Emergence and transmission of the high-risk ST78 clone of OXA-48-producing Enterobacter hormaechei in a single hospital in Taiwan

ABSTRACT Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes blaKPC-2, blaIMP-8, and predominantly blaOXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a blaOXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harbouring a blaIMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel blaKPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.


Introduction
Carbapenem-resistant Enterobacterales has emerged as a significant global healthcare threat over the past decade, presenting challenges in treating infections due to limited treatment options [1].Among the top five nosocomial species of Enterobacterales causing bloodstream infections, the Enterobacter cloacae complex (ECC) stands out prominently.Within ECC, Enterobacter hormaechei emerges as a predominant pathogen associated with various infections [2].An overall genome-related index suggested that E. hormaechei contains at least five subspecies, including oharae, steigerwaltii, hormaechei, hoffmannii, and xiangfangensis [3].Characterized by its ability to acquire multiple antimicrobial resistance (AMR) genes through horizontal gene transfer from other Enterobacterales, E. hormaechei has increasingly been linked to nosocomial outbreaks [4][5][6].
Notably, carbapenemase-producing E. hormaechei (CPEH), including those producing KPC, NDM, GIM, and IMP enzymes, have been documented in various studies [4,[6][7][8].The OXA-48 carbapenemase was initially identified in a carbapenem-resistant Klebsiella pneumoniae strain in Turkey in 2001 and arrived in Taiwan at the end of 2013 [9].Through acquiring a variant of the epidemic bla OXA-48 -carrying IncL plasmid, pOXA-48, ST11_KL64 K. pneumoniae emerged as one of the primary clones of carbapenemase-producing K. pneumoniae, which peaked during 2013-2015 [10] and has shown a constantly increasing prevalence in hospitals in Taiwan [11].Inter-species transfer of pOXA-48 has been suggested as a contributing factor to the emergence and spread of OXA-48-producing E. hormaechei among companion animals and humans, as evidenced by a recent study in Swiss hospitals [12].Through the acquisition of bla OXA-48 -carrying IncL plasmids, high-risk international clones ST66, ST171, and ST78 of ECC have emerged as the etiological agents of bacteremia in a Spanish hospital [13].
In May 2017, we identified the first OXA-48-producing E. hormaechei isolate from a patient with bacteremia in our hospital.In addition to bla OXA-48 , we isolated several carbapenemase-producing E. hormaechei, including KPC-2-and IMP-8-producing isolates.By January 2021, 22 carbapenemase-producing E. hormaechei (CPEH) were identified out of 67 non-duplicated carbapenem-resistant isolates.To elucidate the molecular mechanisms contributing to the emergence and dissemination of CPEH in our hospital, we utilized conventional profiling methods and next-generation whole genome sequencing techniques, including Illumina and MinION sequencing.This comprehensive approach enabled us to characterize the strains and examine their possible phylogenetic relatedness, shedding light on the dynamics of AMR gene acquisition and spread for this clinically significant pathogen.

E. hormaechei isolates.
Positive cultures indicating carbapenem-resistant E. hormaechei were collected from patients admitted to Tung's Taichung Metro Harbour Hospital, a regional teaching hospital with 24 clinical departments and 1,381 beds.Between January 2017 and January 2021, 67 non-duplicate isolates associated with either colonization or infections were included in this study.Species identification was conducted using the Bruker MALDI Biotyper™ and confirmed via whole genome sequencing, which was based on an average nucleotide identity (ANI) of over 96%, using E. hormaechei (CP017186) as the type strain for species delineation.Antimicrobial susceptibility testing was performed using the Phoenix Automatic Microbiology System (BD Diagnostics, MD, USA), with interpretations based on CLSI breakpoints (M100-S27).The minimal inhibitory concentration of colistin was determined using the broth dilution method.
Pulse-filed gel electrophoresis (PFGE), multilocus sequence typing (MLST), and detection of carbapenemase genes, mcr-9, and the replicon region of IncL and IncHI2 plasmid.Following a standardized protocol for the subtyping of Enterobacteriaceae [14], pulse-filed gel electrophoresis (PFGE) was performed with the CHEF-DR III system (Bio-Rad Laboratories Inc, USA) to determine the clonal relatedness of carbapenem-resistant E. hormaechei (n = 67).The profiles of the XbaI macro-restricted fragments of each isolate were analyzed with the Dice coefficients and the unweighted pair group method with an arithmetic mean algorithm with a 1.5% optimization value and 1.5% position tolerance using the analytic tools provided by the GelCompar II 6.5 software (Applied Maths, Belgium).XbaIdigested DNA samples from Salmonella enterica subsp.enterica serotype Braenderup H9812 was used as molecular size markers.Clusters of PFGE-XbaI were determined in the dendrogram using a 75% similarity cut-off.The sequence types (STs) of the representative strains were determined using the MLST scheme established for E. cloacae [15].This scheme is based on the sequences of seven housekeeping genes: aspC, clpX, fadD, icd, mdh, recA, and purA (http://pubmlst.org/ecloacae).Additionally, bla ACT was amplified using polymerase chain reaction (PCR) with specific primers (supplement Table S1) and subjected to Sanger sequencing for genotyping.Genomic DNA extracted from each carbapenemresistant E. hormaechei (CREH; n = 67) was subjected to the detection of carbapenemase-coding genes, including bla KPC , bla OXA-48 , bla IMP , bla NDM , and bla VIM , and mcr-9 gene by PCR with specific primers as previously described [16,17].The carriage of IncL-type and IncHI2-type plasmids was detected using PCR with specific primers targeting the IncL and IncHI2 replicons, respectively (supplement Table S1).
Whole genome sequencing.The genomic DNA of representative carbapenemase-producing E. hormaechei isolates was extracted and subjected to whole genome sequencing by Illumina MiSeq sequencer (Illumina, San Diego, USA) and Nanopore MinION sequencer (Oxford Nanopore Technologies, Oxford, UK), according to the standard protocol provided by the manufacturer, respectively.Qualified reads yielded from Illumina and MinION were assembled with Unicycler v.0.4.8.The final assembly was polished by Unicycler using Illumina reads, and the rate of minor base-level errors was reduced by Pilon.Assemblies with a size ≤ 1000 kb containing For each non-duplicated isolate, features such as isolation time, location, specimen type, and colonization or infection with CREH are presented.In addition to antibiotic susceptibility testing results, the presence of specific carbapenemase genes (bla OXA , bla KPC , bla IMP ) or the mcr-9.1 gene, as well as the carriage of IncL-and IncHI2-type plasmid replicons, was detected by PCR and is indicated in black.The absence of these genetic loci is shown in light grey.Sequence types (STs) were determined by the MLST scheme for E. cloacae (http://pubmlst.org/ecloacae).Genotyping of bla ACT was performed by sequencing PCR products amplified with specific primers (Table S1).Strains with one of the carbapenemase genes (bla OXA , bla KPC , bla IMP ) were classified as CPEH (n = 22) and highlighted with white text on a black background.Colour coding indicates antibiotic resistance: red for resistant, yellow for intermediate, and green for susceptible.Abbreviations for antibiotics are as follows: AN (amikacin), GM (gentamycin), AM (ampicillin), SAM (Ampicillin-Sulbactam), CZ (cefazolin), CMZ (cefmetazole), CTX (cefotaxime), CAZ (ceftazidime), CRO (ceftriaxone), FEP (cefepime), TZP (Piperacillin-Tazobactam), ETP (ertapenem), IMP (imipenem), MPM (meropenem), CL (colistin), CIP (ciprofloxacin), LVX (levofloxacin), TGC (tigecycline), MI (minocycline), SXT (Trimethoprim-Sulfamethoxazole).
plasmid replicons were extracted from the assembly graph with BANDAGE v.0.8.1.The genome assemblies of E. hormaechei strains in this study, which can be downloaded through the link: https://reurl.cc/34aboX, are publicly available in GenBank under Bio-Project PRJNA791797.
Genome profiling and comparative genomic analysis.Genome assemblies were annotated by RAST (https://rast.nmpdr.org/)and manually curated.Antimicrobial genes were identified with ResFinder, and plasmid incompatibility (Inc) groups were assessed with PlasmidFinder from the Centre for Genomic Epidemiology (http://www.genomicepidemiology.org/).Comparative sequence alignments were performed with Geneious Prime 2022.1.1 (Biomatters, New Zealand).Alignment and visualization of plasmids were performed with BLAST Ring Image Generator (BRIG v.0.95) [18].Mauve alignment and comparative genomics analysis were performed with Geneious Prime.cgSNP analysis.Parsnp 1.2, with default parameters [19], was utilized for core genome alignment, SNP calling, and phylogenetic tree construction in the genome assemblies of carbapenemase-producing E. hormaechei, using ST78 E. hormaechei subsp.hoffmannii strain Eh1 (GCA_009834325.1)as the reference.The resulting cgSNP phylogeny tree was visualized in Geneious Prime, with the corresponding metadata, including isolation time, ST, type of plasmids, and the carriage of AMR genes.

Results
Emergence of carbapenemase-producing E. hormaechei.Over a span of four years, we isolated 67 nonduplicate carbapenem-resistant E. hormaechei (CREH; Figure 1).A significant finding occurred in May 2017 when we first isolated a carbapenemaseproducing E. hormaechei (CPEH) carrying the bla OXA-48 gene from a patient with bacteremia.Subsequently, OXA-48-producing E. hormaechei strains were consistently found in various specimens associated with colonization or infections.Additionally, starting in 2019, we observed the co-occurrence of the mcr9 gene with bla OXA-48 in several isolates.By January 2021, out of the 67 CREH isolates, 22 were identified as carbapenemase-producing E. hormaechei (CPEH).These included one KPC-positive, one IMP-positive, one IMP-OXA-48-positive, and 19 OXA-48-positive strains (Figure 2a).The intensive care unit (ICU) was the location where OXA48-and mcr9-positive E. hormaechei isolates were most frequently encountered.Moreover, most OXA-48- positive E. hormaechei were isolated from urine, sputum, or pus, while mcr9-positive isolates were predominantly collected from pus specimens (Figure 2b).
Acquisition of the endemic bla OXA-48 -carrying IncL plasmid by E. hormaechei ST78, ST66, and ST90.Based on the cut-off of 75% similarity of PFGE-XbaI pulsotypes, the majority of the 67 CREH isolates were grouped into two major clusters, I and II (Figure 1).The sequence types correlated to the PFGE cluster I and II were ST78 and ST90, respectively.Of 22 CPEH strains, 18 belonged to ST78, and 2 were in the ST90 cluster.The remaining two CPEH isolates were 17CRE33, classified as ST66 and CRECL48, for which the ST could not be determined due to poor sequence quality.Regardless of the sequence type, all the OXA-48-positive E. hormaechei carried a bla OXA-48 -carrying IncL plasmid, designated as pOXA48-CREH (Figure 3a).This plasmid, sized at 66,276 bp, exhibited close relatedness to the endemic bla OXA-48 -carrying IncL plasmid frequently carried by ST11 K. pneumoniae in Taiwan.Like OXA-48 K. pneumoniae, the IncL plasmid harboured bla OXA-48 within a Tn1999.2transposon in E. hormaechei.An inversion occurred between two copies of IS1999, leading to a duplication of IS1 on pOXA48-CREH (Figure 3b).

Discussion
More than 80% of the carbapenemase-producing E. hormaechei isolates collected in this study were clustered into a group belonging to ST78 (Figure 1).ST78 is recognized as a high-risk international clone with a unique ability to acquire various antimicrobial resistance (AMR) gene-carrying plasmids [6].Notable examples include the bla NDM-7 -IncX3 plasmid in isolates from Spain [6] and the bla IMP-1 -carrying class I integron on IncW and IncFIB plasmids in isolates from Japan [25].The first OXA-48-producing E. hormaechei isolate was identified in May 2017.The bla OXA-48 -carrying IncL plasmid acquired by E. hormaechei was nearly identical to the endemic plasmid (pOXA48-KP) frequently found in ST11_KL64 K. pneumoniae in Taiwan [24].The IncL plasmid exhibits high plasmid stability and strong conjugal transfer ability in vitro [26] and can also be horizontally transferred between enterobacteria within the gut microbiota of colonized patients [27].The conserved conjugal transfer region of pOXA48-CREH (supplement Figure S3a) supports a plausible scenario in which ST78 E. hormaechei acquired the pOXA48-KP plasmid from K. pneumoniae through co-colonization within patients.Subsequently, an ST66 E. hormaechei acquired pOXA48-CREH, potentially transferred from an OXA-48-producing ST78 strain.Nevertheless, clonal expansion of pOXA48-CREH-carrying lineages was predominantly noted in ST78 E. hormaechei, beginning in 2018.By 2019, pOXA48-CREH was transmitted from ST78 to ST90, another high-risk clone of E. hormaechei, but it disseminated on a small scale in the hospital setting.Despite ST66, ST78, and ST90 lineages all pOXA48-CREH, the primary lineage responsible for the clonal dissemination of carbapenemase-producing E. hormaechei in the regional teaching hospital was ST78 (Figure 8).The capability of ST78 E. hormaechei to acquire multi-drug resistance plasmids was further demonstrated by the isolation of 18CRE28 in 2018, which acquired a bla IMP-8 -carrying pIncHI2 plasmid, and the KPC-2-producing strain CRECL55 in 2020.Notably, CRECL55 harboured a novel bla KPC-2 -carrying pIncFII plasmid, likely originating from E. coli and a variant of the pIncC plasmid from ST11 K. pneumoniae (Figure 7).Continuous monitoring and genomic surveillance are crucial for targeted interventions to curb the spread of this high-risk clone.
Since its identification in 2019, mcr-9.1, a plasmidmediated colistin resistance gene, has rapidly spread among clinical carbapenem-resistant Enterobacterales, primarily via IncHI2 plasmids [28].Co-carriage of mcr-9 and bla VIM on pIncHI2 plasmids have been identified in E. hormaechei isolates in Czech Hospitals [29].The acquisition of the mcr-9 gene cassette between IncHI2 plasmids is thought to be mediated by an IS903dependent mechanism.In our study, mcr-9.1 was predominantly detected in ST90 E. hormaechei and occasionally found in ST78 and ST66 isolates.The conserved mcr-9.1 genetic context was carried by pIncHI2 plasmids, which vary in size from 264 to 450 kb and contain a diverse array of AMR genes (Figure 6) as well as genes for conjugal transfer (supplement Figure S2a and S3b).Although the qseBC two-component system is required to express mcr-9.1,all the mcr-9.1-positiveisolates in this study were qseBC-negative and consequently exhibited phenotypic susceptibility to colistin.Despite the minimal phenotypic impact of the mcr-9.1-carryingpIncHI2 plasmids on colistin resistance, the insertion of the bla IMP-8 -containing class I integron on pIncHI2 plasmids in ST78 E. hormaechei (Figures 4 and 6) significantly enhances the advantage of this international high-risk clone under antimicrobial pressure.Co-carriage of mcr-9.1-pIncHI2plasmid with a carbapenemase-encoding plasmid, such as pOXA48-CREH, has also been demonstrated in a clonal outbreak in a tertiary hospital in China where the majority of ST78 E. hormaechei coharboured an IncFII-type plasmid encoding the class B metallo-β-lactamase NDM-1 and the pIncHI2-mcr-9.1 plasmid [5].Regardless of sequence type, all the representative CPEH strains had an AmpC β-lactamase gene bla ACT in their chromosomes.The intrinsic presence of a constitutive AmpC β-lactamase gene in Enterobacter species has been shown to confer resistance to ampicillin, amoxicillin, and first-and second-generation cephalosporins, such as cefazolin and cefmetazole [30].This study revealed a link between specific bla ACT subtypes and the sequence type of E. hormaechei: bla ACT-5 in ST78 and bla ACT-15 in ST90 (supplement Figure S1).This linkage reflected the intrinsic inheritance of the AmpC β-lactamase gene in sub-lineages of E. hormaechei.All representative ST78 and ST90 CPEH strains exhibited resistance to ciprofloxacin and levofloxacin (Figure 1).However, no mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC were identified in their chromosomes.Instead, the plasmid-borne genes qnrA1 and aac(6 ′ )-Ib-Hangzhou, which encode Qnr proteins and an aminoglycoside acetyltransferase variant, respectively, conferred resistance to fluoroquinolones in ST78 or ST90 CPEH strains (Figure 4).
From May 2017 to January 2021, 22 strains were identified as carbapenemase-producing E. hormaechei (CPEH), with the presence of bla KPC-2 , bla IMP-8 , and predominantly bla OXA-48 .Notably, all OXA-48-positive strains carried a bla OXA-48 -carrying IncL plasmid.Phylogenetic and comparative genomic analyses revealed the acquisition of a ∼66-kb bla OXA-48 -carrying IncL plasmid by strains of different sequence types, including ST78, ST66, and ST90.Apart from duplication of IS1, the bla OXA-48 plasmid carried by E. hormaechei was nearly identical to the endemic bla OXA-48 plasmid found in ST11_KL64 K. pneumoniae (Figure 3).Since the end of 2013, OXA-48-producing K. pneumoniae has emerged and disseminated in Taiwan, including this regional teaching hospital [31].The IncL-type bla OXA-48 -carrying plasmid is highly conjugative.The pOXA48-KP-like plasmid was also identified in a recent outbreak of OXA-48-producing Salmonella Goldcoast from December 2020 to January 2021 in central Taiwan [32].Furthermore, a recent study in Switzerland demonstrated a potential transfer of a ∼63-kb bla OXA-48 -carrying IncL plasmid between K. pneumoniae and E. hormaechei isolates from human and animal origins [12].Horizontal transfer of pOXA-48-like plasmids between K. pneumoniae, E coli, and E. cloacae could also occur through co-colonization or co-infection within the same patients [33].These findings collectively indicate the crucial role of this highly transferable plasmid as the main vehicle for the global dissemination of this carbapenemase gene.The inter-species spread of the bla OXA-48 -48-carrying IncL plasmid among Enterobacterales underscores the urgent need for active surveillance of carbapenemase-producing E. hormaechei, which could be isolated not only from hospital settings but also from animal reservoirs and environments.In 2019, an ST90 E. hormaechei strain acquired pOXA48-CREH, probably from the OXA-48-producing ST78 lineage, and disseminated on a small scale.Through acquiring a novel bla KPC-2 -carrying pIncFII plasmid and a variant of the pIncC plasmid, a KPC-2-producing ST78 E. hormaechei was identified at the end of 2020.Echoing the capability of ST78 E. hormaechei in acquiring various types of drug-resistance plasmids, in this high-risk lineage, an IMP-8-producing E. hormaechei was initially found in 2018 after harbouring a bla IMP-8 -pIncHI2 plasmid.Collectively, ST78 E. hormaechei emerged as the primary driving force behind the transmission of carbapenemase genes in this hospital setting.

Conclusion
The high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH in the hospital setting.This lineage has not only acquired various carbapenemase plasmids, such as a bla KPC-2 -carrying pIncFII plasmid and a bla IMP-8 -carrying pIncHI2 plasmid but has also facilitated the transfer of pOXA48-CREH to other lineages, including ST66 and ST90.Our findings highlight the urgent need for genomic surveillance and targeted interventions to control the spread and evolution of this high-risk lineage.

Figure 4 .
Figure 4. Phylogenomic relatedness, plasmidome, and resistome of representative carbapenemase-producing E. hormaechei strains.A phylogenetic tree was constructed based on cgSNP analysis of representative E. hormaechei strains (n = 14) using E. hormaechei Eh1 (GCA_009834325.1)as the reference.The distance between nodes is presented as the substitution rate per site.The plasmid Inc-type and the carriage of antimicrobial resistance (AMR) genes were determined by Plasmid-Finder and ResFinder from the Centre for Genomic Epidemiology (http://www.genomicepidemiology.org/).The absence of the indicated AMR gene is shown in light grey.

Figure 5 .
Figure 5.Comparison of pIncFIB-CREH.(a) Alignment of pIncFIB plasmids in representative E. hormaechei ST78, ST90, and ST66 strains.(b) Alignment of the class I integron-flanked AMR cassettes on the pIncFIB plasmids identified in representative strains in this study against the Tn3-flanked AMR cassette on the pIncFIB plasmid (CP034755.1) of the ST78 reference strain Eh1.

Figure 8 .
Figure 8. Emergence and transmission of carbapenemase-producing E hormaechei during 2017-2020.The index OXA-48producing E. hormaechei strain emerged in May 2017, potentially transferring the bla OXA-48 -carrying IncL plasmid, likely acquired from ST11 K. pneumoniae, to an ST66 E. hormaechei isolate.Clonal expansion of this OXA-48-producing ST78 lineage has occurred since 2018.In 2019, an ST90 E. hormaechei strain acquired pOXA48-CREH, probably from the OXA-48-producing ST78 lineage, and disseminated on a small scale.Through acquiring a novel bla KPC-2 -carrying pIncFII plasmid and a variant of the pIncC plasmid, a KPC-2-producing ST78 E. hormaechei was identified at the end of 2020.Echoing the capability of ST78 E. hormaechei in acquiring various types of drug-resistance plasmids, in this high-risk lineage, an IMP-8-producing E. hormaechei was initially found in 2018 after harbouring a bla IMP-8 -pIncHI2 plasmid.Collectively, ST78 E. hormaechei emerged as the primary driving force behind the transmission of carbapenemase genes in this hospital setting.