engleski

Ertapenem resistance in ESBL-positive Klebsiella pneumoniae from Croatia

Carbapenems are an effective treatment of infections caused by multidrug-resistant Gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and other Enterobacteriaceae. They are considered to be drugs of last resort due to the fact that they are not inactivated by and effectively inhibit most beta-lactamases. As a consequence, carbapenems are frequently used to treat infections with Enterobacteriaceae expressing these enzymes. However, β-lactamase mediated resistance to carbapenems has been reported in Enterobacteriaceae, mostly due to the expression of serin class A β-lactamases (KPC, IMI, SME, NMC) susceptible to the inhibition by clavulanic acid, class B metallo-β-lactamases (IMP or VIM or NDM) or OXA-48 β-lactamase belonging to the class D β-lactamases. Furtermore, carbapenem resistance can be mediated by hyperproduction of ESBLs or plasmid-mediated AmpC β-lactamases combined with porin loss. 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. Twenty strains of K. pneumoniae resistant to ertapenem but susceptible or intermediate susceptible to meropenem and imipenem were collected in different hospital centers in Croatia. The aim of the study was to characterize the mechanisms of ertapenem resistance in these strains. In total 20 ertapenem non-susceptible strains of Enterobacteriaceae 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 phenotypically screened for carbapenemase production by performing combined tests using four disks of meropenem or imipenem one without and the other three with 3-aminophenylboronic acid test for detection of KPC enzymes, with 0.1 M EDTA to screen for MBLs or both 3-aminophenylboronic and EDTA to screen for simoultaneous production of KPC and MBLs 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. Group of CTX-M beta-lactamases was determined by multiplex PCR as described 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. Amplicons were column-purified (Quiaquick DNA purification kit, Inel, Zagreb, Croatia) and sequenced directly using ABI PRISM 377 Genetic Analyser (Applied Biosystems). Genotyping of the strain was performed by PFGE. Results The isolates were uniformly resistant to amoxycillin, cefazoline, ceforoxime, cefotaxime, ceftriaxone, ertapenem and piperacillin/tazobactam but showed variable levels of susceptibility/resistance to ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, gentamicin. All isolates were uniformly susceptible to colistin. Cefotaxime and ertapenem MIC was significantly reduced by clavulanate (for more than three dilutions). DDST was positive indicating production of ESBLs. Modified Hodge test was negative indicating the lack of carbapenemase activity. Combined disk with phenylboronic acid and EDTA were negative showing the absence of group A and group B carbapenemases respectively. Ertapenem resistance was not transferred to recipient strain of E. coli by conjugation. All strains yielded amplicons with primers specific for CTX-M, and SHV β-lactamases while ten were positive for blaTEM genes. PCR Nhe test was negative as typical for SHV-1 β-lactamase. Multiplex PCR revelaled the presence of group 1 CTX-M β-lactamases. Sequecing of CTX-M and TEM amplicons revealed the presence of CTX-M-15 and TEM-1 βa-lactamase. The strains showed distinct PFGE patterns and were not clonally related. Since the first report of carbapenem-resistant K. pneumoniae from Croatia in 2008. twenty strains resistant only to ertapenem were found in many hospital centers in Croatia. The ertapenem-resistance was due to hyperproduction of an CTX-M-15 β-lactamase (as proven by reduction of ertapenem MIC by clavulanate) combined probably with porin loss but the exact mechanism of resistance was not determined. The outer membrane proteins (OmpK 35 and Ompk36) will be characterized in order to determine effect of porin loss on carbapenem-resistance. The fact that the strains were not clonally related points out that there is no common source of isolates.

ertapenem; resistance; Klebsiella pneumoniae

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