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BACKGROUND: Plague, a zoonotic disease caused by Yersinia pestis, was responsible for 3 historical human pandemics that killed millions of people. It remains endemic in rodent populations in Africa, Asia, North America, and South America but human plague is rare in most of these locations. However, human plague is still highly prevalent in Madagascar, which typically records a significant part of all annual global cases. This has afforded an opportunity to study contemporary human plague in detail using various typing methods for Y. pestis. AIM: This review aims to summarize the methods that have been used to type Y. pestis in Madagascar along with the major discoveries that have been made using these approaches. METHODS: Pubmed and Google Scholar were used to search for the keywords: "typing Yersinia pestis Madagascar," "evolution Yersinia pestis Madagascar," and "diversity Yersinia pestis Madagascar." Eleven publications were relevant to our topic and further information was retrieved from references cited in those publications. RESULTS: The history of Y. pestis typing in Madagascar can be divided in 2 periods: the pre-genomics and genomics eras. During the pre-genomics era, ribotyping, direct observation of plasmid content and plasmid restriction fragment length polymorphisms (RFLP) were employed but only revealed a limited amount of diversity among Malagasy Y. pestis strains. Extensive diversity only started to be revealed in the genomics era with the use of clustered regularly interspaced palindromic repeats (CRISPR), multiple-locus variable number tandem repeats (VNTR) analysis (MLVA), and single-nucleotide polymorphisms (SNPs) discovered from whole genome sequences. These higher-resolution genotyping methods have made it possible to highlight the distribution and persistence of genotypes in the different plague foci of Madagascar (Mahajanga and the Central and Northern Highlands) by genotyping strains from the same locations across years, to detect transfers between foci, to date the emergence of genotypes, and even to document the transmission of antimicrobial resistant (AMR) strains during a pneumonic plague outbreak. Despite these discoveries, there still remain topics that deserve to be explored, such as the contribution of horizontal gene transfer to the evolution of Malagasy Y. pestis strains and the evolutionary history of Y. pestis in Madagascar. CONCLUSIONS: Genotyping of Y. pestis has yielded important insights on plague in Madagascar, particularly since the advent of whole-genome sequencing (WGS). These include a better understanding of plague persistence in the environment, antimicrobial AMR and multi-drug resistance in Y. pestis, and the person-to-person spread of pneumonic plague. Considering that human plague is still a significant public health threat in Madagascar, these insights can be useful for controlling and preventing human plague in Madagascar and elsewhere, and also are relevant for understanding the historical pandemics and the possible use of Y. pestis as a biological weapon.
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Peste , Yersinia pestis , Yersinia pestis/genética , Yersinia pestis/clasificación , Yersinia pestis/aislamiento & purificación , Madagascar/epidemiología , Peste/microbiología , Peste/epidemiología , Humanos , Animales , Genotipo , Técnicas de Genotipaje/métodosRESUMEN
Bloodstream infection is a major public health concern associated with high mortality and high healthcare costs worldwide. Bacteremia can trigger fatal sepsis whose prevention, diagnosis, and management have been recognized as a global health priority by the World Health Organization. Additionally, infection control is increasingly threatened by antimicrobial resistance, which is the focus of global action plans in the framework of a One Health response. In-depth knowledge of the infection process is needed to develop efficient preventive and therapeutic measures. The pathogenesis of bloodstream infection is a dynamic process resulting from the invasion of the vascular system by bacteria, which finely regulate their metabolic pathways and virulence factors to overcome the blood immune defenses and proliferate. In this review, we highlight our current understanding of determinants of bacterial survival and proliferation in the bloodstream and discuss their interactions with the molecular and cellular components of blood.
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Bacterias , Humanos , Bacteriemia/microbiología , Factores de Virulencia , Sangre/microbiología , Viabilidad MicrobianaRESUMEN
Enteric yersiniosis, the third most common food-borne zoonosis in Europe, is mainly caused by the pathogen Yersinia enterocolitica. In France, the yersiniosis microbiological surveillance is conducted at the Yersinia National Reference Laboratory (YNRL). Since 2017, isolates have been characterized by whole genome sequencing (WGS) followed by a 500-gene Yersinia-cgMLST. We report here the data of the WGS-based surveillance on Y. enterocolitica isolates for the 2017-2021 period. The YNRL characterized 7,642 Y. enterocolitica strains distributed in 2,497 non-pathogenic isolates from lineages 1Aa and 1Ab, and 5,145 specimens belonging to 8 pathogenic lineages. Among pathogenic isolates, lineage 4 was the most common (87.2%) followed by lineages 2/3-9b (10.6%), 2/3-5a (1.2%), 2/3-9a (0.6%), 3-3b, 3-3c, 1B, and 3-3d (0.1% per each). Importantly, we developed a routine surveillance system based on a new typing method consisting of a 1,727-genes core genome Multilocus Sequence Typing (cgMLST) specific to the species Y. enterocolitica followed by isolate clustering. Thresholds of allelic distances (AD) were determined and fixed for the clustering of isolates: AD ≤ 5 for lineages 4, 2/3-5a, and 2/3-9a, and AD ≤ 3 for lineage 2/3-9b. Clustering programs were implemented in 2019 in routine surveillance to detect genomic clusters of pathogenic isolates. In total, 419 clusters with at least 2 isolates were identified, representing 2,504 of the 3,503 isolates characterized between 2019 and 2021. Most clusters (n = 325) comprised 2 to 5 isolates. The new typing method proved to be useful for the molecular investigation of unusual grouping of cases as well as for the detection of genomic clusters in routine surveillance. IMPORTANCE: We describe here the new typing method used for molecular surveillance of Yersinia enterocolitica infections in France based on a novel core genome Multilocus Sequence Typing (cgMLST) specific to Y. enterocolitica species. This method can reliably identify the pathogenic Y. enterocolitica subspecies and compare the isolates with a high discriminatory power. Between 2017 and 2021, 5,145 pathogenic isolates belonging to 8 lineages were characterized and lineage 4 was by far the most common followed by lineage 2/3-9b. A clustering program was implemented, and detection thresholds were cross-validated by the molecular and epidemiological investigation of three unusual groups of Y. enterocolitica infections. The routine molecular surveillance system has been able to detect genomic clusters, leading to epidemiological investigations.
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Brotes de Enfermedades , Tipificación de Secuencias Multilocus , Secuenciación Completa del Genoma , Yersiniosis , Yersinia enterocolitica , Yersinia enterocolitica/genética , Yersinia enterocolitica/aislamiento & purificación , Yersinia enterocolitica/clasificación , Yersiniosis/epidemiología , Yersiniosis/microbiología , Humanos , Francia/epidemiología , Tipificación de Secuencias Multilocus/métodos , Filogenia , Genoma Bacteriano/genética , Genómica/métodos , Monitoreo EpidemiológicoRESUMEN
Global burden of infectious diseases and antimicrobial resistance are major public health issues calling for innovative control measures. Bacterial NAD kinase (NADK) is a crucial enzyme for production of NADP(H) and growth. In Staphylococcus aureus, NADK promotes pathogenesis by supporting production of key virulence determinants. Here, we find that knockdown of NADK by CRISPR interference sensitizes S. aureus to osmotic stress and to stresses induced by antibiotics targeting the envelop as well as replication, transcription and translation. Thus, NADK represents a promising target for the development of inhibitors which could be used in combination with current antibiotics.
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Antibacterianos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Staphylococcus aureus , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del Gen , Presión Osmótica , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Estrés FisiológicoRESUMEN
Antimicrobial resistance has been considered a public health threat. The World Health Organization has warned about the urgency of detecting new antibiotics from novel sources. Social insects could be crucial in the search for new antibiotic metabolites, as some of them survive in places that favor parasite development. Recent studies have shown the potential of social insects to produce antimicrobial metabolites (e.g. ants, bees, and termites). However, most groups of social wasps remain unstudied. Here, we explored whether Actinobacteria are associated with workers in the Neotropical Social Wasps (Epiponini) of Costa Rica and evaluated their putative inhibitory activity against other bacteria. Most isolated strains (67%) have antagonistic effects, mainly against Bacillus thuringensis and Escherichia coli ATCC 25992. Based on genome analysis, some inhibitory Actinobacteria showed biosynthetic gene clusters (BGCs) related to the production of antimicrobial molecules such as Selvamycin, Piericidin A1, and Nystatin. The Actinobacteria could be associated with social wasps to produce antimicrobial compounds. For these reasons, we speculate that Actinobacteria associated with social wasps could be a novel source of antimicrobial compounds, mainly against Gram-negative bacteria.
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Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.
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Epidemias , Peste , Yersinia pestis , Humanos , Peste/epidemiología , Yersinia pestis/genética , Madagascar/epidemiología , GenómicaRESUMEN
Genomic data on the foodborne pathogen Listeria monocytogenes from Central America are scarce. We analyzed 92 isolates collected during 2009-2019 from different regions in Costa Rica, compared those to publicly available genomes, and identified unrecognized outbreaks. Our findings suggest mandatory reporting of listeriosis in Costa Rica would improve pathogen surveillance.
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Enfermedades Transmitidas por los Alimentos , Listeria monocytogenes , Listeriosis , Humanos , Listeria monocytogenes/genética , Enfermedades Transmitidas por los Alimentos/epidemiología , Costa Rica/epidemiología , Microbiología de Alimentos , Listeriosis/epidemiología , Brotes de EnfermedadesRESUMEN
BACKGROUND: The causative agent of plague, Yersinia pestis, is maintained in nature via a flea-rodent cycle. Western Iran is an old focus for plague, and recent data indicate that rodents and dogs in this region have serological evidence of Y. pestis infection. The purpose of this study was to conduct a large-scale investigation of Y. pestis infection in shepherd dogs, rodents, and their fleas in old foci for plague in Western Iran. MATERIALS AND METHODS: This study was conducted in Hamadan province from 2014 to 2020. Rodents and fleas were collected from various locations throughout this region. Y. pestis was investigated in rodent spleen samples and fleas using culture, serology, and real-time PCR methods. Additionally, sera samples were collected from carnivores and hares in this region, and the IgG antibody against the Y. pestis F1 antigen was assessed using an ELISA. RESULTS: In this study, 927 rodents were captured, with Meriones spp. (91.8%) and Microtus qazvinensis (2.6%) being the most prevalent. A total of 6051 fleas were collected from rodents and carnivores, most of which were isolated from Meriones persicus. None of the rodents or fleas examined tested positive for Y. pestis using real-time PCR and culture methods. Meanwhile, IgG antibodies were detected in 0.32% of rodents. All serologically positive rodents belonged to M. persicus. Furthermore, none of the sera from the 138 carnivores (129 sheepdogs, five Vulpes vulpes, four Canis aureus), and nine hares tested positive in the ELISA test. CONCLUSION: This primary survey of rodent reservoirs shows serological evidence of Y. pestis infection. Western Iran is an endemic plague focus, and as such, it requires ongoing surveillance.
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Infestaciones por Pulgas , Liebres , Peste , Siphonaptera , Yersinia pestis , Animales , Perros , Peste/epidemiología , Peste/veterinaria , Irán/epidemiología , Gerbillinae , Infestaciones por Pulgas/epidemiología , Infestaciones por Pulgas/veterinariaRESUMEN
We report the complete genome sequence of Yersinia pseudotuberculosis strain SP-1303, identified as part of lineage 8 and associated with Far East scarlet-like fever. The genome includes the chromosome, the Yersinia-virulence plasmid (pYV) encoding a type III secretion system essential for virulence, the pVM82 plasmid, and two cryptic plasmids.
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Brucella abortus is a zoonotic pathogen whose virulence depends on its ability to survive intracellularly at the endoplasmic reticulum derived compartment. The two-component system BvrR/BvrS (BvrRS) is essential for intracellular survival due to the transcriptional control of the type IV secretion system VirB and its transcriptional regulator VjbR. It is a master regulator of several traits including membrane homeostasis by controlling gene expression of membrane components, such as Omp25. BvrR phosphorylation is related to DNA binding at target regions, thereby repressing or activating gene transcription. To understand the role of BvrR phosphorylation we generated dominant positive and negative versions of this response regulator, mimicking phosphorylated and non-phosphorylated BvrR states and, in addition to the wild-type version, these variants were introduced in a BvrR negative background. We then characterized BvrRS-controlled phenotypes and assessed the expression of proteins regulated by the system. We found two regulatory patterns exerted by BvrR. The first pattern was represented by resistance to polymyxin and expression of Omp25 (membrane conformation) which were restored to normal levels by the dominant positive and the wild-type version, but not the dominant negative BvrR. The second pattern was represented by intracellular survival and expression of VjbR and VirB (virulence) which were, again, complemented by the wild-type and the dominant positive variants of BvrR but were also significantly restored by complementation with the dominant negative BvrR. These results indicate a differential transcriptional response of the genes controlled to the phosphorylation status of BvrR and suggest that unphosphorylated BvrR binds and impacts the expression of a subset of genes. We confirmed this hypothesis by showing that the dominant negative BvrR did not interact with the omp25 promoter whereas it could interact with vjbR promoter. Furthermore, a global transcriptional analysis revealed that a subset of genes responds to the presence of the dominant negative BvrR. Thus, BvrR possesses diverse strategies to exert transcriptional control on the genes it regulates and, consequently, impacting on the phenotypes controlled by this response regulator.
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Strains of the genus Tsukamurella were isolated from Polybia sp. social wasps from Costa Rica. Draft genome sequences from both isolates were obtained of ~4.5 Mb in length and with 68% GC content.
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Barton et al.1 raise several statistical concerns regarding our original analyses2 that highlight the challenge of inferring natural selection using ancient genomic data. We show here that these concerns have limited impact on our original conclusions. Specifically, we recover the same signature of enrichment for high FST values at the immune loci relative to putatively neutral sites after switching the allele frequency estimation method to a maximum likelihood approach, filtering to only consider known human variants, and down-sampling our data to the same mean coverage across sites. Furthermore, using permutations, we show that the rs2549794 variant near ERAP2 continues to emerge as the strongest candidate for selection (p = 1.2×10-5), falling below the Bonferroni-corrected significance threshold recommended by Barton et al. Importantly, the evidence for selection on ERAP2 is further supported by functional data demonstrating the impact of the ERAP2 genotype on the immune response to Y. pestis and by epidemiological data from an independent group showing that the putatively selected allele during the Black Death protects against severe respiratory infection in contemporary populations.
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Enfermedades Autoinmunes , Enfermedades del Sistema Inmune , Peste , Humanos , Autoanticuerpos , Selección GenéticaRESUMEN
The genus Yersinia includes a large variety of nonpathogenic and life-threatening pathogenic bacteria, which cause a broad spectrum of diseases in humans and animals, such as plague, enteritis, Far East scarlet-like fever (FESLF), and enteric redmouth disease. Like most clinically relevant microorganisms, Yersinia spp. are currently subjected to intense multi-omics investigations whose numbers have increased extensively in recent years, generating massive amounts of data useful for diagnostic and therapeutic developments. The lack of a simple and centralized way to exploit these data led us to design Yersiniomics, a web-based platform allowing straightforward analysis of Yersinia omics data. Yersiniomics contains a curated multi-omics database at its core, gathering 200 genomic, 317 transcriptomic, and 62 proteomic data sets for Yersinia species. It integrates genomic, transcriptomic, and proteomic browsers, a genome viewer, and a heatmap viewer to navigate within genomes and experimental conditions. For streamlined access to structural and functional properties, it directly links each gene to GenBank, the Kyoto Encyclopedia of Genes and Genomes (KEGG), UniProt, InterPro, IntAct, and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and each experiment to Gene Expression Omnibus (GEO), the European Nucleotide Archive (ENA), or the Proteomics Identifications Database (PRIDE). Yersiniomics provides a powerful tool for microbiologists to assist with investigations ranging from specific gene studies to systems biology studies. IMPORTANCE The expanding genus Yersinia is composed of multiple nonpathogenic species and a few pathogenic species, including the deadly etiologic agent of plague, Yersinia pestis. In 2 decades, the number of genomic, transcriptomic, and proteomic studies on Yersinia grew massively, delivering a wealth of data. We developed Yersiniomics, an interactive web-based platform, to centralize and analyze omics data sets on Yersinia species. The platform allows user-friendly navigation between genomic data, expression data, and experimental conditions. Yersiniomics will be a valuable tool to microbiologists.
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Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.
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ADN Antiguo , Predisposición Genética a la Enfermedad , Inmunidad , Peste , Selección Genética , Yersinia pestis , Humanos , Aminopeptidasas/genética , Aminopeptidasas/inmunología , Peste/genética , Peste/inmunología , Peste/microbiología , Peste/mortalidad , Yersinia pestis/inmunología , Yersinia pestis/patogenicidad , Selección Genética/inmunología , Europa (Continente)/epidemiología , Europa (Continente)/etnología , Inmunidad/genética , Conjuntos de Datos como Asunto , Londres/epidemiología , Dinamarca/epidemiologíaRESUMEN
During outbreaks, the lack of diagnostic "gold standard" can mask the true burden of infection in the population and hamper the allocation of resources required for control. Here, we present an analytical framework to evaluate and optimize the use of diagnostics when multiple yet imperfect diagnostic tests are available. We apply it to laboratory results of 2,136 samples, analyzed with 3 diagnostic tests (based on up to 7 diagnostic outcomes), collected during the 2017 pneumonic (PP) and bubonic plague (BP) outbreak in Madagascar, which was unprecedented both in the number of notified cases, clinical presentation, and spatial distribution. The extent of these outbreaks has however remained unclear due to nonoptimal assays. Using latent class methods, we estimate that 7% to 15% of notified cases were Yersinia pestis-infected. Overreporting was highest during the peak of the outbreak and lowest in the rural settings endemic to Y. pestis. Molecular biology methods offered the best compromise between sensitivity and specificity. The specificity of the rapid diagnostic test was relatively low (PP: 82%, BP: 85%), particularly for use in contexts with large quantities of misclassified cases. Comparison with data from a subsequent seasonal Y. pestis outbreak in 2018 reveal better test performance (BP: specificity 99%, sensitivity: 91%), indicating that factors related to the response to a large, explosive outbreak may well have affected test performance. We used our framework to optimize the case classification and derive consolidated epidemic trends. Our approach may help reduce uncertainties in other outbreaks where diagnostics are imperfect.
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Epidemias , Peste , Yersinia pestis , Brotes de Enfermedades , Humanos , Madagascar/epidemiología , Peste/diagnóstico , Peste/epidemiologíaRESUMEN
Yersinia pseudotuberculosis is an enteric pathogen causing mild enteritis that can lead to mesenteric adenitis in children and septicemia in elderly patients. Most cases are sporadic, but outbreaks have already been described in different countries. We report for the first time a Y. pseudotuberculosis clonal outbreak in France, that occurred in 2020. An epidemiological investigation based on food queries pointed toward the consumption of tomatoes as the suspected source of infection. The Yersinia National Reference Laboratory (YNRL) developed a new cgMLST scheme with 1,921 genes specific to Y. pseudotuberculosis that identified the clustering of isolates associated with the outbreak and allowed to perform molecular typing in real time. In addition, this method allowed to retrospectively identify isolates belonging to this cluster from earlier in 2020. This method, which does not require specific bioinformatic skills, is now used systematically at the YNRL and proves to display an excellent discriminatory power and is available to the scientific community. IMPORTANCE We describe in here a novel core-genome MLST method that allowed to identify in real time, and for the first time in France, a Y. pseudotuberculosis clonal outbreak that took place during the summer 2020 in Corsica. Our method allows to support epidemiological and microbiological investigations to establish a link between patients infected with closely associated Y. pseudotuberculosis isolates, and to identify the potential source of infection. In addition, we made this method available for the scientific community.
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Infecciones por Yersinia pseudotuberculosis , Yersinia pseudotuberculosis , Anciano , Niño , Brotes de Enfermedades , Humanos , Tipificación de Secuencias Multilocus/métodos , Estudios Retrospectivos , Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/epidemiología , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.
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Staphylococcus aureus , Factores de Virulencia , Animales , NAD/metabolismo , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Factores de Virulencia/genética , Pez Cebra/metabolismoRESUMEN
Strains of the genera Saccharopolyspora and Streptomyces were isolated from Protopolybia sp. and Metapolybia sp. social wasps in Costa Rica. Draft genome sequences were obtained for six isolates, ranging from 6.4 Mb to 9.1 Mb long and having GC contents of 71 to 73%.
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The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.