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ThesisItem Open Access ASSESSMENT OF ANTIMICROBIAL RESISTANCE IN Escherichia coli AND SALMONELLA ISOLATED FROM INTEGRATED BROILER CHICKEN FARMS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) i ASSESSMENT OF ANTIMICROBIAL RESISTANCE IN Escherichia coli AND SALMONELLA ISOLATED FROM INTEGRATED BROILER CHICKEN FARMS ADDAKAL ANANDRAJ HANNAH JOANNAS; SHARADA. R)A study was conducted to identify the driving factor of AMR by monitoring the phenotypic and genotypic AMR of E. coli and Salmonella isolated from 3 broiler farms (2 integrated and 1 associated). In all, 101 samples were collected at different sampling points in one entire crop cycle. The overall presence of E. coli and Salmonella was 76.23 and 9.09 per cent. Farm 1 samples did not yield Salmonella. Antimicrobial resistance based on disc diffusion assay and MIC revealed that all E. coli and Salmonella isolates were resistant to at least one antibiotic. E. coli and Salmonella showed highest resistance to ampicillin and doxycycline (92 and 87%), followed by enrofloxacin, gentamicin, trimethoprim sulphamethoxazole, amikacin and colistin and lesser resistance to ceftiofur. All the isolates were further screened for the presence of 22 AMR genes belonging to three groups of antibiotics by PCR. Among the AMR genes, highest presence was recorded for qnrB(96.87%), qepA(92.7%) followed by blaTEM(71.96%), blaCTX-M universal(29.56%), qnrS (28.93%), qnrD (28.62%), blaCTX-Mgroup1 (10.8%), mcr3 (6.27%), blaCTX-M group2 (5.64%), oqxAB (5.5%), blaSHV (4%), blaOXA (2.5%), blaCTX-M group 8/25 (1.85%), aac(6’)-Ib-cr (1.5%) and qnrA (0.5%) in E. coli. In Salmonella, blaTEM and blaSHV (100%) followed by qnrD (68.3%), qnrB (61.6%), qnrA (15%) and oqxAB (5%) in Salmonella. Maximum AMR genes in E. coli and Salmonella were found in boot socks, ileal and cecal contents and fecal swabs. Thus, these may be the driving factors for AMR. Also, on day zero high prevalance of AMR genes was found. A significant correlation was observed between phenotypic and genotypic AMR of β Lactams and quinolones but not for colistin. No significant difference was observed in the overall AMR pattern between integrated and associated farms. The findings calls for the need of efficient monitoring and institution of control strategies for effective mitigation of AMR in all the three broiler farms.ThesisItem Open Access EFFECT OF NATAMYCIN ON THE MICROBIOLOGICAL QUALITY OF PANEER(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR-585 401, 2022) K. SREERAMYA; RAMACHANDRA, B.The present study was aimed in increasing the shelf life of paneer by the antifungal agent natamycin. The counts in fresh paneer ranged from 1.00 to 4.20 and in stored paneer the count was from 1.00 to 2.14 log10cfu/g. All the yeast isolates (5 no.) were identified to Rhodotorula sp. and molds isolates were identified as Aspergillus (1 no.), Mucor (1 no.), Penicillium (1 no.) and Rhizopus (2 no.) at 50 ppm natamycin. Rhodotorula sp. produced inhibitory area of 668.03 sq.mm whereas, the molds Aspergillus, Mucor, Rhizopus and Penicillium produced 138.16, 452.16, 668.03 and 452.16 sq.mm. The yeast and molds counts at 20, 30, 40 and 50 ppm incorporated paneer were 4.22, 4.00, 4.10, 3.95 and 3.70 log10cfu/g. Among the dipping, swabbing and spiking the dipping method was found effective in complete inhibition of yeast and molds at 1500 ppm natamycin in both the fresh and stored paneer. The scores given by the judges found that no significant changes in the sensory parameters of the control and natamycin treated samples of both fresh and stored paneer. Then the optimized natamycin treated paneer samples at 1500 ppm by dipping were subjected for sensory, chemical and microbiological analysis during storage at refrigeration temperature (5 ℃). Based on the analysis of moisture, water activity, acidity, FFA, TBC, coliforms, proteolytic, lipolytic and yeast and molds counts the shelf life of fresh and stored paneer were declared. Based on the appearance of yeast and molds the shelf life of fresh and stored natamycin treated paneer at refrigeration temperature were 20 days and 30 days respectively.ThesisItem Open Access CHARACTERIZATION OF DAIRY ORIGIN PROBIOTIC BACILLUS SPECIES(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR-585 401, 2022) K. SIREESHA; PRABHA. RIn the present study, dung sample obtained from KVAFSU dairy farm showed high bacterial, aerobic spore, acid tolerant spore counts of 8.42, 5.42 and 3.4 log10cfu/g, respectively. Fodder of the dairy farm had high bile tolerant spore count of 4.71 log10 cfu/g while Soil, feed, fodder, dung, silage, udder swab, hand swab, pail rinse, can rinse, water, aseptic milk, pail milk, can milk, pasteurized milk and air samples revealed lower counts of aerobic spore, acid and bile tolerant sores. According to specific tests, among 19 Bacillus isolates, 16 isolates were identified as Bacillus licheniformis and one each as B. coagulans, B. subtilis and B. tropicus. Among 19 isolates, B7 and B12 exhibited higher per cents of survivability with respect to acid accounting for 96.20, 99.52 and bile of 99.32 and 98.57, respectively. The hydrophobic nature in xylene of B7 and B12 were 37.7 and 38.7%, respectively. B7 and B12 were genotypically identified as B. subtilis and B. tropicus. B7 and B12 Bacillus strains gave promising growth in urad dhal medium compared to cheese whey. B7 and B12 gave promising growth on urad dhal B7 and B12 were inoculated into sterile urad dhal medium (1 part dhal + 0.7part water + 0.2part tomato juice + 0.1part skim milk powder), fermented at 37°C for 48 h, dried at 35 °C for 20 h, powdered and packed in pouches and kept for shelf-life study at 25°C and 5°C temperatures. At the end of 49th day of storage at 25°C, the count of B7 and B12 were 6.16 and 6.24 log 10 cfu/g from initial counts of 9.73 and 9.82 log 10 cfu/g on 0 day. B7 and B12 at 75th day of storage at 5°C maintained viable count 6.53 log10cfu/g with initial counts of 9.73 and 9.82 log 10 cfu/g, indicating direct use of powder to increase probiotics in gut of human beings.ThesisItem Open Access STUDY ON THE EFFECT OF ANTIMICROBIAL RESISTANT PATHOGENS OF BOVINE MASTITIS AND LACTIC ACID BACTERIA IN DAHI(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR-585 401, 2022) GANESH; PRABHA. RIn the present study 45 isolates of bovine mastitis were obtained from Veterinary Microbiology Department, KVAFSU, Bengaluru. The purified 40 Gram-positive isolates and 5 of Gram negative, were identified as Streptococcus uberis (10), Streptococcus agalactiae (12); one each of Staphylococcus delphini, Staphylococcus carnosus and Staphylococcus gallanarium while 6 each of Staphylococcus equorum & Staphylococcus epidermis and 3 Staphylococcus aureus. Gram negative 5 isolates were identified as E. coli (3), Salmonella enterica (1) and Alcaligenes sp. (1). All the isolates when subjected to antibiotic susceptibility test (18 antibiotics), Streptococcus agalactiae M36, Staphylococcus aureus M35, E. coli M33, Salmonella enterica M27 showed highest resistance for 16, 15, 7, 5 antibiotics, respectively. The highest antibiotic resistant isolates were PCR analysed for their speciation. Lactococcus lactic ssp. lactis 1, Lactococcus lactic ssp. lactis 2, Lactococcus lactic ssp. cremoris were used in dahi preparation alone or in combination along with selected mastitic causing bacteria having public health significance and highly resistant to antibiotics. The prepared dahi had viable count of around 8.6 both in individual and in mixed lactic cultures with lactic acid content of 0.77%. Lactic cultures exhibited resistance to ampicillin, azithromycin, cefoperazone, ceftriaxone, nalidixic acid, penicillin, rifampicin and vancomycin. Mastitic bacterial isolates, showed resistant to all the antibiotics, while the Streptococcus agalactiae and E. coli showed susceptibility to streptomycin. In dahi prepared using mixed lactic culture, there may be possibility of transfer of resistant factor of ceftriaxone to Salmonella enterica ssp. enterica M27 and both ceftriaxone and ofloxacin resistance to Staphylococcus aureus M35 from lactic cultures which were resistant in dahi while pathogens in broth were susceptible.ThesisItem Open Access PRODUCTION OF ENCAPSULATED PROBIOTIC BACTERIAL CONCENTRATES AND THEIR PERFORMANCE IN YOGHURT(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR-585 40, 2022) BAINDLA BHARGAVI; RAMACHANDRA, B.The probiotic cultures, Lactobacillus acidophilus and Bifidobacterium bifidum cells were concentrated by centrifugation process obtained viable count of 10.26 and 10.14. In the present study, 3 % sodium alginate and 0.3 M calcium chloride had considered optimum because of its soft textured beads and the diameter of Lactobacillus acidophilus and Bifidobacterium bifidum were 971 and 941 μm. The 2 % of Lactobacillus acidophilus and Bifidobacterium bifidum in 3 % sodium alginate got higher encapsulation efficiency of 97.1 and 96.4 %. The encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum subjected to acid tolerance test at pH 1.5 and pH 3.0 for 2 h and bile tolerance (0.3 % ox bile) for 6 h resulted in higher survival rate (%) of 72.8, 75.0, 99.2 and 74.8, 79.4, 99.3 than free cells 32.2, 51.1, 83.6 and 39.8, 48.8, 89.6. The yoghurt prepared with combination of 3 per cent of encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum had highest overall acceptability of 8.23. Addition of encapsulated probiotic bacteria reduced acid development in yoghurt compared to free cells and control during storage. The results showed that there was an increase in survival by 1 log cell numbers of encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum in yoghurt during storage. This study revealed that incorporation encapsulated probiotic bacteria do not substantially alter the overall sensory characteristics of yoghurt and microencapsulation helps to retain the viable count of probiotic bacteria (106 log cfu/g) in yoghurt after 30 days of storage at refrigeration temperature (5 ℃).ThesisItem Open Access EVALUATION OF INCORPORATED SOLID STATE FERMENTED (SSF) CULTURES FOR PANEER WHEY BASED IDLI BATTER(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2021-07-01) VISWANATHA ANGADI; PRABHA, R.The present study was undertaken to improve the nutritional quality of idli by incorporation of paneer whey as well isolated lactic cultures through fermented solid substrate, black gram dhal (SSFC). Soaking time of black gram dhal, rice rava were optimised to 4 h and 1 h, respectively in paneer whey at 70:30 (whey: water). The whey-based batter was fermented at 30℃ for 12 h that showed predomination of lactobacilli that ranged from 5.42 to 8.74 log10 cfu/g on MRS agar medium. Among eighteen lactic isolates, four showed better activities were identified as Leuconostoc sp. strain Leu2, Enterococcus sp. strain E1, Lactobacillus brevis strain Lb1 and Lactobacillus casei strain Lb2 based on 16S rRNA sequence. These four cultures were grown on sterile black gram dhal having skim milk powder and tomato juice and inoculated in the dried form as SSFC at 5% rate during batter preparation. As a result, the fermentation time at 30℃ was reduced (by 25%) to 9 h, compared to control (12 h). The idlis made from 5% SSFC with 70 % whey based batter were most accepted with highest overall acceptability score (8.53) compared to control (7.43). SSFC incorporated whey based idli batter showed 0.36% higher total protein, 13.3% lower phytate content and 28% increase in calcium content in comparison with control. Sensory evaluation of idlis from the optimised 5% SSFC batter stored in LDPE pouches at 4℃ for 4days were accepted and considered safe with no coliforms. The SSF cultures showed the viability of 7.04 log10 cfu/g when stored at 4℃ for 4 months.ThesisItem Open Access FORMULATION AND VALIDATION OF SELECTIVE MEDIA FOR THE ENUMERATION OF LACTIC ACID BACTERIA IN FERMENTED MILK PRODUCTS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2021-07-01) SABHAVAT GOPAL; PRABHA R.Market samples of curd, yoghurt probiotic drink and domestic curd samples were enumerated using formulated and readymade media like M17 for lactococci as well as Streptococcus thermophilus and MRS media for lactobacilli incubated in candle jar at 30ºC and 37ºC respectively for 48 h the readymade media gave better recovery of viable cells of lactic acid bacteria. Reduction in lactic counts of fermented milk products was noticed in both M17 and MRS agar after incorporation of calcium propionate and sodium benzoate at 1.5 and 2 per cent each, respectively helped in control of both aerobic spore formers and yeasts. Yeast glucose agar with Bromocresol Purple (BCP) with 0.05 per cent was used which showed better viable counts of lactic acid bacteria than Elliker agar with BCP. In order to reduce aerobic spore forming bacteria and yeasts, calcium propionate of 0.8 per cent and sodium benzoate of 1.2 per cent incorporation to yeast glucose agar with 0.05 per cent BCP led to formation of more than 8 log counts of both yellow and purple colonies. Yellow and purple colonies when subjected for colony morphology demonstrated rods (lactobacilli) and cocci (lactococci), respectively indicating the lactic acid bacteria present in fermented milk products, when inoculated to sterile skim milk set the curd in 8 h with 0.65per cent lactic acid on an average. The correlation coefficient, r was 0.78 between selective M17 of lactococci and selective yeast glucose agar for purple colony of lactic cocci while r was 0.67 when MRS lactobacilli count was compared with yellow colonies of lactobacilli on selective yeast glucose agar indicating good relationship between both the types of selective agar media.ThesisItem Open Access OPTIMIZATION OF BULK CULTURE PROPAGATION FOR THE PRODCTION OF MISTI DAHI(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2021-07-01) PALEPU PAVAN KUMAR; RAMACHANDRA, B.The misti dahi prepared was studied for the effect of sugar, total solids, inoculum levels and caramel flavours on the performance of cultures of Lactococcus lactis ssp. cremoris, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus alone and in combination. Sugar at 12.0 per cent got highest overall acceptability score of 8.58, 8.00 and 8.75 using Lactococcus lactis ssp. cremoris, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus alone and in combination at 1.0 and 2.0 per cent respectively. The misti dahi made with total solids of 31.0 per cent got highest over acceptability score of 8.08, 8.58 and 8.25 using Lactococcus lactis ssp. cremoris, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus alone and in combination at 1.0 and 2.0 per cent respectively. The misti dahi made with 12.0 and 31.0 per cent sugar and total solids at different levels of inoculum 0.5, 1.0, 1.5 and 2.0 per cent of Lactococcus lactis ssp. cremoris scored highest of 8.00 at 1.0 per cent inoculated culture compared to the other inoculum levels, while misti dahi prepared form 2.0 per cent yoghurt culture got overall highest score of 8.16 when compared to the other levels. In case of misti dahi produced from the mixed cultures of Lactococcus lactis ssp. cremoris and yoghurt culture, misti dahi made with yoghurt culture alone scored highest of 8.00. The misti dahi prepared using natural and artificial flavours, the overall acceptability scores of 8.25 misti dahi made using natural was best. The storage stability of misti dahi was analysed at room and refrigeration temperatures. The misti dahi losts its quality in a single day and 15th days at room temperature (27±1 °C) and refrigeration temperature (5 °C) respectively.ThesisItem Open Access PHYLOGENETIC STUDY OF LISTERIA SPECIES ISOLATED FROM DAIRY ENVIRONMENTAL SAMPLES(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR – 585 401, 2021-07-01) P. SUSHMITHA; PRABHA R.In the present study, Listeria count in dairy environmental samples collected from university dairy farm ranged from 0.10 to 4.56 log10cfu/g, out of which soil, dung and udder swab of milch animal showed more Listeria. The pheno and genotypic identity of Listeria revealed as Listeria ivanovii (6 nos; 50.0%), followed by Listeria grayi (5 nos; 41.6 %) and Listeria seeligeri (1 no; 8.40%). Phylogenetic analysis through Neighbour joining method revealed that L. ivanovii L18, L8, L16 and L. grayi L9, L10, L12 were having relatedness about 87% while L. ivanovii L15, L7, L17, L. grayi L11, L13 and Listeria seeligeri L14 had about 92% homology. Non-pathogenic Listeria grayi L13 and pathogenic Listeria seeligeri L14 showed highest DMC of 8.69 and 8.20 log10/ml among 12 isolates and used in further studies. Initial 6 log viable count of Listeria grayi L13 and Listeria seeligeri L14 in sterile skim milk reached to 8.00 and 8.58 log10cfu/ml with 0.64 and 0.62 %LA; protein of 1.00 and 1.62 respectively whereas L14 produced haemolysin of 9mm diameter at 24h of incubation at 37ºC. The viable cells of L13 and L14 were killed when the contaminated milk was subjected to pasteurization, boiling and sterilization. L13 and L14 when inoculated at 6 log counts into sterile whole milk and stored at 5°C, the raise in viable log count was 8.50 and 8.35 whereas titratable acidity reached to 0.19% and oleic acid to 1.28 and 1.24% while protein content declined to 1.00 and 1.36% respectively with storage period of 9 days.
