499 research outputs found
Centre-specific bacterial pathogen typing affects infection-control decision making
Whole-genome sequencing is becoming the de facto standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a Klebsiella pneumoniae and a Vancomycin-resistant Enterococcus faecium, and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall–Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported de novo assemblies, did not result in uniformity of outbreak-cluster composition among centres
Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: an inter-laboratory study.
Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a 'one-stop' test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ('participants') were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories
Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene
Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic
Diseño de un método molecular para la identificación específica de Klebsiella pneumoniae a nivel de subespecie, usando el gen que codifica para la subunidad ribosomal 16S
Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by
K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are
unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective
results and patients must be following up for several years since the infection became chronic.
Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which
encodes 16S subunit ribosomal (rDNA16S).
Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics
programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized.
Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two
reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S
gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally
two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the
rDNA16S gene.
Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation
of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae
rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be
ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical
and reference strains. Introducción: El rinoescleroma es causado por Klebsiella pneumoniae rhinoscleromatis y la ocena por Klebsiella
pneumoniae ozaenae respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior y tienen
una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las dificultades
de establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, porque puede no ser
efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento puede implicar muchos años.
Objetivo: Diseñar un ensayo molecular para la identificación a nivel de subespecie de bacterias del género Klebsiella
basado en restricción de amplicones del gen que codifica para la subunidad ribosomal 16S (ADNr 16S).
Metodología: Se generaron patrones de restricción específicos, utilizando secuencias informadas del gen ADNr 16S y
los programas bioinformáticos MACAW, PFE, GENEDOC y GENE RUNNER. Se estandarizaron las condiciones para la amplificación y restricción para el ensayo experimental.
Resultados: Las predicciones in silico permitieron proponer
un algoritmo para la identificación a nivel de especie y
subespecie de las especies del género Klebsiella. Se incluyeron
dos cepas de referencia y dos aislados clínicos, que se
biotipificaron e identificaron por el método propuesto; los
patrones de restricción obtenidos del gen ADNr 16S evidenciaron
diferencias con respecto a la especie inicialmente
identificada por métodos convencionales. Además se encontraron
dos patrones de bandas en Klebsiella pneumoniae
rhinoscleromatis, indicando la presencia de polimorfismos
en el gen ADNr 16S para esta subespecie.
Conclusiones: Se confirmó la dificultad para identificar
Klebsiella pneumoniae a nivel de subespecie por métodos
convencionales. La implementación de esta técnica podría
permitir la diferenciación temprana entre Klebsiella
pneumoniae ozaenae y Klebsiella pneumoniae rhinoscleromatis
que causan dos infecciones tratadas por lo general
de forma empírica y como consecuencia de esto, la terapia
antimicrobiana suele no ser efectiva, en especial en pacientes
crónicos. Se requiere ampliar los estudios con un número
mayor de cepas de referencia y aislados clínicos
Status and potential of bacterial genomics for public health practice : a scoping review
Background: Next-generation sequencing (NGS) is increasingly being translated into routine public health practice, affecting the surveillance and control of many pathogens. The purpose of this scoping review is to identify and characterize the recent literature concerning the application of bacterial pathogen genomics for public health practice and to assess the added value, challenges, and needs related to its implementation from an epidemiologist’s perspective.
Methods: In this scoping review, a systematic PubMed search with forward and backward snowballing was performed to identify manuscripts in English published between January 2015 and September 2018. Included studies had to describe the application of NGS on bacterial isolates within a public health setting. The studied pathogen, year of publication, country, number of isolates, sampling fraction, setting, public health application, study aim, level of implementation, time orientation of the NGS analyses, and key findings were extracted from each study. Due to a large heterogeneity of settings, applications, pathogens, and study measurements, a descriptive narrative synthesis of the eligible studies was performed.
Results: Out of the 275 included articles, 164 were outbreak investigations, 70 focused on strategy-oriented surveillance, and 41 on control-oriented surveillance. Main applications included the use of whole-genome sequencing (WGS) data for (1) source tracing, (2) early outbreak detection, (3) unraveling transmission dynamics, (4) monitoring drug resistance, (5) detecting cross-border transmission events, (6) identifying the emergence of strains with enhanced virulence or zoonotic potential, and (7) assessing the impact of prevention and control programs. The superior resolution over conventional typing methods to infer transmission routes was reported as an added value, as well as the ability to simultaneously characterize the resistome and virulome of the studied pathogen. However, the full potential of pathogen genomics can only be reached through its integration with high-quality contextual data.
Conclusions: For several pathogens, it is time for a shift from proof-of-concept studies to routine use of WGS during outbreak investigations and surveillance activities. However, some implementation challenges from the epidemiologist’s perspective remain, such as data integration, quality of contextual data, sampling strategies, and meaningful interpretations. Interdisciplinary, inter-sectoral, and international collaborations are key for an appropriate genomics-informed surveillance
Structural Diversity of Class 1 integrons and their associated gene cassettes in Klebsiella pneumoniae isolates from a hospital in China
published_or_final_versio
Klebsiella pneumoniae subsp. rhinoscleromatis; Klebsiella pneumoniae subsp. ozaenae; Klebsiella pneumoniae subsp. pneumoniae; Diagnosis
Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections
caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the
symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot
reach effective results and patients must be following up for several years since the infection became chronic.
Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which
encodes 16S subunit ribosomal (rDNA16S).
Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and
bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were
standardized.
Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification.
Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method.
rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods.
Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms
presence in the rDNA16S gene.
Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation
of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae
subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually
could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more
clinical and reference strains. Introducción: El rinoescleroma es causado por Klebsiella pneumoniae subsp. rhinoscleromatis y la ocena por K.
pneumoniae subsp. ozaenae, respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior
y originan una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las
dificultades para establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, que puede
no ser efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento en el paciente puede necesitar
muchos años. Objetivo: Diseñar un ensayo molecular para la identificación
a nivel de subespecie de bacterias del género Klebsiella
basado en restricción de amplicones del gen que codifica
para la subunidad ribosomal 16S (ADNr 16S).
Metodología: Se generaron patrones de restricción específicos,
con secuencias informadas del gen ADNr 16S y
los programas bioinformáticos MACAW, PFE, GENEDOC
y GENE RUNNER. Se estandarizaron las condiciones para
la amplificación y restricción del ensayo experimental.
Resultados: Las predicciones in silico permitieron proponer
un algoritmo para identificar a nivel de especie y
subespecie los miembros del género Klebsiella. Se incluyeron
dos cepas de referencia y dos aislamientos clínicos, que
fueron biotipificados e identificados por el método propuesto;
los patrones de restricción obtenidos del gen ADNr 16S
evidenciaron diferencias respecto a la especie inicialmente
identificada por métodos convencionales. Además, se encontraron
dos patrones de bandas en K. pneumoniae.
rhinoscleromatis, que indican la presencia de polimorfismos
en el gen ADNr 16S para esta subespecie.
Conclusiones: Se confirmó la dificultad para identificar
K. pneumoniae a nivel de subespecie por métodos convencionales.
La implementación de esta técnica podría permitir
la diferenciación temprana entre K. pneumoniae. ozaenae y
K. pneumoniae. rhinoscleromatis que causan dos infecciones
tratadas por lo general de forma empírica y como
consecuencia de lo anterior, la terapia antimicrobiana suele
no ser efectiva, especialmente en enfermos crónicos. Se
requiere ampliar los estudios con un número mayor de cepas
de referencia y aislamientos clínicos
Integrating whole-genome sequencing within the National Antimicrobial Resistance Surveillance Program in the Philippines
Funding: This work was funded by the Newton Fund, Medical Research Council (UK) grant MR/N019296/1, Philippine Council for Health Research and Development project number FP160007. J.S. was partially supported by research grants RR025040 and U01CA207167 from the National Institutes of Health (NIH). S.A. and D.M.A. were additionally supported by the National Institute for Health Research (UK) Global Health Research Unit on genomic Surveillance of AMR(16_136_111) and by the Centre for Genomic Pathogen Surveillance (http://pathogensurveillance.net).National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor resistance trends and disseminate these data to AMR stakeholders. Whole-genome sequencing (WGS) can support surveillance by pinpointing resistance mechanisms and uncovering transmission patterns. However, genomic surveillance is rare in low- and middle-income countries. Here, we implement WGS within the established Antimicrobial Resistance Surveillance Program of the Philippines via a binational collaboration. In parallel, we characterize bacterial populations of key bug-drug combinations via a retrospective sequencing survey. By linking the resistance phenotypes to genomic data, we reveal the interplay of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying the expansion of specific resistance phenotypes that coincide with the growing carbapenem resistance rates observed since 2010. Our results enhance our understanding of the drivers of carbapenem resistance in the Philippines, while also serving as the genetic background to contextualize ongoing local prospective surveillance.Publisher PDFPeer reviewe
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