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      Raw Cow Milk Bacterial Consortium as Bioindicator of Circulating Anti-Microbial Resistance (AMR)

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          Abstract

          Simple Summary

          Antimicrobials represent useful tools to fight bacterial infections that could harm human and animal health. Antimicrobial resistance occurs naturally or can be induced by the misuse of antibiotics. Its occurrence limits the efficiency of antibiotics and therefore the possibility to treat infections effectively. This can lead to an increasing severity of infectious diseases in humans and animals. Here, we describe the development of a workflow that provides a qualitative representation of the antimicrobial genes that are translated into proteins. Since proteins are ultimately the real effectors, the method herein described demonstrates that those genes are effectively enhancing antimicrobial resistance (AMR). The presented method is independent of any amplification step and provides useful information on the dynamics of the biochemical functions accomplished by the raw milk bacterial consortium.

          Abstract

          The environment, including animals and animal products, is colonized by bacterial species that are typical and specific of every different ecological niche. Natural and human-related ecological pressure promotes the selection and expression of genes related to antimicrobial resistance (AMR). These genes might be present in a bacterial consortium but might not necessarily be expressed. Their expression could be induced by the presence of antimicrobial compounds that could originate from a given ecological niche or from human activity. In this work, we applied (meta)proteomics analysis of bacterial compartment of raw milk in order to obtain a method that provides a measurement of circulating AMR involved proteins and gathers information about the whole bacterial composition. Results from milk analysis revealed the presence of 29 proteins/proteoforms linked to AMR. The detection of mainly β-lactamases suggests the possibility of using the milk microbiome as a bioindicator for the investigation of AMR. Moreover, it was possible to achieve a culture-free qualitative and functional analysis of raw milk bacterial consortia.

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          Most cited references 35

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          Universal sample preparation method for proteome analysis.

          We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry-based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
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            CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database

            The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.
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              Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome

              Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.
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                Author and article information

                Journal
                Animals (Basel)
                Animals (Basel)
                animals
                Animals : an Open Access Journal from MDPI
                MDPI
                2076-2615
                11 December 2020
                December 2020
                : 10
                : 12
                Affiliations
                [1 ]Department of Health Sciences, “Magna Græcia University” of Catanzaro, Campus Universitario “Salvatore Venuta” Viale Europa, 88100 Catanzaro, Italy; tilocca@ 123456unicz.it (B.T.); britti@ 123456unicz.it (D.B.)
                [2 ]Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Roma, Italy; viviana.greco@ 123456unicatt.it (V.G.); andrea.urbani@ 123456unicatt.it (A.U.)
                [3 ]Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo A. Gemelli 8, 00168 Roma, Italy
                [4 ]Department of Veterinary Sciences, University of Messina, 98168 Messina, Italy; egugliandolo@ 123456unime.it
                [5 ]Surgical and Dental Sciences-One Health Unit, Department of Biomedical, University of Milano, Via Celoria 10, 20133 Milano, Italy; alessio.soggiu@ 123456unimi.it (A.S.); luigi.bonizzi@ 123456unimi.it (L.B.); alfonso.zecconi@ 123456unimi.it (A.Z.)
                [6 ]Department of Chemistry, University of Reading, Reading RG6 6DX, UK; r.k.cramer@ 123456reading.ac.uk
                Author notes
                [* ]Correspondence: c.piras@ 123456unicz.it (C.P.); roncada@ 123456unicz.it (P.R.); Tel.: +39-096-1369-4236 (C.P.); +39-096-1369-4284 (P.R.)
                Article
                animals-10-02378
                10.3390/ani10122378
                7763537
                33322611
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                Categories
                Article

                proteomics, amr, raw milk, microbiome, β-lactamase

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