Week 23 - endospores Flashcards by nat r

Endospores

Endospores -> dormant and inert bacterial structures
Produced only by two Gram+ Genera: Bacillus and Clostridium

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Endospore characteristics

Mechanism of survival to harsh environment that would be lethal for the bacteria
->Nutrients depletion
->Environmental stresses (extreme temperature, pH),
->Chemical stresses (antibiotics, disinfectants)
Extraordinarily resistant to desiccation, heat, chemicals, and radiation
Endospores are able to survive for long periods -> centuries / thousands of years

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Sporulation/germination

Sporulation: endospore formation
Germination: endospore returns to vegetative state
Endospore is NOT a form of reproduction -> only one new cell germinates from each endospore

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Sporulation

Sporulation is a process of differentiation induced by stress
->Activation of genes to induce this differentiation into endospores
->Quenching of genes regulating the normal germinative life of bacteria
-7 sequential steps
-Involves the production of new structures (endospores), and disassembly of vegetative cell components

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Endospore structure

An endospore has an ovoid shape and a multi-layered structure, containing:
->Core -> (dehydrated and inactive), with: DNA, ribosomes, essential proteins and calcium
dipicolinate (responsible for dehydration)
->Surrounded by 3-4 different coating layers:
-Core walls: innermost layer
-Cortex: made of peptidoglycan
-Spore coat: thick and impermeable protective
layer to chemicals/antibiotics
-Exosporium: thin covering, not always present
A special stain can be used to detect endospores

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Germination

Process of an endospore that revert back to a vegetative cell very rapidly (even after decades), through removal of the stress inducer
->It also requires an activation step (usually heating to damage the spore coat), which is reversible
->Divided in 3 sequential steps, activation, initiation and outgrowth
->During germination, the core releases calcium dipicolinate becoming is hydrated, spore layers are disassembled, and the bacterial metabolism is restored

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Differences between vegetative bacterial cell and endospores

Vegetative cell : Typically gram+ cell, very few gram- cells
Endospore : Thick spore cortex, spore coat, exosporium

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Endospore resistance

Extraordinary resistance to desiccation, heat, chemicals, and
radiation in comparison to the vegetative bacteria
This makes endospores difficult to eliminate -> resistant to disinfection procedures
Issues in clinical settings or aseptic environments -> increases the risk of healthcare-associated infections (HAIs)

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Microbial control measures

-Sterilisation
-Disinfection
-Sanitisation
-Antimicrobial therapy

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Sterilisation

Completely eliminate any microbial life (including endospores)

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Disinfection

Reduces microbial load to the point where they no longer cause diseases -> not active against bacterial endospores
-Disinfectant: agents applied on inanimate objects/surfaces (toxic to human tissue)
-Antiseptic: agents applied on living tissue

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Sanitisation

Reduces microbe on items to safe levels by public health standards

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Antimicrobial therapy

Use of drugs to prevent or treat infections to SELECTIVELY inhibit growth of microorganisms minimising host tissue toxicity

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Control of bacterial growth

->Prevent infections, microbial spread and spoilage
->Microbes have different resistance to antimicrobial
procedures
Sterilisation = COMPLETE destruction of any viable organisms, INCLUDING endospores
Sterilisation is required for any product destined for
parenteral administration, or for contact with broken skin, mucosal surfaces, or internal organs

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Rate of microbial death

Microbial death progresses in a logarithmic manner

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Treatments to control the microbial death

Effectiveness of the control agents depends on:
-Time of exposure
-Microbial characteristics
-Number of microbes
-Environment (organic matter, temperature, biofilms)

Mechanisms of microbial death

-Alternation of membrane permeability: disrupts the integrity of microbial cells
-Protein denaturation: disrupts enzyme function and cellular processes
-Damage to nucleic acids: prevents replication and proper cellular function

Treatments to control the microbial growth

-Physical
-Chemical

Physical treatments

-Temperature:
->Heat (moist and dry)
->Cold temperatures
-Filtration
-Radiations
-Desiccation

Chemical treatments

-Alcohols
-Halogens
->Iodine
->Chlorine
-Phenolics
-Aldehydes
-Quaternary ammonium compounds
-Sterilizing gases
-Heavy metals

Temperature: heat

Elevated temperatures (> microbial Max temp) to kill microbes
->Moist heat: hot water, boiling water, or steam between 60°C and 135°C (autoclave,
pasteurisation) -> kills microbes by denaturation their proteins
->Dry heat: hot air or a flame (Bunsen Burner, incineration), which ranges from 160°C
to thousands of degrees -> kills microbes by dehydrating the cell & protein denaturation
-Moist heat is more effective
-Boiling water does not have
sterilising power -> direct flaming is the simplest form of dry heat sterilisation

Moist heat - autoclave

Autoclave (moist heat)
->Preferred sterilisation method
->Closed chamber with hot saturated steam under pressure
-121.5 degrees C for 15 minutes, to sterilise microbes/endospores (more time for prions)
-Steam must directly contact material

Moist heat – pasteurisation

Pasteurisation (moist heat disinfection): developed by
Pasteur to prevent the spoilage of beverages by reducing microbes
3 Methods:
1) Classic Pasteurisation: 65 degrees C for 30 minutes
2) Flash Pasteurisation (HTST): Used today -> 72 degrees C for 15 seconds
3) Ultra High Temperature Pasteurisation (UHT): 140 degrees C for 4 sec and quickly cooled in a vacuum chamber -> it is a sterilising method
Except for UHT, the other pasteurisation methods do not sterilise

Cold – low temperatures

Low temperatures (< optimal temp.) slow down microbial growth
->Reduces metabolic rate of most microbes to stop their
proliferation and toxin production, but often do not kill microbes
->Refrigeration temperatures (4-8 degrees C) are used for material preservation

Freezing temperature

Freezing temperature (<0 degrees C) can be used for long-term storage and preservation of some bacterial cultures (useful for analysis) -> Placing solutions in glycerol at -70 degrees C

Filtration

Removal of microbes from a solution by the use of specific pore sizes (< microbe size) as a physical barrier Used for heat-sensitive drugs (vaccines, enzymes/proteins) ->Pore size to exclude most bacteria: 0.2 - 0.45 μm ->Pore size for viruses: 0.01 μm Air filtration using high efficiency particulate air (HEPA) filters in laminar flow biological safety cabinets (0.3 μm)

Desiccation

Based on osmotic pressure and water reduction -Adding salts and sugars in foods is used to dry food and reduce water activity in the environment, creating hypertonic environment -Water leaves the cell by osmosis -Plasma membrane shrinks away from cell wall and bacteria reduce their metabolism (dormant) ->Cell may not die, but usually halts growth

Radiation

Different types of radiations kill microbes UV light: Damages DNA (breaks or mutations) ->Poor penetrating power ->Used only for surface sterilisation Ionising radiations (Gamma rays, electron beams and X-rays): inducing reactive free radicals that lead to microbial cell death ->High penetrating power into objects ->Used to irradiate heat-sensitive pharmaceuticals and medical supplies

Chemical agents

More often employed in disinfection (inanimate objects / surfaces) and antisepsis (human tissues) ->Only a Few chemical agents achieve sterility A number of factors influence the efficacy of a given chemical agent: -The kinds of organisms present -Degree of contamination -Time of exposure -Nature of the material being treated -Concentration of disinfectant

Chemical agents - include;

Chemical disinfectants and antiseptics include: ->Alcohols (60-80%): Ethanol and Isopropanol -> used as an antiseptic before injections, in hand sanitisers and cosmetics -not suitable for open wounds -no effect on nonenveloped viruses ->Iodine: (tincture -> iodine + alcohol) -> used as antiseptic in open wounds during surgery -> must be in contact for 30 seconds ->Chlorine -> used to disinfect drinking water, pools, and sewage They damage plasma membranes, proteins, lipids, or DNA to reduce microbial content

Antimicrobial therapy

Administration of drugs to treat infections, having selective toxicity against specific microorganisms, involved in infections, not host cells Antibiotics -> bacterial infections Antiviral drugs -> virus infections Antifungal drugs -> fungal infections Anthelminthic drugs -> worm infections (parasites) Antiprotozoal drugs -> protozoan infections (parasites) Each group has a different class of drugs, possessing different mechanisms of actions and targeting different specific microbial targets

Targets of different classes of antibiotics

SELECTIVITY - Take advantage of the difference between the structure of the bacterial cell and the host’s cell

Broad / narrow

Considering differences in Gram+ and Gram– bacteria ->Antibiotics have a different spectrum of activity -Broad-spectrum -Narrow spectrum (Gram+ve or Gram–ve, or species -specific) Antibiotics selectively target specific bacteria that are involved in a certain infection, by interfering with specific bacterial targets or steps