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ThesisItem Open Access OCCURRENCE OF ENTEROAGGREGATIVE ESCHERICHIA COLI IN ANIMALS, HUMAN INFANTS AND ASSOCIATED ENVIRONMENTAL SOURCES IN ERNAKULAM(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-09-30) MANJUSHREE T.R.; C. SethulekshmiThe present study was undertaken to find out the occurrence of Enteroaggregative Escherichia coli (EAEC) in young animals, human infants and associated environmental sources in Ernakulam district, Kerala. All the samples procured during a period of 12 months from August 2018 to July 2019. During the study diarrhoeic faecal sample of young animals viz., calves and piglets were collected from individual cattle and pig rearing households of two panchayats in Ernakulam district. To study the environmental association water and soil samples were also collected from the animals rearing areas. Finally to study the occurrence in human beings, infant diarrhoeic stool samples were collected from primary health care units, hospitals, anganwadis and diagnostic laboratories of Ernakulam district. The samples collected were aseptically processed by conventional cultural technique. Molecular confirmation of EAEC was done by performing polymerase chain reaction (PCR) that targeted conserved genus specific 16SrRNA gene and virulence genes viz., astA, Pic, aggR and fimA genes with amplicon size 231, 106, 1111, 251 and 342 bp, respectively. The isolates were subjected to biofilm assay using a 96-well microtitre plate. To understand the antibiotic resistance profile, all the isolates were subjected to standard disc diffusion method for commonly used antibiotics. Thus the study gives information regarding occurrence of EAEC in the Ernakulam district and the risk associated with it. All the procured samples were initially subjected to isolation of E. coli through conventional cultural and biochemical techniques and E. coli could be recovered from 51and 42 samples of calves and piglets respectively. From the environmental samples, 30 soil samples and 62 water samples showed positive for E. coli. Among the human infants samples, 39 samples revealed positive for E. coli. All the 224 E. coli isolates were subjected to a genus specific 16SrRNA which affirmed that all the isolates belong to the genus Escherichia. All these E. coli isolates were further subjected to PCR for the detection of virulence associated genes belonging to EAEC. Four oligonucleotide primers targeting the EAEC viz., heat-stable toxin (astA), mucinase (Pic), transcriptional activator aggregative gene (aggR) and fimbrial subunit gene (fimA) were used in the study for detection of EAEC. On analysis 21.57 per cent, 5.89 per cent, 5.89 per cent and 15.69 per cent of the E. coli isolates from calf diarrhoeal samples carried the astA, pic, aggR and fimA genes respectively. From the piglets’ diarrhoeal samples, astA, pic, aggR and fimA genes were detected from 9.52 per cent, 9.52 per cent, 4.76 per cent and 23.81per cent of E. coli isolates respectively. From water and soil samples of calves rearing areas seven E. coli isolates carried virulence genes. Of the piglets rearing areas only two isolates from water carried the virulence genes where as soil samples remained negative. Out of 39 E. coli isolates from human infant diarrhoeal samples 20.51 per cent, 7.69 per cent, 7.69 per cent and 17.94 per cent carried astA, pic, aggR and fimA genes respectively. To study the biofilm forming ability, all the EAEC isolates were subjected to quantitative biofilm assay through which it was inferred that the 84 and 78.57 per cent of isolates from calves and piglets were low biofilm producers whereas only 10.5 and 7.14 per cent showed high biofilm forming ability. None of the isolates from environmental sources showed high biofilm forming ability. From human infants only two isolates showed high biofilm forming ability whereas 81.2 per cent were low biofilm producers. On antibiotic sensitivity study, EAEC isolates from calves showed highest resistance towards ampicillin (84 per cent) followed by cefotaxime (57.89 per cent). Among the isolates from piglets resistance was observed with cefotaxime and tetracycline (79 per cent). From environmental sources norfloxacin (78 per cent), cefotaxime and tetracycline (77.7 per cent) showed highest resistance. In human infants EAEC isolates showed highest resistance against ampicillin, streptomycin (75 per cent) and azithromycin (68.75 per cent). All the isolates were sensitive to imipenem. Further sensitivity was observed among nitrofurantoin and meropenem. From the study it was concluded that EAEC is an emerging pathogen of public health importance because of its ability to form biofilm and increasing antibiotic resistance towards commonly used antibiotics.ThesisItem Open Access OCCURRENCE AND CHARACTERIZATION OF EXTENDED SPECTRUM β- LACTAMASE PRODUCING ENTERICBACTERIAL PATHOGENS FROM FOOD ANIMALS AND ENVIRONMENT(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-12-20) K L, AKARSHEnteric pathogens are the major threat to public health. Among various enteric pathogens, Escherichia coli and Salmonella are considerably responsible for causing gastrointestinal disorders. Some of them possess Extended spectrum beta lactamase enzyme which are plasmid mediated beta lactamase resistant enzymes and their emergence among the above organisms is a great matter of concern. Hence, the present study is envisaged to know the distribution status of ESBL producing entero bacterial pathogens (E. coli and Salmonella) from food animals and environment in Wayanad district. A total of 460 samples including chicken (100), beef (100), meat (chicken/beef) products (50), animal faecal samples (50), raw milk from milk societies (50), mastitis milk (50), soil (30) and water (30) were analysed for E. coli and Salmonella through conventional culture and molecular method using uidA gene (E. coli) and invA gene (Salmonella). The presence of E. coli was recorded in chicken (32), beef (18), meat products (5), faecal sample (35), raw milk (30), mastitic milk (16), soil (6) and water (50), respectively. Among the recovered E. coli isolates, 68.02 per cent were ESBL producers and the major prevailing gene found was blaCTX-M (53.74 per cent). The presence of Salmonella was recorded in chicken (26), beef (18), cooked meat products (1), raw milk (9), mastitic milk (3), faecal sample (4), water (1) and soil samples (5), respectively. A total of 58.20 per cent of the Salmonella isolates were ESBL producers and the most prevailing gene was blaCTX-M (24 isolates) and blaSHV (23 isolates). Most of the E. coli and Salmonella isolates were found sensitive to Imipenem and were more resistant to cefotaxime antibiotic. Thus, the present study highlights the presence of E. coli and Salmonella among food animals and environmental source, prevalence of ESBL production among the above isolates and its diversified genotypic pattern in WayanadThesisItem Open Access OCCURRENCE OF COMMUNITY- ASSOCIATED AND LIVESTOCK- ASSOCIATED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS FROM COMPANION ANIMALS, LIVESTOCK AND THEIR HANDLERS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-12-21) HAKIM, HAMNAAntimicrobial resistance (AMR) is one of the leading threats in healthcare management system. Staphylococcus aureus is a commensal bacteria commonly seen on skin and nasal passage that can turn as an opportunistic pathogen. Resistance observed in S.aureus is among the first reported cases of AMR. Methicillin Resistant Staphylococcus aureus (MRSA) has spread and proved fatal among human and animal populations around the globe. MRSA was initially reported as healthcare-associated (HA-MRSA), community-associated (CA-MRSA) and livestock-associated MRSA (LAMRSA). Hence, the present study was carried out to evaluate the nasal carriage of CAMRSA and LA-MRSA among apparently healthy companion animals, livestock and their handlers. A total of 220 nasal swabs were collected from dogs (30), cats (30), cattle (40), goats (30), pigs (30) and human handlers (60) including animal owners (20), farm workers (20) and veterinarians (20). All the samples were initially screened for occurrence of S.aureus, which was confirmed by PCR using species specific primer (nuc). All the S.aureus isolates were screened for the occurrence of MRSA, both phenotypically (double disc diffusion using cefoxitin and oxacillin discs) and genotypically (PCR employing mecA and mecC). There was a significant difference in the nasal carriage of S.aureus among animal population ( Chi-square value – 33.206, pvalue < 0.001), which was found as 76.67 per cent, 23.33 per cent, 53.33 per cent, 62.5 per cent, 86.67 per cent, and 71.67 per cent among dogs, cats, cattle, goats, pigs and human handlers respectively. None of the nasal swabs from animals were found positive for MRSA. Among the human handlers, there was no significant difference in nasal carriage of S.aureus (Chi-square value – 1.149, p-value = 0.563). Three human handlers (two farm workers and one veterinarian) were alone positive for MRSA, with a five per cent occurrence rate. All the MRSA isolates were also screened for the presence of virulent gene, pvl. None of the MRSA isolates harboured virulent pvl gene. Later, the molecular typing of MRSA isolates were done by spa typing. Two of the three recovered MRSA isolates belonged to the spa type t18050 and one isolate was spa type t18230. The present study signifies the prevalence of nasal carriage of S.aureus and MRSA among occupationally at risk humans and the absence among domestic animal population of Wayanad district.ThesisItem Open Access OCCURRENCE OF SELECTED ENTERIC BACTERIAL PATHOGENS OF PUBLIC HEALTH SIGNIFICANCE FROM COMPANION ANIMALS AND URBAN DWELLING BONNET MACAQUES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-09-27) N., SUMAZoonotic diseases are a major public health threat that can spillover to human from non-human primates and companion animals due to their close association. The spillover of bacterial enteropathogens such as E. coli, Salmonella spp., Shigella spp. and Y. enterocolitica can cause severe and chronic gastrointestinal illness. Hence, the present study is envisaged to evaluate the occurrence and characterization of above pathogens from dogs, cats and bonnet macaques of Wayanad district and to characterize its virulence potential and ESBL resistance. A total of 150 faecal samples/ faecal swabs from dogs (50), cats (50) and bonnet macaques (50) were analyzed for enteric bacterial pathogens through conventional culture and molecular method using 16S rRNA PCR-RFLP. The occurrence of E. coli, Salmonella, Shigella spp. and Y. enterocolitica was recorded in 107, 31, 6 and 21 samples, respectively and macaques were identified as a major source. PCR-based virulent gene characterization revealed that 20 per cent of isolates were positive for EHEC (eaeA, stx1), 36 per cent of isolates were EAEC (aggR, fimA), 7 per cent were Salmonella (invA, stm), 4 per cent were Shigella (ipaH) and 14 per cent of samples were Y. enterocolitica (ystA). Further, characterization of isolates for ESBL production through phenotypic disc diffusion method and genotypic PCR assays revealed that 77.77 per cent of E. coli isolates were ESBL producers and ESBL gene that could be identified in majority of isolates was blaCTX-M. With regard to the ESBL characterization of other isolates, 22.58 per cent samples were identified as ESBL producers in case of Salmonella spp. and the most common ESBL genes identified were blaSHV and blaTEM. Only few isolates of Shigella spp. and Y. enterocolitica were recovered from dogs and macaques samples, some of which were ESBL producers. Thus, a diverse genotypic pattern of ESBL resistance was observed among isolates. The present study signifies the occurrence of enteric bacterial pathogens among pet and wild animals, its virulence nature and the ability to harboring ESBL resistance genes and its dissemination.
