Non Antibiotics

Anti-microbial Agents
 Types of Antimicrobial Agents
• Disinfectants, antiseptics and preservatives are chemicals which have the ability to destroy or
inhibit the growth of microorganisms.
 
• Disinfectants;
• Disinfection is process of removing microorganisms including potentially pathogenic ones
from the surfaces of inanimate objects.
 
• Different levels of action:
• High-level disinfection; destruction of all microorganisms but not necessarily bacterial spores.
• Intermediate level disinfection; destruction of all vegetative bacteria including
Mycobacterium tuberculosis but may exclude some viruses and fungi & have little or no
sporicidal activity.
• Low level disinfection; can destroy most vegetative bacteria, fungi and viruses, but this will
not include spores and some of the more resistant microorganism.
 
• Liquid chemical sterilant or chemosteriant name give to indicate that they can affect a
complete kill of all microorganisms.
 Types of Antimicrobial Agents
• Antiseptic;
• Antisepsis is destruction or inhibition of microorganisms on living tissues having the effect
of limiting or preventing the harmful results of infection.
 
• Have adequate antimicrobial activity. They must not be toxic or irritating for skin.
 
• Preservatives;
• They are included in pharmaceutical preparations to prevent microbial spoilage of the
product and to minimize the risk of the consumer acquiring an infection when the
preparation is administered.

• Introduced during manufacture and use of non-sterile products such as oral and topical
medications. In sterile products such as eye-drops and multidose injections, preservatives
should kill any microbial contaminants introduced inadvertently during use.
 
• It is essential that a preservative is not toxic.
 Mode of action of non-antibiotic antibacterial
agents
• Drug targets within the bacterial cell:
 
• Cell wall:
• Target for a group of antibiotics that include the penicillins.
• Low concentrations of disinfectant substances cause cell wall lysis, these low
concentrations cause enzymes whose normal role is to synthesize the cell
wall to reverse their role.
 
• Glutaraldehyde reacts with, and provide irreversible cross-linking in, the cell
wall. As a result, other cell functions are impaired. Found especially in Gram-
positive cells.
•  
 Mode of action of non-antibiotic antibacterial
agents
• Cytoplasmic membrane:
• Actions on it may be divided into three categories:
• Action on membrane potentials:
– Bacteria are able to maintain a gradient of electric potential and pH such that the interior of the
bacterial cell is negative and alkaline.
– This potential (or proton motive force) drives a number of energy-requiring functions;
– Synthesis of ATP.
– Coupling of oxidative processes of phosphorylation.
– A metabolic sequence called oxidative phosphorylation.
– Transport and concentration in the cell of metaboilites.
• Certain chemical substances uncouple oxidation from phosphorylation and
inhibit active transport, are named uncoupling agents. e.g.
Tetrachlorosalicylanilide(TCS)
• They are believed to act by rendering the membrane permeable to protons,
hence short-circuiting the potential gradient.
•  
 Mode of action of non-antibiotic antibacterial
agents
• Action on membrane chain:
– Electron transport chain >> Hexachlorophene inhibits the electron transport
chain & all metabolic activities in aerobic bacteria.
– Adenosine Triphosphate >> Chlorohexidine inhibits the membrane ATPase
& anaerobic processes.
– Enzymes with thiol group >> Mercuric chloride or bronopol antibacterial
agents inhibit enzymes in the membrane and in cytoplasm which contain –
SH groups.
 
• Action on general membrane permeability:
• The membrane make dynamic link between transport & metabolism. The
antimicrobial agent like cetrimide, chlorohexidine & phenol break this linkage
making the membrane permeable to K+ ions causing damage of the membrane.
 
 
 Mode of action of non-antibiotic antibacterial
agents
• Cytoplasm:
• Contain number of important organelles. High concentration of disinfectants will coagulate the
cytoplasm.
• Ribosomes;
• Sites of protein synthesis. H2O2 & p-chloro mercuri benzoate will dissociate ribosome into two
parts.
• Nucleic acid;
• Acridine dyes used as antiseptics (like proflavine & Acriflavine) make interfering with nucleic
acid function causing cell death due to fitting into the double helical structure of it. Interfere
with its function and cause cell death.
• Thiol groups;
• Bronopol, chlorine & hypochlorites will oxidize thiol groups.
• Amino groups;
• Formaldehyde, glutaraldehyde & sulphur dioxide reacts with amino group. Cell death will
follow if these groups are essential for metabolic activity.
 
 
 Factors affecting choice of antimicrobial agent
• Properties of the chemical agent;
– Concentration of chemical, temperature, pH, formulation and tissue toxicity. 
• Microbiological challenge;
• Types of microorganism present:
– Vegetative bacteria, Gram negative & Gram positive species.
– Mycobacterium tuberculosis
– Bacterial spores
– Fungi, Candida albicans & Aspergillus niger.
– Viruses, rotavirus and adenoviruses.
• Intended application;
– Chemicals used must not affect the instrument, e.g. cause corrosion of metals.
– Many materials such as fabrics, rubber and plastics are capable of adsorbing certain disinfectants,
e.g. quaternary ammonium compounds are adsorbed by fabrics, while phenolics are adsorbed by
rubber, the consequence of this being a reduction in concentration of active compound.
 
 Factors affecting choice of antimicrobial agent
• Environmental factors;
• Blood, body fluids & milk reduce the effectiveness of antimicrobial agents to varying
degrees.
• Dried organic deposits may inhibit penetration of the chemical agent. Objects to be
disinfected should be thoroughly cleaned.

• Toxicity of the agent;

• Toxic volatile substances should be kept in covered containers to reduce the level of
exposure to irritant vapor. There should be limits governing the exposure of individuals
to such substances.
• Aldehydes may affect the eyes, skin and induce respiratory distress. Face protection
and impermeable nitrite rubber gloves should be worn when using these agents.
• Sampling and analyzing of the atmosphere may need to be carried out on a periodic
basis.
 
 
 Preservatives
• The ideal preservative should be:
• Effective in low concentration & unaffected by pH change and stable.
• Non toxic, non irritant, non sensitizing and non volatile.
• Colorless, odorless & tasteless. Soluble in all vehicles.
• Compatible with product constituent.
• Have a wide range of activity against contaminating organisms.
 
 
 Pharmaceutical preparations which require an antimicrobial preservative:

• Sterile products;
• Such as parentral injections and eye drops. The function of preservative is to
maintain sterility and to ensure the preparation is safe for administration or
use. It should be effective against a wide range of pathogenic bacteria.
 
• Non-sterile products;
• Such as preparations intended for oral or external use. The function of
preservative is to destroy or to prevent the growth of microorganisms which
may be introduced during preparation or use.
 
• These require effective preservation and special care should also be taken to
avoid contamination during manufacture, packaging and dispensing.
 
 
 An ideal disinfectant would have the following properties:

• An ideal disinfectant would have the following properties:

• High potency.
• Ready soluble in or miscible with water.
• Non-caustic with a low degree of toxicity.
• Complete compatibility with other antimicrobial agents being used and with
other components of formulations.
• Stable on storage.
• Cheap.
• No offensive odor, taste or color.
• No ideal disinfectant exists.
 
 
Types of Antimicrobial Agents
– Weak acids and Esters
• Dissociate -----(active un-dissociated form)
• Food preservatives
• Prevent uptake of substrates (PMF)---uncoupling
– Benzoic acid
» Un-dissociated form—active
» In food and pharmaceuticals
– Limitation:
» pH dependent (pH 4.2)
» Pharmaceuticals have pH 5
– Acetic acid
» Vegetables
» Petrochemicals or natural vinegar
 Weak acids and Esters
• Propionic acid
– Baking to inhibit bacterial and fungal growth
• Sorbic acid
– Preservative for gums and syrup
• Lactic acid
– Destroy airborne M.O
• Esters of p-hydroxybenzoic acids (parabens)
– Methyl, ethyl, propyl and butyl esters
– Good preservatives pH 7-8
– Against fungi, less G+ve, poor G-ve, Pseudomonas ----carbon
source
– Use:
• Emulsion, creams, lotions
• Combination of esters (methyl---aqueous, butyl---oily)
Sulpher dioxid, sulphites and metabisulphites
– Sulpher dixoid----food
– Sulphites and metabisulphites---antioxident and preservatives
– Alcohol:
• Disinfections and antiseptic:
– Ethanol and isopropanol
» Against vegetative forms and T. B not sporicidal
» Low penetration of organic matter---clean not dirt
– Methanol
» Poor antimicrobial activity, toxic
– Ethanol
» Widely used
» Water is essential, 100% not effective (60-95%)
– Iospropanol
» Great biocidal but more toxic
» Viricidal -----non-envelop
 Alcohol used as Preservatives
• Benzyl alcohol:
– 2%, in cosmetic, antibacterial, local anesthetic
• Chlorbutol
– 0.5%, antibacterial and antifungal
– Injection and eye drops (unstable at acidic pH)
• Phenylethanol
– 0.25-05%, good against G-ve
• Phenoxyethanol
– 1%, good activity against Pseudomonas aeruginosa
– Combatable with anionic and cationic surfactant, stable at acidic pH
• Bronopol
– 0.01-0.1%, broad spectrum as well as Pseudomonas
– Stable at acidic pH
 Aldehydes
• Formaldehyde and glutardehyde
• Glutardehyde:
– React with enzyme and proteins (alkylation)
– pH dependent
– Prevent dissociation of ribosome
– Broad spectrum, rapid rate of kill
– Vegetative, spore, fungi, virus, TB
– Not affected by organic matter
– pH 8, high activity , low solubility (polymerization)
– Acidic solution, stable but less active
– Supplied as acidic 2% solution (stable)---activation by convert pH to
optimum (alkayating agent)
– Uses:
• Cold, chemical sterilization for medical and surgical materials
 Aldehydes
• Formaldehyde:
– As liquid or gas
– As vapor-----rooms, safety cabinet
– Formaldehyde + low temp steam (LTS) for sterilization of heat sensitive
– React with amine, amide groups of proteins (nucleic acid)---cross linkage
– Highly toxic, carcinogenic
– Humidity at least 70%, temp. above 20oC----optimum activity
– Against fungi, bacteria, virus, sporicidal
– Supplied as solid polymer or liquid
– Formalin—(40%) aqueous solution, gas librated by heating or mixing with
potassium permanganate and water
• Formaldehyde releasing agents:
– Noxythiolin:
• Dry powder--- aqueous reconstitution---release formaldehyde and N-methylthiourea
• Bacteria, fungi due to noxythiolin as formaldehyde
• Local and accessible body cavity
 Aldehydes
– Taurolin:
• One molecule of taurine + three molecules of formaldehyde
• More stable and soluble
• Affinity to bacterial proteins---(selective)
• Peritonitis
• Hexamine:
• Inactive-----acid hydrolysis—formaldehyde
• Antiseptic in UTI
 Biguanides
• Chlorhexidine:
• Activity in pH 7-8
• Base not soluble, salt ----soluble
• In low conc.(< 200µg/ml): inhibit membrane enzymes, leakage of
cellular components(static)
• In high conc.: coagulation of cytoplasmic components(cidal)
• Uses: antiseptics, little antifungal, not used as preservative
• G-ve less senstive
• Polyhexamethylene biguanides:
• Polymeric biguanides linked by hexamethylene chains
• Against G-ve
• Uses: sterilizing agents in food industry
• Disinfectant in swimming pool
 Halogens
• Chlorine:
– A liquid (compressing and cooling gas)
– Potency as ppm
– Hypochlorites:
• Inexpensive
• Rapid killing (fungi and virus, acid fast bacilli and bacterial spores)
• Compatible with anionic and cationic surfactant
• Disadvantages:
– Corrosive
– Inactivated by organic matter
• Factors affecting activity:
– pH (activity decrease as increase in pH), active un-dissociated form
– More activity by increasing conc.
– Organic matter (reduce activity)
• Uses: disinfections of swimming pools
 Organic chlorine compounds
– N-chloro derivatives
– Ex. Chloramine, dichloramieT
– Disinfectant of contaminated drinking water

– Chloroform:
• Narrow spectrum
• Preservative
 Iodine
– Spectrum:
• G+ve, G-ve, spore, T.B, fungi and viruses
• Slightly soluble in water (solution pot. Iodide)
• Activity less dependent as chlorine on pH and temp.
• Avoid alkaline pH
• Inactivated by organic matter
• Disadvantage:
– Staining
– Sensitization of skin and mucous membranes
– Iodophors:
• Iodine carrier
• Slow release of iodine
• Non-ionic or cationic surfactants act as carrier
• Iodine as micellar aggregates, surfactant as carrier increase wetting capacity
• Used in skin and wound disinfections
• Povidone -iodine
 Heavy metal Derivatives
– Mercury and silver
– Zinc, copper, aluminum and tin
– Mercury compounds:
– Thiomersal
– Phenylmercuric nitrate or acetate
– As bacetriostatic and fungistatic

– Merchurchrome:
– Skin disinfectant
– Thiomersal:
– Preservative for eye drops (0.0001-0.004%), lens solutions
– Phenylmercuric salts (0.002%)
– Preservative on eye drops, multidose injection
– Sterilization by heating with bactericidal
 Heavy metal Derivatives
– Sliver compounds:
– Sliver nitrate in eye drops
– Sliver for treatment of burn infections (resistant Pseudomonas)
– React with thiol and sulfahydryl groups of microbial cells and DNA
– Sliver at high concentration cause protein ppt.
– Copper salts:
– Fungicidal and algicidal
– 0.5-3 µg/ml swimming pool algae
 Hydrogen peroxide
– Antiseptic
– Oxidation of organic debris
– Disinfections of soft lens (protozoa)
– 3-6%
– At conc. 30% and increasing temp.-----sporicidal

– Peracetic acid:
– Against bacteria, fungi, T.B , virus and spore
– Disadvantage: corrosive to metals
– Irritant
 Phenols
– Widely used
– Bactericidal but not sporicidal
– Dilution and organic matters reduce activity
– Active in acidic pH
– Disadvantages:
– Caustic effect
– Tar acids:
– Phenol, cresols, xylenols
– Phenols with high boiling points are more active, poor soluble
– Clear soluble fluids (lysol)
– Mixture of cresols
– Vegetative cells as well as T.B killed within 15 min
– Sudol:
– Xylenols and ethylphenols
– Black fluids:
– Solubilized crude phenol
– White fluids:
 Non-coal tar phenols
– Alkyl and halogenation derivatives
– Octylphenol:
– More active than phenol (G+ve), less than (-ve)
– Fungicidal effect
– Uses: preservative for protienaceous (glues and non food gelatin)
– Phenylphenol:
– Antibacterial and antifungal activity
– Preservative
– Disinfections
– Halo and nitrophenols:
– Increase activity but decrease solubility
– Reduced in organic matter
– Trichlorophenol:
– Bactericidal fungicidal and insecticidal
– Preservatives in textile and wood
 Non-coal tar phenols
• Pentachlorophenol
– Insoluble
– Preservative (wood, textile and paper)
• Chlorocreseol:
– Preservative in injection (0.1%)
• Chloroxylenols:
– In skin infection
• Nitrophenols:
– p.-nitrophenol as preservatives in leather industry
• Bisphenols:
– Against bacteria, fungi and algae
• Dichlorophane:
– Bacteria, fungi, algae
– Prevent algae growth in greenhouses
• Hexachlorophane:
– Cosmetics (0.1%)
• Bromochlorophane:
– Less toxic than hexachlrophane
 Surface active agents
• Anionic, cationic and non-ionic
• Anionic and non-ionic less antimicrobial activity but render bacteria spore
to some antimicrobial agents
• Cationic surface active agents:
– Antibacterial, (QACs)
– Active act pH neutral and slightly alkaline, inactive at acidic (pH 3.5)
– Reduce activity by anionic and organic matters
– G+ve, however, G-ve are resistant
– Limited antifungal with no sporicidal
– Antiseptics
– Benzalkonium chloride and cetrimide
• Surgery, urology, gynecology
• With chlorhexidine (Savlon)
• General environmental sanitation (hospitals)
 Surface active agents
• Anionic:
• Weak ant microbial
• Non-anionic:
• No antimicrobial
• Affect permeability of outer membrane of G+ve, increases sensitivity towards
ant microbial agents
• Ex Tweens (sorbitan derivatives)
• Amphoteric agents:
• Both cationic and anionic
• Have both ant microbial and detergent activity
• Ex. Dodocyl ß-alanine
 Dyes
• Crystal violet, brilliant green and malachite green
• Bacteriostatic and fungistatic
• Topical for treatment of skin infection
• Staining
• Acridine dyes:
• Proflavine, acriflavine
• Skin disinfections
• Bacteristatic
 Chelating agents
• EDTA
• Induce lysis of G-ve bacteria increase cell permeability
• No antimicrobial activity, promote the activity of antimicrobial agents (outer
envelope of G-ve)

• Permeabilizers:
• Polycations
• L-lysine
• release, of LPS (increase of sensitivity of antimicrobial agents)
• Lactoferrin and transferrin
• Iron binding proteins
• Release of LPS (g-ve)
• Citric acid and gluconic acids
• Alkaline medium (as permeabliziers)
• Reduce activity in divalent cation
 Aerial Disinfectants
• Properties:
• Dispersed in air
• Maintain effective conc. In air
• Removal of airborne M.O
• Non toxic non irritant
• Hexylresorcinol
• Lactic acid
• Propylene glycol
• Formaldehyde
 Preservative
• Add to inhibit microbial growth and prevent microbial contamination during
package. Storage and use

• Add to sterile (ophthalmic,otic preparations and multi-dose injections)

• Non sterile (topical, oral)
• Access of M.O to products:
• Raw materials
• Water
• Equipments
• Filling and storage
• During use
• Risk of microbial contaminations:
• Pathogenic
• Cause spoilage of pharmaceutical preparations (active ingredients,
SAA, solubilizing agents)
• Change color,odor and taste
 Preservative
• Ideal preservative:
• Stable
• Effective at low conc.
• Wide spectrum
• Compatible
• Non irritant, non toxic no odor
• Resist microbial degradation
• Miscible with water
• Examples:
• Injections:
• Phenylmercuric nitrate (0.001%)
• Eye drops:
• Benzalkonium chloride (0.01%)
• Oral preparations
• Benzoic acid (0.1%)
• Topical preparations:
• Parabens (0.1-0.2%)
• Cetrimide (0.001-0.002%)
 Anti-microbial combinations
• Ex. Chlorhexidine+cetrimide
• Mode of action of anti-microbial agents:
• Interaction of AA with whole cell:
• Adsorption
• Changing electrophoretic mobility
• -ve charge, AA change electrophoretic mobility
• Cell wall
• Lysis of bacterial cells (formalin, hypochlorite)
• Cross linking of cell wall---impair cell function (glutaradehyde)
• Cytoplasmic membrane
• Action on membrane potential
• ECT (electrical potential and pH gradient), PMF
• Uncouple oxidation from phosphorylation (uncoupling agents)
• Render the membrane permeable to proton (short circuiting the
PMF)
• Ex. Pentachlorophenol and phenoxyethanol
 Mode of action of anti-microbial agents
• Action on membrane enzymes:
• ETC
• Ex. Hexachlorophane inhibit ETC
• ATP
• Chlorhexidine inhibit ATPase
• Enzyme with thiol groups
• Mercuric and silver (thioglucolate or cysteine ---reverse action)
• Inhibit thiol group containing enzymes
• Action on general membrane permeability
• Cetrimide, chlorohexidine
• Leak pot. Ions, aa, purines, pyrimidines and pentose
• Action on cytoplasm
• General coagulation
• Phenol and chlorohexidine ---coagulate cytoplasm
• Ribosome
• Hydrogen peroxide
• Dissociate ribosome
 Mode of action of anti-microbial agents
• Nucleic acids
• Acridine dyes
• React with DNA---function and cell death
• Thiol groups
• Heavy metals (mercury)
• Amino groups
• Aldehyde and sulpher dioxide
• React with aa
• Highly reactive compounds:
• ß- propiolactone, ethylene oxide
• Alkalizing amino, imino, hydroxyl and carboxyl groups
• Thiol and disulphide groups
Mode of action of anti-microbial agents
 Resistance to anti-microbial agents
• Relative microbial responses to biocides
• Spores are resistance than vegetative cells
• Non envelop viruses are more resistance than enveloped
• Prions usually highly resistant
• G+ve more sensitive than G-ve
• T.B more resistant
• General mechanisms of bacterial resistance:
• Intrinsic (natural)
• G-ve, T.B, spores
• Acquired (chromosomally or plasmid or transposones)
• Mutation, acquire genes
 Resistance to anti-microbial agents
• G+ve:
• Peptidoglycan and teichoic acid
• G-ve:
• Enterobacteriacae
• Outer membrane (EDTA)
• Removal of permeability barrier (Mg ions)
• Become more sensitive to hydrophobic antibiotics (fucidic acid and
rifampin)
• EDTA and lysozyme remove completely outer membrane and
peptidoglycan----spheroplasts
• Cationic SAA (chlorhexidine and QACs) damage both outer and
cytoplasmic membranes
• EDTA, destabilize outer membrane, increase activity of many anti-
microbial agents (add EDTA+ chlorxylenol)
 Resistance to anti-microbial agents
• G-ve
• Pseudomonas aeroginosa
• Dangerous
• Have intrinsic resistant O- Specific
• High numbers of phosphate in Lipid
n

n= 4-
A (LPS) 40
• Very sensitive to EDTA
• Mg depleted cells are resistant to
EDTA
Core
oligosaccharide
O
OH
P
O
O

Bivalent cations
O
(Mg 2+, Ca2+) H2N
O O O
Endotoxin monomer Endotoxin Micelles O
P
P O
O
P O NH2
(Mw :4x105 –1x106) O O
Detergent O
O
2+
Ca
Cholic acids OH O
2+
Ca O O O
O O HO O P P
P O
O
O O O
O NH O NH
2+ O O O O
Ca O
O O
OH
O O O

Lipid A
2+ O
Ca

2+
Ca

2+
Ca
2+
Ca
 Resistance to anti-microbial agents
• Mycobacterium
• Acid fast
• Resiatance to chlorohexidien, QACs, mercuric cpds
• Phenol may or may not
• Glutaradehyde lethal effect but slow
• Cell wall highly hydrophobic (mycolic acid)
• Bacterial spores:
• Bacillus and Clostridium
• Glutardehyde sporicide
 Resistance to anti-microbial agents
• Acquire bacterial resistance to biocide
• Mutation, acquire genetic elements
• Mutation:
• Serratia marcescens and Pseudomonas aeruginosa
• QACs and chloroxidine respectively
• Plasmid mediated
• Cationic and anionic
• Mercury
• Narrow spectrum (Hg+2)
• Broad spectrum (+ oragnomercuric cpds)
• Encode mercuric reductase
• Hg+2 to Hg metal-----vaporized
• Broad spectrum plasmid (+ hydrolyases) break carbon mercury
bond
 Sensitivity and resistance of fungi
• Moulds are more resistant than yeast (chlorhexidine)
• Higher conc. needed
• Mechanisms of fungal resistance:
• Intrinsic
• Fungal cell wall as barrier
• Degrading enzymes
• Sacchromyces cerevisiae produce H2S (ppt heavy metal)
• Sensitivity and resistance of viruses
• Lipophilic viruses
• Herpes simplex (lipid envelop)
• Hydrophilic viruses
• Poliovirus (non envelop)
• Lipophilic inactivated by lipophilic biocides
 Activity of biocides against prions
• Resistant
• But very high conc. for long time may inactivate prions
..… Thank you