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Subject: Veterinary Public Health × Start date: 1981 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    EFFICACY OF CAMPYLOPHAGES AND SELECTED PHYTOCHEMICALS IN THE CONTROL OF CAMPYLOBACTER BIOFILMS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) VIVEKANANDHAN R; Dr. B. Sunil
    Campylobacteriosis is one of the most common causes of bacterial foodborne disease. Campylobacter spp. are found as commensals in the intestines of a wide variety of animals, particularly pigs utilised for food production. The present study was undertaken to analyse the efficacy of campylophages and selected phytochemicals in the control of campylobacter biofilms. A total of 130 samples including 40 samples each of faecal and rectal swabs and sewage (n=50) were collected from the two unorganised pig farms. An overall occurrence of 26.15 per cent with higher isolation from rectal swabs (57.5 per cent) than faecal and sewage samples (25 per cent and two per cent) were observed. In both the farms, no effective biosecurity measures were followed. In pork processing line, a total of 80 samples including 20 each of carcass swabs, cutting board swabs, knife swabs and sewage samples were collected. An overall occurrence of 12.5 per cent was observed. All the isolates were subjected to biofilm forming ability at three different temperatures 42℃ (10 per cent CO2 condition), 37℃ & 25℃ (aerobic condition) and compared with two media (Dulbecco’s Modified Eagle’s Medium (DMEM) & Muller Hinton Broth (MHB)). Strong biofilm formers were predominant in aerobic condition at 37℃& 25℃ incubated in MHB. Weak biofilm formers were more on stainless steel and nylon fibre. Carvacrol, trans-cinnamaldehyde, eugenol, ethanolic extract of citrus by-product and bleaching powder had minimum biofilm inhibition concentration of 0.078 mg/mL, 0.156 mg/mL, 0.156 mg/mL, 0.321 mg/mL and 0.625 mg/mL, respectively. Trans-cinnamaldehyde significantly (p<0.001) inhibited 99.39 per cent biofilm formation by the campylobacter isolates on polystyrene surface at MBIC of 0.156 mg/mL. Citrus by-product ethanolic extract significantly (p<0.001) inhibited 99.43 per cent biofilm formation by the campylobacter isolates on stainless steel surface at MBIC of 0.312 mg/mL.Carvacrol significantly (p<0.001) inhibited 97.80 per cent biofilm formation by the campylobacter isolates on nylon fibre surfaces at MBIC of 0.078 mg/mL. Carvacrol, trans-cinnamaldehyde, eugenol, ethanolic extract of citrus by-product and bleaching powder had minimum biofilm inactivation concentration of 0.312mg/mL, 2.5 mg/mL, 1.25 mg/mL, 2.5 mg/mL and 5 mg/mL, respectively. Trans cinnamaldehyde inactivated 21.49, 57.89 and 95.92 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively at MBIAC of 2.5 mg/mL on polystyrene surface. Eugenol significantly (p<0.001) inactivated 53.79, 70.87 and 98.90 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively on stainless steel surface at MBIAC of 1.25 mg/mL. Trans-cinnamaldehyde significantly (p<0.001) inactivated 87.71, 93.9 and 99.15 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively on nylon fibre surface at MBIAC of 2.5 mg/mL. Three campylophages were isolated from sewage samples of farm B. Phage 2 and 3 were characterised as Siphoviridae and phage 5 was as Myoviridae by transmission electron microscope. Phage 5 inactivated 30.3 per cent, 30.55 per cent and 29.60 per cent pre-formed biofilms of campylobacter isolates on polystyrene, stainless steel and nylon fibre surface after 24 h of exposure, respectively. Campylophages were effective in inactivating pre-formed biofilms by the isolates which were lysed by the phages. Campylophages were not effective in inactivating biofilms formed by the campylobacter isolates which did not show lytic activity. Cost analysis showed citrus by-product extract as cost effective (Rs 0.41/L) than other phytochemicals.
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    ThesisItemOpen Access
    OCCURRENCE AND MOLECULAR CHARACTERISATION OF CAMPYLOBACTER SPP. IN SEAFOODS AND ASSOCIATED ENVIRONMENT OF KERALA AND CONTROL OF BIOFILM FORMATION
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-07-29) DEEPA JOLLY; B. Sunil
    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.
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    ThesisItemOpen Access
    DETECTION OF DRUG RESISTANT AND BIOFILM FORMING FOODBORNE PATHOGENIC BACTERIA IN BROILER CHICKEN AND MITIGATION USING PLANT EXTRACTS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, 2021-07-05) MATHEW, BINSY; Latha, C.
    The study was undertaken to detect the presence of drug resistant and biofilm forming food-borne pathogens with special reference to E. coli, Salmonella spp. and Campylobacter spp. from retail chicken sold in different parts of Kerala and study the effect of aqueous leaf extracts of Couroupita guianensis Aubl. and Annona muricata on genotypically confirmed isolates of the aforesaid organisms. The districts included in the study were Kozhikode and Palakkad representing northern part of Kerala, Thrissur and Ernakulam representing central part and the southern part represented by Kollam and Pathanamthitta. The prevalence of E. coli, Salmonella spp. and Campylobacter spp. was 54.17, 14.33 and 17.17 per cent, respectively. The positive isolates were subjected to molecular confirmation of virulence genes targeting eaeA, aggR and ipaH for E. coli by standardising a multiplex PCR; invA and spvC for Salmonella spp. using a duplex PCR and mapA and ceuE for Campylobacter spp. using a multiplex PCR. The eaeA, aggR and ipaH were detected in 84.31, 4.31 and 1.54 per cent isolates, respectively. Virulence genes of Salmonella spp. were detected in 100 (invA) and 4.65 (spvC) per cent of Salmonella spp. isolates. The mapA and ceuE genes were detected in 74.76 and 27.18 per cent of Campylobacter spp. isolates, respectively. The antibiotic resistance profiles of the virulent isolates against nine antibiotics: ampicillin, cefotaxime, tetracycline, trimethoprim, streptomycin, ciprofloxacin, nalidixic acid and chloramphenicol were tested for E. coli and Salmonella spp. In addition to these antibiotics susceptibility to erythromycin was also studied against Campylobacter spp. The highest per cent of resistance was detected to nalidixic acid in E. coli and to tetracycline in Salmonella spp. and Campylobacter spp. Multiple drug resistance was seen in 49.64, 25.58, 23.94 and 24 per cent for E. coli, Salmonella spp., C. jejuni and C. coli, respectively. Polymerase chain reaction was used to detect antibiotic resistance genes in antibiotic resistant isolates. Four mismatch amplification mutation assay (MAMA) PCR was standardised for detection of point mutations in gyrA and parC genes in quinolone resistant isolates. In E. coli isolates, gyrA mutation was detected in 64.41 per cent of ciprofloxacin resistant isolates. In Salmonella spp cent per cent of the isolates showed the presence of blaTEM and catA genes. Among the Campylobacter spp. isolates, tetA gene was detected in 80.43 per cent of C. jejuni isolates and cent per cent of C. coli isolates. Biofilm forming ability was detected in all the three organisms. None of C. jejuni and C. coli showed strong biofilm production. The effect of four types of plant extracts: C. guianensis (hot and cold) and A. muricata (hot and cold) was studied on genotypically confirmed resistant isolates. The cold extract of C. guianensis was found to show maximum antibacterial activity in all the three drug resistant organisms. The detection of multidrug resistant foodborne pathogens with biofilm forming ability is a potential threat to human health. Nevertheless, mitigation using plant extracts seems to be a ray of hope in the present scenario of antimicrobial resistance in foodborne pathogens. In addition to the use of plant extracts, a multifaceted one health approach can effectively address the issue of antimicrobial resistance