A 31-year-old woman was admitted to a surgical unit at a public hospital in Benin on March 06, 2019. She presented post-caesarean hematoma and was hospitalized for surgical intervention. During laparotomy, surgical antimicrobial prophylaxis was administered as intravenous ceftriaxone (1 g), and after intervention, empirical antibiotic therapy consisting of intravenous imipenem (500 mg every 8 hours) was initiated for one week. On the tenth day, the patient’s clinical condition worsened and she developed fever (38 °C) and wound suppuration. Preliminary investigation revealed that the patient had no previous history of travel or hospitalization abroad. Intensive programs of environmental cleaning and strict contact isolation precautions were applied. However, following the initial treatment of 500 mg imipenem per 8 hours, the patient preferred to continue with unspecified indigenous treatment due to lack of financial support. As she was no longer in the hospital, the clinical outcome is unknown.
The culture of a pus swab revealed Gram-negative coccobacilli that were glucose-non-fermentative, non-motile, and oxidase-negative. Biochemical identification was performed with the Analytical Profile Index (API 20E, Biomérieux, France) and results were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing was assessed using the modified Kirby-Bauer disc diffusion method and confirmation was done by the microbroth dilution method. The interpretation breakpoints were based on the criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/ast_of_bacteria/). Except for amikacin, colistin and ciprofloxacin, the isolate was resistant to all tested antimicrobial agents with the following Minimum Inhibitory Concentration (MIC) values: ceftazidime (> 16 mg/l), imipenem and meropenem (> 16 mg/l), gentamicin and tobramycin (> 8 mg/l), ceftazidime/avibactam (> 16/4), sulfamethoxazole-trimethoprim (> 8/152). The isolate was therefore considered as extensively drug resistant (XDR) [4]. Additionally, to confirm the resistance pattern, we used the multiplex lateral flow immunochromatographic test, the RESIST-3 O.K.N. ICT (Coris Bioconcept, Gembloux, Belgium), which confirmed the presence of NDM (Fig. 1).
Whole-genome sequencing (WGS) was subsequently performed for detection and characterization of resistance genes, using DNA from a single-colony isolate employing the EZ1 advanced XL biorobot and the tissue DNA kit (Qiagen, Hilden, Germany) with the bacterial card, according to the manufacturer’s instructions. A standard Nextera XT library (Illumina, San Diego, USA) was constructed (Nextera XT DNA library preparation kit, Illumina, San Diego, USA) and subsequently sequenced on an Illumina MiSeq instrument with a 250-bp paired-end protocol (MiSeq v3 chemistry, Illumina, San Diego, USA) according to the manufacturer’s instructions. Data was analyzed as follows. First, reads were trimmed with Trimmomatic 0.36 [12] with the settings ‘NexteraPE-PE.fa:2:30:10’, ‘LEADING:10’, ‘TRAILING:10’, ‘SLIDINGWINDOW:4:20’, and ‘MINLEN:40’. Processed reads were then assembled de novo using SPAdes 3.13.0 [13] with the ‘careful’ option enabled and the ‘cov-cutoff’ parameter set to 10. Contigs smaller than 1000 bases were removed with seqtk seq 1.2 (https://github.com/lh3/seqtk) using the ‘-L’ option. Assembly statistics were determined using Quast 4.4 with default settings [14]. Genome annotations were created using Prokka 1.13 [15] (Table 1).
The NCBI National Database of Antibiotic Resistant Organisms (NDARO) [16], Virulence Factor (full) database (VFDB) [17], and PlasmidFinder database, were used for genotypic detection of genes encoding antimicrobials, virulence factors, and plasmid replicons, respectively, using SRST2 0.2.0 [18] with default settings. The XDR status of the isolate was confirmed by harboring several resistance genes against aminoglycosides (aph(6)-Id, aph(3”)-Ib, ant(3'')-IIa, and aac(3)-IId), beta-lactamases (blaNDM−1, blaOXA−58, blaOXA−558, blaADC−166), macrolide-lincosamide-streptogramin B (msr(E)), macrolide (mph(E)), sulfonamide (sul2), tetracycline (tet(39)), and bleomycin (ble). Additionally, 67 loci encoding different virulence factors were detected including genes related to biofilm formation such as ompA, bfmS, csuE, and a K1 capsular polysaccharide (ABK1) (see Supplementary). No plasmid replicons from the PlasmidFinder database were detected.
Sequence typing was performed with the corresponding regular multi-locus sequence typing (MLST) schemes from Oxford University and Institut Pasteur [19], as described in Bogaerts et al. [20]. MLST analysis detected sequence type 836 (Oxford University scheme) and 388 (Institut Pasteur scheme). No isolates were present for the former, but the latter returned two isolates from Taiwan (from 2012 and 2013) and a single isolate from Norway (year unknown) with the same sequence type. The sample was screened for the presence of the Tn125 transposon by mapping (trimmed) reads against its reference sequence (NCBI KF702386.1) using Bowtie2 2.3.0 [21] with the ‘--sensitive’ setting enabled. In total, 99.55% of the Tn125 transposon reference length of 10,624 bp was covered by at least one read, with three breakpoints however present at ~ 7.7 kb, 8.5 kb, and 9.2 kb, indicating some minor rearrangements. The median depth of coverage of the Tn125 transposon was 98.20X (compared to 83.0X for the whole genome). The alignment and annotation for the Tn125 transposon are visually represented in Fig. 2. An additional de novo assembly was then performed using plasmidSpades 3.13.0 with the ‘--plasmid’ and ‘--careful’ options enabled [22] to reconstruct putative plasmids. This resulted in 21 contigs, with the blaNDM−1 gene located near the center of the largest contig (88,063 bp) and its surrounding region of 7,626 bp aligned to the Tn125 transposon with over 99% sequence identity. Outside of this region, no alignments were found between this putative plasmid contig and the pNDM-BJ01 (NCBI NC_019268.1) plasmid. Additional screening of the putative plasmid contig against the Plasmid Database (PLSDB) online platform [23] (v2020_03_04) with mash was therefore performed and provided four matches: a large plasmid of 78,125 bp (NCBI CP038501.1) that was found in A. baumannii, and three smaller ones (NCBI JQ739158.1, KF220658.1, KR059864.1) with sizes 4,797, 1,634 and 7,865 bp, respectively. Alignments with BLAST showed that the majority of the putative plasmid contig carrying blaNDM−1 aligned to regions on the larger CP038501.1 plasmid (albeit with several rearrangements), except for the region containing the sequence that aligned to the Tn125 transposon (Fig. 3). The three smaller plasmid matches all corresponded to parts of the Tn125 transposon (results not shown).