Molecular interactions of carbapenem antibiotics with the multidrug efflux transporter acrb of escherichia coli

The drug/proton antiporter AcrB, engine of the major efflux pump AcrAB(Z)-TolC of Escherichia coli and other bacteria, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multi-drug resistance (MDR) phenotype. Although hundreds of small molecules are known to be AcrB substrates, only a few co-crystal structures are available to date. Computational methods have been therefore intensively employed to provide structural and dynamical fingerprints related to transport and inhibition of AcrB. In this work, we performed a systematic computational investigation to study the interaction between representative carbapenem antibiotics and AcrB. We focused on the interaction of carbapenems with the so-called distal pocket, a region known for its importance in binding inhibitors and substrates of AcrB. Our findings reveal how the different physico-chemical nature of these antibiotics is reflected on their binding preference for AcrB. The molecular-level information provided here could help design new antibiotics less susceptible to the efflux mechanism

Low cadmium concentration in whole blood from residents of Northern Sardinia (Italy) with special reference to smoking habits

Introduction. The present study was initiated to investigate the cadmium concentrations in whole blood of Northern Sardinian, non-occupationally exposed adult subjects. Sardinia is a large Italian island which differs genetically and environmentally from other mainland Italian areas. Methods. Two hundred and forty-three adults (157 females and 86 males) were selected in the study area from subjects who were undergoing blood collection for laboratory analysis during the period January 2005-May 2005. Whole blood was analysed by graphite furnace atomic absorption spectrometer equipped with a Zeeman-effect background corrector (Perkin Elmer ZL5100) and an auto sampler. The adopted analytical procedure uses the Stabilized Platform Temperature Furnace (STPF) technique. Results. The mean value of Blood Cadmium Concentration (BCdC), expressed as Geometric Mean, was 0.32 mg/l (CI 95%: 0.31-0.34 mg/l) significantly ranging from 0.27 mg/l (CI 95%: 0.26-0.29 mg/l) in non-smokers to 0.34 mg/l (CI 95%: 0.30-0.39 mg/l) in ex-smokers up to 0.47 mg/l (CI 95%: 0.42-0.53 mg/l) in smokers (p inf. 0.0001). Discussion. The results show that BCdC levels in Northern Sar- dinian non-occupationally exposed adults are lower than levels found in many other regions, including those within Italy. Nev- ertheless, similar values have been detected in other European countries and cities. Conclusions. In relation to other reports in which data were analysed by strata for smoking habit and age, we found similar BCdC values among non smokers. However, Sardinian smokers seem to show lower levels of blood cadmium

A framework for dissecting affinities of multidrug efflux transporter AcrB to fluoroquinolones

Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps

Imaging of SNR IC443 and W44 with the Sardinia Radio Telescope at 1.5 GHz and 7 GHz

Observations of supernova remnants (SNRs) are a powerful tool for investigating the later stages of stellar evolution, the properties of the ambient interstellar medium, and the physics of particle acceleration and shocks. For a fraction of SNRs, multi-wavelength coverage from radio to ultra high-energies has been provided, constraining their contributions to the production of Galactic cosmic rays. Although radio emission is the most common identifier of SNRs and a prime probe for refining models, high-resolution images at frequencies above 5 GHz are surprisingly lacking, even for bright and well-known SNRs such as IC443 and W44. In the frameworks of the Astronomical Validation and Early Science Program with the 64-m single-dish Sardinia Radio Telescope, we provided, for the first time, single-dish deep imaging at 7 GHz of the IC443 and W44 complexes coupled with spatially-resolved spectra in the 1.5-7 GHz frequency range. Our images were obtained through on-the-fly mapping techniques, providing antenna beam oversampling and resulting in accurate continuum flux density measurements. The integrated flux densities associated with IC443 are S_1.5GHz = 134 +/- 4 Jy and S_7GHz = 67 +/- 3 Jy. For W44, we measured total flux densities of S_1.5GHz = 214 +/- 6 Jy and S_7GHz = 94 +/- 4 Jy. Spectral index maps provide evidence of a wide physical parameter scatter among different SNR regions: a flat spectrum is observed from the brightest SNR regions at the shock, while steeper spectral indices (up to 0.7) are observed in fainter cooling regions, disentangling in this way different populations and spectra of radio/gamma-ray-emitting electrons in these SNRs.Comment: 13 pages, 9 figures, accepted for publication to MNRAS on 18 May 201

Sardinia Radio Telescope wide-band spectral-polarimetric observations of the galaxy cluster 3C 129

We present new observations of the galaxy cluster 3C 129 obtained with the Sardinia Radio Telescope in the frequency range 6000-7200 MHz, with the aim to image the large-angular-scale emission at high-frequency of the radio sources located in this cluster of galaxies. The data were acquired using the recently-commissioned ROACH2-based backend to produce full-Stokes image cubes of an area of 1 deg x 1 deg centered on the radio source 3C 129. We modeled and deconvolved the telescope beam pattern from the data. We also measured the instrumental polarization beam patterns to correct the polarization images for off-axis instrumental polarization. Total intensity images at an angular resolution of 2.9 arcmin were obtained for the tailed radio galaxy 3C 129 and for 13 more sources in the field, including 3C 129.1 at the galaxy cluster center. These data were used, in combination with literature data at lower frequencies, to derive the variation of the synchrotron spectrum of 3C 129 along the tail of the radio source. If the magnetic field is at the equipartition value, we showed that the lifetimes of radiating electrons result in a radiative age for 3C 129 of t_syn = 267 +/- 26 Myrs. Assuming a linear projected length of 488 kpc for the tail, we deduced that 3C 129 is moving supersonically with a Mach number of M=v_gal/c_s=1.47. Linearly polarized emission was clearly detected for both 3C 129 and 3C 129.1. The linear polarization measured for 3C 129 reaches levels as high as 70% in the faintest region of the source where the magnetic field is aligned with the direction of the tail.Comment: 19 pages, 17 figures, accepted for publication in MNRA

Has VZV epidemiology changed in Italy? Results of a seroprevalence study

The aim of the study was to evaluate if and how varicella prevalence has changed in Italy. In particular a seroprevalence study was performed, comparing it to similar surveys conducted in pre-immunization era. During 2013–2014, sera obtained from blood samples taken for diagnostic purposes or routine investigations were collected in collaboration with at least one laboratory/center for each region, following the approval of the Ethics Committee. Data were stratified by sex and age. All samples were processed in a national reference laboratory by an immunoassay with high sensitivity and specificity. Statutory notifications, national hospital discharge database and mortality data related to VZV infection were analyzed as well. A total of 3707 sera were collected and tested. In the studied period both incidence and hospitalization rates decreased and about 5 deaths per year have been registered. The seroprevalence decreased in the first year of life in subjects passively protected by their mother, followed by an increase in the following age classes. The overall antibody prevalence was 84%. The comparison with surveys conducted with the same methodology in 1996–1997 and 2003–2004 showed significant differences in age groups 1–19 y. The study confirms that in Italy VZV infection typically occurs in children. The impact of varicella on Italian population is changing. The comparison between studies performed in different periods shows a significant increase of seropositivity in age class 1–4 years, expression of vaccine interventions already adopted in some regions

Cryo-EM structure and molecular dynamics analysis of the fluoroquinolone resistant mutant of the AcrB transporter from salmonella

Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance

Chlorpromazine and amitriptyline are substrates and inhibitors of the acrb multidrug efflux pump

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly under-stood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regu-lators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlor-promazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by in-terfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) super-family are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acineto-bacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; how-ever, several issues have thus far hindered all efforts at designing new efflux in-hibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibi-tors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds

Observing galaxy clusters and the cosmic web through the Sunyaev Zel'dovich effect with MISTRAL

Galaxy clusters and surrounding medium, can be studied using X-ray bremsstrahlung emission and Sunyaev Zel'dovich (SZ) effect. Both astrophysical probes, sample the same environment with different parameters dependance. The SZ effect is relatively more sensitive in low density environments and thus is useful to study the filamentary structures of the cosmic web. In addition, observations of the matter distribution require high angular resolution in order to be able to map the matter distribution within and around galaxy clusters. MISTRAL is a camera working at 90GHz which, once coupled to the Sardinia Radio Telescope, can reach $12''$ angular resolution over $4'$ field of view (f.o.v.). The forecasted sensitivity is $NEFD \simeq 10-15mJy \sqrt{s}$ and the mapping speed is $MS= 380'^{2}/mJy^{2}/h$. MISTRAL was recently installed at the focus of the SRT and soon will take its first photons.Comment: To appear in Proc. of the mm Universe 2023 conference, Grenoble (France), June 2023, published by F. Mayet et al. (Eds), EPJ Web of conferences, EDP Science