engleski

Emegence of metallo-beta-lactamases in Dubrovnik-Neretva County, Croatia

Background and aim Carbapenemases involved in acquired resistance to carbapenems in Enterobacteriaceae belong to Ambler class A serin β-lactamases (KPC, GES, SME, IMI, NMC), class B metallo-β-lactamases (MBL) of the IMP, VIM or NDM family and OXA- 48-like β-lactamases belonging to the class D. Recently carbapenem-resistant Enterobacter cloacae isolates were reported for the first time in Dubrovnik-neretva county in Croatia. The aim of this study was to characterize the carbapenem resistance mechanisms and to genotype the isolates. Material and methods In total four carbapenem resistant E. cloacae strains were isolated in Public Health Institute of Dubrovnik neretva county. The antibiotic susceptibility was determined by the broth microdilution method according to CLSI standards. The double disk synergy test (DDST) [23] and the CLSI combined disk test with addition of clavulanic acid were performed to detect ESBLs . Chromosomal or plasmid-mediated AmpC β-lactamases were detected by combined disk test using cephalosporin disks with 3- aminophenylboronic acid (PBA. A modified Hodge test (MHT) and the carbapenem-inactivation method (CIM) were used to screen for the presence of carbapenemases. Additionally, the isolates were tested by combined disk tests with imipenem and meropenem alone and combined with PBA, 0.1 M EDTA or both to screen for KPC, MBLs, or simultaneous production of KPC and MBL, respectively Carbapenemase, other β- lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. The transferability of meropenem resistance was tested by broth microdilution method. Plasmids were characterized by PCR-based replicon typing according to Carattoli et al. The isolates were genotyped by PFGE. Results The isolates were resistant to penicillins, narrow and expanded- spectrum cephalosporins, all three carbapenems (imipenem, meropenem and ertapenem) gentamicin and ciprofloxacin but susceptibile to colistin (Table 1). Hodge test, CIM test and inhibitor based tests with EDTA, PBA and EDTAPCR revealed blaVIM, blaCTX-M and blaTEM genes. Sequencing of representative amplicons identified VIM-1, CTX-M- 15 and TEM-1 β-lactamases as shown in Table 1. Plasmids encoding VIM-1 were found to belong to A/C incompatibility group. PFGE identified one pair of identical isolates and two isolates with distinct PFGE patterns as shown in Figure 1. Conclusions Previous studies in Croatia revealed the spread of VIM-1 MBL among Enterobacteriacae, particularly E. cloacae. In this study we demonastrated spread of MBL positive E. cloacae to Dubrovnik neretva county, a sequestered geographic area in Souteast part of Croatia which was free of carbapenemases until two years ago. Similarily as in the previous study VIM-1 was carried by A/C plasmid and the isolates coharboured ESBLs belonging to group 1 CTX-M- β-lactamases.

metallo-beta-lactamases ; Enterobacter cloacae ; resistance

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