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: Enterobacter cloacae, E. hormaechei and related subspecies remain the most clinically relevant among the Enterobacter cloacae complex (ECC). Carbapenemase-producing ECC strains are increasingly identified in hospital-acquired infections and usually belong to four main multilocus sequence types (MLST STs) named ST114, ST93, ST90 and ST78. Instead, ST182 has been sporadically reported among E. hormaechei strains, and recently, outbreaks of bla_NDM-producing ST182 clonal strains have emerged. Herein, we aimed to investigate the presence of ST182 and explore its evolution and modes of bla_NDM acquisition. Methods: A phylogenetic analysis of 646 MLST STs identified among 4685 E. hormaechei whole-genome sequencing (WGS) assemblies deposited in public repositories was performed, as well as an in silico comparative and phylogenomic analyses for 55 WGS assemblies of ST182. bla_NDM-harboring contigs were also compared to published plasmid sequences. Results: ST182 E. hormaechei strains were recovered from patients on five continents during 2011–2021. They were divided into three major genomic clusters, comprising a separate clonal complex with six other STs. In 30 out of 55 ST182 WGS assemblies, bla_NDM-harboring structures were identified that were similar to the plasmids predominant in Gram-negative bacteria, harboring resistance genes to multiple antibiotic classes and virulence genes. No associations between the genomic clusters and the country/continent of isolation or the presence and the plasmid types of the bla_NDM-harboring contigs were observed. Conclusions: Our findings show that ST182 E. hormaechei strains have been identified in the past decade worldwide; 54.5% of them carried diverse bla_NDM genetic structures, suggesting recent acquisition of the bla_NDM alleles. Thus, bla_NDM-harboring ST182 is an emerging multidrug-resistant and virulent lineage in ECC strains that requires close monitoring.

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.

Recent reports indicate a rise in multidrug-resistant (MDR) Proteus mirabilis in One Health settings, yet global data on its molecular epidemiology remains limited. Herein, an extensively drug-resistant (XDR) P. mirabilis strain was analyzed to determine its genomic make-up and evolutionary history. A clinical isolate, designated Pm GR-164, was consistently recovered from pus specimens obtained from a patient hospitalized at a tertiary care hospital in Athens, Greece. Identification, antimicrobial susceptibility testing, carbapenemase content determination by lateral flow immunoassay and PCR, multilocus-sequence typing, and whole-genome sequencing were performed. Bioinformatic tools were used for prediction of antimicrobial resistance genes, virulence factors, mobile genetic elements, phage sequences, and chromosomal mutations conferring resistance to quinolones. Comparative genomics of the carbapenemase-carrying structures and phylogenomic analyses were also conducted. Pm GR-164 exhibited an XDR phenotype, and its genomic analysis revealed the presence of several acquired antimicrobial resistance, virulence, and phage genes on its chromosome, and one plasmid. The bla_VIM-1 gene and the bla_OXA-48/bla_CTX-M-14-carrying module were located at different chromosomal loci, indicating separate integration events. Mutations in the GyrA, GyrB and ParC subunits were also identified. Pm GR-164 was assigned to ST269, showing a closer phylogenomic relatedness to MDR ST269 strains from Germany compared to those from the USA and UK. This study expands the understanding of the dissemination of MDR P. mirabilis strains. Continuous global genomic surveillance is essential to monitor the emergence and dissemination of XDR P. mirabilis ST269 strains co-producing VIM-1 and OXA-48 carbapenemases, and to characterize the associated genetic structures.

Background: Carbapenemase-producing Enterobacter cloacae complex (ECC) are increasingly identified in hospital-acquired infections. They usually belong to four main multilocus sequence types (STs) named ST114, ST93, ST90, and ST78. Instead, ST182 has been sporadically reported, and recently outbreaks of blaNDM-producing ST182 strains have emerged. Herein, we aimed to investigate the presence of ST182 and explore its evolution and modes of blaNDM acquisition. Methods: A phylogenetic analysis of 646 MLST STs identified among 4,685 E. hormaechei WGS assemblies deposited in public repositories was performed, as well as an in silico comparative and phylogenomic analyses for 55 WGS assemblies of ST182. blaNDM-harboring contigs were also compared to published plasmid sequences. Results: ST182 E. hormaechei strains were recovered from patients in five continents during 2011-2021. They were divided into three major genomic clusters, comprising a separate clonal complex with six other STs. In 30 out of 55 ST182 WGS assemblies, blaNDM-harboring structures were identified similar to plasmids predominant in Gram-negative bacteria, harboring resistance genes to multiple antibiotic classes and virulence genes. No associations between the genomic clusters and the country/continent of isolation, the presence and the plasmid types of the blaNDM-harboring contigs were observed. Conclusions: Our findings show that ST182 E. hormaechei strains were identified the past decade worldwide; 54.5% of them carried diverse blaNDM genetic structures, suggesting recent acquisition of the blaNDM alleles. Thus, blaNDM-producing ST182 is an emerging multidrug-resistant and virulent lineage in ECC that requires close monitoring.