Y. Q. Xiong*, J. Caillon*, X. Y. Zhou*, G. Potel'*, D. Bugnon', P. Le Conte*, F. Le Gallon*, R. Le Floch', D. Baron* and H.

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1 Journal of Antimicrobial Chemotherapy (1995) 35, Treatment of experimental rabbit infective endocarditis due to a raultidrug-resistant Pseudomonas aeruginosa with high-dose ceftazidime alone and combined with amikacin or sulbactam or both Y. Q. Xiong*, J. Caillon*, X. Y. Zhou*, G. Potel'*, D. Bugnon', P. Le Conte*, F. Le Gallon*, R. Le Floch', D. Baron* and H. Drugeon* "Laboratoire cf Antibiologie Clinique et Experimentale, Faculte de Medecine, C.H.U., 1 rue Gaston-Veil, Nantes; b Laboratoire de Microbiologie Medicate, Unwersite Paris VI, 15 rue de I'Ecole de Medecine, Paris, Cedex 06, France Ceftazidime 800 mg/kg iv was effective when given alone as a continuous infusion to rabbits with aortic valve endocarditis 12 h after infection with a multidrug-resistant strain of P. aeruginosa but only in combination with 100 mg/kg amikacin or 400 mg/kg sulbactam, or both drugs when treatment was begun later. Introduction Severe infections due to Pseudomonas aeruginosa are frequently fatal because of the emergence and development of resistance in vivo to the antimicrobial agents most commonly used for treatment (Reyes, Brow & Lerner, 1978; Lagast, Meunier-Carpentier & Klastersky, 1983; Reyes & Lerner, 1983). This poses a therapeutic challenge that cannot be addressed without using animal models to evaluate newer antipseudomonal regimens(zak& O'Reilly, 1991; Fantin& Carbon, 1992). Experimental endocarditis due to P. aeruginosa is considered to be a discriminative model closely resembling human disease and a stringent test of antimicrobial efficacy in vivo. Despite recent advances in the development of antimicrobial agents, the aminoglycosides still continue to play an important role in the treatment of severe infections caused by Gram-negative bacteria. These compounds exhibit concentrationdependent killing activity, a postantibiotic effect (PAE) on both Gram-positive and Gram-negative bacteria and are synergistic with /Mactam agents (Craig & Vogelman, 1987; Fantin & Carbon, 1992). However, recent experiences in treating severe P. aeruginosa infections in both humans and experimental animal models have highlighted the difficulties in achieving cure with an aminoglycoside given a single-agent (Reyes & Lerner, 1983; Bayer, Norman & Kim, 1985) even when the organism is very susceptible in vitro (Jackson & Riff, 1971; Kapusnik et al., 1988) because of the high frequency selection of resistant mutants (Reyes et al., 1978; Reyes & Lerner, 1983). Experimental studies have shown that combining an aminoglycoside with a /Mactam increases bactericidal efficacy and prevents the emergence of drug resistance in vivo Corresponding author. Phone: + 33-{4O)-O83640; Fax: + 33-<40) /95/ S08.00/ The British Society for Antimicrobial Chemotherapy

2 698 Y. Q. Xiong et al. (Fantin & Carbon, 1992). The bactericidal activity against P. aeruginosa in vivo is generally similar to the increased killing rate in vitro, as demonstrated in the endocarditis model (Archer & Fekety, 1977), pneumonia in neutropenic animals (Rusnak et al., 1984; Kapusnik et al., 1988) and septicaemia in rats rendered neutropenic by cyclophosphamide (Lumisch & Norden, 1976; Scott & Robson, 1976). When combined with aminoglycosides, ceftazidime showed frequent bactericidal synergy against aerobic Gram-negative bacilli (Bayer, Eisenstadt & Morrison, 1984). Like most /Mactams, the action of ceftazidime is time-dependent and might therefore be potentially more efficacious if administered as continuous iv infusion (Craig & Ebert, 1992). Sulbactam is a /Mactamase inhibitor that can prevent many /Mactam antibiotics from being destroyed by /Mactamases thereby enhancing efficacy in the treatment of infections due to Eneterobacteriaceae and P. aeruginosa (Less et al., 1986; Hancock & Woodruff, 1988) Being the only /Mactamase inhibitor available as a separate compound we chose sulbactam to help investigate in vitro and in vivo the possibility that failure of treatment with ceftazidime might be mediated by the production of /Mactamase. Our purpose was to compare the activity in vivo of high serum concentrations of ceftazidime alone and when combined with amikacin or sulbactam or both agents together in two phases. In the first phase, ceftazidime, sulbactam and amikacin were administered as a continuous iv infusion in order to mimic the conditions of constant concentrations that obtain in vitro to determine whether there was any correlation between in-vitro and in-vivo efficacy. In the second phase, ceftazidime and sulbactam were administered as a continuous iv infusion because of their time-dependent activity on Gram-negative bacteria while amikacin was administered as a bolus injection twice-daily because of its concentration-dependent activity. Each regimen was evaluated for its ability to reduce bacterial counts in the experimental model following a short period of 24 h therapy to reflect a critical end-point in seriously ill patients. We also investigated the effect of delaying treatment for 12 h and 48 h after the onset. Organism Materials and methods A multidrug-resistant P. aeruginosa strain was used throughout this study and had been isolated from bloodstream infection in a patient in the burns unit of our hospital. During a 6 month-period, four patients had been infected with this strain which was isolated later in other medical units including the Haematology ward and Intensive Care. This epidemic strain was resistant to quinolones and imipenem as well as to rabbit serum. Antibiotics Amikacin (Bristol Laboratories, Paris, France), ceftazidime (Glaxo Laboratories, Paris, France) and sulbactam (Pfizer Laboratories, Orsay, France) were dissolved in sterile water.

3 Endocarditis doe to multiple-resistant P. aeruginosa 699 In-vitro antibiotic susceptibility tests MICs and MBCs of amikacin, ceftazidime and sulbactam were determined by microdilution of the antimicrobial agents in Mueller-Hinton broth (MHB) supplemented with cations (Dougherty, Yotter & Matthews, 1977). Each well contained 100 /il broth and was inoculated with bacteria in the log-phase to yield a final inoculum of 10 s cfu/ml. The MIC was defined as the lowest concentration of the antimicrobial agent to prevent visible growth after 24 h incubation at 37 C. The MBC was defined as the lowest concentration of the antimicrobial agent to kill at least 99-9% of organisms within 24 h and was determined by plating 1 pl of each well showing no growth on to 8 mm thick Mueller-Hinton agar. In vitro bactericidal interaction testing For killing curves P. aeruginosa was grown overnight in MHB and diluted in sufficient fresh MHB to yield two separate inocula of 5 x 10 6 cfu/ml and the higher density of 5 x 10 7 cfu/ml which is regularly achieved within aortic valve vegetations of rabbits with experimental endocarditis (Drake & Sande, 1986; Pangon et al., 1987). Ceftazidime and amikacin were added to achieve final concentrations of 0, 1, 2, 4, 8, 16, 32 and 64 mg/l. Sulbactam was added to achieve a final concentration of 8 mg/l. Every concentration of each antibiotic was inoculated with each of the inocula and incubated at 37 C in a microtube once cultures had been obtained to determine the viable count. Further samples were withdrawn for viable counts after 3, 6 and 24 h of incubation using a semiautomatic dilution micromethod involving an automatic 96-well dispenser and a Steers replicator which had been sterilised by burning off alcohol before distributing 20 ± 5 nl of each dilution on to agar plates. After 24 h incubation, colonies on the first plate to yield 5 to 30 colonies were counted and the result was multiplied by the dilution factor. The standard error of the count was estimated to be 0-2 logio cfu/ml and the limit of detection of the method was 2-4 logi 0 cfu/ml (Drugeon et al., 1987). Synergy was considered present when the combinations caused a > 2 log, 0 decrease in the cfu/ml at 24 h in comparison with the most effective drug alone (Drugeon et al., 1987). fl-lactamase assay Extracts were obtained by sonicating cultures of the P. aeruginosa followed by ultracentrifugation. 0-lactamase activity was determined spectrophotometrically at 30 C with a model 550S double-beam spectrophotometer coupled to a model 561 recorder (Perkin-Elmer, Norwalk, CT, USA) using cephaloridine as the substrate. One unit of /Mactamase activity was defined as the amount of enzyme hydrolysing 1 /imol of cephaloridine/min (Zhou et al., 1993). Experimental endocarditis Female New Zealand white rabbits weighing approximately 2 kg were kept in individual cages and allowed free access to food and water throughout the experiment. In order to establish endocarditis, a polyethylene catheter was introduced into the left ventricle under general anaesthesia induced by 15 mg/kg ketamine until the tip passed through the aortic valve (Perlman & Freedman, 1971; Potel et al., 1991). The catheter remained in place

4 700 Y. Q. Xiong et al. throughout the study. Each animal was inoculated intravenously with approximately 10* cfu of P. aeruginosa 24 h after catheterisation. Treatment and evaluation of therapy Drugs were given as a continuous infusion of 2 ml/h by placing a catheter which was connected to an electric syringe pump into a marginal ear vein. Groups of six to ten rabbits were treated either 12 or 48 h after infection with 800 mg/kg ceftazidime, 400 mg/kg amikacin 800 mg/kg ceftazidime and 400 mg/kg sulbactam, 800 mg/kg ceftazidime and 100 mg/kg amikacin, 800 mg/kg ceftazidime combined with 400 mg/kg sulbactam and 100 mg/kg amikacin each regimen being administered as a 24 h continuous iv infusion. A high dose of ceftazidime was chosen because no antibacterial effect could be expected with serum steady-state concentrations lower than about 8 x MIC. We also studied the effect of a more prolonged treatment with 800 mg/kg ceftazidime together with 100 mg/kg amikacin and 400 mg/kg sulbactam administered as a 48 h continuous iv infusion. In the second phase, amikacin was administered iv as a 50 mg/kg bolus twice daily whilst 800 mg/kg ceftazidime and 400 mg/kg sulbactam were administered as a 24 h continuous iv infusion. In this case 14 animals served as untreated controls. The rabbits that served as controls were killed 12 or 48 h after infection by an iv bolus injection of 100 mg thiopental whereas the treated animals were killed at the end of the continuous iv infusion by an iv bolus injection of 100 mg thiopental. The heart was removed and vegetations were excised, rinsed rapidly in sterile saline, then weighed and homogenised in 0-5 ml of sterile saline. A spiral plater (Spiral system, Interscience, Saint Nom la Breteche, France) was used to count the number of bacteria and spread 50 /i L of undiluted homogenate and 1/100 and 1/10000 dilutions onto Trypticase-Soy agar plates which were then incubated for 24 h at 37 C. No significant carry-over was expected since the concentrations of ceftazidime and amikacin anticipated in serum and in the vegetations were less than 4 x times MIC. The vegetation titre, was defined as logio cfu/g of vegetation and as few as 20 cfu/ml of homogenate could be detected. The weight of the vegetation was taken into account when estimating the mean number of bacteria. Pharmacokinetic studies Blood samples were taken from three animals to determine the plasma concentration-time curve for 800 mg/kg ceftazidime and 400 mg/kg amikacin given as a continuous iv infusion. Blood samples in rabbits receiving a continuous infusion were obtained under local anaesthesia by positioning a catheter in the left femoral artery. Samples were immediately centrifuged and frozen at 20 C until required for assay. Antibiotic assays Serum antibiotic concentrations for amikacin and ceftazidime regimens were determined by microbiological assay with Bacillus subtilis ATCC 6633 and a clinical strain of E. coli respectively. The range of linearity with B. subtilis ATCC 6633 was from 0-25 to 2 mg/l for amikacin (r = 0-98). For ceftazidime, the range of linearity was from 0-5 to 64 mg/l with E. coli (r = 0-99). The supernatant fluid was used to assay the antibiotics in vegetations after they had been weighed homogenised in 0-3 ml of 01 M phosphate buffer, and centrifuged. Concentration of sulbactam was not determined.

5 Endocarditis due to multiple-resistant P. aeruginosa 701 Statistical evaluation Analysis of variance to compare the mean logio bacterial vegetation counts of treatments groups and the untreated controls was performed using the Statview computer programme (Abacus Concepts, Calabasas, CA, USA) with differences between groups only being considered significant when P < 005 by the more conservative Scheffe's test. In-vitro studies Results Antibiotic susceptibility tests. At an inoculum of 10 5 cfu/ml, the MIC/MBC for amikacin, ceftazidime and sulbactam were 16/32, 16/32 and > 256/> 256 mg/l, respectively. The combination of sulbactam with ceftazidime decreased the MIC and MBC of ceftazidime by a single dilution to 8 and 16 mg/l respectively. Killing curves. The combination of 16 mg/l ceftazidime 4 mg/l amikacin and 8 mg/l sulbactam exerted a synergistic activity at 24 h on a low inoculum of 5 x lo'cfu/ml (Figure (a)). Ceftazidime exhibited synergy with amikacin at a 16 mg/l of each drug as did ceftazidime with sulbactam at 64 and 8 mg/l respectively. Ceftazidime and amikacin alone were only transiently inhibitory but exerted no cidal activity even at high o 0 Time(h) Figure. Killing curves of a multidrug-resistant strain. P. aeruginosa. Initial inocula of 5 x 10* and 5 x 10' cfu/ml were incubated with various concentrations of ceftazidime, amikacin and sulbactam. Synergy was observed when the lower inoculum was exposed to combinations of ceftazidime with amikacin, sulbactam and both drugs together (a) but none occurred at the higher inoculum (b). D. Control;, ceftazidime 64 mg/l;, amikacin 32 mg/l; 0. ceftazidime 64 mg/l and sulbactam 8 mg/l; #, ceftazidime 16 mg/l and amikacin 16 mg/l; O, ceftazidime 16 mg/l, amikacin 4 mg/l and sulbactam 8 mg/l.

6 702 Y. Q. Xiong et al. concentrations of 64 and 32 mg/l, respectively. There was no synergistic activity at the higher inoculum of 5 x 10 7 cfu/ml of when the same drug concentrations were used (Figure (b)). fi-lactamase production. The multidrug-resistant P. aeruginosa strain constitutively produced a cephalosporinase which degraded and mu/mg cephaloridine before and after induction by imipenem. The MIC of ticarcillin also declined from 8192 to 1024 mg/l in the presence of clavulanate indicating the production of penicillinase. Animal studies Amikacin alone was ineffective, despite a steady-state concentration 4 x MIC, whether treatment was started 12 h after infection or 48 h after infection (Table). When treatment was begun early, ceftazidime alone caused a single logio reduction in vegetation bacterial counts when compared to controls. The combination of ceftazidime with amikacin with or without sulbactam was more effective than ceftazidime alone, although the difference was not statistically significant. When treatment was started late, only combination regimens led to a significant reduction in vegetation bacterial counts when compared with controls. The results of giving 50 mg/kg amikacin as a twice-daily bolus together with ceftazidime and sulbactam combination were similar to those obtained after continuous infusion of the aminoglycoside nor did extending the infusion from 24 h to 48 h make any difference. None of the regimens sterilised the vegetations. Antibiotic assays A steady-state was reached after 2 h continuous infusion of 800 mg/kg ceftazidime and 400 mg/kg amikacin to yield serum concentrations of 127 ± 42 and 82 ± 13 mg/l, respectively (data not shown), and corresponding concentrations in vegetations of 78 ± 27 and 38 ± 8 /zg/g, respectively. Discussion Thesuccessful treatment of serious infection due to P. aeruginosa depends critically upon giving the most appropriate regimen at the optimal dose. We chose the rabbit model of experimental endocarditis since it represents a serious infection for which treatment fails in 35 to 75% of cases (Reyes et al., 1978; Reyes & Lerner, 1983) and in-vivo efficacy can be assessed in terms of the bactericidal rate after a short period of treatment. Using this model, we were able to demonstrate that early treatment and combination regimens were crucial elements for success therapy. Ceftazidime alone only significantly lowered vegetation counts of the multiply resistant P. aeruginosa when started 12 h after inducing infection. When started later, no reduction in viable counts was observed. Amikacin was ineffective when given alone, even at a high dosage, whether treatment was given early or late, even though serum concentrations were 4 x MIC and the steady-state concentration in the vegetations was approximately twice the MIC. Combined treatment sulbactam with either ceftazidime alone or with ceftazidime and amikacin was more effective than ceftazidime alone in reducing the mean vegetation titres of P. aeruginosa whether or not treatment was started early or late after establishing infection. Stable serum concentrations were attained by giving each of the drugs in a continuous infusion model so that the intrinsic activity of each drug or drug

7 Table. In vivo effect of the early and delayed iv administration of antibiotics by continuous infusion on the experimental model off. aeruginosa endocarditis Regimen Control Ceftazidime 800 mg/kg Amikacin 400 mg/kg Ceftazidime 800 mg/kg -I- amikacin 400 mg/kg Ceftazidime 800 mg/kg + sulbactam 400 mg/kg Ceftazidime 800 mg/kg + amikacin 400 mg/kg + sulbactam 400 mg/kg P < 0.05; k P < 0.01 Schefle's lest;, not determined. Bacterial counts in vegetations early therapy mean (log, 0 cfu/g) ± S.D. (no. of rabbits) 6.8 ± 0.3 (7) 5.7 ± 0.4 (7Y 7.2 ± 0.3 (6) 4.4 ± 0.5 (6/ 4.4 ± 1.2(8)* 4.6 ± 1.2(8)* late therapy mean (logio cfu/g) ± S.D. (no. of rabbits) 8.4 ±0.6 (14) 7.3 ± 1.4(10) 8.2 ± 0.5 (7) 6.3 ± 1.5(6) 6.4 ± 2.2 (6Y 6.9 ± 0.6 (6) ownloaded from at Pennsylvania State University on May 8, 2016 Serum steady-state concentration mean concentration ±S.D. (mg/l) 127 ±42 82 ± 13 Vegetation steady-state concentration mean concentration ±S.D. (^g/g vegetation) 78 ±27 38 ±8 f

8 704 Y. Q. XJong et al. combination could be assessed in vivo (Potel et al., 1992). We established a good correlation between the antibacterial activity of each compound alone and in combination both in vitro and in vivo although our results must be interpreted with caution since the inhibitory activity of many antibiotics, particularly of /Mactams alone and in combination with aminoglycosides, is critically dependent upon the initial density of bacteria in vitro (Bayer, Eisenstadt & Morrison, 1984; Pefanis et al., 1993). The combination of a /Mactam agent with an aminoglycoside generally enhances bactericidal activity in experimental studies (Sande & Johnson, 1975; Bayer et al., 1987). Our data indicate that good in-vivo antibacterial activity can be expected as early as 24 h after beginning treatment even when infection is due to a resistant strain. Similar results were recently obtained with 10-day treatment (Pefanis et al., 1993) although, no regimen is able to sterilise the vegetations in this endocarditis model even after relatively prolonged therapy (Bayer et al., 1987; Pefanis et al, 1993). In our study, ceftazidime and amikacin attained concentrations in vegetations of 61% and 47% of those observed in serum respectively which agrees closely with the results reported by Bayer et al. (1988, 1989). It also did not seem to matter whether or not amikacin was administered as a twice-daily bolus injection or as a continuous infusion method thus ruling out any concentration-dependent activity in vivo at this dose. The role of the interval between the onset of the infection and the beginning of the treatment has been recently underlined by Gerber et al. (1993) who used the mouse thigh model of infection with P. aeruginosa. Their results and ours confirm the critical importance of early treatment of life-threatening infection due to this organism before the inoculum becomes too high at the infective focus. The mechanisms of resistance of ceftazidime and amikacin for this strain are not very clear. A cephalosporinase was produced at a low level by this strain, probably explaining at least in part the increase of the MIC for ceftazidime, and the relative efficacy of the combination with sulbactam, both in vitro and in vivo. Furthermore, it is probable that this strain produced also a penicillinase, as indicated in vitro by the reduction of the MIC of ticarcillin in the presence of clavulanate. Our main objective was to acquire therapeutic data of immediate use to the medical staff of our hospital. Our results indicate that high serum concentrations of ceftazidime (about 127 mg/l in continuous infusion) combined with sulbactam are therapeutically effective, whereas high concentrations of amikacin, combined with ceftazidime and sulbactam, are apparently unsuitable, at least for early antibacterial activity. Finally, early treatment (less than 24 h after emergence of the sepsis syndrome) is potentially an important prognostic factor. Of course, these conclusions can be drawn only for this particular epidemic clinical strain of P. aeruginosa, but might probably not apply to other strains. The strain we used produced low levels of /Mactamase constitutively, the cephalosporinase probably explaining, at least in part, the increase of the MIC of ceftazidime and its reduction in the presence of sulbactam. The strain also appeared to produced a penicillinase since the MIC of ticarcillin declined in the presence of clavulanate. These data suggest that a combination of sulbactam and ceftazidime might prove potentially useful for treating infections due to P. aeruginosa resistant to the cephalosporins unless the strain produces large amounts of /Mactamase as a result of de-repression.

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