First report of multi-drug resistant, VEB-6 producing Proteus mirabilis isolates from Italy

Introduction: The wide diffusion of extended spectrum β-lactamases (ESBLs) in Enterobacteriaceae is a matter of concern, giving their high prevalence in clinical settings and the frequent association with resistance factors active on other antibiotic classes. Even if the majority of clinically relevant ESBLs are those of the TEM-, SHV- or CTX-M- families, several different ESBLs have been reported. VEB-type ESBLs have been initially detected in 1996 in a clinical isolate of Escherichia coli from a Vietnamese infant hospitalized in France. Genes encoding VEB-type enzymes are usually located on integrons, that could explain their diffusion in multiple species including several Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii and Achromobacter xylosoxidans. In this work we describe the first cases of infections caused by Proteus mirabilisproducing a VEB-type ESBL in Italy.Materials and Methods: A total of 37 ESBL+ P. mirabilis isolated from blood cultures of patients admitted to the IRCCS Arcispedale Santa Maria Nuova of Reggio Emilia during the period March 2011 – December 2016 have been analysed. Identification at the species level was obtained by conventional methods. Antimicrobial susceptibility testing was performed by the Phoenix automated instrument and confirmed by reference broth micro-dilution method. Genotyping was carried out by using RAPD and PFGE. Whole genome sequencing (WGS) of the VEB+ isolates was performed using MiSeq and a paired-end approach. Antibiotic resistance genes were detected on WGS data using the ResFinder web-tool. Gap-closing was performed by a PCR approach followed by Sanger sequencing of the obtained amplicons.Results: Four out of 37 ESBL+ P. mirabilis isolates tested positive for a blaVEB-type gene by PCR. These isolates expressed a multi-drug resistant (MDR) phenotype, including penicillins, cephalosporins, fluoroquinolones, aminoglycosides and trimethoprim/sulfamethoxazole. Data from WGS demonstrated that all isolates carried a complex array of resistance genes including qnrA1,dfrA1, sul1 and sul2, several aminoglycoside resistance genes and theblaVEB-6 ESBL gene. PCR experiments and Sanger sequencing revealed that in all isolates the blaVEB-6 was located on a class I integron embedded in the MDR SGI1-V genomic island. Results from both RAPD and PFGE revealed different patterns, suggesting the presence of a multi-clonal population. Conclusions: To our best knowledge this is the first report of VEB-producing P. mirabilis isolates in Italy. The detection of this β-lactamase in four unrelated isolates obtained in a time window of > 4 years suggests the need to set-up surveillance studies to assess the actual prevalence of this resistance gene in invasive isolates of P. mirabilis in our country.