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Bacteriology of diabetic ulcers: effect of sample collection method

Anju Kagal, Uday Kelkar

Most diabetic foot lesions have a polymicrobial aetiology; however, bacterial yield varies with the method of sample collection. This study compared the bacteriology of swab samples and deep tissue samples obtained at the same time from 50 diabetic foot ulcers. The bacterial yield from the deep tissue samples was significantly higher than from the swabs, and provided a comprehensive picture of the pathogens present in the ulcer. As the fate of the lower extremity is dependent on the treatment regimen, which is based on the culture report, it is crucial that no pathogens are missed. Although swabs may be a useful adjunct, they must be accompanied by deep tissue samples to enable health professionals to decide on the most appropriate therapy.

Foot infections are a major cause of morbidity in people with diabetes. Devitalised tissue is the site where the bacteria responsible for the non-healing ulcers inflict damage. The bacteriology of diabetic foot ulcers has been studied by numerous investigators (Sharp et al, 1979; Wheat et al, 1986; Bamberger et al, 1987; Peterson et al, 1989; Lipsky et al, 1990; Gerding, 1995). Most of these lesions have been found to have a polymicrobial aetiology (Louie et al, 1976; Sapico et al, 1984; Wheat et al, 1986.). However, there are several techniques of sample collection, and the bacterial yield may vary with the technique.

Aim of the study
A study was undertaken to assess the influence of sample collection methods on the yield of aerobic and anaerobic bacteria from diabetic foot ulcers.

Method
This study was carried out in a large general hospital at Pune, India, over a one-year period.

Fifty people with diabetes and a foot ulcer of grade II or more (Wagner, 1981) attending surgery clinics were enrolled in the study. Wagner (1981) defines a grade II ulcer as a deep ulcer, often infected, but with no bony involvement. Patients with superficial ulcers or mere abrasions were excluded. 

Sample collection and processing
The ulcer site and size were examined with the patient laying supine on an examination table. Superficial dead tissue or eschar was removed with sterile scissors and a scalpel blade. After local debridement of devitalised tissue, the wound was cleaned with sterile saline.

Samples were then obtained from each ulcer, using two different techniques:

  • Swab samples were collected by rubbing the deepest accessible area of the lesion with a cotton-wool tipped swab moistened with saline. 
  • Samples of devitalised tissue were obtained from the depth of the wound, taking aseptic precautions.

Both samples were transferred to the laboratory in transport media, and processed immediately.

The swab samples were inoculated on culture media. The tissue samples were ground in a sterile mortar and pestle with sterile peptone water. The resulting homogenate was used immediately for inoculation of culture media and smear preparation. The organisms were identified by routine tests using standard procedures (Collee et al, 1989; Koneman et al, 1992).

Results
Swab samples and deep tissue samples obtained from the diabetic foot ulcers of 50 patients were processed.

The swab samples yielded a total of 150 organisms, comprising 125 aerobes and 25 anaerobes (average 3.0 organisms per sample). The deep tissue samples yielded a total of 185 organisms, comprising 145 aerobes and 40 anaerobes – average 3.7 organisms per sample (Table 1; Figure 1). The yield from the deep tissue samples was significantly higher than the yield from the surface swab samples (p<0.01).

Among the aerobic organisms cultured, Staphylococcus aureus was the most common, followed by Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella species and Enterococcus species, in descending order. 

Among the anaerobic organisms cultured, Peptostreptococcus anaerobius was the most common, followed by Prevotella melaninogenica and Prevotella intermedia. Clostridium perfringens was isolated from the deep tissue sample in two patients, but not from the surface swab sample.

Discussion
In diabetic foot ulcers, samples for bacteriological analysis can been obtained by several methods:

  • sterile swabs
  • needle aspiration
  • tissue samples. 

Swab samples are obtained from the base of the ulcer after cleaning with saline and rubbing the swab over the lesion. They can also be taken directly from the purulent exudate.

Specimens obtained by swabs almost always contain organisms that normally colonise the skin and so may not be reliable. Some of the isolates obtained by this method have been labelled ‘false positives’ by Bamberger et al (1987). However, Wheat et al (1986) have demonstrated that these isolates, although commensals, cannot be ignored and may have a role in the aetiology of the ulcer.

Needle aspiration of deep tissue is probably the ideal method of collecting samples, as aspirate contains only the organisms that are colonising the inflamed tissues. Specimens obtained by this method almost certainly exclude the surface contaminants, but pathogens may be missed if the needle is not inserted into the infected portion of the deep tissue. 

Furthermore, Gerding (1995) noted that needle aspirates tend to yield fewer isolates than are actually present in the deep tissue. Thus, although aspirates are highly specific for pathogenic bacteria within the deep tissue of the lesion, they have a low sensitivity. Peterson et al (1989) compared the needle aspiration and swabbing methods, and found no significant difference in quantitative concordance between the two techniques.

In our study, significantly more organisms were isolated from deep tissue samples (185; average 3.7) than from properly collected swabs (150; average 3.0) (p<0.01), indicating that deep tissue sampling is a more sensitive method. Jones et al (1985) and Lipsky et al (1990) also found that culture of specimens obtained by deep tissue sampling is the most sensitive method for detecting pathogens in diabetic foot ulcers.

Conclusion
Obtaining samples from diabetic foot ulcers using sterile swabs in a meticulous manner, although a useful screening method, may miss important pathogens. In a patient with a diabetic foot ulcer, the treatment regimen is based on the culture report, on which the fate of the lower extremity in question may be decided. It is therefore of paramount importance that the culture report does not miss any pathogens.

Deep tissue samples collected from the devitalised tissue provide a comprehensive picture of the pathogens involved, unlike swab samples. Although swabs can be a useful adjunct, they must always be followed by deep tissue samples, so that more rational – and hence more effective – therapy can be commenced.

REFERENCES:

Bamberger DM, Daus GP, Gerding DN (1987) Osteomyelitis in the feet of diabetic patients. American Journal of Medicine 83: 653–60
Collee JG, Fraser AG, Marmion BP, Simmons A (Eds) (1989) Mackie and McCartney Practical Medical Microbiology. 14th edn. Churchill Livingstone, Singapore
Gerding DN (1995) Foot infections in diabetic patients: the role of anaerobes. Clinical Infectious Diseases 20 (Suppl 2): S283–88
Jones EW, Edwards R, Finch R et al (1985) A microbiological study of diabetic foot lesions. Diabetic Medicine 2: 213–15
Koneman EW, Allen SD, Janda WM, Paul CS, Winn WC Jr (Eds) (1992) Diagnostic Microbiology. 14th edn. JB Lippincott, Philadelphia
Lipsky BA, Pecoraro RE, Larson SA et al (1990) Outpatient management of uncomplicated lower extremity infections in diabetic patients. Archives of Internal Medicine 150: 790–97
Louie TJ , Bartlett JG, Tally FP et al (1976) Aerobic and anaerobic bacteria in diabetic foot ulcers. Annals of Internal Medicine 85: 461–63
Peterson LR, Lissack LM, Canter K et al (1989) Therapy of lower extremity infections with ciprofloxacin in patients with diabetes mellitus, peripheral vascular disease or both. American Journal of Medicine 86: 801–07
Sapico FL, Witte JL, Canawati HN et al (1984) The infected foot of the diabetic patient: quantitative microbiology and analysis of clinical features. Reviews of Infectious Diseases 6(1): S171–76
Sharp CS, Bessman AN, Wagner W Jr et al (1979) Microbiology of superficial and deep tissues in infected diabetic gangrene. Surgery, Gynecology and Obstetrics 149: 217–19
Wagner FW (1981) The dysvascular foot: a system for diagnosis and treatment. Foot and Ankle 2: 64–122
Wheat LJ, Allen SD, Henry M et al (1986) Diabetic foot infections. Archives of Internal Medicine 146: 1935–40

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