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IWII CurriculumIWII CurriculumIWII CurriculumIWII CurriculumAll modulesAll 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
PreventionPrevention
Treat & evaluateTIME
interventions
Treat & evaluateTIME
interventions
HealedHealed
Start withThe patientStart with
The patient
Wound Bed Preparation
Care Cycle
Wound Bed Preparation
Care Cycle
Identify wound aetiology
Identify wound aetiology
Perform TIMEAssessmentAgree goals
Perform TIMEAssessmentAgree goals
Dowsett 2004
YesYes
NoNo
Modules 1 - 3
Wound Bed Preparation Care Cycle
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
Modules 1 - 3
Sibbald et al, 2000; Dowsett and Ayello, 2004
Time*‡ - Principles of wound bed preparationClinical
ObservationsProposed
PathophysiologyWBP Clinical
ActionsEffect of WBP
ActionsClinical
Outcome
TISSUE NON-VIABLE OR DEFICIENT
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
INFECTION OR INFLAMMATION
High bacterial counts or prolonged inflammationinflammatory
cytokines protease activitygrowth 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
MOISTURE IMBALANCE
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
EDGE OF WOUND – NON ADVANCING OR UNDERMINED
Non migrating keratinocytesNon 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
Advancing edge of wound
Modules 1 - 3
*Courtesy of International Advisory Board on Wound Bed Preparation 2003Adapted from table 6 - ‡Schultz GS, Sibbald RG, Falanga V et al. Wound Rep Reg (2003)11:1-28Wound Bed Preparation and TIME are clinical concepts supported by Smith & Nephew Medical Ltd
Comparison of commonly used antimicrobials
Modules 1 - 3
Gram +ve Gram -ve Fungi Endospores Viruses Resistance
Chlorhexidine +++ ++ + 0 + +
Honey +++ +++ +++ 0 + 0
Iodine +++ +++ +++ +++ ++ 0
Maggots +++ ++ ND ND ND 0
Silver +++ +++ + ND + +
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
Occasional & short-lived
state following thermal trauma
Usual microbe
start point at wound
initiation
Usual microbe
start point at wound
initiation
Normal state:
normal healing
progress
Normal state:
normal healing
progress
Delayed healing, Wound
deterioration and / or
extension
Delayed healing, Wound
deterioration and / or
extension
Deterioration, spreading cellulitis, systemic signs of
infection
Deterioration, spreading cellulitis, systemic signs of
infection
Abnormal progression for primary intention wounds
Abnormal progression for primary intention wounds
Normal progression for secondary healing wounds
Normal progression for secondary healing wounds
Abnormal progression for all wounds
Abnormal progression for all wounds
Modules 1 - 3
SterilitySterility ContaminationContamination ColonisationColonisation Local infection
Local infection
Systemic infection
Systemic infection
Increasing microbial bioburden; increasing severityIncreasing microbial bioburden; increasing severity
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 ina local UK community
(population = 590,000)
Wound care costs 2005 Percent
Dressings and other materials
Nurse time
Hospital costs
£3.21m
£6.71m
£11.1m
15.3%
31.9%
52.8%
Total £21.02m 100.0%
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
Modules 1 - 3
Wound infections
VirusesInfluenza Tuberculosis Ringworm Malaria
Hepatitis Cholera Thrush Tapeworm
Rabies Tetanus Aspergillosis Schistosomiasis
HIV/AIDS Gas gangrene Cryptosporidium
Polio Gonorrhoea Giardia
Chicken pox Diphtheria
UTI
Bacteria Fungi Parasites
Examples of Clinical DiseaseExamples of Clinical Disease
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
Modules 1 - 3
Gonococcus in urethral pus
Modules 1 - 3
Common wound sampling procedure using a moist swab
Identifying Bacteria in the Wound10 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
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.Levine, Lindberg, Mason, Pruitt. (2004) Advances in Skin & Wound Care)Advances in Skin & Wound Care)
CC
Imagine how many swabs would be generated per wound!
Modules 1 - 3
Should we just sample the infected looking areas?
IWII CurriculumIWII CurriculumIWII CurriculumIWII Curriculum
Module 1 - FundamentalModule 1 - FundamentalModule 1 - FundamentalModule 1 - Fundamental
Goal and ObjectivesAim 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. Host risk factors
2. Levels of bacteria
3. Local or systemic infection
4. Impact on the patient
5. Impact on the wound
Module 1
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
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Modules 1 & 2
Fundamental / Intermediate
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
Additional criteria• Delayed healing• Friable tissue/bleeds easily• Pain• Pocketing at wound base• Abnormal smell• Wound breakdown
Modules 1 & 2
Cutting & Harding 1994
Indications for topical antimicrobials
Wounds with necrotic or poor blood supply
Wounds continually re-contaminated 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
Modules 1 & 2
Melling, Gould and Gottrup 2006
Choosing an antimicrobial agent Factors to consider
Agent• Specificity• Efficacy• Cytotoxicity• Allergenicity
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
Modules 1 & 2
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• Staph. Aureus• Staph. epidermidis• MRSA• Enterococcus faecalis• Streptococcus pyogenes• Pseudomonas aeruginosa• Enterobacter• Escheria coli• Klebsiella spp• Proteus spp
Anaerobic• Bacteriodes spp• Prevotella spp• Peptostreptococcus spp• Clostridia
Modules 1 & 2
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
• Staphyle (Greek for bunches of grapes)
Modules 1 & 2
Staphylococcus aureus – 1882
Sir Alexander Ogston (Scottish Physician)
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
Modules 1 & 2
Lawrence 1999
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
Contamination
Colonisation
Local infection
Spreading Infection
What tips the balance?
This is a dynamic process and depends upon a number of important factors
Modules 1 & 2
Infectious process
Infection
vs colonisation
Immunological status of the
host
Depth and site of
infection
Bacterial population
Nutritional status of the
host
Virulence factors
Environment -dressings
Modules 1 & 2
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Module 2 - Intermediate Module 2 - Intermediate Module 2 - Intermediate Module 2 - Intermediate
Module 2
Biofilms
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Modules 2 & 3Modules 2 & 3
Intermediate / AdvancedIntermediate / Advanced
Modules 2 & 3Modules 2 & 3
Intermediate / AdvancedIntermediate / 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
Modules 2 & 3
Spaccioli et al 2001Yin et al 1999
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.
Modules 2 & 3
McGukin et al (2003)
Debridement
• Mechanical• Enzymatic• Autolytic• Larval• Sharp• Hydrosurgery• Surgical
Modules 2 & 3
Acute vs Chonic WoundsAcute wounds Chronic wounds
Healing Process Regulated Haphazard
Pathology None Underlying
Time to healing Rapid Slow
Inflammatory response Short Prolonged
ExudateReduced 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 durationDo not heal within 4 weeks (Cullum et al 1997)
Recurrence Unlikely to recurCharacterised by episodes of recurrence
Pathophysiology No underlying disease process Multiple underlying pathologies
Healing process Orderly healingDisordered 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)
ExudateThought 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
AetiologySurgical 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 stagesI. 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
Modules 2 & 3
Diagnosis of Infectious Disease
Cultural methods Non-cultural methods
MediaAtmosphereTemperature
Microscopy ToxinDetection
MolecularMethods
Serology
Most bacteria& fungi
TBUTI
Parasites
HepatitisRotavirus
RSV
Cl difficileDiptheria
TSS
ChlamydiaMeningococcus
MRSA
Hepatitis Most
viruses
AntigenDetection
Modules 2 & 3
MicroscopyLight 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
Antimicrobial therapyHistory
• 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
Modules 2 & 3
Sterile culture media in petri dishes
+
++
+++
Modules 2 & 3
Conventional techniques
Typical bacterial growth from clinical specimen
Modules 2 & 3
Modules 2 & 3
Beta-haemolysis on blood agar
Bacterial identification
Modules 2 & 3
Modules 2 & 3
Staph aureus sensitivity by Stokes’ method
Modules 2 & 3
Microorganisms reported causing SSI's for all categories of procedure
MRSAMSSACNSEnterococciStreptococciEnterobacteriacaeP. aeruginosaPseudomonadsAnaerobic bacilliAnaerobic cocciFungi/CandidaOther bacteria
53%
Modules 2 & 3
26
14
983
21
5
43
2 1 4
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
Lag phase (Adaptation) Production of adhesions
Log phase
(Exponential Multiplication)
Production of competitive factors
Stationary phase production of toxins and invasive enzymes
Quorum sensing 10^6 cfu/ml
CONTAMINATION COLONISATION SPREADING INFECTIONLOCAL 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
Quantitative versus qualitative data??
Modules 2 & 3
Should we take punch biopsies to measure invasiveness?
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
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Module 3
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.
Bacterial Shape
Coccus Bacillus (spherical) (rod shaped)
Gram reaction Gram reaction
Gram positive Gram negative Gram positive Gram negative(Staphylococcus Streptococcus)
(Neisseria: gococcus)
(BacillusClostridia)
(‘Coliforms’Pseudomonas)
Basic bacterial classification
Molecular techniques
• Population analysis studies– PCR- Amplify 16s ribosomal DNA– separate using Denaturing Gradient Gel
electrophoresis- (DGGE)– sequence the gene fragments
Module 3
James et al (2008) - Biofilms in Chronic Wounds Wound Repair and Regeneration 16: 37-44Davies 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
Denaturing gradient gel electrophoresis (DGGE)
Module 3
Different coloured bases (ATCG)-capillary sequencing
Module 3
DNA sequencing
Antimicrobial activity depends upon several factors
• Exposure (time)• Concentration• Temperature• pH• Presence of organic matter• Use of permeabilisers
Module 3
Module 3
Break point sensitivity
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
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