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Water and Soil As Reservoirs for Mdr Genes WATER AND SOIL AS RESERVOIRS FOR MDR GENES LUÍSA VIEIRA PEIXE UNIVERSITY OF PORTO . PORTUGAL ESCMID eLibrary 1 © by author Bacteria in the earth - appeared 3,5x109 years ago One gram of soil: up to 1010 bacterial cells & Species diversity of 4x103 to 5x104 species Antibiotics production: tens (daptomycin, vancomycin) to hundreds (erythromycin, streptomycin) of millions of years ago 2 Raynaud ESCMID & Nunan, Pone. 2014 eLibrary © by author Extensive Natural Collection of AMR Genes Antibiotic resistance is ancient: Soil, fresh and marine water phyla contain • TetM and VanA in DNA 30,000-year- old; a huge diversity of ARG genes. • Metallo-b-lactamases emerged one >> More diverse than the clinical ARG pool billion years ago. New MBL in soil Psychrobacter psychrophilus MR29-12 StrepR TetR Permafrost Siberian 15 000-35 000 anos AMR gene is the one that confers protection to a particular antibiotic (increase in MIC) when expressed. Resistome – all resistance genes of a community Network of predicted bacterial phyla for each AMR used in cross- soil comparisons (n=880) (Forsberg et al., Nature. 2014) Without human interference, selection for resistance already occurs naturally in microbial populations in soil, water and other habitats 3 Gudeta et al., FrontiersESCMID Microb. 2016; D’Costa et al, Nature. 2011; Forsberg et al, Nature.2014; Martinez J.L. Science.2010;eLibrary FEMS Microbiol Lett 296.2009; Riesenfeld et al, Envir. Microbiol. 2004 © by author What are AMR genes doing in these Bacteria? Protection against antibiotics ABR genes Physiological functions silencing E.g. detoxification; virulence, signal trafficking, intra- domain communication. 2’ N-acetyltransferase of Providencia stuartii - acetylation of peptidoglycan and gentamycin Antibiotic-producing microorganisms catQ ...Streptomyces...synthesize over half of all known antibiotics... ... Resistance elements clustered in antibiotic biosynthetic operons Others habitants Screening of 480 soil organisms: blaCTX-M-8 qnrS blasfh Co-habitants of tetqnrS sul1 R blaCTX-M cml fosA3 AB producers bla catAsfhI tetM qnrA cml, catA vanA blaIMIrph vanA rph strA blaOXA-48 tet vanA strA Streptomycin - strA vanHAX in Amycolatopsis sp. 4 Surette and Wright.ESCMID Annu. Rev. Microbiol. 2017; D’Costa et al, Curr. Opinion Microbiol eLibrary. 2007; D’Costa et al, Nature, 2011; Fdantas et al, Science. 2008; EMS Microbiol Lett 296. 2009 © by author Is Horizontal Gene Transfer (HGT) shaping natural Resistome ? HGT of R genes is a key driver for acquisition of antimicrobial resistance in the clinical setting, Mobility Elements Phylogeny, Key are Rare role in soil resistome Ongoing metagenomics content studies will contribute to HGT not relevant elucidate HGT frequence in natural soil and water Study of soils bacteria by metagenomic Forsberg et al., Science.2014. 5 Fondi et al, GenomeESCMID Biol Evol. 2016 eLibrary © by author AMR Genes Mobilization and Transfer from Environmental microorganisms to humans/animals bacteria HGT events mainly involve molecules of the same kind (i.e. either plasmids ICE/cTn or chromosomes) R HGT Selective Pressure Selective Bacterial Host R IS/Tn Pathogenic Commensal / R R R Plasmid HGT (co & cross R) R Frequent Rare? Diversity of HGT elements in the environment largely unknown IS - insertion sequence; ICE - integrative conjugative element; Tn - conjugative transposon 6 Fondi et al, GenomeESCMID Biol Evol. 2016 eLibrary © by author Who Are the R Genes Donors? Dantas et al., Science 2008 Different antibiotics added to soil: - Few soil bacterial groups able to survive - Mostly Proteobacteria closely related to human pathogens (e.g. Enterobacteriaceae, Pseudomonas, Burkholderia, Vibrio), but also Actinobacteria and Bacteroidetes. Bacterial Certain environmental bacterial groups (also Host / Firmicutes) more related with opportunistic & human pathogens more prone to act as R gene donors to R those bacteria 7 VazESCMID-Moreira, I. et al. (2014); FEMS Microbiol. Review .eLibrary © by author Origin of relevant horizontally transferred R genes Bacterial Gene Antibiotic Bacterial species MGEs* Host resistance / rd blaCTX-M 3 generation Kluyvera spp. ISEcp1, ISCR1 R Cephalosporins qnrA ↓ susceptibility to Shewanella algae ISCR1, IS26 Fluorquinolones qnrS ↓ susceptibility to Vibrio splendidus IS2, IS26, ISEcl2, Fluorquinolones Tn3-like, mic fosA3 Fosfomycin Kluyvera georgiana (CTX-M-8 origin) IS26 vanA Glycopeptide Paenibacillus thiaminolyticus Tn1546 blaSBM-1** Carbapenems Gene from soil metagenome ISCR1 strA, strB Streptomycin Streptomyces spp. Tn5393 * MGE = Mobile genetic elements; ** just one 8 Guardabassireport L etESCMIDal. AAC 2004; Wachino J et al. AAC 2011; Ohnuki T et al. J Bac 1985eLibrary; Cantón R et al. Front Micr 2012; Hooper DC. Ann N Y Acad Sci 2015 ; Ito R et al. JAC 2018 © by author Origin of relevant horizontally transferred R genes Dispersed host & MGE + Antibiotics ? = R mobilization likely to occur in different locations Gene Antibiotic resistance Bacterial species MGEs cfr Oxazolidinones, Phenicols, Bacillus spp.?/ IS21–558, IS26, Lincosamides, Pleuromutilins, Staphylococcus spp. ISEnfa5, Tn558 Streptogramin A optrA Oxazolidinones, Phenicols Staphylococcus sciuri IS1216, Tn6261 oqxAB ↓ susceptibility to Fluorquinolones Klebsiella Tn3, IS26 pneumoniae mcr-1 Colistin Moraxella novel ISApl1, Tn6330 species qnrB Setting↓ susceptibility where to mobilization Fluorquinolones occurredCitrobacter is unknownspp. ISCR1, IS26, ISEcp1, ISEcp1C, IS3000, IS6100 rd blaCMY-2-like 3 generation Cephalosporins Citrobacter spp. ISEcp1, IS26, IS5, ISkpn26 9 Dai L et al. AAC 2010; Zhang WJ et al, AAC 2015; Wang Y et al. JAC 2015; Sun C et al, JAC 2018; Fan R et al., Vet Microbiol 2017; Rodríguez-Martínez JM et al., Drug Resist Updat 2016; Snesrud EESCMIDet al., MBio, 2018; Ribeiro TG et al., AAC 2015 eLibrary © by author Ribeiro TG et al 5 - CMY Teresa Ribeiro ISEcp1-like blc sugE blaCMY-2 CMY-47 Cluster I Citrobacter portucalensis Cluster II Citrobacter freundii Cluster III Citrobacter europaeus Cluster IV Citrobacter braakii Cluster V Citrobacter werkmanii Cluster VI Citrobacter murliniae Cluster VII Citrobacter sp. (putative novel species) Branch Citrobacter pasteurii Affiliation of all genes encoding CMY-2-like, LAT and CFE β-lactamases. crAmpC, putative new chromosomal blaCMY-2-like identified in a given Citrobacter genome. Chromosomally blaAmpC are shown as underlined, whereas those from Citrobacter type strain are additionally in bold. Acquired bla are not underlined. 10 ≤94.5% identity,ESCMID bootstrap ≥96% eLibraryAmpC © by author Conditions influencing environmental emergence and spread of resistance Sublethal concentrations • Human and animal use Amount/ FQ & tetracyclines: months to years; • Antibiotic Antimicrobial load Diversity/Persistence Mobility production CoMost-selection antibiotics by disinfectantsare excreted & unmetabolized metals* * Persistence of monophasic variant of S. Typhimurium copper tolerant (sil+pco clusters + ABR genes) in pigs & pig environment (Mourão et al., IJAA 2015). Joana Mourão 11 Mourão et al, IJAA.ESCMID 45(6):610-6. 2015; Grave et al, JAC 2014 eLibrary © by author Conditions influencing environmental emergence and spread of resistance Sublethal concentrations • Human and animal use Amount/ FQ & tetracyclines: months to years; • Antibiotic Antimicrobial load Diversity/Persistence Mobility production Co-selection by disinfectants & metals* Survival to environmental stressors Microbes & Mobilome Diversity/spectra composition & density R/amount Contact between H, A and E diverse bacteria Large & diverse pool of MGE Ability to persist in food chain/colonize/infection 12 Mourão et al, IJAA.ESCMID 45(6):610-6. 2015; Grave et al, JAC 2014 eLibrary © by author Conditions influencing environmental emergence and spread of resistance Sublethal concentrations • Human and animal use Amount/ FQ & tetracyclines: months to years; • Antibiotic Antimicrobial load Diversity/Persistence Mobility production Co-selection by disinfectants & metals* Survival to environmental stressors Microbes & Mobilome Diversity/spectra composition & density R/amount Contact between H, A and E diverse bacteria MGE: Host range & maintenance-Fitness cost Other factors Ability to persist in food chain/colonize/infection Lack of hygiene & sanitation Socio-economic Agricultural intensification & land-use changes Extension of R genes environmental Climate change Others contamination Cultural (food habits & open defecation) Emergence of new R genes 13 Mourão et al, IJAA.ESCMID 45(6):610-6. 2015; Grave et al, JAC 2014 eLibrary © by author Where is favoured the emergence and spread of resistance? Increase in Global Antibiotic Consumption Percentage change in antibiotic consumption per capita 2000-2010, by country 65% in 2000-2015 Double the consumption in Low to middle income countries; 6% in high income countries 14 https://www.cddep.org ESCMID/wp-content/uploads/2017/06/swa_edits_9.16.pdf eLibrary © by author Where is favoured the emergence and spread of resistance? Hot spots of AMR environmental contamination Hospital waste Animal waste High level of different antibiotics High level of different Accumulation of R pathogens antibiotics & commensals Accumulation of R pathogens & commensals Low efficiency of composting to concentrations of antibiotics and R bacteria in manure. General sewage waste Antimicrobial Production Waste High Income countries: High levels of Ab,and R genes 30% of wastewater goes untreated downstream discharge site-drugs Urban runoff to beaches WWTP.
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