MEDI1000: Microbial Control

Cleaning

• Removal of visible soils, e.g. food residue, dust, dirt, corrosion, scale, grease

• Microorganisms are removed but not killed

• Often achieved with water and detergent

Sanitation

• Destroys various microorganisms → reducing numbers to meet public quality + health standards

• Usually not effective in presence of organic residues and detergents

• Moist heat (steam), or chemicals (chlorine)

Disinfection

• Removal of pathogens only

• Killing them, or rendering them inert

Sterilisation

Removal of all microbes including bacterial spores

Germacide

An agent that kills pathogenic microorganisms

Disinfectant

Substance that removes or causes of destruction of harmful microbes (not usually spores) from inanimate objects

Antiseptic

Disinfectant for animate objects

Heat (pasteurisation)

• 60-80 degrees for a few minutes

• Kill pathogens, destroy most other bacteria that cause food spoilage

• E.g. preservation

• Developed by Louis Pasteur mid 1800s - prevent spoilage of wine

• Low temp, long time (LTLT) - 63 degrees, 30min

• High temp, short time (HTST) - 72 degrees, 15 sec

• Reduce spoilage bacteria

• Extends shelf life

Radiation

• UV rays (non-ionisation)

• Infrared radiation (non-ionising)

• Ionising radiation (energy to liberate an electron from an atom)

UV

• UV rays (non-ionisation)

• Bactericidal wavelength 200-295nm

• Damages proteins and nucleic acid

• Low penetrating power

• Moderate exposure time

• Reduction of microbes including air, water + surfaces

Ionising

• Most effective: electron beam

• Gamma rays (need lead sheilding)

• X rays

• Damage DNA by disrupting chemical bonds in cells

• Prolonged exposure = sterilisation can be achieved

Methods of Disinfection

1. Heat (pasteurisation)

2. Radiation

3. Heat (boiling)

4. Chemical methods (solutions)

Modes of action for disinfection

• Protein coagulation/denaturation

• Disruption of cell membrane

• Chemical antagonism (inactivation of enzymes)

Examples of Chemical Disinfectants

• Alcohols

• Aldehydes

• Halogens (iodine)

• Heavy metals & their components

• Phenols and phenolic compounds

Examples of disruption of cell membrane for disinfectants

• Surface active agents

• Halogens (chlorine)

• Chorhexidine

• Phenols

Examples of chemical antagonism for disinfectants

• (Inactivation of enzymes)

• Metals

Organic matter modes of inhibition

1. Forms a precipitate with disinfectant - removes disinfectant from contact w bacteria

2. Reacts with the disinfectant to produce a non-bacterial agent(s)

3. Coats bacteria - protects bacteria from the disinfectant

Alcohols

• >60% (70% optimal: osmosis, water and alcohol moves into cell)

• Ethanol and isopropanol

• Akin antiseptic and surface disinfection of objects

• Disadvantages: lengthy exposure, low penetrating power, inactivated by organic substances, low activity against spores and non-enveloped viruses

Aldehydes

• Formaldehyde & gluteraldehyde

• For objects only

• Non corrosive to metals

• Disadvantages: tissue fixatives, strict safety precautions used when handling

Iodine

• Halogens

• Effective against bacteria, fungi, endospores, many viruses

• Iodine tincture, povidone iodine

• Povidone-iodine

Betadine

• Antiseptic for small wounds and infections

• Pre-op, skin disinfection

• Good residual effect

• Effective on a wide range of microbes

• Disadvantages: skin discolouration, hypersensitivity, pseudomonas able to grow

Metals

• Mercuric chloride

• Copper salts

• 1% silver nitrate

Phenols

• High conc. = denature proteins

• Low conc. = damage cell membranes - inactivated by organic material

• Corrosive and toxic (disinfectant use only)

Phenolic compounds

• Skin antiseptic

• Hexachlorphene

• Soap, lotion, cosmetic products scrub

• PHISOHEXXXXX

• Trisoclan

• Chloroxlylenol

Surface active agents (surfactants)

• Quaternary Ammonium Compounds (QUATS)

• Usually regarded as low level disinfectants

• Inactivated by soaps

• Pseudomonas can grow in QUATS

• Some are used as antiseptics in mouth washes, lozenges, eye drops

Chlorine

• Sodium hypochlorite (disinfectant)

• Cheap bleach - 1% for body fluid, spills, 0.1% for contaminated equipment

• Broad spectrum

• Corrosive at high conc.

• Long exposure time to be effective, inactivated by organic material

Chlorhexidine

• Skin antiseptic

• Oral mouth wash

• Anti-bacterial, fungal, viral

Guidelines for disinfectant use

1. Use more reliable sterilising methods - don’t overuse chemicals

2. Clean before disinfection (remove organic matter)

3. Ensure total surface contact

4. Use recommended concentration

5. Correct exposure time

6. Fine after chemical disinfection

7. Antiseptics and ointments - avoid contamination

Reasons for disinfectant failure

1. Wrong conc.

2. Container not clean old solution topped up with new

3. Solution too old

4. Inactivation by chemicals, organic matter, etc

5. Wrong temp or pH

6. Inefficient exposure time

7. Object not completely immersed

Asepsis in clinical practice

• Hand washing: with running water + drying removed most transient material, alcohol-based sanitisers, soap, chlorhexidine with water

• Personal cleanliness: masks, gloves, protective clothing

• Disinfection/sterilisation of work surfaces and equipment

• Use of aseptic techniques

When you should sanitise/wash your hands (5)

1. Before touching patient

2. Before a procedure

3. After a procedure/body fluid exposure risk

4. After touching a patient

5. After touching a patient’s surroundings

Most to least effective controls

Why do we use face masks?

• Protects others from mouth and nasal droplets

• Large droplets w most particles + bacteria trapped - small droplets droplets get thru

• Reduced projectiles spread of small droplets (travel less)

• Reduced inoculum for people in proximity (doesn’t completely stop ALL droplets

• Effective in reducing infection, especially with social distancing

• Sneezing, coughing and scratching under mask will reduce effectiveness

Sterilisation

Process by which all microbes, including endospores are destroyed

When/where is sterilisation required?

• Medical implements used on/in patients/labs - dressings & surgical implements, catheters + prosthetic devices, solutions (saline, antibiotics)

• Culture media, glassware

• Medical waste

• For canned and frozen meals

• Most often achieved by moist heat under pressure (autoclaving)

Bacterial spores

• Most heat resistant and difficult to kill microbial structure

• Thick spore coat protects from radiation + chemicals

• If all spores destroyed → so are all pathogens (except prions)

• Conditions for moist heat sterilisation are based on killing bacterial spores

Prions

• Proteinaceous infectious particles

• Difficult to destroy by standard methods (resistant to ionising radiation, resistant to formaldehyde

• CNS high tissue risk

• Decontamination procedures for non-disposable, heat sensitive instruments

Sterilisation by Moist Heat

• Moist heat (autoclave) more effective than dry heat (hot air oven)

• Moisture = better conductor of heat and better heat penetration

• Mode of action - coagulation & denaturing of proteins

• Temp achieved & exposure time important → 15min @ 121 degrees at 15psi above ambient atmospheric (most common)

• Precautions: loose caking of items to allow steam penetration, loose lids on bottles

Sterilisation indicators

• Autoclave printouts/monitoring

• Biological - spore strips: endospores impregnated on paper strips inside porous envelope and placed inside autoclave, transfer to broth, incubate 55 degrees for 5 days → growth = failed, no growth = efficient yay

• Autoclave tape (Bowie-Dick test): stripes on tape are white, pack with lines still visible = properly sterilised, immediate result, indicates 121 degrees but doesn’t indicate time held

Hot air sterilisation

• 2hrs at 160 degrees or 1hr at 10degrees

• Advantage: glassware, oils, powders, doesn’t blunt shear objects

• Disadvantage: less efficient, dried proteins resist denaturation

Incineration sterilisation

• Bunsen burner and incinerators

• 1 second @ >1000 degrees

• Microbiological loops, Medical waste, Animal carcasses

Sterilisation by gases and cold sterilisation

• Mode of action - denaturation of proteins

• Good for heat sensitive medical plastics, optical instruments

• Whole rooms (e.g. cold rooms) & mechanical apparatus

• E.g. formaldehyde, glutaraldehyde, ethylene oxide, plasma sterilisation

Sterilisation by filtration: fluids and air

• Membrane filtration of fluids

• Filter bacteria out 0.45um or less

• Applies to good quality, under sink water filters

• Doesn’t apply to normal filters (eg. Jug, cartridge)

• Filter out bacteria and virus particles (pores of 0.01um or 10nm)

High efficiency particulate air (HEPA)

• Pre filter: removes large particles, room odour

• Capable of removing 0.2 micrometer particles → 99.97% accurate

• VOC filter - removes volatile organic compounds & chemical substances

• Some have randomly arranged fibres, pre-filters are used to remove large particles that old rapidly clog a HEPA filter,

• Pore sizes usually larger than 0.3um → function relies on diffusion, interception and impaction

Antibiotics

Natural compounds produced by microorganism that kill or inhibit other microorganisms

Can be synthetic or semi-synthetic

Synergism

Compounds that act together to enhance/improve effect

Antagonism

Compounds that act against each other to reduce effect

Bacteriostatic

Compounds that inhibit growth of microorganism (bugs that still viable)

Bactericidal

Compounds that kills microorganisms

Antibiotics - The Beta-Lactam Group

• One of the largest groups of antibiotics

• Inhibit the last stage of the cell wall production process

• Kill bacteria without damaging cells of the host (eukaryotes don’t have a cell wall)

• However, Bacteria express beta-lactamase enzymes - some resistance, multiple different resistance genes are possible

The Beta-Lactam ring structure

• Common to all beta-lactam agents

• Ring must be intact for antibacterial activity - inactivated by beta-lactamase enzymes of the bacteria

• Binds to proteins @ cell membrane interface involved in cross-linking of peptidoglycan stands → cell lysis

• All beta-lactams are Bactericidal

• Not effective against resisting bacteria

Antibiotic resistance

• Over prescription = over exposure = selective pressure

• Not o pelting a course of antibiotics (that work) = electing resistant mutants

• Use in intensive farming of animals - growth promoters → resistant animal strains of bacteria

• Completely resistant and untreatable tuberculosis strains

Antibiotic Creed

Problems with broad-spectrum antibiotics

• Prolonged use = greatly reduced NF at some body sites → increase risk of second infection

• Genital tract and oral cavity → yeast not affective against antibiotics

• GIT → gastrointestinal infections

Anti-fungal treatment

• Antibacterial antimicrobials ineffective against fungi

• Difficult to treat because eukaryotic

• Treatment usually long term + toxic!

• Examples of antifungal drugs: Flucytosine, fluconazole, itraconazole

Antiviral chemotherapy

• Drugs that bind free virus preventing entry

• Prevention of uncoating of virus

• Inhibit viral replication

• Interfere with viral release