microbiological profiling of biosolids used as top soil...

Microbiological profiling of biosolids used as top soil improvers

Federica Gigliucci, NRL for E. coli, Italy

EU RL for E. coli Annual Workshop 2015

Istituto Superiore di Sanità, Rome, Italy. Department of Veterinary Public Health and Food Safety. European Reference Laboratory for E. coli

Biomass originates from farm industry…

Sludges

Natural drying: dried sludge, the hard final remains from a sewage plant.

Manure

Dairy industry

Tomatoes

Eggs

Olive oil production

…From Food industry…

Fruit

Aerobic digestion: • Green compost: food and green wastes,

only • Mixed Compost: food and green wastes,

added with sludges

…From Agriculture…

…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

Biomass: What Use?

Biomass is used for Biogas production (and biofuel to a lesser extent)

Top Soil Improvers (Valuable N and C sources)

Digestates (end of waste)

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

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 < 2X106 MPN/g

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

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: Class A: No restrictions Class B biosolids cannot be used in some States

Restrictions: No restrictions encoded at EU Level Some MS apply restrictions to sludges

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

Real time PCR

Hazard characterisation: Microbiological profiling Resistance to compunds

Metagenomics

Virulence genes

Sample Type Sample name

stx1 stx2 stx2f eae AggR aaiC LT STh STp ipaH

3 Sludge 28 direct - - - - - - - - - -

4 Sludge 29 direct - - - - - - - - - -

5 Sludge 30 direct - - - - - - - - - -

6 Mixed compost 31 direct - - - + + - - - - -

7 Sludge 32 direct - + - + - - - - - -

8 Sludge 33 direct - - - + - - - - - -

11 Mixed compost 11 direct - + - + + - - - - -

12 Mixed compost 12 direct - - - - - - - - - -

13 Mixed compost 13 direct - + - - + - - - - +

14 Mixed compost 14 direct - - - - - - - -

15 Mixed compost 15 direct - - - - + - - - - -

16 Mixed compost 16 direct - - - - - - - - - -

18 Sludge 18 direct - + + + + - - - - -

B1 Sludge 19 direct - + - - + - - - - -

C1 Sludge 20 direct - + + + + - - - - -

E1 Sludge 21 direct - + + + + - - - - +

H1 Sludge 26 direct - - - - - - - - - -

19 Manure (pig) 27 direct - - - - + - - - - -

9 Sludge 9 direct - - - - - - - - - -

10 Mixed compost 10 direct - - - - - - + - - -

C1 Sludge 22 direct - - - - - - + - - -

G1 Sludge 23 direct - + - - - - - - - -

I1 Sludge 24 direct - - - - - - + - - -

H1 Sludge 25 direct - - - - - - + - - -

Pathogenic E. coli virulence genes in DNA extractions from TSI

Virulence genes

Sample Type Sample name

stx1 stx2 stx2f eae AggR aaiC LT STh STp ipaH

3 Sludge 28 Enr - - - - - - - - - -

4 Sludge 29 Enr - - - - - - - - - -

5 Sludge 30 Enr - + - - + - - - - -

6 Mixed compost 31 Enr - - - - - - - - - -

7 Sludge 32 Enr - + - - - - - - - -

8 Sludge 33 Enr - - - - - - - - - -

11 Mixed compost 11 Enr + + - + - - - - - -

12 Mixed compost 12 Enr - - - + - - - - - -

13 Mixed compost 13 Enr - + - + - - - - - -



16 Mixed compost 16 Enr - + - - - - - - - -

18 Sludge 18 Enr - - - + - - - - - -

B1 Sludge 19 Enr - + - + - - - - - -

C1 Sludge 20 Enr - - - + - - - - - -

E1 Sludge 21 Enr - + - + - - - - - -

H1 Sludge 26 Enr - + - - - - - - - -

19 Manure (pig) 27 Enr - + - - - - - - - -

9 Sludge 9 Enr - - - - - - - - - +


C1 Sludge 22 Enr - - - - - - - - - -

G1 Sludge 23 Enr - - - - - - - - - -

I1 Sludge 24 Enr - - - - - - - - - +

H1 Sludge 25 Enr - - - + - - - - - +

Pathogenic E. coli virulence genes in enrichment culture from TSI

18

Sludge

B1-

Sludge

C1-

Sludge

E1-

Sludge

G1-

Sludge

7-

Sludge

11-

MCO

13-

MCO

Copies/rxn 21.5 6.5 6 67 \ \ 1.5 5.5

Copies/g 4.3X103 1.3X103 1.2X103 1.3X104 \ \ 3X102 1.1X103

6- MCO 7- Sludge 8 Sludge 11-

MCO

C1- Sludge

E1-Sludge

Copies/rxn 1.90 \ 33.5 20.5 47 286

Copies/g 3.8X102 \ 6.7X103 4.1X103 9.4X103 5.7X104

STEC and TSI: Quantitative assessment

stx2 gene quantitation

eae gene quantitation

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!!!!

A holistic approach to microbial hazard identification C1-Sludge E1 Sludge

16 Mixed Compost

C1 Sludge 14 Mixed Compost

B1 Sludge 15 Mixed Compost

12 Mixed Compost

KatP katP none astA; bfpA cba; ccl cdtB; cfaC cif; cma cnf1; eae eatA; efa1 espA; espB espC; espF espI; espJ espP; etpD fim41A; ehxA; hlyE; ihaA; ipaH ireA; iroN iss; K88; katP; nleB; nleC; pet 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;

espI iroN espC katP prfB nleB iha prfB fedF nleB tir pet prfB tir ipaH pet vat prfB tir toxB prfB prfB

tir toxB tir tir

Eight samples subjected to metagenomics (Shotgun) DNA extracted from 0,25 g of solid matter using kits specific for soils

Fragmented and sequenced onto an IonTorrent PGM 3.5 M reads obtained from each sample on average with 450 bases lenght

Bowtie2 mapping of reads against the E. coli Virulence genes database (F. Scheutz@SSI)

Metagenomics Analysis of TSI (Shotgun)

Other potential microbiological threats related with TSI

Functional Metagenomics. Diamond Vs“KEGG databse”. ARIES (https://w3.iss.it/site/aries/)

TSI and genes conferring resistance to compounds

Functional Metagenomics. Diamond Vs“SEED databse”. ARIES (https://w3.iss.it/site/aries/)

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

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

microbiological profiling of biosolids used as top soil...

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