ORIGINAL RESEARCH published: 18 November 2021 doi: 10.3389/fmicb.2021.781127 Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Edited by: Klebsiella pneumoniae Karsten Becker, Friedrich Loeffler Institute of Medical Microbiology, University Medicine Carbapenemase-Producing Greifswald, Germany Enterobacterales During a Hospital Reviewed by: Nilton Lincopan, University of São Paulo, Brazil Outbreak in Barcelona, Spain Sara Domingues, Marta Marí-Almirall 1† , Núria Ferrando 1† , Mariana José Fernández 2† , Clara Cosgaya 1 , University of Coimbra, Portugal Katharina Schaufler, Joaquim Viñes 3,4 , Elisa Rubio 2 , Anna Cuscó 4 , Laura Muñoz 1 , Martina Pellice 5 , University of Greifswald, Germany Andrea Vergara 2 , Irene Campo 2 , Laura Rodríguez-Serna 6 , Gemina Santana 6 , Ana Del Río 5 , Olga Francino 3 , Pilar Ciruela 7,8 , Frederic Ballester 9 , Francesc Marco 1,2 , *Correspondence: José Antonio Martínez 5 , Álex Soriano 5 , Cristina Pitart 1,2 , Jordi Vila 1,2 , Ignasi Roca 1* and Ignasi Roca Ignasi.roca@isglobal.org MERCyCAT Study Group † These authors have contributed 1 Laboratory of Antimicrobial Resistance, ISGlobal, Hospital Clínic–Universitat de Barcelona, Barcelona, Spain, 2 Department equally to this work and share first of Clinical Microbiology, Hospital Clínic–Universitat de Barcelona, Barcelona, Spain, 3 Molecular Genetics Veterinary Service, authorship Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Vetgenomics, PRUAB, Universitat Autònoma de Barcelona, Barcelona, Spain, 5 Department of Infectious Diseases, Hospital Clínic–Institut d’Investigacions Biomèdiques August Pi i Specialty section: Sunyer, Universitat de Barcelona, Barcelona, Spain, 6 Department of Preventive Medicine and Epidemiology, Hospital This article was submitted to Clínic–Universitat de Barcelona, Barcelona, Spain, 7 Public Health Agency of Catalonia (ASPCAT), Generalitat de Catalunya, Antimicrobials, Resistance Barcelona, Spain, 8 CIBER de Epidemiología y Salud Pública, Instituto de Salud Carlos III, Madrid, Spain, 9 Hospital and Chemotherapy, Universitari Sant Joan de Reus-Laboratori de Referència del Camp de Tarragona i de les Terres de l’Ebre, Reus, Spain a section of the journal Frontiers in Microbiology Objectives: The study aimed to characterize the clonal spread of resistant bacteria and Received: 22 September 2021 Accepted: 25 October 2021 dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at Published: 18 November 2021 a tertiary hospital in Catalonia, Spain. Citation: Marí-Almirall M, Ferrando N, Methods: Isolates were recovered from surveillance rectal swabs and diagnostic Fernández MJ, Cosgaya C, Viñes J, samples. Species identification was by matrix-assisted laser desorption ionization-time Rubio E, Cuscó A, Muñoz L, time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed Pellice M, Vergara A, Campo I, Rodríguez-Serna L, Santana G, by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Del Río A, Francino O, Ciruela P, Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes Ballester F, Marco F, Martínez JA, Soriano Á, Pitart C, Vila J, Roca I and was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and MERCyCAT Study Group (2021) long-read sequencing were used to characterize resistance plasmids. Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase Associated With Klebsiella (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance pneumoniae Carbapenemase-Producing (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies Enterobacterales During a Hospital identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 Outbreak in Barcelona, Spain. and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from Front. Microbiol. 12:781127. doi: 10.3389/fmicb.2021.781127 different STs. All isolates but one carried the blaKPC- 2 allelic variant. The blaKPC- 2 Frontiers in Microbiology | www.frontiersin.org 1 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC -pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC -pMC-2-1 module was highly conserved. Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC- producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the blaKPC- 2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high- risk clones. Keywords: Klebsiella, outbreak, antibiotic resistance, carbapenemase, epidemiology, plasmid, high-risk clone, KPC INTRODUCTION (NDM) and Klebsiella pneumoniae carbapenemase (KPC), among others. In Spain, OXA-48 is currently the predominant The rapid emergence of bacterial pathogens presenting resistance mechanism associated with the dissemination of carbapenem- to multiple antimicrobial agents (MDR) together with the resistant Enterobacterales (Grundmann et al., 2017). NDM- decreasing trend in the development of new antimicrobial producing bacteria are reported less frequently in Spain but compounds constitute an extremely serious threat to public their presence is increasing and associated with a few clonal health (Roca et al., 2015). Resistance rates are particularly lineages (Marí-Almirall et al., 2021) while KPC-producing alarming among Gram-negative bacteria (GNB) as available Enterobacterales are also sporadic in Spain, and only a treatment options are severely impaired. In 2016 a report few outbreaks have been reported in Madrid and Andalusia commissioned by the UK Prime minister, known as the (Oteo et al., 2016). “O’Neill report,” attributed 700.000 annual human deaths Klebsiella pneumoniae carbapenemase is likely one of the worldwide to microbial infections caused by antimicrobial most common mechanisms of resistance to carbapenems in resistant (AMR) pathogens (O’neill, 2016). The same report K. pneumoniae, and the worldwide spread of the blaKPC estimated that this figure would rise to 10 million annual gene was initially associated with the dissemination of a deaths by 2050 unless there is a global effort to tackle AMR. few K. pneumoniae clonal lineages within the ST258 clonal Likewise, organizations such as the US Centers for Disease group (CG258), a particular plasmid backbone (pKpQIL) and Control and Prevention (CDC), the European Centre for a highly conserved Tn4401 transposon (Chen et al., 2014). Disease Prevention and Control (ECDC) and the World More recently blaKPC has been found on a variety of clonal Health Organization (WHO) are considering infections lineages and plasmids from different incompatibility groups caused by MDR bacteria as an emergent global disease and a (Wyres et al., 2020), and although it is usually associated with major public health problem (Roca et al., 2015). According a composite Tn4401 transposon, some structural variations to their mortality, health-care and community burden, have also been described (Shen et al., 2009; Naas et al., prevalence of resistance and treatability, carbapenem-resistant 2012; Chmelnitsky et al., 2014). In this retrospective study Enterobacterales have been included into the 2017 WHO priority we have examined the rapid dissemination of KPC-producing list of antibiotic-resistant bacterial pathogens and currently Enterobacterales within a tertiary hospital in Barcelona and constitute one of the major public health threats worldwide we have investigated the inter- and intra-species spread of (Tacconelli et al., 2017). carbapenem resistance. Among carbapenem-resistant Enterobacterales, Klebsiella pneumoniae stands out as a formidable nosocomial pathogen causing several infections associated with high mortality rates MATERIALS AND METHODS (Tumbarello et al., 2012). In Europe, carbapenem-resistant K. pneumoniae accounted for almost 16,000 infections and This study included 125 carbapenem-resistant isolates of more than 2,000 deaths in 2015 (Cassini et al., 2018). Enterobacterales collected from July 2018 to February 2019 at Carbapenem-resistance is usually associated with the carriage one tertiary hospital in Barcelona, Spain. Strains were recovered of genes encoding carbapenem-hydrolyzing enzymes, most of from surveillance and clinical samples (Supplementary Table 2). which are commonly located within conjugative plasmids and, Identification at species level was performed by matrix-assisted therefore, are easily disseminated among nosocomial pathogens laser desorption ionization-time time of flight mass spectrometry (Bonomo et al., 2017). Predominant carbapenemases in Europe (MALDI-TOF MS) in a Microflex LT benchtop instrument include OXA-48 oxacillinase, New Delhi metallo-β-lactamase (Bruker Daltonics) operated in linear positive mode. Frontiers in Microbiology | www.frontiersin.org 2 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC Antimicrobial Susceptibility Testing and were determined using the PBRT-2.0 kit (Diatheva, Italy) Detection of Resistance (Carattoli et al., 2005). The genetic environment of blaKPC was determined by PCR using primers listed in Supplementary Antimicrobial susceptibility was assessed by gradient Table 1 and confirmed by long-read sequencing. diffusion (E-test, BioMérieux, Spain) on Muller-Hinton Genomic DNA was extracted using the Wizard Genomic agar plates (Becton-Dickinson, Spain) for the following DNA purification kit (Promega, Spain) and sequenced on a antimicrobials: imipenem, meropenem, ceftazidime, cefepime, MinION instrument (Oxford-Nanopore, United Kingdom) using cefotaxime, amikacin, tobramycin, gentamicin, kanamycin, the rapid barcoding kit (SQK-RBK004) for library preparation tigecycline, ciprofloxacin, levofloxacin, ceftazidime-avibactam and a R9.4.1 flowcell following the manufacturer’s instructions. and fosfomycin. Susceptibility to colistin was assessed by Basecalling was done with Guppy-v3.0.3 and demultiplexing gradient diffusion on Iso-Sensitest agar plates (ThermoFisher, with qcat-v1.1.01 . FASTQ files were mapped using Minimap2- United Kingdom). On note, colistin susceptibility was v2.17 against plasmids from Enterobacterales (Orlek et al., 2017). tested under research use only (RUO) acknowledgment Mapping reads were assembled with Flye-v2.52 . Annotation since the recommended method for in vitro diagnostic was done with Prokka-v.1.12 combined with BLASTP/BLASTN (IVD) is broth microdilution (European Committee on searches against the UniProtKB/Swiss-Prot and RefSeq databases Antimicrobial Susceptibility Testing (EUCAST), 2016). The (Seemann, 2014). MICs were interpreted according to EUCAST breakpoints ResFinder3 , PlasmidFinder4 , and ISFinder5 were used to for Enterobacterales (European Committee on Antimicrobial identify antimicrobial resistance genes, plasmid replicons and Susceptibility Testing (EUCAST), 2021). Escherichia coli ATCC mobile elements, respectively. The presence of virulence genes in 25922 was used for quality control. plasmid sequences was checked against the Pasteur MLST site6 . Production of KPC, OXA-48-like, VIM, IMP or NDM R R Genetic diagrams were drawn using SnapGene Viewer-v5.1.27 carbapenemases was detected with the NG-Test CARBA5 (NG- and CGViewAdvanced-v.0.0.1 (Stothard and Wishart, 2005). Biotech, France) and confirmed by PCR using previously FASTQ files of isolates MC-2-1, MC-2-207, MC-2-230, MC- described primers and conditions (Queenan and Bush, 2007; 2-240, MC-2-251, MC-2-304, MC-2-315, MC-2-316, MC-2-362, Solé et al., 2011; Bogaerts et al., 2013). Amplification products and MC-2-387, were deposited into the NCBI Sequence Read were purified from agarose gels and sent for Sanger sequencing Archive under accession numbers SRX11379706, SRX11379707, (Genewiz, Germany) whenever necessary. The allelic identity of SRX11379708, SRX11379709, SRX11379710, SRX11379711, all genes was determined by sequence alignment with reference SRX11379712, SRX11379713, SRX11379714, and SRX11379715, sequences retrieved from public repositories (PRJNA313047, last respectively; BioProject PRJNA744857. accessed June, 2021). Clonal Relatedness RESULTS Clonality was studied by pulsed-field gel electrophoresis (PFGE) using XbaI (New England BioLabs Inc., United States) genomic Bacterial Isolation and Clonal digestions and a CHEFF-DRIII system (Bio-Rad, Spain) (Marí- Almirall et al., 2021). Molecular patterns were analyzed with Relatedness InfoQuestTM FP-v.5.4 (Bio-Rad, Spain) and the unweighted pair In mid-July 2018, five carbapenem-resistant K. pneumoniae group method with arithmetic mean (UPGMA) to create isolates were recovered from surveillance samples (rectal swabs) dendrograms based on Dice’s similarity coefficient. Bandwidth upon the implementation of a “zero-resistance” surveillance tolerance and optimization values were set at 2% and isolates were program to screen all patients admitted to intensive care units considered within the same PFGE cluster (pulsotype) if their Dice within the hospital. All five isolates produced a KPC-type similarity index was >85%. enzyme. During the following weeks, additional surveillance Multi-locus sequence typing (MLST) was performed samples and an increasing number of clinical samples flagged according to the Pasteur scheme for K. pneumoniae and positive for KPC-producing Enterobacterales. By the end the Achtman scheme for E. coli (Diancourt et al., 2005; of August, 87 KPC-producing isolates had been recovered Wirth et al., 2006). and infection control measures were initiated, including exhaustive room cleaning twice a day, reinforcing surveillance measures, skin cleaning with 2% chlorhexidine wipes, and Plasmid Analysis oral decolonization of patients (amikacin, colistin, nystatin) Conjugation assays were carried out using E. coli MC1061 (Pellicé et al., 2021). resistant to rifampicin and sodium azide as the recipient strain. Transconjugant strains were selected in LB agar plates 1 https://github.com/nanoporetech/qcat supplemented with 1 mg/L of meropenem and 100 mg/L of 2 https://github.com/fenderglass/Flye sodium azide (Sigma-Aldrich, Spain). The location of resistant 3 https://cge.cbs.dtu.dk/services/ResFinder/ genes was determined by S1-nuclease digestion (New England 4 https://cge.cbs.dtu.dk/services/PlasmidFinder/ BioLabs Inc., United States) followed by PFGE and Southern 5 https://www-is.biotoul.fr/ blot hybridization with digoxigenin-labeled PCR-probes against 6 https://bigsdb.web.pasteur.fr/ blaKPC (Bogaerts et al., 2013). Plasmid incompatibility groups 7 https://www.snapgene.com/ Frontiers in Microbiology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC FIGURE 1 | Temporal distribution of KPC-producing K. pneumoniae isolates according to sequence types (STs) as well as KPC-producing E. coli and Enterobacter sp. isolates recovered in this study. During the following months the number of KPC-producing characterization. A total of 24 isolates were eventually selected, isolates rapidly decreased and the outbreak was considered 13 K. pneumoniae, 10 E. coli isolates, and 1 Enterobacter eradicated by November 2018, although some isolates were sp. (Table 1). still recovered up until February 2019 (Figure 1). Overall, 125 isolates (112 K. pneumoniae, 12 E. coli, and 1 Enterobacter sp.) from 101 patients were collected and included for Phenotypic and Molecular further studies. Thirty-two isolates (25.6%) were detected Characterization of Resistance from diagnosis samples and 93 (74.4%) were isolated from The MICs of selected isolates are shown in Table 1. All isolates surveillance samples (Supplementary Table 2). The outbreak were non-susceptible to cephalosporins and carbapenems, but involved the screening of 2031 patients. The Index case could were susceptible to ceftazidime-avibactam, fosfomycin and not be identified. colistin, except for MC-2-285 and MC-22-164, that were highly All K. pneumoniae strains and the single Enterobacter sp. resistant to fosfomycin. isolate were classified into 11 PFGE clusters or pulsotypes Sanger sequencing identified the blaKPC- 2 variant in all isolates (designated as PTk, to differentiate from PTc, derived from except for the MC-2-251 and MC-2-387 isolates, that carried E. coli isolates, see below) with a major cluster, PTk1, that blaKPC- 3 (note that MC-2-387 also carried blaKPC- 2 ). The genes included 103 isolates while all other clusters just contained for CTX-M-group 1 enzymes were also detected in 7 of the 24 singletons. According to MLST, PTk-1 K. pneumoniae isolates strains, while CTX-M-group 2, 9, and 25 were only found in a were identified as ST15, PTk-2 as ST4684, PTk-3 as ST4685, few strains (Supplementary Table 2). CTX-M-group 8 was not PTk-5 as ST147, PTk-6 as ST1564, PTk-7 and PTk-8 as detected. All isolates were negative for the presence of blaOXA- 48 , ST258, Ptk-9 as ST307, PTk-10 as ST393, PTk-11 as ST37, blaNDM , blaVIM , or blaIMP . and PTk-4 corresponded to the single Enterobacter sp. isolate S1-nuclease-PFGE profiles of selected strains revealed the (Supplementary Figure 1). presence of different plasmids (according to size) that contained Among E. coli isolates, PFGE showed higher clonal the blaKPC gene. Eight of the selected K. pneumoniae strains heterogeneity, since the 12 strains were grouped into 10 harbored blaKPC- 2 within a plasmid of circa 110 kb in size clusters (PTc 1-10), with only clusters 1 and 10 containing two (plasmid type A, Figure 2), including the ST15 strains. The strains each and all other pulsotypes corresponding to singletons two ST258 strains MC-2-251 and MC-2-387 carrying blaKPC- 3 , (Supplementary Figure 2). According to MLST, the PTc-1 E. coli however, showed hybridization signals with plasmids of circa isolates were identified as ST101, PTc-2 and PTc-8 E. coli isolates 80 Kb and 120 Kb, respectively (plasmid types C and D, were identified as ST410, PTc-3 as ST38, PTc-4 as ST8576, PTc-5 Figure 2), and the single ST307 strain (MC-2-230) carried as ST1236, PTc-6 as ST1193, PTc-7 as ST131, PTc-9 as ST1431 blaKPC- 2 within a plasmid of circa 300 kb (plasmid type and PTc-10 as ST1642. E, Figure 2). Likewise, the Enterobacter sp. isolate harbored One strain from each cluster of either K. pneumoniae or blaKPC- 2 in a plasmid of approximately 400 Kb (plasmid type B, E. coli were selected for further phenotypic and genotypic Figure 2). Frontiers in Microbiology | www.frontiersin.org 4 November 2021 | Volume 12 | Article 781127 Frontiers in Microbiology | www.frontiersin.org Marí-Almirall et al. TABLE 1 | Antimicrobial susceptibility and molecular characterization of representative KPC-producing K. pneumoniae, Enterobacter sp. and E. coli isolates. Strains Bacterial Species bla gene ST MIC (mg/L) Tn Type P Inc P Size IPM MEM CAZ FEP CTX AMK TOB GEN KAN CST TGC CIP LVX CAZ/AVI FOF MC-2-1 K. pneumoniae KPC-2 ST15 24 >32 32 6 >32 1 0,38 0,38 1 2 1,5 12 4 0,75 24 pMC-2-1 IncFIIk 106 Kb MC-2-16 K. pneumoniae KPC-2 ST15 >32 >32 24 6 >32 1,5 0,5 0,38 1,5 0,094 1 >32 4 0,75 32 pMC-2-1 IncFIIk 106 Kb MC-2-20 K. pneumoniae KPC-2 ST15 >32 >32 24 4 >32 1 0,38 0,38 1,5 1 1,5 16 3 0,75 12 pMC-2-1 IncFIIk 106 Kb MC-2-146 K. pneumoniae KPC-2 ST15 >32 >32 32 48 >32 1 0,38 0,38 1 0,25 1,5 16 6 1 32 pMC-2-1 IncFIIk 106 Kb MC-2-177 E. coli KPC-2 ST410 4 3 192 32 >32 4 16 32 32 0,5 0,5 >32 >32 0,5 1 pMC-2-1 IncFIIk 106 Kb MC-2-196 E. coli KPC-2 ST8576 >32 16 48 >256 >32 1 0,38 0,5 1 0,125 0,75 0,008 0,023 0,125 1 pMC-2-1 IncFIIk 106 Kb MC-2-203 K. pneumoniae KPC-2 ST4684 4 >32 24 >256 >32 1 0,25 0,38 1 0,38 1 0,016 0,047 0,38 8 pMC-2-1 IncFIIk 106 Kb MC-2-207 E. coli KPC-2 ST1236 32 16 192 >256 >32 2 0,5 0,5 2 0,325 0,25 0,016 0,032 0,38 0,5 pMC-2-1 IncFIIk 106 Kb MC-2-216 K. pneumoniae KPC-2 ST4685 3 1 48 4 >32 1,5 2 0,38 4 0,25 1 0,5 0,38 0,25 6 pMC-2-1 IncFIIk 106 Kb MC-2-230 K. pneumoniae KPC-2 ST307 24 >32 24 8 >32 1 0,25 0,25 1 1 0,75 2 2 0,75 16 pMC-2-1 IncFIIk 281 Kb IncN MC-2–240 E. coli KPC-2 ST38 1 1 8 6 32 1,5 0,75 0,5 1,5 0,38 0,19 0,008 0,16 0,125 0,75 pMC-2-1 IncFIIk 190, 50 Kb MC-2-251 K. pneumoniae KPC-3 ST258 >32 >32 >256 >32 >32 32 16 1 >256 0,25 1 >32 >32 2 8 Tn4401 IncFIIk 78 Kb MC-2-285 E. coli KPC-2 ST410 12 8 24 8 >32 1 0,19 0,19 >256 0,25 0,25 >32 >32 0,19 >1024 pMC-2-1 IncFIIk 106 Kb 5 MC-2-303 K. pneumoniae KPC-2 ST37 4 >32 24 12 >32 1 0,19 0,19 0,75 0,38 2 0,125 0,38 1 32 pMC-2-1 IncFIIk 106 Kb MC-2-304 E. coli KPC-2 ST1642 12 1,5 4 3 >32 4 16 128 >256 0,125 >32 >32 >32 0,19 0,75 pMC-2-1 IncFIIk 170 Kb IncU MC-2-306 K. pneumoniae KPC-2 ST147 >32 >32 16 8 >32 2 8 16 16 3 3 >32 >32 1 32 pMC-2-1 IncFIIk 106 Kb MC-2-315 E. coli KPC-2 ST101 >32 >32 192 128 >32 1,5 0,5 0,25 1,5 0,125 0,75 0,125 0,5 0,75 1 pMC-2-1 IncFIIk 101 Kb MC-2-316 K. pneumoniae KPC-2 ST393 >32 >32 16 16 >32 1,5 0,25 0,38 1 0,25 0,75 0,016 0,047 0,75 16 pMC-2-1 IncFIIk 109 Kb MC-2-328 E. coli KPC-2 ST1431 4 4 12 192 >32 1 0,5 0,25 2 0,25 0,5 >32 >32 0,125 0,75 pMC-2-1 IncFIIk 106 Kb MC-2-350 E. coli KPC-2 ST1193 3 24 16 24 >32 2 16 >256 16 0,125 0,38 >32 >32 0,25 0,75 pMC-2-1 IncFIIk 106 Kb MC-2-362 Enterobacter sp. KPC-2 ND >32 >32 48 48 >32 2 12 32 16 1 4 >32 >32 1,5 12 pMC-2-1 IncFIIk 408 Kb IncN IncU November 2021 | Volume 12 | Article 781127 Hospital Interspecies Dissemination of KPC MC-2-382 E. coli KPC-2 ST131 24 24 96 256 >32 6 16 16 16 1 0,25 >32 >32 0,25 1 pMC-2-1 IncFIIk 106 Kb MC-2-387 K. pneumoniae KPC-2,-3 ST258 >32 >32 >256 >32 >32 32 >256 16 >256 0,38 1,5 >32 >32 1 32 pMC-2-1 IncFIIk 120; 28 Kb Tn4401 MC-22-164 K. pneumoniae KPC-2 ST1564 >32 >32 24 64 >32 1 0,19 0,38 1 1 2 1,5 3 1 >1024 pMC-2-1 IncFIIk 106 Kb ST, sequence type; IPM, imipenem; MEM, meropenem; CAZ, ceftazidime; FEP, cefepime; CTX, cefotaxime; AMK: amikacin; TOB: tobramycin; GEN, gentamicin; KAN, kanamycin; CST, colistin; TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; CAZ/AVI, ceftazidime/avibactam; FOF, fosfomycin; Tn Type, type of transposon containing the blaKPC gene; P Inc, Incompatibility group of plasmids containing the blaKPC gene. Inc groups from sequenced plasmids are shown in bold face; P Size, Size of plasmids containing the blaKPC gene; ND, not determined. Marí-Almirall et al. Hospital Interspecies Dissemination of KPC FIGURE 2 | (A) S1-nuclease-PFGE profiles and (B) corresponding southern hybridization membranes with blaKPC probes of selected K. pneumoniae and E. coli isolates in this study. The ST of both K. pneumoniae and E. coli isolates is shown in brackets. Arrows indicate the different arbitrarily designated plasmid types. Lambda ladder PFGE Marker (New England Biolabs, United States). *Enterobacter sp. isolate. **The PFGE plug moved from the loading site, so the band is shifted. ND, not determined. Among selected E. coli isolates, eight isolates also carried antibiotic resistance gene present in the pMC-2-1 plasmid that blaKPC- 2 in plasmids of similar size as that of type A plasmids also harbored several genes involved in conjugative transfer. in ST15 K. pneumoniae (110 Kb), although the MC-2-315 isolate The presence of plasmid-associated virulence genes was not presented a second hybridization signal at circa 180 Kb (plasmid detected in plasmid pMC-2-1 (Supplementary Figure 3). An type F, Figure 2). Two additional strains, MC-2-240 and MC- almost identical plasmid of 106,462 bp was found in the E. coli 2-304, carried blaKPC- 2 in plasmids of different sizes. MC-2-240 strain MC-2-207, and highly similar IncFIIk plasmids of 109,489 showed a hybridization band at approximately 190 Kb, and MC- and 101,915 bp were located in the K. pneumoniae and E. coli 2-304 at 170 Kb (plasmid types G and H, respectively, Figure 2). isolates MC-2-316 and MC-2-315, respectively, both recovered Notably, all E. coli isolates but one and the single Enterobacter from the same patient (Figure 3A), in good agreement with sp. isolate recovered during the outbreak, originated from the carriage of blaKPC- 2 in a plasmid of circa 110 Kb (plasmid patients that previously carried a ST15 KPC-2-producing type A), as identified by S1-digestion. Unfortunately, plasmid K. pneumoniae. On the other hand, only three out of the nine type F of circa 180 Kb, also present in strain MC-2-315 non-ST15 K. pneumoniae isolates were recovered from patients according to S1-digestion, could not be identified by long-read also co-carrying a ST15 isolate (Supplementary Figure 4 and sequencing analysis. Supplementary Table 2). Plasmid replicon typing identified the The K. pneumoniae isolate MC-2-230, the Enterobacter sp. presence of multiple plasmids in some of the strains (Table 1), but isolate MC-2-362 and the E. coli isolate MC-2-304, carried all strains were positive for a replicon belonging to the IncFIIk blaKPC- 2 within IncFIIk plasmids of 281,298, 407,992, and incompatibility group. Conjugation assays using the ST15 isolate 170,594 bp, respectively, much larger than that of ST15 strains MC-2-1 as donor and the E. coli strain MC1061 as recipient, and in good agreement with results from S1-nuclease digestion. showed that the acquisition of a blaKPC- 2 gene was associated with Additional replicon types from IncN and/or IncU incompatibility the transfer of an IncFIIk plasmid (not shown). groups were also identified in these plasmids. Interestingly, the entire 106 Kb sequence that made up for the pMC-2-1 plasmid Plasmid Sequencing was also found inserted within the plasmids harboring KPC in Ten isolates, 5 K. pneumoniae, 4 E. coli, and 1 Enterobacter strains MC-2-304 and MC-2-362, and the 281 Kb plasmid from sp., representative of strains carrying all the different plasmid strain MC-2-230 also carried a 67 kb fragment from pMC-2-1 types identified with hybridization probes, were further selected (Figure 3B). On the other hand, the two ST258 K. pneumoniae for long-read plasmid sequencing. Plasmid sequencing identified isolates (MC-2-251 and MC-2-387) harbored blaKPC- 3 within an IncFIIk plasmid of 106,412 bp carrying blaKPC- 2 in the IncFIIk plasmids of 78,515 and 120,395 bp, and there was no ST15 K. pneumoniae MC-2-1 isolate, designated as pMC-2-1 similarity at all between these two plasmids and those of ST15 (Supplementary Figure 3). The blaKPC- 2 gene was the only strains as there was no resemblance between them either. Frontiers in Microbiology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC FIGURE 3 | (A) Schematic representation of the 106,412 bp IncFIIk plasmid from the ST15 K. pneumoniae strain MC-2-1 (pMC-2-1) carrying blaKPC- 2 within a NTEKPC -pMC-2-1 genetic structure and sequence alignment with similar IncFIIk plasmid backbones from strains MC-2-207, MC-2-315, MC-2-316, MC-2-387, and MC-2-240 (only the aligned region is shown). Arrows are proportional to the lengths of the genes and oriented in the direction of transcription. Red arrows represent resistance genes, orange arrows represent full-length transposon-related genes and ISs, dark blue arrows represent partial or truncated transposon-related genes and ISs, green arrows indicate genes involved in plasmid conjugation and light blue arrows shown genes related to plasmid replication/maintenance. Blue-gray arrows show putative or hypothetical genes and plum arrows show genes involved in other functions. The inner circle shows the GC content. The fully annotated sequence of plasmid pMC-2-1 is shown in Supplementary Figure 3. (B) Schematic representation of the larger IncFIIk plasmids carrying blaKPC- 2 recovered from strains MC-2-230, MC-2-304, and MC-2-363. The location of the genetic structure NTEKPC -pMC-2-1 harboring blaKPC- 2 is shown as well as the region matching the pMC-2-1 sequence. The inner circle shows the GC content. In the E. coli strain MC-2-240 we only detected blaKPC- 2 Notably, in all plasmids blaKPC- 2 was not located within within a 25,058 bp plasmid showing 100% similarity with pMC- the canonical Tn4401 element but inside an IS26-based 2-1, and a similar plasmid of 28,498 bp was also detected in composite transposon of roughly 17 Kb containing an strain MC-2-387, which also carried blaKPC- 3 in the 120,395 bp ISKpn27-blaKPC- 2 -1ISKpn6-korC core structure as well as plasmid (Figure 3A). Tn3-associated sequences upstream from ISKpn27 (Figure 4). Frontiers in Microbiology | www.frontiersin.org 7 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC FIGURE 4 | Schematic drawing showing the genetic elements surrounding the blaKPC genes in the NTEKPC -pMC-2-1 variant and Tn4401, respectively. Arrows are oriented in the direction of transcription. Red arrows show the blaKPC gene, orange arrows represent full-length insertion sequences (IS), dark blue arrows represent partial or truncated transposon-related regions and ISs, and green arrows indicate genes involved in gene regulation. Blue-gray arrows show putative or hypothetical genes and plum arrows show genes involved in other functions. This rearrangement was tentatively designated as NTEKPC - a human patient in 2015 (Piedra-Carrasco et al., 2018) but, to pMC-2-1. The genetic structures surrounding the blaKPC- 3 gene our knowledge, our study constitutes the first hospital outbreak in both ST258 strains, however, did match that of a classical caused by KPC-producing Enterobacterales in this region. Tn4401 element (Figure 4). Specific primers to amplify the Here we report an outbreak caused by KPC-producing structures associated with NTEKPC -pMC-2-1 were designed K. pneumoniae and E. coli in a tertiary hospital in Barcelona (Supplementary Table 1 and Supplementary Figure 5) and used that initiated during the summer of 2018. It involved 125 to verify the carriage of either the canonical Tn4401 transposon Enterobacterales isolates recovered from surveillance (74.4%) or or the NTEKPC -pMC-2-1 variant in all 125 isolates of the diagnosis (25.6%) samples. Outbreak identification was possible outbreak. Interestingly, NTEKPC -pMC-2-1 was identified in all upon the implementation of an active surveillance program in isolates but in the ST258 K. pneumoniae isolate MC-2-251. ICU patients, but it is likely that KPC-producing K. pneumoniae In addition, all plasmid sequences were checked for the isolates had already been circulating in asymptomatic fecal presence of plasmid-mediated virulence genes producing carriers for some time. The outbreak was caused by the rapid salmochelin, aerobactin, the hypermucoviscosity factor and/or spread of a carbapenem-resistant ST15 K. pneumoniae strain other virulence genes (Russo et al., 2018), but none of the carrying blaKPC- 2 in an IncFIIk plasmid of approximately 106 Kb plasmid sequences carrying blaKPC harbored any known and located within a non-Tn4401 genetic element (NTEKPC - virulence determinant. pMC-2-1). Nevertheless, upon the initial stage of clonal spread, we speculate that the IncFIIk plasmid was successfully transferred DISCUSSION first to E. coli isolates in patients either infected or colonized with the original ST15 strain, but later to other strains of The emergence of KPC-producing Enterobacterales has been K. pneumoniae or even Enterobacter spp. (Figure 1 and increasing worldwide during the last decades (Lee et al., 2019). Supplementary Figure 4). This is supported by the fact that Since its first description in 1996 in the United States, KPC has in at least 14 instances the same patient co-carried a blaKPC- 2 - spread through the globe, becoming endemic in areas such as ST15 K. pneumoniae strain together with either a blaKPC- 2 - the United States, Israel, Greece, and Italy, and sporadic spread K. pneumoniae belonging to a different sequence type or a being reported in additional European countries as well as in the blaKPC- 2 -E. coli or Enterobacter sp. strain, but also because Asia-Pacific region (Lee et al., 2019). Many of the descriptions all plasmids carrying blaKPC- 2 in K. pneumoniae or E. coli from European countries have been related to patients traveling strains shared high similarity with the IncFIIk plasmids from to endemic areas. the ST15 K. pneumoniae strains, including the NTEKPC -pMC-2- In Spain, the first KPC report dates back to 2009 and it 1 structure surrounding blaKPC- 2 . Some genetic rearrangements, was associated with the rapid spread of a ST384 K. pneumoniae however, seem to have occurred, mainly in non-K. pneumoniae strain carrying blaKPC- 3 within a Tn4401a structure (Curiao strains where the 106 Kb IncFIIk plasmid either co-integrated et al., 2010). KPC-2 and KPC-3-producing Enterobacterales were with other plasmids or suffered extensive genetic reduction. later reported in the central region of Spain and Andalusia Therefore, intra- and inter-species dissemination of resistance mainly associated with K. pneumoniae isolates belonging to the also contributed to the spread of the outbreak. KPC transfer to CG258 or ST101 (López-Cerero et al., 2014; Porres-Osante et al., other K. pneumoniae strains was not as heavily associated with 2014; Oteo et al., 2016). In Catalonia, a single KPC-3-producing co-carriage within the same patient, as opposite to inter-species K. pneumoniae isolate belonging to ST258 was recovered from dissemination, but such finding may also reflect a selection Frontiers in Microbiology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC bias in the microbiology laboratory. Interestingly though, the possibilities but we expect that results from this study will clonal dissemination of non-ST15 strains was not detected. contribute to a better implementation of WGS pipelines in Recently, San Millán and co-workers proposed a similar hospital our institution. transmission dynamic for pOXA-48, where patient-to-patient transmission was tightly associated with the dissemination of a particular high-risk clone, while intra- and inter-species CONCLUSION transmission of the plasmid was linked to concurrent gut colonization (León-Sampedro et al., 2021). As in the case of We report a hospital outbreak caused by the clonal dissemination OXA-48, this genetic exchange represents an opportunity for the of KPC-producing ST15 K. pneumoniae mainly among colonized resistance gene to rearrange and shuffle into new plasmids and/or carriers but also by the intra- and inter-species transmission of hosts, some of which may become more successful (Millan, 2018). the blaKPC- 2 gene associated with plasmid conjugation and/or The genetic structures associated with the core structure of transposon dissemination. The ST15 clonal lineage is considered NTEKPC -pMC-2-1 identified in this work (ISKpn27-blaKPC- 2 - a high-risk clone and has been associated with KPC-2-producing 1ISKpn6-korC) had already been reported by other studies isolates in Bulgaria and Vietnam (Markovska et al., 2015; and seem to constitute a common rearrangement associated Berglund et al., 2019), and it has also been reported in Portugal with blaKPC- 2 mostly in isolates from China (Wang et al., and Italy (Rodrigues et al., 2016; Fasciana et al., 2019). In 2015; Zhang et al., 2017). In Spain though, this structure Spain, ST15 has been reported but only associated with the has also been identified in IncP-6 plasmids from Citrobacter production of OXA-48 (Madueño et al., 2017). Nevertheless, freundii, Enterobacter cloacae, and Klebsiella oxytoca but, to ST15 has been attributed with a high potential for horizontal our knowledge, have never been associated with K. pneumoniae gene acquisition and dissemination and it is critical that active isolates of human origin, most likely suggesting a recent surveillance strategies are prolonged over time to allow for the acquisition (Yao et al., 2017; Pérez-Vazquez et al., 2019). rapid detection and eradication of these highly resistant and Two sporadic K. pneumoniae strains belonging to the clonal virulent clones (Berglund et al., 2019). group CG258 and carrying the blaKPC- 3 gene within a canonical Tn4401 in two different non-related plasmids were also detected in the study and, most likely, constituted an independent event MERCyCAT STUDY GROUP unrelated to the spread of the outbreak. Nevertheless, one of Pepa Pérez Jove, Emma Padilla, and Mónica Ballestero-Téllez such strains also managed to acquire a partial plasmid sequence (Catlab, Centre Analítiques Terrassa AIE), Yuliya Zboromyrska, containing the entire NTEKPC -pMC-2-1 element. Miguel Ángel Benítez, Raquel Clivillé, Sabina González, and The identification of a carbapenem resistant ST307 Iolanda Calvet (Consorci del Laboratori Intercomarcal de l’Alt K. pneumoniae isolate in this study is also worth mentioning, Pendès, l’Anoia i el Garraf), Carmen Gallés (Corporació de Salut as strains from this epidemic clonal group have recently been del Maresme i la Selva), Goretti Sauca (Hospital de Mataró), responsible for a hospital outbreak in Germany associated Carmina Martí-Sala and Ma Angeles Pulido (Hospital General with multidrug resistance but also with the hypervirulent de Granollers), Anna Vilamala (Hospital General de Vic), Araceli Klebsiella pneumoniae phenotype (hvKp). The hvKp phenotype González-Cuevas (Hospital General del Parc Sanitari Sant Joan de was identified among ST307 isolates upon the acquisition of Déu), Amadeu Gené (Hospital Sant Joan de Déu de Barcelona), several plasmid-mediated virulence genes that merged with a Gloria Trujillo and Joan Lopez Madueño (Hospital Sant Joan de resistance plasmid, hence creating a mosaic plasmid carrying Déu de Manresa), Xavier Raga (Hospital Sant PauI Santa Tecla), both resistance and virulence genes (Heiden et al., 2020). In Frederic Gómez, Ester Picó, and Carolina Sarvisé (Hospital our study the ST307 strain (MC-2-230) carried blaKPC- 2 in a Universitari Joan XXIII de Tarragona), Isabel Pujol and Xesca plasmid of 280 Kb also likely resulting from the merging of Font (Hospital Universitari Sant Joan de Reus). two different plasmids and, hence, the potential carriage of plasmid-mediated virulence genes was investigated. Fortunately, none of the plasmid-mediated virulence factors associated DATA AVAILABILITY STATEMENT with the hvKp phenotype were present in plasmid pMC-2-230 nor in any other plasmid sequenced in this study. Likewise, The datasets presented in this study can be found in online none of the K. pneumoniae strains showed a hypermucoid repositories. The names of the repository/repositories phenotype, which is characteristic (although not exclusive) of and accession number(s) can be found in the hvKp (Lan et al., 2021). article/Supplementary Material. We acknowledge several limitations in our study. First, WGS was performed under a single long-read sequencing approach, and we acknowledge that the use of a hybrid approach would AUTHOR CONTRIBUTIONS have allowed for additional and more accurate comparisons. In addition, only a selected group of isolates were sequenced and MM-A, NF, and MF contributed to the conception, design, sequence similarity was, therefore, assumed for the remaining and implementation of the study, acquisition of laboratory and isolates on the basis of PFGE, MLST and conventional PCR clinical data, analysis of the results, drafting the manuscript, and data. Unfortunately, further WGS analyses were beyond our approval of the final version of the manuscript. CC, JViñ, ER, Frontiers in Microbiology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 781127 Marí-Almirall et al. Hospital Interspecies Dissemination of KPC AC, and LM contributed to the acquisition of laboratory data, 13/02564, respectively, from the Spanish Ministry of Education, analysis of the results, and review and approval of the final version Culture and Sports. IR was supported by the Department of of the manuscript. MP, AV, IC, LR-S, GS, and AD contributed Health, Generalitat de Catalunya, grant SLT002/16/00349. We to the design of the study, acquisition of clinical data, analysis also acknowledge support from the Spanish Ministry of Science, of the results, and review and approval of the final version of Innovation and Universities through the “Centro de Excelencia the manuscript. OF, PC, FB, FM, JM, and ÁS contributed to Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and the design of the study, analysis of the results, and review and support from the Generalitat de Catalunya through the CERCA approval of the final version of the manuscript. CP, JVil, and Program.” The funders had no role in the study design, data IR contributed to the conception, design, and implementation collection, analysis and interpretation of data, decision to publish, of the study, analysis of the results, drafting the manuscript, or preparation of the manuscript. and approval of the final version of the manuscript. All authors critically revised the manuscript for intellectual content and read and approved the final manuscript. ACKNOWLEDGMENTS FUNDING We thank the team of curators of the Institute Pasteur MLST and whole genome MLST databases for curating the data and making This study was supported by Plan Nacional de I+D+i 2013–2016, them publicly available at http://bigsdb.pasteur.fr/. We also thank Instituto de Salud Carlos III, Subdirección General de Redes y the clinical and laboratory staff of the participating hospitals Centros de Investigación Cooperativa, Ministerio de Economía for obtaining clinical samples and the initial identification y Competitividad, Spanish Network for Research in Infectious of microorganisms. Diseases (REIPI RD16/0016/0010); the 2017 Call for Strategic Action on Health (PI17/01932), co-financed by European Development Regional Fund “A way to achieve Europe” and SUPPLEMENTARY MATERIAL the operative program Intelligent Growth 2014–2020; and grant 2017 SGR 0809 from the Departament d’Universitats, Recerca The Supplementary Material for this article can be found i Societat de la Informació, of the Generalitat de Catalunya. online at: https://www.frontiersin.org/articles/10.3389/fmicb. MM-A and CC were supported by grants FPU 14/06357 and FPU 2021.781127/full#supplementary-material REFERENCES in Spain. J. Antimicrob. 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M., Rojo-Bezares, B., Undabeitia, E., this article, or claim that may be made by its manufacturer, is not guaranteed or Torres, C., and Sáenz, Y. (2014). Emergence of a multiresistant KPC-3 and endorsed by the publisher. VIM-1 carbapenemase-producing Escherichia coli strain in Spain. J. Antimicrob. Chemother. 65, 1608–1614. doi: 10.1093/jac/dku055 Copyright © 2021 Marí-Almirall, Ferrando, Fernández, Cosgaya, Viñes, Rubio, Queenan, A. M., and Bush, K. (2007). Carbapenemases: the versatile beta- Cuscó, Muñoz, Pellice, Vergara, Campo, Rodríguez-Serna, Santana, Del Río, lactamases. Clin. Microbiol. Rev. 20, 440–458. doi: 10.1128/cmr.00001-07 Francino, Ciruela, Ballester, Marco, Martínez, Soriano, Pitart, Vila, Roca and Roca, I., Akóva, M., Baquero, F., Carlet, J., Cavaleri, M., Coenen, S., et al. (2015). MERCyCAT Study Group. This is an open-access article distributed under the terms The global threat of antimicrobial resistance: science for intervention. New of the Creative Commons Attribution License (CC BY). The use, distribution or Microbes New Infect. 6, 22–29. doi: 10.1016/j.nmni.2015.02.007 reproduction in other forums is permitted, provided the original author(s) and the Rodrigues, C., Bavlovič, J., Machado, E., Amorim, J., Peixe, L., and Novais, copyright owner(s) are credited and that the original publication in this journal A. (2016). KPC-3-Producing Klebsiella pneumoniae in Portugal linked to is cited, in accordance with accepted academic practice. No use, distribution or previously circulating Non-CG258 lineages and uncommon genetic platforms reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 11 November 2021 | Volume 12 | Article 781127