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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
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 - + - + - - - - - -
14 Mixed compost 14 Enr - - - - - - - - - -
15 Mixed compost 15 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 - - - - - - - - - +
10 Mixed compost 10 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)
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