OCCURRENCE AND MOLECULAR CHARACTERISATION OF CAMPYLOBACTER SPP. IN SEAFOODS AND ASSOCIATED ENVIRONMENT OF KERALA AND CONTROL OF BIOFILM FORMATION
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Date
2021-07-29
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
Distribution of Campylobacter spp. in seafoods of fish catchment areas
(Vizhinjam, Neendakara, Munambam, Chavakkad and Puthiyappa) and it’s related
environment in Kerala and the presence of the organism in humans in central Kerala,
were determined over a period of two years. A total of 2179 samples from fish
catchment areas from the coastal districts, fresh-water bodies and it’s related areas
and human stool samples were subjected to conventional culture technique and
molecular characterization. Molecular characterization was performed by mPCR,
targeting the presence of genus-specific 16S rRNA, C. jejuni specific mapA, C. coli
specific ceuE genes and the virulence gene cadF. Of the overall 250 positive samples,
44.4 per cent expressed cadF, a virulence gene. Among the seafoods, 12.24 per cent
of 245 fresh marine fish, 11.76 per cent of 170 crustaceans, 13.51 per cent of 185
molluscs from the five fish catchment areas were positive for Campylobacter spp.
Presence of the organism in freshwater samples was observed in 21.4 per cent of 70
fishes, 12.1 per cent of 66 crustaceans, and was absent in molluscs. In freshwater
bodies, the occurrence ranged between 6.67- 54.76 per cent, the highest being from
rivers/streams, while 6.67 per cent of seawater from the coastal area revealed it’s
presence. Prevalence in humans was found to be 12.38 per cent. The organism could
be detected from the fish baskets, landing floor, water from boat tank and handwash
of the handlers from many areas. On correlating the presence of the organism with
the physicochemical parameters of water, the organism was more likely to be present
in alkaline waters of lakes, while it’s presence was negatively correlated with
turbidity of rivers/streams and hardness of pondwater. Phenotypic antibiotic
resistance of 62 campylobacter isolates from multiple sources against 36 antibiotics
revealed a higher resistance of the 39 C. jejuni isolates against cefotaxime (94.87 per
cent), aztreonam (87.18 per cent), ampicillin (84.62 per cent), cefuroxime (74.36 per
cent), while sensitivity towards doripenum, gatifloxacin and amikacin was observed
for 92.31, 87.18 and 76.92 per cent of the isolates, respectively. Genotypic resistance
revealed that only 11.29 per cent, 38.71 per cent and 14.52 per cent of the isolates
that were phenotypically resistant to the corresponding antibiotics, showed the
presence of aph-3-1, blaOXA-61 and tet(O), cmeB genes, respectively. The
multidrug resistance of the 39 C. jejuni isolates was 97.44 per cent, whereas that of
23 C. coli isolates was 86 per cent. Multiple antimicrobial resistance (MAR) index of
the isolates was within the range of 0.06-0.89. Survivability studies of Campylobacter
spp. (C. jejuni NCTC 11168 and C. coli isolate) was conducted in sea-water and
river-water samples, at two incubation temperatures, in autoclaved and nonautoclaved
water. Statistical analysis revealed considerable differences in the survivability of the
organism at different storage periods and temperature. Maximum period of survival
of C. jejuni NCTC 11168 at 25°C was 14 days in nonautoclaved sea-water and for C.
coli, in autoclaved sea-water was upto 60 days. In river-water at 4°C it was 60 days
under autoclaved conditions and at 25°C, it was 90 days in nonautoclaved water. The
corresponding values for C. coli at 4°C was 120 days for both autoclaved and
nonautoclaved water, and at 25°C it was 150 days for autoclaved water. Survival
characteristics of the organism points to the degree of adaptability of the organism
under varied environmental conditions. Biofilm formation of the two strains on
abiotic surfaces like stainless steel, fibre and aluminium were studied, in which the
maximum biofilm formation was seen in fibre. Sanitisers were effective against the
C. jejuni/C. coli at various combination treatments. Hot water (75°C for 5 min/65°C
for 3 min), bleaching powder (200 ppm), chitosan (1.5 per cent for 3 min) as well as
aqueous extract (1.00 per cent for 5 min/ 2.00 per cent for 5 min) of Eichhornia
crassipes (water hyacinth) on biofilms of C. jejuni NCTC 11168 and C. coli on the
various surfaces were effective in eliminating the organism. Field Emission Scanning
Electron Microscopical structure of the biofilms on the various surfaces before and
after treatment were also observed. This study unravelled the overall prevalence of
Campylobacter spp. in the state especially in the fish catchment areas and associated
water bodies, which in turn would be helpful in improving the awareness on food
safety among fisher-folk, consumers and the state regulatory authorities.
Description
Submitted in partial fulfilment of the requirement for the degree of
Doctor of Philosophy in Veterinary Public Health