Genomic Characterization of a Clinical NDM-1-Producing Klebsiella michiganensis from Brazil

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.


Introduction
Antimicrobial resistance presents a significant global threat, constraining treatment options for bacterial infections, thereby diminishing clinical effectiveness, escalating treatment expenses, and increasing mortality rates [1].It is known that once resistance genes are successfully established in plasmids, they can spread resistance rapidly through different lineages, species, or even genera [2].Klebsiella michiganensis, initially recovered from a toothbrush holder in 2013, is now recognized as an emerging critical human pathogen [3,4].This species is part of the K. oxytoca complex, consisting of nine related species, including K. oxytoca, K. grimontii, K. huaxiensis, K. michiganensis, K. pasteurii, K. spallanzanii, and three unnamed taxa (taxons 1, 2, and 3), which are challenging to differentiate reliably based on their phenotypic characteristics [5].It has been suggested that K. michiganensis may be more clinically relevant in human-associated infections than K. oxytoca, but differentiation between these two species is difficult, potentially leading to an underestimated real prevalence of K. michiganensis [6,7].The complex can be divided into phylogroups based on variation in the sequence of the intrinsic gene bla OXY , encoding a β-lactamase that confers resistance to amino and carboxy-penicillin, of which K. michiganensis belongs to the phylogroup Ko1 (with Ko5 representing a sub-lineage) [5].The presence of a wide range of accessory genes in the pan-genome of K. michiganensis suggests an open pan-genome, which indicates its ability to adapt to diverse environmental conditions and its potential genomic plasticity [8].
Moreover, the emergence of carbapenemase-producing K. michiganensis poses a new health threat, especially when bla-coding genes have the capability to horizontally spread through mobile genetic elements [3].The resistance in these bacteria primarily involves genes such as bla KPC , bla NDM , and bla IMP , which are associated with plasmids.Strains carrying more than one gene have already been documented in recent years, all co-harboring bla NDM with another gene [9][10][11].
Unlike serine-β-lactamases, metallo-β-lactamases require an active site ion and are not inhibited by the so-called second generation of β-lactamase inhibitors, avibactam and vaborbactam, thus impacting antimicrobial therapy [12,13].Currently, 61 variants of NDM have been identified (according to the BLDL database http://bldb.eu/,accessed on 20 June 2024) [14].In Brazil, the first bla NDM gene was identified in a Providencia rettgeri in 2013 and the variant detected was the NDM-1 [15].According to a recent review, between 2012 and 2021, NDM-producing isolates were identified in 14 bacterial species belonging to eight different genera in Brazil, with 80% belonging to Klebsiella spp.and NDM-1 being the predominant allele (54% of isolates) [16].Another large study identified an increase in the prevalence of NDM-producing organisms in the southern region of Brazil [17].
The bla NDM gene is carried by various Inc-group plasmids, such as IncA/C, IncL/M, IncF, IncX, IncHI1A, IncHI1B, and IncN [18,19].In terms of K. michiganensis, there have been few reported cases of strains carrying the bla NDM gene, with four from China [9][10][11]20], one from Japan [21], and one from South Africa [22], all of them located in the IncFIB or IncX3 plasmids.To the best of our knowledge, however, there are no reported cases of NDM-producing K. michiganensis on the American continents.In the present study, we characterized a clinical bla NDM-1 -producing K. michiganensis strain that was isolated in Brazil and explored the phenotypic and genotypic characteristics of the isolate and the plasmid.

Conjugation Assay and Plasmid Size Determination
A conjugation assay with Escherichia coli K12 strain J53 (sodium azide-resistant) was performed following Fernandes et al.'s (2017) protocol [27].The isolates ID_1060/21 and E. coli J53 were reactivated on TSA and incubated at 35 • C for 24 h.One colony from each sample was inoculated into LB broth and incubated at 35 • C overnight.The optical density of the two samples was measured and equalized to 0.7 on the McFarland scale, and 100 µL of each was inoculated into the same tube with 3 mL of LB broth and placed at 35 • C overnight.Mueller Hinton plates were prepared with 2.0 µg/mL of imipenem and 100 µg/mL of sodium azide and, on the same day, 100 µL of growth was inoculated onto the plates and spread with a Drigalsk loop, and then incubated at 35 • C for 24 h.Colonies grown on the plates were reseeded onto TSA next to a ceftriaxone disk and isolated colonies grown close to the disk were identified, using MALDI-TOF, as E. coli.Of these, a new isolation was performed to obtain pure samples of the conjugate to proceed with the multiplex PCR, to confirm the transference of the resistance gene, and to perform the minimum inhibitory concentration with an E-test with beta lactams.
To determine the size of the plasmid, S1-nuclease pulsed-field gel electrophoresis was performed both in the parental (ID_1060/21) and in the transconjugant strain.The plugs were prepared from a suspension with a transmittance of 4-5%, measured in a spectrophotometer (Analyser, São Paulo, Brazil).Plugs were digested with 0.089 unit of S1-nuclease enzyme (Promega, Madison, WI, USA) for 45 min at 37 • C. For plasmid size determination, a molecular marker, Salmonella enterica serotype Braenderup H9812 [28] digested with 30 U XbaI at 37 • C/16 h, was employed.Electrophoresis was performed on the CHEF DR III (BioRad, Hercules, CA, USA) at 6 V/cm and 14 • C, with an initial time switch of 1.0 s and a final pulse of 40.0 s, for 17 h.The gel was stained with ethidium bromide (5 µg/mL) and visualized in a photo documenter (DNR, Bio Imaging Systems, Jerusalem, Israel).Plasmid size was determined using BioNumerics v.8.0 software (Applied Maths, Sint-Martens-Latem, Belgium).

DNA Extraction and Whole-Genome Sequencing
Genomic DNA of the strain ID_1060/21 was extracted using the commercial Wizard ® Genomic DNA Purification kit (Promega, Madison, WI, USA), starting from an initial volume of 1.4 mL of overnight bacterial growth at 35 • C in LB broth and following the manufacturer's protocol.Purified DNA was initially assessed using a spectrophotometer from NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) and then quantified in a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).The DNA's integrity was verified by E-gel (Invitrogen, Waltham, MA, USA) electrophoresis.
For short reads' sequencing using Illumina technology (Illumina, San Diego, CA, USA), the genomic library was prepared using magnetic beads with the Illumina DNA Prep kit to cleave and tag the DNA.To form clusters, a PCR was performed with NexteraTM DNA CD Indexes.Sequencing was carried out at the Strategic Laboratory, Instituto Adolfo Lutz, Brazil, using Illumina MiSeq ® equipment to generate 75 bp paired-end reads.For the long reads' sequencing, libraries were prepared with the Rapid Barcoding Sequencing kit and sequenced using MinION equipment (Oxford Nanopore Technologies, Oxford, UK).

Genomic Analyzes
FastQC was used to verify the quality of the reads and GC content.The hybrid assembly of the genome's short and long reads was performed using Unicycler and another assembly was performed, using Flye, for only the long reads [29].To confirm the identity of ID_1060/21, an Average Nucleotide Identity (ANI) analysis was per-formed against a local database generated with representative genomes of all Klebsiella species available from the NCBI (Supplementary Table S1).From the assembled genomes, predicted resistance and gene locations were analyzed using ResFinder, PlasmidFinder, and MGEFinder (all using the Center for Genomic Epidemiology services available at http://www.genomicepidemiology.org/services/ accessed on 1 July 2024), and, to analyze the virulence genes, we employed the Virulence Factor Database [29], which compares the query genome with the references' accession numbers: AAF37887, NP_752613, NP_286010, and NP_462662.The online tool PathogenWatch (https://pathogen.watch/accessed on 1 July 2024) was used also for species identification and the determination of sequence length, sequence type (ST), antimicrobial resistance, and the virulence genes' conference.
For the plasmids comparison, genome sequences were annotated with Rapid Annotation using Subsystem Technology (RAST) and the plasmids' sequences were compared with BLAST Ring Image Generator (BRIG) software version 0.95.A partial alignment of the genetic environment of the resistance gene was visualized with Clinker (https://cagecat.bioinformatics.nl/tools/clinkeraccessed on 3 June 2024).
This Whole-Genome Shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number JAOCNS000000000.The version described in this paper is version JAOCNS020000000.

Results
The isolate ID_1060/21 was initially misidentified as K. oxytoca by biochemical tests and MALDI-TOF MS.Nevertheless, ANI analyses identified ID_1060/21 as K. michiganensis, with values ≥ 0.9714 (Supplementary Table S1; Supplementary Figure S1).The presence of chromosomal bla OXY-5 , according to ResFinder, confirms the species' relationship to the variation in the sequence of this β-lactamase.
K. michiganensis ID_1060/21 was resistant to monobactams, penicillins, cephalosporins, tetracycline, and ertapenem, but presented susceptibility to almost all aminoglycosides, fluoroquinolones, phenicol, folate inhibitors, and polymyxin B (MIC 0.5 mg/L) (Table 1).With these phenotypic results, the isolate was classified as having multidrug resistance (MDR) [30].The multiplex PCR assay was positive only for the bla NDM gene.The conjugative transfer of bla NDM was successfully achieved to an E. coli J53 recipient strain, confirmed by conventional PCR.The transconjugant strain showed more than a 60-fold increase in comparison to the wild E. coli J53 (Table 2).S1-PFGE revealed that isolate ID_1060/21 carries three plasmids sized 163.49kb, 105.91 kb, and 50.52 kb, but only the 103.55 kb plasmid was transferred according to the S1-PFGE analysis (Supplementary Figure S2).Hybrid assembly successfully identified the presence of 10 contigs in a chromosome length of 6,071,905 bp.Three additional contigs were assembled, corresponding to the plasmids observed in S1-PFGE.

Discussion
In this study, we report the first case in Brazil of K. michiganensis harboring the blaNDM-1 gene within a transferable IncFIB plasmid.The emergence of carbapenemase-producing

Discussion
In this study, we report the first case in Brazil of K. michiganensis harboring the bla NDM-1 gene within a transferable IncFIB plasmid.The emergence of carbapenemase-producing K. michiganensis highlights its potential role as a reservoir and disseminator of clinically relevant resistance genes [9].Noteworthy cases of K. michiganensis carrying NDM variants have been reported globally in recent years, spanning regions such as China [9][10][11]20], South Africa [22], and Japan [21].
Diverging from our findings, previous reports of carbapenemase NDM in K. michiganensis have identified different Inc-type plasmids.NDM-1 was located in IncX3 plasmids in South Africa [22] and China [10], while NDM-5 was associated with IncX3 plasmids in Japan [21] and China [11].The presence of resistance genes such as bla NDM and its variants in diverse plasmids may elucidate the broad dissemination of these enzymes [17], thus posing a significant public health challenge.
An in-depth analysis of the genetic context of the bla NDM-1 gene revealed a conserved triad of genes (ble MBL trpF, and dsbD) downstream of bla NDM-1 , corroborating the existing literature [31].
According to the phylogenetic tree, ID_1061/21, identified as the new ST418, was closely associated with two isolates from Russia, both classified as ST349.Pan-genomic analyses conducted by Simoni et al. (2023) revealed that 35% of K. michiganensis genes are part of the core genome, emphasizing the existence of a diverse array of accessory genes within this species [8].According to the authors, this suggests potential genomic plasticity linked to adaptation in novel environments, as well as the continuous microevolution of K. michiganensis.An escalating number of reports document K. michiganensis co-harboring genes implicated in carbapenem resistance.In a Chinese study encompassing 25 carbapenem-resistant K. oxytoca complex strains, K. michiganensis emerged as the predominant species in 16/25 (64% of isolates), with 5 harboring bla NDM resistance genes and an additional 10 strains containing bla KPC or bla IMP [32].
Since the initial identification of NDM in Brazil, numerous publications have spotlighted carbapenemase occurrences in Enterobacterales.This study represents the first report of NDM-producing K. michiganensis in the country, elucidating the plasmid and genetic context of the bla NDM-1 gene harbored by a transferable IncFIB plasmid.The increasing incidence of K. michiganensis carrying, accumulating, and disseminating resistance genes globally underscores the critical need for reinforced vigilance, aiming to identify and control the dissemination of carbapenemase-producing pathogens in healthcare settings.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/microorganisms12071408/s1, Figure S1: Heatmap visualization of Average Nucleotide Identity between the Klebsiella spp.genomes available in the NCBI database.A color key located in the upper left corner represents the percentage of identity between the analyzed genomes.The greater the percentage of identity between the genomes, the warmer the color.The clustering of ID_1060/21 with K. michiganensis genomes is observed, as is its distance from other species of the K. oxytoca complex.The database used to construct this figure is presented in Supplementary Table S1; Figure S2: S1-PFGE of ID_1060/21 (lane 1) and transconjugant E. coli J53 with a plasmid of ID_1060/21 (lane 2).It is observed that the wild type of the sample has three plasmids and one of them was transferred to the recipient E. coli J53.The plasmid sizes in the wild strain, from top to bottom, according to Bionumerics, are 163.49kb, 105.91 kb, and 50.52 kb.(M-marker H9812; 1-wild sample ID_1060/21; 2-recipient E. coli J53); Supplementary Table S1:

Figure 1 .
Figure 1.Plasmid hits, according to blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi),against eight sequences in the database from the NCBI.It is possible to see that the blaNDM-1 gene (in red) is aligned with the BD_DM_115 sequence p_kv_NDM1; accession: CP095680.1).Some regions were out of alignment with other sequences, as shown in yellow in the center of the representative circle.

Figure 1 .
Figure 1.Plasmid hits, according to blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi),against eight sequences in the database from the NCBI.It is possible to see that the bla NDM-1 gene (in red) is aligned with the BD_DM_115 sequence (plasmid p_kv_NDM1; accession: CP095680.1).Some regions were out of alignment with other sequences, as shown in yellow in the center of the representative circle.

Figure 2 .
Figure 2. Partial alignment of ID_1060/21 and BD_DM_115 plasmids and the linearized comparison of the bla NDM-1 genetic environment, with bla NDM-1 highlighted in red.This figure was created using the Clinker tool (available at https://cagecat.bioinformatics.nl/tools/clinkeraccessed on 3 June 2024).

Microorganisms 2024 , 11 Figure 3 .
Figure 3. Phylogenetic tree generated from the analysis of the SNPs of 68 global K. michiganensis isolates using CSIPhylogeny, visualized on the Microreact website.The colors represent the different STs and the country of isolation is presented for each strain.Isolate ID_1060/21 is clustered with two isolates from Russia (ST349).

Figure 3 .
Figure 3. Phylogenetic tree generated from the analysis of the SNPs of 68 global K. michiganensis isolates using CSIPhylogeny, visualized on the Microreact website.The colors represent the different STs and the country of isolation is presented for each strain.Isolate ID_1060/21 is clustered with two isolates from Russia (ST349).