Background/Objectives: Antimicrobial resistance of the E. coli O25 ST131 clonal lineage poses a significant therapeutic challenge worldwide, often involving resistance to fluoroquinolones and extended-spectrum beta-lactamase (ESBL) production. This retrospective study compared the dissemination of multidrug-resistant E. coli O25 ST131 isolated from the urine of outpatients at the largest Croatian clinical microbiology department across six years over two study periods. Methods: The E. coli O25 ST131 clonal lineage was detected via a rapid PCR method using pabB and trpA primers after positive agglutination with E. coli serogroup O25 antisera. ESBL phenotypes and antibiotic susceptibility were investigated according to EUCAST guidelines and breakpoint tables. Results: In the first period, there were a total of 45 isolates of E. coli O25 ST131, among which 30 were isolates with proven ESBL production. In the second period, a total of 114 isolates of E. coli O25 ST131 were detected, among which 75 (65.8%) were ESBL-positive (p > 0.05). In ESBL-negative strains, the multidrug-resistant (MDR) phenotype was characterized by simultaneous resistance to ampicillin, co-trimoxazole, and fluoroquinolones (with an equal proportion of 3/15 isolates in the first period and 7/39 isolates in the second period, p > 0.05). There was no statistically significant difference in the frequency of MDR detection across the two study periods (36/45 and 98/114, p > 0.05). This is the first detection of E. coli O25 ST131 in the outpatient population in Zagreb. Conclusions: There was no statistically significant difference in the frequency of detecting the E. coli O25 ST 131 clone across the two study periods. The high frequency of MDR phenotype among ESBL-negative isolates of E. coli O25 ST131 and an equally high proportion of MDR strains among ESBL producers in this clonal lineage, with the total detection of MDR isolates ≥ 80% in both study periods, are the reasons why this bacterial clone poses a public health threat and why further investigation into its metabolic and virulence characteristics is needed in order to estimate its spreading potential among the outpatient population in Zagreb.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.