INTRODUCTON

Antiseptics and disinfectants are used extensively in hospitals and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections (1). Mounting concerns over the potential for microbial contamination and infection risks in the food and general consumer markets have also led to increased use of antiseptics and disinfectants by the general public. A wide variety of active chemical agents (or biocides) are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection, and preservation (2).

DEFINITIONS
Biocide is a general term describing a chemical agent, usually broad spectrum that inactivates microorganisms. Antibiotics are defined as naturally occurring or
synthetic organic substances which inhibit or destroy selective bacteria or other microorganisms, generally at low concentrations. -In general, biocides have a broader spectrum of activity than antibiotics, and, while antibiotics tend to have specific intracellular targets, biocides may have multiple targets.

Disinfectants are products or biocides that are used on inanimate objects or

surfaces such as in hospitals, clinics, homes and schools. They are used to clean surfaces such as toilets, floors, drains, door knobs and garbage cans. Disinfectants aid in maintaining a clean environment to help prevent the spread of harmful bacteria that may cause infections.

Antiseptics are applied to the skin either to sterilize a cut or used for handwashing such as before a surgical procedure or contact with those who are at a high risk of infection such as newborn babies. Antiseptics destroy and inhibit the growth of microorganisms on the skin or mucous membranes. They are not as strong as disinfectants, so they shouldn't be used to clean objects and surfaces (3).

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Although biocides are important chemicals used for a variety of purposes such as killing different types of germs which may cause all kinds of diseases. They may be hazardous waste, irritants and can be harmful to humans, particularly in strong concentrations. Some disinfectants are used to kill crop bacteria and other microorganisms. Some of these disinfectants are capable of causing danger to humans. There is an ongoing controversy over using some disinfectants on crops. Glutaraldehyde is used to kill microorganisms on crops and vegetables. Stronger (4) concentrations could be harmful to humans .

An ideal antiseptic or disinfectant should have following properties:

Should have wide spectrum of activity. Should be able to destroy microbes within practical period of time. Should be active in the presence of organic matter. Should make effective contact and be wettable. Should be active in any pH. Should be stable. Should have long shelf life. Should be speedy. Should have high penetrating power. Should be non-toxic, non-allergenic, non-irritative or non-corrosive. Should not have bad odour. Should not leave non-volatile residue or stain. Should not be expensive and must be available easily. Controlling biofilms.

Such an ideal disinfectant is not yet available. The level of disinfection achieved depends on contact time, temperature, type and concentration of the active ingredient, the presence of organic matter, the type and quantum of microbial load. The chemical disinfectants at working concentrations rapidly lose their strength on standing (5).

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Mechanisms of antibacterial action of antiseptics and disinfectants according to target side:A- Cell wall (Membrane active agent).
1Glutaraldehyde (CH2CHO)2 is an important dialdehyde that has found usage as a disinfectant and sterilant, in particular for low-temperature disinfection and sterilization of endoscopes and surgical equipment and as a fixative in electron microscope. Glutaraldehyde has a broad spectrum of activity against bacteria and their spores, fungi, and viruses (6). - Glutaraldehyde is strongly binding to outer layers of organisms, specifically with unprotonated amines on the cell surface such as E. coli and Staphylococcus aureus. It inhibits dehydrogenase activity and periplasmic enzymes and inhibits spheroplast and protoplast lysis in hypotonic media. It forms protein-DNA crosslinks and inhibit RNA, DNA, and protein synthesis (7, 8).

Mechanism of antimicrobial action of Glutaraldehyde

Target microorganism Bacterial spores Glutaraldehyde action Low concentrations inhibit germination; High concentrations are sporicidal, probably as a consequence of strong interaction with outer cell layers. Action unknown, but probably involves mycobacterial cell wall. Strong association with outer layers of grampositive and gram-negative bacteria; crosslinking of amino groups in protein; inhibition of transport processes into cell. Fungal cell wall appears to be a primary target site, with postulated interaction with chitin. Actual mechanisms unknown, but involve protein-DNA cross-links and capsid changes.
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Mycobacteria

Other non-sporulating bacteria

Fungi Viruses

2Formaldehyde (methanal, CH2O) is a monoaldehyde that exists as a freely water-soluble gas. Its clinical use is generally as a disinfectant and sterilant in liquid or in combination with low-temperature steam. Formaldehyde is bactericidal, sporicidal, and virucidal, but it works more slowly than glutaraldehyde (7, 8). Formaldehyde is an extremely reactive chemical that interacts with protein, DNA, and RNA in vitro. It has long been considered to be sporicidal by virtue of its ability to penetrate into the interior of bacterial spores. The interaction with protein results from a combination with the primary amide as well as with the amino groups. Formaldehyde also reacts extensively with nucleic acid (e.g., the DNA of bacteriophage T2). It forms protein-DNA cross-links in SV40 (Simian Vacuolating Virus 40 T-Ag) , thereby inhibiting DNA synthesis. Low concentrations of formaldehyde are sporostatic and inhibit germination.

B- Cytoplasmic (inner) membrane.

1Chlorhexidine probably the most widely used biocide in antiseptic products (e.g. Cyteal*), in particular in handwashing and oral products (e.g. EZ-care*) but also as a disinfectant and preservative. This is due in particular to its broadspectrum efficacy, persistence on the skin, and low irritation. Despite the advantages of chlorhexidine, its activity is pH dependent and is greatly reduced in the presence of organic matter (9). Chlorhexidine is a bactericidal agent. Its interaction and uptake by bacteria were studied initially by Hugo et al., who found that the uptake of chlorhexidine by E. coli and S. aureus was very rapid and depended on the chlorhexidine concentration (10). -At low concentration, Chlorhexidine damaged to the outer cell layers takes place; the agent then crosses the cell wall or outer membrane by passive diffusion, and attacks the bacterial cytoplasmic or inner membrane or the yeast plasma membrane. Damage to the delicate semipermeable membrane is followed by leakage of intracellular constituents and then cell death (11).
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-High concentrations of chlorhexidine cause coagulation of intracellular constituents. As a result, the cytoplasm becomes congealed, with a consequent reduction in leakage, so there is a biphasic effect on membrane permeability (12).

Mechanism of antimicrobial action of Chlorhexidine

Type of microorganism Bacterial spores Mycobacteria Other non-sporulating bacteria Chlorhexidine action Not sporicidal but prevents development of spores. Inhibits spore outgrowth but not germination (the emergence of cells from resting spores). Mycobacteristatic but not mycobactericidal. Membrane-active agent, causing protoplast and spheroplast lyses. High concentrations cause precipitation of proteins and nucleic acids Membrane-active agent, causing protoplast lysis and intracellular leakage; high concentrations cause intracellular coagulation. Low activity against many viruses; lipidenveloped viruses more sensitive than nonenveloped viruses (Rota and polio viruses); effect possibly on viral envelope, perhaps the lipid moieties.

Yeasts

Viruses

2- The diamidines salts of two compounds, propamidine and dibromopropamidine have been used as antibacterial agents. They are used for the topical treatment of wounds (13). The exact mechanism of action of diamidines is unknown, but they have been shown to inhibit oxygen uptake, generalized membrane damage and induce leakage of amino acids (14).
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3The cationic surfactant agents, as exemplified by quaternary ammonium compounds (QACs), are the most useful antiseptics and disinfectants. They are sometimes known as cationic detergents. QACs have been used for a variety of clinical purposes (e.g., preoperative disinfection of unbroken skin- Salvon* and application to mucous membranes). In addition to having antimicrobial properties and used as eye drop preservative. QACs are also excellent for hard-surface cleaning and deodorization (15). They are not corrosive except at high concentrations cause skin burn; they are stable even in diluted solutions and concentrates, can be stored for a long time without losing their antimicrobial activity(15). Salton proposed the following sequence of events with microorganisms exposed to cationic agents: (i) Adsorption and penetration of the agent into the cell wall. (ii) Reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization (iii) Leakage of intracellular low-molecular-weight material. (iv) Degradation of proteins and nucleic acids. There is thus a loss of structural organization and integrity of the cytoplasmic membrane in bacteria (16). QACs are sporostatic; they inhibit the outgrowth of spores (the development of a vegetative cell from a germinated spore) but not the actual germination processes (development from dormancy to a metabolically active state). Likewise, the QACs are not mycobactericidal but have a mycobacteriostatic action, although the actual effects on mycobacteria have been little studied. The QACs have an effect on lipid, enveloped (including human immunodeficiency virus and HBV) but not nonenveloped viruses.

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4- Phenolic-type antimicrobial agents (Chloroxylenol- e.g. Dettol*) have long been used for their antiseptic, disinfectant, or preservative properties, depending on the compound. They have often been referred to as general protoplasmic poisons, they have membrane-active properties which also contribute to their overall activity (17). Phenol induces progressive leakage of intracellular constituents, including the release of K+, the first index of membrane damage, and of radioactivity from 14Clabeled E. coli. It affected the metabolic activities of S. aureus and E. coli and produced a selective increase in permeability to protons with a consequent an uncoupling of oxidative phosphorylation. The phenolics possess antifungal and antiviral properties. Their antifungal action probably involves damage to the plasma membrane, resulting in leakage of intracellular constituents (18). -Phenol: It is used in dentistry as an analgesic, for dressing of small wounds. In solutions with glycerol, it is used as an antiseptic, and analgesic in mouth ulcers and tonsillitis. -Cresol: It is many times active than phenol and is less damaging to the tissues. It is used for disinfection of utensils, excretory fluids and for washing hands. -Resorcinol : It is less potent than phenol but is keratolytic and antipruritic. It is used for the treatment of various skin disorders like ringworm, eczema, psoriasis, dermatitis.

5- Bis-phenols (17)
The bis-phenols are hydroxy-halogenated derivatives of two phenolic groups connected by various bridges. In general, they exhibit broad-spectrum efficacy but have little activity against P. aeruginosa and molds and are sporostatic toward bacterial spores. Triclosan and hexachlorophene are the most widely used biocides in this group, especially in antiseptic soaps and hand rinses.

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-Triclosan: (2, 4, 4'-trichloro-2'-hydroxydiphenyl ether) exhibits particular activity against Gram-positive bacteria. Its efficacy against Gram-negative bacteria and yeasts can be significantly enhanced by formulation effects. For example, triclosan in combination with EDTA caused increased permeability of the outer membrane. The specific mode of action of triclosan is unknown, but it has been suggested that the primary effects are on the cytoplasmic membrane. In studies with E. coli, triclosan at subinhibitory concentrations inhibited the uptake of essential nutrients, while higher, bactericidal concentrations resulted in the rapid release of cellular components and cell death. Studies with a divalent-iondependent E. coli triclosan mutant for which the triclosan MIC was 10-fold greater than that for a wild-type strain showed no significant differences in total envelope protein profiles but did show significant differences in envelope fatty acids. Specifically, a prominent 14:1 fatty acid was absent in the resistant strain, and there were minor differences in other fatty acid species. It was proposed that divalent ions and fatty acids may adsorb and limit the permeability of triclosan to its site of action. Minor changes in fatty acid profiles were recently found in both E. coli and S. aureus strains for which the triclosan MICs were elevated; however, the MBCs were not affected, suggesting, as for other phenols, that the cumulative effects on multiple targets contribute to the bactericidal activity. -Hexachlorophene: is another bis-phenol whose mode of action has been extensively studied. The primary action of hexachlorophene, based on studies with Bacillus megatherium, is to inhibit the membrane-bound part of the electron transport chain. It induces leakage, causes protoplast lysis, and inhibits respiration. The threshold concentration for the bactericidal activity of hexachlorphene is 10 g/ml (dry weight), but peak leakage occurs at concentrations higher than 50 g/ml and cytological changes occur above 30 g/ml. Furthermore, hexachlorophene is bactericidal at 0C despite causing little leakage at this temperature. Despite the broad-spectrum efficacy of hexachlorophene, concerns about toxicity, in particular in neonates, have meant that its use in antiseptic products has been limited.
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6Anilides (8) have been investigated primarily for use as antiseptics, but they are rarely used in the clinic. Triclocarban (TCC; 3, 4, 4'-triclorocarbanilide) is the most extensively studied in this series and is used mostly in consumer soaps and deodorants. TCC is particularly active against Gram-positive bacteria but significantly less active against Gram-negative bacteria and fungi and lacks appreciable persistency for the skin. The anilides are thought to act by adsorbing to and destroying the semi permeable character of the cytoplasmic membrane, leading to cell death.

C- Effects on DNA and Oxidizing agent.

1- Halogen-Releasing Agents. Chlorine- and iodine-based compounds are the most significant microbicidal halogens used in the clinic and have been traditionally used for both antiseptic and disinfectant purposes and house hold beach. Iodine: Tincture of iodine (2% I2 in 70% alcohol) inactivates proteins and is used as an antiseptic on skin. Iodine is one of the oldest (300 to 400 years) and most effective germicidal agents. It is a broad-spectrum bactericide and a good fungicide with some viricidal action. It will kill spores and is an excellent disinfectant that is effective against protozoa (amebas). It is only slightly soluble in water; iodine is available as a tincture dissolved in alcohol. Problems arise when the alcohol evaporates and the concentration of iodine increases, which can cause burning and staining of skin and fabrics. Iodophors in which the iodine is linked to a carrier molecule that releases free (unbound) iodine slowly. Essentially, four generic compounds may be used as the carrier molecule or complexing agent. These give polyoxymer iodophors (i.e. with propylene or ethyene oxidepolymers), cationic (quaternary ammonium) surfactant iodophors, non-ionic surfactant iodophors and polyvinylpyrrolidone iodophors (PVP-I or povidone-iodine).

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Iodophors work by inhibiting enzyme action and are more effective than iodine. They are nonirritating, good surfactants, and non-staining. Iodophors should be allowed to remain on the skin for 2 minutes to obtain full advantage of the sustained-release iodine. (e.g. Betadine*). The antimicrobial action of iodine is rapid, even at low concentrations, but the exact mode of action is unknown. Iodine rapidly penetrates into microorganisms and attacks key groups of proteins (in particular the freesulfur amino acids cysteine and methionine, nucleotides and fatty acids, which culminates in cell death. Less is known about the antiviral action of iodine, but nonlipid viruses and parvoviruses are less sensitive than lipid enveloped viruses. Similarly to bacteria, it is likely that iodine attacks the surface proteins of enveloped viruses, but they may also destabilize membrane fatty acids by reacting with unsaturated carbon bonds (19) . Surprisingly, some Pseudomonas species survive in the concentrated stock solutions of iodophores. The reasons are unclear, but it may be due to inadequate levels of free iodine in concentrated solutions, the iodine being released from the carrier only with dilution. Pseudomonas species also can form biofilms, which are less permeable to chemicals. Nosocomial infections can result if a Pseudomonascontaminated iodophore is unknowingly used to disinfect instruments. Chlorine-releasing agents (CRAs) are sodium hypochlorite (e.g. Clorox*), chlorine dioxide, and chloramne-T (20). Hypochlorous acid has long been considered the active moiety responsible for bacterial inactivation by CRAs, the OCl2 ion having a minute effect compared to undissolved HOCl. This correlates with the observation that CRA activity is greatest when the percentage of undissolved HOCl is highest. They are highly active oxidizing agents that involve the formation of chlorinated derivatives of nucleotide bases thereby destroy the cellular activity of proteins (21).

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An effective disinfection solution can easily be made by diluting liquid household bleach (5.25% sodium hypochlorite) 1:100 in water, resulting in a solution of 500 ppm chlorine. This concentration is several hundred times the amount required to kill most pathogenic microorganisms and viruses, but it is usually necessary for fast, reliable killing. In situations when excessive organic material is present, a 1:10 dilution of bleach may be required. This is because chlorine also readily reacts with organic compounds and other impurities in water. These compounds consume free chlorine, reducing the germicidal activity of chlorine-releasing compounds. The use of high concentrations, however, should be avoided when possible, because chlorine is both corrosive and toxic (20). Chlorine should not be used on the metal parts of machines that are subject to stress when in use. Do not autoclave chlorine solutions or materials treated with them, as the residual chlorine can vaporize resulting in an inhalation hazard. Do not use chlorine in combination with ammonia, acetylene, butadiene, butane, methane, propane or other petroleum gases, hydrogen, sodium carbide, benzene, finely divided metals, or turpentine. Chlorine may cause irritation to the eyes, skin, and lungs. Wear safety goggles, rubber gloves, aprons, or other protective clothing when handling undiluted solutions (21). 2- Peroxygens. -Hydrogen peroxide (H2O2) is a widely used biocide for disinfection, sterilization, and antisepsis. It is a clear, colorless liquid that is commercially available in a variety of concentrations ranging from 3 to 90%. H2O2 is considered environmentally friendly, because it can rapidly degrade into the innocuous products water and oxygen. Although pure solutions are generally stable, most contain stabilizers to prevent decomposition. H2O2 demonstrates broad-spectrum efficacy against viruses, bacteria, yeasts, and bacterial spores. In general, greater activity is seen against gram-positive than gram-negative bacteria; however, the presence of catalase or other peroxidases in these organisms can increase tolerance in the presence of lower concentrations.

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Higher concentrations of H2O2 (10 to 30%) and longer contact times are required for sporicidal activity, although this activity is significantly increased in the gaseous phase. H2O2 acts as an oxidant by producing hydroxyl free radicals (OH) which oxidize thiol groups in cell components, including lipids, proteins, enzyme and DNA (22). It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted. -Peracetic acid (PAA) (CH3COOOH) is considered a more potent biocide than hydrogen peroxide, being sporicidal, bactericidal, virucidal, and fungicidal at low concentrations ( 0.3%). PAA also decomposes to safe by-products (acetic acid and oxygen) but has the added advantages of being free from decomposition by peroxidases, unlike H2O2. Its main application is as a low-temperature liquid sterilant for medical devices, flexible scopes, and hemodialyzers, but it is also used as an environmental surface sterilant. Similar to H2O2, PAA probably denatures proteins and enzymes and increases cell wall permeability by disrupting sulfhydryl (-SH) and sulfur (S-S) bonds (22).

D- Interaction with thiol groups (23)

-Silver nitrate: The mechanism of the antimicrobial action of silver ions is closely related to their interaction with thiol (sulfydryl,- SH) groups. Lukens proposed that silver salts and other heavy metals such as copper act by binding to key functional groups of fungal enzymes. Ag+ causes the release of K+ ions from microorganisms; the microbial plasma or cytoplasmic membrane, with which is associated many important enzymes, is an important target site for Ag+ activity. In addition to its effects on enzymes, Ag+ produces other changes in microorganisms. Silver nitrate causes marked inhibition of growth of Cryptococcus neoformans and is deposited in the vacuole and cell wall as granules.
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Ag+ inhibits cell division and damages the cell envelope and contents of P. aeruginosa. Bacterial cells increase in size, and the cytoplasmic membrane, cytoplasmic contents, and outer cell layers all exhibit structural abnormalities, although without any blebs. Finally, the Ag1 ion interacts with nucleic acid; it interacts preferentially with the bases in DNA rather than with the phosphate groups. -Silver sulfadiazine: is antibacterial used as a topical burn cream on burns, including chemical burns. It prevents the growth of a wide array of bacteria, as well as yeast, on the damaged skin. AgSD is essentially a combination of two antibacterial agents, Ag+ and sulfadiazine (SD). AgSD has a broad spectrum of activity and, unlike silver nitrate, produces surface and membrane blebs in susceptible (but not resistant) bacteria. AgSD binds to cell components, including DNA. Based on a chemical analysis, Fox proposed a polymeric structure of AgSD composed of six silver atoms bonding to six SD molecules by linkage of the silver atoms to the nitrogens of the SD pyrimidine ring. Bacterial inhibition would then presumably be achieved when silver binds to sufficient base pairs in the DNA helix, thereby inhibiting transcription.

E- Alcohols
Ethyl alcohol (ethanol, alcohol), isopropyl alcohol (isopropanol, propan-2-ol) and n-propanol (in particular in Europe) are the most widely used. Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria), viruses, and fungi but are not sporicidal. Because of the lack of sporicidal activity, alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis (24).

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Many alcohol products include low levels of other biocides (in particular chlorhexidine), which remain on the skin following evaporation of the alcohol, or excipients (including emollients), which decrease the evaporation time of the alcohol and can significantly increase product efficacy (24). The specific mode of action of alcohols, that they cause membrane damage and rapid denaturation of proteins, with subsequent interference with metabolism and cell lysis. Alcohol cause the denaturation of Escherichia coli dehydrogenases and an increased lag phase in Enterobacter aerogenes, speculated to due to inhibition of metabolism required for rapid cell division (25).

Examples: -Ethanol is an effective antiseptic and can be used for a number of purposes in
different concentrations. It can be used as antiseptic, preservative, mild counterirritant solvent, astringent and rubefacient. It acts by precipitating the bacterial proteins. It as an irritant, and should not be applied to mucous membrane, delicate skin or open wound, as it may cause burning sensation(24).

-Isopropanol has been found to be a suitable substitute for ethanol and is used to
disinfect the skin and surgical instruments. It is more potent than ethanol. It cannot be used internally. It is used in a number of pharmaceutical and cosmetic preparations (24).

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Another classification for Antiseptic and Disinfectants (26)

Chemical disinfectants are capable of different levels of action and they are grouped according to their potency: Sterilants can destroy all microorganisms, including their endospores, and viruses. Destruction of endospores usually requires a 6- to 10-hour treatment. These can be used to treat heat-sensitive critical items such as scalpels. High-level disinfectants destroy all viruses and vegetative microorganisms, but they do not reliably kill endospores. Most high-level disinfectants are simply sterilants used for time periods as short as 30 minutes, a time period not long enough to ensure endospore destruction. These can be used to treat semicritical items such as gastrointestinal endoscopes.

Intermediate-level disinfectants destroy all vegetative bacteria including mycobacteria, fungi, and most, but not all, viruses. They do not kill endospores even with prolonged exposure. They can be used to disinfect non-critical instruments such as stethoscopes. Low-level disinfectants destroy fungi, vegetative bacteria except mycobacteria, and enveloped viruses. They do not kill endospores, nor do they reliably destroy naked viruses. Intermediate-level and low-level disinfectants are also called general-purpose disinfectants.

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Classes of Germicidal Chemicals:

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Factors affecting Disinfection Process (26):

1- Types of Microorganisms
Gram negative bacilli are often more resistant than Gram-positive species to many more antibacterial agents because of the outer membrane of Gram-negative bacteria plays an important role in limiting exposure of susceptible target sites to antibiotics and biocides. Some of the most resistant forms of microbes include: Bacterial endospores: The endospores of Bacillus and Clostridium are by far the most resistant forms of life. Only extreme heat or chemical treatment ensures their complete destruction. Chemical treatments that kill vegetative bacteria in 30 minutes may require 10 hours to destroy their endospores. Mycobacterium species: The waxy cell walls of mycobacteria make them resistant to many chemical treatments. Thus, stronger, more toxic disinfectants must be used to disinfect environments that may contain Mycobacterium tuberculosis, the causative agent of tuberculosis. Pseudomonas species are the most resistant Gram negative organism, in some cases can actually grow in some chemical disinfectants. Pseudomonas species are of particular importance in a hospital setting, where they are a common cause of nosocomial infection. Naked viruses such as poliovirus that lack a lipid envelope are more resistant to disinfectants. Hepatitis A is considered to be one of the most resistant viruses to disinfection. Conversely, enveloped viruses, such as HIV and hepatitis B virus (HBV), tend to be very sensitive to heat and chemical disinfectants. Prions are generally considered to be the infectious agents most resistant to chemical disinfectants and sterilization processes. For heat-resistant medical instruments that come into contact with high infectivity tissues or high-risk contacts, immersion in sodium hydroxide (1 N) or sodium hypochlorite (20,000 ppm available chlorine) for 1 hour is advised in WHO guidelines and this must be followed by further treatment including autoclaving, and routine sterilization.
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2- Environmental Conditions
Factors such as pH, temperature, presence of ions in water and presence of fats and other organic materials strongly influence microbial death rates. For example, a solution of sodium hypochlorite (household bleach) can kill a suspension of M. tuberculosis in 150 seconds at a temperature of 50C; whereas it takes only 60 seconds to kill the same suspension with chlorine if the temperature is increased to 55C. The hypochlorite solution is even more effective at a low pH. The presence of dirt, grease, and organic compounds such as blood and other body fluids can interfere with the action of chemical disinfectants either by adsorption or chemical inactivation, thus reducing the concentration of active agent in solution or by acting as a barrier to the penetration of the disinfectant and thus microorganisms are less accessible to the chemical agent. For this reason, it is important to thoroughly clean items before disinfection or sterilization. Also cleaning a product substantially decreases the number of microorganisms, which shortens the time required to sterilize or disinfect the product.

Different forms of Antiseptics and Disinfectants:

- Hand wipes, Hand sanitizers (gels), antiseptic soaps, household sprays and body spray. - Aerosol. - Ointment and creams - Shampoo - Mouthwash and gargle products.

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Bacterial Resistance to Antiseptics and Disinfectants

In recent years, considerable progress has been made in understanding more fully the responses of different types of bacteria to antiseptics and disinfectants. As a result, resistance can be either a natural property of an organism (intrinsic) or acquired by mutation or acquisition of plasmids (self-replicating, extrachromosomal DNA) or transposons (chromosomal or plasmid integrating, transmissible DNA cassettes) (27).

Intrinsic Bacterial Resistance Mechanisms

For an antiseptic or disinfectant molecule to reach its target site, the outer layers of a cell must be crossed. The nature and composition of these layers depend on the organism type and may act as a permeability barrier, in which there may be a reduced uptake or synthesized enzymes may bring about degradation of a compound. Intrinsic (innate) resistance is thus a natural, chromosomally controlled property of a bacterial cell that enables it to circumvent the action of an antiseptic or disinfectant (27).

Examples:
1- Gram-negative bacteria that show a high level of resistance to many antiseptics and disinfectants include P. aeruginosa and Proteus spp.

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The outer membrane of P. aeruginosa is responsible for its high resistance; in comparison with other organisms, there are differences in LPS composition and in the cation content of the outer membrane. The high Mg2+ content aids in producing strong LPS-LPS links; furthermore, because of their small size, the porins may not permit general diffusion through them (28).

2The cell wall of Staphylococci is composed essentially of peptidoglycan and teichoic acid. Neither of these appears to act as an effective barrier to the entry of antiseptics and disinfectants. Since highmolecular-weight substances can readily traverse the cell wall of staphylococci and vegetative Bacillus spp., this may explain the sensitivity of these organisms to many antibacterial agents including QACs and chlorhexidine (29). However, the plasticity of the bacterial cell envelope is a well-known phenomenon. Growth rate and any growth limiting nutrient will affect the physiological state of the cells. Under such circumstances, the thickness and degree of cross-linking of peptidoglycan are likely to be modified and hence the cellular sensitivity to antiseptics and disinfectants will be altered (30). For example, Gilbert and Brown demonstrated that the sensitivity of Bacillus cell to chlorhexidine and 2-phenoxyethanol is altered when changes in growth rate and nutrient limitation are made with chemostat-grown cells (31).

Physiological (phenotypic) adaption as an intrinsic mechanism.

The association of microorganisms with solid surfaces leads to the generation of a biofilm. Biofilms can consist of monocultures, of several diverse species, or of mixed phenotypes of a given species (32). Biofilms are important for several reasons, biocorrosion, reduced water quality, and foci for contamination of hygienic products. Colonization also occurs on implanted biomaterials and medical devices, resulting in increased infection rates and possible recurrence of infection (33).
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Several reasons can account for the reduced sensitivity of bacteria within a biofilm. There may be:(i) Reduced access of a disinfectant (or antibiotic) to the cells within the biofilm, (ii) Chemical interaction between the disinfectant and the biofilm itself. (iii) Production of degradative enzymes (and neutralizing chemicals), or genetic exchange between cells in a biofilm (34).

Examples:1- Gram-negative pathogens can grow as biofilms in the catheterized bladder and are able to survive concentrations of chlorhexidine that are effective against organisms in noncatheterized individuals. Interestingly, the permeability agent EDTA has only a temporary potentiating effect in the catheterized bladder, with bacterial growth subsequent recurring (35).

2- Several instances are known of the contamination of antiseptic or disinfectant solutions by bacteria. For example, Pseudomonas biofilms were found on the interior surfaces of polyvinyl chloride pipes used during the manufacture of povidone-iodine antiseptics (36).

Recently, a novel strategy was described (37) for controlling biofilms through generation of hydrogen peroxide at the biofilm-surface interface rather than simply applying a disinfectant extrinsically. In this procedure, the colonized surface incorporated a catalyst (e.g. phthalocyanine) that generated the active oxygen from a treatment agent only at the biofilm surface interface by the catalytic reaction which means the target cells hidden deep in the biofilm were directly attached.

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Acquired bacterial resistance to biocides

Acquired resistance to biocides results from genetic changes in a cell and arises either by mutation or by the acquisition of genetic material (plasmids, transposons) from another cell 1- Mutation Resistance acquired by mutation can result when bacteria are exposed to gradually increasing concentrations of a biocide e.g. chlorhexidine-resistant Ps. aeruginosa However, in view of the multiple target sites associated with biocide action, it is unlikely that mutation plays a key role in resistance. 2- Plasmids and bacterial resistance to antiseptics and disinfectants 1- Plasmid R124 alters the Omp F (outer membrane protein F) in E. coli, and cells containing this plasmid are more resistant to a QAC (cetrimide) and to other agents (38). Recombinant S. aureus plasmids transferred into E. coli are responsible for conferring increased MICs of cationic agents to the gram-negative organism. For example, a plasmid- borne, QACs resistance determinant from S. aureus cloned in E. coli encodes resistance to QACs, which are expelled from the cells (39). 3MRSA strains often show multiple antibiotic resistances. Furthermore, increased resistance to some cationic biocides (chlorhexidine, QACs, and to another cationic agent ethidium bromide in addition to acridines and the now little used Crystal Violet is found in MRSA strains carrying genes encoding gentamicin resistance). At least three determinants have been identified as being responsible for low-level biocide resistance in clinical isolates of Staph. aureus: - qacA, which encodes resistance to QACs, acridines, ethidium bromide and low-level resistance to chlorhexidine - qacB, which is similar but specifies resistance to the intercalating dyes and QACs
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- qacC which is genetically unrelated to other qac genes, specifies resistance to QACs and low level resistance to ethidium bromide. 4- Bacterial resistance mechanisms to formaldehyde and industrial biocides may be plasmid encoded. Alterations in the cell surface (outer membrane proteins) and formaldehyde dehydrogenase are considered to be responsible. In addition, the so-called TOM (toluene orthomonooxygenase) plasmid encodes enzymes for toluene and phenol degradation (40). 5- Plasmid-mediated efflux pumps are particularly important mechanisms of resistance to many antibiotics, metals and cationic disinfectants and antiseptics such as QACs, chlorhexidine, diamidines, and acridines, as well as to ethidium bromide (41).

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Novel Antiseptic Products. 1- Gendine

contains Gentian Violet and Chlorhexidine, in coating different medical devices, including endotracheal tubes (ETT) and urinary catheters (UC). Gendine-coated UC significantly reduced the amount of viable MRSA or E. coli organisms adhering to their surfaces when compared with silver/hydrogelcoated urinary catheters or control uncoated catheters (P < 0.01). Similarly GND-ETT significantly reduced the adherence of the same organisms as well as P. aeruginosa when compared with control (P 0.02). GND-ETT and GND-UC impregnated using an instantaneous dip method, were shown to have broad-spectrum activity, prolonged antimicrobial durability and high efficacy in inhibiting adherence of organisms commonly causing nosocomial pneumonia and urinary tract infection. Furthermore, these coated devices were shown to be non-cytotoxic.
(42)

2- Dial Complete soap contains triclosan exhibits particular activity against

grampositive bacteria, gram-negativebacteria and yeasts. It has been suggested that the primary effects are on the cytoplasmic membrane, resulted in the rapid release of cellular components and cell death. Reports have also suggested that in addition to its antibacterial properties, triclosan may have anti-inflammatory activity (43).

3- The cell phone antiseptic (44) is used on mobile phone to prevent infection
and remove the dust. It is sprayed on the mobile phone surface to form an invisible antimicrobial film, adsorbing and killing pathogenic microorganisms, which provides mobile phone with long-acting antimicrobial function, maintaining long aseptic condition on mobile phone and infectious routes of microorganisms will decrease.

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Preservatives in Pharmaceutical products.

Preservatives
are included in formulated products as pharmaceuticals, cosmetics and foods to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring an infection when the preparation is administered. Aqueous solutions, syrups, emulsions, suspensions, creams and ointment often provide excellent growth media for microorganisms such as molds yeast and bacteria (typically Pseudomonas, E.coli, Salmonella and Staphylococcus). The increase in microbial activity may cause stability problem such as loss in suspending activity of suspending agents, loss of color, flavor and odor; they must be protected against microbial contamination. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably into non-sterile products such as oral and topical medications during their manufacture and use. In sterile products such as eye drops and multi-dose injections preservatives should kill any microbial contaminants introduced inadvertently during use. Ideally, preservatives should exhibit the following properties: - Possess a broad spectrum of antimicrobial activity encompassing Grampositive and Gram-negative bacteria and fungi - Be chemically and physically stable over the shelf-life of the product - Have low toxicity and should not be Adsorbed on to the container - Should be compatible with other formulation additives. - Tend to be employed at low concentrations - Its efficacy should not be decreased by pH. - In two and three phases emulsion systems in which the preservative may be more solube in the oil phase tha in the aqueous phase. The pH of the preparation must be considered to ensure that the preservative does not dissociate rendering it ineffective or degrade by acid or base catalyzed hydrolysis. The undissociated moiety or molecular form of a preservative
(44)

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possesses preservative capacity because the ionized form is unable to penetrate micro-organisms.

Preservatives often alter cell membrane permeability causing leakage of cell wall
constituents (partial lysis), complete lysis and cytoplasmic leakage and/or coagulation of cytoplasmic constituents (protein precipitation). Other preservatives inhibit cellular metabolism by interference with enzyme systems or cell wall synthesis, oxidation of cellular constituents or hydrolysis. Products such as ophthalmic and injectable preparations are sterilized by autoclaving (20 minutes at 15 pounds of pressure at 120 C followes by dry heat at 180 C for 1 hour) or filteration. However, many of them require the presence of an antimicrobial preservative to maintain aseptic conditions through- out their stated shelf life. -It is clear that Eye lotions intended for use in surgical or first-aid procedures should not contain antimicrobial preservatives and should be in single-use containers. In glaucoma patients the prolonged use of eye drops with preservatives has been associated with changes in the ocular surface accompanied by inflammation. The use of several eye drops at the same time increases exposure to preservatives, since the concentration to which the ocular surface is exposed increases together with the number of applications. Furthermore, repeated doses may result in the accumulation of preservatives (46). -Aqueous formulations in multidose containers shall include the appropriate antimicrobial preservative at adequate concentrations in order to prevent tampering of preparations during the time of use, except in those instances when preparations feature sufficient antimicrobial properties.

*Preservatives are not required for sterile, single-dose products (as

recommended for neonates) when:
The volume to be injected in a single dose exceeds 15mL unless otherwise justified. The preparation is intended for administration by routes where for medical reasons an antimicrobial preservative is not acceptable.
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If the drug formulation itself has sufficient antimicrobial activity

*Also they must avoid access to cuts, scrapes, and burns skin or cerebrospinal fluid and retro-ocular administration (47).

1-

Acids and Esters (48, 49):

Benzoic, sorbic, and propionic acids are weak organic acids that are used as preservatives in many pharmaceuticals and food products such as bread, cheese, and juice as preservative. These are weak acids and will therefore dissociate incompletely to give the three entities HA, H+ and A- in solution. As the undissociated form, HA, is the active antimicrobial agent which alters cell membrane functions and interfere with energy transformation. The low pH at which these acids are most effective is itself sufficient to prevent the growth of most bacteria, so that these preservatives are primarily added to acidic pharmaceutical products and foods to prevent the growth of fungi. ABenzoic acid is included, alone or in combination with other preservatives such as salicylic acid, in many pharmaceuticals. It is advisable to limit use of the acid to preservation of pharmaceuticals with a maximum final pH of 5.0 and if possible < 4.0. It is used in chemically based ointments to treat fungal diseases, such as athlete's foot, ringworm and tinea. A 0.1 percent concentration of benzoic acid is used in pharmaceutical applications. It is used as an antiseptic in mouthwashes and lotions. A disadvantage of the compound is the development of resistance by some organisms, in some cases involving metabolism of the acid resulting in complete loss of activity.

BParabens (alkyl esters of p-hydroxybenzoic acid) are a class of chemicals widely used as preservatives by cosmetic and pharmaceutical industries. Parabens are effective preservatives in many types of formulas. These compounds, and their salts, are used primarily for their bactericidal and fungicidal properties. They can be found in shampoos, commercial moisturizers, shaving gels, personal lubricants, topical/parenteral pharmaceuticals, spray tanning solution, makeup and toothpaste.
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They are active against a wide range of fungi but are less so against bacteria, especially the pseudomonads, which may utilize the parabens as a carbon source. 2Benzyl alcohol: is used as a bacteriostatic preservative at low concentration in intravenous medications. High concentrations can result in toxic effects including respiratory failure, vasodilation, hypotension, convulsions, and paralysis. In Neonates, Benzyl alcohol gasping syndrome may occur due to benzoic acid can accumulate and lead to a decrease in glycine conjugation. Because of this syndrome, the FDA now recommends that drugs containing benzyl alcohol or benzoic acid as preservatives should NOT be used in neonatal nurseries. 3Phenoxy ethanol: is an organic compound which is a glycol ether, frequently used in skin creams and sunscreen. It is recently gaining more attention in the cosmetic industry as a preservative. Phenoxethanol provides a broad spectrum anti- microbial activity against either gram positive or gram negative bacteria, yeast and molds and is used as a stabilizing agent. -It is found in insect repellents, dyes, inks and in many pharmaceuticals. -It acts as a good penetrating agent and is used in detergents, cosmetics and in skin ointments. -It is also used in the perfumes as a fixative by reducing the evaporation rate of the perfumes. -It is used as a substitute for preservatives which releases formaldehyde. -It is also used in vaccines, to help the vaccine remain unchanged when the vaccine is exposed to heat, light, acidity, or humidity. The side effects of Phenoxyethanol can affect the central nervous system and also can induce vomiting and diarrhea and it can also affect the eyes and lungs.

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4Benzalkonium chloride (50) is cationic surfactants, used as preservatives in the formulation of eye drops. The mechanism of bactericidal action is thought to be due to disruption of intermolecular interactions. This can cause dissociation of cellular membrane lipid bilayers, which compromises cellular permeability controls and induces leakage of cellular contents. Other biomolecular complexes within the bacterial cell can also undergo dissociation. Enzymes, which finely control a wide range of respiratory and metabolic cellular activities, are particularly susceptible to deactivation. Benzalkonium chloride solutions are active against bacteria and some viruses, fungi, and protozoa. Bacterial spores are considered to be resistant. Solutions are bacteriostatic or bactericidal according to their concentration. Gram-positive bacteria are generally more susceptible than Gram-negative. Activity is not greatly affected by pH, but increases substantially at higher temperatures and prolonged exposure times. 5Edetate disodium (EDTA) is a chelating agent used in a variety of nonophthalmic products, including hair conditioner, facial cleansers, aftershaves and deodorants. In the medical field, uses include the treatment of acute mercury poisoning, lead poisoning and hypercalcemia. EDTA has gained use in ophthalmic solutions owing to its ability to bind metals. Therapeutically, EDTA has been used to remove calcified plaques that occur in the superficial cornea in band keratopathy. EDTA has also been used in eye washes to aid in neutralization of calcium hydroxide or lime burns to the cornea. Edetate disodium also has preservative effects based on its ability to chelate. When added to topical medicines in low concentrations, EDTA has been shown to inactivate trace amounts of heavy metals, which aids in the preservation of the solution. Ophthalmic solutions that have employed EDTA include Acular (ketorolac tromethamine ophthalmic solution) and Betagan (levobunolol hydrochloride ophthalmic solution USP).

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6Sodium nitrite used as food preservative to add to meat or smoked fish; it prevents the growth of bacteria, particularly Clostridium botulinum, the bacterium responsible for botulism, a potentially deadly disease. Nitrites are being gradually phased out of food processing for two reasons:First, they are themselves toxic in large amounts; a lethal dose of nitrites for a human being is 22 mg per kg of body weight. Second, nitrites in meat can react with the breakdown products of amino acids in the acidic environment of the human stomach to form nitrosamines, substances that are known to be carcinogenic. To be permitted to use sodium nitrite to prevent the growth of C. botulinum in smoked fish or meat, the manufacturer must show that the maximum amount of nitrite in the food will be no more than 200 parts per million (ppm). 7Vitamin C (ascorbic acid) also occurs naturally in many fruits and vegetables, particularly citrus fruits. It is a water-soluble vitamin. The salts of ascorbic acidsodium ascorbate, calcium ascorbate, and potassium ascorbate are also water-soluble and are often added to fresh-cut apples, peaches, and other fruits (Antioxidants) due to it can inhibit the action of the enzyme phenolase which turns surfaces of cut apples and potatoes brown.

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48- Types of Preservatives in Pharmaceutical Chemistry | eHow.com http://www.ehow.com/list_6134102_types-preservatives-pharmaceutical chemistry.html 49- http://www.fda.gov/cosmetics/productandingredientsafety/selectedcosmeticingredient s/ucm128042.htm 50- en.wikipedia.org

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