Biology Reference
In-Depth Information
in being negative for indole production, ornithine decarboxylase activity,
acid production from mannitol, and sorbitol and growth at 42°C. Amaro
and Biosca (1996) concluded that strains of biotype 2 (eel pathogens) are
also opportunistic pathogens for humans.
During 1996-1997, 62 cases of wound infections and bacteremia due to
V. Vulnifi cus
were found to result from contact with purchased inland pond
raised tilapia in Israel (Bisharat et al
.
, 1999). The outbreak was due to a new
marketing policy of selling live fi sh instead of marketing them packed in
ice
post-mortem
. The isolates exhibited fi ve atypical biochemical test results
and were non-typeable by pulsed fi eld gel electrophoresis (PFGE) and
all had the same polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) pattern derived from a 388-bp DNA fragment
of the
cth
gene. Following PCR amplifi cation of this fragment, digestion
was performed with the three restriction nucleases
Dde
I,
Hha
I, and
Hpa
II.
These isolates were distinguishable from biogroups 1 and 2 on the basis
of negative reactions with respect to the utilization of citrate, salicin,
cellobiose, and lactose. The authors designated these isolates as belonging
to a newly established subspecies group biotype 3.
Molecular Methods for Detection and Typing
V. Vulnifi cus
Conventional PCR
developed by Hill et al. (1991) for evaluating the effectiveness of the
PCR in identifying isolates of
V. vulnifi cus
from marine environments. A
total of 13,325 bacterial isolates from seawater, sediments, oysters, and
goby specimens collected along the coastal regions of Tokyo Bay were
metabolically screened. Among these, 713 grew at 40
o
C, required NaCl for
growth, formed greenish colonies on TCBS agar, and were presumptively
identifi ed as
V. vulnifi cus
. The PCR amplifi ed the targeted 519-bp sequence
of the
cth
gene with 61 of these isolates. DNA-DNA hybridization with
the type strain of
V. vulnifi cus
and the API 20E system confi rmed the PCR
results. The authors concluded that the PCR method is useful for rapid
and accurate identifi cation of
V. vulnifi cus
from marine sediments.
Real-time PCR (Rti-PCR)
Panicker et al. (2004) described a SYBR Green I-based real-time PCR assay
for detection of
V. vulnifi cus
in oyster tissue homogenate. A pair of primers
sequence of the
vvh
gene. The minimum level of detection was 100 CFU
(Colony Forming Units) per PCR tube. A 5 h enrichment allowed detection
of 1 CFU/ml of tissue homogenate which is equivalent to 10 CFU/g of
tissue. The assay required 8 h for completion.
