RESUMO
The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.
Assuntos
Genoma Bacteriano , Klebsiella oxytoca , Tipagem de Sequências Multilocus , Tipagem de Sequências Multilocus/métodos , Klebsiella oxytoca/genética , Klebsiella oxytoca/classificação , Klebsiella oxytoca/isolamento & purificação , Humanos , Genoma Bacteriano/genética , Filogenia , Infecções por Klebsiella/microbiologia , Sequenciamento Completo do Genoma , Técnicas de Tipagem Bacteriana/métodos , Genes Essenciais/genética , Reprodutibilidade dos TestesRESUMO
Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.
RESUMO
Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.
Assuntos
Bactérias , Genoma Bacteriano , Técnicas de Genotipagem , Tipagem de Sequências Multilocus , Sequenciamento por Nanoporos , Antígenos de Bactérias/genética , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Vacinas Bacterianas/genética , Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Bordetella pertussis/patogenicidade , Farmacorresistência Bacteriana/genética , Monitoramento Ambiental , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Tipagem de Sequências Multilocus/métodos , Sequenciamento por Nanoporos/métodos , Filogenia , Reprodutibilidade dos Testes , Fatores de Virulência/genéticaRESUMO
Group-living animals are often faced with complex reproductive decisions, namely how to partition within-group reproduction, how to obtain extra-group reproduction and how these two means of reproduction should be balanced. The solutions to these questions can be difficult to predict because ecological conditions can affect the scopes for within-group and extra-group reproduction in complex ways. For example, individuals that are restricted from moving freely around their habitats may have limited extra-group reproductive opportunities, but at the same time, groups may live in close proximity to one another, which could potentially have the opposite effect. The group-living cichlid fish Neolamprologus multifasciatus experiences such ecological conditions, and we conducted an intensive genetic parentage analysis to investigate how reproduction is distributed within and among groups for both males and females. We found that cohabiting males live in "high-skew" societies, where dominant males monopolize the majority of within-group reproduction, while females live in "low-skew" societies, where multiple females can produce offspring concurrently. Despite extremely short distances separating groups, we inferred only very low levels of extra-group reproduction, suggesting that subordinate males have very limited reproductive opportunities. A strength of our parentage analysis lies in its inclusion of individuals that spanned a wide age range, from young fry to adults. We outline the logistical circumstances when very young offspring may not always be accessible to parentage researchers, and present strategies to overcome the challenges of inferring mating patterns from a wide age range of offspring.
Assuntos
Ciclídeos , Animais , Ciclídeos/genética , Feminino , Humanos , Masculino , Reprodução/genética , Caracteres Sexuais , Comportamento Sexual AnimalRESUMO
Klebsiella oxytoca is a ubiquitous bacterium that is increasingly associated with inflammatory diseases. Here, we report the hybrid assembled genome for cytotoxic K. oxytoca strain AHC-6. The genome comprises a total of 5.7 Mbp, with a GC content of 55.2% and 5,258 coding sequences after assembly and annotation.
RESUMO
Group-living animals are faced with the challenge of sharing space and local resources amongst group members who may be either relatives or non-relatives. Individuals may reduce the inclusive fitness costs they incur from competing with relatives by either reducing their levels of aggression toward kin, or by maintaining physical separation between kin. In this field study, we used the group-living cichlid Neolamprologus multifasciatus to examine whether within-group aggression is reduced among group members that are kin, and whether kin occupy different regions of their group's territory to reduce kin competition over space and local resources. We determined the kinship relationships among cohabiting adults via microsatellite genotyping and then combined these with spatial and behavioral analyses of groups in the wild. We found that aggressive contests between group members declined in frequency with spatial separation between their shelters. Female kin did not engage in aggressive contests with one another, whereas non-kin females did, despite the fact these females lived at similar distances from one another on their groups' territories. Contests within male-male and male-female dyads did not clearly correlate with kinship. Non-kin male-male and male-female dyads lived at more variable distances from one another on their territories than their corresponding kin dyads. Together, our study indicates that contests among group members can be mediated by relatedness in a sex-dependent manner. We also suggest that spatial relationships can play an important role in determining the extent to which group members compete with one another.
RESUMO
BACKGROUND: Sex-biased dispersal is a common and widespread phenomenon that can fundamentally shape the genetic structure of the social environments in which animals live. For animals that live in and move between social groups, sex-biased dispersal can result in an asymmetry in the degree of relatedness among cohabiting males and females, which can have strong implications for their social evolution. In this study, we measured the relatedness structure within and across groups of a wild population of Neolamprologus multifasciatus, a highly-social, shell-dwelling cichlid fish endemic to Lake Tanganyika, East Africa. In total, we genotyped 812 fish from 128 social groups at 20 microsatellite loci. Neolamprologus multifasciatus live at high densities, and also experience strong ecological constraints on free movement throughout their habitat. At the same time, they exhibit sex differences in the degree of reproductive competition within their groups and this makes them an excellent model system for studying the factors associated with sex-biased dispersal. RESULTS: Social groups of N. multifasciatus consist of multiple males and females living together. We found that cohabiting females were unrelated to one another (Lynch-Ritland estimates of relatedness = 0.045 ± 0.15, average ± SD), while males shared much higher, albeit variable, levels of relatedness to other males in their groups (0.23 ± 0.27). We uncovered a pronounced decline in relatedness between males living in separate groups as the spatial separation between them increased, a pattern that was not evident in females. Female dispersal was also markedly constrained by the distribution and availability of nearby territories to which they could emigrate. CONCLUSIONS: Our results indicate female-biased dispersal in N. multifasciatus. Our study also highlights how the spatial distribution of suitable dispersal destinations can influence the movement decisions of animals. We also emphasize how sex-biased dispersal can influence the relatedness structure of the social environment in which individuals interact and compete with one another.