Microbicides and sterilization

Websites of Interest:

Companies involved with Microbicidal Development Indevus Pharmaceuticals (HIV microbicidal products)

Introduction/Definitions:

Sterilization:is the killing or removal of all microorganisms, including spores. One can sterilize by the following methods: 1) incineration, 2) filtration, 3) heat such as boiling does not kill spores, dry heat for 180 c for 2 hours. moist-heat autoclaving which is exposure to steam at 121C under pressure of 15 lb/square inch for 15 minutes. flash autoclaving is steam at 134c for 3 minutes (Pasteurization is the heating of liquids to a temperature that inactivates important pathogens, but below that needed for sterilization), 4) gas, ethylene oxide autoclaving is used for plastics and other heat sensitive materials. It uses gas alkylate proteins and nucleic acids 5) radiation, which can include UV and X-ray andchemical. What makes a good sterilization agent or disinfectant? Well the best agent is one which leaves the least number of organisms. The number of survivors (N) is inversely proportional to the concentration of the agent and to the time (T) of application of the agent.

Disinfection: is the killing of many, but not all microorganisms. Disinfectants are generally chemicals used on inanimate objects.

Antiseptics: are disinfectants used on body surfaces.

Antimicrobials exhibit selective or greater toxicity to parasites and have many modes of activity. The Minimum inhibitory concentration (MIC) refers to the tube showing the least amount of agent with no growth after dilutions of the agent in tubes with a constant amount of bacterial isolate. How does one test for antimicrobicity? One way is Disc diffusion where you seed a bacterial isolate over an agar plate and place discs containing antimicrobial agents on the agar. A zone of inhibition develops if the organisms is susceptible to the agent.

Antimicrobial resistance is due to resistant strains found in a small proportion of a population which are selected out. Resistance is mediated primarily by large scale use of antibiotics.

Dessication: Vegetative cells exposed to normal room air gradually become dehydrated or desiccated. Delicate pathogens such as Streptococcus pneumoniae, the spiochete of syphilis or Neiseria gonorrhae can die after a few hours of air drying, but many others are not killed and some are even preserved. Endospores of Bacillus and Clostridium are viable for thousands of years under extremely drug conditions. Staphylococci and streptococci in dred secretions and the tubercle bacillus srrounded by sputum can remain viable in air and dust for lenghty periods. Many viruses (expecially nonenveloped) and fungal spores can also withstand long periods of desiccation. Desiccation can actually be a way to preserve foods because it reatly reduces the amount of water avilable to support microbial growth.

Lyophilization: is the combination of freezing and drying. It is a common method of presrving microorganisms and other cells in a viable state for many eyars. Pure cultures are frozen instantenously and exposed to a vacuum that rapidly removes the water. This method avoids the formation of ice-cyrsatls that would damage the cells.

Products for Disinfection:

Detergents: target the cytoplasmic membrane of bacteria. These agents physically bind to lip layer of the cytoplasmic membrane which opens up the cytoplasmi membrane and allows damaging chemicals to enter the cell and important ions to exit the cell.

Alcohols are protein denaturants. A concentration of 50% and higher dissolve membrane lipids, disrupt cell surface tension and compromise membrane integrity. The rate of evaporation decreases effectivneness and inhaltiton of vapors can affect the nervous system.

Alcohols, namely ethanol and isopropanol, exhibit a broad spectrum of germicidal activity against bacteria, viruses, and fungi. Additionally, they have been used as low-level disinfectants in healthcare settings for many years. Lipid membrane dissolution and protein denaturation are key mechanisms of the antimicrobial action of alcohol, leading to the disruption of the membrane and the inhibition of the metabolism. Alcohol disinfectants are not only used for skin disinfection, but also for inanimate surfaces such as stainless steel, plastic (PET), glass, PVC, and cardboard. See Huang

Alcohols are amphiphilic compounds, as they possess both hydrophilic and lipophilic (hydrophobic) properties that facilitate their entry through the viral envelope. The outermost membrane of SARS-CoV-2 comprises lipids, and the antimicrobial mechanism of alcohol against SARS-CoV-2 and other enveloped viruses is similar to that for bacteria, since both have a lipid-rich outer membrane.

The optimum bactericidal concentrations of alcohols range from 60% to 90% v/v solutions in water but are generally ineffective against most microorganisms below 50% v/v. See Huang

–Ethanol is 70% alchohol and isopropyl is 95%. Activity of alchohol is more active in the presence of water (70% alcohol is more effective than 95% alcohol). A concentraiton of 60-80% alchol is deemed most effective due to the fact that proteins require water in order for denaturation to cocur. A concentration of 70% ethyl alchol requires a10 minutes to kills Staphylococcus aureus, 2 min to kills E coli and 10 minutes to kill poliovius.

Detergents: are polar molecules that act as surfactants. Anionic detergents have limited microbial power but cationic detergents such as quaternary ammonium compounds (“quats”) are effective. The positively cahrged end of the molecule binds the negatively charged bacterial surface proteins and the long, uncharged hydrocarbon chains allows the detergent to disrupt the cytoplasmic mebrane. Detergents are effective against virsues, algae, fungi and gram positive bacterial. They are used to clearn restaurant equipement, surfaces and restrooms. Some quats were banned in consumer products in 2017.

Hydrogen Peroxide. A 3% hydrogen peroxide can kills SA in 12.5 seconds, gonorrheae in 0.3 sec and herpes in 12.6 seconds.

Halogens oxidize. 2 types are 1) iodine and 2) chlorine (5% solution of sodium hypochlorite) 4) surfactants are hydrophobic and hydrophilic groups that solubilize.

–Chlorine: Chlorine can kill TB in 50 seconds, entamoeba cysts in 150 minutes and Hepatitis A virus in 10 minutes. In solution, these compounds combine with water and release hypcholorous acid (HOCl) which denatures enzymes and suspend metabolic reactions. Chlorine is used to disinfect drinking water, sweage and waste water. Hypochlorites are used in health care to treat wounds, disinfect bedding and instruments, sanitize food equipement and in restaurants, pool and spas. Chloramines are alternatives to pure chlorines in treating drinking water.

Despite the introduction of many classes of disinfectants, disinfection approaches that liberate free available chlorine, such as hypochlorous acid and hypochlorite ions, continue to play an important role in improving public health by reducing the cross-transmission of infectious agents via drinking water and environmental surfaces. A large number of antimicrobial chlorine compounds are commercially available, including sodium and calcium hypochlorites, liquid chlorine, and inorganic and organic chloramines.

—-Chlorine dioxide (ClO₂), an alternative disinfectant to chlorine, has been widely used to control a number of waterborne pathogens in water and wastewater treatments. It is an effective disinfectant in both liquid and gas states, making it a versatile biocidal agent. or example, ClO₂ can be safely used in low concentrations around animals and people to control airborne viruses. Compared with chlorine, ClO₂ is less toxic because of the greatly reduced generation of toxic halogenated disinfection products. See Huang

The virucidal mechanism of ClO₂ appears to be different for different types of viruses. One mode of action mainly involves the degradation of the viral proteins which are responsible for interactions with the host cell and injection mechanisms. Therefore, the attachment of the virus to host cells is inhibited, resulting in the inactivation of viruses. It has also been proposed that ClO₂ can act on the viral genome. Specifically, the inactivation by ClO₂ is caused by damage in the 5′ noncoding region within the genome, which is necessary for the formation of new virus particles within the host cell.

—-Chlorhexidine: is a complex organic base containing chlorine and two phenolic rings. It targets bacterial membranes where slective permeability is lost, bacterail cell walls, and proteins resulting in denaturation. Its effects on virsues and fungi are variable. It is often used in hand scrubs, prepping skin for surgery, as an obstetric antiseptic and as a mucous membrane irrigant.

–Iodine: can kill endospores slowly and all other microbes. A 2% iodine and 2.4% sodium iodine (aquoues iodine) is used as a topical antiseptice. A 5% iodine and 10% potassium iodine is used as a dissinfectant for plastica nd rubber instruments as well as cutting blades. Iodine can be extremely irritating to the skin and is toxic when absorbed. Many iodophos were banned in consumer products in 2017.

–Povidone-iodine: Povidone (polyvinylpyrrolidone, PVP) is a water-soluble synthetic polymer used in many products, most famously as a carrier for iodine in the antiseptic povidone-iodine (like Betadine) for skin disinfection and wound care, but also as a binder in tablets, a lubricant in eye drops, and an additive in cosmetics and food. It works by slowly releasing iodine, providing broad-spectrum antimicrobial action against bacteria, fungi, and viruses, while the polymer itself is less toxic than free iodine.

Phenol was the original agent used to disinfect. So the effectivness of all other agents are based on phenol. (i.e., ratio of the [phenol] to the [agent] required to cause the same amount of killing under the same standard conditions. Phenol is a protein denaturant. Two types of phenols are hexachloropheneand chlorhexidine which alters membrane permeability.

–-Iodophor is a complex of iodine and a solubilizing agent or carrier because iodine alone is not stable in water. This formation allows the sustained release of iodine and has powerful microbicidal activity. The most commonly used iodophor is povidone iodine because of its rapid, broad-spectrum antimicrobial activity, even at low concentrations, and due to its established safety profile.

It is free molecular iodine that mediates the antimicrobial activity of iodophor. Iodine rapidly penetrates into microorganisms and reacts with key groups of proteins (in particular, the free sulfur amino acids cysteine and methionine leading to the loss of protein disulfide linkages).

As one of the important medicines on the WHO List of Essential Medicines, povidone-iodine (polyvinylpyrrolidone iodine, PVP-I) is routinely used in surgical procedures, including the disinfection of skin when formulated into scrubs or handwashes and for oral cavities through oral sprays and mouth rinses. The combination of PVP-I with alcohol as a disinfectant shows excellent residual efficacy, and could reduce the amount of alcohol required, plus serve as a useful substitute or supplement to alcohol for disinfecting skin, oral cavities, and fomite surfaces. See Huang

Sodium hypochlorite (“Bleach”): A variety of commercial products used in the home and healthcare facilities contain 1% to 15% sodium hypochlorite, with the most prevalent products being aqueous solutions of 4% to 6% sodium hypochlorite, which are usually called household bleach. The WHO recommends that regular household disinfectants containing 0.1% sodium hypochlorite (1000 mg/L) should be applied to various household surfaces. See Huang

The CDC recommends using 1/3 cup of bleach added to 1 gallon of water for surfaces exposed to COVID-19 patients, which is approximately 64 times diluted and has an available chlorine content of roughly 781 mg/L. A “strong chlorine solution” is a 0.5% solution of hypochlorite (containing approximately 5000 ppm free chlorine) used for disinfecting areas contaminated with body fluids, including large blood spills.

Hypochlorous Acid: Hypochlorite produced by hypochlorite disinfectant can damage the lipids of the membrane and the nucleic acids due to its permeability through membranes and strong oxidizing ability. Moreover, it could inhibit the key enzymatic reactions within the cell and protein denaturation.

Hydrogen peroxide and peracetic acid are strong oxidizing agents and demonstrate broad-spectrum efficacy against a variety of microorganisms including bacteria, yeasts, and viruses.

Hydrogen peroxide is widely used for disinfection, sterilization, and antisepsis due to its ease of handling and expeditious start-up. It is considered environmentally friendly because it can rapidly degrade into innocuous products (water and oxygen) during dissolution, and is therefore a non-pollutant. It is also non-toxic, and is thus safe to use as a disinfectant for medical equipment and surfaces, even skin. Solutions in concentrations varying from 3% for routine disinfection to 25% for high level disinfection have been used. See Huang

Peroxyacetic acid is considered a more potent biocide than hydrogen peroxide against a broad spectrum of pathogens at lower concentrations (<0.3%).

Ozone, a naturally occurring configuration of three oxygen atoms, is a reliable, clean oxidizing agent with a powerful microbicidal effect against bacteria, viruses, fungi, and protozoa. Because ozone can dissolve within solution or be applied in gaseous form, it has been used widely in recent decades. In the disinfection processes, ozone is used in its gaseous or aqueous form depending on the type of decontaminated surfaces. Ozone gas may be used for the disinfection of hospital rooms or transport vehicles, whereas dissolved ozone may be used in water treatment and food disinfection. For wastewater treatment, ozone is a substantial disinfectant that can enhance biological water quality in less time and at a lower concentration with higher efficacy.

Quaternary ammonium compounds (QACs) are among the most commonly used disinfectants in healthcare and food-processing environments, as well as in the home. It was proposed that the following series of events is involved in the mechanism of action of QACs against microorganisms: (1) QACs’ adsorption to and penetration of the cell wall; (2) their reaction with the cytoplasmic membrane (lipid or protein), followed by membrane disorganization; (3) the leakage of intracellular lower-weight material; (4) the degradation of proteins and nucleic acids; and (5) cell wall lysis caused by autolytic enzymes. Thus, QACs, as cationic detergents, are effective against bacteria, yeast, and lipid-containing viruses. QACs are also effective against non-lipid-containing viruses and spores, depending on the product formulation, because they interact with intracellular targets and bind to DNA. See Huang

Common quaternary ammonium compounds (quats) for cleaning include benzalkonium chloride (BAC), didecyl dimethyl ammonium chloride (DDAC), and alkyl dimethyl benzyl ammonium chloride (ADBAC), which are often found in disinfectant sprays, wipes, and sanitizers. You can often identify them by names ending in “ammonium chloride” or by checking the active ingredients list on cleaning product labels.

ultraviolet (UV) radiation: ranges in wavelenght of about 100 to 400 nm. It is most lethal from 240-280 nm with a peak at 250 nm. Because UV radiation passes reapidly through air, slightly through liquids and only poorly through solids, the object to be disinfected must be directly exposed to it for full effect.

UV is a powerful method for destroying fungal cells and spores, bacterial vegetative cells, protozoa and viruses. Bacterial spores are about 10 times more resistant to radiation than are vegetative cells but they can be killed by increasing the time of exposure.

Germicidal lamps can cut down on the concentration of airborne microbes by up to 99%.