G L O B A L S Y M P O S I U MO N S O I L

P O L L U T I O N2 - 4 M A Y 2 0 1 8 | F A O - R O M E , I T A L Y

Animal and anthropogenic pressures on agricultural soils from Shiga/Vero-Toxin producing E. coli (STEC/VTEC): impact on

food safety and food securityGianfranco Brambilla, Federica Gigliucci, Valeria Michelacci, Rosangela Tozzoli and

Stefano Morabito - Istituto Superiore di Sanità, EURL-VTEC

INTRODUCTION

The use of biosolids and remediat-ed water from civil wastewater treatment plants (WWTPs) in agri-culture practices can represent a port of entry of pathogenetic E. coli strains including STEC in the food chain. The latter strains have been proven to cause severe Haemolitic Uremic Syndrome (HUS) out-breaks in children, Enterocolitis in adults and provoked food scandals that hampered food security.Additional to the spreading of pathogens to soil, potential genetic re-assortment between strains of human, animal, and environmen-tal origin, may determine the pres-ence of emerging pathotypes show-ing different combinations of viru-lence and resistance factors:• vtx1, vtx2, eae, aggR aaiC, lt, sth,

stp, and ipaH • Antimicrobial Resistance - AMR

METHODOLOGY

Investigation of virulence factors associated with pathogenic E. coli strains and Antimicrobial resis-tance (AMR) genes in a set of dif-ferent Biosolid samples originat-ing from WWTPs in Italy, using a Real Time Polymerase Chain Reac-tion (RT-PCR) and Whole Metag-enomic Sequencing approaches.

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CO3 CO2 MCO CO1 BSO2 BSO4 BSO3 BSO1

Distributionofthegenetictraitsencodingresistancetocompounds inmetagenomes

Arsenic_resistance Methicillin_resistance_in_Staphylococci Fosfomycin_resistance Multidrug_efflux_pump(CmeABC_operon)

Mercuric_reductase Beta-lactamase Multidrug_Resistance,_Tripartite_Systems Zinc_resistance

Copper_homeostasis:_copper_tolerance Resistance_to_fluoroquinolones Streptothricin_resistance Resistance_to_Vancomycin

Multidrug_Resistance,_2-protein Cobalt-zinc-cadmium_resistance Multidrug_Resistance_Efflux_Pumps Mercury_resistance_operon

Copper_homeostasis Aminoglycoside_adenylyltransferases Streptococcus_pneumoniae_Vancomycin_Tolerance_Locus

Genomic traits related to resistance to compounds, including resistance to heavy metals and antimicrobials

Genetic pool of virulence genes associated with all the pathotypes of Diarrheagenic E. coli.

Positive signals for some virulence genes in some cases appeared only after the enrichment of the samples, suggesting the

presence of viable bacterial cells

Nucleic acid extracted from enriched cultures

vtx1 vtx2 eae aggR aaiC lt sth stp ipaH

MSS 13-0474 - - - - - - - - -MSS 13-0475 - - - - - - - - -MSS 13-0476 - + - + - - - - -DIG 13-0477 - - - - - - - - -MSS 13-0478 - + - - - - - - -MSS 13-0479 - - - - - - - - -MCO 13-0482 + + + - - - - - -MCO 13-0483 - - + - - - - - -MCO 13-0484 - + + - - - - - -GCO 13-0485 - - - - - - - - -MCO 13-0486 - - - - - - - - -GCO 13-0487 - + - - - - - - -MSS 13-0488 - - + - - - - - -MSS 13-0489 - + + - - - - - -MSS 13-0490 - - + - - - - - -MSS 13-0492 - + + - - - - - -MSS 13-0496b - + - - - - - - -MAN 13-0497 - + - - - - - - -MSS 13-0480 - - - - - - - - +MCO 13-0481 - - - - - - - - -MSS 13-0493 - - - - - - - - -MSS 13-0494 - - - - - - - - -MSS 13-0495 - - - - - - - - +MSS 13-0496 - - + - - - - - +

Sample

Representation of a potential super-pathogen E. coli due to the acquisition of virulence and resistance genes from different pathotypes. From Navarro-Garcia F. (2014), modifi ed.

Pathogenic E. coli in human faeces

Animal pathogenic E. coli in manure

Animal pathogenic E. coli in slaughterhouses effluents

Human and Animal Pathogenic E. coli in civil

Waste WaterTreatment Plants

Contaminated sludges Contaminated reclaimed waters

Application on agriculture soil

Irrigation of vegetables

Potential contamination of unprocessed food and well waters

Food exposure leading to Haemorrhagic Colitis in adults and to a severe

Haemolitic Uremic Syndrome in children

Food recall and food insecurity

Potential re-assortment of virulence factors and AMR genes

CONCLUSIONSThe sustainability policies for food production may impact on food safety/food security due to the recycle of former wastes of anthropogenic origin as soil improvers.Pathogenic E. coli should be considered as priority emerging hazards, as far current aerobic/anaerobic fer-mentation to stabilize biosolids can be considered not effective in their abatement.The set up of diagnostic tools based on genomic and metagenomic approaches would be able to identify emerg-ing pathotypes, to design the end-of-waste criteria for the agriculture use of biosolids as well to evaluate the efficacy of the risk management .