(b) Sulfanilamide is an analog of p-aminobenzoic acid, a precursor
of
(c) folic acid, a growth factor
Isoniazid
Isoniazid) is an important growth factor analog with a very narrow
spectrum of activity Effective only against Mycobacterium,
isoniazid interferes with the synthesis of mycolic acid, a mycobacterial cell wall component. A nicotinamide
(vitamin) analog, isoniazid is the most effective single drug used for control and treatment of tuberculosis.
Nucleic Acid Base Analogs
Analogs of nucleic acid bases formed by addition of
a fluorine or bromine atom are shown in. Fluorine is
a relatively small atom and does not alter the overall
shape of the nucleic acid base, but changes the
chemical properties such that the compound does
not function in cell metabolism, thereby blocking
nucleic acid synthesis. Examples include fluorouracil,
an analog of uracil, and bromouracil, an analog of
thymine. Growth factor analogs of nucleic acids are
used in the treatment of viral and fungal infections
and are also used as mutagens.
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Quinolones
The quinolones are antibacterial compounds that interfere with bacterial DNA gyrase, preventing the
supercoiling of DNA, a required step for packaging DNA in the bacterial cell. Because DNA gyrase is found in
all Bacteria, the fluoroquinolones are effective for treating both gram-positive and gram-negative bacterial
infections (Figure
26.13). Fluoroquinolones such as ciprofloxacin are routinely used to treat urinary tract infections in humans.
Ciprofloxacin is also the drug of choice for treating anthrax because some strains of Bacillus anthracis, the
causative agent of anthrax
Antiviral Drugs
Because viruses use their eukaryotic hosts to reproduce and perform metabolic functions, most antiviral
drugs also target host structures, resulting in host toxicity. However, several compounds are more toxic for
viruses than for the host, and a few agents specifically target viruses.
The most successful and commonly used agents for antiviral chemotherapy are the nucleoside analogs). The
first compound to gain universal acceptance in this category was zidovudine, or azidothymidine (AZT)
inhibits retroviruses such as HIV.
AZT (Azidothymidine) is chemically related to thymidine but is a dideoxy derivative, lacking the 3¿-hydroxyl
group. AZT inhibits
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multiplication of retroviruses by blocking reverse transcription and production of the virally encoded DNA
intermediate. This
inhibits multiplication of HIV. Nearly all nucleoside analogs, or nucleoside reverse transcriptase
inhibitors (NRTI), work by the same mechanism, inhibiting elongation of the viral nucleic acid chain by a
nucleic acid polymerase. Several other antiviral agents target the key enzyme of retroviruses, reverse
transcriptase. Nevirapine, a nonnucleoside reverse transcriptase inhibitor (NNRTI), binds directly to reverse
transcriptase and inhibits reverse transcription. Phosphonoformic acid, an analog of inorganic
pyrophosphate, inhibits normal internucleotide linkages, preventing synthesis of viral nucleic acids. As with
the NRTIs, the NNRTIs generally induce some level of host toxicity because their action also affects normal
host cell nucleic acid synthesis.
Protease inhibitors are another class of antiviral drugs that are effective for treatment of HIV . These drugs
prevent viral replication by binding the active site of HIV protease, inhibiting this enzyme from processing
large viral proteins into individual viral components, thus preventing virus maturation.
A final category of anti-HIV drugs is represented by a single drug, enfuvirtide, a fusion inhibitor composed of
a 36-amino
acid synthetic peptide that binds to the gp41 membrane protein of HIV Binding of the gp41
protein by enfuvirtide stops the conformational changes necessary for the fusion of HIV and T lymphocyte
membranes, thus preventing infection of cells by HIV.
Influenza Antiviral Agents
Two categories of drugs effectively limit influenza infection. The adamantanes amantadine and rimantadine
are synthetic amines that interfere with an influenza A ion transport protein, inhibiting virus uncoating and
subsequent replication. The neuraminidase inhibitors oseltamivir (brand name Tamiflu) and zanamivir
(Relenza) block the active site of neuraminidase in
influenza A and B viruses, inhibiting virus release from infected cells. Zanamivir is used only for treatment of
influenza, whereas oseltamivir is used for both treatment and prophylaxis. The adamantanes are less useful
than the neuraminidase inhibitors because resistance to adamantanes develops rapidly in strains of
influenza virus.
Interferons
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Virus interference is a phenomenon in which infection with one virus interferes with subsequent infection by
another virus. Several small proteins are the cause of interference; the proteins are called interferons.
Interferons are small proteins in the cytokine family that prevent viral replication by stimulating the
production of antiviral proteins in uninfected cells. Interferons are formed in response to live virus,
inactivated virus, and viral nucleic acids. Interferon is produced in large amounts by cells infected with
viruses of low virulence, but little is produced against highly virulent viruses. Highly virulent viruses inhibit
cell protein synthesis before interferon can be produced. Interferons are also induced by natural and
synthetic doublestranded RNA (dsRNA) molecules. In nature, dsRNA exists only in virus-infected cells as the
replicative form of RNA viruses such as rhinoviruses (cold viruses) the dsRNA from the infecting virus signals
the animal cell to produce interferon. Interferons from virus-infected cells interact with receptors on
uninfected cells, promoting the synthesis of antiviral proteins that function to prevent further virus
infection. Interferons are produced in three molecular forms: IFN-α is produced by leukocytes, IFN-β is
produced by fibroblasts, and IFN-γ is produced by immune lymphocytes. Interferon activity is host-specific
rather than virus-specific. That is, interferon produced by a member of one species can only activate
receptors on cells from the same species. As a result, interferon produced by cells of an animal in response
to, for example, a rhinovirus, could also inhibit multiplication of, for example, influenza viruses in cells within
the same species, but has no effect on the multiplication of any virus in cells from other animal species.
Interferons produced in vitro have potential as possible antiviral and anticancer agents. Several approved
recombinant interferons are available. However, the use of interferons as antiviral agents is not widespread
because interferon must be delivered locally in high concentrations to stimulate the production of antiviral
proteins in uninfected host cells. Thus, the clinical utility of these antiviral agents depends on our ability to
deliver interferon to local areas in the host through injections or aerosols. Alternatively, appropriate
interferon-stimulating signals such as viral nucleotides, nonvirulent viruses, or even synthetic nucleotides, if
given to host cells prior to viral infection, might
stimulate natural production of interferon.
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ANTIFUNGAL AGENTS:
Mechanism of action of antibiotics
I.Inhibitors of cell wall synthesis: Ex: Penicillin, Bacitracin, and Cephalosporin Cycloserin.
The cell wall of bacteria contain a chemically distinct polymer “mucopeptide “consisting of polysaccaharides
and a highly coss linked polypeptide. The polysaccaharides regularly contain the amino sugars Nacetylglucosamine and N-acetylmuramic acid. To the anino sugars are attached pentapeptide chains the final
rigidity of cell wall is imparted by cross linking of the peptide chains as a result of transpeptidation reactions
carried out by several enzymes. The peptidoglycan layer is much thicker in the gram –positive bacteria cell
wall than gram-negative.
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ANTIBIOTICS
The initial step in drug action consists of binding of the drug to cell receptors (Pencillin binding proteins).
There are 3-6 PBS (mol.wt. 4- 12x 105 ) , some of which are transpeptidation enzymes. They have different
affinities for the drug. Attachment of penicillin to one PBS may cause elongation of the cell , whereas to
another may cause defect in the periphery of the cell wall and thus cause cell lysis. After -lactam drug has
attached to its receptor , the transpeptidation reaction is inhibited and peptidoglycan synthesis is blocked .
the next step is the inactivation of an inhibitor of autolytic enzymes in the cell wall. There fore lytic enzymes
are actinated and results in the lysis if the environment is isotonic.If the cells are placed in a hypertonic
solution (20% sucrose) , the cels change to protoplast and spheroplast.
Pencillin destroying enzymes (-lactamases) open the -lactam ring of penicillin and cephalosporins and
abolish their antimicrobial activity.
II.Antimicrobial action through inhibition of cell membrane: Ex: Amphotericin B, Colstin , Imidazoles,
Nystatin .
The cytoplasmic membrane of bacteria and fungi has a structure different from that of animal cells and can
be more readily disrupted by certain agents.
Polymyxins act on Gram –positive bacteria ( membrane rich in phosphatidylethanolamine and act like
cationic detergent).
Polyenes act on fungal cell membrane ( sterols are present ) . Polymyxins do not act on fungi and polyenes
do not bacteria.
Antifungal imidazoles impair the integrity of fungal cell membrane by inhibiting the biosynthesis of
membrane lipids.
III.Antimicrobial action through inhibition of protein synthesis. Ex: Chlormphenicol , Erythromycin,
Lincomycins , Tetracyclines.
( Aminoglycosides –Amikacin , Gentamycin , Kanamycin , Neomycin , Streptomycin , Tobramycin ).
Bacteria have 70 s ribosomes , whereas mammalian cells have 80 s ribosomes.
The mode of action aminoglycosides is studied in streptomyces.
1) Aminoglycosides attach to a specific receptor ( P12 ) on the subunit.
2) The aminoglycoside blocks the normal activity of the “initiation complex” of peptide formation ( mRNA + formylmethionine + t-RNA ) .
3) M-RNA message is misread ,therefore wrong aminoacid is inserted leading to a non funtional protein
.
4) Aminoglycoside attachment results in the break up of polyenes to form mesosomes. The overall
result is death of the cell.
30s
50s
Tetracyclines-- block the attachment of Charged aminoacyl t-RNA
Chlamphenicol inhibits peptidyl transferase . Therefore
new aminoacids.
interferes with binding of
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Linocomycins and Macrolides ( 23s r-RNA) inhibit peptide chain synthesis or aminoacyl traslocation .
IV.Antimicrobial action through inhibition of Nucleic acid synthesis. Ex: Nalidixic acid, Novobiocin,
Rifampin, Trimethoprin) .
Rifampin inhibits bacterial RNA synthesis by binding strongly to the DNA –depended RNA polymerase.
Nalidixic and Oxolinic acid block DNA gyrase . Therefpre inhibit DNA synthesis.
Many nucleic acid inhibitors are antiviral agents, but they also adversely affect mammalian nucleic acid
synthesis and cell replication. Antimycins and Mitomycins inhibit bacterial as well as animal cells and
therefore not employed in anti bacterial
chemotherapy.Sulphanamides are structurally similar to para aminobenzoicacid ( PABA ) which is used by
the cell in the synthesis of vitamin folic acid . Sulphanamides can prevent the synthesis of folic acid or they
may be incorporated into the folic acid molecule in place of PABA, making the molecule defective and non
functional. Such drugs which are structurally similar to cellular metabolites and compete with the natural
substances for oncorporation into functionally important components of the cell are called antimetabolites.
Other examples are Ethambutol , Nitrofurantoin , Para-aminosalicyclic acid, 5-flurocytosine , Trimethoprim.
Selection of an antimicrobial agent ( antibiotic ) is decided by the 1) dosage ,2) route of administration
,3)whether combination should be used ,4) toxicity of drug, 5) the potential of drug to induce drug
resistance ,6) age of the patient,7)identification of causative organism,8) history of previous allergic
reactions and 9) cost of therapy.
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Factors Influencing Antimicrobial Susceptibility Testing
pH
The pH of each batch of Müeller-Hinton agar should be checked when the medium is prepared. The exact
method used will depend largely on the type of equipment available in the laboratory. The agar medium should
have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear
to lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other agents may appear to have
excessive activity (e.g., tetracyclines). If the pH is too high, the opposite effects can be expected. The pH can be
checked by one of the following means:
*
Macerate a sufficient amount of agar to submerge the tip of a pH electrode.
*
Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup.
*
Use a properly calibrated surface electrode.
Moisture
If, just before use, excess surface moisture is present, the plates should be placed in an incubator (35C) or a
laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation
(usually 10 to 30 minutes). The surface should be moist, but no droplets of moisture should be apparent on the
surface of the medium or on the petri dish covers when the plates are inoculated.
Effects of Thymidine or Thymine
Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides
and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in falseresistance reports. Müeller-Hinton agar that is as low in thymidine content as possible should be used. To
evaluate a new lot of Müeller-Hinton agar, Enterococcus faecalis ATCC 29212, or alternatively, E. faecalis ATCC
33186, should be tested with trimethoprim/sulfamethoxazole disks. Satisfactory media will provide essentially
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ANTIBIOTICS
clear, distinct zones of inhibition 20 mm or greater in diameter. Unsatisfactory media will produce no zone of
inhibition, growth within the zone, or a zone of less than 20 mm.
Effects of Variation in Divalent Cations
Variation in divalent cations, principally magnesium and calcium, will affect results of aminoglycoside and
tetracycline tests with P. aeruginosa strains. Excessive cation content will reduce zone sizes, whereas low cation
content may result in unacceptably large zones of inhibition. Excess zinc ions may reduce zone sizes of
carbapenems. Performance tests with each lot of Müeller-Hinton agar must conform to the control limits.
Testing strains that fail to grow satisfactorily
Only aerobic or facultative bacteria that grow well on unsupplemented Müeller-Hinton agar should be tested on
that medium. Certain fastidious bacteria such as Haemophilus spp., N. gonorrhoeae, S. pneumoniae, and
viridans and ß-haemolytic streptococci do not grow sufficiently on unsupplemented Müeller-Hinton agar. These
organisms require supplements or different media to grow, and they should be tested on the media described
in separate sections.
Variables affecting antibiotic sensitivity in vivo:
Host
Drug
Parasite
1) Distribution of drug
2) Location of organisms
3) Interfering substances and concentrations.
Methods of Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing methods are divided into types based on the principle applied in each
system. They include:
Diffusion
Stokes method
Kirby-Bauer method
Dilution
Minimum Inhibitory Concentration
i) Broth dilution
ii)Agar Dilution
Diffusion&Dilution
E-Test method
Disk Diffusion
Reagents for the Disk Diffusion Test
1. Müeller-Hinton Agar Medium
Of the many media available, Müeller-Hinton agar is considered to be the best for routine susceptibility
testing of nonfastidious bacteria for the following reasons:
*
*
It shows acceptable batch-to-batch reproducibility for susceptibility testing.
It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.
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*
*
It gives satisfactory growth of most nonfastidious pathogens.
A large body of data and experience has been collected concerning susceptibility tests performed with
this medium.
Preparation of Müeller-Hinton Agar
Müeller-Hinton agar preparation includes the following steps.
1.
Müeller-Hinton agar should be prepared from a commercially available dehydrated base according to
the manufacturer's instructions.
2. Preparation of antibiotic stock solutions
Antibitiotics may be received as powders or tablets. It is recommended to obtain pure antibiotics from
commercial sources, and not use injectable solutions. Powders must be accurately weighed and dissolved in
the appropriate diluents to yield the required concentration, using sterile glassware. Standard strains of stock
cultures should be used to evaluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted in 5
ml volumes and frozen at -20ºC or -60ºC.
Preparation of dried filter paper discs
Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are placed in a Petri
dish and sterilized in a hot air oven.
The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter of 2 mm. This delivers
0.005 ml of antibiotics to each disc.
Storage of commercial antimicrobial discs
Cartridges containing commercially prepared paper disks specifically for susceptibility testing are generally
packaged to ensure appropriate anhydrous conditions. Discs should be stored as follows:
*
Refrigerate the containers at 8C or below, or freeze at -14C or below, in a nonfrost-free freezer until
needed.
*
The unopened disc containers should be removed from the refrigerator or freezer one to two hours
before use, so they may equilibrate to room temperature before opening.
*
Once a cartridge of discs has been removed from its sealed package, it should be placed in a tightly
sealed, desiccated container.
*
When not in use, the dispensing apparatus containing the discs should always be refrigerated.
*
Only those discs that have not reached the manufacturer's expiration date stated on the label may be
used. Discs should be discarded on the expiration date.
Turbidity standard for inoculum preparation
To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity standard, equivalent to a 0.5
McFarland standard or its optical equivalent (e.g., latex particle suspension), should be used.
Disc diffusion methods
The Kirby-Bauer and Stokes' methods are usually used for antimicrobial susceptibility testing.
Procedure for Performing the Disc Diffusion Test
Inoculum Preparation
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1.At least three to five well-isolated colonies of the same morphological type are
selected from an agar plate culture,s transferred into a tube containing 4 to 5 ml of a
suitable broth medium, such as tryptic soy broth.
2.The broth culture is incubated at 35C until it achieves or exceeds the turbidity of the
0.5 McFarland standard (usually 2 to 6 hours)
3.The turbidity of the actively growing broth culture is adjusted with sterile saline or
broth to obtain a turbidity optically comparable to that of the 0.5 McFarland standard.
This results in a suspension containing approximately 1 to 2 x 108 CFU/ml for E.coli
ATCC 25922. Visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white
background and contrasting black lines.
Direct Colony Suspension Method
1. As a convenient alternative to the growth method, the inoculum can be prepared by making a direct broth or
saline suspension of isolated colonies selected from a 18- to 24-hour agar plate (a nonselective medium, such
as blood agar, should be used). The suspension is adjusted to match the 0.5 McFarland turbidity standard,
using saline and a vortex mixer.
Inoculation of Test Plates
1.Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is
dipped into the adjusted suspension. The swab should be rotated several times and pressed firmly on the
inside wall of the tube above the fluid level. This will remove excess inoculum from the swab.
2. The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the swab over the entire sterile
agar surface. This procedure is repeated by streaking two more times, rotating the plate approximately 60
each time to ensure an even distribution of inoculum. As a final step, the rim of the agar is swabbed.
3.The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any excess surface
moisture to be absorbed before applying the drug impregnated disks.
NOTE: Extremes in inoculum density must be avoided. Never use undiluted overnight broth cultures or other
unstandardized inocula for streaking plates.
Application of Discs to Inoculated Agar Plates
1. The predetermined battery of antimicrobial discs is dispensed onto the surface of the inoculated agar plate.
Each disc must be pressed down to ensure complete contact with the agar surface. Whether the discs are
placed individually or with a dispensing apparatus, they must be distributed evenly so that they are no closer
than 24 mm from center to center. Ordinarily, no more than 12 discs should be placed on one 150 mm plate
or more than 5 discs on a 100 mm plate. Because some
of the drug diffuses almost instantaneously, a disc should
not be relocated once it has come into contact with the
agar surface. Instead, place a new disc in another
location on the agar.
2.
The plates are inverted and placed in an incubator
set to 35C within 15 minutes after the discs are applied.
With the exception of Haemophilus spp., streptococci and
N. gonorrhoeae, the plates should not be incubated in
an increased CO2 atmosphere, because the interpretive
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standards were developed by using ambient air incubation, and CO2 will significantly alter the size of the
inhibitory zones of some agents.
Reading Plates and Interpreting Results
1.After 16 to 18 hours of incubation, each plate is examined. If the plate was satisfactorily streaked, and the
inoculum was correct, the resulting zones of inhibition will be uniformly circular and there will be a confluent
lawn of growth. If individual colonies are apparent, the inoculum was too light and the test must be repeated.
The diameters of the zones of complete inhibition (as judged by the unaided eye) are measured, including the
diameter of the disc. Zones are measured to the nearest whole millimeter, using sliding calipers or a ruler, which
is held on the back of the inverted petri plate. The petri plate is held a few inches above a black, nonreflecting
background and illuminated with reflected light. If blood was added to the agar base (as with streptococci), the
zones are measured from the upper surface of the agar illuminated with reflected light, with the cover
removed. 2. The zone margin should be taken as the area showing no obvious, visible growth that can be
detected with the unaided eye. Faint growth of tiny colonies, which can be detected only with a magnifying
lens at the edge of the zone of inhibited growth, is ignored.
The sizes of the zones of inhibition are interpreted by referring to Tables.
Advantages: 1. Easy to perform
Disadvantages: False positives due to the variables.
2. No control strains are required.
International Collaborative Study (ICS Method):
It is performed with single, high content discs. Referred to published regression lines that plot the MIC for
many strains of different species against the zone diameter produced by a disc of the strength used in the
test. Four categories Sensitive, two intermediate and resistant grades are obtained.
Comparative Method:
Principle: The test organism is inoculated on the central 1/3 of the plate and the control strain on the 1/3
above and 1/3 below with a gap of 2-3 mm between them. The antibiotic disc is placed on them. The plate is
incubated at 37c for 18 24 hours. The grading is as follows:
1) Sensitive: - Zone size of the test strain is larger than or equal to or not more than 3 mm smaller than
that the control.
2) Intermediate: - the zone size is at least 3mm but also 3mm smaller than that of the control strain.
3) Resistant:- the zone size of the test strain is smaller than 3mm
The control strains used are: - E.coli- ATCC (American type culture Collection) 25922
NCTC (National collection of type culture) 10418
Pseudomonas aeroginosa- ATCC 27853,NCTC10662
Staphylococcus aureus- ATCC25923 ,NCTC 6571
Clostridium perfringens- NCTC11229
Advantages: More accurate since many of the variables likely to affect accuracy are eliminated.
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Disadvantages: Control strains are needed. Differences in the weight of the inoculums or speed of growth
between the two strains may give rise to inaccurate results.
Dilution Methods
Dilution susceptibility testing methods are used to determine the minimal concentration of antimicrobial to
inhibit or kill the microorganism. This can be achieved by dilution of antimicrobial in either agar or broth
media. Antimicrobials are tested in log2 serial dilutions (two fold).
Minimum Inhibitory Concentration (MIC)
Diffusion tests widely used to determine the susceptibility of organisms isolated from clinical specimens
have their limitations; when equivocal results are obtained or in prolonged serious infection e.g. bacterial
endocarditis, the quantitation of antibiotic action vis-a-vis the pathogen needs to be more precise. Also the
terms ‘Susceptible’ and ‘Resistant’ can have a realistic interpretation. Thus when in doubt, the way to a
precise assessment is to determine the MIC of the antibiotic to the organisms concerned.
There are two methods of testing for MIC:
(a) Broth dilution method
(b) Agar dilution method.
Broth Dilution Method
The Broth Dilution method is a simple procedure for testing a small number of isolates, even single isolate.
It has the added advantage that the same tubes can be taken
for MBC tests also.
Reading of result:
MIC is expressed as the lowest dilution, which inhibited growth
judged by lack of turbidity in the tube.
Because very faint turbidity may be given by the inoculum itself,
the inoculated tube kept in the refrigerator overnight may be
used as the standard for the determination of complete
inhibition.
Standard strain of known MIC value run with the test is used as
the control to check the reagents and conditions.
Minimum Bactericidal Concentrations(MBC)
The main advantage of the ‘Broth dilution’ method for the
MIC determination lies in the fact that it can readily be
converted to determine the MBC as well.
Method
Note the lowest concentration inhibiting growth of the
organisms and record this as the MIC. Subculture all tubes
not showing visible growth and incubate at 37oC overnight.
Reading of result:
These subcultures may show
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 Similar number of colonies- indicating bacteriostasis only.
 A reduced number of colonies-indicating a partial or slow bactericidal activity.
 No growth- if the whole inoculum has been killed
 The highest dilution showing at least 99% inhibition is taken as MBC
Advantages of dilution methods:1) to determine the MIC and MBC of an antibiotic.
2) They permit a quantitative result to be reported, indicating the amount of a given drug necessary to kill or
inhibit the microorganism.
Disadvantages: Laborious and time consuming, costly
The Agar dilution Method
Agar dilutions are most often prepared in petri dishes and have advantage that it is possible to test several
organisms on each plate .If only one organism is to be tested e.g M.tuberculosis,the dilutions can be
prepared in agar slopes.The dilutions are made in a small volume of water and added to agar which has been
melted and cooled to not more than 60oC.Blood may be added and if ‘chocolate agar’ is required,the
medium must be heated before the antibiotic is added.
Reading of results
The antibiotic concentration of the first plate showing  99% inhibition is taken as the MIC for the organism.
Dilution and Diffusion
E test also known as the epsilometer test is an ‘exponential gradient’ testing methodology where ‘E’ in E test
refers to the Greek symbol epsilon ().The E test(AB Biodisk) which is a quantitative method for
antimicrobial susceptibility testing applies both the dilution of antibiotic and diffusion of antibiotic into the
medium.. A predefined stable antimicrobial gradient is present on
a thin inert carrier strip. When this E test strip is applied onto an
inoculated agar plate, there is an immediate release of the drug.
Following incubation , a symmetrical inhibition
ellipse is produced. The intersection of the
inhibitory zone edge and the calibrated carrier
strip indicates the MIC value over a wide
concentration range (>10 dilutions) with
inherent precision and accuracy .
E test can be used to determine MIC for
fastidious organisms like S. pneumoniae,
ß-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also be used for
Nonfermenting Gram Negative bacilli (NFGNB) for eg-Pseudomonas sp. and Burkholderia pseudomallei.
Etest can help
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




Determine the MIC of fastidious, slow-growing or nutritionally deficient micro-organisms, or for a
specific type of patient or infection.
Confirm/detect a specific resistance phenotype e.g. ESBL, MBL, AmpC or GISA/hGISA.
Detect low levels of resistance.
Test an antimicrobial not performed in routine use or a new, recently introduced antimicrobial agent.
Confirm an equivocal AST result.
**********
Drug Resistance
Mechanisms of Drug Resistance:
There are many different mechanisms by which microorganisms might exhibit resistance to drugs.
1) Microorganisms produce enzymes that destroy the active drug. Ex. Staphylococci resistant to
penicillin produce -lactomase that destroys the drug.. Gram-negative bacteria resistant to
aminoglycosides produce adenylating , phosphorylating or acetylating enzymes that destroy the
drug. Gram –negative bacteria may be resistant to chloramphenicol if they produce a
chloramphenicol transferase.
2) Microorganisms change their permeability to the drug. Ex. Tetracyclines accumulate in the
susceptible bacteria but not in resistant bacteria. Streptococci have a natural permeability barrier to
aminoglycosides. Polymyxin resistant also due to change in permeability to the drug.
3) Microorganisms develop an altered structural target for the drug. Ex. Erythromycin resistant
organisms have an altered receptor on the 50s subunit of the ribosome, resulting from the
methylation of a 23s ribosomal RNA.
4) Chromosomal resistance to aminoglycosides is associated with the loss or alteration of a specific
protein in the 30s sub unit of bacterial ribosome that serve as a site in susceptible organisms.
Microorganisms develop an altered metabolic pathway that by pass the reaction inhibited by
drug. Ex.Some sulphonamide resistant bacteria do not require extracellular PABA (Para –amino
benzoic acid), but, like mammalian cells can utilize preformed folic acid.
5) Microorganisms develop an altered enzyme that can still perform its metabolic functions but is much
less affected by the drug than the enzyme in the susceptible organisms.Ex. In some sulphanomide
susceptible bacteria, the tetrahydropteric acid synthase has a much higher affinity for sulphanomide
than for PABA. In sulphanomide resistant mutants, the opposite is the case.
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Origin of Drug Resistance
The origin of drug resistance may be genetic or non genetic.
Non-genetic: 1) Active replication of bacteria is usually required for most antibacterial drug action.
Consequently, microorganism that is metabolically inactive may be phenotypically resistant to drugs.
Ex: Mycobacteria often survive in tissue for many years after infection, yet are restrained by host
defenses and do not multiply. Such “persistent” organisms are resistant to treatment and can’t be
eradicated by drugs. Yet, if they start to multiply, they are fully susceptible to the same drugs.
2) Microorganisms may lose the specific target structure for a drug for several generations and thus be
resistant.
Ex. Penicillin susceptible organisms may change to L-forms during penicillin administration. Lacking most
cell wall , they are then resistant to cell wall inhibitor drugs and may remain so for several generations
as” persisters”.
Genetic Origin.
Most drug resistant microbes emerge as a result of genetic change and subsequent selection process by
antimicrobial drugs. The mechanisms are:1) Chromosomal Resistance: This develops as result of spontaneous mutations in a chromosome that
controls susceptibility to a given antimicrobial drug. The presence of the antimicrobial drug serves as a
selecting mechanism to suppress susceptible organisms and favor the growth of the drug resistant
mutants.
Spontaneous mutations occur with a frequency of 10 7 to 1012
emergence of clinical drug resistance in a given patient occurs.
and thus an infrequent cause of
Ex. Chromosomal mutant resistant to rifampicin occurs with high frequency (10 -5) and consequently,
treatment of bacterial infections with rifampicin alone usually fails.
A narrow region of the bacterial chromosome contains structural genes that code for a number of drug
receptor, including those for erythromycin, aminoglycosides and others. The P12 protein in 30s subunit is
the receptor site for streptomycin and mutation in that region causes resistant.
2) Extra chromosomal Resistance.
Bacteria often contain extra chromosomal genetic elements called plasmids.
R factor are a class of plasmids that carry genes for resistance to one and often several antimicrobial
resistance , often control the formation of enzymes capable of destroying the antimicrobial drugs . Thus,
plasmids determine resistance to penicillin and cephalosporins by carrying genes for the formation of lactamases.
Genetic material and plasmids can be transferred by transduction, transformation and conjugation. In
transposition, transfer of short DNA sequences occurs between a plasmid and a portion of bacterial
chromosome within a bacterial cell.
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Cross Resistance: microorganisms resistant to a certain drug may also be resistant to other drugs that
share a mechanism of action. Ex: Polymyxin and -colistin; Neomycin and Kanamycin. It is because of
relationship between agents that are closely related chemically.
Dangers of Antibiotics
 Cause wide spread sensitivity of population.
 Changes in normal flora of the body.
 Masks serious infection without eradication.
 Cause direct drug toxicity.
 Leads to resistance among pathogens
GENERIC NAME: ciprofloxacin
BRAND NAME: Cipro, Cipro XR, Proquin XR
DRUG CLASS AND MECHANISM: Many common infections in humans are caused by single cell organisms,
called bacteria. Bacteria can grow and multiply, infecting different parts of the body. Medicines that control
and eradicate these bacteria are called antibiotics. Ciprofloxacin is an antibiotic that stops multiplication of
bacteria by inhibiting the reproduction and repair of their genetic material (DNA).
SIDE EFFECTS: The most frequent side effects include nausea, vomiting, diarrhea, abdominal pain, rash,
headache, and restlessness. Rare allergic reactions have been described, such as hives and anaphylaxis
(shock).
Ketoconazole is a synthetic antifungal drug used to prevent and treat skin and fungal infections, especially in
immunocompromised patients such as those with AIDS. Due to its side-effect profile, it has been superseded
by newer antifungals, such as fluconazole and itraconazole. Ketoconazole is sold commercially as an antidandruff shampoo
Ketoconazole (kee-toe-KOE-na-zole) is used to treat infections caused by a fungus or yeast. It works by killing
the fungus or yeast or preventing its growth.
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Ketoconazole cream is used to treat:






Athlete's foot (tinea pedis; ringworm of the foot);
Ringworm of the body (tinea corporis);
Ringworm of the groin (tinea cruris; jock itch);
Seborrheic dermatitis;
``Sun fungus'' (tinea versicolor; pityriasis versicolor); and
Yeast infection of the skin (cutaneous candidiasis).
SIDE EFFECTS: Ketoconazole is generally well tolerated. Ketoconazole can cause rash, itching, nausea and/or
vomiting, abdominal pain, headache, dizziness, fatigue, impotence, and blood count abnormalities. Rarely,
ketoconazole has caused a reaction resulting in serious lowering of the blood pressure and shock
(anaphylaxis). Also rarely, ketoconazole has caused severe depression, hair loss, and tingling sensations.
Ketoconazole shampoo has been reported to result in loss of curl of permanently waved hair.
Method of action
Ketoconazole is structurally similar to imidazole, and interferes with the fungal synthesis of ergosterol, the
main constituent of cell membranes, as well as certain enzymes. It is specific for fungi, as mammalian cell
membranes contain no ergosterol.
As with all azole antifungal agents, ketoconazole works principally by inhibition of an enzyme, cytochrome
P450 14-alpha-demethylase (P45014DM). This enzyme is in the sterol biosynthesis pathway that leads from
lanosterol to ergosterol. Fluconazole and itraconazole have been found to have a greater affinity for fungal
cell membrane than ketoconazole, and thus lower doses of these azoles are required to kill fungi
Metronidazole:
Metronidazole (INN) (IPA: [mɛtrəˈnaɪdəzoʊl]) is a nitroimidazole anti-infective drug used mainly in the
treatment of infections caused by susceptible organisms, particularly anaerobic bacteria and protozoa. It is
marketed by Sanofi-Aventis under the trade name Flagyl, and also by various generic manufacturers.
Metronidazole is also used in the treament of the dermatological condition rosacea, where it is marketed by
Galderma under the trade names Rozex and MetroGel.
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Mode of action
Metronidazole is selectively taken up by anaerobic bacteria and sensitive protozoal organisms because of
the ability of these organisms to reduce metronidazole to its active form intracellularly. The nitro group of
metronidazole is chemically reduced by ferredoxin (or ferredoxin-linked metabolic process) and the products
are responsible for disrupting the DNA helical structure, thus inhibiting nucleic acid synthesis.
side effects of metronidazol
an allergic reaction (swelling of your lips, tongue, or face; shortness of breath; closing of your throat; or
hives);
·seizures;
·numbness or tingling;
·dizziness or loss of coordination; or
·severe diarrhea.
• Other, less serious side effects may be more likely to occur. Continue to take metronidazole and talk to
your doctor if you experience
·darkening of your urine;
·nausea, vomiting, or loss of appetite;
·an unpleasant metallic taste in your mouth;
·constipation or mild diarrhea;
·headache; or
·swollen or sore tongue.
• Side effects other than those listed here may also occur. Talk to your doctor about any side effect that
seems unusual or that is especially bothersome.
Amphotericin B (Fungilin®, Fungizone®, Abelcet®, AmBisome®, Fungisome®, Amphocil®, Amphotec®) is a
polyene antimycotic drug, used intravenously in systemic fungal infections. It was originally extracted from
Streptomyces nodosus, a filamentous bacterium. Currently the drug is available as plain Amphotericin B, as
cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The latter formulations have
been developed to improve tolerability for the patient but may show considerable pharmacokinetic
characteristics compared to plain Amphotericin B.
Mode of action:As with other polyene antifungals, amphotericin B associates with ergosterol, a membrane
chemical of fungi, forming a pore that leads to K+ leakage and fungal cell death. Recently, however,
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ANTIBIOTICS
researchers found evidence that pore formation is not necessarily linked to cell death (i.e. Angewandte
Chemie Int. Ed. Engl. 2004). The actual mechanism of action may be more complex and multi-faceted.
SIDE EFFECTS
General (body as a whole): fever (sometimes accompanied by shaking chills usually occurring within 15 to
20 minutes after initiation of treatment); malaise; weight loss.
Cardiopulmonary: hypotension; tachypnea.
Gastrointestinal: anorexia; nausea; vomiting; diarrhea; dyspepsia; cramping epigastric pain.
Hematologic: normochromic, normocytic anemia.
Local: pain at the injection site with or without phlebitis or thrombophlebitis.
Musculoskeletal: generalized pain, including muscle and joint pains.
Neurologic: headache.
Renal: decreased renal function and renal function abnormalities including: azotemia, hypokalemia,
hyposthenuria, renal tubular acidosis; and nephrocalcinosis. These usually improve with interruption of
therapy. However, some permanent impairment often occurs, especially in those patients receiving large
amounts (over 5 g) of amphotericin B or receiving other nephrotoxic agents. In some patients hydration and
sodium repletion prior to amphotericin B administration may reduce the risk of developing nephrotoxicity.
Supplemental alkali medication may decrease renal tubular acidosis.
The following adverse reactions have also been reported:
General (body as a whole): flushing.
Allergic: anaphylactoid and other allergic reactions; bronchospasm; wheezing.
Cardiopulmonary: cardiac arrest; shock; cardiac failure; pulmonary edema; hypersensitivity pneumonitis;
arrhythmias, including ventricular fibrillation; dyspnea; hypertension.
Dermatologic: rash, in particular maculopapular; pruritus. Less frequent reports of Stevens-Johnson
syndrome have been reported during post-marketing experience
Gastrointestinal: acute liver failure; hepatitis; jaundice; hemorrhagic gastroenteritis; melena.
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Hematologic:
leukocytosis.
agranulocytosis;
coagulation
defects;
thrombocytopenia;
leukopenia;
eosinophilia;
Neurologic: convulsions; hearing loss; tinnitus; transient vertigo; visual impairment; diplopia; peripheral
neuropathy; encephalopathy (see PRECAUTIONS); other neurologic symptoms.
Renal: acute renal failure; anuria; oliguria.
PASSWORD: Antibiotics