| dc.description.abstract |
The genus Vibrioof the family Vibrionaceae are Gram negative, oxidasepositive,
rod- or curved- rodshaped facultative anaerobes, widespread in marine
and estuarine environments. Vibrio species are opportunistic human pathogens
responsible for diarrhoeal disease, gastroenteritis, septicaemia and wound
infections and are also pathogens of aquatic organisms, causing infections to
crustaceans, bivalves and fishes. In the present study, marine environmental
samples like seafood and water and sediment samples from aquafarms and
mangroves were screened for the presence of Vibrio species. Of the134 isolates
obtained from the various samples, 45 were segregated to the genus Vibrio on the
basis of phenotypic characterization.like Gram staining, oxidase test, MoF test and
salinity tolerance. Partial 16S rDNA sequence analysis was utilized for species
level identification of the isolates and the strains were identified as V.
cholerae(N=21), V. vulnificus(N=18), V. parahaemolyticus(N=3), V. alginolyticus
(N=2) and V. azureus (N=1). The genetic relatedness and variations among the 45
Vibrio isolates were elucidated based on 16S rDNA sequences. Phenotypic
characterization of the isolates was based on their response to 12 biochemical tests
namely Voges-Proskauers’s (VP test), arginine dihydrolase , tolerance to 3% NaCl
test, ONPG test that detects β-galactosidase activity, and tests for utilization of
citrate, ornithine, mannitol, arabinose, sucrose, glucose, salicin and cellobiose.
The isolates exhibited diverse biochemical patterns, some specific for the species
and others indicative of their environmental source.Antibiogram for the isolates
was determined subsequent to testing their susceptibility to 12 antibiotics by the
disc diffusion method. Varying degrees of resistance to gentamycin (2.22%),
ampicillin(62.22%), nalidixic acid (4.44%), vancomycin (86.66), cefixime
(17.77%), rifampicin (20%), tetracycline (42.22%) and chloramphenicol (2.22%)
was exhibited. All the isolates were susceptible to streptomycin, co-trimoxazole,
trimethoprim and azithromycin. Isolates from all the three marine environments
exhibited multiple antibiotic resistance, with high MAR index value.
The molecular typing methods such as ERIC PCR and BOX PCR revealed
intraspecies relatedness and genetic heterogeneity within the environmental
isolatesof V. cholerae and V. vulnificus. The 21 strains of V. choleraewere
serogroupedas non O1/ non O139 by screening for the presence O1rfb and O139
rfb marker genes by PCR.
The virulence/virulence associated genes namely ctxA, ctxB, ace, VPI,
hlyA, ompU, rtxA, toxR, zot, nagst, tcpA, nin and nanwere screened in V. cholerae
and V. vulnificusstrains.The V. vulnificusstrains were also screened for three
species specific genes viz., cps, vvhand viu. In V. cholerae strains, the virulence
associated genes like VPI, hlyA, rtxA, ompU and toxR were confirmed by PCR.
All the isolates, except for strain BTOS6, harbored at least one or a combination
of the tested genes and V. choleraestrain BTPR5 isolated from prawn hosted the
highest number of virulence associated genes. Among the V. vulnificusstrains,
only 3 virulence genes, VPI, toxR and cps, were confirmed out of the 16 tested
and only 7 of the isolates had these genes in one or more combinations. Strain
BTPS6 from aquafarm and strain BTVE4 from mangrove samples yielded positive
amplification for the three genes.
The toxRgene from 9 strains of V. choleraeand 3 strains of V. vulnificus
were cloned and sequenced for phylogenetic analysis based on nucleotide and the
amino acid sequences. Multiple sequence alignment of the nucleotide sequences
and amino acid sequences of the environmental strains of V. choleraerevealed that
the toxRgene in the environmental strains are 100% homologous to themselves
and to the V. choleraetoxR gene sequence available in the Genbank database. The
3 strains of V. vulnificus displayed high nucleotide and amino acid sequence
similarity among themselves and to the sequences of V. cholerae and V. harveyi
obtained from the GenBank database, but exhibited only 72% homology to the
sequences of its close relative V. vulnificus.
Structure prediction of the ToxR protein of Vibrio cholerae strain BTMA5
was by PHYRE2 software. The deduced amino acid sequence showed maximum
resemblance with the structure of DNA-binding domain of response regulator2
from Escherichia coli k-12 Template based homology modelling in PHYRE2
successfully modelled the predicted protein and its secondary structure based on
protein data bank (PDB) template c3zq7A.
The pathogenicity studies were performed using the nematode
Caenorhabditiselegansas a model system. The assessment of pathogenicity of
environmental strain of V. choleraewas conducted with E. coli strain OP50 as the
food source in control plates, environmental V. cholerae strain BTOS6, negative
for all tested virulence genes, to check for the suitability of Vibrio sp. as a food
source for the nematode;V. cholerae Co 366 ElTor, a clinical pathogenic strain
and V. cholerae strain BTPR5 from seafood (Prawn) and positive for the tested
virulence genes like VPI, hlyA, ompU,rtxA and toxR. It was found that V.
cholerae strain BTOS6 could serve as a food source in place of E. coli strain OP50
but behavioral aberrations like sluggish movement and lawn avoidance and
morphological abnormalities like pharyngeal and intestinal distensions and
bagging were exhibited by the worms fed on V. cholerae Co 366 ElTor strain and
environmental BTPR5 indicating their pathogenicity to the nematode.
Assessment of pathogenicity of the environmental strains of V. vulnificus
was performed with V. vulnificus strain BTPS6 which tested positive for 3
virulence genes, namely, cps, toxRand VPI, and V. vulnificus strain BTMM7 that
did not possess any of the tested virulence genes. A reduction was observed in the
life span of worms fed on environmental strain of V. vulnificusBTMM7 rather than
on the ordinary laboratory food source, E. coli OP50. Behavioral abnormalities
like sluggish movement, lawn avoidance and bagging were also observed in the
worms fed with strain BTPS6, but the pharynx and the intestine were intact.
The presence of multi drug resistant environmental Vibrio strainsthat
constitute a major reservoir of diverse virulence genes are to be dealt with caution
as they play a decisive role in pathogenicity and horizontal gene transfer in the
marine environments. |
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