18-Laboratory Evaluation of Wound Infe Ction v1- 3

8/3/2019 18-Laboratory Evaluation of Wound Infe Ction v1- 3

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Laboratory Evaluation of

Wound InfectionDr. John Warren

Department of PathologyNorthwestern University

Feinberg School of MedicineJune 2007


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Wound Microbiology

Pathophysiology of wound infection

Microbiology of wound infection

Clinical signs of wound infection Wound specimens

Interpretation of stains and cultures

Quality management indicators


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Pathophysiology of woundinfection

Normal function of skin: preventcolonization and invasion of underlyingtissue by potential microbial pathogens

Loss of skin integrity (wound) providesmoist and nutritious environment formicrobial proliferation

Presence of foreign material andnecrotic tissue facilitates microbialproliferation (dirty wound)


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Acute wounds: external damage to intactskin (surgical wounds, bites, burns,gunshots, minor cuts and abrasions)

Chronic wounds: endogenous mechanismscompromising epidermal and dermal tissue(impaired arterial supply or venous drainage,diabetes mellitus, poor nutrition,

immunosuppression, sustained external skinpressure)


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Microbial colonization precedes woundinfection

If tissue devitalized and/or host immunity

compromised, conditions optimal formicrobial growth and invasion followscolonization

Source of microorganisms: exogenous

(environmental), surrounding skin, andendogenous (mucous membranes ofgastrointestinal tract and genitourinary tract,oropharyngeal cavity)


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Predisposing factors for woundinfection

Poor blood perfusion with hypoxia (pO2< 20 mm Hg) inhibits granulation tissueresponse and wound repair

Cell death and tissue necrosis due tohypoxia creates ideal growthconditions for wound microflora

Hypoxia compromises oxygen radicaldependent killing of bacteria bypolymorphonuclear neutrophils


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Density of microorganisms the criticalfactor determining whether or not awound heals

Presence of specific microbialpathogens of primary significance indelayed wound healing

Most likely both factors important indelayed wound healing due to infection


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Healing of decubitus ulcers occurs only whenbacterial load 104 (complex extremity wounds) or>105 cfu/g of wound tissue

Single microorganism on Grams stain reliablypredicts >105 cfus/g of wound tissue

Presence of bacterial cells on Grams stain of burnwounds consistently correlates with >106 organismsper swab specimen

Critical microbial load for wound infection appearsto be 104-106cfu/g wound tissue or ml wound fluid,and 106cfu/wound swab specimen


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Microbiology of wound infection:the Big Three1

Streptococcus pyogenes(capable ofwound infection


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Microbiology of wound infection:Obligate Anaerobic Bacteria1

Bacteroides Porphyromonas(pigmented)

Prevotella(pigmented and non-pigmented)

Fusobacterium

Peptostreptococcus Clostridium21Primarily associated with polymicrobial aerobic and

anaerobic bacterial wound infection2Monomicrobial infection by Clostridiumperfringesin

myonecrosis (gas gangrene) (distinctive Gramsstain showing large boxcar shaped gram-positiverods with a paucity of inflammatory leukocytes


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Polymicrobial wound infection:mechanisms

Oxygen consumption by aerobicbacteria induces tissue hypoxia andfavorable growth conditions for

anerobic bacteria

Nutrients produced by one organismsupports growth of other fastidious

and potentially pathogenic organisms


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Polymicrobial wound infection:mechanisms

Vitamin K production byStaphylococcus aureussupportsgrowth of vitamin K-dependent

Prevotella melaninogenica

Succinate produced by Klebsiellapneumoniaea critical growth factor for

Prevotella melaninogenica


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Microbiology of wound infection:surgical wounds1,2

Staphylococcus aureus(191 patients, 28.2%)

Pseudomonas aeruginosa(170 patients,25.2%)

Escherichia coli(53 patients, 7.8%)

Staphylococcus epidermidis(48 patients,7.1%)

Enterococcus faecalis(38 patients, 5.6%) Anaerobic bacteria (21 patients)1n=672 surgery patients with wound infections2Giacometti et al., JCM 38:918-922 (2000)


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Microbiology of wound infection:surgical wounds1

Superficial wounds, surgical incisions: streptococci,staphylococci

Deep wounds: GI, female genital tract, andoropharyngeal-streptococci, staphylococci, gram-

negative enterics, enterococci, Bacteroides, otheranaerobes; other-streptococci, staphylococci, gram-negative enterics

Gangrenous 24-48 hr after surgery: group Astreptococci, clostridia

Necrotizing >4 days after surgery: polymicrobial(aerobic and anaerobic)

1Nichols and Florman, CID 33(Suppl 2):S84-93 (2001)


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Microbiology of wound infection:diabetic foot infections1

Cellulitis: -hemolytic streptococci (A, B, C, G), Staphylococcusaureus

Infected ulcer (no antibiotic treatment): same as cellulitis, oftenmonomicrobial

Infected ulcer that is chronic or with previous antibiotic: S. aureus, -hemolytic streptococci, Enterobacteriaceae(usually polymicrobial)

Macerated ulcer due to soaking: Pseudomonas aeruginosa(usuallypolymicrobial) Long-duration non-healing ulcers with prolonged, broad-spectrum

antibiotic treatment: S. aureus(MRSA), coagulase-negativestaphylococci, enterococci (VRE), diphtheroids, Enterobacteriaceae(ESBL resistance), Pseudomonas, nonfermentative gram-negatives,possibly fungi (usually polymicrobial)

Fetid foot with extensive necrosis, gangrene, and malodorous:Mixed aerobic gram-positive cocci, Enterobacteriace, nonfermentativegram-negatives, obligate anaerobes

1Lipsky et al., CID 39:885-910 (2004)


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Microbiology of wound infection:burn wound infections1

Staphylococcus aureus(22.9%)

Pseudomonas aeruginosa(20.9%)

Pseudomonasspecies(7.2%)

Escherichia coli(6.7%) Group D Streptococcus(5.0%)

Enterococcus faecalis(4.2%)1Bacteria constituting >4% of organisms that

were recovered from 1,267 burn woundinfections during 1974-1978 (CDC, Mayhall,CID 37:543-550 (2003)


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Microbiology of wound infection:burn wound infections1

Staphylococcus aureus(23.0%)

Pseudomonas aeruginosa(19.3%)

Enterococcusspecies (11.0%)

Enterobacterspecies (9.6%) Escherichia coli(7.2%)

Coagulase-negative Staphylococcus(4.3%)1Bacteria constituting >4% of organisms that

were recovered from 1,234 burn woundinfections during 1980-1998 (CDC, Mayhall,CID 37:543-550 (2003)


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Microbiology of wound infection:human bite infections1,2

Streptococcus(84%)

Staphylococcus(54%)

Prevotella(36%)

Fusobacterium(34%)

Eikenella corrodens(30%)1Bacteria recovered from >30% of 50 patients

with infected human bite injuries.2Talan et al., CID 37:1481-1489 (2003)


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Microbiology of wound infection:animal bite infections1,2

Same as human bites with the additionof:

Pasteurella multocida

Neisseria weaveri

Staphylococcus intermedius1Goldstein, Mandell, Douglas, and

Bennetts Principles and Practice of ID,pp. 3552-3556 (2005)

2Capitini et al., CID 34:e74-74 (2002).


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Clinical signs of wound infection

Purulent discharge

Painful spreading erythema

Failure to heal


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Wound Specimens

Tissue

Wound fluid (purulent exudate)

Superficial swabs Basic principle of specimen collection:

Only wounds with clinical signs of

infection, are deteriorating, or fail toheal should be sampled for Gramsstain and culture


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Grams stain of wound

specimens

Presence of bacteria by Grams stain

indicates 105 to 106 organisms/g woundtissue or ml wound fluid

Types of organisms present on Grams

stain should be correlated with cultureresults to recognize predominant

organisms that dont grow aerobically


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Culture of wound specimens

Facultative anaerobic and aerobic bacteria ofprimary importance in wound infection

Media for aerobic culture of wound

specimens include sheep blood, chocolate,and MacConkey agar

Media for anaerobic culture of woundspecimens include brucella blood agar, laked

blood with kanamycin and vancomycin,Bacteroidesbile esculin, and anaerobiccolistin-naladixic acid


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Correlate growth of facultative anaerobic and aerobic bacteriawith gram-stain morphotypes

If Staphylococcus aureus, -hemolytic Streptococcus, and/orPseudomonas aeruginosapresent in any numbers, identifywith susceptibility testing

If coagulase-negative Staphylococcus, Corynebacterium,and/or Enterococcuspresent in moderate to many numbers,and growth explains gram-stain results, report as genus andfull identification/susceptibility available by request.

If Enterobacteriaceae, or non-fermenters other thanPseudomonas aeruginosapresent in moderate to manynumbers, and growth explains gram-stain results, identify with

susceptibility testing If >4 facultatively anaerobic or aerobic bacteria detected in

culture by these criteria, obtain a technical consult

Report obligate anaerboic bacteria as polymicrobial flora


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If facultative anaerobic and aerobic bacteriarecovered in culture do not correlate with one ormore gram-stain morphotypes, review and repeat theGrams stain

If aerobic cultures still do not explain Grams stain

upon review and repeat, examine anaerobic cultures If obligate anaerobic bacteria in moderate to many

numbers correlate with gram-stain morphotype(s)not explained by aerobic cultures, identify by genusand report susceptibility available by physician

request If > 4 organisms detected in culture by these criteria,

obtain a technical consult


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Genus identification of anaerobicbacteria in wound culture

Bacterioides: growth in 20% bile

Porphyromonas: vancomycin susceptibile,kanamycin resistant, colistin resistant1

Prevotella: vancomycin resistant, kanamycinresistant, colistin susceptibile1

Fusobacterium: vancomycin resistant,

kanamycin susceptible, colistin susceptibile1+/- pigmentation on laked blood


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Wound specimen negative bydirect Grams stain

Review and repeat Grams stain. Ifconfirmed negative, proceed asoutlined below

Identification and susceptibility testingof Staphylococcus aureus, -hemolyticStreptococcus, and Pseudomonasaeruginosaonly

Other culture isolates reported aspolymicrobial flora


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Pure isolate in aerobic and/oranaerobic would

Full identification with susceptibility for

Staphylococcus aureus, -hemolyticStreptococcus, Pseudomonas aeruginosa,and Clostridium perfringensin any amount

of growth, and full identification with

susceptibility for other organismsdemonstrating growth in the second streakof a sheep blood agar plate


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Quality management

Number (%) of wound Grams stains

explained by aerobic culture results


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Reference

Bowler PG, Duerden BI, and ArmstrongDB. 2001. Wound microbiology and

associated approaches to wound

management. Clinical MicrobiologyReviews 14:244-269.

Documents

18-Laboratory Evaluation of Wound Infe Ction v1- 3