Treatment of experimental pneumonia in rats caused by a PER-1 extended-spectrum ß-lactamase-producing strain of Pseudomonas aeruginosa

Olivier Mimoza,*, Najoua Elhelalib, Sophie Léotardb, Anne Jacolotc, Frederic Laurentb, Kamran Samiia, Olivier Petitjeanc and Patrice Nordmannb

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antibacterial activity of imipenem, cefepime and piperacillin-tazobactam alone or in combination with amikacin against a Pseudomonas aeruginosa strain producing an extended-spectrum ß-lactamase (PER-1) were compared using an experimental model of pneumonia in non-leucopenic rats. Animals were infected intratracheally with 8.0 ± 0.4 log 10 cfu of P. aeruginosa, and therapy was initiated 3 h later, by which time animal lungs showed bilateral pneumonia containing >7 log 10 P. aeruginosa cfu/g of tissue. Since rats eliminate antibiotics much more rapidly than humans, renal impairment was induced in all animals to simulate the pharmacokinetic parameters of humans. MICs determined using an inoculum of 4 log 10 cfu/mL were as follows: imipenem, 1 mg/L; cefepime, 8 mg/L; piperacillin-tazobactam, 32 mg/L; and amikacin, 16 mg/L. A noticeable inoculum effect was observed with the four antimicrobial agents tested, which was greatest for cefepime and piperacillin-tazobactam. In-vitro studies indicated that imipenem was the ß-lactam with the greatest bactericidal effect and that amikacin was synergic only in combination with cefepime and imipenem. Cefepime and piperacillin-tazobactam alone failed to decrease bacterial counts in the rats’ lungs 60 h after therapy onset, whereas imipenem and, to a lesser extent, amikacin significantly reduced the number of viable microorganisms. Combination of amikacin with any of the three ß-lactams tested was synergic, despite a high amikacin MIC for the infecting strain. These results paralleled our in-vitro data showing a marked inoculum effect for cefepime and piperacillin-tazobactam. Based on the results of this study, the best treatment for infections caused by this type of extended-spectrum ß-lactamase-possessing strain would be imipenem plus amikacin.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa remains a major pathogen in hospital-acquired infections. In the analysis of the National Nosocomial Infection Study data collected between 1986 and 1989, this bacterial species was one of the two commonest nosocomial pathogens isolated in American hospitals. 1 In Europe, P. aeruginosa has been reported to be involved in 29% of intensive care unit infections and 30% of lower respiratory tract infections. 2

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test organism

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 rat’s 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro studies

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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Table I. In-vitro susceptibility of the PER-1-producing P. aeruginosa strain to the antibiotics studied
 


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Figure 1. Viable counts of PER-1-producing P. aeruginosa RNL-1 with test antibiotics at three drug concentrations. The 7.8 log 10 cfu/mL inoculum was incubated for 24 h with no antibiotic ({blacksquare}), imipenem alone (•), amikacin alone ({diamond}) or combined antibiotic therapy ({circ}) given at the following concentrations: (a) imipenem 64 mg/L and amikacin 64 mg/L; (b) imipenem 8 mg/L and amikacin 16 mg/L; (c) imipenem 1 mg/L and amikacin 2 mg/L.

 


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Figure 2. Viable counts of PER-1-producing P. aeruginosa RNL-1 with test antibiotics at three drug concentrations. The 7.8 log 10 cfu/mL inoculum was incubated for 24 h with no antibiotic ({blacksquare}) cefepime alone (•), amikacin alone ({diamond}) or combined antibiotic therapy ({circ}) given at the following concentrations: (a) cefepime 128 mg/L and amikacin 64 mg/L; (b) cefepime 32 mg/L and amikacin 16 mg/L; (c) cefepime 8 mg/L and amikacin 2 mg/L.

 


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Figure 3. Viable counts of PER-1-producing P. aeruginosa RNL-1 with test antibiotics at three drug concentrations. The 7.8 log 10 cfu/mL inoculum was incubated for 24 h with no antibiotic ({blacksquare}), piperacillin- tazobactam alone (•), amikacin alone ({diamond}), or combined antibiotic therapy ({circ}) given at the following concentrations: (a) piperacillin-tazobactam 256 mg/L and amikacin 64 mg/L; (b) piperacillin-tazobactam 16 mg/L and amikacin 16 mg/L; (c) piperacillin-tazobactam 2 mg/L and amikacin 2 mg/L.

 
Pharmacokinetic analysis

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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Table II. Pharmacokinetics of antibiotics given intraperitonally to non-infected rats with renal impairment induced by uranyl nitrate
 
Therapy efficacy

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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Figure 4. Lung cfu/g of PER-1-producing P. aeruginosain rats at the treatment onset or after 60 h (control and treated groups). The treated groups were with imipenem (IMP) alone, piperacillin and tazobactam (TAZ) alone, cefepime (FEP) alone, amikacin (AMK) alone or with each &bgr;-lactam agent in combination with amikacin. Each mark represents a single animal. The horizontal bar indicates the median in each group. Statistical significance from one treated group to the other is indicated below (each ß-lactam alone versus the combination of the corresponding ß-lactam with amikacin) and above (each ß-lactam alone versus control).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We describe herein a reproducible model of P. aeruginosa pneumonia, the commonest nosocomial infection caused by this organism; this model allows therapeutic studies in animals with normal host defences. Initial mortality was relatively low and mainly the result of trauma from the operation or overwhelming sepsis. Therapy was initiated 3 h after intratracheal inoculation of bacteria, when animal lungs showed bilateral pneumonia, with >7log 10 P. aeruginosa cfu/g of tissue, an usual event in intensive care unit patients. 15 The use of uranyl nitrate to induce a transient impairment of the renal function is a convenient way to study the in-vivo pharmacodynamic properties of ß- lactams and aminoglycosides under pharmacokinetic conditions similar to those observed in humans (elimination half-lives for cefepime and amikacin were c.2 h and those for imipenem and piperacillin-tazobactam c.1 h). 16,17,18,19,20 The selected strain produced an ESBL, PER-1, which is so far the most widespread ESBL-producing P. aeruginosa. Since PER-1-producing strains are antibiotic resistant, the starting point of our study was to look at the in-vivo efficacy of imipenem, cefepime and piperacillin-tazobactam alone or in combination with amikacin. These three ß-lactams are among those used to treat nosocomial infections and are able to clear systemic or experimental infections caused by ESBL-producing Enterobacteriaceae. 21,22,23 Ciprofloxacin, the most usual agent of choice where a ß-lactam-resistant P. aeruginosa infection is suspected or confirmed, was not tested because the strain used in our experiments was highly resistant (MIC value of 8 mg/L, 8-fold higher than the French breakpoint for ciprofloxacin resistance). Moreover, in-vitro viable counts indicated that the combination of a ß-lactam and amikacin was more bactericidal than the corresponding ß-lactam and ciprofloxacin. 24

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


    Acknowledgments
 
This work was supported by a grant in-aid from Smith-Kline Beecham, Nanterre, France and Merck Sharp & Dohme-Chibret, Paris, France.


    Notes
 
* Tel: +33-1-45593219; Fax: +33-1-45593834; E-mail: olivier.mimoz{at}pbr.ap-hop-paris.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 21 October 1998; returned 8 January 1999; revised 1 March 1999; accepted 5 March 1999