IWII Curriculum
All modules
Usage
This slide set belongs to the International Wound
Infection Institute (IWII) and is meant to accompany a
suggested curriculum outline for persons training in the
clinical management of wound infection management.
The IWII gives permission for it to be used by our
members for educational purposes on condition that the
IWII is acknowledged at all times and that this formatting
is maintained.
The IWII is grateful to Jacqui Fletcher, Caroline Dowsett
and Val Edwards-Jones for the provision of this content
Infection
All modules
Cellulitis
All modules
Wound Bed Preparation Care Cycle
Start with
The patient
No
Prevention
Yes
Healed
Wound Bed
Preparation
Identify wound
aetiology
Care Cycle
Treat & evaluate
TIME
interventions
Dowsett 2004
Modules 1 - 3
Perform TIME
Assessment
Agree goals
What is WBP?
• Wound bed preparation (WBP) is a way of
focusing systematically on all of the critical
components of a non-healing wound to identify
the possible causes of the problem….it focuses
on the components of local wound care:
debridement, bacterial balance and moisture
balance
Sibbald et al, 2000; Dowsett and Ayello, 2004
Modules 1 - 3
Time*‡ - Principles of wound bed preparation
Clinical
Observations
TISSUE
NON-VIABLE
OR DEFICIENT
INFECTION OR
INFLAMMATION
MOISTURE
IMBALANCE
EDGE OF
WOUND – NON
ADVANCING OR
UNDERMINED
Proposed
Pathophysiology
WBP Clinical
Actions
Effect of WBP
Actions
Defective matrix and cell
debris impair healing
Debridement (episodic
or continuous)
• Autolytic, sharp
surgical, enzymatic,
mechanical or
biological
• biological agents
Restoration of wound
base and functional
extra-cellular matrix
proteins
Viable wound base
High bacterial counts or
prolonged inflammation
inflammatory cytokines
 protease activity
growth factor activity
• remove infected foci
Topical/systemic
• antimicrobials
• anti-inflammatories
• protease inhibition
Low bacterial counts or
controlled inflammation:
 inflammatory cytokines
 protease activity
 growth factor activity
Bacterial balance
and reduced
inflammation
Desiccation slows
epithelial cell migration
Apply moisture
balancing dressings
Restored epithelial cell
migration, desiccation
avoided
Moisture balance
Excessive fluid causes
maceration of wound
margin
Compression negative
pressure or other
methods of removing
fluid
Oedema, excessive fluid
controlled, maceration
avoided
Non migrating
keratinocytes
Non responsive would
cells and abnormalities
in extracellular matrix or
abnormal protease
activity
Re-assess cause or
consider corrective
therapies
• debridement
• skin grafts
• biological agents
• adjunctive therapies
Migrating keratinocytes
and responsive wound
cells. Restoration of
appropriate protease
profile
*Courtesy of International Advisory Board on Wound Bed Preparation 2003
Adapted from table 6 - ‡Schultz GS, Sibbald RG, Falanga V et al. Wound Rep Reg (2003)11:1-28
Wound Bed Preparation and TIME are clinical concepts supported by Smith & Nephew Medical Ltd
Modules 1 - 3
Clinical
Outcome
Advancing edge of
wound
Comparison of commonly used
antimicrobials
Gram +ve
Gram -ve
Fungi
Endospores
Viruses
Resistance
Chlorhexidine
+++
++
+
0
+
+
Honey
+++
+++
+++
0
+
0
Iodine
+++
+++
+++
+++
++
0
Maggots
+++
++
ND
ND
ND
0
Silver
+++
+++
+
ND
+
+
Modules 1 - 3
Significance of bacteria in wounds
• Bacterial colonisation is of no clinical significance and
should not be confused with wound infection
• A recognised definition of wound infection is if a wound
contains 106 bacteria per gram of tissue
• Chronic wounds may contain 108 without any obvious
signs of infection (host reactions)
Modules 1 - 3
Continuum
Modules 1 - 3
Occasional
& short-lived
state
following
thermal
trauma
Usual
microbe
start point at
wound
initiation
Normal
state:
normal
healing
progress
Delayed
healing,
Wound
deterioration
and / or
extension
Sterility
Contamination
Colonisation
Local
infection
Deterioration,
spreading
cellulitis,
systemic
signs of
infection
Systemic
infection
Abnormal progression for
primary intention wounds
Abnormal progression for
all wounds
Normal progression for
secondary healing wounds
Increasing microbial bioburden; increasing severity
Modules 1 - 3
Uncertainties with swabs
1. Difficulties in removing adherent microbes
2. Uncertainty around the efficiency of recovering
organisms attached to the swabs
3. Change in the local environment during transport may
affect the viability of the organisms
4. Clinical specimens should ideally reach the lab within
4 hours
5. Specialist facilities and expertise are essential for the
characterisation of anaerobes
Modules 1 - 3
The cost of wound care in
a local UK community
(population = 590,000)
Wound care costs
Dressings and other materials
2005
£3.21m
Percent
15.3%
Nurse time
£6.71m
31.9%
Hospital costs
£11.1m
£21.02m
52.8%
100.0%
Total
Drew P, Posnett J, Rusling L. The cost of wound care for a
local population in England. Int Wound J 2007;4:149-155
Modules 1 - 3
Examples of Clinical Disease
Viruses
Bacteria
Fungi
Influenza
Tuberculosis
Ringworm
Malaria
Hepatitis
Cholera
Thrush
Tapeworm
Rabies
Tetanus
Aspergillosis
Schistosomiasis
HIV/AIDS
Gas gangrene
Cryptosporidium
Polio
Gonorrhoea
Giardia
Chicken pox
Diphtheria
UTI
Wound infections
Modules 1 - 3
Parasites
Terminology
• Bacteria are classified in a structured manner and their
names are assigned accordingly
– Eg Families – Enterobacteriacae (coliform) – a group name that
contains a number of different genera!
– Genera – Staphylococcus
– Species – aureus
– Strain (sub-type) – phage type 29/52
– Methicillin resistant Staphylococcus aureus
– Common terms, MRSA, MSSA, VISA, GISA
– EMRSA 15, EMRSA 16
• Epidemic MRSA
Modules 1 - 3
Staphylococci and streptococci
in gram stain of pus
Modules 1 - 3
Gonococcus in urethral pus
Modules 1 - 3
Common wound sampling
procedure using a moist swab
Modules 1 - 3
Identifying Bacteria in the Wound
10 point Method
 Cleanse bed
 Zig Zag and rotate 360o
 Avoid debris and frank pus
Levine Method:
 Cleanse bed
 Rotate over 1cm sq
 Press firmly into tissue
C
Preliminary analyses of 78 study wounds
 Levine’s best of 3 swab techniques in terms of 4 validity parameters: sensitivity .70,
specificity .90, positive predictive value .79, accuracy .81.
www.hsrd.research.va.gov/research/abstracts/NRI_01-005.htm
(Levine, Lindberg, Mason, Pruitt. (2004) Advances in Skin & Wound Care)
Should we just sample the infected
looking areas?
Imagine how many swabs would be generated per wound!
Modules 1 - 3
IWII Curriculum
Module 1 - Fundamental
Goal and Objectives
Aim is to give the student an understanding of microbiology
and epidemiology within the context of wound infection
On completion of the module, students will be able to:
•
Explore how microbes exist and function
•
Differentiate the different types of microbes and their significance in
wounds
• Describe common microbes relevant to wounds (common causative
agents)
• Review the frequency of wound infection in healthcare environment
• Relate this knowledge to your clinical practice
• Describe common terminology related to wound infection
Patient assessment
1.
2.
3.
4.
5.
Module 1
Host risk factors
Levels of bacteria
Local or systemic infection
Impact on the patient
Impact on the wound
When reading the literature a good understanding
of the techniques and their shortcomings is
essential in order to make reasoned comparisons.
Module 1
Host risk factors - systemic
•
•
•
•
•
•
•
Vascular disease
Diabetes
Malnutrition
Immuno suppressed
Patients on corticosteroids
Smoking & alcoholism
Surgery or radiation
Module 1
Host risk factors – local
•
•
•
•
•
•
•
Large wound area
Increased wound depth
Degree of chronicity
Anatomic location: distal extremity, perineal
Foreign body
Necrotic tissue
Reduced perfusion
Using antimicrobials
• Antimicrobials are agents that either kill or inhibit
the growth and division of micro – organisms.
• They include:
– antibiotics - act on specific cellular target sites
– antiseptics, disinfectants and other agents - act on
multiple cellular target sites
Module 1
IWII Curriculum
Modules 1 & 2
Fundamental / Intermediate
What is important?
•
•
•
•
Numbers
Clinical signs and symptoms
Systemic signs
Both
Modules 1 & 2
Recognising infection
Traditional criteria
• Cellulitis
• Redness
• Heat
• Swelling
• Discharge
• Pus
Cutting & Harding 1994
Modules 1 & 2
Additional criteria
• Delayed healing
• Friable tissue/bleeds easily
• Pain
• Pocketing at wound base
• Abnormal smell
• Wound breakdown
Indications for topical antimicrobials
Wounds with
necrotic or poor
blood supply
Wounds
continually recontaminated or
infected
Patients with
specific AB allergy
or AB resistant
infections
Wounds benefiting
from delayed 1o
closure
Systemic antibiotics
may not penetrate
infected ischaemic
tissue at therapeutic
doses; local agents
may be more
successful
High levels of
bacterial
contamination at
wound site delays
healing.
Prolonged AB cover
is undesirable.
Topicals reduce
burden and may
prevent re infection
Particularly where
prolonged systemic
AB therapy has failed
in infected open
wounds
May initially be left
open and treated with
topical antimicrobials.
Usual free form
infection after few
days & can be
cleaned and closed
with prophylactic AB
Melling, Gould and Gottrup 2006
Modules 1 & 2
Choosing an antimicrobial agent
Factors to consider
Agent
• Specificity
• Efficacy
• Cytotoxicity
• Allergenicity
Modules 1 & 2
Dressings
• Absorbency
• Conformability
• Odour management
• Pain management
• Wound size
• Wound location
• Patient preference
• Sustained antimicrobial activity
• Provides a moist wound healing
environment
• Allows consistent delivery over the
entire surface of the wound
• Allows monitoring of the wound with
minimum interference
• Comfortable
• Conformable
Starting point…
There is no evidence that bacteria need to be
removed from wounds in order to make them
heal…
Modules 1 & 2
Definitions
• Antibiotic
– An agent that kills selectively and requires metabolic
activity for it’s action. Antibiotics can be bacteriostatic or
bactericidal
• Antiseptic
– A non selective agent that does not require metabolic
action for efficacy. Always bactericidal and usually surface
acting.
• Antimicrobial
– An umbrella term
Modules 1 & 2
Common bacteria in wounds
Aerobic
Anaerobic
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Staph. Aureus
Staph. epidermidis
MRSA
Enterococcus faecalis
Streptococcus pyogenes
Pseudomonas aeruginosa
Enterobacter
Escheria coli
Klebsiella spp
Proteus spp
Modules 1 & 2
Bacteriodes spp
Prevotella spp
Peptostreptococcus spp
Clostridia
Major wound pathogens
•
•
•
•
•
•
•
•
Staphylococcus aureus
Pseudomonas aeruginosa and other Gram-negative bacilli
Streptococcus pyogenes
Other streptococci
Enterococci
Candida sp and Aspergillus sp
Anaerobes (dependant upon site)
Viruses (Herpes and CMV)
Modules 1 & 2
Diagnostic signs of infection
• Clinical signs
• Pain, redness, swelling, increased exudate, change in
exudate colour, bad wound odour, increased wound
temperature
• Properties of the microbe
• What infects a wound?
– mixed skin flora from the patient
– endogenous pathogens
– exogenous pathogens
Modules 1 & 2
Typical ‘normal’ skin flora
• Highly diverse – hundreds of bacterial species amongst
6 individuals
• Only a few bacteria are common among the individuals
These included
–
–
–
–
Propionibacteria,
Corynebacteria,
Staphylococcus spp,
Streptococcus spp.
Davies CE et al Wound Repair Regen 2001, 9:332-340;
Dekio I et al J Med Microbiol 2005, 54:1231-1238.
Modules 1 & 2
Staphylococcus aureus – 1882
• Staphyle (Greek for bunches of grapes)
Sir Alexander Ogston (Scottish Physician)
Modules 1 & 2
Antimicrobial agents
•
•
•
•
Antibacterial agents
Antifungal agents
Antiviral agents
Antiparasitic agents
Modules 1 & 2
Numbers
• May be expressed as +, ++, +++ or heavy
moderate or light growth.
• Identifies relative proportions of isolates from the
distribution on the primary isolation plates
• Level of detail is usually sufficient to make
clinical decisions if use in conjunction with
clinical signs and symptoms
Modules 1 & 2
Taking a swab
• Gently irrigate the wound ensuring any remnants
of dressings / creams are removed
• Apply the swab in a zig zag motion lightly across
the whole surface of the wound whilst rotating
the swab backwards and forwards or use the
Levine method
• Transport the swab in transport medium
• Store at 40C if processing is likely to take more
than 24 hours
Lawrence 1999
Modules 1 & 2
The Levine Technique
Step 1: Debride the wound
Step 2: Irrigate the wound
Step 3: Rotate swab over 1 sq cm of the wound
bed with sufficient pressure to encourage fluid
to ‘bubble up’ around the swab
Modules 1 & 2
Skin Characteristics
•
•
•
•
•
•
Limited moisture
Acid pH of normal skin
Surface temperature
Salty sweat
Excreted chemicals; sebum, fatty acids and urea
Normal flora
Modules 1 & 2
Skin
• Alterations to the normal flora upsets the
ecological balance and predisposes to infection
• Some microorganisms can overcome the natural
barrier of the skin to cause disease
– arid areas colonised predominantly by Gram positives
– moister areas colonised by numerous species and
also Gram negatives
Modules 1 & 2
Breaches of the skin
• Acute wounds
– minor trauma
• accidental lacerations / superficial
• minor burns
– major trauma
• major burns
• surgery
• Chronic Wounds
• pressure sores
• leg ulcers
• non healing wounds
Modules 1 & 2
Infectious process
Contamination
Colonisation
What tips the
balance?
Local infection
Spreading Infection
Modules 1 & 2
This is a
dynamic
process
and
depends
upon a
number of
important
factors
Immunological
status of the
host
Nutritional
status of the
host
Bacterial
population
Infection
vs
colonisation
Depth and
site of
infection
Modules 1 & 2
Environment dressings
Virulence
factors
IWII Curriculum
Module 2 - Intermediate
Biofilms
Module 2
IWII Curriculum
Modules 2 & 3
Intermediate / Advanced
Minimum inhibitory concentration
(MIC)
• Minimum concentration of silver required to inhibit
bacterial growth / division
• Does not equal death of bacteria
• MIC and zone of inhibition (ZOI) may contribute
to development of resistance
• Use of solutions such as sodium thiosulphate will
also affect the test (use for extracting metal ore,
gives a very stable (i.e. not bioavailable) silver
thiosulphate)
Modules 2 & 3
Minimum bacterial concentration
• Used to assess killing activity
• Data in the literature supports the use of silver
concentrations above 30mg Ag+ / l
• A silver concentration identified as effective in
in vitro killing assays, may represent a threshold
concentration for in vivo activity
Spaccioli et al 2001
Yin et al 1999
Modules 2 & 3
Log reduction
• Measured by: the number of bacteria present at
the start, minus the number of bacteria present
at the end
– e.g.109 – 107 = Log reduction of 2
– 109 – 102 = Log reduction of 7
• Therefore the log reduction of 7 shows a greater
reduction in bacterial count
Modules 2 & 3
Kill time
• Translocation of bacteria occurs if the kill time is
slow
• Gives deep tissue infection
• Gradient of kill, maximum lethal concentration
occurs centrally to the silver ion
• Practically better to overlap the dressing onto the
good skin and gradation occurs here (may get
skin staining)
Modules 2 & 3
Systemic signs
• Raised white cell count
• Raised serum C- reactive protein
Modules 2 & 3
Numbers
• Usually expressed as CFU’s per gram or cm2 of
tissue
• 105 widely accepted as the threshold which
indicates infection if exceeded
• Bowler (2003) suggests treatment should be
based on a host manageable burden
Modules 2 & 3
Clinical signs and symptoms
•
•
•
•
•
•
Several definitions
CDC
NPS
Sepsis
ASEPSIS
WIS
Modules 2 & 3
To diagnose infection laboratory results must be
evaluated in conjunction with local findings such as
erythema, oedema, pain, purulence and
lymphadenitis or systemic factors such as fever,
leukocytosis or glucose intolerance in patients with
diabetes.
Organisms in wound fluid are not necessarily
invading and may only be indicative, not diagnostic
of infection.
McGukin et al (2003)
Modules 2 & 3
Debridement
•
•
•
•
•
•
•
Mechanical
Enzymatic
Autolytic
Larval
Sharp
Hydrosurgery
Surgical
Modules 2 & 3
Acute vs Chonic Wounds
Acute wounds
Chronic wounds
Healing Process
Regulated
Haphazard
Pathology
None
Underlying
Time to healing
Rapid
Slow
Inflammatory response
Short
Prolonged
Exudate
Reduced after 48 hours
Promotes cellular
proliferation
Prolonged Inhibits cellular
proliferation
Bacterial Load
Low
High
Fibroblast proliferation
Active
Inactive
Excoriation/maceration
Infrequent
Frequent
Extracellular matrix
Normal remodelling
Defective remodelling
Vascular network
Good
Poor
Complications
Infrequent
Frequent
Progress
Heal
Fail to heal / recur
Modules 2 & 3
Acute wounds
Chronic wounds
Time bound
Of short duration
Do not heal within 4 weeks (Cullum et
al 1997)
Recurrence
Unlikely to recur
Characterised by episodes of
recurrence
Pathophysiology
No underlying disease process Multiple underlying pathologies
Healing process
Orderly healing
Disordered healing, wound may
improve and then deteriorate in a
cyclical fashion
Inflammatory
response
Progresses through
inflammation to proliferative
phase
Appears to be ‘stuck ‘ in late
inflammatory phase (Hart 2002)
Exudate
Thought to be beneficial,
exerts antimicrobial effect and
contains growth factors
May be detrimental due to imbalance
in production of MMPs and
suppression of TIMPS
Quantity of
exudate
Reduces as healing
progresses
May remain high due to persistent
inflammation
Aetiology
Surgical wounds, traumatic
wounds, burns
Pressure ulcers, leg ulcers, diabetic
foot ulcers, fungating wounds
Modules 2 & 3
Determinants of wound costs
• Dressings and other materials typically
represent 10%-15% of total wound care costs
• Nursing time typically represents 25%-30%
• Hospital inpatient costs represent the largest
single component of cost at 50%+
The key to reducing cost is to prevent hospital
admission and/or delayed discharge by
preventing wound complications
Modules 2 & 3
Wound healing stages
I. Inflammatory phase
II. Proliferative phase
III. Remodelling phase
Modules 2 & 3
Factors that delay healing
•
•
•
•
•
•
•
Age
Social factors
Poor nutrition
Reduced oxygen supply to the wound
Glycated growth factors
Metalloproteases
INFECTION
Diagnosis of Infectious Disease
Non-cultural methods
Cultural methods
Media
Atmosphere
Temperature
Most bacteria
& fungi
Modules 2 & 3
Microscopy
Antigen
Detection
Toxin
Detection
Molecular
Methods
Serology
TB
UTI
Parasites
Hepatitis
Rotavirus
RSV
Cl difficile
Chlamydia
Hepatitis
Diptheria Meningococcus
Most
TSS
MRSA
viruses
Microscopy
Light microscopy
• Uses normal light and stained or unstained preparations.
– Detects parasites, fungi and bacteria.
Fluorescent microscopy
• Uses special stains that fluoresce under UV light.
– Detects fungi and bacteria
Dark ground microscopy
• Uses indirect lighting to visualise difficult organisms.
– Detects special bacteria such as spirochaetes
Electron microscopy
• Uses an electron beam to visualise the smallest particles.
– Detects viruses and large molecules
Modules 2 & 3
Antimicrobial therapy
History
•
•
•
•
•
•
•
Plant extracts
Arsenic compounds
Paul Erhlich 1854-1915, selective affinity for dyes
Domagk, sulphonamides
Fleming 1929, Penicillin
Florey and Chain 1939
Waksman, 1944, soil microbiologist, streptomycin
Modules 2 & 3
Bacterial cell- target sites
Modules 2 & 3
Sterile culture media in petri dishes
Modules 2 & 3
Conventional techniques
+++
++
+
Modules 2 & 3
Typical bacterial growth from
clinical specimen
Modules 2 & 3
Beta-haemolysis on blood agar
Modules 2 & 3
Bacterial identification
Modules 2 & 3
Staph aureus sensitivity by
Stokes’ method
Modules 2 & 3
Modules 2 & 3
Microorganisms reported causing
SSI's for all categories of procedure
53%
3
2 1
4
4
26
5
21
14
3
8
Modules 2 & 3
9
MRSA
MSSA
CNS
Enterococci
Streptococci
Enterobacteriacae
P. aeruginosa
Pseudomonads
Anaerobic bacilli
Anaerobic cocci
Fungi/Candida
Other bacteria
Infection
• When is a wound infected?
– Clinical signs
– Pain, redness, swelling, increased exudate, change in exudate
colour, bad wound odour, increased wound temperature
• Properties of the microbe
• Wounds sterile immediately after trauma but inevitably become
colonised
• What infects a wound?
– Mixed skin flora from the patient
– Endogenous pathogens (from other body sites)
– Exogenous pathogens (from other patients, staff)
Modules 2 & 3
Classifying Infection
• Superficial or localized infection:
– Non-healing, Bright red granulation tissue, Friable and exuberant
granulation, New areas of breakdown or necrosis, Increased
exudate, Bridging of soft tissue and the epithelium, Foul odour
• Spreading infection:
– Deep wound infection: Pain, induration, Erythema (> 2 cm),
Wound breakdown, Increased size or satellite areas, Undermining
or tunnelling, Probing to bone, Flu-like symptoms
• Systemic infection: In addition to deep wound infection,
Fever, Rigours, Chills, Hypotension, Multi-organ failure
Microbial colonisation
• Highly nutritious surface for bacteria to colonise
– Moisture, protein, optimum temperature…
• Incidence of serious infection varies with the size and
depth of the wound
– Non-invasive infection is confined to the superficial layers
• Organism can invade from heavily colonised wounds into
viable tissue
– Destruction of tissue due to extracellular enzymes and toxins
Modules 2 & 3
Stationary phase production of
toxins and invasive enzymes
Log phase
(Exponential Multiplication)
Production of competitive factors
Quorum
sensing
10^6 cfu/ml
Lag phase (Adaptation)
Production of adhesions
CONTAMINATION
COLONISATION
LOCAL INFECTION
SPREADING INFECTION
This is a dynamic process and a number of factors can alter the process
Modules 2 & 3
Chronic wounds –
why do they not heal??
• Static
• Matrix metalloproteases (MMPs)
– endogenous – cellular
– exogenous – bacterial
• Inhibition of growth factors
• Local infection?
– what does this really mean?
Modules 2 & 3
A Biofilm – What is it?
• Complex aggregation of microorganisms marked by the excretion of a
protective and adhesive matrix.
• Often characterized by surface attachment, structural heterogeneity,
genetic diversity, complex community interactions, and an extracellular
matrix of polymeric substances.
• Single-celled organisms generally exhibit 2 distinct modes of behavior:
– planktonic - free floating, form in which single cells float or swim
independently in some liquid medium.
– sessile- an attached state in which cells are closely packed and firmly
attached to each other and usually a solid surface.
• Change in behaviour is triggered by many factors, including quorum
sensing, as well as other mechanisms that vary between species.
Modules 2 & 3
Biofilm
Modules 2 & 3
Interaction with the environment
• Wound dressings
– Increased / decreased TSST-1/protease production
• Buck et al 2004 PhD thesis
• Topical antimicrobial compounds
– Increased / decreased TSST-1 production
– Inhibition of staphylococcal metalloproteases
• Edwards-Jones, V and Foster, H.A 2002. The effects of silver
sulphadiazine on the production of exoproteins by S. aureus J
Med Micro 51(1):50-5.
What affect will superantigens have on the immediate wound environment?
Modules 2 & 3
Should we take punch biopsies to
measure invasiveness?
Quantitative versus qualitative data??
Modules 2 & 3
Virulence factors
• Enzymes
– spreading factors, proteases (8 types including
MMP’s) collagenases, hyaluronidase, elastase
• Toxins
– lecithinase, superantigens , eg TSST-1,
staphylococcal enterotoxins, streptococcal exotoxins,
Pseudomonas exotoxins
• When are these produced??
Modules 2 & 3
Zone of inhibition (ZOI)
• Shows the area across
which spread of bacteria is
inhibited (NB not killed)
• Zones are not proportional
to silver release
• Should not be used as
silver complexes with many
of the components of the
media making the
observations purely
qualitative
Modules 2 & 3
IWII Curriculum
Module 3 - Advanced
Basic bacterial classification
Bacterial Shape
Coccus
Bacillus
(spherical)
(rod shaped)
Gram reaction
Gram reaction
Gram positive
Gram negative
Gram positive
Gram negative
(Staphylococcus
Streptococcus)
(Neisseria:
gococcus)
(Bacillus
Clostridia)
(‘Coliforms’
Pseudomonas)
Coccus (pl. cocci) comes from the Greek and Latin kokkus meaning berry.
Bacillus (pl. bacilli) is from the Latin baculum meaning rod or stick.
Staphylo is from the Latin staphule – a bunch of grapes.
Module 3
Molecular techniques
• Population analysis studies
– PCR- Amplify 16s ribosomal DNA
– separate using Denaturing Gradient Gel
electrophoresis- (DGGE)
– sequence the gene fragments
James et al (2008) - Biofilms in Chronic Wounds Wound Repair and Regeneration 16: 37-44
Davies CE et al (2004) Use of 16S ribosomal DNA PCR and denaturing gradient gel
electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg
ulcers. J Clin Microbiol.;42:3549-57
Module 3
Polymerase chain reaction (PCR)
Increase the number of copies of the genes
Module 3
Denaturing gradient gel electrophoresis
(DGGE)
Module 3
DNA sequencing
Different coloured bases (ATCG)-capillary sequencing
Module 3
Antimicrobial activity
depends upon several factors
•
•
•
•
•
•
Exposure (time)
Concentration
Temperature
pH
Presence of organic matter
Use of permeabilisers
Module 3
Break point sensitivity
Module 3
Module 3
Module 3
Quorum sensing
• Effector molecules are produced by individual
cells and cumulated within the environment
– Homoserine lactones (gram negatives)
– Octapeptides (gram positives)
• At certain concentrations they can switch on (or
off) genes in a coordinated manner
Use as vaccines????
Module 3
Regulatory stimuli
•
•
•
•
•
•
•
•
pH
Temperature
Iron availability
Calcium
Trace metals (including silver, but zinc most common)
Oxygen availability
Carbon dioxide
Accumulation of pppGpp (phosphorylated dinucleotides)
Can we use these to our advantage?
Module 3