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Antibiotic resistance in the aquatic environment promoted by pharmaceutical effluents

This study examines the promotion of antibiotic resistance in the aquatic environment by pharmaceutical effluents, highlighting the negative environmental impacts and the need for sustainable practices.

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Antibiotic resistance in the aquatic environment promoted by pharmaceutical effluents

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  1. Antibiotic resistance in the aquatic environment promoted by pharmaceutical effluents Juan José González Plaza, Ph.D. HRZZ (CroatianScienceFoundation) - EMBO Short-TermFellowship Udiković-Kolić Group; Division for Marine and Environmental Research Ruđer Bošković Institute; Zagreb, Croatia Current: Termite Research Team, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences; Prague, Czech Republic Postdoctoral Fellowship 2016 - 2018

  2. Franz Schubert (1828); Oscar Wilde (1900); Gustav Mahler (1911) • Typhoidfever; bacterialendocarditis; meningitis • Ciprofloxacin; rifampicin, gentamicin, and e.g. vancomicin; treatmentdepending on type ofbacteriacausinginfection Carlet et al. 2011, Lancet 378

  3. Franz Schubert (1828); Oscar Wilde (1900); Gustav Mahler (1911) • Typhoidfever; bacterialendocarditis; meningitis • Ciprofloxacin; rifampicin, gentamicin, and e.g. vancomicin; treatmentdepending on type ofbacteriacausinginfection Carlet et al. 2011, Lancet 378

  4. Franz Schubert (1828); Oscar Wilde (1900); Gustav Mahler (1911) • Typhoidfever; bacterialendocarditis; meningitis • Ciprofloxacin; rifampicin, gentamicin, and e.g. vancomicin; treatmentdepending on type ofbacteriacausinginfection Carlet et al. 2011, Lancet 378

  5. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams

  6. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams

  7. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams

  8. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams 2001: 1st isolationE. colistrain w. reducedsusceptibilitytocarbapenems Miriagou et al. 2003 AntimicrobAgentsChemother (47) 2012: carbapenemresistance has spread throughEurope Canton et al., 2012 CMI (18)

  9. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams 2001: 1st isolationE. colistrain w. reducedsusceptibilitytocarbapenems Miriagou et al. 2003 AntimicrobAgentsChemother (47) 2012: carbapenemresistance has spread throughEurope (CPE: carbapenem-producingEnterobacteriaceae) Canton et al., 2012 CMI (18)

  10. 10 million deaths per year - worst case scenario (2050) • Antibiotic Resistance (AR) – health threat • New mechanisms of resistance appear continuously, spread quickly (global scale) • Carbapenemases – hydrolisisofβ-lactams 2001: 1st isolationE. colistrain w. reducedsusceptibilitytocarbapenems Miriagou et al. 2003 AntimicrobAgentsChemother (47) 2012: carbapenemresistance has spread throughEurope (CPE: carbapenem-producingEnterobacteriaceae) Canton et al., 2012 CMI (18)

  11. Canton et al., 2012 CMI (18)

  12. Levelsofresistancereachstrikinglevels Numberof non-susceptible* isolates (Klebsiellapneumoniae) * Intermediate + resistant Canton et al., 2012 CMI (18)

  13. One Health approaches, to understand AR from different levels: agriculture, clinical, industrial, urban • Our project (WINAR) focuses on the Industry

  14. One Health approaches, to understand AR from different levels: agriculture, clinical, industrial, urban • Our project (WINAR) focuses on the Industry Urban WWTP Agricult. AR Hospitals Industry WWTP: Wastewatertreatmentplants

  15. One Health approaches, to understand AR from different levels: agriculture, clinical, industrial, urban • Our project (WINAR) focuses on the Industry -30% - 90% ingested antibiotic, excreted unchanged (animals and humans) Sarmah et al 2006 Chemosphere (65) -Partial removal in WWTP Giger et al 2003 CHIMIA (57) -Can enter in soil via manure Chee-Sanford et al 2009 Anglais (38) -80% antibiotics in aquaculture go to environment Cabello et al 2013 Environ Microbiol (15) Wecreate a zoneofantibioticenrichment

  16. One Health approaches, to understand AR from different levels: agriculture, clinical, industrial, urban • Our project (WINAR) focuses on the Industry Urban WWTP Agricult. AR Hospitals Industry WWTP: Wastewatertreatmentplants

  17. WINAR Project • Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries

  18. WINAR Project • Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries • Two pharmaceutical companies, Croatia • Company 1 – Macrolide production – river area • Company 2 – Veterinary antibiotics – small creek • Several locations (discharge points, upstream, downstream) • Wastewater and sediments

  19. WINAR Project • Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries • Two pharmaceutical companies, Croatia • Company 1 – Macrolide production – river area • Company 2 – Veterinary antibiotics – small creek • Several locations (discharge points, upstream, downstream) • Wastewater and sediments

  20. Company 1 – Macrolide production – river; Company 2 – formulationofveterinary antibiotics – small creek • Locations: discharge points, upstream, downstream González Plaza et al. 2019 EnvironInt 130

  21. Company 1 – Macrolide production – river; Company 2 – formulationofveterinary antibiotics – small creek • Locations: discharge points, upstream, downstream González Plaza et al. 2019 EnvironInt 130

  22. Company 1 – Macrolide production – river; Company 2 – formulationofveterinary antibiotics – small creek • Locations: discharge points, upstream, downstream González Plaza et al. 2019 EnvironInt 130

  23. Company 1 – Macrolide production – river; Company 2 – formulationofveterinary antibiotics – small creek • Locations: discharge points, upstream, downstream González Plaza et al. 2019 EnvironInt 130

  24. Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries Bielen et al (2017): we described high levels of antibiotics in effluents (up to 10.5 mg/L), high % antibiotic resistant bacteria in effluents

  25. Embriotoxicity assay on zebrafish Danio rerio ; *disclaimer: I did not carry out this experiment

  26. What’sthesituation in sediments?

  27. What’sthesituation in sediments?

  28. What’sthesituation in sediments? Percentage of resistant bacteria in sediments Macrolideantibiotic Tetracyclines and sulfonamides

  29. Whataboutantibioticresistance genes (ARGs)?

  30. Whataboutantibioticresistance genes (ARGs)? ARGs can bestudiedthroughfunctional metagenomic screening. In brief:

  31. Whataboutantibioticresistance genes (ARGs)? • ARGs can bestudiedthroughfunctional metagenomic screening. In brief: • - Build a metagenomic library (small DNA inserts) • Surrogate host sensitivetoantibiotics • Screen in differentantibioticselective media • DNA fragmentscontainingARGsallow host togrow

  32. Whataboutantibioticresistance genes (ARGs)? • Overviewoffunctionalmetagenomic screening: • Is a novel methodologicalapproach • Allowto capture novel ARGs (no limitationsequenceknowledge) • Culture independent, isolateARGsfromany bacteria* • *greatplatecountanomaly > 99% environmental bacteria unculturable • - Implemented in ourlaboratorythroughthisproject

  33. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation

  34. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation

  35. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation

  36. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation

  37. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation

  38. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation LB agar + Screenedantibiotic Library liquidculture 2 h 37 ºC LB broth + Kan orTet (vector selectivemarker)

  39. Percentage of resistant bacteria in functionalscreening

  40. Percentage of resistant bacteria in functionalscreening

  41. Functionalscreening Community DNA isolation > DNA shearing > Cloning > Electroporation LB agar + Screenedantibiotic Library liquidculture 2 h 37 ºC LB broth + Kan orTet (vector selectivemarker) Select clones > isolate vector > evaluaterestrictionpattern > sequence

  42. Genomiclandscape: macrolides Mef: macrolideeffluxpumps Mph: macrolidephosphotransferases (inactivating genes) Msr(E): ABC-F type ribosomal protection protein Genes msr(E)-mph(E) or mef(C)-mph(G) were found in libraries of effluent and receiving river sediment, indicates that industrial effluent is a point source of these gene clusters Some macrolide resistant carried additional genes, such as sul2 (sulfonamide resistance). Co-resistance event due to selection pressure

  43. Genomiclandscape: Sulfonamides Sulfonamide resistance (sul1) aminoglycoside resistance (aac) Dihydrofolate reductase

  44. Genomiclandscape: Sulfonamides Sulfonamide resistance (sul1) aminoglycoside resistance (aac) Dihydrofolate reductase Acetylation of aminoglycosides by acetyltransferases is one of the major mechanisms of acquired resistance to these compounds

  45. Genomiclandscape: Sulfonamides Sulfonamide resistance (sul1) aminoglycoside resistance (aac) Dihydrofolate reductase Acetylation of aminoglycosides by acetyltransferases is one of the major mechanisms of acquired resistance to these compounds IntI: - allow capture and expression of exogenous genes, recombined using the integrase activity - acquisition and expression of genes with minimal disturbance to the existing genome

  46. Total of 82 unique, often clinically relevant ARGs, frequent in clusters, flanked by mobile genetic elements Novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments: Veterinary antibiotics, 66novel ARGs: -dihydrofolatereductases and beta-lactamases (classes A, B, and D) -novel ARGs in upstream sediment (thymidylatesynthases, dihydrofolatereductases, class D beta-lactamase) • Novel macrolide resistance genes (16): • one most similar to a 23S rRNA methyltransferase from Clostridium • gene from upstream unpolluted sediment, similar to a GTPase HflX from Emergenciatimonensis Macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolidephosphotransferases, and transporters

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