Urinary tract infections are among the most common bacterial infections encountered in clinical practice, with
Escherichia coli representing the dominant urinary pathogen. Increasing detection of multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing uropathogens has narrowed empirical treatment options and renewed interest in fosfomycin. However, local long-term surveillance data on fosfomycin susceptibility remain limited in Pakistan. This study evaluated temporal changes in major urinary isolate categories and fosfomycin susceptibility patterns within a diagnostic laboratory network in Pakistan from 2013 to 2025. An exploratory molecular sub-analysis was also performed to assess selected resistance-associated transcript patterns in archived fosfomycin-susceptible and fosfomycin-resistant isolates. A retrospective laboratory-based, isolate-level analysis was conducted using anonymized urine culture records. The source database included 34,230 urine sample records, from which eligible culture-positive urinary isolates with required organism classification and fosfomycin susceptibility data were included in the final analytical dataset. Analyses were performed across predefined mutually exclusive study intervals. Organism categories included non-ESBL
E. coli, ESBL-producing
E. coli, laboratory-coded ESBL
E. coli 24 variant,
Klebsiella spp., and
Enterococcus spp. The ESBL
E. coli 24 variant was treated as a laboratory reporting category, not as a genomically confirmed clone or sequence type. Fosfomycin resistance was evaluated using interval-based comparisons and odds ratios. A selected subset of 24 archived isolates, including fosfomycin-susceptible and fosfomycin-resistant
E. coli and
Klebsiella pneumoniae, was analyzed by RT-qPCR for glpT, uhpT, murA, fosA, fosA3, and blaCTX-M transcript abundance. The final isolate-level analytical dataset included 17,978 eligible urinary isolates. Among urine records with available sex data, female-associated records represented the majority throughout the study period, but this finding reflects laboratory record distribution rather than patient-level UTI prevalence.
E. coli remained the predominant urinary isolate category. Non-ESBL
E. coli declined across study intervals, whereas ESBL-associated
E. coli categories represented a larger proportion of isolates in later years. The laboratory-coded ESBL
E. coli 24 variant increased in later intervals, although this finding requires cautious interpretation because confirmatory molecular typing was not performed. Fosfomycin resistance showed a non-linear temporal pattern: resistance decreased from the early to the middle interval and then increased markedly to 23.8% during 2021–2025, while susceptibility declined to 60.6% in the same interval. Compared with the middle interval, isolates from 2021–2025 had higher odds of fosfomycin resistance (OR = 3.64, 95% CI: 3.23–4.12;
p < 0.001). In the exploratory molecular subset, resistant isolates showed lower transcript abundance of selected uptake-associated genes, particularly glpT and uhpT, and higher expression of selected fosfomycin- and ESBL-associated genes, including fosA, fosA3, and blaCTX-M. These findings represent transcriptional associations in selected isolates and do not establish definitive resistance mechanisms. Urinary isolates in this diagnostic-network dataset showed a temporal shift toward greater representation of laboratory-reported ESBL-associated
E. coli categories and a marked increase in fosfomycin resistance during 2021–2025. The findings support continued local surveillance of urinary pathogens and periodic reassessment of fosfomycin susceptibility for antimicrobial-stewardship guidance. The molecular findings should be interpreted as exploratory transcriptional observations because they were based on a small selected isolate subset and were not supported by genomic, mutational, uptake, or functional validation.
Full article