Detection of VIM, NDM and OXA-48 producing carbapenem resistant Enterobacterales among clinical isolates in Southern Hungary.

Infections caused by carbapenem-resistant Enterobacterales (CRE) present an important therapeutic problem, as there are limited number of effective therapeutic alternatives available. In this study, phenotypic and genotypic methods were used to characterize carbapenemase-production and other resistance-determinants (AmpC and ESBL-production, efflux pump-overexpression) in 50 isolates (Klebsiella spp. n = 35, Escherichia coli n = 12 and Enterobacter cloacae complex n = 3) collected at the Albert Szent-Györgyi Clinical Center (University of Szeged) between 2014 and 2017. Minimum inhibitory concentrations of meropenem, sulfamethoxazole/trimethoprim, tigecycline, amikacin, moxifloxacin, colistin and fosfomycin were also determined. 24% of isolates were AmpC-producers, while 30% carried blaCTX-M ESBL-genes. Carbapenemase-genes were detected in 18 (36%) of the tested isolates: in 2 isolates blaNDM, in 6 isolates blaOXA-48-like and in 12 isolates, blaVIM was detected by PCR. The species-distribution for isolates positive for carbapenemase-genes was the following: Klebsiella pneumoniae n = 11, Klebsiella oxytoca n = 1, E. coli n = 5, E. cloacae complex n = 1. Efflux pump-overexpression based on the PAßN-screening agar was shown in n = 3 of the tested strains. In nine isolates (18%), carbapenemase and ESBL-genes were detected simultaneously. Highest levels of resistance were noted for fosfomycin (74%) and moxifloxacin (70%), while all isolates were susceptible to colistin. Among applied phenotypic tests in this study the modified carbapenem inactivation method (mCIM) proved to be the most accurate one compared to that of PCR results.

Emergence of OXA-162-producing Klebsiella pneumoniae in Hungary.

In August 2012, 2 carbapenem-resistant Klebsiella pneumoniae isolates from the University of Szeged were submitted to the National Reference Laboratory at the National Centre for Epidemiology to confirm the carbapenem resistance mechanism. PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene, a very recently described variant of OXA-48, and the blaCTX-M-15 extended-spectrum ß-lactamase gene. The isolates had indistinguishable PFGE patterns and belonged to sequence type ST15. To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of an OXA-162-type carbapenemase gene in the K. pneumoniae ST15 clone.

Genomic Epidemiology of C2/H30Rx and C1-M27 Subclades of Escherichia coli ST131 Isolates from Clinical Blood Samples in Hungary.

Extended-spectrum ß-lactamase-producing Escherichia coli ST131 has become widespread worldwide. This study aims to characterize the virulome, resistome, and population structure of E. coli ST131 isolates from clinical blood samples in Hungary. A total of 30 C2/H30Rx and 33 C1-M27 ST131 isolates were selected for Illumina MiSeq sequencing and 30 isolates for MinION sequencing, followed by hybrid de novo assembly. Five C2/H30Rx and one C1-M27 cluster were identified. C1-M27 isolates harbored the F1:A2:B20 plasmid in 93.9% of cases. Long-read sequencing revealed that blaCTX-M-27 was on plasmids. Among the C2/H30Rx isolates, only six isolates carried the C2-associated F2:A1:B- plasmid type. Of 19 hybrid-assembled C2/H30Rx genomes, the blaCTX-M-15 gene was located on plasmid only in one isolate, while in the other isolates, ISEcp1 or IS26-mediated chromosomal integration of blaCTX-M-15 was detected in unique variations. In one isolate a part of F2:A1:B- plasmid integrated into the chromosome. These results suggest that CTX-M-15-producing C2/H30Rx and CTX-M-27-producing C1-M27 subclades may have emerged and spread in different ways in Hungary. While blaCTX-M-27 was carried mainly on the C1/H30R-associated F1:A2:B20 plasmid, the IncF-like plasmids of C2/H30Rx or its composite transposons have been incorporated into the chromosome through convergent evolutionary processes.

First description of bla(NDM-1), bla(OXA-48), bla(OXA-181) producing Enterobacteriaceae strains in Romania.

We report the first isolation and characterization of several Enterobacteriaceae strains harboring bla(NDM-1), bla(OXA-48) and/or bla(OXA-181) genes in a Romanian emergency teaching hospital. Between January 2010 and September 2012 nine carbapenemase-producing Enterobacteriaceae strains were identified. The bla(NDM-1) gene was present in two Enterobacter cloacae strains, an Escherichia coli and two Klebsiella pneumoniae strains. One of these K. pneumoniae strains also harbored the bla(OXA-181) gene. Three other K. pneumoniae strains and one Serratia marcescens carried bla(OXA-48).

Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit.

In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum ß-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-ß-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the bla(VIM-4) gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.

Characterization of a rare bla VIM-4 metallo-ß-lactamase-producing Serratia marcescens clinical isolate in Hungary.

A carbapenem-resistant S. marcescens isolate was recovered from a patient with an inflammed pacemaker inplantation pocket from a Cardiac Surgery ward in a Hungarian University Hospital. Phenotypic tests and polymerase chain reaction (PCR) confirmed a very rare gene responsible for production of a carbapenemase ( bla VIM-4 ), which was further characterized by Sanger-sequencing. The characterization of this S. marcescens strain emphasizes the ongoing emergence of novel or rare carbapenemases. Strains expressing a weak carbapenemase like this strain might go unrecognized by routine diagnostics due to low minimum inhibitory concentrations (MICs) for the bacterial strains producing such enzymes.

Countrywide dissemination of a DHA-1-type plasmid-mediated AmpC ß-lactamase-producing Klebsiella pneumoniae ST11 international high-risk clone in Hungary, 2009-2013.

The first plasmid-mediated AmpC ß-lactamase-producing Klebsiella pneumoniae (pAmpC KP) isolate was detected in December 2009 in Hungary. Hungarian microbiological laboratories were asked to send all KP strains showing cefoxitin resistance and decreased susceptibility or resistance to any third-generation cephalosporins to the Reference Laboratories at the National Center for Epidemiology. Investigation was conducted in order to outline spatio-temporal distribution and genetic characterization of pAmpC-KP isolates in Hungary. Between December 2009 and December 2013, 312 consecutive KP clinical isolates were confirmed as producing pAmpCs. All isolates showed resistance to third-generation cephalosporins, aminoglycosides and fluoroquinolones, and 77 % were non-susceptible to at least one carbapenem. Analysis of ß-lactamase genes showed blaDHA-1 in all and additionally blaCTX-M-15 in 90 % of isolates. PFGE typing revealed 12 pulsotypes; of these, KP053 (262/312) and KP070 (38/312) belonged to sequence type ST11 and comprised 96 % of the isolates. The blaDHA-1 and blaCTX-M-15 co-producing KP053/ST11 clone affected 234 patients and spread to 55 healthcare centres across Hungary during the study period. Three KP053 isolates were also resistant to colistin. In two of these, the mgrB gene was truncated by IS10R, while in the third isolate, insertional inactivation of mgrB by ISKPn14 was identified. Hungary is the first European country showing endemic spread of blaDHA-1 facilitated by the international high-risk clone ST11. The rapid countrywide spread of this multidrug-resistant clone seriously endangers Hungarian healthcare facilities and warrants strengthening of infection control practices and prudent use of carbapenems and colistin.