ABSTRACT
The whole-genome sequences of 24 isolates of Neisseria gonorrhoeae with elevated minimum inhibitory concentrations (MICs) to azithromycin (≥2.0 µg/mL) were analyzed against a modified sequence derived from the whole-genome sequence of N. gonorrhoeae FA1090 to determine, by signal ratio, the number of mutant copies of the 23S rRNA gene and the copy number effect on 50S ribosome-mediated azithromycin resistance. Isolates that were predicted to contain four mutated copies were accurately identified compared with the results of direct sequencing. Fewer than four mutated copies gave less accurate results but were consistent with elevated MICs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Neisseria gonorrhoeae/drug effects , RNA, Ribosomal, 23S/genetics , Sequence Analysis, DNA , DNA, Bacterial/chemistry , DNA, Ribosomal/chemistry , Drug Resistance, Bacterial , Gene Dosage , Genome , Microbial Sensitivity Tests , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/isolation & purification , Point MutationABSTRACT
Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ.
Subject(s)
Cephalosporins/pharmacology , Fimbriae Proteins/genetics , Neisseria gonorrhoeae/drug effects , Neisseria gonorrhoeae/genetics , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial , Erythromycin/pharmacology , Gonorrhea/drug therapy , Gonorrhea/microbiology , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Neisseria gonorrhoeae/isolation & purification , Penicillin-Binding Proteins/genetics , Treatment FailureABSTRACT
Semisynthetic analogues of fumagillin, 1, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, 3 (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, 3 underwent ring-opening addition of water and HCl, leading to four products, 4-7, which were characterized in detail. The chlorohydrin, but not the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism. In agreement, chlorohydrin 6 was shown to revert rapidly to 3 in rat plasma. In an ex vivo assay, rats treated with purified acid degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together, the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a pharmacologically useful level of MetAP2 inhibition.
ABSTRACT
This study investigates the performance of a new statistically driven acute ischemia detection algorithm that can process data from two bipolar cutaneous or subcutaneous leads. During a start-up phase, the algorithm processes electrocardiogram signals to determine a normal range of ST-segment deviation as a function of heart rate. The algorithm then generates upper and lower ST-deviation thresholds based on the dispersion of the baseline ST-deviation data. After the start-up phase, persistent ST-deviation that is beyond either the upper or lower thresholds results in detection of acute ischemia. To test the algorithm, we performed long-term (10 day) Holter monitoring in a control group of 14 subjects. We also performed Holter monitoring during balloon angioplasty, and for 2 days after surgery, in 30 subjects who underwent elective percutaneous coronary interventions ("PCI"). We determined the percentage of balloon inflations the algorithm detected without producing false positive detections within the control group 10-day daily life data. The algorithm detected 17/17 LAD occlusions, 7/8 LCX occlusions, and 8/9 RCA occlusions. Our results suggest that automatically generated, subject-specific, heart-rate dependent ST-deviation thresholds can detect PCI induced myocardial ischemia without resulting in false positive detections in a small control group.
Subject(s)
Algorithms , Angioplasty, Balloon, Coronary/adverse effects , Electrocardiography/methods , Myocardial Ischemia/diagnosis , Postoperative Complications/diagnosis , Acute Disease , Female , Heart Rate , Humans , Male , Myocardial Ischemia/etiology , Myocardial Ischemia/physiopathology , Postoperative Complications/etiology , Postoperative Complications/physiopathology , Postoperative PeriodABSTRACT
In 1999, a cluster of gonococcal isolates exhibiting high Minimal Inhibitory Concentrations (MICs), to azithromycin (2.0-4.0 mg/l) were identified in Kansas City, MO. Isolates were characterized by auxotype/serovar class, lipoprotein (Lip) subtyping and sequencing of the mtrR gene, which has been implicated in decreased azithromycin susceptibility in the gonococcus. Isolates were Pro/IB-3 and contained the 17c Lip subtype. Molecular characterization of the mtrR gene revealed a 153 base pair insertion sequence located between the mtrR/mtrC promoter and the mtrC gene. Some isolates also contained a frame shift within the mtrR gene. Transformation of these mutations into an azithromycin-sensitive recipient strain resulted in transformants with MICs as high as 2.0 mg/l and inactivation of the mtrD gene reduced azithromycin MICs 270-fold. These results demonstrated that the mtr mutations were responsible for the increased MICs in these isolates.
