Pharmaceutical Microbiology Revision

Table of Contents

PHARMACEUTICAL MICROBIOLOGY REVISION............................................................................................... 1

BACTERIAL STRUCTURE..........................................................................................................................................1
BACTERIAL GROWTH AND ENUMERATION.................................................................................................................5
BACTERIAL IDENTIFICATION..................................................................................................................................12
BACTERIAL ENDOSPORES.....................................................................................................................................15
STDS AND TB...................................................................................................................................................19
BACTERIAL VIRULENCE........................................................................................................................................21
YEASTS.............................................................................................................................................................25
MOULDS.......................................................................................................................................................... 30
ANTIFUNGALS....................................................................................................................................................31
VIRUSES........................................................................................................................................................... 32
HIV AND AIDS..................................................................................................................................................38
PROTOZOA AND MALARIA....................................................................................................................................43
HELMINTHS.......................................................................................................................................................47
MICROBIOME....................................................................................................................................................50
SOURCES OF CONTAMINATION..............................................................................................................................53
EXPLOITATION OF MICROORGANISMS....................................................................................................................55

Bacterial Structure
Cell Envelope – Gram-Positive
- Outer cell wall (peptidoglycan)
o Chemically fairly inert
o Composed of subunits found
nowhere else in nature
- Plasma membrane
- E.g. Staph aureus, Bacillus subtilis

Cell Envelope – Gram-Negative

- Cell wall (peptidoglycan)
- Plasma membrane
- Cell envelope can produce
endotoxins which cause symptoms
of disease
- E.g. E. coli, Pseudo aeruginosa

Gram Staining
- Distinguish between the different cell envelopes
- Played a major role in classifying bacteria
- From Hans Christian Joachim Gram (1853-1938)
- Gram-positive
o Low lipid conc is important for the retention of the complex
iodine crystal violet
o The cells remain blue
- Gram-negative
o High lipid conc found in outer layers of cell wall is dissolved,
facilitating the release of the iodine crystal violet complex
o The cell goes colourless
- Add safranin stain to see gram negative bacteria under a
microscope

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Cell Wall/Peptidoglycan
- 3D, rigid, multi-layered network around the organism
- Prevents osmotic rupture cell in concentrated ionic solutions
- Consists of 2 major amino sugars
o N-acetyl-muramic acid
o N-acetyl-glucosamine
- They alternate to form a high molecular weight polymer
- A chain of several amino acids is attached to each of the N-acetyl-
muramic acid molecule
- Final assembly outside the membrane by different enzymes
(penicillin binding proteins – PBP)

Cytoplasmic Membrane
- Cytoplasm is bound by plasma membrane
- Chemical composition of the plasma membrane is similar to that of
the mammalian cell: 40% lipid, 60% protein, small amounts of
carbohydrates but sterols are absent
- Semi-permeable – low molecular weight materials can pass through
- Contains many proteins/enzymes essential to the bacterium
- Efflux proteins – pump substances out of cell by active transport
(including antibiotics – antibiotic resistance)

Ribosomes
- Protein factory
- 70S ribosomes (different from 80S
ribosomes of eukaryotic cells)

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 - Made up of 2 parts (30S and 50S)
- Each of these parts is made of 2 different macromolecules
(ribosomal protein and ribosomal RNA)

Nuclear Apparatus
- Lack an organised nucleus but have DNA
- The chromosome exists as a closed circle in all bacteria and is not
surrounded by a nuclear membrane
- Some bacteria also possess a short length of extrachromosomal
DNA in the form of a plasmid
- Plasmids range in size from 0.1-10% of the chromosome
- Plasmids often play an important role in the transfer of genetic
material between bacteria

Infusion Bodies
- Many bacteria form and store granules in their cytoplasm in the
form of high molecular weight polymers
o Glycogen – storage form of both carbon and energy
o Polymer of β-hydroxybutyric acid – storage form of both
carbon and energy
- Phosphate can be stored as granular inclusions of polymeric
phosphate volutin

Flagellum
- Motility
- Usually gram-negative bacteria have this

Pili/Fimbriae
- Hair-like appendages
- Smaller than flagella (0.5µm)

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 - Consist of protein sub-units wound around one another generating
a hollow core
- Number of pili per cell varies from 1 to 400
- Can be separated into a number of types based on their function
o Adherence – to one another and to foreign cells
o Antigenic
o Genetic exchange by conjugation – F or sex pili
o Attachment sites for bacteriophages
o Chemotaxis
o Virulence – toxin

Bacterial Growth and Enumeration

Binary Fission
- How bacteria divide
- Increase of cell mass
- Duplication of genome
- Cell membrane and cell wall separation
- Mother and daughter cells are identical
- A packet of bacteria is formed when the cell divides in 2 or more
planes perpendicular to one another

Bacterial Growth
- Change in the population rather than an increase in size or mass of
an individual bacterium
- Lag phase
o Population remains temporarily unchanged
o Bacterial cells may be growing in
volume or mass, synthesising
enzymes, proteins, RNA, etc., and
increasing in metabolic activity
- Exponential growth phase

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 o Expressed as generation time, or doubling time of the
bacterial population
o Increase in population by geometric progression
o 1, 2 (21), 4 (22), 8 (23), etc. 2n
o N is the no. of generations (assuming no cell death)
- Stationary phase
o Exhaustion of available nutrients
o Accumulation of inhibitory metabolites or end products
o Exhaustion of space

Chemostat
- A bioreactor to which fresh medium is continuously added, while
culture liquid is continuously removed to keep the culture volume
constant
- Control growth rate and optimised production
- Continuous and batch fermentation

Chemical Requirements for Growth

- Energy source
o Chemotrophs – chemical compounds
o Phototrophs – radiant energy (light)
- Electron source
o Lithotrophs – reduced inorganic compounds
o Organotrophs – use of organic compounds
- Carbon source
o Autotrophs – CO2 as their main source of carbon
o Heterotrophs – organic compounds as their carbon source
- Nitrogen source
o Atmospheric nitrogen
o Inorganic nitrogen (nitrate, nitrite, ammonium salts)
o Organic compounds (amino acids)

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 - Oxygen
o Obligate anaerobes cannot grow in the presence of oxygen
o Facultative anaerobes will use oxygen if present but do not
require it
o Microaerophilic bacteria tolerate low levels of oxygen
- Sulphur, phosphorus
- Ions

Physical Requirements for Growth

- Temperature – optimum growth temp allows maximum growth
during a short period of time
o Psychrophiles – 0-20˚C (optimum 15˚C)
o Mesophiles – 25-40˚C
o Thermophiles – >45˚C
o Bacteria that cause diseases in humans grow best at 37-39˚C
- pH – optimum pH for growth is 6.5-7.5
o Limits – 5-9
o Exception – Thiobacillus thiooxidans (0.5-6 [optimum 2-3.5]
and alkaline spring isolate (8-11.4 [optimum 9-9.5])

Prevention of Bacterial Growth

- Temperature
o Deep freeze (-20˚C) – Total Parenteral Nutrition, raw
materials
o 8-12˚C – reconstituted syrups and multi-dose eye drops
(expiry date)
o 80˚C for WFI – regrowth of Gram-negative and release of
toxins
- pH
o Extreme pH – prevent microbial growth
o Neutral pH – bacterial spoilage e.g. antacid mixtures,
flavoured mouth washes, distilled and deionized water

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 o pH>8 – spoilage is rare (soap-based emulsion)
o low pH – spoilage by moulds and yeast e.g. fruit flavoured
syrups
- Water activity (Aw)
o Estimates the proportion of uncomplexed water (just any
water available) available in a formulation
o The greater the solute concentration, the lower the water
activity (more stuff dissolved means less water)
o Most micro-organisms (except halophiles) grow best in dilute
solutions (high Aw)
o Water activity of aqueous formulations can be lowered to
decrease microbial growth
o There are limiting Aw values for each bacterium
o E.g. tablets have no water so no bacteria can grow so long as
it remains dry
o Aw < 0.6 would prevent growth

Sterile Pharmaceutical Products

- Preparations required to be sterile on the dosage form and other
preparations labelled sterile
- Need to comply with test for sterility

Non-sterile Pharmaceutical Products

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 Enumeration – Viable Count
- Colony count
o Single bacterium in the original culture being plates is
assumed to give rise to a single viable colony
o The viable count referred to as the no. of colony-forming
units (CFUs)
o Type of medium used and conditions of incubation will alter
bacterial growth and formation of colonies

- Plate count
o Pour plate method
o Spread plate method
o Any other methods must be validated
o If a lot of bacteria occur and colonies merge together, we
dilute them so they are countable
o Positives
 Simple to use and more sensitive than turbidimetric
 Identification of organism counted
 Easy to count small no. of bacteria in sample
o Negatives
 Time consuming
 Can only count living cells
 Clumps/chains can develop into a single colony
 Colonies can only arise from organisms that can grow
under the provided conditions

- Membrane filtration
o Similar to plate count but for liquids
o Cellulose nitrate filters for aqueous, oily, weakly alcoholic
solutions
o Cellulose acetate filters for strongly alcoholic solutions

- Turbidimetric measurement

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 o Measures loss on intensity of transmitted light
o Positives
 Faster than plate count
 Can be done without spoiling samples
o Negatives
 Requires high number of cells for estimation
 If mass of bacteria increases more than normal, the
light is masked leading to inaccurate results
o Most probable number method can be used with this

- Most probable number (MPN)

o Reserved for enumeration where no other method is
available (e.g. solid or viscous formulations)
o Prepare a series of at least 3 subsequent tenfold dilutions of
the product
o From each level of dilution 3 aliquots of 1g or 1ml are used to
inoculate 3 tubes with 9-10ml of a suitable liquid medium
o Incubate all tubes for 5 days at 30-35˚C and record for each
level of dilution the number of tubes showing microbial
growth

Enumeration – Total Count

- Cell count – directly with microscopy or electronic particle counter
- Cell mass – directly by weighing, measurement of cell nitrogen,
measurement of dry weight (oven)
- Spectrophotometric measurement – turbidity, optical density
- Cell activity – indirectly by relating degree of biochemical activity to
the size of the population

Enumeration – Rapid Microbiology Methods (RMM)

- Growth based methods – detectable signal is usually achieved by a
period of subculture
o Electrochemical methods

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 o Measurement of consumption or production of gas (CO2)
o Bioluminescence (ATP)
o Microcalorimetry (temp)
o Turbidimetry (OD)
- Direct measurement – individual cells differentiated and visualised
o Flow cytometry
o Solid phase cytometry
o Direct epifluorescent filtration technique (DEFT)
- Cell component analysis – expression of specific cell components
offers an indirect measure of microbial presence
o Nucleic acid amplification techniques (NAAT)

Bacterial Identification

Traditional Identification Methods

- Cultivation – microscopy
o Pure culture – isolate a single colony
o Morphology – shape (cocci, bacilli, etc.)
o Growth requirement – temp, oxygen, salt, etc.
o Staining characteristics – endospore production, flagella,
capsule

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 o Does not identify genus or species

- Cultivation – selective media

o Promote growth of target
o Inhibit other bacteria
o Development of indicative colour (of agar and/or colony)
o MacConkey agar (MAC)
 Grows gram-negative bacteria
 Can distinguish lactose fermenters
 E. coli – pink colonies + agar (lactose positive)
 P. aeruginosa – white/brown/green colonies + agar
(lactose negative)
o Vogel Johnson agar (VJA)
 Tellurite and lithium chloride
 Isolates Staph spp which are black colonies with yellow
agar edges
 Tellurite – coagulase enzyme reduces to metallic
tellurium (black colonies)
 Manitol degradation alters pH (yellow halo)
o Cetrimide agar (CET)
 Cetrimide inhibits most bacteria by acting as a
detergent
 Isolate P. aeruginosa which has fluorescent green
colonies + green agar
o Sabouraud agar (SABDEX)
 Used for selective cultivation of fungi, yeasts, moulds,
and aciduric bacteria
 C. albicans has cream colonies

- Biochemical profiling
o Based on growth requirement, enzymatic activities, etc.
o Even closely related microorganisms can be separated
o Colour after 24 hrs tells you if the bacteria have used the
substrate in the wells or not

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 o No. of positive and negative tests gives a profile

- Serological testing
o Uses highly specific antibody antigen interactions
o Can be used to detect certain pathogens
o Enzyme linked immunosorbent assay (ELISA)
 Sensitive – signal amplified by conjugate enzyme
 Quantitative – linked to the conc of antibodies (intensity
of colour depends on antibody conc)
o Positives
 ELISAs are available to test for the most common
bacterial pathogens
 Highly specific
 High sensitivity (signal amplified by conjugated enzyme)
o Negatives
 Expensive
 Cannot identify unknown bacteria
 Complex process (training)
New Identification Methods
- Nucleic acid techniques
o PCR based techniques allow amplification of known gene of
interest for nucleic acid sequencing
o Sequencing of rRNA provides very accurate identification to
genus or species level
o Can be used to identify unknown/uncultivable organisms
o Gene sequence is compared with online database (e.g. NCBI)
o BLAST tool finds regions of similarity between your sequence
and those identified previously
o Positives
 Do not require growth of bacteria for identification
(doesn’t require pure culture)
 Very sensitive
 High accuracy
o Negatives

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  Specialised equipment (expensive)
 Costly reagents
 Time consuming

- Microarrays
o Thousands of ssDNA probes are fixed to a solid support (chip)
o Probes could be rRNA or antibiotic resistance genes
o Labelled (fluorescence) target sample is hybridised with
probes
o Positive match shows as a bright dot

- Whole genome sequencing

o Not just bacterial identification but info about metabolism,
growth requirements, pathogenicity (including antibiotic
resistance)
o Becoming more cost effective
o Vast amounts of data (time consuming analysis)
o Requires expensive equipment and reagents
o Far more expensive than other methods

- MALDI-TOF MS
o Matrix assisted laser desorption ionisation time of flight mass
spectrometry
o Unknown bacteria mixed with matrix material
o Bombarded with laser pulses
o Sample is ionised and passed through electrostatic field
(acceleration)
o Ions pass through flight tube before hitting detector (small
ions travel faster than large ones) (TOF)
o Generates a unique mass spectrum for different bacteria –
check against database
o Positives
 Cost effective
 Short turnaround times

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  Precise identification to species level
o Negatives
 Can’t identify species in mixed bacterial populations
 Reliant on quality and coverage of the database used

Traditional Methods Newer Methods

No specialised equipment
Can be done by most labs
Minimal training
24-72hr for results
Traditional vs New Methods
Specialised equipment (£)
Lower running costs (MALDI-TOF)
Faster (~6hr)
High throughput

Bacterial Endospores
Structure and Function
- Have a very distinctive structure
- Unique structure formed within the vegetative cell
- Enable survival of bacterium (genetic material) during harsh
conditions (lack of food, water, chemical/physical stress)

Sporulation
1. DNA replicates and extends
into an axial filament

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 2. Septum forms near one pole, separating forespore from mother cell
Each gets a chromosome
Asymmetrical separation
3. Mother cell engulfs the forespore surrounding it with a second
membrane
4. Chromosomes of mother cell disintegrate
5. Forespore develops a cortex layer of peptidoglycan between
original forespore membrane and the membrane from the mother
cell
6. Dipicolinic acid is synthesised, and calcium is incorporated into the
spore coat
7. Mother cell releases spore

Germination
- Occurs when environmental conditions will support bacterial
growth
- Can be forced to occur using certain chemicals called germinants
(e.g. D-alanine)
- Germination will not occur, even in good conditions, if the spore is
not activated

1. Spore is activated, it rehydrates, loses resistance to heat and

chemicals
2. Binding of effector molecules activates autolysis that destroys
peptidoglycan of cortex, water is taken up and calcium/dipicolinic
acid is released, typical vegetative cell structures are recovered
3. Functionalities of vegetative cell are recovered (synthesis of DNA,
RNA, proteins), vegetative cell escapes from spore coats, binary
fission occurs again

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Spore Resistance
- Spores can survive a range of physical and chemical reagents that
would kill the vegetative cell (e.g. high temp, deep freeze,
disinfectants, UV, lack of nutrients)
- Spores are used as biological indicators of sterility assurance of
sterilisation processes
- High level disinfection will kill 99.999% of microorganisms including
spores
- Spore coat is a barrier to biocides
- Inner membrane is highly compressed and barrier to biocides and
rehydration
- Cortex is barrier to biocides and give physical pressure to inner
membrane
- Spore coats has SASPs which protect the nucleic acid and has low
water content

C. Difficile Associated Disease (CDAD)

- Clostridium difficile
- C. diff usually lives harmlessly in the bowl
- When you take antibiotics the balance of bacteria can change,
causing an infection
- Use of broad-spectrum antibiotics increases the risk of infection

Impact on Treatment
- Antibiotics should only be prescribed when there is clinical evidence
of bacterial infection

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 - Stop antibiotics if cultures are negative unless otherwise clinically
indicated
- Switch to narrower spectrum antibiotics when possible
- Use short course antibacterial treatment unless otherwise clinically
indicated

STDs and TB
Chlamydia Trachomatis
- Obligate intracellular bacteria – need a eukaryotic host to survive
- Lack the metabolic pathway to produce their own high energy
phosphate compounds
- Exist in 2 forms
o Small (300-400nm) extracellular infection elementary body
(EB), metabolically inactive and highly infectious
o Large (800-1000nm) intracellular non-infection reticulate
body (RB)
- Structure
o Internal and external membrane similar to gram-negative
bacteria
o No peptidoglycan layer

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 o Lipopolysaccharides
o No flagella or pili
- Process
o EB attaches to susceptible cell (e.g. epithelial cell of urogenital
tract) and is ingested by it
o The EB reorganises into an RB inside the phagosome and
binary fission of the RBs occur
o For 24+ hrs there’s a growth phase and then there is
reorganization into EBs
o There are both EBs and RBs in the inclusion
o After 48-72 hrs infectious EBs are released from the cell by
exocytosis
- Treatment
o Inhibited by antibiotics
o Tetracyclines: doxycycline 100mg BD 7days
o Macrolides: Azithromycin 1g single dose
- Prevention
o Safe sex
o Not sharing sex toys
o Appropriate sanitary hygiene
o Contact tracing to treat sexual partners

Mycobacterium Tuberculosis
- Top 10 causes of death worldwide and the leading cause from a
single infection (above HIV/AIDS)
- Multidrug-resistant TB (MDR-TB) remains a public health crisis
- Extensively drug-resistant (XDR-TB) responds to even fewer
medicine

Mycobacteria Structure
- Cell wall
o Highly hydrophobic

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 o Peptidoglycan linked to an arabinogalactan wall (copolymer
of arabinose and galactose) esterified to mycolic acid
structure
- Mycolic acid
o High molecular weight (60-90 carbons) 3-hydroxy fatty acid
o Many types identified in mycobacteria
- Other lipids
o 25% of the dry weight is free
lipids located outside the
outer layers
o Includes waxes, species-
specific mycosides,
lipopolysaccharides and cord
factor (associated with the
parallel alignment of rows of
bacilli)

TB Risk Factors
- Close contacts of an infectious case
- Those who live where TB is common
- Those with weakened immune systems
- People who experience chronic poor health due to lifestyle factors
such as alcoholism, drug abuse and homelessness
- Young children and very elderly people

TB Treatment
- Isoniazid stops lipid production in the cell envelope
- Rifampicin inhibits bacterial DNA-dependent RNA polymerase
- Pyrazinamide inhibits fatty acid synthetase
- Newly diagnosed:
o Initial phase (2 months) – isoniazid + rifampicin +
pyrazinamide + ethambutol
o Continuation phase (4 months) – isoniazid + rifampicin

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 - Re-treatment:
o Initial phase (2 months) – isoniazid + rifampicin +
pyrazinamide + ethambutol + streptomycin
o Continuation phase (5 months) – isoniazid + rifampicin +
ethambutol
- Treatment needs to be fully supervised (Directly Observed Therapy
– DOT) in patients who cannot be relied upon to comply with the
treatment regimen
- Anti-TB drugs are always prescribed in combination to reduce the
risk of TB bacilli becoming resistant to one or more of them
- It is vital the medication is taken as prescribed as taken it in the
wrong dose, intermittently or for not long enough can result in drug
resistance

Bacterial Virulence
Normal Flora and Opportunistic Pathogens
- Some normal flora are able to cause disease at a time of immune
compromise – these are opportunistic pathogens
- E.g. S. aureus, E. coli, C. albicans
- Bacteria that are not normal flora but represent pathogenic
organisms that may be present in a large percentage of the
population without causing disease (asymptomatic carrier)
- E.g. methicillin resistant S. aureus and C. difficile

Virulence
- The degree or intensity of pathogenicity of an organism
- Virulence factor – a microbial product that contributes to virulence
- Example of virulence factors in E. coli:
o Adhesin – colonisation factor
o Endotoxin – lipopolysaccharide (pyrogen)
o Exotoxin – heat-labile (LT) enterotoxin
o Invasin – uropathogenic E. coli

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 o Capsule – evade phagocytosis
- Antibiotic resistance is not a virulence factor

Toxins
- A substance produced by an organism that damages the host
- Exotoxin:
o Usually proteins released by bacteria as it grows and divides
o Neurotoxins, enterotoxins, cytotoxins
o Required in very small amounts to cause disease
o Usually strong antigenic
o Inherently unstable – artificial immunisation
o Often composed of protein subunits
o Mediate their effects outside and inside cells – membrane-
damaging, membrane acting (disrupt signals in receptors),
intracellular
- Endotoxin:
o Bound to bacterium and is released when the bacterium lyses
o Gram-negative bacteria; lipopolysaccharide (lipid A)
o Toxic at high doses compared to exotoxins
o Stable
o Weakly immunogenic
o Capable of producing general system effects

Capsule/Slime Layer/Glycocalyx
- Loose-fitting, gelatinous structure that surrounds some bacteria
- A network of fibrils of polysaccharide-containing material outside
the cell wall
- Has many roles:
o Virulence – prevents phagocytosis
o Protection – contains water which protects bacteria against
desiccation

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 o Protection – exclude bacterial viruses and most hydrophobic
toxic materials such as detergents

Pathogenicity Islands
- Virulence genes can be found in specific regions of bacterial
genome (chromosome, plasmid)
- Specialised for export of virulence factors directly into cells
- Confer a variety of virulence traits (e.g. cell adherence, toxins)
- Provides sudden radical changes in bacterial-host interactions
- Many seem to have been acquired in a single step from a foreign
source

Antibiotic of Choice
- Determinants – selective toxicity, therapeutic dose, toxic dose
- Spectrum of activity – broad, narrow, bacterial type
- Antimicrobial resistance (AMR) – propensity of bacterial isolate to
develop resistance
- Efficacy – bacteriostatic vs bactericidal
- Rout of administration – oral or IV
- Side effects
- Cost – of antibiotic and duration of treatment
- Allergy to penicillin
- CDAD
- Combined treatments

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Therapeutic Index
- Aka therapeutic dosage level
- A comparison of the amount of a therapeutic agent that causes the
therapeutic effect to the amount that causes death (in animal
studies) or toxicity (in human studies)
- Therapeutic Index = toxic dose / therapeutic dose
- Therapeutic Index = TD50 / ED50
- TD50 – dose that produces a toxicity in 50% of the population
- ED50 – minimum effective dose for 50% of the population
- The higher the index the more effective and less toxic the antibiotic

Bacterial Resistance
- Intrinsic resistance – natural property of a micro-organism
o Impermeability barrier (change in permeability)
o Efflux
o Enzymatic inactivation
- Acquired resistance – can be transferred between bacteria
o Decreased uptake/permeability
o Decreased accumulation (efflux)
o Enzymatic inactivation
o Duplication & overproduction of targets
o Modification of target site
o Bypass of a metabolic pathway/enzyme

Yeasts
Mycology
- The study of fungi

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 - Microscopic – unicellular (yeast), multicellular (moulds)
- Macroscopic – toadstools, mushrooms
- General properties
o Eukaryotic
o Spore-bearing
o Absorptive nutrition (e.g. from ground)
o No chlorophyll
o Reproduce sexually and asexually
o Cell wall made from chitin (polysaccharide)
- Saprophytes – live on dead plant and animal material
- Parasites – live on/in living material
- Fungal metabolism
o Aerobic (e.g. ATP from glycolysis)
o Facultative anaerobes – fermentation (make ethanol from
glucose
o Obligate anaerobes (rumen of cattle)
- 90,000 fungal species – only around 50 cause human disease
- Fungal infections are names mycoses

Yeast Structure

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 - Chitin – natural polymer of N-acetyl glucosamine, makes up 80% of
cell wall

- Ergosterol – a unique sterol in fungal cell membrane

- Makes the plasma membrane sensitive to antimicrobial agents
which either block synthesis of ergosterol (e.g. azoles) or bind
specifically to ergosterol (e.g. polyene antifungals)
- Golgi and ER are also part of yeast but aren’t on the diagram

Yeast Reproduction

- Number of times they can divide is limited due to scarring caused

by budding

Patients at Risk
- Immunocompromised patients (cryptococcosis)

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 - Chemo and radiotherapy damages mucosal membranes
(aspergillosis)
- Indwelling catheters (aspergillosis, candidiasis)
- Log-term corticosteroid treatment (aspergillosis)
- Surgery (candidiasis)

Fungal Infections

Contagious Cutaneous Dermatomycoses

- Fungal infections of the keratinous tissues
- Skin, hair, nails (ringworm, athletes’ foot)
- Tinea pedis (foot), Tinea capitis, Tinea corporis (body)
- Trychophyton, Microsporum, Epidermophyton

Non-Contagious Subcutaneous Dermatomycoses

- Sporotrichosis – ulcerating skin lesions (e.g. splinter)
- Chromomycosis – chronic, localised infections
- Eumycetomas – chronic, effects skin

Non-Contagious Systemic Infections

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 - Can be lethal, can spread to other organs
- Aspergillosis – external ear, lungs, eye, brain
- Blastomycosis – lungs, skin, bone, testes
- Cryptococcosis – lungs, meninges
- Histoplasmosis – lungs, spleen, liver, adrenals, lymph nodes
- Sporotrichosis – skin, joints, lungs

Candidiasis
- Candida albicans
- Opportunistic pathogen
- Yeast
- Sabouraud glucose agar, 25˚C
- Most common cause of opportunistic mycoses worldwide
- Causes
o Physiological (pregnancy, age)
o Trauma (infection, burn)
o Haematological (cellular immunodeficiency)
o Endocrinological (diabetes mellitus)
o Iatrogenic (chemotherapeutics, antibiotics, corticosteroids,
catheters, surgery)
o Other (IV drug addiction, malnutrition)
- Strict aerobe – grow with good supply of oxygen
- Can be part of normal microflora (skin, mucosal surfaces) but
doesn’t cause infection due to other good bacteria
- Dimorphic (can change from yeast form to mycelial depending on
environmental factors such as nutrients, CO2, temp, etc.)
- There is other Candida that cause infections – identification is
important so to use correct treatment

- Oral thrush
o White deposit on tongue, oral cavity
o Most common: acute pseudomembranous candidiasis
o Asymptomatic lesions
o Diagnosis:

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  Identify clinical signs and symptoms
 Presence of candida on direct examination
 Biopsy showing hyphae in epithelium
 Positive culture
 Serological tests
- Vaginal thrush
o 75% of woman have at some point in life
o Most common in pregnancy or after antibiotic chemotherapy
o 30% of women in childbearing years get it recurrently
o 1% are almost continuously troubled by it

Moulds
Mould Structure
- Long branched thread-like
filaments of cells (hyphae)
- Hyphae from mycelium
- Hyphae can be continuous or
crossed walled septa
- Spore structure
- Distinct spore structure
(identification)
- One single spore produces
one new colony
(enumeration)

Reproduction – Sexual and Asexual

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Cutaneous Infection
- Onychomycosis
- Common in the general adult population (5-25% depending on age)
- Many people with discoloured or distorted toenails have other
problems, but are treated empirically with antifungal therapy

Systemic Infection – Aspergillosis

- Aspergilloma is a fungus ball that colonises in a healed lung scar or
abscess from a previous disease
- Voriconazole is the preferred treatment for systemic aspergillosis

Pulmonary Aspergillosis

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 - Aspergillus fumigatus
- Opportunistic infection
- Entry is usually respiratory tract
- May cause immediate allergic response (mycotoxin)
- Invasive Aspergillus spp. can spread from the lung to many organs
- Most commonly occurs in severely immunocompromised patients
with neutropenia, usually leukemia patients or those receiving
chemo or corticosteroids

Antifungals
- Fungi are eukaryotes therefore treatment is more difficult
- They have similarities to human, so drugs tend to be more toxic,
need repeated applications, lengthy treatments
- Antifungal drug targets
o Proteins (enzymes) – involved in fungal membrane synthesis
(ergosterol), enzyme chitin synthase (cell wall), beta-(1,3) D-
glucan synthase (cell wall)
o Membranes – drugs that interact with membrane sterols

- Itraconazole
o Active against moulds

31
 o Inhibits cytochrome P450 14α-demethylase (P45014DM)
(enzyme required in sterol biosynthesis pathway – lanosterol
 ergosterol)
o Caution with patients with high risk of heart failure
o Vulvovaginal candidiasis – by mouth, adult = 200mg bd for 1
day
o Systemic candidiasis where other antifungals are
inappropriate or ineffective – by mouth, adult = 100-200mg
od
- Amphotericin B (polyene antifungals)
o Treat systemic candidiasis
o Binds primarily to fungal cell membrane ergosterol
o Binding disrupts osmotic integrity of the fungal membrane –
leakage of intracellular potassium, magnesium, sugars, and
metabolites
o Natural antifungal – Streptococcus nodosus
o Very broad spectrum of activity (yeasts and moulds)
o Toxicity a problem – cardiovascular, renal, hepatic
o Liposomal preparation – Fungizone (IV, 1mg/20min;
250mg/kg daily), AmBisome (IV, 1mg/10min; 1mg/kg daily)

Viruses
- Diverse in size and structure
- Not cells (acellular)
- Carriers of genetic material (DNA or RNA)
- Can be divided into enveloped and non-enveloped
- Requires a host for replication – obligatory intracellular parasites
- Cause diseases (human, animals, plants, insects, bacteria)
- Usually species specific
- Difficult to treat

Virus Transmission

32
 - Airborne – chickenpox (varicella), influenza, measles (rubeola),
mumps, rubella (German measles), smallpox, Coronavirus SARS-
CoV-2
- Direct contact – AIDS (HIV), cold sores (herpes simplex virus type 1),
Human papillomavirus (cervical cancer), genital herpes (herpes
simplex virus type 2), leukaemia (retroviruses), hepatitis B + C
- Food/water – viral gastroenteritis, norovirus, hepatitis A,
poliomyelitis
- Arthropod – yellow fever

Virus Structure
- Viral envelope
o Surrounds the viral capsid in some but not all viruses e.g. HIV,
herpes, influenza
o Derived from portions of the host cell membranes (e.g.
plasma membrane, cell nuclear envelope, organelles such as
the golgi and ER)
o Susceptible to desiccation (low survival in the environment),
physical (heat) and chemical (disinfectant) challenges
o Enveloped viruses can survive longer when mixed with
fomites (blood, mucus, etc.)

- Viral capsid/nucleocapsid
o The capsid denotes the protein shell that encloses the nucleic
acid. It is built of structure units
o Structure units are the smallest functional equivalent building
units of the capsid
o Individual proteins fit together like the pieces of a puzzle to
form a building block called a capsomere
o Inside capsid nucleic acid genome lodges for protection
o Capsid may take the form of a polyhedron (usually
icosahedral), or it may be spiral (helical symmetry), or it may
be more complex

33
 - Viral receptors
o Glycoproteins (virus encoded) on the surface of the envelope
or protruding from the capsid serve to identify and bind to
receptor sites on the host’s membrane

- Viral genome
o Either RNA or DNA but can be single or double stranded
o Circular, linear
o Dictate viral genome expression – translation
o Virus classification

Virus Replication
1. Attachment to cell by receptors
2. Virus enters cell (injection of nucleic
acids, membrane fusion,
endocytosis)
3. Viral capsid is uncoated to release
the genome
4. New viral proteins and genomes are
synthesised
5. Progeny virions are assembled (formation of capsid, envelope,
package of genome)
6. Virus released from the cell

Influenza Virus
- Virus binds to host cell via hemagglutinin proteins
in the cell envelope of the virus
- The virus is endocytosed by the cell
- It fuses with a lysosome
- Viral capsid is uncoated to release genome
- Genome moves in the nucleus
- Replication of the DNA
- Translation of the RNA into proteins
- Budding and release of virus

34
Reassortment
- Antigenic shift – process by which 2 or more
different strains or virus combine to form a
new subtype having a mixture of the surface
antigen of the original strains
- Antigenic drift – small changes (mutations) in
the virus that happen continually over time,
produces new virus strains that may not be
recognised by the body’s immune system,
difficult to vaccinate to long term protection

Herpes Simplex Virus

- Replication same us covered previously
- HSVs may persist in a quiescent (dormant) but persistent form
known as latent infection, notable in neural ganglia
- Infection of epithelial cells in the mucosal surface

35
 o Productive replication, production of progeny virions and
virions spread to infect additional epithelial cells
o Virus enters innervating sensory neurons, and nucleocapsids
are transported to the neuronal cell body
o Viral DNA is released into the neuronal nucleus and
circularises
o Circular viral DNA persists in the neuronal cell nucleus
- Reactivation in the neuronal cell body
o Initiation of viral lytic gene expression
o Newly formed capsids are transported to the axonal termini
o Infectious virus is released from the axon and infects
epithelial cells, resulting in recurrent infection and virus
shedding

Coronaviruses
- Cause significant and economically important illnesses in many
animal species including dogs, cats, cattle, pigs, and chickens
- Cause up to 30% of common colds
- Can undergo rapid genetic change with alterations in clinical
disease and “trans-species” movement to new animal hosts

- Contain single-stranded, positive-sense RNA genome

o Replication of full-length genomic RNA
o Synthesis of mRNAs that are used for translation of viral
proteins
- Use a viral RNA-dependent RNA-polymerase
o High intrinsic error rate with the potential to introduce
multiple nucleotide changes during each genome replication
o “Jump” across the RNA template or between two different
templates, resulting in RNA-RNA recombination
- Viral RNA exonuclease mediates RNA proofreading and regulate
replication fidelity
- Combination of RNA recombination, high polymerase error, and
regulated replication fidelity appears to favour the generation of

36
 recombinant and highly diverse populations of viruses with the
potential to cross species and subsequently adapt to new hosts

SARS – MERS
- SARS-CoV-1
o Spring and summer of 2003: new human coronavirus was the
cause of severe acute respiratory syndrome (SARS)
o Emerged in Guangdong province of south-eastern China
o A zoonotic virus that likely derived from bats
o Transmission was primarily person to person
o 9% of patients with confirmed infection have died
- MERS-CoV
o In 2012: new Middle East Respiratory Syndrome (MERS)
emerged from Saudi Arabia
o Approx. 35% of reported patients with the infection have died
o Dromedary camels may be a major reservoir host for MERS
o Did not seem to pass easily from person to person unless
there is close contact
- SARS-CoV-2
o In 2020: emergence of new human coronavirus causing SARS
o Emerged from Wuhan, China
o A zoonotic virus likely derived from bats
o Transmission primary from close person to person contact
o Pre-symptomatic shredding may be typical among
documented infections – subclinical infections may have been
source of a majority of infections

Factors Affecting Infection Rates

- Healthcare associated infections: environmental disinfection,
identification of infected or colonised patients, hand hygiene,
contact precautions, antimicrobial stewardship
- Community: environmental disinfection, hand hygiene, contact
precautions, identification of infected individuals

37
HIV and AIDS
- Human Immunodeficiency Virus
o Retrovirus
o Primarily infects CD4+ T cells, macrophages, dendritic cells
o Destroys CD4+ T cells
o Severely weakens human immune system
o Associated with body fluids including semen, vaginal and anal
fluids, blood, and breast milk
o Transmission: unprotected sex, sharing needles/syringes or
other injecting equipment, from mother to baby during
pregnancy, birth, or breast feeding
o HIV does not survive outside the body for long
- Acquired Immune Deficiency Syndrome
o Describes life-threatening infections and illnesses that happen
when your immune system has been severely damaged by
the HIV virus
o Defined as an HIV infection with either a CD4+ T cell count
below 200 cells per µL or the occurrence of specific diseases
associated with HIV infection
o If the infection progresses, risk of developing common
infections and tumours increases

HIV Infection
- Primary HIV infection: may be either asymptomatic or associated
with acute retroviral syndrome
- Stage I: Asymptomatic with a CD4+ T cell count (CD4 count) greater
than 500 per µL of blood
- Stage II: Mild symptoms which may include minor mucocutaneous
manifestations and recurrent upper respiratory tract infections. A
CD4 count of less than 500/µL

38
 - Stage III: Advanced symptoms which may include unexplained
chronic diarrhea for longer than a month, severe bacterial
infections including TB of the lung, and a CD4 count of less than
350/µL
- Stage IV or AIDS: Severe symptoms which include toxoplasmosis of
the brain, candidiasis of the esophagus, trachea, bronchi or lungs,
and Kaposi’s sarcoma. A CD4 count of less than 200/µL

HIV Structure

HIV Replication

39
Antiviral Chemotherapy
- Targets
o Virus specific enzymes
o Life cycle processes
o To design a drug, one needs
to understand virus life cycle
- HIV
o Aim of HIV antiviral therapy:
to reduce viral levels (viral
load < 50 copies/ml) and
reduce AIDS-related illnesses
o Highly active antiretroviral therapy (HAART)
o Combination of antivirals – reduce risks of emerging resistant
virus

Nucleotide Reverse Transcriptase Inhibitors (NRTIs)

- Reverse transcriptase is the number one target for drug therapy
- Well-known NRTIs for HIV treatment include zidovudine (AZT) and
tenofovir but also abacavir, didanosine (DDL), emtricitabine (FTC),
lamivudine (3TC), stavudine (d4T)
- AZT: Zidovudine (Azidothymidine): by mouth 500-600mg daily
divided in 2-3 doses
- DNA is synthesised from 4 deoxynucleotide triphosphates
- AZT is a nucleoside analogue – it resembles the nucleotides that are
the building blocks of DNA
- When AZT gets used to build DNA, it prevents any more DNA from
being made by blocking further extension of the growing DNA chain

40
 - When reverse transcriptase is creating new DNA it “accidentally”
inserts a molecule of zidovudine rather than a nucleoside
- This terminate the DNA chain and no more can be appended to it

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

- Also targets the HIV reverse transcriptase
- Attach to the reverse transcriptase and affect the activity of the
enzyme by restricting it mobility and making it unable to function
- Antivirals include
efavirenz, etravirine,
neviparine, rilprivirine
- Efavirenz (Sustiva)
600mg OD

Protease Inhibitors
- Viral proteinase (protease): an enzyme that
cleave peptide/polypeptide/protein chain
- HIV protease inhibitors bind to enzyme
active site
- HIV virus unable to process proteins
- Atazanavir (Reyataz): 300mg daily
- Other example: Darunavir, fosamprenavir, lopinavir, ritonavir

Integrase Inhibitors

41
 - Viral integrase: an enzyme that allows the transfer of HIV cDNA to
cellular DNA
- Dolutegravir (Trivicay): 50mg OD
- Raltegravir (Isentress): 400mg BDS

Entry Inhibitor: Fusion Inhibitor

- Fusion inhibitor: an enzyme that prevents HIV envelope fusion
- Enfuvirtide (Fuzeon): 90mg twice daily (subcutaneous injection)

Entry Inhibitor: Receptor Antagonist

- Receptor antagonist: a negative allosteric modulator of the CCR5
receptor
- C-C chemokine receptor type 5 (CCR5 or CD195) is a protein on the
surface of white blood cells that is involved in the immune system
as it acts as a receptor for chemokine (chemotactic cytokines)
- Maraviroc (Celsentri): 300mg twice daily

Herpes Simplex Virus: Inhibitor of viral DNA polymerase

- Viral DNA polymerase: enzyme that creates DNA molecules by
assembling nucleotides – essential for viral DNA synthesis
- Acyclovir is a guanosine analogue prodrug
- Acyclovir is converted by viral thymidine kinase to acyclovir
monophosphate which is the converted by host cell kinases to
acyclovir triphosphate (ACV-TP)
- ACV-TP competitively inhibits and inactivates HSV specific DNA
polymerases

42
 - Acyclovir (Zovirax): 200mg 5 times a day for 5 days

Influenza Virus: Neuraminidase Inhibitors

- Neuraminidase inhibitor: prevent the release of the virions
- Oseltamivir is a competitive inhibitor of the activity of the viral
neuraminidase
- It is a prodrug as it needs to by hydrolysed in the liver to be active
(oseltamivir carboxylate)
- Oseltamivir (Tamiflu): 75mg OD for 10 days – must be taken within
48 hours of symptoms arising

Protozoa and Malaria

Protozoa
- Eukaryotic micro-organisms
- Generally unicellular
- Heterotrophic – needs organic substances
- Often commensal – live in or on other organisms
- Sometimes parasitic – life stage
- Often live aqueous environments – often motile

Examples of Disease
- Giardia intestinalis – Giardisais
- Entamoeba histolytica – Amoebiasis
- Plasmodium vivax – Malaria
- Leishmania donovani – Leishmaniasis
- Cryptosporidium enteritis – Cryptosporidiosis

43
Malaria Hotspots

Malaria – Plasmodium
- Plasmodium-ameboid intracellular parasites
o P. vivax, P. falciparum, P. malariae
- P. falciparum
o The most prevalent malaria parasite in the WHO African
Region (99.7% of cases in 2018), as well as in the WHO South-
East Asia Region (50%), the WHO Eastern Mediterranean
Region (71%) and the WHO Western Pacific Region (65%)
- P. vivax
o Prevalent in the WHO South-East Asia Region (53%), with the
majority being in India (47%).
o Predominant in the WHO Region of the Americas (75% of
cases)

44
Life Cycle of Malaria

Malaria Prevention
- Mosquito control
o Insecticides (indoor residual spraying)
o Insecticide treated-mosquitoes net
o Breading control
- Prophylaxis – choice based on many factors
o Risk of exposure
o Extent of drug resistance
o Efficacy of recommended drugs
o Side effects of the drugs
o Patient-related factors (age, pregnancy, renal/hepatic,
impairment)

45
 o Prophylaxis a few days to 3 weeks before travel to endemic
areas
o Prophylaxis continues for up to 4 weeks after leaving endemic
area
- Rapid identification
o Rapid diagnosis tests (64% sold for P. falciparum detection)
- Chloroquine
o Prophylaxis not where falciparum malaria endemic (risk of
resistance)
- Mefloquine
o Prophylaxis where falciparum malaria endemic
- Doxycycline (tetracycline)
o Prophylaxis where resistance to mefloquine and chloroquine
- Malarone
o Prophylaxis where falciparum malaria endemic including
resistance to other antimalarial drugs

Malaria Treatments
- Artemisinin-based combination therapy (ACT) delivered by national
malaria programme
- Combination to prevent malaria parasite’s resistance from
developing
- For P. falciparum 2 or more drugs with different modes of action in
combination are now recommended
- Companion drugs include: lumefantrine, mefloquine, amodiaquine,
sulfadoxine/pyrimethamine, piperaquine and
chlorproguanil/dapsone
- Artemisinin derivatives include dihydroartemisinin, artesunate and
artemether

- Co-formulated drugs
- A co-formulated drug is one in which 2 different drugs are
combined in one tablet

46
 - Malarone (proguanil with atovaquone) – proguanil interferes with 2
different pathways involved in biosynthesis of pyrimidines (C, T, U
bases) required for nucleic acid replication and atovaquone inhibits
electron transport chain
- Riamet (artemether with lumefantrine) – interferes with the ability
of the malaria parasites to convert heme into hemozoin. Causes
levels of the toxic heme to rise which kills the blood stages of the
malaria parasites
- Combination treatments depending upon the risk (see BNF)

Cryptosporidiosis
- Cryptosporidium parvum, hominis
- Both aerobic and anaerobic metabolisms
- Opportunistic infections
- Transmission: fecal-oral route (contaminated water – from animals
defecation in reservoir water)
- Symptoms: diarrhoea, abdominal pain, nausea
- Severe cryptosporidiosis in HIV patients
- Diagnosis: stool sample
- Treatment: fluid and electrolyte replacement, no licensed drugs in
the UK

Helminths
Amoebic Dysentery (Amoebiasis)
- Entamoeba histolytica
- 400 million people infected worldwide
- 70,000 deaths per year worldwide
- Infection can be asymptomatic and remain latent
- Transmission: fecal-oral route (contaminated water)
- Symptoms: range from mile diarrhoea to dysentery with blood and
mucus in stool
- Diagnosis: stool sample

47
 - Sever amoebiasis infections occur in 2 major forms:
o Invasion of the intestinal lining causes amoebic dysentery or
amoebic colitis
o Parasite reaching the bloodstream and becoming systemic,
most frequently ending up in the liver which it causes
amoebic liver abscesses
- Treatments: metronidazole, tinidazole, diloxanide fluorate
(asymptomatic patients)

Parasitic Worms
- Flukes
- Tapeworms
- Roundworms
- Threadworms
- Hookworms
- Whipworms
- 1 billion people develop intestinal worm diseases globally each year

Infections
- Schistosomiasis (bilharzia)
- Cysticercosis
- Lymphatic filariasis (elephantiasis)
- Onchocerciasis (river blindness)

Transmission routes
- Schistosome or hookworm larvae directly penetrate the skin from
infected water or soil
- Filarial worms (e.g. onchocerca) transmitted by insect vectors
- Inadequate sanitation is a major risk factor for infection

Symptoms

48
 - Stomach upset (abdominal pain, nausea, vomiting)
- Asymptomatic (a few roundworms)
- Reduced nutritional uptake
- Intestinal obstruction or bleeding
- Rectal prolapse
- Presence of a worm within vomit or stools
- Flu-like symptoms (fatigue, fever, anemia)
- Skin rash
- Breathing difficulties if lungs infected

Tapeworm
- Asymptomatic
- Reduced nutritional uptake
- Intestinal obstruction or bleeding
- Rectal prolapse
- Adult tapeworm is made up of many small segments called
proglottids (contain eggs)
- Can read 10-12m in length
- Transmitted from ingestion of eggs (uncooked meat)
- Diagnosis: presence of worm/eggs in stool

Life Cycle

49
Treatments
- Depends on the worm
- Mebendazole – threadworm, roundworm (ascaris), whipworm,
hookworm – inhibits synthesis of microtubules, glucose uptake
- Piperazine – threadworm, roundworm – agonist effects upon
inhibitory helminths’ GABA receptor which respond to
neurotransmitter, paralysing the parasite
- Niclosamide (named-patient basis – taenicides (tapeworm) –
uncouples oxidative phosphorylation in the tapeworm
- Praziquantel (named-patient basis) – taenicides (tapeworm) –
Increases membrane permeability to calcium ions paralysing the
parasite
- Ivermectin (named-patient basis) – filaricides

Microbiome
Normal Flora

50
Gut Flora
- Actinobacteria
- Firmicutes – important
- Bacteroidetes – important
- Proteobacteria
- Fusobacteria
- Verrucomicrobia

Function of Gut Microbiota

- Metabolism
o Undertake a variety of metabolic functions
o Provide vital biochemical pathways for the metabolism of
non-digestible carbohydrates (cellulose, hemicellulose,
pectins, gums) some oligosaccharides, unabsorbed sugars,
alcohols, and host-derived mucins
o Synthesis of vitamins – enteric bacteria secrete vit K and vit
B12
o Stimulate the development of certain tissues (caecum;
caecum of germ-free animals is enlarged, thin-walled, and
fluid-filled)

- Host protection and immune system development

o Produce antimicrobial compounds – fatty acids and peroxides
to highly specific bacteriocins
o Compete for nutrients and sites of attachment in the gut
lining, preventing colonisation by pathogens (barrier or
competitive-exclusion effect)
o Intestinal epithelium is the main interface between the
immune system and the external environment

51
 o Exposure to intestinal bacteria is also implicated in the
prevention of allergy
o The normal flora stimulates the production of cross-reactive
antibodies
o Host microflora also in oral cavity, skin, vaginal epithelium

- The gut-brain axis

o Bidirectional communication system that integrates neural,
hormonal, and immunological signaling between the gut and
the brain
o Stress influences the composition of the gut microbiota
o Stress influences the integrity of the gut epithelium and alter
peristalsis, secretions, and mucin production

- Mucins
o Glycoproteins that are expressed in cells
o Signal transduction
o Regulation of gene expression
o Cell proliferation
o Embryogenesis
o Cell differentiation
o Immunity
o Apoptosis
o Cancer

Gut and Microbiota in Disease

- Irritable bowel syndrome (Crohn’s disease and ulcerative colitis)
o Gut microbiota alteration linked to low-grade intestinal
inflammation (inflammatory bowel disease)
o Persistence of inflammatory responses in chronic disease:
microbiome plays a significant role in immunomodulation

- Systemic metabolic diseases (obesity and type 2 diabetes)

52
 o Obesity: alteration of firmicutes/Bacteroidetes ration in the
gut microbiota. Obese mice = more firmicutes, lean mice =
more bacteroidetes
o Type 2 diabetes: impacted by the composition of intestinal
microbiota

- Atopic eczema and other allergic disease

o Allergic diseases, specifically those driven by type 1
hypersensitisation
o Infants (1-12 months) with atopic eczema have a significantly
lower bacterial diversity: Bacteroidetes and proteobacteria
o Proteobacteria cell walls contain lipopolysaccharides
o Lipopolysaccharides have ability to elicit a host’s immune
response
o Low exposure to lipopolysaccharides in infancy is linked with
higher risk of atopic eczema

Probiotics
- Microorganisms that are believed to provide health benefits when
consumed
- Mental health, oral health, lung and heart health, digestion, vaginal
health, skin, liver, etc.
- E.g. yogurt
- Main species e.g. Lactobacillus, Bifidobacterium

Long Term Use of Antibiotics and Opportunists

- The inflammation of the colon is pseudomembranous colitis and is
generally caused by antibiotic-associated diarrhoea (AAD)
- C. difficile normally are indigenous bacteria able to proliferate when
most of the normal flora killed by the antibiotic

Sources of Contamination
Water
- Reduction in microbial count from water sample

53
 - Chemical treatment – chlorination, sodium hypochlorite – 0.5-5
ppm of free chlorine for most purposes, 50-100 ppm for storage
and distribution
- Membrane filtration
- UV irradiation – short wavelength (250nm) UVC – UV damages
microbial DNA
- Heating – water storage 80˚C
- Boiling is not an appropriate way to reduce microbial count in water

Operator (You)
- Contamination from skin (e.g. S. aureus) – transient and resident
microorganisms, can be associated with poor hygiene
- Contamination from respiratory tract – coughs and sneezes
- Reducing contamination from operators – gloves, PPE, wash hands,
etc.

Surfaces
- Building – walls and ceilings (moulds, ventilation, disinfectants,
laminated plastic), floors and drains (easily cleaned, drains avoided
in areas where sensitive products are being manufactured),
doors/windows/fittings (prevention of dust, only light entry)
- Packaging – protect product from contamination, maintain product
integrity during shelf-life, deliver product whilst limiting
contamination – rubber can prevent entry of microorganisms in a
multi dose injection vial
- Equipment – e.g. pharmaceutical manufacture, surgical equipment,
critical/semi-critical/non-critical items – sterilisation, disinfection,
single use items

Raw Material

54
 - Products of natural origin – animal (gelatine, growth hormones,
desiccated thyroid), plant (starches, gum acacia)
- Reduction in microbial count from water samples – minimum water
activity (Aw) (spoilage), packaging, liquid and semi-solid raw
materials (sometimes kept at higher temp but extra cost to
company)
- Good manufacturing practice – supplier, quality

Air/Atmosphere
- Filtration – high-efficiency particulate air (HEPA) filters can remove
up to 99.997% of particles greater than 0.3µm in diameter, high
pressure/positive pressure rooms keep atmospheric contamination
out
- Chemical disinfection
- UV irradiation
- Ethylene oxide is used for gas sterilisation
- Heat does not ensure a low microbial count in atmosphere

Exploitation of Microorganisms
Mercury
- Toxicity – absorbed through skin, causes liver and kidney damage
- Naturally occurring – volcanism (up to half atmospheric mercury)
- Large amounts released into environment through industry – fossil
fuel combustion, mining
- Hg0 mercury vapour in atmosphere oxidised to Hg2+
- Enters aquatic environments – absorbs to particulate matter
- Microorganisms – metabolise resulting in methylation –
methylmercury (CH3Hg+) is 100x more toxic
- Several bacteria (P. aeruginosa) can ‘biotransform’ – non-toxic
enzyme mercuric reductase: CH3Hg+ → Hg2+ → Hg0

55
Heavy Metal Contamination
- Used siderophores secreted from some bacteria

Petroleum Degradation
- Hydrocarbons = rich source of
organic material
- Bulk storage – microbial
growth is not desirable
- Oil-oxidising bacteria develop
rapidly on slicks/films
- Eventually decomposes the oil
and disperses the slick
- Bioremediation can be accelerated by addition of nutrients and
oxygen

Xenobiotics – Pesticides
- One of the most widely used chemicals
- Can be attacked by microorganisms – eventually disappear from
soil, prevent toxic accumulations

Xenobiotics – Polychlorinated Biphenyls (PCBs)

- Used as industrial coolants or insulators
- Spills or leakage into environment
- Eventually reaches groundwater
- Toxic, persistent, and bio-accumulative
- Reductive de-halogenation – PCBs can be decomposed by some
microorganisms under anoxic conditions

Xenobiotics – Plastics
- 40 billion kg produced per year
- Half discarded in landfills – remain unaltered for decades

56
 - Biodegradable plastics
o Photo-degradable – structure alters by sunlight (UV) – more
amenable to microbial attack
o Starch-based plastic – starch used to link short fragments of a
second biodegradable polymer

Antibiotics
- Chemicals produced by microorganisms that kill or inhibit growth of
other microorganisms – now includes
substances partly or wholly produced
through chemical synthesis
- Streptomycetes produce >2/3 of clinically
used antibiotics (non-synthetic)

Dextran
- Polysaccharides produced commercially used as a plasma substitute
- Lactic acid bacteria (Leuconostoc) can produce dextrans (from
sucrose)
- Glucose polymer (1,6 α linkages)
- High/variable molecular weight
- Dextran produced depends on the strain used for production

Enzymes
- Usually produced by certain streptococcal strains
- Streptokinase – thrombolysis
o Mammalian blood will clot if allowed to stand
o Streptokinase reduces blood clots
o Administered by IV to treat deep-vein thrombosis
- Streptodornase
o Liquefies pus – breaks down deoxyribonucleoprotein and DNA
o Streptodornase and streptokinase together used topically to
liquefy blood clots and pus in wounds (Varidase)

57
Amino Acids
- Ingredients of infusion solutions for parenteral nutritional food
supplements
- Produced commercially in fermentation processes e.g. glutamic acid
– Brevibacterium flavum, Corynebacterium glutamicum

Vitamins
- Riboflavin (Vit B2)
o Deficiency characterised by enflamed tongue, dermatitis,
‘burning feet’
o Produced commercially in high yields by the mould Ashbya
gossipii and the bacterium Bacillus subtilis amongst others
- Vit B12
o Synthesised in nature exclusively by microorganisms
o Dietary needs satisfied by food intake or absorption of the
vitamin produced in the gut by microbes
o Deficiency – low red blood cell production, nervous system
disorders, dementia, depression
o High yielding strains – Propionibacterium, Pseudomonas

Toxins
- Botulinum toxin
o Clostridium botulinum
o Normally inhabits soil and water
o May contaminate foods before harvest or slaughter
- Botulism
o Severe food poisoning caused by consumption of food
containing the exotoxin produced by C. botulinum
o Most potent biological toxin known
o Causes paralysis, respiratory failure, and death
- Cosmetic Industry

58
 o Minute amounts – muscle relaxant (BOTOX)
- Medically
o Injected into patients suffering from dystonia
o Muscle spasms cause twisting and repetitive movements or
abnormal postures

Insecticide
- Bacillus thuringiensis
o Crystallin inclusion – parasporal body
o δ – endotoxin
o Active against insect larvae (paralysis and death)
o Bt spores produced as a dusting powder
o GM crops contain cry genes – produce δ endotoxin
- Bacillus sphaericus
o 2 proteins (51 and 42 KDa) toxic to mosquito larvae
o Microbe introduced to waters where larvae develop

Vaccines
- https://appliedmicrobiology.org/resource/emi-lecture-2017-
professor-rino-rappuoli.html

Brewing
- Fermentable sugars and nutrients extracted from malt/sugar/hops
- Placed into a fermentation vessel and brewing yeasts added
- Ferment sugars and produces alcohol and CO2
- Top fermenting yeasts – Saccharomyces cerevisiae – ales
- Bottom fermenting yeasts – Saccharomyces carlsbergensis – lager
- Yeasts broken down – full of B vitamins and proteins

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