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Epidemiology and Surveillance

Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016–2017

María Pérez-Vazquez, Jesús Oteo-Iglesias, Pedro J. Sola-Campoy, Hugo Carrizo-Manzoni, Verónica Bautista, Noelia Lara, Belén Aracil, Almudena Alhambra, Luis Martínez-Martínez, José Campos, the Spanish Antibiotic Resistance Surveillance Program Collaborating Group
María Pérez-Vazquez
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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Jesús Oteo-Iglesias
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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  • ORCID record for Jesús Oteo-Iglesias
Pedro J. Sola-Campoy
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
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Hugo Carrizo-Manzoni
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
cLaboratorio de Bacteriología, Micología y Parasitología, Centro de Educación Médica e Investigaciones Clínicas Norberto Quirno, Buenos Aires, Argentina
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Verónica Bautista
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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Noelia Lara
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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Belén Aracil
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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Almudena Alhambra
dServicio de Microbiología Laboratorio Abacid, Hospital Universitario HM Sanchinarro, Madrid, Spain
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Luis Martínez-Martínez
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
eUnidad de Microbiología, Hospital Universitario Reina Sofía, Córdoba, Spain
fDepartamento de Microbiología, Universidad de Córdoba, Córdoba, Spain
gInstituto Maimonides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
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José Campos
aLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
bSpanish Network for Research in Infectious Diseases (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain
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DOI: 10.1128/AAC.02529-18
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ABSTRACT

There is little information about carbapenemase-producing (CP) Klebsiella oxytoca, an important nosocomial pathogen. We characterized CP K. oxytoca isolates collected from different Spanish hospitals between January 2016 and October 2017. During the study period, 139 nonduplicate CP K. oxytoca isolates were identified; of these, 80 were studied in detail. Carbapenemase and extended-spectrum β-lactamase genes were identified by PCR and sequencing. Genetic relatedness was studied by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS), carried out on 12 representative isolates, was used to identify the resistome, to elucidate the phylogeny, and to determine the plasmids harboring carbapenemase genes. Forty-eight (60%) isolates produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1; 4 isolates coproduced two carbapenemases. By PFGE, 69 patterns were obtained from the 80 CP K. oxytoca isolates, and four well-defined clusters were detected: cluster 1 consisted of 11 OXA-48-producing isolates, and the other three clusters included VIM-1-producing isolates (5, 3, and 3 isolates, respectively). In the 12 sequenced isolates, the average number of acquired resistance genes was significantly higher in VIM-1-producing isolates (10.8) than in OXA-48-producing isolates (2.3). All 12 isolates had chromosomally encoded genes of the blaOXY-2 genotype, and by multilocus sequence typing, most belonged to sequence type 2 (ST2). Carbapenemase genes were carried by IncL, IncHI2, IncFII, IncN, IncC, and IncP6 plasmid types. The emergence of CP K. oxytoca was principally due to the spread of VIM-1- and OXA-48-producing isolates in which VIM-1- and OXA-48 were carried by IncL, IncHI2, IncFII, and IncN plasmids. ST2 and the genotype blaOXY-2 predominated among the 12 sequenced isolates.

INTRODUCTION

The prevalence of multidrug-resistant carbapenemase-producing Enterobacteriaceae (CPE), a major public health threat, continues to increase on a global level and is associated with significant morbidity and mortality (1). Frequently, patients with CPE infections cannot be treated with effective antibiotics because of the dearth of alternative drugs (2). Currently, the epidemiological situation regarding CPE in Spain is considered endemic, mainly due to carbapenemase-producing (CP) Klebsiella pneumoniae. However, carbapenemase production is also increasing in other Enterobacteriaceae species, such as Escherichia coli, Citrobacter spp., Enterobacter spp., and Serratia marcescens (3–5).

Klebsiella oxytoca has been recognized as an opportunistic pathogen causing health care-associated infections, such as urinary and respiratory tract infections and sepsis, primarily in immunocompromised and debilitated patients admitted to intensive care units. However, little information about carbapenemase-producing (CP) K. oxytoca is available, since only a few individual cases or nosocomial outbreaks have been reported (6–9).

The aim of this study was to gain insight into the microbiological features and molecular epidemiology of CP K. oxytoca isolates submitted to the Spanish National Reference Laboratory for Antibiotic Resistance.

RESULTS AND DISCUSSION

Bacterial isolates and carbapenemase types.Between January 2016 and September 2017, 139 nonduplicate CP K. oxytoca isolates, submitted from 22 hospitals located in 9 different Spanish provinces, were identified (5.2% of the 2,673 CPE identified by the reference laboratory during the same period of time). Of them, 80 (57.5%) isolates were selected for further studies (see Table S1 in the supplemental material); this selection excluded 59 isolates obtained from rectal swab samples collected in three hospitals with nosocomial outbreaks.

Sixty-one isolates produced clinical infections, as follows: 28 (45.9%) urinary tract infections, 11 cases of bacteremia (18%), 10 (16.4%) respiratory tract infections, 6 (9.8%) wound infections, and 6 (9.8%) abscesses. The patients infected with CP K. oxytoca isolates were mainly males (65.6%) aged 18 to 65 years (59%).

Of the 80 CP K. oxytoca isolates, 48 (60%) produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1. Four isolates coproduced two different carbapenemases: two produced VIM-1 plus KPC-2, one produced VIM-1 plus OXA-48, and one produced VIM-1 plus KPC-3. Carba NP tests yielded positive results for all 80 CP isolates.

As previously described in other Enterobacteriaceae species, such as Citrobacter freundii, Enterobacter spp., and Serratia marcescens (3–5), VIM-1 was the most frequent carbapenemase produced by CP K. oxytoca in this study. However, in K. pneumoniae and Escherichia coli, the most frequent carbapenemases detected in Europe are KPC, OXA-48, and NDM (2). Recently, the clonal spread of an IMP-8-producing K. oxytoca clone was described in a Spanish hospital (9).

Five (6.25%) CP K. oxytoca isolates also coproduced extended-spectrum β-lactamases (ESBLs), as follows: two VIM-1-producing isolates (2.5%) also produced CTX-M-9 plus SHV-12, one VIM-1- and OXA-48-producing isolate and one OXA-48-producing isolate (2.5%) also produced CTX-M-9, and one VIM-1-producing isolate (1.25%) also produced CTX-M-14. The NDM-1-producing K. oxytoca isolate coproduced the plasmid-mediated AmpC (p-AmpC) CMY-4.

Antibiotic susceptibility.The antibiotic susceptibilities of all CP K. oxytoca isolates are detailed in Table 1 and Table S2. In comparison with the VIM-1-producing isolates, the OXA-48-producing isolates were significantly more susceptible to gentamicin, tobramycin, cotrimoxazole, cefotaxime, and ceftazidime (P < 0.05) (Table 1).

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TABLE 1

Antibiotic susceptibility of 80 carbapenemase-producing Klebsiella oxytoca isolatesa

The rates of susceptibility to carbapenems were 36.2% to meropenem, 11.2% to imipenem, and 8.7% to ertapenem (Table 1); all isolates were nonsusceptible to at least one carbapenem antibiotic. These data suggest that ertapenem has the highest sensitivity for the detection of CP K. oxytoca strains, although specificity could not be determined. However, susceptibility to carbapenems varied highly in relation to the carbapenemase types, as follows: the rates of ertapenem, imipenem, and meropenem susceptibility were 0%, 24.1%, and 62.1%, respectively, in OXA-48 producers and 15.9%, 4.5%, and 25%, respectively, in VIM-1 producers. These differences between carbapenemases have been previously described in other species, such as Citrobacter freundii and Escherichia coli (3, 4).

Of the 28 OXA-48-producing isolates that were negative for other carbapenemases, ESBLs, or p-AmpCs, 11 were cefotaxime susceptible but 17 (60.7%) were cefotaxime resistant. Overproduction of chromosomal OXY enzymes can result in reduced susceptibility or resistance to penicillin-inhibitor combinations, cefuroxime, cefotaxime, and aztreonam and has been classically observed in 10% to 20% of clinical isolates of K. oxytoca (10).

Forty-nine isolates (61.3%) were resistant to ceftazidime-avibactam, but all of them produced metallo-β-lactamases. Only two isolates were colistin resistant but were negative for mcr genes. The most frequently described mechanisms of chromosomal resistance to colistin in K. pneumoniae are mutations caused by insertional inactivation or deletion of the mgrB gene and upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) (11).

The percentages of susceptibility to ertapenem, imipenem, and meropenem, according to EUCAST breakpoints (12), varied significantly between VIM-1-producing isolates (15.9%, 4.5%, and 25%, respectively) and OXA-48-producing isolates (0%, 24.1%, and 62.1%, respectively) (P < 0.05) (Table 1). However, all isolates had meropenem MICs of >0.25 mg/liter and therefore would be suspected of producing carbapenemases according to the screening cutoff values proposed by EUCAST (13).

Molecular epidemiology.Pulsed-field gel electrophoresis (PFGE) analysis revealed a high degree of genetic diversity, as 69 different PFGE patterns were obtained from the 80 CP K. oxytoca isolates analyzed (simple diversity index, 86.2%). However, four well-defined clusters with more than two isolates each were detected when a genetic linkage of ≥85% was considered (Fig. S1). The largest cluster (cluster 1) consisted of 11 OXA-48-producing isolates submitted from two hospitals (which sent 9 and 2 isolates, respectively) located in the same province. The other three clusters consisted of five (cluster 2), three (cluster 3), and three (cluster 4) VIM-1-producing isolates, and each of these clusters came from individual hospitals. In addition, five clusters of two isolates each were also detected (Fig. S1).

These PFGE results suggest polyclonal dissemination, since 46 of the 80 CP K. oxytoca isolates showed nonrelated PFGE patterns. However, specific clonal disseminations of CP K. oxytoca either within the same hospital or between different close hospitals were detected.

Resistome of CP K. oxytoca determined by WGS analysis.Whole-genome sequencing (WGS) was performed on 12 selected isolates representing (i) the four main clusters detected by PFGE (five isolates), (ii) the clusters formed by pairs of isolates (four isolates), and (iii) three single isolates: one producing NDM-1, one producing VIM-1 plus KPC-2, and one producing VIM-1 plus KPC-3. According to the PFGE results, these 12 isolates are representative of 33 of the 80 isolates studied (11 isolates of cluster 1, 5 of cluster 2, 3 of cluster 3, 3 of cluster 4, 4 pairs of isolates, and 3 individual isolates).

In the 12 sequenced isolates, the average number of acquired resistance genes was 9.2 (range, 1 to 16), being much higher in the 5 VIM-1-producing isolates (average, 10.8; range, 6 to 15) than in the 3 OXA-48-producing isolates (average, 2.3; range, 1 to 3). A summary of the main antibiotic resistance genes detected is provided in Table 2. The isolate with the highest number of acquired resistance genes (n = 16) was the NDM-1-producing K. oxytoca isolate, which had several genes encoding resistance to β-lactams, aminoglycosides, sulfonamides, quinolones, chloramphenicol, macrolides, rifampin, tetracycline, and trimethoprim; in addition, the genome of this isolate encoded the methyltransferase gene armA, which is of special relevance since it confers high levels of resistance to all clinically important aminoglycosides (14). ArmA has been associated with K. oxytoca in Japan and China (15, 16); however, ArmA has only sporadically been recovered from Enterobacteriaceae isolates in Spain, and these were mainly obtained from animals (17, 18), but it has not been recovered from K. oxytoca. The association of the blaNDM-1 and armA genes in the same plasmid has been described previously (19).

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TABLE 2

Main antibiotic resistance genes and other molecular markers detected by WGS of 12 representative CP Klebsiella oxytoca isolatesa

In addition to carbapenemase genes, the most predominant acquired resistance genes were aadA1 (91.7%), aac(6′)-Ib (75%), sul1 (75%), and catB2 (66.7%).

Concerning the chromosomal blaOXY genes, all 12 isolates had the blaOXY-2 genotype, with three variants detected: blaOXY-2-8 (n = 5), blaOXY-2-2 (n = 4), and blaOXY-2-5 (n = 3) (Table 2). Eight different types of OXY enzymes have been described (10, 20). Of the 68 isolates demonstrating nonsusceptibility to 3rd-generation cephalosporins obtained from 14 hospitals across Europe (10), 42 (61.8%) belonged to phylogroup KoII, which includes isolates of the OXY-2 type (20). Interestingly, in this study, KoII isolates were frequently OXY hyperproducers.

Phylogenetic analysis of carbapenem-producing Klebsiella oxytoca by MLST and core genome MLST.The 12 sequenced isolates belonged to the following types determined by multilocus sequence typing (MLST): sequence type 2 (ST2; n = 5), ST36 (n = 3), ST176 (n = 2), ST20 (n = 1), and ST141 (n = 1) (Table 2; Fig. 1). Of the 12 isolates, 8 (66.7%) had STs belonging to clonal complex 2 (CC2): ST2, ST176 (a double-locus variant [DLV] of ST2), and ST141 (a DLV of ST2). CC2 has been previously described to be the most frequent clonal complex in K. oxytoca (10, 21).

FIG 1
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FIG 1

Minimum-spanning tree of 12 representative carbapenemase-producing K. oxytoca isolates (colored circles). Distance is based on an ad hoc cgMLST of 3,201 genes using the parameters pairwise ignoring missing values and all the K. oxytoca reference sequences available in NCBI. Each circle is named with the MLST type, and colors indicate carbapenemase types; white circles represent control isolates available in NCBI (n = 7) or representative of OXA-48-producing K. oxytoca outbreak isolates from Tunisia (*, n = 3 [unpublished data]).

The genome assemblies of the 12 K. oxytoca isolates were analyzed using a gene-by-gene approach (22), and the allelic distance from the core genome MLST (cgMLST) was visualized in a minimum-spanning tree (Fig. 1). Additionally, the study of single nucleotide polymorphisms (SNPs) was performed as described in Materials and Methods (Fig. S2). ST2 isolates differed by an average of 56 alleles (range, 15 to 88 alleles) and 288 SNPs (range, 35 to 448 SNPs); in ST36 isolates, these differences were 139 alleles (range, 48 to 188 alleles) and 1,013 SNPs (range, 120 to 1,495), and in ST176 isolates, these differences were 134 alleles and 403 SNPs. The most prevalent cgMLST clusters, ST2, ST136, and ST176, included isolates producing different carbapenemase types (Fig. 1).

Characterization of plasmids harboring carbapenemases genes.In 11 of the 12 K. oxytoca isolates (Table 2), the PlasmidID mapping tool was used for the identification and reconstruction of 13 plasmids harboring carbapenemase genes (7 carrying blaVIM-1, 3 carrying blaOXA-48, 2 carrying blaKPC, and 1 carrying blaNDM-1). In the remaining isolate, plasmid reconstruction failed.

In the three isolates producing class D carbapenemases, blaOXA-48 was carried on IncL plasmids, one of ∼63 kb (two isolates) almost identical to plasmid pOXA-48 (GenBank accession number NZ_CP018342.1; average identity, >95%; average coverage percentage, 99.8%) (Fig. S3) and another of ∼48 kb (one isolate) highly similar to the plasmid with GenBank accession number NZ_CP018694.1 (average identity, >95%; average coverage percentage, 94.3%), reconstructed with a low number of contigs (8, 7, and 3 respectively) (Table 2). In all three isolates, blaOXA-48 was flanked by IS1999 sequences. In the two isolates within cluster 1/ST2 (Fig. 1), blaOXA-48 was located in a Tn1999.2 transposon (Fig. S3), and in the ST141 isolate, blaOXA-48 was located in a Tn1999.3 transposon.

In VIM-1-producing isolates, four different plasmid types carrying blaVIM-1 were identified, as follows (Table 2): one IncL plasmid of ∼70 kb identical to the plasmid with GenBank accession number NZ_CP023419.1 in three isolates (Fig. S4); one IncHI2 plasmid of ∼250 kb very similar to the plasmid with GenBank accession number NZ_CP026661.1 in two isolates (average identity, >95%; average coverage percentage, 81.8%) (Fig. S5), one IncN plasmid of ∼50 kb very similar to the plasmid with GenBank accession number NC_014208.1 (average identity, >95%; average coverage percentage, 90.96%) in one isolate, and one IncFII plasmid of ∼70 kb similar to the plasmid with GenBank accession number NZ_CP023419.1 (average identity, >95%; average coverage percentage, 86.4%) in one isolate.

In Greece, a multiclonal epidemic of VIM-1-producing K. pneumoniae isolates mainly due to the spread IncN plasmids of between 50 and 70 kb was described (23). A conjugative IncHI2 plasmid of ∼300 kb has previously been associated with the dissemination of blaVIM-1 among genetically diverse Enterobacter cloacae isolates (24). We identified two different genetic environments for blaVIM-1; in IncN, IncL, and IncFII plasmids, blaVIM-1 was located in the In624 class 1 integron (24, 25) (Fig. S4), and in IncHI2 plasmids, VIM-1 was linked to another similar class 1 integron that was previously described (26) (Fig. S5). In Spain, In624 has been described in VIM-producing E. cloacae (24) and C. freundii (25) isolates, suggesting an important role in the interspecies transfer of blaVIM-1.

Two plasmids carrying blaKPC were identified (Table 2). One of them was an IncP6 plasmid of ∼39 kb identical to the plasmid with GenBank accession number NZ_CP026224.1 with blaKPC-2 as the sole antimicrobial resistance gene in the plasmid (Fig. S6). The genetic environment of the blaKPC-2 gene included ISKpn27 and a truncated blaTEM-1 (Fig. S6) and was the same as that previously detected in the first IncP6 plasmid carrying blaKPC-2 in K. oxytoca (6). The other one was an IncFIB plasmid of ∼103 kb that was similar to the plasmid with GenBank accession number NZ_CP015395.1 (average identity, >95%; average coverage percentage, 88.36%) and that harbored blaKPC-3 in the classical transposon Tn4401 isoform (27, 28), blaTEM-1A, and blaOXA-9 (Fig. S7).

In the NDM-1-producing isolate, blaNDM-1 was harbored by an IncC plasmid of ∼154 kb similar to the plasmid with GenBank accession number NZ_CP029118.1 (average identity, >95%; average coverage percentage, 82.18%) (Table 2; Fig. 2); the genetic environment of blaNDM-1 included the ISAba125 composite transposon Tn125 (Fig. 2), previously described in pM214_A/C2 (29). Additionally, a class 1 integron located adjacent to Tn125 contained several genes associated with resistance to trimethoprim (dfrA14), rifamycins (arr-2), chloramphenicol (clmA5 encoding the efflux MFS transporter), β-lactams (blaOXA-10), aminoglycosides (aadA1), and sulfonamides (sul1) (Table 2, Fig. 2). This IncC plasmid also contained the armA, blaCMY-4, and msrE and mphE genes, encoding resistance to aminoglycosides, β-lactams, and macrolides, respectively. The blaCMY-4 gene was included in an ISEcp1 transposition unit (Fig. 2), which is usually found in A/C plasmids and which is thought to be responsible for the dissemination of the blaCMY cephalosporinase genes (30).

FIG 2
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FIG 2

Overview of the IncC plasmid harboring blaNDM-1 detected in Klebsiella oxytoca in this study. The figure represents the plasmid according to the homology with a highly similar one from the GenBank database (blue outer ring). The graph represents the reads mapped against this reference sequence with a depth of coverage ranging from 0 (red) to 500, with orange indicating values of 1 to 20 reads and green indicating values higher than 200 reads. Gray boxes represent the coding sequence from automatic annotation, with dark and light colors being used when they were found on the forward or the reverse strand, respectively. Colored stripes represent a more detailed annotation that includes antibiotic resistance genes in red, insertion sequences (IS) in blue, and Rep genes in yellow. The homology between the reference plasmid and the assembled contigs is represented in the inner ring, with each contig being colored according to its number.

Conclusions.We observed the progressive emergence of CP K. oxytoca strains in Spain. Our data reveal several remarkable findings of concern that deserve active surveillance: (i) the clonal and mainly polyclonal spread of CP K. oxytoca across different geographical areas and hospitals; (ii) the production of five different carbapenemase types, mainly VIM-1 and OXA-48, but also KPC and NDM; (iii) the identification of a NDM-1-producing isolate presenting a high load of antibiotic resistance genes, including amrA; (iv) the predominance of ST2 and OXY-2 among the studied isolates; and (v) the detection of the plasmid types IncL, IncHI2, IncFIB, IncN, IncC, and IncP6, likely responsible for the dissemination of carbapenemase genes in K. oxytoca isolates in Spain.

MATERIALS AND METHODS

Study design and bacterial isolates.This study was performed by the unrestricted and nonmandatory national Spanish Antibiotic Resistance Surveillance Program, operated by the official public health institute of Spain (Instituto de Salud Carlos III) (3, 4). The number of carbapenemase-producing K. oxytoca isolates referred to this program experienced a constant increase from 2012 (n = 6) (31) to 2015 (n = 35), although their proportion with respect to the total number of carbapenemase-producing Enterobacteriaceae isolates remained stable at about 2% to 3%. The observation of an unexpected increase in this proportion in 2016 (5.2%) prompted the present investigation. In this study, we included all CP K. oxytoca isolates submitted to the Instituto de Salud Carlos III between January 2016 and October 2017; during this period a total of 119 public hospitals (about 35% of Spanish public hospitals) voluntarily participated in the surveillance program.

Only the first isolate per patient was analyzed. Bacterial isolate identification was performed using an API 20E system (bioMérieux) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Leipzig, Germany).

Antibiotic susceptibility of CP K. oxytoca isolates and phenotypic characterization of carbapenemase production.Antibiotic susceptibility testing was performed by microdilution (DKMGN panel; Thermo Fisher Scientific, East Grinstead, UK), and the results were interpreted according to EUCAST breakpoints (12, 13). Susceptibility to colistin was carried out by an in-house microdilution method following the recommendations of the CLSI-EUCAST Joint Polymyxins Working Group (32). According to EUCAST (13), the inhibition of carbapenemase activity was determined by using EDTA, phenyl-boronic acid, and cloxacillin. In addition, all the isolates were tested by the Carba NP method (33).

Characterization of resistance mechanisms.The presence of genes encoding carbapenemases (blaOXA-48, blaKPC, blaVIM, blaIMP, and blaNDM), extended-spectrum β-lactamases (blaCTX-M, blaSHV, and blaTEM), plasmid-mediated AmpC β-lactamases (p-AmpC) (blaCMY, blaDHA, blaMOX, blaACC, blaEBC, and blaFOX), and mcr genes coding for colistin resistance was identified using PCR and DNA sequencing, as previously described (31, 34–36).

Pulsed-field gel electrophoresis.The genetic relationship between the CP K. oxytoca isolates was elucidated by PFGE after total chromosomal DNA digestion with XbaI (34). A simple diversity index (SDI) was calculated in order to determine the population diversity, as follows: (number of different PFGE profiles/total number of isolates) × 100 (37).

WGS, resistome, and core genome MLST analysis.Whole-genome sequencing (WGS) was performed on 12 isolates representative of the main CP K. oxytoca clusters previously detected by PFGE and also on 2 additional isolates coproducing two carbapenemase types each.

DNA was extracted using a QIAamp DNA minikit (Qiagen, Hilden, Germany). Genomic DNA paired-end libraries were generated using a Nextera XT DNA sample preparation kit (Illumina Inc., San Diego, CA, USA). The libraries were sequenced using an Illumina NextSeq 500 sequencer system with 2 × 150-bp paired-end reads (Illumina Inc.).

The quality of the high-throughput sequence data was assessed by the use of FastQC software, and short reads were subsequently assembled de novo into contigs using the SPAdes (version 3.9.0) program (38), testing 5 different kmers under parameters optimized to give the best assembly using the QUAST program (http://quast.bioinf.spbau.ru/) in order to analyze the most relevant statistics (largest contig, N50, and NG50). Scaffolding was performed with the SSPACE program (39), and the GapFiller program was used to close sequence gaps (40). Automatic de novo annotation of draft genomes was performed using the Prokka (version 1.12-beta) program (41).

Antimicrobial resistance genes were analyzed using the ResFinder tool (CGE server, https://cge.cbs.dtu.dk; date last accessed, June 2018) with an identification threshold of 98%, with the exception of β-lactamase variants, which were determined with 100% identity. Additionally, the SRST2 program (42) was used to detect resistance genes and alleles with the ARGannot database (43).

Core genome multilocus sequence typing (cgMLST) was applied; an ad hoc scheme was created using the MLST+ target definer with the default parameters and a reference sequence (K. oxytoca KONIH1, GenBank accession number NZ_CP008788.1). A total of 13 NCBI RefSeq genomes were used as query genomes for validation in a pairwise comparison using the BLAST program. The final cgMLST scheme consisted of 3,201 target genes and 2,380 accessory genes. The percentage of good targets included for distance calculation was 94% (see Table S3 in the supplemental material). A distance matrix among all isolates was calculated with 3,013 target genes to analyze the homology within each cluster; genes with missing values were excluded (Table S4).

Curated Illumina sequence reads were mapped onto the reference sequence (K. oxytoca KONIH1, GenBank accession number NZ_CP008788.1), and putative SNPs were obtained using Lasergene Genomics Suite software (DNAStar Inc., Madison, WI, USA), as described previously (44).

Characterization of plasmids carrying carbapenemases genes.In order to reconstruct the plasmids carrying the carbapenemase genes, an in-house script (PlasmidID, https://github.com/BU-ISCIII/plasmidID) was used to (i) map reads over a plasmid curated database to find those with the higher coverage and de novo assembly of these reads, (ii) make local alignments to localize resistance and replicative genes, and (iii) make a graphic representation of the plasmids identified (45).

Statistical analysis.Differences in the prevalence of antibiotic susceptibility of OXA-48- and VIM-1-producing isolates were assessed using Fisher’s exact test. The null hypothesis was rejected when a P value of ≤0.05 was calculated. Statistical analysis was performed using GraphPad Prism software (version 3.02; GraphPad Software, Inc., San Diego, CA, USA).

Accession number(s).The sequences are available on the ENA website under accession number PRJEB30102.

ACKNOWLEDGMENTS

We thank the Genomics Unit of the Centro Nacional de Microbiología for carrying out the DNA sequencing. We thank Michael McConnell for the revision of the English style and grammar.

The members of the Collaborating Group for the Spanish Antibiotic Resistance Surveillance Program are as follows: Isabel Sánchez-Romero, Beatriz Orden, and Rocío Martínez-Ruiz (Hospital Universitario Puerta de Hierro-Majadahonda, Majadahonda, Madrid); Esteban Aznar (Laboratorio BrSalud, San Sebastián de los Reyes, Madrid); Emilia Cercenado (Hospital General Universitario Gregorio Marañón, Madrid); Pedro de la Iglesia (Hospital de Cabueñes, Gijón, Asturias); Luis López-Urrutia (Hospital Universitario Río Hortega, Valladolid); Santiago Salso (Hospital HM Montepríncipe, Boadilla del Monte, Madrid); José Vicente Saz (Hospital Universitario Príncipe de Asturias); Sagrario Reyes (Hospital Severo Ochoa, Leganés, Madrid), José Cobos (Megalab, Madrid); Luisa García-Picazo (Hospital El Escorial, San Lorenzo del Escorial, Madrid); María Ortega-Lafont and Gregoria Megías-Lobón (Complejo Asistencial Universitario de Burgos, Burgos); Noelia Arenal Andrés and Elisa Rodríguez Tarazona (Hospital Santos Reyes, Aranda de Duero, Burgos); Patricia Álvarez-García (Complejo Hospitalario de Pontevedra, Pontevedra); Dionisia Fontanals (Hospital Parc Taulí de Sabadell, Barcelona); Rafael Carranza (Hospital La Mancha Centro, Ciudad Real); Susana Hernando (Hospital General de Segovia, Segovia); M. Fe Brezmes (Complejo Asistencial de Zamora, Zamora); Luis Moisés Ruiz-Velasco (Hospital Central de la Cruz Roja San José y Santa Adela, Madrid); Paloma Cascales (Hospital de Elda Virgen de la Salud, Alicante); Carmen Guerrero (Hospital JM Morales Meseguer); Yolanda Gil (Hospital de Móstoles, Móstoles, Madrid); Irene Rodríguez-Conde (Hospital Povisa, Pontevedra); and Alicia Saez (Hospital Universitario Santa Cristina, Madrid).

This work was supported by Plan Nacional de I+D+i 2013-2016 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16CIII/0004/0002, RD16/0016/0008), cofinanced by the European Development Regional Fund ERDF (A way to achieve Europe) operative program Intelligent Growth 2014-2020. This work was also supported by a grant from the Instituto de Salud Carlos III (grant number MPY 1135/16) and by the Antibiotic Resistance Surveillance Program of the Centro Nacional de Microbiología (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad) of Spain.

We have no conflicts of interest to declare.

FOOTNOTES

    • Received 12 December 2018.
    • Returned for modification 22 January 2019.
    • Accepted 23 March 2019.
    • Accepted manuscript posted online 1 April 2019.
  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02529-18.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Logan LK,
    2. Weinstein RA
    . 2017. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 215:S28–S36. doi:10.1093/infdis/jiw282.
    OpenUrlCrossRef
  2. 2.↵
    1. Grundmann H,
    2. Glasner C,
    3. Albiger B,
    4. Aanensen DM,
    5. Tomlinson CT,
    6. Andrasević AT,
    7. Cantón R,
    8. Carmeli Y,
    9. Friedrich AW,
    10. Giske CG,
    11. Glupczynski Y,
    12. Gniadkowski M,
    13. Livermore DM,
    14. Nordmann P,
    15. Poirel L,
    16. Rossolini GM,
    17. Seifert H,
    18. Vatopoulos A,
    19. Walsh T,
    20. Woodford N,
    21. Monnet DL,
    22. Koraqi A,
    23. Lacej D,
    24. Apfalter P,
    25. Hartl R,
    26. Glupczynski Y,
    27. Huang T-D,
    28. Strateva T,
    29. Marteva-Proevska Y,
    30. Andrasevic AT,
    31. Butic I,
    32. Pieridou-Bagatzouni D,
    33. Maikanti-Charalampous P,
    34. Hrabak J,
    35. Zemlickova H,
    36. Hammerum A,
    37. Jakobsen L,
    38. Ivanova M,
    39. Pavelkovich A,
    40. Jalava J,
    41. Österblad M,
    42. Dortet L,
    43. Vaux S,
    44. Kaase M,
    45. Gatermann SG,
    46. Vatopoulos A,
    47. Tryfinopoulou K,
    48. Tóth Á,
    49. Jánvári L,
    50. Boo TW
    , et al. 2017. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study. Lancet Infect Dis 17:153–163. doi:10.1016/S1473-3099(16)30257-2.
    OpenUrlCrossRef
  3. 3.↵
    1. Arana DM,
    2. Ortega A,
    3. González-Barberá E,
    4. Lara N,
    5. Bautista V,
    6. Gómez-Ruíz D,
    7. Sáez D,
    8. Fernández-Romero S,
    9. Aracil B,
    10. Pérez-Vázquez M,
    11. Campos J,
    12. Oteo J
    , Spanish Antibiotic Resistance Surveillance Programme Collaborating Group. 2017. Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2. J Antimicrob Chemother 72:3283–3287. doi:10.1093/jac/dkx325.
    OpenUrlCrossRef
  4. 4.↵
    1. Ortega A,
    2. Sáez D,
    3. Bautista V,
    4. Fernández-Romero S,
    5. Lara N,
    6. Aracil B,
    7. Pérez-Vázquez M,
    8. Campos J,
    9. Oteo J
    , Spanish Collaborating Group for the Antibiotic Resistance Surveillance Programme. 2016. Carbapenemase-producing Escherichia coli is becoming more prevalent in Spain mainly because of the polyclonal dissemination of OXA-48. J Antimicrob Chemother 71:2131–2138. doi:10.1093/jac/dkw148.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Oteo J,
    2. Ortega A,
    3. Bartolomé R,
    4. Bou G,
    5. Conejo C,
    6. Fernández-Martínez M,
    7. González-López JJ,
    8. Martínez-García L,
    9. Martínez-Martínez L,
    10. Merino M,
    11. Miró E,
    12. Mora M,
    13. Navarro F,
    14. Oliver A,
    15. Pascual Á,
    16. Rodríguez-Baño J,
    17. Ruiz-Carrascoso G,
    18. Ruiz-Garbajosa P,
    19. Zamorano L,
    20. Bautista V,
    21. Pérez-Vázquez M,
    22. Campos J
    , GEIH-GEMARA (SEIMC) and REIPI. 2015. Prospective multicenter study of carbapenemase-producing Enterobacteriaceae from 83 hospitals in Spain reveals high in vitro susceptibility to colistin and meropenem. Antimicrob Agents Chemother 59:3406–3412. doi:10.1128/AAC.00086-15.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Wang J,
    2. Yuan M,
    3. Chen H,
    4. Chen X,
    5. Jia Y,
    6. Zhu X,
    7. Bai L,
    8. Bai X,
    9. Fanning S,
    10. Lu J,
    11. Li J
    . 2017. First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4, and KPC-2 carbapenemases. Antimicrob Agents Chemother 61:e00877-17. doi:10.1128/AAC.00877-17.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. De Belder D,
    2. Lucero C,
    3. Rapoport M,
    4. Rosato A,
    5. Faccone D,
    6. Petroni A,
    7. Pasteran F,
    8. Albornoz E,
    9. Corso A,
    10. Gomez SA
    . 2018. Genetic diversity of KPC-producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii isolates from Argentina. Microb Drug Resist 24:958–965. doi:10.1089/mdr.2017.0213.
    OpenUrlCrossRef
  8. 8.↵
    1. Simon M,
    2. Melzl H,
    3. Hiergeist A,
    4. Richert K,
    5. Falgenhauer L,
    6. Pfeifer Y,
    7. Gerlach RG,
    8. Fuchs K,
    9. Reischl U,
    10. Gessner A,
    11. Jantsch J
    . 2017. Colistin- and carbapenem-resistant Klebsiella oxytoca harboring blaVIM-2 and an insertion in the mgrB gene isolated from blood culture. Int J Med Microbiol 307:113–115. doi:10.1016/j.ijmm.2017.01.001.
    OpenUrlCrossRef
  9. 9.↵
    1. Vergara-López S,
    2. Domínguez MC,
    3. Conejo MC,
    4. Pascual Á,
    5. Rodríguez-Baño J
    . 2015. Lessons from an outbreak of metallo-β-lactamase-producing Klebsiella oxytoca in an intensive care unit: the importance of time at risk and combination therapy. J Hosp Infect 89:123–131. doi:10.1016/j.jhin.2013.12.008.
    OpenUrlCrossRef
  10. 10.↵
    1. Izdebski R,
    2. Fiett J,
    3. Urbanowicz P,
    4. Baraniak A,
    5. Derde LP,
    6. Bonten MJ,
    7. Carmeli Y,
    8. Goossens H,
    9. Hryniewicz W,
    10. Brun-Buisson C,
    11. Brisse S,
    12. Gniadkowski M
    , MOSAR WP2, WP3 and WP5 Study Groups. 2015. Phylogenetic lineages, clones and β-lactamases in an international collection of Klebsiella oxytoca isolates non-susceptible to expanded-spectrum cephalosporins. J Antimicrob Chemother 70:3230–3237. doi:10.1093/jac/dkv273.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Cannatelli A,
    2. Giani T,
    3. D'Andrea MM,
    4. Di Pilato V,
    5. Arena F,
    6. Conte V,
    7. Tryfinopoulou K,
    8. Vatopoulos A,
    9. Rossolini GM
    , COLGRIT Study Group. 2014. MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin. Antimicrob Agents Chemother 58:5696–5703. doi:10.1128/AAC.03110-14.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    European Committee on Antimicrobial Susceptibility Testing. 2019. Break points tables for interpretation of MICs and zone diameters, version 9.0. European Society of Clinical Microbiology and Infectious Diseases. http://www.eucast.org/clinical_breakpoints/.
  13. 13.↵
    European Committee on Antimicrobial Susceptibility Testing. 2013. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance, version 1.0. European Society of Clinical Microbiology and Infectious Diseases. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms_v1.0_20131211.pdf.
  14. 14.↵
    1. Galimand M,
    2. Courvalin P,
    3. Lambert T
    . 2003. Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother 47:2565–2571. doi:10.1128/AAC.47.8.2565-2571.2003.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Uechi K,
    2. Tada T,
    3. Shimada K,
    4. Nakasone I,
    5. Sonozaki T,
    6. Kirikae T,
    7. Fujita J
    . 2018. Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan. J Infect Chemother 24:68–70. doi:10.1016/j.jiac.2017.09.006.
    OpenUrlCrossRef
  16. 16.↵
    1. Zhang Y,
    2. Zhou H,
    3. Shen XQ,
    4. Shen P,
    5. Yu YS,
    6. Li LJ
    . 2008. Plasmid-borne armA methylase gene, together with blaCTX-M-15 and blaTEM-1, in a Klebsiella oxytoca isolate from China. J Med Microbiol 57:1273–1276. doi:10.1099/jmm.0.2008/001271-0.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. González-Zorn B,
    2. Teshager T,
    3. Casas M,
    4. Porrero MC,
    5. Moreno MA,
    6. Courvalin P,
    7. Domínguez L
    . 2005. armA and aminoglycoside resistance in Escherichia coli. Emerg Infect Dis 11:954–956. doi:10.3201/eid1106.040553.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    1. Hidalgo L,
    2. Gutierrez B,
    3. Ovejero CM,
    4. Carrilero L,
    5. Matrat S,
    6. Saba CK,
    7. Santos-Lopez A,
    8. Thomas-Lopez D,
    9. Hoefer A,
    10. Suarez M,
    11. Santurde G,
    12. Martin-Espada C,
    13. Gonzalez-Zorn B
    . 2013. Klebsiella pneumoniae sequence type 11 from companion animals bearing ArmA methyltransferase, DHA-1 β-lactamase, and QnrB4. Antimicrob Agents Chemother 57:4532–4534. doi:10.1128/AAC.00491-13.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Ho PL,
    2. Lo WU,
    3. Yeung MK,
    4. Lin CH,
    5. Chow KH,
    6. Ang I,
    7. Tong AH,
    8. Bao JY,
    9. Lok S,
    10. Lo JY
    . 2011. Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong. PLoS One 6:e17989. doi:10.1371/journal.pone.0017989.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Fevre C,
    2. Jbel M,
    3. Passet V,
    4. Weill FX,
    5. Grimont PA,
    6. Brisse S
    . 2005. Six groups of the OXY β-lactamase evolved over millions of years in Klebsiella oxytoca. Antimicrob Agents Chemother 49:3453–3462. doi:10.1128/AAC.49.8.3453-3462.2005.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Herzog KA,
    2. Schneditz G,
    3. Leitner E,
    4. Feierl G,
    5. Hoffmann KM,
    6. Zollner-Schwetz I,
    7. Krause R,
    8. Gorkiewicz G,
    9. Zechner EL,
    10. Högenauer C
    . 2014. Genotypes of Klebsiella oxytoca isolates from patients with nosocomial pneumonia are distinct from those of isolates from patients with antibiotic-associated hemorrhagic colitis. J Clin Microbiol 52:1607–1616. doi:10.1128/JCM.03373-13.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Holt KE,
    2. Baker S,
    3. Weill FX,
    4. Holmes EC,
    5. Kitchen A,
    6. Yu J,
    7. Sangal V,
    8. Brown DJ,
    9. Coia JE,
    10. Kim DW,
    11. Choi SY,
    12. Kim SH,
    13. da Silveira WD,
    14. Pickard DJ,
    15. Farrar JJ,
    16. Parkhill J,
    17. Dougan G,
    18. Thomson NR
    . 2012. Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe. Nat Genet 44:1056–1059. doi:10.1038/ng.2369.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Psichogiou M,
    2. Tassios PT,
    3. Avlamis A,
    4. Stefanou I,
    5. Kosmidis C,
    6. Platsouka E,
    7. Paniara O,
    8. Xanthaki A,
    9. Toutouza M,
    10. Daikos GL,
    11. Tzouvelekis LS
    . 2008. Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens, Greece: a prospective survey. J Antimicrob Chemother 61:59–63. doi:10.1093/jac/dkm443.
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    1. Villa J,
    2. Viedma E,
    3. Brañas P,
    4. Orellana MA,
    5. Otero JR,
    6. Chaves F
    . 2014. Multiclonal spread of VIM-1-producing Enterobacter cloacae isolates associated with In624 and In488 integrons located in an IncHI2 plasmid. Int J Antimicrob Agents 43:451–455. doi:10.1016/j.ijantimicag.2014.02.006.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Villa J,
    2. Arana DM,
    3. Viedma E,
    4. Perez-Montarelo D,
    5. Chaves F
    . 2017. Characterization of mobile genetic elements carrying VIM-1 and KPC-2 carbapenemases in Citrobacter freundii isolates in Madrid. Int J Med Microbiol 307:340–345. doi:10.1016/j.ijmm.2017.07.001.
    OpenUrlCrossRef
  26. 26.↵
    1. Giakkoupi P,
    2. Xanthaki A,
    3. Kanelopoulou M,
    4. Vlahaki A,
    5. Miriagou V,
    6. Kontou S,
    7. Papafraggas E,
    8. Malamou-Lada H,
    9. Tzouvelekis LS,
    10. Legakis NJ,
    11. Vatopoulos AC
    . 2003. VIM-1 metallo-beta-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals. J Clin Microbiol 41:3893–3896. doi:10.1128/JCM.41.8.3893-3896.2003.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Leavitt A,
    2. Chmelnitsky I,
    3. Carmeli Y,
    4. Navon-Venezia S
    . 2010. Complete nucleotide sequence of KPC-3-encoding plasmid pKpQIL in the epidemic Klebsiella pneumoniae sequence type 258. Antimicrob Agents Chemother 54:4493–4496. doi:10.1128/AAC.00175-10.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Oteo J,
    2. Pérez-Vázquez M,
    3. Bautista V,
    4. Ortega A,
    5. Zamarrón P,
    6. Saez D,
    7. Fernández-Romero S,
    8. Lara N,
    9. Ramiro R,
    10. Aracil B,
    11. Campos J
    , Spanish Antibiotic Resistance Surveillance Program Collaborating Group. 2016. The spread of KPC-producing Enterobacteriaceae in Spain: WGS analysis of the emerging high-risk clones of Klebsiella pneumoniae ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3. J Antimicrob Chemother 71:3392–3399. doi:10.1093/jac/dkw321.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Sugawara Y,
    2. Akeda Y,
    3. Sakamoto N,
    4. Takeuchi D,
    5. Motooka D,
    6. Nakamura S,
    7. Hagiya H,
    8. Yamamoto N,
    9. Nishi I,
    10. Yoshida H,
    11. Okada K,
    12. Zin KN,
    13. Aye MM,
    14. Tomono K,
    15. Hamada S
    . 2017. Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar. PLoS One 12:e0184720. doi:10.1371/journal.pone.0184720.
    OpenUrlCrossRef
  30. 30.↵
    1. Papagiannitsis CC,
    2. Kutilova I,
    3. Medvecky M,
    4. Hrabak J,
    5. Dolejska M
    . 2017. Characterization of the complete nucleotide sequences of IncA/C(2) plasmids carrying In809-like integrons from Enterobacteriaceae isolates of wildlife origin. Antimicrob Agents Chemother 61:e01093-17. doi:10.1128/AAC.01093-17.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Oteo J,
    2. Saez D,
    3. Bautista V,
    4. Fernández-Romero S,
    5. Hernández-Molina JM,
    6. Pérez-Vázquez M,
    7. Aracil B,
    8. Campos J
    , Spanish Collaborating Group for the Antibiotic Resistance Surveillance Program. 2013. Carbapenemase-producing Enterobacteriaceae in Spain in 2012. Antimicrob Agents Chemother 57:6344–6347. doi:10.1128/AAC.01513-13.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    CLSI-EUCAST Joint Polymyxins Working Group. 2016. Recommendations of the CLSI-EUCAST Joint Polymyxins Working Group. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Recommendations_for_MIC_determination_of_colistin_March_2016.pdf.
  33. 33.↵
    1. Dortet L,
    2. Bréchard L,
    3. Cuzon G,
    4. Poirel L,
    5. Nordmann P
    . 2014. Strategy for rapid detection of carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 58:2441–2445. doi:10.1128/AAC.01239-13.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Oteo J,
    2. Navarro C,
    3. Cercenado E,
    4. Delgado-Iribarren A,
    5. Wilhelmi I,
    6. Orden B,
    7. García C,
    8. Miguelañez S,
    9. Pérez-Vázquez M,
    10. García-Cobos S,
    11. Aracil B,
    12. Bautista V,
    13. Campos J
    . 2006. Spread of Escherichia coli strains with high-level cefotaxime and ceftazidime resistance between the community, long-term care facilities, and hospital institutions. J Clin Microbiol 44:2359–2366. doi:10.1128/JCM.00447-06.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Lima Barbieri N,
    2. Nielsen DW,
    3. Wannemuehler Y,
    4. Cavender T,
    5. Hussein A,
    6. Yan SG,
    7. Nolan LK,
    8. Logue CM
    . 2017. mcr-1 identified in avian pathogenic Escherichia coli (APEC). PLoS One 12:e0172997. doi:10.1371/journal.pone.0172997.
    OpenUrlCrossRef
  36. 36.↵
    1. Pérez-Pérez FJ,
    2. Hanson ND
    . 2002. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153–2162. doi:10.1128/JCM.40.6.2153-2162.2002.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Gastmeier P,
    2. Schwab F,
    3. Bärwolff S,
    4. Rüden H,
    5. Grundmann H
    . 2006. Correlation between the genetic diversity of nosocomial pathogens and their survival time in intensive care units. J Hosp Infect 62:181–186. doi:10.1016/j.jhin.2005.08.010.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    1. Bankevich A,
    2. Nurk S,
    3. Antipov D,
    4. Gurevich AA,
    5. Dvorkin M,
    6. Kulikov AS,
    7. Lesin VM,
    8. Nikolenko SI,
    9. Pham S,
    10. Prjibelski AD,
    11. Pyshkin AV,
    12. Sirotkin AV,
    13. Vyahhi N,
    14. Tesler G,
    15. Alekseyev MA,
    16. Pevzner PA
    . 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi:10.1089/cmb.2012.0021.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Boetzer M,
    2. Henkel CV,
    3. Jansen HJ,
    4. Butler D,
    5. Pirovano W
    . 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. doi:10.1093/bioinformatics/btq683.
    OpenUrlCrossRefPubMedWeb of Science
  40. 40.↵
    1. Nadalin F,
    2. Vezzi F,
    3. Policriti A
    . 2012. GapFiller: a de novo assembly approach to fill the gap within paired reads. BMC Bioinformatics 13:S8. doi:10.1186/1471-2105-13-S14-S8.
    OpenUrlCrossRefPubMed
  41. 41.↵
    1. Seemann T
    . 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi:10.1093/bioinformatics/btu153.
    OpenUrlCrossRefPubMedWeb of Science
  42. 42.↵
    1. Inouye M,
    2. Dashnow H,
    3. Raven LA,
    4. Schultz MB,
    5. Pope BJ,
    6. Tomita T,
    7. Zobel J,
    8. Holt KE
    . 2014. SRST2: rapid genomic surveillance for public health and hospital microbiology labs. Genome Med 6:90. doi:10.1186/s13073-014-0090-6.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Gupta SK,
    2. Padmanabhan BR,
    3. Diene SM,
    4. Lopez-Rojas R,
    5. Kempf M,
    6. Landraud L,
    7. Rolain JM
    . 2014. ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother 58:212–220. doi:10.1128/AAC.01310-13.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Rónai Z,
    2. Kreizinger Z,
    3. Dán Á,
    4. Drees K,
    5. Foster JT,
    6. Bányai K,
    7. Marton S,
    8. Szeredi L,
    9. Jánosi S,
    10. Gyuranecz M
    . 2015. First isolation and characterization of Brucella microti from wild boar. BMC Vet Res 11:147. doi:10.1186/s12917-015-0456-z.
    OpenUrlCrossRef
  45. 45.↵
    1. Sola-Campoy PJ,
    2. Monzón S,
    3. Pérez-Vázquez M
    . 2018. PlasmidID: a mapping based tool for plasmid identification, annotation, typing and visualization, abstr 6606. Abstr 28th Congr Clin Microbiol Infect Dis, Madrid, Spain.
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Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016–2017
María Pérez-Vazquez, Jesús Oteo-Iglesias, Pedro J. Sola-Campoy, Hugo Carrizo-Manzoni, Verónica Bautista, Noelia Lara, Belén Aracil, Almudena Alhambra, Luis Martínez-Martínez, José Campos, the Spanish Antibiotic Resistance Surveillance Program Collaborating Group
Antimicrobial Agents and Chemotherapy May 2019, 63 (6) e02529-18; DOI: 10.1128/AAC.02529-18

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Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016–2017
María Pérez-Vazquez, Jesús Oteo-Iglesias, Pedro J. Sola-Campoy, Hugo Carrizo-Manzoni, Verónica Bautista, Noelia Lara, Belén Aracil, Almudena Alhambra, Luis Martínez-Martínez, José Campos, the Spanish Antibiotic Resistance Surveillance Program Collaborating Group
Antimicrobial Agents and Chemotherapy May 2019, 63 (6) e02529-18; DOI: 10.1128/AAC.02529-18
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KEYWORDS

Klebsiella oxytoca
carbapenemases
cgMLST
plasmids
whole-genome sequencing

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