# Back to the Origin: blaOXA-204 and blaNDM-1 Genes in Shewanella spp. from a Tunisian River

Sana Ncir, Agnese Lupo, Antoine Drapeau, Pierre Châtre, Meriem Souguir,

Sana Azaiez, Jean-Yves Madec, Wejdene Mansour, Marisa Haenni

► To cite this version:

Sana Ncir, Agnese Lupo, Antoine Drapeau, Pierre Châtre, Meriem Souguir, et al. Back to the Origin: blaOXA-204 and blaNDM-1 Genes in Shewanella spp. from a Tunisian River. Microbiology Spectrum, 2022, 10 (5), pp.e0160522. 10.1128/spectrum.01605-22. anses-03954167

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# Back to the Origin: *bla*<sub>OXA-204</sub> and *bla*<sub>NDM-1</sub> Genes in *Shewanella* spp. from a Tunisian River

Sana Ncir,ª,ᵇ Agnese Lupo,ª Antoine Drapeau,ª Pierre Châtre,ª Meriem Souguir,ª,ᵇ Sana Azaiez,ᵇ Jean-Yves Madec,ª Wejdene Mansour,ᵇ Marisa Haenniª

**KEYWORDS** carbapenem, *Shewanella xiamenensis*, *Shewanella bicipitri*, Tunisia, NDM-1, OXA-204, IS91, PER-1

The environmental bacteria *Xanthomonas* spp. and *Shewanella* spp. are progenitors of the *bla*<sub>NDM</sub> and *bla*<sub>OXA-48-like</sub> carbapenemase genes, respectively (1, 2). The most likely hypothesis is that these genes have been mobilized in the environment on mobile genetic elements, and subsequently transferred to Gram-negative bacteria (GNB) of clinical importance. The pathogens (primarily *Escherichia coli* and *Klebsiella pneumoniae*) that acquired these carbapenemase genes then emerged as a major One Health issue and are currently reported not only in clinical settings but also in animals and the environment (3). In January 2019, two carbapenem-resistant (CP-R) *Shewanella* spp. were isolated from fresh water sampled at two different locations along the El Hammam Oued river in Sousse, Tunisia, as part of a pilot study on multidrug-resistant GNB in aquatic environments. This river collects urban treated wastewater but no hospital effluents. The two isolates resistant to ertapenem (MIC ≥32 mg/L) were whole-genome sequenced using short-read (NovaSeq-6000, Illumina) and long-read (MinION, Oxford Nanopore) technologies (all genomic sequences were deposited in DDBJ/EMBL/GenBank under the accession number PRJNA830355). Genomes were assembled with Unicycler and analyzed using tools of the Center for Genomic Epidemiology (https://www.genomicepidemiology.org/).

S. xiamenensis 8A possessed a 4,959,567 bp chromosome and one nontypeable 216,160 bp plasmid (p8A) showing poor or fragmented homologies with published sequences. Except for *bla*<sub>OXA-539</sub>, all resistance genes—conferring resistances to beta-lactams, aminoglycosides, sulfonamides, trimethoprim, rifampicin and erythromycin—were located on p8A (Fig. 1A). Specifically, p8A carried the *bla*<sub>PER-1</sub> extended-spectrum beta-lactamase (ESBL) and the *bla*<sub>OXA-204</sub> carbapenemase genes. *bla*<sub>OXA-204</sub> has already been reported in one S. xiamenensis strain from water in Portugal (4), flanked by the peptidase C15 and *lysR* gene as intrinsically found on the *Shewanella* spp. chromosome (5). Here, the *bla*<sub>OXA-204</sub> gene was surrounded by an *ISEcp1* insertion sequence and a truncated *lysR* gene, as reported in the *K. pneumoniae* KP49 isolated from a Tunisian patient (KP027886) (6). This indicates that *bla*<sub>OXA-204</sub> in p8A is not intrinsic but has transited through another bacterial host before being reintegrated in S. xiamenensis p8A via a transposition event.

*Shewanella bicipitri* 10B possessed a 4,717,299 bp chromosome carrying a *bla*<sub>OXA-48-like</sub> and the *aadA2* resistance genes. The strain additionally presented a nontypeable 160,879 bp plasmid (p10B) sharing conserved regions with the pSA70-3 and pKC-Na-R1 plasmids, respectively, isolated from *Shewanella putrefasciens* and *Shewanella algae* (Fig. 1B) (7). p10B presented genes conferring resistance to beta-lactams, aminoglycosides, sulfonamides, trimethoprim, chloramphenicol, quinolones, and rifampicin, but also to mercury and biocides. CP-R was conferred by the *bla*<sub>NDM-1</sub> gene; *bla*<sub>NDM-1</sub>, together with the *bla*<sub>OXA-10</sub>*sul1*, and *aadA1* genes, was surrounded by two IS91-like that might have mediated its insertion/excision (8). A similar genetic organization was observed in pSA70-3 with only the left

**Editor** Daria Van Tyne, University of Pittsburgh School of Medicine

**Copyright** © 2022 Ncir et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Marisa Haenni, marisa.haenni@anses.fr.

The authors declare no conflict of interest.

**Published** 20 September 2022

<footnote>ªUnité Antibiorésistance et Virulence Bactériennes, ANSES - Université de Lyon, Lyon, France</footnote>

<footnote>ᵇLaboratoire de Biophysique métabolique et Pharmacologie Appliquée (LR12ES02), Faculté de Médecine Ibn Al Jazzar Sousse, Sousse, Tunisia</footnote>
<figure><img src="image_4.png" /><figcaption>**FIG 1** Schematic representation of the p8A plasmid (A). Comparative schematic representation of the p10B, pSA70-3 (CP078040), and pKC-Na-R1 (CP033574) plasmids. Genetic elements were analyzed by BLASTn and visualized using Easyfig 2.2.5_win. The window size used for BLASTn analysis was 2,000 bp.</figcaption></figure>

IS91-like missing (7), while the element was more fragmented in pKC-Na-R1, where only the *sul1-IS91*-like and *aadA1-bla*<sub>OXA-10</sub> genes were conserved upstream the *bla*<sub>VEB-1</sub> ESBL gene.

Environmental bacteria are progenitors of carbapenemases (1, 2), but reacquisition of these genes by environmental bacteria is rarely reported. This study showed that the *bla*<sub>OXA-204</sub> and *bla*<sub>NDM-1</sub> genes, which are highly prevalent in Tunisian hospital settings and effluents (9, 10), can spread back to environmental bacteria. In both cases, resistance genes moved to a *Shewanella* spp. nontypeable plasmid after IS-mediated acquisition, a mechanism that might be more efficient than plasmid transfer for bacterial genera that are not closely related.

## ACKNOWLEDGMENT

This work was funded by the "PHC Utique" program of the French Ministry of Foreign Affairs, the French Ministry of higher education, research, and innovation and the Tunisian Ministry of higher education and scientific research under the CMCU project number 21G0807. This work was also supported by internal funding of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).

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