Laboratory Evaluation of Wound Infection

Dr. John Warren Department of Pathology Northwestern University Feinberg School of Medicine June 2007

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

Pathophysiology of wound infection

Normal function of skin: prevent colonization and invasion of underlying tissue by potential microbial pathogens Loss of skin integrity (wound) provides moist and nutritious environment for microbial proliferation Presence of foreign material and necrotic tissue facilitates microbial proliferation (dirty wound)

Pathophysiology of wound infection

Acute wounds: external damage to intact skin (surgical wounds, bites, burns, gunshots, minor cuts and abrasions) Chronic wounds: endogenous mechanisms compromising epidermal and dermal tissue (impaired arterial supply or venous drainage, diabetes mellitus, poor nutrition, immunosuppression, sustained external skin pressure)

Pathophysiology of wound infection

Microbial colonization precedes wound infection If tissue devitalized and/or host immunity compromised, conditions optimal for microbial growth and invasion follows colonization Source of microorganisms: exogenous (environmental), surrounding skin, and endogenous (mucous membranes of gastrointestinal tract and genitourinary tract, oropharyngeal cavity)

Predisposing factors for wound infection

Poor blood perfusion with hypoxia (pO2 < 20 mm Hg) inhibits granulation tissue response and wound repair Cell death and tissue necrosis due to hypoxia creates ideal growth conditions for wound microflora Hypoxia compromises oxygen radical dependent killing of bacteria by polymorphonuclear neutrophils

Pathophysiology of wound infection

Density of microorganisms the critical factor determining whether or not a wound heals Presence of specific microbial pathogens of primary significance in delayed wound healing Most likely both factors important in delayed wound healing due to infection

Pathophysiology of wound infection

Healing of decubitus ulcers occurs only when bacterial load <106 cfu/ml of wound fluid Acute and chronic wound infection occurs with microbial load >104 (complex extremity wounds) or >105 cfu/g of wound tissue Single microorganism on Grams stain reliably predicts >105 cfus/g of wound tissue Presence of bacterial cells on Grams stain of burn wounds consistently correlates with >106 organisms per swab specimen Critical microbial load for wound infection appears to be 104-106cfu/g wound tissue or ml wound fluid, and 106cfu/wound swab specimen

Microbiology of wound infection: the Big Three1

Streptococcus pyogenes (capable of wound infection <105 cfu/g wound tissue)2 Staphylococcus aureus Pseudomonas aeruginosa 1Associated with monomicrobial and polymicrobial wound infection 2And other pyogenic -hemolytic streptococci

Microbiology of wound infection: Obligate Anaerobic Bacteria1

Bacteroides Porphyromonas (pigmented) Prevotella (pigmented and non-pigmented) Fusobacterium Peptostreptococcus Clostridium2 1Primarily associated with polymicrobial aerobic and anaerobic bacterial wound infection 2Monomicrobial infection by Clostridium perfringes in myonecrosis (gas gangrene) (distinctive Grams stain showing large boxcar shaped gram-positive rods with a paucity of inflammatory leukocytes

Polymicrobial wound infection: mechanisms

Oxygen consumption by aerobic bacteria induces tissue hypoxia and favorable growth conditions for anerobic bacteria Nutrients produced by one organism supports growth of other fastidious and potentially pathogenic organisms

Polymicrobial wound infection: mechanisms

Vitamin K production by Staphylococcus aureus supports growth of vitamin K-dependent Prevotella melaninogenica Succinate produced by Klebsiella pneumoniae a critical growth factor for Prevotella melaninogenica

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 infections 2Giacometti et al., JCM 38:918-922 (2000)

Microbiology of wound infection: surgical wounds1

Superficial wounds, surgical incisions: streptococci, staphylococci Deep wounds: GI, female genital tract, and oropharyngeal-streptococci, staphylococci, gramnegative enterics, enterococci, Bacteroides, other anaerobes; other-streptococci, staphylococci, gramnegative enterics Gangrenous 24-48 hr after surgery: group A streptococci, clostridia Necrotizing >4 days after surgery: polymicrobial (aerobic and anaerobic) 1Nichols and Florman, CID 33(Suppl 2):S84-93 (2001)

Microbiology of wound infection: diabetic foot infections1

Cellulitis: -hemolytic streptococci (A, B, C, G), Staphylococcus aureus Infected ulcer (no antibiotic treatment): same as cellulitis, often monomicrobial Infected ulcer that is chronic or with previous antibiotic: S. aureus, hemolytic streptococci, Enterobacteriaceae (usually polymicrobial) Macerated ulcer due to soaking: Pseudomonas aeruginosa (usually polymicrobial) Long-duration non-healing ulcers with prolonged, broad-spectrum antibiotic treatment: S. aureus (MRSA), coagulase-negative staphylococci, 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, nonfermentative gram-negatives, obligate anaerobes 1Lipsky et al., CID 39:885-910 (2004)

Microbiology of wound infection: burn wound infections1

Staphylococcus aureus (22.9%) Pseudomonas aeruginosa (20.9%) Pseudomonas species (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 wound infections during 1974-1978 (CDC, Mayhall, CID 37:543-550 (2003)

Microbiology of wound infection: burn wound infections1

Staphylococcus aureus (23.0%) Pseudomonas aeruginosa (19.3%) Enterococcus species (11.0%) Enterobacter species (9.6%) Escherichia coli (7.2%) Coagulase-negative Staphylococcus (4.3%) 1Bacteria constituting >4% of organisms that were recovered from 1,234 burn wound infections during 1980-1998 (CDC, Mayhall, CID 37:543-550 (2003)

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)

Microbiology of wound infection: animal bite infections1,2

Same as human bites with the addition of: Pasteurella multocida Neisseria weaveri Staphylococcus intermedius 1Goldstein, Mandell, Douglas, and Bennetts Principles and Practice of ID, pp. 3552-3556 (2005) 2Capitini et al., CID 34:e74-74 (2002).

Clinical signs of wound infection

Purulent discharge Painful spreading erythema Failure to heal

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 to heal should be sampled for Grams stain and culture

Grams stain of wound specimens

Presence of bacteria by Grams stain indicates 105 to 106 organisms/g wound tissue or ml wound fluid Types of organisms present on Grams stain should be correlated with culture results to recognize predominant organisms that dont grow aerobically

Culture of wound specimens

Facultative anaerobic and aerobic bacteria of primary importance in wound infection Media for aerobic culture of wound specimens include sheep blood, chocolate, and MacConkey agar Media for anaerobic culture of wound specimens include brucella blood agar, laked blood with kanamycin and vancomycin, Bacteroides bile esculin, and anaerobic colistin-naladixic acid

Culture of wound specimens

Correlate growth of facultative anaerobic and aerobic bacteria with gram-stain morphotypes If Staphylococcus aureus, -hemolytic Streptococcus, and/or Pseudomonas aeruginosa present in any numbers, identify with susceptibility testing If coagulase-negative Staphylococcus, Corynebacterium, and/or Enterococcus present in moderate to many numbers, and growth explains gram-stain results, report as genus and full identification/susceptibility available by request. If Enterobacteriaceae, or non-fermenters other than Pseudomonas aeruginosa present in moderate to many numbers, 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

Culture of wound specimens

If facultative anaerobic and aerobic bacteria recovered in culture do not correlate with one or more gram-stain morphotypes, review and repeat the Grams 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 genus and report susceptibility available by physician request If > 4 organisms detected in culture by these criteria, obtain a technical consult

Genus identification of anaerobic bacteria in wound culture

Bacterioides: growth in 20% bile Porphyromonas: vancomycin susceptibile, kanamycin resistant, colistin resistant1 Prevotella: vancomycin resistant, kanamycin resistant, colistin susceptibile1 Fusobacterium: vancomycin resistant, kanamycin susceptible, colistin susceptibile 1+/- pigmentation on laked blood

Genus identification of anaerobic bacteria in wound culture

Clostridium: large gram-positive rods with sporulation Peptostreptococcus: kanamycin resistant and sodium polyanethol sulfonate susceptible, or kanamycin susceptibile and sodium polyanethol sulfonate resistant Perform aerotolerance on all anaerobic isolates to be reported

Wound specimen negative by direct Grams stain

Review and repeat Grams stain. If confirmed negative, proceed as outlined below Identification and susceptibility testing of Staphylococcus aureus, -hemolytic Streptococcus, and Pseudomonas aeruginosa only Other culture isolates reported as polymicrobial flora

Pure isolate in aerobic and/or anaerobic would

Full identification with susceptibility for Staphylococcus aureus, -hemolytic Streptococcus, Pseudomonas aeruginosa, and Clostridium perfringens in any amount of growth, and full identification with susceptibility for other organisms demonstrating growth in the second streak of a sheep blood agar plate

Quality management
Number (%) of wound Grams stains explained by aerobic culture results

Reference
Bowler PG, Duerden BI, and Armstrong DB. 2001. Wound microbiology and associated approaches to wound management. Clinical Microbiology Reviews 14:244-269.