Antimicrobial susceptibility and mechanisms of resistance of Greek Clostridium difficile clinical isolates.

OBJECTIVES: This study examined the antimicrobial susceptibility and resistance mechanisms of Clostridium difficile recovered in Greek hospitals during 2012-2015. METHODS: C. difficile isolates (n=88) were collected from clinically-confirmed C. difficile infection from symptomatic patients in 10 Greek hospitals. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by Etest. Isolates were typed by multilocus sequence typing (MLST). Toxin and resistance genes were detected by PCR. Chromosomal mutations in gyrA, gyrB and rpoB were identified by PCR and sequencing. The genetic environment of resistance genes was characterised by Illumina sequencing. RESULTS: The 88 C. difficile isolates comprised 27 sequence types (STs), with ST37 (n=26) and ST11 (n=21) being the most prevalent. All isolates were susceptible to vancomycin and metronidazole, with variable resistance rates to other antimicrobials. Of the 88 isolates, 45.5% were multidrug-resistant and the majority belonged to ST11 and ST37. The presence of chromosomal mutations in gyrA, gyrB and rpoB was mainly observed in high-risk clones such as ST11 and ST37. The antimicrobial resistance genes ermB, mefA, msrA and tetM were identified at different prevalences and combinations. Additionally, cfrB and cfrC were identified for the first time in Greece and were carried by a Tn6218 transposon and a novel plasmid, respectively. CONCLUSIONS: To our knowledge, this is the first study examining the resistance profiles and respective mechanisms of C. difficile recovered in Greek hospitals. Gut commensals such as C. difficile may serve as hubs for further transfer of antimicrobial resistance genes.

First description in Europe of the emergence of Enterococcus faecium ST117 carrying both vanA and vanB genes, isolated in Greece.

OBJECTIVES: An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. METHODS: Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. RESULTS: E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. CONCLUSIONS: This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.

Emergence of sequence type 11 Klebsiella pneumoniae coproducing NDM-1 and VIM-1 metallo-ß-lactamases in a Greek hospital.

Sequence type 11 Klebsiella pneumoniae, coproducing NDM-1 and VIM-1 metallo-ß-lactamases, were isolated in a Greek hospital. blaNDM-1 was part of a Tn125 derivative, located on an ~90-kb plasmid similar to the NDM-1-encoding plasmid pB-3002cz. blaVIM-1 was located in an In-e541-like integron, carried on a multireplicon (IncA/C and IncR) plasmid of ~180kb.

Rapid dissemination of colistin and carbapenem resistant Acinetobacter baumannii in Central Greece: mechanisms of resistance, molecular identification and epidemiological data.

BACKGROUND: Colistin-resistant/carbapenem-resistant Acinetobacter baumannii is a significant challenge for antibiotic treatment and infection control policies. Since 2012, in Central Greece an increase of colistin/pan- resistant A. baumannii has occurred, indicating the need for further analysis. METHODS: A total of 86 colistin-resistant/carbapenem-resistant out of 1228 A. baumannii clinical isolates, consecutively collected between 2012 and 2014 in a tertiary Greek hospital of Central Greece, as well as one environmental isolate from surveillance cultures were studied. Molecular typing and mechanisms of resistance to colistin and to carbapenems were assessed, whereas, epidemiological and clinical data of the patients were reviewed. RESULTS: During the study period, the rate of colistin resistance gradually increased and reached 21.1 % in 2014. All colistin-resistant/carbapenem-resistant A. baumannii belonged to 3LST ST101 clone that corresponds to the international clonal lineage II. Carbapenem resistance was associated with the presence of bla oxa-23-like, while resistance to colistin probably correlated with G54E and R109H amino acid substitutions in PmrA and PmrC, respectively. CONCLUSIONS: Epidemiological data of the patients indicated that the first detection of colistin-resistant/carbapenem-resistant ST101 clone in the University Hospital of Larissa (UHL) was associated with a patient who previously had received colistin, while, the movement of the infected patients into the hospital probably resulted to its spread.

Report on a transborder spread of carbapenemase-producing bacteria by a patient injured during Euromaidan, Ukraine.

Spread of carbapenemase-producing bacteria has been described all over the world. This phenomenon may be accelerated by many factors, including wars and natural disasters. In this report, we described an NDM-1-producing Klebsiella pneumonia ST11 recovered from a patient injured during the Maidan revolution in Ukraine. To our knowledge, this is the first report of a carbapenemase-producing Enterobacteriaceae in Ukraine and one of several reports describing wound colonization/infection of humans injured during war.

Survey of metallo-ß-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008-11.

OBJECTIVES: The objective of this study was to perform a multinational survey of patients' colonization by metallo-ß-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. METHODS: Patients in 18 hospital units across Europe and Israel (n = 17 945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. RESULTS: Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFIIK, IncR + FIIK, IncR + A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFIIK and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. CONCLUSIONS: This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.

Complete nucleotide sequences of two NDM-1-encoding plasmids from the same sequence type 11 Klebsiella pneumoniae strain.

The sequence type 11 Klebsiella pneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other.

Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories.

Carbapenemase-producing bacteria have now spread all over the world. Infections caused by those bacteria are difficult to treat. Therefore, there is an urgent need for accurate and fast detection of carbapenemases in diagnostic laboratories. In this review, we summarize screening methods for suspected isolates, direct assays for confirmation of carbapenemase activity (e.g. the Carba NP test and matrix-assisted laser desorption ionization time-of-flight mass spectrometry carbapenem hydrolysis assay), inhibitor-based methods for carbapenemase classification, and molecular-genetic techniques for precise identification of carbapenemase genes. We also propose a workflow for carbapenemase identification in diagnostic laboratories.

Identification of CMY-2-type cephalosporinases in clinical isolates of Enterobacteriaceae by MALDI-TOF MS.

This study exploited the possibility to detect Citrobacter freundii-derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850-m/z peak, confirmed to represent a C. freundii-like ß-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC- and DHA-like AmpC-type ß-lactamases.

Carbapenemase-producing Klebsiella pneumoniae in the Czech Republic in 2011.

Carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. are increasingly reported in many countries all over the world. Due to the resistance of those bacteria to almost all antibiotics (e.g.beta-lactams, aminoglycosides, fluoroquinolones),treatment options are seriously limited. In the Czech Republic, the incidence of carbapenemase-producing Enterobacteriaceae seems to be low, restricted to only three cases detected between 2009 and 2010.Here, we describe molecular typing of 15 carbapenemase-producing Klebsiella pneumoniae isolates identified in the Czech Republic during 2011. Five VIM-1-producing isolates belonging to sequence type (ST)11 and one VIM-4-producing isolate of ST1029 have been detected. blaVIM-1 and blaVIM-4 as a part of class 1 integrons were chromosomally located or carried by a plasmid belonging to A/C replicon type (blaVIM-4). KPC-3-producing isolates of ST512, recovered from six patients, caused an outbreak. Three more isolates producing KPC-2 enzyme belonged to ST258. Both blaKPCgenes were part of the Tn4401a transposon carried on plasmids of the pKpQIL type. The isolates were resistant to all antibiotics tested except colistin and/or gentamicin.Four of these 15 strains were recovered from patients repatriated to the Czech Republic from Greece and Italy. This is the first report of outbreaks caused by carbapenemase-producing Enterobacteriaceae in the Czech Republic.

Characterization of pKP1433, a novel KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 340.

The nucleotide sequence of pKP1433 (55,417 bp), a blaKPC-2-carrying plasmid from Klebsiella pneumoniae sequence type 340, was determined. pKP1433 displayed extensive sequence and structural similarities with the IncN plasmids possessing the KPC-2-encoding Tn4401b isoform. However, the replication, partitioning, and stability of pKP1433 were determined by sequences related to diverse non-IncN plasmids.

Characterization of pKP1780, a novel IncR plasmid from the emerging Klebsiella pneumoniae ST147, encoding the VIM-1 metallo-ß-lactamase.

OBJECTIVES: To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pKP1780 from Klebsiella pneumoniae ST147 representing a distinct group of IncR replicons. METHODS: The plasmid pKP1780 was from a K. pneumoniae clinical strain (KP-1780) isolated in Greece in 2009. Plasmid DNA was extracted from an Escherichia coli DH5α transformant and sequenced using the 454 Genome Sequencer GS FLX procedure on a standard fragment DNA library. Contig gaps were filled by sequencing of PCR-produced fragments. Annotation and comparative analysis were performed using software available on the Internet. RESULTS: Plasmid pKP1780 (49 770 bp) consisted of an IncR-related sequence (12 083 bp) including replication and stability systems, and a multidrug resistance (MDR) mosaic region (37 687 bp). blaVIM-1 along with the aacA7, dfrA1 and aadA1 cassettes comprised the variable region of an integron similar to In-e541 from pNL194. The mosaic structure also included the strA, strB, aphA1 and mphA resistance genes as well as intact (n = 10) or defective (n = 3) insertion sequences and fragments of various transposons. CONCLUSIONS: The mosaic structure of pKP1780 exhibited high similarity with the acquired region of the IncN plasmid pNL194, indicating the acquisition of the VIM-1-encoding MDR region from pNL194 by an IncR-type plasmid.

Characterization of metallo-beta-lactamase VIM-27, an A57S mutant of VIM-1 associated with Klebsiella pneumoniae ST147.

VIM-27 metallo-ß-lactamase, an Ala(57) → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla(VIM-27) was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most ß-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

Sequence of pR3521, an IncB plasmid from Escherichia coli encoding ACC-4, SCO-1, and TEM-1 beta-lactamases.

The sequence of pR3521, a self-transmissible plasmid from Escherichia coli, was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla(ACC-4), bla(SCO-1), bla(TEM-1b) (two copies), strA, strB, sul2, and aacC2.

Detection of metallo-ß-lactamase genes in clinical specimens by a commercial multiplex PCR system.

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-ß-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting bla(VIM) genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.

Sequence of pNL194, a 79.3-kilobase IncN plasmid carrying the blaVIM-1 metallo-beta-lactamase gene in Klebsiella pneumoniae.

The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla(VIM-1), aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

GES-13, a beta-lactamase variant possessing Lys-104 and Asn-170 in Pseudomonas aeruginosa.

GES-13 beta-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla(GES-13) was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.