a Service d Anesthésiologie, Hôpital Paul Brousse, Assistance Publique-Hôpitaux de Paris, 94804 Villejuif Cédex; b Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, 94275 Le Kremlin-Bicêtre Cédex; c Crépit 93, Centre de Recherche en Pathologie Infectieuse et Tropicale, Hôpital Avicenne, Assistance Publique-Hôpitaux de Paris, 93009 Bobigny Cédex, France
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Abstract |
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Introduction |
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This bacterium is intrinsically resistant to aminopenicillins and first-and second-generation cephalosporins as it produces a chromosomally mediated cephalosporinase. Ureidopenicillins and extended-spectrum cephalosporins such as ceftazidime remain active in the absence of cephalosporinase overproduction. 3 Extended-spectrum ß-lactamases (ESBLs) derived mainly from restricted-spectrum penicillinases (SHV-1, TEM-1 and TEM-2) have been extensively described in Enterobacteriaceae. They can hydrolyse most ß-lactams, including oxyiminocephalosporins, and their activity is inhibited in vitro by inhibitors such as clavulanic acid and tazobactam. Such enzymes have only been reported rarely in Pseudomonasspp.; they include TEM-42, 4 SHV-2 5 and OXA-16. 6 In 1993, we described a novel non-TEM-, non-SHV-derived class A ESBL, PER-1, in a P. aeruginosa clinical isolate in France. 7 This ß-lactamase has spread in Turkey: in a nationwide multicentre study conducted in this country in 1996, PER-1 was detected in 46% of strains of Acinetobacter spp. and in 11% of strains of Pseudomonas spp. isolated in hospitalized patients. 8 The clonal diversity and the high prevalence of the PER-1 type ESBL reported in this study imply that they may no longer be restricted to Turkish hospitals. This ß-lactamase has recently been isolated in an Acinetobacter sp. isolate in France.
Taking into account the possible emerging therapeutic problem caused by such ESBL-producing P. aeruginosa strains, we compared the in-vivo bactericidal activities of antibiotic regimens designed to simulate those used in humans, with cefepime, piperacillin-tazobactam and imipenem alone or in combination with amikacin using an animal model of pneumonia caused by a PER-1-producing P. aeruginosa isolate.
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Materials and methods |
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The infecting strain used in this study was the PER-1-producing P. aeruginosaRLN-1 strain. 7 This strain was originally isolated from the urine of a Turkish patient hospitalized in France. To restore its pathogenicity, this strain was submitted to two subsequent passages in mice inoculated intraperitonally for 24 h. It was stored at -70°C in Mueller-Hinton broth (bioMérieux, Paris, France) supplemented with 10% glycerol. Fresh inocula were prepared for each experiment from cultures grown for 24 h in 10 mL of trypticase soy broth (bioMérieux), then rinsed twice and suspended in normal saline before use.
Antimicrobial agents
Cefepime and amikacin were from Bristol-Myers Squibb (Paris, France), imipenem-cilastatin from Merck Sharp & Dohme-Chibret (Paris, France) and piperacillin-tazobatam from Wyeth Lederle (Paris-La-Défense, France). Antibiotic powders were freshly diluted with saline before each experiment according to the manufacturers instructions.
In-vitro studies
MICs were determined in duplicate by an agar dilution technique on Mueller-Hinton agar
(bioMérieux) using inocula ranging from 4 log
10 to 7 log
10 cfu per spot. In-vitro viable counts of the strain following exposure to each
antibiotic alone and in combination with amikacin were determined in duplicate in flasks
containing
aerated Mueller-Hinton broth. Three concentrations of each drug were chosen to simulate those
obtained in the animals lungs at peak, mid-interval and trough time periods. An overnight culture
was
diluted 1:100 to obtain a final concentration of 7.8 log
10 cfu/mL. Aliquots (2 x 0.1 mL) were removed 0, 2, 4, 6 and 24 h after
antibiotic
addition and quantitatively cultured on trypticase soy agar (bioMérieux) using a Spiral
Système plater (Interscience, Saint-Nom-La-Bretèche, France) after serial
dilutions
(up to 1 in 10
4) to minimize the effect of antibiotic carryover. Preliminary experiments indicated
a
perfect correlation between Spiral Système plating and traditional plating of serial
dilutions. The
lower limit of quantification of viable bacteria was 1 log
10 cfu/mL. In-vitro antibiotic synergy was defined as a 100-fold increase in
bacterial
killing at 24 h with an antibiotic combination as compared with the most active antibiotic alone.
Pharmacokinetics
Preliminary drug-dosing studies were performed in uninfected rats as described elsewhere 9 to determine if the subcutaneous 1 mg/kg uranyl nitrate (Merck, Darmstadt, Germany) dose previously used 9,10 was optimal for impairing the renal function of the rats so as to simulate the pharmacokinetics of cefepime, piperacillin, imipenem and amikacin in healthy humans. Briefly, 4 days after the uranyl nitrate injection, each rat received a single 1 mL intraperitoneal injection of each of the antimicrobial agents studied. Multiple blood samples were collected via a femoral catheter during the 8 h following antibiotic administration and immediately centrifuged to separate the plasma. Plasma samples were stored at -70°C and assayed within 7 days. Individual antibiotic pharmacokinetic parameters were determined using the Siphar software package (Simed, Créteil, France).
Antibiotic assay
Imipenem, piperacillin and cefepime concentrations were determined in plasma using a modified version of the HPLC assays described elsewhere. 11,12,13 The imipenem-containing plasma was, immediately after sampling, mixed 1:1 with stabilizing buffer containing equal volumes of 1 M morphilino-ethane sulphonate and ethylene glycol before freezing. The amikacin concentration was determined using an immunoenzyme assay (Emit; Syva, Dardilly, France). The lower detection limit of the assays was 0.5, 1, 1 and 1 mg/L for imipenem, piperacillin, cefepime and amikacin, respectively.
Pneumonia model
The study was conducted according to standard practices concerning ethics and animal procedures. The animal model used was adapted from one previously developed in our laboratory for another bacterial species, a strain of Enterobacter cloacae. 9,10 Briefly, male Wistar rats weighing 280-300 g were rendered renally insufficient by giving them 1 mg/kg of uranyl nitrate subcutaneously; they were anaesthetized 93 h later with phenobarbital (60 mg/kg) into the peritoneum and each rats trachea was exposed by a vertical midline incision. A 0.5 mL portion of a bacterial suspension containing 8.0 ± 0.4 log 10 cfu of P. aeruginosa was injected intratracheally with a syringe with a 25-gauge needle. Following inoculation, animals were gently shaken for 15 s to help distribution of the inoculum in the lungs.
Treatment regimens
Of the 120 animals used in this study, 90 were still alive 3 h after bacterial inoculation; at that time, six rats were killed by administering a lethal dose of phenobarbital to ensure that pneumonia had been established. The remaining rats were randomly assigned to one control group (i.e. no antibiotic) or seven treatment groups. Treatment groups received ip injections of imipenem-cilastatin alone (30 mg/kg every 6 h), piperacillin-tazobactam (120 mg/kg every 6 h), cefepime alone (60 mg/kg every 8 h), amikacin alone (25 mg/kg od) or a combination of each ß-lactam agent with amikacin given at the same dosages. These dosages were used in order to obtain serum concentrations close to those observed in humans. Therapy was started 3 h after bacterial inoculation and continued for 2.5 days.
Evaluation of antibiotic treatment
At 2.5 days, approximately 4-6 h (13-14 h for amikacin) after the last antibiotic dose, animals were killed by administering a lethal dose of phenobarbital. Blood, obtained by aortic puncture, was placed in a tube containing EDTA and centrifuged; the plasma was stored in two or three aliquots at - 70°C for determination of antibiotic and creatinine concentrations. Plasma creatinine concentrations were determined to document that renal impairment was well established. The two lungs were removed aseptically, gently blotted with sterile absorbent paper to remove blood, weighed, placed in 25 mL of ice saline and homogenized (Ultraturrax; Staufen, Germany). The homogenate was cultured quantitatively after serial dilution on trypticase soy agar using a Spiral Système plater. After overnight incubation at 37°C, viable bacteria were counted; the counts were expressed as log 10 cfu/g of lung. When no bacterial growth was detected, the lower detection limit for the specific animal was entered in the statistical analysis. In-vivo antibiotic synergy was defined as a bactericidal effect of the drug combination significantly greater than the sum of the bactericidal effects of each agent alone. 14
Determination of emergence of imipenem resistance during therapy
Since the emergence of P. aeruginosa resistant to the most active ß-lactam agent, imipenem, would be dramatic, such resistant clones were sought by plating 5 x 200 µL of the lung homogenates from the imipenem-treated rats on to imipenem-containing agar (16 mg/L). After aerobic incubation for 48 h at 37°C, emergence of resistant strain(s) was defined as growth of at least one colony of P. aeruginosa on these imipenem-containing plates. Their number was compared with those obtained from P. aeruginosa isolated from untreated animals. The search for in-vivo-acquired ß-lactam-resistant mutants was limited to imipenem since imipenem-resistant P. aeruginosa strains occur frequently in vivo.
Statistical analysis
Results are expressed as medians and their ranges. Lung bacterial densities in the control and treatment groups were compared using one-way non-parametric analysis of variance (Kruskal-Wallis test); when the value of this test was statistically significant, each treatment group was compared with the control group and each of the other treatment groups using the Mann-Whitney U-test. For all tests, a Pvalue of <0.05 was considered significant.
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Results |
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The susceptibility of the PER-1-producing P. aeruginosa strain to the antimicrobial agents tested in this study is indicated in Table I. According to the French breakpoint concentrations, the strain was susceptible to imipenem, moderately susceptible to piperacillin- tazobactam and cefepime, and resistant to amikacin. As expected, an inoculum effect was observed with the ß-lactam agents; this effect was greatest for piperacillin-tazobactam and cefepime. More surprisingly, such an inoculum effect was also noted with amikacin. Viable counts plotted against time indicated that, at the concentrations tested, imipenem was more bactericidal than cefepime or piperacillin-tazobactam (Figures 1-3). Amikacin showed an incomplete bactericidal effect at the highest concentration tested; it had no killing effect at intermediate and low concentration. Synergy was only observed between amikacin and cefepime or imipenem at the highest and intermediate antibiotic concentrations tested; this synergy was most pronounced with imipenem. At the trough antibiotic concentration, the combination of amikacin with any of the ß-lactam agents was no more effective than the ß-lactam alone (Figures 1-3).
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The pharmacokinetic parameters of the tested antibiotics when given to renally insufficient rats simulated those reported in healthy humans (Table II). Creatinine concentrations in plasma measured 60 h after starting therapy were not statistically different between the study groups, indicating that the degree of renal impairment did not depend on the treatment received (data not shown).
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The six animals killed at the start of therapy presented bilateral pneumonia, with median P. aeruginosa counts of log 10 7.4 (range, 7.0-7.7) cfu/g of lung. Four animals died during the antibiotic treatment period; two of these had received no antibiotic, one had received imipenem alone and one had received cefepime and amikacin in combination. When killed, all untreated animals showed a spontaneous decrease in numbers of bacteria 60 h after the theoretical start of therapy (Figure 4). Bacterial counts in all treatment groups were equal to or lower than those in untreated animals (Figure 4). This decrease was only significant for animals receiving imipenem alone, amikacin alone or the combination of amikacin with either of the ß-lactam agents tested. Bacterial titres were also significantly lower in animals receiving combined antibiotic therapy than in those receiving each corresponding antibiotic alone. In-vivo synergy between a ß-lactam agent and amikacin was observed with the three antibiotics given (Figure 4). No P. aeruginosa isolate resistant to imipenem was detected from imipenem-treated animals.
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Discussion |
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Comparing the bactericidal activity of concentrations similar to those produced by human regimens of various ß-lactam agents and of amikacin over a total treatment period of 60 h, cefepime and piperacillin-tazobactam alone failed to decrease bacterial counts in the lungs, whereas imipenem and, to a lesser extent, amikacin, significantly reduced the number of viable microorganisms. Some authors claim that the combination of piperacillin and tazobactam may be active against enterobacterial species producing ESBLs (TEM or SHV derivatives). 21,22,23 However, our results indicate that a PER-1-producing P. aeruginosa strain is unlikely to be treated safely by piperacillin-tazobactam alone. Similarly, as known for some extended-spectrum cephalosporins and experimental infections caused by ESBL-producing enterobacterial strains, 22 cefepime alone was not efficient in our model. The efficacy of imipenem as compared with cefepime and piperacillin-tazobactam correlated well with our in-vitrodata indicating a greater inoculum effect for the latter two ß-lactams. The in-vivo bactericidal effect observed with amikacin is more surprising. Indeed, an in-vitro bacterial killing was only noted at a high concentration, whereas no bactericidal effect was observed at the intermediate and low concentrations used. Such elevated concentrations would be excepted for only a few hours in plasma of animals. A pronounced postantibiotic effect as well as a better bactericidal activity caused by spontaneous bacterial clearance may explain such results.
In-vivo synergy was noted between amikacin and each of the ß-lactam agents studied. Antibiotic combinations including a ß-lactam and an aminoglycoside have frequently produced an increased bactericidal effect in vivo in experimental models of aerobic Gram-negative bacillary infections which generally parallel an increased rate of in-vitro killing. 14,25 Although these synergic combinations were reported to be advantageous when treating infections in humans or animals with impaired immune defences, especially in neutropenic individuals, 26,27,28 it is not yet known if they are beneficial in patients with normal numbers of circulating polymorphonuclear leucocytes. A combination of a ß-lactam and amikacin is recommended for preventing emergence of antibiotic-resistant mutants in pneumonia caused by P. aeruginosa. 28
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Acknowledgments |
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Notes |
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References |
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Received 21 October 1998; returned 8 January 1999; revised 1 March 1999; accepted 5 March 1999