Characterization of novel extended spectrum beta-lactamase producing bacteria and their bacteriophages from wastewater
Student thesis: Doctoral Thesis
Multidrug-resistant bacteria strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. Bacteria resistant to antibiotics are mostly enteric, they can contaminate the environment and, through ingestion, enter new hosts to cause infections. Therefore, emphasis was put on isolating ESBL-producing bacteria from treated wastewater. The overall aim of this thesis was to isolate, identify and characterise ESBL-producing bacteria from wastewater followed by isolation and characterisation of bacteriophages specific for these bacteria. Bacterial isolates were recovered after growth on selective media and multiplex PCR was used to amplify SHV, TEM, CTX-M and OXA genes. Biochemical test and whole genome sequencing were applied to identify and characterise the isolated strains. The cell-free supernatants were then used to isolate bacteriophages. A high titer lysate was then used to test the specificity of bacteriophages for different bacterial strains. The nature of phage genetic material was established and morphology of isolated phages was determined by the transmission electron microscopy imaging. Two cold-tolerant bacteria isolated harbouring CTX-M gene were studied by 16S rRNA gene sequence analysis and housekeeping gene sequences analysis which revealed that these isolates showed no close similarity to any known member of the Enterobacteriaceae but are related to the members of Rahnella, Rouxiella and Ewingella genera. The phenotypic characteristics of the two isolates were, however, discrete from these 3 genera. Furthermore, two different bacteriophages infected the two newly identified cold tolerant bacterial strains. These bacteriophages were found to be novel viruses, most likely belonging to the Siphoviridae family, based on their characteristics, morphology, and genome size. In conclusion, all the analysis showed that they two isolates belong to a novel genus in the Enterobacteriaceae family. Given the menacing impact of disease outbreaks caused by contaminated water resources, work presented here identifies novel bacteria with harmful capabilities and potentially offers new tools for environmentally safer treatment to overcome this threat.
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