RESUMEN
The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Análisis Costo-Beneficio , Pandemias , Biblioteca de Genes , ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
We describe a multiplex real-time PCR assay for use on the ABI 7500 Fast TaqMan platform to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-ß-lactamases (NDM), and the OXA-48-like family of carbapenemases from bacterial culture lysates or sample enrichment broth lysates.
Asunto(s)
Proteínas Bacterianas/análisis , Monitoreo Epidemiológico , Bacterias Gramnegativas/enzimología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recto/microbiología , beta-Lactamasas/análisis , Proteínas Bacterianas/genética , Bacterias Gramnegativas/genética , Humanos , beta-Lactamasas/genéticaRESUMEN
Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 µg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.
Asunto(s)
Antibacterianos/farmacología , Burkholderia/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Colistina/farmacología , Fosfomicina/análogos & derivados , Triterpenos Pentacíclicos/análisis , Membrana Celular/química , Sinergismo Farmacológico , Fosfomicina/farmacología , Pruebas de Sensibilidad Microbiana , Triterpenos Pentacíclicos/antagonistas & inhibidoresRESUMEN
A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8- and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Complejo Burkholderia cepacia/metabolismo , Permeabilidad de la Membrana Celular/genética , Polimixina B/farmacología , Esteroles/biosíntesis , 1-Naftilamina/análogos & derivados , 1-Naftilamina/metabolismo , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/genética , Membrana Celular/química , Membrana Celular/metabolismo , Colistina/metabolismo , Colistina/farmacología , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana , Biblioteca Genómica , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Mutación , Sistemas de Lectura Abierta , Polimixina B/metabolismoRESUMEN
Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.
Asunto(s)
COVID-19 , SARS-CoV-2 , Colombia Británica , Genoma Viral/genética , Genómica , Humanos , MutaciónAsunto(s)
Colistina/farmacología , Enfermedades Transmisibles Importadas , Farmacorresistencia Bacteriana , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/transmisión , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Viaje , Antibacterianos/farmacología , Canadá , China , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Distinuishing the species of mitis group streptococci is challenging due to ambiguous phenotypic characteristics and high degree of genetic similarity. This has been particularly true for resolving atypical Streptococcus pneumoniae and Streptococcus pseudopneumoniae. We used phylogenetic clustering to demonstrate specific and separate clades for both S. pneumoniae and S. pseudopneumoniae genomes. The genomes that clustered within these defined clades were used to extract species-specific genes from the pan-genome. The S. pneumoniae marker was detected in 8027 out of 8051 (>99.7â%) S. pneumoniae genomes. The S. pseudopneumoniae marker was specific for all genomes that clustered in the S. pseudopneumoniae clade, including unresolved species of the genus Streptococcus sequenced by the BC Centre for Disease Control Public Health Laboratory that previously could not be distinguished by other methods. Other than the presence of the S. pseudopneumoniae marker in six of 8051 (<0.08â%) S. pneumoniae genomes, both the S. pneumoniae and S. pseudopneumoniae markers showed little to no detectable cross-reactivity to the genomes of any other species of the genus Streptococcus or to a panel of over 46â000 genomes from viral, fungal, bacterial pathogens and microbiota commonly found in the respiratory tract. A real-time PCR assay was designed targeting these two markers. Genomics provides a useful technique for PCR assay design and development.
Asunto(s)
Genómica , Infecciones Estreptocócicas/diagnóstico , Streptococcus mitis/genética , Streptococcus pneumoniae/genética , Marcadores Genéticos , Genoma Bacteriano , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Serogrupo , Secuenciación Completa del GenomaRESUMEN
Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98-100% and 85-98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.