Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain

Citation: Pérez-Palacios, P.; Gual-de-Torrella, A.; Portillo- Calderón, I.; Recacha-Villamor, E.; Franco-Álvarez de Luna, F.; Lopez-Cerero, L.; Pascual, A. Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain. Antibiotics 2023,12, 1727. https://doi.org/10.3390/ antibiotics12121727 Academic Editors: Alessandra Piccirilli and Mariagrazia Perilli Received: 15 November 2023 Revised: 1 December 2023 Accepted: 7 December 2023 Published: 13 December 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antibiotics Article Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain Patricia Pérez-Palacios 1,2, Ana Gual-de-Torrella 1, Ines Portillo-Calderón1,2, Esther Recacha-Villamor 1,2,3, Francisco Franco-Álvarez de Luna 4, Lorena Lopez-Cerero 1,2,3,5,* and Alvaro Pascual 1,2,3,5 1 Division of Infectious Diseases and Microbiology, University Hospital Virgen Macarena, 41009 Seville, Spain; [email protected] (P.P.-P.) 2Institute of Biomedicine of Sevilla, 41013 Seville, Spain 3Centro de Investigación Biomédica en Red de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029 Madrid, Spain 4Servicio de Microbiología, Hospital Universitario Juan Ramón Jiménez, 21005 Huelva, Spain 5Departamento de Microbiología, Facultad de Medicina, Universidad de Sevilla, 41009 Seville, Spain *Correspondence: [email protected] Abstract: In 2014–2015, the main CTX-M-15- and OXA-48-producing clone in our region was ST15. Recently, K. pneumoniae ST15 isolates co-producing VIM-1 and CTX-M-15 were detected in several hospitals. The aim was to study the emergence and acquisition of this carbapenemase. Between 2017 and 2019, four hospitals submitted twenty-nine VIM-1- and CTX-M-15-producing K. pneumoniae ST15 isolates to our laboratory. Seven representatives of each XbaI PFGE pulsotype were sequenced using short- and long-read technologies. RAST, CGE databases, and Pathogenwatch were used for resistance determinants and capsule-type analysis. Plasmid comparison was performed with Easyfig2.1. Phylogenetic analysis included other contemporary ST15 isolates from Spain. The 29 isolates were clustered into seven different pulsotypes. The selected genomes, from three hospitals in two different provinces, were clustered together (fewer than 35 alleles) and differed by more than 100 alleles from other ST15 isolates obtained in the region. These seven isolates harbored one IncR plasmid (200–220 kb) with a common backbone and four regions flanked by IS26: one contained bla VIM-1 , another contained bla CTX-M-15 , the third contained bla OXA-1 , and the fourth harbored heavymetal-tolerance genes. The two initial plasmids, from two different centers, were identical, and rearrangement of four regions was observed in the five subsequent plasmids. Our findings showed the first intercenter dissemination of IncR plasmids carrying bla VIM-1 ,bla CTX-M-15 , and metal-tolerance genes mediated by a new lineage of K. pneumoniae ST15. Two different capture events of the bla VIM-1 gene or different IS26-mediated plasmid rearrangements from a common ancestor may explain plasmid variations. Keywords: surveillance; carbapenemase spread; plasmids; K. pneumoniae high-risk clone 1. Introduction Carbapenemase production is a serious health concern in infectious diseases and is becoming a real threat to global health [ 1 ]. An important part of this problem involves the increase in carbapenem resistance in Enterobacterales, which was declared a top priority for action by the WHO in 2017 [ 2 ]. Rapid identification and monitoring of carbapenemresistant Gram-negative bacteria is necessary to tackle and control their spread [ 3 ]. Among Enterobacterales, Klebsiella pneumoniae is characterized by its extraordinary ability to spread in the community, spread in hospitals [ 4 ], and acquire carbapenemase genes. Dissemination has been associated in the last decade with broadly resistant clones with a high capacity for survival and niche colonization, as well as a considerable ability to capture virulence and resistance genes through mobile elements, such as plasmids and transposons, and to transfer them to their progeny or to other species [5]. Antibiotics 2023,12, 1727. https://doi.org/10.3390/antibiotics12121727 https://www.mdpi.com/journal/antibiotics Antibiotics 2023,12, 1727 2 of 11 The ST15 clone is one of the high-risk clones of K. pneumoniae that has worldwide distribution and, together with clones ST11, ST258/512, ST307, and ST147, is responsible for the spread of extended-spectrum beta-lactamases (ESBLs) and carbapenemases [ 6 ]. Associations between plasmids and prevalent bacterial clones are extremely frequent [ 7 ]. These particular associations have been extensively studied for K. pneumoniae ST11 and ST405 in association with the pOXA-48 plasmid [ 8 ], but similar studies for clone ST15 are scarce. In Andalusia, the ST15 and ST11 clones of K. pneumoniae have been one of the main lineages spreading bla OXA-48 and bla CTX-M-15 since 2014 [ 9 ]. Recently, isolates of this clone producing bla VIM-1 , together with bla CTX-M-15 , have been detected in several hospitals in our region. The aim of this study was to characterize these isolates, determine the phylogenetic relationships between them, and analyze the plasmid profile in order to determine whether the increase was due to several independent events or to the geographic spread of a single lineage. 2. Results The 29 K. pneumoniae ST15 strains co-producing VIM-1 and CTX-M-15 were collected from different sources. Nineteen (65.5%) isolates were recovered from clinical samples: seven (24.1%) from urine samples, four (13.8%) from blood cultures, five (17.2%) from skin and soft-tissue samples, and three (10.3%) from respiratory samples. Ten (34.5%) isolates were collected from surveillance samples (rectal swabs) (Table 1). By PFGE, seven pulsotypes were detected: P1 (n= 4), P2 (n= 3), P3 (n= 3), P4 (n= 7), P5 (n= 4), P6 (n= 1), and P7 (n= 7) (Supplementary Figure S1). The first isolate, from a blood culture, was detected in August 2017 at the Hospital Infanta Elena in Huelva. One month later, a second isolate was detected in the same hospital in a urine sample. Both isolates were identical by PFGE. Six months later, in March 2018, a third isolate was detected in a rectal swab sample from a patient, transferred from Hospital Infanta Elena, at the Hospital Virgen Macarena in Seville (Figure 1). This third isolate was assigned to a different pulsotype. During 2018, seven more isolates were detected: six between May and November at the Hospital Infanta Elena, where all patients were admitted to the same medical unit, and the last one in December at the Hospital Virgen Macarena (the reference center for the other hospitals). In 2019, the number of isolates increased to 19, and these isolates were detected in four hospitals in our region: 3 isolates at Hospital Juan Ramón Jiménez (Huelva), 14 at the Hospital Macarena (Seville), 1 at the Hospital Infanta Elena (Huelva), and 1 at the Hospital de la Merced (Seville). The patients admitted to the Hospital Virgen Macarena had also been admitted to the same medical unit (Figure 1). As in the previous year, the origin of the isolates was diverse, and all were distributed among the seven different pulsotypes. The annual increase is shown in Table 1. All isolates were assigned to the KL64 O2v1 capsule type (Table 1). The following control measures were implemented in the four centers: terminal cleaning after patient discharge, isolation of positive patients, and electronic tagging of colonized patients for isolation in case of new admissions. Antibiotics 2023,12, 1727 3 of 11 Table 1. Relevant characteristics of the isolates of K. pneumoniae ST15 producing blaVIM-1 and blaCTX-M-15. Isolate (n◦)Isolation Date Pulse Type bla Source Hospital cgMLST Capsule Type Plasmids ESBL 1CPase 2 2017787 14 August 2017 P1 CTX-M-15 VIM-1 Bloodstream Infanta Elena 3266 KL64/O2v1 IncR, ColpVC 2017917 15 September 2017 P1 CTX-M-15 VIM-1 Urine Infanta Elena N.A 3N.A 20180256 16 March 2018 P2 CTX-M-15 VIM-1 Rectal swab Virgen Macarena 3266 KL64/O2v1 IncR, ColpVC 20180516 24 May 2018 P2 CTX-M-15 VIM-1 Bloodstream Infanta Elena N.A N.A 20180784 5 September 2018 P3 CTX-M-15 VIM-1 Skin and soft tissues Infanta Elena N.A N.A 20180797 15 September 2018 P3 CTX-M-15 VIM-1 Skin and soft tissues Infanta Elena N.A N.A 20180912 5 October 2018 P3 CTX-M-15 VIM-1 Rectal swab Infanta Elena 3266 KL64/O2v1 IncR, ColpVC 20181086 29 November 2018 P1 CTX-M-15 VIM-1 Urine Infanta Elena N.A N.A 20181087 30 November 2018 P1 CTX-M-15 VIM-1 Rectal swab Infanta Elena N.A N.A 20181130 13 December 2018 P4 CTX-M-15 VIM-1 Bloodstream Virgen Macarena 3266 KL64/O2v1 IncR, ColpVC 20190090 31 January 2019 P5 CTX-M-15 VIM-1 Respiratory Juan Ramón Jimenez 3266 KL64/O2v1 IncR, ColpVC 20190095 7 February 2019 P6 CTX-M-15 VIM-1 Urine Juan Ramón Jimenez 3266 KL64/O2v1 IncR, ColpVC 20190096 13 February 2019 P7 CTX-M-15 VIM-1 Urine Virgen Macarena N.A 3N.A 20190175 13 March 2019 P4 CTX-M-15 VIM-1 Urine Virgen Macarena N.A N.A 20190191 22 February 2019 P7 CTX-M-15 VIM-1 Respiratory Juan Ramón Jimenez 3266 KL64/O2v1 IncR, ColpVC 20190260 2 May 2019 P4 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190268 7 May 2019 P5 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190286 12 May 2019 P7 CTX-M-15 VIM-1 Bloodstream Virgen Macarena N.A N.A 20190303 17 May 2019 P4 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190402 1 July 2019 P7 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190403 1 July 2019 P7 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190504 24 July 2019 P4 CTX-M-15 VIM-1 Skin and soft tissues Virgen Macarena N.A N.A 20190523 14 August 2019 P4 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190524 14 August 2019 P4 CTX-M-15 VIM-1 Rectal swab Virgen Macarena N.A N.A 20190526 14 August 2019 P7 CTX-M-15 VIM-1 Skin and soft tissues Virgen Macarena N.A 3N.A 20190643 5 September 2019 P2 CTX-M-15 VIM-1 Respiratory Infanta Elena 5114 KL64/O2v1 IncR, ColpVC 20190693 24 September 2019 P5 CTX-M-15 VIM-1 Skin and soft tissues De la Merced N.A N.A 20190717 14 October 2019 P7 CTX-M-15 VIM-1 Urine Virgen Macarena N.A N.A 20190944 20 December 2019 P5 CTX-M-15 VIM-1 Urine Virgen Macarena N.A N.A 1ESBL: extended-spectrum β-lactamase. 2CPase: carbapenemases. 3N.A: not analyzed. Antibiotics 2023,12, 1727 4 of 11 Antibiotics 2023, 12, 1727 4 of 11 Figure 1. Graphical representation of possible pathways of interhospital spread for this study. Figure 1. Graphical representation of possible pathways of interhospital spread for this study. Antibiotics 2023,12, 1727 5 of 11 Despite the differences observed by PFGE, when the Ridom cgMLST scheme was used, the seven selected representative pulsotypes belonged to the same core-genome sequence (cgST3266) and were detected in three centers (Table 1). A close genetic relationship was found between the seven genomes: fewer than 10 alleles among six genomes recovered from two centers in the same province (Huelva) and the reference hospital (Hospital Virgen Macarena, Seville) and one isolate differed by 27–33 alleles (Supplementary Figure S2). The latter isolate was recovered from a patient admitted to the reference hospital, and an epidemiological link was established with the Hospital Infanta Elena. All seven genomes were different (more than 100 alleles of difference) from other ST15 isolates from other centers in the region, as well from the two other VIM-1 producers detected in Spain in other regions in 2013–2014, indicating a new lineage. All isolates were resistant to piperacillin/tazobactam, cefotaxime, cefepime, ertapenem, meropenem, imipenem, gentamicin, tobramycin, ciprofloxacin, and sulfamethoxazoletrimethoprim and susceptible to amikacin, fosfomycin, and colistin. In addition to the bla CTX-M-15 and bla VIM-1 genes, the isolates also harbored other determinants of resistance: to aminoglycosides (aac(3)-IIa,aac(6 ′ )-Ib-cr, aph(6)-Id, aph(3 ′′ )-Ib, and aph(3 ′ )-Ia), quinolones (qnrB1 and qnrB2), and folate antagonists (sul1,sul2, and dfrA12) (Supplementary Table S1). Plasmid analysis of the seven genomes revealed that the bla VIM-1 and bla CTX-M-15 genes were on the same type of plasmid belonging to the IncR incompatibility group and approximately 200,000 pb in size (Figure 2). Comparative genomic analysis showed that the seven plasmids shared the same backbone and three identical regions flanked by IS26s. The two plasmids from 2017 and 2018 were identical but came from two different centers in Huelva. In the rest of the five plasmids, the order of the four regions within the plasmids was different and showed rearrangements (Figure 2). The bla VIM-1 region (approx. 20,000 bp) was located in a class 1 integron, flanked on each side by IS26. Next to bla VIM-1 , other antimicrobial-resistance genes, to aminoglycosides (aac(6 ′ )-Ib-cr and aadA1), sulfonamides (sul1), and quinolones (qnrB2), were detected (Figure 2). The regions containing blaCTX-M-15 (approx. 28,000 bp) and blaOXA-1 (approx. 12,000 bp) were also flanked on each side by IS26. The bla CTX-M-15 region included bla TEM-1B, and other aminoglycosideresistance genes (aph(3 ′ )-Ib,aph(6 ′ )-Id, and aac(3 ′ )-Ib-cr) and the bla OXA-1 region included the aac(6 ′ )-Ib-cr gene. In addition, a third region of approx. 40,000 bp, also flanked by IS26, carried the copper-resistance operon (copABCDRSE), silver-tolerance (silESRCFBAP) genes, and the arsenic-resistance operon (arsRDABCH) (Figure 2). Antibiotics 2023,12, 1727 6 of 11 Antibiotics 2023, 12, 1727 6 of 11 Figure 2. IncR plasmids harboring bla VIM-1 and bla CTX-M-15 from 7 K. pneumoniae ST15 isolates. bla VIM-1 is shown in red, bla CTX-M-15 , bla OXA-1 and bla TEM-1 are shown in dark green, IS are shown in blue and metal resistance operons are shown in yellow. Figure 2. IncR plasmids harboring bla VIM-1 and bla CTX-M-15 from 7 K. pneumoniae ST15 isolates. bla VIM-1 is shown in red, bla CTX-M-15 ,bla OXA-1 and bla TEM-1 are shown in dark green, IS are shown in blue and metal resistance operons are shown in yellow. Antibiotics 2023,12, 1727 7 of 11 3. Discussion In this study, the characteristics of K. pneumoniae ST15 co-producing bla VIM-1 and bla CTX-M-15 on an IncR plasmid indicate that this is a new lineage within clone ST15 that has been spreading among different hospitals in our region since 2017. This clone was found prevalent in our region during 2014–2015 but was mainly associated with the resistant determinants blaCTX-M-15 and blaOXA-48 [9]. With respect to the specific association between clones and plasmids, about which we have less knowledge in ST15, IncR has been linked to the dissemination of the clone ST15 and CTX-M-15 in Portugal (six isolates) and in four geographical areas of Bulgaria [ 10 , 11 ]. In this study, all isolates carried an IncR plasmid, which, in addition to harboring the bla VIM-1 - and bla CTX-M-15 -resistance determinants,also carried others encoding resistance to aminoglycosides, sulfonamides, and quinolones. The same plasmid was also found to be prevalent among ST15 isolates in our area in 2014–2015 [ 9 ]. These findings suggest that the current VIM-1- and CTX-M-15-producing isolates may have emerged from a population already endemic in our area that carried CTX-M-15. On the other hand, several genes for resistance to copper, silver, and arsenic were also detected in this plasmid. The presence of metal and antibiotic-resistance genes in the same mobile elements could imply contaminated environmental reservoirs exerting positive selection pressure on tolerant bacteria, favoring the co-transfer of carbapenemase and metal genes [12]. In our region, since 2014 and 2015, K. pneumonia ST15 has mainly co-produced bla OXA-48, located on IncM and IncL plasmids, and bla CTX-M-15, located on IncR plasmids [ 9 ]. The presence of bla VIM-1 in a region flanked by IS26 could indicate that the IncR plasmid containing bla CTX-M-15 captured this carbapenemase, which resulted, over time, in different rearrangements within the plasmid. IS26 is a mobile element of great importance in the dissemination of antibiotic-resistance determinants in Enterobacterales, driving rearrangements and remodeling within the plasmid [ 13 ]. Replication of the IS26 transposase, Tnp26, results in the movement of sites flanked by this element, leading to mobilization to new sites and deletion or inversion of adjacent DNA [ 14 ]. The dynamic evolution of plasmids and regions mediated by IS26 has already been described; it has also been suggested that bla SHV , originally on the K. pneumoniae chromosome, was mobilized to a plasmid by an IS26-mediated event [ 15 , 16 ]. Further studies are needed to understand the implications of these genetic events for the evolution and diversity of plasmids containing carbapenem-resistance genes when IS26 is present [17]. Studies describing outbreaks caused by K. pneumoniae ST15 have mainly been associated with bla KPC-2 - and bla VIM-1 -type carbapenemases and have focused on studying its capacity for dissemination in intensive-care-unit patients and on the great difficulty of controlling its spread [ 18 – 20 ]. There has also been one study reporting inter-regional spread of K. pneumoniae ST15, although in that case, it was only associated with ESBL and was the result of transferring patients between different hospitals [ 21 ]. To date, intercenter dissemination of bla VIM-1 - and bla CTX-M-15 -producing isolates has not been described. VIM-1-producing K. pneumoniae ST15 has been detected previously in our country, in a European study conducted in 2013–2014, but those isolates were different from the ones found in this study. In our case, the close genetic relatedness of six of the seven isolates could indicate transmission due to regional spread, although data from an epidemiological survey of all cases are not available. Transmission could have occurred during routine transfers between centers: all the isolates were recovered either from two centers in the same city or from the reference hospital. Although the close relationship is suggestive of intrahospital transmission, genetic proximity could also be due to admissions of already colonized patients from other hospitals with which there is a high frequency of patient exchanges, as was observed in the genetically nearest-neighbor analysis carried out by David et al. [22]. Controlling the spread of multidrug-resistant bacteria is currently one of the challenges facing global epidemiology. Therefore, measures and interventions aimed at preventing interregional transmission are very important [ 23 ]. One measure that has been shown to Antibiotics 2023,12, 1727 8 of 11 have an impact on the control of multidrug-resistant microorganisms is the screening of patients on admission, especially in critical services such as intensive care [ 24 ]. Since 2018, colonized patients are routinely electronically flagged in our local region: any colonized patient is preventively isolated on every new admission to any center in the region, and the alert is maintained for 12 months. Measures of this type could have been the first line of defense to prevent the spread of certain successful clones, although full implementation was delayed in the early years, hindering rapid detection among new admissions and enabling the intercenter spread observed in 2019. A major limitation of this study is that isolates are submitted to the reference laboratory on a voluntary basis and, although the Regional Ministry of Health recommends that all carbapenemase-producing isolates be tested, it is not mandatory. Another important limitation of our study is the lack of epidemiological data on the cases, so there is a possibility of discrepancies between the results obtained in the genetic studies and the epidemiological data. Therefore, in order to have an overall view of the problem of intercenter dissemination, every effort should be made to collect both genetic and epidemiological data. Moreover, the carrier status of the patients was not known prior to admission, so it was difficult to determine the time of acquisition and direction of spread between centers. 4. Materials and Methods 4.1. Bacterial Strains: Collection and Identification Between 2017 and 2019, four hospitals submitted a total of 29 isolates of VIM-1- and CTX-M-1-producing K. pneumoniae ST15 to the Regional Reference Laboratory for the Surveillance and Control of Nosocomial Infections and Prudent Use of Antimicrobials (PIRASOA) program (Hospital Universitario Virgen Macarena, Seville) for the region of Andalusia, Spain. The isolates originated from 4 hospitals located in 2 adjacent provinces (Seville and Huelva) in Western Andalusia: Hospital Virgen Macarena (n= 16) and Hospital de la Merced (n= 1) in Seville and Hospital Juan Ramón Jiménez (n= 3) and Hospital Infanta Elena (n= 9) in Huelva (Table 1). Hospital Virgen Macarena is the reference hospital for several surgeries (cardiac, thoracic, dermatologic, maxillofacial, plastic, and ophthalmic) for the other three hospitals. All isolates were identified by using MALDI-TOF MS (MALDI-TOF Biotyper 3.1; Microflex Bruker, Madrid, Spain). 4.2. Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing of all isolates was performed using the commercial MicroScan NMDRM1 panel (Beckman Coulter, West Sacramento, CA, USA). Assignment to a clinical category was based on EUCASTv21 breakpoints. Preliminary detection of carbapenemase production was carried out by enzymatic assay ( β -carba, Biorad, Marnes-la- Coquette—France) and lateral immunochromatography (NG-Test CARBA-5, NG Biotech, Guipry, France). 4.3. Molecular Typing Clonal relatedness was initially performed by pulsed-field-gel-electrophoresis (PFGE) analysis of XbaI-digested DNA (http://www.cdc.gov/pulsenet accesed on 10 November 2023). Isolates differing by one or more bands by PFGE were assigned to different pulsotypes [ 25 ]. A dendrogram was created using BioNumerics v.7.6 software (Applied Maths, Marcy-l’Étoile, France) using the Dice coefficient with 0.8% optimization and 1% band-position tolerance (Supplementary Figure S1). A representative isolate from each pulsotype was selected for short- and long-read whole-genome sequencing; 3 of the 4 centres were covered. Ridom SeqSphere+ (v8.1.0) was used for core-genome (cgMLST) and whole-genome (wgMLST) genotyping, as well as minimum-spanning-tree construction, comparing 2358 loci and considering sequence types that differed by more than 15 alleles as different. For comparison purposes, 13 contemporary genomes belonging to ST15 from the Andalusian Reference Regional Laboratory and other centers in the region were added (9 CTX-M-15- Antibiotics 2023,12, 1727 9 of 11 producers, 2 hyperproducers of SHV-1, and 2 OXA-48 and CTX-M-15 co-producers), as well as 2 genomes of VIM-1-producing ST15 isolates from other Spanish regions included in a European survey conducted in 2013 and 2014 [ 26 ]. A cut-off threshold of 10 alleles in the wgMLST Ridom scheme was used to define an interhospital transmission event [ 27 , 28 ]. A matrix of allele differences was constructed using the whole genome and the first VIM-1- and CTX-M-15-producing K. pneumoniae ST15 isolate as a reference (Supplementary Figure S2). 4.4. Whole-Genome Sequencing (WGS) DNA extraction was performed with the MagCore HF16 Plus automatic system (RCB Bioscience ® , Nottingham, UK). For short-read sequencing, library preparation was performed using the Nextera Flex DNA library preparation kit (Illumina ® , San Diego, CA, USA). DNA sequencing was performed with the MiSeq Reagent Kit V3 (Illumina ® , San Diego, CA, USA) (600 cycles) and the Illumina MiSeq sequencer (2 × 300 paired-end reads). Raw reads were trimmed, quality filtered, and then assembled de novo with CLC Genomic Workbench v9 (Qiagen, Beverly, MA, USA). Annotation of antimicrobial-resistance determinants was carried out using ResFinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/ (accessed on 14 November 2023)) (Supplementary Table S1). ST was determined using MLSTFinder databases (https:// cge.cbs.dtu.dk/services/MLST/ (accessed on 14 November 2023)) [ 29 ] and capsule loci with Pathogenwatch [30]. 4.5. Plasmid Analysis The plasmid content of the isolates provided by short reads was analyzed using PlasmidFinder 2.1 (https://cge.cbs.dtu.dk (accessed on 14 November 2023)) and multilocus sequence typing with pMLST (Table 1). For complete plasmid analysis, a long-read approach was used. Each representative pulsotype was sequenced using single-molecule real-time (SMRT) technology (PacBio, Sequel II system, San Diego, CA, USA) and Flye (https://usegalaxy.org/ (accessed on 14 November 2023)) for de novo assembly. Further plasmid annotation was performed using the Bakta annotation pipeline [ 31 ] and further polished with the Basic Local Alignment Search Tool (BLAST) and ISfinder. All sequences were deposited in the ENA database (Study ID: PRJEB52486). Plasmid sequences were compared and visualized using Easyfig2.1 [32]. 5. Conclusions Our data indicate the emergence and dissemination of a new lineage of ST15 K. pneumoniae co-producing bla VIM-1 and bla CTX-M-15 in Andalusian hospitals. The acquisition of a new resistance determinant in an area in which CTX-M-15-producing ST15 is endemic may facilitate spread between centers. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antibiotics12121727/s1, Figure S1: Clonal relatedness of Xbal-digested DNA by pulsed-field gel electrophoresis analysis (PFGE); Figure S2: Matrix of 1133 out of 4891 alleles; not taking into account missing values of K. pneumoniae ST15 producing bla VIM-1 and bla CTX-M-15 , the 13 contemporary genomes of ST15 isolates from other centers in the region and the 2 genomes of VIM-1-producing ST15 from Spanish regions other than Andalusia; Table S1: Antimicrobial resistance determinants of K. pneumoniae ST15 producing blaVIM-1 and blaCTX-M-15. Author Contributions: Methodology, P.P.-P.; Validation, A.G.-d.-T. and L.L.-C.; Formal analysis, P.P.-P., A.G.-d.-T., I.P.-C. and L.L.-C.; Investigation, P.P.-P., A.G.-d.-T., E.R.-V., L.L.-C., F.F.-Á.d.L. and A.P.; Resources, A.P.; Data curation, P.P.-P.; Writing—original draft, P.P.-P. and L.L.-C.; Writing—review and editing, A.G.-d.-T., I.P.-C., E.R.-V., F.F.-Á.d.L. and A.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Centro de Investigación Biomédica en Red de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain and Consejería de Salud y Familias, Junta de Andalucía: RH-0136-2020.