Seale, BrentLee, KevinAthuraliya, Deanna Rozanne2025-11-182025-11-182025http://hdl.handle.net/10292/20134Urinary tract infections (UTIs) are one of the most common bacterial infections globally, affecting hospitals and communities with an estimated 150 million cases annually (Wagenlehner et al., 2020; Zagaglia et al., 2022). Infections are common with approximately 50% of females, 12% of men, and 5% of children experiencing a UTI episode in their lifetime (González et al., 2020). These infections are a large burden on affected individuals and healthcare systems due to the high morbidity and high medical costs involved, which are estimated to be more than $5 billion annually in the United States alone (Terlizzi et al., 2017). Escherichia coli is a common facultative anaerobe in the mammalian gastrointestinal tract and can colonise the urinary tract as uropathogenic E. coli (UPEC) (Jaureguy et al., 2008; Kaper et al., 2004). This bacterium is responsible for approximately 90% of community-acquired UTIs (Flores-Mireles et al., 2015). Management of UTIs often uses empirical broad-spectrum antibiotics, making them the second most common cause for hospital antibiotic prescriptions (Pujades-Rodriguez et al., 2019; Zhou et al., 2023). The overprescription of antimicrobials may factor into the emergence of antimicrobial resistant (AMR) microorganisms such as E. coli. As it is one of the most critical AMR bacteria due to its ability to evade antibiotic treatment and transfer resistance genetic material to other species (Zhang et al., 2019). Trimethoprim (TMP) is a synthetic folic acid antibiotic which was commonly used as a first-line treatment for UTIs and is now used in combination with sulfamethoxazole (SMX) as TMP-SMX. However, current global TMP prescription rates have declined due to the high resistance rates observed by recent clinical screenings (Schito et al., 2009; Zagaglia et al., 2022; Zhanel et al., 2006). While several resistance mechanisms have been identified in some key bacterial species throughout international research, the extent of clinical AMR in New Zealand (NZ) and resistance mechanisms are currently underexplored. Antibiotic susceptibility testing (AST) was performed using disk diffusion assays and minimum inhibitory concentration (MIC) analysis to determine the resistance profiles of 106 E. coli isolates obtained from Middlemore Hospital (Auckland, NZ). Using disk diffusion assay AST, each isolate was tested against first-line antibiotics used to treat UTIs such as meropenem, TMP, TMP-SMX, amoxicillin-clavulanic acid, and nitrofurantoin, and the available national resistance data (BpacNZ, 2017; LabPLUS, 2023). One isolate was resistant to meropenem, 98 isolates were resistant to TMP, with 83 of these isolates being resistant to TMP-SMX. Eight isolates were resistant to amoxicillin-clavulanic acid, and two isolates were resistant to nitrofurantoin. Three of the 106 isolates were non-viable during culturing and were not investigated further. The 74 TMP-SMX resistant isolates underwent MIC testing where 24-hour optical density (OD) readings were generated at TMP concentrations ranging from 8-4096 µg/mL. These OD values were used to generate two criteria, based on growth rates features and estimated MIC, to classify isolates into low, medium, and high levels of TMP resistance. Based on these criteria, many of these isolates were classified as being of medium level resistance. Results from MIC testing suggested resistant isolates were able to tolerate a TMP concentration of upwards of 128 times above the clinical breakpoint as demonstrated by OD curves. Therefore, whole genomes of six TMP-SMX resistant isolates were sequenced using Nanopore MinION (Oxford Nanopore Technology), with two isolates selected from each of the three TMP resistance levels generated from the two criteria. This aimed to identify differences in antibiotic resistance mechanisms within each resistant level. OrthoVenn3 and Comprehensive Antibiotic Resistance Database (CARD) were used to investigate novel resistance mechanisms, where efflux pumps and antibiotic target alterations were highlighted as potential mechanisms involved in E. coli TMP resistance, with the presence of the dihydrofolate reductase (dfrA) gene being an established mechanism (Brolund et al., 2010; Grape et al., 2007; Somorin et al., 2022). A total of 152 protein clusters were shared among all six isolates, with known AMR genes detected irrespective of the isolates TMP resistance, each containing two or three copies of the dfrA gene located on plasmids. Overall, these results confirm the presence of TMP resistance in NZ E. coli isolates from UTIs. The level of TMP resistance shows no observable influence on the presence or absence of AMR genes. Future research on the expression levels of these genes to fully understand TMP resistance in UPEC.enThesisOpenAccess