Disinfection and Biosecurity for Veterinary Medicine (PDF)

# Disinfection and Biosecurity For Prevention and Control of Disease in Veterinary Medicine ## Notes for Bachelor's students, School of Veterinary Medicine ## Considerations and assessment for a disinfection action plan Before selecting a disinfectant to use, there are several factors that must...

# Disinfection and Biosecurity For Prevention and Control of Disease in Veterinary Medicine ## Notes for Bachelor's students, School of Veterinary Medicine ## Considerations and assessment for a disinfection action plan Before selecting a disinfectant to use, there are several factors that must be considered - Some disinfectants are effective for routine disinfection protocols at the farm or veterinary clinic level - While others are necessary for outbreak situations. ## Factors Affecting Chemical Disinfectant Efficiency ### Microorganism considerations - Degree of susceptibility to disinfectants **Innate Resistance of Microorganisms** - Microorganisms vary greatly in their resistance to chemical germicides and sterilization processes. - Intrinsic resistance mechanisms in microorganisms to disinfectants vary. For example, spores are resistant to disinfectants because spore coat and cortex act as a barrier, mycobacteria have a waxy cell wall that prevents disinfectant entry, and gram-negative bacteria possess an outer membrane that acts as a barrier to the uptake of disinfectants. **Microorganisms vary in their degree of susceptibility to disinfectants.** - In general, Gram-positive bacteria are more susceptible to chemical disinfectants than Gram-negative while mycobacteria and bacterial endospores are more resistant. - The hydrophilic, non-enveloped viruses (adenoviruses, picornaviruses, reoviruses, rotaviruses) are more resistant to disinfection than lipophilic, enveloped viruses (coronaviruses, herpesviruses, orthomyxoviruses, paramyxoviruses). | Resistance Level | Type | | :------------------------------------------ | :--------------------------------------- | | Most Resistant | Prions | | | Bacterial spores, Cryptosporidium, | | | Mycobacteria (e.g., M. tuberculosis) | | | Nonlipid or small viruses (e.g., poliovirus) | | | Fungi (e.g., Aspergillus, and Candida) | | | Lipid or medium-size viruses (e.g., herpes) | | | Vegetative bacteria (e.g., Staphylococcus,| | | and Pseudomonas) | | Sterilization | Spores: Bacterial, fungal | | | Mycobacteria, TB bacilli | | High Level Disinfection | Hydrophilic viruses | | Intermediate Disinfection | Vegetative fungi & bacteria | | Low Disinfection | Lipophilic viruses | | Least Resistant | Viruses with lipid envelopes | ### Ability to survive or persist in the environment Pathogenic microorganisms also vary in their ability to survive or persist in the environment (i.e., bedding, debris, and feed). - Additionally, some microorganisms are also effective at creating a biofilm that enhances their ability to persist in the environment and avoid the action of disinfectants. - Removal of all organic material prior to application of a disinfectant is essential. - Items or equipment removed from the area, including those used for cleaning (i.e., brooms, shovels, buckets, hoses), must also be decontaminated before reuse or disposal. - Some disinfectants may have some efficacy or residual activity in the presence of organic material (i.e., phenols) and should be considered in circumstances where complete removal of organic debris is difficult. **Biofilms: Bacteria's Elixer of Survival** - Biofilms are the slimy layers formed when aggregates of bacteria encase themselves in a hydrated matrix of polysaccharide and protein. **Biofilm formation:** | Stage | Description | | :---------- | :------------------------------------------------------------------------------------------------------------------------------------------------ | | Attachment | After recognizing that the current surroundings can provide sufficient nutrients, the bacteria first attach to a compatible surface. | | Colonization | They then exude extracellular polymeric substances (EPS) that hold the bacteria together as a community. | | Growth | Once inside the slimy EPS, the bacteria are free to grow, divide, and even disperse individual cells throughout the water. Within their customized microniches, biofilm bacteria can live in a primitive homeostatic and circulatory system. A coat of biofilm can thus help bacteria thrive in unfavorable environments rather than constantly stay in a dormant state. | ## Disinfectant considerations ### Concentration and Potency of Disinfectants - The more concentrated the disinfectant, the greater its efficacy and the shorter the time necessary to achieve microbial kill. - Except lodophors. - The length of the disinfection time depends on the potency of the germicide. - Ex. 70% Isopropyl alcohol destroyed 104 M. tuberculosis in 5 minutes, whereas a simultaneous test with 3% phenolic required 2-3 hours to achieve the same level of microbial kill. - Some disinfectants may be more efficacious at higher concentrations; these levels may be limited by the degree of risk to personnel, surfaces or equipment, as well as the cost of the chemical. - However, over-dilution of a product may render the disinfectant ineffective to the target microorganism. - Use of the proper concentration of a disinfectant is important to the best results for each situation. - Some products will have different dilutions depending on the desired use of the product. - The product label will list the best concentration to use for each situation. ### Application method: - There are a variety of ways to apply disinfectants. - Object surfaces or walls of a building may be treated with a disinfectant solution by wiping, brushing, spraying. - Portable items should be soaked in a container of disinfectant. - Fumigation may be used in some situations but is inefficient in buildings with ill-fitting doors and windows, or damaged roofs. Spraying is much better than applying by brushing ### Contact time - Appropriate contact times are essential. - Disinfectants may vary in the contact time needed to kill versus inactivate microorganisms. For example, 70% isopropyl alcohol can destroy Mycobacterium tuberculosis in 5 minutes, whereas 3% phenol requires 2-3 hours. - The minimum contact time needed is normally stated on the product label. Areas being disinfected should be well soaked with the disinfectant selected to avoid drying before the end of the optimum contact time. Some chemicals may have residual activity (i.e., QAC) while others may evaporate quickly (i.e., alcohols). ### Atomic composition of the molecule - The atomic composition of the molecule influences its germicidal action. - In aliphatic alcohol, bactericidal action increases with the No. of carbon atoms So, Amyl alcohol ($C_5H_{11}OH$) is stronger than Ethyl alcohol ($C_2H_5OH$) which is stronger than Methyl alcohol ($CH_3OH$). - The arrangement of the carbon in the molecule exerts an influence on their germicidal value. - The three isomers of cresol are: Para-cresol (is more powerful) than Meta-cresol (which is more powerful) than ortho-cresol. All these isomers are more powerful than phenol. ### Stability and storage - Some disinfectants (e.g., sodium hypochlorite) lose stability quickly after being prepared for use or when stored over long periods, especially in the presence of heat or light. - To maximize stability and shelf life, products should be stored in a dark, cool location and preferably in stock concentrations. - Use of an outdated or inactivated product may result in the use of a non-efficacious product and will lead to a false sense of bio-security. Disinfectant product labels will list the shelf life of the concentrated product. ### Solvent of the disinfectants - Usually water is the best diluents used for disinfection. Any disinfectant in oily form is less effective or ineffective at all. ### Safety precautions - Most disinfectants can cause irritation to eyes, skin and/or the respiratory tract; therefore, the safety of all personnel should be considered. - Training on proper storage, mixing and application procedures is essential. - Personal protective equipment (PPE) such as gloves, masks and eye protection, should be worn during the mixing or application of disinfectants. - All chemical disinfectant have a Material Safety Data Sheets (MSDS) listing the stability, hazards and personal protection needed, as well as first aid information. This information should be available to all personnel. ### Expense, Economic considerations: - Disinfection protocols are generally a cost-effective means of reducing pathogenic organisms. - Disinfectants cost should always be calculated on a per gallon of use/dilution rather than the cost of concentrate. - For example, a QAC that costs 68.00 $ per gallon of concentrate will cost 0.27 $ per diluted gallon (0.5 ounce concentrate per gallon of water). - Considering a gallon of diluted disinfectant approximately covers 100-150 square feet (10-15 m2), the cost for disinfecting a 500 square foot room is 1.35 $. ## Environmental considerations ### Purpose of disinfection protocol - What are the goals for the disinfection protocol? - Are they to prevent an infectious disease, minimize disease spread, or control an outbreak? - Answers should be addressed to develop an effective disinfection plan. - Ineffective disinfection methods can lead to a false sense of bio-security, which can inevitably lead to further spread of a disease. - Routine practice in biosecurity programmes designed to exclude specific diseases. - Routine sanitary measures employed to reduce disease incidence within farms. - While others are necessary for outbreak situations disease eradication (stamping out) efforts. - The specific reason for disinfection will determine the disinfection strategy used and how it will be applied. ### Organic load - Presence of organic matter: organic matter as blood, serum, faeces, sewage, pus, food residue, milk, vaginal, uterine discharges, soil, bedding, litter...etc - The level of organic material (on an item or in areas to be disinfected can greatly impact the efficacy of a product or protocol. - Organic matter can interfere with the antimicrobial activity of disinfectants in at least two ways: - Organic matter provides a physical barrier that protects microorganisms from contact with the disinfectant. - Most commonly, interference occurs by a chemical reaction between the germicide and the organic matter resulting in a complex that is less germicidal or non germicidal. **How organic matter interferes with disinfectants:** - Organic matter may protect the bacteria by forming a coat on it and so prevent access of the disinfectants. - Disinfectant may react chemically with organic matter giving rise to non-germicidal product. - Organic matter may form insoluble compound and remove it from its germicidal activity. - Fats in serum and milk may inactivate the disinfectant. - Particulate and colloidal matter may absorb the disinfectant and remove it from the solution. ### Surface topography - The type of surface to be disinfected can have a great impact on effectiveness of a disinfection plan. - An ideal surface to be disinfected is smooth. - Porous, uneven, cracked, or pitted surfaces, especially wooden surfaces and earthen floors, can hide microorganisms and are difficult to be disinfected. ### Temperature - In general, most disinfectants work best at temperatures above 68°F. - The activity of most disinfectants increases as the temperature increases...more effective because the penetrating power increases. - However, elevated temperatures may accelerate evaporation of a disinfectant, which can reduce contact time and decrease efficacy. - Furthermore, too great an increase in temperature causes the disinfectant to degrade and weakens its germicidal activity. - Colder temperatures may also reduce the efficacy of some products. ### Relative humidity - Relative humidity is the most important factor influencing the activity of gaseous disinfectants/sterilants. - Ex. formaldehyde - For example, formaldehyde fumigation requires a relative humidity in excess of 70% for effectiveness ### Water hardness - Water hardness (i.e., high concentration of divalent cations) reduces the rate of kill of certain disinfectants because divalent cations (e.g., $Ca^{+2}$, $Mg^{+2}$) in the hard water interact with the disinfectant to form insoluble precipitates. - The water source used when cleaning and diluting disinfectants is also important. Water "hardness" can inactivate or reduce the effectiveness of certain disinfectants (i.e., QAC, phenols). "Hard" water contains calcium ($Ca^{+2}$) and magnesium ($Mg^{+2}$) (leached from limestone and other minerals as groundwater passes over it). These ions can complex with cleaning compounds, leading to residue buildup, which may reduce their cleaning action. However, many detergents have chelating agents, such as EDTA, to help bind these ions. ### PH - An increase in pH improves the antimicrobial activity of some disinfectants (e.g., glutaraldehyde, quaternary ammonium compounds) but decreases the antimicrobial activity of others (e.g., phenols, hypochlorites, and iodine). - Some chemical agents as phenolics, hypochlorite and iodine become less effective at alkaline pH. On the other hand, quaternary ammonium compounds are more effective at alkaline pH. - For example, the efficacy of glutaraldehyde is dependent on pH, working best at pH greater than 7. QACs have the greatest efficacy at pH of 9-10. ### Presence of other chemicals - Presence of other chemicals can affect the efficacy of some disinfectants. For example, iodine agents are inactivated by QACs, while phenols are commonly formulated with soaps to increase their penetrative ability. ### Health, safety and the environment - The health and safety of humans and/or animals should always be a primary consideration when selecting a disinfectant. - Most disinfectants have some level of hazard associated with their use. Some pose a serious threat to human and animal health (i.e., aldehydes, phenols, sodium hydroxide). Some cannot be used when animals are present or must be thoroughly rinsed away with potable water prior to restocking. Personnel training, personal protective measures and safety precautions should always be taken. ### Value of the item to be decontaminated - Equipment will have varying degrees of value and disposability. Items that are reusable will need to be handled differently than those that will be discarded. ## Mode of Action of Chemical Disinfectants - Disinfectants are usually complex formulations of active molecules, sometimes also containing co-solvents, chelating agents, acidic or alkaline agents, or surface-active or anti-corrosive products. - Disinfectants act on microorganisms in two different ways: - Growth inhibition (bacteriostasis, fungistasis) or lethal action (bactericidal, fungicidal or virucidal effects). Only the lethal effects are of interest in disinfection and, as the objects of treatment have no inherent means of defence, lethality is the desired objective. - Understanding the mode of action of disinfectants requires an examination of the structure and functions of the pathogen cell. ## Acidic and Alkaline Compounds - The efficacy of acidic and alkaline agents is linked to the concentration of hydrogen ($H^+$) and hydroxyl ($OH^-$) ions, as follows: - $H^+$ ions destroy the amino-acid bond in nucleic acids, modify the cytoplasmic pH and precipitate proteins. - $OH^-$ ions saponify the lipids in the enveloping membrane, leading to destruction of the superficial structure. - A pH higher than 10.0 disorganises the structure of the peptidoglycane and causes hydrolysis of the nucleotides of the virus genome. - Similarly, the pH must exceed 12.0 to act on mycobacteria. ### Acids **Examples:** - Acetic acid, Citric acid - Acidic disinfectants function by destroying the bonds of nucleic acids and precipitating proteins. - $H^+$ ions destroy the amino-acid bond in nucleic acids, modify the cytoplasmic pH and precipitate proteins. ### Oxidizing agents **Examples:** - Hydrogen peroxide, Peracetic acid, Virkon-S - Oxidizing agents are broad spectrum, peroxide based compounds that function by denaturing the proteins and attack lipids of microorganisms. - **Mode of Action:** **Hydrogen Peroxide** - Hydrogen peroxide works by producing destructive hydroxyl free radicals that can attack membrane lipids, DNA, and other essential cell components. - **Microbicidal Activity**: Hydrogen peroxide is active against a wide range of microorganisms, including bacteria, yeasts, fungi, viruses, and spores. A 0.5% hydrogen peroxide demonstrated bactericidal and virucidal activity in 1 minute and mycobactericidal and fungicidal activity in 5 minutes. - **Peracetic acid** - Peracetic acid is a strong oxidizing agent and is a formulation of hydrogen peroxide and acetic acid. - It is considered bactericidal, fungicidal, sporicidal and virucidal. - It is also effective against mycobacteria and has some activity in the presence of organic material. - **Peracetic Acid Sterilization** - Peracetic acid is a highly biocidal oxidizer that maintains its efficacy in the presence of organic soil. - **Mode of Action**: It is thought to function as other oxidizing agents, i.e., it denatures proteins, disrupts cell wall permeability, and oxidizes sulfhydral and sulfur bonds in proteins, enzymes, and other metabolites. - **Mode of Action**: Peracetic acid denatures proteins and oxidises lipids of microorganisms, leading to disorganisation of the membrane. - **Microbicidal Activity**: Peracetic acid will inactivate gram-positive and gram-negative bacteria, fungi, and yeasts in 5 minutes at <100 ppm. In the presence of organic matter, 200-500 ppm is required. For viruses, the dosage range is wide (12-2250 ppm), with poliovirus inactivated in yeast extract in 15 minutes with 1500 to 2250 ppm. Bacterial spores in suspension are inactivated in 15 seconds to 30 minutes with 500 to 10,000 ppm (0.05 to 1%). ## Gas Sterilization ### Ethylene Oxide (ETO) - **Mode of Action:** The microbicidal activity of ETO is considered to be the result of alkylation of protein, DNA, and RNA. Alkylation or the replacement of a hydrogen atom with an alkyl group, within cells prevents normal cellular metabolism and replication. - **Uses:** ETO is used in healthcare facilities to sterilize critical items (and sometimes semicritical items) that are moisture or heat sensitive and cannot be sterilized by steam sterilization. ### Hydrogen Peroxide Gas Plasma: - Gas plasmas have been referred to as the fourth state of matter (i.e., liquids, solids, gases, and gas plasmas). - Gas plasmas are generated in an enclosed chamber under deep vacuum using radio frequency or microwave energy to excite the gas molecules and produce charged particles, many of which are in the form of free radicals. - A free radical is an atom with an unpaired electron and is a highly reactive species. - **Mode of Action:** The process inactivates microorganisms primarily by the combined use of hydrogen peroxide gas and the generation of free radicals (hydroxyl and hydroproxyl free radicals) during the plasma phase of the cycle. The proposed mechanism of action of this device is the production of free radicals within a plasma field that are capable of interacting with essential cell components (e.g., enzymes, nucleic acids) and thereby disrupt the metabolism of microorganisms. - **According to the manufacturer, the process operates in the range of 37-44°C and has a cycle time of 75 minutes.** - The sterilization chamber is evacuated and hydrogen peroxide solution is injected from a cassette and is vaporized in the sterilization chamber to a concentration of 6 mg/l. - The hydrogen peroxide vapor diffuses through the chamber (50 minutes), exposes all surfaces of the load to the sterilant, and initiates the inactivation of microorganisms. - An electrical field created by a radio frequency is applied to the chamber to create a gas plasma. - Microbicidal free radicals (e.g., hydroxyl and hydroperoxyl) are generated in the plasma. - The excess gas is removed and in the final stage of the process the sterilization chamber is returned to atmospheric pressure by introduction of high-efficiency filtered air. - The by-products of the cycle (e.g., water vapor, oxygen) are non-toxic, so the sterilized materials can be handled safely, either for immediate use or storage. ### Vaporized Hydrogen Peroxide (VHP®): - The method for delivering VHP to the reaction site uses a deep vacuum to pull liquid hydrogen peroxide (30-35% concentration) from a disposable cartridge through a heated vaporizer and then, following vaporization, into the sterilization chamber. - **Microbicidal Activity**: Hydrogen peroxide vapor decontamination has been found to be a highly effective method of eradicating MRSA, Serratia marcescens, Clostridium botulinum spores and Clostridium difficile from rooms, surfaces and/or equipment. Vapor-phase Hydrogen peroxide was shown to possess significant sporocidal activity. VHP offers several appealing features that include: - Rapid cycle time (e.g., 30-45 minutes). - Low temperature. - Environmentally safe by-products ($H_2O$, oxygen ($O_2$)). - Good material compatibility. - Ease of operation, installation and monitoring. ### Ozone: - Ozone has been used for years as a drinking water disinfectant. Ozone is produced when $O_2$ is energized and split into two monatomic (O) molecules. The monatomic oxygen molecules then collide with $O_2$ molecules to form ozone, which is $O_3$. Thus, ozone consists of $O_2$ with a loosely bonded third oxygen atom that is readily available to attach to, and oxidize, other molecules. - Highly unstable (i.e., half-life of 22 minutes at room temperature). - On bacteria; Ozone probably acts by oxidation. The additional oxygen atom makes ozone a powerful oxidant that destroys microorganisms by oxidation. - In viruses; Ozone inactivates bacteriophages F2 and T4 by attacking the protein capsid to release and then inactivate the nucleic acids. ## Alcohol **Examples:** Ethanol, Isopropanol - Alcohols are used for surface disinfection, topical antiseptic and hand sanitizing lotions. - Alcohols are broad spectrum antimicrobial agents that damage microorganisms by denaturing proteins, causing membrane damage and cell lysis. - **Mode of Alcohol Action:** The most feasible explanation for the antimicrobial action of alcohol is due to denaturation of proteins. The bacteriostatic action was believed caused by inhibition of the production of metabolites essential for rapid cell division. - Alcohols are considered fast-acting capable of killing most bacteria within five minutes of exposure but are limited in virucidal activity and are ineffective against spores. - Ethanol is considered virucidal; isopropanol is not effective against non-enveloped viruses. - These alcohols are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria; they also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores. - Their cidal activity drops sharply when diluted below 50% concentration. The optimum bactericidal concentration is 60%-90% solutions in water (volume/volume) - An important consideration with alcohols is the concentration used, with 70-90% being optimum. Higher concentrations (95%) are actually less effective because some degree of water is required for efficacy (to denature proteins). This is supported by the observation that absolute ethyl alcohol, a dehydrating agent, is less bactericidal than mixtures of alcohol and water because proteins are denatured more quickly in the presence of water. - Ethyl alcohol, at concentrations of 60%-80%, is a potent virucidal agent inactivating all of the lipophilic viruses (e.g., herpes, vaccinia, and influenza virus) and many hydrophilic viruses (adenovirus, enterovirus, rhinovirus, and rotaviruses). - Alcohols are not recommended for sterilizing medical and surgical materials (an intensive-care setting) principally because they lack sporicidal action and they cannot penetrate protein-rich materials. - Fatal postoperative wound infections with Clostridium have occurred when alcohols were used to sterilize surgical instruments contaminated with bacterial spores. - Alcohols have been used effectively to disinfect oral and rectal thermometers, scissors, and stethoscopes. Alcohols have been used to disinfect fiberoptic endoscopes, but failures of this disinfectant have lead to infection. - Alcohol towelettes have been used for years to disinfect small surfaces such as rubber stoppers of multiple-dose medication vials or vaccine bottles. Furthermore, alcohol occasionally is used to disinfect external surfaces of equipment (e.g., stethoscopes, ventilators, and manual ventilation bags), CPR manikins, ultrasound instruments or medication preparation areas. ## Halogens **Ex: Chlorine and iodine compounds** ### Chlorine and Chlorine Compounds #### Chlorine gas - Chemical forms of free chlorine: - $Cl_2$ (gas) - $NaOCl$ (liquid) - $Ca(OCl)_2$(solid) - Hypochlorites, the most widely used of the chlorine disinfectants, are available as liquid (e.g., sodium hypochlorite) or solid (e.g., calcium hypochlorite). - The most prevalent chlorine products are aqueous solutions of 5.25%-6.15% sodium hypochlorite. - They have a broad spectrum of antimicrobial activity, are relatively unaffected by water hardness, are inexpensive and fast acting, remove dried or fixed organisms and biofilms from surfaces. - A potential hazard is production of the carcinogen (chloromethyl) ether when hypochlorite solutions contact formaldehyde and. - The production of the animal carcinogen trihalomethane when hot water is hyperchlorinated. **Disadvantages:** - Hypochlorite solutions in tap water at a pH >8 stored at room temperature (23 C) in closed, opaque plastic containers can lose up to 40%-50% of their free available chlorine level over 1 month. - Thus, if a user wished to have a solution containing 500 ppm of available chlorine at day 30, he or she should prepare a solution containing 1,000 ppm of chlorine at time 0. Sodium hypochlorite solution does not decompose after 30 days when stored in a closed brown bottle. - The microbicidal activity of chlorine is attributed largely to undissociated hypochlorous acid (HOCl). The dissociation of HOCl to the less microbicidal form (hypochlorite ion OCl-) depends on pH. The disinfecting efficacy of chlorine decreases with an increase in pH that parallels the conversion of undissociated HOCl to OCl-. **Reactions for free chlorine formation:** $Cl_2 + H_2O \longrightarrow HOCl + Cl + H$ $HOCI \longrightarrow OCl + H$ - HOCl at low PH (acidic) and OCI at high PH. - HOCl more potent germicide than OCI - The actual microbicidal mechanism of chlorine might involve a combination of these factors or the effect of chlorine on critical sites: - Oxidation of sulfhydryl enzymes and amino acids. - Ring chlorination of amino acids. - Loss of intracellular contents. - Decreased uptake of nutrients. - Inhibition of protein synthesis. - Decreased oxygen uptake. - Oxidation of respiratory components. - Decreased adenosine triphosphate production. - Break in DNA and depressed DNA synthesis. - Chlorine is electronegative, and therefore oxidises peptide links and denatures proteins. Exposure of strains of Escherichia coli, Pseudomonas spp., and Staphylococcus spp. to lethal doses of hypochloric acid causes a decrease in ATP production. - Alternative compounds that release chlorine and are used in the health-care setting include demand-release - 1- Chlorine dioxide, - 2- Sodium dichloroisocyanurate. - The advantage of these compounds over the hypochlorites is that they retain chlorine longer and so exert a more prolonged bactericidal effect. #### Chlorine dioxide-based disinfectants - Are prepared fresh as required by mixing the two components (base solution [citric acid with preservatives and corrosion inhibitors and the activator solution sodium chlorite]). - Chlorine dioxide acts on the permeability of the external membrane of E. coli through a primary lethal phenomenon which consists in a substantial leakage of $K^+$ ions; such leakage does not occur for macromolecules. - Sub-lethal doses inhibit cellular respiration due to a nonspecific oxidising effect. #### Sodium dichloroisocyanurate - Sodium dichloroisocyanurate tablets are stable. - The microbicidal activity of solutions prepared from sodium dichloroisocyanurate tablets might be greater than that of sodium hypochlorite solutions containing the same total available chlorine for two reasons. - First, with sodium dichloroisocyanurate, only 50% of the total available chlorine is free (HOCl and OCl-), whereas the remainder is combined (monochloroisocyanurate or dichloroisocyanurate), and as free available chlorine is used up, the latter is released to restore the equilibrium. - Second, solutions of sodium dichloroisocyanurate are acidic, whereas sodium hypochlorite solutions are alkaline, and the more microbicidal type of chlorine (HOCl) is believed to predominate in the acidic media ## Iodine compounds - Understanding the action of iodine-containing antiseptics requires study of the behaviour of iodine in aqueous or alcoholic solution. Iodine-containing products are chiefly used for antisepsis of intact or damaged skin. - **Mode of action:** - **a- Iodine acts by decreasing the oxygen requirements of aerobic microorganisms.** - **b- Iodine interferes at the level of the respiratory chain of the microorganisms by blocking the transport of electrons through electrophilic reactions with the enzymes of the respiratory chain.** - **c- Iodine also interacts preferentially with the proteins of the cytoplasm membrane in a form with a positive ($H_2O+I$) or neutral (I; or HOI) charge. An iodophor is a combination of iodine and a solubilizing agent or carrier; the resulting complex provides a sustained-release reservoir of iodine and releases small amounts of free iodine in aqueous solution. The best-known and most widely used iodophor is povidone-iodine (a compound of polyvinylpyrrolidone with iodine). "Free" iodine ($I_2$) contributes to the bactericidal activity of iodophors and dilutions of iodophors demonstrate more rapid bactericidal action than does a full-strength povidone-iodine solution. The reason for the observation that dilution increases bactericidal activity is unclear, but dilution of povidone-iodine might weaken the iodine linkage to the carrier polymer with an accompanying increase of free iodine in solution. Therefore, iodophors must be diluted according to the manufacturers' directions to achieve antimicrobial activity.** ## Quaternary ammonium compounds - Also known as "quats" or QACS. - **Examples:** Roccal, Zepharin, DiQuat, D-256 - Quaternary ammonium compounds (QACs) have the basic structure $NR_4^+$, possessing R groups with long alkyl chains are known to be especially effective as antimicrobial agents. - This is particularly relevant in the milk industry, as QACs are typically used to disinfect the insides of tanks used for transporting milk from farms to processing plants. - They are classified into cationic, hydrophilic group, surfactants anionic, nonionic, and ampholytic (amphoteric) compounds. - Surface-active agents (surfactants) have two regions in their molecular structures, one a hydrocarbon, water-repellent (hydrophobic) group and the other a water-attracting (hydrophilic or polar) group. - **Synthesis:** - Quaternary ammonium compounds are prepared by the alkylation of tertiary amines with a halocarbon. In older literature this is often called a Menshutkin reaction, however modern chemists usually refer to it simply as quaternization. - The reaction can be used to produce a compound with unequal alkyl chain lengths; for example when making cationic surfactants one of the alkyl groups on the amine is typically longer than the others. - A typical synthesis of benzalkonium chloride from a long-chain alkyldimethylamine and benzyl chloride, $CH3(CH2)N(CH3)2 + CICH₂CH [CH3(CH2)N(CH₃)₂CH₂CH₂] CT$ - Certain quaternary ammonium compounds, especially those containing long alkyl chains, are used as antimicrobials and disinfectants: Examples are benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, dofanium chloride, tetraethylammonium bromide, didecyldimethylammonium chloride and domiphen bromide. - **Mode of Action:** It has been known for many years that QACs are membrane active agents (ie., with a target site predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane in yeasts.) - 1- The antimicrobial action of QACs involves perturbation of cytoplasmic and outer membrane lipid bilayers through association of the positively charged quaternary nitrogen with the polar head groups of acidic phospholipids. - 2- The hydrophobic tail subsequently interacts with the hydrophobic membrane core. - 3- QACs form mixed-micelle aggregates with hydrophobic membrane components that solubilize membrane and lyse the cells. Lethality occurs through generalized and progressive leakage of cytoplasmic materials. - So mode of action can be: (i) Adsorption and penetration of the agent into the cell wall (ii) Reaction with the cytoplasmic membrane followed by membrane disorganization (iii) Leakage of intracellular low-molecular-weight material (iv) Degradation of proteins and nucleic acids; and (v) Wall lysis caused by autolytic enzymes - QACs are cationic detergents that are attracted to the negatively charged surfaces of microorganisms. Irreversibly bind to phospholipids in the cell membrane and denature proteins, impairing cell membrane permeability. - **Microbicidal activity of QACs:** QACs are highly effective against Gram positive bacteria, and have good efficacy against Gram-negative bacteria, fungi and enveloped viruses. QACs are not mycobactericidal but have a mycobacteriostatic action QACs are toxic to fish and should not be discharged into water sources (i.e., streams, ponds, lakes). - QACs are sporostatic but not sporocidal; 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). QACs are more active at neutral to slightly alkaline pH but lose their activity at pH less than 3.5. QACs are considered stable in storage but are, in general, easily inactivated by organic matter, detergents, soaps and hard water. ### Quaternary ammonium compounds - **One product, F10, consists of polihexanide 4g/L., benzalkonium chloride 54g/L, non-toxic ampholytics and sequesterants. Polihexanide is a chemical used as a biocide for control of micro-organisms and algae in swimming pools, spas, disinfectant in veterinary products and as a sanitiser for milk handling equipment. Its efficacy claims are: ** - bacteria (1:500 for 2 minutes) - Fungi, yeasts and moulds (1:500 15 minutes) - fungal spores (1:250 30 minutes) - enveloped viruses (1:500, 10-30 minutes) - non-enveloped viruses-infectious bursal disease virus and parvovirus (1:125, 30 minutes), and - bacterial spores (1:125 for 30 minutes). - **The product has been used safely as an aerosol to treat respiratory infections, including fungi and yeasts (Verwoerd, 2001, Chitty, 2002). No claims are made for circoviruses.** - **There is no evidence at present that F10 inactivates BFDV.** - **QACs are classified as follows:** - **First Generation:** An example is benzalkonium chloride. These have minimal biocidal activity and are commonly used as preservatives; - **Second Generation:** These are substituted benzalkonium chlorides, an example of which is alkyl dimethyl benzyl ammonium chloride. These have high biocidal activity. - **Third Generation:** These are also called "dual QACs" (for example: one contains equal parts of alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl ethylbenzyl ammonium chloride. These QACs have increased biocidal activity, stronger detergency, and increased safety to the user (lower toxicity). - **Fourth Generation:** These are also called "Twin or Dual Chain QACS" Examples are didecyl dimethyl ammonium chloride and dioctyl dimethyl ammonium chloride. They are superior to other QACs in germicidal performance, lower foaming, and have an increased tolerance to protein loads and hard water. - **Fifth Generation:** These are mixtures of fourth generation and second-generation QACs (eg, didecyl dimethyl ammonium chloride alkyl dimethyl benzyl ammonium chloride). They have excellent germicidal performances, are active under more hostile conditions and are safer to use. ## Phenolic compounds **Examples:** One Stroke Environ", TekTrol", Pheno-Tek II - Phenols can be coal-tar derivatives or synthetic formulations and usually have a milky or cloudy appearance when added to water, as well as