Incidence of amyotrophic lateral sclerosis in Chile.

OBJECTIVE: This study aimed to estimate amyotrophic lateral sclerosis (ALS) incidence and survival rates in the Metropolitan region of Chile. METHODS: We conducted a cohort study of ALS cases in the Metropolitan Region from 2016 to 2019. A total of 219 ALS patients were recruited from Corporación ELA-Chile registry, in collaboration with neurologists from Sociedad de Neurología, Psiquiatría y Neurocirugía de Chile. We calculated incidence rates by sex and age and determined median survival from onset and diagnosis. Survival analysis used the Kaplan-Meier statistic, estimating hazard ratios for age, sex, time from symptom onset and from diagnosis using a Weibull regression model. All analyses were done using R 4.1.0. RESULTS: Overall, ALS diagnosis incidence was 0.97 cases per 100,000 inhabitants, peaking in the 70-79 age group and declining thereafter. The male-to-female ratio was 1.23. The median time to death from diagnosis was 2.3 years (95% confidence interval [CI]: 1.9-2.5), and from the first symptom, it was 3.1 years (95% CI: 2.8-3.5). CONCLUSIONS: This is the first population-based study reporting ALS incidence and survival rates in Chile's Metropolitan region. Incidence resembled other Latin American studies. Median survival from diagnosis and from the first symptom were in line with previous findings. Our results corroborated lower ALS rates in Latin America, consistent with prior research.

The effects of cities on quail (Coturnix coturnix) migration: a disturbing story of population connectivity, health, and ecography.

The increasing impact of human activities on ecosystems is provoking a profound and dangerous effect, particularly in wildlife. Examining the historical migration patterns of quail (Coturnix coturnix) offers a compelling case study to demonstrate the repercussions of human actions on biodiversity. Urbanization trends, where people gravitate toward mega-urban areas, amplify this effect. The proliferation of artificial urban ecosystems extends its influence across every biome, as human reliance on infrastructure and food sources alters ecological dynamics extensively. We examine European quail migrations pre- and post-World War II and in the present day. Our study concentrates on the Italian peninsula, investigating the historical and contemporary recovery of ringed quail populations. To comprehend changes in quail migration, we utilize trajectory analysis, open statistical data, and linear generalized models. We found that while human population and economic growth have shown a linear increase, quail recovery rates exhibit a U-shaped trajectory, and cereal and legume production displays an inverse U-shaped pattern. Generalized linear models have unveiled the significant influence of several key factors-time periods, cereal and legume production, and human demographics-on quail recovery rates. These factors closely correlate with the levels of urbanization observed across these timeframes. These insights underscore the profound impact of expanding human populations and the rise of mega-urbanization on ecosystem dynamics and services. As our planet becomes more urbanized, the pressure on ecosystems intensifies, highlighting the urgent need for concerted efforts directed toward conserving and revitalizing ecosystem integrity. Simultaneously, manage the needs and demands of burgeoning mega-urban areas. Achieving this balance is pivotal to ensuring sustainable coexistence between urban improvement and the preservation of our natural environment.

Metaphyseal cones combined with diaphyseal impaction grafting provide good outcomes and survival in a complex revision total knee arthroplasty: a matched comparative analysis.

PURPOSE: To compare clinical and radiological outcomes, implant survivorship at a minimum of 1-year follow-up using metaphyseal trabecular cones with or without impaction grafting in a complex revision TKA. METHODS: A retrospective comparative matched analysis was performed and 15 patients who underwent revision surgery using the combination of porous trabecular metaphyseal cone and diaphyseal impaction grafting (MC-IBG) were matched with a group of 13 patients who also underwent revision surgery using metaphyseal cone (MC) without impaction grafting. All included patients presented severe bone defect (AORI 2,3) and a rotating-hinge prosthesis were implanted. RESULTS: Both groups were comparable regarding the baseline demographic and clinical data. Patients had previously undergone a mean of 4 (range, 2-12) and 3 (range, 2-5) previous procedures in the MC-IBG and MC groups, respectively. The indications for revision were aseptic loosening in 11 (73.3%) and 11 (84.6%) patients; prosthetic joint infection in 4 (26.7%) and 2 (15.4%) in the MC-IBG and MC groups, respectively. There was no significant difference in the mean postoperative Knee Society Score between the study groups (p = 0.806). Overall, 4 patients had further revision. Two patients were revised in the MC-IBG group, one patient for aseptic loosening and the second one after 2 episodes of instability. Two patients in the MC group presented prosthetic joint infection and underwent a two-stage reimplantation. No significant difference was observed between the study groups at comparison (p = 0.92). CONCLUSION: Trabecular metal cones with diaphyseal impaction grafting provide an alternative technique in a complex revision TKA surgery with early clinical and radiographic success.

LAENALS: epidemiological and clinical features of amyotrophic lateral sclerosis in Latin America.

OBJECTIVE: The Latin American Epidemiologic study of ALS (LAENALS) aims to gather data on ALS epidemiology, phenotype, and risk factors in Cuba, Chile, and Uruguay, to understand the impact of genetic and environmental factors on ALS. METHODS: A harmonized data collection protocol was generated, and a Latin-American Spanish language Register was constructed. Patient data were collected in Uruguay in 2018, in Chile from 2017 to 2019, and in Cuba between 2017 and 2018. Statistical analysis was performed using SPSS 25.0.0 software. Crude cumulative incidence, standardized incidence, and prevalence were calculated in the population aged 15 years and older. RESULTS: During 2017-2019, 90 people with ALS from Uruguay (55.6% men), 219 from Chile (54.6% men), and 49 from Cuba (55.1% men) were included. The cumulative crude incidence in 2018 was 1.73/100,000 persons in Uruguay, 1.08 in Chile and 0.195 in Cuba. Crude prevalence in 2018 was 2.19 per 100,000 persons in Uruguay, 1.39 in Chile and 0.55 in Cuba. Mean age at onset was 61.8 ± 11.96 SD years in Uruguay, 61.9 ± 10.4 SD years in Chile, and 60.21 ± 12.45 SD years in Cuba (p = 0.75). Median survival from onset was 32.43 months (21.93 - 42.36) in Uruguay, 24 months (13.5 - 33.5) in Chile, and 29 months (15 - 42.5) in Cuba (p = 0.006). CONCLUSIONS: These preliminary data from LAENALS confirm the lower incidence and prevalence of ALS in counties with admixed populations. The LAENALS database is now open to other Latin American countries for harmonized prospective data collection.

Reconstructing migratory network nodes to improve environmental management and conservation decisions: A case study of the common quail Coturnix coturnix as a biosensor.

Public health risks are related to environmental management practices, and are essential to the understanding of ecosystem dynamics. The expansion of urbanized areas affects migratory bird networks and can impact the population sizes of migratory species and, in parallel, increase the risk of diseases carried by migratory species spreading into urban areas. Taking the European population of the common quail as a case study, we reconstructed its migratory network between Europe and the Maghreb using quail recoveries from the Italian Bird Ringing Scheme. We showed that soils at the central European migratory node have been degraded through urbanization and reforestation, decreasing the number of successful migrations over this area. Conceptual models incorporating the One Health approach, natural and social capital, landscape infrastructure, and ecosystem services could explain the relationships between climate warming and extensively developed, intensively developed, and urbanized ecosystems. Failed migratory flights of quail over central Europe highlight not only the problems of infrastructure design and their impact on ecosystems services, but also on One Health parameters. The damage to migratory network nodes presents a global threat to biodiversity and can increase the spread of diseases. To address this challenge we propose: i) improvements in land quality; ii) transboundary migration monitoring programmes; and iii) management plans for migratory birds - the overall aim being to optimize infrastructures to improve the quality of human life. Lessons drawn from a better understanding of the migratory networks of quail in relation to different ecosystems provide useful tools to improve infrastructure management and political decision making processes.

Ene-adducts from 1,4-dihydropyridines and α,ß-unsaturated nitriles: asynchronous transition states displaying aromatic features.

Hydride transfer reactions involving 1,4-dihydropyridines play a central role in bioorganic chemistry as they represent an important share of redox metabolism. For this class of reactions, direct hydride transfer is the commonly accepted mechanism; however, an Alder-Ene-like pathway has been proposed as a plausible alternative. The reaction between 1,4-ditrimethylsilyl-1,4-dihydropyridine and α,ß-unsaturated nitriles is a solid candidate for this latter pathway. In this work, we perform high level ab initio and density functional theory computations to characterize the mechanism of this reaction, taking into account diverse reaction paths, and evaluating the effect of solvent polarity and variations in the chemical structure. Our analysis explains the stereochemical aspects of the reaction, characterizing the up to now unresolved spatial configurations of the predominant products, and may contribute to the understanding of enzymatic reactions involving NADP(H). The reactions are found to proceed in an asynchronous fashion, with transition states that display significant aromatic features. With this observation in mind, Alder-Ene and direct hydride transfer pathways can be understood as two extremes of a continuous mechanistic spectrum for this kind of reaction, with the analyzed systems located approximately equidistant from both ends.

Prognostic Factors and Midterm Clinical Outcome of Transtibial Pullout and Partial Meniscectomy for Medial Meniscus Posterior Root Tears in Middle-Aged Patients.

Purpose: The purpose of this study was to compare clinical and radiological outcomes of transtibial pullout technique and partial meniscectomy, and to establish prognostic factors in middle-aged patients with mild knee osteoarthritis. Methods: A comparative case-control analysis was conducted. 65 patients between 40 and 60 years of age were included into two groups: 30 patients who underwent transtibial pullout technique (group 1) and 35 patients who underwent partial meniscectomy (group 2). Mean follow-up was set at 27.2 months. Primary clinical outcomes included Knee injury and Osteoarthritis Outcome Score and Lysholm Knee Questionnare. Preoperative MRI and intraoperative arthroscopic findings were recorded. The correlation between these findings and patient-reported subjective outcome were assessed. Results: The transtibial pullout group exhibited significantly greater improvement in clinical outcomes. A univariate model revealed that the presence of preoperative meniscal extrusion, body mass index (> 30), osteochondral defect, and female gender were predictors of poor clinical outcome. Multivariate regression analysis showed meniscal extrusion and osteochondral defect as significant prognostic factors for both study groups. Conclusion: Medial meniscus root repair leads to significantly improved clinical outcomes compared to partial meniscectomy in middle-aged patients with mild knee osteoarthritis. Presence of meniscal extrusion, osteochondral defect, BMI > 30, and female gender were deemed as predictors of poor postoperative patient-reported outcome. Level of Evidence: III therapeutic case-control study.

Intersubunit Coupling Enables Fast CO2-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.

The Latin American Epidemiology Network for ALS (Laenals).

Background: There is evolving evidence of non-uniform distribution of ALS worldwide, with apparently lower incident and prevalent rates outside populations of European origin. However, the phenotype, survival and environmental risk in populations of mixed ancestral origin have not been well established. Large scale population based studies of incidence, prevalence, phenotype and risk factors in admixed populations are necessary to determine the true demography of ALS, and to test the hypothesis of differential risk and phenotype in populations of mixed ancestry. Methods: The Latin American Epidemiological Network of ALS (LAENALS) has been established to perform a comparative analysis of ALS epidemiology between three different Latin American populations (Cuba, Uruguay and Chile), and to test the hypothesis that the demographics, phenotype and outcome of ALS are influenced by ancestral origin, and that environmental and occupational risk factors differ across different ethnicities due to subtle differences in gene- environmental interactions. Recognition and interrogation of these differences is an important step toward novel therapeutic approaches and personalized medicine for all ALS both in the US, and worldwide. Discussion: This work will enable direct and detailed comparative studies between different ancestral populations with varying degrees of admixture, with facility for comparison with a large European reference dataset for ALS, and will provide a unique and rich dataset of admixed populations for later comparative genomic studies.

Crossing artificial obstacles during migration: The relative global ecological risks and interdependencies illustrated by the migration of common quail Coturnix coturnix.

The increase of urban expansion, whereby soils become altered or filled with buildings through human action, presents a global threat to biodiversity and the spread of disease. Many of the factors determining bird migration routes and disease spread are poorly understood. We studied the migration routes of common quail Coturnix coturnix in western Europe. We examined the recoveries of ringed birds to characterize their migration trajectories to understand how this nocturnal migrant crosses artificial areas and predict the risk of migration collapse and disease transmission. We evaluated the possible consequences of quail collisions with human infrastructure elements (i.e., buildings, cranes, overhead cables and wires, and wind farm structures) to assess disease transmission in relation to the amount of urban soil. Our results show that variations in the amount of artificialized soil in central Europe are correlated with the relative absence of quail migratory routes. Conceptual models incorporating environmental ecology showed the relationships between climate warming, agroecosystems, and urban ecosystems as well as human health and economic growth. We predict a drastic loss of biodiversity and spread of disease if we do not curb the spread of land consumption. Taking a broad view of the interrelations discussed here allows predictions of global vulnerability and increased risks to health due to losses of biodiversity and ecosystem services. Lessons drawn from migration route maps of quail in relation to the distribution of urbanized soils provide tools for global conservation political decision making.

A comparison of the transtibial pullout technique and all-inside meniscal repair in medial meniscus posterior root tear: Prognostic factors and midterm clinical outcomes.

INTRODUCTION: There is a paucity of comparative clinical data between arthroscopic all-inside end-to-end meniscal root suture and transtibial pullout technique in medial meniscus posterior root tears (MMPRT). Therefore, this study aimed to compare treatment failure, mid-term clinical and radiological outcomes of all-inside meniscus root repair versus the transtibial pullout technique and to analyze prognostic factors of postoperative clinical and radiological outcomes. MATERIAL AND METHODS: Forty-four patients were included in two therapeutic group: arthroscopic all-inside meniscal suture (MS: 13 knees) and transtibial pullout technique (TP: 31 knees).Primary clinical outcomes at a minimum of 27.2 months postoperatively included Knee injury and Osteoarthritis Outcome Score (KOOS) and Lysholm Knee Questionnare (LKQ). Radiographic assessment of the knee was performed to determine osteoarthritis severity using the Kellgren-Lawrence (KL) grading system.In addition, pre and postoperative MRI scans and intraoperative arthroscopic findings were recorded and the correlations between these findings, as outcome predictors and postoperative patients reported subjective outcome were assessed. RESULTS: No significant differences in postoperative KOOS and LKQ scores were found between the MS and TP group (p = 0.38 and 0.17, respectively).During follow-up one patients (7.7%) in the MS group and two (6.5%) in the TP group underwent total knee arthroplasty. The difference did not reach the statistical significance (p = 0.88).No differences were observed in postoperative MRI findings such as meniscal extrusion, osteochondral defect, chondromalacia patella, and bone morrow edema (p = 0.25, 0.97, 0.97, and 0.88, respectively).A univariate model revealed that the presence of postoperative meniscal extrusion; osteochondral defect; chondromalacia patella; increased BMI; age (>40 years) and longer time lapse between clinical onset to surgery (>6 months) were predictors of poor clinical outcome. The presence of meniscal extrusion, osteochondral defect and chondropatia patella portended worst postoperative outcomes in a multiple linear regression model. CONCLUSIONS: It can be concluded that both techniques can reach good results when performed properly and if the injury pattern allows meniscal suture may be considered a treatment option for the management of MMPRT in well-selected patients.Patients with increased BMI, preoperative meniscal extrusion, chondromalacia patella, and osteochondral defect were at higher risk of poor clinical outcomes.

Atom-Condensed Fukui Function in Condensed Phases and Biological Systems and Its Application to Enzymatic Fixation of Carbon Dioxide.

Local reactivity descriptors such as atom-condensed Fukui functions are promising computational tools to study chemical reactivity at specific sites within a molecule. Their applications have been mainly focused on isolated molecules in their most stable conformation without considering the effects of the surroundings. Here we propose to combine quantum mechanics/molecular mechanics Born-Oppenheimer molecular dynamics simulations to obtain the microstates (configurations) of a molecular system using different representations of the molecular environment and calculate Boltzmann-weighted atom-condensed local reactivity descriptors based on conceptual density functional theory. Our approach takes the conformational fluctuations of the molecular system and the polarization of its electron density by the environment into account, allowing us to analyze the effect of the molecular environment on reactivity. In this contribution, we apply the method mentioned above to the catalytic fixation of carbon dioxide by crotonyl-CoA carboxylase/reductase and study if the enzyme alters the reactivity of its substrate compared with an aqueous solution. Our main result is that the protein environment activates the substrate by the elimination of solute-solvent hydrogen bonds from aqueous solution in the two elementary steps of the reaction mechanism: the nucleophilic attack of a hydride anion from NADPH on the α,ß-unsaturated thioester and the electrophilic attack of carbon dioxide on the formed enolate species.

Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS.

OBJECTIVES: NDM carbapenemases have spread worldwide. However, little information exists about the impact of NDM-producing Enterobacteriaceae in Spain. By WGS, we sought to elucidate the population structure of NDM-like-producing Klebsiella pneumoniae and Escherichia coli in Spain and to determine the plasmids harbouring blaNDM-like genes. METHODS: High-resolution SNP typing, core-genome MLST and plasmid reconstruction (PlasmidID) were performed on 59 NDM-like-producing K. pneumoniae and 8 NDM-like-producing E. coli isolated over an 8 year period in Spain. RESULTS: Five major epidemic clones of NDM-producing K. pneumoniae caused five important nationwide outbreaks: ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1; in contrast, the spread of NDM-producing E. coli was polyclonal. Three blaNDM types were identified: blaNDM-1, 61.2%; blaNDM-7, 32.8%; and blaNDM-5, 6%. Five K. pneumoniae isolates co-produced other carbapenemases (three blaOXA-48 and two blaVIM-1). The average number of acquired resistance genes was higher in K. pneumoniae than in E. coli. The plasmids encoding blaNDM-like genes belonged to IncFII, IncFIB, IncX3, IncR, IncN and IncC types, of which IncF, IncR and IncC were associated with MDR. The genetic surroundings of blaNDM-like genes showed a highly variable region upstream of ISAba125. CONCLUSIONS: In recent years NDM-producing K. pneumoniae and E. coli have emerged in Spain; the spread of a few high-risk K. pneumoniae clones such as ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1 have caused several interregional outbreaks. In contrast, the spread of NDM-producing E. coli has been polyclonal. Plasmid types IncFII, IncFIB, IncX3, IncR, IncN and IncC carried blaNDM, and the same IncX3 plasmid was detected in K. pneumoniae and E. coli.

Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.

Awakening the Sleeping Carboxylase Function of Enzymes: Engineering the Natural CO2-Binding Potential of Reductases.

Developing new carbon dioxide (CO2) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology. Here we used bioinformatics to identify a "sleeping carboxylase function" in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. We show that propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO2 and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO2-binding and kinetic parameters comparable to naturally existing CO2-fixing enzymes. Our results provide a strategy to develop novel CO2-fixing enzymes and shed light on the emergence of natural carboxylases during evolution.

Electronic structure benchmark calculations of inorganic and biochemical carboxylation reactions.

Carboxylation reactions represent a very special class of chemical reactions that is characterized by the presence of a carbon dioxide (CO2 ) molecule as reactive species within its global chemical equation. These reactions work as fundamental gear to accomplish the CO2 fixation and thus to build up more complex molecules through different technological and biochemical processes. In this context, a correct description of the CO2 electronic structure turns out to be crucial to study the chemical and electronic properties associated with this kind of reactions. Here, a systematic study of CO2 electronic structure and its contribution to different carboxylation reaction electronic energies has been carried out by means of several high-level ab initio post-Hartree Fock (post-HF) and density functional theory (DFT) calculations for a set of biochemistry and inorganic systems. We have found that for a correct description of the CO2 electronic correlation energy it is necessary to include post-CCSD(T) contributions (beyond the gold standard). These high-order excitations are required to properly describe the interactions of the four π-electrons associated with the two degenerated π-molecular orbitals of the CO2 molecule. Likewise, our results show that in some reactions it is possible to obtain accurate reaction electronic energy values with computationally less demanding methods when the error in the electronic correlation energy compensates between reactants and products. Furthermore, the provided post-HF reference values allowed to validating different DFT exchange-correlation functionals combined with different basis sets for chemical reactions that are relevant in biochemical CO2 fixing enzymes. © 2019 Wiley Periodicals, Inc.

Catalytic Reaction Mechanism in Native and Mutant Catechol- O-methyltransferase from the Adaptive String Method and Mean Reaction Force Analysis.

Catechol- O-methyltransferase is an enzyme that catalyzes the methylation reaction of dopamine by S-adenosylmethionine, increasing the reaction rate by almost 16 orders of magnitude compared to the reaction in aqueous solution. Here, we combine the recently introduced adaptive string method and the mean reaction force method, in combination with the structural and electronic descriptors to characterize the reaction mechanism. The catalytic effect of the enzyme is addressed by the comparison of the reaction in the human wild-type enzyme, in the less effective Y68A mutant, and in aqueous solution. The influence of these different environments at different stages of the chemical process and the significance of the key collective variables describing the reaction were quantified. Our results show that the native enzyme limits the access of water molecules to the active site, enhancing the interaction between the reactants and providing a more favorable electrostatic environment to assist the SN2 methyl transfer reaction.

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.

OBJECTIVES: There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). METHODS: In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. RESULTS: Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. CONCLUSIONS: We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications.

The Carbapenemase-Producing Klebsiella pneumoniae Population Is Distinct and More Clonal than the Carbapenem-Susceptible Population.

We studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniae isolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group (P < 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers.

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.

OBJECTIVES: We analysed the microbiological traits and population structure of KPC-producing Enterobacteriaceae isolates collected in Spain between 2012 and 2014. We also performed a comparative WGS analysis of the three major KPC-producing Klebsiella pneumoniae clones detected. METHODS: Carbapenemase and ESBL genes were sequenced. The Institut Pasteur MLST scheme was used. WGS data were used to construct phylogenetic trees, to identify the determinants of resistance and to de novo assemble the genome of one representative isolate of each of the three major K. pneumoniae clones. RESULTS: Of the 2443 carbapenemase-producing Enterobacteriaceae isolates identified during the study period, 111 (4.5%) produced KPC. Of these, 81 (73.0%) were K. pneumoniae and 13 (11.7%) were Enterobacter cloacae. Three major epidemic clones of K. pneumoniae were identified: ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3. ST11/KPC-2 differed from ST101/KPC-2 and ST512/KPC-3 by 27 819 and 6924 SNPs, respectively. ST101/KPC-2 differed from ST512/KPC-3 by 28 345 SNPs. Nine acquired resistance genes were found in ST11/KPC-2, 11 in ST512/KPC-3 and 13 in ST101/KPC-2. ST101/KPC-2 had the highest number of virulence genes (20). An 11 bp deletion at the end of the mgrB sequence was the cause of colistin resistance in ST512/KPC-3. CONCLUSIONS: KPC-producing Enterobacteriaceae are increasing in Spain. Most KPC-producing K. pneumoniae isolates belonged to only five clones: ST11 and ST512 caused interregional spread, ST101 caused regional spread and ST1961 and ST678 produced independent hospital outbreaks. ST101/KPC-2 had the highest number of resistance and virulence genes. ST101/KPC-2 and ST512/KPC-3 were recently implicated in the spread of KPC in Italy.