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Jornal Vascular Brasileiro

versão impressa ISSN 1677-5449versão On-line ISSN 1677-7301

J. vasc. bras. v.6 n.3 Porto Alegre set. 2007 



Isolation and susceptibility profile of bacteria in diabetic foot and venous stasis ulcer of patients admitted to the emergency room of the main university hospital in the state of Goiás, Brazil



Ly de Freitas FernandesI; Fabiana Cristina PimentaII; Fernando de Freitas FernandesIII

IPhysician. Specialist in General Surgery, Universidade Federal de Goiás (UFG), Goiânia, GO, Brazil. Specialist in Peripheral Vascular Surgery and Angiology, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil. MSc. student, Graduate Program in Tropical Medicine, Instituto de Patologia Tropical e Saúde Pública (IPTSP), UFG, Goiânia, GO, Brazil. Vascular surgeon, Hospital das Clínicas, UFG, Goiânia, GO, Brazil
IICo-advisor. Associate professor, Department of Microbiology, Immunology, Parasitology and Pathology (DMIPP), Setor de Microbiologia, IPTSP, UFG, Goiânia, GO, Brazil
IIIAdvisor. Associate professor, DMIPP, Setor de Parasitologia-Entomologia, Laboratório de Artropodologia Médica e Veterinária, IPTSP, UFG, Goiânia, GO, Brazil





BACKGROUND: Infected lower limb injuries (diabetic ulcers and venous stasis ulcers) cause great suffering and functional disability with social and economic impact and increase in risk of severe complications.
OBJECTIVE: To characterize the microbiota and determine the antimicrobial susceptibility profile of isolated bacteria in lower limb injuries secondary to the venous stasis ulcer and diabetic foot.
METHODS: Patients with lower limb lesions were included in the study, both diabetics and patients with venous stasis ulcer, receiving care at the emergency service of a university hospital in Goiânia (Brazil) from February 2005 to August 2006. Samples were collected with cotton swab to perform culture and antimicrobial sensitivity test applying standardized techniques.
RESULTS: Presence of bacteria was detected in 88.46% of the samples. Gram-positive cocci were characterized as Staphylococcus aureus and Staphylococcus epidermidis. Among Gram-negative rods, Pseudomonas aeruginosa,Escherichia coli,Proteus mirabilis and Enterobacter sp. were detected.
CONCLUSIONS: Isolated microorganisms of lower limb injuries (diabetic foot and venous stasis ulcer) included Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus,Pseudomonas aeruginosa and Escherichia coli, which werethemost frequent and highly resistant to several kinds of antimicrobial agents.

Keywords: Microbiota, venous stasis ulcer, diabetic foot, infection.




Among the most common lower limb lesions are diabetic and venous stasis ulcers.1 Plantar lesions known as diabetic foot, a chronic and frequent complication of diabetes mellitus,1 result especially from neuropathy and degenerative microangiopathy characterized by alteration in capillary structure and protective endothelial function.2 Increased plantar pressure, skin changes such as dryness, fissures, mycosis, osteoarticular deformities, muscle atrophy and bone prominences, formation of callus3 and repetition traumas can result in skin and subcutaneous tissue infection, abscesses and deep layer phlegms,4 significantly increasing risk of amputation,5 which is also associated with early arteriosclerosis.6

Venous stasis ulcers are also frequent lesions7 and are related to physiopathological mechanisms of chronic venous insufficiency.7 They generate social and economic impact, work disability and expenses associated with treatment.7,8

Microorganisms associated with lower limb lesions mentioned above are part of skin microbiota, and associations of anaerobic and facultative aerobic bacteria are common, resulting in mixed infections.9

Staphylococcus aureus and Streptococcus sp are present in moderate lower limb infections without systemic toxicity, in superficial lesions with cellulitis, moderate ulceration and mild ischemia.10 In severe infections with extensive cellulitis, ulcer, lymphangitis and ischemia, gram-positive cocci are present (Staphylococcus sp, Streptococcus spand Enterococcus sp), anaerobic bacteria, such as bacteroids and facultative gram-negative (Escherichia coli, Enterobacter sp, etc.), and nonfermenting gram-negative rods (Pseudomonas and Acinetobacter) .10 Our aim was to isolate and characterize the microorganisms of lower limb lesions (diabetic foot and venous stasis ulcer), as well as to determine susceptibility profile of isolated bacteria.



The study population was comprised of patients with lower limb lesions (diabetic foot and venous stasis ulcer), who were admitted to a university hospital in Goiânia, Brazil. The study was carried out after approval by the Ethics Committee and signing of a consent form by the patient or responsible. Collection was performed in deep layers using cotton swab after skin disinfection with physiologic solution and Povidine®; , local anesthesia with 2% lidocaine without vasoconstrictor and surgical debridement of devitalized tissues. The samples were conditioned in Stuart medium and sent to the laboratory for culture and antimicrobial sensitivity test (antibiogram).

The samples were sowed in sheep blood agar (5%) and incubated at 37 ºC for 24-48 hours. Colonies were initially identified by gram staining, based on their development in selective and nonselective culture mediums, biochemical/enzymatic tests11 and techniques automated by the MicroScan®; system (Dade Behring – West Sacramento, California, USA). Susceptibility of isolated bacteria was determined by the automated system, and the results were interpreted according to recommendations by the Clinical and Laboratory Standards Institute.12



In this study, 79 cases of lower limb lesions were assessed: 50 diabetic foot and 29 stasis ulcers. A total of 104 cultures were performed, 92 (88.46%) of them being positive. In 65 cultures, gram-negative bacteria were isolated; of these, 42 (45.66%) were enterobacteria, 23 (25%) were nonfermenting rods and 27 (29.34%) were staphylococcus.

The 12 (11.54%) negative cultures corresponded to samples of the first collection from nine individuals with diabetic feet and three with stasis ulcers. In 10 patients with diabetic foot more than one sample was collected for each case, due to unfavorable evolution of the lesion, corresponding to 25 cultures. Figure 1 illustrates a diabetic foot lesion, and Figure 2 shows a stasis ulcer.





Prevalent bacteria in lesions (Figure 3) were: Staphylococcus aureus , Staphylococcus epidermidis and gram-negative rods: Pseudomonas aeruginosa,Escherichia coli,Proteus mirabilis and Enterobacter sp(Table 1). There was prevalence of 70.66% in gram-negative rods isolated from lower limb lesions. In diabetic foot, the most frequent species was Staphylococcus aureus , followed by E. coli and P. aeruginosa;however, in venous stasis ulcer, P. aeruginosa, followed by S. aureus and Enterobacter sp were prevalent (Figure 3).




Clique to enlarger


Table 2 shows the results of cultures obtained in 10 patients with diabetic foot with unfavorable evolution. In the first culture, only one bacterium was isolated, and in 20% there was no microbial development. In subsequent cultures, there was prevalence of S. aureus and P. aeruginosa.In only two cases (A and I), the bacterium detected in the first collection was recovered in subsequent cultures (A and I). Cases A, B, C, D and E progressed requiring lower limb amputation, in which an association of bacteria was isolated in patients A and C, with highlight to P. aeruginosaand S. aureus. In case B, S. epidermidis and P. aeruginosa were isolated, whereas in case E P. aeruginosa and S. aureus were isolated.


Clique to enlarger


Results of the susceptibility profile in the most frequent four bacteria are presented in Figures 4 to 7.S. aureus and P. aeruginosa were prevalent both in diabetic foot lesions and in venous stasis ulcers, whereas the third most isolated bacteria was E. coli in diabetic foot and Enterobacter sp in venous stasis ulcer. All Staphylococcus aureus were sensitive to vancomycin, tobramycin, Synercid(quinupristin-dalfopristin) and linezolid. Sensitivity of S. aureus to gatifloxacin, ampicillin/sulbactam and cefazolin was 80%, whereas it was 77% for rifampicin (Figure 4), with resistance to ampicillin, penicillin, amikacin, cephalothin, amoxicillin/clavulanate and oxacillin. P. aeruginosa was sensitive to meropenem, imipenem e polymyxin B (Figure 5). E. coli was sensitive to imipenem and meropenem, ceftazidime, cefepime, aztreonam, gentamicine and amikacin, and less sensitive to ciprofloxacin (Figure 6). It was resistant to ampicillin, amoxicillin/clavulanate, chloramphenicol and cephalothin. Enterobacter sp was sensitive to amikacin, gentamicine, cefepime, piperacillin/tazocin, ceftriaxone, ceftazidime, meropenem, ciprofloxacin and aztreonam (Figure 7).










A high frequency of S. aureus, P. aeruginosa and enterobacteria was detected in assessed lesions, similar to that reported by Assis et al.13 and Jorge et al.14 Regarding gram-positive cocci, thee was prevalence of S. aureus and S. epidermidis, in agreement with the reports by Goldstein et al.15, Routh et al.16 and Slovenkai et al.17

Selection and dissemination of multiresistant microorganisms have been occurring both in hospitals and in the community and represent a great challenge in therapy.15,18-20 In this study, methicillin-resistant S. aureus (MRSA) had high prevalence (69%), different from the results found by Goldstein et al.15 and Carvalho et al.18, who found rates lower than 20%. Isolation rate of gram-negative bacteria in this study was similar to that found by Carvalho et al.,18 when assessing patients with diabetic foot and mainly isolating enterobacteria.

Rocha et al.21 reported the problem associated with multiresistance of gram-positive and gram-negative bacteria, especially Escherichia coli, in more severe cases. Most staphylococci detected in this study were resistant to amoxicillin/clavulanate, cephalothin, oxacillin and clindamycin, similar to what was described by Unachukwu et al.20 and Rocha et al.21

Due to unfavorable evolution of 10 patients with diabetic foot, subsequent collections were performed (Table 2). There was prevalence of Staphylococcus aureus and Pseudomonas aeruginosa, and the bacteria isolated in the first collection were recovered in only two cases. In four cases that progressed to lower limb amputation, there was presence of P.aeruginosa and/or S. aureus, in agreement with Rocha et al.,21 who considered diabetic foot as the main cause of nontraumatic limb amputation.

The prevalence of gram-negative rods and resistant staphylococci observed in this study makes choice of antimicrobials difficult for empirical treatment. Therefore, culture and antibiogram should be performed; however, if this procedure is not feasible, use of ampicillin/sulbactam in association with piperacillin/tazobactam and ciprofloxacin is recommended when there is no suspicion of infection by Pseudomonas.

Conclusion: A mixed microbiota of lower limb lesions was detected, with gram-positive and gram-negative bacteria, Staphylococcus aureus ,Pseudomonas aeruginosa and Escherichia coli being the most frequent, with high resistance to many antimicrobials and a high rate of MRSA (69%). According to the in vitro results, ampicillin/sulbactam in association with piperacillin/tazobactam could be an option for the in vitro treatment of most cases of lower limb lesions, as well as ciprofloxacin when there is no suspicion of infection by Pseudomonas.



To my advisors.

Laboratório Professora Margarida Dobler Komma.

Laboratório de Análises clínicas do Hospital das Clínicas.

Staff of practitioners and technicians at the emergency room of HC UFG.

José Jurandir de Moraes and Elisa Tiba Gomes for their loyalty in helping provide care for patients at the emergency room.

To CNPq for the partial financial aid to perform this study.



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Fernando de Freitas Fernandes
Departamento de Microbiologia, Imunologia, Parasitologia e Patologia do Instituto de
Patologia Tropical e Saúde Pública da Universidade Federal de Goiás
Caixa Postal, 131
CEP 74605-050 – Goiânia, GO, Brazil

Manuscript received October 5, 2006, accepted June 14, 2007.

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