Antiseptics and disinfectants

Erzsébet Pászti-Gere

AETIOTROPIC AGENTS

ANTISEPTICS AND DISINFECTANTSCHEMOTERAPEUTICS

• Ignaz SemmelweisPuerperal fever could be reduced

in the form of chlorinated lime.

• Joseph ListerHe introduced phenol to sterilize

hands and surgical instruments and to clean wounds.

TERMINOLOGY

Sterilization – a process that destroys all viable microbes, including viruses and endospores ; microbicidalDisinfection – a process to destroy vegetative pathogens , not endospores; inanimate objectsAntiseptic –applied directly to body surfacesSanitization – any cleansing technique that mechanically removes microbesDegermation – mechanically removing microbes from surface (skin) such as surgical hand scrubbing, or wiping skin with alcohol prior to venapuncture

„CID ” vs „STATIC ”

Sepsis – bacterial contamination

Asepsis – absence of significant contamination, employment of techniques, aim: microbe-free environment!

Bacteriocidal (microbiocidal) - -cidal means kill

Bacteriostatic (microbiostatic) - -static means inhibition of growth and multiplication

EFFICACY OF DISINFECTION AND STERILIZATION

– prior cleaning of the object– organic load– type and level of microbial contamination– concentration of the agents and exposure time– physical nature of the object– presence of biofilms– temperature and pH of the disinfection process– relative humidity of the sterilization process (e.g.,

ethylene oxide)

TYPE AND LEVEL OF MICROBIAL CONTAMINATION

• Number and location of microorganisms• Innate resistance

– Spore coat and cortex: spores– Waxy cell wall: Mycobacteria– Outer membrane: Gram- bacteria

• Antibiotic-resistant bacteria

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RESISTANCE OF MICROORGANISMS

SUSCEPTIBILITY OF MICROORGANISMS

Effectiveness of germicides classified as high, intermediate, or low

– High-level kill all pathogens, including endospores

– Intermediate-level kill fungal spores, protozoan cysts, viruses and pathogenic bacteria

– Low-level germicides kill vegetative bacteria, fungi, protozoa, and some viruses

CRITICAL/SEMICRITICAL ITEMS

Mahon: Textbook of Diagnostic Microbiology

BIOFILMS

• thick masses of cells and extracellular materials• up to 1,000 times more resistant• some enzymes and detergents can degrade biofilms

FACTORS AFFECTING THE EFFICACY…

• The more concentrated the disinfectant is, the greater its efficacy is– Exception: iodophors

• QAT and phenol have a concentration exponent effects• temperature, pH• increase in pH improves the antimicrobial activity of:

glutaraldehyde, QAT but decreases the antimicrobial activity of phenols, hypochlorites, and iodine.

• Water hardness• Relative humidity

METHODS OF STERILIZATION

Sridhar Rao www.microrao.com

PHYSICAL METHODS OF STERILIZATION

• Exposure to extremes of heat– Dry heat– Moist heat

• Denaturation of proteins• Interference with integrity of cytoplasmic membrane and cell walls

• Disruption of structure and function of nucleic acids

• Exposure to extremes of cold• Desiccation• Filtration• Radiation

– Non-ionizing rays– Ionizing rays

DRY HEAT

• Types: – read heat – flaming– incineration – hot air oven

• Used for materials that cannot be sterilized with moist heat• Denatures proteins and oxidizes metabolic and structural chemicals• Requires higher temperatures for longer time

MOIST HEAT

• Better conduction of heat• Methods of microbial control using moist heat

– Boiling: Endospores, protozoan cysts, and some viruses can survive boiling!!! time is critical !!!

– Autoclaving– Pasteurization:

• Batch method 30 minutes at 63ºC• Flash pasteurization 72ºC for 15 seconds

– Ultrahigh-Temperature Sterilization: 140ºC for 1 second, then rapid cooling

AUTOCLAVING

Autoclave conditions – 121ºC, 15 psi, 20 minutes•Control:

•BOWIE DICK TAPE•BIOLOGICAL: spores of B. stearothermophilus

RADIATION-NON IONIZING RAYS• UV light causes pyrimidine (thymine) dimers in DNA• UV light does not penetrate well• Suitable for disinfecting air, transparent fluids, and

surfaces of objects

RADIATION- IONIZING RAYS

• Electron beams: sterilization is accomplished in few seconds

• Electromagnetic rays: gamma rays (Co60, Cs137)– More penetrative power– Longer time of exposure– Nucleic acid damage– Impairs the flavour of certain foods

OTHER PHYSICAL METHODS

• Refrigeration and Freezing• Desiccation and Lyophilization• Filtration• Osmotic Pressure

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AN IDEAL DISINFECTANT

Broad spectrum: should have a wide antimicrobial spectrumFast acting: should produce a rapid kill

Not affected by environmental factors:

should be active in the presence of organic matter and compatible with soaps, detergents

Nontoxic: should not be harmful to the user or patient

Surface compatibility: should not corrode instruments and metallic surfaces

Residual effect on treated surfaces: should leave an antimicrobial film on the treated surface

Odorless: should have a pleasant odor or no odor to facilitate its routine use

Economical: should not be prohibitively high in costSolubility: should be soluble in waterStability: should be stable in concentrate and use-dilutionCleaner: should have good cleaning propertiesEnvironmentally friendly: should not damage the environment on disposal

CHEMICAL AGENTS-HOW DO THEY ACT?

• Reaction with components of the cytoplasmic membrane:– Alcohol– Detergent

• Denaturation of cellular proteins:– Alcohol– Phenol

• Reaction with the thiol groups of enzymes:– H2O2– Halogens

• Damage of RNA and DNA:– ETO– Formaldehyde

Classification of disinfectants

GROUPS OF ANTISEPTICS AND DISINFECTANTS

• Halogen-containing compounds– Iodine-containing agents – Chlorine-containing agents

• Biguanides• Acids and Alkalis• Aldehydes and Reducing agents• Alkylating agents• Alcohols• Peroxides and oxidative agents• Surfactants• Phenols and Related Compounds• Metals• Dyes

ALCOHOLS• Ethyl alcohol, isopropyl alcohol and methyl alcohol (not sporicide) • Isopropyl alcohol can be used to disinfect surfaces and to disinfect

clinical thermometers. • Methyl alcohol kills fungal spores (disinfecting inoculation hoods). • Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable • Antimicrobial effect is related to their lipid solubility (damages bacterial

membranes) and their ability to precipitate cytoplasmic proteins. However, they do not destroy bacterial spores.

ACIDS AND ALKALIS

ACIDS• Bacteriostatic at pH 3-6, bactericidal at pH<3• Strong mineral acids but corrosive• Skin contaminated by spores: 2.5% HCl• Weak organic acids can be applied as:

– Food preservatives (benzoic acid)

– Antiseptics (acetic acid 0.25-1%) , at 5% bactericidal to many bacteria (otitis externa)

– Fungicides

ALKALIS• pH>9 it inhibits most bacteria and many viruses• Irritant and caustic effects• 2% solution of soda lye: fowl cholera• CaO and Ca (OH)2

HALOGENS

• Oxidizing agents cause damage by oxidation of essential sulfhydryl groups of enzymes.

• Chlorine compounds and iodine compounds– Tincture of iodine (2% iodine in 70% alcohol) and iodine can be combined with neutral

carrier polymers povidone-iodine.

– Household bleach used in a stock dilution of 1:10.

• Disadvantages: – They are rapidly inactivated in the presence of organic matter.

– Iodine is corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

IODINE COMPOUNDS

• Germicide with a wide spectrum (G+, G-, spores, fungi, viruses) of activity

• 50 ppm iodine kills bacteria in 1 min. and spores in 15 min.

Mode of action: oxidizing agents

inorganic !broad spectrum (sporicide)

organic (iodophor) � antiseptic+disinfectant

povidone-iodine

1:10-1:100 dilution!

warm

Irritation, pain, allergy!

IODOPHORE

• Applications: in teat dips to control mastitis, as dairy sanitizers, and as a general antiseptic or disinfectant for various dermal and mucosal infections.

• They slowly release iodine as an antimicrobial agent and are widely used as skin disinfectants, particularly before surgery.

CHLORINE COMPOUNDS

• potent germicidal effect• undissociated hypochlorous acid (HOCl) effective against most

organisms at 0.1 ppm• irritant to the skin and mucous membranes• used to disinfect water supplies and inanimate objects (e.g.,

utensils, bottles, pipelines) in dairies, and milk houses.

• Inorganic chlorides • sodium hypochlorite solutions (bleach 3-6%): 10X dil corrosive

and against spores, calcium hypochlorite • Organic chlorides:

– Chloramine T– Chloramine B

HYDROGEN PEROXIDEBroad spectrum: ROS damage proteins and DNA (strong solutions

are sporicidal)

Catalase, organic matters, light

Skin disinfection (peracetic acid synergism!) – Antiseptic?

5-20% � 3%

PERACETIC ACID (40%)Sterilant and antiseptic

Broad antimicrobial spectrum

Lack of harmful decomposition products of hydrogen peroxide

Greater lipid solubility

No inactivation by tissue catalase and peroxidases

Corrosive

CATIONIC SURFACTANTS

• alkyl- or aryl-substituted quaternary ammonium compounds• benzalkonium chloride, benzathonium chloride, cetylpyridinium

chloride, benzoxonium chloride /Bradophen®/) • +halogen, such as bromide, iodide, or chloride. • the major site of action: cell membrane• anionic tenzides, hard water, organic material• Not active against spores and viruses

Resist. S.aureus !• aqueous solutions (1:1,000 to 1:5,000) have good antimicrobial

activity, especially at slightly alkaline pH.

PHENOL AND PHENOLIC COMPOUNDS

They denature proteins– Phenol:

• is expensive• is caustic • has a pungent odor

– Derivatives have:• greater germicidal activity • lower toxicity than phenol

– Bisphenols are combinations of two phenol molecules

• They are commonly used in disinfection and antisepsis

PHENOLICS

• good penetrating power • disinfection of equipment or organic materials• irritant and corrosive• cauterize infected areas, e.g., infected umbilicus of

neonates

HEXACHLOROPHEN

• bacteriostatic, Gram+• absorption of the antiseptic to cause spongiform degeneration

of the white matter in the brain • in medicated soaps 1-3%• fungicidal effect

TRICLOSAN

• antibacterial and antifungal agent • risk of bacterial resistance• in soaps (0.10-1.00%)• no extra benefits: anti-gingivitis effect in toothpaste• bind to both human estrogen and androgen receptors in vitro

NO TRICLOSAN IN TOOTHPASTE!!!!

PHENOLS AND RELATED COMPOUNDS

The bactericidal activity is enhanced by - EDTA- warm temperatures

decreased by - alkaline medium- lipids - soaps

- conc. >0.5% exert a local anaesthetic effect , 5% solution is strongly irritating and corrosive to tissues .

Oral ingestion or extensive application to skin can causesystemic toxicity (CNS and cardiovascular effects).

DYES

• Triphenylmethane dyes (e.g. bromocresol green, malachite/brilliant green, crystal/gentian violet, fuchsine)

- active against Gram-positive bacteria. - lower activity in the presence of blood and tissue debris.

- Usage : 0.5-1 % solution: wounds, burns, 2-5% alcoholic solution: navel.

- Malachite green is fungicide and antiparasitic (significant in fish-medicine)

coal tar residue

gentian violet

The Gram staining method

• They alter the 3-D shape of proteins• Low-level bacteriostatic and fungistatic agents• 1% silver nitrate prevents blindness caused by N. gonorrhoeae• Thimerosal (mercury-containing compound) used to preserve

vaccines…problems?• Copper controls algal growth in reservoirs, fish tanks, swimming pools,

and water storage tanks; interferes with chlorophyll.

HEAVY METALS

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ALDEHYDESbroad spectrum, acts through alkylation of amino-, carboxyl and hydroxyl group (sporicide)

FORMALDEHYDE

37% cc, 1-6%

15°C !

above 35°C gas

carcinogenic� USA, EU not allowed

GLUTERALDEHYDE

at 4°C it is effective

in 2% sporicidal effect

spray, on surgace, smoke

organic material

VAPOR-PHASE DISINFECTANTS

• alkylating agents such as formaldehyde, ethylene oxide, and propylene oxide

• they are broad-spectrum biocides • active against bacteria, viruses, and fungi, including spores. • highly reactive gaseous fumigants • used for sterilizing animal feed, human food, surgical equipment that

cannot be autoclaved (e.g., endoscopes, gloves, syringes, catheters, tubing, implantable devices)

• noncorrosive

GASEOUS AGENTS

•Ethylene oxide, propylene oxide, and beta-propiolactone used in closed chambers to sterilize items•Denature proteins and DNA by cross-linking functional groups•Used in hospitals and dental /veterinary offices

BIGUANIDES

CHLORHEXIDINE

cell membrane damage

very broad spectrum (not sporicide!)

incomp: anionic detergent!

organic material

concentration: 1-4%

Chlorhexidinefacts.com

CHLORHEXIDINE

• Gram+, Gram – (10-50X conc)• Not sporicidal, but prevents development of spores• Application:

– Dermatitis (Staphylococcus, Streptococcus)– Otitis externa– Dental hygiene, gingivitis– Hygienic hand disinfection– Udder health

PRACTICAL DISINFECTION

• Disinfection of drinking water and swimming pool– Guideline values: Free chlorine: 0.5-1.5 ppm (mg/l) Combined

chlorine: 0-0.5 ppm (mg/l) Total chlorine: 0.5-1.5 ppm (mg/l) Cyanuric acid: 0-20 ppm (mg/l)

• Disinfecting patient’s skin– Alcohols, iodine compounds

• Disinfection of excretion– Phenolic preparations

• Instrument, surface, room disinfection• Hygienic hand disinfection

– Alcohols

• Surgical hand disinfection– Alcohols combined with iodophores, alcohols

standard disinfection (rubbing into hands 3 ml of isopropanol 60% v/v, 30 seconds, twice)

Target Antiseptic/disinfectant Mode of action

Cell envelope (cell wall,

outer membrane)

Glutaraldehyde Protein cross-linking

EDTA other

permeabilizers

Gr- : LPS ↑ Mg2+

removal

Cytoplasmic membrane QAT membrane damage (PL

bilayers)

Chlorhexidine membrane integrity

Phenols leakage, uncoupling

DNA interaction Acridins intercalation between 2

layers of base pairs in

DNA

Cross-linking of

macromolecules

Formaldehyde,

glutaraldehyde

CL of proteins and

nucleic acids

Interaction with thiol

groups

silver compounds membrane-bound

enzymes

Effects on DNA halogens, H2O2, silver

ions

DNA strans breakage

Oxidizing agents halogens, peroxygens ROSClinical Microbiology Reviews McDonnell 1999