Ceftazidime and amikacin alone and in combination against Pseudomonas aeruginosa and Enterobacteriaceae

Ceftazidime and amikacin alone and in combination against Pseudomonas aeruginosa and Enterobacteriaceae

DIAGNMICROBIOLINFECTDIS 1987;6:59-67 59 Ceftazidime and A m i k a c i n Alone and in Combination against Pseudomonas Aeruginosa and Enterobacteriace...

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DIAGNMICROBIOLINFECTDIS 1987;6:59-67

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Ceftazidime and A m i k a c i n Alone and in Combination against Pseudomonas Aeruginosa and Enterobacteriaceae

Julia A. Moody, Claudine E. Fasching, Lance R. Peterson, and Dale N. Gerding.

The efficacy of ceftazidime alone and combined with amikacin was studied in a rabbit model simulating closed-space infections at locally neutropenic sites. Six strains of Pseudomonas aeruginosa, and six Enterobacteriaceae (two strains each of Klebsiella pneumoniae and Serratia marcescens and one strain each of Escherichia coli and Citrobacter ~reundii) in pooled rabbit serum were each inoculated into separate subcutaneous semipermeable chambers. Intramuscular antibiotic therapy was begun 4 hr later with ceftazidime (50 mg/kg) alone and combined with amikacin (15 mg/kg) for Enterobacteriaceae or ceftazidime (100 mg/kg) alone and combined with amikacin (15 mg/kg)for pseudomonads every 6 hr for 16 doses. Amikacin alone was ineffective for all 12 strains. Ceftazidime alone was successful (>15.5 log~o colony forming units (CFU)/ml decrease from drug-free control) in eliminating five of six Enterobacteriaceae but was not successful against any of the pseudomonads. Ceftazidime plus amikacin was successful against the same five of six Enterobacteriaceae and five of six pseudomonads. The best in vitro tests for the prediction of in vivo outcome were high inoculum (>17 log~o CFU/ml) susceptibility, checkerboard synergism testing, and conventional inoculum time-kill rates at concentrations of antimicrobials simulating extravascular levels obtained in vivo.

INTRODUCTION Ceftazidime is a newer extended spectrum [3-1actam agent that exhibits good in vitro activity against aerobic gram-negative bacilli i n c l u d i n g Enterobacteriaceae and Pseud o m o n a s aeruginosa (Jones et al., 1981; Neu and Labthavikul, 1982). Resistance development with the use of ceftazidime alone has been reported in h u m a n clinical trials (Clumeck et al., 1983; Darbyshire et al., 1983; Eron et al., 1983; Francoli et al., 1983) and in animal models (Bayer et al., 1985; Johnson et al., 1985). Antimicrobial combinations that often i n c l u d e an aminoglycoside are frequently employed for enhanced activity against aerobic gram-negative bacilli and to prevent emergence of resistance. Synergism frequently occurs between ceftazidime and amikacin against From the Infectious Disease Section, Medical Service, and Microbiology Section, Laboratory Service, Veterans Administration Medical Center, Minneapolis, MN. Presented in part at the 86th Annual Meeting of the American Society for Microbiology, Washington, D.C., Abstract A86, March 23-28, 1986. Address reprint requests to: Julia A. Moody, Infectious Disease Section 111F, Veterans Administration Medical Center, 54th Street & 48th Avenue South, Minneapolis, MN 55417. Received June 30, 1986; revised and accepted August 20, 1986. © 1987 ElsevierSciencePublishingCo., Inc. 52 VanderbiltAvenue,New York, NY 10017

0732-8893/87/$3.50

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Enterobacteriaceae and Pseudomonas aeruginosa as reported by Bayer et al. (1984, 1985) The purpose of this study is to (1) determine the efficacy of ceftazidime alone and combined with amikacin under locally neutropenic conditions against P. aeruginosa and Enterobacteriaceae; (2) correlate results of in vitro susceptibility, bactericidal activity over time, and checkerboard synergism with in vivo results; and (3) determine possible causes for failure of ineffective antibiotic regimens. The advantages of using this animal model to assess antimicrobial efficacy include: (1) simulation of closedspace neutropenic infection without animal mortality; (2) the ability to study a relatively large number of isolates under controlled conditions; and (3) ease and uniformity of sample procurement for quantitative measurements of bacterial counts and antimicrobia] concentrations at the infection sites.

MATERIALS AND METHODS Bacteria Six Pseudomonas aeruginosa strains (833, 845, 864, 876, 913, and 915) and six Enterobacteriaceae, (one Escherichia coli [EC1], one Citrobacter diversus [CD10], two Klebsiella pneumoniae [KP85, KP95], and two Serratia marcescens [SM157, SM230]) were selected from clinical isolates at the Minneapolis Veterans Administration Medical Center. All twelve organisms were ~-lactamase producers as determined by the nitrocefin disc test. These organisms were selected on the basis of in vitro susceptibility to ceftazidime and amikacin at concentrations likely to be achievable in vivo in the extravascular chambers, synergism between ceftazidime and amikacin as demonstrated in vitro for some isolates, and prior studies in the same animal model using other antimicrobial regimens against these 12 isolates (Bamberger et al., 1986; Gerding et al., 1985; Peterson et al., 1984).

In Vitro Susceptibility Testing Minimal inhibitory concentrations (MICs) were performed by a broth microdilution method using a Dynatech MIC-20O0 96-channel dispenser (Dynatech Laboratories, Alexandria, VA) in Mueller-Hinton broth (Difco Laboratories, Detroit, MI) supplemented with calcium and magnesium cations. Two log phase inocula were studied: 1 to 5 x 105 CFU/ml (conventional) and 1 to 5 x 107 CFU/ml (high) which were incubated for 18-22 hr at 35°C. The MIC was read as the lowest antimicrobial concentration exhibiting no visible growth. The minimal bactericidal concentration (MBC) was obtained by subculturing the entire contents (0.1 ml) of the MIC, 2 x MIC, and 4 x MIC wells onto single 5% sheep blood agar plates (BAP) as previously described (Shanholtzer et al., 1984). After overnight incubation, the MBC was defined as the lowest drug concentration demonstrating at least a 99.9% reduction in bacterial counts from the starting (MIC) inoculum. Organisms were also tested in a two-drug checkerboard at conventional and high inocula using doubling dilutions of ceftazidime and amikacin. Synergism was defined as at least a fourfold reduction in MIC or MBC for each drug in combination [EFIC or EFBC < 0.5] (Hallander et al., 1982).

Time-Kill Study A time-kill study was performed on all 12 organisms using a starting inoculum of 4.8 to 5.0 _+ 0.6 lOglo CFU/ml in supplemented Mueller-Hinton broth (1-ml volumes) at concentrations equal to mean extravascular chamber antiobiotic levels. Ceftazidime

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(32 p.g/ml), amikacin (8 ~g/ml), and ceftazidime (32 p~g/ml) plus amikacin (16 p~g/ml) were tested against the Enterobacteriaceae; and ceftazidime (32 ~g/ml), amikacin (16 ~g/ml), and ceftazidime (64 ~g/ml) plus amikacin (16 ~g/ml) were tested against the pseudomonads. Bacteria samples (0.1 ml) were quantitated by surface plating on 5% sheep BAP at 0, 3, 6, and 24 hr, spreading the inocula with sterile glass rods and manually counting colonies after 24-48 hr of incubation.

Animal Model The animal model used was one previously described by Peterson and Gerding (1978), for study of extravascular penetration of antibiotics. Briefly, extravascular chambers consisted of Visking regenerated-cellulose tubing (Union Carbide Corp., Chicago, IL) tied off at one end and occluded at the other end by a cork through which tubing from a 21-gauge Butterfly intermittent infusion set (Abbott Hospital Products, Inc., North Chicago, IL) had been passed. These chambers allowed diffusion of molecules below a molecular weight of 15,000. Six chambers (each 3-ml volume) were implanted in the subcutaneous space of the back of anesthetized 3.0-kg female New Zealand white rabbits. Each animal was rested three to four days after implantation before being studied. Four hours before beginning antibiotic therapy, the Visking chambers were filled by percutaneous injection of pooled rabbit serum containing 5 x 104 CFU/ ml of organisms to be tested. One strain was tested in each chamber. Four hours later samples were taken for quantitative bacteriology, and antimicrobial therapy was begun. The Enterobacteriaceae were treated with 50 mg/kg ceftazidime or 50 mg/kg ceftazidime plus 15 mg/kg amikacin; and the pseudomonads with 100 mg/kg ceftazidime or 100 mg/kg ceftazidime plus 15 mg/kg amikacin. Bacterial growth rates in control animals and animals treated with amikacin alone were obtained from two previous studies in this model (Gerding et al., 1985; Peterson et al., 1984). Three animals (each with six chambers) were studied for each regimen. All animals received two intramuscular injections at each dosing period. Sterile phosphate-buffered saline was substituted when a second drug was not being used. Antibiotics were given every 6 hr for 16 doses. Chambers were sampled at 0, 20, 44, and 92 hr after the first antibiotic injection for quantitative bacteriology using Mueller-Hinton agar (Difco) pour plates. Bacterial colonies were counted manually after 24-48 hr of incubation. Organisms that were not eliminated in vivo were aspirated from chambers at 96 hr, passed overnight on BAP, and retested for antibiotic susceptibility. Chambers were sampled at 20, 92, and 96 hr and blood was sampled at 0.5, 1, 3, and 6 hr after dose 13 for measurement of antibiotic concentrations. These times were chosen so as to provide a complete intravascular kinetic curve and also to measure peak and trough antibiotic concentrations in the chambers. No more than 1.1 ml of total sample was removed from each chamber prior to termination of the study (Peterson and Gerding, 1978).

Antibiotic Assays Ceftazidime was a gift from Glaxo, Inc., (Research Triangle Park, NC) and amikacin was a gift from Bristol Laboratories (Syracuse, NY). Assay of ceftazidime was done by a high-pressure liquid-chromatography method (Fasching et al. 1986). Assay of amikacin was done by immunoassay EMIT Amikacin Kit, Syva Co., (Palo Alto, CA) after the methods of Larson et al. (1982) for netilmicin and gentamicin. Protein binding was done by ultracentrifugation (Peterson et al., 1977a, 1977b). Amikacin is 0% bound (Van Etta et al., 1982) and ceftazidime is 17.9% bound (Fasching et al., in press) in pooled rabbit serum.

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TABLE 1. In Vitro Susceptibility of Ceftazidime and Amikacin, Expressed as MIC and MBC in ~g/ml Against Each Strain Enterobacteriaceae strains tested Antibiotic(s)

Inoculum size a EC1

Ceftazidime MIC MBC MIC MBC Amikacin MIC MBC MIC MBC Ceftazidime + amikacin I~FIC EFIC

C H C H

C H

CD10

KP85

KP95

SM157

1 ~0.06-0.125 0.125 1-2 0.125 0.125-0.25 >64 64 8-16 >64 64 8-16 1-2 ~<0.5 40.5-1 1-4 40.5 <~0.5-1 4-8 4 2-4 16 8 4

40.06 1 ~<0.06 1-4 8-16 >64 8-16 >64 40.5-1 4-8 ~<0.5-1 4-8 2-4 64 4 128

0,75 NDb ~<0,125~<0.14

NDb 0.75

NDb 0.75

0.375-0.5 ~<0.125

SM230 0.25-0.5 >4 >64 >64 4-16 16->32 64 128 0.75 NDb

RESULTS

In Vitro Antibiotic Susceptibility Tests. MICs, MBCs, and combination interactions at conventional and high inocula are shown in Table I for all 12 isolates. At conventional inoculum, the Enterobacteriaceae were very susceptible (MIC ~ 1 txg/ml) and the p s e u d o m o n a d s were moderately susceptible (MIC ~ 8 txg/ml) to ceftazidime. MBCs were within two dilutions of MICs except for SM230 for which the ceftazidime MBC to MIC ratio was >8. At high inoculum, n i n e of 12 isolates exhibited ceftazidime MIC increases to ~ 64 Ixg/ml. The other three isolates, KP85, KP95, and PA864 exhibited fourfold or greater increases in ceftazidime MIC to 8-32 ixg/ml. Isolates with ceftazidime MIC/> 64 txg/ml are considered resistant. The amikacin high i n o c u l u m MIC increased fourfold or greater for all six Enterobacteriaceae and one P. aeruginosa. Of these seven isolates only the two Serratia became amikacin resistant (MIC t> 32 Ixg/ml). At c o n v e n t i o n a l inoculum, the ceftazidime and amikacin combination was synergistic against three of 12 isolates: SM157, PA864, and PA876. A combination interaction for CD10, KP85, and KP95 could not be determined because of the panel drug concentration limitations. At high inoculum, three of six Enterobacteriaceae and all six p s e u d o m o n a d s exhibited synergism with the ceftazidime plus amikacin combination.

Animal Studies Against the Enterobacteriaceae, ceftazidime alone and ceftazidime plus amikacin eliminated (/>5.5 lOglo CFU/ml decrease from no drug control) the same five of six isolates (Table 2). Against P. aeruginosa, ceftazidime alone did not eliminate any of the six isolates whereas ceftazidime plus amikacin eliminated five of six isolates. From previous studies, amikacin alone did not eliminate any of the 12 isolates. At the end of the treatment study, isolates were extracted from the chambers, passed one day on 5% sheep blood agar and retested for susceptibility. The MICs of those remaining isolates did not increase significantly (>~fourfold) after treatment with ceftazidime alone or ceftazidime combined with amikacin.

Antibiotic Levels Table 3 shows the m e a n chamber peak (92 hr) and trough (96 hr) antibiotic concentrations for the Enterobacteriaceae and pseudomonads. Differences in ceftazidime

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TABLE 1 (continued) Pseudomonas strains tested Inoculum Size° C H

C

PA833

PA845

0.5 1 >64 >64

2 2 >64 >64

8 16 32 32

8 8-16 8 >32

4-8 8 8 16

0.75 ~0.125

0.5 ~<0.125

2 4 4 16

H C H

1.0 ~<0.129

PA864

PA876

PA913

PA915

4 4 >64 >64

1 2 >64 >64

2 4 >64 >64

16 16 16 32

2 4 8 32

0.5 ~<0.45

1.0 ~<0.078

4 4-8 8 16 0.75 ~<0.094

ac, conventional inoculum 5 log~0CFU/ml;H, high inoculum 7 log~0CFU/ml. bND, not determined.

chamber concentrations alone and in combination with amikacin were c o m p a r e d using two-tailed M a n n W h i t n e y statistics. A m o n g the Enterobacteriaceae, peak ceftazidime chamber levels were not significantly different (P > 0.05), however, trough ceftazidime levels were significantly higher (P < 0.02) for ceftazidime c o m b i n e d w i t h amikacin c o m p a r e d to ceftazidime alone. A m o n g the p s e n d o m o n a d s , ceftazidime chamber levels w h e n used in combination with amikacin were significantly higher at both the peak (P < 0.02) and trough (P < 0.04) than for ceftazidime alone.

Comparison of In Vitro Time-Kill Study with In Vivo Results The in vivo bacterial count decrease (loglo CFU/ml) from no drug control 92 hr after beginning antimicrobial therapy c o m p a r e d to the in vitro 6 hr decrease (log10 CFU/ ml) from no drug control at concentrations simulating extravascular levels against all 12 isolates is shown in Table 2. The lower limit for bacterial detection in vivo is 100 CFU/ml and in vitro is 10 CFU/ml. Efficacy is defined in vivo and in vitro as 1>5.5 lOglo CFU/ml decrease from no drug control. The in vitro and in vivo outcome correlated for five of six Enterobacteriaceae with both ceftazidime and ceftazidime plus amikacin. A m o n g the p s e u d o m o n a d s , the in vitro results correlated with the in vivo outcome for five of six p s e u d o m o n a d s with ceftazidime and amikacin in combination and four of six p s e u d o m o n a d s for ceftazidime alone.

DISCUSSION Previous studies using this same animal m o d e l (Gerding et al., 1985; Peterson et al., 1984) have s h o w n that a m i k a c i n alone was ineffective in eliminating any of the 12 tested strains; ceftizoxime, an e x t e n d e d spectrum third generation cephalosporin, was the best single agent against the Enterobacteriaceae; and against the pseudomonads two agents of different antibiotic classes, a [3-1actam and an aminoglycoside, were required for in vivo success. We found similarities in the current ceftazidime study. Ceftazidime was the most effective single agent against the Enterobacteriaceae. Ceftazidime e l i m i n a t e d five of the six isolates, whereas, ceftizoxime only e l i m i n a t e d three of six. Ceftazidime c o m b i n e d with amikacin eliminated the same five isolates

d~

T A B L E 2. C o r r e l a t i o n of T r e a t m e n t E f f i c a c y b y I n V i v o C h a m b e r R e d u c t i o n I n V i t r o T i m e Kill (Loglo C F U / m l ) R e d u c t i o n f r o m C o n t r o l at 6 h r

Regimen Ceftazidime Ceftazidime ± a m i k a c i n

Regimen Ceftazidime Ceftazidime _+ a m i k a c i n

M e a n drug c o n c e n t r a t i o n in c h a m b e r or in vitro in in in in

vivo vitro vivo vivo

M e a n drug c o n c e n t r a t i o n in c h a m b e r or in vitro in in in in

vivo vitro vivo vivo

(Loglo C F U / m l ) f r o m N o D r u g C o n t r o l at 92 h r a n d

P s e u d o m o n a s logloCFU/ml r e d u c t i o n from control (p~g/ml)

PA833

PA845

PA864

PA876

PA913

PA915

51.3 ± 19.1 32 71.7 ± 31.4, 12.3 ± 3.8 64, 16

2.9 ~6.0 ~6.1 ~6.1

3.8 5.9 ~>6.6 ~6.3

3.3 5.2 ~6.0 ~6.0

2.0 4.6 4.0 ~5.5

3.2 5.2 6.3 ~6.0

3.2 4.9 ~7.3 ~>6.0

Enterobacteriaceae logloCFU/ml r e d u c t i o n from control (p.g/ml)

EC1

CD10

KP85

KP95

SM157

SM230

33.9 ± 17.0 32 33.4 ± 11.5, 12.3 ± 3.8 32, 16

~8.6 5.7 ~8.6 ~7.0

~8.4 6.0 ~>8.4 ~7.3

~8.3 7.0 ~>8.3 ~7.2

~7.6 ~>7.3 ~7.6 ~7.3

~5.5 4.8 ~7.9 4.9

5.0 4.5 5.0 4.7

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TABLE 3. Mean Peak (92 hr) and Trough (96 hr) Antibiotic Concentration (p.g/ml) in Chamber F l u i d for Enterobacteriaceae and P s e u d o m o n a d s for Each in vivo Treatment Regimen Enterobacteriaceae

Pseudomonads

Antibiotics

Mean trough (~g/ml)

Mean peak (~g/ml)

Mean trough (~g/ml)

Mean peak (p.g/ml)

Ceftazidime Ceftazidime + Amikacin

18.0 _+ 2.6 23.0 ± 2.1 9.3 ± 2.3

47.3 _* 6.0 42.3 ~ 4.0 15.2 ± 2.2

37.2 ± 6.7 50.6 ± 5.9 9.7 ± 3.1

66.7 ± 7.8 91.0 ± 8.3 16.9 ± 2.2

and was comparable to the success of mezlocillin plus amikacin (Gerding et al., 1985). Against p s e u d o m o n a d s , ceftazidime plus amikacin was required for in vivo success and was equivalent to a regimen of azlocillin plus amikacin (Peterson et al., 1984). The d e v e l o p m e n t of antibiotic resistance (significant MIC increase) was not found in the P. aeruginosa remaining at the end of the treatment with ceftazidime alone as was found in the previous study of P. aeruginosa treated with ceftizoxime alone (Peterson et al., 1984). The best in vitro p r e d i c t i o n of single drug efficacy in previous studies by Gerding et al. (1985) and Peterson et al. (1984) was high i n o c u l u m MIC testing for the [3lactams (simulating organism numbers present at the beginning of antibiotic therapy in vivo), and anaerobic amikacin MIC testing (simulating the relative anaerobiosis present in the chambers). In this study with ceftazidime and amikacin, correlation of in vitro methods with in vivo efficacy was e x a m i n e d by susceptibility (conventional and high inoculum), checkerboard synergism, and time-kill rates. All 12 isolates were susceptible to ceftazidime (MIC ~< 16 g.g/ml) at conventional inoculum, however, at high i n o c u l u m all MICs increased m a r k e d l y such that nine organisms became resistant to ceftazidime (MIC/> 64 ~g/m]). Similar ceftazidime i n o c u l u m effects have been reported for some gram-negative bacilli (Eng et al., 1985; Neu and Labthavikul, 1982). In vivo treatment outcome correlated with conventional i n o c u l u m susceptibility for five of the 12 isolates and high i n o c u l u m susceptibility for nine of 12. Synergism with ceftazidime c o m b i n e d with amikacin was more frequent with high (9 of 11) than conventional (3 of 9) inoculum. A m o n g the P. aeruginosa, synergism at high i n o c u l u m correlated with in vivo efficacy for five of six isolates and at conventional i n o c u l u m with only one of six isolates. A m o n g the Enterobacteriaceae, ceftazidime alone in vivo was effective against five of six isolates, therefore, synergism with ceftazidime c o m b i n e d with amikacin was not predictive. Taking into account the percent of ceftazidime b o u n d to rabbit serum protein (17.9%), mean total- and free-ceftazidime chamber trough levels w h e t h e r alone or in combination with a m i k a c i n were/>4 × MIC and ~>2 × MBC for 11 of 12 isolates as d e t e r m i n e d by conventional inoculum. However, nine of 12 high i n o c u l u m MICs exceeded mean peak total- and free-ceftazidime chamber levels. A m i k a c i n mean trough levels (when c o m b i n e d with ceftazidime) were equal to or exceeded conventional i n o c u l u m MICs of 10 of the 12 isolates and 9 of 12 isolates at high i n o c u l u m MICs. Bayer et al. (1984) have reported rapid and e n h a n c e d in vitro cidal activity with ceftazidime and amikacin against Enterobacteriaceae. We performed an in vitro timekill study to determine the actual reduction of viable counts at 6 hr at concentrations simulating mean extravascular levels and c o m p a r e d the results to in vivo efficacy.

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For five of six Enterobacteriaceae, the 6 hr in vitro/>5.5 lOglo CFU/ml decrease from drug-free control correlated with in vivo treatment success or failure at 92 hr with ceftazidime alone or combined with amikacin. Among the pseudomonads, in vivo and in vitro results correlated for four of six isolates for ceftazidime alone and five of six isolates for ceftazidime plus amikacin. However, count reductions were considerably greater in vitro than in vivo for ceftazidime alone (Table 2). This study suggests that ceftazidime is an effective single agent against Enterobacteriaceae, the best single [3-1actam agent tested in this model to date, but that ceftazidime c o m b i n e d with amikacin is required for effective therapy against P. aeruginosa. No single in vitro evaluation predicted in vivo outcome. However, high i n o c u l u m susceptibility, synergy testing, and conventional i n o c u l u m time-kill rates at antibiotic concentrations simulating extravascular levels provided the best in vitro prediction of in vivo success. The conclusions to be drawn from this study for treatment of infection in neutropenic patients must be cautious. The model is one of local n e u t r o p e n i a and bacterial counts at the time of antibiotic initiation are quite high. Given this situation in a neutropenic patient infected with Pseudomonas aeruginosa, our data would indicate the need for c o m b i n a t i o n therapy, whereas for infection with Enterobacteriaceae of the type we tested, one might expect a good result from ceftazidime alone. It is reassuring that in neither situation did in vivo resistance to ceftazidime develop.

This study was supported by Bristol Laboratories and the Veterans Administration. We thank Evelyn C. Glatt for typing the manuscript and Leann M. Lissack for technical assistance.

REFERENCES Bamberger DM, Peterson LR, Gerding DN, Moody JA, Fasching CE (1986) Ciprofloxacin, azlocillin, ceftizoxime and amikacin alone and in combination against gram-negative bacilli in an infected chamber model. J Antimicrob Chemother 18:51. Bayer AS, Eisenstadt R, Morrison JO (1984) Enhanced in vitro bactericidal activity of amikacin or gentamicin combined with three new extended spectrum cephalosporins against cephalothin resistant members of the family Enterobacteriaceae. Antimicrob Agents Chemother 25:725. Bayer AS, Norman D, Kim KS (1985) Efficacy of amikacin and ceftazidime in experimental aortic valve endocarditis due to P. aeruginosa. Antimicrob Agents Chemother 28:781. Clumeck N, Van Laethem Y, Gordts B, Jaspar N, Butzler JP (1983) Use of ceftazidime in the therapy of serious infections, including those due to multiresistant organisms. Antimicrob Agents Chemother 24:176. Darbyshire PJ, Williamson PJ, Pedler SJ, Speller DE, Mott MG, Oakhill A (1983) Ceftazidime in the treatment of febrile immunosuppressed children. J Antimicrob Chemother 12(Suppl A):357. Eng RHK, Cherubin C, Smith SM, Buccini F (1985) Inoculum effect of [3-1actam antibiotics on Enterobacteriaceae. Antimicrob Agents Chemother 28:601. Eron LJ, Goldenberg RI, Park CH, Poretz DM (1983) Ceftazidime therapy of serious bacterial infections. Antimicrob Agents Chemother 23:236. Fasching CE, Gerding DN, Peterson LR (1986) High pressure liquid chromatography analysis of BMY-28142 and ceftazidime in human and rabbit serum. J Liq Chromatogr 9:1803. Francoli P, Clement M, Geroulanos S, von Graevanitz A, Luthy R, Regamey C, Stalder H, Vogt M, Woldvogel FA (1983) Ceftazidime in severe infections: A Swiss multicentre study. J Antimicrob Chemother 12(Suppl A):139.

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Gerding DN, Peterson LR, Moody JA, Fasching CE (1985) Mezlocillin, ceftizoxime and amikacin alone and in combination against six Enterobacteriaceae in a neutropenic site in rabbits. ] Antimicrob Chemother 15(Suppl A):207. Hallander HO, Dornsbusch K, Gezelius L, Jacobson K, Karlsson I (1982) Synergism between aminoglycosides and cephalosporins with antipseudomonal activity: Interaction index and killing curve method. Antimicrob Agents Chemother 22:743. Johnson DE, Thompson B, Calia FM (1985) Comparative activities of piperacillin, ceftazidime, and amikacin, alone and in all possible combinations, against experimental Pseudomonas aeruginosa infections in neutropenic rats. Antimicrob Agents Chemother 27:735. Jones RN, Barry AL, Thornsberry CL, Gerlach EH, Fuchs PC, Gavan TL, Sommers HL (1981) Ceftazidime, a pseudomonas-active cephalosporin: in vitro antimicrobial activity evaluation including recommendations for disc diffusion susceptibility tests. J Antimicrob Chemother 8(Suppl B):187. Larson TE, Gerding DN, Peterson LR, Eckfeldt JH (1982) Assay of netilmicin using enzyme immunoassay for gentamicin. Antimicrob Agents Chemother 21:399. Neu HC, Labthavikul P (1982) Antibacterial activity and ~olactamase stability of ceftazidime, an aminothiazolyl cephalosporin potentially active against Pseudomonas aeruginosa. Antimicrob Agents Chemother 21:11. Peterson LR, Gerding DN, Zinneman HH, Moore BM (1977a) Evaluation of three newer methods for investigating protein interactions of penicillin G. Antimicrob Agents Chemother 11:993. Peterson LR, Hall WH, Zinneman HH, Gerding DN (1977b) Standardization of a preparative ultracentrifuge method for quantitative determination of protein binding of seven antibiotics. J Infect Dis 136:778. Peterson LR, Gerding DN (1978) Prediction of cefazolin penetration into high- and low-protein containing extravascular fluid: new method for performing simultaneous studies. Antimicrob Agents Chemother 14:533. Peterson LR, Gerding DN, Moody JA, Fasching CE (1984) Comparison of azlocillin, ceftizoxime, cefoxitin and amikacin alone and in combination against Pseudomonas aeruginosa in a neutropenic-site rabbit model. Antimicrob Agents Chemother 25:545. Shanholtzer CJ, Peterson LR, Mohn ML, Moody JA, Gerding DN (1984) MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques. Antimicrob Agents Chemother 26:214. Van Etta LL, Kravitz GR, Russ TE, Fasching CE, Gerding DN, Peterson LR (1982) Effects of method of administration on extravascular penetration of four antibiotics. Antimicrob Agents Chemother 21:873.