ANTIBIOTICS1

ANTIBIOTICSAntimicrobial 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 andSemi-synthetic-Rifampin from Rifampicin ( Streptomyces mediterranei ), Amikacin (from Kanamycin ).

Classification of AntibioticsClassification 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 antibacterialsa) Active against anaerobic organisms.

Ex: metronidazole, lincomycin

b) for tuberculosisEx: Streptomycin, Isoniazid

b) for Chlamydia , Rickettsia , MycoplasmaEx: Erythromycin, Tetracyline

Group -5 Antifungal, antiviral agents

a) Antifungal agents: polyenes, Nystatin

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b) Antiviral : Idoxuridine , AmantadineAntimalignancy antibiotics: Actinomycin ,Mitomycin

Based on the structure:--Lactam antibiotics – pencillin , Cephalosporins , loe mol.wt. -Lactam.

Aminglycosides, Macrolides, Tetracyclines, Polyenes, Nitrofurans.

Based on their sourceNatural, Synthetic and Semi-synthetic

Based on whether they are cidal or static.

Ex: Bacteriocidal : Pencillin , AminoglycosidesBacteriostatic: Sulphanamides , Tetracyclines.

Based on their mechanism of actions1) Inhibitors of cell wall synthesis.2) Inhibitors of cell membrane.3) Inhibitors of protein synthesis.4) Inhibitors of nucleic acid synthesis.

ANTI MICROBIAL DRUGS-GROWTH 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 antibioticsI.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 N-acetylglucosamine 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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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 ( m-

RNA + 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 Tetracyclines-- block the attachment of Charged aminoacyl t-RNA

50s Chlamphenicol inhibits peptidyl transferase . Therefore interferes with binding of new aminoacids.

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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 bacterialchemotherapy.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 pHThe 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.

MoistureIf, 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 ThymineMedia 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 false-resistance 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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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 CationsVariation 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 satisfactorilyOnly 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 drug2) Location of organisms3) 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 Dilution Diffusion&DilutionStokes method Minimum Inhibitory Concentration E-Test methodKirby-Bauer method i) Broth dilution ii)Agar Dilution Disk DiffusionReagents for the Disk Diffusion Test1. Müeller-Hinton Agar MediumOf 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 AgarMüeller-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 solutionsAntibitiotics 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 discsWhatman 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 discsCartridges 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 8C or below, or freeze at -14C 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 preparationTo 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 methodsThe 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 35C 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 Method1. 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 Plates1.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 Plates1. 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 35C within 15 minutes after the discs are applied. With the exception of Haemophilus spp., streptococci and

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N. gonorrhoeae, the plates should not be incubated in an increased CO2 atmosphere, because the interpretive 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 Results1.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 37c 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- NCTC11229Advantages: 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 MethodsDilution 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 MethodThe 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 MethodAgar 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 DiffusionE 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 ResistanceMechanisms 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

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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.

Origin of Drug ResistanceThe 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 107 to 1012 and thus an infrequent cause of emergence of clinical drug resistance in a given patient occurs.

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 anti-

dandruff 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 metronidazolan 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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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: agranulocytosis; coagulation defects; thrombocytopenia; leukopenia; eosinophilia; leukocytosis.

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