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Research Letter |

Defining Wound Microbial Flora: Molecular Microbiology Opening New Horizons FREE

Yelena M. Frankel, MD, MPH; Johan H. Melendez, MS; Nae-Yuh Wang, PhD; Lance B. Price, PhD; Jonathan M. Zenilman, MD; Gerald S. Lazarus, MD
Arch Dermatol. 2009;145(10):1193-1195. doi:10.1001/archdermatol.2009.246.
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Published online

Chronic wounds cause substantial morbidity.1 Although nonhealing is often attributed to infection, the process for ascertaining bacterial flora within wounds is not standardized. Qualitative swab cultures are characterized as showing few, moderate, or heavy bacterial presence, but the significance of these categories has not been validated.2 A culture is an insensitive test for detecting fastidious organisms. Therefore, to appreciate the role of bacteria in nonhealing wounds, careful characterization and clinical correlation is required.

We evaluated the bacterial ecology of chronic wounds in a prospective series of patients. We developed and standardized a convenient tissue sampling method, compared qualitative and quantitative culture results, and assessed the utility of real-time polymerase chain reaction (RT-PCR) for detecting methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S aureus (MRSA).

This study was approved by the Johns Hopkins institutional review board. Chronic wounds were defined as nonhealing ulcers present for 6 weeks or longer. We enrolled 28 subjects; 9 had multiple visits. One subject had 2 wounds. From 11 wounds, we obtained multiple simultaneous specimens from opposing rims; from 8 of these 11, we also obtained simultaneous specimens from the center.

After receiving the patients' informed consent, we anesthetized the leading edge of the wound with topical lidocaine, 2%, for 5 minutes and washed it with sterile isotonic sodium chloride (saline). A sterile 3-mm curette was placed at the ulcer rim. At a 45° angle, the cutting edge was scraped along the rim of the wound until the curette filled with tissue. The specimen was then placed in nonbacteriostatic saline, immediately transported to the laboratory, minced, and divided. One aliquot was sent for qualitative culture analysis at a CLIA-certified laboratory (Clinical Laboratory Improvement Amendments). Bacterial load was reported as not present, few, moderate, or heavy, and the species of present bacteria were determined. Other aliquots were used for quantitative culture and RT-PCR analysis. Quantitative culture analysis was performed in duplicate, and the results were defined as our referent.

For the RT-PCR analysis, 200 μL of tissue homogenate was processed as described previously3 and assayed for the S aureus (nuc) and methicillin-resistant mec genes in a stepwise approach. To differentiate MRSA from coagulase-negative staphylococcal bacteria, we used RT-PCR to simultaneously target the staphylococcal cassette chromosome mec and the S aureus–specific gene orfX, as previously described.4

Quantitative results were log transformed, and reliability was evaluated using Pearson correlation and linear regression analysis. Qualitative and quantitative results were compared using contingency tables and analysis of variance.

Sixty samples from 41 wound-visits were collected from 28 patients. The minimum amount of tissue required was 20 mg, which was obtained in 1 or 2 curette passes. Duplicate quantitative results were highly correlated, and Pearson correlations for the log-transformed bacterial counts among duplicate pairs ranged from 0.93 to 0.99.

Patients' ages ranged from 33 to 83 years; 16 patients were men (57%). Primary ulcer diagnoses were neuropathic (39%), decubitus and/or pressure (21%), venous (15%), traumatic and/or surgical (11%), and other (14%). Thirty-six of 41wounds were on the lower extremity (88%).

At baseline, 1 or more organisms were quantitatively cultured from 28 of 29 wounds (97%). We excluded Corynebacteria and coagulase-negative staphylococcal bacteria from analysis because these are usually not considered clinically significant. Methicillin-resistant S aureus was isolated in 13 baseline wounds (45%), Pseudomonas aeruginosa in 8 (28%), and group B Streptococcus in 6 (21%) (Figure 1). The titer of MRSA, when found, was high:24 of 29 MRSA-positive specimens had 105 colony-forming units (CFU) or more per gram (85%; 95% confidence interval [CI], 65.5%-94.8%). Other organisms were found in 25 baseline wounds (86%), and 16 wounds had 1 or more organisms (55%). Of the 60 curettings, organisms were identified in 49 (82%). Culture-positive wounds had an average of 2.1 different species of organisms, yielding 104 distinct isolates, which were consistently high titer: 86 had 105 CFU/g or more (83%), and 22 had 108 CFU/g or more (21%). Twelve samples had 3 organisms, each at a titer of 105 CFU/g or higher (20%).

Place holder to copy figure label and caption
Figure 1

Microbial diversity in chronic wounds. MRSA indicates methicillin-resistant Staphylococcus aureus.

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Seven baseline samples were from patients being treated with antimicrobial agents; only 3 of these 7 had organism loads of 105 CFU/g or higher. Six samples were from patients with antibiotic exposure within the previous 2 weeks but not within the previous 24 hours. Of these 6, 5 had 2 or more pathogens at titers of 105 CFU/g or higher, similar to patients without antibiotic exposure. In 7 patients, preantibiotic and postantibiotic samples were available, and analysis showed that despite appropriate therapy, 6 of the 7 had not bacteriologically resolved. Our brief follow-up period and small sample size limited our ability to correlate these findings with the clinical course.

Qualitative findings did not correlate with quantitative assessments (Figure 2). Quantitative cultures identified organisms in 10 of 29 wounds that were qualitatively negative (38%).

Place holder to copy figure label and caption
Figure 1

Comparison of qualitative and quantitative results. The qualitative report categories (not present, few, moderate, and heavy) do not correlate with quantitative assessments. CFU indicates colony-forming units. The larger rectangular boxes represent the interquartile range (distance between the 25th and 75th percentiles of the data points); plus signs inside these boxes, the mean; short horizontal lines inside these boxes, the median; vertical lines issuing from the boxes, minimum and maximum values; smaller boxes in the few and heavy categories, data outliers that were not used in this analysis; and the long dashed horizontal line, the bacterial titer of 105 CFU/g, commonly used as threshold for infection.

Graphic Jump Location

In 11 wound-visits with opposing rim curettings, we isolated 19 distinct cultures: 17 yielded the same organisms at both sites (90%). Sixteen of 17 pairs had bacterial counts within 2 logs (94%; 95% CI, 71.3%-99.9%). The 7 wounds analyzed at rim and center yielded 19 isolates, of which 18 were similar (94.7%): 17 had bacterial counts within 2 logs (94%; 95% CI, 72.7%-99.9%).

The RT-PCR analysis to detect S aureus was both sensitive (35 of 37, 95%) and specific (21 of 23, 91%) and identified 28 of 29 MRSA-colonized wounds (97%). The specificity of RT-PCR is underestimated because it may be more sensitive than culture. For example, we identified MRSA in 3 of 31 MRSA-negative samples quantitatively (10%), whereas 5 of 31 of samples were found to be negative by qualitative culture (15%).

Tissue curettings from the wound rim were easily performed, reproducible, and yielded bacteriological homogeneity within wounds even at multiple locations, similar to reports by Kirketerp-Møller et al.5

Our small sample limits our ability for clinical correlations. Qualitative and quantitative results did not correlate, which might have serious implications for the adequacy of widely used qualitative assays to assess microbial burden. Methicillin-resistant S aureus was common and almost always in high concentrations (≥105 CFU/g). Frequent isolation of group-B Streptococcus was expected because it is common in elderly and diabetic patients.

Analysis by RT-PCR identified S aureus in 3 culture-negative samples. We also assayed by RT-PCR for 10 additional species (Acinetobacter baumannii, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, Streptococcus pneumoniae, and Streptococcus pyogenes) and identified 4 additional pathogens. We therefore used RT-PCR to identify additional pathogens in 7 of 60 samples (12%). Reverse transcriptase–polymerase chain reaction assays can be processed in as short as 6 hours.

Chronic wounds are complex microbiological communities in which bacterial loads might adversely affect healing without showing signs of infection. New analytic methods are needed to identify microbial populations and correlate them with clinical findings to gain greater understanding of the role of bacteria within chronic wounds.

ARTICLE INFORMATION

Correspondence: Dr Frankel, Department of Dermatology, Johns Hopkins Medical Institutions, JHOC, Sixth floor, 611 N Caroline St, Baltimore, MD 21205 (yfrankel@jhmi.edu).

Author Contributions: All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Author Contributions:Study concept and design: Frankel, Melendez, Price, Zenilman, and Lazarus. Acquisition of data: Frankel, Melendez, Price, and Zenilman. Analysis and interpretation of data: Frankel, Melendez, Wang, Price, Zenilman, and Lazarus. Drafting of the manuscript: Frankel, Price, and Lazarus. Critical revision of the manuscript for important intellectual content: Frankel, Melendez, Wang, Price, Zenilman, and Lazarus. Statistical analysis: Wang. Obtained funding: Melendez, Wang, and Zenilman. Administrative, technical, and material support: Frankel, Melendez, Price, Zenilman, and Lazarus. Study supervision: Frankel, Zenilman, and Lazarus.

Funding/Support: This study was supported in part by grant UL1 RR 025005 from the National Center for Research Resources, a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research (Dr Wang), and by the Johns Hopkins Center for Innovative Medicine (Dr Zenilman).

Role of the Sponsors: The sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript.

Bickers  DRLim  HWMargolis  D  et al. American Academy of Dermatology Association; Society for Investigative Dermatology, The burden of skin diseases: 2004 a joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. J Am Acad Dermatol 2006;55 (3) 490- 500
PubMed Link to Article
Davies  CEHill  KENewcombe  RG  et al.  A prospective study of the microbiology of chronic venous leg ulcers to reevaluate the clinical predictive value of tissue biopsies and swabs. Wound Repair Regen 2007;15 (1) 17- 22
PubMed Link to Article
Costa  AMKay  IPalladino  S Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 2005;51 (1) 13- 17
PubMed Link to Article
Huletsky  AGiroux  RRossbach  V  et al.  New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 2004;42 (5) 1875- 1884
PubMed Link to Article
Kirketerp-Møller  KJensen  POFazli  M  et al.  Distribution, organization, and ecology of bacteria in chronic wounds. J Clin Microbiol 2008;46 (8) 2717- 2722
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1

Microbial diversity in chronic wounds. MRSA indicates methicillin-resistant Staphylococcus aureus.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 1

Comparison of qualitative and quantitative results. The qualitative report categories (not present, few, moderate, and heavy) do not correlate with quantitative assessments. CFU indicates colony-forming units. The larger rectangular boxes represent the interquartile range (distance between the 25th and 75th percentiles of the data points); plus signs inside these boxes, the mean; short horizontal lines inside these boxes, the median; vertical lines issuing from the boxes, minimum and maximum values; smaller boxes in the few and heavy categories, data outliers that were not used in this analysis; and the long dashed horizontal line, the bacterial titer of 105 CFU/g, commonly used as threshold for infection.

Graphic Jump Location

Tables

References

Bickers  DRLim  HWMargolis  D  et al. American Academy of Dermatology Association; Society for Investigative Dermatology, The burden of skin diseases: 2004 a joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. J Am Acad Dermatol 2006;55 (3) 490- 500
PubMed Link to Article
Davies  CEHill  KENewcombe  RG  et al.  A prospective study of the microbiology of chronic venous leg ulcers to reevaluate the clinical predictive value of tissue biopsies and swabs. Wound Repair Regen 2007;15 (1) 17- 22
PubMed Link to Article
Costa  AMKay  IPalladino  S Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 2005;51 (1) 13- 17
PubMed Link to Article
Huletsky  AGiroux  RRossbach  V  et al.  New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 2004;42 (5) 1875- 1884
PubMed Link to Article
Kirketerp-Møller  KJensen  POFazli  M  et al.  Distribution, organization, and ecology of bacteria in chronic wounds. J Clin Microbiol 2008;46 (8) 2717- 2722
PubMed Link to Article

Correspondence

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