In this study we described six NDM-4-producing PS-341 E.coli isolates obtained from two patients admitted to an Italian hospital. We also present data on the localization and the genetic environment of the bla NDM-4 gene. Methods Bacterial strains Six E.coli isolated from urine samples of two inpatients at the San Martino-IST University Hospital (Genoa, Italy)
were studied. Isolates were taken as part of standard patient care and informed consent for the use of clinical data has been obtained by both patients. Strain identification, antibiotic susceptibility testing and phenotypic screening for MBL production Routine identification and antibiotic susceptibility testing were carried out using the Vitek-2 automated system (BioMérieux, Marcy-L’etoile, France). In vitro activity of carbapenems, aztreonam, fosfomycin and nitrofurantoin was further determined by the broth microdilution method and interpreted according to the of European Committee on Antimicrobial Susceptibility Testing (EUCAST ) guidelines (Version 4.0, 2014) . To detect metallo-β-lactamase (MBL) production,
a synergy test using imipenem and EDTA discs was used . Pulsed-field gel electrophoresis (PFGE) Genomic DNA was prepared, digested with XbaI (New England Biolabs Inc., MA, USA) and subjected to PFGE with the CHEF DRII device (Bio-rad, Milan, Italy), as described previously . Fingerprinting pattern was interpreted Silmitasertib according to the method of Tenover et al. . Multilocus sequence typing (MLST) MLST was carried out using protocols and conditions described on the E.coli MLST website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html).
Sequence types were assigned using the website interface. Molecular analysis techniques Polymerase chain reaction (PCR) amplification of the bla NDM gene and direct sequencing of the PCR products was performed as previously described . Screening for resistance genes was carried out using primers and conditions previously described [11–13]. Phylogenetic analysis using multiplex PCR method as described previously  was used. PCR experiments were performed to identify the upstream- and downstream-located regions of the bla NDM-4 gene . Mapping of the variable region of class 1 integron was performed by PCR as described previously . The genetic environment of bla NDM-4 was studied by PCR mapping and sequencing Carnitine palmitoyltransferase II as described previously . Conjugation assay and plasmid study Plasmid transfer was attempted by conjugation, using E.coli J53 as the recipient, as described previously . Plasmid DNA, isolated from E.coli, was obtained by the alkaline lysis method and was used as a template in PCR analysis with primers that are specific for bla NDM and bla CTX-M. To rule out chromosomal DNA contamination the template was used to amplify an internal fragment of the house-keeping recA gene. A PCR-based replicon typing method was used to identify the incompatibility group .