Aerobic and Anaerobic Bacteria in Diabetic Foot Ulcers THOMAS J. LOUIE, M.D.; JOHN G. BARTLETT, M.D.; FRANCIS P. TALLY, M.D.; and SHERWOOD L. GORBACH, M.D.; Boston, Massachusetts

Twenty diabetic foot ulcers were cultured using optimal microbiologic techniques. Anaerobic bacteria coexisted with the more commonly recognized aerobic bacteria in 18 specimens. There were a total of 116 isolates with an average of 5.8 species per specimen (3.2 aerobes and 2.6 anaerobes). The principal isolates were Bacteroides species (sp.) (17 strains), peptococci (16), Proteus sp. (11), enterococci (9), Staphylococcus aureus (7), Clostridia (7), and Escherichia coli (6). When antimicrobial therapy is indicated the selection of agents should consider the likelihood of a complex aerobic-anaerobic flora.

prereduced brucella agar base containing 6% sheep's blood and 10 fig/ml menadione (BMB); BMB with 100 fig/ml neomycin; and laked blood agar containing 75 /xg/ml kanamycin and 7.5 tig/m\ vancomycin (Clinical Standards; Torrance, California). Anaerobic media were placed immediately into GasPak jarst for transport to the laboratory where they were transferred to the anaerobic chamber for incubation at 37 °C for 7 days. Aerobic media were incubated in 5% Co2 (BAP) or air. Anaerobic organisms were identified according to the procedures outlined by the VPI Anaerobic Laboratory Manual (4), Enterobacteriaceae were identified by the methods of Edwards and Ewing (5), and nonfermentative bacilli by the method of Pickett and Pedersen (6). Results CLINICAL DATA

A N INFECTED FOOT is the most common septic problem

leading to hospitalization of a diabetic patient ( 1 ) . Ischemia and peripheral neuropathy are the major factors leading to the initial ulcer, a lesion that serves as the portal of entry for soft-tissue, bony, and even systemic infection. Previous studies have stressed the importance of staphylococci, streptococci, and enteric Gram-negative bacilli in these septic complications (1-3). However, certain observations suggest that anaerobic bacteria play a significant role: the exudate from the foot lesion may be putrid, soft-tissue gas is sometimes noted, and aerobic cultures may fail to show a likely pathogen. The present study was undertaken to define the microbiology of diabetic foot ulcers using optimal culture techniques. Methods PATIENT SELECTION

The criteria for inclusion into the study were a foot ulcer greater than 2 cm in diameter and the absence of antimicrobial therapy during the preceding 30 days. Patients were selected from the outpatient diabetic clinic or immediately after hospitalization for foot problems. They were divided into two catagories: [1] uncomplicated stable ulcers, and [2] ulcers with extensive surrounding cellulitis with or without osteomyelitis. BACTERIOLOGIC PROCEDURES

The surface of the wound was prepared by a vigorous saline scrub followed by debridement of superficial exudate using sterile instruments. Specimens were obtained by scraping the ulcer base or the deep portion of the wound edge with a sterile curette. The specimens were inoculated at the bedside onto several microbiologic media. For aerobes and facultative strains, blood agar (BAP), MacConkey agar, and Pfizer Selective Enterococcus Agar* were used. The media for anaerobes were * Pfizer Laboratory; New York, New York. • From the Infectious Disease Section, Tufts-New England Medical Center; Boston, Massachusetts.

Twenty diabetic patients were studied. The mean age was 62.8 years and the mean duration of diabetes was 12.7 years. Nine of the patients were insulin dependent. The ulcer had been present an average of 5.8 months, with a range of 3 weeks to 3 years. Neuropathy was present in all patients. Twelve patients had nonprogressive neuropathic ulcers of long duration and eight had progressively enlarging ulcers associated with extensive cellulitis. Roentgenograms showed three of the latter patients had osteomyelitis and two had soft tissue gas. The clinical status of the diabetes was similar in the group with uncomplicated ulcer and in those with extensive cellulitis with or without underlying osteomyelitis. Fifteen of the 20 ulcers exuded a putrid odor. BACTERIOLOGY

Cultures yielded a combination of aerobic and anaerobic bacteria in 18 of the 20 cases. There was one specimen with multiple aerobic species but no anaerobes and another that produced a pure growth of Clostridium perfringens. Direct Gram stains of exudate confirmed the culture results. A mixture of Gram-positive and Gram-negative bacteria was observed in 19 specimens. The case involving a pure culture of C. perfringens showed only Grampositive bacilli in the direct Gram stain. There were a total of 116 isolates (64 aerobes and 52 anaerobes) representing an average of 5.8 bacterial species per specimen (3.2 aerobes and 2.6 anaerobes). Analysis of cultures from stable ulcers and those complicated by extensive cellulitis showed more bacterial species in the latter group (mean of 4.9 isolates versus 7.3). However, there were no differences in the proportion of anaerobes t BBL; Cockeysville, Maryland.

Annals of Internal Medicine 85:461-463, 1976

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Table 1. Culture Results of Foot Ulcers in 20 Diabetic Patients Aerobic and Facultative Isolates Gram negative Proteus sp. Escherichia coli Klebsiella sp. Pseudomonas aeruginosa Enterobacter sp. Citrobacter freundii Gram positive Streptococcus faecalis Staphylococcus aureus Streptococci (non-group A or D) Staph, epidermidis Micrococcus sp. Bacillus sp. Streptococcus, group D Nonenterococcus

Anaerobic Isolates 29

35 9 7 7 6 3 2 1

to aerobes nor in specific bacterial species that related to the clinical appearance of the ulcer. The most frequent isolate was Peptococcus species (sp.), which was recovered from 16 of the 20 specimens (Table 1). Other major anaerobes were Bacteroides fragilis (9 isolates), B. melaninogenicus ( 7 ) , and Clostridia ( 7 ) . Peptostreptococci were recovered in only two specimens. The predominant aerobic bacteria were Proteus species (11 isolates), enterococci ( 9 ) , Staphylococcus aureus ( 7 ) , and Escherichia coli ( 6 ) . One patient had progressive cellulitis, suppurative thrombophlebitis, septicemia, and septic shock leading to a rapid demise. Exudate from the ulcer yielded B. fragilis subspecies (ss.) distasonis, Peptococcus prevotii, Peptostreptococcus micros, Staph, aureus, enterococci, E. coli, Pr. mirabilis, and Klebsiella sp.; blood cultures grew B. fragilis ss. distasonis. Discussion

Several publications have emphasized the role of Staph, aureus, streptococci, and aerobic Gram-negative bacilli in the septic complications of diabetic foot ulcers (1-3). These reports were based on laboratory methods designed to isolate aerobic microorganisms. With optimal anaerobic methods, we were able to culture anaerobic bacteria from 19 of 20 foot ulcers. In most instances, aerobic bacteria were present in the same specimens. The clinical importance of anaerobes in these infections is supported by several observations. Fifteen of the specimens had a foul odor, a characteristic penetrating stench that is exclusively associated with anaerobic infection ( 7 ) . Soft-tissue gas was noted in two of our patients, and this finding has been reported in as many as 17% of diabetic patients with orthopedic vascular disease ( 3 ) . Previous studies by investigators using adequate anaerobic microbiologic techniques have shown that deep limb infections associated with gas in diabetic patients usually harbor anaerobes, often in association with coliforms (8, 9 ) . Three patients in our series had osteomyelitis; in this regard, Ziment, Miller, and Finegold (10) previously noted that most patients 462

Gram Negative Bacteroides fragilis Bad. melaninogenicus Bact. pnewnosintes Veillonella sp. Gram positive Peptococcus sp. Clostridia C. perfringens C. paraputrificum C. paraperfringens C. innocuum C. ramosum Propionibacterium sp. Eubacterium lentum Peptostreptococci Lactobacillus

11 6 4 4 3 1

18 9 7 1 1 34 16 7 3 1 1 1 1 5 3 2 1

with anaerobic osteomyelitis of the foot had underlying diabetes. Finally, one patient had extension of the foot ulcer to cellulitis, septic thrombophlebitis, and a fatal episode of bacteroides bacteremia. These data suggest that anaerobic bacteria are significant pathogens in deep limb infections in diabetic patients. The role of specific bacteria in causing extension of the localized infection is not readily apparent. When our cases were divided into stable and complicated ulcers, the bacteriology was similar and special pathogenic significance could not be assigned to individual microorganisms. Extension of the process may be governed by noninfectious features such as local care, severity of vascular disease, trauma, and control of the diabetes. The decision to employ antimicrobial agents in the treatment of diabetic foot ulcers depends on the associated clinical findings. Chronic lesions with minimal surrounding cellulitis are unlikely to benefit from systemic antibiotic treatment, and the mainstays of therapy are local care and avoidance of trauma. Indications for antimicrobial chemotherapy are extensive involvement of adjacent soft tissue, osteomyelitis, and signs of systemic infection. In these circumstances antimicrobial agents should be selected on the basis of likely pathogens with due consideration to both aerobic and anaerobic microorganisms. ACKNOWLEDGMENTS: Received 25 March 1976; revisions accepted 4 June 1976. • Requests for reprints should be addressed to Sherwood L. Gorbach, M.D.; 171 Harrison Avenue; Boston, MA 02111. References

1. PRATT TC: Gangrene and infection in the diabetic. Med Clin North AM 49:987-1004, 1965 2. WILLIAMS HGT, HUTCHINSON KJ, BROWN GD: Gangrene of the

feet in diabetics. Arch Surg 108:609-611, 1974 3. BESSMAN AN, WAGNER W: Nonclostridial gas gangrene. Report

of 48 cases and review of the literature. JAMA 233:958-963, 1975 4. HOLDEMAN LV, MOORE WEC: Anaerobic Laboratory Manual,

2nd ed. Blacksburg, Virginia, VPI and State University Anaerobic Laboratory, 1972 5. EDWARDS PR, EWING WH: Identification of Enterobacteriaceae,

3rd ed. Minneapolis, Burgess Publishing Co., 1972

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6. PICKETT MJ, PEDERSEN MM: Nonfermentative bacilli associated

with man. II. Detection and identification. Am J Clin Pathol 54: 164-177, 1970 7. ALTEMEIER WA: The cause of the putrid odor of perforated appendicitis with peritonitis. Ann Surg 107:634-636, 1938 8. ALTEMEIER WA, CULBERTSON WR: Acute non-clostridial crepi-

tant cellulitis. Surg Gynecol Obstet 87:206-212, 1948 9. WILLS MR, REECE MW: Non-clostridial gas infections in dia-

betes mellitus. Br Med J 2:566-568, 1960 10. ZIMENT I, MILLER LG, FINEGOLD SM: Nonsporulating anaerobic

bacteria in osteomyelitis. Antimicrob Agents Chemother 7:77-85, 1968

Louie etal. • Bacteria in Diabetic Foot Ulcers

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Aerobic and anaerobic bacteria in diabetic foot ulcers.

Aerobic and Anaerobic Bacteria in Diabetic Foot Ulcers THOMAS J. LOUIE, M.D.; JOHN G. BARTLETT, M.D.; FRANCIS P. TALLY, M.D.; and SHERWOOD L. GORBACH,...
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