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ThesisItem Open Access Detection of Yersinia spp. in ready-to-eat foods of animal origin.(CSK HPKV, Palampur, 2019) Koundal, Shubham; Panda, A.K.The present study was designed with the aim to determine the incidence of Yersinia spp. in ready-to-eat foods of animal origin. A total of 220 samples; RTE milk products (n=161), RTE meat products (n=21), beverages (n=18) and raw meat samples (n=20) were collected from 13 different places of Himachal Pradesh. In addition, 25 stool samples from hospitalized patients were also screened. Yersinia spp. was not detected in any of the samples tested by culture method using CIN agar. However, 48 other bacterial isolates i.e. Serratia spp. (6.5%, 16/48), Pseudomonas spp. (6.5%, 16/48), Enterobacter spp. (5.7%, 14/48) and Klebsiella spp. (0.8%, 2/48) were recovered from the tested samples. Highest number of isolates were detected in beverages (33%) followed by dahi based products (30%), milk based sweets (21.5%) and paneer based products (13.6%). None of the RTE meat products showed bacterial contamination. All isolates were susceptible to amikacin, tobramycin and doripenem. High level of susceptibility was observed for levofloxacin (95.8%), gentamicin (95.8%), imipenem (93.7%), ciprofloxacin (93.7%), cefepime (89.5%) and ampicillin/sulbactum (81.2%). 5 (10.4%) isolates were found to be multidrug resistant. Three isolates were resistant to imipenem while two isolates were in intermediate susceptibility range for doripenem. 47.9% isolates tested were positive for ESBL and 52.0% isolates as ESBL negative. Only one isolate from raw pork was positive for blaCTX-M-1 gene by molecular testing. In the present study no Yersinia spp. could be detected. However, other bacterial spp. belonging to Enterobacteriaceae were detected and some of them were multidrug resistant strains. Isolates resistant to carbapenem class of antibiotics were also detected which possess serious public health concern.ThesisItem Open Access ISOLATION, IDENTIFICATION AND CULTURAL SENSITIVITY OF Escherichia coli FROM MILK AND MILK PRODUCTS.(CSKHPKV, Palampur, 2019-07-23) Vaishali; Khurana, S. K.In the present study, microbiological quality of milk and milk products was assessed by employing standard plate count and coliform count with special emphasis on Escherichia coli which is a food borne pathogen of public health importance. A total of 205 samples of milk and its products were screened for the presence of E. coli. Based on serotyping maximum prevalence was found in fermented milk products (22.86%), followed by cream based milk product (16.67%), paneer/ channa based product (13.64%), flavoured milk product (4.76%) and processed cheese products (2.70%). Among 17 isolates confirmed by serotyping, 15 isolates belonged to 9 different ‘O’ serogroups viz. O2, O7, O8, O26, O118, O121, O128, O145 and O157 while 2 were untypable strains. On the basis of PCR, out of 25 biochemically characterised isolates 23 were positive for E. coli. All the isolates were characterized in terms of antibiotic resistance/sensitivity. Antibiogram studies of the isolates revealed that 44% of the isolates were multidrug resistant with maximum isolates showing resistance to cefotaxime (88%), followed by cefuroxime (52%), amoxyclav and azithromycin (36%) while 96% sensitivity was observed against gentamycin. Due to ever increasing problem of antibiotic resistance, effect of both methanolic and aqueous extracts of pomegranate peels, rhododendron flowers and drumstick was evaluated against E. coli. The antimicrobial activity of aqueous and methanolic extracts of pomegranate peels and rhododendron flowers was found to be effective against E. coli at the concentration of 10 per cent and 15 per cent.ThesisItem Open Access STUDIES ON PREVALENCE, CHARACTERIZATION AND EFFECT OF ANTIMICROBIAL AGENTS ON Escherichia coli ISOLATES FROM CHICKEN(CSKHPKV, Palampur, 2013-01-14) Choudhary, Shivani; Khurana, S. K.In the present study, microbiological quality of chicken meat, eggs and their products was assessed by employing standards plate count and coliform count with special emphasis on Escherichia coli which is a food borne pathogen of public health importance. A total of 250 samples of raw and ready to eat chicken meat and eggs were screened for the presence of E. coli. Based on serotyping maximum prevalence was found in raw chicken (13.33%), followed by shell eggs (8 %), egg products (6.67%) and chicken products (4.76%). Among the twenty two isolates confirmed by serotyping, 12 isolates belonged to 10 different ‘O’ serogroups viz. O2, O8, O11, O13, O17, O21, O23, O35, O66 and O155 while 5 were rough strains and 5 were untypable strains. All the isolates were characterized in terms of antibiotic resistance/sensitivity, haemolysin production, plasmid profiles and presence of virulent genes. On the basis of PCR, ten out of 22 serotyped isolates revealed the presence of virulence genes with nine isolates showing the presence of eae and bfpA genes and one isolate having bfpA and stx1/stx2 genes. Antibiogram studies of the isolates revealed that 59.09% of the isolates were multidrug resistant with maximum isolates showing resistance to co-trimoxazole (86.36%), followed by tetracycline (68.18%), ofloxacin (40.91%) and piperacillin/tazobactam (40.91%) while cent per cent sensitivity was observed against kanamycin. Plasmid profiling revealed multiplicity and random distribution of plasmid DNA with plasmid bands ranging from 1.2 kb to >10 kb. None of the isolates displayed hemolytic activity. Due to ever increasing problem of antibiotic resistance, effect of both methanolic and aqueous extracts of pomegranate peels, orange peels, curry leaves, radish leaves, seabuckthorn leaves and ginger rhizomes was evaluated against E. coli. Only the aqueous and methanolic extracts of pomegranate peels were found effective against E. coli at the concentration of 8%, 9% and 10%.ThesisItem Open Access DETECTION OF BUFFALO SPECIES IN MEAT AND MEAT PRODUCTS EMPLOYING SEROLOGICAL AND DNA BASED TECHNIQUES(CSKHPKV, Palampur, 2017-10-16) Kashyap, Ishan; Khurana, S. K.The aim of the present study was evaluation of serological and DNA based methods for detection of buffalo meat adulteration in meat and meat products. The AGID and buffalo specific PCR assay using two primer pair based on mitochondrial d-loop and 16S rRNA was employed for detection of buffalo species successfully. The serological methods i.e. AGID, the proteins were extracted from different tissues and then utilized for immunization into rabbit to produce antibodies. Antibodies were reacted for antigen detection using agarose gel immunodiffusion. The results showed serological assay can detect presence of buffalo proteins after extraction and react optimally at a distance of about 7-15 mm. However, further more and specific studies are required for immunological based study for final conclusion for authentic detection of buffalo meat. The buffalo species PCR assay was found to be specific and authentic for detection of buffalo meat adulteration in meat and meat products processed under different processing and heating conditions without any effect in less than 1 percent level of admixing with other meat species used in this study. The PCR assay were found to be specific and repeatable each and every time and can be useful tool for quality assurance of food products containing meat as ingredients. The market survey and laboratory analysis based on the buffalo specific PCR assay revealed that no adulteration of meat and meat products with buffalo species during the study periods.ThesisItem Open Access STUDIES ON ADULTERATION OF MILK AND ITS PUBLIC HEALTH IMPORTANCE IN HIMACHAL PRADESH(CSKHPKV, Palampur, 2018) Palsra, Tanu; Khurana, S. K.The present study was conducted to assess the milk quality with reference to adulteration and its importance on consumers’ health in different areas of Himachal Pradesh. Total 200 raw milk samples were collected directly from consumers. Milk analysis was done firstly to assess the physico-chemical quality attributes and further qualitative analyses of adulterants. The specific gravity of milk samples ranges from 1.010-1.032 with an average value 1.022±0.005. The fat percentage ranges from 1.0-9.2 with an average value 3.5±0.10, SNF ranges from 3.6-12.8 with an average value 7.01±0.10 and Total Solid ranges from 4.6-19.2 with an average value 10.54±0.17. Total 74% milk samples in case of specific gravity, 69.5% samples for fat percentage, 82.5% samples for SNF, and 73.5% milk samples for TS were less than the minimum prescribed standards of FSSAI for specific gravity, fat, SNF, and TS for cow milk in Himachal Pradesh. Further zone-wise determination of physico-chemical parameter of milk samples revealed that there was no significant difference (p>0.05) in specific gravity and %SNF content between Zone I and Zone II whereas, there was significant difference (p<0.05) of Zone III with Zone I and Zone II. No significant difference was observed between Zone I, Zone II, and Zone III in case of fat% and TS. All these samples were analysed for presence of adulterants by using a standard milk adulteration kit. Tests included were alizarin test, urea test, starch test, salt test, skim milk powder test, glucose test, formalin test, sugar test (sucrose), neutralizers test, detergent test, hydrogen peroxide test, maltose test, ammonium sulphate test, boric acid test, nitrate/ pond water test. Assessment of adulteration depicted that water was the most common adulterant (74%) found in the milk samples followed by salt (18%), alizarin (13.5%), skim milk powder (9.5%), detergent (3%), sucrose (1.5%), glucose (1%), formalin (1%), and neutralizers (1%). Other tests performed were negative in all milk samples. None of the individual sample was found positive for all the synthetic ingredients (urea, detergent or soap, sodium hydroxide, vegetable oil, and salt) required for production of synthetic milk. From survey study it could be concluded that majority of the respondents had low awareness towards disease transmission through milk, government regulations against milk adulteration and proper reporting system. Majority of respondents preferred the method of assessment of adulteration was through visual appearance, taste, touch and they perceived that adulterated milk had no harmful effect on their healthThesisItem Open Access STUDY ON INCIDENCE OF BACILLUS SPP. IN READY-TO- EAT FOODS, BEVERAGES AND WATER FROM DIFFERENT TOURIST PLACES OF HIMACHAL PRADESH(CSKHPKV, Palampur, 2018) Rana, Neha; Panda, A.K.The present study was designed with the aim to determine the incidence of Bacillus species in ready-to-eat (RTE) foods, beverages and water from different tourist places of Himachal Pradesh. Bacillus were also analyzed for the presence of toxin genes and antimicrobial susceptibility. A total of 220 samples; RTE milk products (n=80), RTE meat products (n=40), beverages (n=40) and water (n=60) were tested. In addition, 50 stool samples from hospitalized patients were also screened. Bacillus isolates were characterized by cultural and biochemical methods and reconfirmed by amplifying 16S rRNA (1500 bp) through polymerase chain reaction (PCR). Bacillus isolates were characterized for the presence of enterotoxins (hbl encoding hemolytic, nhe encoding non-hemolytic and cytK encoding cytotoxic) and emetic toxin (ces) by multiplex PCR. A total of 11.4 per cent (n=25/220) samples tested were contaminated with Bacillus species. These isolates were identified as B. cereus (76%, n=19/25), B. alvei (12%, n=3/25), B. polymyxa (8%, n=2/25) and B. firmus (4%, n=1/25). RTE milk products were found to have the highest incidence of Bacillus (17.5%, n=14/80) followed by water (8.3%, n=5/60), RTE meat products (7.5%, n=3/40) and beverages (7.5%, n=3/40). None of the stool samples were found positive for Bacillus spp. B. cereus recovery was highest from cheese (25%, n=4/16) followed by khoa (14 %, n=3/21) and paneer (8.6 %, n= 2/23) based items. nhe complex was detected as the predominant (76%) enterotoxin gene, followed by cyt K (40%) and hbl gene complex (28%). ces was not present in any of the tested isolates. All the isolates were resistant to cefixime and penicillin. High level of susceptibility was observed for antimicrobial classes; aminoglycosides, quinolones and phenicols. MDR was found in 28 per cent (n=7/25) isolates of Bacillus spp. Highest number of MDR isolates were recovered from RTE milk products (12%) followed by water (8%), RTE meat products (4%) and beverages (4%).ThesisItem Open Access STUDIES ON INCIDENCE OF AEROMONAS SPP. IN READY-TO-EAT FOODS, BEVERAGES AND WATER FROM DIFFERENT TOURIST PLACES OF HIMACHAL PRADESH(CSKHPKV, Palampur, 2018) Neena Kumari; Panda, A.K.The present study was designed with the aim to determine the incidence of Aeromonas species in ready-to-eat foods, beverages and water from different tourist places of Himachal Pradesh. A total of 220 samples; RTE milk products (n=80), RTE meat/fish products (n=40), beverages (n=40) and water (n=60) were collected from 12 different tourist places. In addition, 50 stool samples from hospitalized patients were also screened. Aeromonas isolates were characterized by cultural and biochemical methods and reconfirmed by amplifying 16S rRNA (953 bp) through polymerase chain reaction (PCR). Enterotoxin genes alt (encoding heat-labile cytotonic enterotoxin), ast (encoding heat-stable cytotonic enterotoxin) and act/hlyA/aer (encoding cytotoxic/β-haemolysin/aerolysin) complex present in characterized Aeromonas isolates were detected by PCR. A total of 20 Aeromonas spp. isolates were recovered from tested samples; 16 from RTE foods, beverages, water and 4 from human stool samples. RTE milk products had highest level of contamination (n=12/80, 15%) followed by beverages (n=3/40, 7.5%) and water (n=1/60, 1.67%). These isolates were identified as A. hydrophila (n=7/20, 35%), A. sobria (n=6/20, 30%), A. schubertii (n=4/20, 20%) and A. veronii (n=3/20, 15%). All Aeromonas isolates carried alt and 25 per cent were positive for act/hlyA/aer complex. None of the isolates was carrying ast. High level of susceptibility was observed for antibiotic classes; nitrofurans, phenicols and tetracyclines followed by quinolones and aminoglycosides. However, all the isolates were resistant to ampicillin. Nine (45%) isolates were multidrug resistant. Highest number of MDR isolates were recovered from RTE milk products (25%) followed by stool samples (15%) and beverages (5%).