Diabetic Feet. Western Society for Clinical Research Annual. 72 h. These cotton swabs were then transferred to

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980, p Vol. 12, No /80/ /08$02.00/0 Quantitative Aerobic and Anaerobic Bacteriology of Infected Diabetic Feet FRANCISCO L. SAPICO, 2* HANNA N. CANAWATI,23 JOYCE L. WITTE,' JOHN Z. MONTGOMERIE,12 F. WILLIAM WAGNER, JR.,24 A ALICE N. BESSMAN'2 Departments ofmedicine,' Pathology,3 and Surgery,4 Rancho Los Amigos Hospital, Downey, California 90242, and School ofmedicine, University of Southern California, Los Angeles, California Quantitative aerobic and anaerobic cultures of deep tissue were performed on amputated infected lower limbs of 13 diabetic patients immediately after surgery. Dissection was made through intact skin distant from any preexisting ulcer. The results were compared with those obtained from: (i) ulcer swabs (pre- and postamputation), (ii) curettage of the ulcer base, and (iii) needle aspiration after normal saline injection. Anaerobic transport media were used for anaerobic cultures before prompt transfer to the anaerobic chamber. A mean of 4.7 bacterial species per specimen was seen (2.3 aerobes, 2.4 anaerobes). Mean logo growth per gram of tissue was as follows: (i) aerobes plus anaerobes = 6.99, (ii) aerobes = 6.42, and (iii) anaerobes = There was poor concordance between the deep tissue culture results and the results from other modalities of culture collection, though the results from other modalities of culture collection, though the results from curettage and saline aspiration were slightly better than those obtained from ulcer swabs. The most frequently isolated organisms were enterococci, anaerobic streptococci, and species of Proteus, Clostridium, and Bacteroides. When antimicrobial therapy is indicated for this patient population, the possibility of the concomitant presence of aerobes as well as anaerobes should be considered. Several microorganisms have been implicated in the bacterial etiology of infected diabetic feet. Staphylococci, aerobic streptococci, and gramnegative aerobic bacilli have been frequently cited as etiological agents in this particular clinical setting (2, 16, 19, 20, 23). Recent studies, using more sophisticated anaerobic bacteriology techniques, have revealed the presence of a variety of anaerobic bacteria in diabetic foot ulcers as well as in the adjacent osteomyelitic bones (4, 8, 18; M. M. Henry, L. J. Wheat, S. D. Allen, J. A. Siders, and A. White, Abstr. Annu. Meet. Am. Soc. Mocrobiol. 1979, C218, p. 346). These studies, however, were made with specimens that were prone to surface contamination, and none of the studies quantitated bacterial density in the infected tissues. We studied the deep tissue bacteriology of 13 patients with diabetic foot infections at Rancho Los Amigos Hospital, Downey, Calif. Quantitation of bacterial growth per gram of tissue was done on specimens obtained immediately after surgical amputation. These tissue specimens were obtained from necrotic areas away from the open ulcer and by incision through disinfected intact skin. The results of these cultures were then compared with those obtained by the more commonly used modalities of culture collection. (This work was presented in part before the Western Society for Clinical Research Annual Meeting, Carmel-by-the-Sea, Calif., 7 February 1980.) MATERIALS A METHODS Diabetic patients who had already been scheduled for lower limb amputations were included in the study. Informed consents were obtained. Blood cultures were drawn before and during surgery. Proximal stump cultures were obtained before surgical closure, using sterile cotton swabs previously stored in loosely capped tubes inside an anaerobic chamber for at least 72 h. These cotton swabs were then transferred to sterile stoppered tubes before removal from the chamber. Immediately after surgery, the amputated extremity was brought to a room adjacent to the microbiology laboratory for dissection. Specimens for culture were then obtained, using the following methods. (i) The base of any preexisting ulcer was swabbed with anaerobic sterile cotton swabs. Swabs of the ulcer were taken before surgery and before preoperative disinfection in some patients. (ii) The base of the ulcer was then vigorously scrubbed with sterile gauze moistened with sterile, nonbacteriostatic normal saline solution to remove overlying debris. The base of the ulcer was then scraped with the edge of a sterile curette for culture. (iii) An area which demonstrated bogginess or fluctuance and which was not in direct communication with any ulcer was chosen for needle aspiration. The 413

2 414 SAPICO ET AL. skin overlying this area was disinfected with an iodophor solution and 70% alcohol, after which aspiration with needle and syringe was attempted. If no pus was aspirated, 4 to 6 ml of sterile nonbacteriostatic saline was then injected and aspirated for cultures. (iv) After disinfection of the overlying skin as in procedure iii, a gangrenous or grossly infected area was dissected, using sterile instruments. Areas in direct communication with an ulcer were avoided. A strip of necrotic material (fat, muscle, or connective tissue) weighing 0.1 to 0.5 g was excised for quantitative tissue culture. Ail specimens for culture were placed in anaerobic transport medium (Anatrans; Carr-Scarborough Microbiologicals, Inc., Stone Mountain, Ga.) immediately after collection. These specimens were then transferred to an anaerobic chamber for processing. The interval between culture collection and transfer to the chamber did not exceed 20 min. Plates for specimens other than deep tissue were streaked in a standard manner that allowed a semiquantitative estimate of the bacteria cultured. Tissue specimens were weighed, diluted with 9 ml of 0.05% yeast extract solution (previously boiled, capped, and stored in the anaerobic chamber), and ground with a mortar and pestle until a reasonably homogenous suspension was obtained. Serial 10-fold dilutions to 10-9 were made with 0.05% yeast extract solution. A hockey stick-shaped sterile glass rod was used to spread 0.1 ml of each dilution evenly over the surface of the agar plate used. Gram stains of the original specimens were routinely performed. For culture of aerobes and facultative strains, 5% sheep blood agar, MacConkey agar, Columbia colistinnalidixic acid agar, and chocolate agar were used. The media for anaerobes included Brucella blood agar with 10,g of menadione per ml, phenylethyl alcohol-blood agar, and laked blood agar with 75,g of kanamycin and 7.5,ug of vancomycin per ml (Clinical Standards, Torrance, Calif.). Original specimens were also inoculated into chopped meat-glucose broth. Aerobic plates were incubated at. 37 C with 5% C02 or in air for 7 days and were inspected daily. Anaerobic plates were incubated at 37 C inside the anaerobic chamber for 10 to 14 days and were also inspected daily. Anaerobic bacteria were identified according to the procedures outlined in the (VPI) Anaerobe Laboratory Manual (7), Enterobacteriaceae were identified by the methods of Edwards and Ewing (3), and nonfermentative bacilli were identified by the method of Pickett and Pedersen (14). Gas-liquid chromatographic studies were performed for anaerobic strains according to the VPI criteria (7). J. CLIN. MICROBIOL. RESULTS Clinical characteristics of the patient population. Thirteen patients were included in the study. All of these patients were diabetics: nine were insulin dependent, and four were diet controlled. Eight patients had had diabetes mellitus for 5 years or longer. No patient was in diabetic ketosis on admission. Eight of the patients were males, and five were females. Twelve of thirteen patients had demonstrable peripheral vascular disease, and six had renal insufficiency (mild in five). Two patients had gangrene without ulcers (one was foul smelling), six had gangrene with ulcers (four were foul smelling), three had ulcers without gangrene (one was foul smelling), one had a nonhealing previous amputation site, and the last patient had foot burns associated with cellulitis. Fever was present in three patients (one with associated history of chills). Five patients had anemia (hematocrit, <35% [vol/vol] blood cells; hemoglobin, <12% [wt/wt]). Six patients had leukocytosis leukocytess, >12,000/mm3) with shift to the left. The erythrocyte sedimentation rates were elevated in all four patients upon whom the procedure was performed (>20 mm/ h, Westergren method). Osteomyelitis of contiguous bones was diagnosed by radiography in six of seven patients who had X rays done. Only one patient had received no antibiotics for at least 2 weeks prior to amputation. Seven patients were receiving one antibiotic each, two were receiving two antibiotics each, three were receiving three antibiotics each, and one was receiving four antibiotics at the time of surgery. The mean duration of antibiotic therapy was 7.8 days (range, 1 to 21 days). Cephalosporins were the most frequently used antibiotics (nine patients), followed by ampicillin (three patients), aminoglycosides (three patients), penicillinaseresistant penicillins (three patients), and penicillin G, clindamycin, chloramphenicol, tetracycline, and vancomycin (one patient each). All but 2 of the 13 patients had been receiving local applications of an iodophor solution to the wounds before surgery. One patient received local hexachlorophene washes, and another received normal saline wet-to-dry dressings. The level of amputation was determined with the help of Doppler ultrasound studies. The types of surgical amputation performed were as follows: (i) below the knee, four; (ii) Symes operation (through the ankle), four; (iii) through the knee, four; (iv) transmetatarsal, two; and (v) great toe amputation, 1. Bacteriological results. Blood cultures preand intraoperatively were sterile in all the nine patients from whom the cultures were drawn. Proximal stump cultures were likewise sterile in these patients. Table 1 shows the numbers of strains isolated per patient according to the different modalities of culture collection used. A total of 4.7 strains per specimen were isolated from deep tissue (range, 3 to 8). An almost equal number of anaerobic and aerobic strains were isolated (2.4,

3 VOL. 12, 1980 TABLE 1. Number of bacterial strains isolated per specimen No. of strains per speci- Type of specimen No. of patients men Aerobes Aonbes Total Ulcer swab, preamputation Ulcer swab, il postamputation Curettage il Saline aspirate Deep tissue TABLE 2. Quantitative bacterial counts of deep tissue specimens in 12 patients No. isolated (mean Type isolated logo/g of tissue ± standard error) Aerobes and anaerobes ± 0.30 Aerobes Anaerobes ± 0.38 Aerobic microorganisms isolated from TABLE 3. deep tissue No. No. Group or species isof Group or species of lates lates Gram-negative bacilli 16 Gram-positive cocci 11 Proteus mirabilis 4 Staphylococcus P. rettgeri... 1 aureus... 3 Escherichia coli.. 3 S. epidermidis... 1 Enterobacter Group D aerogenes... 2 Streptococcus 6 E. cloacae... 1 Enterococcus 5 Klebsiella Nonpneumoniae... 1 enterococcus 1 Serratia Group B marcescens... 1 Streptococcus 1 Citrobacter Gram-positive bacilli 2 freundii... 1 Corynebacterium Pseudomonas sp... 1 aeruginosa... 1 Bacillus Alcaligenes odor- sphaericus... 1 ans (A. faecalis) 1 Fungi 3 Gram-negative cocci 1 Candida albicans 1 Neisseria C. tropicalis... 1 meningitidis... 1 C. parapsilosis... 1 with range of O to 5, for anaerobes and 2.3, with a range of O to 4, for aerobes) from deep tissue. More anaerobic bacterial strains were isolated from deep tissue than from other sites cultured. No aerobic bacteria were isolated from 2 of the BACTERIOLOGY OF DIABETIC GANGRENE 415 TABLE 4. Anaerobic bacteria isolated from deep tissue No. No. Group or species isof Group or species of lates lates Gram-negative bacilli 11 magnus... 3 Bacteroides P. fragilis... 2 asaccharolyticus 1 B. P. prevotii. 1 melaninogenicus 1 Peptostreptococcus B. anaerobius... 2 asaccharolyticus 1 B. ureolyticus... 1 Gram-positive bacilli 13 B. capillosus... 1 Clostridium... 1 sordelii... 2 B. multiacidus Fusobacterium C. perfringens 1 mortiferum... 1 C. sporogenes... 1 F. nucleatum... 1 C. cadaveris... 1 Unidentified bacil- C. ghoni. 1 lus (failed to C. clostridiiforme 1 grow on Propionibacterium subculture) 1 acnes.. 2 Lactobacillus sp. 1 Gram-positive cocci 1 Actinomyces Peptococcus viscosus... 1 TABLE 5. Concordance' with deep tissue culture results No. of specimens Sampling modality with positive concordance/no. done Aerobes and anaerobes Preamputation ulcer swab... 1/8 Postamputation ulcer swab... 2/11 Curettage... 5/11 Needle aspiration... 5/13 Aerobes Preamputation ulcer swab... 2/8 Postamputation ulcer swab... 6/11 Curettage... 7/11 Needle aspiration... 9/13 Anaerobes Preamputation ulcer swab... 3/8 Postamputation ulcer swab... 5/11 Curettage... 7/11 Needle aspiration /13 <"Concordance" was defined as "complete qualitative agreement in the bacteriological results." 13 patients, although yeasts were isolated aerobically from 1 of these patients. Two patients had no anaerobic bacteria isolated from deep tissue. Table 2 shows bacterial growth density per gram of deep tissue. Bacterial growth, in general, was quite heavy (mean logo per gram = 6.99). Anaerobic bacterial growth was heavier than that of aerobes (logio 7.5 versus log1o 6.42, re-

4 416 SAPICO ET AL. J. CLIN. MICROBIOL. TABLE 6. Patient Aerobic microorganisms isolated by using different culture collection techniques from 13 infected diabetic feet Microorganisms isolated by: no. Preamputation ulcer Postamputation ul- Curettage Saline aspiration Deep tissue swab cer swab 1 ` K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa E. cloacae E. cloacae 2 S. aureus S. aureus S. aureus S. aureus P. mirabilis P. mirabilis P. mirabilis P. mirabilis C. freundii C. freundii C. freundii C. freundii 3be S. marcescens S. marcescens Group B beta-he- Group B beta-hemolytic Strep- molytic Streptococcus tococcus Group D Strepto- Group D Streptococcus (entero- coccus (enterococcus) coccus) N. meningitidis N. meningitidis 4h S. aureus S. aureus Group D Strepto- Group D Streptococcus (entero- coccus (enterococcus) coccus) 5 E. aerogenes E. aerogenes E. aerogenes E. aerogenes Group D Strepto- Group D Strepto- Group D Strepto- Group D Streptococcus (entero- coccus (entero- coccus (entero- coccus (enterococcus) coccus) coccus) coccus) Corynebacterium Corynebacterium Corynebacterium Corynebacterium sp. sp. sp. sp. 6 P. rettgeri P. rettgeri P. rettgeri P. rettgeri P. rettgeri A. odorans A. odorans A. odorans A. odorans A. odorans E. aerogenes E. aerogenes E. aerogenes E. aerogenes E. aerogenes S. epidermidis 7 P. mirabilis P. mirabilis P. mirabilis P. mirabilis P. mirabilis Group D Strepto- Group D Strepto- Group D Strepto- Group D Strepto- Group D Streptococcus (entero- coccus (entero- coccus (entero- coccus (entero- coccus (enterococcus) coccus) coccus) coccus) coccus) S. epidermidis S. epidermidis S. epidermidis 8 S. aureus S. aureus S. aureus S. aureus S. aureus E. coli E. coli E. coli E. coli 9 S. aureus Group D Strepto- S. aureus Group D Strepto- Group D Strepto- Group D Strepto- coccus (entero- Group D Strepto- coccus (entero- coccus (enterococcus (entero- coccus) coccus (entero- coccus) coccus) coccus) Group D Strepto- coccus) Group D Strepto- Group D Strepto- Group D Strepto- coccus (non-en- Group D Strepto- coccus (non-en- coccus (non-encoccus (non-en- terococcus) coccus (non-en- terococcus) terococcus) terococcus) terococcus) 10 E. coli E. coli E. coli E. coli P. mirabilis P. mirabilis P. mirabilis P. mirabilis S. epidermidis S. epidermidis

5 VOL. 12, 1980 BACTERIOLOGY OF DIABETIC GANGRENE 417 TABLE 6. Continued Microorganisms isolated by: Patient no. Preamputation ulcer Postamputation ulswab cer swab Curettage Saline aspiration Deep tissue 11 E. coli E. coli E. coli E. coli E. coli P. mirabilis P. mirabilis P. mirabilis P. mirabilis P. mirabilis B. sphaericus B. sphaericus B. sphaericus B. sphaericus B. sphaericus C. parapsilosis C. parapsilosis C. parapsilosis S. epidermidis C. albicans C. albicans C. tropicalis C. albicans Group D Strepto- C. tropicalis C. tropicalis C. tropicalis coccus (enterococcus) C. albicans "', Not done. h Infected diabetic feet of patients 3 and 4 had no ulcer present. spectively). One of the 13 specimens produced no growth when cultured on solid agar, but five different anaerobic species were isolated from the deep tissue specimen that was cultured in chopped meat-glucose broth. Bacterial quantitation could not be performed on this specimen. All the six patients with foul-smelling lesions yielded at least one anaerobe on culture. Thirty-one strains of anaerobic bacteria and 30 strains of aerobic bacteria were isolated from the 13 deep tissue specimens (Tables 3 and 4). Two specimens, in addition, grew three species of fungi: one specimen grew both Candida albicans and Candida tropicalis, and a second specimen grew Candida parapsilosis. The concordance of the results of deep tissue culture with those obtained from (i) preamputation ulcer swab, (ii) postamputation (and postpreoperative disinfection) ulcer swab, (iii) curettage, and (iv) needle aspiration (with saline) was examined (Table 5). There was generally poor concordance of all the modalities of culture collection tested with the deep tissue culture results. However, the results from curettage and from needle aspiration appeared to have slightly better concordance with deep tissue culture results when compared with the results obtained from ulcer swabs. Tables 6 and 7 show the microbiological data obtained from the different modalities of culture collection used in this study. DISCUSSION The importance of Staphylococcus aureus, Proteus sp., enterococci, and aerobic gram-negative bacilli in the septic complications of infected diabetic feet has been emphasized in previous literature (2, 16, 19, 20, 23). The role of anaerobic bacteria in this clinical setting, however, has not been generally recognized. Previous studies have been hampered by the failure to use techniques in specimen collection, specimen transport, and culture that would ensure the isolation of obligately anaerobic bacteria. As early as 1939, however, Meleney stressed the importance of anaerobes in infected diabetic foot ulcers (11). In 1943, Weiss reported an infected stump in a diabetic from which Bacteroides melaninogenicus and anaerobic diphtheroids were isolated together with Proteus and hemolytic streptococci (21). Ziment et al., in 1968, reported diabetic patients with infected feet and osteomyelitis from which a variety of anaerobes were isolated (24). Since then, other publications have similarly implicated anaerobic bacteria in this disease process (4, 8, 15, 18; Henry et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, C218, p. 346). The adequacy and reliability of taking bacteriological cultures directly from the ulcer itself, however, can be questioned. It would be difficult to separate bacteria colonizing the surface from tissue pathogens by using swab specimens of the infected ulcer. Louie et al. used curretted tissue obtained from the ulcer base after a vigorous saline scrub (8). However, they did not compare results obtained from this technique with those obtained from swab specimens. Our study shows poor concordance of results obtained from ulcer swabs with those obtained from uncontaminated deep tissue specimens (Table 5). Previous studies from this institution have shown similarly poor concordance (19, 20). Preoperative disinfection did not appear to alter ulcer culture results. Slightly better concordance with deep tissue results was obtained from cur-

6 418 SAPICO ET AL. J. CLIN. MICROBIOL. TABLE 7. Anaerobic microorganisms isolated by using different culture collection techniques from 13 infected diabetic feet Microorganisms isolated by: Patient no. Preamputation ulcer swab swab Postamputation ul- cer swab ~~~~~Curettage Saline aspiration Deep tissue 1 a p. prevotii P. prevotii P. prevotii P. prevotii 2 3b 4b P. asaccharolyticus 8 9 C. sporogenes 10 C. ghoni C. ghoni B. capillosus B. capillosus C. cadaveris il C. perfringens C. perfringens F. mortiferum 12C F. nucleatum 13 Unidentified gram-negative bacilus a, Not done. F. nucleatum C. clostridiiforme C. sporogenes B. ureolyticus C. ghoni B. capillosus C. cadaveris C. perfringens F. mortiferum B. melaninogenicus Unidentified gram-negative bacillus b Infected diabetic feet of patients 3 and 4 had no ulcer present. 'Growth in broth medium only. Lactobacillus sp. C. sporogenes C. perfringens F. mortiferum B. melaninogenicus Lactobacillus sp. P. anaerobius P. asaccharolyticus C. sporogenes B. multiacidus B. ureolyticus P. anaerobius C. ghoni C. ghoni B. capillosus C. cadaveris C. perfringens F. mortiferum B. melaninogenicus F. nucleatum F. nucleatum F. nucleatum C. clostridiiformis A. viscosus Unidentified gram-negative bacillus Unidentified gram-negative bacillus

7 VOL. 12, 1980 rettage specimens and needle aspirates (Table 5). Our results may have been influenced to some extent by the fact that all patients but one had been receiving systemic antibiotic therapy and local antibacterial therapy immediately before surgery. Antibiotic susceptibility studies were not routinely performed on all deep tissue isolates. Aerobic isolates from surface swabs of seven patients, however, were tested for antibiotic susceptibility by the Food and Drug Administration standardized disk diffusion method (13a). Ail seven patients yielded at least one organism (simultaneously isolated from deep tissue) that was susceptible to at least one antibiotic that the patient was receiving. Heavy growths of anaerobes were seen in the deep tissue of one patient receiving chloramphenicol and in that of another receiving clindamycin. These suggest that the ability to isolate susceptible bacteria in infected diabetic feet was not significantly impaired by concomitant antibiotic treatment. Five different anaerobes and two aerobes were isolated from the deep tissue of the only patient who did not receive any systemic antibiotic immediately before surgery. Local antibacterial therapy may also account for the observation that ulcer swabs did not yield more bacterial strains than did deep tissue specimens. Despite antibiotic therapy, bacterial growth in the infected deep tissue was heavy (Table 2). A noteworthy finding was that anaerobes out- BACTERIOLOGY OF DIABETIC GANGRENE 419 numbered the aerobes by more than 10-fold. This finding further emphasizes the role of anaerobes in diabetic foot infections. Among the aerobes, gram-negative bacilli and enterococci were the most commonly isolated (Table 3). S. aureus was isolated only three times. A curious finding was the isolation of Neisseria meningitidis in the deep tissue of one of the patients. We are not aware of any similar isolation of this organism in infected diabetic feet in the past. The most commonly isolated anaerobes from deep tissue were gram-negative bacilli (11 strains), followed closely by clostridia (9 strains), and anaerobic cocci (7 strains). Bacteroides fragilis was isolated only twice, although other species of Bacteroides were isolated six times. The observation that more anaerobes were isolated from deep tissue than from other culture sources could be explained by the following possibilities: (i) local antibacterial therapy may have killed some of the anaerobes on the surface, (ii) saline injection may have inhibited the growth of some anaerobic bacteria (in the case of saline aspiration), or (iii) the conditions for growth in the deep tissue are more favorable for anaerobic bacteria (i.e., lowered oxidation-reduction potential). Only aerobic bacteria were isolated from two patients, and only anaerobic bacteria were isolated from another two. C. albicans and C. tropicalis, however, were isolated together with the anaerobes in one patient. That diabetic foot infections are polymicrobial in etiology is emphasized by our results: deep tissue cultures yielded a mean of 4.7 species per specimen. Some investigators have alluded to the possible interbacterial synergistic dependence among aerobes and anaerobes in various clincial and experimental infections (1, 5, 9, 10, 12, 13, 17, 22). Our results show that foot infections in diabetics are often polymicrobial in etiology and that anaerobic bacteria are found at least as commonly as aerobic bacteria and with comparable density of bacterial growth in uncontaminated deep tissue. Therefore, when the decision is made to use antimicrobial agents for sepsis, cellulitis, or osteomyelitis associated with infected diabetic feet, initial therapy should be directed at aerobic as well as anaerobic bacteria. ACKNOWLEDGMENT This work was supported by a grant from the Weingart Foundation, Los Angeles, Calif. LITERATURE CITED 1. Altemier, W. A The pathogenicity of the bacteria of appendicitis peritonitis. Surgery 11: Bessman, A. N., and F. W. Wagner, Jr Nonclostridial gas gangrene. J. Am. Med. Assoc. 233: Edwards, P. R., and W. H. Ewing Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. 4. Fierer, J., D. Daniel, and C. Davis The fetid food; lower extremity infections in patients with diabetes mellitus. Rev. Infect. Dis. 1: Fildes, P Tetanus. IX. The oxidation-reduction potential of the subcutaneous tissue fluid of the guinea pig: its effect on infection. Br. J. Exp. Pathol. 10: Finegold, S. 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8 420 SAPICO ET AL. perimental infections with anaerobic streptococci. J. Infect. Dis. 103: a.National Committee for Clinical Laboratory Standards Approved standard: ASM-2. Performance standards for antimicrobial susceptibility tests. National Committee for Clinical Laboratory Standards, Villanova, Pa. 14. Pickett, M. J., and M. M. Pedersen Nonfermentative bacilli associated with man. II. Detection and identification. Am. J. Clin. Pathol. 54: Pien, F. D., R. L. Thompson, and W. J. Martin Clinical and bacteriologic studies of anaerobic grampositive cocci. Mayo Clin. Proc. 47: Pratt, T. C Gangrene and infection in the diabetic. Med. Clin. North Am. 49: Roberts, D. S Synergistic mechanisms in certain mixed infections. J. Infect. Dis. 120: Sanderson, P. J Infection of the foot with Peptococcus magnus. J. Clin. Pathol. 30: Sharp, C. S., A. N. Bessman, F. W. Wagner, Jr., and J. CLIN. MICROBIOL. D. Garland Microbiology of deep tissue in diabetic gangrene. Diabetes Care 1: Sharp, C. S., A. N. Bessman, F. W. Wagner, Jr., D. Garland, and E. Reece Microbiology of superficial and deep tissues in infected diabetic gangrene. Surg. Gynecol. Obstet. 149: Weiss, C The pathogenicity of Bacteroides melaninogenicus and its importance in surgical infections. Surgery 13: Whipp, S. C., I. M. Robinson, D. L. Harris, R. D. Glock, P. J. Matthews, and T. J. L. Alexander Pathogenic synergism between Treponema hyodysenteriae and other selected anaerobes in gnotobiotic pigs. Infect. Immun. 26: Zierold, A. A Gangrene of the extremity in the diabetic. Ann. Surg. 110: Ziment, I., L. G. Miller, and S. M. Finegold Nonsporulating anaerobic bacteria in osteomyelitis, p Antimicrob. Agents Chemother Downloaded from on September 15, 2018 by guest