A Patient With Multiple Carbapenemase Producers Including an Unusual Citrobacter sedlakii Hosting an IncC blaNDM-1- and armA-carrying Plasmid

Background. Patients colonized with multiple species of carbapenemase-producing Enterobacterales (CPE) are increasingly observed. This phenomenon can be due to the high local prevalence of these pathogens, the presence of important host risk factors, and the great genetic promiscuity of some carbapenemase genes. Methods. We analyzed 4 CPE (Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, Citrobacter sedlakii), 1 extended-spectrum cephalosporin-resistant K. pneumoniae (ESC-R-Kp), and 1 carbapenemase-producing Acinetobacter baumannii simultaneously isolated from a patient transferred from Macedonia. Susceptibility tests were performed using a microdilution MIC system. The complete genome sequences were obtained by using both short-read and long-read whole-genome sequencing technologies. Results. All CPE presented high-level resistance to all aminoglycosides due to the expression of the armA 16S rRNA methylase. In C. sedlakii and E. coli (ST69), both the carbapenemase blaNDM-1 and armA genes were located on an identical IncC plasmid of type 1a. The K. pneumoniae (ST268) and P. stuartii carried chromosomal blaNDM-1 and blaOXA-48, respectively, while the ESC-R-Kp (ST395) harbored a plasmid-located blaCTX-M-15. In the latter 3 isolates, armA-harboring IncC plasmids similar to plasmids found in C. sedlakii and E. coli were also detected. The A. baumannii strain possessed the blaOXA-40 carbapenemase gene. Conclusions. The characterization of the genetic organization of IncC-type plasmids harbored by 3 different species from the same patient offered insights into the evolution of these broad-host-range plasmids. Moreover, we characterized here the first complete genome sequence of a carbapenemase-producing C. sedlakii strain, providing a reference for future studies on this rarely reported species.


INTRODUCTION
The spread of carbapenemase-producing Enterobacterales (CPE) represents a major public health issue. To date, KPC-2/-3-and OXA-48-producing Escherichia coli and Klebsiella pneumoniae isolates have been reported worldwide and in some geographic areas their prevalence is alarming [1,2]. In addition, though less predominant, the NDM-producing species are of particular clinical concern because the NDM carbapenemase activity cannot be inhibited by clinically available β-lactamase inhibitors [3]. Moreover, the bla NDM genes show great promiscuity since they can be located in different genetic environments, being either integrated into the chromosome or on extra-chromosomal mobile genetic elements (MGEs) among different bacterial species. In particular, some conjugative plasmids harbor additional antimicrobial resistance genes (ARGs) conferring co-resistances to other antibiotic families, such as the ArmA 16S rRNA methylase enzyme that modifies the target of aminoglycosides resulting in resistance to all clinically-used aminoglycosides, including the most recently developed plazomicin [4][5][6].
In this overall scenario, reports of patients simultaneously infected and/or colonized with multiple species of CPE are becoming a source of real concern. Several cases of interspecies exchange of identical bla KPC - [7,8], bla OXA-48 - [9,10], and bla NDM-1 -carrying plasmids have been described [9,[11][12][13]. In particular, those involving the bla NDM-1 were mainly due to the horizontal spread of broad-host-range IncC plasmids (formerly IncA/C2) [14]. In such cases, 2 to 4 different CPE were isolated from the same subjects: E. coli and K. pneumoniae were usually involved in this phenomenon, but Klebsiella oxytoca, Citrobacter freundii, Proteus mirabilis, or Morganella morganii strains could also be encountered [9,11,13].
In this work, we report a clinical case of a patient being simultaneously colonized by 3 bla NDM-1and one bla OXA-48 -positive Enterobacterales, along with an Acinetobacter baumannii strain possessing a bla OXA-40 carbapenemase gene. Enterobacterales were characterized at the genomic level by implementing both short-read and long-read whole-genome sequencing (WGS) technologies. Above all, we provide here the first genomic characterization of a unique bla NDM-1 -and armA-positive Citrobacter sedlakii isolate.

MATERIALS AND METHODS
Clinical case. In December 2020, a Swiss man in his 20s was admitted at the Inselspital (Bern, Switzerland). The subject was transferred from Macedonia, where he had been hospitalized as a polytraumatized individual for 2 months (further detailed clinical data regarding this hospitalization are not available). For surveillance purpose, a rectal swab to screen for the presence of multidrug-resistant Gram-negative bacteria was withdrawn. Moreover, blood cultures, swabs from skin ulcers, and cultures from vascular catheters were also performed over the course of 14 days ( Table 1). The patient was kept in isolation during these 2 weeks before transfer to another Swiss hospital. The present anonymized case description has been carried out in accordance with the Declaration of Helsinki. The patient has also signed a general consent.

Species identification (ID) and antimicrobial susceptibility tests (ASTs).
ID was routinely obtained using the matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS; Bruker); it was then achieved using WGS data and the implementation of the Type (Strain) Genome Server (https://tygs.dsmz.de/). ASTs were performed using the broth microdilution ESB1F and GNX2F Sensititre panels (Thermo Scientific). Minimum inhibitory concentrations (MICs) for antibiotics were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria (version 9.0, 2019).

Detection of ESBL and carbapenemase-producing (CP) strains.
The Rapid ESBL NP, Carba NP, and CarbAcineto NP colorimetric tests, along with the NG-Test CARBA-5 and the eazyplex Superbug complete B assays, were used to screen strains showing reduced susceptibility to extended-spectrum cephalosporins (ESCs) and/or carbapenems [1,15]. Enterobacterales were further characterized by implementing the WGS (see below), whereas the A. baumannii was typed with a PCR/sequencing approach [16].
The complete genome assemblies of the 5 Enterobacterales have been deposited in GenBank (CP071068-CP071089) under BioProject PRJNA698767.

Samples and bacteria.
Numerous samples taken at the admission of the patient gave positive results for CPE (E. coli, K. pneumoniae, C. freundii complex, and Providencia stuartii) and for a CP A. baumannii. An ESC-resistant K. pneumoniae (ESC-R-Kp) and a carbapenem-resistant P. aeruginosa were also isolated in multiple specimens (Table 1).
To study the features of these MDR-Gram-negatives, 4 representative strains of the CPE species, the ESC-R-Kp, and the CP A. baumannii were selected for further phenotypic and molecular analyses. The antibiotic MICs for these 6 illustrative strains are depicted in Table 2. As expected, the 5 CP strains showed reduced susceptibility to carbapenems, but the 4 CPE also presented high-level resistance to all tested aminoglycosides. Moreover, the ESC-R-Kp showed a phenotype consistent with the extended-spectrum ß-lactamase (ESBL) production. Table 3, the gut flora of the patient was colonized with a sequence type (ST) 69 E. coli strain (named 3347558) possessing numerous ARGs, including the carbapenemase gene bla NDM-1 , the ESBL gene bla CTX-M-15 , and the 16S rRNA methylase gene armA. Of note, the pandemic ST69 lineage is rarely associated with bla NDM-1, and it has never been reported to contain simultaneously both bla NDM-1 and armA [19].

Molecular features of the MDR bacteria. As shown in
In strain 3347558, bla NDM-1 and armA were co-carried on the ARI-A island of a multidrug resistance 169kb IncC plasmid of type 1a (p33477558_1) identical (identity, 99.81%) to pPS-T1 found in Germany (2015) in a P. stuartii strain of human origin ( Figure 1A) [20]. In both p33477558_1 and pPS-T1, the bla NDM-1 was associated with ISAba125 and located between 2 ISCR1 elements in a genetic environment identical to the one reported for the Serbian P. aeruginosa isolate MMA83 [4,5,21,22]. Moreover, armA was positioned upstream of bla NDM-1, and it was organized in a classic element (ISEc28-armA-ISEc29) ( Figure 1B) [23].
The patient also carried 2 K. pneumoniae strains belonging to different STs (Table 3).

Figure 1. BLASTn comparison of IncC-type plasmid sequences. A)
The IncC-type plasmids from the present study were compared to other sequences selected based on high homology in a BLASTn search against the NCBI non-redundant nucleotide collection. Plasmid sequence pPS-T1 was used as reference sequence. Rings were constructed using BRIG (BLAST Ring Image Generator) v.0.95. The colored rings represent similarities to the reference sequence. CDS are depicted as grey arrows in the outermost circle. Mobile genetic elements (MGE) are depicted in white, the bla NDM-1 in blue, armA in red, and all other antimicrobial resistance genes (ARGs) in black. We report GenBank accession, species of isolation, strain name, sequence size, and country of origin. The bla NDM-1 -containing region is bordered with a black line. B) Genetic environment of the bla NDM-1 -and armA-containing region in the reference sequence pPS-T1 (GenBank: MN657252) compared to the IncC plasmids from our study. Hypothetical proteins are depicted as light grey arrows, other CDS are depicted in dark grey.
The CP P. stuartii strain 3347685 possessed bla OXA-48 and armA, consistent with the observed carbapenem and aminoglycoside phenotypic resistance ( Table 2 and Table 3). We note that OXA-48-producing P. stuartii have been rarely reported and none of them co-produced ArmA or other 16S rRNA methylases leading to such pan-resistance to clinically used aminoglycosides.
In strain 3347685, bla OXA-48 was chromosome borne, and it was located within a Tn1999.2 transposon [26]. The armA was located on a 189kb IncC plasmid of type 1b (p3347685_1) in a genetic context identical to the one in the type 1a plasmids p3347558_1 from E. coli and p3347689I_1 from the CP K. pneumoniae ( Figure 1B). However, in contrast to the latter 2 plasmids, p3347685_1 was missing the type 1a patch region and carried the ARI-B resistance island in addition to ARI-A [14].
The CP A. baumannii isolate was resistant to all ß-lactams including high-level resistance to carbapenems and carried the bla OXA-40 gene (Table 3). It was resistant to fluoroquinolones, but it remained susceptible to trimethoprim-sulfamethoxazole and to colistin ( Table 2). It did not produce any additional ESBL or 16S rRNA methylase, remaining susceptible to tobramycin.
Citrobacter sedlakii: genomic and plasmid characterizations. Based on the WGS, the C. freundii complex strain 3347089 II was actually of C. sedlakii species. Moreover, the ANI values (≥98.98%) among our strain and the 6 C. sedlakii genome assemblies currently deposited in the NCBI genome database (accessed on 03/17/2021) confirmed the ID as C. sedlakii (data not shown). A core-genome analysis was performed including C. sedlakii 3347089 II, the 6 deposited genomes, and 3 assemblies deposited as Citrobacter spp. that were highly similar to 3347089 II based on the ANI values (>99%). As a result, no clonal relationship between the deposited assemblies and our C. sedlakii isolate could be observed (Supplemental Figure 1). It should be noted that only 2 bla NDM -positive C. sedlakii isolates were previously reported. However, the genomes of these 2 strains-respectively from Pakistan and Bangladesh-had not been sequenced [27,28].
C. sedlakii 3347089 II carried a 167kb IncC type 1a plasmid and a 44kb IncR plasmid (Table  3). Remarkably, the IncC plasmid (p3347089II_1) was identical (identity, 100%) to the bla NDM-1 and armA carrying IncC plasmid from E. coli 3347558, except for the ∆ISAba14-aph(3')-VI-IS-Aba125 region that was missing ( Figure 1B). Comparison of the 3 IncC type 1a plasmids from our study suggested a common ancestor with a sequence similar to the one of p3347689I_1 from K. pneumoniae, but missing the ISKpn21 and the IncR plasmid-derived insertion sequence. The ISCR1-flanked aph(3')-VI, bla NDM-1 , and sul1 and the bla NDM-1 and sul1 in E. coli 3347558 and C. sedlakii 3347089 II, respectively, were likely acquired by a recombination event with the ISCR1 element ( Figure 1B) [29].
An analysis of the NCBI deposited C. sedlakii genomes revealed that the Citrobacter spp. strain 50677481 (GenBank: GCA_001463265; Supplemental Figure 1) possessed bla NDM-1 and armA together with an IncC plasmid. Therefore, we further analyzed its Illumina-derived WGS assembly. Mapping of the contigs to the plasmid sequence pPS-T1 from the German P. stuartii allowed the reconstruction of the complete plasmid sequence and showed that Citrobacter spp. 50677481 harbored an IncC plasmid identical to p3347558_1 from our E. coli isolate (coverage, 100%; identity, 99.98%) (data not shown). Remarkably, the Citrobacter spp. strain 50677481 was isolated in 2012 from a Norwegian patient with travel history to Serbia, suggesting a persistent and wide distribution of this multidrug resistance plasmid in the Balkan region [30].

CONCLUSIONS
Although reports of CPE from Macedonia are missing, studies from neighboring countries reported concerning levels of NDM producers [31][32][33]. Therefore, the risk of importing CPE through the transfer of patients from these countries to those with a low prevalence is concerning. This phenomenon has been extensively discussed before (eg, in [34][35][36]). The present study underlined the importance of monitoring such cases to prevent the importation of multiple difficult-to-treat pathogens carrying novel antibiotic resistance traits.
Furthermore, we noted that while OXA-40-producing A. baumannii strains have been extensively described [1], the 4 CPE carried by the patient presented unusual patterns of antimicrobial resis-tance. In fact, all CPE were co-resistant to all aminoglycosides due to the production of the ArmA 16S rRNA methylase [6]. More importantly, E. coli 3347558 and C. sedlakii 3347089 II carried the bla NDM-1 and armA ARGs in an identical IncC type 1a plasmid suggesting an in vivo conjugation event. We also noted that this IncC type 1a plasmid was identical to one found in Germany in a P. stuartii isolate and to another one carried by a Citrobacter spp. strain linked to Serbia [20,30].
Overall, our findings emphasize the potential of the IncC plasmids carrying life-threatening ARGs to spread worldwide among different Enterobacterales. The presence of these broad-hostrange MGEs in rare enterobacterial species (eg, C. sedlakii) should be further investigated to better comprehend their origin and future evolution. Days from the hospitalization (admission at our institution in Bern, Switzerland) b. Gram-negatives non-susceptible to carbapenems are reported in bold. "CP" indicates that these strains were carbapenemase producers according to the results of the Rapid Carba NP, CarbAcineto NP, NG-Test CARBA-5 and/or eazyplex assays implemented by the routine laboratory. c. These bacteria were selected for WGS. We show their MIC values and genetic data in Table 2 and Table  3, respectively. d. This strain was extended-spectrum cephalosporin-resistant (ESC-R), but carbapenem susceptible (see Table 2) e. The patient was transferred to another Swiss institution  Note. nd, none detected a The main ARGs are in bold b All sequences were obtained by a hybrid WGS sequencing approach combining Illumina and Nanopore reads c The upstream region of the chromosomal AmpC did not contain mutations able to improve the expression of the bla gene

POTENTIAL CONFLICTS OF INTEREST
The authors report no relevant conflicts of interest to disclose.