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dc.description.abstractEnterococci are no more regarded as GRAS (Generally Recognized As Safe) organism and emerging as an important source of nosocomial infections worldwide. Ecological and epidemiological studies showed that these bacteria enter in the environment via feces and colonize because of their high adaptability. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes (ARGs). This study aimed to examine the prevalence, dissemination, antibiotic resistance and virulence factors associated with enterococci from bulk soil (BS). Total 372 enterococcal isolates out of 500 soil samples were recovered. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E. faecium ( (77%), E. faecalis (E. fl) (10%), E. hirae (E. hr) (4%) and E. casseliflavus (E. cas) (1%) were the major species isolated. Among the identified species, the most prevalent virulent factor was biofilm formation (49%), followed by bacterial antagonism (13%). Gelatinase production was only shown by E. fl isolates (81%) while beta hemolysis was shown by 5% isolates. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E. fl isolates. The E. fm carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E. fm, while enlA (18%) and ef1097 (2.6%) was only detected in E. fl isolates. 50% E. fl and 2% E. fm isolates harbored IS16, while five E. fl harbored both IS16 and espTIM genes providing strong evidence about the presence of espTIM gene on 64kb pathogenicity island (PAI). BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major antibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. The most prevalent antibiotic resistance gene among the isolates was gyrA followed by ermB gene. Although none of the isolate showed resistance against HLG, but 12% and 9% isolates carried aph (3)-IIIa and aac6- aph2 genes, respectively. Presence of multiple antibiotic resistant, virulent and hospital associated ENT in bulk soil represents a potential source for further dissemination to humans and animals and poses potential impact on public health. Use of antibiotics in animal husbandry and manure containing antibiotics in agricultural practices significantly increases ARGs and resistant bacterial population in soil. Current study found out the prevalence, antibiogram pattern and genetic relatedness of ENT from agriculture soil (AS, n=200) of Karachi. Total 118 enterococcal isolates (76% E. fm, 17% E. fl and 3% Page | xxi each E. cas and other enterococcal species) were recovered. Antibiogram analyses revealed high resistance level against bacitracin and kanamycin (74.5%) followed by ciprofloxacin (51.6%). Susceptibility to two heavy metals (i.e. Cu and Zn) were also checked. 91.5% isolates were resistant against Zn metal. 13 AS isolates were high-level gentamicin resistant (HLGR) out of which 12 (92%) contained aac6-aph2 and ermB gene, 8 (61.5%) contained aph (3)-IIIa gene, while gyrA, tetM, tetL and Tn916-1545 was detected in 15%, 77%, 46% and 69% isolates, respectively. Among CL-S (n=30), majority were resistant to kanamycin (96%) followed by gentamicin (80%) and harbored aac6-aph2 (26%), aph (3)-IIIa and parC (30%), ermB (66%), gyrA (36%), tetL/M (50%/53%) genes. Molecular finger printing of all HLGR ENT was analyzed by BOX, ERIC, RAPD and whole-cell proteins SDS-PAGE. ERIC PCR yielded highest diversity, while lowest diversity was shown by SDS-PAGE. Majority of the virulence determinants were detected in E. fl followed by E. fm. Analysis of biofilm formation by semi quantitative 96-well plate method revealed that all the other Enterococcus spp. were biofilm formers followed by E. fl (63%) and E. fm (51%). In contrast, majority of the adherence genes were detected in E. fl with asa being the most prevalent (90%) and ace being the least prevalent (40%) gene. Gelatinase and hemolysis activity was checked phenotypically. 45% E. fl were beta-hemolytic followed by 36% E. fm. Majority of the gelatinase and cytolysin operon genes were detected in E. fl including a variable size product of cylM. 93 (78%) isolates showed lipolytic activity out of which 35 (29%) and 13 (11%) carried lip-fm and lip-fl genes, respectively. IS16 gene, urease and DNase activity were not detected in any of the AS isolates. 43 (36%) isolates showed antagonism against L. monocytogenes and entA, B, P genes were detected with frequencies of 11 (9%), 17 (14%), 26 (22%), respectively. In addition, 30 clinical isolates were also evaluated for their pathogenic potential both in terms of antibiotic susceptibility testing and presence of virulent factors to compare with environmental isolates (BS and AS). In conclusion, this study reflects an eye brow raising situation about diverse antibiotic resistant and virulent enterococcal species in both types of soil (BS and AS) which represents a possible source for further dissemination to humans and poses potential impact on public health. Thus, it is recommended to take practical measures and a routine surveillance towards their eradication.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.publisherUniversity of Karachi, Karachi.en_US
dc.subjectBiological & Medical Sciencesen_US
dc.titleMolecular assessment of virulence determinants, antibotic resistance and bacterial antagonism in environmental enterococcus speciies isolated from soilen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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