Microbiological profile of leg ulcer infections: review study

Rev Bras Enferm. 2021;74(3): e20190763 https://doi.org/10.1590/0034-7167-2019-0763 10 of ABSTRACT Objectives: to analyze the microbiological profile of leg ulcers of patients treated at outpatient clinics and hospitals regarding the type of microorganism, microbiological selection of antibiotics, and techniques for the collection of culture material. Methods: literature review performed on LILACS, IBECS, MEDLINE, and CINAHL databases, resulting in a descriptive analysis of 27 studies. Results: 35.7% of the studies occurred in an outpatient care scenario; and 64.2% in hospitals. There was a predominance of swab (100%) in outpatient care and biopsy (55.5%) in the hospital. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were more common at both levels of assistance. Methicillin-resistant Staphylococcus aureus was isolated in both. Conclusions: the microbiological profile of infections was similar, with the presence of resistant bacteria in both environments. This fact causes concern and raises the need for research to elucidate it. The studies did not compare the effectiveness between biopsy and swab. Descriptors: Leg Ulcer; Culture Techniques; Bacterial Growth; Infection; Nursing.


INTRODUCTION
Leg ulcer is defined as a skin defect below knee level that persists for more than six weeks and shows no tendency of healing after three or more months, and is part of the group of chronic wounds. This condition is not considered a medical diagnosis, but a manifestation of the disease process. It is a relatively common condition among adults, affecting 1% of the adult population and 3.6% of people over 65 years of age. Common causes are venous and arterial diseases, and neuropathy. The causes which are less common are ones related to metabolic and hematological disorders, and infectious diseases (1) . Most patients with chronic wounds will be treated by several professionals in Primary Care (community). Also, its occurrence imposes a substantial economic burden on health care: for example, 5 billion pounds in the United Kingdom, where there are approximately 2.2 million patients with wounds, which makes up 4.5% of the adult population (2) .
Most ulcers cannot be cured in a short period of time. These aspects can be aggravated in the occurrence of an infection, as it results in an increase in the ulcer's healing period and often leads to hospitalization which generates a higher cost of care and treatment, including surgical interventions (3) and eventually prolongs the patient's hospitalization. Infections in leg ulcers can be caused by Gram-positive and Gram-negative bacteria. Among the most frequent bacteria are Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumoniae, and Escherichia coli (4)(5) . These bacteria, although common, can represent a major challenge for therapy when they develop resistance to one or more antibiotics.
The diagnosis of local infection of the wound bed is considered a clinical decision, according to clear criteria of symptoms, a holistic assessment of the patient, and its occurrence requires the timely implementation of appropriate treatment (6) . Early recognition, along with the appropriate and effective immediate intervention, is considered essential in the optimization of patient results, maximizing the management of resources in Primary Care (7) . In the care environment for patients with leg ulcers, the occurrence of an infection and its treatment demand careful attention from professionals, given the absence of specific protocols for the collection of culture material and the proposal for treatment with topical antimicrobials and systemic antibiotics in the majority of health institutions. This context is a challenging issue in clinical practice, since formal guidelines should support professionals in the constructive implementation of clinical diagnosis tools in order to provide an economic and effective service (7) .
Although health professionals in clinical practice identify leg ulcer infection as a complication, this is still a topic that requires investigation to produce answers that help nurses with the assessment, conduct, and microbiological profile in different health services and countries.

OBJECTIVES
To analyze the bacteriological profile of leg ulcers of patients cared for in outpatient and hospital units regarding the type of microorganism and microbiological selection in relation to antibiotics; and techniques for collecting culture material.

METHODS
Integrative literature review, which aims to synthesize and gather scientific evidence through the analysis of multiple studies on a given topic, contributing to critical analysis of professionals in order to support new reflections and respond to knowledge gaps, supporting decision making (8) . The research question, conceived according to the P.I.C.O. strategy, was thus determined: What is the bacteriological profile of leg ulcer infections and the techniques used to collect material for culture?
The inclusion criteria were: studies that necessarily addressed the bacteriological profile of infection in leg ulcers of any etiology, including the diabetic foot or the technique of collecting material from these ulcers for culture; in patients 18 years of age or older, regardless of gender; assisted in Primary Care, outpatient, and hospital level; original studies, available in full, published between 2008 and 2020. The exclusion criteria adopted were: ongoing studies and research protocols; articles that addressed colonization or treatment; studies that dealt with the prevention or cause of these injuries, reports or case studies, specialist consensus.
The search in the electronic databases took place in two phases: the first, between December 2018 and January 2019; and the second, in June 2020. It was carried out by two independent reviewers. for IBECS and CUMED via the BVS, similar strategies were used and found, respectively, 79 and 31 articles, reaching a total of 507. The flowchart of studies selection is shown in Figure 1.
The articles were analyzed according to the etiology of the lesion, the bacteriological profile, and the culture method used to identify the microorganism. The data extracted from the studies were analyzed using descriptive statistics.

Reference
Year  (13) 2014 Brazil Crosssectional n = 77 Microbial culture to assess the bacteriological profile of leg ulcers.
Gram-negatives were more frequent, and infected ulcers had a higher microbial load.
To be continued

Interventions Outcome
Garcia EC, González RG, Albor AR, Salazar-Schettino. Infections of Diabetic Foot Ulcers With Methicillin-Resistant Staphylococcus aureus (14) 2015 Mexico Descriptive n = 100 Microbial culture to assess the bacteriological profile of diabetic foot ulcers. Sonal SM, Rodrigues GS, Vyasc N, Mukhopadhyay C. Antimicrobial susceptibility pattern of aerobes in diabetic foot ulcers in a South-Indian tertiary care hospital (24) 2018 India Crosssectional n = 260 Culture and antibiogram Monomicrobial infections were less common than polymicrobial. Gram-negative bacteria were the most common among the isolates. Staphylococcus aureus and Pseudomonas aeruginosa were the most frequent.
Pseudomonas aeruginosa resistant to classes of cephalosporins, monobactamics, carbapenems, aminoglycosides, chlorophenicol, chylones and fluoroquilones, and β-lactam inhibitors (40) ; Antibioticresistant Escherichia coli such as amikacin, imipinem, tazobactam, cefepime, ceftazidime, levofloxacin, ciprofloxaxine, tobramycin, aztreonam, gentamicin, ampicillin, sulbactam, cefazolin, ceftriaxone, ceftriaxine, cefotethane, furantoin, and sulfamethoxazole (41) . The epidemiological importance of infections caused by multidrug-resistant bacteria is emphasized, because when they occur, they can cause serious complications and consequences for affected patients, as well as financially impact health institutions, in addition to increasing the risk of patients' morbidity and mortality (3,42) . These infections threaten the protection of patients since they minimize the possibilities of therapeutic options against certain species, which lessen the alternatives for the treatment of bacterial infections (42) , prolong the stay in hospitals, treatment, diagnostic procedures and, consequently, increase treatment costs.
The identification of critical colonization as well as the initial signs of infection are essential and must consider the thorough evaluation and the reduction of the bacterial load in the wound bed, involving the techniques for collection of cultures and diagnostic criteria of the infection. Regarding the reduction of bacterial load, in order to minimize critical colonization and possible infection, the importance of a careful cleaning of the wound is highlighted. It can be done using physiological solutions or composed of antimicrobials such as polyhexamethylene biguanide (PHMB); and, when available under pressure, in the form of a jet (43) , it helps to minimize the risk of infection.
In the event of an infection, some signs and symptoms should be carefully evaluated, such as edema of the limb or the edges of the lesion (44) , hardening, erythema, flushing, pain, and local sensitivity, granulation tissue with a friable characteristic, occurrence of fever, chills, odor, high white blood cell count, delayed healing after two weeks, even under adequate topical therapy, and increased volume and changes in exudate characteristics (45) .
In order to facilitate the distinction between clinical signs of infection in the superficial and deep compartments, the bicompartmental model guided by the mnemonics NERDS and STONES was developed in 2007. NERDS was conceived to differentiate "critical colonization" from "infection", being its description: (N) Nonhealing -No wound healing; (E) Exudative -presence of inflammatory exudate; (R) Red and bleeding wound surface granulation tissue -Red and friable granulation tissue; (D) Debris -Debris from tissue; and (S) Smell. STONES reflects the progression from colonization to infection: (S) Size -Increase in the size of the wound; (T) Temperature is increased -Increase in local wound temperature, (O) Os probe to or exposed bone -extent of wound to bone; (N) New or satellite areas of breakdown -Deterioration or new wounds; (E) Exudate, erythema, edema; (S) Smell (46)(47) .
The systematic use of these mnemonics, however, is not common in clinical practice, and the guidelines for such care can be inconsistent and incipient. This fact may favor assistance supported by common sense. Therefore, the involvement of stomatherapist nurses is of fundamental importance, considering their expertise in the assessment and treatment of wounds, as well as Infection Control teams in the hospital environment or Primary Care, aiming

DISCUSSION
The infection of leg ulcers significantly impacts the patient and health services; thus, its occurrence must be identified early. Infections can have a monomicrobial or polymicrobial etiology and still have microorganisms resistant to one or more antibiotics. The predominance of Gram-negative bacteria was similar in outpatient and hospital care, a common finding in studies evaluating the microbiological profile of ulcers (5,30,37) . The most common Gram-negative microorganisms in leg ulcer infections were the specimens Pseudomonas aeruginosa and Escherichia coli, while among the positive ones, Staphylococcus aureus predominated, findings that corroborate other studies (5,38) .
These species also presented microbiological selection to one or more antibiotics. Studies that evaluated the resistance profile of microorganisms isolated from infected ulcers also identified the presence of methicillin-resistant Staphylococcus aureus (14,39) ; at the best therapeutic conduct or its adequacy and monitoring of microbiological profile of the institution.
Regarding the culture method for the identification of microbial isolates, it is worth mentioning that the quantification of the microbial load is the best indicator of the infectious process (48) . The existence of three commonly used techniques is highlighted: biopsy, needle aspiration, and swab. Biopsy is considered the gold standard and consists of the collection of tissue or deep fragments of the wound. However, in some situations, it is not feasible, and swab collection is an acceptable alternative, as it is practical, economical, non-invasive, and allows the identification of infectious bacteria, allowing to guide antibiotic therapy and subsidize sensitivity tests (49) . In this sense, the guidelines of the National Pressure Ulcer Advisory Panel (NPUAP) describe that the determination of the microbial load of pressure injury, by tissue biopsy or swab, is recommended with moderate evidence (48) . There were no differences in the use of the swab and biopsy in the hospital setting; however, the use of the swab in the outpatient clinic predominated. Swabbing is a frequently used method and is therefore recommended using the Levine technique, since it allows for more assertive quantitative culture when compared to the Z technique (49) .
Its execution consists of carefully cleaning the wound with a saline solution, removing the non-viable tissue, after which it is required to wait between two and five minutes (if the bed becomes dry, you must moisten it after that time). Then, in the area in which the tissue appearance appears to be healthier, a sterile swab with a calcium alginate tip must be applied over an area of 1 cm 2 , applying pressure for five seconds (the pressure must be sufficient for an expressive capture of the tissue fluid). Then, the tip of the swab must be broken in the collection device designed for quantitative cultures (48) .
There was a greater occurrence of ulcers due to diabetes, which commonly have a relevant prevalence in health services. In the context of Primary Care, a Brazilian study identified that, among elderly participants, 11.8% had some chronic wound, with 5% being affected by pressure injuries; 3.2%, diabetic ulcers; and 2.9%, due to vasculogenic (50) . It is emphasized that, when complications occur in diabetic foot ulcers, they directly impact the total treatment costs.
Although there are national and international programs and guidelines for diagnostic criteria for infection, the rational use of antibiotics and control of microbial selection, these are still incipient in the context of chronic leg ulcers, which present infection. Therefore, the discussion of the theme, especially on the instrumentalization of professional nurses on the aspects related to the rapid and accurate identification of the infection process, of methods of microorganism identification, is necessary in order to improve the evidence for care at the ambulatory and hospital level, given that these competencies and responsibilities are inherent to the first therapeutic approach, which is usually the responsibility of nurses who care for wounds.

Study limitations
Among the limitations identified for the construction of this review, we highlight the following: even though original articles that evaluated infected ulcers were analyzed, eventually some isolates may correspond to critical colonization, which is not, therefore, clearly described in the studies.
As for the swab collection technique, although Levine's was described, it was not mentioned in all studies, which makes it impossible to identify its use as the first choice at the time of culture collection. Finally, studies that used biopsy and swab to perform culture did not stratify the isolates identified by method, separately, so that this analysis was not possible together.

Contributions to the nursing field
Although the objective of this review does not include the skills of nurses in the care and treatment of infected leg ulcers, it should be noted that these professionals must have skills in the light of suspected infection, clinical-critical judgment in the detection, referrals, and collection of exams. Therefore, the results of this study allow nurses to reflect on the topic for use in clinical practice, regardless of where the care for patients with leg ulcers occurs, since it demonstrates the microbiological profile of chronic wounds, showing the great possibility of bacterial resistance, which can increase patient treatment time and institutional costs.

CONCLUSIONS
The bacteriological profile of leg ulcer infections was similar between outpatient and hospital care, with a predominance of Gram-negative species. However, the profile of microbiological selection for antibiotics was more expressive in the hospital environment. It was possible to observe that many microorganisms presented microbiological selection to one or more antibiotics. The culture method was similar in the two levels of assistance, but a higher occurrence of biopsy was identified in the hospital service; and swab in the outpatient.