Microbiological profiling of biosolids used as top soil improvers

Transcription

1 Microbiological profiling of biosolids used as top soil improvers EU RL for E. coli Annual Workshop 2015 Federica Gigliucci, NRL for E. coli, Italy Istituto Superiore di Sanità, Rome, Italy. Department of Veterinary Public Health and Food Safety. European Reference Laboratory for E. coli

2 Biomass originates from farm industry Sludges Dairy industry Manure Natural drying: dried sludge, the hard final remains from a sewage plant.

3 From Food industry Eggs Tomatoes Olive oil production Fruit

4 From Agriculture Aerobic digestion: Green compost: food and green wastes, only Mixed Compost: food and green wastes, added with sludges

5 From municipal waste Municipal sewage sludge: Sewage sludge refers to the residual, semi-solid material that is produced as a by-product during sewage treatment of industrial or municipal wastewater Organic fraction of the household wastes

6 Biomass: What Use? Biomass is used for Biogas production (and biofuel to a lesser extent) Digestates (end of waste) Top Soil Improvers (Valuable N and C sources)

7 Biomass: What Size? Italy (per year): Sewage: 10,500,000 tons Manure: 6,300,000 tons Agri-Food: 2,500,000 tons Total; 21,300,000 tons Whole Europe: More Than 500,000,000 tons/year

8 Top Soil Improvers: What Rules? USA (USEPA 40 CFR (Part 503): Biosolids class A Compliance with microcriteria Salmonella <3 MPN/4g E. coli counts 1000 MPN/g Enteric viruses 1 PFU/4g Helmint ova <1/4g Biosolids class B fecal coliforms < 2X10 6 MPN/g Restrictions: Class A: No restrictions Class B biosolids cannot be used in some States Europe (ECO-Label criteria): ECO-label: Salmonella absent in 25 g E. coli counts 1000 MPN/g Helmint ova absent in 1.5 g Restrictions: No restrictions encoded at EU Level Some MS apply restrictions to sludges Top Soil Improvers (EU): In some EU MS it can be used as such, in others only after composting, finally in some other MS their use is forbidden

9 Top Soil Improvers: What risks? Chemical: PTEs PCB IPA Dioxins Furans others Microbiological: Pathogens to soils Pathogens to crops AMR genes to soil bacterial communities Risk assessment has not been adequately performed yet Hazard characterisation: STEC-associated genes Pathogenic E. coli associated genes Hazard characterisation: Microbiological profiling Resistance to compunds Real time PCR Metagenomics

10 Pathogenic E. coli virulence genes in DNA extractions from TSI Virulence genes Sample Sample Type name stx1 stx2 stx2f eae AggR aaic LT STh STp ipah 3 Sludge 28 direct Sludge 29 direct Sludge 30 direct Mixed compost 31 direct Sludge 32 direct Sludge 33 direct Mixed compost 11 direct Mixed compost 12 direct Mixed compost 13 direct Mixed compost 14 direct Mixed compost 15 direct Mixed compost 16 direct Sludge 18 direct B1 Sludge 19 direct C1 Sludge 20 direct E1 Sludge 21 direct H1 Sludge 26 direct Manure (pig) 27 direct Sludge 9 direct Mixed compost 10 direct C1 Sludge 22 direct G1 Sludge 23 direct I1 Sludge 24 direct H1 Sludge 25 direct

11 Pathogenic E. coli virulence genes in enrichment culture from TSI Virulence genes Sample Sample Type name stx1 stx2 stx2f eae AggR aaic LT STh STp ipah 3 Sludge 28 Enr Sludge 29 Enr Sludge 30 Enr Mixed compost 31 Enr Sludge 32 Enr Sludge 33 Enr Mixed compost 11 Enr Mixed compost 12 Enr Mixed compost 13 Enr Mixed compost 14 Enr Mixed compost 15 Enr Mixed compost 16 Enr Sludge 18 Enr B1 Sludge 19 Enr C1 Sludge 20 Enr E1 Sludge 21 Enr H1 Sludge 26 Enr Manure (pig) 27 Enr Sludge 9 Enr Mixed compost 10 Enr C1 Sludge 22 Enr G1 Sludge 23 Enr I1 Sludge 24 Enr H1 Sludge 25 Enr

12 STEC and TSI: Quantitative assessment stx2 gene quantitation 18 Sludge B1- Sludge C1- Sludge E1- Sludge G1- Sludge 7- Sludge 11- MCO Copies/rxn \ \ MCO Copies/g 4.3X X X X10 4 \ \ 3X X10 3 eae gene quantitation 6- MCO 7- Sludge 8 Sludge 11- MCO C1- Sludge E1-Sludge Copies/rxn 1.90 \ Copies/g 3.8X10 2 \ 6.7X X X X10 4

13 Pathogenic E. coli risks related with the use of TSI The TSI analysed do contain STEC and other pathogenic E. coli (or their DNA) The load derived from quantitative PCR appears to be high qpcr signals persist after enrichment in many cases, suggesting the presence of live cells TSI host different E. coli pathotypes at the same time, it may favour the emergence of E. coli pathotypes with mixed virulence genes array The use of TSI as C and N sources for soils destined to agricultural uses configure an advantage but also a potential risk for transfer of pathogenic E. coli to crops A complete risk assessment exercise is necessary!!!!

14 A holistic approach to microbial hazard identification C1-Sludge E1 Sludge 16 Mixed 14 Mixed 15 Mixed 12 Mixed C1 Sludge B1 Sludge Compost Compost Compost Compost KatP katp none asta; bfpa espi iron espc katp prfb nleb cba; ccl iha prfb fedf nleb tir pet cdtb; cfac prfb tir ipah pet vat prfb cif; cma tir toxb prfb prfb tir cnf1; eae eata; efa1 espa; espb espc; espf toxb tir tir espi; espj Eight samples subjected espp; etpd to metagenomics (Shotgun) fim41a; ehxa; DNA extracted from 0,25 hlye; g ihaa; of solid ipah matter using kits specific for soils Fragmented and irea; sequenced iron onto an IonTorrent PGM iss; K88; katp; 3.5 M reads obtained from nleb; each nlec; sample pet on average with 450 bases lenght pic; prfb; senb; sepa; stx1a var. d; stx1a var. c; stx1a var. a; stx1b var. d; stx1b var. c; stx2a var. f; stx2a var. e; stx2a var. g; stx2a var. d; stx2a var. a; suba; saa; tccp; tir toxb; tsh; virf; picu; pic; Bowtie2 mapping of reads against the E. coli Virulence genes database (F. Scheutz@SSI)

15 Metagenomics Analysis of TSI (Shotgun)

16 Metagenomics Analysis of TSI (Shotgun)

17 Other potential microbiological threats related with TSI Functional Metagenomics. Diamond Vs KEGG databse. ARIES (

18 TSI and genes conferring resistance to compounds Functional Metagenomics. Diamond Vs SEED databse. ARIES (

19 Final conclusions Biomass used as top soil improvers represent a huge business for the AgriFood sector both in the US and the EU TSI may contain pathogenic E. coli including STEC that may be transferred to crops (not to mention other pathogens!) AMR transfer to bacterial communities in the soil and crops is also a matter of concern TSI that are not awarded the Ecolabel are at highest risk, is needed for a more clear regulatory framework A comprehensive Risk Assessment is needed to come to a sound scientific basis for developing regulations

20 Thank you for your attention Rosangela Tozzoli Stefano Morabito Alfredo Caprioli Valeria Michelacci Roslen Bondì Fabio Minelli Antonella Maugliani Arianna Angeloni Paola Chiani Federica Flamini Clarissa Ferreri