engleski

Diversity of carbapenemases in clinical isolates of Enterobacteriaceae in Ccroatia - the results from the multicenter study

The first carbapenem –resistant strain of Klebsiella pneumoniae was isolated in 2008. in University Hospital Center in Zagreb. It was a metalo-beta-lactamase (MBL) NDM-1. Since that Enterobacteriaceae with reduced susceptibility to one or more carbapenems emerged sporadically in different geographic regions in Croatia. The first group A carbapenemase found in Croatia was KPC-2 identified in K. pneumoniae strain from University hospital Center in Zagreb at the beginning of 2011. A remarkable increase in the in the number of carbapenem resistant isolates was observed in 2011. This observations gave rise to a multicenter study on carbapenem-resistance in Enterobacteriaceae from Croatia. The aim of the study was to characterize the carbapenem-resistance mechanisms of the strain to genotype the strains in oder to analyse the molecular epidemiology of carbapenem-resistance in Enterobacteriaceae from Croatia. In total 32 carbapenem non-susceptible strains of Enterobacteriaceae 21 Enterobacter cloacae, 5 K. pneumonia, 1 K. oxytoca, 4 Citrobacter freundii and1 Escherichia coli were collected during 2011-2012 from five hospital centers located in different geographic regions in Croatia. (University Hospital Center Zagreb, Sisters of Mercy Hospital Zagreb, University Hospital Osijek, University Hospital Split and County Hospital Pula). The strains were identified by conventional biochemical testing and by Vitec automated system. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method in Mueller-Hinton broth and 96 well microtiter plates according to CLSI guidelines. Double-disk-synergy test (DDST) was performed to detect ESBLs and modified Hodge test (MHT) was used to screen for production of carbapenemases. MBL E-test was used to screen for production of metallo-β-lactamases. Additionally the isolates were tested by combined disks tests using four disks of meropenem or imipenem one without and the other three with 3-aminophenylboronic acid test, with 0.1 M EDTA or both 3-aminophenylboronic and EDTA to screen for KPC, MBLs or simoultaneous production of MBL and KPC respectively as described previously. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing E. coli A15R- strain resistant to rifampicin. Transconjugants were selected on combined plates containing imipenem (1 mg/L) to inhibit the growth of recipient strain and rifampicin (256 mg/L) to supress the donor strains. The presence of genes encoding broad and extended-spectrum β-lactamases (blaSHV, blaTEM, blaCTX-M and blaPER-1), plasmid-mediated AmpC beta-lactamases, group A carbapenemases (blaKPC, blaSME, blaIMI, blaNDM), metallo β-lactamases (blaVIM, blaIMP and blaNDM), and carbapenem hydrolyzing oxacillinases (blaOXA-48), was determined by PCR using protocols and conditions as desribed previously. PCR Nhe test was performed to distinguish between SHV-1 and SHV-ESBL. Template DNA was extracted by boiling method. Lysates from reference strains producing TEM-1, TEM-2, SHV-1, SHV-2, SHV-4, SHV-5, CTX-M-15, IMP-1 and VIM-1, NDM-1, were used as positive controls for PCR. Primers for conserved sequences 5’-CS and 3’-CS were used to amplify class 1 integron. PCR assays with primers 5’-CS and 3’-CS combined with forward and revers primers for blaVIM was done to determine the location of blaVIM gene within class 1 integron. Amplicons were column-purified (Quiagen DNA purification kit) (Inel, Zagreb, Croatia) and sequenced directly using ABI PRISM 377 Genetic Analyser (Applied Biosystems). Plasmid DNA was extracted with Qiagen Mini Kit and subjected to PCR with primers specific for ESBLs and KPC. Genotyping of the strain was performed by multilocus sequence typing (MLST) according to Diancourt. Results The isolates were uniformly resistant to amoxycillin alone and combined with clavulante, piperacillin, cefazoline, ceforoxime, ceftazidime, cefotaxime, ceftraxone, ertapenem, piperacillin/tazobactam and gentamicin but uniformly susceptible to colistin. They were intermediate susceptible or resistant to cefepime, imipenem meropenem and amikacin. DDST was positive in all strains indicating the production of ESBLs. Modified Hodge test was consistent with the activity of carbapenemases. Combined disk with phenylboronic acid was positive in one strain while the rest of the strains gave positive results with EDTA indicating the production of MBL. Imipenem resistance was not transferred to recipient strain of E. coli by conjugation. One K. pneumoniae strains from Sisters of Mercy hospital in Zagreb was found to produce KPC beta-lactamase. Twenty eight strains strains from University hospital center Zagreb produced VIM type and three NDM type MBL. VIM and NDM positive isolates harboured also blaTEM-1 genes and blaCTX-M-15 BlaVIM genes were located in classI integron. VIM-positive E. cloacae strains were clonally related. Conclusions Since the first report of carbapenem-resistant K. pneumoniae from Croatia in 2008. 20 strains resistant only to ertapenem were found in many hospital centers in Croatia. They were found to possess only the ESBL combined probably with porin loss but the exact mechanism of resistance was not determined. True carbapenemases emerged in significant number from 2011. Carbapenemases found in this study belong to KPC and VIM family. The first report of KPC beta-lactamases in Croatia originated from 2011. from University hospital Center Zagreb. It was a single K. pnemoniae isolate and due to effective infection control measures the strain did not spread throughout the hospital. The second case was a K. pneumoniae strain from Sisters of Mercy hospital in 2012. The strain was imported from the hospital in Zabok due to a patient transfer KPC-2 β-lactamase with similar properties was previously reported from Switzerland, Israel, Germany Belgium, Italy and China. In Germany and China KPC-2 producing isolates coproduced VIM metallo-β-lactamase. Infection control efforts such as hand hygiene and isolation precautions limited the spread of KPC-producing clone of K. pneumoniae in our hospital so far. It is difficult to explain the origin of the isolate because it was not possible to investigate any connection to USA, Israel, UK, Greece or any other country where the KPC positive isolates were previously reported. VIM producing Enterobacteriaceae were previously reported in Greece, Taiwan, and many other countries. The dominant type of carbapenemase in Enterobacteriaceae from Croatia is VIM. All VIM positive strains originated from University Hospital Zagreb. Haematology unit with bone marrow transplatation was the most important reservoir of blaMBL genes. High consumption of antibiotics, particularly carbapenems in this unit exerts selection pressure which favours spread of multiresistant Gram-negative bacteria and drives the emergence of carbapenem-resistance mechanisms.

carbapenemases; KPC-2 beta-lactamase; VIM-1 beta-lactamase

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