http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/ International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)

1. FAO TCP/INT/3502 “Reducing and managing the risk of
Acute Hepatopancreatic Necrosis Disease (AHPND) of Cultured Shrimp”
Vibrio parahaemolyticus:
genome plasticity, mobile
genetic elements and
bacteriophage therapy
Iddya Karunasagar
Senior International Consultant
Food and Agriculture Organisation of United
Nations
Iddya.Karunasagar@fao.org
Iddya.Karunasagar@gmail.com
6/24/2015
International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer Responses”
22-24 June 2015, Tryp Hotel, Panama City 1

2. Vibrios in the environment
Human pathogenic
Vibrios
Pathogens of aquatic
animals
V. cholerae
V. parahaemolyticus
V. vulnificus
Vibrio harveyi
Vibrio anguillarum
Alivibrio salmonicida
Vibrio penaecida
Vibrio vulnificus
Vibrio owensii
Vibrio harveyi clade includes eleven species:
V. harveyi, V. alginolyticus, V. parahaemolyticus, V. campbellii, V. rotiferianus, V. mytili, V.
natriegens, V. azureus, V. sagamiensis, V. owensii, V. jasicida

3. Vibrio parahaemolyticus
• First reported in 1950 from an outbreak of gastroenteritis
implicating “shirasu” (small half dried sardine) in Japan
involving 272 people
• Mostly reported from sporadic cases involving consumption of
raw or undercooked seafood
• Common inhabitant of estuarine and coastal environments in
the warmer parts of the world.
• Most clinical isolates (over 95%) were found to produce a β
hemolysis in high salt blood agar and this phenomenon was
referred to as kanagawa phenomenon.

4. Vibrio parahaemolyticus
• The hemolysin responsible for kanagawa hemolysin was
designated “thermostable direct hemolysin” (TDH).
• Most environmental strains (about 98%) are kanagawa
negative.
• An outbreak in Maldives associated with TDH negative strains
of V. parahaemolyticus led to the discovery that such clinical
strains produce a TDH-related hemolysin, commonly called
TRH.
• Molecular studies show that the trh gene coding for TRH has
69% similarity with tdh gene coding for TDH.

5. Vibrio parahaemolyticus
• Presently, tdh and trh are considered genetic markers of human
pathogenic strains.
• Characterisation of human pathogenic strains generally involves the
demonstration of the presence of tdh or trh genes by polymerase
chain reaction (PCR) or by probe hybridization assays.
• Variations in tdh gene have been reported and 5 variants (tdh1-
tdh5) have been reported, but most strains producing hemolysis on
wagatsuma agar contain two copies of tdh gene, ie, tdh1 and tdh2.
• tdh4 may be present in a plasmid.
• tdh gene has also been detected rarely in strains of V. mimicus, and
non-O1/139 strain of V. cholerae.

6. Vibrio parahaemolyticus
• Molecular genetic studies show that tdh and trh genes have
been acquired by V. parahaemolyticus.
• tdh and trh genes are present within “genomic islands” in V.
parahaemolyticus.
• They have Guanine + cytosine content different from that in V.
parahaemolyticus genome.
• They are flanked by “direct repeat regions” (DRR), which are
involved in genetic exchange.
• Possibly the genes were acquired by the strains to improve
their fitness in certain niches

7. Vibrio parahaemolyticus
Okada et al., 2009

8.  Mechanism used by bacteria to establish an infection or
symbiotic relationship with a eukaryotic cell by mediating the
injection of effector proteins into the host cell cytoplasm
 Needle-like apparatus- Injects (translocates) proteins into the
cytosol of eukaryotic cells
Activated by bacterial contact with host cell surfaces
 Translocated proteins facilitate bacterial pathogenesis by
specifically interfering with host cell signal transduction and other
cellular processes
Type III Secretion System

9. Type three secretion system
(T3SS) in Gram negative
bacteria
(A) T3SS with the two rings
spanning the membranes and
the needle protruding outside
the bacterium
(B) T3SS act as translocators,
form a pore into the target cell
membrane, and the effectors
are translocated into the cytosol
of the target cell
(Troisfontaines and Cornelis, 2005)

10. T3SS in Vibrio parahaemolyticus
• Two T3SS have been detected in V. parahaemolyticus, T3SS-1
and T3SS-2.
• T3SS-1 gene cluster is found in all V. parahaemolyticus strains.
The G+C content in this gene cluster suggests that this is
indigenous to V. parahaemolyticus.
• T3SS-2 gene cluster is present in only clinical and
environmental strains carrying tdh or trh gene, which are
markers for human virulence. This gene cluster carries the
enterotoxin gene. Two types of T3SS-2 have been identified,
T3SS2α and T3SS2β. The former is found in tdh+ strains and
the latter in trh+ strains.

11. Ham and Orth, 2012

12. Genomic Islands
• Genomic islands are chromosomal genes that are acquired by
horizontal gene transfer that may increase the fitness of the
organism in the environment or in association with their
hosts.
• They could be “pathogenicity islands” or “metabolic islands.
• V. parahaemolyticus genome has been reported to contain 9
genomic islands designated VPaI-1 to VPaI-9.
• These range in size from 10kb to 81kb and are characterised
by the G+C content different from V. parahaemolyticus
genome, presence of phage like integrase, flanked by direct
repeats and are located adjacent to tRNA genes indicating
common mechanism of chromosomal integration.

13. Information on genome,
structural and functional
characteristics of genes
encoding virulent proteins
available
V. parahaemolyticus: Genome Overview
Chromosome 1 Chromosome 2
Length of sequence: 3,288,558 bp 1,877,212 bp
G+C ratio: 45.4 % 45.4 %
Open reading frame (ORF): 3,080 1,752
Protein coding region: 86.9 % 86.9 %
Average length of ORFs 926.9 bp 931.3 bp
Makino et al., 2003

14. Okada et al., 2009

15. T3SS and genomic islands
• T3SS-1 is present in chromomosome 1
• T3SS-2α is present in chromosome 2 in VPaI 7 that also
contains the tdh gene.
• VPaI-7 lacks integrase (unlike other VPaIs), but contains
several transposase genes.
• VPaI-1, VPaI-3, VPaI-4, Vpai-5 and VPaI-6 have been described
to be unique to post-1995 pandemic strains with some
exceptions.
• VpaI-2 is found in pre-1995 strains.

16. Okada et al, 2010

17. Serotype and pathogenicity
• V. parahaemolyticus contain both somatic (O) and capsular (K)
antigens. 11 O antigens and 75 K antigens have been
recognised.

18. Serotype and pathogenicity
• Till 1996, no association between serotype and association
with clinical cases was noticed.
• But in 1996, a serotype O3:K6 was involved in large number of
cases in India and subsequently, infection by this serotype has
been recorded in almost all continents.
• This led to the designation of this strain as “pandemic strain”
• Molecular genetic analysis indicated that these O3:K6 strains
were different from strains of the same serotype isolated in
Asia before 1993.

19. Nair et al, 2007

20. Pandemic Vibrio parahaemolyticus
• Strains isolated from different parts of the world show clonal
nature in molecular typing techniques such as Pulse Field Gel
Electrophoresis (PFGE) or arbitrarily primed PCR (AP-PCR).
• These strains are characterised by the presence of two copies
of tdh gene and a filamentous phage f237.
• They show unique/new ToxR sequence within the ToxRS
operon and this has been used to design a PCR method for
detection of pandemic strains.
• Several serovariants of O3:K6 strain show genetic similarity
with pandemic strains. Some of the common serovariants are
O4:K68, O1:K25, O1:K41 and O1:KUT (untypable)

22. Mobile genetic elements in V. parahaemolyticus
• V. parahaemolyticus genome can carry different types of
mobile genetic elements like:
• Plasmids
• Bacteriophages
• Insertion sequence elements
• Genomic islands

23. Plasmids in V. parahaemolyticus
Plasmids are extrachromosomal self replicating genetic
elements that can be transferred between bacteria.
They play a role in transfer of genes: eg antibiotic resistance
determinants, virulence genes between bacteria

24. Plasmids in V. parahaemolyticus
• Several small plasmids (3.5kb to 4.8kb) have been reported in
V. parahaemolyticus. However they carry only hypothetical
proteins and their role in horizontal gene transfer is not
known.
• Sequence analysis of p22702B, a 28.8kb plasmid indicated
presence of a gene with 98% nucleotide identity with
hypothetical protein encoded by VPaI-6. This suggests
possible role of plasmids in V. parahaemolyticus.

25. Bacteriophages
Filamentous bacteriophages are present in V.
parahaemolyticus genome eg f237 in pandemic strains.
Small bacteriophages (<10kb) and large T4 like phages (eg
KVP40 phage, 244.8kb) have been detected.

26. AHPND causing V. parahaemolyticus
• So far, all the reported AHPND strains lack tdh and trh genes
suggesting that these lack the ability to cause typical V.
parahaemolyticus gastrointestinal symptoms and are not a
food safety concern.
• An interesting feature common to strains from Mexico and
Thailand seems to be the absence of a 140 kbp region
containing genes coding for T3SS.
• Most investigators have found AP3 primers described by
Sirikharin et al (2014) to be highly specific and these have
been reported to be based on the sequence of gene coding
for 12kDa protein that is present in the supernatant of
cultures causing AHPND in experimental studies.

27. AHPND causing V. parahaemolyticus
• Recent studies show that AHPND causing V.
parahaemolyticus strains contain a 69kbp plasmid
(VpA1) encoding Pir (Photorhabdus insect related) -
like toxins.
• A new nested PCR for detection AHPND V.
parahemolyticus based on 1269 bp region of this
plasmid has been reported by Sritunyalucksana et al
(2015).

28. Summary
• V. parahaemolyticus is highly versatile and has been evolving
by acquiring genetic elements by horizontal gene transfer.
• There are strains capable of causing sporadic cases of
gastroenteritis, strains capable of global spread.
• Several genomic islands have been detected in pandemic
strains.
• Horizontal gene transfer has been contributing to the
evolution of V. parahaemolyticus and emergence of new
strains.
• Presence of a characteristic plasmid in AHPND strains suggest
that these strains evolved by horizontal gene transfer.
6/24/2015
International Technical Seminar/Workshop
“EMS/AHPND: Government, Scientist and
Farmer Responses”
28

29. BACTERIOPHAGE THERAPY
• Bacteriophages natural
inhabitants of aquatic
environment
• Play a role in bacterial
population dynamics in
natural environment
• Highly active and specific-
controls only target
bacteria, does not affect
commensal flora
• can act on biofilms
• Autoreplicative

30. Bacteriophages in aquatic environment
• Viruses, most abundant life forms. Most of these are
bacteriophages
• Viral lysis removes 20-40% of the standing stock of
prokaryotes every day

31. LYTIC AND LYSOGENIC STAGES

32. Reported examples of phage therapy in aquaculture
Aquacultured
species
Pathogen Route of
administration
Reference
Yellowtail
(Seriola
quinqueradiata)
Lactococcus
garvieae
Oral Nakai et al.,
1999
Ayu
(Plecoglossus
altivelis)
Pseudomonas
plecoglossicida
Oral Park et al.,
2000
Black tiger
shrimp
(Penaeus
monodon)
Vibrio harveyi Addition to
larval rearing
tank water
Vinod et al.,
2006
Black Tiger
shrimp
(Penaeus
monodon)
Vibrio harveyi
biofilm
Addition to
water
Karunasagar
et al., 2007

33. REGULATORY APPROVAL FOR USE OF
BACTERIOPHAGES- examples
• US EPA has approved use of AgriPhage from
Omnilytics Inc, against plant pathogens
• FDA has approved Listeria LMP 102 from
Intralytics Inc for control of L. monocytogenes
in ready to eat foods
• Intralytics Inc has obtained license for use of
SPLX-1 and PLSV-1 against Salmonella in
poultry

34. COMMERCIAL PRODUCTS ARE AVAILABLE IN SOME
COUNTRIES FOR AQUACULTURE
Commercial products are not endorsed by FAO

35. BACTERIOPHAGE THERAPY: PROBLEMS
• Combination of phages needed to prevent
resistance development
• Possible transfer of virulence factors
Phage A Phage B Phage C Cocktail of
A,B and C
40% 38% 52% 86%
Lysis of V. parahaemolyticus strains isolated from aquaculture
environment (data from Indrani Karunasagar et al., 2014)

36. Summary
• Bacteriophages have potential for application as alternatives
to antibiotics in aquaculture.
• V. parahaemolyticus phages are present in estuarine
environments including sediments.
• There is need for more research on phage therapy for AHPND
V. parahaemolyticus.
6/24/2015
International Technical Seminar/Workshop
“EMS/AHPND: Government, Scientist and
Farmer Responses”
36

37. 6/24/2015
International Technical Seminar/Workshop “EMS/AHPND:
Government, Scientist and Farmer Responses”
37
THANK YOU
Gracias