Evidence of another anthropic impact on iguana delicatissima from the lesser Antilles: the presence of antibiotic resistant enterobacteria

The improper use of antibiotics by humans may promote the dissemination of resistance in wildlife. The persistence and spread of acquired antibiotic resistance and human-associated bacteria in the environment, while representing a threat to wildlife, can also be exploited as a tool to monitor the extent of human impact, particularly on endangered animal species. Hence, we investigated both the associated enterobacterial species and the presence of acquired resistance traits in the cloacal microbiota of the critically endangered lesser Antillean iguana (Iguana delicatissima), by comparing two separate populations living in similar climatic conditions but exposed to different anthropic pressures. A combination of techniques, including direct plating, DNA sequencing and antimicrobial susceptibility testing allowed us to characterize the dominant enterobacterial populations, the an- tibiotic resistant strains and their profiles. A higher frequency of Escherichia coli was found in the samples from the more anthropized site, where multi-drug resistant strains were also isolated. These results confirm how human-associated bacteria as well as their antibiotic-resistance determinants may be transferred to wildlife, which, in turn, may act as a reservoir of antibiotic resistance

Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate

open13noEnterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.openMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, EleonoraMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, Eleonor

Characterization of four novel bacteriophages targeting multi-drug resistant Klebsiella pneumoniae strains of sequence type 147 and 307

Introduction: the development of alternative antimicrobial strategies is deemed to be a high priority to deal with the challenge caused by the spread of multidrug-resistant (MDR) bacteria in clinical settings. according to several international organizations, phages or components thereof are one of these possible options that could be useful to treat bacterial infections. among the drug-resistant bacteria, carbapenem resistant Klebsiella pneumoniae (CR-Kp) are particularly worrisome, given the extensive MDR profiles, their pandemic dissemination and primary role in healthcare associated and life-threatening infections. In this study we isolated and characterised four lytic bacteriophages targeting two major high-risk clones of CR-Kp circulating in hospital environments, i.e., those belonging to Sequence Type (ST) 307 and ST147. Materials and methods: wastewater samples collected from hospitals located in central Italy were screened for the presence of phages by using a previously characterized collection of K. pneumoniae clinical isolates as hosts and the top-agar overlay technique. host specificity and infection efficiency was assessed by spot test and efficiency of plating, respectively. Dynamic of bacterial infections was determined by the one-step growth curve method. Phages were visualized through transmission electron microscopy (TEM) and their genomes were obtained and analysed by a Next Generation Sequencing approach followed by bioinformatics analysis. Results: four bacteriophages, named GP-1, GP-2, GP-4 and GP-5, have been isolated, purified and produced at high titres. collectively, two phages were able to selectively lyse 12/14 K. pneumoniae strains of ST307, while the other two were active only against all the tested K. pneumoniae strains of ST147 (n=12). phages maintain an overall good stability to temperature and pH changes and were characterized by infection cycles having latency periods ranging from 10 to 50 minutes and burst sizes of 10-100 PFU. results from TEM analysis and genome sequencing demonstrated that the four phages were of different families and allowed to rule out the presence of antibiotic resistance genes, virulence factors or toxins. Discussion and Conclusions: Considering their strictly lytic nature and their high selectivity towards two of the major high-risk clones of K. pneumoniae, the isolated phages can be considered as good candidates for their evaluation in animal models as members of cocktails for applications to treat severe infections caused by CR-Kp strains

Genomic epidemiology of carbapenem- and colistin-resistant Acinetobacter baumannii from Greece

Background: Acinetobacter baumannii is a worrisome healthcare-associated opportunistic pathogen that is naturally resistant to several antimicrobial agents and exhibits also a remarkable propensity to acquire new resistance traits. Colistin is used as last-resort agent for treatment of infections caused by carbapenem-resistant A. baumannii, but colistin resistance has also emerged. Materials/methods: A total of 44 carbapenem- and colistin-resistant (colR) A. baumannii isolated during the period 2015-2017 were obtained from 10 Greek hospitals . Antibiotic susceptibility testing was performed by using the Vitek2 automated instrument. Colistin minimum inhibitory concentrations (MICs) were obtained by reference broth microdilution method and interpreted according to the EUCAST susceptibility breakpoint of 2 mg/L. Whole genome sequencing was performed with an Illumina NovaSeq instrument. Results: Antimicrobial susceptibility testing confirmed that all strains were carbapenem-resistant, and 41/44 were also colR (colistin MIC range, 4-64 mg/L). Carbapenem resistance was associated to the production of OXA-23 by all strains. Analysis of the PmrA and PmrB proteins, involved in lipopolysaccharide modifications, revealed that all colR strains had the A226V mutation in PmrB. This mutation has been previously described to be associated with the colR phenotype. Strains carrying this mutation had colistin MICs ranging from 4 to 8 mg/L. In 5 strains, a second mutation in PmrB (either E140F or L178F) or in PmrA (either K172I or D10N) was detected. In these strains colistin MICs were 16 and 64 mg/L, respectively. Interestingly, the PmrB A226V mutation was present in strains of different lineages. Results from MLST analysis performed following the Pasteur scheme demonstrated the occurrence of two major Global Clones (GC1 and GC2), while results from the Oxford MLST scheme further differentiated the GC2 strains in 5 different STs (ST425, ST208, ST195, ST451 and ST436). Conclusions: Genomic analysis of carbapenem-and colistin-resistant A. baumannii isolates from different Greek hospitals revealed a heterogenous population, all sharing the same alteration in PmrB, suggesting a convergent evolution towards the same colR mechanism. This mutation was associated with low level colR. In some strains additional mutations of PmrA or PmrB were likely responsible for higher colistin MICs

Phantom validation of quantitative Y-90 PET/CT based dosimetry in liver radioembolisation

Background PET/CT has recently been shown to be a viable alternative to traditional post-infusion imaging methods providing good quality images of 90Y-laden microspheres after selective internal radiation therapy (SIRT). In the present paper, first we assessed the quantitative accuracy of 90Y-PET using an anthropomorphic phantom provided with lungs, liver, spine, and a cylindrical homemade lesion located into the hepatic compartment. Then, we explored the accuracy of different computational approaches on dose calculation, including (I) direct Monte Carlo radiation transport using Raydose, (II) Kernel convolution using Philips Stratos, (III) local deposition algorithm, (IV) Monte Carlo technique (MCNP) considering a uniform activity distribution, and (V) MIRD (Medical Internal Radiation Dose) analytical approach. Finally, calculated absorbed doses were compared with those obtained performing measurements with LiF:Mg,Cu,P TLD chips in a liquid environment. Results Our results indicate that despite 90Y-PET being likely to provide high-resolution images, the 90Y low branch ratio, along with other image-degrading factors, may produce non-uniform activity maps, even in the presence of uniform activity. A systematic underestimation of the recovered activity, both for the tumor insert and for the liver background, was found. This is particularly true if no partial volume correction is applied through recovery coefficients. All dose algorithms performed well, the worst case scenario providing an agreement between absorbed dose evaluations within 20%. Average absorbed doses determined with the local deposition method are in excellent agreement with those obtained using the MIRD and the kernel-convolution dose calculation approach. Finally, absorbed dose assessed with MC codes are in good agreement with those obtained using TLD in liquid solution, thus confirming the soundness of both calculation approaches. This is especially true for Raydose, which provided an absorbed dose value within 3% of the measured dose, well within the stated uncertainties. Conclusions Patient-specific dosimetry is possible even in a scenario with low true coincidences and high random fraction, as in 90Y–PET imaging, granted that accurate absolute PET calibration is performed and acquisition times are sufficiently long. Despite Monte Carlo calculations seeming to outperform all dose estimation algorithms, our data provide a strong argument for encouraging the use of the local deposition algorithm for routine 90Y dosimetry based on PET/CT imaging, due to its simplicity of implementation

Blocking GM-CSF receptor α with mavrilimumab reduces infiltrating cells, pro-inflammatory markers and neoangiogenesis in ex vivo cultured arteries from patients with giant cell arteritis

Effective and safe therapies are needed for the treatment of patients with giant cell arteritis (GCA). Emerging as a key cytokine in inflammation, granulocyte-macrophage colony stimulating factor (GM-CSF) may play a role in promoting inflammation in GCA.To investigate expression of GM-CSF and its receptor in arterial lesions from patients with GCA. To analyse activation of GM-CSF receptor-associated signalling pathways and expression of target genes. To evaluate the effects of blocking GM-CSF receptor α with mavrilimumab in ex vivo cultured arteries from patients with GCA.Quantitative real time PCR, in situ RNA hybridisation, immunohistochemistry, immunofluorescence and confocal microscopy, immunoassay, western blot and ex vivo temporal artery culture.GM-CSF and GM-CSF receptor α mRNA and protein were increased in GCA lesions; enhanced JAK2/STAT5A expression/phosphorylation as well as increased expression of target genes CD83 and Spi1/PU.1 were observed. Treatment of ex vivo cultured GCA arteries with mavrilimumab resulted in decreased transcripts of CD3ε, CD20, CD14 and CD16 cell markers, and reduction of infiltrating CD16 and CD3ε cells was observed by immunofluorescence. Mavrilimumab reduced expression of molecules relevant to T cell activation (human leukocyte antigen-DR [HLA-DR]) and Th1 differentiation (interferon-γ), the pro-inflammatory cytokines: interleukin 6 (IL-6), tumour necrosis factor α (TNFα) and IL-1β, as well as molecules related to vascular injury (matrix metalloprotease 9, lipid peroxidation products and inducible nitric oxide synthase [iNOS]). Mavrilimumab reduced CD34 + cells and neoangiogenesis in GCA lesions.The inhibitory effects of mavrilimumab on multiple steps in the GCA pathogenesis cascade in vitro are consistent with the clinical observation of reduced GCA flares in a phase 2 trial and support its development as a therapeutic option for patients with GCA.© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ

Timing of surgery following SARS‐CoV‐2 infection: an international prospective cohort study

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4–1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0–2 weeks, 3–4 weeks and 5–6 weeks of the diagnosis (odds ratio (95%CI) 4.1% (3.3–4.8), 3.9% (2.6–5.1) and 3.6% (2.0–5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5% (0.9– 2.1%)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2–8.7) vs. 2.4% (95%CI 1.4–3.4) vs. 1.3% (95%CI 0.6–2.0%), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory. Athena is a versatile observatory designed to address the Hot and Energetic Universe science theme, as selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), X-IFU aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over a hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR (i.e. in the course of its preliminary definition phase, so-called B1), browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters, such as the instrument efficiency, spectral resolution, energy scale knowledge, count rate capability, non X-ray background and target of opportunity efficiency. Finally, we briefly discuss the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, touch on communication and outreach activities, the consortium organisation and the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. The X-IFU will be provided by an international consortium led by France, The Netherlands and Italy, with ESA member state contributions from Belgium, Czech Republic, Finland, Germany, Poland, Spain, Switzerland, with additional contributions from the United States and Japan.The French contribution to X-IFU is funded by CNES, CNRS and CEA. This work has been also supported by ASI (Italian Space Agency) through the Contract 2019-27-HH.0, and by the ESA (European Space Agency) Core Technology Program (CTP) Contract No. 4000114932/15/NL/BW and the AREMBES - ESA CTP No.4000116655/16/NL/BW. This publication is part of grant RTI2018-096686-B-C21 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. This publication is part of grant RTI2018-096686-B-C21 and PID2020-115325GB-C31 funded by MCIN/AEI/10.13039/501100011033

The Urgent Need for Novel Antimicrobial Agents and Strategies to Fight Antibiotic Resistance

Antibiotic resistance in bacterial pathogens has currently reached very high and alarming levels [...