Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs’ genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10⁻⁴ and 10⁻⁵ substitutions per site and year. Recombination was not detected among sequences from this study.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4–8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis.

K. pneumoniae harbors various antibiotic resistance determinants like extended-spectrum and plasmid-mediated AmpC β-lactamases and carbapenemases. In the last three years, in the period of intense population aging, migrations and climate changes in Europe and Croatia as well, we observed changes in antibiotic resistance patters of carbapenem-resistant K. pneumoniae (CRKP) isolates obtained routinely in community and inpatient setting. The aim was to compare and subsequently analyze CRKP hospital and community isolates resistance mechanisms, traits and molecular epidemiology, in order to analyze the dynamic of resistance trends, carbapenemase types and plasmid epidemiology. Disk diffusion and broth dilution method were the methods of choice to determine antibiotic susceptibility. β-lactamases were screened by phenotypic methods and confirmed with PCR. In total 113 isolates were analysed. Resistance to amoxicillin-clavulanate and ertapenem was confirmed in all strains. High resistance rates (over 90%) were observed for extended-spectrum cephalosporins, and ciprofloxacin. OKNV (OXA-48, KPC, NDM, VIM) testing and PCR detected OXA-48 in 106, NDM in 7 and KPC in only one isolate. ESBLs accompanied carbapenemases in 103 isolates. IncL, associated with OXA-48, was the dominant plasmid type. No significant differences in the resistance profile and resistance determinants were found between hospital and community isolates plasmid type. The predominance of OXA-48 carbapenemase is in line with the reports from the neigbouring countries.

Human bocavirus (HBoV) has emerged as a significant pathogen primarily associated with respiratory infections in children. This study aimed to evaluate the clinical relevance of HBoV infection by quantifying viral loads in nasopharyngeal swabs from hospitalized children with acute respiratory infections (ARIs) and investigating correlations with clinical outcomes. A total of 957 children were tested, with HBoV detected in 73 cases (7.6%), either as a sole infection or co-infection with other respiratory viruses. Quantitative polymerase chain reaction (qPCR) was employed to measure viral load, and a threshold of 10⁴ copies/mL was used to differentiate high and low viral concentrations. Results have shown that children with lower respiratory tract infections (LRTIs) had significantly higher viral loads, most notably in cases where HBoV was the sole pathogen. Additionally, children with pre-existing health conditions were more likely to have elevated HBoV concentrations compared to those who were previously healthy. Children with higher viral loads were more likely to require oxygen supplementation and receive empirical antibiotic therapy, indicating a more severe clinical course. This study underscores the importance of considering HBoV viral load in clinical diagnostics, as higher concentrations were associated with more severe presentations, including the need for oxygen support. Future research should focus on refining diagnostic thresholds and exploring HBoV’s role in co-infections to enhance patient care strategies.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

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.