meropenem and carbonyl-3-chlorophenylhydrazone

Hospital infections associated with multidrug-resistant (MDR) Pseudomonas aeruginosa are a worldwide public health problem. Efflux systems and biofilm formation are mechanisms related to resistance to carbapenemics. In this study, quantum dots (QDs) were used to evaluate the effect of carbonyl cyanide-3-chlorophenylhydrazone (CCCP), an efflux pump system inhibitor, on biofilm formation and antimicrobial resistance profile of P. aeruginosa strains. For this, QDs were covalently conjugated to meropenem (MPM) and incubated with a P. aeruginosa resistant isolate (P118) or a control sensitive strain (ATCC Pa27853). P118 was also analyzed with conjugates after previous CCCP efflux inhibitor incubation. Fluorescence microscopy images showed that both sensitive and resistant bacteria were efficiently labeled. Nevertheless, P118 isolates presented fluorescent cell agglomerates, suggesting biofilm formation. The addition of the CCCP changed the labeling profile of the resistant isolate, and the absence of agglomerates was observed, indicating no biofilm formation. Genetic assays revealed the presence of MexA and MexE genes encoding channel proteins from efflux pump systems in both resistant and sensitive strains. Disk-diffusion and broth microdilution tests determined drug susceptibility profiles in the presence and absence of CCCP for P118 isolates. We verified that the CCCP efflux system inhibitor may contribute to P. aeruginosa resistant phenotype reduction for some antimicrobials. This study verified the efficiency of QD-MPM conjugates to trigger and study biofilm formation, or its inhibition, before and after CCCP addition. QDs conjugated to antimicrobials can be used as nanotools to investigate multidrug-resistant bacterial strains on biofilm formation.

Topics: Anti-Bacterial Agents; Biofilms; Drug Resistance, Bacterial; Hydrazones; Meropenem; Pseudomonas aeruginosa; Quantum Dots

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

Topics: Anti-Bacterial Agents; Burkholderia cenocepacia; Chloramphenicol; Ciprofloxacin; Drug Resistance, Multiple, Bacterial; Hydrazones; Kanamycin; Meropenem; Microbial Sensitivity Tests; Novobiocin; Tetracycline; Thienamycins

Acinetobacter baumannii is an emerging pathogen worldwide that is most commonly associated with nosocomial infections and multi-drug resistance. In the present study we determined the mechanisms of carbapenem resistance and clonal diversity of A. baumannii nosocomial isolates in Hospital Civil de Guadalajara, Mexico.. A total of 303 clinical isolates of A. baumannii identified during a period expanding from 2004-2011 were analyzed for carbapenem resistance using several microbiological and molecular methods. Clonal relatedness of these isolates was determined using pulsed-field gel electrophoresis.. Of the 303 isolates, 84% were resistant to meropenem, 71.3% to imipenem and 78.3% the resistant isolates were positive for metallo-β-lactamases as determined by the phenotypic assay. In addition, 49.6% of carbapenem-intermediate or -resistant isolates carried the blaOXA-72 gene and 1.2% carried the blaVIM-1 gene. Efflux pump phenotype was responsible for reduced susceptibility to meropenem in 14.5% and to imipenem in 31.6% of the resistant isolates, respectively in the presence of the efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone. Strains representing different carbapenem-resistant patterns exhibited reduced expression of 22, 29, 33, and 43 kDa OMPs. Among the bacterial collection studied, 48 different clones were identified, two of which were predominant and persistently transmitted.. Carbapenemase production in combination with efflux pump expression, reduction in OMPs expression and the cross-transmission of clones appear to be major contributors to the high frequency of carbapenem-resistance observed in A. baumannii. To our knowledge, this is the first study to define the molecular mechanisms associated with carbapenem-resistance in A. baumannii in Mexico.

Topics: Acinetobacter baumannii; Acinetobacter Infections; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Biological Transport; Carbapenems; Cross Infection; Drug Resistance, Multiple, Bacterial; Electrophoresis, Gel, Pulsed-Field; Hospitals; Humans; Hydrazones; Imipenem; Meropenem; Mexico; Microbial Sensitivity Tests; Thienamycins