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.

Background/objectives: A rise in infections associated with carbapenem-resistant Providencia species (CRPS) has been observed worldwide. This study presents a genomic analysis of CRPS isolates from four hospitals in Croatia and the outpatient setting, in order to determine the extent of the spread of CRPS in Croatia. In the present study, we applied a combination of phenotypic characterization and molecular analysis of resistance traits to determine the mechanisms and the routes of spread of CRPS. Material and methods: The antibiotic susceptibility testing was performed using disk-diffusion and broth dilution methods. The nature of extended-spectrum β-lactamases (ESBLs), carbapenemases, and fluoroquinolone resistance determinants was investigated by polymerase chain reaction (PCR). In order to obtain an insight into the whole resistome, selected isolates were subjected to the Interarray Genotyping Kit CarbaResist and whole genome sequencing (WGS). Results: In total, 30 isolates were collected from four centers, located in different geographic regions of Croatia. There was uniform resistance to piperacillin-tazobactam, cefuroxime, expanded-spectrum cephalosporins (ESCs), imipenem, ertapenem, meropenem, and ciprofloxacin. Immunochromatographic testing and PCR revealed OXA-48 and NDM carbapenemase in 15 isolates, respectively. Phenotypic tests for ESBLs were positive in all OXA-48 and one NDM-positive organism (16 isolates). The isolates were categorized as extensively drug-resistant (XDR). OXA-48-producing isolates were susceptible only to ceftazidime-avibactam, whereas NDM producers were susceptible to cefiderocol and, in the majority of cases, also to amikacin. WGS identified a plethora of genes encoding resistance to aminoglycosides, such as aadA1 and aadA2, (aph(3″)-Ib and aph(6)-Id, sulfonamides sul1 and sul2, trimethoprim dfrA1, dfrA10, and dfrA12, tetracyclines tet(A) and tet(B), and chloramphenicol catA3 and catA5. Conclusions: Providencia spp., in spite of being a rare pathogen, should be included in the surveillance studies across the medical centers in Croatia.

The aim of this study was to compare the virulence-associated characteristics and metabolic activity of fluoroquinolone-resistant (FR) Escherichia coli O25 ST131 isolates recovered from urine samples of outpatients in Zagreb, Croatia, during two study periods, 2011/2012 and 2017/2018. The O25 ST131 clonal lineage was identified by rapid multiplex PCR targeting the O25pabB and trpA genes. PCR was used to detect blaCTX-M, blaCTX-M-15, blaTEM, and blaSHV β-lactamase genes, as well as the virulence-associated genes fimH, papC, hlyA, cnf1, and traT. Metabolic activity was evaluated using the VITEK 2 GN card, and serum resistance was assessed using a serum susceptibility assay. Differences between study periods were analyzed using linear regression and the chi-square test. A total of 4354 and 7717 E. coli isolates were recovered during the first and second study periods, respectively, and 170 FR E. coli O25 isolates were collected. Among the O25 ST131 isolates, 30/45 (66.7%) from the first study period and 75/114 (65.8%) from the second study period were ESBL-positive, with CTX-M-15 predominating. The fimH gene was detected in all 159 O25 ST131 isolates (100%), whereas hlyA, papC, cnf1, and traT were detected in 35 (22%), 43 (27%), 35 (22%), and 123 (77%) isolates, respectively. Compared with the first study period, isolates from the second period showed significantly higher frequency of papC, hlyA, and cnf1, as well as higher serum resistance and metabolic potential (p < 0.01). These findings demonstrate temporal differences in selected virulence-associated characteristics, serum resistance, and metabolic activity among FR E. coli O25 ST131 isolates and support the need for continued surveillance of this clinically important clonal lineage in the community.