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Study on molecular and virulence characterization of aeromonas species isolated from different sources

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Title Study on molecular and virulence characterization of aeromonas species isolated from different sources
 
Creator Rather, Mudasir Ali
 
Contributor Wani, Shakeel Ahmed
 
Subject Aeromonas, Prevalence, Water, Fish, Raw Meats, Human Diarrhea, RFLP, RAPD-PCR, ERIC-PCR, Multiplex PCR, Sequencing, Haemolysis, Vasopermeabilty Reaction
 
Description The present study describes the prevalence and antibiogram of Aeromonads
in foods of animal origin, water and human diarrheal samples. The molecular
epidemiology and in-vitro and in-vivo virulence characterization of the isolated
Aeromonas spp. was also studied. Of the 609 samples comprising, water (182), fish
(172), chicken (57), mutton (83), beef (31) and human diarrheic stools (83) screened
for the presence of Aeromonas spp., 155 (25.45%) were positive. The prevalence of
Aeromonas spp. in water, fish, chicken, mutton, beef and human diarrheal samples
was 26.37, 39.30, 14.03, 22.89, 19.35 and 7.23%, respectively. The isolates of
Aeromonas from all sources were identified as A. hydrophila (23.87%), A. caviae
(20.64%), A. veronii bv sobria (18.06%), A. salmonicida (8.39%), A. popoffii
(6.45%), A. trota (5.16%), A. schubertii (3.87%), A. jandaei (2.58%) and A.
allosaccharophila (2.58%). Thirteen isolates (8.39%) could be identified to genus
level only. A polymerase chain reaction (PCR) used to confirm the Aeromonas genus
detected 153 of 155 isolates identified as Aeromonas phenotypically. The speciation
was studied by restriction fragment length polymorphism of PCR amplified segment of 16S rRNA gene and the expected fragments were generated. Analysis of
restriction enzyme digestion pattern indicated that the fragments generated could be
used to identify the species of Aeromonas barring few exceptions. Molecular
characterization of virulence factors encompassed a multiplex PCR for detection of
three enterotoxin genes (act, alt and ast) of Aeromonas isolates. The act, alt and ast
genes were detected in 104 (67.09%), 98 (63.22%) and 11 (7.06%) of isolated
Aeromonads, respectively. The gene patterns identified included; act/alt (47.09%),
act (13.54%), alt (10.96%), act/alt/ast (5.16%) and act/ast (1.93%). All the isolates
were haemolytic on rabbit blood agar plates while haemolytic activity was shown by
92.25% of the isolates on sheep blood agar. The in-vivo virulence characterization of
isolates was studied by vascular permeability reaction in rabbit skin which were
marked after 12 hr of intradermal injection and ranged between 7 and 19.6 mm in
diameter. Strains having enterotoxin genes produced higher VPR zones compared
with those not carrying the gene. The molecular epidemiology of isolates was
attempted at two different levels by random amplification of polymorphic DNA
(RAPD) and enterobacterial repetitive intergenic consensus sequence PCR (ERICPCR).
The RAPD-PCR did not yield enough amplification products; therefore, the
genetic variability could not be studied by RAPD-PCR. The ERIC-PCR was
however able to type all the isolates and most of the isolates from water were
identical to one other and also to the isolates from fish. The two A. caviae isolates
from human diarrheal samples were identical to A. caviae recovered from water,
indicating water as the most important source of Aeromonas infection. A high degree
of resistance was observed against ampicillin (97.42%), ampicillin/cloxacillin
(89.03%), polymyxin B (87.74%), amoxycillin (72.90%), roxithromycin (70.32%),
erythromycin (61.29%) and streptomycin (54.84%). The isolates were highly
sensitive to enrofoxacin (96.77%), ciprofloxacin (94.19%), ofloxacin (92.9%),
ceftraixone (90.32%), norfloxacin (85.81%), tetracycline (85.81%), gentamicin
(84.52%) and doxycycline (83.23%). The sequence analysis of enterotoxin genes
revealed a high similarity of the genes with sequences available in the GenBank.
 
Date 2016-08-22T13:52:35Z
2016-08-22T13:52:35Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/73294
 
Language en
 
Format application/pdf
 
Publisher SKUAST