Nuevas tecnologas en el estudio de los

contaminantes microbianos en aguas

subterrneas

Rosina Girones,

Slvia Bofill-Mas,

Xavier Fernandez-Cassi, Natalia Timoneda,

Marta Rusiol, Laura Guerrero, Ayalkibet Hundesa

Eloy Gustavson, Josep Abril

III JORNADA DE PUERTAS ABIERTAS DE LA FCIHS 2015

Environmental waters are susceptible to fecal contamination from point and nonpoint sources. Excreted viruses may contaminate water and

food.

Seawater

Shellfish

Rivers and lakes

Bathing water

Ground water

Drinking water

Irrigation water

Vegetables

Water Sources Associated with Drinking Water Outbreaks (N = 33) and Outbreak-related Cases (N = 1,040), Waterborne Disease and Outbreak

Surveillance System, CDC, 2009-2010

Waterborne disease in Norway. H.M.L. Kvitsand and L.Fiksdal. Water Science and

Technology, 2010

Hepatitis A (HAV) Hepatitis E (HEV) Norovirus (NoV)

Family Picornaviridae Hepeviridae Caliciviridae

Genera Hepatovirus Hepevirus Norovirus

Size 27-32 nm 27-34 nm 30-34 nm

Genome 7.5 Kb 7.5 Kb 7.57.7 Kb

Classification Six genotypes 1,2,3: human 4,5,6: simian

Four genotypes 1,2: human

3,4: human, porcine

Five genogroups 1,2,4: humans

3: murine 5: bovine

Transmission Water and food Water ( food) Water and food

Symptoms Hepatitis, mild in young individuals

Hepatitis, severe in pregnant

Gastroenteritis, explosive vomiting

HAV

HEV

NoV

Excreted RNA viral pathogens (HAV, HEV, NoV, SaV, RoV, AstV, and EV).

Human Adenovirus

(HAdV) Human Polyomavirus

Family Adenoviridae Polyomaviridae

Genera Mastadenovirus Polyomavirus

Size 70-100 nm 4245 nm

Genome 36 38 Kb

(lineal double stranded)

5 kb

(circular double stranded)

Classification 7 species

A-G: humans

13 described

JCPyV, BKPyV, MCPyV

Transmission Respiratory, fecal-oral unknown; respiratory,

fecal-oral

Symptoms Gastroenteritis, respiratory

infections , conjunctivitis

Persistent asymptomatic

infections, multifocal

leucoencefalopathy (PML)

HAdV

JCPyV

Excreted DNA viral pathogens.

Methods for the control of viral contamination in water and food

The methods most commonly used present the following steps:

1. Concentration of the viruses from the food or water sample into a

suitable volume

2. Extraction of the RNA or DNA from the target organism;

3. Genomic amplification techniques

4. Detection or quantification of the amplified genomic sequences.

Concentration of viruses from river, seawater, groundwater, using

direct flocculation with skimmed milk

Direct Flocculation Procedure:

A: Detail of the flocculated sample.

B: Image of the sedimented flocs.

Inter-laboratory assays of the SM-flocculation procedure for river water

1 Mean value of 12 samples concentrated in two different days (6 samples per day).

* Brazil, Laboratory of Marize Miagostovich, FIOCRUZ Institut, Rio de Janeiro, Brazil

Viruses Laboratory

localization

1Mean viral

recovery % Range % SD

HAdV Spain 49,95 27,79 - 94,79 24,21

Brazil* 50,25 20,93 - 88,98 36,90

NoV Spain 51,58 33,55 - 92,18 18,46

Brazil* 52,52 21,29 - 73,82 15,59

JCPyV Spain 50,83 37,36 - 71,13 9,27

Viral dissemination in river catchments and

impacted seawater

AVALUACI DE LA DISSEMINACI DE VIRUS EN UNA CONCA MEDITERRNIA I ANLISI EN FUTURS ESCENARIS DE CANVI CLIMTIC

N

2000

150-1000

1000-2000

Corrent marina predominant

Llobregat

- 170Km

- Cabal 690hm3/any

- 4950 Km2

- 5m de persones i activitat industrial

- 51 depuradores (300hm3/any = 43%)

Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas. Calgua B, Fumian T, Rusiol M, Rodriguez-Manzano J, Mbayed VA, Bofill-Mas S, Miagostovich M, Girones R. 2013 May 15;47(8):2797-810.

Viruses in river water

Waste water treatment plant in the area of Barcelona:

Volume of urban sewage: 420 millions of liters per day

representing 2 million population equivalent.

Treatment: Biological treatment with nutrient removal, tertiary

with ACTIFLO process and UV (water reclamation).

Persistence of adenovirus and polyomavirus in wastewater

treatment plants

AVALUACI DE LA DISSEMINACI DE VIRUS EN UNA CONCA MEDITERRNIA I ANLISI EN FUTURS ESCENARIS DE CANVI CLIMTIC

0

1

2

3

4

5

6

7

HAdV JCPyV MCPyV NoVGII EC IE

Log

GC

/L o

NM

P/1

00m

l

Secundari 1.42 1.60 1.57 2.50 1.43 1.79

Terciari 0.87 0.89 0.06 1.12 1.73 1.62

TOTAL 2.29 2.49 1.63 3.61 3.16 3.41

HAdV i FIB al 100% mostres residual crua

JCPyV 85% i MCPyV al 75%

NoV GGII 100% durant els mesos ms freds

HEV es detecta al

13% (5/37) residual crua

12% (4/32) efluents secundaris

0% (0/22) efluents terciaris

Desprs del tractaments secundari i terciari a la

depuradora, encara es detecten virus a un 50-75% de les mostres

DEBAS

T 1ari 2ari 3ari

Caracteritzaci efluents de la depuradora

Logaritmes de reducci

50 de les 51 depuradores aboquen els efluents directament al riu (276hm3) i noms 2 daquestes 50 desprs dun tractament terciari

Highly stable in the environment, host-specificity and high prevalence throughout the year

The quantification by PCR of DNA viruses is robust and has acceptable costs

Viruses have been proposed as Microbial Source

Tracking tools

MST includes a group of methodologies that aim to identify, and in some cases quantify, the dominant sources of fecal

contamination in the environment and, especially, in water

resources.

MST plays a very important role in enabling effective management and remediation strategies.

VIRAL MST tools:

human porcine bovine ovine avian

1. Microbial source tracking studies using

adenovirus and polyomavirus in five river

catchments (Viroclime project).

Rusiol M, Fernandez-Cassi X, Hundesa A, Vieira C, Kern A,

Eriksson I, Ziros P, Kay D, Miagostovich M, Vargha M, Allard A,

Vantarakis A, Wyn-Jones P, Bofill-Mas S, Girones R. Water Res.

2014, 59:119-29

MST 5 case studies:

MA03 MA02

MA01

MA04 MA05

Rio

Negro Rio

Amazonas

Rio

Solimes

Flow: 28.000 m3/s

Basin: 691.000 Km2

Lenght: 2250Km

LL02

LL01

LL03

CR1

Flow: 16,9 m3/s

Basin: 4984Km2

Length 170 Km

Flow:: 792 m3/s

Basin: 156,087 Km2

Length:: 965 km

TI03

TI04

TI06

TI05

TI01

TI02

UM03

UM02

UM01

Flow: 450 m3/s

Basin: 26,814.8 Km2

Length: 470 km

PA03

PA02

PA08

PA04

PA01

PA07

Flow: 1m3/s

Basin: 24 Km2

Length: 98km

Rio Negro (Brasil)

Riu Llobregat (Catalonia)

Umelven (Sweden)

(Greece)

Tisza (Hungary)

Laboratory Sampling

points

Samples

Total Total N of

analyses River Sea

Rio Negro 5 192 - 192 768

Llobregat 3 64 38 122 480

Umelven 3 108 54 162 648

Glafkos 4 100 60 160 600

Tisza 6 110 - 166 664

802 3.160

Samples and analysis. At each sampling point four markers of fecal

contamination were analyzed: 2 human (JCPyV and HAdV), porcine

(PAdV) and bovine (BPyV).

Floculation 50mL sewage or 10L river

5 ml qPCR detection and quantification

2x10 ml

100 ml of the original sample

Extraction 140 ml

2x140 l

Process control: HAdV 35

(HAdV)

(JCPyV)

(PAdV)

(BPyV)

(OPyV)

(Ch/TyPV)

Analysis of viruses

*

*

*

*

*

*

*

*

*

*

* *

*

*

*

*

*

*

*

*

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

0

10

20

30

40

50

60

70

80

90

100

Umealven(SWEEDEN)

Rio Negro (BRAZIL) Glafkos (GREECE) Tisza (HUNGARY) Llobregat (SPAIN)

Me

an

co

nc

en

tra

tio

n (

GC

/L)

Pe

rce

nta

ge

of

po

sit

ive

sa

mp

les

(%

)

Human and animal viruses in diferent river cathments

HAdV

JCPyV

PAdV

BPyV

*

*

*

*

* * *

*

*

*

* *

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

0

10

20

30

40

50

60

70

80

90

100

Umealven (SWEEDEN) Glafkos (GREECE) Llobregat (SPAIN)

Me

an

co

nc

en

tra

tio

n (

GC

/L)

Pe

rce

nta

ge

of

po

sit

ive

sa

mp

les

(%

)

Human and animal viruses in seawater

HAdV

JCPyV

PAdV

BPyV

Groundwater in Catalonia has a great importance in the supply of drinking water and the supply for industry and agriculture. Groundwater constitute

approximately 35% of total water resources used (ACA)

Both point and non-point sources of contamination may affect groundwater

Nitrate is the most widespread groundwater quality problem in many countries, and it is the most frequent cause of a groundwater body failing

to meet good status under the Water Framework Directive in some

countries

The principal nitrogen inputs into groundwater are derived from manure, fertilizers, sewage sludge and crops residues from agricultural areas.

Ground water contamination

Analysis of the Origin of Nitrate Contamination in Ground Water

Human viruses Animal viruses

HAdV JCPyV PAdV BPyV

Site qPCR

nPCR n % qPCR

nPCR n % qPCR

nPCR n % qPCR

nPCR n %

GC/l GC/l GC/l GC/l

1 - NT 4 0 - - 5 0 - - 4 0 - - 4 0

2 - - 4 0 - - 5 0 7,74x102 + 5 100 - - 4 0

3 - NT 4 0 - - 5 0 - - 4 0 - - 4 0

4 - - 4 0 - - 5 0 - - 4 0 9,53x103 + 5 20

n: number of replicates analyzed

%: percentage of positive replicates

NT: no tested

(-): no detected

Departament de Control i

Millora dels Ecosistemes

Aqutics

Specific viruses present in polluted groundwater are

indicative of the source of nitrates and fecal

contamination in agricultural areas.

Slvia Bofill-Mas, Marta Rusiol, Josep Fraile,

Teresa Garrido, Antoni Munn, Rosina Girones.

Metagenomics for the study of viruses in water

Advances in high-throughput, deep sequencing technology make it possible to analyze simultaneously a wide diversity of viruses, to

characterize virome richness, gene functions, and association with

disease

Furthermore, many diseases of unknown etiology are thought to be of viral origin

Jim Pipas

Dave Wang Guoyan Zhao

Paul Cantalupo

Cantalupo PG, et al. 2011. Raw sewage harbors diverse viral populations. mBio 2(5):e00180-

11. doi:10.1128/mBio.00180-11.

Analysis of the excreted virome in urban sewage using high NGS techniques

Overview of the collection and processing of waste water and the subsequent bioinformatic analysis of the waste water metagenome.

10 L

International Locations Waste Water Collection

Viral Concentration by Flocculation

454 Sequencing

1 mL Bioinformatics to classify sequences

Pittsburgh

Barcelona

Addis Ababa

We detected 234 known viruses. Members of 26 different families, including

those with dsDNA, ssDNA, ssRNA(+) and dsRNA genomes, and those with

either enveloped or nonenveloped virions.

Bacteria; 247363

Fungi; 406

Human; 1425

Mouse; 803

Other; 5096

Phage; 37917

Virus; 8491

Unassigned 596146

Total nucleic acid (DNA and reverse-transcribed RNA) was

sequenced and binned according to taxa based on BLAST searches. Most

sequences found within virions do not match the sequences in public

databases.

Plant 90,9%

Human 5,8%

Insect 3,1%

Rat 0,2%

Pig 0,1%

ANNOTATION PIPELINE STATISTICS OF THE RAW SEWAGE METAGENOMES

Addis Ababa Barcelona Pittsburgh

Total 640054 1043224 662246

Unique 52750 577475 307987

% total 8,2% 55,4% 46,5%

Filtered 4346 23778 9950

% total 0,7% 2,3% 1,5%

Low Complexity 225 1114 1152

% total 0,0% 0,1% 0,2%

Quality Seqs 48179 552583 296885

Avg Length 271 bp 295 bp 342 bp

% total 7,5% 53,0% 44,8%

Most virus-related pyrosequencing reads found in raw sewage represent previously

unknown viruses

Detection and quantification of virus by PCR assays in

urban sewage analyzed in metagenomics study

Urban sewage samples

BCN2-GV-

150908 1-ETH_190609 2-ETH_200609

Virus analyzed 33.33 mL 43.75 mL 43.75 mL

HAdV (qPCR GC/mL) 1,01E+04 1,03E+01 8,02E+02

JCPyV (qPCR GC/mL) 1,83E+01 1,78E+02 7,34E+02

HEV (nPCR) - + -

HAV (nPCR) - + +

KV (nPCR) + + +

nASFV (nPCR) - + -

Metagenomics of excreted viruses in sewage samples from

Barcelona using Illumina MiSeq

Sample Reads Mean length

Winter 2828219 231

Autum 796157 243

Spring 1177145 227

Sewage10L

Concentration to 2ml

Nucleic Acid Extraction

DNAse treatment

Sequenase RT-PCR random

+ sequenase

Round B PCR (40 cyles) Clean and concentrator

Nextera

Illumina Miseq

Increase the volume of sample tested (more sensitive) Increase depth (reads) Reduce inhibition

1. Amplification of a region of Hexon protein of adenovirus with degenerated primers

2. Pyrosequencing GS Junior Systems , Life Science (Roche), CCiTUB 3. Bioinformatics Analysis:

A

Quality control Cutadapt / FastQC / Prinseq

B

OTUs determination CDhit

C

Taxonomic assignment ClustalW2 / Genedoc

D

Rarefaction curves and diversity index Dnadist / Mothur

E

Phylogenetic Analysis Fastree / ITOL

NGS Target Enrichment for the identification of the adenoviruses

excreted in the population

Phylogenetic Analysis of human adenovirus species (A-G) and the adenovirus

found in pyrosequencing analysis. Phylogenetic tree reconstruction was obtained

by the neighbor-joining method using ClustalW2 program. Bootstrap analysis was

made with Raxml program and the tree representation was created with iTOL

program.

20

0

l

20

0

l

20

0

l

20

0

l

Pooled samples

according age Sample

homogenization 0.22 filtration Centrifugation

3400 rpm- 15min

DNAse

treatment

Nucleic acid

extraction

RT with

random

primers +

sequenase

PCR

amplification

Nextera +

Illumina Mi-seq

Fecal

samples

PROJECT WATER JPI

METAWATER

New Metagenomics And Molecular Based Tools

For European Scale Identification And Control Of

Emergent Microbial Contaminants In Irrigation

Water

Partners in the project:

Spain: Rosina Girones, Universitat de Barcelona

Maria Jos Figueras, Universitat Rovira i Virgili

Jos Luis Alonso de la Universidad Politcnica de Valencia

Denmark: Charlotta Lfstrm, Anna Charlotte Schultz Technical University

of Denmark

Cyprus: Georgios T. Papageorgiou, State General Laboratory

Germany: Christiane Hller, Bavarian Health and Food Safety Authority,

Michael Seidel, Technische Universitt Mnchen.

.

The final objective is to prevent epidemics and to produce the

scientific bases to support the development of European/national

regulation for water use for irrigation.

Specific objectives are:

1. Developing new bioinformatics tools and novel methods for water-borne

emergent pathogens, NGS systems for nucleic acid detection and novel

sample preparation protocols suitable for irrigation water and

implementation in end-user laboratories. Preparation of internationally

harmonized Standard Operation Procedures for the control of

pathogens in irrigation water.

2. Characterization of viral/bacterial/protozoa communities/pathogens

in critical points of source water and distribution networks, including

antibiotic-resistant bacteria and cyanobacterial toxins.

3. Characterization of microbial communities and pathogens in

wastewater treatment plants using diverse technologies, and

reclaimed water at the point of use.

Specific objectives (continue):

5. Development of the list of microorganisms identified representing a

risk in irrigation water in Europe: virome and bacteriome.

6. Identifying transmission routes, and defining prevalence and behavior

of antibiotic resistant bacteria in irrigation water.

7. Evaluation of microbial removal efficiency in wastewater treatment

plants and risk assessment studies to build new evidence-based

analysis of the suitability of bacterial and viral indicators and current

water regulations for controlling irrigation water quality.

8. To integrate sequence and annotation databases into a web-

based dynamic interface for implementation of the developed

resources at the European level.

Identification of novel viruses analyzed by deep sequencing and emergent pathogenic bacteria in sewage and reclaimed water and evaluation of the risk derived for food safety. Improving deep sequencing techniques for fast identification of viral pathogens in water and food

Acknowledgments:

www.ub.edu/microbiologia_virology

Ayalkibet Hundesa

Marta Rusiol

Laura Guerrero

Natalia Timoneda

Xavier Fernandez Cassi

Eloy Gonzales Gustavson

Slvia Bofill

Natalia Figuerola

Rosina Girones

ComputationalGenomics

Laboratory, UB

Josep Abril