Foundations of infectious disease lecture 1-8

Zaccharias Janssen

• 1590

• develops first useful microscope

Francesco Stelluti

• 1625

• observed bees and weevils using a microscope

Robert Hooke

• 1665

• saw fungal fruiting bodies using a compound microscope that he designed

Antonie van Leeuwenhoek

• 1676

• made first microscopic observations of microorganisms including bacteria and protozoa

Francesco Redi

• 1688

• suggested that all life arose from eggs or parental life

• showed that rotting meat protected from flies will not spontaneously produce maggots

John Needham

• 1750s

• supported spontaneous generation using boiled brooth in loosely sealed or open flasks

Lazzaro Spallanzani

• 1799

• rejected theory using Needham’s (modified) experiment

• boiled flasks for 45 min, sealed, no growth occurred (as long as the flasks remaine sealed)

Louis Pasteur

• 1861

• provided the most convincing evidence against spontaneous generation — experiments with swan-neck flasks

• fermentation is due to yeast

• pasteurisation

• 1881 — anthrax vaccine

• 1885 — Rabies vaccine

Robert Koch

• 1843-1910

• first direct demonstration of bacteria in causing disease (anthrax) — Koch’s postulate

• 1876 — described broths for culturing microbes (nutrient agar and broth)

• 1881 — cultured bacteria on media solidified with gelatine

• 1882 — discovered TB bacillus

• 1884 — reported that Mycobacterium tuberculosis was the cause of TB

• 1887 — assistant Julius Petri modified Koch’s culture technique and developed the petri dish as we know it

3 domains of life

• Bacteria

• Archaea

• Eukarya

bacterial cell components

• cell envelope — cell wall, cytoplasmic membrane

• cytoplasm

• nucleoid region

• ribosomes

• other

cytoplasmic membrane

• thin, pliable, defines the cell, contains cytoplasm

• made of lipids and proteins

• bilayer has two layers of amphipathic phospholipids

• selectively permeable; controls movement of small molecules in and out of the cell

cell wall

• almost all bacteria have some type cell wall — rigid yet permeable layer surrounding the plasma membrane, often peptidoglycan but not always

• peptidoglycan made of two alternatings sugars NAG and NAM

• Polysaccharides have tetrapeptides attached to each NAM

cell wall characteristics

• most bacteria can be classified into Gram Postive or Gram Negative

• groups based on characterisitcs of cell wall which can be shown durig Gram staining process

• thick gram positive cell wall peptidoglycan retains crystal violet/iodine complex — no outer membrane

• thin gram negative cell wall peptidoglycan does not — outer membrane with lipopolysaccharide

outer membrane

• present in only Gram Negative bacteria

• similar composition to plasma membrane but asymmetric

• may have polysaccharide attached

• OM acts as a ‘molecular sieve’

Mycobacterial cell wall

• unlike gram negative or positive

• thin layers of peptidoglycan and arabinogalactan

• thick layer of myolic acids — waxy and hydrophobic. Mycolid acids makes cells difficult to stain

• high lipid contents results in impermeability to stains and resistance to adverse conditions

• stain using Ziehl-Neelsen or Kinyoun’s cold stain

functions of cell wall

• protective and structural function

• provides protection from osmotic shock

• allows passage of nutrients

• gram negative —- outer membrane is a barrier to certain molecules that can pass through Gram positive cell wall

bacterial cytoplasm

• fluid portion of cytoplam contains — soluble enzymes
  and various inorganic and organic compounds

• very granular appearance due to ribosomes

• located between the plasma membrane and the nucleoid

bacterial nucleoid

• contains the bacterial chromosome and a number of proteins

• for most bacteria (and all known archaea) — simply a region in the cytoplasm; not membrane-bound

• genome contains all information for controlling development and metabolic activities of cell

bacterial chromosome

• encodes genes essential for life

• usually consists of single, circular molecule of DNA

• varies in size between species

• folded into tight mass (supercoiled)

Plasmids

• not essential

• Plasmids are extra-chromosomal DNA - replicate autonomously, typically circular but linear plasmids also documented, transferable from one cell to another

• contain non-essential genetic information — not needed for cell survival but useful in certain environments, offer a selective advantage

Ribosomes

• Involved in protein synthesis

• large numbers found in nearly all cells

• 70S in bacteria

endospores

• multi-layered survival structures — enables organism to survive extreme conditions that kill vegetative cells

• endospore formation triggered by starvation

• each vegetative cell produces one endospore

• produced only by a few gram positive genera

three components of taxonomy

• classification

• identification

• nomenclature

Classification of living things

• all life classified into 3 domains — then 23 divisions

• viruses are “non-living”

classification

• grouping microbes into taxa, based on their characteristics

• classical characteristics — includes phenetic

• molecular characteristics — biochemical characteristics and genetic characteristics

• phylogenetic analyses — analyses relationships between isolates using all available information

phylogenetic analyses

• organisms are grouped based on characteristics that reflect evolutionary relationships

• organisms arranged into evolutionary or phylogenetic tree

classical characteristics

• phenetic classification

• the first system used using (1) morphological, (2) physiological and (3) ecological characteristics

• does not necessarily reflect evolutionary relatedness

• still useful for living things but being superseded by molecular methods

Nomenclature

• names assigned to taxonomic groups according to defined rules

• pioneered by Carolus Linnaeus — binomical nomenclature “Genus name + species name”

taxonomic ranks

• Microbes grouped into categories containing smilar organism

• groups at each level share common properties with the group they belong to in higher ranks

• rank names and hierarchy are common to both phylogenetic and phenetic classification schemes

strain

a genetic variant or subtype of a bacterial species

phylotype

an organism identified solely by nucleic acid sequence — lacks sufficient data to confirm a species name but is definitely a real organism

characteristics of fungi

• eukaryotic

• non-vascular

• reproduce by means of spore formation or by vegetative methods

• depending on the species and environmental conditions fungi may produce sexual spores, asexual spores or both

• medically important fungi are typically non-motile

Vegetative body of fungus

• may be unicellular as seen in yeasts

• or multicellular composed of microscopic threads called hyphae with internal divisions called septa and various sporing structures

mycelium

• dense, interconnected network of hyphae

• typically grow hidden within a nutrient source

• visible part of the fungus is the fruiting body

How do fungi eat

• heterotrophic — obtain nutrients from preformed organic material

• produce exoenzymes

• fungi digest first, then absorb nutrients

• store food as glycogen

Superficial Mycoses — Tinea

• Dermatophytes utilise keratin as a subtrate and many affect any superficial body site

• Anthropophilic only affect man

• zoophilic have a reservoir of infection in certain animals

• geophilic are found in soil

Subcutaneous Mycoses

• fungus gains entry to the sub cutaneous tissue usually by a penetrating injury

• sporotrichosis caused by Sporothrix schenckii complex

• usually associated with handling of mould hay or other contaminated organic material

• thermally di-morphic — in 26c have spores on fine denticals that look like a flower, tapered conidiophore

endemic mycoses

• known geographic distribution for each

• inhalation → pulmonary infection → dissemination

• no evidence of transmission among humans or animals

• causative agents: thermally di-morphic fungi

  • coccidioidomycosis, histoplasmosis, blastomycosis

opportunistic mycoses

• causative agents — saprophytes in nature, some found in normal flore

• host — broad spectrum antibiotics

• indwelling catheters/peritoneal dialysis

• therapy

• decreased immune function

• allergy

• i.e candidiasis, cryptococcosis, aspergillosis

clinical laboratory techniques for fungi

• direct microscopy

• culture

• basic morphological identification techniques

• fungal susceptibility testing

general properites of viruses

• genetical material — dna or rna

• proteinaceous shell — capsid

• some viruses have a lipid membrane outer layer envelope containing virus-derived glycoproteins

enveloped viruses

many viruses are surrounded by a membrane called the envelope

• derived from host cell membranes and may also contain proteins encoded by viral genese

two phases of viruses

• extracellular - biochemically inert and cannot reproduce independently of living cells

• intracellular - exist as replicating nucleid acids, induce host cells to synthesize virion components — release from the cell as infectious particles

capsid morphology

• capsids made up of subunits called capsomeres

• protect from environmental danger

• capsids absorb to host cell surfaces during attachment

helical capsid

• hollow tubes with protein walls

• i.e tobacco mosaic virus — composed of single protein, self-associates into spiral arrangement to produce a long rigid tube

• RNA is wound in a spiral inside the capsid

icosahedral capsids

• most efficient method to enclose a space wtih maximal internal voume

• form a ring shaped units called capsomeres, each made with 5 or 6 protomer

• poliovirus is 60 capsomer units (simple) and adenovirus is 250 capsomers (complex)

types of capsomers in icosahedral capsids

• pentons — located at vertices, always 12 pentamers

• hexons — form edges and triangular faces, number varies among virus groups

complex capsids

do not conform to regular symmetry of viruses

viral replication

• attachement: viruses attach to cell membrane

• penetration: via endocytosis or fusion

• uncoating: by viral or host enzymes

• biosynthesis: production of nucleic acid and proteins

• maturation: nucleic acid and capsid proteins assemble

• release: but budding (enveloped viruses) or rupture

baltimore classification

• central theme is “all viruses must synthesze positive-strand mRNAs from their genomes, in order to produce proteins and replicate themselves’

Double stranded DNA viruses

• papillomaviridae

• herpesviridae

• Pox viridae

• hepadnaviridae

viruses with RNA genomes

• most RNA viruses use ssRNA as genetic material

• (+) sense - viral RNA genome identical to viral mRNA

• (-) sense - viral RNA complementary to viral mRNA. Converted to + sense RNA using viral RNA-dependent RNA polymerase (carried by the virus).

(+) stranded RNA viruses

• Picornaviridae

• Togaviridae, flaviviridae

• coronaviridae

(-) stranded RNA viruses

• paramyxoviridae

• orthomyxoviridae

• rhabdoviridae

what are parasites

living organism that acquires some of its basic nutritional requirements through its intimate contact with another living organism

protozoa

• unicellular and small organisms

• cause of major mortality events i.e. malaria, leishmaniasia, chagas disease

• can be intracellular or extracellular

• transmitted by vectors, food/water, or directly

metazoan

• multicellular organism

• very common in rural and tropical regions, less so in developed countries

• often cause chronic debilitating diseases

endoparasite

lives within another living organism

ectoparasite

lives on external surface of living organism

opportunistic pathogen

organisms that are generally not pathogenic or that can reoccur from a quiescent stage

• organisms which are normally harmless to the immunocompetent are lethal to the immunocompromised

direct life cycle

only one host in life cycle

indirect

two or more host required

definitive or primary host

where parasite reaches maturity and undergoes sexual reproduction

reservoir host

can harbour pathogen often with minimal effect

secondary or intermediate host

where the parasite usually undergoes asexual production

transmission of parasites

• direct — passed directly from one infected host to another by some physical means or by direct invasion by the parasite

• food or waterborne

• via an intermediate host or vector — very common amongst parasites

zoonosis

any disease which can be transmitted to animals

anthroponosis

a disease that is spread from humans to humans

ectoparasites

insects and arachnids that feed on human skin or tissues

• mostly due to ticks, fleas and mites

how can cross-infection may occcur?

• aerosols — coughing, irrigation

• direct contact — blood, secretions, skin

• indirect contact — instruments, toilets, bedding

sterilisation

process that kills or removes all living organisms including endospores

disfenction

process that kills or removes the majority of organisms but not endospores

antisepsis

external application of chemical agent to live tissue to kill or inhibit the growth of organisms

dynamics of microbial death

• microbes do not die instantly when exposed to an agent

• microbial death is generally exponential

non-microbial factors

• antimicrobial concentration — inc concentration = inc killing

• duration of exposure (time)

• presence of organic matter i.e saliva, blood, pus

• initial microbial population size

microbial factors

features unique to particular organisms that render them less (or more) susceptible

• endospores

• vegetative cell structure

• efflux of disinfectants/antiseptics

• biofilms

stages of sterilisation

• presterilisation cleaning — manual scrubbing or automated cleaning

• packaging

• sterilisation process

• aseptic storage

sterilisation for items that can tolerate heat

• moist heat

• dry heat

• chemical vapour/high temp

sterilisation for temperature sensitive solutions, equipment and disposables

• chemical vapour/low temp

• radiation

• membrane filtration

autoclave

• sealed chamber with steam generated within chamber at high temp and pressure — the combo of temp and pressure disrupts cell membranes; denatures proteins; degrade nucleic acids

• range of sizes available

disadvantages of autoclave

• items get wet

• cycle cannot be interrupted

• steam may corrode/rust metals or dull blades

• some plastics degrade if autoclaved repeatedly

dry heat sterilisation

• destroys microbes by disruption of membranes, denaturation/degradation of biological molecules

• used for items damaged or impenetrable by moist heat

• low operating costs, non-toxic, no water or pressure

disadvantages of dry heat sterilisation

• long cycle time and higher temperature

• dry air conducts heat poorly

• includes heating, holding an cooling phases

chemical vapour sterilisation

• performed in chemiclave

• uses toxic gases combined with high pressure and temp — unsaturated formaldehyde, alcohols, acetone, ketones

• dry process with low humidity

• kills microbes by combo of heat denaturation/degradation of biological molecules and chemical modification of proteins

• dry process, no corrosion or damage to metals, relatively quick

disadvantages of chemical vapour sterilisation

• toxic fumes

• chemicals may be expensive

gas sterilisation

• chemical vapour combined with low temperature

• similar chemiclave but lower temperature and longer time

• used ethylene oxide gas — kill microbes by chemical modification of proteins

• used to sterilise plasticware, syringes, sutures, catheters

ionising radiation

• gamma radiation

• kills by inducing variety of chemical changes to biological molecules

• used to sterilise — plastics including sutures and other disposables and pharmaceuticals such as antibiotics, hormones

• penetrates well, not affected by temperature or pressure

radiation

• UV radiation

• kills microbes by generating covalent links between adjacent thymine bases (dimers) in DNA

• mostly commonly used the sterilise inside of laminar flow cabinets

disadvantages of uv

• does not penetrate glass, water, other substances

• limited to sterilising air spaces/surfaces

membrane filtration

• removes bacteria and fungi from solutions by passeg

• smallest pore size commonly available = 0.2μm

• does not exclude viruses or mycoplasma spp.

• may be used to sterilise heat-sensitive pharmaceuticals or culture media/laboratory

classification of disinfectants

• low

• intermediate

• high level

low-level disinfectants

• relatively mild, non-irritating — suitable for skin antisepsis

• bisbiguanide compounds

• quaternary ammonium compounds

bisbiguanide compounds

• chlorhexidine

• disrupts cell wall and cell membranes: increased cell permeability → death

• used for skin antisepsis and hard-surface disinfection

• low skin irritation, binds strongly to proteins in skin

• inactivated by hard water and soap

intermediate level disinfectants

• alcohols like 70% ethanol, propanol

• peractic acid and hydrogen peroxide

• halogen compounds

• phenolics

• aldehydes

70% ethanol

• disrupt cell membranes

• used for skin antisepsis

• evaporates quickly without residual activity

• readily inactivated by organic matter

peracetic acid and hydrogen peroxide

• oxidising agents

• sporicidal

• used for hard surfaces

• used in automated machines to chemically sterilise instruments

• vaporised form may be used for fogging/fumigation

• eco friendly; biodegradable

• corrode metal, bleach fabrics, may be irritating to eyes; fumes should be inhaled

halogen compound

• chlorine-containing — hard surface disinfectant, act by oxidising a wide array of biological molecules

• iodine-containing — aka iodophors, used as skin antiseptic

• corrod metal, inacitivated by organic matter

phenolics

• chemical derivatives of phenol

• used widely for decontamination of surfaces

• disrupt cell membranes and denaturing proteins

• remain active for long periods after application

• not readily inactivated by organic matter

• highly irritating to skin

aldehydes

• glutaradehyde, formaldehyde

• kill microbes by chemical modifiction/cross-linking of proteins

• immersion in glutaraldehyde for long time periods may achieve sterilisation

• highly toxic, skin irritant, vapours irritating to eyes and respiratory tract

• use as disinfectant banned in some places due to long term health effects