One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the bla _VIM-2 gene located in a novel class 1 integron composed in its variable part of the bla _VIM-2 - bla _oxa-10 -Δ qacF-aacA4 genes. Metallo-β-lactamase-producing strains belonged to sequence types ST235 and ST111.

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.

The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent^® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.

Background: An increased frequency of extended-spectrum β-lactamase (ESBL)-positive Proteus mirabilis isolates was observed recently in the Clinical Hospital Center Split in Croatia. The aim of this study was the molecular characterization of ESBLs in P. mirabilis isolates from this hospital. Material and Methods: Seven strains showing reduced susceptibility to ceftazidime were investigated. Antimicrobial susceptibility was determined using the broth microdilution method. ESBLs were characterized by PCR and sequencing of blaESBL genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of strains was performed by pulsed-field gel electrophoresis (PFGE). Results: The presence of an ESBL was confirmed in all strains by a double-disk synergy test. All strains were resistant to amoxicillin, piperacillin, gentamicin, ciprofloxacin, chloramphenicol, sulfamethoxazole and trimethoprim, but susceptible to ceftazidime/clavulanic acid, piperacillin/tazobactam, cefoxitin, imipenem and meropenem; PCR sequencing using primers targeting blaESBL genes revealed TEM-52 β-lactamase. PFGE genotyping demonstrated the clonal relatedness of TEM-52-producing P. mirabilis strains isolated from different clinical samples and wards within the hospital. BlaTEM-52 in 3 isolates was carried by a 70-kb conjugative plasmid. Conclusions: Our findings indicate the emergence of the TEM-52 enzyme among P. mirabilis in Croatia.