Staphylococcus aureus is Associated with High Microbial Load in Chronic Wounds

Abstract: The purpose of this study was to describe the association of Staphylococcus aureus with clinical and microbiological indicators of localized infection in a sample of chronic wounds. Sixty-six subjects with chronic wounds were assessed for signs and symptoms of localized infection, and viable wound tissue specimens were obtained for quantitative microbiological analyses. The study wounds were then grouped according to whether or not they contained S. aureus and statistically compared for differences in the expression of clinical signs of infection, microbial load (i.e., number of organisms per gram of tissue), and diversity of species (i.e., number of different species present in the wound.) S. aureus wounds were significantly more likely to contain greater than 105 organisms per gram of tissue than non-S. aureus wounds (Fisher’s exact p=Disclosures: This study was supported by grants from the Department of Veteran’s Affairs, Health Services Research and Development, Nursing Research Initiative (NRI 01-05-1) and from NIH, NINR, (NIH R01 NR07721). A chronic wound is regarded as tissue injury that does not proceed through the repair process in an orderly, timely manner¹. Chronic wounds include pressure ulcers, venous ulcers, diabetic ulcers, arterial ulcers, and other nonhealing secondary wounds². Unlike acute wounds that heal in days or weeks, chronic wounds can persist for months or years. Wound infection is a major factor that contributes to impaired healing^3,4 and can lead to osteomyelitis, bacteremia⁵, and sepsis⁶. Wound infection occurs when the virulence factors of one or more wound organisms overwhelms the host’s resistance resulting in invasion and replication of the organism or production of toxin and local tissue damage⁷. The ensuing inflammatory response to this local tissue injury presents as the classic signs and symptoms of infection: erythema, heat, edema, pain, and purulent exudates. Unfortunately, many chronic wounds do not express signs of clinical infection despite high numbers of organisms⁸, possibly indicating colonization rather than infection⁹. In the absence of clinical signs of infection, the quantity of organisms, or microbial load, is believed to be the best indicator of wound infection^9,10. The gold standard method for examining microbial load is quantitative culture of viable wound tissue. Wound tissue is viewed as the most valid specimen for culture because tissue cultures reflect organisms invading the wound, not those contaminating the wound surface¹¹. Numerous studies^3,12–15 have demonstrated that a microbial load greater than 105 organisms per gram of tissue is the critical level for diagnosing infection. Others have questioned the 105 guideline asserting that the interactions among specific types of pathogens may be more important than microbial load in promoting bacterial growth and infection^7,16. Supporting this assertion is microbiological evidence that chronic wounds contain multiple species of microbial organisms^{5,7,12,15,18–20} and that those that contain four or more different species have poor healing outcomes²⁰. Nonetheless, it is unclear which organisms represent a definitive threat to the wound environment or which interact with others in a synergistic manner⁷. Of the numerous organisms that colonize chronic wounds, wound care experts believe Staphylococcus aureus, Pseudomonas aeruginosa, beta-hemolytic streptococcus, and anaerobes are the most likely bacterial causes of delayed healing and infection⁷. Of these organisms, S. aureus is most commonly isolated from chronic wounds^8,21 with the others occurring at relatively low rates. S. aureus is a facultative, gram-positive coccus. It is a known pathogen with an extensive array of virulence factors including proteases and toxins. As with most bacteria, these factors are primarily expressed at higher densities to enable the organism to further colonize, and subsequently invade surrounding tissue. Such factors are rarely expressed at lower densities where adherence and survival are paramount. The significance of S. aureus in a chronic wound is difficult to ascertain, i.e., whether it is a pathogen or colonizer. Despite the great number of chronic wounds colonized by S. aureus the relationship between S. aureus and other measures of wound bioburden has received little attention^21,22. Since many clinicians rely on microbiological analyses (i.e., wound cultures) to diagnose infection, clarifying the relationship between S. aureus and other indicators of infection will improve clinical decision making and management. The purpose of this study was to describe the association of S. aureus with clinical and microbiological indicators of localized infection in a sample of chronic wounds. The primary aims were to identify the relationship between S. aureus and the following: 1) clinical signs of infection; 2) microbial load (i.e., number of organisms per gram of wound tissue); and 3) diversity of microbial species in the wound (i.e., number of different species isolated per wound). Methods The study employed an observational, cross-sectional design. Patients who met entry criteria with chronic wound problems were assessed for signs and symptoms of localized infection, and viable wound tissue specimens were obtained for quantitative microbiological analyses. Human subject approval was obtained from the University of Iowa Institutional Review Board. Setting and sample. The study population consisted of patients with nonarterial chronic wounds. A Department of Veteran’s Affairs Medical Center and a university-associated tertiary care facility served as settings for the study. Patients were screened and enrolled using the following criteria: 1) 18 years of age or older; 2) presence of a full-thickness, nonarterial chronic wound; 3) white blood cell count (WBC) greater than 1500 cells/mm³ or total lymphocyte count greater than 800 cells/mm³; 4) platelet count greater than 125,000/mm; 5) no clinical coagulopathies; and 6) not on anticoagulation therapy (e.g., heparin infusion, warfarin, clopidogrel, enoxaparin). The wound sample was limited to nonarterial chronic wounds in order to decrease the risk of infection imposed by wound biopsy among wounds with poor perfusion. Patients with arterial ulcers were identified using clinical history and available physical/diagnostic exams (e.g. ankle brachial index [ABI], pulse volume recordings [PVR], Doppler studies, and angiography). The sample was restricted to full-thickness chronic wounds in order to justify acquisition of full-thickness tissue specimens. Patients with low white cell counts were excluded to reduce the risk of wound infection related to wound biopsy. In addition, frankly neutropenic patients are likely to lack the cardinal signs of inflammation. Patients with low platelet counts, coagulopathies or on anticoagulation therapy were excluded to prevent the risk of bleeding associated with wound biopsy. Eligible patients were invited by the PI to enter the study, and informed consent was obtained. Study variables. The primary study variables were the following: 1) clinical signs and symptoms of infection and 2) culture findings based on viable wound tissue specimens. A Clinical Signs and Symptoms Checklist (CSSC) was developed by the first author to measure the presence or absence of the 12 clinical signs and symptoms of chronic wound infection²³ (i.e., pain, erythema, edema, heat, and purulent exudates) and those specific to secondary wounds²⁴ (i.e., serous drainage with concurrent inflammation, delayed healing, discoloration of granulation tissue, friable granulation tissue, pocketing at the base of the wound, foul odor, and wound breakdown). Descriptors for each sign and symptom were submitted to the panel of six chronic wound experts (e.g., one physician, four doctorally prepared nurses, and one masters-prepared nurse) for content validation and were revised based on their comments. The reliability of each item on the checklist has been previously established using 31-paired independent wound assessments²³. Percent agreement ranged from 65 to 100 percent and Kappa statistics ranged from 0.53 to 1.00. For purposes of this study only the classic signs and symptoms of infection were assessed. After completion of the CSSC, the wound was categorized as infected or non-infected based on examiner interpretation of the presence or absence of these classic signs and symptoms of infection. Wound cultures were processed at a single microbiology laboratory. The tissue specimens were weighed, homogenized, serially diluted in tryptic soy broth (TSB; Remel, Lenexa, Kansas), and plated onto Columbia blood agar (Remel), BBL™ CHROM-agar™ Candida (BD, Sparks, Maryland), eosin-methylene blue agar (Remel), and reduced agar media. Plates were incubated under both aerobic and anaerobic conditions at 37C for 48 hours. All organisms isolated were identified using standard microbiologic procedures, which are based on criteria such as colony morphology and gram stain appearance²⁵. For example, S. aureus is identified based on characteristic yellow b-hemolytic colonies on Columbia blood agar, which on stain appear as gram-positive cocci organized into grape-like clusters and which test catalase positive and staph-latex positive. Streptococci were identified to Lancefield group (A, B, C, G, and F) by agglutination with appropriate antisera using the PathoDx kit (Remel). Because dilutions were based on weight of tissue, the plate count multiplied by the dilution factor yielded the number of organisms per gram of tissue. Subject age, gender, and nutritional status were also assessed. Serum albumin levels served as measures of nutritional status. In addition, wound variables known to be risk factors for wound infection were measured including type of chronic wound, wound size, amount of necrotic tissue, and wound tissue oxygen (transcutaneous oxygen measurement). Chronic wound type was categorized based on etiology and included pressure ulcers, venous ulcers, diabetic ulcers, chronic secondary incisions, or chronic traumatic wounds. Wound etiology was determined using clinical history, physical exam, and available diagnostic exams (e.g., ABI, PVR, Doppler studies, angiography, or air plethysmography). Surface area and depth served as measures of wound size. Surface area was measured by tracing the wound edge on transparent plastic film using an indelible marker with a 1mm tip. The wound edge was defined as the line dividing the healed portion from the unhealed portion of the wound. Surface area was determined using a Lasico Planimeter/Digitizer (Lasico, Los Angeles, California), which has a manufacturer’s reported accuracy of ±0.1-percent accuracy. Inter-rater and intrarater reliability of measurements made on 84 ulcers by this four-member research team were assessed using interclass correlation coefficients. The results showed an inter-rater reliability of 0.99 and an intrarater reliability of 0.98 for all four investigators. In order to measure depth, a cotton-tipped swab was placed in the deepest area of the wound and marked at the point level with the surrounding peri-wound skin. The type and amount of wound bed tissue was measured using the necrotic tissue subscale of the Pressure Sore Status Tool (PSST).26 Necrotic tissue was defined as yellow, tan, brown, gray or black tissue in the wound bed after cleansing the surface of the wound bed with saline-moistened gauze, which ensured the necrotic tissue was adherent.27 The category descriptors on the 5-point scale were: 1 = none present; 2 = 50 and Data collection. All study data were collected by a member of the research team and were recorded on a Case Report Form labeled with subject identification number. Demographic data were collected from the patient record. For subjects with more than one eligible chronic wound (i.e., full-thickness and nonarterial), one wound was randomly selected (i.e., random draw) for data collection procedures. Data regarding the type, location, and history of the study wound were collected from direct observation, the patient record and patient/caregiver report. Following calibration of the transcutaneous oxygen monitor, a heated oxygen sensor was applied to prepped skin (i.e., cleansed with alcohol) just proximal to the study wound area and allowed to equilibrate for 10 to 20 minutes. After equilibration, TCPO2 levels were recorded every minute for a five-minute period. The mean of these measurements was taken as the TCPO2. After removing the dressing, the wound margin outline was traced on transparent film with an indelible marker. The depth of the wound was measured using a cotton-tipped swab placed in the deepest portion of the wound. The amount of necrotic tissue in the wound bed was rated using direct observation and the PSST. The presence or absence of the clinical signs and symptoms of infection were assessed using the CSSC and each wound was categorized as infected or noninfected by the rater based on the clinical interpretation of this assessment. After cleansing the wound, a specimen of viable wound tissue was removed sterile aseptically using a 4- to 6mm dermal punch instrument. The larger (i.e., 6mm) punch instrument was used when the wound surface area could accommodate the larger size. The tissue specimen was placed in sterile container and immediately transported to the microbiology laboratory for processing. All specimens were processed within two hours of acquisition in order to minimize alterations in number of organisms related to progression of time. Data analysis. Study wounds were grouped according to whether or not S. aureus was isolated and identified during microbiological laboratory procedures. Those containing any amount of S. aureus were categorized as S. aureus (SA) wounds. Those that were negative for S. aureus were categorized as non-S. aureus (non-SA) wounds. The SA and non-SA wounds were examined for differences in clinical signs of infection using Fisher’s exact tests. Differences between the SA and non-SA groups with respect to microbial load and diversity of species were statistically examined using t-tests for independent groups. An alpha level of 0.05 (two-tailed) was employed. Results Sixty-six subjects completed the study. Fifty-four (82%) were male. The mean subject age was 57.5 (±11.83) years with a mean albumin level of 3.6 (±0.52). Descriptive statistics for the 66 study wounds are presented in Table 1. Most were diabetic foot ulcers with little necrotic tissue present in the wound bed and adequate wound tissue oxygen levels (i.e., TCPO2 levels). S. aureus was the most common organism isolated from the wound sample; S. aureus was isolated from 34 (52%) wounds. P. aeruginosa was isolated from five (8%) wounds. Group A beta-hemolytic streptococcus was isolated from two (3%) wounds, and anaerobes were isolated from one (2%) wound. Five (8%) wounds contained a combination of S. aureus with either P. aeruginosa, Group A beta-hemolytic streptococcus, or anaerobes. There were no significant differences between SA and non-SA wounds with respect to descriptive characteristics (Table 1). Table 2 presents the data on the clinical and microbiological indicators of infection. Twelve (18%) wounds were categorized as clinically infected based on the clinical judgment of the rater assessing for clinical signs and symptoms of infection. Twenty-four (36%) of the study wounds contained more than 105 organisms per gram of tissue, which was used as the critical value for diagnosing wound infection based on quantitative microbiological findings. The mean number of different species per wound was 2.7 (±2.02). There were no statistically significant differences between SA and non-SA wounds with respect to whether or not they appeared clinically infected although the study had negligible power to detect a difference (i.e., power was 0.03). There were also no significant differences between the SA and non-SA group with respect to the presence or absence of each sign and symptom delineated on the CSSC (i.e., pain, erythema, heat, edema, or purulent exudates). However, SA wounds were significantly more likely to contain greater than 105 organisms per gram of tissue than non-SA wounds (Fisher’s Exact, p=<.001 although="" the="" mean="" number="" of="" different="" species="" isolated="" from="" sa="" wounds="" was="" greater="" than="" non-sa="" this="" difference="" did="" not="" reach="" statistical="" significance.="" power="" study="" to="" detect="" a="" s.="" aureus="" predominant="" organism="" contributing="" microbial="" load="" with="" highest="" colony="" forming="" units="" per="" gram="" tissue="" in="" wounds.="" five="" that="" contained="" p.="" aeruginosa="" only="" one="" two="" group="" beta-hemolytic="" streptococcus="" it="" anaerobes="" were="" wound="" anaerobes.="" there="" no="" significant="" associations="" between="" or="" and="" organisms="" discussion="" findings="" indicate="" presence="" chronic="" is="" associated="" clinical="" signs="" infection.="" however="" lack="" infection="" surprising="" since="" sample="" comprised="" has="" been="" noted="" elsewhere="" do="" always="" express="" symptoms="" despite="" high="" loads.8="" consistent="" other="" studies="">21,22, the findings from this study suggest that the presence of S. aureus in wound tissue is not related to whether or not the wound appears clinically infected. Although the association of S. aureus and clinical signs of infection has been explored previously, this is the first study that has examined the relationship of S. aureus and microbial load. The findings indicate that wound tissue containing S. aureus is more likely have a high microbial load (i.e., number of organisms per gram of tissue) than wounds that do not contain S. aureus. Furthermore, it is important to note that while S. aureus was present in 52 percent of the wounds it was the predominant organism in only 29 percent, indicating it was often not the organism contributing most significantly to microbial load. Whether this overgrowth is pathological or not is yet to be determined. It is plausible that overgrowth of normal skin flora serves a protective function limiting the growth of S. aureus. However, five wounds in this sample contained S. aureus in addition to other pathogenic organisms. Microbial synergy may increase the pathogenic potential of all species in a summative manner⁷. Nonetheless, wounds containing S. aureus were not significantly more diverse with respect to microbial flora than wounds that did not contain S. aureus. One possible limitation of the study relates to the technique for microbial isolation of anaerobes. We used a 48-hour time incubation window to culture organisms. Recent evidence suggests that some of the pathogenic organisms require long culture periods²⁸. In that report it took up to 120 hours to culture some pathogens. We may have excluded some pathogens by using the 48-hour cutoff period. Although this is the first study to examine the association of S. aureus with microbial load and diversity, the cross-sectional design limits the ability to draw definitive conclusions. While S. aureus was not related to clinical signs of localized infection, S. aureus may be a risk factor for the development of overt signs of infection associated with advancing systemic infection, such as osteomyelitis. In addition, the relationship between S. aureus and microbial load needs to be examined further using prospective study designs that would allow serial microbiological analyses of chronic wounds and measurement of wound outcomes. The specific role that S. aureus plays in increasing microbial load and its independent contribution to poor wound outcomes could then be more precisely identified. This knowledge would inform the judicious interpretation of chronic wound cultures and more precisely guide treatment decisions.