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Survey of Antimicrobial Resistant Indicator Bacteria and Mechanisms of Resistance from Lake Panorama retention pond and Paremuka Stream in West Auckland.

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Date

2025

Authors

Supervisor

Seale , Brent
Lee, Kevin

Item type

Thesis

Degree name

Master of Science (Research)

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Publisher

Auckland University of Technology

Abstract

Antibiotic resistant bacteria are becoming an alarming trend throughout the world due to overuse and overprescription of antimicrobial compounds. Death attributed to antimicrobial-resistant micro-organisms was estimated to be 1.27 million deaths in 2019 and the economic cost is projected to reach $1 trillion US dollars in health care related costs by 2050 (World Health Organization, 2025). Treatment of bacterial infections is becoming more challenging as more pathogens develop resistance due to extensive use for both human therapy and in animal husbandry (Nikaido, 2009). Due to their ubiquitous usage, antibiotics and resistant organisms are expelled from people and animals into the waste management system. Most methods of monitoring resistance rely on detection of specific genes (Baba et al., 2022). However, the sewer systems used to transport waste and by extension the resistant organisms are prone to leakage during wet weather and accumulate in artificial reservoirs constructed to prevent flooding of urban areas (Carlisle & Mulamoottil, 1991; Tryland et al., 2011). These artificial basins and reservoirs accumulate stormwater from waste leakages and urban runoff during wet weather and discharge into streams and rivers without treatment (Yang & Lusk, 2018). Most antibiotic resistance surveillance is limited to wastewater or the number of indicator bacteria in water, with little attention given to when potentially harmful bacteria escape into the environment. This study is aimed to survey the presence of any antimicrobial resistance, and the types of mechanisms present in the community of indicator organisms from a local West Auckland stormwater pond and its associated stream. Five sample sites along the Paremuka stormwater system were selected. Starting from Lake Panorama stormwater retention pond to downstream and then to the outlet at the Waitamatā Harbour. Samples were also taken before and after rainfall events, as well as different seasons for comparison. The number of indicator organisms such as Enterobacteriaceae and Enterococci spp. was determined using the Most Probable Number (MPN) method and membrane filtration (MF). Presumptive antibiotic resistance was determined using CHROM agar for extended spectrum beta-lactamase (ESBL) Enterobacteriaceae and vancomycin-resistant Enterococci (VRE). Antibiotic resistant positive isolates was further screened against further antibiotics (e.g. ampicillin, amoxicillin with clavulanic acid, cefalexin and meropenem) using antimicrobial disk assay to determine multidrug resistance (MDR). Of the resistant strains isolated, two MDR isolates was selected to undergo whole genome sequencing to determine the genetic bases that contributed to antibiotic resistance. The number of indicator organisms during winter, along with heavy rainfall increased the overall count by 200cfu/100ml at upstream sites and downstream sites became too numerous to count (TNTC). Indicator numbers during the summer in resulted in much higher counts ranging from 300cfu/100ml to TNTC. All isolates were resistant to ampicillin and susceptible to meropenem, with varying degrees of resistance to amoxicillin and cefalexin. Isolates UBr35 and URr35 were the only isolates resistant to ampicillin but showed susceptibility to cefalexin and the combination of amoxicillin and clavulanic acid. Filter isolates, isolate UBd35 and stream isolates showed complete resistance to all tested antibiotics except for meropenem. Estuary isolate BRd42 was the only organism to grow at 42°C that presented resistance to both ampicillin and cefalexin but showed susceptibility to meropenem and amoxicillin. Genome sequencing was conducted on two isolates UBd35 and SBr35, due to both isolates presenting the greatest resistance to the tested antibiotics. Using MinION and reconstructed using Flye, the assembled genomes were submitted to the Comprehensive Antibiotic Resistance Database (CARD), the Kyoto Encyclopaedia of Genes and Genomes (KEGG), the National Center for Biotechnology Information (NCIB) and the Center for Genomic Epidemiology (CDE) mobile element finder to identify potential resistance genes, mobility of detected genes, and to identify the bacterial species. The resistance mechanisms detected in the UBd35 isolate was presumed to be the bacteria H. alvei with the ACC-3 AmpC beta-lactamase gene. Isolate SBr35 was presumed to be a species of Citrobacter with a presumed identity of C. portcalunesis, carrying the CMY-65 class C beta-lactamase gene. The mobile genes detected were insertion sequences and mobile transposons found in SBr35, which was located hundreds of thousands of base pairs away from the CMY-65 beta-lactamase gene, indicating that the CMY gene is not part of the mobile genetic elements. In summary, the results show that regardless of seasonal conditions, the indicator organisms are present at elevated counts. Among the population of indicator bacteria, antibiotic resistance is present in a local retention pond and its associated stream. Of the two bacteria identified and sequenced, both species have multiple mechanisms capable of resisting common antibiotics. However, of those two bacteria sequenced, only one contained mobile genetic elements relating to insertion sequences and mobile transposons, which do not appear to be related to antibiotic resistance.

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http://hdl.handle.net/10292/19852

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