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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?

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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