Comparative Genomics of an Emerging Multidrug-Resistant blaNDM-Carrying ST182 Lineage in Enterobacter cloacae Complex

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 blaNDM-producing ST182 clonal 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 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. blaNDM-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, blaNDM-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 blaNDM-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 blaNDM genetic structures, suggesting recent acquisition of the blaNDM alleles. Thus, blaNDM-harboring ST182 is an emerging multidrug-resistant and virulent lineage in ECC strains that requires close monitoring.

Instead, ST182 ECC isolates have been sporadically reported worldwide from clinical specimens [4][5][6][7][8][9][10].In Europe, an ST182 bla NDM-4 -producing isolate from a Czech patient previously hospitalized in Sri Lanka was reported for the first time in 2012 [11].Later on, in 2016, ST182 caused an outbreak of bla NDM-4 -producing ECC strains in the same country [12].Additionally, a recent study of multidrug-resistant ECC isolates from Lebanon has shown that ST182 was the second most frequent ST accounting for 10.4% of the ECC isolates [13].Lately, the largest European dissemination of ECC NDM producers has been reported in Greece and the outbreak was caused by an ST182 clonal strain [14].Whole-genome taxonomic analysis of two bla NDM-1 -producing strains recovered during the outbreak (EC-ML559 of MLST ST182 and EC-ML621 of ST2143, a single-locus variant of ST182) revealed that both strains were assigned as E. hormaechei [15].In silico prediction of components of the bacterial cell surface and genomic islands showed the presence of various virulence factors and resistance genes to several antimicrobial classes, as well as differences in the plasmids carrying β-lactamase genes [15].
In the present study, we aimed to investigate the presence of whole-genome sequencing (WGS) assemblies of ST182 E. hormaechei in public databases and explore their characteristics, geographic distribution and evolution.For this purpose, we have compared in silico the WGS assemblies of ECC isolates of ST182, including plasmid types, and antimicrobial resistance and virulence genes.Furthermore, the bla NDM -harboring contigs of the WGS assemblies of the isolates were compared with published plasmid sequences to explore the plausible modes of acquiring bla NDM alleles.

Bacterial Strains, Whole-Genome Sequences and Phylogenetic Analysis of ECC Isolates
The WGS assemblies retrieved from public databases have been obtained from E. hormaechei isolates (n = 4685), which belonged to 646 MLST STs.In this dataset, the most prevalent STs were ST171 (n = 396), ST93 (n = 244), ST78 (n = 220) and ST114 (n = 208), whereas ST182 (n = 55) ranked at position 16 (Figure 1a).By implementing the goeBURST algorithm and PHYLOViz analysis based on the MLST allelic profiles, the possible phylogenetic relationships between STs were obtained.Of the 646 MLST STs, 400 MLST STs (3953 isolates) were clustered into 74 CCs, whereas the remaining 246 STs (732 isolates) were singletons (i.e., each group comprised one ST) (Table S1).ST182 comprised a separate lineage in the phylogenetic tree, being in the same CC as ST98, ST710, ST1611, ST1752, ST2143 and ST2608 (Figure 1b).Among the nucleotide sequences of the 55 ST182 E. hormaechei WGS assemblies, there were 4554 SNPs, and the overall mean distance was 0.1013 (Figure S1).Phylogenomic analysis of the WGS assemblies has revealed that the strains were distributed into three genomic clusters (sublineages); cluster A (n = 37), cluster B (n = 10) and cluster C (n = 8) [Figures 2, S1 and S2; Table 1; Table S2].The 55 ST182 WGS assemblies were collected mainly from Asia (n = 17), Europe (n = 17) and North America (n = 14) but also from Africa (n = 4) and South America (n = 3), while 30 of them carried bla NDM genes (Table 1; Table S2).The first WGS assemblies of ST182 strains with no bla NDM genes were identified in the United Kingdom collected in 2002 and 2006, which belonged to cluster B. No bla NDM genes were identified in nine out of ten cluster B isolates, whereas one isolate collected from India carried bla NDM-1 .Both bla NDM carriers and strains with no bla NDM genes were recovered annually from 2011 and onwards (Figure S3).
(n = 4) and South America (n = 3), while 30 of them carried blaNDM genes (Table 1; Table S2).The first WGS assemblies of ST182 strains with no blaNDM genes were identified in the United Kingdom collected in 2002 and 2006, which belonged to cluster B. No blaNDM genes were identified in nine out of ten cluster B isolates, whereas one isolate collected from India carried blaNDM-1.Both blaNDM carriers and strains with no blaNDM genes were recovered annually from 2011 and onwards (Figure S3).

In Silico Identification of Plasmids, Antimicrobial Resistance and Virulence Genes of the ECC NDM-Harboring Isolates
The characteristics and the predictions for the presence of plasmids, antimicrobial resistance and virulence genes of the 30 bla NDM -carrying WGS assemblies of ST182 are shown in Tables S3 and S4.All isolates were predicted to carry plasmids predominant in Gram-negative antibiotic-resistant strains, belonging to several incompatibility groups, such as IncX3, IncFII/IncFIB, IncHI2, IncHI2A, IncL, IncM, IncN, IncN3, IncR, IncX5, Col440I and Col440II replicon-type plasmids.No associations were observed between the country/continent of isolation or the presence of the bla NDM plasmid types and the genetic clusters (Figures 3 and S2, Table S5).

Genetic Background of bla NDM and Plasmid Analysis
In three ST182 strains (M515, MY196 and AZ 664), a bla NDM-1 gene was located on two different contigs of the WGS assemblies.BlastN comparisons of the bla NDMharboring contigs revealed the presence of genetic structures showing 100% identities with regions of six different plasmid types; an IncX3 (pNDM-HN380), three different IncFII (pKOX_NDM-1, pGUE-NDM, pKPX-1), an IncA/C (pM214_AC2) and an IncN2 (pJN24NDM) (Tables S4 and S5; Figures 3, S3 and S4) [24][25][26][27].The most prevalent plasmidic sequences were found in 16 strains and distributed into clusters A and C, which were similar to the IncX3 replicon-type K. pneumoniae pNDM-HN380 from China [24].The bla NDM-4 -encoding plasmid pEncl-922cz of the incompatibility group IncX3 from the Czech Republic has been published previously [11].pEncl-922cz was identical to the respective sequences of bla NDM-4 -encoding plasmids recovered in the same hospital during 2016 (such as strain Encl-44578 included in the present study) [12] but differed by the insertion of a Tn3-like transposon downstream of the topB gene compared with pNDM-HN380 and other IncX3 replicon types, such as the bla NDM-5 -producing K. pneumoniae pNDM-MGR194 from India [28].

Discussion
The ECC mainly comprises six Enterobacter species (E.asburiae, E. cloacae, E. hormaechei, E. kobei, E. ludwigii and E. nimipressuralis); however, the accurate identification of species/subspecies of the genus Enterobacter by routine identification techniques, as well as 16S rRNA and housekeeping genes, has often been inconsistent [1,16].Thus, reclassification of species and subspecies of the genus Enterobacter by phylogenetic studies based on whole-genome DNA-DNA hybridizations and sequencing is challenging and ongoing [1,16,34].A global study of carbapenemase-producing ECC isolates collected during 2008-2014 revealed that the most common identified carbapenemase was VIM MBL, followed by NDM MBL, class A KPC, class D OXA-48 and IMP MBL [8].As observed with other carbapenemase-producing ECC strains, bla NDM -producing ECC strains were also found to mainly belong to four STs, named ST114, ST93, ST90 and ST78.In the present assay, we performed phylogenetic analysis for 646 STs identified among all 4685 E. hormaechei WGS assemblies deposited in public databases, which revealed that ST182 is an emerging lineage in ECC strains.ECC ST182 strains were predicted in silico to harbor plasmids commonly found among multidrug-resistant bacteria, which have acquired antimicrobial resistance and virulence genes, whereas different bla NDM -harboring plasmid types among ST182 ECC strains were distributed in all sublineages.
It has been suggested that global travel has facilitated the rapid spread of NDM from its initial emergence in India [5] to all continents since the importation of NDM producers has been associated with patients having a history of travel [3][4][5][6][7][8]13,[35][36][37].A recent study from Israel has shown that most of the bla NDM -harboring Enterobacterales possessed nine different MGE modules, variably distributed across species and hospitals [35].In another study, the role of mobile genetic elements in the global dissemination of the bla NDM was investigated and it was estimated that bla NDM emerged on a Tn125 transposon before 1985 but only reached global prevalence around a decade after its first recorded observation in 2008 [36].The global dissemination of the bla NDM gene was primarily driven by successive between-plasmid transposon jumps [36].In K. pneumoniae, different trajectories have been shown for the spread of carbapenemase genes, including via one plasmid/multiple lineages (bla OXA-48 -like), multiple plasmids/multiple lineages (bla VIM , bla NDM ) and multiple plasmids/one lineage (bla KPC ) [37].The findings of the current study revealed that E. hormeacei ST182 WGS assemblies deposited in public databases were collected from 2002 to 2021, and during this period, we have identified both WGS assemblies carrying bla NDM and WGS assemblies with no bla NDM genes.No clustering over time was observed for the two groups of strains or the different bla NDM plasmid types, suggesting that strains without bla NDM genes have been distributed globally, and then bla NMD genes were diffused in different genomic clusters.
In a previous study, a common bla NDM genetic structure on plasmid pNDM-U.S. was identified in 14 different ECC clones obtained from six countries spanning four continents [6].Moreover, in some cases, certain mobile genetic elements with carbapenemase genes were found associated with the geographic distribution of clades, clones and species, suggesting that these mobile elements have the ability to move between clones and clades of ECC on a global scale.Several surveys have shown that the bla NDM genes were distributed across a large number of STs in the most prevalent species of Enterobacterales (E.coli, K. pneumoniae and Enterobacter spp.), with no predominant lineages, suggesting that there are no obvious high-risk clones of bla NDM -producing strains [3,4,7].In the current study, bla NDM -harboring contigs showed similarities with six different plasmid types [.The most prevalent IncX3 replicon-type pNDM-HN380-like structures were found in four continents (Asia, Europe, North America and Africa) and diffused into genomic clusters (sublineages) A and C. Similarly, the three different IncFII-type genetic structures were also distributed into different continents and/or genomic clusters: the pKOX_NDM-1 in Asia, Europe, North and South America (cluster A), the pGUE-NDM-like structures in Asia (clusters A and B) and the pKPX-1 in Europe (cluster C).Thus, different bla NDM -carrying plasmids were diffused among strains of the same genomic cluster (sublineage), and on the other hand, the same bla NDM -carrying plasmid could be found in strains belonging to different sublineages of ST182.Therefore, no associations were observed between the genetic clusters and the country/continent of isolation, the presence of the bla NDM alleles and the plasmid types.
Finally, in the present survey, we have performed a phylogenomic analysis and in silico prediction of antimicrobial resistance genes, virulence genes and plasmid types of E. hormaechei ST182 WGS assemblies deposited in public repositories.It should be noted that there are some limitations considering this approach.Firstly, phenotype testing or functional studies are required to determine whether some of the detected genes could confer resistance.Secondly, the existing databases for virulence factors (e.g., Virulence Factor Database-VFDB; available at http://www.mgc.ac.cn/VFs/, accessed on 15 January 2024) do not include data for Enterobacter species.Therefore, there may be additional virulence genes that were not predicted in this survey.Thirdly, plasmid reconstruction was not performed due to short sequencing reads.It should be noted that plasmids are difficult to reconstruct from WGS data.NGS assembly programs tend to return short contigs of heterogeneous origin.On the other hand, alignment-based tools tend to miss diverged plasmids, while learning-based tools often have lower precision.In some studies, the combination of short and long sequencing read WGS strategies has been used [].Another limitation of the present descriptive survey is that it included only sequenced ECC isolates in the NCBI and PubMLST public repositories, which are deposited randomly by users, and there may be a bias towards multidrug-resistant strains; thus, they do not represent the global molecular epidemiology of ECC isolates.Further epidemiological and molecular surveillance studies at a global scale would define the prevalence of the ST182 lineage in ECC strains.

Bacterial Isolates, Genome Sequences and Phylogenetic Analysis
A total of 4685 WGS assemblies of E. hormaechei isolates with available MLST profiles were analyzed.We have retrieved WGS assemblies from the Pathogenwatch database [38] and the PubMLST Enterobacter cloacae database (available at: https://pubmlst.org/organisms/enterobacter-cloacae; accessed on 15 January 2024) [39], which include WGS assemblies from public repositories, such as the European Nucleotide Archive (ENA) and NCBI.Additionally, we have searched the PubMLST database for the presence of the alleles of ST182 (dnaA-49, fusA-20, gyrB-19, leuS-44, pyrG-90, rplB-24, rpoB-32) in other MLST profiles, which were found in 62, 174, 89, 220, 9, 27 and 125 MLST profiles (STs), respectively.Since alleles pyrG-90 and rplB-24 are present in fewer MLST profiles (9 and 27 profiles, respectively) compared with the other MLST alleles of ST182, we have also searched the NCBI database for these alleles (pyrG-90 and rplB-24), and the MLST 2.0 tool (available at: https://cge.food.dtu.dk/services/MLST/(accessed on accessed on 15 January 2024), Center for Genomic Epidemiology, Technical University of Denmark) was used to define the MLST STs so as to retrieve any additional WGS assemblies of ST182.In the final dataset, a total of 55 WGS assemblies of ST182 were included.The genetic relationships and groups of STS were formed by linking all STs that were single-locus variants (SLVs), known as clonal complexes (CCs), by using the goeBURST and the PHYLOViZ version 2.0 software (available at http://www.phyloviz.net/,accessed on 15 January 2024) [40].

Identification of MGEs, Antimicrobial Resistance Genes and Virulence Factors and Plasmid Analysis
BLASTN (available at: https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 15 January 2024), the KmerResistance 2.2 tool and the Mobile Element Finder tool (available at: https://cge.food.dtu.dk/services/;accessed on 25 February 2024) were used to query the sequence assemblies for identification of the bla NDM -harboring contigs, MGEs, plasmids and their relation to antimicrobial resistance genes and virulence factors on the genomes [44,45].k-mer alignment examines the co-occurrence of k-mers between the WGS data and a database of resistance genes and scales well for large redundant databases [21].The GC content of the WGS assemblies was calculated by using the GC-profile (available at: http://tubic.tju.edu.cn/GC-Profile/,accessed on 15 January 2024) [46] and the GCdraw (available at: http://www.endmemo.com/bio/gcdraw.php,accessed on 15 January 2024) online tools.The bla NDM -harboring contigs of the isolates were analyzed with the oriTfinder tool [47] so as to explore the presence of conjugative regions of the self-transmissible MGEs: the origin of transfer site (oriT), the relaxase gene, the gene encoding the type IV coupling protein (T4CP) and the gene cluster for bacterial type IV secretion system (T4SS).A conjugative plasmid must possess all the conjugative regions, whereas a transmissible plasmid must possess at a minimum an oriT and usually a relaxase, but this can be provided in trans [47].. BlastN comparisons of the bla NDM -harboring contigs with plasmid sequences retrieved from the NCBI were performed using the BLAST Ring Image Generator (BRIG) version 0.95 software (available at: https://brig.sourceforge.net/,accessed on 15 January 2024) [48].

Conclusions
In the present survey, we have shown via phylogenetic analysis that the multidrugresistant ST182 is an emerging lineage in ECC strains, representing a distinct clonal complex among bla NDM -carrying ECC strains.E. hormarchei ST182 strains retrieved from public databases were distributed into three genomic clusters (sublineages), which contained strains recovered from five different continents.Both bla NDM -carrying ST182 strains and strains with no bla NDM were diffused into the three ST182 sublineages.Moreover, different plasmid types have been spread among the three genetic clusters of ST182, whereas no associations were observed between the genetic clusters and plasmid types.The diversity of the bla NDM -harboring genetic structures identified among ST182 isolates denotes different routes of bla NDM acquisition into the ST182 clusters worldwide.These findings suggest that ST182 strains without bla NDM genes emerged and spread initially and later on acquired the bla NDM genetic structures via horizontal gene transfer from other bacteria in the recent past.Furthermore, ST182 has already caused outbreaks in the Czech Republic and Greece, and therefore, it has the potential to cause outbreaks worldwide.Vigilance and continuous molecular-typing-based surveillance seem mandatory among ECC strains in order to understand the further expansion of the emerged E. hormarchei ST182 and restrain its dissemination.

Figure 1 .
Figure 1.(a) Phylogenetic relationships of 646 MLST STs (n = 4685 isolates) retrieved by the Phyloviz version 2.0 software.The numbers of the allelic differences are shown on the lines of the branches of the phylogenetic tree.ST nodes colors denote: light green -group founder, light blue -common node, red -selected node (ST182).(b) Assignment of ST182 E. hormaechei into a clonal complex with ST98, ST710, ST1611, ST1752, ST2143 and ST2608 using the goeBURST algorithm.