Isolation, phenotypic, and genotypic characterization of Escherichia coli isolated from environmental wastewater

Date of Publication

4-2025

Document Type

Bachelor's Thesis

Degree Name

Bachelor of Science in Biology major in Molecular Biology and Biotechnology

Subject Categories

Biology | Microbiology

College

College of Science

Department/Unit

Biology

Thesis Advisor

Llewelyn M. Espiritu

Defense Panel Chair

Florabelle D. Querubin

Defense Panel Member

Marigold O. Uba
Yokimiko D. Torrejos

Abstract/Summary

Antimicrobial resistance (AMR) is a pressing issue concerning public health as pathogens take up AMR genes (ARGs) that counter commonly available medications—prompting consistent monitoring. However, regular surveillance of AMR in the Philippines remains inadequate and primarily restricted to select tertiary hospitals and laboratories. This study sought to address the gap in surveillance within urban environments by collecting wastewater samples from three sites within De La Salle University Manila, an institution that represents an urban community equipped with various laboratories, classrooms, clinics, canteens, and offices. Focusing on Escherichia coli as an AMR indicator in environmental settings, 110 isolates were purified from the samples. After a series of phenotypic characterization methods, 58 presumptive E. coli isolates were identified and subjected to antibiotic susceptibility testing against imipenem, meropenem, ertapenem, cefotaxime, and levofloxacin following CLSI standards using the Kirby-Bauer disk diffusion method. Majority of the isolates were susceptible with the exception of the C135 isolate being intermediate to levofloxacin, and the cefotaxime-resistant A241 isolate. Focusing on AMR monitoring, whole genome sequencing was performed on the extracted DNA of A241 isolate to determine underlying genetic mechanisms regarding its resistance. Genome analysis revealed 56 ARGs, 25 mobile genetic elements (MGEs), and 163 virulence genes. The resistome revealed a variety of ARGs for different classes of antibiotics, however only one of them has been phenotypically confirmed as an active ARG—the blaCTX-M-15 gene identified as the genetic determinant of A241’s cefotaxime resistance—suggesting that the other ARGs are not highly expressed, differentially regulated, or dependent on other elements. Analysis of its mobilome revealed the presence of MGEs, specifically plasmids and insertion sequences, like the phage plasmid IncFIB(H89) that contains the blaCTX-M-15 gene which could accelerate the spread of AMR while the virulome disclosed potential pathogenicity mechanisms including secretion systems, nutrient-intake genes, and stress-related genes. The presence of a cefotaxime-resistant or ESBL-producing E. coli in urban wastewater could be an early indicator that AMR can spread in the university’s wastewater, but with one resistant isolate resulting from 58 isolates, the institution could be containing the wastewater adequately. Although cefotaxime-resistant E. coli in the Philippines has been documented in agricultural environments, this is the first recorded occurrence of cefotaxime-resistant E. coli in urban wastewater—highlighting a need for expanded AMR surveillance beyond clinical and agricultural settings as urban wastewater may serve as unmonitored reservoirs for ARG dissemination.

Abstract Format

html

Language

English

Format

Electronic

Keywords

Anti-infective agents; Escherichia coli; Genomes

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Embargo Period

4-14-2025

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