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3.
J Antimicrob Chemother ; 68(12): 2779-85, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23861308

ABSTRACT

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.


Subject(s)
Enterobacteriaceae/drug effects , Gene Expression/drug effects , Gene Transfer, Horizontal/drug effects , Pseudomonas aeruginosa/drug effects , beta-Lactamases/biosynthesis , beta-Lactamases/genetics , Blotting, Southern , DNA Transposable Elements , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enterobacteriaceae/genetics , Humans , Molecular Sequence Data , Plasmids/analysis , Pseudomonas aeruginosa/genetics , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA
4.
Curr Pharm Des ; 19(2): 209-22, 2013.
Article in English | MEDLINE | ID: mdl-22894618

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Humans , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , beta-Lactam Resistance , beta-Lactamases/genetics , beta-Lactamases/metabolism , beta-Lactams/pharmacology
6.
Antimicrob Agents Chemother ; 55(8): 3936-8, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21576436

ABSTRACT

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.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Gene Dosage , Gram-Negative Bacteria/genetics , Transcription Initiation Site , beta-Lactamases/biosynthesis , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , DNA Fingerprinting , DNA, Bacterial/genetics , Enterobacter cloacae/drug effects , Enterobacter cloacae/genetics , Enterobacter cloacae/metabolism , Escherichia coli K12/drug effects , Escherichia coli K12/genetics , Escherichia coli K12/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/metabolism , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , beta-Lactam Resistance , beta-Lactams/pharmacology
7.
Clin Microbiol Rev ; 22(4): 582-610, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19822890

ABSTRACT

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.


Subject(s)
Chromosomes, Bacterial , Drug Resistance, Multiple, Bacterial/genetics , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Humans , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics
8.
J Antimicrob Chemother ; 64(2): 294-300, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19468029

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cystic Fibrosis/complications , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Profiling , Humans , Membrane Transport Proteins/biosynthesis , Membrane Transport Proteins/genetics , Microbial Sensitivity Tests , Phenotype , Pseudomonas aeruginosa/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Saliva/microbiology , beta-Lactamases/metabolism
9.
Postgrad Med ; 120(3 Suppl 1): 46-52, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18931471

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Ciprofloxacin , Drug Resistance, Bacterial , Levofloxacin , Ofloxacin/pharmacology , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/drug effects , Anti-Bacterial Agents/administration & dosage , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , Drug Resistance, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Mutation/genetics , Ofloxacin/administration & dosage , Pneumococcal Infections/epidemiology , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/isolation & purification , United States/epidemiology
10.
Expert Rev Anti Infect Ther ; 5(5): 793-809, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17914914

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Bacterial Infections/drug therapy , Carbapenems/chemistry , Carbapenems/pharmacokinetics , Clinical Trials as Topic , Doripenem , Drug Resistance, Multiple, Bacterial/physiology , Ertapenem , Humans , Imipenem/chemistry , Imipenem/pharmacokinetics , Imipenem/pharmacology , Meropenem , Microbial Sensitivity Tests , Structure-Activity Relationship , Thienamycins/chemistry , Thienamycins/pharmacokinetics , Thienamycins/pharmacology , United States , beta-Lactams/chemistry , beta-Lactams/pharmacokinetics , beta-Lactams/pharmacology
11.
Antimicrob Agents Chemother ; 51(8): 2997-3000, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17517839

ABSTRACT

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.


Subject(s)
Anti-Infective Agents/pharmacology , Bacterial Proteins/metabolism , Ciprofloxacin/pharmacology , Mutation , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Up-Regulation , beta-Lactamases/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Humans , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , beta-Lactamases/genetics
12.
J Antimicrob Chemother ; 57(5): 999-1003, 2006 May.
Article in English | MEDLINE | ID: mdl-16513915

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/drug effects , Imipenem/pharmacology , Levofloxacin , Ofloxacin/pharmacology , Pseudomonas aeruginosa/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Multiple, Bacterial/genetics , Drug Synergism , Microbial Sensitivity Tests , Pseudomonas aeruginosa/genetics
13.
Biochem Pharmacol ; 71(7): 1057-65, 2006 Mar 30.
Article in English | MEDLINE | ID: mdl-16316633

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Drug Design , Drug Evaluation, Preclinical/methods , Pharmacokinetics , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Chemistry, Pharmaceutical , Drug Resistance, Multiple, Bacterial/drug effects , Humans
14.
Antimicrob Agents Chemother ; 49(11): 4763-6, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16251325

ABSTRACT

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.


Subject(s)
Bacterial Proteins/physiology , Imipenem/pharmacology , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Porins/physiology , Pseudomonas aeruginosa/drug effects , beta-Lactamases/physiology , Microbial Sensitivity Tests , Pseudomonas aeruginosa/genetics
15.
Clin Infect Dis ; 40 Suppl 2: S105-14, 2005 Feb 15.
Article in English | MEDLINE | ID: mdl-15712098

ABSTRACT

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.


Subject(s)
Anti-Infective Agents/pharmacology , Drug Resistance, Bacterial/physiology , Imipenem/pharmacology , Levofloxacin , Ofloxacin/pharmacology , Pseudomonas aeruginosa/drug effects , Ciprofloxacin/pharmacology , Drug Synergism , Drug Therapy, Combination , Humans , Microbial Sensitivity Tests , Pseudomonas Infections/microbiology
16.
Diagn Microbiol Infect Dis ; 50(1): 43-50, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15380277

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Drug Resistance, Multiple, Bacterial , Fluoroquinolones/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Base Sequence , Cross Infection/microbiology , DNA, Bacterial/analysis , Female , Gene Expression Regulation, Bacterial , Hospitals, University , Humans , Male , Microbial Sensitivity Tests , Molecular Sequence Data , Polymerase Chain Reaction , Pseudomonas Infections/diagnosis , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sampling Studies , Sensitivity and Specificity , Texas
17.
FEMS Microbiol Lett ; 236(1): 137-43, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15212803

ABSTRACT

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.


Subject(s)
Carbapenems/pharmacology , Porins/genetics , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Amino Acid Sequence , Base Sequence , Drug Resistance, Bacterial/genetics , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Mutagenesis, Insertional , Transcription, Genetic
18.
J Antimicrob Chemother ; 53(2): 297-304, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14729755

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins , Enzyme Inhibitors/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , beta-Lactamase Inhibitors , beta-Lactams/pharmacology , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/therapeutic use , Clavulanic Acid/pharmacology , Colony Count, Microbial , Drug Synergism , Enzyme Induction , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Microbial Sensitivity Tests , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Rabbits , beta-Lactamases/biosynthesis , beta-Lactams/pharmacokinetics , beta-Lactams/therapeutic use
19.
Acad Med ; 78(3): 307-12, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12634214

ABSTRACT

The authors discuss the damaging influence of informal and hidden curricula on medical students and describe a two-week clerkship in palliative care and clinical ethics at their school (Weill Medical College of Cornell University). This required clerkship, begun in 1999, uses reflective practice and a special pedagogic technique, participant observation, to counteract the influences of the informal and hidden curricula. This technique seeks to immerse the participant observer in the context of care. In their role as participant observers, students are relieved of any direct clinical responsibilities for two weeks so they have time for the careful observation and reflection required and also can consider the humanistic dimensions of practice, which are often displaced by the need to master diagnostic and therapeutic skills. Course objectives include identifying psychosocial and contextual factors that influence care, principles of pain and symptom management, and ethical and legal issues at the end of life. Students are expected to learn how to apply ethical norms to patient care, describe methods of pain and symptom management, communicate in an effective and humanistic manner, and articulate models of patient-centered advocacy. The clerkship fosters professionalism in patient care, appreciation of cultural diversity, and the student's ability to assume responsibility for developing competency in these areas. Although it is too early to know whether this clerkship will ultimately affect the practice patterns of students who experience it, short-term evaluation has been very favorable.


Subject(s)
Clinical Clerkship/ethics , Curriculum , Education, Medical, Undergraduate/ethics , Ethics, Clinical/education , Palliative Care/ethics , Professional Practice/ethics , Clinical Clerkship/organization & administration , Clinical Competence , Education, Medical, Undergraduate/organization & administration , Humans , Palliative Care/organization & administration , Professional Practice/organization & administration
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