Resistance to carbapenems in Enterobacterales has become a matter of the highest concern in the last decade. Recently, Enterobacterales harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight Klebsiella pneumoniae and two Enterobacter cloacae complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, β-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel β-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for β-lactam antibiotics: bla_CTX-M-15, bla_TEM, bla_SHV, bla_OXA-1, bla_OXA-2, bla_OXA-9, aminoglycosides: aac6, aad, rmt, arm and aph, fluoroquinolones: qnrA, qnrB and qnrS, sulphonamides: sul1 and sul2 and trimethoprim: dfrA5, dfrA7, dfrA14, dfrA17 and dfrA19. mcr genes were reported for the first time in Croatia. This study demonstrated the ability of K. pneumoniae and E. cloacae to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.

Rapid and accurate serotyping of Salmonella (S.) enterica subsp. enterica serovars is essential for effective public health surveillance, outbreak control, and food safety management. Traditional serotyping, although considered the gold standard, is time-consuming, technically demanding, and costly. This study aimed to evaluate the applicability of artificial intelligence (AI)-enhanced Fourier-transform infrared (FT-IR) spectroscopy using an IR Biotyper (Bruker Daltonics, Bremen, Germany) for the rapid and accurate serotyping of Salmonella enterica subsp. enterica isolates in Croatia. Materials and Methods: A total of 143 isolates representing different S. enterica serovars of human and food origin were analysed using the IR Biotyper. Each strain was tested in three biological and at least three technical replicates. The obtained results were compared with traditional serotyping according to the Kauffmann–White–Le Minor scheme. Isolate identification at the genus level was further confirmed by MALDI-TOF mass spectrometry. Results: The IR Biotyper demonstrated high reproducibility and complete concordance with standard serotyping methods, enabling accurate differentiation of the most prevalent S. enterica serovars in Croatia. Conclusions: Our findings demonstrate the applicability of FT-IR in routine laboratory work, with the potential to reduce typing time, reduce the number of strains, and lower overall costs required for epidemiological surveillance within the One Health approach.

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.

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.