Nosocomial outbreaks of multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) are often reported worldwide, mostly associated with a small number of multilocus-sequence types of E. hormaechei and E. cloacae strains. In Europe, the largest clonal outbreak of bla_NDM-1-producing ECC has been recently reported, involving an ST182 E. hormaechei strain in a Greek teaching hospital. In the current study, we aimed to further investigate the genetic make-up of two representative outbreak isolates. Comparative genomics of whole genome sequences (WGS) was performed, including whole genome-based taxonomic analysis and in silico prediction of virulence determinants of the bacterial cell surface, plasmids, antibiotic resistance genes and virulence factors present on genomic islands. The enterobacterial common antigen and the colanic antigen of the cell surface were identified in both isolates, being similar to the gene clusters of the E. hormaechei ATCC 49162 and E. cloacae ATCC 13047 type strains, whereas the two strains possessed different gene clusters encoding lipopolysaccharide O-antigens. Other virulence factors of the bacterial cell surface, such as flagella, fimbriae and pili, were also predicted to be encoded by gene clusters similar to those found in Enterobacter spp. and other Enterobacterales. Secretion systems and toxin–antitoxin systems, which also contribute to pathogenicity, were identified. Both isolates harboured resistance genes to multiple antimicrobial classes, including β-lactams, aminoglycosides, quinolones, chloramphenicol, trimethoprim, sulfonamides and fosfomycin; they carried bla_TEM-1, bla_OXA-1, bla_NDM-1, and one of them also carried bla_CTXM-14, bla_CTXM-15 and bla_LAP-2 plasmidic alleles. Our comprehensive analysis of the WGS assemblies revealed that bla_NDM-1-producing outbreak isolates possess components of the bacterial cell surface as well as genomic islands, harbouring resistance genes to several antimicrobial classes and various virulence factors. Differences in the plasmids carrying β-lactamase genes between the two strains have also shown diverse modes of acquisition and an ongoing evolution of these mobile elements.

Background/Objectives: Among Enterobacterales, OXA-48-like-producing Proteus mirabilis strains have been scarcely detected. Herein, we characterized a blaOXA-48-harbouring P. mirabilis strain recovered from Greece (Pm GR-1), while phylogenomics and comparative genomics analyses with previously published blaOXA-48 carriers were also assessed. Methods: Characterization of Pm GR-1 was performed by the Vitek® Compact and Mass Spectrometry systems, antimicrobial susceptibility testing, detection of beta-lactamases, multilocus-sequence typing (MLST), and whole-genome sequencing (WGS). In silico prediction of mobile genetic elements (MGEs), genomic islands (GIs), antimicrobial resistance genes (ARGs) and virulence factors (VFs), and phylogenetic, core-genome SNP and comparative genomics analyses were executed using bioinformatic tools. Results: Pm GR-1 was isolated from a urine sample of an outpatient in a Greek hospital. It exhibited a multidrug-resistant phenotype, being susceptible only to amikacin and ceftazidime/avibactam. It co-carried several beta-lactamase genes on the chromosome (blaOXA-48, blaCTX-M-14, blaTEM-1) and a plasmid (blaTEM-2) and several other ARGs, but also mutations associated with quinolone resistance in the DNA gyrase and topoisomerase IV subunits. It belonged to the international clone ST135 that has also been detected among OXA-48-producing P. mirabilis strains from Germany and the USA. Pm GR-1 was genetically related to those from Germany, sharing highly similar MGEs, GIs, ARGs and VFs, including the chromosomal blaOXA-48 genetic structure, the O-antigen locus, the flagella locus, the MR/P fimbriae operon, and the urease gene cluster. Conclusions: To our knowledge, this is the first report from Greece of a blaOXA-48-possessing P. mirabilis strain. The emergence of blaOXA-48 among P. mirabilis strains of the international clone ST135 in different geographical regions is worrying. Close monitoring of these strains is required in One Health settings.