RESUMEN
Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.
Asunto(s)
Borrelia burgdorferi , Enfermedad de Lyme , Humanos , Borrelia burgdorferi/genética , Genotipo , Secuenciación Completa del Genoma , Plásmidos/genéticaRESUMEN
The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.
Asunto(s)
Legionella pneumophila , Legionella , Enfermedad de los Legionarios , Neumonía , Humanos , Enfermedad de los Legionarios/diagnóstico , Estudios Retrospectivos , Legionella pneumophila/genética , Secuenciación Completa del Genoma , Brotes de Enfermedades , ADNRESUMEN
We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype. Even though it had the same spoligotype as M. caprae strains, single-nucleotide polymorphism-based phylogenetic analysis placed the isolate as a sister lineage distinct from M. caprae, most closely related to 5 previously sequenced genomes isolated from primates and elephants in Asia. We propose a new animal-associated lineage, La4, within MTBC.
Asunto(s)
Mycobacterium tuberculosis , Animales , Bangladesh/epidemiología , Genotipo , Humanos , Mycobacterium tuberculosis/genética , Filogenia , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito's reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. RESULTS: We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. CONCLUSIONS: Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.
Asunto(s)
Aedes , Aedes/genética , Animales , Femenino , Expresión Génica , Mosquitos Vectores/genética , Ovario , OviposiciónRESUMEN
Legionnaires' disease, a severe lung infection caused by the bacterium Legionella pneumophila, occurs as single cases or in outbreaks that are actively tracked by public health departments. To determine the point source of an outbreak, clinical isolates need to be compared to environmental samples to find matching isolates. One confounding factor is the genome plasticity of L. pneumophila, making an exact sequence comparison by whole-genome sequencing (WGS) challenging. Here, we present a WGS analysis pipeline, LegioCluster, that is designed to circumvent this problem by automatically selecting the best matching reference genome prior to mapping and variant calling. This approach reduces the number of false-positive variant calls, maximizes the fraction of all genomes that are being compared, and naturally clusters the isolates according to their reference strain. Isolates that are too distant from any genome in the database are added to the list of candidate references, thereby creating a new cluster. Short insertions or deletions are considered in addition to single-nucleotide polymorphisms for increased discriminatory power. This manuscript describes the use of this automated and "locked down" bioinformatic pipeline deployed at the New York State Department of Health's Wadsworth Center for investigating relatedness between clinical and environmental isolates. A similar pipeline has not been widely available for use to support these critically important public health investigations.
Asunto(s)
Legionella pneumophila , Enfermedad de los Legionarios , Análisis por Conglomerados , Biología Computacional , Brotes de Enfermedades , Humanos , Legionella pneumophila/genética , Enfermedad de los Legionarios/epidemiología , New YorkRESUMEN
Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multidrug-resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations that do not confer high levels of RIF resistance, as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF MICs compared to fully susceptible strains but remain phenotypically susceptible by mycobacterial growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here, we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1,779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 2.5-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 µg/ml in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT but were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.
Asunto(s)
Mycobacterium tuberculosis , Preparaciones Farmacéuticas , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/farmacología , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , New York , Rifampin/farmacologíaRESUMEN
Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.
Asunto(s)
Legionella/aislamiento & purificación , Enfermedad de los Legionarios/microbiología , Brotes de Enfermedades , Agua Dulce/microbiología , Humanos , Legionella/clasificación , Legionella/genética , Enfermedad de los Legionarios/diagnóstico , Enfermedad de los Legionarios/epidemiología , New York/epidemiología , Abastecimiento de AguaRESUMEN
During 2016-2017, three rabid terrestrial animals were discovered in the raccoon rabies virus-free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently.
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Vacunas Antirrábicas , Virus de la Rabia , Rabia , Animales , Animales Salvajes , New York/epidemiología , Filogenia , Rabia/epidemiología , Rabia/veterinaria , Virus de la Rabia/genética , Mapaches , ZoonosisRESUMEN
Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.
Asunto(s)
Mycobacterium tuberculosis , Secuenciación de Nanoporos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , FilogeniaRESUMEN
In Escherichia coli, one sigma factor recognizes the majority of promoters, and six 'alternative' sigma factors recognize specific subsets of promoters. The alternative sigma factor FliA (σ28 ) recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, strongly suggest that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription may be a conserved phenomenon, but that specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence bacterial protein evolution and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Evolución Molecular , Salmonella typhimurium/genética , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Mapeo Cromosómico , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Factor sigma/genética , Transcripción Genética/genética , beta-Galactosidasa/genéticaRESUMEN
Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared with traditional diagnostic methods. Sequencing pathogens directly from a primary clinical specimen would help circumvent the need for culture and, in the process, substantially shorten the time to diagnosis and public health reporting. Unfortunately, this approach poses significant challenges because of the mixture of multiple sequences from a complex fecal biomass. The aim of this project was to develop a proof of concept protocol for the sequencing and genotyping of Shiga toxin-producing Escherichia coli (STEC) directly from stool specimens. We have developed an enrichment protocol that reliably achieves a substantially higher DNA yield belonging to E. coli, which provides adequate next-generation sequencing (NGS) data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, assess the STEC versus commensal E. coli population in the samples, and build consensus sequences based on population allele frequency distributions. Side-by-side analysis of WGS from paired STEC isolates and matched primary stool specimens reveal that this method can reliably be implemented for many clinical specimens to directly genotype STEC and accurately identify clusters of disease outbreak when no STEC isolate is available for testing.
Asunto(s)
Infecciones por Escherichia coli/diagnóstico , Heces/microbiología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Genoma Bacteriano/genética , Técnicas de Diagnóstico Molecular/métodos , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , ADN Bacteriano/genética , Monitoreo Epidemiológico , Escherichia coli/clasificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/epidemiología , Enfermedades Transmitidas por los Alimentos/epidemiología , Genotipo , Humanos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genéticaRESUMEN
Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury. In vivo, the effect of OAA was evaluated using the left portal vein ligation (LPVL) model of liver ischemia and a model of warm IRI. Liver injury was evaluated in vivo by serum transaminase levels, liver histology, and liver weight (edema). Levels and activity of caspase 3 were also measured. In vitro, the addition of OAA to hepatocytes kept in a hypoxic environment significantly improved cell viability (P < 0.01), decreased cell injury (P < 0.01), and improved energy metabolism (P < 0.01). Administration of OAA significantly reduced the extent of liver injury in the LPVL model with lower levels of alanine aminotransferase (ALT; P < 0.01), aspartate aminotransferase (AST; P < 0.01), and reduced liver necrosis (P < 0.05). When tested in a warm IRI model, OAA significantly decreased ALT (P < 0.001) and AST levels (P < 0.001), prevented liver edema (P < 0.001), significantly decreased caspase 3 expression (P < 0.01), as well as histological signs of cellular vesiculation and vacuolation (P < 0.05). This was associated with higher adenosine triphosphate (P < 0.05) and energy charge levels (P < 0.01). In conclusion, OAA can significantly improve survival of ischemic hepatocytes. The hepatoprotective effect of OAA was associated with increased levels of liver bioenergetics both in vitro and in vivo. These results suggest that it is possible to support mitochondrial activity despite the presence of ischemia and that OAA can effectively reduce ischemia-induced injury in the liver.
Asunto(s)
Trasplante de Hígado/efectos adversos , Ácido Oxaloacético/administración & dosificación , Sustancias Protectoras/administración & dosificación , Daño por Reperfusión/prevención & control , Isquemia Tibia/efectos adversos , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Cultivo Primario de Células , Ratas , Daño por Reperfusión/sangre , Daño por Reperfusión/etiologíaRESUMEN
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano , ARN Polimerasa Sigma 54/genética , Iniciación de la Transcripción Genética , Sitios de Unión , Proteínas de Unión al ADN/genética , Regiones Promotoras GenéticasRESUMEN
RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.
Asunto(s)
Mycobacterium/genética , ARN Mensajero/genética , Genes Bacterianos , Mycobacterium/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARNRESUMEN
We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in Stony Brook, New York, USA. Initial real-time PCR assay failed to provide a final subspecies identification within the M. tuberculosis complex, but whole-genome sequencing characterized the isolate as M. orygis.
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Genoma Bacteriano , Linfadenitis/diagnóstico , Mycobacterium/genética , Anciano , Emigrantes e Inmigrantes , Femenino , Humanos , India , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Linfadenitis/microbiología , Linfadenitis/patología , Mycobacterium/clasificación , Mycobacterium/aislamiento & purificación , New York , Filogenia , Secuenciación Completa del GenomaRESUMEN
During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires' disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires' disease.
Asunto(s)
Brotes de Enfermedades , Legionella pneumophila/aislamiento & purificación , Enfermedad de los Legionarios/epidemiología , Enfermedad de los Legionarios/microbiología , Abastecimiento de Agua , ADN Bacteriano , Microbiología Ambiental , Genoma Bacteriano , Humanos , Legionella pneumophila/clasificación , Legionella pneumophila/patogenicidad , New York/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Secuenciación Completa del GenomaRESUMEN
The incidence of Legionnaires' disease in the United States has been increasing since 2000. Outbreaks and clusters are associated with decorative, recreational, domestic, and industrial water systems, with the largest outbreaks being caused by cooling towers. Since 2006, 6 community-associated Legionnaires' disease outbreaks have occurred in New York City, resulting in 213 cases and 18 deaths. Three outbreaks occurred in 2015, including the largest on record (138 cases). Three outbreaks were linked to cooling towers by molecular comparison of human and environmental Legionella isolates, and the sources for the other 3 outbreaks were undetermined. The evolution of investigation methods and lessons learned from these outbreaks prompted enactment of a new comprehensive law governing the operation and maintenance of New York City cooling towers. Ongoing surveillance and program evaluation will determine if enforcement of the new cooling tower law reduces Legionnaires' disease incidence in New York City.
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Aire Acondicionado/efectos adversos , Brotes de Enfermedades , Legionella/aislamiento & purificación , Enfermedad de los Legionarios/epidemiología , Microbiología del Agua , Educación Médica Continua , Humanos , Incidencia , Enfermedad de los Legionarios/microbiología , Ciudad de Nueva York/epidemiologíaRESUMEN
Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.
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Farmacorresistencia Bacteriana , Técnicas de Genotipaje/métodos , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Secuenciación Completa del Genoma/métodos , Biología Computacional/métodos , Humanos , New York , Estudios Prospectivos , Estudios Retrospectivos , Tuberculosis/microbiologíaRESUMEN
Supermatrix and supertree analyses are frequently used to more accurately recover vertical evolutionary history but debate still exists over which method provides greater reliability. Traditional methods that resolve relationships among organisms from single genes are often unreliable because of the frequent lack of strong phylogenetic signal and the presence of systematic artifacts. Methods developed to reconstruct organismal history from multiple genes can be divided into supermatrix and supertree approaches. A supermatrix analysis consists of the concatenation of multiple genes into a single, possibly partitioned alignment, from which phylogenies are reconstructed using a variety of approaches. Supertrees build consensus trees from the topological information contained within individual gene trees. Both methods are now widely used and have been demonstrated to solve previously ambiguous or unresolved phylogenies with high statistical support. However, the amount of misleading signal needed to induce erroneous phylogenies for both strategies is still unknown. Using genome simulations, we test the accuracy of supertree and supermatrix approaches in recovering the true organismal phylogeny under increased amounts of horizontally transferred genes and changes in substitution rates. Our results show that overall, supermatrix approaches are preferable when a low amount of gene transfer is suspected to be present in the dataset, while supertrees have greater reliability in the presence of a moderate amount of misleading gene transfers. In the face of very high or very low substitution rates without horizontal gene transfers, supermatrix approaches outperform supertrees as individual gene trees remain unresolved and additional sequences contribute to a congruent phylogenetic signal.
Asunto(s)
Transferencia de Gen Horizontal , Modelos Genéticos , Filogenia , Biología Computacional , Simulación por Computador , Evolución Molecular , Genómica/estadística & datos numéricos , Alineación de Secuencia/estadística & datos numéricosRESUMEN
UNLABELLED: A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations ("outbreaks") that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCE: We demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.