Synergy in polymicrobial infections in a mouse model of type 2 diabetes

Abstract

Human diabetics frequently suffer delayed wound healing, increased susceptibility to localized and systemic infections, and limb amputations as a consequence of the disease. Lower-limb infections in diabetic patients are most often polymicrobial, involving mixtures of aerobic, facultative anaerobic, and anaerobic bacteria. The purpose of this study is to determine if these organisms contribute to synergy in polymicrobial infections by using diabetic mice as an in vivo model. The model was the obese diabetic mouse strain BKS.Cg-m +/+ Lepr(db)/J, a model of human type 2 diabetes. Young (5- to 6-week-old) prediabetic mice and aged (23- to 24-week-old) diabetic mice were compared. The mice were injected subcutaneously with mixed cultures containing Escherichia coli, Bacteroides fragilis, and Clostridium perfringens. Progression of the infection (usually abscess formation) was monitored by examining mice for bacterial populations and numbers of white blood cells at 1, 8, and 22 days postinfection. Synergy in the mixed infections was defined as a statistically significant increase in the number of bacteria at the site of injection when coinfected with a second bacterium, compared to when the bacterium was inoculated alone. E. coli provided strong synergy to B. fragilis but not to C. perfringens. C. perfringens and B. fragilis provided moderate synergy to each other but only in young mice. B. fragilis was anergistic (antagonistic) to E. coli in coinfections in young mice at 22 days postinfection. When age-matched nondiabetic mice (C57BLKS/J) were used as controls, the diabetic mice exhibited 5 to 35 times the number of CFU as did the nondiabetic mice, indicating that diabetes was a significant factor in the severity of the polymicrobial infections.

FIG. 1.

Graph showing the mean values of serum glucose levels (open squares) and masses (open circles) with increasing age in diabetic BKS.Cg-m +/+ Lepr^db/J mice. Also shown are the serum glucose levels (filled squares) and masses (filled circles) of the young and aged nondiabetic C57BLKS/J mice. Blood glucose levels above 200 mg/dl (horizontal bar) were considered evidence of hyperglycemia.

FIG. 2.

Number of CFU isolated from abscesses in young (5- to 6-week-old), prediabetic BKS.Cg-m +/+ Lepr^db/J mice. One hundred-microliter samples of bacterial suspensions (2 × 10⁵ to 3 × 10⁶ CFU), singly or in combination, were injected subcutaneously into the inner thighs of the mice, as described in Materials and Methods. Control mice (data not shown) were injected with PBS alone. Panels A to G correspond to the infection protocols listed in Table 2 as experimental groups 1 to 7, respectively. For panels A to C, the numbers of CFU for individual mice (squares) are shown along with the mean values for E. coli (panel A, circles), B. fragilis (panel B, triangles), and C. perfringens (panel C, diamonds). In panels D to G, only the mean values are shown, with the same symbols used in panels A to C. The horizontal lines represent the lower limit of detection; for some panels, levels less than the lower limit are shown to illustrate the mean calculated values. B. fra, B. fragilis; C. per, C. perfringens.

FIG. 3.

Number of CFU isolated from abscesses in aged (23- to 24-week-old), diabetic BKS.Cg-m +/+ Lepr^db/J mice. The symbols used are described in the legend for Fig. 2.

FIG. 4.

Representative image showing a lesion with granulomatous characteristics from an infected diabetic mouse. A 5-μm-thick section from an abscess area of a BKS.Cg-m +/+ Lepr^db/J mouse was stained with hematoxylin and eosin. The young (5- to 6-week-old) mouse was infected with a combination of E. coli and B. fragilis and euthanized 22 days postinfection, when the area around the lesion was removed and processed to obtain the section shown. Prominent in the figure are giant cells (arrows), epithelioid cells (arrowheads), and high levels of infiltrating polymorphonuclear leukocytes, macrophages, and lymphoid cells (area inside the black boundary line).