Effect of drug treatment options on the mobility and expression of blaKPC.

OBJECTIVES: Both transposition and increases in gene expression have been implicated in the success of KPC-producing pathogens, but the stimulus required for these phenomena are unknown. It is possible that exposure to antimicrobials during patient treatment increases bla(KPC) expression or induces Tn4401 transposition. The purpose of this study was to determine if exposure to carbapenems or other antimicrobial drug classes could stimulate expression of bla(KPC) or the in vitro transposition of Tn4401. METHODS: Five KPC-producing clinical isolates were evaluated in this study. Gene expression of RNA from each isolate exposed to subinhibitory, MIC or suprainhibitory levels of antibiotics was evaluated using real-time RT-PCR. Southern blots were performed on plasmids from isolates exposed to subinhibitory levels of antibiotics. RESULTS: There were subtle changes in bla(KPC) RNA expression following antibiotic exposure that were both strain and drug dependent. Multiple plasmids ranging from ~8 to >200 kb were observed for the Enterobacteriaceae isolates, whereas the Pseudomonas aeruginosa isolate had one ~55 kb plasmid. No changes in hybridization patterns or binding intensity for the bla(KPC) probe were observed after antibiotic exposure. CONCLUSIONS: While the changes in bla(KPC) RNA expression are subtle, the different responses observed suggest both strain- and genera-specific variations in response to different antibiotic treatments.

Mechanisms of ß-lactam resistance among Pseudomonas aeruginosa.

Treatment of serious P. aeruginosa infections becomes more challenging with each passing year. As this pathogen acquires more transferrable resistance mechanisms and continues to rapidly adapt and emerge resistant during the course of antimicrobial therapy, we face the growing threat of pan-resistance. This review has focused on those mechanisms that directly impact the future of ß-lactam antibiotics, including the production of ß-lactamases, porin-mediated resistance, and/or the overexpression of RND efflux pumps. With the pipeline of new anti-pseudomonal agents diminishing, it is essential that novel therapeutic strategies be explored. These include targeting biofilm formation and maintenance, virulence factors, and resistance mechanisms. Furthermore, we must continue to search for effective antibacterial combinations to not only prevent further emergence of resistance but also treat resistant strains already in the environment.

bla(KPC) RNA expression correlates with two transcriptional start sites but not always with gene copy number in four genera of Gram-negative pathogens.

Klebsiella pneumoniae carbapenemase (KPC)-producing organisms are therapeutically and diagnostically challenging. It is possible that bla(KPC) gene expression plays a role in the variability observed in clinical susceptibility testing. bla(KPC) transformants together with 10 clinical isolates representing four genera were evaluated for bla(KPC) copy number and gene expression and correlated with ß-lactam MIC data. The data suggest that mechanisms other than gene copy number and expression of bla(KPC) contribute to variability in susceptibility when testing KPC-producing isolates.

Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms.

Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.

Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype.

OBJECTIVES: Although Pseudomonas aeruginosa from cystic fibrosis patients are well known for their antibiotic resistance, isolates that are highly susceptible to multiple drug classes have also been encountered. In this study, hypersusceptible P. aeruginosa isolates were analysed for changes in intrinsic resistance mechanisms to explain the observed phenotype. METHODS: P. aeruginosa strains PA30 and PA431 were isolated from the sputa of cystic fibrosis patients and susceptibilities were determined by agar dilution. Isolates were genetically unrelated by PFGE analysis. Expression of efflux pumps, porins, a chromosomal cephalosporinase and a gene, glmS, previously implicated in hypersusceptibility were evaluated by real-time RT-PCR, outer membrane protein analysis and beta-lactamase hydrolysis assays. RESULTS: PA30 was hypersusceptible to beta-lactams, fluoroquinolones and antimetabolites, with MICs at least 4-fold lower than those for the prototype strain PAO1, while PA431 was hypersusceptible to beta-lactams and antimetabolites. Both isolates overproduced the porin OprF but showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. PA30 had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance. PA431 showed down-regulation of oprM, the last gene in the mexAB-oprM operon, but overexpressed the mexXY pump. The ampC beta-lactamase was weakly inducible in strain PA30, corresponding to cefoxitin hypersusceptibility. CONCLUSIONS: The changes in expression of several intrinsic mechanisms in the hypersusceptible strains did not correlate with the observed phenotype. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility.

Pharmacodynamics of levofloxacin against characterized ciprofloxacin-resistant Streptococcus pneumoniae.

In a previous study, levofloxacin 750 mg eradicated 4 ciprofloxacin-resistant isolates of Streptococcus pneumoniae from an in vitro pharmacodynamic model (IVPM). However, quinolone resistance-determining region (QRDR) mutations were not detected among those isolates. This study further evaluates levofloxacin 500 mg and 750 mg against S pneumoniae strains with characterized QRDR mutations. Six isolates with levofloxacin minimum inhibitory concentrations (MICs) of 2 to 4 microg/mL were selected for this study. Strains 5401, 5409, and 5437 contained only parC mutations. Three additional strains contained 2 mutations each: strain 5429 (parC and parE ), strain 5442 (parC and gyrA), and strain 5445 (parC and gyrB). Logarithmic-phase cultures (approximately 1 x 10(7) CFU/mL) were inoculated into the peripheral compartment of the IVPM and exposed to peak free-drug concentrations achieved with levofloxacin 500 mg and 750 mg (PO) and ciprofloxacin 750 mg (PO). Elimination pharmacokinetics were simulated and changes in viable counts were measured over 30 h. Ciprofloxacin exhibited very little antibacterial activity against the 6 strains, while levofloxacin 750 mg rapidly killed and eradicated the 3 parC mutant strains and the dual parC/parE mutant strains. Although levofloxacin 500 mg initially decreased viable counts by 4.5 to 6 logs, inoculum regrowth was observed between 12 and 24 h for the 6 strains. Regrowth was not due to the selection of mutant subpopulations. The pharmacodynamics of both levofloxacin doses were substantially diminished against the 2 strains with dual mutations in both parC and gyrA/B. The rapid eradication of single parC and dual parC/parE mutants with levofloxacin 750 mg demonstrates that this dose may slow the emergence of resistance due to these mutations. The decreased efficacy of both levofloxacin doses against the double parC and gyrA/B mutants highlights the importance of preventing the development and spread of double mutants.

Carbapenems in the USA: focus on doripenem.

The threat of antibacterial resistance continues to increase globally, and therapeutic options for the treatment of some serious infectious diseases are diminishing. The carbapenems are a potent class of broad-spectrum drugs, and their stability against hydrolysis by many important beta-lactamases make them an important weapon in the treatment of beta-lactamase-producing bacterial pathogens. This review focuses on four carbapenems of clinical importance in the USA: imipenem, meropenem, ertapenem and doripenem. After a historical review of carbapenem development, these four carbapenems are evaluated based on their mechanism of action, spectrum of activity, potency, pharmacodynamics, clinical pharmacokinetics, clinical profiles and toxicity issues.

Increased expression of ampC in Pseudomonas aeruginosa mutants selected with ciprofloxacin.

Two Pseudomonas aeruginosa mutants exhibiting increased expression of ampC were selected during exposure to ciprofloxacin. These mutants also exhibited significant increases in mexCD-oprJ expression, but further studies failed to show a link between the increased expression of mexCD-oprJ and ampC. Increased ampC expression was not related to mutations within ampR, the ampC-ampR intergenic region, ampD, ampDh2, or ampDh3 or to changes in the levels of expression of these amidase genes. However, ampD complementation restored wild-type levels of ampC expression and ceftazidime susceptibility, suggesting alternative mechanisms of ampC regulation.

Levofloxacin/imipenem prevents the emergence of high-level resistance among Pseudomonas aeruginosa strains already lacking susceptibility to one or both drugs.

OBJECTIVES: Previous studies have demonstrated that a combination of levofloxacin with imipenem could prevent the emergence of resistance during the treatment of susceptible Pseudomonas aeruginosa isolates in a two-compartment pharmacodynamic model of infection. In this study, the efficacy of levofloxacin/imipenem was further evaluated against a panel of characterized P. aeruginosa strains that lacked susceptibility to one or both drugs in the combination. METHODS: Five P. aeruginosa strains with characterized resistance mechanisms were evaluated. Log-phase cultures were inoculated into the peripheral compartment of the in vitro pharmacokinetic model and treated using simulated doses of 750 mg levofloxacin (dosed every 24 h) and 250 mg or 1 g doses of imipenem (dosed every 12 h). Peak levels were adjusted for protein binding. Pharmacodynamic interactions were evaluated by measuring the changes in viable counts over 30 h. To evaluate the emergence of resistance, samples removed at 30 h were plated onto agar containing the drug at 4x MIC, and potential mutants were evaluated for changes in susceptibility. RESULTS: Against strains overexpressing MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps, levofloxacin/imipenem prevented the emergence of resistance and achieved a 5 log total kill of one strain and eradication of two strains. Levofloxacin/imipenem also eradicated an imipenem-resistant strain lacking OprD. Although the combination initially killed 6-7 logs of a dual-resistant strain lacking OprD and overexpressing MexXY, it could not prevent the emergence of resistance when the 250 mg dose of imipenem was simulated in the combination. However, when the 1 g dose of imipenem was simulated with the combination, resistance was suppressed. CONCLUSIONS: These data suggest that levofloxacin/imipenem may be an effective combination for preventing the emergence of resistance among P. aeruginosa, even with strains already lacking susceptibility to one or both drugs in the combination. Clinical evaluation of this combination is warranted.

The role of pharmacodynamic research in the assessment and development of new antibacterial drugs.

Antibacterial resistance continues to increase world wide, with some bacterial pathogens exhibiting resistance to virtually all available drugs. As the plague of antibacterial resistance continues to grow and create serious therapeutic problems, it is essential that the development of new antibacterial agents continue. Pharmacodynamic research plays an important role in the development of new antibacterial agents, as pharmacodynamic data can help define the clinical potential of a new drug and identify the strengths and weaknesses in comparison to other drugs already on the market. Furthermore, pharmacodynamic experiments can help focus the clinical phases of drug development by providing key information on the pharmacodynamic parameters that influence efficacy and the pharmacodynamic targets that should be achieved to optimize clinical success. Characterization of these pharmacodynamic properties for a new drug in development can help direct the design of the best dose and dosing strategy for clinical trials. This review will focus on the tools, methods, and strategies used to characterize the pharmacodynamics of antibacterial agents and aide in their development for clinical use.

AmpC and OprD are not involved in the mechanism of imipenem hypersusceptibility among Pseudomonas aeruginosa isolates overexpressing the mexCD-oprJ efflux pump.

Pseudomonas aeruginosa strains that overexpress mexCD-oprJ become hypersusceptible to imipenem. Disruption of AmpC induction has been suggested to cause this phenotype. However, data from this study demonstrate that hypersusceptibility to imipenem can develop without changes in ampC expression or AmpC activity. Furthermore, hypersusceptibility is not caused by changes in expression of the outer membrane porin, OprD.

Levofloxacin-imipenem combination prevents the emergence of resistance among clinical isolates of Pseudomonas aeruginosa.

A 2-compartment in vitro pharmacokinetic model (IVPM) was used to assess the potential of a levofloxacin-imipenem combination to prevent the emergence of resistance during treatment of Pseudomonas aeruginosa infection. Log-phase cultures (10(8) cfu/mL) of 3 clinical isolates were inoculated into the peripheral compartment of the IVPMs and were treated with simulated human doses of levofloxacin (750 mg) and imipenem (250 mg). Pharmacodynamics and the emergence of resistance were evaluated over the course of 24 h. Resistant mutants were evaluated for transcriptional expression of specific efflux pumps. Initially, rapid killing was observed in association with each regimen. However, with levofloxacin and imipenem alone, rapid regrowth was observed as a result of the selection of resistant subpopulations. Analysis of mutants selected by levofloxacin demonstrated that mexEF-oprN-overexpressing subpopulations resistant to both levofloxacin and imipenem were selected from cultures of all 3 strains. Nevertheless, the levofloxacin-imipenem combination rapidly eradicated all 3 P. aeruginosa strains. These data suggest that levofloxacin-imipenem may be an effective combination for preventing the emergence of resistance among P. aeruginosa strains, even when subpopulations resistant to both drugs are present. Further studies are warranted to evaluate the use of this combination against strains with established resistance to either or both drugs.

Multidrug resistance associated with mexXY expression in clinical isolates of Pseudomonas aeruginosa from a Texas hospital.

Fluoroquinolones and carbapenems are important drug classes used for the treatment of Pseudomonas aeruginosa infections. However, overexpression of the P. aeruginosa efflux pump, MexEF-OprN, can provide dual resistance to both fluoroquinolones and carbapenems. Recently, a hospital in Texas encountered several P. aeruginosa isolates resistant to both of these drug classes. The purpose of this study was to determine whether the mechanism responsible for this multidrug resistance involved the overexpression of MexEF-OprN. To test this hypothesis, 7 clinical isolates from the Texas hospital were analyzed for clonality, antimicrobial susceptibility, expression of the porin, oprD, and four multidrug-resistant efflux pumps (mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY), quinolone resistance-determining region mutations, and beta-lactamase production. Two groups of isolates possessed similar pulse field gel electrophoresis patterns indicating their genetic relatedness. In addition to fluoroquinolone and carbapenem resistance, each isolate also displayed varying degrees of susceptibility to additional beta-lactams tested and resistance to gentamicin. Interestingly, none of the 7 clinical isolates overexpressed mexEF-oprN as determined by semiquantitative reverse transcriptase polymerase chain reaction, but overexpression of mexXY was observed in 6 of the 7 isolates. All 7 isolates showed a decrease of OprD in the outer membrane and a reduction in transcriptional expression of oprD compared to wild-type strain, PAO1. These results demonstrate that multidrug resistance to the fluoroquinolones and carbapenems in these clinical isolates was not a result of the overexpression of the mexEF-oprN pump. Instead, resistance to these two agents seemed to arise through independent mutational events.

Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance.

Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem. These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of oprD transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains. Expression of oprD could only be detected in two of the strains, but expression was very low as indicated by the high number of RT-PCR cycles required to amplify the product. PCR was performed to amplify the oprD gene using primers upstream of the promoter and downstream of the structural gene. Amplified products were sequenced, and sequences were compared to wild-type P. aeruginosa strain PAO1. Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon. The other five isolates provided PCR products that were 1.3-1.6 kb larger than expected, suggesting the presence of large inserts. Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within oprD. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD. Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates. This observation does not explain the observed decrease in transcriptional expression. This is the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by IS elements.

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.

Chromosomally-encoded resistance mechanisms of Pseudomonas aeruginosa: therapeutic implications.

Pseudomonas aeruginosa is an important nosocomial pathogen that presents a difficult therapeutic challenge. Although P. aeruginosa has been shown to acquire resistance mechanisms encoded on plasmids, this pathogen comes armed with multiple chromosomally-encoded mechanisms of resistance that can provide impressive intrinsic resistance, as well as the potential to mutate to high-level multi-drug resistance. Recent analysis of the sequenced genome of P. aeruginosa PAO1 suggested that we have just started to unlock the resistance potential of this pathogen. One of the most serious threats to the usefulness of beta-lactams against P. aeruginosa is the chromosomal AmpC cephalosporinase. When AmpC production increases through mutational events, overproduction of this cephalosporinase provides high-level resistance to all beta-lactams except the carbapenems. Carbapenem resistance typically requires down-regulation of the outer membrane protein (OprD), which serves as the primary route of entry for carbapenems. Perhaps the most threatening of the resistance mechanisms encoded on the P. aeruginosa chromosome are the multi-drug efflux pumps. These pumps have the ability to extrude multiple classes of antibiotics from the periplasmic space, as well as the cytoplasm. Natural expression of efflux pumps in 'wild-type' cells plays an important role in the relatively decreased susceptibility of P. aeruginosa to antibiotics. However, the greatest therapeutic problems occur when these pumps are overproduced in mutants and high-level, multi-drug resistance develops. Although the development of infections with highly resistant strains of P. aeruginosa can present serious therapeutic challenges, the most troublesome threat associated with the chromosomally-encoded resistance mechanisms is the potential for high-level resistance to emerge during the course of therapy. When resistance emerges during therapy, clinical failure can occur and the therapeutic options for second-line therapy can become severely limited. Unfortunately, the emergence of resistance during therapy is not a rare event with P. aeruginosa and these three resistance mechanisms. Therefore, clinicians must be mindful of this threat when choosing an appropriate therapy, and usually appropriate therapy includes a combination of drugs. Since the standard combination of an aminoglycoside and a beta-lactam has been shown to be ineffective in preventing the emergence of some resistance problems, the search for more effective combinations must be a priority.

Pharmacodynamics of 750 mg and 500 mg doses of levofloxacin against ciprofloxacin-resistant strains of Streptococcus pneumoniae.

An in vitro pharmacokinetic model (IVPM) was used to evaluate the pharmacodynamics of the 750 mg and 500 mg doses of levofloxacin against 4 ciprofloxacin-nonsusceptible Streptococcus pneumoniae. Levofloxacin MICs ranged from 1.4 to 3.2 micro g/ml. Log-phase cultures (5 x 10(7) cfu/ml) were inoculated into the IVPM and exposed to the peak free-drug concentrations of levofloxacin achieved in human serum with each dose. Levofloxacin was dosed at 0 and 24 h, elimination pharmacokinetics were simulated, and viable counts were measured over 30 h. The 750 mg dose was rapidly bactericidal against all 4 strains, achieving eradication within 30 h. Against strains with levofloxacin MICs of 1.4 and 1.8 micro g/ml, the 500 mg dose exhibited pharmacodynamics similar to the 750 mg dose. In contrast, against strains with levofloxacin MICs of 2.6 and 3.2 micro g/ml, viable counts never fell below 10(4) cfu/ml. The rapid killing and eradication of these pneumococci by the 750 mg dose warrant the clinical evaluation of this new dose in the treatment of pneumococcal infections.

Pharmacodynamics of gatifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model: impact of area under the curve/MIC ratios on eradication.

Previous studies have demonstrated that fluoroquinolone area under the curve (AUC)/MIC ratios of 30 to 50 are sufficient to eradicate pneumococci from in vitro pharmacokinetic models (IVPMs). However, more systematic studies of the impact of AUC/MIC ratios on the antipneumococcal activities of fluoroquinolones are needed. In the present study, a two-compartment IVPM was used to evaluate the impact of AUC/MIC ratios on the pharmacodynamics of gatifloxacin against four strains of Streptococcus pneumoniae. Gatifloxacin MICs were 0.4 to 1 microg/ml, whereas levofloxacin MICs were 1.8 to 3.2 microg/ml. Since both peak concentration/MIC (peak/MIC) and AUC/MIC ratios affect fluoroquinolone pharmacodynamics, logarithmic-phase cultures (5 x 10(7) CFU/ml) were exposed to gatifloxacin at constant peak/MIC ratios of 2:1 to 3:1 at 0 and 24 h, elimination half-lives were varied to provide a range of AUC/MIC ratios, and changes in viable counts were measured over 30 h. As a comparison, levofloxacin was evaluated at similar peak/MIC ratios and at AUC/MIC ratios of 30 to 38. For each strain, killing rates through 4 to 8 h were similar since peak/MIC ratios were kept constant. However, continued killing and eradication were observed only when gatifloxacin AUC/MIC ratios were 27 to 48. Levofloxacin also provided eradication. In contrast, substantial regrowth was observed in most experiments when gatifloxacin AUC/MIC ratios were 9 to 24. These data provide further support that fluoroquinolone AUC/MIC ratios of approximately 30 or higher can be sufficient for eradication of pneumococci from IVPMs. Furthermore, the overall impact of the AUC/MIC ratio was not influenced by the strain evaluated or its susceptibility to gatifloxacin. Further studies with other fluoroquinolones and pneumococci that exhibit wider ranges of susceptibilities are warranted. In addition, similar studies with higher peak/MIC ratios are needed to better define the impact of AUC/MIC ratios and peak/MIC ratios on the antipneumococcal pharmacodynamics of fluoroquinolones.