CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

Carbapenemases; High-risk clones; Whole genome sequencingCarbapenemasas; Clones de alto riesgo; Secuenciación del genoma completoCarbapenemases; Clons d'alt risc; Seqüenciació del genoma sencerObjectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.This research was supported by grants from the Instituto de Salud Carlos III (numbers PI18CIII/00030 and PI21CIII/00039). It was also supported by Plan Nacional de I + D + i 2013–2016, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (grants RD16CIII/0004/0002, RD16/0016/0001, RD16/0016/0003, RD16/0016/0004, RD16/0016/0006, RD16/0016/0007, RD16/0016/0008, RD16/0016/0010, and RD16/0016/0011). Cofinanced by the European Development Regional Fund “A way to achieve Europe,” Operative Program Intelligent Growth 2014–2020. CIBER – Consorcio Centro de Investigación Biomédica en Red (CB21/13/00095, CB21/13/00012, CB21/13/00049, CB21/13/00054, CB21/13/00055, CB21/13/00068, CB21/13/00081, CB21/13/00084, and CB21/13/00099) (CIBERINFEC) and Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU also supported this work

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE; Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.This study was supported by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16CIII/0004/0002), cofinanced by European Development Regional Fund ERDF “A Way To Achieve Europe,” operative program Intelligent Growth 2014-2020. This study was also supported by a grant from the Instituto de Salud Carlos III (grant 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. The Dutch CPE surveillance was funded by Dutch Ministry of Health, Welfare, and Sports.S

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.

CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA-48 (263/377), bla KPC-3 (62/377), bla VIM-1 (28/377), and bla NDM-1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Table_4_CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.pdf

[Objectives] CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.[Methods] In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.[Results] In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).[Conclusion] This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.Peer reviewe

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Centromeric Satellite DNA in Flatfish (Order Pleuronectiformes) and Its Relation to Speciation Processes

This work was supported by the Spanish Ministry of Science and Innovation (AGL2009-11872), PLEUROGENE (GEN2003) project funded by the Genome Canada-Genoma España joint programme, and the Consolider-Ingenio AQUAGENOMICS Project (CSD2007-00002).Two new centromeric satellite DNAs in flatfish (Order Pleuronectiformes) have been characterized. The SacI-family from Hippoglossus hippoglossus, restricted to this species, had a monomeric size of 334 base pair (bp) and was located in most of the centromeres of its karyotype. The PvuII-family, with a monomeric size of 177 bp, was initially isolated from the genome of Solea senegalensis, and fluorescent in situ hybridization (FISH) localized the repeat to centromeres of most of the chromosomes. This family could only be amplified in 2 other species of the genus Solea (Solea solea and Solea lascaris). Molecular features and chromosomal location indicated a possible structural and/or functional role of these sequence repeats. The presence of species-specific satellite-DNA families in the centromeres and their possible role in the speciation processes in this group of fishes is discussed.Departamento de Genética. Grupo BIO200. Facultad de Ciencias. Universidad de Granad

Prevalence, detection and characterisation of fosfomycin-resistant Escherichia coli strains carrying fosA genes in Community of Madrid, Spain.

The aim of this this study was to describe the presence of different variants of the fosA gene in fosfomycin-resistant Escherichia coli strains in Madrid, Spain. fos genes were searched for in 55 E. coli strains collected from seven representative hospitals located in Madrid. A phenotypic screening test was performed following the disk diffusion method with sodium phosphonoformate added as described by Nakamura et al. Additionally, a molecular study based on PCR was used to confirm the screening results. Positive strains for fos genes were further subjected to whole-genome sequencing (WGS). Phenotypic screening was positive in 9/55 strains (16.4%), although genotypic detection was positive in only 3 (fosA3, fosA4 and fosA6). Thus, the prevalence of fos genes in Madrid was 5.5% (3/55). WGS data were not available for the fosA6-positive strain. One isolate with fosA3 (ST69) carried a blaCTX-M-55 gene and seven virulence genes (air, eilA, iha, iss, lpfA, sat and senB). The fosA4-positive isolate (ST4038) carried the virulence genes iss, lpfA, iroN and mchF. Both fos genes were located between two IS26 mobile elements of a plasmid. We detected the presence of different variants of plasmid-mediated fosA genes in fosfomycin-resistant E. coli strains in Madrid, Spain. Despite the few reports in Europe, it would be of interest to monitor the spread of these acquired resistance genes.S

Nutrient uptake efficiency of five varieties of pistachio (Pistacia vera L.) varieties

Pistacia vera L., whose edible fruit is the pistachio, is an economically important crop. It is cultivated worldwide and over 50 different varieties have been described attending to morphological and phenological characteristics. The selection of a suitable cultivar may affect the profitability of the orchards, and thus requires careful consideration for any given region. The type and concentration of nutrients taken up by the plant affect its development and eventually the quality and quantity of the fruits. It is a matter of discussion whether all varieties have the same capacity to absorb nutrients. In order to clarify this aspect, in this study the efficiency of nutrient uptake has been assessed for five pistachio varieties: three males (M38, G1, and Mateur), and two females (Batoury, and Joley), by measuring the concentration of 30 chemical elements in leaves by Inductively Coupled Plasma (ICP) and C/N micro-elemental analyses. Data were subjected to a non-parametric Friedman test, using a series of Wilcoxon Rank Sum test with a Bonferroni correction as post hocs. Our findings demonstrate that all these varieties have an equal uptake capacity for Al, Cr, Cu, K, Li, Mn, Ni, Pb, S, Sr, Ti, Tl, Zn, N, B, Fe, Mg, Na, and V. No differences related to the gender of the plants were detected. Only Mateur exhibited significantly higher levels of Ca and lower levels of C. Stock plants from an experimental plot were used as material. These plants are not used for productive purposes, and flowering and fruiting are partially restricted by removing potential nutrient-demanding structures. These findings support the contention that the presence/absence of such organs has more influence on the nutrient uptake than does the variety itself.This work was supported by the project AGL 2009-09094, P.J.S.-C. is a FPI scholar, and R.N.-P. a Ramón y Cajal senior scientist (RYC-2011-08653), all funded by Ministerio de Ciencia e Innovación of Spain.Departamento de Genética. Grupo BIO200. Facultad de Ciencias. Universidad de Granad

The Forgotten Myrtle of the Alhambra Gardens of Granada: Restoring and Authenticating World Heritage

In the Alhambra (Granada, Spain), and in other Moorish locations, several individuals of the original variety of myrtle, the emblematic plant of their gardens, have been identified and genetically authenticated. After microsatellite analysis, we differentiated between the wild form (Myrtus communis L.) and two cultivated varieties: the one original to the Alhambra, the Moorish myrtle (subsp. baetica), and the variety introduced in more modern times (subsp. tarentina). The genetic and morphological differences between these two varieties confirm the taxonomic distinctness of the subsp. baetica. With very few individuals known, this Moorish myrtle is on the verge of extinction. The genetic identification offers the opportunity to restore a key element of this 14th-century garden and enhance the authenticity of a World Heritage site.Departamento de Genética. Grupo BIO200. Facultad de Ciencias. Universidad de GranadaDepartamento de BotánicaJardín Botánico de la Universidad de GranadaThis work was been partially supported under the Agreement C-3161-00/ 01, “Botanical Studio, Historiographical and Genetic Variety of Myrtus communis in the Alhambra and Generalife” by the Council of the Alhambra and the Generalife and the University of Granada