Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-48) in environmental samples
In: Environmental science and pollution research: ESPR, Band 24, Heft 7, S. 6710-6714
ISSN: 1614-7499
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In: Environmental science and pollution research: ESPR, Band 24, Heft 7, S. 6710-6714
ISSN: 1614-7499
Sensitization of singlet delta oxygen (O2(1Δg)) by bacteriochlorophyll e (BChle) has been investigated to gain a better understanding of the photoprotection mechanism(s) operating in chlorosomes of green photosynthetic bacteria. The sensitization process has been studied in media where BChle forms monomers (acetone and aqueous solutions containing 0.5% Triton X-100 [TX]) and in systems where BChle aggregates, namely, aqueous solutions containing 0.003% monogalactosyl diglyceride (MGDG) and chlorosomes (control as well as hexanol perturbed) from Chlorobium phaeobacteroides strain CL1401. In Ar-purged acetone, BChle triplets (BChle†) have a lifetime of a few tens of microseconds; however, in air-saturated acetone, quenching of BChle† by ground-state oxygen (O2(3Σ−g)) and formation of O2(1Δg) take place. The O2(1Δg) so formed is susceptible to quenching by BChle0, a ground-state BChle molecule. A Stern–Volmer analysis reveals a linear fit between the decay rate of O2(1Δg) and the BChle concentration. The rate constants for the quenching of O2(1Δg) by BChle0 and for the deactivation of O2(1Δg) by the solvent come out to be kq= (1.4 ± 0.1) × 10^9 M−1 s−1 and k0= (18.5 ± 0.7) × 10^3 s−1, respectively. The absolute quantum yield of O2(1Δg) sensitization by BChle monomers is 0.65 ± 0.15 in air-saturated acetone. In aqueous phase, the triplet lifetime of BChle aggregates in native or hexanol-perturbed chlorosomes shortens by more than two orders of magnitude when compared with the triplet lifetime of BChle monomers in 0.5% TX solution (a few hundreds of microseconds). Quenching by carotenoids (Car) makes only a minor contribution to the decay of BChle† in aggregates. Because O2(1Δg) sensitization by BChle† could be detected neither in MGDG aggregates nor in chlorosomes (control as well as hexanol perturbed), it is concluded that (1) this process is highly likely when BChle is present as a monomer but not when it is tightly packed in artificial aggregates or in chlorosomes; and (2) Car, though vital for the baseplate BChla, are dispensable for BChle. ; We acknowledge financial support from the Research Council of Norway and the European Union (contract FMRX-CT96-0081). ; Peer reviewed
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Este artículo contiene 8 páginas, 5 figuras. ; Antibiotic resistance has become a major Global Health concern and a better understanding on the global spread mechanisms of antibiotic resistant bacteria (ARB) and intercontinental ARB exchange is needed. We measured atmospheric depositions of antibiotic resistance genes (ARGs) by quantitative (q)PCR in rain/snow collected fortnightly along 4 y. at a remote high mountain LTER (Long-Term Ecological Research) site located above the atmospheric boundary layer (free troposphere). Bacterial composition was characterized by 16S rRNA gene sequencing, and air mass provenances were determined by modelled back trajectories and rain/snow chemical composition. We hypothesize that the free troposphere may act as permanent reservoir and vector for ARB and ARGs global dispersal. We aimed to i) determine whether ARGs are long-range intercontinental and persistently dispersed through aerosols, ii) assess ARGs long-term atmospheric deposition dynamics in a remote high mountain area, and iii) unveil potential diffuse ARGs pollution sources. We showed that the ARGs sul1 (resistance to sulfonamides), tetO (resistance to tetracyclines), and intI1 (a proxy for horizontal gene transfer and anthropogenic pollution) were long-range and persistently dispersed in free troposphere aerosols. Major depositions of tetracyclines resistance matched with intensification of African dust outbreaks. Potential ARB mostly traced their origin back into agricultural soils. Our study unveils that air masses pathways are shaping ARGs intercontinental dispersal and global spread of antibiotic resistances, with potential predictability for interannual variability and remote deposition rates. Because climate regulates aerosolization and long-range air masses movement patterns, we call for a more careful evaluation of the connections between land use, climate change and ARB long-range intercontinental dispersal. ; This research is included within the global surveillance activities of the Long-Term Ecological Research node Aigüestortes (LTER-AT) and was supported by Grant ECOSENSOR-BIOCON 04/009 from BBVA Foundation; Grants AERBAC 079/2007, AERBAC-2 178/2010, and DISPERSAL 829/2013 from the Spanish Office for the Environment's- National Parks Research Programme (OAPN); and INTERACTOMA RTI2018-101205-B-I00 from the Spanish Agency of Research (AEIMICINN) and European funding (ERDF) to EOC. JC was supported by PTA2018-016527-I (AEI-MICINN) and XTM by grant INTERACTOMA. CB wish to thank the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ; Peer reviewed
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8 pages, 2 figures, 2 tables. ; Isolated chlorosomes of several species of filamentous anoxygenic phototrophic bacteria (FAPB) and green sulfur bacteria (GSB) were examined by atomic force microscopy (AFM) to characterize their topography and biometry. Chlorosomes of Chloroflexus aurantiacus, Chloronema sp., and Chlorobium (Chl.) tepidum exhibited a smooth surface, whereas those of Chl. phaeobacteroides and Chl. vibrioforme showed a rough one. The potential artifactual nature of the two types of surfaces, which may have arisen because of sample manipulation or AFM processing, was ruled out when AFM images and transmission electron micrographs were compared. The difference in surface texture might be associated with the specific lipid and polypeptide composition of the chlorosomal envelope. The study of three-dimensional AFM images also provides information about the size and shape of individual chlorosomes. Chlorosomal volumes ranged from ca. 35000 nm3 to 247000 nm3 for Chl. vibrioforme and Chl. phaeobacteroides, respectively. The mean height was about 25 nm for all the species studied, except Chl. vibrioforme, which showed a height of only 14 nm, suggesting that GSB have 1–2 layers of bacteriochlorophyll (BChl) rods and GFB have ~4. Moreover, the average number of BChl molecules per chlorosome was estimated according to models of BChl rod organisation. These calculations yielded upper limits ranging from 34000 BChl molecules in Chl. vibrioforme to 240000 in Chl. phaeobacteroides, values that greatly surpass those conventionally accepted. ; This study was supported by The European Union through the TMR Program, Contract No. FMRXCT96‐ 0081. ; Peer reviewed
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We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl–supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and β-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30–40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from ;0.24 for native chlorosomes to ;0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides. ; European Union (Contract FMRX-CT96-0081). Spanish Ministry of Education and Science (Ref. BIO96-1229-002-01). ; Peer reviewed
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Quantitative measurements of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw wastewater have been implemented worldwide since the beginning of the pandemic. Recent efforts are being made to evaluate different viral concentration methodologies to overcome supplier shortages during lockdowns. A set of 22-wastewater samples seeded with murine hepatitis virus (MHV), a member of the Coronaviridae family, and the bacteriophage MS2, were used to characterize and compare two ultrafiltration-based methods: a centrifugal ultrafiltration device (Centricon® Plus-70) and the automated concentrating pipette CP-Select™. Based on the recovery efficiencies, significant differences were observed for MHV, with Centricon® Plus-70 (24%) being the most efficient method. Nevertheless, concentrations of naturally occurring SARS-CoV-2, Human adenoviruses and JC polyomaviruses in these samples did not result in significant differences between methods suggesting that testing naturally occurring viruses may complement the evaluation of viral concentration methodologies. Based on the virus adsorption to solids and the necessity of a pre-centrifugation step to remove larger particles and avoid clogging when using ultrafiltration methods, we assessed the percentage of viruses not quantified after ultrafiltration. Around 23% of the detected SARS-CoV-2 would be discarded during the debris removal step. The CP-Select™ provided the highest concentration factor (up to 333×) and the lowest LoD (6.19 × 103 GC/l) for MHV and proved to be fast, automatic, highly reproducible and suitable to work under BSL-2 measures. ; This work was partially funded by the projects PCI2019-103643, RTI2018-097346-B-I00, and AGL2017-86797-C2-1-R projects, all funded by the Spanish Ministry of Science, Innovation and Universities. S. Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. This study is performed with partial support from the Water Research Institute of the University of Barcelona. Graphical abstract was created with Biorender.com. ICRA authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ICRA is part of the CERCA program. We would like to thank Dr. Zammit for his critical revision of the manuscript. ; Peer reviewed
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