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20223
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Subject: Veterinary Microbiology × End date: 2022 × Start date: 2022 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    ASSESSMENT OF ANTIBIOGRAM AND DETECTION OF GENETIC DETERMINANTS OF SELECTED VIRULENCE FACTORS IN AVIAN PATHOGENIC ESCHERICHIA COLI ISOLATES OF CHICKEN
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-05) LOKESWARI, R; SIVARAMA KRISHNA, G(MAJOR); ANAND KUMAR, P; GANGU NAIDU, S
    The present study was taken up for understanding the pathogenic potential and prevalence of antibiotic resistance pattern of avian E. coli strains, isolated from colibacillosis affected chickens by both traditional and molecular methods. A total of 105 samples were collected from the internal organs of colibacillosis affected chickens. Based on morphological, cultural and biochemical characterization viz., Gram staining, growth pattern on EMB agar, lactose fermentation on MacConkey agar, IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate), Catalase, Nitrate, Oxidase and Urease tests, E. coli was isolated and characterized from 81 samples (77.14%) out of 105 samples collected. The gene coding for 16S rRNA is highly conserved among species/strains of Escherichia genus was detected in all the 81 E. coli isolates by PCR. In congo red dye uptake assay, all the isolates produced orange red to red color colonies, indicating their invasiveness and pathogenic potential. Out of 81 isolates, the production of hemolysin was phenotypically characterized in 74 (91.4%) isolates, of which 39 (48.2%) isolates are ɑ-hemolytic and 35 (43.2%) are Entero-hemolytic on 10 per cent sheep blood agar. The biofilm production was assessed by qualitative tests like Christensen borosilicate glass tube method and polypropylene tube method, and quantitatively by tissue culture plate method (TCP). Among the qualitative methods the polypropylene tube method (89.47% Sensitivity and 91.66% Specificity in comparison to TCP method) was found to be superior to the borosilicate tube method (77.1% sensitivity, 83.3% specificity in comparison to TCP method). In TCP method, 61 isolates (75.3%) were quantitatively characterized as biofilm producers, which is the most reliable method. In Embryo lethality test, based on the % embryo death rate, 52 isolates were characterized as highly pathogenic strains, 24 isolates were characterized as moderately pathogenic and five isolates were characterized as low pathogenic strains. A total of eight genes were targeted for the identification of genetic determinants of virulence factors for all the 81 avian pathogenic E. coli isolates. The hlyF gene was detected in four isolates viz., F6, H1, H2 and H3, iroN gene was detected in five isolates viz., B3, B4, B5, D1 and D3, whereas eaeA gene was detected only in H3 isolate. The genes iss, ompT, stx1, stx2 and hlyA were detected in none of the isolates tested. All the isolates were found to be multidrug resistant with a Multiple antibiotic resistance index (MAR) of more than 0.2 as assessed by disc diffusion method. The occurrence of genes coding for extended spectrum β-lactamase (ESBL) were assessed in multiplex PCR and a prevalence of 86.27 per cent of blaTEM, 3.7 per cent of blaOXA and 0 per cent of blaSHV was reported in the present study. In conclusion, our study might be the first to assess the pathogenic potential of E. coli from colibacillosis affected chickens by both traditional and molecular methods in Andhra Pradesh. The present data on characterization of pathogenic E. coli strains and existence of multidrug resistant strains of E. coli in colibacillosis affected chickens may be useful for developing novel control strategies and vaccines.
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    ThesisItemOpen Access
    ASSESSMENT OF ANTIBIOTIC RESISTANCE PATTERN AND BIOFILM FORMATION IN Pseudomonas aeruginosa ISOLATES FROM BUFFALOES AND DOGS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) RAMA NARAYANA, P; DEEPIKA KUMARI, G (MAJOR); ANAND KUMAR, P; BINDU KIRANMAYI, Ch
    The current research is conducted to assess the antibiotic resistance pattern and biofilm formation ability in Pseudomonas aeruginosa isolates from the clinical samples of buffaloes and dogs. Samples were inoculated in Brain Heart Infusion (BHI) broth and cultured on Pseudomonas isolation agar. Based on pigment production and biochemical tests (catalase, oxidase, IMViC), 52 samples (34.8%) out of 149 collected in the present study were provisionally confirmed for Pseudomonas species. The isolates were further confirmed by Polymerase Chain Reaction (PCR) assay with Pseudomonas aeruginosa species-specific primers (16S rRNA) and all the 52 isolates yielded specific PCR product of 956 bp. One of the main objectives of the present study, antibiotic resistance pattern was determined by subjecting the PCR confirmed 52 P. aeruginosa isolates to 12 different antibiotic discs in vitro by Kirby Bauer method. The results of the test revealed that co-trimoxazole was highly resistant (98.07%), followed by ampicillin (96.15%), clindamycin (92.30%), oxytetracycline (88.46%) and amoxycillin (84.61%). Streptomycin (75%), ceftriaxone (50%) and norfloxacin (36.53%) were moderately resistant. Ciprofloxacin (5.76%), enrofloxacin (9.61%), amikacin (11.53%) and gentamicin (21.15%) were least resistant than others. The Multiple Antibiotic resistance (MAR) indices of P. aeruginosa isolates ranged from 0.33 to 0.91 in the current study, 51 isolates were confirmed as Multi Drug Resistance (MDR) and 1 isolate was found to be Extensively Drug Resistance (XDR). Phenotypic screening for extended spectrum β-lactamases production was carried by employing phenotypic screening test (PST). Isolates were tested with four cephalosporin antibiotics (cefotaxime, ceftazidime, ceftriaxone, and aztreonam). Forty seven out of the 52 PCR confirmed Pseudomonas aeruginosa isolates were found to be resistant against one or more cephalosporin discs, 44 isolates were resistant against cefotaxime, 26 were resistant against ceftriaxone, 9 were resistant to ceftazidime and 5 against aztreonam. Hence they were considered as ‘suspect ESBL producers’ as defined by the CLSI (2018) guidelines. Thereafter, the suspect ESBL producers were confirmed for Extended Spectrum β Lactamases (ESBL) presence by phenotypic confirmatory tests (PCTs). ESBL production was confirmed in 42 isolates by combination disc method (CDM) and 37 isolates exhibited double disc synergy in double disk synergy test (DDST). Further, all the 52 isolates were tested for genetic determinants of antibiotic resistance with oligonucleotide primers specific for extended spectrum genes (blaOXA, blaSHV, blaTEM). A PCR product of 564 bp, 713 bp and 800 bp size was yielded for blaSHV, blaOXA and blaTEM genes, respectively. Out of 52 isolates of Pseudomonas aeruginosa, 10 isolates were positive only for blaOXA, 9 isolates were positive only for blaSHV, 3 isolates were positive only for blaTEM. Both blaSHV and bla OXA were observed in 5 isolates (9.61 %), both blaSHV and blaTEM were observed in 3 isolates (5.76 %), whereas blaOXA and blaTEM together were observed in 6 isolates (11.50 %). All the three genes blaSHV, blaOXA, blaTEM were observed in 3 isolates (5.67 %). No ESBL genes were observed in 13 isolates. The biofilm formation ability was assessed by processing the P. aeruginosa isolates through congo red agar method (CRA) and tube method. CRA method detected that 7 isolates (13.46%) were weak, 26 (50%) as moderate and 19 (36.53%) isolates to be strong biofilm producers, whereas, in tube method 11 (21.15%) isolates were detected weak, 18 (34.61%) moderate, 23 (44.23%) as strong biofilm producers. In conclusion, the current study investigated the presence of genetic determinants of multidrug resistant P. aeruginosa isolates from clinical samples of buffaloes and dogs in Andhra Pradesh. There is a great necessity to pursue further research on multi-drug and extended drug resistance in Pseudomonas aeruginosa isolates from dairy and companion animals
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    ThesisItemOpen Access
    STUDY ON ANTIMICROBIAL EFFICACY OF Lactobacillus SPECIES FROM CHICKEN AGAINST Clostridium perfringens AND E. coli
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) CHANDANA SIVA POOJA, M; SRIVANI, M (MAJOR); RAMANI PUSHPA, R.N.; ASWANI KUMAR, K
    A study was carried out on the isolation, molecular characterization and probiotic potency which includes antibacterial activity of Lactobacillus spp. isolated from healthy chicken against Clostridium perfringens and E. coli. A total of 73 samples from desi and commercial chicken were collected of which 50 were cloacal swabs and 23 were tissues, from which 56 (76.71%) isolates were obtained. Tissues (87.5%) have an increased incidence of Lactobacillus spp. than cloacal swabs (70%). The percentage of autoaggregation of 56 isolates increased significantly over time from 5.03 % - 77.33 % at 4 hours to 10.37 % - 82.29 % after 24 hours of incubation. The cell surface hydrophobicity of the 56 isolates ranged from 6.3 % to 90.68 % for xylene and 18.13 % to 95.84 % for n-hexadecane. Isolates exhibited high percentage of hydrophobicity to n-hexadecane when compared to xylene. Based on autoaggregation and hydrophobicity results 22 isolates were chosen. All the 22 isolates demonstrated viable colonies at all ox-bile concentrations (0.1 %, 0.3 % and 0.5 %) after 2 and 4 hours of incubation, however after 6 hours of incubation, viability varied. In the acid tolerance assay, majority of the isolates showed viability at pH 3 and all the isolates demonstrated viability at pH 6.5. Based on bile and acid viability results 16 isolates were selected for further tests. All these 16 isolates inhibited Clostridium perfringens with zone of inhibition ranging from 11mm-22mm and 10mm-20mm anaerobically and aerobically, respectively. Whereas, the zone of inhibition of E. coli ranged from 10mm-18mm in agar well diffusion assay. In the antibiofilm assay, all 16 isolates developed biofilm and reduced E. coli biofilm formation. The antibiotic resistance pattern of 16 isolates revealed 100% resistance to Nalidixic acid and Vancomycin, 93.75 % resistance to Tetracycline and Streptomycin, 75 % resistance to Ciprofloxacin and Gentamicin, 66.66 % resistance to Cotrimoxazole, 25 % resistance to Nitrofurantoin, 6.25 % resistance to Clindamycin and Cefaclor and 0 % resistance to Chloramphenicol, Ampicillin and Erythromycin. The hemolysis test for the 16 isolates indicated a complete hemolysis for 14, partial hemolysis for one and no hemolysis for one isolate, whereas all the isolates showed negative reaction for gelatin hydrolysis test. Based on the results of probiotic potency and safety assays, one isolate was finally chosen from the 56 isolates. The isolate sequence revealed 96.82% of its similarity to Lactobacillus fermentum. The present study concluded that selected Lactobacillus fermentum has potential probiotic properties with good antimicrobial activity against Clostridium perfringens and E. coli hence, may be tested in vivo in poultry as an alternative to antibiotics.