In-vitro and in-vivo bacterocodal interactions of clindamycin and ceftazidime, by non-beta-lactamase mechanisms, in experimental Pseudomonas aeruginosa endocarditis caused by a constitutive beta-lactamase overproducing strain

We studied a ceftazidime-resistant strain of Pseudomonas aeruginosa (PA-48) stably-derepressed for constitutive beta-lactamase overproduction, and its ceftazidime-susceptible parent (PA-96) in order to characterize the ability of clindamycin to: (1) enhance the in-vitro and in-vivo bactericidal activities of ceftazidime for PA-48; and (2) prevent beta-lactamase induction and spontaneous mutation to the derepressed state by the parental strain (PA-96). In vitro, clindamycin synergistically enhanced the bactericidal activity of ceftazidime vs PA-48. In the experimental aortic endocarditis model, the combination of clindamycin with ceftazidime significantly reduced mean intravegetation bacterial densities of PA-48 versus ceftazidime monotherapy and untreated controls at therapy days 6 and 11 (P less than 0.05). Exposure of growing PA-48 cells to clindamycin did not interfere with the hydrolytic function of extracted periplasmic beta-lactamase. Moreover, clindamycin did not suppress cefoxitin-mediated beta-lactamase induction in the parental strain (PA-96). In vitro, clindamycin prevented spontaneous mutation of PA-96 to the stably-derepressed state for beta-lactamase overproduction and also enhanced reversion of derepressed cells of PA-48 to the ceftazidime-susceptible parental phenotype by approximately 2 log10 cfu/ml. This latter effect was mirrored in vivo during clindamycin+ ceftazidime therapy of experimental endocarditis due to strain PA-48, as the proportion of ceftazidime-susceptible cells with vegetations increased by approximately 1-1.5 log10 cfu/g, versus untreated controls or ceftazidime monotherapy recipients. After clindamycin treatment ceased, vegetations contained predominantly ceftazidime-resistant variants. Clindamycin appeared to enhance bactericidal effects of ceftazidime vs PA-48 through non-beta-lactamase mechanisms probably involving promotion and maintenance of spontaneous reversion to the fully-repressed state. However, the concentrations of clindamycin required to achieve these effects are unlikely to be sustained at normal therapeutic dosage.     

Efficacy of ciprofloxacin in experimental aortic valve endocarditis caused by a multiply beta-lactam-resistant variant of Pseudomonas aeruginosa stably derepressed for beta-lactamase production.

The emergence of multi-beta-lactam resistance is a limiting factor in treating invasive Pseudomonas infections with newer cephalosporins. The in vivo efficacy of ciprofloxacin, a new carboxy-quinolone, was evaluated in experimental aortic valve endocarditis caused by a strain of Pseudomonas aeruginosa which is stably derepressed for beta-lactamase production and is resistant to ceftazidime and multiple other beta-lactam agents. A total of 51 catheterized rabbits with aortic catheters in place were infected with this strain and then received no therapy (controls), ceftazidime (75 mg/kg per day), or ciprofloxacin (80 mg/kg per day). Ciprofloxacin sterilized all blood cultures and significantly lowered vegetation densities of P. aeruginosa by day 2 of treatment versus controls (P less than 0.0005) and animals receiving ceftazidime (P less than 0.0005). This beneficial effect of ciprofloxacin was also noted on therapy days 6 and 11. Ciprofloxacin rendered most vegetations (85%) culture negative over the 11-day treatment period and achieved bacteriologic cure in 73% of animals (P less than 0.0005 versus other therapy groups). Ciprofloxacin prevented bacteriologic relapse at 6 days posttherapy. No ciprofloxacin resistance was detected among Pseudomonas isolates from cardiac vegetations. Ciprofloxacin warrants further evaluation in vivo versus multi-drug-resistant gram-negative bacillary infections.     

Role of beta-lactamase in in vivo development of ceftazidime resistance in experimental Pseudomonas aeruginosa endocarditis.

Two ceftazidime-resistant variants of Pseudomonas aeruginosa (PA-48, PA-60), obtained from cardiac vegetations of rabbits with endocarditis receiving ceftazidime therapy, were studied for mechanisms of resistance. Both resistant variants were stably derepressed for the type Id beta-lactamase, which was ceftazidime inducible in the parental strain (PA-96) used to initially infect the rabbits. There was no evidence of ceftazidime bioinactivation by the resistant strains, and their outer membrane permeabilities were comparable to those of the parental strain. No alterations were observed in patterns of outer membrane proteins or membrane lipopolysaccharides in the resistant variants as compared with the parental strain. Penicillin-binding protein patterns of the resistant variants revealed the absence of penicillin-binding protein 4 in both, with acquisition of a new protein of higher apparent molecular weight in PA-60. Calculation of the rate of appearance of ceftazidime in the periplasm at sub-MICs suggested that slow enzymatic hydrolysis of the beta-lactam, rather than nonhydrolytic trapping, was the major explanation for the induced resistance in vivo in strains PA-48 and PA-60.     

Development of beta-lactam resistance and increased quinolone MICs during therapy of experimental Pseudomonas aeruginosa endocarditis.

The in vivo efficacies of pefloxacin, a new fluoroquinolone, and amikacin-ceftazidime were compared in 50 rabbits with experimental aortic endocarditis caused by Pseudomonas aeruginosa. Animals were randomly chosen to receive 4 or 10 days of no therapy (controls), pefloxacin (40 mg/kg [body weight] per day, intramuscularly [i.m.]), or amikacin (30 or 80 mg/kg per day, i.m.)-ceftazidime (150 mg/kg per day, i.m.). Pefloxacin and both amikacin regimens significantly reduced vegetation bacterial densities compared with controls at days 4 and 10 of treatment (P less than 0.0005). By day 10 of therapy, between 33 and 40% of vegetations from amikacin-ceftazidime recipients contained ceftazidime-resistant bacteria (MICs, greater than 25 micrograms/ml); nitrocefin agar overlay confirmed that these ceftazidime-resistant variants were constitutive overproducers of beta-lactamase. At therapy days 4 and 10, approximately 30% of vegetations sampled from pefloxacin recipients contained bacteria for which pefloxacin MICs were four- to eightfold higher than the MIC for the parental strain used to initially induce endocarditis (MIC, 0.19 microgram/ml). These variants also exhibited increases in ciprofloxacin and ticarcillin MICs, as well as pleotropic resistance to chloramphenicol (but not to amikacin, ceftazidime, or tetracycline). Amikacin-ceftazidime, as well as pefloxacin, was effective in this model of aortic pseudomonal endocarditis. However, in vivo development of ceftazidime resistance and step-ups in pefloxacin MICs among intravegetation isolates were associated with inability to completely eradicate P. aeruginosa from aortic vegetations.     

Effects of inner-membrane-associated beta-lactamase on penicillin-binding protein assays. Study of stably derepressed Pseudomonas aeruginosa strains from experimental endocarditis

We studied penicillin-binding protein (PBP) profiles of two Pseudomonas aeruginosa strains stably derepressed for constitutive, type Id beta-lactamase overproduction. Substantial levels of beta-lactamase were found to be strongly associated with isolated inner membranes of these two strains, as well as from a plasmid-encoded, TEM-2 beta-lactamase-producing control strain of P. aeruginosa. The inner membrane-associated beta-lactamase resulted in significant decreases in the intensity on autoradiographs of PBPs labelled with 35S-penicillin, yielding spurious PBP profiles for these strains. Inner membrane beta-lactamases could be substantially removed by a sonication-ultracentrifugation step, producing the bona fide PBP profiles.     

Evidence for multiple forms of type I chromosomal beta-lactamase in Pseudomonas aeruginosa.

The multiple stages of derepression of the type I chromosomal beta-lactamase in Pseudomonas aeruginosa were examined. Mutants partially and fully derepressed for beta-lactamase were selected from a wild-type clinical isolate. An analysis of the beta-lactamase produced by these mutants and the induced wild type revealed significant differences in the products of derepression at each stage. Beta-lactamase produced by the fully derepressed mutant showed a lower affinity (Km, 0.113 mM) for cephalothin than that produced by the partially derepressed mutant (Km, 0.049 mM). However, due to a very large Vmax, the former possessed a much greater hydrolytic efficiency. Differences in substrate profile were also noted. Only beta-lactamase from the fully derepressed mutant hydrolyzed cefamandole, cefoperazone, and cefonicid. The partially derepressed mutant possessed a single beta-lactamase band with a pI of 8.4. The fully derepressed mutant possessed this band and an additional major band with a pI of 7.5. Induction of the wild type with cefoxitin produced both bands. The changes in physiologic parameters of the enzymes produced in the different stages of derepression suggest a complex system for beta-lactamase expression in P. aeruginosa. This may involve at least two distinct structural regions, each of which is under control of the same repressor.     

Nebulized and intravenous colistin in experimental pneumonia caused by Pseudomonas aeruginosa

Purpose

Emergence of multidrug-resistant strains in intensive care units has renewed interest in colistin, which often remains the only available antimicrobial agent active against resistant Pseudomonas aeruginosa. The aim of this study is to compare lung tissue deposition and antibacterial efficiency between nebulized and intravenous administration of colistin in piglets with pneumonia caused by P. aeruginosa.

Methods

In ventilated piglets, colistimethate was administered 24 h following bronchial inoculation of Pseudomonas aeruginosa (minimum inhibitory concentration of colistin = 2 μg ml^?1) either by nebulization (8 mg kg^?1 every 12 h, n = 6) or by intravenous infusion (3.2 mg kg^?1 every 8 h, n = 6). All piglets were killed 49 h after inoculation. Colistin peak lung tissue concentrations and lung bacterial burden were assessed on multiple post mortem subpleural lung specimens.

Results

Median colistin peak lung concentration following nebulization was 2.8 μg g^?1 (25–75% interquartile range = 0.8–13.7 μg g^?1). Colistin was undetected in lung tissue following intravenous infusion. In the aerosol group, peak lung tissue concentrations were significantly greater in lung segments with mild pneumonia (median = 10.0 μg g^?1, 25–75% interquartile range = 1.8–16.1 μg g^?1) than in lung segments with severe pneumonia (median = 1.2 μg g^?1, 25–75% interquartile range = 0.5–3.3 μg g^?1) (p < 0.01). After 24 h of treatment, 67% of pulmonary segments had bacterial counts <10² cfu g^?1 following nebulization and 28% following intravenous administration (p < 0.001). In control animals, 12% of lung segments had bacterial counts <10² cfu g^?1 49 h following bronchial inoculation.

Conclusion

Nebulized colistin provides rapid and efficient bacterial killing in ventilated piglets with inoculation pneumonia caused by Pseudomonas aeruginosa.     

Characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa.

A clinical isolate of Pseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a beta-lactamase. This beta-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pI of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pI of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km). PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type beta-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum beta-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.     

Left-sided endocarditis caused by Pseudomonas aeruginosa: successful treatment with meropenem and tobramycin

Medical treatment alone is rarely successful in left-sided infective endocarditis caused by Pseudomonas aeruginosa. We report the cure of such a case with high-dose meropenem in combination with tobramycin.     

Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm.

Imipenem induced high levels of beta-lactamase production in Pseudomonas aeruginosa biofilms. Piperacillin also induced beta-lactamase production in these biofilms but to a lesser degree. The combination of beta-lactamase production with other protective properties of the biofilm mode of growth could be a major reason for the persistence of this sessile bacterium in chronic infections.     

Pharmacodynamic study of beta-lactams alone and in combination with beta-lactamase inhibitors against Pseudomonas aeruginosa possessing an inducible beta-lactamase

OBJECTIVES: The antimicrobial efficacies of beta-lactams alone and in combination with beta-lactamase inhibitors were investigated by applying a rabbit tissue cage model against a strain of Pseudomonas aeruginosa with an inducible AmpC (iAmpC) beta-lactamase. METHODS: Two sterilized golf Wiffle balls were surgically implanted in the rabbit dorsal cervical area. After 4 weeks, Wiffle balls had filled with tissue cage fluid (TCF), in which 2 mL of 10(6) cfu/mL of the test isolate were inoculated. To achieve the same T > MIC as in humans, 400 mg/kg of the beta-lactams alone and in combination was administered twice a day via subcutaneous injection. The dosing regimens were as follows: piperacillin alone, 4 g piperacillin/0.5 g tazobactam; ticarcillin alone, 3 g ticarcillin/0.1 g clavulanate; and 3 g ticarcillin/ 0.3 g clavulanate. RESULTS: The changes in bacterial counts (log cfu/mL) after the 3 day treatments were as follows: 1.03 +/- 0.97 (control), -1.31 +/- 0.61 (piperacillin), -2.81 +/- 0.53 (4 g piperacillin/0.5 g tazobactam), -1.61 +/- 0.68 (ticarcillin), -3.42 +/- 0.75 (3 g ticarcillin/0.1 g clavulanate) and -1.65 +/- 1.47 log cfu/mL (3 g ticarcillin/0.3 g clavulanate). AmpC induction by high-dose clavulanate was observed in rabbit TCF, and was confirmed by the in vitro induction study. CONCLUSIONS: The study indicated that tazobactam significantly enhanced the antibacterial activity of piperacillin against iAmpC P. aeruginosa; clavulanate had synergy with the antibacterial activity of ticarcillin at low concentration, but had no effect on ticarcillin at high concentration due to AmpC induction by clavulanate.     

Resistance to beta-lactam antibiotics in Pseudomonas aeruginosa due to interplay between the MexAB-OprM efflux pump and beta-lactamase

We evaluated the roles of the MexAB-OprM efflux pump and beta-lactamase in beta-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from beta-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to beta-lactam resistance in both beta-lactamase-negative and beta-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed beta-lactamase. Overexpression of the efflux pump alone contributes to the high level of beta-lactam resistance in the absence of beta-lactamase.     

Successful therapy of experimental endocarditis caused by vancomycin-resistant Staphylococcus aureus with a combination of vancomycin and beta-lactam antibiotics

VRS1 is the first isolated strain of vancomycin-resistant Staphylococcus aureus (VRSA) found to carry the vanA gene complex previously described in Enterococcus. Under vancomycin pressure, VRS1 makes aberrant cell walls consisting of stem tetrapeptide and depsipeptide that lack the terminal D-Ala-D-Ala residues targeted by vancomycin. Previous data have suggested that this aberrant cell wall is not cross-linked by PBP2a, the enzyme responsible for cell wall transpeptidation in the presence of beta-lactam antibiotics. We examined the efficacy of treating VRS1 with a combination of vancomycin and beta-lactam antibiotics in vitro and in vivo. We found that the MIC of oxacillin for VRS1 decreased from >256 microg/ml to <1 microg/ml in the presence of vancomycin. Using the rabbit model of endocarditis, we treated VRS1-infected rabbits with nafcillin alone, vancomycin alone, or a combination of nafcillin and vancomycin. Treatment with nafcillin in combination with vancomycin cleared bloodstream infections within 24 h and sterilized 12/13 spleens (92%), as well as 8/13 kidneys (62%), following 3 days of treatment. Mean aortic valve vegetation counts were reduced 3.48 log(10) CFU/g with the combination therapy (compared to untreated controls) and were significantly lower than with either vancomycin or nafcillin given alone. VRS1 was extremely virulent in this model, as no untreated rabbits survived the 3-day trial. Treatment of clinical infections due to VRSA with the combination of vancomycin and beta-lactams may be an option, based on these results.     

Efficacy of amikacin and ceftazidime in experimental aortic valve endocarditis due to Pseudomonas aeruginosa.

The in vivo efficacies of amikacin, ceftazidime, and their combination were evaluated in experimental aortic valve endocarditis due to Pseudomonas aeruginosa. Eighty catheterized rabbits were infected with a P. aeruginosa strain susceptible to both amikacin and ceftazidime and then received no therapy (controls), amikacin (15 mg/kg per day), ceftazidime (100 mg/kg per day), or amikacin-ceftazidime. Amikacin-ceftazidime significantly lowered vegetation titers of P. aeruginosa at day 7 of therapy versus other regimens (P less than 0.0005). However, by day 14 of therapy, vegetation titers in animals receiving amikacin or ceftazidime regimens or both were not different from those of untreated controls; this was associated with in vivo development of amikacin resistance in most infected vegetations (79%), a phenomenon not seen at day 7 of therapy. Amikacin resistance was unstable in vivo, being undetectable in vegetations examined 5 days after treatment with amikacin had been completed. In contrast, ceftazidime resistance (first noted at day 7 of therapy in 12% of vegetations) persisted after termination of treatment with this agent. These in vivo observations on loss of amikacin resistance and persistence of ceftazidime resistance were mirrored during in vitro passage studies of amikacin- or ceftazidime-resistant P. aeruginosa strains isolated from cardiac vegetations. Amikacin resistance was no longer detectable by passage 5 in antibiotic-free media; however, ceftazidime resistance was stable despite 15 such passages. In vivo development of aminoglycoside-beta-lactam resistances was associated with poor bacteriologic efficacy in this model.     

Activity of beta-lactamase in Pseudomonas aeruginosa as studied by HPLC

Mutants with increased resistance were selected from a clinical isolate of Pseudomonas aeruginosa using ceftazidime, piperacillin and carbenicillin. The MICs of these antibiotics and of ticarcillin were determined for the parent strain and for the selected mutants. Subsequently, cell-free extracts of the strains were prepared and the rates of hydrolysis of several beta-lactam substrates by the extracts were determined by HPLC procedures. It appeared possible to determine beta-lactamase activities in the crude cell extracts at the low substrate concentrations which may be attainable in the periplasm of Gram-negative bacteria. It is concluded that the increased drug MICs for mutants selected with ceftazidime or piperacillin, but not for those selected with carbenicillin, were caused by increased chromosomal beta-lactamase activity.     

Amikacin + ceftazidime therapy of experimental right-sided Pseudomonas aeruginosa endocarditis in rabbits

We investigated the efficacy of a potent new antipseudomonal beta-lactam agent, ceftazidime, in a model of right-sided Pseudomonas endocarditis in 72 rabbits. Animals received either: no therapy (controls), amikacin (15 mg/kg/day), ceftazidime (100 mg/kg/day) or amikacin + ceftazidime. Amikacin + ceftazidime was significantly more effective than single-drug regimens in terms of reduction of mortality (p less than 0.01), prevention of pulmonary infarction (p less than 0.05), reduction of mean vegetation titers of Pseudomonas aeruginosa (p less than 0.05-p less than 0.0005), sterilization of vegetations (p less than 0.0005) and reduction in prevalence of bacteriologic relapses after therapy (p less than 0.005). There was no development of resistance in vivo to either amikacin or ceftazidime.     

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

The antibacterial activity of imipenem, cefepime and piperacillin-tazobactam alone or in combination with amikacin against a Pseudomonas aeruginosa strain producing an extended-spectrum beta-lactamase (PER-1) were compared using an experimental model of pneumonia in non-leucopenic rats. Animals were infected intratracheally with 8.0 +/- 0.4 log10 cfu of P. aeruginosa, and therapy was initiated 3 h later, by which time animal lungs showed bilateral pneumonia containing >7 log10 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 log10 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 beta-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 beta-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 beta-lactamase-possessing strain would be imipenem plus amikacin.     

Activities of tobramycin and azlocillin alone and in combination against experimental osteomyelitis caused by Pseudomonas aeruginosa.

Azlocillin and tobramycin were used alone and in combination in the treatment of chronic osteomyelitis due to Pseudomonas aeruginosa in rabbits. This combination showed in vitro synergy measured by both the checkerboard technique and time-kill curves. A marked inoculum effect was demonstrated in vitro with azlocillin and the infecting strain of P. aeruginosa. The minimal inhibitory concentration of azlocillin, with an inoculum of 10(5) organisms, was 12.5 micrograms/ml; when the inoculum size was increased to 10(7) organisms, the minimal inhibitory concentration rose to more than 500 micrograms/ml. In therapeutic trials, the combination of azlocillin and tobramycin, given for 28 days, was significantly better than either no therapy or azlocillin alone, but was not significantly better than tobramycin alone. Even after 4 weeks of combined therapy with azlocillin and tobramycin, P. aeruginosa was recovered from the bones of 60% of the treated rabbits.