Polymicrobial Infection in Mice

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1993, P. 1531-1535 0066-4804/93/071531-05$02.00/0 Copyright 1993, American Society for Microbiology Vol. 37, No. 7 Use of Cephalosporins for Prophylaxis and Therapy of Polymicrobial Infection in Mice ITZHAK BROOK Naval Medical Research Institute, Bethesda, Maryland 20814-5055 Received 29 January 1993/Accepted 28 April 1993 Cefoxitin, cefotetan, and cefmetazole were compared in 10-day therapy of intra-abdominal and subcutaneous infections caused by three organisms: Bacteroidesfragilis and Bacteroides thetaiotaomicron combined with either Escherichia colt or Staphylococcus aureus. Intra-abdominal infection was caused by B. fragilis plus B. thetaiotaomicron plus E. coil. Therapy was initiated immediately before inoculation or was delayed for 8 h. Mortality was 14 of 30 (47%) for saline-treated mice, and all survivors developed abscesses. Immediate therapy reduced mortality and the percentage of mice with abscesses (in survivors), respectively, to 17 and 20%o with cefoxitin, 0 and 13% with cefotetan, and 0 and 17% with cefmetazole, and the numbers of all bacteria were reduced by all the cephalosporins. Delayed therapy reduced mortality and abscess formation, respectively, to 20 and 8% of mice with cefoxitin, 10 and 93% with cefotetan, and 7 and 96% with cefmetazole. B. thetaiotaomicron survived in all abscesses treated with cefotetan and cefmetazole. Subcutaneous abscesses were caused by each organism alone or in combinations of one aerobe (S. aureus or E. coli) and one or two Bactetoides species. Early therapy reduced the numbers of all bacteria independent of their in vitro susceptibility. All agents reduced the number of each Bacteroides species with either E. coli or S. aureus. However, when therapy was delayed, cefotetan and cefmetazole were less effective than cefoxitin against B. thetaiotaomicron. Cefotetan was the most active agent against E. coli, and cefmetazole was the most effective against S. aureus. These data illustrate the efficacy of all tested cephalosporins in the prophylaxis of polymicrobial infections. Bacteroides fragilis group organisms mixed with Escherichia coli are frequently isolated from intra-abdominal (i.a.) infections that originate from the gut flora (5, 15). These organisms mixed with Staphylococcus aureus are often recovered from skin and soft tissue infections around rectalvulvovaginal and oral areas (8). The B. fragilis group consists of several subspecies that were elevated to full species level (11). The rates of recovery of the different species of the B. fragilis group differ for various infection sites. The distribution ofb. fragilis group species can affect the management of these infections because of the different antimicrobial susceptibilities of the various group members. B. fragilis group organisms produce a potent cephalosporinase, and their susceptibilities to expanded- and broad-spectrum cephalosporins are not uniform (1). B. fragilis is the most susceptible, while Bacteroides thetaiotaomicron and other B. fragilis group members are more resistant (1, 12, 14). The purpose of this study was to evaluate several expanded-spectrum cephalosporins advocated as single-agent therapy for the prophylaxis and management of polymicrobial infections. Their abilities to prevent and treat mixed infections caused by two members of the B. fragilis group and either E. coli or S. aureus were tested in i.a. and subcutaneous (s.c.) abscess models in mice. In contrast to previous studies in which combinations of two isolates (a B. fragilis isolate plus E. coli or S. aureus) were used, this study employed inoculation of three organisms, which more truly simulates the polymicrobial infection that occurs in patients. (This study was presented in part at the First North American Congress on Anaerobic Bacteria and Anaerobic Infections, Marina Del Ray, Calif., 25 July 1992.) 1531 MATERIALS AND METHODS The experiments reported herein were conducted according to the principles set forth in Guide for the Care and Use of Laboratory Animals (1Sa). Organisms. All aerobic and anaerobic bacteria used in the experiments were recent clinical isolates. These included one isolate each of B. fragilis (NMRI isolate 32), B. thetaiotaomicron (NMRI isolate 54), E. coli (NMRI isolate 8), and S. aureus (NMRI isolate 7). The bacteria were kept frozen in skim milk at -70 C. They were identified by standard criteria (18, 22) and processed as previously described (10). All Bacteroides species were encapsulated (>50% of the organisms had capsules). The encapsulated form was induced by passage of the nonencapsulated form mixed with Kebsiella pneumoniae in s.c. abscesses in mice (17). The presumptive identification of a capsule was established by microscopic examinations only, by electron microscopy after staining with ruthenium red (17), which demonstrated a homogeneous polysaccharide capsule that was external to the cell wall. Bacterial suspensions of organisms for inoculation were prepared as previously described (7). Numbers of CFU were determined by plate count with brain heart infusion agar enriched with vitamin K1 and hemin to support the growth of the Bacteroides species. Animals. Male Swiss albino mice weighing 20 to 25 g each were obtained from the Institute's mouse colony. The mice were raised under conventional conditions. Abscess induction. Abscesses were induced as previously described (10). For induction of i.a. abscesses, mice received intraperitoneally a mixture of 0.1 ml of each bacterial suspension in saline (containing 103 live E. coli cells and 105 live cells of each of the two B. fragilis group isolates) and

1532 BROOK 0.05 ml of sterile human fecal adjuvant solution (total volume, 0.35 ml). For induction of s.c. abscesses, mice were inoculated s.c. in the right groin with 0.1 ml of each appropriate bacterial suspension in saline solution containing 10 of each organism. Cultivation of abscesses. (i) i.a. abscesses. Ten days after abscess induction, the mice were killed by cervical dislocation. For each mouse, the abdomen was sterilely prepared, and a t-shaped incision was used to thoroughly explore the abdomen and pelvis. All i.a. abscesses >2 mm in diameter were excised, placed in prereduced saline, and weighed together for each mouse. (ii) s.c. abscesses. Mice were killed 5 days after inoculation, and the s.c. abscess material was aseptically removed. The number of bacteria in each abscess was determined individually. Quantitation of bacteria. Both i.a. and s.c. abscesses were minced, and the homogenate was serially diluted and quantitatively plated aerobically on McConkey's and blood agars and anaerobically on brain heart infusion-enriched blood agar plates. Selective media containing cefotetan were used to quantitatively determine the numbers of the two phenotypically similar Bacteroides species. Growth was evaluated 24 and 48 h after plating. No attempt was made to inactivate the antimicrobial agents in the homogenized abscess material, since a considerable dilution was achieved before plating. Colonies were counted after incubation at 37 C in an anaerobic chamber for 48 h, and the results were presented as log1o of the viable bacteria per peritoneal cavity (for i.a. abscesses) or per abscess (for s.c. abscesses). The lowest detectable number of organisms per 1 ml of pus was 10. This number was used as a zero value for statistical analysis. Antimicrobial therapy. Doses of antimicrobial agents were selected on the basis of pharmacokinetic studies of uninfected animals. Levels of the antibiotics in serum were measured by agar diffusion assay (18) with Bacillus subtilis ATCC 12432 at 30, 60, 120, and 180 min and at 8 and 12 h after administration of the last dose of antibiotics. Antimicrobial therapy was started in half of the mice 1 h before inoculation of bacteria and in the other half 8 h after bacterial inoculation. The daily doses of cephalosporins were given intramuscularly, diluted in 0.1 ml of saline, in divided doses every 12 h. The control group received 0.1 ml of saline in divided doses every 12 h. The antimicrobial agents used were cefmetazole (Upjohn Company, Kalamazoo, Mich.), cefoxitin (Merck Sharp & Dohme, West Point, Pa.), and cefotetan (Stuart Pharmaceuticals, Wilmington, Del.). Susceptibility tests. The MICs of the test antibiotics against the bacterial isolates were determined by the agar dilution method (18, 22). Experimental design. To determine the effects of prophylactic and therapeutic use of the cephalosporins on the various organisms in i.a. mixed infections, E. coli was inoculated in combination with the two B. fragilis group members. To determine the effect of therapy on s.c. infection, E. coli and S. aureus were inoculated alone or in combination with one or both B. fragilis group isolates. Therefore, in the first set of i.a. abscess experiments, single organisms were inoculated; in the second set of experiments, two organisms were inoculated (E. coli or S. aureus and one of the two Bacteroides species); and in the third set of experiments, three organisms were injected (E. coli or S. aureus and both of the Bacteroides species). Groups of 10 mice were treated with saline or with one of ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. In vitro susceptibilities of organisms (Og/ml) MIC Cefoxitin Cefotetan Cefmetazole B. fragilis 8 8 8 B. thetaiotaomicron 16 64 64 E. coli 2 0.5 0.5 S. aureus 4.0 16 2.0 the three antimicrobial agents. Each i.a. experiment was done three times, and each s.c. experiment was done twice. Statistical analysis was done by using Student's t test of independent means and the chi-square test. RESULTS In vitro susceptibility to antibiotics. B. fragilis was susceptible to cefoxitin, cefotetan, and cefmetazole, but B. thetaiotaomicron was resistant to the last two. E. coli and S. aureus were susceptible to all agents (Table 1). Antibiotic concentrations in serum. The concentrations of antimicrobial agents in uninfected animals were determined. The peak concentrations in serum were at least four times higher than the MICs for all susceptible bacteria (Table 2). Mortality. Mortality occurred within the first 5 days after inoculation. After i.a. inoculation, mortality in control animals was 47% (14 of 30 mice) (Table 3). Mortality in animals starting to receive cephalosporins prior to inoculation was 17% for cefoxitin and 0% for cefotetan and cefmetazole. Mortality in those receiving cephalosporins 8 h after inoculation was 20% for cefoxitin, 10% for cefotetan, and 7% for cefmetazole (P < 0.05 compared with value for control). No mortality was noted after s.c. inoculation. i.a. infection caused by E. coli with B. fragilis and B. thetaiotaomicron (Table 3). Abscesses were observed in all 16 surviving saline-treated mice. In the mice that started to receive cephalosporins 1 h prior to inoculation, mortality and abscess formation (in survivors) were reduced, respectively, to 17 and 20% with cefoxitin, 0 and 13% with cefotetan, and 0 and 17% with cefmetazole. All three cephalosporins significantly reduced the number of E. coli, B. fragilis, and B. thetaiotaomicron organisms in the abscesses. In the mice whose therapy was started 8 h after inoculation, cefoxitin therapy had effects similar to those it had when therapy was started prior to inoculation. Cefotetan and cefmetazole reduced mortality, but abscesses, mostly due to B. thetaiotaomicron, were noted on 93 and 96%, respectively, of the surviving mice. s.c. abscesses. (i) Abscesses caused by single organisms. s.c. abscesses were induced by each of the four organisms, and the CFU of the organisms in saline-treated mice varied from TABLE 2. Antimicrobial agenta Concentrations of cephalosporins in serum in uninfected mice Concn (mean [jlg/mlj ± SD) in serum at": 0.5 h 12 h Cefoxitin 62.0 ± 9.1 3.3 + 1.8 Cefotetan 66.3 ± 8.4 6.3 + 2.5 Cefmetazole 60.4 ± 8.6 7.2 ± 1.2 a Daily dose, 600 mg/kg of body weight. b Time after administration of drug.

VOL. 37, 1993 THERAPY OF B. FRAGILIS GROUP ABSCESSES 1533 TABLE 3. Effects of antimicrobial agents on i.a. infection caused by E. coli (NMRI 8), B. fragilis (NMRI 32), and B. thetaiotaomicron (NMRI 54) No. (%) of mice Log1o CFU of bacteria recovered/mouse (no. of mice infected) Dead With abscesses/total surviving E. coli B. fragilis B. thetaiotaomicron None (control) 14 (47) 16/16 (100) 8.7 ± 0.4 (16) 9.0 ± 0.5 (15) 8.6 t 0.4 (13) Starting 1 h before inoculation Cefoxitin 5 (17) 5/25 (20) 5.4 + 0.8 (5) 4.2 ± 0.3 (4) 4.0 + 0.5 (4) Cefotetan 0 (0) 4/30 (13) 2.3 ± 0.2 (2) 3.5 (1) 4.6 ± 0.6 (3) Cefmetazole 0 (0) 5/30 (17) 2.8 ± 0.4 (3) 3.2 + 0.5 (2) 4.2 ± 0.8 (4) Starting 8 h after inoculation Cefoxitin 6 (20) 2/24 (8) 3.9 ± 0.4 (2) 4.0 (1) 3.8.(1) Cefotetan 3 (10) 25/27 (93) 4.8 ± 0.5 (4) 3.5 ± 0.2 (2) 8.2 ± 0.6 (24) Cefmetazole 2 (7) 27/28 (96) 4.5 ± 0.4 (3) 2.8 ± 0.4 (2) 8.0 ± 0.5 (26) a Thirty mice were included in each group. log10 8.5 to 9.0 (Table 4). A significant reduction in the number of in vitro-susceptible and -resistant organisms was noted in all instances when therapy was started 1 h before inoculation. However, although not statistically significant, for susceptible isolates the reduction was about 10 times larger in the mice that received earlier (prophylactic) therapy. In contrast, a correlation between in vitro and in vivo efficacies was noted when therapy was started 8 h after inoculation. While cefoxitin was effective against all isolates, cefotetan did not reduce the number of S. aureus or B. thetaiotaomicron organisms, and cefmetazole failed to reduce the number of B. thetaiotaomicron organisms. (ii) Abscesses caused by two organisms. The number of each organism in mixed infection in saline-treated mice was significantly (P < 0.05) higher in all instances than when the organisms were inoculated alone and varied from log1o 10.2 Effect of cephalosporin therapy on the decrease in number of organisms in s.c. abscesses induced by B. fragilis, B. thetaiotaomicron, E. coli, and S. aureus" Reduction in no. of organisms/abscess (log1o CFU + SD) Infection typeb Therapy started 1 h before inoculation Therapy started 8 h after inoculation TABLE 4. Cefoxitin Cefotetan Cefmetazole Cefoxitin Cefotetan Cefmetazole Single organism B. fragilis (8.6 ± 0.8) 5.1 ± 0.6 4.8 ± 0.5 5.1 ± 0.8 4.2 ± 0.6 4.3 ± 0.8 3.0 ± 0.6 B. thetaiotaomicron (9.0 ± 0.7) 4.8 ± 0.8 3.8 ± 0.6 3.5 ± 0.2 4.0 ± 0.8 0.9 _ 0.4" 0.7 _ 0.4d E. coli (8.5 ± 0.6) 5.4 ± 1.0 5.8 ± 0.7 5.1 ± 0.8 3.8 ± 0.8 5.2 ± 0.8 4.3 ± 0.7 S. aureus (8.8 ± 0.9) 3.2 ± 0.7 4.6 ± 0.6 6.1 ± 0.5d 2.0 ± 8.6 1.0 ± 0.6 5.2 ± 0.6c Two organisms B. fragilis (10.5 ± 0.5) 6.5 ± 0.7 6.1 ± 0.8 6.4 ± 0.4 4.6 ± 1.0 5.2 ± 0.8 6.2 ± 0.4 E. coli (10.8 ± 0.6) 5.3 ± 0.5 7.8 ± 0.7 6.0 ± 0.6 4.4 ± 0.6 6.3 ± 0.7 5.2 ± 0.2 B. thetaiotaomicron (10.2 ± 0.7) 5.9 ± 0.2 6.3 ± 0.3 4.0 ± 0.8 5.2 ± 0.8 1.2 ± 0.4" 2.2 ± 0.5d E. coli (11.3 ± 0.7) 6.0 ± 0.3 7.6 ± 0.9 6.9 0.5d 5.1 ± 0.5 7.2 ± 1.0" 5.6 ± 0.6 B. fragilis (11.2 + 0.8) 5.8 ± 0.5 6.3 ± 0.9 6.8 ± 0.4 4.3 ± 0.4 5.8 ± 0.7 7.0 ± 0.7d S. aureus (10.3 ± 0.6) 5.2 ± 0.7 4.4 ± 0.4 7.8 ± 0.9c 4.8 ± 0.7 2.8 ± 0.4" 7.4 ± 0.8" B. thetaiotaomicron (11.0 ± 0.8) 6.3 ± 0.8 4.4 ± 0.5 3.9 ± 1.0" 5.4 ± 0.5 1.8 ± 0.5 3.2 ± 0.4 S. aureus (10.5 ± 0.9) 4.8 ± 0.5 3.8 + 0.5 8.2 ± l.c 4.6 _ 0.8 2.0 ± 0.5 7.6 ± 0.6c Three organisms B. fragilis (10.2 ± 7) 7.2 ± 0.6 7.4 ± 0.4 7.5 ± 1.2 6.6 + 1.6 7.6 ± 1.0 6.3 ± 0.9 B thetaiotaomicron (10.6 ± 0.6) 6.8 ± 0.5 7.0 ± 0.6 6.4 ± 0.9 5.8 ± 0.7 2.2 ± 0.5" 0.8 ± 0.4" E. coli (11.6 ± 1.3) 5.5 ± 0.8 7.9 ± 0.3" 7.0 ± 1.1 3.3 ± 0.4 7.0 ± 1.2" 6.8 + 1.1" B. fragilis (10.9 ± 0.6) 7.7 ± 0.7 7.8 ± 0.6 8.0 ± 0.7 7.1 ± 1.2 6.8 ± 0.9 7.2 ± 1.3 B. thetaiotaomicron (11.2 ± 0.7) 6.4 ± 0.8 6.8 ± 0.5 7.1 ± 0.6 6.2 ± 0.8 1.9 ± 0.3" 2.0 ± 0.7d S. aureus (11.8 ± 0.8) 5.2 ± 0.6 4.8 ± 0.4 8.2 ± 0.9" 4.8 ± 0.5 4.3 ± 0.4 8.0 ± 1.2" a Each treatment group contained 20 mice. b Log10 CFU + standard deviations of organisms in saline-treated mice are given in parentheses. c Significant difference compared with reduction by cefoxitin or cefotetan. d Significant difference compared with reduction by cefoxitin.

1534 BROOK to 11.3. Similar to results for abscesses caused by a single bacterial species, a significant reduction in the numbers of all in vitro-susceptible and -resistant isolates was observed when therapy was started early but not when it was delayed. In delayed therapy, cefmetazole was highly effective against S. aureus compared with the other two cephalosporins (P < 0.05). (iii) Abscesses caused by three organisms. For abscesses caused by three organisms, as for infection caused by one or two organisms, all three cephalosporins were independently effective in vivo, in accordance with their in vitro activities, and significantly reduced the numbers of all organisms when therapy was started prior to their inoculation. However, when therapy was started 8 h after inoculation, the reduction of susceptible bacteria was generally decreased by 1 log. While cefoxitin reduced the numbers of all organisms in infections caused by either E. coli or S. aureus with both B. fragilis group isolates, cefotetan and cefmetazole did not reduce the number of B. thetaiotaomicron organisms. Cefmetazole was, however, the most effective agent against the S. aureus component of the infection. DISCUSSION This study illustrates the efficacy of all the cephalosporins tested-cefoxitin, cefotetan, and cefmetazole-in the prevention of polymicrobial infection due to E. coli or S. aureus with B. fragilis and/or B. thetaiotaomicron when therapy is started 1 h prior to inoculation of the infectious organisms. This study illustrates the discrepancies between in vitro and in vivo efficacies of cefotetan and cefmetazole when these agents are given prior to inoculation of bacteria. They were active in vitro against B. fragilis but not against B. thetaiotaomicron. However, when administered prior to bacterial challenge, they were effective in reducing the instances of sepsis and the number of animals with abscesses. This discrepancy between in vitro susceptibility and in vivo efficacy may be due to an inoculum effect in which early antibiotic administration can suppress the relatively small number of bacteria present in the infection site (6). However, when antimicrobial therapy is given 8 h later, after the bacteria have had time to increase in number, their resistance to certain cephalosporins is more apparent. These data highlight the importance of prophylactic antibiotic therapy that is given prior to the occurrence of infection. The present report also confirms the previously observed synergistic relationship between aerobic and anaerobic bacteria (9). Interruption of this synergistic relationship at an earlier stage of the infection by an antimicrobial agent that is effective against only one or two components of the mixed infection may be sufficient to abort the progression of the mixed infection (3). This may be another factor that contributes to the efficacy of cefotetan and cefmetazole against in vitro-resistant organisms when these antibiotics are administered at an earlier stage of the infection. These in vivo data support previous clinical experience of the use of cefotetan (2, 21) and cefmetazole (20) in surgical prophylaxis and our retrospective clinical observations in which we found no difference in the prophylactic uses of cefotetan and cefoxitin for colorectal surgery in over 400 patients at our hospital (6a). Cefotetan and cefmetazole are relatively new cephalosporins with longer half-lives (cefotetan, 3.3 h; cefmetazole, 1.2 h) than cefoxitin (0.68 to 0.98 h). Their prolonged half-lives allow their administration once or twice a day, ANTIMICROB. AGENTS CHEMOTHER. compared with three or four times a day for cefoxitin. They have generally lower MICs against members of the family Enterobacteraceae than does cefoxitin (4), and cefmetazole has improved activity against S. aureus (15). Although cefotetan and cefmetazole are as effective as cefoxitin against B. fragilis, they are less active against the other members of the B. fragilis group (1, 12-14, 16). This study illustrates the efficacy of prophylactic use of cefotetan and cefmetazole in the prevention of infection. It also highlights the efficacy of cefmetazole in the prevention and therapy of infection caused by S. aureus and B. fragilis group organisms. Further studies to investigate the clinical significance of these observations are warranted. ACKNOWLEDGMENTS I thank J. E. Perry for technical assistance and C. H. Dorsey for electron microscopy. REFERENCES 1. Aldridge, K E., C. V. 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