Dilek Ince

Serological Responses to an Avian Influenza A/H7N9 Vaccine Mixed at the Point-of-Use With MF59 Adjuvant: A Randomized Clinical Trial

IMPORTANCE Human infections with avian influenza A/H7N9 have resulted in high morbidity and mortality in China. OBJECTIVE To compare safety and immunogenicity of different doses of influenza A/Shanghai/2/13 (H7N9) vaccine mixed with or without the MF59 adjuvant. DESIGN, SETTING, AND PARTICIPANTS Multicenter, randomized, double-blind, phase 2 trial at 4 US sites enrolled 700 adults aged 19 to 64 years beginning in September 2013; 6-month follow-up was completed in May 2014. INTERVENTIONS The H7N9 inactivated virus vaccine was administered intramuscularly on days 0 and 21 at nominal doses of 3.75, 7.5, 15, or 45 µg of hemagglutinin (actual doses approximately 50% higher) with or without the MF59 adjuvant. A total 99, 100, or 101 participants were randomized to each group (7 groups; N = 700). MAIN OUTCOMES AND MEASURES Proportions achieving day 42 antibody titer of 40 or greater or seroconversion (a minimum 4-fold increase to titer ≥40) with the hemagglutination inhibition assay; vaccine-related serious adverse events through month 13; and solicited postvaccination symptoms through day 7. RESULTS Hemagglutination inhibition antibodies were minimal after participants received an unadjuvanted vaccine. After receiving 2 doses of H7N9 vaccine at a dosage of 3.75 µg plus the MF59 adjuvant, day 42 seroconversion occurred in 58 participants (59%; 95% CI, 48%-68%). The peak seroconversion occurred at day 29 in 62 participants (62%; 95% CI, 52%-72%). The day 42 geometric mean titer was 33.0 (95% CI, 24.7-44.1). Higher antigen doses were not associated with increased response. For the neutralizing antibody assays, after receiving 3.75 µg of H7N9 vaccine plus the MF59 adjuvant, day 42 seroconversion occurred in 81 participants (82%; 95% CI, 73%-89%). The day 42 geometric mean titer was 81.4 (95% CI, 66.6-99.5). There was no statistically significant difference in day 42 hemagglutination inhibition seroconversion after mixing adjuvant with either the first or both 15 µg doses (n = 34 [35%; 95% CI, 25%-45%] vs n = 47 [47%; 95% CI, 37%-58%], respectively; P = .10). Recent receipt of seasonal influenza vaccination and older age were associated with attenuated response. No vaccine-related serious adverse events occurred. Solicited postvaccination symptoms were generally mild with more local symptoms seen in participants who received the adjuvant. CONCLUSIONS AND RELEVANCE Point-of-use mixing and administration of 2 doses of H7N9 vaccine at the lowest tested antigen dose with MF59 adjuvant produced seroconversion in 59% of participants. Although these findings indicate potential value in this approach, the study is limited by the absence of antibody data beyond 42 days and the absence of clinical outcomes. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01938742.

Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

Mechanisms and Frequency of Resistance to Premafloxacin in Staphylococcus aureus: Novel Mutations Suggest Novel Drug-Target Interactions

ABSTRACT Premafloxacin is a novel 8-methoxy fluoroquinolone with enhanced activity against Staphylococcus aureus. We found premafloxacin to be 32-fold more active than ciprofloxacin against wild-type S. aureus. Single mutations in either subunit of topoisomerase IV caused a four- to eightfold increase in the MICs of both quinolones. A double mutation (gyrA and eithergrlA or grlB) caused a 32-fold increase in the MIC of premafloxacin, while the MIC of ciprofloxacin increased 128-fold. Premafloxacin appeared to be a poor substrate for NorA, with NorA overexpression causing an increase of twofold or less in the MIC of premafloxacin in comparison to a fourfold increase in the MIC of ciprofloxacin. The frequency of selection of resistant mutants was 6.4 × 10−10 to 4.0 × 10−7 at twofold the MIC of premafloxacin, 2 to 4 log10 less than that with ciprofloxacin. Single-step mutants could not be selected at higher concentrations of premafloxacin. In five single-step mutants, only one previously described uncommon mutation (Ala116Glu), and four novel mutations (Arg43Cys, Asp69Tyr, Ala176Thr, and Pro157Leu), three of which were outside the quinolone resistance-determining region (QRDR) were found. Genetic linkage studies, in which incross ofgrlA+ and outcross of mutations were performed, showed a high correlation between the mutations and the resistance phenotypes, and allelic exchange experiments confirmed the role of the novel mutations in grlA in resistance. Our results suggest that although topoisomerase IV is the primary target of premafloxacin, premafloxacin appears to interact with topoisomerase IV in a manner different from that of other quinolones and that the range of the QRDR of grlA should be expanded.

Quinolone Resistance Due to Reduced Target Enzyme Expression

We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G-->A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41 degrees C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41 degrees C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin in Staphylococcus aureus

ABSTRACT Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two- to fourfold more active against wild-typeStaphylococcus aureus ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single grlBA mutations caused two- to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in gyrA andgrlA or grlB caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100- to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.

Activity of and Resistance to Moxifloxacin in Staphylococcus aureus

ABSTRACT Moxifloxacin has enhanced potency against Staphylococcus aureus, lower propensity to select for resistant mutants, and higher bactericidal activity against highly resistant strains than ciprofloxacin. Despite similar activity against purified S. aureus topoisomerase IV and DNA gyrase, it selects for topoisomerase IV mutants, making topoisomerase IV the preferred target in vivo.

Topoisomerase targeting with and resistance to gemifloxacin in Staphylococcus aureus.

ABSTRACT Gemifloxacin, a novel quinolone with potent activity against Staphylococcus aureus, was 8- to 16-fold more active against wild-type S. aureus than ciprofloxacin. The two- to fourfold increase in the MIC of gemifloxacin in genetically defined grlBA mutants and the twofold increase in a single gyrA mutant, supported by the low frequency of selection of resistant mutants at twice the MIC (7.4 × 10−11 to 1.1 × 10−10), suggested similar targeting of the two enzymes by gemifloxacin. Dual mutations in both gyrase and topoisomerase IV caused a 64- to 128-fold increase in the MIC of gemifloxacin, similar to that seen with ciprofloxacin. Gemifloxacin also had similar activity in vitro against topoisomerase IV and gyrase purified from S. aureus (50% inhibitory concentrations of 0.25 and 0.31 μg/ml, respectively). This activity was 10- to 20-fold higher than that of ciprofloxacin for topoisomerase IV and 33-fold higher than that for gyrase. In contrast to the in vitro findings, only topoisomerase IV mutants were selected in first-step mutants. Overexpression of the NorA efflux pump had a minimal effect on resistance to gemifloxacin, and a mutation in the promoter region of the gene for NorA was selected only in the sixth step of serial selection of mutants. Our data show that although gemifloxacin targets purified topoisomerase IV and gyrase similarly in vitro, topoisomerase IV is the preferred target in the bacteria. Selection of novel resistance mutations in grlA requires further expansion of quinolone-resistance-determining regions, and their study may provide increased insight into enzyme-quinolone interactions.

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

UNLABELLED The opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa. IMPORTANCE The inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison with Ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus is a major cause of communityand hospital-acquired infections. Although fluoroquinolones have been in use for slightly more than a decade, the development and rapid dissemination of resistance in staphylococci, especially in methicillin-resistant strains, has already limited their use and necessitated the development of newer fluoroquinolones. Gatifloxacin, a newly developed 6-fluoro8-methoxyquinolone with enhanced activity against staphylococci in comparison with older quinolones,[1-3] may be a candidate for treatment of some resistant staphylococcal infections. In this study, we evaluated the activity of gatifloxacin in comparison with ciprofloxacinagainst geneticallydefinedmutantsofS.aureus, determined the frequency of selection of resistant mutants and characterised these mutants to determine the drug target in S. aureus.

Antimicrobial Stewardship Training for Infectious Diseases Fellows: Program Directors Identify a Curriculum Need

A needs assessment survey of infectious diseases (ID) training program directors identified gaps in educational resources for training and evaluating ID fellows in antimicrobial stewardship. An Infectious Diseases Society of America-sponsored core curriculum was developed to address that need.