Background: Urinary tract infections (UTIs) are among the most common hospital- and community-acquired infections, creating a substantial healthcare burden due to recurrence, complications, and rising antimicrobial resistance. Accurate diagnosis and timely antimicrobial therapy are essential. This study aimed to identify trends in the etiology, treatment, and resistance patterns of UTIs through a retrospective analysis of urine isolates processed at the Laboratory of Microbiology at University Hospital St. George in Plovdiv, the largest tertiary care and reference microbiology center in Bulgaria, between 2017 and 2022. Materials and Methods: A retrospective single-center study was performed at the hospital’s Microbiology Laboratory. During the study period, 74,417 urine samples from 25,087 hospitalized patients were screened with the HB&L UROQUATTRO system. Positive specimens were cultured on blood agar, Eosin-Methylene Blue, and chromogenic media. Identification was performed using biochemical assays, MALDI-TOF MS, and the Vitek 2 Compact system. Antimicrobial susceptibility testing included disk diffusion, MIC determination, broth microdilution (for colistin), and Vitek 2 Compact, interpreted according to EUCAST standards. Descriptive analysis and temporal resistance trends were evaluated with regression models, and interrupted time-series analysis was applied to assess COVID-19-related effects. Results: Out of 10,177 isolates, Gram-negative bacteria predominated (73%), with Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis as the leading pathogens. Among Gram-positives, Enterococcus faecalis was the most frequent. In the post-COVID-19 period, ESBL production increased in E. coli (34–38%), K. pneumoniae (66–77%), and P. mirabilis (13.5–24%). Carbapenem resistance rose in K. pneumoniae (to 40.6%) and P. aeruginosa (to 24%), while none was detected in E. coli. Colistin resistance increased in K. pneumoniae but remained absent in E. coli and P. aeruginosa. High-level aminoglycoside resistance in E. faecalis was stable (~70%), and vancomycin resistance in E. faecium rose from 4.6% to 8.9%. Conclusions: Both community- and hospital-acquired UTIs in Southeastern Bulgaria are increasingly linked to multidrug-resistant pathogens, particularly ESBL-producing and carbapenem-resistant Enterobacterales. Findings from the region’s largest referral center highlight the urgent need for continuous surveillance, rational antibiotic use, and novel therapeutic approaches.

Background/Objectives : Microbiological confirmation of suspected complicated urinary tract infections (cUTIs) is challenging, particularly in patients previously exposed to antibiotics or when fastidious organisms are involved. Molecular assays detect microbial nucleic acids independently of bacterial viability and may therefore yield results that differ from conventional culture. This study compared microorganism detection patterns and inter-method agreement between multiplex real-time PCR (mPCR), standard urine culture SUC, and rapid nephelometric screening (Uroquattro HB&L). Methods: In a prospective single-centre study, urine samples from 72 hospitalized patients with clinical suspicion of cUTIs were analyzed using SUC, mPCR (Novaplex™ UTI panel), and the Uroquattro system. Detection rates were calculated for each method. Agreement between paired methods was evaluated using Cohen’s kappa, and paired differences in detection were assessed using McNemar’s and Cochran’s Q tests. Results: mPCR detected microorganisms in 83.3% of samples, compared with 47.2% for SUC and 42.6% for Uroquattro. Agreement between mPCR and SUC was fair (κ = 0.26), whereas SUC and Uroquattro demonstrated high concordance. mPCR identified a broader spectrum of organisms, including fastidious and polymicrobial findings that were not recovered by culture. Correlation between PCR cycle threshold values and colony counts was weak and not statistically significant. Conclusions: mPCR demonstrated a substantially higher microorganism detection frequency than culture-based methods; however, the assays target different biological characteristics, highlight bacterial nucleic acid versus viable growth, and should be interpreted as complementary rather than interchangeable. Conventional culture remains necessary for antimicrobial susceptibility testing and clinical decision-making. Further studies incorporating clinical outcome-based reference standards are required to determine the clinical relevance of molecular detection in cUTIs.