First Report of New Delhi Metallo-β-Lactamase-6 (NDM-6) in a Clinical Acinetobacter baumannii Isolate From Northern Spain

The objective of this study was the phenotypic and genotypic characterization of a carbapenem resistant Acinetobacter baumannii (CRAB) isolate. The isolate, recovered in Northern Spain in 2019, was identified by MALDI-TOF to the species level. Antimicrobial susceptibility testing was performed using the Phoenix BD NMIC-502 Panel, E-test, and broth microdilution methods. The presence of a metallo-β-lactamase (MBL) was verified by PCR and immunochromatographic assays. The genetic location of the MBL was confirmed using S1-pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blot hybridization. Whole genome sequencing (WGS) was completed using the Miseq and MinION platforms, followed by core-genome MLST (cgMLST) and seven-locus MLST analysis. The CRAB was assigned ST85 (Pasteur scheme) and ST957 (Oxford scheme) representing international clone (IC) 9 and harbored the intrinsic β-lactamase OXA-94 with ISAba1 upstream of it, and the MBL blaNDM-6. Hybridization experiments revealed that the blaNDM-6 was encoded on the chromosome. Using WGS the blaNDM-6 environment could be identified arranged in the following order: ISAba14, aphA6, ISAba125, blaNDM-6, bleMBL, trpF, dsbC, cutA, and ISAba14. Downstream, a 10,462 bp duplication was identified, including a second copy of blaNDM-6 in the following genetic composition: ISAba125, blaNDM-6, bleMBL, trpF, dsbC, cutA, and ISAba14. To our knowledge, this is the first description of blaNDM-6 in A. baumannii. The MBL was present in two copies in the chromosome in a new genetic environment associated with IS elements highlighting the contribution of mobile genetic elements in the dissemination of this gene.

The aim of the present study was the phenotypic and genotypic characterization of a CRAB isolate harboring bla NDM-6 recovered from a patient in Northern Spain.

Patient and Bacterial Isolate Data, Species Identification, and Antimicrobial Susceptibility Testing
A 70-74 year-old patient, from Maghreb (Northwest Africa) presented to the Hospital de Basurto (Bilbao, Northern Spain) with dysuria in September 2019. The patient had a positive urine culture with >100.000 CFU/ml of a Gram-negative bacillus and reported a previous hospitalization in his home country due to a prostatectomy. Unfortunately, no further data were available about the country of origin.

S1-Pulsed-Field Gel Electrophoresis and Southern Blot Hybridization
Bacterial DNA embedded in agarose plugs was digested using 50 units S1-nuclease (Thermo Fisher Scientific, Waltham, MA, United States) per plug slice and followed by pulsed-field gel electrophoresis (PFGE). Samples were run on a CHEF-DR II system (Bio-Rad, Munich, Germany) for 17 h at 6 V/cm and 14°C, while initial and final pulses of 4 and 16 s, respectively, were applied. The Lambda PFG and λ DNA-Mono Cut Mix (New England Biolabs, Frankfurt, Germany) were used as markers. Southern blot hybridization was performed to determine the plasmid/chromosomal location by hybridization with digoxigenin-labeled probes (Roche, Mannheim, Germany). A bla NDM-6 specific probe was generated and the chromosomal location was shown by colocalization with a bla OXA-51-like probe.
Signal detection was performed using CDP-Star ® ready-to-use (Roche) chemiluminescent substrate by autoradiography on X-ray film (GE Healthcare, Buckinghamshire, United Kingdom).

Electroporation Experiments
To determine the transferability of bla NDM-like variants, plasmid DNA isolated from AbBAS-1 using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and electroporated into the reference strain A. baumannii ATCC 17978. Selection of A. baumannii transformants was performed on Luria-Bertani agar (Oxoid, Wesel, Germany) supplemented with ticarcillin (150 mg/L). The presence of bla NDM-like in the obtained transformants was confirmed by PCR.

Whole Genome Sequencing
Total DNA was extracted using the MagAttract HMW DNA Kit (Qiagen) according to manufacturer's instructions and used for short-read sequencing. Sequencing libraries were prepared using a Nextera XT library prep kit (Illumina GmbH, Munich, Germany) for a 250 bp paired-end sequencing run on an Illumina MiSeq platform. The obtained reads were de novo assembled with the Velvet assembler integrated in the Ridom SeqSphere+ v. 7.0.4 software (Ridom GmbH, Münster, Germany).
DNA extraction for long-read sequencing was performed using the Genomic-Tips 100/G kit and Genomic DNA Buffers kit (Qiagen) according to the manufacturer's instructions. Libraries were prepared using the 1D Ligation Sequencing Kit (SQK-LSK109) in combination with Native Barcoding Kit (EXP-NBD104; Oxford Nanopore Technologies, Oxford, United Kingdom) and were loaded onto a R9.4 flow cell (Oxford Nanopore Technologies). The run was performed on a MinION MK1b device. Collection of raw electronic signal data and live base-calling was performed using the MinKNOW software and the Guppy basecaller (Oxford Nanopore Technologies). The long-reads were assembled using ONT assembly and Illumina polishing pipeline (Oxford Nanopore Technologies), performing Canu assembly followed by polishing steps, including pilon and BWA mem mapping using the Illumina reads. 3

Molecular Typing, Genome Annotation, Analysis and Visualization
Multi-locus sequence typing (MLST) was performed using the Oxford and Pasteur typing schemes 4 to assign the sequence type (ST). Clonal complexes (CCs) were assigned using the BURST function available at pubmlst.org. The bla OXA-51-like variant combined with the CCs derived from both schemes and coregenome MLST (cgMLST) analysis, using the Ridom SeqSphere+ v. 7.0.4 software, were used to assign the isolate to an IC (Higgins et al., 2017).
The resistome of the bacterial isolate was identified using ResFinder v.3.2.0 (Zankari et al., 2012). Capsular polysaccharidetype (KL-type) and the outer core of the lipooligopolisacharide (OCL-type) were assigned using Kaptive Web (Wick et al., 2018). The motility phenotype was analyzed on 0.5% agarose plates, supplemented with 5 g/l tryptone, 2.5 g/l NaCl, and pH 7.4, inoculated on the surface and incubated overnight at 37°C (Skiebe et al., 2012). Prophage-related sequences were screened using the PHASTER tool and virulence factors using virulence factor database (VFDB; Arndt et al., 2016;Liu et al., 2019). The genome was annotated using Prokka integrated in the Galaxy web platform 5 and partially manually edited. SnapGene and SnapGene Viewer (from Insightful Science; available at snapgene.com) were used to predict open reading frames (ORF) and for genome visualization.

RESULTS AND DISCUSSION
The bacterial isolate AbBAS-1 was identified by MALDI-TOF and WGS as A. baumannii. Antimicrobial susceptibility testing revealed that the isolate was resistant to all tested antimicrobial agents except for amikacin (MIC <=4 mg/L), colistin (MIC <=0.5 mg/L), and tigecycline (MIC 0.5 mg/L; Table 1). Using phenotypical tests, a halo difference of 10 mm between the meropenem disk and both the dipicolinic acid and EDTA disks was observed, suggesting the presence of a MBL. The UNMIC/ ID-409 signaled the presence of a carbapenemase and the NMIC-502 and lateral flow immunochromatography identified it as a class B carbapenemase, while, by lateral flow immunochromatography the MBL was identified as part of the NDM-β-lactamase complex.
Sequencing identified the carbapenemase as bla NDM-6 , which differs from bla NDM-1 in one amino acid substitution (A233V). New Delhi Metallo-β-Lactamase-6 (NDM-6) has a similar hydrolyzing activity as NDM-1 and has been mainly reported in members of the Enterobacterales family to date (Williamson et al., 2012;Rahman et al., 2014;Bahramian et al., 2019). NDM-1-like derivative enzymes have been reported in carbapenem resistant Gram-negative organisms from multiple countries worldwide (Berrazeg et al., 2014;Dortet et al., 2014) including European countries, e.g., Germany, Switzerland, Slovenia, France, Belgium, Czech Republic, and very recently also in Southern Spain (Pfeifer et al., 2011;Bonnin et al., 2012;Fernandez-Cuenca et al., 2020). However, in the region of the Basque Country (Northern Spain), no NDM-like-enzymes have been previously reported. Our isolate AbBAS-1 was resistant to all β-lactams tested but also to fluoroquinolones and aminoglycosides, these findings are consistent with other NDM-producing isolates limiting the therapeutic options to amikacin, colistin, and tigecycline (Bonnin et al., 2012)   never consulted again the Basque Public Health System (Osakidetza). Because up until now NDM-6 has not been identified in Spain, we speculate that the patient acquired the NDM-6-positive CRAB isolate during his previous hospitalization in Northern Africa. The AbBAS-1 isolate has been assigned as ST85 Pas (Pasteur scheme) and ST957 Ox (Oxford scheme) and harbored the bla OXA-51-like variant bla OXA-94 . Furthermore, AbBAS-1 belonged to the clonal complex CC464 Pas and CC1078 Ox and could be assigned to the recently described IC9 (Müller et al., 2019). The novel IC9 was previously identified in A. baumannii isolates recovered between 2012 and 2016 in Belgium (bla NDM-1 -positive), Egypt (bla OXA-23 -positive), Italy (bla NDM-1 -positive), and Pakistan (bla OXA-23 -positive; Müller et al., 2019). ST85 A. baumannii, carrying bla OXA-94 and bla NDM-1 , recovered from Syrian civil war victims were first reported from Lebanon (Rafei et al., 2014). Furthermore, isolates encoding bla OXA-94 and bla NDM-1 were also reported from Southern Spain, Saudi Arabia, and Tunisia, or harboring bla VIM-1 in Egypt, indicating that the novel IC9 clonal lineage has a widespread distribution and was found repeatedly harboring MBLs (Jaidane et al., 2018;Al-Hassan et al., 2019;Al-Hamad et al., 2020;Fernandez-Cuenca et al., 2020). cgMLST analysis using currently available complete genomes of ST85 Pas A. baumannii isolates revealed that the AbBAS-1 isolate is closely related (81 alleles difference) to an NDM-1-positive CRAB isolated in Southern Spain in 2017 (Supplementary Figure 1).
Attempts to transfer the bla NDM-6 by electroporation experiments were not successful, suggesting that the MBL was encoded on the chromosome. S1-PFGE and Southern blot experiments confirmed that bla NDM-6 was encoded on the chromosome. Using WGS the bla NDM-6 environment could be identified arranged in the following order: ISAba14, aphA6, ISAba125, bla NDM-6 , ble MBL (resistance to bleomycin), trpF (phosphoribosylanthranilate isomerase), dsbC (tat twin-arginine translocation pathway signal sequence domain protein), cutA (periplasmic divalent cation tolerance protein), and ISAba14 (Figure 1). By BLASTn, the genetic environment of NMD-6 showed 99% similarity to an ST1089 Ox A. baumannii isolated in India in 2018 (Acc. No. CP038644), a single locus variant of ST957 Ox with bla OXA-94 , and which harbored a single copy of NDM-1. Further downstream, a 10,462 bp duplication was identified including a second copy of bla NDM-6 in the following genetic composition: ISAba125, bla NDM-6 , ble MBL , trpF, dsbC, cutA, and ISAba14 (Figure 1). The genetic environment of the second MBL was missing the type VI aminoglycoside phosphotransferase. The ISAba125 located directly upstream of both copies of bla NDM-6 could indicate that the two copies of the MBL were the result of duplication mediated through the mobile genetic element ISAba125 (IS30 family). IS element members of the family IS30 are known to transpose by a copy-and-paste mechanism (Szabo et al., 2010). Upstream of the aphA6 and bla NDM-6 a gene (colored green in Figure 1) truncated in three fragments was identified and was identical to an ATP-binding protein from an A. baumannii (Acc. No. CP038644.1) with the locus tag E5D09_10165. Particularly, 480 bp of the 5' end of the ATP-binding protein containing the start codon were located upstream of the ISAba33, which is followed directly by the next 44 bp of the ATP-binding protein. The 711 bp of the 3' end were found directly downstream of the second copy of ISAba14. Another copy of the 711 bp of the 3' end was also present 9,7 kb downstream supporting the finding of the duplication of the genetic environment of the bla NDM-6 .
The AbBAs-1 isolate harbored in addition to bla NDM-6 the intrinsic, ADC-158-like (with no insertion element upstream), ant(2'')- mph(E), msr(E) antibiotic resistance determinants, while upstream of the bla OXA-94 ISAba1 was located. The isolate AbBAS-1 also presents an S81L substitution in DNA gyrase subunit A and S84L substitution in ParC, which are known to be associated with fluoroquinolone resistance. By typing the capsular polysaccharide (K and/or O-antigen), which is a critical determinant of virulence, the AbBAS-1 isolate was assigned as KL77, a KL type previously reported in ST2 and ST10 isolates, and OCL6 the third most common OCL type in A. baumannii. In accordance to recent studies that have found K & O-antigen diversity within members belonging to the same clone, capsule typing is a promising epidemiological marker in combination with MLST (Wyres et al., 2020). Of note is the mucoviscous phenotype of the AbBAS-1 colonies when grown on agar plates generating a viscous string >5 mm in length between a colony and a inoculation loop (string test), a phenotype that has been associated with hypervirulent K. pneumoniae strains (Fang et al., 2004). In addition, the A. baumannii isolate exhibited a nonmotile phenotype. Phage analysis identified in the AbBAS-1 two questionable phage regions both similar to Acinetobacter phage YMC/09/02/B1251_ABA_BP (Acc. No. NC_019541.1) and an incomplete phage region similar to Pseudomonas phage nickie (Acc. No. NC_042091.1). Virulence factors known to be associated with A. baumannii have been identified using VFDB and included genes linked with adherence (ompA); biofilm formation (csuE, csuC, csuB, csuA, csuA/B, pgaD, pgaC, pgaB, and pgaA); regulation (bfmR, bfmS, abaI, and abaR); phospholipases (plc, plcD); and iron uptake (basJ, basI, basH, barB, barA, basG, basF, entE, basD, basC, bauB, bauE, bauC, basB, basA, and bauF).
In conclusion, to the best of our knowledge, this is the first report of bla NDM-6 in an A. baumannii isolate. The CRAB isolate encoded two copies of bla NDM-6 in close proximity with ISAba125. The carbapenemase NDM-6 has been detected in a ST85 Pas multidrug-resistant isolate belonging to the recently described IC9. The present study highlights the complexity and diversity of the genetic environment of NDM-1-like enzymes contributing to its dissemination. The emergence of NDM-6 in an A. baumannii clinical isolate highlights the need of surveillance studies and exhaustive control to prevent its spread in the clinical setting. The implementation of infection control measures should also be a priority to fight against multidrug-resistant isolates in the nosocomial environment.

DATA AVAILABILITY STATEMENT
The assembled genome generated in this project has been deposited in the NCBI and we are awaiting for processing to include the accession number in the manuscript.