Detection of a New Resistance-Mediating Plasmid Chimera in a blaOXA-48-Positive Klebsiella pneumoniae Strain at a German University Hospital

Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven blaOXA-48 gene-carrying Klebsiella pneumoniae isolates, which were isolated between 2013 and 2014 at the University Medical Center in Göttingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively; and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All blaOXA-48 genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in K. pneumoniae in Germany.


Introduction
Plasmids play a major role as causative entities of acquired antimicrobial resistance in Gram-negative bacteria. In particular, horizontal spread of antimicrobial resistance is frequently driven by the conjugation-based transmission of plasmids [1]. Although the Microorganisms 2021, 9, 720 2 of 23 replication of resistance-mediating plasmids is associated with fitness costs for bacterial pathogens [2], compensatory mutations alleviate the associated evolutionary disadvantages [1,3]. Adaptive mutations in intergenic regions and selection of genes involved in anaerobic metabolism are thought to specifically stabilize the persistence of plasmidcarrying bacteria in the intestine of colonized individuals [4].
Due to the significant public health impact of plasmid-mediated resistance transfer, bioinformatic solutions for the identification of plasmid sequences from next generation sequence data obtained from bacterial pathogens with acquired resistances were introduced early on [5]. In particular, long read (PacBio) sequencing has proven to be a particularly suitable tool to completely resolve the DNA sequence of extrachromosomal mobile genetic elements like plasmids [6,7]. These methods can be used for the assessment of abundance, quantity, and diversity of plasmids in whole microbial communities.
In microbial species like Escherichia coli and Klebsiella pneumoniae, multidrug-resistance is mainly associated with acquisition and maintenance of plasmids encoding the resistance genes [4]. The geographic distribution patterns of plasmids suggests that special plasmid families are particularly successful in the global spreading of resistance genes. So-called epidemic resistance plasmids comprise plasmids of incompatibility (Inc) groups IncFII, IncA/C, IncL/M, IncN, and IncI1; these carry genes that encode for extended-spectrum βlactamases (ESBLs), AmpC β-lactamases, and carbapenemases. All of these cephalosporin and carbapenem hydrolyzing enzymes have been globally identified in Enterobacterales of different sources and origins [8].
In 2013-2014, the nosocomial transmission of an oxacillinase-48 (OXA-48) carbapenemase producing K. pneumoniae strain of sequence type (ST)147 was detected at the University Medical Center Göttingen, Germany. During these investigations, seven further isolates of four different STs were identified [36]. Here, we characterize the plasmid-based acquired resistome of these seven bla OXA-48 -carrying K. pneumoniae isolates. This explorative assessment will contribute a piece to the puzzle of local resistance epidemiology.

Bacterial Isolates and Clinical Information
Seven bla OXA-48 -positive clinical K. pneumoniae isolates, which were isolated from five patients at the diagnostic laboratory of the University Medical Center Göttingen, Germany between 2013 and 2014, were included in the plasmid-resistome analysis. During a previous epidemiological assessment, multilocus sequence typing (MLST) assigned three isolates from two patients to sequence type ST101, two isolates from one patient to ST11, and two isolates from two patients to ST23 and ST15 [36]. Clinical information provided for the otherwise fully anonymized isolates comprised patient sex, patient age, site of isolation, and underlying medical condition (Table 1).

Species Identification and Resistance
Bacterial species identification was performed using the MALDI (Matrix-Assisted Laser Desorption/Ionization) Biotyper system (Bruker Daltonics, Bremen, Germany). Results with MALDI Biotyper identification score values ≥ 2.000 were assessed as correct.

Whole Genome Sequencing and Bioinformatics
The seven isolates were identified as carbapenemase producers that harbored carbapenemase gene bla OXA-48 , and genome sequence information was generated as described previously [36]. In brief, DNA extractions were subjected to Single-molecule real-time (SMRT) sequencing on a PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA). Using the same DNA preparation, short-read sequencing was performed on a HiSeq 2500 device (Illumina Inc., San Diego, CA, USA). SMRT cell data were assembled independently using the RS_HGAP_Assembly.3 protocol. Briefly, each replicon was circularized independently and the artificial redundancies at the ends of the contigs were removed. The validity of the assembly was checked using the RS_Bridgemapper.1 protocol. Each genome was corrected for indel errors by a mapping of Illumina short-reads onto the SMRT long-read assembled genomes. A consensus concordance of QV60 could be confirmed for all genomes. The assembled genomes were deposited at GenBank. The accession numbers are listed in Table 3 and Table A1. Assembled plasmid contigs were subjected to BLASTN search at the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 17 June 2019). The annotation of the assembled genomes was performed applying 'rapid annotations using subsystems technology' (RAST) (http://rast.nmpdr.org, accessed on 17 June 2019). Annotated genomes were scanned in the SEED viewer (https://seed-viewer.theseed.org/, accessed on 5 June 2016). Spreadsheet charts with protein coding genes (CDS) were obtained and analyzed for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In addition, the completely assembled genomes in fasta format were provided as inputs to the web-based ResFinder 2.1 tool (http://cge.cbs.dtu.dk/services/ResFinder/, accessed on 22 December 2016) and ARGs identities were accepted at an increased identity threshold of >96% and a min length of 60% [36].

Clinical Information
The seven K. pneumoniae isolates were from five male patients between 31 and 53 years of age. The isolates comprised etiologically relevant isolates from urine in case of urinary tract infection (n = 2), from tracheal secretion in case of pneumonia (n = 1) and from wounds in case of wound infections (n = 2). Furthermore, two ST11 isolates were mere colonizers as detected by hygiene-related routine swabbing (Table 1). Of note, the two isolates from patient 3 were isolated in a temporal distance of about one month. Core genome MLST that was performed in a previous study [36] revealed close genetic relationship of the three ST101 isolates and the two ST11 isolates, respectively.

Antibiotic Resistance Assessment and Transferability
All seven isolates were resistant to piperacillin, piperacillin/tazobactam, aztreonam, cefotaxime, ceftazidime, gentamicin, tobramycin, ciprofloxacin, moxifloxacin, and trimethoprim-sulfamethoxazole. Resistance and reduced susceptibility to imipenem (range between 0.5 and >8 mg/L) and meropenem (range between 1 and >8 mg/L) was detected for all seven isolates, and the two ST11 isolates were additionally resistant to colistin ( Table 2). For two isolates (Kp_Goe_121641-ST101 and Kp_Goe_39795-ST15) the broth mating experiment were successful. The obtained transconjugants were positive for bla OXA-48 , were resistant to piperacillin and MICs of 1-2 mg/L were detected for imipenem and meropenem. S1-nuclease pulsed field gel electrophoresis (PFGE) showed the presence of a plasmid of ca. 60 kb size in the transconjugants (Appendix B Figure A1).

Analysis of the Plasmids and Comparison with Phenotypical Resistance
As visualized in Figures 1-3, and in Table 3, numerous resistance genes occurred in various types of plasmids of the lineages IncL/M, IncR, IncF, and IncN. Each isolate had at least two different types of plasmids, one of which was IncL/M with bla OXA-48 (example: Figure 1A).

Analysis of the Plasmids and Comparison with Phenotypical Resistance
As visualized in Figures 1-3, and in Table 3, numerous resistance genes occurred in various types of plasmids of the lineages IncL/M, IncR, IncF, and IncN. Each isolate had at least two different types of plasmids, one of which was IncL/M with blaOXA-48 (example: Figure 1A).        The resistance gene bla OXA-48 , whose abundance was the selection criterion of the seven isolates for this study, was exclusively associated with IncL/M plasmids of variable size; plasmid pKp_Goe_795-2 of ST15 isolate Kp_Goe_39795 is shown exemplarily in Figure 1A. These plasmids, however, further hosted additional antibiotic resistance genes (ARGs) besides bla OXA-48 gene in two out of three isolates of ST101 (Table 3). In five isolates (Kp_Goe_121641, Kp_Goe_821588, Kp_Goe_822917, Kp_Goe_154414, Kp_Goe_39795), the IncL/M plasmids showed 100% identity with the reference plasmid pOXA-48 (GenBank accession number: JN626286). Complete sequences of these plasmids were similar to each other above the 99.9% level, but were also similar to some other plasmids in enterobacterial species collected from the same region and other countries (Appendix A Table A1). In contrast, the IncL/M plasmids of the two ST101 isolates from patient 1 (Kp_Goe_33208 and Kp_Goe_71070) showed 99.59% identity with the K. pneumoniae plasmid pMU407 (GenBank accession number: U27345). The identity between the complete sequences of these two plasmids was 100%. In contrast to the third ST101 isolate from patient 2, further resistance genes (e.g., ESBL gene bla CTX-M-14b and plasmid mediated quinolone resistance determinants qnrS1/9) were located on the IncL/M plasmid with bla OXA-48 (Table 3, Figure 4). The comparative BLAST analysis of complete sequences also revealed 100% identity with a 90% query coverage to a plasmid (GenBank accession number: KP025948) from a Proteus mirabilis strain (Appendix A Table A1) [38]. The resistance gene blaOXA-48, whose abundance was the selection criterion of the seven isolates for this study, was exclusively associated with IncL/M plasmids of variable size; plasmid pKp_Goe_795-2 of ST15 isolate Kp_Goe_39795 is shown exemplarily in Figure 1A. These plasmids, however, further hosted additional antibiotic resistance genes (ARGs) besides blaOXA-48 gene in two out of three isolates of ST101 (Table 3). In five isolates (Kp_Goe_121641, Kp_Goe_821588, Kp_Goe_822917, Kp_Goe_154414, Kp_Goe_39795), the IncL/M plasmids showed 100% identity with the reference plasmid pOXA-48 (Gen-Bank accession number: JN626286). Complete sequences of these plasmids were similar to each other above the 99.9% level, but were also similar to some other plasmids in enterobacterial species collected from the same region and other countries (Appendix A Table A1). In contrast, the IncL/M plasmids of the two ST101 isolates from patient 1 (Kp_Goe_33208 and Kp_Goe_71070) showed 99.59% identity with the K. pneumoniae plasmid pMU407 (GenBank accession number: U27345). The identity between the complete sequences of these two plasmids was 100%. In contrast to the third ST101 isolate from patient 2, further resistance genes (e.g., ESBL gene blaCTX-M-14b and plasmid mediated quinolone resistance determinants qnrS1/9) were located on the IncL/M plasmid with blaOXA-48 (Table 3, Figure 4). The comparative BLAST analysis of complete sequences also revealed 100% identity with a 90% query coverage to a plasmid (GenBank accession number: KP025948) from a Proteus mirabilis strain (Appendix A Table A1) [38].    As indicated in Figure 3 and Table 3, the ST15 isolate Kp_Goe-39795 carried a large 232kb plasmid chimera that has not been described before. This IncF plasmid ( Figure 3) with FIB replicon did not show similarity with any known plasmid in BLAST analysis. It encoded the ARGs blaCTX-M-15, blaOXA-1, blaTEM-1, aac(6′)-Ib-cr, aac(3)-IIa, aph(6)-Id, aph(3′')-Ib, ant(3′')-Ia, catB3, catA1, sul2, tetA, as well as various heavy metal resistance determinants. The mobile genetic elements flanked by these genes are shown in Table 3. Individual parts of this plasmid chimera could be matched to putative origin plasmids (Figure 3). GenBank accession numbers of all detected plasmids in the seven K. pneumoniae isolates are shown in Appendix A Table A1.
In four out of seven isolates, IncF plasmids were present (Table 3); these carried various genes that mediate resistance to β-lactams, aminoglycosides, phenicols, tetracyclines, sulfonamides and trimethoprim. The presence of these genes corresponded with the observed phenotypes. Examples for reconstructed IncF plasmids are given in Figures 1B and  2A. IncFII, IncFIB, and IncFIA replicons were detected on these plasmids (Appendix A  Table A1). The complete sequences of the IncF plasmids of the two ST11 isolates Kp_Goe_821588 and Kp_Goe_822917 were identical to each other. These plasmids carried both FII and FIB replicons, resistance genes blaCTX-M-15, blaOXA-1, aac(6′)-Ib-cr, aac(3)-IIa, aph(3′)-Ia, catB3, dfrA14, and genes mediating resistance to heavy metals, such as arsenic, cobalt, zinc, cadmium and copper (  Figure 5. Comparison of transposons encoding OXA-48. (A) In five of the tested isolates, the bla OXA-48 gene was part of Tn1999; (B) in the two ST101 isolates Kp_Goe_33208 and Kp_Goe_71070 the bla OXA-48 gene was part of a Tn1999.2 transposon, which is characterized by the insertion of IS1R into the upstream IS1999 region. Figure 3 and Table 3, the ST15 isolate Kp_Goe-39795 carried a large 232kb plasmid chimera that has not been described before. This IncF plasmid (Figure 3) with FIB replicon did not show similarity with any known plasmid in BLAST analysis. It encoded the ARGs bla CTX-M-15 , bla OXA-1 , bla TEM-1 , aac(6 )-Ib-cr, aac(3)-IIa, aph(6)-Id, aph(3")-Ib, ant(3")-Ia, catB3, catA1, sul2, tetA, as well as various heavy metal resistance determinants. The mobile genetic elements flanked by these genes are shown in Table 3. Individual parts of this plasmid chimera could be matched to putative origin plasmids (Figure 3). GenBank accession numbers of all detected plasmids in the seven K. pneumoniae isolates are shown in Appendix A Table A1.

As indicated in
In four out of seven isolates, IncF plasmids were present (Table 3); these carried various genes that mediate resistance to β-lactams, aminoglycosides, phenicols, tetracyclines, sulfonamides and trimethoprim. The presence of these genes corresponded with the observed phenotypes. Examples for reconstructed IncF plasmids are given in Figures 1B and 2A. In-cFII, IncFIB, and IncFIA replicons were detected on these plasmids (Appendix A Table A1). The complete sequences of the IncF plasmids of the two ST11 isolates Kp_Goe_821588 and Kp_Goe_822917 were identical to each other. These plasmids carried both FII and FIB replicons, resistance genes bla CTX-M-15 , bla OXA-1 , aac(6 )-Ib-cr, aac(3)-IIa, aph(3 )-Ia, catB3, dfrA14, and genes mediating resistance to heavy metals, such as arsenic, cobalt, zinc, cadmium and copper (Table 3). Kp_Goe_154414 (ST 23) had four IncF type plasmids. Three out of them harbored several ArGs encoding resistance to β-lactams, aminoglycosides, quinolones, phenicols, sulfonamides, tetracycline, and heavy metals (Table 3).
In three ST101 isolates (Kp_Goe_33208 ( Figure 1C), Kp_Goe_71070, and Kp_Goe_121641), IncR plasmids carried a composition of ARGs similar to those found on the IncF plasmids, one example is given in Figure 1B. The backbones of these plasmids were 100% identical with the plasmid pK245 (Gene accession number: DQ449578). The complete sequences of the IncR plasmids of isolates from patient 1 (Kp_Goe_33208 and Kp_Goe_71070) were identical to each other. Their identity with the smaller IncR plasmid of Kp_Goe_121641 was 99.99% with a coverage of 74%. The resistance genes cat, ant(3")-Ia, aac(6 )-Ib, aac(6 )-Ib-cr, aac(3)-IIa, bla TEM-1A , bla OXA-9 , dfrA14, merE, merT, merC were detected in all IncR plasmids (Table 3). In addition to this, tetA, tetD, mph(A), emrE, sul3, cmlA, and floR were located on the IncR plasmids of the isolates Kp_Goe_33208 and Kp_Goe_71070 compared to Kp_Goe_121641 ( Table 3). The additional ArGs on the IncR plasmid of isolate Kp_Goe_121641 included β-lactamase genes bla CTX-M-15 and bla OXA-1 . The bla CTX-M-15 gene was flanked downstream by insA and IS1 and upstream by the tryptophan synthase coding gene and IS1 ( Figure 4B).
An IncN plasmid was detected in one of the two K. pneumoniae-ST11 isolates from patient 3 (Kp_Goe_822917), associated with a number of genes that mediate resistance to macrolides, trimethoprim and ethidium bromide ( Figure 2B, Table 3).

Discussion
The study was performed to contribute to the existing epidemiological knowledge on the distribution of antibiotic resistance-mediating plasmids in carbapenemase producing K. pneumoniae isolates in Germany. The characterized seven isolates represent epidemic clonal lineages of K. pneumoniae (ST11, ST15, ST101, and ST23) that have been described worldwide and are associated with multidrug resistance and/or enhanced virulence [39]. The genetic relationship of these bacterial isolates has been first characterized in detail in a previous study [36]. To allow an unambiguous attribution of the described plasmids to the previously described bacterial isolates, the specific identifier codes Kp_Goe_xxxxx are identical in the previous manuscript [36] and in the present one.
As expected, the so-called epidemic resistance plasmids IncF, IncL/M, and IncN [8] that carry various resistance-mediating genes were identified in these seven isolates. Furthermore, we identified IncR plasmids and a new IncF (FIB) plasmid chimera.
As reported by others, the bla OXA-48 genes waere associated with IncL/M plasmids [10,[18][19][20]. As typical for bla OXA-48 , which is only associated with high-level carbapenem resistance in case of combination with other resistance mediating elements [18], the majority of the seven K. pneumoniae isolates were phenotypically tested susceptible towards carbapenems ( Table 2).
The tracking of carbapenem resistance-associated mechanisms remains an issue of relevance, as infections, due to carbapenem-resistant K. pneumoniae, are globally reported, and are rising [40][41][42]. Thereby, plasmid-encoded carbapenemase production is the major mechanism of carbapenem resistance in K. pneumoniae. While mobile genetic elements such as plasmids, transposons, and insertion sequences readily enable the transmission of carbapenemase-encoding genes, clonal expansion contributes to the globally increasing rates of dissemination of carbapenemase-producing K. pneumoniae [40][41][42]. Next to transmission events within human microbiomes, spread of resistance determinants may also occur in external environments under the selection pressure of pollution with antimicrobial active substances [43].
Our seven study isolates, which included both disease-associated and merely colonizing isolates, were associated with the carriage of resistance-mediating plasmids. As shown for the phylogenetically closely related isolates of the sequence type ST101 (confirmed by cgMLST [36]) indicating likely nosocomial transmission, their accessory genome varied remarkably. Cause of such variance is a different set of resistance mediating plasmids, encoding various resistance determinants ( Table 3) that may create phenotypical variations. Discrepancies in phenotypical resistance does therefore not necessarily exclude nosocomial spread. However, isolates from the same patients showed identical phenotypic resistance in our study, although variations in plasmid content were detected for two ST11 isolates (Table 3).
While carbapenem resistance, or at least reduced carbapenem susceptibility compared to the wild type, was associated with bla OXA-48 in all cases, this resistance gene is not the only one that has been frequently detected in K. pneumoniae. In the early 2000s, three types of carbapenemases, Klebsiella pneumoniae carbapenemase (KPC), oxacillinase-48 (OXA-48) type carbapenemase, and the New Delhi metallo-β-lactamase (NDM), emerged globally and spread rapidly. Nosocomial outbreaks due to carbapenemase producing K. pneumoniae strains became an alarming issue in hospital setting in many countries in Europe and worldwide [40][41][42]. To cite some examples, IncF plasmids with the IncFII K replicon mediated the global spread of KPC-encoding genes throughout the United States, Colombia, Argentina, Israel, Greece, Norway, Sweden, Italy, Poland, Canada, Brazil, Korea, and Taiwan [40][41][42]. Starting at the Indian subcontinent, New Delhi metallo-β-lactamase producing K. pneumoniae have rapidly spread to various countries such as Romania, Poland, Hungary, Denmark, Italy, Spain, Greece, Turkey, China, Australia, Japan, Colombia, South Africa, Algeria, Morocco, Saudi Arabia, and Oman [40][41][42] in association with the plasmids IncA/C, IncFII, IncN, IncH, and IncL/M. In contrast, OXA-48 producing K. pneumoniae, such as those examined in the presented study, were first identified in Turkey in 2001 and showed a rapid global dissemination [40][41][42]44]. Just as shown for the ST11 isolates in our study, bla OXA-48 , encoding the OXA-48 carbapenemase, was first identified as a part of Tn1999 on an IncL/M plasmid, pOXA-48a (GenBank accession number JN626286). As observed for the isolates presented here, the global dissemination of bla OXA-48 in K. pneumoniae has been mainly linked to the epidemic IncL/M plasmid [10,[18][19][20]. The variant of Tn1999, Tn1999.2, which was observed in two of the analyzed ST101 isolates, has been detected in K. pneumoniae carrying bla OXA-48 together with bla CTX-M-14-b and other antimicrobial resistance genes, while other authors reported the association of bla OXA-48 together with bla CTX-M-15 [45]. While the IncL/M plasmid was the genetic element primarily responsible for reduced carbapenem susceptibility or carbapenem resistance in our isolates, the other detected IncF, IncN, and IncR plasmids carried various resistance genes and contributed to their multidrug-resistance phenotype ( Table 3).
The observation of the new plasmid chimera pKp_Goe_795-1 (CP018460, Figure 3) is of particular interest, as it documents "real life evolution" by the assembly of resistance determinants in one, and the same plasmid vector through extensive transposition. Associated with this process, it is worth focusing on the epidemiological background of transposases and other ARG-associated mobile genetic elements (MGEs) found in the genetic information of the plasmid chimera ( Table 3). The reported IS6 insertion sequence family has previously been reported from a multidrug resistance-mediating plasmid in Proteus mirabilis in China [46]. IS6 also allowed migration of a replicative elements carrying ARGs by replicative transposition in Klebsiella pneumoniae in France and in Japan [47,48]. Tn3-like transposons have recently been reported from Enterobacterales including K. pneumoniae from China and Switzerland [49][50][51]. The transition protein-encoding tnp1 has been described from E. coli in China [52]. The insertion sequence IS1380 has been first identified in Acinetobacter pasteurianus [53,54], the insertion sequence IS110 was recently reported from Klebsiella pneumoniae from New Jersey [55], IS1 in K. pneumoniae from China and Russia [56,57], IS481 in K. pneumoniae from Japan [48], IS903 in K. pneumoniae from France and China [58,59], and in Salmonellae from the USA [60]. IS66 is abundant in the worldwide-distributed K. pneumoniae sequence types ST258/ST512 [61]. IS3, using a two-step transposition mechanism to specifically insert into short palindromic repeated sequences, has been reported from K. pneumoniae in Europe and the USA [62][63][64]. IntIPac was described from a IncFIB plasmid found in a clinical Klebsiella variicola isolate in Hong Kong [65]. A new member of the insertion sequence family ISNCY has been reported from K. pneumoniae in California, USA, some years ago [63]. The insertion sequence ISL3 has been linked to the transposition of sequence information for colistin resistance in Europe [66] and for carbapenem resistance in China [67] in K. pneumoniae. Summarized, the observed MGEs are internationally common in Enterobacterales in general and K. pneumoniae in particular, while just the arrangement in the newly described plasmid has not yet been described so far.
The limited number of assessed isolates is the major limitation of this study presented here. Nevertheless, the presented data provide a piece in the puzzle of molecular epidemiology of resistance-mediating plasmids in Germany. It contributes to previous efforts to improve our understanding how bla OXA-48 and other antimicrobial resistance genes spread among K. pneumoniae isolates [36,68,69].

Conclusions
The study demonstrated the abundance of various IncF, IncM/L, IncN, and IncR plasmids in seven OXA-48 producing K. pneumoniae isolates from hospitalized patients in Germany. Long read sequencing enabled the complete plasmid reconstruction and identification of a novel IncF (FIB) plasmid chimera. It further highlighted the association of bla OXA-48 with IncL/M plasmids in K. pneumoniae. This information, once implemented in bioinformatics tools for molecular identification of antibiotic resistance, will help to improve the molecular epidemiology of resistance plasmids and resistance genes. Thus, it contributes to the prediction of phenotypic susceptibilities and improve our understanding of the evolution of resistance gene-encoding plasmids. Future challenges for the goal of achieving broad surveillance of resistance-mediating MGEs comprise ready and affordable availability of sequencing technology and lacking automation of bioinformatic assessments, which is still associated with an unrealistically high work-load for application under routine-diagnostic conditions. Further declines of sequencing costs and increased implementation of artificial intelligence (AI) solutions in order to reduce the required hands-on time of experts may guide the way to achieve molecular near-real-time surveillance in the future.