Background: Treating complicated urinary tract infections (cUTIs) has become more challenging due to escalating antimicrobial-resistance. Analysing the local etiology and antimicrobial susceptibility profile of pathogens causing cUTI is important for promoting evidence based antimicrobial prescribing. Adopting antimicrobial and diagnostic stewardship in clinical and laboratory practice is essential for implementing good practices. Microbiologists can further support optimum treatment protocols by evaluating new or repurposed antimicrobials and assess their effectiveness for providing appropriate recommendations. Aim and Objectives: To analyse the local etiology and antimicrobial susceptibility profile of pathogens causing cUTI and assess the prevalence of ESBL, AmpC and CRE among E. coli and K. pneumoniae while also evaluating different phenotypic tests for the detection of ESBL, AmpC and carbapenemase production in representative isolates. We investigated the antimicrobial activity of revived and novel antibiotics against representative E. coli and K. pneumoniae isolates. In-vitro synergy between aztreonam and ceftazidime-avibactam against NDM-producing bacteria was assessed. Antimicrobial resistance of representative strains producing AmpC β-lactamases and carbapenemases was assessed by whole genome sequencing (WGS). Current knowledge, awareness and practice (KAP) of pre-analytic diagnostic stewardship, infection prevention control, and antibiotic use were assessed among patients and nurses. Methods: The clinical, etiological and antimicrobial susceptibility profile of patients presenting with cUTI in Sultan Qaboos University Hospital (SQUH) between September 2022 and August 2023 were analysed and the distribution of ESBLs, AmpC β-lactamases, and CRE among E. coli and K. pneumoniae was identified phenotypically. A total of 114 non-duplicate E. coli and K. pneumoniae isolates demonstrating ESBL, AmpC and CRE phenotypes were isolated from urine samples of patients with cUTI. The isolates were tested against revived and novel antibiotics by disk diffusion test and E-test diffusion methods. For ESBL detection, potentiation between several cephalosporins and aztreonam with four B-lactam/B-lactamase inhibitor combinations were tested to assess which would be the best combination for phenotypic detection of ESBL. For AmpC detection, the disk approximation assay and the commercial D69C AmpC detection set were used. For carbapenemase detection, the mCIM test and KPC/IMP/NDM/VIM/OXA-48 Combo test kit (Medomics, China) were evaluated. The antimicrobial synergy between aztreonam (ATM) and ceftazidime-avibactam (CZA) was assessed in 6 isolates by disk approximation and Etest cross method. WGS was carried out in representative isolates and the bioinformatic analysis was performed using online tools for identifying multilocus sequence typing, acquired resistance genes and plasmids. Assessment of KAP about pre-analytic diagnostic stewardship, infection prevention control, and antibiotic use among patients and nurses was performed using online questionnaires. Results: The predominant bacterial uropathogens were E. coli (33%), K. pneumoniae (13%) and Enterococcus species (12%). Among routinely tested antibiotics ceftazidime-avibactam, meropenem, imipenem, ertapenem, amikacin, gentamicin and piperacillin-tazobactam achieved >70% susceptibility rate among E. coli and K. pneumoniae. E. coli exhibited a high susceptibility rate to nitrofurantoin (96%). The overall prevalence of ESBL among E. coli and K. pneumoniae was 37.2%, while the estimated prevalence of AmpC was 5.4% and the prevalence of CRE was 6.2%. E. coli was the predominant ESBL and AmpC producer, whereas K. pneumoniae was the major CRE producer. Among revived antibiotics, nitroxoline was the most active drug and all ESBL, AmpC and CRE-producing E. coli and K. pneumoniae were 100% susceptible to nitroxoline, while mecillinam, fosfomycin and cefoperazone-sulbactam exhibited excellent activity against ESBL and AmpC producers with susceptibility rate of >80%. E. coli exhibited higher susceptibility to oral fosfomycin than K. pneumoniae. Among novel antibiotics, cefiderocol showed the highest antibacterial activities and >90% of CRE strains exhibited susceptibility to cefiderocol. The susceptibility rate of imipenem-relebactam and plazomicin in ESBL and AmpCproducing strains was 100%. The antimicrobial activity of eravacycline was higher in E. coli than in K. pneumoniae. For ESBL detection, amoxicillin-clavulanate and ticarcillin-clavulanate were better inducers for aztreonam and 3rd/4th generation cephalosporins than piperacillin-tazobactam and cefoperazone-sulbactam. Cefepime was the best substrate for the detection of ESBL in AmpC co-producers. Using WGS as a reference method, the D69C set and KPC/IMP/NDM/VIM/OXA-48 Combo test kit achieved 100% sensitivity and specificity. ATM and CZA showed synergistic activity in the majority of NDM-producing isolates (5/6). WGS revealed DHA-1 as the predominant plasmid-mediated AmpC gene, while OXA-232 and NDM-5 as the most common carbapenemase genes. All E. coli DHA-1 positive isolates co-harboured the quinolone resistance gene qnrB4 and the sulfonamide resistance gene sul1 while no aminoglycoside resistance genes were detected. The majority of CRE K. pneumoniae carried other β-lactamase genes such as blaCTX-M-15, blaSHV, blaTEM, and all co-harboured the OqxAB efflux pump genes and 77% carried the aminoglycoside resistance gene armA. The analysis of KAP surveys revealed the need for improving pre-analytic diagnostic stewardship among patients and nurses. Conclusions: Nitroxoline, mecillinam and fosfomycin are promising oral antimicrobial choices that can spare the use of cephalosporins and fluoroquinolones for treating cystitis caused by ESBL and AmpC-producing Enterobacterales. Cefiderocol monotherapy and a combination of ATM and CZA appeared to be good colistin-sparing drugs for treating UTI caused by XDR/PDR Enterobacterales if no other antibiotics were effective. For phenotypic detection of ESBL, we recommend the use of DDST which combines clavulanic acid with 3rd generation cephalosporins and cefepime for enhancing the possibility of ESBL detection, the D69C set for routine detection AmpC β-lactamases and the KPC/IMP/NDM/VIM/OXA-48 Combo test kit for detecting the type of carbapenemases. WGS results revealed DHA-1 was the predominant AmpC while OXA-232 and NDM-5 were the circulating carbapenemases. Effective interventions to improve the pre-analytic diagnostic stewardship are essentially needed for better management of UTI.