resistance genes associated with lettuce leaf surfaces grown in - - PowerPoint PPT Presentation

Metagenomic profiling of antibiotic resistance genes associated with lettuce leaf surfaces grown in soils receiving cattle manure-based amendments

Giselle K. P. Guron, Amy Pruden, Monica A. Ponder Virginia Tech IAFP Annual Meeting July 9, 2018

Antibiotic resistance from foods?

• Worldwide

consumption of antimicrobials in livestock is projected to rise from over 60,000 tons in 2010 to over 105,000 in 2030 (Van Boeckel et

• al. 2015)

https://www.cdc.gov/foodsafety

Interdisciplinary Approach Tracking Antibiotic Resistance from Farm-to-Fork

Animal Dosing Composting Soil Application Vegetables Postharvest Education & Outreach

FSMA Regulations for Fresh Produce

• The Food Safety Modernization Act (FSMA) outlines

composting methods to reduce potential foodborne pathogens

• Composting also benefits soil health and nutrient availability
• Composting methods require minimum number of days ≥ 55

°C (21 CFR Part 112.54(b))

Ray et al. 2017 J. Environ. Qual. Pirlimycin Length of Composting (Day) Temperature (°C) Days

Methods to identify ARGs in foods

• PCR-based methods
• Antibiotic susceptibility testing and whole genome

sequencing

• High throughput sequencing (HTS)

Objectives

• To use HTS to characterize ARG profile (or

resistome) of lettuce grown in two different soil types amended with dairy cattle-based manure, compost, or chemical fertilizer

• Identify putative markers of gene transfer co-
• ccurring with ARGs

Cattle dosing and composting

Alfalfa Hay Mulch Sawdust

Greenhouse Set-Up

Soil Type Loamy Sand Silty Clay Loam Amendment Type Fertilizer Control (FC) Manure Static Compost Cattle Type From Control Dairy (DC) From Antibiotic- Treated Dairy (DA)

0.1% peptone (w/v) 0.1% Tween 80 (w/v) 0.22-µm-pore mixed cellulose membrane 1/10 dilution (w/w) Diluent Membrane Rinse & massage

Chloroplast 16S rRNA gene sequences Rarefaction to 12,700,000 reads each sample ARG idenfication:

• CARD v1.2.1, modified (≥ 80% identity, ≥ 75 bp coverage) (Jia et al. 2017)
• ARG-Miner v1.1.6, modified (DeepARG Machine Learning algorithms) (Arango-Argoty et al. 2017)

Arango-Argoty et al. 2016

Direct annotation of reads (average length 101-bp) MetaStorm (https://bench.cs.vt.edu/MetaStorm/)

Results

• CARD: 614 ARGs identified among 23 ARG classes
• ARG-Miner: 3,249 ARGs identified among 26 ARG

classes

• The most genes shared between both databases:

ARG Classes # genes unique to CARD # genes unique to ARGMiner # common genes Aminoglycoside 32 124 44 Beta-Lactam 68 417 47 MLS 15 428 60 Multidrug 28 284 111

ARGs identified using CARD

Multidrug Trimethoprim Triclosan Tetracycline Tetracenomycin Sulfonamide Rifamycin Quinolone Polymyxin Pleuromutilin Phenicol Peptide Nucleoside MLS Glycopeptide Fusidic Acid Fosfomycin Elfamycin Bicyclomycin Beta-Lactam Bacitracin Aminoglycoside Aminocoumarin

n = 2 n = 2 Loamy Sand n = 2 n = 2 n = 2 n = 3 n = 3 n = 3 n = 3 n = 3 Silty Clay Loam Gene copies/16S rRNA gene copies Manure Compost Manure Compost

Total ARGs affected by compost * soil

Multidrug Trimethoprim Triclosan Tetracycline Tetracenomycin Sulfonamide Rifamycin Quinolone Polymyxin Pleuromutilin Phenicol Peptide Nucleoside MLS Glycopeptide Fusidic Acid Fosfomycin Elfamycin Bicyclomycin Beta-Lactam Bacitracin Aminoglycoside Aminocoumarin

n = 2 n = 2 Loamy Sand n = 2 n = 2 n = 2 n = 3 n = 3 n = 3 n = 3 n = 3 Silty Clay Loam Gene copies/16S rRNA gene copies Manure Compost Manure Compost

* *

* p<0.05, Wilcoxon

ARGs identified by ARG-Miner

n = 2 n = 2 n = 2 n = 2 n = 2 n = 3 n = 3 n = 3 n = 3 n = 3 Loamy Sand Silty Clay Loam

Multidrug Tunicamycin Trimethoprim Triclosan Thiostrepton Tetracycline Tetracenomycin Sulfonamide Streptothricin Rifamycin Quinolone Puromycin Polymyxin Pleuromutilin Peptide Mupirocin MLS Kasugamycin Glycopeptide Fusidic Acid Fosmidomycin Fosfomycin Elfamycin Phenicol Beta-Lactam Bacitracin Aminoglycoside Aminocoumarin

Gene copies/16S rRNA gene copies Manure Compost Manure Compost

Total ARGs affected by compost * soil

n = 2 n = 2 n = 2 n = 2 n = 2 n = 3 n = 3 n = 3 n = 3 n = 3 Loamy Sand Silty Clay Loam

Multidrug Tunicamycin Trimethoprim Triclosan Thiostrepton Tetracycline Tetracenomycin # Sulfonamide # Streptothricin Rifamycin Quinolone Puromycin Polymyxin Pleuromutilin Peptide Mupirocin MLS Kasugamycin Glycopeptide Fusidic Acid Fosmidomycin Fosfomycin # Elfamycin Phenicol Beta-Lactam Bacitracin Aminoglycoside Aminocoumarin

* *

Gene copies/16S rRNA gene copies Manure Compost Manure Compost * p<0.05, Wilcoxon; # ARGs significantly different only using ARG-Miner

Soil amendments cause shift in lettuce resistomes (ANOSIM, R>0.25, p<0.05)

Soil Texture (R=0.350) Amendments: DC Compost & DC Manure (R=0.543) DC Compost & DA Compost (R=0.629) DC Compost & FC (R=0.629)

• CARD v1.2.1, modified (≥ 80% identity, ≥ 75 bp coverage) (Jia et al. 2017)

Loamy Sand Silty Clay Loam DA Manure DC Manure DA Compost DC Compost Fertilizer DA Manure DC Manure DA Compost DC Compost Fertilizer Beta-Lactam antibiotic inactivation regulatory system Glycopeptide antibiotic sequestration molecular bypass regulatory system MLS macrolide inactivation streptogramin inactivation antibiotic target modifying efflux pump complex or subunit Multidrug antibiotic target modifying efflux pump complex or subunit regulatory system Polymyxin gene altering cell wall charge efflux pump complex or subunit Quinolone antibiotic target protection efflux pump complex or subunit regulatory system

• Assembled, CARD v1.2.1, modified (≥ 60% identity, ≥ 75 bp coverage) (Jia et al. 2017)

• CARD v1.2.1, modified (≥ 60% identity, ≥ 75 bp coverage) (Jia et al. 2017)
• ACLAME v0.4, modified (≥ 60% identity, ≥ 75 bp coverage) (Leplae et al. 2010)

Aminoglycoside Bacitracin Beta-Lactam Glycopeptide MLS Multidrug Phenicol Polymyxin Rifamycin Sulfonamide Tetracycline Triclosan Trimethoprim Intracellular mobile genetic element

Conclusions

• MLS ARGs and multidrug ARGs that utilize efflux

mechanisms and corresponding regulatory systems were present on all lettuce

• More ARGs were identified on lettuce grown in

composted-amended loamy sand than from silty clay loam

• Composts can shift lettuce leaf resistomes
• More exploration needed for putative transposase

and integrase genes co-localized with ARGs identified from manure-amended lettuce

Thank you!