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      Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae

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          Abstract

          Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

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          16S ribosomal DNA amplification for phylogenetic study.

          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            Bacteroides: the good, the bad, and the nitty-gritty.

            Bacteroides species are significant clinical pathogens and are found in most anaerobic infections, with an associated mortality of more than 19%. The bacteria maintain a complex and generally beneficial relationship with the host when retained in the gut, but when they escape this environment they can cause significant pathology, including bacteremia and abscess formation in multiple body sites. Genomic and proteomic analyses have vastly added to our understanding of the manner in which Bacteroides species adapt to, and thrive in, the human gut. A few examples are (i) complex systems to sense and adapt to nutrient availability, (ii) multiple pump systems to expel toxic substances, and (iii) the ability to influence the host immune system so that it controls other (competing) pathogens. B. fragilis, which accounts for only 0.5% of the human colonic flora, is the most commonly isolated anaerobic pathogen due, in part, to its potent virulence factors. Species of the genus Bacteroides have the most antibiotic resistance mechanisms and the highest resistance rates of all anaerobic pathogens. Clinically, Bacteroides species have exhibited increasing resistance to many antibiotics, including cefoxitin, clindamycin, metronidazole, carbapenems, and fluoroquinolones (e.g., gatifloxacin, levofloxacin, and moxifloxacin).
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              What is a resistance gene? Ranking risk in resistomes.

              Metagenomic studies have shown that antibiotic resistance genes are ubiquitous in the environment, which has led to the suggestion that there is a high risk that these genes will spread to bacteria that cause human infections. If this is true, estimating the real risk of dissemination of resistance genes from environmental reservoirs to human pathogens is therefore very difficult. In this Opinion article, we analyse the current definitions of antibiotic resistance and antibiotic resistance genes, and we describe the bottlenecks that affect the transfer of antibiotic resistance genes to human pathogens. We propose rules for estimating the risks associated with genes that are present in environmental resistomes by evaluating the likelihood of their introduction into human pathogens, and the consequences of such events for the treatment of infections.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                01 February 2021
                February 2021
                : 12
                : 2
                : 213
                Affiliations
                [1 ]Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy; v.milanovic@ 123456staff.univpm.it (V.M.); a.roncolini@ 123456pm.univpm.it (A.R.); f.cardinali@ 123456staff.univpm.it (F.C.); c.garofalo@ 123456staff.univpm.it (C.G.); l.aquilanti@ 123456staff.univpm.it (L.A.); p.riolo@ 123456staff.univpm.it (P.R.); s.ruschioni@ 123456staff.univpm.it (S.R.); lorenzo.corsi1993@ 123456gmail.com (L.C.); n.isidoro@ 123456staff.univpm.it (N.I.); s.ceccobelli@ 123456staff.univpm.it (S.C.); s.tavoletti@ 123456staff.univpm.it (S.T.); f.clementi@ 123456staff.univpm.it (F.C.)
                [2 ]Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy; m.zarantoniello@ 123456pm.univpm.it (M.Z.); i.olivotto@ 123456staff.univpm.it (I.O.)
                Author notes
                [* ]Correspondence: a.osimani@ 123456univpm.it
                Author information
                https://orcid.org/0000-0001-8548-366X
                https://orcid.org/0000-0002-0416-5748
                https://orcid.org/0000-0003-4095-6181
                Article
                genes-12-00213
                10.3390/genes12020213
                7912857
                33535615
                2164dfdd-7690-4f43-8aca-923131548e57
                © 2021 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/).

                History
                : 30 December 2020
                : 29 January 2021
                Categories
                Article

                hermetia illucens,antibiotic resistance genes,rearing substrates,microalgae,coffee silverskin,frass

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