Antimicrobial Resistance in Aquaculture.

An overview of the science behind antimicrobial resistance

1. Antimicrobial Resistance D.D.T.T.D. Senarathna School of Environmental Resources and Management Asian Institute of Technology

2. Outline What is AMR? Antibiotics in Aquaculture AMR in Aquaculture Mechanism of AMR AMR detection methods in Aquaculture AMR in Aquaculture: case studies How to control? Conclusion References

3. Abbreviations bMP - Biodegradable microplastics CEC - Cation Exchange Capacity cMP - Conventional Microplastics HDPE - High-density Polyethylene PBAT- Polybutylene Adipate Terephthalate PE - Linear Low-density Polyethylene PES - Polyester fibres PET- Poly (ethylene terephthalate) PP - Polypropylene RSG %- Relative Seed Germination RShG % - Relative Shoot Growth WHC - Water Holding Capacity

4. What is AMR? 1

5. Antibiotics in Aquaculture Demand is increasing Intensification Gram negative organisms: Aeromonas hydrophila , A. salmonicida , Vibrio anguillarum , V. harveyi , Flavobacterium psychrophilum Gram positive organisms: Streptococcus and Staphylococcus; and fast Mycobacterium sp Inhibit cell wall synthesis Depolarize the cell membrane Inhibit protein synthesis, Inhibit nuclei acid synthesis, Inhibit metabolic pathways FAO (2020) Preena et al. (2019) Therapeutic Prophylactic Metaphylaxis 2

6. AMR in Aquaculture Use of antibiotics in aquaculture Intensification Continuous use Overuse No prescriptions Poor management Selective pressure Poor regulations 80 kg antibiotics per production cycle in a single shrimp hatchery Global antimicrobial consumption in aquaculture (2017) 10,259 tons Asia–Pacific region 93.8% Schar et al. (2020) Hinchliffe et al. (2018) A ntimicrobials used by Chilean salmonid farms, from 143.2 t in 2010 to  382.5 t ons in 2016 3

7. AMR and ARGs in Aquaculture Healthy fish Sick fish Carrier fish Rapidly transport and dilute 70-80% of antibiotics remain in water and sediment Selective pressure on aquatic bacterial diversity Medicated feed Mobile genetic elements ( plasmids, integrons and transposons that can easily move, recombine and mobilize ) ARGs ARGs hotspots Santos and Ramos (2018) 4

8. Selective pressure Alterations of biodiversity Substitute resistant communities in place of susceptible ones Genetic fluctuations in susceptible ones Give rise to the antimicrobial resistant types Once resistance got acquired, the determinants retain within the community even in the absence of responsible antibiotics Enhances the risk of wider dissemination of resistance determinants tetA , tetC , tetH , tetM Preena et al. (2019) 5

9. Antimicrobial resistant fish pathogens associated with cultured fishes Preena et al. (2020) 6

10. Antimicrobial resistance determinants found in fish pathogens and other marine and freshwater bacteria, shared with human pathogens Santos and Ramos (2018) 7

11. Antibiotic Resistance Acquired Natural A trait shared within a bacteria Independent of previous exposure Reduced permeability of outer membrane Natural activity of efflux pumps Acquire through horizontal gene transfer (HGT): transformation, transposition, and conjugation Mutations to its own chromosomal DNA Temporary or permanent Plasmid-mediated transmission of resistance genes - most common Bacteriophage-borne transmission – rare Directly from environment (Acinetobacter spp.) Mutations usually only occur in genes; encoding drug targets , encoding drug transporters , encoding regulators (control drug transporters), and encoding antibiotic-modifying enzymes Reygaert (2018) 8

12. Horizontal gene transfer Transformation Transduction Conjugation Bello-López et al. (2019) 9

13. Horizontal gene transfer

14. Mobile genetic elements of AMR associated with fishes 10

15. Mechanism of AMR Intrinsic resistance Acquired resistance Reygaert (2018) Gram positive Gram negative 11

16. 1. Limiting drug uptake Functions of the LPS layer in G - bacteria provides innate resistance to antimicrobial agents Mycobacteria have an outer membrane that has a high lipid content where Fluoroquinolones have an easier access to the cell, but hydrophilic drugs have limited access Mycoplasma related species, are intrinsically resistant to all drugs that target the cell wall Staphylococcus aureus (G+), recently has developed resistance to vancomycin ( VISA strains) Decrease number and mutations Biofilm Thick, and sticky consistency Sessile Blair et al. (2014) Miller et al. (2014) 12

17. 2. Modification of drug targets Alterations in structure and number of PB Ps ( transpeptidases – PG in cell wall ) Ex. PBP2a in S. aureus by acquisition of the mecA gene van genes – structure of peptidoglycan precursors, decrease binding ability. Mutations in genes ( mprF ) change the charge of the cell membrane surface to positive, inhibiting the binding of calcium R ibosomal: Mutation Subunit methylation involving erm genes Protection M odifications in DNA gyrase (G-: gyrA ) or topoisomerase IV (G+: grlA ) Mutations in enzymes involved in the folate biosynthesis pathway and/or overproduction of resistant DHPS and DHFR enzymes for the drugs that inhibit metabolic pathways . β- lactam drugs (+) Glycopeptides Lipopeptides (-) Target the ribosomal subunits Target nucleic acid synthesis Inhibit metabolic pathways Beceiro et al. (2013) Huovinen et al. (1995) 13

18. 3. Drug inactivation Acetylation is the most widely used method, and it's been linked to: Aminoglycosides Chloramphenicol Streptogramins Fluoroquinolones Actual degradation Transfer of a chemical group Acetyl Phosphoryl Adenyl Most common Chemical groups β-lactamases - drug hydrolyzing enzymes tetX gene - hydrolyzing tetracycline Reygaert (2018) 14

19. 4. Drug efflux Five main families of efflux pumps: ATP-binding cassette (ABC) Multidrug and toxic compound extrusion (MATE) Small multidrug resistance (SMR) Major facilitator superfamily (MFS) Resistance-nodulation-cell division (RND) 15 Reygaert (2018)

20. Antimicrobial resistance mechanisms

21. Antibiotic Resistome Term “ ANTIBIOTIC RESISTOME ” was first coined in 2006 by Gerry Wright’s group Source: http://dx.doi.org/10.1016/j.mib.2014.09.002 Source: https://doi.org/10.1038/s12276-021-00569-z

22. AMR detection methods in Aquaculture Preena et al. (2019) 16

23. Culture based detection methods Antimicrobial susceptibility testing Minimum inhibitory concentration ( MIC ) Division and evaluation of values can be calculated following epidemiologic cut off values based on the frequency of distribution of susceptibility results Data generated based on the antibiotic resistance tests can be interpreted using appropriate statistical tools like WHONET 5 software Phenotypic assays Calculating MIC of antibiotics in the presence and absence of efflux pump inhibitors - the four-fold reduction of MIC in the presence of inhibitor can be considered as significant Production of beta lactamases could be confirmed by Chromogen based nitrocefin hydrolysis methods Preena et al. (2019) 17

24. Minimum inhibitory concentration (MIC) Lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism. AST Kirby-Bauer Method (Disc diffusion method) Dilution method Epsilometer test E-Test Tube dilution Agar dilution Micro-dilution Macro-dilution

25. Broth dilution method and MIC

26. E - Test

27. Minimum Bactericidal Concentration (MBC) L owest concentration of an antimicrobial agent required to achieve bactericidal killing , defined as a 99.9% reduction in the initial inoculum. Determined by subculturing all tubes showing no visible turbidity Tube with highest dilution that fails to yield growth on the subculture plate contains the MBC of antibiotic for the test strain.

28. Minimum inhibitory concentration (MIC) versus minimum bactericidal concentration (MBC). 

29. Disc diffusion method

30. Molecular detection methods Global antimicrobial resistance surveillance system (GLASS) Loop mediated isothermal amplification (LAMP) Lateral flow immunoassay (LFIA) Line probe assay (LPA) Whole genome sequencing (WGS) Next generation sequencing (NGS) Fluorescence in situ hybridization (FISH) Multiplex PCR Coupled multiplex PCR and micro array technique Quantitative PCR (qPCR) Pulsed-field gel electrophoresis (PFGE) Restriction fragment length polymorphism (RFLP), Random amplified polymorphic DNA (RAPD), Amplified ribosomal DNA restriction analysis (ARDRA) DNA microarray technology 18

31. Lee et al. (2015) Multiplex PCR for β- Lactamase Gene

32. List of PCR Primers used for antibiotic resistance gene detection in aquaculture 19

33. Preena et al. (2019) 20

34. Omics based approach Metagenomic approach Bioinformatics resources: AMRfinder , ResFinder, ARG-ANNOT, SRST2, Genefinder , ARIBA, MEGARes, KmerResistance Metagenomic analysis, through High-throughput Illumina sequencing coupled with structured sub database of Comprehensive Antibiotics Resistance Database (CARD) Matrix-Assisted Laser Desorption/ Ionization-Time-Of-Flight (MALDI-TOF) mass spectrometry Preena et al. (2019) 21

35. High-throughput Illumina sequencing coupled with structured sub database (CARD)

36. Rapid detection tools for tracking AMR bacteria Real time PCR kits : NucliSENS EasyQ ® KPC, Xpert ® Carba -R cartridge from GeneXpert® ( bioMérieux ), eazyplex ® system, AID ESBL. Colorimetric methods: employed as a part of rapid visualization using commercial kits like RAPIDEC® CARBA NP kit ( bioMérieux ) Recombinase polymerase assay (RPA) was also successfully validated on programmable digital microfluidics (DMF) platform for the rapid and simultaneous detection of multiple antimicrobial resistance genes 22

37. How to control? 23 Conceptual framework proposed for a One Health approach to antimicrobial resistance surveillance in aquaculture

38. Mitigation of AMU/AMR Farmer training Spatial planning A ssistance with disease identification A pplying better management practices at farm level s tricter regulations Disease-free fish seed and vaccines Enforce rigid monitoring of the quantity Quality of antimicrobials used by farmers Minimize antimicrobial residues in the farmed species and in the environment

39. Implement valid measurement methods at national and international levels Minimize antibiotic uses and furtherance of resistance Effective alternative strategies Vaccination Probiotics Bacteriocins (Ex. Marine bacteriocins: divercins and pisciocins produced by Carnobacterium associated with fish intestine) Immunostimulants (Ex. β-1,3 glucans) Immunomodulation (Ex. Seaweeds: Ceramium rubrum, Gracilaria cornea and Asparagopsis armata ) Quorum sensing inhibition compounds (Ex. halogenated furanones, from marine algae) Metal based nanoparticles 24 Preena et al. (2020)

40. Reference 26 Bello-López, J. M., Cabrero -Martínez, O. A., Ibáñez-Cervantes, G., Hernández-Cortez, C., Pelcastre -Rodríguez, L. I., Gonzalez-Avila, L. U., & Castro- Escarpulli , G. (2019). Horizontal gene transfer and its association with antibiotic resistance in the genus aeromonas spp. Microorganisms , 7 (9), 1–11. https://doi.org/10.3390/microorganisms7090363 C Reygaert, W. (2018). An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiology , 4 (3), 482–501. https://doi.org/10.3934/microbiol.2018.3.482 Lee, J. J., Lee, J. H., Kwon, D. B., Jeon, J. H., Park, K. S., Lee, C. R., & Lee, S. H. (2015). Fast and accurate large-scale detection of β-lactamase genes conferring antibiotic resistance. Antimicrobial Agents and Chemotherapy , 59 (10). https://doi.org/10.1128/AAC.04634-14 Preena, P. G., Swaminathan, T. R., Rejish Kumar, V. J., & Bright Singh, I. S. (2020). Unravelling the menace: detection of antimicrobial resistance in aquaculture. Letters in Applied Microbiology , 71 (1), 26–38. https://doi.org/10.1111/lam.13292 Preena, Prasannan Geetha, Swaminathan, T. R., Kumar, V. J. R., & Singh, I. S. B. (2020). Antimicrobial resistance in aquaculture: a crisis for concern. Biologia , 75 (9), 1497–1517. https://doi.org/10.2478/s11756-020-00456-4 Santos, L., & Ramos, F. (2018). Antimicrobial resistance in aquaculture: Current knowledge and alternatives to tackle the problem. International Journal of Antimicrobial Agents , 52 (2), 135–143. https://doi.org/10.1016/j.ijantimicag.2018.03.010 Schar, D., Zhao, C., Wang, Y., Larsson, D. G. J., Gilbert, M., & Van Boeckel , T. P. (2021). Twenty-year trends in antimicrobial resistance from aquaculture and fisheries in Asia. Nature Communications , 12 (1), 6–15. https://doi.org/10.1038/s41467-021-25655-8 Subasinghe , R., Soto, D., & Jia, J. (2009). Global aquaculture and its role in sustainable development. Reviews in Aquaculture , 1 (1). https://doi.org/10.1111/j.1753-5131.2008.01002.x Thornber , K., Verner-Jeffreys, D., Hinchliffe, S., Rahman, M. M., Bass, D., & Tyler, C. R. (2020). Evaluating antimicrobial resistance in the global shrimp industry. Reviews in Aquaculture , 12 (2), 966–986. https://doi.org/10.1111/raq.12367

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