Whole-genome sequence and resistance determinants of four Elizabethkingia anophelis clinical isolates collected in Hanoi, Vietnam

Four isolates of the opportunistic pathogen Elizabethkingia anophelis were identified for the first time in a Vietnamese hospital and underwent antimicrobial susceptibility testing and genomic characterization by whole-genome sequencing. Complete, fully circularized genome sequences were obtained for all four isolates. Average Nucleotide Identity analysis and single nucleotide polymorphism phylogenetic analysis on the core genome showed that three of the four isolates were genetically distinct, ruling out the hypothesis of a single strain emergence. Antibiotic susceptibility testing highlighted multi-resistant phenotypes against most antimicrobial families, including beta-lactams, carbapenems, aminoglycosides, quinolones, macrolides, amphenicols, rifamycins and glycopeptides. Additionally, in silico genomic analysis was used to correlate the phenotypic susceptibility to putative resistance determinants, including resistance genes, point mutations and multidrug efflux pumps. Nine different resistance genes were located inside a single resistance pocket predicted to be a putative Integrative and Conjugative Element (ICE). This novel ICE was shared by three isolates from two different lineages and displayed similarity with ICEs previously reported in various Elizabethkingia and Chryseobacterium species. The role of such ICEs in pathogenicity, genome plasticity and antimicrobial resistance gene spread within the Flavobacteriaceae family needs to be further elucidated.

medical conditions or COVID-19 infection, are at higher risk of infection 15,17,[24][25][26][27][28] .E. anophelis transmission and global epidemiology remain unclear.Nevertheless, its frequent detection in various areas of the hospital environment (e.g.sinks, faucets, mechanical ventilation equipment, medical devices, healthcare worker hands) and its ability to colonize and persist over long periods of time in water systems (such as tap water pipes) highlight the potential for sporadic outbreak emergence in hospitals 7,19,29 .In the last decade, E. anophelis has been confirmed as the responsible agent of several outbreaks across the world, including in Singapore 25 , the United States 23,30 , Hong Kong 15 and Taiwan 31,32 .Here, we report for the first time the detection of four multi-resistant E. anophelis clinical isolates in a hospital of Hanoi, Vietnam.We carried out antimicrobial susceptibility testing and whole-genome sequencing (short and long reads) to obtain four complete, fully circularized genome sequences.

Bacterial isolates and clinical data collection
In 2020-2021, a cluster of suspected Elizabethkingia infections was detected at the Clinical Institute of Infectious Diseases and the Intensive Care Center and Poison Control of the 108 Military Central Hospital in Hanoi Capital, which is one of the five largest general hospitals in Vietnam (2200 beds and 5000 outpatients/day).Sampling and bacterial isolation were part of the routine laboratory procedures at the Microbiology Department.All procedures were performed in accordance with the ethical standards of 108 Military Central Hospital and the 1964 Helsinki declaration and its later amendments.Therefore, formal consent was not required for this study.Four Vietnamese patients were hospitalized for different reasons: one had been admitted following a traumatic traffic accident (NVH72, aged 55 years old), while the three others (NVB490, NNN508 and VTKC53, aged 67, 84 and 76 years old, respectively) suffered from pre-existing comorbidities that led to multiple secondary infections (including fungal infection, bilateral pneumonia, sepsis and UTI).Four clinical isolates (EAV_NVB490, EAV_NNN508, EAV_VTKC53, and EAV_NVH72) were cultured from bronchial fluid (n = 1) and from sputum (n = 3) on Blood Agar culture medium (Oxoid, UK) at 37 °C.After isolation, isolates were initially identified as E. meningoseptica using the VITEK-2 Compact system (BioMérieux, Inc., NC, USA) when antibiotic susceptibility testing was performed, and as E. anophelis using the VITEK-MS system (BioMérieux, Inc., NC, USA).They were then stored at − 80 °C in Tryptic Soy Broth medium with 50% glycerol, and were subsequently sent to the University of Science and Technology of Hanoi (USTH) for additional genomic characterization.Upon arrival, the four isolates were confirmed to be E. anophelis by 16S rRNA sequencing using the primer pairs 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 518R (5′-ATT ACC GCG GCT GCTGG-3′), followed by whole-genome sequencing (see "Whole-genome sequencing, assembly and annotation").

Whole-genome sequencing, assembly and annotation
Genomic DNA from the four clinical strains was extracted using the DNeasy PowerLyzer Microbial Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.For short-read sequencing, DNA samples were sent to Beijing Genomics Institute (BGI Group, Shenzhen, China), where libraries were constructed and whole-genome sequencing was performed on a BGI-SEQ500 platform using a 2 × 150 paired-end protocol.For long-read sequencing, the Rapid Barcoding Kit (SQK-RBK004) from Oxford Nanopore Technology was used to generate libraries that were loaded and sequenced on a R9.4.1 flow cell (FLO-MIN106D) with the MinION M1kB device (ONT, Oxford, United Kingdom).Base calling, demultiplexing, quality check and trimming were performed by BGI (short reads) and with the default MinKNOW and guppy software programs (long reads).Hybrid de novo genome assembly, combining short and long reads, was carried out with Unicycler v0.4.9 35,36 using default parameters.Statistics about the resulting assemblies were obtained using QUAST v5.1.0 37, and assembly completeness (based on the presence of housekeeping genes for the Flavobacteriales lineage) was checked with BUSCO v5.2.2 38 .Genomic contamination scores were estimated using CheckM v.1.2.2 39 .The four genomes were then annotated with Prokka v1.14.6 40 and the prokaryotic genome Rapid Annotations using Subsystems Technology (RAST) Server [41][42][43] .Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein (Cas) systems were detected using CRISPRCasFinder v4.2.2 44 .Putative antimicrobial resistance genes were predicted in silico using a combination of tools including ResFinder v4.2.3 45 , RGI and the Comprehensive Antibiotic Resistance Database (CARD) v3.1.4 46, and manual inspection of the .gbkfiles from Prokka and RAST annotations.Point mutations were detected by manually aligning the relevant gene sequences against the corresponding sequences of the reference E. anophelis R26 (NZ_CP023401.1).
The Harvest suite 50 was used for core-genome alignment and visualization of a phylogenetic tree based on a Single Nucleotide Polymorphism (SNP)-calling within the core genome.The suite includes the Parsnp tool, highly efficient to align the core-genome of closely related species, and the dynamic visual platform Gingr, used to explore the trees and alignments generated by Parsnp 50 .Three Vietnamese isolates were included in the analysis (EAV_NVB490, EAV_VTKV53 and EAV_NVH72) along with 29 sequences of other E. anophelis isolates, i.e. the totality of the complete genomes that were available on the NCBI repository at the time of writing.The strain CSID_3000521207 (NZ_CP015067.2) was used as the reference core genome.The complete list of the isolates included in the analysis is available in Supplementary Table S1.

Detection and visualization of mobile genetic elements
The genomic islands of the four Vietnamese isolates were predicted by IslandViewer 4 51 , using the .gbkfiles annotated by RAST as input.IslandViewer uses a combination of different methods (IslandPick, IslandPath-DIMOB, SIGI-HMM) to detect genomic regions thought to have horizontal origins, including sequence composition (codon bias, GC content), sequence prediction (mobility genes) and comparative genomics (regions not present in related strains).The prediction of putative Integrative and Conjugative Elements (ICE) was confirmed by manual inspection of the annotation files from prokka and RAST, looking for genes involved in conjugative machineries (such as tra genes, transposases, relaxases, integrases or Type 4 Coupling Proteins) or direct repeats (DR) flanking the integrative element.Linear portions of some ICE were visualized through the Linear Genome Plot tool from Galaxy Version 1.0 by uploading sequencing data on the Galaxy web platform and using the public server (https:// cpt.tamu.edu/ galaxy-pub) to analyze them 52,53 .

Antimicrobial susceptibility testing
The VITEK-2 Compact system (AST card) or/and the Diffusion Susceptibility Test (DDT) method were used to test the susceptibility of the four Vietnamese isolates (EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_NVH72) to 43 antibiotics or antibiotic combinations.The results are detailed in Table 1.Overall, the four E. anophelis isolates were multidrug resistant, as they displayed a resistant phenotype to at least one drug in three or more antimicrobial families.With the VITEK2 Compact system, isolates were resistant to nearly all tested antibiotics: all beta-lactams (including penicillins, cephalosporins, carbapenems and monobactams), fluoroquinolones, aminoglycosides and polymyxins.The only exception was isolate EAV_NVB490 that showed intermediate resistance to the trimethoprim-sulfamethoxazole combination.Similarly, with the DDT method, the four isolates were resistant to nearly all beta-lactams (except the piperacillin-tazobactam combination), fluoroquinolones, aminoglycosides (but not isolate EAV_VTKC53 to amikacin), nalidixic acid, colistin, chloramphenicol and fosfomycin.Conversely, they were sensitive to minocycline and the piperacillin-tazobactam and trimethoprim-sulfamethoxazole combinations.All isolates showed intermediate resistance to teicoplanin, and two isolates (EAV_NVB490 and EAV_NNN508) to vancomycin and tetracycline.Unlike the other three isolates, EAV_VTKC53 was sensitive to amikacin and tetracycline, and was only moderately resistant to erythromycin and rifampicin.Moreover, its growth was inhibited in the presence of ticarcillin, cefoxitin, cefepime, imipenem, gentamicin, levofloxacin and chloramphenicol (asterisks in Table 1).This growth defect did not Vol:.(1234567890 Beta-lactam/Beta-lactamase inhibitor combinations Amoxicillin/clavulanic acid 14 13  15 www.nature.com/scientificreports/result in an inhibition zone diameter wide enough to be considered as sensitive, but was larger compared with the other isolates.
Comparison of the results obtained with the two testing methods highlighted several discrepancies.For example, the trimethoprim-sulfamethoxazole and piperacillin-tazobactam combinations inhibited bacterial growth in the DDT but not in the VITEK2 Compact assay.Similarly, the EAV_VTKC53 isolate was sensitive to amikacin in the DDT assay, but not in the VITEK2 Compact assay.Overall, the resistance profile variability was higher with the DDT assay than with the VITEK2 Compact assay, in which virtually all tested antibiotics displayed high MIC values except for the SXT with EAV_NVB490.

Genome sequences
The complete, circular genome sequences of the four Vietnamese isolates EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_NVH72 were obtained by hybrid de novo assembly.The genome lengths, completeness, GC content and number of coding sequences detected with the two annotation tools (Prokka and RAST) are summarized in Table 2. Genome sizes varied between ~ 4.12 and 4.17 Mb, and completeness scores were > 97% using the Flavobacteriales lineage as reference.Two clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) systems were identified in the EAV_VTKC53 isolate by CRISPRCasFinder, and none in the other three isolates.The four complete, circularized genome sequences and the raw reads have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the bioproject accession number PRJEB49667 (https:// www.ebi.ac.uk/ ena/ brows er/ view/ PRJEB 49667).The isolates EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_NVH72 are represented by the biosamples SAMEA12009938, SAMEA112835271, SAMEA12009939, SAMEA12009940 and the nucleotide accession numbers GCA_927611585.2, GCA_951394135.2, GCA_927611525.2 and GCA_927611535.2, respectively.

Comparative genomics analysis
To confirm the species-level identification of the four isolates and assess their genetic relatedness, we calculated their genome sequence identity (ANI values) relative to the reference genomes of five E. anophelis strains from Table 1.Antimicrobial susceptibility testing of EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_ NVH72 using the VITEK2 Compact system (MIC, in mg/L; left) and DDT (inhibition zone diameter, in mm; right).When possible, the interpretation is indicated between brackets; no interpretation was attempted in the absence of CLSI interpretive guidelines for Acinetobacter spp. or Enterococcus spp.Intermediate (I) and sensitive (S) phenotypes are indicated in bold, but not resistant (R) phenotypes.In DDT, some diameters were marked with an asterisk (*) when the antimicrobial agent had a visible inhibitory effect on bacterial growth, but the inhibition zone was not large enough to consider it as sensitive/intermediate resistant.**MIC of colistin: microdilution assay.various origins in the world, and seven closely related species amongst the Elizabethkingia genus and the Flavobacteriaceae family.The heatmap constructed with the ANI values obtained for each intra-and inter-species pairwise comparison is in Fig. 1 and the matrix of raw values obtained with OrthoANI for each genome pair is in Supplementary Table S2.Using a 95% cut-off value, all four Vietnamese isolates (tagged with a red star in Fig. 1) were confirmed to be E. anophelis (values > 97% with any of the other five E. anophelis strains used as reference).Overall, the ANI values ranged from 70.8 to 99.9% and classified the genomes into three clusters.E. anophelis was clearly separated from all other species, with ANI values between 89 and 92% (for E. occulta, E. miricola, E. bruuniana and E. ursingii that are more closely related) and between 70.8 and 89% (for Chryseobacterium sp.POL2, E. meningoseptica and E. argenteiflava that are more distant).The isolates EAV_NVB490 and EAV_NNN508 displayed an ANI value of 99.99% between them, indicating that they were representing a single strain 54 .However, they displayed ANI values of 97.90% and 98.10% when compared with EAV_NVH72 and EAV_VTKC53.The relationships of the Vietnamese isolates with 29 other E. anophelis genomes were further investigated through core-genome SNP-calling based phylogeny (Fig. 2).Isolate EAV_NNN508 was excluded from the analysis, as it represented the same strain as EAV_NVB490.In the generated phylogenetic tree, EAV_NVB490 was closely related to isolates 2002N07-090 and 090-MNO-R (both recently isolated in Taiwan) and to the cluster of strains CSID (implicated in the Wisconsin outbreak of 2015-2016).EAV_VTKC53 was closer to FMS-007 (isolated in China in 2013) and EAV_NVH72 of 2008N07-201 and 2002C02-176, two other Taiwanese isolates.Overall, Fig. 2 strongly suggests that EAV_NVB490/NNN508, EAV_VTKC53 and EAV_NVH72 originate from separate lineages and have distinct phylogenetic origins.

In silico prediction of resistance determinants and correlation with AST
The genome sequences were used to predict the genetic determinants of antimicrobial resistance in the four E. anophelis isolates by integrating the results of the different annotation and prediction tools.Table 3 lists the identified genes, point mutations and multidrug efflux pumps that could be implicated in the multidrug resistance phenotype of the four isolates.This analysis identified 49, 49, 38, and 52 resistance determinants in EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_NVH72, respectively.Among all the antibiotic resistance genes identified in the four isolates, seven families of genes were involved in resistance to beta-lactams, four in resistance to aminoglycosides, two in resistance to macrolides, tetracyclines and amphenicols, and one in resistance to rifamycins and glycopeptides.Moreover, we identified multiple mutations that could inhibit the action of quinolones and folate pathway inhibitors, as well as a total of 17 different families of efflux pump genes potentially involved in antibiotic transport across the membrane of the four isolates.
All four isolates harbored at least two chromosome-encoded metallo-β-lactamases (MBL), one from the bla B family (subclass B1) and one from the bla GOB family (subclass B3), as well as two class A extended-spectrum β-lactamases (ESBL) (bla CME and bla PER1 ).Intrinsic resistance to beta-lactams within the Elizabethkingia genus is well documented and has been related to the presence of these MBL: BlaB and GOB provide resistance to carbapenems, while CME could be involved in resistance to cephalosporins and monobactams 55,56 .Elizabethkingia spp.members are also the only known bacteria that carry several MBL genes encoded in their chromosome 21 .These MBL display some degree of diversity with an underestimated number of allelic versions in the Elizabethkingia genus 21 .A BLAST analysis of the bla sequences of the four Vietnamese isolates highlighted the presence of the bla B-1 , bla B-11 and bla B-29 , and bla GOB-20 , bla GOB-6 and bla GOB-38 subtypes in EAV_NVB490/NNN508, EAV_VTKC53, The strain CSID_3000521207, used as the reference genome, is highlighted in light-blue in the tree, and the three Vietnamese isolates are tagged with a red star.and EAV_NVH72, respectively.Additionally, EAV_NVB490, EAV_NNN508 and EAV_NVH72 carried the type II MBL ccrA (subclass B1) and the β-lactamase bla OXA-10 (class D), and EAV_NVH72 harbored a unique subclass A2 ESBL.Concerning genes related to resistance to aminoglycosides, aadK was detected in all four isolates, while aacC1 and aacA4 were in EAV_NVB490, EAV_NNN508 and EAV_NVH72, and an additional version of the gene aadK was detected in EAV_NVH72 only.Since EAV_VTKC53 was the only isolate sensitive to amikacin in DDT, it reinforces the hypothetic involvement of aacA4 in amikacin resistance.Two tet(X) genes (encoding two different flavin-dependent monooxygenases that inactivates enzymatically tetracyclines) 57 were identified.
The tet(X) gene has already been detected sporadically in various species of the Flavobacteriaceae family, the potential ancestral source of this tetracycline destructase 58 .EAV_VTKC53, which unlike the others harbored a single version (and not two) of the tet(X) gene, was the only isolate sensitive to tetracycline, with a significantly larger inhibition zone than the other three isolates in DDT.Additionally, the three other isolates also carried the macrolide-resistance tandem genes mefC and mphG that might be involved in the observed resistance to erythromycin: mph(G) is a phosphotransferase that can inactivate macrolides by phosphorylation, and mef(C) is an efflux pump involved in high-level macrolide resistance 59 .It has been shown that the introduction of these tandem genes in E. coli significantly increased the MIC values for several macrolides 59 .EAV_VTKC53, which does not carry these two genes, had a significantly larger inhibition zone diameter compared with the Table 3. Prediction of antibiotic resistance determinants in EAV_NVB490, EAV_NNN508, EAV_VTKC53 and EAV_NVH72, including efflux pumps, point mutations and chromosomally-encoded resistance genes.A short description of the products and the expected resistance phenotypes are also indicated.When relevant, the exact genomic location of a point mutation or the allelic version of a gene (for the blaB and blaGOB families) is given between brackets.The point mutations marked with an asterisk (*) have already been reported to be involved in resistance 16,59,60 .For multidrug efflux pumps, the number of genes identified with this name and annotation in the genome is given between brackets, although they might represent non-identical sequences and therefore different proteins of the same family.
other three isolates (23 mm vs. 0 mm), although this could only be interpreted as an intermediately resistant phenotype.The rifampicin-resistance gene arr, detected in EAV_NVB490, EAV_NNN508 and EAV_NVH72, is a class I ADP-ribosyltransferase that inactivates rifampicin 60 .Arr enzymes are widespread in bacteria and have already been identified in the Bacteroidota phylum 61 .EAV_NVB490, EAV_NNN508 and EAV_NVH72 were resistant to rifampicin but EAV_VTKC53, which lacks arr, was only intermediately resistant.Moreover, these three isolates harbored two cat genes previously detected in the E. anophelis NUHP1, and encoding a type B chloramphenicol-acetyltransferase that is strongly suspected to provide resistance to chloramphenicol by catalyzing its acetylation 62 .Last, all four isolates harbored a vancomycin-resistance gene containing a vanWlike domain that could provide partial or complete resistance to glycopeptides 63 .However, vancomycin usually does not display significant activity against Gram-negative bacteria due to their lipidic bilayer cell membrane.Concerning single-point mutations, all four isolates carried the Ser83Ile mutation in gyrA (encoding the gyrase subunit A) that confers potential resistance to fluoroquinolones 17,64 , and which could explain the observed resistance to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin.For sulfonamides, several point mutations (including Leu10Ile, Thr52Ala, Thr52Ser, Asn171Asp and Met163Ile) were detected in the folP gene that encodes a dihydropteroate synthase, an enzyme necessary for the folate reduction pathway and in which an accumulation of mutations is suspected to increase resistance to folate pathway inhibitors such as the trimethoprimsulfamethoxazole combination 65 .In this study, resistance to SXT was observed in the VITEK2 Compact assay but not in DDT.Among the four isolates, EAV_NVH72 displayed the highest number of folP mutations (n = 9), including three mutations involved in sulfonamide resistance (asterisks in Table 3).
Lastly, approximately 30 putative efflux pump-encoding genes were detected in each isolate, predicted to be multiple copies of 17 different multidrug resistance transporters (Table 3).Several of the main families of membrane transporters were represented, including the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) superfamily, the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance-nodulation-division (RND) family.Although it has not been demonstrated that all these pumps can transport antibiotics, they could contribute at least partly to the observed multi-resistant phenotype of the E. anophelis isolates.

Detection of putative mobile genetic elements
Following assembly and annotation, we predicted genomic islands (GI) and other putative mobile genetic elements (MGE) in the four Vietnamese isolates using IslandViewer (Fig. 3).Three GI larger than 50,000 bp were detected by at least two different prediction methods in isolate EAV_NVB490/NNN508, and two others were predicted in EAV_VTKC53 and in EAV_NVH72.EAV_VTKC53 also harbored four GI of intermediate size (between 20,000 and 50,000 bp).One of the large predicted GI (tagged by a red arrow in Fig. 3) seemed to be located at a very similar location in the genome sequences of both EAV_NVB490/NNN508 and EAV_NVH72, but was absent in EAV_VTKC53.Moreover, nine different antibiotic resistance genes previously described (ccrA, bla OXA-10 , aacC1, aacA4, an additional aadK for EAV_NVH72, tet(X), cat, mefC, mphG and arr) were harbored in this region, ranging from 1,715,338 to 1,742,564 bp in EAV_NVB490/NNN508 and from 1,688,044 to 1,707,580 bp in EAV_NVH72.This close arrangement of multiple resistance genes was indicative of the presence of an Integrative and Conjugative Element (ICE), a type of MGE integrated in the chromosome whose circulation in Elizabethkingia spp.has already been discussed 66,67 .This hypothesis was supported by the identification of a wide range of genes encoding a putative conjugative machinery in the immediate proximity, including an integrase, a relaxase, a Type 4 Coupling Protein and a Type 4 Secretion System formed by multiple Tra proteins (see the annotation files from bioproject PRJEB49667, under accession numbers OX596081.1,OX596082.1,OX596083.1 and OX596084.1).These genes can be used as markers for the detection of putative ICE, because this machinery is required for the ICE excision out of the chromosome and its re-integration inside a new chromosome after transfer via conjugation to a novel recipient strain.It is also their structural arrangement, the type of excision and conjugation mechanism and the integration target site that defines the type of the ICE, as described by Xu et al. 66 in their classification of the ICE of E. anophelis into the three types I, II and III.A linear map of this newly discovered putative ICE in EAV_NVB490/NNN508 and in EAV_NVH72 is illustrated in Fig. 4. The structure and the cargo genes were quite similar in the three isolates, despite some minor differences (e.g. the presence of the additional aadK gene in EAV_NVH72).Furthermore, the architecture of the modular genes was very similar to the type III described in 66 , featuring seven tra genes (traAEGJKMN) flanked by the relaxase (annotated as Mobilization protein BF0132) and the T4CP (annotated as Mobilization protein BF0133).However, an additional gene traF was present between traE and traG in both versions of the ICE detected here.They also shared the same integration site, a gene coding for a transfer RNA (tRNA), in this case specifically a tRNA-Glu-TTC gene.Their integrase was a tyrosine recombinase annotated as XerC, present in two copies.Also, a tandem of transposase genes annotated as ISWz1 and ISMno24 of the IS91 family was present in multiple copies in both ICE (seven times in ICEEaIII(NVB490/NNN508) and three times in ICEEaIII(NVH72)).The presence of this conjugative machinery suggests that this ICE is still capable to excise itself to spread to other bacteria through conjugation.Until further characterisation, the two ICEs were named ICEEaIII(NVB490/NNN508) and ICEEaIII(NVH72), based on their predicted type and the code of the associated isolate.Their size was estimated at 76,039 bp and 68,918 bp, respectively, thanks to the identification of a common direct repeat sequence of 17 bp (5′-ATT CCC CTA CGG GCTAC-3′) that marked the beginning and the end of the ICE in all three genomes.

Discussion
In this study, we presented the antibiotic susceptibility profile and genomic characterization of four clinical E. anophelis isolates identified at 108 Military Central Hospital, Hanoi.To our knowledge, this is the first report of E. anophelis isolated in Vietnam and the first time that complete, fully circularized genomic sequences are obtained in the country.However, several outbreaks of E. anophelis have been reported over the last decade in the region 68,69 , including in Taiwan 26 , Hong Kong 15 , Shanghai 70 and Fujian 71 .Moreover, Elizabethkingia spp.have already been detected in several South-East Asian countries (Singapore, Malaysia, Indonesia, China, Thailand and Cambodia), but never in the Philippines, Brunei, Myanmar, Laos and Timor-Leste 68 .In Vietnam, Hoa and Hai recently reported the detection of an E. meningoseptica isolate in a patient who underwent liver transplantation at the 108 Military Central Hospital 72 , but this is the only previous report of an Elizabethkingia spp.infection in Vietnam, and genome sequencing was not carried out to confirm species identity.
Here, the four isolates were initially identified as E. meningoseptica using the VITEK-2 Compact, and were later re-assigned to E. anophelis.For many years, the default databases of several mass spectroscopy identification systems, including VITEK-MS and Bruker MALDI Biotyper (Bruker Daltonics, Germany), could not accurately distinguish members of the Elizabethkingia genus, leading to the false-positive detection of E. meningoseptica in most cases 15,73 .Recent global retrospective sequencing analyses of archived clinical specimens showed that Elizabethkingia spp-linked bacteremia was predominantly caused by E. anophelis and not by E. meningoseptica 17,[73][74][75] .From our study, we reiterate the need to update the default databases of current automated identification systems for Elizabethkingia spp. as well as the crucial role of sequencing-based methods for reliable clinical diagnostics.
The four Vietnamese patients suffered upon admission from multiple secondary infections (including pneumonia, sepsis and UTI).The routes of transmission of this pathogen remain poorly understood, but infections are mostly detected in patients with underlying conditions and a prolonged hospital stay 76 .Even though no obvious source of E. anophelis contamination has been identified in this study, we suspect the infection by opportunistic E. anophelis to have occurred during their hospital stay, due to the pre-existing co-morbidities and a weakened immune system.Therefore, we cannot exclude the persistence of at least three different E. anophelis populations in the Hanoian hospital environment, leading to a risk of occasional nosocomial transmission.
The isolates EAV_NVB490 and EAV_NNN508 presented similar phenotypic resistance profiles, almost identical genome sizes (4,139,197 bp and 4,139,199 bp), and identical GC content, bla B and bla GOB alleles, point mutations and predicted resistant determinants.Moreover, these two isolates were from samples collected from two patients in adjacent units of the Clinical Institute of Infectious Diseases only five days apart (02/04/2021 and 07/04/2021).Therefore, we hypothesized that these two isolates might represent a single strain, probably resulting from an intra-hospital contamination.The ANI analysis indicated that all four isolates belonged to E. anophelis and that EAV_NVB490 and EAV_NNN508 shared a sequence similarity of 99.99%, confirming that these two genome sequences represented a single strain shared by two patients 54 .Conversely, EAV_NVB490/ EAV8_NNN508 shared only 97.90% similarity with EAV_NVH72 and 98.10% similarity with EAV_VTKC53, suggesting distinct origins.The core-genome SNP analysis (cgSNP) confirmed that these three isolates belonged to separate lineages of the generated phylogenetic tree, ruling out the hypothesis of a single strain emergence.It should be noted that the cgSNP phylogenetic analysis includes only essential genes that are mostly inherited vertically; hence, their mutations rate is lower and confers a stronger signal-to-noise ratio for inferring phylogeny 50 .This type of analysis is therefore more reliable than ANI and quite suitable to the Elizabethkingia genus, because the Flavobacteriaceae species are subject to frequent genomic re-arrangements due to a large diversity of ICE providing a high degree of genome plasticity.
Overall, the four Vietnamese E. anophelis isolates were resistant to most of the tested antibiotic families.A multi-resistant phenotype is not uncommon within the Elizabethkingia spp., and many studies already reported E. anophelis intrinsic resistance to different antibiotic families, such as beta-lactams and aminoglycosides 3,15,17,22,70 .Conversely and in agreement with our findings, E. anophelis is susceptible to minocycline, trimethoprim-sulfamethoxazole and piperacillin-tazobactam.Therefore, these antibiotics are the first choice for multi-resistant E. anophelis infections because most strains exhibit sensitivity to at least one of these combinations 17,23,26,70,73,75 .Importantly, we observed multiple discrepancies between the VITEK2 Compact and the DDT results.The poor concordance between testing methods in the Elizabethkingia genus has already been discussed 34,77 .Susceptibilities determined by disk diffusion, gradient diffusion (E-test) and other automated methods often do not agree with the MIC values obtained using the broth dilution method, especially for some antibiotics including trimethoprim-sulfamethoxazole, piperacillin, vancomycin, tigecycline and ciprofloxacin 34 .As dilution-based methods (e.g.broth microdilution) are considered the gold standard for antimicrobial susceptibility testing in E. anophelis, the DDT results should be considered less reliable and should be interpreted with caution, especially for therapeutic purposes 77 .
The interpretation of the antimicrobial susceptibility testing data was limited also by the fact that neither established breakpoints (MIC values) nor inhibition zone diameter guidelines (disk diffusion assays) are available for Elizabethkingia spp.This lack of standard interpretation criteria has not been addressed yet.Traditionally, antibiotic susceptibility of Elizabethkingia spp.has been reported based on the MIC breakpoints of 'Other Non-Enterobacteriaceae' and on the inhibition zone diameters of Acinetobacter spp.and Enterococcus spp.from  Testing guidelines.However, this is far from optimal and could be misleading.Caution is required when defining Elizabethkingia spp.strains as resistant, intermediate resistant or sensitive because this interpretation is not based on validated criteria and might not reflect the biological reality.We used the traditional standard guidelines for indicative purposes, but these interpretations might be less relevant than the overall differences in inhibition zone diameters among isolates.Following sequencing and assembly, we used genomic data to correlate the AST observations with putative resistance determinants.Even though the lack of interpretive guidelines and the unreliable results of disk diffusion antibiotic sensitivity testing in Elizabethkingia spp.are serious limitations, our results clearly showed that EAV_VTKC53 had a much more sensitive phenotype compared with the other three isolates.Indeed, EAV_VTKC53 was sensitive to amikacin and tetracycline, and displayed intermediate resistance to erythromycin and rifampicin.Moreover, a visible growth inhibition effect and/or a significantly larger inhibition zone diameter was observed for eight additional antibiotics (ticarcillin, cefoxitin, cefepime, imipenem, gentamicin, levofloxacin, chloramphenicol and fusidic acid).Although the inhibition zone diameters were not large enough to characterize EAV_VTKC53 as sensitive to these agents, this isolate was the only one that did not harbor the additional genes potentially involved in resistance to cephalosporins (ccrA), carbapenems (bla OXA-10 ), aminoglycosides (aacC1, aacA4, additional aadK for EAV_NVH72), tetracyclines (additional tet(X)), amphenicols (additional cat), macrolides (mefC, mphG) and rifamycin (arr).
The genomic analysis of these specific genes showed that they were all localized in a small part of the chromosome that matched a predicted GI in EAV_NVB490/NNN508 and EAV_NVH72 sequences.Therefore, we hypothesized the presence of an ICE shared by all isolates except EAV_VTKC53, and providing additional resistance to several antimicrobial agents.The discovery of a complete conjugative machinery (including seven tra genes, a T4CP, a relaxase and an integrase) as well as a 17 bp direct repeat (DR) sequence flanking both ends of the ICE further reinforced this hypothesis.Interestingly, this same DR was previously identified in the ICECspPOL2 in Chryseobacterium sp POL2 (NZ_CP049298.1),another species of the Flavobacteriaceae family closely related to Elizabethkingia spp. 78.Moreover, several genes from the cargo of ICECspPOL2 were also identified inside the putative ICEEaIII(NVB490/NNN508) and ICEEaIII(NVH72), including tet(X), cat, the tandem mph(G)/mef(C) and bla OXA-10 genes.The two new ICEs also displayed structural similarity with several other ICEs type III previously discovered in the Flavobacteriaceae family, including ICEEaIII(5) in E. anophelis NUHP1 (NZ_CP007547.1)and ICEEaIII (10) in E. anophelis NUH6 (ASYJ00000000.1), in addition to ICECspPOL2 78 .Many antibiotic resistance genes described in this study such as aadK, ccrA, cat or tet(X) were reported as well in these ICE, indicating a possible common origin or horizontal gene transfers having taken place between these strains.Interestingly, Fu et al. demonstrated that the ICECspPOL2 in Chrysebobacterium could be transferred experimentally to an Elizabethkingia recipient, highlighting the potential involvement of ICE in the inter-species circulation of antimicrobial resistance genes 78 .Because very few plasmids (as opposed to numerous ICEs) have been identified in the Elizabethkingia and the Chryseobacterium genera, we hypothesize that the involvement of chromosome-integrated genetic elements in the circulation of AMR might have been underestimated, and that ICEs might act as the main vehicle for the transfer of resistance genes (and possibly as well for genes involved in virulence or pathogenicity) within the Flavobacteriaceae family.An example from this study is the detected version of the carbapenem-resistance gene bla OXA-10 , which was previously reported in ICECspPOL2 but had never been reported in E. anophelis before.The newly discovered ICEEaIII(NVB490/NNN508) and ICEEaIII(NVH72), as well as other ICEs present in the four Vietnamese isolates, need to be better characterized to precisely describe their architecture, their transmission mechanism and their transfer frequency, and to trace back their evolutionary history and their circulation worldwide.

Conclusion
This study reported for the first time the detection and isolation of four pathogenic E. anophelis in Vietnam.All four isolates displayed high resistance levels against most of the main antimicrobial agent families; however, the discrepancies observed between antibiotic susceptibility testing methods emphasized the need of appropriate interpretative guidelines for Elizabethkingia spp., especially for clinical decision-making.As this opportunistic pathogen usually displays high lethality rates, its increasingly multi-resistant phenotype is a worrying feature that needs to be closely monitored.A wide range of resistance determinants were detected by WGS analysis, including point mutations, resistance genes and multidrug efflux pumps.However, they were not investigated experimentally and their involvement in E. anophelis resistance phenotype should be precisely assessed.The detection of nine different resistance genes as cargo genes in a single ICE structure suggests that these mobile elements might represent an underestimated vehicle for spreading antimicrobial resistance genes within the Elizabethkingia genus.Two similar versions of this ICE were shared by three isolates of different phylogenetic origins and included resistance genes that had never been reported before in Elizabethkingia spp.Consequently, we cannot exclude the possibility of ICE transmission events that could have occurred between closely related members of the Flavobacteriaceae family.The description of this mobile element has therefore serious implications in terms of global public health, because ICE might play a key role in the future emergence of outbreaks of multi-resistant E. anophelis strains that would be difficult to address.Worldwide monitoring and the use of WGS approaches are critically needed to better understand the spread of the different E. anophelis lineages and the circulation of their mobile genetic elements. https://doi.org/10.1038/s41598-024-57564-3

Figure 1 .
Figure 1.Heatmap showing the ANI values obtained for each pairwise genome comparison.ANI values ranged from 70.80% (light blue) to 99.99% (bright red).A cluster tree displaying the Euclidian distance between sequences is shown on top and on the right.Red stars indicate the four Elizabethkingia anophelis isolates sequenced in this study.

Figure 2 .
Figure 2. Core-genome Single Nucleotide Polymorphisms (cgSNP)-based phylogenetic tree of the Vietnamese isolates EAV_NVB490, EAV_VTKC53 and EAV_NVH72, along with 29 other E. anophelis complete sequences.The strain CSID_3000521207, used as the reference genome, is highlighted in light-blue in the tree, and the three Vietnamese isolates are tagged with a red star.

Figure 4 .
Figure 4. Linear map of the genetic structure of the putative Integrative and Conjugative Elements (a) ICEEaIII(NVB490/NNN508) and (b) ICEEaIII(NVH72).The genes involved in antimicrobial resistance are tagged in red; the genes involved in the ICE conjugative machinery or in genetic mobility are tagged in purple; the other genes (including predicted proteins without annotation) are tagged in grey.