May 31st - June 1st, 2012

Koblenz, Germany

Effect of global warming on Vibrio

spp. in the temperate marine
environment
Luigi Vezzulli1*, Ingrid Brettar2, Elisabetta Pezzati3,
Philip C. Reid4 Rita R. Colwell5, Manfred G. Hfle2, Carla Pruzzo1
1 Department for the Study of Territory and its Resources, University of Genoa, Corso Europa, 26, 16132 Genova
2 Helmholtz Centre for Infection Research, Department of Vaccinology & Applied Microbiology, D-38124 Braunschweig, Germany
3 Department of Pathology, Section of Microbiology School of Medicine, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy
4 Sir Alister Hardy Foundation for Ocean Science, Citadel Hill, The Hoe, Plymouth PL1 2PB, UK
5 Maryland Pathogen Research Institute and Center of Bioinformatics and Computational Biology, University of Maryland, College Park,
MD 20742, USA.

Vibrio spp.

Over 80 species described

Gram negative (comma shaped)
Asporigen
Aerobe/facultative anaerobes
non capsulated
motile (polar flagellum)
Pathogenic for humans and animals

Cholera DFA
counts

Extreme weather events

Eutrophication
Rainfall
Small and large climatic
process

TEMPERATURE
SALINITY
pH
NUTRIENTS
SUNLIGHT

ECOSYSTEM

COMMUNITY

POPULATION
MICROBIAL
INTERACTIONS
ASSOCIATON WITH LIVING
&NONLIVING SUBST.

GOOD
KNOWLEDGE

CELL

TROPHIC INTERACTIONS
OTHER ECOL. INTERACTIONS (INTRA AND INTERSPECIFIC)

cascade effect

RESOURCES and
CONDITIONS

UNDERSTANDING THE
AUTOECOLOGY OF
VIBRIO SPECIES

??

Vibrio cholerae
ENVIRONMENT vs HUMAN
ASSOCIATED FACTORS
Origin of outbreaks and epidemics
Endemicity
Emergence of new virulent strains

RESOURCES and
CONDITIONS

ECOSYSTEM

TEMPERATURE
SALINITY
pH
NUTRIENTS
SUNLIGHT

MICROBIAL
INTERACTION
COMMUNITY
POPULATION

MICROBIAL
INTERACTIONS
ASSOCIATON WITH LIVING
&NONLIVING SUBST.

CELL

TROPHIC INTERACTIONS
OTHER ECOL. INTERACTIONS (INTRA AND INTERSPECIFIC)

cascade effect

CLIMATE CHANGE
HUMAN ACTIVITIES

UNDERSTANDING THE
AUTOECOLOGY OF
VIBRIO SPECIES

Gut

Oral region

Egg sacs

A colonized copepod may contain up to 104 cells

of V. cholerae equal to an infectious dose

Vezzulli L, Pruzzo C, Huq A, Colwell RR (Environ Microbiol Rep, 2010)

Persistence as
Biofilm and VBNC
Natural transformation
(seroconversion)
Heterotrophic
metabolism

Diffusion

Chemotaxis

Infectious dose

Cholera

Protection from
acidic condition

Chitin cycling

Pruzzo C, Vezzulli L, Colwell RR (Environ Microbiol, 2008)

Trophic interactions: marine food chain

INORGANIC AND ORGANIC
NUTRIENTS
PHYTOPLANKTON
ZOOPLANKTON
DOC

Bottom-up

VIBRIOS
Top-down
VIBRIOPHAGES

CHOLERA

RESOURCES and
CONDITIONS

ECOSYSTEM

TEMPERATURE
SALINITY
pH
NUTRIENTS
SUNLIGHT

TEMPERATURE
COMMUNITY

POPULATION
MICROBIAL
INTERACTIONS
ASSOCIATON WITH LIVING
&NONLIVING SUBST.

CELL

TROPHIC INTERACTIONS
OTHER ECOL. INTERACTIONS (INTRA AND INTERSPECIFIC)

cascade effect

CLIMATE CHANGE
HUMAN ACTIVITIES

UNDERSTANDING THE
AUTOECOLOGY OF
VIBRIO SPECIES

CELL

Effects of Temperature on
Vibrio cell physiology

Growth rate
Culturability
Phage sensitivity
Antigenicity
Virulence

A
COMMUNITY

Effect of temperature on
Vibrio-plankton interaction

COMMUNITY

Vibrio choleraes
colonization factors
Stauder et al (2012), Env Microbiol Rep

Temperature-related colonization of
plankton
Temperature related expression of V.
cholerae colonization factors

ECOSYSTEM

Effects of SST on seasonal Vibrio

occurence in aquatic ecosystems

VIBRIOSEA project

MPN/L

Vibrio spp.

V. parahaemolyticus

Seawater
Sediment
Mussel
Plankton
Vezzulli et al (2009), Microb Ecol

V. cholerae

MPN/L

June 2006-September 2007

LIGURIAN SEA

Temperature

THRESHOLD
TEMPERATURE

SST, Vibrio and Cholera

epidemics

Pascual et al (2000), Science

Eastern Tropical Pacific

Vibrio coralliilyticus

SST, Vibrio and Coral

mass mortality

NW Mediterranean Sea

BIVALIFE

SST, Vibrio and Oysters

mass mortality
Temperature (<19C)
Eutrophication

Bivalve
genetics

Bivalve
physiology

Virus
V. splendidus
V. aestuarianus

NE Atlantic Ocean

Global Ocean Warming

Global Upper Ocean Heat Content

Effects of SST on Longterm Vibrio occurence?

C Frank, M Littmann, K Alpers, J Hallauer

Vibrio vulnificus wound infections after
contact with the Baltic Sea, Germany
Volume 11, Issue 33, 17 August 2006

In 2006 several human

infections related to
vibrios were reported
for the North Sea and
Baltic Sea

Y Andersson, K Ekdahl
Wound infections due to Vibrio
cholerae in Sweden after swimming in
the Baltic Sea, summer 2006.
Volume 11, Issue 31, 03 August 2006

FM Schets, HHJL van den Berg, AA

Demeulmeester, E van Dijk, SA Rutjes, HJP van
Hooijdonk, AM de Roda Husman
Vibrio alginolyticus infections in the
Netherlands after swimming in the North
Sea.
Volume 11, Issue 45, 09 November 2006 .

CONCERN EXPRESSED BY THE

2010-2011 MCCIP
Annual Report Card
(www.mccip.org.uk/arc)

2010-2011
Marine vibrios

An experimental-based approach to study

long-term change in Vibrio population:

CONTINUOUS PLANKTON RECORDER

SURVEY (CPR)

CPR is one of the largest running marine biological

monitoring program in the world

CPR
Survey

CPR Archive 1948-2011

(more than 200.000 samples)
Reid et al., 2010

Northern hemisphere

Southern hemisphere

Selected CPR samples (1961-2005)

CPR samples DNA

extraction protocol

Filtering silk
5X

1 cm2 section

25 ml of TE buffer + vortex
to detach particles from silk mesh
24h incubation RT
1h incubation at 56C, 1h incubation at 90C
after lysozyme and proteinase K digestion
QIAamp DNA Micro procedure

3-5 ug Genomic DNA

Fragmentation analysis of DNA extracted from selected

CPR samples collected in front of the Rhine Estuary
1961

1976

1992

2004

A dominant DNA smears in the

200-800 bp size range was found
in genomic DNA recovered from
~50-years old CPR samples

DNA extracted from CPR samples:

good quality and suitable for real-time PCR
A

Real-time PCR amplification of

Vibrio 16S rRNA genes from
genomic collected in the North
Sea off the Rhine estuary.
A.Melting curve analysis
B.Agarose gel

Vibrio relative quantification

by Real Time PCR

Vibrio relative Abundance Index

(VAI)
Small amplicons (~100bp)
maximise the PCR yields even if the DNA was
fragmented.

Similar size amplicons (98bp vs 113bp)

avoid age/formalin induced bias by assuming that
DNA damage over time was the same for the whole gene
pool within a sample.

Validation of VAI index using historic plankton samples

Correlation analysis between culturable Vibrio counts (CFU/10L) associated with
small plankton samples collected in the Adriatic Sea from April 1996 to March
1997 (Montanari et al., 1999) and VAI index (average STD of 5 replicate
measurements) calculated 14 years later on the same samples.

WinCPR

A database of North Sea plankton

free download at: http://cpr.cscan.org/

Vezzulli and Reid (2003). Prog Oceanogr

Increase over 4 decades in

the relative abundance of
vibrios with rising SST
Vezzulli et al (2012), ISME J

Correlation analysis

Vezzulli et al (2012), ISME J

Step-wise multiple regression analysis

Variance Explained by SST 45%

Vezzulli et al (2012), ISME J

Vibrio-plankton interaction
(Rhine Area)
PCA analysis

16SrDNA Deep Sequencing

of the plankton associated bacterial community
About 44,000 PCR amplicons
that span the V6 hypervariable
region of 16SrRNA genes.

CPR 1961
CPR 1972
CPR 1976
CPR 1998

CPR 2004

Rhine estuary

Stringent trimming procedure

reduced dataset by 23%
The number of reads
sample: 6037 - 13000

per

BLAST against V6RefDB

database (Sogin et al. PNAS)

Multiple-comparative analysis in MEGAN of 16S RNA gene

Pyrosequencing data

Vezzulli et al (2012), ISME J

Global Scale Macroecology and Epidemiology

of Human Vibrio pathogens (GLOBALVIBRIO)

10
11

8
7
9

3
4
5

(1940 2010)

1 North Sea
2 Shetland island
3 Iceland Sea
4 Irish Sea
5 Bay of Biscay
6 portugal coast
7 North Atlantic
8 Newfoundland

12

14

9 Nova Scotia
10 Aleutinian
11 Vancouver
12 Japan
13 Tasmanian Sea
14 South Africa
15
15 Antarctica

13

Non-Parametric
Probabilistic Ecological
Niche (NPPEN) model
1960-2008
Beaugrand et al (2011), MEPS

Laboratory of Environmental Microbiology

University of Genoa, Italy

Laboratory Staff:
Carla Pruzzo
Luigi Vezzulli
Elisabetta Pezzati
Monica Stauder
Main Collaborators:
Rita R. Colwell (USA)
Anwar Huq (USA)
Ingrid Brettar (Germany)
Manfred Hofle (Germany)
Philip C. Reid (UK)