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Antibiotic Resistance Genes Online) Resistomics: detection of resistance genes by massive sequencing Jordi Vila © by author Hospital Clinic ESCMIDBarcelona, OnlineSpain Lecture Library Synthesis of folic acid Synthesis of nucleic acids Sulfonamides Metronidazole Trimeth oprim Quinolones Rifampicin PABA DNA DNA gyrase DHFA Topo IV THFA RNA polimerase mRNA Purines/Pyrimidines/Amino acids © by author Cellular wall Synthesis of proteins β-lactams Tetracyclines Glycopeptides Aminoglycosides FosfomycinESCMID Online Lecture Chloramphenicol Library Macrolides, streptogramins Clindamycin Polimixin (membrana externa BGN) Oxazolidinones Daptomycin (GPB) How do bacteria show resistance to antimicrobial agents? © by author ESCMID Online Lecture Library OM = Outer Membrana pe = Periplasmic space Α OM Β IM = Inner Membrane pe pe IM IM Gram - Efflux pump Gram + Α Β 1 = ANTIBIOTIC 4 3 © by author 2 ESCMID Online Lecture Library How do bacteria become resistance to antimicrobial agents? © by author ESCMID Online Lecture Library ACQUISITION OF RESISTANCE • MUTATIONS. • TRANSFERENCE OF GENETIC MATERIAL© by author ESCMID Online Lecture Library TRANSFERABLES GENETIC ELEMENTS • PLASMIDS • TRANSPOSONS • INTEGRONS© by author • RESISTANT ISLANDS ESCMID Online Lecture Library Transposon classe I IS Resistance Gene IS Transposon classe II Resistance gene Plasmid SIR Resistance Gene SIR MOBILOME Integron Resistance gene © by author ESCMID Online Lecture Library MECHANISMS OF TRANSFERENCE OF RESISTENCE GENES A = Transformation B = Conjugation © by author C = Transduction ESCMID Online Lecture Library Resistance gen • Spread of multiresistant bacteria • Spread of resistance genes © by author ESCMID Online Lecture Library How do ARB and ARG spread? © by author ESCMID Online Lecture Library Antibiotics in Antibiotics in animals / food developing countries Antibiotics in the community © by author Health care associated infections ESCMID Online Lecture Library Antibiotics in Hospitals © by author ESCMID Online Lecture Library Fish farms Rivers and streams Wildlife (Birds, Rodents, Insects) Sea / Drinking water Lakes Ground Water Wastewater Industrial Soil Treatment plants Sewage Farm effluents and Fruits and vegetables manure spreading Commercial abattoirs© by / author Food chain Processing plants - Hospitalized Farm animals Humans - Community ESCMIDDirect Online contact Lecture Library Pets Environmental (Dust, Waste) SELECTIVE PRESSURE ANTIBIOTIC ANTIBIOTIC Mutation Susceptible bacterial © by author Resistant bacterial population population ESCMID Online Lecture Library Selection pressure is particularly acute in human waste streams, where resistance are shed, mixed with high concentrations of antibiotics and other slective agents. Definition of resistome • Professor Gerry Wright of McMaster University has coined the term ‘‘antibiotic resistome’’ to describe the collection of antibiotic genes in the environment.© by author Taken as a consideration the role that environmental ESCMID microorganisms Online play as reservoirsLecture of resistance Library genes Nature Review of Microbiology (2007) 5: 175 Definition of resistome • A collection of all the antibiotic resistance GLOBAL genes and their precursors in pathogenic and non-pathogenic bacteria. SPECIFIC NICHE • Antibiotic resistance© by genes author from the pathogens are only a tiny fraction from the resistome •ESCMIDResistome however Online can Lecturebe defined in Library a multiple levels BACTERIA What is the origin of the antibiotic resistant genes? © by author ESCMID Online Lecture Library D’Costa, et al: Antibiotic resistance is ancient. Nature 2011; 457:477 • Metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to b-lactam, tetracycline and glycopeptide antibiotics. • Genes related to vanX, tetM and bla were found. • These results show© conclusively by author that antibiotic resistance is a natural phenomenon that predates the modern selective ESCMIDpressure of clinical Online antibiotic Lecture use. Library Evolution of antimicrobial resistance • Microorganisms producing antibiotics are potential sources of clinical resistance. For instance the functional relationship between aminoglycoside resistance and aminoglycoside producers has been established. • Vancomycin resist©an byce: anauthor elaborate resistance strategy (Nature Reviews in Microbiology (2007)5:175 ESCMID Online Lecture Library Vancomycin resistance • Binds to a ubiquitous cell-wall polymer and not to a protein target – No mutations in the target • They are effective at the exterior of the cell and are not susceptible to efflux or influx resistance mechanisms – Prediction that i was unlikely that resistance to vancomycin© by author would occur • The arrangement of genes is conserved between ESCMIDVRE and vancomycin Online-producing Lecture bacteria, Library which might indicate that antibiotic producers were the origins of vancomycin resistance Evolution of antimicrobial resistance • Microorganisms producing antibiotics are potential sources of clinical resistance. For instance the functional relationship between aminoglycoside resistance and aminoglycoside producers has been established • Antibiotic resistance proteins evolve from proteins with alternative biochemical functions that function as precursors to resistance© by elements author • This can occur either through the evolution of resistance ESCMIDmechanisms in Online the microbial Lecture neighbour, or byLibrary horizontal gene transfer of resistance genes from other bacterial species. What is the origin of the clinically relevant resistant genes? © by author ESCMID Online Lecture Library Model of ESBL emergence and dissemination Emerging model Chromosomal ß-lactamases (Kluyvera sp.) Integration within gene capture units CTX-M-ESBL © by author ESBL Dispersion of genetic elements ESCMID Online Lecture Library Clonal selection? Other examples emergence and dissemination • Qnr - Shewanella algae - Acuatic environment (JAC 2005; 56:1118) • OXA -23 – Acinetobacter radioresistens - Soil environment (AAC 2008; 52:1252) • NDM - Erythrobacter© by author litoralis - Acuatic environment (Protein Cell 2011; 2: 250) ESCMID Online Lecture Library D’Costa, et al: Sampling the antibiotic resistome Science 2006; 311:304 • The soil resistome – Isolate sporing forming bacteria (actinomycetes), ientified by 16S rRNA as belonging to the genus Streptomyces from soil samples (urban, agricultural and forest) – Library of 480 ©strains by screenedauthor against 21 antibacterial agents ThisESCMID study offered Online an informative Lecture sampling of theLibrary resistance burden in the environment outside the clinic and demonstrated that antibiotic resistance is widespread in environmental bacteria. Bhullar K, et al: Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS one 2012; 7 • 93 bacterial strains (31 Gram+ve and 62 Gram-ve) isolated from cultures resembling the carbon sources available in the cave • Four strains of Paenibacillus lautus inactivated daptomycin probably via a metalloesterase that hydrolytically opens the ring structure. © by author ESCMID Online Lecture Library Walsh F and Duffi B: The culturable soil antibiotic resistome: A community of multidrug resistant bacteria PLoS one 2013; 8:e65567 • 412 antibiotic resistant bacteria isolated from agricultural, urban and pristine soils • All isolates were multidrug resistant with 80% resistant to 16-23 antibiotics • Mobile genetic elements© by carrying author ESBL, PMQR or NDM were not found, meanwhile efflux seems to play an important role ESCMIDin the soil resistome. Online Lecture Library Kristiansson, et al: Pyrosequencing of antibiotic- contaminated river sediments reveals high levesl of resistance and gene transfer elements PLoS one 2011; 6:e17038 • Microbial communities in river upstream and downstream from a waste water treatment plant in Sweden and India • Sequence done in Roche Genome Sequencer FLX Titanium • Analysis: © by author – All sequencing reads were compared against the non- ESCMIDredundant databaseOnline (nt) Lectureat NCBI GenBank Library Blast 2.2.18 – Taxonomic affilitation – NCBI Taxonomy database – Functional annotation – Clusters of orthologous genes (COGs) and TIGRFAMS databases using RPSBlast Kristiansson, et al: Pyrosequencing of antibiotic- contaminated river sediments reveals high levesl of resistance and gene transfer elements PLoS one 2011; 6:e17038 • Analysis: – Detection of integrases – INTEGRAll – Detection of insertion sequences – ISFinder – Plasmid analysis: Reads wer aligned with • Plasmid sequences available in the NCBI Ref.Seq database (2138 sequences) © by author • Sequences in GenBank nucleotide database annotated as plasmid – De novo plasmids was performed by concatenating ESCMIDoverlapping Online in a step-wise Lecture fashion until Library the fragment was found to be circular. – Antibiotic Resistance database Szczepanowski, et al: Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology 2009; 155:2306 • Source: sludge or effluent of the wastewater treatment plant • Methodology: 1 liter of sample centrifuged and pellet resuspended in 5 ml of LB broth and 100 mcl spread onto antibiotic selective ©media by author • Plasmid DNA were obtained from isolated resistant colonies with Nucleobond kit (40 to 180 kb) ESCMID• 192 resistance
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