Emergence of New Delhi Metallo-β-Lactamase (NDM-5) in Klebsiella quasipneumoniae from Neonates in a Nigerian Hospital.

Carbapenem-resistant Klebsiella pneumoniae is of global health importance, yet there is a paucity of genome-based studies in Africa. Here we report fatal blood-borne NDM-5-producing K. quasipneumoniae subsp. similipneumoniae infections from Nigeria, Africa. New Delhi metallo-β-lactamase (NDM)-producing Klebsiella spp. are responsible for high mortality and morbidity, with the NDM-5 variant showing elevated carbapenem resistance. The prevalence of NDM-5 in Klebsiella has been limited primarily to K. pneumoniae, with only one isolate being collected from Africa. During an outbreak of sepsis in a teaching hospital in Nigeria, five NDM-5-producing K. quasipneumoniae subsp. similipneumoniae sequence type 476 isolates were identified. Given the increased resistance profile of these strains, this study highlights the emerging threat of blaNDM-5 dissemination in hospital environments. The observation of these NDM-5-producing isolates in Africa stresses the urgency to improve monitoring and clinical practices to reduce or prevent the further spread of resistance.

In 2011, a multidrug-resistant (MDR) Escherichia coli strain isolated in the United Kingdom from a patient returning from a recent hospitalization in India was found to harbor . In comparison to the prevalent NDM-1 allele, NDM-5, a 2-amino-acid variant, conferred elevated carbapenem resistance (3) and has subsequently been identified in isolates from other members of the Enterobacteriaceae family worldwide . Among the Enterobacteriaceae, reports of NDM-5-producing Klebsiella spp. are sporadic (17-19, 21-24, 31), with only one isolate collected from a hospitalized infant in northern Africa (20). Furthermore, the prevalence of NDM-5 in Klebsiella has been limited primarily to K. pneumoniae strains.
In this study, we report on an NDM-5-producing K. quasipneumoniae subsp. similipneumoniae strain representing the sequence type 476 (ST476) clonal group isolated from neonates at the University of Abuja Teaching Hospital, Gwagwalada, Nigeria.

RESULTS AND DISCUSSION
Clinical setting of Klebsiella outbreak. The evaluation of the causative agent of this outbreak was facilitated by one of the surveillance laboratories for the Community-Acquired Bacteremic Syndrome in Young Nigerian Children (CABSYNC) program, which is located at the University of Abuja Teaching Hospital, Gwagwalada, Nigeria, and the diagnostic service was offered at no cost to the parents of these babies. At most health care facilities in Nigeria and, indeed, throughout sub-Saharan Africa, diagnostic microbiology laboratories are not readily available, and where they are available, the service is neither free nor affordable; thus, most septic newborns are treated empirically.
An outbreak of neonatal sepsis occurred during the month of April 2016, when there was a high admission in the special care baby unit of the University of Abuja Teaching Hospital, Gwagwalada, in central Nigeria. This is one of two special care neonatal units in the Federal Capital Territory, both of which cater to a population of over 3 million (National Bureau of Statistics, Nigeria). The bed occupancy rates typically exceed the total number of beds, with babies being nursed on Resuscitaire units (Dräger, Lübeck, Germany) and less critically ill babies occasionally sharing cots. Mechanized respiratory support was limited to continuous positive airway pressure (CPAP). The outbreak prompted an increased level of infection control, such as enforced hand washing, restricted access to the unit, and temporary closing of the unit for 4 days for sanitization, which presumably led to resolution of the outbreak by the beginning of May 2016.
Sequencing of neonatal bloodstream isolates of MDR Klebsiella spp. Illumina NextSeq genome sequencing was performed on seven bloodstream isolates of Klebsiella spp. from babies being treated in the University of Abuja Teaching Hospital, Gwagwalada, in central Nigeria, as part of an ongoing (2012 to 2016) surveillance for community-acquired bacteremic syndromes (CABSYNC). Part of this collection included five isolates obtained during the April 2016 outbreak of neonatal sepsis. The resulting de novo assembly Illumina sequence coverage of five of the seven isolates was between 90-fold (for isolate G4612) and 230-fold (for isolate G4584) across an average of 121 contigs per genome (minimum, 46 for G4704; maximum, 146 for G4612), resulting in average draft genome sizes of between 5.4 Mbp (G4704) and 5.8 Mbp (G4601) ( Table 1). Two representative isolates, G4584 and G747, which received additional Oxford Nano- Taxonomic classification. Phylogenetic characterization via in silico multilocus sequence typing (MLST) and determination of single nucleotide polymorphisms (SNPs) of publicly available Klebsiella species genome sequences (n ϭ 4,963), including Klebsiella species isolates from Nigeria (n ϭ 93), revealed that the Nigerian isolates in this study could be taxonomically classified as K. quasipneumoniae subsp. similipneumoniae (Fig. 1). The taxonomy of all Klebsiella species genomes was confirmed by the average nucleotide sequence identity (ANI). Those phylogenetically characterized as K. quasipneumoniae subsp. similipneumoniae (n ϭ 102) in this study had Ͼ98% ANI to K. quasipneumoniae subsp. similipneumoniae 07A044 T (see Table S1 in the supplemental material). Sequence type 476 (ST476) was identified in six of the K. quasipneumoniae subsp. similipneumoniae isolates collected from the University of Abuja Teaching Hospital, Gwagwalada, Nigeria (Table 1), representing a clonal group with 98.94% to 100% identity by pairwise ANI.
Phenotypic susceptibility characterization. Antimicrobial susceptibility testing of the K. quasipneumoniae subsp. similipneumoniae isolates demonstrated various degrees of resistance to broad-spectrum antibiotics ( Table 2). The April 2016 outbreak isolates, G4582, G4584, G4593, G4601, and G4612, were resistant to virtually every antibiotic tested, including the carbapenems imipenem, meropenem, and ertapenem (MICs, Ͼ32 g/ml). Exceptions for resistance to other antibiotics included intermediate resistance to amikacin (MICs, 24 to 32 g/ml) and, in the case of G4612, also resistance to chloramphenicol (MIC, 16 g/ml). No other K. quasipneumoniae subsp. similipneumoniae isolate obtained as part this study had a high level of resistance to all antibiotics tested, including the carbapenems. G747, which shares the same sequence type, ST476, and five of six plasmids with G4584 but was isolated from the same hospital 3 years earlier, exhibited a similar resistance profile but was sensitive to imipenem (MIC, 0.5 g/ml), meropenem (MIC, 0.125 g/ml), and ertapenem (MIC, 0.064 g/ml), as well as cefoxitin (MIC, 8 g/ml). Similarly, G4704, obtained 3 months later, was also sensitive to carbapenems (imipenem, meropenem, and ertapenem), as well as cefoxitin, ceftazidime, cefepime, piperacillin-tazobactam, and amikacin, and showed intermediate resistance to amoxicillin-clavulanic acid.
Comparative analysis of the NDM-5 genetic environment. IncX3-type plasmids have a narrow host range and are found primarily within the Enterobacteriaceae (41), and IncX3-type plasmids containing bla NDM-5 were found within several members of the Enterobacteriaceae, including E. coli, Salmonella enterica subsp. enterica serovar Typhimurium, K. pneumoniae, K. michiganensis, and K. quasipneumoniae (Fig. 2). Comparative analysis of all fully sequenced IncX3 plasmids containing an NDM-5 allele was performed to assess the genetic context of the NDM-5 gene. Ten different structural forms were identified from a total of 48 plasmid sequences available in GenBank and the five outbreak isolates from this study and are denoted groups A to J (Fig. 2). The plasmid backbone was nearly identical across the groups (conserved region, Fig. 2), with all plasmids carrying genes for replication (pir and bis), partitioning (parA-parB), entry exclusion (eex), maintenance (topB and stpA), and conjugative transfer (type IV secretion system and taxA, taxB, taxC, and taxD). However, there were some structural differences resulting from potential insertions/deletions of components of existing insertion sequence (IS) elements (Fig. 2, blue arrows). For example, group F may have had a second insertion of IS5, disrupting the IS3000 transposase, and groups B, D to G, and J displayed apparent insertions of ISAba125 between IS3000 and IS5 that were lacking in the prototype sequences from group C. It is difficult to determine from the available sequence data whether the group C sequences resulted from deletion of these IS elements or whether the other groups represented novel IS insertions relative to group C. Only group I, represented by the unpublished E. coli plasmid pMTC948, possesses an additional bla gene (bla SHV ). These results suggest that the variable region (Fig. 2) may be highly dynamic, but other than the loss of a promoter from the end of the ISAba125 fragment, previously shown to drive the expression of bla NDM-1 and ble MBL (42), it is unclear if these differences have any effect on the expression of bla NDM-5 , ble MBL , or the accessory genes trpF and dsbC. Both trpF and dsbC appeared to be tightly linked to bla NDM-5 in all sequences examined, with the exception of the sequence of the unpublished plasmid pTBCZNDM01 in group H, which lacked both trpF and dsbC, suggesting a critical role either in the stability, retention, or spread of this element or in facilitating enzyme functionality. The NDM-5-containing plasmid from K. quasipneumoniae subsp. similipneumoniae is structurally similar to members of group E, with two differences: a partial duplication of the IS3000 element and truncation of the accessory replication protein Bis via insertion of the Tn5403 transposon. Inactivation of bis results   in the loss of beta origin replication, but not alpha or gamma origin replication, in the prototypical IncX family plasmid R6K (43). Given that similar IncX3 plasmids possess multiple origins of replication (44), it is unlikely that the loss of Bis will reduce the spread of antibiotic resistance. Transmission of NDM-5. The transmission of a plasmid carrying NDM-5 was evidenced by performing conjugation experiments with donor cells harboring the NDM-5-containing plasmid. Due to the extensive drug resistance of strain G4584, the NDM-5-containing plasmid was electroporated into E. coli DH10B and subsequently transferred to a separate E. coli strain (JW2786-1). JW2786-1 cells harboring the NDM-5-containing plasmid grew on CHROMagar KPC plates supplemented with kanamycin only if the NDM-5-containing plasmid transferred via conjugation from DH10B to JW2786-1. Transfer of the bla NDM-5 IncX3-type plasmid to recipient cells (E. coli JW2786-1) was confirmed by PCR and the acquisition of carbapenem resistance in the recipient strain, which was measured using disk diffusion. The recipient strain turned from Salmonella enterica subsp. enterica serovar Typhimurium; CP024820 from Citrobacter freundii; CP014006, KF220657, KU761328, MH161191, and MH341575 from K. pneumoniae; CP022351 and CP023188 from Klebsiella michiganensis; and MG833406 from Klebsiella oxytoca. Group F represents the sequence with GenBank accession no. KY435936 from E. coli plasmid pNDM5_WCHEC0215. Group G represents the sequence with GenBank accession no. MG591703 from E. coli plasmid pNDM-EC36. Group H represents the sequence with GenBank accession no. MH107030 from K. pneumoniae plasmid pTBCZNDM01. Group I represents the sequence with GenBank accession no. MH349095 from E. coli plasmid pMTC948. Group J contains 5 examples (from this study), sequences with GenBank accession no. NZ_NFXE01000097 (G4582), CP034133 (G4584), NZ_NFXD01000099 (G4593), NZ_NFXB01000102 (G4601), and NZ_NFWY01000105 (G4612), all from K. quasipneumoniae subsp. similipneumoniae. Arrows indicate protein-coding genes (CDSs) drawn to scale and colored as follows: salmon for factors involved in plasmid replication, yellow for factors involved in plasmid maintenance and mobility, red for antibiotic resistance determinants, blue for mobile elements, green for other known proteins, and gray for unknown proteins. Homologous CDSs between adjacent groups are joined vertically by colored lines. resistant to meropenem (decrease in zone diameter, 35 mm to 15 mm), cefoxitin (23 mm to 6 mm), amoxicillin-clavulanic acid (22 mm to 8 mm), and cefepime (36 mm to 12 mm) after acquiring the bla NDM-5 IncX3-type plasmid.

AMP AMC CEF FOX CAZ CRO CTX FEP TZP IPM MEM ETP GEN AMK TOB SXT TET CIP CHL ESBL CAZ-CLA ESBL CTX-CLA
In conclusion, we describe the occurrence of clonal (ST476) NDM-5-producing K. quasipneumoniae subsp. similipneumoniae isolates in Africa with an IncX3-type plasmid highly similar to the plasmids found in other members of the Enterobacteriaceae. The original source and transmission route of these isolates are unclear, but the close proximity of patients within the hospital when this outbreak occurred could have played a role in its transmission to other neonates in the unit. After the introduction of infection control measures, no isolates with NDM-5 were identified. Given the increased resistance profile of these strains and the associated high mortality rate among infected patients, this study highlights the emerging threat of the plasmid-mediated transfer and spread of bla NDM-5 in hospital environments. Furthermore, the increasing pervasiveness of NDM-5 enzymes confirmed in North Africa and the now newly identified occurrence in western Africa stress the urgency to improve monitoring and clinical practices to reduce or prevent further the spread of resistance.

MATERIALS AND METHODS
Participant description. Children were enrolled per a previously published protocol (45,46). Briefly, children less than 5 years old who presented to any of the enrolling clinical facilities in the Federal Capital Territory of Nigeria with clinical symptoms that were suggestive of bacteremia were enrolled following the provision of informed consent by the parent or guardian.
Bacterial isolation and culturing. Blood sampling and processing were as previously described (45,46). Briefly, only aerobic blood culture bottles were utilized, and cultures were held in a Bactec 9050 incubator for a maximum of 5 days. Bacteria were identified by a combination of morphology and biochemical testing for Enterobacteriaceae using an API 20E system (bioMérieux, France). All blood-borne bacterial isolates that were recovered from September 2012 to September 2016 and that were identified as Klebsiella spp. were shipped to the University of Nebraska, where secondary confirmation of their identity was performed using standard biochemical tests. For genomic DNA isolation, Klebsiella isolates were cultured aerobically at 250 rpm in 1.5 ml brain heart infusion (BHI) medium overnight at 37°C. Only one bacterial isolate was processed per participant.
DNA isolation and whole-genome sequencing. Using a 1-ml overnight BHI culture, genomic DNA was isolated using a MasterPure Gram-positive DNA purification kit (Epicentre). The extracted genomic DNA was resuspended in ϳ30 l Tris-EDTA (TE) buffer and quantified using a NanoDrop spectrophotometer. Paired-end 150-bp Nextera XT libraries of whole genomic DNA were sequenced on an Illumina NextSeq sequencer with a target average coverage of 100-fold. All sequences were de novo assembled individually using the SPAdes algorithm (47). The genomes of strains G747 and G4584 were selected for additional sequencing using the Oxford Nanopore minION technology (one-dimensional sequencing on an R9.4 flow cell). G4584 was hybrid de novo assembled using reads from both the Illumina NextSeq and Oxford Nanopore minION sequencers with the Unicycler (v0.4.6) assembler (48). G747 was de novo assembled using the long-read assembler Canu (v1.7.1) (49), and the consensus sequence was generated using the Racon (v1.3.1) program (50). The circular nature of the assembled contigs was determined based on the presence of nearly identical repeats at the contig ends. The redundant regions were trimmed from one end, and the contig orientation and starting position were adjusted such that the first gene of the chromosome and plasmids was dnaA and repA, respectively. The final G747 assembly was polished using the Pilon (v1.22) tool (51) and the Illumina reads. All assembled sequences were annotated with NCBI's prokaryotic genome annotation pipeline (PGAP) (52).
Genomic analysis. In silico MLST of the seven-locus K. pneumoniae Pasteur Institute MLST scheme (http://bigsdb.pasteur.fr/klebsiella/) and identification of virulence factors were performed using the LOCUST typer (53). Resistance Gene Identifier (RGI) software (54) in strict mode was used to predict the antibiotic resistome from whole-genome sequence data using the Comprehensive Antibiotic Resistance Database (CARD) (54)(55)(56). A whole-genome alignment was inferred from SNPs identified by the Northern Arizona SNP Pipeline (NASP; v1.0.2) (57) using the genome of K. quasipneumoniae subsp. similipneumoniae 07A044 T (GenBank accession no. NZ_CBZR010000000) as the reference. The resulting alignment was run through the Gubbins (v2.2.1) program (58) to filter out the effects of recombination on our maximum likelihood phylogenetic tree, generated using the RAxML tool (59) under the GTRCAT model with 100 bootstrap replicates. The resulting tree was rendered with metadata annotated using the Interactive Tree of Life (iTOL) (60-62). Taxonomic assignments were confirmed using the Mash (v1.1.1) program, which is based on ANI (63).