A single Proteus mirabilis lineage from human and animal sources: a hidden reservoir of OXA-23 or OXA-58 carbapenemases in Enterobacterales

In Enterobacterales, the most common carbapenemases are Ambler’s class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

The aim of this study was to characterize at the genomic level a collection of OXA-23-and OXA-58-producing P. mirabilis isolates recovered from human and animal sources from France and Belgium.

Results
A collection of 61 isolates with phenotypes compatible with the production of OXA-23 or OXA-58 was tested using the lateral flow immunochromatographic assay NG-test Carba 5 (NG Biotech, Guipry, France) test, Carba NP test and PCRs. None of the common enterobacterial carbapenemases (OXA-48-like, NDM, KPC, VIM, and IMP) were detected. Nevertheless, among the 61 isolates, 21 were positive for a bla OXA-23 gene and one for a bla OXA-58 gene. These isolates originated from many different areas in France and Belgium (Table 1 & Fig. 1) and were collected over a 4-years period. OXA-23, and OXA-58 CHDLs are weak carbapenem-hydrolyzing enzymes. When they are expressed in E. coli, they confer a slightly reduced susceptibility to carbapenems. Recently, we have identified the first OXA-58-producing P. mirabilis clinical isolate 1091 11 , that was resistant to amoxicillin, ticarcillin and clavulanate-amoxicillin combination. This strain also showed a reduced susceptibility to ertapenem with MIC over the EUCAST screening cut-off for carbapenemase-producing Enterobacterales (CPE) (>0.125 μg/ ml or diameter inhibition zone size <25 mm). Resistance phenotypes of all OXA-producing P. mirabilis are summarized in Table S1. A similar pattern was observed for all isolates with a antibiotic susceptibility pattern of clavulanate-amoxicillin resistance and decreased susceptibility to carbapenem. They were all susceptible to broad-spectrum cephalosporins, fluoroquinolones, tigecycline, fosfomycin and amikacin. Few differences were observed on gentamicin and tobramycin, with few isolates being susceptible to these compounds whereas the others were resistant to both of them.
Resistome of OXA-23-/OXA-58-producing P. mirabilis isolates. WGS of all the OXA-23-and OXA-58-producing P. mirabilis isolates (n = 22) of this study along with OXA-58-producing P. mirabilis 1091, and OXA-23-producing P. mirabilis S4 11,16 were performed using Illumina technology. Resistomes were determined using the Resfinder 3.1 and the CARD database 17,18 . They are summarized in www.nature.com/scientificreports www.nature.com/scientificreports/ of aph(3')-Ia and aac(3')-II), the phenicol resistance gene floR, the lincosamide nucleotidyltransferase gene lnuG, the sulfonamide resistance gene sul2, a streptothricin acetyltransferase gene sat2, and the carbapenem resistance bla OXA-23 gene (Table S2). Accordingly, this strain was selected as reference for further analyses and sequenced using the PacBio technology. Sequencing gave 27,741 reads representing a total of 166 521,740 nucleotides. The genome of P. mirabilis VAC was reconstructed and was 4.08 Mb in size with a GC content of 39% ( Fig. 2A). phylogenetic analysis of bla oXA -containing P. mirabilis isolates. To dive deeper into the understanding of the dissemination of the bla OXA-23 or bla OXA-58 genes, the genome sequences of all sequenced P. mirabilis isolates were compared. In addition, 122 available reference genomes of P. mirabilis from GenBank were also included in the analysis (Table S2). Surprisingly, 22 of the 24 CHDL-producing isolates, including the OXA-23-producing P. mirabilis S4 and the OXA-58-producing P. mirabilis 1091, belonged to the same lineage (Fig. 3). Single nucleotide polymorphisms (SNPs) count revealed that 22 isolates possessed the same background (less than 50 SNPs vs > 2000 SNPs for unrelated clones) confirming that all these isolates belonged to the same lineage. Moreover, despite the fact that three isolates (NEYX, NJFA and LDIU) were branched to OXA-producing lineage, they are not related with an average of 4,200, 4,900 and 5,000 SNPs respectively with the OXA-23/OXA-58-producing isolates ( Fig. 3 and Table S3). Two OXA-producing isolates (P. mirabilis 160A10 and CNR20130297) were not related to the main lineage (>2000 SNPs). Isolate 160A10 and SDUJ01 are close with 185 SNPs (Table S3) whereas isolate CNR20130297 was a singleton.
Of note, a chloramphenicol acetyltransferase gene (cat) and a tetracycline efflux pump encoding gene (tet(J)), both related to the intrinsic resistance to tetracyclines and chloramphenicol of P. mirabilis species were present in all genomes.
the bla OXA-23 gene is carried by a transposon on the chromosome. Attempts to transfer the bla OXA-23 carbapenemase gene from P. mirabilis VAC by conjugation and transformation failed. Genome analysis using P. mirabilis VAC as reference for the dominant OXA-23-producing clone (see above) confirmed that the bla OXA-23 gene was located on the chromosome. Comparative genomics between the P. mirabilis VAC isolate and the fully  susceptible P. mirabilis BB2000 reference strain revealed the presence of genomic islands (GIs) only in the P. mirabilis VAC isolate ( Fig. 2A). Here, GIs refer to large DNA sequences coming from an horizontal transfer and integrated in the chromosome 19 . Among these GIs, GI1 corresponds to the Tn6703 transposon that carries the bla OXA-23 gene. GI3 shares 97% of nucleotide identity with an integrative and conjugative elements (ICE) ICEPmiJpn1 identified in P. mirabilis (KY437729). GI4 another ICE identified in different P. mirabilis isolates as well as in Klebsiella quasipneumoniae strain KPC142 (CP023478), Providencia stuartii strain BE2467 (CP017054) and Morganella morganii strain AR_0133 (CP028956). GI5 is a copy of the class 2 transposon Tn7. Finally, GI6 contains a putative type VI secretion system encoding operon.
In P. mirabilis VAC, the bla OXA-23 gene is carried on GI1 of 55-kb in size. It is bracketed by two copies of IS15∆II, an IS26 point mutant variant belonging to the IS6 family 20 . IS15∆II themselves are bracketed by a target site duplication (TSD) TAATTTCC (Fig. 2B), typical of IS15∆II (as well as IS26) transposition events [21][22][23] . This composite transposon was named Tn6703 according to the transposon registry database (https://transposon.lstmed.ac.uk/). It has been previously demonstrated that at least 6 copies of bla OXA-58 gene were duplicated in tandem in P. mirabilis 1091 11 . Conversely to what was reported in P. mirabilis 1091 isolate, only one copy of the bla OXA-23 gene was present in all isolates of the main clone (ratio bla OXA-23 /housekeeping genes at 1). Analysis of the close genetic structure of bla OXA-23 gene revealed that it was carried by a Tn2008-like transposon named Tn6704 (Fig. S1) 24 . Tn6704 was inserted in a fragment of Tn5393 within a non-coding region between the resolvase and strA genes. Noticeably, a plasmid replicase from Acinetobacter was identified within this Tn6704. However, this replicase encoding gene is interrupted by ISAba125 (bracketed by TSD of 3 bp, TAG). This Tn6704 is, itself, bracketed by TSD of 9-bp (GATGAAGCG) consistent with ISAba1-based transposition (Figs. 2B and S1). Alignment of IR of ISAba1 and the putative IRL found at the left extremity of Tn6704 revealed a weak nucleotide sequence identity with IRL of ISAba1 (Fig. S1). As usually reported, ISAba1 is present upstream of bla OXA-23 gene in Tn6704. ISAba1 is known to be involved in bla OXA-23 gene expression 25 . Downstream of the bla OXA-23 gene, an ATPase-encoding gene was identified, as described in all transposons carrying bla OXA-23 10 . Following this ATPase-encoding gene, a copy of ISAba14 and of ISAba125 were identified. Downstream the Tn6704, a IS15∆II-mediated putative transposon carrying aph(3′)-Ia is present, followed by aac(3′)-II genes. Then, a putative sigma factor sharing homology with σ factor from environmental bacteria was identified (52% ID AA WP_127199220), followed by a copy of ISKpn12, an aph(3′)-Ia gene and another IS15∆II copy (Fig. 2B).
A region covering ca. 20% of the GI1, including the Tn6704, shares 99% of nucleotide identity with A. baumannii genome Ab04-mff (CP012006) (Fig. 2B.). This region contained a copy of ISAba125 followed by two aminoglycoside resistance genes (aph(6′)-Id and aph(3′)-Ib) and two genes involved in plasmid transfer (traA/traD). This structure was identified in all OXA-23-producing P. mirabilis of this study. Intriguingly, close to this region and only in P. mirabilis VAC, a fragment of Tn6260 carrying lnu(G) resistance gene originating from Enterococcus faecalis was identified 26 . The lnu(G) gene was bracketed by two copies of ISCR2, an IS91-like mobile element. Ultimately, two copies of IS15∆II bracketed this MDR GI with a TSD of 8-bp (TAATTTCC) leading to a putative composite transposon. Of note, all isolates of this clone do not share the same resistome ( Fig. 2A.). The alignment of whole genome sequences revealed some differences in this region. This can be explained, for instance, by the presence of the transposon carrying lnuG only in P. mirabilis VAC. Accordingly, this lnuG-carrying transposon was most likely acquired recently. Some aminoglycoside resistance genes are also present in few isolates. The genetic diversity of Tn6703 is not surprising since studied isolates were recovered from different countries, over a long period and from animal or human. They were likely submitted to different selective pressures that might explain this diversity.
In P. mirabilis VAC and other isolates of the same clone, GI1 was inserted within the remnant (15 kb in size) of a prophage sharing 75% nucleotide identity with a prophage identified in Providencia rettgeri RB151 (CP017671). P. mirabilis BB2000 reference strain (CP004022) also harboured this prophage, but neither Tn6703 nor any resistance genes were inserted in it (Fig. 2B.). In the unrelated P. mirabilis 160A10, the bla OXA-23 gene was also part of a Tn6703-like element. However, since 160A10 possessed an intact homolog of the phage Burkho_BcepB1A tail protein-encoding gene (GenBank NC005886). To decipher the genetic context of the carbapenemase gene in this isolate, P. mirabilis 160A10 was sequenced using MinIon technology. In this isolate, the bla OXA-23 gene is carried by a conjugative plasmid of 67 kb in size (Fig. S2). This plasmid carried a full transfer operon and was not typeable using PlasmidFinder v2.1 for replicon typing of Enterobacterales. The bla OXA-23 gene was present within a fragment of Tn6703 carried by the plasmid (Fig. S2) The other resistance genes (aadA1, sat2 and dfrA1) were identified within a class 2 integron carried by a Tn7 transposon (GI5) (Fig. 2C.). This transposon has been identified in many isolates of P. mirabilis 27 . As previously reported, the class 2 integrase gene contains a premature stop codon leading to a pseudo-gene (Fig. 2C.) 28 .
the bla OXA-58 gene might be mobilized by XerC/XerD recombination events. Within P. mirabilis CNR20130297, the bla OXA-58 gene is carried on a plasmid of 6,219 bp that shared 99,9% nucleotide identity (only one SNP), with plasmid p10797-OXA-58 (KU871396). This plasmid has been previously identified in a OXA-58-producing P. mirabilis from Germany 12 . The plasmid replicase showed 51% amino acid identity with a replicase of Stenotrophomonas maltophilia (GenBank accession number WP_029214130.1) and to a lesser extent with another replicase of Acinetobacter lwoffii (50% amino acid identity) (GenBank accession number WP_005102557.1). Analysis of the closed genetic environment of the bla OXA-58 gene revealed that XerC-XerD recombination was likely involved in its acquisition (Fig. 2D.). The process of site-specific recombination can be performed by two chromosomally-encoded tyrosine recombinases (XerC and XerD). These recombinases recognize a 28-bp recombination site named dif and may allow resolution of the recombination event 29 . XerC and XerD recombination sites are composed of two sequences of 11 nucleotides separated by a spacer of 6 nucleotides 30 . In P. mirabilis 1091, the bla OXA-58 gene was bracketed by two fragments of ISAba3, and a gene coding for a cephalosporinase as previously described 11,31 . Bracketing ISAba3-bla OXA-58 -ISAba3, two XerC-XerD sites were identified named XerC3/XerD3 and XerC4/XerD4. Downstream of the bla ampC gene, another site was identified called XerC5/XerD5. In P. mirabilis VAC, only XerC5/XerD5 is present and might be considered as an empty XerC-XerD binding site within a prophage (Fig. 2D.). In P. mirabilis CNR20130297, harbouring the p20130297-OXA-58 plasmid, XerC1/XerD1 binding site is found at the 5' end extremity of the structure whereas a XerC2-XerD2 binding site is present at the 3' end extremity. Analysis of XerC-XerD sites suggests a mobilisation of this structure via XerC-XerD recombinases.

Discussion
OXA-23 is the main carbapenemase identified in Acinetobacter species. The bla OXA-23 gene is now widespread and even endemic in some areas 32 . However, this carbapenemase is very rarely identified in Enterobacterales. Only a few CHDL, other than OXA-48-like carbapenemases, have been reported in Enterobacterales and especially in Proteus spp [11][12][13][14][15] .
Here, we report the first genomic characterization of twenty-one OXA-23-and one OXA-58-producing P. mirabilis isolates from 2013 to 2018. Two reference OXA-producing P. mirabilis isolates were also sequenced: an OXA-23-producer isolated in France in 1996 16 and the OXA-58-producing P. mirabilis 1091 isolated in Yvoir, Belgium, in 2015 11 . This analysis revealed that one clone carrying bla OXA-23 gene is circulating since 1996 and had spread over the last twenty years among humans and animals. Interestingly, the recently described OXA-58-producing P. mirabilis 1091 isolate 11 also belonged to this lineage (Fig. 3). The comparison with genomes recovered from GenBank revealed that this lineage is distantly related to others lineages except a branch represented by three isolates (NEYX02.1, LDIU01.1 and NJFA02.1). Nevertheless, despite being of the same lineage, these three isolates that do not carry any carbapenemase-encoding gene, are not part of this OXA-23/OXA-58-producing "successful" clone (4000 to 5000 SNPs) ( Fig. 3 and Table S3).
The bla OXA-23 gene is part of a Tn6704, which is embedded in a 55-kb DNA sequence bracketed by two copies of IS15∆II, thus forming a composite transposon, named Tn6703. This transposon is bracketed by an 8-bp target site duplication compatible with an IS15∆II-mediated transposition event [21][22][23] . It is unlikely that this structure was acquired in one step since the mapping of reads on GI1 revealed variability of its content among different isolates. Of note, three resistance genes (aadA1, sat2 and dfrA1) were not present in Tn6703 transposon but carried by Tn7 (Fig. 2C.). The class 2 integron, carrying these genes, does not seem to be functional anymore. Indeed, the int2 gene carried a premature stop codon leading to an incomplete integrase. Regarding the bla OXA-58 gene, its acquisition involved a XerC-XerD tyrosine recombinases and it has been identified either on the chromosome or on a Scientific RepoRtS | (2020) 10:9160 | https://doi.org/10.1038/s41598-020-66161-z www.nature.com/scientificreports www.nature.com/scientificreports/ plasmid. XerC-XerD tyrosine recombinases have been involved in the resolution of plasmid co-integrates carrying the bla OXA-58 gene in A. baumannii 33 . Interestingly, this plasmid was reported to replicate in Enterobacterales and in A. baumannii ATCC17978 12 . Accordingly, we might hypothesize that this plasmid might be the shuttle vector between the Acinetobacter genus and P. mirabilis.
Comparative genomics also revealed the presence of other GIs in P. mirabilis VAC as compared to the P. mirabilis BB2000 reference strain. Among the identified GIs, an ICE sharing high homology with ICEPmiJpn1 (KY437729) has been identified (GI3) 34 . Interestingly this ICE was identified in only two isolates of the main lineage (Fig. 3A.). Several other GIs carrying potential virulence genes were identified in P. mirabilis VAC including GI6 carrying a putative type VI secretion system encoding operon. The content of all genomic islands is indicated in Tables S4 and S5. Accordingly, we can speculate that these GIs might be involved in the spread of this clone. Investigations of these elements will be further conducted to decipher their potential role in the spread of this clone.
Here, we described the clonal relationship of OXA-producing P. mirabilis over a twenty-one-year period . The spread of the bla OXA-23 gene is due to a single clone possessing a complex IS15∆II-based composite transposon, Tn6703. This dissemination could be silent, and the prevalence underestimated since bla OXA-23 genes are not targeted by most of the carbapenemase detection assays in Enterobacterales. Amplidiag ® CarbaR+MCR and CarbaR+MCR (Mobidiag, Paris, France) PCR-based assays are the only commercially-available molecular tests targeting the big 5 carbapenemases (KPC, NDM, VIM, IMP, OXA-48-like), and the main CHDLs from A. baumannii (OXA-23, OXA-24/-40, OXA-58, and the over-expressed chromosomally-encoded OXA-51-like β-lactamase associated with an upstream inserted ISAba1). These kits are thus able to detect these carbapenemase producers 35 . Recently, a novel assays either immunochromatographic test targeting OXA-23 in Acinetobacter spp., OXA-23 K-SeT ® test (Coris BioConcept, Gembloux, Belgium), or molecular assays such as Amplidiag ® Carba-R + MCR's that detects the major carbapenemases: KPC, NDM, VIM, IMP, and OXA-48, as well as the main OXA-type carbapenemases from Acinetobacter spp. have been demonstrated to accurately identify OXA-23-producing P. mirabilis isolates 35,36 . The use of these assays might help to decipher the underestimated carriage of these OXA-23/58-producing P. mirabilis. However, the clinical impact and the need to set-up hygiene measures around these OXA-23/58-producing P. mirabilis need to be evaluated since these isolates remain multi-susceptible to most antimicrobials including carbapenems.

Material and methods
Strain collection and reference strains. P. mirabilis resistant to amoxicillin and amoxicillin-clavulanate sent to the French and Belgium National Reference Centres (NRC) for antibiotic resistance as well as isolates collected through the National Monitoring Network for Antimicrobial Resistance in Diseased Animals (Resapath; https://resapath.anses.fr) were screened for the presence of the bla OXA-23 or bla OXA-58 gene. Thus, a total of 61 P. mirabilis isolates (4 from the Belgium NRC; 54 from the French NRC and 3 from the Resapath) were collected with a phenotype compatible with the production of a CHDL (Table 1). A collection of 22 OXA-23-and 2 OXA-58-producing P. mirabilis were included in this study (Table 1 & Fig. 1). Three isolates were recovered from veterinary samples whereas the others were from human origin. All available reference genomes of P. mirabilis from GenBank at the date of November 1 st 2019 (n = 122) were used for phylogenetic or comparative genomic analyses.
The copy number of bla OXA-23 was assessed to identify a potential gene duplication event as observed for bla  . The gene copy number was calculated using the ratio of the coverage of the bla OXA-23 gene and that of distantly located single copy chromosomal genes (rpoB, dnaA and mdh). Insertion sequences were identified using the ISfinder database 44 . transfer of β-lactam resistance determinants. Plasmids were extracted using Kieser's method as previously described 45 . Plasmids were extracted using Kieser's method and subsequently analysed by electrophoresis on a 0.7% agarose gel as previously described 45 , and attempted to be introduced by electroporation into E. coli TOP10. Recombinant E. coli were selected on TSA supplemented with 50 µg/ml of amoxicillin as previously described 39 . Conjugation assays using P. mirabilis isolates as donors and E. coli J53 as recipient strains were performed as previously described 46 . ethic statements. No animal or human experiments were performed in this study. All the human isolates were sent anonymously to the NRCs, and none of the authors had access to any identifying information along with the isolates, and that thus ethical approvals and informed consents were not needed.
Nucleotide sequence accession number. The whole genome sequences generated in the study have been submitted to the GenBank nucleotide sequence database under the accession numbers detailed in Table 1. The nucleotide sequence of the 6-kb plasmid carrying bla OXA-58 in P. mirabilis CNR20130297 was submitted to the GenBank nucleotide sequence database under the accession number MK533136. The genomes of OXA-23-or OXA-58-producing P. mirabilis were submitted to GenBank (bioproject number PRJNA521327).
Transparency declarations: L.D. is co-inventor of the Carba NP Test, which patent has been licensed to bioMérieux (La Balmes les Grottes, France).