ORIGINAL RESEARCH published: 31 January 2020 doi: 10.3389/fmicb.2019.02961 Core/Whole Genome Multilocus Sequence Typing and Core Genome SNP-Based Typing of Edited by: Ruiting Lan, OXA-48-Producing Klebsiella University of New South Wales, Australia pneumoniae Clinical Isolates From Reviewed by: Yvonne Pfeifer, Spain Robert Koch Institute, Germany Jane Fiona Turton, Elisenda Miro 1* † , John W. A. Rossen 2,3 , Monika A. Chlebowicz 2 , Dag Harmsen 4 , Public Health England, Sylvain Brisse 5 , Virginie Passet 5 , Ferran Navarro 1 , Alex W. Friedrich 2 and United Kingdom S. García-Cobos 2* †‡ *Correspondence: 1 Department of Microbiology, Hospital de la Santa Creu i Sant Pau, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Elisenda Miro Pau), Barcelona, Spain, 2 Department of Medical Microbiology and Infection Prevention, University Medical Center emiro@santpau.cat Groningen, University of Groningen, Groningen, Netherlands, 3 ESCMID Study Group for Genomic and Molecular S. García-Cobos Diagnostics (ESGMD), Basel, Switzerland, 4 Department of Periodontology and Restorative Dentistry, University of Münster, s.garcia.cobos@umcg.nl Münster, Germany, 5 Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur, Paris, France † These authors have contributed equally to this work ‡ Presentaddress: Whole-genome sequencing (WGS)-based typing methods have emerged as promising S. García-Cobos, and highly discriminative epidemiological tools. In this study, we combined gene- Reference and Research Laboratory by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) on Antimicrobial Resistance and Healthcare-Associated Infections, approaches to investigate the genetic relatedness of a well-characterized collection National Microbiology Center, Institute of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the of Health Carlos III, Madrid, Spain predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone Specialty section: and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained This article was submitted to from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) Evolutionary and Genomic Microbiology, schemes from Institut Pasteur’s BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 a section of the journal genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) Frontiers in Microbiology were used. Allele differences or allelic mismatches and the genetic distance, as the Received: 10 September 2019 proportion of allele differences, were used to interpret the results from a gene-by- Accepted: 09 December 2019 Published: 31 January 2020 gene approach, whereas the number of SNPs was used for the cgSNP analysis. We Citation: observed between 0–10 and 0–14 allele differences among the predominant ST405 Miro E, Rossen JWA, using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches Chlebowicz MA, Harmsen D, Brisse S, Passet V, Navarro F, when using Institut Pasteur’s BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 Friedrich AW and García-Cobos S and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, (2020) Core/Whole Genome and 2–5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a Multilocus Sequence Typing and Core Genome SNP-Based Typing previously established threshold for epidemiological link) was generally in concordance of OXA-48-Producing Klebsiella with a low number of allele differences (<8) when using the SeqSphere+ cgMLST pneumoniae Clinical Isolates From Spain. Front. Microbiol. 10:2961. scheme. The cgSNP analysis showed 6–29 SNPs in isolates with identical allelic doi: 10.3389/fmicb.2019.02961 SeqSphere+ cgMLST profiles and 16–61 cgSNPs among ST405 isolates. Furthermore, Frontiers in Microbiology | www.frontiersin.org 1 January 2020 | Volume 10 | Article 2961 Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains comparison of WGS-based typing results with previously obtained MLST and pulsed- field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics. Keywords: K. pneumoniae, OXA-48, cgMLST, wgMLST, WGS, molecular epidemiology INTRODUCTION MATERIALS AND METHODS The accelerated spread of multidrug-resistant bacterial pathogens Sample Collection poses an important threat to public health (Zhao et al., 2010). Thirty-seven OXA-48-producing K. pneumoniae isolates were One example is the marked increase of carbapenem-resistant selected from a previous study collection from 2012 and eight Klebsiella pneumoniae producing different carbapenemases such Catalan hospitals, including representative isolates of each MLST as KPC, VIM, IMP, NDM, and OXA-48 (Oteo et al., 2014). and PFGE profile, and antimicrobial resistance profile (Argente Besides, the propensity of this species to cause outbreaks in et al., 2019). These isolates were previously typed by PFGE healthcare institutions is well known (Snitkin et al., 2012). We (Argente et al., 2019) and MLST following the Institut Pasteur need to understand the transmission of resistant pathogens seven-gene MLST scheme1 (Diancourt et al., 2005). Information to rapidly and effectively implement control measures during on phenotypic resistance and genotyping results from PCR and outbreak management (Zhou et al., 2016). Cluster identification Sanger sequencing is summarized in Table 1. The selected isolates and pathogen profiling, including antimicrobial resistance and were from ST405 (n = 31, corresponding to E1–E18 PFGE virulence profile characterization, are crucial for effective patterns), ST101 (n = 3, PFGE patterns A1–A3), ST14 (n = 1, infection control (Leopold et al., 2014; Zhou et al., 2015). PFGE pattern D), ST17 (n = 1, PFGE pattern B), and ST1233 Pulsed-field gel electrophoresis (PFGE) and multilocus (n = 1, PFGE pattern C). The studied isolates did not belong to sequence typing (MLST) have been used as gold standards for an outbreak, and neither had a known epidemiological link; thus, many years to confirm suspected outbreaks. However, PFGE they were considered sporadic cases. results are subjective and difficult to interpret (Leopold et al., 2014), and classical seven-gene MLST is an expensive and time- WGS consuming method (Larsen et al., 2012). In contrast, whole- After DNA extraction using the Ultraclean Microbial genome sequencing (WGS) is a technology with a proven high DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, discriminatory power and has become more accessible due to United States), WGS was applied to the 37 OXA-48-producing K. recent technological advances (Larsen et al., 2012; Maiden et al., pneumoniae isolates. The libraries were prepared using a Nextera 2013; Bialek-Davenet et al., 2014; Leopold et al., 2014; Zhou XT v.01 kit (Illumina Inc., San Diego, CA, United States) and et al., 2015, 2017; Rossen et al., 2018). However, defining criteria sequenced on an Illumina MiSeq sequencer (Illumina Inc., San to interpret the data obtained by WGS in infection control is Diego, CA, United States) to generate 2 × 250 bp paired-end difficult and requires a comparison of available methods on reads. After sequencing, the reads were quality trimmed and de well-characterized sets of isolates. Such criteria are advisable so novo assembled using the CLC Genomics Workbench software that, following an outbreak, differences among isolates can be v7.0.4 (CLC Bio, Aarhus, Denmark), with the default parameters, interpreted easily and accurately. except for “removal of low quality sequence limit,” which was set The objective of this work was to investigate the molecular to 0.01 to increase the quality of the called bases. epidemiology of a well-characterized collection of OXA-48- Assembly quality was assessed using QUAST v4.5 (Gurevich producing K. pneumoniae – the dissemination of which occurred et al., 2013), and a report including assembly metrics was in eight hospitals in Catalonia in 2012 (Argente et al., 2019) – generated (Supplementary Table S1). using different WGS-based typing methods and to compare them with classical MLST and PFGE. We used the following WGS- based typing methods: (i) core-genome MLST (cgMLST), based Core-Genome and Whole-Genome MLST on two different schemes, SeqSphere+ and BIGSdb-Kp (Snitkin (cgMLST, wgMLST), Ridom et al., 2012); (ii) whole-genome MLST (wgMLST), based on the SeqSphere+ Schemes SeqSphere+ scheme (Zhou et al., 2016); and (iii) core-genome The assembled genomes were imported into single nucleotide polymorphism (cgSNP) analysis. In addition, SeqSphere+ software v5.1.0 (Ridom GmbH, Münster, Germany) we investigated and compared phenotypic data and genotypic antimicrobial resistance data obtained using PCR and WGS. 1 https://bigsdb.pasteur.fr/ Frontiers in Microbiology | www.frontiersin.org 2 January 2020 | Volume 10 | Article 2961 Frontiers in Microbiology | www.frontiersin.org Miro et al. TABLE 1 | Phenotypic and genotypic characters of the 37 OXA-48-producing K. pneumoniae strains obtained by conventional methods, PCR, and Sanger sequencing. MLST PFGE N◦ Strain CEF CXM FOX CTX CAZ ATM FEP ERT IMP Blc K T G A N Nm AME NAL CIP 101 A1 CARB115 R R S R R R S R S blaSHV- 1 R R R S S R aac(30 )-IIa, aac(60 )-Ib R R A2 CARB077 R R R blaSHV- 1 S aac(30 )-IIa, aac(60 )-Ib A3 CARB058 S R R blaSHV- 1 R aac(30 )-IIa, aac(60 )-Ib 405 E1 CARB050 R R R R R R I R S blaSHV- 76 R R R S R S aac(30 )-IIa R R E1 CARB009 S R I S blaSHV- 76 I aac(30 )-IIa I E1 CARB007 S S I S blaSHV- 76 S S S S aac(30 )-IIa S S E1 CARB011 S S R S blaSHV- 76 S S S S S I S E1 CARB015 S I S R S blaSHV- 76 S S aac(30 )-IIa I I E1 CARB037 S S I S I S blaSHV- 76 S S S S aac(30 )-IIa, aac(60 )-Ib S S E1 CARB125 I S S S S S I S blaSHV- 76 S S I aac(60 )-Ib E1 CARB102 I S S S S S S I S blaSHV- 76 S S S aac(60 )-Ib S I E2 CARB020 R R R R R R S R S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib R R 3 E3 CARB042 R I I I S S S R I blaSHV- 76 R R R S S S aac(60 )-Ib R R E4 CARB106 R R S R R R I I S blaSHV- 76 R R R R R S aac(30 )-IIa, aac(60 )-Ib R R E5 CARB182 R R R R R R R R R blaSHV- 76 R R R R R S aac(30 )-IIa, aac(60 )-Ib R R E5 CARB056 I S S blaSHV- 76 S S aac(30 )-IIa E5 CARB022 S S S S S blaSHV- 76 S S aac(30 )-IIa, aac(60 )-Ib I I E5 CARB128 S S S S S S blaSHV- 76 S S S S aac(30 )-IIa S S cg/wgMLST of OXA-48-Producing K. pneumoniae Strains E6 CARB065 R R R R R R R R I blaSHV- 76 R R S S S S aac(30 )-IIa, aac(60 )-Ib S I E6 CARB166 I S S S S S S S blaSHV- 76 I aac(60 )-Ib I R E7 CARB038 R R R R R R I R S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib R R E8 CARB100 R R S R R R S R S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(600 )-Ib I I January 2020 | Volume 10 | Article 2961 E9 CARB123 R R S R R R S S S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib S I E10 CARB039 R S I S S S S I S blaSHV- 76 R R R S R S aac(60 )-Ib R R (Continued) Frontiers in Microbiology | www.frontiersin.org Miro et al. TABLE 1 | Continued MLST PFGE N◦ Strain CEF CXM FOX CTX CAZ ATM FEP ERT IMP Blc K T G A N Nm AME NAL CIP E11 CARB184 R R S R R R S S S blaSHV- 76 R R S R R S aac(30 )-IIa, aac(60 )-Ib R R E12 CARB112 R R S R R R S I S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib R R E12 CARB130 blaSHV- 76 R aac(30 )-IIa, aac(60 )-Ib I I E13 CARB096 R R S R R R S R S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib S S E14 CARB010 R R S R I R S R S blaSHV- 76 S S R S S S aac(30 )-IIa, aac(60 )-Ib I I E15 CARB026 R R S R R R S R S blaSHV- 76 R R R S S S aac(30 )-IIa, aac(60 )-Ib I I E16 CARB183 R R S R R R I I S blaSHV- 76 R R S S R S aac(30 )-IIa, aac(60 )-Ib R R 4 E17 CARB044 S S S S S S S S S blaSHV- 76 R R S I I S aac(30 )-IIa, aac(60 )-Ib S S E18 CARB040 R R S R R R S R S blaSHV- 76 R R R S I S aac(30 )-IIa, aac(60 )-Ib R R E18 CARB139 S blaSHV- 76 S R aac(30 )-IIa, aac(60 )-Ib S S 14 D CARB117 R S S S S S S I S blaSHV- 1 S S S S S S S S 17 B CARB098 I S S S S S S S S blaSHV- 11 S S S S S S S S 1233 C CARB122 S S S S S S S S S blaSHV- 42 S S S S S S S S cg/wgMLST of OXA-48-Producing K. pneumoniae Strains In bold, consensus sequence. Blank spaces regarding resistance phenotype indicate same result as consensus sequence, and regarding resistance genes (AME) indicate absence. Blc, beta-lactamase; AME, aminoglycoside-modifying enzymes. R, resistant; I, intermediate; S, susceptible; CEF, cephalotin; CXM, cefuroxime; FOX, cefoxitin; CTX, cefataxime; CAZ, ceftazidime; ATM, aztreonam; FEP, cefepime; ERT, ertapenem; IMP, imipenem; K, kanamycin; T, tobramycin; G, gentamicin; A, amikacin; N, netilmicin; Nm, neomycin; NAL, nalidixic acid; and CIP, ciprofloxacin; MLST, multilocus sequence type; PFGE, pulsed-field gel electrophoresis. All strains were resistant to ampicillin, piperacillin, coamoxiclav [except: CARB128 (E184) and CARB184 (E11)], and piperacillin/tazobactam [except: CARB122 (C), CARB128 (E5), and CARB184 (E11)]. All strains carried OXA-1 [except: CARB117 (D) and CARB122 (C)], TEM-1 [except: CARB042 (E3), CARB166 (E6), CARB117 (D), CARB098 (B), and CARB122 (C)], CTX-M-15 [except: CARB125 (E1), CARB102 (E1), CARB042 (E3), CARB039 (E10), CARB044 (E17), CARB117 (D), CARB098 (B), and CARB122 (C)], and QnrB [except: CARB077 (A2), CARB117 (D), CARB098 (B), and CARB122 (C)]. All strains had the Tn1999.2 genetic platform of January 2020 | Volume 10 | Article 2961 OXA-48, except: CARB106 (E4), CARB166 (E6) CARB184 (E11) CARB112, and CARB130 (E12), and CARB183 (E16). Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains for a gene-by-gene allele calling comparison using a higher Antimicrobial Resistance Genes and number of loci than the already published Institut Pasteur’s Plasmid Analysis BIGSdb-Kp cgMLST scheme (Bialek-Davenet et al., 2014). In addition to WGS typing, resistance genotypes and plasmid A hard defined and stable scheme comprising 2,365 targets families were investigated in the collection of OXA-48-producing or genes of the K. pneumoniae core genome (cgMLST) and K. pneumoniae and compared with those obtained previously 2,526 genes of the K. pneumoniae accessory genome (wgMLST; (Argente et al., 2019). Genome assemblies were uploaded to total of 4,891 targets), available in SeqSphere+, was used for the Center for Genomic Epidemiology to extract information the analysis (targets included in the schemes can be found at on antimicrobial resistance genes (ARGs) and plasmid replicons cgMLST.org). This scheme was developed using the seed genome for K. pneumoniae using ResFinder 2.1 and PlasmidFinder 1.3, K. pneumoniae subsp. pneumoniae NTUH-K2044 (GenBank respectively (Zankari et al., 2012; Carattoli et al., 2014). accession no. NC_012731.1; 15-JUN-2016) and comparing it The de novo assembled genomes were annotated using with 30 query genome sequences that cover the genetic variability Prokka 1.12 (Seemann, 2014) and compared with reference of the whole species complex, i.e., K. pneumoniae, K. variicola, K. pneumoniae plasmid pOXA-48 NC_019154.1 using BLASTn and K. quasipneumoniae, as previously defined (Bialek-Davenet analysis. Circular representation of compared genomes was et al., 2014). In addition, 61 plasmid sequences were used to performed with the Circular Genome Viewer (CGView) exclude chromosomal homologs of plasmid genes. A detailed Comparison Tool program (Grant et al., 2012), in which the description of the scheme development is available in the coding sequences (CDS) in reference and query sequences were software. In addition, a cgMLST complex type (CT) function used. In addition, generated blastn output files were further used was used to identify isolates with very similar cgMLST profiles2 . for visualization using DNAplotter (Carver et al., 2009) and for A cluster alert distance of 15 cgMLST allele differences and a further analysis using the Artemis Comparison Tool (Carver cluster alert quality threshold of at least 90% good cgMLST targets et al., 2005). Similarity matches were filtered based on their were used to detect closely related isolates. These thresholds were lengths (100 kb segments) and percentage similarity scores, and based on retrospective analysis of well-defined outbreaks and only the filtered hits with at least 80% sequence similarity were out-group isolates with the same MLST/MLVA/PFGE profiles. subsequently displayed using the Artemis Comparison Tool. Distance matrices describing pairwise allele differences (ignoring missing values) are included in Supplementary Data Sheet S1 (cgMLST and wgMLST, SeqSphere+). Furthermore, another parameter named the genetic distance RESULTS was calculated for paired isolates as the proportion of allele differences, obtained by dividing the number of allele cgMLST and wgMLST Schemes From differences between the two genomes by the total number of Ridom SeqSphere+ called genes shared by those two genomes. We considered The cgMLST analysis of the 37 isolates resulted in nine CTs: 185, previously described genetic distance thresholds, 0.0035 for 952, and 1149 to 1155 (Figure 1A). Among isolates of MLST type cgMLST and 0.0045 for wgMLST, to discriminate between ST405 (n = 31), three different CTs were assigned: CT185 (PFGE epidemiologically related and unrelated K. pneumoniae isolates subtypes E1, E4, E5, E7, E14, E15, E17, and E18), CT1150 (PFGE (Kluytmans-van den Bergh et al., 2016). subtypes E1, E2, E3, E5, E6, E8, E10–E13, and E16), and CT1153 (PFGE subtype E9). The three ST101 isolates (PFGE subtypes cgMLST, BIGSdb-Kp Scheme A1–A3) had different CTs (CTs 952, 1152, and 1154) (Figure 1A). Assembled genomes were analyzed using the Institut ST14, ST17, and ST1233 isolates (n = 1, each ST) (PFGE profiles Pasteur BIGSdb-Kp3 634-gene strict core-genome MLST D, B, and C, respectively) were assigned to CTs 1151, 1155, and (scgMLST) scheme (Bialek-Davenet et al., 2014). A spanning 1149, respectively. These minority STs (ST14, ST17, and ST1233) tree (BioNumerics v6.7.3, Applied-Maths, Belgium) was were clearly separated from the ST405 and ST101 isolates, with constructed from the allelic profiles of this cgMLST scheme to an allele distance from ST405 isolates of more than 1,875 allele visualize the corresponding diversity of genotypes (figure in differences when using cgMLST (Figure 1A), and more than Supplementary Figure S1). 3,618 alleles when using wgMLST (Figure 1B). Isolates within ST101, CT952 (PFGE-A1), and CT1152 Core-Genome SNP Analysis (PFGE-A2) showed 14 different alleles by cgMLST (Figure 1A The number of single nucleotide polymorphisms (SNPs) in the and Table 2) and differed in two bands by PFGE (97% similarity; core genome (defined as orthologous sequences conserve in all data not shown). However, these isolates had a genetic distance of aligned genomes) was calculated from the de novo assembled 0.0060 indicating no genetic relatedness. Clinical epidemiological genomes for isolates with the same ST (ST405 and ST101) using data of isolates clustering together by cgMLST (<15 allele the Harvest software suite (parsnp), including recombination differences) are described in Table 3. filtration (Treangen et al., 2014). A cgSNP-based tree was built Isolates within ST405 (PFGE subtypes E1–E18, which differed using the Neighbor Joining method. in less than three bands between them) differed by a maximum of 10 alleles by cgMLST and 14 alleles by wgMLST (Figures 1A,B). 2 https://www.ridom.de/u/Core_Genome_MLST_Complex_Type.html All of them had a genetic distance below the threshold (0.0035) 3 https://bigsdb.pasteur.fr/klebsiella and belonged to CT185 or CT1150, except isolates CARB100 Frontiers in Microbiology | www.frontiersin.org 5 January 2020 | Volume 10 | Article 2961 Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains FIGURE 1 | Minimum spanning tree of 37 OXA-48-producing K. pneumoniae isolates obtained from a previous study (Argente et al., 2019). Each circle represents a genotype, and the number of different alleles is indicated on the edges between connected isolates (nodes). In addition, the genetic distance (gd) for specific pairwise comparisons is indicated in blue (see also Table 2). Isolates are presented by their ID, pulsed-field gel electrophoresis (PFGE) profile, sequence type (ST), and hospital origin. Colors indicate the different complex types (CTs). The cluster distance threshold is 15. (A) Distance based on whole-genome multilocus sequence typing (cgMLST) of 2,365 genes using the parameters “pairwise ignoring missing values” during calculation. Groups of isolates (groups a, b, c, and d) with 0 allele differences obtained by cgMLST analysis are highlighted. (B) Distance based on whole-genome multilocus sequence typing (wgMLST) of 4,891 genes (cgMLST of 2,365 core genes and 2,526 accessory genes) using the parameters “pairwise ignoring missing values” during calculation. Isolates of group c that were previously investigated by cgMLST analysis are highlighted. (CT1150) and CARB123 (CT1153), which showed a genetic determined using other WGS analysis tools, confirming the distance of 0.0042 (10 allele differences) by cgMLST (Table 2). In high reproducibility of the seven-gene MLST scheme from contrast, when considering the wgMLST analysis, CARB100 and genomic assemblies. All ST405 isolates had ≤2 allelic mismatches CARB123 had a genetic distance of 0.0031 (14 allele differences) (mostly 0 allelic mismatches) (Table 2; figure in Supplementary indicating genetic relatedness (Table 2). By PFGE analysis, these Figure S1). The three ST101 isolates presented between 2 and two isolates showed 95% similarity (two different bands). 5 allelic mismatches, whereas ST14, ST17, and ST1233 isolates Four pairs of isolates belonging to PFGE-E subtypes, were genetically unrelated with more than 480 allelic mismatches CARB102-E1/CARB183-E16 (group a), CARB130- (figure in Supplementary Figure S1). E12/CARB096-E13 (group b), CARB044-E17/CARB139-E18 We also calculated the genetic distance for this scheme; all (group c), and CARB007-E1/CARB009-E1 (group d), showed paired isolates had a genetic distance below 0.0032, except for no allele differences when analyzed by cgMLST (Figure 1A). CARB115-A1 and CARB058-A3, which had a genetic distance of When using wgMLST only, isolates from group c had zero 0.0080 (five allelic mismatches) (Table 2). allele differences (Figure 1B), whereas isolates from group a showed one allele difference, isolates from group b showed four allele differences, and isolates from group d were genetically Core-Genome SNP Analysis Isolate CARB007-E1 – the oldest isolate – was used as a reference unrelated (Figure 1B). genome for the cgSNP analysis of ST405 isolates (n = 31). The average of core genome alignment was 88.7%. Among ST405 Analysis of Isolates Using the isolates, cgSNP distance ranged between 16 and 61 core SNPs BIGSdb-Kp cgMLST Scheme (Supplementary Figure S2 and Supplementary Data Sheet We obtained identical seven-gene MLST STs for all isolates S2). The four groups of two isolates that had identical allelic when using the BIGSdb-Kp database to those previously profiles, as analyzed by cgMLST (Figure 1A), showed 6, 29, Frontiers in Microbiology | www.frontiersin.org 6 January 2020 | Volume 10 | Article 2961 Frontiers in Microbiology | www.frontiersin.org Miro et al. TABLE 2 | Genetic distance for pairwise comparisons of OXA-48- K. pneumoniae isolates using cgMLST/wgMLST schemes from Ridom SeqSphere+ and cgMLST from BIGSdb-Kp (Institut Pasteur). Ridom SeqSphere+ Schemes BIGSdb-Kp Scheme Sample Hospital Isolation date in PFGE ST CTa Minimum No. alleles No. alleles Genetic Genetic No. alleles Genetic 2012 (month–day) profile spanning tree differences/no. shared differences/no. shared distance distance differences/no. shared distance results genes cgMLSTb genes wgMLSTc cgMLSTd wgMLSTe genes cgMLSTf cgMLSTf 1 CARB115 HGV July-31 A1 101 952 CARB077 HGH May-05 A2 101 1152 14/2322 29/4416 0.0060 0.0066 2/623 0.0032 CARB058 HM July-11 A3 101 1154 54/2328 97/4432 0.0232 0.0219 5/624 0.0080 CARB098 HGG September-10 B 17 1155 CARB122 HM January-19 C 1233 952 CARB117 HGV October-18 D 14 1151 CARB102 HSJDD October-05 E1 405 1150 Group a 0/2332 1/4458 NA 0.0002 0/623 NA CARB183 HSCSP August-21 E16 405 1150 Group a CARB130 HSJDD December-05 E12 405 1150 Group b 0/2334 4/4427 NA 0.0009 0/628 NA 7 CARB096 HSJDD September-06 E13 405 1150 Group b CARB044 HGV February-19 E17 405 185 Group c 0/2358 0/4504 NA NA 0/629 NA CARB139 HGV December-29 E18 405 185 Group c CARB007 HGV January-04 E1 405 185 Group d 0/2306 7/4388 0.0016 NA 1/623 0.0016 CARB009 HGV January-19 E1 405 185 Group d 2 CARB026 HC January-03 E15 405 185 cg/wgMLST of OXA-48-Producing K. pneumoniae Strains CARB050 HGG June-20 E1 405 185 1/2351 6/4488 0.0004 0.0013 0/631 NA CARB040 HGV February-16 E18 405 185 1/2357 1/4505 0.0004 0.0002 0/630 NA CARB011 HGV January-15 E1 405 185 2/2354 8/4480 0.0008 0.0018 0/629 NA CARB022 HGG Mars-21 E5 405 185 2/2357 5/4501 0.0008 0.0011 0/630 NA January 2020 | Volume 10 | Article 2961 CARB038 HGV May-10 E7 405 185 3/2356 9/4496 0.0013 0.0020 1/630 0.0016 CARB015 HGV September-04 E1 405 185 3/2357 4/4500 0.0013 0.0009 1/630 0.0016 CARB010 HGV January-11 E14 405 185 4/2357 8/4497 0.0017 0.0018 0/630 NA CARB106 HGG October-29 E4 405 185 5/2351 11/4474 0.0021 0.0024 0/628 NA CARB042 HGV April-04 E3 405 1150 5/2357 15/4499 0.0021 0.0033 1/629 0.0016 (Continued) Frontiers in Microbiology | www.frontiersin.org Miro et al. TABLE 2 | Continued Ridom SeqSphere+ Schemes BIGSdb-Kp Scheme Sample Hospital Isolation date in PFGE ST CTa Minimum No. alleles No. alleles Genetic Genetic No. alleles Genetic 2012 (month–day) profile spanning tree differences/no. shared differences/no. shared distance distance differences/no. shared distance results genes cgMLSTb genes wgMLSTc cgMLSTd wgMLSTe genes cgMLSTf cgMLSTf CARB037 HGV May-14 E1 405 185 6/2355 11/4493 0.0025 0.0024 0/631 NA CARB112 HGV June-21 E12 405 1150 7/2356 11/4500 0.0030 0.0024 1/631 0.0016 CARB056 HGG July-03 E5 405 1150 8/2349 13/4464 0.0034 0.0029 2/630 0.0032 CARB184 HSCSP October-02 E11 405 1150 CARB042 HGV April-04 E3 405 1150 5/2358 12/4500 0.0021 0.0027 1/629 0.0016 CARB100 HC October-05 E8 405 1150 CARB123 HC September-12 E9 405 1153 10/2357 14/4503 0.0042 0.0031 1/628 0.0031 3 CARB037 HGV May-14 E1 405 1150 CARB065 HSJDD August-19 E6 405 1150 5/2357 11/4493 0.0021 0.0024 0/628 NA 8 CARB182 HSCSP July-19 E5 405 1150 4/2356 14/4492 0.0017 0.0031 0/631 NA CARB020 HGG Mars-06 E2 405 1150 5/2326 9/4419 0.0021 0.0020 1/624 0.0016 CARB020 HGG Mars-06 E2 405 1150 CARB128 HGG Desember-10 E5 405 1150 3/2317 9/4397 0.0013 0.0020 1/623 0.0016 CARB182 HSCSP July-19 E5 405 1150 CARB039 HGV May-08 E10 405 1150 3/2313 10/4411 0.0013 0.0023 0/630 NA cg/wgMLST of OXA-48-Producing K. pneumoniae Strains CARB065 HSJDD August-19 E6 405 1150 CARB166 HSCSP September-19 E6 405 1150 3/2355 7/4489 0.0013 0.0015 0/627 NA a CT,complex type from cgMLST Ridom SeqSphere+. b cgMLST = 2,365 targets including 7 MLST genes (Ridom SeqSphere+). c wgMLST = 2,365 targets of cgMLST + 2329 accessory targets, 4,891 targets in total. d Threshold = 0.0035 (Kluytmans-van den Bergh et al., 2016) (italic indicates genetic distance above the threshold). e Threshold = 0.0045 (Kluytmans-van den Bergh et al., 2016) (italic indicates genetic distance above January 2020 | Volume 10 | Article 2961 the threshold). f cgMLST = 634 genes corresponding to the strict core genome (scgMLST634) scheme of Bialek-Davenet et al. (2014) (28) (described at bigsdb.pasteur.fr/klebsiella). 1 CARB115 isolate used for pairwise comparison with CARB077 and CARB058. 2 CARB026 isolate used for pairwise comparison with CARB050, CARB040, CARB011, CARB022, CARB038, CARB015, CARB010, CARB106, CARB042, CARB037, CARB112, and CARB056. 3 CARB037 isolate used for pairwise comparison with CARB065, CARB182, and CARB020.cgMLST, core-genome multilocus sequence typing; wgMLST, whole-genome multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; HSJDD, Hospital de Sant Joan de Déu (Manresa); HSCSP, Hospital de la Santa Creu i Sant Pau (Barcelona); HGV, Hospital General de Vic (Vic); HGH, Hopsital General de l’Hospitalet (Hospitalet del Llobregat); HM, Hospital de Mataró (Mataró). Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains TABLE 3 | Epidemiological and clinical context of OXA-48-producing K. pneumoniae isolates that clustered together using cgMLST. MLST PFGE WGS groups Isolates Hospital Department Isolation date Clinical sample Baseline pathology Gender Age ST405 E1 a CARB102 HSJdD Internal Medicine 10-05-12 Urine Trasplanted Woman 71 E16 CARB183 HSCSP Oncology 08-21-12 Urine Leukemia Man 60 E12 b CARB130 HSJdD Cardiology 12-05-12 Wound Cardiac dyspnea Woman 98 E13 CARB096 HSJdD Dialysis Unit 09-06-12 Urine Dialysis Man 59 E17 c CARB044 HGV Emergencies 02-19-12 Urine UTI Man 88 E18 CARB139 HGV Emergencies 12-28-12 Urine UTI Man 68 E1 d CARB007 HGV Emergencies 01-04-12 Urine UTI Woman 76 E1 CARB009 HGV Emergencies 01-19-12 Urine UTI Woman 91 ST101 A2 CARB077 HGH Emergencies 05-11-12 Rectal exudate ESBL-carrier Man 82 A1 CARB115 HGV Emergencies 08-25-12 Urine UTI Woman 89 A3 CARB058 HM Emergencies 07-11-12 Urine UTI Woman 87 MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; WGS, Whole-genome sequencing; UTI, urinary tract infection; ESBL, extended-spectrum beta-lactamase; HSJdD, Hospital de Sant Joan de Déu (Manresa); HSCSP, Hospital de la Santa Creu i Sant Pau (Barcelona); HGV, Hospital General de Vic (Vic); HGH, Hopsital General de l’Hospitalet (Hospitalet del Llobregat); HM, Hospital de Mataró (Mataró). 6, and 17 SNPs, respectively, for groups a, b, c, and d. The aac(3)-IId (5.4%; 2/37) (kanamycin, gentamycin, tobramycin, average of core genome alignment for ST101 isolates was 85.4%. and netilmicin resistance). ST101 isolates CARB115-A1 and CARB077-A2 showed 2,427 and We compared the nucleotide sequences of the gyrA, gyrB, 2,673 core SNPs, respectively, compared with isolate CARB058- parC, and parE genes and their amino acid sequences, of A3, which was used as reference (Supplementary Figure S2 the entire isolate collection with those of the ATCC reference and Supplementary Data Sheet S2), whereas the number of strain 13883, a quinolone-susceptible strain (Brisse et al., 1999). core SNPs between CARB115-A1 and CARB77-A2 was 54 Of the 37 isolates, 29 had a ciprofloxacin minimal inhibitory (Supplementary Figure S2 and Supplementary Data Sheet S2). concentration ≥2 mg/L, but mutations in gyrA and parC were only found in 5 isolates. The ST101 isolates CARB058, CARB077, and CARB115 had non-silent mutations in the gyrA Antimicrobial Resistance Genes From gene (Ser83Tyr, Asp87Gly, or Asp87Ala) and the parC gene WGS Data Compared to PCR and (Ser80Ile, Asn304Ser). The ST405 isolate CARB039 had a non- Phenotypic Results silent mutation in the gyrA gene (Ser83Ala). In addition, one The ResFinder tool from the Center for Genomic Epidemiology ST17 isolate (CARB098) showed two new mutations in the detected the presence of multiple resistance genes implicated in gyrA gene (Ala863Val, Thr868Ile); however, the ciprofloxacin β-lactams, aminoglycosides, and quinolone resistance as follows. minimal inhibitory concentration for this isolate was 1 mg/L. In All isolates were positive for blaOXA- 48 , except CARB077, and addition, qnrB66 (81.6%), qnrB1 (27%), and aac(60 )-Ib-cr genes for one of the following blaSHV genes: blaSHV- 1 (n = 4), (78.4%) were present, which could explain a certain level of blaSHV- 11 (n = 4), blaSHV- 42 (n = 1), and blaSHV- 76 (n = 31) quinolone resistance (Table 4). However, seven isolates carrying (Table 1). In addition, 78.4% (29/37) of the isolates contained the qnrB gene (CARB007, CARB011, CARB037, CARB044, blaCTX-M- 15 ; 56.4% (22/37), blaOXA- 1 ; and/or 81% (30/37), CARB096, CARB128, CARB139) and five isolates carrying the blaTEM- 1 genes (Table 4). We observed some discrepancies aac(60 )-Ib-cr gene (CARB037, CARB044, CARB096, CARB128, with results previously obtained by PCR and Sanger sequencing CARB139) were susceptible to ciprofloxacin and nalidixic acid. (Argente et al., 2019). In three isolates (CARB009, CARB050, OqxA/B genes, encoding an efflux pump, were present in and CARB056), the blaOXA- 1 gene was detected by PCR but not all isolates. Nevertheless, this mechanism is associated with by WGS, whereas in two isolates (CARB039 and CARB044), a low level of resistance to several antimicrobials, such as the blaCTX-M- 15 gene was not detected by PCR but detected by conferring low to intermediated resistance to quinoxalines, WGS. In the latter case, the phenotypes were in concordance quinolones, tigecycline, nitrofurantoin, several detergents, and with the PCR results; the isolates were susceptible to third- disinfectants (benzalkonium chloride, triclosan, and sodium generation cephalosporins. Finally, CARB184 isolate was positive dodecyl sulfate). for the blaCTX-M- 15 gene by PCR (in concordance with its Other resistance genes identified by WGS were sul2 (78.4%) antimicrobial susceptibility testing results), but this gene was not and dfrA14 (91.9%) genes (co-trimoxazole resistance), tet(A) detected by WGS. (64.8%) and tet(D) (8.1%) genes (tetracycline resistance), and We observed five acquired genes related to aminoglycoside catB3 (72.9%) (chloramphenicol resistance) and fosA (83.8%) resistance: strA/strB (78.4%; 29/37) and aph(30 )-Ia (2.7%; (fosfomycin resistance). 1/37) (streptomycin resistance), aac(60 )-Ib-cr (78.4%; We defined different resistance profiles (resistomes) based 29/37) (kanamycin, tobramycin, amikacin, netilmicin, and on the combination of acquired ARGs (Table 4). Resistome fluoroquinolone resistance), aac(3)-IIa (70.3%; 26/37), and I included 14 ARGs, whereas resistomes XVI to XVIII only Frontiers in Microbiology | www.frontiersin.org 9 January 2020 | Volume 10 | Article 2961 Frontiers in Microbiology | www.frontiersin.org Miro et al. TABLE 4 | Cluster types (CTs) and resistance genes found in the 37 carbapenemase-producing K. pneumoniae (except one isolate, by ResFinder, all were positive for blaOXA- 48 , and for one of the following genes: blaSHV- 1 , blaSHV- 11 , blaSHV- 42 , and blaSHV- 76 ). ST CT n PFGE profiles Isolate Beta-lactamases Aminoglycosides-modifying enzymes Others Resistome 101 1149 1 A1 CARB115 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (99,88)/strB (99,88); aac(3)-IId (99,88); sul2 (100); catB3 (100); aac(60 )-Ib-cr (100) tet(D) (100) 1152 1 A2 CARB077 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (99,88)/strB (99,88); aac(3)-IId (99,88); sul2 (100); catB3 (100); aac(60 )-Ib-cr (100) tet(D) (100) 1154 1 A3 CARB058 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (99,88)/strB (99,88); aac(3)-IIa (99,77); sul2 (100); catB3 (100); aac(60 )-Ib-cr (100); aph(30 )-Ia (100) tet(D) (100) 405 185 7 E4; E5; E7; E14; CARBa blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); tet(A) (100); I E17; E18 (2) aac(60 )-Ib-cr (100) catB3 (100) 185 3 E1 CARBb blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77) sul2 (100) II 185 1 E1 CARB015 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); tet(A) (100) III aac(60 )-Ib-cr (100) 185 1 E15 CARB026 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(60 )-Ib-cr (100); sul2 (100); tet(A) (100); V catB3 (100) 185 1 E1 CARB011 blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100) sul2 (100) VI 1150 8 E2; E5 (2); E6; E8; CARBc blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); tet(A) (100); I E12 (2); E16 aac(60 )-Ib-cr (100) catB3 (100) 1150 1 E5 CARB056 blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77) sul2 (100) II 10 1150 1 E1 CARB037 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); tet(A) (100) III aac(60 )-Ib-cr (100) 1150 2 E1 (1); E6 (1) CARB125/116 blaOXA- 1 (100) aac(60 )-Ib-cr (100) tet(A) (100): catB3 (100) IV 1150 1 E10 CARB039 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(60 )-Ib-cr (100); sul2 (100); tet(A) (100); V catB3 (100) 1150 1 E1 CARB102 blaOXA- 1 (100) aac(60 )-Ib-cr (100) catB3 (100) VII 1150 1 E13 CARB96 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); catB3 (100) VIII aac(60 )-Ib-cr (100) cg/wgMLST of OXA-48-Producing K. pneumoniae Strains 1150 1 E3 CARB042 blaOXA- 1 (100) aac(3)-IIa (99,77); aac(60 )-Ib-cr (100) tet(A) (100); catB3 (100) IX 1150 1 E11 CARB184 blaOXA- 1 (100) blaTEM- 1B (100) aac(3)-IIa (99,77); aac(60 )-Ib-cr (100) tet(A) (100): catB3 (100) X 1153 1 E9 CARB123 blaOXA- 1 (100) blaTEM- 1B (100) blaCTX-M- 15 (100) strA (100)/strB (100); aac(3)-IIa (99,77); sul2 (100); tet(A) (100); I aac(60 )-Ib-cr (100) catB3 (100) January 2020 | Volume 10 | Article 2961 14 1151 1 D CARB117 17 1155 1 B CARB098 blaOXA- 1 (100) 1233 952 1 C CARB122 In parenthesis, the percentage of homology is indicated with the following genes: blaOXA-1 (J02967), blaTEM-1B (JF910132), blaCTX-M-15 (DQ302097), aph(30 )-Ia (V00359), strA (AF321551), strB (M96392), aac(3)-IId (X51534), aac(60 )-Ib-cr (DQ303918), sul2 (CQ421466), catB3 (AJ009818), tet(A) (AJ517790), and tet(D) (AF467077).All strains showed dfrA14 (99,59) (DQ388123) and oqxA (99,66)/oqxB (98,61) (EU370913) genes. All ST405 strains had qnrB66 (99,07) (KC580655) and fosA (98,81) (NZ_ACWO01000079), and the A3 strain carried qnrB1 (100) (EF682133).CARBa : E4 = CARB106, E5 = CARB022, E7 = CARB038, E14 = CARB010, E17 = CARB044, E18 = CARB040 and CARB139.CARBb : CARB007, CARB009, CARB050.CARBc : E2 = CARB020, E5 = CARB128 and CARB182, E6 = CARB065, E8 = CARB100, E12 = CARB112 and CARB130, E16 = CARB183.ST, sequence type; PFGE, pulsed-field gel electrophoresis. Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains FIGURE 2 | DNA comparison of the reference K. pneumoniae plasmid pOXA-48 (NC_019154.1) with assembled genomes using the CGView Comparison Tool (Grant et al., 2012). The legend on the left lists the 37 genome sequences used for the comparative BLAST-based analysis (genome names from the top to the bottom, and corresponding BLAST rings from the outer to the inner ones). The two outermost rings represent the forward and reverse coding strands of the plasmid reference genome. The color code for the coding strand features is highlighted in the legend on the bottom right. A separate BLAST ring is drawn for each comparison genome toward the inner part of the figure, and region of sequence similarity between the reference sequence and any part of the comparison sequence is shown in gradient color. blaOXA- 48 gene is indicated with an arrow. included 1 ARG each. We observed three different resistomes Inc ColE and FIA plasmids, previously detected by PBRT, were (XIII to XV) in ST101 isolates, in concordance with CTs not detected by WGS analysis since they are not included in the (cgMLST) and PFGE profiles; whereas in ST405 isolates, we PlasmidFinder 1.3 tool (Supplementary Table S2). In addition, observed 12 resistomes (I–XII); the two most predominant ones we found plasmid FIB homology with three previously described (I and II) were shared between CT185, CT1150, and CT1153. plasmids of the same incompatibility group [FIB (PKPHS1) ST405 isolates of two of the three groups that showed no (95.54%), FIB (Mar) (99.77%), FIB (PKPHS1) (96.01%)]. allele differences by cgMLST analysis and that had different PFGE Figure 2 shows a similarity of 84–100% between annotated profiles displayed a different resistance pattern. Isolates from coding sequences of the plasmid pOXA-48 reference genome group a had resistance patterns I/VII, and isolates from group and genome assemblies of OXA-48-producing K. pneumoniae b had resistance patterns I/III, with the difference being the isolates from this study. Plasmid L genetic rearrangements, such presence of tetA gene (Table 4). In contrast, isolates from group c as deletions, are shown in Supplementary Figure S3 and did not that had the same resistome (I/I) and the same CT were identified show correlation with the cgMLST tree. by wgMLST as having identical allelic profiles. Nucleotide Sequence Accession Plasmid Analysis Numbers Plasmid analysis revealed that all but two isolates (CARB039- Raw reads used in this study have been deposited to the European E10 and CARB077-A2) were positive for a plasmid of the Nucleotide Archive of the European Bioinformatics Institute incompatibility group (Inc) L. All isolates also carried plasmids under the project accession number PRJEB27508. of Inc FIB, and all isolates except those of PFGE profiles B (ST17), C (ST1233), and D (ST14) also carried a plasmid of the Inc FIIk (Supplementary Table S2). In addition, two isolates DISCUSSION (ST101; A1 and A2) were positive for an Inc R plasmid, and two (ST405; E4 and E5) had an Inc HI1B plasmid. Inc FIB, WGS has a higher discriminatory power than do conventional FII, and HI1B plasmids were not previously amplified by PCR- typing methods and allows for a more precise outbreak analyses. based replicon typing (PBRT) (Argente et al., 2019). However, This is especially important when analyzing the emergence and Frontiers in Microbiology | www.frontiersin.org 11 January 2020 | Volume 10 | Article 2961 Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains evolution of antimicrobial resistance, such as the threat of a and 29 (group b) cgSNPs. These results are within the range of worldwide spread of carbapenem resistance. 1–77 SNPs that was previously described for ST405 isolates as Previous studies described the molecular epidemiology sporadic (Onori et al., 2015). Other studies reported comparable of carbapenemase- and extended-spectrum beta-lactamase- results of cgMLST and SNP-based phylogeny in KPC-producing producing K. pneumoniae isolates by WGS using a gene-by-gene K. pneumoniae (Lee et al., 2014; Taboada et al., 2017). We comparison and/or SNP analysis (Snitkin et al., 2012; Lee et al., observed 50 times more core SNPs between isolates CARB058 2014; Marsh et al., 2015; Mathers et al., 2015; Onori et al., 2015; and CARB115 (both PFGE-A and ST101) than between isolates Zhou et al., 2015; Kluytmans-van den Bergh et al., 2016; Pérez- CARB058 and CARB077. These results were in concordance with Vázquez et al., 2016; Zhou et al., 2016; Taboada et al., 2017). cgMLST and wgMLST results, demonstrating again the higher Here we combined both approaches, describing for the first time discriminatory power of these two WGS-based approaches the cgMLST scheme included in SeqSphere+ Ridom software. compared to PFGE and MLST. In addition, we compared the results with those obtained with One of the main challenges with the WGS-based typing classical typing methods. For this purpose, we used a collection methods (cgMLST, wgMLST, and cgSNP analysis) is to define cut- of OXA-48-producing K. pneumoniae isolates related to an off values that can be used to identify a single-clone outbreak. increase in carbapenem resistance (ertapenem and imipenem) Previous studies focusing on a gene-by-gene approach have used in Catalonia, which has been observed since 2012. The ST405 different cgMLST schemes (Lee et al., 2014; Treangen et al., isolates were the most prevalent (77.6%) (Argente et al., 2019) 2014; Taboada et al., 2017; Zhou et al., 2017). Moreover, some and represented a predominant clone in Spain (Oteo et al., 2014). authors have developed bioinformatic tools for an interactive Previous bacterial-typing analysis of this collection by PFGE and identification of the most appropriate scheme (Silva et al., MLST methods showed close genetic relatedness between all 2018). Taken together, this suggests that a definition of a ST405 isolates, despite the fact that no clinical epidemiological unique threshold to define clonal relatedness is questionable. relationships were established among patients. A previous study on K. pneumoniae established a maximum First, we performed cgMLST and wgMLST using the BIGSdb- of 10 allele differences to cluster outbreak isolates and to Kp and Ridom SeqSphere+ software tools, using a gene-by- separate unrelated out-group isolates when using a cgMLST gene approach. This reduced the analysis to coding regions scheme of 1,143 genes (Zhou et al., 2017). In this work, but benefited from a stable nomenclature, which ensures we applied two different cgMLST schemes, and both easily interlaboratory reproducibility (Zhou et al., 2017). Second, we identified isolates with close genetic relatedness. However, the analyzed genome assemblies using the previously described number of allele differences/allelic mismatches varied from 0 Institut Pasteur cgMLST scheme (Bialek-Davenet et al., 2014), to 2 when using a 694-gene cgMLST scheme, and from 0 to which already demonstrated its ability to subtype isolates with the 14 when using a 2,365-gene cgMLST scheme (Table 2), which same MLST denominations, and showed that MLST classification indeed suggests that a different cluster alert distance should into traditional clonal complexes can merge members of distinct be applied depending on the cgMLST scheme. No threshold clonal groups (Bialek-Davenet et al., 2014). Last, we performed a has been defined when using cgMLST from BIGSdb-Kp, but cgSNP analysis of isolates of the same ST to investigate whether clearly, genetically related isolates are nearly identical (≤2 the isolates that did not show any allele differences by cgMLST mismatches) when using this scheme. The analysis using the were truly identical. scgMLST BIGSdb-Kp scheme therefore provided complementary We observed two major clusters when using the cgMLST information that may be easier to interpret given the lower SeqSphere+ analysis. Cluster 1 included all isolates of the variation observed using this scheme. ST405/PFGE-E clone and comprised CTs 185, 1150, and 1153. We used two different thresholds in the gene-by-gene Within cluster 1, three groups of two isolates each, each with approach, a cluster alert distance of 15 from SeqSphere+ software different PFGE pattern subtypes E1/E16, E13/E12, and E17/E18 (see text footnote 2) and a genetic distance of 0.0035 for (groups a to c), showed no single allele difference using cgMLST cgMLST analysis described by Kluytmans-van den Bergh et al. and had a genetic distance below the threshold of 0.0035, (2016). Both proved to be useful for the description of confirming a close genetic relatedness (Kluytmans-van den Bergh genetic relatedness. In addition, the latter could be adopted et al., 2016). The PFGE and the cgMLST/wgMLST patterns independently of the cgMLST scheme used, which would were not the same, which may be explained by the fact that also facilitate the interpretation of results, since it considers these two subtyping methods examine and distinguish different the proportion of shared genes by two isolates that are types of genetic mutation events (Zhou et al., 2017). Indeed, being compared. CARB100/CARB123 and CARB077/CARB115 isolates could have mutations in the genome that do not affect isolates, with fewer than 14 allele differences (cluster alert restriction sites, and therefore, different cgMLST/wgMLST allelic distance 15), had a genetic distance above the threshold, profiles could have the same PFGE pattern (Tenover et al., which would have been interpreted as there being no genetic 1995). Furthermore, PFGE diversity could be reflecting mostly relatedness. However, when using the cgMLST BIGSdb-Kp changes in the accessory genome, for example, gain or loss of scheme, the genetic distance was 0.0032 (2 allelic mismatches), mobile genetic elements or recombination events, rather than indicating instead genetic relatedness when applying the same true genealogies (Zhou et al., 2013). threshold. This illustrates that thresholds must be used with In this study, ST405 isolates with identical cgMLST allelic caution and cannot per se provide a definitive answer as to the profiles (SeqSphere+) showed between 6 and 17 (groups a, c, d) genetic relatedness. Frontiers in Microbiology | www.frontiersin.org 12 January 2020 | Volume 10 | Article 2961 Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains A threshold assessment is even more difficult for an analysis, we only showed the presence or absence of the replicon SNP analysis; most authors analyzing K. pneumoniae isolates genes. Nevertheless, the rapid advance in sequencing techniques described the number of different SNPs detected, but no clear will be decisive to describe plasmid features and sequences in cut-off value has been assigned (Snitkin et al., 2012; Marsh accurate detail. et al., 2015; Mathers et al., 2015; Onori et al., 2015; Zhou et al., 2015; Pérez-Vázquez et al., 2016). Moreover, the number of SNPs detected depends on the reference genome used for the analysis CONCLUSION and on the degree to which artifactual SNPs, due to paralogous sequences, for example, are filtered out during the SNP analysis. In conclusion, we observed an overall correlation between Some studies investigated the mutation rate of different species cgMLST and wgMLST, and cgSNP-analysis results to determine in order to define a threshold for the number of SNPs between similarity of OXA-48-producing K. pneumoniae clinical isolates. related isolates (Köser et al., 2012; Zhou et al., 2013; Ruppitsch CgMLST produced high-resolution allelic profiles and different et al., 2015; Moura et al., 2017; Silva et al., 2018). Pérez-Vázquez cgMLST schemes provided complementary results that allowed et al. (2016) described an accumulation of approximately 11 for a better interpretation of results. Furthermore, cgSNP- SNPs per genome per year for OXA-48-K. pneumoniae isolates of based typing was useful to resolve and better discriminate ST405. Public Health England has published a reproducible and OXA-48-producing K. pneumoniae clusters within the same scalable pathogen population SNP-based analysis, including an ST. Differences between classical and WGS-based typing “SNP address” nomenclature (Okoro et al., 2012). methods highlight the different molecular principle of these The advantages of WGS-based typing are undeniable, techniques, and we emphasize the importance to include clinical however, “unique” or “one-fits-all” thresholds, by means of epidemiological data to elucidate clonal spread. either gene-by-gene or SNP analysis, seem to be difficult to establish; it may be not only species-specific but also population-specific, which requires the careful and flexible DATA AVAILABILITY STATEMENT application of proposed thresholds as previously discussed (Dallman et al., 2017). Nevertheless, efforts within the The datasets generated for this study can be found in the ENA. scientific community and public health authorities to unify the parameters and values used to define clonality would facilitate a common practice in data interpretation. This could AUTHOR CONTRIBUTIONS be achieved by adopting a threshold range to be interpreted in combination with clinical epidemiological data and species EM, JR, and SG-C: conception and design. EM, SB, and SG-C: population characteristics. acquisition of data. EM and SG-C: laboratory analysis. EM, MC, Regarding antimicrobial resistance mechanisms and possible VP, and SG-C: data analysis. EM, JR, MC, DH, SB, VP, and vectors for the dissemination of those, we described 18 SG-C: data interpretation. EM and SG-C: writing of the draft ARGs and the presence of up to four plasmids as possible manuscript. EM, JR, MC, DH, SB, VP, FN, AF, and SG-C: review disseminators of these. We established that quinolone resistance of the manuscript. was not determined by mutations in topoisomerases as expected, but was probably due to the interplay between different mechanisms, i.e., plasmid factor QnrB66, the FUNDING aminoglycoside-modifying enzyme AAC(60 )-Ib-cr, and the efflux pump OqxAB. The antibiotic resistance genotype This work was partly supported by Interreg IVa, EurSafety profiles obtained by classical PCR (Argente et al., 2019) were Health-net (III-1-02D73), and EurHealth-1Health projects, mostly congruent with WGS results. Detection of genes by which are part of a Dutch-German cross-border network PCR, but not by WGS, could be explained by the loss of the supported by the European Commission; the Dutch Ministry of plasmid harboring the corresponding gene or by incorrect Health, Welfare and Sport (VWS); the Ministry of Economy, assembly or low coverage of the respective gene (Leopold et al., Innovation, Digitalisation and Energy of the German Federal 2014). Multiple factors could cause a negative PCR result, State of North Rhine-Westphalia; and the German Federal State including inadvertently contamination of PCR tubes with of Lower Saxony. BIGSdb-Kp receives institutional funding from glove power, which can inhibit steps during the PCR process Institut Pasteur. (Schürch et al., 2018). Our findings confirmed the conserved IncL plasmid as the major vector for the dissemination of OXA-48 carbapenemase. ACKNOWLEDGMENTS However, genetic rearrangements, such as deletions and point mutations, have also been observed within our 1-year We want to thank the group of microbiologists at Hospitals isolate collection, reflecting genome plasticity and dynamics Comarcals in Catalonia and the Balearic Islands (http://www. of horizontal gene transfer (Lomas et al., 1992). In addition, scmimc.org/grupstreball02.php) for collecting the clinical isolates WGS allows us to identify the degree of homology between and clinical epidemiological data: Carles Alonso (Hospital plasmid incompatibility groups, whereas by classical PBRT Creu Roja de l’Hospitalet), Frederic Ballester (Hospital Sant Frontiers in Microbiology | www.frontiersin.org 13 January 2020 | Volume 10 | Article 2961 Miro et al. cg/wgMLST of OXA-48-Producing K. pneumoniae Strains Joan de Reus), Carmen Gallés (Corporació de Salut del Maresme FIGURE S2 | (A) SNP-based tree of ST405 isolates using the Neighbour-Joining I la Selva), Carmina Martí (Hospital General de Granollers), method. The genome reference is the first ST405 isolate collected (CARB007-E1). The number of SNPs compared to the reference (*) for the isolates that clustered Montserrat Morta (Hospital Sant Joan de Déu de Manresa), together using a gene-by-gene approach (groups a, b, c and d) is indicated. The Montserrat Olsina (Hospital General Universitari de Catalunya- number of SNPs between these isolates themselves is also indicated in brackets. Quironsalud), Goretti Sauca (Hospital de Mataró), Montserrat (B) SNP-based tree of ST101 isolates using the Neighbour-Joining method. The Sierra (Hospital de Barcelona, SCIAS), Alba Rivera (Hospital genome reference was the first ST101 isolate collected (CARB0058-A3). The de la Santa Creu i Sant Pau), and Anna Vilamala (Hospital number of SNPs compared to the reference (*) is indicated. General de Vic). FIGURE S3 | Phylogenetic tree based on cgMLST-SeqSphere+ (2,365 targets) and BLASTn comparison of de novo assembled genomes with K. pneumoniae plasmid pOXA-48 NC_019154.1 with at least 80% sequence similarity using DNAplotter (Carver et al., 2009) and visualized using Artemis Comparison Tool SUPPLEMENTARY MATERIAL (ACT) (Carver et al., 2005). Information of hospital, isolation, and date of isolation is also included. The Supplementary Material for this article can be found TABLE S1 | Assembly quality using QUAST 4.5. online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02961/full#supplementary-material TABLE S2 | PlasmidFinder v.1.3 and PBRT-PCR results. DATA SHEET S1 | CgMLST distance matrices for pairwise comparisons of 37 FIGURE S1 | Minimum spanning tree (BioNumerics v6,7,3, Applied-Maths, OXA-48 K. pneumoniae isolates. Belgium) based on BIGSdb-Kp scgMLST allelic profile comparisons. Each circle represents a single profile. The number of allelic mismatches among profiles are DATA SHEET S2 | VCF files for ST101, including SNP, indel, and structural given along links (excluding missing data in one or both profiles). The large circle variation calls information generated by Harvest software suuite (parsnp) corresponds to the dominant genotype. (Treangen et al., 2014). REFERENCES genome multilocus sequence typing of extended-spectrum beta-lactamase- producing Enterobacteriaceae. J. 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M., Thomas, P. J., Stock, F., Nisc Comparative Sequencing The remaining authors declare that the research was conducted in the absence of Program, Henderson, D. K., et al. (2012). Tracking a hospital outbreak of any commercial or financial relationships that could be construed as a potential carbapenems-resistant Klebsiella pneumoniae with whole-genome sequencing. conflict of interest. Sci. Transl. Med. 4:148ra116. doi: 10.1126/scitranslmed3004129 Taboada, E. N., Graham, M. R., Carriço, J. A., and Van Domselaa, R. G. (2017). Copyright © 2020 Miro, Rossen, Chlebowicz, Harmsen, Brisse, Passet, Navarro, Food safety in the age of next generation sequencing, bioinformatics, and open Friedrich and García-Cobos. This is an open-access article distributed under the data access. Front. Microbiol. 8:909. doi: 10.3389/fmicb.2017.00909 terms of the Creative Commons Attribution License (CC BY). The use, distribution Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., or reproduction in other forums is permitted, provided the original author(s) and Persing, D. H., et al. (1995). Interpreting chromosomal DNA restriction the copyright owner(s) are credited and that the original publication in this journal patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain is cited, in accordance with accepted academic practice. No use, distribution or typing. J. Clin. Microbiol. 33, 2233–2239. reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 15 January 2020 | Volume 10 | Article 2961