# First report of carbapenemase OXA-181-producing Serratia marcescens

Aziza Messaoudi, Wejdene Mansour, Lamia Tilouche, Pierre Châtre, Antoine Drapeau, Chrifa Chaouch, Sana Azaiez, Olfa Bouallègue, Jean-Yves Madec, Marisa Haenni

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Aziza Messaoudi, Wejdene Mansour, Lamia Tilouche, Pierre Châtre, Antoine Drapeau, et al. First report of carbapenemase OXA-181-producing Serratia marcescens. Journal of Global Antimicrobial Resistance, 2021, 26, pp.205 - 206. 10.1016/j.jgar.2021.06.004. anses-04013104

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Distributed under a Creative Commons Attribution 4.0 International License
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**First report of carbapenemase OXA-181-producing *Serratia marcescens***

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Editor: Professor A Tsakris

Sir,

Among the carbapenemases, OXA-48-like enzymes (Ambler
class D) have disseminated worldwide or even become endemic
in some areas, such as OXA-48 in Tunisia and the whole Mediter-
ranean basin [1]. OXA-181, first described in 2007, has emerged
as the second most frequent OXA-48-like enzyme after OXA-48.
It is commonly acknowledged that OXA-48-like enzymes originate
from *Shewanella* spp., but OXA-48 and OXA-181 followed different
evolutionary paths, with *bla*<sub>OXA-48</sub> genes usually associated with
Tn1999 and IncL plasmids, and *bla*<sub>OXA-181</sub> mostly associated with
Tn2013 and IncX3 plasmids [1]. These enzymes/genes were mostly
reported in *Klebsiella pneumoniae* and *Escherichia coli*, but also in
other Enterobacteriales, notably OXA-48, but not OXA-181, in a few
*Serratia marcescens* isolates [2]. Long considered a harmless sapro-
phyte, *S. marcescens* is now recognised as an important pathogen
presenting intrinsic resistance (narrow-spectrum penicillins, amox-
icillin/clavulanic acid, cephalosporins, macrolides, tetracyclines and
colistin) and having a propensity to survive in clinical environ-
ments [3,4].

Between 2017 and 2019, four temocillin-resistant isolates were
identified as S. marcescens by VITEK® (bioMérieux, Marcy-l'Étoile,
France) at Sahloul Hospital in Sousse, Tunisia. They were mostly
isolated from young patients (three of four) from the cardiol-
ogy and orthopaedic wards (Table 1). Using a specific PCR, the
presence of OXA-48-like enzymes was confirmed in the four iso-
lates [5], which presented reduced susceptibility to imipenem, er-
tapenem and meropenem, with minimum inhibitory concentra-
tions (MICs) of 0.5, 0.25 and 0.125 mg/L, respectively. All four
isolates were submitted to whole-genome sequencing (WGS) us-
ing a NovaSeq instrument (Illumina Inc., San Diego, CA, USA)
(BioProject PRJNA702027). Resistance genes and replicon con-
tent were inferred using ABRicate v.1.0.1 (https://github.com/
tseemann/abricate) on reads assembled using Shovill v.1.0.4. WGS
analysis revealed that isolates 53643 and 53646 were OXA-
48-producers, while isolates 53644 and 53645 were OXA-181-
producers (Table 1). Isolates 53644 and 53646 were further se-
quenced using MinION technology (Oxford Nanopore Technolo-
gies, Oxford, UK), and assembly both of Illumina short reads and
Nanopore long reads was performed using Unicycler [6]. Hybrid
analysis of isolate 53644 showed that *bla*<sub>OXA-181</sub> was located to-
gether with the *qnrS1* gene on an IncX3/ColKP3 plasmid of 51
475 bp. This plasmid showed 100% homology over 30 361 bp
with *E. coli* plasmid pABC381-OXA-181 (GenBank MK412919)
described in the United Arab Emirates [7]. In agreement with
the absence of identification of any known replicon (especially
IncL) in the Illumina data, hybrid analysis of isolate 53646 re-

vealed the presence of a unique contig of 5 191 506 bp, thus
demonstrating the atypical chromosomal location of the *bla*<sub>OXA-48</sub> gene.

To elucidate the phylogenomic distribution of these four iso-
lates in the global OXA-48-like-producing *S. marcescens* epidemiol-
ogy, all publicly available genome assemblies of OXA-48-producing
*S. marcescens* were retrieved from the NCBI database and anal-
ysed (Supplementary Table S1). A total of 15 isolates were found,
which all carried *bla*<sub>OXA-48</sub> and presented an IncL replicon. Two
isolates originated from patients in Madrid, Spain [2], one from a
human sample in Lebanon, and twelve from environmental swabs
collected in a hospital in Pakistan. Assemblies were de novo anno-
tated with Prokka v.1.14.6 and pangenome analysis was performed
with the Roary pipeline v.3.13.0 using a Protein BLAST identity of
80% and a core definition of 90%. A maximum likelihood tree was
constructed from the core gene alignment produced by Roary using
RAxML v.8.2.12 with default parameters. The corresponding phylo-
genetic tree based on the matrix presence/absence of a gene was
visualised using iTOL v.5.5.1 (http://itol.embl.de/itol.cgi) (Supple-
mentary Fig. S1). Pairwise single nucleotide polymorphism (SNP)
distances were calculated from core genome alignments generated
using snp-dists (https://github.com/tseemann/snp-dists). In all, iso-
lates strongly clustered by country, however the paucity of WGS
data of carbapenemase-producing *S. marcescens* isolates prevents
any further interpretation from this phylogenetic tree. Among the
Tunisian isolates, the two OXA-48-positive isolates were identical
(no SNP difference) and the two OXA-181-positive isolates differed
by only 1 SNP, while there was around 95 SNPs difference between
the two groups.

Up to now, OXA-48-like enzymes in clinical settings have been
principally detected in epidemic lineages of *E. coli* and *K. pneumo-
niae*, and more rarely in other Enterobacteriales. However, a signif-
icant circulation of carbapenemase-producing *S. marcescens* clones
was recently reported in a Spanish healthcare institution, similarly
to our report from Tunisia. The two OXA-48-producing isolates
were collected 2 years apart and their close genetic relatedness
suggests long-term persistence of the same clone within the hospi-
tal. Of note, *S. marcescens* is capable of colonising, even silently and
over long periods of time, different surfaces (such as sink traps)
or liquids (such as chlorhexidine) [3,4]. In contrast, the two OXA-
181-producing isolates were collected within a week, most likely
indicating nosocomial transmission between two different wards.
These two situations strongly reinforce the need for stringent mon-
itoring to avoid larger outbreaks. Interestingly, three of the four
patients were children, in line with the description of several *S.
marcescens* outbreaks in paediatric hospitals. Finally, this is the first
description of an OXA-181 enzyme produced by *S. marcescens*, an
association that has most probably been favoured by the efficient
spread of *bla*<sub>OXA-181</sub>-carrying IncX3 plasmids. Preventing the emer-
gence and transmission of carbapenemase-producing *S. marcescens*
will be a challenge in the future because of the persistent charac-
<table><caption>**Table 1**
Characteristics of the OXA-48- and OXA-181-producing *Serratia marcescens* isolates</caption><thead><tr><th>Isolate</th><th>Sampling date</th><th>Sex</th><th>Age (years)</th><th>Pathology</th><th>Ward</th><th>Treatment</th><th>Carbapenemase gene</th><th>Carbapenemase-carrying plasmid</th></tr></thead><tbody><tr><td>53643</td><td>12/10/2017</td><td>M</td><td>0.33</td><td>Cyanogenic heart disease</td><td>Cardiology</td><td>Imipenem, amikacin</td><td>bla<sub>OXA-48</sub></td><td>-</td></tr><tr><td>53644</td><td>02/12/2018</td><td>M</td><td>57</td><td>Ischaemia</td><td>Cardiology</td><td>Imipenem, amoxicillin/clavulanic acid, gentamicin, ciprofloxacin</td><td>bla<sub>OXA-181</sub></td><td>IncX3</td></tr><tr><td>53645</td><td>09/12/2018</td><td>M</td><td>11</td><td>Osteomyelitis</td><td>Orthopaedic</td><td>Cefotaxime, cefixime, sulfonamide/trimethoprim</td><td>bla<sub>OXA-181</sub></td><td>IncX3</td></tr><tr><td>53646</td><td>29/04/2019</td><td>M</td><td>9</td><td>Osteomyelitis</td><td>Orthopaedic</td><td>Cefotaxime, gentamicin, ciprofloxacin, fosfomycin</td><td>bla<sub>OXA-48</sub></td><td>-</td></tr></tbody></table>

M, male.

ter of this bacterial species and the selective pressure due to carbapenem use in hospitals.

## Funding

This work was supported by internal funding of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).

## Competing interests

None declared.

## Ethical approval

Not required.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jgar.2021.06.004.

## References

[1] Pitout JDD, Peirano G, Kock MM, Strydom K-A, Matsumura Y. The global ascendency of OXA-48-type carbapenemases. Clin Microbiol Rev 2019;33 e00102-19.

[2] Pérez-Viso B, Hernández-García M, Ponce-Alonso M, Morosini MI, Ruiz-Garba-josa P, Del Campo R, et al. Characterization of carbapenemase-producing *Serratia marcescens* and whole-genome sequencing for plasmid typing in a hospital in Madrid, Spain (2016–18). J Antimicrob Chemother 2021;76:110–16.

[3] Regev-Yochay G, Smollan G, Tal I, Pinas Zade N, Haviv Y, Nudelman V, et al. Sink traps as the source of transmission of OXA-48-producing *Serratia marcescens* in an intensive care unit. Infect Control Hosp Epidemiol 2018;39:1307–15.

[4] Keck N, Dunie-Merigot A, Dazas M, Hirchaud E, Laurence S, Gervais B, et al. Long-lasting nosocomial persistence of chlorhexidine-resistant *Serratia marcescens* in a veterinary hospital. Vet Microbiol 2020;245:108686.

[5] Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119–23.

[6] Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017;13:e1005595.

[7] Mouftah SF, Pál T, Darwish D, Ghazawi A, Villa L, Carattoli A, et al. Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide. Infect Drug Resist 2019;12:1729–42.

Aziza Messaoudi¹

Laboratoire de Biophysique métabolique et Pharmacologie Appliquée (LR12ES02), Faculté de Médecine Ibn Al Jazzar Sousse, Sousse, Tunisia

Laboratoire de Microbiologie, Hôpital Universitaire Sahloul, Sousse, Tunisia

Wejdene Mansour¹

Laboratoire de Biophysique métabolique et Pharmacologie Appliquée (LR12ES02), Faculté de Médecine Ibn Al Jazzar Sousse, Sousse, Tunisia

Faculté de Médecine Ibn Al Jazzar Sousse, Université de Sousse, Sousse, Tunisia

Lamia Tilouche

Laboratoire de Microbiologie, Hôpital Universitaire Sahloul, Sousse, Tunisia

Pierre Châtre, Antoine Drapeau

Université de Lyon – ANSES Laboratoire de Lyon, Unité

Antibiorésistance et Virulence Bactériennes, 31 Avenue Tony Garnier, 69007 Lyon, France

Chrifa Chaouch

Laboratoire de Microbiologie, Hôpital Universitaire Sahloul, Sousse, Tunisia

Sana Azaiez

Laboratoire de Biophysique métabolique et Pharmacologie Appliquée (LR12ES02), Faculté de Médecine Ibn Al Jazzar Sousse, Sousse, Tunisia

Faculté de Médecine Ibn Al Jazzar Sousse, Université de Sousse, Sousse, Tunisia

Olfa Bouallègue

Laboratoire de Microbiologie, Hôpital Universitaire Sahloul, Sousse, Tunisia

Faculté de Médecine Ibn Al Jazzar Sousse, Université de Sousse, Sousse, Tunisia

Jean-Yves Madec, Marisa Haenni*

Université de Lyon – ANSES Laboratoire de Lyon, Unité

Antibiorésistance et Virulence Bactériennes, 31 Avenue Tony Garnier, 69007 Lyon, France

*Corresponding author. Tel.: +33 4 78 69 65 60
E-mail address: marisa.haenni@anses.fr (M. Haenni)
¹ These two authors contributed equally to this work.
Revised 4 May 2021