Dijana Varda-Brkić

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia.

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia. In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST. Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively. Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.

Epidemic spread of OXA-48 beta-lactamase in Croatia

Purpose. A dramatic increase in OXA‐48 &bgr;‐lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA‐48 carbapenemase in Croatia. Methods. Carbapenemase and other &bgr;‐lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole‐genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. Results. Forty‐eight isolates positive for OXA‐48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty‐three isolates were ESBL positive and harboured group 1 CTX‐M 1 &bgr;‐lactamases. In addition to the &bgr;‐lactam resistance genes detected by PCR (blaSHV‐1, blaOXA‐48 and blaOXA‐1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3‐Ia, fluoroquinolone resistance determinants aac(6)Ib‐c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. Conclusions. The OXA‐48‐positive organisms found in this study showed wide variability in antibiotic susceptibility, &bgr;‐lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM‐1 in 2012–2013 to that of OXA‐48 in the 2015 to 2017.